Edited by K.: Enzyme Catalysis in Organic Synthesis

Enzyme Catalysis in Organic Synthesis
Edited by K. Drauz and H. Waldmann
Second Edition
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A Comprehensive Handbook
Edited by
Karlheinz Drauz and Herbert Waldmann
Second, Completely Revised and Enlarged Edition
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Editors: This book was carefully produced. Nevertheless
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Prot Dr. Karlheinz Drauz information contained therein to be free of er-
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63457 Hanau-Wolfgang
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I'
Foreword
That biological systems are masterful chemists is a fact long appreciated by those
who study how living things build complexity from simple compounds in the
environment. Enzymes catalyze the interconversion of vast numbers of chemical
species, providing materials and energy to fuel cell survival and growth. Enzymes
build the intricate natural products, which, for their potential utility in treating
disease, pose almost unlimited new challenges for ambitious synthetic chemists.
But, unlike most industrial chemical processes, Nature's catalysts generate few
waste products and effect their transformations under mild conditions-in water, at
room temperature and atmospheric pressure. Biocatalysts are models of energy-
efficient, environmentally-consciouschemistry and will play a prominent role in the
21Stcentury's chemicals industry.
The world of biocatalysishas undergone significant change in the eight years since
the first edition of this handbook appeared. Most of the news is good, with enzymes
showing up in many more organic syntheses and a number of important new
industrial processes coming on line. Apart from continuing clever insights into how
to integrate biocatalysis into synthetic chemistry, several forces are accelerating a
move to biocatalytic processes. In the first place, the search for better, enantiomer-
ically pure drugs has forced many chemists to turn to enzymes for assistance in their
preparation. Ever increasing demands for environmentally acceptable processes
push in the same direction. At the same time, rapidly-developing technologies for
making better catalysts through genetic enginering and for discovering new catalysts
are are offering new process opportunities which in the past were either not
economical or not even conceivable. A plethora of new catalysts to choose from, as
well as a high probability that a catalyst can be further improved during the process
design and engineering phases, means that we can respond rapidly to new synthetic
needs with biocatalybc solutions.
The organization of these volumes into specific technologies and transformations
provides a comprehensive coverage of practical biocatalysis that no other single
source provides. The work of experts in each of the fields, the individual chapters
review vast relevant literature and synthesize it in order to present key concepts and
many illustrative examples. This coverage should give organic chemists immediate
access to the wealth of experience that has accumulated in the biocatalysis world and
allow them to identify the most promising ways to use biocatalysts in their own
VI
I Foreword
syntheses. Biocatalysts should feature prominently in the repertoire of synthetic

chemistry, and this handbook deserves a prominent place in the modern chemist’s
library.
Pasadena, January, 2002 Frances Arnold

Preface
Nearly eight years have passed since we the First Edition of ,,Enzyme Catalysis in
Organic Synthesis“ was issued but much of what we had written in its preface then
still applies today. The application of biocatalysis in organic synthesis is a powerful
technique. It has grown steadily and today this field is well-established in both
academia and industry. With increasing application and acceptance the need for a
comprehensive and up to date overview of the state of the art has grown. In addition
numerous colleagues have approached us and asked for an update of “the Hand-
book”.
In response to these demands and in recognition of the new and groundbreaking
strides taken since the first half of the nineties the Second Edition which is now in
the hand of the reader was prepared. In comparing it with the First edition one
discovers that we have not changed the overall arrangement in the volumes.
Therefore we continue to have a part that addresses general principles (Chapters
1-10) and another one which summarizes the application of enzymes in organic
synthesis according to reaction type (Chapters 11-20). This arrangement was very
well received by the readers before and we hope that it will be for the Second Edition
as well.
However, the entire text was streamlined and in many cases regrouped to ensure
for a better presentation. Also a few chapters which in the long run turned out to be
less relevant to organic synthesis were not included again. In contrast other aspects
were now integrated and attention was given to techniques of enzyme evolution,
bioinformatics and enzymatic reactions in low-water media, areas that have devel-
oped with great pace and that we believe to be of major importance in the time to
come.
We hope that the Second Edition of the “Handbook will be a plentiful source of
information just as valuable as the First Edition was eight years ago.
Dortmund and Hanau, February 2002 Karlheinz Drauz, Herbert Waldmann

I IX
Contents
Foreword V
Preface VII
Volume I
1 Introduction 1
Maria- Regina Kulo
1.1 Enzymes as Catalysts 1

1.2 Enzyme Structure and Function 4
1.3 Cofactors and Coenzymes 12
1.4 Enzyme Nomenclature 21
1.5 Enzyme Kinetics 23
1.5.1 Reaction Rate and Substrate Concentration 23
1.5.2 Inhibitors and Efl’ectors 26
1.5.3 Influence of pH and Buffers 27
1.5.4 Temperature 28
1 .G Organic Solvents as Reaction Media 31
1.7 Enzyme Handling: Quality Requirements 32
1.8 Biotransforrnation Using Whole Cells 33
1.8.1 General Aspects 33
1.8.2 Biotransformation with Growing Cells 36
1.8.3 Biotransformation with Resting Cells 37
1.8.4 Biotransforrnationswith Permeabilized or Dried Cells 37
Bibliography 38
2 Production and I d a t i o n of Enzymes 41

Yoshihiko Hirose
2.1 Introduction 41
2.2 Enzyme Suppliers for Biotransforrnation 44
2.3 Origins of Enzymes 45
2.3.1 Microbial Enzymes 45
2.3.2 Plant Enzymes 46
2.3.3 Animal enzymes 46
X
I Contents
2.4 Fermentation of Enzymes 46

2.4.1 Liquid Fermentation 46
2.4.2 Solid Fermentation 47
2.4.3 Extraction of Enzymes 47
2.5 Extraction of Enzymes 47
2.5.1 Microbial Enzymes 47
2.5.2 Plant Enzymes 48
2.5.3 Animal Enzymes 48
2.6 Concentration 48
2.7 Purification of Enzymes 49
2.7.1 Chromatography 49
2.7.1.1 Ion Exchange Chromatography (IEX) 49
2.7.1.2 Hydrophobic Interaction Chromatography (HIC) 54
2.7.1.3 Gel Filtration (GF) 56
2.7.1.4 Reversed Phase Chromatography 58
2.7.1.5 Hydrogen Bond Chromatography 59
2.7.1.6 Affinity Chromatography 59
2.7.1.7 Saltingout Chromatography 62
2.7.2 Precipitation 62
2.7.2.1 Precipitation by Salting out 62
2.7.2.2 Precipitation by Organic Solvents 63
2.7.2.3 Precipitation by Changing pH 63
2.7.2.4 Precipitation by Water-Soluble Polymer 63
2.7.3 Crystallization 64
2.7.4 Stabilization During Purification 64
2.7.5 Storage of Enzymes 64
2.7.5.1 Storage in Liquids 64
2.7.5.2 Storage in Solids 65
2.8 Commercial Biocatalysts 65
References 66
3 Rational Design of Functional Proteins 67

Tadayuki lmanaka and Haruyuki Atomi
3.1 Protein Engineering 67

3.2 Gene Manipulation Techniques in Enzyme Modification 68
3.3 Protein Crystallization 70
3.4 Comparative Modeling of a Protein Structure 73
3.5 What is Needed to Take a Rational Approach? 75
3.6 Examples of Protein Engineering 76
3.6.1 Protein Engineering Studies: Providing a Rational Explanation
for Enzyme Specificity 76
3.6.2 Enhancing the Thermostability of Proteases 78
3.6.3 Contribution of Ion Pairs to the Thermostability of Proteins from
Hyperthermophiles 79
3.6.4 Thermostability Engineering Based on the Consensus Concept 80
Contents I"'
3.6.5 Changing the Optimal pH of an Enzyme 81
3.6.6 Changing the Cofactor Specificity of an Enzyme 82
3.6.7 Changing the Substrate Specificity of an Enzyme 84
3.6.8 Changing the Product Specificity of an Enzyme 85
3.6.9 Combining Site-directed Mutagenesis with Chemical Modification 86
3.6.10 Changing the Catalyhc Activity of a Protein 087
3.7 Conclusions 89
References 90
4 Enzyme Engineering by Directed Evolution 95

Oliver May, Christopher A. Voigt and Frances H.Arnold
4.1 Introduction 95
4.2 Evolution as an Optimizing Process 96
4.2.1 The Search Space of Chemical Solutions 97
4.2.2 The Directed Evolution Algorithm 98
4.3 Creating a Librarr of Diverse Solutions 99
4.3.1 Mutagenesis 99
4.3.1.1 Random Point Mutagenesis of Whole Genes 99
4.3.1.2 Focused Mutagenesis 104
4.3.1.3 Calculation of Mutagenesis Hot-Spots 105
4.3.2 Recombination 107
4.3.2.1 In Vitro Recombination 107
4.3.2.2 In vivo Recombination 110
4.3.2.3 Family Shuffling 111
4.4 Finding Improved Enzymes: Screening and Selection 112
4.4.1 You Get What You Screen For 113
4.4.2 Screening Strategies 113
4.4.2.1 Low-Throughput Screening 114
4.4.2.2 High-Throughpu t Screening 115
4.4.2.3 Choosing Low versus High Throughput 116
4.4.2.4 Analyzing the Mutant Fitness Distribution 117
4.4.3 Selection and Methods to Link Genotype with Phenotype 119
4.5 Applications of Directed Evolution 121
4.5.1 Improving Functional Enzyme Expression and Secretion 122
4.5.2 Engineering Enzymes for Non-natural Environments 127
4.5.3 Engineering Enzyme Specificity 129
4.5.3.1 Substrate Specificity 129
4.5.3.2 Enantioselectivity 131
4.6 Conclusions 132
References 133
XI1
I Contents
5 Enzyme Bioinformatics 139

Kay Hofmann
5.1 Introduction 139

5.2 Protein Comparison 140
5.2.1 Sequence Comparison versus Structure Comparison 140
5.2.2 Substitution Matrices in Sequence Comparisons 141
5.2.3 Profile Methods 142
5.2.4 Database Searches 144
5.3 Enzyme-specificConservation Patterns 145
5.3.1 General Conservation Patterns 145
5.3.2 Active Site Conservation Patterns 146
5.3.3 Metal Binding Conservation Patterns 146
5.3.4 Making Use of Conservation Patterns 148
5.4 Modular Enzymes 149
5.4.1 The Domain Concept in Structure and Sequence 149
5.4.2 A Classification of Modular Enzymes 150
5.4.3 Inhibitory Domains 151
5.5 Enzyme Databases and Other Information Sources 151
5.5.1 E. C. Nomenclature and ENZYME Database 152
5.5.2 BRENDA 152
5.5.3 KEGG and LIGAND database 153
5.5.4 UM-BBD 153
5.5.5 Structural Databases 153
5.5.6 Metalloprotein Databases 154
5.5.7 Databases for Selected Enzyme Classes 154
5.6 Protein Domain and Motif Databases 154
5.6.1 PROSITE 155
5.6.2 PFAM 156
5.6.3 Other Related Databases 156
5.7 Enzyme Genomics 156
5.7.1 Ortholog Search 157
5.7.2 Paralog Search 157
5.7.3 Non-homology Based methods 159
5.8 Outlook 159
References 161
6 Immobilization o f Enzymes 163

James Lalonde

6.2 Methods of Immobilization 164
6.2.1 Non-Covalent Adsorption 165
6.2.2 Covalent Attachment 168
6.2.2.1 Carriers for Enzyme Immobilization 170
6.2.3 Entrapment and Encapsulation 171
6.2.4 Cross-Linking 175
I
Contents xi’i
6.3 Properties of Iminobilized Biocatalysts 175

6.3.1 Mass Transfer Effects 176
6.3.2 Partition 176
6.3.3 Stability 177
6.3.4 Activity of Immobilized Enzymes 177
6.4 New Developments and Outlook 178
6.4.1 Cross-linked Enzyme Crystals (CLEC@) 179
6.4.2 Sol-Gel 181
6.4.3 Controlled Solubility “Smart Polymers” 181
References 182
7 Reaction Engineering for Enzyme-Catalyzed Biotransformations 185

Manfed Biselli, Udo Kragl and Christian Wandrey

7.2 Steps of Process Optimization 186
7.3 Investigation of the Reaction System 190
7.3.1 Properties of the Enzyme 190
7.3.2 Properties of the Reaction System 193
7.3.2.1 Thermodynamic Equilibrium of the Reaction 193
7.3.2.2 Complex Reaction Systems: The Existence of Parallel and Consecutive
Reactions 195
7.3.2.3 Other Properties of the Reaction System 204
7.3.2.4 Application of Organic Solvents 204
7.4 Investigation of Enzyme Kinetics 208
7.4.1 Methods of Parameter Identification 209
7.4.2 The Kinetics of One-Enzyme Systems 210
7.4.2.1 THE Michaelis-Menten Kinetics 210
7.4.2.2 Competitive Inhibition 214
7.4.2.3 Non-Competitive Inhibition 215
7.4.2.4 Uncompetitive Inhibition 216
7.4.2.5 Reversibility of One-Substrate Reactions 217
7.4.2.6 Two-Substrate Re,ictions 218
7.4.2.7 Kinetics of Aminoacylase as Example of a Random
Uni-Bi Mechanism 223
7.4.3 Kinetics of Multiple Enzyme Systems 230
7.5 Enzyme Reactors 232
7.5.1 Basic Reaction Engineering Aspects 232
7.5.2 Reactors for Soluble Enzymes 238
7.5.2.1 Reactor Optimization Exemplified by the Enzyme
Membrane Reactor 241
7.5.2.2 Control of Conversion in a Continuously Operated EMR 249
7.5.3 Reactor Systems for Immobilized Enzymes 250
7.5.4 Reaction Techniques for Enzymes in Organic Solvent 251
XIV
I Contents
Conclusions and Outlook 253

7.6
References 254
8 Enzymic Conversions in Organic and Other Low-Water Media 259

Peter Halling

8.2 Enzyme Form 260
8.2.1 Lyophilized Powders 260
8.2.2 Immobilized Enzymes 261
8.2.3 Cross-Linked Crystals 261
8.2.4 Direct Precipitation in Organic Solvents 262
8.2.5 Additives in Catalyst Powders 262
8.2.6 Solubilized Enzymes 263
8.3 Residual Water Level 264
8.3.1 Fixing Initial Water Activity of Reaction Components 266
8.3.2 Control of Water Activity During Reaction 269
8.3.3 “Water Mimics” 273
8.4 Temperature 274
8.5 Substrate (Starting Material) Concentrations 274
8.6 Solvent Choice 276
8.6.1 Effects on Equilibrium Position 276
8.6.2 “Solvent Effects” that Really are Not 276
8.6.3 Solvent Polarity Trend and Recommended Choices 277
8.6.4 Solvent Parameters 279
8.6.5 Solvent Effects on Selectivity 280
8.6.6 No Solvent or Little Solvent Systems 280
8.7 Acid-Base Conditions 281
8.7.1 pHMemory 281
8.7.2 Processes Erasing pH Memory 282
8.7.3 Systems for Acid-Base Buffering 283
References 285
9 Enzymatic Kinetic Resolution 287

Jonathan M.J. Williams, RebeccaJ. Parker, and Claudia Neri

9.2 Alcohols and their Derivatives 288
9.2.1 Cyanohydrins 289
9.2.2 Other Readily Racemized Substrates 290
9.2.3 Enzyme and Metal Combinations 293
9.3 Carboxylic Acids and their Derivatives 297
9.3.1 Readily Enolized Carboxylic Acid Derivatives 297
9.3.2 Amino-Esters and Related Compounds 301
9.3.3 Reactions of cyclic amino acid derivatives 302
9.4 Reduction of fi-Ketoesters 307
9.5 Conclusion 309
‘Ontents Ixv
References 310
10 Enzymes from Extreme Therrnophilic and HyperthermophilicArchaea and

Bacteria 313
Costanzo Bertoldo and Carabed Antranikian
10.1 Introduction 31 3
10.2 Starch-ProcessingEnzymes 315
10.2.1 Thermoactive Annylolyhc Enzymes 316
10.2.1.1 Heat-StableAmylases and Glucoamylases 316
10.2.1.2 a-Glucosidases 317
10.2.1.3 Thermoactive Pullulanases and CGTases 317
10.3 Cellulose-Hydro1yzing Enzymes 0321
10.3.1 Thennostable Cellulases 321
10.4 Xylan-Degrading Enzymes 324
10.4.1 Thermostable Xylanases 324
10.5 Chitin Degradation 325
10.6 Proteolytic Enzyrnes 326
10.6.1 Stable Proteases 327
10.7 Intracellular Enzymes 329
References 331
Volume I1
11 Hydrolysis and Formation of C - 0 Bonds 335

11.1 Hydrolysis and Fmnation of Carboxylid Acid Esters 335
Hans-joachim Cars and Fritz Theil
11.1.1 Hydrolysis and Formation of Carboxylic Acid Esters 351
11.1.1.1 Hydrolysis of Carboxylic Acid Esters 351
11.1.1.2 Formation of Carboxylic Esters 472
11.1.1.3 Inter- and Intramolecular Alcoholysis 545
References 574
11.2 Hydrolysis of Epoxides 579
Kurt Faber and Romano V: A. Orru
11.2.1 Epoxide Hydrolases in Nature 581
11.2.1.1 Isolation and Characterization of Epoxide Hydrolases 582
11.2.1.2 Structure and Mechanism of Epoxide Hydrolases 584
11.2.1.3 Screening for Microbial Epoxide Hydrolases 587
11.2.2 Microbial Hydrolysis of Epoxides 588
11.2.2.1 Fungal Enzymes 588
11.2.2.2 Bacterial Enzymes 590
11.2.2.3 Yeast Enzymes 591
11.2.3 Substrate Specificity and Selectivity 592
11.2.3.1 Asymmetrization of meso-Epoxides 592
XVI
I Contents
11.2.3.2 Resolution of Racemic Epoxides 592

11.2.3.3 Deracemization Methods 596
11.2.4 Use of Non-Natural Nucleophiles 599
11.2.5 Applications to Asymmetric Synthesis GOO
11.2.6 Summary and Outlook 604
References 605
11.3 Hydrolysis and Formation of Glycosidic Bonds 609
Chi-Huey Wong
1.3.1 Introduction 609
11.3.2 Glycosyltransferases of the Leloir Pathway 611
11.3.2.1 Synthesis of Sugar Nucleoside Phosphates 613
11.3.2.2 Substrate Specificity and Synthetic Applications of
Glycosyltransferases 619
11.3.2.3 In Situ Cofactor Regeneration 626
11.3.2.4 Cloning and Expression of Glycosyltransferases 628
11.3.3 Non-Leloir Glycosyltransferases:Transfer of Glycosyl donors from
Glycosyl Phosphates and Glycosides 631
11.3.4 Glycosidases 633
11.3.4.1 Equilibrium-controlledSynthesis 633
11.3.4.2 Kinetically Controlled Synthesis 634
11.3.4.3 Selectivity 634
11.3.5 Synthesis of N-glycosides 637
11.3.5.1 Nucleoside Phosphorylase 638
11.3.5.2 NAD Hydrolase 639
11.3.6 Biological Applications of Synthetic Glycoconjugates 639
11.3.6.1 Glycosidase and Glycosyl Transferase Inhibitors 639
11.3.6.2 Glycoprotein Remodeling 641
11.3.7 Future Opportunities 642
References 643
11.4 Natural Polysaccharide-degrading Enzymes 653
Constanzo Bertoldo and Carabed Antranikian
11.4.2 Starch 6 5 3
11.4.2.1 Classification of Starch-degrading Enzymes 654
11.4.2.2 a-Amylase (1,4-a-~-Glucan,4-G1ucanhydrolase, E. C. 3.2.1.1) 655
11.4.2.3 P-Amylase (1,4-a-~-Glucan Maltohydrolase, E. C. 3.2.1.2) 656
11.4.2.4 Glucoamylases (1,4-a-~-glucan glucohydrolase,E. C. 3.2.1.3) 656
11.4.2.5 a-Glucosidase (a-6-GlucosideGlucohydrolase, E. C. 3.2.1.20) 657
11.4.2.6 Isoamylase (Glycogen 6-Glucanohydrolase,E. C. 3.2.1.68) 657
11.4.2.7 Pullulanase Type I (a-Dextrin 6-Glucanohydrolase,E. C. 3.2.1.41) 657
11.4.2.8 Pullulanase Type I1 or Amylopullulanase 658
11.4.2.9 Pullulan Hydrolases (Type I, Neopullulanase;Trpe 11, Isopullulanase,
E. C. 3.2.1.57, Pullulan Hydrolase Type 111) 659
11.4.2.10 Cyclodextrin Glycolsyltransferase (1,4-a-~-Glucan 4-a-~-(1,4-a-~-
G1ucano)-Transferase,E. C. 2.4.1.19) 659
11.4.2.11 Biotechnological Applications of Starch-degrading Enzymes 659
=Ontents Ixv''
11.4.3 Cellulose 661
11.4.3.1 Cellulose-degrading Enzyme Systems 663
11.4.3.2 Endoglucanase (I,4-P-~-Glucan-Glucanohydrolase, E.C. 3.2.1.4) 663
11.4.3.3 Cellobiohydrolase (1,4-P-~-Glucan Cellobiohydrolase, E. C. 3.2.1.91) 663
11.4.3.4 P-Glucosidase (P-D-GlucosideGlucohydrolase, E. C. 3.2.1.21) 664
11.4.3.5 Fungal and Bacterial Cellulases 664
11.4.3.6 Structure and Synergistic Effect of Cellulases 665
11.4.4 Xylan 667
11.4.4.1 The Xylanolytic Elnzyme System 668
11.4.4.2 Endoxylanase (1;l-P-D-Xylan Xylanohydrolase, E. C. 3.2.1.8) 670
11.4.4.3 P-Xylosidase (P-c-XylosideXylohydrolase, E. C. 3.2.1.37) 670
11.4.4.4 a-L-Arabinofuranosidase (E. C. 3.2.1.55) 671
11.4.4.5 a-Glucuronidase (E.C. 3.2.1.136) 671
11.4.4.6 Acetyl Xylan Esterase (E.C. 3.1.1.6) 672
11.4.4.7 Mechanism of Action of Endoxylanase 672
11.4.4.8 Biotechnological Applications of Xylanases 672
11.4.5 Pectin 673
11.4.5.1 Classification of Pectic Substances 675
11.4.5.2 Pectolpc Enzymes 675
11.4.5.3 Classification of Pectolytic Enzymes 676
11.4.5.4 Protopectinase 676
11.4.5.5 Pectin Methylesttxase 677
11.4.5.6 Pectin and Polygalacturonate Depolymerizing Enzymes 677
11.4.5.7 Pectin and Polygalacturonate Hydrolase 678
11.4.5.8 Pectin and Polygalacturonate Lyase 679
11.4.5.9 Biotechnological Applications of Pectolytic Enzymes 680
References 681
11.5 Addition of Water to C=C Bonds 686
Marcel Wubbolts
11.5.1 Addition of Water to Alkenoic Acids 686
11.5.2 Addition of Water to Alkene-Dioic Acids 687
11.5.2.1 L- and D-Malic A'cid 687
11.5.1.2 Substituted Malic Acids 688
11.5.3 Addition of Water to Alkene-TricarboxylicAcids 688
11.5.3.1 Citric Acid and Derivatives 688
11.5.4 Addition of Water to Alkynoic Acids 690
11.5.5 Addition of Water to Enols 690
11.5.5.1 Carbohydrates: Addition of Water to 2-Keto-3-Deoxysugars 690
11.5.5.2 Addition/Elimination of Water with Other Enols 691
11.5.6 Addition of Water to Unsaturated Fatty Acids 693
11.5.6.1 CoA and ACP Coupled Fatty Acid Hydratases 693
11.5.6.2 Hydratases Acting on Free Fatty Acids 695
11.5.7 Addition of Water to Steroids 695
References 690
XVlll
I Contents
12 Hydrolysis and Formation of C-N Bonds 699
12.1 Hydrolysis of Nitriles 699

Birgit Schulze
12.1.2 Types of Nitrile Hydrolyzing Enzymes 700
12.1.2.1 Enzymatic Hydrolysis of Organic Nitriles 700
12.1.2.2 Enzymatic Hydrolysis of Cyanide 702
12.1.3 Examples of Enzymatic Nitrile Hydrolysis 703
12.1.3.1 Enantioselective Hydrolysis of Nitriles 703
12.1.3.2 Monohydrolysis of Dinitriles 705
12.1.3.3 Substrate and Product Inhibition of Nitrile Hydrolysis 708
12.1.3.4 Activation and Stabilization of Nitrile Hydratases 710
12.1.3.5 Nitrile Hydrolysis in Organic Solvents 710
12.1.3.6 Large Scale Production of Acrylamide 711
12.1.4 Availability and Industrial Future of Nitrile Hydrolyzing Biocatalysts 713
References 713
12.2 Formation and Hydrolysis of Amides 716
Birgit Schulze and Erik de Vroom
12.2.2 Enzymatic Formation of Amides 716
12.2.3 Enzymatic Enantioselective Hydrolysis of Amides 719
12.2.3.1 Hydrolysis of Carboxylic Amides 719
12.2.3.2 Hydrolysis of Amino Acid Amides 720
12.2.3.3 Hydrolysis of Cyclic Amides 727
12.2.4 Selective Cleavage of the C-Terminal Amide Bond 728
12.2.5 Amidase Catalyzed Hydrolytic and Synthetic Processes in the Production
of Semi-syntheticAntibiotics 729
12.2.5.1 Enzymatic Production of 6-APA, 7-ADCA and 7-ACA Using Amidases:
Hydrolytic Processes 730
12.2.5.2 A New Fermentation-based Biocatalytic Process for 7-ADCA 735
12.2.5.3 Enzymatic Formation of Semi-syntheticAntibiotics:
Synthetic Processes 735
12.2.6 Conclusions and Future Prospects 737
References 738
12.3 Hydrolysis of N-Acylamino Acids 741
Andreas 5.Bommarius
12.3.2 Acylase I (N-AcylaminoAcid Amidohydrolase, E. C. 3.5.1.4.) 742
12.3.2.1 Genes, Sequences, Structures 743
12.3.2.2 Substrate Specificity 744
12.3.2.3 Stability of Acylases 746
12.3.2.4 Thermodynamics and Mechanism of the Acylase-catalyzed Reaction 748
12.3.3 Acylase I I (N-Acyl-L-Aspartate Amidohydrolase,Aspartoacylase,
E.C. 3.5.1.15.) 749
12.3.4 Proline Acylase 1:N-Acyl-L-ProlineAmidohydrolase) 752
Contents I
12.3.5 Dehydroamino k i d Acylases 753
12.3.6 D-Specific Aminoacylases 754
12.3.7 Acylase Process on a Large Scale 757
References 758
12.4 Hydrolysis and Formation of Hydantoins 761
Markus Pietzsch m d Christoph Syldatk
12.4.1 Classification and Natural Occurrence of Hydantoin Cleaving and Related
Enzymes 761
12.4.2 D-Hydantoinases- Substrate Specificity and Properties 773
12.4.3 DN-Carbamoylases - Substrate Specificity and Properties 777
12.4.4 L-Hydantoinases- Substrate Specificity and Properties 784
12.4.5 L-N-Carbamoylases- Substrate Specificityand Properties 786
12.4.6 Hydantoin Raceinases 792
12.4.7 Conclusions 794
References 796
12.5 Hydrolysis and Formation of Peptides 800
Hans-Dieterjakulike
12.5.2 Hydrolysis of Peptides 801
12.5.2.1 Peptide-CleavingEnzymes 801
12.5.2.2 Importance of Proteolysis 813
12.5.3 Formation of Peptides 818
12.5.3.1 Tools for Peptide Synthesis 818
12.5.3.2 Choice of the Ideal Enzyme 822
12.5.3.3 Principles of Enzymatic Synthesis 823
12.5.3.4 Manipulations to Suppress Competitive Reactions 831
12.5.3.5 Approaches to Irreversible Formation of Peptide Bond 840
12.5.3.6 Irreversible C-N Ligations by Mimicking Enzyme Specificity 842
12.5.3.7 Planning and Process Development of Enzymatic Peptide Synthesis 851
12.5.4 Conclusion and Outlook 858
References 859
12.6 Addition of Amines to C=C Bonds 866
Marcel Wubbolts
12.6.1 Addition of Ammonia to Produce Amino Acids 866
12.6.1.1 Aspartic Acid 866
12.6.1.2 Aspartic Acid Derivatives 868
12.6.1.3 Histidine Ammonia Lyase 869
12.6.1.4 Phenylalanine, Tyrosin and L-DOPA 870
12.6.1.5 Serine and Threonine Deaminases 871
12.6.1.6 Ornithine Cyclocleaminase 871
12.6.2 Ammonia Lyases that Act on Other Amines 871
12.6.2.1 Elimination of Ammonia from Ethanolamine 871
References 872
XX
I Contents
12.7 Transaminations 873

J. David Rozzell and Andreas 5.Bommarius
12.7.1 tntroduction 873
12.7.2 Description of Transaminases 875
12.7.2.1 Homology and Evolutionary Subgroups of Aminotransferases 875
12.7.2.2 Mechanism of Transamination 875
12.7.2.3 Protein Engineering and Directed Evolution with
Aminotransferases 876
12.7.3 Use of Aminotransferases in Biocatalytic Reactions 878
12.7.3.1 Synthesis of a-L-AminoAcids 878
12.7.3.2 Synthesis of Enantiomerically Pure Amines 880
12.7.3.3 Other Preparative Applications of Aminotransferases 881
12.7.4 Driving the Reaction to Completion 884
12.7.5 Production of L-Amino Acids Using Immobilized Transaminases 885
12.7.6 D-Amino Acid Transferases 889
12.7.7 Synthesis of Labeled Amino Acids 891
12.7.8 Availability of Enzyme 892
References 892
13 Formation and Cleavage of P - 0 Bonds 895

George M. Whitesides

13.1.1 Enzymes Forming or Cleaving Phosphorous-OxygenBonds 896
13.1.2 Biological Phosphorylating Agents 899
13.2 Phosphorylation 901
13.2.1 Regeneration of Nucleoside Triphosphates 901
13.2.1.1 Regeneration of ATP from ADP and AMP 902
13.2.1.2 Regeneration of other Nucleoside Triphosphates 906
13.2.2 Applications 907
13.2.2.1 Phosphorylations with ATP as a Cofactor 907
13.2.2.2 P- 0 Bond Formation with other Nucleoside Triphosphates than
ATP 909
13.2.2.3 Other Phosphorylating Agents 910
13.2.3 Tables Containing Typical Examples Ordered According to the Classes of
Compounds 918
13.3 Cleavage of P - 0 bonds 918
13.3.1 Hydrolysis of Phosphate and Pyrophosphate Monoesters 919
13.3.2 Hydrolysis of S- and N-substituted Phosphate Monoester Analogs 920
13.3.3 Hydrolysis of Phosphate and Phosphonate Diesters 922
13.3.3.1 Nucleic Acids and their Analogs 922
13.3.3.2 Other Phosphate and Phosphonate Diesters 922
13.3.4 Other P - 0 Bond Cleavages 923
References 926
14 Formation o f C-C Bonds 931
Chi-hey Wong
14.1 Aldol Reactions 931

14.1.1 DHAP-Utilizing Aldolases 931
14.1.1.1 Fructose 1,G-Diphosphate(FDP) Aldolase (E. C. 4.1.2.13) 931
14.1.1.2 Fuculose 1-Phosphate (Fuc 1-P) Aldolase (E.C. 4.1.2.17), Rhamnulose
1-Phosphate (Rh.a 1-P) Aldolase (E. C. 4.1.2.19) and Ragatose
1,6-Diphosphate (TDP) Aldolase 939
14.1.1.3 Synthesis of DihlydroxyacetonePhosphate (DHAP) 943
14.1.2 Pyruvate/Phosp:hoenolpyruvate-UtilizingAldolases 944
14.1.2.1 N-Acetylneurarninate (NeuAc)Aldolase (E.C. 4.1.3.3) and NeuAc
Synthetase (E.C. 4.1.3.19) 944
14.1.2.2 3-Deoxy-~-mann.o-2-octu~osonate Aldolase (E. C. 4.1.2.23) and 3-Deoxy-
~-manno-2-octulosonate &Phosphate Synthetase (E. C. 4.1.2.16) 946
14.1.2.3 3-Deoxy-~-arabiii0-2-heptulosonic Acid 7-Phosphate (DAHP)
Synthetase (E.C. 4.1.2.15) 947
14.1.2.4 2-Keto-4-hydroxyglutarate(KHG) Aldolase (E. C. 4.1.2.31) 948
14.1.2.5 2-Keto-3-deoxy-6-phosphogluconate (KDPG) Aldolase (E. C. 4.1.2.14) 949
14.1.2.6 2-Keto-3-deoxy-1~glucarate (KDG) Aldolase (E. C. 4.1.2.20) 950
14.1.3 2-Deoxyribose 5-phosphate Aldolase (DEW) (E. C. 4.1.2.4) 950
14.2 Ketol and Aldol Transfer Reactions 960
14.2.1 Transketolase (TK) (E. C. 2.2.1.1) 960
14.2.2 Transaldolase (TA) (E.C. 2.2.1.2) 962
14.3 Acyloin Condensation 962
14.4 C-C Bond Forming Reactions Involving AcetylCoA 963
14.5 Isoprenoid and Steroid Synthesis 965
14.6 p-Replacement of Chloroalanine 966
References 966
14.7 Enzymatic Synthesis of Cyanohydrins 974
Martin H. Fechtcr and Hefried Cringl
14.7.1 The Oxynitrilasm Commonly Used for Preparative Application 975
14.7.2 Oxynitrilase Caialysed Addition of HCN to Aldehydes 976
14.7.3 HNL-CatalyzedAddition of Hydrogen Cyanide to Ketones 978
14.7.4 Transhydrocyanation 978
14.7.5 Experimental Techniques for HNL-Catalysed Biotransformations 981
14.7.6 Resolution of Racemates 982
14.7.7 Follow-up Chemistry of Enantiopure Cyanohydrins 985
14.7.8 Safe Handling of Cyanides 985
14.7.9 Conclusions and Outlook 986
References 98.6
XXll
I Contents
Volume 111
15 Reduction Reactions 991
15.1 Reduction of Ketones 991

Kaoru Nakamura and Tomoko Matsuda
15.1.1.1 Enzyme Classfication and Reaction Mechanism 991
15.1.1.2 Coenzyme Regeneration 992
15.1.1.3 Form ofthe Biocatalysts: Isolated Enzyme vs. Whole Cell 995
15.1.1.4 Origin of Enzymes 996
15.1.2 Stereochemical Control 997
15.1.2.1 Enantioselectivity of Reduction Reactions 997
15.1.2.2 Modification of the Substrate: Use of an “Enantiocontrolling”
Group 998
15.1.2.3 Screening of Microorganisms 1000
15.1.2.4 Treatment of the Cell: Heat Treatment 1001
15.1.2.5 Treatment ofthe Cell: Aging 1001
15.1.2.6 Treatment of the Cell: High Pressure Homogenization 1002
15.1.2.7 Treatment of the Cell: Acetone Dehydration 1002
15.1.2.8 Cultivation Conditions of the Cell 1003
15.1.2.9 Modification of Reaction Conditions: Incorporation of an Inhibitor 1004
15.1.2.10 Modification of Reaction Conditions: Organic-Solvent 1005
15.1.2.11 Modification of Reaction Conditions: Use of a Supercritical Solvent 1006
15.1.2.12 Modification of Reaction Conditions: Cyclodextrin 1007
15.1.2.13 Modification of Reaction Conditions: Hydrophobic Polymer XAD 1007
15.1.2.14 Modification of Reaction Conditions: Reaction Temperature 1008
15.1.2.15 Modification of Reaction Conditions: Reaction Pressure 1009
15.1.3 Improvement of Dehydrogenases for use in Reduction Reactions by
Genetic Methods 1010
15.1.3.1 Overexpression of the Alcohol Dehydrogenase 1010
15.1.3.2 Access to a Single Enzyme Within a Whole Cell: Use of Recombinant
Cells 1011
15.1.3.3. Use ofa Cell Deficient in an Undesired Enzyme 1012
15.1.3.4 Point Mutation for the Improvement of Enantioselectivity 1012
15.1.3.5 Broadening the Substrate Specificity of Dehydrogenase by
Mutations 1012
15.1.3.6 Production of an Activated Form of an Enzyme by Directed
Evolution 1014
15.1.3.7 Change in the Coenzyme Specificity by Genetic Methods: NADP(H)
Specific Formate 1014
15.1.3.8 Use of a Mutant Dehydrogenase for the Synthesis of 4-Amino-2-Hydroxy
Acids 1014
15.1.3.9 Catalyhc Antibody 1015
15.1.4 Reduction Systems with Wide Substrate Specificity 1016
15.1.4.1 Bakers' Yeast 1016
15.1.4.2 Rodococcus Erythropolis 1016
15.1.4.3 Pseudomonas sp. !$trainPED and Lactobacillus Kefir 1017
15.1.4.4 Thermoanaerobium Brockii 1018
15.1.4.5 Geotrichurn Candidum 1019
15.1.5 Reduction of Various Ketones 1021
15.1.5.1 Reduction of Fluoroketones 1021
15.1.5.2 Reduction of Fluoroketones Containing Sulhr Functionalities 1024
15.1.5.3 Reduction of Chloroketones 1025
15.1.5.4 Reduction of Ketones Containing Nitrogen, Oxygen, Phosphorus
and Sulfur 1028
15.1.5.5 Reduction of Diketones 1028
15.1.5.6 Reduction of Diary1 Ketones 1029
15.1.5.7 Diastereoslective Reductions (Dynamic Resolution) 1030
15.1.5.8 Chemo-enzymabc Synthesis of Bioaktive Compounds 1031
15.2 Reduction of Various Functionalities 1033
15.2.1 Reduction of Aldehydes 1033
15.2.2 Reduction of Peroxides to Alcohols 1034
15.2.3 Reduction of Sulfoxides to Sulfides 1034
15.2.4 Reduction of Azide and Nitro Compounds to Amines 1035
15.2.5 Reduction of Carbon-Carbon Double Bonds 1036
15.2.6 Transformation of a-Keto Acid to Amine 1037
15.2.7 Reduction of Carbon Dioxide 1038
15.2.7.1 Reduction of COz to Methanol 1038
15.2.7.2 Reductive fixation of COz 1039
References 1040
15.3 Reduction of C=lV bonds 1047
Andreas 5. Bomtmarius
15.3.2 Structural Features of Amino Acid Dehydrogenases (AADHs) 1049
15.3.2.1 Sequences and Structures 1050
15.3.3 Thermodynamics and Mechanism of Enzymatic Reductive
Amination 1050
15.3.3.1 Thermodynamics 1050
15.3.3.2 Mechanism, Kinetics 1051
15.3.4 Individual Amino Acid Dehydrogenases 1052
15.3.4.1 Leucine Dehydrogenase (LeuDH, E. C. 1.4.1.9.) 1052
15.3.4.2 Alanine Dehydrogenase (AlaDH, E. C. 1.4.1.1.) 1053
15.3.4.3 Glutamate Dehydrogenase (GluDH, E. C. 1.4.1.2-4) 1054
15.3.4.4 Phenylalanine Dehydrogenase (PheDH, E.C. 1.4.1.20) 1054
15.3.5 Summary of Substrate Specificities 1056
15.3.6 Process Technology: Cofactor Regeneration and Enzyme Membrane
Reactor (EMR) 1058
15.3.6.1 Regeneration of NAD(P)(H)Cofactors 1058
15.3.6.2 Summary of Processing to Amino Acids 1060
References 1001
XXlV
I Contents
16 Oxidation Reactions 1065
16.1 Oxygenation of C-H and C=C Bonds 1065

Sabine Flitsch
16.1.2 Hydroxylating Enzymes 1066
16.1.2 Hydroxylating Enzymes 1068
16.1.4 Hydroxylation of Non-Activated Carbon Atoms 1069
16.1.4.1 Hydroxylation of Monoterpenes 1069
16.1.4.1 Hydroxylation of Monoterpenes 1075
16.1.4.3 Hydroxylation of Steroids 1078
16.1.4.4 Miscellaneous Compounds 1079
16.1.5 Epoxidation of Olefins 1084
16.1.5.1 Epoxidation of Straight-Chain Terminal Olefins 1084
16.1.5.2 Short-Chain Alkenes 1088
16.1.5.3 Terpenes 1090
16.1.5.4 Cyclic Sesquiterpenes 1096
16.1.6 Conclusions, Current and Future Trends 1097
16.1.7 Cis Hydroxylation of Aromatic Double Bonds 1099
16.1.7.1 Introduction 1099
16.1.7.2 Preparation of cis Dihydrodiols 1100
References 1103
16.2 Oxidation of Alcohols 1108
Andreas Schmid, Frank Hollmann, and Bruno Buhler
16.2.2 Dehydrogenases as Catalysts 1108
16.2.2.1 Regeneration of Oxidized Nicotinamide Coenzymes 1108
16.2.2.2 Dehydrogenases as Regeneration Enzymes 1109
16.2.2.3 Molecular Oxygen as Terminal Acceptor 1111
16.2.2.4 Electrochemical Regeneration 11 12
16.2.2.5 Photochemical Regeneration 1114
16.2.2.6 Oxidations Catalyzed by Alcohol Dehydrogenase from Horse Liver
(HLADH) 1115
16.2.2.7 Alcohol Dehydrogenase from Yeast (YADH) 1120
16.2.2.8 Alcohol Dehydrogenase from Thernaoanaerobiurn brokii
(TBADH) 1120
16.2.2.9 Glycerol Dehydrogenase (GDH, E. C. 1.1.1.6) 1122
16.2.2.10 Glycerol-3-phosphateDehydrogenase (GPDH, E. C. 1.1.1.8) 1124
16.2.2.11 Lactate Dehydrogenase (LDH, E.C. 1.1.1.27) 1125
16.2.2.12 Carbohydrate Dehydrogenases 1126
16.2.2.13Hydroxysteroid Dehydrogenases (HSDH) 1127
16.2.2.14 Other Dehydrogenases 1127
16.2.3 Oxidases as Catalysts 1129
16.2.3.1 General Remarks 1129
16.2.3.2 Methods to Diminish/Avoid H202 1129
16.2.3.3 Pyranose Oxidasa (P20, E.C. 1.1.3.10) 1132
Contents lm
16.2.3.4 Glycolate Oxidase (E.C. 1.1.3.15) 1135
16.2.3.5 Nucleoside Oxidase (E. C. 1.1.3.28) 1138
16.2.3.6 Glucose Oxidase (E. C. 1.1.3.4) 1138
16.2.3.7 Alcohol oxidase (E.C. 1.1.3.13) 1139
16.2.3.8 Galactose Oxidase (E. C. 1.1.3.9) 1141
16.2.3.9 Cholesterol Oxidase (ChOX, E. C. 1.1.3.6) 1142
16.2.4 Peroxidases as Catalysts 1142
16.2.4.1 Introduction 1142
16.2.4.2 Methods to Generate HzOz 1143
16.2.4.3 Chloroperoxidase (CPO, E. C. 1.11.1.10) 1145
16.2.4.4 Catalase (E.C. 1.11.1.6) 1145
16.2.5 Quinoprotein Dehydrogenases (QDH) 1146
16.2.5.1 General Remarks 1146
16.2.5.2 Methanol Dehydrogenase (E.C. 1.1.99.8) 1147
16.2.5.3 Glucose Dehydrogenase (E. C. 1.1.99.17) 1148
16.2.6 Whole-Cell Oxidations 1148
16.2.6.1 Stereoselective Oridation of (-)-Carve01 to (-)-Carvone 1148
16.2.6.2 Sugar Dehydrogenases Applied in Whole Cells 1149
16.2.6.3 Oxidation of Aromatic and Aliphatic Alcohols to Corresponding
Aldehydes and Acids 1150
16.2.6.4 Enantiospecific Reactions 1154
16.2.6.5 Stereoinversions using Microbial Redox Reactions 1157
16.2.7 Miscellaneous I162
16.2.7.1 Biofuel Cells 1162
16.2.7.2 Biomimetic Analogs to Nicotinamide Co-nzymes 1163
References 1164
16.3 Oxidation of Phenols 1170
Andreas Schmid, Frank Hollmann, and Bruno Biihler
16.3.2 Oxidases 1170
16.3.2.1 Vanillyl oxidase (E. C. 1.1.3.38) 1170
16.3.2.2 Laccase (E.C. 1.10.3.2) 1174
16.3.3 Monooxygenases 1176
16.3.3.1 Tyrosinase (E.C. 1.10.3.1) 1176
16.3.3.2 2-Hydroxybiphen1yl-3-monooxygenase (HbpA, E. C . 1.14.13.44) 1179
16.3.4 Peroxidases 11115
16.3.4.1 Oxidative Coupling Reactions 1185
16.3.4.2 Hydroxylation of Phenols 1186
16.3.4.3 Nitration of Phenols 1187
16.3.5 Other Oxidoreductases 1188
16.3.5.1 4-Cresol-oxidorecluctase (PCMH, E. C. 1.17.99.1) 1188
16.3.5.2 4-Ethylphenol Oxidoreductas 1189
16.3.6 In vivo Oxidations 1190
16.3.6.1 Phenoloxidase of Mucuna pwriens 1190
Xxvl
I Contents
16.3.6.2 Monohydroxylationof (R)-2-PhenoxypropionicAcid and Similar

Substrates 1191
16.3.6.3 Biotransformation of Eugenol to Vanillin 1191
References 1192
16.4 Oxidation of Aldehydes 1194
Andreas Schmid, Frank Hollmann, and Bruno 6uhler
16.4.2 Alcohol Dehydrogenases 1194
16.4.3 Aldehyde Dehydrogenases 1196
16.4.4 Monooxygenases 1198
16.4.4.1 Luciferase (E. C. 1.14.14.3) 1198
16.4.4.2 P 4 5 0 ~ ~ . 31199
16.4.5 Oxidases 1201
16.4.5.1 Xanthine Oxidase (E.C. 1.1.3.22) 1201
16.4.6 Oxidations with Intact Microbial Cells 1201
References 1201
16.5 Baeyer-Villiger Oxidations 1202
Sabine Flitsch and Gideon Grogan
16.5.1.1 Steroidal Substrates 1202
16.5.1.2 Aliphatic Substrates 1205
16.5.1.3 Alicyclic Substrates 1207
16.5.1.4 Polycyclic Molecules 1212
16.5.2 Baeyer-Villiger Monooxygenases 1213
16.5.2.1 Type 1 BVMOs 1214
16.5.2.2 Type 2 BVMOs 1216
16.5.2.3 Mechanism of the Enzymatic Baeyer-Villiger Reaction 1216
16.5.3 Synthetic Applications 1222
16.5.4 Models for the Action of Baeyer-Villiger Monooxygenases 1234
16.5.5 Conclusion and Outlook 1238
References 1241
16.6 Oxidation of Acids 1245
16.6.2 Pyruvate Oxidase (PYOx, E.C. 1.2.3.3) 1246
16.6.3 Formate Dehydrogenase (FDH, E. C. 1.2.1.2) 1247
16.6.4 Oxidations with Intact Microbial Cells 1247
16.6.4.1 Production of Benzaldehyde from Benzoyl Formate or Mandelic
Acid 1247
16.6.4.2 Microbial Production of &,cis-Muconic Acid from Benzoic Acid 1248
16.6.4.3 Biotransformation of Substituted Benzoates to the Corresponding
cis-Diols 1249
References 1249
16.7 Oxidation of C-N Bonds 1250
Contents
I
Xxvll
Andreas Schmid, thank Hollmann, and Bruno Buhler

16.7.1 Introduction L!50
16.7.2 Oxidations Catalyzed by Dehydrogenases 1251
16.7.2.1 L-Alanine Dehydrogenase (L-Ala-DH,E. C. 1.4.1.1) 1251
16.7.2.2 Nicotinic Acid Dchydrogenase (Hydroxylase)(E. C. 1.5.1.13) 1252
16.7.3 Oxidations Catalyzed by Oxidases 1254
16.7.3.1 Amino Acid Oxiclases 1254
16.7.3.2 Amine Oxidases 1256
16.7.4 Oxidations Catalyzed by Transaminases 1260
References 1261
16.8 Oxidation at Sulfur 1262
Karl-Heinz van Pke
16.8.1 Enzymes Oxidizing at Sulfur and their Sources 1262
16.8.2 Oxidation of Sulfides 1263
16.8.2.1 Oxidation of Sulfides by Monooxygenasesand by Whole
Organsims 1263
16.8.2.2 Oxidation of Sulfides by Peroxidases and Haloperoxidases 1264
References 1266
16.9 Halogenation 1267
Karl-Heinz van Pke
16.9.1 Classification of lialogenating Enzymes and their Reaction
Mechanisms 1;!67
16.9.1.1 Haloperoxidases and Perhydrolases 1267
16.9.1.2 FADH2-dependent Halogenases 1268
16.9.2 Sources and Production of Enzymes 1268
16.9.2.1 FADHz-dependentHalogenases 1268
16.9.2.2 Haloperoxidases and Perhydrolases 1269
16.9.3 Substrates for Hadogenating Enzymes and Reaction Products 1271
16.9.3.1 Halogenation of ,4romatic Compounds 1271
16.9.3.2 Halogenation of Aliphatic Compounds 1273
16.9.4 Regioselectivity and Stereospecificityof Enzymatic Halogenation
Reactions 1275
16.9.4.1 FADH2-dependentHalogenases 1275
16.9.5 Comparison of Chemical with Enzymatic Halogenation 1277
References 1275
17 lsornerizations 1281
Nobuyoshi Esaki, 'Tatsuo Kurihara, and Kenji Soda
17.1 Introduction 12.81

17.2 Racemizations and Epimerizations 1282
17.2.1 Pyridoxal 5'-phosphate-dependentAmino Acid Racemases
and Epimerases 1283
17.2.1.1 Alanine Racemase (E. C. 5.1.1.1) 1283
Xxvlll
I Contents
17.2.1.2 Amino Acid Racemase with Low Substrate Specificity

(E.C. 5.1.1.10) 1289
17.2.1.3 a-Amino-E-caprolactam Racemase 1292
17.2.2 Cofactor-independent Racemases and Epimerases Acting
on Amino Acids 1293
17.2.2.1 Glutamate Racemase (E. C. 5.1.1.3) 1293
17.2.2.2 Aspartate Racemase (E. C. 5.1.1.13) 1297
17.2.2.3 Diaminopimelate Epimerase (E. C. 5.1.1.7) 1299
17.2.2.4 Proline Racemase (E. C. 5.1.1.4) 1301
17.2.3 Other Racemases and Epimerases Acting on Amino
Acid Derivatives 1301
17.2.3.1 2-Amino-A2-thiazoline-4-carbo~ylateRacemase 1301
17.2.3.2 Hydantoin Racemase 1303
17.2.3.3 N-Acylamino Acid Racemase 1306
17.2.3.4 Isopenicillin N Epimerase 1308
17.2.4 Racemization and Epimerization at Hydroxyl Carbons 1310
17.2.4.1 Mandelate Racemase (E. C. 5.1.2.2) 1310
17.3 Isomerizations 1312
17.3.1 D-Xylose (Glucose) Isomerase (E.C. 5.3.1.5) 1313
17.3.1.1 Properties 1313
17.3.1.2 Reaction Mechanism 1314
17.3.1.3 Production of Fructose 1316
17.3.1.4 Production of Unusual Sugar Derivatives 1316
17.3.2 Phosphoglucose Isomerase (E. C. 5.3.1.9) 1318
17.3.3 Triosephosphate Isomerase (E. C. 5.3.1.1) 1320
17.3.4 L-Rhamnose Isomerase (E. C. 5.3.1.14) 1321
17.3.5 L-Fucose Isomerase (E. C. 5.3.1.3) 1323
17.3.G N-Acetyl-D-glucosamine 2-Epimerase 1324
17.3.7 Maleate cis-trans Isomerase (E. C. 5.2.1.1) 1324
17.3.8 Unsaturated Fatty Acid cis-trans Isomerase 1325
17.4 Conclusion 1326
References 1326
18 Introductionand Removal of Protecting Groups 1333

Dieter Kadereit, Reinhard Reents, Duraiswamy A. Feyaraj, and
Herbert Waldmann

18.2 Protection of Amino Groups 1334
18.2.1 N-Terminal Protection of Peptides 1334
18.2.2 Enzyme-labile Urethane Protecting Groups 1338
18.2.3 Protection of the Side Chain Amino Group of Lysine 1341
18.2.4 Protection of Amino Groups in P-Lactam Chemistry 1341
18.2.5 Protection of Amino Groups of Nucleobases 1343
18.3 Protection of Thiol Groups 1343
18.3.1 Protection of the Side Chain Thiol Group of Cysteine 1343
Contents
I XXlX
18.4 Protection of Casboxy Groups 1344

18.4.1 C-Terminal Protection of Peptides 1344
18.4.2 Protection of the Side Chain Groups of Glutamic and
Aspartic Acid 1352
18.5 Protection of Hydroxy Groups 1353
18.5.1 Protection of Monosaccharides 1354
18.5.2 Deprotection of Monosaccharides 1369
18.5.3 Di- and Oligosaccharides 1378
18.5.4 Nucleosides 1350
18.5.5 Further Aglycon Glycosides 1383
18.5.6 Polyhydroxylated Alkaloids 1386
18.5.7 Steroids 1388
18.5.8 Phenolic Hydroqq Groups 1390
18.6 Biocatalysis in Pclymer Supported Synthesis: Enzyme-labile Linker
Groups 1392
18.6.1 Endo-linkers 13.93
18.6.2 Exo-linkers 1402
18.7 Outlook 1408
References 1400
19 Replacing Chemical Steps by Biotransformations:

Industrial Application and Processes Using Biocatalysis 1419
Andreas Liese

19.2 Types and Handling of Biocatalysts 1420
19.3 Examples 1421
19.3.1 Reduction Reactions Catalyzed by Oxidoreductases (E. C. 1) 1422
19.3.1.1 Ketone Reduction Using Whole Cells of Neurospora crassa
(E.C. 1.1.1.1)~1422
19.3.1.2 Ketoester Reduction Using Cell Extract of Acinetobacter calcoaceticus
(E.C. 1.1.1.1) 1423
19.3.1.3 Enantioselective Reduction with Whole Cells of Candida sorbophila
(E.C. l.l.X.X) 1424
19.3.2 Oxidation Reactions Catalyzed by Oxidoreductases (E. C. 1) 1425
19.3.2.1 Alcohol Oxidation Using Whole Cells of Gluconobacter suboxydans
(E.C. 1.1.99.21) 1425
19.3.2.2 Oxidative Deamin ation Catalyzed by Immobilized D-AminO Acid Oxidase
from T'gonopsis variabilis (E. C. 1.4.3.3) 1426
19.3.2.3 Kinetic Resolution by Oxidation of Primary Alcohols Catalyzed by Whole
Cells from Rhodococcus erythropolis (E. C. l.X.X.X) 1427
19.3.2.4 Hydroxylation of Nicotinic Acid (Niacin) Catalyzed by Whole Cells of
Achrornobacter xylosoxidans (E. C. 1.5.1.13) 1428
XXX
I Contents
19.3.2.5 Reduction of Hydrogen Peroxide Concentration by Catalase

(E.C. 1.11.1.6) 1428
19.3.3 Hydrolybc Cleavage and Formation of C-0 Bonds by Hydrolases
(E.C. 3) 1430
19.3.3.1 Kinetic Resolution of Glycidic Acid Methyl Ester by Lipase from Serratia
rnarcescens (E.C. 3.1.1.3) 1430
19.3.3.2 Kinetic Resolution of Diester by Protease Subtikin Carlsberg from
Bacillus sp. (E. C. 3.4.21.62) 1431
19.3.3.3 Kinetic Resolution of Pantolactones and Derivatives thereof by a
Lactonase from Fusariurn oxysporurn (E. C. 3.1.1.25) 1433
19.3.3.4 Hydrolysis of Starch to Glucose by Action of Two Wnzymes: a-Amylase
(E. C. 3.2.1.1) and Amyloglucosidase (E. C. 3.2.1.3) 1433
19.3.4 Formation or Hydrolyhc Cleavage of C-N Bonds by Hydrolases
(E.C. 3) 1435
19.3.4.1 Enantioselective Acylation of Racemic Amines Catalyzed by Lipase from
Burkholderiaplantarii (E. C. 3.1.1.3) 1435
19.3.4.2 7-AminocephalosporanicAcid Formation by Amide Hydrolysis Catalyzed
by Glutaryl Amidase (E. C. 3.1.1.41) 1436
19.3.4.3 Penicillin G Hydrolysis by Penicillin Amidase from Escherichia coli
(E.C. 3.5.1.11) 1438
19.3.4.4 Kinetic Resolution of a-Amino Acid Amides Catalyzed by
Aminopeptidase from Pseudornonasputida (E. C. 3.4.1.11) 1439
19.3.4.5 Production of L-Methionineby Kinetic Resolution with Aminoacylase of
Aspergillus oryzae (E. C. 3.5.1.14) 1441
19.3.4.6 Production of D-p-Hydroxyphenyl Glycine by Dynamic Resolution with
Hydantoinase from Bacillus brevis (E. C. 3.5.2.2) 1441
19.3.4.7 Dynamic Resolution of a-Amino-E-caprolactamby the Action of
Lactamase (E.C. 3.5.2.11) and Racemase (E. C. 5.1.1.15) 1442
19.3.4.8 Synthesis of P-Lactam Antibiotics Catalyzed by Penicillin Acylase
(E.C. 3.5.1.11) 1444
19.3.4.9 Synthesis of Azetidinone P-Lactam Derivatives Catalyzed by Penicillin
Acylase (E.C. 3.5.1.11) 1444
19.3.4.10 Enantioselective Synthesis of an Aspartame Precursor with Thermolysin
from Bacillus proteolicus (E. C. 3.4.24.27) 1446
19.3.4.11 Hydrolysis of Heterocyclic Nitrile by Nitrilase from Agrobacteriurn sp.
(E.C. 3.5.5.1) 1447
19.3.5 Formation of C-0 Bonds by Lyases 1447
19.3.5.1 Synthesis of Carnitine Catalyzed by Carnitine Dehydratase in Whole Cells
(E.C. 4.2.1.89) 1447
19.3.6 Formation of C-N Bonds by Lyases (E. C. 4) 1448
19.3.6.1 Synthesis of L-Dopa Catalyzed by Tyrosine Phenol Lyase from Enuinia
herbicola (E. C. 4.1.99.2) 1448
19.3.6.2 Synthesis of 5-Cyano Valeramide by Nitrile Hydratase from Pseudornonas
chlororaphis B23 (E. C. 4.2.1.84) 1449
Contents
19.3.6.3 Synthesis of the Commodity Chemical Acrylamide Catalyzed by Nitrile
P'
Hydratase from Rhodococcus rodochrous (E. C. 4.2.1.84) 1450
19.3.6.4 Synthesis of Nicotinamide Catalyzed by Nitrile Hydratase from
Rhodococcus rodcchrous (E. C. 4.2.1.84) 1451
19.3.7 Epimerase 1452
19.3.7.1 Epimerization of Glucosamine Catalyzed by Epimerase from E. coli
(E. C. 5.1.3.8) 1452
19.4 Some Misconceptions about Industrial Biotransformations 1453
19.5 Outlook 1454
References 1454
20 Tabular Survey ofCommercially Available Enzymes 1461

Peter Rasor
Index 1519
List of Contributors
Carabed Antranikian Manfred Biselli

Technische Universitat Harnburg- Fachhochschule Aachen
Harburg Abteilung Julich
Institut fur Biotechnologie Labor fur Zellkulturtechnik
KasernestraBe 12 Ginstenveg 1
21073 Hamburg 52428 Julich
Germany Germany
Andreas 5. Bommarius
Frances H. Arnold School of Chemical Engineering
California Institute of Technology Georgia Institute of Technology
Department of Chemical Engineering 315 Ferst Drive
MC 210-41 Atlanta, GA 30332-0363
Pasadena CA 91 125 USA
USA
Bruno Buhler
Institut f i r Biotechnologie
Haruyuki Atomi ETH Honggerberg, HPT
Department of Synthetic Chemistry and 8093 Zurich
Biological Chemistry Switzerland
Graduate School of Engineering
Kyoto University Nobuyoshi Esaki
Yoshida, Sakyo-ku Institute for Chemical Research
Kyoto, 606-8501 Kyoto University
Japan Uji
Kyoto-fu 611
Japan
Constanzo Bertoldo
Technische Universitat Hannburg- Kurt Faber
Harburg Department of Chemistry
Institut fur Biotechnologie Organic and Bioorganic Chemistry
KasernestraBe 12 University of Graz
21073 Hamburg Heinrichstrasse 28
Germany 8010 Graz
Austria
XXXIV
I List ofcontributors
Duraiswamy A. Feyaraj Peter Halling

Max-Planck-Institutfur Molekulare Department of Chemistry
Physiologie University of Strathclyde
Abteilung Chemische Biologie Glasgow G1 1XL
Otto-Hahn-StraBe11 United Kingdom
44227 Dortmund
Germany Yoshihiko Hirose
Amano Enzyme Inc.
Martin H. Fechter Gifu R & D Center
Institut f i r Organische Chemie 4-179-35,Sue, Kakamighara
Technische Universitat Graz Gifu 509-0108
Stremayrgasse 16 Japan
8010-Graz
Austria
Kay Hofmann
Bioinformatics Group
Sabine Flitsch MEMOREC Stoffel GmbH
Department of Chemistry Stockheimer Weg 1
The University of Edinburgh 50829 Koln
West Mains Road Germany
The King’s Building
Edinburgh EH9 3J J
United Kingdom Frank Hollmann
Institut fur Biotechnologie
ETH Honggerberg, HPT
Hans-Joachim Gais
CH-8093 Zurich
Institut fur Organische Chemie
Switzerland
RWTH Aachen
Professor-Pirlet-StraBe1
52056 Aachen Tadayuki lmanaka
Germany Department of Synthetic Chemistry and
Biological Chemistry
Herfried Criengl Graduate School of Engineering
Institut fur Organische Chemie Kyoto University
Technische Universitat Graz Yoshida-Honmachi,Sakyo-ku
Stremayrgasse 16 Kyoto 606-8501
8010-Graz Japan
Austria
Hans-Dieterjakubke
Cideon Crogan Fakultat fur Biowissenschaften,
Department of Chemistry Psychologie und Pharmazie
The University of Edinburgh Institut fur Biochemie
West Mains Road Universitat Leipzig
The King’s Building TalstraBe 3
Edinburgh EH9 3JJ 04103 Leipzig
United Kingdom Germany
List ofcontributors
I-
Dieter Kadereit Tomoko Matsuda
Johann-StrauB-StraBe18a Department of Materials Chemistry
65779 Kelkheim Faculty of Science and Technology
Germany Ryukoku University
Otsu
Shiga 520-2194
Udo Kragl Japan
Universitat Rostock
Fachbereich Chemie
Oliver May
BuchbinderstraBe 9
Degussa-Huh AG
18051 Rostock
Rodenbacher Chaussee 4
Germany
63457 Hanau
Germany
Maria-Regina Kula
Institut fur Enzymtechnologie Kaoru Nakamura
Heinrich-Heine Universitai Dusseldorf Institute for Chemical Research
im Forschungszentrum Jiilich Kyoto University
Stetternicher Forst Uji
52428 Jiilich Kyoto 611-0011
Germany Japan
Tatsuo Kurihara Claudia Neri

Institute for Chemical Research School of Chemistry
Kyoto University University of Bath
Uji Claverton Down
Kyoto-Fu 611 Bath BA2 7AY
United Kingdom
lapan
Romano A. Orru
lamesJ. Lalonde Division of Chemistry
Altus Biologics Inc. Bio-organicChemistry
625 Putnam Avenue Vrije University Amsterdam
Cambridge De Boelelaan 1083
MA 02139-4807 1081 H V Amsterdam
USA The Netherlands
Andreas Liese RebekkaJ. Parker

Forschungszentrum Jiilich GmbH School of Chemistry
I BT University of Bath
Leo-Brandt-Strage Claverton Down
D-52428 Jiilich Bath BA2 7AY
Germany United Kingdom
xxxvt
I List ofcontributors
Markus Pietzsch Birgit Schulze

Institute for Bioprocess Engineering DSM Food Specialties
University of Stuttgart Nutritional Ingredients
Department of Microbial Physiology P.O. Box 1
Allmandring 31 2600 MA Delft
70569 Stuttgart The Netherlands
Germany
Christoph Syldatk
Reinhard Reents
Institute for Bioprocess Engineering
Max-Planck-Institutfur Molekulare
University of Stuttgart
Physiologie
Department of Microbial Physiology
Abteilung Chemische Biologie
Allmandring 31
Otto-Hahn-Strage 11
70569 Stuttgart
44227 Dortmund
Germany
Germany
Peter Rasor Fritz Theil

Industrial Biochemicals Business ASCA Angewandte Synthesechemie
BB-PS, Roche Molecular Biochemicals Adlershof GmbH
Roche Diagnostic GmbH Richard-Willstatter-Strage 12
Nonnenwald 2 12489 Berlin
82372 Penzberg Germany
Germany
I. David Rozzell Karl-Heinz van Pie

School of Chemical Engineering Institut fur Biochemie
Georgia Institute of Technology Technische Universitat Dresden
315 Ferst Drive Mommsenstrage 13
Atlanta, GA 30332-0363 01062 Dresden
USA Germany
Kenji Soda Christopher A. Voigt

Faculty of Engineering California Institute of Technology
Kansai University MC 210-41
Yamate-cho Pasadena CA 91125
Suita USA
Osaka-Fu 564
Japan
Erik de Vroom
Andreas Schmid DSM Food Specialties
Institut f i r Biotechnologie Nutritional Ingredients
ETH Honggerberg, HPT P. 0. Box 1
CH-8093 Zurich 2600 MA Delft
Switzerland The Netherlands
Herbert Waldmann Jonathan M.J. Williams
Max-Planck-Institutfiir Mcllekulare School of Chemistry
Physiologie University of Bath
Abteilung Chemische Biologie Claverton Down
Otto-Hahn-StraBe11 Bath BA2 7AY
44227 Dortmund United Kingdom
Germany
Chi-Huey Wong
Christian Wandrey Department of Chemistry
Forschungszentrum Julich GmbH The Scripps Research Institute
Institut fur Biotechnologie 10550 Torrey Pines Road
52425 Julich La Jolla, CA 92037
Germany USA
George M. Whitesides Marcel Wubbolts

Department of Chemistry Manager Research & Development
Harvard University DSM Biotech GmbH
12 Oxford Street Karl-Heinz-Beckurts-Strage13
Cambridge, MA 0213&2902 52428 Julich
USA Germany
Karlheinz D r a w and Herbert Waldmann
Copyright 0Wiley-VCH Verlag GmbH, Weinheim 2002
1
Introduction
Maria-Regina Kula
1.1
Enzymes as Catalysts
Enzymes are the catalysts evolved in nature to achieve the speed and coordination of
a multitude of chemical reaction necessary to develop and maintain life. Chemical
reactions are far too slow to be effective under the conditions prevalent in normal
living systems - aqueous environments with neutral pH values and temperatures
between 20 and 40 "C. Even catalysts developed in the chemical industry fall short;
enzymes in comparison achieve up to lo7 - fold faster reaction rates. Mankind has
utilized enzymes empirically since ancient times for the conservation or production
of food, e. g. in cheese making or brewing. A historical background is given in Table
1-1.The catalytic properties of enzymes were recognized long before their chemical
nature was known. We stil~use acceleration of reaction rate to search for unknown
enzymes as well as to measure and quantify enzyme activity.
As catalysts - true to the definition familiar in chemistry - enzymes alter the rate
at which a thermodynamic equilibrium is reached, but do not change that equilib-
rium. This implies that enzymes work reversibly. The acceleration in reaction rate is
achieved by lowering the activation energy of the overall process as shown schemat-
ically in Fig. 1-1.
Enzymes bind their substrates by multiple non-covalent interactions on a specific
surface. This way, a micro-heterogenization occurs and the local concentration of
substrates is increased relative to the bulk solution. In addition, the chemical
potential of specific groups may be drastically changed temporarily compared to
aqueous solutions by the Iexclusion of water in the reactive site upon binding of
substrate. Both aspects contribute to the observed phenomenon of high acceleration
in reaction rate; some examples are presented in Table 1-2. Enzymes often bind the
substrate in the transition state better than in the ground state, which lowers the
activation energy.
Since the pioneering work of Buchner (1897), it has been known that enzymes do
not require the environmept of a living cell to be active. This opened the way to many
applications in food technology, in the production of leather, textiles and paper, in
1 Introduction
2
I Table 1-1. Brief history of enzymes and their applications. -
BC Chymosin from the stomach of young cattle, sheeps and goats
was used for cheese production in many ancient cultures for
approximately 7000 years.
1783 Hydrolysis of meat by gastric juice demonstrated. Spallazani
1814 Starch degradation and sugar production by malted barley Kirchhoff
observed.
1833 The active principle of malt is called diastase and its applica- Payen and Persoz
tion to industrial art described.
1846 Invertase activity observed. Dubonfout
1867 The term enzymes is coined to describe catalytic activity not Kiihne
bound to living cells (unorganised ferments). The name
is extended later also to intracellular catalysts (organised
ferments as defined by Pasteur).
1893 Definition of a catalyst including enzymes is given. Ostwald
1894 Enzyme stereospecificity anticipated. E. Fischer
1894 “Taka diastase” produced commercially with Aspergillus olyzae Takamine
by surface culture
1897 The conversion of glucose to ethanol demonstrated by a cell Buchner
free extract from yeast.
1906 Preparative separation of L-leucinefrom the racemate carried Warburg
out by hydrolysis of the propyl ester with liver extracts.
1908 Synthesis of optically active cyanohydrins described, using Rosenberg
D-oxynirerilasefrom almonds as catalyst.
1908 Application of pancratic enzymes in the leather industry for Rohm
the bating of hides.
1911-1913 Glucoside synthesis in the presence of high concentration of Bourquelot,
ethanol or acetone described. Bride1 and Verdon
1913- 1915 Application of pancreatic enzymes to clean laundry intro- Rohm
duced, first commercial product sold to the public: Burnus.
1916 Immobilization of invertase on charcoal demonstrated with Nelson and Griffin
retention of activity.
1926 Urease from Jack beans crystallized. Sumner
1936 Enzymatic ester synthesis improved using pancreatic lipase in S P
the presence of benzene.
1953 The first primary sequence of a protein (Insulin) established, Sanger
proving the chemical identity of proteins.
1960 Cultivation of Bacillus lichenijomis in submerged culture NOVO
started for protease production on a large scale.
after 1980 Application of genetic engineering techniques to improve many
enzyme production and to alter enzyme properties by protein
engineering and evolutionary design.
1.1 Enzymes as Catalysts
13
I \
\
: \
?
C
a
a
a
71
+
U
C
C
[G
II ----
//
II
-'i\
,I
I
\
\\
\
ES
\\
\
E+P
Course of r e a c t i o n
Figure 1-1. Free-energy profile for the course o f an enzyme-catalyzed reaction involving the
formation o f enzyme-substrate (ES) and enzyme-product (EP) complexes. The reaction pathway
goes through the transition states TS,, TS,2, and TS,3 with standard free energy o f activation
ACc. The rate limiting step would be the conversion o f ES into EP. The schematic profile for the
uncatalyzed reaction is shown as the dashed line. The catalytic effect is due t o the lowering o f t h e
standard free energy o f activation from A C u t o ACc and is not governed by the difference in free
energy between S and P.
diagnostics and food analysis and, last but not least, in the production of chemicals
by biotransformations.
One or more of the following reasons could make enzymes the catalysts of
choice:
- a highly selective operation in complex mictures,
- stereo- and regiospecificity of conversions,
- absence of side reactions leading to simpler separation processes and higher
yields, or
- savings in energy and waste treatment cost owing to mild reaction conditions.
Enzymes have limitations, SI; does any other highly specialized catalyst. Most notable
is one consequence arising from the selectivity of enzymes with regard to the
substrates bound and the type of reaction catalyzed. The price for such selectivity is
that it may be difficult to satisfy the requirement for many special enzymes to cover
the diversity of chemical rextions desired in organic chemistry. The enzyme needed
in a specific case may not be readily available. However, there are new enzymes
discovered all the time and an increasing number can be obtained commercially.
Other limitations with regard to reaction conditions, pH and temperature tol-
4
I 1 Introduction
Table 1-2. Relative rates of enzyme catalyzed and non-catalyzed reactions under conditions
optimal for enzyme reaction.
Enzyme Reaction Ratioa
Triose phosphate 9 3x 108-109
Isomerase @ Y H %=== @ T O H
OH 0
105-1010
Urease 0 1014
H2NKNH2 C02+2NH3
Hexokinaseb > 8 x 10" M

glucose + ATP + glucose G @ + ADP
Alcoholb >2x1O1OM
dehydrogenase
ethanol + NADa -+ acetaldehyde + NADH + Ha
a ratio of enzyme catalyzed to non-catalyzed rate
b bimolecular reactions
@ = Phosphate
erated by enzymes are to some extent predictable by their chemical nature. In this
Introduction, general aspects of enzyme structure, function and nomenclature will
be discussed to guide the reader with little biochemical background into the field of
enzyme application in organic chemistry.
1.2
Enzyme Structure and Function
All enzymes are proteins, with the exception of the recently discovered ribozymes.
Ribozymes are special ribonucleic acids performing catalytic functions in the
processing of RNA which will not be considered here. Proteins are polar macromole-
cules with molecular mass in the range 104-106. They are linear polymers, defined
by the sequence of amino acids, which are linked by peptide bonds:
0
H2N O0
The individual properties of a protein depend on the chemical nature of the side
chains depicted as R in Scheme 1-1.In protein biosynthesis, 20 amino acids are
1.2 Enzyme Structure and Function
15
Table 1-3. Amino acids for protein biosynthesis.
Name Symbol Structure pK, of ionizing side chain"

Glycine GlY (G) /COOH
I
Alanine Ala (A)
Valine Val (V)
Leucine Leu (L)
Isoleucine Ile (I)
Serine Ser (S)
Threonine Thr (TI
Cysteine CYS (C) 9.1-9.5
Methionine Met (M)
Proline Pro (P)
Phenylalanine Phe (F)
Tyrosine Tyr (Y) 9.7
Tiyptophane Trp (W)
Asparagine Asn (N)
Glutamine Gln (Q)
ASP (D)
Aspartate
HonCOOH
0 NH,
4.5
6
I I Introduction
Table 1-3. (cont.).

Name Symbol Structure pK, of ionizing side chain"
Glutamate Glu (E) 4.6
"o&cooe
NH2
Lysine LYS (K) 10.4

H2N-cooH
NH2
a-amino group 6.8-7.9

a-carboxylgroup 3.5-4.3
a The p& values depend on temperature, ionic strength and, especially on the microenvirronment of the
ionizable group
condensed according to information coded in the corresponding genes. The coded

amino acids are summarized in Table 1-3. Some modified amino acids, for example
4-hydroxyproline, 5-hydroxylysine,y-carboxyglutamate,0-phosphorylated serine, or
N-glycosylated asparagine are also found in proteins, usually in minor amounts,
resulting from post-translational modifications. These modifications are important
for the structure and function or the regulation of the activity of certain proteins. For
20 building blocks and a random sequence, the number of possible variations in the
primary structure is 20"; for a protein of average size of 33 000 Da 6 300 amino acids,
lo3" possibilities exist. The number is far beyond our perception, the known cosmic
space is not large enough to accommodate a single copy of each variant. To generate
a specific surface as part of the active center of an enzyme, the protein chain has to
fold. From the observed length and angle of the C=O and C-N bonds in peptides it
can be deduced that the peptide bond possesses partial double bond character,
resulting in a planar arrangement as shown schematically in Fig. 1-2. Movement of
the planes against each other occurs around the a-carbon, which serves as a joint
(Fig. 1-3). Rotation around the C-C and C-N bond is restricted because of steric
influences of the side chain R. By this feature of the peptide bond, two structural
arrangements of a polypeptide become energetically favored: the a-helix and the p-
pleated sheet, which are further stabilized by hydrogen bonds between the peptide
backbone (Fig. 1-4). Helices and pleated sheets are commonly found in proteins;
these secondary structure elements are linked by p-turns or loops to build a domain
or a subunit. This level of organization is called the tertiary structure of proteins,
while the assembly of subunits into homo- or hetero-oligomers or multi-component
systems is called quaternary structure. The hierarchy of structures is depicted in
Fig. 1-5.
17
Figure 1-2. Special features ofthe peptide bond. Dimensions are given in A and
represent average values from X-ray analyses. The peptide group has a rigid and
planar structure.
The mechanism determining the folding of a given protein is presently the topic of
intense research. For this discussion, it is sufficient to state that there exists a unique
tertiary/quaternary structure for each native protein chain, determined as an energy
minimum in aqueous solution. The information to reach this structure is thought to
be encoded in the primary sequence in a way not yet understood completely. The
8
I 7 Introduction
19
Figure 1-5. Hierarchy o f protein struc- Primary structure:

tures. The three-dimensional structure of
sequence of acid, e, g,
- Gly - Glu - Ser - Lys - Phe -
an enzyme is the result ofdifferent levels
of folding and interactions of protein
chains proceeding in ordered fashion from secondary structure:
the primary structure after protein bio- a-helix
synthesis.
p-sheet
tertiary structure:
Single domain or multi domain folding
Quarternary structure:
protein aggregate of like or unlike subunits
folded structure of a protein is stabilized by a network of non-covalent interactions,

most notably hydrogen bonds, hydrophobic and van der Waals interactions, and
ionic bonds. In the folding process, hydrophobic side chains of amino acids are
10
I preferentially oriented towards the interior of the molecule, thereby diminishing the
I Introduction
surface area in contact with water and minimizing the free energy. Polar groups are
preferentially oriented towards the surface interacting with water. In the compact
inner core of a protein, water is virtually excluded or present as single HzO
molecules (!) in defined places. The folding process generates a unique three-
dimensional surface of a protein defined in molecular dimensions by the specific
side chains and the polypeptide backbone.
Substrates and their transition states are also bound by multiple noncovalent
interactions with such a surface. Since the strength of all these noncovalent bonds is
strongly dependent on distance and angle of interaction, a highly selective binding
may result. By a three-point attachment even discrimination between two enantio-
mers is possible. Steric constraints may also contribute to differentiation between
similar structures during binding. The specific binding site of enzymes often is
found in a cleft on an irregularly shaped surface. Substrate recognition is a dynamic
process not only with regard to association and dissociation of the substrate; it may
also involve movements of the polypeptide chain in response to the binding. An
example of the latter is shown in Fig. 1-6.
Carboxypeptidase A hydrolyzes proteins sequentially starting from the free car-
boxyl terminus. The enzyme preferentially cleaves hydrophobic amino acids. Al-
ready in 1967 the three-dimensional structure had been determined with high
resolution by W. Lipscomb and his group. In the meantime much is known about
the catalybc mechanism. The essential features are discussed here briefly to improve
the understanding of how enzymes work. Two aspects of enzymatic catalysis can be
illustrated by this example: 1. Substrate binding may be accompanied by changes in
enzyme structure. 2. Substrate binding induces subtle but important shifts in
electron distribution in the substrate, making it more susceptible to certain reactions
(here hydrolysis).
In Fig. 1-6,the tertiary structure of the free carboxypeptidaseA is presented as well
as an enzyme-substrate complex with glycyl-tyrosine. Changes in the enzyme
structure are most evident by looking at the position of tyrosine-248. The phenolic
hydroxyl group of the side chain moves from a position near the surface of the
enzyme 12 A toward the interior. A distance of 12 A represents about a quarter of the
diameter of carboxypeptidase A. Tyrosine-248then covers the bound glycyltyrosine
(Fig. 1-6 B) and the phenolic hydroxyl group is oriented toward the terminal carboxyl
group of the substrate. The movement of the tyrosine-248 side chain is possible by
rotation of the C - C bond at the j3 carbon. As a consequence of the rotation, the
binding site of carboxypeptidase A is shielded from bulk water. Closer inspection of
Fig. 1-6 A and B shows that the guanido group of arginine-145 as well as glutamate-
270 also move about 2 A upon substrate binding. Both residues are involved in the
catalytic step.
The second important point is the perturbation of the electron distribution in the
substrate by the essential Zn” and specific side chains in the enzyme. During the
binding process the substrate is oriented first by an electrostatic interaction of the
carboxylate group with the positively charged arginine-145;in addition, tyrosine-248
forms a hydrogen bond with the amide nitrogen of the terminal peptide bond. The
carbonyl group becomes coordinated to the Zn” displacing water as a ligand.
Figure 1-6. The structure

of carboxypeptidase A
changes dynamically upon
substrate binding. (A)
Enzyme alone, (B) enzyme
complex with glycyl-tyro-
sine. Tyrosine 248 moves
12 A after binding of sub-
strate. Hydrolysis results as
a concerted action o f ZnZ+,
Glu, Tyr, and Arg side
chains towards the carbonyl
and nitrogen group i n the
susceptible peptide bond
(C).
B
12
I 7 Introduction
The hydrophobic group in the substrate (tyrosine in the example illustrated here)
is bound into an unpolar, large cleft by hydrophobic forces replacing at least 4 water
molecules upon binding and inducing the movement of tyrosine-248 discussed
above. The unpolar lining and the size of the cleft explains the preference of
carboxypeptidase A for bulky, hydrophobic side chains of the terminal amino acid.
The free amino group of glycyltyrosine is hydrogen bonded through a water
molecule to glutamate-270. This bonding of glutamate is thought to slow down
dramatically the hydrolysis rate of glycyltyrosine and related dipeptides (and make
possible the X-ray analysis of the complex). Such a hydrogen bond is not found in
productive enzyme-substrate complexes involving oligopeptides or proteins. The
carboxylate group of glutamate-270 is thought to attack the carbon in the carbonyl
bond of the substrate leading to a mixed anhydride. The carbonyl bond is already
polarized by the Lewis acid Zn2+, the induction of the dipole is favored by the
unpolar surrounding of the Zn2+ion and the tetrahedral intermediate is stabilized by
the positive charge of nearby arginine-127. The hydrolysis of the peptide bond is
completed by transfer of a proton form water to the nitrogen, releasing the C-
terminal amino acid.
Substrate binding in a defined manner is a prerequisite for enzyme catalysis. It
exposes a chemical compound long enough to a unique chemical potential built into
the system, which defines the type of reaction that will proceed, for example,
hydrolysis, oxidation/reduction, or C - C bond formation. The mechanism most
often is the same as that known from solution chemistry, for example, acid-base
catalysis. The close proximity of reactants and the precise orientation, together with
the effect of microheterogenization discussed above, lead to the outstanding per-
formance of an enzyme as catalyst (examplesare given in Table 1-2). Often, transient
covalent bonds are formed between substrate and enzyme or coenzyme (see below)
during a catalyhc cycle. Serine, cysteine,histidine, lysine, aspartate or glutamate may
donate en electron pair to a substrate, forming a covalent linkage as shown in Fig. 1-7
for the well-known charge-relay system in serine proteases. The highly reactive
intermediates formed may be attacked by water or a second substrate to yield the
characteristic products of the reaction.
1.3
Cofactors and Coenzymes
The chemical potential of side chains found in amino acids is limited; for example,
there are no efficient electron acceptors. Therefore, enzyme catalysis incorporates if
necessary additional chemical potential by specific metal ions, for example, Zn2+(see
Fig. 1-6), Fe2+ Co2+, Cu2+ and others Examples are shown in Fig. 1-8 for the co-
ordination of the transition metal ions in protein structures. Besides metal ions,
cofactors or coenzymes serve to activate groups and participate in the catalytic
process. A summary of cofactors and coenzymes is given in Table 1-4;the relation to
vitamins is quite apparent. Chemical structures are presented in Table 1-5. Coen-
zymes and cofactors may act by nucleophilic or electrophilic attack on the sub-
7.3 Cofactors and Coenzymes
113
Tetrahedral Acyl-enzyme
Substrate transition state intermediate
Ser 195
R'
-O\ ,R' 0%
-CH2-O O=C' / C\ C- R'
I
-
I
H. R-N--H -CH2-0 /
N-H -CHz-O
RH R-N-H
d I
I
H
HC\IN-!"
HC,lN +- i H
;-c,cI-lz N-C,
His 57 : I H CH2
0- N-C,
I H CH2
o@- : I
Asp102 0."-
I 0-
I
O,C\
Acyl-enzyme Tetrahedral Acid component

intermediate transition state of the substrate
0% -O\ ,R'
C-R'
C\
-CH2-0 H, /
-CH2-0 0-H
0
I H
..
U I
N-CH
"C:'
N-CH
I/
- lN-iH
HC\\ +
N-C,
H
- II
CH2
HC:'
N-C,
H CHz
N-C,
: I : I
* --
,.
HIS 5 /
H 7H2
I
- --
u, /u
n-
U
C I
0-
I
I o'c\
Asp 102 O S c \
Figure 1-7. The catalytic triad in serine proteases. The reactive serine forms an acyl
enzyme as a covalent intermediate during the proteolytic cleavage o f a peptide bond.
During substrate binding a proton is transferred from serine 195 t o histidine 57, and the
positive charge o f t h e iniidazole ring is stabilized by interaction with the carboxylate side
chain o f aspartic acid 102. The numbering corresponds t o the structure o f chymotrypsin.
strate(s) to initiate a reaction. Cofactors are tightly (covalently)bound to the protein

and may undergo cyclic reactions during the catalytic process but will return to the
ground state at the end. Ifoxygen is the terminal electron acceptor in FAD, FMN or
NAD(P)+ linked reactions,, these cofactors require a second reaction with the co-
substrate oxygen to regenerate the active form. In the older literature cofactors
sometimes are called "prostetic groups". Coenzymes are bound in an association/
dissociation equilibrium to enzymes and have to be present in sufficient concentra-
tion to obtain maximal enzymatic activity. Some are regenerated in the catalytic cycle
14
I 7 Introduction
H H
C"rl
1 3
HisxJ
H
@
,
7
Figure 1-8. Typical co-ordination complexes o f transition metal ions in proteins. 1: M may be
Fe", as i n rubredoxin, or Zn" as in aspartate transcarbamylase and alcohol dehydrogenase, 2:
carboxypeptidase A, 3: carbonic anhydrase, 4: liver alcohol dehydrogenase, 5: azurin, 6: heme
group, L is His and L'either H i s or Met i n cytochromes, 7: deoxy-heme group in hemoglobin and
myoglobin, 8: oxyform o f 7, 9: superoxide dismutase.
R,6CooH
0
Leucine-Dehvdroaenase
-/-E.C. 1.4.1. /
Formate Dehydrogenase
Figure 1-9. N A D H regeneration using for-
mate dehydrogenase (FDH) in a coupled
reaction with leucine dehydrogenase CO2 HCOONH4
(Leu DH). ammonium formate
Table 1-4. Cofactors and coerizymes.

Compound" 1:unction Relation to vitamins
NAD+/NADH+ H' redox reactions and hydrogen transfer vitamin PP, (niacin)
NADP'/NADPH + H' redox reactions and hydrogen transfer vitamin PP, (niacin)
FAD redox reactions and hydrogen transfer vitamin Bz. (riboflavin)
FMN redox reactions and hydrogen transfer vitamin B2, (riboflavin)
Haem iiansfer of electrons -
Coenzyme A transfer of acyl groups pantothenic acid
ATP metabolic energy,
phosphate-,pyrophosphatetransfer,
adenylation
Pyridoxal phosphate transamination, vitamin Bg, (pyridoxine)
(PLP) amino acid decarboxylation
Thiamine pyrophosphate decarboxylation, transfer of Cz units vitamin B1, (thiamine)
(TW
Biotin transfer of COZ biotin
Tetrahydrofolicacid transfer of C1 groups folic acid
S-Adenosylmethionine methylation -
(SAM)
Adenosyl-cobalamine isomerisation (hydrogen-shift) vitamin BIZ
Methyl-cobalamine methylation cyano-cobalamine
a The structure of the various compounds is summarized in Table 5.
while bound to the enzyme, for example, pyridoxal phosphate or thiamine pyro-
phosphate, so that catalytic amounts are sufficient to sustain the reaction. Others
require one or more separate reactions with cosubstrates other than oxygen to
regenerate the starting material. This holds true for example for NAD(P)',
NAD(P)H, SAM, coenzyme A, ATP and other nucleotide triphosphates. In such
instances, the coenzyme is consumed i n stoichiometric relation to product forma-
tion. This relation may render enzymatic synthesis quite expensive unless efficient
coenzyme regeneration cycles can be devised. In situ regeneration processes have
been successfully developed i n recent years, especially for the nicotinamide nucleo-
tides. The stoichiometric relation with product formation is shifted from the
expensive coenzyme to h e conversion of a cheap cosubstrate such as formate, as
shown schematically i n Fig. 9. A detailed discussion of coenzyme regeneration is
found i n another chaptei:
16
I 1 Introduction
Table 1-5. Chemical structures of cofactors and coenzymes.

Nicotinamide nucleotides
NAD' and NADP+and their reduced forms are involved in many dehydrogenase reactions
within the cell. They are water-soluble, and are usually free to diffuse away from the enzyme,
after conversion to the oxidized or reduced form to take part in another dehydrogenase reaction
catalyzed by another enzyme.
R = H: N A D ~
R = P O ~ HNADP
:
@ = Phosphate (PO3H2-group)
Flavin nucleotides
FAD is the coenzyme of a class dehydrogenases calledpauoproteins.The flavin moiety of the
molecule is derived from riboflavin (vitamin Bz). Reduction of FAD involves the two un-
substituted N atoms of the isoalloxazine structure.
7.8-Dimethyl-
isoalloxatine
'
CH,
I
HC-OH
I
Hy-OH
HC-OH OH
,I I
5 CH2-0-P-OR
It
0
Flavin adenine
d
dinucleotide FAD
R = -@-cH
OH OH
Flavin mononucleotide
FMN R=H
117
Table 1-5. (cont.).
The electron transport chain
Enzymes in the electron transport chain split hydrogens into H+ and ec. The electrons are then
camed by enzymes called cytochrornes a, b, c, d.
These enzymes are able to accept an alectron and then pass it on to another cytochrorne. The
iron atom is bound, within the haern a, b, c, d group, to a porphyrin coenzyme identical with
that found in haernoglobin, with the difference that in the cytochrornes the iron undergoes
oxidation and reduction.
Haem b
82
Cytochrome c with haem c
I Cytochrome b
Fe'@ 1
Cytochrome b
Fe ' @
1 Introduction
18
I Table 1-5. (cont.).
CoenzymeA (CaA-SH)
Coenzyme A is a complex molecule which contains a free sulfydryl (-SH) group. This group can
react with a carboxyl group to form a thioester. In acetyl CoA, the thioester linkage can activate
the methyl carbon as well as the acetyl group.
---
Cysteamine P-Alanine Pantoic acid
R =H:
w
CoA-SH
R = Acetyl: Acetyl CoA
Adenine nucleotides
Pantothenic acid
- w
HO-P=O
I
OH
OH
Adenosine-3'-phoshate-5'-diphosphate
ATP, ADP and AMP are coenzymes influencing the direction of flow in metabolic pathways. In
addition ATP often functions as a donor of a phosphate to other molecules in reactions catalysed
by kinases.
CH*-O- @ - @ - @-OH
Table 1-5. (cont.).

Pyridoxal phosphate
Pyridoxal phosphate, a derivativ,: of vitamin B6, acts as coenzyme in transamination and
decarboxylationreactions. In a transamination reaction the aldehyde group of pyridoxal phos-
phate first forms a Schiff base with the amino group of the amino acid, which is then converted
to keto acid. Pyridoxal phosphate is thereby converted to pyridoxamine phosphate which
transfers the amino group to another keto acid to form the corresponding amino acid.
CH2M2
H o e -@-OH CH&- OH
H3C
Pyridoxal phosphate
RYC*H RKCmH
0
NHZ
Thiamine pyrophosphate
All biochemical reactions with participation of thiamine start with C-C-bond cleavage of 2-0x0
carbonyl-compound and proceed with formation of an “activatedaldehyde”, TPD catalyzes
decarboxylationof a-keto acid::, oxidative decarboxylationstogether with lipoic acid, and trans-
ketolase reactions.
Biotin
Biotin containing enzymes catalyze CO2-transfer reactions: these are carboxylation,transcarbox-
ylation and decarboxylations. The carboxy group of biotin is bound to an E - N Hof
~ lysine in an
enzyme protein.
0 R = H : D(+)-Biotin
R = COOH: N-1’-Carboxybiotin
0
20
I 7 Introduction
Table 1-5. (cont.).

Folate coenzymes
The transfer of a Cl-group like methyl, methylene, formyl or formimino often involves folic acid
in one of its substituted forms
2-Amino-4-hydroxy- p-Amino-
6-methylpteridine benzoic acid L-GIu
0 COOH
Tetrahydrofolic acid
Compound Structure Cl-fragment
Tetrahydrofolic acid H H -
4-10\ N
N5-formyl- formic acid
Po-methenyl- formic acid
N5, N"-rnethenyl- formic acid
N5-formimino- formic acid
N5, N"-methylene- formaldehyde
Ns-methyl- methanol
S-Pdenosyl-1-methionine YHz
S-Adenosyl-L-methionineas sulfonium compound could transfer its methyl group as CH3e to

nucleophile centers of substrates in biochemical reactions.
Table 1-5. (cont.).
7.4 Enzyme Nomenclature
I 21
Cobalamine
Adenosyl-cobalaminecatalyzes hydrogen shifts as a special isomerisation reaction. With excep-
tion of reduction of ribonudeotides the H-shift occurs intramolecularly. Methyl-cobalamine and
tetrahydrofolic acid are the coenzymes in methylating homocysteine to methionine.
I
CH,-CH
I
y 2
NH ,CONHI
\
p 2
FH2
CONH2
: Adenosyl-cobalamine
R=CH, : Methyl-cobalarnine
R=CN : Cyano-cobalarnine (Vitamine BI~)
1.4
Enzyme Nomenclature
The IUB has classified enzymes into 6 main classes according to the type of reaction
catalyzed:
22
I 1 Introduction
1. Oxidoreductases
These catalyze oxidation/reduction reactions, transferring hydrogen, oxygen,
and/or electrons, between molecules. In this important class belong dehy-
drogenases (hydride transfer), oxidases (electron transfer to molecular oxygen),
oxygenases (oxygen transfer from molecular oxygen), and peroxidases (electron
transfer to peroxide)
2. Transferases
These catalyze the transfer of groups of atoms, e. g. amino-, acetyl-, phosphoryl-,
glycosyl- etc. from a donor to a suitable acceptor. Reactions covered in class 1 , 3 , or
4 are excluded.
3. Hydrolases
These catalyze the hydrolytic cleavage of bonds. Many commercially important
enzymes belong to this class, e. g. proteases, amylases, acylases, lipases, and
esterases.
4. Lyases
These catalyze the non-hydrolytic cleavage of, for example, C - C, C - 0 or C - N
bonds by elimination reactions leaving double bonds or, in reverse, adding groups
to a double bond. Examples are fumarase, aspartase, decarboxylases, dehy-
dratases, and aldolases; many lyases are important catalysts for organic synthesis.
In older literature class 4 enzymes are often called synthases, e.g. tryptophan
synthase. These should not be confused with synthetases, as class 6 enzymes are
sometimes called.
5. Isomerases
These catalyze isomerization and transfer reaction within one molecule. The
most prominent member of this group is D-xylose ketol-isomerase, commonly
known as glucose isomerase.
6. Ligases
These catalyze the covalent joining of two molecules coupled with the hydrolysis
of an energy rich bond in ATP or similar triphosphates. An example is y-L-
glutamyl-L-cysteine:glycine ligase (ADP-forming),also found under the name
glutathion synthetase. Ligases find limited applications only for synthetic pur-
poses.
The main classes are further subdivided into subclasses and subgroups, as in part
indicated above. A complete ordering system can be found in the publications from
IUB. The systematic name of an enzyme is based on the equation of the chemical
reaction taking place and the type of reaction, followed by the suffix-ase. By
international agreement the catalytic reaction is expressed and identified by 4 groups
of numbers according to the E. C. (enzyme classification)system introduced above.
For example, an enzyme converting an alcohol to an aldehyde (or ketone) using NAD
as coenzyme would be classified as
oxidoreductase main class 1
acting on CH - OH groups sub class 1.1
using NAD+ as acceptor sub group 1.1.1
alcohol NAD+-oxidoreductase E.C. 1.1.1.1
7.5 Enzyme Kinetics
I 23
The last number is the serial number of an enzyme identified by the first three
entries. An alcohol could be converted to similar products also using oxygen as the
electron acceptor by an oxidase.
oxidoreductase main class 1
acting on CH - OH groups sub class 1.1.
using oxygen as acceptor sub group 1.1.3
alcohol: oxygen-oxidoreductase E. C. 1.1.3.13
For newly isolated enzymes the nomenclature committee of IUB assigns the
correct E.C. number to avoid confusion. The last edition (1992) contains 3196
entries. This code system is used in the scientific literature, textbooks and catalogues
to identify an enzyme on the basis of the chemical reaction it catalyzes. For a proper
description the source has to be included. Besides the systematic name IUB also lists
trivial names or recommended names, the two enzymes described above being
better known as alcohol dehydrogenase or alcohol oxidase, respectively. The recom-
mended name is shorter and preferred in discussion after the catalyst has been duly
identified. It should be noted that the classification is not based on the enzyme
source and in general not on a single substrate. The physical properties of the
individual enzyme protein may vary, for example, pH optimum, K, values, stability,
substrate range etc., but the systematic name and the number code are identical as
long as the same type of reaction is catalyzed. Often it is worthwhile to test enzymes
from different sources for the reaction of interest to find the optimal catalyst.
Numerous successful applications of enzymes are described in the following
chapters. Many more opportunities exist for innovative approaches in synthetic
chemistry.
1.5
Enzyme Kinetics
1.5.1
Reaction Rate and Substrate Concentration
An enzymatic reaction rnay be described by the following steps: first, binding of

enzyme E and substrate IS occurs; second, while bound to the enzyme the substrate
will be converted to the product P;finally, the product is released from the enzyme
and free enzyme becomcs available for the next cycle. In the simple case of a one
substrate reaction this can be described by the following equations
[ S l + [El 5
K,-'
[ES] =+[EP] 5 [El+ [PI
Michaelis and Menten derived a mathematical description for the reaction rate of an
enzymatic process from this scheme
24 I a
1 Introduction
2.0 - v,,, ([El = 2.0 u n i t - ---------- -
-
VI
c
.-
C
3
K,, Concentration of substrate IM1

-
b
0.8
C
0
.-
c
0
go6
L
I/ I
0.2
Figure 1-10. Reaction rate as a function o f substrate concentration: a) using two

different enzyme concentrations in the assay, b) comparing low and high affinity
substrates o f t h e same enzyme.
with the assumption that the binding of substrate and enzyme is reversible and fast
compared to product release. Equation (1) represents a hyperbolic curve, relating
reaction rate with substrate concentration as shown in Fig. 1-10. The hyperbola is
described by two parameters: V,, and K,. K, the so-called Michaelis constant, is
defined as the substrate concentration for which the observed reaction rate is half of
V,, The K, value characterizes the affinity between substrate and enzyme and in a
first approximation can be viewed as the dissociation constant of the enzyme-
1.5 Enzyme Kinetics
substrate complex ES. K, is independent of enzyme concentration and usually has

I 25
values between and 3 0-2 M. V, is the maximal reaction rate possible if every
enzyme molecule present is saturated with substrate and is a property of the
particular enzyme. It may be related to the molecular mass of the enzyme and then is
called turnover number, representing the number of substrate molecules converted
per active site of an enzyme molecule per unit of time. The turnover number may
have values between and lo6 s-l; 103-4s-l is commonly found.
Another quantity used frequently for the characterization of an enzyme is the
catalpc activity. The unit for the catalytic activity is the Katal (kat), as defined by the
International Union of Eliochemistry (IUB), 1 kat corresponds to the amount of
enzyme catalyzing the coriversion of one mole of substrate per second at 30 "C under
specified conditions. In the biochemical literature, another quantity is often used,
the international unit (IU);1 IU catalyzes the conversion of 1 pmole of substrate per
minute under specified conditions. From the catalytic activity other values such as
volumetric activity ]kat 1;'; IU ml-'1 or specific activity [kat kg-'; IU mg-'1 are
derived. Catalpc activity can be determined unequivocally even in crude mixtures
and if the molecular prop-rties of the enzyme are unknown. Therefore, enzymes are
quantified measuring tht-ir catalytic activity and sold on the basis of activity. To
ensure reproducible and meaningful results when measuring enzyme activity,
several points have to be taken into consideration. As shown in Fig. 1-10[Eq. (l)], the
reaction velocity depends on substrate concentration; for [S] 2 100 K, the reaction
rate becomes zero order and so no longer depends on substrate concentration. In
special cases, for example, lipases reacting at an interface, the reaction rate depends
on the available interface rather than the concentration. Lipases are therefore
preferentially analyzed in stable emulsions. The catalpc activity has to be deter-
mined at sufficiently high substrate concentration (>10 Km) to ensure pseudo-zero
order rates. This may be difficult to achieve with substrates of low solubility.
Furthermore, it is desirable to measure initial rates, when only a small amount of
total substrate is converted; [S] remains essentially constant during the reaction time
and [PI is small. In reactions involving more than one substrate all concentrations
have to be considered. I f an unknown substrate or reaction is investigated two or
more substrate levels should be employed. At low substrate concentration and high
K, values the observed reaction rate may be small and not easily differentiated from
background noise, while, at high substrate concentration, inhibition by surplus
substrate (see below) may cause a substantial drop in the rate.
The reaction rate is be5 t determined by analyzing product formation as a function
of time by physical methods such as UV/VIS spectroscopy,optical rotation, potentio-
metry, etc. Alternatively, formation of a coproduct produced in stoichiometric
relations may be followed, such as formation of NAD(P)H in dehydrogenase
reactions, which is followed conveniently at 340 nm in a spectrophotometer. Product
formation may be coupled to a second reaction using a surplus of an auxiliary
enzyme producing an easily quantified signal, for example (NAD(P)+ or
NAD(P)H+H+with a dehydrogenase.
glutaminase
L-gluta:mine + H2O 4r-glutamate + NH3
26
I I lntroduction
L-glutamate + NAD' + H2O .

glutamate-dehydrogenase
a-ketoglutarate + NADH + NH4+
A similar approach determines a quinone-imine dye formed by the reaction of

HzOzcatalyzed by peroxidase.
alcohol oxidddase
2 ethanol + 2 0 2 + _ _ _ j 2 acetaldehyde + 2 H202
peroxidase
2 Hz02 + 4-amino antipyrine + phenol quinone-imine dye + 2 HzO
In such coupled systems, care must be taken in choosing reaction conditions, such
that the enzyme of interest is catalyzing the rate-determining step. Special synthetic
colorless substrates converted to colored products have been developed for hydro-
lases (esterases, phosphatases, glycosidases and proteases); 4-nitrophenol or 4-ni-
troanilide are used as the alcohol or amide component, which can be measured
readily around 400-420 nm.
phosphatase
4-nitrophenyl phosphate + HzO L4-nitrophenol + phosphate
N-a-benzoylarginine-4'-nitroanilide')
protease
__+ N-a-benzoyl arginine + 4 nitroaniline
If direct physical measurements are not available or feasible, the enzymatic reaction
can be stopped at predetermined times by rapid heating, acid treatment, or similar
measures and the amount of product present at time t measured by available
analytical techniques such as HPLC, GC, TLC (with or without prior derivatization).
Controls are required to ensure that the conditions employed to stop the enzymatic
reaction do not destroy the product and that the derivatization is complete. It may be
more convenient to follow the decrease in substrate concentration over time as a
measure of enzyme activity. This has the disadvantage that the difference of two
large values is prone to error. If such an approach is adopted it has to be proven by
independent experiments that the anticipated product is actually formed.
1.5.2
Inhibitors and Effectors
Chemical compounds negatively influencing the reaction rate of an enzyme-

catalyzed process are called inhibitors. Irreversible inhibitors might be reactive
substrate analogs forming a covalent linkage to the enzyme after binding and in this
way blocking the reactive site. Usually, such reactions are designed intentionally.
Heavy metal ions present in trace amounts as contaminants in crude substrates may
react with essential sulfhydryl groups and inactivate the enzyme. the situation is
similar to the well known poisoning of a metal catalyst by sulfur compounds. Far
more important for enzymatic processes are reversible inhibitors, forming specific
enzyme inhibitor complexes and thereby influencing the reaction rate. It is im-
portant to note that substrates and especially products might inhibit an enzymatic
Figure 1-11. Reaction rate as a
7.5 Enzyme Kinetics
I 27
function o f substrate concentra-

tion illustrating allosteric regula-
tion o f enzyme activity: a) rate in
the presence o f an allosteric
activator, b) rate in the absence
o f effectors, c) rate in the pres-
ence of an inhibitory effector.
reaction as might substrate analogs. Inhibition by substrate and/or product(s) is

important when considering how much of the activity added actually can be utilized
at a given set of reaction conditions. Such reaction engineering aspects are treated in
more detail in Chap. 4 of this book. Many enzymes may also be activated by
inorganic ions such as Ca2+, K+, or C1- possibly raising V, by stabilizing certain
protein conformations. If such an effect is noted, the activator should be added in
saturating amounts. Special effects are observed in the kinetics of allosteric en-
zymes. A typical sigmoidal curve describing reaction rate as a function of substrate
concentration is presented in Fig. 1-11. Binding of an effector to the regulatory
center alters the reaction rate very efficiently and subtly and is often used in nature to
divert the metabolic flow into different directions at branching points. Such a
response is important for living systems, but rarely will be seen with enzymes
employed in organic synthesis. The complex kinetics may be described by appro-
priate mathematical models, found in the specialized literature.
1.5.3
Influence of pH and Buffer!;
Enzymes contain many polar amino acids at the surface which may be protonated or
unprotonated depending on the pH of the surrounding medium. Typikal pK, values
are included in Table 1-3. Consequently, charges on the protein surface are altered
and K, and V,, will depend on pH. Fig. 1-12 illustrates that an optimum of the
reaction rate is observed ;is a function of pH. The optimal pH may vary slightly for
different substrates, reflecting differential binding energies. The pH optima for the
forward and reverse reaction of the same enzyme are not identical and may differ by
2-3 pH units. In the laboratory, the pH is usually set and maintained using buffers.
Selection of buffer ions may influence the observed reaction rate as shown in Fig.
1-3. The reasons are not well understood and are thought to be related to the polarity
of buffer molecules interacting with the protein, influencing simultaneously hydrati-
zation and solubility of substrates. On the preparative scale, pH is maintained better
by a pH-stat arrangement, saving chemicals and separation cost. Also, on the
28
I 7 Introduction
Figure 1-12. Reaction rate as a function o f pH.

Reductive amination of 2-keto caproate (O),
so0 2-keto-isocaproate ( O ) ,2-keto-valerate (o),
2-keto-4-mercapto-butyrate (A),and 2-keto iso-
valerate g)by leucine dehydrogenase (Bacillus
cereus) are shown as function of pH.
.-
v 300
0
Ex
0,
N
; 200
100
Y I 1 I
Y
7 8 9 10 71
PH
preparative scale, high substrate levels are desired, impossible to buffer sufficiently
in reactions involving release or consumption of protons.
In switching from buffered to pH-stat operation one should be aware of changes
in kinetics as discussed above. The pH of the solution is important not only for
enzyme activity but also for enzyme stability. Unfortunately, the optimal pH values
for enzyme activity and stability are not necessarily identical, as is well documented
in the literature for the hydrolysis of penicillin G by penicillin acylase. In such cases,
the method for controlling pH and mixing behavior of the reactor may become
crucial.
1.5.4
Temperature
Another important factor for enzyme activity is temperature. In general, the reaction
rate will increase with temperature (Fig. 1-14). From an Arrhenius type plot, the
activation energy of the process may be calculated. With increasing temperature,
however, the mobility of protein segments increases while the strength of hydro-
7.5 Enzyme Kinetics
129
-
7
1 I
5 6 8 9 10 11
PH
Figure 1-13. Effesct o f p H on the activity ofsec-alcohol dehydrogenase (Candida

boidinii) during the oxidation o f isopropanol in various buffers in 50 m M concentra-
tion: 0 sodium citrate, 0 potassium phosphate, Atriethanolamine/HCI, ATris/HCI,
W glycine.
phobic interaction decreases. At first, this results in a decrease in catalytic activity,

but, with further rise in temperature, in complete deactivation. Thermally induced
denaturation of proteins often leads to aggregation which is not readily reversible.
Denaturation may be expected in the temperature interval between 30 and 80 "C.
The optimal temperature of operation has to be lowered if long reaction times or
long service life of an enzyme are required. For enzyme assays, a defined (for
example 30 "C) and constant temperature has to be maintained. Enzymes from
extremely thermophilic microorganisms may be almost inactive at ambient tem-
peratures and operate in the temperature interval between 80 and 120 "C.
30
I 1 introduction
1 , 1 I I - *
20 30 40 50 60
Temperature ("C)
t 0
/-
' 0
8
.-2.
c
Y
h 1:
>
._
5 60-
m
(u
;
.-
- LO-
a:
(u
20
2o 1
-
I I I I *
20 30 LC! 50
Temperature ("C)
Figure 1-14. Temperature dependence o f the reaction rate A: L-2-hydrox-

ysisocaproate dehydrogenase (L. confusus) 6: D-lactate dehydrogenase
(L. confusus).
1.6 Organic Solvents as Reaction Media
I 31
1.6
Organic Solvents as Reaction Media
Enzymes as biocatalysts have been developed for aqueous reaction systems. Applica-
tion of enzymes in the preijence of organic solvents is of interest to organic chemists
because substrates may not be sufficientlysoluble in water, or the equilibrium of the
desired reaction may be iinfavorable in aqueous solution. The following general
approaches are used
- to add increasing con'centrations of water miscible solvents to the reaction
system,
- to work in two-phase systems composed ofwater and an immiscible solvent,
- to work in nearly anhydrous organic solvents with minimal necessary amounts of
water.
In the first two cases, the enzyme may be employed either in the soluble state or
immobilized. In nearly anhydrous organic solvents the enzyme is present in the
solid state only. The presence of organic solvents will influence activity as well as
stability of enzymes. In recent years, work of various groups has shown that the
majority of bulk water in a reaction system may be replaced by organic solvents. A
certain low amount of resi dual water is needed for activity; 0.02 % may be sufficient.
Organic solvents influence the dielectric properties of the reaction medium and to
varying degree disrupt ordered water structures. This, in turn,will influence the
non-covalent, weak forces responsible for the ordered structures of an enzyme.
Protein structures may be stabilized by adsorption, crosslinking, or covalent binding
to a hydrophilic surface. Immobilization may also help to avoid denaturation at the
interface in two-phase systems. If an immobilized or solid enzyme preparation is
used, it is important to provide sufficient surface area to catalyze the reaction. In
nearly anhydrous systems, maintaining the pH in the optimal range is a problem. In
such cases the enzyme has to be prepared (dried)under pH conditions providing the
optimal activity. This way the dissociation of charged groups on the enzyme surface
is fixed; there obviously exists a memory effect. The selection of a suitable solvent
with regard to activity and stability may be guided by the log P concept, where Pis the
partition coefficient of thr. solvent in an octanol/water biphasic system. Hydrophilic
solvents with log P value:<< 2 often lead to enzyme deactivation if present in high
concentrations; in contrast, apolar solvents with log P 2 4 are compatible with
enzymes, leaving the essential layer of water molecules on the polar surface regions
unperturbed. The results using solvents with intermediate values of log P (2-4) are
unpredictable and depend very much on the individual case. Solvent selection and
reaction conditions today are optimized empirically. Nevertheless, there are many
examples which clearly demonstrate that enzymes can be employed successfully also
in organic solvents.
32
I 1 Introduction
1.7
Enzyme Handling: Quality Requirements
An increasing number of enzymes are offered on the market by manufacturers and

vendors. Enzymes are produced for different purposes and may differ widely in
purity and price. In general, enzymes are sold on the basis of unit of activity. The
catalogue or data sheet of the supplier should contain information or a reference on
assay conditions and definition of the unit. Special requirements for storage (4 “C or
-20 “C) are recorded on the label. Enzymes are shipped as dry powders, suspensions
or (frozen) concentrated aqueous solutions. The sample may contain undeclared
additives such as inert materials (for example, fillers or filter aids), salts or
saccharides for stabilization, or precipitating agents such as ammonium sulfate or
polyethyleneglycol. Crude preparations may also contain several enzymatic activities,
for instance crude pig liver esterase is a mixture of esterases, lipases and other
enzymes. The presence of other proteins may be inferred from the specific activity
(U/mg protein) of the enzyme preparation, provided the catalytic activity of the pure
protein of interest is known. All common protein assays are relative measurements
only and therefore depend to some extent on the method and the protein used for
calibration. If desired, compounds of low molecular weight can be removed from the
enzyme by dialysis, ultrafiltration or gel filtration. For assay purposes, the necessary
dilution of the catalyst may often be sufficient to avoid interferences. Wetting of dry
proteins is not without problems, resolubilization of (freeze)dried material may take
many hours and appropriate controls should be applied if the expected conversion or
activity is found to be low.
The requirements for purity of an enzyme are not very high from the point of view
of application in the synthesis of fine chemicals. More importantly, enzyme quality
can be accessed fairly accurately by establishing a mass balance. Only such activities
responsible for undesired side reactions have to be removed. As an example,
fumarase (adding water to fumaric acid) should be absent from an aspartase
preparation (adding ammonia to fumaric acid), because the enzymatic reaction is
performed in water. Aspartase, however, is “silent”in a fumarase preparation as long
as ammonium ions are absent. Therefore, aspartase does not necessarily need to be
removed from a catalyst employed for production L-malic acid. Very often one can
exploit the fact that the reaction in synthetic applications is restricted to the available
reactants. Such a situation is entirely different from analytical applications, where
complex mixtures are introduced and the selectivity of the reaction depends solely on
the catalyst. An enzyme should be purified only to the degree necessary for the
application. Absence or low levels of proteases are also desired to protect the enzyme
catalyst from degradation. Stability under operation conditions is usually more
important for production than the initial cost of the enzyme. Stability will allow long
reaction times at low enzyme levels or prolonged service life in continuous
processes.
Genetic engineering techniques can be employed for the production of large
amounts if a certain enzyme is needed for application as a catalyst. In addition,
specialized knowledge is available to bring the desired enzyme to an appropriate
1.8 Biotransformation Using Whole Cells
I 33
level of purity. Such develcipments will lower the cost for the catalyst considerably,
which should be kept in mind when analyzing and evaluating process costs.
1.8
Biotransforrnation Using Whole Cells
1.8.1
General Aspects
Microbial cells can be employed also as biocatalysts to achieve a desired conversion

instead of isolated, cell-free enzymes. In contrast to microbial cultivation exploiting
the complex metabolism of cells to produce, for example, organic acids or antibiotica
from sheap nutrients, biotransformation utilizes only one or a few enzymes to
convert added educts to a desired product. Since many microbial enzymes will accept
non-natural compounds biotransformation also gives access to many products not
found in nature, while microbial cultivation yields only natural primary or secondary
metabolites. The acetic acid generator developed in 1824 by C. Ham represents a
suprisingly modem industrial biotransformation process oxidizing ethanol to acetic
acid using strains of Acetc bacter immobilized on beech shavings in a current of air.
About 100 years later the biotransformation process for the production of R-
l-phenyl-l-hydroxypropane-2-one, the key chiral intermediate in the synthesis of
( l R , 2s) ephedrine was cclmmercialized using Saccharomyces cereuisiae to catalyze a
stereoselective acyloin condensation. At the same time regiospecific oxidation of D-
sorbitol to L-sorbose by Aretobacter suboxydans or related species was developed as a
key step in vitamin C production. Biotransformations have since revolutionized the
production and availability of steroid hormones and steroid related drugs including
contraceptives, cortisone, prednisone etc. The modification of the steroid ring
system as well as other cyclic structures by various microbial cultures is well
documented in the literature. From this information, selection of potentially useful
strains is possible for hydroxylation, oxidation, dehydration, reduction or dehydroxy-
lation reactions. For a successful process development beyond a few grams and
scale-up to an industrial ~~rocess extensive screening and biochemical engineering
will be necessary demanding a close collaboration between biologists, chemists and
engineers.
There are far more rnicroorganisms available from culture collections than
enzymes on the market. A list of important culture collections can be found in Table
1-6. Further information can be found on the internet:
http://wdcrn.nig.ac.jp/wfi:c/wfcc.html
Fortunately, the majority of known microorganisms are classified as non-patho-
genic and harmless to humans and the environment. These can be handled safely in
the normal laboratory. Strains with desirable properties identified from the literature
can often be ordered front catalogues of culture collections,just like chemicals from
a supplier. There may be more than one strain of a given species listed in the
catalogues as there are many “John Brown” found in a telephone directory. It is
34 I 7 Introduction
Table 1-6. Important collections of microorganisms in the public domain.

American Type Culture Collection (ATCC),
12301 Parklawn Drive, Rockville, Maryland 20852, USA
Centraalbureau voor Schimmelcultures (CBS),
P. 0. Box 273, Oosterstraat 1, NL-3740 AG Baarn
Japan Collection of Microorganisms (JCM)RIKEN,
Wako, Saitama 351-01, Japan
Institute of Applied Microbiology (LAM),
University ofTokyo, Yavoi 1-1-1,Bunkyo-ku,Tokyo 113, Japan
Culture Collection of the Institute for Fermentation (IFO),
17-85 Juso-Houmachi 2-chome, Yodogawa-ku,Osaka, Japan
Agricultural Research Service Culture Collection (NRRL), Northern Regional
Research Center, Agricultural Research Service, US Department of Agriculture,
1815 North University Street, Peoria, Illinois 61604, USA
National Collection of Industrial and Marine Bacteria Ltd.,
23 St Machar Drive, Aberdeen AB2 lRY, UK
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM-Z),
Mascheroder Weg lb, D-38124 Braunschweig
advisable to search for the strain identified by the unique catalogue number that was
originally employed. If the particular strain is no longer available or cannot be
identified from older literature on should screen for the desired activity in a number
of strains from the same species to find a good substitute.
Usually strains are mailed as lyophilized culture or as a freshly inoculated agar
slant. The investigator has to master some basic skills to maintain and grow cells
under aseptic conditions if he wants to utilize these biological resources. Detailed
information can be found in text books of microbiology and laboratory manuals. For
aerobic organisms (needing oxygen for growth) or microaerophilic organisms
(tolerant against trace amounts of oxygen) a working knowledge of media prepara-
tion is required (useful compositions are found in the literature or in catalogues of
culture collections) as well as sterilization techniques for media and equipment to
remove or destroy all living cells by heat, microfiltration or chemical treatment
before inoculation with a pure strain. Aseptic conditions have to be maintained
when inoculating sterile media with pure strains and in sampling the culture later.
This means that airborne particles containing contaminating microorganisms have
to be excluded from the growing culture, which can be accomplished working in a
laminar flow hood and using exclusively sterilized equipment in handling. The
laminar flow hood provides a positive pressure of filtered air in the working area and
was especially designed to allow easy handling under aseptic conditions. Aerobic
microorganisms are grown in a capped Erlenmeyer flask with baffles on a rotary
shaker at constant temperatures in the range between 25 and 40 "C. Microaerophilic
cultures are purged with nitrogen and agitated slowly if at all. Growth is observed as
turbidity; the uniformity of the culture is checked using a light microscope. Pure
cultures can be used to prepare seed stocks taking aliquots at the mid or late
I
1.8 Eiotransforrnation Using Whole Cells 35
logarithmic growth phase and freezing the suspension at -20°C or -80 “C if

available. Glycerol (5-10%) or dimethylsulfoxide (2.5-5%) may be added as a
protecting agent. Working cultures can be maintained on nutrient agar in a Petri
dish or vial at 4 “C for some weeks. There are numerous other methods to preserve
and maintain pure cultures. The culture collection or a microbiologist should be
consulted in case of doubt.
Anaerobic microorganisms cannot utilize oxygen as a terminal electron acceptor
during growth and usually are very sensitive to oxygen in the environment. Instead
they use a vast variety of organic and inorganic compounds as electron donors and
acceptors in their energy metabolism. Strictly anaerobic suains are difficult to
handle especially on a small scale without proper training and specialized equip-
ment. Nevertheless anaerobic strains may contain interesting biocatalysts also for
biotransformations, especially unusual redox enzymes.
Some microorganisms produced as starter cultures in the food industry can be
easily obtained without necessitating the ability to grow microbial cultures under
defined conditions in the laboratory. The most notable example is “baker’s yeast”,
Saccharomyces cerevisiae, which can be bought cheaply in large amounts from the
regional supplier of bakeries or in smaller amounts from any local food store.
Baker’s yeast is sold as pressed filter cake (usually containing a certain amount of
starch granules), in the form of blocks or flakes or as a lyophilized preparation. The
latter is rehydrated to a viable culture using water or buffer.
Because of the ready ,availability and cheap price, baker’s yeast has been ex-
tensively used for the stereoselective reduction of carbonyl groups in numerous
examples. Problems may arise from genetic and physiological differences in local
strains of S. cerevisae beyond the control of the organic chemist. This leads to
variation in the stereoselectivity and sometimes poor reproducibility of published
results. The cells often contain more than one dehydrogenase/reductase accepting
the substrate of interest and the result obtained is the sum of all parallel reactions.
Besides, the biosynthesi:; of enzymes in general is strictly regulated in micro-
organisms. The activity oj-an enzyme in a given microbial cell may vary more than
1000-fold and depends 011 available nutrients and inducing agents, growth condi-
tions, time of harvest, condition of storage etc. This biological variability (and the
reasons behind them) should be kept in mind when planning and judging bio-
transformations. The relative merits of using whole cells versus cell-free enzymes as
biocatalyst are summarized in Table 1-7.
Obviously whole cell biotransformations are the method of choice if the enzyme
involved is not stable enough in isolated form or is an integral part of the cell
membrane and utilizes tk e electron transport chain in complex coenzyme regenera-
tion schemes. Whole cell biotransformations are particularly important for hydroxy-
lation and oxidation reactions involving mono- and dioxygenases or epoxidases.
Depending on the type of the reaction investigated, whole cells are employed as
growing culture, as viable resting cells or in a nonviable form simply as a “bag of
enzymes”.
7 introduction
Table 1-7. Advantages and disadvantages using isolated enzymes or whole cell biocatalysts.
Advantages Disadvantages
Isolated Enzymes Catalyst concentration as Limited stability

free process variable
High catalyst concentration Cofactor regeneration needed
possible
No side reaction
Simple product recovery
No transport limitation
Multienzyme reactions possible
Whole cell biocatelysts Unlimited availability exploiting Side reactions

growth
Cofactor recycling by cellular Transport limitation
machinery
Multistep conversions possible Complex product recovery
In general low space time yield
1.8.2
Biotransformationwith Growing Cells
When growing cells are to be used for a biotransformation, e. g. of steroids, the cells
are grown in a suitable growth medium, usually in batch culture. After an initial lag
phase the cell will grow and multiply exponentially as observed by the turbidity of the
culture. After one or more compounds become rate limiting for growth a period of
linear growth may be observed before a stationary phase is reached, where viability of
cells is maintained but growth stops. Eventually cells will die if energy sources are
exhausted. Many h n g i but also bacteria produce spores in the stationary phase,
which are able to withstand adverse conditions and secure the survival of the species
in nature.
The biosynthesis of a desired enzyme is often not parallel with growth: the specific
activity will vary with the growth phase. Therefore timing of educt addition is crucial
for product yield. The optimal conditions are usually determined experimentally. It
may be advantageous to add small amounts of educt in the early logarithmic phase to
help to induce the formation of the desired enzyme(s).Toxic substances are added
best in the late logarithmic growth phase to minimize negative effects on growth and
enzyme production. Repeated dosing helps to maintain low stationary levels of toxic
or inhibitory educts but allow product accumulation to high levels provided the
product is non-toxic. After batch or fed batch biotransformation processes, the
biomass is separated and discarded after the conversion is completed or when side
reactions become prominent. The product has to be isolated from whole broth or the
spent medium depending on the yield. Fresh biomass has to be prepared from
culture stocks for each successive experiment.
I 37
1A.38
Biotransformationwith Resting Cells
Resting cells are non-growing viable cells retaining many enzyme activities of
growing cells. Bakeri yeast, discussed above, essentially consists of resting cells of S.
cerewisiae. In the laboratory, resting cells are obtained by growing the selected
microbial culture under ,appropriate conditions until a high or maximal enzyme
activity is reached in the cells. At this point in the growth cycle, cells are separated
frorn the growth medium by centrifugation or filtration and washed with saline.
Then cells are resuspended in the biotransformation buffer, and the conversion is
followed by suitable analytical techniques.
Cell concentration can be varied and a higher catalyst concentration applied in
comparison with experimlmts using growing cells. The addition of small amounts of
glucose or other energy sources helps to maintain the electrochemical potential
across the cell membrane and the viability of resting cells. If the biotransformation
step requires coenzyme regeneration, co-metabolites such as glucose or glycerol
have to be added in sufficient amounts. Resting cells are convenient to use as one
large cultivation yields a uniform biocatalyst for many parallel biotransformations. It
may also be possible to store the cell cake at 4 "C for some days or weeks without
detrimental losses in actikity. Otherwise resting cells may be conserved by lyophiliza-
tiori using sucrose or trekialose as a protectant. The biotransformation buffer is less
complex than spent medium, and thus product isolation is usually easier than from
growing cells. Once the initial growth is completed the danger of infection is also
less using resting cells because biotransformation buffers often lack essential
nutrients for growth. This makes handling in the laboratory more convenient.
1.8.4
Biotransformations with Plermeabilized or Dried Cells
Microbial cells are separated from their surroundings by complex cell walls and one
or more membranes. The cell wall provides the mechanical strength to withstand
sudden changes is osmotic pressure while the membrane serves as an effective
diffusion barrier. In addition, the membrane is important for the selective inter-
action of the cells with the environment and the maintenance of an electrochemical
potential important for viability. Cell membranes may cause more or less severe
mass transfer limitation:;, hindering educts from reaching biocatalysts inside the
cells and the transport of products out of the cells. For lipophilic, uncharged and
sufficiently soluble educi s, passive diffusion across the membrane@) may provide
satisfymg reaction rates. Detergents or solvents can be used to enhance permeability
and mass transport. This approach should be used with care in case membrane-
bound enzymes or coenzyme regeneration via the electron transport chain are
necessary for the particular biotransformation. Otherwise, the integrity of the
membrane may be of no concern for simple reactions involving only intracellular
enzymes. For example L- malic acid is produced from fumarate using Breuibacieriurn
amrnoniagenes as a biocatalyst. The cells are permeabilized by treatment with bile to
38
I improve transport of the charged reactants, and in addition a side reaction, the
I introduction
conversion of fumarate to succinate by membrane-bound enzymes of the citric acid

cycle, is abolished. Resting cells may be dried by treatment with a large excess of cold
(-20 "C) acetone; the resulting "acetone powder" can be stored at -20 "C for many
months, yielding a convenient enzyme source. It should be noted that acetone
treatment removes lipids from the cell membranes, and therefore membrane-bound
activities are irreversibly damaged. Disturbance of membrane function is the major
factor in the toxicity of solvents towards microbial cells and needs to be considered in
a case-by-caseevaluation.
Bibliography
S. L. Neidleman (1990),The archeology of Enzyme chemistry, (C. J. Suckling, ed)

enzymology, in Biocatalysis (D. Abramovicz, Chapman and Hall, London, 1984.
van Nostrand, eds), pp. 1-23. A.Schellenberger (ed),Enzymkatalyse,
H. Uhlig, Enzyme arbeitenfir uns, Tech- Springer Verlag Berlin, 1989.
nische Enzyme und ihre Anwendung, Carl M. J. Page, 7'he chemistry ofenzymeaction,
Hanser Verlag, Miinchen, 1991. Elsevier, Amsterdam, 1984.
W. N. Lipscomb (1983), Structure and catal- M. J. Page and A. Williams, Enzyme
ysis of enzymes, in: Ann. Rev. Biochem. 52, mechanisms, Royal Society of Chemistry,
17-34. London, 1987.
D. W. Christianson, W. N. Lipscomb (1987), H. Bisswanger, n e o r i e und Methoden der
The complex between carboxypeptidase A Enzymkinetik,Verlag Chemie, 3rd edn,
and a possible transition state analogue, I. 2000.
Am. Chem. Soc. 108,4998-5003. W. W. Cleland, Enzyme kinetics as a toolfor
B. L. Vallee, A. Galdes (1984), The metal- determination ofenzymemechanism, in:
lobiochemistry of zinc enzymes Adv. Enzy- Bernasconi (ed),Investigations of rates and
mol. Rel. Areas Mol. Biol. 56, 284-430. mechanism of reactions, Wiley, 1986, pp.
W. N. Lipscomb (1971), Proc. Robert A. 792-821.
Welch Found. Con$ Chem. Res. 15, 141. A. Cornish-Bowden, Principles ofenzyme
G. E. Schulz, R. H. Schirmer: Principles of kinetics, Butterworth & Co., London, 1976.
protein structure. Springer Verlag, New York, J.A. Robinson, J. Retey, Stereospecijkity i n
1979. organic chemistry and enzymology,VCH
C. R. Cantor and P. R. Schimmel, Biophys- Verlag, Weinheim 1982.
ical chemistry, part I: The conformation of I . H. Segel, Enzyme kinetics, Behavior and
biological macro-molecules. part 111: The analysis of rapid equilibrium and steady state
behavior of biological macromolecules, enzyme systems, Wiley & Sons, New York.
W. H. Freeman and Co., New York, 1980. 1975.
T. E. Creighton, Proteins, struaures and G. N. Wilkinson, Statistical estimations on
molecularproperties, W. H. Freeman and enzyme kinetics, Biochem. J. 80,
Co., New York, 1984. 1009-1012.
K. Buchholz und V. Kasche, Biokatalysatoren A. Fersht, Enzyme structure and mechanism
und Enzymtechnologie, VCH, Weinheim, (2ndedn.), W. H. Freeman & Co., New York,
1997. 1985.
D. Zaks, Enzymes in organic solvents, in: B. E. Kirsop and A. Doyle (eds), Mainte-
Biocatalystsfor Industry (J. Dordick, ed), nance ofmicroorganisms and cultured cells,
Plenum Press, New York, 1991, pp. Academic press, London, 1991.
161-180. J. C. Hunter-Cevera and A. Belt, Isolation of
Enzyme nomenclature, published for the cultures, in: Manual of Industrial Micro-
I U B by Academic Press Orlando, 1992. biology and Biotechnology, (2ndedn.),A. L.
39
Demain and J. E. Davies (eds.), ASM Press,

M. D. Hilton, Small scale liquid fermenta-

I
Washington, DC 1999, pp. 3-;!0. tion, in: Manual oflndustrieal Microbiology
R. L. Monoghan, M. M. Gagliardi and L. and Biotechnology, (2ndedn.), A. L. Demain
Streicher, Culture preservation and inocu- and J. E. Davies (eds.), ASM Press,
lum development, in: Manual ofhdustrial Washington, DC 1999, pp. 49-GO.
Microbiology and Biotechnology (2ndedn.), R. Leon, P. Fernandes, H. M. Pinheiro and
A. L. Demain and J. E. Davies (eds.) J.M.S. Cabral, Whole-cell biocatalysis in or-
ASM Press, Washington, DC 1999, pp. ganic media, EnzymeMicrob. Biotechnol. 23,
29-48. 483-500.1998.
I
41
2
Production and Isolation o f Enzymes
Yoshihiko Hirose
2.1
Introduction
This chapter gives a brief review of the isolation and production of enzymes. More
detailed information can be obtained from various published textbooks and re-
view~['-~]. Most of the industrial enzymes used for chemical synthesis are supplied
in a crude form with an active enzyme content of only a few percent. The other
constituents are inorganic salts, polysaccharides and diatomaceous earth used as
stabilizers and excipients. Purified enzymes for biotransformation are supplied by
some manufacturers in a crystal or immobilized form. These enzymes, though
expensive, are easy to apply for biotransformation in organic media. The use of more
purified enzymes is increasing.
Barriers to the production of industrial enzymes include economic factors, the
availability of optimal enzymes and safety issues. Common fermentation and
purification processes are described in Figs. 2-1 to 2-3. The process differs for
extracellular and intracellular enzymes, liquid and solid culture, and enzyme
application. The fermentation conditions are computer-controlled for optimization,
e. g. of temperature, pH, agitation speed, aeration, demand oxygen etc.
There are no internationally standard assay methods for industrial enzymes and
the definition of enzyme activity unit is also different for each enzyme. The activity of
industrial enzymes is shown by various methods depending on manufacturers. For
instance, commercial lipase activities are measured by the hydrolysis of olive oil
under the various conditions and these figures are not comparable with each other.
When customers apply these biocatalysts for chemical synthesis in organic solvents,
these figure are sometirnes reliable, and sometimes not. Users should not judge
commercial enzymes based only on price and the activity shown in the table the
manufacturer provides. Enzymes should be evaluated based on their practical
performance under the conditions used. Most users of biotransformation are not
experts in measuring enzyme activity, so the establishment of an assay method and
practice are essential if one is to optimize the performance of enzymes.
Several commercial enzymes are powders including diatomaceous earth or
Broth-out
Filtration
Concentration by evaporation or ultrafiltration
Precise filtration
Solvent precipitation
Filtration or centrifugation
Dissolution
Ion-exchangechromatography
Salting-out
Desalting
Hydrophobic chromatography
Gel filtration
Desalting
V Freeze-drying
I
I
2.1 fntroduction 43
Preparation of fermentation medium ----- Starch, sugar, soybean powder,

yeast extracts, minerals, inducer, etc.
Dissolution
Sterilization
Inoculation
Fermentation in flask to in main tank
1 Centrifugation or Filtration
Disruption and extraction
Filtration
I Concentration by evaporation or ultrafiltration
Further purification
(Salting-out, chromatography, etc)
Precise filtration
j. Crystallization or freeze-drying
Product
Figure 2-2. Commori production process for special intracellular enzymes.
Sterilization
Inoculation
Extraction of enzyme
Filtration
Concentration by ultrafiltration
Precipitation
44 2 Production and lsolation of Enzymes
I dextrin. These enzymes should be used after immobilization on a suitable carrier.
The activity of an immobilized enzyme usually is enhanced up to tenfold.
Regulatory assessments for enzymes used in biotransformation are not clearly
stipulated. At present, food assessments of microbial enzymes are provided by
AMFEP, which has suggested microbial enzyme purity and immobilization as given
below.
Purity
A chemical and microbial specification must be given. Based on FCC recommenda-
tions, AMFEP recommended the following.
Arsenic 3 PPm
Lead 10 PPm
Heavy metals <40 ppm
Mycotoxins negative
Antibacterial activity negative
Coliforms <30/g
E. coli negative in 25 g
Salmonella negative in 25 g
Total viable count <5oooo/g
Immobilization
The immobilization system should be described in detail. Tests to indicate the
physicochemical stability of both the system and its carrier and enzyme are
essential.
These regulatory aspects would be acceptable for biocatalysts.
2.2
Enzyme Suppliers for Biotransformation
There are more than 400 companies dealing with enzymes all over the world and
approximately 12 major producers with an increasingly distinct separation of
product ranges. About GO companies produce substantial amounts of a small range
and about 400 companies produce a very limited range of industrial enzymes.
Japanese enzyme producers have a special range for industrial or in-house use and
contribute to 12-15% of world production. There are 24 companies which supply
special enzymes for biotransformation (Table 2-1).
Table 2-1. Main enzyme suppliers for biotransformation.
2.3 Origins of Enzymes
I 45
Company Country
Altus Biologics Inc. USA
Amano Enzyme Inc. Japan
Asahi Chemical Co. Japan
Biocatalysts Ltd. UK
Biozyme Labs Ltd. UK
Calbiochem Corp. USA. Denmark
Christian Hansen AS DK
Diversa USA
Fluka Chemicals Ltd. Germany, UK
Genencor Int. Finland, USA
Genzyme Ltd. UK
DSM (Gist) Holland
Meito Sangyo Co. Japan
Merck KSA Germany
Nagase Biochemicals Japan
Novo Nordisk AS Denmark
Oriental Yeast Co. Japan
Osaka Saiken KK Japan
Roche Diagnostics GmbH Germany
Rohm GmbH Germany
Shin Nihon Chem Co. Japan
Sigma Chemical Co. USA
ThermoGen USA
Toyobo Co. Japan
2.3
Origins of Enzymes
2.3.1
Microbial Enzymes
More than 90% of enzymes are produced by fermentation by microorganisms,

which are used to prepare industrial and special use enzymes. Prokaryotic cells and
eukaryotic cells can be easily grown in culture, and the technology of scale-up is well
established on an industrial scale. Various kinds of fungi, bacteria and yeast have
been screened for the production of special enzymes. Extracellular enzymes, for
instance hydrolytic enzymes, are secreted into liquid and solid culture and are
relatively stable in cultivation media.
Production by genetically modified organisms (GMO)is becoming popular in this
field, and several kinds of GMO have been used to increase the productivity of
biocatalysts. When one uses GMO enzymes for industrial use, one should know the
origin of the microorganisms used and how the production method was changed.
The new enzyme preparation is likely to have a different compositional spectrum of
enzymes and side activities.The regulations for biocatalysts are not severe at present,
but are likely to become more stringent.
46
I 2 Production and /solation ofEnzyrnes
2.3.2
Plant Enzymes
Some proteases, such as papain, bromelain and ficin, lipoxygenases from soy bean
and white germ, and peroxidase from horseradish, are typical plant enzymes. Plant
proteases are extracted and partially purified to give a powder extract. Some are
supplied as digestive enzymes or neutraceutical enzymes. These have the character-
istics of an SH-enzyme (thiol protease) and work in the hydrolysis of racemic esters
as a protease. Lipoxygenases are only available from soybean, but the activity is not
high and the regiospecificity for unsaturated fatty acids is not severe. Lipoxygenases
from other plants are relatively unstable and used in-house only.
2.3.3
Animal enzymes
Porcine liver esterase (PLE), porcine pancreas lipase (PPL) and arginase are well
known as biocatalysts among industrial animal enzymes. PLE catalyzes very well the
hydrolysis of certain kinds of prochiral diesters and is supplied as a suspension with
ammonium sulfate or in liquid form. The substrate specificity of PLE is not wide,
but this is a well-investigated enzyme. PPL is very cheap and a useful biocatalyst in
industry. Commercial PPL is a mixture of many kinds of pancreas enzymes, and the
name pancreatin is well known as a digestive enzyme. Pregastric esterase is applied
for transesterification of triglycerides. Arginase from calf liver is used to produce L-
ornithine from the proteinogenic amino acid-arginene. The use of animal enzymes
seems to be gradually decreasing because of disease and a variable supply. In the
future, animal enzymes will no doubt be replaced by microbial enzymes of
equivalent performance.
2.4
Fermentation of Enzymes
2.4.1
Liquid Fermentation
Liquid fermentation is useful for the production of enzymes as well as antibiotics. It

is good for scale-up and reproduction. There are two types of enzyme produced:
intracellular and extracellular enzymes. With advances in genetic engineering,
Escherichia coli is now being used to produce enzymes. When E. coli is used, the
enzyme is accumulated inside the cell. This method is very popular.
2.5 Extraction of Enzymes
I
47
2.4.2
Solid Fermentation
In Japan, solid fermenta.tion is still used to produce many kinds of enzymes

including lipases, proteases and acylases. Some glycotransferases are also produced
by solid fermentation. In the production of proteases, solid fermentation is often
used to increase the productivity in solids. On changing to liquid fermentation, the
protease is not produced and its properties change. Solid fermentation is old-
fashioned and difficult to scale up because of the expensive facilities needed.
2.4.3
Extraction of Enzymes
In order to improve the erttraction of enzymes, organic solvents and surfactants are
sometimes used.
2.5
Extraction o f Enzymes
2.5.1
Microbial Enzymes
Extraction methods depend on the fermentation conditions and the microorganism,

for example, liquid or solid culture, intracellular or extracellular enzyme, laboratory
or production scale preparation, etc.
Because enzymes are more soluble in buffer solution than water, enzymes in solid
fermentation are extracted by stirring several times in a suitable buffer solution. The
pH of the buffer solution is adjusted based on the stability and the PI of the enzyme.
The solid medium is removed by filtration after extraction and the crude enzyme
solution is concentrated at the next step.
Liquid fermentation produces two types of product, intracellular and extracellular
enzymes. In the case of extracellular enzymes, the crude enzyme solution is
collected by filtration or centrifugation of the microorganism. In the case of
intracellular enzymes, collection of the microorganism and extraction of enzymes
after disruption is required. On a laboratory scale, ultrasound equipment or a French
press is used for disruption of microorganisms. On a large or industrial scale, a
mechanical grinding mill with glass beads (for instance, a Dynamill) is used. It can
treat microorganisms suspended in buffer solution at a rate of 100 L/h. Another
method is enzymatic disruption of microorganism with lysozyme or YL. This
method is easy to apply on an industrial scale because it does not require special
apparatus. The microorganism suspended in buffer is stirred for several hours in the
presence of a suitable amount of lysozyme at room temperature. During the
enzymatic treatment, freezing and thawing of the microorganism is effective for
disruption. In this case, lysozyme should be added before freezing the micro-
48
I 2 Production and Isolation offnzyrnes
organism. The combination of mechanical and enzymatic treatment is much more

effective.
After disruption of the cell wall, enzymes should be extracted with buffer solution.
Sometimes enzymes are adsorbed by or adhered to the cell wall and must be
extracted by adding a small amount of surfactant, such as Triton X-100. The cell wall
is then filtered or centrifuged to obtain the crude enzyme solution.
Recombinant heterogeneous enzyme produced by gene-modified organisms
(GMO) such as E. coli are sometimes obtained as inclusion bodies which form
insoluble aggregates and show less original activity. In order to regenerate the
activity from the inclusion body, it is dissolved in the presence of denaturing agents,
usually a highly concentrated guanidinium salt and urea and reducing agents,
usually thiol compounds. Protein is allowed to refold into its original active
conformation after removal of the agents.
2.5.2
Plant Enzymes
Some proteases, papain and bromelain, are derived from plants and are extracted
from fruits. The fruits are ground by a grinder or cutter and the proteases are
extracted by buffer solution. A diluted cooled buffer is more effective than water for
extraction and the extracted solution including desired enzymes should be cooled
during all treatments. The content varies depending on the time and place as well as
the plant.
2.5.3
Animal Enzymes
Animal organs containing desired enzymes are stored frozen and then ground and
crushed by a homogenizer. Enzyme stabilizers or protease inhibitors are sometimes
added on homogenizing the organs, and a buffer is preferable for extraction. In order
to improve the extraction, the residue is removed by filtration or by centrifugation to
obtain crude extract. Animal organs contain various kinds of enzymes and a large
volume of protein is extracted. Even relatively unstable enzymes retain their activity
in crude extracts, but it is necessary to purify the enzymes step by step.
2.6
Concentration
After the extraction of extracellular enzymes of solid culture or the fermentation of

extracellular enzymes of liquid culture, the fermentation medium including desired
enzymes is centrifuged or filtered to remove the microorganism etc. The next step is
concentration by evaporation under reduced pressure or ultrafiltration (UF) to
reduce the volume of the enzyme solution. It is not easy to evaporate large amounts
of water under reduced pressure. Evaporation should be carried out at <30 "C except
2.7 Purification of Enzymes
149
in the case of thermostable proteins, which can be evaporated at higher tem-
peratures. The concentr,itions of the salt and other soluble materials of the
concentrated solution are increased by evaporation.
The most convenient and simple method for production is ultrafiltration. The
method uses membrane tubes with pore sizes from about 6000 to 50000 A. Small
molecules like salt ions as well as water pass through the pores of the membrane
tube while large molecules like proteins remain inside the tube. The concentration
of the buffer solution is the same before and after ultrafiltration. The leaking of
desired proteins in permeates should be checked during the concentration stage.
Regular maintenance is carried out by using a standard protein. The membrane tube
is made of polyethylene, polypropylene etc., and the irreversible adsorption of
desired proteins should be avoided. The materials for the membrane should be
selected before use. The flow rate of ultrafiltration depends on the facility and the
protein solution applied. The final protein concentration is up to about 100 g/L.
Ultrafiltration is also used for desalting. Smaller membrane tubes are used for this
purpose.
Another concentration method is precipitation using organic solvents or salts as
described in Sect. 2.7.2. Ethanol is especially useful for this purpose. Despite the
volume of organic solvents, it is still frequently used as a first step in the
purification.
2.7
Purification of Enzymes
2.7.1
Chromatography
Chromatography is the major purification method. The choice of technique is

determined by the overall yield, efficiency, speed and convenience. To purify a
desired enzyme in the broth, a combination of different types of chromatography is
an effective approach.
2.7.1.1
Ion Exchange Chromatography (IEX)
Ion exchange chromatography (IEX) 171 is the most typical and frequently used
method for separating enzymes. Some of the advantages of IEX are high resolution
power, applicability, and ease of control and scale-up. There are two types of
exchanger in IEX (Figs. 2-4 and 2-5). Positively charged exchangers have negatively
charged counter-ions (anions) available for exchange and are called anion exchang-
ers. Negatively charged exchangers have positively charged counter-ions (cations)
and are called cation exchangers.
The basic principle of separation in IEX is the reversible adsorption of charged
protein molecules dissolved in a buffer solution by oppositely charged ion-ex-
changed groups on the inatrix (Fig. 2-6).
50
I 2 Production and Isolation ofEnzyrnes
Anion exchanger with counter-ions Cation exchanger with counter-ions

Figure 2-4. Two types o f ion-ex~hanger[~J.
c-
/ Figure 2-5. The principle of ion exchange

equilibrium 141.
Proteins dissolved in buffer solution have different charges depending on the

solution. When the pH of the buffer solution is below the isoelectric point (PI), the
protein has a positive charge, and when the pH is above the PI, the protein has a
negative charge (Figs. 2-7 and 2-8).
An ion exchanger consists of a solid matrix covalently bound to a charged group.
The matrix is made of an organic compound, synthetic resin or polysaccharide, such
as Sepharose and Sephadex. A typical matrix is a round microbead. The character-
istics of the matrix determine its chromatographic properties, for instance, efi-
ciency, capacity, recovery, chemical stability, mechanical strength and flow proper-
I
51
0 000 000
nvo 000
v v 000 000
Substances to be separated
Figure 2-6. The principle of anion exchange chromatography[’].
ties. The properties of the matrix affect its behavior towards biological substances
and the maintenance of hiological activity.
The charged group determines the basic property of IEX, such as the type and the
strength of the ion exchanger. The number of charged substituent groups per gram
of dry ion exchanger or per mL of swollen gel affects its total ionic capacity. It can be
measured by titration with a strong base or acid and is shown as pmol/mL gel.
Typical functional groups are shown in Table 2-2 and are classified in two types. The
functional groups of ani’mexchangers are substituted ammonium groups. Cation
exchangers have sulfoql or carboxyl groups. Ion exchangers with sulfonic and
quaternary amonium grcups are called “strong ion exchangers”.Those with carboxyl
and diethylaminoethyl groups form “weak ion exchangers”. The terms strong and
weak refer to the extent of the variation of ionization with pH and not the strength of
binding. A strong exchanger is ionized over a broad pH range, a weak one over a
narrower range.
52
I 2 Production and Isolation of Enzymes
Ib ( K L J
tange of stability
Attached to anion exchangers
I \ I I I PH
I 2 \ 6 8 10
I t
Figure 2-7. The net charge o f protein as a function o f p H f71.
+ acid PI base
anion exchange
cation exchange
NH2
Figure 2-8. Relation between the charge of proteins and the p H 16]
Experimental Design
The choice of matrix and functional group depends on the pH stability, molecular
size and isoelectric points ( PI) of the protein, and on the requirements of the
application. PI can be measured by electrophoresis or can be checked in the
comprehensive lists of PI for proteins.
Table2-2. Functional groups used on ion exchangers and its structure.
I
53
Ion exchangers Structure
Anion exchangers
Dimethylaminoethyl (DE) -CHzCHzN(CH3)2
Diethylaminoethyl (DEAE) -CHzCHzN(CzHs)z
Quaternary ammonium (QA) -CHzN+(CH3)3
Quaternary ammonium (QAE) -CH2CH2NC(C2H5)2CH2CH(OH)CH,
Quaternary ammonium (QMA) -N'(CH,)s
Cation exchangers
Carboxymethyl (CM)
Phosphate (P)
Sulfonic ethyl (SE)
Sulfonic propyl (SP)
The starting pH of buffer is chosen so that proteins to be bound to the exchanger

are charged. So, the startjng pH is at least more than 1 unit above the PI for anion
exchangers or at least less than 1 unit below the PI for cation exchangers to facilitate
adequate binding. Proteiru begin to dissociate from ion exchangers at about 0.5 pH
units from their PI at ionic strength 0.1 M.
Most proteins have their PI within the acidic range, so they are usually negatively
charged in neutral buffer solution and show the properties of as anion.
Ion exchange separation is carried out using the following three procedures:
column chromatography, a batch method and an expanded bed adsorption. In-
dustrial- scale preparation is used.
Column Separation
Ion exchangers are available for laboratory-scaleseparations, and factors such as cost
and reproducibility etc. are not very important. For industrial separation, however, it
is necessary to optimize the purification conditions.
The DEAE exchanger is the most useful in terms of the PI and stability of most
proteins.
Choice of Exchanger Groujr and Buffer

The choice of ion exchanger and buffer solution is limited by the stability of the
proteins. Because most proteins have their PI in the acidic range, they have a slightly
positive charge below the PI and can be easily absorbed on a cation exchanger (ex.
Chl). On the other hand, they have a negative charge above the PI and an anion
exchanger (ex. DEAE) is used.
Choice ofpH and Ionic Strength

The pH of the buffer depends on the PI of the proteins, and the ionic strength causes
absorption on the ion exchanger and desorption from it. The required concentration
of starting buffer depends on the nature of the buffering substance. It should be at
54
I 2 Production and Isolation of Enzymes
least 10 mM. Suitable ion salts stabilize the proteins in solution and excess salts
cause the denaturation and precipitation of the protein.
Batch Separation
Batch separation is conducted in the same way as column development by a stepwise
elution method. Batch separation is a better method for large sample volumes with
low concentration protein. Large volumes take a long time. The initial conditions for
batch separation are almost the same as for column chromatography, for instance,
buffer pH and ionic strength etc.
It is necessary that the conditions are rather strong so that the proteins completely
bind to the adsorbent. In order to increase the recovery, the pH of the buffer is kept a
couple of units away from the PI of the protein. Batch separation is simple and
useful to concentrate a low protein solution, but the resolution is not high.
Experimental Procedure
Batch separation is a simple method whereby the protein solution is stirred together
with the ion exchanger in an appropriate buffer for 1h. The slurry is collected by
filtration and the ion exchanger is washed with fresh buffer solution. When no
desired protein is observed in the filtrate, a couple of bed volumes of the elution
buffer are added and stirred for 1 h to desorb the desired protein. The solution
including the desired protein is collected by filtration. The change of pH or ionic
strength of the elution is determined by gradient or stepwise chromatography.
2.7.1.2
Hydrophobic interaction Chromatography (HIC)
Hydrophobic interaction chromatographyf8Iis based on the hydrophobic properties

of proteins and the hydrophobic ligands covalently bonded to the matrix. The
chromatography has three forms: hydrophobic chromatography,where both absorp-
tion and desorption are based only on hydrophobic binding, hydrophobic-ionic
chromatography,where absorption is based on hydrophobic binding and desorption
on ionic exclusion by changing the pH of the buffer, and mixed function chromatog-
raphy, based on hydrophobic, ionic and hydrogen binding.
HIC and reversed phase chromatography (RPC)are very similar principles based
on hydrophobic interaction. Adsorbents for RPC are much substituted with hydro-
phobic ligands, such as octadecyl,octyl or phenyl groups (Figs. 2-9 and 2-10).Protein
binding to RPC adsorbents is usually very strong in spite of the concentration of salt,
and some kinds of organic solvents, such as acetonitrile and is0 propyl alcohol, are
used to desorb the protein. So, RPC is carried out for low molecular weight
molecules such as peptides because most proteins are unstable in highly concen-
trated organic solvents. On the other hand, adsorbents for HIC are less substituted
with similar groups, mainly butyl groups. Protein binding to HIC adsorbents
requires a neutral salt like ammonium sulfate in the mobile phase, and the protein is
desorbed by decreasing the concentration of salt. The slope of ionic strength in HIC
is opposite to that of IEX.
2.7 Pur$cation of Enzymes
I 55
f- OCH2CH(OH)CH20-R:
R-
C2H5- Ethyl
C4H9- Butyl I I
Hexyl c4 c6 c8
C6H 13-
Length of the n-alkyl chain
C8H17- Octyl Figure 2-10. The effect of alkyl chain length and
C1oH21- Decyl degree of substitution on binding capacity in HIC['].
C6H5- Phenyl
Figure 2-9. Hydrophobic
ligands attached to matrix.
The surface of a protein is relatively hydrophilic in the lower concentration buffer

solution, but hydrophobic: interaction increases at high ionic strength (Fig. 2-11). It
is estimated that 40-50% of the surface area of a protein is non-polar.
The HIC parameters are type of ligand, degree of substitution, concentration of
salt, and effect of temperature and pH.
The immobilized ligands used are hydrocarbon groups like butyl and octyl groups
and phenyl groups. The polarity of the ligand increases with alkyl chain length and
its degree of substitution. The interaction of the phenyl group is not simple because
of an aromatic effect as well as hydrophobicity.
The most typical salt in HIC is ammonium sulfate. As the concentration of
ammonium sulfate is increased, the amount of protein adsorbed on the ligand
increases linearly up to the precipitation point. The effect of the ion used in HIC the
precipitation of proteins is shown in Table 2-3. Sodium, potassium or ammonium
sulfates have a relatively high saltingout effect and molar surface tension of water
an increasing effect.
Ammonium (1M)sulfate is a good starting point for experiments. If the protein
does not retain the ligand, a more hydrophobic ligand should be selected. The
recovery of protein in H I C should be S O % . When a small amount of miscible
organic solvent is needed, the ligand should be changed to a less hydrophobic one.
Hydrophobic interaction in HIC is diminished by increasing the pH and in-
creased by decreasing the pH. The PI of protein is in the acidic range and the
hydrophilicity increases in the basic range. Hydrophobic interaction strength
changes strongly at a pH below 5 or above 8.5. Also, on increasing the temperature of
HIC, hydrophobicity slightly increases. A small amount of miscible organic solvent
affects the decrease in hydrophobicity of protein and facilitates elution in the buffer
solution.
56
I 2 Production and lsolation of Enzymes
Figure 2-11.The principle o f

hydrophobic chromatography['].
ow
P: Polymer matrix
S: Soluble molecule
L: Ligand attached to polymer matrix
H: Hydrophobic patch on surface of soluble molecule
W: Water molecules in the bulk solution
S: Salt (ammonium sulfate)
Table 2-3. The effect of some anions and cations in precipitating proteins141.
Protein Antichaotropic effect * Chaotropic effect
Collagen SO4'- < CH3COO- < C1- < Br- < NOs- < CI04- < I- < SCN-
Zelatin NH4' < Rb', K', Na', Cs' < Li' < MgZ' < Ca" Ba"
2.7.1.3
Gel Filtration (CF)
Gel filtration"] is a key method in the purification of enzymes as well as biological

macromolecules. It is reliable and simple as a separation technique without
adsorption and interaction on gel filtration media. In gel filtration, the principle of
separation is very simple, and macromolecules in solution are separated based on
differences in their size as they pass through a column (Fig. 2-12). Large molecules
pass through the stationary phase first while smaller molecules move about the gel
filtration medium slowly. Gel filtration is also called molecular-sieve chromatog-
2.7 Purifcation ofEnzyrnes
I 57
0 . . Sample proteins of different molecular size
0 Gel particles
Figure 2-12. The principle of gel filtration.
raphy. Molecules are eluted in the order large to small. Gel filtration is usually used at
the final or latter stage for changing buffer and concentration.
Gel filtration is carried out using a single buffer solution of appropriate pH and
ionic strength. Some gel filtration media have a small number of ionic charged
groups, such as carboxyl and sulfonic groups, which sometimes cause non-specific
adsorption of basic proteins at low ionic strengths. In order to avoid the adsorption,
gel filtration should be carried out at an ionic strength above 0.15 M. Non-ionic
interactions between proteins and gel filtration media are negligible at buffer
concentrations between 0.15 M and 1.5 M. An ionic strength below 0.15 M causes a
slight retardation of basic proteins and exclusion of acidic proteins.
The first gel filtration medium Sephadex, provided by Pharmacia, was a bead-
formed gel prepared by cross-linking dextran with epichlorohydrin. Gel filtration
media with various particle size grades are now available and globular proteins can
be separated between 700 and 4x10' A. The fractionation range of the medium
determines the porosity of the gel and is measured using typical globular biological
molecules or dextrans. The shape (its diameter and length) of biological molecules
affects the theoretical separation. When the molecule is not globular but a linear
string, the separation is quite different.
58
I 2 Production and /solation of Enzymes
Choice ofColumn, Sample Volume and How Rate

There are some factors affecting the choice of column equipment in order to obtain a
good separation. The length of column is necessary more than 30 times the diameter
of column, because the resolution increases at the square root of column length.
That is why a longer column is used for gel filtration especially for an analytical
fractionation. A bed length of more than 1m is not useful and effective for industrial
separation.
The dead volume at the inlet and outlet should be less than 0.1%. A sample
volume of 0.5-5% of the bed volume is recommended for good resolution and
depends on the gel filtration medium. The relationship between sample volume,
medium and resolution is described; however, the actual sample volume should be
determined by experiment. A smaller sample size is not good for resolution. Up to
30 % of the total bed volume can be applied for changing the buffer and salting out.
An effective flow rate for resolution is the order of 5 mL/cm2h and up to about
25 mL/cm2h is allowed for industrial preparations. The length of the column and
flow rate are basically in an inverse relation.
2.7.1.4
Reversed Phase Chromatography
Except for a few specific applications, reversed phase chromatography (RPC)[lo]is

rarely used in biological purification. RPC is commonly used for the purification of
organic compounds and low molecular weight peptides. The principle of RPC is
similar to that of HIC. The ligand is stronger in RPC than in HIC, and includes
octadecyl, octyl, butyl and phenyl groups (Fig. 2-13). The remaining silanols are
quenched with trimethylsilyl groups.
RPC medium consists of hydrophobic ligands chemically bonded to porous
microbeads. The microbeads are made of silica gel or a synthetic organic polymer
like polystyrene.
Figure 2-13. Reacting a silanol with octadecyldimethylsilyl group.

2.7 Purijication of Enzymes
Table 2-4. Solvents used in reversed phase chromatography.

I 59
Solvent Diielectric constant Viscosity bP ("C)

(20 "C) (cP at 20 "C)
Acetonitrile 38.8 0.36 82
Ethanol 24.3 1.20 78
Methan o1 3:i.G 0.60 65
Propanol 20.1 2.26 98
Isopropanol 18.3 2.30 82
Water 80.4 1.00 100
It is necessary to use water miscible organic solvents in order to elute proteins

(Table 2-4). Most proteins are apt to be denatured in that case. RPC is useful for the
purification of small samples or peptides and is usually carried out as high
performance liquid chromatography (HPLC).
2.7.1.5
Hydrogen Bond Chromatography
There are three types of chemical interaction between the ligands and proteins:
ionic, hydrophobic and hydrogen bond interaction. Hydrogen bond chromatography
is not as popular as ionic and hydrophobic chromatography.Precipitation of proteins
is sometimes observed in the presence of a water-soluble polysaccharide and
polyethylene glycol. The complex with proteins easily forms via hydrogen bonding at
high ionic strength. Ionic cellulose, such as DEAE-cellulose and CM-cellurose, as
well as cellulose, is used as a matrix for this purpose. Hydrogen bond - ion
chromatography is complicated because the ionic strength of the buffer solution
used for each of the two methods is opposite to elute proteins.
Protein is adsorbed on DEAD-cellulose in buffer solution with 3 M ammonium
sulfate and is eluted by decreasing the concentration of ammonium sulfate or
adding the releasing reagents, such as urea and sucrose etc. Sodium formate and
sodium acetate are used instead of ammonium sulfate. On the other hand, small
amounts of ethanol, glycerol and ethylene glycol are available for elution.
2.7.1.6
Affinity Chromatography
The principle of affinity cliromatography is shown in Fig. 2-14.

Affinity chromatography["] is used for a biologically specific ligand bound to the
matrix. The protein binds with ligand specifically in an active form and the rest
passes through without adsorption upon washing with a buffer solution.
The ligand should have specificityand reversibility for the protein and release it on
affinity elution or change of ionic strength and pH. Interactions between proteins
and ligands include ionic binding, hydrogen binding and hydrophobic binding. The
factors necessary for iortic binding have been listed before. The effect of ionic
60
I 2 Production and /solation ofEnzyrnes
Adsorption
- A
Desorption
A
___)
If
0 0
00 GI
0 U0 Q
Q
A Affinity absorbent
Q Protein to be separated
0 impure proteins
Figure 2-14. The principle of affinity chromatography.
strength on ionic binding is opposite to that on hydrophobic binding and the

recovery of protein is sometimes not good. The use of a 1-3 M urea solution or
5-20% sucrose is a good idea in such a case.
Affinity chromatography is carried out by the batch and column methods. The
procedure involves (i)equilibration of the adsorbent, (ii) preparation of sample, (iii)
application of the sample, (iv) washing away of unbound materials, (v) elution and
(vi) regeneration of adsorbent.
When the ligand has a simple specificity for protein, about 90% purified protein is
obtained by one-step purification. So, affinity chromatography is a revolutionary
I 61
0 NH:
\
Figure 2-15. Immobilized dye chrornatography[ll].
purification method. Adsorbents are relatively expensive and affinity chromato-

graphy is useful for small scale purification.
There are several types of affinity chromatography.Two typical types, immobilized
dye chromatography and metal chelate affinity chromatography, are described.
lmnzobilized Dye Chromatography

Immobilized dye chromatography is the most useful affinity chromatography. Its
ligands are synthetic polycyclic dyes. These structures are very similar to the
cofactors NAD’ and NADP’ as a dinucleotide analog, which are apt to bind strongly
with a protein like kinases, dehydrogenases, etc. (Fig. 2-15). Some of the proteins
bind biospecifically with the dye because of its stmctual similarity to NAD and
NADP. Some proteins like lipoproteins and albumin bind in a less specific manner
by electrostatic and hydrophobic interactions with the aromatic anionic ligand.
Bound proteins can be ‘eluted by affinity elution using low concentration free
cofactors. On the other hand, non-specifically bound proteins need a much higher
concentrate of cofactors or ionic strength.
Metal Chelate Afinity Chromatography

Metal chelate affinity chromatography is a kind of separation method which has, as a
ligand, a metal ion. Some proteins and peptides are purified on the basis of affinity
for metal ions immobilizl2d by chelation on the adsorbents. Histidine and cysteine
form complexes with the chelated metals around neutral pH. Biological proteins
include many histidines as well as recombinant proteins as polyhistidine fusions: for
instance, His-tag protein:; have a specific metal chelate affinity. The adsorbent is
prepared by coupling a metal chelate ligand with an iminodiacetic acid group, which
forms a chelate with divalent metal ions such as Zn2+,Cuz+,Cd2+,Hg”, Co2+, Ni2+,
Fez+,etc.
Elution is carried out by reducing the pH and increasing the ionic strength of the
buffer or by adding EDTA. to the buffer. The most typical method is to gradually add
sodium chloride (0.5-1.0 M) or imidazole (0-0.0sM). This ligand is very expensive,
so metal chelate affinity chromatography is used only for small scale purification.
62
I 2 Production and lsolation of Enzymes
His-tag proteins produced by a recombinant are easily purified by metal chelating

chromatography. His-tag proteins have about G histidines at the N- or C-terminal site
and the His-tag easily forms a chelate with Ni2+,Zn2+and Cu2+Elution of His-tag
proteins is carried out by increasing the concentration of imidazole in the buffer
solution.
2.7.1.7
Salting-out Chromatography
Salting out is popular for the purification of proteins. Salting-outchromatography is

a precise method based on the same principle; however, it is not popular. Both
positive and negative salting-out chromatography are carried out. The former is a
combination of molecular-sieving chromatography and salting out. Proteins in
buffer solution are applied to a concentration gradient column of salts. With the
latter method, the precipitation of proteins salted out in the presence of celite is filled
in the column and proteins are eluted with a flowing buffer solution by decreasing
the concentration of salt.
2.7.2
Precipitation
Among the methods of purifymg protein, precipitation is the most useful and typical
for both small and large scale procedures. The precipitation methods are classified
into 4 types, salting-out, organic solvent precipitation, pH changing precipitation
and water-solubleprecipitation. The precipitation is usually carried out early and the
total protein concentration should be >0.1mg/mL.
2.7.2.1
Precipitation by Salting out
The solubility of macromolecules like proteins in water generally increases in the

presence of a suitable concentration of salt, so-called salting in. Furthermore,
increasing the concentration of salt further leads to a decrease in the solubility of the
proteins and their precipitation (salting out).
Salting out much depends on the pH and temperature of the solution. Proteins
show minimum solubility around their isoelectric point (PI) in water and a little
lower solubility in buffer solution, in other words, in the presence of salt. Regarding
temperature, the solubility of proteins generally decreases at higher temperature in
buffer solution with higher ionic strength.
Many salts are used for salting out, including ammonium sulfate, sodium sulfate,
potassium phosphate, magnesium sulfate, sodium citrate, sodium chloride etc. The
solubility of these salts is independent of temperature, and the salts do not affect the
denaturation of the proteins. Ammonium sulfate is the most effective salt for salting
out because of its high solubility at any temperature and low cost. Ammonium
sulfate is also a useful stabilizer for proteins.
I
63
2.7.2.2
Precipitation by Organic Solvents
Water miscible organic solvents, such as ethanol, isopropanol and acetone, reduce
the solubility of proteins b'y decreasing the dielectric constant of aqueous solution
and taking away water from around the proteins. Also, these organic solvents can
remove the lipids bound to a protein. Precipitation by organic solvents is affected by
temperature, ionic strength and the pH of the buffer solution.
Common proteins are precipitated at about 40% in ethanol, but proteins with
hydrophobic surface are soluble, like lipases, under the conditions. Alcohol concen-
trations of 80-90 % are necessary to precipitate lipases. The concentration of protein
should be >lmg/mL and that of buffer solution <SO mM. The solution and organic
solvent should be cooled and the mixture kept at below 0 "C during the addition of
organic solvents.
2.7.2.3
Precipitation by Changing pH
There are two types of precipitation by pH change, isoelectricpoint (PI) precipitation

and acidic precipitation.
PI precipitation is suitable for a protein with very low solubility and is more
effective in combination with salting-out and organic solvent precipitation. Anions
bind with proteins more easily than cations, so the PI of proteins shifts a little to the
acidic range.
On the other hand, acidic precipitation is good when the protein is stable, but
impure proteins are unstable in the acidic range.
2.7.2.4
Precipitation by Water-Sohble Polymer
Precipitation by water-soluble polymer is a simple method for the purification and

crystallization of proteins. Many proteins are easily precipitated in the presence of
water-soluble non-ionic polymers such as polyethylene glycols (PEG 2000, 4000,
GOOO), methyl cellulose, pdyvinyl alcohol (PVA) and dextrans (DEX).
These water-solublepolymers take away water from around proteins. The proteins
bind with these polymers via hydrogen bonds, and then the complex precipitates as a
solid or sometimes becomes a viscous liquid. Hydrogen bond chromatography is
based on this principle.
The complex contains .water-solublepolymers, which must be removed by ionic
chromatography,salting out, ethanol precipitation, electrophoresis etc.
64 2 Production and Isolation of Enzymes
I 2.7.3
Crystallization
Relatively purified proteins are easily crystallized at >1%, usually 5-lo%, of the
protein concentration in buffer. So, crystallization is the final stage of purification,
and useful for storage of proteins and X-ray crystal structure analysis. In protein
chemistry, crystallization does not mean the protein is 100% pure even though it is
in crystalline form. As described for salting out, a crystallized protein is in a solid
state together with precipitation aids such as salts, organic solvents, water-soluble
polymers etc.
Freeze drying is one of the crystallization methods; however, denaturation,
deactivation, or a slight change in the three-dimensional structure of a protein is
sometimes observed. It is necessary to check the stability before freeze drying.
2.7.4
Stabilization During Purification
Care must be taken not to lose the activity during purification of the enzyme after
fermentation. Enzymes are affective macromolecules influenced by changing the
pH, temperature, the concentration of buffer and salts, metal ions, detergents,
organic solvents and so on. In order to preserve their activity, enzymes should be
kept under natural physiological conditions such as a low temperature of about 4 "C,
natural pH for the enzyme, physiological buffer solution and concentration, etc.
Some additives for enzyme stabilization are used during purification. Mercaptoetha-
no1 and dithiothreitol work as antioxidants and EDTA works as a chelating agent to
prevent inactivation by heavy metal ions and metalloproeases.
Polysaccharides like dextrin, sugars, sugar-alcohols like sorbitol and mannitol,
glycerol and ethylene glycol are sometimes used as stabilizers. Some peptides and
amino acids are useful excipients for purification. Compounds with a similar
structure to that of the substrate are generally effective as stabilizers and are used as
fillers for storage.
Degradation by proteases derived from the same microorganism or from con-
tamination during purification must be avoided. Once a protease contaminates an
enzyme solution, the desired enzyme is degraded during purification and might
disappear. To prevent degradation by proteases, it is helpful to add protease
inhibitors like PMSF (SH protease) and EDTA (metal protease).
2.7.5
Storage of Enzymes
2.7.5.1
Storage in Liquids
Common enzymes in liquid form should be stored below 4 "C in a refrigerator and
kept with a stabilizer. Most enzymes keep their activity for several years under
suitable conditions, especially thermostable enzymes.
2.8 Commercial Biocatalysts
I 65
Ammonium sulfate (2 h4) is a popular storage solution for commercial porcine
liver esterase (PLE). Ammonium sulfate prevents microbial growth on the solution.
Storage in 50% glycerol is also useful and this glycerol stock can be stored below
0 "C.
2.7.5.2
Storage in Solids
Solid forms for storage are preferred in commercial enzymes. Generally, an enzyme
is much more stable in solid form than in liquid form even without a stabilizer. A
solid form for storage is prepared by precipitation with organic solvents and freeze
drying or spray drying depending on the purification stage. Precipitation using an
organic solvent is convenient, but the purity is not so high. Freeze drying is very
useful but expensive. Spray drying is preferable for commercial enzymes. Spray
drying is commonly carried out at about 140-70 "C for which the enzyme needs
moderate thermostability.
Stabilizers are effective to avoid loss of activity and the typical stabilizers described
above are used during precipitation and crystallization.
Some enzymes in solid form are very stable and can be stored at room temperature
for several years without loss of activity.
2.8
Commercial Biocatalysts
Among biocatalysts, hydrolases like lipases and proteases are the most popular.
There are several types of biocatalysts in commercial products. Immobilized lipases
and cross-linking enzymes are briefly described in this section.
The most popular immobilization method is adsorption on a carrier such as
diatomaceous earth or a synthetic polymer. The advantage of this method is that the
original activity of the enzyme is maintained, but the disadvantage is that the
enzyme cannot be used in an aqueous solution.
Lipases immobilized 011 ceramics modified with a chemical silyl reagent adsorb
strongly and can be used in aqueous solutions as well as organic solvents. The
activity is sometimes ten times the original and the thermostability is increased.
These products can be reused more than ten times depending on conditions.
Cross-linked enzymes are commercial biocatalysts and can be reused in organic
solvent and aqueous solution. They are purchased as crystals derived from a single
cross-linked enzyme.
Some screening kits an- provided for user convenience. Main suppliers are listed
in Chap. 20.
66 2 Production and Isolation of Enzymes
I References
1 M. P. Deutscher (ed), Methods in Enzyrnol- 7 Amersham Pharmacia Biotech (ed), Ion Ex-
ogy, vol. 182, Academic Press, San Diego, change Chromatography: Principles and
1990. Methods, APB, Uppsala, 1999.
2 W. B. Jakoby (ed), Methods in Enzymology, 8 Amersham Pharmacia Biotech (ed), Hydro-
vol. 104, Academic Press, San Diego, 1984. phobic Interaction Chromatography: Princi-
3 T. Godfrey (ed), Industrial Enzymology, ples and Methods, APB, Uppsala, 1999.
Macmillan Press Ltd, London, 1996. 9 Amersham Pharmacia Biotech (ed),Gel Fil-
4 T. Horio (Ed.),Theory and Practice on En- tration Chromatography: Principles and
zymes and Other Proteins, Nankodo, Tokyo, Methods, APB, Uppsala, 1998.
1994. 10 Amersham Pharmacia Biotech (ed),Re-
5 K. Drauz (ed), Enzyme Catalysis in Organic versed Phase Chromatography: Principles
Synthesis, VCH, Weinheim, 1995. and Methods, APB, Uppsala, 1999.
6 Amersham Pharmacia Biotech (ed), Purifi- 11 Amersham Pharmacia Biotech (ed), Affinity
cation for Proteins: Principles and Methods, Chromatography: Principles and Methods,
APB, Uppsala, 1999. APB, Uppsala, 1999.
I 67
3
Rational Design of Fiunctional Proteins
Tadayuki /manaka and Hatuyuki Atomi
3.1
Protein Engineering
One of the ultimate goals of protein engineeringrl] is the ability to design and
synthesize a biocatalyst that meets the demand of any user. The enzyme would have
to satisfy the desired activity, stability, specificity and so on in each individual case.
Unfortunately at the present time, although we have the means to synthesize
proteins of any desired primary structure, we are still a long way away from the de
novo design and synthesis of enzymes. We will need to understand the structure-
function correlation of proteins, and the principles of protein folding and interaction
much better. Even a protein of average size will contain thousands of atoms, and
therefore the number of possible inter-atomic interactions will be in the millions,
and the number of conformations accessible to a protein grows exponentially with
chain length. Although ab initio structure prediction methods (predicting three-
dimensional protein structures from amino acid sequences alone) are steadily
advancing, accurate predictions are still fairly limited to small proteins or structural
domains.
A completely contradictory approach that has been, and is still now a major
method to obtain an ideal biocatalyst, is to simply find it. This strategy leaves most of
the work to nature. Adaptation of a wide variety of organisms to diverse environ-
ments on our planet has led to a massive collection of enzymes from which we can
select. As the number of crganisms identified keeps growing, so does the number of
constituent enzymes. In particular, the recent studies on extremophiles (thermo-
philes, halophiles etc.), have significantly broadened the range of available bio-
catalystsL2].Hyperthermophiles, which grow at temperatures above 90 C, provide a
complete set of thermostable proteins that are sufficient to maintain life at these
temperatures L31. However,this approach does have its limitations. Enzyme activities
towards substrates not found in nature, or properties that are not required for life
such as protein stability against organic solvents, may be difficult to find.
The two methods mentioned above are the extremes, and many methods that
combine the two are now being developed, or have already been applied. One
68
I popular method is the random mutagenesis of
3 Rational Design of Functional Proteins
a gene encoding a particular protein

of interest, for example a lipase. A lipase that most satisfies the demands would be
chosen rationally, to try to optimize the enzyme. Random mutagenesis, or recently,
DNA shufflingF4], of the lipase gene produces a collection of proteins that resemble
the original lipase from which we can select. If the structure of the lipase is available,
it may be possible to define the region that should be subject to mutagenesis
rationally, thereby raising the fraction of improved mutants. The structure may also
allow us to pinpoint rationally one or more particular residues as targets for site-
directed mutagenesis. This is usually followed by biochemical and structural
evaluation of the variant protein, comparison with the wild-type protein, inter-
pretation of the data, and then the designing of a further modification. This will have
to be repeated until the desired changes in a protein are obtained. When affinity to a
particular molecule can be used as a means for selection, phage display['], cell
surface engineering 16], catalytic antibodies "1, the two-hybrid system [I', Profusion
technology['], and so on provide powerful tools for selection of a desired peptide
from a de nouo synthesized library. The simplicity of selection, which in many cases
is basically the binding of the peptide to a molecule immobilized on a matrix, allows
multiple cycles of mutagenesis and selection in a relatively short time. Although this
methodology had been considered an advantage enjoyed mainly by affinity screen-
ing, recent high-throughput technologies have enabled rapid analyses of tens of
thousands of clones for various enzyme parameters such as stability and substrate
specificity. Thus it is now possible to improve various parameters of biocatalysts by
this methodology, called directed evolution which is presented in Chap. 4.
In the present chapter, we will focus on the more rational approaches of enzyme
engineering and design. Basic techniques for site-directed mutagenesis, protein
crystallization,and comparative modeling will also be introduced. Some recent, key
examples of rational protein engineering will be described in a somewhat detailed
manner. There are also very informative reviews in the literature["? 15-171.
3.2
Gene Manipulation Techniques in Enzyme Modification
The repertoire of recombinant gene technology allows us to manipulate foreign or

heterologous genes in a genetically well understood organism. There may be a few
exceptions, but from a general viewpoint, Escherichia coli is the organism of choice
for protein engineering. Thanks to the general availability of easy to use cloning kits
tailor-made for mutagenesis, straightforward experiments can be carried out. Even if
other organisms are superior for the production of a particular enzyme, mutagenesis
procedures will be carried out in E. coli. A variety of multi-purpose plasmids for
mutagenesis and expression are readily available from commercial sources. PCR-
based methodologies now make it possible to incorporate, clone, isolate and confirm
gene mutations within a couple of days. E. coli host cells have also been dramatically
improved, and an abundant collection of strains are now commercially available for
various needs. Some typical techniques will be described here.
3.2 Gene Manipulation Techniques in Enzyme Modification
I 69
Many recombinant gene expression systems have been developed in the past
years. Synthesis of the prot(einis controlled at the transcriptional level as in the well-
known lac and tac systems ["]. Chemicals such as isopropyl-0-D-thiogalactopyrano-
side (IPTG) in the lac and tac systems are used to initiate and induce high levels of
transcription. Although expression of the genes is low when an inducer chemical is
not present, this basal expression may inhibit experiments when the target proteins
are lethal to the host cell. Some systems overcome this problem by controlling gene
expression under the control of a T7 promoter["]. This promoter is specifically
recognized by T7 polymerase, whose gene is introduced into the host cell. Expres-
sion of the T7 polymerase gene is regulated by an upstream lac promoter. When
IPTG is not present, very little T7 polymerase is produced, consequently leading to
minimal expression of the target gene. A lac operator can be inserted in between the
T7 promoter and the target gene in order to achieve higher stringency. Furthermore,
the gene encoding T7 lysozyme, a natural inhibitor of T7 polymerase, can also be
introduced in the host cells to reduce target gene transcription under uninduced
conditions further [l'].
One of the major problems one might encounter when expressing foreign genes
in E. coli is the formation of inclusion bodies when the proteins produced are host-
lethal, or mis-folded. Thi:j will require the unfolding of the protein with various
detergents or denaturants, followed by refolding experiments. Another problem
often seen is the low levels of target gene expression in the host cells when these
genes contain many codons that are not frequently used in E. coli. This is due to the
depletion of rare tRNA :species in the host cells. There are now commercially
available host cells transformed with extra copies of argU, ileY and leuW tRNA genes
to allow high-level expression of genes with rare codons. Many other strategies, such
as inactivatingthe Lon protease gene in the host cell[18],have been applied in order to
maximize the production of diverse recombinant proteins that may be of interest.
Site-directed mutagenesis methodology has also seen many advances in the recent
years. Most strategies are described in detail in reference 18. In essence they all rely
on synthetic oligonucleotides which contain the desired information for a modified
protein sequence, be it replacement, insertion or deletion of amino acids. Classical
cassette mutagenesis techniques are available, along with newly developed strategies
utilizing PCR techniques.
In cassette mutagenesie, synthetic complementary oligonucleotides including the
modified sequence are hybridized to form a double-stranded DNA fragment. This
fragment should span a region including two appropriate restriction enzyme sites on
opposite sides of the mutation. It is then easily possible to exchange the native
sequence with the mociified sequence after restriction enzyme digestion and
ligation. When only a pariicular mutation is required, PCR-based methods should be
less tedious and faster. However, when mutations span a relatively long region, or
require completely different nucleotide sequences compared with the original gene,
or when random sequences are to be introduced into a particular region, this
classical method still has its advantages.
To introduce mutations into genes via PCR four instead of the normal two primers
are needed. The so-called outer primers bind at the beginning and the end of the
70
I 3 Rational Design of Functional Proteins
target region, while the inner primers bind to the site where mutations are desired
and consist of the modified base information. To obtain the complete sequence with
the mutation, two rounds of PCR must be performed. The first reaction amplifies the
DNA fragment from the beginning to the mutated region, the second one from the
mutation to the end. Both PCR products carry the mutation either at the beginning
or at the end of the DNA fragment. After purification of both PCR products the two
amplificates are mixed, denatured and then the PCR will be started (without
primers). To amplify the whole sequence with the mutation the outer primers are
added and the PCR is started again. During the PCR both the mutated strand and the
natural strand are amplified but after only a few cycles will there be much more DNA
harboring the mutation.
There are now also methods that can efficiently introduce mutations in a single
PCR run (Fig. 3-1). The procedure utilizes a double-stranded DNA plasmid with the
target gene isolated from a dam" E. coli strain. Two complementary oligonucleotides
with the desired nucleotide substitutions are used for PCR along with the plasmid as
a template. The product is a mutated plasmid with staggered nicks. This is treated
with DpnI, an endonuclease specific for methylated and hemimethylated DNA. As
dam+ strains methylate plasmid DNA, the parental strain harboring the original
gene will be susceptible to DpnI treatment and digested, markedly enhancing the
efficiency of mutant gene isolation.
The interesting part begins when comparing the original enzyme with the mutant
enzyme. Thus it is advisable to run the experiments in parallel. To interpret the
results various options have to be considered. Either the enzyme activity is un-
changed (a so-called silent mutation) or it is changed for the better or worse. The
interpretation of these changes poses a serious problem. First it has to be asked
whether the mutation has altered the overall enzyme topology or whether it
influenced only the local geometry. Thus besides the usual kinetic analysis some
structural determination is advisable. To date X-ray crystallography and NMR
spectroscopy have given the most detailed picture, CD or IR spectroscopy are of less
value.
3.3
Protein Crystallization
The three-dimensional structure of a protein is the most powerful basis from which
a rational approach can be taken to modify a protein. When the structure of a highly
homologous protein has been determined, one may attempt to obtain structural
information by comparative modeling, or homology modeling. However, the reliabil-
ity of a model is questionable when similarities of the compared proteins are not
high, and we are almost helpless when a structurally novel protein is the one of
interest. Although rapid progress is being made in the use of NMR spectroscopy,the
orthodox methodology in elucidating a protein structure is still protein crystalliza-
tion and X-ray diffraction. As detailed explanations of both methodologies appear in
the literature[1'], we will just touch on some points concerning protein crystalliza-
tion.
3.3 Protein Crystallization
I
71
extension using a
thermostable DNA polymerase
digestion of the parental, methylated

DNA template with Dpn I
introduction into competent cells

and sealing of the nicks
Figure 3-1. A rapid method to introduce mutations into a target gene.

Thick lines represent the metiylated plasmid DNA harboring the wild-type
target gene.
Although there exists a vast number of protein structures in the databases, there is
as yet no rational procedure to crystallize a particular protein. The procedure is still
mainly based on a trial arid error approach. The crystallization process itself is one of
which the protein is slowly and orderly precipitated from a solution. As a general
rule, the purity of the protein is the most important factor to be dealt with before
attempting to crystallize a protein. If possible, care should be taken not only to
remove contaminant proteins, but also to remove any structurally heterologous
populations in the purified protein sample. This may be achieved by discarding tail
72
fractions after chromatography. Ideally, all molecules of the protein should have
identical surface properties, especially in terms of charge distribution, as this will
influence the packing of the molecules in the crystal. SDS-PAGE (sodium dodecyl
sulfate-polyacrylamideelectrophoresis), often used to display the homogeneity of a
purified protein in biochemical studies, will not always provide sufficient informa-
tion. Mass spectrometry can be recommended for a detailed examination of protein
homogeneity.
After the purity of a protein sample is confirmed, an appropriate solvent and
precipitant must be chosen. Again, there are no rational means in deciding these
components. Solvents are usually a water-buffer solution, and detergents or organic
solvents may be added when necessary, such as in the cases of membrane proteins or
lipases with a relatively hydrophobic surface. Typical precipitants are polyethylene
glycol (PEG), ammonium sulfate, sodium or lithium chloride salts, or 2-methyl-
2,4-pentanediol (MPD).As multiple parameters must be considered, the search for
an optimal crystallization condition may be complicated and tedious. Alternatively,
convenient kits with an array of ready-to-usesolutions including various buffers and
precipitants are commercially available. One may screen for an appropriate crystal-
lization condition with these kits, and then optimize the conditions based on the
results.
Various techniques can be applied to crystallize proteins. Vapor diffusion using
the hanging drop method is one of the popular ways to obtain crystals. In this
method, a sample solution of 2-5 pL of protein solution is placed on a siliconized
microscope cover glass. The same volume of precipitant solution is mixed, forming a
small drop on the glass surface. The glass is then placed on a well, with the drop
hanging down from the glass. Prior to this, 1 mL of the precipitant solution is poured
into the bottom of the well, so that the surface does not make contact with the
hanging drop. Vaseline or grease should be applied to the rims of the wells, so that an
air-tight chamber is made when positioning the cover glass. In this example, the
concentration of precipitant in the well is twice that of the drop. Equilibrium is
reached by vapor diffusion, and the precipitant concentration in the hanging drop
will gradually increase, possibly leading to crystallization. The sitting drop method
can also be applied when there is a surface separated from the precipitant solution in
the well. Drops are placed on the surface, and the chamber is sealed. Other methods
include batch crystallization, liquid-liquid diffusion, and dialysis. Approximately
20 pg of protein are used in a single screen, therefore 50 to 100 tests will require
roughly 1 to 2 mg of purified protein. In Fig. 3-2, some examples of protein
crystal^[^^^^] are shown. Figure 3-2 A and B provide a good example of different
crystallization conditions of a single protein, archaeal 06-methylguanine-DNA
methyltransferase, leading to distinct forms of crystals [20, 21]. Crystals of archaeal
DNA polymerase[22,231 and archaeal R ~ b i s c o [251~ ~are
. shown in Fig. 3-2 C and D,
respectively.
3.4 Comparative Modeling ofa Protein Structure
I 73
Figure 3-2. Crystals o f various proteins from the hyperthermophilic archaeon, Thermococcus
kodakaraensis KODl. A, rod-like crystal of O6-methylguanine-DNA methyltransferase (MCMT);
6,plate-like crystal of MCMT; C, bar-like crystal o f DNA polymerase; D, cubic or hexagonal
crystals o f ribulose 1,s-bisphosphate carboxylase/oxygenase (Rubisco).
3.4
Comparative Modeling of ;a Protein Structure
Comparative modeling, or homology modeling, is the most powerful tool when a

rational approach is taker1to engineer a protein with an unknown three-dimensional
structure (the target protein). Through comparative modeling, the three-dimen-
sional structure of the target protein can be predicted based on its alignment to one
or more proteins of kncwn structure (the templates). The rapid accumulation of
known protein structures and the advances in modeling software have significantly
increased the accuracy of comparative modeling. It is now possible to model, with
sufficient accuracy, significant parts of one third of all known protein sequences.
Detailed and informative reviews[26-30] can be found in the literature, and we will
only present a general overview in this chapter.
In order to predict a structural model, similarity between the primary sequences of
the target and template(!;)must be detectable. Furthermore, an accurate alignment
of the two or more sequences must be calculated. If these requirements are met, one
may proceed to model the target. The process of comparative modeling can be
divided into four steps: (i) fold assignment and template selection, (ii) target-
template alignment, (iii) model building, and (iv) model evaluation.
74
I 3 Rational Design ofFunctional Proteins
Templates can be selected using the target sequence as a query for searching
protein structure databases [e.g. Brookhaven Protein Data Bank (PDB): h q : /
/www.rcsb.org/pdb/index.html;Structural Classification of Proteins (SCOP):
scop.mrc-lmb.cam.ac.uk/scop/; DALI: www2.ebi.ac.uk/dali/; Class, Architecture,
Topology and Homologous superfamily classification at CATH: www.biochem.uc1.a-
c.uk/bsm/cath/).
Methods for protein comparison can be divided into three types. BLAST and
FASTA represent the first type, where the target sequence is independently com-
pared with each sequence in the databases, using pairwise sequence-sequence
comparison. The second type is represented by PSI-BLAST, which expands the set of
homologs of the target sequence. In a PSI-BLAST search, an initial set of homologs
against the target sequence is collected, aligned with the target sequence, and a
position-specific scoring matrix is constructed from the alignment. This matrix is
then used to cany out another search for new homologs, and this is subsequently
repeated until no new homologs are identified. It has been reported that PSI-BLAST
identifies homologs of known structure for approximately twice as many sequences
than a BLAST search. The third type of search is the 3D template matching method.
The target sequence is threaded through a library of known three-dimensional
protein folds, and a structure-dependent scoring function predicts the suitability
between the protein and the fold. This method is useful when homologs of the target
sequence cannot be found in terms of primary structure comparison. After a
collection of candidate templates is obtained, one should take into account the
relationship of each template to the target, the quality of the templates, and other
factors (e.g. the presence of convenient protein-ligand structures) before choosing
the template(s) to be used for alignment and modeling. Comparisons of the
relationships between protein sequences can be determined by constructing a
phylogenetic tree among the candidates [CLUSTALW at European Bioinformatics
Institute (EBI): http://www.ebi.ac.uk/clustalw/ or DNA Data Bank of Japan (DDBJ):
http://www.ddbj.nig.ac.jp/htmls/E-mail/clustalw-~.html]. The CLUSTAL programs
can be further used for target-template sequence alignment. When protein se-
quences display over 40 % identity, the alignment is usually correct. When sequence
identity is below 20%, multiple template structures should be used in order to
identify specific regions or secondary structures that can be used as “guides” to
construct an accurate alignment.
Once a target-template sequence alignment has been constructed, a variety of
methods and software is available for model building. Modeling methods are based
on rigid-body assembly, by segment matching or coordinate reconstruction, or by
satisfaction of spatial constraints. We will not introduce the details of each method,
and readers should refer to the indicated literature. When used optimally, all three
methods usually give similar results. Furthermore, the accuracy of the alignment
used in modeling is crucial, as no current comparative modeling method can
compensate for an incorrect alignment. On the other hand, the evaluation of a model
is usually more reliable than the evaluation of an alignment. Therefore, when a
choice among candidate alignments is difficult, one should generate models from
each alignment, and choose the most promising one by evaluating the integrity of
3.5 What is Needed to Take a Rational Approach?
I
75
the three-dimensional models. Information concerning modeling programs such as

Insight I1 and QUANTA are available at http://www.accelrys.com, SYBYL/Base at
www.tripos.com, and Internal Coordinate Mechanics (ICM) software at http:/
/www.molsoft.com. A detailed list of databases and software programs is shown in
reference [261. Various programs are available to the public at the ExPASy Molecular
Biology Server (http://wwvv..expasy.ch/).
After generating a structure model, it is essential to evaluate its accuracy before
one can use it for rational purposes. A wide variety of programs and servers exist that
can be used to evaluate A model. The BIOTECH (biotech.embl-ebi.ac.uk:8400/),
ERRAT and VERIFY3D (http://www.doe-mbi.ucla.edu/Services/), and PROVE
(http://www.ucmb.ulb.ac.l)e/UCMB/PROVE/) are servers available to the public.
Model evaluation programs check various features of the model, including bond
length, bond angles, main-chain and side-chain torsion angles, peptide bond and
side-chain ring planarities, chirality, and clashes between non-bonded pairs of
atoms. After the evaluation step, the generated structure model is ready for use as a
basis for site-directedmutagenesis. The information obtained from the biochemical
evaluation of variant proteins will also contribute to improvements in the model for
the rational design of muiants.
3.5
What is Needed to Take a I?ationalApproach?
It is quite obvious that the more information that is available on a particular protein,
the easier it is to take a raiional approach to improving its performance. Biochemical
analyses of a protein provides valuable information in terms of its activity, specificity,
and stability under various conditions. Kinetic analysis of the enzyme reveals the
kinetic mechanism of the reaction, in other words the order in which substrates
enter and products are released from the enzyme. This gives us an idea as to what
types of intermediates or complexes may be formed during the reaction. Cloning and
sequencing of the genes provides the primary structure of the protein. The number
of sequences available in public databases (e.g. GenBank/EMBL/DDBJ for genes,
SwissProt and PIR for proteins) is enormous, and readily available (Entrez protein or
nucleotide sequence search at NCBI; http://www.ncbi.nlm.nih.gov/entrez/quer-
y.fi:gi). Comparative analysis of these sequences, along with their biochemical
properties, may in some cases provide enough information to modify a protein
rationally through site-directed mutagenesis. Furthermore, there are now an ever
increasing number of three-dimensional structures of enzymes in the databases,
and these provide us wiih the precise architecture of various enzymes. Along with
advances in protein crystallization methods and X-ray diffraction technology, rapid
progress has also been made in solving protein structures by alternative tools, such
as NMR spectroscopy. Using the comparative modeling mentioned above, it is also
possible to predict the three-dimensional structure of a protein using the determined
structure of a closely related protein. Modeling software can also calculate and
predict in silico the local :structuralchanges of a protein after site-directed mutagene-
76
sis, opening the way for rational protein design. In the following sections, we will
introduce some successful examples of rational improvement or alteration of
enzyme biocatalysts based on various degrees of available information.
3.6
Examples of Protein Engineering
3.6.1
Protein Engineering Studies: Providing a Rational Explanation for Enzyme Specificity
Tyrosyl-tRNA-synthetase from Bacillus stearothermophilus is an enzyme (MW =

2 x 47 500 Da) that as a dimer catalyzes the aminoacylation of tRNATrwith tyrosine
in the following two steps.
E + Tyr + ATP = E.Tyr-AMP + PPi
E.Tyr-AMP + tRNA = Tyr-tRNA + AMP
This enzyme was one of the first and best case studies of protein engineering. Based
on the X-ray structure, there are eleven hydrogen bond contacts between the protein
and its reaction intermediate tyro~yl-adenylate[~'I. Of these, eight are hydrogen
bonds involving amino acid side chains, the remaining three are with backbone
C = 0 or N - H. A great deal of detailed information about the performance of these
contacts has been obtained. Thus the amino acids Tyr 34, Cys 35, Gly 36, Asp 38,
His 48, Thr 51, Tyr 169, Gly 192 and Gln 195 are the relevant hydrogen bond
partners for the enzyme-bound intermediate (Fig. 3-3).
The contributions that these individual hydrogen bonds can make have been
(Table 3-1). Thus, deletion of a hydrogen bond donor or acceptor
weakens the substrate-binding energy by 0.5-1.5 kcal mol-'. Deletion of the charged
Asp 38 that binds to the substrate lowers the binding energy by -4 kcal mol-'. Based
on Michaelis-Mentenkinetics, this amounts to the following barrier:
AG*T = RT(ks T/h) - RTln(kca,/KM)
kB = Boltzmann constant
h = Plancks constant
and BAG for the mutant enzyme (mut) compared with the wild-type enzyme (wt):
AA G*T = Rnn{ (kcat/ &)mut/ (kcat/ &I),}
Hydrogen bonding energies in the range of 0.05-1.5 kcal mol-' give discrimination
rates in kCa,/KM from 2-fold to 12-fold whilst 4 kcal mol-' amounts to 1000-fold
discriminations. The latter point explains nicely the enormous specificity for
tyrosine as compared with phenylalanine. Asp 176 in the binding pocket binds to the
phenolic hydroxyl group of tyrosine (Fig. 3-3).Thus from the large amount of data
presented we can learn about the binding energy of hydrogen bond donors and
acceptors in proteins: deletion of a side chain between the enzyme and substrate to
leave an unpaired, uncharged, hydrogen donor and acceptor weakens the binding
energy by only 0.5-1.5 kcal mol-'. However, the presence of an unpaired and
3. G Examples of Protein Engineering
I 77
Figure 3-3. Hydrogen bonds between the tyrosyl-tRNA synthetase and tyrosyl
adenylate.
Table 3-1. Relative binding eiiergies of groups in tyrosyl-tRNA synthetase infered from
comparison between mutant and wilde-type enzymes at 298 K.
Compared residues and their numbering Substrate MGf (kcal mol-')
-
Phe 34 Tyr 34 TYr 0.52
Gly 35 cys 35 ATP 1.14
Ala 51 cys 51 ATP 0.47
Gly 48 Asn 48 ATP 0.77
Gly 48 His 48" ATP 0.96
Ser 35 cys 35" ATP 1.18
Phe 169 Tyr 169" Tyr 3.72
Gly 195 Gln 195" TYr 4.49
Gly 35 Ser 35 ATP - 0.04
Ala 51 Thr 51" ATP - 0.44
* Residues found in the wild-type protein
charged donor or acceptor weakens binding by a further 3 or more kcal mol-'. These
values are much lower than the absolute strength of hydrogen bonds in vucuo and are
the consequence of hydrogen bonding in aqueous solution being an exchange
process.
78
Figure 3-4. (A) Structure o f thermolysin

B
from Bacillus thermoproteolyticus used t o
determine candidate residues for site-di-
rected mutagenesis o f t h e neutral pro-
tease from B. stearothermophilus. The
structure was visualized with Ras-Mol
software and slightly modified. Data were
from M M D B ID: 3638, PDB ID: lLNF,
submitted by Holland, and Matthews.
The two domains are indicated with the
bars on the left, and the Cly 144 residue is
highlighted i n black. (B) Themostability
I
0
1 I
to
I
20
I I I
30 o f t h e wild-type neutral protease (open
Time (min) circles), and the C144A variant (closed
circles).
3.6.2
Enhancing the Themostability o f Proteases
An increase in thermostability of a neutral protease from Bacillus stearothemophilus

(NprT) was achieved from a rational approach by comparing its sequence with the
thermostable thermolysin from B. t h e m o p r o t e o l y t i ~ u s [ The
~ ~ ] . enzymes were 85 %
identical, while the thermostability of NprT at 75 C was significantly lower than that
3.6 Examples of Protein Engineering
of thermolysin. Taking into account the statistical data of various amino acid
I 79
substitutions that increase thermostability, and the three-dimensional structure of

thermolysin, a single mutation G144A was chosen as a candidate to increase the
thermostability of NprT. The glycine residue was supposed to be located in an a-helix
that connected the N- and C-terminal domains of the enzyme (Fig. 3-4A). The
mutation was expected to stabilize the a-helix, and increase internal hydrophobicity
of the enzyme. Furthermore, the G144A mutation introduces only a small methyl
group, minimizing any structural or functional interruption that may be caused by
introduction of a new side chain. Indeed, this single mutation led to a significant
increase in the thermostability of NprT (Fig. 3-4B). This is a good example of the fact
that an increase in internal hydrophobicity of an enzyme and stabilization of a
secondary structure a-helix leads to an increase in the thermostability of a protein.
3.6.3
Contribution of Ion Pairs to the Thermostability of Proteins from Hypertherrnophiles
Proteins found in hyperthermophiles display an astonishing resistance to thermal

denaturation. Some are :stable for hours or even days at temperatures near to the
boiling point. This has attracted much attention as these proteins are promising
candidates themselves as stable biocatalysts, and also provide valuable hints to the
understanding of the mechanisms of protein thermostability.
The present authors have pursued attempts to elucidate the three-dimensional
structures of various proteins from the hyperthermophilic archaeon Thennococcus
kodakaraensis KOD1. These include DNA polymerase[22s231, homing endonuclease
I1 [34], 0'-methylguanine-DNA methyltransferase (MGMT)[20s 211, aspartyl-tRNA
synthetase[351, and ribulose 1,s-bisphosphate carboxylase/oxygenase (Ru-
bisco) [24, 251. MGMT repairs alkylated DNA by suicidal alkyl transfer from guanine
0' to its own cysteine residue. We determined the three-dimensional structure of
MGMT from T. kodakaraensis KODl (Tk-MGMT) at 1.8 A resolution[211.This
structure was compared with its counterpart from Escherichia coli (AdaC, C-terminal
fragment of Ada protein). It has been reported that helical conformation is stabilized
by (i+ 4) or (i+ 3 ) glutamate-lysineintra-helixion-pairs in a short model peptide. We
observed seven intra-helixion pairs in Tk-MGMT, while none were detected in AdaC.
It is presumed that these intra-helix ion-pairs contribute to reinforcement of the
stability of the a-helices. Furthermore, four extra inter-helix ion-pairs not found in
AdaC were observed in the interior of Tk-MGMT, stabilizing the internal packing of
the tertiary structure. The structure of Tk-MGMT strongly indicates that intra-helix
and inter-helix ion-pairs provide a major contribution to the thermostability of the
protein.
As the importance of ion-pairs toward protein thermostability has been stressed in
many cases, addition or removal of an ion-pair should have significant effects. A
clear example is provided by mutagenesis studies of glutamate dehydrogenase from
?: kodakaraensis KODl (Tk-GDH)r3'1. The GDH from Pyrococcusfirriosus (P'GDH)
and Tk-GDH are 83% identical in terms of primary structure. However, while ' P
GDH displays a half-life of 12 h at 100 C, that of Tk-GDH is 4 h. The three-
80
A
7
30 40 50 60 70 80 90 100 110
Temperature (“C) Time (h)
Figure 3-5. Temperature profile (A) and thermostability ( 6 ) o f glutamate dehydrogenase from
T. kodakaraensis KODl and its mutants. Data o f t h e wild-type enzyme (circles), the T138E
mutant protein (squares), and the El58Q mutant protein (triangles) are shown.
dimensional structure of P’GDH has been determined, and exists in a stable

hexameric form. A structural model of Tk-GDH was constructed based on the
structure of P’GDH. A difference was observed between the two structures at the
monomer-monomer interface. In P’GDH, there is a large ion-pair network com-
prised of six residues, Arg 35, Asp 132, Glu 138, Arg 164, Arg 165, and Lys 166.
Glu 138 is located at the center of the network, interacting with Arg 165 and Lys 166.
In the case of 2%-GDH,Glu138 was replaced by a threonine residue. When a T138E
mutation was introduced into Tk-GDH,an increase in both thermostability (2 to 3 h
at lOOC) and optimal temperature (80 to 85C) was observed, confirming the
importance of ion-pair networks (Fig. 3-5). At one of the two-fold axes of the
proteins, Glu 158 is at the center of another ion-pair network, interacting with
Arg 124 and Arg 128. An El58Q mutation would interrupt this network, and is
presumed to destabilize the protein. As expected, the El58Q mutant protein of Tk-
GDH displayed a lower optimal temperature for activity (80 to 60 C), and decreased
thermostability (2 h to 50 min at 100 C, Fig. 3-5).
3.6.4
Thermostability Engineering Based on the Consensus Concept
The examples mentioned above have shown that in many cases, sequence compar-
isons between two homologous enzymes with different thermostabilities provide
valuable clues as to the how to increase protein thermostability rationally. An
interesting observation has recently been made that even a set of amino acid
sequences of homologous, mesophilic enzymes provides sufficient information to
3.6 Examples of Protein Engineering
I 81
allow the rapid design of a thermostabilized variant of the family of enzymes[37].

Using myo-inositol hexakisphosphate phosphohydrolase (phytase) as the target
enzyme, a sequence alignment of 13 homologous fungal phytases was used to
calculate a consensus amino acid sequence. An amino acid that has already been
proven to fit into the structure of at least one of the homologous enzymes used in the
alignment is chosen as a (consensusresidue. A synthetic gene, corresponding to the
consensus phytase sequence was expressed and the recombinant protein, consensus
phytase-1, was characterized. Differential scanning calorimetry revealed that con-
sensus phytase-1 displayed an unfolding temperature (T,) of 78.0 C, which was
15-22C higher than the T, values of all parent phytases used in its design.
Furthermore, by including six more sequences in the alignment, a refined con-
sensus sequence was calculated (consensus phytase-10). Consensus phytase-10
displayed even higher thermostability, with a T, value of 85.4 C. Further optimiza-
tion through site-directed mutagenesis eventually led to consensus proteins with
unfolding temperatures of up to 90.4 C. When the effects of individual substitutions
were evaluated, all single mutations affected the thermostability by less than 3 C.
This suggests that the increases in stability observed in the consensus phytases were
due to the combination of multiple amino acid exchanges distributed over the entire
sequence of the protein. Remarkably, in spite of the increase in thermostability,
catalybc activity at 37 C was not compromised. Although further examination with
other proteins will be necessary, the consensus concept may provide a powerful
alternative as a means to enhancing the thermostability of proteins when the
information available is limited.
3.6.5
Changing the Optimal pH o f an Enzyme
Various thermostable alcohol dehydrogenases have been studied for use in the
industrial production of alcohol. Based on the three-dimensional structure of horse
liver alcohol dehydrogenase and a multiple sequence alignment of alcohol dehy-
drogenases from variou:; sources, the optimal pH of a thermostable alcohol dehy-
drogenase (ADH-T) from Bacillus stearothermophilus NCA 1503 was rationally
The amino acid residues responsible for the catalytic activity ofhorse liver
ADH had been clarified on the basis of its three-dimensional structure. As the
catalytic amino acid residues were fairly conserved in ADH-T and other ADHs,
ADH-Twas presumed to harbor the same proton release system as horse liver ADH,
and confirmed by site-directed mutagenesis. In ADH-T, catalysis was showri to be
performed by a proton release system involving a zinc-bound water molecule, a
hydroxyl group of Thr 40, and an imidazole ring of His 43 (Fig. 3-6)[391.Cys 38,
which interacts with the zinc ion, along with Thr 40, and His 43 were the targets for
site-directed mutagenesis, and C38S, T40A, T40S, and H43A mutants were pro-
duced. The C38S, T40A, and H43A mutations completely abolished the activity of
ADH-T, while the T40S mutant displayed a slightly lower activity than the wild-type
enzyme. As the pK, value of His 43 was presumed to play an important role in
proton release, an H43R. mutation was incorporated in order to alter the optimal pH
82
I 3 Rational Design ofFunGtiona1 Proteins
Active site zinc \ i!! /

I I
Figure 3-6. Mechanism
for the proton release
system of ADH-T. The
Water
H -0 H- - NAD+ system is composed of a
I zinc-bound water mole-
H cule, and the side chains
H of residues Thr 40 and
1 His 43. Proton release is
R -0
induced by NAD' binding.
H
H
R I
H H+
of the enzyme. As expected, the optimum pH of the mutant enzyme H43R was
shifted from 7.8 (wild-type enzyme) to 9.0. Furthermore, at the optimum pH, the
H43R enzyme exhibited a higher level of activity than the wild-type ADH-T.
3.6.6
Changing the Cofactor Specificity of an Enzyme
Nicotinamide adenine dinucleotide (NAD') and nicotinamide adenine dinucleotide

phosphate (NADP') are ubiquitous redox cofactors involved in a huge variety of
enzyme reactions. The two are similar in structure, with NADP' harboring a single
additional phosphate group esterified to the 2'-hydroxyl group of the AMP moiety.
However, enzymes found in nature usually display a clear preference for one of the
two cofactors, providing an interesting example of molecular recognition by en-
zymes.
Many studies have addressed this subject, and it is now possible to engineer and
switch rationally the cofactor specificities of particular enzymes from NADP' to
NAD' and vice versa. Pioneering work has been carried out with glutathione
reductase, a member of the highly homologous flavoprotein disulfide oxidoreductase
family[40].Most members of this enzyme family utilize NADP' as a cofactor with
one exception, the NAD'-dependent dihydrolipoamide dehydrogenases. Using the
three-dimensional structure of glutathione reductase from human erythrocytes (H-
GR), and sequence alignment of various enzymes in the flavoprotein disulfide
oxidoreductase family (Fig. 3-7), the cofactor specificity of glutathione reductase
from E. coli (E-GR,gor gene product) was switched from NADP' to NAD'. From the
structure of H-GR, a Pap "fingerprint" motif was found in the NADP-binding
domain ofthe enzyme. Two arginine residues in H-GR,Arg 218 and Arg 224, bind to
the 2'-phosphate group of the NADP' molecule. These residues are conserved in
virtually all NADP'-dependent enzymes in the flavoprotein disulfide oxidoreductase
family, but not in the NAD'-dependent dihydrolipoamide dehydrogenases. Substitu-
tion of each corresponding arginine residue in E-GR (R198M, R204L) or both,
E-GR 114
3.6 Examples ofprotein Engineering
I
83
H-GR 194
P-GR 172
S-MR 251
P-MR 211
T-TR 195
E-DD
6-DD
Y-DD
H-DD
* * * * * **
180 GGG ILGLEMGTVYHALG---SO I DWEMFDOVIPAAD
183 GGGYIGIELGTAYAhFG---TKVTILEGAGEILSGFE
209 GGGI IGLEMGSVYSRLG---SKVTVVEFQPQIGASMD
220 GAGv IGVELGSWQRIG---ADVTAVEFLGHVGGVG I
*
pjAD+
Figure 3-7. Sequence alignment of various enzymes in the flavopro-
tein disulfide oxidoreductase family. The sequences of the NADP’-
dependent enzymes are the glutathione reductase from E. coli (E-
CR), human (H-CR), Pseudomonas aeruginosa (P-GR), mercuric re-
ductase from Staphylococcus atireus (S-MR), P. aeruginosa Tn 501 (P-
CR), and trypanothione reductase from Trypanosoma congolense
(T-TR). The NAD+-dependent enzymes are dihydrolipoamide dehy-
drogenase from E. coli (E-DD), B. stearothermophilus (B-DD), yeast
(Y-DD), and human (H-DD). Residue positions marked with an
asterisk correspond to those that were targets o f site-directed muta-
genesis in the text.
resulted in a modest fall in the kcat value of the NADP+-dependentactivity, but

caused a large increase in KM toward NADP+ (-25-fold). Drastic effects were not
observed for NAD+-dependentE-GR activity.
Further mutations were introduced, focusing on the G-X-G-X-X-Gmotif, found in
various NAD+-dependentdehydrogenases, including dihydrolipoamide dehydroge-
nase. In NADP+-dependent enzymes, including H-GR and E-GR, the third Gly
residue is usually replaced by an Ala residue (Ala 179 in E-GR).Another Ala residue
is also conserved four resi~duesfurther toward the C-terminus in NADP+-dependent
enzymes (Ala 183 in E-GR), but substituted by a Gly residue in dihydrolipoamide
dehydrogenases. The A179G mutation in E-GR led to a dramatic decrease in the KM
toward NAD’ (-40-fold), with little change in the kcat value. The A183G mutation
had little effect towards N AD+-dependentactivity.
Another set of mutations were introduced centered on the Val 197 residue of E-
GR. In the NAD+-dependent dihydrolipoamide dehydrogenases, this residue is
replaced by a Glu residue, whose negative charge interacts with the 2’-hydroxyl
group of NAD’ via a hydrogen bond. In order to generate such interaction in E-GR, a
V197E mutation, along with K199F and H200D mutations to remove residual
positive charges that may interact with the 2’-phosphate group of NADP’, were
introduced. The mutant protein with seven mutations, A179G/A183G/V197E/
R198M/K199F/H200D/R204P displayed a - 250-fold decrease in kcat/KM value for
NADP+-dependentactivity, while that for NAD+-dependentactivity increased by a
factor of - 70. The ratio of these two contrasting shifts is 18 000, indicating that the
cofactor specificity of the enzyme was rationally switched.
As all mutation sites ‘chosen in this study are limited to the pap “fingerprint”
motif, the strategy appl led is applicable to other NAD+- and NADP+-dependent
dehydrogenases. Indeed, a systematic replacement of amino acid residues in the pap
“fingerprint” motif in tht? NAD’-dependent dihydrolipoamide dehydrogenase from
E. coli converted its cofactor specificity from NAD+ to NADP+L4*]. A similar strategy
84
l has been successfully applied
3 Rational Design of Functional Proteins
on inverting the cofactor specificity of NAD+-depend-

ent malate dehydrogenase from Themusflavus, using the crystal structure of the
NAD+-dependentporcine enzyme and alignment with the NADP+-dependenten-
zyme from chloroplasts[421. The engineered mutant protein displayed a 1000-fold
improvement toward NADP' and a 600-folddecrease in efficiency with NAD'. Other
key examples have been shown with decarboxylating dehydrogenases, isocitrate
dehydrogenase (IDH)[43, 441 and isopropylmalate dehydrogenase (IMDH)[451. Al-
though these enzymes do not bind the nucleotide cofactors in the pap binding motif
mentioned above, conversion of an NADP'-dependent IDH into an NAD+-depend-
ent enzyme (850-foldpreference) has been achieved[43].Engineering the secondary
structure of NAD+-dependentIMDH from Themus themophilus led to a 1000-fold
preference for NADP+L4'].
3.6.7
Changing the Substrate Specificity of an Enzyme
Recently, there have been an increasing number of reports where rational muta-
geneses of enzymes led to a dramatic change in their substrate specificity. One
example is the study on cucumber linoleate 1 3-lipoxygenaseL4'1. Lipoxygenases
constitute a family of non-heme, iron-containing dioxygenases catalyzing the regio-
and stereoselective dioxygenation of polyenoic fatty acids to form hydroperoxy
derivatives. Enzymes from plants are classified into 9- and 13-lipoxygenasesaccord-
ing to their positional specificitytoward linoleic acid oxygenation. Multiple sequence
alignments and structural modeling of enzyme-substrate interaction suggested that
a single residue, His 608, played a key role in the regiospecificity of the 13-lipox-
ygenase. An H608V mutation was introduced, and resulted in an enzyme variant
with specific 9-lipoxygenaseactivity. This was elegantly explained by the fact that an
H608V mutation enables a positively charged guanidino group of Arg 758, masked
by the bulky His 608 residue in the wild-type enzyme, to interact with the carboxyl
group of the substrate linoleic acid. This interaction forces a reversal of the substrate
in the active site. This explanation was strongly supported by the observations that an
R758L/H608V double mutant protein exhibited a lower reaction rate and random
positional specificity. Furthermore, the drastic alteration of positional specificity was
not observed when substrates lacking a free carboxyl group were examined.
Another example deals with the mammalian 3a-hydroxysteroiddehydrogenase14'1.
Mammalian hydroxysteroid dehydrogenases convert potent steroid hormones into
their cognate inactive metabolites and belong to the aldo-keto reductase superfamily.
Although 3a- and 20a-hydroxysteroid dehydrogenases display 67 % amino acid
sequence identity with one another, they differ in their regiospecificity and ster-
eospecificity. 3a-Hydroxysteroid dehydrogenase converts 5-dihydrotestosteroneinto
3-androstanediol, while 20a-hydroxysteroid dehydrogenase converts progesterone
into 20-hydroxyprogesterone, the two enzymes catalyzing the formation of secon-
dary alcohols on opposite ends of steroid hormone substrates. The crystal structure
of 3a-hydroxysteroid dehydrogenase complexed with testosterone indicated that
10 residues located on 5 loop structures were involved in the enzyme-substrate
interaction. Multiple sequence alignment of various hydroxysteroid dehydrogeriases

I
85
displayed that G of these 110 residues were substituted in the 20a-enzyme. Single and
multiple replacements of the 3a-enzyme residues to the 20a-enzyme residues did
not lead to an alteration in regiospecificity. However, when individual loops were
exchanged, a drastic chan:gein regiospecificitywas observed. An exchange of loop A
led to a protein variant with both 3a- and 17P-hydroxysteroiddehydrogenase activity.
A double exchange of loops A and C resulted in 3a- and 20a-activity. Finally, a triple
exchange of loops A, B and C completely converted the specificity of the enzyme into
a stereospecific 20a-hydroxysteroid dehydrogenase with a resultant shift in kcat/ KM
for the desired reaction of'2 x lo1'.
3.6.8
Changing the Product Specificity of an Enzyme
A rational approach can also be used to change the product specificityof an enzyme.
Prenyl diphosphate synthases catalyze the condensations of isopentenyl diphosphate
with allylic diphosphate to give linear hydrocarbons of various lengths and different
stereochemistries. Heptaprenyl diphosphate synthase from B. stearothemophilus is a
member of the medium-chain prenyl diphosphate synthases. The enzyme catalyzes
the consecutive condensation of isopentenyl diphosphate with allylic diphosphate to
produce (all-E)-C35 prenyl diphosphate as the ultimate product. The product
specificity of short-chair1 prenyl diphosphate synthases has been shown to be
regulated by a structure around the first aspartate-rich motif (FARM). Component 11'
of heptaprenyl diphosphate synthase also harbors a FARM, suggesting that this
structure in component 11' may also regulate elongation in this enzyme. Via site-
directed mutagenesis, a relatively bulky isoleucine residue eight positions before the
FARM, was substituted by a small glycine residue (I7GG variant). As anticipated, the
I7GG variant catalyzed condensations of isopentenyl diphosphate beyond the native
chain length of C35. Furthermore, two small residues Ala79 and Ser80 were
individually replaced with the bulky tyrosine and phenylalanine, respectively (A79Y
and S80F variants). In contrast to the I7GG mutation, these variants mainly yielded a
C20 product. The study demonstrates that in the wild-type enzyme, the elongation
reaction is precisely blocked at the length of C35 by the bulky Ile 76 residue, and that
the degree of elongation can be controlled by removal or introduction of a bulky
residue in the enzyme (Fig. 3-8).
A similar approach can be utilized with the geranylgeranyl diphosphate synthase
from Sulfolobus acidocaidarius. The wild-type enzyme yields (all-E)-C20 prenyl
diphosphate as a final product. The three-dimensional model of the enzyme
suggested that the remotal of two bulky residues Phe 77 and His 114 would allow
additional prenyl-chain elongation. F77G, F77G/H114A, F77G/H114G, H114A, and
H114G variants gave C30, C(45), C50, C30 and C40 as the major maximum length
products, respectively L4'1
86
I 3 Rationo/ Design of Functional Proteins
Figure 3-8. Proposed mechanism of the chain-length determination of the wild-type and variant
heptaprenyl diphosphate synthases based on the pocket mechanism. A, Wild-type enzyme;
6, 176C variant; C, A79Y variant; D, SSOF variant.
3.6.9
Combining Site-directed Mutagenesis with Chemical Modification
Combining site-directed mutagenesis strategies with chemical modification is a

popular tool in both enzyme engineering and mechanistic studies. This has often
been applied to the subtilisin from Bacillus lentus (SBL), or savinase. Subtilisins are
I 87
one of the most well-characterized and well-engineered proteins; mutational effects
of more than half of the 275 amino acid residues have been reportedLs0I. A high-
resolution, three-dimensional structure of S BL is also available. Furthermore, wild-
type SBL does not harbor any cysteine residues. Therefore, if a single cysteine
residue were to be introduced by site-directedmutagenesis, treatment with methane-
thiosulfonate reagents would lead to specifically localized modification of the
enzyme. This approach, the chemically modified mutant approach (CMM),has been
utilized in altering the stability,specificity, kinetic properties, and pH profiles of SBL.
The following example displays how the CMM approach can expand the specificityof
the S 1 pocket of SBLL5l1.
Wild-type SBL is known to prefer bulky, hydrophobic P1 residues in its S 1 pocket.
The Phe P1 residue of the standard suc-AAPF-pNA (succinyl-alanyl-alanyl-prolyl-
phenylalanyl-p-nitroanilide)substrate was shown to be preferred by a factor of
9500-fold over the small P1 residue of suc-AAPA-pNA, by a factor of 24-fold
compared with the positively charged P1 residue of suc-AAPR-pNAand by a factor of
522-fold compared with the negatively charged P1 residue of suc-AAPE-pNAA.The
Ser 166 residue, located at the bottom of the S 1 pocket and whose side chain points
inward toward the pocket, was chosen for substitution by cysteine and subsequent
chemical modification. In order to increase specificity toward small uncharged P1
residues such as Ala, bulky moieties, for example benzyl, decyl, cyclohexyl, and
steroidyl groups, were incorporated at Sl66C so as to reduce the volume of the S 1
pocket and induce a betier fit for small P1 groups. Likewise, negatively charged
groups such as an ethylsulfonatomoiety, a dicarboxylic aromatic group, and aliphatic
mono-, di-, and tri-carboxyl groups were incorporated for higher specificity for
positively charged P1 residues such as Arg. A positively charged ethylamino group
was introduced to improve the acceptance of the negatively charged P1 residue Glu.
In the case of a cyclohexyl group, the modified enzyme showed a 2-fold improve-
ment in kcat/& with the suc-AAPA-pNA substrate and a 51-fold improvement in
suc-AAPA-pNA/suc-AAPF-pNAselectivity relative to WT-SBL. The enzymes mod-
ified with mono-, di-, arid tricarboxyl groups displayed improved kcat/& values
toward suc-AAPR-pNA. Furthermore, these values increased in parallel with the
number of carboxyl groups introduced, and led to a 9-fold improvement in kc,,/ KM
for the suc-AAPR-pNA substrate and a 61-foldimprovement in suc-AAPR-pNA/suc-
AAPF-pNA selectivity compared with the wild-type SBL. Conversely, the introduc-
tion of the positively charged ethylamino group led to a 19-foldimprovement in kcat/
KM for the suc-AAPE-pNA substrate and a 54-fold improvement in suc-AAPE-pNA/
suc-AAPF-pNA selectivity relative to the wild-type SBL.
3.6.10
Changing the Catalytic Adivity of a Protein
With the abundant number (> 16 000) of three-dimensional structures in the

Brookhaven Protein Data Bank, a challenging but promising task in protein
engineering is the synthesis of novel biocatalysts by assembling individual func-
tional modules (substrat12 binding sites, catalytic centers etc.), or by introducing a
88
designed functional environment into a known protein template structure. The

following is an example of the latter strategy, taking advantage of the diverse
functions of a protein superfamily sharing a common fold LS21.
The 2-enoyl-CoA hydratase/isomerase enzyme superfamily is comprised of en-
zymes with various specificities and functions, including 4-chlorobenzoyl-CoA
dehalogenase, 2-enoyl-CoA hydratase, carnitine racemase, dihydroxynaphthoate
synthase, 2-ketocyclohexanecarboxyl-CoAhydrolase, A3,A2-enoyl-CoA isomerase,
and even the proteolytic component of Clp protease. Structural comparison of these
proteins indicated the possibility that a majority of the individual active sites were
derivatives of a single active site structure. This environment provides a CoA binding
site, an expandable acyl-binding pocket, an oxyanion hole for binding or polarizing
the thioester carbonyl group, and numerous sites for strategic positioning of catalytic
residues. In the study, the active site of one member of the 2-enoyl-CoA hydratase/
isomerase family, 4-chlorobenzoyl-CoAdehalogenase, was altered by site-directed
mutagenesis to include the two glutamate residues functioning in acidlbase catalysis
in a second family member, 2-enoyl-CoAhydratase. As a result, the syn hydration of
2-enoyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoAdehalogenase, was ob-
served in the engineered protein with kcat and KM values of 0.06 s-l and 50 M ,
respectively. Although the efficiency of the engineered protein is far from the native
2-enoyl-CoA hydratase, the study clearly demonstrates the possibility of exchanging
catalytic functions of two enzymes within a structural enzyme family. It also sends
an encouraging message that if an appropriate template is available, it is possible to
obtain a desired enzyme activity by rationally designing a catalytic environment on
the “template landscape”.
Other studies have explored or resulted in even more drastic alterations in
enzymatic characteristics. Tyrosine phenyl-lyase (TPL) and aspartate aminotransfer-
ase (AspAT) both belong to the a-family of vitamin B6-dependentenzymes. While
TPL catalyzes the p-elimination reaction of L-tyrosine,AspAT catalyzes the reversible
transfer of an amino group between dicarboxylic amino acids and their correspond-
ing 2-0x0 acids. The double mutation R100T/V283R, leading to an AspAT-like
sequence, was introduced into TPL. The protein obtained displayed a 104-fold
increase in p-elimination activity towards dicarboxylic amino acids than the wild-type
TPL. The activity towards L-aspartate was twice as high as that towards the native
substrate L-tyrosine. The created enzyme can be considered a dicarboxylic amino
acid p-lyase, an enzyme that is not found in nature[53].A further study attempted to
design a protein with enzymatic activity, starting from a structurally homologous
non-catalytic protein The nuclear transport factor 2 (NTF2) and scyt-
alone dehydratase both share a common alp barrel structure. Four key catalytic
residues, along with a C-terminal a-helix found in scytalone dehydratase, but not in
NTF2, were introduced into the NTF2 protein. A mutant protein exhibited scytalone
dehydratase activity with minimal kcat and KM values of 0125 min-’ and 800 p ~ ,
respectively. The study is one of the few examples of converting a non-catalytic
protein scaffold into an enzyme.
3.7 Conclusions 89
I
3.7
Conclusions
The examples above represent some of the most successful studies in protein
engineering. They show that it is possible to enhance protein thermostahility
rationally, alter cofactor or substrate specificity, regiospecificity, and even change
catalytic activity. Furthermore, the creation of enzymatic activity from a non-catalytic
protein backbone, and tht creation of a biocatalyst with an unprecedented catalytic
activity not found in nature, have also been achieved. However, the examples
published in the literature are probably only a tiny fraction of the many studies that
have been, or are still, in progress awaiting positive results.
We are still at a premature stage in designating precise rules to engineer a variant
protein with each and every desired property. It is still not easy to predict the
outcome of even a single amino acid residue substitution. However in some cases,
depending on the information available and the property desired, some basic
guidelines are available. Whatever the position, the three-dimensional structure of
the protein, or of a homologous protein is highly desired. Without any structural
information, strategies will be limited, and the sense of rationality of the experi-
ments will be low.
When enhancement of protein (thermo)stabilityis desired, there are a number of
strategies available, taking into account four major interactions within a protein;
covalent bonds via disulfide bridges, ionic interactions, hydrogen bonds, and
hydrophobic interaction (Fig. 3-9). Introducing a covalent disulfide bond in a region
distant from the catalyticcenter of T4 lysozyme was reported to enhance dramatically
the thermostability of the protein[55,561. With human lysozyme, introduction of Asp
residues to generate a Ca2+ binding pocket rationally, and consequently ionic
interactions, led to a calcium binding variant protein with an increase in thermo-
Although performed by a random approach, the effects of hydrogen
bonds on protein thermostability has also been displayed with T4 lysozyme[581.A
single T157I mutation, interrupting a hydrogen bond in the wild-typeenzyme, led to
a temperature-sensitive mutant protein. The importance of hydrophobic interactions
has been mentioned above. Addition of any of these four types of interactions may be
considered in order to enhance the thermostability of a protein. Another alternative
may be to introduce proline residues at P-turn structures (the proline rule). This has
been clearly demonstrated with oligo-1,G-glucosidasesfrom various Bacillus spe-
cies[58-6*1
When the aim is to aher the substrate or cofactor specificity of an enzyme, one
should look for a homologous structure of an enzyme bound with the target
molecule or a structurally similar compound (template structure). This will provide
much more information than the structure of a homologous protein alone, even
when the latter has been determined at a higher resolution. If the (modeled)
structure of the target enzyme is also available, superimposing the structures of the
two proteins will make the examination of the supposed interaction of the target
enzyme and the binding molecule possible. Side chains that sterically or electro-
statically interfere with binding may be identified, and subsequent mutations can be
90
Figure 3-9. Various interactions in

a protein molecule. Increasing
these interactions may enhance
the thermostability of the protein,
as described in some examples in
the text.
dOOH
designed for their removal. On the other hand, residue substitutions that may
possibly enhance affinity or increase specificity can also be designed. Even when a
(modeled) structure of the target protein is not available, an accurate sequence
alignment may also be sufficient, as long as the three-dimensional template
structure is available. In some very well-studied cases, such as the pap binding
motifs for NAD( I?) cofactor binding (mentioned above), primary sequence align-
ment may provide enough information to engineer the binding site.
Recent studies, some mentioned here, convey new strategies and concepts for
protein engineers. Combining rational design with directed evolution has also
become a popular means of obtaining a protein with a desired function. The growing
number of strategies will surely attract more scientists to become engaged in the
field of protein engineering. This will hopefully accelerate the accumulation of
information available to the engineer, ultimately enabling the de novo design of a
biocatalyst.
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within the 2-enoyl-coenzymeA hydratasel niak, S. P. Cook, B. W. Matthews, Contribu-
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I
95
4
Enzyme Engineering by Directed Evolution
Oliver May, Christopher A. Voigt and Frances H. Arnold
4.1
Introduction
Previous chapters have outlined the huge potential of enzymes as tools for organic
synthesis. However, this potential is only slowly being realized in large-scale
industrial applications. The main reason for this is that enzymes are often incompat-
ible with the specific requirements of a synthesis, especially under economic
constraints. Enzyme behaliors such as substrate or product inhibition, stability, and
catalytic efficiency (kc,&,) are all finely tuned by natural evolution to support
efficient reproduction of the organisms that make them. Product inhibition can be
useful in a living cell, where it prevents the accumulation of undesired or even toxic
products. But it is highly undesirable in a synthesis requiring high substrate
concentrations and complete conversion into products. Similarly, an enzyme may
naturally be highly substrate specific so as to prevent undesired side reactions with
other chemically similar metabolites. But such an enzyme can only be used to
synthesize a very limited range of products. Other properties that are highly
desirable for chemical applications, such as long-term stability and activity in organic
solvents, are simply not Iequired in nature and are therefore not found in natural
enzymes. While it is possible to devise effective bioprocess engineering solutions to
some of these problems, it will often be necessary or more effective to engineer the
catalyst itself.
The previous chapter reviews methods for structure-guided enzyme engineering.
A prerequisite for this approach is knowledge of the enzyme structure and detailed
insight into how this structure determines function. Then we must be able to predict
how specific amino acid changes affect the desired properties. Despite rapid growth
in the numbers of enzyme structures solved and the considerable progress made in
cornputational methods, our understanding is still very limited and in most cases
insufficient to obtain the desired features with an acceptable probability of success.
The strategy nature uses to adapt organisms to new demands is evolution.
According to Darwinian iheory, the fantastic diversity of life was created by random
mutation and natural selection'']. The power and simplicity of the evolution
96
I 4 Enzyme Engineering by Directed Evolution
algorithm has tempted scientists and engineers to try to implement this same
approach for biomolecular design. In 1984,long after Eigen’s pioneering work on
the theory of evolution [2231, Eigen and Gardiner suggested the following procedure
that “should allow a new type ofevolutionary biornolecular engineering [41:
10 PRODUCE A MUTANT SPECTRUM OF SELF-REPRODUCINGTEMPLATES
20 SEPARATE AND CLONE INDIVIDUAL MUTANTS
30 AMPLIFY CLONES
40 EXPRESS CLONES
50 TEST FOR OPTIMAL PHENOTYPES
60 IDENTIFY OPTIMAL GENOTYPES
70 RETURN TO 10 WITH A SAMPLE OF OPTIMAL GENOTYPES
Scientists wishing to design useful proteins, peptides, or nucleic acids have picked
up this evolutionary approach, which is now known as directed evolution, applied
molecular evolution, in vitro evolution, or molecular breedingI5-l3I.Directed evolu-
tion combines a high probability of success (the possibility of obtaining an improved
catalyst within months) with no requirement for detailed knowledge of structure,
function or even mechanism. The basic evolutionary engineering approach outlined
in Fig. 4-1has generated impressive results in a few short years, from enzymes that
function in organic solvents[l4land at high temperature[151 to enzymes that are active
towards non-natural substrates[16]or even carry out whole new reactions[17].It is
now clear that directed evolution will drive biocatalysis into a growing number of
commercial settings, including many synthetic applications.
The aim of this chapter is to explain the concepts underlying directed evolution
and to describe its application to engineering useful enzymes. In Sect. 4.2,we
describe the principles of an evolutionary optimization algorithm. The tools and
their implementation in different working strategies of directed evolution are then
described in Sections 4.3and 4.4.The intention is to highlight the main practical and
conceptual differences among the various approaches and to compare their
strengths and limitations. Section 4.5 discusses specific examples of directed
evolution, with a focus on enzymes and properties that are of interest in organic
syntheses. Many other important and highly successful applications of directed
evolution, such as the design of catalytic antibodies and nucleic acids (ribozymes)or
peptides and proteins of pharmaceutical interest, are covered in recent re-
views [13, 18-26]
4.2
Evolution as an Optimizing Process
Without an understanding of the theory of evolution, one may be tempted to

consider in vitro evolution an irrational, trial-and-error approach to protein design.
However, the beauty of the structural architectures and sophisticated functions that
nature has created attest to the power of the evolutionary design strategy. Many
theoretical studies of evolution explain this process based on physical principles. The
4.2 Evolution as an Optimizing Process
Choose parent gene@)

I 97
I
t
/
P
\ -
Create diversity by mutation / recombination
-
-7
Isolate gene(s) 1 , -
Couple genotype to phenotype
e.g. by expression of the genes
z
\
a
in recombinant cells
f
Screen or select for

the property of interest
Figure 4-1. Evolutionary enzyme optimization. One or several parent
genes are chosen and subjected to mutagenesis and/or recombination.
The mutant gene library (genotype) is then expressed in vivo (or in vitro)
where it is linked to the enzyme produced (phenotype). These enzymes
are tested for the targeted property by screening or selection. DNA from
the most fit clone(s) is isolated and subjected to a new cycle of mutage-
nesi:; and screening or selection. This procedure is repeated until the
desired goal is reached or until no further improvements are observed.
principles that emerge are very different from those important in traditional
“rational” design. Rather than trying to fully understand how mutations affect the
structure and function of the enzyme (which is very difficult), the physics of
evolution aims to understand the forces that make systems and problems evolvable.
That is, what makes proteins so apt for evolution? Moreover, how can this be used to
advantage in enzyme desi-gn?
4.2.1
The Search Space of Cherriical Solutions
To describe evolution as a search process, it is necessary to define the search space. It

is convenient to define sequence space as the connected network of all possible
amino acid combinations (for a fixed sequence length) [27].For a protein composed of
A different amino acids and a sequence length of N residues, there are N sequences,
connected by an N(A-1)-dimensionalnetwork. Each point in this vast space has an
associated fitness, representing the combination of properties undergoing selection.
Together, sequence space and a fitness description construct a fitness landscape on
which an enzyme walks towards higher peaks under the influence of mutation and
selection [28-301.
4 Enzyme Engineering by Directed Evolution
98
I
Exhaustively searching all possible solutions is impossible as sequence space is
extraordinarily large. The mass of all amino acid combinations for 285 residues
(about possible sequences) would be lo3’’ times the mass of the universe, thus
creating sequence spaces of a size greater than the power of our
Nature could have explored only a small fraction of the sequence space of proteins
during the age of earth. Nevertheless, excellent solutions to biological and environ-
mental challenges have been found. Similarly, i n uitro evolution experiments have
been successful in finding improved molecules, although a priori success probabili-
ties may seem to be prohibitively small. There are many successful examples where
only a very small fraction of the many possible sequences have to be explored to find
mutants with improved properties. The key is to develop experimental algorithms
that optimize exploration subject to practical limitations.
4.2.2
The Directed Evolution Algorithm
Spontaneous mutations, recombination and selection are the tools of evolutionary

design. Plant and animal breeders who influence properties of the offspring by
choosing parents with the desired traits have been successfully using these tools for
millennia. Following their lead, molecular biologists first employed selection strate-
gies that acted on spontaneously generated bacterial mutants with the goal of
developing new metabolic pathways 132, 331. Solutions found by these approaches
often resulted from complicated changes in regulatory genes, transport proteins, or
the activation of silent genes. Whole pathways are targeted by the evolution
experiment when such solutions are desired. However, if the target is a specific
enzyme, it is not desirable to produce solutions that are not directly related to the
enzyme.
The milestone techniques of cloning and i n uitro recombination of genetic
information[34]and other advances in molecular biology, such as the development of
the polymerase chain reaction (PCR) 135], allow carefully controlled directed evolution
experiments. Researchers are now able to specifically engineer the enzyme of
interest and control the rate of mutagenesis and focus mutations towards specific
regions within the gene. Furthermore, methods are now available to reconstruct
“sexual” recombination i n vitr~[36-381 as well as in recombinant 401. In
addition, using screening and in uitro selection methods, we can control the selection
pressure independently of constraints in living cells, thus allowing acquisition of
properties never required in nature. Technologies are available to create protein
libraries of up to molecules and select them within a few hours or days[41].
All of these tools allow us to implement the evolutionary design algorithm i n uitro
and accelerate it to create molecules with desired properties in a fraction of the time-
scale of natural evolution. The success of evolutionary protein design is highly
dependent on the thoughtful combination of methods for creating diversity and
searching the mutant population that is generated. Optimizing an evolutionary
search has been well studied in the computer science genetic algorithm lit-
erature [42, 431. Some of these results can be applied to directed evolution, including
4.3 Creating a Library ofDiverse Solutions
I 99
determining appropriate mutation and recombination rates, optimal recombination
parameters, and the appropriate screening effort
4.3
Creating a Library of Diverse Solutions
Given the high cost (both in terms of money and time) of analyzing a mutant library,
the goal of the diversity-cn?atingstep is to produce mutant libraries that are rich in
variants with improved properties. To achieve this, the few positive mutations that
might occur on a gene cannot be diluted with many neutral or deleterious mutations.
The level of mutant redundancy also affects the quality of the molecular diversity.
Redundancy must be low because screening or selection efforts are wasted on testing
identical mutants. In this section, we will first describe different approaches to
creating mutant libraries, including mutation and recombination.
4.3.1
Mutagenesis
A commonly used strategy to create mutant libraries is to target the whole gene for
random point mutagenesis. Nucleotide mutations are typically introduced by error-
prone PCR, mutator strains, or by treatment of the isolated DNA with chemicals or
UV light. The success of this approach depends critically on using an appropriate
error rate. If the error rate is too low, inadequate diversity is created and screening is
wasted on large numbers of redundant parent enzymes. On the other hand, if the
error rate is too large, the haction of positive mutants also becomes very low and the
search for improved mutants is wasted on screening inactive clones.
A serious limitation of the random mutagenesis approach comes from the
degeneracy of the genetic code and the biases of available methods, for example the
preference for transitions over transversions. Together, these effects limit the amino
acid substitutions that arc accessible by DNA point mutations. A combination of a
stepwise random mutagenesis approach with methods of focused mutagenesis and
recombination can circumvent some of these limitations. The different require-
ments, limitations, and advantages of the most commonly employed methods are
summarized in Table 4-1.In practice, a good strategy is to use a combination of
methods.
4.3.1.1
Random Point Mutagenesis of Whole Genes
Before the introduction ofthe polymerase chain reaction (PCR)[351, point mutations
were usually produced by UV radiation, by chemical treatmentL4’1 or by using
mutator strains that have an increased mutation rate compared to normal strains
because of defects in their DNA-repair mechanisms [461. Chemical mutagenesis [471,
mutator strains 491, and even spontaneous mutations coupled with selection in a
[483
100
Table 4-1. Comparison of methods for creating genetic diversity for directed evolution.
Requirement Advantage Limitation
Random point mu- None Exhaustive No multiple simultane-
tagenesis ous mutations; re-
quires multiple rounds
to accumulate benefi-
cial mutations
Focused mutagene- Structural informa- Reduced library Misses possible
sis tion or knowledge size; multiple simul- good sites
from previous gen- taneous mutations
erations possible
Recombination
- single gene None Recombine positive No multiple simultane-
mutations; remove ous mutations: recre-
neutral and deleteri- ates large number of
ous ones already known se-
quence
- family shuming Homologous genes “Functional diversi- Not exhaustive: limited
ty”: large jumps in to amino acid diversity
sequence space in parental sequence
space
chemostat are still used for random point mutagenesis in directed

evolution experiments. However, the dominant method is now error-prone PCR
because the protocols are very straightforward, safe, fast and versatile, and allow
simple adjustment of the error level. Point mutations are introduced by PCR because
of erroneous incorporation of a nucleotide during the amplification of the target
gene. Under normal reaction conditions, the error rate of the Tuq DNA polymerase is
about 0.001 % to 0.02 % per nucleotide per replication 521. Although this
error rate is sufficient to create libraries of large genes lS31, it is too low for efficient
mutagenesis of small genes. However, the fidelity of the PCR can be reduced by
changing the reaction conditions (e.g., increasing the concentration of MgC12,
adding MnCl2 to the reaction mixture, increasing and unbalancing the concentra-
tions of the four dNTPs, adding deoxyinosine triphosphate (dITP), increasing the
concentration of Tuq polymerase, or increasing the extension time and cycle
numbers [54-581. These methods can result in error rates as high as 2 % per nucleotide
position. The possibility of using of low fidelity Tuq mutants has been described[59],
but has yet to be explored experimentally.
By changing some of the PCR conditions, the error rate can be adjusted according
to the gene length to produce the desired average number of amino acid substitu-
tions. A frequently used method to estimate the level of mutation (a quality check of
the diversity) is to determine the fraction of inactive clones from small samplings of
the generated mutant libraries L60, 61]. However, the statistical distribution of muta-
4.3 Creating a Library ofDiuerse Solutions I 101
tions (which should be narrow) cannot be estimated by this method, and the relation
between the fraction of inactive clones and average number of amino acid substitu-
tions can differ for different enzymes. Therefore, the statistical distribution of
mutations and the relationship between inactive clones and the number of muta-
tions are determined by sequencing randomly picked mutants.
Another consideration I S the distribution of mutation type. Typically, there is a
strong bias for transitions (A-G or T+C) over transversions (C-G or G+C), which
limits the accessible amino acid substitutions. There are protocols to reduce this
bias, but they do not completely eliminate it[55, 62]. In addition, the structure of
3' '
the genetic code limits the accessible amino acid substitutions. Depending on the
specific codons, only 2 4 4 0 % of the possible amino acid changes are accessible by
single base substitutions 631. Furthermore, the accessible substitutions are more
likely to be conservative with similar physicochemical properties. For large genes
and small error rates in whole-gene mutagenesis, it is very unlikely that two DNA
mutations will occur in tkie same codon, dramatically reducing the possible amino
acid substitutions.
Although little is known of the cost of these constraints in directed (and natural)
evolution, several studies have shown that the best mutations at specific sites
required multiple substitutions in a single ~ o d o n [651.~ ~Methods
, that introduce
diversity at the codon level might therefore be preferable to methods that create point
mutations at the nucleotide level [631. Methods available for codon-level mutagenesis
of a few amino acid positims are unfortunately very cumbersome and expensive for
mutagenizing whole genes, leaving room for future developments of improved
mutagenesis methods.
4.3.1.1.1 Optimal Mutation Rates: Experimental

One rule-of-thumb has been to adjust the error rate according to the number of
targeted amino acid residues and the size of the screen, such that a significant
fraction of the total number of combinatorial possibilities can be sampled["]. For an
enzyme of 300 amino acid residues, there are about six thousand possible single
mutants, sixteen million double mutants, and thirty billion triple mutants. It is
generally within our abi1ii.y to exhaustively screen a single-mutation library, whereas
double-mutation libraries already exceed the throughput of most screening methods
(typically < lo6). Standard selection methods allow a throughput of about lo8
mutants and can therefore sample most double mutants. In vitro selection methods
might push this limit somewhat higher (10"). The assertion that it is desired to
sample most of the conibinatorial possibilities is based on the assumption that
beneficial mutations are rare and the probability is very small that multiple random
amino acid substitutions are beneficial.
An error rate resulting in single or double mutation libraries has been found to be
a good compromise between creating adequate diversity while limiting the screening
effort. In practice (e.g., Table 4-3), significant improvements in enzyme activity,
stability, selectivity, folding, and expression have been achieved by the stepwise
accumulation of single amino acid substitutions. Even large changes in substrate
102
specificity[621and inversion of enantioselectivity["I can be achieved by this con-

servative approach. The obvious disadvantage of this approach is that some proper-
ties will benefit from simultaneous mutation of multiple amino acids. These
solutions will not be found.
Arguments for low error rate mutagenesis were recently challenged by several
researchers. Zaccolo and Gheradi produced p-lactamase libraries with error rates
that they claim generated 5-16 amino acid changes per mutant enzyme per
generation [67]. The libraries (- 104-105 clones) were selected for increased resistance
to the antibiotic cefotaxime over three generations. However, the best mutant
differed in only three effective amino acid substitutions from wild-type and is
therefore much less mutated than would be expected from three generations of 25
amino acid changes per generation. This indicates that the applied average error rate
was actually much lower, and a more suitable average mutation rate would have been
three amino acid substitutions. Another recent study by Georgiou and coworkers
indicates that a high mutation rate was appropriate in improving the affinity of a
single-chain antibodyr6*].When screening for improved affinity, they found that the
most improved mutants were observed in libraries created with moderate to high
mutation rates (3.8-22.5 mutations/gene). In the next section, we discuss the
circumstances under which a higher mutation rate is advantageous.
4.3.1.1.2 Optimal Mutation Rates: Theory

In nature, the spontaneous mutation rate is tightly controlled. During one replica-
tion round, about 3 ~ 1 0 -mutations
~ occur in the genome of human cells, which is
1 . viruses, such as HIV, have a much
about the same level as in Escherichia c 0 l i [ ~ ~RNA
higher mutation rate, typically on the order of one mutation per genome per
replication Such a high mutation rate is crucial for the viruses to survive
the attacks of the immune system. However, there is a maximum mutation rate,
above which the requirement of inheritance for evolutionary optimization breaks
down (referred to in quasi-species theory as the error At the mutation
rate just before this threshold, the speed of evolution is highestL3. 721. Interestingly,it
was demonstrated that the mutation rate of the replication machinery of fast
evolving RNA viruses is indeed close to this error For directed
evolution of enzymes, the optimal mutation rate maximizes the speed of the adaptive
walk and is influenced by the number of mutants that can be screened and the
structure of the fitness landscape. As discussed in the previous section, the general
rule has been to use a mutation rate for which the permutations can be effectively
sampled during screening. However, if the fitness landscape is amenable to a high
mutation rate, fewer mutants must be screened in order to achieve the benefit of a
higher mutation rate. For example, it would require a smaller library than all double
mutant permutations to benefit from a mutation rate of two per sequence. In this
section, we explore how the optimal mutation rate is influenced by the number of
mutants that can be sampled, the fitness of the parents, and the ruggedness of the
fitness landscape.
The optimal mutation rate depends on fitness of the parental sequence. For a sub-
4.3 Creating a Library of Diverse Solutions
optimal sequence, a large mutation rate allows a greater sweep of sequence space.
However, because the probability of finding improved mutations decreases as the
fitness of the sequence increases, adaptation via a large mutagenesis rate is rapid at
first, then slows. If the parent is highly optimized, the probability that a mutation is
deleterious is higher. The accumulation of deleterious mutations is more rapid and
these mutations quickly (erode the few positive mutations that occur. By using a
Markov chain analysis to study genetic algorithms, Miihlenbein found that there
should be approximately one amino acid substitution per sequence for highly
optimized sequences [741. ]His analysis also suggested that that the optimal mutation
rate should decrease as the fitness of the parent increases. In several independent
studies, it was demonstrai ed that the solution of an evolutionary search is improved
whm the mutation rate was decreased over time[75-781.
A higher mutation rate dramatically increases the fraction of mutants in the
library that contain stop codons, requiring a larger screening effort[7g].For instance,
if the average number of DNA mutations per gene is five, over 20 % of the resulting
library will contain stop codons. The quality of the mutant library can also be
degraded by the accumulation of deleterious mutations, an effect that is exacerbated
by the landscape rugged~iess[~~~]. For the mutation of a highly coupled residue to
generate a fitness improvement, it is necessary to optimize all the other residues to
which it is coupled. Ideally, the optimal mutation rate equals that of the maximum
number of residues involved in a single coupled interaction, thus assuring that the
sequence will not become trapped in a local optimum. However, the finite number of
mutants that can be screened imposes an upper limit on the mutation rate.
Therefore, the optimal mutation rate decreases as the landscape ruggedness in-
creases. This observation is similar to the long-jump mutagenesis strategy suggested
by KauffmanIs0I. By making moves that are larger than the correlation length
(smoother landscapes have larger correlation landscapes), more space can be
explored. Quasi-species theory also predicts that smoother landscapes have higher
optimal mutation rates [811.
Because real protein fitness landscapes are undoubtedly highly anisotropic, they
contain many correlation lengths, and different regions of the sequence will have
different optimal mutation rates 82]. A highly coupled region (such as the catalytic
site) has a small correlatlon length; thus a smaller mutation rate is allowed with a
limited mutant library. Based on some simplified simulations, it was found that the
probability of picking a mutant that has a highly coupled mutation decreases
significantly as the sequt-nce increases in fitness iU]. This effect intensifies as the
number of interactions that are coupled to the mutated residue increases. From this
observation, it follows that when the screening effort is limited, uncoupled regions
of the protein should be targeted for mutation. More highly coupled residues require
a larger rearrangement of amino acids than is likely given the limited mutation rate.
Avoiding the regions of high coupling decreases the total number of residues
undergoing mutagenesis. To utilize this observation, it is necessary to have experi-
mental techniques to target specific positions as well as methods that can be used to
predetermine the coupling of each residue. These goals are the subject of the
following two sections.
104
4.3.1.2
Focused Mutagenesis
Focused mutagenesis strategies are used with the intention of enriching a library for
desired mutants. To reduce screening efforts [83-851, the targeted region can be
reduced from 300 to only a few residues (Fig. 4-2). The library of quintuple mutants
has a theoretical size of only -lo6 mutants, compared to 10“ if the entire gene is
targeted. This reduced library can be searched exhaustively with currently available
methods. Focused mutagenesis significantly reduces screening requirements for
libraries of mutants with multiple amino acid substitutions and eliminates the
codon bias of PCR. However, it imposes obvious limitations on the possible
solutions and can fail to explore the most effective mutations.
Targeting single amino acids (“saturation mutagenesis”) is straightforward be-
cause of available strategies that eliminate laborious subcloning steps. Several
commercial kits are available, such as the TransformerTM(CLONTECH Laborato-
ries, Palo Alto, CA, USA), Altered Site@ I1 (Promega, Madison, WI, USA), and
QuickChangeTM(Stratagene, La Jolla,CA, USA) site-directed mutagenesis systems,
which can produce targeted mutant libraries in one day. This approach has been
used to target amino acid positions that random point mutagenesis identified as
important for the targeted enzyme properties [64. 651. Variants with improved proper-
number of simultaneously changed amino acids

Figure 4-2. Plot showing the number of possible protein variants (v) that can be
created given the number of amino acid positions ( M ) changed simultaneously and
the sequence length ( N ) (Y=lg’[N!/(N-M)!M!]). From a given standard screening
throughput (<lo6 clones) one can calculate that it is not possible t o exhaustively
screen protein libraries of all possible double mutants o f an average sized protein.
I 105
ties were identified in saturation mutagenesis libraries that contained mutations not
accessible by random point mutagenesis.
Methods for randomization of specific regions of a gene typically employ random-
ized oligonucleotides or PCR mutagenesis of small stretches of the gene. The
required diversity is introduced by randomized oligonucleotides that are produced
by an automatic (programmable) DNA synthesizer. If only one amino acid position
is targeted, the specific codon can be completely randomized (saturated) by adding
an equal amount of all four bases (A, T, G ,C) during the oligonucleotidesynthesis at
the first two positions of the triplet and with a mixture of G and C at the third
position to exclude stop codons (referred to as NN(G,C)-mutagenesis).
To target multiple amirto acid positions simultaneously within a small region of
the gene, oligonucleotide-cassette mutagenesis is often used. This method was
among the first applied for in vitro evolution of DNA sequences [“I and has also been
used successfully for the evolution of various enzymesL5’, 87-901. When suitable
restriction sites are not already present, they are introduced by site-directedmutage-
nesis adjacent to the targeted region. These restriction sites are then used to
substitute the wild-type region with the synthetic randomized DNA duplex (cas-
sette) [911. Depending on the number of targeted amino acid positions, randomiza-
tion strategies are used to facilitate an exhaustive search of the library. Complete
randomization of all targeted codons is preferred if few amino acid residues are
targeted and can be done as described above for the randomization of single sites by
using NN(C/G)during oligonucleotide synthesis. It was also reported that trinudeo-
tide analogs such as 9-fluorenylmethoxycarbonyl (Fmoc) trinucleotide phosphor-
amidites can be used during DNA synthesis to achieve a codon-based mutagene-
sis 1921.
If several distant positions are targeted simultaneously, cassette mutagenesis is
technically cumbersome. Other methods allow the efficient assembly of several
randomized oligonucleotides to whole genes. For example, recursive PCR1931,the
ligase chain reaction[”, ‘)41, or in uitro assembly of whole genes[”] can be used to
construct targeted mutant libraries. Partial randomization of many positions most
frequently employs spiked oligonucleotides,which are produced by DNA synthesis
using a mixture of the wild-type base and equimolar amounts of all four
The oligonucleotides can also be produced by error-prone PCR. It is possible to
calculate the appropriate compositions of the nucleotide mixture in order to encode
whole sets of amino acid!; at certain
4.3.1.3
Calculation of Mutagenesis Hot-Spots
In focused mutagenesis experiments, the challenge is to identify the residues where

mutagenesis is likely to be beneficial. Indeed, many successful directed evolution
experiments show that mutations occur in regions that would be hard or impossible
to predict (and difficult to explain that they do), even when a high-resolution
structure and much information about the enzyme is available[14,9s1001 . 0ne
possibility is to make use of knowledge gained from early rounds of random point
106
mutagenesis that targeted the whole enzyme[", 101, lo21. The content of the mutant
library can be improved by only mutating sites that do not severely disrupt stability. A
structurally tolerant protein allows more mutations, and therefore more potentially
beneficial ones, making it more likely that there is a connected path in sequence
space of single mutations that leads to regions of higher fitness. By reducing the
evolutionary search to regions of sequence space that retain structure, functional
space can be explored more This concept can also be inverted: if the
goal is to improve stability while retaining functionality, then eliminating the
sequence space inconsistent with the function improves the search.
Several groups have proposed targeting mutagenesis to residues where natural
diversity is observed. Fersht and coworkers reengineered the tumor suppressor p53
by creating a small library of mutants where the hot-spots were determined from a
sequence alignment of 23 homologous proteins [1031. The mutations were made in
the wild-type sequence background, and several were found that improved stability.
Using a similar methodology, Lehmann et al. constructed a thermostable phytase
from the consensus sequence of 13 homologous The mutant phytase
exhibited a 15-22 "C increase in melting temperature.
Alanine scanning has been widely used to identify the residues which are
contributing to various protein properties L105, lo6].Alanine substitutions are made at
various positions and the perturbation in the property of interest is measured. This
has several potential applications to directed evolution. For instance, it can be used to
predetermine which positions are essential to the structure (or function) of the
protein and therefore should be avoided. Conversely, positions that tolerate the
alanine substitutions may be good candidates for saturation mutagenesis. Un-
fortunately, this procedure is tedious. To surmount this difficulty, Kollman and
coworkers proposed a method to determine the effects of alanine substitutions
c~mputationally['~~]. Kollman's method could be used to scan the protein structure
for positions to mutagenize in directed evolution.
The observation that some sequence positions are more tolerant to mutation
initiated the application of information theory to studying the importance of these
residues to structure and function["]. The sequence entropy can be calculated from
the probability distribution of allowed amino acids substitutions at each resi-
due [108-1101 . u sing simulations of evolution on fitness landscapes, Voigt et al.
predicted that beneficial mutations are found by directed evolution at amino acids
that are largely uncoupled to other sites (Figure 4.9) 1441. To test this prediction, they
compared the calculated site entropies with mutations found from previous evolu-
tion experiments on subtilisin E and T4 lysozyme. The sequence space considered in
the subtilisin E computation was enormous: amino acid combinations (274
residues). Seven out of the nine mutations that improved the thermostability of
subtilisin E occur at positions computed to be highly tolerant. Mutations that
improved activity in organic solvent similarly occurred at high-entropy positions.
This calculation may be used to determine the positions where improvement will
likely be found in an evolution experiment.
4.3 Creating a Librmy of Diverse Solutions
I
107
4.3.2
Recombination
Another approach to creating genetic diversity is based on DNA recombination.

Multiple positive variants are used to parent the next generation, which allows for
recombination of beneficial mutations, elimination of deleterious mutations as well
as creation of new diversity. Using recombination also has a practical advantage.
More than one mutant may show improved fitness during a single screening effort.
Allowing only the fittest mutant to continue to the next generation can be wasteful
(Fig. 4-3). For highly non.additive (rugged) fitness landscapes, recombining positive
mutations is less certain to be beneficial because combining individually good
,
mutations can have deleterious effects. In this discussion, we focus on the experi-
mental techniques for recombination and describe the theoretical basis for the
optimal parameters.
4.3.2.1
In Vitro Recombination
In vitro recombination of’ DNA, often referred to as DNA shuffling, was introduced
by Stemmer for evolutionary protein design[3G,371. The method is based on recursive
PCR, which allows for whole gene synthesis from several DNA As
outlined in Fig. 4-4, one or several parental genes are cut by enzymatic digestion
using the endonuclease DNase I in the presence of Mg2+.This generates overlapping
DNA fragments that are randomly distributed over the gene. The isolated DNA
fragments are then reassembled in a PCR-like reaction with denaturation, anneal-
ing, and extension steps, during which recombination occurs through the reanneal-
ing of DNA fragments from different parents.
14-.--;A+B+c B, 6-c+B+;
--wt
-6
-C f=:
-wt
---t
1 2 3 1 2 3
Figure4-3. Comparison o f the progress o f evolution for a random mutage-
nesis approach (A) where the best mutant is used as parent for the next
cycle o f mutagenesis and screening and a DNA-recombination approach (B)
where several improved mutants are used as parents for the next genera-
tions. N o t shown here is any additional screening cost associated with
finding several improved variants in each generation.
108
Diversity is created by combining parental mutations and random point mutations

which are introduced at a rate of about 0.7 % because of the intrinsic error rate of Taq
DNA polymerase [371. A high error rate, however, can mask the relationship between
evolved phenotype and combined parental mutations. Using different DNA poly-
merases and substituting Mg2' with Mn2+during DNase I digestion reduces the
mutation rate to as little as 0.05 % r61]. A high-fidelity protocol is also important if
recombination is used to distinguish between functional and non-functional muta-
tions or for structure-function studies of evolved sequences ['"I.
In normal PCR, recombination can also occur at a low rate. This is caused by
incomplete extension of primer during the extension cycle and annealing to a
different template [112-1141. Increasing the recombination efficiency of incomplete
primer extension motivated development of the staggered extension process
(StEP)[1151. The basis of StEP is repeated switching of the template caused by fast
annealing and extension cycles (Fig. 4-5). During each cycle, the growing oligonu-
cleotide can randomly anneal to different templates and thereby combine informa-
tion from different parents. A further method for in uitro DNA recombination is the
random-priming method (RPR)f3'1, which involves production of gene fragments by
annealing and extension of random-sequence primers. The fragments are re-
assembled as in the Stemmer procedure.
StEP, RPR and Stemmer's method were compared based on their recombination
efficiency of truncated GFP genes[''']. The Stemmer method using small fragments
( 4 0 0 bp) and StEP yielded the highest recombination efficiencies. However, the
efficiencies may differ from gene to gene and are highly dependent on the
experimental conditions chosen. Detailed protocols for RPR, StEP and the Stemmer
method are given in [151.
parent genes -2
- = = =
PCR-Iike
.n-_y;
_ ._- _ .
*
assembly ' ~
. -A' Figure 4-4. In uitro recombination by DNA shuf-
fling as described by Stemmer[37].Parent genes
+ carrying mutations (indicated by X) are digested

with DNase I and randomly reassembled in a
cyclic PCR-like reaction t o yield a library of re-
combined genes. New point mutations are in-
troduced (indicated by underlined X) during the
reassembly reaction.
4.3 Creating a Library ofDiuerse Solutions
PO9
Figure 4-5. In uitro recombination by the stag-
gered extension process (StEP) I”’]. Only one
primer and single strands from two parents are
shown. Fast annealing and extension cycles during
F parent genes
PCR of the parent genes cause template switching

of the extending strand. After .full-sized recombined
genes are synthesized, parent genes are removed
by treatment with Dpnl.
template switching
during annealing
and polyrnerizatior
Dpnl digestion of pareni
It is possible to recombine any number of parent genes with the available

methods, which raises the question of what is the optimal number. Similar to
determining the optimal mutation rate for random mutagenesis, the answer will
depend on the number of screened mutants and the additivity of the combined
mutations. It could be advantageous to screen all the permutations of mutations
from the parents. Assuming independent and additive recombination, the probabil-
ity pd that an offspring has d mutations is given by
p d = - T! M-1 T-d
-
(T- d)!d![ h)*[7)
where M is the number of sequences and T is the total number of mutations[1’71.
Unfortunately, only 25 74, of the sequences in the recombination library have novel
combinations of mutations because there is a statistical disadvantage for the
presence of new combinations of mutations. The probability of creating a mutant
that contains all mutations is only (l/M)? If all mutations from four parents (each
having two mutations) are recombined, the probability of creating an offspring gene
with all the parental mutations is only 1.5~10-~. This probability decreases rapidly as
the number of mutations and parent genes increase. The probability is further
reduced if recombination is accompanied by a high error rate. It is clear that
recombination of large pools of sequences quickly reaches the throughput limitation
for available screening or selection methods if the mutant containing all mutations
needs to be sampled.
One strategy for reducing the screening requirements is to divide the recombina-
tion experiment into multiple generations L1l7]. The following example demonstrates
the advantage of this procedure when the goal is to combine eight mutations onto a
single gene from four dtmble-mutant parents. The experiment is divided such that
110
two libraries are created using two parents each. In each library a mutant is created
that contains all four mutations of its parents. The probability of creating this
quadruple mutant is p = (1/2)4 = 1/16. All recombinant mutants at this step can be
sampled by screening only 32 clones (some oversampling will be required). If the
quadruple mutants from each library are then recombined to create a mutant with all
eight mutations, the probability of creating it is (1/2)'. In sum, screening fewer than
300 clones (16 + 16 + 256) would be sufficient,whereas the simultaneous recombina-
tion of all four mutants to create the same mutant in one recombination experiment
would require screening about 65 000 clones. The success of this procedure also
relies on the additivity of the mutations. If some of the mutations are non-additive,
then combining all mutations is not guaranteed to be optimal. In addition, not all
combinations of mutations are screened. If a particular double or triple mutant is the
fittest, it may not be found (see pooling strategies, Sect. 4.2.3). Note that if the
mutations to be combined were discovered in previous generations, then it is likely
that they are relatively additive (or uncoupled, see Mutagenesis Hot-Spots, Sect.
3.1.3).
All of the recombination methods described above require considerable sequence
identity among the parents for crossovers to occur. For example, in the Stemmer
method, the fragments require a minimum nucleotide sequence identity to reanneal
and form an offspring gene. Non-homologous recombination methods seek to
remove the sequence identity restriction from the recombination process. Os-
termeier et al.["'* describe a method based on generation of N- or C-terminal
fragment libraries of two genes by progressive truncation of the coding sequences
with exonuclease 111 followed by ligation of the products to make a single-crossover
hybrid library. An intrinsic problem of this approach is that random ligation of two
DNA fragments results in two-thirds out-of-frame sequences, yielding mostly non-
functional products. In addition, recombination of more than two parents is hard to
realize. Another approach to create a mutant enzyme library is based on the
permutation of modules or secondary structure units[120.l2'1. It is not yet known to
what extent non-homologous recombination generates usefil genetic diversity, rich
in improved functions.
4.3.2.2
In vivo Recombination
In vivo homologous DNA recombination mechanisms are known for various host
cells such as E. c0li1~~1or Saccharomyces c e r e v i ~ i a e [and
~ ~ ] have long been applied in
protein engineering purposes, for example to shuffle mammalian P-450 substrate
specificitiesf9'1. Because of its simplicity, it is an attractive tool for directed evolution
of enzymes['22,1231 . The most common system is based on the ability of S. cerevisiae
to rescue plasmids with a double-bond break by intermolecular homology-depend-
ent re~ombination1'~~I. A plasmid is cut by restriction enzymes and transformed into
yeast together with different genes or fragments thereof. Recombination at homolo-
gous positions on both sites of the gap and within homologous regions of the
fragments yields a functional, self-replicatingcircular plasmid. The reconstitution of
I
the functional plasmid allows for easy detection of recombination events based on
the selection marker of the plasmid. Besides its simplicity, an important feature of in
vivo recombination is that additional point mutations are extremely rare because of
the high fidelity recombination mechanism in yeast. This is particularly advanta-
geous when the aim is solely to recombine positive mutations or eliminate delete-
rious and neutral ones. IYL vivo methods are unlikely, however, to generate as many
crossovers as i n uitro methods.
Volkov et al. described a hybrid in uitro-in vivo recombination method involving
formation of a heteroduplex between two homologous sequences in uitro and
transformation into bacterial cells [1251. Mismatches present in the heteroduplex are
randomly repaired by the host cell, creating recombinant sequences composed of the
elements of each parent. This approach may be particularly useful in recombining
large genes or entire operons.
4.3.2.3
Family Shuffling
Recombination of homo1ogous parent genes, referred to as “family shuffling” or

“molecular breeding,” ini-roduces an additional dimension to creation of molecular
diversity for directed evolution.]‘’1L While random point mutagenesis and local
recombination explores only the sequence space covered by neighboring mutants,
and focused mutagenesis covers only a small fraction of the whole sequence space of
the protein, family shuffling explores more distant regions of the sequence space
with a lower sampling density. Since the diversity is usually created by a combination
of mutations from pareni s that were previously selected in nature to be functional, it
is suggested that family shuffling provides functional diversity that could accelerate
evolution [”‘I.
Several recent studies ,attest to the power of this . Us ing a family
of four genes from different species, a comparison to single-gene recornbination
suggested an evolutionary advantage for family shuffling[”‘]. Recombination of four
cephalosporinase genes (57%-82 % identity in amino acid sequence) and screening
of 5x104 clones yielded $3 mutant with a 540-fold increased moxalactam resistance
compared to wild-type. The mutant that was generated by family shuffling was an
offspring of three out of the four parent sequences and contained 33 new amino acid
substitutions. The large number of new point mutations raises the question of how
so many could be tolerated. It is unclear whether these mutations contribute to the
improved Ij-lactamasefunction.
An important Characteristic of the family shuffling approach is that the diversity
changes after each generation. During early generations, many different combina-
tions of mutations are tested, and the best combination becomes fixed during
subsequent generations After futation, new diversity is created only by additional
random point mutations inherent to the recombination method. Thus, the most
promising distant regions in sequence space are explored by recombination,
followed by step-wise mutagenesis towards the fitness optimum. This hybrid
methodology might take more than the maximum of four generations of recombina-
112
tion that have been used so far in family shuffling Based on studies
of genetic algorithms it was also proposed that the recombination process with
additional mutations provides a powerful method for finding higher fitne~ses1~~~1.
Family shuffling should gain increasing importance with the greater availability of
homologous genes as a result of the rapid accumulation of new sequences in public
databases. The restrictive licensing policies for the known recombination technolo-
gies might be limiting the commercial use of recombination methods at the
moment. However, this situation and technical limitations of the existing methods
are stimulating the development of new and improved recombination methods.
4.4
Finding Improved Enzymes: Screening and Selection
Given a thoughtful strategy to generate a mutant library, developing a method to

detect positive mutants is probably the single most important step determining
success (or failure) of directed evolution. Screening refers to a qualitative or
quantitative assay of each single clone or few pooled clones of a mutant library,
whereas selection refers to methods that enrich positives in a pool of all members of
a mutant library or, even better, allow growth only of desired variants (extensively
reviewed in[133-135]). In directed evolution, diversity is created on the genotype
(DNA) level, whereas screening and selection acts on the phenotype (protein) level. A
physical link between the genotype and phenotype is required because direct amino
acid sequencing of positive mutants is not feasible and because DNA or RNA is
required for replication of the desired mutants as well as for introduction of diversity
in subsequent cycles of evolution. For the evolution of enzymes, the most simple,
versatile and therefore most commonly used approach to couple genotype and
phenotype employs recombinant cells. Genes are introduced (transformed) into cells
and translated into proteins by the cell's natural transcription-translation machinery.
For organisms such as E. coli, Bacillus subtilis, and S. cerevisiae, efficient transforma-
tion protocols are available that allow for the production of reasonably sized mutant
libraries of 105-109different members. In addition, recombinant protein expression
is well established for those organisms, making them the current preferred choices
for directed evolution experiments. In this section, we focus on general principles for
searching mutant libraries and discuss important characteristics of available systems
such as throughput, error level and versatility. We also discuss theoretical approaches
to determining the required screening effort, analyzing the immense amount of data
that are generated during the screening step and the theoretical advantages of
different screening strategies.
4.4 Finding lmproved Enzymes: Screening and Selection
I 113
4.4.1
You Get What You Screen For
The first rule of directed evolution is “you get what you screen for.” The importance
of establishing appropriate screening or selection methods cannot be overestimated.
Corlditions used for screening or selection should be as close as possible to the
conditions where the enzyme is going to be applied. This includes the actual
substrate, its concentration, pH, buffer, salt, temperature, co-solvents, and any other
conditions that affect the enzyme behavior. For example, the screen can be estab-
lished conveniently using an artificial substrate, the enzymatic products of which
produce color or fluorescence. However, optimizing an enzyme on an artificial
substrate bears the risk that gains in performance will disappear on the desired
substrate. This is true for virtually all enzyme behaviors. If compromises in reaction
conditions or substrates cannot be avoided, the risk of obtaining undesired solutions
during several rounds of directed evolution can be reduced by using a secondary
screen under more “real”reaction conditions or by testing the chosen mutant(s)with
the actual substrate after each cycle of directed evolution[’’]. For in vivo selection,
which uses the indirect measurement of cell growth as an indicator of enzyme
performance, one has to he aware of the immense flexibility (and creativity)of living
organisms to circumvenl selection pressures by inventing new solutions unrelated
to the enzyme and its property we wish to optimize. Many such examples are well
known from studies of metabolic acquisitions through laboratory selection, where
strong selective pressure uncovered solutions to biochemical blocks [32*33, 1361.
Directed evolution experiments using combinatorial mutant libraries have also
found complementation that was caused by activating a novel gene locus rather than
by the mutated enzyme[L37]. Starvation under selection conditions may induce low-
fidelity polymerases and speed up the evolution of new s o l ~ t i o n s1[3’1~. ~ ~ ~
4.4.2
Screening Strategies
Screening methods allow enzyme behavior to be measured independently of

biological function. Novel enzyme properties can be explored, such as activity or
stability in unnatural eiivironments (e.g., extreme pH, temperatures, or organic
solvents) or activity on unnatural substrates. These properties are impossible to
target using in vivo selection methods, because of the cell’s inability to survive under
such biologically harsh conditions. The advantage of higher versatility and better
control of the applied selection pressure comes at the cost of lower throughput
(defined as the number of clones that can be tested in a given time period).
Depending on the screening methods, libraries of about 104-10G clones can be
screened within a few days, which is several orders of magnitude less than the
lO’-lO” clones that can be tested with selection methods. High throughput is
usually accompanied by a high error level in the measurement, which dictates the
minimum change that can be detected. On one hand, the consequence of low
throughput is missing r.ire beneficial mutants because they are not sampled. On the
114
other hand, mutants will also be missed if the method cannot resolve the small
fitness difference between a mutant and the parent (Fig. 4-6). Part of this difficulty
stems from the fact that only a few mutations are made at each generation, and it is
often only over multiple generations that large fitness improvements are observed.
It is important to take both the throughput and the error level of the assay into
account when setting up a screen. The intrinsic error level of a screen can be tested
by screening a number of wild-type clones, which allows an estimate to be taken of
the minimal change that can be resolved. Fig. 4-7 shows typical statistics of a screen
using a 96-well microtiter plate pH-indicator assay, specifically applied for the
evolution of a hydantoina~e[~~]. From the standard deviation (in this case, 5 %) and
maximal deviation (20%), one can estimate that mutants differing in more than 50%
activity can be detected with high confidence. Thus, this screening method is
suitable for detecting small improvements in activity in a mutant library.
4.4.2.1
Low-Throughput Screening
Screening in 96- or 384-well plate formats allows precise fitness measurements. The
accuracy of the detection system, kinetic assays (in contrast to end point assays),the
possibility to normalize activity values (e.g., using measured cell concentrations),
and better control over cell growth and protein expression contribute to the improved
>
clone #
6
Figure46 Throughput (sam-
sampling of large library size pling size) versus error level of a
< P screen. Low error level and small
missed mutant sampling size (A) might miss the
*.
0
* .
......
.
.*. 0
.. ...
.
high
best mutant because it is too
rare to be sampled. Large sam-
piing size and high error level (B)
might miss the mutant because
it cannot be distinguished from
______, wild-type background even
clone # though it was sampled
-
4.4 Finding Improved Enzymes: Screening and Selection
I
I
115
Figure 4-7. Statistics of a experimental data
typical screen using a 96-well gaussian distribution
50
6o
h
microtiter plate pH-indicator
assay which was applied for 0 40
the evolution of a hydantoi-
c standard deviation: 5%
~ ] . # clones = 192.
n a ~ e l ~Total 2D 30 maximal deviation: 20%
The experimental data roughly 2 20
follow a Gaussian distribution. \c
10
0
0.8 0.9 1.0 1.1 1.2 1.3
activity
accuracy of microtiter plate-based screening systems compared to colony-based

screens. Semi-quantitatiw visual screens are usually based on fluorescence[140k1421,
color formation[16,1431, or formation of clear zones (halos)[144, 14’1 of colonies grown
on agar plates (often filter membranes). The time-consuming process of gridding
transformed colonies into microtiter pates is not required. Further, sample prepara-
tion steps are straightforward (and sometimes not necessary at all), which simplifies
and accelerates the screening process. If coupled to digital imaging analysis, visual
screens can be used for 14’1. However, the high throughput and
simplicity of these methods are often balanced by a larger error. Standard analybc
systems such as HPLC, GC or capillary electrophoresis allow very sensitive and
precise measurement and are highly versatile. However, throughput is very limited
to fewer than lo3 samples per week. This is too low for efficient screening of most
mutant enzyme libraries. Further developments, such as integration of HPLC, GC,
or capillary electrophoresis into automatic liquid handling systems, coupling of
mass detection systems, and parallel separations will increase the throughput.
4.4.2.2
High-Throughput Screening
Complete automatic systems are now available that can screen up to a thousand
96-well plates per day (about lo6 samples per week). Systems have been developed to
increase the density of the plates (number of samples per area) by reducing the
required sample volume[’48,14’1. This reduces the cost of screening each mutant and
increases the throughput of the assays. In screening enzyme libraries, the through-
put seems to be more limited by the required step to transfer single clones from agar
plates into the arrays of microtiter plates or silicon wafers rather than by the assay
step. Robotic systems are sometimes used to speed up this transfer. An alternativeto
this step might be a dilution that adjusts cell density to one cell within a certain
liquid volume, which could be transferred into adequate plates (or onto silicon
wafers) much faster. Although the theoretical throughput would be increased, the
statistical problem arises. that a huge fraction of the transferred volume would be
empty or contain more than a single cell. Other ultra-high-throughput systems that
116
can directly analyze single cells or single proteins do not require a transfer step and
thus have a potentially higher throughput. Confocal fluorescence coincidence
analysis (CFCS) can analyze up to lo7 single molecules or cells per week[”0].
Although the reported sensitivity (<lo-” mM can be detected by regular FCS [1511)
and throughput are impressive, applications for directed evolution of enzymes have
not yet been reported. A fluorescence-activated cell sorter (FACS) can be used to
analyze as many as lo’ mutants per week. It is currently the only available screening
tool with a sufficiently high throughput to exhaustivelyscreen mutant libraries for all
possible double mutants in less than a week. So far, most reported applications of
evolutionary protein design using FACS to screen and sort mutant libraries have
been for binding molecules 921. Joo et al. describe a plate-based fluorescence
digital imaging screen with a throughput of -10’ clones per day.
4.4.2.3
Choosing Low versus High Throughput
In this section, we will discuss the critical issues in deciding the balance between
throughput and accuracy in the screen. A minimal screening requirement can be
roughly estimated from the frequency of positive mutants found either in earlier
rounds of directed evolution or from results reported in literature. The frequency of
positive mutants for different enzymes and different properties varies, but is usually
found in the range of about one out of lo2 to lo’ mutants (Table 4-2). However, it
should be noted that the frequency of positive mutants will strongly depend on the
fitness of the parent the property, and the strategy chosen to create the mutant
library.
A theoretical approach to determine the required number of screened mutants is
based on the landscape paradigm. Following this paradigm, several studies have
shown that, when the landscape is additive, the number of mutants that need to be
screened in order to find fitness improvements increases linearly as the wild-type
sequence increases in fitness 12’, 15*1. However, as the landscape ruggedness in-
creases, the number of fitter neighbors decreases more rapidly as the sequence
becomes 159, 160]. Thus, in order to discover improved mutants, the
number of mutants screened has to increase more rapidly on rugged landscapes.
This implies that a protein that is tolerant (corresponding to a smooth landscape) can
undergo more rounds of mutation and improvement.
There is a tradeoff between generating large libraries for a few generations and
generating small libraries for many generations. In other words, if the total number
of mutants that can be screened is fmed, what is the optimal number of generations?
While the improvement in fitness increases with the size of the screening library, the
benefit of accumulating stepwise positive mutations over multiple generations is
compromised. Both experimental and theoretical studies have suggested that the
best method may be short, adaptive walks utilizing small “‘1 . Hu simi
‘
and Aita further studied the effect of the screening cost on the optimal search
strategy[78].
They found that screening multiple generations of small libraries has the
advantage of evolving more rapidly; however, it has a greater potential of being
Table 4-2.
4.4 Finding Improved Enzymes: Screening and Selection
Frequencies of positive mutants found during directed evolution studies.

I
117
Calculation is based on reported mutants and might not represent all positive
mutants. Actual frequencies rnay be higher.
Frequency o f positive mu- Evolved property / rnutagenesis procedure /test Reference
tants
(tested/positive)
3 ~ 1 0 -(1500/5)
~ Tiermostability / PCR / screen
5 ~ 1 0 (2000/1)
-~ Tierrnostability / family shuffling / screen
2xlO-' (1.5x106/34) Therrnostability / chemical / selection
3x10-' (6.4x104/2) Thermal and oxidative stability / PCR / screen
3 ~ 1 0 (103/3)
-~ Activity in organic solvent / PCR / screen
2xlO-' (2x106/35) Activity at elevated temperature / PCR / screen
4 ~ 1 0 (2x105/7)
-~ Activity / cassette rnutagenesis / selection
I x ~ O -(104/124)
~ Activity / family shuffling / screen
3 ~ 1 0 (300/1)
-~ Activity in organic solvent / PCR
2xlO-' (1.7x103/4) Activity / family shuffling
~ X I O - ' (1.2x104/1) Functional expression / PCR / screen
1x 10-2 (10~112) Enantioselectivity/ PCR /screen
1x10-3 (75o/i) Enantioselectivity/ rnutator strain / screen
4xlO-* (150/6) Substrate specificity / focused rnutagenesis / screen
-
trapped in a local optimum. If the cost of screening a mutant is high, then the walk
should tend towards many generations of small libraries, whereas, if it is low, it
should tend towards fewer generations of large libraries. A powerful strategy for
balancing the limitations of throughput and sensitivity is tiered screening (Fig. 4-8).
In this method, a series of screening or selection methods with decreasing through-
put and increasing accuracy are combined. This strategy has been used successfully
for the evolution of subtilisin towards a combination of and for the
evolution of an esterase towards increased enantioselectivity[491.
A computational method, such as the entropy calculation presented in Sect.
4.3.1.3,can be used to eliininate the portions of sequence space where improvement
is unlikely (Figure 4.9)[82 . Pre-screening drastically reduces the number of mutants
that have to be experimeritally screened.
Another approach to increasing throughput is to use a pooling ~trategy["~-'~~I.
This methodology is coiiceptually equivalent to the recombination strategy pre-
sented in Section 4.3.2, in which the recombination load is subdivided into multiple
generations, thus reducing the required screening effort. Most screens, however, are
not sufficiently sensitive to use a pooling strategy to find small improvements.
4.4.2.4
Analyzing the Mutant Fitn'ess Distribution
During the screening, a large amount of fitness data is generated, but only the fitness
information of the improved mutants is used to continue to the next round of
evolution. The large ensemble of less fit mutants provides a view of the local fitness
landscape. By analyzing these data, certain statistical landscape parameters can be
118
FACS, selection Figure 4-8. Strategy o f tiered screening. Series of

screening or selection methods with decreasing
throughput and increasing accuracy are com-
bined.
+
visual screening, microtiter plates
deduced, such as the fitness landscape ruggedness, which can then be used to guide
in setting evolutionary parameters. In this analysis, sequencing is time-consuming
and expensive, so a sequence cannot be assigned to each measured fitness. The lack
of sequencing data means that only the probability distribution of mutant fitnesses
can be analyzed. In this section, we discuss some methods that have been developed
to extract useful information from the mutant fitness distribution.
Several theoretical approaches based on the additivity of mutations have been
proposed to analyze the screening data. Urabe and coworkers developed a model that
captures additive and non-additive mutational effects in directed evolution and fit
their theoretical model to the experimental fitness distribution of catalase I. By
investigating the degree of non-additivity of specific properties, they tuned the
parameters of the experiment to suit the fitness landscape['"]. In a similar approach,
Aita and Husimi proposed that the additive model can be applied to give a rough
estimate for the Hamming distance from the wild-type to the optimum, the fitness
slope near the wild-type, and the height of the optimum['58].They calculated the
expected fitness distribution and compared this to experimental data produced by
the mutagenesis of a region of E. coli lac promoter. Based on a fit between the
theoretical and experimental distributions, they estimated that the Hamming
distance between the wild-type lac promoter and the optimum is 7-10 nucleotide
substitutions and the activity could be improved 100-to 1000-fold.
Mean-field theory can be used to predict the effects of mutation rate, landscape
ruggedness, and parental fitness on the moments of the mutant fitness distribu-
t i ~ n [ ~ 'In
] . this analysis, only the portion of the mutant distribution that is not dead
(zero fitness) or parent (unmutated) is considered. The mutant effects are averaged
over the transition probabilities. In order to obtain the fitness distribution, two sets
of probabilities are required: (1) the probabilities Pi('i(a)that a particular amino acid
identity a exists at a residue i, and (2) the transition probabilities that one amino acid
4.4 Finding lmproued Enzymes: Screening and Selection
will mutate into another, la+,. The probabilities PG(a)can be determined through a
I 119
mean-field approach and the probabilities qa-b are calculated based on the genetic
code["', 166168]. Using the mean-field solution, the change in the mutant fitness
distribution is captured as the sequence ascends the fitness landscape. By increasing
the coupling interactions between residues, the effect of the landscape ruggedness
on the moments is calculated. As the fitness of the wild-type increases, the first and
second moments increas'e (Fig. 4-9). In other words, as the sequence ascends the
fitness landscape, the mutant distribution spreads out (difises) and becomes
skewed towards less-fit rnutants (drifts). In addition, the dependence of the mo-
ments on mutation rate can be predicted. As the mutation rate increases, both the
drift and the diffusion of mutants from the parent increases. Because rugged
landscapes have less correlation between parent and offspring fitnesses, the drift-
diffusion effect becomes exaggerated as the coupling between residues increases.
Through this approach, ii may be possible to model the mutant fitness distribution
to experimentally obtain :#tatisticalparameters that describe the fitness landscape.
4.4.3
Selection and Methods to ILink Genotype with Phenotype
The advantage of high throughput, together with minimal experimental and techni-
cal effort, makes selection an attractive tool for in vitro evolution. The most
commonly used in vivo selection approach links a targeted enzyme property to cell
growth through their contribution to resistance[36,37, 88, 126, 1691 or complementa-
tion of auxotrophy or genetic defects that block the metabolism of a host
strain[", 89. 90. 1"'. Most of these systems allow cells with improved enzymatic
properties to grow, while cells with wild-type properties do not. Such systems are
especially powerful if the selection pressure can be adjusted as the evolution
progresses, for example, by increasing antibiotic concentrations [36, 371, decreasing
substrate concentrations or changing the enzyme expression level [ 6 2 ] .
If the growth of cells with wild-type activity cannot be prevented and positive
mutants contribute only to an increased growth rate, cells with improved variants are
usually enriched by continuous culture techniques, dilution series, or detection of
the size of tested colonies. A problem for selection arises if enzymes are secreted into
the culture medium. This problem was avoided in one case by growing cells in
hollow fibers that limit (cross-feedingbetween neighboring colonies [1701. Another
limitation of in vivo selection methods is that selection conditions must be compat-
ible with the requirements of the host organisms, which are often very different from
conditions under which the enzyme is going to be applied. This is particularly true if
the enzyme is to be use'd in an industrial reactor, where conditions often involve
extreme temperatures and the substrates are suspended in organic solvents. It might
be advantageous to use (other expression hosts that grow under different reaction
conditions than the commonly used organisms (E. coli, S. cerevisiae or B. subtilis).
Thermophilic hosts with reasonable transformation efficiencies, such as Bacillus
steiarothemophilus or Tetrahymena themophilus, have been used for selection for
increased thermostability by growth at elevated temperatures [171-1731. Other in vivo
120
A B
"*"I-
0 05 1 1.5 2 25
Si
Figure4-9. A histogram o f t h e residue entro- at a residue, then s; = 0.0 (marked by the arrow).
pies for the structure o f subtilisin EIg2]. The The connected bar i n the center o f t h e graph
entropy is a measure o f t h e number o f amino marks the mean and standard deviation o f t h e
acid mutations that can be made at a residue histogram. The lines above this bar indicate
without disturbing the structure, as determined where beneficial mutations that improved sta-
using a model o f stabilizing interactions (van bility (top row) and activity in organic solvents
der Waals, electrostatics, hydrogen-bonding, (bottom row) occurred in directed evolution
and the hydrophobic effect). When all amino experiments. Most o f these beneficial muta-
acids are equally allowed at a residues, then s; = tions occurred at residues that are predicted t o
In 20 = 3.0. When a single amino acid is allowed have high entropies.
selection approaches are based on infectivity of phages['74, 17'1 rather than on cell
growth. This method has been applied to select for proteins with improved
thermo~tability['~~I and stability against proteolysis [l7'1. Unfortunately, many of the
targeted enzyme properties and catalyzed reactions cannot be linked to cell growth or
infectivity of phages. Even if selectable traits exist, it remains a tedious task to
guarantee that the targeted enzyme property and not other factors such as substrate
uptake or other metabolic steps is limiting for growth.
Selections also face difficulties in controlling biological complexity. New in vitro
selection methods might reduce some of the limitations imposed by biological
complexity of living cells ['* 19* 241. In addition, much larger mutant libraries can be
searched by in vitro selection methods if they are used together with in vitro
genotype-phenotype coupling systems. However, such methods often select en-
zymes based on single turnover 17'1, binding of transition state ana-
logs[15o,179,1 " ' or suicide inhibitors [lS1]and therefore do not necessarily reflect
enzyme properties of highly active catalysts.
Another in vivo approach to couple the genetic information with a screenable or
selectable phenotype is phage display, which has been extensively reviewed else-
where[lg, 21, 23, 182, 1831. Th e most commonly used approach is to fuse the mutage-
4.5 Applications ofDirected Evolution
nized target genes to a coat protein gene of filamentous bacteriophages. After

I 121
transformation of bacteria with the recombinant DNA, bacteriophages are as-

sembled that display the protein of interest on their surface fused to the coat protein
and carry the genetic information for the displayed enzyme in the DNA. The typical
diversity of a library produced by phage display is high: 107-1011 different sequences.
However, it is difficult to screen for properties other than Furthermore,
the folding of displayed proteins occurs in the periplasm of the bacteria, which is
often problematic for the functional display of cytoplasmic enzymes. In addition,
large and multimeric enzymes are difficult to display. However, a few examples exist
where some of these limitations have been overcome, such as alkaline phospha-
tase [I', P-lactamase glutathione transferase [181] and penicillin G acylase, which
is a 86 kDa heterodimeric enzyme[187].Several theoretical models have been
proposed to capture the &pamics of phage
All in vivo approaches require the transformation of the mutated genes into cells,
which limits the library size that can be produced to a maximum of about 109-1010
members [lo2].In vitro approaches circumvent the transformation step by using cell-
free transcription/translai ion systems that can produce the mutant proteins directly
from the mutated The required link between the gene and the protein
produced can be achieved with ribosome display['"] or mRNA-protein fu-
sions [I9'* 1961. These elegant systems allow the generation of protein libraries that
contain up to 1013different members, which is a 10 000-foldexpansion of accessible
sequence space. Theoretically, such methods can cover all possible triple mutants of
an enzyme with 300 amino acid residues if coupled to a suitable selection system.
Another approach to coupling a phenotype with its genotype is based on In vitro
transcription / translation reactions compartmentalized in water-in-oil emul-
sions [126, 1391. In principle, each aqueous compartment contains one single gene that
is transcribed and translated into protein. In practice, however, many compartments
will be empty or contain inore than one gene. All these approaches require suitable
screening or selection technology to make use of the larger libraries.
4.5
Applications of Directed Evolution
With directed evolution we can engineer enzyme properties rapidly and with a high
probability of success. Many enzymes that have been improved by directed evolution
are listed in Tab. 4-3. This powerful biocatalyst engineering strategy creates new
opportunities in organic synthesis: new and improved bioconversion processes can
be developed and novel compounds that are otherwise inaccessible by classical
chemistry can be synthesized. In addition, the molecules created by directed
evcllution offer an excellent opportunity for improving our still poor understanding
of sequence-structure-function relationships.
The specific applications of directed evolution that are described below focus on
properties that are important for efficient enzyme production as well as on those that
are of special interest for applications in organic synthesis, including enzyme
122
specificity, activity towards non-natural substrates, and function in non-natural

environments.
4.5.1
Improving Functional Enzyme Expression and Secretion
Pharmaceutical or industrial applications of enzymes require their production at a

large scale. This is usually done by overexpressing the enzyme in E. coli, Bacillus sp.,
yeasts or fungi. Many enzymes, however, fail to fold properly in heterologous hosts.
This is a problem particularly for membrane-bound, highly glycosylated or disulfide
bond-containing eukaryotic proteins. Eukaryotic enzymes such as glycosylases,
peroxidases or cytochrome P-450s will require significant improvements in func-
tional expression to make them available in large quantities and at low cost.
Enzyme expression can be affected by mutations in the structural gene that may or
may not change amino acid sequence or by mutations in regulatory elements that
control expression. It was reported that horseradish peroxidase (HRP),a glycosylated
heme enzyme, cannot be expressed in functional form in E. coli or yeast. We found,
however, that we could obtain significant levels of HRP activity in the supernatant of
yeast cultures by accumulating point mutations in the structural gene['56].Fur-
thermore, mutants expressed at high levels in S. cereuisiae were also better expressed
in Pichia pastoris. Although far from sufficient for commercial enzyme production,
the activity level that was achieved enabled us to carry out further generations of
directed evolution to tailor the enzyme for specific applications.
Other reports show that intracellular expression of misfolded or unstable proteins
can be dramatically improved by directed evolution. For example, directed evolution
increased the expression of disulfide-containing antibody fragments in E. coli
50-fold,to reach a level of more than 0.5 g/L[2121. The expression of a wide spectrum
amidase of B. stearothemophilus in E. coli was improved 23-fold by two muta-
tions [210]. And, in uiuo fluorescence of the green fluorescent protein was improved
45-fold by increasing its solubility and native folding in E. c ~ l i [ ' ~ ~ I .
Secretion into the culture medium is preferred for some industrial enzymes
because it can facilitate purification. This is especially true if the protein reaches
high concentrations. Schellenberger's group at Genencor devised a method to select
for improved subtilisin-secreting mutants in Bacillus[170].Mutants secreting up to
five times as much enzyme were found after one round of error-prone PCR and
selection.
Directed evolution can improve functional enzyme expression and even allow
expression of enzymes that are otherwise difficult to produce in recombinant
systems. Results obtained by directed evolution, however, will depend on the
particular system, and high expression will undoubtedly be best achieved by a
combined approach of molecular biology, fermentation optimization and directed
evolution.
Table 4-3. Examples of designed enzymes by directed evolution.
Target enzyme Target function Changeeffected Approach Organism Reference
Kanarnycin nucleoti- Thermostability >200-foldincrease in half-life Mutator strain + selection B. stearothennophilus [167]
dyltransferase at 60-65 "C
Subtilisin E Activity in organic solvents - 170-foldincrease in 60% di- Random mutagenesis + B. subtilis [14, 1971
rnethylformarnide screening
P-Lactarnase activity Activity towards new sub- 32 000-fold greater resistance DNA shuffling + selection E. coli [371
strate to cefotaxime
* ~ A A
r-12 :.----I-- :- ~ - 1 r i ; r . .
Sra'uiiiryin iiir dLxrlie of I W W U - I W I U IIILICa.JC LII I I a u - I u c Loci: remG7ed + cassette E. s?!bti!k [198!
Ca2+ mutagenesis + screening
p-Nitrobenzyl esterase Activity towards pNB es- 60- to 150-foldincrease Random rnutagenesis + E. coli [99, 117, 1991
ters; activity in organic sol- DNA shuffling + screening
vent
Thymidine kinase Substrate specificity (gene 43-fold increase in sensitivity Cassette rnutagenesis + se- E. coli ~ 5 1
therapy) to gancyclovir in hamster cells lection + screening
P-Galactosidase Activity towards new sub- 66-fold increased activity; DNA shuffling + screening E. coli [161
strate; substrate specificity 1000-foldincrease in substrate
specificity
4A
06-alkylguanine- DNA Protection against alkylat- 10-foldincreased protection Cassette rnutagenesis + se- E. coli ~ 4 1 L,
alkyltransferase ing agents (gene therapy) against toxic methylating lection
agent
*
a6,
'
Arsenate detoxifica- Arsenic resistance 12-foldincreased rate of arse- DNA shuffling + selection E. coli 9
[ZOO1 8'
tion pathway nate reduction 2
Arninoacyl-tRNA syn- Aminoacylation of a mod- 55-fold increase in activity DNA shuffling + selection E. coli 1111 9
thetase ified tRNA P
a
Aspartate aminotrans- Activity towards P-branched lo5 increase DNA shuffling + selection E. coli [621 :
ferase amino and 2-0x0 acids
-
4
N
w
Table 4-3. (cont.) b
m
Target enzyme Target function Change effected Approach Organism Reference z
-c
3
Lipase Wash performance Improved performance in Mutagenesis + in vivo re- S. cerevisiae
one-cyclewash combination + screening
Lipase Enantioselectivity in hydrol- Increase in enantiomeric ex- Random mutagenesis + E. coli
ysis ofp-nitrophenyl2-me- cess from 2% to 81 % screening
thyldecanoate
Lipases Activity towards long-chain 3-fold increase In vivo recombination of E. coli
p-nitrophenyl esters homologous genes +
screening
pNB esterase Thermostability 14 "C increase in T, + in- Random mutagenesis + E. coli
creased activity at all tempera- DNA shuffling + screening
tures
Esterase Enantioselectivity of hydrol- Increase in enantiomeric ex- Mutator strain + screening E. coli
ysis of a sterically hindered cess from 0% to 25 %
3-hydroxy ester
Subtilisin E Thermostability 17 "C increase in T,,, + in- Random mutagenesis + B. subtilis
creased activity at all tempera- DNA shuffling + screening
tures
Subtilisin E Thermostability 50 x increase in half-life at DNA shuffling + screening B. subtilis
65 "C
B. lentus subtilisin Expression level (total activ- 50 % increase Random mutagenesis + en- B. subtilis
ity of secreted enzyme) richment in hollow fibers
Subtilisin BPN Activity at 10 "C 2-fold increase Chemical mutagenesis + E. coli
screening
3-Isopropylmalatede. Thermostability 3.4-fold increase in activity at Spontaneous mutations + Th. thermophilus
hydrogenase 70 "C selection
Cephalosporinases Activity towards moxalac- 270- to 540-fold increased re- DNA "family" shuffling + E. coli
tam sistance selection
Chorismate mutase Conversion to monomeric Functional monomeric Oligonucleotide directed E. coli
enzyme (solubility) enzyme codon mutagenesis +
selection
Biphenyl dioxygenases Degradation of Gained activity towards DNA “family” shuffling + E. coli
polychlorinated biphenyls substrates poorly degraded by screening
(PCBs) native enzymes; improved
activity towards various
substrates
FLP recombinase In viwo recombination Improved recombination Random mutagenesis + E. coli 11541
PfiriPnry a t elevated efficiency in E. coli and DNA shuffling + screening
temperatures in E.coli and mammalian cells
mammalian cells; in vitro
thermostability
EcoRV endonuclease Extend recognition site Becomes 10 bp cutter Targeted random E. coli
mutagenesis + screening
Cytochrome P450cam Increased activity in 5- to 20-fold increase Random mutagenesis + E. coli
peroxide shunt pathway, step shuffling + screening
towards naphthalene
Aspartate Substrate specificity 2.lx1OG-foldincrease in cat. DNA shuffling + selection E. coli
aminotransferase efficiency towards valine P
L,
Glutathione Substrate specificity Found range of specificities DNA “family”shuffling + E. coli
transferase screening
Peroxidase Stability to peroxide, 110 x greater thermal stability, Random mutagenesis + in Yeast
thermostability 2.8 x oxidative stability viwo recombination +
site-directed mutants +
screening
B-Glucuronidase Retention of function after More resistant to Random mutagenesis + E. coli
glutaraldehyde glutaraldehyde and DNA shuffling + screening
cross-linking formaldehyde
Subtilisin S41 Improved thermostability 100-fold increase in half-life Random mutagenesis + B. sub&
saturation mutagenesis +
screening
-
Table 4-3. (cont.) A
m
Target enzyme Target function Change effected Approach Organism Reference ?
5
Hydantoinase Enantioselectivity + total ac- Inverted enantioselectivity, 5 x Random mutagenesis + sat- E. coli
tivity increase in total activity uration mutagenesis +
screening
Subtilisins Various properties Improved activity stability DNA “family” shuffling + B. subtilis
screening
Esterase Enantioselectivity Two-fold improved E Random mutagenesis + E. coli
screening
Thymidine kinase Substrate specificity 7-44 fold improved specificity DNA “family” shuffling + E. coli
screening
Catechol2,3-dioxyge- Thermostability 13-26 x more thermostable DNA “family” shuffling + E. coli
nases screening
Lactate dehydrogenase Cofactor (fmctose-1,6-bi- 70-fold activation without DNA shuffling + screening E. coli
sphosphate) requirement cofactor (fully active in the
absence of cofactor)
Phytoene desaturase New carotenoid pathway Production of tomlene in DNA “family” shuffling + E. coli
and lycopene cyclase (substrate and reaction E.coli screening
specificity)
Indole-3-glycerol- Confer new catalytic activity Rational design + DNA E. coli
phosphate synthase (phosphoribosyl anthrani- shuffling + selection
late isomerase)
Kanamycin nucleoti- Thermostability Increase 20 “C DNA shuffling + screen- T thermophilus
dyl transferase inglselection
B. stearothermophilus Increase expression in E. Increase expression (23 x ) Random mutagenesis + E. coli
amidase Coli screening
Horseradish peroxi- Increase activity/ expres- Increase total activity 40 x Random mutagenesis + S . cereivisiae
dase sion in s. cereuisiae screening
Cytochrome P450 Substrate specificities Hydroxylates indole Saturation mutagenesis + E. coli
BM-3 screening
4.5 Applications of Directed Evolution
I
12'
4.5.2
Engineering Enzymes for Non-natural Environments
Bioconversion processes performed in organic solvents or at elevated temperatures

impart such benefits as increased substrate solubility, decreased viscosity of the
reaction medium, altered reaction selectivity and equilibria, higher rates, and
reduced risk of microbial contamination. High thermostability also tends to translate
to resistance to other denaturants and better long-term stability at lower tem-
peratures. Most natural enzymes are poorly suited for function in organic solvents or
at high temperatures, however, and their limited stability and activity in these
environments can be a limiting factor for applications in organic synthesis. These
properties are good targets for engineering by evolution.
Directed evolution has generated a large number of thermostabilized enzymes
(see Table 4-3 for example:<);there are too many reports for a comprehensive review
here. We will discuss only the general picture that arises from those studies; the
interested reader is referred to a recent review that deals with evolution of enzyme
stability in greater detail[2131.
The increase in thermostability imparted by single amino acid substitutions is
usually small and is typically in the range of a 1-2 "C increase in melting
temperature or optimal reaction temperature[152,17', 173, 214-21G]. Larger changes
are possible, but rare. Significant changes in thermostability therefore require the
accumulation of multiple substitutions, e. g., by sequential rounds of mutagenesis or
recombination. This strategy has generated 20 "C and higher increases in thermo-
~tability['~~ "l]. The stabilization mechanisms are consistent with those found in
naturally thermophilic ertzymes and include reduction of surface loop flexibility,
new hydrogen bonds, altered core packing, helix stabilization and acquisition of
surface salt bridges[217].Although the mechanisms are familiar, most of the changes
would have been difficult or impossible to predict.
I n nature, thermophilic enzymes tend to be less active at low temperatures than
their mesophilic counterparts, which in turn are less thermostable (Fig. 4-10). One
popular explanation for this observation is that activity and stability make mutually
exclusive demands on enzyme flexibility. Two properties coupled in this way cannot
evolve independently. However, directed evolution experiments have shown that
these properties can evolve lS2,2021. A 17 "C increase in the
melting temperature of a mesophilic esterase was achieved at the same time as
catalybc efficiency was increased several fold by random mutagenesis and screening
over several generations I'S2, 2131. A similar approach taken with mesophilic sub-
tilisin E generated a 17 "C increase in the temperature optimum and a >200-fold
increase in half-life at 65 oC[202].The thermostable subtilisin was also more active
than wild-typeover the whole temperature range. Most recently, directed evolution of
a psychrophilic subtili~in1~~] led to a 500-fold increase in half-life at GO "C at no cost to
its activity at low temperature. The evolved enzyme is more stable than homologous
mesophilic subtilisins. The stabilized enzymes contained between 7 and 13 amino
acid substitutions. In the studies described above, mutants were screened simultane-
ously for activity and thermostability, and mutations were accepted only when
128
Figure 4-10. Enzymes isolated from organisms growing at different

temperatures often exhibit a tradeoff between thermostability and cata-
lytic activity measured at low temperature. Enzymes that are both highly
thermostable and highly active at low temperatures are rare in nature but
highly desired for various applications and can be obtained by directed
evolution with relatively few
enhanced thermostability came at little or no cost to activity. If the selection pressure

is not maintained, thermostability can easily be lost[218.2191.
Creating enzymes that are both more thermostable and more active is particularly
exciting for industrial applications. In addition, these studies nicely demonstrate that
behaviors of natural enzymes may not necessarily be due to physical limitations
intrinsic to proteins themselves, as is often assumed. Instead they reflect what is
both relevant to the organism and accessible to natural evolution
Directed evolution has also been very effective for increasing enzyme activity in
organic solvents 991. For example, the serine protease subtilisin can catalyze
specific peptide syntheses and transesterification reactions, but organic solvents are
required to drive the reaction towards synthesis. Sequential rounds of error-prone
PCR and visual screening yielded a subtilisin variant with twelve amino acid
substitutions that was 471 times more active than wild-type in GO% dimethylforma-
mide (DMF) [145, 2201; this enzyme is much more effective for peptide and polymer
synthesis.
The production of cephalosporin-derivedantibiotics requires a deprotection step
usually catalyzed by zinc in organic solvents. Since this step produces large amounts
of solvent- and zinc-containing waste material, scientists at Eli Lilly were interested
in using an enzyme. Classic screening identified an esterase that catalyzed the
desired reaction but performed poorly in the solvents required to solubilize the
substrate. Directed evolution was therefore used to try to improve the performance
of the enzyme for efficient hydrolysis of an antibiotic p-nitrobenzyl ester inter-
mediate in aqueous-organic solvent mixtures [991. Four rounds of random mutagene-
sis by error-prone PCR and screening followed by one recombination step improved
the esterase activity 50- to GO-fold in 25 % DMF and yielded mutants that performed
I 129
as well in 30% DMF as the wild-type enzyme in water. None of the six mutations
found in the best mutant were in direct contact with the substrate and some were as
far away as 20 A. Thus, focused mutagenesis in the substrate binding site may have
overlooked important beneficial mutations.
High product concentrations are important in organic synthesis but often detri-
mental to enzymes. Scientist at Celgene reduced product inhibition in trans-
aminases1221] which are valuable for the production of chiral amines or amino acids.
A single round of error-prone PCR and screening of 10000 clones revealed mutants
with better product tolerance that translated to a four-fold increase in volumetric
productivity for a substituted amphetamine.
4.5.3
Engineering Enzyme Specificity
Enzymes are particularly valuable for the production of enantiomerically pure

compounds, as shown in examples throughout this book. However, the narrow
range of substrates that some enzymes accept and the less than impressive
enantioselectivities exhibited by others often frustrate attempts to develop new
synthetic applications and to commercialize existing ones. Directed evolution can
efficiently tune substrate specificity and catalytic efficiency towards non-natural
substrates; it can also tailor enantioselectivity,as illustrated in the examples below.
4.5.3.1
Substrate Specificity
Zhang et al. evolved a fucosidase from a galactosidase[''I. Seven rounds of DNA

shuffling and screening using a chromogenic fucose substrate yielded a mutant with
GG-fold increase in fuco:;idase activity. Kinetic analysis of the purified enzyme
revealed a 10-to 20-fold increase in kc,,&, for the fucose substrate and a SO-fold
decrease for galactose ( a total of 1000-fold increased substrate specificity for
fucose).
Kumamaru et al. reco nbined two biphenyl dioxygenases (96% identical) and
visually screened for mutants whose substrate range differed from the parents'.
These mutants degraded various biphenyl compounds more efficiently and also
exhibited oxygenation act]vity for single-ring aromatic compounds on which neither
parent was active[13'1.
Lanio et al. reported the tailoring of restriction endonucleases EcoRV specific-
it^[*^]. Focused combinatorial mutagenesis was used to make variants that cleave
specific DNA sequences of eight or ten base pairs rather than the six recognized by
the natural enzyme. Twenty-two amino acid residues were targeted by oligonucleo-
tidr-directed mutagenesis within three different regions of the enzyme. Screening a
total of only 500 colonies over three cycles of mutagenesis was sufficient to find
several mutants with high activity and high specificity for AT- or GC-flanked
GATATC cleavage sites.
Aspartate aminotransferase catalyzes amino group transfer between acidic amino
130
acids, aspartate and glutamate, and their corresponding 2-0x0 acids. The wild-type
activity for P-branched amino acids is barely detectable, but was dramatically
increased by directed evolution["! 1'" . The aspartate aminotransferase gene derived
from E. coli was subjected to DNA shuming and introduced into an E. coli host
lacking the branched-chain amino acid aminotransferase gene and therefore allow-
ing selection by complementation with mutant aspartate aminotransferases. The
stringency of the selection was increased during the progression of evolution by
omitting the substrate (2-oxovaline)from the medium, shortening the incubation
time and decreasing the expression level of the mutant enzymes by manipulating the
construction of the plasmid. A mutant with 105-foldincreased catalytic efficiency
(kcat&) for P-branched amino acids and 30-fold decrease for the natural substrate
was created after five cycles of shuffling and This mutant was further
improved to yield a mutant with a remarkable 2.1x10G-foldimproved catalyhc
efficiency compared to wild-type["l'. Analysis of the structure of the mutant enzyme
complexed with a valine analog provided detailed insight into how the mutations
affected substrate binding and demonstrated the importance of cumulative effects of
residues far from the active site.
The P-450 monooxygenase from Pseudomonas putida was evolved for efficient
utilization of hydrogen peroxide in lieu of 0 2 and NADH and for improved activity
towards the non-natural substrate One round of error-prone PCR
and screening of about 200 000 clones by high-throughput digital image analysis [1461
revealed several mutants with increased activity. Subsequent recombination of five
improved mutants yielded several variants with about 20-fold improvements in
naphthalene hydroxylation activity over wild-type using hydrogen peroxide as sole
cofactor.
Fructose 1,6 bisphosphate (FBP) is an allosteric activator of the thermostable L-
2-hydroxyacid dehydrogenase from B. stearothermophilus, which might be useful for
the asymmetric synthesis of chiral compounds. Since FBP is quite expensive, Allen
and Holbrook wished to create an FBP-independent Three rounds of
shuffling and screening produced a mutant L-2-hydroxyacid dehydrogenase with
three amino acid substitutions that is almost as active in the absence of FBP as the
wild-type is in its presence.
Recently, Schmidt-Dannert et al. reported the molecular breeding of carotenoid
biosynthetic pathways in E. coli I2O91. Two different phytoene desaturases were
shuffled and expressed in the context of a carotenoid biosynthetic pathway as-
sembled from different bacterial species. Clones containing mutant desaturases
were visually screened to identify new carotenoid products. One out of approx-
imately 10 000 colonies turned pink and produced shuffled tetradehydrolycopene
instead of lycopene. The new pathway was extended with a second library of shufled
lycopene cyclases. Visual screening identified a cyclase with altered substrate
specificity that produced the cyclic carotenoid torulene for the first time in E. coli.
Complementary to the strategy of creating new polyketides by mixing and matching
subunits in a multi-enzyme 2231, the combination of a rational pathway
assembly and directed evolution is an exciting opportunity to create libraries of
otherwise inaccessible biologically active compounds.
I 131
4.5.3.2
Enantioselectivity
Matcham and Bowen were among the first to apply an evolutionary approach to
improve the enantioselectivity of an enzyme for use in chiral synthesis [2211. The wild-
type enzyme (an s-selective transaminase) converts a particular p-tetralone to the
corresponding amine at only 65 % ee. By screening a mutant library of 10000 variants
in a microtiter plate-based assay, they identified 10 mutants that produced the (S)-
aminotetraline with 80-94 % ee. Sequencing the mutants revealed positions im-
portant for enantioselectivity and, interestingly,the existence of synergistic combina-
tions of mutations.
The lipase from Pseudoinonas aeruginosa (PAL) catalyzes the hydrolysis of 2-me-
thyldecanoic acid p-nitroplienyl ester with only 2 % ee in favor of the (S)-acid.Keetz
and Jaeger used four rounds of error-prone PCK and screening on enantiomerically
pure R and S substrates to generate a more enantioselective variant that produced
the desired (S)-acid with 81% Additional cycles of error-prone PCK in
combination with saturation mutagenesis further improved the enantioselectivity of
this enzyme, which hydra’lyzesthe 2-methyldecanoicacid p-nitrophenyl ester with
91 % ee (E= 25.8) in favor of the (S)-acid[121.
Bornscheuer et al. improved the enantioselectivity of an esterase from Pseudomo-
nasfluore~cens[~’,’071. In their first report, the enzyme was evolved for hydrolysis of a
3-hydroxy ester serving als a building block in epithilone ~ynthesis[~’1.Isolated
plasmids obtained from a mutator strain were transferred into E. coli and plated onto
two different kinds of agar plates. One plate contained a pH indicator which shows
active clones by a color change. The other plate contained a minimal medium and a
glycerol ester as the sole carbon source. Cleavage of the glycerol ester releases
glycerol, which leads to growth of active cells. One clone that produced the desired
enantiomer with 25 % ee was identified, compared to no enantioselectivity for wild-
type. The screen allowed for detection of active clones, but is not sensitive to
enantioselectivity; this mirght explain why further improvements in enantioselectiv-
ity were not reported.
A subsequent report deaicribes the evolution of the same enzyme for the hydrolysis
of 3-phenylbutyric acid re:sorufinester using both a mutator strain and error-prone
PCK[2071.Mutants were sc:reened for improved enantioselectivity based on a micro-
titer plate assay using the optically pure R- or S-esters. Both mutagenesis methods
generated first-generation mutants with higher enantioselectivity (E=6.6 and 5.8
compared to wild-type E=3.5).
Recent results show that directed evolution can also invert enzyme enantioselectiv-
ity“”]. The hydantoinase derived from Arthrobacter sp. shows a substrate-dependent
inversion of enantioselect-ivitywhich limits its use for the production of certain L-
amino acids such as L-methionine (for applications of hydantoinases in organic
syntheses see Chapter 12).By accumulation of mutations through sequential rounds
of error-prone PCR and. saturation mutagenesis, the enantioselectivity of the
hydantoinase was inverted from ee = 40% for the D-enantiomerto ee = 20% for the L-
isomer at 30% conversion. Only one amino acid substitution was required for the
132
inversion of enantioselectivity. Furthermore, mutant hydantoinases exhibiting high

D-selectivity (ee = 90% at 30% conversion) were also found. The L-selective mutant,
whose overall activity was improved 5-fold over wild-type, was co-expressed with a
racemase and L-specific carbamoylase in E. coli. This yielded a recombinant whole-
cell catalyst with an improved hydantoin converting pathway. Application of this
whole-cell catalyst for the production of L-methionine resulted in >S-foldimproved
productivity for 290 % conversion of the racemic substrate into the optically pure
product.
The optimization of whole pathways by directed evolution and their introduction
into recombinant whole-cell catalysts may offer the possibility of substituting
complicated multi-step processes with straightforward single-pot processes. This, of
course, is highly desired for industrial applications and a major advantage of
biocatalysis over other competing technologies used in organic synthesis.
4.6
Conclusions
The power of directed evolution is now well documented. These methods are robust
and are able to improve industrial enzymes in reasonably short times. The first
laboratory-evolvedenzymes are now used commercially in laundry detergents [201];
other commercial applications are on the horizon. Directed evolution may well help
move biocatalysis from an “enabling tool” to a “lowest cost approach. It also offers
new opportunities to engineer multi-enzyme pathways and even whole mi-
crobes 224* 2251, which will lead to straightforward single-pot, multi-enzyme
bioconversions and new fermentation processes based on “green”resources such as
glucose or inexpensive waste materials.
Sixteen years after Manfred Eigen and William Gardiner presented the basic
algorithm for evolutionary molecular engineering; it is worth commenting on the
conclusion of their paper:
“... The clones have to be addressable; the analytical methods must combine parallel
processing and automatic sampling with sensitivity and speed. With such elaboration
and scale, experimental biology might well become ‘Big science’. ’ [751
Today’s tools of evolutionary engineering certainly fulfill these requirements, and
directed evolution has in fact emerged as the method of choice for biocatalyst
improvement. However, we are only beginning to explore the power of evolutionary
design.
The most obvious limitations of these methods are still related to the tools.
Screening or selection methods require significant development time. This might be
reduced by the development of versatile enzyme assays that can be adapted rapidly to
specific conditions. The problem will also be reduced by integrating versatile
standard analytic systems such as mass spectroscopy, HPLC or capillary electro-
phoresis into automatic high-throughput systems.
The finite sampling capacity of most screening methods and the low versatility of
References I 1 3 3
selection methods will Frobably remain significant limitations. This makes it

difficult, if not impossible, to generate surprising new functions that require
multiple simultaneous amino acid substitutions. It is clear that more “rational”
approaches, based on stmcturelsequence comparisons or computation, will be
necessary to target key amino acid positions. Other limitations of directed evolution
are inherent in the current mutagenesis and recombination methods, which
strongly bias the combinatorial libraries. It is not yet clear how best to create
molecular diversity for evolution. What is clear is that many of these questions and
limitations can and will be addressed in the near future.
The field of molecular evolution used to focus on the past and aimed to explain the
existence of today’s fantastic array of biological molecules. Applied molecular
evolution is changing this focus to the future, by creating molecules for a bio-
technology industry of un limited opportunities.
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I 139
5
Enzyme Bioinformatics
Kay Hofmann
5.1
Introduction
Enzymes are a particular class of proteins which, through gradual developments,

have been optimized extensively to catalyze a large variety of chemical reactions ‘1.
The primary metabolism enzymes, whose role is the “housekeeping” catalysis of
metabolically important reactions, are generally optimized for robustness and high
turnover rate. By contrast, the products of secondary metabolism, which comprise
e. g. colorants, odorants, hormones and toxins, frequently have complex chemical
structures and require biosynthetic enzymes highly optimized for stereo- and
regiospecificity. A number of enzymes, many of them from microbial sources, have
already proved useful for ex vivo applications in synthetic chemistry. The main part of
this book gives an extensive overview of biosynthetic applications of enzymes
currently in use. The advent of genome sequencing, both of microbes and other
organisms, has lead to a sharp increase in the information available on their enzyme
repertoire and metabolic pathways. It is to be expected that these additional insights
will soon find their way into biocatalytic applications, leading to a broadened base of
synthetically useful enzymes.
( h e consequence of the increased amount of raw genomic data becoming
available is the requirement for bioinformatical analysis in order to extract useful
information. While there is an extensive literature on bioinformatics algorithms, on
protein bioinformatics in general, and on the analysis of particular protein families,
there are only a few publications dealing with enzyme-specific issues of bio-
informatical analysis. This chapter tries to fill a gap by specifically addressing those
aspects of protein sequence analysis that are important for identifying enzymes in
genomic sequences, for understanding their mode of action, and for predicting
some of their properties.
‘fie problem of understanding the mechanism of an enzyme, particularly when
pertaining to the optimization of catalytic properties, is more suitably addressed by
analysis of the enzyme’:; three-dimensional structure instead of its sequence 12, ’1.
W.hile structural analysis is occasionallyconsidered a subtopic of bioinformatics, this
140
I 5 Enzyme Bioinformatics
chapter will focus exclusively on aspects of sequence analysis. No matter how fast the
currently initiated projects on structural genomics proceed, the availability of a
protein structure will always lag considerably behind the availability of its se-
q~ence[&’~. Thus, any piece of information that can be derived from the sequence
alone will be useful in its own right. Moreover, many tasks that are commonly
believed to require knowledge about an enzyme’s structure, can nowadays be
performed by using the sequence alone, given that the appropriate tools are used and
the analysis is done properly. Examples include the identification of active site
residues or the establishment of extremely distant protein relationships with
sequence similarity way below 20 % identical residues [*I.
Since the major part of enzyme bioinformatics is based on the results of the
comparison of evolutionarily related sequences, the following paragraphs will start
(Sect. 5.2) with a brief survey of protein sequence comparison approaches. Compar-
ison of multiple sequences belonging to a single family usually reveals a specific set
of conserved residues. When dealing with enzymes, the nature and positioning of
the resulting conservation patterns can be indicative of the enzymatic mechanism, of
cofactors involved, or of other properties of that particular enzyme family. Thus,
Section 5.3 will discuss the conclusions that can be drawn from this type of analysis.
Section 5.4 elaborates on the “domain” concept, both in terms of structure and of
sequence. Multi-domain organization of enzymes is frequently associated with
multiple functionalities, which can occasionally be separated and used for over-
coming undesirable regulation mechanisms or even for combinatorial biocatalysis.
As in other areas of bioinformatics, specialized databases are of crucial importance
for the field of enzyme bioinformatics. Section 5.5 provides an overview of publicly
accessible databases that digest and store information on enzymes, pathways and
metabolites and make it available for querying. Section 5.6, which also deals with
databases, puts a focus on collections of pre-classified conservation patterns charac-
teristic of protein families and domains, both of enzymes and non-catalytic proteins.
These databases have become indispensable tools for the recognition and classifica-
tion of novel enzymes, a task frequently encountered when dealing with genome
sequences. Section 5.7 introduces and compares a number of strategies used to mine
microbial and other genome sequences for enzymes. Finally, Sect. 5.8 attemps to
give an outlook on possible future developments and on the impact ofbioinformatics
on the identification and optimization of enzymes for biocatalytic applications.
5.2
Protein Comparison
5.2.1
Sequence Comparison uersus Structure Comparison
It is a widely held tenet that the three-dimensional structure of a protein family is

better conserved than the sequence itself. In general, there is some truth to this
assumption, although the methods of measuring structural or sequence similarity
5.2 Protein Comparison
are merely operational and the results are difficult do compare. Sequence similarity
is fi-equentlyexpressed in terms of “% residue identity”, a measure that cannot be
applied to structural comparisons. Conversely, structural similarity is usually ex-
pressed by the r.m.s. distance, i. e. the root of the mean square distance of atom pairs,
which in turn cannot be applied to sequences. Nevertheless, there are a number of
proteins with identical or related function, whose 3D-structures look similar to the
skilled eye, while there is no apparent similarity in the amino acid sequence, at least
no similarity that would exceed the ‘background noise’ expected from comparing
two random sequences[” This apparent superiority of structural comparison has
pervaded the recent literature and has fuelled the demand for large-scale projects in
structural genomics. Whide such projects undoubtedly have their merits, it should
not be neglected that several recently introduced or improved methods of sequence
analysis come very close to the sensitivity of structural comparisons. Profile- or
Hidden Markov Model-b,ised methods in particular can make use of the enzyme-
specific conservation patterns discussed in Sect. 5.3, and thus are very well suited to
identifylng and classifying even the most distant evolutionary relationship between
enzymes. A comprehensive treatment of issues in protein comparison would be
beyond the scope of this chapter; the interested reader is referred to some recent
reviews on this topic ’I.
5.2.2
Substitution Matrices in Sequence Comparisons
Most sequence comparison methods, including the modern profile techniques, are
based on a “dynamicprograming” algorithm introduced by Smith and Waterman in
1981 [l4I. The method strives to find a mathematically optimal alignment from two
given sequences. The scoring system used for comparing the alignment “quality”is a
compromise between being a good model of biological reality and being computa-
tionally tractable. The airn is to maximize a composite score that is calculated from
all positions in the alignment. The pairing of identical residues makes a positive
contribution to the alignment score; the contribution of non-identical paired
residues depends on their similarity as defined by a generally valid similarity table,
the substitution matrix. Similar residues are associated with positive scores, while
dissimilar residue pairings give a negative contribution to the alignment score.
Insertions and deletions of residues in one sequence with respect to the other are
allowed, but penalized. Given a proper choice of the substitution matrix and the
deletion/insertion penalty, it can be assumed that the resulting mathematically
optimal alignment will be close to an evolutionarily optimal one[”, ‘1 (see Fig. 5-1).
While there are several possible ideas of what constitutes a “biologically correct”
alignment [l’], the context of enzyme comparison would minimally require that
corresponding active site residues of the two sequences are properly aligned.
The concept of using a substitution matrix, i.e. a knowledge-derived table for
judging amino acid sim larities, was introduced by Dayhoff et al. in 1978“’l. Most
types of currently used substitution matrices are derived from the analysis of well
established alignments, by counting which types of residues are frequently substi-
142
I ADE-FGA-KL
5 Enzyme Bioinformatics
ADEFGAKL
I I I I I I I I II
A-EDF-ASKL AEDFASKL
Figure 5-1. Influence o f t h e scoring
system o n the alignment appearance.
The left half o f the figure shows an
unreasonable alignment resulting from
too low deletion/insertion penalties. The
right half shows a better alignment,
although the "% identity" score is worse.
tuted for particular amino acids [17-201. The resulting 20 x 20 table has high positive
values for identical or highly similar residue pairs, since they can be easily exchanged
by evolution without significantly altering a protein's structure or function. Dissim-
ilar residue pairs, by contrast, have negative values, since they are rarely found in
homologous positions of related proteins. It is interesting to note that not all
identical residue pairings have the same positive value. For example, the Trp -Trp
value is very high, while almost all combinations of Trp with non-Trp residues have
negative scores. The most likely interpretation is that tryptophane tends to have a
- -
very specialized role in protein architecture, which can not really be fulfilled by any
other amino acid. By contrast, the Ile Ile value is not nearly as high as the Trp self-
score and is only marginally higher than the Ile Leu score. The likely reason is that
most functions of isoleucine can also be fulfilled by other residues such as leucine.
All commonly used substitution matrices are derived from a large collection of
protein alignments, containing both enzymes and non-enzymes. Thus, favorable
residue groupings tend to reflect a structural compatibility rather than a functional
equivalence. It would be expected that a substitution matrix derived from particular
sets of enzymes would have quite different values for residues that are frequently
found in active sites.
Both the inequality of residue self-matching scores, and the above mentioned
influence of the gap penalties on the alignment appearance show that the "%
residues identity" value is not always a good measure for judging the similarity of
two sequences. First of all, this value only makes sense when based on a biologically
reasonable alignment. Moreover, identical alignment positions containing trypto-
phane or cysteine can be considered better indicators of sequence relatedness than
conserved leucines or isoleucines.
5.2.3
Profile Methods
The profile method, introduced in 1987 by Gribskov et al.12'] and improved more
recently by various groups [22-251, can be considered a generalization of the Smith and
Waterman method. The idea of this technique is to abandon the traditionally equal
treatment of all positions in an alignment. When using profiles, it is possible to
assign a specific weight, a specific substitution matrix and a specific set of gap
penalties to each alignment position. The advantage of the additional degrees of
freedom lies in the possibility to incorporate a priori knowledge into the calculation
5.2 Protein Comparison
of the alignment score. If, for example, a sequence region is known to be very
I 143
important for a protein’s structure or function, it can be assigned a higher weight in

the calculation of the overall alignment score. Similarly, if a position is known to be
part of the active site, a particular substitution matrix could be used for that region. If
the structure of a protein is known, sequence stretches corresponding to solvent-
exposed regions could bc assigned “cheaper” gap penalties, since it is known that
external loops in protein structures can accommodate deletions or insertions more
easily than the structural core.
The most typical field of profile application is the alignment of a single sequence to
an already established protein family. Starting from a multiple alignment of the
protein family, specialized profile construction programs (Table 5-1) look for regions
with high conservation, implying a greater importance for the family’s structure or
function, and assigns high weights to the preferred residues in these positions.
Regions that already harbor gaps in the original family alignment are considered
structurally variable and are assigned lower gap penalties.
A mathematically very different approach, which is formally equivalent to the
generalized profile method, uses so called Hidden Markov Models (HMMs).A more
Table 5-1. Unified resource locators (URLs) for online accessible information sources mentioned
in the text.
Section 2 Profile and HMM construction programs
pfiools http://www.isrec.isb-sib.ch/ftp-server/pfiools
HMMer http://hmmer.wustl.edu
Setzion 5: Enzyme databases
ENZYME http://www.expasy.ch/enzyme
SWISS-PROT http://www.expasy.ch/sprot
BRENDA http://www.brenda.uni-koeln.de
KEGG http://www.genome.ad.jp/kegg
LIGAND http://www.genome.ad.jp/dbget/ligand.html
PDB http://www.pdb.org
Enzymes Structures Databa5.e http://www.biochem.ucl.ac.uk/bsm/enzymes
UM-BBD http://umbbd.ahc.umn.edu
PROMISE http://bmbsgil l.leeds.ac.uk/promise
MDB http://metallo.scripps.edu
MEROPS http://merops.iapc.bbsrc.ac.uk
ESTHER http://www.ensam.inra.fr/cholinesterase
-
Section 6 Domain and motif databases
PROSITE http://www.expasy.ch/prosite
PFAM http://www.sanger.ac.uk/Pfam
SMART http://smart.embl-heidelberg.de
BL.OCKS http://www.blocks.fhcrc.org
PRINTS http://bioinf.man.ac.uk/dbbrowser/PRINTS
INTERPRO http://www.ebi.ac.uk/interpro
PROCAT http://www.biochem.ucl.ac.uk/bsm/PROCAT/PROCAT.html
- - ~ _ _
Section 7: Genome resources
GOLD http://wit.integratedgenomics.com/GOLD
COG http://www.ncbi.nlm.nih.gov/COG
STRING http://www.bork.embl-heidelberg.de/STRING
144
I
extensive coverage of the construction of profiles and HMMs, as well as their
application in sequence comparisons, is given elsewhere[10-131.
5.2.4
Database Searches
A frequently encountered task in sequence analysis is to screen a sequence database

for relatives of a given protein. In general, all sequence comparison methods that
assign alignment scores, including the Smith and Waterman method and the profile
method, can be used to that end. A straightforward way is to compare the query
sequence (or profile) to each single sequence in the database and sort the results by
their respective alignment score.
A major obstacle of this approach is the large amount of computation necessary
for full dynamic programing algorithms, making these database searches very slow
unless running on high-performance computers. Alternative database search meth-
ods, including the well known FASTA and BLAST programs[2G-28], are substantially
faster through their use of heuristical approximations. While these methods cannot
guarantee to find the optimal solution, the minor trade-offin sensitivity is more than
compensated for by the immense gain in speed. The heuristical methods are
nowadays routinely used for database searches, sometimes combined with a true
Smith and Waterman post-processing step for the highest scoring matches.
An additional problem that has to be faced when doing sequence database
searches is the judgement of alignment score significance. Whenever comparing a
query to every sequence in a database and sorting the results by score, it is inevitable
that one database sequence will come out at the top of the list. However, this does not
necessarily mean that the top-scoring sequence is a true relative of the query: it is
quite possible that the database does not contain any relative at all. A number of
strategies have been devised to address this question. A common basis is the
statistical analysis of the score distribution that would be expected if the database
contained only random sequences. For each score obtained in the actual sequence
comparison an “expectation value” or “E-value” is then calculated, which corre-
sponds to the probability that the given score is the result of a chance match alone.
Low E-values are indicative of significant matches, a value of 0.01 would correspond
to a 1 % chance of being a mere coincidence and thus a 99 % chance of being
meaningful.
Heuristical or exact database search methods, combined with a rigorous statistical
analysis of the scores obtained, are very useful tools for identifylng relatives to given
sequences, with the profile and HMM methods being the most sensitive ones. The
two latter approaches have the additional advantage of allowing an “iterative
refinement” process[l1]. In the first step, a profile or Hidden Markov Model is
calculated from a starting family of proteins. In the second step, the resulting profile
or HMM is used for a database search. Database sequences that give significant
scores and are not members of the initial family can, after carrying out some
additional tests, be considered new members of the family. The expanded family
now contains more sequence diversity and thus offers more possibilities to discrim-
5.3 Enzyme-spec$c Conservation Patterns
I 145
inate the important regions from the less important ones. A new profile or HMM
calculated from the expanded family can then be used for further rounds of database
searches, until no new proteins with significant scores are found any more. This
iterative process has becn very successful in detecting even extremely distant
sequence relationships with residue identities €15 %.
5.3
Enzyme-specific Conservation Patterns
An iterative profile refinement search, as discussed in the previous section, fre-

quently starts with a relatively small set of sequences with readily visible sequence
homology. The multiple alignment will typically contain many conserved residues,
some of them being conserved because of their crucial importance for the protein’s
structure or function, and others being conserved just for the reason that the genes
encoding the proteins did not have enough time to diverge. Subsequent runs of the
refinement process will pick up more distantly related proteins, decreasing the
number of “chance conservations”.When the iterative process has finished and the
protein family under study contains a sufficient number of members and sequence
diversity, most of the inkariant residues will have a particular reason for being that
well conserved.
‘fie next paragraphs try to interpret frequently occurring classes of “conservation
patterns’, meaning the set of totally invariant or at least highly conserved positions
in a family alignment. The conservation patterns of enzyme families are different
from those ofnon-catalytic proteins and can be used for enzyme identification and
classification.
5.3.1
General Conservation Patterns
When analyzing a large number of solved three-dimensional protein structures, it

becomes evident that the amino acids buried in the internal regions of the structure
are mainly non-polar and form the so called “hydrophobic core”. By contrast,
residues that are exposed on the surface and thus in contact with the solvent tend to
be hydrciphilic. Data available on structural flexibility also indicate major differences
between the rigid core region and the flexible surface. If the conservation patterns of
typical protein families are analyzed, a further trend becomes visible. Residues
contributing to the hydrophobic core tend to be much better conserved than residues
exposed on the outside. As a consequence, highly conserved residues are mostly
hydrophobic. There are two other classes of residues that are frequently found to be
invariant: glycine and proline. The reason for this preference is again based on the
structure. A number of secondary structure elements, such as p-turns, require a very
small residue like glycine. Proline too is required for particular structural elements
since it introduces some rigidity to the backbone.
In general it can be stated that structural reasons contribute most to the
146
determination of the average protein family’s conservation pattern. Hydrophobic

residues tend to be highly conserved but not invariant, since they can frequently be
replaced by related hydrophobic residues. Glycine, proline and, in the case of
disulfide-bridges,also cysteine tend to be invariant or nearly invariant at structurally
unique positions. The multiple alignment of a typical non-catalytic protein region is
shown in Fig. 5-2A.
5.3.2
Active Site Conservation Patterns
The structure of enzymes is governed by the same principles as that of every other
protein. However, in addition to the structurally important residue conservation,
enzyme families also have the tendency to conserve their active site residues highly.
As will be discussed below, most enzyme families have retained a common reaction
mechanism and thus a common set of catalybcally important residues. As a
consequence, active site positions are not only well conserved but mostly invariant.
The set of residues found in catalytic centers of enzymes consist mainly of amino
acids that can be protonated and/or deprotonated, or those able to form hydrogen
bonds. The exact set of residues depends on the nature of the catalytic mechanism,
but serine, cysteine, histidine and aspartate are particularly frequent. In addition,
lysine, arginine, glutamate, threonine and tyrosine are occasionallyfound. In several
enzyme classes, the high degree of conservation around the active center extends
into a second layer, consisting of residues involved in orienting the catalytic side
chains by forming a network of hydrogen bonds. As an example of a typical
enzymatic conservation pattern, the multiple alignment of the duplicated but very
compact catalytic region of phospholipase D type enzymes[2g]is shown in Fig.
5-2B.
5.3.3
Metal Binding Conservation Patterns
A number of proteins contain metal ions, which may serve either a structural or
functional role, or even In some proteins, the metal is bound by a particular
cofactor, such as haem; other enzymes use the side chains of amino acids for
coordinating the metal ion. While bound metals are not restricted to enzymes, a
substantial proportion of hydrolases contain Zn2+and other heavy metal ions, which
typically contain one unoccupied coordination site that is used for binding and thus
activating the substrate to be hydrolyzed. Similarly, a number of redox enzymes
coordinate metal ions that are able to change their oxidation state, such as Fe2+/3+
or
Cu+l2+.Prominent members among the proteins that bind metals for non-catalytic
purposes are zinc-fingers, which frequently bind to DNA or to other proteins, and
Ca2+-bindingEF-hand proteins, which serve mainly regulatory purposes.
Not all amino acid side chains make good ligands for metal ions. Acidic residues
such as aspartate and glutamate are frequently found to coordinate small metal ions
like Ca” or Mg2+,while cysteine, histidine and aspartate are frequently involved in
5.3 Enzyme-specific conservation Patterns
I 147
Figure 5-2. Typical conservation patterns o f three protein classes. Residues invariant or conserved in
more than 80% o f t h e sequences are printed on a black or grey background, respectively. A Mainly non-
polar conservation in the UBA domain, a small protein domain that interacts preferentially with
u b i q ~ i t i n [ ’ ~ IB:. invariant polar active site residues i n the phospholipase D family[29].C: Nearly invariant
metal-binding residues i n the HtpXpteZ4 family o f Zn-containing metalloproteases.
148
coordinating Zn2+or heavy metal ions. Just as with the amino acids participating in
catalytic conversions, those coordinating metal ions fulfil a specialized role and tend
to be invariant within protein families. If substitutions are observed, they normally
stay within one class of coordinating residues, such as Cys-His or Cys-Asp.
Since all side chain bound metal ions require multiple ligands, the corresponding
protein families usually have a characteristic Conservation pattern consisting of
several invariant positions of the mentioned residue classes. A typical example is
shown in Fig. 5-2 C.
5.3.4
Making Use of Conservation Patterns
From what was said in the previous paragraphs, it appears that the specific
conservation pattern of a protein family can be used to predict whether the proteins
are enzymes, bind metal ions, or rather have a structural or regulatory role. If the
proteins are known to be enzymes, the conservation pattern can be used to predict
which residues are part of the active site, and possibly also which catalytic mecha-
nism is being used. For example, it would be straightforward to submit a family of
structurally uncharacterized proteases to that type of analysis in order to find out
whether they are serine proteases, aspartate proteases, metalloproteases, or if they
belong to a different class. Moreover, it is possible to compare the family’s
conservation pattern with those of other, better characterized enzyme families; this
approach will be discussed in more detail in Sect. 5.6.
There are, however, a number of caveats that apply to the analysis of enzyme-
specific conservation patterns. As mentioned previously, the method can be expected
to work only in those cases where the sequence family contains enough divergent
sequences to discriminate between the important and non-important positions. The
large amount of available sequence data from all phyla, in combination with
sensitive comparison methods like the iterative profile technique, make it possible to
meet this requirement quite frequently. In addition, the analysis is complicated by
the presence of catalytically inactive members of enzyme families. There is a rapidly
increasing number of reports on those “outsider” proteins, which in the course of
evolution have acquired fundamentally different non-catalytic roles. Examples in-
clude the transferrin receptor, which is a metal-free and inactive member of an
ancient metalloprotease and the neuroligins, which are inactive members
of the choline esterase family[32].Those proteins have no selection pressure to
preserve the non-functional active site residues and, as a consequence, they are
typically replaced by various structurally compatible amino acids. The presence of
inactive members in a family alignment means that one can no longer expect a total
invariance of the active site residues. Since the non-catalytic proteins usually replace
not only one active site residue but rather all of them, there is the chance to identify
inactive members or even inactive subfamilies by the concerted loss of conservation
in the presumed catalytic positions.
Finally, there is a small number of cases, where members of an enzyme family
have, in the course of evolution, assumed a different catalytic role, using a different
5.4 Modular Enzymes
I
149
set of active site residues. An example of this situation is the enoyl-CoA isomerasel
hydratase family (or crotonase family). The “inner family” comprises various enoyl-
CoA hydratases, isomerases, epimerases and 4-chlorobenzoyl-CoA dehalogena-
ses[331.While these reactions are catalytically distinct, they all share the feature of
using CoA-activated substrates and all of them utilize the same set of residues for
catalyzing the first common step of the reaction[34.351. However, sensitive sequence
comparisons demonstrate a more distant but nevertheless highly significant rela-
tionship to the ClpP enzymes, a class of bacterial proteases. This latter family does
not use C:oA activated substrates, catalyzes a totally different reaction and uses a
distinct set of active site residues grafted onto a very similar structural core[3G*
371. In
terms of conservation pattern analysis, this case can be treated similarly to the
previous one, i.e. a coordinated loss or change in residue conservation has to be
accounted for.
It has to be said that a11 of the mentioned complications should be considered
exceptions rather than the rule. Overall, an analysis of conservation patterns has
been and will continue to be a valuable tool in the identification and classification of
new enzyme families.
5.4
Modular Enzymes
A survey of known three-dimensional structure of proteins shows that a sizeable

portion of them contain several apparently independent folding units, usually
referred to as “domains”.
5.4.1
The Domain Concept in Structure and Sequence
A protein domain, in the structural sense, is a part of the whole protein that folds
independently from the rest of the structure and has a hydrophobic core of its own.
Residues lying within the domain are mainly in contact with other residues of the
same dornain; there are only few interactions between residues within and outside of
the domain. In evolutionary terms, genes encoding multi-domain proteins can be
explained as fusion products of simpler genes. Nature’s main advantage of using a
multi-domain organization of proteins is the possibility of having different functions
assigned to different domains of a proteins, which can act more or less independ-
ently of leach other. Functional domains that have proven useful can then, by an
evolutionary process involving exon shuffling or gene fission/fusion events, be re-
used in other proteins where they fulfil a similar function[38,391. Apparently, this
modular approach to pmtein structure has been very successful: there are several
functional domains that can, with only minor modifications, be found in more than
100 different proteins of one organism.
While the original definition of a protein domain is based on the structure, it is
also possible to detect “ re-usable modules” in protein sequences. Local regions of
150
sequence similarity, which are typically found in several proteins per organism, are
called “homology domains” and usually correspond roughly to structural do-
mains [401. The self-sufficiency of protein domains makes it possible to insert them
into almost any sequence context, thus giving rise to the sharp drop of sequence
similarity at the domain boundaries. When comparing two sequences, the presence
of a well-conserved homology domain, embedded into a totally unrelated context,
makes it necessary to use “local” alignment methods as opposed to “global” ones.
Local alignment algorithms do not require the total sequences to match with each
other but rather score the best matching region within the sequences. All sequence
comparison methods mentioned in Sect. 5.2 support a local alignment mode.
5.4.2
A Classification of Modular Enzymes
A modular architecture is no particular hallmark of enzymes, the highest degree of

modularity is typically observed in structural proteins of the extracellular matrix and
in proteins involved in intracellular signal transduction. Nevertheless, there are a
number of modular enzymes including some which are of interest for biocatalytic
applications.
A recent review of modular mentions three different but not mutually
exclusive types of modularity: i) the separation of substrate recognition and catalytic
activity on different domains, ii) modularity in multi-substrate enzymes, which use
different domains for binding the two or more substrates that are to react with each
other, and iii) modular enzyme systems that catalyze several consecutive reactions of
a metabolic pathway. When considering the biocatalytic usefulness of modular
enzymes, the first type appears less relevant, since the typical substrate molecules
are too small to allow a true spatial separation of recognition and reaction. In
physiological situations, however, this type of modularity is highly important
wherever the recognition and conversion of macromolecular substrates is con-
cerned. The second type, i. e. modularity in multi-substrate enzymes, might offer
some possibilities to change the components involved. If, in a family of transferases
catalyzing different reactions, donor and acceptor moieties are recognized by
different domains, it could be attempted to swap domains between family members
and thus change the specificity of the enzyme, possibly even to one not observed in
nature. Examples of this situation are the NDP-glycosyltransferases and the bacterial
polyketide synthases.
The third type of modularity, the multi-catalyticenzymes using substrate channel-
ling, are of particular interest for synthetic applications. Prominent members are the
fatty acid synthases, the polyketide synthases and the non-ribosomal peptide
synthases 142-441. In these large proteins, a number of catalytic domains is combined
with accessory domains and allows the catalysis of an entire pathway by a single
polypeptide chain. Multi-catalyticenzymes frequently use a “swinging arm”, which
is covalently attached to the intermediary product of one reaction step, and is
subsequently able to present this molecule to the next catalytic domain for further
5.5 Enzyme Databases and Other Information Sources
I 151
processing 14’1. As an example, a typical non-ribosomal peptide synthase contains
one or more domains forining the “swinging arm”, several domains for activating
specific amino acids, several domains for catalyzing the transfer of the activated
residues onto the growing chain, and one domain each for loading and unloading
the swinging arm. Additional domains that catalyze further enzymatic steps such as
redox reaciions or cyclizations are also found. These enzymes, as well as the bacterial
polyketide synthases, are promising tools for the biosynthesis of antibiotics and
other related natural products. Part of the promise stems from the specificity of the
activation reaction and from the fact that the sequence of the reaction process is
encoded by the domain arrangement. It has been shown that artificially swapped
domains can lead to active enzymes that now synthesize a different 471.
So far, not all domains occurring in those enzymes are fully understood by function,
and not all attempted domain swappings have lead to viable enzymes. Nevertheless,
multifunctional re-prograinable enzymes and other engineered hybrid-enzymeswill
undoubtedly have an interesting future in biocatalyhc applications14’1.
5.4.3
Inhibitory Domains
Besides the three types of modularity mentioned, there is a fourth type that is very
useful in a physiological setting but tends to be undesired ex uiuo. In a living cell, an
uncontrolled enzymatic activity at the wrong place or the wrong time can be
deleterious. To avoid is type of complication,many enzymes have acquired inhibitory
domains, which are encoded by the same polypeptide as the enzyme itself. When-
ever in the biological system the enzymatic activity is needed, the inhibitory region is
cleaved off or is neutralized by other methods, e. g. by binding to an activator protein.
Biocatalytic applications iypically require permanently active enzymes. Thus, it is
desirable to recognize inhibitory domains and remove them before using the
enzyme. ,4s mentioned above, bioinformatic methods such as sequence compar-
isons can help to find those domains and to determine, with some confidence, the
likely domain boundaries.
5.5
Enzyme Databases and Other Information Sources
Now that the principles of enzymatic architecture and the corresponding analysis
strategies have been highlighted and briefly discussed, an overview of the existing
enzyme classes and their properties is needed. Given the more than 4000 different
enzyme types, any attempt at only listing them would be far beyond the scope of this
chapter. Fortunately, there are a number of specialized databases available, which
aim to treat various aspects of enzyme structure and function comprehensively. All
of these databases are accessible via the Internet, and a list of the relevant URL
addresses,is given in Table 5-1.
152
I 5.5.1
E.C. Nomenclatureand ENZYME Database
The widely accepted basis of all enzyme classifications are the recommendations of
the Enzyme Committee (E.C.) of the International Union of Biochemistry and
Molecular Biology (IUBMB) [491. Within this system, enzymatic activities are classi-
fied by a four-level hierarchy and each entry is described by a set of four numbers.
The first number describes the top level and can be either “1”for oxidoreductases,
“2” for transferases, “3” for hydrolases, “4” for lyases, “5” for isomerases or “6” for
ligases. The meaning of the three lower hierarchy levels depends on the top level
group. As an example, glycogen synthase is classified as 2.4.1.11; here, the “2” stands
for transferases, the “4” for glycosyl-transferases,the “1” for hexosyl-transferasesand
the “11”for the particular subfamily.
The ENZYME database[50],maintained by the Swiss Institute for Bioinformatics
(SIB), provides a comprehensive list of all IUBMB classifications, together with
associated information such as systematic and alternative enzyme names, cofactor
requirements, and pointers to the corresponding entry in the SWISS-PROTdatabase
of protein sequences[51].In addition, there is a concise free-text description of the
reaction catalyzed, together with a description of preferential substrates and prod-
ucts. Currently, the ENZYME database holds entries for approximately 3700 en-
zymes.
5.5.2
BRENDA
A much more ambitious database that builds on the IUBMB classification is

BRENDA, maintained by the Institute of Biochemistry at the University of Cologne.
In addition to the data provided by the ENZYME database, the BRENDA curators
have extracted a large body of information from the enzyme literature and incorpo-
rated it into the database. The database format strives to be readable by both humans
and machines. The categories of data stored in BRENDA comprise the EC-number,
systematic and recommended names, synonyms, CAS-registry numbers, the reac-
tion catalyzed, a list of known substrates and products, the natural substrates,
specific activities, KM values, pH and temperature optima, cofactor and ion require-
ments, inhibitors, sources, localization, purification schemes, molecular weight,
subunit structure, posttranslational modifications, enzyme stability, database links,
and last but not least an extensive bibliography. Currently, BRENDA holds entries for
approximately 3500 different enzymes.
From the wealth of information presented, it is clear that BRENDA is a very
important resource for enzymes in organic synthesis.
5.5 Enzyme Databases and Other Information Sources
I 153
5.5.3
KECC and LICAND database
The Kyoto Encyclopedia of Genes and Genomes (KEGG) is an effort to reconstruct

biological pathways from the gene repertoire found in the genome sequencing
The LIGAND database is an associated database of enzymes and their
reactions, which is also hosted by the University of LIGAND consists of
three different but interconnected segments. The COMPOUND section holds 5600
entries of various compound classes with relevance to enzymatic reactions (sub-
strates, products, inhibitlxs etc.). The ENZYME section contains 3400 entries
corresponding to the enzymes themselves. Finally, the REACTION section contains
approximately 5200 reactions. In combination with the KEGG/PATHWAY database,
the data stored in LIGAND are not only presented in static form but can also be used
to calculate biological pathways between a given substrate and product.
5.5.4
UM-BBD
The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD) is a

data repository providing curated information on microbial catabolic enzymes and
their organization into metabolic At present, the UM-BBD stores
information on approximately 100 pathways with 700 reactions, GOO compounds and
400 enzymes. The database does not try to cover every known enzyme but rather
focuses 011 those used for the biodegradation of xenobiotics. UM-BBD is linked to
the ENZYME, BRENDA and KEGG/LIGAND databases mentioned above.
5.5.5
Structural Databases
Although not being in the focus of this chapter, structural databases are a most
useful resource for the slcientist interested in enzymes and reaction mechanisms.
The Protein Data Bank (PDB) is the main repository for all three-dimensional
structures of macromolecules including enzymes ['I. Nowadays, most journals
accepting manuscripts that describe new structures require a simultaneous deposi-
tion of the structural coordinates with the PDB database. In addition to the structure
of single protein molecules, the PDB also contains several entries of multi-plotein
complexes, or proteins bound to small-moleculecompounds.
Of the 14500 entries currently in PDB, there are roughly 7200 enzyme structures.
The Enzymes Structures Database, maintained by University College, University of
London, Focuses on this portion of PDB and offers links between the E.C. nomen-
clature of the IUBMB and the corresponding PDB entries.
154
I 5.5.6
Metalloprotein Databases
As mentioned in Sect. 5.3.3, a number of enzymes contain metal ions that

participate in the catalytic reaction. Two specialized databases store information on
metal ions and other bioinorganic motifs in enzymes. PROMISE (prosthetic centers
and metal ions in protein active sites) is maintained by the University of Leeds and
focuses on six major groups of metal containing proteins: diiron-carboxylate pro-
teins, haem proteins, iron-sulfur proteins, molybdopterin proteins, mononuclear
iron proteins, and chlorophyll containing proteins ["I.
The Metalloprotein Database and Browser (MDB) is maintained by the Scripps
Research Institute and aims to collect quantitative information on all metal contain-
ing sites available from structures in the PDB database[57].The data stored com-
prises both structural and functional information on the metals bound and the
ligands involved. The associated database server allows specific queries for particular
site geometries and functions.
5.5.7
Databases for Selected Enzyme Classes
In addition to the above mentioned databases that try to cover the entire world of
enzymes, there are a number of more topical databases focusing on particular
enzyme families. The MEROPS database, maintained at the Babraham Institute in
Cambridge, provides a catalog and a structure-based classification scheme for all
proteolytic enzymes ["I. In addition to the classification,the database also provides a
digest of published information on the peptidases as well as cladograms and
multiple sequence alignments of the peptidase families.
The ESTHER database, maintained at the INRA-ENSAM in Montpellier, follows a
similar concept but focuses on the a/S fold family of esterases/lipases ["I.
5.6
Protein Domain and Motif Databases
As has been described in Sect. 5.3, the conservation patterns of enzymes are often
indicative of the particular family they belong to and can be used for their
classification. However, the iterative searches and multiple alignment methods used
for their establishment require a certain bioinformatic infrastructure as well as some
experience with these issues. If the goal of the analysis is not the detection of novel
enzyme families, but rather the classification of a novel sequence into one of the
already existing enzyme families, there are a number of protein domain and motif
databases that will be useful in this respect[", "1. These databases do not store the
sequences themselves but rather work with "descriptors" of protein families and
protein domains. These descriptors can consist of the Profiles or Hidden Markov
Models mentioned above, but other types are also being used. With a particular
5.6 Protein Domain and MotifDatabases
I 155
search engine, typically prsovided with the databases, it is possible to scan one or
more unknown protein sequences against large libraries of pre-defined family or
domain descriptors. These search engines are publicly accessible via the Internet;
the relevant addresses are listed in Table 5-1. Currently, none of the available
databases hlas a particular focus on enzymes. Nevertheless, a substantial proportion
of the databases discussed below consist of enzyme families or of catalytic or
regulatory enzyme domains.
5.6.1
PROSITE
The PROSITE database, maintained by the Swiss Institute of Bioinformatics (SIB),

was the first database that tried to catalog functional motifs and domains of
proteinslG21.Nowadays, PROSITE consists of two major parts storing different types
of descriptors: the “pattern”library and the “profile”
The pattern entries of the PROSITE database are based on a regular expression
syntax, which emphasises only the most highly conserved residues in a protein
family, corresponding approximately to what is termed a “conservation pattern” in
Sect. 5.3. I n contrast to the other databases mentioned below, PROSITE patterns do
not attempt to describe a complete domain or even protein, but rather try to identify
the functionally most important residue combinations, which in enzymes typically
correspond to the active site. As an example ofthe PROSITE syntax, “K-x(1,2)-[DE]”
would mean a lysine residue, followed by one or two arbitrary residues, followed by a
residue that is either aspartate or glutamate. When a sequence is compared with a
library of such patterns, (my pattern is found to be either present or absent, no
intermediate scores are assigned. Currently, the PROSITE pattern libraries contains
approximately 1400 entries.
A consequence of the riazid syntax of PROSITE patterns is the restriction that they
work well only with those protein families that really contain invariant or at least
highly conserved positions. When dealing with catalytic sites of enzymes, this
requirement is usually met. However, a large number of protein families and
domains are too divergent to be appropriately described in the framework of a
regular erpression syntax. To circumvent that problem, the PROSITE curators
introduced another secticln of the database using generalized profiles as descrip-
t o r ~ [ As
~ ~mentioned
]. above, profiles are based on preferences for particular amino
acids rather than on strici requirements. Thus, profiles are suited better for highly
divergent protein families and domains, but require a different search engine. An
important factor contributing to the usefulness of PROSITE is the extensive
documentation of the entries, discussing e.g. the active site residues or the
phylogenetic scope of a motif, and also providing links to other databases and to the
literature.
156
I 5.6.2
PFAM
PFAM is a database of Hidden Markov Models of protein families and domains,

maintained at the Sanger Centre in Cambridge[65].The concept of PFAM is
comparable to that of the PROSITE profile section. Similar to the profiles, the
HMMs in PFAM have been derived by the iterative refinement procedure men-
tioned in Sect. 5.2.4. Unlike the PROSITE profiles, which all have been created
manually by the curators, the HMMs in PFAM are generated semi-automatically,
which accounts for a slightly lower sensitivity. However, this lack is more than
compensated for by the facilitated update procedure, allowing the database to grow
much faster than PROSITE and to have a shorter generation cycle. Currently, PFAM
holds 2727 entries.
5.6.3
Other Related Databases
A number of other protein motif databases should not be left unmentioned. The
SMART database is conceptually very similar to PFAM, but the collection of Hidden
Markov Models focuses on proteins involved in intracellular signal transduction (“1.
The PRINTS and BLOCKS databases are similar to PROSITE and PFAM in that they
do not have a thematic focus [67, “1. However, unlike the databases mentioned above,
their motif descriptors recognize short non-gapped regions of the proteins. Several
other protein motif- and domain-databases and their application in the classification
of proteins have been reviewed recently’“, 61]. The INTERPRO consortium, consist-
ing of the curators of various protein domain databases, is currently developing a
non-redundant combination database, offering a common search interface [(jg].
A fundamentally different approach is used by PROCAT, which does not describe
motifs in linear sequence but rather structural motifs, i. e. combinations of residues
that occur in a similar position in the 3D-structure of protein family mem-
bersl70. 711
5.7
Enzyme Cenomics
The last few years have seen a rapid increase in the number of completely sequenced
genomes; an even greater number of whole genome sequences is near completion.
Currently,49 genome sequences have been published in the scientificliterature, and
both their DNA sequence and the protein sequence of the predicted gene products
are in publicly accessible databases. A taxonomic breakdown of the completed
genome sequences shows that five of them belong to eukaryotes,nine to archaea and
35 to eubacteria. So far, the choice of the organism for the genome projects has been
based mainly on the general scientific interest or on their biomedical importance. A
number of organisms selected for their technological interest are being sequenced as
5.7 Enzyme Genomics
I
157
well. However, the driving force behind those genome projects are mainly commer-
cial entities, raising the question of when and to what extent the sequences will be
made known to the public. Databases like GOLD provide information on both
finished genomes projects and those
What is the relevance of genome sequences to the search for biocatalytically
applicable enzymes? At least two different avenues, in this context called “ortholog
search and “paralog search have the potential to yield results that are immediately
useful.
5.7.1
Ortholog Search
A number of enzymes from microbes or other organism are considered useful but
not totally satisfactory for synthetic applications. Frequently encountered problems
include lack of stability,too low catalytic rate, too broad or too narrow specificity, and
poor availability of the naiural or recombinant enzyme. In these cases, it might be
favorable to replace the enzyme by an ortholog from another organism, i. e. by an
enzyme that fulfils exactly the same role in another species and is related to the
original enzyme in the same way as the corresponding organisms. One possible
rationale for this approach is that not all orthologs in a family have exactly the same
properties thus there is a certain likelihood of finding a “better”enzyme in another
species by chance alone. A more targeted approach for finding “better”orthologs can
also be envisaged if e. g. the goal is to increase the thermal stability of an enzyme,
the orthologs from therrriophilic organisms are prime candidates for the desired
improvement [731. In general, different life environments and slight differences in
metabolic pathways give rise to certain variations in an enzyme’s properties, which
can be exploited in the search for optimized enzymes.
An obvious prerequisite for this type ofoptimization is being able to find orthologs
to given enzymes. A second requirement is that the (sequence derived) ortholog pair
has not evolved so far that they catalyze different reactions. When dealing with
completely sequenced geiiomes, the search for othologs is frequently straightfor-
ward. A number of complications have been described [741, the most frequent being
that the gene of interest has been duplicated in one of the lineages. In the cases of
absent one-to-one ortholog relationships, it is more appropriate to speak of ortholo-
gous groups of genes rather than of ortholog genes. The COG database, maintained
at the NCBI, has defined orthology clusters for the publicly available genome
sequences and is updated whenever new genome sequences become available[75].
5.72
Paralog Search
A more demanding problem in the bioinformatic mining of genome sequences is

the search for truly novel enzymes. A possible starting point would be the knowledge
that a particular organism possesses an enzyme with the desired specificity, while
the corresponding protein sequence is elusive. In order to address this type of
158
I question, several approaches are conceivable.One of them is based on the analysis of
conservation patterns and phylogenetic relationships in large, non-orthologous
enzyme families, and will be discussed in the following paragraph. Other methods,
which are not based on sequence homology at all, are highlighted in Sect. 5.7.3.
When analyzing the sequence/function relationship in multiple enzyme families
such as those collected in PROSITE and PFAM, a number of general rules emerge. It
has been mentioned previously that the catalytic mechanism and the active site
residues of an enzyme are better conserved than the overall sequences. In most
cases, the same is true for the region ofthe substrate that is modified in the course of
the reaction, in particular for the type of bond that is being broken or formed. There
is a general trend that related enzymes catalyze identical or closely related reaction
types but not necessarily with related substrates. While there are numerous
examples of this trend, there are only a few counter examples. Almost all members
of the u/p fold lipase family catalyze the hydrolysis of carboxyl-esters (or the reverse
reaction), no matter whether the substrates are lipids or polar compounds. Similarly,
there are several families of phosphoesterases, that act on substrates as diverse as
phospholipids, phosphoproteins and nucleic acids, but invariantly cleave a phos-
phoester bond. Multiple families of acyltransferases exist, which have as a unifying
criterion the nature of the acceptor atom (0,N, S) rather than a common recognition
feature in the substrates. Among the very few counter examples are the enzymes of
fatty acid B-oxidation. As mentioned in Sect. 5.3.4, the enoyl-CoA hydratase and
isomerase catalyze different reactions but use a very similar substrate. This partic-
ular example can, however, be explained by a common activation step in both
reactions.
This knowledge can be exploited to search complete genome sequences for
proteins that encode enzymes of a given specificity. In the first step, the enzymatic
reaction under question has to be analyzed for the nature of the atoms involved and
the bonds to be formed or broken. In the second step, the available knowledge base
of enzymes and enzymatic reactions has to be screened for any relatives. Useful in
this respect are the databases of Sect. 5.5, like ENZYME and BRENDA, which
already have classified enzymatic reactions by the necessary criteria. In addition, the
protein motif databases of Sect. 5.6 might already have assembled a family of
enzymes that catalyze the desired reaction type. If, in this process, a known enzyme
is found to catalyze a reaction with a similar mechanism to the desired one, this
enzyme sequence can be used for a paralog search in the third step. The expression
“paralog” applies to evolutionarily related proteins, either within one species or
between species, that are not “orthologs”,i. e. that do not directly correspond to each
other. Paralog pairs are expected to catalyze similar reactions instead of identical
ones. Finally, in the fourth and last step, the found paralogs can be assumed to be
candidates for the missing enzyme and their activity can be verified experimentally.
Since paralogs are typically more distantly related than orthologs, their detection
frequently requires sensitive protein comparison methods such as profiles or
HMMs. Even the detection of orthologs can, under some circumstances, require
sophisticated database searching methods, e. g. if the corresponding organisms
belong to very distant phyla.
5.8 Outlook 1159
5.7.3
Non-homology Based methods
The methods described in the previous section are all based on homology, i.e. a
recognizable sequence relationship caused by a common evolutionary descent. An
additional approach to identify candidate genes for a given enzymatic function does
not rely on homology, but rather on a peculiarity of bacterial genome organization.
Bacteria tend to have proteins belonging to one metabolic pathway clustered in a
contiguous stretch of the genome, all present in the same transcriptional orientation.
The reason for this clustering is an economy of transcriptional regulation. In most
cases, the components of a pathway have to be expressed in a coordinated fashion.
This regulation is greatly facilitated by the “operon” arrangement, where multiple
bacterial genes are under the control of a single promoter.
Again, this knowledge can be exploited when searching for an unknown enzyme
with a known involvement in a particular pathway. The first step is the identification
of other proteins likely to work in the same pathway as the desired enzyme. In the
second step, the genome cf the target organism is searched for genes encoding those
upstream or downstream components. In the third step, other genes belonging to
the same operon(s) are identified and treated as enzyme candidates unless a
different function can be assigned to them. This “operon-approach”to enzyme
identification is particularly useful in situations were the gene in question cannot be
identified by sequence similarity, e. g. in cases of “non-orthologous gene displace-
ment”. This expression d.escribes a phenomenon that is occasionally observed in
bacterial genome compai isons [74s 761. Here, two organisms use similar pathways,
where most but not all of the genes involved have a clear one-to-onerelationship. The
remaining genes might catalyze exactly equivalent reactions, but are not related at all
because the two organisms have recruited members coming from different protein
families fix an identical task.
Not all bacterial genes in general, and enzymes in particular, are organized in
operons. A prerequisite ior the method described above is a reliable detection of
operons and the participating genes. Again, evolutionary considerations can help: if
related genes, preferably orthologs, occur in a conserved order in several bacterial
genomes, this is a clear indication of an operon organization and thus most likely
also of a functional coupling. Computer databases of genome organization, such as
the STRING system maintained at the EMBL, are useful tools for detecting those
relationships [771.
5.8
Outlook
In the recent years, at least two developments have made major contributions to the
field of enzyme bioinforniatics. One of them, the advent of whole genome sequenc-
ing, is widely recognized for its impact on virtually every field of biochemistry and
moleculair biology. By contrast, the development of sensitive sequence comparison
160
I
methods has remained largely unnoticed, although it has made possible a new level
of understanding genomic data. The most useful databases of protein families and
domains, together with their associated search systems, would not have been
possible without profile and Hidden Markov Model methods.
These two achievements work synergistically.On one hand, the sequence compar-
ison and classification approaches are required for an efficient functional assign-
ment of genome sequences and also for inter-genome comparisons. On the other
hand, the iterative refinement process relies on sequence diversity within protein
families and can make use of the genomic data, even in its raw and functionally
uncharacterized state. At present, only a fraction of known enzymatic domains and
protein families is covered by databases such as PROSITE and PFAM. Within the
next years, this fraction will increase, since more genome data will probably uncover
a large amount of new enzymes, accompanied by only a minor increase in the
number of truly new enzyme families. Eventually, we will see a nearly complete
coverage of enzyme families, which will greatly facilitate the identification and
classification of any new enzyme sequence that becomes available.
A field that will most certainly gain influence in the next years is that of “structural
genomics”. Several attempts have been initiated to elucidate the three-dimensional
structure of an organisms entire protein complement, or at least a substantial
fraction of it. While the results coming from these projects will open a straightfor-
ward path to fold recognition, the value for enzyme bioinformatics might not be as
high as it might seem. The most useful structural information on enzymatic
mechanisms comes from structures where the enzyme is analyzed while binding to
a substrate analog or to an inhibitor. These studies, however, require a priori
knowledge on the enzymatic properties and the nature of the substrate, which is not
available in “blind”high-throughput studies.
Another area of intensive research in the field of applied genomics is the gene
expression analysis by DNA microarrays and similar methods. As of now, most
applications of these techniques are either based on their scientific merits or on
medial/pharmaceutical/toxicologicalapplications. It is probably only a matter of
time until these methods find their way into research on biocatalysis. Possible
applications include the analysis of coordinated regulation of enzymes not linked in
operons, or the identification of new enzymes on the basis of their expression
pattern.
As in all other areas of bioinformatics, databases will play an increasingly
important role in managing and integrating the data coming from various sources. A
database system meant to be useful for the exploitation of enzymes for synthetic
applications would have to encompass information on organisms, their genome
sequences and their metabolic pathways, with a special emphasis on the enzymes
involved, their reaction types and the nature of the substrates and products.
Databases such as KEGG and others have already started to address these questions.
However, none of the currently available genome- and pathway-databases are
focused on biocatalysis, a fact that will certainly change within the next couple of
years.
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6
Immobilization of Enizymes
James Lalonde and Alexey hlargolin
6.1
Introductio'n
Readers of this text are well aware of the promise of enzyme catalysis for the elegant
synthesis of complex molecules. However, the practical application of enzymes as
catalysts for organic synthesis is often limited by the inherent differences between
the way that molecules are synthesized by biological systems and the way they are
prepared on the laboratory bench. Nature has designed enzymes to catalyze reactions
under physiological conditions, most often in aqueous media at ambient tem-
perature and pressure and at neutral pH with dilute concentrations of reactants.
Preparative chemical syntheses, in contrast, usually require high concentrations of
reactants and the use of organic solvents to dissolve organic substrates and to shift
reaction equilibria. Isolatilm of organic products from water can be complicated by
the presence of an amphiphilic protein. While biological systems destroy and
regenerate enzymes as they are needed, catalysts used in chemical manufacture
must often be recovered and reused many times for economic viability. Immobiliza-
tion of an enzyme is the most commonly used strategy to impart the desirable
features of conventional heterogeneous catalysts onto biological catalysts.
By definition, enzyme immobilization is the conversion of an enzyme to a form
with artificially restricted mobility and retention of catalytic function [l]. This re-
stricted mobility allows for containment and recovery of the enzyme and is often
achieved by either conversion to an insoluble form (for example by linking to
insoluble ]particles)or by containment within a semi-permeable barrier (for example
entrapment within an ultrafiltration membrane). In the course of this immobiliza-
tion, enzy:mes can acquire four advantageous properties:
- Immobilized enzymes can be used repeatedly or continuously in a variety of

reactors:.
- They can be easily separated from soluble reaction products and unreacted
substrate, thus simplifjing work-up and preventing protein contamination of the
final product.
G lmrnobilization ofEnzyrnes
164
I - The catalytic properties, pH-activity profile and enzyme stability can be enhanced
in the immobilized form.
- The control of microbial contamination in solid immobilized preparations is often
simpler than for soluble protein.
There are, however, a number of practical limitations on the utility of immobilized

enzymes. First, the yield of protein binding is rarely quantitative. Second, in many
cases, the cost of the carrier can be quite significant and may even exceed the cost of
the enzyme itself. Third, the activity of the resulting immobilized enzyme is usually
reduced because of chemical modification of the protein, steric hindrance and mass
transfer limitations. Finally, the proportion of active enzyme to the carrier material in
immobilized enzyme preparations rarely exceeds 5-10 % w/w, and thus dramatically
reduces catalytic activity per weight of solid.
Despite the limitations, the great success of enzyme immobilization in diag-
nostics, pharmaceutical, food and chemical industries is undeniabler2>'1. The
decision whether one should use a soluble enzyme preparation or an immobilized
enzyme does not have a universal solution and can be decided only on a case by case
basis. Ordinarily, if the cost of an enzyme represents a significant portion of the
overall cost or if isolation of the final product is complicated by the presence of the
soluble protein, the cost of immobilization can be offset by the gains in productivity
and improved product quality. The intent of this section is to describe, in general
terms with illustrative examples, the features and considerations of these broad
classes of enzyme immobilization as they impact their application to biocatalysis.
Detailed experimental protocols are available in the original literature and exemplary
protocols for these methods are offered in many excellent reviews and texts 141.
6.2
Methods of Immobilization
Thousands of publications and patents detail the immobilization of specific enzymes

using an impressive array of strategies. The majority of these immobilization
techniques can be divided into four broadly defined groups:
- non-covalent adsorption of an enzyme onto a solid support;
- covalent attachment of an enzyme to a solid support;
- entrapment of an enzyme in a polymeric gel, membrane or capsule;
- cross-linking of an enzyme with a polyfunctional agent.
The first three classes involve the use of a solid matrix to support or entrap the
enzyme and to confer the desirable mechanical properties of the solid carrier
(Fig. 6-1 A-D). The last method entails covalent linking of the enzyme to itself with
no additional support (Fig. 6-1 E). Each ofthe covalent methods requires one or more
covalent bonds between reactive groups on the enzyme surface with complementary
groups on the carrier, either directly or through the action of a multivalent cross-
linking reagent. Covalent attachment methods result in direct chemical modification
G.2 Methods oflmmobilization
I 165
Figure 6-1. Classification o f Immobilization Methods.
of the protein molecule. Non-covalent methods are based on formation of an

enz yme/c,arrier complex through simple physical confinement or by electrostatic
attraction, hydrogen bonds, van der Waals interactions, and so-called hydrophobic
interactions. Matrix entrapment (Fig. 6-1 C) and encapsulation (Fig. 6-1 D) are both
considered to be methods of entrapment in this chapter. A summary of the
advantages and disadvantages of each of these four classes of immobilization is
given at the end of this section in Table 6-1.
6.2.1
Non-Covalent Adsorption
Adsorption of an enzyme to a solid carrier is characterized by the interaction of a

protein with a solid surface through reversible, non-covalent binding. The inter-
action forces in adsorption processes range from relatively strong ionic and hydro-
gen bondiing to weaker van der Waals forces and “hydrophobic”interactions of the
protein with the support. Electrostatic forces of ionic and hydrogen bonding are
much stronger than purely hydrophobic ones, and so can afford a tightly bound
protein, even in purely aqueous media. Immobilization by adsorption has the
advantage of simplicity, is often inexpensive, and does not usually result in disrup-
tion of the catalytic protein structure. No chemical modification of the protein or
166
I support occurs; however, binding to the carrier is reversible and leaching of the
G Immobilization ofEnzymes
protein can be a problem. Moreover, in cases where the binding forces are weak,
there is little stabilization of the enzyme tertiary structure relative to the solution
form of the enzyme. Interaction of the reaction substrate or products with the
support can cause desorption of the adsorbed enzyme. This reversibility of binding
can in some situations be advantageous; if the protein catalyst has become inacti-
vated from extended use, the resin can be regenerated by a change in pH or solvent
to desorb the deactivated enzyme from the carrier and then fresh biocatalyst added
under binding conditions. While more exotic or expensive proteins often warrant the
use of covalent binding methods, adsorption is most often used in large scale
industrial processes because of the low cost and simplicity.
Electrostatic binding of enzymes to polyionic carriers is operationally simple, once
appropriate binding conditions are identified. The carrier is first equilibrated using
aqueous media at the appropriate pH, solvent composition and ionic strength. An
aqueous solution of the enzyme is then treated with the adsorptive solid under
conditions of protein concentration, pH, ionic strength, and temperature that have
been determined experimentally to give efficient protein binding. If the binding
protocol is relatively selective, the immobilization can also effect purification of the
enzyme from cell debris and fermentation by-products.The immobilized biocatalyst
can be recovered by filtration and then washed. Air drying or washing with a water-
miscible organic solvent can be used to give a dried biocatalyst solid for use in non-
aqueous media. A disadvantage of this method is that the support materials used in
ionic adsorption are polyfunctional and charged and thus can dramatically change
the microenvironment of the protein. Steric hindrance to diffusion of substrate and
product can also be a problem, due to the short protein-support distance in tight
ionic bonding.
Ionic adsorption of proteins is one of the oldest methods of protein immobiliza-
tion and has been used widely in industry. Chibata and co-workers developed one of
the earliest industrial biocatalytic processes using an amino acylase adsorbed on
diethylaminoethyl (DEAE) carbohydrate resin for the kinetic resolution of amino
acids '1. Macroporous synthetic ion exchange resins, based on those originally
developed for chromatography and water treatment, are among the most frequently
used carrier materials. The protein is bound through association with the side chains
of amino acids such as aspartate and glutamate (carboxylate) and lysine (ammo-
nium) through oppositely charged groups on the carrier. The tightness of binding is
dependent on the proximity and charge of the binding residues on the protein
surface and the carrier. Protein binding can be quite tight if factors which affect
ionization such as pH, counter-ion identity, hydrophobicity and ionic strength are
optimal.
A demonstration of the principles of ionic adsorption is found in the use of
glucose isomerase bound to DEAE-cellulose[61 for the conversion of glucose syrup to
high fructose corn syrup. Remarkably little enzyme desorption of glucose isomerase
is observed over many months, despite the use of elevated temperatures and high
flow rates through columns of the resin-bound enzyme. During the lifetime of the
catalyst, 1 g of catalyst converts 15000 g (dry substance) of high fructose corn syrup.
G.2 Methods oflmmobilization
However, once inordinate activity has been lost, the protein can be easily removed by
a simple shift in pH and then the resin regenerated in situ.
Li.pases from Candida cintarctica, Humicola lanuginosa, and Mucor meihei, useful
for enantioselective ester hydrolysis or tranesterification, have also been immobi-
lized by ionic attachment to synthetic resins 1'1. For the interesterification of fats and
oils, macroporous (>lo0A pore diameter) methacrylate resin cross-linked with
divinylberizene gives virtually quantitative binding of the protein. The air-dried resin
can be used to catalyze interesterification of oils in the absence of solvent[']. The
preparatio'n of Candida anarctica B lipase has been widely used for the resolution of
carboxylic acids and alcohols ['I.
Ionic attachment to noriionic surfaces can be effected through the intermediacy of
a polyvalent metal catiori[lO].Chelation of a transition metal by both the carrier
surface and the enzyme results in binding to the surface. Inorganic oxides (such as
silica) or polyhydroxylated biopolymers (such as polysaccharides) are used as solid
supports in cornbination with polyvalent transition metals capable of binding
multiple ligands such as Ti(V).This type of chelation binding is also used extensively
in the isolation of genetically engineered proteins by incorporation of a poly-
histidine tag sequence. The poly-His sequence chelates tightly to Cu(I1) or Ni(II),
providing a selective means for selective recovery of the protein["].
Affinity binding is an important sub-group of ionic protein adsorption methods.
Specific electrostatic and hydrophobic interactions between the target enzyme and
an immobilized ligand allow for extremely tight, selective binding of the protein of
interest. The ligand may be a small molecule or a large protein such as an antibody;
however, the loading capacity tends to decrease with the effective molecular weight
of the ligand. One of the most frequently used affinity binding systems is the
cornbination of biotin with the protein avidin. Avidin is a tetrameric protein which
binds four biotin ligands ! ~ specific
y ion-pair interactions with a dissociation constant
of .about lo-'' M. In a typical embodiment, biotin derivatives that are linked to a
reactive fiinctional group are covalently attached to both the solid surface and to the
protein. The biotinylated solid is first treated with avidin, and this is followed by
treatment with the biotinylated enzyme [I2]. The expense and necessity for extensive
ma.nipulakions make this method of affinity binding practical only for aqueous
systems and those using very highly valued enzymes.
Immobilized enzyme preparations that are bonded only through strictly non-
iordc, physical adsorption are rare; however, in non-aqueous systems physical
adsorption can be a very effective approach. In purely hydrophobic binding, the
protein molecule is not solvated by the bulk reaction solvent sufficiently to overcome
the weak interaction forces with the solid surface, and so the protein does not desorb
from the carrier. In many of these non-aqueous systems, there is thought to be
activation of the protein !JY the support providing a more hydrophobic environment
which facilitates wetting and interaction with the non-polar substrate and by
distribution of the enzynie over a larger surface area. Alternatively, activation of the
lipase enzyme by interaction of hydrophobic regions on the protein with the
hydrophobic surfaces have been postulated. Dispersion of lipases over a high-
surface hydrophobic polymeric carrier such as polypropylene or nylon has been
168
I 6 Immobilization ofhzymes
shown to activate this enzyme in organic solvent media relative to particles of the
untreated protein prepared by lyophilization. Hydrophobic binding of lipases is
sufficiently strong to allow their use in purely aqueous media, presumably because
of the affinity of this protein for waterloil interfaces. Pate1 has reported that a lipase
immobilized on polypropylene could be reused for ten cycles without loss of activity
in the kinetic resolution of a key intermediate for semi-syntheticT a ~ o l [ ~ ~ ] .
Adsorption on polar, nonionic carriers represents the middle ground in non-
covalent attachment, where a combination of hydrogen bonding and dipole inter-
actions helps to bind the protein to the support. The immobilized lipase used in the
upgrading of low value fats and oils by interesterification is a successful example of
this mode of non-covalent adsorption. In one example, an aerosol of an aqueous
solution of the lipase is sprayed onto finely divided silica and then the particles are
agglomerated to give the particulate biocatalyst. The simplicity and effectiveness of
this adsorption process afford a dried immobilized biocatalyst with sufficient
productivity to be used on a manufacturing scale at relatively high temperatures [I4].
6.2.2
Covalent Attachment
The immobilization of enzymes by covalent attachment to a solid carrier involves

formation of a covalent bond between amino acid side chain residues of the protein
with reactive groups on the support surface. Covalent attachment is often the
method of choice where the protein value is high, minimal protein leaching from the
support is required or rational control of the biocatalyst properties is desired.
Because of the stronger carrier-protein linkage, the resulting heterogeneous bio-
catalyst can be much more stable than those prepared by adsorption or entrapment.
The most common protein functional groups involved in covalent bonding are
nucleophilic amino (lysine, hisitidine and arginine), thiol (cysteine) and hydroxyl
groups (serine, threonine and tyrosine) and electrophiliccarboxylate groups (aspartic
acid and glutamic acid). The reactivity of these functional groups can be modulated
through chemical modification, but this can be detrimental to activity and the extra
degree of complexity is not often warranted.
Rational control of properties of the immobilized biocatalyst is possible with
covalent binding; by choice of the reactive functional group on the support and
control of its distribution, the practitioner can control the nature and degree of
protein modification and the microenvironment of the immobilisate. Binding with
minimal loss of catalytic activity is thought best to occur with residues on the surface
of the protein and should involve groups that are remote from the active site of the
enzyme to avoid deactivation. In the preponderance of cases, primary amino groups
on the protein surface are coupled with electrophilicgroups on the support material.
Surface-exposedlysine and arginine residues are allowed to react with electrophiles
via alkylation, conjugate addition, imine formation or acylation. Alkylation and
conjugate addition proceed with retention of the net protein charge. Less frequently,
carboxylate residues on the enzyme are activated for reaction with nucleophilic
functional groups on the carrier. Figure 6-2 depicts some of the more commonly
6.2 Methods oflmmobilization
I
169
Figure 6-2. Examples of Corrimon Carrier Activation Methods.
used combinations of reactive protein groups and activated supports['5]. The choice
of reactive group is important; highly reactive groups may result in non-specificover-
modification, and chemical functionalization that adds or removes charge from the
protein can alter the actikity and stability. There are many examples of more limited
and specific attachment methods using reagents selective for less common amino
acids. No one support and linker is ideal and the large number of supports and
binding method leads to an enormous number of possible permutations.
The preparation of covalently bound immobilized enzymes involves treatment of a
solution of the protein with the reactive support. Judicious choice of conditions
including enzyme concentration, pH, and ionic strength can be used to increase the
yield of bound activity. The loading capacity of the carrier can be estimated from the
manufacturer's specifications or by titration with reagents specific for the reactive
functional group. A competitive inhibitor or high concentration of substrate may be
used during attachment to protect the active site and to maintain the active
conformation of the enzyme. After incubation, the resin is washed repeatedly to
remove unbound protein, and then the free reactive sites are quenched by treatment
with an appropriate nucleophilic or electrophilic reagent (for example, glycine or
acetic anhydride).
Either the solid support or the enzyme may be activated, but to limit disruption of
the enzyme tertiary structure the functional groups of the support material are most
often actiwated. The activation may occur prior to the coupling reaction (pre-activated
supports), or a bi-functional linking reagent may be used to form the bond between
G lmmobilization of Enzymes
Table 6-1. A Comparison of immobilization methods.

Immobilization Method Advantages Disadvantages
Adsorption Simple Weak binding, leaching of

No chemical modification of enzyme
enzyme Little or no stabilization
Reversible Non-specificbinding
Often inexpensive May limit mass transfer
Covalent Tight binding Chemical modification of
Wide variety of supports and enzyme
linkers available Often expensive
Rational control of enzyme Activity diluted by carrier
loading, distribution and mi- May limit mass transfer
croenvironment
Entrapment and Encapsu- No chemical modification of Little or no stabilization
lation enzyme Environmental changes can
Can be simple disrupt network and cause
Efficient for whole cells leakage
Often limits mass transfer
Cross-linking High volumetric activity Chemical modification of
Compatible with elevated tem- enzyme
perature and organic solvents Little control of particle prop-
No carrier required erties (especiallyfor precipi-
Tight binding tate and whole cell)
Efficient for whole cells Requires crystallization o f
enzyme (for CLEC@)
May limit mass-transfer
protein and support. A comparison of the various immobilization methods is given

in Table 6-1.
6.2.2.1
Carriers for Enzyme Immobilization
The physical and chemical properties of protein molecules are often not compatible
with the conditions used in most chemical syntheses, and so fixation to a solid
carrier is one effective strategy to alter the properties of the biocatalyst. By binding of
the protein to a proportionately large amount of a solid carrier, the bulk properties of
the resultant solid biocatalyst are more derived from the carrier than from the
protein. Enzymes are subject to denaturation conditions found in typical chemical
processing such as high concentrations of organic reagents and high shear forces.
Moreover, proteins are water soluble and amphipathic thus causing emulsions on
extraction and being difficult to recover and reuse. The carrier-fixed biocatalyst is
often more resistant to deactivation by organic reactants or shear and can be
recovered by simple filtration. In many cases the recovered biocatalyst maintains
catalytic function and may be reused many times.
An enormous number of carriers are available for the immobilization of enzymes
Table 6-2. Carrier types
I 171
Organic - synthetic polymer Organic - biopolymer Inorganic
Polv.amides Polysaccharide Minerals

Nylon
Cellulose Sand
Polyalkylene
Starch Pumice
Polystyrene
Agarose Metal oxides
Polyacrylates
Dextran Diatomaceous earth
Polyacrylamide
Chitin Clays
Polyethylene
Polyalginate
Polypropylene
Carrageenan
Polyvinyl alcohol ~~~~ ~
Polyvinyl.acetate Proteinaceous Synthetic

Polyvinykhloride
Gelatin Glass, controlled pore glass
Polyethylene glycol
Collagen Zeolites
Polyester
Silk Silica
Polycarbonate
Albumin Sol-gel
Pol)~~rethane
Bone Alumina
Polysiloxane
Metal Oxides
Phenol-fonnaldehyde
Metals
and a wide range of methods have been used for fixation of protein to these carriers.
For rapid preparation of laboratory samples, commercially available pre-activated
macroporous resins are available. Considerations of the desired properties of the
immobilized biocatalyst such as ease of use, mechanical strength, activity density,
stability, intended application, cost, and availability help to determine which carriers
and methods of attachment are appropriate. In most industrial applications, cost of
the support and efficiency of immobilization are paramount, while in biomedical
applications binding efficiency and ability to sterilize can be most important.
Classification of materials used in solid carriers is given in Table 6-2.
When the mass of carrier material is large relative to that of the enzyme, the
physical and chemical properties of the carrier (Table 6-5) will, in large part,
determine properties of the resultant immobilized enzyme. Often, the carrier will
impart mechanical strength to the enzyme, allowing repetitive recovery by simple
filtration of the solid particles and reuse of the enzyme. The degree of porosity and
pore volume will determine the resistance to diffusion and molecular size selectivity
of the biocatalyst. When used in non-aqueous media, dispersion of the enzyme over
a large surface area can greatly increase its activity. Table 6-3 summarizes many of
the key properties and considerations for enzyme carrier materials.
6.2.3
Entrapment and Encapsulation
Entrapment can be defhed as any system in which an enzyme or whole cell is

physically restricted within a confined space or network. This class of immobiliza-
tion is often extended to include systems where a combination of physical entrap-
172 I 6 lrnrnobilization ofhzyrnes
Table 6-3. Summary of properties and considerations for enzyme carriers.

Property Examples or typical range Characteristics and considerations
Binding mode Covalent, ionic or physical ad- Binding strength, enzyme stabiliza-
sorption. Pre-activated or activat- tion, ease of use, protein charge
ed in situ.
Shape Bead, flat sheet or hollow fiber Ease of filtration,
membrane, amorphous aggre- Control of diffusion path length and
gate flow properties
Crystal Simple preparation
Surface area >SO m2/g Binding capacity,volumetric activity
Porosity, 71 ml/g, Resistance to diffusion, molecular
pore size 2-50 nm weight sieving, flow properties,
enzyme retention
Particle size and dis- 1 pm to 1 mm Ease of filtration, sedimentation
tribution velocity
Density Resistance to diffusion, sedimenta
tion velocity
Safety Sterility, toxicity, regulatory approval
for food and drug use, consumer
and worker exposure
Mechanical strength Resistance to shear, compression,
tearing of membranes or particle
fracture
Compressibility Rigid particle to soft gel Ease of filtration and handling,
column packing
Solvent compatibility Swelling, dissolution of carrier,
enzyme desorption, controlled
dissolution
Hydrophobicity and Hydrophobic to polyionic Alteration of substrate selectivity,
charge shift of pH/rate optimum, enzyme
stabilization, binding force and
capacity
Reactive site distribu- Evenly distributed or only on Surface vs. bulk attachment, multi-
tion surface point attachment, stabilization
Loading capacity 0.1% to 10% w/w Enzyme/carrier ratio, volumetric
activity
cost “Free”to 1000s of USD/g Economics, availability for scale-up,
catalyst productivity and lifetime
merit and covalent binding is used. Entrapment immobilization includes enzymes

contained within such diverse systems as polymeric matrices, hollow-fiber ultra-
filtration membranes, liposomes, cross-linked arrays, or cross-linked whole cells.
Depending on the density of the entrapment matrix, the environment of the protein
can be similar to that of the protein in the bulk reaction media, and disruption of
catalybc activity is relatively minor. The pore structure of the matrix used for
I 173
entrapment is such that small molecules (substrates and products) are able to diffuse
in and out of the matrix, while the macromolecular enzyme is maintained within the
network. Mass transfer limitations are almost always an issue with entrapped
enzymes and whole cells, since precise control of pore size is usually not possible.
Often a certain fraction of the enzyme is able to diffuse from the network, and
swelling of these molecular networks by a change in reaction conditions can
accelerate this leakage of protein.
Entrapment of enzymes,or whole cells in a cross-linkedpolymeric network can be
achieved by a number of methods. The most common methods of gelation of a
polymer or pre-polymer include:
Cross-linking of a pre-formed polymer or formation of a polymer network in the
presence of the biocatalyst;
Solvent-,temperature-, 'or pH-induced precipitation;
Addition of multivalent cations to a polyacid.
Polyacrylate and po1yacry:lamide gels have been found to have favorable properties
for the entrapment of enzymes and whole cell biocatalysts116]. These gels are
sufficiently hydrophilic to provide an environment similar to that of the bulk
aqu.eous :solution. Acryla mide or methacrylate monomers, for example, can be
polymerized in the presence of enzymes and polyfunctional cross-linkers to form a
gehentrapped biocatalyst preparation. The stiffness of the gel and pore size can be
controlled by the amount and type of cross-linker used. Higher degrees of cross-
linking and short spacer groups give a stiffer gel, while longer spacer groups give
larger pores. The average molecular weight of the gel can be influenced by the
amount of free radical initiator used, the reaction time and the temperature of
polymerization. The particle size can be controlled by mechanically cutting the
particles to the desired size or by performing the polymerization under emulsion
polymerization conditions.
Rhodoc,occus sp. microorganisms which express high levels of nitrile hydratase
have been entrapped in polyacrylamide and polyacrylate resins for the conversion of
acrylonitrile to acrylamide ["I. Limitations common in cell entrapment such as resin
swelling, deactivation du:ring the entrapment, mass transfer limitations of substrate
and product were addressed by the control of mixing rate during polymerization, gel
density, particle size and resin hydrophobicity. Activation of carboxylate residues in
the polymer matrix by conversion to the hydrazide improved retention of the
enzyme, presumably through the covalent attachment of lysine side chains on the
enzyme surface via amide linkages [*'I.
Gelation of polyanionic or polycationic polymers by the addition of multi-valent
counter-ions is a simple and common method of entrapment of enzymes and whole
cells. In one common embodiment, whole cells or enzymes are entrapped by the
drop-wise addition of an aqueous solution of sodium alginate and the biocatalyst to a
concentrated solution of a Ca2+salt. The cation acts as a cross-linking agent towards
the alginate biopolymer and the droplets precipitate as beads with the biocatalysts
entrapped within the network. Although the beads are relatively soft and unstable,
this method has been one of the preferred methods for entrapment of whole cells. A
G Immobilization ofEnzymes
174
I second commonly used example of this technique, gel formation using K-carragee-
nan in the presence of high concentrations of potassium salts, has been used for the
immobilization of asparatase producing cells for the production of L-asparticacid ['I.
Similarly,carrageenan entrapment of yeast cells has been used on an industrial scale
for the production of malic acid by the action of fumarase on fumaric
Leakage of enzyme is often a problem in these systems, especially on exposure to
ion-complexing agents such as phosphate buffer. The mechanical properties and
enzyme retention can be improved by treatment with glutaraldehyde or other
covalent cross-linking reagents.
The term encapsulation has been used to distinguish entrapment preparations in
which the biocatalyst environment is comparable to that of the bulk phase and where
there is no covalent attachment of the protein to the containment medium (Fig. 6-1
D)C2'1. Enzymes or whole cells may be encapsulated within the interior of a
microscopic semi-permeable membranes (microencapsulation) or within the inte-
rior of macroscopic hollow-fiber membranes. Liposome encapsulation, a common
microscopic encapsulation technique, involves the containment of an enzyme
within the interior of a spherical surfactant bilayer, usually based on a phospholipid
such as lecithin. The dimensions and shape of the liposome are variable and may
consist of multiple amphiphile layers. Processes in which microscopic compart-
mentalization (cf. living cells) such as multienzyme systems, charge transfer
systems, or processes that require a gradient in concentration have employed
liposome encapsulation. This method of immobilization is also commonly used for
the delivery of therapeutic proteins.
Enzyme-membrane reactors represent an interesting subset of macroscopic
enzyme entrapment [22]. A semi-permeable ultrafiltration membrane with a suffi-
ciently low molecular weight cut-off restricts passage of the enzyme to the bulk
substrate and product phase. In these reactors, the soluble enzyme can be used in a
continuous fashion as the product isolation is isolated in a separate vessel from the
enzyme. Progress in this application has been facilitated by the availability of
solvent-resistant membranes with tighter pore size distributions. The membrane
can be used to simply separate the enzyme and bulk substrate and product phases or
to separate the aqueous enzyme phase from an organic phase containing substrate
and product. The resolution of L-methionineby the enantioselectivehydrolysis of N-
acetyl-L-methioninehas been performed on the scale of hundreds of tonnes/year in
a continuous process using soluble amino acylase in a membrane reactor[231. An
extension of this strategy to cofactor restriction was effected by coupling the cofactor
nicotinamide adenine dinucleotide (NAD+) with polyethylene glycol to increase its
molecular weight. Co-entrapment of the pegylated cofactor with the soluble enzymes
leucine dehydrogenase and formate dehydrogenase in the asymmetric reductive
amination of trimethylpyruvate to L-tert-leucine[241 allows thousands of turnovers of
the expensive cofactor. In the synthesis of the key chiral intermediate for Diltiazem,
a lipase entrapped an asymmetric hollow fiber membrane performs the kinetic
destruction of the undesired enantiomer. The membrane serves to maintain an
aqueous environment for the enzyme and an interface between the buffer phase and
that of an organic phase which contains the substrate phenylglycidate ester [251.
G. 3 Properties oflmmobilized Biocatalysts
I 175
6.2.4
Cross-Linking
Imrnobilization by chemical cross-linking without the addition of an inert carrier or

matrix can provide the means to stabilize and reuse a biocatalyst without dilution of
volumetric activity. A major deficiency in all of the aforementioned immobilization
methods is that a substantial amount of a catalytically inert carrier or matrix is used
to bind or contain the biot atalyst. In many cases, the amount of carrier is two orders
of magnihde higher than the protein catalyst. Unfortunately,direct cross-linking of
the enzyme, followed by precipitation of an amorphous solid often results in low
activity arid poor mechanically properties and so this method is not often used.
Recently, however, cross-linked enzyme crystals have been reported to give many of
the desirable properties of immobilized enzymes without the need for a support
material (Sect. 6.4.1).
Chemical cross-linking of an enzyme within its host cell is a simple and
economical method to produce an entrapped or encapsulated biocatalyst, eliminat-
ing the need for isolation or purification of the enzyme. Whole cells may be lysed or
left intact and then chemically cross-linked by the addition of polyfunctional
reagents such as glutaraldehyde or toluene diisocyanate. The mechanical properties
of such preparations are poor, but can be improved by the addition of support
matrices such as gelatin or synthetic organic polymers (which, technically, are
considered to be entrapment methods). Cross-linking of whole cells is an effective
entrapment method for relatively stable enzymes that do not require additional
stabilization of the support matrix. One of the largest industrial biocatalytic proc-
esses, that to produce high fructose corn syrup, can employ the biocatalyst as a cross-
linked whole cell preparation. A patent assigned to Nov0[~'1 describes the im-
mcibilization of glucose isomerase via entrapment of the lysed cells of the host
organisml within a cross linked network of glutaraldehyde and, optionally, an alkyl
diamine.
6.3
Properties of Immobilized Biocatalysts
As with most heterogeneous catalysts, it is often difficult to characterize im-

mobilized enzymes at a molecular level. Most immobilized preparations are often
complex mixtures with a distribution of chemically modified protein species. The
gross caialytic properties observed are a composite of those of a range of differ-
entially modified individual proteins, often irregularly distributed within the sample.
Mass transfer limitations and microenvironment effects further complicate charac-
terization.
176
I G Immobilization ofEnzymes
6.3.1
Mass Transfer Effects
The catalytic behavior of enzymes in immobilized form may dramatically differ from
that of soluble homogeneous enzymes. In particular, mass transport effects (the
transport of a substrate to the catalyst and diffusion of reaction products away from
the catalyst matrix) may result in the reduction of the overall activity. Mass transport
effects are usually divided into two categories - external and internal. External effects
stem from the fact that substrates must be transported from the bulk solution to the
surface of an immobilized enzyme. Internal difhsional limitations occur when a
substrate penetrates inside the immobilized enzyme particle, such as porous
carriers, polymeric microspheres, membranes, etc. The classical treatment of mass
transfer in heterogeneous catalysis has been successfully applied to immobilized
enzymes [271. There are several simple experimental criteria or tests that allow one to
determine whether a reaction is limited by external diffusion. For example, if a
reaction is completely limited by external diffusion, the rate of the process should
not depend on pH or enzyme concentration. At the same time the rate of reaction
will depend on the stirring in the batch reactor or on the flow rate of a substrate in
the column reactor.
The extent of internal mass-transfer is usually expressed by the efficiency coeffi-
cient N,
where V,, and Vsolare the rates of the reaction catalyzed by an immobilized and
soluble enzyme, respectively. In order to find out whether a reaction is limited by
diffusion one can calculate n as a function of the Thiel modulus (Fr)
Where R is the carrier radius, De%the effective diffusion coefficient of the substrate,
E is the enzyme concentration in the carrier, and kcat and K, are the kinetic
parameters of an enzyme. From a practical standpoint it is important to remember
that there are no diffusional limitations as long as substrate concentration S exceeds
K,. This condition normally exists at the beginning of many processes catalyzed by
immobilized enzymes. At the end of the process, when a substrate is depleted and
effective K, may increase because of the product inhibition, the whole reaction may
be limited by diffusion.
6.3.2
Partition
The other important phenomenon that, in addition to the mass transfer, occurs
when enzymes become heterogeneous catalysts, is the partitioning of substrates,
products, inhibitors, metal and hydrogen ions between a bulk solution and a carrier.
An elegant and simple theory describing the effect of microenvironment inside the
particles of immobilized enzymes on their kinetics, has been developed by the group
G.3 Properties oflmmobilized Biocatalysfs
I 177
of K.atchalsky[28].In particular, the theory explains why one often observes shifts in
pH profiles of activity with immobilized enzymes; it is due to the redistribution of
hydrogen ions between a bulk solution and a carrier. As a practical consequence, one
should use a negatively charged carrier if a shift of pH profile to a more alkaline pH
is desired and a positively charged carrier if the opposite shift to an acidic pH region
is necessary. However these electrostatic effects exist in solutions with low ionic
strength and almost disappear when salt concentration increases. In general, the
partitioning of substrates and products between a solution and a carrier may occur
whenever the character ofa carrier (charge, hydrophobicity, etc.) differs from that of
a bulk solution. As a result, the binding constants for substrates (K,) and for
products (,KJ with immobilized enzymes may vary dramatically from those observed
for free enzymes.
6.3.3
Stability
One of the chief benefits of enzyme immobilization is the ability to use them
repeatedly in chemical reactors on a large scale. Usually this cannot be achieved
without a significant increase of enzyme stability. It is clear that attachment of an
enzyme to a solid surface greatly limits deactivation by intermolecular protein-
protein processes such a:: aggregation or proteolysis. In some cases, this is the only
stabilization provided by immobilization. In other cases, immobilization leads to
stabilization of the three-dimensional catalybc structure against intramolecular
protein denaturation under conditions such as elevated temperature, extremes of
pH, organic solvents and oxidants. Protein unfolding can be prevented by multi-
point attachment of a protein to a support; however, it is not clear whether this
increase in rigidity is generally beneficial to catalytic function. As an approximation,
the optimal immobilization is given by the maximum functionalization which
results in minimal activily loss. Over modification of the enzyme often results in loss
of activity and stability. In some specific cases, covalent multipoint attachment of a
prtotein to a solid carrier clearly enhances the resistance to chemical and thermal
An increase in the number of both polar (electrostatic) and
hydrophobic interactions among the protein molecules when a protein goes from a
frce to immobilized environment may also significantly enhance stability of proteins
against heat and other denaturants I3O1 by preventing unfolding, aggregation or
dissociation of the proteins 13'1. Moreover, observed stabilization effects correlate
w th the number of contacts involved[32].The intermolecular cross-linking of
proteins by glutaraldehyde and other cross-linking may, in turn, lead to
additional thermostabilization of proteins by preventing their unfolding.
6.3.4
Activity of Immobilized Enzymes
On the surface the activity assay of immobilized enzymes is quite simple and is not
very dissimilar from measuring the activity of soluble enzymes. In both cases the
G lrnrnobilization ofhzyrnes
178
I Table 6-4. Parameters for characterization of immobilized biocatalysts.
General description Preparation Physical and chemical Kinetics
characterization
Reactionscheme Method * Particle shape, Initial rate versus con-
Enzyme and source Detailed reaction diameter, swelling centration for free and
Carrier type conditions Compression in immobilized enzyme
Method of immobi- Dry weight yield columns or abra- pH and buffer effects
lization Activity left in sion in stirred ves- Diffusional limitations
supernatant sels or sedimenta- Effect of particle size
Enzyme leakage, tion and abrasion in and enzyme loading
conditions fluidized bed Degree of conversion
versus residence time
Storage stability
Operational stability
activity is measured in pmol of substrate per minute per gram of a catalyst under
defined conditions (concentrations, pH, temperature, etc.). Yet, the heterogeneous
nature of immobilized enzymes poses additional challenges. First, special care
should be taken in choosing a representative sample of an immobilized enzyme.
Second, the activity of immobilized enzymes is much more sensitive to external
parameters, such as stirring, and may be limited by diffusion (see above).Third, the
determination of true catalytic parameters is more difficult, since the amount of the
active enzyme attached to the carrier is not easy to measure. One has to realize that
the k,,,, K, and Ki measured for immobilized enzymes often represent the effective
parameters. This is further complicated by surface activation effects in lipases [341.
The complexityof the physical and catalytic properties of immobilized biocatalysts
and the difficulty in comparison of effectiveness based on literature descriptions has
led to the publication of guidelines for the characterization of immobilized bio-
catalyst^^^^]. The authors suggest that description of parameters listed in Table 6-4
should be the minimum required for characterization of an immobilized prepara-
tion.
6.4
New Developments and Outlook
Opportunity for innovation and creativity still exists in the field of biocatalyst
immobilization. Despite the tremendous volume of biocatalyst immobilization
literature, there is no one technology that is universally applicable and no one
technique that can be applied using a generic procedure. The limitations of
individual immobilization techniques have been pointed out in each section.
Operationally simple adsorption methods often are limited by the lack of stabiliza-
tion and by protein leaching, especially under aqueous conditions. Restriction of
diffusion can be severe for entrapped proteins and cells. Covalent methods often
result in protein inactivation and a much higher carrier cost. The combined effects of
G.4 New Developments and Outlook
inefficiency in protein binding, carrier expense, protein inactivation on binding,

I 179
restricted substrate diffusion, enzyme leaching, and enzyme denaturation during

use can result in a tremendous overall activity loss and increase in cost when
compared to the native biocatalyst. For example, with most current carrier-fixation
technologies, Ra~or[~‘] estimates that a 10- to 25-fold overall increase in cost can be
expected in converting an enzyme to its immobilized form. Recent work in the field
of biocatalyst immobilization has focused on the development of more efficient
systems that employ relatively inexpensive support materials (see for example [141),
and in some cases, no support at all (Sect. 6.4.1).
6.4.1
Cross-linked Enzyme Crystals (CLEC@)
Early work in protein X-ray crystallographic structure determination demonstrated

that protein crystals could be stabilized by cross-linking with glutaraldehyde13’1.
More recently, cross-linked enzyme crystals (CLEC@)have been shown to be highly
active and stable heterogeneous biocatalyst preparations 1381. In this method, a
polyfunctional cross-linking agent is allowed to diffuse into a protein crystal such
that the protein is cross-linked throughout the entire particle. In this case the
enzyme is not linked to a carrier, but to adjacent enzyme molecules within the
crystal. The protein itself is thus both catalyst and carrier. Electrostatic and hydro-
phobic contacts within the crystalline lattice, combined with added covalent cross-
linkers, help maintain protein activity and stability in aqueous and organic media. It
has been proposed that a higher degree of chemical functionalization is possible
than with attachment to a two-dimensional surface because the added protein-
protein contacts within the crystal particle stabilize the tertiary structure.
Iinmobilization by cross-linking of enzyme crystals appears to be a generic
method; however, unique protocols must be developed for each individual protein.
Preparation of a CLEC form of many types of proteins and classes of enzymes have
been reported including hydrolases, oxidoreductases, carbon-carbon lyases and
isomerases. Crystallization of the protein is a highly effective purification step, so
Figure 6-3. Graphic Comparison o f 6 A

Zeolite B Channel (A) and 21 A Thermoly-
sin Crystal Pore (B).
180
I G lmmobilization ofhzymes
Figure 6-4. Cross-Linked Enzyme Crystals@ o f

Thermolysin, Average Length 40 pm.
that undesired side activities can also be However, conversion of a

soluble protein to a cross-linked enzyme crystal form requires development of
procedures of protein crystallization that are specific for each protein, so that no
generic protocol can be applied. The high degree of porosity (average of 50%) and
large pore diameter (20-100 A) of most protein crystals allow relatively unrestricted
diffusion of small organic molecules (<2000 Da) within the crystal. This unrestricted
diffusion, combined with the absence of an inert carrier, results in maximal
volumetric activity for CLEC immobilized enzymes. As with all immobilized
biocatalyst particles, mass transfer limitations can become appreciable as the rate of
catalysis increases, as molecular radius increases and when larger crystal particles
are used. The rate of enzyme catalysis in organic media for hydrolases in CLEC form,
long considered to be two or three orders of magnitude lower than catalysis in water,
have been shown in some instances to be equal to or greater than that of the
corresponding hydrolytic reaction f4O]. Cross-linked enzyme crystals of lipases r3’l,
proteases and penicillin acylase [421 have been made on the multi-kilogram scale
and used in industrial processes to produce tonnes of drug intermediate per
Cross-linkedenzyme crystals, in contrast to untreated protein crystals or cross-linked
amorphous precipitate, are mechanically tough. In high shear mixing or repetitive
filtration cycles of the CLEC form of penicillin acylase, there was no observable
particle attrition. Kasche and Tischer have compared the relative benefits of CLEC
immobilization and traditional carrier fixation. The authors conclude that CLEC
enzymes follow the same kinetic laws as traditionally immobilized enzymes and that
the chief benefits are the lack of inert carrier, operational stability and high
volumetric activity
An alternative approach to CLEC technology, the cross-linking of protein in
solution followed by precipitation or freeze drying has been used for some time to
avoid the use of inert carrier (see Sect. 6.2.4). Recently it has been reported by
Sheldon that a cross-linked enzyme aggregate (CLEA)formed by first precipitation
of penicillin acylase using salt or alcohols followed by chemical cross-linking gives
an insoluble enzyme preparation with excellent activity and stability in water and
organic solvents l4’1.
6.4.21
6.4 New Developments and Outlook
I 181
Sol-Gel
Reetz [4G1 and others [471 have found that entrapment of lipases within a hydrophobic
silica sol-gelcan result in ;i biocatalyst whose activity in organic media is enhanced in
comparison to the corresponding lipase powder under the same conditions. A silica
matrix is generated in the presence of an aqueous solution of the lipase by treating
hydrophobic alkyl alkoxysilanes with a catalpc amount of sodium fluoride. The gel
is allowed! to set, then dried and crushed to the desired average particle size.
Optimization of lipase a’ctivity can be achieved through variation of the hydro-
phobicity of the gel by choice of the alkyl group of the silane monomer, the use of
hydrophilk polymeric add-itives (polyvinyl alcohol or inert proteins), control of water
activity and by the waterlsilane monomer ratio. Activation of up to two orders of
magnitude in the rate of fatty acid esterification relative to the suspended protein
powder h.ave been attributed to the combined effects of dispersion over a large
surface area and the interaction of hydrophobic regions of the polysiloxane gel with
hydrophobic domains of the lipase. The volumetric activity of these systems can be
quite low: protein loadings of 0.1-1 % relative to the carrier are necessary for high
activation factors. Moreover, the lipase is for the most part passively entrapped
within the gel, so its use would be limited to non-aqueous systems. Organopolysilox-
anes carrying pendant groups for covalent binding can be employed for immobiliza-
tion catalysts intended foI aqueous systems [481.
More recently, Ballesteros has extended the use of siloxane gel supports by
developing support systems employing glyceryl poly(alkylsi1oxanes)L4’)] and poly-
(hydroxyniethylsiloxanes)for the gel entrapment of lipases. The authors point out
that the higher water solubility of these polymers allow for better control of the
protein-polymer ratio. Mechanical properties and protein retention of the prepara-
tion were improved by entrapping the poly(hydroxylmethylsi1oxane) lipase ad-
sorllates within a solid cross-linked silicone rubber matrix. The versatile chemistry of
silicone polymers allows the tailoring of the hydrophobicity and rigidity of the
support matrix. Lipase loadings of 1-5% are described for the poly(hydroxyme-
thyl)silox;ine/silicone polymer composites. Entrapment efficiencies are apparently
sufficient to retard protein leaching; however, most aqueous reaction systems
reported employed an organic co-solvent which limits enzyme dissolution. These
lipase/polymer composites could be used for the kinetic resolution of rac:emic
carboxylic acids and alcohols via ester hydrolysis or synthesis with negligible loss of
activity over 10 reuse cyclles.
6.4.3
Controlled Solubility “Smart Polymers”
Enzymes are normally used as water-solublehomogeneous catalysts or immobilized

onto water-insoluble solid supports. The many advantages of immobilized systems
have been outlined in this chapter. Yet insoluble immobilized enzyme preparations
can also have serious drawbacks. First, efficiency with macromolecular or insoluble
G
182
I lrnrnobihzation ofEnzyrnes
substrates such as proteins or polysaccharides is often limited by diffusion. Secondly,

in many chemical processes a product of a reaction is less soluble in the reaction
mixture than a substrate. In these cases, the precipitation of a product from the
reaction mixture makes product isolation and reuse of immobilized enzyme diffi-
cult. Thus, it is highly desirable to create a catalyst which combines the advantages of
both water-soluble (homogeneous) and water-insoluble (heterogeneous) catalysts.
To combine the salient features of immobilized and soluble enzymes, immobiliza-
tion can be performed on so-called “smart polymers”[5o]that undergo a reversible
phase separation from water with small changes in the environmental parameters
(pH, ionic strength or temperature). In the most effective systems, these phase
transitions are fast, completely reversible and take place within a narrow range of
environmental parameters. Several pH-controlled solubility systems have been
developed. They include immobilization of enzymes on carboxymethylcellulose,
synthetic polyelectrolytes or on polyelectrolyte complexesrS11. The drawbacks of these
systems stem from the fact that precipitation normally occurs at low pH (5.5 or
lower), which may lead to inactivation of many enzymes. In addition, the repeated
use of acids and bases for pH adjustment leads to accumulation of salts and
subsequent loss of enzyme activity or change of precipitation pattern. In this regard,
temperature-sensitive polymers [521 may be advantageous if the precipitation can be
achieved at temperatures near that of biological systems. One such system, based on
oligomers of N-is0 propyl acrylamide [531, has been successfully used in the laboratory
for repeated solution-precipitation cycling without significant loss of activity.
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1987, 135, 3-30. W. Hartmeier, Immobili- Enzymes and Cells, G. F. Bickerstaff (ed),
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7
Reaction Engineeringfor Enzyme-CatalyzedBiotransformations
Manfied Biselli, Udo Kragl and Christian Wandrey
7.1
Introduction
The application of isolated enzymes to preparative organic synthesis on an industrial

scale is a matter of active research worldwide. Since the late sixties, immobilized
enzymes have been used in amino acid production in continuous processes on a
large scale['* 1' . In the late seventies, the use of soluble enzymes, especially in
membrane reactors, broadened the scope of enzyme technology[3.41 and opened the
way to simultaneous use of more than one enzyme for complex conversions -
especially coenzyme-dependentbiotransformations ['-I. In the early 1980sthe use of
enzymes was extended further to involve organic solvents L8, 'I.
Enzymes are used as catalysts for large scale bioconversions e.g. glucose iso-
merase in the high fructose corn syrup (HFCS) process[''], penicillinase in the
synthesis of semisynthetic penicillins ["I, as well as aminoamidase[12,131 and ami-
noacylase in the production of L-amino acids. Additionally, a variety of processes
for fine chemical synthesis has been developed, e. g. for amino acids, peptides and a
broad spectrum of other optically active substances [15-231.
Based on the classical methods of enzyme isolation and characterization and the
screening for appropriate microorganisms, about 3200 different enzymes are known
today and are listed with E. C. numbers[", 241. Modern methods of genetic engineer-
ing give access to sufficient quantities of enzymes by overexpression in micro-
organisms, thus reducing costs of enzymes [25-281. Enzyme reaction engineering
allows further reduction of the amount of enzyme consumed per kilogram of
product. Therefore, costs of enzymes do not necessarily dominate the overall cost of
production, but they are often, still, a major factor. The productivity of enzyme
reactors typically exceeds the level of 100 g product per litre of reactor volume per day
(extreme values of 25 kgL-ld-l are reported)[*'] and consequently is no longer far
below the productivity of classical chemical synthesis.
The aim of this contribution is to illustrate some basic aspects of a strategy to
optimize an enzymatic process, starting from considerations focusing on the
enzymes used and the reactions involved. Additional investigation of the enzyme
186
I 7 Reaction Engineeringfor Enzyme-Catalyzed Biotransformations
Downstream
processing
El- - Substrate
Figure 7-1.
- Enzyme
Microorganism
Chemical
catalyst
Process design.
-- -
kinetics yields a deeper insight into the process and is the basis of final optimization
of process performance by reaction engineering methods. A detailed process
optimization is of great significance especially for pilot and production scale.
7.2
Steps of Process Optimization
Considerations about process design precede any detailed process optimization.

Process design includes the choice of an appropriate substrate, of a catalyst and of
methods for downstream processing in order to gain the final product in a defined
purity (Fig. 7-1).
The situation may be exemplified by showing the different catalytic methods of
asymmetric synthesis of L-phenylalanine, starting from six different substrates
(Fig. 7-2). Additionally, fermentation processes, using glucose as the carbon and
energy source, have been developed [30-321.
Within the scope of this chapter, only enzyme-catalyzed biotransformations are
considered, presuming
- a defined product (especiallyimportant in the context of this book is the demand
for high optical purity of the product),
- a defined enzyme (criteria for the usefulness of an enzyme are its activity,
selectivity, stability, coenzyme dependency and its kinetic constants; process
optimization studies have to cover these subjects and therefore may influence the
choice of the enzyme, e. g. using an enzyme from a different source),
- defined substrate(s) (the selection of a substrate including the method of enzy-
matic transformation is determined by availability and price of both enzyme and
substrate (see Fig. 7-2).
The aim of process optimization is to find process conditions defined by
- high conversion of the substrate,
- high selectivity of the reaction,
- high optical purity of the product,
7.2 Steps ofprocess Optimization I187
COOH
0
I I
I I
Figure 7-2. Asymmetric syntklesis o f L-phenylalanine.
- high space-time yield (productivity)of the process,

- low enzyme and coenzyme consumption per unit mass of product.
The overall process development basically consists of three steps (Fig. 7-3).
The jrsf step is the investigation of the reaction system. This can be further
classified into two parts:
First of all the enzyme properties have to be examined, considering the following
aspects:
- reaction catalyzed,
- substrates, cosubstrate:;, coenzymes, effectors, inhibitors,
- dependency of enzyme activity on substrate concentration, temperature and pH
value,
- dependency of enzymestability on pH, temperature, oxidizing agents,
- dependency of enzymeselectivity on reaction conditions,
- necessity of immobilization for enzyme stabilization,

- c:onsiderationson the use of organic solvent/cosolvents,
188
1 Investigation of the reaction system
-
Enzyme
properties
Parameters
I
I Properties of
reaction system
activity thermodynamic
*
*temperature I *
equilibrium
' stability
* substrate * complex reaction
selectivity product system
concentration
* other properties
" solvent
J1
Selection of reaction conditions
2 Investigation of reaction kinetics
Measurement and mathematical modelling of
enzyme kinetics
* non-enzymic reaction kinetics
* mass transfer phenomena
Selection of reactor
3 Investigation of reactor kinetics
Q@Ite@
_ _
Parameters ~
* conversion
* residence time
* selectivity
of reactor * concentration of
* productivity performance - substrate(s)
- enzyme(s)
* enzyme/ - coenzyme
coenzyme
consumption
4)
Selection of reactor conditions
Figure 7-3. Steps of process optimization in enzyme reaction engineering

7.2 Steps of Process Optimization
Then the other properties of the reaction system have to be characterized. The
I 189
following topics are of major concern:

- Equilibrium constant of reversible reactions,
- Structure of the reaction system. The term “structure” should include the sum of
the chemical reactions occurring within the reaction system, e. g. parallel reac-
tions, consecutive reactions (see Eq. (4)), coupled reactions such as in the case of
coenzyme regeneration (seeEq. (49)), non-catalyzed reactions occurring alongside
the enzymatic reaction.
- Influence of the reaction conditions (pH, temperature, concentration of sub-
strates, organic solvent/cosolvents)on the reaction system and on the equilibrium
constant,
- Other properties such as pH- and temperature effects of the reaction and
solubility of substrates and products.

There are reciprocal relationships between the parameters summarized above. On
the one hand enzyme stability measurements strongly depend on the concentrations
of substrates, coenzymes, buffers etc. in the assay. On the other hand the choice of an
appropriate concentration ].eve1is a consequence of the enzyme kinetics investigated
afterwards. A compromise has to be found between different optimization criteria
e. g. a lower temperature leads to a reduced enzyme activity but results in a higher
enzyme stability. In the example ofthe oxynitrilase reaction (Eq. (12)) a low pH value
is a prerequisite for high enantiomeric purity of the product but lowers enzyme
activity. As a consequence, only a rough optimization can be carried out at this
level.
The investigation of the reaction system ends with a (preliminary) selection of
reaction conditions. This is a prerequisite for investigation of the reaction kinetics as
it makes no sense to measure kinetics without knowing whether the kinetic assay
conditions are favorable for the final process.
The second step in process optimization is the investigation of the reaction kinetics.
This step aims at a detailed analysis of the kinetics of the reaction system under the
optimized reaction conditions chosen above. The following topics have to be
covered:
- Measurement and modeling of the kinetics of the enzyme-catalyzed reaction@),
- Measurement and modeling of the kinetics of the non-enzyme-catalyzedreac-
tion(s),
- Measurement and modeling of mass transfer phenomena. These will occur in
reaction systems which consist of two or more phases, such as in the case of
immobilized enzymes or in the case of liquid/liquid two-phase systems. This
aspect will not be covered and the reader is referred to the literatu~-e[~~-~’I.
The second step results in ;I kinetic model for the whole reaction system and a choice
of an appropriate reactor based on the reaction kinetics. Criteria for reactor choice
will be disussed in Sect. 7.5.1.
The Jinal step includes tlne reactor design and the prediction of reactor perform-
ance depending on operation conditions. The scope of points to consider is as
follows:
190
I 7 Reaction Engineeringfor Enzyme-Catalyzed Eiotransformations
- Formulation of the reactor kinetics by combining the kinetic model of enzymatic

and non-enzymatic reactions and mass balances of the reactor.
- Description of the reactor performance as a function of parameters such as
substrate concentration, enzyme and coenzyme concentration and residence
time.
- Optimization of reactor performance based on the following criteria: substrate
conversion, selectivity, space-time yield, enzyme and coenzyme consumption.
The extent of optimization in the development of an enzymatic reaction may be
described as follows:
- Studies with one-enzyme systems on a laboratory scale with the aim of small scale
production or verification of the desired concept do not require a detailed kinetic
analysis. The properties of the enzyme and of the reaction system should be
investigated to choose convenient assay and reaction conditions (e.g. temperature,
pH value, substrate and cosubstrate concentrations). Typically some essential
enzyme data are provided by the enzyme supplier or can be taken from hand-
books [411.
- Also some basic kinetic data of the enzyme should be known, especially Michaelis
and inhibition constants. For example, an enzyme suffering substrate inhibition,

when used at a high substrate concentration, will exhibit only a little activity,
whereas at low concentrations the reaction will run quite well.
- Dealing with complex systems (two or more coupled enzymatic reactions or
reactions with coenzyme regeneration) a complete kinetic investigation and

computer simulation of the reaction system is very helpful to achieve the desired
selectivity and yield of reaction (e.g. by choosing a sensible substrate and
coenzyme concentration, enzyme ratio and reaction time). A case study is
available [42, 431 exemplifylng the production of L-tert-leucine by reductive amina-
tion and simultaneous coenzyme regeneration.
- To optimize a production process all topics listed above have to be treated in detail
to achieve the optimum process performance.
7.3
Investigation of the Reaction System
7.3.1
Properties of the Enzyme
The purpose of studies on enzyme properties is to select favorable reaction

conditions for the investigation of enzyme kinetics. The choice of assay conditions
has to be performed very carefully, and it has to be proven that the assay conditions
are as close as possible to the reactor conditions of the final process. This aspect
cannot be stressed too much!
The influence of all relevant reaction parameters on enzyme activity, selectivity and
stability has to be considered. Parameters determining the enzyme properties are
7.3 Investigation ofthe Reaction System
I
191
listed below (the most important of them have been discussed already in Chap.
7.1):
- Substrates, cosubstrates, coenzymes, effectors, inhibitors
- pH value
- Temper(1ture
- Buffer
- Organic solvents/cosohents
- Ionic strength
- Viscosity of the medium
- Enzyme modification by
- immobilization
- covalent modification of the enzyme
- Redox potential (oxygen sensitivity)
- Heavy metal ions
- Influences resulting from reactor conditions
- shear stress
- effects of the reactor material
- surface effects (adsorption on reactor surfaces, liquid/liquid or gas/liquid
interfaces).
Measurement and modeling of the influence of concentrations of substrates,
cosubstrates, coenzymes and inhibitors on enzyme activity form the central subject
of enzyme kinetics and are discussed in Sect. 7.4.
A few of the aspects of practical importance are as follows:
First of all the pH value and the temperature of the assay have to be chosen. The
pH-optimum ofthe enzymc is determined by measuring the effect of pH on activity. It
has to be recognized that the location of the pH-optimum depends on
- the substrate,
- the choice of buffer,
- the reaction medium (organic cosolvents change dissociation equilibria at the
enzyme),
- the ionic strength,
- chemical derivatizatiort or immobilization of the enzyme.
In addition the temperature dependency of enzyme activity must be measured, also

yielding an optimum curve. This “temperature optimum” depends on the assay
corlditions,especially the incubation time, and is not, on its own, useM to identify a
reasonable reaction temperature. Instead of this, the temperature stability of the
enzymehas to be determined. To that end the enzyme is incubated with all relevant
reaction components in test tubes, changing the temperature while keeping all other
parameters constant. The assay conditions have to be as close as possible to the
conditions relevant in the final process. In particular, stability measurements have to
be performed in the presence of a relevant concentration of all substrates and
coenzymes which have a stabilizing influence on the enzyme.
Typically, the remaining activity is plotted versus incubation time and the deactiva-
tion rate can be taken from the slope of a half logarithmic plot (Fig. 7-4).
192
I7 Reaction Engineeringfor Enzyme-Catalyzed Biotransformations
0.5
65°C
0.1 I
0 5 10 15 20
Time (h)
Figure 7-4. Influence o f temperature on enzyme stability; example: a r n i n o a ~ y l a s e l ~ ~ 1
The activation energy of an enzymatic reaction (typicalvalues 20-GO kJ/mol) is far

below the activation energy of thermal denaturation (values between 200 and 600 kJ/
mol) [451. Therefore, by lowering the temperature, the deactivation rate decreases
more rapidly than enzyme activity and more product is obtained during the mean
lifetime of the enzyme. In practice, the temperature is lowered until enzyme stability
is acceptable or other denaturation effects become dominant. Lowering of reaction
temperature is of course limited by the solubilities of the substrates and the freezing
point of water.
Similar measurements of enzyme activity and stability have to be performed,
varying the other parameters mentioned above. The dependency ofenzymestability on
p H and ionic strength are quite important. The same method may also be used for
immobilized enzymes.
In order to identify influences of the reactor operation, enzyme stability has to be
measured under the process Figure 7-5 demonstrates that the deactiva-
tion rate may be greatly enhanced by the conditions of reactor operation and is
influenced by the reactor material.
The decrease in enzyme activity in a continuous reactor causes a decreasing
conversion of the substrate. Only in the case of low conversion (compare to “initial
reaction rate”, see Sect. 7.4.1),the decrease of enzyme activity is proportional to the
decrease of conversion and may be calculated from these data. Alternatively, the
remaining activity in the reactor may be measured by taking samples of the enzyme
(especially when using membrane reactors) or by adding fresh enzyme, until the
initial conversion is re-established. In stirred tank reactors, the product of enzyme
activity and residence time determines the conversion (see Sect. 7.5.1). Therefore,
7.3 Investigation ofthe Reaction System
I 193
0 5 10 15 20
Time (day)
Figure 7-5. Deactivation o f Neu5Ac-Aldolase depending on the reactor material[47].
the flow rate can be lowered until the initial degree of conversion is reached again
and the residual enzyme activity can be calculated from the flow rates.
The question of enzymt: immobilization typically depends on the stability of the
soluble en'zyme under process conditions.
Not only activity and stability but also selectivity of reactions may become the most
important criterion in the selection of the reaction conditions. The impacts of
additional reactions and of selectivity problems will be discussed in the following
section.
7.3.;!
Properties of the Reaction System
7.3.2.1
Thermodynamic Equilibrium of the Reaction
In the case of a hypothetical reaction (Eq. (1)).
kl
S-P
k-I
Enzymes, as catalysts, accelerate the reaction rates (a kinetic factor). The forward
reaction and the back reaction are accelerated to the same degree. The position of
equilibrium (a thermodynamic measure), which is not influenced by the enzyme,
yields information about the maximum conversion and therefore is of basic
importance for process development. Two examples will serve to demonstrate this
principle.
194
Example: Hydrolysis of acetyl-L-aminoacidsby aminoacylase

Aminoacylase stereospecifically cleaves N-acetyl-L-aminoacidsto acetate and L-
aminoacid, a reaction well known for the production of optically pure L-aminoacids
(Eel. (2)).
N-acetyl-L-amino acid -
aminoacylase
L-amino acid + acetate (2)
The equilibrium conversion for hydrolysis of the acetyl-L-aminoaciddepends on the

initial oncentration. Only at zero concentration may one hundred percent conver-
sion be attained (Fig. 7-6).
100
96
92
S
.-0
2
9 88
s
S
84
80
0 100 200 300 400 500
[N-acetyl-L-methionine](mmol L-')
Figure 7-6. Hydrolysis of N-acetyl-L-methionine by arninoacylase: equilibrium conversion as
function of substrate concentration [441.
In the industrial acylase process, an increase of both substrate concentration and

conversion is desired to reduce costs, but, as a thermodynamic principle, in cases of
increasing mole number during the reaction, the equilibrium conversion decreases
with rising substrate concentration.
Example: Synthesis of N-acetylneuraminic acid by aldolase

N-Acetylneuraminic acid aldolase catalyzes the cleavage of N-acetylneuraminicacid
(NeuSAc) to N-acetylmannosamine (ManNAc) and pyruvate (Pyr).The reverse
reaction can be employed to synthesize N-acetylneuraminic acid, which plays an
important physiological role as a terminal sugar residue of glycosylated proteins L4']
(Eel. (3)).
ManNAc+Pyr - NeuSAc-aldolase
- NeuSAc (3)
1.o - _
_. -.- -
-. _ I
’
,
-, , -, I
0.10 mol L-’ ManNAc
-.C.-.
_.-.-.-
.C.
.C.‘
*.-,* 0.01 mol L-’ ManNAc
. C .
O . O . C
0.4 - - 0 ’
25 “C
- 0 .
- /
pH 7.5
0.
0.2 ”
K = 29.7 L mol-’
0.0 . I
Figure 7-7. Synthesis o f N-acetyl-neuraminic acid with NeuSAc-aldolase: equilibrium

conversion as a function of the concentration o f both
To obtain a high conversion, high concentrations of both substrates have to be used

since ManNAc is more expensive than pyruvate, the latter is used in excess (Fig. 7-
7).
Bloth examples demonstrate how the maximum conversion can be influenced by
the choice of appropriate substrate concentrations (without changing the equilib-
riurn constant I&). Additionally, a disadvantageous equilibrium position can be
overcome by
- continuously adding a mbstrate,
- continuously withdrawing a product, e. g. by crystallization, extraction, electro-
dialysis [49-511, complexation (aspartame process [521),
- coupling to an irreversible reaction (see Eq. (49)),
- choosing reaction conditions where kqis changed to a more favorable value:
X,, depends on pH if acids or bases are involved in the reaction,

X
, depends on temperature,
k& may be influenced liy c o s ~ l v e n t s [ ~ ~ ~ .
7.3 2 . 2
Complex Reaction Systems: The Existence of Parallel and Consecutive Reactions
In this chapter, two examples demonstrate that, in addition to the desired enzyme-
catalyzed (conversionof the substrate S to the product PI, other reactions have to be
considered, e. g. a parallel reaction of S to P2 or a consecutive reaction of P1 to P3.For
a hypothetical reaction scheme, see (Eq. 4).
7 Reaction Engineeringfor Enzyme-Catalyzed Biotransformations
196
I
The following definitions are set up (all stoichiometric coefficients are set to one):
Conversion x:
The conversion xs of the substrate S stands for the ratio of substrate consumed to the
initial substrate: ([S]o-[S]= [PI]+[P,]+[P,]).
Selectivity 0:
The selectivity opl,s is defined as the product P1 formed in relation to the substrate S
consumed.
Yield Y:
The yield YpI,s is defined as the ratio of the product PI formed to the initial substrate
so
Sometimes, these definitions are mixed up in the literature. Eq. (8) describes the
relationship between conversion, selectivity and yield:
Yp,,, = X, .(Jq,s (yield = conversion. selectivity) (8)
The values of yield and selectivity become equal at quantitative conversion of the
substrate. The following diagram may elucidate the relation of these three parame-
ters (Fig. 7-8).
Additionally, the following measurements are defined:
Space-time yield:
The space-time yield STYpl is the amount of product PI produced per reactor volume
V and time with the dimension of kg L-ld-’. The space time yield is also referred to
as “volumetricproductivity”.
Enantiomeric excess:
eel,, =- iP,l-[P*l
[PIl+[P21
7.3 Investigation ofthe Reaction System I197
&=
l.O? 1.04 1.c 1.(
1.0
X,=
0.6
c P l s = Ypls= TPlS= YPlS=
0.830.5 0.8( 0.8:
Figure 7-8. Interrelation o f conversion, selectivity and yield in the case ofthe reaction scheme
Eq. (4) (PI being the main product; for definitions see Eqs. (5) to (7)).
The enantiomeric excess eepl of the enantiomer PI is defined as surplus of PI in

relation to the sum of enantiomers.
Kinetic parameters are defined in Sect. 7-4. Two examples will illustrate the
implications of complex reactions in enzyme reaction engineering.
Exumple: Enzyme-catalyzed peptide synthesis

An instructive example ofthe occurrence of selectivity problems is available from the
field of enzymatic peptide synthesis. For instance, the following complex reaction
scheme shows where parallel reactions and consecutive reactions occur. It describes
the synthesis of Tyr-Arg from the electrophile Tyr-OEt and the nucleophile Arg-NHz
by simultaneous use of CarboxypeptidaseY (CPD-Y) and Peptide amidase (PA)from
orange flavedo[54](Eq. (11.)).
Tyt-OEI
"Electrophile"
?=:
[Acetyl-enzyme]
Arg-NH2
(2)
"Nucleophile"
Tyr-OH
Tyr.Arg.NH,-
PA
(3)
Tyr-Arg -
CPD-Y
"Dipeptide"
(4)
(11)
Tyr-OH + Arg OH
The protease CarboxypeptidaseY (CPD-Y)reacts with the tyrosine ester forming an

acylenzyme activated complex, which is further converted in a competitive way.
Aminolysis of the acylenzyme by Arg-NH2 (2) yields the dipeptide amide Tyr-Arg-
NH2. The competing hydrolysis (1)yields the non-activated tyrosine. The dipeptide
amide Tyr-Arg-NHz is deamidated selectively by peptide amidase (PA)to the desired
198
100
80
60
>
.-
c
.->
.I-
0
-a
a 40
cn
20
0
0 10 20 30 40
[Tyr-OEt] (mmol L-’)
40
30
20 2
Y
c
0
0 100 200 300 400 500
[Arg-NH2] (mmol L-’)

Figure 7-9a, b. Kinetics o f t h e synthesis ofTyr-Arg-NHZ by CPD-Y: activity and selectivity as a
function of the concentrations ofTyr-OEt (A) and Arg-NHz (B)154’.
dipeptide, which may be further hydrolyzed by CPD-Y to the amino acids Tyr and
Arg (secondary hydrolysis). Secondary hydrolysis of Tyr-Arg-NHzto Tyr and Arg by
CPD-Y can be neglected.
1 .o
--- Yeld [TyrOEt], = 40 mmol L-’

0.8 -- Selectivity [ArgNH,], = 40 mmol L“ ~-
h
v
I
.-% 0.6
.-
.I-
0
a,
-
Q)
v) 0.4-
5
a, c
c c
- c
c
c
F 0.2- c
c
c
c
c
e
c
c
c
c
0.0--- *
Figure 7-10. Synthesis ofTyr-Arg by CPD-Y and P A selectivity and yield as a function o f
cotwersion for continuous operation in an enzyme membrane
The selectivity s of the formation of the dipeptide Tyr-Arg is reduced by the

undesired parallel (1) and consecutive reaction (4). Measuring the kinetics of the
aminolysi,~and the hydrolysis reactions gives an opportunity for process optimiza-
tion (Fig. 7-9).
As expected, increasing concentration of Tyr-0-Etinfluences the activity of CPD-Y
bui not the selectivity of the competing reactions 1 and 2. On the other hand,
increasing the concentraiion of Arg-NH2results in increased activity and selectivity
favoring the aminolysis reaction compared to the competing hydrolysis reaction.
The data in Fig. 7-9 were measured under initial rate conditions (see Sect. 7.4.1).
Additionally, selectivity and yield were calculated as a function of conversion (Fig. 7-
10).
If CPD-Y and PA are used in one reactor the yield of dipeptide initially increases
wiih conversion whereas the selectivity drops because of the consumption of the
nucleophile (compare to Fig. 7-9B). At higher conversion, the yield reaches a
maximum and selectivity drops steeply because of the subsequent hydrolysis
reaction i(4)(Eq. (11)).At 100% conversion, yield equals selectivity, both being zero.
For this reaction, a steady-state conversion of about 0.8 defines the best reactor
performamce. The above complex reaction is an instructive example of the correla-
tion of selectivity, conversion and yield.
Example: Synthesis ofchiral cyanohydrins

The “enantiomerically pure compound (EPC) synthesis” has become a major
strategic concern in the synthesis of bioactive compounds especially for pharmaceu-
tical and agrochemical use. EPC synthesis can be achieved very efficiently using
7 Reaction Engineeringfor Enzyme-CatalyzedBiotransformations
200
I enzymes as chiral catalysts. Although enzymes mostly show a high degree of
enantio- or diastereoselectivity,non-stereospecificside-reactionsmay occur lowering
the optical purity of the product. In this case, the problem of the selectivity of the
reactions has to be addressed.
A suitable catalyst for the synthesis of (R)-cyanohydrins is the enzyme (R)-
oxynitrilase from bitter almonds. It catalyzes exclusively si-face addition of hydrogen
pD<;
cyanide to benzaldehyde or other aldehydes. A competing non-enzymatic parallel
reaction lowers the enantiomeric excess of the product[55,561.
(R)-Oxynitrilase
(R)-Mandelonitrile
Re-face attack
(12)
"O*,, )CN
NS O
H
,
No enzyme * O c ' \ H + D c . \ H
Hydrogen
cyanide
\ /
Racernic product
Benzaldehyde
This example is very instructive in that it demonstrates methods to suppress a non-

desired reaction. The investigation of the reaction system leads to the question of the
influence of the reaction conditions on selectivity.
Influence ofpH value upon selectivity

The pH value has an influence not only on the activity of enzymes but also on
chemical reactions. The chemical cyanohydrin reaction is base-catalysed, as, com-
pared to HCN, the cyanide ion more easily attacks the carbonyl group. As a result, a
distinct decrease of the reaction rate for the non-enzymatic synthesis of mandeloni-
trile occurs at lower pH values. Also, the enzyme activity decreases but not to the
same extent; therefore, the enantioselective enzymatic reaction becomes dominant
at lower pH (see Fig. 7-11).
Influence of temperature upon selectivity

The investigation of the temperature dependence of the competing parallel reactions
gives information about their activation energy. According to the Arrhenius equation
(Eel. (13112
_E.4_ E.4
k=k,,;e orIn(klk,,)=-- (13)
R. T
R1
25000
7.3 Investigation of the Reaction System
I 201
20000
15000
10000
5000
30
20
10
0
3.0 3.5 4.0 4.5 5.0
PH
Figure 7-11. Initial reaction rate ofthe synthesis of rnandelonitrile as function of pH[". 561.
where
k (s-l) rate constant of the reaction
k,, (s-l) frequency factor
EA (J mol-l) activation energy
R (8#.314J K-lmol-') gas constant
T (K) absolute temperature,
the activation energy EA can be determined from the following plot:
In this case the non-enzymatic reaction, having an activation energy of 7 3 kJ
mol-', dlecreases faster with temperature than the enzymatic reaction (activation
energy 46 kJ mol-'). In general, at a lower temperature the reaction with the lower
activation energy is favored. The enzymatic reaction therefore becomes more
dominant at lower temperature.
Influence of substrate concentration

With respect to benzaldehyde, (R)-oxynitrilaseexhibits saturation kinetics (Michaelis
Menten kinetics, see Sect. 7.4.2.1) and a maximum reaction rate is reached above a
concentration of about 5 mmol L-'. The chemical reaction presents a linear increase
of the reaction rate with increasing benzaldehyde concentration, representing first
order kinetics, when the concentration of HCN is kept constant (see Fig. 7-13). As a
consequence the enzymatic reaction becomes more dominating at lower concentra-
tions of the substrate benzaldehyde (for HCN as substrate the same kinetic behavior
occurs, data not shown). Accordingly an enzyme reactor would be suitable that works
under minimum average substrate concentrations. These requirements are satisfied
by the continuous stirred tank reactor (CSTR). In Sect. 7.5.2.1 this aspect of enzyme
reaction engineering wi I1 be discussed further.
202
I 7 Reaction Engineeringfor Enzyme-CatalyzedBiotransformations
25°C
100
80
\
60
\ 10°C I
40
20
0 Non-enzymatic reaction
Enzymatic reaction
10
0.1 335 0.00340 0.00345 0.00350 0.00355
T-' (K-')
Figure 7-12. Relative reaction rate o f t h e synthesis of mandelonitrile as a function o f t h e
reciprocal temperaturelS71.
]/ 0
I
4000 Enzymatic reaction
2000 -
f
1
[Benzaldehyde] (mmol L-')

Figure 7-13. Initial reaction rate o f synthesis o f mandelonitrile as function o f benzaldehyde
concentration '551.
7.3 Investigation of the Reaction System
Influence oiorganic solvents

There are two arguments for the use of organic solvents in the synthesis of alpha-
hydroxynitriles by oxynitrilase:
- the best substrates of the enzyme are aromatic and straight chain aliphatic
aldehydes, both of which have a limited water solubility (e. g. benzaldehyde
80 mmol L-'). The solubility may be enhanced by organic solvents;
- the non-specific reaction is favored in water and may be suppressed by lowering
the water activity of the medium.
Diflerent methods have been used, employing organic solvents in this reaction
(reviewedin [58, 591):
- the use of water-miscible organic cosolvents such as ethanol or methanol,
- the application of aqueous two-phase systems,
- iimmobilizationof the enzyme and the use of organic solvents (e.g. ethyl acetate or
others) which contain only traces of water to preserve the enzyme's activity,
- the application of biphasic lyotropic liquid crystal systems.
In the oxynitrilase reaction, high optical purities can be reached by the use of organic
solvents. Often a major drawback of these methods is a lowered enzyme activity,
resulting in long reaction times and a reduced enzyme stability in continuous
experiments.
Selection ofreaction conditions

Summarizing the above effects, the high selectivity toward the enzyme-catalyzed
reaction and suppression of the non-specificparallel reaction is achieved by
- lowpH,
- low tewcperature,
- low stationary substrate concentrations,
- additionally a high enzyme activity exclusively favors the enzyme-catalyzedreaction
without influencing the non-enzymatic reaction.
Knowing that the enzyme stability is sufficiently high at pH 3.75 (50% deactivation
after 150 h), water was chosen as the reaction medium and the reaction was
peirformed at room temperature. This selection ofreaction conditions was followed by a
detailed kinetic analysis of the system, the investigation of the reactor kinetics and
the simulation of steady state conditions in continuous experiments [55.
'fie discussion about the choice of an appropriate reactor and optimized operation
coiiditions will be continued in Sect. 7.5.2.1. Anticipating the results, the reaction
can be performed effectively under the above reaction conditions in an enzyme
membrane reactor yielding a product with an enantiomeric purity of higher than
99 %.
204
7.3.2.3
Other Properties of the Reaction System
Other properties that have to be considered in enzyme reaction engineering are:
pH effects on the reaction

A pH effect during the reaction occurs if proton shifts are involved, as is the case in
most enzymatic reactions. As enzyme activity is strongly dependent on pH, a pH
shift induced by the reaction has to be prevented by buffering or titrating the reaction
medium. Problems arise especially in immobilized systems where pH gradients
occur within the enzyme matrix if the buffering capacity of the medium is too
low[61,621. In cofactor dependent reactions the influence of pH on the stability of
NAD(P)+and NAD(P)Hhas to be c ~ n s i d e r e d [ ~ ~ l .
The effect of temperature on the reaction

The effects of the variation of temperature on enzymatic reactions usually do not
require precautions, as in typical small scale reactors the capacity of heat exchangers
is sufficient to maintain a constant temperature.
Solubilities of substrates and products

This information may be useful in cases where a product can be removed con-
tinuously from the reaction mixture by crystallization.
7.3.2.4
Application of Organic Solvents
The use of enzymes in non-aqueous solvents can be an advantageous alternative to

reactions in water, especially for poorly water-solublesubstrates and products, e. g. in
the synthesis of esters, lactones or selected peptides. However, the knowledge of how
solvents influence enzyme activity and selectivity is still not profound. As it is
impossible to cover all relevant aspects within this chapter, the reader is referred to
some selected articles for further reading[*’*66751 . H ere some of the basics will be
discussed briefly; in Sect. 7.5.4 reactor concepts for the use of enzymes in organic
solvents will be presented.
There are advantages in performing enzymatic transformations in non-aqueous
media:
- non-specific side-reactions such as the chemical addition of HCN to aldehydes

during the oxynitrilase reaction may be suppressed by performing the reaction in
an organic solvent. This example has been presented in the previous section.
- the solubility of poorly water-soluble substrates or products may be increased in
organic solvents or at least by addition of water-miscible solvents. If non-water-
miscible solvents are used in combination with water, inhibitory effects such as
substrate or product inhibition may be overcome if the organic phase contains
most of the substrates and products and the enzyme remains in the water phase.
7.3 lnvestigation ofthe Reaction System
A very interesting alternative has been published detailing the use of cyclodextrins
I
205
to enhance the solubility of 2-acetylnaphthalenein aqueous solution. The complex

between the cyclodextrin derivative and the ketone is transformed fast enough to
support high reaction rates in an enzymatic reduction.1'7r
- equilibrium reactions may be shifted toward the desired product for one or both of
the following reasons: (i) lowering of water activity by adding a water-miscible
organic solvent (example: enzymatic peptide synthesis (ii)continuous extrac-
tion of the desired product into a non-water-miscibleorganic phase (compare to
the multiple-compartment enzyme membrane reactor (see later, Fig. 7-
37,[77.781).
Both stabilizing and destabilizing effects of solvents on enzymes have been reported.
A reasonably reliable measure of the compatibility of solvents with enzymes is the
log P value, where P is defined as the distribution coefficient of a solvent between
water and 1-octanolin a two-phase system[64*791. Solvents with a log P value above 4
are suitable (e.g. aromatics, aliphatics)whereas water-misciblesolvents with a log P
value below 2 (short chain esters, DMF, short-chain alcohols) are not suitable for
employment with biocatalysts. The latter solvents interfere with the water at the
boundary of the protein itself and so disrupt the binding forces necessary to
maintain an active form of the enzyme. Surprisingly, tert-butanol has a stabilizing
effect on some oxidative enzymes , I ' ' [ despite its low log P value (0.35).
Fig. 7-14 shows a classification for biotransformations in organic solvents into
one-phase and two-phase systems.
One-phase systems may consist mainly of water, water plus a water-misciblesolvent
or a pure organic solvent. Most water-miscible solvents may be used in concentrations
up to 20% before enzyme deactivation exceeds the benefits obtained by better
solubility of substrates and/or increased selectivity. Using this approach, enzyme
activity and enzyme stability have to be examined carefully to select appropriate
reaction conditions.
Using mainly water non-miscible solvents several approaches are possible. In most
cases, the organic solvent has to be saturated with water in order not to remove the
boundary water surrounding the enzyme, which otherwise results in deactivation. In
such microaqueous systems the pH of this tiny amount of water should be carefully
chosen for optimal enzyme activity. The control of water activity can be achieved by
addition of salts or utilization of saturated salt solutions fS1, 82]. The simplest way of
using an enzyme in organic solvents is to suspend the insoluble enzyme in the
required solvent. This technique was first reported in 1900 [831 and has been extended
over the last few years to encompass many systems (mainly proteases and li-
pases) L7', 84, 8'l. Organic solvents may be replaced by supercritical liquid carbon
dioxide, which exhibits similar properties to hexane .'[ 14'1.
To achieve true homogeneous catalysis, enzyme solubility may be increased by
coupling polyethyleneglycol to its surface LS71. The coupling may alter the stability,
activity and selectivity of the enzyme.
By use of detergents and small amounts of water or buffer, reversed micelles can
be formed containing the enzyme in the water phase while the organic solvent forms
the bulk phase[", "1.
206
Figure 7-14. Classification of enzyme catalysis in the presence of organic solvents.
Just like one-phase systems, two-phase systems may consist mainly of water or
mainly of organic solvent. In an aqueous-based system an insoluble substrate is
dispersed, using a non-miscible solvent (oil-in-wateremulsion). Hydrolysis reactions
of poorly water soluble substrates (e.g. fat hydrolysis by esterases) may be performed
in this way. If the aqueous phase is dispersed in a water-immiscibleorganic solvent a
water-in-oil emulsion is obtained. This type of system may be advantageous if a
condensation reaction has to be performed where the water content has to be kept
low. A special form of a two-phase system involves lyotropicliquid crystals, which are
obtained by mixing a detergent, water and an organic solvent["^ 911. Most of the
water and the detergent form the liquid crystal wherein the enzyme is immobilized,
whereas most of the organic solvent forms the second phase.
Enzymes immobilized on an insoluble support also belong to two-phase systems.
They have been mentioned earlier and are not discussed further at this point.
Very recently ionic liquids emerged as a new class of solvents. They are salts with
melting points below 100 "C and are non-volatile. Some typical structures are shown
in Fig. 7-15. They possess good solvating properties for many substrates and
catalysts[92294]. They can also be used to replace organic solvents in enzymatic
reactions [95-981. For the lipase-catalyzed kinetic resolution of phenylethanol an
improved enantioselectivity at higher temperatures was observed compared to the
selectivity obtained when using methyl-tert-butylether(MTBE) as solvent [", 981.
What has been neglected so far is a consideration of the location of the enzyme
7.3 lnvestigation ofthe Reaction System
I
207
Figure 7-15. Increased enantio- h N G . pFg \NQN, CFW;

selectivity foir lipases by reaction
0 20 40 60 80 100
Temperature ("C)
conditions:
1 mol/L alcohol, 2.5 mol/L vinylacetate, water content 0.3%
12 g/L Chirazyme L6 (fseudomonas sp. lipase)
and hence the enzymatic reaction. Depending on the hydrophobic and hydrophilic
properties of the enzyme it may be enriched in one of the phases (normally the
aqueous one). For lipases, catalysis often occurs at interfaces. Therefore, the surface
area per unit reactor volume available in such a system is an important quantity, not
only for reaction performance but also for determination of kinetic data.
As shown in Fig. 7-16, a larger surface area requires a higher enzyme concentration
to be saturated. But a tenfold increase in the surface area - even if saturated with
enzyme - does not result in a tenfold increase of One reason for this
might be product inhibition.
Wherleas kinetic studies are quite easy to perform in homogeneous solution the
extent of the interface has to be taken into consideration for biphasic systems. The
most reliable way of measuring the interfacial area is by use of Fraunhofer
208
I 7 Reaction Engineering for Enzyme-Catalyzed Biotransformations
0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2
Enzyme Concentration (mg Lipase/g Oil)

Figure 7-16. Saturation ofthe phase boundary surface with lipase.
diffraction[lo0I.All reaction velocities can be given based on the same surface area. A
change of droplet size and surface area, which may occur with change of substrate or
product concentration during the reaction, can be distinguished from true inhibition
effects.
7.4
Investigation of Enzyme Kinetics
In this chapter, some principles of the kinetics of enzymatic reactions are discussed.
A more detailed description of enzyme kinetics is covered in a number of textbooks
and articlesL45, 101-1041. F'irst of all, a few general definitions will be given.
The reaction rate u of any chemical reaction (Eq. (14))
+
nAA n,B + n,C + n,D
is defined as follows (Eq. (15)):
=- d[A1- function of k , [A1 [B], ..

nA dt
u (mmol L-' min-') reaction rate
[A], [B] (mmol L-') concentrations of A, B ...
nA, nB (-) stoichiometric coefficients
k (mmol'-" L"' min-') rate constant (nbeing the order of reaction)
7.4 Investigation ofEnzyrne Kinetics
Eq. (15) is the rate equation of the reaction (also called the "kinetic model'). The
formulahon of such a differential equation for all reacting substances is the basic
step in describing the kinetics of chemical/biochemical reactions. These rate
equations include concentration values of the relevant reaction partners and kinetic
parametcrs such as the rate constant k. An investigation of enzyme kinetics includes
the measurement of reaction rates, the choosing of an appropriate kinetic model and
the identification of the kinetic parameters.
7.4.1
Methods of Parameter identification
Kinetic rneasurements [loSl have to be carried out to examine the dependence of the
reaction rate on the concentrations of all relevant components. As described in a
previous chapter, for measuring enzyme kinetics initial reaction rates v, = j S ] are
determined at optimal reaction conditions, which may be chosen according to the
procedure outlined in Sect. 7.3. The initial reaction rates are measured by varying the
concentration of only one component and keeping all other concentrations (e.g. of
cosubstrates and inhibitors) at a constant level (for an example, see Figs. 7-19 and
7-20). The rate of conversion has to be smaller than 5-10%, essentially to keep all
initial concentration values constant.
The parameters of the kinetic model can be identified by fitting the kinetic data
using methods of non-linear regression such as those described by Rosenbrock or
Nelder Mead['OG 1'" (Fig. 7-17A). Methods of linear regression that are often used
need a rearrangement of rate equations into a linear form (e.g. a double reciprocal
plot according to Lineweaver-Burk ['08]). This gives different weight to the data points
measurcd at different concentration levels 'lo]. For the correct calculation of the
[lo93
regression line this point must be considered, otherwise the Lineweaver-Burk

double reciprocal plot is not acceptable["'].
Initial rates are not significant in large-scale processes where high conversion of
the substrate is desired. With rising conversion, the simultaneous effects of both
substrate S and product P on the reaction rate have to be described. In the case of
equilibrium reactions, the forward reaction and the back reaction have to be
described by one rate equation: they can only be treated separately under initial rate
conditions. The overall rate equation has to describe the reaction rate as aficnction of all
relevant components at all relevant concentration levels. A correct fit of all initial reaction
rate data gives no guarantee that the kinetic model will fit the overall reaction data!
A proper fit of the time-courses of some batch reactor experiments at different
starting concentrations represents an appropriate test of the rate equation. This
implies that numerical integration of the rate equation (e.g. by the Runge Kutta
yielding a simulated time-course, has to fit the data of the measured
time-coiirse over the whole range of conversion (compare to Fig. 7-17 B). Examples
of these methods will be given after the presentation of the basic kinetic models.
A combination of the Runge Kutta method and methods of non-linear regression
allows a parameter identification from the time-course data. This technique starts
with a given set of parameters, performs the numeric integration of the rate equation
210
Figure 7-17. Methods of parameter identification: A by fitting initial rate kinetic data, and B by
fitting the time-course of a reaction.
and compares the simulated with the measured time-course. Then the parameters
are changed and the same steps are repeated until the simulation fits the measured
data (Fig. 7-17 B). This method requires specially designed computer software,which
is commercially available (ScientistTM,MathlabTM,MapleTM).The method is espe-
cially useful if components having an effect on the enzyme kinetics are not available
or if they cannot be measured, but are in a defined stoichiometric relation to a
measurable component.
7.4.2
The Kinetics of One-Enzyme Systems
7.4.2.1
Michaelis-Menten kinetics
In this section the basic kinetic model for enzyme-catalyzed bioconversions is

presented. Understanding this model is the foundation for deriving more complex
models. In their theory of enzyme catalysis, Michaelis and Menter~[''~] postulated
the existence of an enzyme substrate complex (ES), which is built up in a reversible
7.4 investigation ofEnzyrne Kinetics
I*”
reaction of the substrate S and the enzyme E. The dissociation of this E S complex to
-
E and P is assumed to be the rate-determining step (Eq. (16)).
E+S - kl
k-1
- ES
k2
E+P
Rate constant Reaction

kl (L mmol-’min-’) Association of the ES complex
k.1 (min-’) Dissociation of the E S complex into E and S
kz (miin-’) “Turnover number” (see below) (also refered as “kcat”)
If the rate constant k2 is much smaller than the rate constant k.1 of the enzyme, the
substrate and the enzyme-substrate complex are in equilibrium, which is not
disturbed by the decomposition of E S into E and P (“rapid equilibrium-assumption”).
Based on this assumption, Michaelis and Menten derived the following rate equation
(Eq. (17)).
.[SI
- urnax
(17)
Ks + bl
2) (nimol L-’min-’) Reaction rate
urnax (nimol L-lmin-’) Maximum reaction rate
Ks (mmol L-l) Dissociation constant of the E S complex
This is the classic form ofthe Michaelis-Mentenequation. Eq. (17)can be rearranged

to
& (min-’) represents the rate constant of the first order enzyme kinetics
KS (seeFig.7-18)
1+ - bl (-) denominator of the kinetic model :represents a dimensionless
KS “adsorption term” (see below)
B1
- _-
__ - [Ed (.)
represents the dissociation of ES into E and S and may be
Ks [El simply derived from the dissociation equilibrium
The curve u = j S ] belonging to the Michaelis-Mentenfunction is shown in Fig. 7-

18.
As long as [ S ] is well below Ks‘ Michaelis-Menten kinetics transpose to (linear)
first-order kinetics. In this case nearly all enzyme molecules are present in the free
form and saturation of the enzyme with substrate is rate limiting. This degree of
saturation is represented by the “adsorption tern”, which gives the sum of the values
1 + [ES]/[E] + [ESi]/[E] (if more substrates Si or inhibitors or products are involved).
The adsovption term represents the dissociation equilibria of all relevant enzyme-substrate
212
Figure 7-18. Michaelis-Menten kinetics: curve analysis
complexes, and reveals the “weight”of the equilibria involved. For example, for [S] =
Ks the value of the adsorption term equals 2, so that 50% ofthe enzyme is in the ES
form and the other 50% is in the free form. The adsorption term initially grows from
a value of 1 (at [S] << Ks) slowly to become proportional to [S] and transforms the
linear first-order rate law to the curve reflecting Michaelis-Menten kinetics.
At concentration values above Ks it is not enzyme saturation but the catalytic
turnover of the enzyme that becomes more and more limiting. Being proportional to
the concentration of the ES complex, the reaction rate cannot rise above the u,
value when all enzyme molecules are in the ES form. The enzyme kinetics turn to
zero order.
The Michaelis-Menten kinetics, represented by Eq. (18),may be extended to more
complicated reactions by looking at the structure of the adsorption term. This
procedure shown below is valid as long as the “rapid equilibrium” assumption is
made. This is not valid in all cases.
Briggs and Haldane[’I4]did not use the above assumption, but pointed out that
within a very short time after starting the reaction, ES would build up to a nearly
“steady state” level where the following assumption is valid: d[ES]/dt = 0. From this
“steady state assumption” they derived the same rate equation:
K~ (mmol L-’) Michaelis constant

7.4 Investigation ofhzyrne Kinetics
I 213
Here the KM value no longer is a dissociation constant but a kinetic constant which
includes lil, Ll, k2 and for more complex reactions even more rate constants. This
equation is the standard form of the Michaelis-Mentenequation.
Although the derivation of reaction rates using the “steady state assumption” is
more exact, often the rapid equilibrium assumption is used because it allows a
simple derivation of the rate equation from the relevant enzyme-substrate complexes
(see below) and allows fitting of the kinetic data. The following explanations are
based on the rapid equilibrium assumption, and therefore allfollowing constants K are
used as dissociation constants with the component dissociatingfr.om the enzyme as the
subscript, e.g. KA, KB, and the component remaining at the enzyme as second subscript
(e.g. KIs, .see below).
Within Eq. (17)urnaxcan be expressed as shown in Eq. (20):
vmax (mmol L-’min-’) Maximum reaction rate

[Elc,’ (mmol L-’) Total enzyme conzentration in molar value:
[El,’ = [El + [ES]
kz (niin-’) Turnover number
The "turnover number‘‘ represents the moles of product formed per minute and per
mole of active sites of enzyme. Usually the molar concentration of the active sites is
not known, so the weight per volume of the enzyme preparation is used; Eq. (20) is
changed to Eq. (21).
[El0 (g L-7 Total enzyme concentration (in g L-‘)

A,,, (mmol g-’ min-’) Maximum specific activity of the enzyme
Using Eq. (21), Eq.(17)can be converted to Eq. (22).
and, with. Eq. (23),
A (rnmol g-‘ min’) Specific activity of the enzyme

to Eq. (24).
‘4- ha.bl
K , + [SI
Besides .;pecific activity, two other quantities are used for the characterization of
enzymes: the International Unit [U](defined as the amount of enzyme which
214
produces 1 pmol of product per minute) and the katal (kat) (defined as the amount
of enzyme which produces 1mol of product per second).
The following correlations exist between the reaction rate u, the specific activity A
and the definitions of unit U and katal (kat)(Eq.(25)):
U pmol mmol
1-=1- = 1- (reaction rate = volumetric activity)
mL mL.min L.min
U pmol mmol
l-=l- = 1-__ (specific activity)
mg mg.min g.mm
1kat = 6.10’ U (activity)
The use of values for the specific activity of an enzyme is only significant if the
conditions of measurement are specified, especially temperature, substrate concen-
tration and pH value. Additionally, enzyme activities are always measured at initial
reaction rate conditions (see above).
In the following sections the extension of Eq. (18) to more complex reaction
schemes is described. Again the “rapid equilibrium assumption” is used to show how
more complex rate equations are derived from simple Michaelis-Menten kinetics.
Attention is focused on some typical rate equations that are useful to describe
enzyme kinetics with respect to a desired process optimization. The whole complex-
ity of enzyme kinetics is of importance for a basic understanding of the enzyme
mechanism, but it is not necessary for the fitting of kinetic data and the calculation of
reactor performance.
7.4.2.2
Competitive Inhibition
Substances that cannot be converted by the enzyme but are competing with the
substrate for the active site of the enzyme are called “competitive inhibitors”. The
following reaction scheme represents this situation:
KS
E+S===== ES ‘ ’ w E+P
t
llhl
El
I Inhibitor
KI (mmol L-’) Dissociation constant of the EI-complex
The corresponding kinetic model again may be presented in two forms (Eqs. (27
and 28)).
u
~,
max
bl
u =+
1+-+-
Ks K,
7.4 fnvestigation ofEnzyrne Kinetics I215
Compared to Eq. (18)the rate equation (Eq. (27))for competitive inhibitors includes
an additional term [I]/ K1 in the denominator representing the additional dissociation
equilibrium of the EI complex ($ the above discussion about the “adsoqtion term”).
Also, each alternative substrate S, ofa reaction would render such a term [Sn]/Ksnin
the denominator. As a consequence of their affinity to the enzyme, alternative
substrates and inhibitors block a part of the enzyme otherwise available for the
reaction S P.
-+
Thus, the product P of enzymatic reactions is often a competitive inhibitor of the

enzyme leading to “product inhibition” (compare Eqs. (27)and (37)).The influence of
product in the case of reversible reactions will be discussed later.
From Eq. (28) it can be concluded that the effect of a competitive inhibitor is to
increase the apparent Ks value while the urnaxvalue is not affected. For example if [I]
is chosen as KI, in the presence of the inhibitor it would take twice as much substrate
S to reach umax/2as without inhibitor.
7.4.2.3
Non-Competitive Inhibition
“Non-competitiveinhibitors” bind at the enzyme E or at the ES complex and build up a

ternary complex ESI, which cannot be converted to the product P (Eq. (29)):
E+S=
KS
ES A E+P
+ +
I I
EI + S ESI
KI (mmol L-’) Dissociation constant of the EI-complexwith release of I
KIS (mmol L-’) Dissociation constant of the ESI-complex with release of I
Ks I (mmol L-’) Dissociation constant of the ESI-complexwith release of S
For ordinary non-competitive inhibition, the Kl and KIs values are identical and
also the KS and Ks’ values. In this case rate equation (30) is valid:
216
u=
Compared to Eq. (27), the rate equation for non-competitive inhibitor includes
another term for the equilibrium of decomposition of the ESI-complex into E, S and
I.
From Eq. (31) it can be concluded that a non-competitiveinhibitor has no effect on
the KS value but lowers the vmaX value. This is because the inhibitor binds all enzyme
species with the same affinity.
7.4.2.4
Uncompetitive Inhibition
Substances which only bind the ES complex and not the free enzyme E are
“uncompetitive inhibitors”:
t+s-
4 ES ”* E+P
I (32)
11;
u= mu . [SI
s.1 (34)
Ks+[S]+[llj
K,S
Compared to Eq. (30) the rate equation for uncompetitive inhibitors includes the
same term for the equilibrium of decomposition of the ESI complex into E, S and I,
but no term for an equilibrium of an EI complex.
7.4 Investigation of Enzyme Kinetics
I
217
One special form of uncompetitive inhibition is substrate inhibition. Here a second
substrate molecule binds at the ES complex resulting in an inactive ESS complex.
This form of inhibition is often found and will be discussed below (see acylase
kinetics, Fig. 7-20A).
7.4.2.5
Reversibility of One-Substrate Reactions
Because of the “principle of microscopic reuersibility” each molecular process (in

contrast to a macroscopic process) may occur in both forward and backward
directions. As a consequence the end product P of an enzymatic conversion can act
as a competitive inhibitor of the enzyme or, depending on the thermodynamic
equilibrium, be transformed to the substrate S. If the interconversion of the ES to the
EP complex is the rate-determining step the rapid equilibrium assumption is valid
and the rate equation can be derived easily.
KS kF KP
Ek-S- ES - - EP- - E+P
kB
’P (mmol L-’ min-l) Reaction rate for the formation of the product P
U F , ~ (mmol
~ L-’ min-’) Maximum reaction rate of the forward reaction
(mmol L-’ min-’) Maximum reaction rate of the backward reaction
Again the denominator represents the dissociation equilibria of the ES complex
and the EP complexes. Both partial reactions, the forward and the backward
reactions, are catalyzed simultaneously; both “substrates” S and P are competing for
the same enzyme. If the value in Eq. (37) equals zero, the equation reflects
competitive inhibition of the enzyme by the product P (see Eq.(27)).
Eq. (371is a result of the rate equation of the forward reaction reduced by the rate
equation of the backward reaction, both having the same denominator. The numer-
ator represents the first-order kinetics of the forward and the backward reactions. If
the equilibrium of the reaction is reached, the numerator becomes zero. From the
equilibrium condition (Eq. (38)),
a correlation between the thermodynamic parameter “equilibrium constant” and the

kinetic parameters can be derived (Eq. (39)):
218
This relation, representing the ratio of the rate constants of the first order kinetics is
known as the “Haldane equation”. Haldane equations can be formulated for every
kinetic model describing an equilibrium reaction, just by setting the numerator to
zero.
An example may illustrate this principle:
Eq. (37) is the basic kinetic model for isomerizations and racemizations. For
example, the kinetic constants of an alanine racemase of Bacillus stearothermophilus
are found to be
z ) D . A ~ ~ - L . A= 7.02
~ ~ , ~ ~ U mL-’ K D . A =~ ~3.42 mmol L-’
= 11.82 U mL-’ K L . A = ~ ~5.76 mmol L-’
The corresponding rate constants of the first order kinetics are
z ) D . A ~ ~ + L . A/ ~KD.A~=
~ , ~ ~ = 2.052 min-l
z ) L . A ~ ~ - D . A /~ K ~ ~2.052 rnin-l
~ ,L~ . ~A=
These rate constants are identical, resulting in Gq = 1, a predictable result of
racemization of D- or L-alanine.
This example shows that the rate constants of the first order kinetics determine the
position of equilibrium of the reaction. In the case of reversible two-substrate
reactions, the rate constants of the second order kinetics have to be used as shown
below.
7.4.2.6
Two-Substrate Reactions
In most enzyme-catalyzed reactions two or more substrates are involved, such as in

the enzyme-catalyzed aldol reaction, the cyanohydrin reaction or enzyme-catalyzed
peptide synthesis (examples used before). For many reaction schemes kinetic
models have been derived using the steady-state assumption. Some important
reaction mechanisms and the corresponding rate equations are summarized in
Table 7-1. An approach to the steady-state method and a detailed discussion of the
resulting kinetic models is difficult and is not the aim of this chapter.
For a practicable approach, the “rapid equilibrium” assumption is applied and the
structure of kinetic models of two substrate reactions is demonstrated for the case of
a “randombi-uni reaction”.
The binding of two substrates A and B to an enzyme may occur in a compulsory
order or in a random order. If one product (uni) is formed out of two substrates (bi),
the corresponding mechanisms are the “ordered bi-uni“ mechanism and the “random
bi-uni mechanism”,respectively (water is not regarded as a substrate).
For a “random bi-uni” mechanism of an equilibrium reaction A + B +=P the
following reaction scheme is valid (Eq. (40)):
KA -
E+A EA
+ +
B B
7.4 Investigation ofEnzyme Kinetics
I 219
If the interconversion of the central EAB complex to the EP complex is rate
determining and all other reactions are in a “rapid equilibrium”,the following rate
equation for the random bi-uni reaction may be derived according to the above
method:
___.
2, F,max
[A].[B]-’”.””. [p]
K, . K,” KP
P A.B
2), =
A B
I+-+-+-+-
K, K, K, KA.K;
Again the denominator represents all enzyme-substrate equilibria, e. g. Eq. (42):
The numerator represents the second-order kinetics of the forward reaction reduced
by the firs-t-orderkinetics of the back reaction.
A Haldane equation can be formulated by setting the numerator to zero, resulting
in a relation for the equilibrium constant (Eq. (43)):
In the absence of the product P, Eq. (41)may be rearranged by multiplication of

numerator and denominator by KAKBA (respectively K B K A ~ which
, is identical to
&KBA, compare to Eq. (40)).
Then Eq. (44)results:
u p= 2, F.max ‘ [B1
K,.KBA+KBA.[A]+KAB.[B]+[A].[B]
Only if a further identity KA = K.A.~is assumed can Eq. (44) be rearranged to Eq.
(45):
This rate equation is called “Michaelis-Mentendouble substrate kinetics”. It is a

formal multiplication of two Michaelis-Menten models for both substrates A and B.
This model can be used to describe rate kinetics of two substrate reactions in the
absence of the product(s). The kinetic measurements have to be performed by
varying the concentration of one substrate keeping the concentration of the second
substrate at a constant value well above the KM value. The model cannot be used if
back reactions occur and an equilibrium has to be described by an appropriate
Haldane tquation.
220
U
G
a-
%
II
7.4 investigation of Enzyme Kinetics
I**'
w
r Q\
c.
+
222
Footnote to Table 7-1.

The above reaction schemes are described according to the notion of Cleland11161.The substrates binding to
the enzyme are indicated as A and B according to the order of binding; the products are indicated as P and Q,
respectively.
The different mechanisms may be explained in short as follows (for a detailed discussion the reader is referred
to textbooks, listed above).
Random bi bi: Both substrates A and B may bind: also the products P and Q may dissociate from the
enzyme in a random order. (Example: E. coli galactokinase)
Random bi uni: Two substrates A and B and only one product P are involved (water is not considered).
(Example: aminoacylase, see above)
Ordered bi bi: The substrates and products bind to or dissociate from the enzyme in a specific order.
(Example: in most dehydrogenase-catalyzedreactions the coenzyme has to bind first;
hexokinase)
Theorell-Chance: This special case of the ordered bi-bi mechanism occurs if the first product P dissociates
from the enzyme very rapidly and an EAB-, EPQ-complex does not occur in a significant
concentration. (Example: alcohol-dehydrogenase)
Ordered bi uni: Two substrates and only one product are involved. (Example: NeuSAc-aldolase and other
aldolases)
Ping Pong bi bi: In this mechanism a product P is released between the addition of two substrates. This
mechanism is often found in group transfer reactions. (Example: transaminases, yeast
transaldolases)
Above-mentionedand additional examples are given in [117,1181. Looking at the rate equations for the different
mechanisms the following points can be identified:
Corresponding to the above discussion about enzyme kinetics, the numerator is nearly identical for all
different bi-bi-mechanisms (for bi-uni mechanisms, respectively),as the numerator characterizes the
thermodynamic equilibrium of the reaction (which is independent of a kinetic mechanism).
The denominator consists of terms characterizing all enzyme substrate equilibria.
Depending on the mechanism every substrate A, B, P and Q requires one or two kinetic constants
(designated as KTand KM)in order to describe its reciprocal action with the enzyme. According to the steady
-state derivation of these rate equations, KI and KM are no longer simple dissociation constants (compare
discussion about KS and KM). In some cases KI is identical to a dissociation constant as described before, but
most often these steady state parameters are defined by three and more rate constants. A verbal distinction
between an inhibition constant; a Michaelis constant and a dissociation constant does not have a
corresponding mechanistic scenario in all cases.
The random mechanism in both cases is the simplest mechanism requiring only 8 (random bi-bi) and 6
(random bi-uni) kinetic constants. This mechanism should be tried first to fit data of a two-substrate
reaction.
The denominator may be supplemented by additional terms e.g. for inhibitory substances, not being
described by basic mechanism (compare to discussion of the acylase kinetics).
A distinction between the different mechanisms is best done using initial rate kinetic measurements as
described in detail in the literature. For reaction engineering purposes only a proper fit of reactor data is
desired, using a minimum amount of kinetic parameters for statistical reasons.
If the concentrations of the products P and Q in the models summarized in Table

7-1 are set to zero, the resulting rate equations will be identical to Eq. (44) in all cases
with the exception of the “ping-pong”mechanism. This means that the Michaelis-
Menten double substrate kinetics is valid under the above circumstances.
An example of the use of this model will be shown for a two enzyme system (see
Sect. 7.4.3).
7.4 fnvestigation ofEnzyme Kinetics
I 223
7.4.2.7
Kinetics of Aminoacylase as Example of a Random Uni-Bi Mechanism
Aminoacylase kinetics may be used as an example for demonstrating the measure-

ment and modeling of enzyme kinetics. This reaction is of industrial importance in
12 I I I
10 - - _ _ _ ~
2 0 Measured ~
Calculated
I
0
0 50 100 150 200
Calculated
0 I
224
Ic
12
10
8
-h
z
3 6
2
.-
>
.-
2
c
4
0 I I I I I
0 200 400 600 800 1000
[D/L-Alanine] (mmol L-’)

Figure 7-19. Kinetics of aminoacylase: effects on the activity of hydrolysis o f acetyl-~-alanine~”~]
(initial rate measurements).
the production of L-amino acids by hydrolysis of acetyl-L-arninoacids.Since the

maximum conversion is limited by the equilibrium of the reaction (compare with
Fig. 7 4 , kinetic measurements of both hydrolysis and synthesis have been per-
formed and fitted by a kinetic model, using acetyl-L-alanine as an example. Addition-
ally the influence of D-alaninewas measured.
Fig. 7-19 shows influences of acetyl-L-alanine,acetate, D- and L-alanine on the
hydrolysis activity of aminoacylase.
With respect to acetyl-L-alanine, the substrate of the hydrolysis reaction, the
enzyme exhibits Michaelis-Menten kinetics (Fig. 7-19A). The hydrolysis activity is
inhibited by acetate (Fig. 7-19 B) and L-alanine (Fig.7-19C). These are the substrates
of the synthesis reaction (see below) acting as inhibitory products.
Figure 7-20 shows the influences of acetate, L-alanine, D-alanine and acetyl-L-
alanine on the specific activity of arninoacylase synthesizing acetyl-L-alanine.
With respect to acetate as the substrate (Fig. 7-20A)the enzyme exhibits substrate
inhibition (compare Eq. (33). Concerning the second substrate, r-alanine, Michaelis-
Menten kinetics are apparent (Fig. 7-20B). D-Alanine slightly inhibits the enzyme
(Fig. 7-2OC).
On adding acetyl-L-alanine(as the product of the synthesis reaction) to a reaction
mixture as specified in Fig. 7-20 D, the measured enzyme activity rapidly decreases,
indicating product inhibition. Finally the measured activity reaches negative values.
A negative activity here means a negative rate of acetyl-L-alaninesynthesis, that is,
hydrolysis.
7.4 Investigation ofEnzyrne Kinetics 225
A 2.5
2.0
h
7
.5
E
2
$
.-
.-
.o
8
I-,
0.5
0.0
B 3.0
2.5
2.0
r
b
E
2 1.5
.->
c)
.->
ti 1.0
a
0.5
0.0 I I I I
0 500 1000 1500 2000
[L-Alanine] (mmol L')
This cannot be described by product inhibition alone and means the hydrolysis
reaction commences above a concentration of 16.5 mmol L-' acetyl-L-alanine.At this
value the reaction is in its equilibrium position and the measurable enzyme activity
226 7 Reaction Engineeringfor Enzyme-Catalyzed Biotransformations
c 2.0
1.5
I
-
h
F
I
[L-Ala] = 500 mmol L-’
3 1.0 -
[Ac] = 400 mmol L-’
0.5
0 Measured
0.0
0 200 400 600 800 1000
[D-Alanine] (mmol L-’)
D 2.0
[L-Ala] = 500 mmol L-’

1.5
[Ac] = 400 mmol L-’
-i3-J
h
1.0 \
I
E
3
v
0 Measured
0.0
I I
-0.5 I I
3
[Ac-L-Ala] (mmol L-’)

Figure 7-20. Kinetics of aminoacylase: effects o n the activity of synthesis of acetyl-L-alanine
(initial rate measurements) [1151.
equals zero. Of course, the enzyme is not inactivated but there is no macroscopic
I 227
turnover of substances.
Fig. 7-19 and Fig. 7-20 show a number of effects on the activity of aminoacylase,
which cannot be described by a standard kinetic model alone. To derive a kinetic
model for the aminoacylase and for describing all influences, a reaction scheme
based on the rapid equilibrium random uni-bi model was used (compare with Eq.
(40)).This model was supplemented, according to the above measured influences of
substrate inhibition by acetate and inhibition by D-alanine. Consequently, the
reaction scheme Eq. (40)had to be extended to yield equation (46) and the
corresponding rate equation (47),which is modified by the additional equilibria:
E( D-Ala)
L-Ala L-Ala
k, +
Ac
E( Ac)z(L- Ah)
The substrate inhibition of Fig. 7-20A is represented by the complexes E(Ac)2 and
~ ) . means that binding of acetate to all enzyme species besides the
E ( A c ) ~ ( L - A . ~This
E(Ac-L-Ala)complex and the E(D-Ala)-complexis assumed. The rate equation of the
random bi-uni model (Eq. (41)) is enlarged by three terms in the denominator
according to the additional enzyme substrate complexes E(D-Ala), E ( A C ) ~and
E(Ac)$-Ala), whereas the numerator has the same structure as in Eq. (41).
The rate equation corresponding to the above reaction scheme can be written as
follows:
Kinetic model ofaminoacylase
d[Ac --L - Ala] --
dt (47)
[ E ] , . L .[Ac-L-Aka]-[E], “.““.[Ac].[L-Ala]
- KAF&Al. KAc ‘ K : : A l ~
[Ac] [D-Ala] [L-Ala]+ [Ac-L-AlaIl [Ac].[L-Ala]

1+--+--+ ~ +- [ A c ] ~ + [Acy .[L-Ala]
KAC KwAi, K , ~Aia KAc-[.-,a ’ KA, KLA-CAI~ K A ’ K::
’ ~ .K t :
K A ’ ~KL~CAL~
228
Maximum activity of the synthesis of Ac-L-Ala

Maximum activity of the hydrolysis of Ac-L-Ala
Dissociation constant of the EX complex with
X = acetate
X = L-alanine
X = D-alanine
X = acetyl-r-alanine
dissociation constant of the E(Ac)z-complexwith release of
acetate
(mmol L-'1 dissociation constant of the E(Ac)(l-A1a)complexwith re-
lease of L-alanine
This kinetic model contains 8 parameters: one parameter for each of the four
components D,L-alanine, acetate and acetyl-L-alanine,one parameter ( KL.AlaAC) for
the second substrate in the bimolecular synthesis reaction, one parameter describing
the substrate inhibition and two parameters describing the hydrolysis and the
synthesis rate. This is the minimum number of parameters necessary to describe the
above influences on enzyme activity (6 Table 7-1).
Eq. (47) was used for a simultaneous fit of all kinetic data measured under initial
rate conditions (Figs. 7-19 and 7-20). Separate fitting of each curve gives a better
coincidence in every single case, but the optimized kinetic parameter will vary from
fit to fit.
The optimized kinetic parameters are summarized in Table 7-2.
As described above, the rate equation can be set to zero yielding the equilibrium
condition ofthe reaction (Haldane equation). Using the kinetic data, k& is calculated
to be 12.2 mol L-'.
As ultimate proof of the kinetic model, a fit of time-courses of batch reactor

experiments was performed (Fig. 7-21). Initial concentrations of the components
over a significant range were chosen to yield hydrolysis conditions (1)and synthesis
conditions (2) respectively. Additionally, the equilibrium positions of corresponding
experiments A, B and C were chosen to be identical. Figure7-21 shows a good
correlation of calculated and measured data over the whole range of the conversion,
for hydrolysis as well as for synthesis.
Table 7-2. Kinetic parameters o f aminoacylase.
Hydrolysis ofAc-L-Ala Synthesis ofAc-L-Ala

AH.^^ 11.2 Umg-' &.ma 118.1 U mg-'
KA~-L-AI= 8.9 mmol L-' K A ~ ~ 1070 mmol L-'
KkA' 26.9 mmol L-'
K L - A I ~ ~ ~ 1070 mmol L-'
KL-AI~ 177 mmol L-'
KD-AI~ 270 mmol L-'
7.4 Investigation o f h z y r n e Kinetics
I
229
6
50
Time (min)
230
0 50 100 150 200 250 300 350

Time (min)
Figure 7-21. Time-course data o f hydrolysis and synthesis o f acetyl-L-alanineusing initial
conditions as specified in Table 7-3[”’1.
Table 7-3. Starting conditions for the experiments shown in Fig. 7-21.
Concentrations 4’) A(2) B(’) C(’) C(2)

IACIO (mmol L-’) 175 200 150 200 300 400
[D,L-A~~], (mmol L-’) 450 500 1400 1500 1300 1500
[Ac-L-Ala], (mmol L-’) 25 0 50 0 100 0
X [L-Ala], (mmol L-’) 200 200 200 200 400 400
X [~-Ala], (mmol L-’) 250 250 750 750 750 750
7.4.3
Kinetics of Multiple Enzyme Systems
If two or more enzymes are involved in coupled reactions, the influence of all
substances present in the reaction mixture on the activity of all enzymes has to be
studied and considered in the kinetic model. Knowing the kinetic behavior of the
single enzymes, coupling can be done by writing mass balances.
As an example, the reduction of dihydroxyphenylpyruvic acid (DHPP) to dihydrox-
yphenyllacetic acid (DHPL), a precursor of rosmarinic acid, is presented in Eq.
(49)‘lI9’.
I
231
H"E?OH
Dihydroxyphenylpyruvic acid Dihydroxyphenyllactic acid
DHPP OHPL
0
a-Hydroxyisocaproate
HO
PEG-NADH + H"' PEG-NAD")
H e-c (49)
I I
The reaction is catalyzed by D-hydroxyisocaproate dehydrogenase (D-HicDH).The

essential cofactor PEG-NADH is regenerated from PEG-NAD' by a second enzyme,
formate dehydrogenase (FDH).By coupling to water-solublepolyethyleneglycol with
a molar inass of 20 000 g mol-', the cofactor can be retained, together with the
enzymes, by an ultrafiltration membrane, and the whole process may be performed
continuously in an enzyme membrane reactor.
The kinetic models of D-HicDH and FDH are described as follows (Eqs. (50) and
(51)):
Kinetic model of D-HicDH
u - ~-
d[DHPL] [DHPP]
= [D - HicDH], A,-,,,,,,,
I - dr K,, + [DHPP]
.- [PEG - NADH + H+]
Kinetic model of FDH
u d[€or]
= -- - = [FDH], . A,,,,, . [For1 ,
dt K,, + [For1
.
, -
\PEG - NAD' 1 _.
In this case Michaelis-Mentendouble substrate kinetics is chosen (compare to Eq.

(45)). The system involving D-HicDH with DHPP and PEG-NADH as substrates
exhibits Michaelis-Menten kinetics for both substrates and competitive product
inhibition by PEiG-NAD'. FDH also shows Michaelis-Menten kinetics for both
substrates formate and PEG-NAD' and competitive product inhibition by PEG-
232
NADH. Both inhibiting effects are taken into consideration by an additional term in
the denominator. This formal procedure is valid as it allows a correct fit of all initial
rate data (not shown).
For a batch reaction (compare to Sect. 7.5) mass balances of all components of this
system may be formulated as follows (Eqs. (52-54)):
d[DHPL] - d[DHPP]
=v, (52)
dt dt
d[For]
-~ =v * (53)
dt
d[PEG-NADH+H+] - - d[PEG-NADt]
dt dt
=v, -v, (54)
The two enzymatic reactions are coupled by PEG-NADH and PEG-NAD’. This
stoichiometric coupling does not affect enzyme kinetics but has to be considered
when writing the mass balances. The discussion of this system will be continued in
Sect. 7.5 where some implications of coupled enzyme systems on reactor design are
described.
7.5
Enzyme Reactors
7.5.1
Basic Reaction Engineering Aspects
First, some basic terms of chemical reaction engineering will be discussed before
introducing the field of enzyme reactors. For further reading, textbooks are availa-
ble [1*&1261
The mode of reactor operation can be classified as “batchwise”or “continuous”.
Batch reactions are started by filling a reactor with the reaction mixture and stopped
after reaching the desired conversion. A steady state is only reached at equilibrium
conversion of the reaction. A typical batch reactor is represented by the stirred tank
reactor.
Continuous reactions are characterized by a continuous substrate feed and product
output. A residence time of the reaction mixture within the total reactor volume V
can be defined by Eq. (55):
=-
V
F (55)
5 (h) Residence time

V (L) Total reactor volume
F (L h-’) Substrate feed rate
7.5 Enzyme Reacton
I 233
stirred tank plug flow reactor continuous stirred
reactor (STR) (PW tank reactor (CSTR)
&I-- dx
t 4 4
._
L
.-
m
”
c
8
I .
* * *
time time time
+ 4 4
position position position
Figure 7-22. Comparison of stirred tank reactor, plug flow reactor and continuous stirred tank
reactor (Reaction: S + P; asterisks indicate time or position of substrate entering the reactor).
After a certain time a steady state will be reached within the reactor, meaning that
concentrations of substrates and products do not change. Typical continuous
reactors are the plug flow reactor (PFR) and the continuous stirred tank reactor
(CSTR).A comparison of these different reactor types is given in Fig. 7-22.
The differences between the reactors may be described by showing concentration
profiles of substrate and product as a function of reactor position and time
respectively.
- The stirred tank reactor (STR) in batch mode exhibits a decreasing substrate
concentration and increasing product concentration with time, independently of
the position within the reactor (the reactor is “wellmixed”, meaning that there are
no gradients within the reactor).
- The plug flow reactor (PFR) presents the same concentration curve along the
reactor length, which is shown for the tank reactor with reaction time. In the
steady state, the concentrations of substrates and products at distinct positions of
the reactor do not change with time. The reactor has plug flow characteristics,
234
I 7 Reaction Engineerhgfor Enzyme-Catalyzed Biotransformations
implying flow of reaction mixture through the reactor in the form of a plug
without any axial mixing.
- The continuous stirred tank reactor (CSTR) in the steady state exhibits constant
concentrations as a function of both time and position. The exit stream from this
reactor has the same composition as the fluid within the reactor; the CSTR is also
a well-mixed reactor.
A very common variation of the CSTR is a cascade of n CSTRs. With an increasing
number of reaction vessels, the cascade approximates to the plug-flow reactor. The
product concentration increases stepwise from vessel to vessel. For example, a two-
stage cascade can be used to overcome effects of product inhibition, e.g. in the
synthesis of L-tert-leucine [421 or GDP-Man[’44s14’1.
The basis for calculating reactor operation conditions is the formulation of mass
balances for all reaction components for the distinct reactor type. The mass balances
for the above reactors can be formulated as follows:
Stirred tank reactor: as no fluid enters or leaves the reactor the mass balance of
every compound is defined by the reaction rate only. To determine the time t
necessary to reach the desired conversion x the reciprocal rate equation has to be
integrated from zero to the desired conversion x:
With the definition of conversion shown in Eq. (56),
the equation for the reaction time t can be derived from the reaction rate v (Eq.
(57)):
x
r-i r. . .
(57)
at
L J”
2) 2)
PlugJow reactor: The change of reaction rate within a unit volume passing through
the length of the reactor is equivalent to a change corresponding to the residence
time z within the reactor. To determine the residence time z necessary to reach the
desired conversion x, it has to be integrated again over the whole range of
conversion. Eq. (57) can also be used for the PFR simply by replacing t by z (Eq.
(58)).
x
z =[S&.jllu-dx
0
Continuous stirred tank reactor: In the case of CSTR, the change of concentration of a
substrate S within the reactor (“accumulation”)is brought about by two terms:
- The “convection term”, which describes the change of concentration of S within
the reactor as an effect of the influx of the substrate into the reactor, reduced by the
efflux out of the reactor.
- The “reaction term”,which describes the change of concentration of S as result of
the reaction S P.
+
7.5 Enzyme Reactorr
I 235
accumulation = convection + reaction
&I
~~ -
- b3, - bl --u (59)
dt z
At steady state, the concentrations within the reactor will not change, meaning that
the accumulation term equals zero. By using the definition of x (Eq. ( S ) ) , the
residence time T necessary to reach the desired conversion x can be calculated (Eq.
(60)):
T is given by a simple multiplication of x

by the reciprocal reaction rate at this steady
state conversion x. The situation can be further illustrated by the following plot
(Fig. 7-23):
k STR, PFR
CSTR 2 CSTRs in series

1 b
* * *
X X ’ 2
Figure 7-23. Reactor design from l / v = f(x) plots (shadowed area represents r[S]o’; asterisks
represent reactor outlet conditions).
236
If the reciprocal reaction rate is plotted as a function of conversion the shadowed

areas represent the residence time necessary to reach a given conversion x (Eqs. (61)
and (62)).
T
= 11/11 .dx (for stirred tank and plug flow reactor) (61)
[SL
~
2
= I/II . dr (for continuous stirred tank reactor) (62)
[SL
~
Obviously the residence time is higher in the case of the CSTR compared to the STR
and the PFR if the reaction rate continuously decreases with conversion, this most
often being the case in enzymatic reactions.
Minimizing the necessary r value for reaching a defined conversion x (at constant
[Sl0 value) can be done graphically using these l / v = f(x) plots (Fig. 7-23). For
different reactors, the area may be determined according to the above method and
the appropriate reactor is given by the minimal area. For example the r value of a
CSTR can be minimized by using two CSTRs in series. The steady state reaction
mixture of the first reactor (conversionxl) is fed into the second reactor, yielding an
overall conversion x2.
With the relation
11 = [El, .A (compare to Eq. 23)
Eq. (61) and Eq. (62) may be written as
= 11/ A .dx (for stirred tank and plug flow reactor) (64)
[SI, 0
[EL -T
___ = I / A . x (for continuous stirred tank reactor)
[SL
This means that the conversion x of a reaction at a constant [S]o value depends on the
extent of activity during the conversion on the one hand and on the product [El0 . T
on the other. Further, this means that lowering the enzyme concentration by a factor
y and raising the residence time by the same factor y gives the same conversion x.
This is the so-called ‘‘[El0 . r-concept“, valid for all three reactors.
Up to now general reaction engineering principles have been discussed. Now we
turn to enzyme reactor design.
The suitability of different reactors is demonstrated for two typical enzyme kinetic
examples, involving substrate inhibition in one case and product inhibition in the
other (Fig. 7-24).
The kinetics within Fig. 7-24 do not represent initial rate kinetics, but the reaction
rate during the conversion S -+ P plotted versus the remaining substrate concentra-
tion (initial concentration [Slo = 1 mmol L-l).
7.5 Enzyme Reactors
I
237
[Substrate] (mmol L-')
-0:o 0.2 0.4 0.6 0.8 1.o

[Substrate] (mmol L-')
Figure 7-24. Performance of a CSTR depending on enzyme kinetics: A substrate inhibition, and
B product inhibition (bold points indicate the inlet and outlet concentrations o f a CSTR).
- The ideal reactor to overcome substrate inhibition (Fig. 7-24A) is the continuous
stirred tank reactor (possible in form of an Enzyme Membrane Reactor, see
below). In spite of a high feed concentration of substrate a high reaction rate
occurs, as the steady state substrate concentration within the reactor is low.
238
- In the case of product inhibition (Fig. 7-24B),the continuous stirred tank reactor
is not beneficial, as a high reaction rate would occur at high substrate concentra-
tion and low conversion only. With increasing conversion the inhibiting effect of
the product becomes dominating, yielding a reduced reaction rate. The CSTR as a
whole operates at low steady state substrate concentration and therefore low
reaction rate.
In the case of product inhibition, the plug flow reactor is advantageous, as the
concentration of the inhibiting product slowly increases along the reactor length,
whereas in a CSTR the product concentration is at the maximum value in the whole
reactor. Therefore, the average reaction rate is higher in the PFR.
A plug flow reactor may be realized using immobilized enzymes within a column
reactor or using soluble enzymes within a cascade of membrane reactors. A batch or
a repetitive batch process with soluble enzymes (see below) has the same productiv-
ity as the plug flow reactor.
The overall reaction rate not only determines the necessary reaction time to reach
the desired conversion (see Fig. 7-23) but also the enzyme and cofactor consump-
tion, both being strongly influenced by the reactor conditions.
Enzyme and cofactor consumption can be defined as follows:
- Enzyme consumption is defined as the number of units of enzyme consumed per
unit weight of product ( U kg-’);
- Cofactor consumption is specified with the “total turnover number”, defined as
mols cofactor consumed per mol of product formed.
As in most cases reaction rate decreases with conversion, enzyme consumption
increases to the same extent (an exceptional case is when strong substrate inhibition
overcompensates product inhibition). Therefore enzyme consumption is minimal
under initial reaction rate conditions (zero conversion) (Fig. 7-25).Approaching total
or equilibrium conversion the reaction rate approaches zero and enzyme consump-
tion increases rapidly.
In contrast to enzyme consumption, substrate utilization, defined as kg substrate
consumed per kg product produced, increases with increasing steady state conver-
sion.
Besides the classical engineering question of reactor choice, the most important
point in enzyme reactor design is the aspect of enzyme reuse, either by immobiliza-
tion or by separation from the product stream. Batch processes without enzyme
reuse are only possible if the costs of the biocatalyst are negligible. Different reactor
techniques addressing the aspect of enzyme reuse are discussed in the following
sections.
7.5.2
Reactors for Soluble Enzymes
If soluble enzymes exhibit sufficient operational stability, their use is advantageous,

as the effort of immobilization and resulting mass transfer limitations can be
avoided. Different techniques have been developed to retain soluble enzymes.
7.5 Enzyme Reactors
I
239
-cn
h
n
-
h
a
Y
d
w
c
Y
2
C C
.-0 0
.-
c
e
4-
E
3 3
v)
C v)
C
8 0
0
a, a,
2 5
c
c
v) N
C
3 w
cn
0.2
0.0
Figure 7-25.
0.4
Conversion
0.6
- 0.8
Substrate utilization and enzyme consumption as a function o f conversion.

1 .o
For small-scale synthesis enclosure of enzymes in dialysis tubes has been

described for several systems (membrane-enclosed enzyme catalysis or the MEEC
technique In this case mass transport of the low-molecular-weightsubstrates
and products across the membrane becomes rate limiting because mass transport
only occurs by diffusion and not by convection as described below.
For synthesis on a preparative scale, repetitive batch processing has proved to be
an effective and easy-to-handle method[12*].The repeated use of the enzyme is
possible after concentration of the solution by means of commercially available
ultrafiltration equipment and adding fresh substrate solution. Some of the advan-
tages given for the Enzyme Membrane Reactor (see below) are also valid for the
repetitive batch technique.
Compared to batch processes, continuous processes often show a higher space-
time yield. Reaction conditions may be kept within certain limits more easily. For
easier scale-up of some enzyme-catalyzed reactions, the Enzyme Membrane Reactor
(EMR) has been developed. The principle is shown in Fig. 7-26A. The difference in
size between a biocatalyst and the reactants enables continuous homogeneous
catalysis to be achieved while retaining the catalyst in the vessel. For this purpose,
commercially available ultrafiltration membranes are used. When continuously
operated, the EMR behaves as a continuous stirred tank reactor (CSTR) with
complete backmixing. For large-scale membrane reactors, hollow-fiber membranes
or stacked flat membranes are To prevent concentration polarization on
the membrane, the reaction mixture is circulated along the membrane surface by a
low-shear recirculation pump (Fig. 7-26 B).
240
I 7 Reaction Engineerhgfor Enzyme-Catalyzed Biotransformations
A
pressure
substrate o +-J-
product 0
B
-0-
ration module
Figure 7-26. A Principle o f t h e Enzyme Membrane Reactor. 6 Set-up for larger scale including
pump for circulation and filtration module.
Advantages and disadvantages of the EMR are summarized as follows:

Advantages
Working under sterile conditions is easy to achieve;
No loss of enzyme activity by immobilization;
No mass transport limitation;
High volumetric activity achievable;
Use of multi-enzyme systems without mass transport limitation;
Use of coenzymes without mass transport limitation;
Simple addition of fresh enzyme to compensate for enzyme deactivation;
Ultrafiltered (pyrogen-free)product solution.
Disadvantages
- Sometimes limited by enzyme stability in solution;
- Back-mixingis not optimal for enzymes with product inhibition.
Figure 7-27 shows a flow chart of the experimental set-up of a two-stage membrane
reactor cascade. A 10 mL version of the reactor for process development and small-
7.5 Enzyme Reactors
I
241
a
1v
analyiics
1
m 7
@ pressure gauge 4 sterile filter

@ thermometer 5 ultrafiltration module
1 substrate reservoir 6 on-line analytics
2 pump (dosing, circulation) (PH, uv, polarimeter u. a,)
3 bubble trap 7 fraction collector
Figure 7-27. Flow chart o f t h e experimental setup o f t h e EMR.
scale production has been commercialized[1301. Depending on the reactor and

membrane material, sterilization by means of steam or chemicals (peraceticacid) is
possible.
7.5.2.1
Reactor Optimization Exemplified by the Enzyme Membrane Reactor
The performance of the EMR may be calculated by means of the measured kinetics
and the simultaneous calculation of mass balances of each reactant. The steady-state
parameters of the reactor can be estimated by numerical integration of the differ-
ential mass-balance equations by means of the Runge-Kutta method.
Simulation of the reactor performance is a useful tool to find suitable conditions
for production. Therefore it is a method resulting in a saving of both time and costs
by avoiding large numbers of different experiments. But it has to be proven that the
model is able to describe the process within the range of interest.
In the following discussion the method of reactor optimization will be demon-
strated using two enzyme systems introduced earlier, namely the enzymatic synthe-
sis of N-acetylneuraminic acid and the enzymatic synthesis of cyanohydrins using
oxynitrilase.
Exampb: Enzymatic synthesis of N-acetylneuraminicacid

This example has been used earlier to discuss the influence of thermodynamics on
the reaction conditions (Fig. 7-7). It was shown that a great excess of pyruvate is
helpful in order to increase equilibrium conversion, but for practical reasons there is
a limit, because
242
v
1.0 I
0.9
8 0.8
1 I I I I 700
- 600
- 500
x 0.7 - 400
C
.-0
2
a 0.6- - 300 h
3
2 3
6 0.5- - 200 o_
0.3
0.4 1 1 2
7
3
- 100
-0
Ratio [Pyr],:[ManNAc], (-)

Figure 7-28. Steady-state concentration and conversion as functions o f initial substrate ratio in
an EMR [471; conditions: [El0 = 4 g L-', t=3 h, [ManNAcIo = 300 rnmol L-'.
- to reach 91% conversion instead of 86% the amount of pyruvate has to be

increased by 50% (conditions specified in Fig. 7-28);
- with increasing concentration of pyruvate, downstream processing is more
difficult and time consuming (Neu5Ac and pyruvic acid have similar pK, values
(2.0 and 2.4), complicating separation by anion exchange chromatography).
To enable a quantitative description of the system, kinetic measurements of
synthesis and cleavage of Neu5Ac were performed and expressed by a kinetic
The overall reaction rate is given by Eq. (66).
Abbreviations: A=Pyr, B=ManNAc, P= NeuSAc, S=synthesis reaction, C=cleavage

reaction.
2) (kat L-') Velocity of Neu5Ac synthesis
&,ma (kat g-') Maximum specific activity of synthesis
&,ma (kat g-') Maximum specific activity of cleavage
K M ~ (mol L-'1 Michaelis-Mentenconstant of Pyr
K M ~ (mol L-'1 Michaelis-Menten constant of ManNAc
KM" (mol L-') Michaelis-Menten constant of Neu5Ac
KIA (mol L-') Inhibition constant of Pyr
Table 7-4. Kinetic parameters of Neu5Ac-aldolase.
7.5 Enzyme Reactors
I
243
Synthesis of NeuSAc Cleavage of Neu5Ac

AS,nlaX 230.5 pkat g-’ Ac.rnax 141.8 pkat g-’
13.8 U mg-’ 8.51 Umg-’
KMA 0.13G mmol L-’ KMP 9.44 mmol L-’
KMB 402.2 mmol L-’ KIA 1.30 mmol L-’
KIB 23.8 mmol L-’
Kv 1556 mmol L-’
KIB (mol L-*) Inhibition constant of ManNAc

[El0 (g L-7 Enzyme concentration (aldolase)
Kv (L mol-’) Concentration-dependent inhibition
The first term in the denominator of Eq. (66) represents a non-competitive inhibi-
tion (compare to Eq. (31)) of the enzyme by the sum of concentrations of A, B and P.
This non-specific inhibition could be correlated with an increased viscosity of the
reaction medium in the presence of A, B and 1311. As the mutarotation of the
carbohydrates is fast compared to the enzymatic reaction there was no need for a
discrimination between the a,P-anomers or the open-chain form of the mono-
saccharides. A complete set of kinetic parameters was determined, summarized in
Table 7-4.
The mass balances for the substrates and products are given by Eqs. (67-69).
- [ManNac], - [ManNAc] -2)

d[ManNAc] -
dt z
d[NeuSAc] -
- [NeuSAc], - [NeuSAc] +2)
dt 0
At steady state the mass balance of each component N may be rewritten as shown in
Eq. (70).
d“1 - 0 =+
-- 2)
“I,
=-.x
dt z
meaning that the reaction term is equal to the convection term (compare to Eq. (59)).
This identity can be used to determine the conversion of the substrate at a given
residence time from a plot u=f(x) (Fig. 7-29).
The point of intersection of the function u=f(x) and the straight line v = [N]@ . x
(the “convection term”) determines the operating point of the reactor at given
concentration “lo and residence time z. The operating point is characterized by
substrate conversion and reaction rate under steady-state conditions. By changing
the slope of the convection line, e. g. by change of the residence time, other operation
244
250 [ManNAc], = 300 mmol L-' ~
h
r
v)
j 200
7
-
E
'D 150
0
-
v
7
Q)
2 100
-2
S
.-0
50
a,
CT
0
0.0 0.2 0.4 0.6 0.8 1.o
Conversion xManNAc(-)
Figure 7-29. Graphical method o f determination of steady-state operation conditions in an
EM Rl4'1 (bold points indicate steady-state reactor conditions; asterisks indicate steady-state
conversion).
points may be determined. Instead of increasing the residence time the enzyme
concentration may be increased to the same extent, yielding the same result
(compare to the [El . T concept).
This method enables determination of operation conditions of an EMR from batch
reactor data. For that purpose a differentiation of the function x(t) has to be
performed and converted to the above curve v = f(x).
Using the mass balances, optimum operating conditions for continuous product-
ion in the EMR were calculated. Concentrations of 300mmol L-' ManNAc and
GOO mmol L-' pyruvate were found to be the most suitable to allow high conversion
of ManNAc, high space-time yield, and easy product isolation. Figure 7-28 shows
steady-state concentration and conversion as a function of substrate ratio in an
EMR.
With a ManNAc concentration of 300mmol L-' and an equimolar amount of
pyruvate in the feed, G8% conversion is reached and the product solution contains
200 mmol L-' NeuSAc, 100 mmol L-' pyruvate and 100 mmol L-' ManNAc. At this
low conversion, unreacted ManNAc should be recovered, requiring an additional
purification step. Conversion increases with excess of pyruvate to almost 90 % at only
twofold excess of pyruvate, but there is a larger amount of pyruvate remaining in the
product solution which has to be separated.
In Fig. 7-30, conversion and space-time yield for the continuous process in an
EMR are depicted. Enzyme concentration and residence time were chosen to be
within practical limits to ensure a reasonably high conversion, suitable for produc-
tion.
7.5 Enzyme Reactors
I '
I
245
Reactor - 2500
- - p - - -
0 500 1000 1500 2000 2500 3000

[ManNAc], (mmol L')
Figure 7-30. Conversion and space-time yield as function o f substrate ~oncentration1~~1.
The increase of-conversion with rising concentration results from an increase of

equilibrium conversion as depicted in Fig. 7-7. The decrease of conversion at
ManNAc concentrations >500 mmol L-' is due to the increasing effect of non-
specific inhibition (compare to Eq. (66)).The space-time yield decreases when the
decrease of conversion exceeds the increase of ManNAc-concentration.
According to these results a feed concentration of 300 mmol L-' ManNAc was
chosen for NeuSAc production, allowing a high conversion (>85%) and a good
space-time yield (650 g L-*d-'). The latter may be raised by increasing enzyme
concentration and decreasing residence time ([El . T=constant), resulting in the
same conversion within a shorter time.
The model proved to be correct by a comparison of predicted and experimental
conversions for several enzyme concentrations and residence times (Fig. 7-31).
Biocatalyst consumption per unit weight of product was found to be about 6000 U/
kg at a conversion of 78%. For production purposes, enzyme membrane reactors
with a working volume up to 500 mL were employed for the synthesis of approx-
imately 2 kg of N-acetylneuraminic acid and other derivatives such as keto-
desoxynonulosonic acid (KDN) 11*', 13*1. Downstream processing was achieved
mainly by anion exchange chromatography on a 30 L column followed by reverse
osmosis to concentrate solutions before lyophilization.
Example: Enzymatic synthesis of benzaldehyde cyanohydrin

In the case of the enzymatic synthesis of cyanohydrins, enantioselectivity is the most
important criterion. Investigation of the kinetics of the whole system (enzymaticand
non-enzymatic reaction) offers the possibility to optimize reaction conditions to
246
1.o
0.8
2 0.6
C
.-0
r
? 0.4
C
8
0.2 -Calculated -
0 Measured
I
0.0 I I
0 10000 20000 30000 40000
[El a s L-')
Figure 7-31. Comparison o f calculated and experimental c o n ~ e r s i o n ~ ~ ~ ] .
obtain products with high optical purity. The discussion within Sect. 7.3.2.2 is
extended to a consideration of reactor design.
From the kinetics of the enzymatic and the non-enzymatic reactions (Fig. 7-13) it
is concluded that the side-reaction is suppressed very effectively by working with
high enzyme concentrations and at a low benzaldehyde concentration. Benzalde-
hyde may react with amino functions of the enzyme to form Schiff bases resulting in
deactivation of oxynitrilase, so low stationary benzaldehyde concentrations are also
necessary with respect to enzyme stability.
As the EMR behaves as a CSTR, conversion in the whole reactor becomes high and
the stationary benzaldehyde concentration becomes low if the product [El . T is
chosen to be sufficiently high.
Enzyme concentration as well as residence time T have been varied and the
resulting conversion and enantioselectivity calculated. Fig. 7-32 shows enantiose-
lectivity as a function of conversion in continuous experiments.
Different operating conditions were obtained by changing the enzyme concentra-
tion. The benzaldehyde concentration is determined by its maximum solubility. A
fourfold excess of hydrogen cyanide is necessary to reach conversions greater than
90%. A larger excess will favor the non-enzymatic reaction yielding lower enantiose-
lectivities. As already postulated from the initial rate measurements, enantioselectiv-
ity increases with increasing conversion. There is a good correlation between
calculated and measured values, as well as for other substrate ratios not shown here.
The reactor conditions and results are presented for one experimental run.
7.5 Enzyme Reactors
I 247
1.o I I n 1
0
0.8
Results
h
v
I Benzaldehyde 0.048 mol/L
Hydrogen cyanide 0.180 mollL
.-
.
I-
.->
.
I- 0.6 Enzyme concentration 0.38 g/L
0
0
-Q)
Residence time 10 min
v) Max conversion 90 Yo
.-0 0.4
.
I-
Enantiomeric excess >99 Yo
a
C
C
w 0.2 Space-time yield 773 g/(L'd)
I I I
Calculated 0 Measured 1 1
In a production run,the space-time yield was enhanced to 2400 g L-'d-' (R)-

mandelonitrile (ee>99%) at a residence time of just 3.8 min using an enzyme
concentration of 0.95 g L-'.
A similar investigation for batch reactor systems has been published recently[133].
DSM Linz in Austria established a process for the synthesis of (S)-phenoxybenzalde-
hyde cyanohydrin using a cloned (S)-oxynitrila~e~~'~ 1341. The reaction is performed
in a two-phase system with the substrate dissolved in an organic solvent.
Example: Coupled enzyme systems

In Sect. 7.4.3, the reduction of dihydroxyphenylpyruvic acid (DHPP) to dihydrox-
yphenyllactic acid (DHPL)was used as an example for discussion of the kinetics of
multiple enzyme systems (Eq. (49)).The rate equations for the reduction reaction of
DHPP to DHPL (u1) and the regeneration of PEG-NAD' to PEG-NADH (u2) have
been introduced (Eqs. (50) and (51)).
The mass balances of all components in the case of performing the reaction in a
CSTR are formulated as follows (Eqs. (71)-(75)):
d[DHPP] - [DHPP], - [DHPP]
--'u I
dt z
d[DHPL] -
-
[DHPL], - [DHPL]
+-'u I (72)
dt z
d[For] - [For]" -[For]

--
dt z
--2) 2 (73)
248
I d[PEG - NADH+ H’]
=I), -Ill (74)
dt
d[PEG - NAD’]
=I), -v2 (75)
dt
In the case of PEG-NAD’ and PEG-NADH,the convection term equals zero as both
are completely retained within the reactor by the ultrafiltration membrane. The
enzymatic reactions are coupled by PEG-NADH and PEG-NAD’. As stated pre-
viously, this stoichiometric coupling does not affect the enzyme kinetics, but has to
be considered when writing the mass balances.
Considerations about process optimization of coupled systems with coenzyme
regeneration are discussed in the literature[”, 13’1. One aspect may be illus-
42,433
trated here - the question of enzyme ratio within the coupled enzyme system.
Figure 7-33 represents the influence of the ratio of the two enzymes D-HicDH for
the synthesis reaction and FDH for the cofactor regeneration.
The enzyme ratio represents the ratio of the initial volumetric activities of the
enzymes (dimensions U mL-l). For process conditions, enzyme activity under the
actual steady-state reactor conditions is significant, and differs from initial rate
conditions as determined by enzyme kinetics. Therefore, the optimum enzyme ratio,
implying maximum conversion within minimum residence time, is not 0.5.
As evident from Fig. 7-33, it does not make sense to add one enzyme, e.g. the
cheaper one, in excess to the reaction mixture, as, a long way from the optimum of
‘I: = 60 min
0.0 0.2 0.4 0.6 0.8 1.o

Enzyme ratio (-)
D-HiDH/(FDH+D-HicDH)
Figure 7-33. Dependence o f conversion on enzyme ratio a t different residence times[”91.
7.5 Enzyme Reactors
I 249
0 20 40 60 80 100 120
Time (h)
Figure 7-34.Comparison of calculated and measured data for the enzymatic synthesis of
dihydroxyphenyllactic acid in an EMR[”91.
enzyme ratio, only one enzyme determines the overall reaction rate. This situation
might appear in coupled enzyme assays where care has to be taken that the enzyme
of interest is rate determining.
As proof of the kinetic model, fitting of initial rate data or time-course data of batch
reactions have been introduced in Sect. 7.4. Additionally, a proper fit of continuous
reactions in an enzyme membrane reactor (EMR) may serve as confirmation of the
kinetic model. For this coupled enzyme system, calculated and measured conver-
sions at different operating conditions (varying [El and T values, not further
specified) are presented in Fig. 7-34.
7.5.2.2
Control of Conversion in a Continuously Operated EMR
As a consequence of enzyme deactivation, conversion may drop during the con-

tinuous operation of enzyme reactors. To maintain a constant degree of conversion,
two methods can be employed according to the [El . T-concept (see above):
- addition of fresh enzyme,
- reduction of flow rate.
Both can be done very effectively by using methods of online analytics combined
with an appropriate automatic controller. Useful methods for online analysis of
enzymatic processes are
- polarimetry (useful for reactions where chiral reactants are involved)[1361,
- UV spectrometry,
250
- online-HPLC (may be used effectively for controlling complex reactions (e.g in

peptide or carbohydrate synthesis))
7.5.3
Reactor Systems for Immobilized Enzymes
The choice of an appropriate reactor for applications of immobilized enzymes as well

as for soluble enzymes depends on the kinetics of the reaction. Kinetics of
immobilized enzymes are not only a function of enzyme activity but also of substrate
transport to the enzyme, which is affected by the matrix used for immobilization. For
a description of immobilized enzyme kinetics the reader is referred to the compre-
hensive literature in this field 138-1401.
[35-40r Additionally, the use of immobilized
enzymes is treated in Chap. 6 of this book.
A brief overview of enzyme reactors used for application of immobilized bio-
catalysts in the laboratory and on the industrial scale is given in Fig. 7-35. Examples
of industrial processes are given in [2] and [20].
To study the kinetics of immobilized enzymes a recirculation reactor may be used.
This reactor allows one to perform kinetic measurements with defined external mass
transfer effects, reached by establishing a high flow rate near the catalyst, minimiz-
ing mass transfer resistance. The reactor behaves as a differential “gradientless”
reactor allowing initial-rate kinetic measurements to be made.
The fixed bed reactor, behaving as a plug flow reactor, is most often used for
immobilized enzyme reactions. Typically, the reactor is used with an upward
direction of the flow to avoid compression of the bed and to release gas bubbles
generated during the reaction. Reactor design may be done readily without knowing
the detailed enzyme kinetics. Kinetic measurements are performed with a recircula-
tion reactor and the data are plotted in the form l / u = f(x) (see above). From this plot,
the residence time necessary to reach a desired conversion x can be calculated as
described. The different enzyme concentrations in the recirculation reactor and in
the plug flow reactor have to be considered.
Fluidized bed reactors are advantageous if small particles which would give high
flow resistance in a fixed bed reactor are used to minimize external mass transfer
resistances. Often it is useful to install nets at different heights of the reactor to
approach plug flow characteristics.
If the immobilized enzyme particle size is so small that an effective retainment is
not possible in fluidized bed reactors, a slurry reactor may be used. This reactor
guarantees catalyst retainment using a filter or a microfiltration membrane. For a
larger particle size, the use of a stirred tank reactor is not advantageous because the
energy input necessary to give an optimal fluidization of the particles is much higher
than in a fluidized-bedtype reactor.
Besides the immobilization of enzymes on solid particles, enzymes may also be
immobilized on the inner or outer surface of tubular supports such as on hollow
fibers or flat membranes. Enclosure of enzymes by the use of an ultrafiltration or
dialysis membrane is regarded as a form of immobilization.
7.5 Enzyme Reactors
I
251
Fixed bed Fixed bed reactor

recirculation reactor
+ P
Fluidized bed reactor Slurry reactor
71
S
V
0 O O
0
0
0
0 0 0
sieve
Figure 7-35. Reactors for use of immobilized enzymes.
7.5.4
Reaction Techniques for Enzymes in Organic Solvents
The same reactors can be used for dealing with immobilized enzymes in organic
solvents or with one-phase organic systems as for dealing with enzymes in aqueous
solutions. For one-phase systems, the enzyme may be recovered from the solution by
means of membrane filtration. Suspended enzyme particles may be retained in a
slurry reactor (compare to Fig. 7-35) by microfiltration membranes or stainless steel
sieves, whereas in other cases such as reverse micelles, ultrafiltration membranes
have to be used[’’]. For some years ultrafiltration membranes have been available
252
Figure 7-36. Multi-compartment enzyme membrane reactor.
which are stable toward organic solvents (e.g. from polyaramide or cellulose). In
these cases the enzyme membrane reactor, as described earlier for the pure aqueous
system, may be used without modifications, if all materials (sealing rings, tubes) are
stable toward the solvent used.
For two-phase systems, special reactors may be used such as multi-compartment
reactors as shown in Fig. 7-36[43, 14', 1421.
In the latter case, the two phases are separated by a hydrophobic or hydrophilic
membrane (solid supported interface). The enzyme is soluble in the aqueous phase
and substrate is added up to its maximum solubility in the aqueous phase.
Substrates and products are distributed according to their hydrophilic or hydro-
phobic properties. The membrane area has to be large enough to avoid mass transfer
limitations. High membrane areas may be achieved by flat membrane stacks or by
hollow fiber modules.
If lipases are used they may be adsorbed at the interface on a hydrophobic
membrane. Such a system has been developed by Sepracor, USA, for the enantiose-
lective hydrolysis of racemic esters (Fig. 7-37)and is used by Tanabe Seiyaku Co. for
the kinetic resolution of 3-(4-methoxyphenyl)glycidicacid methyl ester, which is an
intermediate in the synthesis of diltiazem [20*77,1431.
Here the racemate is circulated on one side of the membrane while the water
necessary for hydrolysis is picked up from the other side. The resulting acid is
extracted into the aqueous phase, where the pH is kept constant with NaOH. The
enzyme is adsorbed in the pores of the membrane. For the pilot scale production the
following data were obtained: reactor productivity 125 g d - k 2 , enzyme consump-
tion 17 g per kg product, enantiomeric excess of product 99 %, product yield 43 %.
Additional reactor systems, especially reactors for synthesis of oleochemicals are
summarized in [99].
7.G Conclusions and Outlook
I
253
rac-Methyl-methoxy- (PS,BR)-Methyl-methoxy- (ZR,SS)-Ester

phenylglycidic acid methyl ester phenylglycidic acid
(2R, 3s) (2S, 3R)

Ester Acid
f f
rac. --&& Q-J

Ester
Figure 7-37. M e m b r a n e reactor system for resolution o f a diltiazem intermediate.
7.6
Conclusions and Outlook
Some basic aspects of reaction engineering for enzyme-catalyzedbiotransformations

have been presented within this chapter, using examples of processes investigated by
the authors. A strategy was shown starting with considerations focusing on the
appropriate enzymes, continuing with an investigation of enzyme kinetics, and
developing a reactor model. Finally, with the aid of process optimization, the most
favorable reactor conditions can be found in order to achieve a high space-time yield
with high conversion, high selectivity and low enzyme consumption.
Enzyme engineering has reached a status where many limitations have been
overcome by interdisciplinary efforts. New enzymes are being sought and, indeed,
found in natural environments which will perform biotransformations on unnatural
substrates. Huge quantities of enzymes can be produced by genetic engineering
methods, making availability and costs of enzymes no longer a barrier for commer-
cial processes. Much progress has been made in the evolution of biocatalysts to
improve their properties. But still the choice of solvent to perform the reaction and
the configuration of the surrounding reactor are important issues to be considered
as well, to obtain optimum results.
Based on enzyme kinetics well established by biochemists, complex reactions can
be modeled. By utilizing classical reaction engineering methods, a quantitative
254 7 Reaction EngineeringforEnzyme-Catalyzed Biotransformations
I description of process performance can be achieved and an operational status of a
reactor can be calculated, saving experimental effort. Of particular note is the
development of continuous processes, which have the advantage of better con-
trollable reaction conditions, allowing suppression of undesired side-reactions and
yielding better selectivity. Space-timeyields of several kilograms of product per liter
of reactor volume per day have been reached.
The methods of enzyme reaction engineering have already shown their benefits in
numerous industrial processes which are being established successfully. Hopefully
it can be concluded from this article that process development for the application of
enzymes in organic synthesis can be performed on a rational basis.
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125 M. F. Chaplin, C. Bucke, Enzyme technology, 1990,80-137.
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1990. stein, Applied Biochemical Bioengineering,
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I
259
8
Enzymic Conversions in Organic and Other Low-Water Media
Peter Halling
8.1
Introduction
It is now often preferred to carry out enzyme-catalyzed biotransformations in non-

aqueous media. These are commonly based on organic solvents, but a variety of
other low-water media can be useful. Popular reasons for using such systems are
listed in Table 8-1.
When a low-waterbiotransformation is planned, a variety of choices must be made
about the precise reaction conditions to be used. As in all biocatalytic systems, the
rate and yield obtained can be greatly affected by the choices made. Some of the
factors that must be considered are the same as in conventional aqueous media:
temperature, reactant concentrations, the form in which the enzyme is added. New
factors must be taken into account such as solvent selection and the level of residual
water in the system. Other factors become somewhat modified: acid-baseconditions
remain important, but, usually, pH is no longer a useful parameter to characterize
them.
This chapter is written primarily for a reader who wishes to carry out a low-water
biotransformation, and requires some general advice on the selection of reaction
conditions. It will be a long time before our understanding of these systems is
sufficient to predict confidently the optimal conditions for a novel reaction. Of
course, we cannot do this for an aqueous biotransformation, or, for that matter, most
non-enzymic chemical transformations. However, it is possible to give recommenda-
Table 8-1. Common reasons for choosing low-water media for biotransformations.
Reactants don’t have to be (very)water soluble - use different solvent, or just undissolved solids
Changes in solvation alter equilibria and kinetics - e. g. readily available hydrolytic enzymes
catalyse synthetic reactions (including direct reversal of hydrolysis)
Can tune specificityby changing medium etc.
Enzymes can be more stable
Suppression of unwanted processes in aqueous solution, e. g. microbial growth, side-reactions
Better integration of biocatalytic with chemical steps in non-aqueous media
260
I 8 Enzymic Conversions in Organic and Other Low-Water Media
tions that will often lead to a reasonable performance at first attempt, and will guide
the design of experiments to approach closer to the optimum.
In general I will not discuss in detail the evidence that lies behind the recom-
mendations presented here. The reader who wishes to explore this further should
consult the following list of recent reviews of the These form a basis for an
individual assessment of the most useful choices. This chapter is more aimed at
those who wish to rely on my judgments and is written in a rather brief and
prescriptive style in many places, in order to minimize the length.
Undoubtedly some of these recommendations will be found less than ideal in the
light of further investigation. However, I am fairly confident that most of them will
remain good choices.
8.2
Enzyme Form
In aqueous media, the most usual state of the enzyme molecules is dissolved in the
reaction medium. In this case, the previous treatment of the enzyme has little effect
on catalytic activity, provided irreversible inactivation is avoided. In contrast, in low-
water media, the enzyme molecules are usually present in solid particles. The way
the solid biocatalyst is prepared will clearly affect the state of the enzyme molecules,
and hence their catalytic properties. Furthermore, as hydration of the enzyme
molecules is reduced, it is clear that conformational changes can become much
slower. As a result the previous history of the enzyme has important effects, not just
the final conditions. In other words, there may be pronounced hysteresis effects.
Enzymes are normally isolated from their biological source as an aqueous
solution. Hence preparation for transfer to a low-water medium requires removal of
much of the water from the environment of the protein molecules. A variety of
different drying methods can be considered. Although presented below are certain
statements about the relative activity of these different forms, it must be acknowl-
edged that there have been few direct comparisons of the same enzyme prepared in
different ways but then used in the same medium.
8.2.1
Lyophilized Powders
Lyophilization (freeze-drying)is one of the most obvious ways of producing solid

particles from an enzyme solution, and is probably the most used in preparing low-
water biocatalysts. However, I would generally not recommend it. It is of course a
standard and usually reliable method of drying enzymes for storage. However,
lyophilized enzyme powders used directly in low-water media typically show much
lower specific activity (per enzyme molecule) than other preparations. At least two
factors seem to contribute. Many of the enzyme molecules in lyophilized powders
have changed conformations, probably due to the conditions in the concentrated
unfrozen regions during freezing. This (partial) denaturation is reversed on return
8.2 Enzyme Form I261
to aqueous solution, but not in low-water media. Considerable attention has been
given to methods that can increase the specific activity of lyophilized powders by
orders of magnitude, such as drying in the presence of salts or lyoprotectants. But
these large increases reflect primarily the very poor activity of the control lyophilized
powders.
8.2.2
Immobilized Enzymes
Immobilization is an alternative route to solid particles containing the enzyme,

typically by attachment to a pre-existing support. Immobilized forms of many
enzymes are now commercially available. For use in low-water media, the attach-
ment is usually done very simply by adsorption - the support particles are simply
stirred for a time in the aqueous enzyme solution. The strength of the linkage to the
surface is not really an issue, because enzyme desorption will not normally be
possible in low-water media. Much of the water is removed by decanting the
supernatant. The wet solid particles may then be dried further by evaporation in air.
However, a better method seems to involve rinsing with a suitable solvent that is able
to dissolve plenty of water (i.e. usually a water-miscible solvent). This exploits the
hysteresis in enzyme behavior to give a higher specific activity catalyst, at least for
initial rates and with some enzymes. Drying procedures are discussed further below
in the context of water effects.
More complicated immobilization methods have been described, including cova-
lent attachment to a support, entrapment inside particles (such as silica made by a
sol-gel process), and covalent incorporation into polymer particles (“biocatalytic
plastics”). However, it is not clear that any of these methods is superior to simple
adsorption, particularly for use in low-watermedia, where enzyme desorption is not
an issue.
A method first described around 1980 has remained popular - drying a slurry of
support material (commonly celite) in an enzyme solution. The resulting powder
necessarily contains both solid support and enzyme. However, the link between
them is usually very weak, and the method is now usually described as co-drying or
deposition rather than immobilization. At least some of the enzyme is probably
present as large aggregates, not close to the support. My impression is that catalytic
activity is usually poorer than with enzymes immobilized by methods more likely to
produce an even protein layer.
8.2.3
Cross-Linked Crystals
One enzyme form that has received considerable attention is based on enzyme
crystals. Production of protein microcrystals from aqueous solution is often quite
easy, and is increasingly used as a step in the manufacture of enzymes on an
industrial scale. (Many people have the impression that protein crystallization is very
difficult, but this stems from the problems in growing large near-perfect crystals for
262
Protein powder Cross-linked Support immobilized

(e.g. lyophilized) protein crystal
Figure 8-1. Schematic illustration of how enzyme molecules are organized in different biocatalyst
forms.
X-ray diffraction.) The protein molecules in the microcrystals are then covalently
cross-linked by treatment with an appropriate multi-functional reagent, usually
glutaraldehyde. This renders the crystals insoluble on transfer to different aqueous
media. The cross-linked crystals are effectively another form of immobilized en-
zyme, and can be dried for transfer to low-water media by the same methods (again
see further details in the discussion of water effects below). Cross-linked crystals are
available commercially for a number of enzymes. Figure 8-1shows a diagrammatic
representation of the organization of the protein molecules in lyophilized powders,
immobilized enzymes and cross-linked crystals.
8.2.4
Direct Precipitation in Organic Solvents
A good and even simpler method of preparation is available where reaction media
are based on an organic solvent that has the ability to dissolve considerable amounts
of water (or is water-miscible).An aqueous enzyme solution may be mixed directly
with the organic liquid (dry or nearly so). Most of the water in the enzyme solution
forms a molecular solution in the organic liquid. The enzyme and other solutes are
precipitated, usually as fine particles. The catalpc activity obtained is usually quite
good, certainly compared with lyophilized powders. In principle particles containing
active enzyme prepared this way might be transferred to other solvents by centrifu-
gation and re-suspension.
8.2.5
Additives in Catalyst Powders
A great deal of attention has been given to co-drying additives with enzymes for low-
water biocatalysis. Large improvements in rate as well as alterations in selectivity
8.2 Enzyme Form 1263
have been demonstrated. A variety of mechanisms have been suggested for these
effects, including the following.
- Denaturation during the drying process may be prevented (“lyoprotection”);
- The additive may select particular conformational microstates that become fixed
on drying (“imprinting”);
- The environment of the enzyme molecules may be favorably altered, for example
by being made highly polar (salt effects).
There are undoubtedly some interesting effects here. However, in general I cannot
recommend such approaches at present to those persons looking for practical
options for biocatalysis. All the main studies of these effects have been made with
lyophilized powders as the final catalyst. As noted above, these usually give a very
poor specific activity compared with other forms of the same enzyme. This is
probably why it is possible to demonstrate very large enhancements in rate - these
are relative to a very low base for the control lyophilized powders. It would be very
interesting to see whether these additive effects are found with other low-water
catalyst forms. It may be that they have been looked for, not found, and hence the
results are not reported. Because of the low base activity of lyophilized powders, it is
not clear whether the “enhanced rates” resulting from these various additives are
better than (or even as good as) those obtained with other catalyst forms (e.g.
immobilized). It would be useful for some studies to make this comparison
systematically, with the same enzyme and reaction. Until such a comparison has
demonstrated superior performance for the lyophilized powder, I would recommend
the use of other catalyst forms as a simpler and more certain route to good catalyhc
activity. One exception would be where it is wished to exploit the alteration of
specificity brought about by additives.
8.2.6
Solubilized Enzymes
All the enzyme forms discussed so far remain as visible suspended solids in organic
reaction mixtures. However, it is possible to treat enzymes so that they become
solubilized in organic media (or at least no longer form an obvious suspension). The
known methods can be classified into three categories,although these are not clearly
distinct at the boundaries:
- The enzyme molecule can be covalently modified with organic soluble groups;
- The enzyme can form a non-covalent complex with appropriate agents, usually
either surfactants or organic-solublepolymers;
- The enzyme molecules can be contained in the water cores of hydrated reverse
micelles or microemulsion droplets.
These systems have been extensively studied, particularly the last. They have
considerable attractions for fundamental studies, because the more or less trans-
parent and mobile systems permit spectroscopic studies. Thus catalytic activity may
be easily followed by direct spectrophotometric measurement of substrate or product
264
concentrations. Fluorimetry or spectrophotometry can be use to probe the protein

molecules directly. In microemulsions and some of the other systems, it has been
clearly shown that the enzyme is molecularly dispersed. In other cases, however, the
individual units remain aggregates, often quite large, but dispersed in such a way
that the suspension is more or less transparent.
The specific activity of the enzymes is often good, comparable with the best
alternative enzyme forms. The enzyme molecules should all be well accessible to the
medium, and mass transfer limitations avoided. However, solubilized enzymes have
not achieved widespread use by those mainly concerned with applications in
synthesis. An extra step is required to separate the enzyme from the final reaction
mixture containing the products. It may be even harder to separate solubilising
additives, notably surfactants, from the products. Thus I in general would not
recommend solubilized enzymes for synthetic applications. One exception is where
it is wished to attack polymeric or solid-state substrates, where the enzyme mole-
cules may need to be able to move to contact the substrate, rather than vice versa.
8.3
Residual Water Level
The level of water remaining in these systems usually has major effects on behavior.
Some of these phenomena are due to mass action effects of water as a reactant. The
equilibria of hydrolysis reactions become more favorable as water levels rise, and
normally the rates increase as well. Low-water media are commonly selected in order
to use hydrolytic enzymes to catalyze synthetic reactions. Hydrolysis will thus be an
undesirable side reaction, or in many cases the direct reverse of what is wanted.
There are also cases where hydrolysis reactions are wanted, and hence water mass
action should be maximized (while keeping non-aqueous media for reasons of
substrate solubility, for example).
Water levels also have important general effects on enzyme behavior. If too little
water is present, the catalytic activity of most enzymes falls dramatically. On the
other hand, reduction in water levels often leads to an increase in enzyme stability. A
decline in catalytic activity at high water levels is also commonly observed, with
several possible explanations:
- The rate of the monitored reaction may fall as a result of competition from
hydrolysis (as a side reaction or direct reversal);
- Water promotes agglomeration of catalyst particles, leading to mass transfer
limitation of rates;
- Water may act as a competitive inhibitor for any of the substrates.
It is often difficult to prepare systems of reproducible water content just by adding a

known amount. Water from other sources may be significant as well as that added
deliberately. Water may be introduced associated with reactants, solvents or the
biocatalyst preparation. It can also enter from (or escape to) the environment.
Furthermore, if the effects of other parameters are being studied, experiments at
8.3 Residual Water Level
I 265
-~ ~
Water dissolved in solvent
m] Enzyme bound water
Non-polar solvent Polar solvent Polar solvent

(same water (same water
content) activity)
Figure 8-2. Quantities ofwater present in different phases when solvents are compared at equal
total water content or equal water activity. The behavior o f t h e enzyme is likely t o depend only on
the amount ofwater bound t o it.
fixed water content can lead to very misleading conclusions. The water present will
end up distributed between several different phases in the reaction mixture. Some
will be closely associated with the enzyme molecules, and it is this quantity that will
mainly affect their behavior. But some water will be dissolved in the bulk phase of the
reaction mixture (e.g. the organic liquid). More will be associated with other solid
phases present, such as an immobilization support and some may be present as
vapor in a gaseous headspace. Changing these other phases (e.g. changing the
nature of the organic solvent or the immobilization support) will affect the amount
of water they retain, and hence that available to the enzyme. So what may appear to
be an effect of solvent etc. may in fact be an effect of water. Put another way, the
optimal water content (on a mass or volume basis) will change when alterations are
made in several other factors. This is illustrated in Fig. 8-2.
It is increasingly accepted that a better parameter to characterize the water levels in
these systems is its thermodynamic activity. This is defined as 1 in pure water, and
will take on lower values in the various reaction media. Water will tend to transfer
between the various phases present until they all reach equal water activity. Hence
the whole reaction mixture will tend to come to a single equilibrium value of water
activity. This will reflect the amount of water in each phase. In particular, a given
water activity will tend to correspond to a particular quantity of water associated with
the enzyme molecules. Hence their behavior will be most simply related to water
266
activity of the system, and the relationship will often stay the same as other factors
are changed (e.g. the solvent).
Even in terms of water activity, hydration effects are not quite so simple however.
As well as the current value, enzyme behavior depends on the history of hydration to
which the catalyst has been exposed. In other words, there can be strong hysteresis
effects. Nevertheless, water activity values are usually the best basis to define the
previous history reproducibly.
8.3.1
Fixing Initial Water Activity of Reaction Components
The reaction mixture for biocatalysis will be prepared by combining several compo-
nents. To ensure defined water conditions in the final mixture, all these components
should preferably be brought to known water activity beforehand. (It may be safe to
disregard this for a component that has a limited affinity for water and makes up
only a small proportion of the final mixture.)
Often the easiest way to set the initial water activity of components of the reaction
mixture is by pre-equilibration with a saturated salt solution. The relative humidity
or water activity is fixed above a saturated solution of a given salt at a known
temperature. As water equilibrates in or out of the solution, solid salt will tend to
dissolve or crystallize to maintain saturation and hence the futed water activity in the
headspace. Any other material placed in contact with the headspace will eventually
equilibrate to the same water activity. The reaction mixture component can simply be
placed inside a closed vessel together with the salt solution, such that water can
transfer between the two via the vapour phase. Wide-mouth screw cap jars are
convenient, with salt solution over the base and an open vial containing the sample
(Fig.8-3). The rate of equilibration depends on the surface areas exposed and the
amount of water that must be transferred. Typically 1-2 days is sufficient for either
solid biocatalyst preparations or liquid phases based on relatively non-polar organic
solvents. The rate of equilibration may be checked by weighing or Karl Fischer
analysis respectively.
Table 8-2 shows the water activity values generated by a selection of salts we
commonly use, taken from the best literature source. Most of these values are weakly
temperature dependent. However, it is essential to use them at controlled tem-
Jar with reasonable seal

,-
Sample being equilibrated
Saturated salt slush
Figure 8-3. Method of pre-equilibration of water activity of reaction mixture components.
Table 8-2. Saturated salt water activities at 25 O C (frornI'*l).
I
267
LiCl 0.113 KI 0.689

KAC 0.225 NaCl 0.753
MgCb 0.328 KCI 0.843
K2CO3 0.432 KN03 0.936
Mg(N03)2 0.529 K2S04 0.973
NaBr 0.576
perature, as fluctuations can cause the liquid phase to move away from saturation.
The saturated solution is best prepared to have a lot of crystals surrounded only by
thin layers of liquid, which will then re-equilibratewith the solid more quickly. Good
sample purity is important when such large amounts of solid are used.
For water activities below 0.05, drying agents are required rather than salt
solutions. Agents suitable for exhaustive drying are described in many conventional
reference sources. Molecular sieves are popular in applied biocatalysis, but I would
note two cautions. Firstly, if they are reactivated by heating, about 350 "C is required
to obtain maximal drying efficiency. Secondly, if placed in direct contact with a liquid
phase, they can have significant acid-base effects.
With water-miscible solvents, the organic phase can be prepared at the desired
water activity more conveniently by simple addition of water to the dry solvent. The
water concentration required will be significant, and the amount of water added will
be much larger than unintentional exchanges with the environment or residual
water levels in the dried solvent. The relationship between water concentration and
activity will be more or less fked for a given solvent, and little affected by reasonably
low concentrations of reactants. This will not be true for less polar solvents, where
direct addition of water rarely gives reproducible hydration or water activity. Table 8-3
gives water contents of various solvents at different water activities.
A decision must be made about the sequence and timing in which components
are combined to make the final reaction mixture. The choices made can have large
effects on the final hydration conditions and biocatalyst behavior. It is usually best
initially to prepare as separate phases: (i) a non-aqueous solution or mixture of the
reactants; and (ii) the solid biocatalyst preparation (lyophilizedpowder, immobilised
enzyme, cross-linked crystal etc). The best treatment to apply then depends on the
objective of the experiment.
- If the aim is to make a fair comparison of the effect of other factors (e.g. different
solvents),then it is desirable to produce reaction mixtures of defined water activity.
For this purpose, it is best if the two phases mentioned are separately pre-
equilibrated to the target water activity before eventually combining them to start
the reaction. In principle it is possible for the water activity to change somewhat
from the pre-equilibrated value as components redistribute between the two
phases. However, in practice such changes are small if the two phases noted are
chosen. Another option is to pre-equilibrate the biocatalyst particles suspended in
a non-aqueous fluid, and to add one final reactant at time zero. This reactant
should be one added at fairly low concentration to prevent significant changes in
water activity. These two options are illustrated in Fig. 8-4.
8 Enzymic Conversions in Organic and Other Low-Water Media
Table 8-3. Water contents (v/v%) o f various solvents equilibrated at different water activities.
Water activity 0.05 0.1 0.2 0.4 0.6 0.8 1
Ethanol 0.449 0.94 2.06 5.2 11.0 28.5 M

2-Propanol 0.280 0.58 1.26 3.06 6.0 12.2 M
tea-Butanol 0.166 0.34 0.75 1.86 3.7 8.2 M
Dioxane 0.172 0.36 0.82 2.25 5.8 27.3 M
Acetone 0.188 0.39 0.86 2.18 4.56 11.7 M
Acetonitrile 0.154 0.32 0.72 1.86 4.19 15.3 M
Tetrahydrofuran 0.089 0.185 0.402 0.98 1.94 4.02 M
Ethyl acetate 0.066" 0.137" 0.298 0.73 1.44 2.99 -
Methyl iso-Butyl
ketone 0.0529" 0.1061" 0.214 0.43 0.66 0.89 1.12
Methyl tert-Butyl
ether 0.0470* 0.0940" 0.189" 0.38 0.58 0.78 0.98
iso-Propyl ether 0.0178" 0.0356" 0.071" 0.143* 0.216 0.290 0.364
Toluene 0,0015" 0.0029" 0.0058" 0.01174 0.0175" 0.0233" 0.0292"
Hexane 0.0004" 0.0007" 0.0015* 0.0029" 0.0044" 0.0058k 0.0073"
Water contents are given as conventionally in terms of the volumes of pure liquids mixed to reach the required
composition. With water-immiscible solvents, a water activity close to 1 is achieved in the mutually saturated
system (as shown), but for miscible solvents (M),water activity 1means pure water! Water activity ofwater-
miscible solvents are estimated using the correlations derived by Bell et al.1191.For water-immiscible solvents,
they are based on water solubility measurements~20~2*l, and the approximation of constant activity coefficient
up to saturation.
* - It is not advised to try to obtain the water activities shown by adding these small amounts of water to dry
water-immiscible solvents. The values are given purely for use in estimating water quantities present or
required. Apart from usual errors, small water droplets can take a very long time to dissolve in such solvents.
Attempting such a method with solvents like toluene or hexane is particularly disastrous.
10 mM Ac-Tyr-OEt
i'[J--,'] Toluene 1 M PrOH
t d''
Silica-enzyme
, ,,,/-,
1 1
Little effect Of Mix and pre-equilibrate Pre-equilibrate
pre-equilibrating water activity water activity
solid powder
--.-_
-.--_ - _- _- _
- _- - _- - _
._- - _ .
--_
------*
+
Combine to start reaction
Figure 8-4. Choices i n water activity pre-equilibration before a reaction i n organic medium. The
solid and broken arrows indicate two alternative schemes that can be considered. Other conceiv-
able schemes will probably lead t o a final reaction mixture ofwater activity very different from that
used in pre-equilibration.
I 269
- On the other hand, if the aim is to achieve maximal catalytx activity in otherwise
iixed conditions, it is often better to transfer the enzyme catalyst quickly and
directly from an aqueous environment. Excess water can be removed if necessary
by rinsing with a suitable polar solvent. It is best if the polar solvent contains
enough water to bring it to the target water activity of the final reaction mixture. It
is still best to ensure that the non-aqueous fluid phase starts with a defined
hydration level, usually by pre-equilibration.
In either case, it is worth noting that achievement of a desired final water activity
value is much easier in relatively polar solvents, where the substantial water
concentration in the bulk phase will effectively buffer the whole system.
Two factors should be mentioned that can lead to significant unintended (and
rather irreproducible) changes in water activity. Firstly, exchange of water with the
environment. When only very small quantities of water are present (e.g. in media
based on non-polar solvents), significant changes are possible. To avoid them,
reaction vessels should be carefully sealed, and it may even be necessary to sample
through a septum or a similar membrane. Sealing sufficient to prevent noticeable
losses of volatile organic solvent may still allow significant water exchanges,because
of the much smaller total quantities involved. Secondly, water may distill to any cold
surface in contact with the headspace above a reaction mixture. This can lead to
significant removal of water from the liquid phase, especially if the temperature
difference is large. For example, a surface at 20 "C will condense water away from a
reaction mixture at GO "C until its water activity falls to 0.12 (the ratio of saturated
vapor pressures). 1t is best to prevent this problem by eliminating all such cold spots.
Reaction vessels may be surrounded by an air bath (taking account of explosion risk
if flammable solvents escape), or completely immersed in a water bath. If in-
dividually jacketed vessels are used, unjacketed surfaces may be heated above the
circulating water temperature.
8.3.2
Control of Water Activity During Reaction
Some methods offer the possibility of control of water activity during the reaction at
the cost of greater complexity and/or the possibility of interference with the desired
conversion. Most obviously a change in water activity may be due to water being
produced or consumed in the reaction. However, it is also possible by water exchange
with the environment (e.g. during sampling). With non-polar solvents water activity
can also change because conversion of substrates to products will change the
solvation of water. Hence water activity at a fixed concentration will be altered.
One simple and convenient method is the addition directly to the reaction mixture
of suitable pairs of solid salt hydrates. A given salt hydrate will give up its water at a
characteristic water activity, transforming to a lower hydrate or an anhydrous form. If
the pair are placed in a system of water activity below their characteristic transition
value, the (higher) hydrate will tend to give up water to the rest of the system. Water
release will continue until the whole system reaches the transition water activity (or
270
I 8 Enzymic Conversionsin Organic and Other Low-Water Media
the higher hydrate is completely exhausted). If on the other hand the salt pair are
added to a system of high water activity, the form with less (or no) water will tend to
take up water, transforming back to the (higher) hydrate. Once again, this will
continue until the system water activity has been reduced to the transition value, or
the salt form with less water has been completely consumed. In principle these
exchanges should be able to buffer the water activity of the systems at the transition
value of the added salt pair, provided enough is added. Typically, the higher hydrate
will give up water at the start, as the reaction mixture is prepared from dry
ingredients. Later, the lower hydrate may take up water produced in a reverse
hydrolysis reaction. An example system is represented by the equation:
NazS04.10H20 (crystals)G+ Na2S04 (crystals)+ lOH2O (1)
in which equilibrium with both solids present can only be reached at a single water
activity value, 0.80 at 25 "C. Figure 8-5 illustrates the use of this hydrate pair in a
synthetic reaction.
100
Salt hydrate pair:-
80 gives out and takes
s
n
water here it up here \ /
c 60
W
.-0
2
Q)
>
40
0
20
0
0 20 40 60 80
Reaction time (h)
Figure 8-5. Control ofwater activity by adding salt hydrates to reaction mixture. Synthesis of
butyl butanoate catalyzed by Candida rugosa lipase. Control reactions with catalyst relatively wet
(0) or dry (0)initially. Reaction in the presence of Na2S04plus Na2S04.10H20 (0).Kvittingen et
a1.[221.
Table 8-4.
Selected salt pairs found useful for water activity control in biocatalysis.
I
271
Salt pair Equilibrium Rate of Maximum

water activity water transfer temperature (‘C)
NaI.2/0 0.12 fast 68

Na2HP04.2/0 0.16 fast 95
LizSO4.1/0 0.17 slow 233
NaAc.310 0.28 fast c 58
NaBr.2/0 0.35 slow 50
Na&0,.5/2 0.37 slow 48
&Fe(CN)6.3/0 0.45 slow 87
Na4P~07.10/0 0.49 slow c 80
CaHP04.2/0 0.50 slow > 100
Na~HP04.7/2 0.61 fast 48
Na2HP04.1217 0.80 fast 35
Na~S04.10/0 0.80 fast 32
The pairs used are identified by a shorthand notation: Na1.2/0 means a combination of NaI.2H20 and
anhydrous Nal (i.e. OH20).Equilibrium water activity values are for 25 OC.“Fast”water transfer indicates
equilibration in a few minutes, “slow”that several hours may be needed. There is only limited information on
the behavior of hydrate pairs giving lower water activities, though some indication that they generally tend to
equilibrate slowly. From Zacharis et al.‘”’.
All of this describes just the thermodynamically favored directions of water

transfer, for ideal crystalline solids. Many salt hydrate pairs seem to behave
approximately ideally. However, if water activity is to be controlled close to the
transition value, the rates of water release and uptake must be sufficient. Different
salt pairs have very different rates ofwater exchange. It is difficult to give quantitative
values, because the rates will depend on the size and shape of the crystals in each of
the salt hydrate forms. This will depend on how they have been crystallized and
handled subsequently. For example, cycling between hydrate forms, with gain and
loss of water, will usually lead to a reduction in crystal size, and hence more rapid
water exchange in future cycles.
The equilibrium water activity achieved depends on the choice of salt hydrate pair
used and the temperature. In most cases the temperature dependence is higher than
for saturated salt solutions. There is also a maximum temperature at which the
higher hydrate will “melt” to give a liquid phase, so above this the biocatalyst will
probably be seriously affected. Table 8-4gives water activity values for some pairs
that can be recommended for biocatalysis, together with an indication of the rates of
transfer, and the maximum temperature. A compilation from the gives
information on temperature dependence, and notes some other hydrate pairs whose
use has not been (fully)tested.
Many chemists have adopted the direct addition of salt hydrates as a simple
method of water activity control. However, it does require a little thought and care to
make sure the desired water activity is really produced. In particular, it must be
ensured that both solid salt forms really will be present at equilibrium. It is best to
estimate a “water budget” for the system, to ensure that enough of the right salt
forms are being added. Table 8-5 shows an example of this, for a system made up of
272
Table 8-5. Example water budget for the use of salt hydrates.
Initial water content Estimated equilibrium Change (pmol)
Phase (pmol) water content (pmol)
10 mL toluene 55 (0.01% w/v) 128 +73

10 mg immobilised
enzyme (on silica) 500 430 -70
100 mL gas headspace 26 (lab air, 20% RH) 104 +78
Water produced by ester-
ification reaction from
10 mM substrates - +loo
Water is assumed to show ideal dilute behaviour in toluene up to the solubility limit (16 mM). Equilibrium
water content of immobilised enzyme estimated from measured adsorption isotherm (mainly adsorbed by
silica support).
the phases shown, to be controlled at water activity 0.80 at 25 “C using the pair
Na2HP04.12/7. This example has been selected as one in which all four contribu-
tions are significant. More usually, one or two will dominate. In the example, the
added salt hydrates will initially have to supply 73 + 78-70 = 81 pmol water to the
reaction mixture. As the reaction proceeds, this will be all need to be taken up again,
followed by another 19 pmol. Hence 81/5 = 16.2 pmol of Na2HP04.12H20should be
able to supply the water required, transforming to the heptahydrate as it does so. To
take up the last portion of the product water, 19/5 = 3.8 pmol of NazHP04.7Hz0
should also be added at the start. In practice, about 10 mg Na2HP04.12H20 and 2
mg Na2HP04.7H20might be sensible, to ensure excess. Even more might be wise if
the reaction vessel is likely to be opened frequently, allowing loss of water to the
surrounding air.
In some cases, estimation of a water budget may indicate that buffering can be
achieved by adding just one hydrate form, with the other formed in situ as water is
given out or taken up. This is particularly attractive where one of the salt forms is not
available. However, it is clearly wise to adopt this approach only when the direction of
net water exchange with the reaction mixture has been very confidently determined.
If not, the second hydrate form required can often be made fairly easily by hydration
or drying of the one that is available.
Some limitations of the addition of salt hydrates must be borne in mind. In some
cases the added salts may have additional, undesirable effects. They may react
chemically with compounds involved in the enzymic reaction. For example
Na2CO3.1OH20 will neutralize carboxylic acids to their Na salts. More subtly, even
quite weakly basic or acidic salts may exchange H+ with the enzyme molecules,
affecting their behavior (see below).
There have been some cases of confusion in the control of water activity between
saturated salt solutions (see above) and salt hydrate pairs. These can both be useful
methods, but the principles and recommended applications are quite different.
Avoid phrases like “control of water activity using salts”,which do not make it clear
which method is being used.
Water activity can be controlled during the reaction via the vapor phase, as in pre-
8.3 Residual Water level
I 273
equilibration. Once again, saturated salt solutions are the best method of generating
a vapor phase of controlled water activity. However, if the reaction produces or
consumes water at significant rates, simple diffusion via the vapor phase will usually
be too slow to maintain constant water activity. Forced circulation of the gas phase
may give sufficient rates. For best water transfer, it can be bubbled through both the
salt solution and the reaction mixture. There is an alternative method to achieve
faster water exchange between a saturated salt solution and the reaction mixture.
The two may be brought into contact across a membrane, so that only a very short
diffusion path separates them (at the cost of a smaller diffusivity of water within the
membrane). Microporous or ultrafiltration membranes may be best in principle, but
for laboratory use one convenient solution is to use silicone This is
resistant to most organic solvents, and offers reasonable water permeability.
In some reactions the objective may be to remove water as vigorously as possible.
This will lead to a low water activity, which would result in very poor catalytic activity
of many enzymes. However, some enzymes are much more tolerant of low water
activity. In this case, exhaustive dehydration may be the best policy, particularly to
minimize hydrolysis reactions or maximize their reverse. In general, the methods
adopted can be based on those used in conventional synthetic chemistry for handling
water-sensitivematerials. However, many of the most powerful drying agents cannot
be used when they might come into contact with the enzyme, because of catalyst
inactivation. For direct addition to the reaction mixture, the usual choice is molecular
sieve. Type 4A is most commonly used, and is effective because nearly all the
solvents have sufficiently large molecules that they are completely excluded. One
piece of practical information is not as widely known as it should be. If molecular
sieves are to be reactivated after use, very severe treatments are necessary to restore
their full water-adsorbingpower. If heating alone is used, a temperature of 350 "C is
needed. It has recently become clear that molecular sieves can affect enzyme
behaviour by acid-base effects as well as water removal. If the components of the
reaction mixture are all relatively involatile (e.g. in solvent-freeesterification),water
removal by evaporation can be another effective method.
8.3.3
"Water Mimics"
One approach to improving catalfic activity at low water activity should be men-
tioned. Small additions of certain very polar liquids have been reported to greatly
enhance catalFc activity at low water activity. They are usually described as "water
mimics". and seem able to replace at least some of the roles of water in facilitating
enzyme activity. Most of them are strongly hydrogen-bonded associated solvents that
show other behavior analogous to water, such as glycerol, glycols and formamide.
However, strong effects have also been observed with methanol and dimethylsulf-
oxide, for example. Most of the studies with these additives have been made with
lyophilized powders, and hence may in part reflect the low control activities of these
preparations (see Sect. 8.2). However, some significant effects have been reported
with other enzyme forms, so I would recommend that use of such water mimics be
274
considered. They are clearly particularly attractive where very low water activity is
desirable to prevent unwanted side reactions. Obviously the water mimic chosen
must not promote analogous side reactions, such as with its hydroxyl groups.
8.4
Temperature
The pattern of temperature effects is the same as in aqueous media. The initial rate
of reaction increases with temperature, in the usual Arrhenius fashion. However, the
stability of the enzyme will decline with temperature, and at high enough values
catalyhc activity will be lost rapidly before significant conversions are reached.
Hence, for given conditions, there will be an optimum temperature to maximize
product yield after a given time. This is rarely a real fixed optimum for the enzyme,
and for example will usually become higher if the reaction time is reduced.
Progressive enzyme inactivation will have less effect over a shorter reaction time.
One important feature that can be exploited in low-water media is an increase in
stability to temperature. Hence reactions may be carried out at temperatures higher
than would be possible in aqueous media, often by many tens of degrees. It is fairly
clear that the most important factor here is the amount of water in the molecular
environment of the enzyme molecules, as determined primarily by the water activity
of the system. The presence or nature of a solvent has little additional effect. Thus,
beware of statements that “enzymes become more thermostable in organic sol-
vents”. It is the reduction in hydration that increases stability. If anything, the
presence of an organic solvent will be destabilizing (in a comparison at equal water
activity). In an organic solvent at water activity close to 1 (i.e. water saturated), the
stability will be no better than in water. If, however, water activity is reduced to
substantially below 1, a very valuable increase in stability may be achieved.
8.5
Substrate (Starting Material) Concentrations
Substrate concentrations affect catalytic rates in the same general way as in aqueous
solution. At low substrate concentrations the rate is roughly proportional to [S] (i.e.
first order kinetics). At higher concentrations the enzyme becomes saturated with
substrate and the activity approaches a maximum limiting value. The full depend-
ence is often described quite well by the Michaelis-Mentenequation or its analogs for
the more common two-substrate case (general two-substrate model, or the Ping-
Pong model). These equations include a K, parameter for each substrate, with units
of concentration. When the actual substrate concentration is many times larger than
its K, value, the enzyme will be saturated with that substrate. Further increase in its
concentration will then have little effect on the rate of reaction.
When the medium is changed, the K, values will change also. An important
contribution to this change has nothing to do with the enzyme directly, but reflects
8.5 Substrate [Starting Material) Concentrations
a,
I 275
a, t
c
mX 0N a,
3
-
a,
10 1
I
L-
!& : 100
1 0.1 10
u)
9
YE
0.1 0.01
b 1
10 100 1000
s, (mM)
Figure 8-6. Kinetic parameters for subtilisin-catalyzed transesterification o f 2-Ala-
O N p in different solvents. Experimental Km (0)and Vm/Km (0) values are shown
as a function ofsubstrate solubility. The filled symbols show the corresponding
“corrected” values, after allowing for substrate solvation. The variation i n V,/K, is
largely explained by solvation, while the “real” variation in K, is opposite t o the
apparent trend. Reimann et
the changed solvation of the substrates in the different media. Often this effect
accounts entirely for the observed change. A simple quantitative picture is based on
the relationship of K, values to substrate solubility: the ratio of these will be
approximately the same in each different medium. Figure 8-6 illustrates an example
of this effect.
Often experiments to screen different solvents will keep the same substrate
concentration in each. Hence, if a solvent in which the substrate is more soluble is
tested, the K, value will be increased, and the reaction rate may fall, as the enzyme is
more limited by the availability of substrate.
For preparative syntheses, good general advice is to use a saturated solution of the
substrate(s)in any solvent tested. This will only be a poor choice in the relatively rare
cases of substrate inhibition. It will certainly be a good policy to allow identification
of any direct effects of the solvent. An obvious way to ensure that the medium is
saturated with substrates is to include excess in the form of solid particles. This leads
276
towards the mainly solid reaction mixtures mentioned elsewhere, and can be a good
option in practice.
8.6
Solvent Choice
A large number of solvents might be chosen to form the basis of the low-water
medium. The choice of solvent will usually have important effects on the rate and
selectivity of the reaction, and on the stability of the biocatalyst.
8.6.1
Effects on Equilibrium Position
In many biocatalyzed reactions, the position of chemical equilibrium is important,

because it will place a limit on the eventual yield. In such cases, the choice of solvent
will usually have a significant effect on the equilibrium position. Because this simply
reflects the differential solvation of reactants and products, these effects can be
predicted fairly confidently, at least to a reasonable approximation[271.
One of the equilibria most commonly of interest is esterification. It may be desired
to hydrolyze an ester, or reverse this in condensation of an alcohol and acid.
Alternatively the hydrolytic equilibrium may be an undesirable side-reaction during
transesterification. In this case, at a given water activity, the equilibrium position is
quite strongly solvent dependent. The fraction of ester will increase dramatically on
going from a polar solvent to non-polar solvent (Fig. 8-7). Hence alkanes are
preferred solvents for esterification, while acetonitrile, a ketone or tertiary alcohol
would be best for ester hydrolysis. If the equilibrium constant is expressed in terms
of concentrations (including that of water), it is relatively solvent independent.
However, optimal enzyme behavior in the different solvents usually requires
maintaining the same water activity. At futed water activity, the ratio of ester to acid
and alcohol concentrations will be maximized in the least polar solvents.
8.6.2
“Solvent Effects” that Really are Not
Many apparent “solventeffects”reported in the literature are actually due to changes

in the availability ofwater or substrate to the enzyme. It is commonly observed that
activity appears to be highest in the least polar solvents. Sometimes the explanation
will be added that these “have the least tendency to strip water from the enzyme”.
This undoubtedly indicates a common mechanism, but in such cases the “solvent
effect” will disappear completely if experiments are run at equal water activity, as
recommended in the discussion above (Sect. 8.3.1 and Fig. 8-2). Many other
observed “solventeffects” operate via changes in substrate solvation, as explained in
Sect. 8.5. Hence, they are really effects of changing substrate availability when
different solvents are compared with equal substrate concentrations.
8.6 Solvent Choice
I
277
P'
0
Y
CSI
0
2
0 I I I I
-3 -2 -1 0
Figure 8-7. Correlation between equilibrium constant for esterification and solubility o f water in
the solvent. Equilibrium constant was defined as [Ester]/([Alcohol].[Acid]), for reactions at fixed
water activity (close t o 1). Solvents are: bb, butyl benzoate; be, bromoethane; bk, dibutyl ketone;
bp, dibutyl phthalate; bz, benzene; ca, 1 ,1 ,1-trichloroethane; cf, chloroform; ct, carbon tetrachlo-
ride; cy, trichloroethylene; ee, ethyl ether; ek, diethyl ketone; ep, diethyl phthalate; hd, hexadecane;
hx, hexane; mc, methylene chloride; mk, methyl iso-butyl ketone; nm, nitrornethane; oc, iso-octane;
pe, ;so-propyl ether; tl, toluene. Valivety et al.[281.
8.6.3
Solvent Polarity Trend and Recommended Choices
A very commonly observed trend is that activity is highest in the least polar solvents.
In many of these cases this is an effect ofwater or substrate availability, as just noted.
Hexane is regularly identified as the best medium, because the low solubility of
water and most substrates makes them most available to the enzyme, when
comparisons are made at equal concentrations. Nevertheless, even when water and
substrate availability have been allowed for, non-polar solvents seem to offer the
highest activity. The probable explanation involves the tendency for solvent mole-
cules to migrate from the bulk phase into the immediate environment of the
enzyme. The picture is simplest when there is a discrete aqueous phase (albeit of
very small volume) around the enzyme molecules. The more hydrophobic the bulk
solvent, the lower will be the (saturating) concentration in the aqueous phase, which
is what is experienced by the enzyme. Even in the absence of an identifiable aqueous
278
phase, the immediate environment of the enzyme molecules will be more polar than
the bulk.
Hence, it is often best to select a non-polar or hydrophobic solvent, at least for
initial trials. Some reasons why this might not be the best choice are:
- If the reaction wanted is a hydrolysis, the equilibrium will be less favourable than
in a polar solvent (see above);
- The reactants may be only poorly soluble; however, using a suspension of
incompletely dissolved substrates may still be a good policy. Provided the rate of
dissolution and concentration in solution are sufficient, a good reaction rate can
still be achieved.
The following list presents some choices for a more general solvent screening
exercise:
- An alkane: n-hexane is most commonly used, although on safety grounds
cyclohexane,heptane or isooctane would be preferred.
- An aromatic hydrocarbon: toluene would usually be preferred over benzene on
safety grounds.
- An ether: diethyl ether is usually inconveniently volatile, and popular alternatives
are di-iso-propylether or methyl tert-butylether.
- A ketone: methyl iso-butyl ketone or acetone. Being miscible, the latter may not be
suitable if a medium of high water activity is required - this will end up as a high
water content mixture that may dissolve and denature the enzyme.
- A tertiary alcohol: tert-pentanol or tert-butanol. These are useful because they do
not react with most enzymes that accept alcohol substrates.

- A water-miscible but aprotic solvent: one of tetrahydrofuran, dioxane or acetoni-
trile.
- A small alcohol. Either ethanol or 2-propanol is probably best. These solvents
must be avoided for many enzymes, as they will be reactive, for example as
nucleophiles or reductants. Methanol as a pure solvent is often particularly
inactivating.
- Chlorinated solvents can have some distinctive properties but are usually avoided
for two reasons. On safety and environmental grounds, they are increasingly
disfavored for large scale applications. They also tend to be more inactivating to
biocatalysts than other solvents of similar polarity. (In some cases this may in fact
be due to the stabilizers added to most chlorinated solvents.)
Supercritical fluids have advantages as reaction media for large scale applications,
but the need for high pressure apparatus means they will not usually be favoured for
laboratory syntheses. Volatile reactants can be supplied to a catalyst through a gas
phase, and the higher temperature stability under low water conditions makes this
applicable to more cases than might first be thought. However, the increased
complexity of apparatus again makes this more likely to be favored only at an
industrial scale.
8.6 Solvent Choice 279
I
8.6.4
Solvent Parameters
For preparative purposes, the idea of correlation with some qualitative idea of solvent
polarity is often sufficient, as implied here. There are numerous parameters which
can be used to quantify the difference between solvents, but they all show some
correlation with each other. By almost any measure, we would obtain the order:
hexane, toluene, methyl iso-butyl ketone, propanol. However, different parameters
can give different rankings when more similar solvents are compared. For bio-
catalysis in non-aqueous media, there are few effects where the “correct” solvent
scale can be confidently identified. However, it is useful to have an idea of two quite
different classes of solvent scale.
- Most of them describe features of how the bulk solvent behaves and is able to
interact with isolated solute molecules. These will be based on measurements on
or in the solvent as a bulk medium. Different parameters measure different
features of the interaction the solvent may have with solutes, e.g. dielectric,
cohesiveness, acidity, basicity. When the behavior of the solvent as a bulk medium
is being considered, it is appropriate to use scales from this group.
- In contrast, some parameters are properties of individual solvent molecules.
Examples are dipole moment and log P (the octanol-water partition coefficient).
These parameters are appropriate where individual solvent molecules are engaged
in interactions away from the bulk phase. Thus, log P is used sensibly to describe
the tendency of solvents to interact with (and affect the functioning of) the
enzyme molecules. However, these parameters are not good choices when bulk
solvent behavior is important, such as its ability to solvate water or reactants (and
hence affect their availability to the enzyme). Even when such mechanisms are
important, it is quite common to see correlations presented against log P.
However, any relationship probably reflects the correlation of log P with appro-
priate scales of bulk solvent behaviour.
There is a tendency to use two different words that make a related distinction
between different types of solvent parameter. The log P parameter can be called a
measure of solvent “hydrophobicity”,which is an accurate description of what affects
its value. This contrasts with other parameters such as dielectric,which measure the
bulk “polarity”.One illustration of the difference is to consider homologous series of
solvents. Adding extra methylene groups to an alcohol, for example, will cause a
regular increase in hydrophobicity.The effect on polarity will be much less, however,
as the hydroxyl groups can still be oriented to solvate a polar solute. Thus decanol is
more hydrophobic (higher log P) than hexane, but will be more polar by almost any
measure of bulk properties.
One illustration of the difference between these two classes of measure comes in
the treatment of mixed solvents. For parameters that relate to the ability of the
solvent in bulk to interact with solutes, it is meaningful to define and measure a
value for a mixture of solvents. Often this will be a simple function of the mole or
volume fractions and the pure solvent values. However, for parameters that describe
280
I 8 Enzymic Conversions in Organic and Other low-Water Media
the behavior of individual molecules of the solvent, a value for the mixture is
meaningless. The two types of solvent molecule present will behave differently and
essentially independently.
8.6.5
Solvent Effects on Selectivity
Solvent effects on enzyme selectivity or specificity are very important. One of the
attractions of non-aqueous media is the ability to tune these key properties, and
substantial effects can certainly be observed. Unfortunately, it is not yet possible to
give confident predictions in most cases.
Predictions can be offered for the effect on selectivity between two substrates. A
major contribution here comes from differential solvation, and the selectivity at a
fixed concentration ratio will depend on relative solubilities, as noted in Sect. 8.5.
However, these effects are rarely of preparative relevance, as it is not common to use
two competing substrates that differ greatly in solvation. Selectivity between enantio-
mers is often desired, but here solvation effects will not distinguish the two
substrates (unless a chiral solvent is used). Changing the solvent can have important
effects on the selectivity between enantiomers (up to 2 orders of magnitude, with
inversion of stereopreference possible). The effects must by definition be based on
differential solvent interaction with the two diastereoisomeric transition states. A
model based on solvent interaction with exposed portions of the substrate moeities
in these transition states can sometimes make correct predictions of the direction of
the effect, although its generality needs more testing.
8.6.6
No Solvent or Little Solvent Systems
In many cases an attractive option is to use no “solvent”at all. In some cases at least
one of the reactants will be liquid, so can be the basis of a fluid phase for transfer of
reactants. If slightly raised temperatures are used, this condition will be met more
often. (Remember that at reduced water activity, the enzyme will usually be stable to
higher temperatures than in aqueous solution.)
Another option is to abandon the usual idea that most or all of the starting
materials should be dissolved in order to get effective reaction. Attractive reaction
mixtures can be prepared containing mainly undissolved solid particles of sub-
strates. The reaction actually takes place in a liquid phase containing the enzyme, but
this can be totally hidden between the reactant particles. The system formed is
illustrated in Fig 8-8. Usually the liquid phase will be generated by adding a small
amount (e.g. 10% by weight) of a “solvent”.Often the best solvent is water itself, as it
will usually give the highest catalytic activity. In these mainly solid systems, this may
be combined with many of the advantages of non-aqueous media, notably the
reversal of the equilibria of hydrolytic reactions.
8.7 Acid-Base Conditions
Liquid phase
I 281
(e.g. aqueous)
Solid reactants
and products
Figure 8-8. Schematic illustration of mainly solid reaction system. Starting
material crystals will progressively dissolve, while product crystals will grow, as
the enzymic reaction happens in the liquid regions between them.
8.7
Acid-Base Conditions
8.7.1
pH Memory
It is well known that pH has a major influence on the behavior of enzymes in

aqueous media. Most who use enzymes under low-waterconditions are aware of the
phenomenon known as “pH memory”. The activity and other properties of the
enzyme are affected by the pH of the last aqueous phase to which it was exposed
before drying for use in low-water conditions. This phenomenon is usually attrib-
uted to the relative rigidity of enzyme molecules at low hydration, by analogy with
the effects of co-drying with additives. (see Sect. 8.2.5)
However, another picture may give a clearer view of pH memory and when it may
prove insufficient to apply controlled acid-base conditions. In aqueous solution, pH
influences enzymes by affecting the protonation state of acidic and basic groups in
the molecule. At a given pH, the protein molecule will have a characteristic net
charge. Electroneutrality requires that the surrounding solution contain an excess of
oppositely charged counter-ions precisely to balance the protein charge. In aqueous
solution these counter-ions are relatively far away, and their presence and identity
has only limited effects on behavior. However, consider drying this portion of
aqueous medium containing the enzyme. In general, the counter-ions present will
remain, as only water is removed. So the net charge on the counter-ions, and hence
the opposite net charge on the protein will be preserved. The requirement for
electroneutrality means that the only possible changes in protonation state are
internal H’ exchanges between groups in the protein. Each such exchange will create
or destroy a pair of positive and negative groups, without altering the net charge. In
summary, this picture shows that pH memory resides in the behavior of the counter-
ions as much as the protein, and does not require any special rigidity of the latter.
This is illustrated in Fig. 8-9.
Also it should now be clear that pH memory is not a phenomenon unique to non-
282
o\te r.,.
3 0
0 3
0 n 8
High
3
a
0
0 3 dielectric
0 8
0
/ @ c J
(net charge) 0
net charge
3
3 =+c 'barge
\
8 , %%j/
c 0
'barge (net charge]

3s = + c
Aqueous solution Dried Return to
arge
liquid medium
Figures-9. Illustration ofthe relationship between protein net charge and that on the
counter-ions, and how drying and re-suspension or dissolution cannot change it.
aqueous media. If a dried enzyme preparation is placed back into pure water, its
behavior will be determined by the pH value before drying - the ionization state of
the protein, and the counter-ions present will effectively buffer the water back to the
original pH value. Of course, we would not normally think of doing an experiment
like this in aqueous media. The pH value reached would be very weakly buffered,
and might be greatly altered by traces of acid or base from impurities, reactants etc.
In low-water media, it is more common that acid-base conditions will not be
seriously affected by these unintended effects. Hence pH memory may be sufficient
to control behavior. However, there are several common ways in which pH memory
may fail, which at least should be carefully considered before deciding to rely on pH
memory. In addition, there are now established some relatively simple methods to
buffer acid-base conditions in some low-water media, making reliance on pH
memory unnecessary.
8.7.2
Processes Erasing pH Memory
As the picture presented above suggests, the net charge on the protein may be
affected by processes leading to preferential loss of counter-ions of one charge. This
can happen if counter-ions undergo proton exchange reactions with the protein to
produce a neutral species. The exchange may be driven to completion if the neutral
species produced is then removed from the neighborhood of the protein. Such
exchanges may be relatively easy if the counter-ions are derived from weak acids or
bases. If the acid or base is then volatile, the counter-ions can be lost during drying
under vacuum, with changes in protein net charge, as represented by reactions such
as:
8.7 Acid-Base Conditions
I 283
Protein-NH3'.-OOCH --* Protein-NH2.HOOCH --*

Protein-NH2 + HOOCH (gas) (4
Protein-COO-.NH4' -+ Protein-COOH.NH3 Protein-COOH + NH3 (gas) (3)
+
A similar process can occur if the acid or base can be extracted into the bulk phase of
the reaction mixture (e.g. octanoic acid or triethylamine in an organic solvent).
Other counter-ions may be exchanged with the bulk non-polar phase, provided
something is able to solubilize them there. This will usually be in the form of an ion-
pair with a species better solvated by the medium. For example, an acid with a large
hydrophobic group may form a Na' salt with sufficient solubility in the bulk
medium. The protonated acid will carry H' to and from the enzyme in exchange, to
maintain electroneutrality. A similar process with a hydrophobic amine, for example,
can transfer H' and Cl-. Solubilization of the small ion may be aided by complexa-
tion, for example of Na' by a crown ether. The exchanges can be written as:
Protein-COO-.Na' + RCOOH (bulk phase) =+
Protein-COOH + RCOO-.Na' (bulk phase) (4)
Protein-NH3+.C1-+ R3N (bulk phase) G= Protein-NH2 + R3NH'.CI- (bulk phase) (5)
Acidic or basic species in the bulk phase may protonate or deprotonate the enzyme,
becoming the necessary counter-ions in the process. So we might have equilibria
such as:
Protein-COOH + CH3NH2 (bulk phase) =+Protein-COO-.+NH3CH3 (6)

Protein-NH2 + CH3COOH (bulk phase) =+
Protein-NH3'. -0OCCH3 (7)
The protonation state of the enzyme may be affected by acidic or basic reactants
(starting materials or products). These species could act as described by either of the
two sets of equilibria just presented. Acidic or basic impurities in solvents could also
be significant here.
8.7.3
Systems for Acid-Base Buffering
It should be clear that there are several possible mechanisms by which the
protonation state of an enzyme may be altered in low-water media. It will often be
desirable to try to maintain the optimal state by controlling acid-base conditions,
rather than just relying on pH memory. This can be done by the addition to the
reaction system of acid-base buffers, as in aqueous media. However, the details of
these buffer systems and how they work is usually somewhat different.
The equilibria represented by Eqs. (4) and (5) can be employed to set up buffering
based on agents dissolved in the bulk non-aqueous phase. As the equilibria indicate,
the state of ionizable groups in the enzyme will depend on the ratio of buffer forms
added to the bulk phase: the acid and its ion-paired salt with Na' (or another cation);
the base and its ion-paired hydrochloride salt (or similar). Also in analogy to aqueous
buffers, a given pair will only be usable over a given range of acidity/basicity. The
284
conditions where optimal buffering is found (analogous to aqueous pK) will depend
on the solvent used. A number of such organic soluble buffer pairs have now been
identified [29-311.
Identification of buffers that can be dissolved in the bulk phase is restricted by
solubility, usually of the ionized form. An alternative approach is to choose buffers
expected to be almost completely insoluble in the reaction medium, which will
remain as suspended crystals. Convenient choices are zwitterionic solids and their
salts, which will give rise to equilibria as shown in Eqs. (8)and (9).
Protein-COO-.Na' + TES'- (crystals, zwitterionic)=s
Protein-COOH + TES-.Na' (crystals) (8)
Protein-NH3'.Cl- + Lys' (crystals, zwitterionic)+
Protein-NH2 + Lys'.Cl- (crystals) (9)
Since the buffer compounds are now present as crystalline solids, the equilibrium
position is independent of the quantity of each. A given pair sets a characteristic
protonation state of the enzyme. This is analogous to the use of solid salt hydrate
pairs to set a hydration state. Again, to cover the range of acid-base conditions that
might be appropriate for different enzymic syntheses, a series of different buffer
pairs is required. A number have been identified[32-34],but the known range
probably needs extending.
Of course, if such equilibria are to be established, a mechanism is required for the
transfer of H' and counter-ions between the solid buffers and the enzyme mole-
cules. Quite surprisingly, this usually does not seem to be a limitation. Only quite
small quantities of ions must be exchanged, which will make equilibration easier.
Probably traces of acids and bases soluble in the bulk phase can catalyze the transfers
by equilibria such as Eqs. (4) and (5). If rates of equilibration are inadequate,
deliberate addition of such transfer agents should help.
Although the analogy to aqueous acid-base behavior is clear, there are important
differences. In particular, the ionization of acidic and basic groups in the protein
becomes to a considerable extent independent. Both are affected by the availability of
counter-ions as well as of H+, as illustrated in equilibria like those shown in Eqs. (4)
and (5). Hence in principle two different buffering systems should be used to fix the
state of these two categories of protein groups. In a medium that is saturated with a
simple salt (typically NaCl), these two different acid-base parameters become linked
in a fixed relationship. In this case, the system reverts to having only a single acid-
base variable, as in water. Only limited studies have been made so far of systems in
which both classes of buffer are present, so it is not possible to say how often better
performance can be obtained by optimizing both. Hence for the present, I would not
advise those persons looking at practical syntheses to use more than one type of
buffer.
References I285
References
1 E. N. Vulfson, P. J. Halling and H. L. Hol- 19 G. Bell, A. E. M.Janssen and P. J. Halling,

land, (eds.) Methods in Biotechnology: En- Enzyme Microb. Technol. 1997,20,471-477.
zymes in Nonaqueous Solvents. Humana 20 J. A. Riddick, W. B. Bunger, T. K. Sakano,
Press, Totowa, NJ, USA, 2001. Organic solvents: physical properties and
2 A. M. Klibanov, Nature, 2001 409, 241-246. methods of purification, 4th ed, Wiley, New
3 G. Carrea, S. Riva, Angau. Chem. Int. Ed. York, 1986.
Engl., 2000 39,2226-2254. Properties and 21 R. M. Stephenson,J. Chem. Eng. Data 1992,
synthetic applications of enzymes in or- 37, 80-95.
ganic solvents 22 L. Kvittingen, B. Sjursnes, T. Anthonsen
4 P. J. Halling, C u r . Opin. Chem. Biol. 2000, and P. J. Halling, Tetrahedron 1992.48,
4, 74-80. 2793-2802.
5 A. J. Mesiano, E. J. Beckman, A. J. Russell, 23 E. Zacharis, 1. C. Omar, J. Partridge, D. A.
Chem. Rev. 1999,99,623-633. Robb and P. J. Halling, Biotechnol. Bioeng.
6 Y. L Khmelnitsky, J. 0. Rich, Cum Opin. 1997,55,367-374.
Chem. Bid. 1999, 3,47-53. 24 P. J. Halling, Biotechnol. Tech. 1992, 6,
7 M. Erbeldinger, X-W. Ni and P. J. Halling, 271-276.
Enzyme Microb. Technol. 1998, 23, 141-148. 25 E. Wehtje, P. Adlercreutz in: E. N. Vulfson,
8 R. D. Schmid, R. Verger, Angew. Chem. Int. P. J. Halling and H. L. Holland, Eds. (2001)
Ed. Engl., 1998, 37, 1609-1633. “Methods in Biotechnology: Enzymes in
9 K. E. Jaeger, M. T. Reetz, Trends Biotechnol. Nonaqueous Solvents”, Humana Press, To-
1998, 16,396403. towa, NJ, USA. 2001, pp 127-134.
10 J. A. M. de Bont, Trends Biotechnol. 1998, 16, 26 A. Reimann, D. A. Robb and P. J. Halling,
493-499. Biotechnol. Bioeng. 1994,43, 1081-1086.
11 R. Leon, P. Femandes, H. M. Pinheiro, 27 P. J. Halling, Enzyme Microb. Technol. 1994,
J. M. S. Cabral, Enzyme Microb. Technol. 16, 178-206.
1998,23,483-500. 28 R. H. Valivety, G. A. Johnston, C. J. Suckling
12 M. T. De Gomez-Puyou, A. Gomez-Puyou, and P. J. Halling, Biotech. Bioeng. 1991, 38,
Cnt. Rev. Biochem. Mol. Bid. 1998, 33, 1137-1143.
53-89. 29 K. Xu, A. M. Kliban0v.J. Am. Chem. Soc.
13 Y. Okahata, T. Mori, Trends Biotechnol. 1997, 1996, 118,9815-9819.
15, 50-54. 30 M. Dolman, P. J. Halling and B. D. Moore,
14 A M . Klibanov, Trends Biotechnol. 1997, 15, Biotechnol. Bioeng. 1997,55, 278-282.
97-101. 31 N. Harper, B. D. Moore and P. J. Halling,
15 R. Lortie, Biotechnol. Adv. 1997, 15, 1-15. Tetrahedron Lett. 2000,41, 4223-4227.
16 J. Bosley, Biochem. Soc. Trans. 1997,25, 32 E. Zacharis, B. D. Moore and P. J. Halling,
174- 178. J. Am. Chem. SOC.1997,119,12396-12397.
17 A. Koskinen, A. M. Klibanov (eds) Enzy- 33 N. Harper, M. Dolman, B. D. Moore and
matic reactions in organic media. Chapman P. J. Halling, Chem. Eur. J. 2000, 6,
6 Hall, Andover, 1995. 1923-1929.
18 L. Greenspan, J. Res. Nat. Bur. Standards 34 J. Partridge, P. J. Halling and B. D. Moore ‘
1977,81A, 89-96. J. Chem. Sac. Perkin I1 2000, 465-471.
9
Enzymatic Kinetic Resolution
Jonathan M. J. Williams, RebeccaJ. Parker, and Claudia Neri
9.1
Introduction
Conventional kinetic resolution procedures often provide an effective route for the
preparation of enantiomerically enriched compounds. However, a resolution of two
enantiomers will only provide a maximum of 50 % yield of the enantiomericallypure
material. This limitation can be overcome in a number of ways, including inversion
of the stereochemistry of the unwanted enantiomer, racemization and recycling of
the unwanted enantiomer or dynamic kinetic resolution.
A dynamic kinetic resolution reaction involves the interconversion of the enantio-
mers of a starting material under conditions where one enantiomer is converted
selectively into product. This principle is shown in Fig. 9-1, where a conventional
kinetic resolution reaction and a dynamic kinetic resolution reaction are compared.
In both cases enantiomer A reacts to form product B more quickly than enantiomer
A'. However, in the conventional kinetic resolution, enantiomer A' is simply left
behind as unreacted starting material. In the dynamic kinetic resolution, Aand A' are
in equilibrium, which allows for the possibility that all of the starting material will be
converted into product B. The reaction conditions must be chosen that whilst the
starting material enantiomers @/A')undergo rapid equilibration (racemization),the
product B must be inert to racemization.
Dynamic kinetic resolution reactions are not limited to enzyme-catalyzed proc-
esses, and there are reviews available that consider all aspects of such
Conventional Kinetic Resolution Dynamic Kinetic Resolution
A -
+ 0 A - B
A' -X- B' A' + B'

Figure 9-1. Comparison of conventional and dynamic resolution reactions.
288
I 9 Enzymatic Kinetic Resolution
In addition, reviews dealing with aspects of enzyme-catalyzed dynamic resolution

and related processes such as stereoinversion and deracemisation have also been
published [4-71. Details of the kinetic principles of dynamic kinetic resolution
reactions have also been reported17-’]. Interestingly, a dynamic kinetic resolution
reaction can provide a product with higher enantiomeric excess than the correspond-
ing kinetic resolution. In a conventional kinetic resolution, the enantiomeric excess
of the product often decreases as a function of conversion. This happens because as
the reaction proceeds, the proportion of the preferred enantiomer of substrate
decreases. Unless the enzyme is able to discriminate perfectly between the substrate
enantiomers, it will catalyze the reaction of the less preferred enantiomer of
substrate (the proportion of which grows as the reaction proceeds). However, in a
dynamic kinetic resolution where the substrate enantiomers are interconverting
rapidly, the ratio of substrate enantiomers will be constant at 1:l. Consequently, the
enantiomeric excess of the product will not decrease as the reaction proceeds.
The following sections consider dynamic resolution reactions of alcohols (and
their derived esters), acids (and their derived esters) as well as dynamic resolution
involving reaction catalyzed by dehydrogenase enzymes.
9.2
Alcohols and their Derivatives
In order to achieve a dynamic kinetic resolution of alcohols, procedures need to be

found for the in situ racemization of these substrates. The racemization conditions
need to be compatible with the enzyme-catalyzedstep, and the product must be inert
to racemization.
The general principles are identified in Fig. 9-2, where enzyme-catalyzedacylation
selectively converts one of the equilibrating alcohols into the corresponding ester.
Methods for racemization of the alcohol include substrates where the R or R’
group is a good leaving group, or where temporary dehydrogenation to the corre-
sponding ketone can be achieved, as shown in Fig. 9-3.
H, OH Acyl donor
R ,A R’ enzyme
t
HO H
Figure 9-2. Dynamic resolution i n the acylation of
R alcohols.
9.2 Alcohols and their Derivatives
I
8,
289
+ HX
-HX
-
R -
+HX R -HX R
Figure 9-3. Racernization o f alcohols via carbonyl compounds.
9.2.1
Cyanohydrins
Cyanohydrins are readily racemized with base, and this has been exploited by Oda
and co-workers in a dynamic kinetic resolution of these substrates [lo,'*I. In a typical
procedure (Fig. 9 - 4 , the cyanohydrins were formed by transhydrocyanation with
acetone cyanohydrin, catalyzed by the hydroxide form of an anion exchange resin
(Amberlite IRA-904). The reversible nature of the cyanohydrin formation allows
racemization to proceed during the course of the enzyme-catalyzed acetylation, and
the choice of isopropenyl acetate as the acyl donor means that the only by-product is
acetone.
The immobilized lipase from Pseudomonas cepacia (Amano) afforded good en-
antioselectivities for the formation of a range of cyanohydrin acetates derived from
aromatic aldehydes (Fig. 9-5). Polymer-supportedquinidine could also be employed
OH
w,
RACN
1
R
R CN
(a) Acetone cyanohydrin, IRA-904 resin (HO-form)

(b) Pseudomonas cepacia lipase, isopropenyl acetate
3A molecular sieves, i-Pr20, 40 "C,3-6 days
Figure 94. Racernization of cyanohydrins with in situ acylation.
290
OAc OAc OAc
W C N / O " C N < r C N
\ \
CI
84% ee 84% ee 91% ee
96% yield 83% yield 81% yield
OAc OAc OAc
47% ee 85% ee 89% ee

57% yield 88% yield 80% yield
Figure 9-5. Examples o f cyanohydrin acetates formed by dynamic resolution.
as the base for racemization and formation of cyanhydrins, although the reactions
were generally slower than with the Amberlite resin [I2].
9.2.2
Other Readily Racemized Substrates
Kellogg, Feringa and co-workers have achieved successful dynamic kinetic resolu-
tion reactions using cyclic hemiacetals as substrates 1' 3, 141. The enzyme-catalyzed
acetylation of 6-hydroxypyranone shown in Fig. 9-6 has been achieved with reason-
able enantioselectivity with essentially complete conversion. The racemisation of the
hemiacetal is presumed to proceed via reversible ring-opening of the pyran~ne['~I.
The rate of reaction was found to greatly increase when the enzyme, lipase PS
(Pseudomonas sp.) was immobilized on Hyflo Super Cell (HSC).
IIW. OH
Figure 9-6. Dynamic resolution o f cyclic hemiacetals.

I
291
PS-HSC
0 0Ac
O O
cyclohexanel
butyl acetate (1:l) 100% conversion
18 h, r.t. 89% ee
CAL (immobilised)
0 0a 0Ac
?J
I cyclohexane I
18 h, 69 "C
COMe COMe
100% conversion
>99% ee
Figure 9-7. Examples o f dynamic resolution o f furanone and pyrrolinones.
(a) HSR', SiOp

(b) fseudornonas fluorescenslipase, t-BuOMe/vinyl acetate (3:1),
30 "C,4-11 days
Figure 9-8. Dynamic resolution o f hernithioacetals.
The related dynamic resolutions of the furanone and pyrrolinone substrates were
achieved with higher selectivities (Fig. 9-7)[I4]. Again, these substrates underwent
spontaneous racemization under the reaction conditions. Appropriate choice of
enzyme afforded a good example of an essentially perfect dynamic kinetic resolution
process in the case of the esterification of the hydroxypyrrolinone substrate.
Rayner and co-workers have demonstrated that hemithioacetals can be racemized
on exposure to sili~a['~1. In a typical experiment, an aldehyde and a thiol are
combined to give a hemithioacetal. In the presence of silica, the enzyme-catalyzed
acetylation proceeds under dynamic resolution conditions, as shown in Fig. 9-8.
9 Enzymatic Kinetic Resolution
292
I
OAc
Meo
&
, SBu M e O TS-0SiEt3
0 0
63% yield 83% yield
>95% ee 90% ee
OAc OAc
AcO A c O A -OSiEt3
SCH(Me)2 S
65% yield 73% yield
>95% ee >SO% ee
Figure 9-9. Examples of acetylated hemithioacetals.
+
H 0OH A+-&H
75% yield 0OAc
97% ee
\
rneso
intermediate
HO
(a) Et3N, THF

(b) lipase PS, vinyl acetate, 48 h, r.t.
Figure 9-10. Dynamic resolution involving a m e w intermediate
Representative products obtained using this procedure are given in Fig. 9-9. The
acetylated products are inert to racemization under the reaction conditions.
An interesting example of dynamic kinetic resolution of an alcohol has been
reported by Taniguchi and Ogasawara~"].The a-hydroxy ketone in Fig. 9-10 under-
293
I
goes racemization via a rneso enediol intermediate, and one of the enantiomers can
be acetylated selectively with lipase PS (Pseudornonas sp. immobilized on Celite,
Amano). The added triethylamine was required in order for the racemization to take
place. Without triethylamine, the reaction proceeded under conventional kinetic
resolution conditions.
9.2.3
Enzyme and Metal Combinations
Most substrates for enzyme-catalyzed kinetic resolution reactions do not undergo

spontaneous racemization under conditions that are suitable for enzyme activity.
One solution to this problem has been to design mild transition metal-catalyzed
methods for in situ racemization[”]. In order to achieve this goal, the racemisation
method must be able to function without an adverse effect on the enzyme.
Additionally, the enzyme must not inhibit the racemization method.
The first example of the use of enzyme and metal combinations to provide a
dynamic resolution procedure was reported by Allen and Williams in 1996[”]. In this
case, a palladium (11) catalyst was employed that was able to racemize the allylic
acetate substrate, but did not erode the enantioselectivity of the product allylic
alcohol (Fig. 9-11). For example, a cyclic acetate was shown to undergo a simple
kinetic resolution, affording enantiomericallyenriched starting material and product
at approximately 50 % conversion. However, performing the reaction in the presence
of a palladium (11) catalyst facilitated a dynamic resolution by continuously racemiz-
ing the starting material as the reaction progressed.
Similar methodology was applied to an acyclic allylic acetate by the group of Kim,
who used Pd(0) catalysts[I9]. Acyclic allylic acetates are easier substrates for palla-
dium-catalyzed racemization, and these workers were able to effect a dynamic
resolution strategy within a more acceptable time scale (Fig. 9-11). The in situ
racemization with palladium catalysts is limited in scope, since allylic acetates are
required as substrates. In addition, not all allylic acetates are expected to undergo
facile racemization .I‘’[
An alternative enzyme/transition metal combination employs transfer hydro-
genation catalysts that are capable of racemizing secondary alcohols. The racemiza-
tion procedure temporarily converts the alcohol into an achiral ketone, which is
reduced back to the racemic alcohol. Coupling this racemization procedure to an
enzyme-catalyzedacylation reaction affords a dynamic resolution process (Fig. 9-12).
Several enzyme/transition metal combinations have been shown to be effective for
these reactions, although ruthenium complexes 1-3appear to be especially effective
for the in situ racemization of the alcohol. The product esters are not prone to
racemization under the reaction conditions. Early results employing transfer hydro-
genation catalysts to effect the racemization of alcohols required the use of added
ketoner’l, 22]. However, it was subsequently shown that added ketone was not
required when appropriate transition metal complexes were used as catalysts.
Furthermore, the use of 4-chlorophenylacetate as the acyl donor afforded improved
results.
294
I
enzyme
*
H20
OAc OH
1 Pd(0) or Pd(1l)
,PR OAc
Ph Ph
I Pseudomonas fluorescens
lipase
37-40 "C, pH 7.0, 19 days
w
U 0.1M phosphate buffer

5 mol% PdC12(MeCN)2
81% yield
96% ee
Candida antarctica
lipase
OAc i-PrOHTTHF
16-18"C, 24 h
*
kPh then 5 mol% Pd(PPh& Ph
with 15 mol% dppf, 36 h
83% yield (HPLC)
98% ee
dppf = 1,l '-bis(dipheny1phosphino)ferrocene

Figure 9-11. Dynamic resolution using transition metal/enzyme combinations.
Backvall and co-workers have reported successful results for a wide range of
substrates, some of which are identified in Table 9-1. The procedure works well for
secondary alcohols containing aryl and alkyl groups [231, diols [241 and a-hydroxy
esters 12'1. Although catalyst 1 requires no additional base, Kim, Park and co-workers
used triethylamine to facilitate racemization using catalyst 2, Table 9-2 [ 2 6 ] . In their
case, small quantities of oxygen were added to initiate the racemization procedure.
In the case of allylic alcohols, careful choice of racemisation catalyst is required in
order to minimize the amount of conversion of the substrate into saturated or
I 295
vinyl acetatel
CH2C12 (3:l)
* OAc
1 equiv. PhCOMe
2 mol% Rh2(0Ac)4
PhM
'e 6 mol% o-phenanthroline PhAMe
Pseudomonas fluorescens
lipase
72 h, 20 "C
R 1 R'
Ru catalyst 1 - 3
enzyme
(see Figs 14 and 15)
OAc
Figure 9-12. Transition metal-catalyzed racemization of alcohols coupled with

enantioselective enzyme-catalyzed acetylation.
unsaturated ketones. For allylic alcohols, Kim, Park and co-workers have used
catalyst 3, which minimizes the formation of undesirable side products LZ71.
In addition to the use of enzyme and transition metal combinations for the
dynamic resolution of alcohols, there has been a brief report of the use of amines as
substrates. In 1996, Reetz and Schimossek reported the combination of palladium
on carbon with an immobilized lipase (from Candida antarctica) in the dynamic
Tableg-1. Examples of dynamic resolution of secondary alcohols with catalyst 1.

Substrate Product Conditions Yield ("h) ee ("A)
2 mol% Ru cat 1 80 > 99

OH OAc Novozym 435
I
3 equiv pCIC6H40Ac
PhAMe PhAMe toluene
70 "C, 46 h
2 mol% Ru cat 1 77 > 99
m
OH OAc
Novozym 435
3 equiv pCIC6H40Ac
toluene
\ 70 "C, 48 h
2 mol% Ru cat 1 88 > 99

OAc Novozym 435
3 equiv pCIC6H40Ac
P h O 4
PhOJ Me Me toluene
70 "C, 46 h
2 mol% Ru cat 1 80 > 97

Novozym 435
3 equiv pCIC6H40Ac
C 6 b 3M
e
' toluene
qH
70 "C, 24 h
OAc 2 mol% Ru cat 1 77 > 99

Novozym 435 (98:2
3 equiv pCIC6H40AC R,R/meso)
OH toluene
70 "C, 24 h
2 mol% Ru cat 1 80 94
OH OAc PS-C (type 11)
2 equiv pCIC6H40Ac
PhAC02Me Ph n C 0 2 M e cyclohexane
60 "C. 48 h
2 mol% Ru cat 1 80 98
PS-C (type 11)
2 equiv pCIC6H40Ac
cyclohexane
60 "C, 48 h
Novozym 435 is Candida antarctua lipase B (Nova Nordisk A/S)
PS-C (type 11) from Amano is Pseudornonas cepacia lipase
9.3 Carboxylic Acids and their Derivatives
Examples of dynamic resolution o f secondary alcohols with catalysts 2 and 3.

I
297
Table 9-2.
Substrate Product Conditions Yield ("A) ee (%)
5 mol% Ru cat 2 85 96
5 mol% 0 2
OAc
PS-C (type 11)
3 equiv Et3N
PhM
'e PhAMe 3 equiv pCIC6H40Ac
CHzClz, 60 "C, 43 h
5 mol% Ru cat 2 98 99
5 mol% 0 2
PS-C (type 11)
3 equiv EtjN
3 equiv pCIC6H40Ac
CHzC12,60 "C, 4 3 h
4 mol% Ru cat 3 84 > 99

OH OAc PS-C (type 11)
I
- 1 equiv Et3N
1.6 equiv pCIC6H40Ac
P h w Me
CH2C12, r. t., 48 h
4 mol% Ru cat 3 90 95
OAc PS-C (type 11)
1 equiv EtJN
1.6 equiv pCIC6H40Ac
CHzC12, r. t., 48 h
PS-C (type 11) from Amano is Pseudomonas cepacia lipase
resolution of phenethylamine[281.The N-acylated product was obtained with 99 % ee

and with 75-77 % yield.
9.3
Carboxylic Acids and their Derivatives
9.3.1
Readily Enolized Carboxylic Acid Derivatives
Carboxylic acid derivatives that have a-substituents can exist as chiral compounds.
The resolution of the enantiomers of such compounds is a useful process, leading to
the preparation of a-amino acids, a-hydroxy acids and other a-substituted carboxylic
acids and their derivatives in enantiomerically enriched form. In addition, the
racemization of such compounds can be achieved by a deprotonation/reprotonation
sequence, as shown in Fig. 9-13.
The ease with which racemization of the carboxylic acid derivative occurs depends
on the nature of the substrate. Carboxylic acids themselves are slow to racemize,
since the carboxylic acid is initially deprotonated to form a carboxylate anion.
298
I
achiral enolate
Figure 9-13. Racemization o f a-substituted carboxylic acid derivatives by enolization.
S.griseus protease
carbonate buffer
(PH 9.7)
24 h, 22 "C
Ketorolac
100% conversion
76% ee
Figure 9-14. Dynamic resolution i n the preparation of Ketorolac.
Subsequent deprotonation to afford the carboxylic acid enolate requires the forma-
tion of a doubly deprotonated species, which is disfavored relative to the formation of
an ester enolate. In fact, activated esters such as phenyl estersI2'1 or thioesters[30]are
especially prone to racemization, since enolization is easier than for simple esters.
Fulling and Sih reported one of the earliest examples to exploit racemization of
carboxylic acid derivatives in order to achieve a dynamic kinetic resolution[31].The
anti-inflammatory drug Ketorolac was prepared by hydrolysis of the corresponding
ester. Whilst most lipases afforded the undesired enantiomer preferentially, a
protease from Streptornyces griseus afforded the required (S)-enantiomerof product
with good selectivity. The substrate was particularly prone to racemization since the
intermediate enolate is well stabilized by resonance effects, although a pH 9.7 buffer
was required to achieve a useful dynamic resolution reaction. Thus the acid was
formed with complete conversion and with 76 % enantiomeric excess.
Drueckhammer and co-workers have published details of a successful strategy for
dynamic resolution in the hydrolysis of suitable thioesters L30, 321. Trioctylamine was
employed as the racemizing agent, which was effective for the racemization of a
series of a-substituted thiopropionates. Specific examples include the hydrolysis of
an ethylthioester using Pseudomonas cepacia lipase, the transesterification of an a-
aryloxy trifluoroethylthioester with butanol and PS-30, as well as hydrolysis of a
trifluoroethylthioester using Subtilisin Carlsberg (Fig. 9-15).
The ability to achieve dynamic kinetic resolution using thioester substrates has
been recognized by other workers, and reports of dynamic resolution strategies
I 299
PCL
0.5 equiv Oct3N
b
toluene, H20
65 h
>99%conversion
96.3%ee
0
0 Subtilisin Carlsberg
0.5 equiv Oct3N
*
97%conversion
83%ee
lipase (PS-30)
Et3N
SCH2CF3 OBu
toluene, BuOH
Me Me
98%conversion
75% ee
Ar = 2,4-dichlorophenyl
Figure 9-15. Dynamic resolution i n the hydrolysis/transesterification of thioesters.
leading to the anti-inflammatory drugs Napr~xen[~’] and S u p r ~ f e n l ~have

~ I been
published. Trioctylamine is again used as the racemizing agent, as shown in Fig. 9-
16.
In addition to in situ racemization of a-substituted carboxylic acid derivatives by
deprotonation/reprotonation, a procedure involving halide exchange has been devel-
oped[35,3G1. Whilst the a-halo esters undergo racemization at a reasonable rate, the
corresponding carboxylates are almost inert to racemization under the reaction
conditions. Using immobilized phosphonium halide and CLEC (cross-linked en-
zyme crystals),a dynamic resolution procedure has been developed for the hydroly-
sis of a-homo and a-chloro esters (Fig. 9-17).The enantiomeric excess in each case
was similar to that achieved for simple kinetic resolution reactions using the same
enzyme/substrate combinations.
Nitriles can be hydrolyzed by various microorganisms, affording the correspond-
ing carboxylic acids. A method has been reported for the hydrolysis of racemic
mandelonitrile (PhCH(0H)CN) into (R)-mandelic acid using Alcaligenes faecalis
300
Candida rugosa lipase
Me 45 "C, 294h
OH
Me
Naproxen
70% conversion
92% ee
Candida rugosa lipase

*SCH2CFs Oct3N
isooctane
Me 45 "C,20h
OH
Me
Suprofen
100% conversion
95% ee
Figure 9-16. Dynamic resolution in the synthesis o f Naproxen and Suprofen.
Br Polyrner-PPh3+ Br- $r
t
P h A 0 2 M e Candida rugosa lipase (CLEC) PhAC02H

H20, pH 7,4.5 h
80% conversion
79% ee
CI Polyrner-PPh3+ CI- CI
Candida cylidfacea lipase (CLE;) phAco2~

PhAC02Me H20, pH 7,24h
90% conversion
90% ee
Figure 9-17. Racemization of a-haloesters by halide exchange coupled with enzymatic hydrolysis.
9.3 Carboxylic Acids and their Derivatives I
301
Figure 9-18.
Racemization o f
a-aminoesters
NH2 -
SLOW
7
NH2
catalyzed by imine RAC02Me RAC02Me

formation.
R'
R'
I
-
FAST-
RnC02Me
ATCC 8750. An isolated yield of 94% of the enantiomericallypure mandelic acid was
obtained, indicating that a dynamic resolution process is occurring.
9.3.2
Amino-Esters and Related Compounds
Typical a-amino esters only undergo racemization slowly, but methods for accelerat-
ing this process have been devised[37.381. Temporary conversion of the amine to an
imine lowers the pK, of the substrate, such that racemization becomes faster.
A series of a-aminoesters has been hydrolyzed to a-amino acids using alcalase in
the presence of pyridoxal S-pho~phate[~~]. During the course of these reactions, the
amino acids precipitated from the reaction mixture, thereby protecting them from
racemisation. The method was used to prepare enantiomerically enriched phenyl-
alanine, leucine, tryptophan and norvaline with high selectivity (Fig. 9-19).
A related ammonolysis of an amino ester has been reported using either pyridoxal
or salicylaldehydeas the racemizing agent I4l1. The amino ester undergoes racemiza-
tion more quickly than the amino amide, and an effective dynamic resolution could
be achieved at -20 "C.
Re-formed imino-esters have also been used as substrates for dynamic kinetic
resolution reactions [421. The free amino acid precipitated from the reaction mixture
as the reaction proceeded.
a-Azido amides have been subjected to kinetic resolution reactions using whole
cells of E. coli DHSa/pTrpLAP,affording hydrolysis to the corresponding acids[43].In
the case of 2-azidophenylaceticacid amide, the substrate racemized in situ, and the
acid product could be obtained with 98% ee at over 50% conversion.
302
1 9 Enzymatic Kinetic Resolution
Alcalase y 2
20 mot% pyridoxal phosphate
i-PrOHIH20(19:1), pH 8.5 RnCO2-
t-BuOH/t-BuOMe (7:3)
40 "C, 3-4 h
R yield ee
PhCH2 92% 98%
(CH3)2CHCH2CH2 87% 93%
(3-indolyl)CH2 95% 97%
CH3CH2CH2 87% 91%
NH Novozym 435 (CAL-B)

* NH2
pyridoxal
Ph NH3 PhACONH2
t-BuOH/t-BuOMe (7:3)
-20 "C, 66 h 85% conversion
88% ee
N4 chyrnotiypsin y42
*
H20/MeCN(1:19)
PhCH2A C02Et 10 mol% DABCO PhCHzAC02H
added after 48h.
Then 48 h at r.t. 87.5% yield
90% ee
Figure 9-19. Dynamic resolution of amino acids via imine formation.
9.3.3
Reactions of cyclic amino acid derivatives
There are several cyclic amino acids derivatives that are prone to racemization and
have been used as substrates for dynamic kinetic resolution reactions.
Oxazolinones were first used as substrates €or enzyme-catalyzed hydrolysis over
30years It was noted that spontaneous hydrolysis could be quite high,
depending on the amino acid derivative being used and the pH of the reaction
medium [451. Bevinakatti and co-workers demonstrated that oxazolinones could
undergo racemization during a lipase-catalyzed enantioselective ring-opening with
n - b u t a n ~ l [471.~ ~At, 100% conversion, they were able to obtain (S)-butylN-benzoyla-
laninate with 34% ee.
This concept has been developed by the research groups of Sih and Turner. The
oxazolinone derived from phenylalanine was subjected to lipase-catalyzed hydrolysis
with ten lipases 14'1. Whilst several lipases gave good enantioselectivities, the lipase
9.3 Carboxylic Acids and their Derivatives I 303
/
PL(Ferm1ipase)
Phosphate buffer
(PH 7.6)
phcH2H
HN
Ph
)C.
OH
100% conversion
99% ee
NQ
I
Ph
\ PhCH2 r/c”
Lipase AP \ HN OH
Po
\
(from Aspergillus Niger)
Phosphate buffer Ph
(pH 7.6), 17 h
100% conversion
99% ee
Figure 9-20. Dynamic resolution in the hydrolysis of oxazolinones.
phcH2H
HN OH
Ph>o
PhCH2Yfo
P30 Lipase *
5 equiv. MeOH
PhCH2Ho
HN
OMe
Prozyme 6
*
82% yield
295% ee
NYo Ph
t-BuOMe
50°C,48h
Ph
)=’
99% yield
Po
65% ee
Ph
15% yield
>95% ee
Figure 9-21. Two stage hydrolysis of oxazolinones.
from Aspergillus niger (AP) and porcine pancreatic lipase (PL Fermlipase) provided
particularly good enantioselectivities,with an opposite sense of asymmetric induc-
tion from each other (Fig. 9-20).
An additional strategy employed by Sih and co-workers involved sequential
enzyme-catalyzedreactions. Pseudomonas lipases were found to tolerate a wide range
of substrates although the enantioselectivity was generally only moderate. However,
by first performing a methanolysis of the oxazolinone followed by a separate
enzyme-catalyzed hydrolysis under kinetic resolution conditions, a highly enantio-
merically enriched product could be obtained, as shown in Fig. 9-21.1’4
304
"w" Prozyme 6
HN OH
NYs Ph
phosphate buffer
10% MeCN
pH 7.5
25 "C, 7-88 h
Ph>s
R yield ee
Me 67% 90%(NoMeCN)
(CH&CHCH2 78% 97%
CH3SCH2CH2 94% 98%
CH~CHZCH~CH~86% 99%
H2NCOCH2 98% 57% (No MeCN)
Figure 9-22. Dynamic resolution in the hydrolysis ofthiazolinones.
Lipozyme * '""?--fO
HN OBu
NYo Ph
25 mol% Et3N
2 equiv. BuOH
toluene, 30 "C, 5 days ph>o
94% yield
99.5% ee
""w" Novozyme (CAL-B)

HN OBu
NYo Po
25 mol% Et3N
2 equiv. BuOH
toluene, 37 "C
Ph Ph
79% yield
94% ee
i-prHo i-prxo c
Novozyme (CAL-B)
2 equiv. MeOH
HN
OMe
MeCN, 37 "C k o
Ph Ph'
83% yield
97% ee
Figure 9-23. Dynamic resolution i n the alcoholysis o f oxazolinones.
I
305
Pseudomonas sp.
AJ-11220
(whole cells)
c
phosphate buffer H2N OH

(PH a), 30 h
HN0
KNH
94% yield
>99% ee
Agrobacterium radiobacter
pH 8.4,40 "C, 48 h
79% yield
>92% ee
pH 8.4,40 "C, 48 h
71Yoyield
>96% ee
Figure 9-24. Dynamic resolution in the hydrolysis of hydantoins.
Sih and co-workers also reported the dynamic resolution of a range of thiazoli-
nones by enantioselective hydrolysis using proteases [491. In these cases, the product
is the corresponding thioamide. Some of the higher enantiomeric excesses reported
are identified in Fig. 9-22.
Turner and co-workers identified conditions appropriate for the dynamic resolu-
tion of a 4-tert-butylsubstituted 0xazolinone[~~1.
The ring-opened butyl ester could be
obtained with high yield (94%) and enantiomeric excess (99.5%) using Lipozyme -
Mucor miehei and 0.25 equivalents of triethylarnine. Subsequent cleavage of the ester
and amide groups afforded a route to enantiornerically pure (S)-tert-leucine.Whilst
Lipozyme provided high selectivities for the sterically demanding tert-butyl group,
Turner reported that Candida antarctica lipase B (Novozyme) was preferred for
smaller groups, as shown in Fig. 9-23[511.
306
hydantoinase
-
""K"" 0
buffer
pH 8.5,50 "C
HN
0b N H 2
OH
Figure 9-25. Dynamic resolution o f racemic hydantoins.
The other major class of cyclic amino acid derivative used in dynamic resolution
reactions is the hydantoin group. Like oxazolinones, hydantoins readily undergo
racemisation under mild conditions. Systems involving a two step procedure using
D-hydantoinase and a carbamoylase were reported to provide a route to D-amino
acids l S 2 . 531. Dynamic resolution of a p-hydroxyphenyl substituted hydantoin was
reported in 1987[54].Using the intact cells of Pseudomonas sp. AJ-11220,the amino
acid was prepared in over 90% yield, as shown in Fig. 9-24. This hydrolytic
procedure leads directly to the amino acid, and the same enantiomer of product, the
D-amino acid, was obtained independently of the stereochemistry of the substrate.
A similar strategy has been used in the hydrolysis of hydantoins with pendant
ureido groups, using the bacterial culture Agrobucterium r u d i o b ~ c t e r [ ~ ~ ] .
D-Hydantoinaseshave also been isolated from thermophilic micro-organisms, and
applied to the dynamic resolution of racemic hydantoins, where the isolated
products are the N-carbamoyl D-amino acids (Fig. 9-25)[561. Subsequent transforma-
tion into D-amino acids could be achieved chemically or enzymatically. Representa-
tive examples using commercially available hydantoinases D-HYD-1 and D-HYD-2
are shown in Table 9-3. Variously ring-substituted D-phenylglycine derivatives have
also been prepared by hydantoin hydrolysis using D-HYD-1and D-HYD-2,affording
the amino acid with excellent levels of enantioselectivity and good yields LS71.
Table 9-3. Dynamic resolution using hydantoinase enzymes.

R Enzyme Carbamoylateyield (%) Amino acid ee ("7)
Me D-HYD-1 71 94 (D-alanine)
D-HYD-2 73 34
PhCH2 D-HYD-1 67 > 99 (D-phenylalanine)
D-HYD-2 12 > 99
i-Pr D-HYD-1 66 > 99 (D-valine)
D-HYD-2 71 > 99
MeSchzCCHz D-HYD-1 81 > 99 (D-methionine)
D-HYD-2 75 > 99
Ph D-HYD 1 95 96 (D-phenylglycine)
D-HYD-2 90 > 99
9.4 Reduction ofa-Ketoesten
I 307
Baker's yeast
OEt
69% yield
spontaneous
racemisation
Figure 9-26. Dynamic resolution in the reduction o f P-ketoesters.
9.4
Reduction of fi-Ketoesters
The reduction of ketones into alcohols can be achieved using biocatalytic methods.
Amongst the most popular of the available methods is the use of Baker's yeast, BY
(Saccharomyces cerevisiae). The use of P-ketoesters as substrates leads to the corre-
sponding P-hydroxy esters, often with high enantioselectivity. In the particular case
of a-substituted p-ketoesters, the substrates spontaneously racemize, and this
provides the basis for many reports of dynamic resolution reactions, some of which
are described in the following discussion. In 1976, Deol and co-workers showed that
cycloalkyl fbketoesters could be reduced under dynamic resolution conditions
(Fig. 9-26)1('.
In fact, many microorganisms are able to achieve similar reductions on the same
and related substrates. Azerad and co-workers have achieved higher selectivities
using other microorganisms including Geotrichum candidum, Mucor racemosus,
Kloeckera magna and Mucor circinelloides[5q-611.The opposite diastereomer of product
(1S,2S instead of 1S,2R) was obtained using Penicillium chrysogenum and Colleto-
trichum gloeosporoides as the microorganism. A range of cyclic (3-hydroxyestershas
been prepared, some ofwhich are identified in Fig. 9-27. The use of a P-ketothioester
as a substrate has been reported to afford better stereoselectivity["I.
Heterocyclic (3-ketoesters have also been used as substrates for reduction, where
the products often have use in the synthesis of pharmaceutical agents or natural
products. Representative examples of heterocyclic 0-hydroxyesters formed using
Baker's yeast are given in Fig. 9-28[65-711.
Acyclic (3-ketoestersare generally less predictable as substrates than their cyclic
counterparts, with the selectivity depending on the nature of the groups attached to
the dicarbonyl moiety (Fig. 9-29).
Representative examples of acyclic p-hydroxyesters obtained by dynamic resolu-
308
I OH OH
Baker's yeast [62]

X= OMe 93% cis
94% ee
ii
Kloeckera magna [60]
100% cis
oo2
Mucor griseocyanus [60]
88% trans
99% ee 95% ee
a
X= SEt 100% cis
>96% ee
OH OH
OH
02Et
Beauveria bassiana [60] Mucor griseocyanus [60] Baker's yeast [61]
100% cis 100% trans >98% cis

98% ee 88% ee 72% ee
Baker's yeast [63,64]

? ? 100% cis
U 98% ee
Figure 9-27. Cyclic (3-hydroxyestersobtained by dynamic resolution.
tion are provided in Fig. 9-30, where Baker's yeast, as well as other microorganisms,
have been employed in the reduction process [72-801.
Improved stereocontrol has been obtained using recombinant E. coli strains
expressing Gre3p or Gcylp (from Baker's yeast). Since fewer competing enzymes are
present in the recombinant E. coli, the enantioselectivity and diastereoselectivity are
found to be better than using Baker's yeast itself as shown in Fig. 9-31181].
I
9.5 Conclusion 309
OH
C02Et
100% cis [651 100% cis [65,66] 63% cis (671

85% ee >85% ee 100% ee
Boc
Boc 0
100% cis [68,69] >99% cis [70] 98% ee [71]

>90% ee >93% ee
Figure 9-28. Heterocyclic 0-hydroxyesters obtained by dynamic resolution.
OH 0
microorganism* dOR''
R E
RV O
R' R " R'
Figure 9-29. Dynamic resolution o f acyclic fl-ketoesters.
9.5
Conclusion
In summary, dynamic resolution strategies employing biocatalytic methods provide

a useful synthetic route to a range of enantiomerically enriched building blocks.
Over the last few years there has been a growing interest in finding new methods for
the racemization of the starting material. The challenge is to discover racemization
methods that are compatible with the biotransformation. Nevertheless, substrates
that spontaneously racemize, such as (3-ketoesters,still provide the most practicable
starting materials for biocatalytic dynamic resolution reactions.
310
OH 0
Me
Baker'syeast [72] Rhodotorula glutinis [73] Baker's yeast [74,75]
73% de 88% de 98% de
97% ee 97% ee 100% ee
OH S
Me =
Me
Baker's yeast [76] Baker's yeast [77] Geotrichurn candidurn [78]
92% de 79% ee 96% de
>96% ee 91Yoee
Me Cl
Candida albicans [79]I Rhodotorula glutinis [80]
98% de 90% de
97% ee 95% ee
Figure 9-30. Acyclic (3-hydroxyestersobtained by dynamic resolution.
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Me
GCY 1P >98% ee >98% de
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I
313
10
Enzymes from Extreme Thermophilic and Hyperthermophilic
Archaea and Bacteria
Costanzo Bertoldo and Garabed Antranikian
10.1
Introduction
Environments that are considered by man to be extreme, such as those affected by

extremes of temperature, pH and salt content, are colonized by a diverse range of
microorganisms. These include an interesting group which are adapted to growth at
high temperatures[']. In the last two decades it has been possible to isolate
microorganisms which can grow optimally even above 100 oC[2-51.The temperature
range of growth can be used to define organisms as psychrophiles (-5 to 20 "C),
mesophiles (20 to 45 "C), thermophiles (45 to 65 "C), extreme thermophiles (65 to
85 "C) and hyperthermophiles (85 to 110 "C). The majority of the last group, which
thrive above the boiling temperature ofwater, belong to the Archaea. However, some
of these microorganisms also belong to the bacterial kingdom. Based on compar-
isons of partial nucleic acid sequences derived from 16 S and 18 S rRNAs, the two
primary kingdoms (prokaryotes and eukaryotes) are reclassified into three, namely
Bacteria, Archaea and Eukarya[61.Archaea are a newly recognized group of organ-
isms with a distinct evolutionary position and unique physiological,biochemical and
genetic properties. The thermophilic representatives of the bacteria that optimally
live above 65 "C comprise four genera, namely Themotoga, Themosipho, Fervid-
obacteriurn (Thermotogales order) and Aqu@feX (Aquificalesorder). The temperature
optimum for growth of these microorganisms ranges between 65 and 90 "C. On the
other hand the thermophilic representatives of the Archaea comprise more than 20
genera, which belong to the following orders: Sulfolobales, Pyrodictiales, Thermo-
proteales, Thermococcales, Archaeglobales, Thermoplasmales and the methano-
gens Methanobacteriales and Methanococcales. Table 10-1 describes some of the
growth conditions and of the biochemical features of microorganisms capable of
surviving at high temperatures [2-12]. The majority of the microorganisms described
in Table 10-1 are heterotrophic and anaerobic; only a few are strict autotrophes.
Organisms which belong to the Sulfolobales, Aquificales and Thermoplasmales can
also live under aerobic conditions. None of these microorganisms, however, can
grow optimally at 100 "C. An exception is the archaeon Pyrobaculurn aerophilum,
314 I 7 0 Enzymesfiom Extreme Thermophilic and Hyperthermophilic Archaea and Bacteria
Table 10-1. Taxonomy and some biochemical features of bacteria and archaea growing at high
temperaturesa.
Order Genus Optimal growth Heterotrophic (het) Anaerobic (an)
temperature ("C) autotrophic (aut) aerobic (ae)
facultative
autotrophic (f)
BACTERIA
Themotogales Thermotoga 70-80 het an
Thermosipho 70-75 het an
Fervidobacterium 65-70 het an
Aqu$ecales Aqu$ex 90 het ae/an
ARCHAEA
Sulfolobales Sulfolobus 65-80 f ae/an
Metallosphaera 75 f ae
Acidianus 88 aut ae/an
Desulfurolobus 80 het ae/an
Pyrodictales Pyrodictium 100-105 het, aut an
Thermodiscus 88 f an
Hyperthemus 100 het an
Trtermoproteales Thermoproteus 88 het, f, aut an
Themotilum 88 het an
Desulfurococcus 85 het an
Staphylothemus 92 het an
Pyrobaculum 100 het, f ae, an
7'hemococcales Thermococcus 70-87 het an
Pyrococcus 100 het an
Archaeoglobales Archaeoglobus 83 f an
Themoplasrnales Themtoplasma GO het ae/an
a Methanogenic microorganisms (Methanobacteriales and Methanococcales) with thermophilic representa-
tives are not shown.
which also grows aerobically at 100 "C. Most of these exotic microorganisms have
been isolated by Stetter, Zillig and co-workersfrom various geothermal habitats such
as hot springs, sulfataric fields and deep-sea hydrothermal vents. Of great interest
are the enzymes that are formed by extreme thermophilic and hyperthermophilic
microorganisms. Some of the enzymes that have been recently studied are even
active at 140 "C[l3].This short chapter will cover selected enzymes from extreme
thermophilic and hyperthermophilic microorganisms that have been described
recently. The enzymes from methanogens and thermophilic microorganisms that
grow below 70 "C (such as Bacillus, Clostridium and Themus)will not be covered. For
more detailed information of this rapidly developing field the reader should consult
the following reviews] I7-lo, 12* 141.
70.2 Starch-Processing Enzymes
I 315
10.2
Starch-Processing Enzymes
Starch from cultivated plants represents an ubiquitous and easily accessible source
of energy. In plant cells or seeds, starch is usually deposited in the form of large
granules in the cytoplasm. Starch is composed exclusively of a-glucose units that are
linked by a-1,4- or a-1,G-glycosidic bonds. The two high-molecular-weightcompo-
nents of starch are amylose (15-25%), a linear polymer consisting of a-1,4-linked
glucopyranose residues, and amylopectin (75-85 %), a branched polymer contain-
ing, in addition to a-1,4 glycosidic linkages, a-1,&linked branch points occurring
every 17-26 glucose units. a-Amylose chains, which are not soluble in water but
form hydrated micelles, are polydisperse, and their molecular weights vary from
hundreds to thousands. The molecular weight of amylopectin may be as high as 100
million, and in solution such a polymer has colloidal or micellar forms.
Because of the complex structure of starch, cells require an appropriate combina-
tion of hydrolyzing enzymes for its depolymerization to oligosaccharides and
smaller sugars such as glucose and maltose. They can be simply classified into two
groups: endo-acting enzymes or endo-hydrolases and exo-acting enzymes or exo-
hydrolases. Endoacting enzymes, such as a-amylase (a-1,4-glucan-4-glucanohy-
drolase; E.C. 3.2.1.1), hydrolyze linkages in the interior of the starch polymer in a
random fashion, which leads to the formation of linear and branched oligosacchar-
ides.
Exo-acting starch hydrolases include j3-amylase, glucoamalase, and a-glucosidase.
These enzymes attack the substrate from the nonreducing end, producing small and
well-defined oligosaccharides. P-Amylase (E. C. 3.2.1.2), also referred to as a-1,4-~-
glucan maltohydrolase or saccharogen amylase, hydrolyzes a-l,4glucosidic linkages
to remove successive maltose units from the non-reducing ends of the starch chains,
producing p-maltose by an inversion of the anomeric configuration of the maltose
(Fig. 10-1).
a-Glucosidase (E. C. 3.2.1.20), or a-D-glucoside glucohydrolase, attacks the a-1,4
linkages of oligosaccharides that are produced by the action of other amylolytic
enzymes. Unlike glucoamylase, a-glucosidase liberates glucose with an a-anomeric
configuration.
Enzymes capable of hydrolyzing a-l,G glycosidic bonds in pullulan are defined as
pullulanases. On the basis of substrate specificity and product formation, pullu-
lanases have been classified into two groups: pullulanase type I and pullulanase type
11. Pullulanase type I (E. C. 3.2.1.41) specifically hydrolyzes the a-1,G-linkages in
pullulan as well as in branched oligosaccharides (debranching enzyme), and its
degradation products are maltotriose and linear oligosaccharides, respectively.
Pullulanase type I is unable to attack a-l,4-linkagesin a-glucans. Pullulanase type 11,
or amylopullulanase, attacks a-1,G-glycosidiclinkages in pullulan and a-l,4-linkages
in branched and linear oligosaccharides, converting the latter to small sugars
(Fig. 10-1B).
In contrast to the previously described pullulanases, pullulan hydrolases types I
and I1 are unable to hydrolyze a-1,G-glycosidic linkages in pullulan or in branched
316
I 70 Enzymesfiom Extreme Thermophilic and Hyperthermophilic Archaea and Bacteria
substrates. They can attack a-1,4-glycosidic linkages in pullulan, leading to the

formation of panose or isopanose. Pullulan hydrolase type I or neopullulanase (E. C.
3.2.1.135) hydrolyzes pullulan to panose (a-6-D-g~ucosylma~tose). Pullulan hydrolase
type I1 or isopullulanase (E. C. 3.2.1.57) hydrolyzes pullulan to isopanose (a-
6-maltosylglucose).Recently, pullulan-hydrolase type I I1 was described, which at-
tacks a-1,4-aswell as a-1,G-glycosidiclinkages in pullulan (Fig. 10-1).
Cyclodextrin glycosyltransferase (CGTase, E. C. 2.4.1.19), or a-l,4-~-glucan a-4-~-
(a-l,4-~-glucano)-transferase, is an enzyme that is generally found in Bacteria and
was recently discovered in Archaea. This enzyme produces a series of non-reducing
cyclic dextrins from starch, amylose, and other polysaccharides. a-, p-, and y-
cyclodextrins are rings formed by 6, 7, and 8 glucose units that are linked by a-
1,4-bonds,respectively (Fig. 10-1).
10.2.1
Thermoactive Amylolytic Enzymes
10.2.1.1
Heat-Stable Amylases and Clucoarnylases.
Extremely thermostable a-amylases have been characterized from the hyperthermo-

philic Archaea Pyrococcusfuriosus, Pyrococcus woesei and Thennococcus profundus. The
optimal temperatures for the activity of these enzymes are 100 "C, 90 "C and 80 "C,
respectively. Thermoactive amylolybc enzymes have been also detected in hyper-
thermophilic Archaea of the genera Sulfolobus, Therrnophilum, Desulfirococcus, and
Staphylothennus [15-191. Molecular cloning of the corresponding genes and their
expression in heterologous hosts circumvent the problem of insufficient expression
in the natural host. The gene encoding an extracellular a-amylase from P. firiosus
has recently been cloned, and the recombinant enzyme has been expressed in B.
subtilis and E. coli. This is the first report of the expression of an archaeal gene
derived from an extremophile in a Bacillus strain. The high thermostability of the
pyrococcal extracellular a-amylase (thermal activity even at 130 "C) in the absence of
metal ions, together with its unique product pattern and substrate specificity, makes
this enzyme an interesting candidate for industrial application. In addition, an
intracellular a-amylase gene from P.furiosus has been cloned and sequenced. It was
interesting to note that the four highly conserved regions usually identified in a-
amylases are not found in this enzyme. a-Amylases with lower thermostability and
thermoactivity have been isolated from the Archaea Thennococcus profindus, Pyro-
coccus sp. KODl and the bacteria Themtotoga maritima and Dictioglomus themtophi-
lum. The genes encoding these enzymes were successfully expressed in E. coli.
Similar to the amylase from B. licheni$onnis, which is commonly used in liquefa-
ction, the enzyme from 1 maritima requires Ca2+ for activity[2&26].Further in-
vestigations have shown that the extreme hyperthermophilic Archaeon Pyrodictium
abyssi can grow on various polysaccharides and also secretes a heat-stable amylase
(unpublished results).
In contrast to a-amylase, the production of glucoamylase seems to be very rare in
70.2 Starch-Processing Enzymes
I 317
extremely thermophilic and hyperthermophilic Bacteria and Archaea. Among the
thermophilic anaerobic Bacteria, glucoamylases have been purified and charac-
terized from Clostridium themohydrosul&ricum 39E, Clostridium thermosaccharo-
lyticumand Themoanaerobacterium themosaccharolyticurn DSM 571 [27-291. Recently,
it has been shown that the thermoacidophilic Archaea Thermoplasma acidophilum,
Picrophilus tomdus and Picrophilus oshimae produce heat- and acid-stable glucoamy-
lases. The purified Archaeal glucoamylases are optimally active at pH 2 and 90 "C.
Catalpc activity is still detectable at pH 0.5 and 100 "C. This represents the first
report on the production of glucoamylases in thermophilic Archaea (unpublished
results).
10.2.1.2
a-Clucosidases.
a-Glucosidasesare present in thermophilic Archaea and Bacteria. An intracellular a-

glucosidase has been purified from P.ficriosus. The enzyme exhibits optimal activity
at pH 5.0 to 6.0over a temperature range of 105-115 "C; the half life at 98 "C is 48 h.
An extracellular a-glucosidase from the thermophilic Archaeon Thermococcus strain
AN1 was purified and its molecular characteristics determined13']. The monomeric
enzyme (GO kDa) is optimally active at 98 "C. The purified enzyme has a half-life
around 35 min, which is increased to around 215 min in the presence of 1% (w/v)
dithiothreitol and 1% (w/v) BSA. The substrate preference of the enzyme is: para-
nitrophenyl-a -D-glucoside > nigerose > panose > palatinose > isomaltose > maltose
and turanose. No activity was found with starch, pullulan, amylose, maltotriose,
maltotetraose, isomaltotriose, cellobiose and P-gentiobiose. The enzyme is also
active at 130 "C. The gene encoding a-glucosidase from Thermococcus hydrothermalis
was cloned by complementation of a Saccharomyces cereuisiae maltase-deficient
mutant The cDNA clone isolated encodes an open reading frame corre-
sponding to a protein of 242 amino acids. The protein shows 42% identity to a
Pyrococcus horikoshii, unknown ORF, but no similarities were obtained with other
polysaccharidase sequences.
10.2.1.3
Thermoactive Pullulanases and CCTases.
Thermostable and thermoactive pullulanases from extremophilic microorganisms

have been detected in Thermococcus celer; Desulficrococcus mucosus, Staphylothermus
marinus and Thermococcus aggregans. Temperature optima between 90 "C and 105 "C,
as well as remarkable thermostability even in the absence of substrate and calcium
ions, have been observed. Most thermoactive pullulanases identified to date belong
to the type I1 group, which attack a-1,4-and a-1,G-glycosidiclinkages. They have been
purified from P.&riosus, T. litoralis, T. hydrothermalis and Pyrococcus strain ES4[32-371.
Pullulanase type I1 from P.&riosus and P. woesei have been expressed in E. coli. The
unfolding and refolding of the pullulanase from P. woesei has been investigated
using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was
318
I found to be very resistant to chemical denaturation and the transition midpoint for
70 Enzymesfrom Extreme Thermophilic and Hypertherrnophilic Archaea and Bacteria
guanidinium chloride-induced unfolding was determined to be 4.86 i 0.29 M for

intrinsic fluorescence and 4.90 * 0.31 M for far-UV CD changes. The unfolding
process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M
guanidinium chloride) was obtained upon removal of the denaturant by stepwise
dilution; 100% reactivation was observed when refolding was carried out via a
guanidinium chloride concentration of 4 M in the first dilution step. Particular
attention has been paid to the role of Ca2+,which activates and stabilizes this
archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ions
with a Kd of 0.080 * 0.010 mM and a Hill coefficient H of 1.00 i 0.10. This cation
significantlyenhances the stability of the pullulanase against guanidinium chloride-
induced unfolding. The refolding of the pullulanase, on the other hand, was not
affected by Ca" [381. Very recently, the genes encoding the pullulanases from T
hydrothermalis, Desulfirococcus mucosus and 7: aggregans have been isolated and
expressed in mesophilic hosts. Since the latter enzyme attacks a-1,4as well as a-1,G
glycosidic linkages in pullulan, it has been classified as pullulan-hydrolase type 111.
Pullulan is converted to maltotriose, maltose, panose and glucose [40-421. The aerobic
thermophilic bacterium Thermus caldophilus GK-24 produces a thermostable pull-
ulanase of type I when grown on starch. This enzyme debranches amylopectin by
attacking specifically a-l,6-glycosidiclinkages. The pullulanase is optimally active at
75 "C and pH 5.5, is thermostable up to 90 "C, and does not require Ca" for either
activity or stability. The first debranching enzyme (pullulanase type I) from an
anaerobic thermophile was identified in the bacterium Fervidobacterium pennivorans
Ven5, which was cloned and expressed in E. coli. In contrast to pullulanase type I1
from P.woesei (specific to both a-1,Gand a-1,4 glycosidic linkages) the enzyme from
F. pennivorans Ven5 attacks exclusively the a-1,G-glycosidic linkages in polysaccha-
rides. This thermostable debranching enzyme leads to the formation of long-chain
linear polysaccharides from amylopectin IL4'1.
Thermostable cyclodextrin glycosyltransferases (CGTases) are produced by Ther-
moanaerobacter species, Thermoanaerobacterium thermosulfirigenes and Anaerobranca
g0ttschalkii1~"'~1. Recently, a CGTase, with optimal temperature at 100 "C, waspur-
ified from a newly isolated Archaeon, Thermococcus sp. This is the first report of the
presence of a thermostable CGTase in a hyperthermophilic Ar~haeon[~']. The
enzyme from this strain has been cloned and sequenced. The gene of 2217
nucleotides encodes a protein with an MW of 83 kDa. The ability of extreme
thermophiles and hyperthermophiles to produce heat-stable glycosyl hydrolases is
summarised in Table 10-2.
The finding of extremely thermophilic Bacteria and Archaea capable of producing
novel thermostable starch-hydrolyzing enzymes is a valuable contribution to the
starch-processing industry. By using robust starch-modifylngenzymes from thermo-
philes, innovative and environmentally friendly processes can be developed, aiming
at the formation of products of high added value for the food industry. New and
enhanced functionality can be obtained by changing the structural properties of
starch. In order to prevent retrogradation, starch-modifyingenzymes can be used at
higher temperatures. The use of the extremely thermostable amylolytic enzymes can
Table 10-2. Starch hydrolyzing enzymes from extreme thermophilic and hyperthermophilicArchaea and Bacteria.
Enzyme properties
Enzyme Organism' Optimal Optimal Mw Remarks
temperature pH Wa)
a-Amylase Desulfurococcus m u c o s ~ s [ ~ ~ l 100 5.0 - Purified/cloned
100 6.5-7.5 129 Purified/cloned/intracellular
100 7.0 68 Purified/cloned/extracellular
Pyrococcus sp. KODl 90 6.5 49.5 Purified/cloned/extracellular
Pyrococcus woesi['OO1 100 5.5 68 Purified/Extracellular
Pyrodictium abyssi["I 100 5.0 - Crude extractb
StaphylothemtusmarinusIg01 100 5.5 - Crude extract
Sulfolobussolfataricus1881 - - 240 Extracellular
Trtemococcus ceIeP51 90 5.5 - Crude extract
Thermococcus projkndus DT54321"I 80 5.5 42 Purified/doned/"Amy S"
Thermococcus projkndus[801 80 4.0-5.0 42 Purified/"Amy L"
Therrnococcus aggregan~[~~l 95 6.5 - Cloned
Dyctyoglomusthemtophilum Rt46B.1[731 90 5.5 75 Purified/cloned/cytoplasmic fraction d
9
Themtotoga mantima MSB8["I 85-90 7.0 61 Purified/cloned/lipoprotein h,
Pullulanase type I Fervidobacteriumpennavorans Ven517*1 80 6 190 (93) Purified/cloned i?

3
nemotoga mantima MSB81"I 90 6.0 93 (subunit) Cloned/typs Ib
Thermus caldophilus GK24[751 75 5.5 65 Purified/cell associated
:
a
74 Purified/doned
a
2.
Pullulanase type 11 Desul&rococcus ~UCOSUS~~~] 85 5.5
2
pyrococcus woesei['OOl 100 6.0 90 Purified/cloned/cell associated T
Pyrodictium abyssi["] 100 9.0 -
Crude extract 3w
W
0
N
-
u
Table 10-2. (cont.). 0
Enzyme properties 2
!
Enzyme Organism" Optimal Optimal Mw Remarks -c
3
temperature pH (kW a
Thermococcus celerlss1 90 5.5 - Crude extract
3
3
Themococcus lit~ralis['~l 98 5.5 119 Purified/extracell./glycoprotein 9
a
Therrnococcus hydrothermalis~80] 95 5.5 128 Purified/extracell./glycoprotein 2
Pullulan-hydrolasetyp 111 Thermococcus aggregan~['~] 100 6.5 83 Purified/cloned
Glucoamylase Themtoplasma acidophilurn["l 90 6.5 141 Purified
Picrophilus oshimae["l 90 2.0 140 Purified
Picrophilus torridus["] 90 2.0 133 Purified 3
n
4
CGTase Themtococcus SP.['~] 100 7.0 83 Purified I
Thermoanaerobacterium 80 4.0-4.5 68 Purified/cloned/crystallized $
thermosulfurigenes[601 z
Anaerobranca g~ttaschallkii['~~ 70 8.0 6G Purified
3
a-Glucosidase Thermococcus strain A N ~ I ~ ~ I 130 - 63 Purified/extracell./glycoprotein
s-
6.
b
Thermococcushydrothermalis["] - - - Cloned 3
s
a Values in brackets give the optimal growth temperamre for each organism in 'C
9
b Unpublished results; - not determined n
4
10.3 Cellulose-Hydrolyzing Enzymes
I
321
lead to valuable products, which include innovative starch-based materials with
gelatin-like characteristics and defined linear dextrins that can be used as fat
substitutes, texturizers, aroma stabilizers and prebiotics. CGTases are used for the
production of cyclodextrins that can be used as a gelling, thickening or stabilizing
agent in jelly desserts, dressing, confectionery, dairy and meat products. Because of
the ability of cyclodextrins to form inclusion complexes with a variety of organic
molecules, they improve the solubility of hydrophobic compounds in aqueous
solution. This is of interest for the pharmaceutical and cosmetic industries['', 'l1.
Cyclodextrin production is a multistage process in which starch is first liquefied by a
heat-stable amylase, and in the second step a less-thermostable CGTase from Bacillus
sp. is used. The application of heat-stable CGTase in jet cooking, where temperatures
up to 105 "C are achieved, will allow liquefaction and cyclization to take place in one
step.
10.3
Cellulose-Hydrolyzing Enzymes
Cellulose commonly accounts for up to 40% of the plant biomass. It consists of

glucose units linked by P-1,Cglycosidicbonds with a polymerization grade of up to
15000 glucose units in a linear mode. Although cellulose has a high affinity to water,
it is completely insoluble. Natural cellulose compounds are structurally heteroge-
neous and have both amorphous and highly ordered crystalline regions. The degree
of crystallinity depends on the source of the cellulose, and the more highly crystalline
regions are more resistant to enzymatic hydrolysis. Cellulose can be hydrolyzed into
glucose by the synergistic action of at least three different enzymes: endoglucanase,
exoglucanase (cellobiohydrolase)and P-glucosidase. Synonyms for cellulases (E. C.
3.2.1.4) are P-l,4-~-glucanglucano-hydrolases, endo-f3-1,4-glucanasesor carbox-
ymethyl cellulases. This enzyme is an endoglucanase which hydrolyzes cellulose in a
random manner as endo-hydrolase producing various oligosaccharides, cellobiose
and glucose. The enzyme catalyzes the hydrolysis of P-1,4-~-glycosidic linkages in
cellulose but can also hydrolyze 1,Clinkages in P-D-glucans containing 1,3-linkages.
Exoglucanases, P-1,4-cellobiosidases, exocellobiohydrolases or P-1,kellobiohy-
drolases (E. C. 3.2.1.91) hydrolyze P-l,4-~-glycosidic linkages in cellulose and cellote-
traose, releasing cellobiose from the non-reducing end of the chain.
P-Glucosidases (E. C. 3.2.1.21), gentobiases, cellobiases or amygdalases catalyze
the hydrolysis of terminal, non-reducing P-D-glucoseresidues releasing 0-D-glucose.
These enzymes have a wide specificity for P-D-glucosides. They are able to hydrolyze
P-D-galactosides,P-L-arabinosides,P-D-xylosides, and P-D-fucosides.
10.3.1
Thermostable Cellulases
Thermostable cellulases active towards crystalline cellulose are of great biotechno-

logical interest. Several cellulose-degrading enzymes from various thermophilic
322
I 10 Enzymesfrom Extreme Thermophilic and Hyperthermophilic Archaea and Bacteria
organisms have been cloned, purified, and characterized. A thermostable cellulase

from Thermotoga maritima MSB8 has been ~haracterized1~~1. The enzyme is rather
small, with a molecular weight (MW)of 27 kDa, and is optimally active at 95 "C and
between pH 6.0 and 7.0. Two thermostable endocellulases, CelA and CelB, were
purified from Thermotoga neapolitana. CelA (MW of 29 kDa) is optimally active at pH
6 at 95 "C, while CelB (MW of 30 kDa) has a broader optimal pH range (pH 6 to 6.6)
at 106 "C. The genes encoding these two endocellulases have been
Cellulase and hemicellulase genes have been found clustered together on the
genome of the thennophilic anaerobic bacterium Caldocellum saccharolyticum, which
grows on cellulose and hemicellulose as sole carbon sources. The gene for one of the
cellulases (celA)was isolated and was found to consist of 1751 amino acids. This is
the largest cellulase gene described to
A large cellulolytic enzyme (CelA) with the ability to hydrolyze microcrystalline
cellulose was isolated from the extremely thermophilic bacterium Anaerocellum
t h e r m o p h i l ~ m [The
~ ~ ]enzyme
. has an apparent molecular weight of 230 kDa, exhibits
significant activity towards Avicel and is most active towards soluble substrates such
as CM-cellulose (CMC) and P-glucan. Maximal activity was observed at pH 5-6 and
85-95 "C. The thennostable exoacting cellobiohydrolase from Thermotoga maritima
MSB8 has an MW of 29 kDa and is optimally active at 95 "C at pH 6.0-7.5 with a half-
life of 2 h at 95 "C. The enzyme hydrolyzes Avicel, CM-Cellulose and b-glucan
forming cellobiose and cellotriose. A thermostable cellobiase is produced by Thermo-
toga sp. FjSS3-Bl The enzyme is highly thermostable and shows maximal activity
at 115 "C at pH 6.8-7.8. The thermostability of this enzyme is salt dependent. This
cellobiase is active against amorphous cellulose and CM-cellulose.
Recently, a thermostable endoglucanase, which is capable of degrading P-1,4
bonds of p-glucans and cellulose, has been identified in the Archaeon Pyrococcus
firiosus. The gene encoding this enzyme has been cloned and sequenced in E. coli
and has significant amino acid sequence similarities with endoglucanases from
glucosyl hydrolases family 12. The purified recombinant endoglucanase hydrolyzes
P-1,4- but not P-1,3-glycosidiclinkages and has the highest specific activity with
cellopentaose and cellohexaose as substrates 15'1. In contrast to this, several 0-
glucosidases have been detected in Archaea. In fact, archaeal P-glucosidases have
been found in Sulfolobus solfataricus MT4, S. acidocaldarius, S. shibatae and P.
JiLriosus[s8-"1. The enzyme from the latter microorganism (homotetramer, 56 kDa/
subunit) is very stable and shows optimal activity at 102 "C to 105 "C with a half-life
of 3.5 days at 100 "C and 13 h at 110 oC[60].The P-glucosidase from S. solfataricus
MT4 has been purified and The enzyme is a homotetramer (56
kDa/subunit) and very resistant to various denaturants with activity up to 85 "C["l.
The gene for this P-glucosidase has been cloned and overexpressed in E. c01i[63-6s1
(Table 10-3).
Cellulose-hydrolyzing enzymes are widespread in Fungi and Bacteria. Less ther-
moactive cellulases have already found various biotechnological applications. The
most effective enzyme of commercial interest is the cellulase produced by Tricho-
derma sp. [66]. Cellulolytic enzymes can be used in alcohol production to improve
juice yields and effective color extraction of juices. The presence of cellulases in
Table 10-3. Production o f thermoactive cellulases (exoglucanase,B-glycosidase),xylanases (endoxylanase)and chitinase by some representatives of extreme
thermophilic and hvpertherrnoDhilicArchaea and Bacteria.
Enzyme properties
Enzyme Organism" Optimal Optimal Mw Remarks
temperature pH (kW
Endoglucanase Thermotoga mantima MSB8[801 95 6.0-7.5 27 Purified/cloned/cellulase I
Thermotoga neapolitana[801 95 6.0 29 Purified/cloned/Cell A
106 6.0-6.5 30 Purified/cloned/Cell B
Exoglucanase Thermotoga mantima MSB8 95 6.0-7.5 29 Purified/cellulase I1
Thermotoga sp. strain FjSS3-B.1(801 115 6.S7.8 36 Purified/cell-associated
Anaerocellum thermophilum Rt46B. lllool 85 6.5 31 Cloned
Pyrococcus&riosus['Oo1 100 6.0 35.9 Cloned
P-Glycosidase Pyrococcus&riosusl'ool 102-105 - 230/58 Purified/cloned
SulfolobussoEfataricus[881 105 5.3 240/56 Purified/cloned
Thermotoga mantima MSB8 75 6.2 95(47) Purified/cloned
Thermotoga sp. strain FjSS3-B.1[801 80 7.0 lOO(75) Purified/ toga-associated
Endoxylanases Pyrodictium a b y s ~ i [ ~ ~ l 110 5.5 - Crude extract
Dyctyoglomus thermophilum Rt46B.1[73] 85 6.5 31 Purified/cloned
Thermotoga mantima MSB8[801 92 6.2 120 Pur./toga associated/XynA d
105 5.4 40 Pur./toga-associated/XynB LJ

P
Thermotoga sp. strain FjSS3-B.1[80,85] 105 5.3 31 Pur./cloned/ toga-associated
85 6.3 40
2-
Pur./cloned E
Thermotoga neapolitana[801 85 5.5 37 Purified 2
95 5.5-6.0 119 Purified/cloned -=
:
SL
Thermotoga thermar~m['~I 80 6.0 105/150 Pur./toga-associated/Endoxylanase1 a
r'
90-100 7.0 35 Pur./Endoxylanase 2 5.
Chitinase Pyrococcus kodakaraensi~['~] 85 5.0 135 Purified/cloned 4
9
a Values in the brackets give the optimal growth temperature for each organism in "C -z
- not determined 3
2
-
W
W
N
324
I 70 Enzymesfrom Extreme Jhermophilic and Hyperthermophilic Archaea and Bacteria
detergents causes color brightening, softening and improvement of particulate soil

removal. Cellulase (Denimax@Novo Nordisk) is also used for the “biostoning” of
jeans instead of using stones. Other applications of cellulases include the pre-
treatment of cellulosic biomass and forage crops to improve nutritional quality and
digestibility, enzymatic saccharification of agricultural and industrial wastes and
production of fine chemicals.
10.4
Xylan-Degrading Enzymes
Xylan is a heterogeneous molecule that constitutes the main polymeric compound of

hemicellulose, a fraction of the plant cell wall which is a major reservoir of fked
carbon in nature. The main chain of the heteropolymer is composed of xylose
residues linked by P-1,4-glycosidicbonds. Approximately half of the xylose residues
have substitution at 0 - 2 or 0 - 3 positions with acetyl, arabinosyl and glucuronosyl
groups. The complete degradation of xylan requires the action of several enzymes
(for a detailed description see reviews[67] The endo-P-1,4-xylanase (E. C.
3.2.1.8), or P-1,4-xylanxylanohydrolase,hydrolyzes P-1,4-xylosydiclinkages in xylans,
while P-1,4-xylosidase,P-xylosidase, P-1,4-xylan xylohydrolase, xylobiase or exo-p-
1,4-xylosidase(E. C. 3.2.1.37) hydrolyzes P-1,4-xylansand xylobiose by removing the
successive xylose residues from the non-reducing termini. a-Arabinofuranosidase or
arabinosidase (E. C. 3.2.1.55) hydrolyzes the terminal non-reducing a-r-arabinofur-
anoside residues in a-L-arabinosides.The enzyme also acts on a-L-arabinofurano-
sides [a-L-arabinanscontaining either (1,3) or (1,5)-linkages].Glucuronoarabinox-
ylan endo-P-1,4-xylanase,feraxan endoxylanase or glucuronoarabinoxylanP-1,4-xyla-
nohydrolase (E. C. 3.2.1.136) attacks ~-1,4-xylosyllinkages in some glucuronoar-
abinoxylans. This enzyme also shows high activity toward femloylated arabinoxylans
from cereal plant cell walls. Acetyl xylan esterase (E. C. 3.1.1.6) removes acetyl groups
from xylan.
10.4.1
Thermostable Xylanases
So far, only a few extreme thermophilic microorganisms are able to grow on xylan
and secrete thermoactive xylanolybc enzymes (Table 10-3). Members of the order
Thermotogales and Dictyoglomus themophilum Rt46B. 1 have been described to
produce xylanases that are active and stable at high temperatures. The most
thermostable endoxylanases that have been described so far are those derived from
Themtotoga sp. strain FjSS3-B.l, Themtotoga maritima, T. neapolitana and T. ther-
marum. These enzymes, which are active between 80 and 105 “C, are mainly cell-
associated and most probably localized within the toga. Several genes encoding
xylanases have already been cloned and sequenced. The gene from T. maritima,
encoding a thermostable xylanase, has been cloned and expressed in E. coli.
Comparison between the T. maritima recombinant xylanase and the commercially
70.5 Chitin Degradation
I 325
available enzyme, PulpenzymeTMindicates that the thermostable xylanase could be
of interest for application in the pulp and paper industry. A xylanase has been found
in the Archaeon Thermococcus zilligii strain AN1, which grows optimally at 75 "C.
The enzyme has a molecular weight of 95 kDa and a unique N-terminal se-
q~ence["~'1. The pH optimum for activity is 6.0, and the half-life at 100 "C is 8 min.
Another archaeal xylanase with a temperature optimum of 110 "C was found in the
hyperthermophilic Archaeon Pyrodictiurn abyssi.
Xylanases from Bacteria have a wide range of potential biotechnological applica-
tions. They are already produced on an industrial scale and are used as food additives
in poultry, for increasing feed efficiency diets [7G. 771 and in wheat flour for improving
dough handling and the quality of baked In recent years, the major
interest in thermostable xylanases is found in enzyme-aided bleaching of paper [791.
More than 2 million tons of chlorine and chlorine derivatives are used annually in
the United States for pulp bleaching. The chlorinated lignin derivatives generated by
this process constitute a major environmental problem caused by the pulp and paper
industry[79].Recent investigations have demonstrated the feasibility of enzymatic
treatment as an alternative to chlorine bleaching for the removal of residual lignin
from pulp[8o].Treatment of craft pulp with xylanase leads to a release of xylan and
residual lignin without undue loss of other pulp components. Xylanase treatment at
elevated temperatures opens up the cell wall structure, thereby facilitating lignin
removal in subsequent bleaching stages. Xylanases from moderate thermophilic
microorganisms are rapidly denatured at temperatures above 70 "C. Several of the
non-chlorine bleaching stages used in commercial operations are performed well
above this temperature; consequently,the pulp must be cooled before treatment with
the available enzymes and reheated for subsequent processing steps c7'1.
10.5
Chitin Degradation
Chitin is a linear p-1,4homopolymer of N-acetyl-glucosamineresidues and is one of

the most abundant natural biopolymers on earth. Particularly in the marine
environment, chitin is produced in enormous amounts, and its turnover is due to the
action of chitinolytic enzymes. Chitin is the major structural component of most
fungi and invertebratesLs1. 8 2 ] , while for soil or marine Bacteria chitin serves as a
nutrient. Chitin degradation is known to proceed with the endo-acting chitin
hydrolase (chitinase A: E. C. 3.2.1.14) and the chitin oligomer-degrading exo-acting
hydrolases (chitinase B) and N-acetyl-D-glycosaminidase (trivial name: chitobiase;
E.C. 3.2.1.52).
Chitobiase degrades only small N-acetyl-D-glucosamineoligomers (up to penta-
mers), and the released N-acetyl-D-glucosaminemonomers retain their C1 anomeric
configuration.
Chitin and its derivatives exhibit interesting properties that make them a valuable
raw material for several applications[83-871.It has been estimated that the annual
world-wide formation rate and steady state amount of chitin is in the order of lo1' to
326
I 10 Enzymesfram Extreme Thermophilic and Hyperthermophilic Archaea and Bacteria
lo1' tons per year. Therefore, application of thermostable chitin-hydrolyzing en-

zymes (chitinases) is expected for effective utilization of this abundant biomass.
Although a large number of chitin-hydrolyzingenzymes have been isolated and their
corresponding genes have been cloned and characterized, only few thermostable
chitin-hydrolyzingenzymes are known. These enzymes have been isolated from the
thermophilic microorganisms Bacillus lichenformis X - ~ U ,Bacillus sp. BG-11 and
Streptomyces thennoviolaceus OPC-520[", 891.
The extreme thermophilic anaerobic Archeon Thermococcus chitonophagus has
been reported to hydrolyze chitin I.'[ This is the first extremophilic Archaeon which
produces chitinase(s) and N-acetylglucosaminidase(s);however, sequence and struc-
tural information for archaeal chitinases have not yet been reported. Very recently,
the gene encoding a chitinase from a hyperthermophilic archaeon Pyrococcus
kodakaraensis KODl was cloned, sequenced and expressed in E. coli. The purified
recombinant protein is optimally active at 85 "C and pH 5.0. The enzyme produces
chitobiose as the major end product (Table 10-3).
10.6
Proteolytic Enzymes
Proteins are the most abundant organic molecules in living cells and constitute more
than 50% of their dry weight. The molecular weight of proteins that are made up of
one or more polypeptide chains can vary from a few thousands to more than one
million daltons. All proteins are constructed from a basic set of 20 amino acids that
are covalently linked by peptide bonds. The three-dimensional conformation of
proteins may vary. Globular proteins (spherical or globular) are soluble and usually
have dynamic function. Fibrous proteins on the other hand occur as sheets or rods,
are insoluble and serve as structural elements. The enzymes which hydrolyze the
peptide bonds in proteins are defined as proteases. They are also called endopepti-
dases because they hydrolyze peptide bonds inside the polypeptide chain. Exopepti-
dases (either carboxypeptidases or aminopeptidases) on the other hand can split off
the terminal residues of the polypeptide chain. Proteases (endopeptidases) play an
important role in the utilization of proteins by various microbes. They are classified
into four groups depending on the nature of their active center.
I. Serine proteases have a serine residue in their active center and are inhibited by
DFP (diisopropylphosphofluoride)and PM SF (phenylmethylsulfonylfluoride).
11. Cysteine proteases have a SH groups in their active center and are inhibited by
thiol reagents, heavy metal ions, alkylating agents and oxidizing agents.
111. The activity of metal proteases depends on tightly bound divalent cations. They
are inactivated by chelating agents.
IV. Aspartic proteases (acid proteases) are rare in Bacteria and contain one or more
aspartic acid residues in their active center. Inactivation of the enzyme can be
achieved by alkylation of the aspartic acid residues with DAN (diazoacetyl-DL-
norleucine methyl ester) [911.
10.6 froteolytic Enzymes
I 327
10.6.1
Stable proteases
A variety of heat-stable proteases have been identified in hyperthermophilic Archaea

belonging to the genera Desulfirrococcus, Sulfolobus, Staphylothemus, Themococcus,
Pyrobaculurn and Pyrococcus. It has been found that most proteases from ex-
tremophiles belong to the serine type and are stable at high temperatures even in the
presence of high concentrations of detergents and denaturing agents (Table 10-4).A
heat-stable serine protease was isolated from cell-free supernatants of the hyper-
thermophilic Archaeon Desulfirrococcus strain Tok12S1 A cell-associated serine
protease was characterized from Desulfirococcus strain SY that showed a half-life of
4.3 h at 95 oC[93].A globular serine protease from Staphylothemus marinus was
found to be extremely thermostable. This enzyme, which is bound to the stalk of
filiform glycoprotein complex, named tetrabrachion, has a residual activity even at
135 "C after 10 min of incubati~n['~I. The properties of extracellular serine proteases
from a number of Themococcus species have been analyzed["]. The extracellular
enzyme from T. stetteri has a molecular weight of 68 kDa and is highly stable and
resistant to chemical denaturation, as illustrated by a half-life of 2.5 h at 100 "C and
retention of 70 % of its activity in the presence of 1% SDS (961. A novel intracellular
serine protease (perinilase) from the aerobic hyperthermophilic Archaeon Aero-
pyrurn pernix K 1 was purified and characterized. At 90 "C, the pernilase has a broad
pH profile and an optimum at pH 9.0 for peptide hydrolysis. Several proteases from
hyperthermophiles have been cloned and sequenced, but in general their expression
in a mesophilic host is difficult. A gene encoding a subtilisin-like serine protease,
named aereolysin, has been cloned from Pyrobaculurn aerophilurn, and the protein
was modeled based on structures of subtilisin-typeproteases [971. Multiple proteolytic
activities have been observed in P. firriosus. The cell-envelope associated serine
protease of P.firriosus, called pyrolysin, was found to be highly stable, with a half-life
of 20 min at 105 oC[981.The pyrolysin gene was cloned and sequenced, and it was
shown that this enzyme is a subtilisin-likeserine protease["]. A serine protease from
Aqu@x pyrophilus was cloned and weakly expressed in E. coli. The activity of the
enzyme was highest at 85 "C and pH 9. The half-life of the protein (G h at 105 "C)
makes it one of the most heat-stable proteases known to date.
Proteases have also been characterized from the thermoacidophilic Archaea
Sulfolobus solfataricus and S. acidocaldarius. In addition to the serine proteases, other
types of enzymes have been identified in extremophiles: a thiol protease from
Pyrococcus sp. KOD1, a propylpeptidase (PEPase)and a new type of protease from P.
firiosus. An extracellular protease, which is designated aeropyrolysin, was purified
from Aeropyrurn pernix K 1 (JCM 9820). The enzyme activity is completely inhibited
by EDTA and EGTA, indicating that it is a metalloprotease. The enzyme is highly
resistant to denaturing reagents and highly thermostable, showing a half-life of 2.5 h
at 120 "C and 1.2 h at 125 "C in the presence of 1mM CaC12. These results indicate
that this enzyme is one of the most thermostable extracellular metallo-proteases
reported to date. Thermostable serine proteases were also detected in a number of
extreme thermophilic Bacteria belonging to the genera Themotoga and Fervido-
W
00
l4
Table 10-4. Properties of thermoactive proteolytic enzymes from extreme thermophilic and hyperthermophilicArchaea and Bacteria. u
0
Enzyme properties 9
Enzyme Organism" Optimal Optimal Mw Remarks -c
3
temperature pH (kW a
Serine protease Desulfurococcus mucosus 95 7.5 52 Purified
Pyrococcusfuriosus[loo] 85 6.3 124(29) Protease I/purified B
105/80 Pyrolysin/pur./cloned $m
Pyrobaculum aerophilum 19'1 - - - Cloned 2
Themtococcus aggregans L7'1 90 7.0 - Crude extract
Themtococcus celerI8'1 95 7.5 - Crude extract '"z
- Crude extract
-_
-6'
Themtococcuslitoralis[901 95 9.5
Q
3
Themtococcus stetteri [751 85 8.5 68 Pur./doned z
a
Staphylothermus marinus [901 9.0 140 Stable up to 135 "C
Sulfolobus solfataricuds8] - 6.5-8 118(52) Purified
4z
Aeropyrurn Pernix K1 I9O1 90 9.0 50 Purified
Aquij%xpyrophilus19001 85 7.0-9.0 43 Purified a-2
6'
Fervidobacterium pennavorans I7O1 80 10 130 Purified/keratin hydrolysis b
Thermobacteroidesproteolyticus I1' 85 9.0-9.5 - $-
Crude extract f!
s
Thiol protease Pyrococcus sp. KODl [')'I 110 7 44 Purified Q
Acidic protease Sulfolobus acidocaldariu~[~~] 90 2.0 - Cloned

a
m
- - 0
Aminopeptidase I Solfolobus solfataricus[881 > 450 Crude extract H
Aminopeptidase I1 - - 170 Crude extract 2.
Endopeptidase I, 11,111 - - 115,32,27 Crude extract

Carboxypeptidase - - 160 Crude extract
a Values in the brackets give the optimal growth temperature for each organism in "C
- not determined
10.7 lntracellular Enzymes
I
329
bacterium (unpublished results). The enzyme system from Fervidobacteriurn penni-
vorans is able to hydrolyze feather keratin forming amino acids and peptides. The
enzyme is optimally active at 80°C and pH 10.0['09].The amount of proteolytic
enzymes produced worldwide on a commercial scale exceeds that of the other
biotechnological enzymes used. Heat-stable proteases have great potential for
various applications including the textile and pharmaceutical industries. Serine
alkaline proteases are currently used as additives to household detergents for
laundering, where they have to resist denaturation by detergents and alkaline
conditions. Proteases showing high keratinolytic and elastolyhc activities are used
for soaking in the leather industry. Proteases are also used as catalysts for peptide
synthesis using their reverse reaction [100-1091.
10.7
lntracellular Enzymes
A number of intracellular enzymes from extreme thermophilic and hyperthermo-

philic microorganisms have been investigated. The majority of the intracellular
enzymes known to date show slightly less thermostability than the extracellular
enzymes. Some of these enzymes, which have been characterized from Archaea
belonging to the order Sulfolobalses and Themococcales, include alcohol dehy-
drogenase, glucose dehydrogenase, glyceraldehyde-3-phospho-dehydrogenase,
NADH dehydrogenase, j3-galactosidase, citrate synthase, malic enzyme, fumarase, s-
adenosylmethionine synthetase, ATPase, ATP sulfurylase, aspartate aminotransfer-
ase, DNA polymerase, RNA polymerase, topoisomerase and polyphosphate kinase
(for review Other reports are available on extremely thermoactive intra-
cellular enzymes that are even active above 100 "C. Glyceraldehyde-3-phosphate
dehydrogenase from the Archaeon P. woesei was characterized and the gene was
cloned in E. coli. This enzyme is strictly phosphate dependent and utilizes either
NAD+ or NADP+; the half-life ofthe enzyme at 100 "C is 4 4 min['lO].The amino acid
composition of glyceraldehyde-3-phosphate dehydrogenase from P. woesei was
determined and compared with mesophilic and thermophilic Archaea. The primary
structure of this enzyme exhibits a high proportion of aromatic amino acid residues
and a low proportion of sulfur-containing residues. The glutamate dehydrogenase
(GDH)from P. woesei and P.firriosus was purified and characterized. This enzyme is
probably involved in the first step of nitrogen metabolism. GDH from P. woesei was
purified in a single-affinity chromatography step['"]. It utilizes both NAD' and
NADP+ as cofactors with a preference for the phosphorylated form. The purified
enzyme from both strains is a hexamer with identical subunits of 45 kDa
each['1*, "31 .Twenty-four N-terminal residues of GDH were determined and used to
construct gene-specific DNA probes via the polymerase chain reaction ["'I. The
GDH gene was cloned in E. coli. Its nucleotide sequence and amino acid composi-
tion were determined. A highly thermoactive glucose isomerase with maximal
enzymatic activity at 105 "C was purified from T. maritima and ~haracterized["~].
This enzyme could play an important role in the industrial bioconversion of glucose
330
I 70 Enzymesfrom Extreme Thermophilic and Hyperthermophilic Archaea and BaGteria
to fructose. Other remarkable thermoactive enzymes such as hydrogena~e["~],

aldehyde ferredoxin oxidoreductase[1'6],and acetyl-Co A synthetase (ADP forming)
were detected in P . ~ r i o s u ~ ~ ' ~ ~ - ' ~ ~ ] .
DNA polymerases (E. C. 2.7.7.7.) are other important intracellular enzymes that
play a key role in the replication of cellular information present in all life forms. They
catalyze, in the presence of Mg2+ ions, the addition of a deoxyribonucleoside
5'-triphosphate onto the growing 3'-OH end of a primer strand, forming com-
plementary base pairs to a second strand. Thermostable DNA polymerases play a
major role in a variety of molecular biological applications, e. g. DNA amplification,
sequencing or labeling. More than 100 DNA polymerase genes have been cloned and
sequenced from various organisms, including thermophilic Bacteria and Archaea.
One of the most important advances in molecular biology during the last ten years is
the development of a polymerase chain reaction (PCR)['20-1221 . The PCR procedure
first described utilized the Klenow fragment of E. coli DNA polymerase I, which was
heat labile and had to be added during each cycle following the denaturation and
primary hybridization steps. Introduction of thermostable DNA polymerases in PCR
facilitated the automation of the thermal cycling part of the procedure. The DNA
polymerase I from the bacterium Themus aquaticus, called Taq polymerase, was the
first thermostable DNA polymerase characterized[123, 1241 and applied in PCR.
A thermostable DNA polymerase from Themotoga maritimal'251was reported to
have a 3'-5'-exonudease Archaeal proofreading polymerases such as Pwo
pol [1271 from Pyrococcus woesei[128],Pfi pol [1291fromPyrococcus f i r i o s ~ s ~ 'Deep
~~~,
VentTM from Pyrococcus strain GB-D[132] or Vent: 1341 from T h e m o -
coccus litorali~['~~]have an error rate that is up to ten times lower than that of Taq
polymerase. The 9"N-7 DNA polymerase from Themococcus sp. strain 9"N-7 has a
fivefold higher 3'-5'-exonuclease activity than T. litoralis DNA polymerase [1361.
However, Taq polymerase was not replaced by these DNA polymerases because of
their low extension rates, among other factors. DNA polymerases with higher fidelity
are not necessarily suitable for amplification of long DNA fragments because of their
potentially strong exonuclease activity [1371. The recombinant KODl DNA polymera-
se from Pyrococcus sp. strain KODl has been reported to show low error rates
(similar values to those of P'), high processivity (persistence of sequential nucleo-
tide polymerization) and high extension rates, resulting in an accurate amplification
of target DNA sequences up to 6 kb[1381.In order to optimize the delicate competi-
tion of polymerase and exonuclease activity, the exo-motif 1 of the 9"N-7 DNA
polymerase was mutated in an attempt to reduce the level of exonuclease activity
without totally eliminating it [13'. 139. l4Ol. Similarly, the PCR performance was
optimized by site-directed mutagenesis of the DNA binding motif of the DNA
polymerase from Themococcus aggregans and Sulfolobus solfataricus['41].
References I331
References
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ular, and Applied Microbiology. T. D. Brock J. Bid. Chem. 1997,272, 16335-16342.
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11
Hydrolysis and Formation of C - 0 Bonds
11.1
Hydrolysis and Formation of Carboxylid Acid Esters
HansJoachirn Gais and Fritz Theil
Catalysis of the hydrolysis and formation of the C - 0 bond of an ester, lactone or

carbonate by hydrolases are amongst the most useful enzyme-catalyzed reactions in
organic synthesis ( i n vitro) Today hydrolases are established tools for organic
synthesis on a laboratory scale as well as on a industrial scale L3’1, The reason for this
lies in the nature of hydrolases. Hydrolases are chiral catalysts, which are easy to
function without a coenzyme, are commercially available in quite a
number and and frequently feature low substrate specificity, high
enantiotopic selectivity, and enantiomer selectivity. In addition, directed evolution,
chemical modification and most importantly site-directedmutagenesis allow for the
attainment of enzymes with improved activity, selectivity and stability, in particular
toward organic solvents [35f]. This adds considerably to the versatility of hydrolases.
The most important application of hydrolases lies in the field of asymmetric
synthesis, which is therefore solely dealed with in this chapter. For the application of
hydrolases in chemo- and regioselective transformations and in particular in
protecting group chemistry, see Chapter 18. Among the hydrolases, the most widely
used are, in first place, the lipases (E. C. 3.1.1.3),as for example pig pancreas lipase,
Pseudornonas sp. lipases and Candida antarctica lipase (see Sect. 11.1.1.1.5, Tables
11.1-10to 11.1-25), in second place the carboxylesterhydrolases (E. C. 3.1.1.1), as for
example pig liver esterase (see Sect. 11.1.1.1.1., Tables 11.1-1 to 11.1-6 and Sect.
11.1.1.2.3, Table 11.1-27),and in third place the proteases (E.C. 3.4.m.n), as for
example subtilisin (see Sect. 11.1.1.1.4, Table 11.1-8 and Sect. 11.1.1.2.2, Table
11.1-26)and a-chymotrypsin (see Sect. 11.1.1.1.2, Table 11.1-7).Today, lipases are the
most versatile hydrolases, primarily because of their ability to be highly active not
only in water but also in water in the presence of an organic cosolvent and, most
importantly, even in organic solvents of low water content. Another reason for the
versatility of lipases is their accessibility in quite large numbers (see Sect. C). Some
confusion has developed in the literature concerning the origin and names of some
336
I 1 1 Hydrolysis and Formation ofC-0 Bonds
CO,H Scheme 11.1-1.

C0,Me
pig liver esterase Enantiotopos-differ-
entiating hydrolysis
C0,Me HzO, PH 7 C0,Me of dicarboxylic
diesters.
[37-42, 271
298% ee, 98% yield
Ph Ph
1, C0,Me a-chymotrypsin
c
I..'ACO,H
"AC0,Me
Me HZO, PH 7 Me C0,Me
[431
298% ee, 95% yield
microbial lipases; this topic is dealt with in Sect. 11.1.1.1.5. Appropriate substrates
for hydrolases are principally those compounds which bear enantiotopic ester
groups with the prochirality contained either in the dicarboxylic acid (Schemes
11.1-1and 11.1-8)or in the diol part (Schemes 11.1-2 and 11.1-12) ofthe molecule, or
those which carry enantiotopic hydroxyl groups (Schemes 11.1-3 and 11.1-12). A
second and no less important class of substrates is the racemates, as for example
esters of racemic carboxylic acids (Scheme 11.1-4) or esters of racemic alcohols
(Schemes 11.1-5 and 11.1-7),racemic alcohols (Scheme 11.1-6),and racemic hydroxy
carboxylic acid esters (Scheme 11.1-8).
The hydrolase-catalyzedreactions utilized most for the selective transformation of
such substrates are hydrolysis (Schemes 11.1-1,11.1-2, 11.1-4, 11.1-5 and 11.1-11),
acylation (transesterification) (Schemes 11.1-3, 11.1-6 and 11.1-11)and alcoholysis
(transesterification)(Schemes 11.1-7, 11.1-8 and 11.1-15). Hydrolase-catalyzedester-
ification of an alcohol with a carboxylic acid, although highly useful in some cases ["],
has been utilized to a lesser extent. Catalysis of formation and cleavage of the C - 0
bond of an ester or lactone by pig liver esterase, most lipases, a-chymotrypsin and
subtilisin, which are all serine hydrolases, involves the following steps (Scheme
=%:
11.1-9). Formation of an enzyme-substrate complex, attack of the hydroxyl group of
N3,," pig pancreas lipase

H,O,pH7
1441
- N3. I I ,coA OH
91% ee. 85% yield
Pseudomonas cepacia
OAc lipase
OAc HzO,pH7 OAc

145, 461 96% ee, 85% yield
Scheme 11.1-2. Enantiotopos-differentiating hydrolysis of diol diacetates.
7 7 . 7 Hydrolysis and Formation of Carboxylid Acid Esters
I
337
Pseudomonas cepacia lipase
vinyl acetate
- /7/0yoH
uoLOAc
[471 298% ee, 78% yield

*
vinyl acetate
97% ee, 77% yield
*P
HO OAc
1
OCH,Ph OCH,Ph
vinyl acetate
HO
/ "W
HO
197% ee, 89% yield
Scheme 11.1-3. Enantiotopos-differentiating transesteritication of diols.
CI/J3°f02Me
\
CI
~ \o ~ c o z H
87% ee, 49% yield

pig pancreas lipase
+ b +
HzO, PH 7
[50-531
CI
299% ee, 49% yield
Scheme 11.1-4. Enantiotopos-differentiatinghydrolysis o f carboxylic acid esters.
the serine residue in the active site of the enzyme on the carbonyl group of the
substrate or reagent with formation of a covalent acyl-enzyme-productcomplex and
its transformation to the free acyl-enzyme and H-X-R2 [Eq. (l)]["3];reaction of the
acyl-enzymewith a nucleophile, as for example water or an alcohol with formation of
an acyl-enzyme substrate complex, which reacts with deacylation and formation of
another enzyme product complex, which finally gives the free enzyme and the
product [Eq. (2)]. The overall equilibrium, the attainment of which is catalyzed by the
338
I 1 1 Hydrolysis and Formation of C - 0 Bonds
299% ee, 43% yield

Pseudomonas sp. lipase
+ * +
HZO, PH 7
WI
/
SiMe,
298% ee, 46% yield
OAc OH
299% ee, 45% yield
+
Pseudomonas fluorescens lipase
H,O. PH 7
- +
[551
OAc OAc
100% ee, 45% yield
Scheme 11.1-5. Enantiotopos-differentiating hydrolysis of acetates.
enzyme, is depicted in Eq. (3). All steps are in principle reversible. Formation of the
acyl-enzyme and its reaction with a nucleophile involves the enzyme-bound tetra-
hedral intermediates A and B. In these processes a triad of three amino acids of the
active site of the enzyme, Ser, His and Asp(Glu),which are specifically orientated in a
three-dimensional way, together with other amino acids is involved. Crucial to the
catalytic function of the enzyme are, besides the interplay of the residues of these
amino acids, the stabilization of the oxy anion intermediates A and B and the
corresponding transition states through hydrogen bonds provided by amide bonds
or other amino acid residues of the active site.
Hydrolysis of C - 0 bonds of esters and lactones [Eqs. (1)to (3), X = 0 and YR3 =
OH] is usually carried out at room temperature in aqueous solution or in mixtures of
water and either a water-miscible or water-immiscible solvent. Because of the large
excess of water, equilibrium usually is mainly if not completely on the side of the
7 7 . 7 Hydrolysis and Formation ofcarboxylid Acid Esters
Ph
I
339
82% ee, 46% yield

+ +
vinyl acetate
tetrahydrofuran
1561 Ph
297% ee, 39% yield
OH OH
Ph
YPh
295% ee, 43% yield
*
+ - +
vinyl acetate
<
[571
Ph Ph
295% ee, 42% yield
OH OH
297% ee, 44% yield
+ c
+
vinyl acetate
1581
1
OH
Me phy$
Me'
OAc
Me
Scheme 11.1-6. Enantiotopos-differentiating transesterification 297% ee, 26% yield

of alcohols.
340
I 7 1 Hydrolysis and Formation of C-0 Bonds
(11 I
OAc (11I OH
Ph Ph
95% ee, 47% yield

+ +
nPrOH
fed-pentyl alcohol
OAc [591 OAc
fl (‘1
I
OAc (11I
OAc
Ph Ph
Scheme 11.1-7. Enantiotopos-differentiatingalcoholysis
95% ee, 50% yield
of acetates.
Me
nBuOH
-
0 diisopropyl ether
[GO1
93% ee, 90% yield
74% ee
pig pancreas lipase
+ z +
diethyl ether
[Ell
OH
Scheme 11.1-8. Enantiotopos-differentiating lactonization 298% ee

of hydroxy esters.
Rl-CO-X-R2 + enzyme
11.1 Hydrolysis and Formation ofcarbowylid Acid Esters
R1-CO-enzyme + H-Y-R3
I 341
11 11
Rl-CO-X-R2 enzyme R1-CO-enzyme H-Y-R3
11 11
R1-CO-enzyme H-X-R2 Rl-CO-Y-R3 enzyme
11 ci
R1-CO-enzyme + H-X-R2 enzyme + R1-CO-Y-R3
(Eq.1) (Eq. 2)
R1-CO-X-R2 + H-Y-R3 Rl-CO-Y-R3 + H-X-R2 (Eq.3)
-&-Asp-& -&Asp-&
f f
I
H-Nk
k
k
1
H-Nk A
-R1C02H
6
Scheme 11.1-9. Mechanism of hydrolase-catalyzed reaction.
carboxylic acid and the alcohol [Eq. (3), X, Y = 0, R3 = H] and the reaction is
practically irreversible. Low solubility of liquid substrates normally presents no
problem. In fact lipases are designed by nature to work at the liquid-liquid interface.
In the case of crystalline and also liquid substrates of low solubility,solubility may be
342
enhanced by addition of an organic solvent. Most commonly used cosolvents are

alcohols, as for example methanol and tert-butanol, and acetonitrile, acetone,
tetrahydrofuran, ether, dimethylformamide and dimethyl sulfoxide[9, **, 30, 34, 361.
The choice of the cosolvent, miscible or immiscible with water, depends on the
enzyme. Lipases frequently show higher activity and selectivity in an emulsion of
water and tert-butylmethyl ether or diethyl ether than in water. At higher concentra-
tions of the organic solvent, the stability and the activity of the enzyme may be very
low and the presence of the cosolvent can also alter the enantioselectivity of the
enzyme in both directions. Many hydrolases, except lipases, show an interfacial
deactivation. In aqueous solution, the parameters which influence the rate of
hydrolysis most are the pH value and the temperature. In most cases, hydrolysis is
run at room temperature at pH 7.0. However, at lower temperatures the selectivity of
hydrolysis may be higher. For an easy recovery of the enzyme from the aqueous
solution and for other purposes, it can be immobilized by various techni-
q u e ~ [ ~34,
' * 36. 64a1. One of the most popular for laboratory scale synthesis is the
covalent immobilization of the enzyme on Eupergit C, a polymer with reactive
epoxide groups. However, the nature of the polymer and the method of fmation may
greatly influence the stability and activity of the immobilized enzyme. Another very
useful technique, especially for synthesis on a larger scale, is the use of a continuous
flow membrane reactor [64b]. Recovery of the enzyme from aqueous solution in small-
and large-scale batch synthesis can be done by ultrafiltration [64c1. Finally, cross-
linked enzyme crystals (CLECs), which are crystals of a pure enzyme cross-linked
with glutaraldehyde, can be used for this and other purposes because of their
insolubility in water and organic solvents L6&].
Formation of C-0 bonds of esters and lactones is accomplished by exploiting the
transferase activity of hydrolases in acylation and alcoholysis (transesterification)
reactions in organic media of low water content[30,34, 361. Especially lipases show a
high activity in organic solvents, as for example ethers, hydrocarbons, chlorinated
hydrocarbons, vinyl acetate, isopropenyl acetate and ethyl acetate. Lipases are thus by
far the most useful hydrolases for asymmetric synthesis in organic media (Sect.
11.1.1.2.1). Subtilisin (Sect. 11.1.1.2.2.), pig liver esterase (Sect. 11.1.1.2.3) and ct-
chymotrypsin (Sect. 11.1.1.1.2), which also show activity in organic media of low
water content, have been applied only to a lesser extent in asymmetric synthesis
through C - 0 bond formation. Lipases, like other hydrolases, are generally not
soluble in organic solvents. They rather form suspensions of protein aggregates.
Enyzmes are only active if a certain amount of water, which is adsorbed or bound by
the enzyme or dissolved in the organic solvent, is present. Organic solvents with a
high solubility for water are generally not well suited for a hydrolase-catalyzed
transesterification, perhaps because of a dehydration of the enzyme. It is frequently
observed that the enantioselectivity of the acylation of an alcohol, catalyzed by a
lipase, in an organic solvent is higher than the hydrolysis of the corresponding ester
catalyzed by the same lipase in water. To provide for extreme equilibrium positions
in acylation reactions of alcohols catalyzed by lipases in organic media, fatty acid
trifluoro- or trichloroethyl esters, vinyl esters, oxime esters or carboxylic acid
anhydrides have been used with much success. They provide for extreme equilib-
1 7 . 1 Hydrolysis and Formation ofcarboxylid Acid Esten
I 343
rium positions and practically irreversible reactions. Among these acylation rea-
gents, vinyl esters are the most useful. However, the formation of acetaldehyde in
transesterification with vinyl esters may cause a deactivation of the enzyme, most
probably because of a reaction with its lysine amino groups[65,66]. Generally, the
lipases used for the formation of C - 0 bonds under these conditions are crude
preparations which may contain only a few percent of the actual lipase. These crude
lipases usually also contain other proteins (which may be even other enzymes),
additives such as carbohydrates, and salts, which stabilize the enzymes. Usually fair
amounts of these crude materials are used as suspension in organic solvents
together with the acyl transfer reagent. The water content of the system is in most
cases only ill defined, and it can decrease through a competing hydrolase-catalyzed
hydrolysis of the acylation reagent[65,“I. Because of the insolubility of hydrolases in
most organic solvents, catalysis is carried out under heterogeneous conditions,
which restrict not only the mobility of the enzyme but also of the substrate and
products, and can thus cause mass transfer limitations. Immobilization on solid
support, addition of hydrated salts, covalent attachment of methoxpoly(ethy1ene
glycol) (MPEG) residues, lyophilization with organic polymers, cross-linking of
enzyme crystals, sol-gel entrapment, coating with amphiphilic molecules and
entrapment in reverse micelles are the most important techniques that have been
shown to improve the performance of the enzyme[36].Some of these measurements
serve to increase the surface area of the enzyme and thus to enhance its activity.
Among the various types of hydrolase-catalyzed reactions, those involving the
differentiation of enantiotopic ester groups (-COzR), acyloxy groups (-OCOR) and
hydroxyl groups or the differentiation of the enantiomers of esters of racemic
carboxylic acids and racemic alcohols are by far the most important ones. Under
non-equilibrium conditions (e.g. hydrolysis in the presence of a large excess of
water, alcoholysis with a large excess of an alcohol in organic solvents of low water
content, or acylation with a vinyl ester in organic solvents of low water content), the
decisive steps of differentiation are as follows. In the case of differentiation of
enantiotopic ester groups or enantiomeric esters starting with the enzyme it may be
the formation or cleavage of the acyl-enzyme; in the case of enantiotopic acyloxy
groups or enantiomeric esters of racemic alcohols starting with the enzyme it may be
the formation or break-down of intermediate A with generation of the acyl-enzyme;
and in the case of enantiotopic hydroxyl groups or the enantiomers of racemic
alcohols starting with the acyl enzyme it may be the formation or break-down of
intermediate Awith formation of the enzyme.
Numerous rneso-configured or otherwise prochiral substrates, preferentially con-
taining enantiotopic methoxycarbonyl groups, have been converted by a pig liver
esterase- or lipase-catalyzedenantioselectivehydrolysis in water to chiral monoesters
(see Sect. 11.1.1.1.1.,Tables 11.1-1to 11.1-4 and Sect. 11.1.1.1.5, Tables 11.1-10 to
11.1-12). In nearly all cases investigated thus far the pig liver esterase-catalyzed
hydrolysis of the substrate diester S terminates at the stage of the enantiomeric
monoesters P and ent-P. In this case, where the products P and ent-P are not
transformed further, the irreversible enantiotopos-differentiationmay be described
by the process depicted in Scheme 11.1-10[G7“’].
344
I I 1 Hydrolysis and formation ofC-0 Bonds
P Scheme 11.1-10. Hydrolase catalyzed enantiotopos-

differentiating irreversible
S: substrate; P, ent-P: enantiorneric products; k,, kz:
apparent first-order rate constants o f t h e irreversible
process; E: selectivity factor; ee: enantiomeric excess.
enf-P
kl [PI E-1
E=-= ___ ee(P)= __
k2 [ent-P] E+l
Thus, in this case the ee value of the monoester P (or ent-P) is determined by the
selectivity of enantiotopos-differentiation. It is not depended on the extent of the
hydrolysis but only on the ratio of the two apparent first-order rate constants kl and
k2 if one assumes an irreversible reaction and the absence of product inhibition, the
former assumption being reasonable because of the large excess of water. In case of
an insufficient degree of differentiation, selectivity can be raised only by a suitable
temporary or permanent modification of the structure of the substrate S, by choice of
another hydrolase, by addition of an organic solvent or by variation of the tem-
perature or pH value, but not by stopping the reaction at various degrees of
conversion. In practice the situation is somewhat different in the case of meso-
configured or otherwise prochiral diacylated diols having enantiotopic acyloxy
groups. For these diesters, hydrolysis by pig liver esterase and lipases in water
usually does not stop completely at the stage of the enantiomeric monoester P and
ent-P, but proceeds further - although at a significantly lower rate - to the achiral diol
Q (Scheme 11.1-11)[67-G91. Thus enantiotopos-differentiation expressed through the
apparent first-order rate constants kl and k2 is accompanied by an enantiomer-
differentiation as expressed by the apparent first-order rate constants k3 and k4.
In this case the ee value of the monoester P (or eat-P) depends on the extent of the
conversion of the diester S to the monoesters P and ent-P and of the conversion of the
latter to the achiral diol Q, and thus on all four rate constants. From the fact that a
hydrolase usually retains the ( R ) - or (S)-group preference of the enantiotopos
differentiation in the enantiomer-differentiating hydrolysis, i. e. the hydrolysis of the
faster formed monoester P to the diol Q is slower than the hydrolysis of the slower
formed monoester ent-P to the diol Q ( k l > k2 and k4 > k j or vice versa), it follows that
the ee value of the monoester P (or ent-P) can be raised upon carrying the hydrolysis
further to the diol Q, at the expense of the yield. This can be advantageously used to
raise the ee value of the monoester to the point where it can be isolated enantiomer-
ically pure (for practical purposes). The diol can in most cases be converted to the
diester. A mathematical model for the prediction of the ee value of the monoester and
the quantity of the individual products in such a combined enantiotopos- and
enantiomer-differentiating hydrolysis, which allows one to find the optimum in
regard to the ee value and the yield, has been developed on the basis of an irreversible
reaction and the absence of product inhibition (Scheme 11.1-11),[' 67-691 . Required
are the kinetic constants a, E l and E2, which can be derived from a determination of
7 1 . 7 Hydrolysis and Formation ofcarbowylid Acid Esters
Scheme 11.1-11. Hydrolase

I
345
catalyzed enantiotopos- and

enantiomer-differentiating
irreversible transformationsL67-691.
[Q] = [So] - [S]- [PI - tent-PI
[PI -[ent-PI
ee (P) =
[PI + [ent-PI
100
90
80
4- 60
ent-P
[%I 50
40
30
20
10
ee[%I
Figure 11.1-1. Dependence of ee value of monoester (P) on yield o f monoester in combined
enantiotopos- and enantiomer-differentiation with different sets o f kinetic parameters.
346
the amounts of S , P and ent-P as well as the ee values at various stages of the
hydrolysisI7O1. The ee value of the monoester is a function of the conversion, which is
generally expressed in curves as schematic depicted in Figure 11.1-1 for two sets of
different kinetic constants a, E l and E2[67-691. The validity of this has been verified
several times[']. A quite similar situation is encountered in the reverse hydrolysis, i.
e. the hydrolase-catalyzedacylation of a prochiral diol with, for example, vinyl acetate
in an organic solvent of low water content, conditions which render the reaction
irreversible,with formation of a chiral monoester. Here the ee value of the monoester
can also be raised at the expense of the yield through further acylation of the
monoester with formation of the achiral diacylated diol. Normally and not surpris-
ingly the hydrolase exhibits in the hydrolysis of the prochiral diacetate and in the
acylation of the corresponding prochiral diol the same enantiotopic group recogni-
tion despite the fact that chemically different species are involved. This leads to the
synthetically favorable situation that generally, through acylation of a prochiral diol
in an organic solvent and hydrolysis of the corresponding diacetate in water, both
enantiomers of the corresponding monoacetate are accessible with one enzyme
(Scheme li.l-12)11-361.The validity of this approach has been demonstrated in
numerous cases.
Chiral monoesters, obtained either from a prochiral diol or diester, may be
converted by a suitable series of chemoselective transformation to either enantiomer
of a given target compound (enantiodivergentsynthesis) (Scheme 11.1-13)[10~ 401.
Because of the results with numerous prochiral diesters and diols, which have
been subjected successfully to hydrolase-catalyzed enantioselective hydrolysis and
acylation, respectively, and because of the desire to predict the sense of the
asymmetric induction in the conversion of a new substrate, active-site or substrate
models have been developed for the hydrolases pig liver esterase [71-731,pig pancreas
1
pig pancreas pig pancreas
lipase lipase
celite H,O/ether
vinyl acetate pH 7.0
9"
Aco-LfoH
Scheme 11.1-12.
Synthesis o f both
enantiomers of a
"U monoacetate
-
through transesterifi-
298% ee, 89% yield 93% ee, 86% yield cation and hydrolysis
1141 (78% ee, without ether) with a hydrolase.
1. CIC0,Et
7 7 . 7 Hydrolysis and Formation ojcarboxylid Acid Esters
Scheme 11.1-13.
I
347
acozH C0,Me
2. NaN,
3. CH
,, A
NHCO,CHzPh Synthesis o f
both eantiomers
from a given
I Kbutene
4. PhCH,OH C0,Me starting material
(enantiodiver-
gent synthesis).
C0,tBu
C0,Me
1 NaOH
1. CIC0,Et
2. NaN,
C0,tBu
3. xylene, A D I(yCOztBu
aCOzH 4. MeOH -NHCO,Me
[39a-c]
1. Na, EtOH, NH,

2. H+
doo
/
/ 298% ee, 82% yield
C0,Me
298% ee
U
3. H+
1401 298% ee, 92% yield
lipase [741, Pseudomonas cepacia lipase r7', 761, Candida rugosa lipase [761, Candida
antarctica lipase [77], Pseudomonas fluorescens lipase rS7, 78], Pseudomonas aeruginosa
lipase [791, cholesterol esterase [761, subtilisin I,'[ and a-chymotrypsinL1, 'l]. The devel-
opment of such models is greatly aided by X-ray crystal structure analyses of
subtilisin a-chymotrypsin[831, Candida rugosa lipase, pig pancreas lipase, ["I
Candida antartica lipase, '(lb] Pseudomonas cepacia lipase [871, and cholesterol
esterase[86c].To a certain extent these models allow for a rationalization of the
enantiotopic group and enantiomer preferences observed with the various substrates
348
I 11 Hydrolysis and Formation of C - 0 Bonds
and for a prediction in the case of new substrates. Interestingly, X-ray structure
analyses show the active site of some lipases in the crystal to be blocked by a helical
segment, called a lid or flap. In complexes of those lipases with transition state
analogs the lid is opened, permitting access to the active site. Lipases in water usually
show a lower activity toward water-soluble substrates than toward water-insoluble,
liquid substrates. Thus, interfacial activation of lipases may be caused by a opening
of the lid upon contact with a hydrophobic phase["].
One of the most valuable and much exploited features of hydrolases is their ability
not only to differentiate between enantiotopic groups but also to differentiate
between enantiomers [1-361. When, for example, a racemic alcohol or ester is sub-
jected to a hydrolase-catalyzed acylation, alcoholysis or hydrolysis, respectively, a
kinetic racemate separation (resolution)can take place, leading, if the process would
be completely selective, at the point of 50 % conversion, to a mixture of the ester and
the corresponding alcohol or acid of opposite configuration. In such a case both the
unreacted enantiomer (substrate, S) and the newly formed ester, alcohol or acid
(product, P) are enantiomerically pure, and their theoretical yield is 50% based on
the racemic substrate. Hydrolase-catalyzedhydrolysis in water, acylation with vinyl
acetate in an organic solvent of low water content and alcoholysis (provided that a
large excess of alcohol is used) are, all three, practically irreversible, and the
efficiency of the racemate separation only depends on the differentiation by the
enzyme. When the selectivity of the enantiomer-differentiatinghydrolase-catalyzed
transformation is insufficient, the enantiomeric purities of the product and of the
unchanged substrate can be raised to a certain degree by changing the extent of
conversion. Here too a mathematical model for the prediction of the ee value of the
product and the unreacted substrate as function of the degree of conversion and the
yield based on the simple classical homocompetitive model, assuming irreversibility
and the absence of product inhibition, has been developed (Scheme 11.1-14)
[S, 67-70]
By determining the E value from pairs of experimentally determined c and ee(P)

values or c- and ee(ent-P) values, the ee values for the product and the substrate,
depending on the degree of conversion, can be calculated and thus the optimum in
terms of ee value and yield be f o ~ n d [ ~ ~ I . T hequations
ree for E, called the enantio-
meric ratio, allow one to calculate the inherent enantioselectivity of an enzyme, i. e.
its ability to differentiate between enantiomers. Thus, E values can advantageously
be used to compare the inherent enantioselectivitiesof different enzymes. E values
are calculated by one of the equations of Scheme 11.1-14 on the basis of the
determination of the conversion c and the enantiomeric excess ee of the remaining
substrate or of the product. Alternatively, E values may be calculated on the basis of
the ee values of both the remaining substrate and the product. Since ee values are
often more accurately measured than conversion, the third equation is preferred[34].
It should be noted, however, that high E values (> 100)are less accurately determined
than moderate E values, because of the enantiomeric ratio being a logarithmic
function of the enantiomeric excess. Small changes in the measured enantiomeric
purities gives large changes in the E values[34, Figure 11.1-2 indicates how to
proceed practically in cases where the enzyme used exhibits only moderate selectiv-
I
349
k, Scheme 11.1-14. Hydrolase-catalyzed enantiomer-differentiating
s - P irreversible c: conversion.
+
ent-S -k2
ent-P
[SI
In -
[Sol
E=
[ent-S]
In ___
[ent-So]
E=
ln[l (1 + ee (P)]
-c
; E=
In[(l - c)(l - ee (S)]
; E=
1 - ee (P)
1 + (ee(S)/ee(P) 1
In[l - c (1 - ee (P)]
(for c <50%)
In[(l - c)(l + ee (S)]
(for c >50%)
1 + ee ( S )
1 + (ee(S)/ee(P) 1
[S] + [ent-S] -- ee 6)
c=l-
[So]+ [ent-So] ee(S)+ ee(P)
[PI -[ent-PI [ent-S] -IS]

ee ( P ) = ee (S) =
[PI + [ent-PI [ent-S]+ [S]
100
90
80
70
60
30
20
10
0
0 10 20 30 40 50 60 70 80 90 100
conversion [t]
Figure 11.1-2. Dependence of ee value of substrate (S) and product (P) on conversion in
kinetic resolution with different E values.
350
I I I Hydrolysis and Formation ofC-0 Bonds
Scheme 11.1-15.
OH
R O L C I + R O A C I Enatiomer preference in
hydrolysis and transester-
ification by a hydrolase.
88% ee, 43% yield 295% ee, 41% yield
or 295% ee or 295% ee

H,O, pH 7.0
or
nBuOH, diisopropyl ether
I I
R=
OH
R O L C I + R O A C I

vinyl acetate
or
AqO, diisopropyl ether
1
OAc
R O L C I + R O A C I
295% ee, 47% yield 295% ee, 45% yield

[561
ity. The product is isolated at the point of < 40% conversion and the substrate is
isolated at > GO% conversion. In the case of not very high E values, the substrate but
not the product can be obtained enantiomerically pure following this procedure. If
the ee values of the thus obtained remaining substrate and the product are still not
sufficiently high, both compounds may be isolated and subjected to another cycle of
hydrolase-catalyzedtransformations, either hydrolysis or transesterification, guided
by the above principles. Attempts have been made to carry out the second cycle of
resolution without the isolation of the product and substrate obtained in the first
cycle[”]. In the case of an insufficient selectivity of the resolution of Cz-symmetric
substrates, the same approach as for substrates having enantiotopic groups can be
applied (see above). For example, the Pseudomonas cepacia lipase-catalyzedhydrolysis
of racemic trans-l,2-diacetoxycyclohexanein a three-phase system composed of
I
351
water, n-hexane and sodium chloride gave the corresponding (R,R)-diolwith 2 99%
ee in 42% yield and the (S,S)-diacetatewith 2 99% ee in 38% yield. Critical to the
success of this resolution were the following three factors [921. The enzyme showed a
preference for the acetoxy group attached to the (R)-centerin both the diacetate and
in the monoacetate, the monoacetate was completely hydrolyzed to the diol, and an
equalization of the rates of the hydrolysis of the diacetate and the monoacetate was
be achieved through a favorable partitioning of both between water and n-hexane.
A synthetically highly interesting method of converting a racemate completely to
one enantiomer is dynamic kinetic resolution, a topic which is dealt with in Sect.
11.1.1.1.5 (Sect. 11.1.2.1.2,Table 11.1-24)on lipases.
Kinetic resolution of alcohols and esters with hydrolases has opened up a new
dimension for the synthesis of enantiomericallypure alcohols, esters and carboxylic
acids, and in consequence the importance of resolution as a method for the
attainment of enantiomerically pure compounds has been increased considerably.
Hydrolase-catalyzed resolution is amenable to large-scale production [33-351, as was
impressively demonstrated much earlier by the acylase-catalyzed racemate separa-
tion of N-acyl amino acids (not discussed in this chapter)[641.
In lipase-catalyzed racemate separation of alcohols the same enantiomer prefer-
ence is usually observed in acylation and hydrolysis (Scheme 11.1-15)[561.
11.1.1
Hydrolysis and Formation of Carboxylic Acid Esters
11.1.1.1
Hydrolysis of Carboxylic Acid Esters
11.1.1.1.1 Pig Liver Esterase

Pig liver esterase (PLE, E.C. 3.1.1.1) is one of the most useful hydrolases for the
enantiotopos-differentiating hydrolysis of dicarboxylic diesters and diacetates of
diols as exemplified by the hydrolysis of dimethyl cis-cyclohex-4-ene-l,2-dicarbox-
ylate [37-421, which yields the corresponding cyclohexenoid monoester in nearly
quantitative yield with an ee value of 2 98 % even on a 100 mol scale (Scheme 11.1-1
and Table 11.1-1)c4l1. Monoesters of the above type, which are in principle accessible
by enzymatic hydrolysis on a large scale, are very useful chiral starting materials for
the synthesis of biologically active natural and non-natural compounds 19, 13, 271
9
'
including p-amino acids [39a-dl. Up to now approximately 400 substrates for pig liver
esterase have been described in the literature. Pig liver esterase, like other hydro-
lases, does not require a coenzyme, is commercially available, and often combines a
low substrate specificity with high enantioselectivity. Pig liver esterase, which is a
serine esterase, is isolated as a mixture of isoenzymes composed of the three
subunits a (58.2 kDa), p (59.7kDa) and y (61.4 kDa), which behave more or less
differently in regard to substrate specificity, pH dependence, inhibition or activation
by organic solvents or other compounds, and e n a n t i o s e l e c t i ~ i t yCommercially
~~~~~~~.
available are the natural isoenzyme mixture and several isoenzyme mixtures,
enriched in one isoenzyme. These enzyme preparations may contain other proteins
352
and perhaps even other hydrolases. However, despite this variability in composition,
the pig liver isoenzyme mixture has been applied with high success in almost all the
cases reported. Even a rather crude acetone extract of pig liver, called pig liver acetone
powder (PLAP), was successfully applied to enantioselectivehydrolysis. The cloning,
functional expression and characterization of recombinant pig liver esterase has
been de~cribed[’~”]. The recombinant pig liver esterase prepared by this method
seems to be a single isoenzyme. It was reported that recombinant pig liver esterase,
in the kinetic resolution of (l-phenyl-2-butyl)-acetate,shows a much higher selectiv-
ity than the isoenzyme mixture[’7b].Pig liver esterase frequently exhibits a reversal in
enantiotopic selectivity such as changing from a (R)-centerto a (S)-centerester group
preference of hydrolysis within a series of structurally closely related diester
substrates. An active-site model for predicting the sense of the enantiotopos-
differentiation, which accounts for this reversal, has been p r o p ~ s e d [ ~ Usually
~-~~l.
the best results are achieved with the dimethyl esters of dicarboxylic acids and with
the diacetates of diols. Frequently the enantioselectivity of the hydrolysis and the
yield of the monoester may be raised by changing the achiral alcohol component of
the ester from methanol to ethanol or isopropanol. In the case of dicarboxylic
diesters as substrates, pig liver esterase-catalyzedhydrolysis usually stops completely
at the stage of the monoester formed. Further hydrolysis of the monoester to the
dicarboxylic acid is in almost all cases thus far investigated extremely slow. This has
been attributed to the presence of a charged group (carboxylate)in the molecule. In
the case of the diacetates of diols, the rate difference is usually not so great. This,
however, in the case of a moderate selectivity of the enantiotopos-differentiating
hydrolysis, can be used to enhance the ee value of the monoacetate via a subsequent
enantiomer-differentiating hydrolysis if the same stereochemical preference is
maintained for the enantiomeric monoacetates; i. e., the faster formed enantiomeric
monoacetate is more slowly hydrolyzed to the achiral diol, which is usually observed
(Scheme 11.1-11).A mathematical model for the prediction of the ee value of the
monoacetate in such a combined enantiotopos- and enantiomer-differentiating
hydrolyses, which allows one to find the optimum in regard to the ee value and yield,
has been developed for pig liver esterase-catalyzed hydrolyses [67-691. Pig liver
esterase-catalyzed hydrolyses are generally carried out in aqueous phosphate buffer
solution at pH 6-8 at room temperature. Equilibrium is under such conditions well
on the side of the hydrolysis products because of the large excess of water. Normally,
in the case of liquid substrates, low water solubility represents no problem. In the
case of crystalline and liquid substrates of low solubility in water, up to 20% of
organic cosolvents such as acetone, methanol, tert-butanol or dimethyl sulfoxide may
be added[9*8-’001.It should be noted, however, that the enantioselectivity and the rate
of the hydrolysis as well as the yield of the product may be influenced in either
direction by organic cosolvents. Since pig liver esterase is a mixture of isoenzymes, it
has been speculated that in the presence of organic cosolvents one or more
isoenzymes might be deactivated. Organic cosolvents can in some cases be ad-
vantageously used to enhance the ee value of the chiral monoester or monoacetate.
Pig liver esterase can be recovered from the aqueous solution by ultrafiltration[411 or
by immobilizing the enzyme covalently on oxirane-activatedacrylic beads (Eupergit
Table 11.1-1.
7 7 . 7 Hydrolysis and Formation ofCarboxylid Acid Esten
Pig liver esterase-catalyzedenantiotopos-differentiating hydrolysis of prochiral

I 353
cyclic dicarboxylic acid diesters in aqueous solution.
C0,Me C0,Me
1 [1-4, 51 Ph,ot,J 2 [6, 71
C
' 02H C
' 02H
100% ee, 99 % yield 88 % ee, 99 % yield
a""."
C0,Me
Me Me .,,,
4 [I,3, 61
U ~ H
Me
C
' 02H C0,Me
91 % ee, 90% yield 74 % ee, 95 % yield
a""." 4
C0,Me
Me ..,, Ph,,,,
Me C0,Et 5 [GI [61
Ph C0,Me
45 % ee, 80 % yield no hydrolysis
4
C0,nPr
Me .,,,
7 [61 o<Co2H
C0,Me 8 [4,8]
Me C0,nPr
no hydrolysis 31 % ee, 65 % yield
occo2H I
9 [91 N 10 [lo]
C0,Me
H02CP\C02Me
99 % ee, 90 % yield 92% ee
FO,CH,Ph
C0,Me
N
11 [lo] 12 [l-4, 71
H02CP\C02Me C
'0
H
.
38% ee 94 % ee, 98 % yield
IdCozMe
-CO,H
13 [ll]
86 % ee, 96 % yield
doZH C0,Me
9 %, 80 % yield
II
354
I Hydrolysis and Formation o f C - 0 Bonds
Table 11.1-1. (cont.).
C0,Me
72 % ee, 83 % yield
82 % ee, 85 % yield, 10% MeOH
15 [12, 71
oaco C0,Me
88 % ee, 85 % yield
16 [12, 131
60% ee, 78% yield, 10% acetone
C0,Me
17 [12, 14) 18 [13]
CO,H
22 % ee, 63% yield 90% ee, 99% yield
caCozH
c:aCozMe C0,Me
0 % ee, 92 % yield
19 [12]
CO,H
48 % ee, 87 % yield
20 1121
(isolated as ketone) (isolated as ketone)
Hoaco2H C0,Me
21 [12] 22 [12]
80 % ee, 76 % yield 22 % ee, 80 % yield
23 (121 24 [12]
AcO
C0,Me
wCO,H
52% ee, 76% yield 28% ee, 91 % yield
25 (121 26 (121
58 % ee, 88 % yield 84% ee, 76% yield

7 1. 1 Hydrolysis and Formation ofcarboxylid Acid Esters
I 355
73% ee 64% ee
M e T xCO,H 29[14]
30 (151
0 CO,H
C0,Me CH,Ph
6% ee 38 % ee, 71 % yield
31 [15] 32 [15]
no hydrolysis
C0,Me
d I
CO,H
33 [15]
C0,Me
34 1161
34% ee, 82 % yield 42 % ee, 98 % yield
qH C0,Me
46 % ee, 83 % yield
35 (161
C0,Me
17%ee,85%yield
36 117, 18, 191
100% ee, 39% yield, 25% DMSO

100% ee, 10% MeOH
61 % ee, 10% DMSO
39% ee, 10% MeCN
356
‘~‘‘,Cl Ho2c””’,o
,+. CO,Me \$.. CO,Me
Ho2C N 37 [20] Y 38 [20]

I
CH,Ph S0,Tol
44% ee, 28% conversion 14% ee, 31 % conversion

27% ee. 96% conversion 11% ee, 88% conversion
Me%Yo2H
39 [20] 40 1211
Me CO,CH,CF,
COPh
55 % ee, 16 % conversion 295% ee, 62% yield
34% ee, 33% conversion
9
:;
C0,Et
41 [22] 42 [22]
S HN
>C02H
6 % ee, 83 % yield 20 % ee, 83 % yield
92; 43 [22] 44 11, 2, 7, 23,

241
PhCH,N
10% ee, 63% yield
(Icoz
0% ee
C0,Et
45 [25]
C0,Me
298 % ee, 99 % yield
46 11, 23, 24,26,
27, 28, 291
27 % ee, 67 % yield
47 [30]
aCozH C0,nPr
25 % ee, 68 % yield
48 [30]
a C O C0,iPr
z H
2 % ee, 5 % yield
49 [30]
aoZH C0,nBu
13% ee, 18 % yield

50 [30]
7 7 . 7 Hydrolysis and Formalion ofcarboxylid Acid Esters
I
357
C0,Me
C0,Me
51 [12, 311 Mexo""~oH
Me o,.-s 52[32, 331
cC02H CO,H
68 % ee, 84 % yield 295 % ee, 42 % yield
:x;::+
C0,Me
C0,Me
53 [34] : x;: : q C0,Me
CO,H
54 [35]
R = tBu no hydrolysis 60% ee

R = H 95 % ee
Mexo,.,,(yH
55 [36]
Me 0""
C0,Me
72 % ee, 86 % yield 96 % ee, 72 % yield
(isolated as derivative)
C0,Me
57 191 &CO,H C0,Me 58 [38, 391
O a O z H
no hydrolysis 75 % ee, 86 % yield
98% ee, 82% yield

59 [38] 4 C0,Me
C0,Me
no hydrolysis
GO [38]
61 [38] 62 [40]
&02HC0,Me
Me 0
64% ee, 87% yield 0% ee, 10% yield

358
no hydrolysis 36% ee, 77 % yield
0
Me*'OZH 65[43,44] Hz'&' 0
Me 66 [40,45]
CO,R C0,Me
R = Me 80% ee, 10% yield 77 % ee, 96 % yield

R = Et 100% ee, 37% yield
R = nPr 45% ee, 15% yield
R = iPr 39% ee, 22% yield
R = nBu 73 % ee, 4 % yield
o&02H 67 [40, 45, 461 68 [47]
C0,Me C0,Me
77% ee, 100%yield 295 % ee, 99 % yleld
C0,Et
GI) (481
O
&
C0,Et C0,Me
65 % ee, 95 % yield 88%ee
71 [SO, 511
CO,R
I
CiCO),
R = Me 94% ee
R=Et 99%ee
1 P. Mohr, N.Waespe-Sarcevic, C . Tamm, 4 G.Sabbioni, I. B. Jones,J. Org. Chem.1987,52,
K. Gawronska, J. K. Gawronski, Helv. Chim. Acta 4565.
1983,66,2501. 5 H.Ito, N . Imai, S. Tanikawa, S. Kobayashi,
2 G. Sabbioni, M. L. Shea, f. B. Jones,J . Chem. SOL., Tetrahedron Lett. 1996,37, 1795.
Chem. Commun.1984,236. 6 P. Walser, P. Renold, V.N'Goka, F. Hosseinzadeh,
3 M. Schneider, N. Engel, P. Honicke, G. C.Tamm., Helv. Chim.Acta 1991,74,1941.
Heinemann, H. Gorich, Angew. Chem.1984,96, 7 P. Renold, C.Tamm, Biocatal. Biotransform. 1995,
55; Angew. Chem., lnt. Ed. Eng. 1984,23,67. 12,37.
7 7 . 1 Hydrolysis and Formation ofcarboxylid Acid Esters I359
8 D. Habich, W. Hartwig, Tetrahedron Lett. 1987,28, 30 K. Adachi, S. Kobayashi, M. Ohno, Chimia 1986,
781. 40,311.
9 P. Mohr, L. Rosslein, C. Tamm, Helu. Chim. Acta 31 Y. Nagao, M. Kume, R. C. Wakabayashi,
1987.70, 142. T. Nakamura, M. Ochiai, Chem. Lett. 1989,
10 P. Renold, C. Tamm, Tetrahedron: Asymmetry 1993, 239.
4, 2295. 32 j. Zemlicka, L. E. Craine, M. J. Heeg, J. P. Oliver,
11 I. Harvey, D. H. G. Crout, Tetrahedron: Asymmetry J. 0%.Chem. 1988,53,937.
1993.4, 807. 33 E. J, Hutchinson, S . M. Roberts, A. I. Thorpe,
12 H.-J. Gais, B. Biilow, A. Zatorski, M. Jentsch, j . Chem. Soc., Perkin Trans. 1 1992, 2245.
P. Maidonis, H. Hemmerle, J. Org. Chem. 1989, 34 I. C. Cotterill, P. B. Cox, A. F. Drake,
54, 5115. D. M. Le Grand, E. J. Hutchinson, R. Latouche,
13 Y.Nagao, M. Kume, R. C. Wakabayashi, R. B. Pettman, R. J. Pryce, S . M. Roberts,
T. Nakamura, M. Ochiai, Chem. Lett. 1989, 239. M. Stanley,j . Chem. SOC.,Perkin Trans. 1 1991,
14 P. Renold, C. Tamm, Tetrahedron:Asymmetry 1993, 3071.
4, 1047. 35 M. Arita, K. Adachi, Y. Ito, H. Sawai, M. OhnoJ.
15 S. Iriuchijima, K. Hasegawa, B. Tsuchihashi, Am. Chem. SOL 1983, 105,4049.
Agric. Biol. Chem. 1982,46, 1907. 36 P. G. Hultin, F. J. Muesler, J. B. Jones,]. Org.
16 J. B. Jones, R. S . Hinks, P. G. Hultin, Can. Chem. 1991,56,5375.
I. Chem. 1985,63,452. 37 T. Kuhn, C. Tamm, Tetrahedron Lett. 1989, 30, 693.
17 M. Kurihara, S. Kamiyama, S. Kobayashi, 38 R. Bloch, E. Guibe-]ample, C. Girard, Tetrahedron
M. Ohno, Tetrahedron Lett. 1985, 26, 5831. Lett. 1985,26,4087.
18 F. Bjorkling, J. Boutelje, H. Hjalmarsson, K. Huh, 39 G. Guanti, L. Banfi, E. Narisano, R. Riva, S. Thea,
T. Norin, J. Chem. SOC.,Chem. Commun. 1987, Tetrahedron Lett. 1986, 27,4639.
1041. 40 Y Ito, T. Shibata, M. Arita, H. Sawai, M. Ohno,
19 A. Mattson, J. Boutelje, I. Osoeregh, J . Am. Chem. Soc. 1981,103,6739.
M. Hjalmarsson, U. Jacobsson, M. Lindbaeck, 41 M. Ohno, Nucleosides and Nucleotides, 1985,4, 21.
T. Norin, P. Szmulik, K. Hnlt, Bioorg. Med. Chem. 42 P. Metz, Tetrahedron 1989,45,7311.
1994, 2, 501. 43 H.-J. Gais, T. Lied, Angew. Chem. 1984, 96,495:
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Tetrahedron: Asymmetry 1996, 7, 345. 44 K. Adachi, S . Kobayashi, M. Ohno, Chimia 1986,
21 A. Salgado,T. Huybrechts, A. Eeckhaut, J. Van der 40,311.
Eycken, 2. Szakonyi, F. Fulop, A. Tkachev, N. De 45 M. Ohno, Y. Ito, M. Arita, T. Shibata, K. Adachi,
Kimpe, Tetrahedron 2001, 57, 2781. H. Sawai, Tetrahedron 1984,40, 145.
22 Y. Morimoto, K. Achiwa, Chem. Pharm. Bull. 1987, 46 S. Niwayama, S. Kobayashi, M. Ohno,J. Am.
35, 3845. Chem. SOC.1994,116,3290.
23 H:J. Gais, K. L. Lukas, Angau. Chem. 1984, 96, 47 S. Kobayashi, M. Sato, Y. Eguchi, M. Ohno,
140; Angau. Chem., Int. Ed. Engl. 1984,23, 142. Tetrahedron Lett. 1992, 33, 1081.
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Lindner, Liebigs Ann. Chem. 1986,687. Lett. 1988,29,6313.
25 F. Bjorkling, J. Boutelje, S . Gatenbeck, K. Hult, 49 I. C. Cotteril, S. M. Roberts, S. J. 0.William,
T. Norin, Appl. Microbiol. Biotechnol. 1985,21, 16. J. Chem. Soc., Chem. Commun. 1988,1628.
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Tetrahedron Lett. 1984.25, 2557. M. G . Palin, P. McArdle, M. OGara, D.
27 H. A. Sousa, J. G. Crespo, C. A. M. Afonso, Cunningham, Tetrahedron: Asymmetry 1992, 3,
Tetrahedron:Asymmetry 2000, 11, 929. 375.
28 H. A. Sousa, C. A. M. Afonso, J. G . Crespo, 51 J. A. S. Howell, M. G. Palin, G. Jaouen,
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29 H. A. Sousa, ). P. S . G. Crespo, Prog. Biotechnol. P. McArdle, D. Cummingham, M. O'Gara,
1998, 15,673. Tetrahedron: Asymmetry 1996,7, 95.
C) [lol], BrCN-Sepharose['"], silica or in a hollow fiber ultrafiltration mem-

brane[42].The Eupergit C immobilized pig liver esterase retains 68% of the specific
activity of the soluble enzyme. It is easily removed by filtration from the reaction
mixture and can be reused several times when stored at 7 "C. In large-scale
experiments with pig liver esterase in aqueous solution the enzyme can be stabilized,
if necessary, by the addition of inexpensive bovine serum For a
determination of the ee value of the monoester, different methods can be used
360
addition of an enantiomerically pure chiral amine as for example ephedrine r401 or a-

phenylethylamine and 'H NMR spectroscopy of the diastereomeric salts formed
thereby, conversion of the dicarboxylic acid monoester to the a-phenylethylamide
and analysis by 'H NMR spectroscopy or HPLC1'041,or conversion to the tert-butyl
ester and 'H NMR spectroscopy in the presence of a chiral shift
Determination of the ee value of monoacetates has been carried out for example
through conversion to the Mosher-esterand analysis by 'H NMR spectroscopy in the
presence or absence of a shift reagent or by HPLC["'], or more directly either by 'H
NMR spectroscopy in the presence of a chiral shift reagent or by HPLC and GC on
chiral columns [Io7].
Cyclic dicarboxylic acid diesters, which bear enantiotopic ester groups, are sub-
strates par excellence for a pig liver esterase-catalyzedhydrolysis under formation of
the corresponding monoesters (1-71)(Table 11.1-1).The examples listed in Table
11.1-1are a good demonstration of the scope and limitation of pig liver esterase-
catalyzed hydrolysis and illustrate general trends. The enantioselectivity and the
yield can be influenced to a certain extent by the structure of the alcohol moiety of the
diester as exemplified by the heterocyclic monomethyl and monopropyl esters 30
and 31.The corresponding isopropyl ester 32 is not a substrate for pig liver esterase.
Extreme examples for the influence of the alcohol moiety are the cyclohexanoid
methyl ester 44,which is formed with an ee value of 80% and the ethyl ester 45,
which is produced as racemate. Usually the dimethyl esters are the best substrates.
This trend, however, is not general. In the series of the cyclohexenoid monoesters
4650,the methyl ester 46 is the one formed with the highest ee value, and, in the
series of the bicyclic monoesters 65 having a norbornene skeleton, it is the ethyl ester
which has the highest ee value. However, the methyl ester 65 (R = Me) is formed
much faster and is obtained in higher yield. Addition of an organic cosolvent such as
dimethyl sulfoxide or methanol can lead to the monoester with a higher ee value.
"his has been impressively demonstrated in the case of the benzyl-protected
heterocyclic monoesters 36.A generalization of the effects of organic solvents upon
the enantioselectivity of the PLE-catalyzed hydrolysis of diesters is difficult. As
exemplified by the series of cyclopentanoid monoesters 14,15,17,19-29, seemingly
small structural changes may invert the enantiotopic recognition through the
enzyme. This can be advantageously used in certain cases to raise the ee value of a
given target monoester by a suitable substrate modification. Branching in the a-
position of the ester group is no prerequisite for high enantioselectivity. The
monoesters lG and 18,the ester groups of which are separated by a methylene group
from the ring, are obtained with comparable enantioselectivities. Interestingly,
enantiotopic recognition is reversed in the series of diesters corresponding to the
monoesters 15 and 16 but not with the diesters corresponding to the monoester 8,9,
17 and 18.This seemingly unpredictable behavior of pig liver esterase may at a first
glance detract from its use in asymmetric synthesis. However, successful attempts
have been made to rationalize this observation as well as the sense of asymmetric
induction observed with the various substrates within an active-site model of the
enzyme. Furthermore, one should bear in mind the experimental simplicity of a pig
liver esterase-catalyzed hydrolysis and the synthetic advantages gained if the diester
I
7 7 . 7 Hydrolysis and Formation ofcarboxylid Acid Esters 361
Table 11.1-2. Pig liver esterase-catalyzed enantiotopos-differentiating hydrolysis o f prochiral

acyclic dicarboxylic acid diesters in aqueous solution.
A
R' CO,R~
R1 cop4
R' R2 R' R4 ee (%) yield VO) Ref.

1 73" 90-98
2 52" 90-98
3 19b 87
4 58" 90-98
5 46= 90-98
6 87= 90-98
7 88" 90-98
8 16" 90-98
9 16b 61
10 82' 85-100
11 93' 85-100
12 96 -
13 6 37
14 21 86
15 67 90
16 96 90
17 2 95 49
18 46b -
19 84b 76
20 87b 81
21 96b 95
22 4Gb 46
23 84b -
24 no hydrolysis -
25 88b 86
25 98b,' 95
26 15= 90-98
26 20 -
27 loa 90-98
27 8 -
28 25" 90-98
28 38 -
29 10" 90-98
30 5" 90-98
31 81 90
32 86b -
33 92 100
34 82 92
35 78 100
36 97 96
37 97 83
38 -b 81
39 1Ob 92
40 80b 70
41 69 66
362
R' cop3
i(CO,R~
yield (%)
- R' R2 R3 ee (%)
42 80 78
43 78 66
44 69 84
45 79 77
46 CH3 89 87
47 CzHs 94 70
48 C2HS 96 45
49 C2HS 97 98
50 H 19 99
51 H 74 95
52 H 88 97
53 H 92 99
a In the presence of 25 % dimethylsulfoxide
b Absolute configuration not determined
c In the presence of 50% dimethylsulfoxide
Rj TR4 cop3
R' R2 R' R4 ee (%) yield (?A) Ref.
54 CH3 H H CH3 90 86 [20,211

54 CH3 H H CH3 79 94 [221
54 CH3 H H CH3 92d - 1221
55 C2Hs H H CH3 50 67 1221
56 n-C3H7 H H CH3 25 78 P I
57 X3H7 H CH3 H 38 98 1221
58 G-CGHII H H CH3 17 95 1221
59 C6H5 H CH3 H 42 98 P I
60 H p-FC6H4 H CH, 95 86 1231
61 C6HsCH2 H CH3 H 54 95 1221
61 C6HsCH2 H CH3 H 81d. e -
P I
61 C6HsCH2 H CH3 H 73 98 P I
62 C6H5(CHz)z H CH3 H 44 98 ~ 4 1
63 CsHs(CHz)3 H CH3 H 88 97 ~ 4 1
64 (E)-C~HSCH=CH-CH~ H CH3 H 88 95 ~ 4 1
65 CGH~CH~O(CH~)Z H CH3 H 54 100 1241
66 (E)-HOCHz-CH=CH-CH2 H CH3 H 18 100 ~ 4 1
67 (E)-THPOCHz-CH=CH-CHz H CH3 H 74 95 1241
68 (E)-CsHs-CH=CH H CH3 H 93 100 ~ 4 1
69 NH2 H H CH3 41 94 1251
70 CH3CONH H H CH, 93 81 ~ 5 1
71 C2HsCONH H H CH3 6 50 1251
72 n-C3H7CONH H H CH3 15 52 [251
1 1 . 1 Hydrolysis and Formation ofCarboxytid Acid Esters
I 363
F+z:;:
R’
R’ R2 R3 R4 ee (%I yield (“9) Ref.
73 ~L-C~H~CONH H CHj H 2 48 PI
74 i-C3H7CCONH H CH, H 54 55 ~ 5 1
75 (CHs)jCCONH H CHs H 93 50 (251
76 c-C~HIICONH H CH, H 79 52 PI
77 CH3CH=CH-CONH H CH3 H 100 60 (251
78 CbHsCONH H CH, H 72 59 ~ 5 1
79 CHz=CHCONH H H CH3 8 50 ~ 5 1
80 CbHsCONH H CH, H 20 60 [251
81 CzHsCONH H CH3 H 40 70 1251
82 (CH,)3COCONH H CH, H 53 93 ~ 5 1
83 CbHsCHzOCONH H CH, H 93 93 ~ 5 1
84 C~HSCH~NH H CH3 H 33 58 ~ 5 1
85 HO H CH, H 15 100 [18,2631,
321
86 HO CH3 CH3 H 99 62 1211
87 COzC2H5 OH C2Hs H 90 - [401
88 CsHsCHz0 H CH, H 40 87 [27, 321
89 CHsOCHzO H CH3 H 14 100 [28]
90 CcjHsC00 H CH3 H 12 78 I281
91 CHjCOO H H CH3 90 38 [311
92 CH30(CHz)zOCHzO H H CH3 39 92 [27, 321
H SCH2-p-CH30CrjH4 H CzHS 71 81 P I
MeXcozMe
Me
HoXcozH
CO,H
94 [20]
HO C0,Me
95 I201
18% ee, 94 % yield 48% ee, 92 % yield
C0,Me
96 [20, 35, 361 97 [20]
CO,H
Me Me
64% ee, 85 % yield 98 % ee, 95 % yield
d In the presence of 20% methanol; e At -10 “C
364
I 11 Hydrolysis and Formation of C-0 Bonds
99 1371
‘C0,Me
100 1381 MeoXCo2Me
Me0 CO,H
101 1391
82% ee 90 % ee, 90 % yield

(20% MeOH)
102 1401
90-92% ee, 80-85 % yield

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B. Maillard, Tetrahedron Lett. 1990, 31, 653. 24 M. Nakada, S. Kobayashi, M. Ohno, Tetrahedron
12 M. Schneider, N. Engel, H. Boensmann, Angew Lett. 1988,29, 3951.
Chem. 1984,96, 54; Angew. Chem., In!. Ed. Engl. 25 K. Adachi, S. Kobayashi, M. Ohno, Chimia 1986,
1984,23, 66. 40, 311.
13 E. J. Toone, J. B. Jones, Tetrahedron: Asymmetry 26 P. Mohr, L. Rosslein, C. Tamm, Helv. Chim. Acta
1991,2, 1041. 1987,70, 142.
14 K. Imchijima, K. Hasegawa, G. Tsuchihashi, Agnc. 27 L. K. P. Lam, J. B. Jones, Can./. Chem. 1988,66,
Biol. Chem. 1982,46, 1907. 1422.
28 R. Roy, A. W. Rey, Tetrahedron Lett. 1987,28,
11.1 Hydrolysis and Formation of Carbowylid Acid Esters
34 S. Johnson, S. R. Kesten, L. D. Wise,]. Org. Chem.

I 365
4935. 1992,57,4746.
29 E. Baader, W. Bartrnann, G. Beck, A. Bergmann, 35 C. S. Chen, Y. Fujirnoto, C. J. Sih,]. Am. Chem.
H. Fehlhaber, H. Jendralla,K. Kessler, R. Saric, Soc. 1981, 103, 3580.
H. Schussler, W. Teetz, M. Weber, G. Wess, 36 G. Guanti, L. Banfi, E. Narisano, R. Riva, S . Thea.
Tetrahedron Lett. 1988,29, 2563. Tetrahedron Lett. 1986,27,4639.
30 F. C. Huang, L. F. H. Lee, R. S. D. Mittal, 37 M. Mikolajczyk, P. Kielbasinski, R. Zurawinski,
P. R. Ravihmar, J. A. Chan, C. J. Sih, E. Capsi, M. W. Wieczorek, J. Blaszczyk, Synlett 1994,127.
C. R. Eck,]. Am. Chem. SOC.1975,97,4144. 38 D. J. Horgan, J. K. Stoops, E. C. Webb, B. Zemer,
31 E. Santaniello, M. Chiari, P. Ferraboschi, S. Trave, Biochemistry 1969, 8, 2000.
I . Org. Chem. 1988,53, 1567. 39 H. I. Bestmann, U. Ch. Philipp, Angnu. Chem.
32 L. K. P. Lam, J. B. Jones,Can.]. Chem. 1988,66, 1991, 103,78 Angnu. Chem., Int. Ed. End. 1991,
1422. 30, 86.
33 S . Kobayashi, K. Kobayashi, K. Hirai, Synlett 1999, 40 R. Chenevert, B. Ngatcha, R. Tchedarn, S. Yannick,
909. D. Goupil, Tetrahedron: Asymmetry 1998, 9,4325.
in question is a substrate. Cyclic diesters, the ester groups of which are not in the
1,2-position,seem to be less appropriate substrates for pig liver esterase (33-39 and
41-43). Finally, it seems noteworthy that transition metal complexes containing
enantiotopic esters groups are also amenable to a highly selective pig liver esterase-
catalyzed hydrolysis (71). Cyclic monoesters of Table 11.1-1,which can be obtained
with other hydrolases as such or of opposite configuration, are contained in Tables
11.1-7 and 11.1-12.
For synthetic and mechanistic reasons, a large number and variety of prochiral
malonates have been subjected to pig liver esterase-catalyzed hydrolysis with
formation of chiral malonates (1-53) (Table 11.1-2). Hydrolysis of dimethyl or diethyl
malonates bearing a methyl group and another small alkyl or functionalized alkyl
group leads preferentially to the monoester with the (S)-configuration. Upon an
increase in the size of the second group, enantiotopic recognition is inverted and the
monoester with the (R)-configurationis formed. Hydrolysis of dimethyl hydrox-
ymethyl methyl malonate provides an excellent example of the strategy to enhance
the enantioselectivity of pig liver esterase by the introduction of a protecting group
on the substrate. While the parent compound itself yields the corresponding (S)-
configured monoester 13 with an ee value of only 6 %, the introduction of a tert-butyl
or tert-butyldimethylsilylprotecting group allows the isolation of the corresponding
(R)-configuredmonoesters (16 and 17) with ee values of 96% and 95%, respectively.
An equally large number and variety of prochiral glutarates have been subjected to
pig liver esterase-catalyzedhydrolysis with formation chiral glutarates (54-93) (Table
11.1-2). Among the synthetically most useful glutarates are the 3-amino-glutarates.
The parent compound methyl amino glutarate 69 itself is obtained only with an ee
value of 41%. The introduction of an amino protecting group improves the
enantioselectivity of the hydrolysis of the corresponding diester dramatically. Pig
liver esterase-catalyzedhydrolysis delivers methyl N-acetylaminoglutarate 70 of ( S ) -
configuration with an ee value of 93 % and methyl N-crotonylamino glutarate 77 of
the opposite (R)-configurationwith an ee value of 100%.Thus, both enantiomers of
methyl amino glutarate are accessible with one enzyme by a synthetically simple
substrate modification.
To a limited extent and with only moderate success, rneso-configured glutarates
and succinates have been subjected to pig liver esterase-catalyzed hydrolysis with
366 I 7 Hydrolysis and Formation of C-0 Bonds
I Table 11.1-3. Pig liver esterase-catalyzed enantiotopos-differentiating hydrolysis of prochiral
cyclic diol diacetates in aqueous solution.
44% ee, 54% yield 37 % ee, 70 % yield
3 [l,21 4 [l, 3,4-61
OH
4 % ee, 44 % yield 86% ee, 83 % yield
5 PI 0+YH NI
CH2Ph
OAc 6 [71
8 % ee, 40 % yield 90 % ee, 70 % yield
7 (81
OH
13% ee, 75% yield 77% ee
-OH
CCC
4% ee, 31% yield 55 % ee, 60 % yield
10 [1,9]
96 % ee, 78 % yield, tBuOH
11 (10, 111 12 [lo]
OAc OAc
13% ee, 75% yield 295 % ee, 80 % yield
Table 11.1-3.
I 367
EtoaoH
(cont.).
M e a o !,.,NO
H , 13 [lo] NO, 14 [lo]
OAc OAc
295% ee, 70% yield 295 % ee, 60 % yield
me^ '"'NO,
OAc
15 [lo]
f ( eM
Me
vjn.NO
OAc
,
16 [lo]
[alD+ 9.8", 68 % yield [ale - 1.3",60% yield
SgoHNO, 17 [lo] O$oHN02 18 [lo]
OAc OAc
295 % ee, 60 % yield [a]o+ 14.7", 20% yield
maoAc
no hydrolysis
NO,
OAc
19 [lo] @OAC
OAc
NO* 20 [lo]
no hydrolysis
FMe
N3 OH
86 % ee, 88 % yield R=Me

OH
99 % ee, 89 % yield
22 (121
R = CHzPh 99% ee, 100% yield

R = CHzOMe 99% ee, 86% yield
OAc
23 (121 @OMe 24 [21
OH OH
99% ee, 89 % yield 99 % ee, 100% yield
I I Hydrolysis and Formation o f C - 0 Bonds
368
I Table 11.1-3. (cont.).
e M&% ) "
HO
25 [12] MxI~i~e
Me 26 [12]
OH OAc
89% ee, 73 % yield no hydrolysis
OAc OH
Q
OH OAc
92 % ee, 100% yield 99 % ee, 70 % yield
R = nPr, 295 % ee, 83 % yield 87 % ee, 62 % yield

R = Me, slow hydrolysis,
racem at e
Me Me
v
..
31 [17, 18, 191 32 [20]
AcO
+OH
OCOR Me Me
R = Me 96% ee, 86% yield 298 % ee, 92 % yield

R = Et 42% ee, 54% yield
R = iPr 94% ee, 62% yield
R = tBu 55% ee, 72% yield
0
33 [91
P-4 - 34 [22]
OAc
57% ee 33 % ee, 52 % yield

68% ee 47% diacetate
1 K. Laumen, M. Schneider, Tetrahedron Lett. 1985, 4 K. Laumen, M. Schneider, Tetrahedron Lett. 1984,
26, 2073. 25, 5875.
2 J. B. Jones, C. J. Francis, Can. J . Chem. 1984, 62, 5 Y. F. Wang, C. S. Chen, G. Girdaukas, C. J. Sih,
2578. F. S. Ciba, j . Am. Chem. SOL 1985, 11 I, 128.
3 Y. F. Wang, C. S. Chen, G. Girdaukas, C. J. Sih, G K. Laumen, E. H. Reimerdes, M. Schneider,
j . Am. Chem. Sac. 1984, 106,3695. Tetrahedron Lett. 1985, 26, 407.
7 Y. F. Wang, C. J. Sih, Tetrahedron Lett. 1984, 25,
11.1 Hydrolysis and Formation ofcarboxylid Acid Esters
15 H. Suemune, M. Takahashi, S. Maeda, Z. F. Xie,

I 369
4999. K. Sakai, Tetrahedron: Asymmetry 1990, 1, 425.

8 H. Suemune, T. Harabe, 2. F. Xie, K. Sakai, Chem. 16 L. Dumortier, M. Carda, J. Van der Eycken,
Pharm. Bull. 1988, 36,4337. G. Snatzke, M. Vandewalle, Tetrahedron:
9 G. Guanti, L. Banfi, E. Narisano, R. Riva, S . Thea, Asymmetry 1991,2,789.
Tetrahedron Lett. 1986, 27,4639. 17 K. Naemura, N. Takahashi, H. Chikamatsu, Chem.
10 M. Eberle, M. Egli, D. Seebach, Helu. Chim. Acta Lett. 1988, 1717.
1988, 71, 1. 18 H. Estermann, K. Prasad, M. J. Shapiro, 0. Repic,
11 D. Seeach, M. Eberle, Chimia 1986, 40,315. M. J. Hardtmann, J. J. Bolsterli, M. D. Walkinshaw,
12 M. Falk-Heppner,M. Keller, H. Prinzbach, Angew. Tetrahedron Lett. 1990, 31,445.
Chem. 1989,101,1281:Angew. Chem., In!. Ed. 19 K. Naemura, R. Fukada, N. Takahashi, M. Konishi,
Engl. 1989,28, 1253. Y. Hirose, Y. Tobe, Tetrahedron: Asymmetry 1993,4,
13 G. Baudin, B. J. Glanzer, K. S . Zwaminathan, 911.
H. Vasella, Heb. Chim. Acta 1988,71, 20 A. Krief, D. Surleaux, N. Ropsan, Tetrahedron:
1367. Asymmetry 1993,4,289.
14 M. Carda, J. Van der Eycken, M. Vandewalle, 22 D. B. Berkowitz, J:H. Maeng, Tetrahedron:
Tetrahedron: Asymmetry 1990, I , 17. Asymmetry 19967,1577.
formation of the corresponding chiral succinates (94 and 95) and glutarates (96)
(Table 11.1-2). Exceptions are methyl 3-hydroxy-2,4-dimethylglutarate (97) and
methyl 1,2-dimethoxysuccinate (101),which can be obtained with ee values of 98%
and of 90 %, respectively. Most interesting examples are the citric acid derivatives 87
and 102.While the pig liver esterase-catalyzedhydrolysis of triethyl citrate proceeded
with high enantioselectivity but low regioselectivity, that of the diester derivative of
102 occurred with both high regio- and enantioselectivity.
Acyclic monoesters ofTable 11.1-2, which can be obtained with other hydrolases as
such or of opposite configuration, are contained in Tables 11.1-7 and 11.1-12.
meso-Configured mono- and bicyclic diacetates, bearing primary or secondary
acetoxy groups, are frequently hydrolyzed by pig liver esterase under standard
Table 11.1-4. Pig liver esterase-catalyzed enantiotopos-differentiatinghydrolysis o f prochiral

acyclic diol diacetates in aqueous solution.
Me OAc
80 % ee, 36 % yield 29 % ee, 43 % yield
95 % ee, 15% yield 39 % ee, 5 4 % yield
Me
Me”
290% ee, 50 % yield
1 Y. F. Wang, C. S. Chen, G . Girdaukas, C. J. Sih, 2 H. Suemune, Y. Mizuhara, H. Akita, K. Sakai,
J. Am. Chem. SOC.1984,106, 3695. Chem. Pharm. Bull. 1986,34, 3440.
3 D. Seebach, M. Eberle, Chimia 1986,40,315.
370
conditions at pH 7.0 to give the corresponding monoacetates (1-34) in high yields

and with high enantioselectivities(Table 11.1-3).The examples listed in Table 11.1-3
demonstrate once again the low substrate specificityof pig liver esterase. At the same
time the series of nitro substituted cyclohexanoid mono and diacetates 11-20 reveals
that seemingly small changes in the structure of the substrate can suppress
hydrolysis. Strategies to improve the enantioselectivity of pig liver esterase-catalyzed
hydrolysis of a dialkyl dicarboxylate are as follows: a synthetically tolerable and
meaningful substrate modification in the dicarboxylic acid part, a modification of the
alcohol part of the substrate or the addition of an organic cosolvent. These strategies
can also be applied in the case of acylated prochiral diols. While the polycyclic
monoester 29, which bears a butyryl group, is obtained with an ee value of 2 95 %, the
derivative 29, which cames an acetyl group, is formed as racemate. Enantioselectivity
in the pig liver esterase-catalyzedformation of the cyclohexenoid monoacetate 10 can
be dramatically improved if the hydrolysis of the corresponding diacetate is carried
out in the presence of tert-butanol.The heterocyclicmonoesters 31 demonstrate how,
in the case of an diacylated diol, the achiral carboxylic acid part of the substrate
influences the enantioselectivity of the hydrolysis. A series of highly functionalized
cycloheptane derivatives (21-26) have been obtained through a pig liver esterase-
catalyzed hydrolysis of the corresponding diacetates, with the same enantiotopic
group recognition in all cases.
Cyclic monoacetates of Table 11.1-3,which can be obtained with other hydrolases
as such or of opposite configuration, are contained in Tables 11.1-9, 11.1-11 and
11.1-18.
Only a very few acyclic prochiral acylated diols have been subjected with moderate
success to pig liver esterase-catalyzed hydrolysis with formation of the correspond-
ing chiral monoacetates (1-3) (Table 11.1-4).For this kind of compounds, lipases are
the hydrolases of choice.
Acyclic monoacetates of Table 11.1-4,which can be obtained with other hydrolases
as such or of opposite configuration, are contained in Tables 11.1-10and 11.1-17.
Enantiomer-differentiating hydrolysis with pig liver esterase has, as with other
hydrolases, become an important method for resolution (Table 11.1-5). Kinetic
resolution of oxirane mono- and dicarboxylic acid esters with pig liver esterase
proceeds effeciently with good selectivities, as demonstrated in the cases 14 and 15.
Resolution is of course not restricted to enantiomers with central chirality.Axial and
planar chiral racemic ester have been resolved with moderate to good results with pig
liver esterase (33-36).
Resolution of esters, the ester group of which is attached to a carbon atom bearing
three other substituents, even when contained in a bi- or tricyclic ring system (67),
represents no problem (Table 11.1-5).It seems interesting to note that these esters,
which might be otherwise difficult to hydrolyze because of steric hindrance, are
hydrolyzed readily via enzyme catalysis.
The cyclopentanoid esters 65 and 66 nicely illustrate how a seemingly remote
functional group can significantly influence the enantioselectivity.Pig liver esterases
allow for the kinetic resolution of a-hydroxy acids (3-7) and a-amino acids (8-13)
which have a quaternary Ca-atom.
11. 1 Hydrolysis and Formation ofCarboxylid Acid Esters
Table 11.1-5. Pig liver esterase-catalyzed enantiomer-differentiating hydrolysis o f racemic

I 371
carboxylic acid esters and lactones i n aqueous solution (HLE horse liver esterase).
Ho Me Me OH 1b 111
Me&CO,H e
M,O
C, , ) ,e
M
low ee 298 % ee, 12% yield

88 % conversion
low ee 94 % ee, 26 % yield

67 % conversion
HO Me
&
C
H
/O
,
low ee 94% ee, 11% yield
HO C O H Et0,C OH
Ph % Ph & 4b [2, 3,4]
75% ee 64% ee
50 % conversion 50% conversion
“0ywh OH
PhL P h
Ph
38% ee 40% ee
“
Ph O m Ph
80% ee 86% ee
HDXCo,H
7 b I21
Ph Me
51% ee 40% ee
J.
Me Ph Me
HN
, CO,H 8a 151
31%ee,6%yield 5% ee, 67 % yield

I I Hydrolysis and Formation ofC-0 Bonds
Et
9b 151
25 % ee, 28 % yield
10a [5]
-
- Ph
10b [S]
H,NXCO,Et
72% ee, 57% yield 95 % ee, 41 % yield
l l a (51 I l b (51
20 % ee, 50 % yield
nBu Ph nBu
12a (51 121, [5]
HN
, /C
' O,H
93 % ee, 41 % yield 97%ee, 31%yield
ph-Ah
HN
, CO,H
13a [5]
Ph-
H,NXCO,Et
Ph
13b [5]
54% ee, 39% yield 61 % ee, 10% yield
9.
-CO,C,H,
14a [GI 141, [6]
&CO,H
74% ee, 30% yield 82 % ee, 40% yield

50% conversion
9.
15a [7] MeO,C- : ', C0,Me 15b[7]
Me0,C
97 % ee, 40 % yield 295 % ee, 40 % yield
50 % conversion
Me o.,, Me&CO,H 16b (81

16a [8]
+co2Me
Me Me
E=17 E = 17
Me Me
17a [9] L C 0 2 H 171, [9]
G COHO z H
OH
64% ee
50% conversion 63 % ee
7 1 . I Hydrolysis and Formation ofCar6oxylid Acid Esters
I 373
18a [lo] O_o~CozH

\ 18b [lo]
10% ee 7 % ee
19a [lo] &oxc"2H 19b [lo]

\
5% ee 5% ee
5% ee G%ee
26% ee 22% ee
22a [lo] boxco222bH [lo]

CI CI
11%ee 10% ee
24 [ll]
Et
( M ~ o c H , c H , ) , N ~Me0
M~ \e /5
Me0
299 % ee, 25-30 % yield 299 % ee, 25-30 % yield
nPr M e O + W I
(MeOCH,CH,),N~" \ /
Me0
299% ee, 25-30% yield

374 I 7 I Hydrolysis and Formation ofC-0 Bonds
0 0
II 26a [12] I1 26b [12]
/s-CO,Me tB"'S-CO'H
tBu
48 % ee, 53 % yield 38% ee, 38% yield
::
.,,S-CO,Me
27a [12] 271, [12]
Ph
21% ee, 52% yield 34 % ee, 40 % yield
CO
,M
$3
0
,e
II 28a [12] CM
O
,eS
,,
R 28b [12]
To1 ' Tot
80 % ee, 32 % yield 46% ee, 58% yield
0
Ph .,,I1 29a [131 29b [13]
Me/P-CozMe
73% ee, 50% yield 82 % ee, 42 % yield
30a [ 131 30b [13]
96% ee, 45 % yield 81 % ee, 41 % yield
31a [13] 4
HC
O
,, ,P; ' R 31b [13]
Ph
299 % ee, 40 % yield 18 % yield
32a [I31 32b (131
80 % ee, 46 % yield 79% ee, 43%yield
Ph.,;/CO,Me
33a [14] 33b [14]
f7
Me Me
90% ee, 33 % yield 61 % ee, 50% yield
Ph Et Ph C0,Me
L? 34a [15] L:. 34b [15]
Et
r7C0,Me Etf E't
96 % ee, 82 % yield 83 % ee, 80 % yield

I 375
Fe
70% ee 75% ee
C0,Me
@
//c”
85% ee
36a [14]
Fe(CO),
85% ee
36b [14]
40 % conversion GO % conversion
n
37 [18]
86% ee
25 % conversion
38a [19] 38b [19]

M
Me
e$ CO,H C0,Me
Me
36% ee 40% ee
HO,C
60% ee
ye C0,Me
39a 1191
Me0,C’
x
50% ee, 50% yield
C0,Me
39b [19]
A C H N . , , , ~.CO,H
-
97 % ee, 47 % yield
40a [20, 21, 221
AcHN--sr C0,Me
87 % ee, 43 % yield
40b [20,21, 221
a: a
CO,H C0,Me
41a [23] 41b (231
Me Me
297 % ee 297 % ee
376
I I 1 Hydrolysis and Formation ofC-0 Bonds
50% ee, 47 % yield 50 % ee, 50 % yield
44a [25]
OTMe 441, [25]
0 o,COzEt
72% ee, 53% yield 96 % ee, 42 % yield
&OZEt''-.R
45 R=Me 0% ee
46 R=Et 93 % ee, 6G % yield
47 R = n-CsH1, 97% ee, 50% yield
48 R = (CH&CH=CH* 99 % ee, 60 % yield
49 R = ~C9H19 81 % ee, 62 % yield
51 [27]
LJ
299 % ee, 88 % yield 70 % ee, 10% yield
53 [27]
299 % ee, 40% yield 299 % ee, 58 % yield
299% ee, 60% yield

7 7. I Hydrolysis and Formation ofCarboxylid Acid Esters I 377
56a [28] &Me 56b [28]
OMe
32 % 93 % ee, 36% veld
57a [28] 5% [28]
-0Me
96 % ee, 42 % yield, HLE 34%, HLE
0
\LOH
58a [28] 58b [28]
0*OMe 0 OMe
30 % 94% ee, 36% yield
0*OMe
92% ee, 38% yield, HLE 34% yield, E = 65, HLE
0::
C0,Me
a""" Me
60a [23]
Me
601,[23]
a::
22% ee 17% ee
61a [23] 61b [23]
o.,,
297 % ee 297 % ee
....C0,Me
62a 1231 62b 1231
Me
297 % ee 297 % ee
I I Hydrolysis and Formation of C - 0 Bonds
O""
+..C0,Me
63b (231
CH,Br
297 % ee 297 % ee
e 0 o 64a [23] O,-.,Br....CO,Me

64b [23]
297 % ee 297 % ee
C0,Me
65a [29] 65b [29]
C0,Me
59% ee, 43 % yield 49% ee, 54% yield
<. CO,H C0,Me

GGa 1301 661, [30]
O = C l C0,Me C0,Me
95 % ee, 34% yield 95 % ee, 45 % yield

50 % conversion
671,[31]
73 % ee, 45 % yield 299 % ee, 40 % yield
68a [32, 33, 341 C0,Me 68b [32, 33, 341
C0,Me C0,Me
73 % ee, 45 % yield 70% ee, 45 % yield

64 % conversion 32 % ee, 95 % yield
53 % ee, 55 % yleld
WH C0,Me
82 % ee, 45 % yield
69a [32, 331 C0,Me
C0,Me
90% ee, 40% yield
69b [32, 33)
I 379
WozH C0,Me
70a [32, 331 C0,Me
C0,Me
70b [32,33]
82 % ee, 45 % yield 95 % ee, 40 % yield
CO,H
71a [35]
A C0,Me
71b [35b]
36 % ee, 40 % yield 57 % ee, 48 % yield
Et02C&o 72b [36]

72a [3G]
nPen
47 % ee, 35 % yield 96 % ee, 35 % yield
73 [37] 74 [37]
98% e, 41 % yield, HLE 80 % ee, 42 % yield, HLE
(yJ: j, HLE
75 [37]
HLE
76 [37]
47 % ee, 40% yield, HLE 95% ee, 34% yield, HLE
& H
GO% ee, HLE

77 [37]
380
I 1 I Hydrolysis and Formation ofC-0 Bonds
R PLE HLE
78 R=Et 98% ee R 22 R [38]
79 R=Pr 62% ee R 4 R [38]
80 R=nBu 88% ee R 38 S [38]
81 R=nPen 77% ee R 53 S [38, 391
82 R=nHex 33% ee R 90 S [38]
83 R=nHep 60% ee R 60 S 1381
84 R=nOct 65% ee R 63 S [38]
85a [40] 85b [40]
70% ee, 97 % yield, PLE 46 % ee, 79 % yield, PLE

95 % ee, 90% yield, HLE 64% ee, 83% yield, HLE
A
OH
86a [40, 38) 86b 140, 38)
HO,C(CH,), Me
83 % ee, 86 % yield, PLE 44% ee, 78% yield, PLE

76% ee, 80% yield, HLE 47 % ee, 84 % yield, HLE
6 M e
87a (401
HO,C(CH,),
pr ” Me
8% [40]
295% ee, 70% yield, PLE 84% ee, 65% yield, PLE
295% ee, 74% yield, HLE 42% ee, 71% yield, HLE
?H
88a [40] 88b [40]
G M e
295% ee, 78% yield, PLE 295 % ee, 80% yield, PLE
295% ee, 94% yield, HLE 295% ee, 84% yield, HLE
Go Me
299 % ee, 88 % yield, HLE

89a [40]
HO,C(CH,),
JH
Me
299 % ee, 86 % yield, HLE
89b (401
11.1 Hydrolysis and Formation ofCarboxylid Acid Esters
I 381
90 [37] 91 [37]
uC 7 H15
78% ee, HLE 92% ee, HLE

18% ee, PLE
92a [41]
0
Me& Me C0,Me
54% ee, 25% yield 82 % ee, 32 % yield
FO,CH,Ph
H
93a [42] 93b [42]
Me0,C C0,Me Me0,C C0,Me
5
27% ee 28% ee
Ph
>94% ee, 90% yield
94a [43]
Phk
after lactonization
94b [43]
94% ee, 80 % yield

0
mo
[a];' + 31.9
290 % ee
95 [43]
0
\y/ NMe
96a [44]
0 NMe
\\ I/
96b [44]
/ '-. .-.S \
R CH,CO,Me d CH,CO,H
R = Ph, 12% ee, 25% yield R = Ph, 10% ee, 45 % yield
R = Tol, 290 % ee, 12% yield R=Tol, 13%ee, 70%yield
0 NH
\\ 4
97a [44] .S 97b [44]
/ 5 .\
Me R R' Me
R = Ph, 14% ee, 50% yield R = Ph, 41 % ee, 12% yield
382
98a [45] 98b [45]
95% yield
[aIDzs-15.4(1.02,MeOH)
99b [46]
295% ee
1 W. K. Wilson, S. B. Baca, Y. J. Barber, T. J. Scallen, 15 S. Ramaswamy, R. A. F. Hui, J. B. Jones,]. Chem.
C. J. Morrow,]. Org. Chem. 1983,48,3960. Soc., Chem. Commun. 1986,1545.
2 H. Moorlag, R. M. Kellogg, Tetrahedron: Asymmetry 16 T. Izumi, T. Hino, A. Ishihara,]. Chem. Technol.
1991, 2, 705. Biotechnol. 1993, SG, 45.
3 H. Moorlag, R. M. Kellogg, M. Kloosterman, 17 N. W. Alcock, D. H. G. Crout. C. M. Henderson,
B. Kaptein, J. Kamphuis, H. E. Schoemaker, S. E. Thomas, ]. Chem. Soc., Chem. Commun. 1988,
J . 0%.Chem. 1990,55,5878. 746.
4 H. E. Schoemaker, W. H. J. Boesten, B. Kaptein, 18 C. Ariente-Fliche, J. Braun, F. Le Goffic, Synth.
E. C. Roos, Q. B. Broxterman, W. J. J. van den Commun. 1992,22,1149.
Tweel, J. Kamphuis, Ada, Chem. Scand. 199650, 19 M. Schneider, N. Engel, H. Boensmann, Angew.
225. Chem. 1984,96,52;Angew. Chem., In!. Ed. Engl.
5 B. Kaptein, W. H. J. Boesten, Q. B. Broxterman, 1984, 23,64.
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Tetrahedron: Asymmetry 1993,4, 1113. 1987,28,1887.
6 D. Bianchi, W. Cabri, P. Cesti, F. Francacalanci, 21 R. Csuk, P. Doerr, Tetrahedron: Asymmetry 1994.5,
M. Ricci,]. Org. Chem. 1988, 53, 104. 269.
7 P. Mohr, L. Rasslein, C. Tamm, Helv. Chim. Acta 22 R. Dernoncour, R. Azerad, Tetrahedron Lett. 1987,
1989, 30, 2513. 28, 4661.
8 P. Mohr, L. Roesslein, C. Tamm, Tetrahedron Lett. 23 E. J. Toone, J. B. Jones, Tetrahedron: Asymmetry
1989,30, 2513. 1991, 2, 207.
9 C. S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, 24 A. Basak, T. Mahato, G. Bhattacharya,
1. Am. Chem. SOC.1982,104,7294. B. Mukherjee, Tetrahedron Lett. 1997, 38,643.
10 R. Chenevert, 1.DAstous, Can.]. Chem. 1988,GG, 25 M. Tarkov, M. Bolli, B. Schweizer, C. Leumann.
1219. Helu. Chim. Acta 1993,76,481.
11 D. J. Bennett, K. 1. Buchanan, A. Cooke, 0. 26 B. Westermann, 1. Kortmann, Biocatalysis 1994,
Epemolu, N. M Hamilton, E. J. Hutchinson, a. 10, 289.
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362. Tetrahedron: Asymmetry 1993,4, 2119.
12 P. Kielbasinski, Tetrahedron: Asymmetry 2000, 11, 28 C. Tanyeli, B. Sezen, A. S. Demir, R. B. Alves,
911. A. Arseniyadis, Tetrahedron:Asymmetry 1999, 10,
13 P. Kielbasinski, R. Zurawinski, K. M. 1129.
Pietrusiewicz, M. Zablocka, M. Mikolajczyk, 29 Y. Morimoto, Y.Terao, K. Achiwa, Chem. Pharm.
Tetrahedron Len. 1994, 35,7081. Bull. 1987, 35, 2266.
14 M. Pietzsch, 0. Vielhauer, D. Pamperin, B. Ohse, 30 H. Suemune, M. Tanaka, H. Obaishi, K. Sakai,
H. Hopf,]. Mol. Catal. B: Enzym. 1999,G, 51. Chem. Pharm. Bull. 1988,3G, 15.
11. I Hydrolysis and Formation ofcarboylid Acid Esters I383
31 A. J. H. Klunder, W. B. Huizinga, A. J. M. 38 R. Fellous, L. Lizzani-Cuvelier,M. A. Loiseau,

Hulschoff, B. Zwanenburg, Tetrahedron Lett. 1986, E. Sassy, Tetrahedron: Asymmetry 1994, 5, 343.
27, 2543. 39 L. Blanci, E. Guibe-Jampel, G . Rousseau,
32 A. J. H. Klunder, F. J. L. Van Gastel, B. Tetrahedron Lett. 1988,29, 1915.
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33 F. J. C. Van Gastel, A. J. H. Klunder, B. 41 D. Moelm, N. Risch, Liebigs Ann. 1995,1901.
Zwanenburg, Red. Trav. Chim. Pays-Bas 1991 110, 42 P. Renold, C. Tarnm, Tetrahedron: Aqmmety 1993,
175. 4, 2295.
34 J . Van der Eycken, M. Vandewalle, G . Heinemann, 43 P. Barton, M. I. Page,]. Chem. SOC., Perkin Trans. 2
K. Laumen, M. P. Schneider, J. Kredel, I. Sauer, 1993, 2317.
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35 C. H. Tran, D. H. g. Crout, J. Chem. SOC.,Perkin 45 G . H. Hakimelahi, N.-W. Mei, A. A.
Trans. 11998, 1065. Moosavi-Movahedi,H. Davari, S . Hakimelahi,
36 S. Drioli; F. Felluga; C. Forzato; P. Nitti; G . K.-Y. King, J. R. Hwu, Y.3.Wen, J . Med. Chem.
Pitacco; E. Valentin]. Org. Chem. 1998,63, 2385. 2001,44,1749.
37 C. Guibe-Jampel,G . Rousseau, L. Blanco, 46 T. G . Back, K. Nakajirna,I.Org. Chem. 1998,63,
Tetrahedron Lett. 1989, 30,67. 6566.
Phosphorus- and to a lesser extent sulfur-containing racemic esters could be

resolved with pig liver esterase (29-32 and 26-28) as well. Particularly interesting
examples are the P-keto esters (45-54), which were frequently obtained with high
enantioselectivities.The enantiomer, which was hydrolyzed preferentially, suffered a
decarboxylation with formation of the corresponding ketone. Interesting examples,
showing the strategy in the application of hydrolases in kinetic resolution, are the
cyclohexadiene-carboxylates5 6 5 9 . The use of both pig liver esterase and horse liver
esterase (HLE) allows for the attainment of both enantiomers of both the acid and
the ester. In the resolution of the racemic esters 60b-G2b,pig liver esterase shows in
regard to the configuration of the C-atom to which the ester group is bound the same
preference as in the case of the corresponding rneso-diesters (Table 11.1-1).This also
holds true for the resolution of the racemates of esters 63b and 64b, which carry a
bromomethyl group instead of the methyl group. Here, the enantiomer, which
carries the carboxy group, suffers lactonization with formation of lactones 63a and
64a. A most remarkable example of a pig liver esterase-catalyzed reaction is the
apparent enantioselective hydration of alkene 98a with formation of the hydroxy
lactone 98b. The mechanism of this reaction is not known. It seems interesting to
note in this context that reaction of 98a with aqueous ammonia affords racemic 98b.
Finally, the pig liver esterase-catalyzedhydrolysis of the racemic diester derivativesof
99a and 99b shows the remarkable feature that the two enantiomers are hydrolyzed
with different regioselectivities.
A large number of mono- and bicyclic lactones (73-91)have been obtained by
using pig liver esterase in combination with horse liver esterase for the enantiomer-
differentiating hydrolysis of the corresponding racemic lactones. Interestingly, in the
series of methyl-substituted lactones (85-89), both enzymes show toward the seven-
membered lactone (86) the opposite enantiomer selectivity as compared to the other
lactones.
Acids or esters of Table 11.1-5, which can be obtained with other hydrolases as
such or ofopposite configuration, are contained in Table 11.1-13.
Kinetic resolution by pig liver esterase is not restricted to mono- and dicarboxylic
acid ester derivatives. Acetates of racemic secondary alcohols are also excellent
I 1 Hydrolysis and Formation ofC-0 Bonds
384
I Table 11.1-6. Pig liver esterase-catalyzed enantiomer-differentiating hydrolysis of esters of
racemic alcohols i n aqueous solution.
Ph
l a (1,21 l b [l, 21
( Y O A c
98 % ee, 40% yield 98 % ee, 53 % yield
0
CMe,Ph CMe,Ph
<$..OH
G O A C
67 % ee, 36% yield 95% ee, 44% yield
tBu
299 % ee
3a [31
UH
299 % ee
3b [31
after crystallization after crystallization
OCOnPr
93 % ee, 96% yield 72 % ee, 99 % yield
5a [41
nPrCOO A/
94% ee, 77% yield 86 % ee, 79 % yield
76% ee 62% ee
I
J 7. J Hydrolysis and Formation ofcarboxylid Acid Esters 385
4 Me OH
\q \q
98% ee
Me OH ACO Me
98% ee
6 o\
OH
Me Me
295 % ee, 12% yield 295 % ee, 28 % yield
OAc
fiMe Q"'
M& h e
100% ee
10a [7]
Me Me
41 % ee
10b [7]
l l a [8] Ilb (81

MeJ
o
H
f
Me
295 % ee, 43 % yield 288% ee, 40% yield
43% conversion 43% conversion
0U M e
12a [9] 12b [9]
CH, CH,
27% ee 94% ee, 32 % yield

386
Table 11.1-6.
8
(cont.).
13a [lo] 13b [lo]
91% ee, E = 14
14a [ l l , 121 '"'Ph 14b [ l l ,121

A
c~'
84% ee 85% ee
15a [ll, 121 '"'OH 151, [ l l ,121

OH dlAC
78% ee 82% ee
OCOPh
16a [13] 16b 1131
I I
R R
99 % ee, 55 % yield 100% ee, 69% yield

40% ee, 38% yield 95 % ee, 40% yield
OCOPh
17a [13]
I
Me Me
99% ee, 71 % yield 97 % ee, 45 % yield
18a [14, 151 18b (14,151

Me*Me
' I
OH OAc OAc OAc
96 % ee, 43 % yield 86 % ee, 46 % yield

50 % conversion
I
11.1 Hydrolysis and Formation ofCarboxylid Acid Esters 387
+
OAc
31 % ee, 43 % yield
50% conversion
19a [14, 151 $7
AcO OAc
31 % ee, 43 % yield
19b [14, 151
20a [16] 20b [16]

AcO Acokb-OA~
10% ee 13% ee
21a [16] 21b [16]

‘ I
OAC OH
87% ee 85% ee
4 OAc OAc
8% ee
21c [16]
AAC
OAc
15% ee
22a [16]
4
OAcOH
19% ee
221, [16]
23b [16]
AcO &OH 23a [16] Aco&OAc
21 % ee 26% ee
24b [16]
- .- L - - J
OH
36% ee
AH
84% ee
ba,
II
388
I Hydrolysis and Formation ofC-0 Bonds
24c [16]
Ac
OAc
66% ee
&OAC 25a [16] A

0 Ac 2% 1161
OH
I
OAc
87% ee 48% ee
HO &
17% ee
Ac
26a [16] Aco4
17% ee
OAc
26b [16]
4"
82% ee
I
OAc
27a 1161
OAc
85% ee
27b [16]
A
OAc OAc
81 % ee
28a [16]
98% ee
OH
28b [lG]
A
OH
73% ee
OAc
28c [16, 171
A0
OH
43% ee
Ac 29a [l6, 171 kOH
OAc
83 % ee
29b [16, 171
I
71. I Hydrolysis and Formation OfCarboxylid Acid Esters 389
OCOnPr
30a [18] 30b [18]
97 % ee, 36 % yield 99 % ee, 40% yield

35 % conversion 56% conversion
31a [19]
AcOCH, CH,OAc
58 % ee, 48 % yield 58 % ee, 47 % yield
PhTPh
Phy-Ph
OAc OAc
32a 1201
OH OH
32b [20]
84% ee, 43 % yield 92 % ee, 40 % yield

5 % monoacetate
OAc
33a [20] d O A C 33b [20]

OAc OH
295 % ee, 41 % yield 295 % ee, 49% yield
30 % conversion
OH
33c 1201
OH
295 % ee, 10% yield
cri""' OAc
54% ee, 54% yield

34a [20] aoAC OH
50 % ee, 43 % yield
341, [20]
25 % conversion 25 % conversion
34c 1201
( L O HOH
47 % ee, 53 % yield
74% conversion
1 1 Hydrolysis and Formation ofC-0 Bonds
35a [20, 211 35b [20,21]
295 % ee, 33 % yield 0 % ee, 26 % yield

25 % conversion
0::
295 % ee, 41 % yield
35c [20, 211
\,..OAC
122,231 [22,23]
Q O R
3Ga R=Ph 98% ee, 32% yield 3Gb 85% ee, 42% yield
37a R = p-MeCsH4 299% ee, 30% peld 37b 70% ee, 41 %yield
38a R = p-tBuCsH4 299 % ee, 33% yield 38b 90% ee, 36% yield
39a R = p-PhCsH4 299 % ee, 34 % yield 39b 88 % ee, 37 % yield
40a R = o-MeOCJI4 92 % ee, 47 % yield 40b 77 % ee, 45 % yield
41a R = 2,4-Me2C~& 90 % ee yield 41b GO%ee
\/.ON02
42a [24] 421,[24]
QOAc
299% ee, 35% yield, 3d, PLAP 71 % ee, 52% yield, 3d, P M P
66% ee, 55% yield, 6d, PLAP 299 % ee, 36% yield, 6d, P U P
,... OAc
( Y O H 43a [25] 43b [25]
'"'NHPh Q N H F
299 % ee, 48 % 91 % ee, SO%, E 2637
o\
%...OAc
D O " 44a [25] 441,[25]
" N(Me)Ph N(Me)Ph
299% ee, 48% yield 80% ee, 51% yield, E >477
ooH "*,, NH-m-BrC,H,
299 % ee, 38 % yield

45a [25]
0,
(...OAc
H-rn-BrC, H,
57% ee, 61 % yield, E >425
45b [25]
J1.1 Hydrolysis and Formation ofCar60xylid Acid Esters I
391
mO" 0,
*... OAc
4Ga [25] 4Gb [25]
NH-pCIC6H4
96 % ee, 49 % yield, E >989
47a 1251 47b [25]

NH-pN02C6H4
94 % ee, 32 % yield 57 % ee, 63 % yield, E >57
4% [25] 48b 125)

D O "'.NH-pMeOC,
H H,
"',NH-pMeOC, H,
79% ee, 52% yield 97% ee, 46% yield, E >39
0,
....OAc
(yoH ,,NH-eMeC€, H,
49a [25]
NH-eMeOC,H,
49b [25]
73%ee, 37%yield 42 % ee, 56 % yield
50a 1251 50b [25]

( Y O'''.H
NH-P-naphthyl NH-P-naphthyl
299 % ee, 34%yield 52% ee, 62% yield, E >335
0
\...OAc
51a [25] 51b [25]
"" N(Et)Ph N(Et)Ph
95 % ee, 32 % yield 5 1 % ee, 51 % yield, E = 65
52a 1251 52b [25]

( Y O "NHPh
65% ee, 60% yield 87% ee, 40% yield, E = 13
53a [25] 53b [25]

7 I Hydrolysis and Formation ofC-0 Bonds
aoH NHPh
92 % ee, 45 % yield
54a [25]
\,..OAc
0 N H P h
99% ee, 52% yield, E = 126
541,[25]
0"" N(Me)Ph
55a [25]
u N ( M e ) P h
55b [25]
cyoH 0
*...OAc
5Ga 1251 561, [25]
N(Et)Ph
N(Et)Ph
299 % ee, 38 % yield 70% ee, 55% yield, E >419
\...OAc
57a (251 571, [25]
G O H
NHPh
0 N H P h
89% ee, 34% yield 57 % ee, 52 % yield, E = 22
aoH N(Me)Ph
299% ee, 25% yield

58a [25]
39% ee, 50% yleld, E >292

58b (251
59a [25] 59b [25]

(-YOH N(Et)Ph
299 % ee, 25 % yield 37% ee, 73 % yield, E >295
OH OCOEt
TMe
N /
GOa [2G] Gob [26]
88% ee, 44% yield 98% ee, 43 % yield
Gla [27] Glb (271
21% ee, 60% yield 32% ee, 36% yield, E = 1,7


I 393
Me
d O ,,..H
H &TOnBu
62a [27] 621, [27]
I
86 % ee, 48 % yield 83 % ee, 49 % yield, E = 40
nBu
63a [27]
& ,s OAC
30% ee, 54% yield, E = 7,G

63b [27]
@j:H 64a [27]

O
B
un&
;
nBu
64b (271
40 % ee, 40 % yield 30% ee, 44% yield, E = 3,s
sp
nBu
H$)Q ,s..H ,...OAc
65a [27] 65b [27]
39 % ee, 49 % yield 33 % ee, 49% yield, E = 3,8
nBu
H$Q ll*. H e f C O n B u
GGa [27] GGb (271
I
GO % ee, 45 % yield 50% ee, 48% yield, E = 8,s
67 [28] 68 I281
AcO OAc
56% ee 64% ee
69a [28] 69b (281
OH OAc
24% ee 3 1 % ~
394 I
FkH
OAc 70a [29c] 70b [29c]
100% ee, 24 % yield 100% ee, 24 % yield
HO
299 % ee
71a [30]
4
299 %
OAc
71b [30]
after hydrolysis, twofold hydrolysis

acylation and hydrolysis
-5 to -10 "C, MeOH
Me-N Me-N
72a (31, 321 &02Me 72b [31, 321
OCOPh OH
82% ee, 45 % yield 71 % ee, 35 % yield
73a [31, 321 73b [31, 321

C0,Me H O Me0,C
. &
OCOPh
299% ee, 91% yield 95% ee, 85% yield
Me\
C0,Me 74a [31, 321 C0,Me 74b [31,32]
MeO&
OCOPh Meo*OH
82% ee, 35% yield 95 % ee, 3 % yield
Me N
\
N 75b [31, 321
75a [31, 321
M
&
e. C0,Me Meo%OH C0,Me
OCOPh
99 % ee, 60 % yield 99 % ee, 30 % yield
11.1 Hydrolysis and Formation ofCar6oxylid Acid Esten I 395
Me
Me \
N
\
7Ga [31, 321 76b [31, 32)
C0,Me
O
H.$
h 0 C O P h Me0 C02Me
97 % ee, 55 % yield 95 % ee, 26 % yield
1
P
0 HO OH
77a [33] 77b [33]
po
Ph Ph
78% ee 97 % ee
78a [34] 78b [34]
R = H 299% ee
R = F 93 % ee
50%conversion
OH
PhnP(O)(OMe),
79a [35]
Ph
R" P(O)(OMe),
79b [35]
7 % ee, 32 % yield 37 % ee, 3 % yield

0" OH
C0,Et 80a [3G]

OAc
I
C0,Et 8Ob [36]
90% ee, 81 % yield 54% ee
OH OH
82 [37]
93% ee
81 [37]
(3-
98% ee
396
22% ee 96% ee
42% conversion 45 % conversion
85 [37]
G P h
22% ee
48 % conversion
moH 8Ga (381 wocon 8Gb [38]
w
R = Me 36% ee, 55% yield
w
78% ee, 26% yield, E = 5
R = nPr 1%ee, 33% yield 2 % ee, 56 % yield, E = 1
%OHFe 87a [38] 87b [38]
R = Me 91% ee, 43% yield 94% ee, 43 % yield, E = 75

R = nPr
Ph
88a [39] ~I~""oA Ph
88b [39]
75% ee, 50% yield, P U P 87% ee, SO% yield, P U P

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911. 37 C. Tanyeli, A. S. Demir, A. H. Arkin,
20 K. Yamamoto, H. Ando, H. Chikamatsu, /. Chem. 1. M. Akhmedov, Enantiomer 1997, 2,433.
Soc., Chem. Commun. 1987,334. 38 T. Izumi, S. Aratani,/. Chem. Technol. Biotechnol.
22 D. Basavaiah, P. R. Krishna, T. K. Bharathi, 1994,59,403.
Tetrahedron: Asymmetry 1995,2,439. 39 T. Ganesh, S. K. Kamalesh, K. G. L. David, Indian
21 G . Caron, R. J. Kazlauskas, /. Org. Chem. 1991,56, /. Chem., Sect. B: Org. Chem. Incl. Med. Chem.
7251. 1999,388,397,
substrates for pig liver esterase in terms of an efficient resolution. In particular, pig
liver esterase is, as well as lipases, the hydrolase of choice for the kinetic resolution of
secondary cyclic alcohols and in particular of cyclohexane derivatives. This is
impressively demonstrated by the many examples contained in Table 11.1-6. Quite a
number of enantioenriched p-amino alcohols (43-59)having different ring size have
been obtained in this manner. Pig liver esterase seems to be especially well suited for
the kinetic resolution of cyclic 1,2-diols (14,15, 33-42). In the case of 1,2-diols,
having Cz-symmetry, sequential kinetic resolution can be applied for ee enhance-
ment. Resolutions of the racemates of 421,and 88b show the successful use of a
crude extract of pig liver (PUP).Further interesting examples are the resolution of
cocaine derivatives (72-76) and of amino alcohols (4-6). In the case of the resolution
of the racemate of Ga, the remote butyroxy group attached to the aromatic ring is
hydrolyzed. Within the series of racemic acetates which have been subjected to liver
esterase-catalyzed hydrolysis (Table 11.1-6),the cyclohexanoid compounds 1-3 are
particularly interesting since they are valuable chiral auxiliaries. Somewhat puzzling
results were recorded in the case of cyclic 1,z-diacetateswith homotopic acetoxy
groups. Selectivity is lowest in the case of the five-membered compound, and, not
398
I I 1 Hydrolysis and Formation of C-0 Bonds
surprisingly, diol formation is in all cases significant. As observed in the case of

cyclic rneso-dicarboxylic acid diesters (Table 11.1-1), there is a reversal in the sense of
asymmetric induction on going from the four-membered to the six-membered
diacetates. Especially noteworthy is the observation that racemic tertiary acetates are
also amenable to kinetic resolution with pig liver esterase (7, 8, 13, and 30). Kinetic
resolution of esters of acyclic alcohols with pig liver esterase has been studied only to
a minor extent. However, some of the examples described proceeded highly
selectively (60, 62, 81, 82, and 84).
Acylated alcohols and alcohols of Table 11.1-6, which can be obtained with other
hydrolases as such or of opposite configuration, are contained in Tables 11.1-15,
11.1-16, 11.1-20and 11.1-21.
11.1.1.1.2 a-Chymotrypsin
a-Chymotrypsin (CHT, E.C. 3.4.21.1) is one of the most thoroughly studied hydro-
lases['. 4. 9* 12, 21* 23. 26s 28. 33, 341, and its crystal structure has been
It is a serine protease with a pH optimum of 7.8, which acts in vivo as an
endopeptidase and catalyzes with great specificity the hydrolysis of non-terminal
amide bonds adjacent to phenylalanine, tyrosine or tryptophan. The enzyme has
been widely used for the kinetic resolution of racemic amino acid esters. From the
results of these studies and based on the crystal structure of the enzyme a useful
active site model for a-chymotrypsin has been developed['*81]. Hydrolyses catalyzed
by a-chymotrypsin are usually carried out in aqueous buffer solution in a pH range
of 7-8. In the case of a low solubility of the substrate in water cosolvents such as
methanol, ethanol, dimethylformamide or dimethylsulfoxide, up to 20 % may be
used. However, it should be noted that primary alcohols used as cosolvents may react
with the acyl-enzyme intermediate with formation of the corresponding ester
(Scheme 11.1-9).Diethyl ether, even in low concentrations, is a strong inhibitor of
the enzyme. Immobilization of a-chymotrypsin by different methods has been
described and the immobilized enzyme is commercially available. a-Chymotrypsin
has been found to be active in organic solvents of low water content also['08].
A limited number of prochiral malonates and glutarates are hydrolyzed by a-
chymotrypsin to the corresponding monoesters with synthetically useful enantiose-
lectivities(1-9) (Table 11.1-7).
Examples of enantioselective hydrolysis of cyclic diesters by a-chymotrypsin are
comparatively rare (10-14) (Table 11.1-7).Interestingly, the cyclopentanoid and the
cyclohexenoid monoesters 11 and 12 have the opposite absolute configuration to
those obtained by the pig liver esterase-catalyzed hydrolysis of the corresponding
diesters (Table 11.1-1).The keto ester 14, which is a valuable building block for the
synthesis of prostacyclin analogs, has been obtained from the corresponding a,a'-
keto diester via a-chymotrypsin-catalyzed hydrolysis followed by a decarboxylationof
the keto acid.
Monoesters and monoacetates of Table 11.1-7,which can be obtained with other
hydrolases as such or of opposite configuration, are contained in Tables 11.1-1,
11.1-2, 11.1-3,11.1-9, 11.1-11and 11.1-18.
11.1 Hydrolysis and Formation of Carboylid Acid Esters
Table 11.1-7. a-Chymostrypsin-catalyzed enantiotopos-differentiating hydrolysis of prochiral

I
399
cyclic dicarboxylic acid esters, acyclic dicarboxylic acid esters and cyclic diol diacetates and
enantiomer-differentiating hydrolysis of racemic carboxylic acid esters in aqueous solution.
PhCH,;.
Me
x
CO,H
CO,R
1 [I]R = CH3 298% ee, 90-98% yield, DMSO

2 [l]R = C2H5 298% ee, 90-98% yield, DMSO
R’ R2 ee (%) yield (“h) Ref.

3 HO CH3 64 100 [2-51
4 C6HsCOO CH3 84 68 [41
5 CcjHsCH2 CH3 92 86 [41
G CHsOCHz CH3 93 100 (41
7 CH3COO CH3 90 38 [51
3 HO CH3 55 78 [51
6
8 CHsCOO CzHs 95 84 151
9 CHjCONH CzHs 295 79 [GI
OH
42 % ee, 73 % yield 83 % ee, 87 % yield
JfJ
acoZH C0,Me
86 % ee, 90 % yield
HO
40%
nPrOCb]
ee, -
OH
13 [91
14 [lo]
OH
295 % ee, GO % yield
400
I I 1 Hydrolysis and Formation of C - 0 Bonds
R2-CH-C02R3
I
NHR’
(4
R’ R2 R3 L-acid D-ester Ref.
op (“A),yield (“A) op (“A),yield (“A)
15a, b COCH, CH3 295,88 295.68
16a, b CHO e c ~ 295,92 ~

CH3 -
-
17a.b COCH3 HoeCH -

CH3 - 295, ti9
295,50
OH
19a, b H 295.78
HO
20a, b H 295,78
21a, b H >95,40
22a,b H 295, ti0
23a. b H 295,60
Hd
11. I Hydrolysis and Formation ofcarboxylid Acid Esters

I *l
R2-CH-C0,R3
I
NHR’
(4
R’ R2 R’ L-acid D-ester Ref.
yield VO)op (%), yield (“7)
op VO),
24a, b H CH3 295,87 >95,70
a
H
25a.b COCH3 CH3 97,GO - 100
CH3 295,40 295, 50
CH3 high, 42 high, 45
28a, b COCH, 0 CH3 295,53 295, GO
29a, b COCH3
0 C2Hs 84,28 high, 93
30a, b COCH3 D ( C H . , * CH3 >98,61 86,58
CH3 290,GS 295,86
CH3 295,GS 295,81

402
Table 11.1.7. (cont.).
R2-CH-C02R3
I
NHR’
(4
R’ R2 R’ L-acid D-ester Ref.
op (“A), yield VO)op (“A), yield (“A)
~ ~~
33a, b COCH3 CH3 CzH5 >95,91 295,92 P I

34a, b COCH3 C2H50COCH2 C2Hs 295,97 295,83 P I
35a, b COCH3 C2HsOCO(CH2)2 CzH5 >95,74 295,68 ~ 3 1
36a, b COCH3 NO2 CH3 295,- ~ 4 1
R3
R2+CO,R~
R’
(*)
R’ R2 R3 L-acid wester Ref.
op YO),yield YO)op (%), yield (“A)
37a, b OCOCH, 0% H low, 100 low, 40 (251
38a, b CH3 H 295,533 >95,88 P61
39a, b OH CH3 high, 100 75,91 ~ 7 1
40a, b CH3 NH2 >95,72 >95,88 [281
I
H
41a, b CH3 NH2 295,66 295,78 [281
42a, b CH3 NH2 -11.2, 78

(1.0, MeOH)
I
403
CH,-CH-C02Rz
I
OCOR'
(4
R' D-acid (R2 = H) L-ester (R2 = C2H5) Ref.
op (%), yield (77) op (%), yield (%)
43a, b C6Hs high, 28 82,88
44a, b CH3 73,85 73,85
(4
R' D-acid (R2 = H) L-ester (R2 = C2H5) Ref.
op (%), yield (%) op (%), yield (%)
45a, b NHCOCH, 84,28 high, 93
4Ga, b OH high, 100 high, 84
47 [30] 48 [30]
42 % ee, 50% ee
C02Me
49a [30] 49b [30]
5"
44% ee 47% ee
50 [30]
270% ee
50% conversion
KC'
0 C02Me
51b [31]
86% ee, 38%yield 82 % ee, 35 % yield

7 7 Hydrolysis and Formation of C-0Bonds
404
52a [32]
I
OMe OMe
295 % ee, 71 % yield, DMF 295% ee, DMF
no hydrolysis of
the 2-isomer
53 [33] 54 [33]
Me
high op, 36% yield high op, 38% yield
Ph’
91 % ee, tBuOMe
55a [34]
PhAS !’
JH
65% ee, tBuOMe
55b [34]

absolute configuration unknown absolute configuration unknown
R= 56 [35] R=Ph 57 (351
I
H
295 % ee, 27% yield 295 % ee
P C H ,
58 [35]
XCH2 59 [35]
295 % ee 85% ee
GO (351
7 1.1 Hydrolysis and Formation ofcarboxylid Acid Esters
1435
1 F. Bjorkling, J. Boutelje, S . Gatenbeck, K. Hult, 19 Y. Hayashi, W. B. Lawson,]. Biol. Chem. 1969,244,
T. Norin, P. Szmulik, Tetrahedron 1985,41, 1347. 4158.
2 S. J. Cohen, E. Khedouri,]. Am. Chem. SOL.1961, 20 T. N. Pattabiraman, W. B. Lawson, Biochem.
83,4228. J. 1972, 126, 659.
3 P. Mohr, L. Rosslein, Ch. Tamm, Helv. Chim. Ada 21 S. G. Cohen, Y. Sprinzak, E. Khedouri,
1987, 70, 142. 1.Am. Chem. SOC.1961,83,4225. S. G. Cohen,
4 R. Roy, W. Rey, Tetrahedron Lett. 1987.28.4935. S. Y.Weinstein, ibid 1964,86, 725.
5 E. Santaniello, M. Chiceri, P. Ferraboschi, S . 22 S. G. Cohen, 1. Crossley,]. Am. Chem. SOC.1964,
Trave,]. Org. Chem. 1988, 53, 1567. 86, 4999.
G S. G. Cohen, E. Khedouri,]. Am. Chem. SOL.1961, 23 S.G. Cohen, J. Crossely, E. Khedouri, Biochemistry
83, 1093. 1963, 2, 820.
7 K. Laumen, M. Schneider, Tetrahedron Lett. 1984, 24 R. Chenevert, R. Letumeau, Chem. Lett. 1986,
25, 5875. 1151.
8 H.-J.Gais, G . Bulow, A. Zatorski, M. Jentsch, 25 S. G. Cohen, S . Y. Weinstein, J. Am. Chem. SOL.
P. Maidonis, H. Hemmerle,]. Org. Chem. 1989, 1964,86,5326.
54, 5112. 26 S. G. Cohen, A. Milovanovic,]. Am. Chem. Soc.
9 G. Baudin, B. I. Glanzer, K. S. Swaminathan, 1968,90,3495.
A. Vasella, Helv. Chim. Ada 1988, 71, 1367. 27 S. G. Cohen, L. W. Lo,]. Biol. Chem. 1970,245,
10 K. Petzold, H. Dahl, W. Skuballa, M. Gottwald, 5718.
Liebigs Ann. Chem. 1990, 1087. 28 G. M. Anantharamaiah, R. W. Roberts, Tetrahedron
11 H. T. Huang, C. Niemann,]. Am. Chem. SOC.1951, Lett. 1982, 23, 3325.
73, 3228. 29 S. G. Cohen, J. Crossley, E. Khedouri, R. Zand,
12 G. E. Hein, C. Niemann, ]. Am. Chem. SOC.1962, L. Klee,]. Am. Chem. SOC.19G3,85,1685.
84,4487. 30 J. B. Jones, P. W. Man, Tetrahedron Lett., 1973,34,
13 R. L. Bixler, C. Niemann,]. Am. Chem. SOL.1958, 3165.
80, 2716. 31 J. H. Udding, J. Fraanje, K. Gaubitz, H. Hiemstra,
14 G. E. Clement, R. Potter,]. Chem. Ed. 1971,48, W. N. Speckamp, K. Kaptein, H. E. Schoemaker,
695. J. Kamphuis, Tetrahedron: Asymmetry 1993, 4, 425.
15 J. H. Tong, C. Petitclerc, A. D’lorio, N. L. Benoiton, 32 G. Gcan, N. Satyamurthy, J. R. Barrio,
Can.]. Biochem. 1971,49,877. Tetrahedron: Asymmetry 1995, 6, 525.
16 H. T. Huang, C. Niemann, J. Am. Chem. SOC.1951, 33 N. L. Dirlam, B. S. Moore, F. J. Urban,]. Org.
73, 1541. Chem. 1987,52, 3587.
17 D. W. Thomas, R. V. Mac Allister, C. Niemann, 34 C. Cardellicchio, F. Naso, A. Seilimati, Tetrahedron
I . Am. Chem. SOC.1951,73,1548. Lett. 1994, 35,4635.
18 1. B. Jones, T. Kunitake, C. Niemann, G. E. Hein, 35 J. J. Calonde, D. E. Bergbreiter, D.-H. Wong,
I. Am. Chem. SOC.1965,87,1777. /. Org. Chem. 1988,53, 2323.
a-Chymotrypsin has been used most frequently and with much success for the
enantiomer-differentiating hydrolysis of a wide range of natural and non-natural
amino acid ester derivatives (Table 11.1-7), which usually leads in both cases to a
mixture of the L-amino acid derivative and the D-amino acid ester derivative (15-36,
52,and 53). Excellent substrates for a-chymotrypsin are aromatic amino acid ester
derivatives, but those amino acid esters which carry aliphatic or functionalized
aliphatic chains of a certain length are excellent substrates also. Even a methyl or a
nitro group as substituent is tolerated by the enzyme. Upon placement of a
methylene group between the ester group and the stereogenic center, enantiomer
differentiation is reverted and the D-acid derivative and the L-acid ester derivative are
formed (14 vs 45). Interestingly, a-chymotrypsin also exhibits high enantiomer
selectivity toward aromatic amino acids which bear a methyl group at the Ca-atom
(40-42). Nitro analogs of methyl-substituted amino acids were also found to be
suitable substrates for an a-chymotrypsin-catalyzed resolution (5660).Enantiomer-
differentiating hydrolysis of a-hydroxy acid ester derivatives is also feasible (43and
44).As organic cosolvents, dimethyl sulfoxide, dimethylformamide and tert-butanol
have been used without significant deactivation of the enzyme. The enantiomer
6
Table 11.1-8. Acetylcholine esterase-catalyzed enantiotopos-differentiating hydrolysis of prochiral
cyclic diol diacetates and of racemic monoacetates in aqueous solution.
2 [2, 31
.-OAc
OH OAc
62 % ee, 62 % yield 96 % ee, 93 % yield
OH
1
3 141 Me
.ex::;;:$' 4 [51
6
1 OAc
OAc
295 % ee, 95 % yield 98% ee, 795 yield
O
-)(H 5 161
OH OH
295 % ee, 79 % yield 100% ee, 39% yield
7 [71 8 [81
OAc
n o hydrolysis 92 % ee, 77 % yield
&82% ee
50% conversion
9a [91
bS*..OH
71% ee
50% conversion
9b 191
10 [lo]
I
NHC0,Me
72% ee,
11. I Hydrolysis and Formation ofCar6oxylid Acid Esten

I 407
g I
NHAc
OAc
NHAc
11b [lo]
92 % ee, 92 % ee,
after a second hydrolysis
1 H. Suemune, T. Harabe, Z. F. Xie, K. Sakai, Chem. 6 C. R. Johnson, C. H. Senanayke,]. Org. Chem.
Pharm. Bull. 1988, 36, 437. 1989,54, 735.
2 D. R. Deardoff, A. J . Matthews, D. S . Mc Heekin, 7 A. J. Pearson, H. S . Bansal, Y.S. h i , ]. Chem. Soc.,
C. L. Carney, Tetrahedron Lett. 1986, 27, 1255. Chem. Commun. 1987,519.
3 D. R. Deardofi, C. Q.Windham, C. L. Craney, 8 H. E. Schink, J. E. Baeckvall,]. Org. Chem. 1992,
Org. Synth. 1996, 73. 57, 1588.
4 D. M. Legrand, S. M. Roberts,]. Chem. SOC.,Perkin 9 J. V. Allen, J. M. J. Williams, Tetrahedron Lett. 1996,
Trans. 11992, 1751. 37, 1859.
5 C. R. Johnson, T. D. Penning,]. Am. Chin. SOC. 10 M. J. Mulvihill, J. L. Cage, M. J. Miller,]. Org.
1988, 110,4726. Chem. 1998,63,3357.
differentiation by a-chymotrypsin can rather accurately be explained by the active

site model for the enzyme.
11.1.1.1.3 Acetylcholine Acetylhydrolases

Acetylcholine acetylhydrolase (E. C. 3.1.1.7) or acetylcholine esterase is a well
characterized hydrolase [lOgl which is commercially available.
Acetylcholine esterase-catalyzed hydrolyses have been reported only for a small
number of prochiral diacetates (Table 11.1-8).However, several of secondary mono-
acetates, which are valuable synthetic building blocks, have been obtained with high
enantioselectivity (2-6 and 11)by using this enzyme. Acetylcholine esterase should
be considered for the hydrolysis of diacetates which are not substrates for lipases and
pig liver esterase.
Monoacetates of Table 11.1-8,which can be obtained with other hydrolases as such
or of opposite configuration, are contained in Tables 11.1-3, 11.1-7 and 11.1-18.
11.1.1.1.4 Subtilisin
Subtilisins are a family of serine proteases, the most important members of which
are subtilisin Carlsberg (from Bacillus lichenijomis) and subtilisin BPN’ (from
Bacillus amyloliquefaciens)[llol. Both enzymes are alkaline proteases with a pH
optimum of 6-9. Because of their industrial importance, both subtilisin Carlsberg
and subtilisin BPP’ have been studied intensively and are produced on a large scale.
The crystal structures of both subtilisins have been determined[82].Directed evolu-
tion and site-directed mutagenesis and chemical modification of subtilisin were
carried out in order to influence the stability, activity and enantioselectivity of the
enzyme, in particular in organic solvents[111].As in the case of other enzymes,
408 I 7 1 Hydrolysis and Formation ofC-0 Bonds
I
Table 11.1-9. Subtilisin-catalyzedhydrolysis of racernic and prochiral esters.
90% ee, 96 % yield 100% ee, 85% yield
2lJ[1]
\
C0,Me
91 % ee, 95% yield 100% ee, 86 % yield
'UH,
MeACO,H
86 % ee, 98 % yield
4a [31
N~
/
CO,H
93% ee, 50% yield 98 % ee, 50 % yield
%, \
GO,H
5a [31
93% ee, 50% yield 98 % ee, 50 % yield
"2
~a [41
PhnC0,H
97% ee, 93% yield 91 % ee, 100% yield
CO2Et 71, [4]
Me0 OMe
90 % ee, 89 % yield 95 % ee, 101% yield

7 1. I Hydrolysis and Formation ofCar6oxylid Acid Esters
I
409
8 2 % ee, 100% yield 93% ee, 95% yield
NHBoc NHBoc
CO,H CO,Me(Et)
9a [5, 61 R = SO,Na, 295% ee, 47% yield 9b (5, 61 295% ee, 48% yield
10a [5, 61 R = P(O)(OEt)z 295% ee, 40% yield 101, [5, 61 295% ee, 46% yield
l l a [5,6] R = COZCHJ’h, 295% ee, 37% yield 11b [5, 61 295% ee, 40% yield
12a [5, 61 R = COzMe, 295% ee, 49% yield 12b [S, 61 295% ee, 47% yield
13a [S, 6]R = CONHCHzPh, 295% ee, 43% yield 13b [S, 61 295% ee, 46% yield
OH
297 % ee, 96 % yield
Meozs
297 % ee, 99 % yield
M e o \2 s
NHAc
L
15b [8]
~ CO,H 15a (81 C0,Et
OH OH
298 % ee 84% ee
NHAc NHAc
1Ga [8] 1Gb [8]
CO,H C0,Me
96% ee 299 % ee
CLEC-Subtilisin
NHBoc
17a [8] 17b [S]
CO,H C0,Me
299 % ee 85% ee
18a (91 18b [9]

Br CO,H Br
97% ee, 96% ee

410
I I I Hydrolysis and Formation of C - 0 Bonds
MevMe Me
BOC-HN&EyCO2H 19a [lo] BOC-HN&iyC02Me 19111101
0 OCH,Ph 0 OCH,Ph
299 % ee, 50 % yield 299 % de, 50 % yield
starting from a 1:1 mixture starting from a 1:I mixture
20b[11]
CI C0,Et
9G % ee, 45 % yield 45 % yield
Me NHAc
21 [I21 22 [13]
Et0,C X C 0 2 H PhK C I
81 % ee, 290% yield vs/vK = 6.8, dioxane
OAc
$ OH
...
”‘ / OAc
23b [14]
80 % ee, 29 % yield 86 % ee, 25 % yield
OAc
acetone
0
C02Me
24 [15]
CO,H
88 % ee, 85 % yield
90% ee, 95% yield, CLEC-subtilisin
11.1 Hydrolysis and Formation ofCarboxylid Acid Esters I411
k:
CN
25 [16] &k
C0,Et
26 [16]
93% ee 99 % ee, 47 % yield

guanidinium chloride
Me Me
27 [17]R = CHzCeCH, 74% ee, HzO, MeCN

43 % conversion
80% ee, MeCN, H20, trioctyl amine

95 % conversion (withdynamic kinetic resolution)
R o z c D c o z H 29 [18]
28 [17] R = CHzCF3,73% ee, H20, MeCN
35 % conversion Me Me
I
83% ee, H20, MeCN, triodyl amine H
97 % conversion (with dynamic kinetic resolution)
R = (CH,),NH-CO
299% ee, 41 % yield
CO H
A
CO,H 30 [19] Ph&SO,CON~O 31 (19)
Ph&SO,tB” Me Me
298% ee, 298 % ee,
1 S.-T. Chen, K.-T. Wang, C.-H. Wong, J. Chem. Soc., 7 R. Chevenert, S.Thiboutot, Synthesis 1989,444.
Chem. Comrnun. 1986,1514. 8 Y.-F. Wang, K. Yakovlevsky, B. Zhang, A. L.
2 E. E. Ricks, M. C. Estrada-Valchs, T. L. McLean, Margolin, J. 0%.Chem. 1997,62,3488.
G. A. Jacobucci, Biotechnol. Prog. 1992,8,197. 9 M. R. Leanna, H. E. Morton, Tetrahedron Lett.
3 B. Imperiali, T.J. Prius, S. L. Fischer, S. L. Fister, 1993,34,4485.
J. Org. Chem. 1993.58,1613. 10 M. Borgenstatter, W. Steglich, Tetrahedron 1997,
4 J. Morgan, J. T. Pinhey, C. H. Sherry,J. Chem. Soc., 53,7267.
Perkin Trans. 1997,613. 11 A. SalladiXavallo, J. Schwarz, V. Burger,
5 C. Garbay-Janreguibeny, I. MeCort-Tranclepain, Tetrahedron: Asymmetry 1994,5, 1621.
B. Barbe, D. Ficheux, B. P. Roques. Tetrahedron: 12 S. Iriuchijima, K. Hasegawa, G. Tsuchihashi.
Asymmetry, 1992,3,637. Agnc. Biol. Chem. 1982,46,1907.
G K. Baczko, W.-Q. Liu, B. P. Roques, C. 13 G. Ottilina, R. Bovora, S. Riva, G. Carrea,
Garbay-lanreguibeny, Tetrahedron 19%,52,2021. Biotechnol. Lett. 1994,16,923-928.
412
14 F. Theil, H. Schick, P. Nedkov, M. Bohme, 17 P. I. Urn, P. G . Drueckhammer,]. Am. Chem. SOC.

B. Hafner, S . Schwarz,]. prakt. Chemie 1988, 330, 1998, 120, 5605.
893. 18 T.Adachi, M. Ishii, Y. Ohta, T.Ota, T. Ogawa,
15 R. ChPnevert, R. Martin, Tetrahedron: Asymmetry K. Hanada, Tetruhedron:Asymmetry 1993,4, 2061.
1992, 3, 199. 19 S. Doswald, H. Estermann, E. Kupfer, H. Stadler,
16 B. Wirtz, M. Soukup, Tetrahedron:Asymmetry W. Walther, F. Weisbrod, B. Wirz, W. Wostl, Bioorg.
1997, 8, 187. Med. Chem. 1994,2,403.
CLECs of subtilisin have been prepared r1l2]. Autohydrolysis of subtilisin-CLECs

seems to suppressed as compared to subtilisin. Subtilisin has been widely used for
the kinetic resolution of amino acid esters. Hydrolyses catalyzed by subtilisin are
usually carried out in aqueous buffer solution in a pH range of 7-8. In the case of a
low solubility of the substrate in water, cosolvents such as methanol, ethanol,
dimethylformamide or dimethyl sulfoxide may be used. subtilisin is the hydrolase of
choice for the racemate separation of natural and non-natural amino acid esters
(1-18 and 20) (Table 11.1-9). Generally, the L-amino acid ester is preferentially
hydrolyzed. Free amino acid esters as well as N-protected amino acid esters are
substrates for subtilisin. The utility of subtilisin for the synthesis of enantioenriched
amino acids is impressively demonstrated by the highly selective resolution of amino
acid esters, the side chains of which contain functional groups (9-15).Frequently,
subtilisin is preferred in the large scale resolution of amino acid esters rather than
other hydrolases because of its lower price. Not only racemic amino acid esters but
also other racemic carboxylic acid esters have been resolved with subtilisin. Im-
pressive examples in terms of selectivity and efficiency are the hydrolyses yielding
the functionalized esters 25, 26,and 29-31, which were prepared on a large scale.
Activity and selectivity of the enzyme in the kinetic resolution of the racemic esters of
30 and 31 could be improved by addition of dimethyl sulfoxide or guanidinium
chloride, which was used at a concentration of 10 mM. A particularly interesting
example of a kinetic resolution with subtilisin is that of the racemic thioesters
derivatives of 27 and 28.This was carried out in the presence of a base to a ensure a
dynamic kinetic resolution (see Sect. 11.1.1.2.1.2) through base-catalyzed racemiza-
tion of the non-hydrolyzed enantiomeric thioester.
11.1.1.1.5 Lipases
Lipases (triacyl glycerol acyl hydrolases, E.C. 3.1.1.3) are a unique class of hydro-
lases [113-1151 for asymmetric synthesis based on prochiral or racemic substrates. The
application of lipases as biocatalysts has been reviewed emphasizing different
aspects in a number of books[', 21, 22, 2 6 28, 30. 322 34, 351 and jour-
rials[". 14. 18, 19, 24, 25, 27, 29, 31. '171. Lipases are catalytically active in water, in
mixtures of water and water-immiscible or miscible organic solvents, in almost

anhydrous organic solvents, and in supercritical fluids [34, 361 and ionic liq-
UidS["8, 119l . They are available from plants, mammals, and microorganisms in
considerable numbers, which explains in part their versatility for asymmetric
synthesis. Lipases are typical induced-fit enzymes, accepting non-natural substrates
of enormous structural diversity.
There is some confusion in the literature regarding the origin and the name of
I
413
some microbial lipases. The lipase from Pseudomonas cepacia from Amano was
formerly called lipase from Pseudomonas Jluorescens, and was most recently re-
identified as Burkholderia cepacia lipase. Candida cylindracea lipase was re-identified
as C. rugosa lipase and Mucor miehei lipase was re-identified as Rhizomucor miehei
lipa~eI~~1. Candida antarctica produces the two lipases A and B that are/were
available either as a mixture or in both individual forms. In order to avoid any further
confusion in this text, by and large the names from the original papers have been
used, but the special supplier names have been translated into names referring to
the biological origin so far as unambiguously possible.
The lipases most used until now are the commercially supplied pig pancreas lipase
(PPL), Pseudomonas cepacia lipase (PCL) or P. juorescens lipase (PFL), Candida
cylindracea (CCL) or C. rugosa lipase (CRL),Pseudornonas sp. lipase (PSL), increas-
ingly Candida antarctica B lipase (CAL-B) and to a lesser extent further lipases
mentioned in Tables 11.1-10 to 11.1-25, and cholesterol esterase (CE). CAL-B is a
recombinant protein produced in Aspergillus oryzae accepting a broad range of
substrates and conditions. A special group of hydrolases, which are considered as
lipases, are the cholesterol esterases (CE), found in mammals and microorgan-
isms (ll31.
About 70 different lipases are commercially available. Most of these are pre-
sumably serine hydrolases containing a serine residue in their active site and
featuring presumably the triad Ser ... His .... Asp. The crystal structures of the 13
different lipases have been determined[84-871.
The molecular weight of the known lipases in their active, native form ranges from
30 to 65 kDa. Lipases are generally soluble in water and insoluble in organic
solvents, and may be strongly adsorbed at the air/water interface.
Lipases are available and applied as lyophilized powders, in covalently and non-
covalently immobilized form on inorganic or organic carriers, in sol-gel mate-
rial['20plZ1l and as CLECs["', lZ2].Most mammalian lipases exhibit pH optima
ranging from 8 to 9 and most microbial lipases from 5.6 to 8.5. The temperature
range for optimal activity is between 30 and 50 "C. In the case of labile substrates or
insufficient enantiomer selectivity, hydrolysis may be carried out in water-saturated
water-immiscibleorganic solvent such as diisopropyl ether, hexane or cyclohexane.
Most lipases are applied as crude materials consisting of a mixture of proteins that
may even contain other hydrolases together with stabilizing solid supports. Pig
pancreas lipase is a glycoprotein which exists as a mixture of isoenzymes differing in
the glycan moiety of the enzyme. Crude pancreas lipase contains presumably
another carboxyl esterase that may be responsible at least in part for the high
enantioselectivity frequently observed with this enzyme in hydrolysis and esterifica-
tion[44, 123-1253. Therefore, an isolated lipase of the same origin may have different
activities and selectivities depending on the isolation and purification procedures of
the individual suppliers. Some of these problems can be overcome, however, by the
application of purified lipases, which are also commercially available. Lipases exhibit
high catalytic activity in water and an even higher activity in two-phase systems
composed of water and a water-immiscible organic solvent or water and a liquid
substrate. In two-phase systems like water and tert-butyl methyl ether or water and
414
Table 11.1-10. Lipase-catalyzed enantiotopos-differentiating hydrolysis of prochiral acyclic diol

diacetates in aqueous solution (MJL Mucorjauanicus lipase, PFL Pseudomonasfluorescens lipase,
PPL ig pancreas lipase, PCL Pseudomonas cepacia lipase).
R' R2 R3 Lipase ee [%) yield (99) Ref.
1 (CH3)zCH H Ac PPL,crude 37 - 111

1 (CH3)zCH H Ac PPL" 75 91 111
1 (CH3)2CH H Ac PPL, pure very slow hydrolysis (11
2 CHzPh H Ac PPLa 61 65 11, 21
3 CHFCH-CH~ H Ac PPL 95 34 [41
4 CHz=CH-(CH2)2 H Ac PPLa 295 80 121
5 Ph H Ac PPL" 295 91 PI
5 Ph H Ac PFL 94 41 [31
6 C-CBHII H Ac PPLa 60 96 121
7 (E)-n-Pent-CH=CH H Ac PPL 84 49 [5,61
8 (E)-n-Pent-CH=CH H Ac PPLb 95 63 [5,61
8 (E)-n-Pent-CH=CH H Ac PPLc 93 59 [5,61
9 (2)-n-Pent-CH=CH Ac H PPL 50 43 161
9 (2)-n-Pent-CH=CH Ac H PPL' 53 31 [61
9 (2)-n-Pent-CH=CH Ac H PPLb 55 44 PI
10 (E)-i-Pr-CH=CH H Ac PPL 90 70 [GI
10 (E)-i-Pr-CH=CH H Ac PPLc 97 75 [61
10 (E)-i-Pr-CH=CH H Ac PPLb 88 71 [GI
11 (2)-i-Pr-CH=CH Ac H PPL 21 25 161
11 (2)-i-Pr-CH=CH Ac H PPLc 15 20 [61
12 n-Hep H Ac PPL' 70 56 [61
13 i-Pent H Ac PPLc 72 47 [61
14 n-Pent-C=C H Ac PPL 78 57 FI
PPLC 80 50 I61
PPLb 82 61 [61
15 i-Pr-CIC H Ac PPL 82 67 PI
PPL' 85 65 [61
PPLb 88 71 (61
16 H Ac PPL 67 29 161
17 H Ac PPL 2 45 [GI
Me0,C
Hc,, OCOR
18 PhCH,O ;CoAc OH l9
Table 11.1-10. (cont.).
I
415
R Ref. Ref.
nPr, 65 % ee, 29 % yield, PPLd [7] 60 % ee, 40 % yield, PPL [81

nBu, 68 % ee, 36 %yield, PPLd [7] 80 % ee, 40 %yield, PPLd [81
nC5HI1, 65 % ee, 29 %yield, PPLd [7] 88 % ee, 45 %yield, PPLc PI
nC6H13,70 % ee, <17% yield, PPLd [7] 91 % ee, 75 %yield, PFL PI
nC7H15, 84 % ee, 48 %yield, PPLd.'
20 [lo] 21 [lo]
CbzHN
297 % ee,55 %yield, PPL 73 % ee, 46 %yield, PDL

91 % ee, 75 %yield, PPLg
R = ME-(CH*)ls
57 % ee, 66 %yield, PPL
87 % ee, 49 % yield, PPLg
22 [lZ] \o<oconBu 23 (131
OH
89 % ee, 82 % yield, PPL 95 % ee, -, MJL
0
x0 24 [14, 151
298 % ee, 98 % yield, PFL 97 % ee, 83 % yield, PFL
96 % ee, 85 % yield, PFL 299 % ee, 33 % yield, PFL
~ ' " ' . "[211 " ~ ~ OH 2g

28 [20]
&OH
\OAc OAc
78 % ee, 80 % yield, PPLh 98 % ee, yield 63 %, PCL'
FI
95 % ee, yield 65 %, PCL'

416
Table 11.1-10. (cont.).

a Protein fraction from chromatography of e 30 % MeOH
crude PPL f 15 % Tetrahydrofuran
b 10 % tee-BuOH g Hexane
c Diisopropyl ether h 15 % t-BuOH
d Absolute configuration unknown i 30 % Diisopropyl ether
1 G. M. Ramos Tombo, H:P. Schar, X. Fernandez I 11 K. Prasad, H . Estermann, C:P. Chen, 0. Repic,
Busquets, 0. Ghisalba, in: C. Laane, J. Tramper, G. E. Hardtmann, Tetrahedron: Asymmetry 1990, 1,
M. D. Lilly (Eds.), Biocatalysis in Organic Media, 421.
p. 43, Elsevier, Amsterdam, 1987. 12 Y.-F. Wang, C . 4 . Chen, G. Girdaukas, C. J . Sih,
2 G. M. Ramos Tombo, H:P. Schar, X. Femandez 1 J . Am. Chem. SOC.1984, 106, 3695.
Busquets, 0. Ghisalba, Tetrahedron Lett. 1986,27, 13 B. Wirz, R. Schmid, J. Foricher, Tetrahedron:
5707. Asymmetry 1992,3,137.
3 S. Atsumi, M. Nakano, Y. Koike, S. Tanaka, 14 C. Bonini, R. Racioppi, L. Viggiani, G. Righi,
M. Ohkubo, T. Yonezawa, H. Funabashi, J. L. Rossi, Tetrahedron: Asymmetry 1993, 4, 793.
Hashimoto, H . Morishima, Tetrahedron Lett. 1990, 15 C. Bonini, R. Racioppi, G. Righi, L. Viggiani,
31, 1601. J . Org. Chem. 1993, 58, 802.
4 Y.-F. Wang, C . J. Sih, Tetrahedron Lett. 1984, 25, 16 N. Adje, 0. Breuilles, D. Uguen, Tetrahedron Lett.
4999. 1993, 34,4631.
5 G. Guanti, L. Banfi, E. Narisano, Tetrahedron Lett. 17 T. Itoh, H. Ohara, Y. Takagi, N. Kanda,
1989,30,2697. K, Uneyama, Tetrahedron Lett. 1993, 34, 4215.
6 G. Guanti, L. Banfi, E. Narisano, Tetrahedron: 18 Y-B. Seu, Y:H. Kho, Tetrahedron Lett. 1992, 33,
Asymmetry 1990, I, 721. 7015.
7 J. Ehrler, D. Seebach, Liebigs Ann. Chem. 1990, 19 2:F. Xie, H. Suemune, K. Sakai, Tetrahedron:
379. Asymmetry 1993,4,973.
8 D. Breitgoff, K. Laumen, M. P. Schneider, 20 G. Guanti, E. Narisano, R. Riva, Tetrahedron:
J. Chem. Soc., Chem. Commun. 1986,1523. Asymmetry 1997,8,2175.
9 V. Kerscher, W. Kreiser, Tetrahedron Lett. 1987,28, 21 T. Yokomatsu, T. Minowa, T. Murano, S . Shibuya,
531. Tetrahedron 1998, 54, 9341.
10 Y.-F. Wang, J. J. Lalonde, M. Momongan, D. E.
Bergbreiter, C.-H. Wong, J. Am. Chem. SOC. 1988,
110,7200.
hexane, much higher reaction rates and enantioselectivities are most frequently
observed. A synthetically useful alternative to the lipase-catalyzed hydrolysis of an
ester in aqueous solution for the attainment of a chiral carboxylic acid or alcohol is
the lipase-catalyzed alcoholysis of an ester described in Sect. 11.1.1.3.
Generally, through (a) hydrolysis or alcoholysis of a prochiral dialkanoate or
racemic alkanoate by water in homogeneous and heterogeneous aqueous/organic
mixtures or by an alcohol in an organic solvent and (b) acylation of the correspond-
ing prochiral diol or racemic alcohol in an organic solvent, access to both enantio-
mers of the corresponding monoacetate and alcohol, respectively, is provided with
one enzyme [(a)Tables 11.1-10to 11.1-12, 11.1-14 to 11.1-16,and 11.1-22; (b) Tables
11.1-17 to 11.1-211(Scheme 11.1-12).Hydrolysis and ester formation are enantio-
complementary, which means that one and the same lipase for a given substrate
reacts preferentially with the same enantiotopic group or the same enantiomer
irrespective ofwhether it is an ester or an alcohol. Enantioselectivities and yields may
differ in hydrolysis and alcoholysis, respectively, and in acylation. A combination of
these routes may be advantageous in a given case for the attainment of both
enantiomers in high enantiomeric purity (Schemes 11.1-11 and 11.1-14). One
cannot expect, however, that a lipase will transform a non-natural substrate in an
optimal manner. Therefore, different, more or less empirical, strategies have been
7 1.1 Hydrolysis and Formation ofCarboxylid Acid Esters
I
417
developed to improve reactivity and/or selectivity. The substrate structure and the
origin of the lipase mainly determine reactivity and selectivity in a lipase-catalyzed
reaction. Hence, the first step of an envisaged lipase-catalyzed kinetic resolution or
desymmetrization of a given substrate comprises rapid screening of available
lipases [35e1. There is no simple relationship, however, between structural parameters
of a given substrate, the origin of the lipase and the outcome of the reaction. On the
other hand, models have been developed in order to understand and predict the
behavior of certain lipases toward structural properties of substrates with regard to
reactivity and selectivity[57,74-79, 126-1321 that help to optimize the reaction by modify-
ing the substrate structure. As mentioned before, lipase activity and selectivity are
strongly influenced by the medium used for the desired reaction. Having identified a
suitable lipase, variation of the medium - solvent engineering - might be the next
step in order to optimize the outcome of the reaction. An additional fine tuning of
the reaction conditions is achievable by using certain additives, which may have a
beneficial influence on the microenvironment of the lipases and hence on their
selectivity and/or reactivity['33].Finally, an increase of the selectivity of the lipase may
be gained by lowering the temperature of the reaction mixture.
Primary acyclic diacetates are substrates par excellence for lipases (1-24,2630)
(Table 11.1-10).Prochiral 1,3-propane diol derivatives have been most thoroughly
studied in terms of the influence of the substrate structure, the composition of the
reaction medium and the origin and purity of the lipase on the enantioselectivity.A
comparison of the hydrolysis of 2-alkyl substituted 1,%propane diol diacetates with
crude pig pancreas lipase, highly purified commercial pig pancreas lipase and a
carboxyl esterase fraction isolated from crude pig pancreas lipase showed that the
latter gave the monoacetate 1 with higher enantioselectivity and reaction rate than
the first two (Table 11.1-10).Imitating the in vivo conditions for the action of lipase
on triglycerides by addition of diisopropyl ether to the aqueous solution and carrying
out the hydrolysis in the two-phase system leads in some but not all cases to a higher
enantioselectivity and reaction rate, as demonstrated for the monoacetates 7 and 8.
Unsaturation in the alkyl chain frequently leads to the monoacetate of a higher ee
value as exemplified with 16 and 17. Comparison of the enantioselectivitiesof the
hydrolysis of diacetates to the corresponding monoacetates is often complicated by
the lack of information on the amount of diol formed. The later arises from the
hydrolysis of the monoacetate that may proceed under enantiomer differentiation,
and thus the ee value of the monoacetate will be a composite of two enantioselective
processes. Interestingly, upon changing the configuration of the double bond of the
substituent R from (q to (Z) the enantiotopic group recognition by pig pancreas
lipase inverts, as demonstrated by the monoacetates 8 and 9 as well as 11 and 12
(Table 11.1-10).
Group-selective and enantioselective hydrolyses as well as the influence of the
alkyl group of the acyl function and the cosolvent upon the enantioselectivity are
demonstrated by the pig pancreas lipase-catalyzedhydrolysis of the methoxycarbonyl
substituted diacylated propanediol corresponding to the monoesters 18.
Protected glycerol monoacetate 19 can be prepared in acceptable enantiomeric
purity and yield by crude pig pancreas lipase or Pseudomoms Juorescens lipase-
418
Table 11.1-11. Lipase-catalyzed enantiotopos-differentiating hydrolysis of prochiral cyclic diol

dialkanoates in aqueous solution (CCL Candida Gylindracea lipase, PFL PseudomonasPuorescens
lipase, M M L Mucor miehei lipase, CVL Chromobacterium uiscosum lipase, PPL pig pancreas lipase,
M J L Mucorjauanicus lipase, RSL Rhizopus sp. lipase, PCL Pseudomonas cepacia lipase, CCL,
Ceotricum candidum lipase, ANL Aspergillus niger lipase, FSPC Fusarium solani pisi cutinase, CRL
Candida rugosa lipase, CAL-B Candida antarctica B lipase, LIP Pseudomonas sp. lipase-Toyobo, RDL
Rhizopus delemar lipase, MSL Mucor sp. lipase, CAL Candida antarctica lipase, not specified).
R
1
4-11
Me 72 % ee, 94 % yield, PPL [l]
93 % ee, 74 %yield, PPL [2]
90 % ee, 83 %yield, PFL [3]
n-Pr 94 % ee, 84 %yield, PPL [2]
299 % ee, 100 %yield, PPL [4]
3 4
OH
R
Me 88 % ee, 94 % yield, PPL [l] 86 % ee, 57 %yield, PPL [6]
96 % ee, 78 %yield, PPL [2] 297 % ee, 75 %yield, PFL [7]
295 % ee, 87 % yield, PFL [3]
n-Pr 96 % ee, 80 % yield, PPL [l]
5 O C o A OHc 6
R
Me 88 % ee, 94 % yield, DPL [l] 50 % ee, 70 %yield, PPL [5]
89 % ee, 90 % yield, PPL [2] 77 % ee, 78 %yield, PPL [6]
295 % ee, 86 %yield, PFL 131 (purified PPL)
n-Pr 96 % ee, 80 %yield, PPL [l]
C
K:
7 151

11.1 Hydrolysis and Formation of Carborylid Acid Esters
I
419
Table 11.1-11. (cont.).
78 % ee, 77 % yield, PPL 30 % ee, 75 % yield, PPL

(purified PPL)
R’ R’
HO H 68 % ee, 80 yield, PPL 41 % ee, 73 % yield, PPL
0-t-Bu H 26 % ee, 80 yield, PPL 81 % ee, 85 % yield, PPL
OMe H 52 % ee, 65 yield, PPL 98 % ee, 91 %yield, PFL
OEt H 66 % ee, 78 yield, PPL
H OAc 90 % ee, 60 yield, PPL
H C1 88 % ee, 81 yield, PPL
H C1 95 % ee, 88 yield, PPL (purified PPL)
H SPh 96 % ee, 65 yield, PPL
H SOzPh 68 % ee, 72 yield, PPL
H N3 91 % ee, 85 yield, PPL
13 [7-91 14
13 % ee, 70 %yield, PPL R

0 % ee, -, PFL
97% ee, 88 yield, PFL Me 78 % ee, 81 yield, PPL [l]
87 % ee, 67 yield, PPL [2]
50 % ee, 54 yield, PPL [3]
n-Pr 84 % ee, 24 yield, PPL (21
coAc OH

15
[l,101
92 % ee, 90 %yield, PFL [31
R
Me 41 % ee, 35 % yield, PPL
Et 33 % ee, 57 %yield, PPL
i-Pr 32 % ee, 68 %yield, PPL
tBu 16 % ee, 71 %yield, PPL
420
Table 11.1-11. (cont.).
R
Me 80 % ee, 20 %yield, CCL 99 % ee, 84 %yield, PFL
Et 26 % ee, 69 % yield, CCL 99 % ee, 66 % yield, MJL
i-Pr 28 % ee, 75 % yield, CCL 85 % ee, 50 %yield, PPL
t-Bu 4 % ee, 71 %yield, CCL 75 % ee, 37 %yield, CCL
n-Bu 299 % ee, 75 %yield, MJL
68 % ee, 85 %yield, CCL
-CoconPI 20 [S, 141

><
:OAC OH
LOH
55 % ee, 77 %yield, PPL 96 % ee, 79 % yield, PFL
(IoA OH
22 [16-181
83 % ee, 26 % yield, CCL 299% ee, 39% yield, PFL
0
PH
BnOQoH 24 123)
OAc
OAc
50 % ee, 82 % yield, CCL 36 % ee, 21 %yield, ANL

66 % ee, 83 % yield, RSL 18 % ee, 1 3 % yield, PFL
92 % ee, 87 %yield, PPL 84 % ee, 16 %yield, PFL
92 % ee, 84 %yield, PFL
95 % ee, 85 % yield, MML
91 % ee, 76 %yield, CVL
>99 % ee, 85 %yield, MSL
99 % ee, 90 % yield, CAL-B
Table 11.1-11. (cont.).
7 1 . I Hydrolysis and Formation ofCarboxylid Acid Esters
I
421
q
R
R OAc
H 44 % ee, 40 %yield, CCL

25 [24] G
N3
O B
OAc
297 % ee, -, GCL

n 26 [25]
‘b0n
.
Me 100 % ee, 61 %yield, CCL
NM
le
27 [25] 28 [25]
6
AcO OAc
297 % ee, GO % yield, PCL 298 % ee, 100 % yeld, ANL
r O H
29 [26, 271 30 [28]
6
OAc
95 % ee, 90 %yield, PPL 79 % ee, 64 %yield, PCL
0
r O A C
ent-30 [29] 31 [30, 311
OH O
‘H
297 % ee, -, CRL 55 % ee, 98 %yield, PPL
30 % ee. 45 % yield, CCL
32 [32] 33 [32]
6 OCOPh
; O ”’x0
o::b
295 % ee, 94 % yield, FSPC 295 % ee, 87 %yield, FSPC
34 [33] 35 [33]
HO AcO
35 % ee, -, PCL 298 % ee, 89 %yield, PCL
1 1 Hydrolysis and Formation of C-0 Bonds
422
emoH
0 / 3 OAc 36[34]
0
37 [35]
95 % ee, 66 % yield, PPL 70 % ee, 80 % yield, PCL
38 [35] 39 [36]
>98 % ee, 88% yield, PCL 96 % ee, 72 % yield, PFL
OTBDMS
I
40 [371 41 [38]
LOA~
>95 % ee, 89 % yield, PSL >95 % ee, 70 % yield, CAL
HO A
0
86 % ee, 100 %yield,CAL
Ac
42 [38]
AcO & L O
Cbz
I
H
>98 % ee, 82 %yield,ANL

43 [39]
OMOM
I
C0,Me
44 [391 45 [40]
AcO&OH
Cbz HO "" "OAc
>98 % ee, 76% yield, ANL 77 % ee, 39 % yield, PFL
O,,
C0,Me C0,Me
46 1401 47 [40]
HO"" "OAc
Me Me
88 % ee, 49 %yield, PFL >99 % ee, 89 %yield, DSL

Table 11.1-11. (cont.).
7 7 . 7 Hydrolysis and Formation of Carboxylid Acid Esten
I
423
C0,Me C0,Me
48 [40] 48 [40]
Et Et
>99 % ee, 88 % yield, PFL 87 % ee, 49 % yield, PPL
49 [41] 50 [41]
AcO "*
>99 % ee, 53 %yield, LIP >99 % ee, 51 % yield, LIP
"::0$ 51 [42] co(:; 52 [42]
OH OH
>99 % ee, 95 % yield, RDL 95 % ee, 86 %yield, RDL
>99 % ee, 61 %yield, PFL 33 % ee, 73 %yield, PFL
94 % ee, 60 % yield, PPL
rOAc rOAC
53 [42] 54 [42]
95 % ee, 64 % yield, RDL 95 % ee, 95 % yield, RDL

87 % ee, 39 %yield, PPL 16 % ee, 60 %yield, PPL
55 [43] 56 [43]
Ac""''Q I
Bn Cbz
>98 % ee, 73 %yield, PFL >98 % ee, 77 % yield, PFL
57 [441 58 [45]
O
-w
>99 % ee, 70 %yield, CAL-B R: CHz-CH=CHz, CHI-CH=CHCH~(E),
CH2-CH=C(Cl)CH,(E),CH2-CrC-CH3, CHI-Ph
>98 % ee, 62-80 % yield, CCL
40 Y. Zhao, Y Wu, P. De Clercq, M. Vandewalle,
44 F. Theil, S . Ballschuh, M. von Janta-Lipinski, R. A.

I
425
P. Maillos, J.-C. Pascal, Tetrahedron: Asymmetry Johnson,I. Chem. Soc., Perkin Trans. 1 1996, 255.
2000,l I , 3887-3900. 45 P. Renouf, J:M. Poiner, P. Duhamel, ]. Chem.
41 H. Konno, K. Ogasawara, Synthesis 1999, 1135. SOC.,Perkin Trans. 1 1997, 1739.
42 M. Tanaka, Y. Norimine, T. Fujita, H. Suemune, 46 T. Taniguchi, R. M. Kanada, K. Ogasawara,
K, Sakai,]. Org. Chem. 1996,61,6952. Tetrahedron: Asymmetry 1997,8, 2773.
43 B. Danieli, G. Lesma, D. Passarella, A. Silvani, 47 K. Toyama, S. Iguchi, T. Oishi, M. Hirama, Synlett,
/. Org. Chem. 1998.63, 3492. 1995,1243.
catalyzed hydrolysis of the corresponding diacetate. Here, too, enantioselectivity of

the hydrolysis by crude pig pancreas lipase is considerably improved if the reaction is
run in the two-phase system composed of water and diisopropyl ether.
Glycerol diacetate derivatives with chain type substituent in the 2-position are
hydrolyzed with crude pig pancreas lipase in a two-phase system composed of water
and hexane to the monoacetates 21 with good enantioselectivity. Hydrolysis in
aqueous solution alone is much less selective. The 2-benzyloxycarbonylamino-
substituted propanediol monoacetate 20 is also accessible with a high ee value by pig
pancreas lipase-catalyzed hydrolysis. Monoacetates 23-27 can serve as a good
illustration of the scope of lipases because of the number of different species
available. The monoacetate 24 is a notable example since it documents the surpris-
ing ability of Pseudomonas fluorewens lipase to differentiate between enantiotopic
groups located relatively far from the stereogenic ring atoms. Monoacetates 27 and
29 are of opposite configuration compared to those obtained from the same achiral
diacetates via pig liver esterase-catalyzedhydrolysis (Table 11.1-3).
Monoacetates of Table 11.1-10 which can be obtained with other hydrolases as
such or of opposite configuration are contained in Tables 11.1-4 and 11.1-17.
One of the most successful applications of lipases lies in the hydrolysis of cyclic
meso-configured dialkanoates, mainly diacetates, to the corresponding chiral mono-
alkanoates (1-61) (Table 11.1-11). However, the attainment of high enantioselectivity
is not restricted to primary dialkanoates. Cyclic secondary dialkanoates are good
substrates too (Table 11.1-11).There seems to be no restriction in regard to the ring
size. Heterocyclic systems are tolerated by the various lipases. Reversal of enantio-
topic group recognition in a series of structurally closely related substrates as
frequently observed in the case of pig liver esterase-catalyzed hydrolysis is usually
not observed with a lipase. This is illustrated by the series of cyclopentanoid
dimethanol diacetates 7-12. Enantioselectivitycan be enhanced in many cases with a
given lipase by either resorting to hydrolysis in a two-phase system, addition of a
cosolvent, or changing the nature of the acyl group (17). If these measures fail,
resorting to another lipase may lead to success. This is exemplified by the cyclopente-
noid monoacetate 23, which is obtained by Candida cylindracea lipase with an ee
value of 50 %, by Pseudomonasfluorescens lipase with an ee value of 92 % and by Mucor
sp. lipase or by Candida antarctica B lipase with ee values of 2 99%. A frequently
encountered synthetically very attractive situation is illustrated by the synthesis of
the enantiomeric monoacetates 30 and ent-30. The two enantiomers are accessible
with two different lipases.
Tetrahydropyran derivatives 37, 38, 41 and 42 as well as the piperidine derivatives
44, 45, 55 and 56 can be prepared with high enantiomeric purity. Bi- and tricyclic
426
Table 11.1-12. Lipase-catalyzed enantiotopos-differentiating hydrolysis of prochiral acyclic and

cyclic dicarboxylic acid diesters in aqueous solution (CCL Candida cylindracea lipase, PPL pig
pancreas lipase, PSL Pseudomonas sp. lipase, CVL Chromobacterium viscosum lipase,
CE cholesterol esterase).
R?. CO,H
R'
R' R2 R3 Lipase ee ("5) yield (%) Ref.
1 CF, H Me CCl n o hydrolysis - PI

2 F Et Me CCL 99" 87 PI
3 F Me Me cc1 95 74 PI
4 F Me Et CCL 91 87 PI
5 F H Et PPL 61 23 PI
5 F H Et CCL 62a 70 PI
6 F Et Et CCL 93" 87 PI
7 F n-Pr Et CCL 33" 30 PI
8 F n-Bu Et CCL lla 78 PI
a Absolute configuration n o t determined
S/\/CO,H
R~*sc/\ozR~
R' R2
Me 298 % ee, 90 %yield, PSL, CVL [31

CI CH2CONEt2 298 % ee, 90 %yield, CCL [3,41
10 151 11 [51
97 % ee, 97 %yield, PPL 6 % ee, 48 %yield, PPL
PPL, n o hydrolysis
MeoxcozH
Me0 C0,Me
92 % ee, 90 % yield, CCL

13 161
Table 11.1-12. (cont.).
I
427
aNo2
H O z C ~ ~ ~ H z O C O t B u
14 [7-91
HO,C
ens14 [9]
Me Me Me
I I
CH,OMe CH,OMe
299 % ee, 80 % yield, lipase B, 89 % ee, 87 % yield, PSL,

diisopropyl ether/H20 diisopropyl ether/H20
299 % ee, 80 % yield, PSL,
diisopropyl ether/HzO
90 % ee, 95 % yield, CE
1 T. Kitazume, T.Sato, N. Ishikawa, Chem. Lett. G H. J. Bestmann, U. C. Philipp, Angew. Chem. 1991,
1984,1811. 103,78;Angew. Chem. Int., Ed. Engl. 1991,30,86.
2 T. Kitazume, T.Sato, T. Kobayashi, J. Tain Lin, 7 H. Ebiike, Y.Terao, K. Achiwa, Tetrahedron Lett.
j . Org. Chem. 1986,51,1003. 1991,32,5805.
3 D.L. Hughes, J. J. Bergan, J. S. Amato, P. J. Reider, 8 H. Ebiike, K. Maruyama, K. Achiwa, Tetrahedron:
E. J. J. Grabowski, j . Org. Chem. 1989,54,1787. Asymmetry 1992,3,1153.
4 D. L. Hughes, 2. Song, G . B. Smith, I. J. Bergan, 9 Y. Hirose, K. Kariya, J. Sasaki, Y. Kurono,
G. C. Dezeny, E. J . J . Grabowski, P. J. Reider, H. Ebiike, K. Achiwa, Tetrahedron Lett. 1992,33,
Tetrahedron: Asymmetry 1993,4,865. 7157.
5 Y. Nagao, M. Kume, R. C. Wakabayashi, 10 R.Chenevert, R. Martin, Tetrahedron: Asymmetry
T. Nakamura, M. Ochiai, Chem. Lett. 1989,239. 1992,3,199.
derivatives such as 36, 40, 49, and 50 are obtained from the corresponding meso-
diacetates.
The monoacetates 58, 59 and GO (Table 11.1-11)are products of the hydrolysis of
prochiral enol diacetates.
Monoalkanoates of Table 11.1-11which can be obtained with other hydrolases as
such or of opposite configuration are contained in Tables 11.1-3, 11.1-7, 11.1-9 and
11.1-18.
A limited number of acyclic and cyclic prochiral dicarboxylic acid diesters were
found to be good substrates for hydrolysis catalyzed by lipases (Table 11.1-12).
Notable examples which give a good illustration of the potential of hydrolases as well
as of the trial and error approach one relies on to a certain extent are the dithio acetal
derivative 9 and the fluoro alkyl malonates 1-8. The dithio monoester 9 is obtained
with different lipases with high enantioselectivities and yields despite its remote
chiral center. Candida cylindracea lipase is the enzyme of choice for the synthesis of
fluoro alkyl malonates with small alkyl groups. An astonishing observation was
428
Table 11.1-13. Lipase-catalyzed enantiomer-differentiating hydrolysis of racemic carboxylic acid

esters and lactones in aqueous solution (PPL pig pancreas lipase, PSL Pseudomonas sp lipase,
PFL PseudomonasPuorescens lipase, CCL Candida cylindracea lipase, ANL Aspergillus niger lipase,
PCL Pseudomonas cepacia lipase, CAL-A Candida antarctica A lipase, CRL Candida rugosa lipase,
CAL Candida antarctica lipase, not specified).
0 0:&CO,nC,H,
I)\/CO,H l a PI 1b 111
-, - PPL, 60 % conversion 295 % ee, -
0
-_--
H
C
O
,S,,
R
R = Ph, p-NOzCsH4, p-MeOC&h,
PhCH2, c - C ~ H I I
80-100 % ee, 20-30 %yield, PSL
50 % conversion
Me ,,,, CO,Me
(C0,H
3a [3,4]
Mey""."e C0,Me
95 % ee, 47 % v e l d , PPL 296 % ee, -

50 % conversion
\CO H ' C O Me
98 % ee, 45 %yield, PPL __
50 % conversion
...C0,Me
5a 151
Me0 CO,H Me0
95 % ee, 40 % yield, PSL (15 % ee, 55 % yield
42 % conversion
OH
rcoz /
35 % conversion
6a 161
99 % ee, 43 % yield
55 % conversion
Table 11.1-13. (cont.).
I
429
MedC02H
R = OH:79 % ee, 48%yield, PFL 7a 95% ee, 43 % yield 7b [61

50 % conversion
R = F: 69% ee, 53 %yield, PFL 8a 299 % ee, 37 % yield 8b [61
GO % conversion
R = Br: 69% ee, 48% yield, PFL 9a 73 % ee, 40YOyield 9b161
10a [7] CF3 10b [7]
O2N3COzH 02NdC02Bn
298 % ee, -, PFL, 35 % conversion 98% ee, -
ye
lla [8] llb [S]
Me0 DCozH / /
98% ee, -, CCL, 39 % conversion

Me0
63 % ee, -
96 % ee, 43 % yield, CCL 94% ee, 48 % yield

89 % ee, 49 % yield, PPL 299 % ee, 49 % yield
93 % ee, 31 % yield, CRL [I61 94% ee, 46 % yield [161
14a [17] )~).~*‘co2nBu 14b [17]
“‘Me
93 % ee, 35 % yield, CCL 94% ee, 47% yield
50% conversion
430
I 7 I Hydrolysis and Formation of C-0 Bonds
Table 11.1-13. (cont.).
xTC OZH
57 % conversion
15a [171 *‘
C0,nBu
295 % ee, 40 % yield

15b [17]
42 % ee, 38 % yield, CCL 95 % ee, 19 % yield

69 % conversion
ypF ”Me
95 % ee, 41 %yield, CCL 77 % ee, 35 %yield
55 % conversion
18a [6] 18b [6]

HOm C O , H HO
75 % ee, 50 %yield, PFL 299 % ee, 45 % yield

55 % conversion
19a (181 ~ o ~c o z M19b [18]

e
Me
NO2
-, -, PCL 90 76 ee, 52 % yield
55 % conversion
Me
R7?CozCH2CN
0
Et 80 % ee, 49 %yield, PPL 20a 293 % ee, 44 % yield 20b [19]

CsHll 85 % ee, 49 %yield, PPL 21a 298 % ee, 49 %yield 21b [19]
PhCH2 85 % ee, 49 %yield, PPL 22a 295 % ee, 49 % yield 22b [19]
23a [20] 23b [20]
299 % ee, -, PPL 2e99 % ee, -

Table 11.1-13. (cont.).
7 7 . 7 Hydrolysis and Formation of Carboxylid Acid Esten
I
431
OH
24a [2l] 24b [21]
PhL C 0 , H /\/CO,Et
Ph
93 % ee, -, PSL 98 % ee, -
OAc
25a [21] L C O , E t 25b [21]
Ph
94 % ee, -, PSL 96 % ee, -
BnO Me Me OBn
x
R CO,H
x
R CO,H
R
Et 81 % ee, 41 % yield, CCL 26a GO % ee, 54 % yield 26b [22]
n-Pr 95 % ee, 40 %yield, CCL 27a 70 % ee, 18 % yield 27b [22]
ally1 299 % ee, 46 % yield, CCL 28a 82 % ee, 52 % yield 28b [22]
n-CsHt3 299 % ee, 38 % yield, CCL 29a 67 % ee, 35 % yield 29b [22]
n-CgH19 94 % ee, 46 % yield, CCL 30a 67 % ee, 8 % yield 30b [22]
L,,
OAc
HO,C-(CH,), RO,C-(CH,), ‘61
R n
Me 4 28%ee,-,CCL 31a -,- 31b [23]
Me 8 68%ee,-,CCL 32a -,- 32b [23]
n-Bu 8 299%ee,-,CCL 33a -,- 33b [23]
OH
R CO,H R LCOzMe
R
n-Pr 84 % ee, 39 %yield, PPL 34a 75 % ee, 47 % yield 34b [24]
n-Bu 82 % ee, 40 % yield, PPL 35a 77 % ee, 46 % yield 35b [24]
n-C5H1l 74 % ee, 50 %yield, PPL 36a 95 % ee, 40 %yield 36b [24]
n-C6H13 82 % ee, 48 % yield, PPL 37a 93 % ee, 43 % yield 37b [24]
n-C7Hls 85 % ee, 43 %yield, PPL 38a 85 % ee, 45 % yield 38b [24]
n-CsH17 83 % ee, 48 %yield, PPL 39a 88 % ee, 5 % yield 39b [24]
R
R=Et 75 % ee, PPL 40 [25]
R = n-C7H15 76 % ee, PPL 41 [25]
432
Table 11.1-13. (cont.).
Me0,C
>99 % ee, 30 % yield, CAL-A 42a [26]

A 0
GO % ee, 56 %yield 42a [26]

>85 % ee, 54 % yield, CAL-A 42a [27] >99 % ee, 41 %yield 42a [27]
CO,H
-, 60 % conversion, CRL
43a [28]
Me0,C
P
92 % ee, -
43b [28]
44a [29] R o ~ C ~ N H A c 441,[29]
R
>99 % ee, -, CCL n-Bu >99 % ee, 42 % yield
n-Hex >99 % ee, 44 % yield
45a [30] 45b [30]

Ph PhkCoZMe
89 % ee, -, PCL >98 % ee, 46 % yield, PCL
L C O , M e 46b [31]
0
84 % ee, 80 % yield, CCL -, 97 % yield
C0,Et CO,H
p-Me-Ph s''s'/hN
F
47a [32]
p-Me-Ph ACN
F
47b [32]
99 % ee, 41 % yield, CRL 98 % ee, 46 % yield
C0,nOctyl
48a [33] 48b [33]
93 % ee, 19 %yield, ANL

OH
73 % ee. -
(purified)
.nQ 49a [34] l

* 49b [34]
HN
, CO,H H,N C02Et
96 % ee, 40 % yield, lipase L >99 % ee, 50 % yield
1 1 . I Hydrolysis and Formation ofcarboxylid Acid Esters
I
433
d.,
Table 11.1-13. fc0nt.l.
-
"COOH
5Oa [35] A~....x-coo,, 5Ob [35]
do
HO,C
51 1361 51b [36]
89 % ee, -, PPL, 28 % conversion 35 % ee, -

82 % ee. -; CRL, 21 % conversion 22 % ee, -
P
0
II
52 1371 53 [37]
82 % ee, CAL, 47 % conversion 72 % ee, CAL, 59 % conversion
92 % ee, CAL, 50 % conversion

54 [37]
c;-
94 % ee, CAL, 50 % conversion
OH
55 [37]
R O L C 0 2 H RO&CO,Me
R
4-Me0-C6H4 99 % ee, -, PCL 56a 97 % ee, -, PCL 56b [38]
2-AIlyl-C~H4 98 % ee, -, PCL 57a 98 % ee, -, PCL 57b [38]
2-Naphthyl 99 % ee, -, PCL 58a 98 % ee, -, PCL 58b 1381
59a [39] ,
H, C 5 y C 0 , E t 59b [39]
0
98 % ee, 13 %yield, PPL 26 % ee, 67 % yield
93 % ee, 13 %yield, PCL 10 % ee, 80 % yield
-
1 D. Bianchi, W. Cabri, P. Cesti, F. Francalanci, 4 J. Salaiin, B. Karkour, Tetrahedron Lett. 1987,28,
M. R i d , / . Org. Chem. 1988,53,104. 4669.
2 K. Burgess, I. Henderson, Tetrahedron Lett. 1989, 5 J.-P. Barnier, L. Blanco, E. GuiK-jampel,
30,3633. G. Rousseau, Tetrahedron 1989,45,5051.
3 E. Guibe-lamuel,
.~ G. Rousseau, 1. Salaiin, 1. Chem. G P. Kalaritis, R. W. Regenye, J. J. Partridge,
Soc., Chem. Commun. 1987,1080. D. L. Coffen, j . Org. Chem. 1990,55,812.
434
7 T. Yamazaki, T. Ohnogi, T: Kitazume, Tetrahedron: 24 P. Allevi, M. Anastasia, P. Ciuffreda, A. M.

Asymmetry1990, 1, 215. Sanvito, Tetrahedron: Asymmetry1993,4, 1397.
8 Q:M. Gu, C:S. Chen, C. J. Sih, Tetrahedron Lett. 25 L. Blanco, E. GuibC-Jampel,G. Rousseau,
1986,27,1763. Tetrahedron Lett. 1988, 29, 1915.
9 Q:M. Gu, D. R. Reddy, C. J. Sih, Tetrahedron Lett. 26 J. Kingery-Wood, 1. S . Johnson, Tetrahedron Lett.
1986,27,5203. 1996,37,3975.
10 R. Demoncour, R. Azerad, Tetrahedron Lett. 1987, 27 E. W. Holla, H:P. Rebenstock, B. Napierski,
28,4661. G. Beck, Synthesis1996, 823.
11 B. Cambou, A. M. Klibanov, Biotechnol. Bioeng. 28 C. M. Schueller, D. D. Manning, L. L. Kiessling,
1984,2G, 1449. Tetrahedron Lett., 1996, 37, 8853.
12 R. Chhevert, L. D'Astous, Can.]. Chem. 1988,66, 29 R. Csuk,.'l Don; Tetrahedron: Asymmetry1994,5,
1219. 269.
13 2:W. Guo, C. J. Sih,]. Am. Chem. SOC.1989, 111, 30 G. Varadharaj, K. Hazell, C. D. Reeve, Tetrahedron:
6836. Asymmetry1998,9,1191.
14 S.-H. Wu, Z.-W. Guo, C. J. Sih,]. Am. Chem. SOC. 31 Y. R. Santosh h i , D. S. Iyengar, Synthesis,1996,
1990, 112,1990. 594.
15 B. Loubinoux, C. Viriot-Chauveau. J. L. Sinnes, 32 Y. Takeuchi, M. Konishi, H. Hori, T. Takahashi,
Tetrahedron Lett. 1992, 33, 2145. T. Kometani, K. L. Kirk, Chem. Commun.1998,
16 1. J. Colton, S . N. Ahmed, R. 1. Kazlauskas, J. Org. 365.
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17 M. Pottie, J. Van der Eycken, M. Vandewalle,J. M. R. J. Kazlauskas,]. Org. Chem. 1994,59, 2075.
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5319. 7398.
18 S . KnesoviS, V. Sunjit, A. Lhai, Tetrahedron: 35 A. Bhaskar Rao, H. Rehman, B. Krishnakumari,
Asymmetry1993,4,313. J. S . Yadav, Tetrahedron Lett. 1994, 35,2611.
19 L. Blanco, G. Rousseau, J:P. Bamier, E. 36 G. Pitacco, A. Sessanta o Santi, E. Valentin,
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783. 37 K. Shioji, A. Matsuo, K. Okuma, K. Nakamura,
20 R.-L. Gu, C. J. Sih, Tetrahedron Lett. 1990, 31, 3283. A. Ohno, Tetrahedron Lett. 2000, 41,8799.
21 N. W. Boaz,]. Org. Chem. 1992,57,4289. 38 K. Wiinsche, U. Schwaneberg, U. T. Bornscheuer,
22 T. Sugai, H. Kakeya, H. Ohta,]. Org. Chem. 1990, H . H . Meyer, Tetrahedron: Asymmetry1996,7,
55,4643. 2017.
23 U. T. Bhalerao, L. Dasaradhi, P. Neelakantan, 39 F. Benedetti, C. Forzato, P. Nitti, G. Pitacco,
N. W. Fadnavis,J . Chem. SOC., Chem. Commun. E. Valentin, M. Vicario, Tetrahedron: Asymmetry
1991.1197. 2001, 12, 505.
made in the case of the dihydropyridine ester 14 and ent-14. Both enantiomers are
obtained with high ee values and in high yields by Pseudomonas sp. lipase-catalyzed
hydrolysis merely upon changing the reaction medium from diisopropyl ether to
cyclohexane, both saturated with water. The limitations of the lipase-catalyzed
hydrolysis of carboxylic acid esters are evident too. Whereas the cyclohexenoid
diester 10 is obtained through pig pancreas lipase-catalyzed hydrolysis with high
enantioselectivity,the cyclopentanoid monoester 11 is formed only with low selectiv-
ity and the cyclopentanoid diester 12 is not a substrate for pig pancreas lipase. An
interesting example for the use of a cholesterol esterase is the cyclopentanoid
monoester 15.
Monoesters of Table 11.1-12 which can be obtained with other hydrolases as such
or of opposite configuration are contained in Tables 11.1-1,11.1-2 and 11.1-7.
The usefulness of lipases for the enantiomer-differentiating hydrolysis of car-
boxylic acid esters and lactones is impressively demonstrated by examples 1-59 of
Table 11.1-13. This broad substrate spectrum is covered mainly by lipases from
Candida cylindracea (rugosa), pig pancreas and several Pseudomonas sp. lipases.
Carboxylic acid esters having the alkoxycarbonyl group attached to a secondary,
tertiary or even quaternary carbon atom are substrates. Thus, in contrast to
I
7 7 . 7 Hydrolysis and Formation ofcarboylid Acid Esters 435
Table 11.1-14. Lipase-catalyzed enantiomer-differentiating hydrolysis of esters of racemic primary

alcohols in aqueous solution (PPL pig pancreas lipase, PCL Pseudomonas cepacia lipase, PCL-A
Pseudomonas cepacia lipase, Sumitomo, PSL Pseudomonas sp. lipase, PAL Pseudomonas aeruginosa
lipase, HLL Humicola lanuginosa lipase, CAL-B Candida antarctica B lipase, CRL Candida rugosa
lipase).
C I T O A c
CI
90 % ee, -, pancreatin
60 % conversion"
NHC0,Et
Me&OAc
Me
90 % ee, 30 % yield, pancreatin 295 % ee. -
4 [31
O P o H
ClLlHZ,
295 % ee, 32 % yield, PPL 295 % ee, 20 % yield, PPL
60 % conversion", 20 % conversiona,
OCOnPr ~[4]
MeToconPr
295 % ee, -, PPL 295 % ee, -, PPL
60 % conversiona 58 % conversiona
Me
, + o c o n P r 8 [41
nPrToConPr Me
56 % ee, -, PPL 73 % ee, -, PPL
60 % conversion" 60 % conversion"
E t F n P r
T O C o n P r
77 % ee, -, PPL 82 % ee, -, PPL
60 % conversion" 60 % conversiona
GOAC
295 % ee, 30 %yield", PPL
436
Table 11.1-14. (cont.).

-
12a [6]
+Ac 1Zb [GI
\OAc
91 % ee, 45 %yield, PSL 295 % ee, 47 % yield
.-“OH
n
,-,KO
0 0
R =H 97 % ee, -, PAL 13a 99 % ee, - 13b [7]
R= i-Pr 297 % ee, -, PAL 14a 297 % ee, - 14b [8,91
R = t-Bu 297 % ee, -, PAL 15a 297 % ee, - 15b [8, 91
R = Ph 96 % ee, -, PAL 1Ga 100 % ee, - 1Gb [7]
nProco”‘,*~Q
17a [lo] 17b [lo]

I I
Boc Boc
94 % ee, -, PCL 91 % ee, -
18a [Ill
61 % ee, 54 % yield, CAL-B 99 % ee, 37 %yield
b
N\
87 % ee, -, PCL, 46 %
19a [12] TOnPr
ND:::::;)
>98 % ee, -, PCL, 61 %

19b [12]
conversion conversion
20a [12] <OConPr 20b [12]
87 % ee, -, PCL, 47 % >98 % ee, -, PCL, 60%

Table 11.1-14. (cont.).
7 7.7 Hydrolysis and Formation of Carboxylid Acid Esters
I
437
OH OCOnPr
21a [12] 21b [12]
90 % ee, -, PCL, SO % 90 % ee, -, PCL, 50 %

/OConPr
22a [12] 22b [12]
N
oJ
):! "0
93 % ee, -, PCL, 46 % >98 % ee, -, PCL, 55 %

bOH
>99 % ee, 11 %yield, PCL
23a [13] p
H . .
,OAc
16 % ee, 85 % yield
23b [13]
95 O41 %yield,
K % ee, H PCL
24a [14] broAc ...".,.,
96 % ee, 41 % yield, PCL

24b [14]
R R
91 % ee, 35 % Me 25a [15] >98 % ee, 27 % Me 25b [lS]
yield, PCL yield
86 % ee, 29 % n-Pr 2Ga [15] 92 % ee, 34 % n-Pr 2Gb [15]
yield, PCL yield
88%ee, 31 % n-Bu 27a [15] 9G % ee, 33 % n-Bu 27b [lS]
yield, PCL yield
438
H O Y S R 28a [16] A c O T S R
28b [16]
OAc OAc
48->96 % ee, 34-50 % yield, 47-939 % ee, 3 4 5 7 % yield,

PCL PCL
Q \CO,Me -CO,Me \CO,nBu
NHCbz
+CO,Me OEt
>99 % ee, 43 % yield, PPL >99 % ee, 50 %yield, PPL
30a [18] 30b [18]
iPr0,C
~ ; ; A C
H H
96 % ee, 50 % yield, HLL 98 % ee, 45 % yield
31a [19] 31b [19]

..H
,O
,, AcO
91 % ee, 26 % yield, PPL 37 % ee, 72 %yield, PPL

Table 11.1-14. (cont.).
7 1 . 7 Hydrolysis and Formation of Carboylid Acid Esters
I
439
R' R2 lipase
96 % ee, 23 % yield H COzMe 32a[20] PCL 33%ee, 72%yield 32b[20]
>98 % ee, 21 %yield PSL 44 % ee, 72 %yield
>98 % ee, 34 % yield H H 33a 1201 PCL 51 % ee, 57 % yield 33b [20]
>98 % ee, 25 % yield PSL 34 % ee, 73 % yield
95 % ee, 28 % yield OMe H 343 [20] PCL 49 % ee, 65 %yield 34b [20]
97 % ee, 27 % yield PSL 40 % ee, 67 % yield
92 % ee, 12 % yield R' = 35a [20] PCL 18 % ee, 72 %yield 35b [20]
92 % ee, 28 % yield PSL 52 % ee, 61 %yield
kMe
?U?
R ~ = H
OH OAc
/
(
36a [211 (--J ,,,OMe 3Gb [2l]
NHCOCF, NHCOCF,
>98 % ee, 37 % yield, PPL,
38 % conversion
-,-, PPL, 70 % conversion 198 % ee, 29 % yield
79 % ee, -, PSL, 36 % __
conversion
-,-, PSL, 69 % conversion >98 % ee
NHCOCl,H,5 NHCOC,,H,,
37a [22] 3% [22]
H 0 A / C 1 3 H 2 7
OAc OH
49 % ee, 54 % yield, PCL-A 98 % ee, 7 % yield
96 % ee, 41 % yield, PCL-A
(immobilized)
Table 11.1-14. [cont.).
NHCOC,&,
37c [22]
Ac0-?13H27 OAc
87 % ee, 38 %yield, PCL-A

G9 % ee, 58 %yield, PCL-A
(immobilized)
38a 1231 38b [23]

HOr Q - 0
AcO
/'+,'
79 % ee, -, PCL, 50 % 84 % ee, -
conversion
39a [23] 391, [23]
HO AcO
89 % ee, -, PCL, 53 % 94 % ee, -
conversion
40a [24] 40b [23]
/"""
HO AcO
96 % ee, -, PCL, 50 % 95 % ee, -
conversion
.(3;. 41a [25] 41b [24]

Ph-OH PhAOAc
78 % ee,-, PCL 53 % ee, -
82 % ee, -, CAL-B 78 % ee, -
"17 R2
O K0N d o H
R R' RZ lipase
75 % ee, 42 % Et Ph H 42a [25] PCL 70%ee, 46%yield, 42b [25]
yield
62 % ee, 50 % t-Bu Ph H 42a [25] PCL 67 % ee, 47 %yield 431, [25]
yield
89 % ee, 51 % Et H Ph 44a [25] PCL 93 %ee, 42 %yield 44b[25]
yield
Table 11.1-14. (cont.).
7 7 . 7 Hydrolysis and Formation ofCarboxylid Acid Oten
I
441
87 %ee, 4 2 % Et H Ph 44a PSL 90 % ee, 46 %yield 44b [25]

yield
97%ee,44% n-Pent H Ph 44a PCL 92 % ee, 50 %yield 45b [25]
yield
94%ee,43% Et H Bn 46a PCL 91 % ee, 46 %yield 461,[25]
yield
72%ee,46% Et H Bn 46a PSL 76 % ee, 44 %yield 461,[25]
yield
69%ee, 52% Et H Et 47a PCL 98 % ee, 40 %yield 47b [25]
yield
97%ee, 50% Et H Et 47a PCL 87 % ee, 47 % yield 47b [25]
yield, 0 "C
1 48a [26] 1 481,[2G]

F F
89 % ee, -, CRL 87 % ee, -
90 % ee, -, lipase MY 77 % ee, -
96 % ee, 54 % yield, lipase OF-360 84 % ee, 59 % yield
91 % ee, -, CCL 80 % ee, -
a Theotherproduct (alcoholor ester) wasnot isolated. b Acetate was hydrolyzed.
1 S. Iruchijima, A. Keiyu, N. Kojima, Agric. Bid. 14 H. Nakano, K. Iwasa, Y. Okuyama, H. Hongo,

Chem. 1982,46,1593. Tetrahedron: Asymmetry1996,7, 2381.
2 F. Francalanci, P. Cesti, W. Cabri, D. Bianchi, T. 15 0. Goj, A. Burchardt, G. Haufe, Tetrahedron:
Martinengo, M. Foi,J. Org. Chem. 1987,52,5079. Asymmetry1997,8,399.
3 D. Bianchi, W. Cabri, P. Cesti, F. Francalanci, 16 S. Brand, M. F. Jones, C. M. Raper, Tetrahedron
F. Rama, Tetrahedron Lett. 1988, 29, 2455. Lett. 1997, 38, 3595.
4 W. E. Ladner, G. M. Whitesides, J. Am. Chem. SOC. 17 G. D. Gamalevich, B. N. Morozov, A. L. Vlasyuk,
1984,106,7250. E. P. Serebryakov, Tetrahedron 1999,55, 3665.
5 F. Van Middlesworth, D. V. Patel, J. Donaubauer, 18 B. Schnell, U. T. Strauss, P. Verdino, K. Faber,
P. Gannett, C. J. Sih, J. Am. Chem. Soc. 1985, 107, 0.C. Kappe, Tetrahedron:Asymmetry2000, 11,
2996. 1449.
6 J. Van der Eycken, M. Vandewalle, G. Heinemann, 19 S. V. Ley, S. Mio, B. Meseguer, Synlett 1996,787.
K. Laumen, M. P. Schneider, J. Kredel, J. Sauer, 20 H. Hongo, K. Iwasa, C. Kabuto, H. Matsuzaki,
J. Chem. Soc., Chem. Commun.1989, 306. H. Nakano,j. Chem. Soc., Perkin Trans. 1, 1997,
7 S. Hamaguchi, H. Yamamura, J. Hasegawa, 1747.
K. Watanabe, Agric. Biol. Chem. 1985,49, 1509. 21 S. Erbeck, X. Liang, R. Krieger, H. Prinzbach, Eur.
8 S. Hamaguchi, M. Asada, J. Hasegawa, J. Org. Chem. 1998, 481.
K. Watanabe, Agric. Biol. Chem. 1985,49, 1661. 22 M. Bakke, M. Takizawa, T. Sugai, H. Ohta, J. Org.
9 S. Hamaguchi, M. Asada, J. Hasegawa, Chem. 1998,63,6929.
K. Watanabe, Agric. Biol. Chem. 1984, 48, 2331. 23 H.-J. Ha, K:N. Yoon, S:Y. Lee, Y.3. Park, M.-S,
10 B. Wirz, W. Walter, Tetrahedron:Asymmetry1992, Lim, Y.G. Yim,J. Org. Chem. 1998,153,8062.
3, 1049. 24 J. Pietruszka, T. Wilhelm, A. Witt, Synlett, 1999,
11 H:J. Gais, 1. von der Weiden, Tetrahedron: 1981.
Asymmetry1996,7, 1253. 25 H. Wakamatsu, Y. Terao, Chem. Pharm. Bull. 1996,
12 M. De Amici, C. De Micheli, G. Carrea, S. Riva, 44,261.
Tetrahedron: Asymmetry1996, 7, 787. 26 V. Khlebnikov, K. Mori, K. Terashima, Y. Tanaka,
13 H. Tanimoto, T. Oritani, Tetrahedron: Asymmetry M. Sato, Chem. Pharm. Bull. 1995,43, 1659.
1996, 7, 1695.
442
uncatalyzed ester hydrolysis, steric hindrance, at least for the known examples 14-17
and 26-30 in the enzyme-catalyzed hydrolysis, poses no problem. In substrates
containing two alkoxycarbonyl groups, one attached to a secondary carbon and the
other one to a tertiary carbon, the former is hydrolyzed more readily, as shown for
3-5. Esters with the alkoxycarbonyl group attached to quaternary carbon are readily
hydrolyzed, as demonstrated for 17,26-30,47,49,51 (Table 11.1-13).
Group selectivity is also observed between an ester group and a thioester group or
an ester and a lactone moiety, as exemplified by 12 and 51, respectively. Acyclic as
well as cyclic carboxylic acid esters are substrates for enantiomer-selectivehydrolysis
catalyzed by lipases. High enantioselectivitiesare observed not only for those esters
having a chiral center in a-position but also for those having the chiral center in p-
position. A spectacular example in this regard is the acetoxy-substituted carboxylic
acid 33,where the chiral center is separated by eight methylene groups from the
carboxyl group. This acid is obtained by a Candida cylindracea lipase-catalyzed
hydrolysis of the corresponding racemic butyl ester with very high enantioselectivity.
Surprisingly, the hydrolysis of the corresponding methyl ester proceeds with a much
lower enantioselectivity. Lipase-catalyzed enantiomer-differentiating hydrolysis has
been utilized with much success for the synthesis of a-hydroxy and a-acetoxy
carboxylic acids (6,7, 24 and 25).A series of vinylogous a-hydroxy carboxylic acids
34-39 is also accessible.
The two a-amino acids 48 and 49 with unprotected amino groups are hydrolyzed
with high enantioselectivity. The series of methyl-substituted seven-membered
lactones 52-55 (Table 11.1-13) are converted in the presence of Candida antarctica
lipase yielding the slow-reactinglactones with ee values between 72 and 94%.
Acids, monoesters and lactones ofTable 11.1-13which can be obtained with other
hydrolases as such or of opposite configuration are contained in Table 11.1-5.
Lipase-catalyzed enantiomer-differentiating hydrolysis of acylated racemic pri-
mary alcohols covers a broad range of substrates (1-48) summarized in Table
11.1-14,including epoxy alcohols (3-lo),amino alcohols (2,17,36,37)and acylated
y-hydroxymethyly-lactones (38-40).By means of incorporating the amino and the
secondary hydroxyl group into a heterocyclic ring system, selectively protected
amino diols are accessible by Pseudomonas aeruginosa lipase-catalyzed hydrolysis
(13-1G).3-Hydroxymethyl-D2-isoxazoline butyrates 19-22 (Table 11.1-14) have been
resolved with high selectivity in the presence of Pseudomonas cepacia lipase.
Monoacetates and alcohols of Table 11.1-14 which can be obtained with other
hydrolases as such or of opposite configuration are contained in Table 11.1-19.
Given the experimental simplicity and the potential scale of reaction, lipase-
catalyzed enantiomer-differentiating hydrolysis of racemic acylated secondary alco-
hols is today one of the best methods for the synthesis of optically active secondary
alcohols. From the list of the tabulated examples 1-170 (Table 11.1-15)one gets the
impression that there is almost no restriction in regard to the substrate structure.
Because of the number of lipases available either as isolated enzymes or contained in
the various organisms, it seems possible to find the right lipase for almost every
substrate. Highly enantiomer-selectivehydrolysis and alcoholysis of esters of a wide
structural range of secondary alcohols by the different lipases are possible. Not only
Table 11.1-15. Lipase-catalyzedenantiomer-differentiating hydrolysis of esters of racemic acyclic

secondary alcohols in aqueous solution (CCL Candida cylindracea lipase, PSL Pseudomonas sp.
lipase, PFL Pseudomonasfluorescens lipase, PAL Pseudomonas aeruginosa lipase, ASL Alcaligenes sp.
lipase, ANL Aspergillus niger lipase, PCL Pseudomonas cepacia lipase, ROL Rhizopus oryzae lipase,
M M L Mucormiehei lipase, CAL-B Candida antarctica B lipase, LIP Pseudomonas sp. lipase -
Toyobo, HSL Hurnicola sp. lipase).
RIAMe
OH
R' RZ
R' x,"'
lipase
la 90 % ee, 39 % yield Et n-Pr CCI 88 % ee, 40 % yield
2a 299 % ee, 48 % yield Ph Me PSL 299 % ee, 48 % yield
3a 97 % ee, 47 % yield 4-Me-C& Me PSL 99 % ee, 45 %yield
4a 80 % ee, 46 % yield 4-MeO-CsH4 Me PSL 80 % ee, 47 %yield
5a 95 % ee, 47 % yield PhCH2 Me PSL 97 % ee, 48 %yield
Ga 95 % ee, 46 % yield 4-Pyridyl Me PSL 89 % ee, 47 %yield
7a 299 % ee, 43 % yield 2-Naphthyl Me PSL 299 % ee, 46 %yield
8a 95 % ee, 50 %yield Ph CHzCl PSL 9G % ee, 44 %yield
m = 0, n = 1 99 % ee, 37 %yield, PFL 9a 95 % ee, 40 % yield 9b I31

46 % conversion 95 % ee, 40 % yield lob [3]
m = 0, n = 2 99 % ee, 35 %yield, PFL 10a
46 % conversion 95 % ee, 40 % yield l l b [3]
m = 1,n = 1 95 % ee, 36 %yield, PFL lla
48 % conversion 95 % ee, 40 % yield 12b [3]
m = 1, n = 2 99 % ee, 35 %yield, PFL 12a
48 % conversion
n=O 299 % ee, -, PSL 13a [4a] 96 % ee, - 13b [4]

n=l 299 % ee, -, PSL 14 [4] -
n=5 299 % ee, -, PSL 15 14) -
n = 10 299 % ee, -, PSL 16 [4] -
7
OH OCOCH,CI
Me0,C , Me MeozC&Me
OW0
93 % ee, -, PSL 17a [5] 299 % ee, - 1% [51
444
Table 11.1-15. (cont.).
jl"
OAc
R CF,
R-CF,
R
Ph 57 % ee, -, CCL 18a [6] -,- 19b [6]
CHzPh 94% ee, -, CCL 19a [6] 98% ee, -
(CH2)zPh 98 % ee, -, CCL 20a[6] -, -
2-styryl 93 % ee, -, CCL 21a [6] -,-
CHzCO2Et 96 % ee, -, CCL 22a[6] -,-
CHZCOZHex 90% ee, -, CCL 23a [6] -, -
28-50 % conversion - -
R
c1 100 % ee, 24 % yield, PSL 24a 100 % ee, 29 %yield 24b [7]
Br 94% ee, 24 %yield, PSL 25a 100 % ee, 11 %yield 25b [7]
50 % conversion
92 % ee, -, PSL 26a 299 % ee, - 26b [8]
98% ee, -, PSL 27a 299 % ee, - 27b [8]
97% ee, -, PSL 28a 98% ee, - 28b [8]
42-53 % conversion
OCOR'
R ' A O T s
R' RZ
Me 299 % ee, 40 % yield, PAL 29a Me, Pr299 % ee, 35 % yield 29b [9]
Et 299 % ee, 46 % yield, PAL 30a Me, Pr299 % ee, 44% yield 30b [9]
CHzCl 299 % ee, 46 % yield, PAL 31a Me, Pr299 % ee, 45 %yield 31b [lo]
OAc
OH 32a [ll] 32b [Ill
Ph*CN PhACN
- - 298% ee, 42 %yield, PSL
oH 33a [ll,121 33b [ll,121
~o'o""N
/
98% ee, -
/
98 % ee, 40 %yield, ASL
(PH 4-5)
87 % ee, -, PSL
Table 11.1-15. (cont.).
I
34a [13] 34b (131
- 87 % ee, 39 %yield, PSL
OCH,SMe
R L C N
R
&
nCe.H, 7
CI
C0,U
Me 298 % ee, -, PFL 35 [14] 94 % ee, 31 %yield, PFL 39 [15]

Ph 298 % ee, -, PFL 36 (141
60-64 % conversion
Ph(CH2)z 298 % ee, -, PFL 37 [14]
2-styryl 298 % ee, -, PFL 38 [14]
OAc
R LC02Me R -CO,Me
R = Me 295 % ee, 37 %yield, PFL 40a [16] 91 % ee, 39 %yield 40b [16]
R = Et 295 % ee, 44 %yield, PFL 41a [ l G ] 295 % ee, 45 %yield 41b [16]
OH
ThexylMe,SiO- . ’ C0,Me ThexylMe,SiO &CO,Me

72 % ee, 57 % yield, PFL 42a (161 295 % ee, 35 % yield 421, [lG]
C0,Me
97 % ee, 41 %yield, PFL 43a [17]

(yJ
96 % ee, 26 %yield
C0,Me
43b [17]
OH COnPr
R&CO,Me C0,Me
R
R
Et 74 % ee, -, CCL 44a 42 % ee, - 44b I181
ClIH23 84 % ee, -, CCL 45a 75 % ee, - 451, [18]
(CH2)4CH(CdHs)z 92 % ee, -, CCL 46a 50 % ee, - 46b [l8]
40 % conversion GO % conversion
446
Table 11.1-15. (cont.).
OH OAc
.I&,, C0,Me
R’ W O z M e
R R2
R’ R2 R’
47a 66 % ee, 56 % yield, ANL 2-fury1 H Me 299%ee, 33%yield 47b(19]
48a 67 % ee, 64 %yield, ANL 2-thiophenyl H Me 91 % ee, 35 %yield 48b [19]
49a 64 % ee, 51 % yield, ANL 2-(2-butenyl) H Me 299 % ee, 38 % yield 49b [19]
50a 75 % ee, 51 % yield, ANL 2-fury1 Me H 98%ee, 32%yield 5Ob[19]
51a 85 % ee, 53 %yield, ANL 2-thiophenyl Me H 299 % ee, 47 %yield 51b [19]
52a 79 % ee, 51 % yield, ANL 2-(2-butenyl) Me H 299 % ee, 44 % yield 52b [19]
R’ R2 R3 lipase
- t-Bu H H CCL 298 % ee, -, ANL 53b (201
- Ph H H PFL 298 % ee, -, ANL 54b [20]
- Et Et H PFL 298 % ee, -, ANL 55b (201
5Ga [21] 298 % ee, - Et H Et PFL
57a[21] 298%ee,- n-Pr H n-Pr PFL
30-40 % 52-60 % conversion
conversion
other product not isolated
R = Et 100 % ee, 50 % yield, PPL 58a (211

R = n-Pr 60 % ee, 50 %yield, PPL 59a (211
R = (CHZ)zCO*Et 56 % ee, 22 %yield, PPL GOa (211
h L O N E t ,
OH
Gla [22] WF OAc
CONEt, 61b [22]
58 % ee, -, CCL 96 % ee, -
GZa (231 G2b [23]
Cbz Cbz
>97 % ee, 58 %yield, CCL 297 % ee, 42 % yield
I
4 7
q
Table 11.1-15. (cont.).
OH
63a [24] ’’ OAc 63b [24]
O h C I O L C l
297 % ee, 41 % yield, PCL 88 % ee, 43 % yield
OH
C, 3H27AMe
52 % ee, 40 % yield, PCL 64a R = Ac
C13H27
x:,“
54 % ee, 41 % yield 64b 1251
95 % ee, 23 % yield, PCL 65a R = OCHzCCl3 35 % ee, 42 %yield 65b 1251
295 % ee, 27 % yield, PCL -, 48 % yield

(hydrolysiswith PFL gives the (S)-
alcohol with 295 % ee)
\ G O C 6 H 4 0 M e
674271 ,
b OAc
OC,H,OMe
67b [27]

(buffer : acetone = 9 : 1)
R’ R2 R’
298 % ee, -, PCL H 0-t-Bu CHzCl 68a 298 % ee, - 68b [28,29]
298 % ee, -, PCL Me 0-t-Bu CH2Cl 69a 298 % ee, - 69b [28,29]
88 % ee, -, PCL H SPh Me 70a 90 % ee, -, 70b [28, 291
298 % ee, -, PCL Me SPh Me 71a 91 % ee, - 71b [28, 291
296 % ee, -, PCL H SPh CHzCl 72a 296 % ee, - 72b [28,29]
296 % ee, -, PCL Me S-t-Bu CHzCl 73a 296 % ee, - 73b [28,29]
296 % ee, -, PCL n-Pi S-t-Bu CHzCl 74a 296 % ee, - 741, [28,29]
296 % ee, -, PCL n-Pent S-t-Bu CHzCl 75a 296 % ee, - 75b [28,29]
296 % ee, -, PCL n-Non S-t-Bu CHzCl 76a 296 % ee, - 76b [28,29]
OH OCOC15H3,
T s O h O R R
o,,!,T~,o
R
99 % ee, 44 %yield, PCL C16H31 77a 295 % ee, 46 %yield 771, [30]
99 % ee, 47 %yield, PCL CloHzl 78a 295 % ee, 42 % yield 78b [30]
99 % ee, 45 % yield, PCL C4H9 79a 295 % ee, 43 %yield 79b [30]
448
Table 11.1-15. (cont.)
OAc
80 [31]
96 % ee, 27 % yield, PPL other product not isolated
boxcl 81b (321
w
(R = H, Me, OMe, NO*, ally], 0-allyl, c-C5HI1 67-99 % ee, 31-52 %yield
39-99 % ee, 31-54 %yield, PSL
OH
82a [33] 82b [33]
Ph-CI Ph
97 % ee, 50 % yield, PSL 299 % ee, 50 % yield
83a [34]
&Me"
(2-4)
295 % ee, 37-55 %yield, PSL 295 % ee, 37-45 % yield
OCOCH,CI
84a 1351 84b [35]
OAc
R' R2
299 % ee, 31 %yield, ROL Ph Me 85a 90 % ee, 35 %yield 85b [36]
299 % ee, 37 % yield, ROL Ph i-Pr 86a 86 % ee, 46 % yield 86b [36]
50 % ee, 21 %yield, ROL Me Me 87a 9 % ee, 29 %yield 87b (361
nPrOCO
&o
R
R
299 % ee, -, CCL i-Pr 88a [37] 299 % ee, 43 %yield 88b (371
299 % ee, -, CCL Ph 89a[37] 299 % ee, 43 % yield 89b (371
Table 11.1-15. (cont.).
7 1.1 Hydrolysis and Formation of Carboxylid Acid Esten
I
449
OH
9Oa [38) 90b [38]
&CI
tBu tBu
OH
P h O A R P h O z R
R
92 % ee, 31 %yield, PSL C1 91a 97 % ee, 41 % yield 91b [39]
86 % ee, 46 %yield, PSL Br 92a 97 % ee, 41 % yield 92b [39]
91 % ee, 48 %yield, PSL N3 93a 84 % ee, 48 % yleld 93b [39]
0 OCOEt
94a [40] 94b [40]
OBn OBn
32 % ee, 66 %yield, CCL 299 % ee, 22 % yield
(isolated as alcohol obtained
with NaBH4)
OCOR2
,&OTBDMS
R
R' R2
295 % ee, -, PSL Me n-Pr 95a 295 % ee, - 95b [41]
295 % ee, -, PSL Et n-Pr 9Ga 295 % ee, - 9Gb [41]
295 % ee, -, PSL CH2CI n-Pr 97a 295 % ee, - 97b [41]
295 % ee, -, PSL CH=CH2 ClCH2 98a 91 % ee, - 98b [41]
295 % ee, -, PSL CH20CH=CH2 ClCHz 99a 51 % ee, - 99b [41]
lOOa [41] OCH,CI lOOb [41]
Me Me-OTBDMS
295 % ee, -, PSL 295 % ee, -
lOla [42] /',n..,(yOCOEt l0lb[421

PhCO,
P h C 0 2 q g"'OH S
-,-,MML 76 % ee, 14 % yield
450 I 7 7 Hydrolysis and Formation of C - 0 Bonds
Table 11.1-15. (cont.).
0 0
could not be
Me\o+s\R
be\O%\R] isolated
OAc
R
-CH2CH(OMe)2 >95 % ee, 45 %yield, PFL 102 [43]
-CHzCH(OEt)z >95 % ee, 49 %yield, PFL 103 1431
-CH2CH(OBn)2 65 % ee, 48 %yield, PFL 104 [43]
-(CH2)2CH(OMe)2 >95 % ee, 45 %yield, PFL 105 [43]
n-Bu >95 % ee, 47 %yield, PFL 106 [43]
-(CH2)20SiEt3 81 % ee, 47 %yield, PFL 107 [43]
-(CH2)30SiEt3 85 % ee, 48 %yield, PFL 108 1431
>95 % ee, 44 % yield, PCL >95 % ee, 42 %yield, PCL
R
Ph 98 % ee, -, PCL llOa 99 % ee, -, PCL 1lOb [45]
Ph 99 % ee, -, CAL-B 99 % ee, -, CAL-B
Bn 95 % ee, -, PCL llla 97 % ee, -, PCL lllb (451
Bn 97 % ee, -, CAL-B 97 % ee, -, CAL-B
-(CH2)2Ph 98 % ee, -, CAL-B 112a 97 % ee, -, CAL-B 112b [45]
OR NPht
; H I
OHH NPht
113a [4G]
Et
R = CO(CH,),Me
>99 % ee, 49 % yield, ASL >99 % ee, 50 % yield
OH
R O A C I R O L C I
R
2-Naphthyl 95 % ee, -, CAL-B 114a 95 % ee, 47 %yield 114b 1471
4-AcNH-GH4 95 % ee, -, CAL-B 115a 79 % ee, 40 %yield 115b [47]
4-[MeO-(CHZ)2]-C6H495 % ee, -, CAL-B 116a 70 % ee, 37 %yield llGb (471
C6H4
Table 11.1-15. (cont.).
I
451
F
/~ C N 117a [481 ‘WcN
F
F
117b [48]
93 % ee, 40 %yield, PFL 82 % ee, 37 %yield, PFL

>98 % ee, 41 % yield, LIP >99 % ee, 521 % yield, LIP
OH
RL C l
R
R
Ph 89 % ee, -, PCL 118a 80 % ee, -, 118b [49]
4-F-C& 93 % ee, -, PCL 119a 78 % ee, -, 119b [49]
4-t-BU-C& >95 % ee, -, PCL 120a >95 % ee, -, 120b [49]
MeoL \
. C02Me
121a [50]
C0,Me
121b [SO]
OH OAc
94 % ee, 51 %yield, ANL >99 % ee, 48 % yield
F*N
/
F
F F
R
Me 94 % ee, 39 %yield, LIP 122a 85 % ee, 39 %yield 122b [51]
Et 97 % ee, 38 % yield, LIP 122a 92 % ee, 46 %yield 123b [51]
Ph 89 % ee, 32 % yield, LIP 122a 96 % ee, 43 %yield 124b [51]
Aco*J5
OAc OH
“R2O r ’ R‘
not separated
R’ R2
Me H 96%ee,CAL-B 97 % ee 125a,b >98 % ee, 42 % yield 125c [52]
Et H >98%ee,CAL-B 93% ee 12Ga,b >98 % ee, 37 %yield 12Gc [52]
i-Pr H >98%ee,CAL-B >98 % ee 127a,b 63 % ee, 39 %yield 127c 1521
Me Me >98%ee,CAL-B >96 % ee 128a,b 96 % ee, 38 % yield 128c [52]
Table 11.1-15. (cont.).
OH
129a [531 o<Le 129b [53]
O r M e \
85 % ee, -, CAL-B 93 % ee, -
52 % conversion
OAc
130a [53] O r M e 130b [53]
?
‘Me
Me Me
96 % ee, -, CAL-B 92 % ee, -
49 % conversion
O
-R -OR
OH OCOCH,CI
R
SiMezThex 97 % ee, -, PCL 131a 99 % ee, - 131b [54]
94 % ee, -, CAL-B 95 % ee, -
Bz 94 % ee, -, HSL 132a 97 % ee, - 1321, [54]
W O T s 133a 1541 T O T S 133b [54]

OH OAc
99 % ee, -, PCL 99 % ee, -, PCL
OAc
““T“
134a [55] 134a (551
/
HA H2N
LI CI
99 % ee, 42 % yield, CAL-B 96 % ee, 42 % yield
OAc
4 C0,Et
>99 % ee, 19 % yield, PSL

135a [56]
46% ee, 33 %yield

135b [56]
136a (571 13Gb [57]

J O
: T C0,Et
B D M S
OH OCOCH,CI
>99 % ee, 43 % yield, PCL 90 % ee, 46 % yield
Table 11.1-15. (cont.).
I
453
87 % ee, 39 %yield, PPL 80 % ee, 45 % yield
Ph
138a[58] wph I : 138b [S8]
>96 % ee, 40 %yield, PPL 70 % ee, 48 % yield
1- N a p h - O Y O M e 13% [58] 1-Naph-O/\/\OMe 13% [59l

OH OAc
82 % ee, 47 % yield, CAL-B 90 % ee, 38 % yield
0
Me\o+o.R
OAc OH
abs. config. unknown racemic
R
-(CHz)7CH3 >95 % ee, 43 % yield, PFL 140 [GO]
-CHZCH=CHz >95 % ee, 32 %yield, PFL 141 [60]
-(CHz)*CH=CH2 90 % ee, 37 % yield, PFL 142 [GO]
-CHZCH=CHPh >95 % ee, 39 %yield, PFL 143 (601
-CHZPh >95 % ee, 35 %yield, PFL 144 [GO]
-(CHZ)C02Et >95 % ee, 27 %yield, PFL 145 [60]
-CHzCH(OEt)z >95 % ee, 37 %yield, PFL 146 (601
* d
R* R2
R'
R' R2
Et Et
>99 % ee, 38 %yield, PCL 146a 94 % ee, 42 % yield 14Gb [61]
>99 % ee, 37 % yield, CAL 147a 59 % ee, 57 %yield 147b [61]
>99 % ee, 22 % yield, CRL 148a 39 % ee, 48 % yield 148b [61]
>99 % ee, 36 %yield, ASL 149a 81 % ee, 40 % yeld 149b 1611
Me Me >99 % ee, 36 % yield, PCL 150a 91 % ee, 54 % yield 150b [61]
>99 % ee, 23 % yield, CAL 151a 73 % ee. 21 %yield 151b [61]
454
Table 11.1-15. (cont.).
R’ = aromatic, heteroaromatic, R2 = Me, Et, LPr, R’ =Me, CH2CI

14-95 % ee, ANL or ROL 9-70 % ee
OH OCOR~
153b (631
Rl Ap/(0R’), 153a[63] =
RlAp/(OR’)2
II I1
0 0
in some cases R in some cases S
R’ = alkyl, R2 = Me, Et, LPr, R’ = Me, CHlCl
7.5-98 % ee, ANL or ROL 3-80 % ee
154a [64] OAc OEt 1541, [64]

a O E t OEt
>98 % ee, 41 %yield, PSL
OAc
&CO,Et I I
U
95 % ee, 38 %yield, PCL
156a[66] 9AC 1561, [66]
OAc
R’+cop2
Cl
R’ R2
Me Et >99 % ee, 29 %yield, PCL 157a -, 30 %yield 15% 1671
Et Me 96 % ee, 29 %yield, PCL 158a -, 43 %yield 158b 1671
Et Et 86 % ee, 29 %yield, PCL 159a -, 22 %yield 159b [67]
n-CsHI7 Et 98 % ee, 29 % yield, PCL lGOa -, 18 %yield lGOb [67]
n-C8H17 Me 87 % ee, 35 % yield, PCL 161a -, 31 %yield 161b 1671
n-CgH17 Et 94 % ee, 31 %yield, PCL 162a -, 22 % yield 162b [67]
Table 11.1-15. (cont.).
I
455
4-MeO-C6H, & OH
C0,Et
OH 164b [69]
P h M C l
95 % ee, 42 %yield, CAL-B
165a[70] 165b [70]
OH OCOPh
91 % ee, -, CRL 97 % ee, -
48 % conversion
OH
166a[70] NCL
, 166b [70]
OCOPh
83 % ee, -, CRL 80 % ee, -
49 % conversion
167a[70] 167b [70]
OH OCOPh
84 % ee, -, CRL 65 % ee, -
43 % conversion
168a [71] 168b [71]
Me0 Me0
>99 % ee, 29 %yield, 48 % ee, 67 % yield
PCL/Celite
98 % ee, 44 % yield, PCL/ 81 % ee, 52 % yield
ENTP-4000 (prepolymer)
456
Table 11.1-15. (cont.).
168a [71] 1681, [71]
OCOCH,CI
Me0 Me0
95 % ee, 49 %yield, PCL/ 94 % ee, 48 % yield
ENTP-4000 (prepolymer)
R = H, 2-Me, 3-Me, 4-Me, 3-F, 4-F, 4-CN

80-97 % ee, 30-50 %yield, 80-99 % ee, 30-50 % yield
PSL
OCOR
CF, 4; CN
R R’
95 % ee, 23 %yield, Me Me 170a [73]
CRL
99 % ee, 37 % yield, n-Bu Me 170b [73]
CRL
97 % ee, 36 % yield, n-Pent Me 170c [73]
CRL
99 % ee, 30 % yield, n-Hept Me 170d [73]
CRL
99 % ee, 40 % yield, Me Et 170e [73]
CRL
1 B. Cambou, A. M. Klibanov, Biotechnol. Bioeng. 10 S . Hamaguchi, T. Ohashi, K. Watanabe, Agnc. B i d .
1984,26,1449. Chem. 1986,50,375.
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1988,29,4927. Biocatalysts i n Organic Synthesis: Amsterdam,
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6 J . T. Lin, T. Yamazaki, T. Kitazumi, /. Org. Chem. 5533.
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7 A. Kutsuki, I. Sawa, J. Hasegawa, K. Watanabe, 1991,56, 1521.
Agric. Biol. Chem. 1986, SO, 2369. 15 T. Itoh, Y Tagaki, Chem. Lett. 1989, 1505.
8 K. Mori, R. Bernotas, Tetrahedron: Asymmetry 16 K. Burgess, I. Henderson, Tetrahedron: Asymmetry
1990, I , 87. 1990, I , 57.
9 S. Hamaguchi, T. Ohashi, K. Watanabe, Agric. B i d . 17 H. Suemune, Y. Mizuhara, H. Akita, K. Oishi,
Chem. 1986,50,1629. Chem. Pharm. Bull. 1987,35,3112.
1 7 . 1 Hydrolysis and formation ofCarboxylid Acid Esters
18 C. Feichter, K. Faber, H. Griengl, Tetrahedron Lett. 47 I. L. Bermudez, C. del Campo, L. Salazar, E. F.

I
457
1989, 30, 551. Llama, J. V. Sinisterra, Tetrahedron: Asymmetry,

19 H. Akita, H. Matsukara, T. Oishi, Tetrahedron Lett. 1996, 7, 2485.
1986,27,5241. 48 T. Sakai, T. Takayama, T. Ohkawa, 0. Yoshio,
20 E. Foelsche, A. Hickel, H. Honig, P. T. Ema, M. Utaka, Tetrahedron Lett. 1997, 38, 1987.
Seufer-Wasserthal.J. Org. Chem. 1990, 55, 1749. 49 D. Bianchi, P. Moraschini, A. Bosetti, P. Cesti,
21 B. A. Marples, M. Rogers-Evans, Tetrahedron Lett. Tetrahedron Lett. 1994,5,1917.
1989, 30, 261. 50 H. Akita, C. Y. Chen, S. Nagumo, Tetrahedron:
22 T. Tsukamoto, T. Yoshiyama, T. Kitazume, Asymmetry 1994,51207.
Tetrahedron: Asymmetry 1991.2, 759. 51 T. Sakai, Y. Miki, M. Nakatani, T. Ema,
23 T. R. Nieduzak, A. L. Margolin, Tetrahedron: K. Uneyama, M. Utaka, Tetrahedron Lett. 1998,39,
Asymmetry 1991,2,113. 5233.
24 H. S. Bevinakatti, A. A. Baneji, ]. Org. Chem. 52 W. Adam, M. T. Diaz, C. R. Saha-Moller,
1991,56,5372. Tetrahedron: Asymmetry 1998, 9, 589.
25 Y. Naoshima, Y. Munakata, S. Yoshida, A. Funai, 53 W. Adam, M. T. Diaz, C. R. Saha-Moller,
J. Chem. Soc., Perkin Trans. 11991, 549. Tetrahedron: Asymmetry 1998, 9, 791.
26 T. Yamazaki, N. Okamura, T. Kitazume, 54 T. Ziegler, F. Bien, C. jurisch, Tetrahedron:
Tetrahedron: Asymmetry 1990, I , 521. Asymmetry 1998,9,765.
27 S. Takano, M. Setoh, K. Ogasawara, Tetrahedron: 55 S. Conde, M. Fierros, M. I. Rodriguez-Franco,C.
Asymmetry 1993,4,157. Puig, Tetrahedron: Asymmetry 1998,9, 2229.
28 U. Goergens, M. P. Schneider,]. Chem. Soc., 56 N. Hayashi, K. Yanagihara, S, Tsuboi, Tetrahedron:
Chem. Commun. 1991,1064. Asymmetry 1998,9,3825.
29 U. Goergens, M. P. Schneider, Tetrahedron: 57 T.Akeboshi, Y. Ohtsuka, T. Sugai, H. Ohta,
Asymmetry 1992,3,1149. Tetrahedron 1998,54, 7387.
30 R. ChCnevert, R. Gagnon,J. Org. Chem. 1993.58, 58 CY
: . Yu, 0. Me&-Cohn, Tetrahedron Lett. 1999,40,
1054. 6665.
31 M. Treilhou, A. Fauve, J.-R. Pougny, 1.-C. Prom&, 59 L. Salazar, J. L. Bermudez, C. Ramirez,
H. Veschambre,]. Org. Chem. 1992,57, 3203. E. F. Llama, J. V. Sinisterra, Tetrahedron:
32 U. Ader, M. P. Schneider, Tetrahedron: Asymmetry Asymmetry, 1999, 10,3507.
1992, 3, 521. GO S . j. Fletcher, C. M. Rayner, Tetrahedron Lett. 1999,
33 M. P. Schneider, U. Goergens, Tetrahedron: 40,7139.
Asymmetry 1992,3,525. 61 T. Itoh, K. Kudo, N. Tanaka, K. Sakabe, Y. Takagi,
34 R. Seemayer, M. P. Schneider, Tetrahedron: H. Kihara, Tetrahedron Lett. 2000,41,4591.
Asymmetry 1992,3,827. 62 G.Eidenhammer, F. Hammerschmidt, Synthesis,
35 S. Liang, L. A. Paquette, Tetrahedron: Asymmetry 1996,748.
1990, I, 445. 63 M. Drescher, F. Hammerschmidt, H. Kahlig,
36 Y:F. Li, F. Hammerschmidt, Tetrahedron: Synthesis, 1995, 1267.
Asymmetry 1993,4,109. 64 M:J. Kim, I.T. Lim, Synlett, 1996, 138.
37 M. Banziger, J. F. Mc Gamty, Th. Meul, J. Org. 65 M. Kamezawa, M. Kitamura, H. Nagaoka,
Chem. 1993,58,4010. H. Tachibana, T. Ohtani, Y.Naoshima, Liebigs.
38 A. Chadha, U. Goergens, M. P. Schneider, Ann. Chem. 1996,167.
Tetrahedron: Asymmetry 1993,4, 1449. 66 K. Mori, H. Ogita, Liebigs Ann. Chem. 1994,
39 U. Ader, M. P. Schneider, Tetrahedron: Asymmetry 1065.
1992, 3, 205. 67 S. Tsuboi, J. Sakamoto, H. Yamashita, T. Sakai,
40 K. Matsumoto, N. Suzuki, H. Ohta, Tetrahedron M. Utaka,]. Org. Chem. 1998,63,1102.
Lett. 1990, 31,7163. 68 S. B. Desai, N. P. Argade, K. N. Ganesh,]. Org.
41 U.Goergens, M. P. Schneider,]. Chem. Soc., Chem. 1996,61,6730.
Chem. Commun. 1991,1066. 69 H:L- Liu, B. H. Hoff, T. Anthonsen,J. Chem. Soc.,
42 R. P. C. Cousins, M. Mahmoudian, P. M. Youds, Perkin Trans. 1 2000, 1767.
Tetrahedron: Asymmetry 1995, 6, 393. 70 F. Bellezza, A. Cipiciani, G.Cruciani,
43 J. Milton, S. Brand, M. F. Jones, C. M. Rayner, F. Fringuelli,J. Chem. Soc., Perkin Trans. 1 2000,
Tetrahedron: Asymmetry 1995, 6, 1903. 4439.
44 B. Charpentier, 1.-M. Bemardon, P. Diaz, M. Vion, 71 H. Akita, I. Umezawa, H. Matsukura, Chem.
C. Millois, B. Bernard, B. Shroot, Bioorg. Med. Pharm. Bull. 1997,45, 272.
Chem. Lett. 1995,5,2801. 72 C. Waldinger, M. Schneider, M. Botta, F. Corelli,
45 B. H. Hoff, V. Waagen, T. Anthonsen, Tetrahedron: V. Summa, Tetrahedron: Asymmetry 1996, 7,
Asymmetry 1996,7,3181. 1485.
46 M. Seki, T. Furutani, T. Miyake, T. Yamanaka, 73 K. Konigsberger, K. Prasad, 0. Repic, Tetrahedron:
H. Ohmizu, Tetrahedron: Asymmetry 1996, 7 , 1241. Asymmetry 1999, 10,679.
458
secondary alcohols of the aryl alkyl or dialkyl type are accessible but also those
containing all kinds of functional groups in the various positions. An inspection of
Tables 11.1-15 and 11.1-13 reveals that in cases where an alkoxycarbonyl group is
present as well as the secondary hydroxyl group, two possibilities for enantiomer-
differentiation may exist, hydrolysis of the acylated alcohol or hydrolysis of the
carboxylic acid ester. Changing the acyl group from acetate to butyrate, chloroacetate,
ethylthioacetate or hexadecanoate may have a beneficial effect on the enantioselectiv-
ity of the hydrolysis. The use of chloroacetates in many cases facilitates the
separation of the ester and the alcohol formed. A series of cyanohydrin acetates have
been prepared. Isolation of the cyanohydrin itself is usually not possible because of
the alkaline pH. With Alcaligenes sp. lipase, which has its pH optimum between 4
and 5, isolation of the cyanohydrin acetate 331, as well as the cyanohydrin 33a
becomes possible.
Enantiomer separation of a-benzyloxy ketones can be accomplished via lipase-
catalyzed enantiomer-differentiating hydrolysis of the corresponding enol esters
with formation of a mixture of the resulting ketone and the unchanged enol ester
(94a,b).
a-Acetoxysulfides (102-108), a-acetoxyethers (140-146) and a-acetoxyphospho-
nates (152-153) (Table 11.1-15)are useful substrates for lipases too.
Acylated alcohols and alcohols of Table 11.1-15 which can be obtained with other
hydrolases as such or of opposite configuration are contained in Tables 11.1-20 and
11.1-22.
A broad structural range of racemic secondary mono-, bi- and tricyclic acylated
alcohols are substrates in lipase-catalyzed enantiomer-differentiating hydrolysis as
the examples 1-90 of Table 11.1-16 reveal. A large number of cis- and trans-
cycloalkanols bearing a functional group in 2-position (1-20, 25, 26, 58-62) is
thereby available in enantiomericallypure form. Enantiomer selectivity in the case of
cyclic allylic alcohols where the double bond bears no other substituent in the a-
position is frequently low. Through a temporary substrate modification such as
mono- or dibromination, enantiomerically pure cyclic allylic alcohols may also be
obtained in these cases (51, 52).
Prochiral diketones or racemic ketones, like enol esters, are also amenable to a
hydrolase-catalyzed asymmetric transformation. The enol acetates and ketones 63
and 64, respectively, may be obtained by Pseudomonas cepacia lipase-catalyzed and
Candida qlindracea lipase-catalyzed hydrolysis of the corresponding racemic enol
esters or prochiral bis enol ester, respectively, with high enantioselectivity and yield.
A variety of allylic monocyclic alcohols (50, 54, 56, 57, 68-70,77-79 and 81) (Table
11.1-16)have been obtained mainly by Pseudomonas cepacia lipase-catalyzedhydroly-
sis. The planar chiral [2,2]paracyclophane87 was readily resolved by two different
lipases, yielding both enantiomers in almost enantiomerically pure form.
The Candida cylindracea lipase-catalyzed, Candida rugosa lipase-catalyzed and
cholesterol esterase-catalyzed hydrolyses of acetates 88b-1021, are examples of the
utilization of a remote phenolic ester group as the site of enzymatic attack. For such
cases, cholesterol esterase seems to be particularly well suited.
Acylated alcohols and alcohols of Table 11.1-16which can be obtained with other
Table 11.1-16. Lipase-catalyzed enantiomer-differentiatinghydrolysis of esters of racemic cyclic

I 459
secondary and tertiary alcohols in aqueous solution (PFL PseudomonasPuorescenslipase, PSL

Pseudomonas sp. lipase, CCL Candida cylindracea lipase, ABL Arthrobacter sp. lipase, PCL
Pseudomonas cepacia lipase, CRL Candida rugosa lipase, CE cholesterol esterase).
e;
OCOR2
,,st R'
R' R2 lipase
OAc Me 299 % ee, 30 %yield la PFL 30 % ee, 65 %yield l b [l]
COzEt Me 299 % ee, 42 % yield 2a PFL 90 % ee, 50 % yield 2b [l]
N3 n-Pr 92%ee,44%yield 3a PFL 298%ee,- 3b PI
0-
OH OAc
0
1
C0,Et
>99 % ee, 43 %yield, PFL 95 % ee, 42 % yield
OCOR2
R' R2 lipase
5a 299 % ee, 33 % yield OAc Me PFL 48% ee, 51 %yield 51, [31
Ga 84 % ee, 48 % yield OAc Me PSL 94 % ee, 41 %yield ~b [41
7a 299 % ee, 41 % yield C02Et Me PFL 55 % ee, 59 %yield 7b [31
8a 96 % ee, 40 % yield N3 n-Pr CCL 298%ee,- 8b PI
9a 298 % ee, 40 % yield NO2 n-Pr CCL 85%ee,- 9b 121
10a 93 % ee, 40 % yield CN n-Pr CCL 93%ee,- 10b [2]
lla 298 % ee, 38 % yield CN n-Pr PSL 95%ee,- l l b [2]
1h 95 % ee, 44 % yield Ph CHzCl PSL 97 % ee, 43 %yield 12b 141
13a 295 % ee, 47 % yield PhCH2 Me PSL 295 % ee, 45 %yield 13b [4]
14a 98 % ee, 45 % yield OMe Me PSL 96 % ee, 49 % yield 14b [4]
15a 299 % ee, 42 % yield OPh Me PSL 96 % ee, 45 % yield 15b [4]
5c02Et
OAc
0
>99 % ee, 32 %yield, PFL
1Ga [3]
** * co*Et
70 % ee, 63 % yield
16b [3]
460
Table 11.1-16. (cont.).
W W
R' R2 lipase
17a 299 % ee, 45 % yield OAc Me PFL 55% ee, 55 %yield 17b [3]
18a 299 % ee, 38 % yield COzEt Me PSL 68 % ee, 58 %yield 18b [3]
19a 89 % ee, 40 %yield N, n-Pr CCL 91 %ee,- 19b [3]
OAC
>99 % ee, 36 % yield, PFL 45 % ee, 64 % yield
bR'
HO
R'
acetate was not isolated
99 % ee, -, ABL R' = CH2C=CH, R2 = Me 21 [5-71

98 % ee, -, ABL R' = CH2CH=CH2'R2 = Me 22 [5-71
79 % ee, -, ABL R' = CH~CGCH,R2 = H 23 [S-71
30 % ee, -, ABL R' = R2 = H 24 [S-71
20-50 % conversion
6
OCOnPr
sYN3
/N3 25a [2] 25b 121
88 % ee, 40 % yield, CCL 298 % ee, -
OCOnPr

2Ga [2]
(YN3
94 % ee, -
261, [2]
7 1.1 Hydrolysis and Formation ofcarboxylid Acid Esten I461
Table 11.1-16. (cont.).
6iMe2
27a [8]
OAc
OiMe2 2% PI
57 % ee, 50 % yield
HO AcO
28a [9, 101 28b [9, 101
298 % ee, 46 % yield, PFL 298 % ee, -

(further hydrolysis of the acetate)
29a [lo] 29b [lo]
81 % ee, -, PFL
H 30 [31
OH
299 % ee, 13 %yield, PFL acetate was not isolated
03
OH
31a [ll] 31b [ll]

/
OH
03 03
OAc
1
32a [ll] 32b [ll]
/

11 Hydrolysis and Formation o f C - 0 Bonds
462
OCOnPr
1
295 % ee, 43 %yield, CCL R = H 33a [12] 295 % ee, 40 %yield 33b [I21
295 % ee, 40 %yield, CCL R = Me 34a [12] 44 % ee, 69 %yield 34b [12]
OH
Y ConPr
R
31 % ee, 48 %yield, CCL R = H 35a [12] 36 % ee, 46 %yield 35b (121
84 % ee, 37 %yield, CCL R = Me 3Ga [12] 77 % ee, 50 %yield 3Gb [12]
x.3
OH
37a [12] >:5npr 371, [12]
OCOnPr
1
38b 1121
OCOR
x-Y 2 R
39a 90%ee,- CH=CH CH2 Me 296%ee,- 39b [13]
40a 88%ee,- CH=CH CH2 n-Pr 89%ee,-, PSL 40b[14]
40a 97%ee,- CH=CH CH2 n-Pr 40b 1141
41a 93%ee,- CH=CH 0 n-Pr 297 % ee, - 41b I151
42a 75%ee,- CH2-CH2 CHI Me 52%ee,- 42b [ll,141
43a 94%ee,- CH-CH CH2 n-Pr t97%ee,- 43b [ll,141

J 1.1 Hydrolysis and Formation of Carboxylid Acid Esters
Table 11.1-16. (cont.).
44a CH2 n-Pr 83%ee,- 44b (171
4Sa 0 n-Pr 85 %ee,- 45b [15]
4Ga 22 % ee, -
x
0
~~
0
CH2 n-Pr 14%ee,- 46b [14]
all CCL CH-CH
MeomNMe
OH
47a 1161 4% [16]
Me0 Me0
Meo&NMe
Ac2L
48a [17] 48b [17]
96 % ee, - 95 % ee, -
(further hydrolysis of reacylated alcohol), CCL (further hydrolysis of acetate)
49a [18] 49b [18]
AcO ACO
OCOnPr
298 % ee, 46 %yield, PCL

Sla [20]
oBr
298 % ee, 46 % yield
51b [20]
464
OCOnPr

52a [20]
ogr
298 % ee, 45 % yield
52b [20]
54b [22]
HO AcO
55a 1231 >'r)OB" 55b 1231

BnO
C0,Et C0,Et
btoH hoAc
99 % ee, 44 % yield, PCL n=1 56a [24] 100 % ee, 45 %yield 56b [24]
57b [24]
100 % ee, 43 %yield, PCL n=2 57a [24] 91 % ee, 52 %yield
HobR R
Aco'GR
93 % ee, 45 %yield, PSL Ph 58a [25] 298 % ee, 42 %yield 58b [25]
90 % ee, 43 % yield, PSL PhCHz 59a [25] 93 % ee, 45 %yield 59b [25]
Table 11.1-16. (cont.).
11.1 Hydrolysis and formation ofCarboxylid Acid Esters
I 465
84 % ee, 45 % yield, PSL PhO 6Oa [25] 298 % ee, 45 %yield Gob [25]
80 % ee, 43 %yield, PSL PhCH20 61a [25] 298 % ee, 40 %yield Glb [25]
51 % ee, 38 %yield, PSL OAc 62a [25] 46 % ee, 45 %yield 62b [25]
0 OAc
II I
63b [26]
Aco&c 64 [27]
298 % ee, 80 % yield CCL
0gHoH
H ~ 0s m 65a [28]
O
- -
d
65b [28]
>99 % ee, 37 % yield, CAL-B

G6a 1291
hc
>99 % ee, 42 % yield
661,[29]
(in the presence of

PdClZ(MeCN)zand air)
.OAc
67a [30] 67b [30]
99 % ee, 35 %yield, PFL 99 % ee, 31 %yield

466
AcO
68a [31] 68b [31]

NO2
OAc
G9a [31] G9b 1311
NO2
>99 % ee, 32 %yield, PCL >99 % ee, 43 % yield
70a (321 70b 1321

TBDMSO TBDMSO""
Me,N.,?H ?H
nPrOCO
71a [33] 71b [33]

, %O
89 ee, -, q
CRL, o
28 % M e
conversion 37 % ee, -
72a [33] nPrOCO*OMe 72bI331

+HO
M Me
>98 % ee, 40 % yield, CRL >98 % ee, 40 % yield
73a [33] 73b[33]

33 % conversion
>99 % ee, 35 % yield
60 % conversion
Table 11.1-16. (cont.).
11.1 Hydrolysis and Formation of Carboxylid Acid Esters
I
467
ROA A c
R
2-Me-naphthyl >99 % ee, 46 % yield, 74a [34] >99 % ee, 46 % yield 74b [34]
PCL
CHZPh >99 % ee, 48 % yield, 75a [34] >99 % ee, 49 %yield 75b [34)
PCL
TBDMS >99 % ee, 48 %yield, 76a [34[ >99 % ee, 48 % yield 76b [34]
PCL
OAc
&SPh

77a [35]
WPh 77b [35]
100 % ee, 46 % yield
eph 78a [35]
us'"
OAc
78b [35]
fj
Bra..,,
Br
OAc
90 % ee, 47 % yield, PCL 79a [36a] >98 % ee, 26 % yield 79b [36a]
>99 % ee, 38 % yield, PPL, 79a [36b] >99 % ee, 38 % yield, 79b [3Gb]
after recrystallization after recrystallization
80a [37] 80b [37]

0h A c
Q /
81a [38] rn 81b [38]
OH OAc
94 % ee, 46 %yield, PCL >99 % ee, 45 % yield
468
Table 11.1-16. (cont.).
.,,\OH
0 AcO
n
1 >99%ee, 42 %yield, PCL 82a [39] >99% ee, 40 % yield 82b [39]
2 >99% ee, 44 % yield, PCL 83a [39] >99% ee, 48 % yield 83b [39]
H H
>99 % ee, 30 %yield, PFL _ -
85b [41]
>99 % ee, 49 % yield, PCL >99 % ee, 51 % yield
AcOo-&R HOA.,.,,,,R
8Ga [42] 8Gb [42]
EtO Etd
R = n-Bu, n-C5HI1,n-C6H13, Ph unstable
95-98 % ee, 17-35 % yield, PCL
AcO A.
. .,, R ent-8Ga [42] HO',/&R
ent-8Gb [42]
E td EtO
R = ~ - B u ,n-CsH11, n-C6H13, Ph unstable
>99 % ee, 32-49 % yield, CAL-B
>98 % ee, 46 % yield, CCL 87a [43] >98 % ee, 43 % yield 87b [43]
90 % ee, 51 %yield, CRL 87a [43] >99 % ee, 44 % yield 87b [44]
Table 11.1-16. (cont.).
I
469
"
n
O D & Aco
""a.
1 13 % ee, 55 %yield, CCL 88a [45] 95 % ee, 13 %yield 88b [45]
2 20 % ee, 65 % yield, CCL 89a [45] 98 % ee, 20 % yield 89b [45]
90a [4G] 90b [4G]

\ \
Me O R
55% ee, 50% yield, CE 76% ee, 42 % yield, CE
R= Me
OH R
@ \ Me
""Ph
91a [47] 91b [47]

99 % ee, 42 % conversion G1% ee, 42% conversion
CE (porcine pancreas), CE (porcine pancreas),
sodium taurocholate sodium taurocholate
53 % ee, 52% conversion 49 % ee, 52 % conversion

CE (porcine pancreas), sodium CE (porcine pancreas), sodium
taurocholate taurocholate
H? AcO
49 % ee, 40 % conversion 33 % ee, 40 % conversion

470
Table 11.1-16. (cont.).
Act? Ac?
94b [47]
43 % ee, 51 % conversion 44% ee, 51 % conversion

OH 0 R
95a [48] 95b [48]
E = 10-15, CE (porcine E = 10-15, CE (porcine

pancreas), pancreas),
ba,..CHCI 9Gb [48]
E = 4.6, CE (porcine pancreas), E = 4.6, CE (porcine pancreas),

AcO
97b [48]
nBu
E = 14, CE (porcine pancreas), E = 14, CE (porcine pancreas),

98b [48]

g:
Table 11.1-16. (cont.).
I
471
s's*,,ph AcO
+jPh,,:
/ 99a [48] 99b [48]
\ \

moH moAC
lOOa [48] 1OOb [48]
OAc OAc
E > 400, CE (porcine pancreas), E > 400, CE (porcine pancreas),

lOla [48] 101b [48]

OAc OAc
&OH
E > 10, CE (porcine pancreas),

c=b"
E > 10, CE (porcine pancreas),
-
0
I
N3 0-
I
.3
102a 1491 102b [49]
....Ph
HO AcO
83 % ee, 51 % conversion 80% ee, 51 % conversion
CE (Pseudomonaspurorescens) CE (Pseudomonaspurorescens)
51 % ee, 35% conversion 96% ee, 35 % conversion
CE (porcine pancreas) CE (porcine pancreas)
1 Z.-F. Xie, H. Suemune, K. Sakai,]. Chem. Soc., 3 Z.-F. Xie, 1. Nakamura, H. Suemune, K. Sakai,
Chem. Commun. 1987,838. J. Chem. SOC.,Chem. Commun. 1988,9GG.
2 H. Honig, P. Seufer-Wasserthal, F. Fiilop,]. Chem. 4 K. Laumen, D. Breitgoff, R. Seemeyer, M. P.
Soc., Perkin Trans. 1 1989,2341. Schneider,]. Chem. Soc., Chem. Commun. 1989,
148.
472
I 5 S. Mitsuda, S. Nabeshima, H. Hirohar, Appl. 27 P. Duhamel, P. Renauf, D. Cahard, A. Yebga,
Microb. Biotechnol. 1989, 31, 334. 1. M. Poirier, Tetrahedron:Asymmetry 1993,4, 2447.
6 H. Danda, A. Maehara, T. Umemura, Tetrahedron 28 A. K. Gosh, Y Chen, Tetrahedron Lett. 1995, 36,
Lett. 1991, 32, 5119. 505.
7 H. Danda, T. Nagatomi, A. Maehara, T. Umemura, 29 H. Nagata, K. Ogasawara, Tetrahedron Lett. 1999,
Tetrahedron 1991, 47, 8701. 40, 6617.
8 K. Fritsche, C. Syldatk, F. Wagner, 30 L. Aribi-Zouioueche, J.-C. Fiaud, Tetrahedron Lett.
H. Hengelsberg, R. Tacke, Appl. Microbiol. 2000,41,4085.
Biotechnol. 1989, 31, 109. 31 J. Doussot, A. Guy, R. Garreau, A. Falguisres,
9 N. Klempier, K. Faber, H. Griengl, Biotechnol. Lett. C. Ferroud, Tetrahedron: Asymmetry 2000, 11, 2259.
1989,685. 32 K. Sugawara, Y. Imanishi, T. Hashiyama,
10 N. Klempier, P. Geymayer, P. Stadler, K. Faber, Tetrahedron: Asymmetry 2000, 1I , 4529.
H. Griengl, Tetrahedron: Asymmetry 1990, I, 111. 33 H:J. Gais, C. Griebel, H. Buschmann,
11 K. Laumen, M. P. Schneider,]. Chem. Soc., Chem. Tetrahedron: Asymmetry 2000, 11, 917.
Commun. 1988,598. 34 T. Taniguchi, M. Takeuchi, K. Kodata, A. S.
12 L. Dumortier, J. Van Eycken, M. Vandewalle, EIAzab, K. Ogasawara, Synthesis 1999, 1325.
Tetrahedron Lett. 1989, 30, 3201. 35 S. Takano, 0. Yamada, H. lida, K. Ogasawara,
13 G. Eichberger, G. Penn, K. Faber, H. Griengl, Synthesis 1994, 592.
Tetrahedron Lett. 198G, 27, 2843. 3G a) C. R. Johnson, M. W. Miller,]. Org. Chenr. 1995,
14 T. Oberhauser, M. Bodenteich, K. Faber, G. Penn, 60,6674 b) 0. Block, G. Klein, H.-J. Altenbach,
H. Griengl, Tetrahedron 1987, 43, 3931. D. J. Brauer, ]. Org. Chem. 2000, 65, 716.
15 R. Saf, K. Faber, G. Penn, H. Griengl, Tetrahedron 37 K. Kadota, A. S. ElAzab, T. Taniguchi,
1988,44,389. K. Ogasawara, Synthesis 2000, 1372.
16 0. Hoshino, K. Itho, B. Umezawa, H. Akita, 38 M. Takahashi, R. Koike, K. Ogasawara, Chem.
T. Oishi, Tetrahedron Lett. 1988, 29, 567. Pharm. Bull. 1995,43, 1585.
17 Y Hirose, M. Anzai, M. Saitoh, K. Naemura, 39 7. Taniguchi, R. M. Kanada, K. Ogasawara,
H. Chikamatsu, Chem. Lett. 1989, 1939. Tetrahedron: Asymmetry 1997. 8. 2773.
18 K. Naemura, T. Matsumara, M. Komatsu, 40 R. A. MacKeith, R. McCague, H. F. Olivo, S. M.
Y. Hirose, H. Chikamatsu, Bull. Chem. Soc.]pn. Roberts, S. J. C. Taylor, H. Xiong, Bioorg. Med.
1989,62, 3523. Chem. 1994,2, 387.
19 S. Takano, M. Suzuki, K. Ogasawara, Tetrahedron: 41 H. Nagata, N. Miyazawa, K. Ogasawara, Synthesis
Asymmetry 1993,4,1043. 2000, 2013.
20 A. K. Gupta, R. J. Kazlauskas, Tetrahedron: 42 B. Westermann, B. Krebs, Org. Lett. 2001, 3, 189.
Asymmetry 1993,4,879. 43 A. Cipiciani, F. Fringulli, V. Mancini, 0. Piermatti,
21 Z:F. Xie, H. Suemune, K. Sakai, Tetrahedron: A.M. Scappini, Tetrahedron 1997,53,11853.
Asymmetry 1990, I , 395. 44 D. Pamperin, C. Schulz, H. Hopf, C. Syldatk,
22 P. Washausen, H. Grebe, K. Kieslich, M. Pietzsch, Eur.]. Org.Chem.,1998, 1441.
E. Winterfeldt, Tetrahedron Lett. 1989, 30, 3777. 45 J. Y. Goujon, F. Zammattio, B. Kirschleger,
23 X. Chen, S. M. Siddiqi, S. W. Schneller, Tetrahedron: Asymmetry 2000, I I , 2409.
Tetrahedron Lett. 1992, 33, 2249. 46 E. Mizuguchi, M. Takemoto, Tetrahedron:
24 S. Takano, T. Yamane, M. Takahashi, Asymmetry 1993,4,1961.
K. Ogasawara, Tetrahedron: Asymmetry 1992, 3, 47 A. N. Serreqi, R. J . Kazlauskas, ]. Org. Chem. 1994,
837. 59, 7609.
25 R. Seemayer, M. P. Schneider, J. Chem. Soc., Chem. 48 A. N. Serreqi, R. J. Kazlauskas, Can. J. Chem. 1995,
Commun. 1990, 2359. 73, 1357.
26 T. Izumi, F. Taura, K. Sasaki, Bull. Chem. Soc. Jpn. 49 X. Yang, A. R. Reinhold, R. L. Rasati, K. K. C. Liu,
1992,65,2784. Org. Lett. 2000, 2, 4025.
hydrolases as such or of opposite configuration are contained in Tables 11.14 and

11.1-16.
11.1.1.2
Formation o f Carboxylic Esters
11.1.1 2.1 Lipases

Of the many hydrolases known, only the lipases, subtilisin and to some extent a-
chymotrypsin, pig liver esterase, and thermolysin [G4aJ show a sufficiently high
I
473
catalytic activity in organic solvents of low water content to be of practical value for
asymmetric synthesis through acylation of prochiral or racemic alcohols, alcoholysis
of prochiral or racemic acylated alcohols and prochiral anhydrides, and cyclization of
racemic hydroxy carboxylic acids. Lipases, as stated previously, are unique for
organic synthesis, since they exhibit not only a high catalytic activity in water or in
two-phase systems composed of water and a water-immiscibleorganic solvent or the
liquid substrate, but most importantly also in water-miscibleor immiscible organic
solvents of low water content. This allows for the attainment of favorable equilibria
not only in asymmetric hydrolysis but also in esterification reactions.
In the formation of carboxylic esters in an anhydrous organic solvent, its
hydrophobicity and the water activity have a major influence on the reac-
ti0n[~'9 36* 1341. Hence, the organic solvent used can significantly influence the
selectivity of a lipase-catalyzedenantiotopos- or enantiomer-differentiating reaction.
Furthermore, the acyl donor may influence reactivity and selectivity.
Lipases are most advantageously used for the acylation of prochiral diols or
racemic alcohols and for the alcoholysis of racemic acylated alcohols. Generally,
through acylation of a prochiral diol or racemic alcohol in an organic solvent such as
diethyl ether, diisopropyl ether, tert-butyl methyl ether, tetrahydrofuran, dichloro-
methane, pentane, hexane, toluene or tert-pentyl alcohol with acylating reagents
such as vinyl acetate, vinyl butyrate, vinyl propionate, vinyl laurate, vinyl palmitate,
vinyl chloroacetate,isopropenyl acetate, oxime esters, ethyl acetate, ethyl propionate,
trifluoroethyl butyrate, trichloroethyl butyrate, trifluoroethyl acetate, ethyl octanoate,
ethyl methoxy acetate, ethyl thiooctanoate, acetic anhydride, succinic anhydride or
2-phenyloxazolin-5-one and hydrolysis of the corresponding prochiral diacetate
(dipropionate,dichloroacetate) or racemic acetate (chloroacetate)in water or in water
and a water-immiscible organic solvent, access to both enantiomers of the corre-
sponding monoacetate and alcohol, respectively, is provided with one enzyme
(Tables 11.1-10 to 11.1-12 and 11.1-18).This is because of the same enantiotopic
group and enantiomer recognition shown in general by the enzyme in both reactions
(Scheme 11.1-12), and favorable opposite equilibria.
In many cases vinyl acetate, isopropenyl acetate, ethyl acetate and propionyl
acetate not only serve as acylating reagents but also as solvents.
For the acylation of prochiral diols, ee values of monoacetates (about 90%) can be
raised considerably in most cases by a higher degree of conversion at the expense of
a lower chemical yield to the point where an enantiomer-differentiating formation of
the diacetate can take place (Scheme 11.1-11,Figure 11.1-l),because in most cases
the enzyme preferentially catalyzes the acylation of the minor enantiomer. The
enantioselectivity and thermostability of lipases is frequently enhanced in organic
solvents of low water content. A minimum amount of water is required for the
catalyhc activity of the lipase. In most cases lipase preparations with a residual water
content of approximately 1 % in anhydrous organic solvents are employed. Fre-
quently in organic solvents of low water content the thermostability of lipases is
much higher than that in aqueous solution [361.
The use of lipase in other forms than lyophilized powders, as for example on
different kinds of solid supports, entrapped in sol-gelmaterials or as CLECs, has the
474
I Table 11.1-17. Lipase-catalyzedenantiotopos-differentiating acylation of prochiral acyclic diols in
organic solvents (CCL Candida cylindracea lipase, PFL PseudomonasPuorescens lipase, PPL pig
pancreas lipase, CVL Chromobacterium viscosum lipase, PSL Pseudomonas sp. lipase, RJL
Rhizomucorjavanicus lipase, A N L Aspergillus niger lipase, CAL Candida antarctica lipase, not
specified, PCL Pseudomonos cepacia lipase, CRL Candida rugosa lipase).
R' R2 R3 Lipase Acyldonor ee yield Ref.

("A) ("A)
1 Me H Ac PFL vinyl acetate 60 70 [l]
2 CHFCH-CH~ AC H PFL vinyl acetate 81 89 [I]
3 CHz=CH-(CH2)2AC H PPLa ethyl acetate 90 70 [2]
4 Ph Ac H PPL" ethyl acetate 92 98 [2]
5 CHzPh Ac H PPLa ethyl acetate 13 90 [2]
5 CHzPh Ac H PFL vinyl acetate 294 100 [3]
PFL vinyl acetate 97 96 111
G CH, Ac H PFL vinyl acetate 86 93 [3]
I L PFL vinyl acetate 90 95 [l]
7 i-Pr Ac H PFL vinyl acetate 61 85 [I]

8 C-CGHLI Ac H PPLa ethyl acetate 58 90 [2]
9 c-CaHiICH2 AC H PPL" ethyl acetate 10 90 [2]
10 Cbz Ac H PPL vinyl acetate 97 77 [4]
11 OCH2Ph H Ac PFL isopropenyl 96 53 [4]
acetate
11 OCH2Ph H Ac PFL vinyl acetate 92 92 [5]
11 OCH2Ph H Ac PFL phenyl acetate 90 88 [5]
12 OEt H Ac PFL phenyl acetate 90 90 [5, 61
0011 0 B n
AcO-OH 13 [71 X O 14 PI
OBn HOAOAc
295 % ee, 70 % yield, CCL 298 % ee, 51 %yield, PFL
vinyl acetate vinyl acetate
R. r O H
Si
M~/LOCOiPr
R = Ph 70 % ee, 80 %yield, CCL 15 [9] 70 % ee, 50 % yield, CVL ent-15 [9]

methyl isobutyrate methyl isobutyrate
R = n-octyl 75 % ee, 63 %yield, CCL 16 [9] 76 % ee, 70 %yield, CVL ent-16 [9]
methyl isobutyrate methyl isobutyrate
Table 11.1-17. (cont.).
1 1. I Hydrolysis and Formation ofcarboxylid Acid Esters
I
475
MeO-0
CZ;OCQH,, 17 [lo] 18 [ll]
MeO-0 OTBDMS
95 % ee, 90 % yield, PPL 97 % ee, 94 % yield, CRL,

C~HI~COCH~CCI~ vinyl acetate
89 % ee, 80 % yield, PPL, >99 % ee, 76 % yield, PPL,

HO*OAC 21 (131
AcO
0
98 % ee, 86 %yield, PFL, 98 % ee, 75 % yield, PCL,

22 [14]
92 % ee, 55 %yield, PPL, 94 % ee, 86 %yield, PPL,

methyl acetate vinyl acetate
25 [16]
t B u 0 / R \ c O AOH
C
99 % ee, 86 %yield, PSL, R = H , 91 % ee, 97 % yield, 26 [17]

vinyl acetate PFL, vinyl acetate
R = Me, 88 % ee, 80 % yield, 27 [17]
PFL, vinyl acetate
OH 28 [18] 29 [19]
Me,PhSi BZO
>99 % ee, 98 % yield, PFL, vinyl 96 % ee, 63 % yield, PPL,

acetate vinyl acetate
95 % ee, 98 % yield, PCL, vinyl
acetate
11 Hydrolysis and Formation ofC-0 Bonds
Table 11.1-17. (cont.).
; + N 30 [20] 9 0Ac 31 [20]
OH
96 % ee, 84 % yield, PPL, vinyl 98 % ee, 81 % yield, PPL, vinyl

acetate acetate
q 0Ac 33 (211
&O Ac
\
OH OH
84 % ee, 21 % yield, ANL, 32 1211 97 % ee, 46 %yield, PPL,
98 % ee, 42 % yield, CAL, ent-32
vinyl acetate [211
WoAc OH
2
CH2 99 % ee, 97 %yield, PCL, 34 [22] 87 % ee, 95 %yield, PCL, 36 [22]
CF2 99 % ee, 82 % yield, PCL, 35 [22]
vinyl acetate
37 [23] &b / 38 [23]
85-92 % ee, 35-78 % yield, 61 % ee, 84 % yield, CRL,

CRL, 1-ethoxyvinyl2-hroate 1-ethoxyvinyl2-furoate
abs. config. not determined abs. config. not determined
Ph , .Ph 39 [24]
Ph Ph
ent-39 [24]
HO
HOAc AcO
nOH
90 % ee, 20 %yield, RJL, 90 % ee, 28 % yield, CCL,
Table 11.1-17. (cont.).
I
477
R
Me >99 % ee, 26 % yield, 40 [25]
PCL
Et phenyl acetate 40 [25]
94 % ee, 43 % yield,
PCL
phenyl acetate
HO
3
AcO
N’
I
R’
R’
R‘ R2
Me Me 97 % ee, 42 % yield, PCL 41 [25]
vinyl acetate
Me Me 96 % ee, 77 % yield, PCL 41 [25]
phenyl acetate
Et Et 96 % ee, 52 %yield, PCL 42 [25]
vinyl acetate
Et Et 84 % ee, 43 %yield, PCL 42 [25]
phenyl acetate
a purified PPL
1 K. Tsuji, Y. Terao, K. Achiwa, Tetrahedron Lett. 11 R. ChCnevert, G.Courchesne, Tetrahedron:
1989,30,6189. Asymmetry1995,6,2093.
2 G.M. Ramos Tombo, H:P. Schar, X. Femandez 12 T. Bando, Y. Namba, K. Shishido, Tetrahedron:
I Busquets, 0. Ghisalba, Tetrahedron Lett. 1986, 27, Asymmetry1997,8,2159.
5707. 13 C. Bonini, R. Racioppi, L. Viggiani, Tetrahedron:
3 S. Atsumi, M. Nakano, Y. Koike, S. Tanaka, Asymmetry1997,8,353.
M. Ohkubo, T. Yonezawa, H. Funabashi, 14 J. C. Anderson, S. V. Ley, S. P. Marsden,
J. Hashimoto, H. Morishima, Tetrahedron Lett. Tetrahedron Lett. 1994, 35, 2087.
1990,31,1601. 15 C. J. Bamett, T. M. Wilson, S. R. Wendel, M. J.
4 Y. F. Wang, J. J. Ialonde, M. Momongan, Winningham, J. B. Deeter,]. Org. Chem. 1994,59,
D. E. Bergbreiter,C.-H. Wong. ]. Am. Chem. Soc. 7038.
1988, 110,7200. 16 A. Avdagit, M. Gelo-PujiC, V. Sunjit, Synthesis
5 Y.Terao, M. Murata, K. Achiwa, T Nishio, 1995,1427.
M. Akamtsu, M. Kamimura, Tetrahedron Lett. 17 F:R. Alexandre, F. Huet, Tetrahedron :Asymmetry
1988,29,5173. 1998, 9, 2301.
6 M. Murata, Y.Terao, K. Achiwa, T. Nishio, K. Seto, 18 B. Danieli, G. Lesma, S. Macecchini, D. Passarella,
Chem. Phann. Bull. 1989,37,2670. A. Silvani, Tetrahedron: Asymmetry1999, 10,
7 K. Burgess, I. Henderson, Tetrahedron Lett. 1991, 4057.
32, 5701. 19 V. B6dai. L. Novik, L. Poppe, Synlett 1999, 759.
8 C. Bonini, R. Racioppi, L. Viggiani, G. Righi, 20 G. Guanti, E. Narisano, R. Riva, Tetrahedron:
L. Rossi, Tetrahedron: Asymmetry1993,4, 793. Asymmetry1997,8,2175.
9 A:H. Djerourou, L. Blanco, Tetrahedron Lett. 1991, 21 L. Banfi, G . Guanti, A. Mugnoli, R. Riva,
32,6325. Tetrahedron: Asymmetry1998, 9, 2481.
10 H. J. Bestmann, U. C. Philipp, Angau. Chem. 1991, 22 T. Yokomatsu, T. Minowa, T. Murano, S. Shibuya,
1 0 3 , 7 8 Angew. Chem., Int. Ed. Engl. 1991,30,86. Tetrahedron 1998, 54,9341.
478
I I 7 Hydrolysis and formation ofC-0 Bonds
23 S . Akai, T. Naka, T. Fujita, Y. Takebe, Y. Kita,Chem. 25 K. Takabe, Y. Iida, H. Hiyoshi, M. Ono, Y. Hirose,
Commun. 2000,1461. Y. Fukui, H. Yoda, N. Mase, Tetrahedron:
24 G. Nicolosi, A.Patti, M. Piatelli, C. Sanfilippo, Asymmetry 2000,II,4825.
Tetrahedron: Asymmetry 1994,5, 283.
advantage of easy recovery by filtration and reuse. Furthermore, these lipases have
higher stability, and, most importantly, their activity and selectivity are often much
higher than with the lyophilized powders.
One should bear in mind, however, that in nearly all cases lipase preparations are
used, which contain, as well as a large amount of mostly unspecified material such as
proteins, carbohydrates and solid support materials, only a minor amount of the
lipase and in several case even additional mostly unidentified hydrolases. The solid
material contained in the crude lipase preparation may have an important stabilizing
function in organic solvents, in which the lipase preparation is insoluble. Crude
lipase preparations supplied commercially contain up to 7 % of water. Drying the
solid material in vacuum may reduce the water content. Acylating reagents such as
vinyl acetate and isopropenyl acetate are very useful since they allow for an extreme
equilibrium position in acylation because of the tautomerization of the vinyl and
isopropenyl alcohol formed to acetaldehyde and acetone, respectively. The possible
harmful effect of acetaldehyde on the enzyme with the crude lipase preparation used
poses practically no problem in most cases because the low price of the enzyme
enables relatively large amounts of it to be used. Synthetically, lipase-catalyzed
acylations are convenient to carry out and, in contrast to the corresponding
hydrolyses, catalysts are easy to recover and can be reused.
A series of alkyl, alkoxy or acylamino 1,3-proanediol derivatives substituted in
2-position have been subjected to lipase-catalyzed acylation, and the monoacetates
(1-12,19, 20, 23-38, 40-42) were obtained with moderate to high enantiomeric
excess (Table 11.1-17).For the monoacetates 1-12,reactions with and in ethyl acetate
are usually slower than those with and in vinyl acetate. As in the hydrolysis of the
corresponding diacetates, much higher selectivities were recorded with the yet
unidentified carboxyl esterase from crude pig pancreas lipase. An excellent lipase for
the enantioselective acylation of 3-benzyloxy-l,3-propanediol is Pseudomonas Juor-
escens lipase, which gives high selectivity with vinyl acetate, isopropenyl acetate and
ethyl acetate. By carrying the acylation further, to a certain extent to the diacetate, the
enantiomerically pure monoacetate should be obtainable.
Sterically demanding substituents in 2-position such as in 20, 25,26,28,29 and
34-36 guarantee high enantioselectivity and yield for the monoacetates.
Sila propanediol derivatives (15,16),and butanediol, pentanediol, hexanediol and
heptanediol derivatives (17,18, 21,22)(Table 11.1-17)have also been prepared.
Monoacetates of Table 11.1-17which can be obtained with other hydrolases as
such or of opposite configuration are contained in Tables 11.1-4and 11.1-10.
Cyclic dimethanol derivatives have been extensively studied not only in lipase-
catalyzed hydrolysis (Table 11.1-11) but also in lipase-catalyzed enantioselective
acylation for synthetic and mechanistic reasons (1-16,20, 30, 32, 33, 37, 40, 45,
47-53, 57-62, 66,72) (Table 11.1-18). Generally, enantioselectivities in acylation of
the diol and hydrolysis of the corresponding diacetate yielding enantiomeric com-
Table 11.1-18.
Lipase-catalyzedenantiotopos-differentiatingacylation of prochiral cyclic diols in

I
479
organic solvents (PPL pig pancreas lipase, PFL Pseudomonasfluorescens lipase, PCL Pseudomonas
cepacia lipase, CCL Candida cylindracea lipase, MSL Mucor sp. lipase, CVL Chrornobacteriurn
viscosum lipase, CCL Ceotrichum candidum lipase, CRL Candida rugosa lipase, M M L Mucormiehei
lipase, CAL-B Candida antarctica B lipase, LIP Pseudomonas sp. lipase-Toyobo).
1 111 2 111
295 % ee, 82 % yield, PFL, vinyl acetate 88 % ee, 87 % yield, PFL,vinyl acetate
295 % ee, 82 % yield, PFL,ethyl acetate
3 I11
295 % ee, 85 % yield, PFL vinyl acetate 94 % ee, 64 % yield, PPL,vinyl acetate
25 % ee, 52 % yield, PPL,vinyl acetate 298 % ee, 87 %yield, PPL,vinyl acetate

vinyl acetate
R?,,
R'
R'
OH
a::c R2
H
8 [2, 31
PPL,vinyl acetate
98 % ee, 92 %yield
Ph3CO H 45 % ee, 72 % yield
H c1 296 % ee, 84 % yield
H SPh 296 % ee, 85 % yield
H SO2Ph 68 % ee, 69 % yield
H N3 295 % ee, 86 % yield
no?""
W'o&OAc
9 [31 10 PI
77 % ee, 91 % yield, PPL,vinyl acetate 7 % ee, 44 % yield, PFL, vinyl acetate

298 % ee, 78 % yield, PFL,vinyl acetate
9 % ee, 71 % yield, PFL,acetic anhydride
f OAc
80 % ee, 60 % yield, PFL,vinyl acetate 100 % ee, 32 % yield, CCL, vinyl acetate
7 7 Hydrolysis and Formation of C - 0 Bonds
480
I Table 11.1-18. (cont.)
80 % ee, 80 % yield, GCL, vinyl acetate, 99 % ee, 68 %yield, PPL, ethyl acetate
CHzClz 99 % ee, 92 %yield, PPL, vinyl acetate
95 % ee, 72 % yield, GCL, vinyl acetate, EtzO
0
0
6
96 % ee, 71 % yield, CCL, isopropenyl acetate 8 % ee, 38 % yield, PPL, vinyl acetate
76 % ee, 70 %yield, CCL, vinyl acetate 87 % ee, 72 % yield, CCL, vinyl acetate
17[7-111 I 18 [lla]
r - i OH
rofl
OH
98 % ee, -, PFL, vinyl acetate R = n-Pr, n-C7H15’ CHlCl
98 % ee, 52 % yleld, PPL, vinyl acetate >99-80 % ee, 58-39 % yield, pancreatin,
295 % ee, 48 % yield, pancreatin, trichloroethyl alkanoate
trichloroethyl acetate
>99 % ee, 50 % yield, PPL, trichloroethyl acetate
>99 % ee, 65 % yield, pancreatin, vinyl acetate
94 % ee, 85 %yield, MSL, vinyl acetate
84 % ee, -, PPL, vinyl acetate 95 % ee, 80 %yield, PFL, vinyl acetate
AcO
1
59 % ee, 60 %yield, PSL, vinyl acetate 295 % ee, 95 %yield, PCL, isopropenyl acetate
Table 11.1-18. (cont.).
I
481
AcO
1
23 [16] @OMe(OBn) 24 [16]
A "0H
HO N3
297 % ee, 89 % yield, PCL, vinyl acetate no acylation with vinyl acetate and several
1ipases
fj 25 1171
/
26 1181
OH AcO
95 % ee, 51 % yield, PCL, vinyl acetate 299 % ee, 38 %yield, PPL
94 % ee, 75 % yield, PCL, vinyl acetate
"?:I,
AcO
.::0 27 [18]
AcO
2 % ee, 60 %yield, MSL, vinyl acetate 299 % ee, 94 % yield, PPL
299 % ee, 87 % yield, DCL, vinyl acetate
/-OH
AcO
\
29 [18] 30 1191
HO O
' AC
86 % ee, 75 % yield, PPL, vinyl acetate 97 % ee, 68 % yield, CCL
18 % ee, 57 %yield, PPL, vinyl acetate
31 [20]
o\;b
OAc
295 % ee, 90 % yield, PCL, isopropenyl acetate 100 % ee, 80 % yield, PCL, vinyl acetate
Gc;c;;
a
Fe 33 [21] 34 [22]
HO
100 % ee, 80 % yield, CVL, vinyl acetate 298 % ee, 46 % yield, PCL, isopropenyl acetate
482
b
Table 11.1-18. (cont.).
+o 35 [22] 36 [22]
HO AcO
84 % ee, 92 %yield, PCL, vinyl acetate 17 % ee, -, PCL, isopropenyl acetate
38 [24]
%OH
HO "*'
299 % ee, 81 % yield, PFL, vinyl acetate 299 % ee, 87 % yield, PCL, vinyl acetate
C0,Et OAc
299 % ee, 96 % yield, PSL, vinyl acetate 95 % ee, 98 % yield, CCL, vinyl acetate
41 [27]
98 % ee, 70 % yield, CRL, 299 % ee, 99 %yield, PSL, vinyl

vinyl acetate acetate
91 % ee, 64 %yield, PCL,
vinyl acetate
43 [29]
AcO bOH 44 [30]
>98 % ee, 90 % yield, PCL, R = Cbz: >98 % ee, 91 % yield,

isopropenyl acetate PCL, isopropenyl acetate
R = Boc: >98 % ee, 92 % yield,
PCL, isopropenyl acetate
Table 11.1-18. (cant.).
I
483
45 [31] 46 [32]
U O H
>99 % ee, 88 % yield, PPL, 298 % ee, 89 %yield, PCL, vinyl

ethyl acetate acetate
95 % ee, 93 %yield, MML, vinyl
acetate
70 % ee, 89 % yield, CAL-B, vinyl
acetate
RO OR
R
TBDMS >99 % ee, 58 %yield, PCL, vinyl acetate 47 [33]
>99 % ee, 65 % yield, PSL, vinyl acetate
TIPS 299 % ee, 77 %yield, PSL, vinyl acetate 48 [33]
CH2Ph 299 % ee, 81 %yield, PSL, vinyl acetate 49 [33]
50 [33] 51 [33]
HO
92 % ee, 92 %yield, PSL, 91 % ee, 82 %yield, PSL, vinyl

AcO Ro$2:
R = 4-MeOCbH4CHz
52 [33] HOO
+
Bn
Ac
>99 % ee, 65 % yield, PPL,

53 [34]
94 % ee, 76 %yield, PSL, vinyl acetate

vinyl acetate
a O OAc
H
54 [35] aoAc OH
88 % ee, 73 %yield, PSL, n = 2: >98 % ee, 94 %yield, 55 [35]

vinyl acetate MML, vinyl acetate
n = 3 : 80 % ee, 85 %yield, 56 [35]
PSL, vinyl acetate
Table 11.1-18. (cont.).
OR
1
R = Boc: >98 % ee, 74 % 57 [36] R = TBDMS: >98 % ee, 70 %yield, 59 [37]

yield, PFL, vinyl acetate CAL-B, isopropenyl acetate
R = Cbz: >98 % ee, 78 % 58 [36] R = MOM: >95 % ee, 68 %yield, GO [37]
yield, PFL, vinyl acetate CAL-B, isopropenyl acetate
C0,Me
Cbz
AcO'"' 0. I
R
"OH
R = H: 95 % ee, 80 %yield, 61 [38] R = Me: >99 % ee, 42 %yield, PSL, 63 [39]

CAL-B,vinyl acetate vinyl acetate
R = OMOM: 96 % ee, 83 % 62 [38] R = Me: 93 % ee, 50 %yield, PSL,
yield, CAL-B, vinyl acetate vinyl acetate 64 [39]
R = Et: >99 % ee, 53 %yield, PSL,
C0,Me vinyl acetate
66 [40]
HO 'I.' "'OAc 65 [39] &F
>99 % ee, 97 %yield, PPL, >95 % ee, 89 %yield, PSL, vinyl
68 [41]
HO"'
w
>99 % ee, 88 % yield, LIP, >99 % ee, 82 % yield, LIP, vinyl
R
Bn 93 % ee, 93 % yield, LIP, vinyl acetate 69 [42]
4-MeOCsH4CHz 84 % ee, 85 % yield, LIP, vinyl acetate 70 [42]
2-NaphthylCHz 93 % ee, 93 %yield, LIP, vinyl acetate 71 [42]
I I, I Hydrolysis and Formation of CarboxylidAcid Esters
Table 11.1-18. (cont.).
72 [43]
92 % ee, 47 % yield, PPL,

vinyl acetate
>98% ee, 75 %yield, PCL
90 % ee, 73 %yield, PSL
1 U.Ader, D. Breitgoff, P.Klein, K. E. Laumen,
~~
23 M. Tanaka, M. Yoshioka, K. Sakai, Tetrahedron:

M. P. Schneider, Tetrahedron Lett. 1989,30,1793. Asymmetry1993,4,981.
2 H. Hemmerle, H.-J. Gais, Tetrahedron Lett. 1987, 24 S. Takano, M. Moriya, Y. Higashi, K. Ogasawara,
28,3471. 1.Chem. Soc., Chem. Commun. 1993,177.
3 H. Hemmerle, Ph. D. 7'hesis,Universitat Freiburg 25 N. Toyooka, A. Nishino, T. Momose, Tetrahedron
1990. Lett. 1993,34,4539.
4 M. Ihara, M. Suzuki, K. Fukumoto, C. Kabuto, 26 M. Sato, H. Ohuchi, Y. Abe, C. Kaneko,
1.Am. Chem. SOC.1990,112,1164. Tetrahedron: Asymmetry1992,3,313.
5 M.Murata, S. Ikoma, K. Achiwa, Chem. Pharm. 27 M. Sato, T.Hirokawa, H. Hattori, A. Toyota,
Bull. 1990,38,2329. C. Kaneko, Tetrahedron: Asymmetry1994,5,975.
6 C. Andreu, J. A. Marco, G . Asensio, /. Chem. Soc., 28 K. Toyama, S. Iguchi, T. Oishi, M. Hirama, Synlett
Perkin Trans. 1 1990,3209. 1995,1243.
7 S.-H. Hsu, S.4. Wu, Y.-F. Wang, C.-H. Wong, 29 C. R. Johnson, L. S . Harikrishnan, A. Golebiowski,
Tetrahedron Lett. 1990,31,6403. Tetrahedron Lett. 1994,35,7735.
8 K. A. Babiak, J. S . Ng, J. H. Dygos, C. L. Weyker, 30 C. R. Johnson, S . J. Bis,/. Org. Chem. 1995,60,
Y.-F. Wang, C.-H. Wong,]. 0%.Chem. 1990,55, 615.
3377. 31 B. Danieli, G.Lesma, M. Mauro, G. Palmisano,
9 F. Theil, S . Ballschuh, H. Schick, M. Haupt, D. Passarella,]. Org. Chem. 1995.60,2506.
B. Hafner, S. Schwarz, Synthesis1988,540. 32 A. Patti, C.Sanfilippo, M. Piatelli, G. Nicolosi,
10 G. Jommi, F. Orsini, M. Sisti, L. Verotta, Gazz. /. Org. Chem. 1996,61,6458.
Chim. Ifal. 1988,118,863. 33 T. Oishi, M. Maruyama, M. Shoji, K. Maeda,
11 a) F. Theil, H. Schick, M. A. Lapitskaya, K. K. N. Kumahara, S . Tanaka, M. Harima, Tetrahedron
Pivnitsky, LiebigsAnn. Chem. 1991,195;b) F. 1999,557471.
Theil, H. Schick, D. Weichert, K. Tannenberger, 34 a) G. Guanti, R. Riva, Tetrahedron: Asymmetry
G. Klappach,]. Prakt. Chem. 1991,333,497. 1995,G,2921;G. Guanti, R. Riva, Tetrahedron:
12 H. Pottie, J . Van der Eycken, M. Vandewalle, Asymmetry2001,12,605.
H. Roper, Tetrahedron: Asymmetry1991,2, 329. 35 G. Nicolosi, A. Patti, M. Piatelli, C. Sanfilippo,
13 K. Naemura, A. Furutani,]. Chem. SOC.,Perkin Tetrahedron: Asymmetry1995,6,519.
Trans. 11991,2891. 36 B. Danieli, G. Lesma, D. Passarella, A. Silvani,
14 C. R. Johnson, A. Golebiowski,T. K. McGill, /. Org. Chem. 1998,63,3492.
D. H. Steensma, Tetrahedron Lett. 1991,32,2597. 37 R. ChCnevert, D. Goupil, Y. S . Rose, E. Bedard,
15 C. R. Johnson, A. Golebiowski, D. H. Steensma, Tetrahedron: Asymmetry1998,9,4285.
1. Am. Chem. SOC.1992,114,9414. 38 R. ChCnevert, G. M. Ziarini, M. P. Morin,
16 C.Hoenke, P. Kliiwer, U. Hugger, R. Krieger, M. Dasser, Tetrahedron: Asymmetry1999,10, 3117.
H. Prinzbach, Tetrahedron Lett. 1993,34,4761. 39 Y. Zhao, Y.Wu, P.De Clerq, M. Vandewalle,
17 K. J. Hams, Q.-M. Gu, Y.-E. Shih, G. Girdaukas, P. Maillos, 1.C.Pascal, Tetrahedron: Asymmetry
C. J. Sih, Tetrahedron Lett. 1991,32,3941. 2000,11,3887.
18 F. Theil, H. Schi&, G. Winter, G. Re&, 40 M. Ranchoux, J.-M. Brunel, G. Iacazio, G. Buono,
Tetrahedron 1991,47,7569. Tetrahedron: Asymmetry1998,9,581.
19 M. Mekrami, S. Sicsic, Tetrahedron: Asymmetry 41 H. Konno, K. Ogasawara, Synthesis1999,1135.
1992,3,431. 42 T.Taniguchi, K. Ogasawara, Tetrahedron Lett. 1999,
20 C. R. Johnson, P. A. PI&,J. P. Adams, /. Chem. 40,4383.
Soc., Chem. Commun.1991,1006. 43 C.Cinquin, 1. Schaper, G. Mandville, R. Bloch,
21 G. Nicolosi, R. Monone, A. Patti, M. Piatelli, Synlett, 1995,339.
Tetrahedron: Asymmetry1992,3,753.
22 S.I. Bis, T. Whitaker, C. R. Johnson, Tetrahedron:
Asymmetry1993,4,875.
486
pounds differ but not to a large extent (Tables 11.1-11and 11.1-18).In many cases the
enantioselectivity of acylation is higher than that of the hydrolysis. Acylation of the
three-, four- and five-membereddimethanol derivatives proceeds uniformly with the
same enantiotopic group recognition to the monoacetates 1-8 with good to high
enantioselectivity and yield. Acylation of the cyclohexanoid dimethanol system is
erratic, giving 10 with low enantioselectivityand low yield. The cyclohexenoid system
11 however is obtained with the same lipase with good enantioselectivity.Acylation
of cyclopentanoid dimethanol derivativeswith a functional group in %positionby pig
pancreas lipase has been intensively investigated (4-8).The enantioselectivitycan be
influenced (5 and 6) by the choice of the appropriate protecting group. The
heterocyclic dimethanol monoacetate 9, which is a derivative of the parent com-
pound meso-butane tetrol, is obtained with high enantioselectivity by Pseudomonas
Jluorescens lipase instead of pig pancreas lipase. Acylation of meso-exo-oxa-norbor-
nane dimethanol with pig pancreas lipase and with Candida cylindracea lipase
provides access to both enantiomeric monoacetates 14 and 15.A further example of
the attainment of both enantiomers by changing the lipase is provided by the
acylation of 1,2-bis(hydroxymethyl)ferrocene with vinyl acetate catalyzed either by
Pseudomonas cepacia lipase which gives the (S)-enantiomer 32 or by Chromobacte-
rium viscosurn lipase which gives the (R)-enantiomer 33. Acylation catalyzed by
lipases is, as in the case of the hydrolysis of the corresponding acetates, not restricted
to substrates containing primary hydroxyl groups, as demonstrated by the successful
synthesis of the monoacetates 17-19, 22-29, 31,34-36, 38,39, 41-44, 46,63-65.
These examples give a good illustration of the scope of lipases as catalysts.
Comparison of the bicyclo[3.l.0]cyclohexane derivatives 28 and 29 shows that
changing the configuration of the cyclopropane ring is accompanied by a switch of
enantiotopos-selectivity under identical reaction conditions.
Lipases are the hydrolases of choice for the kinetic enantiomer separation of
racemic primary, secondary and tertiary alcohols through acylation. Acylation of the
racemic alcohols is complementary to the hydrolysis or alcoholysis of the corre-
sponding esters.
Monoacetates of Table 11.1-18 which can be obtained with other hydrolases as
such or of opposite configuration are contained in Tables 11.1-3, 11.1-7, 11.1-9 and
11.1-11.
A large number of enantiomerically pure primary alcohols carrying additional
nitrogen, oxygen and sulfur functionalities can be prepared by lipase-catalyzed
enantiomer-differentiatingacylation with the usual acylating reagents (1-130)(Table
11.1-19).Most remarkably, a series of primary alcohols whose chiral center bears
only alkyl or alkenyl groups (23-30)has been obtained with high enantioselectivity
through Pseudomonas Jluorescens lipase-catalyzed acylation with vinyl acetate in
dichloromethane. For the attainment of chiral primary alcohols, lipase-catalyzed
acylation seems to be more efficient in terms of selectivity and yield than lipase-
catalyzed hydrolysis of the corresponding esters. A comparison ofTables 11.1-19and
11.1-14 shows that enantiomer-differentiating hydrolysis of acetates and enantio-
mer-differentiating acylation of the corresponding alcohols catalyzed by one and the
same lipase are complementary. Enantiomer-differentiatingacylation with succinic
7 1. I Hydrolysis and Formation of Carboxylid Acid Esters
Table 11.1-19. Lipase-catalyzed enantiomer-differentiating acylation of racemic acyclic primary

I
487
alcohols in organic solvents (PPL pig pancreas lipase, PFL PseudomonasPuorescens lipase,
PCL Pseudomonas cepacia lipase, CCL Candida cylindracea lipase, M M L Mucor miehei lipase,
PSL Pseudomona sp. lipase, CAL-B Candida antarctica B lipase, CAL Candida antarctica lipase, not
specified, CLL Candida lipolytica lipase, SM L Serratia marcensens lipase, HLL Humicola lanuginosa
lipase).
NHCbz NHCbz
RL O A c R&OH
R
Me 73 % ee, - la 85 % ee, -
Et 78 % ee, - 2a 83 % ee, -
n-Pr 99%ee,- 3a 99 % ee, -
n-Bu 95%ee,- 4a 95 % ee, -
all PPL, ethyl acetate
Me
-0Ac RA O H
R
R = PhS 98 % ee, - 5a 98 % ee, - 5a PI
R = PhS0298 % ee, - Ga 98 % ee, - Ga 121
all PCL,vinyl acetate 60 % conversion
40 % conversion
u Fi
R=H 26 % ee, -, PCL, vinyl 7a 299 % ee, 37 % yield 7b I31
acetate
R = OMe 81 % ee, 43 %yield, 8a 83 % ee, 44 % yield 8b [41
PCL,acetic anhydride
OH 9a [51 &OH 9b 151

-0COnPr
tributyrin
NHC0,Et
R&OH
R = Me 90 % ee, 31 %yield 1Oa 295 % ee, 30 % yield lob [6]

R = Et 295 % ee, 31 %yield lla 92 % ee, 32 % yield I l b [6]
all PPL,ethyl acetate
488
Table 11.1-19. (cont.).

~~~~ ~
0
3
, r O .’,,,,, OCO(CH,),CO,H R&
fOH
R = Me 60 % ee, 40 % yield 12a 61 % ee, 40 % yield 12b [7]

R = Ph 92 % ee, 41 % yield 13a 70 % ee, 32 % yield 13b [7]
all PFL, succinic
anhydride
..**-OCO(CH,),CO,H
n’
, I 14a [7] 14b [7]
npr”yo npr/Nyo
0 0
succinic anhydride
n 15a [8] 15b [8]

R/NKO R 4 f 0
0 0
R = t-Bu, i-Pr 295 % ee, 42-45 % yield
295 % ee, 40-43 %yield, PFL
acetic, propionic or butyric
anhydride
(\/OAc
R
‘0 eoH
R
R = CIO& PPL 1Ga 295 % ee, 31 % yield 161, [9, 101

R = (CH&CHMez ethyl acetate 17a 295 % ee, 36 % yield 17b (9, 101
R
Lon,
0
RE O H
R = PhCH2 298 % ee, 32 % yield 18a 298 % ee, 34 % yield 18b [lla]
R = C9H19 96 % ee,38 % yield 19a 96 % ee, 36 % yield 19b [lla]
R = Vinyl- 99 % ee, 38 % yield 2Oa 98 % ee, 38 % yield 20b (llb]
(CH2)3 all PFL, vinyl acetate 60 % conversion
40 % conversion
I
489
Meo
Table 11.1-19. kont.).
M e o ~ o A c
OH OH
94 % ee, 23 %yield, PSL 21a [12] 97 % ee, 27 %yield 21b [12]

isopropenyl acetate
NHC0,Et
22a [ 131 22b 1131
XZ:OCH,NHCOPh A O H
93 % ee, -, MML 63 % ee, -,
Ph
2
R' R2
n-Pr Me 98 % ee, 17 % yield all PCL 23 [14]
n-Bu Me 99 % ee, 22 % yield vinyl acetate. 24 [14]
n-Bu Et 97 % ee, 23 % yield The 25 [14]
n-Hex Me 96 % ee, 20 % yield corresponding 26 [14]
(CHs)2CHCH2 Me 98 % ee, 26 % yield acetates were of 27 [14]
n-Oct Me 98 % ee, 26 % yield low ee. 28 [14]
CH3CH=CHCH2 Me 96 % ee, 33 % yield 29 [14]
Ally1 Me 97 % ee, 25 % yield 30 [14]
OAc
R
H
.O\/\/OAc
79 % ee, 46 % yield 31a

boLoAc
85 % ee, 48 % yield 31b [15]
2-Me 80 % ee, 48 % yleld 32a 93 % ee, 45 % yield 32b [16]
3-OMe 95 % ee, 47 % yield 33a 91 % ee, 49 % yield 33b [15]
4-OMe 94 % ee, 52 %yield 34a 96 % ee, 48 % yield 34b [15]
4-C1 92 % ee, 49 % yield 35a 94 % ee, 48 % yield 35b [15]
4-t-Bu 93 % ee, 50 % yield 36a 99 % ee, 50 % yield 36b [15]
all PCL, vinyl acetate
490
I J J Hydrolysis and Formation ofC-0 Bonds
Table 11.1-19. (cont.).
B 9
R
H 82 % ee, 48 % yield 37a 297 % ee, 39 % yield 37b [17]
Me 90 % ee, 46 % yield 38a 90 % ee, 48 % yield 38b [17]
i-Pr 84 % ee, 47 % yield 39a 294 % ee, 38 % yield 39b [17]
F 79 % ee, 40 % yield 40a 90 % ee, 33 % yield 40b [17]
OMe 90 % ee, 48 % yield 41a 298 % ee, 42 % yield 41b [17]
04
all PSL, vinyl acetate
. .
A,..' 42a [18] 421, [ 181
X O A c
HO
86 % ee, 52 %yield, PCL vinyl acetate 99 % ee, 44 % yield
OAc
R A O A c R Z O H
R
i-Pr >98 % ee, 37 % yield 43a 57 % ee, 53 %yield 43b [19]
t-Bu >98 % ee, 39 % yield 44a 81 % ee, 52 %yield 44b [19]
Ph >98 % ee, 34 % yield 45a 67 % ee, 57 % yield 45b [I91
all PFL, vinyl acetate
OAc
&OAc 4Ga [20] PhL O A c 46b [20]
Ph
96 % ee, 48% yield, PCL, 87 ee, 52% yield
vinyl acetate
47a [21] 47b [21]
F
R = 4-OMeC6H4CH2
>99 % ee, 33 % yield, lipase OF
(Meito Sangyo), vinyl acetate
Table 11.1-19. (cont.).
I
11.1 Hydrolysis and Formation ofCarboxylid Acid Esten 491
OAc ,OH
48b [22]
OH
97 % ee, 20 % yield, PCL 27 % ee, 76 %yield

vinyl acetate

vinyl acetate
93 % ee, 35 %yield, PSL >99 % ee, 41 %yield
vinyl acetate
OH
50a[24] " 0 2 Sob [24]
"OTBDMS f OTBDMS
90 % ee, 51 %yield, CAL-B, -, 49 % yield

isopropenyl acetate
PhE 1 0 A c
R
R = CH2Ph: >97 % ee, -, PSL 51a >97 % ee, - 51b 1251
vinyl acetate, 50 % conversion
R = 4-MeCsH4CHz:94 % ee, -, 52a 90 % ee, - 52b [25b]
PSL, vinyl acetate, 49 % conversion
R = 4-MeC6H4CHz: 73 % ee, -, 53a >99 % ee, - 53b [25b]
PSL, vinyl acetate, 58 % conversion
R = CH2I: 94 % ee, -, PSL 54a 70 % ee, - 54b [26]
55a [26] +OH 55b [26]

Ph Ph
94 % ee, -, PSL, vinyl acetate >97 % ee, -
51 % conversion
492
Table 11.1-19. (cont.).
'it
R2
R' = C1, R2= Ph: 95 % ee, -, PPL, 56a 67 % ee, - 56b (261
R' = I, R2= Ph: 96 % ee, -, PPL, 57a >97 % ee, - 57b [26]
R' = I, R2= CH2-CH2Ph 55 % ee, -, 58a >97 % ee, - 58b [2G]
PPL, vinyl acetate, 64 % conversion
R' = I, R2= %Me3:94 % ee, -, PPL, 59a >97 % ee, - 59b [26]
R' = I, R2= n-Bu: 89 % ee, -, PPL, GOa 88 % ee, - Gob [26]
R' = I, R2= n-Hex: 51 % ee, -, PPL, Gla 93 % ee, - Glb [26]
R' = I, R2= t-Bu: >97 % ee, -, PPL, 62a 94 % ee, - 621, [26]
R = 4-MePh: 72 % ee, 55 %yield, 63a >99 % ee, 42 % yield 631, [27]

CRL, vinyl acetate
R = Me: 62 % ee, -, PSL, 64a 96%ee,- 64b [28]
OAc OH
O+i
OR OR
R = Me: 90 % ee, 36 %yield, G5a 69 % ee, 54 %yield 65b [29]
PCL, vinyl acetate
R = Me: 90 % ee, 38 % yield, 96 % ee, 45 % yield
PSL, vinyl acetate 66a 661, [29]
R = MOM: 94 % ee, 40 % yield, 75 % ee, 51 %yield
PCL, vinyl acetate
R = MOM: 97 % ee, 46 %yield, 99 % ee, 48 % yield
PSL, vinyl acetate
Table 11.1-19. (cont.).
I
493
...\'\ OAc
\
OAc
X = C1>95 % ee. 43 % yield, 67a >95 % ee, 37 % yield 6% (301
PCL, vinyl acetate
X = F: >95 % ee, 44% yield, 68a 295 % ee, 33 % yield 68b [30]
PCL, vinyl acetate
X = H: >95 % ee, 39% yield, 69a >95 % ee, 33% yield 69b [30]
PCL, vinyl acetate
70a 1301 70b [30]
"-OAc
-, 50% yield, PCL, vinyl acetate >95 % ee, 43% yield
0"" " N HBOC

71a [31]
NHBoc
925 % ee, 38% yield
71b [31]
91 % ee, 45% yield, PCL,

vinyl butyrate
72a [32] 72b [32]

( T O"OAc
A c
90 % ee, 51 %yield, PCL, vinyl acetate >99 % ee, 48 % yield
R
k R2
OKNdoAc
0
R' R2
Ph H 73 % ee, 47 % yield, 73a 78 % ee, 42 % yield 73b [33]
PCL,
vinyl propionate
H Ph 92 % ee, 43 %yield, 74a 81 % ee, 48 % yield 7413 [33]
PCL,
vinyl propionate
H Ph 99 % ee, 44 % yield, 75a 74 % ee, 51 % yield 75b [33]
PCL,
vinyl propionate
H CHzPh 71 % ee, 52 %yield, 76a 87 % ee, 43 %yield 76b [33]
PCL,
vinyl propionate
1 1 Hydrolysis and Formation o f C - 0 Bonds
494 I Table 11.1-19. (cont.).
IFoAc
vinyl acetate
89 % ee, 40 % yield
89 % ee, 31 %yield, PSL, 91 % ee, 40 % yield

vinyl acetate
0
78a [35] 78b [35]
mlOAc C O H
99 % ee, 40 % yield, PCL, 97 % ee, 36 % yield
vinyl butyrate
COAc
A3(4): 99 % ee, 34 % yield, 79a 94 % ee, 32 % yield 79b [35]
PCL,vinyl butyrate
A4(5):99 % ee, 34 %yield, 80a 97 % ee, 25 % yield 80b [35]
PCL,vinyl butyrate
96 % ee, 47 % yield, PCL, 81a [36] -, 42 %yield 81b [36]

succinic anhydride
51 % ee, 61 %yield, PSL, >99 % ee, 36 % yield

vinyl acetate
Table 11.1-19. (cont.).

I
495
R I I
Me 92 % ee, -, CRL, vinyl 83a -, - 83b [38]
acetate, 59 % conversion 84b [38]
CH2Ph 96 % ee, -, CRL, vinyl 84a -. -
50 % conversion
>97 % ee, 52 % yield, 85a -, 22 %yield 85b [39]

CLL, Ac2O
88 % ee, 49 % yield, 8Ga -, 25 %yield 8Gb [39]
CLL, Ac~O
>95 % ee, 46 %yield, olipase 87a -, 34 % yield 8% [39]
4SD (Amano),AczO
73 % ee, 50 % yield, 88a -, 38% yield 88b [39]
CLL, AczO
LOAC
75 % ee, 55 %yield, PFL, vinyl 89a 96 % ee, 42 % yield 89b [40]
acetate
98 % ee, -, PFL, vinyl acetate, 89a >99 % ee, -, 57 % 89b [41]
39 % conversion conversion
R A O A c
R:
a b C d
R = a: 97 % ee, -, PCL, vinyl 90a _- 90b [42]

acetate, 39 % conversion
R = b: 95 % ee, -, PCL, vinyl 91a -_ 91b [42]
R = c: 97 % ee, -, PCL, vinyl 92a __ 92b [42]
R = d 94 % ee, -, PCL, vinyl 93a _ - 93b (421
496 I Table 11.1-19. (cont.).
Boc Boc
I I
W O A C
-, -, PCL, vinyl acetate 58 % conversion 96 % ee, -
O
, Ac OH OH
YBn OAc
88 % ee, 50 % yield, PCL,

95a
d - B n
\
OAc
78 % ee, 21 %yield
95b &N--Bn
\
OH
>99 % ee, 29 % yield
95c
[&a]
vinyl acetate
80 % ee, 21 %yield, PPL, 83 % ee, 26 %yield, >99 % ee, 53 %yield [&b, c]
vinyl acetate for ent95b
91 % ee, 18 %yield, PCL, 24 % ee, 64 % yield

vinyl butyrate
R OAc
X R
0 Ph >99 % ee, -, CAL, vinyl 97a 46 % ee, - 9% [46]
0 4-Br-Ph 98 % ee, -, CAL, vinyl 98a 59 % ee, - 981,1461
0 Me 91 % ee, -, PPL, vinyl 99a 38 % ee, - 99b [46]
NMe Ph 82 % ee, -, CAL, vinyl lOOa >98 % ee, - 1OOb [4G]
N
lOla [47] +
O
,.,H 101b [47]
Ph&OAc Ph
vinyl acetate, -50 "C
"US 0
',-OAc
'..
102a[48]
0
OH
102b [48]
62 % ee, 55 %yield, PFL, 94 % ee, 40 % yield

vinyl acetate
7 7.7 Hydrolysis and Formation of Carboylid Acid Esters I
497
Table 11.1-19. (cont.).
R
Me 73 % ee, -, CAL-B, vinyl 103a 92 % ee, - 103b [49]
CHzPh 81 % ee, -, CAL-B, vinyl 104a 99 % ee, - 104b [49]
Me
PhA O A c 105a [SO] Me<OH 105b [SO]
Ph
95 % ee, 26 %yield, PCL, 70 % ee, 31 %yield
vinyl acetate
1OGa [511 Ho-)\,, 10Gb 1511

HZ!3C,, COzMe
83 % ee, 25 %yield, PFL, 54 % ee, 52 %
vinyl acetate
OAc
R O A O A c R O L O A c
R
C16H33 80 % ee, -, PSL, vinyl acetate, 107a 94 % ee, - 10% [52]
55 % conversion, 4 "C
C18H37 30 % ee, -, PSL, vinyl acetate, 108a 89 % ee, - 108b 1521
(9Z)-C&35 24 % ee, -, PSL, vinyl acetate, 109a 93 % ee, - 109b [52]
OH
A c O & ~ ~ ~ 11Oa [531 A C O + ~ ~ ~ 1lOb [53]
N3 N3
91 % ee, -, PCL, vinyl acetate, >99 % ee, -
50 % conversion
111b [54]
89 % ee, 39 % yield, PFL, 80 % ee, 44 % yield

vinyl acetate
86% ee, 44 % yield, PCL, 92 % ee, 42 % yield
vinyl acetate
Table 11.1-19. (cont.).
-OH a O H
112 1551 &OH ent-112 [55]
\ \
acylated enantiomers acylated enantiomers

not shown not shown
>99 % ee, 29 %yield, SML, >99 % ee, 45 % yield, HLL,
vinyl hexanoate vinyl hexanoate
(CH,),-OAc (CH,),-OAc
-h
P ’ / -h
P I Ph*
HO-,(H,C) HO-,(H,C) AcO-,(H,C)

absolute configuration absolute configuration absolute configuration
unknown unknown unknown
n = 2: 113a 113b 113c [SG]
90 % ee, 41 % yield, 35 % ee, 27 %yield 99 % ee, 32 % yield
CAL-B, vinyl acetate
n = 3: 114a 114b 114c (561
>99 % ee, 9 % yield, >98 % ee, 18 % yield 30 % ee, 73 % yield
PSL, vinyl acetate
I--\
HOH,C CH,OH
(P)-115a [57] (M)-115b (571 (M)-115~[57]
98 % ee, 45 % yield, 80 % ee, 38 % yield 95 % ee, 13 % yield
PCL, vinyl acetate
(M)-115a [57]
92 % ee, 44 % yield,

I
499
~ C H , O A C
&
R
Me 81 % ee, -, MML, vinyl llGa 81 % ee, - l l 6 b [58]
84 % ee, 47, CAL-B, vinyl 83 % ee, 47 % yield
Ph 88 % ee, -, CAL-B, vinyl 117a 72 % ee, - 11% [58b]
propionate, 45 %
conversion
t-Bu 90 % ee, -, MML, vinyl 118a 48 % ee, - 118b [58b]
119b (591
119a[591 Fe CH,OH
& & '
89 % ee, -, CAL-B, vinyl 96 % ee, -
92 % ee, 49 % yield, 90 % ee, 51 %yield

PCL, vinyl acetate
121a [61] 121b [61] 121c [GI]

>97 % ee, 22 % yield, 10 % ee, 55 %yield >97 % ee, 18 % yield
PCL, vinyl acetate
122a [61] 122b [61]
*OH
(oz> OAc
>95 % ee, 42 %yield, 58 % ee, 59 % yield
PCL, vinyl acetate
500

isopropenyl acetate
The two further regioisomers
show lower selectivity.
,OH
/OAC
OAc
94 % ee, -, PCL, vinyl acetate, 56 % ee, -
38 % conversion
97 % ee, -, PFL, vinyl acetate, 60 % ee, -
38 % conversion
OH
125a[64] 125b [64]
LOA~ -0Ac
91 % ee, 44 %yield, CAL-B, 91 % ee, 44 %yield
vinyl acetate
nPrOCO
4
BocHN
89 % ee, 51 %yield, CAL-B, 99 % ee, 36 % yield
126b [65]
vinyl butyrate
A 127b [65]
127a [65]
nPrOCO 4
BOCHN'
&OH
NHBoc
95 % ee, 18 %yield, PPL, 96 % ee, 43 % yield,
vinyl butyrate. 37 % 5 3 % conversion
conversion
128a [65] 128b [65]
95 % ee, 40 %yield, PPL, 99 % ee, 36 % yield,

vinyl butyrate, 44 % 56 % conversion
conversion
7 7 . 7 Hydrolysis and Formation ofcarboxylid Acid Esters I
501
Table 11.1-19. (cont.).
92 % ee, 22 % yield, PPL, 90 % ee, 32 % yield,

vinyl butyrate, 30 % 53 % conversion
conversion

vinyl acetate
1 S. Fembndez, R. Brieva, F. Rebolledo, V. Gotor, 18 J. Weidner, F. Theil, A. Kunath, H. Schick, Liebigs
/. Chem. Soc., Perkin Trans. 1 1992, 2885. Ann. Chem. 1991,1301.
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Asymmetry1993,4,85. Tetrahedron: Asymmetry1999, 10, 1927.
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Asymmetry1993,4,773. 31 M. Peter, J. Van der Eycken, G . Bernith, F. Fiilop,
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Tetrahedron: Asymmetry1991,2, 1031. 35 J. K h a n , E. Forr6, F. Fiilop, Tetrahedron:
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Tetrahedron: Asymmetry1993,4, 845.
502
36 J. A. Hyatt, C. Skelton, Tetrahedron: Asymmetry 52 G. G. Haraldsson, P. Thordarson, A. Halldorsson,

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Tetrahedron 1994,50,10849. 56 F. Theil, H. Sonnenschein, T. Kreher, Tetrahedron:
40 F. Bracher, T. Papke, Tetrahedron: Asymmetry 1994, Asymmetry 1996,7,3365.
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42 0. Nordin, B.-V. Nguyen, C. Vorde, E. Y. Kitahara,j . Chem. Soc., Perkin Trans. 1 1998,
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43 F. Schieweck, H:J. Altenbach, Tetrahedron: Tetrahedron Lett. 1996, 37, 127; b) A. Patti,
Asymmetry 1998,9,403. D. Lambusta, M. PiatteUi, G. Nicolosi, P. McArdle,
44 a) M. P. Sibi, J. L. Lu, Tetrahedron Lett. 1994, 35, D. Cunningham, M. Welsh, Tetrahedron 1997,53,
4915; b) G . Guanti, R. Riva, Tetrahedron: 1361.
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Tetrahedron: Asymmetry 2001, 12,605. The given Tetrahedron:Asymmetry 1998,9, 3073.
absolute configurations in b and c are GO A. Patti, G. Nicolosi, Tetrahedron:Asymmetry 1999,
contradictory. 10, 2651.
45 M. Lemaire, J. Bolte, Tetrahedron: Asymmetry 1999, 61 G. Nicolosi, A. Patti, M. Piatelli, ].Org. Chem.
10,4755. 1994,59, 251.
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3423. 37, 5293.
47 T. Sakai, I. Kawabata, T. Kishimoto, T. Ema, 63 Y. Kawanarni, N. lizuna, Ke. Maekawa, Ky.
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48 M. Pallavicini, E. Valoti, L. Villa, 0. Piccolo, J. Org. 2001,57, 349.
Chem. 1994,59,1751. 64 1. Izquierdo, M. T. Plaza, M. Rodriguz, J. A.
49 H.-J. Gais, I. von der Weiden, Tetrahedron: Tarnayo, A. Martos, Tetrahedron: Asymmetry 2001,
Asymmetry 1996,7,1253. 12, 293.
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Asymmetry 1997,8,399. Tetrahedron:Asymmetry 2001, 12,625.
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Chem. 1997,62,3496. 2001, 12, 643.
acid anhydride (12-14) instead of the usually employed vinyl acetate may facilitate
the separation of the remaining substrate and the ester formed because of the
carboxyl group in the latter.
Mixed primary secondary diols are very often separated into their enantiomers in a
sequential two-step acylation wherein the first step - acylation of the primary
hydroxyl group - shows high regio- but very poor enantioselectivity. The useful
enantiomer-differentiating step is realized in the second step by acylation of the
already monoacylated diol (31-36, 46, 72, 107-110, 120,125).On the other hand, as
expected, mixed primary tertiary diols are not acylated at the tertiary hydroxyl group
(47, 48, 51-54, 5 6 6 2 , 106).
Regarding the structural diversity of the primary alcohols, which have been
successfully resolved, there seems to be almost no limitation, as demonstrated by the
axial-chiral diols 112-114, the ferrocene alcohols 116122 and the chiral chromium
carbonyl complex 123, and most remarkably the helicenediolll5 (Table 11.1-19).
11.1 Hydrolysis and Formation ofCar6oxylid Acid Esters
Table 11.120. Lipase-catalyzed enantiomer-differentiating acylation o f racemic acyclic secondary

I 503
alcohols in organic solvents (PSL Pseudomonas sp. lipase, PPL pig pancreas lipase, PFL
PseudomonasPuorescens lipase, PCL Pseudomonas cepacia lipase, CCL Candida cylindracea lipase,
CAL-B Candida antarctica B lipase, CAL-A Candida antarctica A lipase, LIP Pseudomonas sp.
lipase-Toyobo, BSL Burkholderia sp. lipase, GLL goat liver lipase, RML Rhizomucor sp. lipase, CRL
Candida rugosa lipase).
OAc 1 [ l , 21
R*CN
R ee (“A) Yield (“A)
89 80
96 84
84 83
91 64
91 81
85 88
70 70
Amberlite IRA-904, aldehyde, acetone cyanohydrin, all PSL, vinyl acetate (dynamic kinetic
resolution)
504
4 2
Table 11.120. (cont.).
2a [31 2b [31
EtCO;""' Me
Me
295% ee, -, PPL, EtCOzMe 35 % ee, -
3a [31 3b [31
OCOEt OH
298% ee, -, PPL, EtCOzMe 18% ee, -
OCOR2
R ' ~ M ~ R'Me
'
R' R2
Et n-Pr 93% ee, 38% yield, CCL, 4a 89 % ee, 35 % yield 4b [4, 51
tributyrin
95 % ee, 35 % yield, PPL, 5a 90 % ee, 30 %yield 5b [6]
C13CCHzOCOnPr
92 % ee, 41 % yield, CCL, 5a 95 % ee, 38 %yield 5b [7]
tributyrin
299 % ee, 44 % yield, PPL, Ga 95 % ee, 44 % yield Gb [6]
C13CCHzOCOPr
98 % ee, 42 %yield, PPL, 7a 299 % ee, 43 %yield 7b [6]
Cl3CCHZ0COPr
-, -, PPL, vinyl acetate 8a 298%ee,- 81, [71
YCH* Me
80 % ee, 38 % yield, PPL, 9a 297 % ee, 43 %yield 9b [8]

YCH* C11H23 F~CCHZOCOCIIHZ~
n-PR 87 % ee, 31 %yield, PPL, 10a 92 % ee, 26 %yield l o b [6]

C13CCHzOCOnPr
Me
Ph Me 299 % ee, 45 %yield, PSL, lla 93 % ee, 41 % yield l l b [9]

vinyl acetate
Ph Me 295 % ee, 39 %yield, PCL, lla 295 % ee, 43 %yield 11b [lo]
acetic anhydride
PhCHz Me 299 % ee, 30 % yield, PSL, 12a 66 % ee, 43 %yield 12b [9]
vinyl acetate
PhCH2 Et 295 % ee, 39 % yield, PCL, 13a 92 % ee, 43 % yield 13b [lo]
propionic anhydride
1-Naphthyl n-Pr 95 % ee, 47 %yield, 14a 295 % ee, 46 %yield 14b [ll]
PPL, C13CCHZOCOnPr
2-Naphthyl Me 299 % ee, 41 %yield, PSL, 15a 95 % ee, 48 %yield 15b [9]
vinyl acetate
I 1.1 Hydrolysis and Formation of Carboxylid Acid Esten
Table 11.1-20. (cont.).

I
505
OAc
1Ga [9, 101 1Gb [9, 101
&Me
Ph Ph
acetic anhydride
R' RZ
n-BuO Me 295 % ee, 41 % yield 17 [12]
n-BuO Pr 295 % ee, 23 %yield 18 [12]
n-Bu Me 295 % ee, 23 %yield 19 [12]
PhCH2CH2 Me 295 % ee, 41 %yield all PCL, 20 [12]
vinyl acetate
NTBr
B;
21 [13]
97 % ee, -, PSL, C F ~ C H ~ O C O C I I H Z ~
R 1 Me
R
n-C6Hls 297 % ee, - 22a 298%ee,- 22b [14]
Ph 97 % ee, - 23a 98%ee,- 23b [14]
c-C6HI1 95 % ee, - 24a 298%ee,- 24b [14]
all CAL-B, C7H1sCOSEt
OH
R LC02Me R-C02Me
R
Me 91 % ee, 39 %yield 25a 295 % ee, 38 % yield 251, [15]
Et 295 % ee, 45 % yield 26a 295 % ee, 44 % yield 26b [15]
n-Pr 74 % ee, 54 % yield 27a 295 % ee, 42 % yield 27b [15]
all PSL, isopropenyl acetate
OAc
R -CO,Me RLCO,Me
R
i-Pr 19 % ee, 43 % yield 28a 295 % ee, 38 % yield 28b [16]
Me2ThexSi- 295 % ee, 35 %yield 29a 72 % ee, 57 % yield 29b [16]
O(CH2)z all PSL, isopropenyl acetate
7 J Hydrolysis and Formation ofC-0 Bonds
506
OCOCI, OH
PhO-NHPh 30a [17] 301, [17]
PhO NHPh
68 % ee, 26 % yield, PPL, 96 % ee, 28 % yield
(cc13c0)20
MeR
-
OAc OH
R
SiMeJ 80 % ee, 34 % yield 31a 295 % ee, 38 % yield 31b [18]
SiMeztBu 295 % ee, 25 % yield 3h 295 % ee, 44 % yield 32b [18]
SiMezPh 295 % ee, 30 % yield 33a 295 % ee, 42 % yield 33b [18]
all PSL,vinyl acetate
34a [19] 34b [19]

OAc
O A C I
vinyl acetate or acetic
anhydride
OAc OH
35a [20] ACO,H 35b [20]
C,H,~/YO,H CzzH45
77 % ee, 55 %yield, PCL, vinyl 299 % ee, 45 % yield
acetate
OCOnBu OH
R'ASn(R2), R' n S n ( R z ) ) ,
R' RZ
Me Me 97 % ee, 31 %yield 36a 71 % ee, 42 %yield 36b [21]
Et Me 99 % ee, 36 % yield 37a 56 % ee, 36 % peld 371, [21]
n-Pr Me 97 % ee, 7 % yield 38a 68 % ee, 7 % yield 38b [21]
Me Et 99 % ee, 35 % yield 39a 51 % ee, 47 %yield 39b [21]
Et Et 97 % ee, 14 %yield 40a 57 % ee, 14 %yield 40b [21]
all PPL, n-BuCOzCH2CF3
4
OH
Ph
41a [22]
v Ph
41b [22]
295 % ee, 47 %yield, PSL, vinyl acetate 295 % ee, 32 % yield
OH
42a 1221 42b [22]
MeL p h Me-Ph
vinyl acetate
Table 11.1-20. (cont.).
1 1.1 Hydrolysis and Formation of Carboylid Acid Esters
I 507
Me UHi7
31 % ee, 46 %yield, PSL,
43a [22]
Me
56 % ee, 35 % yield
43b [22]
vinyl acetate
OH
44a [22] 44b [22]
Me=C8H,, Me CP,,
vinyl acetate
OH
45a [22] 45b [22]
d p h w p h
vinyl acetate
OH
& Ph
295 % ee, 49 %yield, PSL,
4Ga [22] P-h
295 % ee, 41 % yield
46b [22]
vinyl acetate
47a [22] 4% [22]

Ph

vinyl acetate
q Ph
48a [22]
\ Ph
48b [22]
-
vinyl acetate
49a [22] *. OH
' Ph
49b [22]

vinyl acetate
Me&.)
50a [22] 50b [22]
SiMe, SiMe,
295 % ee, 38 %yield, PSL, 81 % ee, 31 %yield
vinyl acetate
508
Table 11.1-20. (cont.).
OH
R ‘ q R2
R’ R2
Me Ph 295 % ee, 48 % yield 51a 295 % ee, 47 %yield 51b [22]
Me n-Bu 87 % ee, 41 % yield 52a 295 % ee, 31 %yield 52b [22]
Me n-CSH17 295 % ee, 50 %yield 53a 295 % ee, 30 %yield 53b [22]
Me SiMej 295 % ee, 45 % yield 54a 295 % ee, 27 % yield 54b [22]
Me CH20CH2- 78 % ee, 48 %yield 55a 295 % ee, 46 %yield 55b [22]
CGH40Me
Et n-Bu 82 % ee, 36 % yield 5Ga 295 % ee, 46 % yield 5Gb 1221
C(Me)=CH2 C(Me)=CH2 295 % ee, 47 %yield 57a 295 % ee, 36 % yield 5% [22]
OH
R
CHZPh 77 % ee, 52 % yield 58a 95 % ee, 31 % yield 58b [22]
n-CsHlt -, 49 %yield 59a 23 % ee, 24 % yield 59b [22]
R’
R’
PR2 R2
Me SiMe3 295 % ee, 20 % yield GOa 295 % ee, 26 % yield Gob (221
Et Ph 50 % ee, 63 % yield Gla 295 % ee, 33 %yield 61b [22]
Me Et 295 % ee, 44 % yield 62a 54 % ee, 21 %yield G2b [22]
Et Et 38 % ee, 33 % yield G3a 61 % ee, 27 %yield G3b [22]
\dPh G4a [22]

OH
Ph
G4b [22]
72 % ee, 54 % yield, PSL, 295 % ee, 34 % yield

vinyl acetate
OH
G5b [23]
<
\OCH
,O
, Me [231 \ L O C 6 H 4 0 M e
vinyl acetate
OCOnPr OH
Ph02S\j\Me GGa [24] GGb [24]
P h 0 2 S dMe
65 % ee, 20 % yield, PPL, 95 % ee, 25 % yield
CC13CH20COnPr
I
509
OAc
PhOS
, -R Ph0,S A
\ I R
R
Me 295 % ee, 47 % yield 67a 298 % ee, 46 % yield 6% [25, 261
Et 295 % ee, 49 % yield 68a 298 % ee, 49 % yield 68b 125, 261
i-Pr 295 % ee, 48 % yield 69a 94 % ee, 46 % yield 69b [25, 261
n-C6H13 295 % ee, 47 % yield 70a 298 % ee, 48 % yield 70b (25, 261
PhCHZO(CH2)Z295 % ee, 47 %yield 71a 88 % ee, 48 % yield 71b [25, 261
all PPL, vinyl acetate
0C0nC1,H3j
T s O A O R Tso L O R
R
n-Cl6H33 295 % ee, 45 % yield 72a 295 % ee, 43 % yield 72b [27]
n-CloH21 295 % ee, 43 % yield 73a 294 % ee, 42 % yield 73b [27]
n-Bu 295 % ee, 43 % yield 74a 296 % ee, 45 % yield 74b [27]
all PSL, (C1~H31C0)20
OH
I
R = H, F, C1, Br, OMe 299 % ee, -

OH
RE O C P h , &OCPh,
R
R
ClCHz 298 % ee, 43 % yield 76a 72 % ee, 54 % yield 76b [29]
Me 295 % ee, 37 % yield 77a 78 % ee, 40 % yield 77b [29]
Et 298 % ee, 43 % yield 78a 298 % ee, 48 % yield 78b [29]
n-Pr 70 % ee, 44 % yield 79a 56 % ee, 52 % yield 79b [29]
OAc
R DoAcl 80a (301
R
R = H, Me, OMe, Allyl, c-CsHI1, CHZCN,NOz, 0-Ally1
80b [30]
83-87 % ee, 36-53 % yield 86-96 % ee, 38-48 % yield

PSL, vinyl acetate
OH
81 [31]
PhACO,nBu
vinyl acetate
510
Table 11.1-20. (cont.).
OAc
82a [32] 82b [32]

QMe
6-41
295 % ee, 41-50 %yield, PSL, 295 % ee, 33-38 % yield
vinyl acetate
297 % ee, 23 % yield 83a 297 % ee, 38 %yield 83b [33]

297 % ee, 41 % yield 85a 297 % ee, 34 % yield 85b (331
92 % ee, 15 % yield 8Ga 18 % ee, 56 % yield 86b [33]
B~~NH-
OAc
i , 89a
BocNH
& 89b [34]

vinyl acetate
OAc OH
PhO&OCOR PhO&OCOR
R
Me 98 % ee, 49 % yield, PCL, 90a 93 % ee, 51 % yield 90b [35]
vinyl acetate
n-C7H15 92 % ee, 45 %yield, PCL, 91a 82 % ee, 49 % yield 91b [35]
vinyl acetate
n-ClsH31 98 % ee, 48 %yield, PCL, 92a 89 % ee, 52 % yield 92b 1351
vinyl acetate
i-Pr 96 % ee, 42 % yield, PCL, 93a 52 % ee, 58 % yield 93b [35]
vinyl acetate
Ph 95 % ee, 17 %yield, PCL, 94a 19 % ee, 78 %yield 941, [35]
vinyl acetate
7 7 . 7 Hydrolysis and Formation ofCar6oxylid Acid Esters I 511
Table 11.1-20. (cont.).
OAc
R " O A C I RnO&CI
R2 = Naphthyl R3 =
Ac
/
R4 = +(CH,),OMe R5= O N H n P r
R' >98 % ee, 45 %yield, PSL, 95a 91 % ee, 50 % yield 95b [36]
vinyl acetate
RZ 95 % ee, -, CAL-B, 96a 27 % ee, - 961, [37]
vinyl acetate
R' 95 % ee, -, CAL-B, 97a 18 % ee, - 9% (371
vinyl acetate
R4 95 % ee, -, CAL-B, 98a 20% ee, - 98b [37]
vinyl acetate
R5 95 % ee, -, CAL-B, 99a 28 % ee, - 99b [37]
vinyl acetate
cy
OAc
O&C02Me R( y 0 L C O * M e
R
4-OMe 99 ee, -, PCL, vinyl acetate, lOOa 99ee,- 1OOb (381
49 % conversion 99 ee, -
2-Ally1 99 ee, -, PCL, vinyl acetate, lOla 101b [38]
49 % conversion 49 ee, -
2,3-C&4 99 ee, -, PCL, vinyl acetate, 102a 102b [38]
33 % conversion
R
F O T s R y o T s
OAC OH
R
CH=CH2 96 % ee, -, PCL, vinyl acetate 103a 98%ee,- 103b [39]
95 % ee, 48 % yield, PCL, enf-103a 84 % ee, 49 % yield enf-103b [40]
Me vinyl acetate 104b [39]
CHzCl 93 % ee, -, PCL, vinyl acetate 104a 99%ee,- 105b [39]
Et 92 % ee, -, PCL, vinyl acetate 105a >99 % ee, - lO6b [39]
80 % ee, -, PCL, vinyl acetate lO6a 98%ee,-
T O B n m O B n
OAc OH
79 % ee.45 %yield, PCL, vinyl 107a 70 % ee, 55 % yield 10% [40]
acetate
512
I Table 11.120. (cont.).
P h W y
Ph
qR
OAc OH
R
CH20Bz 98 % ee, 43 %yield, PCL, 108a 88 % ee, 49 % yield 108b [41]
vinyl acetate
COzMe 98 % ee, 47 % yield, PCL, 109a 98 % ee, 47 %yield 10% [41]
vinyl acetate
OMe OH QMe OH
I I
Me0 Me0
.eCN
llOa [42] 1101, [42]
>99 % ee, 42 % yield, PFL, 72 % ee, 58 % yield
vinyl acetate
F FwcN F
/
>99 % ee, 45 % yield, LIP, vinyl

acetate
l l l a [43]
F F
>99 % ee, 42 % yield

111b [43]
OAc
HZN NcTr /
CI
90 % ee, 46 % yield, PCL, vinyl

l l 2 a [44]
H2N
CI
97 % ee, 44 % yield
112b [44]
acetate
OAc
113a [45] O2N)+Br 113b [45]
BnO BnO
r
vinyl acetate
OH
114a [46] 114b 1461
F
F / "'
93 % ee, -, PCL, isopropenyl 90 % ee, -
I
513
d
l
115b [47]
R l i2
R
R' = Ph, 1-naphthyl, 2-naphthyl, benzyl, n-hexyl, R2= Me; R', R2= I
50-99 % ee, 39-48 %yield, PCL.
PSL, CAL-B,diketene 95-99 % ee, 30-43 % yield
OAc
R+cI RI .&
R n
Ph 2 92 % ee, 49 % yield, PCL, llGa 99 % ee, 44 % yield llGb [48]
isopropenyl acetate
Ph 2 97 % ee, 31 % yield, CAL-B, llGa 96 % ee, 33 % yield llGb [49]
vinyl butanoate
4-t-Bu-C~H4 3 99 % ee, 47 % yield, PCL, 117a 99 % ee, 47 % yield 117b [48]
isopropenyl acetate
4-t-BU-c~H4 3 >95 % ee, -, PCL, vinyl 117a >95 % ee, - 117b [SO]
1-o-Naphthyl 1 89 % ee, 41 %yield, PCL, 118a 99 % ee, 44 % yield 118b [48]
isopropenyl acetate
Ph 3 79 % ee. -, PCL, vinyl 119a >95 % ee, - 1191, [SO]
4-F-CsH4 3 295 % ee, -, PCL, vinyl 120a >95 % ee, - 120b [SO]
4-F-CsH4 3 97 % ee, 44 % yield, PCL, 120a 85 % ee, 48 % yield 1201,[51]
vinyl acetate
R
R
OAc
A CX,
X
R 1 CX,
2-Naphthyl F 85 % ee, 37 %yield, PCL, 121a >99 % ee, 51 % yield 121b [52]
vinyl acetate
2-Naphthyl H 97 % ee, 37 %yield, PCL, 122a 99 % ee, 43 % yield 122b [S2]
vinyl acetate
1-Naphthyl H >99 % ee, 32 %yield, PCL, 123a 69 % ee, 40 % yield 123b [52]
vinyl acetate
OCOCH(CH,)=CH,
124a [53] 124b [53]
A+ Ar
85-97 % ee, -, PCL, 2,3- 87-95 % ee, -
butanedione monooxime
methacrylate, 43-47 % conversion
514 I 11 Hydrolysis and Formation of C - 0 Bonds
Table 11.1-20. (cont.).
OAc
Ph, i\
X CO,H
X
(CH2)2 84 % ee, 35 % yield, PCL, 125a >99 % ee, 45 %yield 125b [54]
(E)-CH=CH vinyl acetate
94 % ee, 34 % yield, PCL, 12Ga >99 % ee, 42 % yield 12Gb [54]
vinyl acetate
Ph 2
127a [55] Ph-
92 % ee, 48 % yield
OH
- / 1271, [55]
vinyl acetate
>99 % ee, 49 % yield, PCL, 98 % ee, 51 % yield
vinyl acetate, 1,4,8,11-
tetrathiacyclo-tetradecane
as additive
R i q R ‘
R’ :
OAc OH
R’ R2
Ph Me 95 % ee, -, PSL, isopropenyl 128a 89%ee,- l28b [56]
Me Ph 98 % ee, -, PSL, isopropenyl 129a 92%ee,- 129b [56]
OAc 0
130a [57] 130b [57]
Ar Ar
>96 % ee. 26-44 % yield, CCL, 33-70 % ee, 55-73 %yield
3
vinyl acetate
R ’ V O M e R’ OMe
OCOR‘ OH
R’ RZ
Ph Me 93 % ee, 45 %yield, PCL, 131a 95 % ee, 50 % yield 131b [58]
vinyl acetate
Ph Ph >99 % ee, 12 %yield, lipase SL 132a 61 % ee, 41 %yield 131b [58]
(Meito),vinyl benzoate
Alkyl Me 30-98 % ee, 42-74 %yield, PCL, 133a 53-99 % ee, 17-43 % 133b [SO]
vinyl acetate yield
I
515
phvoMe OAc
134a [58] Ph=OMe.
OH
>99 % ee, 46 %yield
134b [58]
vinyl acetate
2
97 % ee. 31 %yield, lipase SL 42 % ee, 46 % yield
(Meito),vinyl acetate
P h vOAc
O M e 135a [58] 135b [58]
Ph OMe
OH
vinyl acetate
$6
OAc OH
RnACO,Et RnACO,Et
/ / tBu & @/ / MeM

% 0
Me tBu OH
R' R2 R3 R4 R5
R' 97 % ee, 46 %yield, PCL (PS-C), 13Ga -, - 13Gb [GO]

R2 97 % ee, 41 %yield, CRL, 137a -,- 13% [GO]
R3 95 % ee, 45 %yield, PCL (PS-C), 138a -, - 138b 1601
R4 97 % ee, 48 % yield, CRL, 139a -,- 139b 1601
Rs 97 % ee, 40 %yield, PCL (PS-C), 140a -, - 140b [60]
141b [Gl]
>98 % ee, 42 % yield, lipase >94 % ee, 40 % yield

TL, vinyl acetate
516
Table 11.120. (cont.)
OAc
142a [62] F
F WN142b [62]
/
Nc*F
F F
98 % ee, 50 %yield, LIP, 96 % ee, 46 % yield
vinyl acetate
143a [63] 143b 163)

OH

vinyl acetate
‘GH13 a4- [63] i 144b[63]

6Ac OH
94 % ee, 42 %yield, CAL-B, 81 % ee, 51 %yield
vinyl acetate
R
Me >99 % ee, -, CAL-B, vinyl 145a 33%ee,- 14% [64]
Et 97 % ee, -, CAL-B,vinyl 146a 52%ee,- 146b [64]
n-Pr >99 % ee, -, CAL-B, vinyl 147a 33%ee.- 147b [64]
n-Bu >99 % ee, -, CAL-B, vinyl 148a 12%ee,- 148b [64]
CHzOPh >99 % ee, -, CAL-B, vinyl 149a 25%ee,- 14% [64]
n‘&C02Me
6 94 % ee, 48 % yield, PCL, 150a 66 % ee, 35 %yield 150b [65]

acetic anhydride
7 90 % ee, 38 % yield, PCL, 151a 54 % ec, 44 %yield 151b [ G S ]
acetic anhydride
7 1. I Hydrolysis and Formation ofcarboxylid Acid Esters
I
517
OCOnC,H,, OH
R l i2 R R"R2
R' R2
n-C6H13 Me 97 % ee, -, CAL-B, 152a 98 % ee, - 152b [66]
n-C7H1=,COSEt, 50 % conversion
n-CsHl;r C=CH 95 % ee, -, CAL-B, 153a 96 % ee, - 153b [66]
n-C7HIsCOSEt,50 % conversion
n-C6H1, CH=CH2 96 % ee, -, CAL-B, 154a 93 % ee, - 154b 1661
n-C7HI5COSEt,49 % conversion
~-C~HI, ET 96 % ee, -, CAL-B, 155a >99 % ee, - 155b [66]
AcO-
156a [67] 156b [67]
j--k
SiMe,
-, 39 % yield, PCL,vinyl acetate
SiMe,
295 % ee, 43 % yield
SiMe, SiMe,
157a (681 HO 8,.
157b (681
98 % ee, -, BSL, vinyl acetate, 93 % ee, -,

49 % conversion
AcoynprH o d n P r
Me Br 158a [69] Me Br
158b [G9]
94 % ee, 30 % yield, PSL, vinyl 84 % ee, 46 % yield

acetate
AcO HO, d i e
159a (691 159b [69]
Me Br Me Br
vinyl acetate
OCOnC,H,,
X d R
X R
c1 Me 97 % ee, -, CAL-B, l6Oa 71 % ee, - lGOb[70]
n-C7HISCOSEt,42 % conversion
Br Me 98 % ee, -, CAL-B, l6la 88 % ee, - 1611, (701
Br Et 96 % ee, -, CAL-B, lG2a 41 % ee, - 1621,1701
n-C7H15COSEt,30 % conversion
518
Table 11.1-20. (cont.).
OCOnC,H,,
/R Me$ p J R
Me$
R
Me 99 % ee, -, CAL-B, 163a 95%ee,- 163b [70]
n-C7Hi&OSEt
Et - 81-96 % ee, 24-49 % yield, 164a 36->98 % ee, 164b [71]
n-CI3H2, PCL, vinyl acetate 41-71 %yield
165b [72]
71 % ee, -, PSL, succinic 98 % ee, -

anhydride, 58 % conversion
yo? 0 OH
l66b[73]
>99 % ee, 45 % yield, PCL,

vinyl acetate
167a [74] 167b [74]

OH
>98 % ee, -, CAL-B, isopropenyl 94 % ee, -
foEt OAc
168a [75] 168b [75]
Ph
99 % ee, 35 % yield, BSL, 99 % ee, 36 % yield
vinyl acetate
QOEt q O E t
R/ '"OAc R*OH
R
Me 87 % ee, 39 %yield, BSL, 169a 99 % ee, 35 %yield 169b [76]
vinyl acetate
Et 98 % ee, 38 %yield, BSL, 170a 98 % ee, 42 % yield 170b [76]
vinyl acetate
Table 11.1-20. (cont.).
I
519
RvCO2H
"Y
OAc
Co2H OH
R
(CH2)13Me 98 % ee, -, BSL, vinyl acetate, 171a 91 % ee, - 171b (771
48 % conversion
CHzPh 98 % ee, -, BSL, vinyl acetate, 172a 89 % ee, - 172b [77]
48 % conversion
173a [78] 173b [78]

&NO2 -NO,
91 % ee, 31 %yield, GLL, __
vinyl acetate
1 i
C0,Me C02Me
174a [79] 174b [79]
nPrOCO C0,Me HO "" C0,Me
82 % ee, 49 % yield, CAL-A, 96 % ee, 35 % yield
vinyl butanoate
175a [80] 175b [80]
>99 % ee, 35-44 %yield, PCL, 73-99 % ee, 47-56 % yield

CAL-B, PFL, vinyl acetate
OCOEt
CI,&C02R
R
Et 96 % ee, 29 % yield, RML, vinyl 17Ga -, - 17Gb [81]
propionate
CHzPh 77 % ee, 43 %yield, RML, vinyl 177a 96 % ee, 29 %yield 177b [81]
propionate
c-C6H11 89 % ee, 45 %yield, RML, vinyl 178a 96 % ee, 44 %yield 178b [Sl]
propionate
t-Bu >97 % ee, 48 % yield, RML, 179a 99 % ee, 42 % yield 179b [Sl]
vinyl propionate
180a [82] 180b [82]

o f y o 0
OAc OH
95 % ee, -, CAL-A, vinyl 32 % ee, -
acetate, 25 % conversion, 55 "C
83 % ee, -, CAL-A, vinyl >99 % ee, -
acetate, 56 % conversion, 22 "C
520
OH
. /
S0,Ph BocNH- S0,Ph
181a [83] 1811, [83]
>99 % ee, 49 % yield, PCL, >99 % ee, 46 % yield
vinyl acetate
Rq cop
R
Me 97 % ee, 43 %yield, PCL, 182a >97 % ee, 42 %yield 1821, [84]
isopropenyl acetate
Et 299 % ee, 37 % yield, PCL, 183a 70 % ee, 50 %yield 183b [84]
Jg
vinyl acetate
ph
0 'R
R
H >97 % ee, 24 % yield, PCL, 184a 41 % ee, 69 % yield 1841, [85]
vinyl acetate
PMP >97 % ee, 29 % yield, PCL, 185a 44 % ee, 61 %yield 18% [85]
yPhgPh
vinyl acetate
: H HI
0 R 0 R
R
H >97 % ee, 49 % yield, PCL, 186a >97 % ee, 47 % yield 186b [85]
vinyl acetate
TBDMS >97 % ee, 42 %yield, PCL, 187a >97 % ee, 49 % yield 1871, [85]
vinyl acetate
R&R
EtOCO HO
R' R=
CF3 TMS >99 % ee, -, CAL-B,vinyl 188a 72 % ee, - 188b [86]
propionate, 42 % conversion
CHFz TBDMS >99 % ee, -, CAL-B, vinyl 189a 53 % ee, - 189b [86]
propionate, 36 % conversion
C2F5 TMS 98 % ee, -, LIP, 190a 22%ee,- 190b (861
Table 11.1-20. (cont.).
I
521
191a [87] ~ 0 :
O A C 191b [87]
OAc OH
82 % ee, 52 % yield, PCL, >99 % ee, 47 % yield
vinyl acetate
OAc OH
R' = H,R2 = N 0 2 , X = C1 192a 89% ee, 43 %yield 192b I881
vinyl acetate
R'= H, R2 = NO2, X = F 193a 95 % ee, 43 % yield 193b [88]
>98%ee, 41 % yield, PCL,
vinyl acetate
R' = H, R2 = NO2, X = Br 194a 39% ee, 45 % yield 194b [88]
94% ee, 21 %yield, PCL,
vinyl acetate
R'= NO2, R 2 = H, X = C1 195a 95 % ee, 43 % yield 195b [88]
92% ee, 36 % yield, PCL,
vinyl acetate
R'= NO2, R 2 = H, X = F 196a 48 % ee, 45 % yield 196b [88]
96% ee, 34 %yield, PCL,
vinyl acetate
OAc OH
1
s v R
)-N Me
Mi Me
R
CH2CH=CH2 -, -, PSL, vinyl acetate 197a 88 % ee, 46 %yield 19% 1891
CHzCsCH -, -, PSL, vinyl acetate 198a 94 % ee, 40 % yield 198b (891
CH=CH2 -, -, PSL, vinyl acetate 199a 90 % ee, 48 % yield 199b [89]
OAc
200a [90] S&-J 200b [90]
93 % ee, -, PFL, vinyl acetate, 42 % ee, -

31 % conversion
522
a
201a [91] Me / 201b[91]

vinyl acetate
202a [92] 202b [92]

R' R'
OAc OH
R' = H, Br, Me, TBDMS-OCHz, TrOCH2, Ph, 2-Py
R2 = Me, Et, Vinyl

92-99 % ee, 31-49 % yield, 78-99 % ee, 43-58 %yield
R = Me, n-Pr, n-C5HII, n-C7H15

98->99 % ee, 22-48 %yield, 203a [93] 35-96 % ee, 25-45 %yield 203b [93]
PCL or CAL-B, vinyl alkanoate
M
Hz >98 % ee, 44-46 % yield, 204a 89->98 % ee, 36-47 % 204b [94]
PCL, CAL-B or LIP, yield
vinyl acetate
Zn >98 % ee, 12-24 %yield, 205a 16->95 % ee, 72-82 % 205b [94]
PCL, CAL-B or LIP, yield
vinyl acetate
Table 11.1-20. (cont.).
1 7 . 1 Hydrolysis and Formation ofcarboylid Acid Esters
I
523
n c 5 H 1 1 ~20Ga R[95] n c 5 H 1 1 ~ R
20Gb [95]
OAc OH
n = 1-3, TBDMS, TBDPS
94-95 % ee, 35-42 % ee, CRL, 58-77 % ee, 33-46 % ee
vinyl acetate
AA
0 0
0
1
R' +i2
R' R2
Ph Me 96 % ee, 41 % yield 207a 98 % ee, 45 % yield 20% [96]
Ph(CH& Vinyl 92 % ee, 42 %yield 208a 96 % ee, 44 % yield 208b [9G]
PhCH=CH Me 97 % ee, 41 % yield 209a 90 % ee, 38 % yield 209b[9G]
2-Naphthyl Me 93 % ee, 40 % yield 210a 96 % ee, 46 %yield 210b [96]
all CAL-B, methyl
acetoacetate
OAc
211b [97]
R = H. 2-Me, 4-Me, 4-C1,4-Br, 35-94 % ee, 41-62 %yield

2,3-C4&, 3,4-C4H4
89- >99 % ee, 34-55 % yield,
PCL, vinyl acetate
Meog ?Me

212a [98]
Meo$
?Me
47 % ee, 68 % yield
212b [98]
vinyl acetate
213a [98] 213b [98]

vinyl acetate
524
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1605. 2000, 11,4485.
30 U. Ader, M. P. Schneider, Tetrahedron: Asymmetry 59 S. Tsuboi, N. Yamafuji, M. Utaka, Tetrahedron:
1992, 3, 521. Asymmetry1997,8,375.
GO W. Zhang, P. G. Wang, J. 0%.Chem. 2000,65,
78 D. Kalita, A. T. Khan, N. C. Barua, G. Bez, Tetra-

I
525
4732. hedron 1999,55,5177.

61 Y. Aoyagi, N. Agata, N. Shibata, M. Horiguchi, 79 A. Liljeblad, L. T. Kanerva, Tetrahedron: Asymmetry
R. M. Williams, Tetrahedron Lett. 2000,41, 1999,10,4405.
10159. 80 I. Petschen, M. P. Bosch, A. Guerrero, Tetrahedron:
62 T. Sakai, Y. Miki, M. Tsuboi, H. Takeuchi, T. Ema, Asymmetry2000,11,1691.
K. Uneyama, M. Utaka,J. 0%.Chem.2000,65, 81 B. H. Hoff, T. Anthonsen, Tetrahedron: Asymmetry
2740. 1999,10, 1401.
63 K. Morishita, M. Kamezawa, T. Ohtani, H. Tachi- 82 B. Henkel, A. Kunath, H. Schick, Liebigs Ann.
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K. Nakamura,J. 0%.Chem. 1996,61,2332. 84 N. Hayashi, K. Yanagihara, S . Tsuboi, Tetrahedron:
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66 C. Orrenius, N. Ohrner, D. Rotticci, A. Mattson, F. Prati,]. Chem. Soc., Perkin Trans. 1 1999,2489.
K. Huh, T. Norin, Tetrahedron: Asymmetry 1995,6, 86 M. Takeda, T. Ishizuka, K. Itoh, T. Kitazume,
1217. /. fluorine Chem. 1995,75,111.
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68 W. Adam, C. Mock-Knoblauch,C. R. Saha-Moller, 88 R. Skupin, T. G. Cooper, R. Frohlisch, J. Prigge,
Tetrahedron: Asymmetry1997,8,1441. G. Haufe, Tetrahedron: Asymmetry 1997,8,2453.
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70 D. Rotticci, C. Orrenius, K. Hult, T. Norin, Tetra- 90 L. Di Nunno, C. Franchini, A. Scilimati, M. S .
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Asymmetry1997,8,93. 91 J. A. Fuentes, A. Maestro, A. Testera, J. M. Bbiiez,
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Asymmetry1998,9,4429. 92 J. Uenishi, T.Hiraoka, S . Hata, K. Nishiwaki,
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hedron: Asymmetry1999,10,315. Lett. 1998,39,6311.
75 W. Adam, P. Groer, C. R. Saha-Moller,Tetrahedron: 95 J. S. Yadav, S . Nanda, A. Bhaskar Rao, Tetrahedron:
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76 W. Adam, P. Groer, H.-U. Humpf, C. R. Saha-Mol- 96 A. Chdova, K. D. Janda,J. Org. Chem. 2001,66,
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77 W. Adam, M. Lazarus, A. Schmerder, H.-U. 97 A. Kamal, G.B. R. Khanna, Tetrahedron: Asymme-
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Chem. 1998,2013. 98 E. Brenna, C. Fuganti. P. Grasselli, S . Serra, Eur.
/. Org. Chem. 2001,1349.
11.1-14.
For a wide structural range of racemic secondary alcohols, lipase-catalyzed
enantiomer-differentiatingacylation has been reported (1-213) (Table 11.1-20).The
results show that this is a general method for the attainment of enantiomerically
pure secondary alcohols that is complementary to the lipase-catalyzedhydrolysis of
the corresponding acylated alcohols (Table 11.1-15). It is especially worth mention-
ing that secondary alcohols of the alkyl-alkyl, alkyl-aryl or alkyl-heteroaryltype, but
also those bearing the various firnctional groups including stannylated derivatives,
are accessible too. Acylation has been utilized in depth for the synthesis of allylic,
homoallylic, propargylic and homopropargylic and allenylic alcohols (17-20,25-27,
526
Table 11.1-21. Lipase-catalyzed enantiorner-differentiating acylation of racernic cyclic secondary

alcohols in organic solvents (CCL Candido cylindracea lipase, PSL Pseudomonos sp. lipase, CAL-B
Candida antarctica B lipase, PPL p i g pancreas lipase, PCL Pseudomonos cepacia lipase, LIP
Pseudomonas sp. lipase-Toyobo, ASL Alcaligenes sp. lipase, PFL PseudomonasPuorescens lipase,
BSL Burkholderia sp. lipase, CRL Candida rugosa lipase, MML Mucor miehei lipase).
U
95 % ee, 48 % yield, CCL, triacetin 96 % ee, 48 % yield
OAc
n
1 299 % ee, 46 % yield 2a 95 % ee, 48 % yield 2b I21
2 97 % ee, 49 % yield, 3a 299 % ee, 44 % yield 3b 121

vinyl acetate

vinyl acetate
6
OCOnC,H,, OH
..+Me
97 % ee, -, CAL-B, n-C,H&OSEt

Ga[51 uMe
97 % ee, -
6
OCOnPr OH
87 % ee, - 98 % ee, -
95 % ee, -, (triple resolution)
PSL,vinyl acetate
P P j % 8P ‘aJ % L67
HO
[6 ‘81 9f I
JlelaJe 1Xup
‘13d ‘PIJj % LE ‘aJ % L67
16 ’81 921
PPP % 8P ’Ja % L67
HO
0 Yd
PIJd % LE ‘JJ % LG7
avo
[6 ‘81 901
b Y d
P I J j % LP ‘JJ % L67
HO avo
PlJlX % GE ‘JJ % 8667
HO
Ho<
[LI 98
$%03v
Table 11.1-21. (cont.).
14a [S, 91 14b [8,91

Me
phQ Me
OAc OH

vinyl acetate
15a [8, 91 15b [8, 91

Me
phQ Me
OAc OH

vinyl acetate
n~*~'.OSitBuPh, 0sitBuPh,
16a [lo] 16b [lo]
"OAc OH
vinyl acetate
H O AQ 0
17a [ll] 17b [ll]
100 % ee, 48 % yield, CCL, 98 % ee, 51 % yield

acetic anhydride
PQ
OH
96 % ee, 50 % yield, CCL, 100 % ee, 48 % yield

acetic anhydride
I 529
FO,Et C0,Et
19a [12, 131 &H 19b [12, 131
298 % ee, 48 % yield, PCL, 298 % ee, 48 %yield

vinyl acetate
C0,Et C0,Et
bss.oActYH
20a [13] 20b [13]
99 % ee, 40 %yield, PCL, 95 % ee, 53 %yield

vinyl acetate
A OCOnPr
87 % ee, -, PPL,
21a [14]
4 OH
87 % ee, -
21b [14]
n-PrC02CH2CC13
HO- .*-R
0%
22a 1151 22b [15]
" O K R
0 0
R = Ph, 3-MeoC~H4,3,4-(MeO)&H3, 3,4-(methylenedioxy)CGH3
299 % ee, -, PCL, 299 % ee, -
vinyl acetate
OCOR OH
R
Me 99 % ee, 38 % yield 23a 71 % ee, 51 %yield 23b [16]
n-Pr 99 % ee, 47 %yield 24a 94 % ee, 48 % yield 24b [16]
PCL, vinyl acetate or
R
butyric anhydride
OH
.,.'"\ 25a (171 cTo~..Ms 25b [17]
OTBDMS
vinyl acetate
530 I Table 11.1-21. (cont.).
SiMe, SiMe,
:T5
1 93 % ee, -, PCL, vinyl 2Ga
Ho,8*b
95 % ee, - 2Gb [18]
acetate, 50 conversion
97 % ee, -, BSL, vinyl >99 % ee, -
2 99 % ee, -, BSL, vinyl 27a 98 % ee, - 2% [18]
28b [19]
R = Me, Et, n-Pr, n-CsH11,

n-C7H15, n-CgH19, ClCH2
70-97 % ee, 41-51 %yield, PCL, 69-95 5 ee, 40-51 %yield
vinyl alkanoate
OCOR OH
1
29b [19]
R = Me, n-Pr, n-CgH19, ClCH2, Ph

98- >99 % ee, 40-46 % yield, 67- >99 % ee, 50-58 % yield
PCL, vinyl alkanoate
AcO
Rb
R
Ph >95 % ee, 45 %yield, 30a >95 % ee, 49 % yield 30a [20]
PCL, isopropenyl acetate
PMP 87-90 % ee, 40 %yield, PCL, 31a -, 41 %yield 31b [20]
isopropenyl acetate
t-Bu 97 % ee, 48 % yield, 32a 91 % ee, 53 % yield 32b 1211
PCL, vinyl acetate
76 % ee, 51 %yield, 32a 98 % ee, 40 % yield 32b [23b]
CMezPh pancreatin, vinyl acetate
>99 % ee. 43 % yield, 33a >99 % ee, 50 % yield 33b [22]
TBDMS PCL, vinyl acetate
98 % ee, 48 % yield, 34a 98 % ee, 47 % yield 341,1231
Bn pancreatin, vinyl acetate
30 % ee, 74 % yield, 35a 98 % ee, 20 % yield 35b (23bj
THP pancreatin, vinyl acetate
91 % ee, 45 %yield, 36a 94 % ee, 50 % yield 3Gb [23b]
pancreatin, vinyl acetate
Table 11.1-21. (cont.).
1 1.1 Hydrolysis and Formation ofCarboxylid Acid Esters
I
531
HO
R
Me 91 % ee, 30 %yield, 37a 63 % ee, 59 % yield 3% [24]
PSL, vinyl acetate
n-CsH19 96 % ee, 20 % yield, 38a 40 % ee, 75 % yield 38b [24]
CHzPh PSL, vinyl acetate
91 % ee, 21 %yield, 39a 43 % ee, 63 % yield 39b [24]
PSL, vinyl acetate
ACooNHCbz
92 % ee, 40 % yield, PSL,
40a [25] HO,,..
__
40b [25]
vinyl acetate
A C o o FN
N e ’ 41a [26] H O , , . . O&
,“.I,‘ FN 41b [26]
“Y
“H,
“Y”NH,
>90 % ee, 35 %yield, PCL, >90 % ee, 33 % yield
vinyl acetate
OnP(0)(OEt), OnP(0)(OEt),
42a [271 HO,,,,b 42b [27]
AcoQ OH OAc
vinyl acetate
OAc
1
n
1 93 % ee, 50 % yield, PCL, 43a 100 % ee, 47 % yield 43b [28]
vinyl acetate
2 98 % ee, 48 % yield, PCL, 44a 100 % ee, 47 % yield 44b [28]
vinyl acetate
11 Hydrolysis and Formation of C - 0 Bonds
NHCbz NHCbz
"OAc OH
n
1 98 % ee, 47 % yield, PCL, 45a >99 % ee, 57 % yield 45b [29]
vinyl acetate
2 >99 % ee, 31 % yield, PCL, 46a 49 % ee, 63 % yield 46b[29]
vinyl acetate
NHCbz
Q N H C"OAc
b2
OH
n
1 99 % ee, 50 % yleld, PCL, 47a 99 % ee, 50 % yield 4% [30]
vinyl acetate
2 99 % ee, 47 % yield, 48a 70 % ee, 47 % yield 48b [30]
CAL-B, isopropenyl acetate
( W N R 2
"OAc OH
n R
1 Me 93-98 % ee, -, CAL-B 49a 97-98 % ee, - 49b [31]
or PCL, vinyl acetate
2 Me 98 % ee, 42 % yield, 50a 96 % ee, 34 %yield 50b [32]
CAL-B,vinyl acetate
3 Me 95 % ee, -, CAL-B or 51a 92-95 % ee, - 51b [31]
PCL, vinyl acetate
1 -(CH45- 97-99 % ee, -, CAL-B 52a 99 % ee, - 52b [31]
or PCL, vinyl acetate
2 -(CH2)5- 99 % ee, 46 % yield, 53a 97 % ee, 49 %yield 53b [32]
CAL-B,vinyl acetate
2 CH2Ph >99 % ee, 40 %yield, 54a 99 % ee, 50 % yield 54b [32]
PCL, vinyl acetate
(oc"". OH
n R
1 Me 95-99 % ee, -, PCL or 55a 94-99 % ee, - 55b [31]
2 Me 99 % ee, 38 % yield, 56a 96 % ee, 49 % yield 56b 1321
PCL, vinyl acetate
2 Me 94-96 % ee, -, PCL or 57a 37-53 % ee, - 57b [31]
2 -(CH2)5- 98 % ee, 44 % yield, 58a 97 % ee, 46 %yield 58b 1321
PCL, vinyl acetate
I
533
Table 11.1-21. (cont.).
Me
I
(aN"HBoc
n
"OAc
OH
1 91 % ee, 45 % yield, 59a 99 % ee, 31 %yield 59b [33]

CAL-B,vinyl acetate
2 99 % ee, 20 % yield, 60a 99 % ee, 22 % yield Gob [33]
NHBoc
'"OAc OH
n
1 99 % ee, 46 % yield, PCL, 6la 99 % ee, 27 % peld 6 l b [33]
vinyl butyrate
c
2 >99 % ee, 43 % yield, PCL, G2a 98 % ee, 45 % yield G2b [33]
vinyl acetate
Ho
3 C0,Et
63a 1341 G3b [34]
AcO SPh
Ho SPh
>99 % ee, 42 % yield, PSL, 299 % ee, 48 % yield
vinyl acetate
(yPh " OAc

G4b [35]

vinyl acetate
()'TLoph 65b 1361
aO"
>99 % ee, 44 % yield, PCL, >99 % ee, 48 %yield
vinyl acetate
G6a [37] G6b [37]

C0,Et
>99 %, 45 %yield, PFL, >99 % ee, -

534
I 1 1 Hydrolysis and Formation of C- 0 Bonds
Table 11.1-21. (cont.).
,-G.l " OAc

R' R2
Me H 91 % ee, -, CAL-B, vinyl acetate 67a __ 67b [38]
t-Bu H 93 % ee, -, CAL-B, vinyl acetate 68a - - 68b [38]
c-C6HI1 H 85 % ee, -, CAL-B,vinyl acetate 69a __ 69b [38]
Ph H 89 % ee, -, CAL-B, vinyl acetate 70a _- 70b 1381
-(CH2)3- 95 % ee, -, CAL-B, vinyl acetate 71a __ 71b [38]
-(CH2)4- 96 % ee, -, CAL-B,vinyl acetate 72a __ 72b [38]
-(CHz)s- 98 % ee, -, CAL-B, vinyl acetate 73a __ 73b [38]
OH
95 % ee, -, PPL, vinyl acetate,

44 % conversion
74a [39]
oBr
75 % ee, -
74b [39]
99 % ee, -, CRL, isopropenyl 60 % ee, -

OH
hCozEt
7Ga [41] ~ c o ~ E t 7Gb [41]
99 % ee, 40 % yield, PCL, vinyl 95 % ee, 53 %yield

acetate
& OAc
Br
77a[42] bBrpBr
OH
. Br
77b[42]
OH
" Br
77c[42]
OAc OAc OH
>98 % ee, 10 % yield, 298 % ee, 37 % >98 % ee,
MML, vinyl acetate yield 48 % yield
I
535
Table 11.1-21. (cont.).
0
AcO H? H
@ OAc OH
96 % ee, 38 %yield, PCL, 78a [43] 98 % ee, 44 %yield 78b [43]
vinyl acetate
>99 % ee, 32 % yield, 78a (431 55 % ee, 57 % yield 78b [44]
pancreatin,
AcOCH2CCls
6
R'OCO
R3
R' R2 R3
Me H H 84 % ee, -, CAL-B, 79a 98 % ee, - 79b [45]
54 % conversion
Me H Me SG%ee,-,CAL-B, 80a 99%ee,- 80b[45]
53 % conversion
Me Me H 90 % ee, -, CAL-B, 81a 97 % ee, - 81b [45]
52 % conversion
n-C5HI1 H H, alkyl, 87-- >99 % ee, 43-51 % 82a 75- >99 % 82b [4G]
CH#h yield, CAL-B, ee, 41-53 %
isopropenyl hexanoate yield
OAc OH
83a [47] 83b (471
94 % ee, -, CRL, vinyl acetate

(f%
83 % ee, -
&o 84a[48] &o 84b [48]
nBuOCO HO
vinyl butanoate, double
resolution
536
AcO
&co2Me
c; H

vinyl acetate
AcO
& H
86a [SO]
HO
H
861,[SO]

vinyl acetate
vinyl acetate
n
1 86 % ee, -, PCL, vinyl 87a [51] 95 % ee, - 87b[S1]
2 79 % ee, -, PSL, vinyl 88a [Sl] 99 % ee, - 88b [Sl]

vinyl acetate
>96 % ee, 44% yield, PCL,
89a [52]
89a [53]
05- /
>99 % ee, 48 %yield
>96 % ee, 46 % yield

89b [52]
89b [53]
isopropenyl acetate
m.,, /
NHCbz
I ,OAc
>99 % ee, 43 % yield, PSL,

g0a'541 78
&OH
NHCbz
% ee, 28 % yield
901, [ 541
vinyl acetate
Table 11.1-21. (cont.).
I
537
NHCbz NHCbz
~ - ~ ~ ~ O A c

vinyl acetate
OH
d N H C b z
>99 % ee, 41 %yield, PSL, 96 % ee, 38 % yield

vinyl acetate
OH
d
Ii NHCbz
95 % ee, 38 %yield, 20 % ee, 40 % yield

PSL, isopropenyl acetate
QAc OH
94b [55]
D / B r
93 % ee, 35 % yield, CAL-B, 100 % ee, 31 %yield

vinyl acetate
OMe
I
95a [56] 95b [56]
HO

vinyl acetate
9Gb [57]
HO"'
92 % ee, 41 % yield, LIP, >99 % ee, 46 % yield
vinyl acetate
538
Table 11.1-21. (cont.).
97a [SS] 97b I581
>99 % ee, 44 %yield, CAL-B, 98 % ee, 47 % yield

vinyl acetate
n
1 99 % ee, 42 %yield, LIP, 98a [59] 95 % ee, 43 %yield 98b I591
vinyl acetate
2 94 % ee, 36 %yield, LIP, 99a [59] 55 % ee, 43 %yield 99b [59]
vinyl acetate
h
lOOa [60] 1OOb 1601
pOAc
\.
HO
299 % ee, 49 % yield, PCL, >99 % ee, 50 % yield
vinyl acetate
HO
n
1 299 % ee, 50 % yield, 1Ola [61] >99 % ee, 49 %yield 1Olb [61]
PCL, vinyl acetate
2 >99 % ee, 46 % yield, 102a [61] >99 % ee, 49 %yield 102b [61]
s
PCL, vinyl acetate
103a [62] 103b [62]
CI
CI CI
>95 % ee, -, CRL, vinyl acetate, 77 % ee, -
44 % conversion
I
539
@O.TO 104a (631 104b [63]
OAc OH
vinyl acetate
OAc
lO5a [64] 105b [64]
94 % ee, 41 % yield, PFL, 97 % ee, 34 % yield

isopropenyl acetate
lOGa [65] 1OGb [65]

I
Ph Ph
vinyl acetate
107a [66] 107b [66]

LNFrnoc
vinyl acetate
uoAc
Y
108a [67]
I
1081,[67]
Cbz Cbz
vinyl acetate
YJNH 109b [68]
OAc OH
50 % ee, 53 %yield, lipase PL, 98 % ee, 35 % yield
vinyl acetate
540
Table 11.1-21. (cont.).
R2
" g N - B n
OAc OH
R' R2
H H >99 % ee, 50 %yield, PCL 110a >99 % ee, 48 % yield llOb [69]
or CAL-B, vinyl acetate
Me H >99 % ee, 49 %yield, CAL-llla >99 % ee, 48 % yield l l l b [69]
B, vinyl acetate
&..
0
1121, [G9]
112a [69] &-Bn
OAc OH
vinyl acetate
TBDMS
78 % ee, 52 % yield, PCL, vinyl acetate
113a [70]
TBDMS-'."'
o'soH
o
97 % ee, 40 % yield
113b [70]
78 % ee, 54 %yield, PSL, vinyl acetate 99 % ee, 42 % yield
phsL-cx
AcO
>99 % ee, 49 %yield, PCL,
114a [71]
HO ""
>99 % ee, 49 % yield
114b [71]
vinyl acetate
115b [72]
62 % ee, 52 %yield, DCL, 93 % ee, 96 % yield

vinyl acetate
llGa [73] "'yyJR3 llGb [73]
I 1 I i
R2 OAc R2 OH
R' = H, Me, R2 = H, OMe, R' =
H, Me
71-100 % ee, -, CCL, 22-100 % ee, -
vinyl acetate, 19-57 %
conversion
Table 11.1-21. (cont.).
I 541
AcO
I
Ts
117a(74] -r\
I
Ts
I
11%[74]
96 % ee, 25 %yield, ASL, 32 % ee, 75 %yield

vinyl acetate
&o 118a [75] 118b [75]
H H
vinyl acetate
119a [76] 119b [76]

RO RO
R = 2-Naphthylmethyl,CHZPh, TBDMS >99 % ee, 47-49 % yield
>99 % ee, 47-50 % yield, PCL,
vinyl acetate
120a[77] 0 12Ob [77]
a
B H
>99 % ee, 48 % yield, PSL, >99 % ee. 47 % yield
vinyl acetate
Ack
H? H
121a[78] l 2 l b [78]
0 ; o
H H
acetic anhydride
dQ
0
OAc
122a[791 d,D l22b [79]
OH
>99 % ee, -, PCL, vinyl acetate, _-
45 % conversion
542
I 1 7 Hydrolysis and Formation o f C - 0 Bonds

~~
&cOAc
/
123a [86]
6 /0
OH (OAc)
123b [86]
c (OH) do.
123c [80]
100 % ee, 38 % yield 100 % ee, 36 %yield 100 % ee, 10 %yield
PCL, vinyl acetate
Brv
OAc
Br"'" R
dAc OAc
R
H >98 % ee, 50 % yield 124a 92 % ee, 50 % yield 124b [81]
Br 94 % ee, 49 % yield 125a 87 % ee, 50 % yield 125b [81]
Me 87 % ee, 50 % yield 12Ga 85 % ee, 50 % yield 126b [81]
~ A c OH
"'-4
Br
OAC OTBDMS
>98 % ee, 47 % yield, CAL-B
127a [81]
AAC ATBDMS
92 % ee, 50 % yield
vinyl acetate
128a [82] 128b [82]
HO
96 % ee, 49 % yiel, PCL, 95 % ee, 49 % yield
vinyl acetate
OAc
>99 % ee, 45 %yield, CAL-B,
129a [83]
cdj OH
>99 % ee, 46 % yield

129b [83]
vinyl acetate, 50 "C

-
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5 H. F r y h a n , N. Ohmer, T. Norin, K. Hult,
11. I Hydrolysis and Formation ofcarboxylid Acid Esters
32 E. Forro, L. T. Kanerva, F. Fiilop, Tetrahedron:

I
543
Tetrahedron Lett. 1993,34, 1367. Asymmetry 1998,9,513.

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544
I 1 I Hydrolysis and Formation of C - 0 Bonds
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937.
28, 29, 31-33, 41-65, 67-71, 83-89, 107-109, 127, 143, 144, 153, 154, 156, 157,
163-170,181-183,201) (Table 11.1-20).A very good illustration for the potential of
enantiomer-differentiating acylation catalyzed by lipases is provided by the high-
yield synthesis of a series of aromatic cyanohydrin acetates (la-g) from aldehydes,
acetone cyanohydrin and vinyl acetate in the presence of Pseudomonas cepacia lipase
and a basic anion-exchangeresin in diisopropyl ether which proceeds under kinetic
resolution coupled with in situ formation and racemization of the cyanohydrin
representing a dynamic kinetic resolution. For further examples see Table 11.1-24.
To the secondary aliphatic alcohols, which have been resolved into their enantio-
mers, belong a variety of hydroxy carboxylic esters and acids (35,100-102,125,126,
131-140, 150,151, 166,168-172, 174,182,183),some hydroxy ketones (128-130,
141) and a crown ether derivative (203)(Table 11.1-20). Even the tetraphenylpor-
phyrin derivatives 204 and 205 were substrates for different lipases.
Diketene is useful acyl donor also, yielding acetoacetates with very high enantio-
meric excess (115,207-210).
11.1-15.
Table 11.1-21 lists cyclic secondary alcohols that have been synthesized by lipase-
catalyzed enantiomer-differentiating acylation (1-1 29).The compounds that have
been obtained by the alternative route of hydrolysis are listed in Table 11.1-16. The
complementary nature ofthe two routes is obvious. For the series ofthe glycals 9-15,
Pseudomonas cepacia lipase-catalyzedacylation works with good to high enantiomer
selectivity and yield. myo-Inositol derivatives 17 and 18 may be prepared enantiomer-
7 7 . 7 Hydrolysis and formation ofCarboxylid Acid Esters
I 545
ically pure by Candida cylindracea lipase-catalyzedacylation with acetic anhydride in
diethyl ether not only with high enantiomer but also with high group selectivity.
Axial-chiral enantiomerically highly enriched binaphthols 4, which are highly
useful chiral auxiliaries, are accessible either through acylation of the racemic diol
with vinyl acetate or deacylation of the racemic diacetate with butanol (Table 11.1-22),
both catalyzed by Pseudomonas cepacia lipase.
Among the many other cyclic secondary alcohols that have been obtained by
lipase-catalyzed enantiomer-selective acylation with high enantiomeric excess are
aminohnctionalized cycloalkanols (40, 45-62, 75), bicyclo[3.3.0]octanoIs (78,
84-86), different types of tri- and tetracyclic alcohols (96104),substituted indanols
(87-94,123),hydroxy lactams (106,109-112) and brominated cyclohexenol deriva-
tives (74,77,124-127) (Table 11.1-21).
11.1-16.
11.1.1.3
Inter- and Intramolecular Alcoholysis
Hydrolase-catalyzed enantiomer-differentiating alcoholysis of esters of racemic

alcohols with achiral alcohols in organic solvents of low water content is a valuable
alternative to hydrolysis (Table 11.1-22).
Lipase-catalyzed enantiomer-differentiatinginter- and intramolecular alcoholysis
of acylated alcohols and lactones in organic solvents may most advantageously be
used instead of hydrolysis in aqueous solution in those cases where insufficient
stability, high solubility or low functional group selectivity is observed or may be
anticipated in the latter case (1-16)(Table 11.1-22).
For lipase-catalyzed intermolecular alcoholysis as alcohols, by and large more
lipophilic ones such as n-propanol, n-butanol, n-hexanol, n-octanol, cyclohexanol or
benzylalcohol are used whereas methanol (44)or ethanol (52,53,61) are used rarely.
Typical solvents are n-hexane, diisopropyl ether, tert-pentyl alcohol, toluene, tetra-
hydrofuran or acetonitrile. In many cases the enantioselectivity and yield are higher
for the alcoholysis than for the hydrolysis catalyzed by one and the same lipase,
provided that a large excess of the alcohol is used. Enantiomer-differentiating
alcoholysis of an acylated thiol (10)has also been described.
Alcoholysis of y-and p-lactones gives access to enantiomerically pure y-hydrox-
yesters and y-lactones (17-24)and P-hydroxyesters and P-lactones (55-63),re-
spectively. Enantiomerically pure enol acetates 67a and the y-acetoxybutenolide
51/ent-51 have been obtained by hydrolysis of the corresponding racemic sub-
strates.
As well as the above-described intermolecular alcoholysis of esters, the intra-
molecular version has been successfully utilized for the synthesis of lactones from
racemic hydroxy carboxylic acid esters (25-41,64-66) (Table 11.1-22).High selectiv-
ity in the pig pancreas lipase-catalyzedenantiomer-differentiating lactonization of y-
hydroxy carboxylic acid esters with formation of butyrolactones substituted in
546
Table 11.1-22. Lipase-catalyzedenantiorner- and enantiotopos-differentiatinginter- and

intramolecular alcoholysis o f esters and lactones in organic solvents (PCL Pseudomonas cepacia
lipase, PSL Pseudomonas sp. lipase, PPL pig pancreas lipase, M M L Mucor miehei lipase, HLL,
Humicola lanuginosa lipase, PFL PseudomonasPuorescens lipase, CCL Candida cylindracea lipase,
CAL-B Candida antarctica B lipase, CRL Candida rugosa lipase, PRL Penicillium roqueforti lipase,
CAL-A+BCandida antarctica A+B lipase).
295 % ee, 45 % yield BuOH 90 % ee, 45 % yield

295 % ee, 45 % yield BuOH/i-Pr20 295 % ee, 45 % yield
295 % ee, 45 % yield H2O 88 % ee, 45 % yield
all PCL
OH 2b [*I
&NHCOnPr Phr r H C O n P r
Ph
295 % ee, 34 % yield BuOH, t-pentanol 295 % ee, 33 %yield
295 % ee, 39 % yield BuO H , toluene 295 % ee, 40 % yield
295 % ee, 42 % yield HexOH, toluene 295 % ee, 46 % yield
295 % ee, 43 % yield OctOH, toluene 295 % ee, 41 % yield
295 % ee, 45 % yield BuOH, n-Bu2O 295 % ee, 39 % yield
295 % ee, 36 % yield HexOH, n-Bu2O 295 % ee, 41 % yield
295 % ee, 44 % yield BuOH, THF 79 % ee, 45 % yield
295 % ee, 43 % yield BuOH, MeCN 85 % ee, 46 % yield
all PCL
3a [31 PhN? OAc 3b [31
95 % ee, 47 %yield, PCL n-PrOH 95 % ee, 50 %yield

CCL, t-pentanol, i-PrzO
OCOEt
R L O T S R A O T s
R
ClCH2 96 % ee,-, PFL, BuOH, n-hexane 4a 96 % ee,-, 4b [41
n-Bu 90 % ee,-, PFL, BuOH, n-hexane 5a 31 % ee,- 5b [41
n-CloH21 298 % ee,-, PFL, BuOH, n-hexane Ga 43 % ee,- 61, [41
Ph 95 % ee,-, CCL, BuOH, n-hexane 7a 70 % ee,- 7b [41
PhOCH2 84 % ee,-, HLL, BuOH, n-hexane 8a 42 % ee,- 8b 141
Table 11.1-22. (cont.).
I 547
R = H, F, C1, Br, OMe

97-99 % ee, CCL, BuOH, i-Pr2O
(fromthe butyrate)
10a [7] lob [7]

Me Me
88 % ee, 39 %yield, n-PrOH, PPL, 95 % ee, 42 % yield
hexane
" O S R
R
v
CICH2C00.,,
Oh
H 79 % ee. 44 %yield lla 84 % ee, 45 % yield Ilb [S]
Me 72%ee,- 12a 95 % ee,- 12b [8]
Et 80 % ee,- 13a 91 % ee,- 13b [8]
n-Bu 86 % ee,- 14a 90 % ee,- 141,[8]
n-CbH13 66 % ee,- 15a 86 % ee,- 15b [8]
all MML, PrOH, i-PrzO
16b [9]
98 % ee, 47 %yield, PSL, BuOH 96 % ee, 50 % yield
R RQO
R
n-C5HII 70 % ee, - 17a 55 % ee, - 1% [lo]
n-C6H13 90 % ee, - 18a 78 % ee, - 18b [lo]
n-C7H15 93 % ee, - 19a 80 % ee, - 19b [lo]
n-CsH17 298 % ee, - 20a 77 % ee, - 20b [lo]
n-C9Hl9 298 % ee, - 21a 71 % ee, - 21b [lo]
n-CloHzl 298 % ee, - 22a 74 % ee, - 22b [lo]
n-C11H23 298 % ee, - 23a 76 % ee, - 23b [lo]
n-ClzHzs 298 % ee, - 24a 77 % ee, - 24b [lo]
all PPL,n-PrOH
548
298 % ee, - 25 [11]

88 % ee, - 26 [ l l ]
82 % ee, - 27 [ l l ]
91 % ee, - 28 [ll]
92 % ee, - 29 [11]
94 % ee, - 30 [ l l ]
88 % ee, - 31 [ll]
94 % ee, - 32 [11]
all PPL, Et20 or hexane or
THF (intramolecular
lactonization of the methyl ester)
78 % ee, -, PPL, Et2O R = Me, Et, PhCHz

(from methyl ester) 295 % ee, -, PPL, Et2O
R
n-C5HI1 86 %ee, 25 %yield 35 [13]
n-C15H,, 298 % ee, 15 %yield, 36 [13]
PPL, Et20 (from the
pentyl ester)
R
Ph 298 % ee, 35 % yield 37 [14]
c-C6HI3 298 % ee, 35 % yield 38 [14]
all PPL, Et2O
I 549
U
x-Y
CHzCHz 299 % ee, 14 %yield 39 (151
(E)-CH=CH 299 % ee, 18 %yield 40 [15]
(2)-CH=CH 98 % ee, 20 %yield 41 I151
PSL,isooctane,
molecular sieves
GBr /
421, [lG]

cyclohexanol
03
NHAc
43a (171 43b [17]
,mttOH OAc
/
>99 % ee, 43 %yield, CAL-B, >99 % ee, 48 % yield

n-BuOH
44b [18]
>99 % ee, 44 % yield, PCL, 80 % ee, 55 % yield

MeOH
/
R
3-OPh >99 % ee, -, PCL, n-BuOH, 45a 97 % ee, - 45b [19]
49 % conversion
3-OPh 98 % ee, -, CAL-B, n- 45a 79 % ee, - 45b [20]
PrOH, 45 % conversion
R = H, 3-Me, 4-Me, 3-OMe, 4-OMe, 3-C1,4-C1
82-98 % ee, -, CAL-B, n- 46a 79- >9 % ee, - 46b [20]
PrOH, 45-53 % conversion
550
Table 11.1-22. (cont.).
4% [21]
Ph
F
82 % ee, -, CAL-B, n-BuOH,

34 % conversion
? 48a [22] 48b [22]

H 0 A o AcO

n-PrOH
NcwB
~ A c
NcTBr
H*N
/
CI
49a [23]
H2N
CI
49b [23]
86 % ee, 50 % yield, CAL-B, n-BuOH 99 % ee, 42 % yield
50 [24]
>95 % ee, 93 %yield, PSL, n-BuOH
AcO ' " ' G O
51a [25] 51b [25] ent-5lb [25]

racemate 70-98 % ee, -, CCL, lipase
PSL, PRL, CRL, PCL, n-
BuOH, 49-61 % conversion
"$N-Bn : "R2g N - B n
OH OAc
R' 'R
H H >99 % ee, 45 %yield, PCL, 521 90 % ee, 49 %yield 52b (261
EtOH 43 % ee, 62 % yield
Me H >99 % ee, 26 % yield, PCL, 53a 53b [26]
EtOH
1 1 . 7 Hydrolysis and Formation ofcarborylid Acid Esters
I
6 6
551
Table 11.1-22. (cont.).
*\\OAc 54a [27] *“oAc 54b [27] 6 O A c 5 k [27]
I OAc : OAc “‘OAc

OAc OH OAc
>98 % ee, 24 % -, 18 %yield 68 % ee, 58 %
yield, CCL, n-BuOH yield
R’T C 0 2 R 2
6H
R’ R2
Me CH2Ph 85 % ee, 51 %yield, PPL, 55a 96 % ee, 36 % 55b [28]
PhCH20H yield
n-Pr CH2Ph 69 % ee, 45 %yield, PCL, 56a 75 % ee, 42 % 56b [28]
PhCHzOH yield
i-Pr CH2Ph 90 % ee, 43 %yield, PCL, 56a 95 % ee, 56b [28]
PhCHzOH 41 % yield
57a [28b] 5% [28b]

(-‘CO,Bn
OH
PhCHzOH
R’
R2Y C O 2 B n
OH
R’ R2
Me n-Pr 87 % ee, 34 % yield, PCL, 58a 92 % ee, 50 % 58b [28b]
PhCHzOH yield
n-Pr Me 98 % ee, 24 % yield, PCL, 59a 79 5% ee, 16 % 59b [28b]
PhCH2OH yield
6Ob [28b]

PhCHzOH
552
Et 81 % ee, 40 % yield, Gla 97 % ee, 43 %yield Glb [29]

PCL, EtOH
n-C6Hls 73 % ee, 36 %yield, Gla >99.9 % ee, 39 %yield Glb [29]
PCL, n-CGH13OH
9, OH
C0,Bn
G2b [30]
95 % ee, -, CAL-B, 99 % ee. -

PhCHzOH, 51 % conversion
131 OH
: CO,Bn
G3b [30]
99 % ee, -, CAL-B, >99 % ee, -

PhCH20H, 50 % conversion
" O U 0
R R
R
E-CH=CH-Ph 80 % ee, 33 %yield, G4a -, 47 % yield G4b [31]
pancreatin, >99 % ee,
double resolution
C=CH-Ph 61 % ee, -, CAL-A+B, 98 % 65a 70 % ee, 30 % 6% [32]
ee, by crystallization from the yield
reaction mixture
GGb [33]
I I
Ph Ph
96 % ee, 28 % yield, - -
CAL-A+B
Table 11.1-22. (cont.).

I
553
6N
100 % ee, 38 % yield (Ar =
Ph), 30 %yield (Ar = 3,4-
67a [34]
Ar%N
A 67b [34]
C12Ph),PFL, n-BuOH
HOA O A c
R
Ts 95 % ee, 76 %yield, PCL, n-BuOH 68 [35]
72 % ee, 56 % yield, CCL, n-BuOH
Cbz 98 % ee, 68 % yield, PCL, n-BuOH 69 [35]
49 % ee, 30 %yield, PPL, n-BuOH
70a [36] 70b [36]

O G O H '-8OAc
racemic >99 % ee, 65 %conversion,

PCL, n-BuOH, n-hexane
O O O A C enb70b 1361
>99 % ee, 57 % conversion,

CCL, n-BuOH, n-hexane
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S. Miyano, Chem. Lett. 1989, 1135; b) M. Inagaki, 10, 2501.
554
18 K. Tanaka, K. Ogasawara, Synthesis 1995,1237. S. Ageishi, H. Isobe, Y. Hayashi, Y. Yamamoto,

19 J. Roos, U. Stelzer, F. Effenberger, Tetrahedron: /. Chem. Soc., Perkin Trans. 12000, 71.
Asymmetry 1998,9,1043. 29 T. Ito, M. Shimizu, T. Fujisawa, Tetrahedron 1998.
20 U. Hanefeld, Y. Li, R. A. Sheldon, T. Maschmeyer, 54, 5523.
Synktt 2000, 1775. 30 W. Adam, P. Groer, C. R. Saha-Moller,Tetrahedron:
21 R. Morrone, G. Nicolosi, A. Patti, M. Piatelli, Asymmetry 1997,8,833.
Tetrahedron:Asymmetry 1995, 6. 1773. 31 B. Henkel, A. Kunath, H. Schick, Liebigs Ann.
22 M. Pallavicini, E. Valoti, L. Villa, 0. Piccolo, /. Org. Chem. 1992,809.
Chem. 1994,59,1751. 32 B. Henkel, A. Kunath, H. Schick, Tetrahedron:
23 S. Conde, M. Fierros, M. I. Rodriguez-Franco, Asymmetry 1994,5,17.
C. Puig, Tetrahedron: Asymmetry 1998, 9, 2229. 33 B. Henkel, A. Kunath, H. Schick, Tetrahedron:
24 M. Ranchoux, J.-M. Brunel, G. lacazio, G. Buono, Asymmetry 1993,4,153.
Tetrahedron: Asymmetry 1998, 9, 581. 34 a) A. J. Carnell, M. L. Escudero Hernandez,
25 H. van der Deen, R. Hof, A. van Oeveren, A. Pettman, J. F. Bickley, Tetrahedron Lett. 2000,
B. L. Feringa, R. M. Kellog, Tetrahedron Lett: 1994, 41, 6929: b) G. Allan, A. J. Carnell, M. L. Escudero
35, 8441. Hernandez, A. Pettman, j . Chem. Soc., Perkin
26 K. Takabe, M. Suzuki, T. Nishi, M. Hiyoshi, Trans. 12000, 3382.
Y. Takamori, H. Yoda, N. Mase, Tetrahedron Lett. 35 K. Fuji, T. Kawabata, Y. Kiryu, Y. Sugiura,
2000,41,9859. Tetrahedron Lett. 1990, 31, 6663.
27 C. Sanfilippo, A. Patti, G. Nicolosi, Tetrahedron: 36 M. van den Heuvel, A. D. Cuiper, H. van der
Asymmetry 1999,10,3273. Deen, R. M. Kellogg, B. L. Feringa, Tetrahedron
28 a) Y. Koichi, K. Suginaka, Y. Yamamoto,j . Chem. Lett. 1997, 38, 1655.
Soc., Perkin Trans. 1 1995, 1645; b) N. Sakai,
4-position and the unchanged y-hydroxy carboxylic acid esters of opposite configura-
tion were observed (25-34). Pig pancreas lipase in diethyl ether is the combination of
choice. Formation of the corresponding monosubstituted y-valerolactones was
unselective. y-Valerolactoneswith a hydroxyl group in 4-position however could be
obtained with high selectivity from the corresponding dihydroxy carboxylic acid
pentyl or methyl ester (35-38). In order to suppress the competition between the
methanol formed during lactonization and the intramolecular hydroxyl group,
reactions were run in the presence of molecular sieve. Otherwise, conversion and ee
value of the lactone were poor because of the reversibility of the reaction. Inter-
estingly, macrocyclic lactones may be prepared by this method too. Treatment of
racemic ricinoleic acid methyl ester, its racemic trans-isomer and the saturated
racemic derivative with Pseudomonas sp. lipase in isooctane in the presence of
molecular sieve gave the corresponding (R)-configurated 13-membered lactones
39-41 (Table 11.1-22) in fair yields with high ee values.
Acylated alcohols, alcohols and lactones of Table 11.1-22 which can be obtained
with other hydrolases as such or of opposite configuration are contained in Tables
11.1-14to 11.1-16and Tables 11.1-19 to 11.1-21.
Lipase-catalyzed enantiomer- and enantiotopos-differentiating alcoholysis may
also be extended to carboxylic acid esters, anhydrides and oxazolin-2-ones (1-22)
(Table 11.1-23).Alcoholysis of methoxy malonic acid dimethyl ester with benzyl
alcohol catalyzed by Candida cylindracea lipase gave, at 50 % conversion, the mixed
diester 2 with high enantioselectivity. At higher conversion the ee values are lower
because of the reversibility of alcoholysis.The enantiomeric mixed diester ent-2 may
be obtained by methanolysis of the corresponding dibenzyl ester. Through catalytic
hydrogenolysis the monobenzyl ester can be converted into the corresponding acid.
It remains to be shown if this is an alternative to the pig liver esterase or lipase-
catalyzed hydrolysis of the corresponding prochiral diester (Table 11.1-2).
Table 11.1-23.
Lipase-catalyzed enantiomer- and enantiotopos-differentiating alcoholysis of

I
555
carboxylic acid esters and anhydrides, alcoholysis or hydrolysis of oxazolin-2-ones, and

esterification of carboxylic acids (PPL p i g pancreas lipase, PCL Pseudomonas cepacia lipase, ANL
Aspergillus niger lipase, CSL Candida sp. lipase, Candida cylindracea lipase, CAL-B Candida
antarctica B lipase, CRL Candida rugosa lipase).
CI,CH,OOC Po
PEG-O,C’ ,
Me0,C /J 1b PI
296 % ee, 43 % yield, PPL 289 % ee, 46 % yield

PEG, i-PrzO
Me0
HxCozMe
C0,Bn
2 121 Me0 HxCozBn
C0,Me
ent-2 [2]
absolute configuration absolute configuration

unknown unknown
296 % ee, -, CCL 90 % ee, -, CCL
PhCHZOH, hexane equlibrium (70 %)
( f r o m the dimethyl ester) MeOH, hexane, 50 %
conversion
(from the dibenzyl ester)
R’ 8 PI
HO,C A C O , n B u CO,H C0,iBu
R’ R2
Me H 93%ee 3 [3, 41 90 % ee, 72 % y i e l d
Et H 87%ee 4 [3,4] CSL, i-BuOH,
n-Pr H 60%ee 5 [3,4] C-C~HI~
i-Pr H 76%ee 6 [3,4]
H C1 62%ee 7 [3,41
65-95 % y i e l d
PCL, BuOH, i-Pr20 ,PI h
f
V Ph
9 [GI
ent-9 [6]
99 % ee
556 I 7 7 Hydrolysis and Formation ofC-0 Bonds
Table 11.1-23. (cont.).
Ph11 N
H
CO,Me(H)
Ph
R
MezCH 77 % ee, 47 % yield (H2O) 10 [7-91
MezCHCHz 78 % ee, 82 % yield 11 [7-91
MeS(CH2)2 82 % ee, 31 %yield (H2O) 12 [7-91
2-Naphthylmethyl 75 % ee, 90 % yield 13 [7-91
4-MeCsH4CHz 66 % ee, 86 % yield 14 [7-91
Ph 75 % ee, 46 %yield (H20) 15 [7-91
PhCHz 69 % ee, 93 % yield 1 G 17-91
Ph(CHz)z 93 % ee, 61 %yield (H20) 17 [7-91
Ph(CHz)3 84 % ee, 91 % yield 18 [7-91
all PCL, MeOH ( or HzO),
t-BuOMe
19a [lo] 19b [lo]

iBu0,C +f CO,H
HO,C C0,iBu
74 % ee, 30 % yield, CAL-B, 92 % ee, 29 % yield
2-methylpropanol
20b [11]
HO,C
"'YY C0,iBu
20a [11]
iBu0,C C0,iBu
90 % ee, 40 % yield, CAL-B, 90 % ee, 48 % yield
2-methylpropanol
21a 1121 21b [12]

iBu0,C CO,H HO,C C0,iBu
2-methylpropanol
22a [13] /\/CO,CH=CH, 221, [ 131

/yC02nC6H13
Ph
Ph
n-hexanol
1 23b [14]
23a [14] &CO,H
R
82-90 % ee, -, CRL, 30-50 % ee, -
hexadecan-1-01,25-39 %
conversion. esterification
Table 11.1-23. (cont.).
I
557
24b [14]
RnCO,nC,,H,,
85-93 % ee, -, CRL,
hexadecan-1-01, 15-33 %
conversion. esterification
64 % ee, 58 %yield, CAL-B, >98 % ee, 39 % yield

n-PrOH, HC(OnPr),
2Gb [15]
-CO,nBu
53 % ee, 65 %yield, CRL, >97 % ee, 35 % yield

n-BuOH, H C (0nBu)S
1 J. S. Wallace, K. B. Reda, M. E. Williams, 9 H. S. Bevinakatti, R. V. Newadkar, A. A. Baneji,
C. J. Morrow,]. Org. Chem. 1990,55,3544. j . Chem. Soc., Chem. Commun. 1990,1091.
2 A. L. Gutman, M. Shapira, A. Boltanski,]. Org. 10 R. Ozegowski, A. Kunath, H. Schik, Liebigs Ann.
Chem. 1992,57,1063. Chem. 1996,1443,
3 Y. Yamamoto, K. Yamamoto, T. Nishioka, 1. Oda, 11 R. Ozegowski, A. Kunath, H. Schick, Liebigs Ann.
Agric. Bid. Chem. 1988,52,3087. Chem. 1994,215.
4 Y. Yamamoto, T.Nishioka, J. Oda, Tetrahedron Lett. 12 R. Ozegowski, A. Kunath, H. Schick, Liebigs Ann.
1988,29,1717. Chem. 1994,1019.
5 R. Ozegowski, A. Kunath, H,Schick, Tetrahedron: 13 H. Yang, E. Henke, U. T. Bornscheuer, j . Org.
Asymmetry 1993,4,695. Chem. 1999,64,1709.
6 R.-L. Gu,1 . 4 . Lee, C. J. Sih, Tetrahedron Lett. 1992, 14 B.-V. Nguyen, E. Hedenstrom, Tetrahedron:
33, 1953. Asymmetry1999,10,1821.
7 J.2. Crich, R. Brieva, P. Marquart, R:L. Gu, S. 15 R. Morrone, M. Piatelli, G. Nicolosi, Eur.]. Org.
Flemming, C. 1. Sih, j . Org. Chem. 1993,58,3252. Chem. 2001,1441.
8 H. S. Bevinakatti,A. A. Baneji, R. V. Newadkar,
A. A. Mokashi, Tetrahedron: Asymmetry1992,3,
1505.
Alcoholysis of prochiral glutaric anhydrides under the usual conditions gives, with
moderate selectivities, the monoesters 3-8.
Lipase-catalyzed enantiomer-differentiating hydrolysis of racemic phenyl benzyl
oxazolin-2-one in aqueous solution in combination with an uncatalyzed in situ
racemization of the unchanged enantiomer of the heterocyclic system, with two
different lipases, gives access to D- and L-N-benzoyl-phenylalanine9 and ent-9,
respectively. Enantiomer-differentiating alcoholysis and in situ racemization in
organic solvents in the presence or absence of added water under the catalysis of
lipase can in some cases furnish amino acid derivatives (10-18)with good selectiv-
ities and yields.
558
During alcoholysisof racemic substituted glutaric acid anhydride one is faced with
regio- and enantioselectivity.These two processes may not cooperate in a matching
sense. Despite this fact, the monoalkyl glutarates 19-21 have been obtained with
moderate to good enantiomeric excess by lipase-catalyzed alcoholysis of the corre-
sponding anhydrides in the presence of Candida antarctica B lipase with 2-methyl-
propanol.
Alcoholysis of alkyl carboxylates is due to the competition of the two alcohols
characterized by reversibility and associated with low conversion and poor enantiose-
lectivity. The alcoholysis of vinyl carboxylates in the presence of Candida antarctica B
lipase with n-hexanol as demonstrated for 22 can be regarded as an alternative in
order to overcome these difficulties.
Esterification of carboxylic acids (25, 26) (Table 11.1-23) in the presence of an
orthoester as water-trapping agent may have advantages.
11.1.2.1.2 Dynamic Kinetic Resolution

The success of an enzyme-catalyzed kinetic resolution is limited by the maximum
chemical yield of 50% for each enantiomer. However, this drawback can be
overcome by a process called dynamic kinetic resolution. The key idea of this
principle is to racemize the slow reacting enantiomer continuously reproducing the
faster one. In an ideal case at the end of the conversion one enantiomer is formed in
100% yield with 100% of enantiomeric excess [135-1371. The kinetic requirements for
a dynamic kinetic resolution are shown in Scheme ll.l-1G[8b].
The in situ racemization can be achieved by different means either spontaneously
or catalytically. Due to their chemical properties certain substrates may racemize
spontaneously under the reaction conditions. Useful catalysts could be ordinary
chemicals such as bases, transition metal complexes and in theory another type of
biocatalyst. Having identified a suitable enzyme promoting the enantiomer-differ-
entiating process by hydrolysis or alcoholysis of a carboxylic ester or by acylation of
an alcohol one has to find the appropriate racemizing catalyst. Lipase and catalyst
must tolerate each other; they must work under identical conditions. The product
must be chemically and configurationally stable in the presence of the catalyst.
Table 11.1-24 lists lipase-catalyzeddynamic kinetic resolutions by different means.
4-Substituted oxazolin-5-ones racemize spontaneously by hydrolysis or alcoholysis
caused by enolization to yield amino acid derivatives as outlined in the transforma-
tions (I), (2) and (3). Triethylamine may promote this type of transformations as
(S)-substrate - kS
(S)-product
kR ’ks kr,, ”k R krac ”ks Scheme 11.1-16. Dynamic kinetic resolution

Table 11.1-24.
11.1 Hydrolysis and Formation ofCar6oxylid Acid Esters
Lipase-catalyzeddynamic kinetic resolution (PCL Pseudomonas cepacia lipase,

I
559
PPL pig pancreatic lipase, A N L Aspetgillus niger lipase, MML Mucor miehei lipase, CAL-B Candida
antarctica 8 lipase, PSL Pseudornonas sp. lipase, PFL Pseudomoasfluorescens lipase, CAL Candida
antarctica lipase, not specified).
Reaction Type Racemization
Ph
R = i-Pr, Me2CHCH2,MeSCHzCH2,
2-Naphthyl-CH2,4-MePhCH2, Ph, PhCH2,
Ph(CH2)2,Ph(CHz)3
R = Me2CHCH2:78 % ee, 82 % yield,
PCL, MeOH; 90 % ee, 85 % yield, MeOH+H20
R = Ph(CHz),: 84 % ee, 91 %yield, PCL, MeOH
95 % ee, 76 % yield, MeOH+H20
hydrolysis spontaneous
0
Ph
99 % ee, -, PPL
Bn
- p h I i q
0
H hydrolysis spontaneous
*
Ph
99 % ee, -, ANL
- 4 H alcoholysis NEt3
,Yo
Ph P A 0 CoznBu
>99 % ee, 67 % yield, MML, n-BuOH, toluene
-
Bnwo B")iH
HN CO,R alcoholysis NEt3 ( 5 ) [41
NYoPh Ph AO
R = Me: 94 % ee, 79 % yield
R = Et: 97 % ee, 82 % yield
R = n-Pr: 97 % ee, 83 % yield
560
I I J Hydrolysis and Formation ofC-0 Bonds
Table 11.1-24. (cont.).

~ ~~

R = n-Bu: 95 % ee, 81 %yield
all CAL-B, toluene
R = Me: 97 % ee, 71 %yield, CAL-B, THF
R = Me: 98 % ee, 88 % yield, CAL-B, MeCN
Hop
- R2 R' R2
AcO ...,
R'
acylation spontaneous (6)(51
R' = R2 = H; R' = R2= Me; R' = Me, R2 = H;

R'= H, R2 = Me
78-86 % ee, 100 % yield, PCL, vinyl acetate
A$:o&++: OH OAc OH a: acylation
OH
a: acetate 290 % ee, 45 %yield, alcohol >90 % ee. 39 % yield, PCL, vinyl acetate
b: 98 % ee, 100 %yield, PCL, n-BuOH, CHzCl2
spontaneous (8) [71
76 % ee, >99 % conversion, PCL, vinyl acetate

79 % ee, 93 conversion, PSL, vinyl acetate
-
I I
H o e R ' C 0 , e acylation spontaneous ( 9 ) (81
at >40 "C
R' = Me, Et, R2 = COMe, COEt

>99 % ee, 100 %yield, CAL-B,
n-hexane/CHzClz
7 7. 7 Hydrolysis and Formation ofcarboxylid Acid Esters 1 561
Table 11.1-24. (cont.).

Reaction TYPe Racernization
OH OAc
RACN --- R
R = 3-PhOCsH4, Ph, 4-Cl-CsH4,
~ C N
acylation basic ion-exchange resin (10)[9]
~,~-OCHZO-CG 2-naphthyl,
H~, 1-naphthyl
70-96 % ee, 6 6 8 8 % yield, PSL, vinyl acetate
OH
- R’vS,~~
OAc
acylation silica gel
R’ = COzMe, BnOCH2, AcOCHr

R2 = n-Bu, Et3SiO(CH2)2,i-Pr, n-Octyl
87->95 % ee, 63-87 % yield, PFL,
vinyl acetate, t-BuOMe
,,+- SEt phs+oH hydrolysis N(n-C8H17)3
96 % ee, >99 % conversion, PCL,

HzO/toluene
n = 1: 97 % ee, 75 %yield, PCL, vinyl acetate

n = 2: 99 % ee, 67 % yield, PCL, vinyl acetate
OCOR
Mitsunobu inversion (14) [13]
R2/iR1
R = n-Pr, R’ = Me, R2 = n-C6H1:+

94 % ee, 100 % yield, PPL, vinyl propionate acylation
R = Me, R’ = Me, R2 = Ph
97 % ee, 97 % yield, PCL, vinyl acetate acylation
R = n-Pr, R’ = Ph, R2 = CH2NCOnPr,
97 % ee, 100 %yield, PCL, n-PrOH alcoholysis of
the (S)-acetate
R = Me, R’ = Aryl, R2 = CN alcoholysis of
61-97 % ee, 68-92 %yield, PCL, n-PrOH the (S)-acetate
562
Table 11.1-24. (cont.).

Reaction Type Racernization
Ph Ph
hydrolysis PdClz(MeCN)z (15) ~ 4 1
OAc
acylation (16)[151
Ph Ph\i
80 % ee, 76 % conversion, PFL, vinyl acetate, [Rh(cod)C1]2
ortho-phenanthroline, PhCOMe, KOH
98 % ee, GO % conversion, PFL, vinyl acetate, &(OAc)4
ortho-phenanthroline, PhCOMe
OAc
acylation A
Ph Ph\i
A
>99 % ee, 92 % yield, CAL-B, 4-C1-C6H40Ac,
PhCOMe, t-BuOH
OAc
Rlx",,-
R'
,lA
R
2
= 4-Br-C6H4, R2 = Me; R' = 1-Naphthyl, acylation A cf(17)

R2 = Me; R' = 2-naphthy1, RZ = Me; R' =
PhOCH2, R2 = Me; R' = c-C6H11,R2 = Me; R'
= n-C&13, RZ = Me; R' = Ph, R2 = E t
>98 % ee, 65-80 %yield, CAL-B,
4-CI-CsH40Ac, PhCOMe, t-BuOH
R' = 4-OMe-C&4, R2 = Me
91 % ee, GO % yield, CAL-B, 4-Cl-CsH4
acetate, PhCOMe, t-BuOH
R' = PhOCH2, R2 = CHiCl
79 % ee, 68 % yield, CAL-B, 4-C1-C6H40Ac,
PhCOMe, t-BuOH
Table 11.1-24. (cont.).
1 1 . 7 Hydrolysis and Formation of Corboxylid Acid Oters
I
563
Type Racemization
n = 1, 2: >99 % ee, 65 and 77 %veld, CAL-B,

4-C1-C6H40Ac,PhCOMe, t-BuOH
OH
rac/meso-mixhlre (R,R/meso)
(-5O:SO) (7G26100:O)
(3852 for X = CH2)
X = CH2, (CH2)2,(CH2)3,(E)-CH=CH,1,3-
C6H4, 1,4-C6H4,2,6-pyridylene,CH2N(Bn)CH2
>96->99 % ee, 47-90 % yield, CAL-B,
4-Cl-C6H40AC,tOlUene
R
OH
L C O , E t - R
OAc
i\/CO,Et acylation A, cf (17)
R = Ph: 95 % ee, 73 %yield

R = 4-OMe-C&: 99 % ee, 69 %yield
R = PhCH2: 96 % ee, 75 % yield
R = C-C~HII: 70 % ee, 71 %yield
all PCL, ~ - C ~ - C ~ H ~ cyclohexane
OAC,
alcoholysis Pd(PPh3)4dppf (23)[211

R
R = Ph, 4-cl-C~H4,4-Me-c~H4,2-Fu~1,
1-Naphthyl
97->99 % ee, 70-87 % ee, CAL or PCL,
GPrOH, THF
564

B
R = Ph, 4-Cl-C6H4,4-Me-C&4, 4-OMe-CsH4,
2-Fury1, 1-naphthyl, C-C~HII,t-Bu,
CHz-CHMe2,i-Pr, n-Pr
95->99 % ee, 8 4 9 1 %yield, PCL,
4-Cl-CsH40Ac,CHzClz
OH
PhAC02H
- OAc
Ph*CO,H
acylation racemase (25)~ 3 1
>98 % ee, 80 %yield, 1. PSL, vinyl acetate,

2. mandelate racemase
1 J. 2. Crich, R. Brieva, P. Marquart, R.-L- Gu, 11 D. S . Tan, M. M. Giinter, D. G. Drueckhammer,
S. Flemming, C. J. Sih, J. Org. Chem. 1993, 58, J. Am. Chem. SOC. 1995, 117,9093.
3252. 12 T. Taniguchi, R. M. Kanada, K. Ogasawara,
2 R.-L. Gu, 1:s. Lee, C. J. Sih, Tetrahedron Lett. 1992, Tetrahedron: Asymmetry 1997, 8, 2773.
33, 1953. 13 E. Vanttinen, L. T. Kanerva, Tetrahedron:
3 N. J. Turner, J. R. Winterman, R. McCague, Asymmetry 19956,1779.
J. S. Parratt, S. J. C. Taylor, Tetrahedron Lett. 1995, 14 J. V. Allen, J. M. J. Williams, Tetrahedron Lett. 1996,
36, 1113. 37, 1859.
4 S. A. Brown, M.-C. Parker, N. A. Turner, 15 P. M. Dinh, J . A . Howarth, A. R. Hudnott,
Tetrahedron: Asymmetry 2000, 11,1687. J. M. J. Williams, W. Harris, Tetrahedron Lett. 1996,
5 J. W. J, F. Thuring, A. J. H. Klunder, G. H. L. 37, 7623.
Nefkens. M. A. Wegman, B. Zwanenburg, 16 A. L. E. Larsson, B. A. Persson, J:E. Backvall,
Tetrahedron Lett. 1996, 37,4759. Angew. Chem. 1997, 109,1256; Angew. Chem. lnt.
6 J. W. J. F. Thuring, G. H. L. Nefkens, Ed. Engl. 1997, 36, 1211.
M. A. Wegman, A. J. H. Klunder, B. Zwanenburg, 17 B. A. Persson, A. L. E. Larsson, M. Le Ray, J.-E.
J. Org. Chem. 1996, 61, 6931. Backval1.J. Am. Chem. SOC.1999, 121, 1654.
7 M. van den Heuvel, A. D. Cuiper, H. van der 18 B. A. Persson, F. F. Huerta, J:E. Backval1.J. Org.
Deen, R. M. Kellog, B. L. Feringa, Tetrahedron Lett. Chem. 1999,64,5237.
1997, 38. 1655. 19 F. F. Huerta, Y R. S. Laxmi, J.-E. Backvall, Org.
8 A. D. Cuiper, M. L. C. E. Kouwijzer, P. D. J . Letters 2000, 2, 1037.
Grootenhuis, R. M. Kellog, B. L. Feringa, J. Org. 20 F. F. Huerta, J:E. Backvall, Org. Letters 2001, 3,
Chem. 1999,64,9529. 1209.
9 a) M. Inagaki, J. Hiratake, J , Oda,J. Am. Chem. 21 Y. K. Choi, 1. H. Suh, D. Lee, I.T. Lim, 1.Y. lung,
SOC. 1991, 113,9360 b) M. Inagaki, J. Hiratake, M:J. Kim,J. Org. Chem. 1999, 64, 8423.
J. Oda. j.Org. Chem. 1992, 57, 5643. 22 D. Lee, E.A. Huh, M.-J. Kim, H. M. Jung,
10 S. Brand, M. F. Jones, C. M. Raper, Tetrahedron J. H. Koh, J. Park, Org. Letters 2000, 2, 2377.
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1999, 10,4079.
I 565
shown for (4) and (5). The hemiacetal and hemiaminal derivatives formed by the
reactions (6)-(9) have been obtained by lipase-catalyzed acylation of the configura-
tionally unstable hemiacetals or-aminal, respectively, or by alcoholysis of an acylated
hemiacetal as shown for (7).The latter case is not a dynamic kinetic resolution but a
normal kinetic resolution (acylation) followed by a spontaneous epimerization by
lipase-catalyzed alcoholysis. Remarkably, the non-acylated hemiaminals are config-
urationally stable at temperatures below 40 "C and dynamic kinetic resolution
proceeds according to reaction (9) at higher temperatures. Cyanohydrins are unsta-
ble under basic conditions regenerating the starting materials aldehyde and hydro-
gen cyanide. This property was used to prepare enantiomerically enriched cyanohy-
drin acetates according to transformation (10) by reaction of the corresponding
aldehydes with acetone cyanohydrin in the presence of vinyl acetate, Pseudomonas sp.
lipase and a strong basic ion-exchange resin as racemizing catalysts. The latter
transformation demonstrates that lipase-catalyzed-acylationof one of the cyanohy-
drin enantiomers is faster than conversion of the cyanohydrin into aldehyde and
hydrogen cyanide.
The 2-acetoxysulfides are obtained according reaction (11)by in situ formation of
the configurationally unstable hemithioacetals and subsequent lipase-catalyzed
acylation. Racemization of the hemithioacetals was achieved by silica gel. The
2-phenylthiocarboxylicacid was formed by hydrolysis of the corresponding thioester
in the presence of trioctylamine [reaction (141.
The formation of the 2-acetoxy ketone in the reaction (13)was achieved by shifting
an enediol-hydroxyketoneequilibrium with triethylamine.
The formation of the enantiomericallyenriched or pure esters by reaction (14)was
not a result of a dynamic process but a one-pot two-step procedure consisting of
lipase-catalyzedresolution and a subsequent inversion of the slow reacting enantio-
meric alcohol by Mitsunobu reaction.
Enantiomerically pure allylic alcohols were prepared by a dynamic kinetic resolu-
tion in the transformations (15) and (23)by lipase-catalyzedhydrolysis or alcoholysis
of the corresponding acetates in the presence of palladium complexes racemizing
the slow reacting enantiomeric acetates. 1-Phenylethanol was converted in an
acylation process into the corresponding acetate in reaction (16) by two different
types of rhodium catalysts. On the other hand, 1-phenylethanoland a variety of the
further secondary alcohols were obtained with high enantiomeric excess under in
situ racemization with the ruthenium catalystA with very high efficiency [reactions
(17)-(19)].Moreover, this catalyst was used also for the racemization/epimerization
procedure (20) converting an approximately 1:1 mixture of racernic/rneso-diols into
the corresponding enantiomeric diacetates under consumption of the meso-diol as
well as for the dynamic kinetic resolution of 2- and 3-hydroxy carboxylic esters as
shown in the reactions (21) and (22), respectively, under acylation conditions. The
ruthenium catalyst was not compatible with vinyl acetate and therefore, 4-chloro-
phenyl acetate was found to be the acylating agent of choice. A redox process can
explain the racemization of the slow reacting enantiomeric alcohol.
Another ruthenium catalyst was used for the dynamic kinetic resolution of allylic
alcohols [reaction (24)]by acylation yielding allylic acetates. Again a redox process
should be responsible for the racemization.
566
Table 11.1-25. The beneficial influence of additives on lipase-catalyzed enantiorner- and

enantiotopos-differentiatingreactions (PCL Pseudomonas cepacia lipase, CCL Candida cylindracea
lipase, CAL Candida antarctica lipase, not specified, LIP Pseudomonos sp. lipase-Toyobo,PFL
Pseudomoasjuorescens lipase, CAL-B Candida antarctica B lipase, M M L Mucor rniehei lipase).
Product(s) Additive Influence ofthe additive
HO
I
AcO
pancreatin, AcOCH2CC13, THF NEt3 reaction rate
no reaction without NEts
pancreatin, vinyl acetate, no further solvent NEt3 selectivity
from 72 to >99 % ee
RY O T s ' Y 0 T . s NEt3 selectivity 2 [21

OAC OH
PCL, vinyl acetate, t-BuOMe
R = Vinyl from E = 50 to >200
R = CH2Cl:from E = 39 to > 200
R = Et: from E = 7 to 60
Me0 OMe Me0 OMe
2,6-lutidine selectivity 3 [31

or KHC03
AcO OH
CCL, Ac20, toluene
from E = 19 to 180with 2,6-lutidine
from E = 19 to 240 with KHCO3
NEt3 selectivity 4 [41
CAL, vinyl acetate, cyclohexane
NEt, reaction rate 5 [51
OH
LIP, vinyl acetate, THF
from 10 d to 3 h
Table 11.1-25. (cont.).
1 1 . 7 Hydrolysis and Formation OfCarboXylid Acid Esters
I 567
Produdlsl Additive Influence ofthe additive
NEt3 selectivity 161
AcO
PFL on sawdust, vinyl acetate, THF
Me0 NEb selectivity 7 [71
OH OAc
PCL on Hyflo Super Cell@,vinyl acetate,
t-BuOMe, from 85 to 93 % ee
(CH,),-OH (CH,),-OAc
NEt, selectivity and 8 [81

reaction rate
HO-,(H,C) AcO-,(H,C)
absolute configuration
unknown
CAL-B,vinyl acetate, THF
RxH
NEt3 selectivity and 9 191

reaction rate
Ph1 0
MML or CAL-B, R'OH, n-hexane or toluene
R = t-Bu, R' = n-Bu: from 80 to >99 % ee
OAr OAr Dextromethor- selectivity and 10 [lo]

phan (DM) or reaction rate
AC02H ACO,Me its enantiomer
Levomethor-
phan (LM)
Me
Table 11.1-25. (cont.).

CCL, phosphate buffer pH 7
Ar: 2-Me-4-Cl-C~Hj: from E = 1 to 37 with DM
and to E = 81 with LM
OH OAc Dextromethor- selectivity 11 [ll]

phan (cf 10) 01
EtL C N Et/\/CN (2S)-2-amino-
4-methylthio-
1-butanol
PCL, aqueous buffer, pH 7.2
OH
Ph Ph-
: / Cf3 U
selectivity 12 [12]
PCL, vinyl alkanoates, n-hexane or i-PrzO
cr3
OAc selectivity and 13 [13]
reaction rate
RE C N R&CN U
and further
crown ethers
PCL, acetone/water
Triton X-100 selectivity 14 1141
CCL, aqueous buffer pH 7.2
CaClz selectivity 15 [ l S ]
nC8H,,AC0,H nC8H,,n
,\lC8H7
,
CCL, aqueous buffer pH 8
NaCl selectivity 16 [16]
PCL, HzO
Table 11.1-25. (cont.).
J1.l Hydrolysis and Formation ofcarboxylid Acid Esterz
I 569
H
0
"'A
C0,nBu aqueous LiCl selectivity a n d 17 [17]
reaction rate
I I
X X
CCL, n-BuOH, i-PrzO
X = Et: from E = 3.8 to 201
X = CF,: from E = 1.3 to 56
1 a) F. Theil, S. Ballschuh, H. Schick, M. Haupt, Brown, M.-C. Parker, N. A. Turner, Tetrahedron:
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In reaction (25) racemization was realized by madelate racemase. However, this

transformation is still a process carried out in two batches and therefore, not a
dynamic kinetic resolution but certainly the starting point for further investigations
by combining a lipase- and a second enzyme-catalyzed reaction in order to perform
real dynamic kinetic resolution.
Enhancement of Selectivity and Reactivity of Lipases by Additives

11.1 2 . 1 . 3
It has been shown that additives have a great potential for fine-tuning the reaction
conditions for lipase-catalyzed reactions (1331. Certain additives such as tertiary
amines, thiacrown ethers or inorganic salts may increase the selectivity and/or
reaction rate. The reason for these effects are little understood and only a few
systematic investigationshave been undertaken. From a synthetic chemist's point of
570 I 1 1 Hydrolysis and Formation of C - 0 Bonds
view treatment of the reaction mixture with an additive is a convenient way to

improve the outcome of the reaction.
Table 11.1-25 list examples in which certain additives have an unambiguous
beneficial influence on selectivity and/or reaction rate. The enantiomerically en-
riched or pure compounds 1-9 have been prepared under the influence of mainly
triethylamine or other bases. In case of 1 for the acetylation with 2,2,2-trichloroethyl
acetate there was no reaction without triethylamine. For the formation of 5 the
reaction time was shortened dramatically from ten days to three hours for 100% of
conversion. In most cases there is no rationale for the effects of bases except the
formation of ion-pairs between the added bases and traces of acids present in the
reaction mixture. Only for the synthesis of 9 a systematic investigations demon-
strates that triethylamine besides its racemizing properties (cf. Table 11.1-24)has a
significant influence on the water activity of the reacting mixture['38].In other cases,
triethylamine has been used as an additive without comparing its influence with the
results in its
Table 11.1-26. Subtilisin-catalyzedacylation of racernic alcohols in organic solvents.
R ,r Me
R = Et, VS/VR = 3.9, dioxane

PI
1
R = nBu, E = 28 2
R = (CH2)zCH=CMe2,E = 11 3
R = nHex, VS/VR = 100, dioxane 4
R = nDec, E = 100 5
R = Ph, VS/VR = 50, dioxane 6
R = 2-naphthyl, VS/VR = 58, 7
dioxane
vinylbutyrate, vinylacetate
Me=Me
8a [2]
Me
u L e 8b [2]
298% ee 54% ee
vinylacetate
OH
9b PI
WMe
\
40% ee
9a [21
92% ee
30% conversion 30% conversion
vinylacetate
1 P. A. Fitzpatrick, A. M. Klibanov,J. Am. Chem. SOC. 2 Y.-F. Wang, K. Yakovlevsky, B. Zhang,
1991,131,3166. A. L. Margolin,/. Org. Chem. 1997, 62, 3488.
I
571
The enantiomerically pure tertiary bases dextro- and levomethorphan were used
for the preparation of 2-aryloxy propionic acid derivatives 10 by increasing the
selectivity dramatically based on the enantioselectiveinhibition of the slow reacting
enantiomer.
The kinetic resolutions yielding the enantiomers 12 and 13 were conducted in the
presence of thiacrown and some further crown ethers. Inorganic salts as shown for
15-17 were suitable modulators of selectivity and/or reaction rate. Particularly, in
case of 17 a strong increase of the enantiomer-selectivitywas found by the addition of
a defined amount of aqueous lithium chloride solution to the reaction mixture. The
E value was increased by factors between ten to fifty depending on the substrate
structure.
11.1.1.2.2 Subtilisin
A beneficial feature of subtilisin, and in particular subtilisin-CLECs, is their high
catalytic activity in polar and non-polar organic solvents, allowing for transesterifica-
tions of alcohols in the presence of small amounts of water. Transesterifications
catalyzed by subtilisin were mostly done with vinyl acetate. Apparently, the acet-
aldehyde formed during transesterification is not harmful to the enzyme as it is in
the case of some lipases and pig liver esterase. Although resolution of such alcohols
either through hydrolysis of the corresponding esters or transesterification is the
domain of lipase, in some cases useful selectivities were achieved with subtilisin
(1-9) (Table 11.1-26).
11.1.1 2 . 3 Pig Liver Esterase

Pig liver esterase-catalyzedenantioselectiveacylation of prochiral or racemic alcohols
in organic solvents has not nearly gained the importance of the lipase-catalyzed
acylation method. This is due to the fact that pig liver esterase shows only very low
activity in organic solvents. The esterase differs considerably in this respect from
lipases and subtilisin, which are both highly active in organic media. Attempts to
confer activity to pig liver esterase in organic solvents by entrapment in water-filled
porous supports 11391, covalent attachment of MPEG residues [140, 14'] , immobiliza-
'
tion on and E ~ p e r g i t [ ' ~'@I,~ , or entrapment in polymers were

met with various degrees of success. It was found, however, that colyophylizationof
pig liver esterase with MPEG significantly enhances the activity and stability of the
enzyme in organic solvents of low to medium polarity and low water con-
tent[", 14', 147j. The colyophilizate of pig liver esterase and MPEG was successfilly
applied to the kinetic resolution of racemic glycerol derivatives through acylation
with vinyl and isopropenyl esters in toluene containing less than 1% water (1, 3,and
5) (Table 11.1-27).Medium selectivities were recorded for alcohols having a primary
hydroxyl group, and a high selectivity was found in the case of the glycerol derivative
with a secondary hydroxyl group. Pig liver esterase-catalyzed hydrolysis of the
corresponding racemic esters in water occurred with lower selectivities (2, 4 and 6 ) .
A number of functionalized secondary alcohols have been resolved with high
572
Table 11.1-27. Pig liver esterase-catalyzed acylation of racemic alcohols in organic solvents
E = 24
vinyl propionate,
toluene
E = 3-5
hydrolysis in
water
E = 30
vinyl propionate,
toluene
E=Z
hydrolysis in
water
E >lo0
vinyl propionate,
toluene
E=l
hydrolysis in
water
I 573
Table 11.1-27. (cont.).
OCOEt y 7b [1,2]
M e O A P h 7a [ l , 21 M e O w P h
E = 100
vinyl propionate,
toluene
E >lo0
vinyl propionate,
toluene
E = 50
vinyl propionate,
octane
OCOEt
10b [ l , 21
E t S A P h
E >lo0
vinyl propionate,
toluene
OCOEt
l l b [ l ,21
F A P h l l a [l,21 F A P h
E >lo0
vinyl propionate,
toluene
OCOEt
12b 11.21
F A P h 12a [ l , 21 F L P h
E = 50
vinyl propionate,
toluene
OCOEt y
13a [ l , 21 &OPh 13b [ l , 21
&OPh
Me Me
E-100
vinyl propionate,
toluene
574 I Table 11.1-27. (cont.).
OCOEt OH
14a [ l , 21 14b [ l , 21
M e O h O P h MeO-OPh
E >lo0
vinyl propionate,
toluene
1 H:J. Gais, M. lungen, V. ladhav,J. Org. Chem. 2 M. Jungen, H:]. Gais, Tetrahedron: Asyrnrnstry
2001,66,3384. 1999,10,3747.
selectivity by pig liver esterase-catalyzed acylation with vinyl propionate in toluene.

As in the case of lipases, a competing pig liver esterase-catalyzedhydrolysis of vinyl
propionate and a partial deactivation of the enzyme by the acetaldehyde formed in
transesterification had been observed. Critical to the activity and selectivity of pig
liver esterase in the presence of MPEG in organic solvents is the water content of the
system, which should be lower than 1 %. In general, the activity of pig liver esterase
in the presence of MPEG in organic solvents is lower than that of lipases and
subtilisin under comparable conditions. The colyophilizate of pig liver esterase and
MPEG can be recovered from organic media with a minor loss of activity through a
spontaneous immobilization on an ultrafiltration membrane placed in the reaction
mixture r6'1.
Acknowledgement
The authors thank Carsten Griebel and Gabriele Bertrand for their help in the
preparation of the manuscript.
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1 1.2 Hydrolysis offpoxides I 579
11.2
Hydrolysis of Epoxides
Kurt Faber and Romano V A. Orru
Chiral epoxides and 1,2-diols,which are central building blocks for the asymmetric
synthesis of bioactive compounds, can be obtained via the asymmetric hydrolysis of
epoxides using enzymes - i.e. epoxide hydrolases (EHs) [EC 3.3.2.Xl. Enzymes from
mammalian sources - such as rat liver tissue - have been investigated in great detail
for several decades during detoxification studies L1]; however, their application for
biotransformations on a preparative scale was hampered because of the limited
supply of these enzymes, and, as a consequence, the examples reported rarely
surpass the millimolar range[*, 1’. During the past few years, highly selective epoxide
hydrolases were identified from a wide range of microbial sources, which allows for
an (almost) unlimited supply of these enzymes for preparative-scale applications.
These valuable biocatalysts have recently gained considerable attention, and their
scope and limitations have been reviewed[”*]. Microbial epoxide hydrolases were
found to be more abundant than previously expected, and numerous sources,
predominantly among bacteria, fungi and (red) yeasts are known to date.
The mechanism of enzymatic hydrolysis of epoxides can be compared to that of
base-catalysis, i.e. it resembles an SNZ-type opening of the epoxide by the nucleo-
phile (i.e. water), which leads to the formation of the corresponding trans-con-
figurated 1,2-diol. Any chiral center present in the substrate oxirane can be
“recognized”, thus effecting kinetic resolution or asymmetrization of racemic or
meso-epoxides, respectively. The data available to date indicate that the enantiose-
lectivities of enzymes from certain microbial sources can be correlated to the
substitutional pattern of various types of substrates: red yeasts (Rhodotorula or
Rhodosporidiurn sp.) give best enantioselectivities with monosubstituted oxiranes;
hngal cells (e.g. from Aspergillus and Beauveria sp.) are best suited for styrene oxide-
type substrates, whereas bacterial enzymes (in particular from Actinornycetessuch as
Rhodococcus and Nocardia sp.) are the biocatalysts of choice for more highly
substituted 2,2- and 2,3-substituted epoxides. In order to overcome the disadvantage
of the classic kinetic resolution pattern, i.e. the formation of two enantiomers in
each 50 % yield, various deracemization methods based on chemo-enzymatic or
purely enzymatic protocols have been developed. The latter led to the highly
desirable formation of a single stereoisomer of the diol in 100% theoretical yield.
The synthetic potential of epoxide hydrolases for asymmetric synthesis has been
proven by the preparation of a number of bioactive compounds.
Chiral epoxides and vicinal diols (employedas their corresponding cyclic sulfate or
sulfite esters as reactive intermediates) are extensively employed high-value inter-
mediates in the synthesis of chiral compounds because of their ability to react with a
broad variety of nucleophiles (Figs. 11.2-1 and 11.2-2). In recent years, extensive
efforts have been devoted to the development of chemo-catalytic methods for their
production['^ “1. Thus, the Sharpless methods allowing for the asymmetric epoxida-
580
I Figure 11.2-1.
R' SR' R
NHR' nucleophiles.
R -4 N3
R
/q N3
RA
OH
H 3
\
Rl-0-
OH
11 OR'
OH
tion of allylic alcohols [''I and the asymmetric dihydroxylationof alkenes [lo]are now
widely applied reliable procedures. In addition, asymmetric catalysts for the epoxida-
tion of non-functionalized olefins [l2-l4]have been developed more recently. Al-
though high stereoselectivityhas been achieved for the epoxidation of cis-alkenes,the
results obtained with trans- and terminal olefins were less satisfactory using the
latter method. More recently, two highly selective methods for the opening of
terminal mono- and 2,2-disubstitutedepoxides have been published. These methods
are both based on a kinetic resolution using cobalt-salen complexes and water[15]or
chromium-salen complexes and azide respectively and have great potential in
asymmetric synthesis [171.
On the other hand, a number of biocatalytic methods have been reported to
provide a useful arsenal of methods as valuable alternatives to the above-mentioned
techniques [18-231. Prochiral or racemic synthetic precursors of epoxides, such as
halohydrins, can be asymmetrized or resolved using hydrolytic enzymes [24, 251. In
particular, esterases and lipases have been used for such a enantioselective ester
hydrolysis or esterification. This methodology is well developed, and high selectiv-
ities have been achieved in particular for esters of secondary alcohols, but it is
impeded by the requirement of regioisomerically pure halohydrins. Furthermore, it
is known that a-haloacid dehalogenases catalyze the SN2-displacementof a halogen
atom at the a-position of carboxylic acids with a hydroxy function. This process leads
to the formation of the corresponding a-hydroxy acid with inversion of configura-
tion[26].However, a-haloacid dehalogenation incurs two drawbacks: (i)the instability
of the substrates, particularly the a-bromoacids, in aqueous systems, and (ii) the
limited substrate tolerance, as only short-chain haloacids are accepted [271. Asym-
metric biocatalyhc reduction of a-keto-acids12'] using D- or L-lactate dehydrogenase
or a-keto-alcohols[291 by glycerol dehydrogenase provides access to chiral a-hydrox-
yacids or 1,2-diols,which can be converted into the corresponding epoxides using
conventional chemical methodology. Although excellent selectivities are generally
11.2 Hydrolysis of Epoxides
I 581
r OH \
I
R u 0 4 cat TosCVPy
NalO
L 0
U,O
,x5
Figure 11.2-2. Syntheses from chiral 1,2-diols.
achieved, the need for the recycling of redox-cofactors such as NAD(P)H has
restricted the number of applications. Likewise, biocatalytic asymmetric epoxidation
of alkenes catalyzed by mono-oxygenasescannot be performed on a preparative scale
with isolated enzymes, because of their complex nature and their dependence on a
redox cofactor such as NAD(P)H. Thus, whole microbial cells have to be used
instead. This method is not trivial and requires high bioengineering skills [301. On the
other hand, haloperoxidases are independent of nicotinamide-cofactors, as they
produce hypohalous acid from H202 and halide, which in turn yields a halohydrin
from an alkene. These enzymes are rare in Nature and exhibit usually low
selectivities due to the fact that the formation of halohydrins can take place not only
in the active site of the enzyme but also without enzyme Similar low
selectivities have been observed with halohydrin epoxidases, which act like a
"biogenic chiral base" by converting a halohydrin into the corresponding e p o ~ i d e [ ~ ~ ] .
On the other hand, peroxidases, such as chloroperoxidase (CPO), are cofactor-
independent and can be used in isolated form for the enzymatic epoxidation of
alkenes [33-351.
An attractive alternative to the methods mentioned above is the use of cofactor-
independent epoxide hydrolases, which are readily available from microbial sources
in sufficient quantities.
11.2.1
Epoxide Hydrolases in Nature
In eukaryotes,microsomal and cytosolic epoxide hydrolases mainly play a key role in

the detoxification of mutagenic, poisonous and carcinogenic epoxidesf3', 371, which
are formed by the action of P4so-dependentmonooxygenases[381. In addition, they are
involved in the biosynthesis of hormones (e.g. leukotrienes). In plants, epoxide
hydrolases are responsible for the generation of chiral aroma compounds and, in the
biosynthesis of cutin, a wax-type polyester, which protects plants against microbial
attack[3']. Insect epoxide hydrolases degrade juvenile hormones and pheromones
bearing an oxirane moiety L31. On the other hand, in microorganisms these enzymes
582
are multi-functional: (i) they can function as detoxifylng agents, (ii) they can play a
role in biosynthetic routes of complex (secondary)metabolites, or (iii) they may be
crucial for the degradation of epoxides during the metabolism of alkenes and
aromatics I4OI.
The degradation of aromatics in eukaryotes occurs via two different pathways
(Fig. 11.2-3):(i) dioxygenase-catalyzedcycloaddition of molecular oxygen to the C=C
bond yields a (putative) dioxetane species, which is then detoxified via reductive
cleavage of the 0-0 bond yielding a physiologically more innocuous cis-1,2-diol; (ii)
The formation of a highly reactive arene oxide via the introduction of a single 0 atom
(from molecular oxygen) into the aromatic system is catalyzed by a mono-oxygenase.
The latter epoxy species is further metabolized via hydrolysis catalyzed by an epoxide
hydrolase to yield a transb1,2-diol.
In lower organisms, alkenes can be metabolized in an analogous fashion, i. e. via
an epoxide intermediate. In an analogous fashion, this intermediate is hydrolyzed to
the corresponding 1,2-diolby an epoxide hydrolase. The latter product is degraded
either by oxidation or by elimination of water under catalysis of a diol dehydratase,
yielding an aldehyde [411. Alternatively, such aldehydes are obtained via direct
rearrangement of the epoxide catalyzed by an epoxide i~omerase[~'].
For a long time it was generally assumed that epoxide hydrolases are predom-
inantly found in mammals[" 1', although epoxide hydrolase activities had been
detected in bacteria[43.441 or fungi i4', 461 quite some years ago. This early view was
certainly too simplistic, and enzymes of this type have now been detected in many
bacteria [47-491, fungi and red yeasts ['*I. Moreover, epoxide hydrolase activity has
been demonstrated in plants ['*Iand insects FS31.
11.2.1.1
Isolation and Characterization o f Epoxide Hydrolases
Several membrane-bound and soluble epoxide hydrolases from mammalian origin

have been purified and (at least partially) sequenced. Some of them have also been
cloned and overexpressed, which is the case for the soluble EH from rat liver which
has been overexpressed in Escherichia "I. This enzyme (as well as its
c0li['~2
microsomal analog) was shown to share an amino acid sequence similarity to a

region around the active center of a bacterial haloalkane dehalogena~e['~]], an
enzyme with known three-dimensional structure that belongs to the a/ P-hydrolase
f0ld-family['~1.Rat soluble EH forms a dimer from two complete structural mono-
meric units, both possessing a distinct active site. The EH activity is known to be
located close to the C-terminal unit, while the function of the N-terminal unit
remains u n k n o ~ n [ ~ ~ l .
To date, several epoxide hydrolases from microbial sources have been purified. For
instance, from Bacillus megaterium La], Corynebacterium sp. 471 and Pseudomonas
but also from dematiaceous fungi such as Ulocladium atrum and Zopfcella
sp. [48, 6ol,
karachiensis["I. However, some of these were only partially purified, or their
enantioselectivitieswere low or not investigated. In contrast, highly enantioselective
epoxide hydrolases from Rhodococcus sp. NCIMB lK!lC~[~'land Nocardia sp. EH1 ["I
11.2 Hydrolysis ofEpoxides I
583
bk Oxygenase dioxetane
Rkx: cis -diol
-
further
u:
+
metabolism
R R
Epoxide
Oxygenase Hydrolase
f * -
H20
arene-oxide trans -diol
1
OH
Epoxide
Hydrolase
I RLoH
Diol-
-
H20 H20 4 D e h y d r a t a s e
Mono-
Oxygenase
R
/= PI
~ further
metabolism
Epoxide
p lsponf.
Isomerase
R T o
H
Figure 11.2-3. involvement of epoxide hydrolases in the biodegradation
of aromatics and alkenes.
were purified to homogeneity. Both (monomeric) proteins exhibit several common

features: they are of similar size (= 34 kDa) and do not possess any metal ion or any
UV-absorbing prosthetic group. The catalyhc power of both enzymes was found to be
in the same range (=ZOO0pmol mg-' h-'), as was the optimum temperature
(33-37 "C) and pH (7.5-9.0). The only notable difference between the two enzymes
is the high instability of the Nocardia epoxide hydrolase (which completely loses its
activity within hours, even when stored at -18 "C), whereas the Rhodococcus enzyme
was shown to be relatively stable. It should be noted that the former enzyme could be
stabilized by immobilization through ionic binding onto DEAE-cellulose. This
resulted in a doubling of the activity (compared to the native enzyme) albeit at a
584
I slight reduction in enantioselectivity
["I. The poor stability of the Nocardia enzyme
and the fact that the N-terminus of the Rhodococcus epoxide hydrolase was un-
specifically blocked precluded their N-terminal sequencing. In contrast, an epi-
chlorohydrin-degrading epoxide hydrolase from Agrobacterium radiobacter AD1
could be isolated, characterized and sequenced after cloning and overexpression in
E. coli BL21 (DE3)["]. This enzyme showed an amino acid sequence similarity to
eukaryotic epoxide hydrolases, haloalkane dehalogenase and bromoperoxidase,
which indicated that it belongs to the a/P-hydrolase fold family. Most epoxide
hydrolases from pro- or eukaryotic sources seem to belong to this group of enzymes.
Another bacterial epoxide hydrolase from this family has been isolated from
Corynebacterium sp. C12 when grown on cyclohexene oxide["]. The purification to
homogeneity was achieved in two steps. The enzyme is (partly) membrane bound
and multimeric (probably tetrameric) which is in contrast to the enzymes described
above. The subunit-size is ca. 32 kDa and amino acid sequence comparison showed
that it is related to mammalian and plant (soluble) EH. Furthermore, it showed
striking similarilties with the Agrobacterium enzyme, particularly around the cata-
lytic site. Epoxide hydrolases from fungal sources were purified recently: an epoxide
hydrolase from Aspergillus niger was purified to homogeneity["] and appears to be a
tetramer composed of four identical subunits of molecular mass 45 kDa. The N-
terminus was blocked, the pH optimum lies at 7.0 and the temperature optimum at
~ ~ ] Rhodosporidium toruloides
40 "C. From red yeasts (Rhodotorula g l ~ t i n i s [ and
CBS0349 [68]), two epoxide hydrolases have been purified. Both membrane-bound
enzymes are medium-sized (45 and 54 kDa, respectively)and are structurally related
to other microsomal epoxide hydrolases. They probably belong to the a/ P-hydrolase
fold family as well. An epoxide hydrolase with an unprecedented low molecular
mass (only 17 kDa) was isolated from Rhodococcus erythropolis DCL14[69].The
cofactor-independent enzyme is efficiently induced when the microorganism was
grown on monoterpenes, such as limonene, reflecting its special role in the
limonene degradation pathway. The low molecular mass, the unusually broad pH-
optimum (6.0-11.0) and the elevated optimum temperature (50 "C), together with
the fact that the N-terminal amino acid sequence revealed no homology with any
other protein, led to the conclusion that this protein does not belong to the alp-
hydrolase fold family.
11.2.1.2
Structure and Mechanism of Epoxide Hydrolases
The first X-ray structure of an epoxide hydrolase (from Agrobacterium radiobacter

AD1) has been reported recently (Fig. 11.2-4)f7O1. The nearly globular protein
consists of a core-domain with typical features of a/P-hydrolase fold enzymes and a
so-called "cap-domain",which is located on top of the core domain.
All epoxide hydrolases known to date require neither any prosthetic group nor a
metal ion, and the mechanism by which these enzymes operate was long debated.
Formerly, it was assumed that a direct nucleophilic opening of the oxirane ring by a
histidine-activatedwater molecule would be the key step C7l1. However, convincing
17.2 Hydrolysis ofEpoxides I
585
il
Figure 11.2-4. X-Ray structure o f Agrobacterium radiobacter epoxide hydrolase (PDB-1EHY).
The catalytic residues (Asp107 and His275) are located on top o f t h e core-domain: at some
distance Asp246 is shown, which is presumably involved in proton transfer. The a-helices
at top left constitute the “cap-domain”, which is covering the active site.
evidence was later provided which showed that the reaction occurs via a covalent
glycol-monoester-enzymeintermediate[72,731 (Fig. 11.2-5). For the Agrobacterium
enzyme, the proposed active-site residues (Asp107 and His275) are located in the
predominantly hydrophobic internal cavity between the core- and cap-do-
mains[64,701. Furthermore, a tunnel filled with water molecules has been located,
which leads to the back of the active site cavity. It is perfectly suited to deliver the
catalytic water molecule within hydrogen-bonding distance to His275. Since the
water is positioned at the back, the epoxide probably enters the active site from the
front. In addition, Asp246 has been proposed as the third member of the catalytic
triad (not shown in Fig. 11.2-S), since replacement of AsplO7, His275 and Asp246
resulted in a dramatic loss of activity[“].
586
cyHO
H H OH
’glycol-monoester
intermediate’
T T
o/ A0-
.. ..
H H H
‘alkyl-enzyme intermediate’
Figure 11.2-5. Schematic representation of the mechanism of epoxide hydrolase
and of haloalkane dehalogenase.
Structure and mechanism show striking similarities to that of haloalkane dehalo-

genase from Xanthobacter autotrophicus (whose structure and mechanism has been
substantiated by X-ray crystallography)[74, 781. Both enzymes have an Asp-His-Asp
catalytic triad which superimpose very well, their side chains point in a similar
relative direction and they form analogous hydrogen bonds. In haloalkane dehaloge-
nase, it has been shown that a halide is displaced from the substrate by an aspartate
residue via a nucleophilic attack, thus leading to an “alkyl-enzyme intermediate”
which is further hydrolyzed in a second step[75,761. Similar mechanisms have been
proposed for other epoxide hydrolases belonging to the a/P-hydrolase fold fam-
ily[% 72, 771
A consequence of the above-mentioned mechanism is a trans-specific opening of
the epoxide with one oxygen from water being incorporated into the product diol[60].
For instance, (+)-trans-epoxysuccinatewas converted into meso- (not D/L-) tartrate by
an epoxide hydrolase isolated from Pseudomonas putida[601. In a complementary
fashion, cis-meso-epoxysuccinategave D- and L-tartrate (with a Rhodococcus sp.) albeit
in low optical purity[’’]. The fact that only one 0-atom originates from water was
proven by 180-labelling experiments using bacterial, fungal i80] and mammalian
epoxide hydrolases I8l]. Although two cases for reactions proceeding via a formal cis-
7 7.2 Hydrolysis of Epoxides
I 587
4" S
"3,
k
rac
L Inversion
+
R R
(S) -Enantiomer reacts faster R R!
Figure 11.2-6. Microbial hydrolysis o f epoxides proceeding with retention or inversion
of configuration.
hydration process have been reported in the mid-1970s[**.831, they seem to be rare
exceptions and - given the present knowledge of the enzyme mechanism - attempts
to explain this phenomenon remain rather speculative[83].It is interesting to note
that several P-glycosidases act via formation of a covalent glycosyl-enzyme inter-
mediate by retaining the configuration at the anomeric centre[84].This suggests that
these enzymes may also be mechanisticallyrelated to epoxide hydrolases.
The above-mentioned facts have important consequences for the stereochemical
outcome of the kinetic resolution of asymmetrically substituted epoxides. In the
majority of enzymatic transformations following a kinetic resolution pattern (e.g. by
ester hydrolysis and synthesis using lipases, esterases and proteases) the absolute
configuration at the stereogenic centre(s) always remains the same throughout the
reaction, since it is not directly involved in the reaction. In contrast, the enzymatic
hydrolysis of epoxides may take place via attack on either carbon of the oxirane ring
(Fig. 11.2-6) and it is the structure of the substrate and of the enzyme which
determine the regioselectivity of the process 8s-891 . A s a consequence, the
absolute configuration of both the product and the substrate from a kinetic resolution
of a racemic epoxide has to be determined in order to elucidate the stereochemical
pathway. To facilitate the determination of this regioselectivity, a mathematical
approach has been suggested, which only necessitates the study of the biohydrolysis
of the racemic mixture ["I.
11.2.1.3
Screening for Microbial Epoxide Hydrolases
In spite of the considerablevalue of epoxide hydrolases for fine chemical synthesis, it

was only recently that a detailed search for epoxide hydrolases from microbial
~ ~ Faber[23s79, 911, bearing
sources was undertaken by the groups of F ~ r s t o s s [ ~and
in mind that the use of microbial enzymes allows an (almost) unlimited supply of
biocatalyst. The screening was based along the following considerations: on the one
hand, the catabolism of alkenes often implies the hydrolysis of an epoxide inter-
588
mediate and, on the other hand, detoxification of the highly reactive epoxy-
intermediates is achieved via hydrolysis. As a consequence, it was anticipated that
bacteria and fungi which were known to be able to epoxidize alkenes in an efficient
manner should also possess a matching epoxide hydrolase activity. This proved to be
true for the fungi Aspergillus niger and Beauueria bassiana, which were able to achieve
the enantioselective hydrolysis of different types of epoxides derived from geraniol,
limonene [)'I or substituted styrene derivatives 931. In an extensive follow-up
study, seven additional fungal strains (from a total of 42) were selected for exhibiting
promising epoxide hydrolase activity[94].The guidelines mentioned above were also
successfully applied to the screening of bacteria, and strains were selected after a
careful literature search based on the capabilityfor alkene-epoxidation.Following the
work described above, the occurrence of epoxide hydrolases in yeasts has been
in~estigatedl~'. "1. From a screening of 187 different yeast strains belonging to 25
different genera, 8 strains (Trichosporon, Rhodotorula and Rhodosporidium sp.) were
identified by using 1,2-epoxyoctane as substrate [971. The membrane-associated
epoxide hydrolases from these yeasts show good enantioselectivitiesand high initial
rates, especially for monosubstituted aliphatic epoxides L9'1.
It is noteworthy that, in contrast to mammalian systems, the majority of bacterial
and fungal strains exhibited sufficient activity even when the cells were grown on a
non-optimized standard medium. Since enzyme induction is still a largely empirical
task, cells are usually grown on standard media in the absence of inducers.
Furthermore, all attempts to induce epoxide hydrolase activity in Pseudomonas
aeruginosa NCIMB 9571 and Pseudomonas oleovorans ATCC 29 347 by growing the
cells on an alkane (decane) or alkene (decene) as the sole carbon source failedL4].
Epoxide hydrolases from Corynebacterium["] and Rhodococcus DCL4['"] seem to be
exceptional with respect to their inducibility.
11.2.2
Microbial Hydrolysis o f Epoxides
1 1 2.2.1
Fungal Enzymes
One of the first observations on microbial epoxide hydrolysis on a preparative scale

was reported from the terpene field: thus, racemic geraniol N-phenylcarbamate was
efficiently hydrolyzed by the fungus Aspergillus niger, yielding 42 % of the remaining
((5s)-epoxidein 94% ee. Interestingly, from the preparative point of view, this could
easily be conducted on 5 g of substrate using a 7 L fermentor r91'.
Similar results were obtained with styrene oxide, which was again very efficiently
hydrolyzed by A. niger, thus affording the (S)-epoxide in 99% ee within a few
hours[85].In contrast, the fungus Beauveria bassiana (formerly B. sulfirescens)
showed opposite enantioselectivity,leading to the (R)-epoxidein 99% ee (Fig. 11.2-7).
In addition, interesting information concerning the mechanism implied in these
transformations [*I' and the scope of the substrates admitted could be established.
Thus, it was shown that cyclic styrene analogs like para-substituted styrene oxide
17.2 Hydrolysis ofEpoxides
I
589
e.e. >99% e.e. 62%
OH
d n . A. niger +
B.b.
&G
B. bassiana
89% e.e., 92% yield
rac
Beauveria bassiana
(inversion)
e.e. >99% e.e. 84%

Figure 11.2-7. Resolution and deracernization o f styrene oxide by fungal cells.
derivatives[991 or P-substituted analogs [921 were accepted by one - or both - of these

fungi. During a subsequent study, seven additional fungi were tested on more than
ten styrene oxide derivatives bearing various substituents[lOO]. It was shown that an
increase of the size of the substituent resulted in a selectivity-enhancement,e. g.,
from E = 3 for styrene oxide to E = 39 for para-nitrostyrene oxide. However, a methyl
substituent at Ca did not improve the enantioselectivity of the reaction.
Racemic epoxyindene was rapidly hydrolyzed when submitted to a culture of B.
sulfirescens, leading to a 20% yield of recovered enantiomerically pure (ee >98%)
(lR,ZS)-epoxide,and to a 48% yield of the corresponding (lR,ZR)-trans-diolshowing
G9 % ee [loll. The latter product is of considerable importance for the synthesis of the
HIV protease inhibitor indinavir. This prompted Merck Co. to perform a more
extensive study of this biotransformation [Io2. lo3],during which 80 fungal strains
were evaluated for their ability to enantioselectivelyhydrolyze racemic epoxyindene.
In a similar fashion, epoxydihydronaphthalenewas successfully hydrolyzed to the
corresponding (1R,2R)-diol in excellent enantiomeric purity [lo'].
Many of these fungal epoxide hydrolases were found to be soluble enzymes, which
could be obtained as crude cell-free extracts and which could be stored at +4 "C
without significant loss of activity. In this way, easy-to-use water-soluble catalysts
were developed,which circumvented the problems often encountered when working
with whole-cellmycelia ['04, 1"'.
590
I
large
large
- Lyophilized
Bacterial Cells
buffer pH 7-8
+v
small
c)
0-
small
large
rac
Figure 11.2-8.
0
small
Resolution o f 2,2-disubstituted oxiranes by bacterial cells.

OH
11.2.2.2
Bacterial Enzymes
The use of bacterial cells for preparative biotransformations is particularly attractive

for the following reasons: (i) they do not tend to form dense mycelia, which may
impede agitation of large-scale reactions when whole-cell (fungal) systems are
employed, and (ii) cloning of bacterial enzymes is generally less problematic.
However, disappointingly low selectivities were observed with monosubstituted
aliphatic epoxides such as 1-epoxyoctane( E 4) or benzyl glycidyl ether ( E <2) 831.
On the other hand, the sterically more demanding 2,2-disubstitutedoxiranes turned
out to be much better substrates (Fig. 11.2-8,Table 11.2-1). Especially the substrates
bearing a straight alkyl chain were transformed with virtually absolute selectivity,
and functional groups such as a C=C-double or a terminal bromo-
group [lo7]were well tolerated. As a consequence of the exquisite enantioselectivity,
the reactions ceased and did not proceed beyond a conversion of 50%.
Interestingly, the enantiopreference was found to depend on the substrate struc-
ture, but not on the strain When the epoxide bears a synthetically useful
phenyl moiety (mimicking a masked carboxyl function) at the o-position of the alkyl
chain, the selectivity was slightly reduced but still in a useful range (Table 11.2-1, last
entry, E = 123)['07,lo9]. Unexpectedly, when the carbon chain was extended by an
Table 11.2-1. Selectivities in the resolution of 2,2-disubstituted oxiranes by bacterial cells

(see Fig. 11.2-8).
Small substituent Large substituent Biocatalyst Selectivity (E)
CH3 n-CSH11 Nocardia sp. EH1 > 200

CH3 n-C4Hs Nocardia sp. TB1 > 200
CH3 (CHz),CH = CH2 Nocardia sp. EH1 > 200
CH3 n-C7H15 Rhodococcus equi I F 0 3730 > 200
CH3 n-CgH19 Mycobactenurn paraflnicum NCIMB 10420 > 200
CH3 (C&)4-Br Nocardia sp. H8 > 200
CH3 CHzPh Nocardia sp. EH1 123
Table 11.2-2. Selectivities i n the resolution of 2,2-disubstituted oxiranes by whole cells of

I 591
Rhodococcussp. NClMB11216 (seeFig.11.2-8).

Small substituent Large substituent Selectivity (E)
2.Sa
105
7
125
> 200
111
9.5
a With opposite absolute configuration.
additional CHz-unitthe selectivity declined (Table 11.2-2, last entry, E = 9.5). From an
extensive study on Rhodococcus sp. NCIMB 11216 it was concluded that the
enantioselectivity largely depends on the relative difference in size of the two alkyl
substituent groups (Table 11.2-2). Increasing this size difference resulted in en-
hanced selectivities. The fact that the substrates bearing a phenyl group behave
differently might be attributed to electronic effects, such as n-n-stacking.All of these
biohydrolyses can be performed on a multigram-scale.
In contrast to the rather flexible bacterial epoxide hydrolases mentioned above,
limonene-1,2-oxide hydrolase from Rhodococcus erythropolis DCL14, an enzyme
involved in the limonene degradation pathway, has a rather narrow substrate
specificity. Of the compounds tested, only the natural substrate limonene-1,2-oxide
and several highly substituted (alicyclic) epoxides were substrates for the enzyme.
The enantioselectivities were usually low, except for the natural substrate [l1O1.
Styrene oxide, various derivatives thereof and phenyl glycidyl ether were obtained in
high ee and reasonable yield using a recombinant epoxide hydrolase from Agrobacte-
rium radiobacter ADl["']. Interestingly, the Tyrl52Phe and Tyr215Phe mutants
showed a considerableincrease in stereoselectivity.For example, the Evalue for para-
chlorostyrene oxide increased fourfold from E = 32 for the wild-type enzyme to E
>130 for the Tyr215Phe mutant enzyme[78].
11.2.2.3
Yeast Enzymes
Yeasts are generally very sturdy microorganisms which are easy to cultivate on a
large scale. These features make them interesting for preparative use r9'1. Enantiose-
lective epoxide hydrolysis by (red) yeasts has been studied only recently; the first
report demonstrated the epoxide hydrolase activity of Rhodotorula gl~tinis['~] on
several aryl, alicyclic and aliphatic epoxides. In follow-up studies, additional yeast
strains exhibiting good activities and sufficient enantioselectivitieswere found, and
the application of these biocatalysts has great potential[97.981.
Given the data available to date, it seems to be a general phenomenon that epoxide
hydrolases from fungi and from bacteria generally possess an opposite enantio-
preference. Whereas epoxide hydrolases from fungi of matching opposite enantio-
592
I
preference are not known, an extensive screening showed that bacteria seem to be
more flexible in this respect[’’*]. This allows one to control the stereochemical
outcome by a simple choice of the appropriate microorganism.
112 . 3
Substrate Specificity and Selectivity
1 1 2.3.1
Asymmetrization o f meso-Epoxides
The asymmetrization of a meso-epoxide via regioselective attack at one of the

(enantiomeric) stereocenters of the oxirane would be an elegant application of
epoxide hydrolases since it leads to a single trans-diolin 100% theoretical yield. Such
asymmetrization reactions have been demonstrated with epoxide hydrolases from
dio1[113.1141. u nfortunately, few such reactions have been reported with microbial
mammalian origin, which afforded the enantiomerically enriched corresponding
enzymes. For instance, cyclohexene oxide was hydrolyzed using Corynesporium

cassiicola cells yielding trans-cyclohexane-l,2-diol,albeit with disappointingly low ee
(27%) It was only after further metabolism involving an oxidation-reduction
sequence by dehydrogenases present in the cells that the reaction product was
transformed into optically pure (S,S)-cyclohexane-1,2-diol. In a related experiment,
asymmetric hydrolysis of cis-epoxysuccinate using a crude enzyme preparation
derived from Rhodococcus sp. led to D- and L-tartaric acid in almost racemic form[79].
Similar discouraging results were obtained using baker’s yeast [IiG].
Recently, encouraging progress was made in the hydrolysis of cyclopentene oxide
and cyclohexene oxide using the yeast Rhodotorula glutinis[”]. The corresponding
(R,R)-trans-diolswere obtained in over 90% optical and chemical yields. However,
asymmetric hydrolysis of meso-epoxides by bacterial and fungal epoxide hydrolases is
still impeded by insufficient selectivities.
11L3.2
Resolution of Racemic Epoxides
Monosubstituted Epoxides. Monosubstituted oxiranes [Fig. 11.2-9(a), Table 11.2-31

represent highly flexible and rather “slim”molecules, which make chiral recognition
a difficult task. As a consequence, the majority of attempts using epoxide hydrolases
from bacterial and fungal origin to achieve highly selective transformations
failed[i1s]with one exception [li7].Most interestingly, the only selective enzymes
were found among red yeasts, such as Rhodotorula araucarae CBS 6031[”], Rhodo-
Table 11.2-3. Enzymatic hydrolysis of monosubstituted epoxides, see Fig. 9(a).
I 593
R Selectivitya Enantiopreference Enzyme Sourceb Reference
f R BEH 117
- n. d. BEH 8
- R BEH 23
- R FEH 50
+ R YEH 51
- to f S YEH 51
++ R YEH 97
++ R YEH 96
a Selectivity denoted as (-) =low (E < 4), (3=moderate (E = 4-12), (+) = good (E = 13-50), (++) =excellent
(E > SO).
b BEH = bacterial epoxide hydrolase; FEH = fungal epoxide hydrolase; YEH =yeast epoxide hydrolase.
n. d. = not determined.
sporidiurn toluloides CBS 0349197],Trichosporon sp. UOFS Y-01118[971 and Rhodotorula

glutinis CIMW147 ["I. These yeasts' epoxide hydrolase seem to have a preference for
monosubstituted oxiranes with a chain length of approximately six carbon atoms or
more (E up to 200). Furthermore, olefinic side chains are sometimes hydrolyzed
selectively (E up to 100) as well[98].Based on the rules of the kinetic resolution of a
racemate, diols of high ee could only be obtained at low conversions[95].With few
exceptions, the enantiopreference for the (R)-configuratedoxirane was predominant
regardless of the enzyme source['18].
Styrene Oxide-Type Epoxides. Styrene-oxide-type oxiranes [Fig. 11.2-9(b), Table
11.2-41have to be regarded as a special group of substrates, as they possess a benzylic
carbon atom. This facilitates the formation of a carbocation which is stabilizedby the
adjacent aromatic moiety. As a consequence, nucleophilic attack at the benzylic
position is electronically favored. On the other hand, the benzylic position is
sterically more demanding, which favors the non-benzylic position. As a con-
sequence, either oxirane carbon atom is easily attacked in this class of substrates and
mixed regiochemical pathways are common. Since this results in reactions occur-
ring with inversion and retention of configuration, E-values reported on these type of
oxiranes have to be regarded with great caution whenever the regioselectivityhas not
clearly been elucidated. In order to achieve optimal enantioselectivities, the bio-
catalysts of choice for styrene oxide-type oxiranes are derived from red yeasts such as
Rhodutorula glutinis CIMW 147 951 and particularly from fungal epoxide hydro-
1"s
lases, e.g. Aspergillus niger LCP 521 and Beauveria bassiana ATCC 7159['". '"]. The
first entry in Table 11.2-4is given solely for reason of comparison, since mammalian
hepatic epoxide hydrolase was used. This enzyme source is not applicable to
preparative-scale reactions.
Interestingly, the bacterial epoxide hydrolase from Agrobacteriurn radiobacter AD 1
seems to hydrolyze para-substituted styrene oxides with opposite enantiopreference
when compared to EHs from fungi or yeast["8]. Although initial selectivities were
594 I Table 11.2-4. Enzymatic hydrolysis of styrene oxide-type epoxides, see Fig. 9(b).
Ri RP R3 X Selectivitf Enantio- Enzyme Reference

preference sourceb
CH3, CzHs, n-C3H7, H H H ++ 1 S‘ mEHd 127
n-C4H9, n-C6HI3
H H Hp-CH3, o-C~, * R BEH 111
p-c1
H H CH3 H * R BEH 111
H H H p-F,p-Cl, + S YEH 93
p-Br, p-CH3
H H H p-CH3 + S YEH 93
H H H o-CH3, o-Hal - n. d. YEH 93
H H H H f R YEH 51
H CH3 H H ++ 2s YEH 51
H H H H ++ S FEH 92
H H CH3 H - n. d. FEH 92
indene oxide, + 2s FEH 92
dihydronaphthalene oxide
H CH3 H H ++ 2R FEH 92
a Selectivity denoted as (-) =low ( E < 4). (i)= moderate ( E = 4-12), (+) =good ( E = 13-50), (++) = excellent
( E >50).
b BEH =bacterial epoxide hydrolase; FEH = fungal epoxide hydrolase; mEH = microsomal epoxide hydrolase
from liver tissue; YEH =yeast epoxide hydrolase. n.d. = not determined.
c Enantioconvergent process (i.e. a single stereoisomeric diol was formed as the sole product).
d Performed on a microgram-scale only.
rather low[”1], the E-values could be significantly increased by using specific

mutants of the Agrobacterium enzyme[”].
Disubstituted Epoxides. Among the sterically more demanding substrates, 2,2-dis-
ubstituted epoxides were hydrolyzed with virtually absolutly enantioselectivities (E
>200) using enzymes from bacterial sources [Fig. 11.2-9(c),Table 11.2-51.In partic-
ular, Actinomycetes such as Rhodococcus and (closely related) Nocardia sp. are the
biocatalysts of choice for this class of oxiranes [”*I. Epoxide hydrolases from
Chryseomonas l u t e ~ l a [ ’and
~ ~ ]several 941 were less useful. Also for yeasts a
2-alkyl substituent resulted in a dramatic decrease in enantioselectivity[”].In several
cases, the regioselectivityof the reaction has been determined to be absolute. Attack
occurs exclusively at the less hindered unsubstituted oxirane C-atom with complete
retention at the stereogenic center. Most bacterial epoxide hydrolases showed a
preference for the (S)-enantiomer.Only recently, it was shown that several methylo-
trophic bacterial strains exist, which show an opposite preference (i.e. for the ( R ) -
epoxy enantiomer), albeit in moderate selectivitiesI l l 2 ] .
On the contrary, mixed regioselectivities were common when 2,3-disubstituted
oxiranes were hydrolyzed and ring opening ocurred at both positions of the oxirane
ring at various ratios (Table 11.2-6)[lls]. This is understandable, bearing in mind that
the steric requirements are similar at both positions. As a consequence, E-values are
not applicable to the description of stereoselectivities. Again, Actinomycetes were
found to be the catalysts of choice for this group of Most remarkably,
in selected studies, it was proven that the hydrolysis proceeded in an enantio-
Table 11.2-5.
7 1.2 Hydrolysis ofEpoxides
Enzymatic hydrolysis o f 2,2-disubstituted epoxides, see Fig. 9(c)

I
595
Ri Rz Selectivitya Enantio- Enzyme Reference

preference Sourceb
n. d. BEH 117
f Ror Sc FEH 50
f S BEH 8
f S BEH 8
+ S BEH 8
++ S BEH 8
++ S BEH 8
++ S BEH 8
a Selectivity denoted as (-) =low (E < 4). (3=moderate (E = 4-12), (+) = good (E = 13-50), (++) =excellent
IE > 50).
I ,
b BEH = bacterial epoxide hydrolase; FEH = fungal epoxide hydrolase; YEH =yeast epoxide hydrolase.
c Depending on the strain, the enantiopreference varied. n. d. = not determined.
Table 11.2-6. Enzymatic hydrolysis of 2,3-disubstituted epoxides, see Fig. 9(d).

Ri Rz R3 R4 Selectiviw Enantio- Enzyme Reference
preference Sourceb
H n-C4H9, n-CSH17 H n-C&17, n-CloH21 ++ 2s mEH 137

H n-C4H9 H (CH2)ioOH ++ 2s mEH 137
H n-CsH17 H (CH2)7C02H ++ 2s mEH 137
H CH3,C2H5 H n-C4H9, n-CsH11 ++ 2s mEH‘ 138
H (CHz)20H H n-CsHl1 ++ 2s mEHC 138
H (CH2)zOCH3 H n-C5H11 + 2s mEH‘ 138
H CH3 H n-CsH11 f 2s FEH 50
CH3 H H n-CsH11 f 2R/2Sd FEH 50
H CH3 CH3 H ++ S YEH 51
H CH3 H CH3 ++ S YEH 51
H C2HS H n-C4Hr, f 2s BEH 8
C2HS H H L3H7 f 1s BEH 8
H CH3 H n-C4H9 ++ 2s BEHC 89
CH3 H H n-C4Hg, n-CsHiI, ++ 1s BEH 8
n-C~Hi3
a Selectivity denoted as (-) =low (E c 4), (3=moderate (E = 4-12), (+) = good (E = 13-50), (++) =excellent
(E > 50).
b BEH =bacterial epoxide hydrolase; FEH =fungal epoxide hydrolase; mEH = microsomal epoxide hydrolase
from liver tissue; YEH =yeast epoxide hydrolase.
c Enantioconvergent process (i.e. a single stereoisomeric diol was formed as the sole product).
d Depending on the strain, the enantiopreference varied.
convergent fashion, and only one stereoisomeric diol was formed as the sole
product. In contrast, fungi seem less appropriate for the hydrolysis of 2,3-dis-
ubstituted oxiranes Lg4], whereas Rhodotorula glutinis was more effective on the cis-
configurated analogs of this substrate class[’’]. Interestingly, in contrast to the
bacteria, this yeast seems to operate via a classic kinetic resolution rather than an
enantioconvergent pathway. In this way, the simple choice of the appropriate
microorganism gives access to either the optically pure epoxide (yeast) or the
optically pure diol (bacteria).
596
Table 11.2-7. Enzymatic hydrolysis of trisubstituted epoxides, see Fig. 9(d).

Ri R2 R3 Rq Selectivitya Enantio- Enzyme Reference
preferenceb Source'
H (CHZ)~C(OAC)(CH~)C CH3 CH3 + 1s BEH 119

H=CH2
H Ph CH3 CH3 - - FEH 92
1,2-limonene oxide ++ S YEH 51
1-methylcyclohexeneoxide ++ 2s BEH 121
a Selectivity denoted as (-) =low (E < 4), (+) =moderate (E = 4-12), (+) =good (E = 13-50), (++) = excellent
(E > 50).
b Configuration of preferentially attacked oxirane carbon atom.
c BEH =bacterial epoxide hydrolase; FEH = fungal epoxide hydrolase; YEH =yeast epoxide hydrolase.
Trisubstituted Epoxides. To date, only a limited set of data are available on the
enzymatic hydrolysis of trisubstituted epoxides (Table 11.2-7).Regardless of their
steric bulkiness, however, they seem to be accepted by epoxide hydrolases from
bacterial['". '*'I, fun gal["^ 941 and yeast["] sources, as long as the access to one side
of the substrate is not too severely restricted (e.g. a 2,2-dimethyl-3-alkyloxirane).
Further data are required to depict a general selectivity pattern within this group of
substrates.
11.2.3.3
Deracemization Methods
In contrast to the asymmetrization of meso-epoxides, which would lead to the highly

desirable formation of a single stereoisomeric vicinal diol in 100% theoretical yield,
the kinetic resolution of racemic epoxides by fungal and bacterial cells has proven to
be highly selective (see above). However, this latter technique forms both the
unreacted epoxide and the corresponding vicinal diol in equal amounts. This so-
called classic kinetic resolution pattern of the biohydrolysis is often regarded as a
major drawback, since the theoretical chemical yield can never exceed 50% based on
the racemic starting material. As a consequence, methods that offer a solution to this
intrinsic problem are highly advantageous[120]. Several procedures which overcome
this drawback have been reported in the last few years.
For instance, based on the finding that styrene oxide could be resolved by whole
cells of Aspergillus niger and Beauveria bassiana via two different pathways showing
matching enantio- and regioselectivities a deracemization was developed thus,
combination of both biocatalysts in a single reactor led to (R)-phenylethane-l,2-diol
as the sole product in 98% ee and 8 5 % isolated yieldLssl (Fig. 11.2-7).
Another strategy for the achievement of an enantioconvergent process was set up
using the combination of bio- and chemo-catalysis[107r*09, 12', 1221 . For instance,
2,2-disubstituted epoxides were selectively resolved by lyophilized whole cells of
Nocardia sp. The biohydrolysis proceeds via attack at the less substituted C-atom
with excellent regioselectivity thus leading to retention of configuration at the
stereogenic center. On the other hand, acid-catalyzed hydrolysis of such epoxides
usually proceeds at the more substituted oxirane carbon with inversion. Careful
11.2 Hydrolysis ofEpowides
I 597
Lyophilized cells of
Nocardia sp. EH1 or H8
*
R
O A +
:A R'
(S) -diol
E >lo0 (R)-epoxide
rac e.e. >95%, yield >90%
I 4
HpSO cat. I dioxane I trace H 2 0
Figure 11.2-10. Resolution-inversion sequence f o r t h e deracemization of

2,2-disubstituted oxiranes involving t h e remaining epoxide.
combination of both catalytic steps (Fig. 11.2-10) in a resolution-inversion sequence

yields the corresponding (S)-1,2-diolsin virtually enantiopure form and in high
yields (> 90%) [Io7. In a similar fashion, racemic para-nitrostyrene oxide was
deracemized using a crude enzymatic extract from Aspergillus niger (Fig. 11.2-11). In
this case a 4:l water-DMSO solvent mixture was used, showing that this enzyme is
operative in the presence of water miscible organic solvents. The resolution step was
followed by the careful addition of acid, leading to (R)-para-nitrostyrenediol in good
yield (94%) and ee (80%).Because of the reduced enantioselectivity and the fact that
racemization occurred to a certain extent during the acidic hydrolysis, it was
necessary to tune both catalytic steps very carefully. A mathematical method was
therefore developed that made it possible to select the optimum conversion at which
the acid hydrolysis step should be initiated[122.1231. Careful mechanistic analysis of
the acidic hydrolysis reaction, using different solvents and mineral acids, made it
possible to select general conditions for the resolution-inversionprocedure['071.As a
consequence, large scale deracemization became feasible
The compatibility of microbial epoxide hydrolases with organic solvents deserves a
special comment. It has been reported that in the majority of cases, the addition of
water-miscible or -immiscible organic (co)solvents has negative effects on the
activity. This is particularly true for bacterial enzymes, which showed total deactiva-
-- fl- fl
~ ]the
t i ~ n [ ' ~On . other hand, several epoxide hydrolases from yeasts and fungi seem
enzymatic
hydrolysis
recryst.
chemical
hydrolysis
yield 78%
O2N O2N
e.e. 80% e.e. 99%
yield 94%
cyclisation
yield 89%
( R)-nifenalol e.e. 99%

overall yield 58%, e.e. 99%
Figure 11.2-11. Deracemization ofpara-nitrostyrene oxide by a chemoenzymatic process.

Application t o t h e synthesis o f (R)-Nifknalol@.
598
R1
p 4
rac-trans
- Lyophilized
Bacterial Cells
R'
HO OH
(R,S)- diol
Figure 11.2-12. Resolution and
deracernization of 2,3-disubstituted
oxiranes by bacterial cells.
Lyophilized
Bacterial Cells
R1 R'
rac-cis (R,R)-diol
to be less sensitive and are able to tolerate water-misciblecosolvents, such as DMSO

at a low
Cases for a non-classic deracemization of racemic epoxides using one single
biocatalyst impose high requirements on matching regio- and enantioselectivities,
and are therefore rare. For instance, the enantioconvergent hydrolysis of
(*)-3,4-epoxytetrahydropyran I'[ and several cis-p-alkyl substituted styrene ox-
ide~[''~]by hepatic microsomal epoxide hydrolase has been reported on an analytical
scale. Similarly, soybean epoxide hydrolase converted (*)-cis-9,1O-epoxy-l2(Z)-octade-
cenoic and (~)-cis-12,13-epoxy-9(Z)-octadecenoic acid into the corresponding (R,R)-
dihydroxy acids as the sole products [lzsl. However, enantioconvergent hydrolysis on
a synthetically useful scale was only reported recently. Thus, the fungus Beauveria
bassiana transformed (*)-cis-P-methylstyrene oxide in an enantioconvergent manner
to afford (lR,2R)-l-phenylpropane-1,2-diol in 85% yield and 98% eeI9'1. In a related
fashion, 2,3-disubstituted epoxides were hydrolyzed by using the Nocardia EH 1
(Table 11.2-8)[89s12'1 . Thus, the biohydrolysis of cis-2,3-epoxyheptanefurnished
(R,R)-threo-2,3-heptane diol in 79% isolated yield and 91 % ee on a gram scale. In the
latter study, the four stereochemical pathways and the enzyme mechanism were
elucidated by "0Hz-labeling experiments. The hydrolysis was shown to proceed by
attack of a (formal) hydroxyl ion at the (S)-configuratedoxirane carbon atom with
concomitant inversion of configuration at both enantiomers with opposite regioselectivity.
In addition, a mathematical model for the kinetics which allows the optimization of
such enantioconvergent processes in preparative applications was developed.
Table 11.2-8. Selectivities in the deracemization of 2,3-disubstituted oxiranes by bacterial cells,

see Fig. 9(d).
Ri Rz R3 Rq Biocatalyst Configuration ofdiol ee [%]

n-C4H9 H H CHI NocardiaEHl 2R, 3 s 90
n-C3H7 H H C2H5 Arthrobacter sp. DSM 312 2R, 3s 63
n-CSHi1 H H CH3 Rhodococcus SP. NCIMB 11 216 2R, 3s 77
n-GjH13 H H CH3 Rhodococcus SP. NCIMB 11 216 2R, 3 s 78
n-C4H9 H CH3 H Nocardia EH1 2R. 3R 97
n-C,H7 H C2H5 H NocardiaTBl 2R, 3R 77
11.2.4
I 599
Use of Non-Natural Nucleophiles
In reactions catalyzed by hydrolytic enzymes of the serine-hydrolase type, which

form covalent acyl-enzyme intermediates during the course of the reaction, it has
been shown that the “natural” nucleophile (water) can be replaced with “foreign”
nucleophiles [1301 such as an alcohol, amine, hydroxylamine, hydrazine and even
hydrogen peroxide. As a consequence, a wealth of synthetically useful reactions,
which are usually performed in organic solvents at low water content, can be
performed in a stereoselective manner. Although one requirement is fulfilled by
epoxide hydrolases - i. e. a covalent enzyme-substrate intermediate is formed - the
sensitivity of epoxide hydrolases to most of the water-miscible or -immiscible
organic solvents [49, 1241 poses a general problem in the use of non-natural nucleo-
philes in enzymatic epoxide hydrolysis. However, two types of transformations, i.e.
the aminolysis and azidolysis of an epoxide have been reported for selected cases
(Fig. 11.2-13).
When racemic aryl glycidyl ethers were subjected to aminolysis in aqueous buffer
catalyzed by hepatic microsomal epoxide hydrolase from rat, the corresponding (S)-
configurated amino-alcohols were obtained in 5 1 4 8 % On the other hand,
when azide was employed as nucleophile for the asymmetric opening of 2-methyl-
1,2-epoxyheptane in the presence of an immobilized crude enzyme preparation
derived from Rhodococcus sp., which contains an epoxide hydrolase activity, the
reaction revealed a complex The (S)-epoxide from the racemate was
hydrolyzed (as in the absence of azide), and the less readily accepted (R)-enantiomer
was transformed into the corresponding azido-alcohol (ee >GO %). Although at
present only speculations can be made about the actual mechanism of both the
aminolysis and azidolysis reaction, in both cases it was proven that the reaction was
catalyzed by a protein and that no reaction was observed in the absence of biocatalyst
“7
Ph
Ph
R =ti, CI e.e. 51-88%
rac
e.e. >60%
Figure 11.2-13. Enzyme-catalyzed arninolysis and azidolysis of epoxides.

600
or by using a heat-denatured preparation. However, a recent related report on the

aminolysis of epoxides employing crude porcine pancreatic l i p a ~ e lmay~~~ likewise
]
be explained by catalysis of a chiral protein surface rather than true lipase catalysis,
since the latter enzyme - being a serine hydrolase - is irreversibly deactivated by
epoxides. In view of these facts, it remains questionable whether the use of non-
natural nucleophiles will be of general applicability with epoxide hydrolases.
11.2.5
Applications to Asymmetric Synthesis
Although the use of an epoxide hydrolase for the asymmetric hydrolysis was
reported for industrial synthesis of L- and meso-tartaric acid as early as 1969 r60], it was
only recently that applications to asymmetric synthesis appeared in the literature.
This fact can be attributed to the limited availability of these biocatalysts from
sources such as mammals or plants. Since the production of large amounts of crude
enzyme is now feasible, preparative-scaleapplications are getting within reach of the
synthetic chemist. For instance, fermentation of Nocardia EH1 on a 70-L scale
afforded >700 g of lyophilized cells ['*I.
One of the first applications of the microbial hydrolysis of epoxides for the
synthesis of a bioactive compound is based on the the resolution of a 2,3-dis-
ubstituted epoxy-fatty acid having a cis configuration (Fig. 11.2-14).Thus, by using
an enzyme preparation from Pseudomonas sp., the (9R,lOS)-enantiomerwas hydro-
lyzed in a trans-specificfashion (i.e. via inversion of configuration at C-10) yielding
the (9R,lOR)-threo-diol.The remaining (9S,lOR)-epoxidewas converted into (+)-dis-
parlure, the sex pheromone of the gypsy moth in >95%
Another illustration of the use of such a biocatalytic approach was the synthesis of
either enantiomer of a-bisabolol. One of the stereoisomers is of industrial value for
the cosmetic industry. This approach was based on the diastereoselective hydrolysis
of a mixture of oxirane-diastereoisomers obtained from ( R ) - or (S)-limonene
(Fig. 11.2-15) r9O]. Thus, starting from (S)-limonene,the biohydrolysis of the mixture
of (4S,8RS)-epoxidesled to unreacted (4S,8S)-epoxideand (4S,8R)-diol.The former
-CO*H
rac-cis
- Pseudomonas sp.
buffer
Ho&co2H
HO
9R
+ .
(+)-Disparlure
Figure 11.2-14. Resolution of a cis-2,3-disubstitutedepoxide and synthesis of disparlure
I 601
(-)-(4S) -1irnonene
e.e. 99%
(p
(4S, 8 R)
yield 63%
(-)-(4 S,8S)-a-bisabolol
I d.e. 94% e.e. 99%, d.e. 94%
steps
I
Aspergillus
OH
(4 S, 8 R) (4s,8S) CulnHF (-)-(4 S ,8R)-epi -a-bisabolol

d.e. 94% d.e. 98% e.e. 99%, d.e. >95%
Figure 11.2-15. Chemoenzymatic synthesis ofa-bisabolol using fungal epoxide hydrolase.
72% 29%
1) Nocardia EH1 OH 1 ) Pd +2fcat./CuCIq/DME

L
'1, o,
2) H2SO,, dioxane
2) HCI, r.t.
89%
99% e.e. (S)-(-)-frontalin

99% e.e
Figure 11.2-16. Chemoenzymatic synthesis of (S)-(-)-frontalin using bacterial epoxide hydrolase.
showed a high diastereomeric purity (de >95%) and was chemically transformed into
(4S,SS)-a-bisabolol. The formed diol (de >94%) could be cyclized back to the
corresponding (4S,BR)-epoxide,thus affording access to another stereoisomer of a-
bisabolol. In addition, the two remaining stereoisomers of bisabolol could be
prepared in a similar manner starting from (R)-l'imonene.
Based on the deracemization of (*)-3-methyl-2-(4-pentenyl)-oxirane(Fig. 11.2-16)
using Nocardia EH1 and sulfuric acid in dioxane containing a trace amount ofwater
(see above), (S)-2-methyl-hept-6-ene-1,2-diol
was obtained in 97% yield and 99%
ee["']. This intermediate was successfully applied in a short synthesis of (S)-
602
I Figure 11.2-1 7.
Rhodococcus Synthesis of cis- and
OAc NCIMB 11216 OAc
trans-linalool oxide.
epoxide hydrolase
OH +
I I
9 (R R)
d.e. 98% d.e. 98%
MeOH,
MsCI,
Et3N, 0 OC K2C03
reflux
OH
I OH
trans- linalool oxide cis- linalool oxide
d.e. 94% d.e. 98%
(-) -frontalin, a central aggregation pheromone of pine beetles of the Dendroctonus

family [loGI.
Enantiopure cis- and trans-linalooloxides are found in several plants and fruits and
constitute the main aroma components of oolong and black tea. These compounds
were prepared from 2,3-epoxylinalyl acetate (Fig. 11.2-17)[1191. The key step consists
of a separation of the diastereomeric mixture of the starting epoxide by employing an
epoxide hydrolase preparation derived from Rhodococcus sp. NCIMB 11216, which
furnished the product diol and remaining epoxide in excellent diastereomeric excess
(de >98%). Further follow-up chemistry gave both linalool oxide isomers on a
preparative scale in excellent diastereomeric and enantiomeric purities.
Both enantiomers of the biologically active Bower's compound, a potent analog of
insect juvenile hormone[135],were prepared using Aspergillus sp. cells in 96% ee
(Fig. 11.2-18).Subsequent biological tests showed that the (6R)-antipodewas about
ten times more active than the (GS)-counterpartagainst the yellow meal worm
Tenebrio molitor.
Aspergillus niger was the biocatalyst of choice for the biohydrolysis of para-
nitrostyrene oxide (see above).A selective kinetic resolution using a crude enzyme
extract of this biocatalyst, followed by careful acidification of the cooled crude
reaction mixture, afforded the corresponding (R)-diolin high chemical yield (94%)
and good ee (80%).This key intermediate could then be transformed via a four-step
sequence into enantiopure (R)-nifenalol,a molecule with p-blocker activity, which
was obtained in 58% overall yield (Fig. 11.2-11) r8', 1221.
The natural (R)-(-)-isomer of mevalonolactone, a key intermediate in a broad
spectrum of cellular biological processes and their regulation, was synthesized via
7 7.2 Hydrolysis ofEpoxides
1a3
&OR Aspergi//us niger
k rac e.e. 96%

yield 36%
e.e. 70%
yield 48%
e.e. 96%
Bower‘s compound
Figure 11.2-18. Synthesis of Bower’s compound.
1) Nocardia EHl
TRIS-buffer pH 7.5 P H TsCW,
_____)
OH
2) H2S04 cat.
rac H20/dioxane e.e. 94%
v C o 2 H 1) LiAIH4TTHF ,
2) AC 20lDMAP
ho
HO
1) Na1O4/RuCl3 cat./
H O p C W o A c 1) K2C03/MeOH
MeCN/CCI4/H 20 ,.’ OAc ____)
_____)
2) HCI aq.
2) HCI aq.
(-)-( R)-rnevalonolactone
e.e. >99%
Figure 11.2-19. Synthesis of (R)-(-)-mevalonolactone.
eight steps in 55% overall yield and >99% ee (Fig. 11.2-19). In the key step, the
aforementioned enantioconvergent chemoenzymatic deracemization route was ap-
plied. Thus, 2-methyl-2-benzyl-oxiranewas deracemized on a 10 g scale using
lyophilized cells of Nocardia EH1 and sulfuric acid. The product (S)-diolwas isolated
in 94% chemical yield and 94% optical During the scale-up of this
604
I
external recycling:
$- ,& J. 1) HBr/AcOH
2) KOH/MeOH
Aspergillus niger + ~
OH OH , .
0 cell-free extract
0 0
i-Bu i-Bu i-Bu
KMn04/H SO,
i-Bu ' (S)-ibuprofen

Figure 11.2-20. Chemoenzymatic synthesis o f (S)-ibuprofen
biotransformation it was observed that the increase of the substrate concentration

led to a fourfold enhancement of the enantioselectivity as compared to analytical
scale test reactions
Finally, a chemoenzymatic enantioconvergent procedure led to (S)-ibuprofen in
four steps and 47 % overall yield (Fig. 11.2-20). The latter compound is a widely used
antiinflammatory drug and pain remedy and is one of the top ten drugs sold
worldwide[loo]. In the key step, the conditions for the enantioconvergent hydrolysis
of para-iso-butyl-a-methylstyreneoxide was optimized (elevated substrate concentra-
tion at +4 "C) to afford the non-reacted epoxide in 295 % ee[l3']. After separation from
the epoxide, the formed diol (70% ee) was recycled via a two-step sequence via the
corresponding bromohydrin, which was cyclized back to give (+)-epoxide.The latter
material was subjected to repeated biocatalyhc resolution in order to improve the
economy of the process.
11.2.6
Summary and Outlook
Over the past few years, an impressive array of epoxide hydrolases has been
identified from microbial sources. Due to the fact that they can be easily employed as
whole-cellpreparations or crude cell-free extracts in sufficient amounts by fermenta-
tion, they are just being recognized as highly versatile biocatalysts for the preparation
of enantiopure epoxides and vicinal diols. The future will certainly bring an
increasing number of useful applications of these systems to the asymmetric
synthesis of chiral bioactive compounds. As for all enzymes, the enantioselectivity of
References I605
microbial epoxide hydrolysis depends on the substrate structure and the type of
enzyme involved. The data available to date indicate that the enantioselectivites of
enzymes from certain microbial sources can be roughly correlated to the substitu-
tional pattern of various types of substrates: red yeast give best selectivities with
monosubstituted oxiranes, fungal cells are suited for styrene oxide-type substrates
and bacterial enzymes are the catalysts of choice for more highly substituted 2,2- and
2,3-disubstituted epoxides. Since the first three-dimensional X-ray structure of an
epoxide hydrolase has recently been solved, more will follow, which will improve the
predictability of stereoselectivities. Given the data presented above, possible in-
dustrial applications of microbial epoxide hydrolases can be anticipated in the near
future.
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7 7.3 Hydrolysis and Formation ofclycosidic Bonds

I 609
11.3
Hydrolysis and Formation of Clycosidic Bonds
Chi-Huey Wong
1.3.1
Introduction
Carbohydrates are found in nature as components of a broad range of molecular

structures [l-sO1. Attached to cell surface glycoproteins and glycolipids,they play vital
roles in cellular communication processes ['-231, function as points of attachment for
proteins such as antibodies, and serve as receptor sites for bacteria and viral
particles [12, 13, 521. For example, the sialyl-Lewis X tetrasaccharide mediates the
adhesion of neutrophils to the endothelial layer, an initial event in the inflammatory
responsel". 45. 5 3 - 5 6 ] . ~1ycoprotein glycans can modulate protein folding and are
involved in the sorting and trafficking of proteins to appropriate cellular

sites[l. 21, 40, 41, 57, 581
Nature employs two groups of enzymes in the biosynthesis of oligosaccharides,
namely those of the L e l ~ i r [ ~ ' -and ~ ~ ]non-Leloir pathways. Leloir enzymes are
responsible for the synthesis of most glycoproteins and other glycoconjugates in
mammalian systems. Glycoprotein glycans are typically classified as either N-linked
or 0-linked, based on the linkage between the carbohydrate and the protein. N-
linked glycans are characterized by a j3-glycosidic linkage between GlcNAc and an
asparagine 8-amide nitrogen. The majority of 0-linked glycans contain an a-
glycosidic linkage between GalNAc and a serine or threonine hydroxyl group. The
addition of oligosaccharide chains to glycoproteins occurs post- or co-translationally
in the endoplasmic reticulum and the Golgi apparatus r60]. N-linked oligosaccharides
all contain the same basic core structure composed of GlcNAc and mannose
residues. N-linked glycan biosynthesis involves the initial construction of a dolichyl
pyrophosphoryl oligosaccharide intermediate in the endoplasmic reticulum cata-
lyzed by GlcNAc-transferases and mannosyltransferases. This structure is then
glucosylated, presumably as a signal for transfer of the oligosaccharide to the
polypeptide. The entire oligosaccharide moiety is then transferred en bloc to an Asn
residue of the growing peptide chain catalyzed by oligosaccharyltransfer-
ase[60. 6 3 , 641. ~h e Asn is typically part of the amino acid sequence Asn-X-Ser(Thr),
where X#Pro or Asp[3o,60. 65-671 . B efore transport into the Golgi apparatus, trim-
ming of the glycan by glucosidases I and I1 and a mannosidase reveals a core
pentasaccharide (peptide-A~n-(GlcNAc)2-(Man)~). This structure is further proc-
essed by mannosidases and glycosyltransferases in the Golgi apparatus, resulting in
either a high-mannose, complex, or hybrid type oligosaccharide. Sequential addition
of monosaccharides then provides the fully-elaborated oligosaccharide chain. In
contrast, the more structurally diverse 0-linked glycans are assembled within the
Golgi apparatus by glycosyltransferases[*lS60]. In the most common route, GalNAc
is initially appended to serine or threonine catalyzed by a UDP-Ga1NAc:polypeptide
GalNAc-transferase. Monosaccharides are then added individually to the growing
oligosaccharide chain by glycosyltransferases.
610
R HOO o G Ho
&AcHN 0
AcHNo-b(-O-)(-O 0
I I
0- 0-
Dolichylpyrophosphate-linked oligosaccharide
R = oligosaccharide, n = 9-15
R20
HO
HO OH OH
OH
Ganglioside GMI: R' = Gal 1,SGalNAc -, R2 = H
Ganglioside GM2: R' = GalNAc -, R2 = H
Ganglioside GM3: R' = H, R2 = H
Ganglioside GD3: R' = H, R2 = NeuAc - How&
; o& OH
AcHN OR
HO OH +OH NHAc
Sialyl Lewis X antigen OH

HO
Gal 1,2Gal 1,6Gal 1 Glycosyl phosphatidylinositol

Gal 1 f 2
j-;o&)Ho-..-&& NHAc COZH -03SHN0

OH
Heparin pentasaccharide oso3
Fuc
NeuAc 2,3Gal 1.4GlcNAc 1,PMan 1
\6 .N 6
GlcNAc 1 - - - - - + 4Man 1,4GlcNAc 1.4GlcNAc 1N A s n
NeuAc 2,3Gal 1,4GlcNAc 1,PMan 1 y 3 Typical structure of N-linked complex glycan

Figure 11.3-1.
7 1.3 Hydrolysis and Formation of Glycosidic Bonds
All mammalian cells, with the exception of erythrocytes, contain the necessary
I
elements for glycosylation. In certain secretory cells, however, the preponderance of
glycosyltransferases is greater fG81. The structures of some typical naturally-occurring
glycoproteins, glycolipids,and oligosaccharides are illustrated in Fig. 11.3-1.
The major classes of cell-surfaceglycolipids include the glycosphingolipids (GSLs)
and glycoglycerolipids. Gangliosides IG91, or sialic acid-containingglycosphingolipids,
are especially abundant on neural cell surfaces I7O]. These compounds play a role in
the differentiation of cell types and in the regulation of cell growth. Additionally,
sphingosine, the lipid component of GSLs, has been suggested to function as an
intracellular second messenger [711.
The mammalian glycosyltransferases of the Leloir pathway utilize monosacchar-
ides activated as glycosyl esters of nucleoside mono- or diphosphates as glycosyl
donor substrates Primarily eight nucleotide sugars serve as glycosyl donors for
the synthesis of most oligosaccharides: UDP-Glc, UDP-GlcNAc, UDP-Gal, UDP-
GalNAc, GDP-Man, GDP-Fuc, UDP-GlcUA, and CMP-NeuAc (Fig. 11.3-1). Many
other monosaccharides, such as the anionic or sulfated sugars of heparin and
chondroitin sulfate, are also found in mammalian systems, but usually are the result
of post-glycosyl transfer modifications 272 72a,b1. Non-Leloir glycosyltransferases
typically employ glycosyl phosphates as activated donors. A diverse array of mono-
saccharides (e.g. xylose, arabinose, KDO) and oligosaccharides is also present in
microorganisms, plants, and invertebrates[33* G2, 73-7G1 .The enzymes responsible for
their biosynthesis, however, have not been extensively exploited for synthesis,
though the same principles as in mammalian systems apply. Some sugar nucleo-
tides used by enzymes of other pathways are also shown in Fig. 11.3-2.
Chemists have employed glycosyltransferases from the Leloir and non-Leloir
pathways for the synthesis of oligosaccharides and glycoconjugates[77-831. Glycosi-
dases have also been exploited for synthesis [77-841. The function of glycosidases in
vivo is to cleave glycosidic bonds; however, under appropriate conditions, they can be
useful synthetic catalysts. Each group of enzymes has certain advantages and
disadvantages for synthesis. Glycosyltransferasesare highly specific in the formation
of glycosides, but the availability of many of the necessary enzymes is limited.
Glycosidases have the advantage of wider availability and lower cost, but they are not
as regio-specificor high-yieldingin synthetic reactions. Therefore the chemist must
choose the enzyme which is best suited for the application at hand. Other enzymatic
methods used to synthesize glycoconjugateswill also be discussed.
11.3.2
Clycosyltransferases o f the Leloir Pathway
Glycosyltransferases are highly regiospecific and stereospecific with respect to the

formation of new glycosidic linkages. Although also usually substrate-specific,
minor chemical modifications are tolerated on both the donor and acceptor compo-
nents. The preparative use of glycosyltransferaseshas been somewhat limited in the
past because of a lack of enzyme availability. Additionally, because glycosyltrans-
ferases are membrane-bound enzymes, they are relatively unstable and can be
612
n 0
NHz
HO OH HO OH
a-UDP-Glucose a-GDP-Mannose
(UDP-Glc) (GDP-Man)
HO
HobUDP
HO&
HO
AcHN
OUDP
VD
HO OH
a-UDP-Galactose a-UDP-KAcetylglucosarnine P-GDP-L-Fucose

(UDP-Gal) (UDP-GIcNAc) (GDP-FUC)
HO
HO $
+
HoOUDP
AcHN
HO OH a-UDP-KAcetylgalactosarnine a-UDP-Glucuronic acid
(UDP-GalNAc) (UDP-GlcUA)
P-CMP-N-Acetylneurarninic acid
(CMP-NeuAc)
0
HO OCMP
HO OH
P-TDP-L-Rharnnose b-CMP-KDO a-UDP-N-Acetylrnuramicacid
Figure 11.3-2.
difficult to handle in solution. However, the recent isolation of many of these

enzymes, as well as advances in genetic engineering and recombinant techniques,
are rapidly alleviating these drawbacks.
Glycosyltransferases utilize nucleotide sugars as activated glycosyl donors [Go].
Most of these sugar nucleoside phosphates are biosynthesized in uiuo from the
corresponding monosaccharides (Fig. 11.3-3). The initial step is kinase-mediated
phosphorylation to produce a glycosyl phosphate. This glycosyl phosphate then
reacts with a nucleoside triphosphate (NTP), catalyzed by a nucleoside diph-
osphosugar pyrophosphorylase, to afford an activated nucleoside diphosphosugar
[Eq. (l)]. Some sugar nucleoside phosphates, such as GDP-Fuc and UDP-GlcUA, are
biosynthesized by further enzymatic modification of existing key sugar nucleotides.
11.3 Hydrolysis and Formation of Clycosidic Bonds
I
- 613
--
Gal Gal-I-P
Glc - Glc-6-P Glc-1-P - - 4DP-Xyl
1-
Fru-6-P Man-6-P -Man-I-P I--
1 1
GkN-6-P Man
1
GlcNAc-6-- GIcNAc
ManNAc-6-P-N&AC-~-P
Figure 11.3-3.
In contrast, CMP-NeuAc is formed by the condensation of NeuAc with CTP [Eq. (2)].
Some of the enzymes involved in the biosynthesis of sugar nucleotides also accept
unnatural sugars as substrates. In general, however, the rates are quite slow, thus
limiting the usefulness of this approach.
Sugar-1-P+ NTP NDP-Sugar + PPi
-+
(1)
NeuAc + CTP CMPNeuAc + PPi
+
(4
11.3.2.1
Synthesis of Sugar Nucleoside Phosphates
Chemical syntheses of some sugar nucleoside phosphates have been reported[85].

Most of these methods involve the reaction of an activated NMP[8G”]with a glycosyl
phosphate to produce a sugar nucleoside diphosphate (Fig. 11.3-4). Of the com-
monly used activated NMP derivatives, phosphoramidates such as phosphorimida-
zolidates [92-941 and phosphoromorpholidates [86-911 are considered the most effec-
tive. A recent improvement in coupling methodology employing 1 H-tetrazole as
catalyst has been reported[”]. These activated NMPs may also be used to prepare
614
.OH
0 0 0
I1 I1 I1
-0-P -0 - P -0 -P - 0
0-
I I
0. I
0. d
XMP *-
Figure 11.3-4.
XTP
XDP *
*
OP 0
XTP
Jco,
0 OP
A 0 ; A o 2
1. Adenylate kinase (EC 2.7.4.3, X = A,C,U)
Guanylate kinase (EC 2.7.4.8, X = G)
Nucleoside monophosphate kinase (EC 2.7.4.4, X = U)
2. Pyruvate kinase (EC 2.7.1.40) Figure 11.3-5.
NTPs by reaction with pyrophosphate (Fig. 11.3-5)[88].A number of chemical

methods are available for the synthesis of glycosyl phosphates. Reactions of phos-
phates with activated glycosyl donors [", 961 or chemical phosphorylation of anome-
ric hydroxyl groups [89-92, 971 have proven to be convenient. Additionally, routes via
glycosyl phosphites are useful["]. Enzymatic procedures include employing glyco-
gen phosph~rylase['~] and sucrose phosphorylase["'] for the production of a-
glucose-1-phosphate. Phosphoglucomutase can also be used to prepare glucose-
1-phosphate from glucose-G-phosphate[lO1], the latter generated from glucose by
hexokinase catalysis.
Preparative-scale synthesis of nucleoside triphosphates. Nucleoside triphosphates are
utilized as substrates for the biosynthesis of sugar nucleoside phosphates. Practical-
scale biosynthesis-based enzymatic preparation of NTPs for use in glycosylations is
therefore required.
Most preparative-scale enzymatic syntheses of NTPs use commercially available
NMPs as starting materials. Alternatively, NMPs can be obtained from yeast RNA
digests at low cost["*], or can be easily prepared chemi~ally~''~]. In general,
enzymatic methods involve the sequential use of two kinases to transform NMPs to
NTPs, via the corresponding NDPs. Several kinases have been utilized to synthesize
NTPs from the corresponding NDPs, each employing a different phosphoryl donor:
pyruvate kinase (E. C. 2.7.1.40) uses phosphoenolpyruvate "1' as a phos-
phoryl donor, acetate kinase (E. C. 2.7.2.1) uses acetyl phosphate, and nucleoside
11.3 Hydrolysis and Formation ofClycosidic Bonds
I 615
XTP
1 phospho-
glycero-
mutase
chemical
synthesis
diphosphate kinase (E. C. 2.7.4.6) uses ATP. Pyruvate kinase has generally been the
enzyme of choice because it is less expensive than nucleoside diphosphate ki-
and because PEP is more stable and provides a more thermodynam-
94, lo7],
ically favorable driving force for phosphorylation than does acetyl phosphate
(Fig. 11.3-5). A recently described polyphosphate kinase uses polyphosphate as
donor, providing a potentially cheaper kinase route [lo6].
The preparation of NDPs from NMPs is more complicated, and requires different
enzymes for each NMP. Adenylate kinase (E. C. 2.7.4.3) phosphorylates AMP[88]and
CMP[1081,and also slowly phosphorylates UMP. Guanylate kinase (E. C. 2.7.4.8)
catalyzes the phosphorylation of GMP. Nucleoside monophosphate kinase (E. C.
2.7.4.4) uses ATP to phosphorylate AMP, CMP, GMP, and UMP; however, the
enzyme is relatively expensive and Both CMP and UMP kinases exist
but are not commercially available. For those kinases requiring ATP as a phosphor-
ylating agent, ATP is usually used in a catalFc amount and recycled from ADP
using pyruvate kinase/PEP or acetate kinaselacetylphosphate[77, lo9].Phosphoe-
nolpyruvate may be prepared chemically from p y r u ~ a t e [or ~ ~generated
~] enzymat-
ically from D-3-phosphoglycericacid['''] (Fig. 11.34).
When chemical and enzymatic methods for NTP synthesis are
enzymatic techniques provide the most convenient route to CTP and GTP, whereas
chemical deamination of CTP is the best method for preparing UTP[941.ATP is
relatively inexpensive from commercial sources, although it has been synthesized
enzymatically from AMP on 50 mmol scale. Mixtures of NTPs can be prepared from
RNA by sequential nuclease PI, polynucleotide phosphorylase, and pyruvate kinase-
catalyzed reactions [llO]. This mixture can be selectively converted to a sugar nucleo-
tide using a particular sugar nucleoside diphosphate pyrophosphorylase['lo].
UDP-glucose ( UDP-Glc) and UDP-galactose ( UDP-Gal). UDP-glucose has been
prepared from UTP and glucose-1-phosphate under catalysis by UDP-glucose
pyrophosphorylase (Fig. 11.3-7)[94, 2' ' ''13, '141. UDP-Ga1 can be synthesized in an
analogous fashion using UDP-Gal pyrophosphorylasellO1],or from UDP-Glc by

epimerization of C-4 with UDP-glucose epimerase['O'l (Fig. 11.3-7). Though the
epimerase equilibrium favors UDP-Glc, the reaction can be coupled to an in situ
glycosylation with galactosyltransferase to shift toward UDP-Gal production. The
latter process has been applied to large-scale synthesis of N-acetyllactosamine
(L~CNAC)['~']. UDP-Gal has been prepared from UMP and Gal using dried cells of
616
I
HO
HO /& Ho 0PO32.
UTP,
UDP-Glc
DvroDhosDhorvlase ~
HO
K = 0.17 ROH, GalT

HO 7
UDP-Glc HO OR
4-epimerase OH
Figure 11.3-7.
E2: pyruvate kinase

E3: phosphoglucomutase
Pyruvate PEP Ed: UDP-Glc pyrophosphorylase
E5 inorganic pyrophosphatase
Figure 11.3-8.
Towlopsis candida["'l. In this system, Gal-1-phosphate and UTP were generated in

situ as substrates for UDP-Gal pyrophosphorylase. Gram quantities of UDP-Gal, as
well as the 2-fluoro-UDP-Gal derivative have been synthesized by an enzymatic
method employing Gal-1-P uridyltransferase
UDP-N-acetylglucosamine ( UDP-GlcNAc). UDP-GlcNAc has been synthesized by
reaction between GlcNAc-1-phosphate and UTP, catalyzed by UDP-GlcNAc pyr-
ophosphorylase [lll].Although this enzyme is currently not commercially available, a
whole-cell process using baker's yeast can be employed ["'I. Another procedure
exploits UDP-Glc pyrophosphorylase to catalyze a condensation between UTP and
glucosamine-1-phosphate (GlcN-I-P) to afford UDP-glucosamineI'121 (Fig. 11.3-8).
The product UDP-GlcN can then be selectively N-acetylated to provide UDP-GlcNAc.
GlcN-1-Phas been synthesized from GlcN by phosphorylation of the 6-position with
hexokinase to give GlcN-G-P, followed by a phosphoglucomutase-catalyzed iso-
merization to provide GlcN-1-P. UDP-GlcNAc also serves as an acceptor for p1,4-
GalT to provide UDP-L~CNAC[~"].
UDP-N-acetylgalactosumi~e ( UDP-GalNAc). The biosynthetic enzymes UDP-Gal-
NAc pyrophosphorylase and UDP-GlcNAc 4-epimerase are not readily available for
facile synthesis of UDP-GalNAc.An alternative synthetic procedure based on UMP
exchange between UDP-Glc and GalN-1-P, catalyzed by commercially available
UDP-Glc: galactosylphosphate uridyltransferase (E. C. 2.7.7.12) has been reported
(Fig. 11.3-9)[62. 'I3]. Galactose-1-P is the natural substrate for the enzyme, but
2-deoxygalactose-l-P, 2-deoxyglucose-l-P, and galactosamine-1-P are also tolerated.
11.3 Hydrolysis and Formation ofclycosidic Bonds I617
HHO O G + Hr$ -- El H HO O G + Hr$

HoOUDP H2N
E, = UDP-G1c:galactosylphosphate
0PO32.
lE2 lACZO
H00P032- H2NOUDP
uridyltransferase HO OH
E2 = phophoglucomutase H HOO G o H Ho%

HO AcHN
OUDP
Figure 11.3-9.
As the equilibrium constant for the exchange reaction is close to unity, phosphoglu-
comutase was required to relieve product inhibition and shift the equilibrium. The
UDP-GalN thus produced was then chemically acetylated to give UDP-GalNAc.
A modification of the latter procedure has been adapted to large-scalesynthesis of
UDP-GalNAc["8]. In this procedure, UDP-Glc was regenerated in situ from UTP and
the product Glc-1-P under catalysis by UDP-Glc pyrophosphorylase. This also shifts
the equilibrium toward the formation of UDP-GalN. Alternatively, UDP-GalNAc can
be prepared from UMP and sucrose employing sucrose ~ y n t h a s e [ ~Large-scale
~~].
production of UDP-GalNAc in yeast has also been accomplished[120].
GDP-mannose (GDP-Man).GDP-mannose has been prepared from Glc and GMP
using dried baker's yeast cells ['"I. The procedure involves the biocatalytic conver-
sion of glucose to Man-1-P and subsequently to GDP-Man using GDP-Man pyr-
ophosphorylase. A cell-free extract from baker's yeast has also been used to
synthesize GDP-Man from mannose"'']. A direct synthesis from chemically-
prepared Man-1-P and GTP, catalyzed by GDP-Man pyrophosphorylase (E. C.
2.7.7.13) is useful for large scale producation (Fig. 11.3-10)Lg4]. Alternative strategies
for continuous GDP-Man production["'], the synthesis of GDP-Man directly from
mannose and other routes have also been pursued[124].
GDP-ficose (GDP-Fuc). GDP-fucose is biosynthesized in vivo from GDP-Man by
an NADPH-dependentoxidoreductase enzyme system. Such systems have also been
utilized for in vitro syntheses of GDP-Fuc. For example, the synthesis of GDP-Fuc
was accomplished using a crude enzyme preparation from Agrobacterium radio-
ba~ter[l'~].NADPH was regenerated in situ from NADP using glucose-6-phosphate
dehydrogenase and Glc-6-P[12G]. Employing a similar procedure, GDP-Fuc has been
Baker's
Glc + GMP yeast HFq HO

[o*EGDP] NADPH
VDP
OGDP
t
- A
GTP, GDP-Fuc
GDP-Man pyrophos-
Man-1-P DvroDhosoholvlase fucokinase p-tucose-,-p ho lase
Fucose
GTP pp,
Figure 11.3-10.
618
Figure 11.3-11.
HO
2 NADH
L-LDH
-HraoH
OH
H HO O h o H HO- Ho ~ A El0 2 - * H o
AcHN a C O z H
NHAc HO OH
El: NeuAc aldolase

EZ CMP-NeuAc synthetase XCDP
CTP f
E4
E3 Adenylate
Pyruvate kinase
kinase
Pyruvate PEP H
z@
H
o:
AcHN
HO OH
Figure 11.3-12.
generated in situ for use in a glycosylation reaction with a-1,3-f~cosyltransferase~'~~].

Enzymes from a minor biosynthetic pathway which synthesize GDP-Fuc from L-
fucose have also been exploited for Fucose was phosphorylated by
fucokinase (E. C. 2.7.1.52)to produce Fuc-1-P, which subsequently underwent a
GDP-fucose pyrophosphorylase-catalyzed reaction with GTP to provide GDP-Fuc.
Several practical chemical syntheses of GDP-Fuc have also been reported[95,12'].
UDP-glucuronic acid ( UDP-GkUA).UDP-Glucuronic acid is biosynthesized by C-
6 oxidation of UDP-Glc with UDP-Glc dehydrogenase, an NAD-dependent enzyme.
Enzyme preparations from bovine liver have been employed for gram-scale synthe-
ses of UDP-GlcUA (Fig. 11.3-11)[944. 1291. The NAD cofactor was regenerated with
lactate dehydrogenase in the presence of pyruvate. Additionally, extracts from guinea
pig liver have been used to generate UDP-GlcUA in situ for use in enzymatic
glycosylations with glucuronyltransferases [1301.
CMP-N-acetylneuraminic acid (CMP-NeuAc). CM P-N-acetylneuraminic acid has
been prepared enzymatically on small scales (> 0.5 mmol) from CTP and NeuAc,
under catalysis by CMP-NeuAc synthetase (EC 2.7.7.43)[l3l].An improvement in this
procedure, involving in situ production of CTP from CMP under adenylate kinase
and pyruvate kinase catalysis, is suitable for multigram-scale synthesis Adeny-
late kinase catalyzes the equilibration of CTP and CMP to CDP, which is subse-
quently phosphorylated by pyruvate kinase to provide CTP. A one-pot synthesis of
CMP-NeuAcbased on this procedure involves the in situ synthesis of NeuAc from N-
acetylmannosamine and pyruvate, catalyzed by sialic acid aldolase (Fig. 11.3-12)[1081.
Chemical syntheses of CMP-NeuAc have also been
17.3 Hydrolysis and Formation of Glycosidic Bonds
The gene encoding E. coli CMP-NeuAc ~ y n t h e t a s e “ 1341

~ ~ ,has been cloned into the
Lambda ZAP vector and overexpressed in E. coli at a level 1000 times that of the wild
type[307,3081. The enzyme from calf brain has also been cloned and overexpressed.
CMP-NeuAc synthetase was shown to accept several NeuAc derivatives as substrates.
For example, 9-deoxy-,7,9-dideoxy-,and 4,7,9-trideoxy-NeuAcare all converted to the
corresponding CMP-NeuAc derivatives[137]. On the other hand, the 4-ox0,~-0xo, and
8-0x0 NeuAc derivatives are not substrates for CMP-NeuAc syntheta~e[~’~I. However,
the enzyme accepts a variety of modifications at the 9-position, and the hydroxyl
group can be replaced with several different groups with little effect on the KM
value[139-141].CMP-NeuAc can also be obtained on the large scale by fermenta-
tion [1431 or by coupling of metabolically engineered bacterial cells I1&].
11.3.2.2
Substrate Specificity and Synthetic Applications of Clycosyltransferases
For each sugar nucleotide glycosyl donor, many glycosyltransferases of varying

substrate specificities exist. These enzymes are generally considered to be specific
for a given glycosyl donor and acceptor, as well as for the stereochemistry and the
linkage position of the newly formed glycoside bond. This specificity has led to the
“one enzyme-one linkage” concept[28,142, lG1] . I n other words, the specificity of the
glycosyltransferasesensures fidelity in oligosaccharide sequences in vivo without the
use of a template scheme. Though systematic investigations of the in vitro substrate
specificity of most glycosyltransferases have not been carried out, some deviations
from this picture of absolute specificity have been observed in the tolerated
modifications of both glycosyl donors and acceptors. Additionally, studies toward the
design of inhibitors of glycoprotein biosynthesis[2051have also shown that the
specificities of glycosyltransferases are not absolute.
Galactosyltransferase (GalT). Because of its availability, P1,4-GalT (E. C.
2.4.1.22)[lG21471 is one of the most extensively studied mammalian glycosyl-
transferases with regard to synthesis and substrate specificity. The X-ray crystal
structure of the bovine enzyme has recently been reported[148].P1,4-GalT catalyzes
the transfer of galactose from UDP-Gal to the 4-position of p-linked GlcNAc residues
to produce the Galpl,4GlcNAc (LacNAc) structure. In the presence of lactalbumin,
however, both a- and P-linked substrates are allowed, and glucose is the preferred
acceptor. P1,4-GalT has been employed in the in vitro syntheses of LacNAc and
glycosides thereof, as well as other g a l a c t o s i d e ~ [(Table
~ ~ ~ ]11.3-1).
P1,4-GalT also tolerates 2-deoxyglucose,D-xylose, 5-thioglucose,N-acetylmuramic
acid, and myoinositol as acceptor substrates [147]. Modifications at the 3- or 6-position
of GlcNAc are also accepted. For example, Fuca1,GGlcNAc and NeuAca2,bGlcNAc
are substrates Acceptor substrates which are derivatized at the 3-position
include 3-0-methyl-GlcNAc 3-deoxy-GlcNAc,3-O-allyl-GlcNAc~OBu, and 3-0x0-
G ~ C N A C D-Mannose,
[~~~]. D-allose, D-galactose, D-ribose, and D-xylose do not serve as
substrates. Monosaccharides which have a negative charge, such as glucuronic acid
and a-glucose-1-phosphate, are also not accepted. Fig. 11.3-13 illustrates several
disaccharideswhich can be synthesized with p1,4-GalT[’47]. A particulary interesting
620
Table 11.3-1. Products of galactosyltransferase reactions.

UDP-Gal (or analogs) + CalT Scale" Ref.
GalPl,4Glc C [147]
Galpl,4GlcNAc A [142,1451
Galp1,4GlcNAc-Agarose C [147]
Gal~l,4GlcNAc-hexylarnine C 11471
Galp1,4GlcNAcpl,4Gal C (1471
Gal~l,4GkNAcpl,6Gal C [150]
Galpl,4GlcNAcp1,3Gal C (1501
Gal~1,4Glc~OCH2C~H~(NO~)-CONH-Polymer D 11511
Gal~1,4Glc~l,4Glc~OCH~C~H~(NO~)-CONH-Polymer D [151]
Gal~l,4Glc~1,4Glc~OCH~NH-~-Phe-CONH-Polymer D [151]
Gal~1,4GlcNAc~l,3(Gal~l,4GlcNAc~l,G)Gal~l,4Glc~OMe C [152]
Gal~1,4GlcNAc~1,6(GlcNAc~l,3)Gal~l,4Glc~OMe C [152]
Galp1,4(Fucal,6)GlcNAc~O(CH~)~CO~Me D [150]
Ga~~l,4(NeuAc(OMe)a2,G)GlcNAc~O(CHz)sCOZMe D [lSO]
Galpl,4GlcNAcpR;R = N-Ac-Asn(0Me) C [153]
Gal~1,4GlcNAcpl,4GlcNAc C [153]
Gal!.31,4GlcNAc~1,4GlcNAc~R;
R = N-Ac-Asn(0Me) C I1531
G~~~~,~G~CNAC~O(CH~)~CO~M~ D [154]
GalNAcpl,4GlcNAc~O(CH~)&O2Me D [154]
GalNAc~l,4GlcNAc~1,2Man~O(CH~)~CO~Me D [154]
Ga~NAc~l,4GlcNAc~l,2Manal,6(GalNAc~1,4GlcNAc~1,2Manal,3)
D [154]
Manp0(CH~)sCOzMe
GlcNAc~1,4GlcNAc~O(CH~)~CO~Me D [154]
Galpl,4GlcNAcpR R = GlyGlyAsnGlyGly or N-Alloc-PheAsnSerThrlle C [155]
Galpl,3Galp1,4Glc D [156]
Galal,3Galp1,4GlcNAc D 11561
a A, > 1 g; B, 0.1-1 g; C, 10-100 mg; D, < 10 mg
HO
HoOUDP
+ HO NHAcOR p1,4-GalT,Mn" * H:go& HO Ho
NHAc
OR
OH
H Y HO ~
G Ho
& H OH ":Go--&
HO Ho NHAcOR
HO OH
.:go& HO R' NHAcOR
R' = H, OH, OCH3,OCH&H=CH,
Figure 11.3-13.
example is the P,P-l,l-linked disaccharide, in which the anomeric hydroxyl of

3-acetamido-3-deoxyglucose serves as the acceptor m ~ i e t y ~ 'The
~ ~ ]acetamido
. func-
tion apparently controls the position of glycosylation.
I621
I
Fmoc-Thr(a-0GlcNAc)-COOH Ac-Lys-Pro-Pro-Asn-Thr-Thr-Ser-Ala
* *
1 SPPS, 2. deprotection 0
H(G O Y'NHAC
'COOH
1. pl,4-GalT,
2. a2,3-SiaT,
3. a1, ~ - FucT
4. Pd(PPh3)4
Figure 11.3-14.
AcO 1. SPPS (RINK Amide Resin) HOHO

&o:h
NHAc AcHN
2 TFNH20
*
GIu-NH,
Fmoc-HN OH 3. NaOH, or Ac-Tyr-Asp-Phe-Leu-Pro-Glu-HN
I
0 SOs-pyr, then NaOH OR 0
1. p1,4-GalT,
2. a2,3-SiaT,
3. al,S-FucT
OH
HA OH Ac-Tyr-Asp-Phe-Leu-Pro-Glu-HN
GIu-NH,
I
OR 0
Figure 11.3-15.
NHAc
Protein
Figure 11.3-16.
Table 11.3-2. Relative rates of bl.4-CalT catalyzed transfer of donor substrates.

Donor substrate Relative Rate Ref.
UDP-Gal 100
UDP-Glc 0.3 1165, 1541
UDP-4-deoxy-Glc no
5.5 ~ 5 1
UDP-Ara
HoOUDP
UDP-GalNAc
UDP-GlcNAc HO 0
UDP-GlcN HO%
HO 0.09
HzNOUDP
UDP-5-thio-Gal H O q 5
HoOUDP
pl,4-GalT has also been employed in solid-phase oligosaccharide synthesis, and

has been used to galactosylate gluco and cellobio subunits of polymer-supported
oligosaccharides and polysaccharides[160]. The resulting oligosaccharides can then
be removed from the support by either a photochemical cleavage or a chymotrypsin-
mediated hydrolysis. GlcNAc-amino acids and peptides have also been used as
substrates for p1,4-GalT to afford galactosylated glycopeptides (Fig. 11.3-
1 6 ) [ 1 5 3 , 155, 160. 1611 ~h
. e carbohydrate chain can then be further extended with other
glycosyltransferases, such as SiaT and F u c T ~ ’ ~ ~ , “‘1,
“‘9 as was shown in the
enzymatic solid-phase synthesis of glycopeptides from MAdCAM-1 (Fig. 11.3-
14)[“lb]. Furthermore, solid- and solution-phase techniques can be employed
I 623
together for the synthesis of complex sulfated glycopeptides such as those from
PSGL-1 (Fig. 11.3-15)['"d]. In terms of glycolipids, P1,4-GalT was utilized in the
preparation of a ceramide-linked LacNAc glycoside that was further enzymatically
sialylated to provide a GM3 analog['55r162].
With regard to the donor substrate, P1,4-GalT also transfers glucose, 4-deox-
ygalactose, arabinose, glucosamine, galactosamine, GalNAc, and 2-deoxyglucose
from their respective UDP-derivatives.This flexibility provides an enzymatic route to
oligosaccharides which terminate in Pl,4-linked residues other than galactose[326],
such as 5-thiogalactose[1641 (Table 11.3-2).Although the rate of enzyme-catalyzed
transfer for many of these unnatural donor substrates is quite slow, this method is
useful for milligram-scale synthesis. Besides P1,4-GalT, other GalTs are also of
interest synthetically.Recently, al,3-GalT has received a heightened focus because of
its role in xenotransplantation studies. Several studies of substrate specificity and
synthetic potential have also been carried
Sialyltransferase (SiaT). a2,G- and a2,3-~ialyltransferasehave been used for oligo-
saccharide synthesis [166168] . Sialyltransferases generally transfer N-acetylneura-
mink acid (NeuAc)to either the 3- or 6-position of terminal Gal or GalNAc residues
Table 11.3-3. Products of sialytransferase reactions.

CMP-NeuAc + a2,6-SiaT Scale" Ref
D
D
C
NeuAca2,6Galp1,4GlcNAc~OMe
NeuAca2,6Gal~1,4GlcNAc~l,3GaI~l,4Glc
NeuAca2,6Gal~l,4GlcNAc~1-N-Asn
NeuAca2,6Gal~l,4GlcNAc~l,2ManaOMe
NeuAca2,6Ga~~1,4GlcNAc~1,3(Ga~~1,4GlcNAc~1,6)Cal~l,4Glc~OMe
NeuAc(9-O-Ac)a2,6Gal~1,4GlcNAc
NeuAca2,6Galpl,4GlcNAc~R R = OH, NHs,GlyGlyAsnGlyGly or
N-Alloc-PheAsnSerThrIle
NeuAca2,6Ga~~l,4G1cNAc~l,4(NeuAca2,6Gal~l,4GlcNAc~l,2/3)Gal~O
D
(CH2)5C02Me
CMP-NeuAc + a2,S-SiaT
NeuAca2,3Galpl,4GlcpOMe D [166]
NeuAca2,3Gal~l,4GlcNAc~OMe D [166]
NeuAca2,3Galpl,3GlcNAc~OR;
R = Me,Ph,(CHz)sC02Me D [166]
NeuAca2,3Gal~1,3GlcNAcpl,3Gal~l,4Glc D [166]
NeuAca2,3Gal~l,3GlcNAc~l,3Gal~O(CH2)8C02Me D [254]
NeuAca2,3Gal~l,3GlcNAc~1,6Gal~O(CH2)8C02Me D [254]
NeuAca2,3Galpl,3GlcNAcpOR (R= Et) C [I771
(R= H,(CH2)5COzMe) D 11671
NeuAca2,3Galp1,3(NeuAca2,6)GalNAc~OPh D [178]
3-O-Me-Gal~l,4Glc~l,6(NeuAca2,3Gal~l,4)GlcNAc~l,3Gal~l
,4Glcp1,6- D [179]
(NeuAca2,3Gal~1,4)GlcNAc~OMe
a A, > 1 g; B, 0.1-1 g; C, l(L-100mg; D,< 10 mg
(Table 11.3-3)[1691. Some SiaTs accept CMP-NeuAc analogs which are derivatized at
the 9-position with amino, fluoro, azido, acetamido, or benzamido
groups[139, 140. 142c,d, 168-1701 A
. zido-, phthalimido-, carbamate, and pivaloyl analogs
of LacNAc and Galpl,3GalNAc are also substrates for the enzymes[172].Sialyl-
transferases have been used to append NeuAc to galactose on the terminus of
glycopeptides [‘“I, glycolipids and glycoproteins [‘“I.
Fucosyltransferase (FucT). Fucosyltransferases are involved in the biosynthesis of
many blood-group substances and tumor-associated antigens. al,3-FucT L-fucosy-
lates the GlcNAc 3-position of LacNAc and sialyla2,3LacNAc to provide the Lewis X
and sialyl Lewis X structures, respectively Several other acceptor substrates with
modifications in the GlcNAc residue [lactose, Galpl,4Glucal, Galp1,4(5-thioGlc)]can
also be fucosylated by various FucT isozymes (Table 11.3-4)[182]. a-1,3/4-FucT
fucosylates either the GlcNAc 3-position of Galpl,4GlcNAc or the GlcNAc 4-position
of Galpl,3GlcNAc (as well as the sialylated versions) to afford (sialy1)Lewis X or
(sia1yl)Lewis A, respectively[174,17’, . Furthermore, a1,3/4-FucT will transfer a
fucose residue which is substituted on C-6 by a very sterically demanding structure.
Notably, a synthetic blood group antigen can be attached, and the resulting “oligo-
saccharide” can be transferred to an acceptor from its GDP derivative[’86].This
approach has been employed to alter the antigenic properties of cell-surface
glycoproteins.
The Lewis A al,4-FucT has been used to transfer unnatural fucose derivatives-
from their GDP esters. 3-Deoxyfucose and L-arabinose are transferred to LacNAc-
pO(CH2)8C02CH3 at a rate of 2.3% and 5.9%, respectively, relative to ~ - f u c o s e [ ’ ~ ~ ] .
Moreover, al,3-FucTs have been extensively employed as the final step in an
enzymatic cascade for the synthesis of complex oligosaccharides [lS71, glycopep-
tides and glycoproteins [1811 in which the sLeX structure is formed. N-Acet-
ylglucosaminyltransrase (GlcNAcT).In viuo, the N-acetylglucosaminyl transferases
control the branching pattern of N-linked glycans[188, . Each of these enzymes
transfers a p-GlcNAc residue from UDP-GlcNAc to a high mannose-based acceptor.
The GlcNAc transferases I-VI, which catalyze the additon of the GlcNAc residues to
Table 11.3-4. Products of fucosyltransferase reactions

CDP-Fuc + a1,2 or a1,3/4 FucT Scale” Ref.
Fucal,2GalpOR;R = CH2CH3,(CH2)6NH2 C [184]

R = H,(CH2)6NH2
Fucal,2Gal~l,4GlcNAc~OR; C [184]
Fucal,3(NeuAca2,3Gal~l,4)GlcNAc~O(CH2)~C02Me C,D 1175,1831
Fuca1,3(Galp1,4)-5-thio-Glc C [183]
Fucal,4(Gal~l,3)GlcNAc C [183]
Fuca1,3(NeuAca2,3Gal~l,4)Glucal D [163]
Fucal,4(NeuAca2,3Ga~~l,3)GlcNAc~l,6Gal~O(CH~)~CO~Me D [lSS]
Fucal,4(NeuAca2,3Gal~l,3)GlcNAc~l,3Gal~O(CH2)~CO~Me D [185]
Fucal,4(Gal~l,3)GlcNAc~O(CH~)~CO~Me D [185]
3-deoxy-Fucal,4(Gal~1,3)GlcNAc~O(CH~)~CO~Me D [184]
S-desmethyl-Fucal,4(Gal~1,3)GlcNAc~O(CH~)~CO~Me D [lSS]
a A, > 1 g; B, 0.1-1 g; C, 10-100 mg; D,< 10 mg
Table 11.3-5.
Products of ClcNAc-transferase reactions.

I
625
~ ~~
UDP-ClcNAc (or analogs) + ClcNAcTase Scale” Ref.

~ ~~ ~
UDP-GIcNAc + GlcNAcT I
G~cNAc~1,2Manal,3(Manal,G)Man~O(CH~)~CO~Me C [59, 1921
3-deoxy, 4-deoxy, or G-deoxy-GlcNAc~l,2Manal,3(Manal,G)Man~O D [190]
(CH2)8C02Me
UDP-CICNAC+ CICNACTII
GlcNAc~l,2Manal,G(GlcNAc~l,2Manal,3)Man~O(CH,~)~CO~MeD [192]
UDP-CICNAC+ CICNACT
G~cNAc~l,G(Gal~l,3)GlcNAc D [193]
UDP-CIC+ CICT
GlcPOR; R = C H ~ C H J(CH&NH2
, C,D [194]
a A, > 1 g; B,0.1-1 g: C, 10-100 mg; D,< 10 mg
the core Asn-linked pentasaccharide of glycoproteins (Fig. 11.3-16), have been

identified and characterized[Iss, lS9l.
GlcNAc transferases have been utilized for the synthesis of natural and non-
natural oligosaccharides (Table 11.3-5).In addition to transferring GlcNAc, GlcNAcT
I from human milk catalyzes the transfer of 3-, 4-, or 6-deoxy-GlcNAc from its
respective UDP derivative to Man al,3(Manal,6)Man~O(CH2)sC02CH3[”o~ 19‘1.
The 4- and 6-deoxy-GlcNAcanalogs can also be transferred by GlcNAcT 11, although
UDP-3-deoxy-GlcNAcis not a substrate for this The core 2 GlcNAcT can
employ UDP-trifluoro-GlcNAcas a s~bstrate1”~I. GlcNAcT has also been used to
attach the terminal GlcNAc of GlcNAcpl,4GlcNAca dolichyl pyrophosphate, a
substrate of oligosaccharyltransferase[’gll.Mannosyltran~ruse(ManT). Various
mannosyltransferases have been shown to transfer mannose and 4-deoxymannose
from their respective GDP adducts to acceptors [1971. al,2-ManT transfers mannose
to various derivatized a-mannosides and a-mannosyl peptides to produce the
Manal,2Man structural This method has also been extended to whole cells
as a source of al,2-ManTL”’]. Mannosyltransferases from pig liver accept
GlcNAcpl,4GlcNAc phytanyl pyrophosphate, an analog of the natural substrate in
which the phytanyl moiety replaces dolichol [200]. Overexpression of P1,CManT has
also been instrumental in the synthesis of an N-glycan core structure L2011 as well as
the bacterial 0 p1,CManT is especially valuable synthetically, as p-
mannosyl glycosides are exceedingly difficult to form chemically.
Sucrose synthetase. The fructose derivatives 1-azido-1-deoxy-,1-fluoro-1-deoxy-,
6-deoxy-,6-fluoro-6-deoxy-,and 4-fluoro-4-deoxy-fructosehave been used as glycosyl
acceptors in the sucrose synthetase-catalyzed synthesis of sucrose analogs [*031.
6-Deoxy- and 6-fluoro-6-deoxy-fructose were generated in situ from the correspond-
ing glucose derivativesunder catalysis by glucose i s o m e r a ~ e [ ~Sucrose
~ ~ I . synthetase
has also been extensively employed in the synthesis of nucleotide sugars l2O4l.
626
I
* OHo
H O W OOH H NHAc
HO
OH f
E j : pl,4-GalT
E2: pyruvate kinase
E3: UDP-Glc pyrophosphotylase
E4: UDP-Glc 4-epimerase
E5: pyrophosphotylase
E6:phosphoglucomutase
Figure 11.3-17.
11.3.2.3
In Situ Cofactor Regeneration
Though analytical and small-scale synthesis using glycosyltransferases is extremely

powerful, the high cost of sugar nucleotides and the product inhibition caused by the
released NMP or NDP present major obstacles to large-scale synthesis. A simple
solution to both of these problems is to regenerate the sugar nucleotide in situ from
the released NDP[2051. The first example of this strategy was the pl,4-GalT-catalyzed
synthesis of LacNAc[2011(Fig. 11.3-17). Only a catalyhc amount of UDP-Gal is
initially used to glycosylate GlcNAc. However, UDP-Gal is regenerated from the
product UDP and galactose using an enzyme-catalyzed reaction sequence which
requires stoichiometric amounts of a phosphorylating agent. Several oligosacchar-
ides have been prepared using routes based on this concept[150]. Another regenera-
tion system for UDP-Gal, which is based on the use of galactose-1-phosphate
uridyltransferase, has also been A third, which employs sucrose
synthetase for recycling of UDP-Glc/UDP-Gal from sucrose and UMP has recently
been A very recent approach to recycling systems employs coupling
metabolically engineered bacterial cells for large scale sugar nucleotide production,
to date including UDP-Gal (Fig. 11.3-18)L2O9I and CMP-NeuAcI'441.
In situ cofactor regeneration offers several advantages. First, a catalytic amount of
NDP and a stoichiometric amount of monosaccharide are used as starting materials
rather than a stoichiometric quantity of sugar nucleotide, thus tremendously
reducing costs. Second, product inhibition by the released NDP is minimized
I
7 7.3 Hydrolysis and Formation ofClycosidic Bonds 627
UTP- UDP C- UMP
Globotriooe
-0&
HO
OH
OH
Figure 11.3-18.
because of its low concentration in solution. And third, isolation of the product is
greatly facilitated.
A multi-enzyme regeneration system for CMP-NeuAc is illustrated in Fig. 11.3-
19[135,2081, n'IS system follows the same basic principles as the UDP-Gal recycling
system. A CMP-NeuAc synthetase/a2,3-SiaTfusion enzyme with increased stability
has also been applied to this The development of these regeneration
systems, as well as those for G D P - M ~ ~ I [ ~GDP-FucL'~~],
'~], and UDP-G~CUAL~~']
should facilitate the widespread use of glycosyltransferases for oligosaccharide
synthesis. Notably, when UDP-GlcUA and UDP-GlcNAc recycling systems are
combined with hyaluronic acid synthase, HA polymers can be New
/ "n NHAc
CTP c HO OH
Aco;
E, : a2,3-sialyltransferase; E,: nucleoside monophosphate kinase;

E,: pyruvate kinase; E,: CMP-NeuAc synthetase; E5: pyrophosphatase
Figure 11.3-19.
628
HO
AcNH
0 0
It II
o=s-0-P-
I I
0- 0-
PAPS
0 O H \ / o=po
/ Sulfotransferase IV \
Figure 11.3-20.
systems for the recycling of PAPS for the synthesis of complex sulfated carbohy-
drates have also recently been developed (Fig. 11.3-20)f2l2I.
11.3.2.4
Cloning and Expression o f Clycosyltransferases
While many glycosyltransferases catalyze similar reactions and use the same donor
substrate, there appears to be little sequence homology among the different enzymes
of this class (i.e GalT vs SiaT, etc.). There is, however, a significant cross species
homology for the same glycosyltransferase. For instance, one finds 86% identity
when comparing the P1.4-GalT protein sequence from humans to that from rat. The
different glycosyltransferases do exhibit some similarity in that their cDNA se-
quences encode regions consistent with a short N-terminal tail, a hydrophobic
transmembrane sequence, a short stem sequence, and a large C-terminal catalytic
domain [2131. In addition to the membrane-bound form of the glycosyltransferases,
soluble enzymatic forms have also been identified in body fluids such as blood, milk,
and colostrum. Indeed, these fluids have been sources for the purification of specific
glycosyltransferasesL2lk2l7].A comparison of the cDNA sequences of these soluble
enzymes with full-length glycosyltransferasegenes suggests that the stem region has
been cleaved to release the large catalytic domain from the membrane. Presumably,
this theme of signal sequence cleavage is consistent for all the glycosyltransferases
(Fig. 11.3-21)[2191.
The amount of a glycosyltransferase that can be isolated from natural sources is
often limited by the low concentrations of these enzymes present in most tissues and
7 1.3 Hydrolysis and Formation ofclycosidic Bonds
Figure 11.3-21.
I 629
body fluids. The purification of glycosyltransferases is further complicated by their

relative instability["]. For this reason, a great deal of interest has been directed
toward the cloning of glycosyltransferase genes into convenient expression sys-
t e m ~ [2191~ ~(Table
, 11.3-6).The general strategy involved is outlined in Fig. 11.3-22.
The glycosyltransferase gene must first be identified and isolated from the mRNA
pool via the cloning of the cDNA to make a cDNA library. This library is then
screened to identify the glycosyltransferase gene of interest among - lo6 different
sequences present. Once identified, the gene is sequenced and a more complete
cloning strategy is developed in order to incorporate the gene into an expression
vector. This laborious path has been successfully employed by several groups, many
ofwhom are referenced in Table 11.36 The nuances to the general cloning scheme
used by these groups are discussed below.
Among the organs that have been used for the isolation of glycosyltransferase
mRNA are the liver [220, 2211, placenta [222], mammary gland[223],testis [2241, and
Table 11.3-6. Cloned glycosyltransferaseso f the glycoprotein and glycolipid pathways.

Enzyme Source Ref.
UDP-Glucuronosyltransferase murine liver POI

rat liver PI1
Mannosyltransferase yeast ~321
a2,G-Sialyltransferase rat liver ~291
a2,3-Sialyltransferase porcine submaxillary gland 12331
~1,4-Galactosyltransferase bovine placenta P I
bovine mammary gland ~231
murine mammary gland WI
bovine liver PSI
murine F9 cells 12361
bovine kidney epithelial cells WI
murine testes ~241
human placenta ~381
~1,3-Galactosyltransferase calf thymus 12251
murine F9 cells WI
a1,2-Fucosyltransferase human A431 cells ~ 7 1
al,3/4-Fucosyltransferase human A431 cells 12391
630
(liver, testis, placenta,

mammary gland, thymus,
with DNA probes,
antigenic response, cDNA insert
or lectin screenings)
A
Extraction
of mRNA
I Subclone into
‘ I Restriction map
and redesign of
u
- / expression system
1
expression system
I
L I I
Synthesis
of cDNA
/
Figure 11.3-22.
In addition, tissue cultures have been used in place of the organ[22G,

2271.
From these sources, cDNA is synthesized, and the double stranded cDNA is ligated
in h phage via a convenient linker and packed into bacteriophages. The bacter-
iophages are then plated onto a lawn of E. coli, and screened for the desired gene or
gene product. Identification of the glycosyltransferase gene has most frequently
been achieved by the hybridization of the gene to specific radiolabeled DNA
probes [220-2231. Screening in this manner obviously requires a previous knowledge of
the gene sequence - information that in some cases may be obtained by extrapola-
tion from a partial protein sequence or from the DNA sequence of the glycosyl-
transferase from a related source. Two other approaches have been used to screen
glycosyltransferase cDNA libraries, both requiring successful transcription and
translation of the gene product. In the cloning of the aZ,(i-SiaT from rat liver,
Weinstein et al. used polyclonal antibodies raised to the purified enzyme to screen
the X The approach used by Larsen et al. alleviated the need for a
previous knowledge of the sequence[226]. This method made use of the specificity of
a lectin that recognizes the surface-expressed glycoconjugate product of al,3-GalT.
The transfected cells were then panned in dishes coated with the lectin. The
adherent cells were isolated and re-panned for further purification. Each of these
techniques makes use of libraries in which there are very few copies of the desired
gene. A greater chance of success may be possible if the number of copies of the
genes could be amplified. The introduction, in 1985, of an in vitro amplification
method based on the polymerase chain reaction (PCR)fulfilled this need[229,2301. Of
course PCR (and ECPCR)[2301, like the hybridization screening, requires a specific
knowledge of the sequence.
Once identified, the genes are sequenced using standard procedures. Recloning of
the gene into an expression vector is then used to develop an expression system. This
recloning has been performed on only a few of the glycosyltransferases.Toghrol et al.
have inserted the mouse liver GlcUAT gene into the yeast vector pEVPll and
expressed the enzyme in Saccharomyces cerevisiae[2201.The rat liver GlcUAT, on the
7 7.3 Hydrolysis and Formation of Clycosidic Bonds
other hand, has been expressed in COS cells using the SV40 Expression
in COS cells using SV40 was also applied to the cloning of bovine j31,4-GalT[222]. A
noteworthy approach toward the expression of glycosyltransferases in E. coli has
been developed by Aoki et al. to obtain human j31,4-GalT[231].A unique RsrII
restriction site in the P1,4-GalT gene allowed the dissection of the sequence at the
location of signal peptidase cleavage. The cohesive terminus was digested with
Klenow fragment, and the blunt end ligated to pINIII-ompA2[232] at a Klenow
fragment treated EcoRI site. This generated the code for a soluble fusion protein of
j31,4-GalT with the ompA signal sequence. Transcription and translation of this
sequence in E. coli produced an active enzyme that was released into the periplasmic
space. Purification and N-terminal sequencing of the enzyme verified the expression
of the soluble form of j31,4-GalT with an additional tripeptide N-terminal tail. The
kinetic parameters of this enzyme appear to be identical to the isolated native
enzyme.
To date, over a hundred glycosyltransferaseshave been clonedLZ1l.Expression and
production in quantities sufficient for enzymatic synthesis is, however, another
matter. Only a handful of glycosyltransferases are currently commercially available.
Given the advantages of enzymatic synthesis of oligosaccharides over traditional
schemes, research into the overexpression of glycosyltransferases will undoubtedly
continue to be developed.
11.3.3
Non-Leloir Clycosyltransferases: Transfer o f Clycosyl donors from Clycosyl Phosphates
and Glycosides
Oligosaccharides can also be prepared using non-Leloir glycosyltransferases. Phos-

phorolysis is reversibly catalyzed by glucan phosphorylases for the synthesis of
polysaccharides. For example, and t r e h a l ~ s e [ ~have
~ ~ ”been
] synthesized
by the corresponding p h o s p h ~ r y l a s e [ ~Sucrose
~ ~ ” ] . phosphorylase has also been used
in the recycling of UDP-Ga1[241].Other enzymes of this class are involved in the
synthesis of dextrans and levans [2421.
Modified polysaccharides may provide materials with more desirable physical and
biological properties than their natural counterparts. Approaches to controlling
glycopolymer characteristics have included the control of genes encoding the
enzymes responsible for their production, regulation of the activity of these en-
zymes, or the influence of their in vitro synthesis [2431. Potato phosphorylase has been
used in vitro to prepare maltose oligomers,[”] as well as a family of linear, star, and
comb-shaped polymers [2441. This enzyme will synthesize polysaccharides in the
presence of primers
A coupled potato phophorylase/sucrose phosphorylase system, where glucose-
1-phosphate is generated in situ from sucrose and inorganic phosphate, has been
employed for polysaccharide synthesis [loo].The inorganic phosphate liberated by
potato phosphorylase is used by sucrose phosphorylase to drive the formation of
polymer, thereby increasing the yield. Regulation of the molecular weight of the
polysaccharide product can be controlled by the concentration of the primer.
I
4
632 7 1 Hydrolysis and Formation of C-0 Bonds
HO
HO
Sucrose Potato
phosphorylase phosphorylase
+ HOOP032-
HO OH Primer
--Pnrner
OH OH HoYxonOH n
Sucrose, P,,
sucrose
ln>61
Figure 11.323.
Unnatural primers bearing functional groups can also be used to prepare tailor-
made polysaccharides for further manipulation, e. g. attachment to protein or other
compounds (Fig. 11.3-23).
Cyclodextrin al,4-glucosyltransferase (CD a1,4-GlcT, E. C. 2.4.1.19) from Bacillus
macerans catalyzes the cyclization of oligomaltose to form a-, 0- and &cyclodextrin,
n'
and the transfer of sugars from cyclodextrin to an acceptor to form oligosacchar-
ides[245. 2461. is enzyme can transform a-glucosylfluoride into a mixture of a- and
P-cyclodextrinsand malto-oligomers[2471. When immobilized on a silica gel support,
CD a1,CGlcT was very stable, with no loss of activity observed after 4 weeks when
stored at 4 "C. This type of enzymatic catalysis may provide a new route to unnatural
cyclodextrin analogs and novel oligosaccharides, as glucose analogs are also sub-
strates. For example, oligoglucosyl deoxynojirimycin and N-substituted derivatives
were produced under CD al,4-GlcTcatalysis. Subsequent hydrolysis by glucoamy-
lase gave glycosylazasugars like 4-O-a-~-glucopyranosyl deoxynojirimycin in - GO %
yield (Fig. 11.3-24)[2481 The N-methyl derivative was reported to be a potent inhibitor
of glucosidase.
In spite of the progress that has been made, several difficulties limit the use of cell-
free enzymes for the synthesis of polysaccharides. The major problem is the
complexity of many polysaccharide-synthesizing systems. Isolation, purification,
and stabilization of the required enzymes is often difficult, as many enzymes lose
activity when they are no longer membrane-associated. Enzyme isolation from
eukaryotic sources is tedious, because of low cellular enzyme concentration. It is
unlikely that cell-free enzymatic synthesis will provide better routes to most natural
polysaccharides than do fermentation and isolation. The use of genetic engineering,
OH
+ (glucose), 1. Cyclodextrin
glucosyltransferase * Ho%&R
HO
HHO o e R
OH 2. Glucoamylase HO
OH
Figure 11.3-24.
633
7 7.3 Hydrolysis and Formation ofClycosidic Bonds
I
HO
HO & OH
X
ROH, glycosidase *&
HO
HO OH
OR
Equilibrium conditions: X = OH
Kinetic conditions: X = F, o or p N 0 2 , OR’
Figure 11.3-25.
both using classical genetics and recombinant DNA technology, is an approach

being used to prepare modified carbohydrate polymers [2501.
11.3.4
Clycosidases
Glycosidases catalyze the hydrolysis of glycosidic linkages, typically with retention of

configuration at the anomeric carbon (P-galactosidaseand lysozyme),but sometimes
with inversion (trehalase and (3-amylase)[2511. Enzymatic hydrolysis is thought to be
mechanistically similar to acid-catalyzedhydrolysis of glycosides. Both proceed via
an oxonium ion intermediate or a transition state having oxonium ~haracter[~”]. A
proximal carboxylate in the enzyme active site appears as a common structural motif
among glycosidases, and presumably acts to stabilize this intermediate or transition
state. Whether the oxocarbenium ion exists as a stabilized ion pair or is trapped by
the carboxylate to form a glycosyl ester has been the subject of debate. However, an a-
glycosyl enzyme intermediate has been observed by ”F NMR in the P-glucosidase-
catalyzed hydrolysis of 2-deoxy-2-fluoro-~-glucosyl fluoride, and was shown to be a
catalytically competent species[2521.
Glycosidase-catalyzedglycoside synthesis is quite analogous to protease-catalyzed
peptide synthesis. As with proteases, glycosidases may be used under either
equilibrium or kinetically controlled conditions for synthetic purposes (Fig. 11.3-
25) [84. 2531
11.3.4.1
Equilibrium-controlledSynthesis
The obvious approach to glycosidase catalyzed synthesis of glycosidic linkages

involves reversing the catabolic role of the enzyme. Indeed, examples of equilibrium-
controlled synthesis were reported by Bourquelot at the early part of this century[253].
Synthesis by this approach involves an endergonic process with the free energy
change under ambient aqueous conditions favoring bond cleavage by approximately
4 kcal/mol. Reaction conditions must therefore be manipulated in order to drive the
reaction to produce glycoside.
In efforts to shift the equilibrium toward product, the addition of water-miscible
organic cosolvents was investigated, but this usually results in enzyme inactivation
and a decrease in K, for the glycoside acceptor[254].The use of high substrate
concentrations and elevated reaction temperatures have also been explored. Jo-
hansson et al. [2551 reported the synthesis of mannose disaccharideswith jack bean a-
mannosidase, while Ajisaka et al. [2561 utilized almond P-glucosidase with glucose
634
concentrations as high as 90 % w/v to obtain glucose disaccharides. Carbon-celite[2573

and active carbon columns have been developed as molecular traps which selectively
absorb the product as the reaction mixture is circulated through the column. Yields,
however, are still only about 15%. Though quite simple in theory, the equilibrium
approach generally provides poor yields and the formation of side products, which
make purification difficult.
11.3.4.2
Kinetically Controlled Synthesis
Kinetically controlled synthesis relies on the trapping of a reactive intermediate

generated from an activated glycosyl donor to form a new glycosidic 2541.
The trapping agent is generally an exogenous nucleophile. Suitable glycosyl donors
for this transglycosylation reaction include di- or oligosaccharides, aryl glycosides,
and glycosyl fluorides (Table 11.3-7).The reactive intermediate must be trapped by
the glycosyl acceptor more rapidly than by water[258].Under the proper conditions,
glycoside formation may be favored kinetically, but hydrolysis is always favored
thermodynamically. The reaction must therefore be carefully monitored, and ar-
rested when the glycosyl donor is consumed in order to minimize subsequent
glycoside hydrolysis. Recently, mutant glycosidases have been engineered to avoid
competing product hydrolysis. Because these enzymes lack a catalytic nucleophile in
the active site, they can synthesize but not hydrolyze glycosides [293g*h1.
In a comparative study of kinetically vs thermodynamicallycontrolled synthesis of
Galpl,GGalNAc, the kinetic approach afforded of 10-fold increase in product yield
(20% vs 2 %) [2591. Yields in kinetically controlled synthesis generally range from 20
to 40%. Although addition of organic solvent has not generally been observed to
increase product yields, increase of acceptor or donor concentration seems to be
quite effective. As an exception, though, polyethylene glycol-modified P-galactosi-
dase is soluble in organic solvents and seems to be suitable for transglycosyla-
tion [2601.
The kinetically controlled approach has primarily been applied to the retaining
glycosidases. However, using glycosyl fluorides as glycosyl donors, an inverting
glycosidase has been used to afford products having the configuration at the
anomeric position which is opposite to that of the For example, the a,a-
linkage of a-D-glucopyranosyl-a-D-xylopyranoside has been prepared utilizing 0-
glucosylfluoride and a-trehalase
11.3.4.3
Selectivity
The primary goal of enzymatic glycoside formation or oligosaccharide synthesis is to

achieve selectivity which is difficult to achieve by chemical methods. Glycosidase-
catalyzed chemoselective reaction of one hydroxyl group of an unprotected sugar
with the glycosyl donor has been observed, although the selectivity is not necessarily
absolute or predictable. Kinetically controlled synthesis has been more successful
Table 11.3-7.
11.3 Hydrolysis and Formation ofclycosidic Bonds
Synthesis o f oligosaccharides and other glycosides using glycosidases.

I
635
Substrate Produd Scalea Ref.

aCaladosidase [252, 4621
Raffinose + CHI = CHCHzOH GalaOCH2CH = CHZ A [263]
Gala0-p-PhNO2+ GalaOCHZCH = CHz Galal,3GalaOCHzCH = CHZ B [263]
+ Gal(a or p)OMe Galal,3/6GalaOMe B [263]
Gala0-o-PhNO2+ Gal(a or P)O-p-PhNOz Galal,2/3GalaO-p-PhNOz C [263]
fi-Caladosidase[252, 4631
GlcPOPh + ROH (R = alkyl) GlcPOR C [267]
GalP1,4Glc + GlcNAc-R (R = O H or SEt) Galpl,3GlcNAcR B [265]
GalP1,4Glc + GlcNAc GalPl,4GlcNAc A [269]
+ GlcNAc Galp1,3-GlcNAc B [259,268]
+ GalNAc GaQ31,G-GalNAc B [259, 2681
+ ROH (R = allyl, benzyl, GalPOR A, B [263]
TM S (CH2 ) 2 ) GalP1,3/6GalPOR B (2631
b
GalPOPh + B [270]
R' 1. R', R4 = H , R2 = OH, R3 = CHJ (Gitoxigenin)

2. R', RZ, R4 = H, R' = CH3 (Digitoxigenin)
3. R', RZ= 0, R' = CH', R4 = H (16P, 17P-epoxy-17a-digitoxigenin)
4. R', R2 = H , R' = CHO, R4 = OH (Strophanthidin)
GalPO-o-PhNOZ + GalaOMe GalP1,bGalaOMe C [263]

+ GalPOMe Galpl,6/3GalPOMe C [263]
GalbOPh + ROH (R = alkyl) GalPOR B [271]
GlcPOPh + BnOH GlcPOBn B [271]
GalP1,4Glc + sucrose Galbl,6al,2Fru E [272]
GalP1,4Glc or GalPODh +
E [273]
HO n=lor2 GalPO (89-90% de)
E [273]
R = (CH& or GalPO (75 % de)
Ho CH=CH "O
E [273]
(50% de)
636
Substrate Product Scale” Ref.

fi-Calactosidase [252,463]
Galpl,4Glc or GalPOPh + ROH GalPOR A, B [274, 2751
GlcPOPh + ROH GlcPOR B [275, 153,

1541
Galactal + ROH 2-deoxy-GalPOR E [159]

Galactal + Galactal 2-deoxy-Galpl,3/6Galactal+2-deoxy- C (1601
Gal~l,3-2-deoxy-Gal~l,6Galactal
GalpO-o-PhNO2 + 2-Ser-OR GalPO-2-Ser-OR C [lSS]
GalPO-o-PhNO2 + Ser GalpOSer E [279]
GalP1,4Glc + 2-Ser-OMe GalpO-2-Ser-OMe B 12801
a-Mannosidase[252,464]
ManaO-p-PhNO2 + ManaOR Manal,2/6ManaOR B [263]
(R = Me or p-PhNO2)
a-Glucosidase [252,465]
Glc + Fru Glca1,lFru D 12631
Glcp1,4Glc + HO C [154, 2671
HO x) GlcaO
fi-Glucosidase[252,4651
Glc Glcpl,4/6Glc C [284]
Glcpl,4Glc Glc~l,4Glc~l,4Glc C (2841
8-N-Acetylhexosaminidase [252, 46151

Gal(Glc)NAcpO-p-PhNOz+ Glc Gal(Glc)NAc~l,3/4Glc(NAc)POMe C [285]
(NAc)POMe
Gal(Glc)NAcpO-p-PhN02+ Glc Gal(Glc)NAc~l,4/6Glc(NAc)aOMe C [285]
(NAc)aOMe
a-Fucosidase [252,467]
FucaO-p-PhNOz + Gal(a or p)OMe Fucal,3Gal(a or p)OMe E ~ 4 1
Neuraminidase[252,468]
NeuAca-p-PhN02 + Gal(a or p)OMe NeuAca2,3/6Gal(a or p)OMe D [289]
NeuAca-p-PhN02 + Galpl,4GlcNAc NeuAca2,3/6GalPl,4GlcNAc D [289]
a A, > 1 g; B, 0.1-1 g; C, 10-100 mg; D, < 10 mg; E, not reported
I 637
chitinase
HO polymerization
Figure 11.3-26.
L NHAc 1"
than thermodynamicallycontrolled synthesis in achieving selectivity. In general, the
primary hydroxyl group of the acceptor reacts preferentially over secondary hydroxyl
groups, resulting in a 1,G-glycosidic linkage. Some control of selectivity has been
demonstrated by the selection of an appropriate donorlacceptor combination. For
example, the a-galactosidase-catalyzed reactions of a-Gal-OPh-p-NO2with a-Gal-
OMe and P-Gal-OMeform predominantly a-1,3 and a-l,G linkages, re~pectivelyl~"].
The substituent at the anomeric center of the acceptor controls the position of
glycosylation to some extent. aGal-OPh-p-NO2'acting both as donor and acceptor,
forms preferentially the a-1,3 linkage, whereas the ortho-nitrophenylglycoside reacts
in a similar fashion to form predominantly the a-1,2 linkage [2631. With P-galactosi-
dase, P-1,3-linkeddisaccharides were formed preferentially when benzyl or ally1 p-
galactosidewas used as acceptor[84.2631. The use of glycals as acceptors has also been
employed as a means of controlling ~electivityI~~~1.
One can also use glycosidasesfrom different species to control the regioselectivity.
For example, the 0-galactosidase from testes catalyzes the formation of Galpl,3Glc-
NAc[2651 from lactose and GlcNAc. The minor products produced in this preparation
were then hydrolyzed by the E. coli p-galactosidase, which preferentially hydrolyzes
P-1,G-linked galactosyl residues. The overall yield of the P-1,3-linked disaccharides
was around 10-20 %.
Synthesis of polysaccharides based on kinetically controlled glycosidase reactions
have been accomplished, as exemplified by the cellulase-catalyzed reaction of p-
cellobiosyl fluoride to form cellulose, with degree of polymerization c 22L266]. In
another strategy, employing a chitin hydrolysis transition state analog, chitinase
catalyzed polymerization was accomplishedwithout competing hydrolysis (Fig. 11.3-
26) [2491. Glycosyl transfer to non-sugar acceptor has also been demonstrated. These
reactions are especially interesting with chiral, racemic, or meso alcoholic acceptors,
as one might expect some degree of diastereoselectivity due to the asymmetric
microenvironment of an enzyme active site. Such selectivity has indeed been
observed, with diastereoselectivities ranging from moderate to exceptional, as
illustrated in Table 11.3-7.
11.3.5
Synthesis of N-glycosides
Nucleosides and their derivatives are ubiquitous in nature, and are involved in a
myriad of biochemical phenomena, most notably the storage and transfer of genetic
information. Interest in this class of compounds has been stimulated by the efficacy
638
of certain nucleosides as antiparasitic [2971 and antiviral agents [298, 2991. Nucleosides
have traditionally been prepared by chemical methods (3001 requiring multiple pro-
tecting group manipulations and glycosyl activation procedures. Problems encoun-
tered include control of anomeric configuration and regiospecific C-N glycoside
formation when there are several possible nucleophilic groups in the purine or
pyrimidine base.
11.3.5.1
Nucleoside Phosphotylase
Enzymatic preparations of both natural and unnatural nucleosides have been

reported using nucleoside phosphorylases as catalysts[3011. These enzymes catalyze
the reversible (but highly favorable) formation of a purine or pyrimidine nucleoside
and inorganic phosphate from ribose-1-phosphate (R-1-P) and a purine or pyr-
imidine base. Nucleoside synthesis has relied on the transfer of the ribose moiety of
a readily available nucleoside to a different purine or pyrimidine base or analogs
through the intermediacy of R-1-P. This work has been done primarily with isolated
but whole cells have also been employed in a few cases[3o3].The
deleterious hydrolases present in whole cells could be largely neutralized by
conducting the reactions at 60 "C, a temperature at which the nudeoside phosphor-
ylases maintain < 70 % of their activity for 3-5 days [3031.
The first synthetic strategy toward nucleosides employed involves isolation of R-
1-P,which can be prepared in good yield from a nucleoside in the presence of a high
concentration of phosphate[3o4]. The isolated R-1-P is then used as the glycosyl donor
in an enzymatic coupling reaction with added purine or pyrimidine bases or analogs.
By this method, generally any heterocycle which is a substrate for a nucleoside
phosphorylase can be glycosylated. The second strategy involves a one-pot exchange
of one base for another in the presence of a catalytic amount of inorganic phosphate
without isolation of R-1-P. At best, this procedure results in an equilibrium mixture
of substrate and product nucleosides, from which the product must be isolated. In
less favorable cases, the natural purine or pyrimidine base released from the glycosyl
donor may be a potent competitive inhibitor versus the purine or pyrimidine analog.
For example, competitive inhibition by hypoxanthine ( K , = 5.6 mM) was the cause
for the lack of glycosylation of 1,2,4-triazole-3-carboxamide (TCA, the aglycon
component of virazole, K, = 167 mM) when inosine was used as the ribosyl donor
and purine nucleoside phosphorylase (PNPase) as the catalyst [25G1. It was, however,
possible to synthesize virazole by isolating R-1-P and subsequently using it as the
ribosyl donor l3O5]. An alternative way to circumvent the inhibition problem is to
employ a pyrimidine nucleoside as the glycosyl donor and a purine (or purine
analog) as the acceptor, since the released pyrimidine base does not inhibit the
purine nucleoside phosphorylase[306]. By this method, both pyrimidine nucleoside
phosphorylase and purine nucleoside phosphorylase are required. Direct purine-to-
purine exchange reactions have been conducted without isolation of R-I-P using
activated purine derivatives as the ribosyl donors r3071.
The nucleoside phosphorylases accept a wide range of nucleoside analogs as
I
639
substrates, with modifications in both the base and glycosyl components. The use of
unnatural bases has met with success using both natural and unnatural glycosyl
donors. However, a few limitations have been observed, such as loss of appropriate
regio-specificitywith unnatural bases I3OG]. The synthesis of sugar-modified nucleo-
sides has made use of glycosyl donors which are prepared by chemical modification
of readily available nucleosides, such as uridine and cytidine. Good yields of
arabino r3O3] and 2'-amino-2'-deoxyribonucleosides L3091 have also been obtained enzy-
matically, although the enzymatic synthesis of 3'-amino-2',3'-dideoxyribonucleosides

has given only low yields L3lo, 3111. The synthesis of ribosides of unnatural purine and
pyrimidine bases and the synthesis of nucleosides containing modified glycosyl
moieties are summarized in Table 11.3-8.Most of these reactions have been carried
out in one step without isolation of the intermediate sugar phosphate, although
involvement of the sugar phosphate intermediate has been demonstrated.
In summary, the nucleoside phosphorylases provide a regio- and stereo-specific
route for nucleoside synthesis which is applicable to nucleoside analogs which are
modified in either the base or the sugar moiety. These processes provide good yields
of products in most cases without the extensive protection and deprotection steps
involved in traditional chemical synthesis of nucleosides. Application of this strategy
to the synthesis of 2'-deoxy-and 2',3'-dideoxynucleosides was reported with the use
of N-deoxyribosyltransferase from Lactobacillus species1301, 3121.
11.3.5.2
NAD Hydrolase
The enzyme NAD glycohydrolase has been used in exchange reactions for the
preparation of NAD analogs (3181. The enzyme accepts nicotinamide analogs with
modification at the amide functionality as substrates. Depending on the structure of
the nicotinamide analogs used, the reaction may be either reversible or irreversible.
NADH and its 6-hydroxyl derivative are not substrates for the enzyme. When
4-amino, 4-methylamino, or 4-dimethylamino nicotinamide or nicotinate was used
as substrate, the product NAD analog existed as a 1,4-dihydro-typet a ~ t o m e r [ ~ ~ ' ] .
11.3.6
Biological Applications of Synthetic Clycoconjugates
11.3.6.1
Clycosidase and Clycosyl Transferase Inhibitors
Carbohydrate analogs and derivatives are valuable in studying the biosynthesis and
modification of oligosaccharides: deoxynojirimycin, swainsonine, and castanosper-
mine inhibit trimming of the N-linked oligosaccharides of glycoproteins[3201; tunica-
mycin and streptovirudin inhibit protein glycosylation in the Leloir pathway[32'];
acarbose inhibits amylase [3221. These inhibitors provide a way of exploring cell-
surface oligosaccharide chemistry, a topic of central interest in differentiation,
development, and disease. Most are relatively easily understood as transition state
640
Table 11.3-8. Nucleoside phosphorylase-catalyzed synthesis with various heterocycles as

acceptors or sugar-modified nucleosides as donors.
Donor Acceptor Method" Yield Ref.
(%)b
~~ ~~
Uridine X = MeS, Y = H B 59-76 [313, 3141

X = NHz, Y = C1
Thymidine X = Me2N, Y = H B 81 [313, 314)
7-N-MethylGuanosine B 100 [307]
Inosine X = C ~ H I I SY, = H A 59 13131
X
Uridine X = NH2, BnNH B 18-79 [313, 3061
Thymidine B 18-71 [313,306]
7-N-Methyl Guanosine B 53 [307]
Uridine X = OH, PhCONH B 23-63 [313, 3151

k
Inosine
7-N-Methyl Guanosine
.GN N."
A
B
47
44
[305]
[307]
X = NHz, Y = H, NH2, C H j
N X = OH, Y = C1, H , NH2, CH, B 3 6 9 2 [316]
HO
,!+X = S H , Y = N H z
1-(P-o-arabinosyl)uracil
X = NH2, Y = H B 20-50 [308, 309,

HO NH2 X=OH,Y=H,NHz,CI 3171
2'-amino-2'-deoxyuridine
0
B 7-29 [310]
deoxythymidine
H G e "
HO R
B 12-17 [306]
R = H (5'-deoxyuridine)or ?.
R = OH (5'-deoxythymidine)
a Method A: a-Glycosyl-1-phosphate generated and isolated prior to addition of acceptor heterocycle
Method B: In situ generation of a-glycosyl-1-phosphate.
b Yields are based on the initial amount of heterocycle acceptor.
analogs, and the design of new sugar analogs to inhibit other glycosidases and
glycosyltransferases [Is2. 3231 can be accomplished.
The syntheses of these types of structures are not straightforward using classical
synthetic methods. Enzymatic methods have already been proven to be very useful in
I 641
syntheses of deoxynojirimycinand related materials [3241, and are widely applicable to

other similar structures.
11.3.6.2
Clycoprotein Remodeling
A number of the proteins employed as human pharmaceuticals (tissue plasminogen

activator, juvenile human growth hormone, CD4, EPO) are glycoproteins. There is
substantial interest in developing methods that will permit modification of oligo-
saccharide structures on these glycoproteins by removing and adding sugar units
(“remodeling”) and in making new types of protein-oligosaccharide conju-
gates [325. 32G1. Modification of the sugar components of naturally occurring or
unnatural glycoproteins might increase serum lifetime, increase solubility,decrease
antigenicity, and promote uptake by target cells and tissues.
Enzymes are plausible catalysts for manipulating the oligosaccharide content and
Fmoc-AA-0
11 0
t
Fmoc-SPPS
I! PG removal
TFA,
Frnoc
Sugar H--f+
PEPTIDE 0
TFA cleavage
Frnoc+T+o Sugar
1 Pd(O),
nucieophile
Engineered Subtilisin.
Fmoc removal (morpholineIDMF)
PS = polystyrene
PG = acid-sensitive protectin
Figure 11.3-27.
642
Expression as a
C-terminal intein- Intein-mediated
"."-:'
HS
COOH fusioytein thioester formation t
H.N- L N k y k corn
0
Protein lntein
Protein of interest H.N-E;;~COOH
structure of glycoproteins. The delicacy and polyfunctional character of proteins and

the requirement for high selectivity in their modification indicate that classical
synthetic methods will be of limited use. Major problems in enzymatic glycoprotein
remodeling and generation are the unavailability of many of the glycosyltransferases
and the uncertainty in glycosyltransferase specificity on the surface of novel
proteins. Recent advances in this area have provided several new methods for the
synthesis of homogeneous glycoproteins. Proteases have been utilized for glycopep-
tide bond ligation (Fig. 11.3-27)[3271, specifically in the generation of a homogeneous
RNase glycoform. Endo-glycosidases are capable of transforming heterogeneous
glycans to homogeneous species in a single trans-glycosylation reaction 13281. Fur-
thermore, intein-mediated splicing reactions allow modification of a protein C-
terminus with carbohydrates or other molecular probes (Fig. 11.3-28) [3291.
11.3.7
Future Opportunities
In general, the development of carbohydrate-derived pharmaceutical agents has

occurred at a slower pace than that of other biomolecules, undoubtedly because of
difficulties in their synthesis and analysis. However, distinct areas of biology and
medicinal chemistry have directed attention at carbohydrates. Interfering with the
assembly of bacterial cell walls L7, 3301 remains one of the most successful strategies
for the development of antimicrobials. As bacterial resistance to antibiotics of last
resort (i. e. vancomycin) becomes more widespread, interest in developing new anti-
pathogenic agents is increasing. Those based on carbohydrate components of the
cell wall, such as KDO, heptulose, and Lipid A, represent novel targets. Cell-wall
constituents are also relevant to vaccines and as leads toward non-protein im-
munomodulating compounds. Furthermore, cell-surface carbohydrates are central
to differentiation and development, and may be relevant to abnormal states such as
those characterizing some malignancies L71. The broad interest in diagnostics has
begun to generate interest in carbohydrates as markers of human health. In
addition, there are a number of other possible applications of carbohydrates, for
example as dietary constituents, in antivirals, or as components of liposomes for
drug delivery. Enzymatic methods of synthesis, by rendering carbohydrates more
accessible, will contribute to further research in all of these areas.
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I 1.4 Natural Polysaccharide-degrading Enzymes
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11.4
Natural Polysaccharide-degrading Enzymes
Constanzo Bertoldo and Garabed Antranikian
11.4.1
Introduction
Polymeric substrates such as starch, cellulose, hemicellulose and pectin are abun-
dant in nature and provide a valuable and renewable source of carbon and energy. A
diverse range of fungi, yeast, bacteria and archaea are capable of attacking such
complex polymeric substrates by producing extracellular enzymes with a wide range
of specificity. In this chapter we summarize the current state of knowledge on
polysaccharide-degradingenzymes, and attempts are made to show their biotechno-
logical significance.
11.4.2
Starch
Starch is the most economically important reserve polysaccharide in the plant

kingdom and is in addition the major source of carbohydrates in human nutrition.
In contrast to non-starch reserve polysaccharides, which are outside the cell and the
plasmalemma, starch is located in the so-called plastids or in vacuoles within the
plant cells"]. In seeds, the highest starch content can be found in the endosperm,
whereas its content in the embryo and the pericarp is very low. In general, the starch
content of seeds or fruits varies with the degree of maturation[']. Starch occurs in
semicrystalline form in granules. The size and the shape of the granules is
dependent on the plant species and may reach about 175 mm.
654
Structure ofthe
Figure 11.4-1.
branching point in arnylo-
pectin.
linkage
a-1,4linkage Y 0;
...
Starch is composed of amylose (15-25%) and amylopectin (75-85%). Amylose is

a linear macromolecule consisting of 1,4-linked a-D-glucopyranose residues. The
chain length varies from several hundred to GOO0 residuescl].The direction of the
chain is characterized by the reducing and the non-reducing end. The reducing end
is formed by a free C-1 hydroxyl group. Like amylose, amylopectin is composed of a-
1,Clinked glucose molecules, but in addition branching points with a-1,G linkages
occur. The branching points occur at every 17-26 glucose molecules, so that the
content of a-l,Glinkages in amylopectin is about 5%131.With its molecular mass of
10' to lo', amylopectin is one of the largest biological molecules (Fig. 11.4-1).
The iodine-binding capacity of starch is dependent on the degree of polymeriza-
tion (DP). Amylose forms with iodine a helical inclusion complex with an intense
blue colour, which possesses an absorption maximum at wavelengths between 620
and 680 nm. Amylopectin has much less iodine-binding capacity because of its
branched character, leading to a red-violet colour with absorption maximum of 540
nm i31.
11.4.2.1
Classification of Starch-degrading Enzymes
Starch-degradingenzymes can be divided into two classes according to their reaction

mechanism: the glucosidases and the glycosyl-transferases. The first class, the
glucosidases, are classified as hydrolases, which catalyze an irreversible hydrolytic
cleavage of the glycosidic bond. The group of glucosidases is further subdivided,
according their point of attack, into endoacting and exoacting enzymes. Endoacting
enzymes hydrolyze linkages in a random manner in the inner part of the starch
molecule, releasing linear and branched oligosaccharides with various chain length.
a-Amylaseis classified as an endoacting enzyme. In contrast to endoacting enzymes,
11.4 Natural Polysaccharide-degrading Enzymes
I 655
the exoacting enzymes hydrolyze linkages from the non-reducing end of the
polysaccharide chain. This group includes P-amylase, glucoamylase and a-glucosi-
dase. Isoamylase, pullulanases type I and pullulanase type I1 are classified as
debranching enzymes.
Glycosyltransferases transfer glycosyl groups from a starch chain to an acceptor.
The acceptor may be another starch molecule, phosphoric acid or nucleotides. Most
enzymes in this class catalyze reversible reactions; some enzymes are involved in the
starch biosynthesis. The only glycosyltransferaseresponsible for starch degradation
is the cyclodextrin glycosyltransferase.
11.4.2.2
aAmylase (l14-a-~-CIucan14-CIucanhydro~asel
E.C. 3.2.1 .l)
Amylases are widely distributed in plants, mammalian tissues and microorganisms.

The endoacting enzymes produce oligosaccharides and glucose as end products by
hydrolyzing the a-1,4-glycosidiclinkages in a random manner. The enzyme catalyzes
multichain attack as well as multiple attack on the same chaini41. Amylose is
hydrolyzed to maltose and glucose. The anomeric carbon in all products formed has
the a - configuration.
~ a-Amylase is not able to attack a-1,6 linkages in amylopectin
and glycogen. The a-1,4 linkages in the vicinity of branching points are also not
attacked by this enzyme[']. In spite of this, the enzyme is capable of bypassing the
branching points. Therefore, the action of a-amylase on branched substrates results
in the formation of a-limit dextrins. The structure of the a-limit dextrin is dependent
on the source of a-amylase. a-Amylases are also described as liquefying and
saccharifylng enzymes. The saccharifying a-amylases reduce the viscosity less than
liquefymg enzymes and attack the substrate repetitively[']. Most enzymes have an
absolute requirement for calcium ions, and the temperature optima as well as the
temperature stability of a-amylases are significantly enhanced in the presence of
calcum ions and substrate. a-Amylases are widely distributed among microorgan-
isms, including aerobic and anaerobic bacteria and archaea as well as actinomyces,
fungi and yeasts. a-Amylases are produced by a variety of Bacillus species, like B.
amyloliquefaciens, B. cereus, B. circulans, or B. s ~ b t i l i s [ ~ - The
' ~ ] . Bacillus enzymes are
characterized by a wide range of temperature and pH optima [l].The a-amylase from
B. acidocaldarius shows optimal activity at pH 3.5 and 75 "C; the enzyme from
Bacillus sp NRRL B2881 prefers alkaline conditions (pH optimum at pH 9.2) and
50 "C [11, 12]. Anaerobic microorganisms belonging to the genera Clostridium, Ther-
moanaerobacter, Themoanaerobiurn and Themobacteroides have also been reported to
synthesize extracellular, amylolytic enzymes [131. Also the a-amylases from archaea
have been characterized (e.g.: Pyrococcusfiriosus, Themococcusprofindus). Some of
these enzymes are optimally active above 100 0C[14,"1.
656
11.4.2.3
fi-Amylase (1,4-a-~-GlucanMaltohydrolase, E. C. 3.2.1.2)
P-Amylases occur in most higher plants and a number of microorganisms, and are
absent in mammalian tissues. The exoacting P-amylaseshydrolyze a-1,4linkages by
the stepwise removal of maltosyl residues from the non-reducing end of polysaccha-
rides [16].During the hydrolysis an inversion of the anomeric configuration occurs
leading to P-maltose as end product. Unlike a-amylase, P-amylasecannot bypass the
a-l,Glinkages in branched substrates and stops two or three glucose units before the
branching point. In amylopectin or glycogen, hydrolysis occurs only in the outer
chains, and therefore maltose and a-limit dextrins of high molecular weight are the
endproducts. p-Amylase is produced by few Bacillus species. The pH optima
determined for the B. megateriurn and the B. polymyxa enzymes are in the neutral
and slightly alkaline region and the enzymes are unstable above GO "C. The 0-
amylase from Clostridium themosulfirogenes ATCC 33 743 was characterized as a
thermoactive enzyme with a temperature optimum of 75 "C So far, this is the
only P-amylaseproduced by an anaerobic microorganism.
11.4.2.4
Glucoarnylases (1,4-a-o-glucan glucohydrolase, E. C. 3.2.1.3)
Glucoamylases, also termed amyloglucosidase or y-amylase, are produced predom-

inantly by fungi, especially by species of Aspergillus, Rhizopus and Endomyces. They
are rare in procaryots and absent in plants or in mammalian tissues.
The enzyme acts similarly to p-amylase, but attacks a-1,4as well as a-l,Glinkages
from the non-reducing end. 0-D-glucose is released as an end product. Glucoamy-
lases are not specific for a-1,4 and a-l,Glinkages; hydrolysis of a-1,3linkages has also
been reported["]. The enzyme prefers polysaccharides for rapid hydrolysis and has
lower affinity to oligosaccharides or maltose. Because of the lower affinity to a-1,G
linkages, the rate of starch hydrolysis decreases subsequentially. In practice, a
complete degradation of amylopectin or branched substrates could not be ob-
served["]. Pullulan hydrolysis from the non-reducing end by glucoamylases to
glucose was also reported[21,22]. (For a description of pullulan see 1.2.1.5.). The
glucoamylases produced by Aspergillus species and yeast are active in the acidic range
(pH 4-5 for the fungi and pH 2.5-5.5 for the yeast).The enzymes are unstable above
GO "C. The presence of glucoamylase in the thermophilic anaerobic bacterium
Clostridium thermosaccharolyticum was reported by Specka et al. (1992) and very
recently in the thermoacidophilic archaea Picrophilus oshima, Picrophilus torridus and
Themoplasma acidophil~m['~]. These enzymes are optimally active at 90 "C and
pH 2.0.
I 7.4 Natural Polysaccharjde-degrading Enzymes
I
657
11.4.2.5
a-Clucosidase (a-Wlucoside Clucohydrolase, E. C. 3.2.1.20)
a-Glucosidase catalyzes the hydrolysis of terminal a-1,4 linkages from the non-
reducing end in different substrates. The product released is a-D-glucose. The
enzyme prefers short-chain oligosaccharides as substrates and has very low affinity
to polysaccharides. In addition, many a-glucosidases show activity towards maltose,
acylglucoside, alkylglucoside and isomaltose [241. aGlucosidases are produced by
many Bacillus and Aspergillus strains and may be present in several industrial
enzyme preparations as side activities.
Intracellular a-glucosidases are produced by many microorganisms and are also
widely distributed among animals and plants. a-Glucosidases formed by various
Bacillus species (B. subtilis, B. amyloliquefaciens, B. cereus), Pseudomonas (P. amylode-
ramosa, P. Jluorescens W) and lactic acid bacteria ( Lactobacillus acidophilus, Strepto-
coccus pyogenes) are active at slight acidic pH at temperatures up to 75 "C. (for review
a-Glucosidases produced from thermophilic Clostridia and archaea are
extremely thermostable and thermoactive. The highest activity for clostridial and
archaeal enzymes is determined from 65 to 9O"C, and from 105 to 115"C,
respectively. a-Glucosidases vary in their substrate specificity. In addition to a-
1,4-hydrolyzingactivity, some enzymes show low a-1,G-hydrolyzing activity and are
capable of hydrolyzing isomaltose. Interestingly, the a-glucosidase from B. t h e m o -
glucosidasius KP lOOG is unable to hydrolyze maltose but attacks isomaltose with high
affinityr2']. The enzymes from B. cereus and from P. amyloderamosa are reported to
hydrolyze besides a-1,4and a-l,Glinkages also a-1,2 and a-1,3glucosidic bonds[28].
11.4.2.6
Isoamylase (Glycogen 6-Clucanohydrolase, E.C. 3.2.1.68)
Isoamylase hydrolyzes with high specificity the a-l,Glinkages in branched substrates

such as amylopectin or glycogen. The enzyme cannot catalyze a complete degrada-
tion of a- or P-limit dextrins, although the smallest subtrate is 26-a-D-mah0
triosylmaltose r21. Branched substrates are completely debranched, but isoamylase is
unable to attack pullulan. Pullulan is an a-D-glucan synthesized by the yeast
Aureobasidium pullulans and consists of about 480 maltotriose units linked by a-1,6-D
bonds (Fig. 11.4-2). Pullulan is used as a model substrate for starch debranching
enzymes, because the a-l,G linkages seem to imitate to a certain degree the a-
1,&branching points in substrates like amylopectin. Isoamylase has higher affinity
to large branched polysaccharides. The enzyme is very rare among microorganisms
and has been detected in Pseudomonas amyloderamosa and Cytophaga sp. [29, 301.
11.4.2.7
Pullulanase Type I (a-Dextrin 6-Clucanohydrolase, E.C. 3.2.1.41)
Pullulanase type I hydrolyzes a-l,G linkages in amylopectin, pullulan or limit

dextrins with high specificity. Pullulan is completely degraded in a random fashion
*
658
OH
f- a-1,6 linkage
* +-.- a-1,6 linkage
Figure 11.4-2. Structure o f pullulan.
to maltotriose, whereas native glycogen is not attacked by the enzyme. Substrates

with short branches such as P-limit dextrin are hydrolyzed at a higher rate than
amyl~pectin[~~l. Pullulanase I requires at least two a-1,4linked glucose units in the
vicinity of the a-1,6 The smallest substrate for pullulanse I was reported
by Marshall to be 26-a-~-maltosylmaltose[211. Pullulanase I catalyzes a condensation
reaction in the presence of maltotriose or maltose at high enzyme concentration; the
condensation products contain a-1,6 Pullulanases type I are predo-
minantly formed by mesophilic microorganisms such as Klebsiella pneumoniae,
Bacillus acidopullulyticus, B. cereus var mycoides, B. macerans ' B. polymyxa and
Streptomyces mitisi2].Fervidobacterium pennivorans is one of the few anaerobic
bacteria which produce heat-stable pullulanase type I [14].
11.4.2.8
Pullulanase Type II or Amylopullulanase
Unlike pullulanase I, pullulanase I1 hydrolyzes a-1,6linkages in pullulan and in

addition is capable to cleave a-1,4linkages in a m y l ~ s e [351.~ ~These
, enzymes with
dual specificity belong to a new class of pullulanases, termed pullulanase type I1 or
amylopullulanase~'3~ 36, 371. Pullulanase I and 11 are absolutely unable to hydrolyze
substrates like dextran or isomaltotriose, which contain exclusively a-1,6linkages.

They possess an absolute requirement for a-1,4linkages in the vicinity of the a-1,6
linkages [13, 26]. Pullulanase type I1 is widely distributed in anaerobic microorgan-
isms including species of the genera Clostridium, Dictyoglomus, Thermoanaerobacter,
Thermoanaerobium, Thermobacteroides and Pyrococcus. These enzymes are extremely
7 1.4 Natural Polysaccharide-degrading Enzymes
I 659
thermostable and are optimally active in the temperature range between 75 and
105 0 ~ 1 1 4 .381.
11.4.2.9
Pullulan Hydrolases (Type I, Neopullulanase; Type II, Isopullulanase, E.C. 3.2.1.57,
Pullulan Hydrolase Type 111)
Pullulan hydrolase type I (neopullulanase) and pullulan hydrolase type I1 (iso-

pullulanase) hydrolyze the a-l,4 linkages in pullulan, liberating panose and iso-
panose, respectively. Both enzymes are unable to hydrolyze a-1,6 glycosidic bonds in
branched substrates or pullulan. Due to this fact, the classification of pullulan
hydrolases into the group of debranching enzymes is misleading. Pullulan hydro-
lases type I have been described from B. stearothemophilus and B. stearothemophilus
KP 1064 and pullulan hydrolase type I1 from Arthrobacter globij-omis T6[3g,581.
Recently, pullulan-hydrolasetype I11 from T.aggregans has been detected cloned and
expressed in mesophilic hosts. This enzyme attacks a-1,4 as well as a-1,6 glycosidic
linkages in pullulan, producing maltotriose, maltose, panose and glucose f5'1.
11.4.2.1 0
Cyclodextrin Clycokyltransferase (1,4-a-~-Clucan4-a-~-(1,4-a-~-C~ucano)-Transferase,
E.C. 2.4.1.19)
Cyclodextrin glycosyltransferases are produced predominantly by Bacillus species

(B.circulans, B. stearothemophilus, B. macerans, B. megaterium), Klebsiella pneumoniae
and Micrococcus sp. (for review see[40]).The extracellular enzymes produced by
Bacillus macerans and B. megaterium catalyze the transformation of linear chains of
starch into cyclic oligosaccharides, the Schardinger cyclodextrins. The glucose
residues in cyclodextrins are linked by a-1,4-glycosidicbonds and, because of the
ring structure, reducing ends are absent. a-,p- and y-cyclodextrinsconsist of 6,7 and
8 glucose units, respectively. The specificity, the source and the type of the enzyme
are responsible for the ratio of different cyclodextrins formed. In principle, all
cyclodextrin glycosyltransferases produce a-, p- and y-cyclodextrins simultane-
o u ~ l y571.[ ~Thermostable
~~ cyclodextrin glycosyltransferases (CGTases) are produced
by Themoanaerobacter species, Thermoanaerobacterium thermosul&rigenes and Anae-
robranca gottschalkii. Recently, a CGTase, with optimal temperature at 100 "C, was
purified from a newly isolated Archaeon, Thermococcus sp. This is the first report of
the presence of a thermostable CGTase in a hyperthermophilic A r ~ h a e o n [ ~ ~ ] .
The occurrence of different starch-degradingenzymes in microbes is summarized
in Table 11.4-1.
11.4.2.1 1
Biotechnological Applications of Starch-degrading Enzymes
Starch-degrading enzymes are applied in the starch bioprocessing, sugar, alcohol

and brewing industries. The commercially most important application of starch-
660
Table 11.4-1. Occurrence of different starch hydrolyzing enzymes in microorganisms.

Enzymes Substrate Products Organisms
(enzyme action)
a-Amylase starch a-limit dextrins Bacillus amyloliquefaciens

(endoacting a-1,4) branched oiligo- B. cereus
saccharides, glucose Tnermotoga maritima
maltose, linear Pyrococcusfuriosus
oligomers
P-Amylase starch p-maltose Bacillus megaterium
(exoactinga-1,4) limit dextrins B. polymyqa
Clostridium thermosulfurogenes
Glucoamylase polysaccharides 0-D-glucose Aspergillus niger, A. oryzae

[exoactinga-l,4,(a-l,6)] Rhizopus nodosus
Clostridium acetobutylicum
Picrophilus torridus
a-Glucosidase oligosaccharides a-D-glucose Bacillus subtilis

[exoactinga-l,4,(a-l,6)] B. cereus
Streptococcus pyogenes
Thermococcus strain AN1
'I: hydrothermalis
Isoamylase branched polysaccharides linear polysaccharides Pseudomonas amyloderamosa

[endoacting a-1,6] Flavobacterium odorratum
Pullulanase pullulan, branched maltotriose, linear Klebsiella pneumoniae

Type I polysaccharides oligosaccharides Bacillus acydopullulyticus
[endoacting a-1,6] Fervidobacterium pennivorans
Thermotoga maritima
Pullulanase pullulan, branched maltotriose, linear B. subtilis

Type I1 polysaccharides oligosaccharides C. thermohydrosulfuricum
[endoacting; a-1,6 in Pyrococcus woesei
pullulan; a-1,6 + a-1,4 Desulfurococcus mucosus
in branched poly- and
oligosaccharide]
Pullulan- pullulan [a-1,4] panose B. stearothermophilus
hydrolase
Type I
Pullulan- pullulan [a-l,4] isopanose Atthrobacter globi&ormis
hydrolase
Type I1
Pullulan- pullulan [a-1,4] glucose, maltose Thermococcus aggregans
hydrolase maltotriose
Type I11
Cyclodextrin- branched a-P-y-cyclodextrin B. circulans
glycosyl- polysaccharides B. macerans
transferase [endoacting a-1,4] Anaerobranca gottschalkii
Thermococcus sp.
*Values in brackets () indicate low enzyme affinity
17.4 Natural Polysaccharide-degrading Enzymes
I
degrading enzymes is the production of syrups and sweeteners. The conversion of
corn starch to fructose begins with a liquefaction step carried out with a-amylase
from B. lichenifomis at 105-115 "C and 90-95 "C at pH 6 . Amylose and amylopectin
are hydrolyzed to dextrins and some oligosaccharides. The saccharification follows
the liquefaction in the presence of glucoamylase from Aspergillus niger and debranch-
ing enzymes. The process conditions in the saccharification step have to be changed
since the enzymes are optimally active at pH 4-4.5 and 55-65 "C. A dextrose solution
of 95% results from this step. The dextrose solution can be crystallized or subse-
quently further isomerized. The isomerization from glucose to fructose again
requires variation of the process conditions (55-GO "C, pH 7-8) r4lS421. The finding of
different amylolytic enzymes that are active under the same conditions will certainly
improve the starch bioconversion process. Recently, it was found that hyper-
thermophilic microorganisms are a good source of such enzymes. a-Amylase,
pullulanase and a-glucosidasefrom Pyrococcus sp. are optimally active at pH 4-5 and
100-110 "C'2GI.
In the baking industry a-amylase from fungi is used in order to release dextrin and
fermentable sugars for yeast metabolism. Exhaustive dextrin formation, however,
will lead to undesirable properties like loaf stickness and dark color.
In the process of fuel alcohol production different grains and tubers serve as raw
material in the fermentation process. The liquefaction and saccharification steps are
carried out in the presence of a-amylase and glucoamylase, respectively. This
saccharified feedstock forms the substrate for the ethanol fermentation with
yeastsL4'].Pullulanases are also used in the production of "light beer", which has low
carbohydrate content. During the fermentation process the pullulanase is added
together with fungal a-amylase or glucoamylase to the The production of
branched and more water-soluble cyclodextrins can also be carried out with pull-
ulanase. The pullulanase catalyzes the transfer reaction of malto-oligosaccharidesto
cyclodextrins. Branched cyclodextrins are more water-soluble than linear cyclodex-
CGTases are used for the production of cyclodextrins that can be used as a
gelling, thickening or stabilizing agent in jelly desserts, dressing, confectionery,
dairy and meat products. Because of the ability of cyclodextrins to form inclusion
complexes with a variety of organic molecules, cyclodextrins improve the solubility
of hydrophobic compounds in aqueous solution. This is of interest for the pharma-
ceutical and cosmetic industries. Cyclodextrin production is a multistage process in
which starch is first liquefied by a heat-stable amylase, and in the second step a less
thermostable CGTase from Bacillus sp. is used. The application of heat-stable
CGTase in jet cooking, where temperatures up to 105 "C are achieved, will allow
liquefaction and cyclization to take place in one step.
11.4.3
Cellulose
Cellulose is the principal component of plant cell walls, and thus represents the
worlds most abundant organic polymer, with an annual production of 4 x 10"
tonnes per year. Cellulose is found in nature as an unbranched insoluble polymer
662
Endoglucanase
[Cellulose]
n
OH OH
OH
Cellobiohydrolase n
n
B-Glucosidase
Figure 11.4-3. Action of cellulolytic enzymes on cellulose.
(Fig. 11.4-3) containing up to 14000 glucose units linked together by p-1,4-~-

glycosidic bonds 15’), ‘1. The hydrogen capacity between individual chains in cellulose
is quite high, with each residue contributing up to three OH groups. The individual
chains of cellulose tend to form microfibrils as a result of inter- and intramolecular
hydrogen bonding. The microfibrils associate in a similar way to form fibers [l,“. “1.
Cellulose contains both crystalline and amorphous regions. The term “crystalline”
refers to those regions in which a high degree of order is found within and between
the fibrils. In the amorphous regions, however, a lesser degree of order is pre-
dominant [62, 631. Crystalline regions are more resistant to degradation than amor-
phous regions [64, 651. Thus, the fraction of crystalline regions found in cellulose is an
important factor affecting the rate and enzymatic hydrolysis of cellulose 16’, “1.
Cellulose is found in nature as a principal structural element in cell walls of higher
plants, in association with hemicellulose, lignin and other polysaccharidesiG7].
Cellulose is also found in some seaweeds and can be synthesized by some
bacteria[“]. Cellulose occurs in an almost pure form (98%)in cotton fibers, while in
flax (SO%), jute (GO-70%), wood (40-SO%), and forages ( 2 4 3 6 % ) a less pure form
of cellulose is found[’, 6 2 , 691.
1 1.4 Natural Polysaccharjde-degrading Enzymes
I 663
11.4.3.1
Cellulose-degrading Enzyme Systems
Cellulases are a group of enzymes capable of hydrolyzing insoluble cellulose to its

monomer glucose L7O]. Because of the crystalline and insoluble nature of cellulose, its
degradation is very slow. Cellulose degradation requires a multienzyme complex
involving at least three major enzymes, namely 1,4-P-~-glucan glucanohydrolase
(endoglucanase, E. C. 3.2.1.4), l,CP-~-glucan cellobiohydrolase (exoglucanase, E. C.
3.2.1.91), and P-D-glucoside glucohydrolase (P-glucosidase, E. C. 3.2.1.21) [711
(Fig. 11.4-3). Mainly two types of enzyme systems have been recognized to be
involved in cellulose hydrolysis. The first is the non-aggregating system, in which the
three main cellulolyhc enzymes are produced and mainly found to be secreted into
the growth medium as separate entities. In this system, endoglucanase, cellobiohy-
drolase and P-glucosidase act in synergy[129* 721. The second enzyme system is
referred to as the aggregating system. This system is found mainly in anaerobic
bacteria, where cellulases are secreted as a high molecular weight multienzyme
complex. This complex is generally found on the cell surface, where it mediates the
attachment between the cells and the substrate[129]. The most studied system is that
of Clostridiurn thermocellum [731; the complex is named “cellulosome”.
11.4.3.2
E. C. 3.2.1.4)
Endoglucanase (1,4-fJ-~-Clucan-Clucanohydrolase,
Endoglucanase hydrolyzes cellulose randomly, producing oligosaccharides, cello-

biose and glucose as end products (Fig. 11.4-3). Endoglucanase attacks mainly the
amorphous regions in cellulose and soluble derivatives of cellulose [741. The action of
endoglucanase results in a decrease in the chain length of carboxymethylcellulose
(CMC),acid-swollen cellulose and soluble barley glucan, producing mainly glucose,
cellobiose, cellotriose and other oligosaccharideslG4,75, 7G]. Substrates like p-ni-
trophenyl-P-D-cellobiosideand methylumbelliferyl-P-o-cellobiosideare hardly at-
tacked by endoglucanase. Low activity is also observed with microcrystalline cellu-
lose[77].In contrast to this, the endoglucanase of Trichoderma viride shows high
activity towards crystalline cellulose but only weak activity towards CMC I8O].
11.4.3.3
Cellobiohydrolase (1 ,Cfi-~-ClucanCellobiohydrolase, E. C. 3.2.1.91)
Cellobiohydrolases are exoglucanases that attack the non-reducing end of the

cellulose polymer chain to produce cellobiose (Fig. 11.4-3). Recent reports have
indicated that the attack on cellulose is not restricted to the end of the chain. Thus,
the cellobiohydrolase I from Trichoderma reesei is capable of degrading the p-glucan
from barley in a manner typical of an endoglucanase[81].Cellobiohydrolases com-
prise the major part of fungal cellulase systems that are capable of degrading
Up to 80% of microcrystalline cellulose can be degraded by
crystalline cellulo~e[~~1.
this enzyme[”]. Earlier studies have indicated the absence of cellobiohydrolases in
664
bacterial cellulase systems [751. However, Langsford et al. [821 reported the presence of
this enzyme in Cellulomonasjmi. This enzyme has also been found in Ruminococcus
albus and R. Jlavefaciens[841. Bacterial cellobiohydrolases are capable of hydrolyz-
ing model substrates such as p-nitrophenyl-P-D-cellobioside and methylumbelliferyl-
P-D-cellobioside. They release cellobiose from microcrystalhe cellulose and show
low activity towards CMC [771.
11.4.3.4
fi-Clucosidase (b-d-ClucosideClucohydrolase, E. C. 3.2.1.21)
Cellobiase or P-glucosidase acts mainly on cellobiose and cellodextrins (up to DP of

6 ) to produce P-glucose (Fig. 11.4-3); cellulose and higher cellodextrins are not
hydrolyzed by this enzyme[“]. P-Glucosidase acts also on sophorose and cellobiose
to produce monosaccharides. In addition, model substrates such as p-nitrophenyl-P-
D-glucosides or methyhmbelliferyl-~-D-glucoside are attacked. Because of the action
of P-glucosidase,the inhibitory effect of cellobiose on cellobiohydrolaseand endoglu-
canase can be removed. As shown for the P-glucosidase from Penicillium finiculo-
sum, this enzyme also acts synergistically with endoglucanases and cellobiohy-
drolases [”I.
11.4.3.5
Fungal and Bacterial Cellulases
Most of the studies on cellulases have been conducted using fungal cellulolytic
systems. Relatively few cell-free cellulases have been reported to degrade crystalline
cellulose. Such fungal systems contain extracellular endoglucanase and cellobiohy-
drolase activities that convert crystalline cellulose to cellobiose[861. The conversion of
cellobiose to P-glucose is catalyzed by 0-glucosidase,which has been found in the
cultures of T’choderma T. reesei[881,7: k~ningii[~’]
and Talaromyces emerson-
nii[’OI (Table 11.4-2).Compared to fungal systems, cell-free supernatants from
cultures of cellulolytic bacteria seems to lack activity against crystalline cellulose f8‘I.
Several cellulolyhc bacteria have been isolated, but their cellulases have not been
fully characterized[”, 921. The system from Cellulornonas sp. is one of the most
studied cellulolytic systems in bacteria[93,941. Many species which belong to the
genera Bacillus, Pseudomonas, Streptomyces, Thermoactinomyces and Thermomono-
spora are capable of producing cellulolytic enzymes [911. Several endoglucanases were
detected in the culture fluid of many of these microorganisms. However, cellobiohy-
drolase has not been detectedrS6l.The hydrolysis of cellulose by bacteria involves the
action of cellulolytic enzyme complexes consisting of different multicomponents.
These complexes are associated with the cell wall of the bacterium and are often
tightly bound to the cellulosic substrate I7Ol. They are released into the culture fluid
only after extensive hydrolysis of cellulose. The most thoroughly studied cellulolytic
enzyme complex, referred to as “cellulosome”, is that of Clostridium thermo-
~ e l l u m 1951.
~~-
Table 11.4-2. Microbial cellulolytic enzymes

I
665
Organism Endoglucanase Cellobiohydrolase fi-Clucosidase
Fungi
Aspergillus niger
Humicola insolens -
Tricoderma koningii +
1: reesei +
1: viride +
Bacteria:
Cellulornonas frmi
Clostridiurn thermocellum
C. stercorarium
Cytophaga sp.
Fibrobacter succinogenes
Ruminococcus albus
Trtermotoga maritima
‘I?termotoga neapolitana
Archaea
Pyroccusfuriosus + +
Suljblobus solfataricus - +
11.4.3.6
Structure and Synergistic Effect of Cellulases
11.4.3.6.1 The “Cellulosome” Concept

The cellulolytic enzyme sytem of bacteria forms aggregates, which are associated
with the cell wall forming catalytically active “protuberances”. Electron microscopy
studies revealed that these “protuberances” are found on the surface of all cellulo-
lytic bacteria studied, whereas they are absent on the surfaces of non-cellulolytic
bacteria[73,96, 971. In addition, they are not present during growth in the absence of
~ e l l u l o s e [ The
~ ~ ~ best
. characterized aggregation system is the cellulosome of
Clostridium thennocellum~”~9 5 , 731. The cellulosome binds to the substrate and is
active towards crystalline cellulose. A 200 kDa polypeptide seems to be responsible
for the substrate binding. In the early stages of growth the cellulosomes of
C. therrraocellum form polycellulosomes, which appears as protuberances on the cell
surface. In the late growth phase the cellulosomes are released into the culture
Cellulases present in the culture fluid seem to represent only
fragments of cellulosomes and polycellulosomes.I‘’[ Other cellulolytic bacteria
which express cellulosome-like structures are Ruminococcus albus, R. flavefaciens,
Fibrobacter succinogenes [96, 981, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Clo-
stridium cellobioparum, C. cellulovorans and Cellulomonas sp. [96, 991. Cellulose-degra-
ding enzymes from various thermophilic organisms (Thennotoga maritima, Thenno-
toga neapolitana, Caldocellum saccharolyticum and Anaerocellum thermophilum) have
been cloned, purified, and characterized. Recently, a thermostable archaeal endoglu-
666
canase which is capable of degrading p-1,4 bonds of P-glucans and cellulose has been
characterized from Pyrococcus&riosus [l4I.
11.4.3.6.2 Multiple Forms o f Cellulases

The cellulolytic enzymes from bacteria and fungi (endoglucanase,cellobiohydrolase
and 0-glucosidase)exist in multiple forms. Multiple forms of these enzymes seem to
arise through post-translationalmodification by physiologically regulated processing
activity or through post-secretional modification by proteolytic digestion["]. Di-
versity of endoglucanases and cellobiohydrolases have been reported by several
investigators[G4, 'O0, loll. However, Penicillium notatum and Stereum sanguindentum
produce a single cellulase and are still able to degrade cellulose['02.'031. Wilson[104]
isolated five different endoglucanases from a protease negative mutant of Thermomo-
nospora fisca; cellobiohydrolase activity, however, was not detected. Similarly, Shoe-
maker and B r ~ w n [ ' ~identified
~l four endoglucanases from Trichoderma uiride.
Further studies with T. viride proved the presence of six endoglucanases (Endo I, 11,
111, IV, V and VI), three cellobiohydrolases (Ex0 I, 11 and 111) and one P-glucosi-
dase L8O1.
11.4.3.6.3 Synergism
It has been recognized that the rate of hydrolysis of crystalline cellulose by the
combination of endoglucanase and cellobiohydrolaseis much faster than the sum of
the individual actions of the components[70].The rationale for the synergy of
cellulase has been postulated as follows. The attack is initiated by a randomly-acting
endoglucanase in the amorphous areas of the cellulose creating numerous new non-
reducing ends that are attacked by cellobiohydrolase, resulting in the release of
cellobiose. The P-glucosidase is needed for the removal of cellobiose, a strong
inhibitor of both endoglucanase and cellobiohydrolase[72, loGI. Studies with the
cellulases from T. reesei and bacteria showed that cellobiohydrolase 11 was only able
to attack one end of the microcrystalline cellulose. However, in the presence of their
endoglucanase, several sites of cellobiohydrolase attack at the amorphous region
were ob~ervedI'~~1. Another observation showing the synergism between endogluca-
nase and cellobiohydrolase has been reported with the fungus Neocallimastix
jontalis. Heat inactivation of the cellulases of this fungus resulted in loss of its ability
to degrade crystalline cellulose. Interestingly, the endoglucanase activity was still
measurable. The additon of cellobiohydrolasefrom Trichodema koningii restablished
the ability ofthe system to degrade cotton Synergism between P-glucosidase
and cellobiohydrolase or endoglucanase has also been observed. P-Glucosidase
produced by T. koningii has shown synergism with cellobiohydrolase but not with
endoglucanase['08].Synergism between a-glucosidase and cellobiohydrolase can be
explained by the ability of P-glucosidaseto hydrolyze cellobiose, a strong inhibitor of
cellobiohydrolase[log].
I 667
11.4.3.6.4 Biotechnological Applications o f Cellulases
Cellulase preparations have found different biotechnological applications in several
industrial processes. The most effective commercial cellulase is the one produced by
Trichodema species. Other cellulases of commercial interest are obtained from
strains of Aspergillus, Penicillium and Basidomycetes. Fungal cellulases have been
recommended for use in alcohol production. The alcohol yield from cassava is
significantly increased if cellulases from Trichodema sp. are added [‘lo]. Cellulolytic
enzymes can also be used to improve juice yields and effective color extractions of
juices. Cellulolytic enzymes also improve the silage-making process [1301. The cellu-
lase from Trichodema reesei has been reported to accelerate the rate of ensilage
processing when treating grass, lucerne and red clover[’”]. The presence of
cellulases in detergents causes colour brightening, softening and improved partic-
ulate soil removal^"*]. A novel application of cellulases in textil industry is the use of
Denimax (Novo Nordisk) for the “biostoning”of jeans instead of the classical stones
in stone-washed jeans [1131. Another application of cellulases includes the pre-
treatment of cellulosic biomass and forage crops to improve nutritional quality and
digestibility, enzymatic saccharification of agricultural and industrial wastes and
production of fine chemicals [130].
11.4.4
Xylan
Hemicelluloses are non-cellulosic low molecular weight polysaccharides that are

found together with cellulose in plant In the cell walls of land plants,
xylan is the most common hemicellulosic polysaccharide, representing more than
30% of the dry weight[132].Most xylans are heteropolysaccharides which are
composed of 1,Clinked 0-D-xylopyranosylresidues [133, 134, 13’1 .Th’is backbone chain
is substituted with acetyl, arabinosyl, and glucuronosyl residues Homoxylans,
on the other hand, consist of xylosyl residues exclusively and have been isolated from
esparto grass tobacco stalks [I3’], and guar seed husk[138].
The xylan of hardwoods (0-acetyl-4-0-methylglucuronoxylan) consists of at least
70 P-xylopyranose residues (average degree of polymerization between 150 and 200)
linked by P-1,4-glycosidicbonds (Fig. 11.4-4) Every tenth xylose residue carries a
4-0-methylglucuronic acid attached to the C-2 of xylose [1311. In addition, hardwood
xylans are highly acetylated;e. g. birchwood xylan contains more than 1mol of acetic
acid per 2 mols of xylose [1401. Acetylation occurs usually at the C-3 rather than the C-2
position of xylose. Acetylation at both positions has also been 14’1 . Th e
presence of these acetyl groups is responsible for the partial solubility of xylan in
water”33].The alkali extraction of xylan leads to the deacetylation of this sub-
strate [I4’].
Softwood xylans (arabino-4-0-methyl-glucuronoxylans) are composed of shorter
chains with a degree of polymarization between 70 and 130 (Fig. 11.4-5). Unlike
hardwood xylan, the softwood xylan has a higher content of 4-0-methyl-glucuronic
acid. The acetyl groups are replaced by a-L-arabinofuranoseunits, which are linked
by a-1,3-glycosidicbonds to the C-3 position of xyl0se1~~~1.
668
I
a-4-0-Me-GlcA
Ac Ac Ac
4 4 r' 1
3 3 2 2
4-D-Xylp1~4-B-Xylp-1~4-B-Xyl~14B-Xyl~l~4-~-Xyl~l~4-B-Xyl~l~4-R-Xyl~l~4-RXyl~l
2 3 2
t t t
Ac Ac Ac
Figure 11.4-4. 0-Acetyl-4-0-methyl-glucuronoxylan from hardwood.
cli'
HOH,C
OH
0
HOH,C
OH
a-4-0-Me-GlcA a-4-0-Me-Glc A
1 1
1 1
a-Araf a-Araf
Figure 11.4-5. Arabino-4-0-methyl-glucuronoxylanfrom softwood.
11.4.4.1
The Xylanolytic Enzyme System
Because of the heterogeneity of xylan, its hydrolysis requires the action of a

xylanolytic enzyme system which is composed of P-1,4-endoxylanase (E. C. 3.2.1.8),
P-xylosidase (E. C. 3.2.1.37), a-L-arabinofuranosidase (E. C. 3.2.1.55), a-glucuroni-
dase (E.C. 3.2.1.-) and acetylxylan esterase (E. C. 3.1.1.6) activities (Table 11.4-3).The
concerted action of these enzymes converts xylan to its constituent sugars (Fig. 11.4-
6). Xylan-degrading enzymes have been reported to be present in marine and
Table 11.4-3. Microbial xylanolytic enzymes.
7 1.4 Natural Polysaccharide-degradingEnzymes
I
669
Organism Endoxylanase B-Xylosidase a-L-Arabino a-Glucuro- Acetyl xylan

furanosidase nidase esterase
Fungi
Aspergillus awamori + + i +
Furiarum oxysporum - + - +
Tricoderma reesei + + +
Bacteria:
Bacillus subtilis + + - +
Streptomyces + + + +
olivochromogenes
Thermoactinomyces N.D. + N.D. +
vulgaris
Thermoanaerobacter + + N.D. +
saccharolyticum
Thermonosporafusca + + N.D. +
Thermotoga maritima N.D. N.D. N.D. N.D.
Thermotoga neapolitana + N.D. N.D. N.D. N.D.
Archaea
Thewnococcuszilligii + N.D. N.D. N.D. N.D.
N. D.: not determined
A
a-Araf a-Me-GlcA a-Araf
ArabinofuranosidaseCZ)i
1 Ac
1 iQ a-Glucuronidase i
3 3 2 3
-+4~-Xylp-14-B-Xyip-14-~-xylp-l4B-Xylp-l-.4-B-Xylp-l4B-Xytp-l~B-Xylp-l
a 0
--f
Ac
t Acetyl Xylan Esterase
Endoxylanase
2
t
Ac
I'r
Endoxylanase
B
BXylosidase
Figure 11.4-6. (A) Action ofxylanolytic enzymes on an hypothetical xylan structure. (B) Action o f
0-xylosidase on xylobiose. Ac, acetyl residue; a-Araf, a-L-arabinofuranose; a-Me-ClcA, 4-O-rnethyl-
o-glucuronic acid; fl-Xylp, 0-D-xylopyranose.
1 1 Hydrolysis and Formation o f C - 0 Bonds
670
I terrestrial bacteria, rumen and ruminant bacteria, fungi, marine algae, protozoa,
snails, crustaceans, insects and seeds of terrestrial plants [la]. Among the different
functions of xylanases is the utilization of xylan as a carbon and energy source,
degradation of cell wall components and degradation of xylans during germination
of
11.4.4.2
Endoxylanase (1,4+~-Xylan Xylanohydrolase, E. C. 3.2.1.8)
P-1,4-Endoxylanase cleaves the internal glycosidic linkages of the heteroxylan back-

bone, resulting in a decreased DP (degree of polymerization) of the substrate
(Fig. 11.4-GA). The attack of the substrate is not random, and the bonds to be
hydrolyzed depend on the nature of the substrate, e.g. length, presence of sub-
stituents and degree of branching[145].During the early course of hydrolysis of xylan
the main products formed are xylooligosaccharides.As hydrolysis proceeds, these
oligosaccharides are hydrolyzed to xylotriose, xylobiose and x y l ~ s e [ ~ ~Diffe-
~~~'].
rentiation of endo-acting xylanases has been made according to the end products
formed i. e. xylose, xylobiose and xylotriose, and/or arabinose. Thus, xylanases may
be classified as non-debranching (arabinose non-liberating) or debranching (arabi-
nose-liberating) enzymes [1452 1461 . M any organisms are able to produce both de-
branching and non-debranching xylanases, resulting in a maximum efficiency of
xylan hydrolysis "'I. The production of multiple forms of xylanases has been
reported for many organisms such as Aspergillus niger and Fibrobacter succino-
genes[150*l5l]. The endoxylanase I from F. succinogenes possesses debranching
activity and liberates arabinose from xylan. This is followed by the action of
endoxylanase 11, which converts unbranched xylans to xylooligosaccharides['"I. This
may indicate that the removal of the arabinose substituents, which act as a
hindrance, is a requirement to permit the access of endoxylanase to the xylan
backbone. This also demostrates the synergistic relation between debranching and
non-debranching xylanases. Arabinose-cleaving endoxylanases have been purified
from Streptornyces roseiscleroticus [1521 and T'chodema k~ningii["~I.
11.4.4.3
fi-Xylosidase (fi-o-Xyloside Xylohydrolase, E. C. 3.2.1.37)
P-D-Xylosidases are exo-glycosidases that hydrolyze short xylooligosaccharidesfrom

the non-reducing end forming xylose as end (Fig. 11.4-GB). 0-Xylosi-
dases appear to be mainly cell-associated (found in the cytosol) in bacteria and
yeast [1341. However, extracellular P-xylosidases have also been reported [1541561. In
the yeast Cryptococcus albidus, xylooligomers (xylobiose and xylotriose)enter the cells
through a P-xylosidepermease transport system and are converted by 0-xylosidase to
x y l o ~ e [ ' ~P-Xylosidases
~]. are in most cases unable to hydrolyze xylan. However, there
are some reports of xylosidases that are capable of attacking xylan and producing
xylose Such exo-xylanases would have a limited hydrolysis activity towards
heteroxylans, as their action would end at the branch points [I4']. 0-Xylosidase activity
may play a role in relieving the end product inhibition of endoxylanase. This has
I
671
been reported for the enzyme system of Themtornonospora&sca [ls71. Transferase

activity is a typical feature of most P-xylosidases, resulting in products of higher
molecular weight than the Transfer reaction may result in the
formation of both p-1,3 and p-1,4 bond^['^^-'^^].
11.4.4.4
a-L-Arabinofuranosidase(E. C. 3.2.1.55)
a-L-Arabinofuranosidasesare active against branched arabinoxylans, arabinans,

arabinose-substituted xylooligosaccharides and p-nitrophenyl-a-L-arabinofurano-
side. Their action on arabinoxylan results in the release of arabinose residues
(Fig. 11.4-6A).The production of a-L-arabinofuranosidasein several actinomycetes
seems to be induced among others by xylan, arabinan, and wheat bran['", 161].a - ~ -
Arabinofuranosidases from A. niger and S. purpurascens are also capable of hydrolyz-
ing both 1,3-and 1,s-a-L-arabinofuranosyl linkages in arabinanr'", 163].TheAspergil-
lus niger enzyme attacks first the a-~-1,3-linkedarabinofuranosyl residues to the
extent of 30% and then proceeds with a slow attack of the a-L-l,S-arabinan['"].
Synergism between a-L-arabinofuranosidaseand endoxylanase has been reported. A
significant increase in xylose, xylobiose and arabinose production was observed
when both enzymes are used simultaneously['"].
11.4.4.5
a-Glucuronidase (E.C. 3.2.1.136)
a-D-Glucuronidasesare required to hydrolyze the a-1,2linkages between glucuronic

acid and xylose residues in glucuronoxylan (Fig. 11.4-GA). Because of the lack of a-
glucuronidase activity in many fungal hemicellulase preparations [13'1, this enzyme
was not described until 1986[165].Only a few a-glucuronidases have been purified so
far; these include the enzymes from Trichoderrna reesei, Tnemoascus aurantiacus and
Agaricus bisporus[166].Thus, most of the studies on a-glucuronidases have been
performed using partially purified enzymes. These enzymes release 4-0-methyl-
glucuronic acid from 4-0-methyl-glucuronicacid-substituted xylooligomers,but not
from the polymer [13'1. Simultaneous hydrolysis of acetyl-4-0-methyl-glucuronoxylan
with the endoxylanase from A. oryzae and the acetyl xylan esterase from T.
longibrachiaturn resulted in the production of non-substituted xylan fragments as
well as substituted xylooligomers. These products were further treated with a P-
xylosidase from T. reesei and an a-glucuronidase from A. bisporus. The a-glucu-
ronidase was not active against these oligomers, indicating that the acetyl groups
next to the glucuronosyl substituent may hinder the action of the a-glucur-
onidase [1431.
672
11.4.4.6
Acetyl Xylan Esterase (E.C. 3.1.1.6)
Acetyl xylan esterase removes the 0-acetyl substituents at the C-2 and C-3 positions
of xylose residues in acetylxylan (Fig. 11.4-GA). The importance of acetyl xylan
esterase in the hydrolysis of xylan was demonstrated recently[l6'I. It is mainly due to
the fact that most of the xylan preparations used to study xylanolyhc enzymes
systems are alkali extracted xylans. Under these conditions mainly deacetylated
xylans are obtained['68].Nowadays, acetyl xylan esterase activity has been recognized
as a part of the xylanolytic enzyme system of many organisms such as T reesei, T.
uiride, A. niger, Schizophilum commune['69]and Streptomyces sp. [161]. The importance
of this enzyme in the hydrolysis of xylan has been clearly demonstrated. Incubation
of endoxylanases with acetylated glucuronoxylan resulted in the production of small
amounts of xylose, xylobiose, xylotriose and large amounts of substituted oligomers.
The addition of acetyl xylan esterase to the hydrolyzed mixture significantly increases
the production of xylotriose and xylotetr~se['~~]. Similarly, an enzyme mixture of
endoxylanase and p-xylosidase results in a limited hydrolysis of acetylated xylooligo-
mers. The addition of acetyl xylan esterase enhanced xylose Thus,
complete hydrolysis of acetylated xylans by xylanases will require the deacetylation of
the substrate by acetyl xylan esterases
11.4.4.7
Mechanism o f Action of Endoxylanase
Most of the studies on the mechanism of action of endoxylanase arise from the work
of Biely et al. [172,1731 using the yeast Cryptococcus albidus. The reaction of the enzyme
with 5 mM [ U-14C] xylotriose resulted in a constant product ratio of xylobiose to
xylose throughout the reaction. However, when the concentration of [ U-I4C]
xylotriose was increased, the major product formed was xylobiose. Xylotetrose is
cleaved at the middle glycosidic bond to form xylobiose. Xylopentose when present
in low concentrations is converted to xylobiose and xylotriose in a ratio of 2:1.
However, at higher concentrations xylotetrose is also produced. The action of
endoxylanase on xylotriose, xylotetrose and xylopentose is usually accompanied by
the formation of xylooligosaccharides larger than the original substrates. These
studies also revealed that xylose and xylobiose can act as acceptors for the transferase
reaction of xylanase. Although the acidic endoxylanase produced by Aspergillus niger
differs from that of C. albidus, the mechanism of action of the enzyme is similar to
the yeast enzyme. The mechanism of action of endoxylanase appears to be analogous
to that reported for lysozyme and a-amylase
11.4.4.8
Biotechnological Applications o f Xylanases
Plant polysaccharides are a major source of renewable substrates for the chemical,
pharmaceutical and feed industries [12'1. Xylan-degrading enzymes have considera-
7 1.4 Natural Polysaccharjde-degrading Enzymes
ble potential in several biotechnological applications. Two main areas for the
I 673
application of xylanolytic enzymes have been discussed by Biely['341.The first is the

use of xylanolytic enzymes in the presence of cellulolytic enzymes for the effective
conversion of paper pulp and agricultural wastes into xylose, for the clarification of
juices and must, and for the pre-treatment of cellulosic biomass to improve
digestibility of ruminant feeds or to facilitate c o m p ~ s t i n g [ ~The~ ~ second
]. area of
application involves the use of xylanolytic enzymes in the absence of cellulases
Most attention has been paid to the incorporation of xylanases as pre-bleaching
agents for kraft pulps. Here the use of xylanases will help in reducing the kappa
numbers (measure of residual lignin) of the pulp, thus reducing the requirement for
chlorine during pulp b l e a ~ h i n g [ ' ~Most
~ l . of the studies on the effect of xylanases in
the pre-bleaching of pulp have been conducted with enzyme preparations from
Trichodema sp. The reduction of chlorine required during chlorination of pulp has
been reported to be 35-41 % for hardwoods and 10-26 % for softwoods [17'3. Addi-
tional applications of xylanases are as flour improvers for bakery products, in the
extraction of coffee, plant oils and starch[177],for the saccharification of biomass, and
in the production of fuel and chemical feedstocks[173,17', '*'I.
11.4.5
Pectin
Pectic substances are widespread in the plant kingdom. The dry substance of
primary cell walls of plants consists of up to 90% polysaccharides and their
derivatives. These polysaccharides are composed of approximately equal parts of
cellulose, hemicellulose and pectic substances. The exact proportion depends on the
kind of plant (plant species) and the plant texture['*']. In fruits and vegetables, pectic
substances are often found between the cells in intercellular regions. To the large,
heterogenous group of pectic substances belong rhamnogalacturonans, galacturo-
nans, arabinans, galactans and arabinogalactans [182].Pectins are designated as
rhamnogalacturonane with the structure shown in Fig. 11.4-7:molecules of ga-
lacturonic acid are linked by a-1,4glycosidic linkages forming a helically wound
chain. This chain is interrupted by rhamnose molecules which are bound by u-1,2
glycosidic linkages to the galacturonic acid [lS3,lS41.
The number of galacturonic acid molecules varies according to the origin of the
pectin. For instance, between two rhamnose molecules in citrus pectin there are 25
galacturonic acid molecules, whereas in tomato pectin there are 16 galacturonic acid
Pectic substances have no definite molecular weight. The molecular
weight may range from 23 000 for citrus pectin to 360 000 for apple or lemon
pectin['85. 18'1 . Th e break of the galacturonic acid chain by rhamnose leads to a break
in the regular helical structure. In these regions, molecules are substituted to a high
degree. The C-2 or C-3 atoms of the galacturonic acid and the C-4 atom of the
rhamnose molecules are preferentially substituted. The substituents are acetate, L-
arabinose, L-rhamnose, L-fucose, D-galactose, D-xylose or D-glucose. These sub-
stituents give to the pectin a complex and branched configuration['87, 188].Fur-
thermore, the main galacturonic acid chain is substituted with polymers of L-
674
I
0
R O ORq
a(l,:o+ 0
G
OR
R': H (= Polygalacturonic acid), CH3 (=Pectin) 0

I COOR'
R : H, Acetate, L-Arabinose, L-Fucose,
D-Galactose, D-Glucose, D-Xylose
Araban. Galactan
Rh: Rhamnose
G : D-Galacturonic acid
Roq
Figure 11.4-7. Structure o f pectin.
arabinose (1,s-linked arabinan) and D-galactose (p-1,4- or p-1,3-linked galactans).

Also, arabinogalactan I, which contains p-1,4galactan, has been reported to form
side chains[']. These various side chains account for the complexity of pectic
substances. The degree of substitution and the kind of substituents is dependent on
the source of the pectin. In addition to the modifications on the C-2/C-3 of
galacturonic acid and the C-4 of rhamnose, a large number of carboxyl groups of the
galacturonic acids are esterified with methanol [1891. The degree of esterification
varies with the source of the pectin. Apple pectin is esterified to the extent 80-90 %
and citrus pectin to 45-60 %[l9O].
I
-
675
I 1Cellusose fibers
1
Xyloglucan
sugar side chains of pectin
Rhamnogalacturonan (pectin)
Figure 11.4-8. Structure of protopectin
11.4.5.1
Classificationof Pectic Substances
Protopectin is composed of water-insolublepectic substances, which are fixed to the

middle lamella and primary cell walls of plant cells. The neutral sugar side chain of
the pectin is attached to the xyloglucan residues, which are bound to the cellulose
fibers. The protopectin includes polyvalent such as calcium (Fig. 11.4-8). Protopectin
is present in unripe fruits. During the maturation process of fruits or after
harvesting, the protopectin is converted to soluble pectin [1851. The insolubility of
protopectin may be due to the polymerization of the molecule and to the cross-
linking with divalent cations [186].
Pectin (pectinate) consists of rhamnogalacturonan molecules that are modified
with neutral sugar side chains. The carboxyl groups of the galacturonic acid
molecules are partially esterified with methanol. The concentration of pectin in
fruits varies with the degree of ripeness and the storage conditions. The average
pectin concentration in fruits (not citrus fruits) varies between 0.5 and 1%[186]. The
completely demethoxylated pectin is designated as polygalacturonic acid (polyga-
lacturonate) or pectate.
1 1.4.5.2
Pectolytic Enzymes
Pectolytic enzymes are widespread in nature, as they have been found in plants,
fungi, insects, nematodes, protozoa and bacteria. During fruit development, ripen-
ing and leaf abscission, pectin-degrading enzymes play an important role [192-1951.
Furthermore, plant pectinases are important in the defensive mechanisms prevent-
ing attack of the plant by pathogenic microorganisms.
Microorganisms, especially plant pathogenic microorganisms, produce a wider
spectrum of pectolybc enzymes than plants themselves. Many of these extracellular
enzymes occur in multiple forms, which enhance the adaptation of the plant
pathogens to different hosts[196,19’1 . The most important enzyme in the plant
pathogenesis process is the endo-polygalacturonase(for review see [1981). Pectinases
synthesized by microorganisms also take part in symbiotic processes and in the
676
Protopectin
methylesterase
Polygalacturonic acid
Pectin
(PGA)
hydrolase lyase
Methyloligo- unsaturated oligo- and mono- unsaturated oligo-

galacturonates methyloligo- galacturonates and digalacturonates
galacturonates
Figure 11.49. Action of pectolytic enzymes.
rotting of plant material. Therefore, pectolytic enzymes are widespread in patho-

genic, symbiotic microoganisms, saprophFc soil bacteria and rumen bacteria. To
this group belong members of the genus Envinia, Pseudomonas, Xanthomonas,
Agrobacteriurn, Corynebacterium, Lactobacillus, Arthrobacter, Bacillus, Flavobacterium,
Azospirillum, Actinomyces, Yersinia, Klebsiella, Clostridium, Cytophaga, Bacteroides and
Lachnospira [199-205, 231-2341
11.4.5.3
Classification o f Pectolytic Enzymes
One can distinguish between three different types of enzymes acting on pectic
substances (Fig. 11.4-9):protopectinases, which degrade protopectin, pectin methy-
lesterases, which release methanol from the galacturonic acid, and depolymerizing
enzymes. The group of depolymerizing enzymes is further divided into four
subgroups according to the reaction mechanisms (hydrolases and lyases) and the
substrates being used (pectin and polygalacturonic acid).
11.4.5.4
Protopectinase
Protopectinasesare enzymes acting on the water-insolubleprotopectin. By the action

of protopectinases the protopectin is solubilized, and water-solublehighly polymer-
ized pectin is released. These enzymes were first described by Sakai and Okushima
in 1978[206]. Further investigations of protopectinases have been r e p ~ r t e d [ ~ ~ ~ - ~ ~ ’ I .
Protopectinases (or pectin-liberating enzymes) have two points of attack in the
protopectin (Fig. 11.4-8):the polygalacturonic regions of the protopectin (A-type of
protopectinases) and the sugar side chains, which connect the protopectin to the
xyloglucans and to the cellulose fibers of the cell walls (B-type of protopecti-
nases) [2101.
A-type protopectinases are produced by yeast, Kluyveromycesfiagilis, Galactomyces
reesei I F 0 0288 and Trichosporon penicillatum SNO 3. Some of these extracellular
enzymes have been purified from the concentrated culture broth[211,212]. Basedon
1 1.4 Natural Polysacchan'de-degrading Enzymes
its ability to hydrolyze the polygalacturonic acid backbone, protopectinase A is

I 677
classified in the group of endo-polygalacturonases(E. C . 3.2.1.15, see also 4.2.5.1.).

The protopectinase A hydrolyzes the glycosidic linkages in polygalacturonic acid if at
least three unmethoxylated galacturonic acid molecules are present at a short
distance. According to this, the molecular mass of pectic products increases with the
increasing degree of esterification of the glucoronic acid residue l2l0].
B-type protopectinases, on the other hand, are unable to degrade the polyg-
alacturonic acid chain. These enzymes were first detected in the culture filtrate of B.
subtilis I F 0 12113 by Sakai and Ozaki in 1988[2131.Many strains of Bacillus species,
including B. amyloliquefaciens, B. cereus, B. circulans, B. coagulans, B. firmus, B.
lichen$ormis, B. macerans and B. pumilus, have been found to be good sources of B-
type protopectinases[210].The production of B-type enzymes is repressed in the
presence of glucose and enhanced in the presence of starch and soybean flour extract
containing arabinogalactan.
11.4.5.5
Pectin Methylesterase
Pectin methylesterases (E. C. 3.1.1.11) deesterify the galacturonic acid methylester in

pectins liberating pectic acid and methanol (Fig. 11.4-10a).The hydrolysis is charac-
terized by high specificity and a high yield (98%)L2l4]. The deesterification proceeds
from the reducing end of the pectin molecule in a linear mode along the chain"].
Pectin methylesterases are produced by molds, yeasts and bacteria [1851. In general,
pectin methylesterases are active in the pH range 5.0-8.0. In contrast to fungal
enzymes, which are active at low pH, the bacterial esterases prefer alkaline
conditions. In fruits and vegetables, especially in citrus fmits and tomatoes, high
pectin methylesterase activities have also been found.
11.4.5.6
Pectin and Polygaladuronate Depolyrnerizing Enzymes
The activity of pectin-depolymerizing hydrolases, especially endoacting enzymes,

can be followed by a rapid decrease in the viscosity of the pectin solutions. By the
cleavage of only 2-3 % of the glycosidic bonds the viscosity diminishes to about 50%.
In addition, the increasing amount of reducing ends can be determined. The last
stage of pectin depolymerization on an industrial scale is proved by the alcohol test.
The depolymerizing reaction is complete when the addition of 50 % alcohol to the
reaction mixture does not lead to flocculationL2l4]. The activity of trans-eliminases
(lyases) can be followed photometrically by measuring the UV adsorption of
4,5-dehydrogalacturonicacid at 232 nm[2151.
678
+ n20
r
a. Pectin methylesterase
0 -
n
+
HO
=OH OH
H
O
Qn
+ H,O
%
HO
-%
HO
?
HO
q0 -
b. Pectin and polygalacturonicacid hydrolase
0
HO
HO
O W O H
+
OH
HO % ?
H O 4
c. Pectin and polygalacturonic acid lyase (R'= H: polygalacturonicacid; R'= CH3: pectin)
Figure 11.4-10. Reaction mechanisms of pectolytic enzymes.
11.4.5.7
Pectin and Polygalacturonate Hydrolase
Pectin hydrolase and polygalacturonate hydrolase (polymethylgalacturonase, polyg-

alacturonase) catalyze the cleavage of the polysaccharide backbone of pectin and
polygalacturonate. Pectin hydrolases prefer pectin, and polygalacturonases prefer
polygalacturonic acid as substrates (Fig. 11.4-lob). According to the mode of action,
these enzymes can be defined as endo- or exoenzymes. Exoenzyrnes are able to split
mono-, di- or trimers from the reducing end of the polysaccharide chain (pectin or
polygalacturonic acid). Endoacting enzymes, on the other hand, attack the complex
polysaccharide in the inner part of the chain backbone, resulting in a rapid decrease
of viscosity of pectin- or polygalacturonate solutions. Endoacting enzymes prefer
long polysaccharide chains of pectin or polygalacturonic acid. The activity decreases
with decreasing chain length.
Endopolygalacturonatehydrolases (E. C. 3.2.1.15) are widespread in fungi, in most
plant pathogens, in some bacteria, in plant organs and in the digestive tracts of some
insects [*lG]. The enzyme catalyzes the random hydrolytic cleavage of a-1,4linkages of
galacturonan and requires free carboxyl groups for their catalytic activity. The activity
I 679
therefore decreases with increasing degree of esterification of the polygalacturonic

acid substrate [2171. Endopolygalacturonases have been purified from several plant
and microbial sources and are optimally active under acidic conditions
(PH 2.5-6.5).
Most exopolygalacturonases release D-monogalacturonic acid from the non-reduc-
ing end of the chain (E. C. 3.2.1.67). The enzymes produced by Erwinia aroideae and
Pseudomonas sp. (E. C. 3.2.1.82) are able to release digalacturonic 218].
Exopolygalacturonases from fungi exhibit optimal activity between pH 4.0 and pH

6.0, whereas the enzymes from Clostridium multijirmentans show highest activity at
pH 7.2.
In addition to exo- and endopolygalacturonases a number of microorganisms
produce oligogalacturonases which hydrolyze oligogalacturonate chains forming
short oligomers and galacturonate. The oligogalacturonases have higher affinity to
low molecular weight oligogalacturonates than to polygalacturonates. The activity
decreases with increase of the chain length of the substrate. The oligogalacturonases
from Bacillus species and A. niger attack the substrate from the non-reducing end,
whereas the enzymes produced by Erwinia carotovora and E. aroideae hydrolyze the
substrate from the reducing end['8G].
11.4.5.8
Pectin and Polygalacturonate Lyase
The reaction mechanism of lyases is characterized by a trans-elimination reaction

resulting in 6 4,5-unsaturated galacturonic acid molecules. The lyases are calcium
dependent and attack either pectin (pectin lyases) or pectic acid (polygalacturonate
lyases) from the non-reducing end (Fig. 11.4-1Oc).
Endopolygalacturonate lyase (E. C. 4.2.2.2) has been detected in many bacteria and
some pathogenic fungi. These enzymes show highest activity under alkaline condi-
tions in the pH range 8-10. The enzyme activity depends exclusively on the presence
of calcium ions and decreases with decreasing chain length of the polygalacturonic
acid.
Exopolygalacturonatelyases (E. C. 4.2.2.9) have been detected in only a few bacteria
which belong to the genera of Clostridium, Erwinia, Streptomyces and Fusarium['861.
The majority of these enzymes are active under alkaline conditions (pH 8-9.5) and
require calcium ions for activity. Erwinia carotovorans and E. aroideae have been
found to synthesize oligogalacturonate lyase (E. C. 4.2.2.6) [219, 220]. The enzyme
releases unsaturated monomers from the reducing end of the oligogalacturonate
substrates.
Endopectin lyases (E.C. 4.2.2.10) are widespread in fungi and prefer long poly-
methylgalacturonate chains (pectin) as substrates, resulting in decreasing activity
with decreasing chain
The distribution of different pectolytic enzymes in microorganisms is shown in
Table 11.4-4. For review see [lSG.23G1.
680
Table 11.4-4. Occurence of different pectolytic enzymes in microbes.

Organism PME Pectin Pectin PCA PCA Ref.
hydrolases lyases hydrolases lyases
Fungi:
Aspergillus niger + - + + + [664]
Aspergillus alliaceus - + i - - [665]
A.flavus - + + - - [666]
A. fumigatus - + i - - [666]
Botrytis cinerea + + - + - [667, 6681
Fusarium tricinctum + - - + + [669]
Yeasts:
Candida pseudotropicalis - - - + - [670]
Saccharomyces vini + - - + - [671]
Bacteria:
Clostridium pectinofmentans + - + + - [672]
C. thermosulfurogenes 4B + - - + - 16731
C. thermosaccharolyticum + - - + - [674]
Bacillus stearothemophilus - - - - + [675]
Corynebacterium michiganenese - - + - + [676]
Enuinia chrysanthemi - + + + + [677]
Pseudomonas marginalis - - + - - [678]
Streptomyceskadiae - - - - + [679]
Xanthomonas campestris - + - + + 16801
PME: Pectin methylesterase; PGA: Polygalacturonicacid.
11.4.5.9
Biotechnological Applications of Pectolytic Enzymes
Enzymes with pectolytic activity have been used since 1930 in the clarification of
fruit juices. In freshly pressed apple juice, pectin acts as a stabilizing colloid for the
insoluble cell debris. After hydrolysis of the pectin, the insoluble particles floc out.
Also, in white wine production, a clarification process for the removal of insoluble
particles suspended in the grape must is The commercial enzyme
preparations for industrial application may contain, as well as pectolytic enzymes,
cellulases, hemicellulases, xylanases and proteases. All these enzymes solubilize the
cell wall constituents to form soluble products such as galactose, mannose, rham-
nose, arabinose, galacturonic acid and methanol [222* 2231. Similar processes are in
use for the maceration of vegetables and the extraction of olive oil. The preincubation
of sugar beet with pectolytic enzymes (1-2 h at 54 "C or 6-8 h at 18 "C) before the
pressing procedure improves the yield significantly[224]. Pectin methylesterases are
also used in the production of apple cider. After demethoxylation of pectin, the
product formed (polygalacturonic acid) can be easily removed from the fermenting
apple juice by precipitation with calcium ions['"].
Pectolytic enzymes are also involved in natural fermentation processes. The coffee
seeds (coffeebeans) are directly surrounded by the so-called seed coat or silver skin,
followed by the endocarp (hull), the mesocarp (mucilage layer) and the exocarp
(skin). One of these envelopes, the mesocarp, consists of 30% pectic substances.
References I681
Table 11.4-5. Microorganisms used for the commercial production of pectolytic enzymes.
Organism Pectin Pectin Pectin PCA PCA Oligo-
methylesterase hydrolase lyase hydrolase lyase galacturonase
A. niger +
Bacillus sp. -
Penicillium sp. +
Rhizopus sp. - - - + - -
PGA: Polygalacturonic acid.
This polysaccharide is degraded by the pectolytic enzymes that are produced by the
epiphyhc microbial flora of the coffee fruits, i.e. Envinia and Enterobacter spe-
~ ' ] . 1-4 days the digestion is complete and a mechanical depulping step of
c i e ~ [ ~ After
the coffee fruits can take place. Also, by the cocoa fermentation during the first
1-2 days, pectolytic enzymes from yeasts aid in the maceration of the cocoa pulp and
the draining of the fluid. The fermentation of cocoa and coffee fruits can be
enhanced by the addition of commercial enzyme preparations containing pectin-
depolymerizing enzymes [2351.
Protopectinases are also used in the production of pectin from mandarin orange
peel. Pectin can be used as an additive in the food and cosmetic industries[22G].
In all applications described above involving conventional pectolFc enzymes, the
rhamnogalacturonan backbone of pectic substances is not degraded ~ o m p l e t e l y [ ~ ~ ~ l .
It has been reported that Aspergillus aculeatus produces an enzyme complex consist-
ing of 10 to 15 different enzymes. This enzyme complex has the potential for the
complete hydrolysis of complex polysaccharides and may support liquefaction
processes with plant material, fruits or vegetables[2271. For the commercial produc-
tion of pectolytic enzymes, Aspergillus niger or related species are mainly used. In
these fermentations, low value agricultural products containing pectin are used as
substrates [228-2301. Table 11.4-5shows some of the microorganisms that are used for
the industrial production of pectolytic enzymes.
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141 H. 0. Bouveng, Acta Chem. Scand. 1961, 15, 161 C. R. MacKenzie, D. Bilous, H. Schneider,
96-100. K. G. Johnson, Appl. Environ. Microbiol.
142 B. Lindberg, K:G. Rossell, S. Svensson, 1987,53,2835-2839.
Svensk. Papperstidn. 1973, 76, 30-32. 162 A. Kaji, K. Tagawa, Biochem. Biophys. Acta
143 J. Puls, J. Schuseil in Hemicelluloses and 1970,207,456-464.
Hemicellulases M. P. Coughlan, G. P. Hazle- 163 K. Komae, A. Kaji, M Sato, Agnc. Bid.
wood (eds),Portland Press, London, Eng- Chem. 1982,46,1899-1905.
land, 1993, pp. 1-97. 164 K. Poutanen,/. Biotechnol. 1988,7, 271-292.
144 M. Zinbo, T. E. Timell, Svensk. Papperstid. 165 J. Puls, K. Poutanen, 0. Schmidt, M. Linko
1967,70,597-606. in Proc. 3rd Int. Con5 Biotechnol. Pulp Paper
145 P. J. Reilly in Trends in the Biology ofFernen- Industry K. E. Eriksson, P. Ander
tations A. Hollaender (ed), Plenum Press, (eds),STFI, Stockholm, Sweden, 1986, pp.
New York, USA, 1981, pp. 111-129. 93-95.
146 R. F. H. Dekker, G. N. Richards, Adv. Carbo- 166 J. Puls in Xylans and Xylanases J. Visser,
hydr. Chem. Biochem. 1976,32, 277-352. G. Beldman, M. A. Kusters-van Someren,
147 K. K. Y. Wong, L. U. L. Tan, J. N. Saddler, A. G. J. Voragen (eds), Elsevier, Amsterdam,
Microbiol. Rev. 1988, 52, 305-317. The Netherlands, 1992, pp. 213-224.
148 M. Debeire-Gosselin, M. Loonis, E. Samain, 167 P. Biely, C. R. MacKenzie, J. Puls,
P. Debeire in Xylans and Xylanases J. Visser, H. Schneider, Bio/TechnoL 1986,4,
G. Beldman, M. A. Kusters-van Someren, 731-733.
A. G. J. Voragen (eds), Elsevier, Amsterdam, 168 M. Tenkanen, K. Poutanen in Xylans and
The Netherlands, 1992, pp. 463-466. Xylanases J. Visser, G. Beldman, M. A. Kus-
149 M. Debeire-Gosselin, J. P. Touzel, P. De- ters-van Someren, A. G. J. Voragen (eds),
beire in Xylans and Xylanases J. Visser, G. Elsevier, Amsterdam, The Netherlands,
Beldman, M. A. Kusters-van Someren, A. G. 1992, pp. 203-212.
J. Voragen (eds),Elsevier, Amsterdam, The 169 P. Biely, J. Puls, H. Schneider, FEBS Lett.
Netherlands, 1992, pp. 471-474. 1985, 186,80-84.
150 S. Takenishi, T. Tsujisaka, Agnc. Bid. Chem. 170 J. Puls, M. Tenkanen, H. E. Korte, K. Pouta-
1975,39,2315-2323. nen, Enzyme Microb. Technol. 1991, 13,
151 A. Matte, C. W. Forsberg, Appl. Environ. Mi- 483-486.
crobiol. 1992, 58, 157-168. 171 K. Poutanen, J. Puls, A C S Symp. Ser. 1989,
152 A. C. Grabski, T. W. Jeffries, Appl. Environ. 388,630-639.
Microbiol. 1991, 57, 987-992. 172 P. Biely, 2. Kratky, M. Vrsanska, Eur.
153 T. M. Wood, S. 1. McCrae, Carbohydr. Res. /. Biochem 1981, 119,559-564.
1986, 148,321-330. 173 P. Biely, M. Vrsanska, 2. Kratky, Eur.
154 F. Deleyn, M. Claeyssens, J. Van Beeumen, /. Biochem. 1981, 119, 565-571.
C. K. De Bruyne, Can. /. Biochem. 1978,56, 174 M. Vrsanska, 1. V. Gorbacheva, 2. Kratky,
43-50. P. Biely, Biochem. Biophys. Acta 1982,704,
155 W. Zimmermann, B. Winter, P. Broda, 116122.
F E M S Microbiol. Lett. 1988, 55, 181-186. 175 L. Viikari, J. Sundquist, J. Kettunen, Paperi
156 J. Dobberstein, C. C. Emeis, Appl. Microbiol. j a Puu 1991,73,384-388.
Biotechnol. 1991, 35, 10-215. 176 Cultor, Albazyme@,Pulp and Paper Enzymes
157 S. L. Bachmann, A. J. McCarthy, Appl. En- (product data sheet, Cultor, UK Ltd.), 1991.
viron. Microbiol. 1991, 57, 2121-2130. 177 P. Biely, A C S Symp. Ser. 1991, 460,408-416.
158 D. Conrad, W. Noethen in Proc. 3rd Eur. 178 M. Linko, K. Poutanen, L. Viikari in Enzyme
Congr. Biotechnol. 1984, 2, pp. 169-177. Systemsfor Lignocellulose Degradation M. P.
159 M. Claeyssens, E. Van Leemputten, F. G. Coughlan (ed), Elsevier Applied Science,
Loontiens, C. K. De Bruyne, Carbohydr. Res. London, England, 1989, pp. 331-346.
1966, 3, 32. 179 K. Poutanen, M. Ratto, J. Puls, L. Viikari,/.
160 M. Claeyssens, F. G. Lonntiens, H. Ker- Biotechnol. 1987, 6, 49-60.
sters-Hilderson, C. K. De Bruyne, Enzymo- 180 Y.-E. Lee, E. E. Lowe, J. G. Zeikus, Appl. En-
logia, 1971,40,177-198. viron. Microbiol. 1993, 59, 763-771.
686
11.5
Addition of Water to C=C Bonds
Marcel Wubbolts
The addition of water to carbon-carbondouble bonds is a reaction that is catalyzed by

lyases belonging to the subclass of the hydro-lyases (E. C. 4.2.1.-),which have been
grouped under the carbon-oxygen lyases. Not all members of this subgroup are
capable of water addition to carbon-carbon double bonds. Nitrile hydratase (E. C.
4.2.1.84, discussed in Section 12.1) for instance, is categorized in this subclass and
catalyzes the addition of water to nitriles. The nomenclature of the hydro-lyases
subgroup, which contains hydratases and dehydratases, does not preclude any
direction of the reaction, but rather reflects the context in which the enzyme was
originally discovered.
The addition of water to carbon-carbon double bonds is very common to biology,
and a large variety of enzymes from different sources representing almost a hundred
different hydro-lyase types have been characterized biochemically. Hydro-lyases are
for instance involved in the metabolism of a variety of carbohydrates and play a
prominent role in fatty acid synthesis and degradation as well. Despite the abundant
presence of hydro-lyases in nature, however, applications of these enzymes in
organic chemical synthesis are not as widespread. This is mainly due to the limited
availability of these enzymes and the fact that many of the enzymes cannot easily be
stably maintained during catalysis.
11.5.1
Addition of Water to Alkenoic Acids
The catabolic enzyme 2-oxopent-4-enoatehydratase (E. C. 4.2.1.80) is involved in L-

phenylalanine metabolism and in the degradation of a number of aromatic hydro-
carbons as well[']. It catalyzes the selective addition ofwater to a terminal C-C double
bond of cis-2-hydroxypent-2,4-dienoic acid and forms 4-hydroxy-2-oxopentanoic acid.
The enzyme also accepts cis-2-hydroxyhex-2,4-dienoic acid as a substrate, but is not
active on the trans-isomer121.
D-Tartaric acid dehydratase (E. C. 4.2.1.81) and the stereochemical counterpart L-
tartaric acid dehydratase (E.C. 4.2.1.32) are able to catalyze the conversion of
oxaloacetic acid to D- and L-tartaric acid respectively. The actual addition of water to
the C-C double bond is most likely to occur at the enol tautomer, and the resulting
tartaric acid has the 2S,3S (D-stereo isomer made by E.C. 4.2.1.81) or 2R,3R (L-
tartaric acid dehydratase)configuration. Despite the stereochemistry of the reactions
catalyzed, the lack of available enzyme and the instability of the enzymes in presence
of oxygen[3]have hampered their application in organic synthesis thus far.
Furnarate
11.5 Addition of Water to C=C Bonds
I
687
-
-
hydratase
HOOC-COOH 1
H O O C YCOOH 2
Maleate OH
hydratase
H O O C T
3 -
- HOOC -
-
-
COOH
COOH Citraconate OH
HOOC/\\/ 5 -
-hydratase
H O O C T C o o H
COOH 0
Mesaconate OH
hvdratase
3-isopropylmalate
dehydratase
COOH 10
HOOC HOOC
-
COOH dH
Dirnethylrnaleate
I hydratase
Scheme 11.5-1.
11.5.2
Addition of Water to Alkene-Dioic Acids
11.5.2.1
L- and D-Malic Acid
The production of L-malic acid (2)from fumaric acid (1)is carried out by the enzyme
fumarate hydratase (E.C. 4.2.1.2). which is part of the tricarboxylic acid cycle and
ubiquitous in nature (Scheme 11.5-1). The product is used in food, pharmaceutical
and cosmetic industries and is produced at a multi-ten-tonne scale. Although the
enzyme can be applied in isolated form, as performed by Tanabe, the use of whole
cells of Corynebacteriurn glutarnicurn has been reported by Amino GmbH as well[4].
688
The conversion of fumaric to L-malic acid is brought to completion by forcing the

product to precipitate as calcium salt [41.
The synthesis of D-Malic acid (4) from maleic acid (3) by maleate hydratase (E. C.
4.2.1.31) has been described as early as 1969, using an enzyme from rabbitI5].
Maleate hydratase from various other, more accessible sources such as Pseudomo-
have been used for the same purpose. The combined use of calcium-counter
ions and maleate hydratase (E. C. 4.2.1.31) from Pseudomonas pseudoalcaligenes has
been an elegant method to produce on multi-kilogram scale D-malate that in
complex with calcium precipitated out from solution, thereby eliminating the
reverse reaction[6]. He et al. used a similar enzyme from Arthrobacter pascens
DMDCl2, which is called (R)-2-methylmalatedehydratase, citraconate hydratase or
citraconase (E.C. 4.2.1.35), to produce D-malate as well as D-citramalate or ( R ) -
2-methylmalic acid (6) from 2-methylmaleate ( S ) , using an enzyme membrane
reactor The demand for D-malate is limited: it merely serves as a general
synthetic building block for chiral 1' and as a resolving agent.
11.5.1.2
Substituted Malic Acids
The enzyme of opposite selectivity relative to citraconase (E. C. 4.2.1.35) mentioned

above, is (S)-2-methylmalatedehydratase or mesaconate hydratase (E. C. 4.2.1.34),
which has, among others, been found in Clostridium t e t a n o m ~ ~ h u m [Pseudomo-
~]],
nasl"], Citrobacter and Morganella["], and is of use to convert 2-methylfumarate (7)
to the (S)-isomerof citramalate (8). Interestingly, both citraconase and mesaconate
hydratase have a broader substrate range and are also able to produce the respective
stereo-isomers of malic acid and 2-ethylmalic acid [', 91. The 3-iso-propylmalate
dehydratase (E. C. 4.2.1.33) from Neurospora crassa and numerous other prokaryotic
strains are involved in synthesis of L-valine, L-isoleucine and L-leucine. The enzyme
accepts the iso-propyl group as a substituent during the reaction and converts 2-iso-
propylmaleate (9) to 3-iso-propylmalate(10)[l21.
Dimethylmaleate hydratase (E. C. 4.2.1.85) has been described as the enzyme that
catalyzes the addition of water to dimethylmaleate (11) to yield a molecule with two
chiral centers, (2R,3S)-2,3-dimethylmalate (12)[131.
11.5.3
Addition o f Water to Alkene-Tricarboxylic Acids
11.5.3.1
Citric Acid and Derivatives
Other C-0 lyase enzymes include aconitate hydratase or aconitase (E.C. 4.2.1.3), an
enzyme that catalyzes two tricarboxylic acid cycle steps from isocitric acid to citrate
(14)[141 or vice versa, via the intermediate cis-aconitate(13). Citrate dehydratase (E. C.
4.2.1.4) is only capable of converting citrate to cis-aconitate and does not act on
isocitrate (15)[151.
11.5 Addition of Water to C=C Bonds I 689
COOH Citrate dehydratase
VooH 13 -
or Aconitase
. H O O C T c o o H 14
\COOH COOH
OH
- I
HoocY
Aconitase
13 15
COOH
-
Homo-aconitate
hydratase
.
H O O C ~ c o o H
l""OO" 16 17
COOH
COOH
2-Methylcitrate COOH
\7 COOH
dehydratase
18 19
\COOH \COOH
2-Methylisocitrate COOH
de hydratase
18
20
\COOH
4-Oxarnesaconate
hydratase
Hooc
Hooc-=
21 22
COOH COOH
Scheme 11.5-2.
A similar reaction is catalyzed by homoaconitate hydratase (E. C. 4.2.1.36),which

is an enzyme from the L-lysine synthesis that forms homocitric acid (2-hydrox-
ybutane-1,2,4-tricarboxylic acid, 17) from homo-cis-aconitate (16)[16]. The enzyme
2-methylcitratedehydratase (E. C. 4.2.1.79) catalyzes the addition of water to (Z)-but-
2-ene-1,2,3-tricarboxylic acid (18) to yield 2-methylcitric acid (2-hydroxybutane-
690
I 1,2,3-tricarboxylicacid,19) [l']. 2-Methylisocitrate dehydratase (E. C. 4.2.1.99) from
Yarrowina lipolytica does not accept isocitrate (15) as substrate, but rather acts on (2)-
but-2-ene-1,2,3-tricarboxylic acid (18) to produce 2-methylisocitrate(20)["I.
Lastly, 4-carboxy-2-oxohexenedioatehydratase (4-oxamesaconate hydratase, E. C.
4.2.1.83) adds water to (E)-4-oxobut-l-ene-l,2,4-tricarboxylic acid (21) and results in
the formation of 2-hydroxy-4-oxobutane-1,2,4-tricarboxylic acid (22)[191 (Scheme
11.5-2).
115.4
Addition o f Water to Alkynoic Acids
Interestingly,two enzymes have been described that catalyze the addition of water to
alkynes, resulting in the formation of alkenols: acetylene carboxylate hydratase from
Pseudomonas (E. C. 4.2.1.71), which converts propynoic acid to 3-hydroxyprope-
noate f20]. The latter tautomerizes to malonic semialdehyde. Acetylene dicarboxylate
hydratase (E. C. 4.2.1.72) converts acetylene dicarboxylic acid to 2-hydroxyethylenedi-
carboxylic acid, which spontaneously decarboxylatesto pyruvate 121].
11S.5
Addition o f Water to Enols
11.5.5.1
Carbohydrates: Addition o f Water to 2-Keto-3-Deoxysugars
Hydro-lyasesplay a prominent role in the metabolism of sugars and of sugar-derived

carboxylic acids in particular. The eliminationladdition ofwater to sugar carboxylates
proceeds via an enol intermediate[22],as depicted in Scheme 11.5-3.The elimination
or addition of the water molecule is highly specific, and a large variety of hydrolyases
have been characterized examples include Pseudomonas saccharophila D-arabinoate
dehydratase (E. C. 4.2.1.5) [231, Pseudomonas sp. and E. coli galactonate dehydratase
(E. C. 4.2.1.G)[241,E. coli altronate dehydratase (E. C. 4.2.1.7)[251,E. coli mannonate
dehydratase (E. C. 4.2.1.8) L2' ] , L-arabinoate dehydratase (E. C. 4.2.1.25) from Rhizo-
bium[2G1, phosphogluconate dehydratase (E. C. 4.2.1.12) from various organisms [271,
gluconate dehydratase (E. C. 4.2.1.39) from various organisms 12'], D-fuconate hydra-
tase (E. C. 4.2.1.67) from Pseudornonas sp. [291, Mammalian L-fuconatehydratase (E. C.
4.2.1.68) i3O1, D-xylonate dehydratase (E. C. 4.2.1.82)I3l ], and fungal L-rhamnonate
dehydratase (E. C. 4.2.1.90).
The elimination of water from glucarate, a 1,G-dicarboxylic hexose, by glucarate
dehydratase (E. C. 4.2.1.40) results in the formation of 5-dehydro-4-deoxy-~-glucar-
ate[32].The reaction is however identical to that of the other dehydratases and the
seemingly different specificity is only due to IUPAC rules (Scheme 11.5-3).The
enzyme belongs to the enolase superfamily, and the structure of the enzyme from
Pseudomonas putida has been resolved [331. Similarly, galactarate dehydratase from E.
coli (E. C. 4.2.1.42) produces 5-dehydro-4-deoxy-~-galactarate [321.
7 7.5 Addition of Water to C=C Bonds
I
691
-
Dehydratase HO
HokCOOH
O F C O O H
~
4
-
F
R t R
C O O H
/
R
-
Galactonate
Dehydratase
f:-
HO HO
CHzOH
I-OH
CHpOH CHpOH
2-dehydro-3-deoxy-
D-Galaconate - 'enol' D-Galactonate
-
COOH
Glucarate
Hoji
Dehydratase
=
COOH
COOH - COOH
5-dehydro-4-deoxy-
D-Glucarate 'enol' D-Glucarate
Scheme 11.5-3.
11.5.5.2
Addition/Elirnination of Water with Other Enok
Dihydroxyacid dehydratase (E.C. 4.2.1.9) is a ubiquitous enzyme that is involved in

the biosynthesis of the branched-chain amino acids (Ile, Leu and Val) and of
pantothenic acid and coenzyme A. The enzyme catalyzes the elimination of water
from 2,3-dihydroxyalkanoic acids (23) to 2-hydroxy-2-alkenoic acids (24), which
tautomerize to 2-ketoalkanoic acids (25).The enzyme from spinach has the highest
activity towards 2,3-dihydroxy-3-methylbutanoic acid (Val precursor, Scheme 11.5-4)
but also accepts other substrates [341. Thus, 2,3-dihydroxybutanoic acid, 3-cyclopropyl-
2,3-dihydroxybutanoic acid as well as 2,3-dihydroxy-3-methylpentanoic acid are
substrates. With the latter substrate a slight preference for (2R,3S)-2,3-dihydroxyy-
692
I Dihydroxyacid
JCOOH dehydratase [<COOH\ ~ ~
HO
23 24 25
Enolase
26 p l o ~ C O O H 27
Scheme 11.5-4.
3-methylpentanoate over the (2R,3R)-2,3-dihydroxy-3-methylpentanoate was ob-

served [341.
The glycolytic enzyme phosphoenolpyruvate (PEP) hydratase (enolase, E. C.
4.2.1.11) catalyzes the addition of water to 2-phospho-~-glycerate(26). The enzyme
from E. c0li1~~1 also accepts 3-phospho-~-erythronate(28) and thereby forms phos-
phoenol-4-deoxy-3-tetrulosonate (29 in Scheme 11.5-4).Both PEP and phosphoenol-
4-deoxy-3-tetrulosonaterepresent “fixed enolates that can be isolated.
The enzymes 1,2-propanedioldehydratase (E. C. 4.2.1.28) and glycerol dehydratase
(E. C. 4.2.1.30) from the facultative anaerobic microorganism Klebsiella pneumoniae
[36, 371 and other sources have recently gained interest, since these enzymes can be of
use for the synthesis of 1,3-propanediol (PDO) starting from glycerol. PDO is of use
for the synthesis of polyesters, and Dupont is currently developing a biological
production method based on fermentation. Glycerol dehydratase (E. C. 4.2.1.30)
catalyzes the elimination of water from a number of polyols: ethylene glycol to
acetaldehyde, glycerol to 3-hydroxypropanal and 1,2-propanediol to propionalde-
hyde, all of which reactions proceed via an enol intermediate.
693
11.5.6
I
Addition ofwater to Unsaturated Fatty Acids
11.5.6.1
CoA and ACP Coupled Fatty Acid Hydratases
Hydratases that add water to unsaturated fatty acids coupled to coenzyme A (CoA)or
acyl carrier protein (ACP) cannot be used in vitro, and consequently have to be
applied in whole-cellbiotransformations. Prohibitive as this may seem to production
on a commercial scale, Kanegafuchi has developed a process, making use of whole
cells of Candida rugosa, to produce (R)-2-hydroxybutanoicacid (31) from butanoic
acid (30) (Scheme 11.5-5).The series of reactions catalyzed by these cells include
coupling of butanoic acid to CoA, desaturation of butyryl-CoA to 2-butenyl-CoA and
water addition catalyzed by enolyl-CoA hydratase (enoylase, unsaturated enoyl-
Y
COOH
30 32
Candida rugosa
fatty acid metabolism
ll Enoyl-CoA hydratase
Thioesterase
1
' L C O O H 33
Scheme 11.5-5.
694
Agrobacterium
fatty acid metabolism
35
tl Carnitine
de hydratase
Carnitine
dehydrogenase
I
36 SCoA 37
T hioesterase
38
Scheme 11.5-6.
coenzyme A hydratase, E.C. 4.2.1.17). Removal of the CoA group liberates the p-
hydroxy acid, which is of use for the synthesis of carbapenems[381.Similarly, the
Candida rugosa system has been used by Kanegafuchi to produce (R)-2-hydrox-
yisobutyric acid (33),an intermediate for the synthesis of the ACE inhibitor Captopril
from the starting compound isobutync acid (32)r4, 38, 391.
The production of L-carnitine by Lonza is also camed out by whole cells that make
use of CoA-coupled fatty acid degradation or the p-oxidation pathway. L-Carnitine
(38),or (R)-3-hydroxy-4-trimethylaminobutyric acid, serves as a fatty acid carrier and
plays an important role in the metabolism of fats. In addition to clinical applications,
such as for the treatment of disorders in fat metabolism, it is also a popular over-the-
counter product in fitness and anti-obesity formulations. Lonza carries out the
production of L-carnitineon a multi-tonne scale, in a whole-cellprocess. The whole-
cell process utilizes intact Agrobacteriurn cells that are fed with glucose and
4-butyrobetaine (34)as a precursor. The key enzyme that catalyzes the addition of
water to crotonobetaine is L-carnitine dehydratase (crotonobetainyl-CoAhydratase,
E. C. 4.2.1.89),which adds a water molecule to the fermentation product, crotonobe-
tainyl-CoA (35,see Scheme 11.56).The resulting product, L-carnityl-CoA (36)is not
oxidized to the corresponding B-keto acid 37, since the cells lack the enzyme
carnitine dehydrogenase. Instead, the CoA coenzyme is hydrolyzed off by a thioester-

I 695
ase resulting in the release of L-carnitine (38) in the medium[40].

Despite the fact that numerous enzymes have been characterized that catalyze the
addition of water to unsaturated fatty acids that are coupled to CoA or ACP, such as
methylglucatonyl-CoA hydratase (E. C. 4.2.1.18), lactoyl-CoA dehydratase (E. C.
4.2.1.54), 3-hydroxybutyryl-CoA dehydratase (E. C. 4.2.1.55), itaconyl-CoA dehy-
dratase (E. C. 4.2.1.56),isohexenylglutaconyl-CoA hydratase (E. C. 4.2.1.57),farnesyl-
CoA dehydratase (E. C. 4.2.1.57), long-chain enoyl-CoA hydratase (E. C. 4.2.1.74),
3-hydroxydecanoyl-ACP dehydratase (E. C. 4.2.1.60) and 3-hydroxypalmitoyl-ACP
dehydratase (E. C. 4.2.1.61), these enzymes are seldomly applied in organic synthe-
sis.
An unusual coenzyme A-coupled hydratase reaction occurs during the anaerobic
degradation of benzoic acid by Thauera aromatica, where cyclohexa-1,5,-diene-
1-carboxylate CoA hydratase (E.C. 4.2.1.100) adds a water molecule to the cyclohex-
adiene functionality, resulting in the formation of 6-hydroxycyclohex-1-enecarbonyl
COAI4l1.
11.5.6.2
Hydratases Acting on Free Fatty Acids
The enzyme oleate hydratase (E. C. 4.2.1.53) from Pseudomonas catalyzes the elim-
ination ofwater from (R)-10-hydroxystearateor the addition ofwater to a number of
free unsaturated fatty acids, yielding (R)-10-hydroxyfatty acids. Substrates that have
been identified include linoleic acid, oleic acid and palmitoleic acid, which are
converted to the corresponding 10-hydroxy-fattyacids [421.
11.5.7
Addition o f Water to Steroids
Hydratation of unsaturated carbon-carbon bonds in the steroid nucleus has been

ascribed to hydrolyases such as 5-a-hydroxysteroiddehydratase (E. C. 4.2.1.62) from
Saccharomyces cerevisiae, which catalyzes the interconversion of 5-a-ergosta-
7,22-diene-3+-5-diol and Similarly, the formation of 16-a-hydro-
progesterone from 16-dehydroprogesteroneis catalyzed by 16-dehydroprogesterone
dehydratase. The same enzyme catalyzes the addition of water to 16-a-hydroxy-
progesterone or 16-a-hydroxy-pregnenolone, yielding the corresponding 16,17-dide-
hydroprogesterone and 16,17-didehydropregnenolone (E. C. 4.2.1.86 or E. C.
4.2.1.98 La]).
696
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5 J. S. Britten, H. Morell, J. V. Taggart, Bio- 1986,132,2733-2742,
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6 M. J. van der Werf, W. J. van den Tweel, Enzymol.1982,89,98-101.
S. Hartmans, Eur. J. Biochem. 1993,217, 28 R. Bender, G. Gottschalk, Anal. Biochem.
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7 B. He, T. Nakajima-Kambe,T. Ozawa, T. 29 A. S . Dahms, R. L. Anderson,]. Biol. Chem.
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47-414. 30 R. Yuen, H. Schachter, Can.J. Biochem.
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58,2854-2860. 90 302-305.
9 C. C. Wang, H. A. Barker, J. Biol. Chem. 32 B. K. Hubbard, M. Koch, D. R. J. Palmer,
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12 S. R. Gross, H. E. Umbarger, Biochemistry 34 M. C.Pinung, C. P. Holmes, D. M. Hor-
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16 G. Weidner, B. Steffan, A. A. Brakhage, Microbiol. Rev. 1999,22, 553-566.
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17 T. Tabuchi, H. Aoki, H. Uchiyama, T. Naka- ker Inc., New York 1993.
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2823-2929. Noda, H. Kawaharada, K. Watanabe, /. Fer-
18 J. V. Schloss, M. H. Emptage, W. W. Cle- ment. Echnol. 1982,60, 501-508.
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19 K. Maruyama, Biochem. Biophys. Res. Com- len, F. H. Hoeks, in: Chirality i n Industry.
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20 E. W. Yamada, W. B. Jakoby,/. Biol. Chem. (eds), John Wiley and Sons, New York,
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21 E. W. Yamada, W. B. jakoby,J. Biol. Chem. 41 Hanvood CS, Gibson J,J. Bacteriol. 1997,
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I
699
12
Hydrolysis and Formation of C-N Bonds
12.1
Hydrolysis of Nitriles
Birgit Schulze
12.1.1
Introduction
Organic nitriles are used extensively industrially as precursors for the production of
a wide variety of amides and acids by chemical synthesis. In recent years, consider-
able attention has been paid to enzymatic hydrolysis of nitriles as an alternative route
to the chemical synthesis of amides and carboxylic acids. Conventional chemical
conversion of nitriles suffers from several disadvantages,including the requirement
for highly acidic or basic reaction conditions, high energy consumption, formation
of undesirable by-products, low yields and environmental problems due to the
generation of waste salts.
Biocatalysis, on the other hand, may be performed under mild conditions (low
temperatures, neutral pH) thus affording high conversion yields and selective
hydrolysis of the -CN functionality of compounds containing acid or base labile
groups. Furthermore, enzyme hydrolysis is, in some instances, enantio- and/or
regioselective.
This chapter does not aim to give a complete treatise on the extensive literature on
nitrile bioconversions but rather aims at presenting a brief overview of enzymatic
nitrile hydrolysis with a selection of examples. Several reviews on the bioconversion
of organic nitriles and its potential technological application have been pub-
lished [1-71.
700
I 72 Hydrolysis and Formation ofC-N Bonds
12.1.2
Types of Nitrile Hydrolyzing Enzymes
12.1.2.1
Enzymatic Hydrolysis of Organic Nitriles
A variety of organic nitriles, such as cyanoglycosides and cyanolipids, occur naturally

in biological materialrl, 1' . It has been shown that nitrile-hydrolyzing activity is
widespread in bacteria and fungi but has recently also been identified in insects and
humans 1('. In microorganisms the hydrolysis of organo-nitriles (hereafter denoted
as nitriles) is effected by two distinct enzymes, nitrilases or nitrile hydratases.
Nitrilase (E. C. 3.5.5.1.) catalyzes the hydrolysis of nitriles to the corresponding
acids and ammonia in a one step reaction without the formation of a free amide
intermediate:
Scheme 12.1-1.
whereas nitrile hydratase (E. C. 4.2.1.84) catalyzes the hydrolysis of nitriles to the
corresponding amides:
0
Scheme 12.12.
Microorganisms which produce a nitrile hydratase also seem to synthesize one or

more amidase enzymes [linear amide hydrolase (E. C. 3.5.1)] thus enabling them to
hydrolyze nitriles to the corresponding acids in a two-step reaction:
0 0
R-cN + H20
nitrile
hydratase)
-,K,, lN
R am?
Hi0
+ NH3
Scheme 12.1-3.
Nitrilases and nitrile hydratases are distinct enzymes, apparently differing both with
respect to prosthetic groups and reaction mechanisms.
12.1.2.1.1 Nitrile Hydratases

In recent years nitrile hydratases have been studied intensively. Enzymes from a
wide range of microorganisms have been isolated and characterized and the
corresponding genes have been cloned and overexpressed. Nitrile hydratases can be
subdivided into two classes: enzymes requiring Fe3' (for example: from Rhodococcus
sp. 19-121, Pseudomonas chlororaphis B23 [13] and Cornamonas testosteroni NII [I4]) and
12.1 Hydrolysis of Nitriles
I 701
OH@
Figure 12.1-1. Two o f the mechanisms discussed for nitrile hydratase c a t a l y s i ~ [ ~ ~ - ~ ~ ] .

(I) Direct coordination o f t h e substrate t o the metal ion in the active site. (11) Attack o f
a water molecule activated by a metal-bound hydroxide ion.
enzymes requiring Co3+(for example: Rhodococcus rhodocrous J 1[15,1G1 and Pseudomo-

nasputida NRRL 18GG8[171)for catalysis. The metal clusters of the former group have
been studied intensely by EPR, ENDOR, EXAFS and other spectrophotometric
techniques, revealing a unique coordination of a non-heme iron cluster [18-241.
Nitrile hydratases show a high degree of homology. They consist of from two up to
twenty subunits L2] containing one metal ion per a,p-dimer with the only exception
described to date being the nitrile hydratase from P.chlororaphis which appears to be
a hom~tetramer[~~l.
The first X-ray structure of a nitrile hydratase was determined for the enzyme from
Rhodococcus sp. R312[25].In this study an a&-tetramer conformation has been
found in the native enzyme. Characterization of the highly related Rhodococcus sp. N-
771 nitrile hydratase, however, revealed a dimeric species in solution for this
enzyme[l21.
Based on the X-ray structure mechanisms have been proposed which are depicted
in Fig. 12.1-1 [25-271. Earlier studies suggested the involvement of the cofactor
pyrroloquinoline quinone (PQQ) X-ray studies and spectral characteriza-
tion [24, 251 have, however, recently discarded this.
12.1.2.1.2 Nitrilases
Nitrilases have been studied less than the nitrile hydratases. The enzymes appear as
homomultimers, exhibiting a wide range of molecular masses. The reaction mecha-
nism depicted in Fig. 12.1-2 has been proposed recently by Kobayashi et al.[281.
Several nitrilases have been found to be inhibited by reagents which bind to thiol
groups, indicating that sulfhydryl groups are essential for the catalytic activity of
72 Hydrolysis and Formation of C-N Bonds
702
I
R-CGN
Enz-SH
0
4
R-C\
OH
Figure 12.1-2. Reaction mechanism proposed for nitrilase catalysisIz8I.
these enzymes. This has been confirmed by the work of Kobayashi et al. who cloned
the nitrilase gene from Rhodococcus rhodocrous J 1 and have proved that Cys 165 is
crucial for nitrilase activity. The group also found that the nitrilase does catalyze the
formation and hydrolysis of amides, although with lower activity[28].
All the nitrile hydrolyzing enzymes described so far are intracellular and differ
considerably with respect to substrate specificity, stereoselectivity,molecular mass
and substrate and product inhibition characteristics.
12.1.2.2
Enzymatic Hydrolysis of Cyanide
Microorganisms appear to have evolved separate metabolic pathways for the hydroly-
sis of inorganic cyanide. Thus most nitrilases and nitrile hydratases investigated so
far do not display activity towards cyanide. However recently, as a first exception to
this, a nitrilase has been reported that hydrolyses potassium cyanide as well as
organic nitriles 12’* 301.
Enzymes catalyzing the hydrolysis of cyanide had already been identified earlier.
Here too, two different types can be distinguished. The enzyme cyanide hydratase
[formamide hydrolase (E. C. 4.2.1.66)] can be found in various fungi[31].It catalyzes
the hydration of cyanide to formamide:
0
Scheme 12.1-4.
A direct hydrolytic activity on cyanide, yielding formate and ammonia without

forming formamide as an intermediate, has been identified in different bacte-
fia[32,331.
HCN +
Scheme 12.1-5.
2H20 -,AoH
cyanidase
0
+ NH3
72.1 Hydrolysis ofNitriles
This new type of enzyme, tentatively named ‘cyanidase’,has also recently been
I 703
cloned, overexpressed and characterizedP4*351. Cyanidase tolerates high cyanide

concentrations and is able to deplete cyanide in aqueous solutions to less than 0.01
PPm.
Cyanide hydrolyzing enzymes (i.e. reaction schemes 12.1-4and 12.1-5) have, with
the exception mentioned above, not been reported to hydrolyze organic nitriles and
thus appear to be highly specific for inorganic cyanide. Therefore, they are mainly of
interest in waste water treatment as a biological alternative to conventional chemical
detoxification of cyanide by alkaline chlorination and will not be treated further.
Comprehensive reviews on biological cyanide transformations may be found in
the literature [36, 371. Reviews on microbial cyanide metabolism have been published
by Knowles [38, 391 and Knowles and Bunch[40].
12.1.3
Examples of Enzymatic Nitrile Hydrolysis
12.1.3.1
Enantioselective Hydrolysis of Nitriles
Several groups of investigators have reported on the production of optically active

acids from racemic mixtures of nitriles. A comprehensive overview has recently been
presented I4l].
Fukuda and coworkers have described one of the first applications of the enantio-
selective hydrolysis of nitriles (Scheme 12.14). Using a whole cell biocatalyst
optically pure a-hydroxy acids (1-a-hydroxyisovalericacid and L-a-hydroxyisocaproic
acid) have been prepared from the racemates of the corresponding a-hydroxyni-
triles [421.
Scheme 12.1-6.
In recent years, the enantioselectivehydrolysis of nitriles has been studied in more

detail. Whereas in the past only whole cell catalysts had been investigated, it is now
possible to assign the activities to specific enzymes occurring in the cell. These
enzymes are nitrilases, nitrile hydratases and/or amidases.
12.1.3.1.1 Enantioselective Nitrilases

First indications of stereoselective nitrilases have been given by Macadam and
Knowles describing the production of L-alanine from racemic a-aminopropionitrile
by a stereoselective nitrilase produced by an Acinetobacter sp. [43j.
Bhalla et al. [441 reported the stereoselective hydrolysis of a-aminonitriles by a
704
nitrilase from Rhodococcus rhodochrous PA-34. The nitrilase exhibited relaxed sub-
strate specificity hydrolyzing both mono- and dinitriles and its stereospecificity
differs from that of the Acinetobacter sp. since it produced D-alanine from D,L-c~-
aminopropionitrile.
Enzymatic production of (S)-(+)-ibuprofen,an anti-inflammatory drug, from
racemic 2-(4'-isobutylpheny1)propionitrileusing various bacterial strains has been
published by Yamamoto et al. (Scheme 12.1-7)[45. 461. One of the strains, Acineto-
bacter sp. AK226 produced (S)-(+)-ibuprofenby means of a nitrilase in whole cell
experiments. High optical purity of the product (about 95 % ee) was obtained at a low
percentage of hydrolysis but the optical purity of the product decreased at the later
stages of the reaction. This was in accordance with other results which showed that
(R)-(-)-Ibu-CNwas indeed also hydrolyzed by the nitrilase, although at a 108-fold
lower rate than the (S)-nitrile. Also Rhodococcus butanica ATCC 21 197 has been
shown to produce a nitrilase suitable for the enantioselective hydrolysis of racemic
2-aryl-propionitriles;however, selectivitieswere rather low [471.
Scheme 12.1-7.
An enantioselectivenitrilase has also been shown to be applicable in the dynamic

kinetic resolution of mandelonitrile. Using the nitrilase produced by Alcaligenes
faecalis ATCC 8750 Yamamoto et al. showed that they could derive (R)-(-)-mandelic
acid from mandelonitrile in 91 % yield with an ee of 100%. Under the reaction
conditions used non-reacting (S)-mandelonitrile undergoes spontaneous racemiza-
tion leading to the high yield (see Scheme 12.1-8)L4'I. Currently (R)-mandelic acid
and (R)-chloromandelicacid are produced using nitrilases on an industrial scale by
the Mitsubishi Rayon Corp.
Scheme 12.1-8.
12.1.3.1.2 Nitrile Hydratases

Enantioselective hydrolysis of nitriles by the nitrile hydratase/amidase system has
often been attributed to the combination of a non-selective nitrile hydratase and a
selective amidase. However, more recently several enantioselectivenitrile hydratases
have also been identified and studied in detail. For example, a nitrile hydratase from
Pseudomonas putida NRRL-18668 has been found to hydrate 2-(4-chlorophenyl)-

I
705
3-methylbutyronitrilewith high enantioselectivity (Scheme 12.1-9).The whole cell

preparation was found to transform the (S)-enantiomer preferentially until approx-
imately 90 % has been consumed. Successively the (R)-enantiomer also reacted but
at a 6-fold lower ratel4’]. Using a purified enzyme preparation on the enantiomer-
ically pure substrates the reaction rate towards the (S)-enantiomer has been
determined to be more than 50 times higher than for the (R)-enantiomer[”l.
Recently the enzyme has also been cloned and overexpessed in E . ~ o l i [ ~and
~ l Pichia
pa~toris[~~].The whole cells from Pseudomonas putida NRRL-18668 have also been
investigated in the hydrolysis of a-substituted arylpropionitiriles. A stereoselective
hydrolysis was found. However, in these cases the enantioselectivity must be
attributed to the combined reactions of the hydratase and the amida~e[~’]]. This is
also the case for several other whole cell catalysts containing the hydrataselamidase
system. Hence the enantioselectivehydrolysis of a-substituted phenylacetonitrilesby
Rhodococcus sp. AJ 270[52]and by Rhodococcus sp. (SP361)[531 is based on the highly
(S)-selective amidases. However, evidence for enantioselective hydratases has also
been provided by studying the transformations using a whole cell catalyst in the
presence of amidase inhibitors. Thus, the hydratase of Rhodococcus equi exhibits a
preference for (S)-nitriles whilst the hydratase from Agrobacterium tumefaciens is
selective towards the (R)-nitriles[54, 551.
Scheme 12.1-9.
12.1.3.2
Monohydrolysis o f Dinitriles
Several investigators using enzymes of different microbial origin have reported on

the monohydrolysis of dinitriles to the corresponding cyano-carboxylic acids in high
yields. Selective hydrolysis of one cyano group of a dinitrile is very difficult to carry
out by chemical hydrolysis and is, therefore, an intriguing aspect of nitrile bio-
conversion.
Using whole cells of Rhodococcus rodochrous NCIB 11216 Bergis-Garber and
Gutman demonstrated complete monohydrolysis of fumaronitrile and almost com-
plete monohydrolysis of succinonitrile, 1,3-dicyanobenzeneand 1,4-dicyanobenzene
(Scheme 12.1-10,12.1-11) rS6, ’1.
Analysis of the reaction products showed that during conversion of succinonitrile
into 3-cyanopropionic acid, succinamic acid (H~NOC-CH~CHZ-COOH) was de-
tected as a free intermediate in the reaction mixture, suggesting that enzymatic
activities other than nitrilases (i.e. nitrile hydratase) were present in the cell
I
-
706 72 Hydrolysis and Formation ofC-N Bonds
Nc*CN
0
Scheme 12.1-10.
CN CN
Scheme 12.1-11.
preparation. In parallel experiments with the same bacterial strain glutaro-, adipo-
and pimelodinitriles were hydrolyzed further to glutaric, adipic and pimelic acid,
respectively rS71. Kobayashi et al. ['*I on the other hand obtained complete conversion
of glutarodinitrile into cyanobutyric acid without any formation of glutaric acid using
a nitrilase from another strain of Rhodococcus (Rodococcus rodochrous K22). Turner et
al. [591 have studied the hydrolysis of aromatic dinitriles using an immobilized
preparation of Rhodococcus sp. (Novo SP 361). Only after esterification of the
carboxylic acid formed was the second nitrile group hydrolyzed by repetitive use of
the catalyst (Scheme 12.1-12).
g g
0 OH
CN sp361_ CN CN OH
0
Scheme 12.1-12.
The chemoselecivity of nitrile hydrolyzing enzymes has also been used by Tani
and coworkers to examine the formation of trans-4-cyanocyclohexane-1-carboxylic
acid (t-MCC) from trans-1,4-dicyanocyclohexane (t-DCC) using a resting cell system
of Corynebacteriurn sp. C5 (Scheme 12.1-13)[601.The reaction was shown to be
12.7 Hydrolysis ofNitriles I 707
catalyzed by the sequential action of a nitrile hydratase and an amidase, both of

which were purified and characterizedIG1]. The nitrile hydratase exclusively hydro-
lyzed truns-l,4-dicyanocyclohexane (t-DCC) into trans-4-cyanocyclohexane-1-carboxy-
amide (t-MCMA), which could be detected in the reaction mixture[G1].The nitrile
hydratase did not attack the nitrile groups in t-MCC and t-MCMA. This conversion
has also been described using the nitrile hydratase from Rhodococcus rhodochrous
AJ270 giving a 99% yield[62s63].
nitrile H&rz amidase H a H

hydratase)
CN CN CN
Scheme 12.1-13.
In addition to the outstanding chemoselectivity,high regioselectivity can also be

found in the hydrolysis of dinitriles and has been used for example in the chemo-
enzymatic production of lactams from aliphatic a,w-dinitriles. Using a nitrilase from
Acidovoraxfacilis or a nitrile hydrataselamidase system from Comamonus testosteroni
high yields of the lactams have been achieved (see Scheme 12.1-14)cG4].
Scheme 12.1-14.
The chemo- and enantioselectivity of nitrile-hydrolyzing enzymes gives rise to

products of high optical purity with a theoretical yield of 100%, when starting from
prochiral substrates. Thus several whole cell catalysts have been studied in the
conversion of 3-substituted glutaronitriles. The enantioselectivity was found to be
highly dependent on the substituent at the 3-position (Scheme 12.1-15, Scheme
12.1-16) 651. Using this technology (R)4-hydroxy-S-cyanopentene,a precursor for
the protected lactone moiety of the 6-lactone pharmacophore of the mevinic acids,
becomes available in high yields [GG].
Recently, an industrial application of the chemoselective hydration of a dinitrile,
adiponitrile, has been introduced. A Pseudomonas chlororuphis B23 nitrile hydratase
N C A C N -SP361
NC-COOH
9
R= OBn e.e. = 83% 73% yield

OBz e.e. = 84% 25% yield
OH e.e. = 22% 52% yield
OMEM' e.e. = 61% 19% yield
OAc e.e. = 0 45%yield
*MEM = methoxyethoxymethyl
Scheme 12.1-15.
708
I 72 Hydrolysis and formation ofC-N Bonds
R= Bn e.e. = 29% 59% yield

OBn e.e. = 90% 68% yield
OBz e.e. = > 99% 71% yield
Scheme 12.1-16.
immobilized in alginate beads is used for the production of 5-cyanovaleramide.The

biocatalyst is extremely stable and has been used in almost GO consecutive batches
producing more than 13 metric tons in the production of the precursor of a new
herbicide
12.1.3.3
Substrate and Product inhibition of Nitrile Hydrolysis
Substrate and/or product inhibition may seriously reduce the productivity of nitrile-
hydrolyzing enzymes. Already nitrile concentrations higher than 200-500 mM have
been reported to be inhibitory, often causing rapid and irreversibleinactivation of the
biocatalyst[Gg-741. Substrate inhibition may be overcome by running the enzymatic
reaction constantly at a low substrate concentration using periodic or continuous
feeding of the substrate. Product inhibition/inactivation, on the other hand, is
considerably more difficult to tackle in a large scale industrial process and may
prevent implementation of enzymatic hydrolysis for a particular reaction. Thus it
appears that the success of the commercial acrylamide process of the Mitsubishi
Rayon Corp. (the former Nitto Corp.) is the result of extensive and elegant efforts
within the areas of process optimization and the development of improved bio-
catalysts which are less susceptible to product inhibition. Currently the acrylamide
production is run optimally using a highly efficient nitrile hydratase catalyst at low
temperature (5-10 "C) thereby avoiding substrate inhibition which occurs at higher
751. For details see Sect. 12.1.3.5.
The same whole cell catalyst can be used in the hydration of 3-cyanopyridine to
nicotinamide (Scheme 12.1-17).This vitamin, broadly applied in animal feeding, is
currently produced biocatalybcally on an industrial scale (> 3000 t/a) by the Lonza
AG. For this substrate Yamada and Kobayashi showed that the whole cell catalyst of
Rhodococcus rhodocrous J 1, containing a nitrile hydratase induced with crotonamide,
can even tolerate substrate concentrations up to 12 M [ ~(see
] Fig. 12.1-3).
Mauger et al. also succeeded in achieving high final product concentrations of
various amides when using the Rhodococcus rhodochrous J1catalyst (see Table 12.1-1).
Scheme 12.1-17.
I
I I
709
Figure 12.1-3. Conver-
sion o f 3-cyanopyridine
after 5(*), 9 (B) and 22 h
-
g 90 (A)of incubation at vari-
C ous substrate concentra-
.O
(?
80 tions.
70 1
0
60
7 9 11 13 15
substrate concentration [MI
Table 12.1-1. Nitriles hydrolyzed by Rhodococcus rhodocrous J l 1’. 761.

Substrate Amide Product concentration
(g L-7
3-Cyanopyridine nicotinamide 1465
4-Cyanopyridine isonicotinamde 1099
2,G-Difluorobenzonitrile 2,G-difluorobezmide 30G
2-Cyanopyrazine pyrazinamide 985
2-Cyanopyridine picolinamide 977
2-Cyanothiophen 2-thiophencarboxamide 210
3-Indolylacetonitrile indole-3-acetamide 1045
Benzonitrile benzamide 489
2-Cyanofuran furanecarboxamide 522
The hydrations were carried out either at low substrate concentrations with slow
feeding of the substrate (for example: benzonitrile, 2,G-difluorobenzonitrile and
3-indoleacetonitrile)or, in the case of less toxic substrates, by direct incubation at
high substrate concentrations (for example: 3-indolylacetonitrile and 2-cyanopyr-
azine[2. 7611.
In addition, high substrate levels have been used in the industrial production of
5-cyanovaleramide(see Sect. 12.1.3.2)using the nitrile hydratase from Pseudomonas
chlororaphis B23. Starting at a substrate concentration of 1.5 M, high above the
solubility level (0.45 M). The hydration was carried out in a two phase system. The
nitrile hydratase showed outstanding stability at these high substrate concentrations.
Sequential addition of the substrate, instead of starting at a high concentration, only
slightly improved the stability. Increased stability could be achieved by the addition
of butyric acid to the medium. However, the higher stability has to be traded off with
a lower activity caused by the inhibition of the nitrile hydratase by butyrate (see Sect.
12.1.3.4).
710
12.1.3.4
Activation and Stabilization of Nitrile Hydratases
Iron-dependent nitrile hydratases, for example from Rhodococcus R3 12 or Pseudomo-

nus chlororuphis, exhibit a remarkable dependency on light.
The enzymes, after being inactivated by aerobic incubation in the dark, regain
their activity when exposed to light irradiation[12,771. Using different spectrophoto-
metric techniques (ENDOR, EXAFS, FTIR, UV-VIS and X-ray) this phenomenon has
been studied extensively in recent years. It has now been confirmed that the
deactivation is caused by the reversible binding of nitric oxide to the non-heme iron
center in a 1 :1 stoichiometric complex. Upon irradiation the complex is destroyed
and the activity of the nitrile hydratase is restored[‘4, 27, 78-811.
Another interesting characteristic of the iron-dependent nitrile hydratases is their
stabilization during purification and storage by alcanoic acids such as butyric acid,
hexanoic acid and valeric acid. The effect has already been described by Nagasawa et
al. in 1987[13].However, only in recent years has the role of the acids been clarified by
spectroscopic studies. Studying the EPR signals of the nitrile hydratase from
Brevibucterium R312, Kopf et al. showed that butyric acid interacts with the iron in
the active site of the nitrile hydratase, stabilizing the enzyme but, at the same time
acting as a competitive inhibitor[82].
12.1.3.5
Nitrile Hydrolysis in Organic Solvents
Most nitrile bioconversions published have been conducted in aqueous media and
consequently few data are available on the effect of solvents on enzymatic nitrile
hydrolysis. Such studies seem highly justified in order to investigate the effects of
different solvents or co-solvents on substrate specificity, conversion rate, ster-
eoselectivity, and catalyst half-life.
‘w Rhodococcussp.
NCIMB 12218
.
Scheme 12.1-18.
De Raadt et al. reported on the inhibition of nitrile hydrolysis by various

solvents [831.
However, production of 2,G-difluorobenzamide(Scheme 12.1-18)was effected in
99.5 % n-heptane using the nitrile hydratase from Rhodococcus sp. NCIMB 12 218[84].
The enzymatic reaction was found to be activated by light (see 12.1.3.4). More
recently, Layh and Willetts have studied nitrile transformations in various organic
solvents and biphasic mixtures using a nitrilase from Pseudowonus sp. DSM 11 387
and a nitrile hydratase from Rhodococcus sp. DSM 11 397[”]. The enzymes exhibited
good stabilities in biphasic mixtures with hydrophobic solvents when dispersed in
12.1 Hydrolysis ofhlitriles
the buffer-saturated higher alcohols 1-hexanol,1-heptanol, 1-octanoland 1-decanol,

I 711
respectively. The nitrilase still retained 58 %, 49 %, 44% and 47 % activity, while the
nitrile hydratase only showed low activities (2-5 %).
12.1.3.6
Large Scale Production o f Acrylamide
Acrylamide monomer is an important chemical commodity produced on a multi-

hundred thousand ton scale for the production of polymers and copolymers. The
preferred manufacturing process is by the catalytic hydration of acrylonitrile at
70-120 "C using reduced Raney copper as the catalyst; the initial concentration of
acrylonitrile being around 4 M. There are several shortcomings to this process,
among which are the high level of acrylic acid formed and byproduct forma-
tionI2. '1.
An enzymatic acrylonitrile hydration was first patented in 1981["I. Many nitrile
hydratases of different origin have been shown to be able to convert acrylonitrile into
acrylamide. However, a major problem associated with biocatalysis for production of
acrylonitrile is the short half-life of the enzyme due to substrate and product
inhibition. Acrylonitrile is a strong alkylating agent which reacts by Michael addition
with the sulfhydryl groups of proteins[", 691.
The Mitsubishi Rayon Corp. (the former Nitto Chemical Industry Co.) established
the industrial production of acrylamide in 1985 using immobilized cells of Rhodo-
coccus sp. N-774L3.". 741. In 1988 a hyperproducing mutant strain of Pseudornonas
chlororaphis B23 was chosen for production. As in Rhodococcus sp. N-774, the active
Table 12.1-2. Operating conditions for acrylamide production.

Reaction conditions Productivity
pH 7.5-8.5 Conversion acrylonitrile > 99.9%

Temperature 0-5 "C Yield of acrylamide > 99.9%
Acrylonitrile concentration Acrylamide concentration 27-30%
in the reactor 1.5-2.0% from the reactor
Table 12.1-3. Comparison of enzyme data ofthree types of nitrile hydratases.

Parameter Rhodococcus sp. Pseudomonas Rhodoccus
N-774 chlororaphis 823 rhodochrous J l
Tolerance to acrylamide (%) 27 40 50

Acrylic acid formation very little barely detected barely detected
Cultivation time (h) 48 45 72
Activity of culture broth (units mL-I) 900 1400 2100
Specific activity (units per mg cells) 60 85 76
Cell yield (g L-') 15 17 28
Acrylamide productivity (g per g cells) 500 850 > 7000
Total amount of production (t per year) 4000 6000 30.000
Final concentration of acrylamide ("h) 20 27 40
First year of production scale 1985 1988 1991
712 I 12 Hydrolysis and Formation of C-N Bonds
Table 12.1-4. Comaprison of enzyme data ofthree types of nitrile hydratases.

Parameter Rhodoccus sp. Pseudomonas Rhodoccus
N-774 chlororaphis 823 rhodochrous I1
Molecular mass 70.000 100.000 505.000
Subunit molecular mass a 27.000 a 25.000 a 26.000
p 27.500 p 25.000 p 25.000
Metal Fe"' Fe"' co
Optimum temperature ("C) 35 20 35-40
Heat stability ("C) 30 20 50
Optimum pH 7.7 7.5 6.5
pH stability 7.0-8.5 6.0-7.5 6.0-8.5
Substrate specificity aliphatic nitriles aliphatic nitriles aliphatic and
aromatic nitriles
Activation by light irradiation + - -
Formation type constitutive inducible inducible
(methacrylamide) (urea)
Biocatalyticalprocess
immobilization
of microorganism
1
Acrylonitrile
iLI
Water
Spent catalyst
Cu-catalyticprocess
Acrylonitrile
Water
Figure 12.1-4. Comparison of the biocatalytic and the conventional chemical process
for acrylamide production.
biocatalyst in Pseudomonas chlororaphis B23 is also a nitrile hydratase containing

ferric ion as the cofactor[2s741.
Current acrylamide production at Mitsubishi using bioconversion is around
40 000 tonnes per year. Using a highly improved cobalt-containingnitrile hydratase
from Rhodococcus rhodochrous 71, final product concentrations of around 700 g L-'
References 1 713
can be obtained[87].The reaction is performed at 5-10 “C in order to reduce cell

degradation and enzyme inhibition. No data have been published on the half-life of
the Rhodococcus rhodochrous J1 nitrile hydratase under production conditions. A
good summary of the biocatalyhc production of acrylamide has been given by
Yamada and c o - ~ o r k e r s [871~ ~ , Tables 12.1-2 to 12.1-4).
(see
In Fig. 12.1-4 a comparison is presented of the enzymatic and the conventional
chemical processes for acrylamide production.
12.1.4
Availability and Industrial Future o f Nitrile Hydrolyzing Biocatalysts
Although nitrile-hydrolyzingenzymes have attracted considerable interest as prom-

ising “green catalysts”,none of these enzymes are presently available as commercial
products. Thus studies on nitrile biotransformations have been conducted with a
variety of enzyme preparations ranging from resting cells, immobilized whole cells,
cell-free extracts, immobilized enzymes and pure soluble enzymes. However, nowa-
days several nitrile hydratases and nitrilases have been cloned and overexpressed,
giving rise to highly efficient and well defined catalysts 71. This not only provides
12
‘
commercial access to even more interesting catalysts, but also opens the way for the
application of modern molecular biological methods for further optimization.
Within the recent years several industrial processes based on nitrile hydrolyzing
enzymes have been introduced, as has been discussed above. This number is now
expected to increase rapidly, due to the better availability of these biocatalysts.
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12.2
Formation and Hydrolysis of Arnides
Birgit Schulze and Erik de Vroom
12.2.1
Introduction
Organic amides and acids are versatile precursors to the production of various
commercial products; nowadays these compounds are mainly produced chemically.
However, owing to environmental considerations and the increasing demand for
chiral amides and acids there is a strong tendency to explore and develop biocatalytic
production processes.
This section gives a global overview of the potential of microorganisms and
enzymes to catalyze the regio- and enantioselective hydrolysis and formation of
amides.
The biocatalytic hydrolysis of amides and also the enzyme catalyzed formation of
amides and the synthesis of (semisynthetic)antibiotics is included in this section.
12.2.2
Enzymatic Formation of Arnides
Currently two biocatalytic methods are known for the production of amides:
The hydrolysis of nitriles using nitrile hydratases (for example: acrylamide pro-
duction).
The enzymatic ammoniolysis of carboxylic esters and amidation of carboxylic
acids.
In the previous Section 12.1 the formation of amides from the corresponding nitriles
is well addressed. The latter method, the enzyme catalyzed reaction of carboxylic
esters or acids with ammonia or amines yielding amides, has only recently been
studied in depth. Encouraging results have been described, especially in the field of
amidation of esters, a technology now being used by BASF to produce optically pure
amines (vide infa).
Lipases and esterases comprise a versatile group of enzymes that catalyze the
hydrolysis of esters, esterifications and transesterifications via an acyl enzyme
intermediate (Chapter 11). Various other nucleophiles can attack this acyl enzyme
complex in addition to water.
In recent years several authors have also shown that NH3 and amines can act as
nucleophiles leading to the formation of amides 1' .
Initially De Zoete et al. showed that fatty acid esters could be converted into the
corresponding amides by bubbling gaseous NH3 through the reaction mixture
containing lipase B from Candida antarctica (SP 435 from Novo-Nordisk) in tee-butyl
alcohol. As shown in Scheme 12.2-1 very good yields can be achieved. This enzyme
has also been used in related studies by other authors. A conclusive summary can be
found in L31.
72.2 Formation and Hydrolysis of Amides
I 717
Conversion 100%
95% Octanarnide
5% Octanoicacid
L O E t SP435
Conversion 100%
95% Hexanarnide
5% Hexanoic acid
Scheme 12.2-1.
Further to this kinetic approach, the thermodynamic ammoniolysis has also been
studied. Here the amide is formed directly by the reaction of a carboxylic acid with
ammonia. Because these reactions are governed by the equilibrium concentrations
of substrates and products, the solubility of the reactants is of major importance. The
optimal conditions for an efficient ammoniolysis of butyric acid and oleic acid have
now been reported. Oleamide, which has been reported to have a pharmacological
application as a sleep inducing has been derived from the acid by direct
ammoniolysis in an efficient manner with good yields (Scheme 12.2-2) 1’1.
94% yield
Scheme 12.2-2.
An interesting effect was found whilst studying the enantioselectivity of these

reactions. Although several esters (ethyl-2-chloropropionate, ethyl lactate, ethyl-
2-hydroxy hexanoate and ethyl-2-methylbutyrate) were converted into the amides
with only low to moderate ee values, the ammoniolysis of ibuprofen (2’-chloro-
ethy1)ester was highly enantioselective.At 56% conversion the ee of the remaining
(S)-esterwas 96% (Scheme 12.2-3),corresponding with an Evalue ofthe ammoniol-
ysis of 28.
In comparison, the ester hydrolysis of ibuprofen (2’-chloroethy1)ester
catalyzed by
the same enzyme proceeded with an E value of only 3.5 [*I. The same phenomenon
has been observed in the ammoniolysis of 4-methyloctanoic acid. Here an E value of
76 was determined for ammoniolysis, whereas in the transesterification reaction an
E-value of only 23 was found[’, 1‘.
718
I 72 Hydrorysis and Formation ofC-N Bonds
(R) (-)-amide (S)(+)-ester
56% conversion e.e. = 96%
Scheme 12.2-3.
It has now been reported that the amidation of esters will be used by BASF in the
industrial production of amines. A broad range of amines become available in their
optically pure form by kinetic resolution using a lipase from Pseudomonas sp. DSM
8246 in the amidation of methoxyacetic acid ethyl ester (see Scheme 12.2-4) 171.
- -hydrolysidracemisation
- - - - - -
I
I
0
I _ _ _ _ _ _ I
racernisation
Scheme 12.24.
A comparitive study of a variety of lipases and lipase preparations in such

alkoxycarbonylation reactions of amines has been presented by Sinisterra and co-
workers [I'.
72.2 Formation and Hydrolysis of Amides I719
12.2.3
Enzymatic EnantioselectiveHydrolysis of Amides
12.2.3.1
Hydrolysis o f Carboxylic Amides
Although amidase activities have been known for quite some time, it is only in recent
years that the increasing demand for chiral drugs and herbicides has triggered their
exploitation as biocatalysts to a great extent. Many, but not all, amidases have been
identified in microorganisms also exhibiting nitrile hydratase activity. In some cases
where the enantioselectivetransformation of the nitrile to the acid can be observed,
the selectivity is based on the high selectivity of the amidases rather than on the
discrimination by the nitrile hydratase. Thus, using these enzyme combinations,
both the (R)-amides and the (S)-acids can be obtained by such a double enantio-
meric selection. The first catalytic step is carried out by a nitrile hydratase with a
slight preference for the (R)-enantiomer. At high conversions this will lead to a
mixture of (S)-nitrile and (R)- and (S)-amide, the latter being subsequently
hydrolyzed with high selectivity by an (S)-selectiveamidase to yield the ( S ) - a ~ i d [ ~ ~ , ~ ~ l
Scheme 12.2-5).
(S)-selective
amidase
Scheme 12.2-5.
Other examples of (S)-selectiveamidases are described for the production of (S)-

2-(4'-chlorophenyl)-3-methyl butyric acid 191, (S)-ibuprofen[lo],(S)-naproxen and
L-carnitine[I2'131.
In addition to the more common (S)-amidase activities, (R)-specificenzymes have
also been identified. Thus (S)-ketoprofenamidehas been derived from the racemic
mixture using a biocatalyst from Cornamonas acidovorans KPO 2771-4 to hydrolyze
the (R)-enantiomer with high selectivity['4](see Scheme 12.2-6).
Lonza AG has reported on the use of enantioselectiveamidases for the resolution
of piperazine-2-carboxamide and piperidine-2-carboxamide using whole cell bio-
catalysts from Klebsiella terrigena, PseudornonasJluorescence and Burkholderia sp., the
last containing an (R)-selective amidase (Scheme 12.2-7)[''I. Furthermore, several
amidases exhibiting high selectivities [either (S)- or (R)-] towards 2-arylpropiona-
720
C.
KPO-2771-4
acidovorans &c02H \
49% yield e.e. = 99%
Scheme 12.2-6.
(SJ-spec.
Q\ 4
H amidase c X
H C O O H 99.4% e.e. 0 Klebsieiia
41% yield terrigena DSM 9174
H
t H
(;I...
CoNH2
(R)-spec.
amidase
99.0% e.e. @Burkho\deria sp.
H "'"COOH 20% yield DSM 9925
(A COOH 97.3% e.e.

20% yield
Bpseudornonas
DSM 9924
Scheme 12.2-7.
mides have been identified. A good overview has been given by Wieser and
Nagasawa [791.
In recent years the availability of several amidases has been improved by cloning
and overexpression[16191 resulting in biocatalysts of high activities which can readily
be used for industrial purposes. Furthermore, homology studies have been carried
out to identify the common features of this class of enzymes[20].
12.2.3.2
Hydrolysis o f Amino Acid Amides
The conversion of amino acid amides into chiral amino acids has been the subject of
a large number of monographs and reviews [21-29]. In this section information will be
given on amidases and aminopeptidases that have been reported for the stereo-
selective hydrolysis of amino acid amides.
12.2 Formation and Hydrolysis ofAmides
12.2.3.2.1 Production of Chiral a-H-a-Amino Acids

At DSM a very efficient and universally applicable industrial process for the
production of both optically pure L- and D-amino acids has been commercial-
i ~ e d [ ~271.
’ , Pivotal in this process is the enantioselective hydrolysis of D,L-aminoacid
amides. The stable D,L-amino acid amides are prepared efficiently under mild
reaction conditions starting from simple raw materials (Fig. 12.2-1). The reaction of
an aldehyde with hydrogen cyanide in ammonia (Strecker reaction) affords the
amino nitrile. The amino nitrile is converted in a high yield into the D,L-amino acid
amide under alkaline conditions in the presence of catalytic amounts of acetone. The
HCNlNH3
NH2
R&N
ketoneloH
1) NH3
3) OH-(pH=13)
racemisation
4) H30+
*IPhCH0 r- R 4 N H 2
NH2
I
DL-amino acid amide
L-specific
a
aminopeptidase
1) OH-(pH=13)
racemisation
2) H30+
(Pseudornonas putida)
0
I
L-amino acid D-amino acid amide
PhCHO
pH=8-11
D-amino acid H
Figure 12.2-1. DSM’s chemo-enzymatic route for the production of chiral a-H-amino acids.
722
Table 12.2-1. Substrates by Pseudomonas putida cells.
R R R R
H3C-
H3C-CH2-
resolution step is accomplished with permeabilized whole cells of Pseudornonas

putida ATCC 12 633; a stereoselectivityof nearly 100% (E > 100) on hydrolyzing only
the r-amino acid amide is combined with a very relaxed substrate specificity (see
Table 12.2-1)[27, 2g-311.
Not only the smallest optically active amino acid, for example alanine, but also
valine, leucine, several (substituted) aromatic amino acids, heterosubstituted amino
acids (methionine, homomethionine and thienylglycine) and even an imino acid,
proline, are obtainable in both the L- and the D-form. Furthermore, this biocatalyst
has recently been reported to hydrolyze azido amino acid amides with high
enantioselectivitiesas well (vide in@) [321.
No enzymatic side effects are observed and substrate concentrations up to 20 % by
mass can be used without affecting the enzyme activity. The biocatalyst is active in a
broad pH-range and can be used in soluble form in a batchwise process; thus poorly
soluble amino acids can be resolved without technical difficulties.
Re-use of the biocatalyst is possible. A very simple and elegant alternative to the
use of ion-exchangecolumns or extraction to separate the mixture of D-amino acid
amide and the L-amino acid has been elaborated at DSM. Thus addition of one
equivalent of benzaldehyde (with respect to the D-amino acid amide) to the
enzymatic hydrolysate results surprisingly in the formation of a water insoluble
Schiff base with the D-amino acid amide which can be easily separated. Acid
hydrolysis (H2S04'HHal, HN03 etc.) results in the formation of the n-amino acid
amide, which can be hydrolyzed by cell-preparations of Rhodococcus erythropolis,
yielding the D-amino acid. The amidase from this organism lacks stereoselectivity.
This option is very useful for amino acids that are highly soluble in the neutralized
reaction mixtures obtained after acid hydrolysis of the amide.
Process economics dictate the recycling of the unwanted isomer. Path A in
Fig. 12.2-1 illustrates that racemization of the D-N-benzylideneamino acid amide is
facile and can be carried out under very mild reaction conditions. After removal of
72.2 Formation and Hydrolysis ofAmides I723
N3
+
-
amino peptidase
from Pputida N3
--
N3
NH2
spontaneous racemisation
Scheme 12.2-8.
the benzaldehyde the D,L-amino acid amide can be recycled. This option means that
100% conversion into the L-amino acid is theoretically possible. A suitable method
for racemization and recycling of the r-amino acid (path B, Fig. 12.2-1) comprises the
conversion of the L-amino acid into the ester in the presence of concentrated acid,
followed by addition of ammonia, resulting in the formation of the amide. Addition
of benzaldehyde and racemization by base (pH 13) gives the D,L-amino acid amide.
In this way 100% conversion into the D-amino acid is possible. For the production of
2-azidophenylaceticacid an even more elegant way of achieving 100% yield of one
enantiomer has been reported. Under the conditions used for the resolution a
spontaneous racemization of the substrate is achieved, resulting in a dynamic
kinetic resolution with a theoretical yield of 100% (Scheme 12.2-8).The distinct
advantages of the aminopeptidase process are:
The substrate for the enzymatic hydrolysis is a precursor of the amino acid; the
number of chemical steps can be kept to a minimum.
The use of relatively cheap whole cell biocatalysts contributes to the economical
feasibility of the procedure.
Both L- and D-amino acids can be prepared with a very high optical purity.
The aminopeptidase from Pseudomonas putida ATCC 12 633 has also recently been
cloned and overexpressed in E. coli resulting in a highly efficient whole-cell
biocatalyst for industrial applications[*'I. The specific activity of this new biocatalyst
is substantially increased (25 times) compared with the specific activity of the P.
putida wild type cells without changing the other positive characteristics of the
aminopeptidase. Even though the aminopeptidase from Pseudomonas putida exhibits
the relaxed substrate specificity described above, an a-hydrogen atom in the
substrate is an essential structural feature for the enzymatic activity. Therefore this
enzyme can not be used for the resolution of higher substituted amino acids.
Recently, a new biocatalyst with a broad substrate spectrum of L-specific amidase
activity has been identified at DSM. Of 125 microorganisms that were able to use a-
hydroxy acid amides as the sole nitrogen source, Ochrobactrum anthropi NCIMB
40 321 was selected for its ability to hydrolyze racemic amides with high L-selectivity.
The substrate specificity of whole Ochrobactrum anthropi cells is remarkably wide
and ranges from a-H-a-amino,a-alkyl-a-amino,and N-hydroxy-a-aminoacid amides
to a-hydroxyacid amides[331. After 50% conversion, both the L-acids formed and the
724 I 12 Hydrolysis and Formation ofC-N Bonds
Table 12.2-2.Substrate specificty of Ochrobactrum anthropi cells. L-selective hydrolysis" of amides

by Ochrobactrum anthropi.
Substrate Relative activity ("h)
o,r-a-valine amide 25
D,L-a-methylamide 5
D,L-a-methylleucineamide 15
D,r-tert leucine amide 1
D,L-a-cinnamylalanineamide 17
D,L-phenylglycineamide lOOb
D,r-a-methylphenylglycineamide 2
D,L-a-ethylphenylglycineamide 4
D,L-a-propylphenylglycine amide 1
D,L-a-allylphenylglycineamide 4
D,L-a-benzylphenylglycine amide 0
D,L-N-hydroxyphenylglycineamide' 25
D,L-mandek acid amide (MAA) 5
a Activities were measured at pH 8.0 (100mM phosphate buffer) and 40 "C using 3.0 g L-' of amide.
b A relative activity of 100 corresponds to 2000 nMol min-' (mg dry mass)-'. c Incubation performed under
anaerobic conditions by flushing with nitrogen.
residual D-amides were present in 99% enantiomeric excess and ammonia

accumulated in stoichiometric amounts. The substrate specificity is illustrated in
Table 12.2-2. Using this biocatalyst a new route to thiamphenicol, a synthetic analog
to the antibiotic chloramphenicol has been developed (Scheme 12.2-9).A precursor
of the biologically active (I R,2R)-enantiomer,the (2S,3R)-para-substituted3-phenyl-
serine is obtained by the enzymatic resolution. The residual enantiomer can be
efficiently recycled via separation by Schiff base formation with the corresponding
para-substituted benzaldehyde and subsequent transformation into the racemic
threo-amides[341.
A D-aminopeptidasehas been identified at the Sagami Research Institute by Asano
et al. 13'1. The group was successful, by using an enrichment culture technique, in
selecting a microorganism (Ochrobactriurn anthropi SCRC C1-38)with D-aminopepti-
Scheme 12.2-9.
dase activity from a soil sample. The enzyme, which hydrolyzes D-alanine amide,
I 725
was purified about 2800 fold. The molecular mass of the native enzyme was
approximately 122000 Da, with two identical subunits having a molecular mass of
about 59000 Da each. Remarkably, D-valine amide is hydrolyzed very slowly.
Generally, the enzyme has higher affinity towards peptide substrates than towards
amino acid amides. It does not act on peptides bearing an L-amino acid at the NH2-
terminus. Thus it exhibits a mode of action typical of aminopeptidases. The optimal
pH for activity was 8.0. The immobilized enzyme was also active in organic solvents
(benzene, butyl acetate, l,l,l-trichloroethane)[361. ~-Alanine-(3-aminopentyl) amide
was quantitatively synthesized in an amination reaction from D-alaninemethylester
and 3-aminopentane within 1 h.
Asano et al. have also purified a D-stereospecificamino acid amidase from another
Ochrobactrum anthropi isolate[37.381. Recently, a new amidase from Comamonas
acidovorans has been reported that exhibits a broad substrate specificity and also D-
amino acid amidase In addition, a D-specific amidase has been identified
in Arthrobacter sp. NJ-2G[401. In contrast to the D-selective enzymes of Ochrobactrum
sp. and Cornomonas acidovorans, the D-amide hydrolase identified in Arthrobacter sp.
NJ-26 was very substrate specific: a good hydrolysis rate was only observed for D-
alanine amide.
12.2.3.2.2 Synthesis of a-Alkyl-a-Amino Acids

Within the pharmaceutical industry a-alkyl-a-aminoacids are regarded as valuable
building blocks. An example of this class is ~-a-methyl-3,4-dihydroxyphenylalanine
(L-methyl-Dopa),which is used as a drug to treat patients suffering from high blood
pressure. More recently, medicinal chemists became interested in bio-active peptides
containing a-alkyl-a-amino acids since they tend to freeze specific conformations
and slow down enzymatic degradations Nowadays, many a-alkyl-a-
amino acids have been found in nature. For example, L-isovaline is found in
peptaibol antibiotics. Their influence on the conformational behavior of peptides is
presently under active investigation. Several routes to enantiomerically pure a-alkyl-
a-amino acids have been elaborated in recent 42, 431. At DSM a Mycobacte-
rium neoaurum biocatalyst has been obtained in a screening, which hydrolyzes a
broad range of a-alkyl-a-amino acid amides with high enantioselectivities (Table
12.2-3).
The basis of the process leading to the enantiomericallypure acid is essentially the
same as that for a-H-a-aminoacids. However, in this case, a ketone is used as the
starting material which undergoes a Strecker reaction, followed by hydrolysis of the
resulting aminonitrile to form the racemic a-alkyl-a-aminoacid amide. Enzymatic
hydrolysis results in the formation of the L-a-alkyl-a-aminoacid (Fig. 12.2-2).
Some characteristics of the process are:
Using this process both L- and D-a-alkyl-a-aminoacids can be produced.
Permeabilized whole cells of Mycobacterium neoaurum ATCC 25795 or crude
enzyme preparations can be used.
726
I 12 Hydrolysis and Formation of C-N Bonds
Table 12.2-3. Substrate specificity of the amidase activity of Mycobacterium neoaurum cells.
Products formed
c recycle
Figure 12.2-2.
+OH
R
NH2
L-a-alkyl-amino acid
I
D,L-a-alkyl-amino acid amide
L-amidase
(Mycobacferium neoaurum)
E+NH2fiH2
D-a-alkyl-amino acid amide
D-a-alkyl-amino acid
DSM's chemo-enzymatic route for the preparation of a-alkyl-a-amino acids.
Very high stereoselectivity (> 98% ee) and a remarkably relaxed substrate specifici-
ty are observed (table 12-7).
The enzyme is active in the pH range from 6.5 to 11,with a broad optimum from
pH 8.0-9.5
Recently the enzyme has been purified and thoroughly characterized. It was
identified as an amino acid amidase, most probably belonging to the group of
metallocystein hydrolases [291. In addition to DSM, Ube company reported an

I 727
analogous biocatalytic route to a-methyl phenylalanine. A Pseudomonas Juorescens

( I F 0 3081) showed the highest conversion (94%) but the stereoselectivity was
relatively low (ee 93.4%)[441.
12.2.3.3
Hydrolysis of Cyclic Amides
Cyclic amides can also be hydrolyzed in a highly selective fashion using enzymes. A
well known example in this respect is the biocatalytic production of L-lysine from D,L-
a-amino-s-caprolactam(D,L-ACL)[45-471. This process is based on the combination of
two enzymatic reactions: the enzymatic enantiospecific hydrolysis of L-a-amino-s-
caprolactam to 1-lysine and the simultaneous racemization of the residual D-a-
amino-s-caprolactam (Scheme 12.2-10).
D-ACL L-ACL
hydrolase
L-Lysine
Scheme 12.2-10.
In this way L-lysine is produced from D,L-a-amino-s-caprolactam,with a yield of

almost loo%, by incubating the racemate with microbial cells of Cryptococcus
laurentti, which possess L-a-amino-s-caprolactamase activity, together with cells of
Achromobacter obae, which possess a-amino-E-caprolactam racemase activity.
The enzymatic hydrolysis of other cyclic amides was also investigated in order to
obtain chiral precursors for antibiotics and/or HIV inhibitors. Using isolates capable
of growth on a range of N-acyl compounds as the sole carbon and energy source, two
strains were selected for the enantioselective hydrolysis of (*)-2-azabicyclo-
[2.2.l]hept-5-en-3-one(Scheme 12.2-11). Rhocococcus equi NCIMB 40 231 selectively
hydrolyzed the (-)-enantiomer, yielding (+)-lactam with > 98% ee (45% yield),
whereas, Pseudomonas solanacearum NCIMB 40 249 hydrolyzed the opposite enantio-
mer with great selectivity yielding (-)-lactamwith > 98% ee (45%yield)f4',491. In
72 Hydrolysis and Formation ofC-N Bonds
these biotransformations the relatively low concentration of enzyme (6 g dry mass

L-l), the high concentration of substrate (50 g L-'), and the speed of the reaction (3 h)
are worth noting. Moreover, mutant strains have been constructed which hyper-
express the amidase activity. Subsequently it was shown that Rhodococcus equi cells
can also be applied for the enantioselective hydrolysis of 6-azabicyclo[3.2.0]hept-3-en-
?'-one, yielding a precursor for the antifungal agent cispentacin f5O].
Evidently, the use of the whole cell biocatalyst enables an efficient biotransforma-
tion with high substrate concentrations.
/
O D H
\
Scheme 12.2-11.
Recently, BASF has described the enantioselective hydrolysis of substituted

lactams. Using strains of Pseudomonas aeruginosa and Rhodococcus erythropolis
obtained from soil samples, both enantiomers of 5-vinylpyrrolidinon can be de-
rived ["I.
12.2.4
Selective Cleavage of the C-Terminal Amide Bond
In peptide synthesis selective deprotection of C- or N-terminal groups is common in

most methods of chain elongation. The amide groups offer some advantages for C-
terminal protection (enhanced chemical stability and increased solubility in water).
However, selective cleavage of this amide bond in the C-terminal position was
previously impossible. Both chemical and biochemical methods also led to internal
peptide bond hydrolysis, giving rise to difficult separation problems. Consequently
the amide group has been rather unattractive for C-terminal protection in peptide
synthesis.
Now, however this situation has changed. Steinke and Kula have isolated an
unusual peptide amidase from orange flavedo, which is very selective for the
hydrolysis of the C-terminal amide bond of peptides[52].The peptide amidase is free
I
72.2 Formation and Hydrolysis ofAmides 729
of any proteolytic activity, which would either hydrolyze internal peptide bonds of
substrate peptides or side chain amide bonds.
The substrate spectrum of this enzyme includes protected and unprotected
peptide amides and N-protected amino acid a m i d e ~ ' ~The ~ ] . chain length of the
substrate peptide amide, as well as the amino acid composition, including the C-
terminal amino acid side chain, are of minor importance. The amidase activity is
stereoselective with regard to the C-terminal position, only L-amino acid amides are
accepted. Unprotected amino acid amides are not hydrolyzed by this novel enzyme.
The broad application of this enzyme is further extended by its broad pH activity
range, from 6.5 to 9. Evidently, a new and usehl biocatalyst is now available for
selective deaminati~n['~]. Recently the same group has also shown that the reverse
reaction, the ammoniolysis of peptides, is catalyzed by this enzyme. Reducing the
water activity by carrying out the reaction in acetonitrile containing 5 % of water, Z-
Gly-Phe-NH2has been derived in a thermodynamic ammoniolysis in yields of up to
35 % [55'.
12.2.5
Amidase Catalyzed Hydrolytic and Synthetic Processes in the Production of
Semi-synthetic Antibiotics
Since the discovery of the P-lactam antibiotic penicillin G (Fig. 12.2-3)by Fleming in
1929, the use of antibiotics against pathogenic bacteria has increased dramatically.
Penicillin G was initially used, which must be applied intravenously because of its
instability in the stomach, but now penicillin V, which can be administered orally,
has been introduced. However, as a result of the increasing resistance of bacteria,
new antibiotics had to be developed. The semi-synthetic antibiotics, which often
possess a broad spectrum of antibacterial activity, were produced by altering the side
chain of penicillin G through acylation of the amine function of 6-aminopenicillanic
acid (6-APA)[561.
The first commercial semi-synthetic antibiotic was ampicillin, which was in-
troduced by Beecham in 1961[57].A few years later a new class of antibiotics, the
cephalosporins, was marketed. Some of the semi-synthetic cephalosporins are
prepared from 7-aminocephalosporanic acid (7-ACA),others from 7-aminodesace-
toxycephalosporanic acid (7-ADCA).7-ACA is an intermediate that can be obtained
from the fermentation product cephalosporin C; 7-ADCA is an intermediate that
was discovered by Morin et al. ["I using chemical ring expansion of the penicillin
nucleus (Fig. 12.2-4).The only difference between the two molecules is the absence
of an acetoxy moiety in 7-ADCA. Today, the main intermediates for semi-synthetic
cephalosporins (SSCs) and penicillins (SSPs), 7-ADCA and 6-APA, respectively, are
produced in quantities of many thousands of tons annually in biocatalytic processes
using penicillin amidases.
The coupling of the side chains to 6-APA and 7-ADCA is still performed
chemically. However, in order to obtain improved coupling yields and to overcome
the use of toxic and hazardous chemicals and solvents, several leading producers are
730
O A O H @OH
Penicillin G Penicillin V
Cephalosporin C
Figure 12.2-3. The three main fermentatively derived fi-lactams.
currently investigating so-called“green routes” again utilizing penicillin amidases to

perform this coupling reaction enzymatically.
12.2.5.1
Enzymatic Production o f 6-APA, 7ADCA and 7-ACA Using Amidases: Hydrolytic
Processes
Owing to the increasing importance of semi-synthetic antibiotics, commercially

feasible routes are of the utmost importance and several methods have been
developed.
About a decade ago 6-APA and 7-ADCA were mainly produced by chemical
deacylation of penicillin G, penicillin V or phenylacetyl 7-ADCA, the last of which
was derived from chemical ring expansion of oxidized penicillin G. As a result of the
fact that these processes were rather complex and employed hazardous reagents, for
example pyridine, phosphorus pentachloride, nitrosyl chloride and dichlorome-
thane, alternative processes have been developed. Penicillin amidases (E. C. 3.5.1.11)
catalyze the hydrolysis of the linear amide bond in penicillin molecules producing
both the p-lactam nucleus, 6-APA and the corresponding side chain without
affecting the p-lactam amide bond in the four-membered ring. Based on their
substrate specificity the penicillin amidases are grouped into three classes [’I:
1. Amidases that preferentially hydrolyze penicillin V (phenoxymethylpenicillin).
2. Amidases which are primarily active against penicillin G.
3. Amidases which are most active using ampicillin as the substrate.
12.2 Formation and Hydrolysis ofArnides I 7 3 1
H H
0-OH OAOH
Penicillin G Phenylacetyl7-ADCA
I Penicillin amidase I
O>cH
H2N% /3 CH3
0-OH OAOH
6-APA 7-ADCA
Figure 12.2-4. Enzymatic production o f 6-APA reaction in which a molecule ofwater is ex-
and 7-ADCA. 6-APA is produced from penicillin cluded. Again, penicillin amidase is used for the
G (or V) using the enzyme penicillin amidase. hydrolysis o f phenylacetyl 7-ADCA into 7-ADCA.
For the production of 7-ADCA, penicillin C is Both the production o f 6-APA and o f 7-ADCA
transformed chemically into phenylacetyl involve the liberation o f phenylacetic acid, a
7-ADCA. This transformation involves oxidation molecule that can be recycled into the fermenta-
of penicillin C followed by a ring expansion tion process.
Penicillin G amidases, in contrast to penicillin V amidases, display a fairly relaxed

substrate specificity. Consequentlypenicillin G amidases can also be used for various
other applications 611.
Major breakthroughs that facilitated enzyme application on an industrial scale
were improvements in the area of enzyme isolation, purification and immobiliza-
tion. Thus, the development of genetically engineered microorganisms accounted
for the high yield production of penicillin amidases. Also, the introduction of
immobilized enzyme systems, both for whole cell systems and for the isolated and
purified amidases [’’)- 62, 631, resulted in prolonged enzyme stability enabling reuse
and continuous process modes. As a result of this, the enzymatic routes currently
display far better economics for both 6-APA and 7-ADCA production (Fig. 12.2-4)
compared with their chemical counterparts.
Nowadays, excellent penicillin amidases from various sources are being used on
732
t W
6-APA
Figure 12.2-5. Schematic representation of industrial production of 6-APA using
column packed immobilized penicillin amidase.
an industrial scale for producing either 6-APA or 7-ADCA[’’. 631. These biocatal@c
processes are generally performed batchwise at 35-40 “C and pH 7.5-8.5[622. 64,651.
Upon formation of 6-APA and 7-ADCA the side chain acid is liberated, which causes
a drop in the pH of the reaction mixture. This pH change results in a decrease in the
reaction velocity. Since a higher starting pH is not desired because of enzyme
deactivation and P-lactam ring hydrolysis, a strict control of the pH is necessary
during the process. Generally, pH adjustment occurs separately from the im-
mobilized enzyme, either by packing the immobilized enzyme in columns, as
outlined in Fig. 12.2-5, or by cycling the contents of the enzyme reactor through a
sieve, retaining the immobilized biocatalyst over a small pH control vessel. Cur-
rently, immobilized penicillin amidases can be reused up to 600 times [”]. After
completion of the hydrolcc reaction the immobilized biocatalyst is separated from
the liquid and the products 6-APA or 7-ADCA are precipitated at their iso-electric
points and collected by filtration. After washing and drying an extremly pure product
is obtained[65].
In addition to 7-ADCA, 7-ACA is also a very useful intermediate for the production
of other SSCs (for example cefazolin, cefotaxime, ceftriaxone and cefuroxime).Until
recently, 7-ACA was produced chemically from cephalosporin C using the phospho-
rus pentachloride process or the “Delft Cleavage”[65].As a result of the good
experiences with penicillin amidases and the increasing concern about the amount
I
733
NH3 0
I
Cephalosporin C
chemical deamination followed by

decarboxylation
I hydrolysis by glutaryl amidase
7-ACA
Figure 12.2-6. Cherno-enzymatic production of 7-ACA from cephalosporin C.
of waste being produced in chemical side chain cleavage processes, several com-
panies are engaged in the development of enzymatic processes for the production of
7-ACA. Several years ago Asahi commercialized a chemo-enzymatic process in
which cephalosporin C is oxidatively deaminated to glutaryl-7-ACA, which is
734
Cephalosporin C
D-amino acid oxidase; 0, +

H,O;
0 H
I1 I
HO
0 0
K0
CH3
2-Ketoadipyl-7-ACA
H
I
K0
CH3
4
Glutaryl-7-ACA
glutaryl-7-ACA amidase;
oy&
glutaric
acid
HZN
K 0
CH3
Figure 12.2-7. Two-enzyme bio-
catalytic process for production of

7-ACA 7-ACA from cephalosporin C.
subsequently hydrolyzed enzymatically using a glutaryl amidase from a Pseudorno-

I 735
nas species (Fig. 12.2-6)IG5I.Currently,the first step of this process is also carried out
enzymatically. Using a D-amino acid oxidase cephalosporin C is oxidized to the
corresponding a-ketoadipyl derivative. This latter compound spontaneously dec-
arboxylates to give glutaryl-7-ACA (Fig. 12.2-7)rG5, G71.
So far no direct deacylation of cephalosporin C has been commercialized,
although enzymes have been identified that can indeed catalyze this one-step
hydrolysis[‘*I. After optimization of the production of this biocatalyst, and possibly
improvement of its intrinsic properties, it is very likely that a one-step enzymatic
hydrolysis of cephalosporin C will be industrialized.
12.2.5.2
A New Fermentation-based Biocatalytic Process for 7-ADCA
With the aid of metabolic pathway engineering a large step forward has now been
realized in the production of 7-ADCA by adapting processes within penicillin
producing organisms. Thus, the conversion of the five-membered penicillin ring
into the six-membered cephalosporin ring can now be performed within the
microorganism as outlined in Fig. 12.2-8. By modifymg the responsible gene, the
penicillin producing mould can be set to produce a 7-ADCA derivative directly. Thus,
several chemical steps from penicillin via penicillin oxide to 7-ADCA can be
omitted[G9].
Because of a newly introduced gene, the substrate specificity of the engineered
strain changed. Now, dicarboxylic acid is used as the externally added side chain,
instead of phenylacetic acid as in penicillin G.
Later in the process this side chain is removed enzymatically, using an enzyme
quite similar to the glutaryl amidase from Pseudomonas sp. as in the enzymatic
production of 7-ACA. For the production of 7-ADCA and the dicarboxylic acid
amidase, new plants are currently under construction at DSM in The Netherlands.
Compared with the old process for the production of 7-ADCA, the major advantages
of this process are higher purity of the end product, much greater energy efficiency
and almost complete absence of organic solvents.
12.2.5.3
Enzymatic Formation of Semi-synthetic Antibiotics: Synthetic Processes
The chemical synthesis of semi-syntheticantibiotics (SSAs)from a p-lactam nucleus

(such as 6-APA, 7-ACA, or 7-ADCA) and a side chain (such as D-(-)-phenylglycineor
an aminothiazoleiminoaceticacid derivative) is difficult to carry out in a single step
since both reactants have functional groups that can easily form undesired covalent
bonds.
In order to obtain the desired product in high yield it is necessary to activate the
carboxyl function of the acylating agent, to temporarily protect interfering amino
functions, to effect the formation of the amide bond and to remove the protecting
groups. Moreover, this condensation should be performed under conditions that will
736
~ ~~
Sugar
Fermentation Fermentation Fermentation

1 1
Penicillin G
Chemistry
1
Chemistry
1
!
Penicillin G sulfoxide
Chemistry Chemistry Biocafalysis
Phenylacetyl-7-ADCA
Chemistry Biocatalysis
I
7-ADCA
Chemistry Biocatalysis Biocatalysis

1 1 1
Cefadroxil, Cephalexin, Cephradine
Figure 12.2-8. Production of 7-ADCA has undergone remarkable changes. In the

early days (left-hand side), chemical ring expansion o f penicillin C resulted in the
formation of the cephalosporin nucleus. The phenylacetyl moiety was then re-
moved chemically. Later on, this last step was replaced by a biocatalytic step using
penicillin arnidase (middle). On the right hand side, a completely new route is
presented. Dicarboxyl-7-ADCAis obtained directly by fermentation. A dicarboxyl
amidase is used to remove the dicarboxyl group.
preserve stereochemical integrity and leave the fragile four-membered p-lactam ring
intact.
Today, nearly all SSAs are produced using the methodology described above.
However, because of the relative complexity of these chemical processes and the use
of toxic reagents and solvents, the application of biocatalysis has promising possibil-
ities here too. Indeed, several biocatalytic processes have been developed and are
now being introduced on a production scale. A noteworthy advantage of these
processes is that protection of functional groups is not a prerequisite because of the
mild reaction conditions and the high selectivity of the enzymes involved.
So far, the major focus of research has been directed at enzymatic coupling of D-
(-)-phenylglycine methylester or D-(-)-phenylglycine amide with either 6-APA or
7-ADCA yielding ampicillin and cephalexin, respectively, and at the coupling of D-
(-)-4-hydroxyphenylglycine derivatives with either 6-APA or 7-ADCA leading to
amoxicillin and cefadroxil, respectively (Fig. 12.2-9) [ 6 3 , 7&751 . During this kinetically
controlled condensation, the activated 4-hydroxyphenylglycineforms an acyl-en-
zyme complex with the penicillin amidase L7'1. Subsequently, this acyl-enzyme
complex is deacylated by a nucleophile, the p-lactam nucleus 6-APA or 7-ADCA, or
I
737
OAOH OAOH
I Penicillin amidase I
J. f
Ampicillin Cephalexin
Figure 12.2-9. Enzymatic conversion o f 6-APA and 7-ADCA into ampicillin and cephalexin using
penicillin amidase. The side chain is introduced using an activated form o f D-(-)-phenylglycine,
either the amide (R = NHp) or the ester (R = OCH3, OCzHs).
water. In the first case this leads to product formation, whilst in the second case the
activated side chain is hydrolyzed. By carefully tuning reaction conditions and
downstream processing sequences for every individual product, yields of up to 90%
based on 6-APA or 7-ADCA have been obtained. However, as a result of the
competing hydrolysis of the acyl-enzyme complex by water the yield with respect to
the ester or amide was quite low (approximately 30%)[72,731. By performing this
condensation in the presence of an alcohol, as a result of which the activated
phenylglycine is 'recycled' in situ, the yield based on phenylglycine could be
improved[75].The latest breakthroughs are the development of immobilized bio-
catalysts with improved performance, application of low temperatures and using
high substrate concentrations L7'1. It is these improvements that make enzyme-
catalyzed synthesis of SSAs in a purely aqueous environment competitive in-
dustrially with traditional chemical synthesis.
12.2.6
Conclusions and Future Prospects
As indicated in the preceding sections, amides and their derivatives are important
versatile building blocks for the (agro)chemicaland pharmaceutical industry. Owing
to the selectivity of amidases (both regio- and enantioselectivity) and the fact that
738 I Table 12.2-4. Reduction of waste volumes resulting from introducing penicillin acylase
biocatalysis in antibiotics production.
Process Waste reduction factor
Penicillin G + 6-APA
Penicillin G --t 7-ADCA
6-APA -t semi-synthetic penicillins
7-ADCA --t semi-synthetic cephalosporins
these conversions can be achieved under very mild conditions, several biocatalytic
processes based on amidases have recently been commercialized. The use of these
biocatalysts in the chemical industry is expected to increase in importance in the
near future as environmental restrictions become more pronounced. The benefits in
terms of reduction of waste can be enormous, as can be judged from that already
achieved in the production of antibiotics (Table 12.2-4).
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72.3 Hydrolysis of N-Acylarnino Acids
12.3
I
741
Hydrolysis of N-Acylamino Acids
Andreas 5.Bornrnarius
12.3.1
Introduction
The enzymatic hydrolysis of N-acylamino acids has been known for a century and
was first detected in aqueous kidney preparations ['I. Based on the finding that this
enzymatic hydrolysis proceeds enantiospecifically[2],Greenstein and coworkers
developed a general and very attractive procedure for the resolution of a vast number
of racemic N-acylated amino acids to the corresponding L-amino acids catalyzed by
aminoacylase (E. C. 3.5.1.14) whereas the N-acetyl-D-amino acid does not reactr31
(Fig. 12.3-1).
These initial investigations on a laboratory scale subsequently lead to industrial
processes for the production of L-amino acids on a multi-ton scale applied by
Tanabe["'I and Deg~ssa['-'~~. The first work on the isolation and characterization of
aminoacylases came from Greenstein and coworkers. Fractionation of hog kidney
homogenates with ammonium sulfate and acetone revealed that two distinct
enzymes were present in the crude preparati~nl'~]. One was found to hydrolyze a
large number of N-acetylamino acids and was designated acylase I (E.C. 3.5.1.14)
whereas the other was found to hydrolyze preferentially N-acylated L-aspartic acid
and was designated acylase I1 (aspartoacylase, E. C. 3.5.1.15). Additionally, a third
aminoacylase, which acts preferentially on N-acylated aromatic amino acids, was
found in kidney homogenates and was designated acylase 111[14, 151. Besides the L-
DL-Met -
- HCN + NH3 + ow+ H3C-SH
?;" 0
Racernization
Figure 12.3-1. Enantiospecific hydrolysis of N-acetyl-o,L-amino acids
catalyzed by aminoacylase I.
742
specific enzymes from kidney preparations, L- as well as D-specific aminoacylases

have been isolated from a variety of microorganisms [16-331.
Although the physiological function of these enzymes is not known with certainty,
it is assumed that they may be involved in the degradation of N-acylated amino acids
occupying the N-termini of many proteins and are subsequently formed in the
catabolic metabolism of proteins [I7, 34, 351.
12.3.2
Acylase I (N-Acylamino Acid Amidohydrolase, E.C. 3.5.1.14)
Since acylase I has a wide substrate specificity and high enantioselectivity, it is a

broadly applicable enzymatic catalyst for the kinetic resolution of most of the natural
Table 12.3-1. Substrate specificity of acylase I (E.C.3.5.1.14) f r o m hog kidney["].

Substrate Relative activity
X = acetyl X = chloroacetyl
N-X-D,L-rnethionine looa 413b

N-X-D,L-ethionine 64 -
N-X-D,L-norvaline - 167
N-X-~,~-aminobutyric acid - 139
N-X-D,L-norleucine 59 126
N-X-D,L-aminoheptanoicacid - 117
N-X-D,L-leucine 22 68
N-X-DJ-alanine 13 48
N-X-D,L-serine - 48
N-X-D,r-glutarnicacid 13 52
N-X-o,L-arninocaprylicacid - 32
N-X-D,L-valine 7 21
N-X-D,L-arninocyclohexylacetic
acid - 19
N-X-D ,L-aminophenylaceticacid - 19
N-X-glycine - 11
N-X-D,L-allothreonine - 11
N-X-D,L-threonine - 3
N-X-D,L-isoleucine 2 -
N-X-D,L-arginine 2 -
N-X-D,r-alloisoleucine 1.o -
N-X-o,L-histidine 0.6 -
N-X-D,r-phenylalanine 0.6 -
N-X-o,r-diaminopropionic acid - 1.9'
N-X-D,r-arninocydohexylpropionic acid - 1.4
N-X-D,L-tyrosine - 1.4
N-X-D,L-ornithine - 1.3'
N-X-D,L-lysine - 0.6'
N-X-D,L-arninocyclohexylbutyricacid - 0.5
N-X-D,r-S-benzylcysteine 0.4 -
N-X-D,L-tryptophan < 0.1 < 0.1
N-X-D,L-asparticacid < 0.1 < 0.1
N-X-D,L-proline < 0.01 < 0.1
N-X-D,r-phenylserine - 0
a 400 pmoles x min-' per mg of N at 38 "C. b Dichloroacetyl.c Not determined.
12.3 Hydrolysis ofN-Acyhmino Acids
Table 12.3-2. Comparison of the kinetic and chemical properties o f pig kidney and Aspergillus
I
743
oryzae aminoa~yIase[~'1.
Aspergillus oryzae Pig kidney

Property aminoacylase aminoacylase
Molecular mass 73 200 85 500
Subunits 2 2
Metal ions (Zn2+) 6
SH-groups 0
Inhibition by N-a-ptosyl-r-lysine -
Chloromethyl ketone HC1
Inhibition by diethylpyrocarbonate + +
Inactivation by metal chelating agents completely reversible completely reversible
pH-optimum (chloroacetylalanine) 8.5 8.0
112 Cystine residues 4 12
Tryptophan residues 6 12
KM x lo3 mol L-' (chloroacetylalanine) 6.3 6.6
Spec. activity U mg-' (chloroacetylalanine) 319 (pH 8.0) 250 (pH 7.8)
Peptidase activity - +
CI-Ac-Glu-PABAhydrolysis + -
Heat stability 60 "C: denaturation 60 "C:denaturation
Activation by Co" + +
as well as unnatural and rarely occurring a-amino acidsl3>361. Thus it is the most
important and mostly frequently used aminoacylase in the chemoenzymatic synthe-
sis of L-amino acids from the corresponding racemic N-acetylated precursors (Table
12.3-1).
The numerous investigations on acylase I have been reviewed on several occa-
sions [361. Acylase I has been isolated and characterized from kidney prepara-
tions [13, 37-39] as well as the fungi Aspergillus oryzae[16. 17, 35* 401 (AA) and Aspergillus
melleus (AM).The enzymes from the two Aspergillus species are virtually
A comparison of some kinetic and chemical properties of pig kidney acylase (PKA)
and the mold enzyme from Aspergillus olyzae (AA) is shown in Table 12.3-2. Both
enzymes are dimeric, zinc-containing metalloproteins of roughly the same size and
which are strongly activated by external addition of cobalt ions['7, 35, 40, 42-441; Zn2+
is essential for activity[40].The Co2+/acylase-dissociationconstants of PKA and AA
are similar with 10-7.5M i4O] und M [431, respectively, the respective constants for
Zn*+/acylaseare identical at 10-l' M L40, 451. They differ in the amount of zinc ions
bound per subunit and in the number of SH-groups as well as cysteine and
tryptophan residues essential for catalyhc activity. The properties of acylase I from
Aspergillus oryzue are summarized in Table 12.3-3[461.
12.3.2.1
Genes, Sequences, Structures
The DNA and protein sequences of eight aminoacylases are now known, as of March
2001. Sequences from ~ o m supiens
o [47, 481, pig [49, "1, ~ucillusstearothermophilus
and Lactococcus lac ti.^[^^] are known for aminoacylase I, sequences from Homo
744
Table 12.3-3. Properties of acylase I from Aspergillus o r y ~ a e ‘ ~ ~ ~ .

~~~
Parameter Quantity Reference
Molar mass Da; no. of subunits 73 200; 2 (identical) WI

Specific activity (pure enzyme, U mg-’) 319 (C1Ac-Ala, pH 8.0) I351
T; pH optimum 55 “C; pH 7.5 1151
Substrate(s)[KM-value(s), mM] ClAc-Met [1.5], ClAc-Phe (0.7) [35]
Activators (0.5 mM, in %), pH 6 CO” (151),Zn2+(loo),Mg” [35]
(97), Mn2+(37), Ni2’ (27)
Inhibitors Cd”, Cu2+,chelators 1351
Sequences and structure:

protein sequence: not known DNA sequence: not known
expression system: not known 3D crystal structure: not known
sapiensLS3,541 and Mus musculus (house mouse)[”] for aminoacylase I1 and se-
quences from Achromobacter xylosoxidans A-6 rS61 and Alcaligenes faecalis [”I for D-
aminoacylase. The DNA sequences of several other acylases, notably r-acylases I,
from organisms such as Arabidopsis thaliana, Streptomyces coelicolor, Bacillus subtilis
and from two human genome project groups have been annotated as aminoacylases
but have not been confirmed to possess aminoacylase activity. Regarding three-
dimensional structures, as of the beginning of March 2001, no structures of
aminoacylases were known or under review according to the Protein Data Bank
(PDB).
12.3.2.2
Substrate Specificity
An extensive study of the substrate specificity of both enzymes (AA, PKA), especially
for the resolution of unnatural and rarely occurring amino acids has been conducted
by Whitesides and (Fig. 12.3-2).
Both enzymes have an unusually wide substrate specificity with a preference for
hydrophobic substrates. N-acylated aliphatic straight-chain amino acids are the
preferred substrates for both enzymes, however, the corresponding aliphatic
branched-chain amino acids are also readily accepted, especially by the fungal
enzyme[’3, 351. N-acylated amino acids with an aromatic side chain are significantly
hydrolyzed only by the fungal enzyme[17](Table 12.3-4).The substrate spectrum of
AA was even broader than anticipated[’’]. Sulfur- and selenium analogs react at
comparable rates, often even faster than the carbon analogs; four to five atoms are
the optimum length of the side chain. S-Methyl-L-cysteine gained significance
recently as building block for HIV-protease inhibitors [59, “1, L-selenomethionine
was described as part of a suitable treatment for Alzheimer’s disease and Parkinson’s
syndrome [61]. Another striking difference is that the renal enzyme hydrolyzes
dipeptides whereas the mold enzyme does
Acylase I has not only been used for the enantioselective resolution of N-acetyl-D,r-
amino acids to the corresponding L-amino acids but also for substrate-selective
resolution of N-acetyl amino acids using the different activity of the enzymatic
72.3 Hydrolysis of N-Acylamino Acids
R2
I
745
R'eCOOH
A
HNYR3
0
NH,O
HO
K H N ~
CH2'\=~
C H ~
,f H,N
K NH(CH&,' CH3g CH3CHzCH(CH3)g poor, 0.01-1 %
d' 0
NHCH2, H~N(CH~)H
R2, R') are expressed relative to the reactivities of the corresponding

a Reactivities of substituents (R',
substituents of the model substrate, N-acetylmethionine (40 mM racemic: R' = CH'SCH2CH2, R2, RZ = H, R'
= CH3. b AA only; reactivity with PKA is fair. c PKA only: reactivity with AA is fair. d AA only: reactivity with
PKA is poor. e AA only; no reactivity with PKA. f Data for PKA only. g PKA only: no reactivity with AA.
Figure 12.3-2. Reactivities of substituents of acylase
746
Table 12.3-4. Comparison o f the relative activity of Aspergillus and pig kidney aminoacylase with
different substrates [351.
Substrate Conc. Aspergillus Pig kidney
(rnM1 arninoacylase arninoacylase
N-chloroacetyl-r-alanine 7.1 100“ looa

N-chloroacetyl-L-methionine 7.1 400 480
N-chloroacetyl-D,r-norleucine 2.1 207 120
N-chloroacetyl-r-leucine 2.1 26 96
N-chloroacetyl-L-phenylalanine 3.5 325 5
N-chloroacetyl-L-tryptophan 2.1 125 0
N-acetyl-L-glutamic acid 8.2 0 21
N-acetyl-L-aspartic acid 8.2 0 0
N-acetyl-L-glutamine 8.2 13 4
N-acetyl-L-alanine 8.2 14 7
N-acetyl-r-lysine 8.2 3 0
N-dichloroacetyl-glycine 4.1 0 1
N-dichloroacetyl- L-leucine 4.1 0 3
N-dichloroacetyl-D,L-norleucine 4.1 4 69
N-dichloroacetyl-L-alanine 4.1 0.7 2
a V, = 4.2 N M s-‘.
catalyst towards different N-acetyl-r-amino acids. Martens and Weigel used kidney
acylase for the separation of N-acetyl-L-leucineand N-acetyl-L-isoleucine[631.
12.3.2.3
Stability of Acylases
Acylase I from both sources is very stable as a lyophilized powder. In aqueous

solution, the resting stability of acylase from Aspergillus oryzae was found to depend
much more on pH than on concentration: while at room temperature (25 “C) and
standard pH (7.0) the half-life ~ 1 1 was
2 around GO d for concentrations of between 30
and 120 g crude enzyme L-l, the 5 1 / 2 dropped to 45 d at pH 6.5 and to about 30 d at
pH 6.0[46].Also, in solubilized form, the fungal enzyme is fairly stable whereas the
pig kidney enzyme is sensitive to auto oxidation and therefore should be kept under
nitrogen if stored in a solubilized Tests for operating stability in repeated-
batch mode L9, 17, 36s 641 reveal that acylase from Aspergillus oryzae again fares much
better than the porcine kidney enzyme. Tests for operating stability in a continuous
reactor with the acylase from Aspergillus 0ryzae[‘~1again demonstrated:
- superior stability of AA (616 U kg-’ L-methionine) over PKA (6000 U kg-I L-met),
both measured with [Co”] at S X ~ O -M,~
- tighter binding of Zn” vs. Co2+at 5 x M (308 vs. 616 U kg-’ L-met),
- that loss of metal, commonly Zn”, is responsible for activity loss and
- possibility of reconstitution over a timescale of several hours, whereas the time
constant of leaching is on the order of 48 h, as well as
- the option of pulsing divalent metal addition resulting in 477 U kg-’ L-met at
[Zn”] of 4 x M.
W-
40 -
20 - C
20-
d
Porcine kidney acylase seems to have a different spectrum of activationrG4]: although

Co2+activates PKA most strongly, Ca2+is not far behind whereas Zn2+,just like Mg2+
or Fe", does not seem to exert a strong effect. Both enzymes have moderate
therrn~stability~~~]and moderate stability in the presence of organic cosolvents13'1
(Fig. 12.3-3).
The behavior of aminoacylase both from porcine kidney and Aspergillus sp.
towards a wide range of water-miscible cosolvents was investigated by Iborra et
al.["I. They found that enzymatic activity can be correlated with the denaturing
capacity of the water-cosolvent system.
In 1993,a thermostable aminoacylase from Bacillus stearothermophilus was charac-
terized by Sakanyan et al.rs1].The enzyme hydrolyzes N-acyl derivatives of aromatic
amino acids preferentially and even has some dipeptidase activity. Its optimal
reaction temperature is 70°C; after incubation for 15 min, 90% of the original
activity was retained. The authors write that the similarity ofthe B. stearothermophilus
enzyme sequence with that of other enzymes such as aminoacylase I, acetylornithine
deacetylase and carboxypeptidase G2 suggests a common origin. The aminoacylase
from B. stearothermophilus is well characterized: the gene has been completely
sequenced[51],cloned into E. coli and overexpres~ed[~~~ G71 and studied for catalytic
and stability properties["]: the intrinsic one Zn2+ion per subunit seems to have a
predominantly structural role and activity can be restored to the apo-enzymeby Co2+
and particularly by Cd2+(3-foldactivity!) but not by Zn2+.
748
Table 12.3-5. Thermodynamics o f the N-acetyl amino acid Conditions: pH 7.5, J =

25 "C; xeq calculated at [So] of0.5 M , K,,-data determined from synthesis and hydrolysis reaction.
Amino acid Keq Xeq
Glycine 4.5 90.8%

Alanine 5.6 92.4%
Aminobutyric acid 5.6 92.4%
Norvaline 10.5 95.6%
Norleucine 12.5 96.3%
Methionine 3.7 89.2%
Another thermostable acylase, aminoacylase SK-1, was reported by the Amano

Pharmaceutical Comp. The enzyme is isolated from B. stearothemophilus I F 0
12983. It possesses an optimal temperature for reaction at 60 "C and is stable at
70 "C for at least 30 min. The preferred substrates are dipeptides besides the N-acyl
derivatives of Met, Phe and Tyr. K. Soda's group has isolated and characterized a
thermostable aminoacylase from Bacillus them~glucosidius~~~] which has many
similarities to the Aspergillus enzyme, such as metal content and requirements,
activity and specificity profile as well as high stability at elevated temperatures and
high content of organic solvents and denaturants. Judged by the identity of the
organism used for culturing, of the specificity profile and of some enzyme properties
(both are identical dimers with molecular mass of 86000 Da), aminoacylase SK-1
and the aminoacylase from Bacillus therm~glucosidius[~~] seem to be the same
enzyme.
12.3.2.4
Thermodynamics and Mechanism of the Acylase-catalyzed Reaction
Equilibrium
The hydrolysis reaction of N-acetyl amino acids is equilibrium-limited, however, the
equilibrium is well on the side of the hydrolysis so that at low substrate concentra-
tions conversion is almost quantitative. For the case of N-acetyl methionine,
Wandrey and Flaschel determined the equilibrium constant K defined as in Eq. (1)
acetate][^-Met]
K=[
[N-Ac-L-M~~]
and found K = 2.75 M at 37 "C and pH 7"l. Then, at 37 "C and [So] = 100 mM,
equilibrium conversion x, is 96 % (basedon N-Ac-L-Met),at [SO]= 500 mM, x, = 86 %.
The enthalpy of reaction is 7.9 kJmol-' 191. Data for other substrates are listed in Table
12.3-5. More physicochemical data on the N-acetyl amino acid acylase reaction can be
found in ref. [641.
pti-Dependence
The Michaelis constant for hydrolysis is independent of pH in the pH range 6.0-9.5
whereas the pH-dependence of maximum velocity has a bell-shaped profile with the
maximum at pH 7.5 and inflection points at pKa values of 6.7 and 8.9[69].
Table 12.3-6. Substrate specificityof acylase II (aspartoacylase; E.C. 3.5.1.15) from hog
kidney[”].
Substrate Relative activitv
X = acetyl X = chloroacetyl
N-X-D,L-asparticacid looa 526
N-X-D,L-ghtamic acid -b 22
N-X-D,L-methionine 33 -
N-X-D ,I-alanine - 19
N-X-D,L-leucine - 26
N-X-D,L-serine - 11
a 0.45 pmolesxmin-’ per mg of N at 38 “C. b Not determined
Mechanism
The mechanism of acylase-catalyzed reaction has been studied, particularly for
porcine kidney acylase [34, 6g--711 . Wh’ile the mechanism of action was contested for
some time between a linear mechanism (random uni-bi)[34, 701 and a double- “3
displacement, “ping-pong”,mechanism involving a stable intermediate l7ll, it now

seems to have been decided that base-catalyzed attack of the carbonyl carbon by
water is the rate-determining step followed by a linear sequence involving an E-P1P2-
complex[34, 69, 701 E 9. (2):
I
Recent work on the non-competitive inhibition of both porcine and fungal aminoacy-
lase by a- and p-fluoro- and -hydroxy acids indicated that the active site of the fungal
enzyme should interact with the a-substituent of a substrate via an acidic group
while the porcine enzyme has a basic group in the corresponding position with
which to recognize substrates 17’1.
Enantiospecijicity
Acylase I acts on racemates in a highly enantiospecific way to yield L-amino acids
exclusively, with ee values in almost all cases, especially with N-acetyl substrates,
exceeding 95 %. According to Cahn-Ingold-Prelogrules, L-amino acids correspond to
the (S)-configuration,with the exception of L-cysteine which is in the (R)-configura-
tion owing to first stereochemical priority of the thiomethyl group. In general, the
amino acid amide enantiomer bearing the larger substituent in the pro-(S)-position
is hydrolyzed preferentially [731.
12.3.3
Acylase II (N-Acyl-L-Aspartate Amidohydrolase, Aspartoacylase, E. C. 3.5.1.1 5)
Apart from acylase I, another aminoacylase was found in kidney preparations by

fractionation of hog kidney homogenates with ammonium sulfate and acetone [I3].
Whereas acylase I could be enriched and thus partially purified by this procedure the
main activity with N-acylated-L-aspartic acid as the substrate was found in another
750
Figure 12.3-4. Enantioselective hydroly-

sis of N-acetyl-DL-prolineto L-proline
catalyzed by proline a~ylase1~~1.
0
N-Acetyl-D,L-proline
L-Proline II
0
N-Acetyl-D-proline
Table 12.3-7. Comparison of some kinetic and chemical properties of proline acylases from three
different microorganisms~'g-221.
Property PS.spec. 1' 1' Rh. rubraPO] Corn. Testosteroni"'. *'I
Enantiospecificity L L L
Molecular mass Da 597 000 560000 380 000
+/- 12000 +/- 40000
No. of subunits 10-12 -a 8
Molecular mass of subunits (Da) 55000 - 45000 +/- 15000
Isoelectric point 5.0 - -
pH-optimum 6.0 6.0 6.8
pH-stability 7.0-8.0 6.0-10.0 5.0-10.0
(30 min, 35 "C) (4weeks, room temp.)
Temp. optimum - 50 "C 65 "C
Temp. stability 40 "C 40 "C 70 "C
(10 min, pH 8.0) (30 min, pH 7.5)
Activation by divalent cations none none none
Inhibitors phosphate PCMB phosphate
EDTA 2-mercaptoethanol
o-phenanthroline o-phenanthroline
2,2-dipyridyl PCMB, PHMB
Hg2+> Cu2' cu2+ Fe" > Hg2+
> ZnZ+> Fe3+ > cu2+> Zn2+
> Ni2+> Pb2+ > Sn2' > Fe3'
Reactivation of the apoenzyme
by divalent cations Mn" > Ca2+ co2+> Zn2'
Pb2+> co2+
Zn" > Ba2+
a Not determined.
fraction of the ammonium sulfate precipitates. To distinguish between these two

I 751
activities, the former fraction was designated acylase I, and the latter acylase II[13]. In
contrast to acylase I, acylase [I has a very narrow substrate specificity. Among the N-
acetyl derivatives of the twenty proteinogenic amino acids only N-acetyl-L-aspartic
acid is hydrolyzed significantly (Table 12.34). Therefore, acylase I1 from kidney
preparations was designated as aspartoacylase or N-acyl-L-aspartateamidohydrolase
(E.C. 3.5.1.15) and is the enzyme of choice for the resolution of racemic aspartic
acid.
Comparison ofthe substrate specificity of proline acylases from four different

Table 12.3-8.
microorganisms "8-211.
Relative activity
Substrate Alc. spec. ['I R. spec. [''l Rh. rubra Po] Corn. testost. 12'1
N-acetyl-L-Pro 100" 1OOb 100 100'

N-acetyl-D-Pro 0 0
N-acetyl-L-Ala 0.03 9
N-acetyl-o,r-Ser 0 0.2
N-acetyl-L-Val - 0.2
N-acetyl-D,r-Val 0 -
N-chloroacetyl-L-Pro 172 362

N-chloroacetyl-r-Met - 17
N-chloroacetyl-L-Val - 14
N-chloroacetyl-r-Leu - 2
N-chloroacetyl-L-Phe - 1
N-chloroacetyl-L-Tyr - 1
N-chloroacetyl-L-Ile - 0.5
N-acetyl-L-Hyp - 10
N-formyl-L-Pro - 18
N-propionyl- L-Pro 13 29
N-butyryl-L-Pro 24 14
N-valeryl-L-Pro - 15
N-caproyl-L-Pro 33 9
N-capryloyl-L-Pro 23 -
N-caprinoyl- L-Pro 59 -
N-myristoyl-L-Pro 7 -
N-palmitoyl-r-Pro 0.6 -
N-benzoyl-L-Pro 61 -
Gly-L-Pro 123 4
N-Z-L-Proe 24 0
N-2-Gly-L-Pro 269 11
N-2-L-Ala-L-Pro 2 -
N-2-Gly-L-Ala 1 -
N-2-Gly-r-Pro 217 -
N-2-Gly-L-Pro
L-Leu-Gly-L-Pro 101 -
a 142 pmoles x min-' x mg-'. b 410 pmoles x min-' x mg-I. c 85 pmoles x min-' x mg-'.
d Not determined. e Z: benzyloxycarbonyl.
752
12.3.4
Proline Acylase (N-Acyl-L-ProlineAmidohydrolase)
The acylase-catalyzed resolution of N-acyl-D,L-amino acids has some limitations.

Although acylase I from porcine kidney and Aspergillus oryzae has a broad substrate
specificity and high enantioselectivity, the enzyme does not accept N-acylated
substrates where the hydrogen atom at the amide nitrogen is replaced by an alkyl
group. Therefore, N-acylated secondary amines such as N-acetyl-proline and N-
acetyl-N-alkyl-aminoacids are not hydrolyzed by this enzyme[13,36, 37, 74, 751 as well
as aminoacylases from other This gap in the substrate specificity of
aminoacylase I was successfully closed with the isolation of acylases which act
specifically on N-acetyl-L-prolineand its derivatives[18-22] (Fig. 12.3-4).
The enzyme has been isolated from Alcaligenes sp. [18, 21], Pseudomonas sp. [I9],
Rhodotorula rubru(20]and Comamonas testosteroniIZ1.2 2 ] . Some kinetic and chemical
properties of proline acylases from three different microorganisms are listed in Table
12.3-7. A comparison of the substrate specificity of proline acylases from four
different microorganisms is shown in Table 12.3-8.
Proline acylase is a relatively large protein with a molecular mass in the range of
380-600 kDa consisting of 8-12 subunits with a molecular mass of 45-55 kDa. The
Table 12.3-9.Substrate specificity of proline acylases from Cornomonas testosteroni towards

N-acyl-amino acids [771.
Substrate Relative activity Conversion (“A)
N-acetyl-L-praline 100 100

N-acetyl-o,L-proline 78 54
N-acetyl-~-thiazolidine-4-carboxylicacid 255 99
N-acetyl-~-azetidine-2-carboxylicacid < 50 100
N-acetyl-D,L-pipecolicacid 58 49
N-chloroacetyl-L-proline 308 100
N-chloroacetyl-o,L-proline 290 52
N-chloroacetyl-~-thiazolidine-4-carboxylic acid 357 100
N-chloroacetyl-~-azetidine-2-carboxylic acid 437 100
N-chloroacetyl-o,r-pipecolicacid 507 54
N-chloroacetyl-o,~-indoline-2-carboxylic acid 0 0
Table 12.3-10. Substrate specificity of proline acylases from Comamonas testosteroni towards
N-alkyl-amino acids [781.
Substrate Relative activity Conversion (“A)
N-chloroacetyl-L-praline 100 100
N-chloroacetyl-N-methyl-L-alanine 175 100
N-chloroacetyl-N-methyl-o,L-alanine 115 49
N-chloroacetyl-N-ethyl-o,L-alanine 82 -
N-chloroacetyl-N-propyl-o,L-alanine 27 -
N-ch~oroacetyl-N-methyl-~,~-2-aminobutyric
acid 10 50
N-chloroacetyl-N-ethyl-o,~-2-aminobutyric
acid 2 -
a Not determined.
12.3 Hydrolysis ofN-Acylamino Acids I
753
u
R'
H20 CH3COOH
R2 hCooH -
acylase I
HN KCH3
0
enarnine N H ~
IA
N-acetyldehydroamino acid
R' NH3 H20 R'

I1 spontaneous
Z%COOH _2/ $+
COOH
spontaneous
0 NH
a-keto acid irnine
R1
R F ! C O O H
NH2
L-amino acid
Figure 12.3-5. Coupled enzymatic reaction of a dehydro amino acid acylase
with a amino acid dehydrogenase (from[821).
enzyme is not activated by cobalt ions and has a relatively narrow substrate
spectrum. The enzyme from Cornamonas testosteroni preferentially hydrolyzes N-
acylated L-proline and the N-acetyl derivatives of other cyclic imino acids [771 (Table
12.3-9)and opens for the first time the route to resolution of racemic N-acylated N-
alkyl-aminoacids [781 (Table 12.3-10).Among the proteinogenic amino acids, only the
N-acetyl derivatives of r-proline and L-alanine are hydrolyzed to a significant
efient[21s22, 771
12.3.5
Dehydroarnino Acid Acylases
A new acylase was found in strains of Breuibacterium sp. by H ~ m m e l [ ~1' ' ' . in 1987,
catalyzing the hydrolysis of acetamidocinnamate (ACA) and was named acet-
amidocinnamate acylase (ACA acylase).A similar, just as enantiounspecific acylase,
N-acetyldehydroleucine acylase (ACL acylase), catalyzing N-acyl hydrolysis of
branched-chain dehydroamino acids (N-acetyl-dehydrovaline, -1eucine and -iso-
leucine) was isolated and characterized from Zoogloea ramigera by Kittelmann and
Kular8'. 82]. The hydrolysis product in both cases, an enamine, first undergoes
754
Table 12.3-11. Comparison of the substrate specificityO f D-, and L-aminoacylasein Streptornyces
tuirus and Streptomyces olivaceus[28].
Relative activity
5. tuirus 5. olivaceus
Substrate D- L- D- L-
N-acetyl-phenylglycine 100 - 100

N-acetyl-leucine 1130 70 957 120
N-acetyl-phenylalanine 1004 8 723 15
N-acetyl-methionine 682 167 448 208
N-acetyl-tyrosine 522 0 307 2
N-acetyl-valine 314 29 261 61
N-acetyl-tryptophan 117 0 68 0
N-acetyl-alanine 102 38 69 92
N-acetyl-glutamicacid 69 0 38 10
N-acetyl-asparticacid 20 0 0 21
N-acetyl-arginine 14 32 5 72
N-acetyl-proline 0 0 0 0
a Not determined.
spontaneous rearrangement to the ketimine which is then deaminated sponta-

neously to the a-keto acid. The dehydroamino acid acylase reaction can be coupled
with reductive amination by amino acid dehydrogenases such as PheDH, LeuDH or
even AlaDH, respectively, to produce L-amino acids[821 (Fig. 12.3-5).
L-Phenylalaninehas been produced continuously from ACA with the help of ACA
acylase in an enzyme membrane reactor (EMR) with a space-time-yieldof 277 g L-'
d-'[831.With ACL acylase, L-Ieucine was produced at 123-180 g L-' d-' in the same
reactor set-up [82].The dehydroamino acid substrates can be prepared conveniently,
either from 2-halogencarboxylic acid esters [841,or, specifically in the case of ACA, via
the acetamidomalonic ester route by reaction with benzyl halogenides I*'].
Apart from the L-specific acylases from kidney and Aspergillus strains it has been
shown that similar aminoacylases are widely distributed in microorgan-
i s m ~ [ ~"1. ~ However,
- ~ ~ , from the viewpoint of costs, those acylases which are
practically employed for large scale industrial purposes, are restricted to the enzyme
from Aspergillus oryzae (see Sect. 12.3.7).
12.3.6
D-Specific Aminoacylases
D-Specific aminoacylases have been found in Pseudornonas sp. [33, 87-901 , Streptomyces
sp. [28] and Alcaligenes sp. [29-32, 'l]. The first investigations on the use of D-specific
8"
aminoacylases for the synthesis of D-amino acids were carried out by Kameda and
coworkers. They demonstrated that a strain of the genus Pseudomonas hydrolyzed N-
benzoyl-D-amino acids in addition to N-benzoyl-L-aminoacids [871. The partially
purified enzyme was employed to synthesize D-phenylalanine from N-benzoyl-D,L-
phenylalanine ["I and D-phenylglycine was synthesized from N-chloroacetyl-D,L-
phenylglycine with the crude enzyme preparation
Sugie and Suzuki conducted an extensive screening among soil samples as well as
Table 12.3-12.
12.3 Hydrolysis of N-AcylarninoAcids
Comparison of the substrate specificityof purified D-aminoacylasesfrom three

I 755
30v 321.
strains ofAlcaligenes SP.[~’*
Relative activity of strain
DA1 DA181 MI-4
Substrate D- L- D- L- D- L-
N-acetyl-methionine 100 100

N-acetyl-phenylalanine 65 80
N-acetyl-norleucine - 38 (DJ)
N-acetyl-leucine 52 17
N-acetyl-tryptophan 14 5
N-acetyl-alanine 14 1
N-acetyl-asparagine 8 0
N-acetyl-all0isoleucine - 1
N-acetyl-valine 6 1
N-acetyl-phenylglycine 3 0
N-acetyl-tyrosine - -
N-acetyl-asparticacid - 0 (DJ)
N-acetyl-glutamicacid - 0
N-acetyl-lysine - -
N-acetyl-arginine - -
N-acetyl-histidine - -
N-acetyl-serine - 0 (D,L)
N-acetyl-glycine - 0
N-chloroacetyl-phenylalanine - 68
N-chloroacetyl-norleucine - 66 (W)
N-chloroacetyl-isoleucine - 40 (D,L)
N-chloroacetyl-alanine - 38
N-chloroacetyl-valine 33 18
N-chloroacetyl-serine - 5 (DJ)
N-formyl-methionine - 56 (DJ)
N-formyl-phenylalanine - 35
N-benzyloxycarbonyl-methionine - 2
Glycyl-leucine
. . - 20
a Not determined.
among 420 strains of the genus Streptomyces and 16 strains of the genus Streptoverti-
cillium from type culture collections and isolated four Streptomyces strains producing
a D-specific aminoacylase suitable for the production of D-phenylglycine[”I. Since
the bacteria also produced an t-aminoacylasethe D-aminoacylase had to be separated
from the L-specific enzyme by ion exchange chromatography prior to use. Thus, D-
phenylglycine could be produced from N-acetyl-D,L-phenylglycinein 99.9 % optical
purity. Table 12.3-11lists the substrate specificity of the D- and L-aminoacylases from
two Streptomyces species.
Microbial D-aminoacylases have also been found in different species and strains of
the genus Alcaligenes. The enzyme has been isolated, purified and characterized
from Alcaligenes denitii..cans subsp. x y l o s ~ x y d a n s [30,
~ ~321,, Alcaligenes denitnfcans[”]
and Alcaligenes f a e ~ a l i s [ ~Several
~]. companies, all of them Japanese, have filed
applications for D-aminoacylases The substrate specificity of the D-
aminoacylases from these strains is shown in Table 12.3-12.
756
Table 12.3-13. Enantioselective deprotection of N-protected D,L-aminoacids by D-aminoacylase

from Alcaligenesfaecalis DA-1
Substratea Reaction time Conversion e e of D-amino

(h) (W acid (77)
N-Ac-D,L-methionine 2 50.0 100
N-Ac-D,L-methionine(in 50% DMSO) 15 53.0 30
N-Ac-D,L-leucine 2 49.3 100
N-Ac-D,L-leucine(in 50% DMSO) 15 30.7/48.9 100
N-Ac-D,L-phenylalanine 2 49.9 100
N-Ac-glycine 2 10 -
N-n-Butyl-D,L-methionine 2 45 100
N-Bz-D,L-methionine 10 47.2 89
N-Bz-D, L-leucine 10 48.1 99
N-Bz-D,L-phenylalanine 10 50 100
N-Bz-D,L-norleucine 10 43.9 53
N-Bz-~,~-2-amino-n-butyric
acid 10 33.8 80
N-Z-D,r-methionine 10 32.6 99
N-Z-D,r-leucine 10 32.6 100
N-Z-D,L-norleucine 10 12.8 51
N-Z-~,~-2-amino-n-butyric
acid 10 15.8 77
a Ac; acetyl; Bz; benzoyl; 2; benzyloxycarbonyl.
As with the D-aminoacylases from Streptomyces sp. the enzymes from Alcaligenes
strains have a preference for hydrophobic N-acetyl-aminoacids. In this respect, they
are similar to the L-specific acylase I from kidney preparations and Aspergillus sp. The
Alcaligenesfaecalis enzyme prefers the N-acyl-D-aminoacid derivatives from Met, Phe
and Leu ["I. If a high-affinity substrate residue occupies the hydrophobic side-chain
pocket the enzyme even deacylates D-Met methyl esters or N-Ac-D-Met-Xaadipeptide
derivatives.
Two D-aminoacylases have been described that resemble the L-specific acylase I1
from kidney, which only hydrolyzes the N-acyl derivatives of L-aspartic acid. The D-
specific counterpart of acylase 11, N-acetyl-D-aspartate deacetylase, has been isolated
from Alcaligenes xylosoxydans subsp. xylosoxydans L3lI. The same strain produces an
aminoacylase which specifically hydrolyzes N-acyl derivatives of D-glutamic acid [311.
The latter N-acetyl-D-glutamatedeacetylase has also been found in Pseudomonas
sp. (331.
All microorganisms producing D-aminoacylases commonly produce L-aminoacy-
lases as well. Therefore, to reach high optical purity of the D-amino acids produced
from the respective N-acetyl-D,L-amino acids, the D-aminoacylases have to be
separated from the r-aminoacylases (Table 12.3-13). However, this is a disadvantage
in view of an industrial application since additional purification steps lead to more
expensive enzymes and thus add costs to the whole production process. This is one
of several reasons why it is widely accepted today that the production of D-amino
acids by enzyme-catalyzedhydrolysis of D,r-hydantoinsseems to be more promising
than the ~-aminoacylaseroute via N-acetyl-D,L-aminoacids. The enzyme-catalyzed
synthesis of D-amino acids from the respective D,L-hydantoins is described in
Chapter 12.4.
Proteinogenicamino acids
Non-proteinogenic amino acids

I
757
Y O O H
Alanine qCooH a-Aminobutyric acid
NH2 NH2
WHrooH yCooH
/
Phenylalanine
NH2
Norvaline
/tCOOH
Valine TcooH
Norleucine
NH2 NH2
Leucine ~ 0 ~ " " " 0-Benzylserine

"
/ NH2
Methionine o""'ycooH S-Benzylcysteine

/ NH2
Q-J-yCOOH Tryptophan
NH2
N
H
HO mHYH
Figure 12.3-6.
Tyrosine
L-amino acids prepared in bulk quantities by acylase I resolution

of N-acetyi-DL-aminoacids.
12.3.7
Acylase Process on a Large Scale
The most established method for enzymatic L-amino acid synthesis is the resolution
of racemates of N-acetylaminoacids by acylase I from Aspergillus o r p e fungus. The
N-acetyl-r-aminoacid is cleaved to yield L-amino acid whereas the N-acetyl-D-amino
acid does not react. After separation of the L-amino acid through ion exchange
chromatography or crystallization, the remaining N-acetyl-D-amino acid can be
758
racemized by acetic anhydride in alkaline solution or by adding a racema~e['~] to

achieve very high overall conversions into the L-amino acid. N-acetyl-D,L-aminoacids
are conveniently accessible on a laboratory as well as an industrial scale through
acetylation of w-amino acids with acetyl chloride or acetic anhydride in a Schotten-
Baumann reaction['8]. As was demonstrated in the synthesis of 13C-~-methionine,
the acylase process has a virtually closed material balance because almost 99.5 % of
the amino acid components can be retrieved after
The acylase is relevant for enzyme reaction engineering along two different lines
as follows. With the aminoacylase process, Tanabe Seiyaku commercialized the first
immobilized enzyme reactor system ever in 1969 after running the process in batch
mode since 19541'. '1. Enzyme from Aspergillus oryzae fungus was immobilized by
ionic binding to DEAE-Sephade~[~l. In a fued-bed reactor, the reaction is carried out
at elevated temperature to produce r-methionine, L-valine, and L-phenylalanine.
Costs are significantly lower than in a batch process with native enzyme. Tanabe
started up more fixed-bed reactor processes with immobilized enzymes: L-aspartic
acid with aspartase in 1973 and L-malic acid with fumarase one year
At Degussa, several enzyme membrane reactor (EMR) set-ups are in operation
covering six orders of magnitude from laboratory via pilot stage to full production
scale; the process has been scaled up to an annual production level of several 100
tons of enantiomerically pure a-amino acids, mostly L-methionine and 1-valine['*I
(Fig. 12.3-6).The enzyme membrane reactor is a recycle reactor operated as a CSTR
with a recycle ratio Frecycle/Finflux of up to 200. For both pilot and large-scale
operation, the necessary membrane area is configured into polysulfone hollow-fiber
modules with a molecular-weightcut-off of 10 kDa resulting in a rejection rate of the
73 kD-acylase far in excess of 99.9%.
Aminoacylase has also been immobilized on a nylon membrane ["I. While the
half-life as measured by thermal stability, of 161 d is superior to the data for
immobilized acylase (65d) 1'1 or soluble enzyme in an EMRr'l, reactor productivity at
0.136 L-valine kg/L-'d-' is lower than that for DEAE-Sephadex-immobilizedacylase
(0.5 kg/L-'d-')['] or that for a membrane reactor (0.35 kg/L-'d-l)['].
Results on operational stability of both acylases in a recycle reactor at constant
conversionF41 with reaction conditions close to intended large-scale conditions
demonstrated much better stability of the Aspergillus enzyme, while renal enzyme is
not stable enough for long-term operation [64.651. Moreover, on the process scale
achieved today the supply of renal acylase is insufficient, so that fungal acylase is
used almost exclusively nowadays, especially since the price per unit is comparable.
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12.4 Hydrolysis and Formation ofHydantoins I

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12.4
Hydrolysis and Formation of Hydantoins
Markus Pietzsch and Christoph Syldatk
12.4.1
Classification and Natural Occurrence of Hydantoin Cleaving and Related Enzymes
Abbreviations
Cit citrulline
DTT dithiothreitol
EDTA ethylenediaminetetraaceticacid
HIC hydrophobic interaction chromatography
MTEH methylthioethylhydantoin
IEX ion exchange chromatography
IMH indolylmethylhydantoin
Phg phenylglycine
SEC size exclusion chromatography
Thienylala thienylalanine
0-Me-Ser 0-methylserine
The compound hydantoin was discovered by von Baeyer in 1861 by reduction or

hydrogenation of allantoin, which is a naturally occurring cyclic amide in many
plants [l]. The systematic terms for “hydantoin” are “imidazolidine-2,4-dione” or
“2,4-diketotetrahydroimidazole”.In the literature, a wide spectrum of various
5-mono- and 5,5’-disubstitutedhydantoin derivatives of industrial and pharmaco-
logical interest is described, of which 5-monosubstituted hydantoins may be re-
garded as cyclic ureides of a-amino acids. They are obtained by Strecker synthesis
and are important precursors, e. g. in the industrial production of D,L-a-aminoacids.
The 5,5’-disubstitutedhydantoin derivatives have been of pharmacological interest
since the 1930s,e. g. for the treatment of Parkinson’s disease. Figure 12.4-1gives a
survey of the different methods for the chemical synthesis of hydantoins. Detailed
reviews on their chemical syntheses and applications are given in references[*]
762
R-CHO + HCN
+
(NH,),CO,
Strecker Synthesis
R’YcooE:
NCO
II
0
R-CHO +
H,N
,f(, NH,
+ CO n=O.1
* = Lor D or D.L
Figure 12.4-1. Chemical syntheses of hydantoins.
and13],on their structures in solution, and in the solid state in With the
increasing interest in new amino acid derivatives, recent investigations on their
chemical synthesis concentrates on the development of “one-pot-syntheses”of the
corresponding hydantoin derivatives, e. g. by carbonylation of aldehydes in presence
of urea derivatives[’I.
Many of the hydantoin derivatives are substrates for enzymatic reactions. It has
been known since the 1940s that some microorganisms are able to grow on D,L-
5-monosubstituted hydantoins as the sole C- and/or N-source in a mineral salt
medium, often hydrolyzing only one enantiomer of a racemic mixture, and that even
enzymes from plant and animal sources are able to hydrolyze and close the
hydantoin ring. Various enzymes, so called hydantoinases, facilitate the hydrolysis of
the hydantoin ring system in an initial reaction step. The biosynthesis of these
enzymes often has to be induced by adding specific compounds during the growth of
the microorganisms. The so-formed hydantoinases may have different substrate
specificities and in general are selective in forming L- or D-N-carbamoylamino acids
(= hydantoic acids). The hydantoinases can often be found in combination with
highly stereoselective N-carbamoylamino acid amidohydrolases (N-carbamoylases),
which catalyze the further hydrolysis of the hydantoic acids to the free amino acids in
an irreversible reaction. In some cases a hydantoin-racemase is involved as a third
enzyme. Together, these three enzymes accomplish the total conversion of racemic
~,~-5-monosubstituted hydantoin derivatives into the corresponding enantiomer-
ically pure D- or L-amino acids. This cascade of reactions, whether located in whole
cells or carried out using isolated enzymes is called the “hydantoinase-process”.
72.4 Hydrolysis and Formation of Hydantoins
I
763
L-specific
E
-
a
@ 4
(L-carbamoyl-aminoacid I Il-hydantoin( IPhydantoinI ID-carbamoyl-aminoacic
dH b a
R ~ c o o CO2,NHa m o y l a s e l
C02,NHs N"t
[ L-amino acid1 ID-amino aid1
Figure 12.62. Reaction scheme for the enzymatic cleavage of o,L-S-monosubstituted

hydantoin derivatives to the corresponding D- or L-amino acids.
Figure 12.4-2 shows the general reaction scheme for the enzymatic cleavage of D,L-
5-monosubstituted hydantoin derivatives to the corresponding D- or L-amino acids.
The great advantages for industrial use of the hydantoinase-process are based on
the fact that potentially 100% conversion and a 100% optically pure amino acid can
be obtained at the same time if a racemic substrate is used. Until the mid 1990s in
most cases, wild type strains, resulting from traditional screening methods (for a
review see: reference L6]), were used as whole cell biocatalysts. Detailed reviews on the
use of free or immobilized whole cell systems for hydantoin cleavage were given in
references [3, '. 1'. More recent activities are summarized in this chapter and concen-
trate on the use of recombinant free or immobilized enzymes (see Sect.
12.4.2-12.4.G), fusion proteins (see Sect. 12.4.7), specially designed recombinant
whole cell biocatalysts (see Sect. 12.4.4) or the optimization of enzyme properties by
directed evolution (see Sect. 12.4.7).
The hydantoinases belong to the E.C. 3.5.2 group of cyclic amidases"], which is
shown in Table 12.4-1. Of this group, four enzymes are original hydantoinases,
because their substrates are naturally occurring hydantoin derivatives: carbox-
ymethylhydantoinase (E. C. 3.5.2.4), allantoinase (E. C. 3.5.2.5), l-methylhydantoi-
nase (E. C. 3.5.2.14), and carboxyethylhydantoinase. All other enzymes listed have
natural occurring cyclic amides as substrates (e.g. barbiturate, 5,G-dihydrouracil,
5,6-dihydroorotate).
From recent investigations on DNA- and amino acid sequences of the different
cyclic amidases and subsequent phylogenetic analyses, it is known today that most of
these enzymes not only share a number of highly conserved regions and invariant
amino acid residues["], but form a protein superfamily and are the product of a
divergent evolution["]. Although most of them only share limited sequence homol-
ogy (identity < 15%) and therefore are only distantly related, it can be shown:
764 I 12 Hydrolysis and Formation ofC-N Bonds
Table 12.4-1. Hydantoinases and cyclic arnidases[’].

Recommended name Other names Systematic name E. C.-number
Barbiturase barbiturate amidohydrolase 3.5.2.1
Dihydropyrimidinase D-hydantoinase 5,6-dihydropyrimidine 3.5.2.2
amidohydrolase
Dihydroorotase carbamoylaspartic acid ~-5,6-dihydro-orotate 3.5.2.3
dehydrase amidohydrolase
Carboxymethylhydan- ~5-carboxymethylhydantoin 3.5.2.4
toinase amidohydrolase
Allantoinase allantoin amidohydrolase 3.5.2.5
Penicillinase p-lactamase, penicillin amido-a-lactam 3.5.2.6
Cephalosporinase hydrolase
Imidazolone propi- 4-imidazolone-5-propionate 3.5.2.7
onase amidohydrolase
5-Oxoprolinase pyroglutamase 5-oxo-~-proline 3.5.2.9
(ATP-hydrolyzing) amidohydrolase
Creatininase creatinine amidohydrolase 3.5.2.10
r-Lysine-lactamase 3.5.2.11
6-Aminohexanoate-cyclic 3.5.2.12
dimer hydrolase
2,s-Dioxopiperazine 3.5.2.13
hydrolase
1-Methylhydantoinase 1-methylhydantoin 3.5.2.14
(ATP-hydrolyzing) amidohydrolase
Carboxyethylhydantoinase L-5-carboxyethylhydantoin
amidohydrolase
Indolylmethylhydanto- 5-indolylmethylhydantoin
inase amidohydrolase
1. that most of them are members of a broad set of amidases with similarities to
ureases and build u p into a protein superfamily[’’, ‘*I, whereas
2. the ATP-dependent hydantoinases (see Fig. 12.4-3)are not related, and
3. that they share a metal-binding motif consisting of conserved histidine residues,
which seems to have a n important role to play i n structure and activity“’. “8 131.
The differences i n enantioselectivity, often used for the classification of hydantoi-

nases based o n their biotechnological value, therefore do not reflect the evolutionary
relationship of the different hydantoinases, which are forming a more diverse group
of enzymes than was assumed earlier (for more details see reviews : referencesIl4I
and [131). This protein superfamily probably has its origin i n the prebiotic conditions
of the primitive earth, where N-carbamoyl-u-amino acids rather than free a-amino
acids are supposed to be the first synthons for prebiotic peptides i n the evolution
today[”].
This section will have a detailed look at the occurrence of the different cyclic
amides i n nature and their physiological role i n various metabolic pathways.
Allantoin is widely distributed in nature and is a n important metabolite in the
degradation of purine nucleotides (see Fig. 12.4-4).Allantoin occurs i n all organisms
that do not have uric acid as the final product of their purine degradation pathways,
and is the substrate for the enzyme allantoinase or 5ureidohydantoinase (E. C.
72.4 Hydrolysis and Formation ofHydantoins I
765
-
Relations based on:
Structure homology A Sequence homology
Superfamily of 'Amidases involved Family of:
I
in nucleotide metabolism' ATP-dependent cyclic amidases'
Hydantoinase no relationship N-Methyihydantoinase

from Arthrobacteraursscens DSM 3145
Dihydropyrimldlnase L-Oxoprolinase
Allantolnase
Dihydroorotase
urease
others: Adenine deaminase NOsequence information available

Adenosine deaminase about:
Aminoacylase
AMP deaminase
Aylphosphatase
Cytosine deaminase
Chlorohydrolase
Formylmethyldehydrogenase Carboxyethylhydantoinase
imidazolonepropionase
Phosphotriesterase
Figure 12.4-3. The evolutionary relationship of hydantoinases derived from sequence

and structural similarity. Enzymes i n bold letters are h y d a n t ~ i n a s e s " ~ ] .
3.5.2.5), which can be found in microorganisms, plants and animals, either in

combination with an allantoicase (E. C. 3.5.3.4) or an allantoate amidohydrolase
(E. C. 3.5.3.9). The latter hydrolyzes allantoin to urea and glyoxylic acid, which are the
final products of purine degradation in fishes. A recent paper describes the
purification of this enzyme from Bacillusfastidiosus[l6].
In the 1960s, different groups[17.1" described the microbial enzyme as inducible
and (+)-specific.Besides allantoin other inducers are compounds with a free ureido
group such as N-carbamoyl-L-asparagine,N-carbamoyl-L-aspartate(the correspond-
ing D-compounds were ineffective), hydantoate (i.e. N-carbamoylglycinate) and
diureid~methane[~']. Information on the substrate specificity of allantoinases for
other hydantoin derivatives is limited but D,L-S-arninohydantoin was shown to be
accepted, albeit poorly, as a substrate I2O]. Non-stereoselectiveallantoin hydrolysis and
association of the allantoinase with a cofactor-independentallantoin racemase (E. C.
5.1.99.3) has been reported[20,211, so that some microorganisms are also able to use
(-)-allantoinas a substrate. An excellent review of these purine as well as pyrimidine
degrading enzymes was given by Vogels and van der Drift[22].
The natural function of the carboxymethylhydantoinase(E. C. 3.5.2.2) is postulated
to be the hydrolysis of 5-carboxymethylhydantoin,which is described to be the
product of a non-enzymatic cyclization of N-carbamoyl-L-aspartic acid [23, 241 and to
occur as a side-productin the metabolism of the pyrimidine nucleotide dihydroorotic
acid[25].This enzyme often occurs in combination with a ureidosuccinase (E.C.
3.5.1.7)["I, which catalyzes the cleavage of the resulting N-carbamoyl aspartic acid to
L-aspartic acid (see Fig. 12.4-5). L-5-Carboxymethylhydantoinwas first isolated after
incubating orotic acid, a six-membered cyclic amide, with crude cell extracts of the
anaerobic bacterium Clostridium oroticum 12', 26].
766
A third naturally occurring hydantoin, ~-S-carboxyethylhydantoin, was first iso-

lated by Brown and Kies[271from the urine of rats, monkeys and humans after being
fed '4C-histidine, and it was postulated to be a by-product in the histidine degrada-
tion pathway shown in Fig. 12.4-6. A k a m a t ~ u [proved,
~ ~ ] by induction experiments,
that the L-carboxyethylhydantoinasefrom a Bacillus brevis strain, also described by
Adenine Guanine
0 0
bNA" Xanthine
H dehydrogenase
Hypoxanthine Xanthine
+H201
2 [HI
Xanthine
dehydrogenase
Uric acid
Oz+ 2Hz0
Uricase
C 0 2 + H,O,
H2N
o""0
HNKNH
0
(-)-Allantoin
- Racemase
Allantoin-
H2N
p)-,fo
HNKNH
0
(+)-Allantoin
Allantoinase
NH3+ CO, H,O
72.4 Hydrolysis and Formation ofHydantoins
I
767
A Hz:”FCOOH
1
Ureidoglycine 0
Allantoic acid
A = Allantoate
amidohydrolase
NH3 Hzo Allantoicase
urea
S-Ureidoglycolicacid
R-Ureidoglycolicacid
R-Ureidoglycolase
or Allantoicase \ H
OACOOH
I ~
2 NH, + CO,
Urease or
Allophanate pathway
Figure 12.4-4. Purine degradation pathway via allantoin i n
Tsugawa et al. 12’] and Hassall and GreenbergL2’I for the formation of L-glutamic acid
from ~,~-S-carboxyethylhydantoin, was not able to hydrolyze L-carboxymethylhy-
dantoin and consequently it is not identical to the former enzyme described above.
This enzyme has no E. C. number at present.
The six-membered ring systems 5,G-dihydropyrimidine, 5,G-dihydrouracil and
5,G-dihydrothyminecan be hydrolyzed by the enzyme dihydropyrimidinase (E. C.
3.5.2.2),which is involved in the degradation of pyrimidine nucleotides. This widely
spread, inducible catabolic enzyme is strictly D-selective in contrast to the L-selective
dihydroorotase (E. C. 3.5.2.3),which is involved in the opposite anabolic pathway (see
above). Another name often used in the literature for the dihydropyrimidinase is D-
hydantoinase, because it is also able to hydrolyze ~,~-S-monosubstituted hydantoin
derivatives with high activity. Both reactions are shown in Fig. 12.4-7.
Natural cyclic amides such as 5,G-dihydrouracil, uracil and 5,G-dihydrothymineas
well as hydantoin, 5-methylhydantoin and 5-hydroxymethylhydantoinare effective
inducers for enzyme biosynthesis (for a more detailed review on induction experi-
ments see referencef3I).In some cases, the dihydropyrimidinase (D-hydantoinase)is
associated with an N-carbamoyl-D-aminoacid amidohydrolase (D-carbamoylase)and
a hydantoin racemase [301. The previously proposed identity of the D-N-carbamoylase
with the P-ureidopropionase(E. C. 3.5.1.G),which was assumed to be responsible for
the hydrolysis of N-carbamoyl-P-alanine (see Fig. 12.4-7)[31-351 is no longer valid
since the investigations of Ogawa et al. on different aerobic bacteria showed that the
768
HN 0,
H
0AOrotic
2 Cacid
O O H 1 i
Methylene blue
f...',. Cytochrome C
L-5,6-Dihydroorotic acid
Carboxymethyl-
hydantoinase
Hooc-)---COOH HZ0
Hooc-kf
HN K0
NH2 '6
Non enzymatic
HNYNH
0
N-Carbamoyl-L-aspartic acid cyclization L-5-Carboxymethylhydantoin
NH, + CO,
H20 i Ureidosuccinase
L-Aspartic acid
Figure 12.4-5. Metabolism of orotic acid and dihydroorotic acid[22.241.
I 769
L-Histidine Urocanate
H+NH
A
Hooc-fcooH H2O Hoocw HN\//N
L-Formiminoglutamicacid L-4-Imidazolone-5-propionicacid
L-5-Carboxyethylhydantoin
J.
HooC-YCooH
0N H z
HNK
NCarbamoyl-glutamic acid
Figure 12.4-6. Histidine degradation pathway and carboxyethyl hydantoinase-catalyzed
reaction.
770
"w"
""KNH 0
5,6-Dihydrouracil D,L-5-rnonosubstituted
hydantoin
("'
5,6-Dihydropyrirnidinase
or
" D-Hydantoinase"
RvCOOH
P C O O H
HN
0 KNH2
0
P-Ureidopropionic acid NCarbarnoyl-D-amino acid
Figure 12.4-7. Analogy between dihydropyrirnidinase- and D-hydantoinase-
catalyzed reactions.
L-specific carbamoylase from Pseudomonas putida I F 0 12 996 also hydrolyzes p-

ureidopropionate[14, 3 G ] . The enzyme from Pseudomonas putida IF0 12996 was
shown to be strictly L-selective and to be active on L-N-formyl-and also on L-N-acetyl-
alanine13']. In this context it may be of interest that Runser and Meyer described a D-
hydantoinase with no dihydropyrimidinase activityL3'] and Ogawa et al. reported on
the occurrence of a D-N-carbamoylasewith no relation to a ~-hydantoinase[~*~.
Nevertheless, the dihydropyrimidinase seems to be closely related to the barbitur-
ase (E.C. 3.5.2.1), which is able to hydrolyze barbituric acid[39](Fig. 12.4-8).
The difference between barbituric acid and the natural compounds uracil and
thymine is the presence of a keto-group instead of a methyl- or a hydrogen-group in
the 6-position of the ring. Barbiturase was first detected by Hayashi and K ~ r n b e r g r ~ ~ ]
in bacteria of the genera Mycobacterium and Corynebacterium and postulated to
catalyze a sidereaction in the degradation of pyrimidines. Unfortunately, there are no
further data in literature on the substrate specificity and the stereoselectivity of this
enzyme, which would allow comparison with the D-hydantoinase, but Kautz and
Barbituric acid Malonic acid Urea

Figure 12.4-8. Barbiturase catalyzed reaction[39].
ATP
I 771
rCOOH
H3CyN).( NH2
1 -WMethyl-
NH 0 hydantoinase 0
Creatinine I -NMethylhydantoin NCarbamoylsarcosine
NCarbamoyl-
sarcosine-
hydrolase
CO, + NH,
rCOOH
H
H3C0N)(NH2 C
O
H, N
,,
H3C
NH
Creatine Sarcosine
Figure 12.4-9. 1-Methyl hydantoinase- and N-carbamoylsarcosine-amidohydrolase-catalyzed

reactions in creatinine metabolism in bacteria.
Schnackerz were able to show that beef liver dihydropyrimidinase is also able to
hydrolyze barbituric acid, although only with low activityIm1.
Two other hydantoinases are described in the literature, which have not yet been
listed in the Enzyme Nomenclature[’]. Siedel et al.L4l],Yamada et al.[42,431 and
Ogawa et al. found a new ATP-dependent 1-methylhydantoinasewith additional
nucleoside-triphosphatase activity [451 in different bacteria. This inducible enzyme,
which was also shown to act on unsubstituted hydantoin and 5-methylhydantoin14’1,
is involved in the degradation of creatinine after its deimination in the 2-position to
I-methylhydantoin, resulting in N-carbamoylsarcosine (N-carbamoyl-N-methylgly-
cine) [42,431 (see Fig. 12.4-9).It is associated with a so-called D-N-carbamoylsarcosine
hydrolase [431, which eventually hydrolyzes N-carbamoylsarcosine to free sarcosine.
Both enzymes can be used for monitoring creatinine levels in blood L4l].
Nishida et al.[46],Syldatk et al.l4’. 481, Yamashiro et al.L4’, ”I, and Yokozeki et
al. [51-531 found new L-5-arylalkylhydantoinases and a N-carbamoyl-L-aminoacid
amidohydrolases (L-N-carbamoylase),which are involved in the L-selective cleavage
of 5-arylalkylhydantoinsand could be most favorably induced by D,L-S-indolylme-
thylhydantoin or its N-3-methylated derivative(1’. The natural functions of these
enzymes are not yet known, while one of the associated N-carbamoyl-L-amino acid
amidohydrolases (L-N-carbamoylase)was also shown by Syldatk et al. to be reactive
on N-formyl-L-aminoacids [541. In this strain both, hydantoinase and L-N-carbamoy-
lase were shown to occur in combination with a hydantoin racemase[’, 55. 561.
Resting cells were used for the industrial production of L-amino acids from D,L-
5-monosubstituted hydantoin derivatives as shown in Fig. 12.4-2LS71.
Concerning their structure, cyclic imides are closely related to dihydropyrimidines
and hydantoins. The metabolic transformation pathway for cyclic imides in micro-
organisms (see Fig. 12.4-10)was studied by Ogawa et al.[”, I’ in Blastobacter sp. and
772
lrnidase
I
HOOC
nCOOH
Succinate
I TCA cycle
Acetyl-CoA - COOH
Pyruvate
in different aerobic bacteriaL6']. The enzyme involved in this reaction, a so called

imidase, was also found to hydrolyze dihydropyrimidines [I4].
Activity for the enzymatic cleavage of disubstituted hydantoins useful in the
synthesis of a-,a-disubstituted amino acids was recently detected in crude enzyme
extracts from the plant Lens esculenta[6'. 62] and in papain by Rai and Taneja[63].
Of all the enzymes described above, at present only the D-hydantoinase- and the L-
arylalkylhydantoinase processes are of significance for use in organic synthesis, in
particular for the production of natural and non-natural optically pure D- and L-
amino acids, and will be discussed in more detail in the following sections.
12.4.2
I 773
D-Hydantoinases- Substrate Specificity and Properties
Since the early 1950s it has been known that the inducible catabolic enzyme
dihydropyrimidinase (E. C. 3.5.2.2) plays an important role in pyrimidine metabo-
lism[23s31, 33, 39, G4-GG] and is widespread in nature. The natural substrates of this
enzyme, which were also reported to be inducers, are 5,G-dihydrouracil and 5,G-dihy-
drothymine. Both compounds are important intermediates in the degradation of
pyrimidine nucleotides. The dihydropyrimidinase-reactionis described to be strictly
D-specific and to have a wide substrate specificity (see Fig. 12.4-11). In 1970 and
-0
do -sw
HNYNH HNYNH
0 0 HNYNH
0
H O
""K""0
W \ O
,"YE
0
h
* ""YNH
0
C I
HNYNH0
W
Q
O W
""YNH0
0
%-)+
""IfE
0
w
""If"" 0
O
\>O
w
""K""
02"W
""If"" ""If""
/
""YNH
0
0
0 ' ""f 0
9;
0
Q Q
0$ 0 oA,Xo O++O oA,Xo
H H H
JX0 ox>o H
Figure 12.4-11.Substrates accepted by different
D-hydantoinase preparations from mammalian and
microbial "1.
774
1973, Dudley et al. were the first to publish on the D-selective cleavage of 5-phenyl-
hydantoin to N-carbamoyl-D-phenylglycineby a mammalian enzyme and on the
spontaneous in vivo racemization of the residual isomer['^, "1. In 1975, Cecere et
al."1' published on the enzymatic production of other N-carbamoyl-D-aminoacids
starting from chemically synthesized ~,~-5-monosubstituted hydantoin derivatives
using a partially purified fraction of the dihydropyrimidinase from calf liver. They
were the first to stress that this enzyme might find an industrial application for the
preparation of optically active D-amino acids as the so called "D-hydantoinase" (see
Fig. 12.4-7). In 1978, the same group published on the production of various N-
carbamoyl-D-amino acids using an immobilized calf liver dihydropyrimidinase
preparation[70r711. Other publications have reported on the occurrence of D-hydan-
toinases in plant cell cultures[72].Rai and Taneja published on the use of a plant
enzyme from Lens esculenta immobilized to DEAE-cellulose for the same pur-
In other publications, Wallach et al. ["I, Brooks et al. [731 and Kautz and Schnack-
erz14'] gave detailed reports on the isolation and characterization of the dihydro-
pyrimidinase from beef liver. Table 12.4-2 gives a short overview of the purification
procedures and characteristic properties of these mammalian enzymes. The beef
liver dihydropyrimidinase consists of four subunits and every active enzyme mole-
cule contains four Zn(")~ations[~~] which are tightly bound (& > 1.33 x lo' M-'). In
addition to 5,G-dihydrouracil, glutarimide, thiohydantoin and barbituric acid are also
accepted as substrates, but with low reaction rates I4O1.
In the late 1970s the group of Yamada et al. in Japan postulated that in
microorganisms the reason for the wide spread ability to hydrolyze D-selectively D,L-
5-monosubstituted hydantoin derivatives was the existence of an enzyme called "D-
h y d a n t o i n a ~ e " 751.
[ ~ ~With
~ the increasing interest in the production of D-phenyl-
glycine and D-p-OH-phenylglycine,since then several publications have described D-
selective hydantoinases isolated from various microorganisms as Pseudomonas
~triata[~'], Pseudomonasfluorescens DSM 84["], Pseudomonas sp. AJ-l1220[35], Arthro-
bacter crystallopoietes AM2[77],Agrobacterium sp. IP-I 671[37. 781, in anaerobic micro-
organism~[~'], Pseudomonas sp. KBEL 101["], Agrobacterium turnefaciens["I, thermo-
philic microorganisms Pseudomonas d e s m o l y t i ~ u m [ ~Bacillus
~], sp, Cs41, Bacillus
stearothermophilus SD-1 Is', "1 and Bacillus circulans LS71. Runser and co-workers
described a D-hydantoinase of an Agrobacterium sp. with remarkably high tem-
perature and pH stability but no dihydropyrimidinase "1. Soong et al.
were recently able to show that D-hydantoinase from Blastobacter sp.Al7p-4 also is
able to hydrolyze cyclic imides with bulky substituents to the corresponding half-
amides and postulated that this enzyme may also function in cyclic imide metabo-
lism in addition to pyrimidine metabolism ["I. New screening methods for isolation
of n-hydantoinase-producing microorganisms were described by Morin et al. using a
continuous cultivation systemI,'[ and by LaPointe et al. using a polymerase-chain-
reaction-amplifiedDNA probe to detect D-hydantoinase-producingmicroorganisms
by direct colony hybridization['l].
A survey of the isolation and some characteristic data on some of the bacterial
enzymes, which seem to be rather similar to the dihydropyrimidinases from
mammalian tissues (Table 12.4-2) and plants, is given in Table 12.4-3.
Table 12.4-2. Purification and characteristicproperties of D-hvdantoinasefrom animal cells

I
775
Source Acetone powder Catalase fraction Acetone powder

from beef liver from beef liver from beef liver
Reference WI [731 ~401
Purification steps acid and heat hydrophobicchroma- heat treatment,

treatment, ammonium tography or prepara- ammonium sulfate
sulfate and acetone tive electrophoresis precipitation,chro-
precipitation matography on
chelating and DEAE-
Sepharose
Yield (%) 25 13 44
Purification factor 200 24.2 186
Purity 80 % homogeneous homogeneous
Optimal pH 8.2 no data given 8-10
Metal ion requirements Mn2’ and Mg2+(only Zn2+and Co2+ one Zn” per subunit
when dihydrouracilis
the substrate!)
Molecular mass 226 000 Da 217 000 Da
Subunits 4x56 500 Da 4 x 5 4 000 Da
Figure 12.4-11 gives a survey of the substrates accepted by the different dihy-
dropyrimidinase or n-hydantoinase preparations The differences between the en-
zyme preparations from mammalian and microbial sources are discussed in more
detail in reference L3], but D-hydantoinases or dihydropyrimidinases, respectively,
seem to have the following in common: (i) a wide substrate specificity, (ii) metal
dependence and (iii) that they are strictly D-specific. Preferably, cyclic amides are
hydrolyzed at pH values around 8.5. Furthermore, most of the enzymes are also
described to be able to catalyze the hydantoin formation: the optimal pH of this
reaction is neutral or weakly acidic.
In 1983 the first gene sequence of a D-hydantoinase derived from thermophilic
Bacillus sp. LU 1220 and its overproduction in Escherichia coli HB 101 was
published[”]. Not until 1994 were cloning, sequencing and expression of a D-
hydantoinase gene from Pseudomonas putida DSM 84 in Escherichia coli reported[”],
shortly followed by a paper on cloning, sequencing and expression of a thermostable
D-hydantoinase from Bacillus stearothermophilus NS 1l22A[”4]. The same was de-
scribed for the strain Bacillus stearothermophilus SD-1 by Lee et al. in 1997[951.The
same group reported that the C-terminal region of the D-hydantoinase was not
essential for catalytic activity but affected the oligomeric structure of the
In 1998, Chien et al. described the cloning, sequencing and expression of the D-
hydantoinase gene from Pseudomonas putida CCRC 12857 in Escherichia coli[”].
Molecular cloning and sequencing of a cDNA encoding dihydropyrimidinase from
rat liver was reported by Matsuda et al. [981, and the complete sequencing of a 24.6 kB
segment of yeast chromosome XI including homologies to D-hydantoinases by
Tzerma et al. “J91.
D-Phenylglycine and n-p-OH-phenylglycineare important side chain moieties in
the synthesis of semisynthetic penicillins and are produced in several thousand tons
per year using the hydantoinase loo].The different methods that this
776
I
HO
1
D,L-5-pHydroxyphenylhydantoin - co,
Snamprogetti-Process Kaneka Process

1 ’ + HO
,
Recordati-Process
- Immobilized dihydropyrimidinase - Immobilizedresting cells of - Immobilized resting cells of
from calf liver Bacillus brevis with Agrobacteriurn radiobacter with
D-hydantoinaseactivity D-hydantoinaseand L-Ncarba-
moylase activity
- Reaction conditions: pH 8.0, 30°C - Reaction conditions: pH 9.0, 30°C - Reaction conditions: pH 9.0, 30°C
I HO
NGarbamoyl-D-p-hydroxyphenylglycine
D-p-Hydroxyphenylglycine
Figure 12.4-12. Industrial production of D-4-hydroxyphenylglycine acids

by t h e D-hydantoinase process.
reaction has been realized in industrial application in recent years can be seen in
Fig. 12.4-12.
In the 1970s, the company Snamprogetti first reported on the use of the beef liver
dihydropyrimidinase immobilized on an ion exchanger for the continuous produc-
tion of D-phenylglycine[70, 71], while the company Kanekafuchi was reported to use
I 777
resting cells of a Bacillus sp. containing D-hydantoinaseactivity only[’001.Because of
missing D-N-carbamoylase activity or the instability of this enzyme in resting
microbial cells, the decarbamoylation of the resulting D-N-carbamoylaminoacid is
often performed chemically by treatment with HN02. Because of the high stability of
the D-hydantoinase it is possible to use immobilized resting cells, which can be
applied repeatedly.
With the increasing interest in products other than D-phenylglycine and D-POH-
phenylglycine, the companies Recordati and Degussa reported on the use of resting
cells of an Agrobacterium radiobacter with high activities for both the D-hydantoinase
and D-N-carbamoylase[loo,loll. The advantage of this process in comparison with the
methods mentioned above is not only the environmental friendly “one pot produc-
tion’’ of D-amino acids without use of HN02‘ but the possibility of also producing D-
amino acids, which are unstable against treatment with this acid (e.g. D-tryptophan,
D-citrullineor D-pyridylalanine)(for the production of D-citrulline from L-ornithine
see Fig. 12.4-13).
Nevertheless, the main problem of using resting cells in a “one pot process” still
seems to be the stability of the D-N-carbamoylase (see e.g. reference[”]), which is
discussed in Sect. 12.4.3.Therefore, a series of papers from the 1990s concentrated
on: the optimization of the chemoenzymatic D-hydantoinasecatalyzed production of
D-N-carbamoylphenylglycine and ~-N-carbamoyl-4-hydroxy-OH-phenylglycine as
the enhanced chemical decarbamoylation of D-N-carbamoylphenylglycine by its
interfacial solubilization under micellar conditions; the repeated use of a commer-
cially available covalently immobilized D-hydantoinase at high substrate concentra-
tions [Io2],the repeated use of a thermostable D-hydantoinase from Bacillus stear-
othermophilus SD-1 immobilized on DEAE-cellulose resin[’03],the mass production
of the same enzyme in Escherichia coli using a constitutive expression system[95];the
application of numerical modeling for optimization of a complex medium for D-
hydantoinase production from Agrobacterium radiobacter NRRL B 11291 [‘041; the
modeling, simulation and kinetic analysis of a heterogeneous reaction system for the
conversion of ~,~-4-hydroxy-phenylglycine to the corresponding D-N-carbamoyl
amino the use of a so called “pressure swing reactor” for the same
as well as on the racemization of the remaining substrate enantio-
mers [lo7I.
12.4.3
D-N-Carbamoylases - Substrate Specificity and Properties
In some cases, D-hydantoinases are described as being associated strictly with D-

specific N-carbamoyl-D-aminoacid amidohydrolases (D-N-carbamoylases).One nat-
ural role of these enzymes was discussed as being the P-ureidopropionase (E.C.
3.5.1.6), which catalyzes the decarbamoylation of P-ureido propionic acid in pyr-
imidine metabolism (see Fig. 12.4-7), but with the recent information on its
stereo~electivity[~~]and its DNA and amino acid sequences, this previously proposed
h o m o l ~ g y [is~no
~ longer
~ ~ ~ ]clear.
Various D-N-carbamoylases were purified from rat liver as well as from microbial
U
U
0
-
d
Table 12.4-3a. Purification and characteristic properties of microbial D- and L-hydantoinases. t
u
Source Arthrobacter crystallo- Arthrobacter aurescens Pseudomonasjluorescens Pseudomonas striata Pseudomonas sp. AJ -=z
Q
poietes DSM 20 117 DSM 3745 11 220 a
Reference 177, 1591 1126, 127. 128, 1301 [761 1751 I351
Inductor dihydrouracil, hydantoin N-3-Methyl-o,~-S-indo- Hydantoin 5-cyanoethyl-hydantoin

and various w-5-mono- lylmethylhydantoin F
substituted hydantoins
Purification protamine sulfate and DEAE-Streamline, HIC (Phenylsepharose), protamine sulfate and IEX (DEAE-Toyopearl) .:3
ammonium sulfate HIC (Phenylsepharose), SEC (Sephacryl S-400) ammonium sulfate pre- S
precipitation, IEX (DEAE- Mono Q and preparative electro- cipitation, IEX (DEAE- s9
cellulose), HIC (Phenyl- phoresis cellulose), hydroxyl apa- 2
sepharose), Mono Q , gel tite- and SEC (Sephadex m
S
filtration G 200), crystallization >
Yield (“A) 5 77 1 3 63
Purification factor 20 52.6 0.53 300 27
Purity homogenous homogeneous homogeneous homogeneous crude enzyme
Optimal temperature (“C) 50-60 50 55 45-55 55
Temperature stability (“C) 4 0 <40 <GO
Optimal pH 8.2-9.2 8.8-9.3 9.0 8.0-9.0 8.0
pH stability 6.5 5.5-8.5 6.0-7.0
Metal ion requirements Zn2’ 10 mol Zn2’per mol of Fe” Fe”, Co”
active enzyme, but
Mn” and Co2+Enhance
the enzymatic activity
Molecular mass (Da) 257 000 200 000 230 000 190 000
Subunits (Da) 4 x 60 000 4x49680 4x60 000
Table 12.4-3b. Purification and characteristic properties o f microbial D- and L-hydantoinases.
Source Bacillus stearothermophilusSD-1 Bacillus circulans Blastobacter sp. A1 7p-4 Agrobacterium sp. IP-l 671
Reference [85. 861 ~ 7 1 [89, 1601 137. 881
Inductor hydantoin methylthioethylhydantoin uracil uracil

Purification ammonium sulfate fractionation, heat treatment, Sephadex DEAE-Sephacel,HIC protamine sulfate and am-
Q-Sepharose, heat treatment, HIC G-50, DEAE-cellulose,HIC (phenylsepharose), SEC monium sulfate precipita-
(phenylsepharose, preparative gel (phenylsepharose), Fractogel (Sephacryl S-200 HR), tion, heat treatment, IEX
electrophoresis) Mono Q and superose-12 (DEAE-Sephadexand Trisa-
cyl, HIC (octyl-Sepharose)
Yield (%) 1.5 12.4 3 9
Purification factor 50 243 30 965
Purity homogenous homogeneous homogeneous homogeneous
Optimal temperature (“C) 65 75 60 60
Temperature stability (“C) <60 <60 <60 < 70
Optimal pH 8.0 8.0-10.0 10.0 10.0
pH stability 5.5-11.0 8.5-9.5 5.0-10.0 7.5-10.5
Metal ion requirements Mn” Mnzf. C02+.Ni2+
4
enhance the enzymatic activity Mg2+,Mn”, Co”, Ni2’ lu
enhance the enzymatic activity Ni2’, Mg” P
Molecular mass (Da) 126 000 212 000 200 000 250 000 I
-=0.
Subunits (Da) 2 x 54.000 4x53 000 4x53 000 4x62 000 $
U
\o
U
780
L-Ornithine 0
H2"
""KN"
0
""K""
0
4
CO, + NH,
D-Citrulline
Figure 12.4-13. Production o f o-citrulline from L-ornithine by means o f

Agrobacterium radiobacter.
cells. In contrast to D-hydantoinases (see above), induction and stability of these

enzymes seem to be problemati~[~I. Meyer and Runser reported that both D-
hydantoinase and D-N-carbamoylase were found to be highly inducible by the
addition of non-metabolizable thiolated hydantoins or pyrimidines to the culture
medium of Agrobacterium sp. 1-671[lo'].
Rat liver D-N-carbamoylase was isolated by Caravaca and G r i ~ o l i a [ ~ The
~ ] .same
authors also found it in the supernatants of liver homogenates of dogs, pigeons and
rabbits. In microorganisms, D-N-carbamoylase activity was detected in various
strains of Agrobacterium sp.[32, 109-1111 , B1astobacter sp. A17p-4[ll2I,Clostridium
u r ~ c i l i c u m [ ~Cornamonas
~1, acid~vorans[~'],
Pseudomonas putida 77 [431 and Pseudomo-
nus sp. AJ-l1220[75]. Induction of enzymatic activity during growth was done either
by addition of N-carbamoyl amino acids or pyrimidine and hydantoin derivatives.
Some of the enzymes were purified and characterized as shown in Table 12.4-4.
The enzyme isolated from rat liver and the inducible Clostridium D-N-carbamoy-
lase are both postulated to be involved in the degradation of pyrimidines [331. With
only a few compounds having been tested as substrates for these enzymes, they are
Table 12.4-4a. Purification protocols and characteristic properties of microbial D-N-carbamoylases.
Source Agrobocterium radiobacter Agrobacterium sp. KNK712 Agrobacterium sp. Clostridium uracilicum Comamonos sp. E 222c
NRRL B11291 (after expression in (BEECHAM-strain)
Escherichia colr)
1113, 1161 11111 11101 1331 1381
Reference
Inductor N-carbamoyl- urea, N-carbamoyl-phenyl- strain was genetically N-carbamoyl-P-alanine N-carbamoyl-0-alanine
o-phenylglycine glycine, N-carbamoyl-phe- engineered - no data
nylglycine (for the wild are given on this
strain)
Purification Q-Sepharose FF, chelating heat treatment, HIC (Phe- clarified crude extract MnC12, ammonium sul- ammonium sulfate
Sepharose, Superose 12 nylsepharose), ammonium was used for experi- fate and acetone precip- precipitation, IEX
(procedure for the recom- sulfate precipitation, ments itation, hydroxyl apatite (DEAE-Sephacel,
binant enzyme) DEAE-Sepharose (data for chromatography MonoQ), HIC
the recombinant enzyme) (Phenylsepharose)
Yield ("5%) 34 (for the recombinant 12.3 (data for recombinant clarified crude extract 18 36
enzyme) enzyme)
Purification factor 20 (for the recombinant 3.9 (data for recombinant clarified crude extract 119
enzyme) enzyme) -
h,
Purity homogenous clarified crude extract crude enzyme homogeneous A
Optimal temperature ("C) GO 65 52 30-35 40 "C -=z

9
Temperature stability < 40 "C < 55 "C < 45 "C < 40 a
Optimal pH 7.0 7.0 7.4-7.6 7.4-7.8 8.0-9.0
pH stability 7.0-9.0 7.0-9.0 6.2-9.0 6.5-9.5 0
2
Metal ion requirements none no details given none none none a
Molecular mass (Da) 68 000 no details given 84 000 (determined by no details given 111000 on
native gel filtration) 3
Subunits (Da) 34 000 34 285 (calculated) no details given no details given 3 x 4 0 000
2.
3
4
z
%
2
2
2.
5
-
-4
m
Purification protocols and characteristic properties o f microbial

Table 12.4-4b.
o-N-carbamoylases.
Source Blastobacter sp. A1 7p-4 Pseudomonas sp. Pseudomonas putida
AJ-11220 77
Reference 1351 1431
Inductor 5-cyanoethylhydan- 1-methylhydantoin

toin
Purification ammonium sulfate pre- IEX (DEAE Toyo- ammonium sulfate
cipitation, DEAE-Sepha- pearl) precipitation, IEX
cel, HIC (Phenylsephar- (DEAE-Cellulose),
ose), Sephadex G150, crystallization
Mono Q
Yield (%) 2.3 36 63.2
Purification factor 37 17 27.4
Purity homogeneous crude enzyme homogeneous
Optimal temperature ("C) 55 55 37
Temperature stability ("C) < 50 < 40
Optimal pH 8.0-9.0 7.0 7.0-8.0
pH stability 6.0-9.0 6.0-7.0
Metal ion requirements no details given no details given no details given
Molecular mass (Da) 120 0000 no details given 102 000
Subunits (Da) 3x40 000 no details given 4 x 2 7 000
obviously different from the other microbial D-N-carbamoylaseslisted in Table

12.4-4.
The N-carbamoylsarcosine amidohydrolase from Pseudomonas putida 77 is re-
ported to have its biological function in creatinine metabolism [431. The D-N-
carbamoylases of the various Agrobacterium sp. and the Pseudomonas sp. Aj-11220
are likely to be identical. They have a wide substrate specificity in common, for a
survey see Fig. 12.4-14, and hydrolyze only the D-enantiomers of aliphatic and
aromatic hydantoic acids r3', '131 . The main problems of this enzyme seem to be (i) its
instability and its rapid inactivation in absence of a reducing agent["'] probably
caused by an oxidation of an SH-gro~p["~] and (ii) its inhibition by ammonium
i ~ n s [ ' ' ~Grifantini
]. et al. were able to prove the role of the cysteine 172 out of five
cysteines for enzyme activity by site-directed mutagenesis while Nanba et al.
were able to obtain a more thermotolerant D-N-carbamoylase by substitution of Pro
203 by Leu in the gene from Agrobacterium sp. KNK712 before expression in
Escherichia c~li[~''I.For stabilization, the same group immobilized the enzyme by
glutaraldehyde coupling to Duolite A-568, a macroporous phenol formaldehyde
Kim and Kim tried to overcome limitations in the production of D-p-OH-
phenylglycine with resting cells of Agrobacteriurn sp. 1-671by adsorptive removal of
the ammonium ions with a silicate c0rnplex[''~1and proposed the optimized ratio
between D-hydantoinaseand D-carbamoylase of about 1 : 3 based on mass for further
process optimization('"].
As discussed before, there is a lot of interest in microbial biocatalysts with highly
active D-hydantoinase- and D-N-carbamoylase-activity for the direct synthesis of
HNOz-sensitiveD-amino acids used as chiral synthons in the production of pharma-
12.4 Hydrolysis and formation ofkfydantoins
I 783
h
rcooH
Y C O O H \o>COOH W C O O H Y C O O H Ho*COOH
HNTNH2
HNyNH2 HNyNH2 HNyNHz
HNyNHz HNyNHz
rCOOH b C O O H
-S
>COOH COOH
clq I
COOH
-0
\o&)COOH
ANTNH2 HNKNH2
6
HNKNHz
6 HNyNH2HNyNH2 HNTNHz
Q
CI
COOH
HNyNH2HNyNH2 HNTNHz HNTNH2

)--GOOH T C O O H ""\COOH COOH O Y C O O H
I
"KNH2 HNyNHz
\GOOH H o q C O O H COOH
HNyNH2 HNyNH2 HNKNH2
6
H
HOOC
HNyNHz
\COOH
HNTNH2 HNyNHz HNyNH2 HNyNHz HNyNHz
Ho-$COOH >COOH HO
R C O O H COOH f h C O O H
+GOOH C
-:O -
'"OH O q
HNTNHzHNyNH2HNyNH2 HNyNH2HNTNHz
H
0
~ '\COOH
N ~
COOH "-COOH &COOH
HNyNH2
Figure 12.4-14. Substrates accepted by microbial o-N-carbamoylases

of Agrobacterium radiobacter and Pseudomonos sp. AJ-11220[35f' 1 3 ] .
ceutical drugs and intermediates. For the synthesis of peptides in particular, a great
variety of D-amino acids and derivatives are highly desirable molecules. Recently, cell
free extracts of Blastobacter sp. A17p-4 were used for the preparation of optically
active D-p-trimethylsilylalanine from the corresponding D,L-carbamoyl amino
acid[118]and several biocatalysts (isolated enzymes as well as whole cells) have been
compared with respect to stereoselectivityfor the hydrolysis of D,L-S-trimethylsilylhy-
~ ] . free extracts of Blastobacter sp. A17p-4 were shown to distinguish
d a n t ~ i n " ~Cell
stereoisomers of hydantoins not only at the a-carbon but also at the 0-carbon of N-
carbamoyl-a,0-amino acids [120].
784
12.4.4
L-Hydantoinases - Substrate Specificity and Properties
In the 1960s, Tsugawa et al.[28]were able to isolate strains of Pseudomonas,

Micrococcus, Aerobacter, Achromobacter, and Bacillus that were capable of producing L-
glutamic acid from ~,~-5-carboxyethylhydantoin by L-5-carboxyethylhydantoinase.
Bacillus breuis ATCC 8185 was the first microorganism used for bioconversion of a
racemic hydantoin derivative to an L-amino acid in the case of L-glutamic acid with a
yield of 90%. In 1988, Yamashiro et al.I4', 501 reported on an L-hydantoinase from
Bacillus brevis AJ 12299. This Bacillus L-hydantoinase requires ATP and Mg2+,Mn"
or K' as cofactors and acts selectively on r-configured substrates. The optimal
reaction conditions for the hydantoin cleavage are pH 8.0 and 50 "C. Only a few
substrates have been investigated as shown in Fig. 12.4-15, so it is not clear whether
this enzyme may be identical to the L-5-carboxyethylhydantoinase described before
(see Sect. 12.4.1).
Watabe et al. reported on the cloning and sequencing of genes for an L-
hydantoinase deriving from Pseudomonas sp. NS 671 able to convert L-selective D,L-
5-MTEH, a precursor of methionine['*']. Production of L-methionine from the
corresponding hydantoin derivative was also described by Ishikawa et al. for resting
cells of Bacillus stearothermophilus NS1122A['221after growth of this strain on a
medium containing D,L-5-MTEHas an inducer. The resting cells were described to
be stimulated by addition of cobalt and manganese ions, while copper and zinc ions
caused a strong inhibition of the enzymatic activities. Wagner et al. described the use
of an Arthrobacter sp. DSM 7330 for the production of L-methionine and were able to
obtain product concentrations of up to 12Og L-' using a special feed-batch technique
for feeding of the hydantoin substrate[123].
From the data available, the three L-hydantoinases from Bacillus breuis and Bacillus
stearothermophilus and the enzyme from Pseudornonas mentioned above seem to have
a preference for hydantoin derivatives containing aliphatic side chains and therefore
differ distinctly from those enzymes found in Arthrobacter sp. by Cotoras et al.
M0 w -sy
""If"" ""If""
""K""
0 ""If""
0 0
0
Figure 12.4-15. Substrates accepted by the L-hydantoinase

o f Bacillus breuis AJ 12 29914', 501.
12.4 Hydrolysis and Formation ofHydantoins I 785
-0
ffo
""Y""
0
+ ""K""
0
)-w"
""K""
0
'OW0
""Y""
0
Figure 12.4-16. Substrates accepted by the L-hydantoinases of Arthrobader
Yokozeki et a1.[51-53]and Syldatk et al.r7. 1251 as well as in FZavobacteriurn sp. by

Nishida et al. [461. These so called "L-5-arylalkylhydantoinases"have comparable
substrate specificities and are especially active towards the hydrolysis of hydantoin
derivatives with aromatic substituents, as can be seen from Fig. 12.4-16. They could
only be induced by D,L-S-indolylmethylhydantoin or the corresponding N-3-methyl
derivative of a variety of hydantoins and natural cyclic amides 53* 124* 1251.
1'9
The L-hydantoinase from Havobacteriurn sp. was reported to be L-selective. Its

optimal pH of 9.7 is remarkably high and its optimal temperature is 40 0C[4G].
The enzyme from Arthrobacter aurescens DSM 3745, which has been crystallized
and used for initial X-ray analytical studies['26],was described in detail by May et
al.['27-'301. The active enzyme is a tetramer consisting of four identical subunits,
each with a molecular mass of 49 (570 Da[lz7l,containing 10 mol of zinc per mol of
active enzyme, which could be detected by atomic absorption spectrometry and
inductive coupled plasma-atomic emission spectrometry['28].By kinetic studies of
metal/chelator enzyme inactivation and by identification of specific metal binding
ligands, the role of the zinc atoms was found to be in the catalFc activity as well as in
the stabilization of the quaternary structure of the hydant~inase[~~']. A reaction
mechanism was proposed by Syldatk et al. [131along the lines published for ureases
by Jabri et a1.[131]and is shown in Fig. 12.4-17.
786
Hydrophobic Electrophile (e. g. Zn2*?)

interaction ........ Electrophile (e. g. Zn*+?)
,/
,o;-
......... ........
H
-
.'".
b w\ ,o, .......... His62

h2+-
""Y""
0 ""YNH ('H \ Asp274?
0
Nucleophile
(e. g. Asp ?)
6 -
Electrophile
I Electrophile
,../
.......
o", ..'."
R t l /
'
2t
YCOOH * Zn -His62
HNKNH2
0
K
HN
0
H,,, Asp274?
Nucleophile Nucleophile
Figure 12.4-17. Proposed reaction mechanism atom, generating a tetrahedral intermediate.

catalyzed by the hydantoinase: after binding o f The tetrahedral intermediate undergoes ring-
the substrate, an electrophilic residue (or zinc) opening, assisted by protonation of the ring
stabilizes the negative charge of the carbonyl The proposed residues are derived
oxygen. Zinc-bound water is activated and per- from a conserved sequence pattern and their
forms a nucleophilic attack on the C4 carbon respective function in urease['"I.
The enantioselectivity of the enzyme was shown to be strongly dependent on the

substrate used[127]:while the enzyme is strictly L-selective for the cleavage of D,L-
5-IMH, it appears to be D-selective for the hydrolysis of D,L-S-MTEH~~*~]. As part of
these investigations, a method based on enzyme activity stain was developed for the
detection of hydantoinases with respect to their enanti~selectivity['~'].The isolated
enzyme from Arthrobacter aurescens DSM 3745 was recently used for the chemoenzy-
matic production of optically pure ~-(trimethylsilyl)alanine[l')]. A good stability for
the continuous conversion of ~,~-5-indolylmethylhydantoin to N-carbamoyl-L-trypto-
phan of tIl2 >720 h was first achieved after immobilization of the enzyme by
covalent binding to Eupergit CL1321. Further optimization of the immobilization of
hydantoin cleaving enzymes has been subsequently carried out [133, 1341.
12.4.5
L-N-Carbamoylases - Substrate Specificity and Properties
In contrast to the D-route, N-carbamoyl-L-aminoacid amidohydrolases (L-N-carba-

moylases) were identified in all L-hydantoinase containing microorganisms dis-
cussed in Section 12.4.4 (see above). In this section, L-N-carbamoylases from twelve
bacterial strains will be discussed with respect to their enzymatic properties and
substrate specificities (Table 12.4-5).
The biological function of these enzymes is still unknown, with the exception of
the Mn2+/Fe2+-dependent
L-selective P-ureidosuccinasefrom Clostridium oroticum (=

I
787
Zymobacterium oroticum) found by Lieberman and Komberg in 1955 and postulated

to play a role in the degradation of orotic acid[26].This hydrolase works best at pH 7.8
to 8.5 and its biological function is postulated to be the conversion of N-carbamoylas-
partic acid into L-aspartic acid. It has not been investigated from the biotechnological
aspects as yet.
The twelve L-N-carbamoylasesderive from seven genera of bacteria: Alcaligenes (1),
Arthrobacter (l),Bacillus (4),Blastobacter (l),Clostridium (l),Havobacterium (l),and
Pseudomonas (3). Only four of the twelve enzymes have been purified to homoge-
neity, making a comparison of enzymatic properties difficult. Two of the Bacillus
strains have been reported to be thermophilic and the enzymes enriched from these
strains have been found to possess optimal temperatures approximately 10 to 20 "C
higher than most of the other enzymes (Table 12.4-5).The pH-optima of all L-N-
carbamoylases are between pH 7.5 and 8.5. Whereas hydantoinases are not always
strictly L-specific a strictly L-specific carbamoylase, responsible for the optical
purity of the amino acid produced with resting cells, has been identified in each
strain.
The L-N-carbamoylases from Alcaligenes, Arthrobacter, Bacillus brevis A J-12299,
Bacillus stearothermophilus NS 1122A and the Pseudomonas putida I F 0 12996 and
Pseudomonas sp. NS 671 enzymes have been reported to be (hyper-)activated by one
or several of the following heavy metal ions: Mn2+,Co2+,Fe2+Ni2+.
In addition to N-carbamoylamino acids some enzymes are able to hydrolyze N-
formyl- or N-acetylaminoacids L3', 135-1371. As with the hydantoinases, N-carbamoy-
lases accept N-protected amino acids of unnatural origin. The enzymes of the
different genera differ significantly in their substrate specificities. Aliphatic N-
carbamoylaminoacids are preferentially hydrolyzed by the enzymes from the genera
Alcaligenes, Bacillus, and Pseudomonas. Only the N-carbamoylasefrom Pseudomonas
strain NS 671[13*1 accepts aromatic amino acids as well as aliphatic ones. Aromatic L-
N-carbamoylamino acids are preferentially hydrolyzed by the enzymes from the
genera Arthrobacter and Havobacterium. The substrates hydrolyzed by these enzymes
are shown in Fig. 12.4-18. Interestingly, the L-N-carbamoylase from Pseudomonas
putida I F 0 12 996 accepts N-carbamoyl-j3-alanine[36] as a substrate, which is an
intermediate of the dihydropyrimidine metabolism (see Fig. 12.4-7).In contrast, p-
ureidopropionate is not at all converted by the enzymes from Alcaligenes, Arthro-
bacter, Bacillus, and Pseudomonas sp. NS 671 and is converted by Havobacterium only,
with a very low relative activity.
As has been shown by HPLC, whole cells of Alcaligenes xylosoxidans were able to
distinguish not only the configuration of the a- but also that of the p-carbon of N-
carbamoyl-o-methylphenylalanine:from the mixture of the four diastereoisomers
only threo-L-p-methylphenylalanine was produced [120, 1391.
The enzymes from Arthrobacter, Bacillus stearothermophilus NCIB 8224 and
NS 1122A, and Pseudomonas sp. NS 671 have been cloned and expressed in E. coli.
The enzymes from Bacillus and Pseudomonas share approximately 38 % sequence
identity with the Arthrobacter enzyme whereas the 20 amino acids known from the
N-termini of the enzymes from Alcaligenes and Pseudomonas putida I F 0 12996 are
Table 12.4-5. Comparison of L-specific carbamoylases (modified from
Micro- Alcaligenes Arthrobacter Bacillus brevis Bocillus Bacillus steoro- Bacillus stear- Blastobocter Clostridium Flavobacterium Pseudomonos Pseudomonas Pseudomonas
organism xqlosoxidans aurescens AJ-12299, Mu- brevis thermophilus othermophilus sp. A17p-I oroticum sp. AJ-3912 putido I F 0 sp. AJ-11220 sp. NS671
DSM 3747 tant No.102 ATCC 8185 NClB 8224 NS 1122A 1mqfi
Reference 11351 11361 1501 1281 11371 11531 11121 1261 1531 1361 1351 11211
Purification homogeneous homo- partial whole cells crude extract partia1l”‘l crude whole cells parhal homogeneous crude extract homogeneous
status geneous extract ““1 (recombinant
enzyme) 11381
.MWSDS 65000 44 000 Da 44 000 44.000 45 000 45 000
Pa) (calc. 43993) (calc. 44120)
.MW natlve 134 000 Da 93 000 95 000 109 000
(Da) (2 subunits) (2 subunits) (2 subunits) (2 subunits)
Optimal Tem- 35 50 50 60 60 40 60 40
perature (“C)
Optimal pH 8.0-8.3 8.5 7.5
(‘C)
Cloning and rec. in E. coli rec. in E. coli rec. in E. coli rec. in E. coli
Expression
Sequence- no 38 38 no 37
identity with
Arthrobacfer
1-N-carbamoy.
lase (%)
Micro- Alcaligenes Arthrobacter Bacillus brevis Bacillus Bacillus stearo- Bacillus stear- Blastobacter Clortridium Flauobacterium Pseudomonar Pseudomonar Pseudomonas
organism xylosoxidans aurescens A)-12299, Mu- brevis therrnophilus otherrnophilus sp. A17p-4 oroticum sp. A)-3912 putida I F 0 sp. AJ-11220 5p. NS671
DSM 3747 tant No.102 ATCC 8185 NClB 8224 NS 1122A 12996
References 11351 11361 I501 1281 11371 1124 11121 1261 1531 134 139 11211
Substrates b aliphatic aliphatic b b b b
accepted' aliphatic aliphatic aliphatic C-a-AS: C-a-ASC: aliphatic aliphatic aliphatic aliphatic
C-a-AS: C-a-AS: C-a-AS: C-r-Met (100) C-L-Met (97) C-a-AS: C-a-AS: C-a-AS: C-a-AS:
C-UAla (100) C-r-Met (17) C-r-Val (100). C-o,L-Ala (183) C-Gly (71) C-r-Met (24) C-Gly (16) C-r-Val (100) C-r-Met (100)
C-Gly (75) C-r-Leu (102), C-L-GIU(112) C-DL-Ala (100) C-D.1-0-Me-Ser C-r-Ala (118) C-r-Met (47) C-o,r-Ala (102)
C-r.Va1 (28) C-r-Ile (84). C-Gly (77) C-DL-Val(100) (13) C-r-Ser (34) C-r-Ala (44) C-D,r-Val (106)
C-r-Leu (9) C-r-Met (73). C-r-Leu (28) C-r-Leu (94) C-r-Ser (5) C-or-a-ami- C-r-Leu (98) C-r-Leu (118)
C-r-Met (12) C-r-Ala (48) C-r-lle (55) C-Gly (5) nobutyrate C-L-GIU(3) C-r-Ile (97)
C-r-lle (5) C-Dr-Ser (86) C-r-Leu (3) (31) C-r-Asn (2)
C-o,r-2-ami- C-DL.Thr (94) C-i-Ile (2) C-2-aminova-
nohexanoic C-L-G~U (56) C-r-Val(2) lerate (9)
acid (24) C-L-ASD(52) C-r-Gln (1) C-o.r-Thr (1)
C-o,r-Ser (19) C-1-Asn (1) C-o,r-Asp
C-o,r-Thr (9) C-r-Ala (0.5) (0.1)
C-r-Asn (64) C-L-GIU(0.3)
C-r-Asn (1.6)
aromatic aromatic aromatic aromatic aromatic aromatic aromatic aromatic
C-a-AS: C-a-AS: C-a-AS: C-a-AS: C-a-ASC: C-a-AS: other: C-a-AS: C-a-AS:
C-r-Phe (5) C-i-Trp (100) C-r-Phe (86). C-D,r-Phe (< 0.1) C-D,r-Phe (25) C-~,r-3,4-me- J3-ureidopro- C-r-Phe (10) C-D.r-Phe (94)
C-r-Thienyl- C-L-T,T(45) C - L - T(~c~0.1) C-i-Trp (trace) thylenedioxy- pionate (100) C - ~ T y (9)
r C - L - T(60)
~~ 1
Other: ala (316) C-i-Tyr (< 0.1) C-L-TY(3) Phe (100) y-ureidobuty- .b
formyl-D,L-Ala C-r-Phe (98) C-r-Phe (82 rate (290) .cI
(13) C-r.Tyr (127) other: C-L-TY (59) p-Ureidoiso- 1:
fOnTlyl-D,L- Acetyl-Met (38) C-r.Trp (55) butyrate (43)
-=a
Leu (5) other: Acety-Glu (7) C-o.r-3,4-dime- formyl-D,L-Ala a
fOImyl-D,L- Formy1-o.~- thoxy-Phe (24) (75)
Met (5) Trp (98) C-D,L-O-benzyl- Acetyl-D,r-Ala
Acetyl-L-Phe serine (15) (6) D
(0.7) a
kcetyl-o, L- other: 3
2-aminohexa- P-ureidopro-
noic acid pionate (3)
3
4
(0.06) 5
a relative acuvines ~n[%Iare given in brackets () except'. b additional data on substrates not hydrolyzed are given in the cited hterahlre. c isolated yleld m [%] after 24 h ~n brackets ()
9
1
U
0
W
790
rCOOH
HNKNHz
0
\
O Y C O O H
0
i.;L
c COOH
HNKNHz
0
\ I
HNKNH
0
COOH
)-COOH -'>COOH c l a C O O H h C O O H
"KNH2
0 HNKNH2
0 HNKNHz
0 HNKNHz
0
-0.
)-tCOOH Ho>COOH
HNKNHz
0 HNKNHz
0 HNKNHz
0
0
5 C O O H F-COOH O>COOH
HNKNHz
0 HNKNHz
0 HNKNH
HNKNHz
0
\COOH 0
H2N+COOH z'N-@)- COOH S ° C O O H
HNKNHz
0 HNKNHz
0 HNKNHz
0 HNKNH
0
+COO, f\COOH COOH
HNKNH2
0 H N 70f N H z HNKNH2
0
Figure 12.4-18. Substrates accepted by the L-N-carbarnoylases of

Atthrobacter sp.['] and Flauobacterium sp.[46, 51-531.
completely different. In contrast to the D-N-carbamoylases (see Sect. 12.4.3),the L-N-

carbamoylase of Arthrobacter sp. DSM 3747 is induced by N-3-methylated D,L-
5-indolylmethylhydantoin,which cannot be hydrolyzed by the cells 17].
Resting cell L-hydantoinase processes were first developed for the industrial
production of L-tryptophan by the companies Ajinomoto and Tanabe146,51-531. In
1992 the Riittgers company tried to enter the amino acid marked with a resting cell
I 791
process for the production of unnatural aromatic L-amino acids using Arthrobacter
sp. DSM 3745 or DSM 3747, which both contain an L-hydantoinase, hydantoin
racemase and L-N-carbamoylase. However, the productivities obtained (see Fig. 12.4-
19 and for details referenceLs7I)seemed to be too low to fulfill economic require-
ments.
In recent years, new developments have been published, which could overcome
these problems:
1. the L-N-carbamoylase from Arthrobacter aurescens DSM 3745 and 3747 could be
produced as recombinant enzymes in high cell density culture in Escherichia coli
using an expression system based on the Escherichia coli rha-BAD-promoter[1401,
2. purification of the recombinant L-N-carbamoylases could be optimized by expres-
sion of enzymes carrying different tags, making the purification protocols much
easier[’41]and,
3. the hydantoin-cleaving enzymes from Arthrobacter aurescens DSM 3747 could be
stabilized significantly by immobilization 1341.
Reaction rate (“A) Molar conversion (“A) L-amino acid

after 1 h after 2 6 h
HNyNH
100 > 90 tryptophan
140 - 160 > 90 phenylalanine
20 - 40 > 70 Obenzylserine
0-
I
150-200 > 80 pchloro-phenylalanine
170 - 200 > 80 pfluoro-phenylalanine
50 - 70 > 80 pnitro-phenylalanine
15-20 > 70 1‘-naphthylalanine
25 - 30 > 70 2 -naphthylalanine
-0 25 - 30 > 80 3,4-dirnethoxy-
phenylalanine
170 - 200 > 80 2’ 4hienylalanine

Q-
Figure 12.4-19. Industrial production of unnatural aromatic L-amino
792
All these developments, together with the directed evolution of the hydantoinase
towards a more L-selective enzyme with higher activity['42]will possibly lead to an
economically viable production process in future.
Additionally, an Escherichia coli whole cell biocatalyst has been constructed
containing the genes of hydantoinase, hydantoin racemase and L-N-carbamoylase
from Arthrobacter aurescens in optimal proportions, so that during the reaction no L-
N-carbamoylamino acid occurs as an intermediate product any longer['43].
12.4.6
Hydantoin Racemases
During enzymatic hydrolysis of 5-monosubstituted hydantoin derivatives in some

cases the remaining, non-hydrolyzed enantiomer is racemizing chemically under
alkaline reaction conditions. The velocity of this chemical racemization is strongly
dependent on electronic factors ofthe substituent in the 5-position (seeTable 12.44).
High velocities of racemization are observed particularly for 5-phenyl-and 5-I)-OH-
phenylhydantoin.
From reports in the early literature resting cell bioconversions of hydantoin
derivatives, which do not racemize with high velocities, indicated an enzymatic
racemization and the presence of a hydantoin racemase. In addition, the chemical
and the enzymatic racemization proceed via the keto-enol tautomerism, which is
shown in Fig. 12.4-20. Stabilizing effects on the enolate structure such as electroneg-
ative substituents are responsible for the velocity of the racemization[2' 1' . Increased
racemization rates can be also seen at more alkaline pH-values and with increased
temperatures "1.
The first hydantoin racemase acting on a cyclic amide substrate reported in the
literature was the allantoin racemase (E.C. 5.1.99.3) (Fig. 12.4-4). This enzyme
enables several bacteria to use both allantoin enantiomers as substrates [20-22].
Racemic mixtures of allantoin, e. g. from plant materials, can be completely metabo-
Table 12.4-6. Racemization rate constants k,,, and corresponding half-live times t,,z,rac for
various hydantoins at pH 8.5 and 40 "C. Values were calculated from first order rate law:
In ([al/[a10)= - krac.t; = In 2/krac.
5-Substituted hydantoin Correspondingo-amino acid k,,, (ti') tbmc (h)

Substituent:
Phenyl D-Phg 2.59 0.27
Hydroxymethyl D-Ser 0.43 1.60
Benzyl D-Phe 0.14 5.00
Methylthioethyl D-Met 0.12 5.82
1'-Hydroxyethyl D-allo-Thr 0.11 6.41
3 '-Ureidopropyl D-Cit 0.049 14.26
1'-Methylethyl D-allo-Ile 0.044 15.84
Imidazolylmethyl D-His 0.043 16.09
Isobutyl D-Leu 0.032 21.42
Methyl D-Ala 0.020 33.98
Isopropyl D-Val 0.012 55.90
Figure 12.4-20. Keto-

I 793
R
‘yo enol-tautomerism o f
-““K
5-monosubstituted
NH hydantoin derivatives.
0 0
L-Hydantoin Enol 0-Hydantoin
lized by various bacteria using a sequence of the L-specific allantoinase and allantoin
racemase (see Sect. 12.4.1).Although the natural function of this allantoin racemase
is not clear, because allantoin racemizes with high velocities under physiological
conditions.
The fast and total conversion of r-5-isopropylhydantointo D-valine by resting
microbial cells led Battilotti et al.[30]to the suggestion that a hydantoin racemase
might be responsible for the racemization of the L-enantiomer.The first hydantoin
racemase to be described in detail was a 5-arylalkylhydantoinracemase, which was
isolated and purified from Arthrobacter sp. DSM 3747[’’* 1442 14’1. Its substrate
specificity is shown in Fig. 12.4-21.
As can be seen from Fig. 12.4-21, only some aliphatic and aromatic hydantoin
derivativesare accepted by the enzyme out of a variety of substrates. The enzyme was
recently cloned and heterologously expressed in Escherichia coli [1461. The gene
encoding the hydantoin racemase, designated hyuA, was identified upstream of an L-
N-carbamoylase gene in the plasmid pAWl6 containing genomic DNA of Arthro-
HN)N
fR
..
Substrate
R1 R2 Relative activity Ri R2 Relative activity

(%) (%)
-H 100.0 -H 9.8
\f\
I
/s- -H 20.4
HO-
-H 76.7 w, -H 0
HO- -H 0
-H 62.7
HOCC- -H 0
Figure 12.4-21. Substrate specificity o f the hydantoin racemase from

Arthrobacter sp. DSM 3745 [”. 1441.
794
bacter aurescens. The matrix assisted laser desorption ionization spectrum (MALDI)
of the purified racemase gave a peak at a molecular mass of 25 078.7. This is in good
agreement with the calculated value of 25 085 Da for the racemase monomer. On a
calibrated column of Superose 12 HR, the relative molecular mass of the native
enzyme was estimated to be approximately 170 kDa + 25, so that the native enzyme
is suggested to be either a hexamer, heptamer or octamer. The optimal conditions for
racemase activity were pH 8.5 and 55 "C with L-5-benzylhydantoinas the substrate.
The enzyme was completely inhibited by HgClz and iodoacetamide and stimulated
by addition of dithiothreitol, while no effect was seen with EDTA. Kinetic studies
revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhy-
dantoin. Enzymatic racemization of 0-5-indolylmethylenehydantoinin DzO and
NMR analysis showed that the hydrogen at the chiral center of the hydantoin is
exchanged for solvent deuterium during the racemization.
Comparative analysis of h y u A with various protein databases indicated homology
to hydantoin racemases. This hydantoin racemase shared 47.2 % identity in amino
acid sequence with the hydantoin racemase of Pseudomonas sp. NSG71 and lower
identities to putative hydantoin racemases of Schizosaccharomyces pombe (SwissProt
accession no. 409921) and Saccharomyces cerevisiae (SwissProt accession no.
P324GO). The multi-alignment of the enzymes showed that the N-terminal region in
particular is highly conserved. No significant similarity to the various amino acid
racemases or any other racemases deposited was found in the data bases.
The hydantoin racemase from Pseudomonas sp. NS 671 is able to racemize both
enantiomers of 5-(2-methylthioethyl)hydantoin,5-isopropylhydantoin,S-isobutylhy-
dantoin and 5-ben~ylhydantoin[~~~I. All together, the presence of hydantoin race-
mases in resting cells used in industrial processes is of importance for a fast and
total conversion of hydantoins which racemize chemically very slowly. In future
there might be a combination of hydantoin racemases from L-selective micro-
organisms with D-hydantoinases and D-N-carbamoylaseswhen designing optimal
processes leading to D-amino acids. For industrial use, the fast racemization of
5-monosubstituted hydantoin derivatives under mild conditions in the presence of
ion exchangers [144, 14'1 could prove more significant, as this procedure also enables
fast and total conversion of D,L-S-monsubstitutedhydantoins without enzymatic
racemization.
12.4.7
Conclusions
The hydantoinase method has become of significant interest for preparative organic
chemistry: total conversion of racemic hydantoins, synthesized by well-established
chemical methods to nearly 100% optically pure products is possible using free or
immobilized microbial cells or enzymes. Further, it is possible to prepare a wide
range of optically pure D- as well as L-amino acids by this method.
Of course there are many factors which influence the competitiveness between
enzymatic processes and chemical processes, for example, costs of substrates, costs
for production/isolation of enzymes, possible space-time yields and costs for
I 795
isolation of the products. These factors are strongly dependent on the desired
product and therefore there is no single best process for the production of amino
acids. For D-p-hydroxyphenylglycine, which is the most important compound pro-
duced by the hydantoinase process on an industrial scale (> 1000 tons) at the
moment, a first comparison of the feasibility of different methods was given by
Tramper and Luyben in the 1 9 8 0 ~ [ ' ~However,
~]. it has already been shown that the
hydantoinase process can be employed for the production of many unnatural amino
acids which are components of promising pharmaceuticals[l5'1. If these pharmaceu-
ticals reach the market, there will be an augmented demand for these amino acids,
which could lead to an increased importance of the hydantoinase process in the
future.
With the availability of recombinant enzymes, one could expect that the hydantoi-
nase method will also become an important tool in biotransformation of simple
precursors to L- and D-amino acids.
Some of the current reports on hydantoinase processes focus on isolation and the
recombinant expression of thermostable enzymes[84*86* 87, 95* . Processes at an
elevated temperature would increase the solubility and racemization rate of hydan-
toins. Therefore, the increased thermostability of these enzymes is very useful, if the
specific activities are still high.
Another main advantage of the recombinant expression of the hydantoin cleaving
enzymes is to decrease the costs of catalysts, which might contribute to the
competitiveness of the hydantoinase processes, which to date do not employ
recombinant enzymes. The Kanekafuchi company have published a patent for the
production of D-N-carbamoyl-aminoacid from 5-substituted hydantoin, using a
recombinant hydantoinase derived from a strain of Pseudomonas, Agrobacterium or
Bacillus['52].This might indicate that highly active recombinant Escherichia coli cells
could replace the wild-type cells in the near future. Furthermore, the recombinant
expression of hydantoinases (and of course carbamoylases[153, 1541) allows enzyme
properties such as stability or stereoselectivity to improve by means of protein
design. If an X-ray structure was solved, this could be done by a rational protein
design[lS5]or, lacking knowledge about a structure, by evolutionary protein de-
sign['S6]. May et al. are already able to improve the stereoselectivity of a L-
hydantoinase for the conversion of ~,~-S-methylthioethylhydantoin[~~~~, while Kim et
al. have shown the possibility of using fusion proteins of D-hydantoinase and D-N-
carbamoylase for the production of D-amino lS81.
Future work will show the impact of these methods on the biotechnological
application of hydantoinases. Besides the applied research on hydantoinases for the
production of amino acids, the natural functions and genetic organization of distinct
hydantoinases, related hydantoin racemases and N-carbamoylasesare still unknown
and are of great interest for basic research.
796
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800
12.5
Hydrolysis and Formation of Peptides
Hans-DieterJakubke
12.5.1
Introduction
Peptides and proteins play a fundamental role in the formation and maintenance of
structure and function of living systems. Peptides comprise a variety of biologically
active linear and cyclic compounds with diverse functions. The different classes of
peptides include, for instance, hormones and other signalling or regulatory factors,
antibiotics, alkaloids, toxins, enzyme inhibitors, and sweeteners. There is perma-
nently great interest in pharmaceutically active peptides and proteins since they have
many applications and great potential in medicine, such as in cardiovascular
diseases, mental illness, connective tissue diseases, the therapy of cancer, regulation
of fertility and growth, and the control of pain. The demand for peptides and proteins
is enormous, and rising all the time.
In a peptide chain amino acids are linked together by bonds between the carboxyl
group of one and the amino group of another amino acid, known as peptide bonds.
This amide or peptide bond has some characteristics of a double bond: it does not
rotate freely and is shorter than other C - N bonds. Nature provides a wide range of
special enzymes, the proteol$c enzymes or correctly designated as peptidases,
which can cleave these bonds in peptide and protein substrates. In contrast, for
catalyzing the formation of peptide bonds the number of efficient enzymes is rather
low. Peptidases catalyze a single reaction, the hydrolysis of a peptide bond. The
ubiquitous distribution among all life forms and their enormous diversity of
function makes the peptidases one of the most fascinating families of enzymes. As a
result of complete analysis of several genomes it has been shown that about 2 % of all
gene products are proteolytic enzymes. In biological and biochemical research
proteolytic enzymes play a contrary role: some researchers either love them or other
hate them. In the first case, the only good peptidase is a dead one, no longer capable
of degrading the desired protein during isolation and purification. Irreversible
inhibition of any contaminating proteolytic enzyme is the best way to solve this
problem. However, for most purposes proteolytic enzymes are of great importance.
Owing to the special physiological functions, some proteolytic enzymes are active in
degrading proteins for digestive and nutritional purposes. These enzymes act both
extracellulary (e.g. in the intestine of animals) and intracellulary (in the hydrolytic
subcellular organelles, preferentially in liver and kidney cells). Other peptidases are
responsible for controling processes, e. g. they can act to cause limited proteolysis of
peptide and protein substrates. In limited proteolytic processes a single susceptible
peptide bond may be cleaved followed by a dramatic change in the biological activity
of the products formed. Physiological functions are a result of proteolytic conversion
of inactive precursors into biologically active proteins, e. g. in blood coagulation,
prohormone or proenzyme activation. Pancreatic peptidases frequently exist as
72.5 Hydrolysis and Formation of Peptides
zymogens, a special inactive proenzyme arrangement that ensures that the pancreas
I
does not digest itself. These enzymes have their function outside cells and will be
activated by another peptidase at the place of action. The number of peptidases
within the cell are more numerous but much more difficult to investigate in
comparison with the extracellular enzymes"]. A much smaller group are the cell-
surface peptidases which are specialized in the hydrolysis of relatively simple
peptides rather than proteins. This group of peptidases does not need activation.
Usually the biological function is the inactivation of signalling peptides in order to
terminate a hormonal or neuropeptide signal but sometimes they activate peptide
substrates, e. g. the conversion of angiotensin I to angiotensin 11F2, 1' .
Contrary to the well-known native function of peptidases the reverse reaction, the
peptidase-catalyzed peptide bond formation, can only be successfully carried out by
manipulating the reaction conditions, the enzyme or the substrate. Besides enzy-
matic techniques, classical chemical synthesis in solution, solid-phase synthesis and
recombinant techniques belong to the most important methods of peptide synthe-
sis.
The main aim of this chapter is to give an overview of the present importance of
proteases in the technology of peptide synthesis.
12.5.2
Hydrolysis of Peptides
12.5.2.1
Peptide-CleavingEnzymes
12.5.2.1.1 Introduction and Terminology

More than 500 proteolytic enzymes are known and, in a general sense, they all
catalyze the same reaction: hydrolysis of peptide bonds. An excellent handbook c41
provides a ready reference to the approximately 500 proteolytic enzymes known up to
the end of the 1990s. These enzymes are classified as peptidases or proteases. In the
past there has been widespread uncertainty about the exact meaning of the terms
proteases, peptidases and proteinases, as well as proteolytic enzymes. There is no doubt
that proteolytic enzymes was the most generally understood term in the current
usage. However, this is ambiguous since many of the enzymes which are capable of
hydrolyzing peptide bonds do not accept proteins as substrates. The Nomenclature
Committee of the International Union of Biochemistry and Molecular Biology (NC-
IUBMB) recommends the term peptidase as the general term for all peptide bond-
hydrolyzing enzymes. The E. C. List can be found in its revised version on the World
Wide Web (www) at http://www.chem.qmw.ac.uk/iubmb/enzyme/index.html.
The acceptable terms for the major types of peptidases are shown in Fig. 12.5-1.
The meanings of the words below are described by the italicized semi-systematic
terms. The terms in bold type are preferred, whereas the terms in parentheses have
historical precedence and are satisfactory when used in the correct context. Most of
the peptidases fall into one of two categories, depending on the positional specificity
of the peptide bond cleavage process. An enzyme is said to be an endopeptidase when
802
Peptide bond hydrolase

Peptidase
(=Protease)
I
I
Endo-acting peptide bond hydrolase Exo-actingpeptide bond hydrolase
Endopeptidase Exopeptidase
(=Proteinase) (=Carboxy- and Aminopeptidases)
Figure 12.5-1. Proposed terms for the major types of peptidases.
L3 I T
Site of endopeptidase action
9’ Y
H,N-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH
T5 Figure 12.5-2.
t t Scheme o f the
A
aminopeptidases carboxypeptidases action o f endo-
peptidases and
L S i t e of exopeptidase action exopeptidases.
the susceptible peptide bond is an internal one in a peptide or protein. In contrast, an

enzyme is termed an exopeptidase when the susceptible peptide linkage is at the
carboxyl terminus or at the amino terminus of the substrate. In the E. C. List there
are also terms for subtypes of exopeptidases and endopeptidases. Exopeptidases
acting at the free N-terminus liberating a single amino acid residue (aminopeptidases)
or a dipeptide or a tripeptide (dipeptidyl-peptidases and tripeptidyl-pepidas), whereas
those acting at the free C-terminus liberate a single residue (carboxypeptidases) or a
dipeptide (pepidyl-dipeptidases). Furthermore, other exopeptidases are specific for
dipeptides (dipeptidases)or remove terminal residues which are substituted, cyclized
or linked by isopeptide bonds (omegapeptidases). Endopeptidases act on bonds in the
middle of the peptide chain (see Fig. 12.5-2). The term oligopeptidase is used to refer
to endopeptidases that act optimally on oligopeptide substrates rather than on
proteins.
peptidases differ in the specificities that they display in a hydrolysis reactions. It is
somewhat simplistic to designate a peptidase on the basis of a single amino acid
residue at the active site. Near the active site of the peptidase is a “pocket” in the
surface of the enzyme molecule which is specific for amino acid side chains of the
substrate. Owing to different interactions in this region there are great differences in
the so-called primary specificity of the peptidases. Trypsin, for example, cleaves only
those peptide bonds adjacent to the amino acids lysine or arginine which carry a
positive charge and are hydrophilic. In the binding pocket of trypsin a negatively
charged aspartic acid unit is at the back, holding the positively charged lysine or
arginine side chain in the pocket by electrostatic forces. Despite the fact that this
pocket for specific side chains is obviously important for binding, it is not the only
binding site. It has been followed from kinetic studies that the binding of substrates
(and inhibitors) involved interactions at a number of subsites on either side of the
Figure 12.5-3. Simplified represen-
Protease tation of the DeDtidase sDecificiW
s3 s, s, s; s; s; ger[2551.The amino acid residues of
HN
, p3 pz pi p; p; p; COOH thecorrespondingsand S'subsites
pair of residues containing the peptide bond to be hydrolyzed. The enzyme and
substrate must be fixed at several points, so that the susceptible bond is oriented at
the active site in optimal configuration.
In 1967, a system of nomenclature to describe the interaction of peptidases and
their substrates was introduced by Schechter and Berger[2551. According to this
system the binding site for a peptide substrate in the active site of a peptidase is
envisioned as a series of subsites S which interact with the amino acid building
blocks P ofthe peptide or protein substrate (see Fig. 12.5-3).The amino acid residues
of the substrate are denoted by P and P', respectively, which interact with the
corresponding S and S' subsites within the active site of the peptidase. The sites are
numbered from the catalytic site, S1....S, towards the N-terminus of the peptide
substrate, and S1'. ...S', towards the C-terminus. In analogy, the residues which they
accommodate are numbered PI.. .. P,, and PI'....P,,', respectively. The arrow
indicates the site of enzymatic cleavage of the substrate between the residues PI -PI'.
With the increasing knowledge of the amino acid sequences of peptidases and
particularly when the three-dimensional protein structure began to emerge, a
functional division of peptidases became possible. Detailed mapping of the active
sites has provided a better understanding of the interaction of substrate and
peptidase and has permitted both the design and synthesis of highly specific
inhibitors as well as a useful prediction of the outcome of the reverse peptidase
action in peptide synthesis (see Sect. 12.5.3.3).
The general stoichiometry for the hydrolysis of a peptide bond is shown in
Fig. 12.5-4.
Water attacks the electron-deficient carbonyl atom targetting first a tetrahedral
adduct, which then eliminates the amine fragment and produces the acid. The
process is characterizedby transferring the aminoacyl moiety of the peptide to water.
In this type of group-transfer reaction the nucleophilic co-substrate is water; 55.5 M
water is the most nearly ubiquitous weak nucleophile in degradative enzymatic
->
R4-NH-R'
'?I
H,O
+- [ R ~ ~ H . 1 + R? It'
OH
+ HZNR'
peptide tetrahedral adduct acid amine fragment

Figure 12.5-4. The general mechanism for the hydrolysis o f a peptide bond.
804
processes in the cell. Under physiological conditions the hydrolysis of peptide bonds
will proceed in the absence of peptidases, but only at an exceedingly low rate. The
reactants only rarely attain the high internal energy required for the hydrolysis
process.
In contrast, enzymes allow the reaction to follow a different pathway from the
substrate to the products, and, therefore, reduce the energy barriers. In the course of
the reaction new intermediate states of highest energy appear, with energy lower the
internal energy barriers, e. g. the high-energy transitions between one intermediate
and the following one.
Proteolysis is functionallyirreversible,since energy is liberated in the hydrolysis of
peptide bonds. From the overall change in energy it follows that the ionized
hydrolysis products are thermodynamically more stable.
On the other hand, aminoacyl-grouptransfer is involved in protein biosynthesis.
As a result of the ionized state of amino acids at physiological pH, the attack by the
amino group of another amino acid to form a peptide bond would involve formal
expulsion of 0 z 2 - . This species is very instable and, therefore, would not proceed to
any reasonable extent. In protein biosynthesis the carboxylate must be chemically
modified so that an oxygen atom can be eliminated with a low energy activation. The
key concept in protein biosynthesis is that the aminoacyl group from an activated
intermediate is transferred to the specific nitrogen of the amino group catalyzed by
the ribosomal peptidyltransferase. The reaction takes place via the transfer of a
peptidyl residue from peptidyl-tRNAin the ribosomal P site to the amino group of
the aminoacyl-tRNA in the A site.
Despite many years of intensive research, the nature and the basic mechanism of
the ribosomal peptidyltransferase reaction is still largely unknown. Recently, Zhang
and Cech[’] demonstrated that an in vitro-selected ribozyme can catalyze the same
type of peptide bond formation as a ribosome. The ribozyme resembles the
ribosome in such a way that a very specific RNA structure is necessary for substrate
binding and catalysis, and both amino acids to be coupled are attached to nucleo-
tides. Despite the presence of many different possible peptidyltransferase ribo-
zymes, one of these must be strikingly similar in sequence and secondary structure
to the “helicalwheel” portion of 2 3 s rRNA implicated in the activity of the ribosomal
peptidyltransferase.These results from Cechs group demonstrate that a ribozyme is
capable of catalyzing peptide bond formation analogous to the action of the
ribosome, providing evidence that RNA itself can make peptides and support the
“RNA world” hypothesis in biological evolution.
Since the ribosomal peptidyltransferaseactivity is not suitable for practical use as a
simple C - N ligase and, in addition, the multienzyme complexes involved in bacterial
peptide synthesisr6Ido not seem to possess a general applicability,only the reverse
catalFc potential of peptidases can be considered as valuable supplement to
chemical coupling methods (cf. Sect. 12.5.3). In addition, peptidases have been used
successfully for enzymatic manipulation of protecting groups in peptide synthe-
sis 17-91.
72.5 Hydrolysis and Formation ofpeptides
12.5.2.1.2 Catalytic Mechanism[”*

The overall process of peptide bond scission is identical in all classes of peptidases
and differences between the catalyhc mechanisms are rather subtle. The attack on
the carbonyl group of the peptide bond requires a nucleophilic agent, either oxygen
or sulfur, in order to approach the slightly electrophilic carbonyl carbon atom. To
remove a proton from the attacking nucleophile, general base catalysis will assist this
process. Furthermore, some type of electrophilic action on the carbonyl oxygen
increases the polarization of the C - 0-bond.
Generally, the four classes of peptidases (serine, cysteine, aspartic and metallo-
peptidases) differ in the groups that perform nucleophilic attack, general base
catalysis, and electrophilic assistance. Also, different groups are involved in the
breakdown of the tetrahedral intermediate which is formed in the initial nucleophilic
attack, requiring general acid catalysis to promote the departure of the amine
fragment. The four types of peptidases are based on the different catalytic mecha-
nisms, which were first recognized by the use of some group-specificinhibitors.
The reactive serine residue in the active site of serine peptidases (but also in other
serine hydrolases, such as acetylcholine esterase) react in an irreversible step with
organophosphate compounds, e. g. diisopropyl phosphofluoridate (DFP or DipF)
resulting in the death of the appropriate enzyme. Owing to the high toxicity of DFP
other reagents, e. g. phenylmethylsulphonylfluoride (PMSF) and 3,4-dichloroisocou-
marin (3,4-DCl)have been used in its place. The reactive cysteine residue of cysteine
peptidases is susceptible to oxidation and can react with various reagents: iodoace-
tate, N-ethyl-maleimide,heavy metals (for example Hg) and with the highly selective
inhibitor N-[~-3-tr~~~-carboxy0xiran-2-carbonyl-~-leucyl-amido(4-guanidino)butane]
(E-64).The highly acidic pH optima led to the first recognition of aspartic peptidases.
Later, with pepstatin A from a strain of Streptomyces, a specific inhibitor was found.
Chelating agents, e. g. EDTA and 1,lO-phenanthroline are prone to inhibit metal-
lopeptidases.
Serine PeptidasesI’21
These form the most studied class of peptidases. They have a reactive serine residue,
e. g. the hydrolysis of a peptide substrate involves an acylenzyme intermediate in
which the hydroxyl group of Ser19’ (from the chymotrypsin numbering system) is
acylated by the acyl moiety of the substrate, releasing the amine fragment of the
substrate as the first product. The formation of the acylenzyme is the slow step in
peptide bond hydrolysis, but the acylenzyme often accumulates in the hydrolysis of
ester substrates. The acylenzyme thus formed will be the same for a series of
substrates which differ in their leaving group.
The catalytic mechanism of serine peptidases will be given in terms of chymo-
trypsin (Fig. 12.5-5). After chymotrypsin has bound the substrate to form the
Michaelis complex, the attack of Ser”’ on the peptide bond of the substrate forms a
high energy tetrahedral intermediate. At the same time the proton of the serine
hydroxyl group is transferred to the nearby His”, the serine hydroxy group forms a
covalentbond with the carbonyl atom of the peptide bond to be cleaved. The liberated
proton is taken by the imidazole ring of Hiss7thereby forming an imidazolium ion
806
/His
Substrate
polypeptide lN-?\
H Lo
Michaelis complex Tetrahedral intermediate
I2
Ser q<: 0-.
0
-
Acyl-enzyme intermediate
(His (His
- 4
1:F
-.O-C-R
+ IR
P-c,
H O
Tetrahedral intermediate Active enzyme
Figure 12.5-5. Scheme o f the catalytic mechanism o f serine proteases
(chymotrypsin numbering).
(general base catalysis). This process is supported by the polarizing effect of the
unsolvated carboxylate ion of Asp"' which is hydrogen bonded to Hiss7 in the sense
of electrostatic catalysis. Mutagenic replacement of Asp'04 by Asn in trypsin, for
example, did not changed the K M substantially at neutral pH. On the other hand, kcat
72.5 Hydrolysis and formation ofpeptides I807
was reduced to < 0.05% of its wild-type value. Furthermore, neutron diffraction
studies have shown that Asp104 remains as a carboxylate ion rather than a proton
being abstracted, as from the imidazolium ion of HisS7 to form an uncharged
carboxylic moiety. The active site of serine peptidases is complementary in structure
to the transition state of the reaction, a structure which is very close to the tetrahedral
adduct of Ser19’ and the carbonyl carbon of the peptide substrate. Indeed, transition
state binding catalysis provides the catalytic power of the appropriate serine
peptidase.
In the course of the formation of the tetrahedral intermediate a conformational
distortion causes the carbonyl oxygen of the scissile peptide bond to move deeper
into the active site to occupy the oxyanion hole. The resulting oxyanion is hydrogen-
bonded to the backbone of NH groups of G l ~ l ’and ~ Ser’”, whereas the NH group of
the peptide bond preceding the scissile bond forms a hydrogen bond to the backbone
carbonyl of Glyl”. The decomposition of the tetrahedral intermediate forming the
acylenzyme intermediate and the amine product occurs under the driving force of
proton donation from the N3-atom of Hiss7 through general acid catalysis. The N-
terminal part of the cleaved peptide chain (amine product) will be released in the
next step and replaced by a water molecule forming a second tetrahedral inter-
mediate. The latter decomposes to the reaction’s carboxyl product (C-terminal
portion of the cleaved peptide chain) and the active enzyme. Generally, all the serine
peptidases employ the same catalytx three amino acid units to hydrolyze peptide
bonds. The diversity of serine peptidases results entirely from the way they
accommodate their specific substrates.
Cysteine Peptidases l1 1’
Other terms for cysteine peptidases are cysteine-type peptidases, thiol peptidases or
sulfhydryl peptidases. They are peptidases in which the attacking nucleophile is the
sulfhydryl group of a cysteine residue ( C Y Sin~ the
~ papain numbering system). The
mechanism of catalysis is similar to that of serine peptidases because a covalent
intermediate is formed. Beside the cysteine nucleophile a proton donorlgeneral base
is required, which in the majority of cysteine peptidases is a His residue (His”’).
Despite the fact that in some families of cysteine peptidases a third amino acid
residue is required to orientate the imidazolium ring of the histidine moiety in the
course of the catalytic process, in general, only a catalytic dyad is necessary.
The archetypal cysteine peptidase is papain which was isolated from the latex of
the tropical papaya fruit (Carica papaya)[’3.141. It is a single protein of 212 amino
acid residues containing three disulfide bonds and the three-dimensional structure
is known with 1.65 8, resol~tion[~’]. The catalytic amino acid residues have been
identified as Cys2’, His”’ and Asn”’, whereas Gin’’ helps to stabilize the oxyanion
hole. A second group of cysteine peptidases which is very diverse in sequence is the
“papain-like’’endopeptidasesof RNAviruses containing only the catalytic dyad Cys/His
without any additional residues being involved in the catalytic mechanism. The
same is true for caspases, a group of ten cytosolic endopeptidases with strict
specificity for cleavage of aspartyl bonds. Clostripain from the anaerobic bacterium
Clostridium histolyticum is a heterodimeric protein of 526 amino acid residues. The
808
heavy chain ( M ,- 43000 Da) and the light chain ( M , - 15398 Da) are held together by
strong noncovalent forces rather than by disulfide bridges. Cys4’ of the heavy chain
was identified as the catalytic residue of the active site. This peptidase is well known
for the selective cleavage of arginyl bonds, whereas lysyl bonds are hydrolyzed at a
lower rate. The catalytic mechanism of the adenovirus endopeptidase is similar to that
of papain, the difference being that four amino acids His, Glu (or Asp) Gln and Cys
are involved in it.
Last but not least, the caspases with a strict specificity for cleavage of aspartyl bonds
should be mentioned as the last family of cysteine peptidases. Members of this
family transmit the events leading to apoptosis of animal cells.
Aspartic Peptidases
The aspartic peptidases comprise peptidases which catalyze the hydrolysis of peptide
bonds without the use of nucleophilic attack by a functional group of the enzyme.
The nucleophile attacking the scissile peptide bond in this case is an activated water
molecule and no covalent intermediate will be formed between the enzyme and a
fragment of the substrate. The name of this group of peptidases is based on the
catalytic domain which consists of two aspartic acid side chains (Asp32and Asp215of
the porcine pepsin numbering system) activating the water molecule directly. These
two side chain carboxyl groups are close enough to share a hydrogen bond between
two of their oxygens holding the water in place. However, there are not two Asp
residues in the catalytic dyad in all members of aspartic peptidases. An endopepti-
dase from nodavirus has an Asp and an Asn as catalytic residues, and in a related
tetravirus endopeptidase the Asp residue is replaced by Glu. It is interesting to note
that all the enzymes so far described are endopeptidases.
Metallopeptidases
As with the aspartic peptidases, metallopeptidases do not form covalent inter-
mediates and the nucleophilic attack on the peptide bond to be cleaved is mediated
by a water molecule. The latter is activated by a divalent metal cation, usually Zn2+
but sometimes also Co2+or Mg2+.In order to assist in attack of a water molecule the
metal ion provides a strong electrophilic “pull”. The metallopeptidase has a water
molecule coordinated to the fourth tetrahedral site. Beside the metal ion the other
ligands are two histidine building blocks and a glutaminic acid residue in thermoly-
sin and carboxpeptidase A. The enzymes of this family can be divided in two groups
depending on the number of metal ions necessary for catalysis. In many cases only
one zinc ion is required, but often two metal ions act cocatalytically.All the enzymes
which contain cobalt or manganese require two metal ions, but zinc-dependent
enzymes are also known in which two zinc ions act in a cocatalytic manner. Enzymes
known to date containing cocatalytical metal ions are exopeptidases, whereas those
with one catalyhc metal ion belong to exopeptidases or endopeptidases. His, Glu,
Asp or Lys are known metal ligands in metallopeptidases. Together with the metal
ligand very often a Glu residue is engaged in the catalytic process. In the leucyl
aminopeptidase Lys or Arg fulfill this function.
12.5.2.1.3 E.C. Classification

As shown above, based on the chemical groups that are responsible for their catalytic
activity, peptidases have been classified into four distinct groups. Recommended by
the International Union of Biochemistry and Molecular Biology (1992) [16] all
hydrolases are designated as E.C. 3., and the peptidases as E.C. 3.4. defining the
main classes of peptidases by a third numeral (11to 24) as indicated in Table 12.5-1.
The sub-subclasses are not further divided. The enzymes are listed in arbitrary order
within each of them. Unfortunately, the molecular structures and evolutionary
relationships are not taken into account in the E. C. classification. In this E. C. list the
exopeptidases are mainly classified based of their action. Generally, only peptides
with an unblocked terminus are attacked. The only exception are so-called omega
peptidases which comprise a very small number that are capable of releasing certain
modified terminal residues. To this group belong, for example, acylaminoacyl
peptidases which release acetyl or formyl moieties from the N-terminus, and the
pyroglutamyl peptidase, capable of releasing the cyclic residue. An isopeptide bond
will be cleavaged by the P-aspartylpeptidase. Other omega peptidases are directed to
the substituted C-terminus, e. g. the peptidyl glycinamidase releasing a C-terminal
glycine amide, and the y-glutamyl carboxypeptidase which splits a C-terminal
glutamic acid linked by an isopeptide bond.
12.5.2.1.4 Peptidase Families and Clans

Starting with the earlier work of Rawlings and Barret1l7]and improved in the
handbook[4]another level of sophistification to the classification of peptidases has
been developed. Evolutionary considerations can be taken into account due to the
relative ease by which cDNA-derived sequences can now be obtained. According to
this principle of classification a family of peptidases is defined as a group in which
Table 12.5-1. Principles of peptidase classification according to the Enzyme Commission (E. C.)
of the international Union of Biochemistry and Molecular Biologyl’61.
E.C. Number Type of peptidase Type of cleavage

Exopeptidases
3.4.1 1.- Aminopeptidase N-terminal residue
3.4.14.- Dipeptidase Dipeptides only
3.4.14.- Dipeptidyl peptidase N-terminal dipeptide
Tripeptidyl peptidase N-terminal tripeptide
3.4.15.- Peptidyl dipeptidase C-terminal dipeptide
3.4.16.- Carboxypeptidase (serine) C-terminal residue
3.4.17.- Carboxypeptidase (metallo) C-terminal residue
3.4.18.- Carboxypeptidase(cysteine) C-terminal residue
3.4.19.- Omega peptidase Terminal modified residue
Endopeptidases
3.4.21.- Serine endopeptidase
3.4.22.- Cysteine endopeptidase
3.4.23.- Aspartic endopeptidase
3.4.24.- Metalloendopeptidase
3.4.99.- Endopeptidase with unknown mechanism
810
every member indicates a statistically significant relationship in the amino acid

sequence to at least one other member of the family in the part of molecule which is
responsible for peptidase activity. Applying strict statistical criteria implies con-
fidence that any two peptidases that are placed in the same family have evolved from
a common ancestor and thus are homologous according to the definition of Reeck et
al. ['*I. Each peptidase family is named with a letter that denotes the catalpc type (S,
T, C, A, M or U, for serine, threonine, cysteine, aspartic acid, metallo- or unknown),
followed by an arbitrarily assigned number ( see Table 12.5-2). The term clan is used
for defining a group of families the members of which have evolved from a single
ancestral protein, but have diverged so far that their relationship can no longer
proved by comparison of the primary structures. Clan-level relationships between
families can at best be made evident by similarities in three-dimensional structures.
The name of the clan is formed from the letter for the catalytic type (in analogy to
families) followed by an arbitrary second capital letter.
About 40 families of serine- and threonine-type peptidases can be distinguished
on the basis of sequence comparison. However, only a few known families of
threonine-dependent peptidases are included therein. By comparing the tertiary
structures and the order of the catalytic residues in the sequence most of these
families can be grouped into seven clans (cf. Table 12.5-2).
The serine peptidases and their clans can be used to demonstrate this type of
classification in more details. In clan SA with the order of the catalytic triad His, Asp,
Ser the tertiary structure is characterized by a p sheet-based two-domain structure.
Each domain contains a p barrel and between the domains the active site cleft is
located. The largest family S 1 of trypsin consists of more than 70 sequenced
proteins. Well-known members of the family S2 are, e. g. streptogrisin A, glutamyl
endopeptidase, and lysyl endopeptidase (Achromobacter). Togavirin (S3), IgAl-spe-
cific serine-type prolyl endopeptidase (SG), flavivirin (S7), hepatitis C polyprotein
peptidase (S29), helper component proteinase (S30), pestivirus NS2-3/NS3 serine
peptidase and arterivirus serine endopeptidase (S32) complete the families of clan
SA. The order of the catalytic triad of clan S B is Asp, His, Ser and the tertiary
structure contains both p sheets and a helices. This clan contains only the subtilisin
family (S8) including peptidases from archaea, bacteria and eukaryotes.
Clan SC contains peptidases with the alp hydrolase fold bearing the catalytic triad
in the order Ser, Asp, His. This clan includes the families (characteristic member in
parentheses) S9 (prolyl oligopeptidase), S10 (carboxypeptidase C), S15 (Xaa-Pro
dipeptidyl -peptidase), S28 (lysosomal Pro-Xaa carboxypeptidase), S33 (prolyl amino-
peptidase), and S37 (StreptomycesPS-10 peptidase). The characteristic catalytic dyad
Ser, Lys of clan SE is represented by the motif Ser-Xaa-Xbb-Lys,and the fold consists
of helices and an a+p sandwich. The families of this clan S 1 1 (penicillin-binding
protein 5), S12 (Streptomyces RG1 D-Ala-D-Ala carboxypeptidase), S13 (penicillin-
binding protein 4) are involved in the biosynthesis, turnover and lysis of bacterial cell
walls.
The catalytic residues in clan S F (catalyticdyad Ser, Lys or Ser, His) are more widely
spaced in comparison with clan SE. The families of this clan include only endopepti-
dases from bacteriophages, bacteria, archaea and eukaryotes with the members: S24
Table 12.5-2. Evolutionary classification o f peptidases into families and

I 8”
clans based on primary and teriary structure.

Class Families Clans (families) Catalytic residues
(E.C. list)
Serine SA (Sl-3,6,7,29-32,35,43) His, Asp, Ser
(E. C. 3.4.21.)
SB (S8) Asp, His, Ser
SC (S9,10,15,28.33,37) Ser, Asp, His
SE (Sll-13) Ser, Lys
SF (S24,26,41,44) Ser, Lys, (His)
SH (S21) His, Ser, His
TA (S42) Thr
SX (14,16,18,19,34,38,39,43)
Cysteine CA (C1,2,10,12,19) Cys, His, Asp (Asn)
(E.C. 3.4.22.)
CB (C3,4,24,30,37,38) His, Cys
CC (C69,1G,21,23,27-29,31-36, Cys, His
41-43)
CD (C14) His, Cys
CE (C5) His, Glu(Asp),Gln, Cys
CX (C11,13,15,22,25,2G,39,40)
Aspartic AA (A1-3,9,10-18) Asp, Asp
(E.C. 3.4.23)
AB (AG, 21) Asp, Asn
Metallo MA (M1,2,4,5,9,13,30,36,48) His, Glu, His
(E.C. 3.4.24) (HEXXH)
MB (M6-8,10-12) His, His/Asp
(HGXXHXXGXXH/D)
MC (M14) His, Glu, His
(HXXE/H)
MD (M15) His, His, Asp
(HMYGHAAD)
ME (M16,44) His, Glu, His
(HXXEH)
MF (M17) LYS, Asps, Glu
(NTDAEGRL)
MG (M24) Aspz, His, Gluz
MH (M18,20,25,28,40,42) His, Asp,, Glu
MX (M3,19,22,23,26,27,29,32,34-38,
41,43,45,47)
(Lex A repressor), S2G (signal peptidase I), S41 (TSP protease), and S44 (tricorn
protease). All known members of clan SH (catalytic triad: His, Ser, His) are
endopeptidases from DNA viruses which are involved in virus prohead assembly.
The clan includes only the family S21 (Cytomegalouirusassemblin). Clan TA with the
catalyhc residue Thr, Ser or Cys, and an a,p,a,p sandwich fold includes a number of
peptidases whose only proteolytic activity is self-activation.Important families of this
clan are T1 (proteasome),and S42 (y-glutamyltranspeptidase).
Other families (clan SX) of serine peptidases including S14 (endopeptidase Clp),
S1G (endopeptidase La), S18 (omptin), S19 (cell wall-associated endopeptidase of
812
Trichophyton), S34 (HflA endopeptidase), S38 (Treponema chymotrypsin-like endo-

peptidase), S39 (cocksfoot mottle virus endopeptidase), S43 (porin) cannot yet be
assigned to clans, since neither the tertiary structure nor the order of catalytic
residues are known.
The cysteine peptidases comprise the clans CA, CB, CC, CD, CE and CX. The last
includes a number of other families of cysteine peptidases for which tertiary
structures are unknown and virtually nothing is known about the specificity of the
catalytic machinery.
The clan CA contains papain and its relatives. Papain was the first clearly studied
cysteine peptidase. From the crystal structure of papain and a few closely related
peptidases of the family C1, it could be concluded that the catalybc residues are Cys,
His and Asn in that order of sequence. Further members of C1 are the cathepsine B,
H, K, L and 0, the dipeptidyl peptidase I, and glycyl endopeptidase. The C2 family
contains various calpains, whereas streptopain belongs, to C10 ubiquitin C-terminal
hydrolyse PGP 9,s to C12, and the isopeptidase T to C19.
Clan CB contains viral “chymotrypsin-like” cysteine peptidases that process the
viral polyproteins, and in clan CC are listed viral “papain-like’’endopeptidases. The
only family of clan CD (C14) comprises a number of cytosolic endopeptidases which
cleave aspartyl bonds with high specificity. This family of caspases consists of ten
members from which caspase-1 and caspase-3 are best known. The mature caspase-
1, processed from a single-chain precursor by presumably autocatalytic cleavage of
four aspartyl bonds, is a heterodimer of a 22 kDa heavy chain and a10 kDa light
chain [I9]. This peptidase was formerly known as interleukin 1P-converting enzyme
(ICE) since it mediates, among other things, the processing of interleukin l p at
aspartyl bonds. Human caspase-3 is also a heterodimer consisting of the subunit p12
(11896 Da) and the subunit p17 (16617 Da) with a tertiary and quarternay structure
similar to caspase-1 L20]. This peptidase appears to function in order to proteolytically
inactive proteins which are involved in cellular repair and homeostasis during the
effector phase of apoptosis.
Clan CE contains only the adenovirus endopeptidase[”]. A catch-all clan CX
comprises all other families of cysteine peptidases which could not been classified
up to now due to the lack of necessary data of structure and catalytic maschinery.
Aspartic peptidases have so far been described for all endopeptidases. Unfortu-
nately, the tertiary structure has only been elucidated for four families. Endopepti-
dases of the family A 1 consist of two lobes, with the active site between them. One
lobe has been derived from the other by gene duplication. In the active site each lobe,
with very similar three-dimensional structures, bears one Asp residue of the catalytic
dyad. It is interesting to note that the crystal structure of retropepsin from family A2
of clan AA showed a single lobe with one catalytic Asp residue with structural
similarity to one lobe of the pepsin from family Al. Retropepsin is only active as a
homodimer forming the catalytic site between the two monomeric molecules. There
is evidence that the peptidases of families A1 and A2 have evolved from a common
ancestor. Unfortunately, a number of other families could not yet been assigned to
any clan.
Metallopeptidases are allocated to eight clans. A couple of families could not yet be
I 813
assigned to these clans since, in particular, the metal ligands have not been
biochemically characterized. Zinc-dependent metallopeptidases,both exopeptidases
and endopeptidases, with the HEXXH motif are listed in the clan MA. The family
M4 contains along with thermolysin, and elastase (Staphylococcus) well-known
peptidases. The tertiary structure has been determined for members of this family
showing a two-domain structure with the active site between the domains. The N-
terminal domain contains the HEXXH motif and includes both a helices and /3
sheets as dominating structure elements and shows some similarities to the domain
structure of clan MB.
In the C-terminal domain are five helices in a closed bundle. This characteristic
fold is typical of thermolysin-like peptidases. Clan MC contains metallocarbox-
ypeptidases which belong to only one family (M14) which is divided into the
subfamilies A, B and C. Typical for this clan is that one zinc ion is tetrahedrally
coordinatedby a water molecule, two histidine and one glutamate residues. Clan MF
includes aminopeptidases that require cocatalyhc zinc ions for their enzymatic
activity. The well-known leucyl aminopeptidase has a two-domain structure bearing
the active site in the C-terminal domain. Whereas exopeptidases of clan MG require
cocatalytic ions of cobalt or manganese, clan MH contains the third group of
metallopeptidases that also require cocatalytic metal ions, but here these are all zinc
ions. The third clan in which cocatalytic metal ions are necessary is clan MF with
zinc or manganese. Only one catalytic zinc ion is required for peptidases of clans
MA, MB, MC, MD and ME.
12.5.2.2
Importance of Proteolysis
Historically, enzymatic proteolysis has generally been associated with protein diges-
tion. Therefore, the digestive peptidases of the pancreatic and gastric secretions are
among the best characterized peptidases and much of the current knowledge of
structure and function has been derived from investigations of those proteolytic
enzymes. Activation of the pancreatic digestive enzymes is initiated by enterokinase,
an enzyme secreted by the mucous membrane of the stomach. It converts some
trypsinogen into active trypsin, which then activates all the proenzymes, including
more trypsinogen. The function of the digestive proteases is merely to breakdown all
the proteins they encounter.
Later, it became evident that peptidases play regulatory roles in a great variety of
physiological processes [22* 231. These include processing and molecular assembly of
nascent polypeptide chains, and the processing of protein hormones, developing
enzyme precursors to mature enzymes, fertilization, many other proteolytic proc-
esses important for cellular functions, and the regulation of the programmed cell
death (apoptosis).The last is a mechanism that regulates cell number and is vital
throughout the life of all animals. Apart from various biochemical events involved in
apoptosis, the most fundamental one is the participation of members of the caspase
family in both the initiation and execution phases of cell death. The mechanism of
activation of the caspases constituting the different apoptosis-signaling complexes
814
can be explained by an unusual capability of the caspase zymogen to autopress to an

active
Proteolytic processing occurs in many different ways and is triggered by different
proteases. Limited proteolysis is the key to this selectivity which depends on the
accessibility of the scissile peptide bond to the acting peptidase and on its specificity.
In this cases proteolysis is directed and limited to the cleavage of specific bonds in
the target protein. A wide variety of prokaryotic and enkaryotic proteins are
synthesized as larger pre- or pre-proforms. Some of these are biologically inactive
and become activated upon limited proteolysis. Lysomal enzymes, mitochondria1
proteins, membrane proteins, secreted proteins etc. undergo intracellular proteolytic
maturation.
Various viruses code for specialized peptidases which are essential for virus
A couple of viral peptidases are interesting therapeutic targets. An
extremely large number of publications have been dedicated to the aspartic pepti-
dases, especially to the enzyme of the human immuno deficiency virus (HIV),which
is a key target in the treatment of AIDS. HIV-1 protease (HIV-1 PR), more exactly
named as human immunodeficiency virus 1 retropepsin (HIV-1retropepsin; E. C.
3.4.23.16), has become the most thoroughly investigated system in the history of
peptidases. The biological function of the retroviral peptidase is to cleave the
polyprotein precursor into its constituent functional units such as the matrix, capsid,
and nucleocapsid structural proteins of HIV to permit assembly. For this reason, the
great interest in HIV-1 retropepsin has centered on the development of compounds
that selectively inhibit the viral enzyme and not the related human aspartic
peptidases. Useful principles of inhibition have been combined by several com-
panies to produce antiviral compounds that have achieved approval from the Food
and Drug Administration (FDA) in the USA (cf.review[25]). Despite the development
of extremely strong and selective inhibitors which have been demonstrated to be
effective in human trials one major problem remains: the extremely rapid develop-
ment of forms of the virus that are resistant to the drugs containing the inhibitors.
Secretory proteins are usually synthesized as precursors bearing an aminotermi-
nal extension. The signal peptide is removed co-translationallyby signal peptidases
during translocation across the membrane. In the next step precursors of protein
hormones, growth factors and certain polycistronic precursor proteins are processed
by specific enzymes. In contrast to consecutive zymogen activation consecutive pre-
pro-cleavage reactions are regulated independently. The pathway of processing of
many pre-proteins is known, but many of the maturation peptidases can not yet be
characterized. For this reason, the application of molecular cloning techniques will
be helpful in the near future for the sequence elucidation of pro-proteins as well as
the cDNA and genomic sequences for maturation enzymes. The structure changes
range from the relatively simple alterations in zymogen activation to more complex
processing events in multidomain peptidase precursors, such as prothrombin or
plasminogen. Generally, proteolpc processing induces intramolecular rearrange-
ments required for the expression of biological response. Like blood coagulation,the
complementary system is triggered by a signal that activates several consecutive
zymogen activation reactions. This later system takes part in the immune reaction
directed against foreign organisms of tissues. Several components of the com-

plementary system are serine peptidases.
Peptidases as integral components of cells have only been partly explored, e. g.
lysosomal peptidases, granulocyte serine peptidases, membrane-bound peptidases,
and enzymes of specialized tissues, such as the reproductive tract, skins, lens,
muscle, pituitary, adrenals etc. Various ATP-dependent peptidases have been iso-
lated.
The proteasome is a large multifunctional protease complex that degrades intra-
cellular proteins. The name is derived from protease (“protea-”) and large particle
(“-some”) This complex is an exception among peptidases as regards the nucleo-
philic residue and the general structure. Both in eukaryotes and archea, the
proteasome is a multisubunit complex comprising four stacked rings each contain-
ing seven subunits (M, -20-30 kDa). In eukaryotes the 20s proteasome (E.C.
3.4.99.46; also named multicatalytic proteinase, macropain and prosome) has the
form of a hollow cylinder (length 148 A, diameter 113 A). It shows several different
catalytic activities and contains 14 different but homologous subunits, whereas in
archea there are just two different kinds of subunits, and the enzyme complex
possesses only one catalytic activity.
In bacteria the proteasome is built up of two rings of six subunits. One of the two
different subunits is related to the eukaryote and archaen proteasome subunits, the
other is an ATPase. The 26s protea~ome[’~,”1 ( M , - 2100 kDa) consists of the 20s
proteasome and at least one other multisubunit regulatory protein known as PA700,
19s cap, p-particle, ball, and ATPase complex. It was first found in extracts of rabbit
reticulocytes by its capabilityto degrade ubiquitinated proteins in an ATP-dependent
manner. Since this complex can also degrade various nonubiquitinated proteins the
older designation ubiquitin-conjugate degrading enzyme (UCDEN) is probably
inappropriate. The 20s proteasome subcomplex of the 26s proteasome containing
multiple catalytic sites with distinc specificities is responsible for the whole proteo-
lytic activity. In addition, the PA700 regulatory complex displays further enzymatic
activities, such as ATPase activity, isopeptidase activity and seems to contain a
substrate protein unfolding activity. The ATPase activity is necessary for assembly of
the 26s proteasome from the 20s proteasome and PA700 subcomplexes and also for
the degradation process. Since peptide bond hydrolysis is not energy dependent, the
hydrolysis of ATP might be required for unfolding protein substrates and/or for
translocation of the unfolded peptide substrate into the central channel of the
proteasome. The proteasome is responsible for turnover of most cellular proteins in
mammalian cells and the selective degradation of proteins with abnormal structures.
Last but not least, the proteasome is involved in the production of antigenic peptides
for presentation by MHC class I complexes. The generation of antigenic peptides
seems to be performed by a specific subpopulation of proteasomes containing two or
three subunits encoded in the major histocompatibility complex.
Considerable attention has been paid to a group of intracellular serine peptidases
associated with granulocytes as well as leukocytes and mast cells as mentioned
above. These peptidases are stored in granulas and released in response to in-
flammatory or allergic stimuli. Many of the peptidases are relevant to human health
816
and disease r2’1, some as natural components of the human body, and others because
they are important in species which provide us with food, or cause diseases.
In oder to understand proteolytic activity in biological processes, knowledge of the
contribution of the natural peptidase inhibitors to the regulation of the activity is
Inhibitors are as diversified as the proteases themselves. Generally, they
can be divided into two main classes: (a) active site-specific low-molecular-mass
inhibitors, and (b) naturally occuring protein peptidase inhibitors. Examples of the
first group are the serine peptidase inhibitors diisopropyl phosphofluoridate (DFP)
and phenylmethanesulphonyl fluoride (PMSF).Both react with the active site serine.
Many of the naturally occuring peptidase inhibitors, isolated from animal, plant and
bacterial organisms, behave as pseudosubstrates. They combine essentially irreversi-
bly with the active site and are converted into a modified form in which a peptide
bond, related to the primary substrate specificity of the peptidase, is cleaved.
Of special physiological interest are inhibitors which react with mammalian
plasma serine peptidases, especially those involved in blood coagulation. In princi-
ple, such inhibitors have both protective and regulatory functions. Approximately
10% of the nearly 200 proteins in blood serum are peptidase inhibitors. ?lie al-
proteinase inhibitor secreted by the liver, for example, inhibits leukocyte elastase
which is thought to be part of the inflammatory process. Furthermore, special
variants of this inhibitor with reduced inhibiting power are associated with pulmo-
nary emphysema. The latter is a degenerative disease of the lungs which results from
the hydrolysis of its elastic fibers. Interestingly, certain plants release peptidase
inhibitors in response to insect bites in order to inactivate the digestive enzymes of
the attacking insect.
Peptidases are valuable tools in the study of the primary and higher-order
structure of proteins r3l]. Proteolysis of proteins for sequence analysis and peptide
mapping can be carried out according to different Based on the extent
of proteolytic reaction, it is allowed to reach completion or it is prevented from
reaching completion. In the first case the products constitute an equimolar set of
peptides whose composition will not be influenced by further digestion with same
enzyme. Depending on the restriction imposed by the primary specificity of the
peptidase used, a protein will be fragmented to varying degrees. The fragments can
subsequently be separated and sequenced. Combining these data with sequence data
of other overlapping sets which are generated with different peptidases allows the
reconstruction of the sequence. If proteolysis is prevented from reaching completion
a different set of data is obtained. Inhibition or removal of the peptidase are desirable
interventions to determine the initial cleavage products.
Furthermore, peptidases are also structural probes of conformation of soluble
proteins [331. Although X-ray crystallography[34] and two-dimensionalN M R L3’1 are the
methods of choice for the determination of the three-dimensional structure of
globular proteins, some weaknesses of these techniques demand alternative meth-
ods even if these will provide structural information at a lower level of resolution. For
example, limited proteolysis can be used to probe the structure and the dynamics of
proteins in solution, which provide experimental data that are easy to obtain and
complement well those results derived from the techniques mentioned above. The
I 817
goal of investigations are soluble proteins in their native or near-native states.
Limited proteolysis occurs in this case at the level of only one or very few peptide
bonds which leads to the formation of “nicked proteins. This species of proteins
consists of rather large fragments which remain associated in a stable and often also
functional complex. Usually, a nicked protein is much more labile than the native
form. Therefore, the unfolding leads to a suitable substrate for an extensive
proteolytic degradation to small peptides, and further proteolysis is much faster in
comparison with the initial peptide bond cleavage at the level of the native protein.
Consequently, in this case, during proteolysis the intact protein and small proteins
are present in the incubation mixture, without intermediate sized products. In the
case where nicked proteins are sufficiently stable, they may resist further extensive
proteolytic degradation and can be isolated and characterized.
It is assumed that the limited proteolysis phenomenon derives from the fact that a
specific polypeptide chain segment of the compact, folded protein substrate is
exposed and flexible so that it can fit the active site of the appropriate peptidase for an
efficient and selective limited hydrolysis. There is no doubt that enhanced chain
flexibility or segment mobility is the key feature of the site of peptide bond hydrolysis
demonstrated by a clear-cutcorrelation between sites of proteolytic attack and sites of
enhanced chain flexibility. The present availability of automatic, efficient and
sensitive techniques of protein sequencing and, particularly, the recent dramatic
advances of mass spectrometry[36]in the analysis of peptides and proteins, allows a
more systematic use of the limited proteolysis approach as a simple first step in the
elucidation of structure-dynamics-function relationships for novel proteins which
are only available in minute amounts.
Since a growing number of newly discovered peptidases are specifically expressed
in single tissues, especially, at low expression levels or often only at certain
development stages, it is very complicated to isolate the enzymes in sufficient
quantities using classical biochemical procedures. Therefore, the only alternative is
the cloning and expression of these peptidases. In addition, recombinant techniques
allow directed structural alterations in order to program mechanistic or functional
features. Peptidases can be expressed in most of the developed expression systems
(yeast, viral, bacterial, insect cells and mammalian). It is not usually easy to predict
which expression system is the method of choice. For functional expression of
recombinant peptidases various examples have been presented [37].
Last but not least, it should be mentioned that a couple of peptidases have
industrial importance. In particular, since subtilisins have a broad substrate specific-
ity and are highly stable at neutral and alkaline pH they are of considerable industrial
interest as protein-degrading additives to detergents. These reasons combined with
their large data base make subtilisins attractive for protein engineering. Extensive
engineering studies have been carried out on the Bacillus subtilins and more than
500 site-directed mutants have been produced to alter specific enzyme properties,
such as pH profile, thermal stability or substrate specificity (see e.g. refer-
enceSL37-391 ).
818
12.5.3
Formation of Peptides
12.5.3.1
Tools for Peptide Synthesis
Although the origins of peptide chemistry are usually traced back to the early 20th
century when Emil Fischer obtained the simplest dipeptide glycyl-glycineby cleavage
of the appropriate diketopiperazine, the first peptide bond in a chemical laboratory
was synthesized by the young Theodor Curtius in the laboratory of Hermann Kolbe
at Leipzig University in 1881. Despite the fact that Emil Fischer with co-workers in
Berlin made basic contributions to peptide synthesis, the productive epoch of
peptide chemistry began some decades later in the1950s. Wieland and Bodan~zky[~']
have written an excellent account of the history of peptide synthesis.
Peptides belong to an increasingly important class of bioactive molecules in
physiology, biochemistry, medicinal chemistry and pharmacology. They act as
hormones, neurotransmitters, cytokines, growth factors etc. However, it is not only
naturally occuring physiologically relevant peptides that are the subjects of interest.
Peptide analogs possessing agonist or antagonist activity are also useful tools in
investigations when searching for suitable drugs. Radiolabeled analogs and mole-
cules bearing affinity labels have been applied for the characterization and isolation
of receptors. Furthermore, peptides are useful as substrates of peptidases, kinases,
phosphatases and special transferases in investigations on enzyme kinetics, and
mechanisms of action. In the preparation of polyclonal and monoclonal antibodies
peptides play an important role as synthetic antigens, and epitope mapping using
synthetic peptides has been developed as a valuable approach for the identificationof
specific antigenic peptides for the preparation of synthetic vaccines, and also for the
determination of protein sequence regions which are important for biological
function. In addition, the design of small peptide mimetics of protein function or
structure, and the development of various peptidomimetics in drug development are
further goals in peptide chemistry. In particular, in the last ten years the number of
known peptides has doubled and besides the development of efficient chemicals for
peptide synthesis methods, the field of peptide and protein chemistny has been
opened up to molecular biology and genetic engineering.
The classical chemical peptide synthesis is a synthesis in a homogeneous
s o l ~ t i o n [ ~ ~Even
- ~ ~ in
] . the 1950s this approach had started to gain industrial
importance followed by the solid-phasetechnique in the early 19GOs, invented by the
Nobel laureate Bruce Merrifield147-501. The most fundamental time-consuming
operations in chemical peptide synthesis (sometimes not free from undesirable side
reactions) are the selective protection, and after synthesis the deprotection of the a-
amino function, the carboxyl group and the various side chain functionalities of
trifunctional amino acids. Despite the development of numerous efficient protection
methods based on chemical techniques, the whole process is rather slow as all
intermediate products have to be purified and characterized after each reaction step.
The formation of each peptide bond requires the activation of the carboxylic acid
function of the carboxyl moiety.
An important point in selecting a coupling method is its degree of safety from

racemization, since all synthetic operations carried out at a center of chirality have
this permanent risk. Therefore, the synthesis of peptides with a multitude of chiral
centers continues to be a formidable chemical effort. The existence of more than 150
chemical variations for peptide bond formation indicates that an ideal coupling
method does not exist, e.g. a fast procedure without racemization or other side
reactions to realize quantitative coupling of equimolar amounts of the carboxyl and
amine components. There is no doubt that the use of well known strategies and the
application of activation methods with well established safety steps to protect from
racemization in simple model systems does not assure the loss of optical purity
during a multitude of coupling steps in the synthesis of medium-sized and long
peptides. For the chemical peptide synthesis in homogeneous solution, which still
plays an important role in the production of large quantities of peptides for
pharmaceutical use, highly skilled personel are required. In this manner multi-
kilogram quantities or even tons of peptides consisting of the range of 2-30 amino
acid residues can be produced.
Since peptides for research purposes are usually required in only mg tog
amounts, the time-saving solid-phase peptide synthesis can be used.
The strategy is in principle similar to that in solution, with the difference that there is
no need for isolation of the intermediate products. As the growing peptide chain is
synthesized on a suitable resin the whole procedure lends itself to automation. The
drawback is that every reaction step at the resin has to be forced to give an almost
100% yield. In practice, this cannot be accomplished,with the consequence that the
desired product must be isolated from a mixture of side-products by the final,
normally HPLC, purification procedure which is sometimes difficult to perform and
also expensive. Peptides of up to - 50 amino acid residues are now readily accessible
using stepwise solid-phaseprocedures C5O1.
An alternative for the preparation of larger polypeptides and proteins is the
biotechnological production (geneticengineering, recombinant DNA technology,) in
bacteria, yeast, or cultured mammalian cells [51-531. In principle, this is an economic
way to produce peptides of more than 50 amino acid residues and even small
proteins with complicated glycosyl or other groups attached to amino acid side
chains. Compared with the problems connected to the chemical synthesis strategies,
recombinant techniques provide quite a different set of problems. Whereas the
principle of the expression of a gene in host cells through the normal biosynthetic
and genetic machinery of the host cell using a suitable expression vector is relatively
simple, putting this technique into practice poses some problems: Selection of the
appropriate expression strategy, and the host cell system as well as the optimal vector
system, the control of the stability of mRNA and also of the translated protein,
isolation and purification of the product, scale-up,downstream processing etc.
The development of cloning vectors which propagate in eukaryotic hosts, e.g.
yeast or cultured animal cells, has in particular eliminated many of the problems
associates with the synthesis of eukaryotic proteins. It should be noted that post-
translational processing may also vary among different eukaryontes. It is an
advantage that shuttle vectors are available that are capable of propagating in both
820
Figure 12.5-6. Principle of native

-/(o>s->-] chemical ligation according to Dawson
SR H2N et al.[551.
Step 1
0
1Trans thioesterification
Peptide 2
H2N
Step 2
iS to N Acyl transfer
yeast and E. coli and thus transfer genes between these two cell types. Recombinant
protein production is of great medical, agricultural, and industrial importance [541.
For example, human insulin, human growth factor, erythropoietin, various types of
colony-stimulating factors, blood clotting factors are typical examples of recombi-
nant proteins which are in routine clinical use.
Despite the fact that heterologous expression of recombinantly cloned genes is by
far the most commonly employed method of to engineering proteins this approach
is only applicable to naturally occurring amino acids. This limitation is in principal
overcome by unnatural amino acid mutagenesis [s41 and some other chemistry-
driven approaches. Among the various chemical ligation methods the so-called
“native chemical has proved to be a useful route to fully synthetic
proteins 155, 56-60] . A s shown in Fig. 12.5-6 this procedure relies on the reaction that
occurs between a peptide fragment possessing an essential N-terminal cysteine
residue (peptide 2; Fig. 12.5-6), which can be expressed in principal using re-
combinant DNA procedures, and a second peptide fragment possessing an a-
thioester group (peptide 1; Fig. 12.5-6).In an initial intermolecular, chemoselective
reaction a thioester-linked intermediate is formed (step 1) which spontaneously
rearranges via S+ N acyl transfer to the final amide-linked product (step 2). The
rearrangement step corresponds mechanistically to an intramolecular S+ N acyl
transfer reaction described by Wieland et al. r61] in 1953.
Pulling together protein splicing (for a review see reference[”]) and native
chemical ligation led to “expressed protein ligation” (EPL) [631 or also termed “intein-
mediated protein ligation (IPL) [641. As shown in Fig. 12.5-7 the protein fragment of
interest is expressed in E. coli as an intein-CBD (chitin binding domain) fusion
protein. The chitin binding domain allows protein affinitity purification using chitin
Figure 12.5-7. Principle of

I
Expressed protein ligation
expressed protein ligation
Clone gene into intein vector according t o Muir et a1.[63].
Express in
E. coli
0 HSJ
N 1 Recomb. protein ~--J--NH-C~~-/GH-
Affinity
purification
k Contaminants
0 HSj
N+ Recomb. protein P N H - C y s
N to S Acyl
4
transfer
0
N Recomb. protein
Synthetic peptide
(both in large excess)
Native chemical
ligation QUICK
Semi-synthetic protein
beads. The necessary expression vector is commercially available. The N-t S acyl
transfer results in a thioester-linked intermediate. In the next step a large excess of a
suitable thiol agent (for example thiophenol) generates, by trans-thioesterification in
situ, the protein a-thioester which reacts quickly with the simultaneously added
synthetic amine component. The latter has to bear an N-terminal cysteine residue.
Customized peptides containing N-terminal cysteine residues are available from a
variety of sources. There is no doubt that the extension of the native chemical
ligation to EPL led to significant progress in protein semi-synthesi~[~~I, despite the
remaining requirement of an N-terminal cysteine residue in the amine fragment to
822
be coupled. Apart from these advantages it must keep in mind that direct reaction of
a thiophenyl ester with the amine component could result in partial epimerization of
the C-terminal amino acid residue of the protein a-thioester.
12.5.3.2
Choice of the Ideal Enzyme
Enzymes have become valuable tools in medium to large-scale synthetic organic

Owing to the fact that hydrolases possess a wide substrate spectrum
and do not usually need cofactors for their catalpc function, they are at present the
enzymes most widely used as biocatalysts in preparative organic chemistry. Among
the hydrolases the huge family of peptidases plays an important role in various
processes of proteolysis as shown above.
Unfortunately, a universal C - N ligase with a high catalytic efficiency for all
possible combinations of the 21 proteinogenic amino acids both as C- and N-
terminal amino acid residues, respectively, in fragments to be coupled could not be
developed during evolution. Such heavy demands on specificity could not even be
solved by nature. Therefore, protein biosynthesis has been developed as a step-wise
strategy starting with the N-terminus of the growing peptide chain and catalyzed by
the ribosomal peptidyltransferase. Limited proteolysis of the biosynthesis precursor
molecules and posttranslational modifications provide the bioactive peptides and
proteins. In nature the peptide bond formation is accomplished on the ribosome and
takes place via the transfer of a peptidyl residue from the peptidyl-tRNA in the
ribosomal P site to the amino group of aminoacyl-tRNA in the A site. Despite
intensive investigations in recent years, the nature and the basic mechanism of the
peptidyltransferase reaction within the ribosome is largely unknown. According to
recent studies from the Nobel laureate Thomas R. Cech and coworkers[5]an in uitro
selected ribozyme is capable of catalyzing the same type of peptide bond formation
as a ribosome, e.g. its sequence and secondary structure seems to be strikingly
similar to the “helical wheel” portion of 23s rRNA implicated in the activity of the
ribosomal peptidyltransferase. These results provide evidence for the feasibility of
the “RNA world” hypothesis by demonstrating that RNA itself is capable of
catalyzing peptide bond formation.
Furthermore, from these findings the idea that the rRNA has a catalpc function
in the ribosomal peptide bond formation is supported. It can be assumed that the
selection of the individual aminoacyl-tRNAfor the A site is mostly attributed to the
specificity of the appropriate aminoacyl-tRNA synthetase together with the specific
codon-anticodon interactions, whereas the 2 3 S rRNA participates in catalyzing
peptide bond formation but without side-chain specificity for the amino acid
esterified to the tRNA’s 3’-terminalnucleoside. In comparison with the prerequisites
for specificity of peptidases the peptidyltransferase seems to be an old unspecific
ribozyme in accordance with its function in evolution as precursor to extant life.
It is of interest to note that in a recent paper a possible mechanism for peptide
bond formation on ribosome without the mediation of peptidyltransferase has been
These authors assume, by analysis of the energetics using a semi-
72.5 Hydrolysis and formation ofpeptides
I
823
empirical method for the formation of a cyclic intermediate, that the peptide bond
formation through the tetrahedral intermediate in an S-configurationmay not need
assistance from an enzyme or ribozyme. From the tetrahedral intermediate a cyclic
intermediate will be formed, where the 2’-OHof the ribose sugar of the P-site tRNA
is a member of the ring, which produces a free tRNA and a tRNA attached to a planar
peptide unit. Since the free 2’-OH group of the peptidyl-tRNA was proposed to be
involved in peptide bond formation, it has been argued that the appropriate tRNA
may be acting as a biocatalyst (enzyme or ribozyme).
Even in the case it should be possible to separate ribozyme activity from the
ribosome or to isolate an in vitro selected ribozyme that can catalyze the same type of
peptide bond formation as a ribosome, however such a biocatalyst seem does not to
be suitable for simple practical use rather than using a chemical coupling reagent. In
principle, this conclusion is also valid for the nonribosomal poly- or multienzymes
which are involved in the biosynthesis of peptide antibiotics[721. Up to now, they have
only found application in the synthesis field of cyclosporin, gramicidin S, special p-
lactam antibiotics and analogs.
At the end of this short assessment only those enzymes that usually act as
hydrolases catalyzing the cleavage of peptide bonds remain to be discussed. The
fundamental suitability of peptidases for catalyzing the formation of peptide bonds
is based on the principle of microscopic reversibility that was predicted by van? Hoff
in 1898[731. On the last page of his contribution he had proposed the basic idea of
peptidase-catalyzedformation of the peptide bond, as follows:
“Die Frage ist berechtigt, ob . ... auch nicht das Trypsin imstande ist, unter Umstanden,
durch die Gleichgewichtslage gegeben, Eiweiss zu bilden aus den Spaltprodukten, die es
selber bildet”.
The concept of van’t Hoff of the equilibrium constant of a reversible chemical
reaction, along with the function of a catalyst (including biocatalysts) for accelerated
achievment of the equilibrium according to O ~ t w a l d [ ~is~ Ithe , theoretical back-
ground of enzyme-catalyzed peptide synthesis. However, about 40 years elapsed
before the first experimental proof of van’t Hoff‘s prediction became evident through
the first clear-cut peptidase-catalyzed synthesis of an amide bond carried out by
Bergmann and Fraenkel-Conrat[”].Before this approach gained any industrial im-
portance another 40 years had elapsed, and in recent decades considerable efforts
have been made to find the optimum conditions for peptidase-catalyzed peptide
synthesis as can be seen in various reviews [7G-981.
12.5.3.3
Principles of Enzymatic Synthesis
As shown in Fig. 12.5-8the equilibrium of a peptidase-catalyzedreaction is normally

shifted to the thermodynamically more stable cleavage products. In contrast to
proteolysis, the peptide bond formation is a two-substrate reaction and requires not
only a specificity-dependentinsertion of the carboxyl component into the S-subsites
of the active site, but also an optimal binding of the amine component in the S’
region. To shift the equilibrium in favor of fragment product formation various
824
Responsiblefor cleavage specificity
[Primary specificity] ?
R3 R2 R' R1' R2' 73'

I I I I I
-NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-CO-
p3 p2
oe
t 1 P'l
Protease
R"
P'2 P'3
T3
-NH-CH-CO-NH-CH-CO-NH-CH-C<
R2
I
R1
I
+
( 8 1
R2'
I
R3'
I
H3N-C H-CO-N H-C H-CO-N H-C H-CO-
p3 p2 PI O P'l P'2 P'3
t
Responsiblefor the primary
t
Important for the nucleophile
specificity in synthesis efficiency in kinetically controlled
peptide bond formation
Figure 12.5-8. Peptidases function in vivo as hydrolases rather than as ligases.
manipulations are necessary which also differ mechanistically. The approaches to

peptidase-catalyzedpeptide bond formation are generally classified into basic strate-
gies (see below) according to the type of carboxyl component used. In the equilib-
rium-controlled approach the carboxyl component bears a free carboxyl group as
shown in Fig. 12.5-9, p. 826, whereas in the kinetically controlled approach the
carboxyl component is employed in a slightly activated form, mainly as an alkyl ester.
Both strategies are fundamentally different due to the energy required for the
conversion of the starting components into the peptide products. Before interpreting
the two mechanisms in more detail some general considerations of reversing
proteolysis must be discussed.
12.5.3.3.1 General Manipulations in Favoring Synthesis

Looking at the equilibrium for the reversal of proteolysis, under normal conditions
the equilibrium is shifted towards the hydrolysis products. For example, a synthesis
of a dipeptide from its constituent free amino acids is, from the energetic point of
view a very unfavorable process because of considerable increase in the free enthalpy
involved. Under these circumstances it is not possible to accomplish peptide bond
formation by simple reversal of hydrolysis, even using high concentrations of the
starting amino acid zwitterions. Energetically more favorable is the reaction of an
anion and a cation using an P-protected amino acid as a carboxyl component and a
P-blocked amino acid as an amine component, respectively. According to the
underlying thermodynamic principles, the outcome of peptide synthesis in aqueous
solution depends on (a) the value of the equilibrium constant, (b) the ionization
constants of the selectively protected starting compounds and (c) the initial concen-
trations of the ionized and nonionized forms of the carboxyl and amine component.
I 825
The thermodynamic parameters only allow statements relating to the free enthalpy
change between the start and the end of the reaction, e. g. the equilibrium of the
reaction. Only the velocity with which the equilibrium is reached depends on the
catalytic action of the enzyme used.
According to the law of mass action the product yield is proportional to the starting
component concentration. Using the least expensive starting component in excess,
manipulations described in the following make it possible to transform the other
starting component almost quantitatively into product.
The formation of insoluble products is a useful way of shifting the equilibrium
towards synthesis.The reaction medium must be designed so that both starting
components on the left-hand side of the equation are soluble in the medium while
the peptide product on the right-hand side is insoluble. Under these conditions the
product is continuously removed from the reaction medium by precipitation and
sometimes an almost quantitative product yield can be obtained. A second way of
reversing the proteolysis reaction can be performed by product extraction, a concept
quite close to the solubility-controlled process of precipitation. The reaction is
carried out in a biphasic system where the product is much more soluble in the
organic phase and is continuously removed from the aqueous phase where the
starting components and the enzyme are soluble. In both approaches to product
removal the benefits of the appropriate organic solvent must be taken into considera-
tion, which will be discussed later. Last but not least, in special cases the formed
product can be separated from the equilibrium by molecular traps, where the desired
product will removed by specific complex formation, as demonstrated, for example,
in the course of clostripain-catalyzed fragment condensation of the ribonuclease
(RNase)fragments 1-10 with 11-15 using RNase S(21-124) as a trap["].
12.5.3.3.2 Equilibrium-controlledSynthesis
This equilibrium-controlledor thermodynamic approach (see below) represents the
direct reversal of proteolysis. Consequently, all peptidases, independent of their
mechanisms, can be used. Apart from this advantage the necessary high enzyme
requirement and the low reaction velocity are drawbacks of this approach.
Preceding the conversion, determined by Ken, is an ionization equilibrium Kion:
+ KLmm IL."
RCOO- + H3NR' + RCOOH + HzNR' RCO- NHR' + HzO (1)
If the water concentration is taken into the equilibrium constant Eq. (2) is obtained:
+
K s p = Kion . &on = [RCO-NHR']([RCOO-][H3NR'])-' (2)
The reaction medium, especially the pH, determines the constants for a given pair of
reactants. To obtain an equilibrium that is shifted in favor of peptide product
formation the ionization equilibrium must be manipulated. One efficient method is
the addition of water-miscible organic solvents to the aqueous reaction mixture
thereby lowering the dielectric constant of the medium, reducing the acidity of the
carboxyl group, and to a lesser extent the basicity of the amino group of the
nucleophilic amine component [loo,l o l l . The use of biphasic systems (for a review
826
see reference[102]),e.g. solvent systems consisting of an aqueous phase and a

nonmiscible phase (nonpolar organic solvents)does not damage the enzyme since it
is localized in the aqueous phase. Under ideal conditions the reactants diffuse from
the organic phase into the aqueous phase and after peptide bond forming step the
product diffuses back into the organic phase. Only the insufficient solubility of the
reactants in nonpolar organic solvents limits the general application of the biphasic
approach, particularly for the condensation of longer segments.
For the direct reversal of catalybc hydrolysis of peptides, discussed in this chapter,
the term equilibrium-controlled approach should be preferred. Because of the
thermodynamic control of both equilibria in Eq. (1)the reversal of proteolysis is
often denoted as a thermodynamic approach. In order to increase the product yield
of this endergonic process various manipulations are required. In addition to those
mentioned above, reverse micelles I1O3], anhydrous media containing minimal water
concentrations [lo41,water mimics [*''I, and reaction conditions promoting product
precipitation as discussed in first part of this chapter are often employed.
12.5.3.3.3 Kinetically Controlled Synthesis

In contrast to the equilibrium-controlled approach the peptidase-catalyzed kinet-
ically controlled peptide synthesis (for a review see needs much less
enzyme, the reaction time to reach maximal product yield is significantly shorter,
and the product yield depends both on the properties of the enzyme used and the
substrate specificity. Kinetic control means that the product appearing with the
highest rate and disappearing with the lowest velocity would accumulate. Whereas
the equilibrium-controlled approach ends with a true equilibrium, in the kinetic
approach the concentration of the product formed goes through a maximum before
the slower hydrolysis of the product becomes important. The product will be
hydrolyzed if the reaction is not stopped after the acyl donor ester is consumed and
true equilibrium is allowed to be reached.
In Fig. 12.5-9 both approaches are compared schematically. The kinetic approach
(b)requires the use of an acyl donor ester as a carboxyl component and is limited to
a R-COOH + H2N-R' b R-CO-X + H,N-R'
R-CO-NH-R'
41
+ H,O
1
R-CO-NH-R' + HX
t - t -
Figure 12.5-9. Comparison of the equilibrium (a) and the kinetically controlled approach
(b) of peptidase-catalyzed peptide synthesis.
I
827
Ac-E..HN EH + Ac-N
Figure 12.5-10. Kinetics of peptidase-catalyzed acyltransfer reaction.
EH =enzyme, Ac-X = acyl donor ester (carboxyl component), H N =
nucleophile (amine component), H X = leaving group o f t h e acyl
donor ester, Ac-OH = hydrolysis product, Ac-N = peptide product;
E..Ac-X = enzyme-substrate complex (Michaelis complex), Ac-N..HN
= acylenzyme-nucleophile complex.
peptidases which rapidly form an acylenzyme intermediate, e. g. serine and cysteine

peptidases. The peptidase acts as a transferase catalyzing the transfer of the acyl
moiety to the amino acid- or peptide-derived amine component. Specifically, the
acylenzyme reacts, in competition with water, with the nucleophilic amine compo-
nent to form the peptide bond. The ratio of formation of aminolysis and hydrolysis
products is of decisive importance for successful preparative peptide synthesis.
Figure 12.5-10 describes the kinetics of the peptidase-catalyzedacyl transfer reaction.
First, the acylenzyme is formed via the Michaelis-Menten complex, which binds the
amine component to the acylenzyme. The resulting acylenzyme-nucleophile com-
plex can undergo aminolysis as well as hydrolysis. The acyl transfer efficiency of the
peptidase for the corresponding substrates is determined by the ratio of the
aminolysis and hydrolysis product formed, which is also denoted as selectivity.
12.5.3.3.4 Prediction of Synthesis by S’ Subsite Mapping.

Serine and cysteine peptidases are not perfect acyltransferases.Therefore, it is useful
to have a method for the prediction of the outcome of the kinetically controlled
peptide synthesis. In order to get a simple efficiency parameter we decided to
introduce the partition value p1106]analogous with the definition of the Michaelis
constant according to Eq. (3), where Pz = Ac-OH, P3 = Ac-N, and N = HN.
The p value corresponds to the nucleophile concentration at which hydrolysis and

aminolysis of the acylenzyme proceeds with the same velocity. The advantage of p is
that the definition is not based on a particular kinetic scheme. Furthermore, p allows
[w
a rapid estimation of the yield of any acyl transfer reaction. A concentration of the
nucleophilic amine component >> p is necessary for peptide formation in high
yield. Assuming an equilibrium between the acylenzyme and the acylenzyme-
828
I 12 Hydrolysis and Formation ofC-A! Bonds
nucleophile complex, Eq. (4) and (5) can be derived from Fig. 12.5-10 for the
velocities of hydrolysis and aminolysis of the acylenzyme, where E = EH, EA = Ac-E,
A = Ac-X, and EAN = Ac-E.. HN.
k
V H = [EA]k3 + [EAN] KN
k4
VA = [EAN] -
KN
Eq. (6) results from combination Eq. (4) and (5).
It follows from Eq. (6)that a linear correlation between the partition value p and the
nucleophile concentration is obtained. The quotient ks/k4 corresponds to the ratio of
hydrolysis and aminolysis of the EAN complex whereas the term kNk3/k4 is a
measure of the nucleophile efficiency.
The partition value p can be determined by differnt m e t h ~ d s [ ' ~ ~ - 'In
~ ' ~the
.
presence of a large excess of nucleophile ([W >> [Ale) the decrease in the nucleophile
concentration during the reaction course can be ignored. Under these conditions vH/
V A = [P2]/[P3].
The determination ofp can be established out from the product ratio
obtained by HPLC analysis according to Eq. (7).
In the preparative application of acyl transfer reactions, however, a large excess of the
nucleophile is not useful because a complete turnover of both reactants is desired.
For this reason, we developed the determination of p from the integrated rate
eq~ation[~~'1according to Eq. (8).
A plot of [Pz]/[P3]versus ln([Nlo/([Nlo- [P3]))/[P3] gives a straight line with the slope
KN k3/k4 and an intercept with the y axis at kslk4. Since this method permits the
determination of p under the conditions employed in preparative peptide synthesis it
should be useful for the optimization of the reaction conditions.
An understanding of the molecular interactions between the acylenzyme and the
attacking nucleophilic amine component allows an optimization of the acyl transfer
efficiency. The efficiency of the nucleophilic attack of the amine component depends
essentially on an optimal binding within the active site by S ' - P' interactions
(Fig. 12.5-11). Consequently, more information on the specificity ofthe S ' subsites of
serine and cysteine peptidases are useful, which can be obtained by systematic acyl
transfer studies using libraries of nucleophilic amine components. According to the
definition of the p value (see above) small values of p indicate high S' subsite
specificity for the appropriate amine component in peptidase-catalyzed acyl transfer
reactions.
I
829
Figure 12.5-11. Schematic representation o f subsite-substrate interactions in the

course o f t h e acyl transfer from the acylenzyme t o the nucleophilic amine
component catalyzed by a serine peptidase.
We have studied a couple of different serine peptidases (for a review see

reference ["I), the cysteine peptidases papain [I' and clostripain['", 'I2], respec-
tively, and the prolyl endopeptidase from Hauobacterium meningosept~m[~'~I, and
have determined p values for various series of nucleophilic amine components.
Apart from clostripain none of the enzymes under investigation catalyzed acyl
transfer to nucleophilic amine components with P'1 = Pro or D-amino acids. The
efficiency of chymotrypsin-catalyzedacyltransfer decreases in the order of positively
charged > aliphatic > aromatic > nagatively charged PI1 side chains. The specificity of
chymotrypsin for P'1= Arg and Lys is attributed to electrostatic interactions between
these side chain moieties and Asp3' and Asp36in the active site. A statistical analysis
of proteolysis data confirmed that chymotrypsin possesses a specificity for peptide
bonds bearing Arg or Lys at the Po1position, whereas Leu-Asp bonds of proteins
were cleaved by this enzyme considerably less frequently than one expects from the
frequency of occurence of this peptide bond['l4]. Our results confirm this statistical
evaluation exactly. Furthermore, remarkably chymotrypsin prefers arginine residues
at the P'1 and PI3 positions, which offers an interesting option for using chymo-
trypsin in the sense of a restriction peptidase for peptide-catalyzed processing of
recombinant proteins (cf. Fig. 12.5-27 ).
The selectivity of the S' subsites of different peptidases is reflected by the broad
range of data obtained as shown for simple amino acid amides in Table 12.5-3. The
values demonstrate the preference of basic and hydrophobic P'' residues for
chymotrypsin and also for papain. In the case of chymotrypsinthe strongly basic side
chain of arginine amide gives rise to a higher efficiency than all other nucleophiles.
Despite the difficulties in catalyzing Xaa-Pro bonds, we have studied the clostripain-
catalyzed acyltransfer using a large number of proline-containing peptides as well as
830
Table 12.5-3. Comparison ofp values of selected amino acid amides H-Xaa-NH2 i n acyltransfer
reactions catalyzed by various serine and cysteine peptidases according t o Schellenberger and
Jakub ke Ig51.
~ ~~
Enzyme P
Xaa Arg Leu Val Met
EndoproteinaseGlu-C V8 > 500 16 117 64
Endoproteinase Glu/Asp-C 30 132 n. d. 382
Chymotrypsin 0.11 4.2 6.7 3.3
Tiypsin 66 72 130 12
Elastase 16 62 69 34
Papain 1.3 0.41 3.9 1.5
Ala-Xaa dipeptides and amino acid amidesL"" 'I2] . The efficiency of clostripain-
catalyzed acyltransfer, using Bz-ArgOEt as the acyl donor to amino acid amides
decreases in the order Leu > Lys > Gly > Arg > Gln > Ser > Pro > Thr > Ala > Asn >
Asp > Glu. S' subsite mapping using an Ala-Xaa library led to the result that
clostripain prefers PIz residues with positively-charged side chains, followed by
proline, whereas negatively-charged side chains of Asp and Glu are weak nucleo-
philic acceptors. In the pentapeptide series, containing only one proline residue, the
efficiency decreases in the order Pro-Pt3> Pro-P'2 > Pro-Ptl.Surprisingly, PAPAG,
PPAAG and PF-NH2 act as very weak nucleophilic acceptors. The variety of different
conformations of proline-containing peptides should be the reason for the extreme
differences in enzyme-nucleophile interactions.
12.5.3.3.5 What Approach Should be Preferred?

As mentioned above, the equilibrium-controlledapproach has the advantage that all
peptidases can be used. However, the high enzyme requirement and the low reaction
velocity are serious drawbacks. Owing to the endergonic process the reaction
conditions must be manipulated in order to increase the product yield. The addition
of high concentrations of water-miscibleorganic solvents to decrease the pK value of
the carboxyl component very often decreases the catalytic activity of the peptidases.
Furthermore, by carrying out equilibrium-controlled synthesis in aqueous media
using reactants with unprotected side chain functions, the specificity-determining
amino acid residue should again not occur in the segments to be coupled.
In the kinetic approach, the serine or cysteine peptidase rapidly reacts with a
suitable acyl donor ester to form the acylenzyme intermediate, which can be
deacylated competitivelyby the added nucleophilic amine component and water. The
ratio between aminolysis and hydrolysis of the acyl donor ester is of great importance
for the outcome of the synthesis route.This selectivity is essentially determined by
the S' subsite specificity of the enzyme as shown above. To establish an optimum
synthesis strategy, it is useful to know the basic kinetic parameters for the reaction
course, in particular those obtained by S' subsite mapping are of great importance
for planning and optimization of the enzymatic synthesis.
Depending on the specificity of the peptidase used, pH and solvent conditions, the
I 831
peptide product formed in the kinetic approach is quite stable since the amidase
activity of most enzymes is lower than the esterase activity. In addition, the esterase
activity can be positively manipulated by varying the type of leaving group, as shown
later. For preparative peptide synthesis such a manipulation is very important as it
allows complete conversion of the acyl donor ester before the product is hydrolyzed.
There is no doubt that the course of kinetically controlled protease-catalyzedpeptide
synthesis can be influenced more efficiently than the equilibrium approach.
Although the kinetic approach should be preferable, the decision must depend on
the overriding total synthesis concept. The largest industrial scale application of the
equilibrium approach is probably the enzymatic synthesis of Z-Asp-Phe-OMe,the
precursor of the peptide sweetener The best known use of trans-
peptidation technology is the large scale conversion of porcine insulin into human
insulin by trypsin['lG1or Achrornobacter lyticus protease [ll'l.
12.5.3.4
Manipulations to Suppress Competitive Reactions
The most important factors which limit the widespread routine application of
peptidases in kinetically controlled peptide synthesis are undesired hydrolysis of the
acyl donor ester and proteolysis of both the starting segments to be coupled and the
final peptide product, respectively (Fig. 12.5-12). An elimination or minimization of
these undesired reactions can be performed by various manipulations concerning
the reaction medium, the enzyme and the substrate as well as on mechanistic
features of the process. In particular, an efficient leaving group of the acyl donor
ester can provide high reaction rates in combination with a decreasing danger of
possible proteolysis of the starting segments and the final product.
12.5.3.4.1 Medium Engineering With Organic Solvents

In peptidase-catalyzed peptide synthesis the solubility of the starting components
dramatically influences the course of the synthesis. From the ideal medium, water,
the spectrum of solvents ranges from water-miscibleorganic solvents and aqueous-
organic biphasic systems to monophasic organic solvents with trace amounts of
Avoidance of hydrolysis
Cleavable
J :
Y-NH-C-OH
Enzyme
H,Of
?
Hydrolysis
NH,UR'
\?
Y-NH*C-(S,O)R $Y-NH-ll-C-Enzyme ---)-Y - N H I I ) C - N H D R '
H(S,O)R NH,UR'
T
Leaving group (Thio-)acyl enzyme Arninolysis Peptide product
Figure 12.5-12. General course of the kinetic approach to fragment condensation
catalyzed by serine or cysteine peptidases.
832
Reaction Advantages Drawbacks Alternatives

medium
Water
ideal medium for poor solubility for use of solubilizing
enzymes partially protected protecting groups
reactants
optimal ecological kinetic approach
conditions only promotion of
hydrolysis
WaterNater-
Miscible
Organic
Solvents
increased reactant reduzed enzyme use of chemically or
solubility activity genetically modified
enzymes
promoting equili- difficult product
brium-controlled isolation
approach
Watermater- [Biphasic
Nonmiscible Systems]
Organic Solvents
prevention of enzyme higher enzyme use of chemically or
activity reauirement genetically modified
enzymes
easy product isolation limitation of reactant
solubility lowering of
velocity
Monophasic prevention of reduced enzyme use of chemically or
Organic hydrolysis activity genetically modified
Solvents enzymes
no solubility problems change of stereo-
of partially protected and regiospecificity
reactants
adjusting media higher enzyme
between chemical requirement
and enzymatic
strategies
-
Figure 12.5-13. Influence of the reaction medium on peptidase-catalyzedpeptide synthesis
water necessary for the catalytic activity of the enzyme (Fig. 12.5-13). Not only for
ecological reasons, but water should be the preferred reaction medium for enzymatic
processes, since it is i n vivo the medium of choice for enzymes anyway.
Solubilizing Protecting Groups

These are the only alternative way of bypassing the poor solubility of most amino
acid-derived starting components, and synthesis of peptides can only be performed if
one or both reactants bears such a solubility-promoting group. A successful
synthesis of kyotorphin (Tyr-Arg)in a continuous large scale procedure using highly
solubilizing P-protecting groups was carried out by Fischer et a1.[1181.They used
maleyl (Mal-, 3-carboxyacryloyl-),a group which increases both the solubility of the
tyrosine ethyl ester as well as the activity of chymotrypsin. This procedure was
performed with concentrations of Mal-Tyr-OEt of up to 1.5 mol L-' and an equimolar
I 833
concentration of H-Arg-OEt.A 72.7 mol procedure resulted in 12 kg of the diacetate
of Tyr-Arg which corresponds to an overall yield of 50.4% including protecting group
removal, purification by ion exchange chromatography, and final product isolation
by spray drying. Further large scale procedures using solubilizing protecting groups
were carried out by Florsheiner et al. and Hermann et al. [120]. It was also reported
that carboxypeptidasesare capable of coupling N-terminally unprotected amino acid
esters (50 mM) to unprotected amino acids as well as amino acid derivatives (0.2-1.5
M) in one step at room temperature in aqueous solution[12'1.This synthesis principle
is more generally applicable to other esterolpc endopeptidases or lipases [122-1241.
The reduced stereoselectivityallows synthesis of D,L-dipeptides in higher yields than
the corresponding L,L-dipeptides [12'1. The chymotrypsin-catalyzed coupling of H-
Phe-OMe with nucleophilic amine components in a frozen aqueous
starting from lower acyl donor/nucleophile ratios should be mentioned as an
interesting alternative, and enzyme-catalyzed synthesis in frozen-aqueous systems
will discussed later in more detail (see Sect. 12.5.3.4.2).
Water/Water-Miscible Organic Solvent Systems

Such systems promote the solubility of partially protected starting compounds and
increase the pKvalue of the carboy1 component in equilibrium-controlledprocesses
thereby promoting this synthesis course. However, reduced enzyme activity in the
presence of high portions of organic solvents and difficulties in product isolation are
sometimes serious drawbacks. Despite these limitations such media with a small
organic solvent content are preferred in enzymochemical peptide synthesis. The
application of more stable immobilized enzymes as well as chemically or genetically
engineered enzymes offers advantages in cases of high contents of organic solvents,
as will discussed below.
Biphasic Aqueous/Organic 12*1

These have been developed as an alternative to water/water-miscible organic
solvents systems. This approach leads to preservation of enzyme activity and allows
simple product separation, an advantage which is counteracted by prolonged
reaction times where additional partition equilibria are most likely to be the rate-
determing steps. The general use of biphasic systems is mostly limited by the
solubility of the starting components in the nonpolar organic phase. This alternative
to the use of water-miscibleorganic solvents has been used with various peptidases
and good yields were obtained using no more than two equivalents of the nucleo-
philic amine component (for a review see
Synthesis in Reversed Micelle~('~'~l3O]
This is principially very similar to the approach discussed above. After adding small
amounts of water and a surfactant to a hydrocarbon, the polar ends of the surfactant
form a sphere which contains the water. Since the lipophilic group of the surfactant
is facing outside into the surrounding hydrocarbon, the reverse structure of a
normal micelle is formed. Liposome-assisted selective polycondensation of amino
acid and peptides shows an interesting continuation along this line [l3'1.
834
Monophasic Organic Solvents['32]

The ultimate way of preventing undesired hydrolpc side reactions in the course of
peptide synthesis is offered by these solvents. Trace amounts of water between
approximately 0.3 to about 1% are necessary to maintain the catalytic activity of the
enzyme. Although it has been generally assumed that higher concentrations of
water-miscible organic solvents significantly reduce the catalytic activity of the
peptidases, few papers have demonstrated successful enzymatic peptide synthesis
performed in some hydrophilic organic solvents, such as aliphatic alcohols and
. Generally, enzyme specificities change dramatically in organic
a~etonitrile['~~-'~~1
solvents. Higher enzyme requirement and reduced rates should be noted. It is
interesting to mention that peptidases also catalyze esterification and transesterifica-
tion reactions in organic solvents when the appropriate alcohol is added.
Chemically or Genetically Modijied Peptidases

They provide a useful alternative for peptide synthesis in high concentrations of
organic solvents since they are more stable than the native enzymes. Various
possibilities for modification are known.
Immobilized Enzymes
Such enzymes can be used in a very simple way for enzymatic peptide synthesis as
first reported by Jakubke and coworkers['*', 137-1391 at the beginning of the 1980s.
The effort involved in immobilizing an enzyme is mostly compensated for by the
possibility of its repeated use. Immobilized biocatalysts have almost the same
efficiency as the native enzymes. The peptidase is covalently linked or adsorbed to an
insoluble gel or resin. The water content in these systems plays an important role in
modulating the catalytic properties of the immobilized peptidase. The presence of
water molecules on the enzyme is required in order to retain the catalytic activity.
The measurement and control of the thermodynamic water activity is necessary to
quantify the water effect on enzyme activity and the intrinsic influence of other
variables such as support, solvent and e d u ~ t s [ 1411. ' ~ ~The
~ advantage of these
systems have been demonstrated in the synthesis of various biologically active
peptides 1421 .The effect of water-miscible aprotic solvents on kyotorphin synthe-
sis catalyzed by immobilized chymotrypsin was studied by Lozano et a1.['43] Of
special technical interest are the continuous synthesis of the aspartame precursor Z-
Asp-Phe-OMewith thermolysin immobilized on amberlite XAD-7 in a plug flow
type reactor and the conversion of porcine insulin into human insulin catalyzed
by Achromobacter lyticus protease I immobilized on SiOz-polyglutamicacid['45].
Solvent-Modijkd Enzymes
These are named as enzymes which are modified, for example, with polyethylene
glycol (PEG) allowing synthesis in monophasic organic solvents as described, e. g.
for chymotrypsin[146r 14'1, papain [1481 and thermolysin [14')1. Using PEG-modified
enzymes in monophasic organic solvents undesired proteolytic reactions can be
almost completely eliminated. However, owing to the solubility properties the use of
hydrophobic organic solvents makes the application for the synthesis of longer
I
835
peptides very complicated and often impossible. Insoluble cross-linked chymo-
trypsin can be obtained using glutaraldehyde concentrations several times
higher in contrast to the procedure for soluble polymeric preparations of chymo-
trypsin[”’I. Insoluble cross-linked chymotrypsin was used in a medium with GO%
(v/v) dimethylformamide (DMF) for successful synthesis of short peptides. High
amounts of powdered suspensions of peptidases in DMF have been used for peptide
synthesis [1521. An very interesting synthesis approach has been described using cross-
linked enzyme crystals (CLECs)[153* 1541.
Chemically Modijed Enzymes

Enzymes are often prepared with the aim of reducing the peptidase activity with
some of the esterase activity remaining, thus preventing the hydrolyhc cleavage of
peptide bonds [*‘I. Methyl-chymotrypsin(MeCT) obtained by N-methylation of His57
shows a significant change in the enzymatic catalysis. MeCT is less active than native
chymotrypsin by a factor lo4 to lo5 but it is virtually without any peptidase
activity[155].Owing to the low activity more activated cyanomethyl ester is used
instead of methyl ester. Subtilisin can also be changed to an acyltransferase via
modification of the active site serine to cysteine (thiol subtilisin with low amidase
activity[’5G1)
or seleno subti1isin[’’’1. Successful synthesis of various L,D-dipeptides
using [Met(0)”2]chymotrypsin[’58~ were carried out as well as the synthesis of Ac-
Tyr-OEt from Ac-Tyr-OH and ethanol catalyzed by hexyl-chymotrypsinin a biphasic
system[’’’].
Genetically Engineered Enzymes

They have elevated solvent tolerance and also owing to the lowering amidase activity
have been successfully used for synthetic purposes [lG01. Enzyme engineering de-
scribes a range of techniques from deliberate chemical modification as shown above
to remodeling a wild-type enzyme by gene technology. The aim of engineering
peptidases to generate peptide ligases by conversion of serine and cysteine pepti-
dases via site-directed mutagenesis, is to make enzymes more stable and favor
aminolysis rather than hydrolysis. Using multiple site-directed mutagenesis sub-
tilisin can be converted into a mutant which allows kinetically controlled synthesis to
be performed in the presence of high concentrations of DMF. An ingenious
combination of chemical and enzymatic steps should promote the progress in
peptide and protein synthesis as was demonstrated with subtiligase,a double mutant
of subtilisin BNP’. This variant was prepared by protein design and used in a further
total synthesis of Ribonuclease A (RNase A) [‘“I by combining solid-phase synthesis
for fragment synthesis and enzymatic coupling of these fragment to form the
protein (cf. 12.5.3.7.2, p. 856). The selection for improved subtiligases by phage
display results in the identification of two new mutants that increased the activity of
subtiligase[IG2].
836
Enzyme engineering Enzyme engineering

1 T
Promoting equilibrium approach Media adjusting between chemical
(reducted enzyme activity) and enzymatic strategies
Preventionof enzyme activity (reduced enzyme activity and

(limited application) changed specificity)
Organic-aqueoussolvent Biphasic systems

Micro-aquousmonophasis
mixtures organic solvents
f f t
Addition of organic solvents
Advantages: Disadvantages:
* Ideal medium for enzymes tI promotion of hydrolysis
- optimal ecological conditions
Water
*
* bad reactant solubility
[Solubilizing protecting groups]
Reducing of water concentration
I
Solvent-free micro-aqueoussystems Freezingthe reaction mixture
(Diffusion-controlledsynthesis; using (Frozen-stateenzyme-catalyzed
sonification and fluidization, resp.) peptide synthesis)
Figure 12.5-14. Extended approaches to medium engineering in
enzymatic peptide synthesis1961.
12.5.3.4.2 Medium Engineering by Reducing Water Content

Competitive reactions in enzymatic peptide synthesis are, as mentioned above,
mainly undesired hydrolysis of the acyl donor ester in the kinetic approach, and
undesired proteolytic side reactions in both the starting components in fragment
condensation as well as the final product. It can be demonstrated that side reactions
of these types can be largely, but not completely, avoided by synthesis in organic
solvents of controlled water activity. However, since the main drawbacks caused by
organic solvents are enzyme deactivation and changes in specificity, which can only
partly be improved by enzyme engineering, new strategies (Fig. 12.5-14)in reducing
the water concentration without substitution by organic solvents have been de-
scribed (for a review see reference["]).
Enzymatic Peptide Synthesis in Frozen Aqueous Systems

This is based on observations by Grant and Alburn['"l that trypsin-catalyzed
hydroxylaminolysis of amino acid esters was favored over hydrolysis in frozen
reaction mixtures (for a review see Hansler and Jakubke('"1). In 1990 Schuster et
I
837
al. [lG5]
first reported on the influence of freezing on peptidase-catalyzed kinetically
controlled peptide synthesis. The peptidase is added to the reactants precooled to
0 "C in a polypropylene tube and immediately inserted into liquid nitrogen. After
20 s the tube is transferred into a cryostat at - 15 "C or similar temperature. Amino
components that are considered to be inefficient nucleophiles in enzymatic synthe-
sis at room temperature gave substantially higher yields in frozen reaction mixtures.
Later these results could be explained on the basis of the so-called freeze-concentra-
tion model[16G] and were confirmed by other investigators(1G71.
In frozen aqueous systems the endopeptidase chymotrypsin is capable of acting as
a reverse carboxypeptidase catalyzing coupling of free amino acids as amino
components (168]. Various amino acids were acylated under catalysis of chymotrypsin
starting from 2 mM Mal-Phe-OMeand 50 mM (50% as free base) of the appropriate
amino acids at - 25 "C in unexpectedly high yields (% given in parentheses): Met
(75), Val (58),Ser (52), Ile ( 3 5 ) ,Thr (30),Asn (29),Leu (2G), Lys (GO). Tougu et a1.[169]
described similar results on coupling Mal-Tyr-OEt with free amino acids. The
surprising catalytic behavior of chymotrypsin under frozen state conditions is
demonstrated in Table 12.5-4. N"-unprotected amino acid esters as well as dipeptide
esters, even containing unusual amino acids, can be coupled in frozen aqueous
systems in high yields indicating both reverse aminopeptidase and dipeptidylpepti-
dase activities. Furthermore, cysteine proteases, with the exception of clostripain,
were capable of catalyzing peptide synthesis in high yields using amine components
with low efficiency at room temperature in frozen reaction mixtures. The specific
properties of peptide synthesis in frozen solutions such as changes in specificity
observed in serine and cysteine peptidase-catalyzed reactions strongly suggest that
factors other than concentration of the reactants are probably involved in yield-
enhancement by freezing. This assumption is supported by investigations reported
by Jakubke et a1.[171]who determined the amount of unfrozen water in frozen
samples at - 15 "C using the 'H-NMR-relaxation time technique and obtained an
apparent concentration factor of 50. Synthesis experiments carried out under these
concentration conditions at room temperature gave substantially lower yields com-
Table 12.5-4.Comparative model peptide synthesis catalyzed by chyrnotrypsin in frozen aqueous

systems and at room temperature.
Acyl donor Amino component Peptide Yield ("A) Reference
Ice 25 "C
Mal-Tyr-OMe H-p-Ala-Gly-OH 79
Mal-Tyr-OMe H-D-Leu-NHZ 73
Mal-Phe-OMe H-Lys-OH 60
H-Tyr-OEt H-Lys-OH 71
H-Phe-OMe H-Leu-NH2 94
H-4-fluoro-PheOMe H-Leu-NH2 90
H-2-naphtyl-Ala-OMe H-Leu-NH2 93
H-Leu-Phe-OMe H-Ala-Ala-OH 88
H-Asp-Phe-OMe H-Ala-Ile-OH 91
H-Gly-Phe-OMe H-Ala-Ile-OH 85
838
pared with frozen reaction mixtures and, therefore, could not simulate the reaction
conditions in ice.
In addition to the freeze-concentration effect, a catalytic role for ice crystals, a
favorable orientation of substrate and biocatalyst, the markedly lower dielectric
constant of ice compared with water, and the high proton mobility in ice, have been
discussed as further factors that possibly influence reactions in frozen systems. In
summary, the reverse action of hydrolases provides an attractive alternative to the
chemical synthesis of peptides but this approach could also be verified for the
synthesis of oligosaccharides and oligonucleotides using glycosidases and ribonu-
cleases,
Peptidase-catalyzed Synthesis in Solvent-jee Micro-aqueous Systems

Such systems show a second route to reducing water concentration without
substitution by organic solvents. This interesting development allows the application
of reaction systems with partly undissolved reactants and is based on an extensive
theoretical treatment of the equilibrium position described by Halling et al. [1721. The
principle of a solid-to-solidconversion is illustrated graphically in Fig. 12.5-15 and
selected examples of its experimental implementation are illustrated in Table 12.5-5.
The application of solid phase substrate pools combines the equimolar (or nearly
equimolar) supply of starting compoments with high obtainable yields, easy work-up
procedures and compatibility with chemical standard procedures. The key parameter
for obtaining high product yields via acyltransfer reactions is the ratio of aminolysis
and hydrolysis favored by high nucleophile concentrations. In combination with
solid phase acyl donor pools, this approach allows an equimolar supply of starting
materials without any addition of organic solvents. The synthetic potential of
systems with partly unsolved reactants was proven by pilot scale synthesis of Z-His-
Phe-OMe and the low calorie sweetener precursor of Z-Aspartame in the thermody-
namic approach [1731 and by kinetically controlled synthesis of enkephalin deriva-
tives Furthermore, Halling and co-workers have studied the effect of water and
A B A + B P
n n
before ____,
n n e
in equilibrium
m
substrates substrates product

Figure 12.5-15. General principle of application o f “equilibrium shift” towards the
product by solid-phase substrate pools (botton) compared with synthesis starting
from
Table 12.5-5.
Selected examples for peptide synthesis in water-based solid-liquid systems

I 839
according to Eichhorn et aI."73]and Jakubke et al.[g'l.

Carboxyl component Amine component Peptide yield (%) Time (h) Enzyme
2-Ala-OH H-Leu-NHz 95 0.5 Thermolysin
2-Asp-OH H-Phe-OMe 90 7 Thermolysin
2-Gln-OH H-Leu-NHZ 94 4 Thermolysin
2-Phg-OH H-Leu-NHz 89 2 Thermolysin
2-Ser-OH H-Leu-NH2 89 2.5 Thermolysin
2-His-OH H-Leu-NH2 95 3 Thermolysin
Z-Phe-OH H-Met-NHz 88 24 Thermolysin
Ac-Tyr-0E t H-Arg-NH2 90 1 Chymotrypsin
Ac-Tyr-OEt H-Gly-Gly-OH 63 2 Chymotrypsin
2-His-Phe-OBzl H-Arg-Trp-NHz 95 2.5 Chymotrypsin
2-Ser-OCam H-His-ONb 85 2.5 Papain
2-Gly-His-ONb H-Lys-NHZ 90 1.5 Chymotrypsin
2-Arg-His-ONb H-Gly-NH2 55 6 Subtilisin
enzyme concentration of thermolysin-catalyzed solid-to-solid peptide synthesis in

and reviewed the recently developed approach to enzymatic synthesis with
mainly undissolved substrates at very high concentrations
12.5.3.4.3 Substrate Engineering

In the case where undesired subsequent reactions occur during kinetically con-
trolled synthesis it is of minor importance which bond is cleaved by the enzyme.
These side reactions underline the issue that the specificity of the enzyme for the
acyl donor ester does not lie sufficiently above its specificity for the peptide product.
Since the sequence of the starting components cannot be changed, the only practical
alternative to suppress such competitive reactions is to use a highly specific leaving
group of the acyl donor ester. As a simple model peptide with a highly sensitive
cleavage site for chymotrypsin Schellenberger et al. [1771 used the chromogenic
chymotrypsin substrate Mal-Leu-Phe-pNA (Ma1 = maleyl), which is formed by
chymotrypsin-catalyzed coupling of Mal-Leu-OY with H-Phe-pNA. Table 12.5-6
shows that the leaving group moiety Y is of major influence on the reaction course.
When Mal-Leu-OMe is employed as the carboxyl component, the velocity of the
product cleavage reaches the rate of its formation after a short time. By using the
Table 12.5-6. Influence of the specificityconstants of acyl donor esters on the yield ofthe
chymotrypsin-catalyzedsynthesis of Mal-Leu-Phe-pNA"starting from Mal-Leu-OYwith varying
leaving groups Y and H-Phe-pNA according to Schellenberger et al.[1771,
Leavinggroup Y KM kcat kcat/KM Reaction time Yield
(mM) (s-7 (M-l S-l) (min) ("A)
Me(Methy1) 120 f 30 5.6 i 0.4 4.7x 10 10 -3
Bzl(Benzy1) 1.7 f 0.4 5.4 f 0.4 3.21 ~0' 5 65
Nb~~~inobenrv~~ 0.38 f 0.1 5.9 f 0.4 1.5x lo4 5 80.5
~ M-'s-'
a k,,,/K, of Mal-Leu-Phe-pNA1 . 2 10 (Mal; maleyl).
840
more specific acyl donor esters (higher specificity constants kcat/KM) a clear product
accummulation is attained. With Mal-Leu-ONbor ester of similar or higher specific-
ity, starting components containing highly protease-labile cleavage sites can be
coupled even in a homogeneous phase in high yields. According to this finding
Bongers et al. published a two-step enzymatic semisynthesis of the superpotent
analog of human growth hormone releasing factor [deNHzTyr',D-Ala2, Alal']
GFR(1-29)-NH2 from the amine component [Ala1']GRF(4-29)-NH2 and the car-
boxyl component deNH2Tyr-D-Ala-Asp-OY(Y = Et or 4-NOzBz1, respectively) cata-
lyzed by V8 protease and Glu/Asp-specific endopeptidase (GSE) from Bacillus
lichenijomis, respectively. Using the 4-nitrobenzyl leaving group compared with the
ethyl moiety results in a higher yield without the undesired proteolytic side
reactions. The state-of-the-artof substrate engineering is without doubt the substrate
mimetic-mediated C - N ligation strategy which allows irreversible peptide bond
formation and will be presented separately (see Sect. 12.5.3.6).
12.5.3.5
Approaches to Irreversible Formation of Peptide Bond
Despite the development of various possibilities to suppress competitive reaction, as

shown in the preceding section, an absolute avoidance of proteolytic cleavage of the
peptide bond formed cannot be guaranteed. The only alternative seems to be the use
of biocatalysts that do not have the catalytic potential to hydrolyze peptide bonds.
12.5.3.5.1 Use of Nonpeptidases

Nonpeptidases are supposed to possess favorable prerequisites for the formation of
peptide bonds because undesired proteolytic cleavages in the starting components
and the product can be ruled out.
Enzymes involved in protein synthesis also possess potential, e. g. aminoacyl-
tRNA-synthetase-aminoadenylate complex['7g~l, the arginyl-tRNA: protein arginyl-
transferase1'80], and nonribosomal poly- or multienzyme complexes [721 which re-
quire ATP or GTP to activate the carboxyl group of an amino acid, and seem to accept
various amino acid nucleophiles for peptide bond formation. However, the applica-
tion ofthese enzyme systems for generally practical peptide bond formation is rather
limited.
Furthermore, lipases ['", lx2I containing the catalytic triade typical for serine
peptidases have been used for peptide synthesis as well as pig liver esterase[ls3. 1841.
These enzymes accept both D- and L-amino acid derivatives as weak acyl donors or
nucleophilic acceptors, but concerning the practical importance the situation is
similar to the enzyme systems reported previously.
Particularly promising from the theoretical point of view seems to be the
developments in catalytic antibodies. It could be established that antibodies raised
against suitable transition state analogs are capable of catalyzing the formation of
peptide bonds['85, lS6l. At present the practical importance is rather low, but the
development of more tailor-made catalytic antibodies for peptide bond formation
could change the situation in the future.
12.5.3.5.2 Use of Proteolytic Inactive Zymogens

I
841
In 1994, it was firstly established that zymogens, the catalytically inactive precursors
of various peptidases, can be used as biocatalysts for practically irreversible peptide
bond formation[91,96, 97, lS71. The capability of reacting slowly with site-specific
reagents indicated that such reactions proceed via the formation of an acyl-zymogen
intermediate [lS8.lS9l. Although, the second-order rate of ester hydrolysis is 106-107
times slower than by the appropriate active enzyme, the deacylation rates of
zymogen and active enzyme do not differ significantly.Therefore, it can concluded
that the conversion of a zymogen to an enzyme should not be the activation of an
inert zymogen, but the potentiation of catalytic activity intrinsic to the zymogen.
Based on this feature Jakubke’s group has used the zymogens of the well studied
serine peptidases trypsin and chymotrypsin, respectively, in peptide synthesis
experiments, and has surprisingly observed catalysis of peptide bond formation by
the zymogens trypsinogen and chymotrypsinogen. In several cases S’ subsite
mapping studies showed significant differences in the deacylation of the acyl
enzymes compared with the corresponding acyl zymogens, based on acyl transfer to
various peptide derivatives.Although the zymogens possess the same catalytic triad,
which is necessary for the formation of the appropriate covalent acyl intermediate,
the non-optimal formed substrate binding cleft prevents proteolysis. In particular,
Glylg3is distorted and is not capable of forming a hydrogen bond to the carbonyl
oxygen of the substrate which is necessary for the stabilization of the oxyanion-
h ~ l e ~ ”However,
~]. because of the high flexibility in this region, a principle oxyanion
stabilization takes place, although not in an ideal manner. To confirm true zymogen
catalysis it was essential to prove that the zymogen preparations were not contami-
nated with traces of the appropriate active enzyme. Based on the significantly
different affinity of both enzyme and zymogen to the basic pancreatic trypsin
inhibitor (BPTI) it was possible to analyze the esterase activity of the zymogen, which
is an efficiency parameter used in estimating their peptide bond forming potential.
Since the differences in L t / K cover ~ a range of about 5 orders of magnitude, for
general use of zymogen catalysis it is essential to improve the acylation rate.
The application of zymogens for irreversible fragment condensations was studied
by coupling a synthetic tetrapeptide methyl ester with a recombinant 24-peptide
according to the procedure of Cerovsky et al.L9’]. A comparison of the coupling
reactions (Fig. 12.5-16)was carried by dropping the acyl donor ester 1into a solution
of the amine component 2 and, alternatively,under batch conditions. The first way
was chosen in order to minimize the undesired ester hydrolysis of 1and, in addition,
to manipulate a large excess of the amine component 2. In this case 5.4 mg
(0.002 mmol) of 2 was coupled with 4 mg (0.008 mmol) of 1 in the presence of 0.5
mg of chymotrypsinogen and resulted, after 400 min, in the complete conversion of
the acyl donor ester in GO % yield to the desired product 3. The batch procedure led to
a product yield of 52 %.
In order to avoid any undesired zymogen activation by limited proteolysis, e. g. of
the L y ~ * ~ - I peptide
l e ’ ~ bond in the case of trypsinogen, it would be useful to prevent
this reaction by chemical means. The guanylation of trypsinogen by l-guanyl-
3,s-dimethylpyrazolecauses a stable zymogen because of the conversion of all lysine
842
Z-AGGF-OMe + H2N-G’ KLSQELHKL”QTYPRTDVGA2’ GTPA-OH
1 1 chymotrypsinogen 2
Z-A’ GGFGKLSQE’’ LHKLQTYPRT” DVGAGTPA-OH
3
Figure 12.5-16. (4+24)-Fragrnent condensation catalyzed by chymotrypsino-
gen according t o Cerovsky et al. (cf. reference[98]).
residues into homoarginine (Har), including the crucial Lys”. Peptide synthesis
with the guanylated zymogens led to very surprising results. Using dipeptides with a
free carboxyl group as the amine components they are much more effectively
accepted by the the guanylated species Lgll. From molecular modelling studies it can
be concluded that there is an interaction between the carboxyl group of the dipeptide
with the only lysine within the active site (LysG’). The conversion of LysG1 to
homoarginine increases the pK of the side chain and therefore the basic character.
12.5.3.6
Irreversible C-N Ligations by Mimicking Enzyme Spe~ificity”~’]
The synthetic importance of peptidases as biocatalysts for peptide synthesis is

undisputed due to a couple of advantages over pure chemical coupling methods. The
mild reaction conditions and the high degree of regio- and stereospecificity guaran-
tees both freedom from partial epimerization and that there is no need for temporary
protection of side-chain functions. On the other hand, there are some serious
drawbacks of the classical peptidase approach which has been discussed below in
detail. Most important is the fact that the formed peptide bond formed can be
cleaved in the course of the catalytic process by the same enzyme. There are no
differences in the requirements of the specificity in both the peptide bond forming
step and cleavage step, respectively. Since the specificity is manifested by the side-
chain of the PI amino acid residue, e.g. Arg or Lys in the case of trypsin, an
irreversible peptide bond formation seems not to be possible according to the
classical concept of reversal of proteolysis.
In ribosomal peptide bond formation the mechanism is based on an acyl transfer
of the acyl moiety from the peptidyl-tRNA (or Met-tRNA at the start of the
prokaryotic biosynthesis) located at the P site of the ribosome to the amino group of
the aminoacyl-tRNA in the A site catalyzed by the side-chain unspecific ribozyme
peptidyltransferase. Learning from nature our philosophy was that mimicking
specificity is the only way to make the peptidase-catalyzed peptide bond forming step
irre~ersibleI”~~ Ig3, lg71. Since from the mechanistic point of view the kinetic
approach with serine and cysteine peptidases is also an acyl transfer process, the idea
arose of to transfering the specificity moiety of the PI amino acid side chain to the
leaving group of the acyl donor ester. In this manner the enzyme should recognize
the acyl donor ester. However, after the acylation of the enzyme the leaving group
with the specificity determinant is released from the enzyme with the consequence
I 843
that the peptide bond formed cannot be cleaved by the enzyme due to the lack of
specificity for recognizing this bond. In 1991 we were able to confirm this assump-
tion by model peptide synthesis catalyzed by trypsin using various nonspecific W -
protected amino acid 4-guanidinophenyl esters (OGp) as acyl donors and various
amino acid and peptide derivatives as nucleophilic acyl acceptors [192, 1931, and later
extended by further examples from another 19’1. At that time this type of
acyl donor ester was named an inverse substrate according to time-dependent
irreversible inhibitors of trypsin and trypsin-likepeptidases, such as 4-amidino- and
4-guanidinophenyl esters which were found to be hydrolyzed by these peptidases
virtually idependently of their acyl 19’1. Although this fact was first
published in 1973 by Wagner and Horn[’96],very little was known about the basic
mechanism of the hydrolysis of these inverse esters. In 1997 an extension of this new
approach to irreversible peptide segment condensation with other peptidases was
described and the term substrate mimeticswas introduced by Bordusa et al.
12.5.3.6.1 Mechanism o f Substrate Mimetic Hydrolysis

The most striking structural differences of W-protected amino acid or peptide
4-guanidinophenyl esters compared with common peptide substrates are the non-
specific acyl residue and the highly specific leaving group. It was established by
Bordusa’s group [19’1 that all 4-guanidinophenyl esters, independently of structure
and chirality of the acyl moiety, are hydrolyzed despite the lack of trypsin-specificacyl
moieties, with the exception of the lysine derivatives (Table 12.5-7).This behavior is
in contrast to common trypsin substrates. According to the familiar model, conven-
tional trypsin substrates bind with their acyl residue to the S-binding site of the
enzyme having the leaving group at the S’-subsite and the scissile bond between
attacked by Ser”’.
Table 12.5-7. Steady-state kinetic parameters for the hydrolysis of Boc-Xaa-OCp by trypsin”
according to Thormann et a1.[’98].
r-Ala 0.206 32.4 1.6 x 105

D-Ala 0.161 0.61 3.8 x 10’
GlY 0.087 23.5 2.7 x 105
L-Leu 0.146 38.8 2.7 x 105
o-Leu 0.035 0.85 2.5 x lo4
L-Gln 0.239 35.2 1.5 x 105
o-Gln 0.071 0.68 9.6 x lo3
L-Phe 0.211 66.1 3.1 x lo5
o-Phe 0.249 9.0 3.6 x lo4
L - G ~ 0.071 5.5 7.6 x 104
D-GIu 0.039 0.43 1.1 x lo4
L-LYS 0.107 270 2.5 x 10‘
D-LYS 0.314 15.7 5.0 x lo4
a Conditions: 25 m M Mops, pH 7.6,100 m M NaC1.5 m M CaC12,25 “C: errors less than 15%.
844
cleavage site Figure 12.5-17. Schematic compari-

son of the binding of a peptide 4-gua-
: m
Protease ~ % nidinophenyl ester and a common
S3 S2 S1 $ S'q S> S; trypsin substrate t o the active site o f
the enzyme according t o the conven-
tional binding model.
As shown schematically in Fig. 12.5-17 applying the same binding principles for
the acyl moiety of the substrate mimetics leads to a catalytically unproductive
binding. The acyl residue binds at the S-subsite of trypsin, but the scissile bond
would be far away from the active site and, therefore, and cannot be attacked by
Ser"5. However, docking calculations show that the specificity-bearing OGp group
binds to the S1-bindingpocket like the side chain of L-arginineof commom peptide
substrates. Surprisingly, this holds even for the substrates Boc-L-Arg-OGpand Boc-
L-Lys-OGpdespite the presence of the S1 specific arginine and lysine residues, thus
indicating a higher S1-specificityfor the 4-guanidinophenylmoiety. Indeed, all L- and
D-substrate mimetics realize an arrangement in such a way that the scissile bond is
very close to the hydroxyl group of the active Ser195.Furthermore, the carbonyl group
of the scissile ester bond of the appropriate substrate mimetic is located at exactly the
same position as the carbonyl group of the scissile peptide bond between P1-Lysl5
and PI'-Ala16in the trypsinogen-BPTI complex. This implies a possible attack by
trypsin, which was confirmed by the hydrolysis studies.
How does it work from the mechanistic point of view? Contrary to common
trypsin substrates, the acyl residues of these enzyme-substrate mimetic arrange-
ments bind to the s'-subsiteof trypsin (Fig. 12.5-18). For this reason, all binding sites
beyond S1 are only of minor importance for the substrate mimetics. Furthermore,
the acyl residues of the substrate mimetics do not reflect the specificity of the S-
-
Ac-X
- 0
HX
Hz0 W C O O H
Ac-OH
Figure 12.5-18. Schematic representation o f the new extended kinetic model o f peptidase-
catalyzed hydrolysis o f substrate mimetics according t o Thormann et
EH, free enzyme; Ac-X, substrate (substrate mimetic); [E..Ac-XI, Michaelis-Menten complex; HX,
leaving group; E-Ac, acyl enzyme intermediate located i n S'-region; Ac-E, acyl enzyme inter-
mediate located i n S-region; KR, rearrangement equilibrium constant; Ac-OH, hydrolysis product.
binding site of the enzyme. Since the direction of the peptide backbone chain is
I 845
reversed, the S’-subsitespecificity is also not reflected. Therefore, substrate mimetics

show a unique specificity behavior.
The deacylation step, however, requires an unoccupied S’-subsitesince water can
only attack the acyl enzyme from this site without hindrance. Hence, the flipping
acyl moiety acts like a “sliding window” within the active-site, spanning the primed
and unprimed subsite regions. The extended kinetic model requires a rearrange-
ment step between the two arrangements (E-Ac and Ac-E) of the acyl enzyme
described by the equilibrium constant KR (Fig. 12.5-18).From the experimental data
of Table 12.5-7it follows that D-configured substrates exhibit lower k,,, values, which
might be related to lower KR values. Exploring the dynamic behavior by molecular
dynamics simulations of Boc-L-Ala-trypsinand Boc-D-Ala-trypsinindicated that the
flip of the D-Ala complex to the S-subsite takes about 1.5 ns, much more than in the
L-Ala complex (300 ps).
For an experimental study of the S’-subsite accessibility, S’ mapping studies (cf.
Sect. 12.5.3.3.4) are suitable. By their specific S-binding capacity, peptide nucleo-
philes should be capable of pushing aside the acyl moiety from the S’ region more
efficiently than water. Therefore, the aminolysis of acyl enzymes bearing the acyl
moiety in S’ should proceed at higher rates compared with their hydrolysis. Indeed,
from the mapping studies it follows that the p-values for the deacylation of Bz-D-Ala-
trypsin are dramatically lower than for Bz-L-Ala-trypsin. Consequently, the experi-
mental data of aminolysis also support this unique catalysis mechanism for
substrate mimetics.
12.5.3.6.2 Cationic Substrate Mimetics

The Na-protected amino acid 4-guanidinophenyl ester was the first example of
substrate mimetics for Arg-specific peptidases used for irreversible peptide bond
formation[192,1931. Apart from the guanidino group linked at various aromatic and
aliphatic spacers, also the amidino moiety is also suitable as a specificity-determing
residue in the leaving group of cationic substrate mimetics 1192-1951 . After the basic
studies with trypsin we could also establish that other Arg-specific peptidases such
as thrombin and clostripain are suitable enzymes for peptide synthesis using
cationic substrate mimetics [1971. Im particular, clostripain has been very useful in
substrate mimetic-mediated fragment condensation. As shown in Fig. 12.5-19 the
(3 + 5) fragment condensation provided a product yield of over 90% within a few
minutes and the product formed remains unchanged after 72 h. The course of this
synthesis clearly proves the irreversibility of this model C - N ligation.
For synthesis planning, clostripain has an additional decisive advantage due to the
extremely low PI’ specificity for the N-terminal amino acid residue of the amine
component. Firstly, Bordusa and co-workers[19’1 demonstrated impressively the
capability of the cysteine peptidase clostripain as a biocatalyst for the synthesis of
peptide isosteres. These authors have investigated the function of clostripain for
acylating aliphatic noncyclic and cyclic amines varying in chain length and ring size
using the trypsin standard acyl donor ester Bz-Arg-OEt. Furthermore, using a model
846
I BOC-Phe-Gly-Gly-OGp 1 + H-Ala-Phe-Ala-Ala-Gly-OH 2
122 mg (0.174 mM) 157 rng (0.286 mM) Tos x H,O
Clostripain
Boc-Phe-Gly-Gly-Ala-Phe-Ala-Ala-Gly-OH 3
177 mg (91% yield); 3 x 2 5 TFA x 2 H,O
MALDI-TOF. m/z calc for [M+Na+]= 819.36, found. 819.29
Figure 12.5-19. Clostripain-catalyzed (3 +5) fragment condensation of Boc-Phe-Cly-Cly-
OCp and H-Ala-Phe-Ala-Ala-Cly-OH['"I. Conditions: 50 mM HEPES-buffer, pH 8, 100 mM
NaCI, 10 mM CaC12, 25OC, [Clostripain]: 1.6 p ~ .
substrate mimetic, clostripain was capable to catalyze the reaction with noncoded
and non-amino acid-derived amines. The results of these investigations indicate that
the substrate mimetic approach may extend outside of peptide synthesis.
In a recent paper Bordusa's group presented a novel enzymatic approach to the
synthesis of carboxylic acid amides using substrate mimetics and clostripain as a
biocatalyst [200]. This unexpected peptidase-mediated approach to the coupling of
non-coded and non-amino-acid-derivedamines with pure organic esters could only
be realized by the combination of the substrate mimetic strategy with the use of
clostripain that possesses a broad tolerance towards amines. Selected examples of
the clostripain-catalyzedcoupling of Bz- P-Ala-OGp and the 4-guanidinophenyl ester
of 4-phenylbutyricacid (Pbu-OGp)with various amino acid amides and peptides are
summarized in Table 12.5-8. Furthermore, the broad tolerance of clostripain toward
non-coded amino acids and even simple amines, such as aliphatic, aromatic, or
substituted amines including unnatural amino acids, and diamines as acyl acceptors
is demonstrated by the results of appropriate syntheses compiled in Table 12.5-9.
The substrate mimetic approach has opened a new range of synthesis applications
beyond peptide synthesis offering efficient and selective organic amide bond
formation under extraordinarily mild reaction conditions.
Table 12.5-8. Clostripain-catalyzed coupling of 4-guanidinophenyl esters o f 4-phenylbutyric acid

(Pbu-OCp) and benzoyl-P-alanine (Bz-P-Ala-OCp),respectively, with various amino acid amides
and peptides according to Cunther et al.[1991.
Acyl donor ester Acyl acceptor Product Yield ("96)
Pbu-OGp H-Leu-NH2 Pbu-Leu-NHz 98
Pbu-OGp H-LYS-NH~ Pbu-Lys-NHz 96
Pbu-OGp H-Ala-Pro-OH Pbu-Ala-Pro-OH 93
Pbu-OGp H-AFAAG-OH Pbu-AFAAG-OH 92
Bz-P-Ala-OGp H-Leu-NH2 Bz-0-Ala-Leu-NHz 98
Bz-P-Ala-OGp H-Lys-NHZ Bz-0-Ala-Lys-NHz 93
Bz-P-Ala-OGp H-Ala-Pro-OH Bz-P-Ala-Ala-Pro-OH 91
Bz-P-Ala-OGp H-AFAAG-OH Bz-P-Ala-AFAAG-OH 93
Clostripain-catalyzed coupling of non-amino acid-derived carboxyl and amine

Table 12.5-9.
I
847
componentsa according t o Gunther and 6 0 r d u s a ' ~ ~ ~ .

Acyl donor Acyl acceptor Product Yield ("h)
Pbu-OGp PbuNH- 81
Pbu-OGp WUNH-J" 80
Pbu-OGp H2N P~UNH- 53
Pbu-OGp H2N-OH mu N H - O ~ 65
Pbu-OGp wuNH'-"OH 78
Pbu-OGp P~UNH-"'OH 70
Pbu-OGp
NHTH 92
Pbu-OGp
Pbu NH 2 95
Pbu-OGp wu-o- n. s.
Bz-OGp BZ-NH-+J 82
Bz-OGp BZ NH--"' 76
Bz-OGp BZ NH- 56
Bz-OGp BZ-N H-O 57
Bz-OGp Bz-NH-OH 84
Bz-OGp H2N-OH BZNH-OH 70
Bz-OGp
NHTH 82
2
Bz
Bz-OGp 94
Bz-NH
Bz-OGp Bz-0- n. s?
-
a Conditions: 0.2 M HEPES-buffer (pH 8.0),0.1 M NaCl, 0.01 M CaCL 5 % DMF, 25 "C, (acyl donor): 2 m M ,
(acyl acceptor): 12 m M ; b n. s., no synthesis.
12.5.3.6.3 Anionic Substrate Mimetics

Owing to the general validity of the concept of substrate mimetics Giinter and
Bordusa[*'*]have expanded this strategy to anionic leaving groups in the appropriate
mimetic structures based on the specificity determinants of Glu-specific endopepti-
dases. Since the leaving group moiety of a substrate mimetic binds in place of the
specificity-determining amino acid side chain, for the strong Glu-preferred V8
protease from Staphylococcus aurens a carboxylate function linked with a suitable
spacer was chosen as the ester leaving group. Unfortunately, the so far unknown 3D
structure of this enzyme allows only the design of suitable mimetic structures by
848
I Z-Pro-Leu-Gly-SCm
1 + H-Leu-Ala-Phe-Ala-Lys-Ala-AspAla-Phe-Gly-OH 2
2 mM 10 mM
i V8 protease
Z-Pro-Leu-Gly-Leu-Ala-Phe-Ala-Lys-Ala-Asn-Ala-Phe-Gly-OH 3
Yield 55% (determined by analytical RP-HPLC)
MALDI-TOF m/z calculated for [M+Na+] = 1433 71, found 1434 17
Figure 12.5-20. V8 protease-catalyzed (3 +lo) fragment condensation o f Z-Pro-Leu-Cly-Leu-

Ala-Phe-Ala-Lys-Ala-Asp-Ala-Phe-Cly-OH[*"I. Conditions: 0.2 M HEPES-buffer, p H 8.0, 37 "C,
[V8 protease] = 4 9 FM.
empirical structure-function relationship studies. Apart from other structures, the

carboxymethyl thioester moiety in particular was selected as a potentially suitable
leaving group for imitating the Glu residue in PI' position. The capability of the
carboxymethyl thioester group to act as an artificial recognition site for the V8
protease was initially studied by steady-state hydrolysis kinetic studies. As a general
result it can be summarized that the carboxymethyl thioester moiety was found to
mediate specific hydrolysis of all carboxymethyl thioester substrate mimetics inde-
pendently of the PI' amino acid residue. This also holds for Pro and even for D-Ala,
which causes only a slight decrease in specificity compared with the L-enantiomer.
Generally, a one to four orders of magnitude lower specificity compared with the
common substrate Z-Glu-SMe ( k c a t / K ~= 1 . 1 2 ~ 1 M-'
0 ~ s-l) was found.
In contrast to the non-specificity of the V8 protease for the acyl part, the negative
charge of the leaving group is essential to mimick substrates. Lacking this charge in
Z-Phe-Scam ( -S-CH2-CONH2instead -S-CH2-COOH)a complete loss of specificity
results since no hydrolysis of Z-Phe-Scam could be observed. The utility of
carboxymethyl thioester for V8 protease-catalyzed peptide synthesis could be demon-
strated both by model acyl transfer reactions using amino acid and dipeptides as acyl
acceptors and fragment condensation, respectively. Fig. 12.5-20 indicates a semi-
preparative (3 + 5) model fragment condensation. After 2 h the enzymatically
nonoptimized coupling reaction of 1with an excess of 2 in HEPES-buffer containing
5 % DMSO was stopped by the addition of diluted trifluoroacetic acid and resulted in
a yield of 55 %.
In addition to carboxymethyl thioesters, Bordusa's has investigated
further types of thioesters and phenylester bearing the carboxyl group, e. g. carbox-
yethyl thioester, 2-carboxyphenyl thioester, 3- and 4-carboxyphenyl ester, which also
mediate acceptance by V8 protease. It is surprising to note that despite the lower
degree of structural similarities, the aromatic part of the leaving group led to even
higher specificity constants than found for the aliphatic counterparts. In addition,
these studies have been expanded to the use of the not so expensive but equally Glu-
specific endopeptidase from Bacillus licheni$obmis (BL-GSE),which can easily be
purified from alcalase in good yields.
I 849
12.5.3.6.4 Hydrophobic Substrate Mimetics
In addition to the enzymes mentioned above with a high specificity for positively and
negatively charged P1’ amino acid residues, a third important class of enzymes are
represented by peptidases with specificity for aromatic and hydrophobic functional-
ities. Well-known representatives of this family are the serine peptidases chymo-
trypsin and subtilisin, which have application in classical enzymatic peptide synthe-
sis and, therefore, they should also be interesting biocatalysts for the substrate
mimetic approach. Both enzymes primarly prefer bulky hydrophobic and aromatic
P1‘ amino acid residues. In addition, the S1 binding pocket of subtilisin contains a
carboxylic acid moiety (G~U’’~) which causes additional activity towards Arg and
Lys r2031. For this reason, aromatic leaving groups with additional positively charged
substitutions, e. g. 4-guanidinophenyl ester should fit the natural specificity of these
peptidases.
Parallel to an empirical design of specific mimetic structures, the well-known 3D
structures of the two enzymes allow the use of rational approaches such as the
computer-assisted protein-ligand docking approach. Using the latter to predict the
function of the 4-guanidinophenyl ester functionality, Bordusa and co-worker
selected Boc-Ala-OGp as a model ligand and docked it towards the enzyme[”’].
Fig. 12.5-21shows the arrangement of the ligand Boc-Ala-OGp at the active site of
chymotrypsin in the lowest energy complex (A) in comparison with that found for
trypsin (B) [lg81. In analogy to the natural specificity of chymotrypsin, hydrophobic
contacts between the phenyl moiety of the ester group and the residues Cys”’ and
Val213of the enzyme predominante. Interestingly, the guanidino functionality favors
this binding mode by formation of additional hydrogen bonds with three serine
residues which are located at the bottom of the S1 binding pocket. This specific
binding pattern, specifically, the orientation of the carbonyl oxygen to Glylg3
Asp 1
A
191
--+
Asp189
Figure 12.5-21. Arrangements o f Boc-Ala-OCp at the active site o f chymotrypsin (a) and trypsin
(b), respectively according to Cunther, Thust, Hofmann and Bordusa (see e. g. r e f e r e n ~ e ” ~ ’ ~ ) .
850
Figure 12.5-22. Chy-

motrypsin-catalyzed
peptide synthesis
10 80 using 4-guanidino-
phenyl esters o f vari-
ous non-specific and
non-coded acyl moie-
H-Ala-Ala-NH
-Gly-Leu-NHp
(oxyanionhole), the distance between the carbonyl C-atom of the scissile ester bond
and the active Ser'", and the reversed binding of the acyl moiety fulfill the
conditions for the binding and catalytic mechanism of substrate mimetics. Indeed,
acyl 4-guanidinophenyl ester was hydrolyzed by chymotrypsin, and also peptide
bond formation using various 4-panidinophenyl esters with nonspecific coded and
non-coded acyl residues could be successfully performed as shown in Fig. 12.5-
22[191]. The yields obtained are in the same range as the yield obtained using the
normal-type acyl donor Bz-Phe-OMe.
Furthermore, phenyl ester are also suitable substrate mimetics for chymotrypsin-
catalyzed peptide synthesis, as was established by Bordusa's group and will demon-
strated by sophisticated fragment condensations in Sect. 12.5.3.7.
12.5.3.6.5 Enzymochemical Substrate Mimetic Approach

In order to synthesize longer polypeptides and proteins the condensation of the
initial fragments is an essential prerequisite. Despite different chemical ligation
techniques in the field of protein semisynthesis (cf. 12.5.3.1,p. 820-821) enzymatic
C - N ligation seems to be the only way to avoid partial epimerization, which cannot
be completely eliminated in the course of a chemical fragment coupling reaction.
Consequently, the application of the substrate mimetic strategy for the peptidase-
mediated condensation of peptide fragments indisputably needs to be combined
with the solid-phase peptide synthesis approach. Since a peptide ester can be
achieved using of the oxime resin ~tartegy[~'~, 2051 Cerovsky and Bordusa[2061
developed a procedure for the synthesis of peptide fragments in the form of
substrate mimetics esterified as 4-guanidinophenyl-,phenyl- and mercaptopropionic
acid esters. The synthesis protocol involves covalent attachement of the first N"-Boc-
protected amino acid to the oxime resin, blocking free hydroxylic groups by acetic
a) Figure 12.5-23. General

approach t o fragment
substrate mimetics via
the oxime resin strategy
(a) and substrate mimet-
ic-supported peptide
-
fragment condensation
b)
(b) catalyzed by specific
+ Protease peptidases according t o
-A
00000
Cerovsky and Bordu-
sa [2061.
a protecting group 0individual amino acid A specific leaving group
anhydride, deprotection of the N*-amino group of the attached amino acid, followed
by successive chain elongation according to the well-known SPPS methodology.The
generation of the peptide fragment in the form of the substrate mimetic can be
performed by aminolysis of the oxime ester linkage between the peptide and resin,
with the appropriate free amino acid substrate mimetic ester as shown schematically
in the upper part (a) of Fig. 12.5-23. After deprotection of side-chain functions of the
amino acid residues, and if necessary also those of the ester leaving group, the only
Nu-protected peptide ester can be coupled with an amine component using the
suitable peptidase (b).Some examples of model fragment condensations using this
approach with catalysis from three different peptidases are given in Fig. 12.5-24. The
coupling reactions were performed on a preparative scale using 1 : 2 ratios of acyl
donor ester to the nucleophilic acyl acceptors (in the case of trypsin 1 : 2.5) resulting
in product yields between GO-70 %.
12.5.3.7
Planning and Process Development of Enzymatic Peptide Synthesis
The high enantio- and diastereoselectivity in peptidase-catalyzed peptide synthesis

allows, in constrat to most chemical coupling methods, the formation of peptide
bonds without partial epimerization in the C-terminal amino acid residue of the
carboxyl component. Furthermore, owing to the regiospecificity of the enzymes,
tedious protection/deprotection steps are not problems in the enzymatic approach.
Using serine and cysteine peptidases a further point needs to be decided; namely,
should the carboxyl component be used as the acylamino acid or should an ester be
used in order to favor acylation of the enzyme. Enzymatic synthesis using peptide
esters or amino acid esters as substrates has the clear advantage of proceeding at a
high rate, thereby demanding a low concentration of enzyme and, furthermore,
being completely independent of the solubility of the starting materials and product.
Although the kinetically controlled synthesis would be preferable, the decision
should depend on the total synthetic concept. An unfavorable nucleophile specificity
may be better taken care of in an equilibrium-controlledreaction with the necessary
manipulations of conditions. In spite of some limitations the equilibrium-controlled
approach has proved to be worthwhile in the trypsin-catalyzed semisynthesis of
852
a)
Boc-Tyr(Bz1)-Pro-Ser(Bz1)-Ala-Leu-0-P + H-Ala-OGp(Z)z
Boc-Tyr(Bzl)-Pro-Ser(Bzl)-Ala-Leu-Ala-OGp(Z)2
1
1
HZlPd
I
+
Boc-Tyr-Pro-Ser-Ala-Leu-Ala-OGp H-Met-Ala-Ala-Ala-GIy-OH
2 I 3
trypsin
Boc-Tyr-Pro-Ser-Ala-Leu-Ala-Met-Ala-Ala-Ala-Gly-OH
4
b)
I
Boc-Trp-He-He-Leu-0-P + H-Gly-SCe
Boc-Trp-He-He-Leu-Gly-SCe + H-Leu-Ala-Ala-Ala-GIy-OH
5
1 6
V8 protease
Boc-Trp-lle-lle-Leu-Gly-Leu-Ala-Ala-Ala-Gly-OH
7
c)
Boc-Leu-Asn-Lys(Z)-Ile-0-P + H-Val-OPh
i
I
Boc-Leu-Asn-Lys(Z)-He-Val-OPh
8
HdPd
Boc-Leu-Asn-Lys-lle-Val-OPh + H-Arg-Ala-Ala-Ala-Gly-OH
9 10
1
chymotrypsin
Boc-Leu-Asn-Lys-He-Val-Arg-Ala-Ala-Ala-Gly-OH
11
Figure 12.5-24. Combination of solid-phase peptide synthesis and sub.
strate mimetic-supported segment condensations with different pepti-
dases and substrate mimetics according t o Cerovskyy and Bordusa[2061.
human insulin as well as the industrial aspartame synthesis using therrnolysin. In

order to overcome poor solubility of the starting components the introduction of
solubilizing protecting groups is frequently necessary.
I 853
12.5.3.7.1 Stepwise Chain Elongation
Contrary to chemical synthesis, enzymatic stepwise chain building may start either
from the N-terminus or from the C-terminus. In chemical synthesis, incremental
chain lengthening from the N-terminus, as performed in ribosomal protein synthe-
sis, is normally not recommended under preparative conditions, since the efforts
needed to avoid the permanent risk of partial epimerization outweigh the potential
gain. Despite these principal limitations, investigations on solid-phase peptide
synthesis in an N-to-Cdirection, called inverse synthesis, has been performed using
HOBt salts of the amino acid 9-fluorenylmethyl esters L2O71. Unfortunately, the
racemization problem could not be excluded. Furthermore, Mitin and Ryadnov[208]
have described inverse peptide synthesis in order to exclude deprotection reactions at
every solution synthesis stage. This could be realized using the high solubility of free
amino acids in dimethylformamide containing Ba(C104)2, Ca(C104)2 or Ca(N03)2.
An attempt to solve the extensive exclusion of racemization was tried using
copper(n)ions (CuC12)during activation of the carboxyl group with ethyldimethyla-
minopropylcarbodiimide (EDC) as the coupling reagent in the presence of
HOBt [2091.
In a general sense exopeptidases should be the enzymes of choice for stepwise
chain assembly since once formed the internal peptide bonds of the growing chain
can no longer be proteolytically cleaved from this type of peptidase. Carbox-
ypeptidase exhibit superior properties for the stepwise synthesis, especially, carbox-
ypeptidase Y (CPD-Y)[2101 or other serine peptidases of this type. In principle,
aminopeptidases can also be used starting from the C-terminus. Because under
these conditions not only the carboxyl component but also the amine component has
a free a-amino function, product isolation is more difficult, particularly, if one
component is used in excess. Otherwise, stepwise synthesis from the C-terminus is
not a problem in chemical peptide synthesis.
A classical example for a kinetically controlled synthesis starting from the N-
terminus and using CPD-Y as an enzyme for all coupling steps was described by
Widmer et al. f211] for [Metlenkephalin(Tyr-Gly-Gly-Phe-Met). Bz-Arg-OEt was cou-
pled with H-Tyr-NH2 at pH 9.6 giving the Bz-dipeptide amide in 85% yield. The
CPD-Y-catalyzeddeamidation at pH 9.6 provided Bz-Arg-Tyr-OHin 90 % yield. After
chemical esterification with EtOH/HCl, the resulting Bz-Arg-Tyr-OEtwas coupled
with H-Gly-OEt at pH 9 to give the protected tripeptide derivative (yield: GO%),
followed by the successive addition of the other amino acid derivatives in the same
manner. Amino acid amides were preferred as the amine components, since free
amino acids (except Met) only give low yields and amino acid esters give rise to side
reactions that are difficult to control.
Finally, the protecting group for the P-amino function of Tyr, the Bz-Arg moiety,
was easily removed with trypsin. The disadvantage of this synthesis strategy seems to
be the complicated route of selectively removing the C-terminal amide grouping by
means of CDP-Y. This step followed by chemical esterification of the peptide had to
be resorted to before it was possible to use the intermediate in the next coupling
reaction as the carboxyl component.
A second step-by-steppeptide synthesis from the N- to C-terminus was described
Tyr-Arg-Ser-OH from N- t o
SBzl H OEt C-terminus using clostripain and
1 a) chymotrypsin, respectively, as bio-
Yield: 89% catalysts according t o Bordusa et
Z OEt H OPr
3 al.[208].a) and c): Clostripain; b):
b)
Yield: 84% chymotrypsin; d): catalytic hydro-
Z OPr H OBZ'
5 genation using 10% Pd/C; -OPr,
C)
Yield: 83% propyl ester; -SBzl, thiobenzyl ester.
Z OBzl
7 4
Yield: 100%
H OH
8
by Bordusa et al. 1212] for the model tetrapeptide H-Lys-Tyr-Arg-Ser-OHbut using the
endopeptidases clostripain and chymotrypsin as biocatalysts (Fig. 12.5-25). The
synthesis could be performed without side chain protection for all trifunctional
building blocks and the only nonenzymatic reaction was the final catalytic hydro-
genation for cleavage the terminal blocking groups.
As a rule, peptidases can only make a meaningful contribution to a synthesis
strategy if the full advantagee of the enzymatic reactions can be utilized. An a priori
completely unrealistic position is the comparison of a stepwise peptidase-catalyzed
assembley of a peptide chain with the automatic solid-phase technique.
On the other hand, selected di- and tripeptides can be synthesized enzymatically
using solubilizing protecting groups on a large scale, even in a continuous proc-
ess[118-120]
(cf. 12.5.3.4.1, p. 832-833). In addition, the solid-to-solidconversion has
proven to be a very useful method for the synthesis of selected short peptides which
fulfil the requirements for this special synthetic procedure (cf. 12.5.3.4.2, p.
838-839).
12.5.3.7.2 Fragment Condensation

This approach has some advantages over the stepwise strategy. Firstly, if small
fragments are combined to make one which is larger its isolation is more easily
facilitated in contrast to a stepwise synthesis, and, secondly, the fragment condensa-
tion approach offers the possibility of synthesizing a set of related analoges with
variabel sequences in a region. In principle it is possible to synthesize peptides using
enzymes both for protection/deprotection procedures as well as for the formation of
the peptide bonds. Fig. 12.5-26 shows the fully enzymatic synthesis of the tert.-butyl
ester of Leu-enkephalin12131 using both equilibrium and kinetically controlled
coupling steps. In order to obtain the unprotected Leu-enkephalin, the C-terminal
protecting group must be split off by chemical means.
Although, in principal it is possible to perform totally enzymatic synthesis of
peptides, in practice combined chemical and enzymatic steps are preferred. For the
classical enzymochemical synthesis of polypeptides and even small proteins, the
optimum approach is usually synthesis of fragments using the SPPS methodology
Hl
I 855
1
G~Y
p
Phe
h A ~
Leu
~
OBU'
Figure 12.5-26. Fully
enzymatic synthesis of
~ [Leulenkephalin
~ ~ tert-butyl
PA.. DeniciIIin
.
acylase; CT, chymotrypsin;
n i ~ ~ OBU' P,
P papain; PhAc,
h phenyla-
~ ~ ~ ~
.;iH
PhAc OMe ~
PhAc OMe OBu' cevl.
P
PhAC 1 I I I
for enzymatic conjunction in a overall divergent strategy. In a given synthesis project

initially it is necessary to separate the whole sequence into segments containing
favorable combinations of amino acids which permit peptidase-catalyzed segment
coupling according to the eluciated S'-subsite specificity. Since the kinetic parame-
ters of the enzymatic synthesis course are often not available, they can be estimated
from the data for similar substrates and nucleophilic amine components.
Based on such estimates an optimum synthesis strategy can be established. In
Table 12.5-10selected examples of enzymatically synthesized peptides are compiled.
During the last decade in particular, remarkable efforts have been made to find
optimum conditions for peptidase-catalyzedpeptide synthesis including the develop-
ment of new reaction conditions and new biocatalyts. Once the optimal synthesis
conditions have been recognized, kg amounts of biologically active peptides can be
produced. The synthetic biotransformations can normally be achieved with commer-
cially available enzymes which are easy to handle. In addition, owing to the
application in only catalytic amounts the higher costs of the enzymes used are
usually insignificantly in comparison with highly sophisticated chemical coupling
reagents plus the financial expense of the reagents necessary for protection/
deprotection procedures in chemical synthesis.
As a model system for peptidase-catalyzed modification of peptides produced by
recombinant DNA technology Schellenberger et al. [243, 244] developed a new ap-
proach to the production of peptides based on chemical synthesis and peptidase-
catalyzed processing (Fig. 12.5-27). First, they produced an artificial substance P
precursor as a P-galactosidase (1-459) fusion protein containing nine copies of the
sequence H-Arg-Leu-Arg-Argl-Pro-Lys-Pro-Gln-Gln-Phe7-OH. The sequence of the
peptide precursor was designed to meet the specific requirements of chymotrypsin
and papain, respectively, used in conversion reactions as the complete amino acid
sequence should be regenerated by addition of the appropriate dipeptide derivatives.
After isolation and purification of the fusion protein, which was accumulated in E.
coli as inclusion bodies, the dodecapeptide ester H-Arg-Leu-Arg-Arg'-Pro-Lys-Pro-
Table 12.5-10. Selected examples of enzymaticallysynthesized peptides.

Peptide/Protein Synthesis route* References
Angiotensin I1 (analog)
Aspartame
Calcitonin (salmon)
Calcitonin (dicarba analogs)
Caerulein
Caerulein (analog)
Cholecystokinin-8
Cholecystokinin-8 (analogs)
Delta sleep inducing peptide (DSIP)
Dynorphin-(1-8)
EGF (3-14,21-31, 33-42)
EGF (29-44)
Eledoisin (611)
Eledoisin
[Met/Leu]Enkephalin
[MetIEnkephalin
[LeuIEnkephalin
[LeuIEnkephalinderivatives
Growth hormone releasing factor (human) analog
Hepatitis B S antigen (122-137)
Ht31(493-515) peptide
Insulin (human)
Kyotorphin
LH-RH
[D-Phe6]LH-RH
MSH (5-8,9-12,13-16)
Oxytocin (1-9)
Ribonuclease A
Somatostatin
Substance P (6-11,7-11)
Vasopressin (1-6)
* E, equilibrium approach: K, kinetic approach; total, totally enzymatic coupling; part, partly enzymatic
coupling
Gln-Gln-Phe-Gly'-OMe was formed by chymotrypsin-catalyzed transpeptidation in

the presence of H-Phe-Gly-OMe. In a papain-catalyzed acyl transfer reaction and
subsequent tryptic cleavage, the resulting dodecapeptide ester was converted into
substance P. These results indicate that peptides can be readily produced by a
combination of recombinant DNA technology and peptidase-catalyzed conversion
with the advantage of possible incorporation of groups other than coded amino acids
into the recombinant product.
The chemoenzymatic synthesis of RNase A['"] using a mutant of subtilisin BNP',
called subtiligase, underlines the progress of enzyme-catalyzed fragment condensa-
tions in the course of the synthesis of a small protein. The fragments (98-124,
77-97,64-76,52-63,21-51 and 1-20) were synthesized by standard SPPS method-
ology. The choice of the fragments was solved in such a way that the C-terminal
residues of the appropriate fragments (Tyr", Tyr", Val63and Alazo)were the closest
Linker Sequence
12.5 Hydrolysis and Formation ofPeptides
I
857
Helper Protein
c <Substance P-(l-7)] \
00000 Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Arg-Leu-Arg-[ SP1-7],00000
+ ff-Phe-Gly-OMe
Chymotrypsin-catalyzedtranspeptidation
00000 Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gfn-Gln-Gln-Phe-Phe-Gly-OMe
Papain-catalyzedacyltransfer
1 + /+-/-eu-Met-Nff2
00000 Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH~
1
Trypsin-catalyzedcleavage
n H-Arg-Pro-Lys-Pro-GIn-Gln-Phe-Phe-Gly-Leu-Met-NH2
Substance P
Figure 12.5-27. Peptidase-catalyzed modification of an artificial substance P precursor
protein according to Schellenberger et a/. [2431.
to matching the substrate specificity of the subtilisin mutant. Using a considerable

excess of the fragments bearing a Phe-NH2-modified carboxamido methyl ester
ensured that most of the side reactions could be suppressed. Starting with the C-
terminal fragment (98-124) the total yield after five fragment condensations was
15%, and after folding the final protein could be obtained in 8% yield. In a similar
manner the three analogs of RNase A were synthesised in which the two residues
His” and His’19 of the active center were exchanged individually and simultane-
ously for L-4-fluorohistidine.
Despite this impressive example of five successful enzyme-catalyzed fragment
condensations with average yields of roughly 75 % in the course of the synthesis of
RNase A all the peptide bond forming steps could not be performed irreversibly.
Even though the new C - N ligation strategy based on the substrate mimetic concept
(cf. Sect. 12.5.3.6)has not as yet been proved for the synthesis of a similar protein
target, it guarantees the irreversibility of the enzymatic coupling reaction, as can be
demonstrated by the chymotrypsin-catalyzed (8 + 16) fragment condensation of the
Ht 31(493-515) peptide derived from the human protein kinase A anchoring protein
(sequence 493-5F1)[~~~]. The synthesis of the 24-peptide was accomplished by the
chymotrypsin-catalyzed fragment condensation at a nonspecific Ser-Arg peptide
bond via the substrate mimetics strategy (Fig. 12.5-28). The fully protected carboxyl
component 1 was synthesized on Kaiser’s oxime resin and was released from the
support by aminolysis with H-Ser(Bz1)-OPaccording to the procedure described on
p. 850-851. After side-chain deprotection by catalytic hydrogenation, 2 was coupled
with the unprotected amine segment 3 catalyzed by chymotrypsin, leading to the
complete conversion of both peptide segments. Finally, the N-terminal Boc group
was cleaved by TFA giving the desired Ht 31 (493-515) peptide 5.
858
Boc-Asp(OBzl)-Leu-lle-Glu-(OBzl)-Glu(OBzl)-Ala-Ala-O-oxime resin
ITFA.Ser(Bzl)-OPh
Boc-Asp(OBzl)-Leu-lie-Glu-(O6zl)-Glu(OBzl)-Ala-Ala-Ser(Bzl)-OPh 1
I H,IPd
Boc-AspLeu-Ile-Glu-Glu-Ala-Ala-Ser-OPh 2
H-Arg-lle-Val-AspAla-Val-lle-Glu-Gln-Val-Lys-Ala-AIa-Gly-Ala-Tyr-OH 3
I
I
1
Chyrnotrypsin
Boc-Asp-Leu-lle-Glu-Glu-Ala-Ala-Ser-Arg-lle-Val-Asp 4
-Ala-Val-lle-Glu-Gln-Val-Lys-Ala-Ala-Gly-Ala-Tyr-OH
1 TFA
H-Asp-Leu-lle-Glu-GIu-Ala-Ala-Ser-Arg-ile-Val-Asp- 5
-Ala-Val-lle-Glu-Gln-Val-Lys-Ala-Aia-dy-Ala-Tyr-OH
Figure 12.5-28. Chymotrypsin-catalyzed (8+16) segment synthesis of the Ht
31 (493-51 5) peptide via substrate mimetic strategy[233].
12.5.4
Conclusion and Outlook
Despite the fact that chemical methods are popular for the synthesis of peptides a
huge number of papers has been published in recent decades dealing both with
enzymatic formation of peptide bonds and enzymatic manipulation of protecting
groups. Enzymatic methods have several advantages over chemical procedures but at
present more peptides are synthesized by chemical synthesis than in peptidase-
catalyzed processes. The use of peptide synthesizers, in addition to recent new
developments in the field of chemical ligation procedures, still favor chemical
methods compared with the enzymatic approach. However, there is no doubt that
enzymatic methods have advantages, including the prevention of racemization, no
need for time-consuming and expensive protection/deprotection procedures of side-
chain functions, the reduced use of problematic (toxic) solvents and reagents and
possible reuse of the biocatalysts. The question should not be whether to use a
chemical or an enzymatic approach in peptide synthesis; an ingenious combination
of chemical and enzymatic steps should promote the general progress in peptide
synthesis.
It could be demonstrated that after establishing the optimal synthesis conditions,
kg amounts of biologically active peptides and analogs can be obtained using
enzymatic coupling methods. The semisynthetic synthesis of human insulin and the
References I859
production of aspartame in a ton-scale underline the industrial importance of the

enzymatic approach. However, the enzymatic approach does not have the versatility
of chemical synthesis methods and suffers from some limitations. The main reason
seems to be the lack of a universal enzyme which is capable of catalyzing peptide
bond formation for all possible combinations of the 21 proteinogenic amino acid
residues located both as C- and N-terminal building blocks in peptide fragments to
be coupled. Such an enzyme could not be developed during evolution due to the
extremely high specificity requirements. In ribosomal protein synthesis nature
prefers the stepwise synthesis from N- to C-terminus followed by maturation
procedures based on limited proteolysis and further modifications. The only bio-
catalyst involved in ribosomal synthesis, the peptidyl transferase, seems to be an old
ribozyme without any specificity for the PI side chain functions of the amino acids,
only catalyzing the acyl transfer reaction of the selected aminoacyl-tRNAs. Since
such a biocatalyst has no practical importance in peptide synthesis in a peptide
laboratory, the only alternative for this purpose is the reverse catalytic hydrolysis
potential of proteases.
The advantages and drawbacks of peptidases used for catalyzing peptide bond
formation have been demonstrated in this contribution. An ingenious combination
of chemical and enzymatic strategies as demonstrated in a new synthesis of RNase A
should be the state-of-the-artin this field at present. Furthermore, using the new C -
N ligation strategy based on the substrate mimetic concept, irreversible peptide
bond formations catalyzed by high specific peptidases can be performed for the first
time. In combination with peptidase mutants which lack amidase activity, this new
C - N ligation approach will contribute to significant progress in enzymatic peptide
synthesis, especially in clear-cut fragment condensations using recombinant poly-
peptide thioester as the substrate mimetics with chemically synthesized or re-
combinant fragments. This specific programming of enzyme specificity by molec-
ular mimicry corresponds in practice to a conversion of a peptidase into a C - N
ligase, a biocatalyst which could not developed by nature during evolution.
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866
12.6
Addition of Amines to C = C Bonds
Marcel Wubbolts
The ammonia lyases (E. C. 4.3.1.x),which catalyze the addition of amines to carbon-
carbon double bonds, belong to the class of carbon-nitrogen lyases. The reactions
catalyzed by ammonia lyases are in equilibrium and depending on reaction condi-
tions the reaction can be directed either towards ammonia addition or in the
direction of elimination of ammonia.
Ammonia lyases in their natural role are involved in the metabolism of amino
acids and also play a role in, for instance, the degradation of amino sugars, but only a
limited amount of these enzymes have been characterized biochemically. Applica-
tion of a broad range of different ammonia and lyases in organic chemical synthesis
on an industrial scale has thus far not occurred, which is due to both their limited
commercial availability and their lack of stability under process conditions. Excep-
tions are the commercially applied aspartase, which is an ammonia lyase that is
utilized for the synthesis of 1-aspartic acid from fumaric acid, and phenylalanine
lyase. The latter is an example of a commercial application of an ammonia lyase in a
process for the production of L-phenylalanineand more importantly L-phenylalanine
derivatives.
12.6.1
Addition of Ammonia to Produce Amino Acids
12.6.1.1
Aspartic Acid
L-aspartic acid ammonia lyase, or aspartase (E.C. 4.3.1.1) is used on a commercial

scale by Kyowa Hakko, Mitsubishi, Tanabe and DSM to produce L-aspartic acid,
which is used as a building block for the sweetener Aspartame, as a general acidulant
and as a chiral building block for synthesis of active ingrediants [ll. The reaction is
performed with enzyme preparations from E. coli, Breuibacterium Jauum or other
coryneform bacteria either as permeabilized whole cells or as isolated, immobilized
enzymes. The process is carried out under an excess of ammonia to drive the
reaction equilibrium from fumaric acid (1)in the direction of r-aspartic acid (L-2)
(see Scheme 12.6-1) and results in a product of excellent quality (over 99.9% e. e.) at a
yield of practically 100%.The process is carried out on a multi-thousand ton scale by
the diverse producers of L-aspartic acid. Site directed mutagenesis of aspartase from
E. coli by introduction of a Cys430Trp mutation has resulted in significant activation
and stabilization of the
Since maleic acid is a cheaper starting material than fumaric acid, the process that
is probably the most economical makes use of both a maleate isomerase (E.C.
5.2.1.1) and aspartase (E. C. 4.3.1.1), Scheme 12.6-1. Mitsubishi has succeeded in
12.G Addition ofAmines t o C = C Bonds I 867
Aspartase
+COOH
H O O C T
L-2 NH:,
-
Maleate Aspartase
COOH lsomerase
3 L C O O H
’ H O O C T
COOH
L-2 NH2
-
Aspartase Fumarate
H O O C T
COOH
-
+------
H OO C , - HOOC
, - ,
-
hydratase
+ I.c-- H O O C T
OH
COOH
NHZ NHz
rac 2- D-2 4
Aspartase Aspartate-p
HOOC
&COOH
___L
4
- L-2 -
decarboxylase
Y C O O H
1 NH2
Scheme 12.6-1.
combining both activities in a Brevibacteriumjavumrecombinant for the large-scale

production of L-aspartic acid13].
Mitsubishi has also developed a process for production of D-asparticacid (D-2)and
L-malic acid ( 4 ) by incubation of racemic aspartic acid with the exclusively L-selective
aspartase in combination with fumarase, thereby preventing the reaction going
backwards by conversion of the generated fumaric acid into L-malic acidI4].
The combined utilization in a single reactor of both aspartase from Brevibactevium
flavum and aspartate-P-decarboxylase from Pseudomonas dacunhae, thereby catalyz-
ing the reaction from fumaric acid via L-aspartic acid to L-alanine (S),has also been
developed by Mitsubishi[’I.
Another combination reaction is the biocatalytic production of the herbicide
phosphinotricin [ ~-2-amino-4-(hydroxymethylphosphinyl)butyric acid, (7)in Scheme
12.6-21 by the company Meiji Seika, whereby an amino-transferase that acts on
4-(hydroxymethylphosphinyl)-2-oxo-butyric acid and that utilizes aspartic acid as the
amino donor was used in combination with aspartase to generate the amino donor
from fumaric acid and ammonia[‘].
868
0 Aspartase
Amino transferase
OH 0
6 OH 7
Scheme 12.6-2.
12.6.1.2
Aspartic Acid Derivatives
The enzyme methylaspartate ammonia lyase (P-methylaspartase, E. C. 4.3.1.2) is

involved in the metabolism of branched pentanoic acids. The enzyme catalyzes the
addition of ammonia to mesaconic acid (8) to yield ~-threo-3-methylaspartate(9) as
depicted in Scheme 12.63. The enzyme has been shown to be induced under
anaerobic conditions in facultative anaerobes such as Citrobacter, Proteus, Escherichia
coli and Enteroba~ter[~.81 and has been applied for the synthesis of 3-substituted (S)-
aspartic acid derivatives, such as (2S,3S)-3-methylasparticacid (9), (2S,3S)-3-ethylas-

partic acid (ll),and (2R,3S)-3-chloroasparticacid (13)"1. In addition, a process for
the preparation of dialkyL(2S,3S)-3-ethylaspartatesusing methylaspartate ammonia
lyase has been developed by Merck['I].
Bear et al. have been using methylaspartate ammonia-lyase from Clostridium
tetanomorphum to produce optically active pure precursors [3-methyl-,3-ethyl and
-
Methylaspartate
Ammonia Lyase
HOOCL C O O H
7
HOOCL C O O H ___)
-c--.---
10
HOOCL C O O H ____)
7
HOOC+
12 13 NH2
HOOCL C O O H ____)
P
14
12.6 Addition ofAmines to C = C Bonds
I
869
RT
Rp
rcoo Phenylalanine
Ammonia
P
_____) Lyase
NH2
19
e0/
R = NOz, CI. NH2. OH, CH3 at o. rn and p position
C;3.iUr
Phenylalanine 0
Ammonia Lyase
y
3,4-Dihydroxy-
q-cooH
phenylalanine
Ammonia Lyase COOH
HO = HO 23
OH OH
Scheme 12.6-4.
3-iso-propylaspartic acids, (15)] for the synthesis of benzyl 3-alkylmalolactonates,

which are suitable building blocks for semi-crystallinepolyesters ("1.
12.6.1.3
Histidine Ammonia Lyase
Histidine ammonia lyase (HAL, histidinase, histidine-a-deaminase, E. C. 4.3.1.3) is

capable of abstracting ammonia from L-histidine (17),resulting in the formation of
urocanoic acid [Scheme 12.6-4, (G)], an intermediate in the metabolism of L-
histidine("]. HAL has also been identified as a key enzyme in the synthesis of
secondary metabolites such as Nikkomycin in Streptomyces teradae['21. The mecha-
nism of the enzyme has been investigated and seems to proceed via the carbanion
intermediate [l', 131. Synthetic applications of HAL are difficult to achieve, partic-
ularly as the enzyme is sensitive to oxygen[13].The utility of HAL is limited to niche
applications such as the synthesis of radiolabeled urocanic acids as tracers of
histidine metabolism [ll].
870
I L-Serine
Deaminase
NH2 0
L-Threonine
Deaminase
[ "r:cooH]
- /\ljCOOH
0
NH2
Scheme 12.6-5.
12.6.1.4
Phenylalanine, Tyrosin and L-DOPA
Phenylalanine ammonia lyase (PAL, E. C. 4.3.1.5)is an enzyme of relaxed substrate

specificity that accepts both trans-cinnamic acid (Scheme 12.6-4: (18),R = H) and p -
coumaric acid [(19),R = OH] as substrates and thus results in the formation of the
natural amino acids L-phenylalanineand L-tyrosine.The enzyme plays an important
role in the synthesis of alkaloids, flavenoids and lignin in plants. The reaction has
been exploited by Mitsui[14,"1, Great Lakes/NSC[lGland others to implement
synthetic routes for non-natural substituted derivatives of L-phenylalanine starting
from trans-cinnamic acids, for instance using the PAL enzymes from Rhodotorula
rubra, Rhodotorula glutinis or Rhodosporidium toruloides. The PAL mediated synthesis
of a variety of L-phenylalanine derivatives, carrying aromatic ring substituents such
as nitro-, chloro-, amino-, hydroxy- and methyl groups at the 2, 3 and 4 position have
thus been described116181. Also, the synthesis of N-heterocyclicmolecules, derived
from phenylalanine by PAL has been shown['7, 19, I.' The direct synthesis of
phenylalanine methyl ester [Scheme 12.6-4, (21)] as a building block for aspartame,
from trans-cinnamyl methyl ester (20) by PAL from Rhodotorula glutinis further
illustrates the synthetic versatility of PAL'", 1'. Radioactive tracers derived from L-
phenylalanine have also been made with the aid of PAL[23s241s
An enzyme that is related to PAL, dihydroxy-L-phenylalanine ammonia lyase (E. C.
4.3.1.11), is capable of synthesizing L-DOPA (23) from 3,4-dihydroxy-trans-caffeic
acid (22), but this starting material is not as readily available as catechol, pyruvate,
and ammonia are. As a result, the tyrosine phenol-lyase (TPL, E.C. 4.1.99.2) of
Envinia herbicola is the enzyme of choice for biocatalytic L-DOPAproduction[25,"1,
particularly as productivity has been increased since the TPL encoding gene from
Enuinia herbicola was cloned and has been overexpressed successfully[25].
12.G Addition ofAmines t o C = C Bonds
I 871
12.6.1.5
Serine and Threonine Deaminases
Both the L- and D-serine deaminase catalyze the elimination of the amino function-
ality of both L- and D-serine,but the mechanism proceeds via the initial elimination
of water and these enzymes are thus classified as hydrolyases (L- and D-serine
dehydratases E. C. 4.2.1.13 and E. C. 4.2.1.14, respectively)[27, *‘I. The aminoacrylate
generated is unstable and subsequent elimination of the amine results in the
formation of pyruvate. Similarly, threonine deaminase is in effect a dehydratase that
converts L-threonine into 2-oxobuturate, water and ammonia (E. C. 4.2.1.16)
(Scheme 12.6-1).
12.6.1.6
Ornithine Cyclodeaminase
Ornithine cyclodeaminase (E.C. 4.3.1.12) is an ammonia lyase that is not ubiqui-

tously present but which has been identified in genera such as Rhizobium,Agrobacte-
hum, Pseudomonas, Rhodobacter and Clo~tridium[~”~~]. Ornithine cyclodeaminase,
which contains NAD that is tightly bound to the enzyme, catalyzes the conversion of
L-ornithine, an intermediate in the metabolism of L-arginine, into L-proline. The
reaction is peculiar among the ammonia lyases in that it involves a deamination of
the amino group at the a-position followed by attack of the &amino group to give
2-0x0-5-aminopentanoicacid to form proli line[^^]. Conversions other than that from
L-ornithine to L-proline have not been described.
12.6.2
Ammonia Lyases that Act on Other Amines
12.6.2.1
Elimination of Ammonia from Ethanolamine
The elimination of ammonia from ethanolamine to give acetaldehyde, which

involves vitamin BIZ and which has been demonstrated to proceed via a radical
anionL34,351, is catalyzed by ethanolamine ammonia lyase (EAL, E.C. 4.3.1.7).
Genetic and biochemical analysis of the ethanolamine ammonia lyase isolated from
Salmonella tphimurium and Rhodococcus sp. have been carried 371 and the
enzyme appears to belong to a class of BIZ dependent enzymes that catalyze similar
rearrangements, such as diol dehydratase and methylmalonyl-CoA mutase [351.
Ethanolamine ammonia lyases are induced under anaerobic conditions, which is
required since the radical reaction intermediates are highly reactive with dioxy-
gen[38].Despite the interesting chemistry, we did not come across synthetic applica-
tions of ethanolamine ammonia lyases, other than the observation that the enzyme
of Acetobacterium catalyzes the elimination of ammonia from triethanolamine in
addition to ethanolamine r3’1.
872
References
1 A. Liese, K. Seelbach, C. Wandrey, Industrial 16 W. Liu, Synthesis ofoptically Active Dhenyl-

Biotransformations, Wiley-VCH Verlag alanine Analogs using Rhodotorula grami-
GmbH, Weinheim, 2000. nis, Great Lakes Chemical Corp., 1997, US
2 S. Murase, J. S. Takagi, Y. Higashi, H. Im- 598 1239.
aishi, N. Yumoto, M. Tokushige, Biochem. 17 J. S. Zhao, S. K. Yang, Huaxue Xuebao 1997,
Biophys. Res. Commun. 1991, 177,414-419. 55,196-201.
3 K. Hatakeyama, M. Goto, M. Terasawa, H. 18 J . S. Zhao, J. Q. Cao, S. K. Yang, YaoxueXue-
Yukawa, Fermentative Manufacture ofr-As- bao 1995,30,466-470.
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nia, Mitsubishi Chemical Industries Ltd., 1993, 13,486-489.
1997, J P 103 13889. 20 M. Yanaka, D.Ura, A. Takahashi, N. Fuku-
4 M. Terasawa, S. Nara, H. Yamagata, H. Yu- hara, Manufacture ofa-Substituted Alanines
gawa, Manufacture of D-Aspartic Acid and/or with L-PhenylalanineAmmonia-Lyase,Mit-
r-Malic Acid with Aspartase and Fumarase, sui Toatsu Chemicals, 1992, J P 061 13870.
Mitsubishi Petrochemical Co., 1991, 21 G. B. D’Cunha, V. Satyanarayan, P. M. Nair,
JP06014787. Enzyme Microb.Techno1. 1996, 19,421-427.
5 M. Gotoh, T.Nara, M. Terasawa, and H. 22 G. B. D’Cunha, V. Satyanarayan, P. M. Nair,
Yukawa, Single Reactor Microbial Manufac- Enzyme Microb.Technol. 1994, 16, 318-322.
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6 S. Imai, M. Takahashi, S. Fukatsu, Y. 24 J . Jemielity,M. Kanska, R. Kanski, Isot. En-
Ogawa, New Processfor the Production ofr- viron. Health Stud. 1998, 34, 335-339.
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ric Acid, Meiji Seika Kaisha, 1989, EP viron. Microbiol. 1993, 59, 3070-3075.
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7 Y. Asano, Y. Kato, Biosci., Biotechnol., Bio- Kumagai, Appl. Environ. Microbiol. 2000, 66
chem. 1994,58,223-224. 4764-4771.
8 Y. Kato, Y. Asano, Arch.Microbiol. 1997, 168, 27 A. E. M. Hofmeister, S. Berger, W. Buckel,
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Preparation ofN-protected dialkyl (2S,3S)- P. F. Cook,J. Bid. Chem. 1999,274,
3-ethylaspartates, Merck, 1990, DE 36 935-36943.
4007038. 29 M. J. Soto, P. van Dillewijn, J. Olivares, N.
10 M. M. Bear, V. Langlois, M. Masure, P. Toro, FEMS Microbiol. Lett. 1994, 119,
Guerin, Macromol. Symp. 1998, 132, 209-214.
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1 1 T. Furuta, H. Takahashi, H. Shibasaki, Y. Caballero, F. Castillo, FEMS Microbiol. Lett.
Kasuya, ]. Bid. Chem.,1992, 267, 1993,114,333-337.
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12 U.Roos, S. Mattern, H. Schrempf, C. Bor- crobiol. 1991, 137, 2911-2918.
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13 J. D.Galpin, B. E. Ellis, M. E. Tanner,J. Am. 33 W. L. Muth, R. N. Costilow,]. Bid. Chem.
Chem. Soc. 1999,121,10840-10841. 1974,249,7463-7467.
14 N. Naito, R. Taneda, M. Koito, H. Ito, N. 34 T. T. Harkins, C. B. Grissom, Science 1994,
Fukuhara, Manufacture of L-Phenylalanine 263,958-966.
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171,3316-3323.
12.7 Transaminations
I 873
12.7
Transaminations
J. David Rozzell and Andreas 5.Bomrnarius
12.7.1
Introduction
Given their critical role in biological systems, it is not surprising that numerous
applications for amino acids have developed, particularly in the pharmaceutical
industry. Fourteen of the twenty common proteinogenic L-amino acids are essential
in human diets, which has led to the development of a significant market for these as
components in intravenous feeding solutions. L-Glutamic acid is used as a flavor
enhancer in foods with annual sales estimated at greater than one billion dollars. L-
Lysine, D,L-methionine,and L-threonine have already become established as large-
volume additives to animal feeds that require enrichment in certain deficient amino
acids, and L-tryptophan is developing a similar application. L-Phenylalanineand L-
aspartic acid have very important markets as key components in the manufacture of
the high-intensity sweetener aspartame. A competitive product in development,
alitame, is synthesized from D-alanine.
The importance of non-naturally occurring amino acids can be seen from the
increasing number of pharmaceutical products that incorporate one or more such
compounds as intermediates. Numerous chiral drug candidates are synthesized
from various natural and non-natural amino acid building blocks and have been
submitted for biological testing. Inevitably, applications for amino acids, both
naturally-occuringand non-natural, will result from this activity. There are already
numerous examples. The synthesis of two thrombin inhibitors, Tirofiban from
Merck & Co. and Inogatran from Astra-Zeneca,is based on analogs of L-tyrosine and
D-cyclohexylalanine, respectively. D-2-Aminoadipicacid is one of the amino acids
found in the tripeptide that is converted biologically into the p-lactam nucleus, and
its use as a precursor for producing semi-synthetic penicillins and cephalosporins
has been suggested. The L-antipode is also a common component of combinatorial
synthesis approaches that incorporate non-naturally occurring amino acids. Fluorine
substitution is also becoming increasingly common in the preparation of peptide
analogs. In particular, p-fluoro-L-phenylalanineis a good choice as a non-naturally
occurring amino acid for such work because it is almost isosteric with L-phenyl-
alanine, but contains a strongly electron withdrawing fluorine atom to modify its
dipole moment.
In particular, the non-naturally occurring amino acid r-tert-leucine has received
significant attention due to several pharmaceutically active compounds into which it
is incorporated[']. HIV-protease inhibitors developed by Novartis and Abbott are
based on L-tert-leucine[ 2 , 1' . Roche has developed the anti-arthritic compound Ro
31-9790 based on its potent inhibition of collagenase14]and a key component in the
synthesis of Ro 31-9790 is the methylamide of L-tert-leucine. Boehringer Ingelheim
developed a series of compounds that inhibit the ribonucleotide reductase of Herpes
874
simplex virus; several of the most active structures contained ~-tert-leucine[~]. As a

result, a market for L-tert-leucine as a pharmaceutical intermediate is developing. In
addition, the derivative L-tert-leucinolis widely used as a chiral auxiliary['].
The markets for non-naturally occurring amino acids can be substantial. In-
hibitors of angiotensin-converting enzyme, or the so-called ACE inhibitors, have
developed strong markets as anti-hypertensivedrugs. One of the most successful is
the product Enalapril, which has achieved sales of more than $1 billion annually.
Marketed by Merck, Sharpe and Dohme, Enalapril is due to go off patent soon,
leading to the emergence of generic competitors. Other similar ACE inhibitors
include Ramipril, Benazapril, Lisinopril, Zestril, Trandolopril, and Quinipril. A key
component in all of these compounds is L-4-phenyl-2-amino-n-butanoic acid, or L-
homophenylalanine. As price competition for generic ACE inhibitors intensifies, L-
homophenylalanine will probably become an important non-naturally occurring
amino acid product. Other large volume products include D-phenylglycine and D-p-
hydroxyphenylglycine,key intermediates in the synthesis of ampicillin and amox-
icillin, respectively, D-penicillamine, a chelator used to treat cystinuria and severe
arthritis, D-valine, a building block for the synthetic pyrethroid Fenvalerate, and
phosphinothricin, an important herbicide marketed by AgrEvo. Additional commer-
cial opportunities exist for the production of isotopically labeled amino acids,
particularly 15N, 15N/13C, and 15N/13C/2H amino acids for use in medical
research, with a larger potential market in magnetic resonance imaging.
Various methods have been developed for the production of amino acids. Most
naturally-occurring, proteinogenic amino acids can be produced by fermentation,
although chemical synthesis, isolation from hydrolyzed proteins, and enzymatic
conversion are used in a few instances. For the production of non-proteinogenic or
non-natural amino acids for which no metabolic pathways exist, traditional fermen-
tation methods cannot be used without re-engineering of the metabolic pathways in
the cell. For these types of amino acids, various chemical and enzymatic synthetic
methods have become increasingly common.
Among the various enzymes capable of producing optically-active amino acids,
transamination reactions, catalyzed by enzymes known as aminotransferases or
transaminases, have broad potential for the synthesis of a wide variety of enantio-
merically pure (R)-and (S)-compounds containing amine groups. Indeed, various
examples of the use of aminotransferases for the production of D- and L-amino acids,
both naturally-occurring and non-natural, have been published "I.[' In addition,
certain aminotransferases have been found to act on amines, and methods for the
production of enantiomerically pure amines by transamination have been de-
scribed[1G21]. This method allows for yields of up to 100% whereas routes based on
hydrolases require external racemization to reach such yield levels. In this section we
will focus on the application of aminotransferases.
I
875
12.7.2
Description of Transarninases
12.7.2.1
Homology and Evolutionary Subgroups o f Aminotransferases
About one third of all known sequences of vitamin BG-dependent enzymes belong to
aminotransferases which in turn can be divided into four subgroups based on
sequence homology: the most common species such as aspartate, tyrosine, or
phenylalanine aminotransferase belong to subgroup I, subgroup I1 takes (acet-
yl)ornithine, o-amino acid and y-aminobutyrate aminotransferases, subgroup 111
comprises the D-amino acid transferases, and subgroup IV the (phospho)serine
aminotransferases[221.Only 4 of the about 400 amino acid residues proved to be
invariant among all aminotransferase sequences: Gly 197, Asp/Glu 222, Lys 258,
and Arg 386. Apparently, aminotransferases form a group of homologous proteins,
the chemistry of which already existed very early in evolution.
12.7.2.2
Mechanism of Transamination
Aminotransferases are key enzymes in a number of metabolic pathways, and as a

result, enzymes from this class are widely distributed in nature. The first evidence
for the presence of an enzyme catalyzing a transamination reaction was published by
Needham and Szent-Gyorgyi and co-workerswho noticed a relationship between the
L-glutamic acid, L-aspartic acid, and oxaloacetic acid levels in pigeon breast mus-
~le[~ Banga
~ ] . and Szent-Gyorgyi demonstrated the reversibility of glutamic-pyruvic
transaminase (E. C. 2.6.1.2, alanine aminotransferase) by chemically isolating the
amino acid products L-glutamate and ~ - a l a n i n e [251.~ ~Since
. that time, a large
number of different aminotransferases have been discovered and characterized,
including aminotransferases, capable of catalyzing the transamination of all natu-
rally-occurring amino acids. There are now more than 2500 sequences of amino-
transferases known, compared with 51 sequences in 1993L221. As of the middle of
February 2001, the Entrez databank contained 121 3D-structures of 9 amino-
transferases from 13 organisms.
The mechanism of the reaction is well understood as a result of the detailed
studies of Meister [26,271. Aminotransferases catalyze the transfer or an amino group
from an amino acid donor to a 2-ketoacid acceptor (Fig. 12.7-1).This amino group
transfer is mediated by the cofactor pyridoxal phosphate, which is reversibly bound
to the enzyme through a Schiff-baselinkage to the epsilon-amino group of an active-
site lysine. Mechanistically, the reaction catalyzed by an aminotransferase can be
thought of as the result of two discrete steps. The first step is the transfer of an amino
group from the amino group donor to pyridoxal phosphate, generating a 2-ketoacid
byproduct that dissociates from the enzyme and an enzyme-bound pyridoxamine
phosphate intermediate. The second step involves the transfer of the amino group
from the enzyme-bound pyridoxamine phosphate to the 2-ketoacid acceptor, and
876
Figure 12.7-1. General

R1\ ,COOH R’\ rr OCOOH
2%H
C reaction catalyzed by
II H,N
arninotransferases.
0
2-Ketoacid Acceptor L-Amino acid
4%
H,N H
L-Amino acid Donor 2-Ketoacid Coproduct
4%
H NH,
2-Ketoacid Acceptor D-Amino acid
+ - L
D-Amino acid Donor 2-Ketoacid Coproduct
producing the corresponding amino acid product and regenerating the pyridoxal
phosphate cofactor for another catalyhc cycle. As a result, aminotransferases
characteristically exhibit ping-pong kinetics r2’].
12.7.2.3
Protein Engineering and Directed Evolution with Aminotransferases
Aminotransferase (AAT), the enzyme catalyzing the reversible transformation of

aspartate and glutamate into the respective 0x0 acids, has been studied most among
the vitamin B6-dependentenzymes. An X-ray crystal structure is now known for the
aspartic-glutamic aminotransferase from E. coli r2’1. Active site residues have been
identified, laying the groundwork for further detailed mechanistic studies and
modification of the enzyme by specific mutagenesis. Several workers have been
successful at changing the relative activity of aminotransferase towards different
groups of substrates or even different reactions through structure-based protein
engineering and directed evolution.
I
877
12.7.2.3.1 Structure-based Protein Engineering

Multiple active-site site-specific mutations of AAT led to an increase in P-decarbox-
ylase activity with the double mutant Y225R/R386A (1380-fold)f3O]. Coupled with a
decreased transaminase activity by a factor of 500 in the single mutant R292KL3lI,
workers found a combined 20 000-fold decrease in the rate of transamination in the
triple mutant Y225R/R292K/R386A [321. In fact, the triple mutant catalyzed p-
decarboxylation %fold faster than transamination, a change of ratio from the wild-
type enzyme by a factor of 25 million. The observed changes in substrate specificity
were rarely additive however, because triple mutants containing R292X, i. e. muta-
tions to amino acids other than lysine, were mostly completely inactive towards p-
decarboxylation even though they contained the double mutant Y225RJR386A
eliciting p-decarboxylase activity.
Previously, AAT had been transformed into an L-tyrosine aminotransferase (TAT)
by site-specificmutation of up to six amino acid residues lining the active site of wild-
type TAT. The hexuple AAT-mutant achieved kinetic data towards the transamination
of aromatic substrates such as L-phenylalaninewithin an order of magnitude of wild-
type TAT[33].
12.7.2.3.2 Directed Evolution o f Aminotransferases

Meanwhile, directed evolution methods that combine mutagenesis of genes with
high-throughput screening of functional gene products have developed rapidly.
In a selection strategy based on the substrate 1-phenyl-n-propylamine(PPA)as the
sole source of nitrogen in a chemostat, a recombinant Pseudornonas putida strain
carrying the R-transaminase gene, a single amino acid change, Y112F, presumably
at or near the active site, improved enantioselectivity of the reaction of racemic
1-phenyl-n-propylamineto (S)-1-phenyl-n-propylamine and propiophenone to 37.8 %
e. e. from 6.5 % e. e. in the wildty~e[~’].
Further site-directedmutagenesis of position
112 yielded 99.4% e.e. in the mutant Y112L. In a related example, a single mutant
T51S, generated by error-prone PCR in about 10000 samples, both improved
tolerance of (R)-transaminasetowards the reaction product, a substituted I-phenyl-
2-propylamine (an amphetamine), from 85 to 105 mM as well as reaction rate[”I.
Lastly, a p-tetralone was converted into the corresponding (S)-aminein 65 % e. e. by
wild-type (S)-transaminase (Fig. 12.7-2)[‘’I. Random mutation followed by activity
screening for the colored ketone starting from the enantiomerically pure amine,
produced a number of single mutants such as M245V, P247L, and F407L with higher
enantioselectivity,up to 84% e. e., at similar level of activity. It was further found that
+wo
combination of advantageous mutants through site-directed mutagenesis around
*-(S)-Transaminase .-mNH
2-amino- methylethylketone
butane
Figure 12.7-2. Conversion oftetralone-2 to 2-arninotetraline by (S)-transarninase.
878
sensitive sites such as 245-247 and 405-407 improved enantioselectivity further, up

to 94%.
Efforts to evolve aminotransferases with improved activity on new ketoacid
substrates have been initiated with encouraging results [341. Using directed evolution,
the substrate specificityof AAT has been changed to one favoring P-branched amino
acids and their respective oxoacids, effectively converting AAT into a branched-chain
aminotransferase (BCAT). By employing an E. coli auxotroph deficient in the
branched-chain aminotransferase (BCAT) gene, ilvE, the authors set up a stringent
selection system which provided a powerful advantage for cell growth to the mutated
AAT systems[35,3Gl. The resulting evolved aminotransferases had 13 [351 and 1713']
amino acid substitutions and showed 10S-fold and 2x10G-foldimprovement in
catalytic efficiency (kcat/&), respectively, towards the unnatural substrate, valine,
and between 10- and 100-fold decrease towards the natural substrate, L-aspartate,
compared with the wild-type. A high degree of conserved amino acid substitutions
was found in most active mutants. Interestingly, only one mutated amino acid
residue in each case is located at a distance to the substrate that would allow
interactions, the remainder were mutated far away from the active site. This work
demonstrates that 1OG-foldshifts in substrate specificity can be achieved when
employing directed evolution methods, that combinatorial or evolutionary methods
are probably superior to rational design methods when changing substrate specific-
ity, and most importantly, that remote residues and their interactions with the active
site environment are important determinants of enzyme activity and specificity.
Such remote residues act cumulatively, possibly by remodelling the active site, by
altering the subunit interfaces, or by shifting different enzyme domains.
12.7.3
Use o f Arninotransferases in Biocatalytic Reactions
12.7.3.1
Synthesis o f a-L-Amino Acids
Aminotransferases (transaminases) have been studied as potentially useful bio-

catalysts for the production of a wide range of different amino acids. The general
reaction catalyzed by aminotransferases is shown in Fig. 12.7-1.An amino group is
transferred from a donor amino acid to a 2-keto acid acceptor. As described earlier, a
cofactor, most commonly pyridoxal phosphate, is involved in the catalysis. The
cofactor, which is only required in concentrations of 50-100 PM,is reversibly bound
to the enzyme through a Schiff-baselinkage to the epsilon-amino group of active-site
lysine[2628].Using an aminotransferase, a desired amino acid can be produced from
a given 2-keto acid precursor using an inexpensive L-amino acid as the amino group
donor. As a co-product of the reaction, a second 2-keto acid corresponding to the
amino acid donor is produced along with the desired amino acid product in
equimolar amounts.
Among the advantages of transaminases as biocatalysts for the production of
optically pure amino acids are as follows:
I 879
Aminotransferases have high stereoselectivity for a given enantiomer. Optically

active L- or D-amino acids are produced stereoselectively; the process is a chiral
synthesis, not a resolution.
The catalytic rates of these enzyme-catalyzed reactions are generally relatively
rapid.
Capital costs for such a biocatalytic process are low; in contrast to the situation with
fermentations, already existing chemical process equipment can be used for
performing the enzyme-catalyzed reaction.
A large number of the required 2-keto acid precursors are accessible through
chemical synthesis, expanding the range of potential products.
Aminotransferases are potentially applicable to the production of a wide range of
amino acids, because enzymes are available for D- and L-amino acids. In addition, a
wide range of aminotransferases with side-chain specificity are known, including
enzymes for the production of amino acids with aromatic side chains, acidic side
chains, branched alkyl side chains, etc.
In some cases, the 2-keto acid by-products may also have significant value. For
example, important markets exist for pyruvic acid, 2-ketoglutaric acid, and other
similar compounds.
One of the simplest examples of an efficient transamination process is the produc-
tion of L-alanine and 2-ketoglutarate from the precursors L-glutamate and pyruvic
acid (Fig. 12.7-3). Porcine glutamic-pyruvic transaminase is available commercially,
and this enzyme was used as a model system for studying the transamination on a
preparative scale. The equilibrium constant was measured for this reaction and
found to be 1.86, slightly favoring the formation of r-alanine and 2-ketoglutarate.
Glutamic-pyruvic transaminase was immobilized on porous aminopropyl glass
using water-soluble carbodiimide as a coupling agentL71. At a loading of 20 mg of
total protein bound per gram of glass, the activity of the biocatalyst when assayed or
the production of L-alanine was 400 units per gram of biocatalyst. The enzymatic
activity retained after immobilization was 40%, and the immobilized enzyme was
used for the continuous production of L-alanine and 2-ketoglutarate from pyruvate
L-Alanine
Pyruvic acid Glutamate-pyruvate
+ - aminotransferase
L
+
L-Glutamic acid 2-Ketoglutarate

Figure 12.7-3. Transamination using glutarnic-pyruvic arninotransferase.
880
@ + L-alanine - (S)-Transaminase - @ + pyruvate
acetophenone (S)-phenylethylamine
Figure 12.7-4. Enantiomerically pure (S)-amines via w-transaminases.
and L-glutamate over a three-month period with less than 40% loss in activity.
Volumetric productivity was 200 gL-lh-l of L-alanine.
12.7.3.2
Synthesis of Enantiomerically Pure Amines
While most methods for the synthesis of enantiomerically pure amines have
employed kinetic resolution with the help of lipases or esterases, a method
independent of kinetic resolution has been developed using the transamination of
ketones catalyzed by o-transaminases (w-TA), shown in Fig. 12.7-4 with acet-
ophenone as an example[20*211.
The w-transaminases can be employed in two ways to produce both enantiomers
in a pure form [l81:
a racemic mixture can be separated, by kinetic resolution, into the corresponding
ketone and the remaining amine enantiomer, which is typically obtained in high
enantiomeric excess, the ketone can be recycled as a starting material for the
racemic amine;
the same o-transaminase can be employed to synthesize the enantiomer of the
opposite configuration straight from the ketone.
Both ( R ) -and (S)-aminotransferasehave been employed at Celgene for the synthesis
of enantiomericallypure amines from racemic amines. Degrees of conversion were
at or close to 50 % for resolutions and enantioselectivitiesnormaly reached > 99 %
e. e. for the amine product from both resolutions or syntheses from ketones [18, 19].
The donor for resolutions of amine racemates was usually pyruvate whereas either
isopropylamine or 2-aminobutane served as donors for reduction of ketones. The
amine products ranged from phenylethylamine and tetramines with the amine
group at activated benzylic sites or in a cyclic structure, to phenylisoproylamines
(amphetamines) or phenoxyisopropylamines where the amine group is hardly or not
activated at all. A selection of products synthesized with o-aminotransferase technol-
ogy is shown in Fig. 12.7-5.The Celgene process has been scaled up to the 500 kg
level [I9].
The (S)-w-TA from Vibrio fluvialis was found to catalyze the reduction of acet-
ophenone to (S)-a-methylbenzylaminewith the concomitant oxidation of L-alanine
to pyruvate. The enantiomeric excess was always > 99% e.e. As thermodynamic
equilibrium strongly favors the reverse reaction, however, high yields were achieved
only when an excess of acetophenone was added and upon removal of pyruvate:
I
&
+WOH UOJ
/ / R=H.Me /
oy
RO
MeO NHz
i"' I
R = H, Me. CI, Br, NOp

R
Figure 12.7-5. List o f amines produced by o-arninotransferase technology

(both enantiorners produced i n each case).
yields of z 90 % were achieved with acetophenone and benzylacetone in the presence

of a 10-fold excess of L-alanine if pyruvate was removed by using whole cells. The
reaction suffers from strong inhibition by both products, pyruvate and (S)-a-
methylbenzylamineI2O]. Interestingly,the authors found a linear correlation between
the reactivities of amino acceptors and the inverse reactivity of amino donors[2'].
12.7.3.3
Other Preparative Applications o f Arninotransferases
12.7.3.3.1 Preparative Applications: L-Phosphinothricin

L-Phosphinothricin, the active ingredient of the broad-spectrum herbicide Basta
(AgrEvo),can be obtained through enzymatic transamination of the corresponding
oxoacid, 2-0x0-4-[(hydroxy)(methyl)phosphinoyl]butyric acid, in a coupled system
with aspartate aminotransferase (AAT) and 4-aminobutyrate:2-ketoglutaratetrans-
aminase (E.C. 2.6.1.19) from E. coli (Fig. 12.7-G)[371.In solutions containing 10%
substrate, 85 % conversion was reached with only i3 % amino acid by-products. For
882
pyruvate + CO
t L-phosphinothricin
oxaloacetate
glutarnate- P-ketoglutarate-
oxaloacetate 4-arninobutyfate
transarninase transaminas6
(GOT)
L-AsP
glutarate
Figure 12.7-6. Coupled process for the herbicide ingredient L-phosphinothricin

with transaminases.
this process, a new AAT from B. stearothermophilus has been screened and charac-
terized (Topt= 95 "C, pH,,, = 8.0) before being cloned and overexpressed in E. coli.
12.7.3.3.2 Synthesis of an Omapatrilat Building Block with L-Lysine

E-am hotransferase
&-0x0-L-norleucineacetal is a key intermediate for the synthesis of Omapatrilat
(BMS-186716), a novel dual-action vasopeptidase inhibitor under development at
Bristol-Myers-Squibb(BMS). The BMS researchers developed a novel synthesis of a
key building block of Omapatrilat, the bicyclic compound BMS-199541-01, by
oxidation of the &-groupof L-lysine in the N-protected dipeptide N-Cbz-L-homo-cys-L-
lys with a newly found L-lysine ~-aminotransferase[~~]. The enzyme was isolated
from Sphingornonas paucimobilis and was cloned and overexpressed in E. coli (2
kUL-I). It is an 81 kDa homodimer with a specific activity towards the product BMS-
199541-01 of 1.68 Umg-' of protein; the enzyme requires a-ketoglutarate as a co-
substrate which is recycled back into the process after oxidation of the L-glutamate
back to a-ketoglutarate by glutamate oxidase (isolated from Streptomyces noursei). L-
Lysine E-aminotransferasewas found to be one of the most important rate-limiting
enzymes in cephalosporin biosynthesis 13'1.
The process scheme (Fig. 12.7-7) starts from the N-protected dipeptide dimer [L-
lys-~-homocys]~ disulfide which, after reduction of the S - S bond, is oxidized
enzymatically to N-Cbz-L-homo-cys-L-lys-&-aldehyde. Under acidic conditions, the
aldehyde group is present as a gem-diol, attacks the a-N and closes the ring to the
aminol. After nucleophilic attack of the S - H group, the hydroxyl group acts as a
leaving group and affords closure of the 1,3-thiazepinering.
In the biotransformation process to BMS-199541-01, yields of 65-70 mole-%were
achieved without recycling of the L-glutamate resulting from the reduction of a-
ketoglutarate yields were substantially lower. L-Lysine &-aminotransferasealso cata-
lyzes the oxidation of N-a-protectedL-lysines as well as L-lysine peptides such as N-
protected L-met-L-lys.
I
883
Dithiothreitolor
Tributylphosphine
c
SH
Dipeptide Monomer
a-keto glutarate
Dipeptide Dimer L-Lysine-E-aminotransferase Glutamatt

BMS-201391-01 from Sphingomonas paucimobilis Oxidase
or rec E. coli
L-Glutamate
PGHN
-
- Acid
SH
cb-
PGH%
(protecting group)
BMS-199541-01
Figure 12.7-7. BMS process to the bicyclic intermediate BMS-199541-01 via L-lysine
~-aminotransferase[~~’.
884
12.7.4
Driving the Reaction to Completion
There is one major disadvantage to most of the transamination technology as

presented above: because the transamination reaction involves an amino acid
reacting with a 2-keto acid to generate products which consist of a 2-keto acid and an
amino acid, the equilibrium constant is often close to unity. As a result, the net
conversion of substrates to products is thermodynamically limited. The key to the
development of an efficient transamination technology lies in overcoming the
problem of incomplete conversion of the 2-keto acid precursor to the desired amino
acid product.
One approach to this problem is the coupling of the transamination reaction to a
second reaction that consumes the keto acid by product in an essentially irreversible
step; this drives the transamination reaction to completion. By using an amino-
transferase that can utilize aspartic acid efficiently as the amino group donor (instead
of glutamic acid), the corresponding 2-keto acid by product is oxaloacetate (rather
than 2-ketoglutarate).Oxaloacetate is a P-ketoacid and can be easily decarboxylated to
pyruvate. This decarboxylation occurs spontaneously in aqueous solution, catalyzed
R, ,COOH
RKcooH
0 H,N
C
4%
H
2-Ketoacid L-Amino acid
+ - L
+
H O O C VCOOH
L-Aspartic acid Oxaloacetic acid Pyruvic acid
Acetolactate synthase (ALS)
\R- - co,
3---
acetoin
Figure 12.7-8. Driving the transamination reaction to completion.
12.7 Transaminations I 885
Table 12.7-1.Time course for the transamination o f phenylpyruvate t o L-phenylalanine i n the

presence and absence o f oxaloacetate decarboxylase.
Reaction time (min) Transaminase alone Transaminase & Oxaloacetate decarboxylase
Phenylpyruvate (mM) Phenylpyruvate(mM)
0 200 200
10 184 140
20 166 116
45 124 44
80 116 6
by various metal ions and amines, and can be accelerated chemically, as shown in
Fig. 12.7-8, or enzymatically using the enzyme oxaloacetate decarboxylase. The
important feature of the process is that the essentially irreversible decarboxylationof
oxaloacetate to pyruvate drives the entire process to completion, allowing the
transamination of 2-keto acids to amino acids in yields approaching 100% of the
12, 131. I mportantly, this method of driving the reaction to completion
8'
may be used for the production of either D-amino acids or L-amino acids.
The decarboxylation reaction catalyzed by the enzyme oxaloacetate decarboxylase
has been examined using enzymes from four different sources: Pseudornonas putida,
Micrococcus luteus, and two strains of Azotobacter vinelandii. The highest rates were
obtained with the oxaloacetate decarboxylase isolated from Pseudornonas, a Mg2+-
requiring enzyme[7. l21.
The effectiveness of decarboxylation in driving the reaction to completion was
demonstrated in a coupled enzymatic process by using phenylpymvate as the
starting 2-keto acid. In this experiment, phenylpyruvate sodium salt and L-aspartate
were incubated with E. coli broad-range transaminase at room temperature and pH
7.5 in both the presence and absence of oxaloacetate decarboxylase from Ps. putida.
Magnesium ion, which is cofactor for the decarboxylase, was also present in both
reaction mixtures at a concentration of G mM. The transamination reaction was
monitored by following the disappearance of phenylpyruvate. The results are
summarized in Table 12.7-1. As demonstrated by the data, when oxaloacetate
decarboxylase was included in the mixture the reaction proceeded to completion
much more rapidly than in the case when the decarboxylase was omitted [I2].
Other methods can also be used for driving the transamination reaction to
produce amino acids in high yields. For example, if L-lysine or L-ornithineare used as
the donor in the two-enzyme process shown in Fig. 12.7-9, the cyclization of the
aldehyde is strongly favored, creating an essentially irreversible reaction that can
lead to high yields of a desired amino acid from the corresponding 2-keto-
acid[lO,40, 411
12.7.5
Production of L-Amino Acids Using Immobilized Transaminases
Continuous decarboxylationof oxaloacetate as it is formed is an important part of an

efficient, high-yielding transamination process. This decarboxylation occurs readily
886
HOOcTcOO
HooC7fCooH NH, 0
+ -Transaminase
- +
R, ,COOH
RKcooH
0
HN
,
4%
H
HOOcTcO
Hooc7fcooH NH,
0
L-Glutamic acid
+ - Lysine
Aminotransferase
2-Ketoglutarate
L-Lysine Cyclized lmine Coproduct

Figure 12.7-9. Coupled reactions using L-lysine for driving the transamination of
2-ketoacids to amino acids.
in aqueous solution, and may be accelerated enzymatically as described above, or

chemically using a metal ion such as Mg2*in sufficient concentration. Immobiliza-
tion of the enzyme allows reuse of the enzyme or continuous production of amino
acid in a flow reactor system.
Immobilization of the E. coli broad-range transaminase has been accomplishedby
covalent attachment using glutaraldehyde PVC-silica support matrix that had been
activated with a polyamine[13].In the example described in Table 12.7-1,4 L of cell
lysate containing 61.6 g of enzyme (activity of 5.2 million international units) were
clarified by centrifugation at 13 000 g for 30 min and recirculated through a pre-
activated support matrix for 1.5 h. After washing, 57 g or 93% of the enzyme
remained bound to the support. Bound activity was 4.2 million units. The retained
activity of the enzyme after immobilization was approximately 89 %.
The pH-rate profile for the reaction catalyzed by the E. coli broad-range trans-
aminase was determined using the immobilized transaminase with p-fluorophe-
Table 12.7-2.Concentrations of reactants for the production of L-p-fluorophenylalanineby
I
transamination ofp-fluorophenylpyruvate.
Reactant Concentration (mM)
Sodium p-fluorophenylpyruvate 100

r-Aspartate 110
Pyridoxal phosphate 0.1
MgCl2 50
nylpyruvate as the keto acid and L-aspartate as the amino group donor. The
transamination reaction displayed a fairly broad useful pH range; the immobilized
transaminase had a pH optimum of approximately 7.5, but retained activity in the
range of pH 6.0-9.5. At pH 5.0 and 10.0, activity fell to less than 20% of that
measured at pH 7.
For continuous production of L-p-fluorophenylalanine,a typical set of operating
conditions is shown in Table 12.7-2.L-Aspartate is used at a 10% molar excess to the
starting 2-ketoacid. The cofactor pyridoxal phosphate is added to the reaction
mixture to achieve a final concentration of 0.1 mM. The initial pH of the feed
solution is 7.2. Mg2+ion was used to accelerate the decarboxylationof oxaloacetate to
pyruvate. The reaction was maintained with a temperature range of 37-40 "C. Under
these conditions using an immobilized broad-range aminotransferase, the volu-
metric productivity of the reactor for the production of L-phenylalanine at 85%
conversion was 20 gL-lh-'.
One of the main advantages of the transamination system is its applicability to a
range of other L-amino acids, including non-naturally occurring amino acids. For
example, broad-range aminotransferase (encoded by the aspC gene) will efficiently
transaminate the 2-keto acids corresponding to L-phenylalanine,p-fluoro-L-phenyl-
alanine, L-tyrosine, rn-hydroxy-L-phenylalanine, L-tryptophan,L-methionine,~-1iorrio-
phenylalanine, L-2-aminoadipicacid and a number of others. Using other amino-
transferases, the transamination of other 2-ketoacids to the corresponding amino
Table 12.7-3. Amino acids produced by transamination
Amino Acid Aminotransferase
r-Phenylalanine Broad-range, aromatic
r-Tyrosine Broad-range, aromatic
L-Tryptophan Broad-range, aromatic
L-p-fluorophenylalanine Broad-range
L-meta-tyrosine Broad-range
r-Homophenylalanine Broad-range, aromatic
L-2-Aminoadipicacid Broad-range
L-2-Aminopimelicacid Broad-range
r-Valine Branched-chain
r-Leucine Branched-chain
r-tert-leucine Branched-chain
D-Alanine D-broad-range
D-Leucine D-broad-range
D-Tyrosine D-broad-range
D- Phenylalanine D-broad-range
888
HOOc-fcOOH
Hooc7fCooH NH, 0
+ - Branched-chain
Transarninase
L
H3C. 22,I ,COOH

CH, C
II
0
Trirnethylpyruvate L-tert-Leucine
HOOcTc
HOOc-TfcOOH 0 NHZ
2-Ketoglutarate L-Glutarnic acid
Broad-Range
+ I
L
+
Transarninase
co,
COOH
HOOC/YcooH
NHZ
H O O C V
0
H3c7fC02H
0
L-Aspartic acid Oxaloacetic acid Pyruvic acid
Net: L-Aspartate + Trirnethylpyruvate

Figure 12.7-10.
- L-tert-Leucine+ Pyruvate + CO,
Coupled arninotransferases for the production o f L-tert-leucine.
acids can be carried out. A list of amino acids that have been produced by
transamination is shown in Table 12.7-3.
The broad-range aminotransferase has low catalytic activity for the group of
branched-chain amino acids, including L-leucine, L-isoleucine and L-valine. To
enable production of this group of L-amino acids, another transaminase, the so-
called branched-chain amino acid transaminase (BCAT), has been used. This
enzyme has also been shown to catalyze the transamination of trimethylpyruvate to
produce the commercially interesting unnatural amino acid L-tert-leucine,although
the rate of the reaction is significantly less than that for L-valine. Unlike the broad-
range transaminase, the branched-chain aminotransferase is not active with L-
I
aspartate as the amino donor. L-Glutamate is used for efficient transamination using
this enzyme.
To drive this reaction, a coupled transamination reaction was established with both
the broad-range and branched-chain aminotransferases acting together as shown in
Fig. 12.7-8 for the production of L-tert-leucine.In the first reaction, the branched-
chain aminotransferase catalyzes the reaction of L-glutamate with trimethylpyruvate
to produce L-tert-leucineand 2-ketoglutarate.The second reaction catalyzed by broad-
range aminotransferase converts L-aspartate and 2-ketoglutarate into oxaloacetate
and L-glutamate.The donor L-aspartate is present in stoichiometric amounts relative
to 2-ketoisovalerateand is used to continuously recycle the 2-ketoglutarateformed in
the first step to L-glutamate as the reaction proceeds. Oxaloacetate is decarboxylated
to pyruvate in an essentially irreversible reaction, driving the entire sequence of
reactions to completion. The net reaction is the transamination of trimethylpyruvate
to L-tert-leucine with L-aspartate using 2-ketoglutarate as an intermediary amino
transfer agent. This sequence of reactions has also been used to produce L-leucine
and L-valine in the laboratory (Fig. 12.7-10).
In laboratory-scaleexperiments, solutions containing 200-GOO mM keto acid were
transaminated to the corresponding branched-chain L-amino acid, with a concentra-
tion of L-glutamate between 50 mM and 100 mM and a 1.1 molar excess of L-
aspartate. Yields obtained for the branched-chain amino acids have typically been in
the range of 80-90% based on starting with a 2-keto acid[''].
Another example of a coupled enzyme reaction demonstrates the versatility of the
transaminase system in biocatalysis. Using a racemic D,L-amino acid mixture as the
starting material, the enzyme D-amino acid oxidase from Trigonopsis variabilis will
convert the D-amino acid in the mixture selectively into the corresponding 2-keto
acid. The 1-amino acid of the D,L- pair is neither a substrate nor an inhibitor of D-
amino acid oxidase. If a transaminase is present in the same reaction mixture, the
2-keto acid can be transaminated in the presence of L-aspartate to the corresponding
L-amino acid. The entire reaction can be driven to completion as described pre-
viously by decarboxylation of the oxaloacetate. Thus, in a single pot, racemic D,L-
amino acids can be convened directly into optically active L-amino acids (Fig. 12.7-
11).
12.7.6
D-Amino Acid Transferases
The aminotransferase reaction can be utilized for the synthesis of D-amino acids as
well as the better-known route to L-amino acids (Fig. 12.7-11).Regarding sequence
similarity, D-aminotransferases form a distinct subgroup among the transferases,
however, it has been found, with the help of crystal structures[42-431that some
striking similarities exist between L-amino acid aminotransferases with respect to
active site structure and to branched-chain aminotransferase (BCAT) with respect to
sequence. D-Aminotransferases utilize the same PLP chemistry as L-aminotrans-
ferases to effect tran~amination[~~l.Mutagenesis of a distant interdomain loop of D-
arninotransferase to produce enhanced conformational flexibility (proll9-argl20-
890
Figure 12.7-11. Conver-
RYCooH
NH, RYcooH NH,
sion of racemic amino
acids into L-amino acids
with D-amino acid oxidase
L-Amino acid and an L-aminotransferase.
+ D-Amino Acid Oxidase

m +
Racemic Amino acid 2-Ketoacid
,R ,COOH
RKCooH
0 H,N
4%
H
L-Amino acid
+ L-Transaminase
m +
L-Aspartic acid Pyruvic acid + CO,
pro121 to gly-gly-gly)resulted in higher catalytic constants towards most D-amino

acid substrates [&I.
D-Amino acids can also be produced directly by transamination using a D-
aminotransferase. Since these enzymes require a D-amino acid donor, we developed
a coupled enzymatic reaction with aspartate racemase to generate D-aspartic acid in
situ from inexpensive L-aspartic acid. The reaction scheme is shown in Fig. 12.7-12.
Aspartate racemase, cloned from Streptococcus thermophilus and expressed in E. coli,
is used in conjunction with a D-aminotransferaseto produce D-amino acids from
corresponding 2-ketoacidsin a reaction that is analogous to that for the production of
L-amino acids. Oxaloacetate,produced from D-aspartateduring the transamination,
is decarboxylated to pyruvate, driving the reaction to completion as with the L-
transamination. Significant amounts of D-alanine are produced using the D-amino-
transferase cloned from Pseudomonas sphaericus ATCC 10208 as it has activity toward
pyruvate. Directed evolution efforts are in progress to develop an enzyme having
reduced D-alanine production, resulting in a cleaner product mixture. For the
synthesis of D-glutamate,a two-enzyme system consisting of of glutamate racemase
and D-aminotransferasehas been found in B. s p h a e r i c u ~ [ ~ ~ ] .
R + ~ Figure
~ 12.7-12.~ ~
Production of
I
RIYcooH --
0
GH,
D-amino acids by transamination
2-Ketoacid Acceptor D-Amino acid
NH,
R2Yc02H
0
D-Amino acid Donor 2-Ketoacid Coproduct
12.7.7
Synthesis of Labeled Amino Acids
Isotopically labelled amino acids are particularly amenable to production by trans-

amination. Because the reaction catalyzed by aminotransferases transfers a specific
amino group from the donor, amino acids highly enriched in isotopes such as 15N
can be produced. For example, 15N L-tyrosinehas been produced in greater than 90 %
yield from "N L-aspartate and p-hydroxyphenylpyruvate using the broad-range
aminotransferase from E. coli. The reaction is shown schematically in Fig. 12.7-13.
Analysis of "N-isotope incorporation was carried out by mass spectrometry by
Cambridge Isotope Laboratories. The samples showed incorporation of 98.4 % "N,
which was almost identical to the isotopic purity of the starting L-aspartic acid. Only
the L-isomer of tyrosine was detectable by chiral HPLC. This result established the
feasibility of the production of 15N amino acids by transamination by meeting three
important criteria for success:
there was no detectable loss of isotopic purity in the transfer of the amino group
from 15N aspartic acid to the 2-ketoacid,
HO mooH HO
COOH
p-Hydroxyphenylpyruvic acid 15N L-Tyrosine
+ Transaminase
* +
A ,COOH
HOOC
c
15NH,
,3H
H3CKC00H
0 + co2
15NL-Aspartic acid Pyruvic acid

Figure 12.7-13. Production of "N-labeled amino acids by transamination.
892
the stereochemical fidelity of the transamination reaction was perfect within

detection limits, and
The yield of conversion of the 15N aspartic acid (the most costly starting material in
this reaction) was high (> 90%).
12.7.8
Availability of Enzyme
In order to facilitate the production of adequate amounts of transaminase at low cost,

the genes encoding aminotransferases have been cloned and overexpressedin E. coli.
Two examples are the aspC and ilvE genes from E. coli. The expression of these genes
has been described previously, with the levels of aminotransferase enzyme reaching
approximately 30-40% of the total cell protein['*! More recently, the genes encoding
other aminotransferase genes have been c l ~ n e d [leading
~ ~ ~to~the
~ ,availability of a
broader group of aminotransferase enzymes for evaluation. Given the high reaction
rates observed and the potential for wide applicability for the production of amino
acids, both D and L, natural or unnatural, transamination reactions should prove to
be useful method for the chemist.
References
For a review on tert-leucine, see: A. S. Bom-

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Wiley, New York, 1992, Chap. 9,209- miyama,]. B i d . Chem. 1999, 274(4),
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mica Oggi 1996 (6), 20-24. Environ. Microbiol. 1996, 62(10), 3794-3799.
20 J . 4 . Shin, B.-G. Kim, Biotech. Bioeng. 1998, 38 R. N. Patel, A. Banerjee, V. B. Nanduri, S. L.
60(5), 534-540. Goldberg, R. M. Johnston, R. L. Hanson,
21 J.-S. Shin, B.-G. Kim, Biotechnol. Bioeng. C. G. McNamee, D. B. Brzozowski, T. P.
1999,65,206-211. Tully, R. Y. KO, T. P. LaPorte, D. L. Cazzu-
22 P. K. Mehta, T. I. Hale, P. Christen, Eur. ]. lino, S. Swaminathan, C.-K. Chen, L. W.
Biochem. 1993, 214(2), 549-61. Parker, J. J. Venit, Enzyme Microb. Technol.
23 D. M. Needham, Biochem. J. 1930,24,208. 2000,27(6), 376-389.
24 E. Annau, I. Banga, A. Blazo, V. Bruckner, 39 L.-H. Malmberg, W.4. Hu, D. H. Sherman,
K. Laki, F. B. Staub, A. Szent-Gyorgi,2. Appl. Microbiol. Biotechnol., 1995,44,
Physiol. Chem. 1936, 224, 105. 198-205.
25 I. Banga, A. Szent-Gyorgi,2. Physiol. Chem. 40 I. G. Fotheringham, D. P. Pantaleone, P. F.
1937,248, 118. Taylor, Chimica OggilChemistry Today, 1997,
26 A. Meister. Adv. Enzymol. 1955, 16, Sept.-Oct., 33-36.
185-246. 41 K. Soda, Biochemistry 1968,7,4102-4109.
27 A. Meister, Annu. Rev. Biochem. 1956,25, 42 S. Sugio, G. A. Petsko, J. A. Manning, K.
29-56. Soda, D. Ringe, Biochemistry 1995, 34,
28 P. Christen, D. E. Metzler, Transaminases 9661-9669.
1985, John Wiley & Sons, New York. 43 D. Peisach, D. M. Chipman, P. W. Van
29 D L. Smith, D. Ringe, W. L. Finlayson, J. F. Ophem, J. M. Manning, D. Ringe, Biochem-
Kirsch,]. Mol. Biol., 1986, 191, 301-302. istry 1998, 37(14), 4958-4967.
30 R. Graber, P. Kasper, V. N. Malashkevich, E. 44 A. Gutierrez, T. Yoshimura, Y. Fuchikami,
Sandmeier, P. Berger, H. Gehring, J. N. Jan- K. Soda, N. Esaki, Protein Eng. 1998, 11(1),
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31 R. A. Vacca, S. Giannattasio, R. Graber, E. lor, /. Bacteriol. 1998, 180(16), 4319-23.
Sandmeier, E. Marra, P. Christen, ]. Biol. 46 J. D. Rozzell, unpublished results.
Chem. 1997,272(35),21 932-7. 47 P. V. Warren, R. V. Swanson, Transaminases
32 R. Graber, P. Kasper, V. N. Malashkevich, P. and Aminotransferases, 1998, U. S. Patent
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I895
13
Formation and Cleavage o f P - 0 Bonds
George M. Whitesides
13.1
Introduction
The use of isolated enzymes to form or cleave P - 0 bonds is an important

application of biocatalysts. Restriction endonucleases, (deoxy)ribonucleases,DNA/
RNA-ligases, DNA-RNA-polymerases, reverse transcriptases etc. are central to
modern molecular biology[']. Enzyme catalyzed phosphoryl transfer reactions have
also found important applications in synthetic organic chemistry. In particular, the
development of convenient cofactor regeneration systems has made possible the
practical scale synthesis of carbohydrates, nucleoside phosphates, nucleoside phos-
phate sugars and other natural products and their analogs. This chapter gives an
overview of this field of research.
Hundreds of potentially useful enzymes are available in nature. It is often
worthwhile to survey enzymes for applicability in the synthesis of a specific
compound, but how to find the best enzyme? Enzymes have been reviewed and
classified by many schemes [2-41. Enzymes involved in reactions at phosphoryl
groups are, unfortunately for the synthetic chemist, spread almost over all classes.
Without a good knowledge of enzymology, it is not easy to find the enzyme classes of
interest for a particular transformation. This review links the compound classes and
enzyme classification systems in Section 13.1.1 to help overcome this barrier.
Most synthetically useful phosphorylating enzymes require nucleoside triphos-
phates as cofactors. The central importance of cofactor regeneration, and the most
used regeneration methods for these cofactors, are discussed in Section 13.2.1. The
end of Chapter 13 includes tabular surveys of the most important applications,
classified in compound or structural classes (see Sections 13.2.2 and 13.3.3), to
facilitate the search for relevant enzymes and procedures.
896
I 73 Formation and Cleavage of P - 0 Bonds
13.1.1
Enzymes Forming or Cleaving Phosphorous-Oxygen Bonds
Phosphoesters are ubiquitous in biochemistry and serve several functions 1'1. Genetic
information is stored in DNA and RNA. In cellular control mechanisms, phosphory-
lation of proteins is an important mechanism for regulating protein activitiesr6].
Phosphorylation can activate metabolites or change solubility properties. Enzyme-
catalyzed formation and cleavage of P - 0 bonds are central to the cellular energy
balance l71, Biosynthesis depends heavily on phosphorylated intermediates.
A useful classification for enzymes involved in phosphoryl transfers was in-
troduced by Knowles[*](see Fig. 13-1).This classification, based on enzyme func-
tions and mechanisms, differentiates primarily between two groups of enzymes. The
first group contains only enzymes that accept phosphoric monoesters as substrates
(type A and B). The second group includes all enzymes catalyzing reactions at
phosphoryl groups of phosphodiesters (type C-E). Table 13-la and 13-lb link
Knowles' classification and the enzyme classification recommended by the Inter-
national Union of Biochemistry (IUB; compare Chapter 1)121.The IUB classes give a
direct access to the specific enzymes in reference works and to the CA registry
numbers necessary for an efficient literature searchI2*1'.
Tables 13-la and 13-1b list only the most important categories of enzyme classes
(E. C.'s). Some enzymes that are involved in reactions at phosphorus are hidden in
other classes. For example glyceraldehyde-3-phosphatedehydrogenase,which cataly-
ses the oxidative phosphorylation of glyceraldehyde-3-phosphateto 1,3-diphospho-
glycerate, is classified under E.C. 1.2.1.12 and 1.2.1.13. Neither the name of the
enzyme nor its IUB-classification,gives information about the phosphorylating step.
Identifying enzymes potentially useful in synthesis that have been ambiguously
classified is difficult for those outside of biochemistry because no complete reference
is available connecting enzymatic activity with synthetic applicability.
A second important point is that many enzyme catalyzed reactions are reversible.
Some hydrolytic enzymes can be used in enzyme catalyzed phosphorylation reac-
R-*Of[-0
?-
-
4- ?-
-O-P-'j-O-ZfOfL-Nu
4-
R-O-$[-O-R'
4-
to
A "I B OC
t 1"
D
to
E
Figure 13-1. Classes o f enzymes involved in reaction at phosphorus. A and B
represent enzyme types that handle phosphoric monoesters and related com-
pounds ("0 may be an oxygen o f a hydroxyl, carboxyl, or phosphoryl group, or
the nitrogen o f a guanidine group. For simplicity, displacements at t h e y -
phosphoryl groups o f nucleosides triphosphates were classified with these
reaction). C , D and E represent the enzymes that catalyze transformations o f
phosphoric diesters (displacements at a or fi phosphorous groups o f nucleo-
side triphosphates and transfer of pyrophosphates were classified with the
reactions of phosphoric diesters).
Table 13-la. Enzymes accepting phosphoric monoesters as substrates.
13. I Introduction
I
897
Enzyme Functional Fundion' IUB classes with titles, containing

type" classb such types of enzymesd
Phosphomutases Phosphoryl group transfer, 2.7.5. Phosphomutases
for which the acceptor is 5.4.2. Intramolecular phospho
another functional group transferases
on the donor molecule.
Phosphorylases Formation of a P - 0 bond 2.4.1. Hexosyltransferases
under phosphorolytic 2.4.2. Pentosyltransferases
cleavage of a C-Hetero-
atom bond.
Nucleotidases Phosphoryltransfer from 3.1.3. Phosphoric ester hydrolases
a nucleotide to water as an (3.1.4 Phosphoric diester hydro-
acceptor molecule. lases)
(Nucleotides are cleaved
hydrolytically).
Phosphatases Phosphoryl group transfer 3.1.3. Phosphoric ester hydrolases
from a phosphoric mono- 3.6.1. Hydrolases acting on acid
ester to water as an accep- anhydrides in phosphorous-
tor molecule. (Phosphoric containing anhydrides
monoesters are cleaved
hydrolytically).
Phosphokinases Phosphoryl group transfer: 2.7.1. Phosphotransferaseswith an
Nucleoside triphosphate is alcohol group as acceptor
and the donor and some other 2.7.2. Phosphotransferaseswith a
molecules than HzO are carboxyl group as acceptor
Phosphotransfera- the acceptors. Compounds 2.7.4. Phosphotransferaseswith a
ses different than nucleoside phosphate group as acceptor
triphosphates are the
donor and some other
molecules than HzO are
the acceptors.
ATPases Phosphatases which are 3.6.1.3 ATPases
responsible for the cou-
pling of ATP cleavage to
other metabolic processes.
a See figure 13-1; b functional classes bases on ref."'; c see ref."' and 14'; d see ref."'
tions. Alkaline phosphatase (E.C. 3.1.3.1), for example, was used in enzyme-
catalyzed phosphorylation of glycerol with inorganic phosphate (1' . In some cases
enzymes may catalyze unexpected reactions with unnatural substrates: aminoacyl
tRNA synthetases (ARS) were used to synthesize p',p4-di(adenosine 5'-)tetraphos-
phate ( A p d ; l),a natural inhibitor of human platelet aggregation["] (Fig. 13-2).
Here, in the first step an amino acid (AA) reacts reversibly with ATP and ARS and
forms an aminoacyl-AMP-ARS complex and PPi; the back reaction of this inter-
mediate with ATP leads to the desired product A P ~ [ " - ' ~ ] .
898
I 73 Formation and Cleavage ofP-0 Bonds
Table 13-lb. Enzymes accepting phosphoric diesters as substrates.

Enzyme Functional Function' IUB classes with titles, containing
type" classb such types of enzymesd
C Pyrophosphokin- Pyrophosphate group 2.7.6. Diphosphotransferases

ases transfer from ATP to an
acceptor molecule other
than water.
D Nucleotidyl Transfer of nucleotidyl 2.7.7. Nucleotidyltransferases
transferases moieties
D Nucleotidyl Nucleoside triphosphate 4.6.1. Phosphorous oxygen lyases
cyclases cyclisation under forma-
tion of pyrophosphate
E Triphosphohydro- Triphosphate transfer 3.1.5. Triphosphoric monoester
lases from a nucleoside triphos- hydrolases
phate to water as an
acceptor molecule.
E Polynucleotide Responsible for the link- 6.5.1. Ligases forming phosphoric
synthetases age of two poly- or oligo- ester bonds
nucleotide moieties to
form polynucleotide
chains
E Phospholipases Hydrolytic cleavage of 3.1.4. Phosphoric diester hydrolases
phosphoglycerides
(essentiallyphospholipase
C and D)
E Nucleases Phosphonucleotide trans- 3.1.4. Phosphoric diester hydrolases
fer from a polynucleotide 3.1. Endo- and exonucleases
to water as an acceptor
molecule. (Polynucleotides
are cleaved hydrolytically).
E Phosphodiester- Phosphomonoester trans- 2.7.8. Transferases for other
ases fer from a phosphodiester substituted phosphate groups
other than polynucleotide 3.1.4. Phosphoric diester hydrolases
to water as an acceptor
molecule. (Phospho-
diesters are cleaved hydro-
lytically).
a See figure 13-1;b functional classes bases on ref.? c see ref."' and r4'; d see ref.['].
One of the most important criteria in the evaluation of a new process is the
availability of an (see Chapter 20: Tabular Survey of Commercially
Available Enzymes). If the enzymes are not commercially available, their isolation
and purification can be expensive and time consuming (see Chapter 2: Production
and Isolation of Enzymes). The importance of the product to be synthesized may
sometimes justify the additional effort.
Mechanistic aspects of L O bond formations and cleavages have been reviewed["]
and are outside the scope of this work. The use of enzymes catalyzing the formation
I
t
ARS ATP
AA + AT? AA-AMP-ARS
pi AA + ARS
HO OH HO OH
1
Figure 13-2. Enzymatic synthesis of p’,p4-di(adenosine 5’-)tetraphos-
phate (Ap4A 1) with aminoacyl tRNA synthetases (ARS). AA can be
leucine, for example, and ARS leucyl t-RNA synthetase[”].
of P-N bonds - for example, phosphorylations of amino acids (E.C. 2.7.3) - are
discussed only briefly. Enzymes dealing with the formation of aminoacyl tRNA (E. C.
6.1.1),acyl-CoA derivatives (E. C. 6.2.1) or peptides (E. C. 6.3.2) are also not covered,
even if cleavages of nucleoside phosphates are involved.
13.1.2
Biological Phosphorylating Agents
To compare the ability of different compounds to transfer a phosphoryl group,

phosphorylation of water was chosen as a standard reaction[’7].The free energy of
hydrolysis of a phosphorus compound (AG2ydr) is called its phosphorylating poten-
tial. Table 13-2 summarizes the phosphorylating potentials of the most important
biological compounds (Fig. 13-3)having phosphoryl donor abilities.
By far the most important strong biological phosphorylating agent is adenosine
5’-triphosphate (ATP, 8). ATP is ubiquitous and plays a central role as cofactor in
anabolic and catabolic processes. Moreover, many enzymes involved in the forma-
tion of P-0 bonds are ATP dependent. The biologically active form of ATP is, in
most cases, the magnesium salt MgATP2-[221. Other nucleoside triphosphates have
similar phosphorylating potentials but they are rarely used as phosphoryl group
donors[23,241; usually GTP, CTP and UTP act as nucleoside or nucleoside phosphate
donors (see Section 13.2.2.2).
Creatine- and arginine phosphate (7 and 9) play important roles in the storage of
phosphorylating potential in vertebrates and invertebrates, respectively[25* 26]. In
living cells, these N-phosphoguanidine derivatives are formed by phosphoryl group
transfer from ATP, and in the reverse reaction ADP is the only acceptor for 7 and 9.
1,3-Diphosphoglycerate(5) and phosphoenolpyruvate (2) are important phosphor-
ylating agents of ADP in the glycolytic pathway. P~lyphosphate[~~], phosphor-
amidate 12’] and pyrophosphate[”] are involved in the biochemical phosphorylation
900
Table 13-2. Free energies of hydrolysis of some important biological phosphorus

"1.
Compound (R-OPOs2-) PH [kcal/mol] [kJ/mol]

Phosphoenolpyruvate (2) 7.0 12.8 53.5
Methoxycarbonylphosphateb (3) 7.0 12.4 51.8
Carbamyl phosphate (4) 9.5 12.3 51.4
1,3-Diphosphoglycerate ( 5 ) 6.9 11.8 49.3 strong
Acetyl phosphate (6) 7.0 10.3 43.1 phosphorylating
Phosphocreatine (7) 7.0 10.3 43.1 agents
ATP (8) (+ ADP + Pi)' 7.4 7.3-9.6 30.5-40.1
ATP (8) (+ AMP + PPi) 7.0 7.7 32.2
Arginine phosphate (9)
Pyrophosphate' (PPi)
Glucose 1-phosphate (10)
Glucose 6-phosphate (11)
Glycerol-1-phosphate (12)
8.0
7.0
7.0
8.5
7.7
4.5-8.0
3.3
5.0
2.2
32.2
18.8-33.4
13.8
20'9
9.2
1 rl?:phorylating
agents
aThe standard free energies are bases on a standard state of IM total stoichiometric concentration of reactants
and products, except hydrogen ion, and on an activity of pure water of 1.0: see ref."8]; Hydrolysis of ATP
and PI', depend strongly on the concentration of Mg2+in solution and on pH[1s211,
2 3 4 5 6
I 8 9
/OP
HO
OP Ho*HO
OH OH P O L O H
OH
10 11 12
Figure 13-3. Structures of the most important biological phosphorylating agents. P =
phosphate.
of D-glucose, hexoses and L-serine respectively in some organisms. Carbamylphos-

phate (4) and acetylphosphate (6) have high phosphorylating potentials (see Table
13-2), but nature uses them mainly as donors of ~arbamyl[~'] or acetyl groups[31].
Only in a few cases do they act as phosphoryl donors[30,321.
Phosphorylations with low-potential phosphorylating agents are thermodynam-
ically not favorable. In biological systems, these processes are made possible by
13.2 Phosphorylation
I
coupling them to a thermodynamically more favorable process. Examples of weak
phosphorylating agents are sugar phosphates such as glucose- and ribose phos-
phates, which can transfer their phosphate group to other sugars[32]or to nucleo-
sides like riboflavin [331. Phosphate sugars are formed when polysaccharides are
cleaved with a phosphorylase and inorganic phosphate[34].
13.2
Phosphorylation
Chemical phosphorylations usually involve many protection and deprotection steps.

Enzymatic phosphorylations can make synthesis more efficient by eliminating many
of these steps. In addition, enzyme-catalyzedintroduction of phosphoryl groups can
be diastereo-[351 or enantiospecific[36, 371.
One of the major challenges in enzyme-catalyzedphosphorylation reactions is, as
mentioned above, the choice of the most convenient enzyme. The other major
difficulty is the availability of the coenzymes. Cofactors act as biological phosphoryl
donors and in enzyme-catalyzed synthesis, they have to be added in stoichiometric
amounts or coupled to an efficient regeneration system.
13.2.1
Regeneration of Nucleoside Triphosphates
In enzyme-catalyzed synthesis, adenosine 5'-triphosphate (8) is the cofactor most

often used as phosphoryl group donor. Other nucleoside phosphates, UTP, or CTP
are used principally as donors of a nucleoside phosphate moiety to form activated
intermediates in biological pathways (see Section 13.2.2.2). For example: UTP
precedes the activated from a glucose, UDP-glucose, in the Leloir synthesis of
polysaccharides, CTP precedes CDP-choline in the synthesis of phospholipids and
CMP-NeuAc in the formation of glycosides of sialic acids (see Chapter 11.3). The
costs for a mole CTP, GTP or UTP vary from $ 32000 to 90000 (as research
biochemicals)[381. The high price of these cofactors precludes their large-scale use in
stoichiometric quantities and makes cofactor regeneration necessary. Even with
ATP, one of the least expensive cofactors used in organic synthesis[3', 381 and
available through mole scale synthesis from RNA r4OI regeneration remains of central
importance. The use of a cofactor regeneration system not only eliminates the need
for stoichiometric quantities of cofactor but it can also favorably influence the
position of the reaction equilibrium and prevent the accumulation of cofactor by-
products that may inhibit the forward process. Product isolation is simplified as
well.
A nucleoside phosphate regeneration system must meet several specifications to
be practical. To be economical, a regeneration method must be capable of recycling
the cofactor 102-106 times [391. All materials should be readily available, inexpensive,
easily handled, stable under reaction conditions and compatible with the rest of the
reaction system. The transfer of phosphate should be thermodynamically and
902
kinetically favorable and it should be regioselective in forming a high-energy P - 0

bond.
13.2.1.1
Regeneration ofATP from ADP and AMP
At the scale required for synthesis of fine chemicals, the major problems of ATP
regeneration have been solved13'. 41, 421 . Three strategies have been applied: chem-
ical synthesis; biological methods including whole cells, organelles, and fermenta-
tion processes: and cell-free enzymatic catalysis. Chemical methods often lack the
necessary specificity and are not compatible with biochemical transformations.
Biological and enzymatic systems provide the most efficient ATP regenerating
systems 13'1. The use of cell-free enzymes requires a greater initial effort or expense
than do the biological methods, but are more specific than biological systems and
often generate fewer by-products (see ref. r3'1 and references cited therein).
a) From ADP. Several procedures for the large-scaleregeneration of ATP from ADP
using isolated enzymes as catalysts are 391. These methods have in
common the characteristic that phosphoryl groups are transferred from a high-
energy phosphoryl donor to ADP (compare Section 13.1.2). The advantages and
disadvantages of these methods are summarized in Table 13-3.
In practice, for most synthetic applications, either acetyl phosphate/acetate kinase
or phosphoenolpyruvate/pyruvatekinase are used to regenerate ATP. Because of the
ease of preparing AcP, AcP/AcK is the most economical method for large-scalework.
Its application is, however, limited to fast phosphorylation reactions where the
hydrolysis of AcP is not important. The PEP/pyruvate kinase system is used in
instances where the requirement for a strong, stable phosphorylating reagent
outweighs the relative inconvenience of preparation of PEP.
Phosphoenolpyruvate/pyruvate kinase. Phosphoenolpyruvate(PEP; Z)/pyruvatekinase

(PK; E.C. 2.7.1.40) is the most efficient system for the regeneration of ATP from
ADP. The phosphorylating agent PEP can be prepared in a mole Starting
from crude pyruvic acid, the crystalline monopotassium salt PEP-K' is synthesized
in a three-step procedure. For transformations on a scale <1 mol, PEP can be
prepared from commercially available 3-phosphoglyceric acid in an enzyme-cata-
lyzed reaction["]. This method is more expensive than the chemical preparation, but
is more convenient because it requires less time and produces less organic waste
(see Section 13.2.1.2; Fig. 13-5).
Pyruvate kinases are commercially available from multiple sources F3*1. The en-
zyme generally used in ATP regeneration - from rabbit muscle - has high specific
activity (-500 U per mg of protein), is inexpensive (Is 2-4/1000 U), and is stable
when immobilized[Gs5-G7]. This regeneration system can be used in membrane
enclosed enzyme catalysis (MEECtechnique) kG8, "1 as well.
The stability of PEP in solution and its strength as a phosphoryl donor (Table 13-2)
make PEP particularly convenient for use in slow and thermodynamically un-
Table 13-3. Properties o f ATP-regeneration systems.
PEP/PK AcP/AcK MCP/AcK CP/CK CrP/CrK Pn/PnK Pi/glycolysis
costs [$/loo0 U], 2.5 (rabbit 378 (B. stearo- 378 ( B . stearo- 10 (Strepto- 2.6 (rabbit isolated from 66 (from diff.
(so~rce)[~~l muscle) thermophilusj thermophilusj coccusfaecalisj muscle) E. coii[44~ sources)a
product inhibition/ pyruvate acetate HCOj- C creatinelZsJ - -
KL[mMlb 10, c 400, NC 500, NC 6-40d, NC
Enzyme spec. activity 30&500 400-1200 2 140b 400-900 150-250 15&250[44] 8h
properties [u/mg pr~tein][~'J
stability ++++e. 1471 ++++e. [481 +++f, +I301 +++f. 1491 ++++e, [*I +a. f
Km (ADP)'[mM] O.lb 0.41501 - O.OSC 0.05[25] 0.17[441 -

K, (P-donor) [ m ~ ] 0.07b 0.4b l.Gb 0.1' Sf, b 0.003[441
ease of preparation/ +I471 +++I51 ++b +++[52J +WI +++++[S4J
availability
P-donors AG (P-transfer)g - 5.5; - 23.0 - 3.0; - 12.6 - 5.1; - 21.3 - 5.0; - 20.9 - 3.0; - 12.6 2 - 0.7; - 2.9* - 16; - 67.5k
properties [kcal;kJ/mol]
half life for hydrolysis - lo3 21 0.3 2.2 - 1o2I -
(25 "C, pH 7)b
a calculated from 1000 U of each enzyme; see ref.l4'I and 1381: b see ref."*l C = competitive, NC = non-competitive; c carbamate kinase kinetics 1s complex, inhibition plays an -
'~;
important rolel4'I; d K,depends strongly on the anions present in ~ o l u t i o n ~e~immobilized enzyme($ fvalue(s) for the free enzyme(s);g calculation based on the values from Table
13-2 (AG(P-transfer)= AG$, (P-donor)- A d , , , (ATP); AG;,,, (ATP)= - 7.3 kcal/mol; 30.5 kJ/mol);h calculation based on the sum of all enzymes present; i based on Table 13-2,values
I calculated from data in ref.ls51.
for PP,; k the driving force of this process is the transformation of glucose to 2 equivalents of lactate (AG = - 197 kJ/mol)lz2~; I!
-5-
13 Formation and Cleavage of P - 0 Bonds
904
I favorable phosphorylation reactions. It is also the method of choice for the regenera-
tion of ATP at low concentrations of ADP, since the Michaelis constant for PK is
smaller (K,(MgADP) = 0.1 mM)ll'l than for acetate kinase (K,(MgADP) = 0.4
mM) [SO].
The PEP/PK regeneration method has two minor disadvantages. First, the synthe-
sis of PEP [64. 471 requires more effort and expense than does the synthesis of AcP rS71.
Second, pymvate is a strong inhibitor of PK (see Table 13-3). The reactions are
therefore carried out in dilute solutions to keep the pyruvate concentration low, and
pyruvate is either removed from the reaction solution or PEP is used at high
concentrations to minimize the effects of inhibition.
Acetyl phosphatelacetyl kinase. Acetyl phosphate (AcP; G)/acetyl kinase (AcK; EC

2.7.2.1) is the most widely used large scale ATP-regeneration system. AcP is
modestly stable in aqueous solutions and is a phosphoryl donor of intermediate
strength (Table 13-2and 13-3).Diammonium acetyl phosphate can be prepared from
ketene and anhydrous phosphoric or, more easily, from acetic anhydride and
anhydrous H3P04[57].However, the use of the diammonium salt in ATP regenera-
tion has three disadvantages. First, NH; reacts with acetyl phosphate in solution.
Second, it forms an insoluble precipitate with Mg2+ under reaction conditions.
Third, its preparation involves several steps that require careful experimental control
and that are difficult to carry out at large scale. Preparation of aqueous solutions of
acetyl phosphate as its sodium r5l] or potassium salt [l'] circumvents these draw-
backs.
Two types of commercially available 13'1 acetate kinases, from Escherichia coli ["I
and from Bacillus ste~rothe~ophilus~~'1, have been used in ATP regeneration. The
latter kinase is more expensive but it is preferred for synthetic use because it is
thermostable and it is stable to auto~idation[~'1.Both enzymes have acceptable
specific activities (150-300 and 400-1200 U respectively per mg protein) and can be
stabilized by imm~bilization[~', ", G662]. Acetate is a weak inhibitor of AcK, but
product inhibition is not a serious problem (see Table 13-3)unless reaction solutions
have acetate concentrations greater than 1 M [ ~ ~The ] . relative instability of AcP in
solution compared with PEP is the major disadvantage of the AcP/AcK system. The
contribution of the enzymes to the total cost of the process in generally low when
they are recycled, making the slightly higher cost of AcK compared with PK a minor
disadvantage [391. Polymer bound ATP was regenerated in a membrane reactor with
AcP/AK Ib31.
Methoxycarbonyl phosphate/acetate kinase. Methoxycarbonyl phosphate (MCP; 3) was

designed to replace AcP as phosphoryl donor['']. It is comparable to PEP in its high
phosphorylating strength (see Table 13-2),but resembles acetyl phosphate in its ease
of synthesis. Aqueous solutions of MCP are prepared from aqueous phosphate and
methyl chloroformate and used in ATP regeneration without purification. The
reaction product after phosphoryl transfer is methyl carbonate, which hydrolyses
rapidly to form C 0 2 and MeOH. Product isolation is simple and bicarbonate
inhibition can be avoided by purging the reaction mixture.
13.2 Phosphoryhtion
I 905
MCP is accepted as an unnatural substrate by AcK (E. C. 2.7.2.1) and CK (E. C.
2.7.2.2) but not by PKL1'l.The principal disadvantage of methoxycarbonyl phosphate
as phosphorylating agent in ATP regeneration is MCP's rapid spontaneous decom-
position. The half-life of MCP in aqueous solution is only 0.3 h (25 "C, pH 7)''').
Because of this short half-life, MCP is only used in a few cases where high
phosphorylating potentials are required to push the phosphorylation reaction to the
product side (see Table 13-4, entry 20).
Others. ATP has been regenerated from ADP with propionylphosphate/acetate

kinase, but propionylphosphate is a poorer substrate than AcP[*'].
Carbamoyl phosphate (CP; 4)lcarbamyl kinase (CK E. C. 2.7.2.2) was described as
a regeneration method for ATP in 1973 but it has seldom been used[70].CP is a very
strong phosphorylating agent (see Table 13-2) and can easily be prepared from
aqueous potassium cyanate and KH2P04[52]. Rapid decomposition of carbamyl
phosphate generates ammonium ions and magnesium ammonium phosphate
complex is formed as a gelatinous precipitate. This precipitation lowers activity, both
by precipitating the magnesium(I1) required for activity of the kinases, and because
it occludes enzymes.
ADP phosphorylation with polyphosphate (P,) and polyphosphate kinase (P,K
E. C. 2.7.4.1) has also been demonstrated[44].The cheap, stable polyphosphates and
the stability of P,K are highly attractive. Unfortunately, P, has a low phosphorylating
potential and P,K is not readily available. These facts may explain why this
regeneration system has not found any practical application.
Another very interesting but little-used regeneration method is based on phos-
phocreatine (PC; 7) and creatine kinase (CrK E. C. 2.7.3.2)r7l].PC is comparable in
its phosphorylating potential to AcP, but it is more stable in aqueous solutions (Table
13-3). CrK is inexpensive and fairly stable. The current lack of an efficient and simple
laboratory scale synthesis for PC seems to have limited the applications of this
method to a few syntheses of sugars r7lI and nucleosides [721.
Recently, a promising regeneration system based on a multienzyme system from
the glycolytic pathway was described[45](Fig. 13-4). Glucose and inorganic phos-
phate were used as low-energy phosphorylating agents. Eleven enzymes are used to
convert glucose to lactate. Two equivalents of inorganic phosphate are consumed and
two equivalents of ATP are formed. The overall process of this regeneration system
has a favorable free energy (see Table 13-3), which can be useful in the synthesis of
compounds with high phosphorylating potentials (see Table 13-4, entry 20). The
main backdraw of this method is the complexity of this multienzyme system and the
poor stability of some of the enzymes used.
CHpOH
Figure 13-4. ATP regeneration via the glycolytic Eleven enzymes are used t o
catalyze the conversion of glucose t o lactate.
906
b) From A M P . In biochemical processes, ATP may be converted to either ADP or

AMP. The regeneration of ATP from AMP is slightly more complicated than from
ADP. Methods coupling one of the above mentioned regeneration systems and
adenylate kinase (AdK; E. C. 2.7.4.3)have been used extensively in the production of
ATP[48s431. AdK catalyzes the formation of 2 ADP from ATP and AMP. AcP/AcK and
PEP/PK are the methods most often used to convert ADP to ATP (vide infra).
13.2.1.2
Regeneration o f Other Nucleoside Triphosphates
The preparation of nucleoside triphosphates (NTP) from nucleoside diphosphates

(NDP) follows the same regeneration systems described above (Section 13.2.1.1).
AcK and PK have broad substrate specificities and recognize all of the N D P s [ ~731.
~,
The efficient generation of NDP from nucleoside monophosphates (NMP)has been
solved on preparative scale[74].Adenylate kinase was used in the preparation of
cpdine S'-triphosphate (CTP) and uridine 5'-triphosphate (UTP) from the corre-
sponding nucleoside monophosphates. Nucleoside monophosphate kinase (NMP;
E. C. 2.7.1.4)was used in the synthesis of UTP. Guanosine 5'-triphosphate (GTP)was
prepared with guanylate kinase as catalyst, coupled to a conventional ATP regenera-
tion system. Best results were achieved when 3-phosphoglyceric acid served as the
ultimate phosphorylating agent, and a multienzyme system was used as transfer
catalyst (Fig. 13-5).
XMP
li
- ii OP
A 0 0
0
Aoo-
Figure 13-5. Enzymatic synthesis of nucleoside triphosphates. i) Phospho-
glycerate mutase (E.C. 2.7.5.3); ii) enolase (E.C. 4.2.1.1 1); iii) pyruvate
kinase (E.C. 2.7.1.40); iv) adenylate kinase (E.C. 2.7.4.3, X = A , C, U),
guanylate kinase (E.C. 2.7.4.8, X = C) or nucleoside monophosphate kinase
(E.C. 2.7.4.4, X = U). P = phosphate. See
I
907
13.2.2
Applications
13.2.2.1
Phosphorylationswith ATP as a Cofactor
The most widely used and best developed enzyme-catalyzed phosphorylations are
the ones that are coupled to ATP regeneration systems. Sugar phosphates, nucleo-
side phosphates and glycerides are the major classes of compounds prepared with
these methods.
Kinases are the enzymes most often used for the phosphorylation of saccharides
(Table 13-4,entries 7-19).For example, glucose-6-phosphate(ll),a useful reagent for
the regeneration of nicotinamide cofactors (see Chapter 15), was prepared from
glucose in a one-step reaction by phosphorylationwith ATP 17'1. ATP was regenerated
with AcP/AcK and the phosphoryl transfer was catalyzed with hexokinase, (HK;E. C.
2.7.1.1), and enzyme with broad substrate specificity. Both enzymes were im-
mobilized and reused after product isolation. Alternatively, fluorinated hexopyranose
phosphates and glucose phosphate analogs, with sulfur or nitrogen in the ring, were
prepared with PEP/PK and HK[761 (Table 13-4,entry 9). The synthesis of 11 starting
from polysaccharides or from fructose 1,6-diphosphate[771 was demonstrated, but
the former method is less convenient and the latter is more expensive than the
procedure starting from D-glucose.
5-phospho-D-ribosyla-1-pyrophosphate (PRPP; 13) is a key intermediate in the
biosynthesis of various nucleotides and other natural products. An interesting
application of an ATP/AMP regeneration system is demonstrated in the synthesis of
PRPP from D-ribose in a multienzyme (Table 13-4,entries 13 and 14). In
the first step, ribose was phosphorylated with ATP using ribokinase ( R K E.C.
2.7.1.17) as catalyst. In the second step, PRPP-synthetase(E. C. 2.7.6.1) catalyzed the
transfer of a pyrophosphate group from ATP to ribose 5-phosphate (Fig. 13-6).
The preparations of ATP, GTP, CTP and UTP have been discussed in Section
13.2.1. Kinases are the enzymes most popular for the synthesis and regeneration of
nucleoside triphosphates from their mono- and diphosphates. Nucleoside phosphate
analogs have been synthesized using the same enzymes. For example, ribavarin
triphosphate (RTP; 14), a compound with anti-viral properties, was prepared from
ribavarin monophosphate with adenylate kinase (E. C. 2.7.4.3) and pyruvate kinase
(E. C. 2.7.1.40) as catalysts with PEP as ultimate phosphorylating agent (Fig. 13-7)[481.
Here PEP/PK has proved to be more useful as regeneration system for ATP than
AcP/AcK (see Section 13.2.1.1) in a typical example of a kinetically unfavored
reaction. RMP is one of the rare unnatural substrates accepted by adenylate kinase.
Other nucleotide analogs - for example ATP-a-S and ATP-)I-S- have also been
synthesized using kinases (Table 13-5, entries 4 and 5) 17', 791.
Glycerol kinase (GK; E. C. 2.7.1.30) catalyzes the enantiospecific phosphorylation
of glycerol to form sn-glycerol-3-phosphate, an important intermediate for the
synthesis of phospholipids. The enzyme is inexpensive and stable when im-
mobilized. Studies with enzymes from a variety of microbial sources have shown
I
i-203pw
908 73 Formation and Cleavage of P - 0 Bonds
HO
*OH i
pyruvate
HO OH HO OH
1
ATP
AMP + ATP
PYmate Fw
13
Figure 13-6. Coupled enzymatic synthesis o f PRPP from ~ - r i b o s e [ ' ~
i) ]ribokinase; ii) pyruvate
kinase; iii) PRPP-synthetase; iv) adenylate kinase.
RMP
RTP RDP p- p- p-
H
'0-P-0-P-0-P-0
HO OH
pymvate PEP
14
Figure 13-7. Enzymatic synthesis of ribavarin 5'-triphosphate (RTP; 14) from
ribavarin 5'-monophosphate (RMP) 1481. i) adenylate kinase (EC 2.7.4.3); ii) pyr-
uvate kinase (EC 2.7.1.40).
that glycerol kinase accepts a wide range of glycerol analogs as substrate (Table 13-4,
entries 3 - 5 ) [361. sn-Glycerol-3-phosphate(12) and analogs were synthesized in gram
scales, using glycerol kinase as catalyst and PEP/PK or AcP/AcK as ATP regeneration
system. The phosphorylation of racemic mixtures produced chiral organic phos-
phates with enantiomeric excess (ee) >90-95% and yields of 75-95%. The un-
phosphorylated enantiomers of the chiral substrates were recovered in yields of
30-40% (80-90% ee). Alkaline phosphatase was used to hydrolyze the phosphory-
lated enantiomer and to provide enantiomerically pure unphosphorylated mate-
For example, ~-3-Chloropropane-l,2-diol (15) was prepared from a racemic
mixture in a two step procedure with a 53 % overall yield (97% ee) (Fig. 13-8).
Another application of glycerol kinase is the monophosphorylation of dihydroxy-
HO H
I 909
CI &OP03'-
ATP ADP
j ii
HO H
C I A O H
15
Figure 13-8. Enzyme catalyzed separation of a racemic mixture of ~,~-3-chloropropane-
1,2-diol. i) glycerol kinase; ii) alkaline phosphatase.
Dihydroxyacetone phosphate (DHAP),an important intermediate in the

aldolase catalyzed synthesis of monosaccharides (see Chapter 14), was prepared in a
0.4 mol scale using AcP/AcK for the regeneration of ATP (Table 13-4; entry 6 ) .
Guanidine derivatives were phosphorylated with ATP as well. The syntheses of
arginine- and creatine phosphate (7 and 9), two relatively strong biological phospho-
rylating agents, are not economical, but they demonstrate the potentials of different
ATP regeneration systems (see Table 13-4, entries 20 and 21). Further applications of
ATP regeneration systems are given in Table 13-4 and 13-5.
13.2.2.2
P - 0 Bond Formation with Other Nucleoside Triphosphates than ATP
The use of stoichiometric quantities of nucleoside triphosphates or their regenera-

tion from the corresponding mono- or diphosphates have found important applica-
tions in the synthesis of activated natural products; for instance, nucleoside phos-
phate sugars are important biological intermediates in the synthesis of complex
carbohydrates, glycoproteins, glycolipids and proteoglycans. All of the eight nucleo-
side phosphate sugars, used in vivo by mammalian glycosyltransferases in the Leloir
pathway, are accessible today by practical enzymatic or chemoenzymatic approaches
(see reviews ref.[''] and [81]). The use of these activated monosaccharides in the
enzymatic preparation of oligosaccharides and glycoconjugates is discussed in
Section 11.3. The enzymatic synthesis of sucrose is, however, discussed here, to
illustrate the efficient in situ generation of UDP-glucose from UDP in a complex
multienzyme reaction (Fig. 13-9). In a typical example, a nucleoside triphosphate is
recycled to regenerate a nucleoside phosphate sugar The synthesis, starting from
glucose-1-phosphateand fmctose, used sucrose synthetase (E. C. 2.4.1.13), pyruvate
kinase (E. C. 2.7.1.40) and UDP-glucose pyrophosphorylase (E. C. 2.7.7.9). Inorganic
pyrophosphatase (E. C. 2.6.1.1) was used to keep the pyrophosphate concentration in
the reaction mixture low and to drive the equilibrium to the product side.
910
HO
HO OH
D-fructose
UDP-glucose UDP
4
a-D-glucose 1-phosphate
Figure 13-9. General scheme for an enzyme-catalyzed synthesis o f sucrose with UDP-
glucose[821.i) sucrose synthetase; ii) pyruvate kinase; iii) UDP-glucose pyrophosphorylase;
iv) inorganic pyrophosphatase.
13.2.2.3
Other Phosphorylating Agents
Agents other than ATP are rare in enzyme-catalyzed formation of P - 0 bonds.

Inorganic phosphate, pyrophosphate and polyphosphates were used to prepare
phosphorylated monosaccharides, alcohols, polyols and phenols rS41. The yields
were poor and the reactions lack specificity (Table 13-4, entries 1, 2 and 4). Glycerol-
1-phosphate was prepared from glycerol, for instance, using inorganic phosphate
and alkaline phosphatase (E.C. 3.1.3.1): 75 g of the product was isolated in a 41%
yieldL9I. The reaction was regio- but not stereospecific. Phosphorylases were used
with isotopically labeled phosphate or with inorganic thiophosphate to prepare
(thi0)phosphorylated monosaccharides from oligo- or polysaccharides (see Table
13-4, entries 18 and 15). 32P-labelingof phospholipase with [32P]Piwas used to study
the biological function of this enzyme complex[85].p-Nitrophenyl phosphate was
employed as phosphoryl group donor in the synthesis of allo-uridine, using a
phosphotransferase as catalyst (see Table 13-5, entry 6 ) .
Table 13-4. P - 0 bond formation at non-nucleoside compounds.
~~ ~~ ~
Entry Starting Product Enzyme P-Source Cofactor References

material regeneration
Aliphatic and Aromatic Alcohols
1 Monosaccharides, Phosphorylated Alkaline Phosphatase PPi
alcohols, polyols products (E.C. 3.1.3.1)
2 R = PO: (45 %) Alkaline Phosphatase Pi
?R
(E.C. 3.1.3.1)
R=H
Glycerol, GlycerolAnalogs, Dihydroxyacetone
3 Glycerol Kinase ATP PEP/PK or
J/YH xJ,um:- + non (E.C. 2.7.1.30) AcP/AcK
(rat)
phosphorylated enantiomer
X = C1, SH, OCH3, (29-95 %)
CHIOH, Br, CHzCH3,
OH; Y = 0 or NH
4 X=OH;Y=O (rac)-glycerolphosphate Alkaline Phosphatase Pi, PPi ~ 3 1 [91
.
(E.C. 3.1.3.1)
5 Y Z Y Z Glycerol Kinase ATP PEP/PK 13'1; see also "[I
d
(E.C. 2.7.1.30)
R CH2XH RxCN2XPO:- h,
w
X = 0, NH, H; (45-96 %) D
3-
Y OH, H, CH20H, a
-Q
F, SH, NHz; Z = H, s-
CH3, CH2CH3; i
Y=Z=O. 2.
3
-
2
d
2
N
-
Table 13-4. (cont.).
-
(u
F
Entry Starting Product Enzyme P-Source Cofactor References 3
material regeneration s.
3
0 GK (E.C. 2.7.1.30) ATP PEP/PK or [431; see also [")I Q
HOAOP0:- AcP/AcK SL
DHAP (83%)
9
f!
6
%
Monosaccharides
Hexokinase ATP AcP/Ac
9
(E.C. 2.7.1.1) 0
P
HO m
OH 3
OH OH B
11 (65%)
ibid 11 (80%) y-Glutamyl-Cysteine ATP AcP/AcK 1631; see also

I131, IS91. [771
Synthetase
(E.C. 6.3.2.2)
Hexokinase ATP PEP/PK I7'j1; see also
(E.C. 2.7.1.1)
OH OH
(and other glucose (86%)
analogs)
ATP PEP/PK
HO
D O H
HO HO
D-arabinose (63%)
Entry Starting Product Enzyme P-Source Cofactor References
material regeneration
11 Om;- Fucose Kinase ATP PEP/PK 1911
H3C77-&?
H 3 c F o H (E.C. 2.7.1.52)
HO OH HO OH
(80%)
12 0PO;- OPO,” Phosphoribulokinase ATP PEP/PK
Ho &OH (E.C. 2.7.1.19) (82%) or
Ho &OH
om:- AcP/AcK
0 OH (6G %)
ribulose 5-phosphate ribulose 1,s-biphosphate
13 Ribokinase ATP PEPIPK
(E.C. 2.7.1.17)
2-03p0T2- HO OH
HO OH
D-ribose D-ribose 5-phosphate
(74%)
14 PRPP Synthase ATP AcP/AcK
2-03polp& 0 0 (E.C. 2.7.6.1)
0-7-0-7-0
HO OH
HO OH 0- 0-
2-03p0w
D-ribose 5-phosphate PRPP (75%)
15 Glycogen [35P]glucose1-phosphate Phosphorylase a [=PIPi none
16 Fructokinase ATP none P
(E.C. 2.7.1.11) and
a
2- Phosphoglucoisomerase
-.,
3
L
03psPoH 0PO:-
OH HO
(E.C. 5.3.1.9)
1
Table 13-4. (cont.). bJ
Entry Starting Product Enzyme P-Source Cofactor References $

material regeneration 2.
3
17 Galactose Galactose 1-thiophosphate (25%) Galactokinase ATWS) none 1971 n
5
(E.C. 2.7.1.6) n
18 Sucrose Glucose I-thiophosphate (55 %) Sucrose Phosphorylase NasSP03 none [971; see also ["I g
(E.C. 2.4.1.7) 2
Hexokinase ATP PEP/PK 1991
19 R = PO:
(18%) (E.C. 2.7.1.1)
OH B
R=H B
Guanidine derivatives
Creatine Kinase ATP AcP/AcK LSSI
20 NH; NH;
(E.C. 2.7.3.2) (24%)
H2NKTVo- bo$'HNKT/Ko- MCP/AcK [I81
CH3 0 CH, 0 (55%)
creatine phosphocreatine Glucose, [451
Pi + Mutienzyme
system
21 r-Arginine Phospho-arginine Arginine Kinase ATP PEP/PK 11001
(E.C. 2.7.3.3) (67 %) or

AcP/AcK
(31%)
Enzyme
1851
22 Phospholipase C Phosphorylated Phospholipase Acid Phosphatase Pi
(E.C. 3.1.3.2)
Table 13-5. P - 0 bond formation at nucleosides.
Entry Starting Material Product Enzyme P-Source Cofactor References

regeneration
Nudeotides and Analogs
dAMP (DNA) dATP (67%) Adenylate Kinase ATP PEP/PK 1941
(E.C. 2.7.4.3)
NMP (RNA) ATP, GTP, CTP, UTP NMP-Kinase ATP
(E.C. 2.7.4.4)
RMP RTP (14) (93%) Adenylate Kinase ATP
(E.C. 2.7.4.3)
ATP-a-S (53%) AdK (E.C. 2.7.4.3)lPK ATP PEP/PK 1781;
(E.C. 2.7.1.40) see
HO OH
(AMPS)
ATP-.I-S (80%) Phosphoglycerate- 1-(Thiophosph0)- Dihydroxyacetone, [791
Kinase 3-phosph0- NaZHSP03,
(E.C. 2.7.2.3) glycerate Multienzyme System
0 R = PO:- (31%) Phosphotransferase p-Nitrophenyl none [103].
(Malt Sprouts) phosphate see a l ~ ~ [ ~ ~ ~ l
(u
Table 13-5. (cont.). -
F
Entry Starting Material Product Enzyme P-Source Cofactor References 3
regeneration 4
6'
Oligonucleotidesand Analogs Q
7 Single Stranded DNA+ Phosphorothioate + DNA Polymerase 1 11051 SL
Mixture of Deoxynucleoside Analogs of DNA (E.C. 2.7.7.7) ;

5 ' - 0 - (1-thiotriphosphate) and T4 DNA Ligase z
phosphorothioates (E.C. 6.5.1.1) %
4
8 Polynucleotide
F1F1 P
0
-O-P-O-P-CH,-CHz Phosphorylase m
I t
(E.C. 2.7.7.8) 3
B
0 OH
O- O- WA pppoYYA \ o
HO OH P+
-0' \
A=Adenine
cHz-C;l* HO OH
(-90%)
9 ATP A p A (98%) Leucyl t-RNA ATP

Synthetase
(E.C. 61.1.4)
Nucleoside Phosphate Sugars

10 UTP + Glucose UDP-Glucose (95%) UDP-Glucose
1-Phosphate Pyrophosphorylase
(E.C. 2.7.7.9)
Entry Starting Material Product Enzyme P-Source Cofactor References
regeneration
11 Galactose 1-thiophosphate Uridine-5'-0-(2- Galactose-1-Phosphate [971
and UDP-glucose thiodiphosphogalactose) Uridyl Transferase

(UDP@S)-Galactose (E.C. 2.7.7.12)
(50%)
NAD(P)+
12 ATP + Nicotinamide NAD+ (> 90%) NAD ATP AcP/AcK AdK [lo81
Mononucleotide Pyrophosphorylase
(E.C. 2.7.7.1)
13 NAD+ NADP+ (quant) NAD Kinase ATP AcP/AcK [I081
(E.C. 2.7.1.23)
918
13.2.3
Tables Containing Typical Examples Ordered According to the Classes of Compounds
Sugars, nucleosides and their analogs are the classes of compounds most often
involved in enzyme catalyzed phosphorylation. Typical carbohydrate phosphoryla-
tions are included in Table 13-4, together with the phosphorylation of other non-
nucleosidic compounds. Table 13-5 gives an overview of the enzyme catalyzed
phosphorylation reactions of nucleosides and their analogs. A few representative
examples of nucleoside sugars are listed, for more detailed information consult the
review. refs [74, 'l].
13.3
Cleavage of P - 0 Bonds
In vivo, cleavage of P - 0 bonds are performed by enzymes such as phosphatases,

phosphodiesterases, phosphohydrolases, nucleases, DNases and RNases (see Sec-
tion 13.1.1). In vitro, cleavage of a P - 0 bond is often a trivial synthetic step. Even for
an easy step, enzymes attract increasing attention. The enzymatic reactions are
preferred when regio- or stereoselectivity is required, and when the substrates are
temperature or pH sensitive. Many phosphate analogs have been tested as substrates
of enzymes that hydrolyze phosphoryl groups. These analogs are often accepted as
substrates for the enzymes, and such reactions could be synthetically valuable.
Typical examples are presented in the tables.
-
Table 13-6. Hydrolysis of phosphate and pyrophosphate monoester.
R-O-PO:- R-OH
R-O-P(OP-)-PO;- --t R-OH
Entry R-O Enzyme References
1 Polyprenol (phosphates Acid Phosphatase (E.C. 3.1.3.2) or 1110-1161
and pyrophosphates) Alkaline Phosphatase (E.C. 3.1.3.1)
2 Acid Phosphatase (E.C. 3.1.3.2) or [l1'1
Alkaline Phosphatase (E.C. 3.1.3.1)
Acid Phosphatase (E.C. 3.1.3.2) [1181.see a ~ s 0 [ 1 1 9 - w
q0 OH
[1231
4 KDO 8-Phosphate Phosphatase
HO
H
13.3 Cleavage ofP-0 Bonds
I
919
Entry R-O Enzyme References

5 5'-Ribonucleotide phosphohydrolase 11241
(E.C. 3.1.3.5)
I I
HO OH
Alkaline Phosphatase (E.C. 3.1.3.1) [1031
or Acid Phosphatase (E.C. 3.1.3.2)
Alkaline Phosphatase (E.C. 3.1.3.1) I1041
[ I 2 5 1261
Alkaline Phosphatase (E.C. 3.1.3.1) or
Acid Phosphatase (2-Phases System)
(E.C. 3.1.3.2)
Alkaline Phosphatase (E.C. 3.1.3.1) (1271
0 in 3 or 6
10
9
0-7-0-
Inorganic Pyrophosphatase
(E.C. 3.6.1.1)
PSI
0-
13.3.1
Hydrolysis of Phosphate and Pyrophosphate Monoesters
Both acid and alkaline phosphatases have been used to cleave aliphatic and aromatic
phosphate monoesters. Table 13-6 shows typical examples ordered according to the
substrate class. This table includes an example where the enzymatic reaction was
run with a sensitive substrate (entry l), and examples where regio- or a ste-
reoselectivity was required (entries 2 and 5, respectively).
Polyprenyl phosphates and pyrophosphates have been hydrolyzed by acid and
alkaline phosphatases (Table 13-6, entry 1). For this hydrolysis, classical chemical
methods are inadequate as the reaction products decompose under acid condi-
ti~n~[~~~]].
920
I 13 Formation and Cleavage ofP-0 Bonds
A regioselective dephosphorylation was used in the synthesis of 2'-carboxy-~-

arabinitol 1-phosphate (Table 13-6, entry 2), a natural inhibitor of ribulose 1,s-
bisphosphate carboxylase. Either acid or alkaline phosphatases can be used for the
selective hydrolysis of the 1-phosphoryl group of 2'-carboxyl-~-arabinitol 1,s-
bisphosphate. With acid phosphatase, the conversion was essentially quantitative
yielding exclusively the 1-phosphate derivative (cleavage of the 5-phosphoryl group).
On the other hand, hydrolysis with alkaline phosphatase gave a 4 : 1mixture of the 1-
and 5-phosphate derivatives.
Many natural and unnatural monosaccharides have been prepared by aldolase
catalyzed condensation. The synthesized sugars were often dephosphorylated in situ
by an acid phosphatase (Table 13-6,entry 3). These reactions illustrate multienzyme
synthesis. In this case, no isolation of the phosphate intermediate is required: both
enzymatic reactions are run in the same pot after adjustment of the pH value.
One of the best examples of an enzymatic dephosphorylation for a synthetic
purpose is shown in the entry 5 ofTable 13-6.A 5'-ribonucleotidephosphohydrolase
was used in the synthesis of (-)-aristeromycin, a carbocyclic analog of adenosine.
The (-)-enantiomer of aristeromycin shows some cytostatic and antiviral activity,
while the (+)-enantiomeris inactive. The racemate (*)-5'-phosphorylatedaristero-
mycin was resolved by selective hydrolysis of the (-)-enantiomer with the hydrolase.
The (-)-alcohol and the (+)-S'-phosphatederivative were separated easily on a silica
gel column. Hydrolysis of the (+)-enantiomer with calf intestinal phosphatase
yielded pure (+)-alcohol.
Phosphorylated p-nitrophenol was hydrolyzed with an alkaline phosphatase['29]l.
This hydrolysis was also performed in a two-phase system with an acid phospha-
tase [lzsI.
The naphtol derivative, Table 13-6, entry 9, is dephosphorylated by an alkaline
phosphatase. The resulting naphtol decomposes with chemiluminescent emission
and can be used in bioassays to generate a chemiluminescence signal proportional to
the concentration of an alkaline phosphatase label.
Inorganic pyrophosphate may be considered as a particular case of a phosphate
monoester. The enzymatic decomposition of pyrophosphate by inorganic pyro-
phosphatase (Table 13-6, entry 10) can be used to drive a multienzyme synthesis
(see[351).
13.3.2
Hydrolysis of 5- and N-substituted Phosphate Monoester Analogs
Enzymatic hydrolysis of oligonucleotide-analogs containing modified phosphoryl

moieties have been examined extensively to study their resistance to the enzymatic
hydrolysis.
Thiophosphates (Table 13-7) were subjected to hydrolysis with both acid and
alkaline phosphatases. Most authors claimed that these compounds are substrates
for alkaline phosphatases, but the reaction rate is much lower than with the
corresponding phosphates [12'. N e ~ m a n n [ ~ ~however,
'], reported that these
same S-substituted analogs are resistant to alkaline phosphatases but hydrolyzed by
acid phosphatases.
73.3 Cleavage of P - 0 Bonds
I 921
Table 13-7.
R-O-PS0;-
Entry R-O
-
Hydrolysis ofthiophosphates.
R-OH
Enzyme References
1 H,C-0 Alkaline Phosphatase (E.C. 3.1.3.1) [12'. 1291
2 Alkaline Phosphatase (E.C. 3.1.3.1)

(126, 1291
or Acid Phosphatase (E.C. 3.1.3.2)
Alkaline Phosphatase (E.C. 3.1.3.1) [1281

D N = N e O
Only the alkaline phoshatases have been used with phosphorothioates (Table
13-8).The presence of sulhr between the phosphoryl moiety and the residue does
affect the enzymatic reaction with alkaline phosphatases.
Imidodiphosphates are also potential substrates for phosphoryl hydrolyzing en-
zymes (see Table 13-8, entry 7). They have been used less often than the S-substituted
phosphate analogs.
Another goal of these studies involving analogs with modified phosphoryl groups
or isotopicallylabeled nucleotides was mechanistic elucidation of the stereochemical
course of the r e a c t i ~ n I *13',~ ~1331.
- Hydrolysis of phosphorothioates and irnidodiphosphates.

Table 13-8.
R-S-POf R-SH
R-NH-PO:--+ R-NH2
Entry R-S or R-NH Enzyme References
1 HZN-CH,-CH2-S Alkaline Phosphatase (E.C. 3.1.3.1) [12g1
2 Alkaline Phosphatase (E.C. 3.1.3.1)

? [lZ9l
H3C-C-NH-CH,-CH,-S
3 -0OC-CHZ-CH2-S Alkaline Phosphatase (E.C. 3.1.3.1) (1291
co, Alkaline Phosphatase (E.C. 3.1.3.1) [1301
4 1
5
=to; S
Alkaline Phosphatase (E.C. 3.1.3.1)
or Pyruvate Kinase (E.C. 2.7.1.40)
[1301
6 Alkaline Phosphatase (E.C. 3.1.3.1) [961

fS
OH
7 R a Alkaline Phosphatase (E.C. 3.1.3.1) [l3lI
Ae
HO OH
0-p-0-p-NH
0- 0-
922
13.3.3
Hydrolysis of Phosphate and Phosphonate Diesters
13.3.3.1
Nucleic Acids and their Analogs
Endo- and exonucleases have been used successfully with nucleic acids and their
analogs for organic synthetic purposes. For example, ATP was synthesized from
AMP for use in cofactor recycling (Table 13-9, entry 1).The AMP was obtained from
yeast RNA by cleavage with the nuclease P1 yielding a mixture of nucleoside
monophosphates [lo*].In another report[73],nucleoside diphosphates were obtained
by hydrolysis of RNA with nuclease PI and a polynucleotide phosphorylase (the
diphosphates are preferred because the diphosphates were more easily transformed
to the nucleoside triphosphates than the monophosphates).
Similarly, dATP was synthesized from dAMP, obtained by cleaving herring sperm
DNA with DNase I and nuclease PI (Table 13-9, entry 2). Selective phosphorylation
was obtained with adenylate kinase in the presence of pyruvate kinase and phos-
phoenol pyruvate.
Synthetic oligonucleotide analogs are interesting in applications in which they
suppress translation of mRNAs by hybridization (antisense technology). A good
antisense agent would be resistant to nucleases, and able to maintain its biological
activity for substantial periods in living organisms [13'1. Oligonucleotide analogs
modified at the phosphodiester linkage with a phosphorothioate group are the
subject of numerous papers (see Table 13-9). Other oligonucleotide analogs have
been tested as substrates for endo- and exonucleases. The natural substrates were
modified at either the base residues (Table 13-9, entry 4) or at the sugar moieties
(Table 13-9, entries 5, G and 7).
The tetraphosphate Ap& and its analogs are other examples of a cleavage of a
phosphodiester (Table 13-9, entry 8).
13.3.3.2
Other Phosphate and Phosphonate Diesters
Enzymes have often been used as mild catalysts to hydrolyze phosphate and
phosphonate diesters.
Cyclic phosphate diesters can be hydrolyzed selectively with RNases and phospho-
diesterases to give the corresponding phosphate monoesters (Table 13-10,entries 1
and 2).
Phosphodiesterases have been used to deprotect phosphonate diesters (Table
13-10, entries 3-5). This method is especially useful for sensitive compounds (see
Table 13-10, entry 6: a P - 0 bond could be cleaved selectively in the presence of a P -
N bond).
Table 13-9.
13.3 Cleavage o f P - 0 Bonds
Hydrolysis of nucleic acids, nucleosides and their analogs.

I 923
Entry Starting material Product Enzyme References

RNA Nucleoside Nuclease P 1 (E.C. 3.1.30.1) [lol,731
Monophosphates or Nuclease P1
or Nucleoside (E.C. 3.1.30.1) and
Diphosphates Polynucleotide
Phosphorylase (E.C. 2.7.7.8)
denatured DNA Deoxy Nucleoside DNase I (E.C. 3.1.21.1) P41
Monophosphates Nuclease P1 (E.C. 3.1.30.1)

Phosphorothioate Endo- (E.C. 3.1.30.1)and [lo5, 13+1401
Substituted Exonudeases (E.C. 3.1.4.1)
Nucleic Acids (E.C. 3.1.16.1)
Nucleotide Analogs Restriction Endonucleases [14'1
Containing Modified
Bases
Nucleic Acid Analogs Exonucleases 11421
Containing r-Ribose
cytidine + allo-uridine RNase A (E.C. 3.1.27.5) l1O31
6'-phosphate RNase T2 (E.C. 3.1.27.1)

Nuclease S 1 (E.C. 3.1.30.1)
0 OH
Lo- ?
HO OH
Nucleotide Analogs 1143. 1441
Nucleases
Containing Acyclic Phosphodiesterases
Sugar Analogs (E.C. 3.1.16.1)
[145, 1071
Thiophosphate Analogs Ap4 Hydrolases
of APPPPA (E.C. 3.6.1.17)
(E.C. 3.6.1.41)Ap.+A
Phosphorylase
(E.C. 2.7.7.53)
13.3.4
Other P - 0 Bond Cleavages
Phosphate and phosphonate esters can also be cleaved enzymatically to give

products different from those obtained by enzymatic hydrolysis.
The formal migration of a phosphoryl group between the CG and the C1 of glucose
is catalyzed by phosphoglucomutase. Mechanistic studies were performed with the
924
Table 13-10. Hydrolysis of phosphate and phosphonatediesters.

R-O-PO,Ri--*
R-CRi-PO3R2
Entry
-
R-O-PO3H R'
R-CRi-PO3Hz or R-CRi-PO3HR"
Starting Material Product Enzyme
~ ~
References
~~
Acoa
o=p-0-
OH
RNase TI (E.C. 3.1.27.3)
and RNase Tz
(E.C. 3.1.27.1)
0-
2 HzF-YH-CHzB H,F-FH-CH,B RNases or 11471
9P OH Phosphodiesterase
A o=y-o-
0 0- 0-
B = Base
3 0 R = R' = H Phosphodiesterase I l4'I
II
(EtO),HC-CH=CH--CH,-P-OR' (E.C. 3.1.4.1)
I
OR
R = R' = Et
4 TFA-Ala.AspNH. CHCOOEt R =H Phosphodiesterase I (1481
I
CHFH (E.C. 3.1.4.1)
CHzP03R,
R = Et
4
'
5 R= H Phosphodiesterase I 11501
R2°3P (E.C. 3.1.4.1)
L N
PN7P03R,
0
R = Et
R=H Phosphodiesterase I [1481
(E.C. 3.1.4.1)
R = Et
thiophosphate analog of glucose 6-phosphate 16. In the presence of phosphogluco-

mutase, this analog yields 6-thioglucose 1-phosphate 17; albeit at a slower rate than
the natural substrate (Figs. 13-10).
Aminolysis of phosphonate diester derivatives have been used to form organo-
~~
phosphorus analogs of peptides (18) with phosphatases and phosphodiestera-

ses[151, 1521
The equilibrium between phosphoenolpyruvate and phosphonopyruvate (19,

Fig. 13-10)is catalyzed by a phosphomutase. The mechanism of the transformation
of a phosphoryl into a phosphonoyl group has been studied with labeled and S-
substituted analogs of the natural substrate [153-1581.
13.3 Cleavage o f P - 0 Bonds
I
925
HO
&OH OH
s ~ o ~ ~ Phospho-
-
glucomutase
- HO
&HO op032-
16 17
YOOEt YOOEt
Phospho-
AcNH-FH ?i3 diesterase AcNH-FH cH3
H2C-CHzr- OEt 4 H,C-CH~~-N~H qH3
0 CH~-CH2~r-NHCHzC0OEt
0
18
OP032- Phosphomutase 9
A C O O * o
-'3pJ
,-(
coo-
19
Figure 13-10. P - 0 bond cleavages with hydrolytic enzymes, not leading t o the products o f
hydrolysis.
Figure 13-11. Phosphorylase catalyzed formation of polysaccharides and modified

polysaccharides. i) phosphorylase.
Numerous analogs of carbohydrate polymers (i.e., amylose, glycogen) have been

prepared from modified monosaccharide 1-phosphateswith phosphorylase (Fig. 13-
11 shows the natural substrates) 1159-1621.
Abbreviations
AcK: acetate kinase; AcP: acetyl phosphate; AdK adenylate kinase; AP,,A: pl,pn-
di(adenosine 5'-) n-phosphate; ARS: aminoacyl tRNA synthetase; ATP, ADP, AMP:
adenosine 5'-tri-, di-, monophosphate; ATP-a-S: (&)-adenosine 5 ' - 0 - ( I-thiotriphos-
phate), ATP-y-S: adenosine 5'-0-(3-thiotriphosphate); CK carbamyl kinase; CP:
carbamyl phosphate; CrK: creatine kinase; CTP, CDP, CMP: cytidine Sl-tri-, di-,
monophosphate; dATP, dAMP: deoxyadenosine S'-tri-,monophosphate; DNA: de-
oxyribonucleic acid; AG: change in free energy; GK glycerol kinase; GTP, GDP,
GMP: guanosine 5'-tri-, di-, monophosphate; HK: hexokinase; IUB: International
Union of Biochemistry; MCP: methoxycarbonyl phosphate; NTP, NDP, NMP:
nucleoside Sl-tri-, di-, monophosphate; PC: phosphocreatine; PEP: phosphoenol
pyruvate; Pi: orthophosphate; PK: pyruvate kinase; P,: polyphosphate; P,K poly-
926
I 13 Formation and Cleavage o f f - 0 Bonds
phosphate kinase; PPi: pyrophosphate; PRPP: S-phospho-~-ribosyla-l-pyrophos-

phate; RNA: ribonucleic acid; tRNA: transfer RNA; RK: ribokinase; RTP, RMP:
ribavarin tri-, monophosphate; U: one unit: the amount of enzyme that catalyzes the
formation of 1 pmol/minute; UTP, UDP, UMP: uridine Sl-tri-, di-, monophos-
phate.
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I 931
14
Formation of C-C Bonds
Chi-Huey Wong
14.1
Aldol Reactions
The aldol reaction is one of the most powerful methods for carbon-carbon bond
formation, and its catalytic asymmetric variants have great potential in contemporary
organic synthesis [‘I. Aldolases are enzymes which catalyze reversible and irreversi-
ble asymmetric aldol condensations in r ~ a t u r e [ ~ via , of two distinct reaction
- ~ ]one
mechanisms 1‘1. Type I aldolases activate the donor/nucleophilic substrate via Schiff
base formation with an active-sitelysine residue. These enzymes are predominantly
found in animals and higher plants, and do not require metal cofactors. Type I1
aldolases activate both donor and acceptor substrates via chelation to an active-site
Zn”, and are found mainly in microorganisms. Aldolases can be conveniently
classified into groups according to their natural donor substrates, i. e. dihydrox-
yacetone phosphate (DHAP), pyruvate/phosphoenol pyruvate (PEP), glycine, acet-
aldehyde, and a small number of other molecules. The ability of aldolases to accept a
variety of unnatural acceptor substrates, and to generate new stereocenters of known
absolute and relative stereochemistry reliably, has made them powerful tools for
asymmetric synthesis.
14.1.1
DHAP-Utilizing Aldolases
14.1.1.1
Fructose 1,dDiphosphate (FDP) Aldolase (E.C. 4.1.2.13)
FDP aldolase catalyzes the reversible aldol addition reaction of DHAP and D-
glyceraldehyde3-phosphate (D-G~Y 3-P) to form D-FDP(Fig. 14.1-1).The equilibrium
constant for this reaction has a value of - lo4 M-’ in favor of FDP formation. The
enzyme has been isolated from a variety of eukaryotic and prokaryotic sources, both
in type I and type I1 forms [7-211. Generally, the type I FDP aldolases exist as tetramers
(M. W. - 160 KDa), while the type I1 enzymes are dimers (M. W. - 80 KDa). For the
932 74 Formation of C-CBonds
I 0
PO&OH + H+op FDPaldolase
c _
OH OH OH
DHAP
(PO = phosphate) D-G’y3-p D-FDP
Figure 14.1-1. Aldol addition reaction catalyzed in uivo by FDP aldolase.
type I enzymes there is a high degree of sequence homology (<SO%), with the active
site residues being highly conserved through evolution[12-22]. However, significant
differences identified in the C-terminal regions may control substrate specificity[221.
No sequence homology between type I and type I1 aldolases, or between different
type 11 enzymes, has been identified. Mechanistic studies have mainly been carried
out on FDP aldolases from rabbit muscle (RAMA)[231 and yeast[241,and the X-ray
structures of the enzymes from rabbit muscle (2.7 A and human
muscle (3 A resolution) [251 have been determined. Some of the type I aldolases are
commercially available, inexpensive, and have useful specific activity (-GO U mg-I).
These enzymes are not particularly air-sensitive,though there is an active site thiol
group. The free enzyme has a half-life of - 2 days in aqueous solution at pH
7.0[26r 271, but this is lengthened by immobilization or enclosure in a dialysis
membrane. The type I aldolase from rabbit muscle has been cloned and expressed in
E. c ~ l i [ ~The
l . equivalent enzyme from Staphylococcus carnosus is much more stable
for synthesis The type I1 aldolases from several microbial sources have
recently been cloned and overexpressed[”. 27s 29. 301 . D espite the small degree of
homology in primary sequence between the enzymes from E. coli and rabbit muscle,
studies have shown that they possess almost the same substrate
To date, FDP aldolase, especially the commercially available RAMA, is the most
widely-used aldolase in organic synthesis. A few studies which compare the stability
and lunetic parameters of RAMA vs. bacterial fmctose-1,G-bisphosphate aldolases
have been 331, and FDP aldolase from spinach leaves has also been
employed for synthesis purposes. RAMA accepts a wide range of aldehyde acceptor
substrates, with DHAP as the donor, to generate vicinal diols with D-threo ster-
eochemistry reliably L5. 26, 27, 35-441. Suitable acceptors include unhindered aliphatic
and a-heteroatom substituted monosaccharides, and derivatives
thereof 1441. Aromatic, sterically hindered aliphatic, and a, p-unsaturated aldehydes
are generally not substrates [261. The specificity for the donor substrate is much more
stringent. Initially, only three DHAP analogues were shown to be substrates, but
they were so weak (- 10% cf. DHAP),that their general use in organic synthesis was
451. However, recently, a DHAP phosphonate analog has been shown to
be a good substrate for FDP aldolases from rabbit and S. carnosus, as well as Rha 1-P
aldolase from E. c 0 l i [ ~ ~ 1 .
FDP aldolase exhibits kinetic diastereoselectivity with unnatural chiral aldehyde
acceptor substrates. However, even though there is significant discrimination
(- 20 : 1)between the D- and L-enantiomersof the natural substrate Gly 3-P[261, this is
usually not the case with unnatural aldehydes. In fact, resolutions of racemic
aldehydes are normally only successful if carried out under thermodynamic control.
Often the aldol products can cyclize via formation of a hemiketal, leading to
933
0
74.7 A h / Reactions
1 FDP A. DHAP
I
NHAc
1.0,
2.reductlon ,LNHA,
HO
2 Pase
3 Hz,PdC
i 3
NHAc
P h v N H 2
N3
HbNHAc
PhvNHAc N3
2. reduction
N3
1. FDP A, DHAP
2. Pase
3. Hz, Pd/C *
NHAc
Figure 14.1-2. Preparation o f optically active aldehyde acceptors for FDP aldolase.
significant energy differences between the two diastereomeric products, and ulti-
mately favoring one product after equilibration. For example, with racemic p-
hydroxybutyraldehyde[26, 371 as a substrate, only a single diastereomer was obtained,
with the methyl group in the more stable equatorial position.
Synthetically, FDP aldolase has been employed in the production of I3C-lab-
eled r3’, 46, 471, nitrogen-containingr27* 38-40], deoxy-[3’-371, fluoro-136, 481, and high-
carbon sugarsL342 35, 37, 431. Most of these syntheses require the preparation of the
aldehyde acceptor. In cases where the aldehyde is optically active, this necessitates
either asymmetric synthesis of the required enantiomer, or use of a racemic
aldehyde, with subsequent separation of diastereomeric products. In general,
ozonolysis of a terminal olefin (Fig. 14.1-2)r4’1 and acid-catalyzedacetal deprotection
are convenient routes to the acceptor aldehydes. a-Chiral aldehydes have also been
prepared by ring opening of readily-available ( R ) -and (S)-glycidaldehydeacetal, or
the corresponding thirane and aziridine, by appropriate nucleophiles c4’1. Both
enantiomers of glycidaldehyde acetal may be prepared by lipase-catalyzedresolution
of 3-chloro-2-hydroxypropanal diethyl acetal r4’1. Alternatively, tandem use of Sharp-
less asymmetric dihydroxylation (AD) and aldolase-catalyzed condensation allows
quick and facile synthesis of carbohydrates with complete stereocontrol (Fig. 14.1-
3 ) [SO].
A 1 : 4 mixture of deoxynojirimycinand deoxymannojirimycin was obtained when
racemic 3-azido-2-hydroxypropanal was used as a substrate for RAMA [38, 391, indicat-
ing that the D-aldehyde is a better substrate for the enzyme. A similar result was
obtained with FDP aldolase from E. c0lir~~1.Since both deoxynojirimycin and
.,
.o. EtO N, 1. FDP A, DHAP
Sharpless 2. Pase
HOfiOR
epoxidalion ~0-0~ 3.Hz. Pd/C ~ - : H
HO
OH OH
HO nOH
.
.o.
.~ Porcine
Pancreas
Lipase HO A O
..
.o.
i
C O R -
LoH
EtO
El0 I
1.FDPA.DHAP
2.Pase
3. H, Pd/C *
HO 0%
&&NR
OH HO
R = protecting group,
R‘ = H, Bn. or OBn
Figure 14.1-3. Chernoenzyrnatic stereo-controlled synthesis o f azasugars.

74 Formation of C-CBonds
934
I
+
CH3
HO
HO co2
H a o H H%&
OH HO OH
DAHP 6H
- .
(+)-exo-brevicomin 1-deoxynojirimycin
cyclilol
+H
r HO
OH
aza-suaar analoa
C-glycosides of ManNAc
Figure 14.1-4. Various classes o f m o l e c u l e s synthesized u s i n g FDP aldolase.
deoxymannojirimycin are potent glycosidase inhibitors, each compound was also

prepared in an optically pure form from the respective optically pure azidoalde-
hydes L2'1. Both (R)-and (S)-azido-2-hydroxypropanal were obtained via LP-80 cata-
lyzed resolution of the racemic acetal precursor .]'41 Similar strategies have been
employed to prepare the P-glycosidase inhibitors P-1-homonojirimycin,P-1-homo-
mannojirimycin and the azasugars corresponding to N-acetylglucosamine and
N-acetylmannosamine(Fig. 14.1-4)[491.
Similarly, employing 2-azidoaldehydes as RAMA substrates allowed the prepara-
tion of polyhydroxylated pyrrolidines (Fig. 14.1-5)[38, 52, 531. 1,4-Dideoxy-l,4-imino-
D-arabinitol was synthesized from azidoacetaldehyde, and both (2R,5R)- [491 and
(2S,5R)-bis(hydroxymethyl)-(3R,4R)-dihydro~~olidine were synthesized from
racemic 2-azido-3-hydroxypropana1, respectively. In the latter case, the kinetic
product of the aldol addition was transformed into the (ZR,SR)-stereoisomerof the
pyrrolidine, while the thermodynamic product gave the (2S,SR)-stereoisomer.Fur-
thermore, pyrrolidines structurally related to GlcNAc have been prepared ster-
eoselectively by a similar transformation from lipase-resolved aldehyde precur-
sors [541.
2. Pase OH HO O
'H
thermodynamic product
kinetic product
Figure 14.1-5. Synthesis of polyhydroxylated pyrrolidines u s i n g RAMA.

'u I
1. AcSK
AcSH
EtO
___)
1. DHAP,
RAMA
2. Pase
..JGJS"
0
-..
OH
-..
EtO& '0 2. HCI * OH OH OH
tl
AcO
P
Et,SiH,
BF3*Etz0 A,..&
AcO
~
HO
OAc HO OAc HO OH
Figure 14.1-6. Preparation of deoxy-thio sugars.
The 6-deoxyazasugars and their analogs can also be easily prepared by direct
reductive amination of the aldol products prior to removal of the phosphate
group rS51. Studies using glucose 6-phosphate (Glc 6-P) indicate that the phosphate
group is probably reductively cleaved from the imine 6-phosphate rather than the
azasugar 6-phosphate. Use of 3-azido-4-hydroxy aldehydes results in the formation
of homoaza sugars[51,"I. The optically pure aldehydes can be obtained either by
Sharpless epoxidation of the olefins[51]or enzymatic resolution of the epoxides[57].
The lipase-resolvedmaterial was also used to prepare another class of glycosyl cation
mimics, the tetrahydropyrimidineslsS,591. These compounds exist in equilibrium
with their guanadinotetraose forms which predominate at low pH. The tetra-
hydropyrimidines are potent inhibitors of a-galactosidase,due to their close resem-
blance to the transition state half-chair conformation of the enzymatic reaction.
Interestingly, an inhibitor with an OBn group attached to the nitrogen has a much
lower pK, and inhibits a-galactosidasein the region of a physiological pH ["I.
Similar to the synthesis of azasugars, a series of deoxy-thiosugars was prepared by
aldol condensation of thioaldehydes with DHAP followed by reduction of the
resulting thioketoses (Fig. 14.1-6)I.'[ Regioselective ring opening of the (S)-glyci-
daldehyde diethyl acetal with potassium thioacetate introduced the thio function.
RAMA-catalyzed aldol condensation followed by dephosphorylation gave the corre-
sponding thioketose[''], which was then acetylated and reduced to the 1-deoxy-5-thio-
D-glucopyranose peracetate I.'[ Also, in a similar manner, l-deoxy-5-thio-~-man-
nopyranose was obtained from the other aldehyde enantiomer, while a Fuc-1-P
aldolase-catalyzed reaction provided 1-deoxy-5-thio-galactopyranoseand l-deoxy-
5-thio-altropyranose, and Rha-1-P aldolase catalyzed reaction produced l-deoxy-
S-thio-~-mannopyranose I.'[
With other aldolases in place of FDP aldolase, a wide range of other poly-
hydroxylated piperidines and pyrrolidines have been synthesized (vide in@) LS31.
Aldolase-catalyzed condensation followed by reductive amination has become a
general strategy for the synthesis of 5-["], 6- LG2, G31, and 7-membered[G4] cyclic imine
sugars. The resulting compounds have become the gold standard template for
glycosidase inhibitor design.
Use of racemic methyl N-acetylaspartateP-semialdehydeas a substrate for RAMA
provides a precursor to 3-deoxy-~-arabino-heptulosonic acid 7-phosphate (DAHP,
936 C-CBonds
I 74 Formation of
Fig. 14.1-4) L4l]. This compound is an important intermediate in the shikimate

pathway for the biosynthesis of aromatic amino acids in plants. The RAMA reaction
produced the desired D-threo stereochemistry, and chemical reduction of the keto
group gave the desired (GR)-stereoisomerin GO% diastereomeric excess. Other
analogs of DAHP are also potentially available by this route, due to the broad
substrate specificity of RAMA.
The use of pentose and hexose phosphates as RAMA substrates provides a route to
high-carbon sugars, including analogs of sialic acid and KDO[44,65, 66]. 0ther
carbohydrate derivatives prepared by RAMA include unsaturated C8-C9 sugars [671,
phosphonic acid derivatives["], fluorescently-labeled fructose derivatives["], per-
fluoroalkylated sugars L7O], and those protected by thioacetals L7'1. Furthermore, the S.
carnosus enzyme has been employed for the synthesis of bicyclic sugars [721 and
disaccharide mimetics [731.
Complex xylulose structures can also be synthesized by RAMA[741. Employing a
one-pot, three-enzyme system with RAMA, triose phosphate isomerase, and
1-deoxy-D-xylulose-5-phosphate synthase, l-deoxy-~-xylulose-5-phosphate could be
obtained in 47% Furthermore, a four-enzyme, one-pot system employing
FDP-aldolasefrom S. carnosus furnished 5-deoxy-5-ethyl-~-xylulose [761.
The synthesis of (+)-exo-brevicomin(Fig. 14.1-4) was the first example ofthe use of
RAMA to synthesize a non-carbohydrate RAMA was employed to
catalyze the key aldol addition step, in which the two chiral centers of the target
molecule were established. RAMA has also been employed for the synthesis of a key
fragment of (+) a s p i ~ i l i n [ ~and
~ I ,for that of acyclic polyols [791. Single aldol condensa-
tion on remote dialdehydes has also been achievedI.'[
Other molecules synthesized by FDP aldolase include C-glycosides[43, 1' and
cyclitols (Fig. 14.1-4) LS31. Cyclitols are an interesting class of bio-active compounds,
and the use of aldolases provides a chemo-enzymatic strategy towards their synthe-
sis. An example is the synthesis of nitrocyclitolswhich was accomplished by an FDP
aldolase catalyzed reaction with nitroaldehyde, followed by a non-enzymatic intra-
molecular nitro-aldol reaction (Fig. 14.1-7) [83a1. A one-pot synthesis of cyclitols has
been reported, involving an FDP aldolase-catalyzedreaction between a phosphonate
aldehyde and DHAP. The aldol product cyclized in situ via an intramolecular
Horner-Wadsworth-Emmonsolefination to give the polyhydroxylated cyclopentane
1. FDP A, Ac20,
DHAP O z N V BF3-Etz0 *
2. Pase * OH
OH
HO OH
;02N--f5 c] "2' * OzN -$- + ozNq-Oo;c

AcO OAc OAc OAc
(1:l)
Figure 14.1-7. Preparation o f nitro-cyclitols.

I
/
NC NC
Figure 14.1-8. One-pot synthesis of cyclitols.
0 0 OEt 0 OH OEt OH OH H
+ H U O E t 1. RAMA “U
POAOH o
l.IDH,NADH * E
HO&~
s ;
t ~
2. Pase 2. H+
I
DHAP OH OH
L-xylose
NaBH(0Ac)s
H o
OH OH OEt
a O E t
1. IDH, NADH
2.H+
- H
OH OH H
O
R ;
U O
OH OH
2-deoxy-D-
arabino-hexose
Figure 14.1-9. Use of the “inversion strategy” t o synthesize L-xylose and

2-deoxy-D-arabino-hexose.
(Fig. 14.1-8)[83b1. Using this approach, different functionalized cyclitols may become
easily accessible.
FDP aldolase is a useful catalyst for the direct synthesis ofketose monosaccharides
and their analogs (vide suprcz). However, a number of the important naturally-
occurring carbohydrates are aldoses. Various FDP aldolase products can be iso-
merized to a mixture of the ketose and aldose, and subsequently separated with Ca2+
or Ba2+treated cation exchange resins. Another strategy involves the use of glucose
isomerase (GI)LS41, which catalyzes the isomerization of fructose (Fru) to glucose
(Glc),and is used in the food industry for the production of high fructose corn syrup.
GI also accepts analogs of Fm with modifications at positions 3, 5 and 6 as
substrates L3‘1. Aldose analogs including 6-deoxy, 6-fluoro, 6-0-methyl and 6-azido-
glucose have been synthesized using this FDP aldolase/GI 371.
However, not all FDP aldolase products are substrates for GI, and in the case of
5-deoxy-~-fructose,the equilibrium lies completely in the favor of the ketose.
Furthermore, in the inversion strategy (Fig. 14.1-9)[421, monoprotected dialdehydes are
used as substrates for FDP aldolase, generating protected aldehyde ketoses. The
ketone group is then chemically or enzymatically stereoselectivelyreduced, and the
aldehyde subsequently deprotected to produce the aldose. The strategy also places
the vicinal diol produced in the aldol reaction in a position other than C3/C4. One
enzyme suitable for the reduction is the NADH-dependant iditol dehydrogenase
938
I 14 Formation ofC-C Bonds
Table 14.1-1. Products prepared from FDP aldolase-catalyzed reactions with DHAP.
[a1 [bl [c] R=H, [d] R = P032-

0 OH OH 0 OH OH 0 OH OH
O-H - - PO
O
-H
PO PO
O
-R
.
. -
-
OH OH OH OH OH OH
[el [f,g] R=H, [h-k] R = P03'. [h]

PO&OH .
. .- P
O+
o
R P
O+
o
p
OH OH OH OH OH OH OH
If1 [h-kl If1

0 OH OH 0 OH OH OH OH
O
H
+' ' p
o
+
OP poO
*P
OH OH OH OH OH OH OH OH OH
If1 [h] R=H,OH,NH2 [hl

0 OH OH 0 OH OH OH 0 OH OH OH
PO OP PO OP
OP
[hl [I1 [ml [bl

OH OH OH 0 0 OH
OH OH OH
PO- R2 PO& 0 OH R2 P0-R' P 0 0A OH 3

R
~.
- -
OH R OH R' OH R OH
R' R2 Ref. R' R2 Ref. R' R2 Ref R3
H THPO
H BzO
H CHJCHZ
H CH3
H (Et0)zPO
H Ph
H OHC(CHz),
H HOCH2
H CbzNH
H CbzNHCH2
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h M. D. Bednarski, H. J. Waldmann, G. M. White-

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o F. Effenberger, A. Straub, Tetrahedron Lett. 1987,28, L C. H. von der Osten, A. J. Sinskey, C. F. Barbas Ill,
1641. R. L. Pederson, Y.-F. Wang, C.-H. Wong, J. Am. Chem.
p A. L. Lehninger, J. Sice, J. Am. Chem. SOC. 1955,77, SOC.1989,111,3924.
5343.
q J. R. Dumachter, D. G. Drueckhammer, K. Nozaki,
H. M. Sweers, C.-H. Wong,]. Am. Chem. SOC. 1986,
108,7812.
(IDH) from Candida utilis, also known as sorbitol or polyol dehydrogenaseL3’>851.

Reduction of the ketone occurs to give the alcohol with (S)-stereochemistry.The
corresponding (R)-alcohol was obtained by non-stereoselective reduction of the
ketone with NaBH(OAc)3‘ and the (S)-epimer was selectively removed by IDH-
catalyzed oxidation. L-Xyloseand 2-deoxy-~-arabino-hexose were synthesized by each
of these two processes, respectively.
Other aldose/ketose isomerases with different substrate specificity have been
cloned and overexpressed[86], including Fuc isomerase (Fuc I, EC 5.3.1.3) and Rha
isomerase (Rha I, EC 5.3.1.14). Fuc isomerase, in combination with Fuc 1-P aldolase
or Rha 1-P aldolase, has been used to prepare L-glucose, L-galactose, L-fucose, and
derivatives from the corresponding L-glyceraldehydederivatives and DHAP iS71.
RAMA has been the most popular synthetic aldolase, due to its commercial
availability. Notably, no significant differences in substrate specificity or ster-
eoselectivity between FDP aldolases from different sources have been observed[88].
However, it is still important to verify this, especially for the type I1 aldolases which
operate by a different mechanism. In fact, the type I1 aldolase from E. coli, which has
been subcloned and o~erexpressed[~~], has the potential to supplant RAMA as the
FDP aldolase of choice for synthesis. It has enhanced stability compared with RAMA
(vide supra),and is available from a microbial as opposed to an animal source.
Table 14.1-1 illustrates products prepared from FDP aldolase-catalyzed reactions
with DHAP.
14.1.1.2
Fuculose 1-Phosphate (Fuc 1-P) Aldolase (E.C. 4.1.2.17), Rhamnulose 1-Phosphate
(Rha 1-P) Aldolase (E. C. 4.1.2.1 9) and Ragatose 1,C-Diphosphate (TDP) Aldolase
Fuc 1-P aldolase, Rha 1-P aldolase, and TDP aldolase also use DHAP as the donor
substrate in aldol condensation. Fuc 1-P aldolase catalyzes the reversible condensa-
940
I 14 Formation of C-C Bonds
PO&OH
0
+
0
,,kCH3 - FUC 1-p aldolase, PO*CH3
0 OH
OH OH OH
DHAP L-lactaldehyde FUC1-P

Figure 14.1-10. Aldol addition reaction catalyzed in vivo by Fuc 1-P aldolase.
DHAP L-lactaldehyde Rha 1-P

Figure 14.1-11. Aldol addition reaction catalyzed in vivo by Rha 1-P aldolase.
0 0 OH
&
‘‘OH + H G
OH
O P - TDP aldolase
* O
‘+OP
OH OH
DHAP D-GIY3-P D-TDP
Figure 14.1-12. Aldol addition reaction catalyzed in vivo by TDP aldolase.
tion of DHAP and L-lactaldehyde to provide L-FUC1-P (Fig. 14.1-10). With the same
substrates, Rha 1-P aldolase produces L-Rha 1-P (Fig. 14.1-11). Both of these
enzymes are type I1 aldolases and are found in many microorganisms rS91. Both E. coli
enzymes have been cloned, overexpressed in E. coli, and purifiedI”O-’*I. TDP
aldolase, a type I aldolase involved in the galactose metabolism of cocci, catalyzes the
reversible condensation of D-G~Y 3-P with DHAP to give D-TDP(Fig. 14.1-12), and
has also been cloned and overe~pressed[’~l.
Both Fuc 1-P and Rha I-P aldolase show specificity with regard to the aldehyde
component, generating vicinal diol units of D-erythro and L-threo configurations,
respectively (Fig. 14.1-13) [90-931. While the stereospecificity for the absolute (3R)-
configuration is mechanism-based, the configuration at C4 is somewhat substrate
dependent. However, these two aldolases also show significant kinetic preference for
the L-enantiomer of 2-hydroxyaldehydes (c95 : 5), facilitating resolution of racemic
mixtures of these compounds (Fig. 14.1-14) LS6]. Both enzymes have been used in the
synthesis of rare ketose l-phosphates[86], azasugars, and deoxyazasu-
gars[54.64, 951. Rha 1-P has also been employed in the synthesis of bicyclic
943
carbohydrate structures L9‘l.

Fuc 1-P and Rha 1-P aldolases have also been utilized in whole cell systems with
DHA and catalyhc inorganic aresenate [971. With L-lactaldehydeas the substrate in the
Rha 1-P aldolase reaction, the aldol product L-rhamnulose was subsequently iso-
merized to L-rhamnose, catalyzed by rhamnose isomerase. No such isomerization
was observed with L-xylulose, the corresponding aldol product using glycolaldehyde
as the substrate. Recent studies have since shown that both rhamnose and fucose
isomerase require fixed stereochemistry only up to C3 for aldohexose substrates;
14.7 Aldol Reactions
Diastereomeric Ratio Relative

I
941
Aldehyde substrate Product Aldolase trans ; cis Rate (%)
glycolaldehyde Fuc 1-P <3 : 97 38

OH Rha 1-P >97 : 3 43
. c
L-lactaldehyde 3 H Fuc I - P <3 : >97 100
OH OH Rha I - P >97 ; <3 100
D-Giy Fuc I - P <3 : > 9 7 28

H 0 a o H
OH OH Rha 1-P >97 . <3 42
L-Gly Fuc I - P c3 : 297 17

H OOH GOH o H
Rha I - P >97 : <3 41
OH 0
3-hydroxy- Fuc I - P <3 : >97 11
proplonaidehyde HO
OH Rha 1-P >97 : <3 29
Formaldehyde ,,AoH Fuc I-P 44

22
OH Rha 1-P
acetaldehyde H3C=OH . Fuc I - P 5 95 14

OH Rha 1-P 69 31 32
ebutyraldehyde Fuc I - P 30 70 20
Rha 1-P 97 3 22
Figure 14.1-13. Acceptor substrate specificity and diastereoselectivity o f Fuc 1-P

and Rha 1-P aldolase.
FUC1-P
aldolase/
HO *OH
O / (L)
CHO + H O A O P
OH DHAP
(L)
Figure 14.1-14. Kinetic resolution of 2-hydroxyaldehydes using Fuc 1-P
and Rha 1-P aldolase.
(2R,3R) for rhamnose isomerase and (2S,3R) for fucose isomerase186].A sequential
application of Fuc 1-P aldolase and fucose isomerase was employed for the prepara-
tion of L-fucose analogs [981.
TDP aldolase has been isolated from several sources [991. The enzyme from E. coli
has a narrow pH profile with an optimum at pH 7.5, but still displays acceptable
942
o + HO&OP
0
TDP aldolase
OH 0 OH 0
R +OP + R+oP
DHAP bH bH
L-erythro D-threo
<lo% >go%
Figure 14.1-15. Diastereoselectivity of TDP aldolase.
R .,,, L
Rs w O H protection J 1protection OH OH
,..-Rs
OH OH
\ AD-mix-a H O
OH OH
W RL
OH OH 0 0 OH OH
O
$
*H sL;H
*o
OH OH 'H : OH 6H
OH OH 0
) $ , ; " ,
OH OH OH 6H
OH OH / \ ; ;++o
H 0 OH OH
OH OH OH OH
Figure 14.1-16. Product stereochemistries generated by the four complementary

DHAP aldolases.
activity within pH 6.5-7. TDP aldolase accepts a variety of substrates, including

glycoaldehyde, D- and L-glyceraldehyde, acetaldehyde, and isobutyraldehyde['OO].
However, a diastereomeric mixture of products is generally formed. Also, only with
the natural D-substrate does the major product (D-TDP)have the tagatose configura-
tion (Fig. 14.1-15). Owing to this lack of stereoselectivity, TDP aldolase is not as
synthetically useful as the other FDP aldolases. However, with suitable protein
engineering, this may change in the future.
The four DHAP-utilizingaldolases generate all four stereochemical permutations
of the vicinal diol at C3/C4 of the ketose product, and can be used to generate all four
stereoisomers of a desired product (Fig. 14.1-16). In this manner, these enzymes
have been utilized for the synthesis of sLeXmimetic side chains[81],among other
targets.
I
14.1.1.3
Synthesis of DihydroxyacetonePhosphate (DHAP)
All four aldolases described previously use DHAP as the donor substrate, and several
approaches have been taken towards its synthesi~1'~'-~~~]. Enzymatic in situ genera-
tion of DHAP from FDP can be accomplished using FDP aldolase and triosephos-
phate isomerase (TI)[341. FDP aldolase catalyzes the retro-aldol reaction of FDP to
y and DHAP, and TI catalyzes the conversion of ~ - G l 3-P
give ~ - G l 3-P y into DHAP.
However, the overall reaction may not go to completion, depending on the thermody-
namic stability of the product compared with FDP. The presence of FDP may also
complicate product isolation. Another enzymatic method involves glycerol kinase-
catalyzed phosphorylation of dihydroxyacetone (DHA) using ATP, with in situ
regeneration of ATP [34* 1"' . This procedure generates DHAP directly in high yield,
but may be expensive for large-scale synthesis. Another method involves the
generation of DHAP from phosphatidyl choline and DHA using phospholipases C
and DI1OG]. DHAP and the corresponding phosphoramidate and phosphorothioate
have been generated in situ enzymatically from the reduced form and used as
substrates [104b].
DHA dimer can be phosphitylated chemically with (PhCH20)2PNEt2'and subse-
quently oxidized to the phosphate with H202 (Fig. 14.1-17)['021,or it can be
phosphorylated directly with either POC13[lo3]or (PhO)2POC1['04a].Alternative
efficient chemical syntheses of DHAP have also been These phosphate
derivatives are all subsequently transformed into the stable dimer precursor of
DHAP, which can easily be converted into DHAP by acid hydrolysis. The main
drawback to preparing DHAP chemically is the lengthy synthetic procedure.
Alternatively, DHAP can be replaced by a mixture of DHA and inorganic
* reacts reversibly with inorganic arsenate (k - 2.4 x
a r ~ e n a t e [971.~ ~DHA M-'s-')
to form dihydroxyacetone arsenate, an analog of DHAP and a donor substrate for
aldolases. In fact, in the presence of RAMA, triose phosphate isomerase, and
inorganic arsenate, DHA was converted into D - F ~ uin almost quantitative yield["].
However, reaction rates tend to be very slow, and low yields of aldol products are
often obtained. Inorganic vanadate and DHA has also been investigated,but cannot
be successfully utilized as a DHAP analog[97].
OH
OH 1. (PhCH20)2PNEt2,
then H202
EtO$$Ilt H+ P O 0
A O H
,H
,O
"J 0 EtoH' H+ EtO$$OEt
0 2.HzlPd PO 0
DHAP
HO HO
dihydroxy-
acetone dimer
Figure 14.1-17. Chemical synthesis of DHAP.
944
14.1.2
Pyruvate/Phosphoenolpyruvate-UtilizingAldolases
14.1.2.1
N-Acetylneurarninate (NeuAc) Aldolase (E.C. 4.1.3.3) and NeuAc Synthetase
(E.C. 4.1.3.19)
NeuAc aldolase, or sialic acid aldolase, catalyzes the reversible condensation of

pyruvate with N-acetylmannosamine(ManNAc)to form NeuAc (sialicacid; N-acetyl-
~-am~no-3,~-d~deoxy-~-g~ycero-~-ga~acto-2-nonu~opyranoson~c acid) (Fig. 14.1-
18)[108, 1091.
The a-anomer of NeuAc serves as the aldolase substrate, even though the p-
anomer predominates in solution. The initial products of aldol cleavage are a-
ManNAc and pyruvate["o]. In uiuo, the enzyme has a catabolic function, with an
equilibrium constant for the retro-aldol reaction of 12.7 M-'. However, for synthetic
purposes, the production of the aldol product can be achieved using excess
pyruvate['O'I. NeuAc aldolase is a Schiff base-forming type I aldolase and has been
isolated from both bacteria and animals. The optimum pH for activity is 7.5,
although it retains activity between pH 6 and 9, and is stable under oxygen["'. 'l'].
The enzymes from Clostridia and E. coli are now commercially available (Toyobo),
and that from E. coli has been cloned and overexpressed in E. c01i["~].It can be used
free in solution, in immobilized f ~ r r n [ ~ ' ~,-or
~'~ I
enclosed in a dialysis mem-
brane['l'].
Good conversion (-90%) of ManNAc to NeuAc has been achieved using the
isolated enzyme, and purification can be achieved by decomposing excess pyruvate
with pyruvate decarboxylase['"I, or acid-catalyzedesterification of the products [ll'l.
The need for excess pyruvate and purification of NeuAc can be circumvented by
coupling the synthesis of NeuAc to a more thermodynamically stable process. For
example,the NeuAc aldolase reaction can be coupled to a sialyltransferasereaction to
produce sialyloligosaccharides[I2']. Another variant of this process used a mixture of
ManNAc and GlcNAc, whereby the GlcNAc was epimerized to ManNAc chem-
ically[121.1221 or enzymatically['23.1241.
Extensive substrate specificity studies have indicated that only pyruvate is accept-
able as the NeuAc aldolase donor substrate"'']. However, this enzyme has broad
acceptor specificity, and over s x t y aldoses have been characterized as substrates.
Substitutions at C2,4 and 6 of ManNAc are allowed,with only a slight preference for
absolute stereochemistry at C4, 5 and 6[11&120s125-1301 . Some pentoses and their
"s
analogs are also substrates, although 2 and 3 carbon molecules are not accepted.
HO
OH NeuAc aldolase
- H6 OH
OH * H o G
H6
C
OH
0 2 H
D-ManNAc a-NeuAc P-NeuAc
Figure 14.1-18. Aldol addition reaction catalyzed in uivo by NeuAc aldolase.

0
14. I Aldol Reactions
H?
I 945
OH 0 OH OH 0
HO+H NeuAc aldolase H

C
O
-; - HO OH
OH OH OH OH
D-arabinose KDO
Figure 14.1-19. Synthesis of KDO using NeuAc aldolase.
n NeuAc
0
HO
OH
subtilisin BPN'
*
HO
- aldolase,
pyruvate
OH
1. HCI
0
2. dansyl-CI,
B O c - H N ~ o ~ Na2C0,
w
AcHN C02H
HO OH HO OH
/
Figure 14.1-20. Synthesis of 9-0-acetylNeuAc by combined use of subtilisin
and NeuAc aldolase.
The stereoselectivity of NeuAc aldolase is unusual, as it is thermodynamically

controlled. With the natural substrate D-ManNAc, attack occurs exclusively on the si
face of the carbonyl group. However, a mixture of KDO and 4-epi-KDOwas obtained
when D-arabinose was used as a substrate[1281, and a complete reversal of diaster-
eoselectivity was observed with L-mannoseand 6-deoxy-~-mannose[~~'. '19, 13'1 . W'lth
these latter aldehydes, exclusive attack on the re face of each carbonyl group gives the
more thermodynamically favored products. NeuAc aldolase has been investigated as
a catalyst for the condensation of various pentoses and hexoses, and enzyme
stereoselectivity characterized['32].Furthermore, KDN has been produced on a 100
gram scale from pyruvate and D-mannose in a crystallized yield of 75 %[1331.
NeuAc, derivatives thereof, and polysialic acids play important roles in cell-cell
adhesion and communication in bacterial and mammalian 13'1 . Th e
wide substrate specificity and ready availability of NeuAc aldolase provide the
opportunity for the synthesis of many sialic acid derivatives. Azasugar analogs of
NeuAc were synthesized using the 3-deoxy-3-azidoanalogs of ManNAc, mannose,
and glucosamine as acceptor substrates Similarly, KDO was produced using D-
arabinose as the acceptor (Fig. 14.1-19).
A facile synthesis of 9-0-acetyl-NeuAc has been accomplished via regiose-
lective irreversible acetylation of ManNAc catalyzed by subtilisin, followed by NeuAc
aldolase-catalyzed condensation of the resulting 6-0-acetyl-ManNAcwith pyru-
vateI"'1. A 9-0-glycyl-NeuAcderivative was prepared in a similar fashion, and was
further converted into a fluorescent derivative (Fig. 14.1-20)[13'1.
A large number of NeuAc derivatives modified at the 5-amino the C7-
or the C9-p0sition[~~", l 4 O ] have been reported. Many other compounds
946
R' R2 R3 R4 R5 Ref.
AcNH H OH H CHzOH, CH~OAC, 12.3,
CHzN,, CHzF, CH,OMe, 5,11
CHzOCOCHOHCH,
OH H OH H C W H , CHZOAC,H 7,4,10
OH H H H, F CHzOH, CHpF 83
H H,OH OH H CHzOH 10,8
Ph H OH H CHzOH 10
N, H OH H CH2OH 8
Figure 14.1-21. NeuAc analogs synthesized using NeuAc aldolase.
have been prepared using this aldolase (Fig. 14.1-21)[47.141-1441, including an a-

methyl ketoside of 5-amino-NeuA~['~~1, and polyacrylamides bearing pendant a-
sialoside groups [1431 or C-linked sialosides ll#]. The latter strongly inhibit agglutina-
tion of erythrocytes by the influenza virus.
The synthesis of NeuAc in vivo is accomplished using NeuAc synthetase["ll. This
aldolase catalyzes the irreversible condensation of PEP and N-acetylmannosamine.
Although this enzyme has not yet been isolated and characterized, it may prove
synthetically useful in the future.
14.1.2.2
Aldolase (E.C. 4.1.2.23) and
3-Deoxy-~-manno-2-ocu~osonate
8-Phosphate Synthetase (E. C. 4.1.2.16)
3-Deoxy-~-manno-2-octulosonate
3-Deoxy-~-manno-2-octulosonatealdolase, also known as 2-keto-3-deoxyoctanoate

(KDO) aldolase, catalyzes the reversible condensation of pyruvate with D-arabinose
to form KDO (Fig. 14.1-22). KDO and its activated form CMP-KDO are key inter-
mediates in the synthesis of the outer membrane lipopolysaccharide (LPS) of Gram-
negative bacteria[145].Inhibitors of LPS biosynthesis or LPS binding
therefore serve as antimicrobial agents [147-1511. KDO aldolase has a catabolic func-
tion, with an equilibrium constant for degradation of 0.077 M. It has been isolated
and purified from E. coli [1521 and Aerobacter cloacae [1531. Preliminary investigations
on this enzyme showed high specificity for KDO in the direction of cleavage,
whereas the condensation reaction proceeded with some flexibility. Several unnatu-
ral substrates, including D-ribose, D-xylose, D-lyxose, L-arabinose, D-arabinOSe
5-phosphate and N-acetylmannosaminewere reported to be weak substrates (relative
rate >5% cf. ~-arabinose['~~]). Studies on the substrate specificity of KDO aldolase
from Aureobacterium barkerei strain KDO-37-2, have indicated that this enzyme
OH 0 KDO aldolase OH OH 0
- H O W C O Y
OH OH OH OH
D-arabinose pyruvate KDO
Figure 14.1-22. Aldol addition reaction catalyzed in vivo by KDO aldolase.
I 947
-0pc A
0
+ O
+H
,
0
R
OH
OH
- KDO aldolase
-02C-OH
0 OH OH
R OH
- RHO 4c0
HO,,,.
OH
pyruvate R = OH, D-ribose R = OH, 57%
R = H, 2-deoxy-~-ribose R = H, 47%
-o,cJ--- + H b o H - KDO aldolase

- O
0
&
OH
W O H - - H 0 H q c 0 2 -
OH OH OH
11%
D-GIY
Figure 14.1-23. KDO aldolase-catalyzed synthesis of carbohydrates.
OP p?
.- .
I " KDO 8-P : I
I OH synthetase OH C
o-arabinose 5-P KDP 8-P

Go;
Figure 14.1-24. Aldol addition reaction catalyzed in uiuo by KDO 8-Psynthetase.
widely accepts trioses, tetroses, pentoses and hexoses as substrates['54].The best

substrates have (R)-configurationat C3, with the substituent at C2 having little effect.
Several aldol addition reactions have been conducted on a preparative scale,
including the synthesis of KDO itself, which was obtained in 67% yield (Fig. 14.1-
23). In each case, attack of the pyruvate took place on the re face of the carbonyl
group of the acceptor substrate. Excess pyruvate can be decomposed with pyruvate
decarboxylase to simplify the i~olationI'~~1.
3-Deoxy-~-manno-2-octulosonate 8-phosphate synthetase, also known as phospho-
2-keto-3-deoxyoctanoate(KDO 8-P) synthetase, catalyzes the irreversible aldol reac-
tion of PEP and D-arabinose 5-phosphate to give KDO 8-P (Fig. 14.1-24)[155]. The
enzyme has been isolated from E. coli B[15'1 and Pseudomonas aer~ginosa('~~1, and the
E. coli enzyme has been cloned and overexpressed in E. coli and Salmonella
typhirnuri~m['~~]. It has been used in the synthesis of KDO 8-P, using D-arabinose
5-phosphate generated either by hexokinase-catalyzed phosphorylation of arabi-
nose 11521, or an isomerase-catalyzed reaction of D-ribose 5-phosphate Studies
indicate KDO-8-P is very specific for its natural substrates, although some KDO
analogs may be accessible.
14.1.2.3
3-Deoxy-~-arabino-2-heptulosonicAcid 7-Phosphate (DAHP) Synthetase (E.C. 4.1.2.1 5)
In vivo, DAHP synthetase, also known as phospho-2-keto-3-deoxyheptanoate

synthe-
tase, catalyzes the synthesis of DAHP from PEP and D-erythrose 4-pho~phate['~~]].
DAHP is a key intermediate in the shikimate pathway for the biosynthesis of
0
948
OP
Ao2-*- Aco2-
ATP ADP PEP
3 OH 0 OH OH 0
D-fructose upo&OH 1 p o M C 0 2 -
OH OH OH OH
0-fructose 6-P D-erythrose 4-P DAHP
1. Hexokinase,2. Pyruvate kinase, 3. Transketolase + D-ribose 5-P, 4. DAHP synthetase
Figure 14.1-25. Multi-enzyme synthesis of DAHP.
aromatic amino acids in plants['60].The enzyme has been cloned['"] and used to
synthesize DAHP (Fig. 14.1-25) In this synthesis, D-erythrose4-phosphate was
generated in situ from Fru 6-P, catalyzed by transketolase in the presence of D-ribose
5-phosphate. Fru 6-P was generated from D-FWand ATP, catalyzed by hexokinase in
the presence of an ATP regeneration system. In general, it is more efficient and
economical to use whole cells containing a DAHP synthetase plasmid['"]. Such a
system also provides the necessary enzymes for the synthesis of DHAP. Recently,
DAHP synthase purified and overexpressed in E. coli has been characterized with
respect to substrate specificity, and catalyzes the condensation of PEP with ribose-
5-phosphate, deoxyribose-5-phoshate,and arabinose-5-phosphate This enzyme
has also been employed as a component of a biocatalytic process for large-scale
production of vanillin from glucose [1651.
14.1.2.4
2-Keto-4-hydroxyglutarate (KHC) Aldolase (E. C. 4.1.2.31)
In viuo, KHG aldolase catalyzes the reversible condensation of pyruvate and glyox-
ylate to form KHG (Fig. 14.1-26)W6* 1671. This enzyme participates in the terminal
step of mammalian catabolism of L-hydroxyproline The enzymes isolated and
purified from bovine liver and E. coli are both type I aldolases. Limited substrate
-0pc '
pyruvate
+
0
HKC02.
glyoxylate
-
KHG aldolase -
-02c
Other pyruvate analogs which are donor substrates for KHG aldolase
0
KHG
OH
-02cLR Hk Et02C
0
R = CH3, CHpCO;, Br, OH, SH, Ph, imidazole, PhOH, CO;
Figure 14.1-26. Aldol addition reaction catalyzed in vivo by KHC aldolase and
the donor substrate specificity of this enzyme.
I 949
specificity studies on KHG aldolase from bovine liver indicate that it accepts both
enantiomers of KHG equally well, and also cleaves 2-keto-3-deoxyglucarate, 2-keto-
4,5-dihydroxyvalerate, and oxaloacetate [1671. In the condensation direction, this
enzyme is relatively specific for glyoxylate, although it does accept other pyruvate
derivatives[l6'I. The enzyme from E. coli prefers the natural substrate [KHG with (S)-
configuration]and also cleaves 2-keto-4-hydroxybutyrate and oxaloacetate [1691. Using
the E. coli enzyme, both L- and D-4-hydroxy-2-ketoglutarate have been prepared on a
millimole scale[170].In the condensation reaction, glyoxylate can be replaced with
glyoxaldehyde, formaldehyde, acetaldehyde, and formic acid, while pyruvate can be
substituted by a-ketobutyrate and bromopyruvate.
14.1.2.5
2-Keto-3-deoxy-dphosphogluconate (KDPC) Aldolase (E. C. 4.1.2.14)
In vivo, KDPG aldolase catalyzes the reversible condensation of pyruvate with D-G~Y
3-P to form KDPG (Fig. 14.1-27).The equilibrium constant lies in favor of the aldol
addition (K- lo3 M - ~ ) .KDPG aldolase accepts a number of unnatural acceptor
aldehydes, although at rates much lower than the natural Various
sources of KDPG aldolase have been investigated as C - C bond forming catalysts in
organic synthesis [17*1, such as for the synthesis of non-carbohydrate components of
the nikkomycin natural products The related enzyme KDPGal aldolase has also
been utilized for similar purposes sp[1741. Unlike other aldolases, simple aliphatic
aldehydes are not KDPG aldolase substrates. However, other than the presence of
polar functionality at C2 or C3, there appears to be no other structural requirement
for the acceptor aldehyde. These studies also demonstrate that KDPG aldolase
stereospecifically generates the new stereocenter at C4 with (S)-configuration.
Furthermore, by using the technique of directed evolution, KDPG aldolase has been
altered with respect to its acceptor enantioselectivity and phosphate requirement to
accept non-phosphorylatedenantiomeric aldehydes [1751.
0 0 OH 0
PO
OH
~ + -
ACO;
KDPG aldolase
- PO--,J-%Oi
OH
D-GIY3-P pyruvate KDPG
Other acceptor substrates of KDPG aldolase
Acceptor VEI Acceptor Vrd

nitropropanal 200 erythrose 1.5
chloroacetaldehyde 120 glycoaldehyde 1.5
D-glyceraldehyde 100 benzaldehyde 0
D-lactaldehyde 27 butyraldehyde 0
ribose 5-P 5 ribose 0
Figure 14.1-27. Aldol addition reaction catalyzed in vivo by KDPC aldolase and the
acceptor substrate specificity of this enzyme.
950
14.1.2.6
(KDC) Aldolase (E. C. 4.1.2.20)
2-Keto-3-deoxy-~-glucarate
In vivo, KDG aldolase catalyzes the reversible reaction of pyruvate and tartronic acid
semialdehyde to form KDG (Fig. 14.1-28).This aldolase has been found in various
bacteria and the enzyme from E. coli has been isolated and purified['76]. KDG
aldolase accepts several other aldehyde acceptor substrates, including glycoaldehyde,
glyoxylate, and D- and L-glyceraldehyde. It has been used to synthesize 2-keto-
3-deoxy-~-gluconate on a preparative scale[1771.
14.1.3
2-Deoxyribose 5-phosphate Aldolase (DERA) (E.C. 4.1.2.4)
DERA[1781is unique among the aldolases, in that the donor of the aldol reaction is an
aldehyde, rather than a ketone. In vivo,the enzyme catalyzes the reversible condensa-
tion of acetaldehyde and ~ - G l y3-P to form D-2-deoxyribose5-phosphate, with an
equilibrium constant in the cleavage direction of 2 x M (Fig. 14.1-29).It is a type
I aldolase, and has been isolated from animal tissues [17q1 and microorganisms [180].
The E. coli gene encoding DERA has been sequencedIls1I, subcloned, and the
enzyme overexpressed in E. c01i[182-184].At 25 "C and pH 7.5, DERA is fairly stable
(70% activity retained after 10 days).
A number of unnatural substrates are accepted by DERA (Fig. 14.1-29), and it
generates (R)-configuredchiral centers. DERA from L. p l a n t a r ~ r n [accepts
~ ~ ~ 1 various
acceptor substrates including L-G~Y 3-P, D-erythrose 4-phosphate, glycoaldehyde
phosphate, D-ribose 5-phosphate, D,L-glyceraldehyde, D-erythrose,and D-threose [lS6].
Only propionaldehyde can weakly replace acetaldehyde as the donor. The E. coli
enzyme[lS2]accepts acetaldehyde, propionaldehyde,acetone, fluoroacetone, aliphatic
aldehydes, sugars, and sugar phosphates as acceptor substrates. However, the rates
of the aldol reactions are very slow (0.4-1 % CJ the natural substrates). More recently,
DERA has been used to obtain key intermediates in the synthesis of the epothilone
class of natural products['88].Several syntheses of azasugars conducted using DERA
are illustrated in Fig. 14.1-30.
When acetaldehyde is used as the donor, the products from the DERA-catalyzed
reaction are aldehydes, capable of being acceptor substrates for a second aldol
condensation (Fig. 14.1-31)[lS71. For example, when a-substituted acetaldehydes
were employed as substrates, products of the first aldol condensation could not
cyclize to a hemiacetal, and the products reacted with a second molecule of
acetaldehyde to form 2,4-dideoxyhexoses. These products could then cyclize to stable
-02cL + H&coi - KDG aldolase -

0
-02c+co2
OH
OH OH
PYrUVate tartronic acid KDG
semialdehyde
Figure 14.1-28. Aldol addition reaction catalyzed in vivo by KDC aldolase.
I
951
acceptor donor product
OH
R = H, F, CI, Br, OH, or CH,
1 OH
HO
R = CH,. CH,OH, or Ph
P
Hoa N3
OH OH
P
OH OH HoTsT-oH
OH OH
OH
I
9
Figure 14.1-29. Aldol
addition reaction cat-
alyzed in vivo by
DERA, and reactions
with unnatural sub-
strates.
952
Figure 14.1-30. Synthe-
/J ses of azasugars using
-
N3 : DERA.
donor OH P product Hz,Pd/C azasugar
OERA
9 N3v0H OH
OH
P " ' POH O

CH3 H OH
A OH
HO
DERA OH 0 DERA OH 0
I'
R R R
I]
R = CH3, MeOCH,.
MOMOCH,, CICHz.
N3CHn
CH,CH,COOH,
'm ~ Br,/HZO
BaCO,
72%
t
'Tor
-
OH
CH,0HCH20P OH OH
Figure 14.1-31. Sequential aldol reactions catalyzed by DERA.
hemiacetals, thus stopping the polymerization after two sequential aldol reactions.
Conversion to chiral lactone derivatives of mevinic acids, which are active as
cholesterol-loweringagents, could then be accomplished. The best substrate for the
DERA-catalyzed sequential reaction appeared to be succinic semialdehyde (R =
CH2CH2COOH) in which the carboxylic acid mimics the Gly 3-P phosphate
group [1841.
One-pot sequential aldol reactions were performed by combining DERA with FDP
Figure 14.1-32. One-pot

I 953
aldol reaction employing

OH RAMA and DERA.
HO OH
R = MeOCH,,
MOMOCH,, HO R&
CICH,, N&H, OH OH
R
87- DERA OH 0 DERA
1\1111
OH OH 0 Figure14.1-33. Tandem
use of DERA and NeuAc
P R
P R aldolase.
R = CH, MeOCH,,
MOMOCH,, CICH,.
NsCHz,
CHZCHZCOOH,
Rr
R,oroH ~ Br2/H,O
BaCO,
72%
v
CHzOHCH2OP OH OH
aldolase (Fig. 14.1-32)[18’), ”)‘I . The products of these reactions are 5-deoxy ketoses
with three substituents in axial positions. Owing to the formation ofthese thermody-
namically unfavored products at long reaction times, some inversion of the usual
stereochemistry of both DERA and FDP aldolase was observed. Combination of
DERA and NeuAc-aldolase catalysis gave sialic acid derivatives (Fig. 14.1-33)[1891. In
this case, however, one-pot synthesis was not possible, due to the incompatibility of
the reaction conditions for the two aldolases.
Glycine-dependentAldolases
The glycine-dependent aldolases, including serine hydroxymethyltransferases
(SHMT) and threonine aldolases, are pyridoxal 5-phosphate-dependent enzymes
which catalyze the reversible aldol reaction of glycine with an aldehyde acceptor to
form a p-hydroxy-a-amino In vivo SHMT (EC 2.1.2.1) catalyzes the con-
densation of glycine and formaldehyde to give L-serine, and requires the cofactor
tetrahydr~folate[”’~].SHMT has been used for the resolution of racemic erythro p-
hydroxy a-amino acids, the large-scale synthesis of ~-serineI’”>1931, and the produc-
tion of 2-amino-3-hydroxy-l,6-hexanedicarboxylic acid [1941. Although SHMT is se-
lective for the L-configuration at the a-center, it generally displays poor erythro-threo
discrimination, resulting in product mixtures [195,1961.
Threonine aldolases catalyze the reversible aldol reaction between glycine and
acetaldehyde to give threonine (Fig. 14.1-34),and both D- and L-Thr aldolases have
been reported. The substrates for the L-threonine aldolases (E.C. 4.1.2.5) are also
substrates for L-SHMT (vide supra). Many threonine aldolases also accept allo-
C-CBonds
I
poH- -
954 74 Formation of
,P
L-threonine OH 0 OH 0
aldolase
+ R
NHp NHz
erythro threo
R Yield (%) Ratio (elythro:threo)
CH, - 38 93 : 7
Ph - 87 60 : 40
N&Hz - 45-75 70 : 30 to 100 : 0
BnOCH, - 78 92 : 8
BnOACH2 - 53 53 : 47
BnO-0,
CHp - 45 92 : 8
PhS-CHZ- 80 50 : 50
Figure 14.1-34. Reaction catalyzed in vivo by L-Thr aldolase, and unnatural

substrates.
threonine derivatives as substrates, sometimes preferably over compounds with the

threo configuration "'l.
Threonine aldolases have been used extensively for the resolution of racemic p-
hydroxy a-amino acids. For example, with a L-threonine aldolase isolated from
Streptornyces arnakusaensis, several racemic mixtures of 3-@-substituted-phenyl-
)serines were resolved to give the enantiomers with the D-threo stereochemistry in
>95 % ee['99*2001 . Recently, both D-[~''] and L-Thr aldolases ['01* 2021 have been used in
the preparation of novel P-hydroxy-a-aminoacids. In addition, D-threonine aldolase
has been utilized to prepare a small molecule that acts as a gelator of organic
solvents [2031. L-Threonine aldolase has been employed in the synthesis of fragments
of the mycestericin class of natural as well as peptidic RNA mim-
e t i c ~ [ ~L-Threonine
~~]. aldolase (E. C. 4.1.2.5) from Candida hurnicola has been
and has been investigated for use in condensation reactions['"]. The
enzyme accepted a broad range of aldehydes, but in general mixtures of L-erythro and
L-threo products were obtained, with the L-erythro configuration being the preferred
one (Fig. 14.1-34).
When hydroxyaldehydes are employed as L-Thr aldolase substrates, complex
product mixtures result. Protection of the hydroxyl groups prevents this, and allowes
the preparation of CCprotected L-threonineand L-allothreoninederivatives.Acceptor
aldehydes with an oxygen functionality at the a-position gave high erythro/threo
I 955
&
‘OH
OH .!.
Figure 14.1-35. Use of L-Thr aldolase in the preparation of sLe” mimetics.
ratios, a ratio which was reduced when the oxygen was in the S-position.Although
a,S-unsaturated aldehydes did not serve as substrates, several thiophenol derived
aldehydes were accepted, providing a route toward unsaturated amino acids. One L-
Thr aldolase product, the 4-hydroxy-~-allothreonine derivative,has been used as a key
intermediate in the synthesis of potent sialyl Le” mimetics (Fig. 14.1-35)C2O71.
Other known aldolases whose substrate specificity remains to be examined are

summarized in Table 14.1-2.
Catalytic Antibodies
In recent years, catalytic antibody technology has provided methods for developing
new protein catalysts[208]. Monoclonal antibodies (mAbs)elicited against “transition-
state” haptens catalyze reactions with remarkable rate accelerations. By appropriate
antigen design, functional groups that perform general acid/base catalysis, nucleo-
philic/electrophilic catalysis, and catalysis by strain or proximity effects can be
induced into the binding site of an antibody. Even reactions which are unfavorable or
otherwise unattainable have been achieved using the catalyhc antibody approach.
Aldolase catalytic antibodies developed recently have the ability to match the
efficiency of the natural aldolases while accepting a more diverse range of sub-
strates.
Initial catalytic antibodies were developed to bind a primary amine cofactor as a
mimic of the type I aldolases. The hapten designed mimicked the transition state the
iminium ion, resulting in the production of an antibody that catalyzed the aldol
condensation of acetone and aldehyde acceptors (Fig. 14.1-36) [2091. Even though no
stereochemical information was built into the transition-state mimic, the antibody
catalyzed stereoselective addition to the si face of the aldehyde.
The subsequent development phase, namely reactive immunization L2l0], involved
OH 0 OH 0
acetone,
AcHN AcHN
pH 9.0
> 95% de 65% de
1 : 2.8
Ar
Figure 14.1-36. Aldol reaction catalyzed by catalytic antibody 72D4, and a

transition-state hapten.
956
Table 14.1-2. Other aldolases and the reactions they catalyze in uiuo.
0 ketotetmse pimphate
P O L O H
&
O
'H
" OH
+
aldolase (EC 4.1 2.29) [a]

DHAP
0
phospho-5-keto-Zdeoxy-
PO
&
JO
i.z
+
'
HIccoz. gluconate aldolase (EC 4.1.2.29) [b]
OH
'bop
0 OH
[i
2-keto-3.deoxy-S-phospho-
'02C
pyruvate
+ OH
galactonate aldolase (EC 4.1.2.21) -02c+oP
6H
4hydroxy-2-keto4rnethyl
+
'OZC glutarate aldolase (EC 4.1.3.17) [c]
+ H K O H - 2-keto-3deoxy-c-pentanoate
aldolase (EC 4.1.2.28) [c]
2-keto9deoxy-~-pentanoate 0 OH
+ Hj C O H '0,C &OH
aldolase (EC 4.1.2.18) [d]
-0zc
0 OH OH
Nacetylneurarninate(NeuAc)
synthetase (EC 4.1.3.19) -0zc
WOH
PEP ACHN OH
serine hydmxymelhyl
-0,c R O H
transferase (EC 2.1.2.1) [el
OH
dlhydroneopledn
HoT)$lNH2
+ HKoH aldolase (EC 4.1.2.25) [fj HoG:;7Jl NHZ
PO K + H b
OH
O p + HzO
phosphoketolase
(EC 4.12.9) [g]
H O 0 A OHO P + Pi
0 OH OH OH
+ H
+H20 fructose-6-phosphate - + Pi
phosphoketolase(EC 4.1.2.22) [h] 0 OH
OH
OH
@ pH
17a-hydmmrwestemne -
+
aldolase (EC 4.1 2.30)[i]

-0 0
0 ketopantoaldolase 0
-ozc+ + (EC 4.1.2.12) 1 .OzC?OH
a Isolated from rat liver, see: F. C. Charalampous, b W. A. Andeson, B. Magasanik, J. Bid. Chem. 1971,
Methods Enzymol. 1962, 5, 283. Acetaldehyde, glycoal- 246, 5662.
dehyde or glyceraldehydecannot replace formalde- c W. A. Wood in: The Enzymes (Ed.: P. D. Boyer),Aca-
hyde. demic Press, New York, 1970;Vol. VII, p. 281.
d This enzyme also catalyzes the aldol addition of
with acetaldehyde to give L-allothreonine.originally

I
957
pyruvate with formaldehydeto give 4-hydroxy-2-ox- thought to be catalyzed by I-allothreonine aldolase

obutyrate, originally thought to be catalyzed by hy- (E.C. 4.1.2.6).
droxyoxobutyrate aldolase (E. C. 4.2.1.1). Phenylpyru- f I. B. Mathis, G. M. Brown,]. Bid. Chem. 1970,245,
vate is also a donor substrate, while acetaldehyde, 3015. The reaction requires thiamine pyrophosphate
benzaldehyde and crotonaldehydeare not acceptor and favors cleavage.
substrates, see: H. Hift, H. R. Mahler,]. B i d . Chem. g E. C. Heath, J. Hunvitz, B. L. Horecker, A. Ginsberg,
1952,198,901. /. B i d . Chem. 1958,231, 1009. The reaction favors the
e L. Schirch, Adv. Enzymol. 1982,53,83. A multicopy cleavage of ~-xylulose-5-phosphate.The enzyme from
plasmid containing the E. coli serine hydroxymethyl Leuconostoc msenteroides also accepts fructose-6-phos-
transferase was introduced to Klebsiella aerogenes for phate, hydroxypyruvate and glycoaldehyde as sub-
overexpression of the enzyme. The enzyme requires strates.
tetrahydrofolate (THF) and pyridoxal phosphate. THF h E. Racker, Methods Enzymol. 1992,5,276. The reac-
first reacts nonenzymaticallywith formaldehydeto tion favors degradation.
form NS,NlO-methyleneTHF which is then accepted i D. E. lohnston, Y:B. Chiao, 1. S. Gavaler, D. H. Van
by the enzyme to form serine, see: B. K. Hamilton, Thiel, Biochem. Pharm. 1981,30, 1827.
H. Y. Hsiao, W. E. Swanm, D. M. Anderson, J. Dele- j W. K. Maas, H. J. Vogel,]. Bacterial. 1953,65,388;
nte,]. Trends Biotechnology, 1985, 3,64. This enzyme E. N. McIntosh, M. Purko, W. A. Wood,]. B i d . Chem.
also catalyzes the reversible aldol reaction of glycine 1957,228,499.
raising antibodies against a 0-diketone “chemical trap” to imprint the lysine-

dependent type I aldolase mechanism in the active site (Fig. 14.1-37)[211!The E-
amino group of a lysine side chain reacts with the 0-diketoneto give a 0-ketoimine,
which tautomerizes to the stable vinylogous amide. By using this method, two
catalyix antibodies with aldolase selectivity, 38C2 and 33F12, were identified and
subsequently shown to have remarkable scope[212]. The structure of 33F12 has been
determined and shown to have the Schiff base forming Lys residue buried in a
hydrophobic pocket at the base of the binding site[211].
Unlike natural aldolases, catalytic antibodies accept a wide range of ketone donor
substrates (Fig. 14.1-38A). Small aliphatic ketones are well tolerated, but mixtures of
products result with unsymmetrical ketones, due to reaction at both a-positions. a-
Heteroatom-substituted ketones show much higher levels of regioselectivity, with
reaction occurring almost exclusively at the carbon atom bearing the heteroatom.
Interestingly, the regiochemistry of the reaction of fluoroacetone is opposite to that
observed with the natural aldolase DERA, thus providing a complementary ap-
proach.
A wide variety of aldehydes serve as acceptors (Fig. 14.1-38B),including those that
Figure 14.1-37. Reactive immunization strategy. H

A.
958
A it 1, FJ ,),OeM
0
OH, OMe I
R = NHAc, NO2
AH AcHN
J H
n = 3, 4, 5
Figure 14.1-38. A, Catalytic antibody ketone donor substrates. B, Catalytic antibody aldehyde
acceptor substrates.
resemble the hapten, and simple aliphatic aldehydes. Polyhydroxylated aldehydes,

such as glyceraldehyde, glucose, and ribose, are not substrates, most likely because
of the hydrophobic nature of the active site. In contrast to the natural aldolases,
aromatic and a$-unsaturated aldehydes are excellent substrates.
The stereochemistry of the addition is donor dependent. When acetone is used as
the donor substrate, addition occurs from the si face of the carbonyl group; with
hydroxyacetone, addition occurs from the re face. The stereoselectivity is generally
quite high, with ee values greater than 99 % commonly observed. As a general rule,
high enantioselectivity is observed with acceptors having an sp2 center in the a-
position, and lower enantioselectivitiesare observed for a-position sp3 centers.
The utility of catalytic antibodies was demonstrated with the antibody-catalyzed
aldolase approach to the brevicominssp[213]and the epothilones l2l4l (Fig. 14.1-39).
Antibody 38C2 is commercially available and has recently been used as a catalyst to
activate prodrugs i2l5].Generic, drug-masking groups can be selectively removed by
sequential retro-aldol and retro-Michael reactions catalyzed by 38C2 (Fig. 14.1-40).
The antibody was also used in the enantioselective retro-aldol reaction of tertiary
aldols containing heteroatom-substituted quaternary carbon centers F21G]. This gave
enantiomerically enriched tertiary aldols, most with ee values greater than 95 %.
Synthesis of enantiomerically pure tertiary aldols using the catalytic asymmetric
aldol reaction with ketone acceptors represents a significant challenge. Compounds
prepared in this study have been used in the synthesis of (+)-frontalin,the side chain
of saframycin H, and mevalonolactone.
In order to increase the repertoire and efficiency of the aldol reaction further, and
$ I
74. I Aldol Reactions 959
OH 40%,96%ee
Ar Ar
(*)
+;flcHo
51%, 75%ee
Figure 14.1-39. Use of catalytic antibody 38C2 for the preparation o f epothilone intermediates.
0 OH 0
O
*H / / "'OH 3*c2
*
Me0 0 OH 0 retro-
aldol Me0 0 OH 0
pro-drug drug
Figure 14.1-40. Retro-aldol reaction catalyzed by Ab 38C2 for the unmasking o f pro-drugs.
to develop antibodies with complementary enantioselectivity, a P-diketone sulfone

' ~ I 14.1-41).
was employed as the h a ~ t e n [ ~ (Fig. The tetrahedral geometry of the
sulfone moeity in this hapten mimics the rate-determining tetrahedral transition
state of the C-C bond forming reaction. It is thus expected to facilitate nucleophilic
attack of the enaminone intermediate on the acceptor aldehyde. It was indeed
demonstrated that catalytic antibodies with broad reaction scope can be generated
using this approach. In addition, antibody 93F3 was more efficient (k,,, - 3min-l)
than and enantiocomplementary to 38C2, providing the unreacted (S)-aldolwith
>96% ee.
The mechanism-based approach to eliciting catalytic antibodies combined with
the rapid, immune-selection process as illustrated in these studies provides a new
and exciting direction for catalyst design and development.
960
5-
antibody * +[>7&N-Ab
R
R' 6'
transition state
R o s 2 + H~N/\/\/~~
transition-state
analog
H
reactive immunization
hapten, R = - O 2 C V N
0 1
-
CH3
Figure 14.1-41. fi-Diketone sulfone as hapten for reactive immunization.
14.2
Ketol and Aldol Transfer Reactions
14.2.1
Transketolase (TK)(E.C. 2.2.1 .l)
TK is one of the enzymes involved in the oxidative pentose phosphate pathway, and
requires the cofactors thiamine pyrophosphate (TPP)[21'1and Mg2+[2181.It reversibly
transfers the C1-C2 ketol unit from D-xylulose 5-phosphate to D-ribose 5-phosphate,
and generates D-sedoheptulose7-phosphate and D-GIY3-P. D-Erythrose 4-phosphate
also functions as an acceptor of the ketol unit from D-xylulose 5-phosphate, to
produce Fru 6-P and ~ - G l y
3-P (Fig. 14.2-1).TK from baker's yeast is commercially
available, and the enzyme can also be isolated from spinach[220.221]. TK from E. coli
has been overexpressed and prepared on a large scale[222].
In ketol transfer reactions,
OH 0 0 OH OH OH
$ , ) , OP + H*op - TK - P O A H +Ho>\+op
OH OH OH 0 0 OH OH
D-XylUlOSe 5-P D-ribose 5-P D-GIY3-P D-sedoheptulose 7-P
OH 0 0 OH OH OH OH
H
o, - ,k- .O
P
+ + H+oP - TK - PO+H + Ho++.OP
OH OH 0 0 OH
D-xylulose 5-P D-erythrose 4-P D-GIY3-P D-Fru 6-P
Figure 14.2-1. Ketol transfer reactions in the oxidative pentose phosphate pathway
catalyzed by TK.
0
14.2 Ketol and Aldol Transfer Reactions
I
961
+ HL O H TK * H O L o H
HOJLoz
HPA OH
R = CHZOH, CH3, N3, CHzCH3
0 OH OH 0 OH OH
H+OH TK + HOO
*H .
.
.
.
OH OH OH OH OH
0 OH 0 OH
OH OH OH
0 OH
TK
H& -* H -.
O -
. L\
OH OH OH
OH 0
TK * HOQH
OH 0 OH OBn
Figure 14.2-2. Acceptor substrate specificity o f TK.
the enzyme isolated from yeast shows a higher diastereoselectivity(- 100%)[221] than
that from spinach (- 95 %), with the newly-formed hydroxymethine chiral center
always possessing an (S)-configuration. TK also accepts p-hydroxypyruvic acid
(HPA) as a ketol donorlzz3],and an efficient multi-enzyme synthesis of D-xylulose
5-phosphateemploying FDP aldolase and E. coli transketolase has been reported[224].
The ketol unit is transferred to an aldose acceptor with an activity of 4 % compared
with D-xylulose 5-pho~phate[~~']. This has been an invaluable discovery for the use of
TK in synthesis, as the decarboxylation of HPA and subsequent loss of carbon
dioxide, render the overall condensation reaction irreversible. A wide range of
aldehydes are ketol acceptors, including aliphatic, a,P-unsaturated, aromatic, and
heterocyclic aldehydes, although some are relatively poor substrates (Fig. 14.2-
2)[225, 2261. The presence of a hydroxyl or an oxygen atom at C2 and/or C3 has a
positive effect on the rate, while steric hindrance near the aldehyde exerts a negative
effect. P-D-Hydroxy aldehydes (and not L-) are substrates, producing vicinal diol
products of D-threo configurationLzz5, 2271. This allows efficient resolution of alde-
hydes epimeric at C2 by transketolase. The enzyme appears to have no preference for
configuration beyond C2.
962
0 OH
OH OH OH OH OH
P O 4 H +H o ~ ~ TAo
======= H o p~ , ; r / O P + H+OP
0 0 OH OH 0 OH OH
D-GIY3-P D-sedoheptulose7-P D-Fru 6-P D-erythrose4-P
Figure 14.2-3. Aldol transfer reaction in the oxidative pentose phosphate pathway
catalyzed by TA.
7~7;
phosphoglucose
Starch phosphorylasea D-Glc ,-p phosphoglucomutase D-Glc 6-p isomerase
- D-Fru 6-P
TA
D-Fru
__j__j______ u
D-G~Y D-GIY3-P
phosphate - ~~~
3-phosphoglycerate
phosphatase
Figure 14.2-4. Multi-enzyme synthesis of D-Fru from starch.
TK has been used to catalyze the key step in the synthesis of the naturally
occurring beetle pheromone (+)-exo-brevicomin[228] and the azasugar 1,4-dideoxy-
1,4-imino-~-arabinitol [391. Both syntheses involve the condensation of H PA with
racemic 2-hydroxyaldehydes,whereby the ketol unit is diasteroselectivelytransferred
to only the D-enantiomer of the aldehyde. In addition, transketolase has been
employed in the synthesis of complex heptuloses [2291, fructose analogs [2301, and
other sugars [231j. Erythrulose has been continuously produced through trans-
ketolase-catalysisin a membrane reactor [2321.
14.2.2
Transaldolase (TA) (E.C. 2.2.1.2)
TA is also an enzyme ofthe oxidative pentose phosphate It catalyzes the

transfer of the C1-C3 aldol unit from D-sedoheptulose7-phosphate to ~ - G l 3-P,
y and
produces D-FI-U6-P and D-erythrose 4-phosphate (Fig. 14.2-3).TA forms a Schiff base
intermediate and does not require any co-factors. This enzyme is commercially
available, and was used in a multi-enzyme synthesis of D - F ~ ufrom starch (Fig. 14.2-
4)[2331.Here, it accomplished transfer of an aldol moiety from Fru 6-P to D-
y and D - F ~ .
glyceraldehyde,and formed ~ - G l 3-P
14.3
Acyloin Condensation
Acyloin condensation catalyzed by yeast was first observed in the early part of the
twentieth century[234,2351 . Yeast-catalyzed acyloin condensations between acetalde-
hyde and benzaldehyde derivatives have since been reported, giving products with a
(R)-configurationin all cases (Fig. 14.3-1)[ 2 3 G , 2371. The acyloin formed from benzal-
dehyde alone has been used in the industrial manufacture of It is
$fi
74.4 C-CBond Forming Reactions lnvolving AcetylCoA
0
I 963
FyH
7-
R2
JH +
acetaldehyde
AcyloinYeast
condensation-
R2
R’
benzaldehyde
derivative
Products obtained from yeast-catalyzed acyloin condensation
CI OH
Me0 Me0 HO
Figure 14.3-1. Acyloin condensation between acetaldehyde and benzaldehyde
derivatives catalyzed by yeast.
probably the enzyme a-carboxylase (E. C. 4.1.1.1)that is responsible for catalyzing

the acyloin reactions, as the carboxylase-catalyzed reaction of pyruvate and benzalde-
hyde in the presence of the cofactor thiamine pyrophosphate gives the correspond-
ing acyloin product [2391. Pyruvate decarboxylase in highly purified [2401 or partially
purified catalyzes acyloin condensation to give products of the ( R ) -
configuration.
14.4
C-C Bond Forming Reactions InvolvingAcetylCoA
Enzymatic reactions which utilize coenzyme A thioesters as substrates are involved

in the biosynthesis of steroids, terpenoids, macrolides, fatty acids, and other natural
products. Owing to the high cost of CoA, these enzymes can only be practically used
in organic synthesis if the CoA thioesters can be recycled. AcetylCoA can be
efficiently regenerated by using one of several enzymatic systems [242-2441 . Phospho-
transacetylase (E. C. 2.3.1.8)/acetylphosphate, carnitine acetyltransferase
(E. C. 2.3.1.7)/acetylcarnitine,and acetylCoA synthetase (E. C. 6.2.1.1)/ATPhave all
been employed for this purpose. These enzymatic recycling systems have been
coupled to the synthesis of citric acid catalyzed by citrate synthetase. An interesting
non-enzymatic regeneration of acetylCoA utilizes phase transfer catalysts in a two-
phase aqueous-organic system (Fig. 14.4-1) L2451. Citric acid was efficiently prepared
using this procedure, and this method also offers the potential to prepare many
different acylCoA derivatives for use as substrates of CoA-dependent enzymes.
AcetylCoA is also involved in the biosynthesis of poly-p-hydroxybutyrate(Fig. 14.4-
2, x = 0). Many whole cell systems have been used to synthesize this polymer and
other interesting materials in this class [2461. For example, copolymers consisting of
(R)-3-hydroxybutyland (R)-3-hydroxyvalerylunits (Fig. 14.4-2, x = 0 and 1, respec-
tively) were prepared by feeding propionate to whole cells of A. e ~ t r o p h u s [ ~ ~ ~ ] .
964
aqueousphase organic phase
acetylCoA
H02C
i.
Figure 14.4-1. Chemical regeneration of acetylCoA using a phase transfer catalyst.
0 0 0 OH 0
thiolase reductase synthetase
RASCOA acetylCoA- RuSCoA - RuSCoA
acylCoA P-ketoacylCoA p-hydroxyacylCoA poly-p-hydroxyester

R = CHS(CH~), n = 500-15,000
X = 0-7
Figure 14.4-2. Enzyme-catalyzed reactions involved in the whole-cell synthesis o f

poly-p-hydroxyesters.
AcetoacetylCoA thiolase (E. C. 2.3.1.9),acetoacetylCoA reductase (E. C. 1.1.1.36),and

polyhydroxybutyrate ~ y n t h e t a s e I ~
are
~ ~the
1 enzymes involved in polyester synthesis.
AcetoacetylCoA thiolase catalyzes the head-to-tail Claisen condensation of two
acetylCoA molecules. In this reaction, the active site cysteine attacks acetylCoA to
form a thioester enzyme intermediate, which then reacts with the enolate derived
from enzymatic deprotonation of the other acetylCoA. Mechanistic studies have
been performed on this enzyme from Zooglea ramigera, which has been cloned and
overexpres~ed[~~~I. It has been established that the thiolase will form acyl enzyme
intermediates with a number of acylCoA substrates, but will only accept acetylCoA as
the nucleophile. After subsequent reduction, this results in all polymer units
possessing a fi-hydroxy group. These polymers are also useful sources of (R)-fi-
hydroxy acids [2481.
14.5
14.5 lsoprenoid and Steroid Synthesis
I
965
lsoprenoid and Steroid Synthesis
Enzymes involved in the biosynthesis of isoprenoids and steroids have been used in
organic synthesis [2491. 2,3-Oxidosqualenelanosterol cyclase was used to synthesize a
number of lanosterol analogs (Fig. 14.5-1)[2sG2531. When using an enzyme suspen-
sion from baker’s yeast containing this cyclase, ultrasonic irradiation proved very
effective in promoting catalysisr2”, 2521 . An interesting
. property of lanosterol cyclase
was utilized during the synthesis of C30 functionalized lanosterols, whereby the
enzyme rearranged a vinyl group rather than the usual hydrogen or methyl
group L2”1. This product was subsequently converted into (+)-3O-hydroxylanosterol
and the corresponding aldehyde. These compounds are natural receptor-mediated
feedback inhibitors of HMG-CoA reductase, and therefore are of interest in the
design of hypocholesteremic drugs [2s41.
Both enantiomers of 4-methyldihomofarnesol were synthesized using farnesyl
diphosphate synthetase from pig liver, the (S)-enantiomer being a precursor of
juvenile hormone (Fig. 14.5-2)[2s51. Alkyl group homologs of isopentenyl diph-
osphate have also been examined as substrates for farnesyl diphosphate syn-
thase [2sGl.
&R3 \ ? ~ ~ ~ ~ ~ baker’s
(from $ ‘ , ” , ” , ” _ yeast) \ .
R2 I HO %
...
O R’ R’ R2 R3 R’ R~ lanosterol analogs
H H CH3
H H CO2CH3
OH H CH3
H OH CH,
Figure 14.5-1. Synthesis of lanosterol analogues using 2,3-oxidosqualene lanosterol cyclase.
uopp farnesyl diphosphate

(Sj-4-methyldihomofarnesol juvenile hormone
qthetase
(R)-4-rnethyldihomofarnesol
Figure 14.5-2. Synthesis o f both enantiomers o f 4-methyldihomofarnesol using

farnesyl diphosphate synthetase.
966
tryptophan RH = PhCHZSH, CH3(CH&SH,

YH2 synthase, RH YH2
c 1 4 c o g * R -0ZCCHzSH
*coz
0-chloroalanine 0-substituted
a-amino acids H
tyrosine phenol
c'4co,
YH2 base.
~. RH
- R*C-,-
__
NHZ
RH =
0-substituted
0-chloroalanine R'
a-amino acids R' =OH, CI, alkyl
Figure 14.6-1. Synthesis o f fi-substituted a-amino acids from fi-chloroalanine
using tryptophan synthase and tyrosine phenol lyase.
14.6
6-Replacement o f Chloroalanine
Methods have been developed for the synthesis of unnatural amino acids using
pyridoxal phosphate-dependent enzymes 12571. These enzymes usually catalyze trans-
aminations, a$-eliminations, a,y-eliminations, and decarboxylations of amino ac-
ids. However, using fbchloroalanine as a substrate, unusual amino acids are
produced by P-replacement. Tryptophan synthase (E. C. 4.2.1.20) from E. coli cata-
lyzes the formation of tryptophan and analogs. This enzyme has been employed to
incorporate various heteroatoms into tryptophan, such as selenium [258J, ~ulfurI~~'1,
chloride[261],and Notably, tryptophan synthase could
be used to catalyze exchange of the a-proton from Asn, Glu, Ser, Ala, Phe, and Met as
well as that of Trp[262].Tyrosine phenol lyase (E.C. 4.1.99.2) (Fig. 14.61)has been
utilized to synthesize tyrosine, DOPA, and rneth~latedL~~~1, fluorinated[264],and
azido-tyrosineanalogs [2651.
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S. Thompson, J. T. Davis, S. F. Williams, 257 T. Nagasawa, H. Yamada, Appl. Biochem.
0. P. Peoples, A. J. Sinskey, C. T. Walsh, J. Biotech. 1986, 13, 147.
Am. Chem. Soc. 1989, 111, 1879. 258 M. Welch, R. S. Phillips, Bioorg. Med. Chem.
248 a) D. Seebach, M. F. Zuger, Tetrahedron Lett. Lett. 1999, 9, 637.
1984,25,2747; b) D. Seebach, M. F. Zuger, 259 R. S. Phillips, L. A. Cohen, U.Annby,
Helv. Chirn. Acta 1982, 65,495. D. Wensbo, S. Gronowitz, Bioorg. Med.
249 K. S. Kyler, M. J. Novak, NATO A S 1 Ser., Ser. Chem. Lett. 1995, 5, 1133.
C 1992, 381, 3. 260 M. J. Sloan, R. S. Phillips, Bioorg. Med.
250 E. E. van Tamelen, E. C. Leopold, S. A. Mar- Chem. Lett. 1992, 2, 1053.
son, H. R. Waespe,]. Am. Chem. SOC.1982, 261 M. Lee, R. S. Phillips, Bioorg. Med. Chem.
104,6479. Lett. 1992, 2, 1563.
251 a) J. Bujons, R. Guajardo, K. S. Kyler,]. Am. 262 J. J. Milne, J. P. G. Malthouse, Biochem. SOC.
Chem. Soc. 1988, 110,604; b) J. C. Medina, Trans. 1996, 24, 133s.
K. S. Kyler, J . Am.Chem. Soc. 1988, 110, 263 K. Kim, P. A. Cole, Bioorg. Med. Chem. Lett.
4818. 1999, 9, 1205.
252 J. C. Medina, R. Guarjardo, K. S. Kyler,]. 264 R. S. Phillips, R. L. Von Tersch, J. G.
Am. Chem. Soc. 1989, 111, 2310. Fletcher, A. H. Lai, Amino Acids: Chem. Biol.
253 B. J. Robustell, I. Abe, G. D. Prestwich, Et- Med. 1990, 166.
rahedron Lett. 1998, 39,9385. 265 D. Hebel, D. C. Furlano, R. S. Phillips,
254 S. M. Grundy, Nav Eng.]. Med. 1988,319, S. Koushik, C. R. Creverling, K. L. Kirk, Bio-
24 and references therein. org. Med. Chem. Lett. 1992, 2, 41.
14.7
Enzymatic Synthesis of Cyanohydrins
Martin H . Fechter and Hefried Criengl
In the last decade, optically pure cyanohydrins (a-hydroxynitriles) have become a

versatile source for the synthesis of a variety of chiral building blocks. Diverse
methods for the enantioselective synthesis of cyanohydrins have been published and
reviewed Ill. Besides enzyme catalyzed methods, hydrocyanation or silylcyanation of
aldehydes or ketones controlled by chiral metal complexes or cyclic dipeptides, as
well as diastereoselective hydrocyanation of chiral carbonyl compounds, have been
applied with moderate success.
However, the most advantageous preparations of optically active cyanohydrins,
with respect to the obtained enantioselectivities, are the enzymatically controlled
approaches discussed in the present chapter. Two common enzyme systems are
described and reviewed['-16]: firstly, esterases or lipases, which have been employed
14.7 Enzymatic Synthesis ofcyanohydrins
Figure 14.7-1. Cyanohydrin forrna-

I 975
R’\ A PH tion: R’ = alkyl, cycloalkyl, aryl,

C=O + H C N
R’/f\CN heteroaryl; RZ = H, alkyl.
R2’ R
for the resolution of racemic cyanohydrins or alkoxynitriles, and secondly, oxy-

nitrilases - also known as hydroxynitrile lyases (HNLs),which catalyze the reversible
formation of cyanohydrins (Fig. 14.7-1),using HCN and aldehydes or ketones.
About 3000 plant species are known to release HCN from their tissues, a process
which is known as cyanogenesis[”. “1. Storage compounds are cyanohydrins where
the hydroxy function is glycosylated to a carbohydrate or protected as a fatty acid
ester. The plant defence mechanism in the case of sugar compounds is a two-step
reaction. Initially a glycosidase liberates the cyanohydrin moiety, which is cleaved
either spontaneously by base catalysis or enzymatically by the action of oxynitrilases
to release the corresponding carbonyl compound and HCN [I9].
The application of an HNL was the subject of one of the earliest reports in the field
of biocatalysis, namely the synthesis of mandelonitrile from benzaldehyde and
hydrocyanic acid using a crude enzyme preparation obtained from almonds (termed
“emulsin”)f2O1. However, little attention was paid to this d i s c o ~ e r y [ ~until
~ - ~ ~the
I
1960s, when this enzyme (E. C. 4.1.2.10) was isolated, characterized12”261,and used
for the preparation of enantiomerically enriched (R)-cyanohydrinsfrom aromatic
and aliphatic aldehydes [27-291. The first examination of an (S)-oxynitrilasein millet
seedlings (Sorghum bicolor, E. C. 4.1.2.11) [3s331 revealed that this enzyme only
accepts aromatic substrates. At this time, the best enantiomeric excess obtained was
87% for the formation of (R)-mandelonitrile; other aldehydes gave even lower
enantiomeric ratios.
14.7.1
The Oxynitrilases Commonly Used for PreparativeApplication
At present, the oxynitrilases from eleven cyanogenic plants (from six plant families)
have been purified and characterisedL9. lo1.Theproperties of a selection of these are
outlined in Table 14.7-1.The oxynitrilases E. C. 4.1.2.10 from Rosaceae (e.g. Prunus
sp.) contain the cofactor FAD. However, the latter is not involved in redox reactions.
Instead, it seems to have a structure-stabilizing effect, and its presence might be
explained on evolutionary grounds [34-3G1. Some of these enzymes are glycosylated
and most of them are constructed from several
Recently the crystal structure of the oxynitrilase from Hevea brasiliensis (E.C.
4.1.2.39) was r e p ~ r t e d [ ~ ’ -The
~ ~ ]enzyme
. was found to contain a large j3-sheet which
is surrounded by a-helices and a cap region on both sides. The active site is deeply
buried inside the protein and connected to the surface by a narrow channel. Similar
discoveries were published very recently for the (S)-HNLfrom Manihot esculenta
(E.C. 4.1.2.39)[42.431. A big step forward, toward further applications of the Prunus
amygdalus HNL, was achieved by the Kratky group by elucidating the crystal
structure of this
976 I 14 Formation ofC-C Bonds
Table 14.7-1. Oxynitrilases available for organic synthesis.

Plant Enzyme Natural substrate Substrate acceptance Stereo-
availability for syntheses selectivity
Pnrnus amygdalus Almonds (R)-Mandelonitrile All R’ and R2 (4
Linum usitatissi- Flax seedlings Acetone cyanohydrin Aliphatic aldehydes (R)
mum overexpression (R)-2-Butanonecyano- and ketones
hydrin
Sorghum bicolor Millet seedlings (S)-4-Hydroxymandel- Aromatic aldehydes (S)
onitrile
Hevea brasiliensis Rubber tree Acetone cyanohydrin All R’ and R2 (S)
leaves overex-
pression
Manihot esculenta Manioc leaves Acetone cyanohydrin All R’ and R2 (S)
overexpression
Until quite recently, all HNLs had to be isolated from natural sources. To supply
the industrial demand, enzymes from Hevea b r a ~ i l i e n s i s [461~ ~ , Manihot escu-
l e n t ~ ~ [and - ~ ~ ] usitatissimum (E. C. 4.1.2.37)
~ ~ Linum have been successfully
overexpressed in several microorganisms. Presently, the (S)-cyanohydrinof 3-phe-
noxybenzaldehyde is used as an intermediate for various pyrethroid type in-
secticides; this reaction is catalyzed by overexpressed (S)-HNLfrom H. brasiliensis
and the cyanohydrin is produced on the hundred ton per year scale[53].
In contrast to the HNLs from H. brasiliensis and M.esculenta, where aliphatic and
aromatic aldehydes or ketones function as substrates, the HNL from Sorghum bicolor
only catalyzes the formation and cleavage of aromatic (S)-cyanohydrins[54-591. The
most convenient natural sources of enzymes yielding products with (R)-ste-
reochemistry are almonds (Prunus amygdalus)I ‘ ‘ [ and almond meal r6’]. In addition to
Linum usitatissimum[621,other sources of (R)-HNL have also recently been re-
ported[63,641. Concerning the substrate spectrum, the P.amygdalus HNL catalyzes
the HCN addition to aliphatic and aromatic carbonyl moieties, the L.usitatissimum
oxynitrilase accepts only aliphatic ketones or aldehydes I6’1.
14.7.2
Oxynitrilase Catalyzed Addition of HCN to Aldehydes
(R)-Hydroxynitrile lyases. For preparative applications, (R)-HNLfrom almonds has

been extensively investigated. Brussee et al. [“, 671 showed that without enzyme
purification a crude extract from almond meal in aqueous methanol using in situ
HCN generation from a solution of KCN in an acetate buffer affords cyanohydrins in
up to 93 % ee. Apple meal, in the form of unpurified enzyme preparations, accepts
sterically hindered aldehydes (e.g. pivalaldehyde) as substrates, leading to ( R ) -
cyanohydrins with high enantiomeric purity (usually ee > 90 %) [63, “1. A purified
enzyme from Prunus amygdalus supported on cellulose using nonaqueous systems
was employed for the first time by Effenberger and co-workersLG9]. Optimal results
were obtained by almost completely suppressing the non-enzymatic HCN addition
74.7 Enzymatic Synthesis of Cyanohydrins
using ethyl acetate as solvent. In this manner enantiomeric purity could be

I 977
improved. Besides crystalline cellulose (Avicel), other hydrophobic enzyme im-

mobilization systems such as Celite were used[70,711. Utilizing the natural support,
unpurified almond meal in organic solvents with small amounts of aqueous phase
(4%), provides products with ees of up to 99%rG1, "3 72-751. Similar results were
achieved with so-called "microaqueous systems" In order to reduce the amount
of racemic cyanohydrin produced by chemical conversion, low concentrations of
HCN were used by employing a relatively safe and convenient source of this reagent:
acetone c y a n ~ h y d r i n [73,
~ ~77-791.
, Kanerva has developed a method where HCN
diffuses into the reaction mixture from a second flask[74].Wandrey used an enzyme
membrane reactor for the continuous production of product employing an (R)-HNL.
In a production run the volumetric yield was increased to 2400 g (R)-mandelonitrilel
L x day with a residence time of just 3.8 min. The enzyme consumption was 17000
U/kg product["]. Applying a biphasic system a second industrial scale procedure was
Based on these findings, four parameters (pH, concentration of HCN
and benzaldehyde, temperature) were optimized to obtain a throughput of 6700 g
(R)-mandelonitrile/Lx day. A novel synthesis of (R)-cyanohydrinswas described
based on the use of cross-linked and subsequently polyvinyl alcohol-entrapped (R)-
oxynitrilases. These immobilized lens-shaped biocatalysts have a well-defined mac-
roscopic size in the mm range, show no catalyst leaching and can also be efficiently
recycled. Furthermore, this immobilization method is cheap, and the entrapped ( R ) -
oxynitrilases gave similar results to those using free enzymes. Accordingly, ( R ) -
cyanohydrins were obtained in good yields and with high enantioselectivitiesof up to
ee > 99 % [821.
Some substrates, e. g. acrolein, gave only low optical purity with the P. amygdalus
HNL. The catalytic capability of (R)-specific HNL from L. usitatissimum for the
preparation of aliphatic cyanohydrins was investigated L50, 51, 651 and gave encourag-
ing results (ee of up to 99 %).
(S)-Hydroxynitrile lyases. As already mentioned, the (S)-hydroxynitrilelyase from
Sorghum bicolor adds HCN only to aromatic and heteroaromatic aldehydes. Initial
investigationswere performed on the natural substrate 4-hydroxybenzaldehyde,and
rather promising results concerning the enantiomeric excess were found[83].These
results were confirmed and extended using a suspension of enzyme immobilized on
Avicel ~ellulose['~~ "1 or etiolated shoots of S.bicolor[861 in diisopropyl ether. The
Sorghum enzyme was one of the first recombinant hydroxynitrile lyases[871,overex-
pressed in Escherichia coli. In parallel to this work the H . brasiliensis HNL was also
o~erexpressed[~~], giving access to sufficient quantities of this enzyme both on a
preparative scale and for industrial use. To date only a few preparative applications
for Sorghum HNL[74]are known because of the narrow substrate range.
A similarly broad substrate range to that for the (R)-HNLfrom Prunus amygdalus is
revealed by the (S)-HNLs from Manihot esculenta and Hevea brasiliensis (E.C.
4.1.2.39). Detailed sequence studies have revealed high homologies between both
enzymes (M. e s ~ u l e n t a ["I;~ ~H
, . brasilien~is[~~~
451). This result was confirmed by the
crystal structures. The latter was solved for H. brasiliensis in Grazc3'] and for the M.
esculenta enzyme in S t ~ t t g a r t [ ~Expectations
~]. that these enzymes would be similar
978
I with respect to substrate specificitywere established by experimental data from both
groups.
The cyanoglycoside Linamarin was found in 1965 in the seeds of the rubber tree
(Hevea brasiliensis) [891.Tw0decades later the corresponding hydroxynitrile lyase was
described[”. 911. Studies regarding the synthetic potential of this enzyme with
respect to the preparation of optically pure cyanohydrins started with the wild
type113s92-941 . A s already mentioned groundbreaking results were obtained with the
synthesis of the (S)-cyanohydrinof 3-phenoxybenzaldehyde,this being a precursor
for some important synthetic pyrethroids 95-971.
HNL from Manihot esculenta Crantz (termed E.C. 4.1.2.37 at this time because
E. C. 4.1.2.39 was not created earlier than 1999[”])was purified to homogeneity from
young leaves of the cyanogenic tropical crop plant cassava in 1994[471.First experi-
ments demonstrated a broad substrate range, but only unsatisfactory optical purities
were obtained[”]. The overexpression of the cloned M.esculenta HNL gene in E. coli
increased the accessibility and specific activity of the biocatalyst as well as the ee of
produced cyanohydrins [871.
A selection of substrates with typical enantioselectivitiesof the obtained cyanohy-
drins, from the respective HNLs, is shown in table 14.7-2.
14.7.3
HNL-Catalyzed Addition of Hydrogen Cyanide to Ketones
Preparative elaboration of (R)-cyanohydrinsof ketones employing oxynitrilase from

Prunus amygdalus was first investigated in organic solvents l1O71. Alkyl methyl ketones
were obtained in moderate yields and in high optical purity, whereas with alkyl ethyl
ketones the chemical and optical yields were reported to be lower[lo8].The alteration,
working with almond meal instead of purified enzyme, resulted in an astonishingly
high enantiomeric excess [‘*I. Similar results with 98 % ee for the (R)-cyanohydrinof
butyl methyl ketone, were obtained[lo9I.
(R)-Oxynitrilasefrom Linum usitatissimumhas been used for the synthesis of ( R ) -
butan-Zone cyanohydrin on a preparative scale I”].
Concerning (S)-ketonecyanohydrins, impressive results were gained on aliphatic
or aromatic ketones, e. g. acetophenone cyanohydrin. The latter was obtained using
the oxynitrilase from H . brasiliensis. (40% conversion, 99 % ee)I‘[ or M . esculenta
HNL (87% conversion, 98% ee) [‘lo].
In table 14.7.3.the results gained by HNL-catalyzed conversions of selected methyl
ketones to the corresponding cyanohydrins are shown.
14.7.4
Transhydrocyanation
The transcyanation (exactly termed transhydrocyanation ) of aromatic and aliphatic

aldehydes with acetone cyanohydrin, catalyzed by (R)-oxynitrilaseto give cyanohy-
drins (see Fig. 14.7-2.), was first performed in This innovative
method avoids the use of free HCN as the cyanide source and is mostly accompanied
Table 14.7-2.
Aldehydes R-CHO as substrates for oxynitrilase-catalyzed cyanohydrin formation.

I 979
R HNL Source Conversion 1%1 ee I%] Reference
Ph R P. a. 100 99
S S. b. 97 97
S H. b. 96 99
S M. e. 100 98
(El-PhCH=CH R P. a. 54 87
S H. b. 93 98
3-PhO(CsH4) R P. a. 99 98
S S. b. 93 96
S H. b. 99 99
PhCH20CH2 S H. b. 92 12
PhCHz R P. a. 83 88
S H. b. 44 99
PhCHzCH2 R L. u. 10 10
S H . b. 88 93
2-CH30(C&) R P. a. 65 96
S H. b. 61 77
3-CH@(C&) R P. a. 85 98
S S. b. 93 89
S H . b. 80 99
4-CH3 0 (c6H4) R P. a. 47 99
S S. b. 54 71
S H. b. 49 95
S M.e. 82 98
2-fury1 R P. a. 96 99 (S)"
S S. b. 80 80(R)"
S H. b. 95 98(R)"
3-fuvl R P. a. 96 99
S S. b. 88 87
S H . b. 98 98
S M. e. 98 92
2-thienyl R P. a. 71 99 (S)"
S S. b. 64 91(R)"
S H. b. 98 99(R)"
S M . e. 85 96(R)*
3-thienyl R P. a. 95 99
S S. b. 95 98
S H. b. 49 99
S M. e. 98 98
CH4H R L. u. 100 74
S H. b. 92 98
S M.e. 70 56
(E)-CHjCH=CH R P. a. 99 98
S H . b. 80 86
S M. e. 100 92
(E)-CH,(CH2)4CH=CH S H . b. 96 99
R HNL Source Conversion ["h] ee ["h] Reference
(E)-CH3(CH2)2CH=CH S H . b. 46 95
S M . e. 82 97
(Z)-CH,(CHz)*CH=CH S H . b. 35 80
CH~(CH~)~CGC S H . b. 88 80
3-cyclohexenyl R P. a. 86 55
S H . b. 87 99
P. a. 90 99
H.b. 95 99
M . e. 100 92
P. a. 82 96
H. b. 35 85
P. a. 72 97
H.b. 81 96
P. a. 99 98
L. u. 91 98
H . b. 80 80
M . e. 70 88
P. a. 99 83
L. u. 100 93
H . b. 80 81
M . e. 91 95
P. a. 58 92
L. u. 100 89
H.b. 80 67
M. e. 80 94
-L Change of product configuration owing to a priority replacement according CIP rules
Abbreviations: HNL, hydroxynitrile lyase; P. a., Prunus amygdalus; S. b., Sorghum bicolor; H . b., Hevea brasi-
liensis; M. e., Manihot esculenta; L. u., Linum usitatissimum.
Figure 14.7-2. Principle o f transhydrocyanation: R' = alkyl, cycloalkyl,

aryl, heteroaryl; R2 = H, alkyl.
by a slight decrease in ee compared to standard conditions. It was optimized in

Turku [721 by comparing the feasibility of powdered almond meal as a catalyst to that
of a purified enzyme preparation in an organic solvent.
The attempt to use racemic 2-methyl-2-hydroxyhexanenitrile as the cyanide donor
was rewarded by obtaining aliphatic o-bromo cyanohydrins from the corresponding
aldehydes in 90-97% ee[781. As a biocatalyst, (R)-oxynitrilasewas used.
Table 14.7-3.
Methyl ketones R-CO-Meas substrate for oxynitrilase-catalyzedcyanohydrin

I
981
formation.
R HNL Source Conversion I"/.] ee rh] Reference
R P. a. 80 76
R L. u. 100 95
S M . e. 91 18
R P. a. 70 97
R L. u. 100 93
S H . b. 99 74
S M . e. 36 69
R P. a. 73 99
S H.b. 59 99
S M . e. 58 80
R P. a. 54 90
S H.b. 99 98
R P. a. 57 98
S H . b. 86 99
S M.e. 69 91
S H. b. 49 78
S M.e. 81 28
R P. a. 14 90
S H. b. 40 99
S M . e. 87 98
S H. b. 74 95 . .
Abbreviations: HNL, hydroxynitrile lyase; P. a,, Pnrnus amygdalus; L. u., Linum usitatissiwum: H. b., Heuea
brasiliensis; M. e., Manihot esculenta.
14.7.5
Experimental Techniques for HNL-Catalyzed Biotransforrnations
HNL catalysis in aqueous medium. Reaction in aqueous solution is performed with an

appropriate acidic component and alkali cyanide for in situ development of the
required HCN. The following procedure is a typical e~arnple['~I.
To a stirred solution of 1 mmol aldehyde in 1.7 mL of 0.1 mol/L sodium citrate
buffer (pH 4.0),1 mL of a crude cytosolic extract of (S)-HNLfrom Heuea brasiliensis
(100IU/mL) was added and the mixture was cooled down to 0 "C. Subsequently,
2 mmol of potassium cyanide adjusted to pH 4.0 with cold 0.1 moljL citric acid
(17 mL) were added in one portion. After stirring for 1 h at 0-5 "C, the reaction
mixture was extracted with methylene chloride (3 x 50 mL). The combined organic
layers were dried over anhydrous sodium sulfate and the solvent was removed to give
the crude cyanohydrin. This was then purified by column chromatography on silica
gel using petroleum ether / ethyl acetate acidified with trace amounts of anhydrous
HCl as the eluent.
H N L catalysis in organic medium. A significant advancement in cyanohydrin
production was made by performing the transformation in organic solvents im-
miscible with water. It has been observed that there is virtually no spontaneous
982
I chemical addition of HCN to the carbonyl moiety[48,
69-71, ", lo'. A

lo7, 111-1141.
representative protocol for cyanohydrin formation in organic solvents with im-

mobilized oxynitrilase is the
A suspension of Avicel cellulose (0.5 g) in 0.05 mmol/L phosphate buffer (pH 4.5,
10 mL) containing ammonium sulfate (4.72 g) was stirred for 1 h, and a solution of
(S)-HNLfrom Sorghum bicolor (50 pL, 1000 IU/mL, specific activity 70 IU/mg) was
added. The mixture was stirred at room temperature for 10 min and filtered, and the
immobilized enzyme was suspended in diisopropyl ether (10 mL). After addition of
aldehyde (2 mmol) and dry liquid HCN (300 pL, 7.5 mmol), the mixture was stirred
until all aldehyde had reacted. After removal of the immobilized enzyme, the filtrate
was concentrated to yield the crude cyanohydrin.
H N L catalysis in biphasic medium. Biphasic solvent mixtures were reported
employing (R)-oxynitrilase[812 11'1 as well as (S)-HNLfrom Hevea b r a ~ i l i e n s i s ~
"*I.l ~ ~
A typical procedure is as follows Is'].
Freshly distilled benzaldehyde (37.1 g, 0.35 mmol), HCN (12.2 g, 0.45 mmol) and
(R)-oxynitrilase(78 mg) were dissolved in 225 mL of methyl t-butyl ether (MTBE)
and 250 mL of citrate buffer (50 mmol/L, pH 5.5) at 22 "C. After stirring for 20 min
the MTBE layer was separated and the aqueous layer was extracted once with 25 mL
of MTBE. The combined organic layers were dried over MgS04, filtered and
concentrated under reduced pressure. Yield: 45.2 g (97%), purity 98%, ee 98%. The
aqueous layer was reused in a series of four consecutive experiments using the same
amounts of reagents in the organic phase. A total of 185.5 g of benzaldehyde was
converted into 226 g of (R)-mandelonitrileusing 78 mg of (R)-oxynitrilase (0.035
wt%).
Transhydrocyanation for HCN generation. An alternative method of employing
organic solvents that allows the safe use of HCN is transhydrocyanation L7', 73,
77-79, 'I6, 'I7]. An example of cyanohydrin formation using acetone cyanohydrin as
the cyanide source is given in the following procedure[77].
To a solution of 120 mg (1 mmol) of phenylacetaldehyde and 110 mg (1.3 mmol)
of acetone cyanohydrin in 11 mL of diethyl ether at 23 "C, 0.5 mL of (R)-oxynitrilase
buffer solution (10mg/mL, 0.4 mol/L acetate buffer, pH 5.0) was added. The mixture
was stirred for 18 h at 23 "C and diluted with 50 mL of ether. The aqueous phase was
extracted with 2 x 10 mL of ether and the combined organic phases were dried over
anhydrous magnesium sulfate. Evaporation of solvent gave a pale amber liquid
which was chromatographed on a flash silica gel column in 1 : 30 : 50 ethyl acetate /
benzene / dichloromethane to afford 122 mg (83%) of cyanohydrin, ee 88%.
14.7.6
Resolution of Racemates
Oxynitrilase as catalyst. It is possible to treat a racemic cyanohydrin with a (R)-or (S)-

HNL to decompose selectively one enantiomer of this mixture (exemplified in Fig.
14.7-3.).The (R)-HNLfrom Prunus amygdalus was used for the resolution of racemic
cyanohydrins. Employing a biphasic system, namely citrate buffer/diisopropyl ether
(40:l) at 39"C, catalytic amounts of PhNH2 and semicarbazide were added for
14.7 Enzymatic Synthesis ofCyanohydrins I983
Figure 14.7-3. Enantioselective HNL catalyzed decomposition of racemic

cyanohydrins: R’ = alkyl, cycloalkyl, aryl, heteroaryl; R2 = H, alkyl.
Figure 14.7-4. Lipase-catalyzed formation of optically enriched cyanohydrins: R’ = alkyl,

cycloalkyl, aryl, heteroaryl; R2 = acyl; R’ = H, acyl.
aldehyde capture. In this manner the (S)-cyanohydrinof 3-phenoxybenzaldehyde

was obtained with 91 % ee at 50% conversion[”’].
Recently, almond meal was used for the resolution of rac-2-hydroxy-2-phenyl-
propanenitrile. Under the optimized conditions, (S)-2-hydroxy-2-phenylpropaneni-
trile, as the less reactive enantiomer, was obtained in 98-99% ee at approximately
50% conversion[’18].In a similar way the (S)-cyanohydrinwas afforded from racemic
2-methyl-2-hydroxyhexanenitrilewith P. amygdalus HNL in more than 90%
ee~73, 781
Esterase or lipase as catalyst. Application of hydrolytic enzymes is realized in three

different systems: enzymatic hydrolysis or transesterification of racemic cyanohy-
drin esters (see Figure 14.7-4.)as well as enzymatic acylation of racemic cyanohy-
&ins [W 1201
A series of cyanohydrin acetates with an e.e. up to 98% has been prepared by
enzymatic hydrolysis of their racemic acetates in the presence of an esterase from
Pseudomonas sp. [1371. Lipoprotein lipase from Pseudomonas sp. catalysed irreversible
transesterification using enol esters was applied to the resolution of different
aromatic cyanohydrins[13’, 139].
The enantioselectivehydrolysis of the racemic acetate by Arthrobacter lipase gave
the optically pure (S)-3-phenoxybenzaldehyde cyanohydrin. The unhydrolysed (R)-
acetate was reracemised by heating with triethylamine and submitted again to
enzymic hydrolysis[l4O]. In addition, the resolution of the racemic acetate ester of the
cyanohydrin of 3-phenoxybenzaldehydeusing a highly enantioselective lipase from
Pseudomonas sp. was carried out recently with an e.e. of >9G%[1411.Both the
cyanohydrin esters and the free cyanohydrins (which are prone to racemization) can
be isolated as enantiomers with high optical purity (ee 97%) on a preparative scale by
the hydrolysis of the racemic butyrates with Candida cylindracea lipase and Pseudo-
monas sp. lipase11211.
A one-pot synthesis of optically active cyanohydrin acetates from aldehydes has
been accomplished by lipase-catalyzed kinetic resolution coupled with in situ
formation and racemization of cyanohydrins in an organic solvent. Racemic cyano-
hydrins, generated from aldehydes and acetone cyanohydrin in diisopropyl ether
under the catalysis of basic anion-exchangeresin, were acetylated stereoselectivelyby
a lipase from Pseudomonas cepacia (Amano)with isopropenyl acetate as an acylating
984
-
HH
H R' TBDMSOR HH HoR PZR2 NHR3
j y
- g
H o ~ NH,
Y XYMS r
A CN
H CHO
1.c"
g
R& NH2 \i
lq
grsCN
P
COOR2
\I
X H
COOH th
&R
CN - CR2k
CN to %
T COOR'
H
KY \
A7Y
H
x; CN
CN
Figure 14.7-5. Follow-up reactions of optically pure cyanohydrins: a) TBDMSCl/imidazole[66,671;
b) R2MgX/ether,NaBH4, H30+[84f1251. , c) CH3Mgl/ether,H J O + [ ~d) ~ ]R2CH2Mgl/ether,
; MeOH,
R3NH2,NaBH4[126];e) IAH[84];f) H30+['O71;g) R20H/CHC13/wolfatite[127];h), R3CI/Nal/
CHsCN/pyr/O 0C[1271;i) DIBALH/hexane/ -78 "C, conc. HCI/MeOH [1271; j) R2S02Cl/pyrL1021; k)
KN3/DMF, (inversion) [12']; I) LiAIH4/Et20/-80 "C, phosphate buffer pH 7.0/-70 "C['28];m)
potassium phtalimide/DMF, (inversion) ["'I; n) KOAc/DMF/r.t., (inversion)[lo2.12', l2'I; 0)conc.
HCl/r.t. or lipase['02a12', lZ91; p) Me3SiCl/pyr/Et20/0to +25 0C[1301;q) diethylaminosulfurtri-
fluoride (DAST)/CH2CI2/-80to +25 "C['301;r) DIBALH/CHzC12/ -78 "C, 1 N H2S04"3'1.
reagent. The (S)-cyanohydrinwas preferentially acetylated by the lipase, while the

unreacted (R)-isomer was continuously racemized through reversible transhy-
drocyanation catalyzed by the resin. These processes consequently led to a one-pot
conversion with up to 94% ee in 63-100% conversion yields[122,1231.
The Pseudomonas aeruginosa lipase (immobilised on hyflo Super-Cel) catalysed
kinetic resolution of (~ac)-2-(acetyloxy)-2-(pentafluorophenyl)acetonitnlegave en-
antiomerically pure cyanohydrin and its antipodal ester [142-1441.
14.7 Enzymatic Synthesis ofCyanohydrins I 985
14.7.7
Follow-up Chemistry of Enantiopure Cyanohydrins
Optically pure cyanohydrins are important synthetic building blocks[', 2 . 4. ',

9. Is* 1241, as can be seen from Fig. 14.7-5. in selected examples. Both func-
tional groups, the hydroxy and the cyanide moiety, can be easily converted into a
large range of other chiral intermediates such as a-hydroxy acids and esters, a-
hydroxy aldehydes and ketones, p-amino alcohols and a-fluorocyanides. These
structural moieties are present in a large number of industrially valuable products
such as drugs, agrochemicals,flavorings and fragrances.
14.7.8
Safe Handling of Cyanides
Hydrogen cyanide smells like bitter almonds, although many people cannot smell it
Cyanide is a fast acting poison in the human body; it affects the ability of all
at all[132].
cells to breathe. Severe breathing difficulties develop very rapidly when cyanide is
swallowed, inhaled, or absorbed through the skin. Cyanide poisoning symptoms in
the early stages indude: general weakness, breathing difficulty, headache, nausea,
giddiness, vomiting, the victims breath smelling like bitter almonds, and irritation of
the nose, mouth, and throat. Hydrogen cyanide is liberated by the addition of acid to
cyanide compounds.
The TLV (threshold limit value) for HCN is 11 mg/m3 or 10 ~ p m [ ' ~These~ ] . limits
include the potential contribution of skin absorption to the overall exposure. Proper
gloves should be worn when handling dry sodium cyanide. Rubber gloves and
splash-proof goggles should also be worn when substantial amounts of sodium
cyanide solution are used. All reaction equipment in which cyanides are used or
produced should be placed in well-ventilated hoods, and it should be determined
immediately whether anyone has been exposed to cyanide vapors or liquid splash-
Vapor-detector tubes sensitive to 1 ppm of HCN are available commercially. The

presence of free cyanide ion in aqueous solution may be detected by treating an
aliquot of the sample with ferrous sulfate and an excess of sulfuric acid. A precipitate
of Prussian blue indicates that free cyanide ion is present. More sophisticated for
continuous warning is the use of electrochemical sensors for HCN detection.
Waste solutions containing cyanides treated with sodium hypochlorite are con-
verted to harmless cyanate, which can be further processed to ammonia and carbon
dioxide by addition of diluted sulfuric acid to pH 7. Surplus HCN gas can be
neutralized by aqueous sodium hydroxide and then oxidized. Caution has to be
advised with liquid hydrogen cyanide because bases including sodium hydroxide
and sodium cyanide may initiate a violent polymerization[133].
Explosive hazards can occur on exposure of HCN to air in the presence of sources
of ignition (flammable limits in air: 5.640% v/v) including heat (polymerizes
explosively at 5 0 4 0 "C)and when HCN is stored for long periods of time.
986
I 14.7.9
Conclusions and Outlook
The enzymatic synthesis of enantiopure cyanohydrins has been brought to a high

stage of development. Both (R)- and (S)-cyanohydrins are accessible for a broad
variety of substrates in as a rule excellent yield and enantiopurity. Following recent
progress in overexpression, HNLs are also available in quantities needed for
industrial production. The procedures for safe handling of cyanides are well
established so that they do not restrict the exploitation of HNLs.
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I991
15
Reduction Reactions
15.1
Reduction of Ketones
Kaoru Nakamura and Jomoko Matsuda
15.1.1
Introduction
15.1.1.1
Enzyme Classfication and Reaction Mechanism
Research on the asymmetric reduction of ketones by biocatalysis is expanding, and

its practical applications to organic chemistry have resulted in success in the
enantioselective synthesis of pharmaceuticals, agrochemicals and natural pro-
ducts [1-41. It is attracting increasing attention because of the following advantages:
- providing a green and sustainable process (natural catalysis),
- high enantio-, regio- and chemo-selectivity compared with most man-made
reagents and catalysts,
- achiral ketones can be transformed into the corresponding alcohols with 100%
yield and 100% ee theoretically, whereas kinetic resolution of racemic substrates
by hydrolytic enzymes such as lipases yields only 50% of products to achieve
100% ee.
- the resulting alcohol functionality can be easily transformed, without racemiza-
tion, into other useful functional groups such as halides, thiols, amines, azides,
etc.
Dehydrogenases, classified under E.C.l.l., are enzymes that catalyze reduction and
oxidation of carbonyl groups and alcohols, respecti~ely[~]. The natural substrates of
the enzymes are alcohols such as ethanol, lactate, glycerol, etc. and the correspond-
ing carbonyl compounds, but unnatural ketones can also be reduced enantiose-
lectively. To exhibit catalytic activities, the enzymes require a coenzyme; most of the
dehydrogenases use NADH or NADPH, and a few use flavin, pyrroloquinoline
quinone, etc. The reaction mechanism of the dehydrogenase reduction is as follows:
992
I 15 Reduction Reactions
Step 1 A holoenzyme (an enzyme with its coenzyme) binds a ketone.

Step 2 A hydride on the coenzyme is transferred to the ketone to produce an alcohol.
(With concurrent oxidation of the coenzyme)
Step 3 The enzyme releases the product alcohol.
Step 4 The oxidized coenzyme is transformed back into the reduced form. (With
concurrent oxidation of an auxiliary substrate)
There are four stereochemical patterns in the transfer of the hydride from the
coenzyme, NAD(P)H,to the substrate (Step 2) as shown in Fig. 15-1.With E l and E2
enzymes, the hydride attacks the si-face of the carbonyl group, whereas with E3 and
E4 enzymes, the hydride attacks the re-face, which results in the formation of R and
S alcohols, respectively. On the other hand, E l and E3 enzymes transfer the pro-R
hydride of the coenzymes, and E2 and E4 enzymes use the p r o 3 hydride. Examples
of the El-E3 enzymes are as follows:
E l : Pseudornonas sp. alcohol dehydrogenaseL6]
Lactobacillus kefir alcohol dehydrogenase171
E2: Geotrichurn candidurn glycerol dehydrogenase[8-101
Mucorjavanicus dihydroxyacetonereductase [11]
E3: Yeast alcohol dehydrogenase[l2]
Horse liver alcohol dehydrogenase[l3-l61
Moraxella sp. alcohol dehydrogenase[I'
E2 Figure 15-1. Stereo-

chemistry ofthe hydride
transfer from NAD(P)H
to the carbonyl carbon on
the substrate (S is a
small group and L is a
large group).
-ry
ADPR sifac re face I
ADPR
15.1.1.2
Coenzyme Regeneration
Reduction of the substrate accompanies the oxidation of the coenzyme (Step 2).
Before the next cycle of the reduction of the main substrate can occur, the coenzyme
has to be reduced (Step 4). Many methods for the regeneration of the reduced form
of coenzyme [NAD(P)H]have been developed, so that only a catalytic amount of the
coenzyme is required for the reaction. The coenzyme regeneration methods can be
classified into two types:
- two-enzmye system: different enzymes reduce the substrate and NAD(P)+,
- one-enzyme system: the substrate and NAD(P)+ are both reduced by the same
enzyme.
IS. 1 Reduction ofKetones
I
993
(a) (b)
0 Enzvme 1 OH 0 Enzyme 1
u
main substrate
for asymmetric for asymmetric
reduction reduction
NADH NAD' NAD(P)H NAD(P)*
A l
COZ
Enzyme 2
HCOzH
auxiliary
substrate
A U Enzyme2 x
auxiliary
substrate
Figure 15-2. Regeneration o f NAD(P)H: (a) Two-enzyme system using a formate dehy-
drogenase as an auxiliary enzyme and formic acid as an auxiliary substrate; Enzyme
1 = Enzyme for the reduction o f t h e main substrate
Enzyme 2 = Formate dehydrogenase
(b) one-enzyme system using 2-propanol as an auxiliary substrate.
Enzyme 1 = Enzyme 2 For example alcohol dehydrogenase from Thermoanaerobium
brockiil"* "1, Pseudomonas sp. 1'1, Lactobacillus kefirlq, and Ceotrichum candidum I2O, "1.
One of the examples of the two-enzymesystem uses a formate dehydrogenase for the
recycling of coenzyme [Fig. 15-2(a)]['*3, 22-24] . It catalyzes oxidation of HC02H to
C02 in order to drive the reduction of NAD' to NADH. The system is one of the most
widely used due to the advantages such as: 1)the enzyme is commercially available,
2) COZ can be easily removed from the reaction, 3) formate is strongly reducing,
therefore no back reaction occurs, and 4) C02 and HCOzH are innocuous to
enzymes. For example, the reduction of ethyl 4-chloro-3-oxobutanoateby a carbonyl
reductase from Rhodococcus eruthropolis uses NAD'/formate dehydrogenase as
shown in Fig. 15-3["I. As exemplified, the system is very useful for the recycling of
NADH. However, it does not accept NADP', so it cannot be used for the direct
reduction of NADP+. For the reduction of NADP' using formate dehydrogenase,
catalytic amounts of NAD' and NAD(P)' transhydrogenase are required. Changing
the coenzyme specificity of a formate dehydrogenase using genetic methods is
discussed in Sect. 15.1.3.7.
Two-enzyme systems using glucose dehydrogenase or glucose-6-phosphatedehy-
drogenase (commercially available enzymes) have also been widely employed [2G-311.
Carbonyl reductase from

0 Rhodococcus erythropolis OH
* CI &COzEt
cIdk/cozEt NAD+ HC02H
formate dehydrogenase Conv. 100%. ee >99% (R)
Carbonyl reductase from

Rhodococcuseiythropolis OH
COpEt * A C O z E t
NAD' HCOpH
formate dehydrogenase
Conv. 49%, de 95%, ee>95% (2R. 3s)
Figure 15-3. Examples of reduction using the formate/formate dehydrogenase
N A D H recycling system[251.
994
0 OH
HLADH
CI&COpEt t C I A C O Z E t
NAD' Glucose
Glucose dehydrogenase yield '*% ee "%(5)
from Baci//uscereus
Figure 15-4. Example o f reduction using glucose/glucose dehy-
drogenase NADH recycling
yield 90%
ee >99% (S)
F Photosynthetic microorganism
F Synechococcussp. PCC 7942 F
Figure 15-5. Utilization o f light energy for an efficient
They oxidize glucose or glucose-6-phosphate to form gluconolactone or glucono-

lactone-6-phosphate, respectively, which is spontaneously hydrolyzed to give
gluconic acid. Both NAD' and NADP' act as substrates for these enzymes. For
example, a thermostable glucose dehydrogenase form Bacillus cereus was used to
recycle NADH in the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by
horse liver alcohol dehydrogenase (HLADH)as shown in Fig. 15-4[261.
Another example of a two-enzyme system involves molecular hydrogen and a
hydrogenase[lI.Hydrogenases catalyze the reduction of NAD' or other redox dyes by
dihydrogen. The system is attractive because dihydrogen is inexpensive, strongly
reducing and innocuous to enzymes and NAD(H), and no by-product is formed.
However, a drawback is the extreme sensitivity of the hydrogenase enzymes to
inactivation by dioxygen, preventing this system from being widely used.
To provide an environmentally friendly system, photochemical methods have been
developed, which utilize light energy for the regeneration of NAD(P)H['. 32, 331.
Recently, the use of cyanobacterium, a photosynthetic biocatalyst, for the reduction
was reported where the effective reduction occurred under illumination (Fig. 15-
5) [321. When a photosynthetic organisms is omitted, the addition of a photosensitizer
is necessary. The methods utilize light energy to promote the transfer of an electron
from a photosensitizer via an electron transport reagent to NAD(P)+['].
One-enzyme recycling systems are also well developed. One of the most frequently
utilized is the alcohol-alcohol dehydrogenase system as shown in Fig. 15-2(b).The
system does not need an auxiliary enzyme, but an auxiliary substrate is necessary.
Ethanol or 2-propanol is frequently used as an auxiliary substrate. For example,
HLADH uses ethanol as shown in Fig. 15-G[13-16]and Thermoanaerobium
brockii"'. *'I, Pseudomonas sp. 16], Lactobacillus kefid71, and Geotrichum can-
didum[20.21] alcohol dehydrogenases recycle NAD(P)H by employing an excess of
2-propanol.A detailed investigation of the type and amount of the auxiliary substrate
needed by G. candidum revealed that it can use 2-alkanols from 2-propanol to
2-octanol (and cyclopentanol as well), and 15-20 equivalents of the supplementary
alcohol are necessary to shift the equilibrium (between the oxidation and reduction)
towards the reduction of the main substrate. Because a much higher concentration
15.1 Reduction of Ketones
0 Figure 15-6. Reduction of

I
995
heterocyclic ketones by
HIADH using ethanol as
%, an auxiliary
Yield 11% Yield 29% Yield 35% s u b ~ t r a t e [ l6].
’~~
ee 100% ee 100% ee36%
H H
Yield 26% Yield 48%

ee >97% ee 60%
of the auxiliary substrate to that of the main substrate is required, 2-propanol is

deemed most suitable for synthetic purposes due to its high volatility.
Electrochemical regeneration of NAD(P)H represents another interesting
method [34-361. The system involves electron transfer from the electrode to the
electron mediator such as methyl viologen or acetophenone etc., then to the NAD(P)+
(which is catalyzed by an electrocatalyst such as ferredoxin-NADP’ reductase or
alcohol dehydrogenase, etc.) [34]. Other methods involve the direct reduction of NAD’
on the Both one-enzyme systems and two-enzyme systems have been
reported.
15.1.1.3
Form ofthe Biocatalysts: Isolated Enzyme vs. Whole Cell
Enzymes in a pure form, in a partially purified form, and in the whole cell can be
used for organic synthesis, and each has advantages and disadvantage^[^]. The
proper choice of the form of the biocatalyst is important because it affects the
enantio-, regio- and chemo-selectivities,the requirement (or not) of a coenzyme and
an auxiliary enzyme, the ease of catalyst preparation and work up procedures, etc. as
shown in Table 15-1.
The most widely used whole cell biocatalyst is bakers’ yeast. Since it has many
different kinds of enzymes, many kinds of substrate can thus be reduced, and
various types of the reactions are expected. For example, 0-keto esters, aromatic,
aliphatic, cyclic and acyclic ketones can be reduced with high yield[’. 37-391. There-
fore, it is a versatile “all-round”reagent. However, since bakers’ yeast contains many
kinds of dehydrogenases, some of them may be S selective, while others are R
selective, so that the enantioselectivities can be low to high depending on the
substrate structure. Further degradation of the product may also be a problem, again
associated with the fact that there are many kinds of enzymes in the cell.
Not only the enzymes but also the cellular components such as coenzymes and
carbohydrates are conserved in the cell, which makes the whole cell processes
favorable. For example, the addition of an expensive coenzyme and an auxiliary
enzyme for coenzyme regeneration is not necessary, which makes the system simple
and economical when comparing with the equivalent isolated enzyme process.
996
Table 15-1. The form of biocatalyst: whole cell vs. isolated enzyme
Parameter Whole Cell Isolated Enzyme
Kinds of enzymes Many One

Kinds of reactions Many One
Regio- and enantioselectivity Low to high High
Coenzyme Unnecessary Necessary
Catalyst preparation Easy Difficult
Work up Difficult Easy
Example Bakers' yeast Horse liver alcohol dehydrogenase
However, the product isolation may be complicated due to large amounts of biomass
and metabolites.
On the other hand, isolated enzyme processes also have many advantages. The
problem associated with the product isolation and overmetabolism can be avoided
using an isolated enzyme. More importantly, chemo-, regio-, and enantioselectivities
of isolated enzyme systems are usually higher than that of whole cell processes
because two competing enzymes with different stereoselectivities are not present.
One of the most widely used isolated enzymes is horse liver alcohol dehydrogenase
(HLADH) which reduces, for example, S-heterocyclic ketones to give the corre-
sponding tetrahydrothiopyran-4-01with 100% ee [Fig. lS-G(a)]['I. However, when
the selectivity is so high, the substrate specificity is not wide; thus HLADH can
reduce cyclic ketones with excellent enantioselectivity but cannot reduce acyclic
ketones.
Another advantage of the isolated enzyme system is that the reaction pathway can
be understood and predictions made. For example, for HLADH, the crystal struc-
ture[4s421 and the active site (diamond lattice) model[13,141 are available to under-
stand the reduction, whereas, in a whole cell process, even the catalytic species itself
may not be clear.
In summary, whole cell and isolated enzyme biocatalysts both have various
advantages and disadvantages. Using a recombinant yeast having the gene of a
requisite enzyme is the way to access a single predominant enzyme in a micro-
organisms, a strategy which will be further discussed in Sect. 15.1.3.2.
15.1.1.4
Origin of Enzymes
Enzymes from various sources have been used for asymmetric reductions in organic
synthesis. Microorganisms are the most important sources. There are a huge
number of species (mostly in soil), containing a variety of enzymes. Commercially
available microbial dehydrogenases are alcohol dehydrogenases from yeast, Ther-
moanaerobium brockii (TBADH),and the hydroxysteroid dehydrogenase from Pseu-
domonas testosteroni.
One of the most attractive kinds of microorganisms for organic synthesis is a
thermophilic microorganism such as Themoanaerobium brockii['8, l91, or T h e m o -
anaerobacter ethanolicus, etc. (43-491. The thermostability of the dehydrogenase en-
zymes from these microorganisms is very high: TBADH is stable even at 86 "C [I8*
I
997
and an alcohol dehydrogenase from Thermoanaerobacter ethanolicus can be used at

50-60 'T[",471. Since the enzymes with high thermostability usually have a high
tolerance to organic solvent or substrates, the enzymes from thermophilic micro-
organisms are most suitable for organic synthesis.
Another interesting class of biocatalyst encompasses the photosynthetic micro-
organisms, the algaeL3** "]. Owing do the high growth rate, a large amount of the
biomass for use as the biocatalyst is available. Importantly, such organisms can use
light energy as power for coenzyme recycling as described in Sect. 15.1.1.2, so an
environmentally friendly system can be constructed using them.
The second most widely studied source of enzymes are mammalian enzymes as
exemplified by horse liver alcohol dehydrogenase (HLADH).Detailed investigations
on this enzyme have been reviewed elsewhere[13,1'.
The third and least studies source is from plant cell cultures, which have only
recently been used in biocatalysis[51-571. Although the number of species available
are much less than microorganisms, plants possess a much larger gene. More
importantly since plants can effect photosynthesis, different types of enzymes exist
in plants to those of microorganisms. Therefore, different enzymes which catalyze
unique reactions with man-made substrates may be expected. Despite the strong
possibility of the discovery of interesting enzymes, plant cell cultures have not been
fully investigated for use in biocatalysis due to their relatively slow growth rate.
15.1.2
StereochemicalControl
15.1.2.1
Enantioselectivity of Reduction Reactions
The synthesis of enantiomerically pure compounds is becoming increasingly im-

portant for research and development in chemistry and especially
in the pharmaceutical industry, as chiral drugs now represent close to one-third of all
pharmaceutical sales world wide["]. In most of the cases, one enantiomer is more
effective as a drug than the other. The influence on the environment is also different
between the enantiomers; different enantiomers of chiral pollutants in soils are
preferentially degraded by microorganisms in various environments [Go]. Therefore,
synthetic methods exhibiting extremely high enantioselectivitiesare necessary.
The enzymatic reactions occurring in Nature involving natural substrates usually
show very high enantioselectivities. On the other hand, with man-made substrates
the enantioselectivity can also be high (> 99% ee) but this is not always the case as
shown in Fig. 15-7. Low enantioselectivity results when the catalyst is a low
selectivity enzyme [Fig. 15-7 (C)] and/or when there are more than two competing
enzymes with different enantioselectivities [Fig. 15-7 (D)]. In case (C), either an
enzyme or substrate has to be changed. On the other hand, in case (D), a change in a
microorganism or substrate as well as a change in reaction conditions may be
effective in improving the enantioselectivity. In case (D), by choosing the proper
75 Reduction Reactions
998
I OH OH
&R+ RAR’
(high]: OH OH
Figure 15-7. Enantioselectivity of the product and improvement methods.
Inhibitor of R-enzyme or Inhibitor of S-enzyme or

0 OH
-
QH activator of S-enzyme activator of R-enzyme
RAW
(3
Figure 15-8. Synthesis of both enantiomers using one microorganism by

choosing appropriate conditions.
conditions, both enantiomers can be synthesized by using only one microorganism;

when a selective inhibitor for an S-directing enzyme or on R-directing enzyme is
added to the reaction mixture, the (R)-alcohol or (S)-alcohol will be enantiose-
lectively produced, respectively, as shown in Fig. 15-8.
15.1.2.2
Modification of the Substrate: Use of an “Enantiocontrolling” Group
The enantioselectivity of a biocatalytic reduction can be controlled by modifying the

substrate because the enantioselectivity of the reduction reaction is profoundly
affected by the structure of substrates. For example, in the reduction of 4-chloro-
3-oxobutanoate by bakers’ yeast, the ester moiety can be used to control the
stereochemical course of the reduction 161-631. When the ester moiety was smaller
than a butyl group, then (S)-alcoholswere obtained, and when it was larger than a
pentyl group then (R)-alcoholswere obtained as shown in Fig. 15-9.
After the reduction, the ester moiety can be exchanged easily without racemiza-
tion, so both enantiomers of an equivalent synthetic building block are obtained
using the same reaction system by changing an “enantiocontrolling”group, the ester
moiety. The “enantiocontrolling”group can also be introduced into the keto esters at
the a- or a‘-positions. For example, sulfur functionalities such as methyl- and
15.1 Reduction ofKetones
I 999
S n=1-4 yeast cell R n=5-12
-100 I , , , , , ,
0 2 4 6 8 1012
n
Figure 15-9. Stereochemical control on yeast-catalyzed reduction by changing the
ester group[61-631.
0
/ICC02Me
Bakers' yeast
- ee87% QH
ee ,96% hCozM
; 0 OH
' e*
%%%
j Bakers'yeast b +CO2Me f
t SR SR
0 OH OH
Bakers' yeast
Ph02S&C02Me PhOzS&C02Me _c ee 98%
M
eO
C, , - )
2
Figure 15-10. Stereochemical control on yeast-catalyzed reduction by introducing sulfur

function aIities "1.
lS43
....*
Bakers' yeast
l B a k e r s ' y e a s t OH
~0 ~ -i ee69%
ee96%
p
OH
I
Figure 15-11. Improvement of enantioselectivity by substituting iodide at the para
position; yeast reduction followed by dehalogenation (dh) [651.
phenylthiorG4land phenyl~ulfonyl[~~1 groups can be used to improve the enantio-

selectivities as shown in Fig. 15-10.
Other types of ketones can also be modified to improve the enantioselectivities,
and various functionalities can be used to modify the substrate to produce the
corresponding alcohol with higher enantioselectivities.For example,the reduction of
acetophenone by yeast results in the formation of phenylethanol in 69 % ee, whereas
the reduction of p-iodoacetophenone followed by the dehalogenation results in a
product of96% ee (Fig. 15-11)[G51.
As shown above, the substrate modification and "de"modification steps can be
used to improve the enantioselectivity, although on the negative side the strategy
may introduce extra steps into a synthetic route.
IS Reduction Reactions
Table 15-2. Screening for the synthesis of important chiral building blocks.
Reactions Microorganisms Result Reference

screened
Candida magnoliae
90 g/L, 96.6% ee 67
(99% ee after heat treatment)
H o- go
191actinomycetes
59 bacteria
45 mg/mL stoichiometric yield
230 yeasts Rhodoforula minufa IF0 0920:86% ee
81 molds Candida parapsilosis IF0 0708:87% ee 68
0
(D' 42 basidiomycetes Aspergillus niger IF0 4415:87% ee
Klebsiella pneumoniae IF0 3319

C02Et - OH
-CO2Et 450 bacteria 99% de, >99% ee, 99% yield
(2Kg in 200 L fermentor)
70
(2R,3s)
15.1.2.3
Screening of Microorganisms
Screening for a novel enzyme is a classical method and one of the most powerful
tools available to find the system to convert a selected ketone into a desired
alcoh01[~"~~1. It is possible to discover a suitable enzyme or microorganisms by the
application of the newest screening and selection technologies that allows rapid
identification of enzyme activities from diverse sources 166]. Enzyme sources for
screening can be soil samples, commercial enzymes, culture sources, a clone bank,
etc. From these sources, enzymes which are regularly expressed and enzymes which
are not expressed in the original host can be tested to establish whether they are
suitable for the transformation of certain substrates [661. For example, 400 yeasts were
screened for the reduction of ethyl 4-chloro-3-oxobutanoate, and Candida magnoliae
was found to be the best one as shown in Table 15-2[", 7 2 , 731. For the reduction of
ketopantoyl lactone, various kinds of microorganisms were screened, and several
microorganisms which produce D-pan pantoy lactone stoichiometrically at a concen-
tration of 45 mg mL-' with high enantioselectivity were found['*]. For the reduction
of ethyl 2-methyl-3-oxobutanoate,out of 450 bacteria, Klebsiella pneumoniae I F 0 3319
and 4 other strains were found to give the corresponding (2R, 3S)-hydroxyesterswith
more than 98 % de and > 99 % ee["].
Screening techniques have also been applied for the purpose of drug synthesis.
For example, a key intermediate in the synthesis of the anti-asthma drug, Mon-
telukast, was prepared from the ketone 1 by microbial transformation as shown in
Fig. 15-12[711. The biotransforming organism, Microbacterium campoquemadoensis
(MB5614),was discovered as a result of an extensive screening programme.
15. I Reduction of Ketones
I ’Ool
-
-
- Montelukast (Singulair)
Figure 15-12. Reduction of a ketone by Microbacterium carnpoquemadoensis (MB5614) in a

synthesis of the anti-asthma drug, M o n t e l ~ k a s t [ ~ ’ ] .
Table 15-3. Control on diastereoselectivity by heat treatment74.
QH OH
COzEt Yeast /\/COZEt +
ee , 9 5 2 1 ee >95%
Yeast cell Svn (%) Anti (%)
No heat treatment
50 OC, 30 min
heat + inhibitor
15.1.2.4
Treatment of the Cell: Heat Treatment
Treatment of the cell before the reaction is sometimes an effective method of

controlling the selectivity of some biocatalysts. When reducing with a whole cell and
the selectivity is not as is desired due to the presence of plural enzymes with different
selectivities, heat treatment of the cell to selectively deactivate one or more enzymes
can change the selectivity of the reduction. For example, the diastereoselectivity in
the yeast reduction of 2-allyl-3-oxobutanoatewas changed from anti-selectivityto syn-
selectivity by pre-treatment of the yeast before the reaction as shown in Table 15-3[741.
In this case, the diastereoselectivityis further improved to 96 : 4 by using an enzyme
inhibitor.
Another example is the use of heat treatment as a supplement to the screening
process. The enantioselectivity of the reduction of ethyl 4-chloro-3-oxobutanoateby
Candida magnoliae was improved from 96.6% ee (S) using untreated cells to 99% ee
(S) with heat treated cells rG71.
15.1.2.5
Treatment of the Cell: Aging
When a whole cell system is used for a reduction, the substrate is usually added to
the cultivation medium after a certain growth period, or to the mixture of the
1002
medium and freshly harvested cells. However, when the mycelium of a local strain of
Geotrichum candidum was not used immediately after growth, but filtered and
preincubated by shaking in deionized water for 24 hours at 27 "C ("agedmycelium"),
then used for the reduction of ethyl 3-oxobutanoate, the stereochemistry of the
product alcohol was different from that obtained from the reduction using fresh
mycelium [75-781. When fresh mycelium was used, the enantioselectivity and the
absolute configuration of the product shifted from S (26% ee) to R (58% ee) on
raising the substrate concentration from 1 to 20 g L-'. When aged mycelium was
used, the absolute configuration was always R and showed constant enantioselectiv-
ity (ca. 50% ee) regardless of the substrate concentration, although the reduction
proceeded at a slightly slower rate.
In the aging process, an S-forming activity, was lost, leaving unaffected either one
low-specificityreducing enzyme with major R-forming activity, or several enzymes
having opposite enantioselectivitiesbut similar KM values.
15.1.2.6
Treatment of the Cell: High Pressure Homogenization
High pressure homogenization is a new technology in food processing. It was found

that the same technology can be applied to effect the microbial reduction of chemical
compounds[791.The cell culture with substrate (such as acetophenone, 5-hexen-
z-one, etc.) was poured into the high pressure homogenizer, and then it was
incubated for 48 h and the enantioselectivity of the product was evaluated. During
the process the reaction mixture was forced under pressure through a narrow gap
where it was subjected to rapid acceleration [l (blank experiment), 500, 1000, 1500
bar] after which it undergoes an extreme drop in pressure. Various strains of
Saccharomyces cerevisiae and Yarrowia lipolytica are utilized in the reduction processes
and higher enantioselectivitieswere generally achieved albeit in lower yields than the
standard process.
15.1.2.7
Treatment of the Cell: Acetone Dehydration
A dried cell mass is often used as a biocatalyst for a reduction, since it can be stored
for a long time and can be used whenever needed, without cultivation. One of the
useful methods to dry the cell mass is acetone dehydration["]. For example, the cells
of Geotrichum candidum I F 0 4597 were mixed with cold acetone (-20 "C) and the
cells were collected by filtration[20.21]. The procedure was repeated five times and
then the cells were dried under reduced pressure. The dried cells (acetone powder of
G. candidum I F 0 4597: APG4) were obtained; they can be stored for a long time in
the freezer.
The drying of the cell not only aids the preservation of the cell but also contributes
to the stereochemical control as shown in Table 15-4.The reduction of acetophenone
catalyzed by G. candidum I F 0 4597 resulted in poor enantioselectivity [28% ee(R)].
When the form of the catalyst was changed from wet whole-cell to dried powdered-
Table 15-4. Acetone treatment o f Ceotrichum candidum for the improvement of

enantioselectivity", ".
Untreated whole cell Acetone dried cell (APG4) oH

P'h * Ph NAD' or NADP' * j\Ph
28% ee (RJ 2-propanol or cyclopentanol >99% ee (S)
Catalyst Coenzyme Additive Yield ("h) ee (%)
Untreated whole cell none none 52 28(R)

Acetone dried cell (APG4) none none 0 -
Acetone dried cell (APG4) NAD' 2-propanol 89 >99(S)
Acetone dried cell (APG4) NAD' cyclopentanol 97 >99(S)
Acetone dried cell (APG4) NADP' cyclopentanol 8G >99(S)
cell (APG4), no reduction was observed, which would indicate the loss of the
necessary coenzyme(s) and/or coenzyme regeneration system@)during the treat-
ment of the cells with acetone. Addition of coenzyme, NAD', did not have a
significant effect on the yield. Addition of 2-propanol resulted in only a small
increase in the yield, but a significant improvement in the enantioselectivity was
observed. Surprisingly, addition of both NAD' and 2-propanolprofoundly enhanced
both chemical yield and enantiomeric excess. Addition of NADH, NADP' or
NADPH instead of NAD' and addition of cyclopentanol instead of 2-propanol also
gave enantiomericallypure alcohol in high yield.
The improvement in the enantioselectivity from 28 % (R) to > 99 % (S) was due to
the suppression of every enzyme which reduces the substrate, followed by the
stimulation of an S-directingenzyme by the addition of the coenzyme and an excess
amount of 2-propanol, agents which push the equilibrium towards the reduction of
the substrate.
It was confirmed, by separating the enzymes in the powder, that many S- and R-
directing enzymes exist in the biocatalyst. The addition of coenzyme and cyclopenta-
no1 stimulates only one particula S-enzymebut not other S-enzymesand R-enzymes
because the spec& S-enzyme can oxidize cyclopentanol [concomitantly reducing
NAD(P)'], while other S- or R-enzymes cannot use cyclopentanol as effectively["].
This is a very interesting case where the reduction with a cell initially having both S-
and R-directing enzymes was modified and resulted in excellent S enantioselectiv-
ity.
15.1.2.8
Cultivation Conditions of the Cell
The dependence of enantioselectivity in microbial transformations on the cultivation

conditions of the microorganisms has also been investigated[82-8G1.The enzymes
induced during the growth phase and during starvation are certainly different,
therefore the enantioselectivity of the product may be different when two competing
enzymes with different enantioselectivitiescatalyze the reduction. Since the enzyme
1004
reducing the non-natural substrate is not usually known, cultivation conditions

which induce the desired enzyme have to be found by trial and error.
For example, the effect of cultivation time and different carbon sources on the
enantioselectivity of the reduction of sulcatone by some anaerobic bacteria has been
Another example is the investigation on the effect of the medium
concentrations for cultivation of Geotrichum candidurn I F 0 4597 on the enantiose-
lectivity of the reduction of acetophenone derivatives. The yield of R-alcohol (the
minor enantiomer) increased with the medium concentration; therefore, the me-
dium concentration was kept low, optimally to produce the S-enantiomer[**]. The
effect of the aeration during cultivation on the enantioselectivity of bakers' yeast
production of 3-hydroxyesters has also been reported["]. Inducers such as a
substrate analog may also induce the desired enzyme to improve the enantiose-
lectivity.
15.1.2.9
Modification of Reaction Conditions: Incorporation o f an Inhibitor
In the case of the observation of poor overall enantioselectivity due to the presence of
two competing enzymes with different enantioselectivities,one of the most straight-
forward methods to improve the enantioselectivity is the use of the inhibitor of the
unnecessary enzyme(s). Ethyl chloroacetate, methyl vinyl ketone, allyl alcohol, allyl
bromide, sulfur compounds, Mg2+,Ca2+,etc. have been reported as inhibitors of
enzymes in yeast [87-971.
For example, the low enantioselectivity in the yeast reduction of P-keto ester was
improved by addition of ethyl chloroacetate or methyl vinyl ketone as described in
Fig. 15-13. The enzymes inhibited and those not inhibited were identified by
enzymatic studies using purified enzymes["]. The mechanism of the inhibition is
reported to be non-competitive.
These inhibitors were also used to improve the enenatioselective reduction of
inhibitor
0
inhibitor
L-enzyme-l
without
any inhibitor
i
OH
R A C H 2 C O 2 R ' low ee
Figure 15-13. Improvement o f t h e enantioselectivity by using an inhibitor of undesired

enzymes187. 88. 971
I
uR r:
1005
Allyl alcohol or
methyl vinyl ketone OH
UR Bakers’ yeast F3c
OH 0
F3C
Allyl bromide F,C aR (R)
Figure 15-14. Stereochemical control using an inhibitor[89].
fluorinated diketones (Fig. 15-14). By applying a suitable inhibitor, both enantiomers

of the alcohol can be obtained using only one kind of microorganism, namely
bakers’
15.1.2.10
Modification of Reaction Conditions: Organic Solvent
Organic solvents have been used widely for esterifications and transesterifications
using hydrolytic enzymes to shift the equilibrium towards esterification by avoiding
hydrolysis. Organic solvents can also be used for reductions using dehydroge-
nases [98-1091 . They can be used to control the overall enantioselectivity of the
reduction, when there are more than two competing enzymes with different
enantioselectivities,K M and V.,
Enzymatic reactions follow the Michaelis-Menten equation, therefore, the rate of
the enzyme catalyzed reaction depends on the substrate concentration. When an
organic solvent is introduced, most organic substrates usually dissolve in the organic
phase, and the effective substrate concentration in the aqueous phase around the
enzyme decreases. The change in substrate concentration by the addition of the
organic phase causes the change in the enzyme species catalyzing the reduction. For
example, as shown in Fig. 15-15, if the K M for an S-directingenzyme is much smaller
than that for an R-directing enzyme, and V, for the S-directing enzyme is much
smaller than that for the R-directing enzyme, then when the substrate concentration
is low, the S-enzymewill dominate, whereas at high substrate concentration, the R-
enzyme will dominate the biotransformation.
In fact, when the yeast reduction of ethyl 2-oxohexanoatewas conducted in water,
’ R-enzyme
velocity
€enzyme
Figure 15-15. Effect of
the substrate concentra-
tion on enantioselectivity
of the reduction with the
system having both an
IS1
Substrate concentration S-enzyme with small KM
and small V, and an
R-enzyme with large KM
At low substrate conc. the At high substrate conc. the
Senzyme predominates. R-enzyme predominates. and large V,.
1006
(9
Further,,
BuKCO,Et decomposition
\
yeast
in benzene OH
B U L C o 2 E t (4
Figure 15-16. Stereochemical control by using an organic solvent.
Table 15-5. Mechanism of stereochernical control using benzene: kinetic parameters of yeast
a-keto ester reductases (YKERs)'". 'lo.
Enzyme Enantioseledivity KM (mM) t a t (s-') VmaX(U kg-' yeast)

YKER-I R 8.40 1.53 37.7
YKER-IV R 0.142 4.59 41
YKER-V s 5.72 27.8 649
YKER-VI R 1.03 2.10 1774
YKER-VII S 27.3 127 501
both (R)- and (S)-alcoholswere produced and the (S)-alcohol was obtained as the
major product as a result of the further enantioselective decomposition of the (R)-
enantiomer (Fig. 1S-16)['00slo9]. H owever, when the biotransformation was con-
ducted in benzene, then the (R)-alcoholwas formed selectively in high yield.
KM and V,, for all enzymes existing in yeast and catalyzing the reduction were
determined and it was found that an R-enzyme, YKER-IV, has a KMwhich is smaller
than other enzymes by an order of magnitude (Table 15-S), and, therefore, predom-
inantly catalyzes the reduction in benzene"". llo, lll].
15.1.2.1 1
Modification of Reaction Conditions: Use of a Supercritical Solvent
Supercritical fluids, materials above their critical pressure and critical temperature
(Fig. 15-17),have been attracting attention as solvents with the advantages of gas-like
low viscosities and high diffusivities coupled with their liquid-like solubilizing
power r112]. Supercritical carbon dioxide (SCCOZ) has the added benefit of an envir-
onmentally benign nature, nonflammability, low toxicity, ready availability, and
ambient critical temperature (T, = 31.0 "C) that is suitable for biotransformations.
The attraction of combining natural catalysts with a "natural" solvent has been the
driving force behind a growing body of literature on the stability, activity and
specificity of enzymes in SCCOZ.The first report on biotransformations in super-
critical fluids was in 1985[113-115],and the benefit of using supercritical fluids for
biotransformations has been demonstrated, e. g. through improved reaction rates,
etc.[116. 1171
Recently the alcohol dehydrogenase from Geotrichum candidum was found to

Figure 15-17.
Phase diagram o f carbon dioxide.

I 1007
_______------
-100 0 100
Temperature (OC)
I: OH
Immobilized Geotrichum candidurn cell
R ~ R '
Supercritical CO?
- R-R'
R = CH, CH,F, etc. -

Yield 11 96%
R' = Ph, 0- , rn- or pfluorophenyl, Ph-(CH,),- etc. ee 96 - 299%
Figure 15-18. Reduction of fluoroketones by Ceotrichum can-

didum I F 0 5767 in supercritical C O Z " ' ~ ~ .
catalyze the reduction of fluoroacetophenones etc. in scCO2 at around 100 atm and
35 "C (Fig. 15-18)[''81. The enantioselectivity obtained was equivalent to the system
using an organic solvent.
15.1.2.12
Modification of Reaction Conditions: Cyclodextrin
Cyclodextrin has also been used to control the enantioselectivity of bioreduc-

tion~['~'-'~'1.When added to a reaction mixture, the substrate can reside in the
cyclodextrin, which decreases the effective substrate concentration around the
enzyme and results in the domination of reactions involving enzymes with low KM.
The effect can be demonstrated by the reduction of ketopantoyl lactone by yeast. The
enantioselectivity was improved from 7 3 % to 9 3 % by adding P-cyclodextrin to the
reaction mixture. The improvement in enantioselectivity of the reduction in the
presence of enzymes with different enantioselectivitiesand KMvalues by decreasing
the substrate concentration was confirmed by the ineffectiveness of a-cyclodextrin
which is too small to include the substrate. It was also confirmed by dilution of the
reaction mixture, which improved the enantioselectivity in the absence of cyclodex-
trin.
15.1.2.1 3
Modification o f Reaction Conditions: Hydrophobic Polymer XAD
A decrease in the effective substrate concentration around the enzyme but not in the
bulk can also be achieved using hydrophobic polymer XAD instead of using
cyclodextrin or an organic solvent['22-'26].For example, the technique was used in
the reduction of methyl benzyl ketone by Zygosaccharomyes rouxii for the synthesis of
LY300164, a noncompetitive antagonist of the AMPA subtype of excitatory amino
acid The adsorption properties of the resin on both substrate and
1008
Zygosaccharomyces rouxii
a ketone loading 5K2,3-benzodiazepine

(LY300164)
Ydrl.7 ,,,+ - " croui?$ I

I'
Adsorbed species:
2 g/L of product alcohol
Initial: 80 g/L of ketone

final: 75 glL of product alcohol *OH
0 0
Y Y
Figure 15-19. Decrease in the effective substrate concentration around the enzyme by using
hydrophobic polymer XAD[12z].
product allowed a ketone loading of 80 g L-l, while limiting the effective solution
concentration of both substrate and product to sublethal concentrations of 2 g L-'
(Fig. 15-19).
The hydrophobic resin has also been used for the purpose of controlling selectiv-
ity[123, 1241. E~ antioselectivity, chemoselectivity and space-time yields of the yeast
reduction of a,P-unsaturated carbonyl compounds were impressively enhanced. The
distribution of substrates and products between the resin and the water phase
showed that the improved selectivity could be attributed to the control of substrate
concentration.
The powerful influence of the hydrophobic resin was also demonstrates in the
Geotrichum candidum catalyzed reduction of simple aliphatic and aromatic ke-
tones [126]. For example, the enantioselectivity of the reduction of 6-methylhept-5-en-
2-one was improved from 27 % ee ( R ) to 98 % ee (S).
15.1.2.14
Modification o f Reaction Conditions: Reaction Temperature
Reaction temperature is one of the parameters that affects the enantioselectivity of a

r e a ~ t i o n [ ~ ~For
- ~the
~ ] oxidation
]. of an alcohol, the values of k,,,/KM were determined
for the ( R ) -and (S)-stereodefining enantiomers; E is the ratio between them. From
the transition state theory, the free energy difference at the transition state between
(R)-and (S)-enantiomers can be calculated from E [Eq. (2)], and AAG is in turn the
temperature function [Eq. (3)) The racemic temperature (T,) can be calculated as
shown in Eq. (4).With these equations, T, for 2-butanol and 2-pentanol of the
Thertnoanaerobacter ethanolicus alcohol dehydrogenase was determined to be 26 "C
and 77 "C, respectively.
Moraxella sp. TAEl23
alcohol dehydrogenase
0°C NADH
E = (kcat / KM)R/ ( k a t /
--OH
ee >99% (SJ
Figure 15-20. Reduction of 2-butanone by

the alcohol dehydrogenase from Moraxella
sp. TAE123 a t 0 0C[171.
from transition state theory

- RTln(E) = AAGI
AAGI = AAHI - TAASI
When
AAGI = 0, T, = AAHI 1 AASI (4)
Since the transition state for alcohol oxidation and ketone reduction must be
identical, the product distribution (under kinetic control) for reduction of 2-buta-
none and 2-pentanone is also predictable. Thus, one would expect to isolate (R)-
2-butanol if the temperature of the reaction was above 26 "C. On the contrary, if the
temperature is less than 26 "C, (S)-2-butanolshould result. In fact, the reduction of
2-butanone and 2-pentanone at 37 "C resulted in 28 % ee (R)- and 44% ee (S)-alcohol,
respectively, as expected [431.
The temperature range that can be used for a biocatalytic reduction is very wide
because alcohol dehydrogenases from various types of microorganisms (thermo-
philic and psychrophilic) are available. The extremely high stability of enzymes from
thermophilic microorganisms are discussed in Sect. 15.1.1.4. On the other hand,
conducting reactions at temperatures as low as 0 "C is also possible using an
Antarctic psychrophile [171. For example, the reduction of 2-butanone, which is an
extremely challenging substrate for enantioselective reduction, with alcohol dehy-
drogenase from Moraxella sp. TAE123, at 0 "C afforded (S)-2-butanol in > 99% ee
(Fig. 15-20).
15.1.2.15
Modification of Reaction Conditions: Reaction Pressure
The effect of high hydrostatic pressure (400 bar) on microbial reductions of the
ketones such as acetophenone, etc. has been examined using various strains of
Saccharomyces cerevisiae and Yarrowia lipolytica. Higher enantioselectivitiesare gen-
erally achieved together with lower yields compared with the results obtained at
atmospheric pressure as in the case of treatment of cells with high pressure
homogenation [791. Although the enantioselectivity obtained here is not as high as
> 99% ee, this finding added pressure as an adjustable parameter to control the
enantioselectivity of the bioreduction.
1010
15.1.3
Improvement of Dehydrogenasesfor use in Reduction Reactions by Genetic Methods
15.1.3.1
Overexpression o f the Alcohol Dehydrogenase
Recent developments in molecular biology have contributed to the development of

useful biocatalysts. Overexpression as well as rational and random mutations of
many alcohol dehydrogenases have improved the function of enzymes so that they
can be useful in organic synthesis[22,127-1341 . E xamples of overexpressed enzymes
are introduced here, and Sect. 15.1.3.2-15.1.3.8 will describe the improvement of
catalytic functions achieved by using genetic methods. Although the non-genetic
chemical modifications of enzymes can also be important in order to improve a
bio~atalyst['~~],
they are not mentioned here.
Example 1: The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secon-
dary alcohol dehydrogenase was overexpressed in Escherichia coli to form more than
10% to total protein[136].The recombinant enzyme was purified by heat treatment
and precipitation with aqueous (NH4)2S04and isolated in 67 % yield. Enzymes with
mutation@) around the active site residues were also created to examine the
catalytically important zinc binding motif in the proteins.
Example 2: The gene encoding a phenylacetaldehyde reductase with a unique and
wide substrate range was cloned from the genomic DNA of the styrene-assimilating
Corynebacteriurn strain ST10[137-139J . The enzyme was expressed in recombinant E.
coli cells in sufficient quantity for practical use and purified to homogeneity by three
column chromatography steps [140]. The amino acid residues assumed to be three
catalytic and four structural zinc-binding ligands were characterized by site-directed
mutagenesis of two zinc-binding centers within the enzyme
Besides these examples, many other important enzymes for biocatalytic reductions,
such as the NADPH-dependent carbonyl reductase from Candida r n a g n ~ l i a e [ ~the
~~I,
ketoreductase from Zygosaccharornyces rouxii and the aldehyde reductase from
Sporobolomyces salmonicolor AKU4429 [1441, etc. have also been expressed in E. coli etc.
and shown to be active.
The availability of sufficient quantities of enzymes for crystallization studies has
led to the crystal structures been obtained for several dehydrogenases. For example,
two tetrameric NADP+-dependentbacterial secondary alcohol dehydrogenases from
the mesophilic bacterium Clostridiurn belj'erinckii and the thermophilic bacterium
Thermoanaerobium brockii have been crystallized in the apo- and the holo-enzyme
forms, and their structures are available in the Protein Data Bank[145]. The crystal
structure of the alcohol dehydrogenase from horse liver is also available [40-421.
15.1.3.2
I
Access to a Single Enzyme Within a Whole Cell: Use of Recombinant Cells
The advantages and disadvantages of using whole cell and isolated enzymes are
described in Sect. 15.1.1.3. Here, genetic methods are used to build the systems with
the advantages of both whole cells and isolated enzymes; the technology enables one
to access essentially a single enzyme within a whole cell[127].
For example, to improve a low enantioselectivity due to the presence of plural
enzymes in a cell with overlapping substrate specificities but different enantiose-
lectivities, a recombinant cell with only the enzyme possessing the desired enantio-
selectivity was used (Fig. 15-21). Isolation of the enzyme, of course, improves the
enantioselectivity.However, the requirement of a laborious enzyme isolation process
and expensive cofactor with its associated regeneration enzyme (if necessary) have
limited the practical utility of isolated enzyme processes. However, once the gene
encoding the enzyme with high enantioselectivity has been overexpressed in E. coli,
then the essentially single enzyme system can be accessed within the whole cell.
Since it is a whole cell system, it can be cultivated to supply an appropriate amount
without involving a laborious process for the isolation of an enzyme. The fact that
there is no coenzyme requirement is also a merit for the system. Because it has only
one enzyme which transforms the substrate, the problems of overmetabolism or low
selectivity are also resolved. Using E. coli expressing Gcylp and E. coli expressing
Gre3p, various 0-keto esters and a-alkyl-0-ketoesters were reduced with excellent
enantio- (up to > 98% ee) and diastereo-selectivities (> 98% de) [*'*I.
I Yeast catalyzed blotransfonnations

~ - ...... Isolated enzyme catalyzed
WPH .- - -
:S-enqmel
- ---- biotransformation
catalyst= r-------,
Isolation of
an enzyme
A
I I I i
Sometimes result in low selectivity Coenzyme will be necessary
Enzymes with overlapping substrate specificities Limited supply due to the
but ditferent enantioselectiiitiespresent
Creation of engineered E. coli

strains expressing an enzyme
I Recombinant cell expressing the enzyme of interest from yeast

catalyzed biotransformation
I
Single catalytic species (high enantio- and chemoselectivity, no overmetabolism)
No coenzyme necessary
No laborious process for the isolation of an enzyme
Figure 15-21. Advantages and disadvantages of whole cell, isolated enzymes and recombinant
cell as biocatalysts.
1012
I I5 Reduction Reactions
Figure 15-22. Use o f FAS deficient

yeast t o improve the diastereoselectiv-
ity of a
cis (3R,4S) trans (3R,4R)

Commercial yeast 48 : 38
FAS deficient yeast 36 3
15.1.3.3.
Use o f a Cell Deficient in an Undesired Enzyme
This is a similar approach to that described above. Use of a yeast strain deficient in
fatty acid synthase (FAS) suppressed formation of the undesired trans-diastereomer
of a 0-lactam as shown in Fig. 15-22['*'].
15.1.3.4
Point Mutation for the Improvement of Enantioselectivity
Point mutation of enzymes has played an important role in determining those

amino acid residues involved in catalytic activities. It has also been used to improve
the enantioselectivity of dehydrogenases. For example, even a single point mutation
of a secondary alcohol dehydrogenase from Themoanaerobacter ethanolicus can
change substantially the enantioselectivity for the reduction of 2-butanone and
2-pentanone as shown in Table 15-6[451.
15.1.3.5
Broadening the Substrate Specificity of Dehydrogenase by Mutations
Developments in molecular biology enable us to change the substrate specificity of

enzymes; the enzymes can be engineered to be more suitable for the requisite
substrate. For example, variations have been made to the structure of the NAD'
dependent L-lactate dehydrogenase from Bacillus stearothemophilus (LDH)[1301. Two
regions of LDH that border the active site (but are not involved in the catalytic
Table 15-6. Control o f enantioselectivity by a single mutation (serine-39 t o threonine) of the

secondary alcohol dehydrogenase from Thermoanaerobacter e t h a n ~ l i c u s ~ ~ .
Parameter Wild type Mutant (S39T)
( M ' s-') for oxidation at 55 "C of:

kat/K~
(R)-2-butanol 3.1 x i 0 5 2.8 x10'
(S)-2-butanol 1.1 x105 0.29~ lo5
(R)-2-pentanol 0 . 8 7 ~105 3.5 x10'
(S)-2-pentanol 1.3 xi05 2.1 X l O S
Ee of the reduction at 55 "C of:
2-butanone
2-pentanone
Table 15-7.
Broadening the substrate specifity o f L-lactate dehydrogenase from Bacillus

I
1013
stearothermophilus by rational protein engineerit~g'~'.
Wild Type CH3 250 0.06 4200000
-
102-'oSGlnLysPro MetValSer
CHiGH(CH3)z
CH3
CH2CH(CH3)2
0.33
66
0.67
6.7
0.16
1.9
410000
353
50
23GZ37AlaAla+ GlyGly CH3 167 4 42 000

CH2CH(CH3)2 1.74 15.4 110
'02c'05GlnLysPro + MetValSer CH3 32 4 8000
/236237AlaAla GlyGly
+ CH;?CH(CH3)2 18.5 14.3 1300
reaction) were altered in order to accommodate substrates with hydrophobic side

chains larger than that of the naturally preferred substrate, pyruvate. The muta-
tions [102-'051GlnLy~Pr~ --* MetValSer and [23G-2371AlaAla -+ GlyGly were made to
increase to tolerance for large hydrophobic substrate side chains as shown in Table
15-7.The five changes together produced a broader substrate specificity LDH, with a
55 fold improved k,,, for a-keto isocaproate [R = CH2CH(CH+].
The substrate specificity of isocitrate dehydrogenase (IDH) has also been rede-
signed by genetic methods [13'1. Despite the structural similarities between isocitrate
(ISO) and isopropylmalate (IPM),wild type isocitrate dehydrogenase (IDH) exhibits
a strong preference for its natural substrate (ISO). The substrate specificity of IDH
was changed to that of isopropylmalate dehydrogenase (IPMDH) using a combina-
tion of rational and random mutagenesis. Three amino acids of IDH (S113, N115,
V l l b ) were changed and the chimeric enzyme ETV (S113E, N114T, V116V) showed
Table 15-8. Redesigning the substrate specificity of isocitrate dehydr~genase'~'.
0 OH
~~z~Xr,.~ OH
HOzc&co2H
OH
isocitrate (ISO) isopropylmalate (IPM)
Enzyme IDH position Lt/KM IPM kcat/KM IS0 kcat/b IPM

113 115 116 (tW1S-') (W' S-') kcat/Khn IS0
Wild Type IPMDH E L L 1.4~10 0 -
Wild Type ID H S N V 1.7x10-" 1.6~10 1.0~10-~
EVG E V G 1.1~10-~ 1.1~10-~ 1.0
ENA E N A 1 . 5 lo-'
~ 5.9~ 2.5
ETV E T V 1.8~10-~ 3.9x10-' 4.6
1014
Table 15-9. Elimination of the cofactor requirement by “blind” directed e v o l ~ t i o n ’ ~ ~ .
Bacillus stearothermophillus Cofactor Kt.APYruvate
lactate dehydrogenase (Fructose 1,dbisphosphate) (mM )
Wild + 0.05
Wild - 5
Mutated (R118C, 203L, N307S) + 0.05
Mutated (R118C, 203L, N307S) - 0.07
/ [kCat/K~ISO]
a preferred substrate specificity for IPM over ISO; [kcat/K~IPM] of
ETV was 4.6 while that of wild type IDH was 1.0 x
15.1.3.6
Production of an Activated Form of an Enzyme by Directed Evolution
One of the drawbacks of using alcohol dehydrogenases as catalysts for organic

synthesis (comparing them with hydrolytic enzymes) is the cofactor require-
ment For example, Bacillus stearothermophillus lactate dehydrogenase is activated
in the presence of fructose l,G-bi~phosphate[~~*]. The activator is expensive and
representative of the sort of cofactor complications that are undesirable in industrial
processes. Three rounds of random mutagenesis and screening produced a mutant
which is almost fully activated in the absence of fructose 1,G-bisphosphate as shown
in Table 15-9.
15.1.3.7
Change in the Coenzyme Specificity by Genetic Methods: NADP(H) Specific Formate
Dehydrogenase
Formate/formate dehydrogenase is one of the most useful coenzyme regeneration

systems as has been described in the Sect. 15.1.1.2. However, the known wild type
formate dehydrogenases only accept NAD+; NADP’ is not the substrate. Multipoint
site-directed mutagenesis was used to create a formate dehydrogenase which was
able to accept NADP+.This mutant enzyme was then coupled to the reduction using
the alcohol dehydrogenase from Lactobacillus sp as shown in Fig. 15-23t2*l. The
activity of the NADP(H)-specific mutant (with NADP’ as substrate) is about GO% of
the activity ofwild type formate dehydrogenase (with NAD’ as substrate).
15.1.3.8
Use of a Mutant Dehydrogenase for the Synthesis of 4-Amino-2-HydroxyAcids
The usefulness of a mutant dehydrogenase was demonstrated in a practical synthe-

sis of 4-amino-2-hydroxy acids, which themselves are valuable as y-turn mimics for
investigations into the secondary structure of peptides [1461. Chemoenzymatic synthe-
sis of these compounds were achieved by lipase catalyzed hydrolysis of a a-keto
esters to the corresponding a-keto acids followed by reduction employing a lactate
dehydrogenase in one pot. Wild type lactate dehydrogenase from either Bacillus
15. I Reduction ofKetones
I
1015
0
)cph Lactobacillus sp. alcohol dehydrogenase
~ - APh
OH Figure 15-23. Recycling o f
NADPH with protein engi-
neered formate dehydroge-
nase[”].
NADPH NADP’
protein engineered formate dehydrogenase

from Pseudomonas sp.101
Table 15-10. The use o f a mutant dehydrogenase for the synthesis of 4-amino-2-hydroxyacids’46.
Dehydrogenase R Reaction Time Yield (“A)
Wild type a:CH3 4 days 67

Staphylococcusegidemidis b:CH(CH3)2 no reaction -
lactate dehydrogenase C: CH2CH(CH3)2 no reaction
d: CHzPh no reaction
H205Q mutant of a:CH3 4h 85
Lactobacillus delbrueckii bulgaricus b: CH(CH3)z 5h 90
D-hydroxyisocaproatedehydrogenase c: CHzCH(CH3)z 4h 78
d: CHzPh 5h 85
stearothermophilus (BS-LDH)or Staphylococcus epidermidis (SE-LDH)could be used

specifically to reduce the ketone of the alanine derived a-keto acid, 2a, giving the (S)-
and (R)-2-hydroxyacids, respectively, in good yields.
However, more bulky a-keto acids 2 b 2 d were not substrates for these enzymes. In
contrast, the genetically engineered H205Q mutant of Lactobacillus delbrueckii
bulgaricus D-hydroxyisocaproate dehydrogenase proved to be an ideal catalyst for the
reduction of all the a-keto acids 2a-2d, giving excellent yields of the CBZ-protected
(2R, 4S)-4-amino-2-hydroxyacid as a single diastereomer (Table 15-10).This geneti-
cally engineered oxidoreductase has great potential value in synthesis, not only due
to its broad substrate specificity but also due to the high catalytic activity. For
example, reduction of 1mmol of 2a took just 4 h with the H205Q mutant, whereas
with SE-LDH the reaction required 4 days.
15.1.3.9
Catalytic Antibody
Nakayama and Schultz have developed antibodies to carry out the catalyhc enantiose-
lective reduction of an a-keto amide using NaBH3CN as the r e d ~ c t a n t “ ~Mono-
~].
clonal antibodies raised to phosphonate 3 were prepared (Fig. 15-24), and one
antibody showed activity for the enantioselective reduction of a chiral keto amide 4.
I5 Reduction Reactions
1016
I
hapten 3
‘“W;t antibody
NaBH3CN ~ 0zNq;h+
0 CH3 0 EH3
4 (2s)
Figure 15-24. Reduction of a ketone by a catalytic antibody”47!
Reduction with the antibody gave the 2s product with a diastereomeric excess
greater than 99 % (oppositeto the stereoselectivityof the uncatalyzed reaction which
afforded the 2 R product).
15.1.4
Reduction Systems with Wide Substrate Specificity
15.1.4.1
Bakers’ Yeast
Many methods for asymmetric reduction have been developed and some of these are
used for the synthesis of optically active alcohols on a preparative scale. Bakers’ yeast
is one of the most widely used microorganisms due to its commercial availability and
its wide substrate specificity, which enables the non-expert in biochemistry to use the
biocatalyst as a reagent for organic synthesis. Detailed reactions will not be described
in this text since there are many reviews and original reports on this sub-
ject[’. 37-39, 148-1621 . H owever, one of the most important and useful reactions using
yeast, the reduction of a hydroxymethyl ketone, is featured here due to the excellent
enantioselectivity obtained even on a large scale (Fig. 15-25).1163-1661 . For example,
1-hydroxy-2-heptanone(50 g) was reduced to the corresponding (R)-diol in an
optically pure form in 56% yield [Fig. 15-25 (b)][1641. Another example [Fig. 15-25(c)]
is the reduction of a sulphenyl hydroxyketonewith yeast in the synthesis of a natural
product [166]. Products isolated from the mandibular glands of the oriental hornet
were synthesized using yeast reduction of an S-substituted hydroxyketone.
15.1.4.2
Rodococcus erythropolis
A carbonyl reductase isolated form Rhodococcus erythropolis accepts a broad range of

substrates, including a variety of compounds useful for synthetic chemistry, as
shown in Table 15-1112s1.Reduction of all the carbonyl compounds tested yielded
(S)-configuredhydroxyl compounds with high enantioselectivities.
I 1017
(a) OAc Yield 97%

ee >95%
0 OH
yeast
Yield 56%(isolated)
large scale (509)
(c)
0
H O A S P h A
yeast
OH
H O A S P h
- 0
&SPh
-
-
-
Yield 90% ee 78%
Yield 63% ee 100%
(after recrystallization)
Figure 15-25. Reduction of hydroxyketones by bakers’ yeast[’63, 164, 1661.
Table 15-11. Kinetic constants of the R. erythropolis carbonyl reductase2’.
Substrate V, KM Substrate V, KM
Pmg-1 (mM) (U mg-’1 (mM)
A 3.5 330 0.46 18
L 3.5 260 & 1.4 7.3
An 4.8 59 M0/ 2.6 16
0 0
L 7.7 3.8
u
0
0
0
-
5.5 3.1
10.4 9.9
0.59 C I A O - 4.2
10.3 0.42 &04 7.6 8.3
A 10.8 0.34 10.6 0.039

0
11.1 0.54 1.7 3.8
15.1.4.3
Pseudomonas sp. Strain PED and Lactobacillus kefir
The substrate specificities of the alcohol dehydrogenases from Pseudomonas sp.

strain PED and Lactobacillus ke$r have been investigated. It was reported that they
reduce wide varieties of ketones [6, ’1. Both reactions use 2-propanolfor the regenera-
tion of coenzyme and produce (R)-alcoholsas depicted in Table 15-12. However, they
require different coenzymes. The alcohol dehydrogenase from the Pseudomonas sp.
uses NADH and transfers to pro-R hydride of NADH to the si-face of carbonyl
compounds as shown in Sect. 15.1.1.1. The mechanism is ordered bi-bi with the
coenzyme binding first and released last. On the other hand, the enzyme from
1018
I I5 Reduction Reactions
Table 15-12. Enantioselectivities of the alcohol dehydrogenases from Pseudornonas sp. strain PED
and Lactobacillus kefiP, '.
ee (%) Product ee (%%I

Product
Pseudomonas Lactobacillus Pseudomonas Lactobacillus
sp. strain PED kefr sp. strain PED kefr
OH 0 OH -
98
PhACF, 92 > 99 \o&cl
OH - 97 > 99
94
Phi
OH
OH
86 CI4 93 > 97
P h / K
0 OH
OH 45
Ph+', 98
27
0
Lactobacillus kejr uses NADPH and transfers the pro-R hydride from the cofactor to
the si-face of carbonyl compounds.
15.1.4.4
Thermoanaerobium brockii
The alcohol dehydrogenase from Themoanaerobium brockii is very suitable for the
reduction of aliphatic 191. Even very simple aliphatic ketones can be
reduced enantioselectively.An interesting substrate size-induced reversal of enantio-

selectivity was observed. The smaller substrates (methyl ethyl, methyl isopropyl or
methyl cyclopropyl ketones) were reduced to the (R)-alcohols, whereas higher
ketones produced the (S)-enantiomers.
This example and the next one (Sect. 15.1.4.5) using G. candidurn show that the
biocatalytic reduction system is very beneficial for the reduction of aliphatic ketones
over a non-enzymatic system where no report on highly enantioselective (> 99% ee)
reduction of unfunctionalized dialkyl ketones can be found, to the best of our
knowledge.
-
Table 15-13. Asymmetric reduction o f aliphatic ketones with the alcohol dehydrogenase from
Therrnoanaerobiurn brockii".
Product Relative rate ee VO)Config. Product Relative rate ee (%) Config.
12.0 48 R OH
0.9 99 s
OH
3.0 8G R OH
w 0.2 95 s
XLl
OH
0.8 44 R oH
/+/.v-/ 0.G 97 s
3.3 79 s
0.3 99 s
1.0 96 S
OH
0.3 98 S
0.3 95 s OH
0.1 99 s
0.1 81 2S,3R OH
L C l 1.5 98 S
0.9 97 s
15.1.4.5
Ceotrichum candidum
Reductions using an acetone powder of G. candidum (APG4), NAD' and 2-propanol

exhibit one of the widest substrate specificities together with very high enantiose-
lectivities (Table 15-14) 21].Various ketones such as acetophenone derivatives can
be reduced with APG4 with excellent enantioselectivities(> 99% ee). The nature and
electronegativityof substituents on the phenyl ring did not affect the enantioselectiv-
ity although the yield was slightly lower for para derivatives than for the correspond-
ing ortho and meta derivatives.
Reduction by APG4 of several aromatic ketones having different length alkyl
chains demonstrated the scope and limitations of the substrate specificity. The
phenyl moiety of acetophenone can be replaced by a benzyl or even by a 2-phenyl-
ethyl group with slightly better results in terms of chemical yield without any
decrease in enantioselectivity.However, when the methyl moiety of actophenone was
replaced by an ethyl, isopropyl or methoxymethyl group, the yield decreased
dramatically, although the enantioselectivity remained high (z 99% ee). When the
alkyl chain was elongated to a propyl or enlarged to a t-butyl group, the reaction was
observed scarcely to proceed.
The versatility of the APG4 reduction system is further exemplified by the use of p-
keto esters as substrates. 3-Oxobutyrates involving methyl, ethyl, t-butyl, or neo-
pentyl esters are reduced to the (S)-hydroxyesterswith > 99% ee and in quantitative
yield. Moreover, simple aliphatic ketones from 2-octanoneto 2-undecanone, as well
1020
Table 15-14. Reduction ofvarious ketones by the acetone powder of C. candidum, NAD' and
2-propanoIz0.21.
Product Yield ("A) ee ("A) Product Yield ("A) ee ("A)
X=H 89 >99(S) R = Et 41 > 9 9 (S)

O-F > 99 > 99 (S) Pr 0 -
OH m-F 95 > 9 9 (S) OH i-Pr 12 99(S)
pF 74 >99(S) RAPh t-Bu 1 -
0-CI >99 >99 (S) CHzOMe 8 >99(R)
X m-CI 95 99 (S) CHzCl 80 98(R)
p-C1 62 > 9 9 (S)
o-Br 97 >99 (S) R=Me >99 >99(S)
m-Br 92 >99(S) Et >99 >99 (S)
p-Br 95 > 9 9 (S) t-Bu >99 > 99 (S)
o-Me 96 > 99 (S) neo-Pentyl > 99 > 99 (S)
m-Me 86 >99(S) OH 0
p-Me 78 >99(S) *o.., 72 >99 (S)
o-Me0 84 >99(S)
m-Me0 90 > 99 (S) OH R=me 87 >99 (S)
p-Me0 29 > 9 9 (S) W R Et 87 >99 (S)
o-CF~ 6 97 (S) Pr 85 >99 (S)
m-CF3 96 >99(S) Bu 60 >99 (S)
p-CF, 73 >99 (S)
1',2',3',4',5'-Fs 62 > 99 (S) 90 99 (S)
OH OH
&Ph 96 > 99 (S) 92 99 (S)
OH
*Ph 93 >99 ( S )
as 6-rnethyl-S-heptene-2-one and 5-chloro-2-pentanoneare also reduced by the APG4

system to the corresponding (S)-2 alkanols giving high yields with 99% ee.
In summary, a detailed investigation of substrate specificity for the acetone powder
of a G. candidum system reveals that as long as there is a methyl group at the a-
position of the carbonyl group, high yield and enantioselectivity can be obtained
regardless of the substituent on the other side of the ketone moiety.
Apart from acetone-dried G. candidum I F 0 4597, intact whole cells of various
strains of G. candidum have been found to be useful for asymmetric reduc-
tions[75-78, 101. 126, 1G7-1711 . For example, methyl 2-acetylbenzoatewas reduced by G.
candidum ATCC 34614, I F 0 5767 or I F 0 4597 as well as by other microorganisms
such as Mucor javanicus, Mucor heimalis, Endomyces magnusii, Endomyces reessii and
bakers' yeast to afford phthalide derivatives (Fig. 15-26)which have various pharma-
cological profiles such as relaxant, antiproliferative or antiplatelet effects, e t ~ . [ ' ~ ~ ] .
75.7 Reduction ofKetones
I
lo2'
Figure 15-26. Asymmetric reduction by G. candidurn ATCC 34614 for the

synthesis of a bioactive phthalide d e r i ~ a t i v e " ~ ' ] .
15.1.5
Reduction of Various Ketones
15.1 S.1
Reduction of Fluoroketones
The biocatalytic reduction of fluoroketones is useful in order to gain an insight

into the enzyme recognition of fluorinated groups, and is also very important due to
the high synthetic values of the products, optically active fluorinated alco-
hols [lc.O, 172-1851. Sometimes the monofluorinated substrate can be a straightforward
mimic of the unsubstituted counterpart, but with difluorinated and trifluorinated
substrates, different recognition patterns compared with unfluorinated or mono-
fluorinated substrates and with each other are often observed. For example, the
enantioselectivity of yeast reduction is definitely affected by the fluorination pattern
on the One of the most prominent effects of the fluorination of a
substrate is seen in the reduction of acetophenone derivatives by the acetone powder
of Geotrichum candidurn (APG4) as shown in Fig. 15-27[173,1741. Reduction of methyl
ketones afforded (S)-alcohols in excellent ee, whereas the reduction of trifluoro-
methyl ketones gave the corresponding alcohols of the opposite configuration, also
in excellent ee. Monofluoroacetophenone and difluoroacetophenone were also
reduced under the same conditions. The reduction proceeded quantitatively for both
substrates. As expected, the stereoselectivity shifted from the acetophenone type to
the trifluoroacetophenone type according to the number of fluorine substituents at
the a-position as shown in Fig. 15-28.
The replacement of the methyl moiety with a trifluoromethyl group alters the
bulkiness and electronic properties: the effect on the enantioselectivity has been
examined. No inversion in stereochemistry was observed for the reduction of
hindered ketones such as isopropyl ketone, while the stereoselectivity was inverted
for the reduction of ketones with electron-withdrawingatoms such as chlorine. The
mechanism for the inversion in stereochemistry was investigated in further studies.
Several enzymes with different enantioselectivities were isolated; one of them
OH
H3CAph
- 0
X3CKPh -
acetone powder G. candidurn
OH Figure 15-27. Reduction of
acetophenone and trifluoroa-
cetophenone by an acetone
(s) NADP+ (9 powder of Geotrichum candid-
Cyclopentanol
Yield ,99%
urn, NADP' and cyclopenta-
Yield 90%
ee >99% X=HorF ee 98% no11' 73. 1741.
1022
OH 100
Configuration =
X3CAPh
50
ee of product
0
(%)
-50
OH
Configuration = b
X3CA Ph -1 00 I I I
JPh FJPh F$Ph

F L$Ph
F
Substrates
Figure 15-28. Effect o f introducing a fluorine atom or atoms at the a-position

of acetophenone on the stereoselectivity in the reduction by C. candidurn
acetone
&cx3 & cx3

X=HorF
Figure 15-29. Substrates used for the exam-
ination o f t h e stereodirecting effects o f
trifluoromethyl and methyl
catalyzed the reduction of methyl ketones, and another, with the opposite enantiose-
lectivity, catalyzed the reduction of trifluoromethyl ketones.
The differing abilities of trifluoromethyl and methyl groups to direct enantioselec-
tion in the reduction of carbonyl substrates has also been analyzed using various
other microorganisms including different strains of G. candidum, Hansenula anom-
ala, Saccharomyces cervisiae, Streptomyces, e t ~ . [ ’ ~The
~ ] . reduction of the cyclic ketone
and enones shown in Fig. 15-29was investigated. The differences in the electronic
and steric properties of the trifluoromethyl and methyl residues resulted in different
chemo- and enantioselectivities in the reduction of the phenylbutenones, while the
cyclohexanones showed similar enantioselectivities.
Many synthetically valuable reactions involving reductions of fluoroketones have
been reported as shown in Fig. 15-30[17G-1781. Various monofluoroketones are
reduced with yeast; some of them proceeded with high diastereoselectivity.
Chiral trifluoromethyl benzyl alcohols are useful synthons for ferroelectric liquid
crystals. Therefore, Fujisawa et al. investigated the asymmetric reduction of the
corresponding ketones using bakers’ yeast [179. l8’]. The enantioselectivity of the
bakers’ yeast reduction of trifluoroacetylbenzene derivatives was improved by the
introduction of some functional groups at the para-position to give the correspond-
ing (R)-trifluoromethyl substituted benzylic alcohols in high chemical and optical
yields as shown in Fig. 15-31.The “enantio-controlling”functional group at the para-
position was then used in further transformations.
Yeast and G. candidurn acetone powder (APG4) are complementary to each other
in the reduction of various trifluoromethyl biphenyl ketones. Yeast reduction affords
the (R)-alcohol, whereas G. candidurn reduction affords the (S)-alcohol (Fig. 15-
32) [181].
I 1023
OH
yeast Diastereomeric ratio up to 72/28
Ri...yRA.y ee up to 86%
F F
R = Me, Et, Pr, Bu
R, = Me, Et, Pr, Bu

R, = Me, Et
yeast
F3C LR
-
R = Ph, Pr, Bu
F3C
Figure 15-30. Reduction of fluorinated ketones by y e a ~ t [ ’ ~ ~ - ’ ~ ~ ~ .
OH
F 3 C h - yeast
F 3 C m E
- ferroelectric liquid crystals
R R
upto 92% ee
R = C02H, CO Me, NH, NHBz. NHTs,
N H A ~OH,
. o d e , OAC, O T ~OBZ
,
Figure 15-31. Asymmetric reduction of trifluoroacetylbenzene derivatives by

bakers’ yeast[’79’ 180].
OH
yeast *-3“‘ up to 96% ee
OH ’ R
G. candidurn
R = H. Br. OMe, OH, acetone powder F3c% up to 99% ee

C02H, C0,Me. NH2
’ R
Figure 15-32. Reduction of trifluoromethyl biphenyl ketones: bakers’ yeast vs
C. candidurn acetone
Moreover, various optically pure fluorinated alcohols are produced by employing

G. candidurn reductions as shown in Table 15-15(174].Monofluoroacetophenoneand
difluoroacetophenoneare reduced to (R)-alcoholsby the acetone powder, NAD’ and
1024
Table 15-15. Synthesis o f chiral fluorinated alcohols by the reduction with acetone powder and
isolated enzymes of Ceotrichurn candidurn I FO 4597’74.
Product Yield (“A) ee (“A) Product Yield (“A) ee (“A)
X=H 84 98 (S) OH 93 > 99 (R)

x=c1 81 >99(s) ‘ d p h
F3C$
X=Br 80 >99(S)
X F x p h 91 > 9 9 (S)”
F)“
a The isolated enzyme was used for the reduction.
2-propanol, and to (S)-alcohols by a constituent enzyme previously separated by

anion-exchange chromatography and using glucose-6-phosphate/glucose-6-phos-
phate dehydrogenase as the cofactor recycling system. Both enantiomers of mono-
fluorophenylethanol can be obtained with excellent ee using only one micro-
organism.
15.1 S . 2
Reduction of Fluoroketones Containing Sulfur Functionalities
As the demand for optically active fluorinated compounds increases, the importance
of the development of asymmetric synthetic methods for fluorinated building blocks
grows. On the other hand, sulfur functionalities such as phenylthio and dithianyl
groups have been used as useful reactive units for a variety of chemical transforma-
tions. Therefore, various trifluoromethyl ketones containing a sulfur functionality
have been reduced with various microorganisms [182-1851.
For example, several microorganisms have been employed for the reduction of
a,a,a,-trifluoromethyl a’-sulphenyl ketones (Fig. 15-33). Some of them produce the
corresponding alcohols in high diastereo- and enantioselectivities;the high conver-
SPh
Candida sake CBSl59 PhScF3 - Phk.,\CF,

(2& 0
de 94%. ee 84%
Rq C F 3
0 _____z_________L
Candida lypolytica CBS 2074 R
TCF3
OH
(2R.3R)
R = Ph de 92% ee >96%
R = CH2CH2Ph de >96% ee >96%
Figure 15-33. Reduction o f sulphenyl ketones followed by epoxide forrna-

tion[’821.
1) KHMDS, BnBr
75.7 Reduction $Ketones
I 1025
G. candidurn n-Bu,NI, THF
acetone powder
* F3c% 2) EtOH
Raney Ni (W-2; F3c
Yield 88% (4.16 g) ee >99% (3
ee >99% (Rj
0 G. candidurn OH
acetone powder CHzCH2CH2SPh 78 >99 (9
F3CAR
3-Thienyl D99
1,J-Dithian-P-yl 42 >99
Figure 15-34. Asymmetric reduction o f trifluorornethyl ketones containing a sulfur

functionality by the acetone powder o f C. ~ a n d i d u r n ~ ’ ~ ~ ] .
sion into a single enantiomer is secured by the racemization of starting ketones

under the biotransformation conditions. Transformation of the resulting sulphenyl
trifluoromethyl alcohols into trifluoromethyl epoxides was also
The acetone powder of G. candidurn (APG4)has also been used for the reduction
of sulfur containing trifluoromethyl ketones (Fig. 15-34) [lS3].This reaction can be
scaled up easily without the loss of enantioselectivity. For example, the reduction of
trifluoro(2-thieny1)ethanoneon the gram scale proceeded quantitatively and yielded
the optically pure (R)-alcohol in 88% yield after purification (4.16g, ee > 99%). The
thienyl alcohol can be further transformed into a fluorinated aliphatic alcohol
without racemization.
15.1.5.3
Reduction o f Chloroketones
The reduction of chloroketones has been widely investigated since it can produce
versatile chiral intermediates. For example, reduction of an a-chloroketone results in
the formation of a chlorohydrin, which can easily be transformed into an epoxide on
treatment with a base. On recently published example involves the reduction of
3,4-dichlorophenacylchlorideby Rhodotorula mucillaginosa CBS 2378 or Geotrichum
candidurn CBS233.76to give the (R)- or (S)-chlorohydrinwith > 99% ee and > 98%
ee, respectively, as shown in Fig. 15-35[18G1. The (S)-enantiomer was transformed
into the corresponding epoxide and then into a dichlorophenylbutanolide, an
intermediate in the synthesis of (+)-cis-lS,4S-sertraline,which is an antidepressant
drug of the selective serotonin reuptake inhibitor (SSRI) type.
There are also many other examples of the reduction of a-halomethyl ketones as
shown in Table 15-16[187-1891 . Vanous
. microorganisms are able to reduce fluoro-,
chloro- and bromoketones [161, 19s192’. However, reduction of iodoacetophenone
usually results in a poor yield, producing, mainly, acetophenone or phenylethanol.
Another example of the reduction of a-chloroketone involves dynamic kinetic
resolution. The reduction of an a-chloroketo ester by M. racernosus and R. glutinis
resulted in optically active syn- and anti-chlorohydrin, respectively, as shown in
IS Reduction Reactions
1026
I OH Rhodotoru/a Geotrichum OH
muci//aginosa cI
CBS 2378 CBS233.76
*
CI XAD-1180 CI
Figure 15-35. Reduction of a chloroketone followed by epoxidation for the synthesis

o f sertraIine”861.
Table 15-16. Reduction o f a-halogenated acetophenones
- xv
x+ Biocatalyst OH
Catalyst X Yield” (%) ee (“A) Reference
c1 80 100 187
Cryptococcus macerans
Br 95 93 187
F 67 97 188
c1 37 90 188
Br 9 97 188
Bakers’ yeast
F 55 35 189
c1 6 (40) 68 189
Br 0 (15) - 189
F 65 75 189
Geotrichum candidum sp. 38 C1 86 87.4 189
Br 15 (25) 94 189
OH RCONH
0
M racemosus ph&C02H
/ CI
R = Ph
OH
0 side chain of taxol
Ph%COpEt or Diltiazein3
CI R = fert-butoxy
-A- Ph
K C O 2 E t ~ 0
p.\\C02Et
side chain of taxotere
Ph
~
Cl
Figure 15-36. Enantio- and diastereo-selective reduction o f a c h l o r ~ k e t o n e [ ’lg4].
~~~
Fig. 15-36[1931. The syn-isomer was transformed into the corresponding epoxide,
followed by conversion into the side chain of taxol and taxotere[’”].
One of the most studies a-chloroketones is ethyl 4-chloro-3-oxobutanoate.
( R ) -and
(S)-enantiomers of the corresponding alcohol were produced by various micro-
Table 15-17. Comparison of various microorganisms for the reduction of ethyl

I
1027
4-chloro-3-oxobutanoate.
0
C I A C O 2 E t -
Microorganism OH
Cl&C02Et
(s)
Microorganism Yield (“A) ee (“A) Reference
Geotrichum candidum 98 96 170
Bakers’ Yeast 100 90 90
Bakers’ Yeast 55 61
Lactobacillus kefr 100 100 195
Candida magnoliae 88 100 142
(recombinant and overexpressed
in Escherichia coli)
0 Microorganism OH
CI A C O 2 E t Cl*CO,Et
(R)
Microorganism Yield (“A) ee (%) Reference
Dancus carota 42 52 196
Sporobolomycessalmonicolor 95 86 197
Lactobacillusfermentum 70 98 195
Saccharomyces cerevisiae 55 16 63
(FAS (P-keto reductase) negative)
organisms as shown in Table 15-17. The (R)-enantiomer is a promising chiral

building block for the synthesis of L-carnitine,an essential factor for the P-oxidation
of fatty acids in mitochondria.
As shown in Fig. 15-37, a chiral intermediate for a human immunodeficiency
vims protease inhibitor (HIVPI) was also synthesized by the reduction of an a-
chloroketone with a Streptomyces strain [1981.
Another example of the reduction of chloroketone is the reduction of 5-chloro-
2-pentanone by TBADH as shown in Fig. 15-38[19].Using this biotransformation in
the synthetic pathway, a naturally occurring heterocycle isolated from the glandular
secretion of the civet cat (Viverru civettu), was prepared.
COEN cI StreptomycesnodosusSC 13149

*
H O H OH
Figure 15-37. Synthe.

sis of a chiral inter-
mediate for an HIV-
EMS-186318(Antiviralagent) pi 11981.
1028
0
- TBADH
A
OH
C I -
-
-a m
Figure 15-38. Reduction o f 5-chloro-2-pentanone by TBADH for
natural product
Yield 65% de 88% sex attractant of the pine saw-fly

ee>96% (2R3.5~
Figure 15-39. Reduction o f ketones containing sulfur or nitrogen

f u n ~ t i o n a t i t y ~ *191.
'~~.
15.1.5.4
Reduction of Ketones Containing Nitrogen, Oxygen, Phosphorus and Sulfur
Functionalities
Ketones with useful heteroatomic functional groups containing n i t r ~ g e n [ ' ~ ~ - ~ ' ~ ] ,

oxygen[lG3, 213-2171
phosphoms 12181 and sU]fUr1154s 184, 219-2271 h ave been reduced by
biocatalysts. For example, an intermediate in the synthesis of P-lactam antibiotics
was obtained by microbial reduction of a P-keto ester as shown in Fig. 5-39(a)['"],
while yeast reduction of a 0-keto dithioester afforded an easily separable mixture of
P-hydroxy-dithioesters, the major component of which was converted enantiose-
lectively into a sex attractant of the pine saw-fly as shown in Fig. 15-39(b)r2191.
15.1.5.5
Reduction of Diketones
Regio- and enantioselective reduction of diketones can be achieved readily by using a

b i o ~ a t a l y s t [. ~A ~
s a~ result,
- ~ ~ ~ optically
~ active hydroxyketones and diols have been
synthesized successfully.
For the reduction of a-diketones, the selectivity between the reduction to diol and
to hydroxyketone can be controlled using a diacetyl reductase from Bacillus stear-
othemophilus (Fig. 15-40)[233]. When a one-enzyme system was used for the coen-
zyme recycling using endo-bicyclo[3.2.0]hept-2-en-G-ol ( S ) , both carbonyl groups
were reduced selectively to produce a diol. On the other hand, a-hydroxyketones
were obtained using a two-enzyme system glucose 6-phosphate/glucose 6-phosphate
dehydrogenase for coenzyme recycling. The synthetic potential of both systems has
been illustrated by the synthesis of the male sex pheromone of the grape borer
Xylotrechus pyrrhoderus, identified as a two-component mixture of the reduction
products, G and 7.
I One-enzvme svsteml [Two-enzymesystem]
15. I Reduction ofKetones
I 1029
Bacillus sfearafherrnophilus Bacillus sfearofherrnophilus

oH OH
diacetyl reductase
"%"' 0
diacetyl reductase
glucose 6-phosphate/
glucose 6-phosphate dehydrogenase 0
NADt NADH
Bacillus stearofherrnophilus
I Product Yield (%) ee (%) 1

OH R=Me 40 >98(S,S)
eR
6~
Pr
Ph
Pentyl(6)
92
80
82
>98(S,S)
>98(S,S)
>98(S,S)
OH
80 95(S,S)
OH
Figure 15-40. Reduction o f a-diketones by diacetyl reductase from Bacillus s t e a r ~ t h e r m o p h i l u s ~ ~ ~ ~ ~ .
Regio- and enantioselective reduction of P-diketones may be carried out using

biocatalysts. For example, a diketo ester 8 was reduced by the alcohol dehydrogenase
from Lactobacillus brevis, to provide the corresponding hydroxyketo ester with 99.4 %
ee in 78% yield; this was used as an intermediate for the synthesis of dimeric
metabolite vioxanthin of Penicillium citreo-viride in order to develop an assay system
to monitor phenol oxidative coupling in lignan formation [Fig. 15-41(a)][228]. Yeast
reduction also proceeds regio- and enantioselectively with aliphatic diketones pro-
ducing hydroxyketones with perfect selectivities as shown in Fig. 15-41(b)[2321. The
yeast reduction also proceeds satisfactorily with 2,2-disubstitutedcycloalkanediones,
producing hydroxyketones with excellent enantio- and diastereoselectivities as
shown in Fig. 1 5 - 4 1 ( ~ ) [ ~ ~ ~ !
15.1S . 6
Reduction o f Diary1 Ketones
Bulky ketones such as diaryl ketones can be also reduced by biocatalysts. For
example, a rice plant growth regulator, (S)-N-isonicotinoyl-2-amino-5-chlorobenzhy-
drol, was prepared by microbial reduction of 2-amino-5-chlorobenzophenone with
Rhodosporidium toruloides followed by isonicotinoylation as shown in Fig. 15-
42(a)[2431. A phosphodiesterase 4 inhibitor was also prepared by microbial reduction
of a diaryl ketone 9 with Rhodotorula pilimanae, which was found by the screening of
310 microbial strains [Fig. 15-42(b)][244].
1030
alcohol dehydrogenase
from Lactobacillus brevis OH
Dimeric metabolite vioxanthin of

PeniciNium citreo-viride
OH o
(b) ,y,0
,.
,
) 0
- yeast
Yield 42%
ee>99%
( 2 s 3s)
Figure 15-41. Regio- and enantioselective reduction of

diketones[228. 231. 2321
(a) ,
0 NH2 Rhodosporidium
toruloides (J"f$ -QH NH2
/
OH N H C O ~ N
CI CI CI
Yield 60% ee 99%(s) Rice plant growth regulator
)fo&
(b)
9 OMe
Rhodorola pilimanae - a a /
/ OMe
Yield 10% ee96%(S)
P N
Figure 15-42. Reduction of diary1 ketones for the synthesis of bioactive

compounds[243,2441.
15.1.5.7
Diastereoslective Reductions (Dynamic Resolution)
Enantio and diastereoselective reduction (dynamic resolution) of keto esters and

ketones can be achieved using yeast and other microorganisms[55* 70r 74, 245-2531 . As
shown in Fig. 15-43, when the racemization rate of the keto ester is faster than that
for the yeast reduction, and the product hydroxyester is not racemized under the
reaction conditions, then the yeast reduction may proceed enantioselectively and
15.1 Reduction ofKetones I1031
2s
&C02R - yeast
OH
/YCOzR' +
Figure 15-43. Diastereo-
c o p ~ ' selective reduction.
Rk R h R h
2s, 3s 2S, 3R
faster racemization rate
than reduction rate
2R
2R,35 2R, 3R
Kloekera
rnagna or
Rhizopus Cunninghamella QH 0
- okoEt
arrhizus 8 O E t echinulata
(yo&-
gOEt
(1 s, 25) (1 s, 2R)
_OH 0
0
Mucor Rhodotorula
racernosus glufinis
....&oEt
(1 s, 25) (1s, 2R)
Figure 15-44. Diastereoselective reduction of cyclic keto esters[245].
diastereoselectively;thus only one stereoisomer out of the four possible ones can be
obtained in one step. Actually, when bakers' yeast was used for the reduction of
neopentyl2-methyl-3-oxobutanoate(R = Me, R = neopentyl), then the ratio of (2R,
3s) : (2S, 3R) : (2S, 3s) : (2R, 3R) products was found to be 96 : c 1 : 4 : c 1[2471. When
an enzyme was isolated from the yeast, then the diastereoselectivitywas improved to
> 99 : 1, and only a single isomer was obtained[248]. Another example is the large
scale reduction of ethyl 2-methyl-3-oxobutanoate by Klebsiella pneurnoniae I F 0
3319I7']. On a 200 L scale, 2 Kg of the substrate were converted into the (2R, 3S)-
hydroxyester with 99 % de, > 99 % ee, and 99 % chemical yield as shown in Table
15-2.
Enantio- and diastereoselective reduction of cyclic keto esters are also achieved
using various microorganisms (Fig. 15-44)[2451. By selecting a suitable organism, syn-
and anti-hydroxyestersmay be synthesized enantio- and diastereoselectively.
15.1.5.8
Chemo-enzymaticSynthesis of Bioactive Compounds
Ketones with various functionalitis, containing F, C1, N, S , 0, etc., have been shown
to be reduced by a biocatalyst, and by using the biocatalytic reduction as a key step,
the chemoenzymatic synthesis of many bioactive compounds have been re-
*
Figure 15-45. Synthesis o f all four isomers o f t h e western corn rootworm
sex pheromone [2341.
Figure 15-46. Synthesis o f natural products from a key intermediate obtained by yeast reduction.
15.2 Reduction ofvarious Functionalities I 1033
ported['229 '2% '99. 228-230. 7-34! 235, 243, 254-2741 For example, 2,8-nonandione can be
reduced enantioselectivelyby TBADH to furnish the corresponding diol, from which
all four isomers of 8-methyldec-2-ylpropanoate, the western corn rootworm sex
pheromone, were prepared (Fig. 15-45)[2341.
One of the most versatile key intermediates discovered to date is the hydroxy-
ketone 10 which is synthesized by the yeast reduction of the corresponding
diketone [229, 2301. Starting with 10, many terpenes have been enantioselectively
synthesized by Mori et al., as shown in Fig. 15-46.
15.2
Reduction of Various Functionalities
15.2.1
Reduction of Aldehydes
Many aldehyde reductases transform both aldehydes and ketones 275, 2761.
For example, phenylacetaldehyde reductase from a styrene-assimilating Corynebacte-

riurn strain, ST-10, reduces aldehydes and ketones as shown in Table 15-18[138].
Other aldehyde reductases such as one from Sporobolornyces salrnonicolor also reduce
aldehydes as well as ketones['&, 2751.
Organometallicaldehydes can be reduced enantioselectivelywith dehydrogenases.
For example, optically active organometallic compounds having planar chiralities
were obtained by biocatalytic reduction of racemic aldehydes with yeast [277, 2781 or
H L A D H [ 2 7 9 1 as shown in Fig. 15-47.
The dynamic resolution of an aldehyde is also possible as shown in Fig. 15-48[280j.
The racemization of the starting aldehyde and enantioselective reduction of a
carbonyl group by bakers' yeast resulted in the formation of tertiary chiral carbon
centers. The ee of the product was improved from 19% to 90 % by changing the ester
moiety from the isopropyl group to the neopentyl group.
Table 15-18. Examples of substrates of phenylacetaldehyde reductase from Corynebacteriurn

strain, ~ ~ - 1 0 ' ~ ~ .
Substrate (mM) Relative activity Substrate (mM) Relative activity
(aldehyde) (%I (ketone) (W
Acetaldehyde (3) 0 Acetone (3) 0
n-Valeraldehyde (3) 181 2-Hexanone (3) 207
n-Hexyl aldehyde (3) 1220 2-Heptanone (3) 760
Phenylacetaldehyde (3) 100 Acetophenone (3) 35
3-Phenylpropionaldehyde (1) 364 4-Phenyl-2-butanone (3) 29
15.2 Reduction ofvarious Functionalities I 1033
ported['229 '2% '99. 228-230. 7-34! 235, 243, 254-2741 For example, 2,8-nonandione can be
reduced enantioselectivelyby TBADH to furnish the corresponding diol, from which
all four isomers of 8-methyldec-2-ylpropanoate, the western corn rootworm sex
pheromone, were prepared (Fig. 15-45)[2341.
One of the most versatile key intermediates discovered to date is the hydroxy-
ketone 10 which is synthesized by the yeast reduction of the corresponding
diketone [229, 2301. Starting with 10, many terpenes have been enantioselectively
synthesized by Mori et al., as shown in Fig. 15-46.
15.2
Reduction of Various Functionalities
15.2.1
Reduction of Aldehydes
Many aldehyde reductases transform both aldehydes and ketones 275, 2761.
For example, phenylacetaldehyde reductase from a styrene-assimilating Corynebacte-

riurn strain, ST-10, reduces aldehydes and ketones as shown in Table 15-18[138].
Other aldehyde reductases such as one from Sporobolornyces salrnonicolor also reduce
aldehydes as well as ketones['&, 2751.
Organometallicaldehydes can be reduced enantioselectivelywith dehydrogenases.
For example, optically active organometallic compounds having planar chiralities
were obtained by biocatalytic reduction of racemic aldehydes with yeast [277, 2781 or
H L A D H [ 2 7 9 1 as shown in Fig. 15-47.
The dynamic resolution of an aldehyde is also possible as shown in Fig. 15-48[280j.
The racemization of the starting aldehyde and enantioselective reduction of a
carbonyl group by bakers' yeast resulted in the formation of tertiary chiral carbon
centers. The ee of the product was improved from 19% to 90 % by changing the ester
moiety from the isopropyl group to the neopentyl group.
Table 15-18. Examples of substrates of phenylacetaldehyde reductase from Corynebacteriurn

strain, ~ ~ - 1 0 ' ~ ~ .
Substrate (mM) Relative activity Substrate (mM) Relative activity
(aldehyde) (%I (ketone) (W
Acetaldehyde (3) 0 Acetone (3) 0
n-Valeraldehyde (3) 181 2-Hexanone (3) 207
n-Hexyl aldehyde (3) 1220 2-Heptanone (3) 760
Phenylacetaldehyde (3) 100 Acetophenone (3) 35
3-Phenylpropionaldehyde (1) 364 4-Phenyl-2-butanone (3) 29
1034
Yield 53% ee78% (s) Yield 32% ee >99% (R)
CHO
I
Yield 33% ee 91%(s) Yield 51% ee 81%(R)

Figure 15-47. Reduction of organometallic aldehydes to produce alcohols
with planar chiralities [277-2791.
OHC-C0zR yeast HOH$2,COzR

c
ee (%)
ti =//
OHCYC02R HOHzCyC02R I -CH2C(CH3)3 90
Figure 15-48. Reduction o f aldehyde with dynamic resolution1280].
15.2.2
Reduction of Peroxides to Alcohols
Horseradish peroxidase has been used for the reduction of peroxide to alco-
hol [2*1-*84l . The enzyme selectively recognizes sterically uncumbered (R)-alkyl aryl
hydrogenperoxides,which allows kinetic resolution to provide (Rj-alcohol and (S)-
peroxide. However, poor enzyme recognition is observed with hydroperoxides
possessing larger R2 groups such as a propyl or an isopropyl moiety as shown in
Fig. 15-49. This reaction can be performed on a preparative scale conveniently to
provide optically pure hydroperoxides.
15.2.3
Reduction of Sulfoxides to Sulfides
Asymmetric synthesis of sulfoxides can also be achieved by biocatalytic reduction.

One example is the reduction of alkyl aryl sulfoxides by intact cells of Rhodobacter
sphaeroides f sp. denitrijcan~[~~~I.
In the reduction of methyl p-substituted phenyl
sulfoxides, (S)-enantiomers were exclusively deoxygenated while enantiomerically
pure (R)-isomers were recovered in good yield. For poor substrates such as ethyl
phenyl sulfoxide, the repetition of the incubation after removing the toxic product
was effective in enhancing the ee of recovered (R)-enantiomers to 100% as shown in
Table 15-19.
QOH
15.2 Reduction of Various Fundonahies
OH
I
1035
R2% Horseradish peroxidas: -2" + %%

Guaiacol
\ \
Ri R1 R1
ee (Oh)
(9 (Rj
R1 '2 (-)-(S)-ROOH (+)-(Rj-ROH
SiMezPh Horseradish peroxidase M

Guaiacol
OOH
if,-e2Ph+
E = 14.2
- 5 SiMe2Ph
QOH OOH
Horseradish peroxidase
+R1 + *Rl
Guaiacol
R2 OH R2 OH
OOH
I Me Me 2
Figure 15-49. Reduction o f peroxides t o alcohols [281-2841.
Table 15-19. Reduction o f sulfoxide to obtain optically pure ( R ) - s u l f o ~ i d e ~ ~ ~ .
? 4 9'.
R"\A~
Rhodobactersphaeroidesfsp. denitrificans
- R's\Ar + *{ -<<-
R's'.Ar
R Ar Yield (%) ee (%) (R)-sulfoxide
Me Ph 46 100
Me pMe-C6H4 40 100
Me pBr-C6H4 43 100
Me pMeO-C&, 47 >99
Me PhCH2 41 90
Et Ph 41 100
&Pr Ph 54 21
15.2.4
Reduction ofAzide and Nitro Compounds to Amines
Bakers' yeast catalyzes the reduction of azides and nitro compounds to ami-
nes [2862911. For example, it catalyzes chemoselective reduction of azidoarenes to
arenamines as shown in Fig. 15-50[286*2871. Excellent yields are obtained for various
aromatic compounds on reaction at room temperature. Aromatic nitro compounds
1036
R-N3
Bakers' yeast
- R-NHz Yield U P to 90%
Figure 15-50. Reduction of azide to amine
by bakers!yeast[286. 2871
x = H, MeCO, MeO, CI. Br, I, Me, NO,, OH, etc
containing 0-,rn-, or p-electron withdrawing groups, such as carbonyl, halogen and

nitro, were also selectively and rapidly reduced to their corresponding amino
derivatives in good yields using bakers' yeast in basic solution[288].
N-oxides can also be reduced. For example, the microbial deoxygenation of a series
of aromatic and heteroaromatic N-oxide compounds, including quinoline N-oxides,
isoquinoline N-oxides, 2-aryl-2H-benzotriazole1-oxides, benzo[c]cinnoline N-oxide
and azoxybenzene, has performed with bakers' yeast-NaOH to afford quinolines
and pyridines [2911.
15.2.5
Reduction of Carbon-Carbon Double Bonds
The reduction of carbon-carbon double bonds to single bonds has been studied with
various substrates ['l, 124, 292-3061 . For example, Ohta et al. demonstrated that the
reduction of a number of 1-nitro-1-alkenesby fermenting bakers' yeast was enantio-
selective, resulting in the formation of optically active 1-nitroalkanes as shown in
Fig. 15-51(a)[294s 2951 . 0n the other hand, Fuganti et al. reduced a,P-unsaturated-&
lactones to produce enantiomerically pure (+)-(R)-goniothalamins [Fig. 15-51(b)],
which show CNS activity. They also performed the kinetic resolution of the
corresponding embryotoxic epoxide with yeast [2961.
One of the most studied enzymes for the reduction of carbon-carbon double
R2 ee
(a) ,
1 Ph H
Yeast or 1 Ph Me 98
R1 ,JL NO2
old yellow enzyme
N
R
O
,2-?
.l,
pCI-Ph
pBr-Ph
Ph
Me
Me
Et
89
94
97
Ph n-Pr 89
Ph Hexyl no reaction
Hexyl Me (E) 83
Me Hexyl (Z) 66
2-Thienyl H
etc
ee 99% ee 77%
(+)-(R)-goniothalamins
Figure 15-51. Examples of substrate specificity of yeast reduction of

olefins 1294-2961
15.2 Reduction of Various functionalities
I
6
1037
0, +,o o;,+,o-
old yellow enzyme old yellow enzyme
Figure 15-52. Mechanism of the reduction of nitro olefin by “old

yellow enzyme” from y e a ~ t [ ~ ’ ~ - ~ ’ ~ ] .
&R
S
-
Nicotiana tabacum
p90 reductase
Q
11
R
Nicotiana tabacum
p44 reductase
R
R
ee
n-Pr 95
Figure 15-53. Reduction o f carbon-carbon double bonds by reductases

from plant cell
bonds is the “old yellow enzyme’’ from y e a ~ t [ ~ ’ ~ -which

~ ’ ~ ] has been shown
efficiently to catalyze the NADPH-linked reduction of nitro olefins. The reduction of
the nitro-olefin proceeds in a stepwise fashion (Fig. 15-52). The first step involves
hydride transfer from the enzyme-reduced flavin to the P-carbon of the nitro-olefin
which forms a nitronate intermediate that is freely dissociable from the enzyme. The
second step, protonation of the nitronate at the a-carbon to form the final nitroalkane
product, is also catalyzed by the enzyme.
Photosyntheticmicroorganisms and plant cell cultures are very important sources
of enzymes for the reduction of ole fin^['^, 2981. For example, Hirata et al. found that
reduction of enone 11 with Nicotiana tabacum p90 reductase and Nicotiuna tabacum
p44 reductase affords (S)- and (R)-alkylcyclohexanones,respectively, with excellent
enantioselectivities as shown in Fig. 15-53. They also found two enone reductases
from Astasia longa, a nonchlorophyllous cell line classified in Euglenales, and studied
the mechanism. Both catalyzed enantiospecific trans-addition of hydrogen atoms to
carvone from the si-face at the a-position and from the re-face at the P-position.
15.2.6
Transformation o f a-Keto Acid to Amine
A dehydrogenase can also be used for the transformation of an a-keto acid to an

amine (Fig. 15-54). The chiral intermediate for an antihypertensive drug was
prepared by reduction of an a-keto acid with glutamate dehydrogenase from beef
liver. The cofactor NADH was regenerated using glucose dehydrogenase from
Bacillus sp. [3071.
1038
Glutamate dehydrogenase
NHp
HO dco2H
from beef liver
NADH NH3
Glucose I
- HOC
-OpH
yield 92% ee>99%
glucose dehydrogenase
-
-
- Antihypertensive drug
Figure 15-54. Reduction of a-keto acid to amine[307].
15.2.7
Reduction of Carbon Dioxide
15.2.7.1
Reduction of C 0 2 to Methanol
Syntheses using C 0 2 as a carbon source are attracting growing interest. The

development of environmentally benign methods to utilize C02 is very important
due to the abundance of COz. For this purpose, dehydrogenases have been success-
fully utilized. Formate, formaldehyde and alcohol dehydrogenases are used for the
reduction of C 0 2 to methanol as shown in Fig. 15-55[333 308-3111.
For the efficient production of methanol, electrochemical methods have been used
(Fig. 15-55)[33,308, 310, 3111. Electrochemically, C02 was converted into formate by
formate dehydrogenase with the aid of methyl viologen or pyrroloquinolinequinone
as a mediator. Methanol dehydrogenase was used to reduce formate to formaldehyde
and methanol with the same system[308,310. 3111.
An approach for the conversion of COz into formic acid which combines a
semiconductor photoelectrode with formate dehydrogenase is very interesting [331.
Electrons in the semiconductor can be produced with light of wavelengths shorter
than 900 nm. Then, the photogenerated electrons were transferred to C02 through
methyl viologen to produce formic acid as shown in Fig. 15-5G[331.
Another highly efficient process involves the immobilization of three enzymes in a
silica ~ol-gel[~~’]. Since the process consists of a sequential reaction of in situ
generated substrates with three different enzymes, the confinement of the system in
a porous matrix resulted in an enhanced probability of the reactions as shown in
Fig. 15-57 due to an overall increase in local concentration of reactants within the
nanopores of the sol-gel processed glasses [3091.
Formate Formaldehyde Alcohol
COZ
dehydrogenase
* HCOpH -
dehydrogenase
HCHO
dehydrogenase
* CH3OH
dehydrogenase
dehydrogenase
e- I electron mediator
Figure 15-55. Reduction of COZto methanol with dehydrogenases.
MV2+'
75.2 Reduction of Various Functionalhies
I
1039
a
'ght M e - N w N - M e
Me-N-N-Me
Figure 15-56. Photoelectrochemical pumping of enzymatic COz reduction[33].
Sol-gel
Methanol (pmol)
10 Solution
0 ' Figure 15-57. Effect of the confinement of

0 50 100 150 200
the three enzymes in a porous matrix on
NADH (pmol) methanol productionISo9I.
0 Malic enzyme
+ cop * FCOzH
AC02H NADP', NADP' reductase CO,H
methyl viologen
e-
OH
lsocitrate dehydrogenase
* HO,C+CO,H
HOpCJ C O p H + methyl viologen
COpH
e'
Figure 15-58. Electrochemical reductive fixation o f COzPi2~
3131.
15.2.7.2
Reductive fixation of COZ
Reductive fixation is an another important process. Malic enzyme and isocitrate

dehydrogenase catalyze both the reduction of the carbonyl group in an a-keto acid
and fixation of C02 at the a-position with the aid of an electric power source and an
electron mediator (Fig. 15-58)1312. 3131. Uniquely, the reaction using isocitrate dehy-
drogenase does not require the use of NADP+. When C02 is reductively fixed in an
organic molecule, the enzyme is oxidized;the oxidized enzyme is ultimately reduced
back to its original form by methyl viologen cation radicals[312!
1040
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75.3 Reduction ofC=N bonds
G. Fronza, G. Fogliato, C. Fuganti, S . La-

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15.3
Reduction of C=N bonds
Andreas 5.Bommarius
15.3.1
Introduction
Enantiospecific reduction of C=N bonds is of interest for the synthesis of a-amino

acids and derivatives such as amines. While nonenzymatic reductive amination has
been known since 1927['],only recently have enzymatic procedures to L-amino acids
became established. The reduction can be achieved by different enzymes following
different mechanisms, e. g. by pyridoxalphosphate (PLP)-dependenttransaminases
(E.C. 2.6.1, discussed in Chapter 12.7)or by amino acid dehydrogenases (E.C. 1.4.1)
using NADH or NADPH as the cofactor. The synthetic usefulness of the trans-
aminase reaction is diminished by the location of the equilibrium (&, often is close
to one), so that complex mixtures result, which are often laborious to separate (for
solutions to this problem, see Chapter 12.7). For this reason, this chapter focuses on
the reduction of C=N bonds by reductive amination with amino acid dehydroge-
nases, AADHs.
Reductive amination of a-keto acids to a-amino acids is similar to the reduction of
C = 0 bonds to the corresponding a-hydroxy acids. In an equilibrium reaction, a-
keto acids can be reductively aminated to a-amino acids or, vice versa, a-amino acids
can be oxidatively deaminated:
RyCooH+
NAD+ + H20
NH2
A very promising process route is the reductive amination of prochiral a-keto acids
to a-amino acids with AADHs and the cofactor NADH and its regeneration by co-
oxidation of formate to COz by formate dehydrogenase (Fig. 15.3-1).
This asymmetric synthesis route possesses a number of advantages rendering it
attractive in today's context of seeking environmentally benign processes:
compact synthesis of a-keto acid substrates,
formation of harmless and easily separable COZ as the only co-product,
extreme enantioselectivity of amino acid dehydrogenases, and
yields of up to 100% with respect to a-keto acid, resulting in no undesirable
enantiomers and other by-products.
RKCooH Ry
CooH-aGGiGq
+h [ L-dehydrogenase I
OH
or
RyCooH
NHZ
pziGcq
[NADH + H@I
FDH
co2 HCOOH or HCOONH4

Figure 15.3-1. Schematic o f enzymatic reductive amination with cofactor
regeneration.
75.3 Reduction of C=N bonds
Table 15.3-1. List of NAD(P)+-dependent amino acid dehydrogenase~(~1.

I 1049
~~~~
E.C. Number Enzyme Coenzyme Sourcea

1.4.1.1 Alanine DH NAD+ B (Bacillus, Streptomyces, Halobacterium)
1.4.1.2 Glutamate DH NAD+ B, F, Y,P
1.4.1.3 Glutamate DH NAD(P)' A, F, Etrahymena
1.4.1.4 Glutamate DH NAD' B, F, Y,Chlorella
1.4.1.7 Serine DH NAD' P
1.4.1.8 Valine DH NAD(P)+ B (Alcaligenes,Streptomyces),P
1.4.1.9 ucine DH NAD' B (Bacillus,Clostridium)
1.4.1.10 Glycine DH NAD' B (Mycobacterium)
1.4.1.11 3,5-Diaminohexanoate DH NAD' B (Clostridium)
1.4.1.12 2,4-Diaminopentanoate DH NAD+ B (Clostridium)
1.4.1.15 Lysine DH NAD' Human, B (Agrobacterium)
1.4.1.16 Diaminopimelate DH NADP' B (Bacillus, Colynebacteriurn)
1.4.1.20 Phenylalanine D H NAD' B (Brevibacterium,Bacillus, Rhodococcus)
1.4.1.- Tryptophan DH NAD(P)+ P
a Abbreviations: B: bacterium; F: fungi; Y: yeast; A: animal; P: plant; DH: dehydrogenase
With three exceptions (AlaDH from Phormidium lapideum, L-lysine-&-dehydrogenase

and meso-a,&-diaminopimelateDH) all of the AADHs (Table 15.3-1) catalyze
reduction of prochiral keto acids to the L-amino acids [(S)-configuration].The natural
function of L-AADHsis not known. The D-AADHsthat have been found appear to be
iron-sulfur membrane-associated flavoenzymes which seem to catalyze the oxidative
reaction from keto acids to amino acids only; artificial dyes and the coenzyme Q
analog serve as electron acceptors but not AADHs have been screened
from a variety of organisms (Table 15.3-l),the most important enzymes for synthesis
are alanine dehydrogenase (AlaDH, E. C. 1.4.1.1), phenylalanine dehydrogenase
(PheDH, E. C. 1.4.1.20), and particularly leucine dehydrogenase (LeuDH, E. C.
1.4.1.9). The ubiquitous glutamate dehydrogenase (GluDH, E. C. 1.4.1.2.-4), how-
ever, is still the most studied member of the group.
Reviews on AADHs: Apart from early review articles on individual amino acid
dehydrogenases by Schiitte et al. (1985; LeuDH from B. cereus)I6],Ohshima et al.
(1985a; LeuDH from B. species)17]and Hummel et al. (1987; PheDH from Rh.
rhodocrous) [8],comprehensive reviews have been published by Hummel and Kula
(1989)['], Ohshima and Soda (1989 and 1990)[5~'o~1'] and by Brunhuber and Blan-
chard (1994)['21.
15.3.2
Structural Features of Amino Acid Dehydrogenases (AADHs)
Most of the AADHs possess hexameric structure, although octamers, tetramers,

dimers and even monomers have been found. The subunits are usually of similar
size: for instance, most bacterial AADHs are hexamers with a molecular weight of
around 49 000 per subunit.
1050
I Table 15.3-2. Identities o f protein sequences of different amino acid dehydrogenases
(in per cent) [221. The data were calculated via BLAST search i n the database 'Swissprot'[231.
Protein LeuDH, LeuDH, B. PheDH, Rh. PheDH, Th. CIuDH, C.
B. cereus sphaericus rhodocrous intermedius symbiosum
LeuDH, B. stearothemophilus 82.5 79.9 32.0 45.6 12.6

LeuDH, B. cereus - 76.9 31.5 44.5 13.4
LeuDH, B. sphaericus - 31.7 41.8 14.0
PheDH, Rh. rhodocrous - 26.4 12.4
PheDH, 'I: intemedius - 14.2
15.3.2.1
Sequences and Structures
Several amino acid dehydrogenases have been screened from a variety of micro-
organisms, the preparatively most important are phenylalanine dehydrogenase
(PheDH, from Rhodococcus sp. M4) and leucine dehydrogenase (LeuDH, from
Bacillus stearothemophilus and Bacillus cereus). As of the end of February 2001, more
than 20 gene and protein sequences for AADHs except GluDH (which more than
triples the number) and 3D crystal structures from five different AADHs have been
deposited (GluDH from Clostridium s y m b i ~ s u r n [ ~LeuDH
~], from B. sphaeri~us['~I,
AlaDH from Phomidium lapideum[l5I, PheDH from Nocardia sp 239L''l and PheDH
from Rhodococcus sp. M4[178'81). Sequence homologies and similarities of 3D
structures of the members of several organisms are so high that amino acid
dehydrogenases can be termed a single superfamily, generated through divergent
evolution [19-211 (Table 15.3-2).
Remarkable, on one hand, is the high degree of identity of the three leucine
dehydrogenases, and on the other hand the sequence of glutamate dehydrogenase,
which bears no homology to the other dehydrogenases. Although overall sequence
homology varies from around 20 % up to 80 %, the residues essential for the three-
dimensional structure of a subunit, for nicotinamide cofactor binding, and for
catalysis have been conserved[*']. While a complex between NAD' and GluDH from
Clostridium symbiosum left the overall conformation unaltered [241, a drastic con-
formational change (hinge movement) was observed on binding of the gl~tamateI'~1.
15.3.3
Thermodynamics and Mechanism of Enzymatic Reductive Amination
15.3.3.1
Thermodynamics
For reductive amination, basically no thermodynamic limitation exists: for the

leucine/ketoleucine reaction at pH 11.0, equals 9x1012[251,
for phenylalanine/
phenylpyruvate at pH 7.95 a k& of 2 . 5 ~ 1 0has
~ been thus, the maximum
degree of conversion is very close to 100%. Coupling of the reductive amination
reaction with cofactor regeneration via the FDH/formate reaction, which is irreversi-
ble, further helps to pull the equilibrium towards the amino acid product.
I 1051
15.3.3.2
Mechanism, Kinetics
As will be elucidated below, the mechanism of reductive amination and the geometry
of the active center[13,18,3'' *'* 271 cause the (S)-configuredamino acid products of
the reaction to be completely enantiomerically pure, an important criterion for a
large-scaleapplication.
The catalytic mechanism of AADHs has been studied most thoroughly with
GluDH from C. s y r n b i o ~ u r n [ ~and
~ , ~with
~ I PheDH from Rhodococcus M4["1. The
mechanism was found to be remarkably similar in both cases so that the prediction
by Stillman et al.[131seems to have been borne out. In Fig. 15.3-2, the study on
PheDH is illustrated[18]:
Following the scheme in Fig. 15.3-2, which depicts oxidative deamination, in a
clockwise fashion starting from the top left, the a-N-protonated L-Phe molecule is
stabilized by the &-groupof LysGG at the carbonyl group as well as by the &-groupof
Lys78 via a water molecule, the carbonyl group of Pro117 and the P-carboxyl group of
Asp118 at the a-amino group. The first intermediate is the protonated imine after
steps (2) and (3)in which Lys78 picks up the proton from the a-N-group of L-Phe and
delivers a hydrogen to the Si face of the cofactor NAD' with deprotonation of Lys78.
Accompanied by another Lys78 protonation, the water molecule adds to the imine
-K78
H' H
' 0
Figure 15.3-2.Proposed mechanism for amino acid dehydrogenases

(with PheDH as an example) [la].
1052
carbon to form the carbinolamine, the second intermediate [step (4)].The Lys78
proton is picked up by Asp118 [step (S)] and in turn by the amino group [step (G)] of
the substrate to liberate NH3 and with the formation of phenylpyruvate. The keto
group is stabilized by the protonated c-sidechain of Lys78 as well as a by a proton
from Gly40. The positioning of Lys78 and Gly40 also prevents the oxidation of
phenyllactate, so that PheDH cannot act as a HicDH.
A similar mechanism had already been proposed for GluDH from C. syrnbio-
the only major difference seems to be the attribution of the initial deprotona-
tion of the amino acid molecule to Asp165 (which corresponds to Asp117 on
PheDH) instead of Lys125 (Lys78 in PheDH). The Lys125 in GluDH is known to
have a low pK value[28],which causes this residue to act as a proton shuttle more
easily.
The optimum degree of protonation and catalybcally important amino acid
residues can be determined from a log V,,-pH on the acidic and
alkaline side of the optimum pH, log V,, decreases nearly linearly with pH, the two
slopes intersect at the optimum degree of protonation, which is also the optimum
point of activity. The experimentally observed optimum pH value of 9.2-9.3 for
LeuDH l3O], corresponding to two pK values of around 8.7 and 10.0 for amino acid
residues participating in the catalytic step, can be linked to lysine residues, corrobo-
rating the results of Rife and Cleland (1980)rZ6] and Sekimoto et al. (1993)["I for the
case of GluDH. Brunhuber et al. in their study of PheDH assigned their pKas values
of8.1 and 9.4 to Asp118 and Lys78, respectively["]. The influence ofpH on reductive
aminations with AADHs can also be explained by the dissociation equilibrium of
ammonia (pK, value 9.25). Only an uncharged ammonia molecule can be accepted
by LeuDHIz6,301 so that a minimal pH of around 7.5 has to be kept throughout the
reaction.
15.3.4
Individual Amino Acid Dehydrogenases
15.3.4.1
Leucine Dehydrogenase (LeuDH, E. C. 1.4.1.9)
Isolation and characterization of LeuDH has been pioneered by Hummel et al.131]

(from B. sphaericus), Schiitte[6](from B. cereus), and by Ohshima and Soda (from
mesophilic Bacillus sphaericus and from moderately thermophilic Bacillus stear-
othemophilus['O, 321). The biochemical data for the last two enzymes, however, do
8"
not differ much, as Table 15.3-3reveals.

The LeuDH from B. stearothemophilus as compared with the B. sphaericus enzyme
has an extended pH range of activity (5.5-10 vs. 6.5-8.5), a higher heat stability (70
vs. 50 "C after a heat treatment of 5 min), a longer half-life (several months vs. six
days at pH 7.2 and G "C), and much greater stability against organic solvents and
denaturants [I'.
LeuDH from B. stearothemophilus had already been cloned and overex-
pressed['" 331 during early studies. Recently, the production of recombinant enzyme
from B. cereus even on a large scale has been demonstrated[34.351.
Table 15.3-3.
Properties of LeuDH from Bacillus sphaericus and Bacillus stearothermophilus['O1.

I
1053
~ ~~~
Source B. sphaericus B. stearothermophilus

M, (kDa) 245 000 300 000
Subunit (M,) 41 000 49 000
hexamer hexamer
Optimum pH: deamination 10.7 11.0
amination 9.0-9.5 9.0-9.5
Coenzyme NAD (KM 0.39 mM) NAD (KM 0.49 mM)
Substrate specificity (in % of L-leucine)
Deamination: L-leucine 100 (KM 1.0 mM) 100 (KM4.4 mM)
L-valine 74 (1.7) 98 (3.9)
L-isoleucine 58 (1.8) 73 (1.4)
L-norvaline 41 (3.5) -
L-a-aminobutyrate 14 (10)
L-norvaline 10 (6.3)
D-leucine 0 0
Amination: a-ketoisocaproate 100 (0.31) 100
a-ketoisovalerate 126 (1.4) 167
a-ketovalerate 76 (1.7) 86
a-ketobutyrate 57 (1.7) 45
a-ketocaproate 46 (7.0)
The substrate specificity of LeuDHs, catalyzing mainly branched-chain a-keto

acids to the a-amino acids, has been investigated by Zink and Sanwal(1962)[361 and
subsequently by Schiitte et al. (1985: B. cereus)161,Ohshima and Soda (1989; Bacillus
stearothemophilus and Bacillus sphaericus) f51, Nagata et al. (1990; Bacillus DSM
7330)[371, Misono et al. (1990; Corynebacterium pseudodiphtheriticum) 13*1 and by
Bommarius et al. (1994; Bacillus stearothemophilus) i3')1. In addition to the proteino-
genic amino acids valine, leucine, and isoleucine, unnatural amino acids such as tert-
le~cine[~'] or L- P-hydroxy-valinek4l1 can be synthesized.
The kinetic parameters of several leucine dehydrogenases show a similar pH-
profile. The opposite tendency of V, and KM for all substrates is remarkable: the
dimethyl-substituted substrates show values above 10 mM (Vm, values are
between 0.2 and 30% of the reactivity of 2-0x0-4-methyl-pentanoicacid, the base
case), whereas KM values below 1 mM are typical for good substrates (V,, = 100%
compared with the base case, 2-0x0-4-methyl-pentanoic acid).
15.3.4.2
Alanine Dehydrogenase (AlaDH, E.C. 1.4.1.1)
AlaDH has been isolated and characterized from both mesophilic (B. subtilis and B.
sphericus) [421 and thermophilic (B. stearothemophilus) [431 organisms. For cloning and
purification of AlaDH, see ref.[*]. The narrow substrate specificity of AlaDH[421
renders the enzyme useful for synthesis of L-alanine and analogs only, such as [15N]-
L-alanine[451, 3-fluoro-L-alanine1461, and 3-chloro-~-alanine [471.
1054
NADH NAD’
Figure 15.3-3. Synthesis of 6-hydroxy-~-norleucine

with CluDH/glucose DH(’’1.
15.3.4.3
Clutamate Dehydrogenase (CIuDH, E. C. 1.4.1.2-4)
GluDH has been investigated by the groups of Engel and Rice since the 1980s so that
more is known about GluDH, especially from C. syrnbiosurn, than about any other
AADH. Although there is no sequence identity to other AADHs beyond random
similarity (Table 15.3-2),site-directed mutagenesis of two amino acids residues,
K89L and S380V, led to similar activity levels towards glutamate, norleucine and
methionine and demonstrated the importance especially of the K89L muta-
tion[48, 491. Stud’
ies on GluDH from the same source define the knowledge base
regarding conformational change of the enzyme upon binding of the substrate but
not upon the preceding binding of the cofactor. These conformational changes also
seem to be responsible in part for substrate spe~ificity[’~1.
Just as with other AADHs, GluDH has potential as a catalyst in synthesis: beef
liver GluDH was the best catalyst for the reductive amination of 2-keto-6-hydroxy-
hexanoic acid Na salt to 6-hydroxy-~-norleucine,a potentially important building
block for the vasopeptidase Vanlev (BMS) (Fig. 15.3-3)[511. The reaction of 95 mM
substrate (2: 1 mixture of 2-keto-6-hydroxy-hexanoicacid Na salt in equilibrium with
2-hydroxy-tetrahydropyran-2-carboxylic acid) was complete in 3 h, resulting in an
amino acid product of 89-92 % chemical yield and >99% optical purity. As the keto
acid substrate is very cumbersome to synthesize, an alternative way of providing the
keto acid substrate was the separation of D,L-6-hydroxynorleucine, which can be
prepared easily from 4-hydroxybutylhydantoir1,by D-amino acid oxidase to L-amino
acid and keto acid where the latter in turn was reduced by G~uDH/NADH[~*]. Both
FDH/formate and glucose DH/glucose were employed for cofactor regeneration.
15.3.4.4
Phenylalanine Dehydrogenase (PheDH, E. C. 1.4.1.20)
An enzyme catalyzing the reductive amination of phenylpyruvate to the desired L-

Phenylalanine was first found by Hummel et al.[52]in a strain of Brevibacteriurn and
later in Rhodococcus sp. ,’[ 531. Table 15.3-4 summarizes the microbiological and
kinetic data [’)I.
Table 15.3-4.
Comparison of PheDH from Brevibacteriurn and Rhodococcusspecies[9].

I
1055
Parameter Brevibacteriurn Rhodococcus

Microbiological data:
enzyme yield (U L-I) after addition of 1% of
L-phenylalanine 210 15200
L-histidine 120 1800
L-phenylalaninamide 3500
L-isoleucine 0 0
D-phenylalanine 204 0
DL-phenylalanine 214 0
9.0 9.25
Enzymological data:
pH optimum
reductive amination
oxidative deamination 10 10
0.11 0.16
KM (mM)
phenylpyruvate
phydroxypyruvate 0.24 2.4
indolepyruvate 8.0 7.7
2-0x0-4-methylmercaptobutyrate 3.0 2.1
100 100
V, (relative to phenylpyruvate)
phenylpyruvate
p-hydroxypyruvate 96 5
indolepyruvate 24 3
2-0x0-4-methylmercaptobutyrate 59 33
47 130
KM (pM) NADH
KM (mM) N H 2 431 387
4-8h 10 d
Stability:
stored at 4 "C (tip)
deactivation ("3 d-I) under operation 26 5
Reference 8 53
Table 15.3-5. Substrate specifity of different P ~ ~ D H S [ ~ ' ] .

Rhodococcus rhodocrous B. sphaericus
Substrate' VmaX(U m L-') KM (mM) Rel. activity (%) Rel. activity (%)
Ketoisocaproate 4.2
Keto-methionineb 50 2.1 33 6.0
Phenylpyruvate 150 0.16 = 100 = 100
p-OH-phenylpyruvateb 7.5 2.4 5 138
Indolepyruvateb 4.5 7.7 3 n. d.'
Keto-4-phenylbutyrate 96 0.01 64 1.9
Keto-5-phenylvalerate 46 0.65 30 1.5
a Conditions: pH 8.0, T = 25 "C, 1.31= 0.1 M; comparison: LeuDH from 5.cereus: 2-0x0-4-methyl-pentanoic
acid = loo%, 2-0x0-4-phenylbutyrate= 0.2% B. sphaericus data fromis5]
b As in a except for a pH of 8.5'''
c Not determined
1056
[:mCo
w
PheDH NH,
L-allysine ethylene
NADH NAD+ acetal
cop NH,HCOO
Figure 15.3-4. Synthesis o f allysine ethylene acetal with PheDH/FDH1581.
Apart from L-phenylalanine, the homolog L-homophenylalanine (L-Hph), im-

portant as a component in ACE inhibitors, can be obtained from 2-keto-4-phenyl-
butyrate with PheDH [541. The substrate specificity of PheDH from Bacillus sphaericus
has been investigated by Asano et al.rS51.Table 15.3-5compares the activities of two
PheDH from Rhodococcus rhodocrous['I and Bacillus s p h ~ e r i c u sfor
~ ~ the
~ ] transforma-
tion of aromatic and aliphatic keto acids.
Sequencing, cloning, and heterologous expression of PheDH from Rhodococcus
was first described by Brunhuber et al.[sGl.A double mutation G124A/L307V was
created by site-directed mutagenesis of PheDH from Bacillus sphaericus to change
the substrate specificityfrom a PheDH closer to a LeuDH. This led to a mutant with
decreased activity towards L-phenylalanineand enhanced activity towards almost all
aliphatic amino acid substrates, thus confirming the predictions made from molec-
ular modeling [57J.
PheDH from Trtermoactinornyces intemedius ATCC 33 205 was utilized recently to
synthesize allysine ethylene acetal [( S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid
(2)] from the corresponding keto acid with regeneration of NAD' cofactor by FDH/
f ~ r m a t e [ ~(Fig.
* ] 15.3-4);the specific activity towards the keto acid was 16% com-
pared to the standard substrate phenylpyruvate.
The system was used in three different configurations: (i) the system with heat-
dried cells from Trt. intermedius (PheDH) and C. boidinii (FDH) yielded on average
only 84 M% and could not be scaled up owing to lysis of the 7%. intemedius cells; (ii)
a similar system with recombinant PheDH from E. coli improved the yield to 91 M%;
(iii) heat-dried Pichia pastoris containing endogeneous FDH and expressing re-
combinant PheDH from Th. intemedius yielded 98 M% with an optical purity of
>98%.
Altogether, more than 200 kg of allysine ethylene acetal have been produced.
15.3.5
Summary of Substrate Specificities
The most comprehensive investigation of substrate specificity of LeuDH and

PheDH has been conducted by Krix et al. (1997)[30!Table 15.34 lists the relative
rates of various substrates.
15.3 Reduction of C=N bonds I1057
Table 15.3-6. Relative V,,, values of keto acid substrates ofvarious LeuDHs and PheDHP’].
Keto acid B. stearo- B. cereus B. sphaericus Rhodococcus
thermophilus LeuDH LeuDH Rhodocrous
LeuDH PheDH
Specific activity (u mg-‘ of protein) 120 15.9 3.3 54.8
2-Oxobutyricacid 48 74 6G 72
2-0x0-3-methylbutyricacid 113 152 205 96
2-0~0-3,3-dimethylbutyric acid 31 74 51 8
2-Oxopentanoic acid 63 81 102 157
2-0x0-3-methylpentanoicacid 110 114 88 193
2-0x0-4methylpentanoicacid” = 100 = 100 = 100 = 100
2-0~0-3,3-dimethylpentanoic acid 2 11 5 4
2-0~0-4,4-dimethylpentanoic acid 7 14 11 54
2-Oxohexanoicacid 15 63 75 250
2-0x0-4-methylhexaoicacid 22 19 n. d? 296
2-0x0-4-ethylhexanoicacid 1 11 n. d? 79
2-0xo-4,4-dimethylhexanoic acid 0.5 1.2 0.2 146
2-0xo-5,5-dimethylhexanoic acid 0.8 0.3 n. d? 257
2-0x0-3-cyclohexylpropanoic acid 0.8 0.1 0.3 140
2-Oxooctanoicacid 0.2 n. d.b n. d.b n. d.b
2-Oxo-3-(l-adamantyl)propanoic acid 0 n. d? n. d? 16
a AllV, values refer to 2-0x0-4-methylpentanoic acid (= loo%),pH 8.5, T = 30 “C. Absolute activity of
LeuDHs with 2-0x0-4-methyl-pentanoic acid (ketoisocaproic acid) were 120 U mg-’ (B. stearothemophilus),
15.9 U mg-’ (B. cereus) and 3.3 U mg-’ (B. sphaericus) as well as 54.8 U mg” with PheDH (Rh.rhodocrous).
b Not determined
LeuDHs from B. cereus, B. sphaericus and B. stearothermophilus display a re-

markably similar substrate spectrum:
LeuDHs accept 2-oxoacids with hydrophobic, aliphatic, branched and unbranched
carbon side chains of up to six C atoms as well as some alicyclic keto acids as
substrates, however, not the adamantyl group, where the geometric limit seems to
be reached. 2-0x0-3-methylpentanoicacid is the preferred substrate, the preferred
chain length is C5.
The keto acid substrate should have at least four C atoms; pyruvate is only
converted at less than 3 % of standard. Short-chainketo acids with branching at the
C3 position are only preferred by the enzyme from B. sphaericus.
The different amino acid dehydrogenases differentiate substrate side chains
mainly based on steric parameters in the C3 and C4 position of branched
ketoacids.
Functionalized keto acids such as ketoglutarate are not accepted (activity < O . l % of
the base case). Phenylpyruvate as a model compound of an aromatic substrate was
inert[’’].
A correlation of LeuDH activity with van-der-Waals volumes [601 or hydrophobici-
ties[“] for different C atom configuration of side chains only yielded a moderate
correlation [39, ‘*I.
PheDH differs markedly from all LeuDHs, as it can convert not only aromatic
substrates but also the aliphatic substrates typical for LeuDHs. Owing to the high
1058
intrinsic specific activity of PheDH from Rhodococcus, in many cases the enzyme
actually registers higher specific activity with many sterically demanding a-keto acid
substrates than LeuDH. The substrate specifity of PheDH from Rhodococcus rhodoc-
rous and Bacillus sphaericus seems to vary more between the two PheDHs than the
specificity between the different LeuDH species. PheDH from B. sphaericus mainly
converts (substituted) phenylpyruvates whereas the enzyme from Rhodococcus sp.
displays a fairly high degree of activity in the presence of a phenylalkyl group in the
substrate.
15.3.6
Process Technology: Cofactor Regeneration and Enzyme Membrane Reactor (EMR)
15.3.6.1
Regeneration of NAD(P)(H) Cofactors
Enzymatic reductive amination with NADH as the cofactor can only be operated on a
large scale if the cofactor is regenerated. Wandrey and Kula have developed a
regeneration scheme using formate as the reductant of NAD' generated upon
reductive amination (Fig. 15.3-1). The formate is oxidized irreversibly to COz by
formate dehydrogenase (FDH, E.C. 1.2.1.2) [62].
For soluble reactants and products, enzymes are preferentially immobilized in an
enzyme-membrane reactor (EMR). To prevent the cofactor from penetrating through
the membrane, it can be enlarged with polyethyleneglycol (PEG)[631.
L-leucine was produced in an EMR with LeuDH from both B. ~phaericus[~'] and B.
stearothermophil~s[~~1.LeuDH has also been employed successfully for the synthesis
of L-tert-leucine in batch pr0cesses[~~1 and in its continuous version[40b* 651. L-tert-
leucine is an important building block for several novel pharma developments["* 671
as well as being on intermediate for templates for asymmetric L-Phe
was produced in an EMR with PheDH starting from phenylpyruvate [Fig. 15.3-5,
(i)]["]. Owing to the instability and high cost of this compound, two additional
processes were devised generating phenylpyruvate in situ (Fig. 15.3-5): (ii) inter-
mittent oxidation of DL-phenyllactate with D- and L-hydroxyisocaproate DH
(HicDH)[691, or (iii) hydrolysis of acetamidocinnamic acid (ACA) with ACA acy-
lase["]. For productivities of all processes, see Table 15.3-7.
Another regeneration scheme for NADH from NAD' utilizes glucose which is
oxidized to gluconic acid with the help of glucose dehydrogenase (see Fig. 15.3-3for
an example)[51].Regeneration to NADPH from NADP' can be afforded by glucose-
6-phosphate dehydrogenase with glucose-6-phosphate as the 721; the
system, however, has not found widespread use yet, probably owing to the higher
price of NADP' vs. NAD' and the cost associated with the generation of glucose-
6-phosphate from glucose.
With the advantage of the potentially quantitative use of a keto acid substrate and
with suitable processes of cofactor regeneration, reductive amination of keto acids is
an interesting route to a-amino acids worthy of consideration in comparison with
more established routes.
I
mCo0"
1059
r\ PheDH W H f i " " "
u
Phenylpyruvate
NADH+H@ NAD@
co2 FDH HCOO@N@
L-HicDH
m H C o o H D-HicDH+
D,L-Phenyllactate
,COOH
NAD@ NADH+H@
t I
0
Acetamido-cinnamate
COOH
PheDH
I f
Figure 15.3-5. Enzymatic routes to L-phenylalaninevia phenylpyruvate[''. (i) Reductive

amination o f phenylpyruvate by PheDH with simultaneous NADH regeneration using
FDH.
(ii) Oxidation o f DL-phenyllactate with D- and L-2-hydroxy-4-methylpentanoate (HicDH)
and simultaneous reductive amination of the phenylpyruvate formed in situ with
PheDH. NADH is "substrate-coupled" regenerated from phenyllactate. (iii) In situ
formation of phenylpyruvate by enzymatic deacetylation o f N-acetamidoocinnamic acid
by the respective acylase followed by simultaneous reductive amination with PheDH.
1060
Continuous production of L-amino acids with the aid of dehydrogenases in an

Table 15.3-7.
enzyme membrane reactor(g].
AADH Regeneration Precursor Product Product Degree s.t.y. Enzyme con- Ref.
enzyme(s) conc. ofcon- g/(Lxd) sumptions
(mM) version (U kg-7
LeuDH FDH oxomethyl- L-leu 80 80 250 300/300 40c
pentanoate (LeuDH,FDH)
LeuDH D-HmpDH DL-OH- L-leu 70 70 72 730/350/650 40a
L-HmpDH methyl-pen- (LeuDH,
tanoate D-HmpDH,
L-HmpDH)
LeuDH D-HmpDH D,L- L-met 240 60 143 40c
L-HmpDH OH-methi-
onine
LeuDH FDH trimethyl- L-tle 425 85 640 1000/2000 40c
pyruvate (LeuDH, FDH)
AlaDH D-LDH D,L-lactate L-ala 184 46 134 4700/2600 40a
L-LDH (LeuDH, FDH)
PheDH FDH phenyl-pyru- L-phe 114 95 456 1500/150 68
vate (PheDH, FDH)
PheDH D-HmpDH D,L-phenyl- L-phe 22 43 28 69
L-HmpDH lactate
PheDH FDH acetamid- L-phe 70 88 277 1170/1770/ 70
+ ACA ocinnamate 400 (acylase,
acylase PheDH, FDH)
HmpDH: 2-hydroxy-4-methylpentanoate-DH;LDH: lactate dehydrogenase; L-tle: L-tert-leucine
15.3.6.2
Summary of Processing to Amino Acids
The production of L-tert leucine on a multi-100 kg scale and of L-neopentylglycine on

a 30 kg scale with LeuDH from B. stearothemophilus demonstrates the suitability of
enzymatic reductive amination on a large scale and even for slow substrates. The
economics of the process is influenced decisively by the retention and regeneration
of both production (AADH) and regeneration enzyme (FDH). If yields of less than
100% are acceptable enzyme consumption can be lowered by running the process in
a continuous Owing to the broad substrate specificity of AADHs,
reductive amination can be utilized especially for the synthesis of hydrophobic
amino acids. LeuDH and PheDH feature complementary specificities for aliphatic
and aromatic L-amino acids. Both enzymes are enantioselectiveto the highest degree
and stable in a coupled process with FDH. As non-enzymatic processes of reductive
amination often lead to low yields and enantioselectivities[73-761, enzymatic schemes
are superior to chemical ones. Additionally, enzymatic reductive aminations are
conducted solely in water so that organic solvents can be avoided.
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53 W. Hummel, E. Schmidt, C. Wandrey, M.-R. 4th Eur. Conp. Biotechnol. 1987, 189-191.
Kula, Appl. Microbiol. Biotechnol. 1986, 25, 71 C.-H. Wong, G. M. Whitesides, j . Am.
175-185. Chem. SOC. 1981, 103,4890-99.
54 C. W. Bradshaw, C.-H. Wong, W. Hummel, 72 B. L. Hirschbein, G. M. Whitesides, /. Am.
M.-R. Kula, Bioorg. Chem. 1991, 19, 29-39. Chem. SOC.1982, 104,4458-GO.
5 5 Y. Asano, A. Yamada, Y. Kato, K. Yama- 73 U.Groth, T. Huhn, B. Porsch, C. Schmeck,
References I1063
U . Schollkopf, Liebigs Ann. Chem. 1993, Zhang, Chin. Chem. Lett. 1992,3 (4),
71 5-7 19. 237-238.
74 U. Groth, C. Schmeck, U. Schollkopf, Lie- 76 G.C. Cox and L.M.Hanvood, Tetrahedron:
bigs Ann. Chem. 1993, 321-323. Aqmm. 1994,5,1669-1672.
75 2. X. Shen, J. F. Qian, W. J. Qiang, Y.W.
16
Oxidation Reactions
16.1
Oxygenation of C-H and C=C Bonds
Sabine Nitsch, Cideon Grogan and D. Ashcroft
16.1.1
Introduction
Reactions catalyzed by oxygenase enzymes (mono or dioxygenases) are interesting

for applications in organic synthesis. There are numerous examples of such
reactions in biological systems, yet there are few chemical reagents or catalysts that
can compete with biocatalysts. Examples of monoxygenases catalyzed biotransfor-
mations are shown in Figure 16.1-1. These include heteroatom oxygenation, aro-
matic hydroxylation, Bayer-Villiger oxidation, double-bond epoxidation and hydrox-
ylation of nonactivated hydrocarbon atoms. The latter can occur with regio-, stereo-,
I
I
Monoxygenases
Figure 16.1-1. Some examples of monooxygenase-catalyzed

biotransformations.
1066
I 76 Oxidation Reactions
and in some cases enantioselectivity that is difficult to achieve using conventional

chemistry. There is a large body of literature describing the exploitation of these
oxygenase enzymes for synthetic applications, and the current chapter will only give
a few representative examples of what has been done.
There are few reports on biotransformations using isolated oxygenase enzymes
because of several problems with cell free enzymes. Many of the oxygenases are
membrane-bound, and require a complex set of co-factors and co-proteins. Despite
the fact that a large number of their genes have been identified, the enzymes
themselves are difficult to isolate and quite unstable. Thus, oxidative bioconversions,
especially on an industrial scale, generally use whole-cellbioconversion techniques,
which makes them less accessible for use by organic chemists in organic synthesis
laboratories. However, very recently new techniques have been developed for the
isolation, cloning and over-expression of oxygenases in heterologous expression
systems, and the number of reports using isolated systems is increasing. These new
developments will be discussed at the end of the chapter, and point to the possibility
of overcoming technical difficulties that have hampered the application of oxygenase
systems in biocatalysis in the past.
A number of excellent reviews with comprehensive coverage on the literature of
biooxidations have appeared in journals and books['-*]. In this chapter we will only
try to highlight some of these biotransformation reactions, in particular hydroxyla-
tion of non-activatedcarbon atoms and double-bond epoxidation reactions.
16.1.2
Hydroxylating Enzymes
Hydroxylation reactions in nature are generally catalyzed by monooxygenase, a sub-

class of the oxidoreductase enzyme group. These enzymes are very important and
ubiquitous proteins found in almost all living cells, ranging from bacterial to
mammalian. One of the most important groups of this type of enzyme is the
cytochrome P450 family. These are heme-dependent monooxygenases whose essen-
tial role, among other functions, is to ensure detoxificationof exogenous compounds
by rendering these very often lipophilic molecules water soluble, thus facilitating
their excretion. Because of this essential function, mammalian monooxygenases
have been thoroughly studied in the context of drug metabolism[1, 9-141.
The majority of the cytochrome P450 systems reported to date are multi-
component, requiring the involvement of additional proteins for transport of
reducing equivalents from NAD(P)H to the terminal cytochrome P450 component.
Increasing attention is given to microbial monooxygenases, in particular in their
application for biotransformations. One of the earliest and most important industrial
applications of microbiologicallymediated bioconversions is the 11-a-hydroxylation
of progesterone using Rhizopus arrhizus cells (Figure 16.1-2) [''I. Microbial mono-
oxygenases tend to be soluble enzymes that can be purified fom cell free extracts, and
a number of crystal structures of microbial monooxygenases are now available[", 171.
The first X-ray structure for P450 was that of cytochrome P450,,,, which was
isolated from Pseudomonas putida, and catalyzes the 5-exo hydroxylation of its natural
substrate D-camphor to 5-exo hydroxycamphor as shown in Figure 16.1-2.
16.7 Oxygenation of C-H and C=C Bonds
11067
Progesterone 11-a-hydroxyprogesterone
Pseudomonas
putida * Ok0”
Camphor 5-exo-hydroxycamphor
Figure 16.1-2. Regio- and stereoselective microbiological hydroxylation of
progesterone and D-camphor.
The P45OC,, enzyme has served as a model system for general studies of
cytochrome P450 enzymes in terms of structure, function and mechanism[16,‘*I.
The exquisite regio- and stereoselectivitycan be explained by the active site geometry
of the P45OC,,, which shows several van der Waals interactions with hydrophobic
side chains and a key hydrogen bond between tyrosine 96 and the carbonyl oxygen of
the substrate. Removal of the tyrosine-96hydroxyl group by site-directedmutagene-
sis or removal of the carbonyl oxygen by using camphane results in loss of
selectivity[”J.
There are also an increasing number of non-P450 type biohydroxylases. Examples
are the n-octane o-hydroxylaseof Pseudomonas oleovorans and the n-decane hydrox-
ylase of Pseudomonas denitriicans, which have been shown to be also responsible for
epoxidation of 1-octeneand for O-demethylation of heptyl methyl
Similarly, the progesterone 9-a-hydroxlase from Norcardia sp., one of the first
microbial hydroxlases obtained in crude form, was shown not to be a cytochrome
P450 Interestingly, this enzyme allows for the functionalization of the
steroid skeleton, thus opening the way to production of the C-11-oxygenated
corticosteroids[26] as shown in Figure 16.1-3.
Other very important examples of non-cytochrome P450 enzymes are methane
monooxygenases. These appear to be more reactive than P450 oxygenases and are
able to catalyze the conversion of methane to methanol, chemically one of the most
difficult steps [271. These enzymes have been shown to reductively activate dioxygen
for incorporation into a wide variety of hydrocarbon substrates, including alkanes,
alkenes and alicyclic or aromatic hydrocarbons. The enzyme harbors a hydroxy-
bridged dinuclear iron cluster in its active site, and its structure has been determined
by X-ray crystallographyL2*].
do
IG
1068
I Oxidation Reactions
Nocardia
sp. o-&o OH
/
0 /
Progesterone I
Figure 16.1-3. Use of9-a-hydroxylation of progesterone as a way to

corticosteroids.
16.1.2
Hydroxylating Enzymes
The active site of P450 monooxygenases contains an iron-heme center that is directly
involved in the oxidation process by activating molecular oxygen. The catalpc cycle
by which cytochrome P450-mediated alkane hydroxylation occurs is by now well
studied[*,181. The reaction cycle of cytochrome P450,,, is outlined in Figure 16.1-4.
This mechanism involves (i) reversible substrate binding which converts the six-
coordinate, low spin form of the protein to the penta-coordinate high-spin form, (ii)
electron reduction of the ferric substrate-enzyme complex by flavoprotein NADPH-
I
Fe3'ROH
k 2H+l
Figure 16.1-4. The catalytic
cycle of cytochrome P450
enzymes.
16. I Oxygenation ofC-H and C=C Bonds
cytochrome P450 reductase leading to the ferrous enzyme, (iii)binding of molecular

oxygen to give the six-coordinateiron-dioxygen intermediate, (iv) + (v) reduction of
this species with a second electron and addition of two protons, thus leading to an
activated oxygen intermediate, (vi) insertion of an oxygen atom into the substrate,
and (vii)release of the iron atom in its original ferrous state. Apart from the products
of steps (iv) and (v), all these intermediates have now been investigated by
crystallography using trapping and cryocrystallographymethods [lS1.
The exact mechanistic details of oxygen insertion into the C-H bond are still the
subject of intense discussion. One of the most popular proposals appears to be that
of the so-called “rebound mechanism”, which proceeds by an initial hydrogen
abstraction from the alkane (RH) by the active oxygen intermediate to form a radical
R and a hydroxo-iron species as intermediates. The radical then rebounds on the
hydroxy group and generates the enzyme-product species.I’[ Alternative proposals
involve cationic intermediates f3O1 or two-state reactivity with multiple electromer
species for epoxidations L3l].
It should be noted that the same cytochrome P450 enzyme is able to achieve
reactions as different as double-bond epoxidation or heteroatom demethylation.
Thus it appears clear that the chemo-, regio- and stereoselectivity of the reaction is a
function of the nature and the fit of the substrate, or, more properly, of its transition
state with the protein, rather than being governed by enzyme reaction specificity.
It is obvious that all these very powerful enzymes are of high synthetic value for
the organic chemist since they prove to be able to achieve, at normal temperature
and in aqueous media, reactions which are very difficult, if not impossible, to
perform using conventional chemistry. One additional bonus offered by these
biological tools is their generally high selectivity. It is thus understandable that a
variety of oxygenative biotransformations have been explored using numerous
substrates. We will focus in the following pages on two such particularly interesting
reaction types, namely the hydroxylation of non-activated carbon atoms and the
stereospecificepoxidation of “isolated double bonds.
16.1.4
Hydroxylation of Non-Activated Carbon Atoms
16.1.4.1
Hydroxylation of Monoterpenes
Because of their involvement in the flavor and fragrance industry, monoterpenes are
one type of natural compounds which have been considered as interesting substrates
for biohydroxylation studies. For instance, geraniol, nerol and linalool were studied
by different groups and were shown to lead to the 8-hydroxylatedproducts with the
fungus Aspergillus r ~ i g e r [ ~as~ ]well as with four strains of Botrytis ~ i n e r e a [ ~ ~ ] .
Interestingly, the same 8-hydroxylated products were found starting from the
corresponding acetates, which were shown to be hydrolyzed to the starting alcohol by
the fungus Aspergillus niger prior to hydroxylation [341. This C-8 regioselectivity has
also been observed in hydroxylations of geraniol and nerol with reconstituted
76 Oxidation Reactions
1070
I
-I_-
Figure 16.1-5. Retention of
configuration during the
OH
hydroxylation at C-8 o f isotopically
labeled geraniol.
P450cath 'H '''[3H]
(R)-( 1 -'Hl)[l -3Hl]
+OH +OH
$H "H +,OH
[%I 'H
(S)-(1-'Hl)[l -3Hl]
hydroxylating enzyme systems from rabbit l i ~ e r [ ~ ~and- ~ from

' ] plant cells like Vinca
rosea as well as Catharanthus roseus (L.) G.
In this last case, incubation of different I3C- and 2H-labeledgeraniols revealed that
hydrogen abstraction is completely regioselective in favor of the CH3 group trans to
the chain at C-6, i. e. at position C-8. An intramolecular isotope effect of KH/KD 8.0
was determined, suggesting that the hydrogen abstraction is one of the major rate
determining steps. Furthermore, Fretz and Woggon [411 have studied incubation of
the (R)(8-'H1) (8-3H1)and (S)(8-2H1)(8-3H1)geraniols (Fig. 16.1-5).This resulted in
the formation of the chiral 8-hydroxy products, thus indicating clearly retention of
configuration during the allylic hydroxylation process. Interestingly, in neither of
these cases has an allylic radical rearrangement (migration of the double bond) been
observed.
Bioconversions of (+)-limonene,the major constituent of citrus essential oils, have
also been studied in recent years in order to afford biotechnological routes to
interesting products of natural source valuable for the perfume and/or flavor
industries. For instance, (+)-limonenewas shown to be transformed by Pseudornonas
gladioli to (+)-a-terpineol(one of the most commonly used products in fragrances
and flavors i. e. lemon, nutmeg, orange, ginger, peach and spices), which is resistant
to further degradation by the bacterium, and to (+)-perillicacid, which is further
The corresponding levorotatory limonene antipode is known as
being the primary olefinic constituent of the volatile oils of immature Mentha piperita
(peppermint), Mentha spicata (spearmint) and Perrilla f i t e s c e n s leaves, whereas
(-)-menthone, (-)-camoneand (-)-perilly1aldehyde, respectively, are the major oxy-
genated compounds. The enzymatic hydroxylation of (-)-limoneneat C-3, C-6 and C-
7 to the corresponding derivatives has been studied using light membrane prepara-
tions from leaves of each of these plants. It has thus been shown that they lead to the
1 6 1 Oxygenation of C-H and C=C Bonds
A
Pseudornonas
gladioli
c:
T O H
A
COOH
(+)-lirnonene (+)-a-terpinol (+)-perillic acid
(OH ro
(-)-carvone (-)-trans-carveol (-)-lirnonene (-)-PeWl (-)-perilly1
alcohol aldehyde
M I : Mentha spicata
M2: Perilla frutescens
M3: Mentha piperita
(-)-trans-isopiperitenol (-)-rnenthone
A.
+
cellulosae
19% 5% 12% 21%
Figure 16.1-6. Bioconversion of limonene using various biocatalysts.
corresponding oxygenated compounds in a mutually exclusive manner in each

species. This suggests very strongly that different forms of cytochrome P450 are
present in each type of plant, each one showing an exclusive regiochemistry of
oxygen as shown in Fig. 16.1-6.
Another terpene interesting for flavor chemistry is 0-ionone (Fig. 16.1-7). Its
microbiologically mediated transformation has been explored in order to afford a
mixture of derivatives that is utilized as an essential oil of tobacco, used for tobacco
flavoring at the ppm 451. One of the best microorganisms capable of
converting 0-ionone to the desired mixture of its useful derivativeswas an Aspergillus
niger strain. This process has been recently improved using bioconversion in the
presence of organic solvents and immobilization techniques. Thus, this fungus
1072
I 1G Oxidation Reactions
go k0k0
p-ionone bH (4R) (25)
M : A . niger or 45% 30%

A. awamo 46% 11Yo
$o Qo
/
OH OH
(R)-(+)-pulegone 58% 10% 4% 6%
Sfreptomyces +
albus
OH HO
sulfone of 24% 20%

rnyrcene
Figure 16.1-7. Biohydroxylations o f various monocyclic terpenes
could be repeatedly used for microbial conversion of p-ionone in the presence of

isooctane for more than 480 hr4'1. Another Aspergillus strain, A. awarnori, has also
been shown recently to achieve hydroxylation of p-ionone. This led to a mixture of
two alcohols, the major product being a building block usable for further synthesis of
abscisic acid (an important phytohormone) analogs 1471.
Other monocyclic terpenes like for instance R-(+)-pulegone (a mint-like odor
monoterpene ketone which constitutes the main component of Mentha pulegium
essential oil)L41' menthols, terpinolene and carvotanacetone r4'] have been investi-
gated. All these substrates proved to be transformed by various Aspergillus strains,
including A. nigec leading essentially to monohydroxylation. Also, monocyclic
1 6 I Oxygenation ofC-H and C=C Bonds
I 1073
A. niger
30°C,pH 7.2
(+)-fenchone (+)-6-endo-hydroxyfenchone
Acetobacter
mefhanolicus
(+)-a-pinene (+)-frans- (+)-verbenone (+)-trans-

verbenol Sobrerol
OH
(+)-camphor 60% 7% 9%
v Y
Bacillus
cereus
OH
1,&cineole 74%
Figure 16.1-8. Biohydroxylation of various bridged bicyclic terpenes.
sulfoxide derivatives of the linear terpenes myrcene and ocimene were shown to be
good substrates for several bacteria and fungi, whereas they were themselves only
very poorly
Finally, some bicyclic monoterpenes have also been recently described to be
subject to microbiological hydroxylation (Fig. 16.1-8).For instance (+)-fenchonewas
transformed to (+)-6-endo-hydroxyfenchoneby A. niger[’l], and a-pinene led to the
predominant metabolites (+)-trans-verbenol,(+) -verbenone and (+)-trans-sobrerolby
the action of several strains of the methylotrophic species Acetobacter methanoli-
C U S [ ~ ~(+)-Camphor
]. is transformed to the major metabolite 6-endo-hydroxy-camphor
by cytochrome P450,,, enriched intact cells of Streptomycesgri~eus1~~1, and 1,S-cineole
(the major component of the oil from leaves of Eucalyptus radiata var.) is hydroxy-
lated to 6-(R)-exo-hydroxy-l,&cineoleby the bacterium Bacillus cereus following a
high yielding (74%) and stereospecific route [541. Interestingly, this same bacterium
had been previously described to be able to achieve hydroxylation of 1,4-cineole
yielding good yields of essentially pure 2-(R)-endoand 2 4 R)-exo-hydroxy-l,4-ci-
neole[551.
IG
1074
16.1.4.1
Hydroxylation of Monoterpenes
Similarly to monoterpenes, several studies aimed at the microbiological hydroxyla-

tion of sesquiterpenes have been described, and a review has summarized the most
interesting results obtained in this area Since then, additional examples have
been published. Thus, three germacrone-type sesquiterpenoids, (+)-germacrone-
4,5-epoxide, germacrone and (+)-curdione,were described as being transformed by
Aspergillus niger[57].The interesting feature of these results is the fact that they
q
essentially led to hydroxylated guaiane-type sesquiterpenoids (together with allylic
alcohols and spirolactone) which arise from transannular cyclization of the carbon
ReHo+ + HO
1
(+)-gerrnacrone
4,5-epoxide 3% 4%
__c
nger
/ ' HO
(+)-germacrone 3% 4%
(+)-curdione R1 =OH,R2 = H 9%
R1 = H, R2 = O H 10%
; H
Beauvaria
sulfurescens
H' \ H" \
OCONHPh OCONHPh
Phenylcarbarnoyl derivative
15%
of dihydroarternisinin
Figure 16.1-9. Biohydroxylation of various higher terpenes.
1 6 1 Oxygenation of C-H and C=C Bonds I1075
skeleton as shown in Fig. 16.1-9. The biohydroxylation of an N-phenylcarbamoyl

derivative of dihydroartemisinine by the hngus Beauveria sulfirescens has been
described[58].This allowed the preparation of new and novel derivatives of artemisi-
nine, a drug known to be active against Plasmodiumfalciparum, the strain responsi-
ble for malaria, which claims more than one million lives a year. The C-1O-N-
phenylcarbamoyl derivative of dihydroartemisinine, a highly oxygenated sesqui-
terpene, is thus converted into its 1Chydroxymethyl derivative in 15% yield.
Although this yield can be considered as rather modest, this biohydroxylation is
interesting since it allows us to prepare derivatives which retain the peroxide group
required for biological activity of these drugs. Also, it emphasizes the possibility to
favor biohydroxylation by introducing an amide or urethane group into a substrate,
as already observed on other model substrates (Fig. 16.1-9)[59* 60].
A series of biotransformations of GP-santonin and of some of its derivatives,
achieved by Cuwularia lunata and Rhizopus nigricans cultures, have also been
described (Fig. lG.l-lO)[G1]. Depending on the strain used and on the starting
substrate, several metabolites were obtained, including products resulting from
hydroxylation as well as double bond and/or carbonyl reduction. The same group
also described biotransformations of several 1,G-difunctionalizedeudesmanes lead-
ing to 12-hydroxyderivatives which are interesting intermediates for the synthesis of
G,l2-e~desmanolides[~~]. Similarly, starting from GP-acetoxyeudesmanone,biohy-
‘0
6P-santonine 19%
6p-acetoxy-l p-4p- 12%

p& ’ OAc
1 10%
dihydroxyeudesmane
p&
0 0
HO ” OAc
6p-acetoxyeudesmanone
Figure 16.1-10.
deans- h0;
HO OAc
72%
$J),,Jy
HO
15%
OAc
Further examples of biohydroxylationsof bicyclic natural compounds.

1076
R'=OH, R2=H, R3=CH,0H

Gibberellin
20 -19-lactone R'=H, R'=OH, R3=CH20H
R'=H, R2=OH, R3=CH0
Figure 16.1-11. Some examples o f diterpenes hydroxylation by Cibberellafujiikuroi
droxylation was achieved at C-11 by the fungus Rhizopus nigricans, thus opening
another way to the synthesis of the same lactone targetsFG3].
Several diterpenes have also been described recently to be subject to microbio-
logical hydroxylations. Thus, as shown in Fig. 16.1-11, a compound prepared from
gibberellin A13 was transformed by the fungus Gibberella fijikuroi, affording three
metabolites, two of these arising from hydroxylation at its 3a- and 19-p0sitions[~~].
Similarly, the same fungus was shown to transform isoatisene derivatives into
rearranged isoatisagibberellin derivatives[G51.
Sclareol, a natural product first isolated from the essential oil of Salvia sclarea L.
(Labiatae) in 1931, is used for diverse applications in the perfumery and flavoring
industries and in folk medicine. This diterpene has been described recently to be
hydroxylated by three strains, i. e. Cunninghamella sp., Septomyxa afinis[661and
Mucor p l u m b e ~ s [68],
~ ~leading
, essentially to hydroxylation reactions on the A ring of
this compound (Fig. 16.1-12).Some of these metabolites could be used for further
synthesis of some biologically active targets or as mammalian metabolism models.
Amazingly, as in the case of sciareol, the presence of an oxygenated function on
the C ring position of grindelic acid again orients the hydroxylation process towards
the A ring, since mainly 3P-hydroxylation is observed [691. This is to be compared with
the results obtained in studying the behavior of Rhizopus and Aspergillus strains as
potential hydroxylating species for kaurene sesquiterpenes 1"' (Fig. 16.1-13).Inter-
IG. 1 Oxygenation of C-H and C=C Bonds
I
1077
Cunninghamella
sp. NRRL 5695
R' = OH, R2 = CH3 36%

R' = H, R2 = CHzOH 50%
OH
Septomyxa
affinis
R' = H, R2 = R3 = 0 49%
Scalerol
R' = R3 = H, R' = OH 3%
R'=OH,R2=R3=H 27%
Mucor
plumbeus
k3
R1 = OH, R2 = R3 = H 84%
R1 = R3 = H, R 2 = OH 6%
R1 = R 2 = H, R3 = OH 6%
Figure 16.1-12. Hydroxylation of natural sclareol using different
microorganisms.
estingly, the fact that the starting substrate bears an oxygenated function on the A
ring now orients the oxidations catalysed by R. nigricans toward the C ring (and in
particular to the C-13 position). This nicely resembles the results previously observed
in the steroid family by Jones and and once more emphasizes the role
of a preexisting oxygenated function which operates as an anchoring and thus a site-
directing entity inside the hydroxylating active site.
By modifylng the location of this function on the starting material, it should
therefore be possible to orient the hydroxylation locus differently. This is nicely
exemplified by hydroxylation of stemodine, a diterpene bearing a preexistent OH
group at position 2 of the A ring. Hydroxylation by C. elegans and by Polyangium
cellulosum then orients the hydroxylation process toward positions 17 and 19[721.
1078
t mixture
nigsr H O W
Grindelic acid
+ mixture
:ans
I \ I
OAc OAc
Kaurene derivative 22%
Stemodin
R' = R 3 = R 4 = H, R'= a-OH

M: C. elegans R' = R3 = R 4 = H, R'= P-OH
R' = R2 = R 4 = H, R 3 = O H
M: I? celldosum
I R2 = R 3 = R 4 = H, R' = O H
R2 = R 3 = H, R' = R 4 = OH
Figure 16.1-13. Effect of a preexisting oxygenated function on the

orientation of a biohydroxylation process.
16.1.4.3
Hydroxylation of Steroids
Because of their utmost importance as bioactive molecules, steroids have been the
most thoroughly studied family as far as microbiological hydroxjlations are con-
cerned.
The most important features and references have been put together by Holland in
his important monograph'73].At the present time, one could presumably almost
consider that one or even several strains are known which are able to introduce a
hydroxyl group at every carbon atom of the steroidal framework. Obviously, however,
further work will have to be achieved in order to improve the selectivities and yields
16.7 Oxygenation ofC-H and C=C Bonds
I 1079
&OH Curvularia
lunata o#oH
fl op
0
Cortexolone
C E CHH3 ZEZH3
Curvularia
/ lunata
0
Norethisterone acetate
Figure 16.1-14. Examples of steroid hydroxylations by Curvularia lunata.
of these bioconversions. Thus, the course of the Ilp-hydroxylation of cortexolone by

Curuularia lunata, a hydroxylation of considerable commercial importance, has been
more recently reexamined. The work described by Chen and Wey[741focused on the
improvement of this process by studying the characteristics of mycelial growth as
well as the role of substrate addition time and dissolved oxygen tension. These
studies have provided some more insight into the fundamental aspects of this
biotransformation. The 110-hydroxylation of norethisterone acetate by the same
microbial strain has also been described[75](Fig. 16.1-14).
16.1.4.4
Miscellaneous Compounds
Although being initially the major part of literature concerning microbiological

hydroxylations,natural compounds of the terpene or steroid families have not been
the only ones to be studied in this context. Indeed, more recently studies have
focused on using biohydroxylationsto provide interesting synthetic intermediates, in
particular chiral intermediates.
Linear or branched-chain alkanes have been shown previously to undergo hydrox-
ylation by various microorganisms, and can lead for instance to fatty acids,
hydroxyacids or a-dicarboxylicacids of commercial importance. Pseudornonas oleovor-
am is one of the strains capable of achieving such transformations, but this process
suffers from the fact that the monocarboxylic acids formed in the first step are
submitted to P-oxidation and thus used as a source of energy and carbon. Such
problems can now be overcome using heterologous expression of P450 genes in
microorganism, which will be discussed later on.
An interesting aspect of biohydroxylations is that the biocatalyst can in principle
generate one single enantiomer starting from a prochiral substrate - a reaction
which could be defined as “enantiogenic”1“‘. This enantiogenic hydroxylation can
lead to enantiopure compounds by stereospecific attack of one single enantiotopic
1080
n=l yield = 26%, ee = 20%

n = 2, 3, 4 yield > 65%, ee > 98%
Montierellc
isabellina
chroman yield = 1070,ee > 98%

Figure 16.1-15. Examples of regio- and stereoselective benzylic hydroxylation.
face of the starting substrate. Biohydroxylation reaction can therefore be used to

prepare high value chiral synthetic intermediates from low value prochiral starting
materials, and there are now a number of reports of such reactions.
Thus, benzocycloalkenes have been described to undergo bacterial hydroxylation
by the Pseudomonas putida strain UV4. As shown in Fig. 16.1-15, this yielded
exclusively hydroxylation at the benzylic position, and also one single enantiomer,
i. e. the (R)-alcohol.The biotransformation of benzocyclobutene proved, however, to
be different from that observed for higher benzocycloalkenes, presumably because
of its particular chemical A similar result has been observed by Holland
and coworkers in the course of chroman biotransformation by the fungus Mortierella
isabellina [781, which leads, although in low yield, to the benzylic (R)-alcohol.
Another interesting example of asymmetric synthesis from a prochiral substrate is
the preparation of (S)-naproxen,a non-steroidal anti-inflammatory drug. It has been
shown that several strains are able to regioselectively oxidize one of the enantiotopic
methyl groups of the isopropyl moiety. This allows the preparation of the corre-
sponding acid, which is obtained with high enantiomeric purity (Fig. 16.1-16).
Next to Aspergillus niger the fungus Beauueria bassiana (previously classified as
Sporotrichum sulfirescens and B. sulfirescens) is one of the most frequently used
fungal biocatalystl2). In particular, hydroxylations of piperidine and pyrrolidine
derivatives have been studied by several groups, and interesting regio- and ster-
pro R
CH3
Figure 16.1-16.
Selective hydroxyla-
Organism ee (%) tion o f one enantio-
topic methyl group
Cordyceps rnihfaris 99
Graphinium fructicola 96
as an approach t o
Exophiala rnansonni 68 optically pure
Exophialajeanselmei 66 Naproxene.
1 6 1 Oxygenation ofC-H and C=C Bonds
I 1081
COPh
Beauveria
sulfurescens
-
&
HO
COPh
Figure 16.1-17. Hydroxylation
versus epoxidation of two
spiro-bicyclic amides.
83%
Beauveria
sulforescens *
\
0
\
COPh
74% CoPh
eoselectivities have been rep0rted[~'-'~1.It should be noted that the ring nitrogen
generally needs to be protected for a successful biohydroxylation.This can be used to
advantage since the choice of protecting group can influence the regio- and
stereochemistry of the hydroxylati~n[~~]. Some examples of hydroxylations of spir-
obicyclic amides are shown in Fig. 16.1-17.Similarly to previous results described by
Fonken and coworkers[". 861 and by Furstoss and coworkers[87* "I, these led to good
yields of hydroxylated products. In all these cases, the regioselectivity of the reaction
is partly or even exclusively oriented toward the C-9 carbon atom, a result which
could have been predicted on the basis of the previously described results. Inter-
estingly, a similar substrate bearing a double bond at carbon C-9 led to the
corresponding epoxide.
Because they constitute partial structures of various higher terpenes and/or
steroids, enantiomers of different substituted hexahydronaphthalenones are pivotal
intermediates in the total synthesis of these target compounds. Therefore, several
differently substituted octalone derivatives have been studied for microbiological
hydroxylations. These substrates were prepared in optically active form by chemical
synthesis from (S)-(+)-Wieland-Miescher's ketone and were submitted for screening
with nine strains known to hydroxylate polyterpenic or steroidal substrates. Thus,
submitted to a culture of Rhizopus arrhizus, these substrates led to allylic hydroxyla-
tion at the B ring, as shown in Fig. 16.1-18f8',"1. Similar results were obtained by
Azerad and coworkers ['*I in the same series, starting from differently substituted
octalones. These authors have investigated the biotransformation of their substrates
with a variety of fungal strains. For most of these strains, the (R)-enantiomer of
hydronaphthalenone led to the 8-hydroxyenone as the main product, i.e. again a
product of allylic hydroxylation, which is quite disappointing since this product is
easily accessible by (e1ectro)chemicaloxidation. However, the fungus Mucor plum-
beus produced another hydroxylated metabolite, the 6a-hydroxyl derivative. Inter-
estingly, the S-enantiomer ofthe starting substrate only led to the 8-hydroxyenonein
this last case. Introduction of an additional methyl group on the carbon framework
of the starting octaenone also led to different regioselectivities of the hydroxylation.
Hydrindane derivatives, which bear a five-membered B ring (instead of a six-
membered ring in the decalones derivatives) have also been examined for bio-
hydroxylation by the fungus Rhizpous a r r h i ~ u s [ ~All
~ Ithe
. hydroxylations observed
now occur at position 3, to the a$-unsaturated ketone. This can be considered as
R’=OH, RLH 42% 27%
R’=H, $=OH 55% 9%
0 0 0
HO @ Rhizopus
arrhizus
* HO@OH
63%
+ HO@
9%
54% 16%
n’/>(j
- -
6
,-,\OH
0 403 Mucor
plumbeus
~
0 ;a
bH
+
22%
aotBu
66%
Rhizopus
arrhizus
0
ofio +
58% OH 8% bH
Figure 16.1-18. Microbiological hydroxylation of differently substituted octalones.
being formally analogous regioselectivity as compared to the results obtained on

decalones. In this case, however, reactivity is identical in both antipodal series, and
led almost quantitatively but with moderate or low stereoselectivityto the formation
of the epimeric alcohols. Interestingly, these biohydroxylations prove to be com-
plementary to lead tetraacetate oxidation of these substrates, which affords the
6-acetyl substituted products.
Stmctually much more complex molecules have also been submitted to re-
gioselective enzymatic hydroxylation. Two such examples have been described
involving milbemycin, a sixteen-membered macrolide which exhibits broad-spec-
trum insecticidal and acaricidal activity, and monensin, a carboxylic polyether
antibi~tic”~. 941. Milbemycin (Fig. 16.1-19) was thus regioselectivelyhydroxylated at
the 130 position (followed eventually by a C-29 hydroxylation) to afford the 138,29-
I 1083
H H
Monensin
Sebekia /
benih/
<
Figure 16.1-19. Regioselective hydroxylation o f structurally complex substrates.
hydroxylated product by a strain isolated from solonized brown Mallee oil (collected
in Adelaide in Australia) and identified as Streptomyces cavourensis. It is interesting to
emphasize here the high regioselectivity observed for this hydroxylation of a rather
complex and multifunctional compound.
Even more complex is the structure of monensin, a compound which has been
extensively used as an anticoccidial agent for poultry and shown to improve the
efficiency of feed utilization in ruminant animals. When submitted to a culture of
Sebekia bevihana, monensin was first quantitatively converted by enzymatic reduc-
tion of the 6-hydroxy-ketone(which is in equilibrium with its hemiketal tautomeric
form) and was regioselectively further hydroxylated at the C-29 methyl group as well
as at the nearby ethyl group ~ubstituent[”~].
All the previously described examples exemplify the ability of various mono-
oxygenase enzymes to achieve, often with good to reasonable yields and interesting
7G Oxidation Reactions
1084
I regioselectivities, the hydroxylation of non-activated carbon atoms which are in-
accessible using conventional chemistry. This thus allows one-step syntheses of
these metabolites, which can in certain cases be of high enantiomeric purity.
Another type of oxygenation reaction which is of interest is the stereoselective
epoxidation of double bonds, the essential aim being in this matter the access to
epoxides of high enantiomeric purity. This will be the subject of the following part of
the discussion.
16.1.5
Epoxidation of Olefins
As discussed previously, monooxygenases provide highly activated oxygen inter-

mediates that can oxidize a wide range of functional groups. One of the most studied
among these has been the epoxidation of olefins [95-971. This epoxidation is partic-
ularly interesting when applied to prochiral double bonds. Spectacular success has
been obtained in the field of asymmetric chemical epoxidation, notably using
Sharpless epoxidation catalysts for ally1 alcohols and Jacobsen catalysts for aryl
olefins, which has made epoxides key intermediates in the synthesis of chiral
compounds. However, these chemical catalysts often have a limited “substrate”
range, and biocatalysts can provide access to complementary structural motifs.
Without attempting to be exhaustive, we will try in this chapter to focus on results
allowing us to directly oxidize olefins to their corresponding epoxide, using micro-
bial cells. Other sources of monooxygenases, such as mammalian cells (micro-
somes) or plant cells, have been studied in this respect. However, these will not be
considered in this review.
16.1.5.1
Epoxidation of Straight-Chain Terminal Olefins
One of the earliest observations implicating the formation of epoxides during

microbial olefin metabolism was the report by Bmyn in 1954 that Candida lipolytica
grown on I-hexadecene produced 1-hexadecanediol (about 5 % of the hydrocarbon
consumed was accounted for as the diol)[98].Molecular “ 0 was shown to be
incorporated into this diol, and the 1,2-epoxide was identified as one of the by-
products of this metabolism[”. “1‘. Several further reports confirmed that enzy-
matic systems are able to achieve epoxidations. For instance, Van der Linden showed
in 1963 that Pseudomonas aeruginosa grown on n-heptane and resuspended in a
buffer solution produced the epoxide from 1-octene (Fig. 16.1-20)110’1. This led the
authors to conclude that this epoxide was formed by enzymes already present in the
alkane-growncells and that epoxidation might be catalyzed by the same hydroxylases
that would normally oxidize alkanes. A similar conclusion was reached by Maynert
and coworkers [lo2],who demonstrated that epoxides are obligatory intermediates in
the metabolism of simple olefins in rat liver microsomes.
However, the real breakthrough in the study of enzymatic epoxidations is due to
Abbot and coworkers I1O3] and to May and coworkers [lo41,who established unequivo-
1G. 1 Oxygenation ofC-H and C=C Bonds I 1085
M * W&
’o %
+ H
O
- /
H
1-octene (R)
ally1 benzene
M: Pseudomonas oleovorans
Figure 16.1-20. Stereospecific epoxidation of straight-chain terminal olefins.
cally that epoxides are formed from terminal olefins by the bacterial strain Pseudomo-
nas oleovorans (Fig. 16.1-20).They showed that 1-octene is epoxidized to 1,2-epox-
yoctane of (R)-configuration(ee 70%) or hydroxylated to 7-octen-1-01.The 1,7-dieneis
exclusively epoxidized, affording 7,8-epoxy-l-octene,which can be further processed
to the corresponding diep~xide[’~~]. It was shown later that this monoepoxidation
was stereospecific, leading to the R(+)-7-epoxideshowing an ee of about 80%.
Furthermore, the diepoxide was shown to be essentially of (R,R)configuration. This
interestingly indicates that the configuration of the monoepoxide formed at one end
of the molecule profoundly affects the stereochemicalcourse of the reaction. Indeed,
the authors showed that when starting from racemic monoepoxide, the diepoxide
was essentially formed from the (R)-monoepoxide.Interestingly it was observed that
olefins bearing an allylic (or homoallylic) hydroxyl were not epoxidized, but were
converted instead to the corresponding saturated ketones.
One of the most useful characteristics of this work is the fact that these epoxides
could be routinely produced at yields approaching (at best) 1 g L-’ after simple
overnight shaking using whole-cell or even crude cell-free systems. Thus, these
results clearly opened the way to a new type of biotransformation which should be
very useful for organic synthesis.
The enzymatic system involved in hydroxylation reactions of long-chain alkanes
had been previously studied by Coon and coworkers, who isolated an enzyme system
from P. oleovorans that catalyzes co-hydroxylation of alkenes and fatty ac-
106-1151. This was resolved into three protein components: rubredoxin (an
iron-sulfur protein of molecular weight 19 000), an NADH-rubredoxin reductase (a
flavoprotein of molecular weight 55 000) and an “o-hydroxylase”(characterized as
being a non-heme iron protein, with one iron atom and one cysteine per polypeptide
1086
I chain). Interestingly,it was shown that this same enzyme system is responsible for
the conversion of terminal olefins to their corresponding 1,2-epoxides[lo4].This leads

to a competition between the two types of biotransformations, which results in a
specific pattern for each type of substrate. Thus, further investigation demonstrated
that this monooxygenase can produce epoxyalkanes with from six to twelve carbon
atoms containing terminal alkenes. As a result of the influence of carbon chain
length on epoxidation versus hydroxylation it was shown that hydroxylation predom-
inates for the “short” substrates propylene and 1-butene, but that epoxidation activity
falls off much less readily than hydroxylation for “long”substrates. For the “medium”
length substrates, like for instance 1-octene, both reactions do occur. Thus, this
substrate is epoxidized to 1,2-epoxyoctaneor hydroxylated to 7-octen-1-01,while for
1-decene epoxidation largely predominates. Interestingly, the epoxidation reaction
exhibits a specificity far different from that expected for chemical reactivity. Indeed,
terminal olefins are epoxidized exclusively even in the presence of more highly
substituted (electron-rich)double bonds. Thus, cyclic and internal olefins were not
epoxidized. This indicated that the substrate specificity pattern observed severely
moderates the inherent reactivity of the activated oxygen species involved in these
transformations. Methyl imidoesters as well as sodium cyanide were found to be
inhibitors of enzymatic epoxidation, and the potency of a homologous series of
imidoester inhibitors was examined.
In the reaction with dienes, 1,s-hexadiene to 1,ll-dodecadiene were epoxidized
while dienes with a smaller number of carbon atoms were hydroxylated to the
corresponding unsaturated alcohols[116]. The reactivity was shown to be maximal for
octadiene (leadingto 0.3 to 0.4 g of diepoxyoctaneper liter) and falls off rapidly as the
carbon chain is shortened, but decreases only slightly as the chain is lengthened. In a
further study, it was shown that a very efficient conversion of 1,7-octadiene to
7,8-epoxy-l-octeneand 1,2-7,8-diepoxyoctane could be obtained by incorporating a
high concentration of cyclohexane into the conventional fermentation medium.
Thus, a 90% yield of product was achieved within 72 h, instead of a 18.5 % yield in
the absence of cyclohexane, when a 20% (v/v) amount of cyclohexane was used.
Clearly, this is an early example of the use of organic solvents applied to microbial
transformations l1”1.
A similar result was obtained later on using the 1-octene substrate itself as the
organic phase (20% v/v), leading to comparable results (70% ee) [118]. Interestingly it
also has been shown in the course of this work that, when n-hexadecane (which is
not metabolized by the cells) is used as a solvent, racemic epoxide is enantiose-
lectively degraded by the “a-hydroxylation” enzymatic system of P. oleovorans,
leading to an enrichment in (S)-1,2-epoxyoctane.
Further work by Wynberg and coworkers was aimed at even increasing the yield of
1,2-epoxyoctaneusing an optimized two-phase system and a cell renewal proce-
d~re‘’~’]. Thus, yields up to 150 mg 1,2-epoxyoctaneper mL I-octene and up to
20-25 mg 1,2-epoxyoctaneper mL culture was obtained. Some other substrates were
tested in this optimized system. Of these, 1-decenewas converted into (R)-1,2-epox-
ydecane (GO % 0. p.), while allylbenzene was converted to the corresponding epoxide.
However, no effort was made to determine the absolute configuration and the optical
purity of this product.
16.1 Oxygenation ofC-H and C=C Bonds
I 1087
H R. equi 95.4
lM
NClB 12035
f! putida 98
NClB 9571
I
/? oleovorans 98.4
Chemical AT CC 29347
f! aeruginosa 98.8
NClB 8704
R = CH2CH20CH3: Metropolol
R = CH2CONH2 : Atenolol (M: f! oleovorans 0.p. = 97%)
Figure 16.1-21. Microbiological epoxidation as a way t o optically
pure fl-blocker drugs.
All these results led to an interesting application for asymmetric organic synthesis.
Thus, P. oleovorans has been used, among some other microorganisms, for ster-
eospecific epoxidation of some arylallylethers into (+)-arylglycidylethers (Fig. 16.1-
21). These intermediates were chemically converted into (S)-(-)-3-substituted-1-alky-
lamino-2-propanols, which are the physiologically active components of the p-
adrenergic receptor blocking drugs. This method has been used to synthesize (S)-
(-)-Metoprolo1 and (S)-(-)-Atenolo1with enantiomeric purities of 95.4-98.8 % and
97 % respectively['201.These applications are of great industrial interest, since it has
been shown that (S)-(-)-Metoprolo1is 270-380 times more active than its anti-
pode[121].
Microorganisms screened for epoxidation activity were selected from bacteria
belonging to the genera Rhodococcus, Mycobacterium, Nocardia and Pseudomonas.
Species of Pseudomonas gave the best activities, but there were variations between the
individual members, and P. oleovorans was the most active organism. The activity
was further enhanced by carrying out the transformation in the presence of a
cosubstrate such as glucose.
This pioneering work on microbial expoxidationof straight-chain terminal olefins
has triggered several further studies aimed at preparing enantiopure epoxides via
biotransformations. Thus, a number of alkene-utilizing microorganisms have been
described in the literature. In the context of aliphatic substrates these efforts have
been developed essentially along two lines: epoxidation of long-chain olefins and
epoxidation of short (CI-C,) chain compounds. Thus, for instance, it was shown that
Corynebacteriurn equi ( I F 0 3730) grown on n-octane is able to oxidize 1-hexadeceneto
give the corresponding optically pure (R)-(+)-epoxide(41% yield based on consumed
substrate) 123J. This strain also assimilated other terminal olefins and produced
IG Oxidation Reactions
1088
I the corresponding epoxides from substrates which have a carbon chain longer than
fourteen, although in very low yields (less than 1%). Production of 7,8-epoxy-
1-octenefrom 1,7-octadieneby non-growing Pseudomonas putida species using two-
phase transformation has also been achieved [1241. Similarly, a gaseous hydrocarbon-
assimilating microorganism Nocardia c o r a l h a B-276 grown on I-alkenes (C3,C4 and
c13-c18) was described as being able to produce the corresponding 1,2-epoxy-
alkanes. One of the products, 1,2-epoxytetradecane,was shown to be optically active.

Glucose-grown cells could also transform styrene and C2.C18 1-alkenes to their
epoxyalkanes[12'1. Similarly, production of epoxides from c&10 I-alkenes and
styrene was shown to be enhanced by using n-hexadecane as an additional solvent,
while this led to a decreased rate for epoxidation of longer chain l-alkenes1126].
Epoxidation of unsaturated fatty acids such as palmitoleic acid by Bacillus rnega-
teriurn has also been reported [1271. Here again, experiments indicated that epoxida-
tion and hydroxylation were catalyzed by the same soluble cytochrome P450-
dependent enzymatic system.
16.1.5.2
Short-Chain Alkenes
Short-chain alkenes are another type of substrates which have been studied for
microbiological epoxidation during the last thirty years. In this context, an extensive
study has been conducted by De Bont and coworkers in order to prepare epoxides
from gaseous olefins. Thus, a Mycobacteriurn sp. (E 20) was isolated from soil and
shown to excrete ethylene oxide when grown on ethylene['28, 1291 . Stud'ies carried out
using ' * 0 2 showed that a monooxygenase was involved in these epoxidations, as
proved by incorporation of only one l80into the product. Another Mycobacterium
(Py 1) was also shown to achieve this reaction. Experiments were performed in a gas-
solid reactor to prevent accumulation of the toxic ethylene oxide in the immediate
vicinity of the biocatalyst[1301. An experimental set-up, allowing for automatic gas
chromatography analysis of circulation gas in a batch-reactor system, was also
described allowing on-line monitoring of the microbial oxidation of the gaseous
alkenes propene and 1-butene (Fig. 16.1-22)[1311. Optimization was achieved by
studying the influence of various organic solvents on the retention of immobilized
cell a~tivityIl~~1.
High activity retention was favored by a low polarity in combination
with a high molecular weight. Using chiral gas chromatography (at that time recently
described by Schurig and Biirkle [1331), eleven strains of alkene-utilizingbacteria were
screened with respect to the stereospecific epoxidation of propene, 1-butene and
3-chloro-1-propene. The results obtained showed that seven of these bacteria
strongly resembled each other, in that they all produced 1,2-epoxypropane and
1,2-epoxybutanemainly in the (R)-form(93 and 85 % ee respectively).Several of these
strains were also able to epoxidize stereoselectively l-chloro-2,3-epoxypropane, thus
leading to the synthetically very useful (S)-epichlorohydrin (ee > 95 %). Stereose-
lective epoxidation of 4-bromo-1-butene and of 3-buten-1-01was similarly studied
using three strains. The results showed that the epoxides were again obtained
predominantly in the (R)-formbut that their enantiomeric purity depended on both
16.1 Oxygenationof C-H and C=C Bonds
Figure 16.1-22. Short-chain alkene

epoxidation.
X = Br, CI M: Mycobacteria
Nocardia &O
corallina CnH2n+1
n=34 e.e. = 76-90%
(-)-fosfomycin (90%)
the strain used and on the substrate studied(87].Inactivation of the alkene oxidation
enzymatic system by the produced epoxide was also investigatedin view of setting up
a biotechnological procedure for producing these epoxides Modeling the effects
of mass transfer on the kinetics of propene epoxidation was also achieved by the
same authors[135,13'1, and they showed that product inhibition can be reduced by
absorbing the epoxide in the gas phase in cold di-n-octylphthalate['37].
In addition to the Mycobacterium species, several other strains have been reported
to achieve epoxidation of olefins. Thus, three distinct types of methane-grown
methylotrophic bacteria (Methylosinus trichosporium, Methylobacterium capsulatus and
Methylobacterium organophilum) were shown by Hou and to be able to
oxidize terminal C2 to Cq n-alkenes to their corresponding 1,2-epoxides,which
accumulated extracellularly. Results from inhibition studies indicated, as in the case
of the previously discussed o-hydroxylation system of P. oleovorans, that the same
monooxygenase enzyme was responsible for the hydroxylation of methane and the
epoxidation of alkenes. Further work achieved by the same group showed that whole
cells of Methylosinus sp. CRL 31, immobilized by adsorption on glass beads, were
able to convert propylene to propylene oxide for several hours until the reduced NAD
cofactor was depleted. This could be regenerated by periodic addition of methanol.
These authors also observed that attempts to immobilize the cells by covalent
binding or entrapment in polyacrylamide gel led to complete loss of propylene
1090
epoxidation activity(139]. However, no mention is made in this work of the enantio-

meric purities of the obtained epoxides. Further studies carried out by Sub-
ramanian1'401 revealed that these were nearly racemic compounds, and also that the
major problem of these biotransformations was again product (epoxide)inhibition.
More information about the reaction mechanism of the epoxidation achieved with
whole-cell M. trichosporium was gained by Okura and coworkers1l4l],who showed
that the configuration of the double bond was retained during the epoxidation of cis-
2-butene. This result was further confirmed by studies of the epoxidation of
1,2-deuterated-cis-propene. A concerted insertion of oxygen was postulated to ac-
count for this result Oxidation of propylene to propylene oxide by Methylococcus
capsulatus (Bath) was studied in order to optimize the biotransfomation for a
possible industrial production. However, the high rates obtained could only be
sustained for 3-4 min before loss of biocatalytic activity 0ccurred[l~~1.
Similar results were obtained by Wyngard and in the course of a
study aimed at exploring how immobilization of the whole cells on solid supports
would influence the rate and duration of the epoxidation of propylene by the strain
Nocardia corallina B-27G initially isolated by Furuhashi and coworkers [I2'. 12'] . Here
again the results suggested that entrapment in a hydrophobic matrix might be a
favorable system, but that loss of activity was quite rapid with time. The same
Nocardia strain has been shown to be able to epoxidize branched chain terminal
olefins in an asymmetric manner leading to (R)-epoxidesshowing optical purities of
7G-90% depending on the chain length. These epoxides were used as chirons for
further synthesis of prostaglandin a-chains. The same strain was shown by these
authors to be also able to epoxidize trifluoromethylethylene (75% ee) [1451.
Some newly isolated Xanthobacter sp. were recently shown to be able to accumulate
1,2-epoxyethanefrom ethene or, when grown on propene, to accumulate 2,3-epox-
ybutane from cis- or trans-2-butene but with apparently low yields [14'1. Similarly,
Rhodococcus rhodochrous, a propane-oxidizingstrain, was shown to produce 1,2-epox-
yalkanes from short-chain terminal alkenes. Interestingly, its oxygenase enzyme
appeared to be capable of tolerating high levels of product without inhibiti~n"~'].
Finally, a very useful and industrially interesting epoxidation which deserves
special attention is the stereospecificepoxidation of cis-propenylphosphonate.Eight-
een species of Penicillium, one of Oidium and one of Paecilomyces were found to
effect this reaction, which affords directly (-)-fosfomycin,a broad-spectrum antibi-
otic. Using the strain Penicillium spinulosum MB 2843 at optimum culture condi-
tions, a 90% efficiency (based on olefin charged, 0.5 g L-l) was obtained after G days,
leading to a product claimed to be optically pure [1481.
16.1.5.3
Terpenes
Besides the extensive studies aimed at preparing optically active epoxides starting
from short or long straight-chain alkenes, another area of investigation has been the
microbiological epoxidation of various natural substrates, essentially in the terpene
and steroid area. Interestingly enough, it appears that terminal olefins (and only
76.7 Oxygenation ofC-H and C=C Bonds I 1091
"
9H
i +
Methyl geranate
w1-m
e.e - 100%
P
S.
albus OH
HO
Linalool
7
D.
gossypina
+(&
HO
bH
(-)-Linalool (SR,GR)-Linalool oxide
Figure 16.1-23. Some examples of olefinic terpene epoxidation.
these) are epoxidized almost exclusively by bacteria, and lead to accumulation of the
corresponding epoxide in the culture. On the other hand, more substituted double
bonds are often preferentially oxidized by higher organisms like fungi. The product
is generally the corresponding vicinal diol arising from further metabolism (hydroly-
sis) of the primarily formed epoxide. Numerous publications describe microbial
transformations of various terpenes [14', lS0l. However, there are few cases of an
accumulation of intermediates in sufficient amounts for further use in synthe-
sis [w.
One of the first examples of such a transformation has been described by Marumo
and coworkers (Fig. 16.1-23)I1'*l. Their investigations, aimed at preparing optically
active insect juvenile hormone, showed that methylgeranate was metabolized by the
fungus Colletotrichurn nicotianae, leading to 19.6% of S(-)-methyl-6,7-epoxygeranate
and to 15.6% of R(+)methyl-6,7-dihydroxygeranate after 9 h incubation. Longer
incubation times (24 h) produced only the optically pure glycol with an isolated yield
as high as 85%, showing that the first epoxidation step had to be stereospecific.
Unfortunately, this analpcal study was not pursued on a preparative scale, and no
accurate results concerning the stereochemical and kinetical aspects of these
interesting biotransformations have been described.
A similar microbial oxidation of the isoprene double bond has been studied by
1092
I Veschambre and coworkers starting from l i n a l o 0 1 [ ~Thus
I6 Oxidation Reactions
~ ~ ~ . Streptomyces albus, a
strain which synthesized nigericine, transforms each enantiomer of linalool, as well
as the racemic compound, into a mixture (10-20% yield) of two diastereoisomeric
linalool oxides. In this case, the epoxide formed primarily is trapped by an
intramolecular cyclization. Based on the reported proportions of these products, one
can deduce that the ee of the formed epoxide was about 35 %. Further work achieved
using several other microorganisms showed that Beauveria sulfirescens gave similar
yields (15-20 % analytical) of an equimolar mixture of linalool oxides [lS4].Botyris
cinerea, a fungus which participates in the formation of flavors in sweet wines, was
also checked for linalool biotransformation. This led to several metabolites including
linalool oxides, presumably arising from prior epoxidation of the olefinic bond [1551.
Interestingly, these products were also detected in the Carica papaya fruit flavor,
together with the diastereoisomeric epoxides [1s61. It was also observed by Abraham
and coworkers that (-)-linalool is processed by Diplodia gossypina exclusively to a
mixture of trans-(3R,G R)-linalool oxide and to the corresponding tetrahydropyran.
These were proposed to arise by intramolecular cyclization of the intermediate G(S)-
epoxide. Some other similar substrates have been studied in the course of this study,
but they generally led to low yield mixtures of products. Comparable results were
obtained from linalool using the strain Streptomyces cinnamonensis [15*l. Similar
transformations were observed starting from 2-methyl-2-heptene-6-one[1s91. How-
ever, because of the number of metabolites formed and the low yields obtained, these
biotransformations cannot be usefully employed for organic synthesis.
Myrcene and trans-nerolidol were also shown by Abraham and Stumpf to be
transformed by two fungi (Diplodia gossypina and Corynespora cassiicola respectively)
into a mixture of several products including vicinal diols arising from oxidation of
the isoprenyl double bond. These were shown to be further degraded, presumably
via an acyloin-splittingmechanism [l6O1. During the course of the fermentation, the
diol occurred at first in the culture medium followed by the nordiols and the
trialcohols. So, the formation of these compounds from diols seemed to be very
likely. Some other related substrates were also studied in the same context, and it was
shown that both strains revealed a pronounced and almost opposite substrate
selectivity. Much more impressive is the result obtained by the same groupL'"1,who
conducted a broad screen of 800 various microorganisms using both the ( S )(-)- and
the (R)(+)-limoneneenantiomers as a starting substrate, as well as some other
terpenes which were tested with the best suited strains (Fig. 16.1-24).The most
interesting results were observed with Diplodia gossypina (ATCC 1093G), which
afforded 380 mg of a diol which was found to be the ( l R , 2R, 4S)-8-p-menthen-
1.2-diol from 1 g of (S)(-)-limonene. Similarly, Corynespora cassiicola (DSM 62474)
was described to yield l.lg of (lR, 2R)-3-p-menthen-l,2-diol from 1.8g of a-terpene.
(R)(+)-limonenewas shown to afford (lS, 2S, 4R)-p-8-menthene-1,2-diol.
Because of the interest of these products in flavor chemistry, the preparative-scale
transformation of this enantiomer by the fungus Diplodia gosvpina has been
undertaken: thus 1300 g were transformed, yielding 900 g of the (IS, 2s) diol
showing high optical purity[1621. Interestingly, these strains convert the substrates
fast with only negligible amounts of side products. Also, it is noteworthy that the
2 y2 gossypina
Diplodia - 30%
I
1093
(S)-(-)-Limonene ( I R , 2R, 4S)-8-p-Menthene-l,2-diol
,,,\OH
\ Diplodia
gossypina
-1
(R)-(+)-Limonene
,
corynespora
cassiicola
55%
/
OH
,,,,\OH
+
f'
/
0.6%
+
pJ
/
OH
a-Terpinene 49% 2% 1Yo
Figure 16.1-24. Stereoselective oxidation of rnonocyclic terpenes.
obtained diols are almost exclusively of trans configuration. No indication is provided

concerning the determination and the values of the obtained products' optical
purities. It was suggested that these trans-diols were formed via an intermediate
epoxide, which could be further cleaved enzymatically to the obtained diols.
Surprisingly, both these microorganisms were shown not to attack 3,3,5,5-tetra-
methyllimonene[1631.However, geranylacetone, nerylacetone, trans-nerolidol, cis-
nerolidol, farnesol and 2,5-dimethyl-1,3-hexadiene were transformed by these
strains to the corresponding glycols in yields of up to 70%[1643 16'1 and interesting
optical purities of up to 98%. Using (+)-trans-nerolidolas a substrate, the strain

Nocardia a k a DSM 43 130 was shown to be lacking an epoxide hydrolase, thus
leading to a 27 % yield of the corresponding (S)-epoxidewhich accumulates in the
culture medium [lS71.
Also, the ability of the monensin-producing organism Streptomyces cinnamonensis
to convert the cis and trans isomers of nerolidol has been investigated['58].However,
here again this led to a low-yield mixture of several products.
Much more useful in that sense are the results obtained by Furstoss and coworkers
in the course of their study of biooxygenation of geraniol derivatives (Fig. 16.1-25).
Indeed, it has been described in a first paper that, if the N-phenylcarbamate of
geraniol is used instead of geraniol itself, its transformation by the fungus Aspergillus
niger leads to a 49 % isolated yield of the 6,7-dihydroxylatedproduct. Moreover, this
diol proved to be of (6s) absolute configuration and was shown to possess an
enantiomeric excess of about 95%[76, This diol, which is a very versatile
substrate for further organic synthesis, can thus be obtained without problem in
1094
I
A. niger
18
02
R = CONHPh
(6R) ee = 95%
Figure 16.1-25. Stereoselective pH-dependent oxidation of geraniol N-phenyl
carbamate.
gram-scale quantities (1g substrate treated for 36 h in 1 L culture afforded 550 mg

pure diol). Further work aimed at exploring the influence of the culture conditions
showed that a unique stereochemical control could be achieved simply by modulat-
ing the pH of the medium. Thus, although when the culture was at pH 2 the diol of
(S)-configurationwas obtained, at pH 6-7 the diol of opposite ( R )absolute configura-
tion was isolated in similar yields and with an ee again as high as 95%. This
interestingly showed that the fungus A. niger not only is able to convert the substrate
across the pH 2-7 range, but that the (6s)-epoxide must be the primarily formed
metabolite. This can then be further hydrolyzed in acidic medium (following the
classical acid-catalysismechanism) to afford the (6s)-diolor, at pH 6, be hydrolyzed
enzymatically to the (6R)-diol by attack on the less substituted oxirane carbon
Experiments conducted in the presence of " 0 confirmed this hypothesis.
When the incubation was carried out at pH 2, the distribution of the " 0 label in the
obtained diol was 95 % on C-6 and 5% on C-7.This ratio was inverted at pH 7. These
results show clearly that, whatever the pH, molecular oxygen is involved in these
oxygenations but only one labeled oxygen atom is incorporated into the diol, leading
to an epoxide which is differently hydrolyzed, depending on the pH of the medium.
Very interestingly as far as organic synthesis is concerned, these biooxygenations
can be conveniently performed on a scale of several grams (5 g), thus allowing easy
preparation of either enantiopure diol. These can be conveniently used as "chirons"
for the synthesis of various natural or non-natural products. For instance they can be
cyclized to the optically pure linalool oxides [16'] or the corresponding tetrahydropyr-
anols [1691.
Biooxygenation of some other similar compounds, i. e. 7-geranyloxycoumarin,
citronellyl N-phenylcarbamate and sulcatol N-phenylcarbamate were studied
. The reaction was shown to be operative in all these cases,
(Fig. 1G.1-26)['70-'721
leading, for instance to either enantiomer of marmin (a member of the umbellifer-
one family). Moreover, this result opens the way to an easy preparation of either
:I ,<\OH
OH
(-)-marmine
A. niger
PH 2
60%
7-geranyloxycournarin
PH 6
43%
7 6 7 Oxygenation of C-H and C=C Bonds I1095
A.niger
OH
(+)-marmine
0 0
>,
1.: p
+
,:::o
(G‘R),i”epoxyaurapten (G’S),i”epoxyaurapten
* AP?
85% A60%
P E .
cR
(3RS)
ee=90%
$ A.niger
PH 2
73%
~
(3R)-citronellyl
N-phenylcarbamate
\,\OH
R + $R
,\\OH
(3R96R)
ee=92%
Sulcatyl (2R 5s) P S , 55)

N-phenylcarbamate
Figure 16.1-26. Application of the pH-dependent oxidation of geranyl derivatives
to the synthesis of some natural products.
enantiomer of 6‘,7’-epoxyauraptenand of 3’,6‘-epoxyaurapten,both these com-

pounds being natural products isolated from various sources. Similar results were
obtained from both commercially available citronellol enantiomers, leading to the
corresponding diols showing ee’s as high as 90 and 92 %.
Bioconversions conducted at pH 2 on racemic sulcatol N-phenylcarbamate led to a
73 % yield of a 1/1mixture ofthe two expected diastereoisomeric diols, which can be
readily separated by flash chromatography. They both show ee’s > 95 %, indicating
that the first (epoxidation) step again occurred in a highly stereospecific manner.
Interestingly in this case, it was also possible to avoid hydrolysis of the intermediate
epoxide by changing the preculture conditions and performing the reaction at
neutral pH. This intermediate can thus be obtained directly with high enantiomeric
purity. Using this chiron allows the four-step synthesis of optically pure pityol, a
IG
1096
(25) (2S,5S) (2R,SS)-pityol

Figure 16.1-27. A four-step synthesis o f (ZR,SS)-pityol using microbiologically mediated
steps.
male-specific attractant of the bark beetle Pityophtorus pityographus. Thus, prochiral

6-methyl-hept-5-en-2-onewas reduced with baker’s yeast to the corresponding
alcohol (60% yield, 98.5% ee). This was converted to its N-phenylcarbamate,which
was subsequently subjected to epoxidation using A. niger, thus affording a 50%
preparative yield of the corresponding enantiopure epoxide. In a final step, treatment
of the epoxycarbarnate with an alcoholic NaOH solution led to the natural (2RS.S)-
pityol(7.5% overall yield, 100% ee, 98% de) (Fig. 16.1-27).
16.1 S.4
Cyclic Sesquiterpenes
Various cyclic sesquiterpenes have also been studied in order to explore the
possibility of achieving their microbiological transformations. Very often these were
shown to lead to epoxidation processes when one (or several) double bonds were
present in the starting substrate (Fig. 16.1-28).
Thus germacrone, which is thought to be the precursor of a variety of bicarbocyclic
sesquiterpenoids, was shown to be transformed by the fungus Cunninghamella
blakesleena. This led primarily to regio- and stereoselective epoxidation of one of the
intracyclic double bonds of this prochiral triene, thus affording two epoxides. The
third product isolated from this experiment was due to subsequent epoxidation of
the remaining intracyclic double bond. Interestingly, the exocyclic olefinic bond
conjugated to the carbonyl function appeared resistant to oxidation [1731,
Valencene, another olefinic sesquiterpene, has been studied in the same context
using microorganisms isolated from It was observed that these bio-
transformations led in reasonable yields to a mixture of three main metabolites,
including an epoxide and nootkatone, an interesting flavoring compound.
The microbial transformation of humulene, a substrate showing a structure
similar to that of germacrone, was studied by Abraham and Stumpf using a screen of
about 300 strains[175]. This led the authors to select the fungi Diplodia gossypina and
Chaetonium cochlioides for preparative scale experiments. It was thus observed that
the main reaction path starts with the epoxidation of the 1,2-doublebond, as shown
by direct biotransformation of this monoepoxide obtained by chemical synthesis.
This is then further oxidized to yield a multitude of products including diepoxides
and hydroxy-epoxides (Fig. 16.1-28).
Comparable results were obtained from caryophyllene, a compound similar to
humulene. Again, the biotransformation of this substrate with cultures of Chaeton-
76.7 Oxygenation of C-H and C=C Bonds
I
1097
p+- C.b
Germacrone
my-- -
Soil +
-
Valencene
(- Nookatone 12%
D. gossypina ............
* mixture
C.cochlioides
D. gossypina
mixture
C. cochlioides
Carophyllene
Figure 16.1-28. Epoxidation steps i n the course of sesquiterpene biotransforrnations
i u m cochlioides as well as of Diplodia gossypina give a broad spectrum of products,

resulting from an initial epoxidation of the 1-2 double bond followed by additional
epoxidation or hydroxylation processes (Fig. 16.1-28)[176, 1771.
16.1.6
Conclusions, Current and Future Trends
This review has illustrated the very broad range of biohydroxylations and epoxida-
tions that can be achieved using monoxygenase enzymes. In fact, one can propose
1098
l that almost all organic compounds are potential substrates for these enzymes. Since
each substrate can lead to many different oxidized products, the range of compounds
that can be generated is clearly enormous.
Finding new enzymes with novel substrate specificities and selectivities of
reaction has in the past been achieved by screening organisms and substrates and
has very much been down to good luck. Current and future work is focused on
finding methods to make this process faster and more rational and predictable. This
is now possible because of new technologies in genetics, molecular biology and
structural biology, of which a few highlights are discussed below.
More and more P450 monoxygenases have been sequenced and cloned into
heterologous expression systems. This can have the advantage of higher turnover
yields because of higher expression of the enzyme in the host or because higher cell
mass can be obtained when using easy growing organisms such as E. coli as
hosts[’78,1791. Heterologous expression can also overcome problems of loss of
product because of further metabolic degradation[l8’I as in the case of the alk gene of
P oleovorans. Such expression systems also allow the facile generation of chimeric
enzymes and mutants with more desirable biocatalytic properties, such as increased
activity towards a particular substrate[lS1,182]. Some of the popular organisms for
biohydroxylationssuch as Beauvaria bassiana also might contain several endogenous
P450 enzymes that can interfer with selectivity of one enzyme and make predictions
of reactions very difficult [1831.
The rapid emergence of whole genome sequences has made a major impact on the
study of P450 monooxygenases [lS41, since they are often easily identifiable by small
conserved consensus sequences, in particular around the heme binding site. We
now know that Mycobacterium tuberculosis contains probably twenty different P450
monoxygenases; Bacillus subtilis contains seven. The a priori prediction of substrate
specificity and selectivity from gene sequence is at the moment impossible and
presents a great challenge to the researcher. However, there has been some success
in prediction of substrate specificity by “in silico screening” based on available three-
dimensional structures of P450-monooxygenases[1851. Thus, substrate docking algo-
rithms were used to predict substrate suitability for P450cam and its L244A mutant
from a library of commercially available compounds.
The most practical way of using P450-based biocatalysts is still in whole-cell
systems, because of cofactor requirements and problems with enzyme stability.
However, some P450 monooxygenases, such as the P450cam, can be isolated in
sufficient quantities and reconstituted for cell-free preparative scale biotransforma-
tions[1821.This might be particularly useful for substrates that cannot penetrate cell
walls, are toxic to the organism or are unstable in the organism. One solution for
overcoming co-factor requirements might be the use of electrochemical methods,
and is has indeed been shown that P450cam can be immobilized on an electrode and
can take up electrons from the electrode[186].
Another novel area of intense research is the application of mutagenesis (random
and directed) to obtain desired changes in substrate specificity. Thus P45Ocam,
which is highly selective for camphor and closely related analogs, was subjected to
site-specific mutagenesis, changing the tyrosine in position 96 to a phenylala-
1G. 1 Oxygenation of C-H and C=C Bonds
which resulted in about a 20-fold increase in the reactivity towards

I 1099
naphthalene. The P450 monooxygenase was independently subjected to random

mutagenesis by Arnold and co-workers[18’1, and mutants were screened for in-
creased activity towards naphthalene. Similar improvements to those observed by
specific mutagensis were obtained. However, interestingly, the mutations that were
found to be responsible for improved activity were not at position 96, but were
distant from the active site of the enzyme. Such a “directed evolution” approach has
great promise in quickly generating desired biohydroxylationcatalysts, provided that
a suitable screening system for the product can be found. The method has also been
recently used by the same group on P450BM3 [188].
In conclusion, the application of biocatalysts in biohydroxylations and epoxida-
tions is rapidly expanding in terms of practicality, substrate range and selectivity. A
vast diversity of P450 genes is generated by genomics programmes and mutagene-
sis. Methods for screening such oxidation catalysts are becoming more rapid, and
one can forsee a future where designer biooxidation catalysts, tailored for a specific
substrate and even for selectivity of reaction, can be generated within short time
spans using a combination of rational and screening methods.
16.1.7
Cis Hydroxylation of Aromatic Double Bonds
16.1.7.1
Introduction
The microbial dioxygenation of aromatic compounds 1 has been known for over
thirty years through the pioneering efforts of D. Gibson et al., who characterized the
metabolic pathway of toluene degradati~n[’~’]. In lower organisms, the chiral cis
glycol intermediates 2 are rapidly oxidized by dihydrodiol dehydrogenase, involving
rearomatisation to the diol3, which is further oxidized by ring cleavage dioxygenase
to give dicarboxylic acid 4, which can be channeled into the organism’s normal
metabolic pathways (Scheme 16.1-1)[190-1921.
R
1
dioxygenase
02
R
aoH 2
OH
NADP+ \I dihydrodiol
dehydrogenase
~ ‘ C O ~ H ring cleavage
Scheme 16.1-1. Oxidative
/C02H dioxygenase
R OH degradation of aromatic
4 3 compounds by microorganisms.
7 G Oxidation Reactions
1100
I
The use of certain strains of Pseudomonas putida, most notably the mutant 39 D
with blocked dehydrogenase allows accumulation of the chiral glycols in
the fermentation medium associated with high stereospecificity while the substrate
tolerance remains high with respect to ring substituents. The enzymology of
dioxygenases has been and refinement of the mechanistic details of
the dioxygenases c o n t i n ~ e s [ ~ 'but
~ J , only those enzymes and applications of rele-
vance to the preparative biotransformations will be considered here.
16.1.7.2
Preparation o f cis Dihydrodiols
An impressive number of substituted aromatic compounds 5 have been converted

by mutant strains of Pseudomonas putida into the corresponding chiral cis glycols G
with often excellent stereosele~tivity[~~~~ 1961 . The remarkable substrate range and
selectivity of this dioxygenase system for the aromatic ring have been demonstrated
by the conversion of a series of substituted benzenes and of alkenyl benzenes with
the side chain double bond being left intact (Scheme 16.1.2) ' . An analogous
191
product was obtained from para-fluorotoluene, but the dihydrodiols from para-
chloro- and para-bromotoluenes were found to be racemic [1991. Unlike the substrates
shown in Scheme 16.1-2,benzoic acid, toluic acid, and their halogenated analogs, for
example 7, undergo enzymatic dioxygenation by Alcaligenes eutrophus B 9 and two
strains of Pseudomonas, for example JT 103, mainly at the 1,2-position (Scheme
16.1-3)[200]. However, with other strains of Pseudomonas putida, for example JT 106,
enantiospecific cis 2,3-dihydroxylationis possible, too [201].
The structure of the substrates is not necessarily restricted to monocyclic aromatic
compounds such as those shown in Scheme 16.1-2. The dioxygenase activity of
Pseudomonas putida and Belj'erinckia species has been used exclusively for the
synthesis of cis dihydrodiols from polycyclic [202] and heterocyclic[2031 derivatives,
Such products have been obtained from naphthalene, anthracene, phenanthrene,
benz[a]pyrene, benz[a]anthracene, and methylsubstituted benz[a]anthracenes, and
R ?
Pseudomonas putida
JTI 03
OH
5 6
R = H, Me, Et, nPr, nBu, BU,

EtO, nPrO, halogen, CF,, Ph, PhCH,
PhCO, CH(OH)CH,, COCH,, CHz=CH, CHz=CHCHz, CH$H=CH Scheme 16.1-2. Synthesis of cis
CH3C=CHCH3, HC-C, CF,, CN, COP!+ SiMe, diols by Pseudomonas putida.
HOZC, OH
Pseudomonas putida
JTI 03
Scheme 16.1-3. Cis-hydroxyla-
F F F
tion of aromatic carboxylic acids
7 by Pseudomonas putida JT 103.
a
1 G. 7 Oxygenation of C-H and C=C Bonds
Scheme 16.1-4.
I
'lol
Beijerinckia
\ 0 8/36
monas putida.
x=o.s
03
Pseudomonas
putida
33%
Ho""
a
Pseudomonas
putida
\ \ N 47% HON
Q
9 OH
Pseudomonas
putida
55%
10
Pseudomonas
putida
45%
many of the enzymes responsible have been identified and ~haracterized[~'~].

Benz[a]anthracene,for example, is converted to three cis dihydrodiol regioisomers by
Beijeierinckia B 8/36['04]. This organism has also been reported to produce dihy-
drodiols from dibenzofuran [2051 and dibenzothiophene (Scheme 16.1-4)['06]. The
ability of a Pseudomonas putida mutant to metabolize heteroaromatic compounds is
demonstrated by the bioconversion of quinoline 8, isoquinoline 9, quinazoline 10,
and quinoxaline 11 L2O7]. Attack occurred exclusively in the carbocyclic ring
(Scheme 16.1-4).
The impact of the genetic revolution has been greater in the area of dioxygenase-
catalyzed reactions than in many other areas of bioconversion, largely because of the
bacterial origin of the enzymes concerned. The bacterial oxidation of aromatic
double bonds to cis diols in an enantiospecific manner leads to highly interesting
synthons for organic chemistry. For example, the diene may be subjected to Diels-
Alder reactions, as well as Michael-type addition reactions. Alternatively, oxidative
cleavage of the cyclohexadienering leads to open chain products, which further react
to yield cyclopentanoids. The large synthetic potential of chiral cis glycols is
illustrated in Scheme 16.1-5.
T. Hudlicky et al. efficiently synthesized the prostaglandin PGE2 12 through an
oxidative ring cleavage of the methyl-substituted diol G (R = CHS)[208], the vinyl
I6 Oxidation Reactions
1102
I
HO
OH
21
1‘ R=CH3 // \ . -OH
HO“’ .
OH 15
19
16
HO
18
HO’ .
Et6
17
Scheme 16.1-5. Syntheses of natural products from substituted cyclohexadienediols.
derivative G (R = CH = CHI) was used for the construction of the plant metabolite
(-)-zeylena13r209), and the chloro-substituted diol for the synthesis of the alkaloid
trihydroxyheliotridane 14[2101 and the carbohydrates L-ribonolactone 15i 2 1 1 ] and D-
erythrose 16[’12].Hudlicky et al. also prepared the sesquiterpene specionin 17L2l3],an
antifeedant to the spruce budworm, and the narcissus alkaloid lycoricidine 18i214]in
only nine steps.
Biologically active polyols like pinitol 19L2”, D-myo-inositol ZO[217],conduritol
CF218]and conduritol E r2”)1 were obtained from diol6 in both enantiomeric forms in
only a few steps using this approach.
Futhermore C. R. Johnson et al. synthesized (-)-shikimic acid 21, the biosynthetic
precursor of the benzene moiety of aromatic amino acids [220].
In the case of the cyclohexadienediols 6, the current development promises to
complement the traditional and rather arduous use of carbohydrates as starting
materials from the chiral pool. The popularity of diol-based methods will, therefore,
be directly proportional to their ready commercial availability and to the operational
References I
1103
ease of their transformations for the stereocontrolled introduction of further

functionalities.
Several supply houses are now providing some simple chiral diols of type 6,and
further applications will assuredly follow.
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16.2
Oxidation o f Alcohols
Andreas Schmid, Frank Hollmann, and Bruno Bijhler
16.2.1
Introduction
The enzymatic oxidation of alcohols is catalyzed by different oxidoreductases. Here,

examples of dehydrogenases, oxidases, and peroxidases are discussed. Single en-
zymes were selected based on representative or demanding reactions that are
catalyzed, or because of interesting reaction engineering solutions applied. Reac-
tions catalyzed by whole microbial cells are described in a separate chapter. A focus is
put on presenting or introducing enzyme catalysts and their substrate spectra in
order to give the reader a basis for designing his or her own, new reactions with
sterically or electronically similar compounds or with such compounds which are
compatible with a certain reaction mechanism.
Biocatalysis usually exploits advantageous features of enzymes such as chemo-
selectivity, regioselectivity, enantioselecivity and substrate spectrum of a certain
broadness as depicted in Fig. 16.2-1. These points are addressed in examples in the
following chapters.
16.2.2
Dehydrogenasesas Catalysts
16.2.2.1
Regeneration of Oxidized NicotinamideCoenzymes
Regeneration of NAD(P)' from NAD(P)H is a redox reaction involving the transfer

of two electrons and a proton (successivelyor at once as hydride ion H') to a suitable
acceptor. Most commonly these acceptors are carbonyl functions, molecular oxygen
or the anode. Apart from a few exceptions the direct hydride transfer is slow or
disadvantageous so that catalytic procedures have to be applied. Here we selected
representative examples to give an overview. Excellent review articles are available,
tooil-3. 101
16.2 Oxidation ofAlcohols
I
1109
A
H R-0 B
PH
t -.A-
G C
.COOH
E
Figure 16.2-1. Enzyme-catalyzed oxidations o f 16.2.3.4); E, F: Enantioselectivity (e. g. Sects.
alcohols. Reactions are grouped according t o 16.2.5.2 and 16.2.6.4); C: Non-natural sub-
the feature mainly exploited in the preparative strates (e.g. Sect. 16.2.2.3); H: Complex struc-
application. A-C: Chemoselectivity (e. g. Sects. tures from simple starting materials (e.g. Sect.
16.2.2.3, 16.2.2.6, and 16.2.2.11); C, D: Regiose- 16.2.2.3).
lectivity (e.g. Sects. 16.2.2.9, 16.2.2.10, and
16.2.2.2
Dehydrogenases as Regeneration Enzymes
Today, the utilization of a dehydrogenase-catalyzed reduction reaction is still the

most widespread approach for the regeneration of oxidized NAD(P)+.Its principle is
displayed in Fig. 16.2-2.
Most commonly, alcohol dehydrogenase (E. C. 1.1.1.1) from yeast (YADH), horse
liver (HLADH), or Themzoanaerobiurn brockii (TBADH) as well as glutamate dehy-
drogenase (E.C. 1.4.1.2.) or lactate dehydrogenase (E.C. 1.1.1.27) are used for
NAD(P)+regeneration (Table 16.2-1).Thus, the reduction equivalents are transferred
to an aldehyde or ketone as terminal electron acceptor yielding the corresponding
alcohols.
The drawbacks of this approach result from the necessity to use a second enzyme,
whose optimal reaction conditions may differ significantly from those of the actual
production enzyme, and the presence of cosubstrates and coproducts. Furthermore,
1110
I 7 G Oxidation Reactions
NAD(P)+ NAD(P)H
red. regeneration enzyme OX.
Figure 16.2-2. Enzymatic regeneration of oxidized NAD(P)+.
Table 16.2-1. Comparison of commonly used dehydrogenases for NAD(P)' regeneration.

Regeneration Cosubstratel Specific Activity Stability Coenzyme E'o M vs.
enzyme coproduct [U mg-'I
NHE" [11
YADH Acetaldehyde/ 300 Low, sensitive NAD' - 0.199
ethanol to 0 2
TBADH Acetone/ 30-90 Thermostable NADP' - 0.286
isopropanol
Glutamate DH a-Ketoglutaratel 40 High NAD' and - 0.121
glutamate NADP'
Lactate DH Pymvate/lactate 1000 High NAD' - 0.185
a NHE: normal hydrogen electrode.
1 H. K. Chenault, G. M. Whitesides, Appl. Biochem. Biotech. 1987, 14, 147-197.
the thermodynamical driving force is low because the formal redox potential of the
cosubstrate/coproduct couple is often close to that of the NADH/NAD' couple.
Some of these problems can be addressed using the following regeneration con-
cepts:
16.2.2.2.1 Enzyme-Coupled Regeneration

Since dehydrogenase catalysis is reversible, the production enzyme can be used to
perform the regeneration reaction of NAD(P)+using a suitable cosubstrate as
electron acceptor. In this case, the regeneration enzyme in Fig. 16.2-2 is identical
with the production dehydrogenase. However, conversion rates in this set-up tend to
be low because of a given reaction equilibrium, which requires an efficient method
to withdraw the products and coproducts.
1 6.2.2.2.2 lntrasequential Regeneration

One elegant way of in situ product removal is to use the product of a first
dehydrogenase reaction as substrate for a subsequent enzymatic reaction, thus
recycling the oxidized nicotinamide coenzyme (Fig. 16.2-3). Various NAD(P)-de-
pendent enzymes can be applied as regeneration enzymes in this cascade reaction.
I
1111
dehydrogenase
substrate
NAD(P)+ intermediate
product I
regeneration enzyme
Figure 16.2-3. lntrasequential regeneration of NAD(P)'. The strategy
applied is the synthetic coupling of a dehydrogenase-catalyzed oxidation
and a regeneration reaction yielding the final product and NAD(P)
regeneration.
If the regeneration enzyme is a second dehydrogenase, an overall redoxisomeriza-

tion takes place. But also monooxygenases are reported as regeneration enzymes
thus yielding an overall double oxidation of the substrate (see Sect. 16.2.2.6.1).
16.2.2.3
Molecular Oxygen as Terminal Acceptor
The application of molecular oxygen as oxidant is favorable for several reasons. It is

cheap and easily applicable. Furthermore, the high redox potentials of the 02/H20 or
0 2 / H 2 0 2couples (in acidic solution + 1.23 V and + 0.682 V, respectively) result in a
strong thermodynamic driving force for the regeneration reaction. Since direct
oxidation of NAD(P)H by molecular oxygen is very the electron transfer has
to be accelerated via enzymatic or chemical techniques.
NADH oxidases (NADH dehydrogenases, E. C. 1.6.99.x) from several organisms
have been characterized in recent years[']. Two types of NADH oxidases can be
distinguished, namely those reducing molecular oxygen to water and those perform-
ing the reduction to hydrogen peroxide. Interestingly, few examples are found in
literature employing NADH oxidases for the regeneration of NAD', probably
because of stability reasons. However, an NADH oxidase from Themus aquaticus
was reported to be stable at 80 "C for at least 1 hL6],which might allow small scale
applications.
FMN reductase (NAD(P)H dehydrogenase (FMN), E. C. 1.6.8.1) catalyzes the
transhydrogenation from NAD(P)H to FMN r71, yielding the oxidized nicotinamide
coenzyme and FMNH2, which reacts spontaneously with molecular oxygen
(Fig. 16.2-4). The reaction might be coupled to the catalase reaction in order to
decrease the degree of enzyme inactivation over longer reaction times. Compared to
the non-catalyzed hydride transfer from NAD(P)H to FMN 1'1, up to 1000-fold
increases in the transhydrogenation rate are reported, which is not very high when
applied synthetically. In this respect also the operational stability of FMN reductase
has to be optimized. Besides the native substrate, cheaper alloxazine-based analogs
are also accepted"].
1112
I
Figure 16.2-4. Transhydrogenation catalyzed by F M N reductase.
Among the chemical mediator systems especially o-pinones are capable of

accepting the hydride equivalent from reduced nicotinamides. The oxidized media-
tors are regenerated by molecular oxygen. Since these mediators can also be recycled
electrochemically,they are discussed in the following chapter.
16.2.2.4
Electrochemical Regeneration
A very elegant method to regenerate NAD(P)+from NAD(P)His to use the anode as

terminal electron acceptor. The most common approaches are summarized in
Figure 16.2-5.
Direct electrochemical N A D ( P J H oxidation (Fig. 16.2-5 A)

The easiest way to oxidize NAD(P)H is to withdraw the excess electrons anodically.
Although the formal potential of the NADH/NAD' couple is - 320 mV [- 324 mV for
NADP] vs NHE["], overpotentials as large as 1 V are required to achieve significant
oxidation rates at bare electrodes[", '*I. The number of enzymes, substrates, and
NAD(P)H
Figure 16.2-5. Electrochemical regeneration of NAD(P)'. A: direct anodic

oxidation; 8:indirect electrochemical oxidation; C: diaphorase-accelerated
indirect electrochemical oxidation.
IG.2 Oxidation ofAlcohols
products that can withstand this oxidizing power is limited. In addition, direct
oxidation is often accompanied by electrode fouling, which is attributed to the
formation of NAD dimers or stable adducts [12,131.
Indirect electrochemical NAD(P)H oxidation (Fig. 16.2-5 B,C)

The high overpotentials needed for NAD(P)Hoxidation can be considerably lowered
by the use of redox mediators. Organic compound (such as ortho- and pura-
substituted quinones [I4lSl, diimines I2O1, and organic dyes 121-221) undergoing two-
electron transfer processes were found to be ideal fpr NAD(P)' regeneration.
Amongst these, l,lO-phenanthroline-5,6-diones [23, 241 are probably the most potent
mediators. Furthermore, quinoid mediators can be generated in the surface of

carbon electrodes by oxidative pretreatment ["I.
Besides these hydride acceptors, single-electron-transfermediators (e.g. transition
metal complexesr2', 22], viologene derivatives12'* 271, ferrocenes 12*1, heteropolyan-
ions[2g1,conducting polymers[3o]or ABTS C3l1 are also capable of oxidizing NAD(P)H.
Examples for one- and two-electron acceptors are listed in Table 16.2-2.
These mediators have been applied mostly freely diffusing but also immobilized at
the electrode surface. A great variety of immobilization techniques have been used
for the preparation of these modified electrodes - the mediator molecules have, for
example, been directly adsorbed onto electrode surfaces, incorporated into conduct-
ing polymers or covalently linked to functional groups on electrode surfaces.
Often the electron transfer between the reduced nicotinamide coenzyme and the
mediator is rather slow because of kinetic limitations. In many of these cases
electron transfer catalyzed by diuphoruse (E. C. 1.6.99.x) results in a drastic enhance-
ment of the reaction rate (Fig. 16.2-5C). Diaphorase-catalyzedNAD(P)+regeneration
was reported for example with methylene blue[32],PQQ[33](under aerobic condi-
tions), ferrocene 12'1, N-methyl-p-aminophenol1341, N,N-dimethylindoaniline, 2,G-di-
chlorophenol indophenol (DCIP), [ F e ( c N ) ~ ] ~ -viologenes
[~~l, or several quinoid
structures f3'1.
Many of the quinone-based mediators react in their reduced states with molecular
oxygen. This aerobic regeneration has the advantage that no additional electro-
chemical equipment is necessary to perform NAD(P)+regeneration. On the other
hand, reactive oxygen species are generated, which might inactivate enzymes and
which therefore need to be removed from the reaction mixture.
It should be mentioned at this point that most of the mediators described here
were developed for analytical purposes. Only a few systems were applied to
electrochemically driven dehydrogenase-catalyzed oxidations. This is partially be-
cause some systems exhibit moderate half-life times.
In conclusion it can be said that, for each individual case, a mediator with a good
performance and stability under the given production conditions has to be found.
7 6 Oxidation Reactions
1114
I Table 16.2-2. Selection of frequently used mediators for indirect electrochemical regeneration of
NAD(P)'.
One-electron acceptors
SO,NH,
2,2'-azino-bis-(3-ethylbenzothiazoline-6- [Os(bpy)z(PVI)ioCl]'
sulfonic acid)-diammonium salt (ABTS)
GD
R = Me: methyl viologene Ferrocenes
R = Bz: benzyl viologene
Two-electron acceptors
orthoquinone (and various derivatives) para-quinone (and various derivatives)
16.2.2.5
Photochemical Regeneration
Various methods for photosensitized oxidation of NAD(P)Hhave been developed[37].

Photochemical methods are based either on the light-induced excitation of a
mediator enabling it to oxidize NAD(P)H (reductive quenching mechanism) or on
the light-induced excitation of the already reduced mediator, thus facilitating its re-
oxidation (oxidative quenching mechanism) (Fig. 16.2-6).
For reductive quenching, photosensitizers such as tin porphyrins r3'1, methylene
blue[3'], and other dyes L4O] are reported (Fig. 16.2-7).Ruthenium(11) tris bipyridine
complexes in combination with viologenes are used for oxidative quenching. After
7 6 2 Oxidation ofAlcohols
NAD(P)H Figure 16.2-6. Electron transfer

from NAD(P)H to acceptors (A) via
photosensitizers (S) facilitated by
*-
photochemical activation.
NAD(P)+
reductive quenching oxidative quenching
0 I
R z CH, rnethylene blue

5 H thionine
-N+/ / \ /
c) I
O CI
+ b - / \0
-
Sn(ll)-rneso-tetrarnethylpyridiniurnporphyrin 2,&dichlorophenol indophenol (DCP/P)

Figure 16.2-7. Photosensitizers used for photochemical regeneration of
NAD(P)'from NAD(P)H.
the oxidation of NAD(P)H,the reduced Ru complex is excited by light. The resulting

powerful reduction agent transforms methyl viologene into the radical cation. The
electrons from NAD(P)Hare usually transferred to molecular oxygen, protons or the
402 411
Next to soluble photosensitizers, semi-conductors were reported for NAD' re-

generationI4*I.The advantage of these photochemical systems is that some of them
utilize visible light, pointing towards the possibility of using sunlight for driving
organic reactions. Disadvantageous, however, are the still low performances (TTN
and TF of the photosensitizers and coenzymes) and the fact that photoexcitation
results in the formation of strong oxidizing agents and the formation of free reactive
radicals. Therefore, photochemical regeneration has not become one of the standard
procedures, yet[37].
16.2.2.6
Oxidations Catalyzed by Alcohol Dehydrogenase from Horse Liver (HLADH)
HLADH is certainly one of the most prominent and widely used oxidoreductases.
The NAD-dependentenzyme is a dimer consisting of two almost identical subunits,
1116
which both contain two zinc atoms[43, The 3-dimensional structure was eluci-
dated via X-ray analysis c4'. 461.
HLADH exhibits a unique combination of a very broad tolerance for primary and
secondary alcohols (or aldehydes and ketones in the reductive direction) with an
481. HLADH exhibits tolerance
almost invariable and predictable stereospe~ificity[~~,
to many organic solvents[49]and is active even in water-saturated organic sol-
vents[", 421. Even though HLADH exhibits a rather poor specific activity in the range
of 1-2 U mg-', it is commercially available at reasonable prices ($ 570/1000 U,
Sigma 2001) and, more importantly, is fairly stable even in oxygen-containing
mediaf4'].Also because of that, HLADH has been studied extensively during the last
few decades.
16.2.2.6.1 Regeneration of NAD' in HLADH-catalyzed Reactions

Various concepts for the enzymatic regeneration of NAD' in combination with
isolated HLADH have been reported, ranging from a second dehydrogenase such as
glutamate dehydrogenase['I, 521 to enzyme-coupledor intrasequential approaches.
A Baeyer-Villiger monooxygenase was applied to oxidize cyclic ketones produced
in situ by HLADH with concomitant regeneration of NAD' (Fig. 1G.2-8)[531. Even
though yields and enantiomeric excesses are moderate, this concept has synthetic
significance and should be optimized in future.
A very elegant reaction sequence was reported by Tanaka and coworkers[54].
HLADH was used for the kinetic resolution of a series of racemic 0-hydroxysilanes
yielding one enantiomer in ee values ranging from 20 to 97 % in reasonable yields
and the corresponding P-ketosilane.This P-ketosilane hydrolyzes spontaneously and
drives the regeneration of NAD' catalyzed by HLADH (Fig. 16.2-9).
Other NAD' regeneration approaches are based on the transfer of hydride either
to PQQ (catalyzedby diaphorase) [331, directly to flavins [55-571, or to flavins via FMN
reductase catalysis["]. Direct hydride transfer to flavins has the advantage that the
alloxazine acceptor can be chosen freely, e. g. cheap riboflavin instead of FAD. On the
HO,,,
HLADH
H2O 0 2
Figure 16.2-8. lntrasequential regeneration o f N A D with HLADH and a

Baeyer-Villiger monooxygenase (BVO) from Acinetobacter calcoaceticus.
SiMe,R
HLADH
+
NAD+
RMe,Si-OH
HLADH
Figure 16.2-9.lntrasequential NAD' regeneration for HLADH-driven kinetic
racemate resolution of P-hydroxysilanes.
other hand, the spontaneous hydride transfer suffers from sluggish kinetics (k = 0.2
M-' s-'; turnover rates ranging between 0.06 and 1.8 h-I) which is app. 1000-fold
slower than the values reported for enzymatic regeneration. For this reason, high
excesses of the acceptor have to be applied in order to achieve acceptable regenera-
tion rates. Introduction of FMN reductase accelerates this reaction remarkably.
Electrochemical methods utilizing quinoid mediators [23, 241 or ferrocenes["I as
well as photo~hemical[~~1 methods have also been applied to regenerate NAD' in
combination with HLADH. Especially the electrochemical variants utilizing quinoid
shuttle systems proved to be very efficient, with mediator performances as high as
130 catalytic cycles per hour and quantitative yields.
substrate product
NAP NADH
Figure 16.2-10. HlADH-catalyzed oxidations i n two-liquid phase systems

(in the case o f buffer-saturated organic solvents, the aqueous phase is
limited t o a layer around HLADH).
1118
I Table 16.2-3. Synthetic application o f HLADH in organic solvents.
Substrate(s) Product(s) Solvent Remarks/Ref.
Plugged-flowreactor for
Hexane continuous production
o^""" o^""
Geraniol Geranial
ao""
c:::
Isopropyl ether [31
Cinnamylalcohol Cinnamylaldehyde
&+A
Hexane (41
&&
Racemic
Ethyl acetate,
chloroform,
Isopropyl ether,
HLADH immobilized on
glass beads
[51
butyl acetate
HLADH in
Hexane polyacrylamide particles
[GI
2 R. Lortie, I. Villaume, M. D. Legoy, D. Thomas, 5 J. Grundwald, B. Wirz, M. P. Scollar, A. M. Kliba-
Biotech. Bioeng. 1989, 33, 229-232. nov, J. Am. Chem. SOC. 1986,108.6732-6734.
3 T. Kawamoto, A. Aoki, K. Sonomoto, A. Tanaka, 6 C. Gorrebeck, M. Spanghoe, G. Lanens,
J. Fern. Bioeng. 1989, 67, 361-362. G. L. Lerniere, R. A. Dornrnisse, J. A. Lepoivret,
4 J. R. Matos, C.-H. Wong. J. Org. Chem. 1986, 51, F. C. Adlerweireldt, Rec. Trav. Chim. Pays-Bas 1991,
2388-2389. 110,231-235.
16.2.2.6.2 HLADH in Organic Media

Several applications of HLADH in organic/aqueous media have been reported (Table
16.2-3).The concept of these two liquid-phase reaction systems is shown schemati-
cally in Fig. 16.2-10.This approach is especially suitable for substrates and products
with low solubility in aqueous media. Furthermore, the organic phase serves as a
sink for products, thus decreasing problems resulting from product inhibition or
back reactions.
16.2.2.6.3 Kinetic Resolution of Alcohols using HLADH

Because of its high enantioselectivity,H LADH has found widespread applications in
the kinetic resolution of racemic alcohols and a-amino alcohols. Total turnovers of
up to 10' for HLADH and 800 for NAD were reported with 90% residual activity,
yielding the corresponding aldehydes in enantiomeric excesses up to 96%. The a-
hydroxy aldehydes were metabolized in situ by an aldehyde dehydrogenase to the
corresponding a-hydroxy acids (Fig. 16.2-11)["I.
Examples of further kinetic resolutions of racemates via regioselective oxidation
using HLADH are given in Fig. 16.2-12.
162 Oxidation ofAlcohols
I 1119
RE O H
I I
NAD’ NADH NAD‘ NADH
~ -NA< f”
HLADH
Figure 16.2-11. HLADH as enantioselective catalyst in the kinetic resolution

of uic-diols (A) and a-amino alcohols (6). R = CHzOH, CHzF, CHzCI, CHZBr, CH,, CH=CH2,
C ~ H SCH2NH2,
, (CH3)z.
k OH
HLADH
k OH
+
‘7
OH
HLADH
Figure 16.2-12. Chemo- and stereoselective oxidations of sec-alcohols.

1120
16.2.2.6.4 HLADH for the Oxidation o f meso-Compounds

Probably the most prominent application of HLADH is the oxidation of rneso-diols to
homochiral lactones. Both 1,4-and 1,s-diolsare accepted as substrates (Table 16.2-4).
The overall 4-electron oxidations proceed via two successive steps (tandem oxida-
tion). The enantiomeric excesses often exceed 97 %.
16.2.2.7
Alcohol Dehydrogenase from Yeast (YADH)
Even though the primary sequences differ significantly, YADH exhibits almost the
same quaternary structure as HLADH I['. Nevertheless, far fewer applications in
biocatalflc processes are known for YADH than for HLADH. In part this is due to
its low overall stability and its low resistance towards organic solvents[61].Fur-
thermore the substrate spectrum of YADH is limited to primary alcohols and
2-hydroxyalkanes["]. It has been used in a few oxidative applicati~ns''~,G41. On
account of its high specific activity (about 300 U mg-') together with its very low
price (less than 1.2 $/lo00 U, Sigma, 2001), YADH has been used as a regeneration
enzyme for NADHrG51.In this approach it is a problem that both ethanol and
acetaldehyde as cosubstrate and coproduct of the regeneration reaction inactivate
YADH and also other enzymes at low concentrations. This problem can be
addressed by elegant techniques such as the use of gas membranes. Only volatile
compounds such as ethanol or acetaldehyde can pass into the gas phase. This
concept has been applied for lactate dehydrogenase (Fig. 16.2-13) 1"- G71. Hazardous
acetaldehyde is removed and even recycled to form ethanol by treatment with
sodium borohydride in the gas phase. Cycle numbers of over 10 000 are reported.
16.2.2.8
Alcohol Dehydrogenase from Thermoanaerobium brockii (TBADH)
TBADH is a NADP-dependent dehydrogenase with remarkable thermostability up

to 65 oC[G8].
Neither HLADH nor YADH are able to convert linear secondary alco-
gaseous interphase
Figure 16.2-13. Regeneration of N A D H with YADH. Acetaldehyde diffuses through the gaseous
interphase into the second liquid phase where it is regenerated chemically to ethanol.
Table 16.24. Examples of H LADH-catalyzed enantioselective oxidations of meso-diols.
HO
HLADH
* n
HO H O ======= 0 OH
8
Meso-diol Lactone Yield [%] e e I"'%] References
HO'ri OH
0 99 > 97 171
HO fiOH 0
90 95 181
NDa > 97 191

0
68 > 97
0
90 > 97
0
P 0
55 99
(2
w
95 "100"
0
> 99 > 99
0
ko 0
70 99
a ND:not determined
1122
7 G.Hilt, B. Lewall, G. Montero, I. H. P. Utley, 11 M.-E. Gourdel-Martin, C. Cornoy, F. Huet, Tetra-

E. Steckhan, Liebigs Ann.lRecueil1997, 2289-229G. hedron: Asym. 1999, 10, 403-404.
8 T. Osa, Y.Kashiwagi, Y. Yanagisawa, Chem. Lett. 12 R. N. Patel, M. Liu, A. Banejee, S. L., Ind.].
1994,3G7-370. Chem. 1992,318,832-836.
9 K. Mori,M. Amaike, J. E. Oliver, Liebigs Ann. 13 Y. Yamazaki, K. Hosono, Tetrahedron Lett. 1988, 29,
Cham. 1992,1179. 57G9-5770.
10 Y Yamazaki, K. Hosono, Tetrahedron Lett. 1989, 30,
5313-5314.
hols. TBADH fills this gap: its activity is highest for secondary alcohols,being low for
primary alcohols[48].Because of this rather narrow substrate spectrum, TBADH is
mostly used for the regeneration of NADPH. Only a few synthetic applications are
reported[24,"1. Figure 16.2-14gives one example where YADH was used simultane-
ously as an oxidizing enzyme and a NADPH regeneration enzyme (intrasequential
cofactor regeneration).
16.2.2.9
Glycerol Dehydrogenase (GDH, E.C. 1.1.1.6)
GDH was isolated from various bacterial strains, especially from Schizosacchar-
omyces pombeC7'. 711 and Cellulomonas SP.['~,731.
It displays a somewhat complementary substrate specificity to HLADH. While
HLADH oxidizes meso-diols with secondary hydroxyl groups rather badly, they are
readily oxidized by GDH to the corresponding (S)-a-hydroxyketones1'1. Furthermore,
the natural substrate glycerol is transformed to achiral dihydroxy acetone by GDH
while HLADH produces optically active (S)-glyceraldehyde.In many cases GDH
seems to prefer secondary hydroxyl groups (Table 16.2-S), although this rule of
thumb has some exceptions.
In aqueous buffers GDH exhibits only low enantioselectivity, e. g. for the kinetic
resolution of l-phenyl-l,2-ethanediol (which is most probably due to spontaneous
racemization via enolization)[74]; furthermore, it suffers from pronounced product
inhibition, accounting for low yields. Both problems (product inhibition and
TBADH
0
Figure 16.2-14.
lntrasequential
regeneration of NADP
with TBADH and a
Baeyer-Villiger mono-
oxygenase (BVO) from
Acinetobacter calcoaceti-
GUS.
7 6.2 Oxidation ofAlcohols
Table 16.2-5. Alcohol oxidations catalyzed by glycerol dehydrogena~e['~].

Substrate Product
HOTOH
HOTOH
OH
- 0
H O T O ' H O T 0
OH 0
H O G 'HO
OH 0
H
OH
O
OH
T . 0
0
14 J. H.Marshall, I. W.May, J. Sloan,J. Gen. Microbiol. 1985,131, 1581-1588.
++ &
a
/ /
NAu
in situ extraction into hexane
via hollow fibre module
-
+COOH
OH LDH I I OC O O H
Figure 16.2-15. Deracernization of roc 1-phenyl-1,Z-ethandiol coupled t o in situ

product extraction via a hollow fiber module.
IG
1124
K13CN+HCH0 d - YH
H0<C~0P03
2-
- - GPDH R
HO<CVOPO,
2-
2-
phosphatase H07y0p03
“’OH HO”” “’OH
OH OH
Figure 16.2-16. Synthesis of”C-labeled sugars in a tandem reaction of CPDH,

aldolase, and phosphatase.
Figure 16.2-17. Pired anodic

regeneration of NAD’ coupled to
cathodic reduction ofpyruvate.
racemization) can be solved by in situ extraction into a second (organic) phase

(Fig. 1G.2-15)[741.
This biphasic system yielded higher ee values (99% instead of 58%) at maximal
theoretical conversions (50% instead of 38 %) in significantly shorter reaction times
(GO h instead of 170 h) compared to the solely aqueous system.
Since GDH contains autooxidizable thiol groups, it is necessary to perform such
reactions in media essentially free from oxygen.
16.2.2.10
Glycerol-3-phosphate Dehydrogenase (GPDH, E. C. 1.1.1.8)
GPDH has been isolated from various organisms. The enzyme from rabbit muscle is
commercially available. Its synthetic applications are limited because of its very
narrow substrate spectrum it almost exclusively accepts ~-glycerol-3-phos-

phate [75, “1. The product 3-hydroxyacetone phosphate, however, is an essential
substrate of aldolases and therefore can serve as a building block in the enzymatic
synthesis of non-native sugars and polyols. Although the redox equilibrium of
GPDH favors the reduced substrates even more than in the case of GDH, it has been
employed in the synthesis of radioactively labeled carbohydrates starting from
K13CN and formaldehyde (Fig. 16.2-16)[77, 781. Depending on the substrates, single-
or double-labeled glucose, fructose or sorbose are available by the sequence outlined
in Fig. 16.2-16.
16.2.2.1 1
Lactate Dehydrogenase (LDH, E.C. 1.1.1.27)
LDH was used to catalyze the deracemization of lactate in a very elegant electro-
chemical approach. The driving force of the endergonic reaction was supplied by
anodic regeneration of NAD’ and cathodic reduction of pyruvate (Fig. 16.2-
17)17’), 80]. Thus, both LDH products were removed efficiently, avoiding product
inhibition. The electrochemical reduction of pyruvate leads to racemic lactate,
producing 50% of the desired product and 50% of “new” substrate for LDH. An
A o o -
Figure 16.2-18. Electrical wiring of lactate

e- dehydrogenase (LDH).
1126
I interesting approach to direct “electricalwiring” of LDH to an electrode was reported
recently (Fig. 16.2-18)[*‘I.
NAD was covalently linked via a PQQ spacer to a gold electrode. This modified
electrode is capable of binding LDH over the exposed nicotinamide groups. Upon
oxidation of lactate to pyruvate the excess electrons tunnel from NADH in the active
site to PQQ and eventually to the anode. Thus, a kind of electrical linkage between
the enzyme and the electrode is established. The enzymes were crosslinked, as LDH
is a homotetramer and might dissociate during the reaction. This approach is not
only useful for electrochemical biosensors but might be transferred to other
oxidoreductase reactions.
16.2.2.12
Carbohydrate Dehydrogenases
Many so-called polyol dehydrogenases have been reported in literature, for example
various glucose dehydrogenases, mannitol dehydrogenase, fructose dehydrogenase,
and uridine-5‘-diphosphoglucose dehydrogenase. Glucose dehydrogenase (E. C.
1.1.1.47)was applied for the production of D-gluconic acid in a plug-flowreactor with
direct electrochemical regeneration of NAD+[82].Glucose-6-phosphate dehydroge-
nase (E.C. 1.1.1.49) is a common regeneration enzyme for NADPHIG9]. Most polyol
dehydrogenases are not specific for their native substrate, but also catalyze the
oxidoreduction of various carbohydrates. Thus, they can be applied for the produc-
tion of (non-)naturalsugars which are especially valuable in the sweetener industry.
Yet their applications are limited compared to the polyol oxidases (see Sect. 16.2.3)
COOH
0 “‘OH “OH
HO’”
Figure 16.2-19. Regioselective oxidation o f cholic acid by hydroxysteroid dehydrogenases.

HO'"
NAD+ NADH NADH NAD'
Figure 16.2-20. 3a- and 3fi-hydroxysteroid dehydrogenase (HSDH) catalyzed

stereoinversion in steroids.
16.2.2.13
Hydroxysteroid Dehydrogenases (HSDH)
The hydroxysteroid dehydrogenases comprise another group of synthetically inter-

esting dehydrogenases. For many hydroxylated positions of the steroid backbone,
individual NAD(P)' dependent dehydrogenases exist, which selectively oxidize the
respective residue.
For example, the three hydroxy groups of cholic acid in the 3-,7-,and 12-positions
can all be oxidized regioselectively(Fig. 16.2-19)[83-851.
In addition to the regioselective oxidation of the hydroxy groups in virtually every
position, a discrimination of the absolute stereochemistry can be achieved by various
a- or P-selective HSDHs. Thus, the stereoinversion of various steroids was achieved
by successive oxidation at position 3 with 3a-HSDH and subsequent reduction with
3P-HSDH (Fig. 16.2-20) Hydroxy functions in other positions were not modified,
and the products at the end of the sequence were essentially pure. Because of the low
solubility of the reactants, biphasic systems with ethyl (butyl) acetate as organic
solvents were used as reaction media.
16.2.2.14
Other Dehydrogenases
In addition to the alcohol dehydrogenases mentioned above, ADHs from various

other sources were examined, especiallywith respect to increased stability,resistance
to organic solvents, and catalpc properties.
A NAD+dependent ADH isolated from Sulfolobus solfataricus was found to exhibit
better thermostability than HLADH [tl/z (GO "C) = 20 h] together with a distinctive
preference for (S)-alcohols(complementaryto HLADH) ["I. The enzyme has a broad
substrate specificity that includes linear and branched primary alcohols and linear
and cyclic secondary alcohols[48].The highly purified enzyme exhibits a specific
activity of 4 U mg-' (for benzyl alcohol at 65 "C)18', 1'. To date, this enzyme is not
commercially available.
Hummel et al. established a new route to enantiomerically pure alcohols by the
1128
I
R. erythopolis
NAD(P)t NAD(P)H
NADH-oxidase
Figure 16.2-21. Deracemization of 1-phenyl-

0 2
HZOZ 1-ethanol. The ADHs from R. erythropolis and L.
*
kefir exhibit complementary steroespecificity.
)/ Hzo Combination o f both in an oxidation-reduction
catalase sequence yields the desired enantiopure alcohol.
A
&R' + L
,R
OH OH 0
SY"
OH NADH 48-50 (RR)
% >99 % ee (9
3-20 YO299% ee
o o -u
AOH LDH Aoo o -
++ OH
QoH
OH
&OH
OH
OH
Figure 16.2-22. Kinetic resolution o f racemic syn-diols by Bacillus stearothermophilus diacetyl
reductase (BSDR). A: reaction with LDH-catalyzed regeneration of NAD+; B: selection
of syn-diols applied.
7G.2 Oxidation ofAlcohols I1129
combination of a (R)-specific,NADP-dependent ADH from Lactobacillus keJr and a

(S)-specific,NAD-dependentADH from Rhodococcus erythropoli~[~~~. In a first step, a
kinetic resolution yielded 50 % of the desired alcohol. Subsequently the ketone was
reduced with the suitable ADH, finally yielding the desired optically pure enantio-
mer in 100% yield (Fig. 16.2-21).
Recently, diacetyl reductase (Acetoin reductase, E. C. 1.1.1.5) from Bacillus stearo-
themophilus (BSDR) was reported to be a powerful catalyst in the oxidative kinetic
resolution of vic-diols (Fig. 16.2-22)[901. All syn-diols tested yielded the enantiopure
(R,R) diols in almost maximum theoretical yields, a-hydroxy ketones were largely
further oxidized to the corresponding diketones. Oxidation of vic-anti diols only gave
ee values in the range of 62-76 %.
16.2.3
Oxidases as Catalysts
16.2.3.1
General Remarks
Oxidases utilize molecular oxygen as terminal electron acceptor. This can be

considered as aerobic regeneration of the prosthetic group of the oxidase. At first
glance, this seems to offer a simpler enzymatic oxidation procedure compared to the
coenzyme-dependent dehydrogenases or monooxygenases. However, with few ex-
ceptions such as cytochrome c oxida~e”~1, some NADH oxidases”’] or laccases[”],
which reduce molecular oxygen directly to water in an overall four-electron transfer
step, O2reduction generally leads to hydrogen peroxide (transfer of two electrons) or
to the superoxide radical anion (transfer of one electron) as primary reduction
products.
16.2.3.2
Methods to Diminish/Avoid H202formation
Autoregeneration of oxidases with concomitant catalase-catalyzed disproportiona-

tion of hydrogen peroxide is a simple and effective regeneration method (Fig. 16.2-
23); it is quite commonly used with oxidase reactions.
oxidase catalase
(E.C. 1 .x.3.y) (E.C. 1.11.1.6)
Figure 16.2-23. Coupling o f oxidase autoregeneration and catalase for dismutation of

hydrogen peroxide.
1130
I
Figure 16.2-24. Indirect electrochemical regeneration of an oxidase.
Hydrogen peroxide, however, is highly reactive and irreversibly inhibits enzyme

activity (also catalase) even in low concentrations.
Hydrogen peroxide can be avoided if excess electrons are transferred to the anode.
However, direct electron transfer between enzymes and solid electrodes is usually
very slow because the enzymatic active sites are often deeply buried within the
protein shell and therefore inaccessiblefor the electrode (the tunneling probability of
electrons is a function of distance). In order to accelerate the electron transfer, low
molecular weight redox active substances can be used to shuttle the electrons
between the enzyme and the electrode. This indirect electrochemical enzyme
regeneration is represented schematically in Figure 16.2-24.
For the anaerobic electrochemical regeneration of a given oxidase, a suitable
mediator can be chosen from various organometallic complexes, especially ferro-
cenes [94'021, but also bipyridine/phenanthroline, terpyridine, or hexacyano com-
plexes [Io3, Also, quinoid salts such as TTF/TCNQ (tetrathiofulvalene/tetra-
cyanoquinodimethane)['05. 1"' as well as benzoquinones [lo7] and redox dyes such as
phenazine and phenothiazine derivatives (MPMS, thionin, azure A, and azure
C) ['08] proved to be useful redox agents for indirect electron transfer. Even incorpora-
tion of oxidases into conducting polymers made of polypyrrole or polythiophene
derivatives proved to function for electrochemical regeneration ['091.
It should be mentioned at this point that most of the research in the field of
electrochemical oxidase regeneration concentrates on analccal applications, in-
spired by the search for electrochemical biosensors ['lo].
However, it was demonstrated that indirect electrochemical methods are suitable
for prolonging oxidase operational stability. In a particular example, glucose oxidase
(E.C. 1.1.3.4) was immobilized on a carbon felt anode and regenerated with the
benzoquinone/hydroquinone redox couple (Fig. 16.2-25)[lo7].Thus, the operational
stability of glucose oxidase could be increased at least 50 times compared to the use
of molecular oxygen as oxidant. Productivities as high as 1OOg h-' L-' were
reached.
One disadvantage of the electrochemical methods is the need for rather elaborate
equipment. Recently, Baminger et al. proposed a novel concept of enzymatic
regeneration of a range of redox mediators including quinones and various redox
dyes [931. Instead of reoxidizing these mediators via the anode, laccases are employed.
Laccases (E. C. 1.10.3.2)are multi-copper oxidases['"] that are found in various trees
and fungi["2. '131 . Laccases catalyze the oxidation of various structurally diverse
162 Oxidation of Alcohols
I
n
1131
glucose oxidase HO'"' "OH H20 JoHO

f;!
I-g$
D-glucose
OH
gluconic acid
0 OH
Figure 16.2-25. Indirect electro-enzymatic oxidation of glucose using glucose oxidase.
flavoprotein laccase
Figure 16.2-26. Laccase-based regeneration concept for oxidized flavoproteins

(oxidases).
substances with concomitant reduction of molecular oxygen to water['14], thus

avoiding the generation of hazardous hydrogen peroxide (Fig. 16.2-26).
This regeneration concept was tested with pyranose oxidase (P20, E.C.
1.1.3.10)~933].
Interestingly, it was found that P20 shows higher affinity for some
mediators than for 0 2 (KM value for 1,4-benzoquinone is 120 m M compared to 650
mM for 02)with otherwise comparable activities yielding a 6 times higher kCat/KM
value. Preparative scale biotransformations could be performed with two-fold
volumetric productivities. The TTNs were 1.1x lo6 for P20, with a residual activity of
85 %, and 800 for 1,4-benzoquinone. Similar results were obtained with the enzyme
cellobiose-dehydrogenase (E. C. 1.1.99.18),which is incapable of autoregeneration,
in combination with ABTS or DCIP and laccase.
1132
H HO o
R
S O
OH
H r~ P20 HoHO
R
0
OH
0,
R = CH,OH
H
Figure 16.2-27.
HzO,
catalase
-
Oxidation of carbohydrates specifically at C-2 by pyranose oxidase (P20).
o, + H,O
16.2.3.3
Pyranose Oxidase (P20, E.C. 1.1.3.10)
P20 is common among wood-degrading basidiomycetes [ll5].It has been isolated

and characterized from various microorganisms Although the substrate speci-
ficity vanes to some extent among the P20s isolated from different fungi, P20s have
some properties in common, such as the homotetrameric structure with covalently
bound FAD. The main metabolic role of P 2 0 appears to be as a constituent of the
fungal ligninolytic system that provides the lignin-degrading lignin peroxidase and
manganese peroxidase with hydrogen peroxide l1 171.
Natural substrates of P 2 0 are probably D-glucose, D-galactose, and D-xylose,which
are abundant in lignocellulose and which are oxidized to the corresponding 2-keto
sugars. In addition, P20 exhibits significant activity with a number of other
carbohydratesIll8]. During such oxidations, electrons are transferred to molecular
oxygen, yielding hydrogen peroxide. In addition, benzoquinones, 2,G-dichloroindo-
phenol, as well as ABTS were reported to function as electron acceptors[’3, ll’l.
Interestingly, up to 11-fold increased reactivity (compared to molecular oxygen as
electron acceptor) was found.
P20 is currently used in various analytical applications, e. g., in clinical chemistry
for the determination of 1,S-anhydro-D-ghcitol,an important marker for glycemic
control in diabetes patients or in amperometric biosensors for the detection of
monosaccharides [120, ‘’1 . For the last two decades, P20 has received increased
attention as the key catalyst in several biotechnological applications. Only a few can
be mentioned here.
The essential structural requirements of substrates for P20 are the six-membered
ring of pyranoid saccharides and an equatorially orientated 2-OH group [1221. In some
cases regioselective oxidation at C-3 was observed[”81. The general reaction scheme
is given in Fig. 16.2-27. Table 16.2-6 gives a selection of preparative oxidations
reported with P20.
The “Cetus process”

P 2 0 is involved in the so-called “Cetus process”, in which D-fructose is produced
from cheap D-glucose (Fig. 16.2-28).
Table 16.2-6. Substrates and oxidation products of pyranose oxidase.
I
1133
Substrate Product Yield p4" Activity [%la References
Oxidation of monosaccharides
100 100
D-Glucose o-Glucosone
94 40
HO"' "OH
OH OH
D-Allose ~-Ribo-hexos-2-ulose
H O T : I I I l
70 8
HO HO
OH OH
D-Galactose ~-Lyxo-hexos-2-ulose
5 very low
HO"' . HO"
OH OH
D-Ribose ~-Erythro-pentos-2-ulose
100 96
HO" " OH HO"
3d-~-Glucose 3d-~-Erythro-hexos-2-ulose
100 92
HO'"
OH
100 75
HO" HO"'
OH OH
1,5-Anhydro-~-glucitoI
1134

[%r Activity [%I"
~~ ~ ~ ~~~~
Substrate Product Yield References

Oxidation o f Disaccharides
HO HO
Hobob:
HO
6H HO OH
100 N D ~ P
I
OH
Allolactose Allolactulose
HO
HO
OH
OH
Meliobiose Meliobiulose
100 N D ~ (161
OH OH
Gentiobiose Gentiobiulose
HO
OH 6H
Isomaltose Palatinose
a expressed as percentage ofyield and activity of o-glucose oxidation: b ND: not determined
15 S . Freimund, A. Huwig, F. Giffhom, S . Kopper, 16 C. Leitner, P. Mayr, S. Riva, J. Volc, K. D. Kulbe,

Chem. Eur. J. 1998,4,2442-2455. B. Nidetzky, D. Haltrich,J. Mol. Cat. B: Enzymatic
2001,11,407-414.
Hydrogen peroxide is not merely dismutated by catalase, but used as substrate in a

second enzyme cascade reaction producing propylene oxide['23-125] . In an alternative
process 1126] the reduction step was performed enzymatically using aldose reductase
and formate dehydrogenase for NADH regeneration. Thus, essentially glucose free
D-fructose was obtained.
I
1135
H A
HCI
‘I- i. fi haloperoxidase
halohydrine
epoxidase
Figure 16.2-28. lsornerization of D-glucose to o-fructose with pyranose oxidase (P20) and
coupling o f hydrogen peroxide t o a synthetic reaction (Cetus process).
Table 16.2-7. Kinetic resolution of some racernic 2-hydroxy acids t o the (R)-2-hydroxy acids and
the corresponding 2-keto acids[”1.
Substrate Yield [“/.I e e I%]
9H
49-50 > 98
*OOH 1,2.4,7
50 > 99
OH
&/trans
-0-COOH
47 86
c
OH
17 W. Adam, M. Lazarus, 9. Boss, C. R. Saha-Moller,H.-U. Humpf, P. Schreier, /. 0%.Chem. 1997,62,
7841-7843.
16.2.3.4
Glycolate Oxidase (E. C. 1.1.3.1 5)
Glycolate oxidase is a peroxisomal enzyme that is found in the leaves of many green
plants and in the liver of mammalians. The enzyme isolated and for economic
reasons only partially purified from spinach (Spinacia oleracea) was applied to the
enantioselective oxidation of various 2-hydroxy acids yielding the corresponding
2-keto acid and the remaining ( R ) Enantiopure 2-hydroxy acids are
valuable building blocks in the synthesis of glycols [‘’I, haloesters [12’1 or ep-
Unless the steric demand of the substituents close to the alcohol function
is too big, the oxidation proceeds smoothly to the full theoretical conversion with
enantiomeric excesses of the alcohols usually in the range of 98-99% (Table
16.2-7).
7G Oxidation
1136
I Reactions
0
glycolate oxidase
Rq~~ 0
. q O H
('
Figure 16.2-29.
Deracemization
racemic o facids
2-hydroxy
in a combination of
0 2 glycolate oxidase and
lactate dehydrogenase
(LDH).
H*O
NAOH NAD'
co2 HCO,
FDH
Table 16.2-8. Conversion of racemic 2-hydroxy acids into (R)-2-hydroxy acids by the combined
action of glycolate oxidase and D-lactate dehydrogenase~'81.
Substrate Oxidase [U] Dehydrogenase[U] Reaction time [h] Yield ["/.I e e pi]
~ ~~
2 450 66 100 > 99
210 100 94
One unit (U) is defined as the amount of enzyme which converts 1 pmol of substrate per minute.
18 W. Adam, M. Lazarus, C . R. Saha-Moller, P. Schreier, Tetrahedron Asymmetry 1998.9,351-355.
Kinetic resolutions have a maximum yield of only 50%. Therefore, a second

enzymatic process was added after completion of the glycolate oxidase-catalyzed
kinetic resolution['31! By addition of D-lactate dehydrogenase (E. C. 1.1.1.28) to-
gether with formate dehydrogenase for NADH regeneration, enantiospecific reduc-
tion of the 2-keto acid was achieved. Overall, a quantitative transformation (derace-
mization) of the racemic 2-hydroxy acid into the corresponding (R)-2-hydroxyacid
was achieved (Fig. 16.2-29).
Unfortunately, this process cannot be performed in a more elegant and more
efficient one-pot synthesis. On the one hand, the pH optima for the three enzymes
are not compatible with each other, and on the other, lactate dehydrogenase is air
sensitive. In addition to this, glycolate oxidase also catalyzes the reverse reaction
under aerobic conditions, thus lowering the ee-value.Therefore, the reaction mixture
is filtered (glycolate oxidase can be reused) and, after pH adjustment, the second
enzymatic transformation is performed. Table 16.2-8 shows some results of this
procedure.
Glycolate oxidase has been studied thoroughly not only for specific oxidation of
I
1137
HO-OH m Homo b H O T o
OH
ethyleneglycol glycolaldehyde glycolic acid
I
0-0 D
OH
glyoxal glyoxylic acid
Figure 16.2-30. Sequential oxidation of ethylene glycol t o glycolic acid.
glyoxylic acid
Vf4
glycolate oxidase
Figure 16.2-31. Synthesis o f

glyoxylic acid by glycolate
oxidase. The undesired side-
reactions (A) with hydrogen
peroxide and (B) overoxida-
CO,
1
+ HCO,H + OH
tion by glycolate oxidase are
prevented by in situ forrna-
tion o f an irnine.
(S)-2-hydroxypropionic acid (lactate)[1321 and for the kinetic resolution of racemic

2-hydroxy acids[127,l3l],but also for selective oxidations of 1,Zdiols such as ethylene
glycol (Fig. 16.2-30).
Reports on the specific conversion of glycolic acid into glyoxylic acid are numer-
ous. Isobe et al. Introduced an in vivo system utilizing Alculigenes sp. isolated from
media containing 1,2-propanediol.By carefully adjusting the pH, a yield of 95 % was
obtained f 1331.
DiCosimo and coworkers optimized the in vitro production of glyoxylic acid from
glycolic acid with glycolate oxidase from spinachll3'1. Improvements in operational
stability as well as in productivity were achieved by enzyme immobilization either
onto a solid or in permeabilized, metabolically inactive cells of Pichia
pastoris or Hansenula polymorphu, containing overexpressed glycolate oxidase from
spinach together with catalase. The undesired oxidation of glyoxylic acid by hydrogen
1138
Figure 16.2-32. Non-natural substrates for nucleoside oxidase from Pseudomonas sp.
These compounds are converted selectively t o their corresponding 5'-carboxylic acids.
peroxide (yielding formate and carbon dioxide) and further metabolization by

glycolate oxidase could be prevented by trapping the aldehyde function of glyoxylic
acid as imine (Fig. 16.2-31)[13'1.
16.2.3.5
Nucleoside Oxidase (E.C. 1.1.3.28)
Nucleoside oxidase is produced by Pseudomonas species and related Gram negative

bacteria [1371. The hetero-tetramer with covalently bound FAD oxidizes the 5'-hy-
droxyl group of purine and pyrimidine nucleosides to the corresponding carboxylic
acids. It has found application in the analytical determination of nucleosides (e.g. in
assessing food freshness)[1381. At Glaxo Wellcome R&D it found attention as key step
in the production of anti-inflammatory compounds [139-1411. Several non-natural
substrates were selectively converted on multi-gram scale into their 5'-carboxylic
acids (Fig. 16.2-32).
The operational stability of the enzyme was improved by immobilization onto a
solid matrix and especially by substitution of molecular oxygen as the primary
electron acceptor by stoichiometric amounts of hydroquinone.
16.2.3.6
Glucose Oxidase (E. C. 1.1.3.4)
The most prominent of the alcohol oxidases is glucose oxidase. The dimeric
flavoenzyme catalyzes the oxidation of P-D-glucoseto D-glucono-&lactone,a reaction
that has attracted the attention of generations of analytical chemists because of its
I62 Oxidation ofAlcohols 1139
possible applicability in glucose sensors for diabetes The reaction of the

I
stoichiometrically formed hydrogen peroxide with various dyes can be used as the
analpcal More elegant variants (that at the same time avoid the
formation of hazardous hydrogen peroxide) utilize anaerobic, electrochemical re-
generation with a suitable mediator. Thus, the catalytic current becomes the
analpcal signal. Several approaches have been reported, e. g. the utilization of freely
diffusible quinones [Io7], the incorporation of glucose oxidase in a conducting
polymer (produced from 1,4-hydroquinones and soybean peroxidase), or the im-
mobilization of several mediators in the vicinity of the prosthetic redox cen-
ter['*. 991
Because of the high substrate specificity of glucose oxidase, which almost
exclusively accepts glucose (other substrates such as D-maltose, D-xylose, or L-
sorbose are converted with less than 6% of the activity on glucose[lU*14'1), this
oxidase has not found any synthetic application, but it is frequently used in the food
industry to remove traces of molecular oxygen from vacuum sealed products.
Immobilized glucose oxidase is also used for the deoxygenation of juices and
beer [1461.
16.2.3.7
Alcohol Oxidase (E. C. 1.1.3.1 3)
The aliphatic alcohol oxidase, a FAD-dependent enzyme, catalyzes the oxidation of

primary short-chain alcohols to the corresponding aldehydes. Dioxygen can be
replaced by synthetic acceptors such as dichlorophenolindophenol or phenazine
methosulfate [14'1.
By utilizing an alcohol oxidase from Pichia pastoris or Candida sp.[l4'I, almost
complete conversion of ethylene glycol into glyoxal (Fig. 16.2-30) was observed.
These enzymatic routes were shown to be superior in terms of reaction conditions
and yields compared to the chemical variants that make use of metal catalysts or even
nitric acid for the oxidation of ethylene glycol.
Recently, aliphatic alcohol oxidase was applied as dehydrated enzyme in a gas-solid
bioreactor an excess amount of catalase was added to prevent oxidase inactiva-
tion.
HO
GAOX HO "'0H
OH OH
Figure 16.2-33. Galactose oxidase (GAOX) catalyzed oxidation of a-o-galactose
t o meso-galactohexodialose.
Table 16.2-9. Substrates and products o f galactose oxidase.

Substrate Product References
OH
rneso-Galactohexodialdose
OHOH
OH OHOH
OH
UDP-[14C]-Galactose UDP-[14C]-Galacturonicacid
HO{OH
HO
D,L-Threitol D-Threose + L-Threitol
HO
)
HO
"03-OH
HO
Xylitol
Hot
HO
OH
OH OH
HO
OH
OH
OH
L-Glucose + D-Glucitol
"$OH
HO
OH
811 OH
L-Galactose + D-Galactit01
7 G.2 Oxidation ofAlcohols I1141
Substrate Product References
OH
HOA O H HO-0
L(-)Glyceraldehyde
H O L C I
(S)-Halodiol + (R)-Aldehyde
19 S. S. Basu, G. D. Dotson, C. R. H. Raetz, Anal. 21 A.M. Klibanov, B. N. Alberti, M. A. Marletta,
Biochem. 2000,280,173-177. Biochem. Biophys. Res. Commun. 1982,1982,
20 D. G . Drueckhammer, W. J. Hennen, R. L. Ped- 108.
erson, D. F. Barbas, C. M. Gautheron, T.Krach,
C. H. Wong, Synthesis 1991,7, 499-7525.
Table 16.2-10. Substrates and products in the kinetic resolution of allylic alcohols with
cholesterol oxidaselZ21.
Substrate (R = H, OH) Product
d
l
HO
HO J35 0
+-'F No product detected

HO
22 S . Dieth, D. Tritsch, J:F. Biellmann, Tetrahedron Lett. 1995,36,2243-2246.
16.2.3.8
Galactose Oxidase (GAOX, E. C. 1.1.3.9)
Galactose oxidases belong to the group of copper-dependent oxidases. For the GAOX
from Dactylium dendroides the existence of covalently bound pyrroloquinoline
quinone (PQQ)could be shown['45].It catalyzesthe specific oxidation of the hydroxyl
group in position 6 of galactose (Fig. 16.2-33)[l5O].
The enzyme regeneration can be performed aerobically or utilizing mediators
7 B Oxidation Reactions
1142
I Figure 16.2-34. Ferric protoporphyrin IX as prosthetic group in
most peroxidases.
such as ferrocene["'], tetracyano-iron-1,lO-phenanthroline, or cobalt tert-pyridine

complexes[lo31.
GAOX stereospecifically oxidizes a broad range of substrates (Table 16.2-9). In
synthetic applications, the oxidation of racemic or meso-polyolssuch as D,L-threitol or
xylitol to the non-native sugars are of special interest['51, lS2]. In addition to the
monosaccharides represented in Table 16.2-9, GAOX also converts di- or oligo-
saccharides[lS31.
16.2.3.9
Cholesterol Oxidase (ChOX, E. C. 1.1.3.6)
ChOX from Rhodococcus erythropoliswas applied for the kinetic resolution of racemic
mono- and bicyclic ally1 alcohols (Table 16.2-10)(lS41.Although the substrates tested
were much smaller than the native substrate cholest-4-en-3P-01,reasonable enantio-
selectivities (E) in the range of 7-20 were found for the (S) alcohols.
Both enantiomers of the alcohol (entry 1)were oxidized with moderate enantiose-
lectivities ( E = 7) for the (S) enantiomer. For bicyclic alcohols, the position of the
hydroxyl group with respect to the methyl group is essential. Only at a relative trans
configuration of both substituents significant oxidation occurred.
By utilizing organic redox dyes as primary electron acceptors and concomitant
reoxidation at a glassy carbon electrode, amperometric biosensors for cholesterol
based on cholesterol oxidase were developed[108].
16.2.4
Peroxidases as Catalysts
16.2.4.1
Introduction
Peroxidases (E. C. 1.11.1.7) are ubiquitously found in plants, microorganisms, and

animals. Most peroxidases studied so far contain ferric protoporphyrin IX (proto-
heme, Fig. 16.2-34) as the prosthetic group[1ss].However, some peroxidases also
contain selenium (glutathione peroxidase)(1561, vanadium (bromoperoxidase)[1571,
I 1143
D
E 2 H++ 0,
Figure 16.2-35. Methods of generating appropriate hydrogen peroxide concen-

trations for chloroperoxidase reactions, (A) enzymatically with glucose oxidase
and (B) electrochemically by cathodic reduction of molecular oxygen.
manganese (manganese peroxidase)[1581,and flavin (flavoperoxidase)[1591 as pros-

thetic groups.
Most peroxidases accept a variety of peroxides, such as hydrogen peroxide or alkyl
hydroperoxides, as oxidizing agents. The mechanism includes the activation of
oxygen in a high valence iron-oxo specie^['^^^ "'1.
16.2.4.2
Methods to Generate HZOZ
At a first glance, utilization of cheap hydrogen peroxide as electron acceptor seems

appealing. The major drawback, however, is the sometimes rapid inactivation of
peroxidases by their substrate. For example, chloroperoxidase (CPO, E. C. 1.11.1.10)
exhibits a half-life time of 38 min even at an H202 concentration of 50 pM [1611.
Several approaches to controlling hydrogen peroxide at a constant low concentra-
tion have been reported. In aqueous/organic emulsions, the use of tert-butyl
hydroperoxide is beneficial. On the one hand, the peroxide concentration is limited
according to the partition coefficient, and on the other hand, tert-butanolwas shown
to exert a stabilizing effect on CPO['"].
The slow continuous addition of hydrogen peroxide results in better CPO
performance [lG3], which can be even further improved by sensor-controlledaddition
of H202[1G2], increasing the CPO total turnover number for indole oxidation more
than 20-fold to ca. 860 000.
IG
1144
Table 16.2-11. Chloroperoxidase-catalyzed oxidation of some alcohols to the corresponding

aldehydes.
Substrate Yield I"/.] Remarks and reference
94 H 2 0 2 or tert-butyl hydroperoxide as oxidants
95 H20z or tert-butyl hydroperoxide as oxidants [231
92 H202 or tert-butyl hydroperoxide as oxidants
0""" quantitative
3 times higher activity with tea-butyl hydroperoxide in
biphasic systems compared to H202 in b~ffer['~I
O
-H 81
Production in gram-scale; low, non-enzymatic cis/trans

/\/=\/OH 95
isomerization observed (*'I
O
-H 99
OH 97
0
50 (40% ee) Production in gram-scale, low yield with cis-isornerl2'1
&OH
46 (45% ee) 1251
92
Quantitative conversion; significant amounts of acid as

74
the product of overoxidation were found[z61
+
O
.H
\
23 S. Hu, L. P. Hager, Biochem. Biophys. Res. Com- 25 E. Kiljunen, L. T. Kanerva,J. Mol. Cat. B: Enzy-
mun. 1998,253, 544-546. matic 2000, 9, 163-172.
24 B. K. Samra, M. Anderson, P. Adlercreutz, Biocat. 26 M. P. J. van Deurzen, F. van Rantwijk, R. A. Shel-
Biotran$l999, 17, 381-391. don,J. Carbohydr. Chem. 1997, 16,299-309.
I 1145
COOH
COOH 2e- 2 H + ~ H o o c ~ C O O H
-
0 0 HO OH
Figure 16.2-36. Pyrroloquinoline quinone (PQQ) in its oxidized and reduced form
as prosthetic group for most quinoprotein dehydrogenases.
However, external HzOz addition still has the disadvantage that locally high
concentrations occur at the entry points, resulting in CPO inactivation at these hot
spots. This can be circumvented via in situ generation of hydrogen peroxide. Two
promising approaches have been reported so far: (i) another enzymatic reaction
producing HzOz e. g. with glucose ~ x i d a s e [ and
~ ~ ~(ii)
] , electrochemicalreduction of
molecular oxygen (Fig. 16.2-35)["l, "'1. In both approaches, drastic increases of the
number of CPO catalytic cycles up to 1.1 x 10' were achieved.
16.2.4.3
Chloroperoxidase(CPO, E. C. 1.1 1.1.lo)
Publications on CPO-catalyzed oxidations of alcohols are rare. However, some

selective oxidations of aliphatic, allylic, propagylic and benzylic alcohols to the
aldehyde stage have been reported (Table 16.2-11).
16.2.4.4
Catalase (E. C. 1.11.1.6)
Most commonly, catalase is applied for the dismutation of hydrogen peroxide[16G].

On reaction of catalase with one molecule of hydrogen peroxide, the intermediate
high valence iron-oxo species is generated. This species, however, is a potent oxidant
and readily reacts not only with a second molecule of hydrogen peroxide (yielding
water and molecular oxygen) but has been reported to oxidize various other
compounds such as methanol or nitrite [166].
Klibanov and coworkers enlarged the substrate spectrum by including a variety of
alcohols that were oxidized to the corresponding aldehydes. Depending on the
substrate and the reaction medium, high enantioselectivitiesare reported
The generation of reactive catalase in its oxidized stage can also be achieved by
direct electrochemical oxidation (transfer of electrons from ferric protoporphyrin IX
to the electrode). Thus, catalase immobilized on graphite electrodes has been used
for the hydrogen peroxide-free oxidation of phenol [lG81.
1146
I
QHDH
glycidol
QHDH
'>( C. testosteroni '>(

_____)
+ O W 0
O q O H <O
'H COOH
solketal
Resolution o f alcohols by enantioselective oxidation using
Figure 16.2-37.
quinohemoprotein dehydrogenases (QHDH) from different microorganisms.
16.2.5
Quinoprotein Dehydrogenases (QDH)
16.2.5.1
General Remarks
Quinoproteins constitute a class of dehydrogenases distinct from the nicotinamide-

and flavin-dependent oxidoreductases They use different quinone cofactors to
convert a vast variety of alcohols and amines into their corresponding carbonyl
products [1701. Proteins containing the cofactor pyrroloquinoline quinone (PQQ)
(Fig. 16.2-36)form the largest and best-characterizedsub-group.
QDHs are independent from classical coenzymes like NAD(P)+.The substrate
electrons are preferentially transferred to organic acceptors (quinones) and non-
native redox mediators such as phenazine derivatives, DCPIP, Wursters blue[171],
ferrocene[lO1],ferricyanide 1731, osmium complexes or direct contact to an
electrode[17'1.
One advantage of the PQQ-dependent dehydrogenases over the NAD(P)-depend-
ent dehydrogenases is the more positive redox potential of the PQQJPQQH, couple
(+ 90 mV/pH 7[1761compared to - 320 mV[177,17811.
Similarly to the flavin-dependent reactions, several mechanisms have been dis-
cussed, including covalent substrate-PQQ intermediates or hydride transfer [179-1811.
The most important QDHs are methanol (alcohol) dehydrogenase (E. C. 1.1.99.8)
and glucose dehydrogenase (E. C. 1.1.99.17), which will be discussed briefly.
1 G.2 Oxidation of Alcohols
I
1147
EP
Figure 16.2-38. A'-dehydrogenation of 6-a-methyl-hydrocortisone-21-acetate with

polyurethane-entrappedArthrobacter simplex cells in buffer-saturated 1-decanol.
The dehydrogenase (DH) activity is largely increased on addition o f quinoid
electron acceptors (EA).
16.2.5.2
Methanol Dehydrogenase (E. C. 1.1.99.8)
In addition to PQQ, the methanol dehydrogenases from Comamonas testosteroni and

Gluconobacter suboxydans contain a heme group, which is indicated in their synonym
quinohemoprotein dehydrogenase.
The regeneration of these enzymes has been achieved by anodic reoxidation of
ferricyanide [1731, 0s-modified anodes [1741, or even direct contact to the anode [17'1.
Quinohemoprotein dehydrogenases (from Comamonas testosteroni and Glucono-
bacter suboxydans) have been reported to oxidize the alcohols solketal and glycidol
(Fig. 16.2-37)enantio~electively['~~~.
Alcohol oxidases from various strains, and especially NAD(P) dependent dehy-
drogenases (except HLADH together with thio-NAD+[1821), were found to be ex-
tremely inefficient for the oxidations in Figure 16.2-37,a fact, which is attributed to
the significantly lower redox potential of the NAD(P)+/NAD(P)Hredox system[172].
The QDH from C. testosteroni was further characterized[1831. It oxidizes stereo-
specifically the (R) enantiomer of secondary alcohols. Both, kcat/KM and E increased
with the substrate chain length. In vitro, ferricyanidewas used as sacrificial electron
acceptor. In uivo, the excess electrons are most probably transferred to molecular
IG
1148
NAD+ NADH
R. eryfhropolis metabolism
Figure 16.2-39. Enantiospecific oxidation of racemic carveol t o (-)-cawone and
(-)-cis carveol using whole cells of Rhodococcus erythropolis.
oxygen via the respirator chain. This process is considerably accelerated (by a factor
of 12) upon addition of external quinoid electron acceptors such as vitamin K (that
are capable of autoregeneration) (Fig. 16.2-38)[ls41.
16.2.5.3
Glucose Dehydrogenase (E.C. 1.1.99.17)
So far, a m e m b r a n e - b o ~ n d and
[ ~ ~ a~ soluble
] glucose dehydrogenase[l’G]have been
identified. The latter oxidizes a wide range of mono- and disaccharides[’8G].In
addition to cytochrome b562, regeneration with artificial acceptors such as DCPIP or
ferr~cene[”~, 188] is effective and unproblematic, as no autoregeneration with
molecular oxygen (producing reactive 0-species) is possible. It has commercial
interest as a component of glucose test strips for diabetes c0ntrol[~”1.
16.2.6
Whole-Cell Oxidations
16.2.6.1
Stereoselective Oxidation of (-)-Carve01 to (-)-Carvone[’901
By using whole cells of Rhodococcus erythropolis DCL14, a racemic mixture of

(-)-carve01was converted to (-)-carvone and (-)-cis-carveol(Fig. 16.2-39).The system
was optimized using the two-liquid concept, in which a second organic phase serves
as substrate and product reservoir. (-)-Carvone is an important flavor compound.
The enzyme responsible for this bioconversion, catalyzed by wild-type cells of
Rhodococcus erythropolis DCL14, is carveol dehydrogena~e[~”I. A high enantiose-
lectivity and no further conversion of (-)-carvone was obtained. Carveol dehy-
drogenase has a broad substrate specificity and prefers substituted cyclohexanols as
substrate^["^]. The regeneration of the cofactor NAD’ was accomplished by the use
of living cells.
I 1149
$1
CH,OH
Enterobacter
agglomerans
- $ CH,OH Figure 16.2-40. Production of the
low calorie sweetener tagatose from
D-galactitol by whole cells of
Enterobacter agglomerans.
CH,OH CH,OH
Gluconobacter
oxydans OH
Figure 16.2-41. Oxidation of
LOH N-protected 1-amino-D-sorbi-
tol to 6-amino-L-sorbose
1-amino-D-sorbitol 6-amino-L-sorbose using Gluconobacter oxydans.
The use of a two-liquid phase system consisting of a 1:1 mixture of phosphate

buffer and dodecane resulted in an increase of the initial (-)-trans-carveolconversion
rate by 70% (to 26 nmol per minute and per mg protein). The production was
increased from 4.3 to 208 pmol (-)-carvone formed per mg protein as compared to
the aqueous system. A simple downstream process consisting of phase separation,
methanol extraction, evaporation, and separation of (-)-cis-carveol and (-)-carvone
over a silica gel column, was developed.
In another study, Rhodococcus globendus PWD8 was found to oxidize D-limonene
regio- and enantioselectivelyvia (+)-trans-carveolto (+)-carvone[”*].
16.2.6.2
Sugar DehydrogenasesApplied in Whole Cells
Cofactor regeneration by the cell metabolism is the main advantage of whole cells in
polyalcohol oxidations. The induction of whole-cell biocatalyst activity is dependent
on the nature of the growth substrate. An example is the production of the low
calorie carbohydrate sweetener tagatose from D-galactitol (Fig. 16.2-40). As bio-
catalysts, wild-type strains of Enterobacter agglomerans and Gluconobacter oxydans
DSM 2343, in which sugar dehydrogenases catalyze the reaction of interest, were
described 1193, 1941
In the case of Enterobacter agglomerans, cells growing on 1% glycerol plus 1%
erythritol resulted in the best biocatalcc performance. In 30 h, galactitol (50 g/L)
was converted with a tagatose yield of 86 %. Immobilization and storage at - 20 “C
are possible.
With Gluconobacter oxydans, growing cells were found to be more effective than
resting cells. Furthermore, galactitol adaptation gave a notable increase in tagatose
yield.
Another example is the oxidation of 1-amino-D-sorbitol(N-protected)to 6-amino-~-
sorbose (Fig. 16.2-41)[’95! This reaction was published as a step in the synthesis of
1150
I
fl-0
1 organic phase
buffer
Figure 16.2-42. Selective oxidation of linear and branched aliphatic alcohols t o
6
the corresponding aldehydes using P. pastoris i n aqueous/organic reaction
mixtures.
<OH
Figure 16.2-43. Oxidation of benzylic alcohol

t o benzaldehyde using whole cells o f P. pas-
toris in organic/aqueous emulsions or with
purified alcohol oxidase. In vitro hydrogen
peroxide was removed by catalase.
1-desoxynojirimycin.Derivatives of 1-desoxynojirimycinare pharmaceuticals for the

treatment of carbohydrate metabolism disorders (e.g. diabetes mellitus). Suspended
whole cells of Gluconobacter oxydans were used as the biocatalyst, in which D-sorbitol
dehydrogenase is responsible for this biotransformation.
To prevent undesired follow-upreactions of G-amino-L-sorbosein water, the amino
group has to be protected by, for example, a benzyloxycarbonyl group (R).Cells are
produced by fermentation on sorbitol and used for the bioconversion step as resting
cells in water without added nutrients. The biotransformation is carried out by Bayer
in a 10 000 L reactor with 90 % yield.
16.2.6.3
Oxidation of Aromatic and Aliphatic Alcohols to Corresponding Aldehydes and Acids
Havin-containing alcohol oxidase combined with catalase in peroxisomes of Pichia

pastoris naturally catalyzes the oxidation of methanol to formaldehyde. In vivo and in
vitro applications are possible. The alcohol oxidase has a broader substrate specificity
than the subsequent enzymes of the methanol degradation pathway. Therefore,
16.2 Oxidation ofAfcohols
I
1151
Figure 16.2-44. Oxidation of alcohols by whole cells ofAcinetobacter. In aqueous media

the oxidation proceeds until the acid stage, whereas the aldehyde is accumulated in the
presence of organic solvents.
NAD+ NADH
Figure 16.2-45. Preparation of isovaleraldehyde using an alcohol de-
hydrogenase (ADH) in whole cells o f Cfuconobacter oxydans.
products other than formaldehyde are not degraded further. The spectrum of
alcohols oxidized by whole cells of Pichia pastoris includes aliphatic Cl-Cs alcohols
(saturated, unsaturated or branched). In biphasic media, Pichia pastoris also oxidizes
c&1 alcohols, phenylethyl alcohol and 3-phenyl-1-propanol[1961.
Up to 70 g/L acetaldehyde was produced from e t h a n ~ l [ ~ ” ~ Here,
” ~ ] . competitive
product inhibition was partially overcome by high Tris buffer concentrations. Tris is
able to bind acetaldehyde and markedly improve reaction yields. In a biphasic system
consisting of 97 % hexane and 3 % aqueous phase, hexanol(l1 g/L) was converted to
hexanal (Fig. 16.2-42)within 24 h at a yield of 9G%[1961.In another example, benzyl
alcohol was oxidized by whole cells of Rchia pastoris and purified alcohol oxidase
(Fig. 16.2-43)[2001.For this reaction the importance of solute partitioning in the
biphasic reaction system was studied[201].With immobilized cells in organic (xy-
lene)/aqueous media, benzaldehyde concentrations up to 30 g/L were reached in the
organic phase[”*]. With purified alcohol oxidase, up to 45 g/L benzaldehyde was
produced within 8 h and with an enzyme concentration of 0.94 g/L.
Dehydrogenases of Acinetobacter and Gluconobacter strains catalyze the oxidation of

various alcohols to corresponding aldehydes and acids in uivo. Substrates tested
NAD+ NADH
Figure 16.2-46. Preparation o f 2-phenyl acetaldehyde with Acinetobacter sp. in
organic/aqueous emulsions.
1152
Table 16.2-12. Oxidations catalyzed by Acinetobacter sp. in aqueous and biphasic medial27].
Water Water/isooctane (vol/vol l / l )
Substrate Acid yield ["A] Time [h] Aldehyde yield r/.1 Time [h]
> 97 3 74 1
> 97 3 90 1
T O H
> 97 3 87 1
)+=fOH > 97 24 72 4
0""" 25 24 <5 24
rO" > 97 3 90 45 min
> 97 93 45 min
> 97 77 45 min
40 ((S)-alcohol:
24 <5 24
95% ee)
racemic
27 R. Gandolfi, N. Ferrara, F. Molinari, Tetrahedron Lett. 2001, 42, 513-514.
include ethanol, propanol, butanol, 2-methyl-1-butanol,3-methyl-1-butanol,l-penta-

nol, 1-hexanol, geraniol, 2-phenylethanol, 2-phenylthioethanol, cinnamyl alcohol,
benzyl alcohol and (R,S)-2-phenyl-l-propanol (Tables 16.2-12 and 16.2-13)[202. 2031.
The molecular structure of substrates and products as well as physicochemical
conditions significantly influence bioconversions of short-chain aliphatic and aro-
matic alcohols into acids[204.2051 . Y. ields depend on the toxicity of the alcohol
(different inhibitory concentrations for different alcohols), since product inhibition
is often the major limiting factor L2O4]. Furthermore, dissolved oxygen concentrations
and pH conditions are important factors for improving such bioconversions.
Depending on strain and substrate (specificity of dehydrogenases), the reaction is
directed to aldehyde or acid accumulation (Fig. 16.2-44).In principle, acid accumula-
tion is favored in aqueous media, whereas aldehydes preferentially accumulate in
biphasic media[203].
Table 16.2-13.
7G.2 Oxidation ofAlcohols
Oxidations catalyzed by Gluconobacterasaii in aqueous and biphasic systems1271.

I
1153
Water Water/isooctane (vol/vol 1/1)

Substrate Acid yield [%] Time [h] Aldehyde yield [%] Time [h]
> 97 4 93 45 min
> 97 4 90 1
T O H
> 97 3 91 45 min
>-3-'OH 16 24 29 5
0""" <5 24 <5 24
> 97 5 85 2
T O H
> 97 5 96 1
20 24 24 4
33 24 <5 24
racemic
27 R. Gandolfi, N. Ferrara, F. Molinari, Tetrahedron Lett. 2001,42, 513-514.
OH OH
0
(3-2-phenylpropanoic acid
Figure 16.2-47. Resolution of racemic (R,S)-2-phenylpropionic alcohol with whole cells of
Gluconobacter oxydans yielding (S)-2-phenylpropanoic acid and (R)-2-phenylpropionic alcohol.
An example of aldehyde formation is the production of isovaleraldehyde by

Gluconobacter oxydans R (Fig. 16.2-45) [202, 2061. Glycerol-grown Gluconobacter oxy-
duns slowly oxidizes 3-methyl-1-butanolto isovaleraldehyde,with yields of over 90%.
The product was recovered by bisulphite trapping or cold Extractive
bioconversion in a hollow-fiber membrane bioreactor allowed continuous produc-
1154
/
\
C.parapsilosis IF0 1396
oxidative
:11267
P”,,H -OH
(@-I ,3-butanediol (S)-l,3-butanediol
Figure 16.2-48. Preparation of both enantiomers of 1,3-butanediol with whole cells of

K. /actis and C.parapsilosis either by enantioselective oxidation o f 1,3-butanediol
(oxidative) or enantioselective reduction of 4-hydroxybutanone (reductive).
tion of isovaleraldehyde at overall productivities of 2-3 g L-l h-’ 1206]. Yields between
72 and 90 % were reached.
Another example of a synthesis is the production of phenylacetaldehyde using
Acinetobacter strains (Fig. 16.2-46) 20sl. Different two-liquid phase systems were
tested for their ability to remove the aldehyde into the organic phase before its
further conversion to acid. In an optimized two-liquid-phase process, in which
isooctane (at a volume fraction of 50%) was used as the organic carrier solvent,
product concentrations of 9 g/L were reached in 4 h of reaction, corresponding to a
yield of 90 %[208].The production strain Acinetobacter sp. ALEG showed satisfactory
long-term stability, being able to perform the transformation with 80 % of the
original activity after 3 days of contact with the solvent.
Besides the multigram-scale production of different aliphatic carboxylic acids by
biocatalytic alcohol oxidation, especially the enantioselective oxidation of racemic
2-phenyl-1-propanol to (S)-2-phenylpropanoic acid with Gluconobacter oxydans
(Fig. 16.2-47)is another good example of acid production from alcohols~209~.
After optimization of the parameters temperature, pH, substrate concentration,
and agitation speed using a simplex sequential method, the resolution involving two
oxidation steps yielded 45 % product with an ee of 98 %.
16.2.6.4
Enantiospecific Reactions
Two ways of producing optically pure 1,3-butanediol via microbial resolution have been
reported: the oxidation of a racemic mixture of 1,3-butanediolyielding one enantio-
I
n
1155
ADH OH 0
CI
0-
ethyl-4-chloro-3-oxobutanoate (R)-ethyl-4-chloro-3-hydroxybutanoate
NADH NAD+
Figure 16.249. Asymmetric reduction of ethyl-4-chloro-3-oxobutanoate catalyzed by an

alcohol dehydrogenase (ADH) in recombinant E. coli. The necessary reduction equivalents
were derived from the oxidation o f isopropanol with the same enzyme.
K Rhodococcus ~
erythropolis
<O
'H OH COOH
Figure 16.2-50. Enantioselective oxidation of isopropylideneglycerol utilizing
Rhodococcus erthyropolis.
mer and 4-hydroxy-2-butanone,and the reduction of the 4-hydroxy-2-butanone

yielding one enantiomer of 1,3-butanediol (Fig. 16.2-48). (R)-1,3-butanediolis an
important chiral synthon for the synthesis of various optically active compounds
such as azetidinone derivatives, which are intermediates in the production of
antibiotics, pheromones, fragrances, and insecticides.
From a screening procedure, Kluyveromyces lactis I F 0 1903 and Candida para-
psilosis I F 0 1396 were found to be effective in the enantioselectiveoxidation of (R)-
1,3-butanedioland (S)-1,3-butanediol,respectively, and in the asymmetric reduction
of 4-hydroxy-2-butanone to (R)-1,3-butanediol and (S)-1,3-butanediol, respec-
tively[2101.
The equilibria between ketones and alcohols are catalyzed by secondary alcohol
dehydrogenases. The secondary alcohol dehydrogenase of C. parapsilosis I F 0 1396
OH
-
enantiospecific 0 OH
oxidation
RIAR2 + RlAR2
racemic alcohol desired enantiomer
asymmetric
reduction
Figure 16.2-51.
1
Stereoinversion cata-
lyzed by two different
alcohol dehydrogenases
R1 R2 via enantiospecific oxi-
dation followed by an
desired enantiomer asymmetric reduction.
1156
I
was purified and characterized as an NAD’-dependent dehydrogenase with a broad
substrate specificity (secondary alcohols > primary alcohols)r2111. The alcohol dehy-
drogenase gene of C. purupsilosis was cloned and expressed in recombinant E. coli
JMlO9, which showed more than twofold higher specific alcohol dehydrogenase
activity than C. purupsilosis[212].
Resting cells of C. parupsilosis were used for the large-scale (2000 L) production of
(R)-1,3-butanediol(94% ee) from racemic 1,3-butanediol.After down-stream proc-
essing 3.1 kg product was isolated (overall yield: 15.5%), and a chemical purity of
Table 16.2-14. Biocatalytic stereoinversions with Ceotrichum candidum [**l.
Without allyl alcohol With allyl alcohol (33 mM)

Substrate Yield [“A] e e [“A] Configuration Yield [“A] e e [“h] Configuration
d O H 96 99 fR) 94 98 fR)
28 K.Nakamura, Y. Inoue, T. Matsuda, A. Ohno, Tetrahedron Lett. 1995,36.

6
I
1157
enantioselective
&j$
oxidation
c
& + OH
/ OH OH / 0 OH
OH
asymmetric reduction
Figure 16.2-52. Synthetic application of the stereoinversion concept using Candida sp.
and Pichia sp.
98.8 %was reached[213]. Resting cells of recombinant E. coli were reported to produce
(R)-1,3-butanediol(93.5% ee, 94.7 % yield) from the racemate without any additive to
regenerate NAD’ from NADHL2”I.
In another application, recombinant E. coli produced 36.6 g/L ethyl-(R)-4-chloro-
3-hydroxybutanoate (99% ee) from 40 g/L ethyl-4-chloro-3-oxo-butanoate r2l0].Here,
the secondary alcohol dehydrogenase served as both synthetic (asymmetric reduc-

tion) and regenerating (NADH-regeneration via isopropanol oxidation) enzyme
(Fig. 16.2-49).
Enzymatic resolution of (R/S) isopr~pylideneglycerol[~’~~

2151
Whole cells of Rhodococcuserythropolis were used for the selective oxidation of the (S)-
enantiomer of isopropylideneglycerol (Fig. 16.2-50).With a 50 % conversion of the
racemate, an ee value of over 98% was reached for (R)-isopropylideneglyceroland of
over 90 % for (R)-isopropylideneglycericacid.
(R)-Isopropylideneglycerolis a useful C3-synthon in the synthesis of (S)-P-block-
ers; e. g. (S)-metoprolol.(R)-Isopropylideneglycericacid can also be used as starting
material for the synthesis of biologically active compounds.
16.2.6.5
Stereoinversions using Microbial Redox Reactionsp14
Racemic mixtures of secondary alcohols can be resolved completely by enantiospe-

cific enzyme-catalyzed oxidation resulting in one enantiomer of the alcohol and the
ketone followed by asymmetric enzyme-catalyzedreduction of the ketone (Fig. 16.2-
51). For oxidation and reduction, two separate microorganisms [217-2191 or two
different enzymes in a single microorganism[22&222] may be used.
An example of a suitable biocatalyst is Geotrichum candidum, harboring both an
oxidizing and a reducing enzyme activity. Table 16.2-14shows the catalFc perform-
ance of Geotrichum candidum towards different substrates [220] when the biocatalyst is
incubated for 24 h with 27 mM substrate. Ally1 alcohol effectively shifts the
stereoselectivityof the reduction. It is presumed to inhibit enzyme@)that reduce aryl
1158
m- /
1
x mo OH
"OH
chemical MO
Figure 16.2-53. Chemoenzymatic synthesis of 2-hydroxy-1-indanone.The racemic syn and
anti diols were prepared by chemical dihydroxylation of indane. Asymmetric induction was
achieved by microbial oxidation (MO) of these diols.
Table 16.2-15. Substrate spedificity ofA\rthrobacter and Pseudomonas strains[29].

Taxonomy Substrate specificity (no substrates)
Arthrobacter sp. strain 1HB cis-(lS,2R)-diol > trans-(lS, 2s)-diol >>

(cis-(1R, 2s)-diol, trans-(1R, 2R)-diol)
Arthrobacter sp. strain 1HE cis-(lS,2R)-diol>>trans-(lS,2S)-diol>>
(cis-(lR,2s)-diol, trans-(1R, 2R)-diol)
Pseudomonas aeruginosa strain I N trans-(IR, 2R)-diol>cis-(lS,ZR)-diol>
cis-(IR, 2S)-diol>>(trans-(lS,2S)-diol)
29 Y. Kato, Y.Asano, /. Mol. Cut. B: Enzymatic 2001, 13, 27-36,
Table 16.2-16. Microbial stereoselective oxidation of cis- and trans-l,2-indandiol~[~~1.

Strain Substrate Product Reaction time [h] Yield rh] e e [%]
Arthrobacter sp. 1HB Cis R 4 46 > 99.9

Trans S 12 35 > 99.9
Arthrobacter sp. 1HB Cis R 4 47 > 99.9
Trans S 24 8 > 99.9
P. aeruginosa IN Cis R 5 7 82.5
Trans R 24 40 > 99.9
29 Y. Kato, Y. Asano, /. Mol. Cut. B: Enzymatic 2001, 13, 27-36,
methyl ketone to (S)-1-arylethanol.The inhibition of yeast reductases by ally1

alcohols has been reported[223].
Another example is the deracemization of (RS)-1-{2',3'-dihydrobenzo[b]furan-
4'-yl}-ethane-1,2-diolby biocatalytic stereoinversion (Fig. 16.2-52)[2241. In order to
find an appropriate biocatalyst to accomplish such a deracemization, different
microorganisms were screened. Several microorganisms belonging to the genera
Candida and Pichia allowed yields of 60-70% with 90-100% enantiomeric excess.
Substrate dissolved in DMF was added to the biotransformation mixture consisting
of resting cells suspended in phosphate buffer (pH 7). The presence of glucose
generally increased the yield but lowered the enantiomeric excess. Different micro-
organisms can be suitable for a given stereoinversion and the optimal biocatalyst
should be chosen by screening.
I
1159
nu -
oxidations
cholesterol androst-2-en-3,i 7-dione
asymmetric
reduction
I
testosterone
Figure 16.2-54. Selective oxidation o f cholesterol to testosterone by whole cells o f
Mycobacteriurn sp NRRL 8-3805.
Stereoselective oxidation of racemic 1,2-indandiol~[*~~1

Kato et al. described the stereoselective microbial synthesis of both enantiomers of
2-hydroxy-l-indanone,selecting cis- or trans-diol as the substrate (Fig. 16.2-53).Cis-
1-amino-2-indanolis an important synthon in organic chemistry (for example in the
synthesis of the leading HIV protease inhibitor Crixivan) and can easily be synthe-
sized from optically active 2-hydroxy-1-indanone[2261.
Microorganisms degrading indane derivatives were screened for stereoselective
oxidation of racemic cis- or trans-l,2-indandiol. Three promising strains specifically
oxidizing the benzylic hydroxyl group were found (see Table 16.2-15).
All strains produced inducible enzymes responsible for the oxidation reaction,
recognizing the stereochemistry of the 1-or 2-positionsof the diol regardless of their
cis and trans geometry. By using the resting cells of the strains, both enantiomers of
2-hydroxy-1-indanonewere synthesized in enantiomerically pure form simply by
selecting cis- or trans-1,2-indandiol as the substrate. Growth conditions were opti-
mized to promote cell growth and the formation of 1,2-indanediol-oxidizing activity.
The biocatalyst activity was optimally induced with 0.05 % indanol. Carefully choos-
ing appropriate carbon and nitrogen sources is crucial for optimal biocatalyst activity
and cell growth.
Table 16.2-16 shows the stereoselective oxidation of racemic cis-diol or trans-diol
into optically active 2-hydroxy-1-indanoneat a 2 mL scale with 50 mg dry cells per
ml.
1160
A I
Figure 16.2-55. Regioselective three-step oxidation of ebastine (A) t o carebastine (B)
using Cunninghamella blakesleeana.
Production of testosteronefrom cholesterol using Mycobacterium sp. [2271

In this multistep reaction the microbial degradation of sterol side chains combined
with the reduction of an intermediate thereof is used to accumulate testosterone
from cholesterol. A cholesterol-assimilatingand androst-2-en-3,17-dione-accumulat-
ing mutant of Mycobacteriurn sp. NRRL B-3805 oxidizes cholesterol through multiple
steps of the sterol side chain degradation pathway, also involving alcohol oxidations,
to androst-2-en-3,17-dione(Fig. 16.2-54).This multistep oxidation is followed by the
reduction of androst-2-en-3,17-dioneto testosterone by the NADH requiring activity
of 17P-hydroxysteroiddehydrogenase. This activity is dependent on the presence of
glucose as the carbon source. After the glucose in the fermentation culture is
completely consumed, most testosterone is oxidized to androst-2-en-3,17-dione.
Adding a larger amount of glucose prevents this oxidation.
On a 2.5 L scale a yield of 51 %was reached in 120 h of cultivation. Here, the initial
substrate concentration amounted to 0.1 % (w/v).
Microbial oxidation of ebastine [228]

Ebastine is a new generation antihistaminic drug with fewer side-effects. The
microbial three-step oxidation of ebastine, using whole cells of the mold Cunning-
hamella blakesleeana as biocatalysts, involves an alcohol and an aldehyde oxidation
step and results in the formation of carebastine, which is the pharmacologically
active The initial step in the oxidation of ebastine is hydroxylation by
a cytochrome P-450-dependent monooxygenase to the corresponding alcohol. The
two consecutive oxidations are catalyzed by oxidoreductases,which are not further
characterized, and lead via the aldehyde to the corresponding carboxylic acid
carebastine (Figure 16.2-55).
Growth in a complex medium containing soybean-peptone and yeast extract is
necessary for biocatalyst activity. A component of soybean-peptone, genistein, is
thought to act as an inducer of cytochrome P-450 enzymes. Growing cells provide a
higher yield than resting cells. Addition of 1% poly(viny1 alcohol) was found to
prevent pellet formation and thereby to guarantee constant mass transfer rates.
From a 3 L batch fermentation, 270 mg carebastine was isolated (yield: 45%).
P1
co2 \f-
FDH
NADH -
Q
~ Diaphorase I
Figure 16.2-56. Enzymatic three-step oxidation o f methanol t o carbon dioxide

in the anodic compartment o f a biofuel cell.
A HO'" "OH
-P-
0s=
E
B
Figure 16.2-57. Mediated electron transfer steps in the electroenzymatic oxidation

o f glucose (A) and reduction o f 02.
IG
1162
Therefore, after 24 h of cultivation, GOO mg ebastine was added and the incubation
was continued for 68 h. .
16.2.7
Miscellaneous
16.2.7.1
Biofuel Cells
In recent years, biofuel cells have gained tremendous attention. The use of methanol
instead of dihydrogen as the oxidizable substance offers special advantages as it is
readily available and easy to store and handle. At the same time, the theoretical cell
voltage of an MeOH/02 cell (1.19 V) is near that of H2/02 (1.23V).
Whitesides and coworkers recently developed a biofuel cell based on the step-wise
enzymatic oxidation of methanol to carbon dioxide (Fig. 16.2-56)[2301. In the anodic
compartment of the biofuel cell, methanol is oxidized to carbon dioxide in three
steps: by an alcohol dehydrogenase, an aldehyde dehydrogenase, and ultimately
formate dehydrogenase. In each of these enzymatic steps, one equivalent of NADH
is produced. NADH itself transfers its electrons via diaphorase to viologene and in
the end to the anode. The redox potential of the reducedloxidized viologene couple
(- 0.55 V) is only slightly less negative than MeOH/C02 (- 0.64 V) and NADH/
NAD' (-0.59 V). Thus, the loss in cell potential was minimized. The catholyte
consisted of platinum gauze in an 02-saturated buffer ( 0 2 + 4e-+4Hf 2H20). An
+
open-circuit potential of 0.8 V and a maximum power output of 0.67 mW cm-2 was
achieved.
Another biofuel cell concept is based on the oxidation of glucose to gluconolactone
catalyzed by glucose oxidase (Fig. 16.2-57)[231. 2321. Because of the slow kinetics of
the electron transfer to 02,dioxygen is usually reduced at a potential several hundred
millivolts more negative than its formal potential, thus lowering the power density of
a fuel cell. Utilizing laccase to catalyze this reaction can circumvent that. ABTS is a
suitable mediator between the electrode and laccase because of its quite positive
redox Wiring laccase reduction to the electrode via an osmium-
modified electrode also facilitates the electroreduction of molecular oxygen. The
same modification serves as the conductor between glucose oxidase and the anode.
-PEG
-V - OH OR
Figure 16.2-58. NAD modified with polyethylene glycol (PEG).
Table 16.2-17. Kinetic constants of different dehydrogenases for NAD(P)' and PEG-NAD(P)+.
Native cofador PEG-bound cofador
NAD'-dependent enzymes KM KM [@I VmaX[as % o f NAD']a
FDH 15 82 57
Glutamate DH 175 444 53
YADH 154 1310 64
HLADH 62 1150 72
LDH 182 142 21
3a-HSDH 29 647 66
Glucose DH 9G 2030 3
NADP+-dependentenzymes KM [@I KM bM1 V,,, [as % o f N A D V ]
Glutamate DH 160 425 96

Malic enzyme 5 12 86
TBADH 13 28 84
a 100% correspond to V, values of the dehydrogenases determined with native coenzymes.
A miniaturized cell was constructed which exhibited a power output of 0.137

mW cm-2. After 72 h of operation, 75 % of the initial power output was still present.
Even though biofuel cells are generally considered to be in their infancy[234],
their
potential, which is based on non-hazardous, easy-to-handlesubstrates and electro-
lytes (especiallythe moderate temperatures compared to those of conventional fuel
cells: 80-1000 "C) cannot be neglected. Even photosynthetic biofuel cells (converting
light energy into electrical energy) have been shown to work in principle[235].
16.2.7.2
Biomimetic Analogs to NicotinamideCoenzymes
For large-scale applications of NAD(P)-dependent enzymes, continuous-flow re-

actors with ultrafiltration membranes have been proposed[236]. In order to retain low
molecular weight nicotinamide cofactors in the reactor, charged membranes have
been used, retarding the overall negatively charged nicotinamide coenzymes by
electrostatic repulsion[237,2381. Retention rates of approx. 99% and TTNs (NAD) of
up to 10 000 were reported.
Another approach makes use of polymer-modified NAD [modification with
polyethylene glycol (PEG; MW = ZOOOO)], thus retaining it on account of its
drastically increased size (Fig. 16.2-58)[239-2411. The polymer modification usually
leads to a drastically increased KM value, whereas the V,, value is generally over
50% of that of low molecular weight NAD(P) (Table 16.2-17).
Another area of research deals with synthetic analogs of NAD(P) coenzymes.
Besides the lower costs, these analogs may offer better stability or easier regenera-
tion and may add new functionalities to known enzyme systems (e.g. thio-NAD
together with HLADH [ls2I). Some artificial redox coenzymes were developed mim-
icking the "shape" of native nicotinamide coenzymes (Fig. 16.2-59) 244]. Activity
with various NAD-dependent enzymes was found, even though the activity was only
1164
Figure 16.2-59. Synthetic analogs

of NAD.
so3-
CL4
blue N-3
in the region of less than 10% of that with the native cofactor. However, it was shown
that these analogs could have at least some potential.
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16.3
Oxidation of Phenols
16.3.1
Introduction
Several classes of oxidoreductases accept phenols and their derivatives as substrates

for oxidation reactions. A broad range of products can be obtained depending on the
substrates and enzymes applied (Fig. 16.3-1). Several monooxygenases catalyze the
hydroxylation of the aromatic ring specifically ortho or para to the existing phenolic
alcohol function (Fig. 16.3-1 A). Oxidases can be used to catalyze the stereospecific
benzylic hydroxylation of aliphatic side chains to (R) or (S) alcohols and the further
oxidation of benzylic alcohols to corresponding ketones or aldehydes; furthermore,
elimination to (2)or (E') alkenes can be obtained if desired (Fig. 16.3-1 B). Laccases
and peroxidases generate phenoxy radicals which - depending on the reaction
conditions - can react further with phenols to structurally complex dimers or
conducting polymers (Fig. 16.3-1 C). Even nitration reactions are reported (Fig. 16.3-
1 D). Thus, enzymatic modification opens up new possibilities for synthetic
chemistry with aromatic compounds under mild and non-toxic conditions.
16.3.2
Oxidases
16.3.2.1
Vanillyl-alcohol oxidase (E.C. 1.1.3.38)
The enzyme vanillyl-alcohol oxidase (VAO, E.C. 1.1.3.38) was examined in detail
with respect to mechanism, structural properties, and biotechnological applications
by van Berkel and coworkers, giving an excellent example of how detailed bio-
chemical studies provide a basis for preparative biocatalytic applications (for recent
reviews see['. '1). The homooctamer with a monomer mass of 65 kDa was isolated
and purified from Penicillium simplicissimum. The catalytic mechanism of VAO-
catalyzed oxidation of para-alkyl phenols was studied in detail r3-'1. After initial
hydride abstraction from the Ca atom, a binary complex of the intermediate para-
quinone methide and reduced FAD reacts with molecular oxygen, regenerating the
76.3 Oxidation of Phenols
I 1171
I--\
/
HO
OMe R R
Figure 16.3-1. Enzyme-catalyzed oxidations of phenols. A ortho- and para-hydroxyla-

tions catalyzed by monooxygenases (Sects. 16.3.3.2and 16.3.6.2);B: oxidation at the
benzylic position catalyzed by oxidases (Sects. 16.3.2.1and 16.3.5);C:coupling reac-
tions catalyzed by peroxidases and laccases (Sects. 16.3.4.1and 16.3.2.2);D: nitration
reactions catalyzed by peroxidases (Sect. 16.3.4.3).
oxidized prosthetic group. Depending on the nature of the aliphatic side chain, the
para-quinone methide is hydroxylated to (chiral) benzylic alcohols (short aliphatic
side chains) or rearranges yielding benzylic alkenes (long aliphatic side chains)
(Fig. 16.3-2).Table 16.3-1 shows a selection of reactions catalyzed by VAO as well as
the kinetic constants thereofl3, 1'.
OH
- r"
HOq - R
0, + H,O
HO rRVAO-FAD
0
VAO-FADH
R = short chain
%
0
HzO2
HO
/o^'"
cis+trans
Figure 16.3-2. Reaction mechanism ofvanillyl oxidase (VAO). R = medium chain
1172
Table 16.3-1. Substrate spectrum and kinetic constants of vanillyl oxidase.

Substrate Product(s)' KM [ W ] kat [S-'1 k,t/K~ [W3][s-' M-'1
76 % alcohol
HO /o^ 24% alkene
9 2.5 280
68 % alcohol
HO Xy- 32 % alkene
4 4.2 1050
90 % alcohol
HO
6 4.9 820
10% alkene
d-
OMe
20 % alcohol
16 1.3 81
HO 80 % alkene
HOd 26 % alcohol
74 % alkene
1 % alcohol
72 0.5 7
-o^" 2 1.2 600
r
HO 99 % alkene
100%alkene 8 0.3 38
HO
10^"/"
100% alkene 42 < 0.001 < 0.02
HO
40%
65 1.4 21
HO
HO
16% alcohol
60%ketone 77 0.5 7
HO 24 % alkene
4% alcohol
2 % ketone 94 0.7 7
HO 94% alkene
16.3 Oxidation ofphenols
I 1173
Substrate Product(s)" K, [pu] kcat[sd] kcot/Khn [lo-'] [s-' M-'j
QH
HO /o^ 100 % ketone 222 0.7 3
HO q-bMe
100 % ketone 4.9 13.0 2700
HO /o^" HO&OH
4.8 6.5 1400
HO qJ- -9""
OMe
HO
OMe
290 5.4 19
HO
HO -0"" 240 1.3 5.4
OMe bMe
HO /o"""' HO $yo 65 5.3 82
a Beside s the structure shown the products formed include benzylic alcohols, benzylic alkenes
and benzylic ketones.
VAO exhibits a remarkable activity towards 4-alkylphenols, bearing aliphatic side

chains of up to seven carbon atoms. The maximum chain-length of 7 is in
accordance with structural data obtained from X-ray crystallography"1. Short-chain
4-alkylphenols are mainly hydroxylated at the Ca position, whereas medium-chain
4-alkylphenols are dehydrogenated to 1-(4'-hydroxyphenyl)alkenes (Fig. 16.3-2)['I.
The hydroxylation reaction is highly stereospecific, producing the (R)-enantiomer
with ee values of up to 94 % 1'1. Furthermore, VAO also catalyzes the further oxidation
of the alcohols to the corresponding ketones. Here, the VAO-catalyzed oxidation of
(S)-alcoholsis far more efficient than the oxidation of (R)-alcohols,promoting a
possible application in kinetic resolution reactions. Substrates with more space-
consuming alkyl side chains are dehydrogenated by the action of VAO. With para-
methyl phenols (e.g. cresol), a very low conversion rate is found which is due to the
formation of a stable intermediate formed through a nucleophilic attack of the
reduced FAD on the para-quinone methide, yielding a covalent bond[']. Since the
rate-limiting hydrolysis of this intermediate is acid-catalyzed,the pH optimum of the
reaction shifts from alkaline to acidic values. The formation of such a covalent
1174
HO /
liver microsmomes
HO
qNH2 VAO -c
HO
OMe OMe OMe
capsaicin vanillin
Figure 16.3-3. Potential biotechnological production route to vanillin from

natural components with vanillyl oxidase.
intermediate is supposed to be more unlikely with increasing length of the aliphatic

side chain, because of increasing steric hindrance.
Much attention has been paid to the shift from hydroxylation to dehydrogenation
with increasing length of the side chain. The product ratio between alcohols and
alkenes is strongly influenced by the extent of hydratation of the intermediate, para-
quinone methide. Thus, by using organic media with a low water content the overall
alkene yield could be significantly increased. The same is true for monovalent anions
such as CP, Br-, or SCN-, which bind to the active site, thereby decreasing the water
concentration at the active site[']. By enzyme engineering based on the three-
dimensional structure [71, the ratio between hydroxylation products and dehydroge-
nation products could be shifted either in favor of the alcohols, when Asp170 was
exchanged with Glu, or in favor of the alkenes, when Asp170 was exchanged with
Ser["I. Double mutants of VAO (D170S/T457E and D170A/T457E) were produced
based on the same rational approach, thus inverting the stereospecificity of the VAO-
catalyzed hydroxylation of 4-ethyl phenol from (R) to (S) (ee = 80%) [I1].
The VAO-catalyzed production of vanillin is of special synthetic interest. In
particular, a route starting from capsaicin that is readily available from red hot
pepper has some biotechnological potential. Here, vanillylamine is obtained by
hydrolysis of capsaicin using rat liver microsomes and further oxidized by VAO
(Fig. 16.3-3). Furthermore, a one-pot synthesis using carboxylesterase for capsaicin
hydrolysis is proposed [''I.
16.3.2.2
Laccase (E. C. 1.10.3.2)
Recently, laccases found some interest for synthetic application. Laccases are widely
distributed in plants and fungi['3].The copper-containing enzymes are some of the
few oxidases so far reported to reduce molecular oxygen to water (aside from
cytochrome c oxidase and others). This ability was recently exploited in a novel
regeneration concept for flavin-dependentenzymes (see Chapter 16.2) [I4].
Purified laccase oxidizes various phenolic compounds via hydrogen abstraction.
The resulting phenoxy radical undergoes various dimerization and oligomerization
reactions. Even though the synthetic potential of such reactions has to be considered
as moderate, in some cases interesting products (such as complex coumaran type
compounds) can be obtained in reasonable yields from simple phenols["1.
Laccases alone are not able to oxidize benzyl alcohols. Bourbonnais and Paice["]
Table 16.3-2. Laccase/ABTS-catalyzed oxidations to corresponding aldehydes.
16.3 Oxidation ofPhenols
I
1175
Catalned reaction Yield I"?] Literature
P\ O
/ " - Po
\ / 94 PI
92 PI
Me0 Me0
P O
CI
H - Po CI
92 PI
*OH- \ / \ /
-0 -0
CI
p-p0 CI
89 [21
1 A. Potthast, T. Rosenau, C. L. Chen, I. S . Gratzl, 2 A. Potthast, T. Rosenau, C. L. Chen, J. S. Gratzl,

I . Mol. Cat. A.: Chemical 1996,108, 5-9. I. Org. Chem. 1995,60,432&4321.
were the first to report that laccase in the presence of a specific compound, usually
called a "mediator", is able to catalyze the oxidation of benzyl alcohols. Mostly ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), HOBT (l-hydroxybenzotria-
zole) [l71,and NHAA (N-hydroxyacetanilide)[18] have been used as mediators so far.
The actual role of the mediator is not yet fully understood, although Potthast et al.
recently found evidence that laccase produces reactive radical species of ABTS and
1176
I
-OH 0 2
*
catecholase activity nun
II I
R creolase activity RwoH ” I,
Figure 16.3-4. Oxidation of phenols catalyzed by tyrosinase displaying so-called

creolase and catecholase activities.
HOBT, which perform the actual oxidations [”]. Nevertheless, some preparative
oxidations of various benzylic alcohols are reported (Table 16.3-2).
It should be pointed out here that the laccase-mediator system still is far from
being economically feasible.
16.3.3
Monooxygenases
16.3.3.1
Tyrosinase (E.C. 1.10.3.1)
Tyrosinases (synonyms: phenol oxidases, poly-phenolases or polyphenol oxidases)

are copper-containing monooxygenases, which catalyze two consecutive reactions
with molecular oxygen as cosubstrate, namely the ortho-hydroxylation of phenols and
the oxidation of the resulting catechols to ortho-quinones (Fig. 16.3-4).
The initial (phenol-hydroxylating)activity is usually referred to as creolase activity,
whereas the second (catechol-oxidizing)activity is most commonly called catecholase
activity[”]. The classification of tyrosinases (polyphenol oxidases) is somewhat
ambiguous; enzymes exhibiting monophenol oxidase activity are classified as E. C.
1.14.18.1., but those with catechol oxidase activity as E. C. 1.10.3.2. However, many
enzymes exhibit both activities, and a more appropriate classification of all two-
electron-acceptingcopper monooxygenases as E. C. 1.14.18.1was proposed[”].
In animals, tyrosinase is involved in the formation of melamines, and in plants,
tyrosinase leads to the well-known browning of open surfaces of
Much attention has been paid to the mechanism[20,22]. In the active site, two
copper(1) ions bind molecular oxygen. Upon binding of the phenolic substrate, the
ortho-position is attacked electrophilically by one of the activated oxygen atoms. The
resulting copper-bound catechol serves as an internal electron donor and leaves the
active site as ortho-quinone.Figure 16.3-5 illustrates this mechanism.
In order to prevent rapid quinone polymerization in aqueous media, the quinones
are usually reduced to the catechols (most commonly by ascorbic acid) (Fig. 16.3-6).
Several tyrosinase-catalyzed oxidations of phenols have been reported; some of
these are presented in Table 16.3-3.
Tyrosinase was reported to hydroxylate and oxidize tyrosine residues in pro-
teins [23], which is important in the production of moisture-resistant adhesives. In
fact, tyrosinase has been used for the production of synthetic glues with similar
compositions to those of naturally occurring adhesives such as mussel glue [24].
An interesting cascade reaction was reported by Waldmann et al.[252 2 G ] . Tyr-
I
1177
0 2
- Q
0 0
,cut
\
cu
+/ \ /2+ \
f
:2
\ ,C", o/cy
0
Figure 16.3-5. Reaction mecha-
nism for the oxidation o f phenols
by tyrosinase.
OH
HO* o - Ho*o
Figure 16.3-6. Ascorbic acid-
HO OH 0 0 driven reduction o f quinones.
R
PH 1
L A
Figure 16.3-7. Chemoenzymatic Diels-Alder reactions. Ortho-quinones (dienes), derived from
phenols by oxidation with tyrosinase, spontaneously react with dienophils.
osinase, immobilized on glass beads, was used to oxidize several phenols in

chloroform as the organic medium. The products of the enzymatic oxidation step,
the ortho-quinones, served in situ as dienes in a Diels-Alder reaction (Fig. 16.3-7).
Table 16.3-4 summarizes some phenols (dienes after enzymatic oxidation) and
dienophiles with which such a reaction cascade was observed.
1178
Table 16.3-3. Oxidations of phenols catalyzed by tyrosinase.

Substrate Product References and remarks
---b Electron-rich phenols are

preferred131
R R OH
R = OCH3, OCzHs, CH3,
-
C(CH3)3,Halogen, etc.
,O/\bNHz (4, 51
HO HO
R = H, CH3
L-DOPA production[6]
HO HO
NH, Possible agent in melanoma

H ° F O H treatment 1'. 8l
HO HO
F F
OH Coumestans L91
OH
S . Passi, M. Nazzaro-Porro, Brit. /. Dematol. 1981, 7 M. E. Rice, B. Moghaddam, C. R. Creveling,

104,659. K.R. Kirk, Anal. Chem. 1987,59, 1534.
M. Jimenez, F. Garcia-Carmona, F. Garcia-Cano- 8 R. S . Phillips, 1.G. Fletscher, R. L. Von Tersch,
vas, 1. L. Iborra, ].A. Lozano, F. Martinez, Arch. K. L. Kirk, Arch. Biochem. Biophys. 1990,276,
Biochem. Biophys. 1984,235,438. 65.
M. Jimenez, F. Garcia-Carmona, F. Garcia-Cano- 9 U.T.Bhalearo, C. Muralikrishna, G. Pandey,
vas, J. L. Iborra, ].A. Lozano, Int. J. Biochem. 1985, Synth. Commun. 1989,19,1303.
17,891. 10 0. Toussaint, K. Lerch, Biochem. 1987,26,
G . M. Carvalho, T. L. M. Alves, D. M. G. Freire, 8567.
Appl. Biochem. Biotech. 2000,84-86,791-800.
By this reaction sequence, highly functionalized bicyclo-[2.2.2]-octenescan be

obtained from simple phenols and alkenes as starting materials. The overall yields
reported are usually satisfactory (> 70%). The Diels-Alder products are racemic,
probably because the Diels-Alder reaction proceeds in the bulk organic phase
without involvement of tyrosinase.
Table 16.3-4. Substrates for the reaction cascade including tyrosinase catalyzed oxidation of
I 1179
ohenols and a Diels-Alder-reaction[ ' l , '*I.

Phenols Dienophiles
Via a tyrosinase catalyzed reaction the phenols are transformed to dienes, which subsequently react with the
dienophiles in a Diels-Alder-reactionas shown in Figure 16.3-7.
11 G. H . Miiller, H. Waldmann, Tetrahedron Lett. 12 G. H . Miiller, A. Lang, D. R. Seithel, H. Wald-

1996,37,3833-3836. mann, Chem. Eur.]. 1998,4,2513-2522.
16.3.3.2
2-Hydroxybiphenyl-3-monooxygenase(HbpA, E. C. 1.14.13.44)
The flavin-dependent,homotetrameric HbpA is the first enzyme in the biodegrada-

tion pathway of 2-hydroxybiphenylin Pseudomonas azelaica HBPl L2'1. HbpA cata-
lyzes the selective ortho-hydroxylation of a broad range of phenols to the correspond-
ing catechols, utilizing NADH as cofactor (Fig. 16.3-8and Table 16.3-5).
Compared to the chemical synthesis of ortho-substitutedcatechols (ortho-hydroxy-
lation and aromatization procedures)[28-311, such an enzymatic approach is superior
with respect to the number of steps involved as well as simplicity, selectivity, and
yield. The resulting ortho-substitutedcatechols are valuable building blocks [321.
HbpA is an excellent example of in vivo as well as in uitro biocatalysis. Since the
desired catechols are rapidly degraded via the D. azelaica rneta-cleavage pathway by
two catechol-2,3-dioxygenases, the gene coding for HbpA was expressed in E. coli
JM109, which served as a biocatalyst accumulating the desired products [331. Draw-
backs such as inhibition by substrate and product can be overcome by continuous
substrate feeding and in situ recovery of the catechol products with solid adsorbents
0 2
NADH NAD'
R = Ph, 2-OH-Ph, 2,3-(OH),Ph, F, CI, Br, Me, Et, Pr, iPr, But
Figure 16.3-8. Reaction scheme for the ortho-hydroxylation o f phenol
derivatives catalyzed by 2-hydroxybiphenyl-3-monooxygenase (HbpA).
1180
I Table 16.3-5. Substrates and relative activities o f 2-hydroxybiphenyl-3-monooxygenase
(HbpA) [131.
Substrate Product Relative activity I"?]"
dp HO
HO
69 HO
100
34
HO OH HO OH
49
Native substrate
OH
24
O
"&
OH
3G
OH
10
OH
Ho$cl 20
HO
& 33
a Relative activities were determined polarographicallywith whole cells of recombinant E. coli containing
HbpA. 100%corresponds to the HbpA-dependent specific oxygen uptake rate of whole cells incubated with
2,2'-dihydroxybiphenyl.
13 A. Schmid, H:P. E. Kohler, K.-H. Engesser,]. Mol. Cat. B: Enzymatic 1998,5,311-316
in such a way that substrate and product concentrations can be kept below toxic
levels [321. Thus, several 3-substituted catechols were produced in gram amounts with
satisfactory to high yields (Table 16.3-6).
The in uivo processes are based on a recombinant E. coli as catalyst[33].Optimized
space-time yields of up to 0.39 g L-' h-' for the formation of 3-phenyl catechol from
2-phenyl phenol can be reached[34].
The enzyme itself was purified and characterized in 351. Based on this
knowledge and via directed evolution, HbpA characteristics were modified (Meyer,
Schmid and Witholt, unpublished results) yielding HbpA variants with improved
7 G.3 Oxidation of Phenols
Table 16.3-6. Preparative-scale production of 3-substituted catechols using E. coli J M l O l

I 1181
containing 2-hydroxybiphenyl-3-rnonooxygena~e~’~].
Product Product recovered 191 Molar yield
Ho& 8.1 94
&
HO
2.1 95
HO OH
0.6 71
HO, ,OH
2.2 77
1.7 85
HO OH
M
0.9 71
2.1 71
14 M. Held, W. Suske, A. Schmid, K. Engesser, H. Kohler, B. Witholt, M. Wubbolts, j.Mol. Cut. B: Enzymatic
1998,5,87-93.
catalytic properties and changed substrate spectrum. For example, a new mutant
with drastically decreased unproductive NADH oxidation and concomitant forma-
tion of hydrogen peroxide was developed. This so-calleduncoupling reaction is quite
common amongst flavin-dependent monooxygenases, and represents the major
mechanism of autoregeneration amongst oxidases. Furthermore, the activity toward
several substrates that are poorly converted by native HbpA, such as 2-sec-butylphe-
no1 (30 % activity increase), 2-tert-butylphenol (fivefold activity increase) or guaiacol
(more than eightfold increase in KM/kCat), could be The HbpA
substrate spectrum could be enlarged even more via directed evolution. Recently, an
HbpA mutant was found that initiated the production of indigo stating from indole.
It is assumed that HbpA converts indole into the 2,3-epoxide,which spontaneously
dimerizes to indigo (Fig. 16.3-9)13’1.
In vitro application of HbpA (and monooxygenases in general) offers some
advantages over whole-cell biotransformations. For example, toxic effects on cell
metabolism and further metabolization of the desired product can be avoided, and
experimentally demanding in vivo set-ups are not necessary (beneficial for organic
chemists). The major challenge in in vitro biotransformations is the efficient
0 2
H
indole
NADH NAD' I
Figure 16.3-9. Proposed reaction sequence

catalyzed by 2-hydroxybiphenyl-3-monooxyge-
H
nase (HbpA) for the formation o f indigo from 0
indole. indigo
- --
m S I ~ Usubstrate JPCKJ
NADH
B
C0,-
-
\ J FDH
HC0,H
cathode
12+
Figure 16.3-10. Formation o f 3-phenylcatechol from 2-phenylphenol catalyzed by partially purified

2-hydroxybiphenyl-3-monooxygenase(HbpA) in organic aqueous emulsions. Regeneration o f
N A D H was achieved in situ with formate dehydrogenase (FDH) (A) or indirectly electrochemically
with [Cp*Rh(bpy)(H20)I2+ (B).
Table 16.3-7. Substrates and products of peroxidase - catalyzed oxidative di- and
I 1183
oligomerizations of phenols.
Substrate Products References and applications
Alkaloid synthesis [ I 5 ]
Me0 OMe
OH
#
OH OH
Alkaloid synthesis [I'

Me0 J$ HO /
OH
OMe
.:WlH Antimicrobial compounds ( I 6 ]
'0H
HO@
HO '
"3 '
\
/
o @
/ Phytoalexin activity~"1
C A
COOCH,
COOCH,
regeneration of reduced nicotinamide coenzymes. The general strategies are de-

scribed in Chapter 7. Furthermore, the production enzyme must be easily available
in large amounts. HbpA was obtained in gram amounts from recombinant E. coli in
a one-step operation via expanded bed adsorption Limitations
1184
Substrate Products References and aDdications
HO
Melanin synthesis [l9I

HO
ti
HO
aoH Racemic ['I
%OH, ' OH
' ,
woH
a OH
Racemic I2O1
Quest Int. Naarden, The

Netherlands, R = arrabinoxy-
lan, carbohydrate gel which
RO RO
retains water
0
Me
15 A. R. Krawczyk, E. Lipkowska, J.T. Wrobel, Coll. 18 A. E. Goodbody, T. Endo, J. Vukovic, J. P. Kutney,

Czech. Chem. Commun. 1991,561147. L. S. L. Choi, M. Misawa, Planta Med. 1988,136.
16 A. Kobayashi, Y. Koguchi, H. Kanzaki, S.I. Ka- 19 M.dlschia, A. Napolitano, K. Tsiakas, G . Prota,
jiyama, K. Kawazu, Biosci. Biotech. Biochem. 1994, Tetrahedron 1990,46,5789.
58, 133. 20 M. M. Schmitt, E. Schiiler, M. Braun, D. Haring,
17 D. M.X. Donelly, F. G. Murphy, J. Polonski, P. Schreier, Tetrahedron Lett. 1998,39,2945-2946.
T. Prang6, J. Chern. Soc. Perkin Trans. 1 1987,2719.
due to low solubility of substrates and products can be overcome in biphasic reaction
systems (Fig. 16.3-10).HbpA exhibits significant activity in the presence of various
organic solvents such as 1-decanol,hexadecane or heptaner3'1.
Thus, the synthetic in vitro application of HbpA was done via an emulsion process.
Several regeneration strategies for NADH were reported (Fig. 16.3-10).
In the emulsion process, a high 3-phenylcatechol concentration in the organic
phase and the same or higher productivities (up to 0.45 g L-' h-') as in the in vivo
process were achievedL4']. Here, formate dehydrogenase and formate served as the
coenzyme regeneration system (Fig. 16.3-10 A). The benefits of this regeneration
I
1185
Hooclf;oH
HO HooI'IOOH
@OH
R
Figure 16.3-11. Hydroxylation of phenols t o catechols catalyzed by

horseradish peroxidase (HRP).
system are described in Chapter 16.6. Even electrical power could be used as a source
of reduction equivalents (Fig. 16.3-10B) l4l].
16.3.4
Peroxidases
16.3.4.1
Oxidative Coupling Reactions
Phenols are typical substrates for peroxidases. Quite similarly to the laccase-
mechanism (described earlier in this chapter), peroxidases catalyze phenol oxida-
tions via hydrogen abstraction. The radicals thus generated leave the active site and
Table 16.3-8. Selected hydroxylation reactions of phenols catalyzed by horseradish peroxidase.
Suberate Product Literature
"&OH
HO
Tyrosine L-Dopa
OH
OH Adrenaline
21 A.M. Klibanov, 2. Berman, B.N. AlbertiJ. Am. Chem. SOC.1981,103,6263-6364.
7 G Oxidation Reactions
1186 I Table 16.3-9. Selected nitration reactions of phenols catalyzed by soybean peroxidase.
Substrate Product(s), Yield I"/.]

ortho nara
O Z N a o H
/ O
*H NO2
OH
58 27
OH OH
I
OH
22 25
0 0
D O H o&oH41
20
yl VNoz
0 OH
25
react with other aromatic compounds (depending on the reaction conditions) to

form dimeric and polymeric products[42].A selection of dimeric products is
presented in Table 16.3-7.
Recently, peroxidases, especially horseradish (HRP) and soybean peroxidase,
found increasing interest in resin manufacturing. The peroxidase-catalyzed coup-
ling of phenols [431, catechols [441, hydroquinones C4'1, or anilines [46, 471 is a potential
substitute for the conventional production of phenolic resins using toxic formal-
dehyde [481. The resins find applications as conductive polymers [45, 4gl.
16.3.4.2
Hydroxylationof Phenols
As early as 1961, Mason and coworkers reported that HRP, in the presence of
dihydrofumaric acid as cofactor, catalyzes the hydroxylation of arenes (Fig. 16.3-
11)'501.
Also lignin peroxidase was found to catalyze the oxidation of phenol, cresol, and
tyrosine ['ll.
Table 16.3-10. Oxidation reactions o f arylamines catalyzed by peroxidases.

I
1187
Substrate Product References and remarks
y 2
Bromoperoxidase1")
Chloroperoxidase [23]
CI CI
Aminopyrrolonitrin Pyrrolonitrin
R 0 R 0 Chloroperoxidase
R = 0-, m-,pC1; pCH3; p-COOH
22 N. Itoh, N. Morinaga, T. Kouzai, Biochem. Mol. 24 V.N. Burd, K:H. van Pee, Bioorg. Khim. 1998,24,
Bid. 1993, 29,785-791. 462-464.
23 S. Kirner, K.-H. van Pee, Angew. Chem. Int. Ed.
1994, 33, 352.
16.3.4.3
Nitration of Phenols
Khmelnitsky and coworkers recently reported a rather unusual application of

soybean peroxidase. In the presence of nitrite and hydrogen peroxide, phenols are
nitrated. The nitration of tyrosine has been reported earlierrS2*s31. The substrate
spectrum was enlarged by various phenolic compounds (Table 16.3-9).Thus, such an
enzymatic nitration represents an alternative to chemical nitration (especially for
acid-labile phenols, which cannot by nitrated chemically).
Other peroxidases such as HRP or CPO were also able to perform such reactions.
Another approach to the production of nitroarenes with peroxidases is based on
the CPO (or bromoperoxidase)-catalyzedoxidation of arylamines. Table 16.3-10 gives
a selection of peroxidase-catalyzedconversions of aniline derivatives to correspond-
ing nitroarenes.
For example, aniline was converted into nitrobenzene by a bromoperoxidase from
Pseudornonas p ~ t i d a ( ' ~ 1and
, aminopyrrolonitrin was converted into the antibiotic
pyrrolonitrin by a CPO from P. p y r r ~ c i n i a [ ~ ~ ] .
1188
Table 16.3-11. Substrates and products of 4-cresol-o~idoreductase~~~~

26].
Substrate Product Substrate Product
6- 4
6 bH 6
OH 0
’ OH 0
’
OH $o / OH
/ @
OH
OH OH OH
OH
25 W. Mclntire, D. J. Hopper, T. P. Singer, Biochem.j. 26 W. Mclntire, D.J. Hopper, J. C. Craig, E.T. Ever-
1985,228,325-335. hart, E.V. Webster, M. J. Causer, T. P. Singer, Bio-
chem.J 1984,224,617-621.
16.3.5
Other Oxidoredudases
16.3.5.1
4-Cresol-oxidoreductase (PCMH, E. C. 1.17.99.1)
This enzyme shares structural and mechanistic properties with VAO[’ll. In contrast
to VAO it is not an oxidase as regeneration of the covalently bound FAD with
molecular oxygen is not possible. It is a flavocytochrome enzyme. The reduction
equivalents from the substrate are transferred to a type c cyto~hrome[’~, 571. In
7 6.3 Oxidation of Phenols
Table 16.3-12. Oxidations of 4-alkylphenols catalyzed by 4-ethylphenol 0xidoreductase1*~].
Substrate Relative conversion rate [%r

p-Cresol 44
4-Ethylphenol 100
4-Propylphenol 112
4-Butylphenol 114
4-Pentylphenol 116
4-Heptylphenol 52
4-Nonylphenol 14
a 100% corresponds to the 4-ethylphenol conversion rate.
27 C. D. Reeve, M.A. Carver, D. J. Hopper, Biochem.J. 1990,269,815-819
addition to a cytochrome c / cytochrome c oxidase regeneration sy~tem['~1, chemical

reoxidation agents such as phenazine methosulfate, dichlorophenol indophenol[57],
and ferrocenes[6G621 have been used.
The reaction mechanism is quite similar to the one of VAO and also includes an
intermediate, the para-quinone methide. Like VAO, 4-cresol-oxidoreductase also
exhibits a high enantioselectivity for (S)-l-(4'-hydroxyphenyl)alkylalcohols
["I.
This enzyme accepts a broad range of substrates; para-methylphenols are prefera-
bly oxidized to the corresponding aldehydes, whereas the oxidation of para-alkylphe-
nols results in the formation of significant amounts of (S)-alcohols (Table
16.3-11) 631.
16.3.5.2
4-Ethylphenol Oxidoreductase
4-Ethylphenol oxidoreductase from Pseudomonas putida JD1 is structurally almost

identical to 4-cresol oxidoreductase,but catalyzes the hydroxylation of para-alkylphe-
nols with longer aliphatic chains (Table 16.3-12). The hydroxylation reactions
enantioselectively produce (R)-alcohols[64, "1. The regeneration properties of this
enzyme are quite similar to 4-cresol oxidoreductaselG11.
1190
I
2-aminotetralines 9-hydroxy N-(n-propyl) hexahydronaphthoxazine

Figure 16.3-12. Substrates for phenol oxidase from Mucuna pruriens.
5-, 6-, or 7-Hydroxylated 2-aminotetralins with R = H or C,H7 and
9-hydroxy-N-(n-propyl)-hexahydronaphthoxazine are substrates for the
phenol oxidase.
Figure 16.3-13. Formation o f 7,g-dihydroxy N-(di-n-propyl)-2-aminotetralin

with Mucuna-phenoloxidase. Quinone formation is prevented in situ with
ascorbate as reductant.
16.3.6
In vivo Oxidations
16.3.6.1
Phenoloxidase of Mucuna pruriens
Like other phenoloxidases, this enzyme has a low substrate specificity and is able to
ortho-hydroxylate a whole range of para-substituted monocyclic phenols. The cate-
chols produced belong to groups of fine chemicals and pharmaceuticals["]. Fur-
thermore, also bi- and tri-cyclic phenols were converted into catechols (Figurel6.3-
12) [G71. 2-Aminotetralines, on the basis of their dopaminergic properties, are com-
pounds of pharmaceutical interest.
Phenoloxidase (monophenol monooxygenase, E. C. 1.14.18.1) introduces one
atom of molecular oxygen into the substrate and was used in alginate-entrappedcells
or in partially purified form. The pharmaceutical 7,8-dihydroxy-N-(di-n-propyl)-
2-aminotetralin was produced continuously using a phenol oxidase suspension in
dialysis tubing in an airlift fermenter coupled to an aluminium oxide column for
selective product isolation (Figure 16.3-13)["j. A product concentration of 130 mg/L
and a yield of 25 % were reached.
I
1191
J3
aoycooH“y
(R)-2-phenoxypropionic acid
0, I
Beauveria bassiana
HO
(R)-2-(4-hydroxyphenoxy)propionic acid
Figure 16.3-14. Regioseledive para-hydroxylation o f (R)-2-phenoxypropionic

acid catalyzed by Beauveria bassiana (HPOPS process).
16.3.6.2
Monohydroxylation of (R)-2-PhenoxypropionicAcid and Similar Substrates[69.701
The product is a frequently used intermediate for the synthesis of enantiomerically

pure aryloxyphenoxypropionic acid type herbicides. The enzyme catalyzing the
hydroxylation of the phenolether is an oxidase, which is not further characterized.
The biocatalyst Beauvena bassiana was found by an extensive screening of micro-
organisms for regioselective hydroxylation of (R)-2-phenoxypropionicacid and for
substrate tolerance. This fungal strain was improved by random mutagenesis and
screening, which resulted in strain LU 700. The hydroxylation is not growth-
associated and the ee is increased during oxidation from 96% for the substrate to
98% for the product. After process optimization, a productivity of 7 g L-l d-’ was
reached. The biotransformation is carried out in a 120 000 L reactor at BASF in
Germany.
The biocatalyst has a broad substrate spectrum. A compound needs the structural
elements of a carboxylic acid and an aromatic ring system to be a substrate for the
oxidase. Hydroxylation primarily takes place at the para position if it is free. If an
alkyl group is in the para position, only the side chain is oxidized. In systems with
more than one ring, the most electron-rich ring is hydroxylated.
16.3.6.3
Biotransformation of Eugenol to Vanillin L7’]
The biotechnological production of vanillin is of interest because there is a large

demand for vanillin originating from so called “natural” sources. Possible strategies
for the biotechnological production of vanillin are reviewed by Priefert et al. [72].
One synthetically interesting strategy is the production of vanillin from eugenol.
Here, a part of a catabolic pathway is used to accumulate an intermediate of this
pathway. This was achieved by the knock-out of the enzyme catalyzing the further
conversion of the putative product.
For the accumulation of vanillin from eugenol, the catabolism of eugenol in
Pseudornonas sp. Strain HR199 (DSM7063) was used. In order to prevent further
degradation of vanillin, the gene enconding vanillin dehydrogenase, responsible for
the oxidation of vanillin to vanillic acid, was inactivated by insertion mutagenesis.
In a non-optimized biotransformation using growing cells in an aqueous mineral
salts medium containing gluconate as a source of carbon and energy and 6.5 mM
eugenol, vanillin accumulated up to a concentration of 2.9 mM, corresponding to a
FoMe
I
qoHqo
1192
hydroxylas:
eugenol ~$$;Yla~ L
/
OMe OMe
I
OH OH OH
eugenol coniferyl alcohol coniferyl aldehyde
coniferyl aldehyde
qo
dehydrogenase
0
%
I enovl-CoA-
hydratase/aldolase F r o A feruloyCCoA-
synthetase
0 M ; T - ' OMe OH
OMe
OH acetyl-CoA OH
vanillin feruloyl-CoA ferulic acid
Figure 16.3-15. Multistep biotransformation of eugenol to vanillin catalyzed
by whole cells o f Pseudomonas sp. HR 199.
molar yield of 44.6%. The major drawback of the process is the degradation of
vanillin by the action of coniferyl aldehyde dehydrogenase when coniferyl aldehyde
is depleted from the medium.
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37 A. Schmid, 2001. T. P. Singer, Biochem.]. 1985, 228, 337-345.
38 J. Lutz, B. Krummenacher, B. Witholt, A. 58 W. McIntire, C. Bohmont. In de Gruyter,
Schmid. "2-Hydroxybiphenyl3-Monooxyge- Havins and Havoproteins, Berlin, 1987, pp.
nase: Large Scale Preparation and Cell Free 677-686.
1194
59
W. McIntire, D. J. Hopper, J. C. Craig, E. T. 67N. Pras, G. E. Booi, D. Dijkstra, A. S. Horn,

Everhart, E.V. Webster, M. J. Causer, T. P. T. M. Malingre, Plant Cell, Tissue and Organ
Singer, Biochem.J. 1984,224,G17-621. Culture 1990,21,9-15.
60 H. A. 0. Hill, B. N. Oliver, D. J.Page, D. J. 68 N. Pras, S. Batterman, D. Dijkstra, A. S.
Hopper, J. Chem. SOC.,Chem. Commun. Horn, T. M. Malingre, Plant Cell, Tissue and
1985,1469-1471. Organ Culture 1990,23,209-215.
61 B. Brielbeck, M. Frede, E. Steckhan, Bio- 69 C. Dingler, W. Ladner, G. A. fiei, B. Cooper,
catalysis 1994,10,49-G4. B. Hauer, Pesticide Science 1996,4G,33-
62 E. Steckhan, Electroenzymatic Synthesis. In 35.
Top. Curr. Chem.; Springer-Verlag:Berlin; 70 B. Cooper, W. Ladner, B. Hauer, H. Siegel.
Heidelberg, 1994;Vol. 170;pp. 84-111. Verfahren zur fermentativen Herstellung
63 W. McIntire, D. J. Hopper, T. P. Singer, Bio- von 2-(4-hydroxyphenoxy-)propionsaure,
chem.]. 1985,228,325-335. 1992,EP0465494Bl.
64 C. D. Reeve, M. A. Carver, D. J. Hopper, Bio- 71 J.Overhage, H. Priefert, J. Rabenhorst, A.
chem. J. 1989,263,431-437. Steinbiichel, Appl. Microbiol. Biotech. 1996,
65 C. D. Reeve, M . A. Carver, D. J. Hopper, Bio- 52,820-828
chem. J . 1990,269,815-819. 72 H. Priefert, J. Rabenhorst, A. Steinbiichel,
66 N. Pras, H. J. Wichers, A. P. Bruins, T. M. Appl. Microbiol. Biotech. 2001,56, 296-314.
Malingre, Plant Cell, Tissue and Organ Cul-
ture 1988,13,15-26.
16.4
Oxidation of Aldehydes
16.4.1
Introduction
To date, few reports on synthetic enzymatic oxidations of aldehydes have been

published. Preparative applications reported include bioconversions of natural
products such as retinal (Fig. 16.4-1 A) and various aliphatic and unsaturated
aldehydes (Fig. 16.4-1 B). A broad range of aromatic acids can be obtained from their
corresponding aldehydes (Fig. 16.4-1 C). Another reported reaction type is the
production of olefins from aldehydes by oxidative removal of formic acid from the
substrate (Fig. 16.4-1 D).
16.4.2
Alcohol Dehydrogenases
Alcohol dehydrogenases are generally applied for the interconversion of alcohols and
aldehydes. Yet, these enzymes have also attracted interest due to their ability to
oxidize aldehydes[lI. HLADH was shown to oxidize butanal12]. This reaction,
however, shows no potential for synthetic application unless a very efElcient NAD'
regeneration system is applied (Fig. 16.4-2).The catalyhc activity of HLADH for the
reduction of the aldehyde is more than 100 times higher than that for aldehyde
oxidation (examined for benzaldehyde)f31. As a result, the initially formed NADH is
76.4 Oxidation ofAldehydes
11195
RAOH
Figure 16.4-1. Selected enzymatic oxidations o f aldehydes. A oxidation o f

complex natural products such as retinal; 6:oxidation of aliphatic and
*
a$-unsaturated aldehydes; C: oxidation o f (hetero)arylic aldehydes; D: oxidative
cleavage of the aldehyde-carbon atom yielding terminal alkenes.
-OH NAD' fi- NADH O

' NADH NAD+
Figure 16.4-2. Oxidation activity for aldehydes exhibited by horse liver alcohol
dehydrogenase (HLADH). Only minor amounts of acid are produced because of the
higher HLADH activity for aldehyde reduction.
-0 + HO
, JOH 0-
TBADH * NAD+ TBADH * NADH
/\OH
Figure 16.4-3. Aldehyde dismutase acitivity of Thermoanaerobium brockii alcohol
dehydrogenase (TBADH). A high affinity o f the TBADH-NAD' complex for hydrated
acetaldehyde is proposed, explaining the stochiometric acetaldehyde dismutation.
7G
1196
used for aldehyde reduction, yielding a dynamic equilibrium between alcohol and
aldehyde.
TBADH also exhibits the so-called aldehyde dismutase activity[41.In contrast to
HLADH, stochiometric dismutation of acetaldehyde into one equivalent of ethanol
and acetic acid has been reported. A gem-diol mechanism was proposed for this
reaction (Fig. 16.4-3).
16.4.3
Aldehyde Dehydrogenases
Several aldehyde dehydrogenases have been reported for biocatalytic applications.

Recently, aldehyde dehydrogenase (E. C. 1.2.1.5) from yeast was applied to oxidize
(Z,Z)-nona-2,4-diena1[’1. Recycling of NAD’ was achieved in situ by addition of an
alcohol dehydrogenase, reducing (Z,Z)-nona-2,4-dienalto the corresponding alco-
hol. Since both reactions are stochiometrically linked via NAD, this corresponds to
an overall dismutation of the aldehyde (Fig. 16.4-4).This concept was extended to
industrially relevant metabolites of linoleic acid (detergents and polymer building-
blocks) (Fig. 16.4-5). No isomerization of the double bonds and yields up to 90%
were reported[’].
Figure 16.4-4. Enzy-
A
matic transformation of
(Z,Z)-nona-2,4-dienal t o
the corresponding alco-
hol and acid catalyzed by
/AD? NADH
NAhidDH
\ an alcohol and an alde-
hyd e d ehyd rogenase
from yeast.
OH
I /-
lipoxygenase
H
y
&
&
I
hydroperoxide lyase
O\J
AldDH A \“.‘Am
C
o*Oo* H
C
>
o
O
OH
Figure 16.4-5. Enzymatic cleavage o f linoleic acid t o o-hydroxy

and dicarboxylic acids.
16.4 Oxidation of Aldehydes
I 1197
Table 16.41. Kinetic constants o f bovine kidney aldehyde dehydrogenase for different
substrates 1’1.
Substrate Vm, [%I” KM bM1
100 9.1
758 1
A
H
-0 855 1.5
-
0 1960 30
1683 33.9
3026 8.2
a The V, values are relative to retinal as substrate.
1 P. V. P. Bhat, L., Wang, X. L., Biochem. Cell Bid. 1996.74,695-700.
Figure 16.4-6. Mechanism proposed for light emission in the course o f the
luciferase reaction.
Another NAD’-dependent aldehyde dehydrogenase (from bovine kidney) was

characterized with respect to its activity toward retinal and other aldehydes (Table
16.4-1)[GI.
1198
I Oxidation of aldehydes t o corresponding carboxylic acids catalyzed by P450
Table 16.4-2.
rnonooxygenases.
0
R
H' *- RKOH
0,, NAD(P)H H,O, NAD(P)*
Substrate Reference
Aliphatic aldehydes ~ 3 1
[31
RA H
&
0
Losartan
~~~
\
171
2 Y. Terelius, C. Norsten-Hoog, T. Cronholm. M. In- 5 K. Watanabe, T. Matsunaga, I. Yamamoto, H. Ya-
gelman-Sundberg, Biochem. Biophys. k s . Commun. shimura, Drug. Metab. Dispos. 1995, 23, 261-265.
1991, 179,689-694. G K. Watanabe, S . Narimatsu, T Matsunaga, I. Yama-
3 K. Watanabe, T. Matsunaga, S. Narimatsu, 1. Yama- moto, H. Yoshura, Biochem. Qhamacol. 1993,46,
moto, H. Yoshimura, Biochem. Biophys. Res. Com- 405-41 1.
mun. 1992,188, 114-119. 7 R. A. Steams, P. K. Chakravarty, R. Chen,
4 S. Tomita, M. Tsujita, Y. Matsuo, T. Yubisui, Y. S.-H. L. Chiu, Drug. Metab. Dispos. 1995, 23,
Chikawa, 1nt.J. Biochem. 1993,25,1775-1754. 207-215.
16.4.4
Monooxygenases
16.4.4.1
Luciferase (E.C. 1.14.14.3)
Probably the most prominent oxidation reaction of aldehydes is the well-known

luciferase reaction. The flavin-dependentluciferase is present in a number of marine
and terrestrial species[" 1'. Light of about 490 nm (blue-green) is emitted as a by-
16.4 Oxidation ofAldehydes
Table 16.43. Oxidations and subsequent decarboxylations of aldehydes catalyzed by P450

I
1199
monooxygenases.
NAD(P)H. 0, NAD(P)+,H,O
Substrate Reference
8 E. S. Roberts, A. D. N. Vaz, M . J. Coon, Proc. Natl. 10 A. D. N. Vaz, K. J. Kessel, M. J. Coon, Biochern

Acad. Sci USA 1991,88,8963-8966. 1994,33,13651-13661.
9 A. D. N. Vaz, E. S. Roberts, M. 1. Coon,]. Am.
Chem. Soc. 1991,113, 5886-5887.
product of the oxidation of aliphatic aldehydes. Excited flavin species are discussed
as emitters (Fig. 16.4-6)1'- lo].
16.4.4.2
Cytochrome P 4 5 0 ~ ~ . 3
The oxidation of an aldehyde to the corresponding carboxylic acid with P450 systems
is reported for various substrates (Table 16.4-2).In some cases oxidative decarboxyla-
tion is observed yielding formic acid and an olefin, one carbon atom shorter than the
substrate (Table 16.4-3).
Several o-0x0 fatty acids are transformed to the corresponding a,w -dicarboxylic
acids, whereas o-formylesters of fatty acids are decarboxylated to the o-hydroxy fatty
acids and carbon dioxide["]. For several w-0x0 fatty acids turnover frequencies
(measured as O2consumption) between 1.8 to 25 s-l were found. Many P450 systems
are multi-component enzymes with small protein cofactors such as putidaredoxin
performing the electron mediation between NAD(P)H and the active site of the
enzyme. Vilker and coworkers recently were able to show that NADPH can be
omitted from the catalyhc cycle by direct electrochemical reduction of putidar-
7G
1200
Table 16.4-4. Kinetic constants ofxanthine oxidase"'].
Substrate KM [mM] vmax Is-']
141.5 22.2
130 100
A H
430 23.3
142 2.4
J H
0.34 3.4
0.046 2.7
0
1.7 4.2
1.03 7.7
0.068 15.7
0.085 1.8
1 1
2 0.1
11 F. F. Morpeth, Biochim. Biophys. Acta 1983,744,328-334.
edo~in['~-'~I, thus oxidizing styrene or camphor. Other approaches utilize Co

sepulchrate as reducing agent, which can be regenerated either chemically (via
Zn) ['I or electrochemically['G.l71.
References I1201
16.4.5
Oxidases
16.4.5.1
Xanthine Oxidase (E.C. 1.1.3.22)
Xanthine oxidase was examined for its catalyhc applicability for the oxidation of
aldehydes as early as 196711*].In addition to 02,xanthine oxidase was reported to
accept e. g. methylene blue, PMS or ferricyanide[”I as electron acceptors. Table
16.4-4gives kinetic data for some substrates L2O].
16.4.6
Oxidations with Intact Microbial Cells[*’]
Burkholderia cepacia was reported to transform aromatic aldehydes into the corre-
sponding acids. Vanillin, para-hydroxybenzaldehyde,and syringaldehyde were con-
verted to corresponding acids with high yields of 94%, 92 %, and 72 %, respectively
(Fig. 16.4-7)[22].
The acid produced is not further metabolized as long as the aldehyde still is
accessible to the cells. The enzyme responsible for aldehyde oxidation in Burk-
holderia cepacia was not further characterized. However, the gene of an NAD-
dependent vanillin dehydrogenase of Pseudomonas sp. strain HR199 was cloned and
characterized[23].Recombinant E. coli containing this vanillin dehydrogenase trans-
formed vanillin to vanillate at a clearly higher rate than Burkholderia cepacia.
Burkholderia
R cepacia *
Figure 16.4-7. Oxidation of aromatic

aldehydes by Barkholderia cepacia TM1.
References
1 L. P. Olson, J. Luo, 0. Almarsson, 4 S. Trivic, V. Leskova, G. W. Winston, Biotech.

T. C. Bruice, Biochemistry 1996, 35, Lett. 1999, 21, 231-234.
9782-9791. 5 A. Nunez, T. A. Foglia, G. J. Piazza, Bio-
2 G. T. M. Henehan, N. J. Oppenheimer, Bio- technol. Appl. Biochem. 1999, 29, 207-212.
chemistry 1993, 32, 735-738. 6 P. V. Bhat, L. Poissant, X. L. Wang, Biochem.
3 G. L. Shearer, K. Kim, K. M. Lee, C. K. Cell Bid. 1996,74,695-700.
Wang, B. V. Plapp, Biochemistry 1993, 32, 7 T. 0. Baldwin, M. M. Ziegler. In Chemistry
1118611194. and Biochemistry of Flavoenzymes, CRC
1202
Press, Boca Raton, 1992, Vol. 111, pp. 16 R. W. Estabrook, K. M. Faulkner, M. Shet,
467-530. C. W. Fisher, Application of Electrochem-
8 A. Palfey, V. Massey, Flavin-Dependent En- istry for P450-CatalyzedReactions. I n Meth-
zymes. I n ComprehensiveBiological Catalysis. ods in Enzymology, Academic Press. San Di-
M. Sinnott (ed),Academic Press, San Di- ego, London, Boston, New York, Sydney,
ego, London, 1998, Vol. 111, pp. 83-154. Tokyo, Toronto, 1996, Vol. 272, pp. 44-51.
9 C. T. Walsh, Y.C.J. Chen, Angav. Chem. 17 K. M. Faulkner, M. S. Shet, C. W. Fisher,
1988,100,342-352. R. W. Estabrook, PNAS 1995,92,
10 P. Macheroux. S. Gishla, Nachr. Chem. Tech. 7705-7709.
Lab. 1985, 33, 785. 18 F. Dastoli, S. Price, Arch. Biochem. Biophys.
11 S. C. Davis, 2. Sui, J. A. Peterson, P. R. Or- 1967, 118,163-165.
tiz de Montellano, Arch. Biochem. Biophys. 19 G. Pelsey, A. M. Klibanov, Biochim. Biophys.
1996,328, 35-42. Acta 1983,742,352-357.
12 M. P. Mayhew, V. Reipa, M. J. Holden, V. L. 20 F. F. Morpeth, Biochim. Biophys. Acta 1983,
Vilker, Biotechnol. Prog. 2000, 16, 610-616. 744, 328-334.
13 V. Reipa, M. Mayhew, V. L. Vilker, PNAS 21 M. Tanaka, Y. Hirokane, /. Biosci. Bioeng.
1997,94, 13554-13558. 2000,90, 341-343.
14 V. L. R. Vilker, Vytas; Mayhew, Martin; Hol- 22 S. Adachi, M. Tanimoto, M. Tanaka, R. Mat-
den, Marcia J.,/. Am. Oil Chem. SOC.1999, suno, Chem. Eng./. 1992,49, B17-B21.
76,1283-1289. 23 H. Driefert, J. Rabenhorst, A. Steinbiichel,
15 U. Schwaneberg, D. Appel, J. Schmitt, R. D. 1. Bacteriol. 1997, 179, 2595-2607.
Schmid,/. Biotech. 2000,84, 249-257.
16.5
Baeyer-VilligerOxidations
Sabine Flitsch and Cideon Crogan
16.5.1
Introduction
The enzymatic Baeyer-Villiger oxidation continues to receive attention from syn-

thetic organic chemists a s it offers advantages of regio- and enantioselectivity still
rarely exhibited by reagents such a s meta-chloroperbenzoic acid (m-CPBA). S o m e
recent advances have resulted in abiotic catalytic reagents capable of i n d u c i n g
modest enantioselectivity in the Baeyer-Villiger reaction but these reactions are
outside the scope of this section. The most encouraging examples of enantioselective
Baeyer-Villiger reactions a r e still those catalyzed by microorganisms and enzymes
and the extensive research in this area over the last decade has been covered in a
number of recent reviews[”’).
16.5.1.1
Steroidal Substrates
I t h a d been known for m a n y years t h a t Baeyer-Villiger-type processes occur during

the catabolic transformations of natural compounds. In 1953, it w a s described that
the C17 side chain of steroids c a n be cleaved by several microorganisms including
16.5 Baeyer-Vdliger Oxidations
Fusarium, Penicillium, Cylindrocarpon, Aspergillus and Gliocladium speciesf8-'']. One

example reported was the conversion of progesterone into A'*4-androstadien-3,17-di-
one in 84% yield as illustrated in Fig. 16.5-1 181.
Since these reports, many others describing the microbiological Baeyer-Villiger
oxidation of various steroids have been publi~hed[~~-'~I. Interestingly, it has been
shown that depending on the microbial strain used, further oxidation may occur
leading to incorporation (of an oxygen atom into the D-ring, thus affording the
corresponding lactone. In general, these oxidations are restricted to this ring. This
selectivity may be due to the fact that the A-ring bears an a, P-unsaturated ketone
moiety, which appears to display a different reactivity compared with the other
carbonyl functions [l51. Introduction of a A' double bond also often occurs during
these processes. Other eicamples involving oxidation of the A ring have been
described with a Glomerellujkaroides strain[lGland with Gymnoascus r e e ~ i i l ~Thus,
~].
eburicoic acid affords a 30% yield of A-secoacid whereas the steroidal alkaloid
tomatidine leads to the corresponding ketone as the major product, but a smaller
amount of A-seco acid is also obtained. This could well be due to hydrolysis of the
lactone which would be formed from Baeyer-Villiger oxidation of the parent ketone
Fig. 16.5-2.
The mechanism of these reactions has been studied by several groups. Fonken and
coworkers[18]first showed using 21-14C labelled progesterone, that the testosterone
acetate formed during degradation of progesterone by Cladosporium resinae is not an
artefact but is indeed an intermediate in the degradation pathway. Further work by
Prairie and Talalay['9]using the strain Penicillium liliacinum established the involve-
ment of two enzymes, a 6.l-dehydrogenaseand an NADPH-dependent oxygenase.
They also showed that I8O2 molecular oxygen is incorporated as the ring oxygen
atom of testololactone. Rahim and Sih[20]succeeded in showing that an oxygenase
(requiring the presence of oxygen) as well as an esterase were involved in the
degradation of the progesterone side-chain. In other studies using the 17a-labelled
substrate, Singh and Rahkit[21]showed that retention of the deuterium label at the
C17 position occurs and that the molecular oxygen is incorporated into the product
(Fig. 16.5-3). More recently, a gene from Rhodococcus rhodochrous has been cloned
and expressed[22],which encodes for a steroid monooxygenase that inserts an atom
of oxygen between the C15 and C20 carbons of progesterone, forming testosterone
acetate.
Fusarium sp.
progesterone A1~4-androstadien-3,17-One
Figure 16.5-1. Biotransformation of progesterone using Fusarium spp.

1204
Glornerella
fusaroides
HO
*
eburicoic acid 30%
~ Gyrnnoascus reesii H 0 2 C e
HO
Figure 16.5-2. A-ring cleavage by Glornerellafusaroides and Cyrnnoascus reesii.
Figure 16.5-3. Retention of the deuterium label and oxygen incorporation during
the side-chain degradation of progesterone.
All these results led to the conclusion that a process similar to the Baeyer-Villiger
oxidation must occur during these degradations. The general scheme for the
formation of testololactone from progesterone can thus be described, as shown in
Fig. 16.5-4. It involves four successive steps; first a Baeyer-Villiger oxidation of the
steroid sidechain leading to a testosterone acetate, secondly an esterase hydrolysis,
thirdly oxidation of the C17 hydroxyl leading to the corresponding 3,17-dione and
finally a second Baeyer-Villiger oxidation of this diketone at the D-ring leading to the
corresponding 8-lactone. It has been shown in the fungus Cylindrocarpon radicicola
that one bifunctional enzyme is involved in these transformations, which is able to
catalyze oxygenative esterification of 20-ketosteroids as well as oxygenative lactonisa-
tion of 17-ketosteroid~[~~~ 241. It is noteworthy that all the above investigations into
steroid substrates for lactonization were conducted on single enantiomers and thus,
no reference to the enantioselectivity of the processes had been recorded.
I
16.5 Baeyer-ViUiger Oxidations 1205
L O I PAC
0 4 -& progesterone
0
testosterone acetate
I
androstenedione testosterone
testololactone
Figure 16.5-4. Mechanism of the biotransformation of progesterone
into testololactone.
16.5.1.2
Aliphatic Substrates
Baeyer-Villiger oxidation has also been reported for aliphatic ketones. Several strains
able to grow on various aliphatic or alicyclic substrates have been isolated, and it has
been shown that their degradation often involves a Baeyer-Villiger oxidation. For
example, it has beeen observed that Pseudomonas multivorans, Pseudomonas aerugi-
nosa, Pseudomonas cepacia and Nocardia sp. are able to grow on tridecan-
2-one P-281.
Forney and Markovetz isolated undecyl acetate directly from growing cultures of
Pseudomonas aeruginosa. They showed that all early intermediates in the pathway
arise biologically and sequentiallyfrom their precursors, indicating involvement of a
Baeyer-Villiger type oxidation. In a further study they also showed that cell-free
1206
tridecanone
J. undecyl acetate
-0
O
H- undecanol
undecanoic acid
- 0 undecyl undecanoate
Figure 16.5-5. Degradation of tridecan-2-one with a crude cell-free preparation

from a Pseudomonas aeruginosa strain.
preparations obtained from methylketone grown Pseudomonas aeruginosa, when

supplemented with NADH or NADPH in the presence of 0 2 , carry out a reaction
sequence visualized in Fig. 16.5-5.
Using Pseudomonas cepacia grown on tridecan-&one, Markovetz and coworkers[28]
later showed that experiments conducted with I8O2 led to 84% incorporation of
into the C - 0 - C linkage, rather than into the carbonyl function, indicating the
occurrence of a Baeyer-Villiger type process. They also observed that the undecyl
esterase involved in the degradation process is able to hydrolyze both aliphatic and
aromatic acetate esters. They also reported that this enzyme is strongly inhibited by
organophosphates such as tetraethylpyrophosphate (TEPP), as well as by other
esterase inhibitors like p-chloromercuribenzoate1271.
A similar degradation pathway was described for oxidation of tetradecane and
1-tetradecene with Penicillium sp. L2’I. Similar mechanisms were proposed for the
degradation of other aliphatic substrates such as butan-2-one[281, acetol l3O1, acet-
ophenone 13’1 and l-phenylethan~lr~~]. Interestingly,cell extracts of Nocardia sp. LSU
169 grown on butan-2-onewere also shown to be capable of oxidizing tridecan-2-one.
Generally,the Baeyer-Villiger reaction was followed by an esterase catalyzed hydroly-
sis [331.
16.5 Baeyer-Villiger Oxidations
Figure 16.5-6.
-&
Degradation of Z-heptyl-
Pseudomonas sp.
w,, cyclopentanone by a
Pseudomonas sp.
C7H,,
5%
16.5.1.3
Alicyclic Substrates
Baeyer-Villiger oxidation is also a common feature during the catabolic degradation

of a variety of other compounds, including monocyclic, bicyclic or polycyclic
molecules. For monocyclic compounds, one of the first reports describing formation
of a lactone from racemic a-substituted cyclopentanone by various Pseudomonas sp.
was by This could be regarded as the first indication that these reactions
were to prove of interest for asymmetric synthesis since the lactone product
displayed some optical activity (Fig. 16.5-6).
Further studies showed that other substrates such as cyclopentanolr3’1, cyclohex-
ane [36391, cyclohexanol [40-421, cyclohexan-l,2-diol[43-451, cycloheptanone [461 and,
more recently, cycl~dodecane[~~] were degraded via analogous pathways. These were
studied using bacterial strains including Pseudomonas sp. NCIMB 9872 L3’, *I,
Nocardia globerula CL1 14’1, Acinetobacter TD 63 [431, Acinetobacter calcoaceticus NCIMB
9871L3’1, Xunthobacter sp. L3’1 and Rhodococcus
All these degradation pathways were shown to involve a Baeyer-Villiger oxidation
of a cycloalkanone that led to formation of the corresponding lactone. Further
degradation then occured via hydrolysis of this lactone by a lactone hydrolase which
has, in some cases, been isolated. As an example, the reaction sequence for the
degradation of cyclopentanol by Pseudomonas sp. NCIMB 987213’] is shown in
Fig. 16.5-7.
A pathway for the degradation of (-)-menthol and menthane-3,4-diol by a
bacterium classified as a Rhodococcus sp. was proposed by Shukla and coworkers.
Again, the proposed scheme involves formation of the corresponding lactone by a
Baeyer-Villiger process[49].Interestingly, an identical process has been shown to
occur in the degradative pathway of menthol and menthone in peppermint (Mentha
piperita) rhizomes .I‘’[ Rhodococcus erythropolis DCL 14[”] has also been reported to
degrade menthone in addition to 1-hydroxy-2-0x0-limonene and dihydrocarvone via
an enzymatic Baeyer-Villiger reaction.
Some other monocyclic compounds bearing ketonic side chains have also been
shown to undergo degradation processes involving Baeyer-Villiger type oxidation.
For example, oxidation of p-ionone by Lasioplodiu the~bromae[’~] affords, among
other products, the alcohols shown in Fig. 16.5-8.In this case, the loss of two carbons
from the sidechain has been attributed to a contribution of Baeyer-Villiger oxidation
followed by ester hydrolysis and reduction.
Similar results were described by Nespiak and coworkers in the course of their
study of cyclopentyl ketones by Acremonium roseum (Fig. 16.5-9).When R = CH3 or
1208
NA< y:
cyclopentanol
dehydrogenase
NADPH
cyclopentanone oxygenase
NADP’
valerolactone
?OH hydrolase
CH,OH 4T
H-0
L
hydroxyvalerate dehydrogenase
and oxovalerate dehydrogenase
-
NADPH
(CH)--CozH Acetyl-CoA
3 \ ~ ~ 2 ~
Figure 16.5-7. Reaction sequence for the oxidation o f cyclopentanol by Pseudomonas

sp. NCIMB 9872.
C2H5, the alcohol formed via Baeyer-Villiger oxidation and ester hydrolysis was the
only product isolated after 2 days. However, higher esters (R = n-or i-C3H7, R = n-
butyl) have also, if not predominantly, some amount of allylic oxidation product. In
this study, it was shown that the (S)-enantiomer of the substrate methyl ester was
oxidized more rapidly than the (R)-isomer and that the reaction proceeded with
retention of configuration at the chiral center. Thus, by using short incubation times
(2 days) the racemic substrate led to the (S)-alcohol,but the optical purity was low
(around 20 %). Although interesting, this apparent enantioselectivity could also be
due eventually to an enantioselectivehydrolysis of the intermediate ester or to some
other catabolic pathway. However, the butyl ketone led to the (R)-alcoholshowing
100% optical purity.
An extensive study by Fuganti and coworkers [54-571 showed that the metabolism of
4-(4-hydroxyphenyl)butan-2-one (“raspberryketone”) by the fungus Beauveria bassi-
ana unexpectedly yielded tyrosol, through insertion of oxygen via a Baeyer-Villiger
reaction and subsequent acetate (Fig. 16.5-10).Only a narrow range of
1 6 5 Baeyer-Villiger Oxidations 11209
S-ionone
esterase
koH
Figure 16.5-8.
--&?
reductase
Biotransformation o f 6-ionone by Lasiodiploida theobromae.
Acremonium roseum
R alcohol
Me (+)-s o.p.= 20%
Bu (-)-R O.P. = 100%
Figure 16.5-9. Transformation of cyclopentyl ketones by Acremonium roseum.
substrates was converted in this manner, The authors were able to show
via deuterium incorporation experiments, that the configuration of the migrating
carbon-carbon bond was retainedIS61,this being a defining characteristic of the
peracid-catalyzedBaeyer-Villiger process.
Camphor and its analogs are the most studied bicyclic substrates for the biological
Baeyer-Villiger r e a c t i ~ n [ ~It has
~ ~ been
~ ] . shown by Gunsalus and coworkers that, in
the early steps of D-(+)-camphoroxidation by Pseudomonas putida C1, both alicyclic
rings are cleaved by lactonization reactions: Thus, the conversion of (+)-camphorto
5-keto-1,Zcampholide involves three reactions; hydroxylation, oxidation and lactoni-
zation. Using a different Pseudomonas strain, the non-hydroxylated campholide has
been isolated, suggesting that in this case lactonization occurs prior to hydroxyla-
t i ~ n [ ~ *ItI was
. also shown, by analysis of extracted metabolites, that an analogous,
enantiocomplementary pathway existed for the metabolism of L-(-)-camphor. Sev-
eral further studies have been devoted to clarifylng these steps. Interestingly, it has
been shown that P. putida does not express lactone hydrolases that are active towards
1210
Beauveria bassiana
ATCC 71 59
HO
raspberry ketone
I
tyrosol
Figure 16.5-10. Biotransformation of raspberry ketone to tyrosol by Beauveria
bassiana ATCC 7159.
the lactone intermediate. The intermediate bicyclic lactone is unstable under

reaction conditions, and spontaneously opens to a cyclopentenone. This is then
again oxidized via a Baeyer-Villiger reaction to the corresponding lactone. The
degradation ofthe enantiomers of camphor is shown in Fig. 16.5-11.Three enzymes
catalyzing the Baeyer-Villiger reaction, i. e. 2,s-diketocamphane 1,2-monooxygenase
[which forms the bicyclic lactone analog of (+)-camphor][641, 3,G-diketocamphane
1,G-monooxygenase[which forms the bicyclic lactone analog of (-)-camphor]16'1 and
2-0x0-A3-4,5,5-trimethylcyclopentenylacetyl-Co-A monooxygenase (which catalyzes
the lactonization of the monocyclic intermediate) [631 from Pseudornonas putida ATCC
17453 have been purified to homogeneity and thoroughly characterized.
Enzymatic Baeyer-Villiger oxygenations are not restricted to microbial cells. It has
been shown that (+)-camphor,a major constituent of the volatile oil of immature
sage (Salvia oficinalis L.) leaves, is converted into a water soluble metabolite via
enzymatic lactonization to 1,2-campholide, followed by conversion into the 8-D-
glucoside-6-0-glucoseester of the corresponding hydroxy acid[", 691.
The oxidation of racemic fenchone by a Corynebacteriurn SP.[~'] (reclassified as
Mycobacteriurn rhodochrous), an organism which grows at the expense of either (+)- or
(-)- camphor, has also been reported. This was shown to lead, in a 45% yield, to a
90/10 mixture of 1,2 and 2,3-fencholides, as shown in Fig. 16.5-12. This result
contrasts with the chemical oxidation of fenchone with peracetic acid, where
2,3-fencholide is the major product in a 40/60 mixture. Accumulation of these
lactones is a priori surprising as compared with the total degradation of the
structurally similar camphor substrate. However this may simply be due to the fact
that this lactone, unlike that formed from camphor, is chemically stable in the
medium. Of course, one has also to assume that, here again, the strain is devoid of
any lactone hydrolase. This bioconversion was the first gram-scalepreparative report
(+)-camphor (-)-camphor
7 6.5 Baeyer-Villiger Oxidations
I
1211
4 Pseudomonas putida C1
J.
HO so &
'OH
$.
diketocamphane
monooxygenases +
0P \COSCoA
J
2-A3-4,5,5-trimethylcyclopentenylacetate
Co-A
ester monooxygenase
0 & -- I
COSCoA
Figure 16.5-11. Metabolism of both enantiomers of camphor by Pseudomonas putida
C1 (= NCIMB 10007=ATCC 17453).
of a non-steroidal product, yet no indication of any enantioselectivity for this reaction

was presented.
Similarly, it has been shown that 1,8-cineoleand 6-0x0-cineoleare degraded via the
scheme shown in Fig. 16.5-1317*1.As in the case of camphor, the first step involves a
1212
9 1
fenchone 1,2-fencholide 2,3-fencholide
Figure 16.5-12. Oxidation of racemic fenchone to the corresponding fencholides.
hydroxylation, followed by oxidation of the alcohol to form 6-oxocineole.This is then

processed via a Baeyer-Villiger reaction leading to a lactone which is spontaneously
opened to the hydroxy acid.
16.5.1.4
Polycyclic Molecules
Enzymatic Baeyer-Villiger reactions have also been described in the metabolic

processing of larger, polycyclic non-steroidal molecules (Fig. 16.5-14).This is the
case for the biosynthesis of aflatoxin B 1 where it has been demonstrated that
formation of versiconal acetate intermediate from avemfin occurs via such a
Similarly, aflatoxin G1 was shown to be formed from aflatoxin B[731
whereas degradation of the anthraquinone questin to desmethylsulochrin was
shown to imply a Baeyer-Villiger process [741.
Furthermore, biological Baeyer-Villiger reactions have been reported in the bio-
synthesis of polyketides such as DTX-417’] and the aureolic acid antibiotics such as
mithramycin 1762 771. The oxygenase MtmOIV from Streptomyces argillaceus responsi-
ble for cleavage of the fourth ring of premithramycin B is unique amongst those
responsible for biological Baeyer-Villiger reactions, in that it displays sequence
homology not with other “Baeyer-Villiger monooxygenases” (vide infa), but with
flavin-type hydroxylases encoded in polyketide synthase gene clusters from other
Streptomyces spp. [761.
1
6-0x0 cineole
* c o 2 y &cozH Figure 16.5-13.by Degradation

6-0x0-cineole a Rhodococcuso f
HO
SP.
w I
7 G. 5 Baeyer-Villiger Oxidations
1213
averufin
HO \ /
0
0
Aspergillus parasiticus
HO
*: versiconal acetate
HO 4P \
0
/
questin
1 Aspergillus terreus
desmethylsulochrin
HO CO,H
Figure 16.5-14. Involvement of enzymatic Baeyer-Villiger processes in the degradation
of non-steroidal polycyclic compounds.
16.5.2
Baeyer-Villiger Monooxygenases
The reactions described above illustrate that there are numerous metabolic routes
wherein biological Baeyer-Villiger reactions have been implicated. The synthetic
potential of the enzymatic Baeyer-Villiger reaction has dictated that intensive
1214
I Figure 16.5-15. Riboflavin
derivatives: the coenzymically
active forms o f flavoprotein.
HO R =H riboflavin
research efforts have been devoted to studying the nature of the enzymes that
catalyze these reactions.
Enzymes that catalyze the Baeyer-Villiger reaction are a subset of the flavin
monooxygenases. In the mechanism of oxidation catalyzed by such enzymes one
atom of molecular oxygen is incorporated into the substrate, whereas the other is
reduced to HzO.Two cofactors are required for catalyticactivity. The first is a reduced
flavin (FAD or FMN) bound non-covalently in the active site. The riboflavin moiety
of flavin monooxygenase holoproteins is shown in Fig. 16.5-15; the second is a
reduced nicotinamide cofactor (NADPH or NADH),which is required to furnish the
enzyme with electrons to reduce the flavin.
Several Baeyer-Villiger monooxygenases (BVMOs) have been purified and in rare
cases, the relevant genesm cloned and expressed. Some of these are listed in Table
16.5-1. There appear to be two types of BVMOs. Type 1are homogeneous; both flavin
reduction and substrate oxygenation are carried out on a single polypeptide, these
are most usually FAD and NADPH dependent. Type 2 are heterogeneous, a
substrate oxygenating subunit appears to require a separate flavin reductase/NADH
dehydrogenase in order to generate reduced flavin. Type 2 BVMOs are usually FMN
and NADH dependent.
16.5.2.1
Type 1 BVMOs
Cyclopentanone monooxygenase, which catalyzes the conversion of cyclopentanone

to valerolactone, has been isolated from Pseudomonas sp. NCIMB 9872[46,781. This
has been shown to be made up of three identical subunits, each using one FAD
equivalent, and to be NADPH dependent. Cyclohexanone monooxygenases have
been purified from Acinetobacter calcoaceticus NCIMB 9871 and Nocardia globerula
CL1 r4'1 and Rhodococcus copr~philus[~']. These enzymes were shown to be single
polypeptides and to be FAD and NADPH dependent. Tridecanone monooxygenase
from Pseudomonas cepacia is a dimer of two identical subunits, however, but is also
FAD plus NADPH dependent["]. A cyclohexanone monooxygenase from Xantho-
bacter sp. is unusual in that it is dependent on FMN, but NADPH as a nicotinamide
cofactor[82].Steroid monooxygenase from Rhodococcus rhodochrous [831 and mono-
cyclic monoterpene ketone monooxygenase from Rhodococcus erythropolis DCL 14[511
Table 16.5-1. Characteristicsof various Baeyer-Villigermonooxygenases.
Enzyme and source Number o f Subunit structure Cofictor Native molecular Mole offlavin/ Optimum pH Reference
proteins specificity mass x 1000 Da mole of protein
Cyclopentanone monooxygenase 1 3-4 identical NADPH 200 (54-58 each) 1 FAD 7.7 1481
Pseudomonas NCIMB 9872 subunits per subunit
Cydohexanone monooxygenase 1 single polypepetide NADPH 59 1 FAD 9.0 1811
Acinetobacter calcoaceticus
NCIMB 9871
Cyclohexanonemonooxygenase 1 single polypeptide NADPH 53 1 FAD 8.4 1811
Nocardia globurela CL 1
2-Tridecanonemonooxygenase 1 2 identical subunits NADPH 123 (55 each) 1 FAD 7.8-8.0 1801
Pseudomonas cepacia
2-0x0-A3-4,5,5-trimethylcyclopentenyI 1 2 identical subunits NADPH 106 1 FAD 9.0 ~ 3 1
acetyl Co-A monooxygenase
Pseudomonas putida ATCC 17453
2,s-Diketocamphane 1,2- 2 2 identical substrate NADH 78 (39 each) 1 FMN 7.2 1641
monooxygenase oxygenating per subunit
Pseudomonas putida ATCC 17453 subunits + NADH
dehydrogenase
3,6-Diketocamphane1,6- 2 2 identical substrate NADH 72 (36 each) 1 FMN
monooxygenase oxygenating per subunit
Pseudomonas putida ATCC 17453 subunits + NADH
dehydrogenase
Steroid monooxygenase 1 2 identical subunits NADPH 115 (56 each) 1 FAD 7.8
Cylindrocarpon radicicola ATCC 11011 per subunit
Cyclohexanonemonooxygenase 1 single polypeptide NADPH 50 FMN 8.8
Xanthobacter sp.
Monocyclic monoterpene ketone 1 single polypeptide NADPH 60 FAD 9.0
monooxygenase
Rhodococcus erythropolis DCL 14
Cyclohexanonemonooxygenase 1 single polypeptide NADPH 58 FAD
Rhodococcus coprophilus
Steroid monooxygenase 1 single polypeptide NADPH 60 FAD
Rhodococcus rhodochrous
1216
I are also Type
1 BVMOs as is the 2-oxo-A3-4,5,5-trimethylcyclopentenyl acetyl Co-A

monooxygenase from Pseudomonas putida ATCC 17453 [631.
Cyclohexanone monooxygenase (CHMO, E.C. 1.14.13.X) is by far the most
studied Type 1 BVMO and has been used extensively for as a model for mechanistic
studies and as a catalyst in synthesis (vide inpa). CHMO was purified from
Acinetobacter calcoaceticus NCIMB 9871 grown on cyclohexanol as the sole carbon
source, by Tmdgill and coworkers["]. It was found to be active as a monomer and to
contain one non-covalently bound FAD molecule per monomer. The gene was
cloned and the protein expressed in Escherichia c0li['~1 and more recently in
Saccharomyces cerivi~iae[~~I.Each subunit is a polypeptide of 542 amino acids and,
although no definitive structure of a BVMO has yet been published, a potential flavin
binding site at the N-terminus was identified, in addition to a potential NADP
binding site. Analysis of the sequence reveals that the N-terminus of the enzyme
bears strong homology with the FAD binding domain of other flavoproteins such as
glutathione reductase from Escherichia coli.
16.5.2.2
Type 2 BVMOs
The diketocamphane monooxygenases (DKCMOs) from Pseudomonas putida ATCC

17453 involved in camphor degradation, are FMN plus NADH dependent and are
heterogeneous, consisting of two identical substrate oxidizing polypeptides and an
NADH dehydrogenase. The enzymes have been purified, extensively character-
ized["* 671 and their N-terminal amino acid sequences determined[86].These data
showed the oxygenating subunits of the DKCMOs to have homology with the NADH
plus FMN dependent luciferase of Vibrio ha~eyi['~], an enzyme which catalyzes the
Baeyer-Villiger oxidation of dodecanal to dodecanoic acid with the release of a photon
of light. The application of the DKCMOs enzymes to synthesis has also been
investigated (vide inja) and, whilst the genes encoding these proteins have not been
identified, preliminary X-ray crystallographic data on 3,G-diketocamphane-l,Gmono-
oxygenase has been reported["].
16.5.2.3
Mechanism of the Enzymatic Baeyer-Villiger Reaction
The mechanism of the enzymatic Baeyer-Villiger oxidation, with reference to

CHMO, has been studied by the group of W a l ~ h I"1~ ~who . proposed the scheme
shown in the top cycle in Fig. 1G.5-lG.The tricyclic isoalloxazine ring is the center of
catalysis. Initially, the exogenous reductant NAD(P)H acts as the electron donor to
afford the reduced flavin. This can be readily reoxidized by both one-electron or two
electron processes in the presence of 0 2 to yield a 4-a-hydroperoxyflavin.This
intermediate undergoes an 0-0 bond fission upon nucleophilic attack on an
electrophilic ketone substrate, a mechanism similar to the chemical Baeyer-Villiger
oxidation of ketones by peracids. This initially affords the 4-a-hydroxyflavinwhich,
by loss of H20 regenerates the starting FAD for a subsequent catalytic cycle.
7G.S Baeyer-Villiger Oxidations I1217
U
Figure 16.5-16. Proposed mechanisms for the enzymatic Baeyer-Villigeroxidation
of cyclohexanone.
1218 I 16 Oxidation Reactions
However, the FAD-4-a-OOHcan also break down directly via liberation of H202.
A variation on this model has recently been proposed by Kelly et al.l7] who
suggested that the hydroxy group of the Criegee intermediate could not be im-
mobilized in such a mechanism, and that unreasonable steric constraints would be
imposed for many of the substrates transformed reported for these enzymes. A new
tautomer of the the flavin hydroperoxide was proposed as part of an alternative
scheme (lower cycle, Fig. 16.5-16) in which an intermediate trioxane decomposes to
yield the lactone and flavin hydrate.
In addition to ketone substrates, the 4-a-hydroperoxyflavin can also react by
nucleophilic attack on other molecules. Thus, boronic acid substrates were trans-
formed into the corresponding alcohols via the intermediate borate esters as
hydrolytically labile initial enzyme products r90* 911.
However, the 4-hydroperoxyflavin,acting in these cases as an electrophile, is also
able to oxidize other nucleophilic substrates and in particular heteroatoms such as
sulfur[911,selenium1"~ 921, and phosphorous 1911. Indeed, CHMO oxygen-
- NADPH, 0,
enz-FAD
0
0
NADPH, 0, NADPH, 0,
____)
Ca
enz-FAD enz-FAD
NADPH, 0,
_____)
enz-FAD $1
(5 NADPH, 0,
enz-FAD
-
(5S
Q
f
S 0-s f O"-sf
I
NADPH, 0,
____) Figure 16.5-17. Studies on
heteroatom oxidation using
enz-FAD
purified cyclohexanone mono
I I oxygenase
76.5 Baeyer-VilligerOxidations
ates trimethyl phosphite to trimethyl phosphate, sulfides to sulfoxides (one equiva-

lent) or sulfones (two equivalents). If 3- or 4-thiocyclohexanones were used as
substrates, these were converted exclusively into the lactone products, showing that
Baeyer-Villiger oxidation is preferred in these cases (Fig. 16.5-17).The synthetic
applications of heteroatom, notably sulfur, oxidation by BVMOs have been thor-
oughly explored and and reviewed L94.
These results illustrate that reactions performed by BVMOs are similar to those of
peroxide containing reagents (hydrogen peroxide, alkyl hydroperoxides or peracids),
which are able to deliver either a formally nucleophilic or a formally electrophilic
oxygen atom to a substrate. Indeed, whereas Baeyer-Villiger oxidation or boronic acid
oxygenation involve initial attack of a nucleophilic oxygen, the sulfide, selenide or
phosphite ester oxygenations require the transfer of an electrophilic oxygen to a
nudeophilic electron pair of the substrate. Interestingly, no epoxidation of olefinic
double bonds by BVMOs have been reported
The substrate selectivity of CHMO was first explored by Trudgill and cowork-
ers [70, 81],who demonstrated that the enzyme processes C4-C8 cyclic ketones. The
migratory aptitude of the enzymatic oxygen insertion process was probed initially
with two types of substrates. First, in an attempt to explore the stereochemical mode
of these reactions, Schwab et al. studied the Baeyer-Villiger oxidation of (2R)-
deuterated cyclohexanone. Detailed NMR multinuclear spectroscopic studies led to
the conclusion that CHMO catalyzes the conversion of cyclohexanone to E-capro-
lactone with complete retention of configuration at the migrating carbon center ["I, a
result identical to the chemical route (Fig. 16.5-18).To eliminate the possibility of an
enolization and/or rearrangement route, 2,2,6,6-tetradeuterocyclohexanone was also
incubated with the enzyme. The fact that no loss of deuterium was observed by GC
again militates in favor of a mechanism similar to that proposed for chemical
Baeyer-Villiger oxidation.
In an elegant further study["^, these authors confirmed their preliminary proposal
of the (R)-absolute configuration of the starting 2-deuterocyclohexanone as well as
the occurrence of a total retention of configuration of the CHMO catalyzed Baeyer-
(EnzFAD-0-\OH Enz-FAD-OH
0. ? D
Figure 16.5-18. Stereochemical studies using deuterated cyclohexanone.

<
-
1220 IG Oxidation Reactions
I cyclohexanone + 0,
NADPH
6-phosphogluconolactone
Figure 16.5-19.
cyclohexanone
oxygenase
G-6-PDH
1-0xepanone + H,O
NADP’
glucose-6-phosphate
NADPH recycling in the course of CHMO catalyzed oxidation.
Villiger reaction, Interestingly, they described for the first time that the efficient
conversion of ketone into lactone could be brought about in the presence of a
catalytic amount of NADPH, with cofactor recycling accomplished by the glucose-
6-phosphate dehydrogenase as shown in Fig. 16.5-19.
In order to test enzyme regio- and enantioselectivity rigorously, Schwab and co-
workers also studied the asymmetric substrate 2-methylcyclohexanone. A “virtual
racemate” made up of equivalent quantities of (2R)-2-[methyl-2H3]methylcyclohex-
anone and of (2S)-[methyl-13C]methylcyclohexanone (each one prepared by different
methods) was studied, using a multinuclear NMR technique. The conclusions from
this experiment were two fold. First, they confirmed that 6-methyl-~-caprolactone
the first time a two-fold rate difference between transformation of the two enantio-
mers of cyclohexanone. This was an interesting result that suggested that CHMO
could show far greater discrimination toward enantiomers of a substrate that bore a
is
the only reaction product, thus indicating a total regioselectivityof oxygen insertion
into the “more substituted carbon-carbon bond. Second, these results showed for
far bulkier C2 substituent. Finally, the measurement of the reaction kinetics for each
one of the substrate enantiomers showed that, after about 50 % reaction, there is a
progressive decrease in both the degree of enantioselectivity as well as the absolute
rate of lactonization of the two substrate enantiomers. However, the reasons for
these diminishing rates of reaction were not clear.
Further work exploring the migratory aptitude of different substituents has been
described[”]. Phenylacetone is converted into benzyl acetate, showing exclusive
benzyl migration in accordance with the chemical reaction achieved with tri-
fluoroacetic acid. Different results were observed with phenacetaldehyde, where an
inverted preference is seen as compared with the peracidic reactions.
Using purified CHMO from A. calcoaceticus NCIMB 9871, Taschner and cowork-
ers r9‘, 971 showed that several prochiral substrates including some 4-substituted
cyclohexanoneswere efficiently converted into their corresponding lactones, each of
them showing very high enantiomeric purities (Fig. 16.5-20).Thus, CHMO prove to
Substrate Product
% yield ('77 e.e.)

I
1221
M e 0 0 80 (>98)
73 (>98)
27 (>98)
d
25 (>98)
76 (75)
Me0
88 (~98)
*oLoH 0
73 (9.6)
Figure 16.5-20. CHMO catalyzed oxidation o f various prochiral substrates.
be extremely effective at discriminating between the two sides of the carbonyl

function of such prochiral substrates. However, the presence of an alcohol or methyl
ether function at position 4 leads unexpectedly to products of lower ee values.
IG
1222
16.5.3
Synthetic Applications
With the exception of steroid type substrates, the results described up to now have
dealt with small-scaleanalytlcal studies. However, in view of the potential of BVMOs
for regio- and even enantioselective transformations of various substrates, studies
into the scale-up of these transformations began in earnest soon after these earlier
investigations, followed by considerations of their application in chiral organic
synthesis.
In this context, Abril et al.[”] examined a variety of readily available ketones in
order to determine the substrate selectivity, regioselectivityand enantioselectivity of
CHMO immobilized in a polyacryalamidegel. They also used the NADPH recycling
system previously desribed by Schwab for in situ regeneration of this cofactor. These
experiments showed that 2-norbornanone, L- and D-fenchone, (+)-camphor and
(+)-dihydrocarvoneare processed by CHMO. In a typical experiment, 10.2 g of
racemic 2-oxabicyclo[3.2.l]octan-3-one were obtained from 11.4 g of 2-norborna-
none, using 1.7 g of NADP cofactor. The authors concluded that the enzyme did not
display a useful degree of enantioselectivity,therefore offering no major advantages
over chemical oxidation.
One major drawback of employing CHMO as a catalyst is the necessity to
regenerate the expensive nicotinamide cofactor NADPH. One strategy for circum-
venting this problem is use of whole-cellpreparations of microorganisms for Baeyer-
Villiger oxidations. One early example of this technique involved the oxidation of
2,2,5,5-tetramethyl-1,4-cyclohexanedione to the optically pure (S)-ketolby Cuwularia
lunata described by Azerad and coworkers[’9]. They showed that during the fungal
reaction of the dione, as shown in Fig. 16.5-21, the already formed (S)-ketolwas
isomerized to its five-membered isomer. Moreover, when submitted to appropriate
culture conditions, the racemic ketol afforded the (S)-lactone(81% ee) as well as the
unchanged (R)-lactol of 97% ee The remaining substrate could then be further
treated by rn-chloroperbenzoic acid to afford the (R)-hydroxylactoneenantiomer.
Extensive studies have been performed on the microbial Baeyer-Villiger oxidation
of bicyclic [3.2.0] ketones and analogues. These studies were prompted by the
important findings of Furstoss and coworkers[’00],who determined that the oxy-
genation of bicyclo[3.2.0]hept-2-en-G-one using Acinetobacter sp. TD 63 led to two
regioisomeric lactones in equal quantities and almost quantitative yield. The first
arises from the “normal” oxygen insertion mode into the more substituted carbon-
carbon bond, whereas the second is the result of an oxygen insertion into the less
substitiuted bond leading to the so-called “abnormal” lactone. Moreover, both these
lactones were of high optical purity i. e. showing a 98% ee for the (-)-(l S, 5R) isomer
and a 95% enantiomeric excess for the (-)-(1R. 5s) enantiomer. These results
appeared to suggest that biological Baeyer-Villiger oxidations could indeed be used
for the large-scale preparation of optically active lactones.
In the case of bicyclo[3.2.0]hept-2-en-G-one, each one of the substrate enantiomers
reacts with a different and divergent regioselectivity for the oxygen atom insertion.
This result is noteworthy since it describes for the first time such an almost perfect
76.5 Baeyer-Villiger Oxidations I
1223
4Y
Figure 16.5-21. ’ Baeyer-Villiger oxidation o f
2,2,5,5-tetramethyl-1,4-cyclohexane-dione
by Curvularia lunata.
Curvularia lunata
$ OH
4 OH
regio- vs. enantioselectivity for the Baeyer-Villiger oxygenation. A more complete

study[lol],aimed at exploring the synthetic potential of these reactions, confirmed
that this enantiodivergent selectivity is not restricted to one particular substrate but is
a general phenomenon within a series of similar compounds. Two strains of
bacteria, Acinetobacter sp. TD 63 and A. calcoaceticus NCIMB 9871 were used
throughout this study and led to almost identical results. In most cases, both
“normal” and “abnormal” lactones were obtained in approximately 1: 1 ratios and
with almost quantitative yields. Also, it was observed as shown in Fig. 16.5-22 that
the “abnormal” lactone, which is not accessible using conventional Baeyer-Villiger
oxidation, always shows very high ee values, whereas the enantiomeric purity of the
“normal” lactone is somewhat lower for the substrate bearing a saturated six-
membered ring. Both of these lactones are interesting chiral synthons; the “normal”
one being an important chiron for prostaglandin synthesis. It is noteworthy that all
lactones of a particular type are formed from the same enantiomer of the starting
ketone: thus, the substrate enantiomer bearing an (S)-configurationat the bridge-
head carbon atom a to the carbonyl group leads to the “normal” lactones, whereas
the (R)-configuration affords the “abnormal” ones.
Similar results were obtained in the course of a study conducted on bicyclic
1224
A. calcoaceticus
NCIMB 9871
,ao 44%
> 95% e.e.
42%
> 95% e.e.
36% 31yo
> 95% e.e. > 95% e.e.
43% 37%
> 95% e.e. > 95% e.e.
41yo 36%
86% e.e. > 95% e.e.
52% 28%
60% e.e. > 95% e.e.
Figure 16.5-22. Oxidation of various In.2.01 bicyclic ketones with Acinetobacter calcoaceticus
NCIMB 9871.
substrates bearing an oxygen atom in the five or six-membered ring[’02](Fig. 16.5-

23). Here again, equivalent ratios as well as high ee values were obtained for both the
‘normal’and “abnormal”lactones. Since the lactones are unreported in the literature
in their optically inactive form, detailed studies using circular dichroism were
conducted in order to attribute the absolute configuration of the products.
Whilst the whole-cell approach has proved invaluable, the associated problems of
overmetabolism and side reactions can be encountered. Another way to counter the
problems of high cost in using isolated BVMOs is to use an NADH dependent
enzyme, as NADH retails at approximately one tenth of the cost of NADPH. The
Type 2 DKCMOs from Pseudornonas putida ATCC 17453 (= NCIMB 10007) are
NADH dependent, and Grogan et al. were successful in applying a complement of
these enzymes, termed M 0 1 , to the transformation of bicyclo[3.2.0]hept-2-en-6-one,
to yield another enantiodivergent mix of lactones enantiomeric to those obtained
76.5 Baeyer-Vihger Oxidations
A. calcoaceticus
NCIMB 9871 * ("3::::Fo +
35% 32%
91% e.e. > 99% e.e.
35% 35%
99% e.e. 97% e.e.
34% 42%
98% e.e. > 99% e.e.
33% 33%
72% e.e. 97% e.e.
60% 18%
35% e.e. > 99% e.e.
Figure 16.5-23. Oxidation of various 0x0-[n.2.0] bicyclic ketones with Acinetobacter

calcoaceticus NCIMB 9871.
with A. calcoaceticus NCIMB 9871/TD 63. The use of NADH dependent enzymes is
also important in this context, as it allows use of the NAD dependent formate
dehydrogenase/sodium formate recycling strategy for cofactor regeneration[lo3I,
reducing costs still further. Interestingly, the separated isoenzymes, 2,s-diketocam-
phane 1,2-monooxygenase and 3,G-diketocamphane 1,G-monooxygenase were
shown to have different selectivities for this transformation, compromising the
result obtained with M 0 1 [lo41(Fig. 16.524). Further transformations of this ketone
by luminescent bacteria containing NADH dependent luciferases (also Type 2
BVMOs) have also been reported [lo5],although characterization of cell-free systems
employing these enzymes has not been investigated further.
The biotransformation of bicycl0[3.2.0]hept-2-en-G-one using whole cell suspen-
sions of the fungus Cylindrocarpon destructans gave not only different ratios of both
lactones depending on the degree of conversion, but also no enantioselectivity was
1226
NADH-dependent BVMOs 0
0 from Pseudomonas putida
NADH NAD’
a /
co* Na+O,CH
‘M01‘ 63%, 60% e.e. 37%, 95% e.e.
2,5-DKCMO 57%, 82% e.e. 43%, 100% e.e.
3,6-DKCM0 17%, 10% e.e. 13%, 72% e.e.

Biotransformation of bicyclo[3.2.0]hept-2-en-6-one by NADH dependent
Figure 16.5-24.
BVMOs from camphor grown Pseudomonas putida ATCC 17453.
observed [‘06]. Further fungal biotransformations described by Carnell and Willetts

showed that a series of dematiaceous fungi were also able to lactonize the same
substrate [‘071. These included various Cuwularia and Dreschlera species. Some of
these fungi produced both regioisomeric lactones with a high degree of ster-
eoselectivity, whilst others produced mostly the 3-oxa lactone. The test strains of
Curvularia lunata and Dreschlera australiensis gave lactones with equal and almost
opposite degrees of regio- and stereoselectivity. Importantly, the biotransformation
of bicyclo[3.2.0]hept-2-en-G-one by another fungus, Cunninghamella echinulata NRRL
3655, is unique in that it results in a resolution of the parent substrate to yield only the
“abnormal” (-)-(lR, 5S)-3-oxalactone in 30% yield and 95% ee[108].This chiral
synthetic intermediate has been used to synthesize both single enantiomer cyclo-
sarkomycin[’08]and the marine brown algae pheremones (+)-multifidene and
(+)-viridiene[lo’](Fig. 16.5-25).
Further reports by Furstoss and coworkers concerned Baeyer-Villiger oxidation of
a-substituted cyclopentanones[’lo].Using the same two Acinetobacter strains used
previously, this study aimed to explore the possibility of synthesising optically active
6-lactonesbearing aliphatic chains, these compounds being of particular interest as
chiral synthons. This study showed that various lactones of (S) configuration can be
obtained in fair yields with moderate to excellent ee values depending on the chain
length and on the conversion ratio. Using Acinetobacter calcoaceticus NCIMB 9871 it
was, however, necessary to run these biotransformations in the presence of tetra-
ethylpyrophosphate (TEPP), a well known inhibitor of hydrolases. This was neces-
sary in order to avoid hydrol$c degradation of the 6-lactonesformed. The use of this
inhibitor was, however, unnecessary when using the Acinetobacter sp. TD 63 strain
which is known to lack a lactone hydrolase. One interesting application of this study
was the preparative two-step synthesis of both enantiomers of 5-hexadecanolide, a
I 1227
Cunninghamella
echinulata 03- steps
(-)-(1 R, 5S)-cyclosarkomycin
35%
(+)-(3R,4S)-viridiene
95%
steps
(+)-(3S,
4S)-multifidene
Figure 16.5-25. Biotransformation of bicyclo[3.2.0]hept-2-en-6-one by Cunninghamella
echinulata NRRL 3655 and synthetic targets.
CllH23 A. calcoaceticus
lh
> ko
I
A. calcoaceticus
Figure 16.5-26.
Baeyer-ViIIiger
oxidation of
mCPBA a-undecylcyclo-
* pentanone: synthesis
o f either enantiomer of
$c~,Ha hexadecanolide.
pheromone isolated from the oriental hornet Vespa orientalis. As shown in Fig. 16.5-
26, Baeyer-Villiger oxidation of racemic undecylcyclopentanone with A. calcoaceticus
NCIMB 9871 led to a 25% isolated yield of (S)-5-hexadecanolideshowing an ee of
74%. Interestingly, a 30 % yield of remaining (R)-2-~ndecylcyclopentanone of 95 %
optical purity can also be isolated using a longer incubation time, thus allowing
direct access, via chemical Baeyer-Villiger oxidation, to the (R)-(+)-5-hexadecanolide
known to be the sole bioactive enantiomer.
The biotransformation of a-substituted cycloalkanones using the BVMOs from
camphor grown Pseudomonas putida has also been investigated in depth. Whilst the
NADPH dependent activity corresponding to 2-0x0-A3-4,5,5-trimethylcyclopenteny-
lacetyl-Co-A monooxygenase (and termed M 0 2 ) resolved a series of a-alkyl cyclo-
pentanones with good selectivity, poorer resolution of these compounds was per-
1228
I
R
M01 or M02 from camphor
'VR grown Pseudomonas putida
cl
ATCC 17453
M01
R Yield ketone e.e. ketone Yield lactone e.e. lactone
C4H9 14 9 16 58
&HI3 48 48 34 74
%HI7 35 22 11 90
R Yield ketone e.e. ketone Yield lactone e.e. lactone

C4H9 26 40 95
&HI3 51 75 35 92
%HI7 44 59 29 95
M02 from camphor grown

Pseudomonas putida
ATCC 17453
+ b R + bR
M02
R Yield ketone e.e. ketone Yield lactone e.e. lactone-
%HI3 30 65 36 72
c8H17 49 61 34 77
CHZCQEt 43 89 30 93
CH~CH~OAC 13 75 34 83
Figure 16.5-27. Biotransformation o f 2-substituted monocyclic ketones by BVMOs from

camphor grown Pseudomonas putida ATCC 17453.
formed by the NADH dependent M 0 1 complement[104](Fig. 16.5-27). An extension

to this study revealed that M 0 2 could be used to resolve a series of a-substituted
cyclohexanones wherein the subsituents consisted of esters, acetates and common
protecting groups [''I. This led to the development of a chemoenzymatic synthesis
of (R)-(+)-lipoicacid incorporating a BVMO catalyzed resolution as the key step
(Fig. 16.5-28). Interestingly, the preferred selectivity of cyclopentanone monoox-
ygenase from Pseudomonas sp. NCIMB 9872, is opposite to that of M 0 2 , and in a
I 6 5 Baeyer-Villiger oxidations
0
I 1229
M 0 2 from camphor grown
Q----
Pseudomonas putida
ATCC 17453
JJ
steps, including Mitsunobu inversion
of chiral centre
0
II
M e o w
S-S
Figure 16.5-28. Chemoenzymatic synthesis of (+)-lipoic acid incorporating a BVMO
catalysed resolution as the key step.
separate investigation,it was suggested that this enzyme be used in the place of M 0 2
to eliminate the need for the Mitsonobu inversion in the chemoenzymatic synthe-
sis [1121.
The biological Baeyer-Villiger oxidation has also been applied, in a variety of
forms, to the production of optically active lactones from prochiral 3-substituted
cyclobutanones. A series of cyclobutanones was subjected to oxidation by Acineto-
bacter sp. and to the M 0 1 and M 0 2 enzyme preparations derived from camphor-
grown Pseudornonas putida ATCC 17453['13]. The results are summarized in
Fig. 16.5-29.In general, the reactions performed with Acinetobacter sp. displayed
better enantioselectivities, but the value of a multi-biocatalyst approach was illus-
trated by the fact that certain BVMOs from P. putida displayed opposite enantiose-
lectivity. A further series of cyclobutanone substrates was oxidized by Acinetobacter
sp. and by the fungus Cunninghamella echin~lata[l'~I(Fig. 16.5-30). The lactoniza-
tion of 3-(4'-chlorobenzyl)-cyclobutanone was performed by this fungus to yield (R)-
lactone of 99 % ee in 30 % yield, which was used in a chemoenzymatic synthesis of
baclofen [lls1, a lipophilic derivative of y-aminobutyric acid. The Cunninghamella
strain was also used to oxidize 3-(benzyloxymethyl)-cyclobutanoneto the optically
pure (R)-(-)-y-butyrolactone, which was used in enantiodivergent chemoenzymatic
syntheses of (R)-and (S)-proline["'I .
The oxidation of either enantiomer of menthone and dihydrocarvone by Acineto-
bacter sp. were also reported['l71. (-)-Menthone is not metabolized but (+)-menthone
leads to the expected lactone, whereas both enantiomers of dihydrocarvone are
oxidized. Thus (-)-dihydrocarvone leads to the expected lactone, whereas (+)-dihy-
drocarvone afforded the unexpected 'abnormal' lactone product (Fig. 16.5-31).Both
enantiomers of dihydrocarvone are also transformed by MMKMO ["I from Rhodo-
coccus erythropolis DCL 14,which in contrast to Acinetobacter sp., also transforms
both enantiomers of menthone.
Taschner and coworkers described the oxidation of cis-3,5-dimethylcyclohexanone
by whole-cell preparations of A. calcoaceticus NCIMB 9871 [118], which led directly to
1230
Baeyer-Villiger monoxygenase
or whole cell catalyst
R R
R Conversion Yield lactone e.e. lactone

Bu 95 68 (s)-,17%
Bu' 98 56 (R)-, 84%
CHpPh 100 57 (6-, 82%
100 83 (R)-, 95%
100 89 (q-,
55
I Mnl
R Conversion Yield lactone e.e. lactone
Bu 100 nd (R-,69
Bul 78 nd (R)-,91
CHnPh 58 40 (q-, 15
48 38 (q-37
Figure 16.5-29. Biotransformation o f prochiral 3-substituted cyclobutanones using BVMOs.
the corresponding optically active lactone and thence to the hydroxyacid, which was
converted into the methylester by reaction with diazomethane. This methylester,
which was shown to be optically active, is a key intermediate in the synthesis of the
polyether antibiotic ionomycin.
In addition, several bridged bicyclic compounds have been examined as potential
substrates (Fig. 16.5-32). In contrast to the regiodivergent behaviour of the [n.2.0]
bicyclic compounds, in these cases, only one lactone product is usually obtained.
This high selectivity compares favorably with the chemical Baeyer-Villiger oxidation
of compounds of this type, which often afford regiomixtures[119].In addition, the
I
1231
1, R = Ph
2, R = pFC,H,
go Organism ~ do 3, R =p-CLC,H,
4, R =p-MeGH,
5, R = C $ c o )
R R ' 0
6, CH,C,H,-p-OMe
7, CH,OCH,Ph
8, CH,Ot-Bu
AcinetobacterTD63 90 (R)-,25
8 C. echinulata 25 98
A. calcoaceticus 43 89
AcinetobacterTD63 15 88
obtained bridgehead lactones are often described to be of high optical purity. The
benzyloxy derivative is known to be an important intermediate for prostaglandin
synthesis. The residual fluorinated bicyclic ketone of high enantiomeric excess was
used to synthesize an antiviral carbocyclic nucleoside['201. In this last case, detailed
studies showed that the first formed product is the corresponding alcohol (about
80% conversion) and that over the next 3 h period, the alcohol concentration
decreased, the amount of ketone rose and the production of lactone This
observation led to an elegant closed-loop recycling procedure, as shown in Fig. 16.5-
33, where the alcohol dehydrogenase from Thermoanaerobium brockii was used in
conjunction with the purified monooxygenase from A. calcoaceticus NCIMB 9871. In
1232
A. calcoaceticus or
*
Acinetobacter TD 63
n
rac-rnenthone (-)-menthone
A. calcoaceticus or
*
Acinetobacter TD 63
A
racdi hydrocarvone
Figure 16.5-31. Oxidation o f dihydrocarvone enantiomers with Acinetobacter
calcoaceticus NCIMB 9871 and Acinetobacter sp. TD63.
this case, the substrate alcohol also serves as a co-substrate for the NADPH recycling
reaction. Thus, endo-bicyclo[2.2.1]heptan-2-01was transformed using catalytic
amounts of NADP. An analogous recycling loop was set up using the NAD
dependent alcohol dehydrogenase from Pseudomonas sp. NCIMB 9872 and the
NADH dependent M 0 1 isozyme complement from Pseudomonas putida ATCC
17453, for the oxidation of 7- endo-methylbicyclo[3.2.0]hept-2-en-6-ol[1221.
A further series of prochiral bicyclic [2.2.1] substrates have also been studied by
Taschner and coworkers and lead generally to lactones of high enantiomeric purity.
One of these is a valuable precursor for chorismic acid synthesis [971.
The transformation of a series of norbornanone derivatives (Fig. 16.5-34) was
studied by Roberts and coworkers who determined that both the M 0 1 complement
of NADH dependent BVMOs from Pseudomonas putida ATCC 17453 and the
NADPH dependent fraction M 0 2 were successful in the resolution of hydroxy,
acetoxy and benzyloxy norbornanones [1231. Interestingly 25DKCMO and 36DKCMO
when separate, displayed notably different reactivity toward the hydroxy and acetoxy
derivative, again emphasizing their complementary nature as potential individual
biocatalysts. The benzyloxy lactone is an intermediate in the synthesis of the insect
antifeedant azadirachtin.
Further studies also been performed on the bicyclo[3.2.0]heptan-6-oneseries of
compounds [124* 12’1. These results are summarised in Fig. 16.5-35.Oxidation of this
ketone with Pseudomonas NCIMB 9872 gave the (lS, 5R)-lactoneoflow optical purity
(23% ee) with only small amounts (5%) of the isomeric lactone, whereas its
oxidation with an Acinetobacter sp. gave these lactones in a 9: 1 ratio and a modest
yield, a result quite different from the one described previously. However, oxidation
of 7-endo-methylbicyclo[3.2.0]hept-2-en-6-one using either Pseudomonas sp. or Acine-
tobacter sp. produced optically pure (ee > 96 %) of both lactones in equal quantities
1 6 5 Baeyer-Villiger Oxidations
I
1233
Pseudomonas sp.
NCIMB 9872
0
38 : 1
4 0
Cylindrocarpon
destructans
F
0% e.e.
Acinetobacter
A d NCIMB 9871 * A d &Lo+F $ Y o

OAc
11%
Acinetobacter
NCIMB 9871
*ao
BzO--
O
H
&:
h
‘0
26%, 95% e.e..
F F
Acinetobacter
0
NCIMB 9871 * &Ao+
F$7° Br
36%, 95% ex.
Figure 16.5-32. Baeyer-Villiger oxidation of various [2.2.1] bicyclic substrates.
dehydrogenase
NADP+ NADPH + H+ 0 Figure 16.5-33.

Closed-loop
recycling proce-
dure for NADPH
recycling using the
substrate alcohol
as the reducing
monooxygenase agent.
1234
NADH-dependent BVMO
l,R=H
2, R = O H
* R&o 3,R = OAC
*R 0 4, R = OBn
(1 S,5S, 6R)-
Enzyme Substrate Conversion (%) Lactone e.e. (%)

25DKCMO 1 20 60
36DKCMO 1 48 >90
25DKCMO 2 0
36DKCMO 2 33 >95
25DKCMO 3 35 >95
36DKCMO 3 0
‘M01’ 4 39 >95
Figure 16.5-34. Biotransformation of norbornanone derivatives using NADH dependent

BVMOs from camphor grown Pseudomonas putida ATCC 17453.
(combined yields 50-55 %). Surprisingly, 7,7-dimethylbicyclo[3.2.0]hept-2-en-6-one

was oxidized by the Acinetobacter strain to give exclusively one lactone of 29% ee, a
very low enantioselectivity. The bromohydrin obtained from this substrate led to
similar results, yielding the same type of oxidation. This can be considered as being
the “normal” lactone since substitution with two methyl groups makes this carbon-
carbon bond the more substituted one. Again, the M 0 1 isozymic complement from
Pseudomonas putida was successful in generating the complementary enantiomers
from endo-methyland dimethyl derivatives with good enantiomeric excess [*031.
16.5.4
Models for the Action of Baeyer-Villiger Monooxygenases
The results of biological Baeyer-Villiger oxidations have been, in some cases

unpredictable and surprising, and, in the continued absence of a structure of one of
these enzymes, several groups have attempted to explain the various observations of
selectivity with an increasingly complex series of models.
Initially, some workers proposed that enantiodivergent biotransformations of the
type witnessed in the oxygenation of bicyclo[3.2.0]hept-2-en-6-one by, for instance
CHMO and 25DKCMO could be due to the presence in either of these preparations
of two separate enzymatic activities. Whilst this was once and indeed still is, a
reasonable assumption in the light of results obtained with whole-cell preparations,
the use of highly purified preparations of the two named enzymes to effect this
biotransformati~n[’~~* 1‘’‘ have eliminated this possibility in these cases. The
phenomenon of enantiodivergence has therefore been addressed with respect to one
enzyme active site.
I
76.5 Baeyer-Villiger Oxidations 1235
Pseudomonas
NCIMB 9872
* 0:::::Fo +
75%, 23% e.e. 5%
ofo
0::::yo
0
Acinetobacter -
/
NCIMB 9871 +
96% e.e. 96% e.e.
Acinetobacter
* +
NCIMB 9871
29% e.e.
Ho,q,,,qo -“111
Acinetobacter D
NCIMB 9871
0
Br
98% e.e.
Figure 16.5-35. Baeyer-Villiger oxidation of various [n.2.0] bicyclic compounds.
The first model was proposed by Furstoss and coworkers, based on steric and
stereoelectronic considerations. In this model, shown in Fig. 16.5-36, the 4-a-
hydroxyperflavin is considered as being the oxygen transfer agent, according to the
hypothesis of Walsh and coworkers[841. The enantioselectivity of the reaction would
be due to a different positioning of each intermediate in the active site. It is
supposed, primarily, that the attack of the hydroperoxyflavin should take place on the
least hindered face of the ketone. On the other hand, the migrating C-C bond of the
peroxidic intermediate should be antiperiplanar to the peroxidic bond and to a non-
bonded electron pair of the hydroxide group, as suggested for chemical Baeyer-
Villiger oxidations. Thus, the cycloalkyl part of the (S,S)-enantiomer of the ketone
(the one leading to the “normal”lactone) could be accommodated in only one region
of the active site (position 1).Position 2 would never be adopted due to some steric
hindrance with the active site (dotted cube). Similarly, in the case of the (R,R)-
enantiomer, position 4 would be favored over position 3 leading to the “abnormal”
lactone. This model was augmented by further work by the inclusion of results
obtained with both monocyclic monterpene 3-substituted cyclobutanone sub-
strates [1131 and a-substituted cyclohexanones
1236 I 1 G Oxidation Reactions
Position 1 Position 3
i
;
::
I .
Position 2 Position 4
Figure 16.5-36. Furstoss model for the active site o f cyclohexanone
monooxygenase from Acinetobacter calcoaceticus NClMB 9871.
Taschner and coworkers proposed a similar model based on two other fla-
voenzymes; the human and E. coli glutathione reductase. The FAD binding domain
of glutathione reductase and p-hydroxybenzoate hydroxylase have been shown to
resemble each other closely via comparison of their respective X-ray crystal struc-
tures. Extrapolating this information to CHMO leads to the proposal that the
hydroperoxide is attached to the re-face of the isoalloxazine ring and that the ketone
substrates approach the hydroperoxide from the direction of the dimethylbenzene
Further stereochemical and stereoelectronic considerations lead to a
hypothesis explaining the observed stereoselectivities.
In the model of Furstoss and coworkers, stereoselectivityof CHMO is determined
by the differentiation of groups of different sizes in the active site. A different model,
proposed by Kelly and coworker^[^^^-^^^^, extends Taschner’s idea that the source of
stereoselectivity might be the flavin cofactor itself. It was suggested that the
stereoselectivityof oxygen insertion arises solely as a result of the flavin face, re- or si-,
from which the hydroperoxide attacks. This would lead to two distinct Criegee
intermediates of opposing absolute configuration (Fig. 16.5-37). Hence it was
I 1237
S or si- Ror re-
Non-migratinggroup Non-migratinggroup
Migrating group RI-o\O~!*A

R21 0 Migrating group
CHMO 25DKCMO or 36DKCMO

Figure 16.5-37. Schematic representation o f enantiomeric Criegee
intermediates for the enzymatic Baeyer-Villiger reaction.
CHMO, NADPH
Sor si
Ror re
25DKCMO or 36DKCMO
NADH
Figure 16.5-38. Enantioselective Baeyer-Villiger oxidation of a tricyclic
ketone by Type 1 and Type 2 BVMOs.
demonstrated that for the tricyclic ketone shown in Fig. 16.5-38 for which attack
from only the exo-face is possible, pure preparations of BVMOs always resulted in
lactones of >95 % ee Interestingly, all Type 1, FAD plus NADPH dependent BVMOs
yield lactone from the (R)-configurationof the intermediate, and all Type 2, NADH
plus FMN dependent BVMOs yield lactone from the (R)-intermediate. Substrate
interaction with the topology of the active site must also be considered however, as
the enantiocomplementary DKCMOs, both proposed to catalyze oxygen insertion via
(R)-Criegee intermediates, catalyze complementary resolutions of racemic cam-
phor [671.
This additional dependence on active site topology for selectivity in CHMO was
carefully considered by Ottolina et al. who developed a sophisticated cubic space
model for the active site of CHMO (Fig. 16.5-39).This group was able to show that,
for example, for the biotransformation of 7-endo-methylbicydo[3.2.0]hept-2-en-
6-one, of the eight possible intermediates in oxidation, the only two “allowed by the
model were the two which led to the lactones observed by experiment. The model
was successfully applied to a series of other ketones and also predicts the ster-
eoselectivity of sulfur oxidation by this The group of Colonna estab-
lished in a series of reports that CHMO was able to catalyze the oxidation of a range
of alkylaryl sulfides, benzyl alkyl sulfides, functionalized sulfides and 1,3-dithioace-
tals with absolute configuration and enantiomeric excesses being highly dependent
1238
Side
\
Front Top nP I
Lj
Side
HS
Figure 16.5-39. Cubic space filling model o f the active site o f cyclohexanone monooxygenase
from Acinetobacter calmaceticus NClMB 9871, based on the results o f the oxidations o f a series o f
bicyclic ketones. The catalytic oxygen is circled. The main (M) hydrophobic large (HL)and
hydrophobic small (Hs) pockets are depicted. The correct arrangements o f the Criegee inter-
mediate are also shown.
on the structure of the substrate['3]. This group has also recently reported the first
asymmetric oxidation of tertiary amines using CHM0['331.
The ability of BVMOs to oxidize sulfur was also exploited by Beecher and Willetts
in order to construct space filling cubic models of the active site of the DKCMO
enzymes from Pseudomonas putida ATCC 17453 (Fig. 16.5-40). They note that the
more relaxed enantiospecificity of 3GDKCM0, at least in terms of sulfoxidation,
appears to be due to an overall larger 3D cubic space available in the active
3GDKCMO appears to be the best candidate for a first X-ray structure of a BVMO, as
preliminary crystal data have been
16.5.5
Conclusion and Outlook
It is apparent from the many application of BVMOs in synthesis, that these enzymes
currently represent the most valuable method of effecting the enantioselective
I G.5 Eaeyer-Villiger Oxidations
Figure 16.5-40. Cubic

I
1239
space filling models

o f active sites o f :
right, 3,6-diketocam-
phane 1,6-monooxy-
genase; and left,
2,S-di ketocamphane,
1,2-monooxygenase
based on results o f
sulfoxidations o f a
series of sulfide
substrates.
R
‘engineered’ Saccharomyces cerivisiae
expressing CHMO
> +
R
1
+
R b 2
R Ratio 1:2 e.e. lactone 1 (%) e.e. lactone 2 (%)

Combined yield
lactones
Me 13:87 9 36
95%
Et 80:20 33 19
80%
n-Pr 83:17 33 60
44%
n-Bu 99:1 38
34%
moct 99:1 16
19%
Figure 16.5-41. Biotransformation o f 3-alkylcyclopentanones by “engineered”

Saccharomyces cerivisiae expressing CH MO.
Baeyer-Villiger reaction. The primary sources of BVMO enzymes carry associated

disadvantages that must now be addressed, although recent biotechnological ad-
vances suggest that BVMOs will be more accessible to the synthetic organic chemist
in the future.
Type 1 BVMOs -
m
B
1 Steroid monooxygenase %
5.
Cylindrocarpon radicicola 2 A-E-W-A-E-E-F-D-V-L-V-V-G-A-G-A-G-G-
2 CHMO
P
2
Rhodococcus coprophilus 2 A-Q-T-I-H-G-V-D-A-V-V-I-G-A-G-F-G-G-I-Y-A-V-H-K- 8'
2
3 CHMO
Acinefobacter NCIMB 9871 1 M-S-Q-L-M-D-F-D-A-I-V-I-G-G-G-F-G-G-L-Y-A-V-K-K-
4 CPMO
Pseudomonas NClMB 9872 1 4 -N-S-V-N-D-K-L-D-V-L-L-I-G-A-G-F-
5 Steroid monooxygenase
Rhodococcusrhodochrous 1 M-N-G-Q-H-P-R-V-V-V-A-A-P-D-A
Type 2 BVMOs
6 2,5-DKCMO
Pseudomonas putida 1 -M-Q-A-G-F-F-G-T-P-Y-D-L-P-T-R-T-A-R-Q-M-
7 3,6-DKCMO
Pseudomonas putida 1 A-M-E-T-G-L-I-F-H-P-Y-M-Y-P-G-K-S-A-A-Q-
Figure 16.5-42. N-terminal amino acid sequence alignment ofType 1 BVMOs (1-5) and Type 2
BVMOs (6 and 7). Conserved residues are marked in bold.
References I1241
this problem has been the cloning and expression of the gene encoding CHMO in
Saccharomyces ceri~isiae[*~1. In a series of reports by Stewart and coworkers[135-1371,
the “designer yeast” was shown to catalyze many of the reactions which had
previously been shown to be catalyzed by either whole cells of Acinetobacter sp. or
CHMO in addition to some new ones (Fig. 16.5-41). Recently, a similar strategy has
seen whole-cell preparations of Escherichia coli expressing recombinant CHMO for
the same purpose[138].It remains to be seen whether constraints on the use of
genetically engineered microorganisms of this type will render these strains as
“difficult”to manipulate as the wild-type strains.
The use of purified enzyme would circumvent the need for whole-cell contain-
ment procedures, and indeed, amounts of CHMO are now available from Fl~ka[~~’]].
However, the attendant costs associated with cofactor recycling must be addressed if
this approach is to prove viable. The recent production of a formate dehydrogenase
suitable for use in NADP/NADPH recycling s y ~ t e m s I ~ should
~ ~ 1 prove attractive in
this regard, as should the further investigation of NADH dependent enzymes. The
practicalities associated with the industrial scale up of biological Baeyer-Villiger
reactions are currently being investigated [14’1.
New sources of enzyme will also become important and with the advent of
genomic science, paralogs of genes that encode CHMO-like proteins are being
identified amongst whole bacterial genomes, most recently those of Pseudomonas
aerugin~sa[’~~] and Mycobacterium tubercul~sis~’~~]. The availability of gene and amino
acid sequence data for BVMOs will prove useful in identifylng more new activities in
this manner. BVMOs of the same Type (1 or 2) exhibit sequence homology within
their N-terminal amino acid sequences although homology between types is not
(Fig. 16.5-42). In the hture, the “tailoring”of enzyme characteristics
by either rational redesign or so-called “directed” evolution approaches could also
doubtless be applied to BVMOs. Fundamental to these studies would be the
development of an efficient, rapid screen for BVMO activity. Rational redesign would
require more knowledge of the 3D structure of these enzymes. This is one reason
why the acquisition of a complete X-ray crystal structure of a BVMO must be
considered of fundamental importance to the ongoing development of this area.
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7 G. G Oxidation of Acids
PEDANT http://pedant.mips.biochem.
I
1245
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16.6
Oxidation of Acids
Andreas Schmid, Frank Hollmann, Bruno Buhler
16.6.1
Introduction
At a first glance, synthetically relevant oxidations of carboxylic acids, except for

oxidations at positions other than the carboxylate group, can hardly be found in
literature. However, some preparative applications in whole cell catalysis were
reported and will be discussed in the following (Fig. 16.6-1A,B,C). In vitro, the high
thermodynamic driving force for the oxidation of fonnate and pyruvate [l?(formatel
COZ)= - 0.42 V"]; l? (pyruvate/(acetate, COZ)) = - 0.70 V[']] are used for the
regeneration ofcoenzymes such as NAD(P)H or, indirectly, ATP (Fig. 16.6-1 D,E).
NAD(P)H regeneration
t
D
a C O O H
-C - R-COOH - E - ATP regeneration
6""
Hoot,, OH
C
\
COOH
COOH
Figure 16.6-1. Synthetic and preparative applications of oxidations of acids.

A, 6:Oxidations of benzoic acid initiated by dihydroxylation (Sects. 16.6.4.2
and 16.6.4.3); C: oxidative decarboxylation (Sect. 16.6.4.1); D,E : energy
coupling for the regeneration of coenzymes (Sects. 16.6.2, 16.6.3).
1246
0 2 H A
Figure 16.6-2. Oxidative phosphorylation o f pyruvate by pyruvate oxidase
(PYOX).
16.6.2
Pyruvate Oxidase (PYOx, E. C. 1.2.3.3)
PYOx from Lactobacillus plantarum L3, 4l or Streptococcus s a n g u i ~ [catalyzes

~] the
decarboxylative phosphorylation of pyruvate to acetylphosphate, or the homologous
arsenylation (Fig. 16.6-2).
Acetylphosphate is an important substrate for the enzyme acetate kinase (E.C.
2.7.2.1), which catalyzes the phosphorylation of various nucleotide diphosphates
such as ADP, GDP, TDP, IDP, or UDP to the activated triphosphates["'I. This
reaction can be applied to regenerate ATP in ATP-dependent enzymatic in vitro
reactions (Fig. 16.6-3).
In a recent example, PYOx-catalyzed regeneration of ATP was coupled to in vitro
protein biosynthesis (e.g. for human lymphotoxin)['I. Under aerobic conditions, no
external regeneration system for PYOx has to be applied; catalase however has to be
added in order to destroy harmful hydrogen peroxide. An alternative to this
autoregeneration approach (Fig. 16.6-3A) was reported by Steckhan and coworkers
for cases where hydrogen peroxide formation has to be prevented (Fig. 16.6-3B) [lo].
n
~ow.2- acetate krnasa
.AtOOH +
PYOX PYOX rerl * D n T t o H
\ L
A
W
Fa.
GF
a
-?
Br/AJ
-R
0
w
FB
H A
0,
Eatarass
\further enzymetic reactions
e-
Figure 16.6-3. Decarboxylative phosphorylation o f pyruvate by pyruvate oxidase
as driving force for the regeneration of ATP; A: aerobic regeneration;
B: indirect electrochemical regeneration.
f"T I
1 G. G Oxidation of Acids
1247
Figure 16.6-4. Regeneration of NADH using the formate
NAD' "I-I dehydrogenase (FDH) reaction.
HCOO CO,
Here, the anode, together with the mediation by ferrocene, removes excess electrons
from the PYOx active site.
Another possible application of the PYOx-catalyzed production of acetylphosphate
lies within the in uitro regeneration of acetyl-CoA [ll].
16.6.3
Formate Dehydrogenase (FDH, E. C. 1.2.1.2)
Probably the most prominent oxidation of a carboxylic acid is catalyzed by the

enzyme formate dehydrogenase (FDH, E. C. 1.2.1.2). FDH was isolated from various
bacteria, yeasts, and plants, where its physiological role is the regeneration of
NADH I1*l.
FDH catalyzes the oxidation of formate to carbon dioxide, concomitant with the
reduction of NAD' to NADH (Fig. 16.6-4). Because ofthe favorable thermodynamic
equilibrium of the reaction and the volatility of the reaction product, the enzyme is
commonly applied for in situ regeneration of NADH during asymmetric synthesis of
chiral compounds [I3].
FDH from Cundida boidinii is mostly used as regeneration enzyme. It found
industrial application at Degussa-Huls AG in a leucine dehydrogenase-catalyzed
reductive amination of 2-keto acids yielding various amino acids (e.g. tert-leu-
Native FDH is very selective for NAD'. Recently a new FDH was
developed by site-directed mutagenesis that shows all advantages of the NAD+-
dependent enzymes and additionally accepts NADP' as substrate [I7]. The activity of
the mutant with NADP' is about GO% of the wild-type FDH with NAD+['8].
16.6.4
Oxidations with Intact Microbial Cells
16.6.4.1
Production of Benzaldehydefrom Benzoyl Formate or Mandelic Acid
Benzaldehyde can be produced from benzoyl formate with whole cells of Pseudomo-
nas putidu ATCC 12633 as biocataly~tI'~~ 201 (Fig. 16.6-5). Alternatively, but less
effectively, mandelic acid can be used as starting material. A pH of 5.4 was found to
be optimal for benzaldehyde accumulation. At this proton concentration, partial
inactivation of the benzaldehyde dehydrogenase isoenzymes and activation of the
benzoyl formate decarboxylase are reported. Fed-batch cultivation prevented sub-
strate inhibition. In situ product removal is necessary to prevent product inhibition.
1248
mandelate mandelate benzoyl formate benzaldehyde

racemase dehydrogenase decarboxylase dehydrogenase
isoenzymes
COOH COOH COOH
__c
Figure 16.5-5. Degradation of tridecan-2-one with a crude cell-free preparation

from a Pseudomonas aeruginosa strain.
Activated charcoal served as a solid-phase adsorption device [201. Thus, benzaldehyde

and thiophene-2-carboxaldehydewere obtained from benzoyl formic acid and
thiophene-2-glyoxylicacid respectively, in final concentrations of up to 4.8 g L-’ and
molar yields exceeding 85 %.
16.6.4.2
Microbial Production of cis,cis-Muconic Acid from Benzoic Acid
Significant effort was put into the oxidation of benzoic acid to cis,cis-muconic acid via
a multi-step reaction catalyzed by whole microbial cells [21-241. Cis,cis-muconic acid is
used as raw material for the synthesis of resins and polymers (precursor of adipic
acid). Furthermore, it is widely used as building block in the synthesis of pharmaceu-
ticals and agrochemicals.
As biocatalyst, growing cells of a mutant Arthrobacter strain (lacking cis&-
muconate derivatization activity) was used. The reaction cascade (Fig. 16.6-6) is
initiated by a dioxygenation of the benzylic ring followed by decarboxylationyielding
catechol, which is transformed to the product via dioxygenase-catalyzedring cleav-
age.
benzoate 0 dehydrogenase
p” aoH
1,2-oxidoreductase
&HoH
OH ~
_. .
OH
NADH NAD+ NAD+ NADH +
02
catechol 1,2-
dioxygenase
C1COOH
\ COOH
Figure 16.6-6. Sequential oxidation of benzoate to (cis,cis)-muconic acid

catalyzed by Arthrobocter sp.
References I1249
Figure 16.6-7. Dioxygenation of ben-

COOH
zoate to corresponding cis-l,2-diols.
O,+NADH NAD+
Benzoic acid was fed continuously to the fermentation medium. The space-time
yield of the process including downstream processing amounts to 70 g L-’ d-l.
16.6.4.3
Biotransformationof Substituted Benzoatesto the Corresponding cis-Diols
Enantiopure 1,2-cis-dihydroxycyclohexa-3 ,S-diene carboxylic acids have considerable

synthetic potential as building blocks in chiral synthesis. Such cis-diols can be
produced from benzoic acid derivatives by the action of toluate-1,2-dioxygenaseof
Pseudomonasputida mt-2F2’]or homologous enzymes of a different origin (Fig. 1G.G-
7).
Growing cells or recombinant Pseudomonas oleovorans GPol2 containing toluate-
1,2-dioxygenaseefficiently transform a whole range of meta- and para-substituted
benzoates to the corresponding cis-diols, which are not further degraded by the
Pseudomonas host. In the ortho position only hydrogen and fluorine were accepted as
substituents. Toluate-l,2-dioxygenaseactivity is induced by ortho-toluate or the
substrates themselves.
Similar reactions were reported for the broad-substrate-specificbenzoate dioxyge-
nase of Rhodococcussp. strain 19070[2Gl. Recombinant E. coli containing this enzyme
transform benzoate and anthranilate to catechol and 2-hydro-1&dihydroxybenzoate,
respectively.
References
1 D. D. Woods, Biochem. J. 1936,30,515. 9 D.-M. Kim, J. R. Swartz, Biotech. Bioeng.

2 K. Burton, Ergeb. Physiol. 1957,49, 275. 1999,66,180-188.
3 B. Sedewitz, K. H. Schleifer, F. Gotz,J. Bac- 10 E. Steckhan. Kontinuierliche enzymatische
teriol. 1984, 160, 273-278. Synthesen enantiomerenreiner organischer
4 B. Sedewitz, K. H. Schleifer, F. Gotz,]. Bac- Zwischenprodukte durch elektrochemische
t e n d . 1984, 160, 462-465. Aktiviemng von Redoxenzymen in elektro-
5 J. Carlsson, U. Kujala, FEMS Microbiol. Lett. chemischen Enzymmembranreaktoren -
1985,25,53-56. Final report for the period 01.03.1998 to
6 H. Vigenschow, H.-M. Schwarm, K. Kno- 31.08.2000 on the Research Project 11 556
bloch, Bid. Chem. 1986,367,951-956. N/1; AiF: Bonn, 2000.
7 K. Suzuki, H. Nakajima, K. Imahori, Meth- 11 U. M. Billhardt, P. Stein, G. M. Whitesides,
ods Enzmol. 1982,90,179-185. Bioorg. Chem. 1989, 17, 1-12.
8 J. S. Nishimura, M. J. Griffith, Methods En- 12 V. 0. Popov, V. S . Lamzin, Biochem. J . 1994,
zmol. 1981,71,311-316. 301,625-643.
1250
13
I
M.-R. Kula, U. Kragl, Dehydrogenases in 21 S. Mizuno, N. Yoshikawa, M. Seki,
synthesis of chiral compounds. In Stereose- T. Mikawa, Y. Iamada, Appl. Microbiol. Bio-
lective Biocatalysis. R. N. Patel, (ed) Marcel tech. 1988, 28, 20-25.
Dekker, New York, 1999, pp. 839-866. 22 N. Yoshikawa, S. Mizuno, K. Ohta,
14 A. Liese, K. Seelbach, C. Wandrey. In In- M. Suzuki,]. Biotech. 1990, 14, 203-210.
dustrial Biotransfomations,Wiley- 23 N. Yoshikawa, 0. Ohta, S. Mizuno,
VCH,Weinheim, 2000, pp. 125-128. H. Ohkishi, Production of cis,cis-muconic
15 A. S. Bommarius, M. Schwarm, K. Drauz,J. acid from benzoic acid. In Industrial Appli-
Mol. Cat. B: Enzymatic 1998, 5, 1-11. cation oflmmobilized Biocatalysts. A. Tanaka,
16 U. Kragl, D. Vasic-Racki,C. Wandrey, Bio- T. Tosa, T. Kobayashi (eds), Marcel Dekker,
proc. Eng. 1996,14,291-297. New York, 1993, pp. 131-147.
17 K. Seelbach, B. Riebel, W. Hummel, M.-R. 24 A. Liese, K. Seelbach, C. Wandrey,
Kula, V. I. Tishkov, A.M. Egorov, C. Wan- Oxygenase of Arthrobactersp. In Industrial
drey, U. Kragl, Tetrahedron Lett. 1996, 37, Biotransfmations. A. Liese, K. Seelbach,
1377-1 380. C. Wandrey (eds), Wiley-VCH,Weinheim,
18 V. I. Tishkov, A. G. Galkin, G. N. March- 2000, pp. 137-138.
enko, Y. D. Tsyganov, H. M. Egorov, Biotech. 25 M. G. Wubbolts, K. N. Timmis, Appl.
Appl. Biochem. 1993, 18, 201-207. Environ. Microbiol. 1990, 56, 569-571.
19 J. Simmonds, G. K. Robinson, Enz. Microb. 26 S. Haddad, D. M. Eby, E. L. Neidle,
Tech. 1997,21, 367-374. Appl. Environ. Microbiol. 2001, 67,
20 J. Simmonds, G. K. Robinson, Appl. Micro- 2507-2514.
biol. Biotech. 1998, 50, 353-358.
16.7
Oxidation of C-N Bonds
16.7.1
Introduction
Enzymatic oxidations of carbon-nitrogen bonds are as diverse as the substances

containing this structural element. Mainly amine and amino acid oxidases are
reported for the oxidation of C-N bonds. The steroespecificity of amine-oxidizing
enzymes can be exploited to perform resolutions and even deracemizations or
stereoinversions (Fig. 16.7-1 A). Analogous to the oxidation of alcohols, primary
amines are oxidized to the corresponding imines, which can hydrolyze and react
with unreacted amines (Fig. 16.7-1 B). In contrast to ethers, internal C-N bonds are
readily oxidized, yielding substituted imines. This can be exploited for the produc-
tion of substituted pyridines (Fig. 16.7-1 C). Furthermore, pyridines can be oxidized
not only to N-oxides but also to a-hydroxylatedproducts (Fig. 16.7-1 D).
16.7.2.1 and 16.7.3.1);
A R B: preparation of alde-
hydes (and subsequent
formation o f imines) by
oxidation of primary
R 16.7.3.2); D: hydroxyla-
16.7.2
Oxidations Catalyzed by Dehydrogenases
16.7.2.1
L-Alanine Dehydrogenase (L-Ala-DH, E.C. 1.4.1.1)
L-Alanine dehydrogenase (1-Ala-DH,E. C. 1.4.1.1) catalyzes the specific deaminative

oxidation of L-alanine and thus can potentially be exploited for the resolution of
racernic alanine (e.g. derived from the Strecker-synthesis).However, the oxidation of
secondary alcohols and amines is thermodynamically unfavorable ['I, so that the
equilibrium of the reversible dehydrogenase reaction is on the substrate side.
Therefore, an additional thermodynamic driving force has to be introduced into the
system in order to drive the desired reaction towards completion. Moiroux and
coworkers recently introduced such a system (Fig. 16.7-2)[-'I.
The general philosophy of their approach is the utilization of electrical power to
remove the dehydrogenase products NADH and pyruvate (which is in situ trans-
formed into the corresponding irnine), thus driving the equilibrium reaction
towards completion. The electrochemical oxidation and reduction reactions produce
NAD' and racemic alanine, respectively, as substrates for the dehydrogenase
reaction. Using this procedure, not only a racemate resolution (with maximum 50 %
yield) but a deracemization (100% yield) is achieved. The overall rate-limiting step is
the slow, non-enzymatic formation of the imine. Consequently, the process is very
slow (at best, the complete conversion of a 10 mM solution of r-alanine required
140 h).
1252
TI
L-Ala-DH
Figure 16.7-2. Stereoinversion o f L-alanine t o o-alanine catalyzed by L-alanine dehydrogenase

(L-Ala-DH) in an electrochemical reactor.
16.7.2.2
Nicotinic Acid Dehydrogenase (Hydroxylase) (E.C. 1.5.1.13)
The membrane-bound molybdoenzyme[G1 nicotinic acid dehydrogenase catalyzes

the first step in the microbial degradation of nicotinic acid by inserting a hydroxyl
function a to the nitrogen atom (Fig. 16.7-3).A possible mechanism for this reaction
is given in Fig. 16.7-417].
The inserted hydroxyl function originates from water, which was confirmed by
H2180 experiments [,' 1' . While nicotinic acid dehydrogenase does not accept NAD'
as electron acceptor, artificial mediators such as benzyl viologene and 2,3,5-triphe-
nyltetrazolium dyes can replace NADP+[']. Various bacterial strains have been
reported to convert a broad range of nicotinic acid derivatives (Table 16.7-1)[lo,12].
An industrial process (according to the first entry in Table 16.7-1) was set up by
* c
QCOOH HOQCOOH ~ c citric acid cycle
Figure 16.7-3. Microbial mineralization o f nicotinic acid.
Figure 16.7-4. Proposed mechanism for enzymatic hydroxylation o f nicotinic

acid (A = acceptor). The reaction scheme is based on the so-called arine
mechanism.
16.7 Oxidation ofC-N Bonds I1253
Table 16.7-1. Microbial a-hydroxylationof substituted pyridines.

Reactions catalyzed by whole cells Final product Enzymes and reference
concentration [g L-'1
74 Dehydrogenase [11
191 Dehydrogenase ['I
301 Dehydrogenase 131
6.4 Dehydrogenase 14]
98 Dehydrogenase ['I
Akdgenesop UK21
coon Z O O C O O H
N R" Dehydrogenase and
decarboxylaseIG]
Rhmbiurn Sp LA17
NRa Dehydrogenase [71
COOH coon
oCN 45 DehydrogenaseI['
40 Nitrilase and
Dehydrogenase [91
55 Nitrilase and
Dehydrogenase1'
40 Nitrilase and
Dehydrogenase [lo]
8 Dehydrogenase [''I
a N R not reported.
1 H. Kulla, Chimia 1991,45,81-85. 7 T. Yoshida, T. Nagasawa, Biosci. Biotech. Biochem.

2 T. Nagasawa, B. Hurh, T. Yamane, Biosci. Biotech. 2000,89,111-118.
Biochem. 1994,58,665-668. 8 M. Yasuda, T. Sakamoto, R. Sashida, M. Ueda,
3 B. H u h , M.Ohshima, T. Yamane, T. Nasagawa,]. Y.Morimoto, Biosci. Biotech. Biochem. 1995,59,
Fern. Bioeng. 1994,77,382-385. 572.
4 M. Ueda, R. Sashida,]. Mol. Cat. B: Enzymatic 9 A. Kiener. USP5266469 (1993).
1998,4,199-204. 10 M. Wieser, K. Heinzmann, A. Kiener, Appl. Micro-
5 A. Kiener, R. Glockler, K. Heinzmann, /. Chem. bid. Biotechnol. 1997,48,174.
Soc. Perkin Trans. I1993,1201-1202. 11 A. Kiener, Y. van Gameren, M. Bokel.; USP
6 T. Yoshida, A. Uchida, T. Nagasawa. "Regiospe- 5,284,767, 1994.
cific ..."; Annu. Meet. SOC. Biosci. Bioeng., 1998,
Japan.
IG
1254
HO
QCOOH
-
___) H./ i"-y-(/ xNoz
CI N
Figure 16.7-5. 6-Hydroxynicotinic acid as synthon for the pesticide
Imidachloprid.
Lonza AG, Switzerland. 6-Hydroxynicotinicacid is precipitated from the fermenta-

tion broth as magnesium salt in the so-called pseudocrystal process, thus enabling
not only easy downstream processing but also continuous fermentation [I3]. 6-Hy-
droxynicotinic acid is the key building block in the synthesis of Imidachloprid
(Fig. 16.7-S), an effective pesticide against hemipterans and other sucking in-
sects [10, 111
16.7.3
Oxidations Catalyzed by Oxidases
16.7.3.1
Amino Acid Oxidases
Among the enzymes catalyzing oxidations of carbon nitrogen bonds, the amino acid
oxidases (AAO, E. C. 1.4.3.x) are the most interesting for synthetic applications.
Compared to some specific amino acid oxidases such as aspartate oxidase or
glutamate oxidase, the two D- and L-amino acid oxidases (E. C. 1.4.3.2 for L-AAOand
E.C. 1.4.3.3 for D-AAO) are advantageous on account of their broad substrate
NH2
R ~ C O O H
L-AA
NH \ HO
,, + NH, +
0
RA k O H R ACOOH
L-AAO
\ NH
R AO
: OH
D-AA
Figure 16.7-6. Resolution of racemic amino acids (AA) catalyzed by
(D)- and (L)-specific amino acid oxidases (AAO).
7 G. 7 Oxidation of C-N Bonds
HowNHz HO, YH,
P O O H + <COO,
NWNH NVNH
1
0, HO
,, + NH,
bleomycine
Figure 16.7-7. Resolution of D,L-erythro-P-hydroxyhistidine as the enantiospecific
step i n bleomycine synthesis.
L-amino acid
t
R = CH,CH,SCH, methionine FDH

CH,CH(CH,), leucine
Figure 16.7-8. Enzymatic deracemization of amino acids catalyzed by

D-amino acid oxidase (D-AAO). Leucine dehydrogenase (LeuDH) transforms
the oxidation product o f the undesired amino acid enantiomer in situ into the
racemic amino acid. Regeneration o f N A D H is performed by formate
dehydrogenase (FH D).
spectrum and their strict stereo~pecificity~'~.

'I. Therefore, AAOs are most com-
monly used for the resolution or deracemization of racemic amino acid mixtures
(Fig. 16.7-6).
The approach outlined in Fig. 16.7-6 was used for example to remove traces of D-
methionine from 99% pure t-methionine[16,"1 or to transform racemic phenyl-
alanine quantitatively into D-phenylalanineand phenylpyruvic acid ["I. Coimmobili-
zation with catalase on a solid matrix (EupergitB) resulted in largely increased D-
AAO stability. In an enzyme-membrane-reactor,space-time-yieldsas high as 90 g L-l
d-' were reached. In another example, a racemic mixture of D,L-erythro-P-hydroxy-
histidine was converted into the ketoacid and L-erythro-j3-hydroxyhistidine[191. The
1256
I
L-pipecolic acid
Figure 16.7-9.One-pot chemo-enzymatic deracemisation of pip pipe colic acid catalyzed by

D-amino acid oxidase (D-AAO).Utilization of catalase was not reported.
latter compound is a key intermediate in the synthesis of the anti-tumor agent

bleomycine (Fig. 16.7-7)
Simple racemate resolutions have a maximal yield of 50% for the desired
compound. Furthermore, additional (potentially laborious) separation steps are
necessary. As a consequence, alternativeprocesses that involve the stereoinversion of
the undesired enantiomer are gaining increasing interest[*']. One approach for these
so-called deracemization processes is to reconvert the oxidation product either
enzymatically (Fig. 16.7-8)or chemically (Fig. 16.7-9)to the racemic substrate.
The enzymatic variant of this concept was reported for the deracemization of D,L-
methionine or D,L-leucine (Fig. 16.7-8)[I7]. Soda and coworkers developed a chemo-
enzymatic racemization procedure utilizing boron hydrides for non-enantioselective
reduction of the undesired D-AAO product (Fig. 16.7-9)[213 221. Using the same
procedure, the authors achieved conversion of D-proline into L-proline["I. Fur-
thermore, D,L-lactate and 2-hydroxy butyric acid were deracemized by utilizing L-
lactate oxidase[231.
The D-AAOcatalyzed oxidative deamination of cephalosporin C found industrial
application (Hoechst Marion Roussel, Germany) as the first step in the so-called
7-aminocephalosporanicacid (7-ACA)process (Fig. 16.7-10)[24-261.
Using this process, this application of heavy metals and chlorinated hydrocarbons
can be avoided, and the volumes of waste-gas as well as of mother liquors are
drastically reduced L2'1.
16.7.3.2
Amine Oxidases
16.7.3.2.1 Monoamine Oxidase (MAO, E.C. 1.4.3.4)

The flavoenzymes monoamine oxidase A and B (MAO-A, MAO-B)[271 catalyze the
oxidative deamination of various primary and secondary amines and the oxidation of
tertiary amines. Their physiological role, as the various synonyms such as epineph-
16.7 Oxidation of C-N Bonds
Figure 16.7-10. Enzymatic reaction

I
1257
YgP&oy
K'
sequence for the production o f 7-ACA
0 from cephalosporin C.
cephalosporin C COOH 0
D-MO
H202
NH3
""""miY
N&ou
K:
0
COOH 0
spontaneous
H O O C - - T i ~ &0
oy
COOH 0
glutaryl amidase
E.C. 3.1.1.41
K~~~CvCOOH
b $ L o ,
0
COOH 0
7-aminocephalosporanicacid (7-ACA)
INH2
serotonine
(i' /
phenetylamine milacemide
, . +O
H
HO Figure 16.7-11. Various neurotransmit-

HO
ters as substrates for mitochondria1
noradrenaline adrenaline monoamine oxidase (MAO).
1258
U MA0
(HAT or SET)
R
I
FAD FADH FADH ' FADH,
HP, 0,
Proposed mechanisms for the oxidation of primary and secondary
Figure 16.7-12.
amines by monoamine oxidase (MAO).
MA0
I I I
Figure 16.7-13. Oxidation of l-methyl-4-aryl(heteroaryl)-l,2,3,6-tetrahydro-
pyridines catalyzed by rnonoamine oxidase (MAO).
rine oxidase, serotonin oxidase, tyramine oxidase, or adrenaline oxidase suggest, is

the transformation of neurotransmitters via oxidative deamination (Fig. 16.7-11) as
well as the detoxification of xenobiotics[28-301.
The mechanism of M A 0 is still a topic of hydrogen atom transfer
(HAT)[321 or single electron transfer (SET)[331 are discussed as initial oxidation steps
in the overall mechanism (Fig. 16.7-12).
Various substrates have been specified for M A 0 with respect to synthetical,
mechanistical and biochemical purposes. Castagnoli and coworkers elucidated
structural requirements of MAO-B with various substituted l-methyLl,2,3,G-tetra-
MA0
HO HO
dopamine
HO
spontaneous
Picet-Spengler
condensation
HO
HO norlaudanosine
Figure 16.7-14. Preparation of norlaudanosine initiated by the oxidation of dopa-
mine by monoamine oxidase (MAO) (A). The oxidation product reacts sponta-
neously in a Picet-Spengler condensation with unreacted dopamine (B).
Table 16.7-2.
167 Oxidation ofC-N Bonds
Oxidation o f various amines catalyzed by monoamine oxidase in n-octane (0.5 %

I 1259
v/v water) ["I.
0""'
Substrate Product Yield I"/.]
99
0"""' 99
P" 69
6 Q
12 7. C. G. Woo, X. Wang, R. B. Silverman,J. Org. Chem. 1995,60,6235-6236.
14
hydropyridines to produce dihydropyridines that are further oxidized to pyridinium

structures (Fig. 16.7-13)[31* 341.
M A 0 was used in viuo and in uitro as a catalyst for the production of norlaudano-
sine from dopamine (Fig. 16.7-14)r3'1. Norlaudanosine is an important synthon for
benzylisoquinoline alkaloids, providing the upper isoquinoline portion of the
morphinan skeleton. In uitro and in viuo yields were in the range of 20%.
MAO-B was also tested in low water content organic media such as ether,
tetrachloromethane, octane, benzene and cyclohexane. Under optimized conditions
quantitative conversions of various substrates were achieved (Table 16.7-2)f3'1.
16.7.3.2.2 Diarnine Oxidase (E.C. 1.4.3.6)

The copper-containing amine oxidases (copper amine oxidases, diamine oxidases)
possess either a topaquinone or a 6-hydroxydopamine cofactor (Fig. 16.7-15), gen-
erally integrated in the oxidase primary structure. Tyrosine residues of the enzyme
backbone in the active site are discussed as precursors for the prosthetic group[37].
As the name suggests, diamine oxidase catalyzes the oxidative deamination of
diamines. Preferably a,w-diamines such as putrescine (1,4-diaminobutane) or
cadaverine (1,s-diaminopentane) (the names already suggest their smell), but also
various derivatives are readily converted. Quite often cyclic imines are obtained via
internal nucleophilic attack by the unreacted amino function (Fig. 16.7-16)[38-401.
1260
I Figure 16.7-15. Topaquinone
and 6-hydroxy-dopamine as pros-
COOH thetic groups of diamine oxida-
o$ H_ 2e-2H+ - H o d ses.
OH
OH OH
topaquinone 6-hydroxydopamine
Figure 16.7-16. Applica-

tion of diamine oxidase in
H2N (-3b) the synthesis of different
n
diarnine oxidase N azaheterocycles.
n = 1,2,3
X = 0, S , CH(CH,)
In the presence of suitable nucleophiles (such as benzoyl acetic acid) the primary
imines can be spontaneously further modified in situ. A convenient approach to
obtain phenacyl-derivatives,building blocks in the synthesis of certain alkaloids, was
reported r3’]. In some cases, diamine oxidases exhibit activities complementary to
monoamine oxidases. For example vanillylamine is far more efficiently converted
into vanillin by a diamine oxidase from Aspergillus niger than by the monoamine
oxidase from E. coli[”].
Even enantioselectiveoxidations of some alkyl-,benzyl-, or phenylethyl- (arylethyl-
) amines were reported with diamine oxidase from pea settlings[41].Porcine kidney
diamine oxidase was used for the oxidative transformation of Nitruria alkaloids such
as na~lininI~~1.
For the conversion of poorly water-soluble amines (and to avoid product inhibi-
tion), diamine oxidase can also be applied in non-aqueous media[43].
16.7.4
Oxidations Catalyzed by Transaminases
Transaminases are generally not considered to be enzymes catalyzing redox reac-

tions, which is obvious considering the meaning of the E. C. code for transferases
(E. C. 2.6.1.x = transferring amino groups). Nevertheless, the exchange of an amino
functionality between an amino acid and an a-keto acid implies the oxidation of the
amino acid. Transaminases are described elsewhere in this book (Chapter 12).
References 11261
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174-188. Org. Chem. 1995,60,6235-6236.
15 P. Wikstrom, E. Szwajcer, P. Brodelius, K. 37 N. K. Williams, J. P. Klinman, J. Mol. Cat. B:
Nilsson, K. Mosbach, Biotechnol. Lett. 1982, Enzymatic 2000,8,95-101.
4,153-158. 38 J. E. Cragg, R. B. Herbert, M. M. Kgaphola,
16 K. Parkin, H. 0. Hultin, Biotech. Bioeng. Tetrahedron Lett. 1990, 31, 6907-6910.
1979,939-953. 39 A.M. Equi, A. M. Brown, A. Copper, S. K.
17 N. Nakajima, D. Conrad, H. Sumi, N. Esaki. Ner, A. B. Watson, D. J. Robins, Tetrahedron
C. Wandrey, K. Soda, Ferment. Bioeng. 1990, 1991,47.
70, 322-325. 40 E. Santaniello, A. Manzocchi, P. A. Biondi,
18 R. Fernandez-Lafuente, V. Rodriguez, J. C. Secchi, T.Simonic, J. Chem. Soc., Chem.
Guisan, Enz. Microb. Tech. 1998, 23. Commun.1984,803-804.
19 S. M. Hecht, K. M. Rupprecht, P. M. Jacobs, 41 A. R. Battersby, J. Staunton, M. C. Sum-
J. Am. Chem. Soc. 1979, 101,3982-3983. mers, J . Chem. Soc., Chem. Commun.1974,
20 W. Kroutil, K. Faber, Tetrahedron: Asym. 465,548-549.
1998,9,2901-2913. 42 E. Cheng, J. Botzem, M. J. Wanner, B. E.
21 J. W. Huh, K. Yokoigawa, N. Esaki, K. Soda, B u m , G.-J. Koomen, Tetrahedron 1996, 52,
J . Ferment. Bioeng. 1992,74, 189-190. 5725-6732.
22 J. W. Huh, K. Yokoigawa, N. Esaki, K. Soda, 43 J. A. Chaplin, C. L. Budde, Y. L. Khmel-
Biosci. Biotech. Biochem. 1992, 56, nitsky,J. Mol. Cat. B: Enzymatic 2001, 13,
2081-2082. 69-75.
lG Oxidation Reactions
1262
I
16.8
Oxidation at Sulfur
Karl-Heinz van Pee
16.8.1
Enzymes Oxidizing at Sulfur and their Sources
The oxidation at sulfur is catalyzed by a number of different enzymes produced by a

variety of organisms. They have been isolated from a fungus"], soybeanr2],rat, pig
and rabbit liver L3-'1, horseradish r61, bacteria ['-I, milk[10],and human white blood
cells ~ ' 1 .
The enzymes catalyzing oxidation reactions at sulfur belong to two different
classes of enzymes: monooxygenases, including cytochrome P-450monooxygenases
and FAD-containing monooxygenases, and heme-containing peroxidases (Figs.
16.8-1and 16.8-2, Table 16.8-1).
Some of these enzymes such as chloroperoxidase from Caldariomyces $mago,
horseradish peroxidase, lactoperoxidase from bovine milk, and myeloperoxidase
from human white blood cells are commercially available.
Others such as pig liver microsomal FAD-containing monooxygenase have to be
isolated from tissue with very low yieldsI41or like hydrocarbon monooxygenase from
Pseudomonas oleovorans [12-131 require several protein components and cofactors,
substantially limiting the use of these enzymes for the production of oxidized sulfur
compounds.
monooxygenase
S-CH, SOCH,
H A
(R) or (S)
Figure 16.8-1. Oxidation of methyl p-tolyl sulfide to methyl p-tolyl sulfoxide by a
monooxygenase. The product can either be ofthe R- or S-configuration depend-
ing on the monooxygenase used.
peroxidase/haloperoxidase
SOCH,
HZOZ
(R) or (S)
Figure 16.8-2. Oxidation of methyl p-tolyl sulfide to methyl p-tolyl sulfoxide by a peroxidase
or haloperoxidase in the presence of hydrogen peroxide. The product can either be
predominantly of the R- or S-configuration depending on the peroxidase or haloperoxidase
used.
Table 16.8-1.
168 Oxidation a t Sulfur
Classification o f enzymes oxidizing at sulfur and their sources.

I
1263
Enzyme class Source Reference

Monooxygenases pig liver microsomes 14
rat liver microsomes 3
rabbit liver microsomes 16
bovine adrenals 17
Pseudomonas oleovorans 12,13
Acinetobacter sp. 15
Peroxidases soybean 2
horseradish 23
Haloperoxidases Caldariomyces&mago 1
Ascophyllumnodosum 27
Corallina ojicinalis 28
human white blood cells 30
bovine milk 25
16.8.2
Oxidation of Sulfides
16.8.2.1
Oxidation of Sulfides by Monooxygenasesand by Whole Organsims
Fujimori et al. [I4] used pig liver microsomal FAD-containing monooxygenase and
phenobarbital-induced rabbit liver microsomal cytochrome P-450 to catalyze the
oxidation of unsymmetrical sulfides to the corresponding optically active sulfoxides
with varying degrees of enantiomeric excess (12-96%). Comparison of the oxy-
genation of racemic 2-methyl-2,3-dihydrobenzo[b] thiophene showed that the en-
antiotopic, diastereotopic, and enantiomeric differentiating abilities of the FAD-
containing monooxygenase are higher than those of the cytochrome P-450 mono-
oxygenase. They found that the oxygenation with the FAD-containing mono-
oxygenase is sterically much more highly controlled than that with cytochrome
I?-450. Whereas higher ee-values are observed in the oxygenation of smaller sulfides
with the FAD-containing monooxygenase, the oxygenation of large sulfides by the
cytochrome P-450 monooxygenase results in higher ee values than those of sulfides
bearing small substituents.
Hydrocarbon monooxygenase from Pseudomonas oleo~orans[~-'*I also catalyzes '9
the stereoselective sulfoxidation of methyl thioether substrates [131 with up to 80% ee.
The products obtained with this enzyme are probably ofthe R-configiration.
The (S)-(-)-sulfoxideis predominantly produced (82 % S, 18 % R) from p-tolyl ethyl
sulfide when cyclohexanone monooxygenase from Acinetobacter sp. NCIB 9871[I'
was used, whereas the the FAD-containing monooxygenase from hog liver micro-
somes oxidizes p-tolyl ethyl sulfide to yield the (R)-(+)-sulfoxideenantiomer as the
major product (95 % R, 5 % S) Llsl.
The enzymatic oxidation of various diaryl, dialkyl, and aryl alkyl sulfides by
cytochrome P-450 from rabbit liver resulted predominantly in the formation of the
sulfoxides with the R-configuration[16].
1264
I
The S-(-)configuration was predominantly obtained when two cytochrome P-450
isoenzymes from rat liver were used for the oxidation of p-tolyl ethyl
Oxidation of phenyl2-aminoethyl sulfide by dopamine b-hydroxylase from bovine
adrenals in the presence of ascorbate as the electron donor resulted in the formation
of phenyl 2-aminoethyl sulfoxide. The product was probably of the S-configura-
tion[l7I.
Holland et al. [18] obtained the (R)-sulfoxidesfrom various para-substituted phenyl
3-chloropropyl and phenyl 3-hydroxypropyl sulfides by biotransformation with the
fungus Mortierella isabellina with an enantiomeric excess of 82-88%. The (S)-
sulfoxides were produced using the fungus Helrninthosporiurn sp. and the bacterium
Acinetobacter calcoaceticuswith ee values of > 95 % and 94%, respectively.
16.8.2.2
Oxidation of Sulfides by Peroxidases and Haloperoxidases
A number of peroxidases were investigated for their use in oxidizing organic

sulfides. p-Substituted thioanisols were oxidized by partially purified soybean sulfox-
idase using 13(S)-hydroperoxylinoleicacid as the peroxide. Methyl p-tolyl sulfide
gave the (S)-sulfoxidewith about 90 % ee 12].
The sulfoxidation of organic sulfides by chloroperoxidase from Caldariomyces
firnago was investigated by different groups [19-261. Colonna et al. ['I compared the
oxidation of sulfides by this enzymes with that catalyzed by horseradish peroxidase.
Chloroperoxidase catalyzed the formation of sulfoxides with tert-butyl and other
peroxides with an R absolute configuration in up to 92% ee, whereas horseradish
peroxidase gave racemic products. When sterically hindered oxidants such as cumyl
hydroperoxides and chloroperoxidase were used, racemic or almost racemic prod-
ucts were obtained. tert-Butyl hydroperoxide also had the advantage of giving higher
yields and higher ee.
Using vanadium bromoperoxidases from marine algae the (S)- or (R)-sulfoxides
can be obtained from methyl phenyl sulfide derivatives, respectively, depending on
the source of the enzyme. While bromoperoxidase from Ascophgllum nodosum
produces the (R)-sulfoxidewith 91% ee1271,the (S)-enantiomer is obtained with
bromoperoxidases from CoralZina o#cinalis and C. piluli&ra[281.
When investigating the substrate selectivity using a series of aryl, alkyl, dialkyl,
and heterocyclic sulfides, it was found that p-substitution led to higher enatioselectiv-
ity and higher chemical yields with respect to o-substitution[*'].A similar influence
of the p-substitution was found for sulfoxidation catalyzed by bromoperoxidase from
the marine alga ~scophyZLum~ o ~ o s u ~ ~ ~ I .
Benzyl methylsulfide, thioanisol, and thiobenzamide were oxidized by chloroper-
oxidase, lactoperoxidase, and horseradish peroxidase to the respective sulfoxides.
Whereas lactoperoxidase and horseradish peroxidase had low activities towards
benzyl methylsulfide, thiobenzamide was efficiently oxidized by lactoperoxidase.
Chloroperoxidase had high activity in halide-independent reactions towards all three
substrates['']. This enzyme was also used for the asymmetric sulfoxidation of a
series of cyclic sulfides. In all cases the (R)-sulfoxideswere obtained. In the case of
Table 16.8-2. Products and absolute configuration obtained in the oxidation of various sulfides by different enzymes.
Sulfide Predominant configuration Enzyme or organism Reference
of sulfoxide obtained
Methyl phenyl R chloroperoxidase 19-21
R vanadium bromoperoxidase 27
S vanadium bromoperoxidase 28
Methyl p-tolyl S soybean hydroperoxide- 2
dependent oxygenase
Ethyl p-tolyl S rat liver cytochrome P-450 3
cyclohexanone monooxygenase 15
FAD-containingmonooxygenase 15
Methyl alkyl alkane monooxygenase 13
Diaryl, dialkyl, aryl alkyl rabbit liver cytochrome P-450 16
Phenyl2-aminoethyl dopamine P-hydroxylase 17
Phenyl3-chloropropyl Mortierella isabellina 18
Phenyl3-chloropropyl Helminthosporiurn sp. 18
2,3-Dihydrobenzo[b]thiophene chloroperoxidase 29
Q
1266
I
2,3-dihydrobenzo[b]thiophenethe yield was 99.5 % with an ee of 99%[*'1. Table
16.8-2shows some examples of sulfides oxidized to sulfoxides by different enzymes
and the absolute configuration of the products. When using peroxidases, care has to
be taken, as the peroxidase-catalyzed oxidation is in competition with the sponta-
neous oxidation of the sulfides by the oxidant.
Depending on the enzyme used for oxidation of organic sulfides, sulfoxides with
S- or R-configuration can be obtained with high ee, whereas at present there is only
one chemical oxidation method which leads to high ee in alkyl aryl sulfoxides. This
method uses chiral titanium complexes and cumene hydroperoxide for the oxidation
of organic sulfides L2'].
References
1 D. R. Morris, L. P. Hager,]. B i d . Chem. Kim, T. Iyanagi, S. Oae, Bull. Chem. Soc.

1966,241,1763-1768. Jpn. 1983,56,2300-2310.
2 E. Blee, F. Schuber, Biochemistry 1989, 28, 17 S. W. May, R. S. Phillips, J . Am. Chem. SOC.
4962-4967. 1980,102,5983-5984.
3 D. J. Waxrnan, D. R. Light, C. Walsh, Bio- 18 H. L. Holland, J.-X. Gu, A. Kerridge, A.
chemistry 1982,21,2499-2507. Willetts, Biocat. Biotrans. 1999, 17, 305-317.
D. M. Ziegler, L. L. Poulsen, Methods Enzy-
4 19 S. Colonna, N.Gaggero, A. Manfredi, L.
mol. 1978, Vol. 52, 142-151. Casella, M. Gullotti, /. Chem. Soc., Chem.
5 Y. Irnai, R. Sato, Biochem. Biophys. Res. Com- Commun. 1988,1451-1452.
m u n . 1973, 60, &14. 20 S . Colonna, N. Gaggero, A. Manfredi, L.
6 L. M. Shannon, E. Kay, J. Y. Lew, J. Biol. Casella, M. Gullotti, G. Carrea, P.Pasta,
Chem. 1966,241,2166-2171. Biochemistry 1990, 29, 10465-1048.
7 A. G. Katopodis, K. Wimalasena, J. Lee, 21 S. Colonna, N. Gaggero, L. Casella, G. Car-
S. W. May,]. Am. Chem. SOC.1984, 106, rea, P. Pasta, Tetrahedron: Asymmetry 1992,
792&7935. 3,95-106.
8 S. W. May, L. G. Lee, A. G. Katopodis, J.Y. 22 L. Casella, S. Colonna, G. Carrea, Biochem-
Kuo, K. Wirnalasena, J. R. Thowsen, Bio- istry 1992,31,9451-9459.
chemistry 1984,23,2187-2192. 23 S. Kobayashi, M. Nakano, T. Goto, T. Ki-
9 N. A. Donoghue, D. B. Norris, P. W. Trudg- rnura, A. P. Schaap, Biochem. Biophys. Res.
ill, Eur. J. Biochem. 1976, 63, 175-192. Commun. 1986, 135, 166-171.
10 C. Dumontet, B. Rousset,]. Biol. Chem. 24 S. Kobayashi, M.Nakano, T. Kimura, A. P.
1983,258,14166-14172. Schaap, Biochemistry 1987,26, 5019-5022.
11 J. Schultz, Reticuloendothel. Syst. 1980, 2, 25 D. R. Doerge, Arch. Biochem. Biophys. 1986,
231-254. 244,678-685.
12 S. W. May, A. G. Katopodis, Enzyme Microb. 26 D. R. Doerge, N. M. Cooray, M. E. Brewster,
Technol. 1986,8, 17-21. Biochemistry 1991,30,8960-8964.
13 A. G.Katopodis, H. A. Smith, Jr., S. W. May, 27 H. B. ten Brink, H. L. Holland, H. E. Shoe-
/.Am. Chem. SOC.1988, 110,897-899. maker, H. van Lingen, R. Wever, Tetrahe-
14 K. Fujirnori, T. Matsuura, A. Mikami, Y. dron: Asymmetry 1999, 10,4563-4572.
Watanabe, S. Oae, T. Iyanagi, /. Chem. Soc. 28 M. A. Anderson, S. G. Allenmark, Tetra-
Perkin Trans. 1 1990, 1435-1440. hedron 1998,54,15293-15304.
15 D. R. Light, D. J. Waxrnan, C. Walsh, Bio- 29 S . G. Allenmark, M. A. Anderson, Chirality
chemistry 1982, 21, 2490-2498. 1998, 10,246-252.
16 T. Takata, M. Yarnazaki, K. Fujirnori, Y. H. 30 M.-F. Tsan,/. Cell.Physiol. 1982, I l l , 49-54.
76.9 Halogenation
I
1267
16.9
Halogenation
Karl-Heinz van Pee
16.9.1
Classification of Halogenating Enzymes and their Reaction Mechanisms
16.9.1.1
Haloperoxidases and Perhydrolases
The only type of halogenating enzymes known until 1997 were peroxidases and
perhydrolases which catalyze the formation of carbon halogen bonds using halide
ions, hydrogen peroxide and an organic substrate activated for electrophilic attack.
According to the halide ions they can utilize they are arranged into three groups:
iodoperoxidases, bromoperoxidasesand chloroperoxidases. Iodoperoxidases catalyze
the formation of carbon-iodine bonds, whereas bromoperoxidases catalyze iodina-
tion and bromination reactions, and chloroperoxidases catalyze the iodination,
bromination, and chlorination of organic substrates. As haloperoxidases are oxidor-
eductases using hydrogen peroxidase as the oxidant for the oxidation of halide ions
producing hypohalogenic acids, the existence of fluoroperoxidases can be ruled out.
The overall reactions catalyzed by haloperoxidases and perhydrolases are shown in
Fig. 16.9-1. All haloperoxidases isolated until 1984 were heme-containing en-
zymes[’]. The first non-heme haloperoxidase was isolated by Vilterr2]. Instead of
heme, vanadium is responsible for the halogenating activity of this algal en-
zyme 41. Non-heme and non-metal “haloperoxidases”were isolated from bacte-
139
ria however, elucidation of the three-dimensional structure and the reaction
’) ‘-+ H’
a) heme-haloperoxidase
b) vanadium haloperoxidase
HOX
2) R-H + HOX * R-X+ H,O
1) CH,COOH + HO
,,
c) perhydrolase
- CH,COOOH
2) CH,COOOH + X- * CH,COOH + HOX
3)HOX + R-H * R-X+H,O
X- = CI-, Br, I-
Figure 16.9-1. Overall reaction catalyzed by (a) heme-IS’]and (b) vanadium-
containing haloperoxidases[5’’ and (c) perhydrolases []’‘.
1268
I mechansim of this type of halogenase showed that they are not real haloperoxidases.
They are actually perhydrolases which produce hypohalogenicacids via the oxidation
of halide ions by enzymatically formed peracetic acid[10-121.Thus, in addition to
grouping the haloperoxidases according to the range of halide ions oxidized, they can
be classified according to their prosthetic group into heme type and non-heme type
haloperoxidases L1 '1.
The heme type haloperoxidases are inactivated during the halogenation reaction,
because the heme group of these enzymes is attacked by the hypohalous acids
produced by the enzymes [l].Thus, heme type haloperoxidases have the disadvantage
that the reaction velocity slows down considerably during the course of the reac-
ti0n['~1.With non-heme type haloperoxidases this does not seem to be the case. They
are not inactivated during the halogenation reaction and are very stable under
reaction conditions [l41. However, the disadvantage of inactivation is partly compen-
sated for by the fact that some of the heme type haloperoxidases have much higher
specific activities than non-heme type haloperoxidases.
Some of the non-heme haloperoxidases are very stable with respect to organic
solvents[15]which is of great importance when the substrates that are to be
halogenated are not very soluble in water. In these cases water missible organic
solvents can be added to the reaction mixture or a two phase-system can be used.
16.9.1.2
FADH2-dependent Halogenases
In 1997 the existence of a novel class of halogenating enzymes was reported[161.

These halogenases showed no relationship to any of the known haloperoxida-
ses[l7.1" and did not require hydrogen peroxide for halogenating activity. Initially
these new halogenases were thought to require NADH [16],but more detailed studies
showed that they actually require FADHZ['~, 1" which is produced by NADH-
dependent flavin reductases. Figure 16.9-2 shows the hypothetical reaction mecha-
nism of FADH2-dependent halogenases.
16.9.2
Sources and Production of Enzymes
16.9.2.1
Although FADH2-dependent halogenases seem to be present in many bacteria

producing halometabolites[19z21, 22], only one example of this new class of haloge-
nases has been isolated to homogeneity until now. This enzyme, tryptophan
7-halogenase, is produced by several Pseudomonas strains producing the antibiotic
pyrrolnitrin such as Pseudomonas fluorescens and P. aureofaciens and by Myxococcus
f i l ~ u s [ ~Monodechloroaminopyrrolnitrin-3-halogenase,
~]. another FADH2-depend-
ent halogenase from pyrrolnitrin-producing Pseudomonas strains has so far only
been purified partially [ I 6 . 2ol.
1G. 9 Halogenation
flavin reductase
FAD + NADH + H+ FADH, + NAD+
QTC
C
&
O:OH
H
FADH,, 0,
halogenase ~
0
07 C
!{;iOOH + FAD + H,O
CI-, H+ halogenase
PTC&:COOH - halogenase
- H,O
QTC
C
&
O:OH
HO
CI CI
Figure 16.9-2. Hypothetical reaction mechanism of FADH2-dependent tryptophan
7-halogenase as an example of FADH2-dependent halogenases[201.
From biosynthetic and hybridization studies it is known that FADH2-dependent

halogenases are involved in the biosynthesis of many halometabolites produced by
bacteria['', 221 and it can be expected that other FADH2-dependent halogenases
"9
will be purified and characterized in the near future.
16.9.2.2
Haloperoxidases and Perhydrolases
Iodoperoxidases such as horseradish peroxidase [241 and thyroid peroxidase LZ51 can be
isolated from many different organisms. Bromoperoxidases have been obtained in a
pure form from mammals (lactoperoxidase)[26], sea urchin (ovoperoxidase)[I',
marine algae 128-301, fungi (lignin peroxidase)[321 and bacteria [33, 341. Chlor-
operoxidases have been found in mammals (myeloperoxida~e[~~1 and eosinophil
peroxidase [36j), a marine worm [371, and fungi [3s401. Several perhydrolases have been
isolated from bacteria L5-', 4Ll.
Chloroperoxidase can be produced in batch culture at concentrations of 280 mg
L-1[421 and 20 mg of lactoperoxidase can be isolated from 1 L of bovine milk[Z6].The
sources for these two enzymes, bovine milk and culture broth of Caldariomyces
IG
1270
I filmago,
Oxidation Reactions
are easily obtained. Chloroperoxidase can also be obtained in larger

quantities from the fungus Curvularia i n a e q u ~ l i s [ ~Thus,
~ ] . a number of different
haloperoxidases from various sources are available in quantities necessary for the
enzymatic halogenation of organic compounds.
tryptophan
tryptamine indole-3-acetonitrile
3-methylindole 5-methylindole
rnonodechloroamino- aminopyrrolnitrin
pyrrolnitrin
Figure 16.9-3. Substrates accepted by tryptophan 7-halogenase: (a) indole

derivatives, (b) phenylpyrrole derivatives; the positions of chlorination are
indicated by
1G. 9 Halogenation
I 1271
16.9.3
Substrates for Halogenating Enzymes and Reaction Products
16.9.3.1
Halogenation of Aromatic compounds
The recently detected FADHz-dependenthalogenases are substrate specific. Trypto-

phan 7-halogenase catalyzes the chlorination and bromination of D- and L-trypto-
phan to 7-chloro-or 7-bromotryptophan, This enzyme also accepts a
number of other indole derivatives such as tryptamine, indole-3-acetonitrile,3-me-
thylindole and 5-methylindoleas substrates (Fig. 16.9-3a)[431. In addition to indoles,
aminophenylpyrrole derivatives are also chlorinated by tryptophan 7-halogenase
(Fig. 16.9-3b)[431.
Monodechloroaminopyrrolnitrin 3-halogenase catalyzes the regioselective chlor-
ination of the aminophenylpyrrole derivative monodechloroaminopyrrolnitrin to
aminopyrrolnitrin ["l, however, nothing is known about the substrate specificity of
this enzyme.
In contrast to FADH2-dependent halogenases, haloperoxidases have no substrate
specificity. The enzymatic iodination, bromination, and chlorination of a number of
different aromatic compounds by haloperoxidases have been reported in the last few
years. All aromatic substrates halogenated successfully by haloperoxidases are
aromatic compounds activated for electrophilic substitution (Table 16.9-1).
Phenols and phenol ethers are very good substrates for haloperoxidases. The first
aromatic substrate to be used in enzymatic iodination was tyrosine. This substrate
was iodinated using chloroperoxidase from Caldariomyces fimago[441and thyroid
pero~idase[~'l. Horseradish peroxidase and lactoperoxidase have been used to lable
proteins with radioactive isotopes of iodide 1'1 and bromoperoxidase from Penicillus
capitatus has been employed to lable human serum albumin with the radioactive
isotope of bromine 14'4.
Phenolsulfonephthalein (Phenol Red) is brominated to 3,3',5,5'-tetrabromophe-
nolsulfonephthalein (Bromophenol Blue) by many haloperoxidases fl, "1. This reac-
tion has been used for the detection of halogenating enzymes by different
groups 19, 471.
Corbett et al.[481obtained 2,6-dibromo-4-chloroaniline or 2,4,6-trichloroaniline
when they incubated 4-chloroaniline with chloroperoxidase in the presence of
hydrogen peroxide and bromide or chloride, respectively.
Several obscurolides, secondary metabolites produced by Streptomyces viridochro-
mogenes T7 [491, were brominated using perhydrolase from Streptomyces aureofaciens
Tu24[501.The obscurolides were monobrominated in the 2-position and dibromi-
nated in the 2,4-positions of the aromatic ring system of the obscurolides (Fig.
16.9-4). In the case of dibromination, the hydroxymethyl group was replaced by
bromine. No bromination of the olefinic double bond could be detected.
A number of aromatic heterocycliccompounds have been halogenated by different
haloperoxidases.
Franssen et al. Is']used chloroperoxidase from Cddariomycesfimago to chlorinate
4
c:
N
-
FADHz-dependenthalogenases, haloperoxidases and perhydrolases used for biotransformation of aromatic compounds and their sources. d
Table 16.9-1. m
Enzyme (type) Source Substrate (halide) 0
Reference 3.
Tryptophan 7-halogenase pyrrolnitrin-producing indole derivatives (Cl-, Br-) 20,43 %
8'
(FADHz-dependent) Pseudomonads phenylpyrroles (Cl-, Br-) 43
Monodechloroaminopyrrolnitrin 3- pyrrolnitrin-producing monodechloroamino- P
halogenase (FADHz-dependent) Pseudomonads pyrrolnitrin (Cl-, Br-) 20 48'
Chloroperoxidase (heme) Caldariornycesfumago phenol ether (Cl-, Br-) 67-68 a
phenols (Cl-, Br-, I-) 44
anilines (Cl-, Br-) 48
pyrazoles, pyridines (Cl-) 51
nucleic bases (Cl-, Br-, I-) 14
Lactoperoxidase(heme) bovine milk phenols (I-) 72
estrone (I-) 73
Thyroid peroxidase (heme) thyroid glands phenols (I-) 45
Chloroperoxidase (heme-flavin) Notomastus lobatus phenols (Cl-, Br-) 37
Bromoperoxidase (non-heme) Asophyllum nodosum phenols (Br-, I-) 74
phenol red (Br-) 15
Bromoperoxidase (non-heme) Corallina pilulijera nudeic bases (Br-, I-) 14
phenols (Br-) 75
Perhydrolase (non-heme, non-metal) Streptomyces aureofaciens nikkomycin (Br-) 52
phenylpyrroles (Cl-, Br-) 56
obscurolide (Br-) 50
phenol red (Br-) 9
Perhydrolase (non-heme, non-metal) Pseudomonas pyrrocinia indole (Cl-, Br-) 29
phenylpyrroles (Cl-, Br-) 7, 54, 56
7G. 9 Halogenation
I 1273
Figure 16.9-4. Bromination of obscurolide A3 by perhydrolase from Streptornyces

aureofaciens Tu24 [501.
pyrazole, 1-methylpyrazole and 3-methylpyrazole to their corresponding 4-chlor-

oderivatives. The same enzyme was used to produce 5,7-dibromo-8-hydroxyquino-
line from 8-hydroxyquinoline.2-Aminopyridinewas regiospecifically chlorinated to
2-amino-3-chloropyridine.
The chlorination, bromination, and iodination of various nitrogen-containing
heterocycles catalyzed by chloroperoxidase from Caldariomyces &mago and bromo-
peroxidase from Corallina pilulijira were compared by Itoh et al. [141.
The nucleoside antibiotic nikkomycin Z was brominated using the perhydrolase
from Streptomyces aureofaciens Ti24[521.Bromination occurred at the 6-position and
at the 4,G-positions of the pyridine system of nikkomycin Z.
The antifungal antibiotic pyrrolnitrin [3-chloro-4-(2-nitro-3-chlorophenyl)pyrrole]
was brominated at the 2-position of the pyrrole moiety by bromoperoxidase from
Streptomyces phaeochromogenes ts31. Pyrrolnitrin was chlorinated at the 2-position and
at the 2,5-positions of the pyrrole system by perhydrolases from Pseudomonas
pyrrocinia and Streptomyces aureofaciens. The corresponding bromo-derivatives were
also obtained with these enzymes [541.
Another phenylpyrrole compound, 2-(3,5-dibromo-2-methoxyphenyl)pyrrole was
brominated to 2-bromo-,2,3-dibromo-,3,4-dibromo-,2,3,4-tribromo-5-(3,5-dibromo-
2-methoxypheny1)pyrroleby perhydrolase from Streptomyces aureofaciens Tu24[”1.
When the same substrate was chlorinated using the perhydrolases from Pseudomo-
nas pyrrocinia and Streptomyces aureofaciens Tu24, 2-chloro-, 3-chloro-, 4-chloro-,
2,3-dichloro-, 2,4-dichloro, and 3,4-dichloro-5-(3,5-dibromo-2-methoxyphenyl)pyr-
role could be isolated (Fig. 16.9-5)[56].
16.9.3.2
Halogenation of Aliphatic Compounds
Haloperoxidases catalyze the halogenation of a wide range of alkene substrates.

Ethylene was iodinated, brominated, and chlorinated to the corresponding 2-haloe-
thanol by chloroperoxidase from Caldariomycesfimago. Using the same enzyme and
propylene as the substrate the 1-halo-2-propanolsand the 2-halo-1-propanolswere
obtained. 1,3-Butadienewas converted into 1-bromo-3-butene-2-01,2-bromo-3-bu-
tene-1-01and 1,4-dibrom0-2,3-butanediol by lactoperoxidase (Fig. 16.9-6). Bromina-
tion of allene by chloroperoxidase from Caldariomyces f i m a g o resulted in 2-bromo-
2-propen-1-01~~~1.Propylene, allyl chloride and allyl alcohol were halogenated to yield
halohydrins and dihalogenated products [’*I. When several halide ions were present
in the reaction mixture, heterogeneous dihalides were obtained[’”].The chlorination
1274
I
B r p perhydrolase
H,O,,ACOH,CI- ''"
" c v
c c l + $+$",Brv
r v cc
' + B l
r pe
/ / / /
Br Br
br Br 8r
Br
Br Br Br
Figure 16.9-5. Chlorination o f 2-(3,5-dibromo-2-methoxyphenyl)pyrrole by perhydrolase

from Streptornyces aureofaciens Tu24 [561.
w/ chloroperoxidase
H,O, B r
~ &+++Rr
Br OH Br OH
Figure 16.9-6. Bromination o f 1J-butadiene by lactoperoxidase from bovine
H3C A chloroperoxidase
HO
,, CI-
CI OH
Figure 16.9-7. Chlorination o f methyl cyclopropane by
chloroperoxidase from Caldariornycesfirnago 16'].
bromoperoxidase
H,O, Br
Figure 16.9-8. Bromination o f monochlorodimedone, the

substrate used for the search for halopereoxidasesl'].
of propenylphosphonic acid resulted in the formation of 1-chloro-2-hydroxypropyl-

phosphonic acid[60].Phenyl acetylene was brominated to a-brornoacetophenone and
a-dibromoacetophenone by lactoperoxidase. Chloroperoxidase from Culdariomyces
&mugo was used to chlorinate methyl cyclopropane to 4-chloro-2-hydroxy-butane
(Fig. 16.9-7)l6I1.
Monochlorodirnedone, the substrate used for the detection and isolation of
haloperoxidases and perhydrolases (Fig. 16.9-8), and other p-diketones such as
barbituric acid[''] is brominated at the 2-position by all known haloperoxidases and
perhydrolases. Oxooctanoic acid and other p-ketoacids form mono- and dihalogen-
ated ketones and carbon dioxide["]. When p-alanine and taurine were used as
substrates for myeloperoxidase the corresponding N-chloroamines could be de-
tected [G6G51.
As can be seen from the number of substrates halogenated by the different
1G.9 Halogenation
I 1275
haloperoxidases and perhydrolases, these enzymes show no substrate specificity.
Examples of aliphatic substrates halogenated by haloperoxidases and perhydrolases
are shown in Table 16.9-2.
16.9.4
Regioselectivityand Stereospecificityof Enzymatic Halogenation Reactions
16.9.4.1
FADHz-dependenttryptophan 7-halogenase shows regioselctivity which is depend-

ent on the substrate used. With tryptophan, the enzyme is highly regioselective and
catalyzes only halogenation at position 7 of the indole ring. However, with other
indole derivatives halogenation occurs at positions 2 and 3 of the indole ring
(Fig. 16.9-3a).Chlorination of aminophenylpyrrole derivatives by tryptophan 7-halo-
genase also proceeds with relaxed regioselectivity (Fig. 16-3b)[431.
Nothing is know about the regioselectivity of monodechloroaminopyrrolnitrin
3-halogenaseand other FADH2-dependenthalogenases, but biosynthetic investiga-
tions suggest that many of these halogenases catalyze halogenation reactions
regio~e1ectively['~~ 'j'j].
So far no investigations on the stereospecificity of this type of halogenating

enzymes have been reported.
Haloperoxidases show very poor regioselectivity. There are only very few reports
on regioselective reactions catalyzed by haloperoxidases. Franssen et al. [ ' j 2 ] reported
the regioselective chlorination of 2-aminopyridine to 2-amino-3-chloroaminopyr-
idine by chloroperoxidase from Caldariomycesfimago. When anisole was brominated
using chloroperoxidase from Caldariomyces fimago Walter and Ballschmitter ['j71
found a para-preference for the bromination reaction with a para :ortho ratio of 16
compared with 9 for the normal electrophilic bromination. The para: ortho ratio for
the chlorination of anisole with the same enzyme obtained by Brown and Hager ['j8]
was 1.9. This discrepancy could be due to the different reaction conditions used.
Walter and Ballschmitter ['j71 used a 50 times higher anisole concentration and only
about half the amount of chloroperoxidase compared with Brown and HagerL"]. If
one takes into consideration that anisole could reach the active site of the enzyme
and is present at a high concentration, a relatively large part of the substrate could be
chlorinated at the active site with a certain orientation and only a smaller part would
be chlorinated by enzymatically produced hypochlorous acid. This effect could be
amplified by smaller amounts of the enzyme and thus lower concentrations of
hypohalous acid produced. However, this would mean that halogenation occurring
at the active site showed a higher degree of regioselectivity, even without a specific
binding site for the organic substrate.
Similar results were obtained by Itoh et al. [I4, 'j91 for the halogenation of different
substrates using bromoperoxidase from Corallina pilulifera and chloroperoxidase
from Caldariomycesfimago.
In addition to poor regioselectivity, haloperoxidases also show poor stereospeci-
4
U
N
m
-
Table 16.9-2. Haloperoxidases used for biotransformations of aliphatic compounds and their sources. -
m
Haloperoxidase (type) Source Substrate (halide) Reference
Chloroperoxidase (heme) Caldariomycesfumago alkenes (Cl-, Br-, I-) 57-60,70,75
B
a.
2
9(11)-dehydroprogesterone(Br-) 76 3
2-hydroxymethylene 77
testosterone (Br-)
?iF:
8'
2-hydroxymethylene-17p-hydroxy- 78 ,3
androstan-3-one (Br-)
glycals (Cl-, Br-, I-) 71
alkynes (Cl-, Br-, I-) 61
cyclopropanes (Cl-,Br-) 61
p-diketones (C1-, Br-) 44,70,79-81
Lactoperoxidase (heme) bovine milk alkenes (Br-, I-) 57,59,82-84
alkynes (Br-) 61
cyclopropanes (Br-) 61
Myeloperoxidase(heme) mammals alanine, taurine (Cl-) 65
0-alanine (Cl-) 64
Bromoperoxidase (heme) Penicillus capitatus a-amino acids, peptides (Br-) 85
Bonnemasoinia harngera
Bromoperoxidase (non-heme) Ascophyllum nodosum barbituric acid (Br-) 62
lG.9 Halogenation
I 1277
ficity. Kollonitsch et al. Ib0] obtained optically inactive erythro-dimethyl l-chloro-
2-hydroxypropylphosphonatefrom trans-propenylphosphonicacid using chloroper-
oxidase from Caldariomyces@mago. Ramakrishnan et al.I'[ investigated the bromi-
nation of racemic 2-e~o-methylbicyclo-[2.2.l]hept-5-ene-2-endo-carboxylic acid to the
S-lactone and racemic bicyclo-[3.2.0]hept-2-en-G-one to the 2-exo-bromo-3-endo-hy-
droxybromohydrin. The products were obtained in racemic form. 2-Methyl-4-pro-
pylcyclopentane-1,3-dionewas chlorinated to 2-chloro-2-methyl-4-propylcyclopen-
tane-1,3-dione. Here the product was obtained as a 40: GO ratio of the racemic
diastereomers. From these findings they concluded that active site chlorination by
chloroperoxidase from Caldariomyces &mago proceeds without appreciable ster-
eoselectivity.
On the other hand, Liu and W ~ n g ( ~ described
*] the stereoselective bromohydra-
tions of D-galactal and L-fucal to 2-bromo-2-deoxy-~-ga~actose (P/a= 3) and 2-bromo-
2-deoxy-~-fucose(p/a = 2), respectively. They also obtained the corresponding
chlorinated products, however, in much lower yields.
16.9.5
Comparison of Chemical with Enzymatic Halogenation
NADH2-dependenttryptophan 7-halogenasecatalyzes the incoporation of a chloride

atom into the indole ring at a position were direct chemical chlorination is not
possible. The structures of metabolites containing halogenated indole rings suggest
that similar halogenases exist which catalyze halogenation reactions at positions 2-7
of the indole ring. These enzymes are certainly very promising candidates as tools in
organic synthesis, especially as they catalyze the incorporation of the halide atoms as
nucleophiles, which allows regioselective and possibly stereoselective halogenation
reactions.
Enzymatic halogenation catalyzed by haloperoxidases and perhydrolases involves
the oxidation of halide ions to a halonium ion species which leads to the formation of
hypohalous acids (Fig. 16.9-1). The products obtained by enzymatic halogenation
with these enzymes are the same as the products obtained by chemical electrophilic
halogenation with hypohalous acids. The differences in the para :ortho ratios in the
halogenation of some aromatic compounds could be due to a mixture of halogen-
ation at or near the active site and in solution.
The major advantage of enzymatic halogenation using haloperoxidases and
perhydrolases is that the enzymes have a very low substrate specificity and that no
free halogen is needed which makes halogenation catalyzed by these enzymes less
hazardous than chemical halogenation.
Some of the non-heme haloperoxidases and perhydrolases are very stable, even
against organic solvents, and easy to use as they do not need any cofactors. However,
care has to be taken not to use too high concentrations of hydrogen peroxide, as this
could lead to oxidation of the substrate.
1278
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I
lZ8’
17
lsomerizations
Nobuyoshi Esaki, T. Kurihara and K. Soda
17.1
Introduction
Isomerases catalyze the isomerization of substrates, and are classified into five
groups as follows:
Racemases and epimerases (E. C. class 5.1)

They are defined as enzymes that catalyze the isomerization of a substrate through
stereochemical reverse rearrangement of a substituent bound to a chiral center
(usually a chiral carbon) in the substrate molecule. Racemases act on molecules
containing only the asymmetric center concerned in the reaction. Epimerases act on
substrates containing one or more asymmetric centers in addition to the reactive
chiral center.
cis-trans-Isomerases (E. C. class 5.2)

They catalyze the interconversion of cis-trans geometrical isomers.
Sugar isomerases, tautomerases, A-isomerases, etc. (E. C. class 5.3)

Sugar isomerases catalyze the interconversion between aldose and ketose. Tauto-
merases catalyze a keto-enol tautomerization. A-Isomerases catalyze the shift of a
double bond. The reactions catalyzed by these enzymes proceed through intra-
molecular oxidation and reduction.
Mutases (E. C. class 5.4)

They catalyze the transfer of a substituent to produce a structural isomer.
Cycloisornerases (E. C. class 5.5)

They catalyze the ring formation through an intramolecular lyase reaction.
Isomerizations catalyzed by most of these enzymes proceed through 1,1-,1,2-, or

1,3-hydrogen shifts (Table 17-1), while mutases catalyze exchange of a hydrogen
1282
I 77 lsomerizations
Table 17-1. Enzyme-catalyzedisornerizations classified as hydrogen shifts.

Type Examples Category
1,l-Shifts
R,+H
Rl
R,
__ H+R*
Rl
R3
Epimerases, Racemases
H
ir
F=O
I
H-:-OH
1,2-Shifts H-C-OH C=O Aldose-ketoseisomerases
I I
1,3-Shifts
atom with particular functional groups such as amino, hydroxy, and a-amino-a-
carboxymethyl groups attached at neighboring carbon atoms of the substrates
through homolytic cleavage.
Here we describe enzymological properties of representative racemases, epi-
merases, and isomerases, and their application to production of various optically
active compounds.
17.2
Racernizations and Epirnerizations
Since the discovery of enzymatic racemization of lactate by lactic acid bacteria '1,
Clostridium acetobutyri~um[~],and CE. butyri~um1~1, a variety of racemases and
epimerases have been demonstrated, and they are classified into the four groups as
follows:
Amino acid racemases and epimerases catalyzing racemization and epimerization
at the chiral center containing an NH2 or NH group (E. C. class 5.1.1);
Mandelate racemase, lactate racemase, and others acting at the chiral center
containing an OH group (E.C. class 5.1.2);
Various carbohydrate epimerases such as UDP-~-glucose-4'-epimerase (E. C. class
5.1.3);
Methylmalonyl CoA epimerase and some others, in whose substrates a CH3 group
is bound to the chiral centers (E. C. class 5.1.99).
Racemases and epimerases have been used for production of various optically active
compounds from cheaply-availableracemic substrates by combination of enzymes
that act specifically on one of the isomers of the racemates to catalyze hydrolysis,
oxidation, reduction, elimination, replacement, and other reactions. The racemases
and epimerases used act exclusively on the substrates, but not on the products of the
77.2 Racemizations and Epimerizations
reaction. Thus, total conversion of the racemic substrates into the desired optically-
active compounds is achieved. Here we describe enzymological characteristics of the
representative racemases and epimerases, and their application to production of
optically active compounds.
17.2.1
Pyridoxal 5'-phosphate-dependentAmino Acid Racemases and Epimerases
17.2.1.1
Alanine Racemase (E.C. 5.1.1.1)
Alanine racemase is a bacterial enzyme that catalyzes racemization of L- and D-

alanine, and requires pyridoxal 5'-phosphate (PLP) as a cofactor. The enzyme plays
an important role in the bacterial growth by providing D-alanine,a central molecule
in the peptidoglycan assembly and cross-linking,and has been purified from various
The enzyme has been used for the production of stereospecifically
deuterated NADH and various D-amino acids by combination of L-alanine dehy-
drogenase (E. C. 1.4.1.1),D-amino acid aminotransferase (E. C. 2.6.1.21), and formate
dehydrogenase (E. C. 1.2.1.2) [I7, "1.
17.2.1.1.1 Gene Cloning and Primary Structure

Two distinct alanine racemase genes were cloned from the Salmonella typhimurium
chromosome. One mapped at minute 37 on the chromosome is termed dadB, and
the other mapped at minute 91 is termed the alr The dadB alanine racemase
is formed inducibly and functions in the catabolism of L-alanine: the alr enzyme is
synthesized constitutively, and functions in the anabolic assembly of peptidogly-
can Alanine racemase genes were also cloned from Bacillus stearothemophi-
lus['OI, Bacillus subtilis [''I, Bacillus p~ychrosaccharolyticus['~~,
and Aquijkx pyrophi-
US['^]. Two distinct alanine racemase genes were assigned in the genome sequences
of Escherichia coli["], B. subtilis, Pseudomonas aeruginosa, and Vibriocholerae, but only
a single one occurs in the other bacterial genomes whose complete nucleotide
sequences were determined as shown at internet sites such as http://www.geno
me.ad.jp/kegg/catalog/org-list.htm1.
Uo et al. [16]have found that fission yeast, Schtzosaccharomyces pombe, has also the
alanine racemase gene, which is involved in the catabolism of D-alanine in S. pombe
in the same manner as dadB of S. typhimurium. The yeast enzyme only shows any
high degree of similarityto the alanine racemases of y-proteobacteria(gram-negative
phylum). Therefore, the gene of S. pombe has possibly been acquired from y-
proteobacteria through some events of horizontal gene transfer such as conjugation:
S. pombe is known to be a recipient of the genes from E. coli through direct
conjugation.
D-Alanine occurs in various natural compounds produced by fungus. For example,
cyclosporin A contains D-alanine as a component and is produced by Tolypocladium
niveum[121. Alanine racemase is involved in the biosynthesis of D-alanine in this
fungus.
I7 lsomerizations
1284
I 17.2.1.1.2 Stability
The native dadB and alr racemases from Salmonella typhimurium are readily
inactivated by digestion with a-chymotrypsin, trypsin, and subtilisin [221. However,
the Bacillus stearothermophilus enzyme is stable even after fragmentation into two
pieces [232 241. A. pyrophilus, a hyperthermophilic bacterium, produces extremely
stable alanine racemase ["I. It maintains catalytic activity in the presence of organic
solvents as well. On the other hand, Bacillus psychrosaccharolyticus, a psychrophyilic
bacterium, produces a thermo-labile enzyme[14].However, it shows high catal9c
activity at low temperatures, such as at 0 ° C . Similar cold activity and thermal
instability was found in the enzyme from a psychrophile isolated from raw milk,
Pseudomonasfluorescens I"].
17.2.1.1.3 Reaction Mechanism

Reaction of alanine racemase proceeds through the steps shown in Fig. 17-1. PLP
bound with the active-sitelysyl residue (A) reacts with a substrate to form an external
Schiff base (B) through transaldimination. The subsequent a-hydrogen abstraction
Figure 17-1. Mechanism o f the alanine racemase reaction. A, An internal aldirnine o f PLP with a
lysyl residue; 6, an external aldimine o f PLP with o-alanine; C, a quinonoid intermediate formed
after removal o f a hydrogen from alanyl external aldimines B or D; D, an external aldimine o f PLP
with L-alanine. Reprinted from Watanabe et al.'"'.
results in the formation of a resonance-stable deprotonated intermediate (C). If

reprotonation occurs at the a-carbon of the substrate moiety on the opposite face of
the planar intermediate (C), then an antipodal aldimine (D) is formed. The &-amino
group of the lysine residue is substituted for the isomerized amino acid through
transaldimination, and the internal aldimine (A) is regenerated. According to
* ] , Ca-H - bond to be broken is positioned perpendicularly to the
D ~ n a t h a n [ ~the
plane of the conjugated x-system of the external Schiff base intermediate, in order to
achieve maximum orbital overlap with the 7c electron system of the complex,
resulting in a substantial rate enhancement for the cleavage of that bond.
The racemization reaction proceeds via either a one-base[2G]or tw~-base[~']
mechanism. The one base mechanism is characterized by the retention of the
substrate-derived proton in the product (internal return) [2G1. By this criterion,
reactions catalyzed by a-amino-&-caprolactamracemase 12*] and amino acid racemase
with low substrate specificity (E. C. 5.1.1.10) L2]' have been considered to proceed
through the one-base mechanism. However, such internal returns were not observed
in the reactions catalyzed by alanine racemases from E. c0li[~~1], Bacillus stear-
otherm~philus[~'], and Salmonella typhimurium (dadB and alr) [291. The internal return
is not expected to occur in the reactions of two-base mechanism. In fact, kinetic
analyses 1301indicated that the alanine racemase reaction proceeds through a two-
base mechanism: proton donors and proton acceptors are situated on both sides of
the planar intermediate (Fig. 17-1, C) and accomplish removal and return of the a-
hydrogen of the substrate amino acid. X-ray crystallographic studies [31* 321 suggested
that Lys 39 and Tyr 265 of alanine racemase from B. stearothemophilus serve as the
bases (Fig. 17-2).Watanabe et al. [331 showed that the lysyl residue binding PLP in the
racemase (Lys 39) acts as the base catalyst specific to the D-enantiomer of alanine.
The crystal structure of the enzyme complex with R-1-aminoethylphosphonic
acid[32],a tight-bind inhibitor of the enzyme[341,demonstrated that the phenolic
oxygen of Tyr 265 is appropriatelyaligned for proton abstraction from an L-isomer in
the active site of the structure: Tyr 265 is the second base specifically acting on the L-
alanyl-PLP aldimine.
Tyr 265' Tyr 265'
Figure 17-2. Stereodia-

gram o f the aldimine
formed from l-amino-
ethylphosphonate and
PLP viewed perpendicu-
lar to the plane o f the
PLP ring. The catalytic
residues Tyr 265' and
Lys 39 Lys 39 Lys 39 are shown. Re-
printed from Stamper
et aI.'32J.
7 7 lsornerizations
1286
I A
D-[tx-'H] Ala
D/L-Ala
AlaDH
@roR)
L-[a-'H] Ala [u-'H]-L-Aia 7-
[4R-2H]NAD2H+ Pyruvate + NH3
[C4-' HINAD'
Oxidized Form Oxidized Form

of Substrate of Substrate
[2H] Reduced Form ['HI Reduced Form

of Substrate of Substrate
Figure 17-3. A, Preparation of [4S-'H]-NADH by coupling of alanine racernase and L-alanine

dehydrogenase. B, In situ determination o f stereospecificity of H-transfer by 'H-NMR. AlaR,
AlaDH, and D H represent alanine racernase, L-alanine dehydrogenase, and dehydrogenase,
respectively.
17.2.1.1.4 Production of Stereospecifically Deuterated NADH

NAD-linked dehydrogenases show either pro-S or pro-R-stereospecificityfor hydro-
gen removal from the C4 position of the nicotinamide moiety of the reduced
coenzymes. The stereospecificity of hydrogen transfer is examined by means of
stereospecifically C4-deuterated NADH, which is prepared enzymatically from
NAD' and deuterated substrates by tedious procedures.
Esaki et al. developed a simple method to produce the stereospecificallydeuterated
NADH by an NAD'-dependent dehydrogenase by combination with amino acid
racemase 13'1. L-Alanine dehydrogenase transfers deuterium of [2-'H]-~-alanineto
NAD' to produce [4R-2H]-NADH[3G1.Alanine racemase catalyzes the C2-deuteration
of D and L-alanine in 2H20110], and [4R-2H]-NADH was produced from D-alanine and
NAD' by coupling of the reactions catalyzed by alanine racemase and r-alanine
dehydrogenase in 2H20 (Fig. 17-3A).Furthermore, this finding led to development
of a simple procedure for the in situ analysis of stereospecificity of hydrogen transfer
of NADH by an NAD-dependent dehydrogenase by means of 'H-NMR (Fig. 17-
3B)C3'1.
17.2.1.1.5 Production of D-Amino Acids

Considerable attention has been paid to multi-enzyme reaction systems as a means
to the stereospecific production of L-amino Wichmann et al. have developed
a continuously operated membrane reactor for production of L-leucine from a-
ketoisocaproate13'1. The system is also applicable to the production of several other
aliphatic L-amino acids such as L-valine, 1-tert-leucine and ['SN]-~-leucine.The
Formatex
FDH
NAD’ yDH xATA L-Alanine aR
:
T====== D-Alanine Keto acid
COP NADH \ Pyruvate D-Amino acid

+H+ NH3
Figure 17-4. Synthesis o f o-amino acids from a-keto acid, formate, NAD+, D-alanine, and
ammonia by coupling of L-alanine dehydrogenase (AlaDH), formate dehydrogenase (FDH),
alanine racemase (AlaR), and D-amino acid aminotransferase (D-ATA).
process has been successfully scaled-up for industrial production of these L-amino
acids [391. A similar system has been developed for production of L-phenylala-
411 and ~-P-chloroalanine[~~~~1. However, little attention has been paid to the
stereospecific production of D-amino acids by means of multi-enzyme reaction
systems, although D-amino acids have been paid considerable attention [&I. For
example, substantial amounts of D-serine,D-aspartateand other D-amino acids occur
in mammalian l ~ r a i n [ ~ ’ -and
~ ~ ]13N-labeled
, D-amino acids are expected to be useful
for the study of their metabolism in brain[481.
A simple procedure was established for the synthesis of various D-amino acids by
means of four types of thermostable enzymes: alanine racemase, D-amino acid
aminotransferase [49* ”1, L-alanine dehydrogenase [’I, and formate dehydrogenase
(Fig. 17-4)[171. The commercial preparation of formate dehydrogenase from Cundida
boidinii used by Wichmanri et a1.19 is not sufficiently stable. However, Galkin et
al.[52]cloned and expressed the gene of thermostable formate dehydrogenase in
E. coli.
D-Phenylalanine and D-tyrosine, which are the poor substrates for D-amino acid
aminotransferase, were synthesized in an optical purity of essentially 100 %, but with
yields of lower than 50 %. However, the yields were increased by addition of excess
amounts of the D-amino acid aminotransferase (Table 17-2)[171. Selenium is an
essential micronutrient for mammals, fish and several bacteria, although it is toxic at
a high c~ncentration[’~, 531. D-Selenomethionine was produced in an 80% yield
based on 2-0~0-4-methylselenobutyrate[’~]. Norvaline, valine, and a-aminobutyrate
were also produced with high yields. However, a-aminobutyrate was synthesized as a
racemic mixture. D-Norvaline was obtained at an enantiomeric excess of only 30%.
The low optical purity is probably due to the action of L-alanine dehydrogenase: a-
ketobutyrate and a-ketovalerate are reduced by t-alanine dehydrogenase at rates of
79 and 6.6% relative to that of pyruvate, respectively[”]. Moreover, alanine racemase
also racemizes a-aminobutyrate and norvaline, though very sl0wly[~~1. Thus, this
method is not applicable to the stereospecificproduction of D-a-aminobutyrateand
D-norvaline. The preparations of D-valine, D-methionine and D-norleucine also
suffered contamination by the antipodes at concentrations of 4, 3, and 1%,
respectively, due to the action of L-alanine dehydrogenase on the a-keto analogs of
these amino acids [’ll. However, D-glutamate, D-phenylalanine and D-tyrosine were
efficiently produced in the system. The final concentration of D-glutamateproduced
1288
I 7 7 lsornerizations
Table 17-2. Synthesis of o-amino acids from a-keto acids by combination offour purified
enzymes: alanine racemase, L-alanine dehydrogenase, formate dehydrogenase, and o-amino acid
aminotransferase.
Substrate Product Yield (“h)” ee (“A)

a-Ketoglutarate D-glutarnate 98 100
a-Ketoisocaproate D-leucine 80 >99
a-Ketocaproate D-norleucine 82 98
a-Keto-y-thiornethylbutyrate D-rnethionine 95 94
a-Ketoisovalerate D-valine 90 92
a-Ketovalerate n-norvaline 92 30
a-Ketobutyrate a-aminobutyrate 93 0
Phenylpymvate D-phenylalanine 72b 100
Hydroxyphenylpyruvate D-tyrosine 70b 100
a The yields were determined after an 8 h incubation.
b The amount of 0-amino acid aminotransferase used (30 units) was 10-foldhigher than that in other systems
was only around 0.3 M, limited because of the equilibrium of the D-amino acid
aminotransferase reaction. The method is most suitable for stereospecific conver-
sion of a-keto acids into the corresponding D-amino acids, in particular labeled
compounds, for example with 13N by means of 13N-NH3.
The industrial use of the above-mentionedsystems depends predominantly on the
cost of the enzymes, although the intact cells of microorganisms containing the
enzymes can be used as catalysts in order to decrease costs[56].In most cases,
however, additional genetic improvements through metabolic engineering are
required, thereby new functional combinations are made by the rational transfer of
pathways from one organism to another[”]. The transfer of the ethanol pathway
from Zymomonas mobilis to other enteric bacteria represents an example of this
approach[58].In the above-mentioned system, various D-amino acids can be pro-
duced from the corresponding a-keto acids, if four functional genes are introduced
into one microorganism. The simultaneous expression of all enzymes in a single cell
Table 17-3. Synthesis of D-amino acids from a-keto acids by E. coli cells harboring pFADA which
codes for four enzyme genes: alanine racemase, L-alanine dehydrogenase, formate
dehydrogenase, and D-amino acid aminotransferase.
Substrate Product Yield (“A)” ee (“A)
a-Ketoglutarate D-glutamate 85 100
a-Ketoisocaproate o-leucine 76 >99b
a-Ketocaproate D-norleucine 70 88
a-Keto-y-thiornethylbutyrate D-methionine 80 90
a-Ketoisovalerate D-valine 85 92
a-Ketovalerate D-norvaline 90 35
a-Ketobutyrate a-arninobutyrate 95 0
Phenylpymvate D-phenylalanine 15 ND‘
H ydroxyphenylpyruvate D-tyrosine 5 ND
a The yields were determined after a 12 h incubation
b The optical purity determined by HPLC is >99.9%
c ND, not determined.
17.2 Racernizations and Epirnerizations
4 I I
Hindlll BamHl Sphl Bgfll Pstl I Nsil EcoRl
plac ptac sm ATG FDH T G A ~ S D ~

ATG AlaDH T G A S~ D ATG
~ DAAT TAA HSD~ ATG AlaR T A A ~ ~ F A D A
Figure 17-5. Construction of the plasmid used for the production o f o-amino acids by expression
in E. coli cells; formate dehydrogenase (FDH), L-alanine dehydrogenase (AlaDH), alanine
racernase (AlaR), and D-amino acid aminotransferase (DAAT).
- I-
E f
g 0,2 Figure 17-6. Time
_1 course for the produc-
Ec
C -
tion o f D-glutarnate
0
with E. coli cells con-
0 0,l- D-Glutamate taining pFADA.
a-Ketoglutarate was
- I a-Ketoglutarate added after 4 and
10 h o f incubation at
provides additional benefit for industrial applications:the intracellular pool of NAD’

(supplied by the cell itself) could be used for NADH regeneration without any
additional supplies.
Galkin et al. I‘[ constructed plasmids containing, in addition to the thermostable
formate dehydrogenase gene, all three genes required for the synthesis of D-amino
acids (Fig. 17-5). D-Enantiomers of glutamate and leucine were produced at high
optical purity and high conversion rates with the recombinant E. coli cells harboring
the plasmid for coding of the four heterologous genes (Table 17-3). a-Keto acids,
particularly branched-chain and long-chain a-keto acids, are toxic, inhibiting the
growth of E. coli when added at concentrations of only 15-30 mM. Therefore, Galkin
et al. used the resting cells of the recombinant E. coli instead of growing ones.
Moreover, the isolation of products in the resting-cell system is much easier than
when using growth media containing complex ingredients such as yeast extracts.
The final concentration of D-glutamateproduced was around 0.3 M (Fig. 17-6).
17.2.1.2
Amino Acid Racemase with Low Substrate Specificity (E.C. 5.1.1.10)
An amino acid racemase which shows very broad substrate specificity was dis-
covered in Pseudoinonas striata (= Ps. putida), purified, and characterized[”1. The
enzyme catalyzes racemization of various amino acids except aromatic and acidic
1290
I 7 7 lsomerizations
amino acids. A similar enzyme also occurs in Aerornonas punctata[60].Arginine

racemase, which also shows a broad substrate specificity, has been demonstrated in
Pseudomonas graveolens (= Pseudornonas taetrolens) ['*I. These amino acid racemases
do not act on threonine, valine and their analogs, whose P-methylene group is
substituted. Recently, Lim et al.L621 found, in Ps. putida ATCC 17642, a new amino
acid racemase catalyzing not only racemization of various amino acids but also
epimerization of D- and L-threonineby stereoconversionat the a-position: it catalyzes
epimerization of L-to D-allo-and also of D- to L-allo-threonine.
Amino acid racemase with low substrate specificity catalyzes racemization of
leucine and various other amino acids, which are also a-deuterated in 2 H z 0during
their ra~emization[~']. Therefore, [4S2H]-NADHwas produced in the same manner
as described above with the racemase and L-leucine dehydrogenase (E. C. 1.4.1.9),
which is pro$
Amino acid racemase with low substrate specificityof Ps. putida ATCC 17642 does
not racemize aromatic and acidic amino acids. However, phenylalanine and phenyl-
glycine undergo a-hydrogen exchange with deuterium from the solvent when
incubated with the racemase in 2H20. Lim et al. [641 found that each enantiomer of
both a-deuterated phenylalanine and phenylglycine are produced stereospecifically
with retention of the C2 configuration. This a-hydrogen exchange reaction is
applicable to the production of a-deuterated phenylalanine and phenylglycine.
Makiguchi and coworkers established a method to synthesize L-tryptophan from
D,L-serine and indole by means of tryptophan synthase (E. C. 4.2.1.20) from E. coli
and the amino acid racemase with low substrate specificity of Ps. striata (= Ps.
putida) [651. Both D,L-serine and indole are cheaply available by chemical synthesis.
Tryptophan synthase catalyzes the p-replacement reaction of L-serine with indole to
produce L-tryptophan, and the amino acid racemase with low substrate specificity
converts unreacted D-serine into L-serine. Because the racemase does not act on
tryptophan, almost all D,L-serine is converted into optically pure L-tryptophan.
Makiguchi et al.["] succeeded in producing L-tryptophan in a 200 L reactor using
intact cells of E. coli and Ps. put id^[^^]. Under the optimal conditions established,
110 g L-l of L-tryptophan was formed in molar yields of 91 and 100% for added D,L-
serine and indole, respectively, after 24 h of incubation with intermittent indole
feeding. Continuous production of L-tryptophan was also achieved using im-
mobilized cells of E. coli and ps. putida. The maximum concentration of L-tryptophan
formed was 5.2 g L-' (99% molar yield for indole).
S-Adenosyl-L-methionine is the important methyl donor in biological trans-
methylation to form S-adenosyl-L-homocysteine,which is hydrolyzed to adenosine
and homocysteine by S-adenosyl-L-homocysteinehydrolase (E. C. 3.3.1.1) in vivo.
However, equilibrium of the S-adenosyl-L-homocysteinehydrolase reaction favors
the direction toward synthesis of S-adenosyl-L-homocysteine.Shimizu et al. devel-
oped a simple and efficient method for the high yield preparation of S-adenosyl-L-
homocysteine with S-adenosyl-L-homocysteinehydrolase of Alcaligenes faecalis, in
which the cellular content of S-adenosyl-L-homocysteinehydrolase was about 2.5 %
of the total soluble protein. S-Adenosyl-L-homocysteinewas produced at a concentra-
tion of about 80 g L-' with a yield of nearly However, when racemic
17.2 Racernizations and Epimerizations
I
1291
HO
1 OH OH 2 OH OH
OH YH2
Figure 17-7. Structures of adenosine and related nucleosides which serve as
substrates for S-adenosyl-L-homocysteinehydrolase. 1, Adenosine; 2, formycin A
3, neburalin; 4, adenosine "-oxide; 5 , 2-chloroadenosine; 6, tubercidine; 7, N6-
methyladenosine; 8, inosine; 9, 1-methyladenosine.
homocysteine was used, the D-enantiomerremained unreacted. When Ps. striata (=

Ps. putida) cells were used as the catalyst, D-homocysteine was converted into S-
adenosyl-r-homocysteine:the amino acid racemase with low substrate specificity
acts on homocysteine, but not on S-adenosylhomocysteine.A. faecalis is better than
Ps. striata in showing higher S-adenosyl-r-homocysteinehydrolase and lower adeno-
sine deaminase activities than those of Ps. striata. Therefore, a mixture of both
bacterial cells was used to produce 70 g L-l of S-adenosyl-r-homocysteinefrom D,L-
homocysteine and adenosine with a molar yield of nearly 100%~G6]. S-Adenosyl-L-
homocysteine hydrolase acts on various adenosine analogs, and the corresponding
S-nucleotidyl-L-homocysteines(Fig. 17-7) were synthesized from the analogs and
D,L-homocysteineby means of both bacterial cells LG7].
1292
I 7 7 lsornerizations
17.2.1.3
a-Amino-E-caprolactamRacemase
a-Amino-E-caprolactam (ACL) is a chiral heterocyclic compound synthesized from

cyclohexene, which is a by-product in the industrial production of nylon. Fuku-
mura168-701established an enzymatic method to produce L-lysine from D,L-ACL. The
process is composed of two enzyme reactions: the selective hydrolysis of L-ACL to L-
lysine, and the racemization of ACL (Fig. 17-8).The L-ACL-hydrolyzing enzyme (a-
amino-e-caprolactam hydrolase (E. C. class 3.5.2) is distributed in the cells of
Cryptococcus laurentii and other yeasts (68-701, and its synthesis is induced by D,L-ACL.
The enzyme purified to homogeneity from a cell extract of C. laurentii has a
molecular weight of about 185000, and is activated by MnC12 and MgC12[71].L-ACL
is the only substrate of the hydrolase: D-ACL and e-caprolactam are not hydrolyzed.
ACL racemase has been found in the cells of Achromobacter obae and other
bacterial7*],and is a unique enzyme among racemases in acting exclusively on cyclic
amides derived from a,o-diamino acids. Ahmed et al.[731purified the enzyme to
homogeneity from the cell extract of A. obae, and characterized it. The enzyme is
composed of a single polypeptide chain whose molecular weight is about 50 000, and
contains 1 mol of PLP per mol of enzyme as a coenzyme. In addition to both isomers
of ACL, D- and L-a-amino-6-valerolactamalso serve as effective substrates [741. The
enzyme catalyzes the exchange of the a-hydrogen of the substrate with deuterium or
tritium during racemization in deuterium oxide or tritium By tritium-
incorporation experiments, the enzyme was shown to catalyze both inversion and
retention of configuration of the substrate with a similar probability in each hrnover.
When [a-2H]-~-ACL and unlabeled D-ACLwere converted into the L-isomer by ACL
racemase in water and in deuterium oxide, respectively, in the presence of excess L-
ACL hydrolase, a-hydrogen (or a-deuterium) was retained significantly in the
product[28].Therefore, a single base mechanism has been proposed for the racemiza-
tion catalyzed by ACL racemase. The ACL racemase gene has been cloned from the
chromosomal DNA of A. obae, and its complete nucleotide sequence determined,
which revealed that the enzyme consists of 435 amino acids and that its molecular
weight is 45 568[751.
H
:"4 ""NH;! -
ACL Racemase
I
D-ACL L-ACL
A C L Hydrolase
H z N ~ c o o Figure
H 17-8. Total conversion
of racemic ACL into L-lysine
NHZ by coupling of ACL racemase
L-Lysine and ACL hydrolase reactions.
7 7.2 Racemizations and Epirnerizations
11293
17.2.2
Cofactor-independent Racemases and Epimerases Acting on Amino Acids
17.2.2.1
Glutamate Racemase (E.C. 5.1.1.3)
D-Glutamateas well as D-alanineis an important component of the peptidoglycan of

bacterial cell walls [761, and is produced by glutamate r a ~ e m a s e c1'.~ ~ ,Lactic acid
bacteria show high activity of the enzyme[7'], and glutamate racemase was first
purified from Pediococcuspentosaceus 811.
17.2.2.1.1 Gene Cloning

Nakajima et al. cloned the glutamate racemase gene of Pediococcus pentosaceus["!
Glutamate racemase genes have been also cloned from various other sources:
Bacillus purnilu~[~~1,
Lactobacillus fermenti 831, Lactobacillus bre~is['~],E. ~oli['~],
and Bacillus subtilis[88*891.
Aquijix pyrophilu~[~~I,
17.2.2.1.2 Enzymological Properties

The glutamate racemase gene from Pediococcuspentosaceus was over-expressedin the
recombinant cells, but formed an inclusion body[81]. However, the enzyme was
solubilized with G M urea, renatured by dialysis to remove urea, and purified to
homogeneity with a high overall yield["]. The amount of enzyme produced by the
clone cells corresponded to about 38 % of the total insoluble proteins. However, the
glutamate racemase gene was solubilized in vivo in an active form when it was co-
expressed with the gene of chaperonin GroESL["]. Choi et al. isolated the active
enzyme and purified it The enzyme is composed of a subunit with a
molecular mass of about 29 kDa. The enzyme acts specifically on glutamate with KM
values of 14 and 10 mM for D- and L-glutamates,respectively. None of other amino
acids occurring in proteins including aspartate, asparagine, and glutamine are
racemized. Other glutamate analogs (homocysteate, a-aminoadipate, glutamate y-
methyl ester, N-acetylglutamate,a-hydroxyglutarate, and cysteine sulfinate) are also
inert. However, L-homocysteine sulfinate, a y-sulfinate analogue of glutamate, is
racemized at a rate of about 10% of that of L-glutamate. Amino acid racemases
generally require PLP as a cofactor, but glutamate racemase is dependent on neither
PLP nor on any other cofactor[80,'l]. Proline racemase (E. C. 5.1.1.4) [921, diaminopi-
melate epimerase (E. C. 5.1.1.7) and hydroxyproline epimerase (E. C. 5.1.1.8)
also require no coenzyme.
Glutamate racemase from E. coli is unique because it is activated about 100 fold in
the presence of UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala),the pre-
cursor of peptidogly~an~~~].UDP-MurNAc-L-Alais ligated to D-glutamate,a product
of the glutamate racemase reaction, by the catalysis of UDP-N-acetylmuramoy1-L-
alanyl-D-glutamate synthetase (E. C. 6.3.2.9). Thus, the activation of the E. coli
glutamate racemase by UDP-MurNAc-L-Ala has a physiological importance in the
1294
I I 7 lsornerizations
regulation of peptidoglycan biosynthesis[951. In contrast, glutamate racemases of

Gram-positive bacteria such as Lactobacillus fernenti, Lactobacillus breuis, Bacillus
pumilus are not activated by UDP-MurNAc-L-Ala,though these enzymes show about
30 % sequence similarities to the E. coli enzyme. The predominant difference
between the E. coli enzyme and the glutamate racemases of the Gram-positive
bacteria is that the former has a 21-amino acid extension at the N-terminus as
compared with the latter enzymes: the N-terminal region is responsible for the
activationL9'1.
Glutamate racemase produced in cell extracts of Bacillus subtilis, an abundant
producer of poly-y-glutamate,is a monomer with a molecular mass of about 30 kDa
containing no cofactor[881.It almost exclusively catalyzes racemization of glutamate
and is mainly concerned in D-glutamate synthesis for poly-y-glutamate production.
B. subtilis produces another isozyme of glutamate racemase encoded by the YrpC
Ashiuchi et al. cloned both enzyme genes and compared their enzymo-
logical properties [88, 891. Enzymological properties of YrpC, such as the substrate
specificity and optimum pH, are similar to those of the other glutamate racemase
(Glr).The thermostability of YrpC, however, is considerably lower than that of Glr. In
addition, YrpC shows higher affinity and lower catalytic efficiency for L-glutamate
than Glr [891.
17.2.2.1.3 Structure and Mechanism

Glutamate racemase contains one essential cysteine residue per mol of enzyme,
whose chemical modification results in complete inactivation c9l1. Choi et al. deter-
mined the amount of tritium incorporated into the substrate and product enantio-
mer during incubation with the enzyme in tritium water, and found that tritium is
exclusively incorporated into the product enantiomer regardless of the configuration
of the substrate used["l. This is compatible with a model in which two different
bases participate in abstraction and return of a-hydrogen of the substrate. One of the
two bases involved in catalysis is suggested to be the essential cysteine residue: a
thiolate from one of the cysteines abstracts the a-proton, and the other cysteine thiol
delivers a proton to the opposite face of the resulting carbanionic intermediate["].
Kim et al.[871cloned the glutamate racemase gene from Aqufifex pyrophilus, a
hyperthermophilic bacterium, and expressed it in E. coli. The enzyme shows strong
thermostability in the presence of phosphate ion, and it retains more than half of its
original activity after incubation at 85 "C for 90 min. Hwang et al.["] crystallized the
glutamate racemase of A. pyrophilus and determined the tertiary structure of the
enzyme by X-ray crystallography.The enzyme is composed of two identical subunits,
and each monomer consists of two alp fold domains. Hwang et al. has also proposed
a mechanism in which two cysteine residues are involved in the catalysis (Fig. 17-
9) [9G].
Glavas and Tanner replaced the two cysteine residues, Cys 73 and Cys 184, by
serine, and analyzed the reactions catalyzed by the mutant enzymes: the elimination
of water from a substrate analog, N-hydroxyglutamate,through a one-base requiring
reaction[97].The C73S mutant was a much poorer catalyst than the wild-typeenzyme
I
1295
Ser 8 - 0
H
H
fyHiS
H
I8O Ser 8-0 I
H
(""'
(YHiS
H
180
CYS70-S-
HO
o F G I u 147
H-S-Cys 178
-
L
Cys 70-S-H
HO
D-Glu \ I
L-Glu
OA0- \\
0
i Asp73 o F G l ~
147
HO
: Cys70-SH HS-CYS 178
Figure 17-9. Mechanism o f glutamate racemase reaction. Cys 70 and Cysl78 serve as the bases
t o abstract an a-proton from the substrate, and a carbanion intermediate is formed. Alternatively,
the racemization may proceed through a concerted mechanism. Reprinted from Hwang et a1.(95].
toward D-N-hydroxyglutamate, whereas the C184S mutant was better than the wild-
type. When L-N-hydroxyglutamate was used as a substrate, C73S was better but
C184S was poorer than the wild-type.Thus, Glavas and Tanner concluded that Cys73
is responsible for the deprotonation of D-glutamate and Cys 184 is responsible for
the deprotonation of L-glutamate
17.2.2.1.4 Synthesis of D-AminoAcids with Clutamate Racemase

Nakajima et al. [981 have developed an efficient method for the synthesis of various D-
amino acids from the corresponding a-keto acids and ammonia by coupling of four
enzyme reactions catalyzed by D-amino acid aminotransferase ["I, glutamate race-
rnase 17'), 911, glutamate dehydrogenase and formate dehydrogenase (Fig. 17-10).
Various D-amino acids are produced by this method. Under the optimum conditions
established by Nakajima et al. ("1, D-enantiomersof valine, alanine, a-aminobutyrate,
1296
I
Formate 1
I 7 lsomerizations
FDH
NAD+ xuL~~utamate
GluR
======= D-GlutamateXA;;Keto acid
co2 NADH
+ H+
a-Ketoglutarate
+ NH3
- D-Amino acid
Figure 17-10. Enzymatic synthesis o f D-amino acids by combination o f glutamate racemase,

glutamate dehydrogenase, D-amino acid aminotransferase and formate dehydrogenase reactions.
Table 17-4. Production ofvarious D-amino acids by means o f f o u r purified enzymes: glutamate
racemase, D-amino acid aminotransferase, glutamate dehydrogenase, and formate
dehydrogenase".
DAmino acids Molar yield ("9)
D-Valine 100
D-Alanine 100
o-a-Aminobutyrate 100
D- Aspartate 100
u-Leucine 84
D-Methionine 80
D-Serine 50
D-Histidine 36
D-Phenylalanine 28
D-Tyrosine 13
a Reprinted from N. Nakajima et al."981.
leucine, methionine and aspartate are synthesized from their a-keto analogs with a
molar yield higher than 80% under the conditions used (Table 17-4)1981.D-Histidine
and a few other D-amino acids, which are poor substrates of D-amino acid amino-
transferaseI"1, are produced in a yield lower than 40% under the same conditions.
However, Bae et al.I1OOlestablished an efficient method for production of D-
phenylalanine and D-tyrosine by feeding a-keto acid intermittently in order to keep
its concentration at less than 50 mM, above which the productivity decreased greatly
(Fig. 17-11). By running the multi-enzyme system for 35 h, 48 g L-' of D-phenyl-
alanine and 60 g L-' of D-tyrosine were produced with 100% of optical purity from
the equimolar amounts of phenylpyruvate and hydroxyphenylpyruvate,respectively.
An enzyme-membrane reactor system containing polyethyleneglycol-NAD' devel-
oped by Wandrey and associatesI"'1 is probably applicable to this system. The
production level of D-aminoacids are mainly dependent on the stability of glutamate
racemase. Therefore, thermostable glutamate racemases produced by A. pyrophi-
1 ~ ~B.
1 ~ 4 ~ and 1 subtilis[881are probably usehl as catalyst of this multi-enzyme system.
Yagasaki et al. (lo21 developed a new method for the synthesis of D-glutamatefrom
L-glutamate by means of E. coli recombinant cells harboring a plasmid containing
glutamate racemase gene from L. brevis ATCC 8287. r-Glutamate was first racemized
to D,L-glutamate at pH 8.5, and L-glutamate was then decarboxylated at pH 4.2 by
glutamate decarboxylase, which was inherently produced by the E. coli host cells.
17.2 Racemizations and Epimerizations I 1297
Figure 17-11. Production o f

D-phenylalanine by successive
feeding o f phenylpyruvate. Phe-
nylpyruvate (U)was added
intermittently. The dotted line
indicates the expected produc-
tivity o f D-phenylalanine )(. on
the basis o f the initial produc-
tion rate. Reprinted from Bae et
aI.["'O].
K
0
C
8 0
' 5 10 15 20 25 30 35
Time (h)
Starting from 100 g L-' of L-glutamate, they obtained 50 g L-' of D-glutamate in a

15 h reaction. D-Glutamate can be produced successively from L-glutamate with L.
breuis ATCC 8287 cells because this strain produces both glutamate racemase and
glutamate decarboxylase simultaneously. Thus, 50 g L-' of optically pure D-gluta-
mate was produced from 100 g L-' of glutam am ate['^^]. Oikawa et a1.['O4I replaced
glutamate decarboxylase by glutamate oxidase because the oxidase has optimum pH
values similar to that of glutamate racemase. They developed a bioreactor consisting
of two columns sequentially connected and containing immobilized glutamate
racemase from B. subtilis and L-glutamate oxidase from Streptomyces sp. X119-6: L-
glutamate was racemized by the glutamate racemase column, and then L-glutamate
was oxidized by the L-glutamate oxidase column. D-Glutamate was produced in
about 90 % of the theoretical yield I1O41.
17.2.2.2
Aspartate Racemase (E.C. 5.1.1.13)
D-Aspartate occurs in the peptidoglycan layer of bacterial cell walls, and is produced
from L-aspartate through an aspartate racemase (E. C. 5.1.1.13) reacti0n1~~'l.The
enzyme has been demonstrated as being present in various Lactobacillus and
Streptococcus strains [*06] such as Lactobacillus &rmenti['051 and Streptococcus faeca-
lis1'"I. Recently, archaea such as Desulfirococcus strain SY [lo'] and Themococcus
strains["'] were shown to produce aspartate racemase. It is interesting to note that
various other archaea such as Pyrobaculum islandicum, Methanosarcina barkeri and
Halobacterium salinarium produce D-amino acids, although their function is not yet
known ["I'.
Okada et al. purified the enzyme to homogeneity from the cell extract of S.
thermophilus, the specific activity of the crude extract of which was elevated
3400-f01d[~~~].The gene encoding aspartate racemase was cloned from S. thermophi-
lus, and overexpressed in E. coli['ll]. The amount of the enzyme produced reached
1298
I 17 lsomerizations
about 20% of the total soluble proteins of the E. coli clone cells. Thus, the enzyme
was efficiently purified to homogeneity from the clone cells['*'! The enzyme is a
homodimer of a subunit with a molecular weight of about 28000. In addition to
aspartate, cysteate and cysteine sulfinate are the only substrates of the enzyme: they
are racemized at a rate of 88 and 51 %, respectively, of that of L-aspartate['''I. The
presence of the acidic group at the fi-carbon is essential; none of asparagine,
cysteine, serine, and alanine are the substrates. Both isomers of glutamate are also
inert. The KM values for L- and D-aspartate are 35 and 8.7 mM, respectively.
Aspartate racemase requires no cofactors and contains an essential cysteine
residue in the same manner as glutamate racemase[80].When L- or maspartate was
incubated with aspartate racemase in tritiated water, tritium was incorporated
preferentially into the product enantiomer. This is consistent with the results of
glutamate racemase as described above["].
Yamauchi et al.['12] concluded that aspartate racemase also uses two bases to
remove and return the a-proton of the substrate. Aspartate racemase contains three
cysteine residues: Cys 84, Cys 190 and Cys 197, and only Cys 84 is essential for the
enzyme activity. The alkylation of one cysteine residue/dimer with 2-nitro-5-thiocya-
nobenzoic acid results in a complete loss of activity. Therefore, the enzyme shows a
ha1f-of-the-sites-reactivity[l1'1.Yamauchi et al. suggested that the enzyme has a
composite active site formed at the interface of two identical subunits in the same
manner as proposed for proline ra~emase['~].
Kumagai and c o w ~ r k e r s [ ' ~developed
~l an enzymatic procedure to produce D-
alanine from fumarate by means of aspartase (E.C. 4.3.1.1), aspartate racemase, and
D-amino acid aminotransferase (Fig. 17-12). Aspartase catalyzes conversion of fu-
marate into L-aspartate, which is racemized to form D-aspartate. D-Amino acid
aminotransferase catalyzes transamination between D-aspartate and pyruvate to
produce D-alanine and oxalacetate. This 2-0x0 acid is easily decarboxylated sponta-
neously to form pymvate in the presence of metals. Thus, the transamination
proceeds exclusively toward the direction of D-alanine synthesis, and total conversion
of fumarate into D-alanine was achieved.
Fumarate
J Aspartase
L-Aspartate
1 ASPR
z
A 1
D-Aspartate
\f Pyruvate /--co2
Figure 17-12. Enzymatic production of
D-alanine by combination of aspartase,
aspartate racemase, and D-amino acid
D-Alanine D-ATA
Oxalacetate aminotransferase reactions.
7 7.2 Racemizations and Epimerizations
I 1299
1 7.2.2.3
Diarninopirnelate Epirnerase (E. C. 5.1.1.7)
meso-a,&-Diaminopimelateis the direct precursor of L-lysine, and is an essential

component of the cell wall peptidoglycans in Gram negative bacteria. meso-a,&-
Diaminopimelate is formed from L-a,&-diaminopimelateby diaminopimelate epi-
merase. The enzyme gene (dupF) was mapped at 85 min on the E. coli chromo-
s0me['~~1. Richaud et al. isolated an E. coli mutant lacking diaminopimelate epimer-
ase activity by insertional mutagenesis, and showed that the mutant does not require
meso-a,&-diaminopimelate in a minimal medium l1l4]. Thus, meso-a,E-diaminopime-
late epimerase encoded by the dapF gene is not essential for E. coli, but meso-a,&-
diaminopimelate still occurs in the mutant cells. Richaud et al. proposed that E. coli
has another enzyme with diaminopimelate epimerase
The diaminopimelate epimerase gene (dupF) was cloned from E. c0li['~~1, and the
amino acid sequence of the enzyme was deduced from the nucleotide
The enzyme was purified to homogeneity from the wild-type E. coli cells and the
recombinant E. coli cells carrying a plasmid coding for dapF gene[116].The enzyme is
composed of two identical subunits with a molecular weight of about 32 000. The
enzyme is independent of PLP or of any other cofactors. The enzyme shows a V,
value of 132 pmol min-' per mg of protein, and a I& value of 0.24 mM for L-a,E-
diaminopimelate. The thiol group of Cys 73 of the enzyme is specifically labeled by a
mechanism-based inactivator, 2- (4-amino-4-carboxybutyl)-2-aziridine carboxylic acid.
Higgins et al. discovered an interesting similarity in amino acid sequences around
the catalytically essential cysteine residue of proline racemase 19*1, hydroxyproline
epimera~e['~], and diaminopimelate epimerase (Cys 73), and proposed that PLP-
independent racemases/epimerases derive from a common evolutionary origin (l16].
However, no significant similarity in the entire amino acid sequence was found
between diaminopimelate epimerase and glutamate racemase, and also between
diaminopimelate epimerase and aspartate racemase.
Cirilli et al. [11'1 cloned the gene of diaminopimelate epimerase from Huemophilus
injuenzae, and purified and crystallized the enzyme. The enzyme is monomeric and
has a unique protein fold, in which the amino terminal and carboxyl terminal halves
of the molecule fold into structurally homologous and superimposable domains
(Fig. 17-13).Cys 73 of the amino terminal domain is found in the disulfide linkage,
at the domain interface, with Cys 217 of the carboxy terminal domain["']. Thus, it is
most conceivable that these two cysteine residues stay in reduced form in the active
enzyme and function as the acid and base in the mechanism. Koo and Blanchard['l8I
explored a number of kinetic and isotope approaches to clarify the mechanism of the
enzyme. However, which of the two cysteine residues is responsible for proton
abstraction from the two enantiomeric Ca-H bonds is not yet known.
1300 I I7 lsomerizations
,----(208 312 201,-I
130 BB 14b'
Figure 17-13. Top: Ribbon diagram of diamino- Bottom: Topology of the secondary structural
pimelate epimerase from Haernophilus influen- elements of diaminopirnelate epimerase. The
zae. The disulfide bridge between Cys 73 and position of pseudo-2-fold symmetry axis is indi-
Cys 21 7 connects domain I (residues 1-1 17 and cated by the black dot between p-strands B7
263-274) and domain II (residues 118-262). and B8. Reprinted from M. Cirilli et
7 7.2 Racernizations and Epirnerizations
I
1301
17.2.2.4
Proline Racernase (E. C. 5.1 .1.4)
Proline racemase occurs in Clostridium sticklandii, which produces 6-aminovalerate

from L-proline. Proline racemase and D-proline reductase are responsible for the
conversion: L-proline is racemized by proline racemase to form D-proline,which is
converted into F-aminovalerate by D-proline reductase (E. C. 1.4.4.1).
Rudnick and Abeles purified proline racemase to 95 % homogeneity from Clostrid-
ium sticklandii, and characterized The enzyme is composed of two identical
subunits with a molecular weight of about 38000, and is independent of any
cofactors or metals. Most amino acid racemases require pyridoxal 5'-phosphate,
which labilizes the bond between the a-hydrogen and the chiral center by aldimine
formation with the a-amino group of the substrate. However, PLP is not involved in
the reaction of proline racemase acting on an a-imino acid. The enzyme also acts on
2-hydroxy-~-prolineand 2-allo-hydroxy-~-proline although slowly: they are epimer-
ized at a rate of 2 and 5 % of the rate of r-proline racemization, respectively. L-Proline
and D-proline showed KM values of 2.9 and 2.5 mM, respectively["'l.
Pyrrole-2-carboxylate is a competitive inhibitor of proline racemase, and stoi-
chiometrically binds with the enzyme (1mol per dimer). Thiol groups of the enzyme
are alkylated by iodoacetate at a stoichiometry of 1 mol of cysteine residue per mol
subunit. However, the enzyme is inactivated completely by modification of only one
cysteine residue per dimer. Thus, Rudnick and Abeles proposed a reaction scheme in
which the active site is located at the interface of two identical subunits, each of
which furnishes one of the two active site thiol groups positioning appropriately at
the composit active site: a thiolate anion derived from one thiol group abstracts the a-
proton from the substrate, and another thiol group protonates the intermediate
derived from the substrate from the opposite face["]. They proposed occurrence of
two forms of free proline racemase: one binds with D-proline and the other binds
with L-proline. According to their proposed mechanism, the product enantiomer is
released much faster than the release of the substrate-derived proton. The proton
release also proceeds much faster than the interconversion of the two forms of the
enzyme. Knowles and coworkers defined the energetics and delineated the complete
free energy profile for the proline racemase reaction [119-1251.
Yagasaki and Ozaki[1261developed a method for production of D-proline from L-
proline using the recombinant proline racemase of Clostridiurn sticklandii. L-Proline
was degraded by Candida sp. PRD-234, and optically pure D-proline was obtained.
17.2.3
Other Racernases and Epirnerases Acting on Amino Acid Derivatives
17.2.3.1
2-Arninod2-thiazoline-4-carboxylateRacernase
Sano et al. [12'1 have found several bacterial strains that are capable of producing L-
cysteine from ~,~-2-amino-2-thiazoline-4-carboxylate (ATC),an intermediate in the
1302
I 7 7 lsomerizotions
. .
hydrolase I
H2N
),sT~~~~ hydrolase II
HS
C
(--ooH
H2Ng & C O O H NH2 NH2
L-2-amino-A2-thiazoline- S-carbamoyl-L-cysteine L-cysteine
4-carboxylate
li racemase
H2N L> "'/C0OH
D-2-amino-A2-thiazoline- Figure 17-14.Enzymatic synthesis of L-cysteine from

4-carboxylate ~,~-2-amino-A*-thiazoline-4-carboxylate.
chemical synthesis of D,L-cysteine. These include several Pseudornonas species

isolated from soil and other strains belonging to different genera such as E. coli,
Bacillus breuis, and Micrococcus s~denensis['~~].
Three enzymes are probably involved
in this pathway: L-ATChydrolase, S-carbamoyl-L-cysteinehydrolase and ATC race-
mase (Fig. 17-14). Pseudornonas thiazolinophilurn isolated from soil was shown to
have the highest activity of the enzymes that produce L-cysteine from D,L-ATC.The
enzymes are inducibly formed in the bacterial cells by addition of D,L-ATCto the
growth medium.
Degradation of L-cysteine by cysteine desulfhydrase or other PLP enzymes present
in the cells was successfully prevented by addition of hydroxylamine or semi-
carbazide to the incubation mixture. A mutant strain of Ps. thiazolinophilum lacking
cysteine desulfhydrase was isolated and used to produce L-cysteine from D,L-ATCin
a molar yield of 95% and at a product concentration of 31.4 g L-'['28]. Pseudornonas
desrnolytica AJ 3872, one of the L-cysteine producers isolated was found to lack the
ability to convert D-ATC into L-cysteine: it is an ATC racemase-deficient strain['*'].
However, little is known about the enzymological properties and function of the
racemase.
Among the three enzymes participating in L-cysteine production, L-ATChydrolase
was found to be the least stable['30].However, the stability of L-ATChydrolase was
sharply enhanced as water activity decreased from 0.93 to 0.80. In the absence of
sorbitol, the stability of L-ATChydrolase increased in proportion to ionic strength.
Thus, Ryu et al. succeeded in enhancing the half life of L-ATChydrolase by 10-foldto
20-fold in sorbitol-saltmixtures [1301.
7 7.2 Racemizations and Epimerizations I 1303
17.2.3.2
Hydantoin Racemase
5-Substitutedhydantoin derivatives have been used as precursors for D- and L-amino

acids in chemical synthesis. However, they are hydrolyzed enantioselectively by the
enzymes named hydantoinases: some act specifically on D-5-substitutedhydantoins,
and others on the r-isomers. N-Carbamoyl amino acids formed are also hydrolyzed
enantiospecifically by N-carbamoyl amino acid amidohydrolases to produce D- or L-
amino acids (Fig. 17-15). Since the Kanegafuchi Chemical Industry, Japan, commer-
cialized an enzymatic procedure for the production of D-p-hydroxyphenylglycine,
which is a building block for the semisynthetic j3-lactam antibiotic amoxycillin,
various processes for amino acid production by means of hydantoinases have been
devel~ped[~~~-'~~].
Subsequent to the discovery that hydantoin is hydrolyzed by extracts of mammal-
ian livers [1341 and plant seeds [1351,various microorganisms have been shown to
utilize D- and L-5-substituted hydantoins as a sole carbon or nitrogen source by
means of D- as well as L-specific hydantoinases inducibly formed [131-1331.
Distribution of D-hydantoinase in microorganisms has been shown by Yamada
and coworker^^^^^]. The enzyme is identical to dihydropyrimidinase (E. C. 3.5.2.2),
and is widely distributed in bacteria, in particular in Klebsiella, Corynebacterium,
Agrobacteriurn, Pseudornonas, and Bacillus, and also in actinomycetes such as Strepto-
rnyces and Actinoplanes. The enzyme activity occurs also in eukaryotes: yeasts, molds,
plants and mammals. Pseudomonas putida was found to be the best strain, which
produced D-hydantoinasemost abundantly and inducibly by addition of 5-methylhy-
dantoin. Most of D-hydantoinase producers form N-carbamoyl D-amino acids from
the corresponding 5-substituted hydantoins. Accordingly, to obtain free D-amino
acids, N-carbamoyl amino acids need to be isolated and hydrolyzed chemically or
enzymatically. However, a few bacterial strains produce N-carbamoyl D-amino acid
amidohydrolase in addition to D-hydantoinase. Thus, optically pure D-amino acids
were produced from D-hydantoinswith these bacterial cells. Olivieri et al. [1371 found
that Agrobacteriurn turnefaciens cells grown on uracil as a sole nitrogen source
catalyze the complete conversion of racemic hydantoins into D-amino acids. Hartley
et a1.[138] obtained a mutant strain which expresses both the hydantoinase and N-
carbamoylamino acid amidohydrolase in the absence of an inducer. In contrast,
other bacterial strains belonging to the genera of Flavobacteri~m['~'],Arthro-
Chemical or
R H O Hydantoinase R y c o o H enzymatic
hydrolysis
H20 * HNKNH2
H20
* RyCooH
NH2
+ + COz NH3
HNKNH
0 0
R = aryl, alkyl * LOrD
Figure 17-15. Enzymatic synthesis of D- or L-amino acids from 5-substituted
D,L-hydantoinsthrough N-carbamoyl-D- or L-amino acids.
1304
I b a ~ t e r [ ' ~Pseud~monas['~'~
7 7 lsomerizations
~], 1421, and B a ~ i l l u s [ ' ~convert

~ - ~ ~ ~whole
] racemic 5-substi-
tuted hydantoins into the corresponding L-amino acids. In these bacteria, 5-substi-
tuted hydantoins are hydrolyzed by L-hydantoinase to form N-carbamoyl L-amino
acids, which are hydrolyzed further to L-amino acids by N-carbamoyl L-amino acid
amidohydrolase in the same manner as described above except that the enzymes
involved show opposite stereospecificity. 5-Mono-substituted hydantoins can race-
mize spontaneously under weakly alkaline conditions, and this chemical racemiza-
tion participates at least partly in the total conversion of the racemic hydantoins into
free L- or D-amino acids. However, if chemical racemization proceeds only
a hydantoin racemase was suggested to occur and participate in the total
conversion[146, 1471.
Watabe et a1.[148]isolated a plasmid which is responsible for the conversion of
5-substituted hydantoins into the corresponding L-amino acids from a soil bacte-
rium, Pseudomonas sp. NS 671, which is able to convert racemic 5-substituted
hydantoins into the corresponding L-amino acids. The genes involved in the
conversion were cloned from the Pseudomonas plasmid into E. coli, and functions of
four genes were identified and named hyuA, hyuB, hyuC and hyuE. Both hyuA and
hyuB are required for the conversion of D- and L-5-substitutedhydantoins into the
corresponding N-carbamoyl-D- and N-carbamoyl-L-amino acids, respectively, al-
though the individual reactions catalyzed by the gene products have not yet been
identified. HyuC codes for an N-carbamoyl-L-aminoacid amidohydrolase, while
hyuE is a hydantoin racemase gene[l4'I. Significant nucleotide sequence similarity
was found between hyuA and hyuC (43%), and also between hyuB and hyuC (46%).
Watabe et al. suggested that these genes have evolved from a common ancestor by
gene However, no proteins registered in NBRF and SWISS protein
data bases showed similarity with the deduced amino acid sequences of the four
genes.
Wagner and associates purified hydantoin racemase from Arthrobacter aurescens
DSM 3747 and characterized Watabe et al. [14'1 also purified the enzyme from
E. coli clone cells harboring a plasmid coding for the enzyme gene derived from
Pseudomonas sp. NS 671. The Pseudomonas enzyme is a hexamer composed of a
subunit with a molecular weight of about 32000, which is consistent with the value
deduced from the amino acid sequence. The D- and L-isomers of 5-(2-methylth-
ioethy1)hydantoin and 5-isobutyrylhydantoinare racemized effectively. ~-5-(2-Me-
thylthioethy1)hydantoinis racemized at a V,, value (79 pmol min-' mg-') which is
about 2.5 times higher than that for the L-isomer. Wiese et al.['501 cloned the
hydantoin racemase gene from Arthrobacter aurescens DSM 3747 and purified the
enzyme to homogeneity. The Arthrobacter enzyme has a molecular mass of 25.1
kDa['50] and acts on aromatic and aliphatic hydantoin derivatives such as 5-in-
dolylmethylhydantoin, 5-benzylhydantoin, 5-(p-hydroxybenzyl)hydantoin,5-(2-me-
thylthioethyl)hydantoin, and 5-i~obutylhydantoin['~~l, although hydantoins with
arylalkyl side chains are preferred substrates [1591. Free amino acids, amino acid
esters and amides are inert, but the enzyme suffers from inhibition by aliphatic
substrates such as L-5-methylthioethylhydantoin. The hydrogen at the chiral center
of a substrate, ~-5-indolylmethylenehydantoin, is exchanged with solvent deuterium
during racemizati~n['~~]. Pietzsch et al.
established a method for the synthesis of

I
1305
optically pure ~-3-trimethylsilylalaninefrom ~,~-5-trimethylsilylmethylhydantoin in

88 % yield and 95 % enantiomeric excess with whole resting cells of Agrobacterium
sp. IP I 671, immobilized in a Ca-alginatematrix. On the other hand, L-3-trimethylsi-
lylalanine was also prepared from the racemic substrate by enantiomer-specific
hydrolysis of the L-form in the presence of L-N-carbamoylase from Arthrobacter
aurescens DSM 3747[1521.
Watabe et al. found that the Pseudornonas enzyme is inactivated by a substrate, L-
5-methylhydantoin,during ra~emization~'~']. However, the enzyme was not affected
by the D-isomer. Both enantiomers of 5-isopropylhydantoininactivated the enzyme
to the same extent. Interestingly, divalent sulfur-containing compounds such as
methionine, cysteine, glutathione, and biotin protected the enzyme effectively from
inactivation. E. coli cells expressing the racemase are capable of racemizing all of
these hydantoin derivatives: the enzyme is protected from inactivation by divalent
sulfur compounds occurring in the cells. Watabe et al. concluded that the protective
effect by the divalent sulfur-compounds is not due to their reducing Both
Pseudom~nas['~~] and Arthr~bacter['~~] enzymes are inhibited strongly by Cu2+.The
Arthrobacter enzyme is completely inhibited by HgCl2 and iodoacetamide, and
stimulated by addition of dithiothreitol [1501. Therefore, the enzyme may contain
essential cysteine residues, which are possibly modified by some activated inter-
mediate derived from the particular substrates leading to the enzyme inactivation.
E. coli cells canying a plasmid coding for hyuA, hyuB, hyuC, and hyuE convert only
~ - 5 2methylthioethyl)hydantoin
-( into L-methionine. On the other hand, E. coli cells
harboring a plasmid coding for only hyuA, hyuB, and hyuC first convert the L-
hydantoin, then the D-isomer is hydrolyzed slowly when the L-isomer is depleted.
Therefore, Watabe et al. believe that ~-5-(2-methylthioethyl)hydantoin is only con-
verted into L-methionine in the presence of the hydantoin racema~e['~'].The
mechanism of stereospecific conversion of D,L-S-substituted hydantoins to the
corresponding L-amino acids by Pseudomonas sp. strain NS 671 has been clarified by
Ishikawa et al. ~,~-S-substituted hydantoins are converted exclusively into the L-
forms of the corresponding N-carbamoylamino acids by the hydantoinase in combi-
nation with hydantoin racemase, and then the N-carbamoyl-t-amino acids are
converted into L-amino acids by N-carbamoyl-t-aminoacid amidohydrolase (Fig. 17-
16).
By directed evolution May et al. succeeded in inverting the enantioselectivity of
D-hydantoinase from Arthrobacter sp. DSM 9771 into an L-selective enzyme. The
improved hydantoinase also acquired a five-fold increase in activity. The recombi-
nant E. coli cells expressing three heterologous genes (i. e. the evolved L-hydantoi-
nase, L-N-carbamoylase, and hydantoin racemase) were found to produce 91 mM L-
methionine from 100 mM ~-5-(2-methylthioethyl)hydantoin in less than 2 h[1541.
1306
“yCooH
= HNyNHz
HNKNH 0
ATP ADP
Acid
KCarbamyl-L-Amino*
Amidohydrolase
RYCooH
NH2
0 0
L-5-Substituted
Hydantoin
II
/I
U
N-Carbamyl-
L-Amino Acid
L-Amino Acid
L-preferential
Hydantoin Hydantoinase
Racemase
D-5-Substituted N-Carbamyl-
Hydantoin D-Amino Acid
Figure 17-16. Stereospecific conversion of o,~-5-substitutedhydantoins into the
corresponding L-amino acids by Pseudomonos sp. NS 671. Reprinted from
lshikawa et al.11531.
17.2.3.3
N-Acylamino Acid Racemase
~-Aminoacylases(E. C. 3.5.1.14) catalyze the hydrolysis of the amide bond of various

N-acyl-L-amino acids, such as N-acetyl-, N-chloroacetyl- and N-propionyl- amino
acids [1551, and is widely distributed in animals [155-1571, plants [lS8, and micro-
organisms [lGo, l6l1. Greenstein[”’] first studied the reactivity of pig kidney enzyme,
and showed its application to the optical resolution of racemic amino acids. Chibata
et al. [IG2] found that L-aminoacylase is produced abundantly by fungal species
belonging to the genera Aspergillus and Penicillium. L-Aminoacylaseswere purified
from pig kidney and A. oryzae, and their reaction mechanism and physiological
function were studied[l“. 163-1Gs1 . Cho et a]. [‘“I showed that various thermophilic
Bacillus strains produce thermostable L-aminoacylase, and purified it to homoge-
neity from Bacillus themoglucosidius DSM 2542, which produces the enzyme most
abundantly. L-Aminoacylases of pig kidney, Aspergillus oryzae and B. thermoglucosi-
dius share many features with each other: they contain Zn” as a prosthetic metal, are
strongly activated by Co2+,and have a pH optimum in the range of 8.0-8.5.
Sugie and S u ~ u k i [ ~ ”demonstrated
] the occurrence of D-aminoacylase, which
specifically hydrolyzes the amide bond of N-acyl-D-aminoacids, in actinomycetes,
and applied the enzyme to the production of ~-phenylglycine.Recently, a new D-
aminopeptidase was found in Alcaligenes denitnicans, and shown to act on various N-
acyl-~-amino acids including N-acetyl-D-methionine[“*. lG9].
I
1307
N-Acylamino acids are usually racemized much more readily than the correspond-
ing free amino acids. Therefore, by combination of chemical racemization and
enantioselective hydrolysis of N-acylamino acids, racemates of N-acylamino acids
can be fully converted into the desired enantiomer of the free amino acids according
to the stereospecificity of the aminoacylases used. For example, L-tryptophan is
produced industrially by combination of chemical racemization of N-acetyltrypto-
phan and enantiospecific hydrolysis of its L-isomer with the Aspergillus L-aminoacy-
lase, which shows high reactivity towards N-acyl derivatives of aromatic L-amino
acids. When N-acetyl-D,L-tryptophanis incubated with the fungal enzyme, N-acetyl-
L-tryptophan is selectively hydrolyzed to L-tryptophan, which is then crystallized
from the solution. N-Acetyl-D-tryptophan in the mother liquor is racemized with
acetic anhydride, and the racemate is again used as a starting material. In principle,
D- and L-amino acids can be produced from their corresponding N-acyl derivatives in
the same manner, provided that N-acyl derivativesof the desired amino acids serve as
the substrates of the available aminoacylases,and are racemized chemically without
any major loss by decomposition. However, the chemical racemization can be
achieved only under extreme conditions in order for the aminoacylases to be
inactivated, and the enzymes are usually required to be saved for the subsequent
cycles for reasons of economy. Therefore, the antipode of the substrate is separated
from the enzyme and preferably from the product in order to avoid its possible
racemization. Tosa et al. have developed a continuous method to produce L-
tryptophan, which is now utilized in industry, by means of the Aspergillus L-
aminoacylase immobilized on DEAE-Sephadex[17'].
Takahashi and Hatano of Takeda Chemical Industries, Japan, succeeded in finding
a racemase that acts on N-acylaminoacids, but not on the corresponding free amino
acids, and named it acylamino acid racema~e["~]. They have established a method of
producing optically active a-amino acids from the corresponding D,L-N-acylamino
acids by means of the acylamino acid racemase and aminoacylases.
Acylamino acid racemase occurs widely in various actinomycete strains belonging
to the genera of Streptomyces, Actinomadura, Actinomyces, Iensenia, and Amycola-
to psi^["^]. The enzyme was purified to homogeneity from Streptomyces atratus Y-53,
which shows the highest enzyme activity among the strains tested['73].The enzyme
is composed of G subunits with identical molecular masses (about 41000), and
shows a molecular mass of 244000 in the native state. Tokuyama and Hatan0['~'1
purified thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60
and purified it to homogeneity. The molecular masses of the native enzyme and the
subunit are 300000 and 40000, respectively. The enzyme is stable at 55 "C for 30
min. The enzyme catalyzes the racemization of N-acylaminoacids such as N-acetyl-
L- or D-methionine, N-acetyl-L-valine,N-acetyl-L-tyrosineand N-chloroacetyl-L-valine
(Table 17-5). In addition, the enzyme also catalyzes racemization of dipeptide L-
alanyl-L-methionine.By contrast, N-alkylamino acids and methyl and ethyl esters of
N-acetyl-D- and L-methionine are not racemized. The apparent KM values for N-
acetyl-L-methionine and N-acetyl-D-methionine are 18.5 mM and 11.3 mM, re-
spectively. The enzyme activity is markedly enhanced by the addition of divalent
metal ions such as Co2+,Mn2+ and Fe2' and inhibited by addition of EDTA and p
1308
I 17 lsornerizations
Table 17-5. Substrate specificity of acylamino acid racemasea.

Substrate Relative activity
N-Acetyl-o-methionine 100
N-Acetyl-r-methionine 100
N-Formyl-D-methionine 40
N-Formyl-L-methionine 63
N-Acetyl-D-alanine 33
N-Acetyl-L-alanine 21
N-Benzoyl-o-alanine 14
N-Acetyl-D-leucine 37
N-Acetyl-L-leucine 74
N-Acetyl-n-phenylalanine 64
N-Acetyl-L-phenylalanine 84
N-Chloroacetyl-D-phenylalanine 90
N-Chloroacetyl-L-phenylalanine 112
N-Acetyl-D-tryptophan 10
N-Acetyl-L-tryptophan 8
N-Acetyl-D-vahe 35
N-Acetyl-r-valine 19
N-Chloroacetyl-D-vahe 80
N-Chloroacetyl-L-valine 105
N-Acetyl-D-alloi s o h c i n e 33
a Inert: D- and r-methionine, D- and r-alanine, D- and L-leucine, wand L-phenylalanine.
D- and L-tryptophan,D- and L-valine.
chloromercuribenzoate. The gene of N-acylamino acid racemase was cloned from

Amycolatopsis sp. TS-1-60[1751, and overexpressed in E. coli host cells with T7
promoter[’76].The gene codes for a protein of 368 amino acids with a molecular
mass of 39411 Da. Palmer et al.[177]found that N-acylamino acid racemase of
Amycolaptosis sp. TS-1-60 is similar to an unidentified protein encoded by the
Bacillus subtilis genome. N-Acylamino acid racemase efficiently catalyzes an 0-
succinylbenzoate synthase reaction, which is responsible for menaquinone bio-
synthesis.
Tokuyama et al. [1721 found that most of acylamino acid racemase-producing strains
produce not only acylamino acid racemase but also aminoacylases; one of either D- or
r-aminoacylase or both of them. Moreover, acylamino acid racemase shows the
optimum pH at around 8.0, which is close to that of aminoacylases. Therefore, N-
acylamino acid can be converted as a whole into L- or D-amino acids in one step by
means of microbial cells of appropriate strains producing either L- or D-aminoacylase
in addition to acylamino acid racemase.
17.2.3.4
lsopenicillin N Epimerase
Isopenicillin N is a precursor of penicillin, and synthesized from 6-(L-aminoadipoy1)-

L-cysteinyl-D-valineby isopenicillin N synthetase Isopenicillin N is then con-
verted into penicillin N by isopenicillin N epimerase. Penicillin N is ring-expandedto
deacetoxycepharosporin C by penicillin N expandase. The latter compound is
sCOOH
Hs$H
isopenicillin N isopenicillin N
synthetase epimerase
7-T-
02
i
H20
O H2N ICOOH H2N COOH
&(L-a-aminoadipoyl)- isopenicillin N penicillin N

L-cysteinyl-0-valine
SI$COOH
-N S%COOH
I-N
deacetoxy-
penicillin N cepharosporin C
expandase hydroxylase
Ir
02 02
a-ketoglutarate a-ketoglutarate
deacetoxycepharosporin C deacetylcepharosporin C
Figure 17-17. Biosynthetic pathway for cepharosporin C.
hydroxylated to form deacetylcepharosporinC by deacetoxycepharosponnC hydrox-

ylase. These reactions proceed sequentially in the biosynthesis of cepharosporin C in
Streptomyces clavuligerus, a producer of various p-lactam antibiotics 180] (Fig. 17-
17). However, in Cepharosporium acremonium, conversion of penicillin N into

deacetoxycepharosponn C is catalyzed by a bifunctional enzyme, penicillin N
expandaseldeacetoxycepharosporin C hydroxylase in Cepharosporium acremon-
ium11811.
Isopenicillin N epimerase activity, demonstrated in the extract of Cepharosporiurn
acremonium protoplasts was found to be very unstabIe['821. Usui and Yu11831,
however, succeeded in purifying the enzyme to homogeneity after development of a
simple assay procedure of the enzyme. They studied its enzymological proper-
ties[183].The enzyme has a monomeric structure with a molecular mass of 47000.
The enzyme contains 1 mol of PLP per mol of protein. The enzyme shows a V,,
value of 3.93 pmol min-' per mg and a KM of 0.30 mM for isopenicillin N, whereas it
shows a V,, of 9.47 pmol min-' per mg and a KM of 0.78 mM for penicillin N. The
Gqvalue for the conversion between isopenicillin N and penicillin N is 1.09, which
is in good agreement with the theoretical value. In addition to isopenicillin N and
penicillin N, deacetoxycepharosporin C was epimerized only slowly: the rate relative
I7 lsomerizations
1310
I
to isopenicillin N is about 1%. However, the following penicillin derivativesare inert:
deacetylcepharosporin C, ceparosporin C, &(L-a-aminoadiopoyl)-L-cysteinyl-D-va-
line, L-a-aminoadipate,and D-a-aminoadipate.The enzyme is inhibited strongly by
thiol reagents such as p-chloromercuribenzoate[1831.
17.2.4
Racemization and Epimerization at Hydroxyl Carbons
Various epimerases acting on carbohydratederivatives and acyl-CoA derivativeswere

demonstrated, purified, and characterized as reviewed previously['84].Lactate race-
mase (E.C. 5.1.2.1) is the first racemase to he The mechanism of
lactate racemase reaction was studied with the enzyme preparations partially
purified from Clostridium b u t y r i c ~ r n [ ' ~ ~Hiyama
]. et al. [186] highly purified the
enzyme from Lactobacillus sake, but little is known about its enzymological proper-
ties. In contrast, mandelate racemase (E. C. 5.1.2.2) is the enzyme best characterized
among various racemases and epimerases: its tertiary structure and functional
groups that participate directly in catalysis has been clarified.
17.2.4.1
Mandelate Racemase (E.C. 5.1.2.2)
Mandelate racemase catalyzes the racemization of mandelate, which is the first step
of the mandelate assimilation pathway in Pseudomonas putida. Although the man-
delate pathway occurs widely in various bacteria, fungi and yeasts, most of them
utilize one enantiomer or the other of mandelate in a benzoate-forming pathway. A
few strains such as Acinetobacter calcoacetic~s[~~~] and Aspergillus nigar[188]are
capable of using both enantiomers with two complementary dehydrogenases with
different stereospecificities. However, a single strain of Pseudomonas putida produc-
ing mandelate racemase can utilize both enantiomers [1891.
In Pseudomonas putida, D-mandelate is converted into L-mandelate by mandelate
racemase, then oxidized to benzoylformate by mandelate dehydrogenase (Fig. 17-
18). Benzoylformate decarboxylase is the second enzyme of the pathway and
catalyzes decarboxylationof benzoylformateto form benzaldehyde,which is oxidized
to benzoate by NAD- and NADP-linked benzaldehyde dehydrogenases. The genes
encoding these five enzymes constitute an operon that is induced by either
enantiomer of mandelate [l9O]. Stecher et al. ["I' established large-scale production of
mandelate racemase by Pseudomonas putida ATCC12633 by optimization of enzyme
induction: both glucose and mandelate were added to the culture right from the start
as the carbon source. Thus, about 300-fold enhancement in the enzyme production
was achieved. Strauss et al. [1921 showed that immobilized mandelate racemase is an
efficient biocatalyst used for repeated batch reactions to produce (R)-mandelatefrom
(S)-mandelateunder mild conditions.
Kenyon and coworkers purified mandelate racemase to homogeneity, and charac-
terized it[ls9].Divalent metal ions such as Mg2+,Mn2+,Co2+,and Ni" were required
for the catalysis. In addition to mandelate, p-hydroxymandelate and p-(bromome-
H : hOH mandelate Hoh:o*
7 7.2 Racemizations and Epirnerizations
mandelate O6;OoH
I
1311
racemase dehydrogenase
L
6
-.. P
\ \ \
benzoylformate
dehydrogenase*
O H
benzaldehyd
dehydrogenase, d ‘OH p-keto adipate
pathway
acetylCoA + succinate
Figure 17-18. Mandelate assimilation pathway in Pseudomonas putida.
thy1)mandelate serve as the substrates. p(Bromomethy1)mandelate is decomposed

to p-(methy1)benzoylformateand bromide by action of the enzyme. The KM values
for D- and L-mandelateare 0.23 and 0.26 mM, respectively.
Ransom et a1.[193]cloned the gene for mandelate racemase from Pseudomonas
putida in Pseudomonas aeruginosa on the basis of the inability of the latter strain to
grow on D-mandelateas a sole carbon source. The amino acid sequence was deduced
from the nucleotide sequence, and the predicted molecular mass of the enzyme was
38750[1931.The enzyme is composed of eight identical subunits. The crystal
structure of mandelate racemase has been solved and refined at 2.5 A re~olution[”~1.
The secondary, tertiary and quaternary structures of mandelate racemase are quite
similar to those of muconate lactonizing enzyme[”’, 1961 . Mandelate racemase is
composed of two major structural domains and a small C-terminal domain. The N-
terminal domain has an a + p structure, and the central domain has an a/P-barrel
topology. The C-terminal domain consists of an L-shaped loop.
Divalent metal ions, which are essential catalykally, are ligated by three distal
carboxyl groups of Asp 195, Glu 221, and Glu 247, all of which occur at the central
domain[194].The active site location was determined by analysis of a complex
between mandelate racemase and p-iodomandelate, whose iodine atom has high
electron density and contributes greatly to the analysis. The active site of the enzyme
is located between the two major domains. The ionizable groups of Lys 166 and
His 297 are located at the positions interacting with the chiral center of the substrate
(Fig. 17-19).Neidhart et al. proposed that they participate in general acid/base
catalysis: Lys 166 abstracts the a-proton of r-mandelate, and His 297 abstracts the a-
proton from D-mandelate. Landro et al. [19’] then replaced His 297 by asparagine,
analyzed the crystal structure of the H297N mutant enzyme at 2.2 A resolution, and
studied the mechanism of catalysis of the mutant enzyme. Although the mutant
enzyme has no mandelate racemase activity, it catalyzes the stereospecific elimina-
tion of bromide from p-(bromomethy1)-L-mandelateat a rate equivalent to that
catalyzed by the wild-typeenzyme. Moreover, the mutant enzyme catalyzes exchange
of the a-hydrogen of L- but not D-mandelatewith deuterium in deuterium oxide at a
rate 3.3 times less than that of the wild-type enzyme. Thus, Landro et al.[”’, ”)‘I
concluded that the mandelate racemase reaction proceeds through a two-base
1312
I 17 lsomerizations
139SEA f\
GLU GLU
GLU
Figure 17-19. Models o f the mandelate racemase active site with complexed
substrate, p-iodomandelate. Reprinted from Neidhart et al.[194].
mechanism in which Lys 166 abstracts the a-proton from L-mandelateand His 297
abstracts the a-proton from D-mandelate (Fig. 17-20). In fact, the X-ray crystal
studies of mandelate racemase inactivated by (R)-a-phenylglycidaterevealed that the
E-amino group of Lys 166 is covalently bound to the distal carbon of the epoxide
ring[”’]. KlGGR mutant enzyme catalyzes the stereospecific elimination of bromide
ion from (R)-p-(bromomethy1)mandelate to form p(methy1)benzoylformateat a rate
similar to that catalyzed by the wild-typeenzyme[200], while H297N acts stereospecif-
ically on (S)-p-(bromomethy1)mandelate [2011. This is compatible with the mechanism
that Lys 166 and His 297 participate as the (S)- and (R)-specificcatalyst, respectively.
Bearne and Wolfenden[2021 proposed that the complementary nature of the struc-
tures of mandelate racemase and its substrate is optimized in the transition state
otherwise the general acid-generalbase catalysis will not become an efficient mode
of catalysis.
17.3
lsomerizations
We describe here the enzymological characteristics and application of isomerases,

especially D-xylose (glucose) isomerase, phosphoglucose isomerase, triose phos-
phate isomerase, L-rhamnose isomerase, L-fucose isomerase, maleate cis-trans iso-
merase, and unsaturated fatty acid cis-trans isomerase. i%ketyl-D-glucosamine
2-epimerase is not an isomerase, but for convenience we will also describe the
characteristics and use of the enzyme because this section deals with sugar-
metabolizing enzymes.
17.3 lsomerizations
I
1313
I o y G l 317
~
LYs 166-NH3 H%...H-O Figure 17-20. Mechanism o f t h e
.
I. reaction catalyzed by mandelate
HO.
N H ~
. ,,\O---HaN-Lys 164 racemase with concerted general acid-
general base through an enolic inter-
i-==/Nt 'Mi2+
mediate. Reprinted from Mitra et
His 297 aI. [' 981.
17.3.1
D-Xylose (Glucose) lsomerase (E. C. 5.3.1.5)
D-Xylose isomerase catalyzes the interconversion between D-xylose and D-xylulose

(Fig. 17-21). Since this enzyme acts on D-glucose to produce D-fructose, it is often
referred to as glucose isomerase (Fig. 17-21). The isomerization of glucose to
fructose by this enzyme is a very important process for the industrial production of
high fructose corn syrup. This enzyme is also applicable to the synthesis of many
aldoses and ketoses because of its wide substrate specificity. The enzyme gene has
been cloned from various microorganisms, and the enzyme has been overexpressed,
purified, and characterized. Their three dimensional structures have also been
determined [203-20G1.
17.3.1.1
Properties
Xylose isomerases have been purified from various microorganisms, such as

Lactobacillus brevis, Streptomyces sp., Bacillus stearothemophilus, and Actinoplanes
1314
I 77 lsomerizations
CHO YH20H
YH2OH H-?-OH c=o
YHO
H-C-OH c=o HO-C-H HO-I;-H
d
I I I
HO-C-H ___)L HO-C-H H-C-OH H-C-OH
I
H-C-OH ~ - 6 - 0 ~
H-+-OH H-?-OH I I
CH2OH CH20H CH2OH CH20H
D-Xylose D-Xylulose D-Glucose D-Fructose
Figure 17-21. Reactions catalyzed by D-xylose isomerase.
rnissouriensis[207-2101. They consist of four identical subunits whose molecular mass

are in the range 42 000-51 000. The optimum pH usually ranges from 7.0 to 9.0. The
cDNA for barley (Hordeurn uulgare) enzyme gene has been cloned, and the re-
combinant enzyme characterized[211]. It is unique because it is a dimer composed of
a subunit with a molecular mass of 53620, which is much larger than those of
microbial enzymes. Thermostable xylose isomerases were purified and charac-
terized from many thermophilic bacteria f204, 205, 212-2221 . The enzyme isolated from
Themotoga neapolitana is extremely thermostable, with the optimal activity being
above 95 oC[21Gl. The catalytic efficiency (kcat/ht) of the enzyme is essentially
constant between GO and 90 "C, and decreases between 90 and 98 "C primarily
because of a large increase in KM. Xylose isomerase requires divalent metal cations,
usually Mg2', Mn2+,or Co2+for the maximum activity and thermal stability. The
enzyme has a wide substrate specificity[223]: glucose and fructose derivatives mod-
ified at the 3-, 5- or 6-position are isomerized by the enzyme as will be described
later.
17.3.1.2
Reaction Mechanism
The reaction mechanism of xylose isomerase was proposed based on X-ray crys-
t a l l ~ g r a p h y [and
~ ~ molecular
~] mechanical and molecular orbital studies [2251.
The a-pyranose form of the substrate binds to the active site of the enzyme, and
the reaction is initiated by ring-opening involving hydrogen transfer from the first
hydroxyl group to 0 5 (Fig. 17-22).After extension of the substrate, a water molecule
abstracts the proton from the hydroxyl group at 0 2 of xylose and transfers it to
Asp 257 in the second step. The following hydride shift causes isomerization. The
01 atom of the ketose is negatively charged and most probably abstracts a proton
from Asp 257. The stable cyclic conformation is then formed.
This hydride shift reaction mechanism is quite different from the base-catalyzed
enolization mechanism proposed for phospho sugar isomerases such as triosephos-
phate isomerase which generally do not require a metal ion for activity[226].
B
17.3 lsomerizations
I 1315
Glu 217 H
), (yHis220
P - O\\ .N
t . ’ -..-o
*’
H
,‘
,Mg*i .\ --_ - F A s p 2 5 5
‘M$T?F
,I
N=r\
\
‘. “0
’0
His54 I OH OH H
Asp 57
bf
+
H N 7
NH
His 54
Asp 57
Asp 257
GIU217, H
His 54
Asp 57
Asp 257
Figure 17-22. Reaction mechanism for xylose-xylulose conversion by o-xylose
isomerase through ring opening (A) and hydride shift (B). Reprinted from
Fuxreiter et al. [2251.
1316
I 77 /sorner;zations
17.3.1.3
Production o f Fructose
Xylose isomerase derived from various microorganisms, such as Actinoplanes

missouriensis, Streptomyces griseofiscus, Havobacterium arborescens, Streptomyces phae-
chromogenes, Bacillus coagulans, Streptomyces murinus, Streptomyces rubiginosus, and
Streptomyces oliuochromogenes, is utilized in the annual conversion of 3 million tons
of glucose into fructose for use as high fructose corn syrup. The enzyme is
immobilized by glutaraldehyde cross-linking or adsorption on an insoluble resin for
the fixed bed isomerization process [2271.
The isomerization is reversible, and the final fructose content depends on the
reaction temperature. The reaction is usually carried out in the region of 60-65 "C.
However, a higher temperature gives a higher fructose content. It is reported that the
degree of conversion is raised from 42 %, which is the normal fructose content of the
syrup, to 55 % by isomerization with xylose isomerase at about 95 0C[2271.Therefore,
the thermostability of the enzyme is an important issue. Recently, several thermo-
stable xylose isomerases were found and 205. 212-222]. It is also
reported that the thermostability of the enzyme is enhanced by site-directed
mutagenesis [22sl.
a-Amylasesand xylose isomerases with low optimum pH values are expected to be
useful for fructose production from cornstarch because raw cornstarch solutions
have an acidic pH of around 4.5 and the glucoamylase reaction, the second step in
the process, prefers an acidic pH. Fructose can be produced from cornstarch without
pH adjustment throughout the process at acidic pH values by means of such
acidophilic a-amylases and xylose isomerases. Takasaki et al. [2291 found an acid-
ophilic a-amylase in a Bacillus licheni&mis strain isolated from soil, and showed that
the enzyme is suitable for digestion of cornstarch at an acidic pH of 4.5-5.0.
Acidophilic xylose isomerases have been demonstrated in Thermoanaerobacterium
sp. JW/SL-YS[2171 and Streptomyces sp. SK[22'], and purified and characterized. Both
ofthese have optimum pH values around G.5, but are highly active at acidic pHs such
as 5.0. Since they are highly thermostable, they are expected to be useful for fructose
production.
17.3.1.4
Production o f Unusual Sugar Derivatives
Xylose isomerase has a wide substrate specificity, and 3-, 5-, or 6-substituted glucose
and fructose are isomerized by this enzyme. Since this enzyme requires the 4-OH
group for hexoses to be substrates, phosphoglucose isomerase instead of xylose
isomerase is used for the synthesis of kubstituted fructose as described below.
17.3.1.4.1 Preparation of Glucose Derivatives Modified at Position 3 or 6

Bock and coworkers [2301 showed that D-glucose derivatives bearing modifications at
the C 3 or CG position are converted by xylose isomerase from Streptomyces sp.
17.3 lsomerizations
Figure 17-23. Conversion

I 1317
by xylose isomerase of
A (ZR,3R)-configuredaldote-
I I OH trose modified at C5 into
OH OH open-chain 2-ketoses (A),
X=F and L-erythrose into L-ery-
X = N3 thrulose (B). Reprinted
from Ebner and S t u t ~ [ ~ ~ ~ ] .
However, epimers of D-glucose are inert as substrates of the enzyme: D-mannose, D-

allose, and D-galactose. Various 5-modified D-glucofuranoses are quantitatively
converted into the corresponding D-fructopyranoseswith the enzyme [2311. Ebner and
S t i i t ~ [ ~showed
~ * ] that various (2R,3R)-configuredaldofuranoses such as D-erythrose
and CS-modified D-ribose derivatives serve as substrates of the enzyme: D-erythrose
is quantitatively converted into D-glycero-tetrulose,with D-ribofuranoses being the
corresponding open-chain 2-ketoses (Fig. 17-23).L-Erythrose, the enantiomer of D-
erythrose, is also isomerized quantitatively by the enzyme to L-erythrulose(L-glycero-
tetrulose) (Fig. 17-23).Fructose bisphosphate aldolase catalyzes a stereospecific aldol
condensation between dihydroxyacetone phosphate and a number of aldehydes to
form hexoketose 1-phosphates, the phosphate groups of which are removed by
hydrolysis. The resultant hexoketoses are converted stereospecifically into hex-
oaldose derivatives by xylose isomerase. Thus, unusual hexoaldose derivatives such
as 3-deoxy-~-glucose, 6-deoxy-~-glucose,6-O-methyl-~-glucose and 6-deoxy-6-fluoro-
D-glucose were prepared by this method[223,2331.
17.3.1.4.2 Preparation o f Fructose and Sorbose Derivatives Modified at Position 5

Xylose isomerase converts a wide range of D-glucose as well as L-idose derivatives
modified at position 5 into the corresponding ketose. 5-Deoxy-5-fluoro-D-xylulose
and a variety of 5,6-dimodified open-chain analogs of D-fructose, namely the
5,6-diazido-S,G-dideoxy, 6-azido-S,6-dideoxy, 6-azido-5,6-dideoxy-5-fluoro,5,6-di-
deoxy-5-fluoro, 5.6-dideoxy-6-fluoro and 5,6-dideoxy-5,6-difluoro
derivatives were
prepared with glucose isomerase (Fig. 17-24)[234, 23s1.
17.3.1.4.3 Preparation of Sucrose Derivatives with Modified Fructose Moieties

Xylose isomerase is also used for the synthesis of modified sucroses, which is
important in the study of the topographical aspects of the binding of sucrose to a
sucrose carrier protein [23G1. 6-Deoxy- and 6-deoxy-6-fluoroglucosechemically synthe-
sized are isomerized to the corresponding 6-substituted fructose by xylose iso-
merase. The resultant substrates are subsequently condensed with UDP-glucose by
sucrose synthase. Although the equilibrium of the first step lies towards the glucose
1318
I 77 fsomerizations
F OH 0
A w o H h F + o H
OH OH
OH 0
B OH Y
+
O
H
OH X OH
X = H, Y = F
X = H, Y = N3
X=Y=F
X = F, Y = N3
X = F, Y = H
X=Y=N3
X=Y=H
Figure 17-24. Production of 5-deoxy-5-fluoro-o-xylulose and 5,6-dimo.
dified open-chain analogs o f D-fructose with xylose isomerase.
Reprinted from Hadwiger et al.[235].
derivatives, this problem is overcome by coupling the isomerization reaction with

the sucrose formation, which is irreversible. The second reaction completely drives
the isomerization reaction almost to completion. Incubation of 6-deoxy- or 6-deoxy-
6-fluoroglucose and UDP-glucose with both the xylose isomerase and sucrose
synthase afforded 6'-deoxy- and 6'-deoxy-6'-fluorosucrose in 73 and 53 % isolated
yield, respectively.
17.3.2
Phosphoglucose Isomerase (E.C. 5.3.1.9)
Phosphoglucose isomerase catalyzesthe interconversion of glucose 6-phosphate and

fructose 6-phosphate. This enzyme is involved in the gluconeogenesis, glycolytic
pathway, and pentose phosphate cycle. Since thermostable enzymes are generally
useful for industrial application, thermostable phosphoglucose isomerase was
purified from Bacillus stearothem~philus[~~'] ~ ~stear-
and Bacillus ~ a l d o t e n a d ~B. l.
othemophilus produces two isozymes of phosphoglucose isomerase, and they were
overexpressed in E. coli, purified to homogeneity, crystallized[239], and the X-ray
structure of the enzyme was 2411 . The structure of the rabbit muscle
enzyme complexed with a competitive inhibitor D-gluconate 6-phosphate was also
determined by X-ray crystallography[242,2431. The enzyme is a dimer with two a/P-
sandwich domains in each subunit. Lys 518 and His 388 are located at the active
center and are probably involved in the catalytic mechanism. Since gluconate
6-phosphate occurs predominantly in its cyclic form, phosphoglucose isomerase
probably catalyze the opening of the hexose ring to give initially its straight chain
form with Lys 518 and His 388. Then the enzyme undergoes isomerization of the
Arg 272 Arg 272 Arg 272
I
17.3 lsornerizations 1319
I I I
H OH
glucose-6-
phosphate /" O V 0
I I
O Y O
fructose-6-phosphate
His 3 8 8 - x y N H Glu 357 Glu 357 Glu 357
Figure 17-25. Mechanism o f phosphoglucose enediol is formed. Then, a proton is transferred

isomerase reaction. His 388 and Clu 216 cata- from the protonated Clu 357 to the C1 position
lyze the ring opening. The side-chain o f Glu357 ofthe intermediate. Reprinted from Jefferyet
abstracts a proton from the C2 position of the ai. [2421.
open chain form ofthe substrate, and the cis-
substrate through formation of a cis-enediol intermediate with the double bond

between C 1 and C2 (Fig. 17-25). Glu 357 transfers the proton from the C2 of glucose
6-phosphate to its C1 position. The side chain of Arg 272 stabilizes the negative
charge of the intermediate (Fig. 17-25).
Xylose isomerase requires the 4-OH group for glucose derivatives to be sub-
strates [2301. On the other hand, phosphoglucose isomerase can act on 4-substituted
phosphoglucose. Therefore the latter enzyme is applicable to the preparation of
glucose or fructose derivatives modified at position 4. For example, 4-deoxy-
4-fluorofructose was prepared from 4-deoxy-4-fluoroglucosewith phosphoglucose
isomerase because xylose isomerase cannot isomerize 4-deoxy-4-fluoroglucose[23G1.
4-Deoxy-4-fluorofructosewas then converted into 4'-deoxy-4'-fluorosucrose, which is
useful for the analysis of the interaction between sucrose and a sucrose carrier
protein, with fructose-6-phosphatekinase [2361.
Fructose 1,6-bisphosphatehas attracted attention due to its important applications
in the field of medicine, and is produced from glucose in three step by enzymatic
reactions catalyzed by glucokinase, phosphoglucose isomerase, and phosphofructo-
kinase. ATP is regenerated by acetate kinase (Fig. 17-26). Ishikawa and coworkers
established an efficient method for production of fructose 1,G-bisphosphate in a
Glucose + ATP - GK
Glucose-6-phosphate (G6P) + ADP Figure 17-26. Synthe-
sis o f fructose 1,6-
PGI bisphosphate from
G6P -2- Fructose-6-phosphate (F6P) glucose by combina-
tion o f glucokinase
F6P + ATF' - PFK

FDP + ADP
(CK), phosphoglucose
isomerase (PCI),
ADP + Acetyl phosphate -AK

ATP + Acetic acid
phosphofructokinase
(PFK), and acetate
kinase (AK) reactions.
1320
I 17 lsomerizations
Figure 17-27.
-
0 Triosephosphate QH
O
H
,k,P
O
-.O
.~
isomerase
L
o, ! opog- Reaction catalyzed by
triosephosphate
Dihydroxyacetone D-Glyceraldehyde isomerase.
phosphate 3-phosphate
Ix,
Glu 165 Glu 165
$
-0
Glu 165
Glu 165 Glu 165
x
I
0i \o c? ' 0
Figure 17-28. Triosephosphate isomerase reaction through a cis-enediol intermediate. The pro-R
proton is removed from C1 o f dihydroxyacetone phosphate by the side chain of Clu 165, and the
carbonyl group o f t h e substrate is polarized by the side chain of His 95. Reprinted from Harris et
a~.[*49~.
batch reactor system using the purified enzymes[244] and the crude extract of Bacillus
stearothermophilus cells[245].The yield of fructose 1,G-bisphosphatedepended on the
activity of glucokinase in the reactor[246].
17.3.3
Triosephosphate lsomerase (E.C. 5.3.1.1)
Triosephosphate isomerase is involved in the glycolybc pathway, and catalyzes the

interconversion of dihydroxyacetone phosphate and D-glyceraldehyde phosphate
(Fig. 17-27).The refined three-dimensional structures of chicken, yeast, and trypano-
17.3 lsornerizations
I
1321
OH OH
Figure 17-29. Synthesis of [3',4'-''Cz]-thymidine from [2',3'-'3Cz]-dihydroxyacetone phosphate
with triosephosphate isomerase (TPI) and D-2-deoxyribose-5-phosphate(DHAP). Asterisks in-
dicate the positions selectively labeled with "C. Other positions that can be isotopically substi.
tuted are marked with ', 4and 0.Reprinted from Ouwerkerk et al.[2511.
soma1enzymes have been elucidated[247]. The reaction is thought to proceed through

a cis-enediolintermediate with Glu 165 and His 95 as acid and base catalysts (Fig. 17-
28)[248,24ql. The side chain of Glu 165 removes the pro-R proton from the C1 of
dihydroxyacetonephosphate, and that of neutral His 95 polarizes the carbonyl group
of the substrate. Fructose 1,6-bisphosphate, a precursor molecule for sugar synthe-
sis, can be prepared from dihydroxyacetone phosphate with this enzyme and
ald0lase1~~~1.Triosephosphate isomerase has been used for various other purposes.
For example, [3',4'-'3C2]-thymidinehas been prepared from [13C2]-aceticacid
through [2',3'-'3C2]-dihydroxyacetonephosphate and ~-[3',4'-'~C2]-2-deoxyribose-
5-phosphate with triosephosphate isomerase and D-2-deoxyribose-5-phosphate aldo-
lase (E.C. 4.2.1.2) (Fig. 17-29)12511.
17.3.4
L-Rharnnose Isomerase (E.C. 5.3.1.14)
L-Rhamnose is an important component ofbacterial cell walls, and is metabolized in

E. coli through a pathway similar to that of glucose 6-phosphate in glycolysis.
Rhamnose isomerase catalyzes the first reaction in the pathway to produce L-
rhamnulose from t-rhamnose (Fig. 17-30).The enzyme gene was cloned from E. coli
and o~erexpressed[~~*], and the enzyme was purified and
Rhamnose isomerase is composed of four identical subunits with a molecular
mass of about 47 kDa. It has the maximum activity around 7.6, and requires Mn2' to
provide the highest activity. The enzyme shows no significant sequence similarity to
any other ketol isomerases including xylose isomerase. However, rhamnose iso-
merase was found, by X-ray crystallography, to be most similar to xylose iso-
m e r a ~ e l ~The
~ ~monomer
], of rhamnose isomerase is composed of (P/a)s-barrels,
and the structure and arrangement of the barrel are very similar to those of xylose
isomerase. However, each of them has an additional a-helical domain, which is
involved in subunit assembly and differs from each other only in its structure. The
1322
L-R hamnose L-Rhamnulose

(6-Deoxy-L-mannose) (6-Deoxy-L-fructose)
Figure 17-30. Reaction catalyzed by rhamnose isomerase. Since
both substrate and product occur in cyclic forms, L-rhamnose
isomerase catalyzes ring opening before isomerization. Reprinted
from Korndorfer et aI.[2521.
Figure 17-31. Superposition o f the metal binding sites o f rhamnose isomerase (residues named
and drawn with thick bonds) and zylose isomerase (thin bonds). Reprinted from Korndorfer et
al. [2521.
residues surrounding the catalyix Mn2+ site (Asp 302, Asp 304 and His 270) are
conserved in the two structures (Fig. 17-31). Therefore, the reaction catalyzed by
rhamnose isomerase is thought to proceed through a metal-mediated hydride-shift
mechanism in the same manner as xylose isomerase [2521.
Bhuiyan et al. 12531 immobilized L-rhamnose isomerase from Pseudomonas sp.
LL172 on chitopearl beads, and used it to produce L-mannose from L-fructose. The
immobilized enzyme was found to be stable: it retained about 90% of the initial
activity after five repeated batch reactions. The concentration of L-mannose relative
to L-fructose was about 3:7 at equilibrium. D-Allose was also produced from D-
psicose with the immobilized L-rhamnose isomerase. Since D-psicose is readily
produced from D-fructose with D-tagatose 3-epimerase, D-allose can be produced
from D-fructose by combination of the two enzymes immobilized on chitopearl
beads. Bhuiyan et al. [2541 found that the reaction progresses steadily until 40% of the
D-psicose is converted into D-allose. The immobilized D-tagatose 3-epimerase was
also stable even after repeated uses, and D-allose was produced efficiently in the
system.
I
1323
17.3.5
L-Fucose Isomerase (E. C. 5.3.1.3)
Fucosylated oligosaccharides are important components of glycoproteins and glyco-

lipids which are useful for cancer diagnosis and immunotyping. Therefore, efficient
production methods for L-fucose and its analogs would be useful.
L-Fucose isomerase acts on D-arabinose, which was known as D-arabinose iso-
merase in earlier literatures. L-Fucose is metabolized through a pathway similar to
that of D-glucose in glycolysis, and L-fucose isomerase corresponds to glucose
6-phosphate isomerase. However, none of the aldose-ketose isomerases including
glucose 6-phosphate isomerase shows sequence similarity to L-fucose isomerase. L-
Fucose isomerase shares the common characteristics with other aldose-ketose
isomerases acting on unphosphorylated substrates: the requirement of metal ions
such as Mn2+for L-fucose isomerase. Aldose-ketose isomerases acting on phos-
phorylated substrates generally require no metal ions with the exception of phospho-
mannose isomerase (E. C. 5.3.1.8) which requires Zn" for its activity.
Seemann and Schulz [2551 determined the three-dimensional structure of L-fucose
isomerase from E. coli, a hexamer from a subunit with a molecular mass of 64 976
Da. The enzyme shows no structural similarity to any other aldose-ketoseisomerases
analyzed thus far. However, Seemann and Schulz, on the basis of the tertiary
structure, suggested that the r-fucose isomerase reaction proceeds through an ene-
diol intermediate [2551.
Fessner et a1.[2561 developed an efficient method for the synthesis of L-fucose
analogs modified at the nonpolar terminus by means of L-fucose isomerase and L-
fuculose 1-phosphatealdolase from E. coli. Various L-fucose analogs bearing linear or
branched aliphatic side chains were prepared in about 30% overall yield with
hydroxyaldehyde precursors and dihydroxyacetonephosphate as the starting materi-
als (Fig. 17-32).
OH 0
R'&o
R2
+ HO&OPOi:- 3
RL
R2
R'
OH OH
op0:-
R' R2
CH3 H
CH2-CH3 H
R2
R'
%OH - Fucl
R@
' "'
R2
OH
CH=CHz
CECH
CH3
H
H
CH3
OH OH HO OH CF3 H
Figure 17-32. Enzymatic synthesis o f L-fucose analogs with L-fucose
1-phosphate aldolase (FucA), phosphatase (P'ase), and L-fucose iso-
merase (Fucl). Reprinted from Fessner et al.[2561.
77 lsomerizations
1324
I 0
ACOOH
\
J"""
HO
Neu5Ac
2-epimerase
Ho&
HO
AcNH OH
GicNAc
Figure 17-33. Synthesis o f N-acetylneuraminate (Neu5Ac) from N-acetyl-
D-glucosamine (ClcNAc) and pyruvate through N-acetyl-D-mannosamine (ManNAc)
with N-acetylneuraminate and N-acetyl-D-glucosamine2-epimerase. Reprinted from
Maru et aI.[259].
17.3.6
N-Acetybglucosamine 2-Epimerase
N-Acetylneuraminate is a sialic acid with various biological functions that is widely

distributed in animals. It has been prepared only from natural resources such as
colominic acid, edible birds nests, milk or eggs. Alternatively, it has been prepared
enzymatically from N-acetyl-D-mannosamine and pyruvate with N-acetylneurami-
nate lyase as the catalyst[257,25s1.However, N-acetyl-D-mannosamine is expensive,
and the method is not suitable for large-scale production of N-acetylneuraminate.
Maru et a1.[2591developed an elegant method for the enzymatic production of N-
acetylneuraminate from the inexpensive N-acetyl-D-glucosamine and pyruvate by
means of N-acetylneuraminate lyase and N-acyl-D-glucosamine2-epimerase, whose
genes were cloned from E. c0li[~"1 and pig kidney[261],respectively (Fig. 17-33).
Simultaneous use of these enzymes and feeding of appropriate amounts of pyruvate
to the reaction mixture enabled production of N-acetylneuraminate from N-acetyl-D-
glucosamine with a 77% conversion rate, and 29 kg of N-acetylneuraminate were
obtained from 27 kg of N-acetyl-D-glucosamine.
17.3.7
Maleate cis-trans lsomerase (E. C. 5.2.1.1)
Maleate cis-trans isomerase catalyzes the conversion of maleate into fumarate. This
enzyme is applicable to the production of L-aspartateby coupling with the aspartase
reaction as shown in Fig. 17-3412", 2631.First, maleate is isomerized to fumarate by
Maleate cis-trans
I
1325
OOCHCOO isomerase coo Aspartase

O O C ~ c o o
H H -OOCHH NH4 _NH3
Maleate Fumarate L-Aspartate
Figure 17-34. Synthesis of L-aspartate using maleate cis-trans isomerase and aspartase.
cis-trans isomerase, and then the fumarate formed is aminated to L-aspartate by

aspartase. In this procedure, the resting cells of Alcaligenesfaecalis containing both
enzymes can be used as a catalyst. Thermostable maleate cis-trans isomerase was
purified from Bacillus stearothermophilus MI-102 and characterized, and the enzyme
gene was cloned and Two cysteine residues, Cys 80 and Cys 198,
among the three conserved cysteines were found by site-directed mutagenesis
studies to be catalytically important, although their catalytic roles are not yet
known.
17.3.8
Unsaturated Fatty Acid cis-trans lsomerase
trans-Unsaturated fatty acids occur in membrane phospholipids of some bacterial

genera such as Pseudomonas and Vibri~[~"].They are produced by cis-trans isomerase
from cis-unsaturated fatty acids in response to environmental stresses such as
elevated temperatures, increased salt concentrations, and the presence of organic
solvents such as toluene [2662691. The structural gene for the cis-transisomerase was
cloned from Pseudomonas putida P8[270].The E. coli recombinant cells carrying the
gene were shown to produce trans-unsaturated fatty acids in response to the organic
solvent, although E. coli has no inherent ability to produce these fatty acids [2701.
Okuyama et a1.1271]purified the cis-trans isomerase from Pseudomonas sp. E-3 and
characterized the enzyme catalyzing cis-trans isomerization toward 9-hexadecenoate.
It catalyzes the cis-to-transconversion of a double bond of cis-mono-unsaturatedfatty
acids with carbon chain lengths of 14, 15, 16, and 17 at positions 9,10, or 11,but not
at 6 or 7: the enzyme shows a strict specificity for both the position of the double
bond and the chain length of the fatty acid. A similar enzyme was also discovered by
Witholt and coworkers, which was purified from the periplasmic fraction of
Pseudomonas oleovorans[2721. Not only 9-cis-hexadecenoatebut also 1l-cis-octadece-
noate were found to serve as substrates of the enzyme. Moreover, the enzyme acted
only on free unsaturated fatty acids and not on esterified fatty acids in contrast to the
enzyme from Pseudomonas sp. E-3.Therefore, the Pseudomonas oleovorans enzyme
differs from the enzyme of Pseudomonas sp. E-3 in substrate specificity, although
both are monomeric enzymes with a molecular mass of about 80 kDa. The cis-trans
isomerases are expected to be useful for biotransformation of unsaturated fatty
acids.
1326
17.4
Conclusion
Total conversion of racemic starting materials into a particular stereoisomer of a

desired compound is very useful in the chemical industry. Half or more of the
starting materials can be saved and steps for the laborious separation of the products
from the starting material remaining reduced. Thus, racemases and epimerases are
very useful in the chemical industry, when their reactions are coupled with some
stereospecific reactions. Isomerases are also powerful catalysts for the production of
particular enantiomers or diastereomers of interest from cheaply-availablestarting
materials especially in the field of carbohydrate chemistry. Various new racemases
and isomerases useful for industrial applications will no doubt be discovered from
microorganisms at some point. However, established and well-known enzymes can
be remodeled in order to expand their uses by various protein engineering technolo-
gies such as directed evolution. A good example for this is L-specific hydantoinase
derived from D-specific hydantoina~e('~~1. The engineered enzymes can be incorpo-
rated into metabolic engineering studies in order to develop powerful microbial
cells.
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222 C. Chang, H. K. Song, B. C. Park, D. S. Lee, Ogino, H. Nakajirna, H. Ishikawa,]. Biosci.
S. W. Suh, Acta Crystallogr. Sect D Bid. Crys- Bioeng. 1999, 87, 611.
tallogr. 1999, 55,294. 245 A. Widjaja, M. Yasuda, H. Ogino, H. Naka-
1332
jima, H. Ishikawa, J. Biosci. Bioeng. 1999, 259 I. Maru, J. Ohnishi, Y. Ohta, Y. Tsukada,
87, 693. Curbohydr. Res. 1998,306,575.
246 A. Widjaja, H. Ogino, M. Yasuda, K. Ishimi, 260 Y. Ohta, Y.Tsukada, T. Sugimori, K. Murata,
H. Ishikawa,J. Biosci. Bioeng. 1999,88, 640. A. Kimura, Agric. B i d . Chem. 1989,53,
247 R. K. Wierenga, M. E. M. Noble, /. Mol. Biol. 477.
1992,224,1115. 261 I. Maru, Y. Ohta, K. Murata, Y. Tsukada,].
248 T. C. Alber, R. C. Davenport, G. K. Farber, Biol. Chem. 1996,271,16294,
D. A. Giammona, A.M. Glasfeld, W. D. 262 Y. Takamura, I. Kitamura, M. Iikura, K.
Horrocks, M. Kanaoka, E. Lolis, G. A. Pet- Kono, A. Ozaki, Agric. Bid. Chem. 1966, 30,
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1989,392, 34. 263 Y. Takamura, I. Kitamura, M. Iikura, K.
249 T. K. Hams, R. N. Cole, F. I. Comer, A. S. Kono, A. Ozaki, Agric. Bid. Chem. 1966,30,
Mildvan, Biochemistry 1998, 37, 16828. 345.
250 C. H. Wong, G. M. Whitesides, ]. Org. 264 K. Hatakeyama, M. Goto, Y. Uchida, M.
Chem. 1983,48,3199. Kobayashi, M. Terasawa, H. Yukawa, Biosci.
251 N. Ouwerkerk, J. H. van Boom, J. Lugten- Biotechnol. Biochem. 2000, 64, 569.
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252 I. P. Korndorfer, W. D. FessnerB. W. Mat- 266 N. Morita, A. Shibahara, K. Yamamoto, K.
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253 S. H. Bhuiyan, Y. Itami, K. Izumori,]. Fer- teriol. 1993, 175, 916.
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10 138. 181, 3256.
I
1333
18
Introduction and Removal of Protecting Groups
Dieter Kadereit, Reinhard Reents, Duraiswamy A.Jeyaraj and Herbert Waldmann
18.1
Introduction
The proper introduction and removal of protecting groups is one of the most
important and widely carried out synthetic transformation in preparative organic
chemistry. In particular, in the highly selective construction of complex, polyfunc-
tional molecules, e. g. oligonucleotides, oligosaccharides, peptides and conjugates
thereof, and in the synthesis of alkaloids, macrolides, polyether antibiotics, prosta-
glandins and other natural products, regularly the problem arises that a given
functional group has to be protected or deprotected selectively under the mildest
conditions and in the presence of functionalities of similar reactivity, as well as in the
presence of structures that are sensitive to acids, bases, oxidation and reduction.
Numerous classical chemical methods have been developed for the manipulation of
protecting groups [1-31. Nevertheless, severe problems still remain caused by the need
to introduce or remove selectively specific blocking functions which can not, or only
with great difficulties,be solved by using classical chemical tools only. However, the
arsenal of the available protecting group techniques has been substantially enriched
by the application of biocatalysts. In addition to their stereodiscriminating proper-
ties, enzymes offer the opportunity to carry out highly chemo- and regioselective
transformations. They often operate at neutral, weakly acidic or weakly basic pH
values and in many cases combine a high selectivity for the reactions they catalyze
and the structures they recognize with a broad substrate tolerance. Therefore, the
application of these biocatalysts to effect the introduction and/or removal of suitable
protecting groups offers viable alternatives to classical chemical methods ["I.
1334
I 18 Introduction and Removal of Protecting Groups
18.2
Protection of Amino Croups16121
18.2.1
N-Terminal Protection of Peptides
The selective protection and liberation of the a-amino function, the carboxy group
and the various side chain functionalities of polyfunctional amino acids constitute
some of the most fundamental problems in peptide chemistry. Consequently,
numerous efficient protective functions based on chemical techniques have been
developed to a high level of practicability. 13, 141 However, since the mid-1970s,a
systematic search for blocking groups being removable with a biocatalyst has been
carried In addition to the mild deprotection conditions they promise,
protecting groups of this type are expected to be particularly useful for the
construction and manipulation of larger peptide units, i. e. for transformations
which, for solubility reasons, in general have to be carried out in aqueous systems.
Also applications in the reprocessing of peptides obtained by recombinant DNA
technology are foreseen (for an interesting appropriate example see Chapter 12.5).
Initial attempts to introduce an enzyme-labile amino protecting group involved
the use of chymotrypsin for the removal of N-benzoylphenylalanine(Bz-Phe)from
the tripeptide Bz-Phe-Leu-Leu-OH("1. The desired dipeptide H-Leu-Leu-OH was
obtained in 80% yield under mild conditions (pH 7.3, room temperature). Chymo-
trypsin, however, is an endopeptidase with a rather broad substrate tolerance,
catalyzing the hydrolysis of peptide bonds on the carboxy groups of hydrophobic and
of aromatic amino acid residues. Since such amino acids appear widely in peptides,
and since no method is available to protect them against attack by the enzyme during
the attempted deprotection, the use of chymotrypsin is problematic. Its use is
therefore limited to special cases [16] in which no danger of competitive cleavage at
undesired sites has to be feared. A protease of much narrower specificity is trypsin
which catalyzes the hydrolysis of peptide bonds at the carboxylic group of lysine and
arginine. These amino acids carry polar, chemically reactive side chain functional
groups which can be protected by various techniques 141. The high specificity of
trypsin together with the possibility of hiding the critical amino acids which function
as primary points of tryptic cleavage allowed for the development of a broadly
applicable system for the protection of the a-amino group of peptides [12, l7-l91. I n
several studies the application of trypsin-labileprotecting groups, along with suitable
blocking functions for the side chains of arginine and lysine were d e ~ c r i b e d [ l ~ - ~ ~ ] .
Thus, for instance Z-Arg-OH served as the enzymatically removable protecting
group in a stepwise synthesis of deamino-oxytocin 1 (Fig. 18-1)[18, 191.
Starting with a pentapeptide the amino acid chain was elongated with Z-Arg-
protected amino acid p-nitrophenyl esters. The N-terminal Z-Arg protecting group
was successively removed in moderate to high yield and without attack on the other
peptide bonds by treatment with trypsin. Unfortunately, the preparation of the
protected arginine p-nitrophenyl esters is difficult,thus preventing this method from
becoming generally useful for the stepwise assembly of larger peptides. The trypsin-
H-Asn-Cys(Acm)-Pro-Leu-Gly-NH2
18.2 Protection ofArnino Groups
I
1335
I
1) 2-Arg-AA-ONp 2) trypsin
t
ONp =
(iterate)
Mpr -fffxf%tAsn-Cys-Pro-Leu-Gly-NH2
I
-s-s-
1 deamino-oxytocin
Bz-GIy-His-He-Glu BLeu-AspmTyr-Thr-Cys(Acm)-NHEt
2 21-31 fragment of murine
epidermal growth factor
n= N-terminally deprotected by enzymatic removal of Z-Arg (1)

or Bz-Arg (2) with trypsin
Figure 18-1. Construction of oligopeptides via removal of N-terminal arginine
residues with trypsin.
labile blocking groups have, however, proven to be very useful for the construction of
oligo- and polypeptides via condensation of preformed peptide fragments. An
illustrative example consists of a chemoenzymatic construction of the 21-31
fragment 2 of murine epidermal growth factor (Fig. 18-1). In the course of this
synthesis the deblocking by trypsin was applied twice[1G]. The enzyme first liberated
the N-terminus of a tetrapeptide and subsequently of a heptapeptide. In a synthe-
sis [241 of human p-lipotropin an Ac-Arg-residue was introduced by a solid-phase
technique at the N-terminus of the 29 C-terminal amino acids of the desired
polypeptide. After cleavage from the resin and protection of the side chain function-
alities, the arginine moiety was removed with trypsin, leaving the peptide chain
intact. Finally, coupling of this 61-89 fragment to a partially protected 1-GO segment,
and subsequent deprotection delivered P-lipotropin. Further examples are found in
syntheses of oxypressin [I2], Met-enkephalin[251 and Glu4-oxytocin
In addition to chymotrypsin and trypsin, the collagenase from Clostridiurn histolyti-
cum has been proposed as a catalyst for the removal of N-terminally attached dummy
amino acids from peptides [26]. The enzyme recognizes the tetrapeptides Pro-X-Gly-
Pro and cleaves the X-Gly bond. The use of this biocatalyst permitted the construc-
tion of des-pyroglutamyl-[15-leucine]humanlittle gastrin I by selective hydrolysis of
the dipeptide Pz-Pro-Leu (Pz = 4-phenylazobenzyloxycarbonyl)from the N-terminus
of the octadecapeptide Pz-Pro-Leu-Gly-Pro-Trp-Leu-(Glu)s-Ala-Tyr-Gly-Trp-Leu-Asp-
Phe-NH2. Transformations of this type are analogous to the naturally occuring
conversion of prohormones into hormones and may prove to be useful for the
processing of peptide factors produced by recombinant DNA technology.
1336
I 78 lntroduction and Removal offrotecting Croups
Despite the impressive syntheses that have been made possible using proteases,
the use of these enzymes is always accompanied by the danger of a competitive (and
sometimes unexpected and unforeseeable) cleavage of the peptide backbone at an
undesired site. At a minimum, complex protecting group schemes may become
necessary if the amino acid which serves as the recognition structure for the protease
occurs several times in the peptide chain to be constructed. This disadvantage can be
overcome if a biocatalyst devoid of peptidase activity is used for the liberation of the
N-terminal amino group. This principle has been illustrated by the application of
penicillin G acylase from E. c0li[~~-@1 in industry for the large scale synthesis of
semisynthetic penicillins and by using a phthalyl imidase from Xanthobacter agi-
lis[45-473(vide infiu).Penicillin G acylase attacks phenylacetic acid (PhAc)amides and
esters but does not hydrolyze peptide bonds. The acylase accepts a broad range of
protected peptides as substrates and selectively liberates the N-terminal amino group
under almost neutral conditions (pH 7-8, room temperature) leaving the amide
bonds as well as the C-terminal methyl, allyl, benzyl and tert-butyl esters unaf-
f e ~ t e d [ ~ ~381.- ~ The
' , PhAc group is easily introduced into amino acids by chem-
icall4'I or enzymatic[49]methods and is stable during the removal of the C-terminal
protecting groups employed [29-321.
Recently, it has been shown that a phthalyl amidase isolated from Xanthobacter
agilis is able to deprotect a variety of phthalimido substrates once the substrates are
partially hydrolyzed to their monoacids (Fig. 18-2)[45-471. The phthalyl group is
commonly used for amine protection, because it completely blocks this functionality
by double acylation[2s '1. The enzymatic phthalyl removal proceeds via a two step
process ofweakly basic hydrolysis to yield the monoacid 4 and subsequent treatment
with the phthalyl amidase (Fig. 18-2).Because the hydrolysis of the phthalimide 3 to
the corresponding monoacid 4 can be catalyzed by imidases such as the rat liver
imidase,I'[ this procedure in particular represents a powerful alternative to the
classical phthalyl deprotection which requires relatively drastic conditions and toxic
reagents. However, the general applicability of the enzymatic phthalyl removal is yet
to be investigated.
If the construction of PhAc- or phthalyl-peptides is carried out by chemical
activation of the PhAc-amino acids, the application of the non-urethane blocking
group results in ca. 6% racernizati~n[~', 301. However, this disadvantage can be
overcome by forming the peptide bonds enzymatically,e. g. with tryp~in['~I, chymo-
tryp~in['~] or carboxypeptidase Y [39, "1, or by using urethane-type protecting groups
(vide infa). For such condensation reactions and the subsequent enzymatic removal
ofthe PhAc group, a continuous process was developed which has the potential to be
transferable to a larger scale [391.
@N-(R2
3
0
0
~1
-@&;-
Base
CH&N/H20
or
0.2 M buffer
(PH 8.0) 4
C02H
O
y 2
R
R'
Phthalyl
arnidase C02H
5
+
R'
HWR2
6
Figure 18-2. Enzymatic removal of the phthalyl group.
s-s
78.2 Protection of Amino Croups
I 1337
PhAC-Gly I I OH
?S ?S
PhAc-Phe Lys-OH
7 (PhAc),insulin PhAcHN
I
8 leucine enkephalin
n= N-terminally deprotected using penicillin G acylase
Is-sl
Mpr-Tyr-Phe-Glu-Asn-Cys-Pro-Lys-Gly-NH2
mN
j
/
penicillin G acylase.
pH 7,37"C, 74%
H
9 1-deamino-Lyse-vasopressin
Figure 18-3. Application ofthe phenylacetamido (PhAc) group as an
enzymatically removable amino protecting group.
The applicability of the penicillin acylase-catalyzed deprotection for the constmc-

tion of larger peptides has been demonstrated by the complete deprotection of the
porcine insulin derivative 7 carrying three PhAc presumably at the N-
terminal glycine of the A-chain, the N-terminal phenylalanine of the B-chain and the
side chain of the lysine in position 29 of the B-chain (Fig. 18-3). The enzymatic
hydrolysis proceeded to completeness and the peptide backbone was not attacked. A
further interesting example is given by a recent biocatalyzed synthesis of leucine
enkephalin tert-butyl ester 8[381 in which all critical steps are performed by enzymes,
two of them through the agency of penicillin G acylase: i) phenylacetates are
introduced as N-terminal protecting groups of the amino acid esters by using
penicillin G acylase, ii) the elongation of the peptide chain is carried out with papain
or a-chymottypsin, iii) the deprotection of the N-terminal amino group is achieved
again by means of penicillin G acylase. These examples and also the application of
this technique for aspartame syntheses[28*40, 41), as well as the deprotection of
glutathione derivatives[351 demonstrate that penicillin G acylase can be used ad-
vantageously for the N-terminal unmasking of peptides. In addition, the enzyme has
1338
I 78 Introduction and Removal of Protecting Croups
been used for the liberation of the side chain functionalities of lysine and cysteine, as
well as in p-lactam, nucleoside and carbohydrate chemistry (vide infia).
18.2.2
Enzyme-labile Urethane Protecting Groups
The enzyme-labile N-protecting functions described so far are simple acyl groups
which typify the danger of razemization during chemical peptide syntheses. This
problem can, in general, be overcome by the use of urethane blocking functions.
However, so far only few examples of a biocatalytic removal of classical urethane
protecting groups such as the Z- and Boc-group are known['*]. Apparently, the
enzymatic attack on the urethane carbonyl group, which would initiate the cleavage
process, is too inefficient to be useful for synthetic purposes. To overcome this
problem, two different strategies were developed. Both concepts have in common
the fact, that the enzyme-labile bond is no longer part of the urethane. However, the
first approach includes the introduction of a spacer (the AcOZ- and PhAcOZ
groups), while the second strategy relies on the cleavage of a glycosidic C - 0-bond of
a glycoside urethane by the respective biocatalyst, e.g. a glucosidase (the BGloc
group).
Through the introduction of a spacer between the group which is recognized by
the enzyme and the urethane, the substrate is kept at a distance from the enzyme
during the reaction (Fig. 18-4). Therefore, any steric effects caused by the bulk of
certain amino acids are expected to be minimal and, as the amino acid sequence
does not influence the reactivity, this concept should be generally applicable to the
synthesis of peptides and peptide conjugates. An additional advantage of the
introduction of the spacer is the option to choose the group that is recognized by the
enzyme and thus the enzyme itself.
This concept was first realized by using p-hydroxybenzyl alcohol as a spacer in the
p-(acetoxy)-benzyloxycarbonyl (AcOZ) group which encorporates an acetic acid ester
as the enzyme-labile bond (Fig. 18-4).Accordingly, the AcOZ group can be removed
under conditions typical for acetyl ester hydrolysis, for instance by treatment with
lipases or esterases [53-551. As lipases display a broad specificity, other esters present
in the substrate molecule might be hydrolyzed during the AcOZ removal. Thus, the
p-(phenylacety1)benzyloxycarbonyl (PhAcOZ) group was developed, which takes
advantage of the high selectivity of penicillin G acylase for the phenylacetyl group
(Fig. 18-4). The versatiliy of this enzyme-labile urethane protecting group was
demonstrated by the synthesis of phosphorylated [56-601, glycosylated [56-601 and
lipidated['l] peptides.
A second approach takes advantage of a characteristic property of glycosidases. It
is well known that glycosidases hydrolyze their substrates by cleaving the glycosidic
bond via nucleophilic attack at the anomeric carbon atom. Therefore, a carbohydrate-
derived urethane protecting group would provide the desired enzyme-lability. In
additional, such sugar derivatives have increased solubility in aqueous solutions, a
necessary requirement for all biotransformations. This concept was success-
fully realized by using glucose and galactose as the carbohydrate component
group which /s
18.2 Protection of Amino Groups
Figure 18-4. Principle of the spacer-

I 1339
recognized by based protecting groups AcOZ and

the enzyme PhAcOZ.
J- 0
enzyme-la bile
linkage
gmup which undergoes spontaneous
fragmentation upon cleavage of the
enzyme-labile linkage
Jenzyrnatic cleavage
1 fragmentation
0 0 + GO2+ H2N-Peptids
0
PhAcOZ =
(Fig. 18-5)['*, G31. During the synthesis the carbohydrate hydroxy functions are
blocked by either benzyl ethers in the tetra-0-benzyl-D-glucopyranosyloxycarbonyl
(BGloc) group or acetyl groups in the tetra-0-acetyl-D-glucopyranosyloxycarbonyl
(AGloc) or the tetra-0-acetyl-0-D-galactopyranosyloxycarbonyl (AGaloc) protecting
groups. The removal of these carbohydrate-basedprotecting groups proceeds via a
two step process by removing the hydroxy blocking function in a first step followed
by treatment with a glucosidase (AGloc, BGloc) or galactosidase (AGaloc), re-
spectively. I n the case of the acetyl derivatives AGloc and AGaloc a sequential two
step process as well as a one-pot procedure were developed for the deprotection
reaction, allowing for a convenient deprotection protocol as demonstrated for
dipeptide 11 (Fig. 18-5)LG2].
1340
I 78 introduction and Removal ofprotecting Groups
I AGloc BGEoc AGaloc
Galactose X=O-PG, Y=H

Glucose X=H, Y=O-PG 0
PG=Bn, Ac PG’
Peptide or
Peptide
Conjugate
Galactose X=OM, Y=H

Glucose X=H, Y=QH
I
Enzyme-labile )
Glycosidic Bond
Glycosidase
HHO
O S , ,
1) Lipase WG, 5% MeOH
OH
,OAc 0.07 M phosphate buffer
H pH 6.0,37 ‘C, 16 h
OAc Q
2) alp-glucosidase, 24 h
10 11
64% over two steps
Figure 18-5. Carbohydrate-based urethane protecting groups.
18.2 Protection ofArnino Groups
I 1341
18.2.3
Protection ofthe Side Chain Amino Group of Lysine
During chemical peptide syntheses and if trypsin is used for the construction of the
peptide bonds or N-terminal deprotection, the side chain amino group of lysine
generally has to be protected to prevent side reactions[13*141. This goal can be
achieved enzymatically by applying the penicillin G acylase-catalyzed removal of the
PhAc group (vide supru)[G4]. Thus, the first application of the PhAc group in peptide
chemistry was a synthesis of l-deamino-Lys'-vasopressin from the protected con-
gener 9, during which the lysine side chain was masked as the phenylacetamide
(Fig. 18-3).After the peptide chain had been assembled and the disulfide bond was
formed by oxidative cyclization, the PhAc group could be removed enzymatically in
74% yield without side reaction. A further interesting example which demonstrates
that this technique can be applied advantageously to the synthesis of even larger
peptides is found in the complete deprotection of (PhA~)~porcine insuline (vide
supru, Fig. 18-3)LZ71 and modified insuline fragments I['. Since penicillin acylase is
commercially available and devoid of peptidase activity["], this method appears to be
generally useful for the construction of lysine-containingoligopeptides.
In addition to the PhAc group, pyroglutamyl amides (Glp) were proposed as
enzymatically removable blocking functions for the lysine side chain LZ31. Their
removal was achieved with pyroglutamate aminopeptidase from calf liver. Thus, all
N-protecting groups were split off from the protected RNAse 1-10 fragment Glp-
Lys(Glp)-Glu-Thr-Ala-Ala-Ala-Lys(Glp)-Phe-Glu-Arg-OH and from a model dipep-
tide. The general usefulness of this method remains to be demonstrated, however.
18.2.4
Protection of Amino Groups in fi-Lactam Chemistry
The enzymatic removal of acyl groups plays an important role in the industrial
production of semisynthetic penicillins and cephalosporins.To this end, penicillin G
12 (R = CH2-Ph) and penicillin V 12 (R = CH2-0-Ph),or the respective cephalospor-
ins are first deacylated by means of penicillin acylases (Fig. 18-6)[", 68]. The
6-aminopenicillanic acid and the 7-aminocephalosporanic acid thus obtained are
subsequently acylated by non-enzymatic or enzymatic methods to give the semi-
synthetic antibiotics 13.
The manu.facture of therapeutically important cephalosporins from penicillin G
and V includes a chemical ring expansion of the thiazolidine ring to a dihy-
drothiazine. In the course of this sequence the amino group remains protected as
phenylacetyl or phenoxyacetyl amide, which is finally removed using penicillin G or
V acylase. Of particular importance is the choice of a suitable protecting function for
the COOH group. It must be stable during the ring expansion but removable
without damaging the ceph-3-em nucleus. As an alternative to chemical methods,
the use of the phenylacetoxymethyleneester was suggested for this purpose[41, It
is easily introduced and is stable during the construction of the cephalosporin
framework (Fig. 18-6).Together with the phenylacetamidethe ester can eventually be
1342
I 18 introduction and Removal of Protecting Groups
R = Ph-CH2-
penicillin G
acylase
R = Ph-0-CH2-
'"m non-enzymatic
or enzymatic R*
methods
- 0
bHNm 0
penicillin V
12 acylase 13
I
'COOH COOH
semisynthetic semisynthetic
penicillins cephalosporins
kHNX& --
Ph 0
ring
0 0 expansion
0&o-()J$
Ph Ph
14
-
penicillin G
acylase
70-90%
H 2
0
N E +
COOH
-
1) cholesterol OTBDMS
esterase,
PH 7
2) Jones
oxidation
16
0
Figure 18-6. Enzymatic deprotection of amino- and carboxy groups in B-lactam chemistry.
removed in high yield from penicillin G and the cephalosporins 14 by penicillin G

acylase. The formaldehyde formed in the deprotection is not harmful to the
enzyme.
18.3 Protection ofThiol Croups
I 1343
In a new approach to the well known versatile 0-lactam building blocks, an
enzymatic deprotection of an acylated methylol amide was applied with advantages
(Fig. 18-6)[70j. Thus, the dibenzoate 15 was regioselectively saponified by cholesterol
esterase at pH 7 giving rise to a monoacylated aminal. After Jones oxidation and
subsequent loss of formaldehyde, the azetidinone 16 was obtained, which can be
transformed into various enantiomerically pure penem and carbapenem building
blocks.
As an alternative to the well established phenylacetyl group in p-lactam chemisq,
recently a biocatalyzed procedure for the removal of phthalyl irnide has been
described (Fig. 18-2)F4', 711. Its general usefulness remains to be demonstrated,
however.
18.2.5
Protection o f Amino Groups o f Nucleobases
In general, the amino groups of the nucleobases adenine, guanine and cytosine in
general must be protected during oligonucleotide synthesis to prevent undesired
side reactions. To this end, they usually are converted into amides which are finally
hydrolyzed under fairly basic conditions. If the amino functions are, however,
masked as phenylacetamides, the protecting functions can be cleaved off by again
employing penicillin G acylase (Fig. 18-7)[72-781. The enzyme, for instance, se-
lectively liberates the amino groups of the deoxynucleosides 17 without attacking the
acetates in the carbohydrate parts and without damage to the acid-labile N-glycosidic
bonds.
The biocatalyzed phenylacetylremoval can be carried out using both solubilized or
immobilized substrates [771. The latter methodology has been developed using
controlled pore glass (CPG) as a solid support (Fig. 18-7).
18.3
Protection ofThiol Groups14-6, *' 12]
18.3.1
Protection o f the Side Chain Thiol Group o f Cysteine
The liberation of the P-mercapto group of cysteine was also achieved by means of the
penicillin G acylase mediated hydrolysis of phenylacetamides[33-351. To this end, the
SH group was masked with the phenylacetamidomethyl(PhAcm)blocking function
(Fig. 18-7).After penicillin acylase-catalyzed hydrolysis of the amide incorporated in
the acylated thioaminal (see, e.g. 18),a labile S-aminomethyl compound is formed
which immediately liberates the desired thiol. This technique was for instance
applied in a synthesis of glutathione which was isolated as the disulfide 19. In a
related glutathione synthesis the method was used for the simultaneous liberation of
the SH- and the N-terminal amino function of g l ~ t a m i n e [351.
~~*
1344
I 18 fntrodudion a n d Removal ofProtecting Croups
AcowBphAc 17
2'-deoxyguanosine 2'-deoxyadenosine 2'-deoxycytidine
O-3,d(TGPhAcGPhAc
G PhAc
G )PhAc
5 '
1) conc. NH3
3'd(TGGGG)5' WN&O-3'd(TGGGG)5'
H 0
Boc-GlU-OtBU
I
a
CYS-Gly-OH 1) CFjCOOH
I 2) penicillin G
fH2 acylase,
s, N PH 8
\ H J 31H202
PhAcm
18 19 glutathione 77%
Figure 18-7. Enzymatic deprotection o f t h e amino groups o f nucleobases and the

mercapto group ofcysteine by means o f penicillin G acylase. The shaded balls represent
controlled pore glass (CPG).
18.4
Protection of Carboxy croup^[^^* 12, 791
18.4.1
C-Terminal Protection o f Peptides
As in the enzymatic liberation of the N-terminus of peptides, initial attempts to

achieve an enzyme-catalyzed deprotection of the corresponding carboxyl groups
78.4 Protection of Carboxy Groups
I
1345
concentrated on the use of the endopeptidases chymotrypsin[8&821, trypsin[81983s 841
and thermolysin PSI, a protease obtained from Bacillus themoproteolyticus which
hydrolyzes peptide bonds on the amino side of hydrophobic amino acid residues
(e.g. leucine, isoleucine, valine, phenylalanine). This latter biocatalyst enables the
cleavage of the “supporting” tripeptide ester H-Leu-Gly-Gly-OEt from a protected
undecapeptide to take place (pH 7, room temperature). The octapeptide thereby
obtained was composed exclusively of hydrophilic amino acids. Owing to the broad
substrate specificity of thermolysin and the resulting possibility of unspecific peptide
hydrolysis this method can not be regarded as being generally applicable.
The exploitation of the esterase activities of chymotrypsin and trypsin opened
routes to the hydrolysis of several peptide methyl, ethyl and tert-butylesters at pH 6.4
to 8 and room temperature[80,81]. The transformations are not only successful with
peptides carrying the respective enzyme-specificamino acids at the C-terminus, but
in several cases different amino acids were also tolerated at this position. However,
severe drawbacks of this methodology are that numerous peptides are poor sub-
strates or are not accepted at all. Moreover, a competitive cleavage of the peptide
bonds occurs if the peptides contain trypsin- or chymotrypsin-labile sequences.
Therefore, these proteases appear not to be generally useful for a safe C-terminal
deprotection as well.
The disadvantages of using by the endopeptidases can be overcome by using
carboxypeptidase Y from baker’s yeast [25, 8G, 871. This serine-exopeptidase also has
esterase activity and is characterized by quite different pH-optima for the peptidase
and the esterase activity (pH >8.5). Even in the presence of various organic
cosolvents the enzyme selectively removes the carboxy protecting groups from a
variety of differently protected di- and oligopeptide methyl and ethyl esters[25,871
without attacking the peptide bonds. An additional attractive feature is, that its
esterase activity is restricted to a-esters, consequently j3-and y-esters of aspartic and
glutamic acid, respectively, are not attacked. Carboxypeptidase Y was used ad-
vantageously for the stepwise C-terminal elongation of the peptide chain in aqueous
solution employing a solubilizing poly(ethy1ene glycol) derived polymeric support as
the N-terminal blocking In a further remarkable synthesis which did not
include the use of a polymeric N-protecting group, Met-enkephalin 20 was built up
employing carboxypeptidase Y for C-terminal deprotection of intermediary gen-
erated peptide amides as well as for the formation of the peptide bonds (Fig. 18-
8) (251.
The additional opportunity to hydrolyze selectively C-terminal peptide amides
with carboxypeptidase Y is of particular interest if, as is demonstrated in the above
mentioned example, enzymatic methods are applied to the formation of the peptide
bonds, because amino acid amides are often the nucleophiles of choice in these
biocatalyzed processes. For this purpose a peptide amidase from the flavedo of
oranges shows very promising p r o p e r t i e ~ 1 ~ The
~ ~ ~enzyme
1. is equipped with a
broad substrate specificity and accepts Boc-, Trt-, Z- and Bz-protected and N-
terminally unprotected peptide amides (Fig. 18-8). The C-terminal amides are
saponified in high yields at pH 7.5 and 30°C without affecting the N-terminal
blocking groups or the peptide bonds. A noticeable advantage of this biocatalyst is
1346
H m G l y - G l y *Met-OH
20 methionine enkephalin
[7=C-terminally deprotected by enzymatic saponification of the

peptide arnide with carboxypeptidase Y;Tyr was N-terminally
deprotected by removal or Bz-Arg with trypsin
amidase from the

flavedo of oranges
pH 7.5,30°C
PG-peptide-NH2 PG-peptide-OH
Tyr-Ser
Leu-Val
Gly-Leu-Val
Gly-Gly-Leu 100
Figure 18-8. C-terminal deprotection o f peptide amides by carboxypepti-

dase Y and an amidase from the flavedo of oranges.
that N-deprotected amino acid amides, in contrast to the respective peptide amides,
do not belong to its substrates. They can, therefore, be used as nucleophiles in
peptide syntheses catalyzed by this enzyme, i. e. the formation of the peptide bond
together with the subsequent C-terminal deprotection is achieved in a single step.
A further possibility for the enzymatic removal of C-terminal blocking groups is
opened up by the application of enzymes which generally display a high esterase/
protease ratio. Such a biocatalyst is the alkaline protease from Bacillus subtilis DY
which shows similarities to Subtilisin Carlsberg. For this enzyme the ratio of
esterase to protease activity is >lo5. It selectively removes methyl, ethyl and benzyl
esters from a variety of Tit-, Z- and Boc-protected di- and tripeptides and a
pentapeptide at pH 8 and 37 "C (Fig. 18-9)L9l1.
The N-terminal urethanes and the peptide linkages are left intact. A further
protease which fulfills the requirements for a successful1 application in peptide
chemistry is alcalase, a serine endopeptidase from Bacillus lichenijomis whose major
component is subtilisin A (Subtilisin Carlsberg)[92-941. It can advantageously be
employed with advantage to selectively saponify peptide methyl and benzyl esters
(Fig. 18-9).In a solvent system consisting of 90% tert-butanol and 10% buffer (pH
8.2) even highly hydrophobic and in aqueous solution insoluble Fmoc peptides were
accepted as substrates and deprotected at the C-terminus without any disturbing side
reactions. A selective classical alkaline saponification of methyl esters would be
impossible due to the base-sensitivity of the Fmoc group.
alkaline protease from
78.4 Protection of Carboxy Groups
I 1347
Bacillus subtilis DY
PG-peptide-OR w PG-peptide-OH
pH 8,37"C
Tyr(tBu)-Glu-Leu
Boc Leu-Glu-Val Bzl 85
Ala-Glu-Asp-Leu-Glu Bzl 80
alcalase, pH 8.2.35°C
PG-peptide-OR t PG-peptide-OH
90 vol% tert-butanol,
10 vol% buffer
PG peptide R yield [%.I
Fmoc Ala-Val-lle Me 85
Fmoc Asn-Phe Bzl 90
Boc Met-Leu-Phe Me 80
Z Met-Asp(0Me)-Phe Me 90
Figure 18-9. C-terminal deprotection of peptide esters by the alkaline

protease from Bacillus subtilis DY and alcalase.
A very promising and unusually stable biocatalyst is thermitase, a thermostable

extracellular serine protease from the thermophilic microorganism ntermoactino-
rnyces vulgaris whose esteraselprotease ratio amounts to >lo00 : 1. The enzyme
shows a broad amino acid side chain specificity and cleaves methyl, ethyl, benzyl,
methoxybenzyl and tert-butyl esters from a variety of Nps-, Boc-, Bpoc- and Z-
protected di- and oligopeptides in high yields at pH 8 and 35-55 "C (Fig. 18-
10)[33, 34, 95-971. In addition, it is specific for the a-carboxygroups of Asp and Glu. To
enhance the solubility of the substrates, furthermore, up to 50 vol% of organic
cosolvents such as DMF and DMSO may be added which also serve to reduce the
remaining peptidase activity to a negligible amount [34. 971.
In the discussion of the protease-catalyzed cleavage of the N-terminal protecting
groups it has already been pointed out that the use of biocatalysts belonging to this
class of enzymes in general, i. e. also for the C-terminal deblocking, may lead to an
undesired hydrolysis of peptide bonds. In particular, this has to be expected if the
respective ester or amide to be hydrolyzed turns out to be only a poor substrate,
which is only attacked slowly, an experience not uncommon if unnatural substrates
are subjected to enzyme mediated transformations. This undesired possibility
would, however, be overcome if enzymes were used which were not able to split
amides at all. This principle has been realized in the development of the heptyl
1348
thermitase, pH 8, 55°C
PG-peptide-OR * PG-peptide-OH
10-60 vol% organic cosolvent
PG peptide R yield [%I
Z Leu-VaCGlu(tBu)-Ala Me 92
Boc Pro-Gly Me 73
Bpoc Tyr(tBu)-Glu-Leu Me 55
NPS Ser(Bzl)-His(Dnp)-Leu- Me 90
Val-Glu(tBu)-Ala
lipase from
Rhizopus niveus
PG-peptide-OR + PG-peptide-OH
pH 7,37"C
21 R = (CHZ)&H3
22 R = (CH&Br
PG peptide R yield [%I

Boc Ser-Thr Hep 95
Z Thr-Ala Hep 85
Aloc Met-Gly Hep 90
2 Ser-Phe EtBr 84
Boc Val-Ala EtBr 95
F moc -Met tG'y#xtPro-

23 C-terminal pentapeptide of the N-Ras protein Figure 18-11. C-terminal
n= C-terminally deprotected by employing lipase from

Rhizopus niveus
deprotection of peptide
esters by lipase from
Rhizopus niveus.
(Hep),[+,' 31, 32, 98-1001 the 2-bromoethyl (EtBr)IG6, 3 1 s 32s "1' and the p-nitrobenzyl
(PNB) esters[lo21as carboxy protecting groups for peptide synthesis which can be
enzymatically removed by means of lipases or esterases, respectively (Fig. 18-11).
The Hep-esters proved to be chemically stable during the removal of the N-
terminal Z-, Boc- and the Aloc-group from the dipeptides 21. The selective removal
of the Hep-esters was achieved by a lipase-catalyzed hydrolysis. From several
enzymes investigated, a biocatalyst isolated from the fungus Rhizopus niveus was
superior to the others with respect to substrate tolerance and reaction rate. The
enzyme accepts a variety of Boc-, Z- and Aloc-protected dipeptide Hep-esters as
substrates and hydrolyzes the ester functions in high yields at pH 7 and 37 "C
18.4 Protection of Carboy Croups
I 1349
without damaging the urethane protecting groups and the amide bonds (Fig. 18-
11)[98* 991. Z- and Boc-dipeptide-2-bromoethyl esters 22 are also attacked, at a
comparable or in some cases even higher rate. In the presence of either one of the
enzyme-labile protecting groups the N-and C-terminal amino acid can be varied
considerably. With increasing steric bulk and lipophilicity of the amino acids, in
particular the C-terminal one, the rate of the enzymatic reactions decreases. If the C-
terminal amino acid is proline, the enzymatic reaction does not take place. The
lipase-mediateddeprotection of peptides was for instance successfully applied in the
construction of the C-terminal pentapeptide methyl ester 23 of the N-Ras-protein,
which is localized in the plasma membrane and which plays a vital role in cellular
signal transduction (Fig. 18-11)[lo3].
The use of lipases for the removal of protecting groups from peptides in addition
to the absence of protease activity has several advantages. Various enzymes belong
ing to this class and stemming from different natural sources (including mammals,
bacteria, fungi and thermophilic organisms) are commercially available and fairly
inexpensive, This variety provides the opportunity of replacing a chosen biocatalyst
by a better one if a particular substrate is only attacked slowly (videinf;a). The lipases
are not specific for L-amino acids but also tolerate the presence of the D-enantio-
mer['041. A noticeable feature is that, in contrast to proteases and esterases, they
operate at the interface between water and organic solvents[105]. This is particularly
important if longer peptides, which are composed of hydrophobic amino acids and/
or carrying side chain protecting groups, and that do not dissolve well in the aqueous
systems, have to be constructed.
The full capacity of the lipase mediated technique for C-terminal deprotection was
demonstrated by the synthesis of complex base-labile phosphopeptideslaI and 0-
glycopeptides, which are sensitive to both acids and bases [loG, lo7]. To this end, e. g.
the serine glycoside 24 was selectively deprotected at the C-terminus by lipase from
the fungus Mucorjavanicus (Fig. 18-12).
The carboxylic acid 25 liberated thereby was then coupled with an N-terminally
deprotected glycodipeptide and after subsequent enzyme-mediated deprotection the
glycotripeptide carboxylic acid 26 was obtained in high yield. This compound was
finally condensed with a tripeptide to give the complex diglycohexapeptide27, which
carries the characteristic linkage region of a tumor-associated glycoprotein antigen
found on the surface of human breast cancer cells. In the course of these enzymatic
transformations, the N-terminal urethanes, the peptide bonds, the acid- and base-
labile glycosidic linkages and the acetyl protecting groups, being sensitive to bases,
were not attacked. In these cases lipase from Rhizopus niveus which was the enzyme
of choice for simple peptides only attacked the substrates slowly, so that a different
biocatalyst had to be used. This demonstrates the above mentioned advantage of
being able to apply several catalytic proteins of comparable activity but different
substrate tolerance for the solution of a given synthetic problem.
The viability and the wide applicability of the principle of using enzymes for the
removal of individual protecting groups from complex multifunctional compounds
such as lipo- and glycopeptides is furthermore proven by the finding that proteases
can also be used for this purpose. Thus, by means of thermitase-catalysis the C-
1350
I 78 Introduction and Removal ofprotecting Groups
H O H O
z' N&- zSN?OH
AcO & 0= lipase from

Mucor javanicus A c
0
'
O ~
I
88% AcO OAc
AcO OAc
24 25
1) chain elongation 2) lipase from
Mucor javanicus
A::q 76%
AqGalNHAc
Z-ker-Thr-Ala-Pro-Pro-Ala-OHep
I
AqGalNHAc
-
chain
elongation
AcHN
Z-Ser-Thr-Ala-OH
AcHN 0
27 characteristic linkage region of a Ace@

AcO OAc
26
tumor associated antigen
Teoc-Ser-A l a I K ] H o g e r m
H
HO o d HO papain,
AcHN therrnitase, OH pH 6.6,
** pH 7.5,45"C,
20% DMF, 86%
29 quant.
Boc-Asnm HO&-peptide+OMe I
AcO
AcO @ HO 31: peptide = Ser-Gly
AcHN papain? HO subtilisin, pH 7, 68%
pH 6.6, 32:peptide = Gly-Ser
3o 96% subtilisin pH 7, 65%
Figure 18-12. Construction of acid- and base labile glycopeptides via enzyme-mediated
C-terminal deprotection.
terminal tert-butyl ester was removed from the glycopeptide28 (Fig. 18-12)[34s"'I. In
a different study, this enzyme was also used for the cleavage of methyl and p -
nitrobenzyl esters[lo9I.From the serine glycoside 29["', 'l1I and from the aspar-
agine conjugate 30['121the methyl esters could be cleaved offwithout disturbing side
reactions by using papain as the biocatalyst. Similarly, the liberation of the C-
terminal carboxy group of the glycosylated dipeptides 31 and 32 was achieved by
means of subtilisin-catalyzedhydrolysis[1131. However, in these cases papain could
not be used since this protease preferably cleaved the peptide bonds. This example
again highlights the danger associated with the use of a protease for the removal of
protecting groups from peptides.
78.4 Protection ofcarboxy Croups
I
1351
A problem arising regularly in the enzymatic deprotection is the poor solubility of
the fully blocked peptides in the required aqueous media, resulting in a limited
accessibility of the substrates to the enzymes. To overcome this difficulty, in many
cases solubilizing organic cosolvents are added, however, a more general and viable
approach consists of the introduction of solubilizing protecting groups, e. g. in the
enzyme-mediated formation of peptide bonds (see Chapter B 2.5) [l14]. An enzymati-
cally removable solubilizing ester protecting group could be found in the ethylene
glycol derived esters such as the methoxyethyl (ME) estersL7**'151, and the methox-
lipase
PG-peptide-0 PG-peptide-OH
pH 7,37"C
n=1: methoxyethyl (ME)

33 n=2: methoxyethoxyethyl(MEE)
Boc-peptide-0
34
--
+
NMe3
B r-
Cho
butyrylcholine
esterase from
horse serum
pH 6.5, r.t.
* Boc-peptide-OH
o=l;.,.
H-Ser-GIy-Asp(0H)-OH HO
HO-
,HE\
H-Thr-Gln-Thr-Ser-Ser-Ser-Gly-OH
OH o k w HI,
j
adenovirus 2 nucleoprotein serum response factor (SRF)
Aloc-Cys-Met-Gly-Leu-Pro-Cys-OMe
SJ
O\
G,-,-proten
i N-Ras protein
Boc-Phe-Cys-Asp-Phe-OH
I
-
' 0 Figure 18-13. Use o f hydrophilic esters as solubilizing
enzymatically removable protecting groups for the
human Y, receptor synthesis o f characteristic protein fragments.
78 htroduction and Removal ofprotecting Groups
1352
I peroxidase
H or r
'0
i
tyrosinase
PG-peptide-NMN PG-peptide-N"No
H pH 7.37 "C
35 spontaneous
fragmentation
Figure 18-14.
PG-peptide-OH
Phenylhydrazide as a carboxy protecting group.

+ N2 +
0
1
yethoxyethyl (MEE) 115-1171 and in the choline esters (Fig. 18-

13)[58,59, 76, 78, 118-1211 . ~he ME and MEE esters serve both as hydrophilic analogues
of the heptyl esters discussed above and can therefore be removed by the same
biocatalysts such as the lipase from Mucor javanicus. Their increased solubility in
aqueous media has been used successfully in the synthesis of small peptides and
peptide conjugates including glyco-[115-1171 and nucleopeptides F7'1.
Similarly, the respective dipeptide choline esters 34 are readily soluble in purely
aqueous media (i.e. without added cosolvent) and are converted into the correspond-
ing carboxylic acids under the mildest conditions, and without side attack on the
peptide bonds and the N-terminalurethanes, by means of the commercially available
butyrylcholine esterase from horse serum. The increased hydrophilicity of peptide
choline esters was used advantageously used for the synthesis of peptides and very
sensitive peptide conjugates such as lipidated peptides [118-121], phosphorylated and
glycosylated peptides Is', "1 and nucleopeptides (Fig. 18-13) [76, 781.
Recently, phenylhydrazide has been introduced as an enzyme-labile carboxy
protecting group[122,1231 . Th'is protecting group can be removed by mild enzymatic
~ ~ ] 18-14).
oxidation using a peroxidase[122.1231 or mushroom t y r ~ s i n a s e [ ' (Fig.
18.4.2
Protection ofthe Side Chain Groups of Glutamic and Aspartic Acid
The stepwise removal of arginine methyl ester by proteases has been investigated as
a possibility for the enzymatic deprotection of the side chain carboxylate groups of
the aminodicarboxylic acids aspartic acid (Asp)and glutamic acid (Glu).To this end,
Z-Asp(ArgOMe)-NHzand Z-Glu(ArgOMe)-NHzwere converted into Z-Asp(0H)-
NH2 and Z-Glu(OH)-NH2by subsequent treatment with trypsin, which hydrolyzes
the arginine methyl esters, and with porcine pancreatic carboxypeptidase B, which
splits off the arginines[125].Since the second step is slow and requires high
concentrations of the carboxypeptidase, this method can, most probably, not be
applied routinely in peptide synthesis because it introduces too much of a danger of
competitive side reactions.
However, enzymatic transformations have proved to be useful for the synthesis of
selectively functionalized aspartic and glutamic acid derivatives. For instance,
18.5 Protection of Hydroxy Croups
I 1353
alcalase selectively hydrolyzes the a-benzyl esters of H-Asp(Bz1)-OBzl and H-
Glu(Bz1)-OBzl in 82% and 85% yield, respectively, on a decagramm scale[’261.
Similarly, aspartyl- and glutamylpeptides can be deprotected selectively at the C-
terminus by this enzyme, however, in these cases an undesirable attack on the
peptide bonds may occur[’27].In addition, Z-Asp(OAl1)-OAll is converted into Z-
Asp(OAl1)-OH in quantitative yield by Also a lipase from Candida
cylindracea is able to differentiate between the two carboxylic acid groups of glutamic
acid. From the respective di-cyclopentylester it preferably (ratio 20 : 1)removes the
y-ester in 90% yield[12’]. In addition, the enzyme thermitase and the alkaline
protease from Bacillus subtilis (vide supra) also have great potential for the selective
manipulation of dicarboxylic amino acids.
The examples given in Sections 18.2 to 18.4 demonstrate that the selective
deprotection of peptides can be achieved advantageouslyby making use of enzymatic
reactions. In the light of the increasing number of available biocatalysts it appears
that in the near future a host of new and superior enzymatically removable blocking
groups for the synthesis of peptides will be developed. However, these techniques
will definitely not be used for the preparation of simple small peptides in the
laboratory. Most probably they will be applied to the synthesis of sensitive polyfunc-
tional compounds and long oligopeptides, the construction of which is cumbersome
by standard chemical methods. Furthermore, they offer significant advantages if a
technical process for the manufacturing of a given peptide has to be developed.
Finally, together with the recently developed methods for the biocatalyzed formation
of peptide bonds (see Chapter 12.5) (l3Ol, enzymatic protecting group techniques
could prove to be the tools of choice for the construction of peptides in aqueous
solution, the practical development of which has been tried for several de-
cades [131,1321
18.5
Protection of Hydroxy Groups [4-93 ’ 33-1 361
Mono- and oligosaccharides,alkyl- and arylglycosides and various other glycoconju-

gates generally include a multitude of hydroxyl groups of comparable chemical
reactivity. Also, the synthesis of oligonucleotides and nucleosides, B-lactams, alka-
loids, steroids and peptides often requires the selective protection of one or more
alcoholic functions. Consequently, for the directed construction of polyhydroxy
compounds these functional groups have to be manipulated selectively, in general
making cumbersome protection and deprotection steps necessary. Although numer-
ous chemical techniques are available to mask or to liberate hydroxyl groups, the
development of enzymatic methods for this purpose has been progressing steadily
and appears to complement the arsenal of classical tools. In addition, the enzymatic
protection of hydroxy goups (and vice versa of carboxy groups) in racemic com-
pounds as well as their enzyme-catalyzeddeprotection has been used extensively for
the separation of enantiomeric alcohols and carboxylic acids (see Chapter 11).
1354
I 18 fntroduction and Removal of Protecting Croups
18.5.1
Protection of Monosaccharides[133f 1371
The selective protection and deprotection of carbohydrates can be achieved with

various classical chemical techniques '
38-1401. In addition, however, owing to the
synthetic challenge the multifunctional carbohydrates pose, enzymatic techniques
for the introduction of blocking groups into sugars and/or their subsequent removal
offer further, different opportunities.
The enzymatic acylation of sugars in aqueous solution has been reported but gives
low yields as the equilibrium for the reaction favors hydrolysis. However, enzymatic
acylation in dry organic solvents has shown substantial success. While direct
enzymatic esterification of alcohols with acids is often not practical, good to excellent
yields have been obtained using transesterification techniques (Table 18-1).The
displacement of the equilibrium toward products has been accomplished by using
an excess of the acyl donor and by using activated, irreversible acyl donors such as
trihaloethyl esters [l4l], enol esters [1421, acid anhydrides or oxime esters [134, l3'1. In
particular,the enol esters have the advantage that the liberated enol tautomerizes to a
ketone or an aldehyde, thereby shifting the equilibrium toward the desired products
and consequently giving higher yields. This technology, however, is not restricted to
carboxylic acid derivatives being the acyl donor. Organic carbonates [1431, either
activated as the or, even better, as an 0xime[~~'1 derivative, allow for the
enzyme-catalyzed synthesis of carbonates such as the methoxycarbonyl, the benzy-
loxycarbonyl (2) and the allyloxycarbonyl (Aloc) carbonate. The last two examples
can later be removed by non-enzymatic means.
The high polarity of sugars and their derivatives requires that polar solvents be
used to dissolve them. Solvents found to be suitable include pyridine, DMSO, DMF
and dimethylacetamide. However, these solvents also often inactivate enzymes,
although some enzymes, for instance the lipases from the porcine pancreas (PPL),
from Candida antarctica (CAL), from Candida GyEindracea (CCL, later renamed
Candida rugosa) and the lipase from Pseudomonas cepacia (PSL) as well as the
proteases subtilisin and proleather, maintain their inherent acitvity [14G]. A less polar
solvent such as THF allows the use of a broader variety of lipases, but does not
dissolve unmodified pyranoses. Nevertheless, it should be noted that even glucose
suspended in THF has been successfully acylated by using lipase of Candida
antar~tica[~~'].
To remain active in an organic solvent, the enzyme must contain a small amount
of water which is required for maintaining the correct protein structure. In the
absence of this essential water, highly polar compounds such as carbohydrates form
excessively tight enzyme-product complexes. This inhibits association and dissocia-
tion of substrates and products from the active site and thus slows down the reaction.
Accordingly, the addition of drying agents such as zeolite CaA not only influences
activity of the the biocatalyst but also its selectivity. For instance, the acylation of 1-0-
methyl 0-D-glycopyranoside49 catalyzed by lipase SP 435 (an immobilized lipase
from Candida antarctica) in ethyl butanote as the solvent and acyl donor led to
acylation predominantly in the G-po~ition['~~, 1491. If zeolite CaA was added, a
18.5 Protection ofHydroxy Groups
I
1355
mixture of 2,6- and 3,6-bisacylatedpyranosides (95 : 5) was formed. In the presence
of zeolite CaA and tert-butanolas a cosolvent, again monoacylation in the 6-position
was observed.
Alternatively, precipitation of the enzyme from aqueous solution at its optimum
pH prior to its use in an organic solvent has also been reported to increase the
enzyme’s activity greatly.
The results of enzymatic acylation of several pyranose and furanose sugars are
shown in Table 18-1. Other lipophilic carbohydrate derivatives such as alkyl glyco-
sides also display a higher solubility in less polar organic solvents, in which most
lipases tend to be more stable than in polar solvents.
A further interesting finding is that heat stable lipases are capable of transferring
long-chain fatty acids to the 6-hydroxy group of ethyl glucoside on a kilogram-scale,
utilizing the molten fatty acids themselves as solvent^^'^^]. On a somewhat smaller
scale, the acylation of glucose has also been carried out using only a minute amount
of solvent or in supercritical CoZ 1741.
The regioselectivity observed in the acylation of underivatized pyranoses in
principle parallels that recorded for the classical chemical introduction of acyl groups
into carbohydrates. However, if the 6-OH groups are protected first or deoxygenated,
in the corresponding enzymatic reactions selectivities are observed which can not be
realized with classical chemical methods. By careful choice of solvent and lipase, it is
possible to rnodifiy selectively a number of C6 protected pyranoses at the secondary
hydroxy groups (Table 18-2).
By combination of enzymatic with non-enzymatic protection group chemishy,
carbohydrates can be selectively modified in the primary and secondary hydroxy
positions. To demonstrate this versatility, the straightforward synthesis of differently
mono-acylated glucose derivatives is described in Fig. 18-15. For instance, 6-0-
butyrylated glucose GGa (R = n-butanoyl; prepared enzymatically, see Table 18-1)is
converted into the 3,6-dibutanoate 93 by lipase from Chromobacterium uiscosum
(CVL) or from Aspergillus niger (ANL). The 2,6-dibutanoate 94 can conveniently be
built up with the lipase from porcine pancreas (PPL; Fig. 18-15)[1641.Similar
observationswere reported for n-octylglucoside,but for the corresponding galactose-
and mannose 6-esters the selectivity was lower. In contrast, the chemical butyrylation
of glucose derivative GGa with the acid anhydride in pyridine gave a complex mixture
of various diesters without any significant regiodiscrimination. The enzymatic
approach was also used to convert the 6-0-tritylglucose GGb (R = Trt) into the
3-butanoate 95 by a chemoenzymatic approach with lipase from Chromobacterium
uiscosum (CVL), and the 6-tert-butyl-diphenylsilylated glucose GGc (R = TBDPS) could
be acylated exclusively at the 2-position when employing lipase from Candida
cylindracea (CCL) From the disubstituted glucoses obtained by the enzyme-
catalyzed reactions, the protecting functions in the 6-position could be split off
chemically or enzymatically, thus making the glucose esters 95 and 96 carrying a
single acyl group in the 2- or the 3-position available in a convenient way (Fig. 18-
15).
The monoacylated saccharides used in these studies dissolve in several organic
solvents, of which tetrahydrofuran and methylenedichloride were found to be
Table 18-1. Selective acylation of the primary hydroxy group in monosaccharides. Y
0
Compound No. Structure Enzyme" Solvent Acyl Donor Position Yield ("7) Ref.
-z
a
PPL pyridine RC02CH2CC13 6 19-35 ~411 2
CAL dioxane ROC02N=CMe2 6 15-72 [I451 g.
36 Hi- CAL TH F RC02CH=CH2 6 [I471 2
OH OH PS L pyndine MeCOzCH2CC13 6 79 ~461 a
PSL pyridine EtC02CH2CC13 6 29 ~461 a
proleather pyridine PhC02CH2CC13 6 33 ~461 0
subtilisin DMF PrC02CH2CC13 6 60 11501 5
subtilisin pyridine PrC02CH2CCl3 6 64
11501 %a
optimase M-440 pyridine Boc-l'he-0CH2CF~ 6 11511 n
'OI
u
PPL pyridine MeC02CH2CCl3 6 57 ~411 3'
og
PSL pyridine RC02N=CMe2 6 70-85 ~521 a0
CAL dioxane ROC02N=CMe2 6 43-68 P451 5
PPL pyridine MeCOlCH2CC13 6 36 ~411
38 CCL benzene/pyridine 2:1 MeC02CH=CH2 6 ~421
PSL pyridine RC02N=CMe2 6 65-80 ~521
CAL dioxane ROC02N=CMe2 6 44-53 [I451
protease N DMF MeC02C(Me)=CH2 6 40 WI
CCL benzene/pyridine 2:1 MeC02C (Me)=CH2 6 11421
39 protease N DMF MeC02C(Me)=CH2 6 73 P531
subtilisin 8399 DMF MeC02CH=CH2 6 92 P541
H subtilisin BNP' 97% DMF BOC-Gly-OCHzCN 6 65 (1551
CAL pyridine RC02N=CMe2 6 45-83 [1561

40 PSL dioxane RC02N=CMe2 6 50-72 11561
18.5 Protection ofHydroxy Croups
EZ
I 1357
In
z
In
ziz
In*
3.
. . IIn
d 66 6
13
m
6
d In
B
el cl
vl c
c c
D;
-
I I
0
r
0
I
0
tI
l
F I P
d'
d N
d d rn
d u
d
4
w
U
00
-
00
Compound No. Structure Enzyme" Solvent Acyl Donor Position Yield ("A) Ref.
-2
CAL acetone/pyridine 3:l CiiHz3C02H 6 67
CAL 6 51
%; H
47b OH 0
CCL benzene/pyridine 2:1 MeCOzCH=CH2 6

CAL THF/pyndine (4:l) MeCOZCH=CHz 3,6
CAL PrCOzEt/tBuOH (1:l) PrCOzEt 6
49 Hi% CAL CHz=CHCOZEt/ tBuOH (l:l)CH2=CHCOzEt 6

OH CAL THF/pyridine (41) MeCOzCH=CH2 6
OMe
CAL THF MeC02CH=CH2 6

H
&
;OC8H
,i CAL PrCOzEt/tBuOH (1:l) PrC02Et 6
CAL tBuOH Ph(CHz),COzH 6 52
OH
CVL THF PrC02CH2CC13 6;3,6 (1:l)
ANL THF PrC02CH2CC13 6;3,6(1O:l)
51 CAL 6
OH
OCEHi7
I
1359
5
a
3
I
m
rn m
0 h
in
N
I
el el el
a a
m m m
fi
ln
N d
ln W
ln
4
W
0
m
-
0
Compound No. Structure Enzyme” Solvent Acyl Donor Position Yield (“h) Ref. s
-
.b
a
PSL MeC02CH=CHz/THF MeC02CH=CH2 6 94 [W $s
n
57 0
‘cog sl.
OH
B
;
D
PPL pyridine 6 81 ~ 5 1
58 Hi=3 3P
OMe 0,
5.
oq
CI
a
2
59 HowoMe PPL THF 5 77 [I681
HO OH
GO HO
PPL TH F 5
voMe 77
HO
PPL TH F 5 84
G1 “VMe OH
Compound No. Structure Enzyme' Solvent Acyl Donor Position Yield (%) Ref.
PPL THF MeC02CH2CF3 5 39

3 17
62
HO
HvMe
CCL EtOAc 6 90
63 kOH
CCL EtOAc 6 93
THF MeCOzCH=CH* 9 60 ~701 u

Po
m
D
2
F
a Many enzymes were usually screened for activity, only the best results are listed. CAL Candida antarctica lipase; CCL lipase from Candida cylindracea (later renamed Candida
rugosa): PPL porcine pancreas lipase: PSL Pseudomonas cepacia lipase.
g.
3
2
W
m
N
-
Table 18-2. Selective acylation o f secondary hydroxy groups in monosaccharides. 00
--
Compound No. Structure Enzymea Solvent A q l Donor Position Yield (“h) Ref. 5
ANL THF PrC02CH2CC13 3 (GGa) 11641
66 CVL THF PrC02CH2CC13 3 (GGa) 80 11641 2
8’
PPL THF PrC02CH2CC13 2 (GGa) 51 ~641 0
CVL THF PrC02CH2CC13 3 (6Gb) 88 ~ 4 1 9

PFL MeC02CH=CH2 MeC02CH=CH2 2 (GGb) F751 ?
a: R=butyryl 2 (GGc)
CCL CHzClz PrC02CH2CC13 45 [I641 $
b: R=trityl
C: R=TBDPS
67 CVL TH F PrC02CHzCC13 2 20
3 31
0
68 CVL TH F PrC02CH2CC13 2 13 11641
3 52
69 lipase from MeC02CH=CH2 MeCOzCH=CH2 2

Mucor miehei
70 PFL MeC02CH=CH2 MeC02CH=CH2 3

O
M
?-€; lipase from MeC02CH=CH2 MeCOzCH=CHz 2
OH ft2Mucor miehei
Compound No. Structure Enzyme” Solvent Acyl Donor Position Yield (“h) Ref.
PSL 2 (71a)
2 (71a)
71 phT% HO PSL 2 (71a) 98
PFL 2 (71a) 94
a: R=OMe R PFL 2 (71b) 73
b: R=SR 2 (71c)
c: R=OPh 76
PSL 3 (72a)
3 (72a)
- \
PSL 3 (72a) 86
a: R=OMe OH PFL 3 (724 86
b: R=SEt PFL 3 (72b) 86
PPL THF/pyridine (41) 2 93
PPL THF/pyridine (41) 2 (74a) 84

PFL THF/pyridine (4:l) 2 (74a) 81
CCL CHzClz/pyridine (4:l) 2 (74a) 80
PFL MeC02CH=CH2 2 (74b)
a: R=butyryl PFL MeC02CH=CH2 2 (744
b: R=trityl
c: R=benzyl
PFL MeC02CH=CH2 MeC02CH=CH2 3
d
W
8
4
5
-
Table 18-2. (cont.). 1
00
Compound No. Structure Enzymea Solvent Acyl Donor Position Yield (“h) Ref.
-
3,
a
2
phTo PSL MeCOzCH=CH2 MeCOzCH=CH2 91 2
76 3 (7Ga) 11771 s
D
4
O
HO h R PFL MeC02CH=CH2/THF MeCOZCH=CHz 3 (7Gb) 10 [180] $
OH
?
a: R=OAII %
b: R=SEt 9
9
77 MeCOzCH=CH* MeCOzCH=CHz 2 90
bMe
78 <rnOMe PFL MeCOzCH=CH2 MeCOzCH=CH* 3 11751
79 PSL RC02CH=CHz RCOzCH=CH2 3 92 11771
Pr
80 o< PPL THF PrC02CHzCF3 4 65
.:WoMe PFL THF PrC02CHzCF3 4 68
OH
18.5 Protection of Hydroxy Groups
I 1365
z7
Ln
d
8
a
h a
4
Ln *
0
m m N N N
el 44
&r&
-1 -1
m m r&
fi fi & & fi
-
00 N
00 d
00 In
00
-
d
Table 18-2. (cont.). 00
Compound No. Structure Enzymea Solvent Acyl Donor Position Yield (“h) Ref. -1
a
a
PSL MeCN MeC02CH=CH2 3,4
C * H l 1
87 H R S O OH PSL Hexane MeCOzCH=CH2 2,4 70
3,4 28
88 CAL dioxane RC02N=CMe2 4 70-72

CAL dioxane MeOCOzN=CMe2 4 42
PSL MeC02CH=CH2 MeC02CH=CH* 4
OH OH CAL PrCO2EtltBuOH PrCOzEt 4; diester
89 HO m0 CAL PrC02Et PrCOzEt 4
90 p:H PSL MeC02CH=CH2 MeCOzCH=CHz 4 1187,1891
6H
I 1367
18 Introduction and Removal ofprotecting Croups
1368
I
R=Trt
95
1) CVL, THF, 2, H+ CCL
TCE-But 88% 85%
R = n-But R = n-But OBut
HHO O q ~ HEo% THF

ButO OH TCE-But
HO OH TCE-But
OH
51% 80%
94 66a:R = n-But 93
66b:R = Trt
66c:R = TBDPS
R = TBDPS
I)CCL 2) Bu~NF/AcOH
TCE-But = CHZCIZ -5O"C, THF
0 TCE-But 75%
~ O ^ C C I , 75%
96
Figure 18-15. Selective enzymatic introduction of protecting groups into partially acylated
hexoses.
particularly suitable for the enzymatic reactions. This was also observed in the lipase-
mediated acylation of the methyl glycosides of both D- and L-fucose and -rhamnose,
Using lipase from Pseudomonasfluorescence (PFL), both D-carbohy-
drates were converted into the 2-monobutanoates with high regioselectivity. The
naturally occurring L-enantiomers of these 6-deoxysugars, however, were esterified
preferably at the 4-hydroxy groups. These results contrast favorably with chemical
derivatizations, since the 4-hydroxy groups of the 6-deoxy-L-carbohydrateshave only
slight reactivity toward chemical acylating reagents. In addition, methyl-L-fucoside
can be converted into the 3-butanoate with lipase from Candida cylindracea. The
introduction of an acyl-substituent into the 6-positions of the D-fucoside and the L-
rhamnoside does not influence the regioselectivity of the enzymatic acylation
Finally, it should be mentioned, that some attempts were made to differentiate
between the hydroxy groups of fructose by enzymatic methods, however, with lipases
as well as with subtilisin, only mixtures of the 1- and 6-isomers were ob-
I
1369
tained [141, 150, 1921. Regioselectively monosubstituted fmctoses can, however, be
obtained by an enzymatic approach from sucrose (vide infu).
18.5.2
Deprotection of Monosaccharides['33.l37I
Initial attempts to apply lipases for the enzymatic removal of acyl groups from
glucose pentaacetate only resulted in low levels of selectivity[1932 'O4I. However, later
on lipase from porcine pancreas (PPL) was found to hydrolyze exclusively the
anomeric acetate from peracetylated pyranoses while the esterase from Rhodospo-
rium tomloides (RTE)F1O5I releases the primary hydroxy group in preferance (Table
18-4).On the other hand, if the anomeric center is derivatized as a methyl glycoside,
the regioselective enzymatic liberation of the 6-OH group becomes feasible with a
number of hydrolytic enzymes [168,195-1991 . Thus, from methyl a-D-glucose tetra-
octanoate 97a and the corresponding tetrapentanoate 97b, lipase from Cundidu
cylindruceu (CCL) removes only the primary ester group in yields of ca. 75%.
Similarly, the a-D-gdactoside103,as well as the corresponding mannoside 104b and
the 2-acetaniido-2-deoxy-mannoside105 were converted into the 6-deprotected
pyranosides in 29-50 % yield (Table 18-3), but the 2-acetamido-2-deoxy-glucoside
was only a poor substrate. In the latter cases the regioselectivitywas less pronounced
and the 4,G-dideoxyderivatives were also formed in ca. 20 % yield. In addition to this
class of compounds, lipases also accept hexopyranosides carrying several different
functionalities (e.g. acetals [1971, enol ethers [160, I'*' and, in particular, 1,G-anhy-
dropyranoses as substrates (Tables 18-3and 18-4).In all cases the reaction conditions
are so mild that the acid sensitive structures of these compounds remain unaffected.
Particularly remarkable is the regioselectivity displayed by lipase from Pseudomonas
cepucia (PSL:)in the deprotection of the glycall31 [16', 2001. The biocatalyst exclusively
attacks the 3-acetate and leaves the primary ester intact. The enzymatic deprotection
strategy can also be used to synthesize carbohydrates carrying a single acyl group in
selected positions. Thus, 3,G-dibutyryl glucose 93 (prepared by enzymatic acylation
of glucose) was converted into the 3-butanoate 95 by lipase mediated hydrolysis of
the 6-ester (Fig. 8-15)[1641. The principles and the enzymes mentioned above which
allow the regio- and chemoselective protection and deprotection of the various
pyranoses to be carried out were also successfully applied to the enzymatic manip-
ulation of acyl groups in furanoses. Of particular interest in this context is the
finding that the five-membered rings can also be handled by the biocatalysts with a
pronounced regioselectivity, although furanoses can adopt more flexible conforma-
tions with similar energies in solution.
The cleavage of the primary acetyl groups from the furanosides 106-111 could be
carried out in high yields with lipase from Cundida cylindruceu (Table 18-3)[lG8]. For
the 2-deoxy-a-~-ribofuranoside and the a- and the P-xylo-compoundsthe hydrolysis
was less selective. From the peracetylated furanoses 125 and 126 the anomeric acyl
group was removed with total selectivity by means of lipase from Aspergillus niger
(Table 18-4).
1,G-Anhydropyranosesserve as convenient starting materials for various synthetic
a
W
U
0
-
Table 18-3. Selective deacylation of primary hydroxy groups in monosaccharides. -
oa
Compound No. Structure Enzyme" Solvent Position Yield ("A) Ref.
-5
97 CCL 0.1 M phosphate buffer 6 78 (97a) [1681 %
CCL 0.1 M phosphate buffer 6 75 (9%) [16531 2.
R;;g CCL 0.1 M phosphate buffer G 90 ( 9 7 4 ~961
CCL 0.1 M phosphate buffer, BuzO (10%) 6 (974 [I971 a
RoOMe PEG-modifiedCCL Cl,CCH, 6 27 (97c) [198] f
a: R=octanoyl 4,6 48 (97c)
b: R=pentanoyl CRL 0.1 M phosphate buffer G 91 (97c) [I991
s%
c: R=acetyl RTE citrate buffer 6 77 (97c) [I951 9
98 CCL 0.1 M phosphate buffer G 77

OR 0
R=octanoyl a
a
99 PPL 0.1 M phosphate buffer, acetone 1O:l 6 90

0
OMe
PPL 0.1 M phosphate buffer, acetone 10:1 6 82
AcO
101 ) 0( o g PPL 0.1 M phosphate buffer, acetone 1O:l 6 75
AcO
Compound No. Structure Enzyme" Solvent Position Yield ("h) Ref.
CCL 0.1 M Tris.HC1 6 85 ~641
103 RORoe RTE citrate buffer 6 85 (103a) [195]
Ro OMe CCL 0.1 M phosphate buffer 6 29 (103b) [168]

a: R=acetyl
b: R=pentanoyl
104 CRL 0.1 M phosphate buffer 6 94 (104a) [199]

RTE citrate buffer 6 70 (104a) [195]
a: k a c e t y l OMe
CCL 0.1 M phosphate buffer 6 33 (104b) [168]
b: R=pentanoyl
CCL 0.1 M phosphate buffer 6
106 AcoQoMe CCL 0.1 M phosphate buffer, 10% DMF 6 85
AcO OAc
4
W
U
N
-
0
Compound No. Structure Enzyme" Solvent Position Yield YO) Ref. P

-
0.1 M phosphate buffer, 10% DMF
AcO bAc
.
%
108 A c o ~ ~ M e CCL 0.1 M phosphate buffer, 10% DMF 5 98
Acb
109 A C 0 q g O M e CCL 0.1 M phosphate buffer, 10% DMF 5 50

3 30
OAc
CCL 0.1 M phosphate buffer, 10% DMF 5 40

3 50
ll1 AcowoMe CCL 0.1 M phosphate buffer, 10% DMF 5 63
AcO
a Many enzymes were normally screened for activity, only the best results are listed. Candida rugosa;CRL); PPL: porcine pancreas lipase; RTE: Rhodosporiurn toruloides
ANL Aspergillus niger lipase: CCL lipase from Candida cylindracea (later renamed esterase.
I
1373
w
m *- 00
0 r.w
Ink In
00
3 2"
IL
4 elw 4
&I-
a
F&
& & 2
N
3
m
f3 I
3
n ua
3
3 3
3 3 3
d
w
U
P
-
d
Table 18-4. (cont.). co
Compound No. Structure Enzyme” Solvent Position Yield (“h) Ref.

-a
a
118 hog kidney acylase phosphate buffer, DMF (1O:l) 2 93 [2061 2
Aspergillus niger pecti- phosphate buffer, DMF (1O:l) 3 27 PO61 9.
S
nase
0
3
AcOO B 0OAc
. e ANL phosphate buffer, DMF (1O:l) 4 11 12061 4
a
PPL 0.05 M phosphate buffer, 10% DMF

RTE citrate buffer
6Ac
120 A ;aoMe ANL 0.1 M phosphate buffer, 10% acetone 3 61
‘ACO
PPL 0.05 M phosphate buffer, 10% DMF 1 88 P681
PSL tAmyl-OH 4 84 ~071

PEG-modifiedCCL Cl3CCH3 4 82 ~981
PPL 0.05 M phosphate buffer, 10% DMF 1 54 11681
OAc
Compound No. Structure Enzyme‘ Solvent Position Yield (“h) Ref.
~~
0.05 M phosphate buffer, 10% DMF
ANL 0.1 M phosphate buffer, 10% DMF

125 Aco?a’”cAcO OAc
126 Aco*oAC ANL 0.1 M phosphate buffer, 10% DMF 1 50
RJL 0.1 M phosphate buffer 2 47 (127a) [208]

4 15 (127a) u
Po
WGL 0.1 M phosphate buffer 3 [208] cn
127 OR OR 67 (127a)
PLE 0.1 M phosphate buffer 4 69 (127a) [208] D
a
F7
a: R=acetyl
PPL
CVL
0.05 M citrate-phosphate buffer
0.1 M phosphate buffer
4
4
42 (127a)
91 (12%)
[209]
[210]
P
9.
b: R=butyvl 3
CCL 0.1 M phosphate buffer 2.4 77 (127b) [210]
%
-=I
Q.
CCL 0.1 M phosphate buffer 4 85-90

alcalase 0.1 M phosphate buffer 2 82
128 OAc
F7 OAc
d
W
Ul
U
4
W
U
m
-
0
--
Compound No. Structure Enzyme" Solvent Position Yield ("A) Ref. 5
CCL 0.1 M phosphate buffer 2

12g But0 CCL 0.1 M phosphate buffer 2 90
PPL 0.1 M phosphate buffer 2
mo OBut 4
2,4
16
19
65
WGL phosphate buffer, DMF (1O:l) 2 60
OAc
PSL 0.25 M phosphate buffer 3 90

acetyl esterase from 0.15 M NaCl buffer 3,4 24 (74,2131
the flavedo of oranges 3,4,6 22
OPhAc
PGA 0.1 M phosphate buffer 3 80-85
OAc
a Many enzymes were normally screened for activity, only the best results are listed. liver esterase; PPL: porcine pancreas lipase; PSL Pseudomonas cepacia lipase; RJL
ANL Aspergillus niger lipase; CAL Candida antarctica lipase; CCL lipase from Candida Rhizopusjaponicus lipase; RTE: Rhodosporium tomloides esterase; WGL wheat germ
cylindracea (later renamed Candida mgosa; CRL); PGA: penicillin-G-acylase; PLE: porcine lipase.
18.5 Protection of Hydroxy Groups
I
1377
HO OR
133a R = acetyl
133b R = butyryl
R = acetyl R = n-butyryl
PPL or PLE lipase from
63-69% Chromobacterium CCL
viscosum (CVL), 77%
Pseudomonas sp. or
Mucor miehei (MML)
91%
R = acetyl
chymotrypsin R = n-butyryl
CCL, 47%
RO OR :;Jgk RO OR HO OH
127a R = acetyl 134
127b R = n-butyryl
R = acetyl, RJL, 47%
7 0
135
RO OH
Figure 18-16. Selective enzymatic removal o f protecting groups from 1,6-anhydropyranoses.
purposes in carbohydrate chemistry. Therefore, the directed manipulation of their

hydroxy groups is of particular interest. Each of the three OH-groups in 1,G-anhy-
droglucopyranose can be liberated selectively making use of enzymatic reactions
(Fig. 18-16, Table 18-4)[208-210s 2121. Thus, the 4-protecting group was split off from
the triacetate 127a using lipase from porcine pancreas (PPL)[2091or pig liver esterase
(PLE)[208, 209]. The acetate in the 3-position could be attacked preferentially using
c h y m ~ t r y p s i n [or
~ ~lipase
~ I from wheat germ (WGL)[zos],and the 3,4-diacetate 135
was obtained by hydrolysis with lipase from Rhizopusjavanicus (RJL)[208! In each
case, however, other derivatives were formed as undesired by products. High yields
could be obtained from the tri-n-butanoate 12%. It was converted into the 2,3-dibuta-
noate 133b in 91 % yield by means of several lipases, but the enzyme from Candida
cylindracea (CCL) removed two acyl groups successivelyto yield the monobutanoate
134. Similarly, the analogous 3-azido-1,G-anhydropyranose 128 is regioselectively
deacylated at 0 2 and 0 4 by means of lipase OF from Candida cylindracea and
1378
alcalase, respectively[2111. Of particular importance is the stereochemistry at C4 of

the bicyclic substrates. If the alcohol at this position is equatorial, as for instance in
the corresponding 1,6-anhydrogalactopyranose129 and the analogous lactone 130,
several enzymes act only in a random fashion or not at However, the acyl
group in the 2-position seems to be preferred (Table 18-4).The results obtained from
these studies indicate that the reactivity of acyl protecting groups in 1,6-anhydropyr-
anoses toward hydrolysis by lipases decreases in the order C4, > C2, > C3, >>
c4q.
The above mentioned investigations revealed that the lipase-mediated hydrolysis
proceeds at higher reaction rate and, in many cases with better selectivity, if
butanoates or pentanoates are employed as substrates instead of acetates. However,
the use of enzymatic deacylations is by no means restricted to simple alkanoates. An
illustrative and impressive example is found in the hydrolysis of generally base-
stable carbohydrate pivaloylates using an esterase from rabbit serum (ERS) [214-2171.
For instance, the biocatalyst selectively splits off the 6-pivaloyl group from a-methyl
3,4,6-tripivaloyl-2-acetamido-2-deoxy-glucoside. On prolonged incubation the com-
plete removal of pivaloylates from carbohydrates is also possible. Of particular
significance is, that the enzyme does not have to be purified, but that crude serum
preparations are sufficient for the preparative purposes. A further enzyme which
allows the chemo- and regioselective unmasking of different carbohydrate deriva-
tives to be carried out is acetyl esterase from the flavedo of oranges, a biocatalyst
which preferably hydrolyzes acetic acid 218]. It can be applied for the
synthesis of selectively deacylated pyranoses. Thus, from pentaacetylglucose 112 the

2,3,4,6-tetraacetateis obtained by means of the regioselective saponification of the
1-acetate.If the hydrolysis is allowed to proceed further, the 6-acetate is also cleaved
and the 2,3,4-triacetatebecomes available in ca. 40% yield. If tri-0-acetyl-glucall31
is subjected to the enzymatic hydrolysis, at 40% conversion the 6-acetate is the main
product.
By introducing acyl groups which are specifically recognized by certain enzymes
into carbohydrates, not only the regioselectivitybut also the chemoselectivity of the
biocatalysts can be exploited. This can, for instance, be achieved by the selective
saponification of phenylacetates catalyzed by penicillin G acylase [3G321. The enzyme
liberates the 2-OH group of 1,3,4,G-tetraacetyl-2-phenylacetyl glucose without affect-
ing the acetic acid esters. In this case, moreover, an ester of a secondary hydroxy
function is chemoselectively hydrolyzed in the presence of the chemically more
reactive acetates at the 6-position and at the anomeric center. This approach was also
adopted for the enzymatic deprotection of the glucal 132. Thus, its 3-OH group was
liberated without cleaving the acetates that were present [1901.
18.5.3
Di- and Oligosaccharides[’37]
For enzymatic protecting group manipulations on di- and oligosaccharides in

particular the use of subtilisin together with dimethylformamide as the solvent is
advantageous.As has already been pointed out, the use of DMF is often critical, since
I
1379
its dissolving ability is high enough to solubilize even highly polar polyhydroxy
compounds (comparable experiments with pyridine as the solvent generally
failed)[l4l].Only a few reports about the successful use of other solvents such as
pyridine L2l91 or tert-butanol[220] have been published.
subtilisin accepts several disaccharides as substrates and transfers butyric acid
from ethyl or trichloroethyl butanoate to the primary G'-hydroxy functions of the
nonreducing monosaccharide of the p-(l-3)-linked cellobiose 136 and the respective
maltobiose (Fig. 18-17) 220]. For lactose the regioselectivity was less pronounced,
however, methyl and benzyl p-D-lactoside 137 were converted into the 6'-butanoates
in 71-73% Rutinose in which the primary hydroxy group of the glucose
moiety is blocked (see also 149, Fig. 18-19),is selectively substituted in the 3-posi-
tion [2221. In addition, higher maltooligomers could also be acylated in the 6-position
of the terminal nonreducing carbohydrate.For instance, 6"-0-butyrylmaltotriosewas
isolated in 29 % yield, but also the corresponding tetra-, penta-, hexa- and heptamer
were substrates for the biocatalyst. These enzymatic esterifications open a route to
discriminating between the primary hydroxy groups in di- and oligosaccharides in a
convenient and straightfonvard way. Classical chemical one step methods of compa-
rable selectivity are not available for this purpose[139* l4O], and multistep sequences
usually have to be carried out if the selective protection of a specific primary hydroxy
group in a di- or oligosaccharide is desired.
Owing to its great commercial importance as a renewable resource, sucrose 138
has been subjected to several enzymatic hydroxy group manipulations. This non-
reducing disaccharide turned out to be a substrate for subtilisin In contrast
to chemical acylations in which the most reactive OH-groups are found in the 6- and
the G'-position, the enzyme selectively transfers various acyl functions to the
1'-alcohol(Fig. 18-17)[lSoS 192, 2 2 3 ] . This acylation was usually carried out in DMF as a
solvent, but the use of anhydrous pyridine gave similar results[219].The mono-
acylated disaccharides 139 thereby obtained could then be further transformed
enzymatically. On the one hand, with the lipase from Chromobacteriurn viscosum
(CVL) the free primary 6-OH group was acylated in 31 % yield. On the other hand,
the 1'-esters 139 are substrates for yeast a-glucosidase which hydrolyzes the
glycosidic bond and thus makes the 1-0-acylfiuctoses 140, potentially useful as
chiral synthons, available [1921. Alternatively, the 6'-OH-group in sucrose 138 can be
selectively acylated,if the carbohydrateis converted into the 2,1':4,6-bisacetalprior to
the treatment with a lipase (NovozymTM435) [2241.
On considering hydrolysis, several enzymes were investigated[225-2291. Depending
on the biocatalyst used, acetyl groups from different positions of octaacetyl sucrose
141 could be removed selectively in usehl yields. For instance, alcalase and protease
N preferably attack the acetate on Ol'[226s2301, the lipase from Candida cylindracea
preferably liberates the OH-group on C4'of the furanoid ring[225,2301 and wheat
germ lipase preferentially liberates the 1'-, 4'- and 6'-OH-groups (Fig. 18-
17)[223* 2311.
The deacylation of the octaacetates of cellobiose, lactose, maltose and melibiose
with Aspergillus niger lipase leads to the formation of the respective carbohydrate
heptaacetates with a free anomeric OH-group at C1 in high yield[230,2321 . Wlth
1380
HHOo eHOo * HO OH H
HO oHO @HO o ~ OR
HO
136 cellobiose 47% 137 lactosides 71-73% R = Me, Bzl
subtilisin, trichloroethyl butyrate, DMF
subtilisin, DMF
K
I
138 R OCH,CX,
12-64%
X = CI, F a-glucosidase
L
O HO
HO V TOH
alcalase or
protease N 140
65-74%
AcO 54%
AcO OAc
141
Figure 18-17. Selective enzymatic protection and deprotection of disaccharides.
prolonged reaction times, the acetates at C1 and C2 are hydrolyzed from cellobiose
and lactose octaacetate in 51 % or 42% yield, respectively.
18.5.4
Nucleosides1’35,2331
The directed protection of nucleoside functional groups is a fundamental problem in

nucleoside and nucleotide chemistry. Although several chemical methods are
available for the regioselective acylation of the nucleoside carbohydrates, enzymatic
methods offer significant advantages with respect to yield, regioselectivity and the
number of synthetic steps which have to be carried out.
Earlier studies focussed on the use of the dihydrocinnamoyl group as an enzyme-
labile nucleoside protecting function which can be removed through the agency of a-
~ h y m o t r y p s i n [2351.
~ ~ ~Although
. the enzyme shows an interesting tendency to attack
preferably the S'-position, this technique was not exploited further. Highly re-
giodiscriminating biocatalyzed acyl transfer reactions to the carbohydrate parts of
various nucleosides could be carried out again employing the protease subtilisin
together with dimethylformamide as solvent. In particular, a mutant of this enzyme,
obtained via site specific mutations appears to display advantageous properties. It
transfers the acetyl group from isopropenyl acetate to the primary hydroxy functions
of various purine and pyrimidine nucleosides and 2'-deoxynucleosides142 in high
yields (Fig. 18-18)[2361. Commercially available subtilisin (protease N from Amano)
provided the same compounds with identical yields and selectivities, however, five
times more enzyme is required for this purpose. In addition, in the transfer of
butyric acid from trichloroethyl butanoate to adenosine and uridine, carried out
earlier I'[, this biocatalyst showed inferior properties with respect to regioselectivity
and yields.
The selective introduction of protecting groups into the hydroxy functions of
different nucleosides can also be achieved by means of lipases. Thus, unprotected
pyrimidine and purine 2'-deoxynucleosides143 (X = H) are selectively converted into
the 3'-O-aqlated derivatives 144 in 6 6 8 2 % yield making use of lipase from
Pseudornonas cepacia (PSL) and employing oxime carbonates as acyl donors (Fig. 18-
18)[237-2391. Similarly, by applying oxime esters or acid anhydrides, different ester
functions can be selectively introduced into the 3I-position of nucleotides by using
the lipases from Candida cylindracea (CCL),porcine pancreas (PPL) or Pseudomonas
cepacia (PSJd)[240-244].If lipase from Candida antarctica (CAL) is used, however, the
esters and carbonates are predominantly generated at the primary 5'-OH group of
(deoxy)nucleotides[238, 239, 241* 242, 244-2471. Furthermore, in the case of ribonucleo-
tides, complete regioselectivity can be achieved by using the same methodology[241].
The regioselectivity of the CAL-catalyzed alkoxycarbonylation is profoundly influ-
enced significantly by the structure of the starting oxime In the
alkoxycarbonylation of thymidine the use of the phenyl derivative leads to almost
exclusive formation of the 5' carbonate, while the corresponding ally1 carbonate is
introduced without any regioselectivity.
An investigation of the enzyme-catalyzed acylation of a-, xylo-, anhydro-, and
arabino-nucleosidesshowed that in these cases the primary 5'-hydroxygroup can be
selectively acylated using lipase from Candida antarctica (CAL)[249-2511. A selective
derivatization of the 3'-OH-group,however, was unsuccessful.
When acylations of nucleosides with acid anhydrides in the presence of lipase
from Pseudomonaspuorescence (PFL) in DMF or DMSO as the solvent first pro-
ceeded, the regioselectivity was However, this lipase together with
subtilisin can be utilized to effect highly specific deacylations of various pyrimidine
nucleosides 145 (Fig. 18-18)[2531. Thus, lipase from Pseudomonas Jluorescence (PFL)
preferably attacks the hexanoyl group on the secondary hydroxy function of the N-
1382
subtilisin 8350
or
protease N
HO R
JOk,
DMF
HO R
142
I R H OH H OH H OH
I
I yield [%I quant. 90 80 80 80 65
X=H,OH base x = H,
PSL, THF 60°C
oxime carbonaL
carbonate HO
HO X
143 0 144
45-68% 64-82%
0
base = A, U,T
R = alkyl. vinyl, oxime carbonate = R O K O / N
ally1
su btilisin X b 3 ' X = H, Br, F, CH3

12-31%
Pseudomonas fluorescence
58-74%
145
Figure 18-18. Selective enzymatic protection and deprotection of the carbohydrate parts o f
nucleosides.
I
1383
glycosides, giving rise to the 5-esters in good yields. On the other hand, subtilisin
gives rise to the $esters with moderate results. It should be noted, however, that in
both cases from considerable to large amounts (6-71%) of the completely depro-
tected nucleosides were also formed. Subtilisin in phosphate buffer also selectively
hydrolyzes the 5'-acetate of purine and pyrimidine triacetylated esters to give the
corresponding 2',3'-diacetylribonucleosidesin 40-92 % A similar prefer-
ence was observed for the lipase from porcine pancreas, but with poorer selectivity
and a slower reaction rate. This enzyme, however, deacetylated the deoxynucleoside
3',5'-di-O-acetylthymidine at the 5'-position in almost quantitative yield[255! In
contrast, if lipase from Candida cylindracea (CCL) was used in the catalysis, the
3'-ester of this diacetate was preferentially hydrolyzed [2551.
Using acetyl esterase of the flavedo of oranges, bisacylated purine deoxynucleo-
tides can be selectively deprotected at the 3'-hydroxy group in 31-40 % yield [741.
Interestingly, by introducing a phenylacetyl group for amino protection in the purine
moiety the regioselectivity of the acetyl removal is reversed. Now the primary acetate
is hydrolyzed by acetyl esterase in 22-52 % yield.
In addition, the complete hydrolysis of an anomeric mixture of peracetylated
2'-deoxynucleosidesby wheat germ lipase or porcine liver esterase has been used to
synthesize the pure p-anomer of e.g. thymidine, this being the only completely
deprotected product [2561. The alcoholysis peractylated uridines catalyzed by Candida
antarctica lipase leads to the formation of the completely deprotected nucleotide[2571.
Although this reaction can be stopped after removal of the first acetyl group, no
regioselectivity was observed for the formation of di-0-acetyluridine.
18.5.5
Further Aglycon Clycosides
In addition to nucleosides, several other naturally occurring carbohydratederivatives

can be selectively protected/deprotected by means of enzymatic techniques. For
instance, salicin 146, a wood component that contains a primary hydroxy group
located in a glucose moiety and a second one in a benzylic position, was butyrylated
exclusively at the 6-OH of the monosaccharide in 35 % yield by applying subtilisin
and trichloroethyl butanoate in DMF (Fig. 18-19)I'[. Under the same conditions, in
riboflavin (vitamin B2) 147 only the primary alcohol was esterified in 25 % yield["'],
and colchicoside 148a as well as a thio analog 148b were converted into the
6'-butanoates by treatment with trichloroethyl butanoate in pyridine in the presence
of subtilisin [2581. The corresponding 6'-acetatesof 148a,bwere obtained by treatment
with vinyl acetate in the presence of Candida antarctica lipase as the biocatalyst
(Fig. 18-19) Similarly, the carbohydrate parts of flavonoid disaccharides were
regioselectively functionalized. Thus, for instance in the disaccharide rutin 149 and
the related hesperidin only the 3"-OH group of the glucose moiety was esterified
upon treatment with trifluoroethyl butanoate and subtilisin in 53 % yield (Fig. 18-
19)[2221. In the presence of lipase from Candida antarctica, however, both the 3"- and
the 4"'-positions were acetylated['621. If only the glucose moiety is present in the
molecule, as in the related isoquercitrin 150, the regioselectivty in the subtilisin-
18 lntroduction and Removal of Protecting Groups
1384
I
HHOO G o
HO
X=O: 148a
X=S: 148b
R fq
R = Me 0
Me0
. XMe
0
HAc
subtilisin, pyridine, 86%
0
-OCH2CF3
or CAL, vinyl acetate
f-amyl alcohol
subtilisin, pyridine, 53 %
0- 0
-OCH2CF3
W H + O R HO Y HO
or CAL, vinyl acetate
f-amvl alcohol. 91 %
149 rutin
H o/ e : : CAL,
f-amylvinyl
alcohol, 79 %
acetate
B
HO 0 or CAL, vinyl cinnemate
acetone, 68 %
150 isoquercitrin
Figure 18-19. Selective enzymatic acylation of aglycon glycosides.
catalyzed reaction was less pronounced[259].However, in the presence of lipase from

Candida antarctica the 3",6"-bisacylatedproduct is formed if vinyl acetate is used as
the acyl donor['62]. Interestingly, by using vinyl cinnamate as the acyl donor, this
biocatalyst only acylates the primary 6"-hydroxy group[260].Naringine 151 was
converted into the 6-glucosyl ester in the presence of subtilisin (Fig. 18-20). In all
cases the rhamnose and the phenolic hydroxyls remained unattacked (for the
protection of phenolic hydroxy groups in flavonoids see Sect. 18.5.8).
The steroidal glucoside ginsensoside Rgl 152 can be selectively monoacylated in
high yields at the 6'-position using Candida antarctica lipase as the biocata-
lyst1261,2G2]. In this case, similar results were obtained with different acyl donors
such as vinyl acetate, dibenzyl malonate and bis(trichloethy1)malonate (Fig. 18-20).
Two impressive examples of selective enzymatic deacylations of complex sub-
I
1385
I
subtilisin, pyridine, 49 %
0
40CH2CF3
151 naringin
CAL, f-arnyl alcohol
vinyl acetate (87 %)

dibenzyl rnalonate (85 %)
bis(2,2,2-trichloroethyl)rnalonate (71 %)
OH
152 ginsenoside Rgl
Figure 18-20. Selective enzymatic acylation of aglycon glycosides.
strates consist in the removal of all acetates from the peracetylated fi-D-glucopyr-
anosyl ester 153 of abscisinic acid[263]
and of the gibberellinicacid derivative 154[264],
containing one glucose tetraacetate glycosidically bound and a second one attached
as an ester (Fig. 18-21).In both cases the removal of the acetyl groups by chemical
methods in particular was complicated by an undesired cleavage of the ester linkages
to the glucoses. However, the four acetyl groups present in 153 could be hydrolyzed
chemoselectively by means of helicase, an enzyme occurring in the seeds of
Helianthus annus, whereby the unprotected glucose ester was formed in 82 % yield
without destroying the ester bond between abscisinic acid and glucose. Similarly, the
biocatalyst removed all acetates from 154.In this case the yield reached only 8%, it
should, however, be kept in mind that ten acetic acid esters had to be cleaved in the
enzymatic process and that the aglycon is rather complex.
In conclusion, the various enzyme-mediated protecting group manipulations
carried out on numerous carbohydrate derivatives indicate that biocatalysts can be
used advantageously in the protecting group chemistry of carbohydrates. In partic-
ular, subtilisin and several lipases from different sources (from porcine pancreas,
from Candida cylindracea, Aspergillus niger, Chromobacterium viscosum, Mucor jav-
anicus, Pseudomonas fluorescence and from wheat germ) allow the chemo- and
regioselective acylation and deprotection of various saccharides, the structures of
1386
"+"'/=<?i-')_.OAc
helicase
153 82%
w.
AcO 0
0 AcO
-0 H
154 8%
--0 OAc helicase
H& 0 O
AcO Figure 18-21. Enzymatic deprotection of complex glucosyl esters.
which differ widely, to be carried out. A general principle that emerges from these
studies is that the enzymes exhibit a predominant preference toward primary
hydroxy groups. If these functionalities are not present or protected, the biocatalysts
are capable of selectively manipulating secondary hydroxy groups or the esters
thereof. In the introduction and removal of acyl groups, the regioselectivitydisplayed
by the enzymes often parallels the findings recorded for classical chemical trans-
formations, although it is significantly higher in many cases. Furthermore, in several
cases regioselectivities were observed in the biocatalyzed processes which can not or
only slightly be achieved by means of chemical methods. Finally, it should be
realized that subtilisin and the lipases are capable of introducing specific acyl groups
into the carbohydrates which can later be removed selectively by different enzymatic
or chemical methods.
18.5.6
Polyhydroxylated Alkaloids
The plant alkaloid castanospermine 155 and the related piperidine alkaloid l-deox-
ynojirimicin 160,like several other polyhydroxylated octahydroindolizidines,piper-
idines and pyrrolidines, are potent glycosidase inhibitors. These nitrogen bases are
of considerable interest for the study of biosynthetic processes and, in addition,
castanospermine and some of its derivatives may be of clinical value as antineo-
plastic agents and as drugs in the treatment of AIDS.
In the light of the analogy between the structures of these alkaloids and glucose,
some of the above mentioned enzymatic methods for the selective functionalization
of carbohydrates were applied to prepare several acyl derivatives of 155 and 160.
Thus, subtilisin transfers the acyl moieties from several activated esters to the 1-OH
group of the bicyclic base in moderate to high yields (Fig. 18-22)[2G52 266] . A gain,
18.5 Protection ofHydroxy Croups
I 1387
157
R = CH3
= CHTPh
} R = CH=CH2 R = n-C3H7
CVL
R = Ac-L-Phe
R = (CH2hCI ~ O C H ~ C C ~ ~
Ac-D-Ala THF 72%
-
subtilisinl subtilisinl
pyridine HO
HO HO
HO 1.5 equiv. HO 6 equiv.
OH trichloroethyl OH trichloroethyl
butyrate butyrate
161 56% 160
162 77%
Figure 18-22. Selective enzymatic protection of polyhydroxylated alkaloids.
pyridine had to be used as the solvent for the polyhydroxy compound. The
monoesters 156 obtained by this technique, like the monoesters of hexoses could
subsequently be dissolved in THF and were further acylated by means of different
enzymes, e. g. to the 6-butanoate 157 and the 1,7-dibutanoate158. Finally, the 1-ester
was removed from 158 by subtilisin in aqueous solution to deliver the 7-butanoate
159 in 64% yield.
In contrast to castanospermine, 1-deoxynojirimicine 160 contains a primary
hydroxy group as well as a much more nucleophilic amino function. If a small excess
of trifluoroethyl butanoate is employed, subtilisin converts this alkaloid preferably
into the 6-monoester 161 (Fig. 18-22)I2"]. However, with 6 equiv. of the acylating
agent, the 2,G-diester 162 is formed in 77% yield. This diester 162 may be
subsequently deacylated regioselectively at the 6-position by means of several
different enzymes.
1388
I 18 lntroduction and Removal offrotecting Croups
It should be noted that under the conditions of the enzymatic acylation the amino
group is not derivatized, an observation which has also been made in related
cases [266. 2671, e. g. N-terminally deprotected serine-peptides.
18.5.7
Steroids
Enzymatic acyl transfer reactions are also practical processes for the acylation of
hydroxy groups in steroids. The lipase from Chrornobacteriurn viscosurn (CVL) for
instance selectively transfers butyric acid from trifluoroethyl butanoate to equatorial
(B) C3-alcoholic functions that are present in a variety of sterols, e. g. 163 and the
respective 5,6-didehydrocompound (Fig. 18-23)[268].Axially oriented alcohols at C3
and secondary alcohols at C17 or in the sterol side chains are not derivatized. In
addition to the equatorial alcohols, the compounds being accepted as substrates by
the lipase must have the A/B-ring fusion in the trans configuration. In the B-ring a
double bond is tolerated, in the A-ring, however, it is not. Similarly, lipase from
Candida antarctica acylates the 3-hydroxy group in steroids such as 163 and its
5,6-didehydro derivative[269].Interestingly, acylation in this position is preferred
regardless of the orientation of the hydroxy group. For instance, treatment of 164
with vinyl acetate in the presence of Candida antarctica lipase leads to the formation
of corresponding 3-acetylated derivative in 82 % yield. In contrast, subtilisin does not
recognize the hydroxy group at C3 of the steroid nucleus, but rather transfers the acyl
moiety to alcoholic groups in the 17-position or in the side chains (Fig. 18-23).
Changes in the A- or in the B-ring do not dramatically influence the selective mode
of action of this biocatalyst. This behavior is the same as that determined for the
lipase of Pseudomonas cepacia, which was recently used for the regio- and ster-
eoselective acylation of steroids r2’O]. Thus, using these enzymes, the completely
regioselective protection of either alcoholic group in several steroid diols is possible.
This feature opened a route to a new chemoenzymatic process for the oxidation of
selected positions of the steroid framework via an enzymatic protection/oxidation/
deprotection sequence. Chemoenzymatic approaches of this type are expected to
provide attractive alternatives to the currently utilized enzymatic oxidation of
steroids by hydroxysteroid dehydrogenases.
A further biocatalyst comes into play when bile acids serve as starting materials,
e. g. deoxycholic acid methyl ester 165[271].The cis-configuration of the A/B-ring
fusion prevents the application of lipase from Chromobacteriurn viscosurn (CVL) and
the aliphatic chain hinders the esterification of the C12a hydroxy group by subtilisin.
The lipase from Candida cylindracea (CCL) has proved to be the most suitable
enzyme for the enzymatic acylation of bile acids. In hydrophobic solvents, i.e.
hexane, toluene, butyl ether, benzene, etc. (except acetone) and employing tri-
chloroethyl butanoate as the acyl donor, the 3a-0-butanoyldeoxycholic acid methyl
ester 166 is formed in 80% yield without any by-products, suggesting that the
enzyme is ineffective towards 12a-OH. In addition, the 7a-OH and the 7p-OH,
present in 167 and 168 are not esterified by the enzyme. In both cases, the
3-butanoate is also formed (Fig. 18-23).
I
1389
CCL, trichloroethyl butyrate, hydrophobic solvent

163 164 R = R' = R2 = OH, R3 = OH
CVL: 3-monoburyrate 83% 165R = R1 = RZ = H, R3 = OH
CAL: 3-monoburyrate
subtilisin: q7-monobutyrate 60% 166 R = But. R1= R2 = H, R3 = OH 80%
167 R = But. R1 = H. R2 = OH. R3 = H
168 R = But, R1 = OH, R2 = H, R3 = H
171
CCL: CCL:
169 R = 3a-OAc, R = 17P-OAc no reaction 3.1 7a-dihydroxyestradiol 60%
170 R = ~P-OAC, R " = 17p-OAc 3-hydroxy-I 7a-acetoxyestradiol 25%
-+ SP-OH, R = 17P-OAc 79%
172 R1=R3=OAc,R2=(0), R4=R5=H 175 R ~ R~=OAC.

= R~=(o)
173 R'=R5=OAc, R2=(0), R3=R4=H 176 R1=OAc, R2 =C(0)CH3, R3=H
174 R1=R2=R3=OAc,R4=R5=H
Figure 18-23. Selective enzymatic protection of steroids.
Saponification of steroid esters can also be steered with Candida cylindracea lipase
(CCL)[272. 2731. This process occurs in the presence of octanol in organic solvents and
is characterized by a pronounced stereospecificity and regioselectivity. Thus, the 3a-
1390
I 18 Introduction and Removal ofprotecting Croups
esters of 3a,17P-diacetoxysteroid 169 resisted liberation, whereas the 3g-isomer 170

is transformed into the corresponding alcohol in 79% yield. The 17a-acetate of
3,17a-diacetoxyestradioll71 is also saponified, but at a slower rate than the 3-acetate
(Fig. 18-23). In the case of the androstane derivatives 172 and 175 different
selectivities of Candida antarctica lipase (CAL) and CCL were Thus, the
alcoholysis of 172 in the presence of CAL afforded the C3 deprotected product in
75% yield whereas CCL led to the removal of the acetate at C1G in 66% yield.
Treatment of 173,174 and 176 with CCL led to the cleavage of the C3 acetate in 79%,
87% and 83 % yield, 2741.
18.5.8
Phenolic Hydroxy Groups
Polyphenolic compounds occur widely distributed in nature and may possess a

variety of interesting biological properties, e. g. antibiotic, antiviral and antitumor
activity. The synthesis and further elaboration of these compounds often requires the
selective protection or deprotection of specific phenolic hydroxy groups. To achieve
this goal, the methods highlighted above for the various aliphatic polyols can also be
applied successfully.
For example, for the the enzyme-catalyzed acetylation of phenols six different
lipases was initially screened for activity[275. 2761. Out of these, only the lipase from
Chromobacterium viscosum (CVL) showed significant activity. In a subsequent study,
the lipase from Pseudomonas cepacia (PSL) turned out to be a more efficient
biocatalyst, which was succesfully used for the regioselective acylation of various
aromatic dihydroxycarbonyl compounds [2771, and (+)-catechin[2781. Thus, by using
PSL as the biocatalyst the dihydroxy aldehydes and ketones 177, 178 and related
compounds were selectively acetylated in conversions ranging from 20 to 97 % using
vinyl acetate as the acyl donor (Fig. 18-24)[2771. (+)-Catechin179 was also subjected to
irreversible acyl transfer conditions. In this case, both the 5- and 7-monoacetates
were obtained in 40% and 32% yield, respectively[278]. Interestingly,the inability of
the lipase from Aspergillus niger to acylate aromatic hydroxy groups has consequently
been used for the selective acylation of primary aliphatic hydroxy functions in
molecules containing both aromatic and aliphatic OH-groups [2791. In fact, even PSL
preferentially acylates primary aliphatic hydroxy groups if they are present in the
compound [280].
In the deprotection of peracetylated polyphenolic compounds a somewhat differ-
ent scheme has emerged. In this area, a broader spectrum of lipases has been used
successfully. For example, the pentaacetyl derivative of catechine 179 was treated
with PSL under alcoholysis conditions (THF, n-butanol) to give the 3,3',4'-trisacetate
in 50% yield after 12 On longer exposure to the biocatalyst, the
3-monoacetylderivative was isolated in 95 % yield.
Thus, the coumarine 180, the chromanone 181, the chalcone 182, the flavanone
183 as well as several flavones, e.g. 183 and 185 were regioselectivelydeacylated by
employing different lipases in organic solvents (Fig. 18-24). Porcine pancreatic lipase
(PPL) predominantly attacks one of the phenolic acetates present in 180-183 with
I
1391
0
&R OH
HO ' OH OH
177a R=H (78 Yo) 178a R=H (20%) OH
177b R=CH3 (97 Yo) 17Bb R=CH3 179 (+)-catechin
wo
(20 %)
1 7 7 RzCHZCH3
~ (93 Yo) 178C R=CH,CH3 (22%)
(only conversion given) (only conversion given)
AcO
\ \
AcO
0
CH3
180 PPL, 65% 181 PPL, 73% 181 PPL,50%
AcO 0
183 PPL,55% 184 PCL,55%
185 PPL, 78% 186 PCL, 95% 187 PPL, 75%

Figure 18-24. Selective enzymatic protection and deprotection o f polyphenolic
compounds.
good to high regioselectivity and produces the respective selectively protected

compounds available in good yields [281-2831 . The flavone acetates 184 and 186 can be
partially deacylated with high regioselectivityby transesterification using lipase from
Pseudomonas cepacia (PSL) and n-butanol in THF. [284s2851 However, in other cases the
positional specificity displayed by the enzyme was less pronounced. This technique
has allowed for an efficient construction of a selectively 0-methylated flavonoid [2841.
In addition, aryl alkyl ketones which are important starting materials for the
synthesis of polyphenolic natural products may be manipulated selectively by
making use of an enzymatic saponification[283,285-2871 . 1n general, in these cases the
sterically better accessible ester groups are cleaved, as for instance in 185[2851.All of
these examples have in common the fact that a carbonyl group is either directly or
vinylogously attached to the aryl moiety. Without such a function present in the
1392
I 78 introduction and Removal of Protecting Groups
molecule, the biocatalysts failed to differentiate the ester groups or completely

deacylated the substrates. However, by using the lipases from porcine pancreas
(PPL)or Candida cylindracea (CCL)immobilized on microemulsion-basedgels it was
possible to monodeacylate resorcinol and related diesters such as 187 in high
yields [lSs1.Alternatively, by using tert-butyl methyl ether saturated with water as the
solvent, it was possible to monodeacetylate diacetoxynaphthalenes selectively~288~.
The influence of the solvent was exemplified by charging the solvent system to
acetone/buffer: under such conditions only completely deacylated products were
obtained.
18.6
Biocatalysis in Polymer Supported Synthesis: Enzyme-labile Linker Groups
Combination chemistry and parallel synthesis of compound libraries on polymeric

supports are efficient methods for the generation of new substances with a
predetermined profile of propertie~[~*~-~’~1. The anchoring of one reactant to a
polymeric support has the advantage that an excess of reagent may be used, while
purification is kept manageable. This is particularly important if the reaction is to be
carried out with several reactants in the same reaction vessel. Solid phase synthesis
involves the use of linkers between the compounds to be varied combinationallyand
the solid supports which are stable during the reactions. These linkers have to be
cleavable as desired, usually at the end of the synthetic sequence, with high
selectivity and in good yield, without affecting the structure(s) of the product(s) that
are released from the polymeric supports.
Linkers have previously usually been cleaved by classical chemical methods, for
instance using strong acids. Such conditions often restrict the application of the
linkers, i. e. acid-sensitivelinkers are not suitable for acid-labile compounds, such as
carbohydrates. Specific linkers have therefore been developed for acid-labile com-
pounds, such as silylether linkages, thioether linkages [2921, and ester linkages [2931.
Although such linkers may be cleaved in the presence of acid-labile groups, they
have the disadvantage that they are themselves quite labile to common chemical
reagents that one might want to employ on the solid phase. For example, esters and
silylethers are unstable to bases and thioethers are unstable in the presence of
oxidants, such as rn-chloroperbenzoic acid, and to electrophilic reagents, such as
alkylating agents.
In principle linker groups are polymer-enlarged versions of blocking functions
used in regular solution phase chemistry. Therefore, enzymatic transformations that
may be employed for the removal of protecting groups in solution in principle may
also open up alternative opportunities for releasing compounds from polymeric
supports. The linkers developed so far can be divided into exo- and endo-linkers
(Fig. 18-25) cleavable by exo- endo-enzymes, respectively, as proposed by Flitsch et
al. [2941.
Exo-linkers are composed of three units: (i)a group providing the site for enzyme
catalyzed hydrolysis (R1); (ii) a site for attachment of the target molecule (R3); and
(iii) a site for attachment to a further optional spacer (R2).
18.6 Biocatalysis in Polymer Supported Synthesis: Enzyme-labile Linker Groups
I 1393
Exo-linkersd e a d by exo-enzymes Enddinkers cleaved by endhemymes
R’: group providingthe site for enzyme catalyzed hydrolysis,

R‘: optional intermediatelinked to a solid support,
R3: residue to be synthesized and varied in the course of a
synthesis on the support,
X: 0, N(H). N(R”). C(O)O, S, C(O)N(H) or C(O)N(R),
R” is a noninterfering substituent. Y: 0 or NH.
Figure 18-25. Graphical representation of exo- and endo-linkers.
Endo-linkers are linkers in which the target molecule (R3), the group, which
provides a site for enzyme catalyzed hydrolysis (R’) and a further optional spacer (R’)
are attached to the polymeric support in a linear arrangement. By means of enzyme
mediated dissection, the target molecule, in many cases tagged with the functional
group recognized by the enzyme, is released.
Examples of endo-cleavablelinkers have been reported (Table 18-5).However, in
many cases the product is tagged with part of the linker. For instance, the endo-
peptidase chymotrypsin cleaves endo-linkers towards the middle of a peptide-chain
or “internally”.Not only does this limit the methodology to a very small number of
enzymes, but it may also restrict the structure of molecules that can be generated.
For instance, this method will typically (but not necessarily, see Figs. 18-26 and
18-27)generate compounds containing C-terminal aromatic amino acids, which are
necessary for recognition by chymotrypsin. By contrast, exo-linkers do not restrict
the structure of the reactant and can be cleaved by more readily available exo-
enzymes, which act at the end of a chain or “externally”(Table 18-5). Furthermore
exo-cleavable linkers yield untagged products upon cleavage from the solid support.
18.6.1
Endo-linkers
For a better overview, examples of endo-linkers and the enzymes used for the
cleavage of the product from the solid phase which have been described in the
literature so far are given in Table 18-5.
Wong and coworkers[’”I introduced a silica-based solid support with a specific
enzymatically cleavable linker for the synthesis of glycopeptides and oligosacchar-
ides. They found that styrene- and sugar-based polymers tend to swell which leads to
a low coupling yield. Their choice of solid support is aminopropyl silica based on the
1394
Table 18-5. Examples o f endo-linkers and the appropriate cleavage enzymes.

Linker Enzyme Examples Ref.
a-Chymotrypsin Oligosaccharide [297-2981

synthesis
Ceramide glycanase Oligosaccharide [299]

synthesis
R O
oAo;,oa Phosphodiesterase Peptide synthesis [301]

0 HO 0
facts that: (a) it is compatible with both aqueous and organic solvents, (b) it has a
large surface area accessible to biomolecules, and (c) it has sufficient density of
functional groups.
A hexaglycine spacer was attached to the solid support to give a substitution of
0.2 mmol g-' of dry silica and the excess amino groups were then capped using
acetic anhydride. In the next step a selectively cleavable, a-chymotrypsin sensitive,
phenylalanine ester 189 was implemented for the release of the products from the
solid support under mild conditions. Then it was transformed to 190 followed by
reactions with glycosyl transferases to yield 191. Finally, the desired glycopeptidewas
cleaved from the solid support in high yield by treatment of 191 with a-chymotrypsin
(Fig. 18-26).
Nishimura and coworkers1296-2971 described a novel method for the enzymatic
synthesis of oligosaccharide derivatives employing an a-chymotrypsin sensitive
linker. The synthesis of the water soluble GlcNAc-polymer 197, sensitive to a-
chymotrypsin, is shown in Fig. 18-27. Oxazoline derivative 193 was coupled with
6-(N-benzyloxycarbonyl-~-phenylalanyl)-amino-hexanol-l (194) followed by N-depro-
tection of the phenylalanine and subsequent condensation with 6-acrylamidocaproic
acid 195. De-0-acetylationgave the polymerizable GlcNAc derivative 196. Finally, co-
polymerization of acrylamide and monomer 196 in the presence of ammoniumper-
sulphate (APS) and N,N,N,N-tetramethyl ethylene diamine (TMEDA) gave the
( 1 ) 25% TFA (CH2C12)
(2) Boc-Gly-OH ((7 eq),
BOPIHOBt,DIEA
(3) 25% TFA (CH2C12)
(4) Boc-Asn(G1cNAcp)-OH
0 -
190
glactosyl transferase,
sialyl transferase
PH
(1) a-chymotrypsin, H20. pH 7.0
(3) a-l.3-fucosyltransferase,
. GDP-Fuc (2.5 eq),

0.1 M HEPES (pH 7.0)
!I
0
192
1396
Figure 18-27. Oligosaccharide

synthesis and a-chymotrypsin
catalyzed release from the solid
support.
(1) Z-Phe-NH-(CH2)6-OH(194)
CSA, (CHCI&, 70" C
(2) H, Pd/C, MeOH. 50" C
(3) CH2=CHCONH(CH,),COOH (195)
EtOH-C,H,
(4) MeONa (cat.), MeOH/THF
CH,=CHCONH,
TMEDA. APS
Ig7 X : Y = 1 : 4
Galactosyl transferase
Sialyl transferase
OH
/- :v0 (cH
HZN
2)5N3:
198 C6H5
I a-chyrnotrypsin
Tris-HCI buffer
X:Y=1:4
PH 7 . a , 4 v c
199
I 1397
A;i&+Co.o+
OAc
I
-N
H
OAc
NHCOOCHpC6H,
(1) Pd/C, MeOH
(2) 195, EEDQ,
EtOH-C&
(3) MeONa
nu OAc
bH
OH
0
0
4
CH*=CHCONH2
TMEDA. APS
DMSO-H20,50° C
,
HO
OH
OH
,,
202 0
X:Y=1:5
1
CMP-NeuAc
a -2,3-sialyltransferase
BSA, MnCI2,ClAP
50 nM sodium cacodylate
.OH buffer, pH 7.49
HO bH
OH
1
ceramide H N K0( C H 2 ) 5 ' 0
ceramide glycanase X:Y=1 :5
Triton CF-54
sodium citrate buffer
pH 6.0, 37" C
(GM3) 204
Figure 18-28. Ceramide glycanase mediated release by transglycosylation.
1398
I 18 Introduction and Removal ofProtecting Croups
polymer 197 in high yield. The polymer 197 was then subjected to galactosylation
and subsequent sialylation with the corresponding glycosyl transferases to yield 198.
The final product 199 was cleaved from the water-soluble support by treatment with
a-chymotrypsin at 40 "C for 24 h in 72 % overall yield from 197.
Nishimura and Yamada [29s1introduced a water-solublepolymeric support having
a linker recognized by ceramide glycanase for a synthesis of ganglioside GM3 (204).
Synthesis of the polymerizable lactose derivative 201 with a ceramide glycanase
sensitive linker is shown in Fig. 18-28. The lactosyl ceramide (LacCer) mimetic
glycopolymer 202 is obtained from the monomeric precursor 201 by co-polymeriza-
tion with acrylamide.
This solid support 202 was converted into the intermediate product 203 by
sialylation using PGa1l-t 3/4GlcNAc a-2,3-sialyltransferase.Finally, the polymeric
support was cleaved by transglycosylation with leech ceramide glycanase in the
presence of excess ceramide as the acceptor to give the desired product 204 in high
yield (Fig. 18-28).An advantage of the water-solublepolymer is that the transfonna-
tion can be monitored by NMR spectroscopy during the enzymatic glycosylation
steps.
Arrays of up to 1000 peptide nucleic acid (PNA) oligomers of different sequence
were synthesized by Jensen et al. on polymer membranes (Fig. 18-29)[2991. The PNA
chain was linked to the peptide spacer glutamic acid-(y-tee-butylester)-(c-aminohex-
anoic acid)-(c-aminohexanoic acid) (Glu[OtBu]-~Ahx-cAhx) via an enzymatically
cleavable Glu-Lys handle. The Glu[OtBu]-~Ahx-~Ahx spacer was coupled to the
amino-functionalized membrane by standard Fmoc-Chemistry. Then the mem-
branes were mounted in an ASP 222 Automated SPOT Robot and a grid o€ the
desired format was dispensed at each position. The free amino groups outside the
spotted areas were capped and further chain elongation was performed with Fmoc-
protected PNA monomers to synthesize the desired PNA oligomers. After comple-
tion of the synthesis, the PNA oligomers were cleaved from the solid support by
incubation with bovine trypsin solution in ammonium bicarbonate at 37 "C for 3 h.
One of the very first papers concerning endo-linkers was published by Elmore et
al. (Fig. 18-30)[3001. They described a new linker containing a phosphodiester group
for solid phase peptide synthesis using a Pepsyn K (polyacrylamide) resin. After
completion of coupling and deprotection cycles, the phosphodiester 207 was cleaved
with a phosphodiesterase. In this way p-casomorphin, Leu-enkephalin and a col-
*05 AOOH \ trypsin

Figure 18-29. Trypsin mediated cleavage of a peptide bond in PNA oligomer synthesis.
78.G Biocatalysis in Polymer Supported Synthesis: Enzyme-labile Linker Groups
I 1399
206 I Synthesis via symmetrical anhydrides

of N-Fmoc amino acids
Fmoc-Ala-Pro-Gly-Leu-Ala-Gly-0
T L O \ II C O A N
H /qzbHN-@
0
*07 1
1
Phosphodiesterase,pH = 5.7,7d (83%)
Fmoc-Ala-Pro-Gly-Leu-Ala-Gly-OH
208
Figure 18-30. Synthesis of a collagenase substrate on a phosphodiesterase-scissile linker.
lagenase substrate were synthesised in high yields. In the context of enzymatic

cleavage of linkers on polymeric supports particular attention was paid to the general
question of whether enzymatic transformations on resins are viable and high
yielding. An in-depth treatment of this problem is beyond the scope of this review.
However, a few examples for the application of biocatalyzed transformations on solid
supports will serve to illustrate that such transformations can indeed be employed
advantageously for various purposes.
Meldal et al. described the proteolytic cleavage of the alanine-tyrosine bond in a
resin-bound decapeptide by treatment with the 27 kDa protease subtilisin BNP’ to
demonstrate the accessibility of the interior of the newly designed S P 0 C C - r e ~ i n [ ~ ~ ~ ]
to enzymes L3021.
Furthermore, enzymatic hydrolysis of model isopeptides F-oligo(L-methiony1)-L-
lysine from Bio-beads L3O3] by pepsin, chymotrypsin, cathepsin C (dipeptidyl pepti-
dase IV) and intestinal aminopeptidase N was investigated using high-performance
liquid chromatography to identify and quantify the hydrolysis products L3O4].
Larsen et al. reported the enzymatic cleavage of a desB30 insulin B-chain from a
presequence (LyS(B0C))a. This spacer shifts the conformation of the growing peptide
chain from a p-structure to a random coil conformation and reduces peptide-chain
aggregation, which otherwise causes serious synthetic problems. Novasyn KA-
[3051 was employed as a solid support, but unfortunately, no information about the
enzyme used was reported L3O61.
Barany et al. were the first to exploit the different enzyme accessibilities of surface
and interior areas of a given bead and the resulting differentiated bead was used to
synthesize a peptide library on the surface and the code for this on the interior
simultaneously[307]. This clever strategy is illustrated in Fig. 18-31. Selective cleavage
of short Na-protectedpeptide substrates with chymotrypsin from the surface area of
1400
I 78 introduction and Removal ofProtecting Groups
*<-e
Cleavage of the peptide
r P’
bonds on the surface
209 210
1
1. Boc-cleavage
2.Coupling of Boc-C
Attachment
of Fmoc -S
1
Fm0c-S- ‘
FITDGS-P ’ :* --
212 211
I.Fmoc- and
Boc-cIeavage
2. Receptor
sereeninu
Bead Selection
a
Hits
a Code Analysis
Structure
Figure 18-31. Peptide encoded combinatorial peptide libraries via enzyme-mediated spatial
segregation. P-P’: substrate with a scissile bond between P and P’; S : terminal residue o f t h e
screening structure, C: terminal residue o f t h e coding structure.
TentaGel-AM-beads209 leaves the majority of the peptide attachment sites in the

interior uncleaved to afford 210 (“shaving”methology).The first residue is attached
using orthogonal FMOC-chemistryto provide 211. Coding is done by using standard
BOC-chemistry on the interior of the bead to yield 212. Repetition of this process
furnishes a surface peptide, which is encoded internally (213).
This generation of two structures on the same bead allowed the investigation of
the synthesized peptide library (1x lo5 members) with different receptors (anti-p-
endorphin antibody, streptavidin and thrombin). After the staining procedure had
been carried out, the beads that showed a color were selected for sequencing and the
coding peptides present within the bead were used to deduce the binding structures.
This screening led to the discovery of a new thrombin ligand, which binds with an
affinity one order of magnitude higher than the natural motif.
I 1401
OH
1. P-Galactosidase
2. Precipitation
+
HO
3. Filtration
214 OH OH
NP = o-Nitrophenyl , . o*O
H HO
OH
215
HO Lo, I
- GIU
OH
*+&I
HO
H o & - o . ,
OH OH 216b
OH
OH 217b
i
OH OH
HO Q
+&.
+ OHHOHO HO
Cleavage of the linker 218a OH
+&/+&I
____)
HO HO
OH
OH 218b
Figure 18-32. General strategy for the liquid-phase synthesis o f disaccharides using glycosidases.
1402
I 18 fntroduction and Removal ofProtecting Groups
Fernandez-Mayoralas and coworkersL3081 used the high substrate specifity of

enzymes in their synthesis of galactose-glucose-disaccharides(218) on an MPEG-
support[3o9].After galactosylation of glucose immobilized on the soluble support
(215)using B-galactosidase,the unreacted monosaccharide glucose was removed by
the combined use of a- and j3-glucosidases to obtain only MPEG-bound disaccha-
rides (216, Fig. 18-32).Finally the disaccharides 218 obtained were released from the
support by ethanolysis.
Schmitz and Reetz described the solid phase enzymatic synthesis of oligonucleo-
tides on Kieselguhr-PDMA-resinsvia T4 RNA ligase. Concomitantly,they found that
RNase A selectively cleaves the last bound nucleotide at the ribose sugar leaving a
3 ' 5 ' - diphosphorylated oligomer behind on the resin, but application in actual
synthesis has not yet been undertaken[31o].
18.6.2
Exo-linkers
An exo-linkeraccordingto Fig. 18-25must contain an enzyme labile group R1, which

is recognized and attacked by the biocatalyst. Possible combinations could be:
phenylacetamidelpenicillin amidase, ester/esterase, monosaccharide/glycosidase,
Table 18-6. Examples o f exo-linkers and the appropriate cleavage enzymes.

Linker Enzyme Examples Ref.
Lipase Picet-Spengler reaction, [313]

nucleoside immobili-
R X y O G Z m zation
0 0
Penicillin acylase Palladium cat. [318, 3191

RX C-C-couplings, Mit-
H
sunobu- and Diels-Alder
reactions, 1,3-dipolar
0 cycloadditions
Penicillin acylase Immobilization of [312, 3131

alcohols (e. g. Fmoc
protected serine methyl
ester, glycosides) and
0 amines (e.g. phenyl-
alanine)
18.6 6ioc:atalysis in Polymer Supported Synthesis: Enzyme-labile Linker Groups
phosphate/phosphatase, su.lfate/sulfatase and peptides/peptidases L3l1].Up till now

I 1403
only the following systems have been worked out (Table 18-6).
In independent and simultaneous investigations Flitsch and coworkers
r311, 3121 and Waldmann and coworkers[313, 314] developed a selectively cleavable exo-
linker, which can be cleaved with penicillin G acylase, a commercially available and
widely used enzyme[77].
Penicillin acylase catalyzes the hydrolysis of phenylacetamides and has been used
in peptide synthesis for the cleavage of protecting 3151. In linker 219
~ ~(Fig.
developed by Flitsch and c ~ w o r k e r s [3121 * ~ 18-33) -XR represents the alcohol or
amine group of the target molecule. Hydrolysis of the phenylacetamide moiety
generates the hemiaminal221 which readily fragments in an aqueous medium and
thereby releases the desired products, RXH. The thioethyl group present in the
anchor group of 219 was activated by treatment with N-iodosuccinimide (NIS)
followed by displacement with a variety of alcohols (223-225). To prove the possible
application of this linker i n solid phase carbohydrate synthesis protected glycosides
226 and 227 were coupled to linker 219 and released enzymatically. Flitsch et al. also
described the immobilization and enzymatic cleavage on a variety of a r n i n e ~ [ ~ l ~ ] .
Nevertheless, the application of this enzyme-labile linker group in multi-step
syntheses on the solid phase and subsequent enzyme-initiated release from the
polymeric support has not been described yet.
Waldmann and coworkers described designed exo-linker 228 r313, 3141 . Theanchor
group comprises a 4-acy:loxy-3-carbo~benzyloxy group, which is recognized and
attacked by the biocatalyst, so that a spontaneously fragmenting intermediate is
generated, thereby releasing the desired compound (Fig. 18-34)[53, 54, 571. The linker
228 is attached as an amide to the solid phase. Cleavage of the acyl group by a lipase
generated a phenolate 229, which fragments to give a quinone methide 230 and
releases the product 231. The quinone methide remains on the solid phase and is
trapped by water or an additional nucleophile.
Following on from this cleavage principle, amines (bound as urethanes), alcohols
(bound as carbonates), arid carboxylic acids (bound as esters) can be detached from
the polymeric carrier. The substrate specificity of the enzyme guarantees that only
the intended ester is cleaved. TentaGelS-NH2was chosen as the polymeric support,
i. e. a polystyrene resin equipped with terminally NH2-functionalized oligoethylene-
glycol units. It has a polar surface and swells in aqueous solutions allowing the
biocatalyst access to the polymer
The applicability of the enzyme-labile anchor group was demonstrated by the
synthesis of tetrahydro.+carbolins 237 employing the Pictet-Spengler reaction
(Figure 18-35).The benzylic alcohol group of the linker 232 was first esterified with
Boc-L-tryptophan, and after its N-terminal deprotection the support-bound trypto-
phan 233 was reacted with aliphatic and aromatic aldehydes to give imines 234,
which cyclized immediately in reasonable to high yields to the tetrahydro-0-carbolins
235. Lipase RB 001-05 :selectively attacked the acetate incorporated into the linker
and generated the corresponding phenolate 236, which then fragmented sponta-
neously. Following these multistep transformations the desired tetrahydro-p-carbo-
lins 237 were obtained in 70-80 % yield.
1404
I 18 Introduction and Removal ofprotecting Groups
Penicillin acylase
I f+:-;b 1
222
FrnocHN
223
225
Figure 18-33. Loading and cleavage of a penicillin acylase scissile linker.

0
I 1405
Lipase -RC02H
* N H y 2 3 0 HO 1 X-R
X = NH: amines (-COP)
X = CR2:
0: alcohols
carboxylic acids
(-COP)
231
Nu e
Figure 18-34. Principle for the development of the enzyme-labile
4-acyloxy-benzyloxy linker group.
Waldmann and coworklers developed a second exo-linker following a new ap-

proach[317,3181 which makes use of a safety-catchlinker. It is based on the enzymatic
cleavage of a functional group embodied in the linker. In this way an intermediate is
generated, which subsequently cyclizes intramolecularly according to the principle
of assisted removal L2, 319-3221 and thereby releases the desired target compounds
(Fig. 18-36). The linker group is immobilized as a urethane on the amino-function-
alized carrier 238. It facilit,atesthe attachment of a variety of molecules such as alkyl
halides, alcohols or amines bound as carboxylic acid esters and amides. According to
the safety-catchprinciple, the separation of the desired products proceeds in a two-
step process. First, penicillin G acylase hydrolyzes the phenylacetamide with
complete chemo- and regioselectivity, under exceptionally mild conditions (pH 7.0,
room temperature or 37 "C) [30, 72*741 . Then the activated intermediate generated,
1406 I 18 introduction and Removal of Protecting Groups
1) Boc-trypothan
DIC, DMAP
2) CF3COOH, r.t.
68
232 \OH
233
R-CHO, molecular sieves,

CHZC12, 50°C
OAc
1
vow 234
'7
R
H
N
I
lipase RB 001-05,
R = Ph, 4-N02C6H4,i-Pr
50 mM MES buffer/ CH30H
55-85 %
(60/40), pH 5.8, 30°C
p6::--]
0
0
237 R
70-80 %
Figure 18-35. Solid phase synthesis of tetrahydro-6-carbolins and subsequent detachment by

enzyme initiated fragmentation of the anchor group.
i. e. benzylamine 239, cyclizes to polymer-bound lactam 240 and releases the desired
target molecule 241.
POE 6000 was used as the polymeric support, a soluble polyethyleneglycol
derivative functionalized at both termini with an amino group and with an average
molecular mass of 6000 Da [323-3241. After completion of the homogeneous reactions
I 1407
X=O,NH,NR
- 1
pmH Penicillin Gacylase
239
Figure 18-36. Principle of the enzyme-labile safety catch linker.
it can be precipitated, filtered off, and washed with diethyl ether, thereby facilitating
the separation of surplus reagents and the side products. Furthermore it allows for
NMR spectroscopic monitoring of the reactions [3251. Most importantly, it is soluble
in aqueous solutions, thereby allowing efficient access of the enzyme to the polymer-
fixed linker group.
The suitability of the polymer-linker conjugate was examined for a variety of
transformations, in particular Pdo-catalyzedreactions. For instance, the polymer-
bound aryl iodide 242 was transformed quantitatively in a Heck reaction to a
cinnamic acid ester 243 and to biphenyl 245 in a Suzuki reaction. It gave an alkine
244 in a Sonogashirareaction (Fig. 18-37).The desired benzyl alcohols 246248 were
released by incubation of the corresponding polymer conjugates 243-245 with
penicillin G acylase at pH 7 and 37 "C in high yields and isolated with a purity of
>95 % by simple extraction with diethyl ether.
Furthermore, the applicability in a Mitsunobu esterification reaction and a Diels-
Alder reaction was proven (Fig. 18-38).The polymer-bound benzyl alcohol 249 was
1408
I 78 Introduction and Removal ofProtecting Groups
242
OMe
I
245
Penicillin G acylase Penicillin G acylase

pH 7.0, 10 % MeOH, pH 7.0, 10 % MeOH,
/ / /
\
OtBU
246 247
Figure 18-37. Pdo-catalyzed reactions on enzyme labile linker-conjugates.
reacted with 4-acetamidophenol in the presence of the Mitsunobu reagent to give

phenyl ether 250 in quantitative yield. It was released from the polymeric support in
high yield. For the Diels-Alder reaction, polymer-bound acrylic acid ester 252 was
treated with cyclopentadiene. The cycloaddition product 253 was formed with an
endolexo ratio of 2.5 : 1 and with quantitative conversion. The subsequent enzy-
matic release delivered the corresponding alcohol (251,254) in high yield and
purity.
18.7
Outlook
During recent decades substantial progress was achieved in the development of

enzymatic protecting group techniques. In particular, it was demonstrated that these
methods offer viable alternatives to classical chemical approaches. Not only do the
biocatalyzed transformations complement the arsenal of chemically removable
protecting groups, but in many cases they additionally offer the opportunity to carry
out useful functional group interconversions with selectivities which can not or only
barely be matched by chemical techniques. However, the overwhelming majority of
the investigations carried out in this area has restricted themselves to the study of the
protection and deprotection of model compounds. Complex synthetic schemes were
nearly always avoided. Whereas this appears to be particularly true in the area of
carbohydrates, noticeable examples which demonstrate the capacity of these bio-
catalyzed processes were recorded in peptide and peptide conjugate chemistry, i. e. in
References I1409
249
0
H
DEAD, PPh3
HO nNr , THF, 60°C
I
250
penicillin G acylase, penicillin G acylase,

pH 7.0,37"C, 48h (81 %) pH 7.0,37"C, 48h (75 %)
+ t
Nr
H
I HOo-
0
H o a o
251 D 254
Figure 18-38. Mitsunobu and Diels-Alder reaction on enzyme labile linker-conjugates.
the synthesis of lipo-, glyco and nucleopeptides. The data and observations high-
lighted above, however, provide a solid basis for the application of biocatalysts in the
handling of protecting group problems in complex multistep syntheses.
On the other hand, the use of biocatalystsin protecting group chemistry in the sense
of a general method deserves and is certainly awaiting further intensive develop-
ment. Numerous applications of the known enzymes appear to be possible in all
areas of preparative chemistry. In addition, the use of catalpc proteins which have
not yet been applied to carry out protecting group manipulations and of biocatalysts
unknown today or which will be developed in the future, e.g. by evolutionary
approaches, will create new opportunities for improved organic syntheses.
References
1 H. Kunz, H. Waldmann in: (Ecls.: B. M. 3 P. J. Kocienski, Protecting Groups,

Trost, I. Flemming, E. Winterfeldt), Com- Georg Thieme Verlag, Stuttgart,
prehensive Organic Synthesis Vol. 6, Perga- 1994.
mon Press, Oxford 1991, pp. 6.31-701. 4 H. Waldmann, Kontakte (Darmstadt) 1991,
2 T. W. Greene, P. G. M. Wuts, Protective 2, 33-54.
Groups in Organic Synthesis, Wiley and 5 A. Reidel, H. Waldmann, J . Prakt. Chem.
Sons, New York, 1999. 1993,335,109.
1410 78 Introduction and Removal of Protecting Groups
I
6 H. Waldmann, D. Sebastian, Chem. Rev. 26 E. Wiinsch, L. Moroder, W. Gohring, P.
1994,94,911-937. Thamm, R. Scharf, Hoppe-Seyler’s 2. Physiol.
7 T. Kappes, H. Waldmann, Liebigs Ann. Chem. 1981,362,1285-1287.
Chem. 1997,803-813. 27 Q.-C. Wang, J. Fei, D.-F. Cui, S.-G. Zhu, L.-
8 T. Pathak, H. Waldmann, Curr.Opin. Chem. G. Xu, Biopolymers 1986, 25, 109-114.
Bid. 1998, 2, 112-120. 28 C. Fuganti, P. Grasselli, P. Casati, Tetra-
9 B. Sauerbrei, T. Kappes, H. Waldmann, Top. hedron Lett. 198627,3191-3194.
Cur. Chem. 1997,186,65-86. 29 H. Waldmann, Tetrahedron Lett. 1988,29,
10 D. Kadereit, J.Kuhlmann, H. Waldmann, 1131-1134.
ChemBioChem 2000, I , 144169. 30 H. Waldmann, Liebigs Ann. Chem. 1988,
11 D. Kadereit, H. Waldmann, Monatsh. Chem. 1175-1180.
2000,131,571-584. 31 H. Waldmann, P. Braun, H. Kunz, Biomed.
12 J. D.Glass in: The Peptides (Eds.: S. Uden- Biochim. Acta 1991,50,243-248.
fried, J. Meienhofer) Academic Press, San 32 H. Waldmann, A. Heuser, P. Braun, M.
Diego, 1987, pp. 167-184. Schulz, H. Kunz in: Microbial Reagents i n
13 E. Wiinsch, Methoden Org. Chem. (Houben- Organic Synthesis (Ed.: S. Servi), Kluwer,
Weyl), Vol. xv/r and rr, Thieme, Stuttgart, Dordrecht, 1992, pp. 113-122.
1974. 33 P. Hermann, Wiss. 2. Univ. Halle 1987, 36,
14 M. Goodman, A. Felix, L. Moroder, C. To- 17-29.
niolo, Methods of Organic Chemistry (How 34 P. Hermann, Biomed. Biochim. Acta 1991,
ben-Weyl), Vol. E22a-c, Thieme, Stuttgart, 50,19-31.
2000. 35 G. Greiner, P. Hermann in: Peptides 1990
15 R. W. Holley,J. Am. Chem. Soc. 1955,77, (Eds.: E. Giralt, P. Malon), Escom Science
2552-2553. Publishers, Leiden, 1991, pp. 277-278.
16 F. Widmer, S. Bayne, G. Houen, B. A. Moss, 36 A.L. Margolin, V. K. Svedas, I. V. Berezin,
R. D. Rigby, R. G. Whittaker, J. T. Johansen Biochim. Biophys. Acta 1980, 616,283-289.
in: Peptides 1984 (Ed.: U. Ragnarsson) Alm- 37 R. J. Didziapetris, V. K. Svedas, Biomed. Bio-
quist Sr Wiksell, Stockholm, 1985, pp. chim. Acta 1991,50,237-242.
193-200. 38 R. Didziapetris, B. Drabnig, V. Schellenber-
17 J. D. Glass, C. Meyers, I. L. Schwartz, R. ger, H.-D. Jakubke, V. Svedas, FEBS Lett.
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I 1419
19
Replacing Chemical Steps by Biotransformations:
Industrial Application and Processes Using Biocatalysis
Andreas Liese
‘Bacteria are capable of briiaging about chemical reactions of amazing variety and sublety
in an extremely short time ... Many bacteria are ofvery great importance to industry where
they peform tasks which would take much time and trouble by ordinary chemical
methods.’
Sir Cyril Hinshelwood, 1956L11
19.1
Introduction
Starting with big promises and many expectations in the seventies biocatalytic
processes have left the status of a lab curiosity together with many prejudices far
behind and are now established on an industrial scale[2].Product examples range
from amino acids, sugars, chiral alcohols and amines, and highly functionalized
building blocks for pharmaceuticals to bulk chemicals such as acrylamide or
propane-1,3-diol. When speaking about biotechnological processes one has to
distinguish between fermentation processes and biotransformations. In a fermenta-
tion process the desired product is synthesized from nutrients and trace elements by
either microorganisms (bacteria, yeasts, fungi) or higher cells such as mammalian
or plant cells. The phrase “biotransforrnation”or “biocatalysis”is commonly used to
describe a one-step or multi-step transformation of a precursor to the desired
product using whole cells and/or (partly)purified enzymes. Whole cell processes are
often used for redox reactions using the metabolism of the living cell for cofactor
regeneration. In some cases the cell is used as a compartment containing the
enzymes in a confinement allowing easier separation of the entire biocatalyst using
centrifugation or microfiltration. If one has to deal with membrane-bound enzymes,
whole cell biocatalysts are to be preferred.
Numerous authors have given overviews over biotransformations used in in-
du~try[~-”]. A very recent monograph summarizes almost 100 processes including
many details on reaction conditions, screening of the biocatalyst or the product
application[2].The use of biocatalysis from the viewpoint of a chemist in the
laboratory is also summarized in several books. Recent ones are[12-14].
1420
I 7 9 Replacing Chemical Steps by Biotransformations
In this contribution we shall focus on those examples where the biocatalytic step
has distinct advantages over the corresponding chemical method, or even has
replaced or is about to replace other methods. The reasons may be better regio-,
stereo- or chemoselectivity,better product purity or simplified downstream process-
ing. Often the incorporation of biocatalytic steps reduces the amount or toxicity of
waste.
19.2
Types and Handling of Biocatalysts
This chapter tries to give a brief introduction to the types of biocatalysts, their
requirements and methods of handling them. A more detailed treatment can be
found in other chapters of this book or in the literature [15-171.
The biocatalyst may be a whole cell or a partly purified enzyme. In the first case the
cell may be regarded as a mini-reactor with all necessary cofactors and enzymes to
catalyze multiple steps concentrated in one cell. In the second case the main
catalytically active species is isolated and purified.
For the whole cell systems either prokaryotic cells such as Escherichia coli or
eukaryotic cells such as Saccharomyces cereuisiae or Zymomonas mobilis are used.
Prokaryotic cells do not posses a nucleus. The nuclear material is contained in the
cytoplasm of the cell. Therefore introduction and processing of foreign DNA to
obtain a genetically engineered strain is simple. They are relatively small in size
(0.2-10pm) and exist mostly as single cells. Eukaryotic cells are higher micro-
organisms and have a true nucleus separated by a nuclear membrane. They are
larger in size (5-30 pm) and sometimes form more complex structures. For both
types the bioreactor has to fulfill certain requirements. An adequate supply of
nutrients as well as oxygen into the bioreactor has to be assured. Parameters such as
pH, oxygen, feed rate and temperature in the bioreactor must be kept within certain
limits in order to guarantee optimum growth and/or metabolic activity of the cells.
Especially when recombinant microorganisms are employed genetic stability during
cultivation has to be observed carefully. Substrates, products and/or solvents re-
quired may be toxic for the cells and may therefore have to be added in low amounts
to secure a low stationary concentration. The example of a ketone reduction with
whole cells of Zygosaccharomyces rouxii shows one possible solution. The toxic
substrate is adsorbed on XAD-7 resin (80 g/L resin, resulting in a concentration of
40 g/L reaction volume), and the resin is added to the fermentation broth. The
equilibrium concentration in the aqueous phase is approximately 2 g/L. The product
is adsorbed on the resin as well, thus providing integrated downstream process-
ingw191.
When purified enzymes are used, basically the same requirements have to be met.
The purification may cause additional costs, but contrary to a biochemical character-
ization it is not necessary to purify the protein to homogeneity. On the contrary, the
remaining protein content in a partly purified enzyme may increase its stability. The
only requirement is to have a functional pure enzyme, meaning that activities
79.3 Examples I1421
catalyzing undesired side reactions have to be absent. This is the major advantage of
purified enzymes over whole cell processes: side reactions may be easily avoided, and
substrates that are toxic for the cell or which may not be able to enter the cell can be
converted. For enzymes the thermal deactivation or deactivation by interphases
(liquid-liquid,liquid-gas) may be limiting.
For industrial biotransformations, catalyst recovery and reuse are major issues.
This may be desirable either for reasons of downstream processing or for repeated
use in order to reduce the specific catalyst costs per kg of product produced. A very
simple method is the use o f membrane filtration. Because of the increasing number
of membranes from difft2rent materials (polymers, metal or ceramics) this is an
attractive alternative. Whereas for whole cells microfiltration or centrifugation can be
applied, for the recovery ‘of soluble enzymes ultrafiltration membranes have to be
Often immobilization on a support is chosen to increase the catalyst’s
stability as well as to facilitate its recovery. The main advantages of immobilization
are:
easy separation,
often increased stability,
use of fixed or fluidized bed reactors (for continuous processes).
Disadvantages are:
loss of absolute activity due to the immobilization process,
mass transport limitations,
There is no general or best method of immobilization; the protocol has to be
developed individually for each catalyst.
The most common methods for the immobilization are entrapment in matrices
such as alginate beads, cross-linking, and covalent or adsorptive binding to a carrier.
A very recent method is the development of cross-linked enzyme crystals (CLECS)
(see also Chapter 6 ) [231. A survey of different immobilization methods can be found
in in[24. 251
19.3
Examples
The examples presented here are taken from 12]. Only those biotransformations were
chosen where a classical chemical step was replaced. The enzymes involved are
mainly from the group:; of oxidoreductases (E. C. class 1) and hydrolases (E. C.
class 3). There are a few examples of lyases (E.C. class 4) and one example of an
isomerase (E. C. class 5). The processes involving oxidoreductases mainly use whole
cells because of the problem of cofactor regeneration. The examples are sorted in the
order of the main classes of the Enzyme Commission (E. C.). The big letter E denotes
the biotransformation in the syntheses schemes.
1422
I 19 Replacing Chemical Steps by Biotransformations
19.3.1
Reduction Reactions Catalyzed by Oxidoreductases (E. C. 1)
19.3.1.1
Ketone Reduction Using Whole Cells of Neurospora crassa (E.C. 1.1.1.1) [26-291
The key step in the synthesis of Trusopt@,which is a topically active treatment for
glaucoma, is the enantioselective reduction of S,G-dihydro-G-methyl-4H-thieno[2,3b]
thiopyran-4-one-7,7-dioxide (Fig. 19-1).
The biological route overcomes the problem of incomplete inversion of the cis-
alcohol in the chemical synthesis (Fig. 19-2).The reaction is carried out below pH S
to prevent epimerization of the (6s)-methylketosulfone in aqueous media.
The (R)-3-hydroxy-butyrate(Fig. 19-l),which is responsible for the stereochem-
istry of the methyl group in the sulfone ring, can be produced by depolymerization of
biopolymers, e. g. Biopol from Zeneca. This is a natural polyester produced by some
microorganisms as a storage compound.
1) pyridinel
twyl chloride CH30ZC
COOCH,
3) H202,NaWOT
LiS
1 2
1 = biologically derived homopolymer

2 = (Rp3-hydroxy-rnethylbutyrate
3 = 5,6-dihydro-6-methyl-4H-thieno(2,3b]thiopyran-4-one~7,7-dioxide
4 = 5,6-dihydro-4-hydroxy-6-methyl-4H-thieno[2,3b]thiopyran-7,7-dioxide
5 = trusopt, MK-0507
E = alcohol dehydrogenase,whole cells from Neurosporia crassa
Figure 19-1. Synthesis ofTrusopt@ (5) via enzymatic ketone reduction (Astra-Zeneca)
79.3 Examples I1423
noMe
- pyridinel
tosyl chlorie
OTS 0
uOMe
cH30Zca,) conc. HCI ~
NHCOCH NHCOCH VHCHZCH,
m s o z c i ~ ~ ~ s o z BH~DMS,
N TH z H ~
oAo S
,.”.., o4 “o
Figure 19-2. Chemical synthesis of Trusopt@.
19.3.1.2
Ketoester Reduction Using; Cell Extract of Acinetobacter calcoaceticus
(E.C. 1.1.1.1)[30-321
The biotransformation (Fig. 19-3)is an alternative to the chemical synthesis via the
chlorohydrine and selective hydrolysis of the acyloxy group (Fig. 19-4). After final
fractional distillation this synthesis has an overall yield of 41 %. The biotransforma-
tion has a yield of 92%. The diketoester can be obtained as shown in Fig. 19-5.
G-Benzyloxy-(3R,5S)-dihydroxy-hexanoic acid ethyl ester is a key chiral inter-
mediate for anticho1estei:ol drugs that act by inhibition of hydroxy methyl glutaryl
coenzyme A (HMG CoAl reductase.
1 (3R,5s)-2
1 = 6-benzyloxy-3,5-dioxo-hexanoic;mid ethyl ester

2 = 6-benzyloxy-3,5-dihydroxy-hexar1oic
acid ethyl ester
E = alcohol dehydrogenasefrom Ac,inetobactercalcoaceticus
Figure 19-3. Synthesis of key intermediate of anticholesterol drugs (BristoCMyers Squibb)

1424
OH 0
CH&OO'BU ~
LiHMDS/ - 78 "C
cluok 1. Et,EOMe/NaBH,
THFiMeOH/78 "C
2. H,OflaOH
I /
("Eu),NOCOMe
___)
CSA NMP/ 85 "C/ 9 h
Figure 19-4. Chemical synthesis of key intermediate of anticholesterol drugs.
a o J c , -pyridine
- HCI
+HN(OMe), I
94 % o\
62 %
Figure 19-5. Synthesis of starting material 6-benzyloxy-3,S-dihydroxy-
hexanoic acid ethyl ester.
19.3.1.3
Enantioselective Reduction with Whole Cells of Candida sorbophila (E.C. 1.l.X.X) 133-351
Here the biotransformation (Fig. 19-6) is preferred over the chemical reduction with
commercially available asymmetric catalysts (BH3- or noble-metal-based), since
with the chemocatalysts the desired high enantiomeric excess (ee > 98%, 99.8% after
purification) is not achievable. Since the ketone has only a very low solubility in the
aqueous phase, I kg ketone is added as solution in 4 L 0.9 M H 2 S 0 4to the bioreactor.
The bioreduction is essentially carried out in a two-phase system, consisting of the
aqueous phase and small droplets made up of substrate and product. The down-
stream processing consists of multiple extraction steps with methyl ethyl ketone and
precipitation induced by pH titration of the pyridine functional group (p& = 4.66)
with NaOH. The (R)-aminoalcohol is an important intermediate for the synthesis of
P-3-agonists that can be used for obesity therapy and to decrease the level of
associated type 11diabetes, coronary artery disease and hypertension.
7 9.3 Examples
I 1425
1 NHzOH .HCI 1 EtOK. EtOH

2 TsCI, pyndine 2 HCI, MTBE
EDC. THF. H20.pH 5.5,

pnilrophenyl acetic acid
--
1 BHo-SMen.
THF. 1 M H,SO,
2 purifyvia
phosphate salt,
ElOH
HCI
92 %
then free base

75 %
1 = 2-(4-nitro-phenyl)-N(2-oxo-2-pyridin-3-ethyl)-acetamide
2 = (R)-N-(2-hydroxy-2-~ridin-3-yl-ethyl)-2-(4-nitro-phenyl)-acetamide
3 = p-3-agonist
E = dehydrogenase, whole cells 01 Candida sorbophila
Figure 19-6. Synthesis of key intermediate of P-3-agonist (Merck Research Laboratories).
19.3.2
Oxidation Reactions Catalyzed by Oxidoreductases (E.C. 1)
19.3.2.1
Alcohol Oxidation Using Whole Cells of Cluconobacter suboxydans
(E. C. 1.1.99.21) [36-381
In 1923 the bacterium Acinetobacter suboxydans was isolated and, starting in 1930,
was used for the industrial oxidation of L-sorbitol to L-sorbose in the Reichstein-
Griissner synthesis of vitamin C 13'1. Bayer uses the same type of reaction, but instead
of Acinetobacter the bacterium Gluconobacter suboxydans is used in the oxidation of N-
protected 6-amino-~-sorbi to1 to the corresponding 6-amino-~-sorbose, which is an
intermediate in miglitol production (Fig. 19-7).1-Desoxynojirimycinis produced by
chemical intramolecular reductive amination of 6-amino-~-sorbose. In contrast, the
19 Replacing Chemical Steps by Biotransformations
HOfi T$
:izi ~ HO$i protection HOfH OH
OH
OH OH OH
D-glucose 1-amino-D-sorbitol 1
R'
HO.
1) deprotection
2) NaBH4 HO{\
HO&
HO
OH
1-desoxynojirimycin
2 90% yield
rniglitol
1 = 1-amino-D-sorbitol (N-protected)
2 = 6-arnino-~-sorbose(N-protected)
E = D-sorbitol dehydrogenase, whole cells of Gluconobacter oxidans
Figure 19-7. Synthesis of key intermediate for miglitol (Bayer).
published chemical synthesis of 1-desoxynojirimycin and its derivatives requires

multiple steps and laborious protecting-group chemistry. Miglitol and derivatives
thereof are pharmaceuticals for the treatment of carbohydrate metabolism disorders
(e.g. diabetes mellitus).
19.3.2.2
Oxidative Deamination Catalyzed by Immobilized D-Amino Acid Oxidase from
Trigonopsis uariabilis (E. C. 1.4.3.3) [40-421
This oxidative deamination catalyzed by immobilized enzymes is part of the

7-aminocephalosporanic acid (7-ACA) process. Ketoadipinyl-7-aminocephalo-
sporanic acid decarboxylates in situ in the presence of H202,which is formed by the
7 9.3 Examples
I
1427
3
1 = cephalosporin C
2 = a-ketoadipinyl-7-aminocephalosponnicacid
3 = glularyl-7-aminocephalosporanicacid (7-ACA)
E = 0-aminoacidoxidase, immobilizedenzyme from Trigonopsis variablis
Figure 19-8.Synthesis of glutaryl-7-arninocephalosporanic acid (7-ACA)

(Hoechst-Marion-Roussel).
biotransformation step yielding glutaryl-7-ACA. The reaction solution is directly

transferred to the 7-ACA production (see Sect. 19.3.4.2 for details and a comparison
with the chemical synthesis).
19.3.2.3
Kinetic Resolution by Oxidation of Primary Alcohols Catalyzed by Whole Cells from
Rhodococcus erythropolis (E. C. 1.X.X.X) [43-45]
(R)-Isopropylideneglycerol is a useful C3-synthon in the synthesis of (S)-P-blockers,

e. g. (S)-metoprolol.Also, (R)-isopropylideneglycericacid may be used as the starting
material for the synthesis of biologically active products. The resolution is carried out
by selective microbial oxidation of the (S)-enantiomer (Fig. 19-9). The chemical
synthesis of (R)-isopropylideneglycerolstarts either from unnatural L-mannitol or
from L-ascorbic acid (Fig. 19-10). In comparison to the biotransformation, here
stoichiometric quantities of lead tetra acetate are needed.
( WS)-1
1 = isopropylideneglycerol
2 = isopropylideneglycericacid
E = oxidase, whole cells from Rhodococcuserythropolis
Figure 19-9. Synthesis of (R)-isopropylideneglyceroland (R)-isopropylideneglyceric acid

(International BioSynthetics).
1428
From L-mannitol:
HO
HO
OH
-
Pb(0Ack
2 0 - 2 0
CHO
HO
(W-1
From L-ascorbic acid:
Figure 19-10. C h e m i c a l synthesis of (R)-isopropylideneglycerol.
19.3.2.4
Hydroxylation o f Nicotinic Acid (Niacin) Catalyzed by Whole Cells ofAchrornobacter
xylosoxidans (E.C. 1.5.1.1 3) 146-481
6-Hydroxynicotinate is a versatile building block used chiefly in the synthesis of

modern insecticides. The 6-hydroxynicotinate-producingstrain (Fig. 19-11) was
found by accident, when in the mother liquor of a niacin-producing chemical plant
precipitated white crystals of 6-hydroxynicotinatewere found. The second enzyme of
the nicotinic acid pathway, the decarboxylating 6-hydroxynicotinate hydroxylase
becomes strongly inhibited at niacin concentrations higher than 1%, whereas the
operation of niacin hydroxylase is unaffected. In contrast to the biotransformation,
the chemical synthesis of 6-substituted nicotinic acids is difficult and expensive
because of the necessity for the separation of by-products that are produced by non-
regioselective hydroxylations.
(Jcoo- **
+H20 2[H]
2
1
1
> 90% yield
'12 02
HvO Figure 19-11. Syn-

thesis of 6-hydrox-
1 = niacin = nicotinic acid = pyridine-3-carboxylate
2 = 6-hydroxynicotinate= 6-hydroxy-pyridine-3-carboxylate ynicotinate
E = nicotinic acid hydrolase, whole cells from Achromobacterxylosoxidans (Lonza).
19.3.2.5
Reduction of Hydrogen Peroxide Concentration by Catalase (E. C. 1.1 1.1.6) [491
During oxidative coupling to dinitrodibenzyl (DNDB),hydrogen peroxide is formed

as a by-product. It is not possible to decompose H202by adding heavy-metal catalysts
19.3 Examples I
1429
0 2 +2 pJM-
e @ (t-BuOK) + H202
NO2 /
NO2 I
1 2
+'
1 = nitrotoluene
2 = dinitrodibenzyl (DNDB)
E = catalase, enzyme from microbial source
Figure 19-12. Degradation o f hydrogen peroxide (Novartis).
catalase
Figure 19-13. Flow scheme ofdinitrodibenzyl synthesis.
because only an incomplete conversion is reached. Additionally, subsequent process

steps with DNDB are problematic because of contamination with heavy-metal
catalyst. The biotransforination is the only relevant method of decomposing the
undesired side product Hz02 (Fig. 19-12).The enzyme of choice is catalase derived
from a microbial source, which has advantages compared to beef catalase since the
activity remains constant over a broad pH range from 6.0 to 9.0, temperatures up to
50 "C are tolerated, and salt concentrations up to 25% do not affect the enzyme
stability. The reaction is carried out in a cascade of three continuously operated
stirred tank reactors (Fig. 19-13).The H 2 0 2concentration is reduced from 7000 ppm
to < 200 ppm in the product solution. The third vessel is aerated with nitrogen to
degas the product solu-tion. The dinitrodibenzyl is used as a pharmaceutical
intermediate.
1430
19.3.3
Hydrolytic Cleavage and Formation o f C - 0 Bonds by Hydrolases (E.C. 3)
19.3.3.1
Kinetic Resolution of Clycidic Acid Methyl Ester by Lipase from Serratia rnarcescens
(E.C. 3.1.1.3)[50-53]
Trans-(2R,3S)-(4-methoxyphenyl)glycidic acid methyl ester is an intermediate in the

synthesis of diltiazem, a coronary vasodilator and a calcium channel blocker with
6
enzymatic process chemical process
CICH,COOMe
NaOMe
Meon aspo
a::2
PCOOMe
w
COOMe
CHO
I
D,L-threo
I ’
1‘ hydrolysis
O
M
/.e--A
-
0 COOMe COOH
I
(2S,3R)-2
99 9% ee
40-45% yield
NaHSO,
-co2
S0,Na
3
COOH
1 (+)-ihreo
reduction
4 NMe, .HCI I
1 = tramp-methoxyphenylmethylglycidale(MPGM)
2 = trans-p-melhoxyphenylglycidic acid
3 = bisuifite adduct after decarboxylalion
4 = diltiazem
E = lipase, enzyme from Serraiia marescens
Figure 19-14. Comparison o f chemical and biocatalytical route t o diltiazem

(Tanabe Seiyaku Co., Ltd.).
19.3 Examples
I
1431
antianginal and antihypersensitive activity. It is produced worldwide in excess of
> 100 t a-l.
In comparison to the chemical route, only 5 steps (instead of 9) are necessary with
the biotransformation (Fig. 19-14).The kinetic resolution is carried out in an earlier
step with a lower molecular weight compound during the synthesis, resulting in a
reduction of waste. By redesigning the synthesis route using a biotransformation,
the manufacturing costs of diltiazem were decreased to two thirds of those of the
original process including a chemical resolution rs41.
The lipase from Serratia rnarcescens has a high enantioselectivity (E = 135) for the
(ZR,3S)-(4-rnethoxyphenyl)glycidic acid methyl ester, which acts as a competitive
inhibitor. The formed acid (hydrolyzed (+)-methoxyphenylglycidate)is unstable and
decarboxylatesto give 4-methoxyphenylacetaldehyde;this aldehyde strongly inhibits
and deactivates the enzyme. It is removed by transfer to the aqueous phase by
formation of a water-soluble adduct with sodium hydrogen sulfite added to the
aqueous phase. The bisulfite acts also as a buffer to maintain constant pH during
synthesis.
The enantioselective hydrolysis is carried out in an organic-aqueous two-phase
reactor (toluene/water), where the phase contact is established by a hydrophilic
hollow-fiber membrane (polyacrylonitrile).The lipase is immobilized onto a spongy
layer by pressurized adsorption. The productivity is about 40 kg trans-(ZR,3S)-
(4-methoxypheny1)glycidic: acid methyl ester m-* a-l. This process has been operated
since 1993.
19.3.3.2
Kinetic Resolution of Diestier by Protease Subtilisin Carlsberg from Bacillus sp.
(E. C. 3.4.21.62)*'si 561
(R)-(2-Methylpropyl)-butanedioic acid 4-ethyl ester is used as a chiral building block

for potential collagenase inhibitors (e.g. Ro 31-9790) in the treatment of osteoar-
thritis. The diester is reacted as a 20% emulsion in 30 mM aqueous NaHC03 using
Protease@L 660 or Alcal.ase@2.5 L (9% each, with respect to the racemic diester)
(Fig. 19-15).The unconverted (S)-diestercan be extracted in a solvent such as toluene
and racemized by heating the anhydrous extract with catalytic amounts of sodium
ethanolate. The resulting racemic diester can be recycled, thus improving the overall
organic phase aqueousphase
1 = (2methylpropyl)butanedioicacid diethylether
2 = (2methylpropyl)butanedioicacid 4-ethyl ester, Na-form
E = hydrolase, subtiiisin Carlsberg from Bacillus sp.
Figure 19-15.Synthesis o f (F')-(2methylpropyl) butanedioic acid diethylether

(Hoffmann La-Roche).
1432
I yield from 45
19 Replacing Chemical Steps by Biotransforrnations
% to 87 %. The reaction was repeatedly carried out on a 200 kg scale

with respect to the racemic diester. The enzyme is highly stereoselective even at high
substrate concentrations (20%). The chemoenzymatic route starting from the cheap
bulk agents maleic anhydride and isobutylene replaced the existing chemical
research synthesis for bulk amounts (Fig. 19-16).
drug discovery synthesis process research synthesis
=A
H~N-COOH
i;"""
COO'BU
EtOO d COOEt
JE
EtOOO
dH
'BuOO
r
A
-?- i.
Ro 31-9790
Figure 19-16. Comparison of drug discovery and process research route of Ro 31-9790.
7 9.3 Examples
I
1433
D,L-1 D-2 crystallization D-1
t +
L$ racemization 0
L- 1
1 = pantolactone
2 = pantoic acid
E = lactonase, whole cells from Fusarium oxysporum
Figure 19-17. Synthesis o f D-pantolactone (Fuji Chemical Industries).
19.3.3.3
Kinetic Resolution of Pantolactonesand Derivatives thereof by a Lactonase from
Fusarium oxysporum (E. C. 3.1.1.25) ["I
Pantenoic acid is used as a vitamine BZ complex. D- and L-pantolactoneare used as

chiral intermediates in chemical synthesis. The enantioselectivehydrolysis is carried
out in the aqueous phase with a substrate concentration of 2.69 M = 350 g L-'
(Fig. 19-17).For the synthesis whole cells are immobilized in calcium alginate beads
and used in a fured bed reactor. The immobilized cells retain more than 90 % of their
initial activity after 180 days of continuous use. At the end of the reaction L-
pantolactone is extracted and reracemized to D,L-pantolactone, which is recycled to
the reactor. The D-pantenoic acid is chemically lactonized to D-pantolactone and
extracted. By applying cells from Brewibacteriurn protophormia the L-lactone is
available. The biotransformation eliminates several steps that are necessary in the
chemical resolution process (Fig. 19-18).
19.3.3.4
Hydrolysis of Starch to Glucose by Action ofTwo Enzymes: a-Amylase (E.C. 3.2.1.1)
and Arnyloglucosidase (E. C. 3.2.1.3) [5s-601
The process is part of the production of high fructose corn syrup. After several
improvements, this process (Fig. 19-19)provides an effective way for an important,
low-cost sugar substitute derived from grain. At various stages enzymes are applied
in this process[", '*I. The corn kernels are softened to separate oil, fiber and proteins
by centrifugation. The enzymatic steps are cascaded to yield the source product for
the invertase process after liquefaction in continuous cookers, debranching and
filtration (Fig. 19-20). Since starches from different natural sources have different
compositions, the procedure is not unique. The process ends, if all starch is
completely broken down to limit the amount of oligomers of glucose and dextrins.
Additionally, recombination of molecules has to be prevented. The thermostable
1434
I I9 Replacing Chemical Steps by Biotransformations
chemical process enzymatic process
-tgo
enzymatic
-
hydrolysis
1
--tre_: KO
resolution
I +
0 - 0
%
HO
I
extraction lactonization
-tgo - -tgo -
Figure 19-18. Comparison o f chemical and biocatalytic route for the enantioselective
synthesis o f pantolactone.
CHzOH CH,OH
&o&o&o&o,
El
+HzO * oligomer units
\O
OH OH OH OH
CH2OH
1 = starch OH
&OH OH
2 = glucose
E l = a -amylase, enzymefrom Bacillus licheniformis
E2 = glucoamylase, enzyme from Aspergillus niger 2
Figure 19-19. Synthesis of glucose (Several companies)
19.3 Examples 11435
‘
0 o
+
*o
,
OH OH OH OH
so* +
centrifuge
1. liquefaction 2. liquefaction
105-115°C 90-95°C
pH-6
protein, fat
4
\
\
OH \ J
Figure 19-20. Flow scheme for the hydrolysis of starch to glucose.
enzyme can be used up to 115 “C. The enzymes need Ca2+ions for stabilization and
activation. Since several substances in corn can complex cations, the cation concen-
tration is increased requiring a further product purification, i. e. making it necessary
to refine the product. There is no alternative industrial chemical process for starch
liquefaction. The worldwide production is about 10’ t a-’,
19.3.4
Formation or Hydrolytic Cleavage of C-N Bonds by Hydrolases (E.C. 3)
19.3.4.1
Enantioselective Acylation of Racemic Amines Catalyzed by Lipase from Burkholderia
plantarii (E.C. 3.1.1.3)[63-651
The lipase catalyzes the kinetic resolution of racemic amines, e.g. l-phenyl-
ethylamine (Fig. 19-21)[ll]. Products are intermediates for pharmaceuticals and
pesticides. They can also be used as chiral synthons in asymmetric synthesis. As
acylating agent ethylmethoxyacetate is used, because the reaction rate is more than
100 times faster than that with butyl acetate. Probably an enhanced carbonyl activity
induced by the electronegative a-substituents accounts for the activating effect of the
methoxy group. The lipase is immobilized on polyacrylate. The lowered activity
caused by use of in organic solvent (tert-methylbutylether= MTBE) can be increased
1436
NHZ NH Lo,
2 (q-3
> 93% ee
1 = I-phenylethylamine
2 = ethylmethoxyacetate
3 = phenylethylmethoxyamide
E = Iipase, enzyme from Burkholdenaplantanr
Figure 19-21. Kinetic resolution o f phenylethylarnine (BASF).
(about 1000 times and more) by freeze drying a solution of the lipase together with
fatty acids (e.g. oleic acid). Because of the use of MTBE a high starting material
concentration of 1.G5 M 1-phenylethylaminecan be established. The enantioselectiv-
ity is greater than 500. The (R)-phenylethylmethoxyamidecan easily be hydrolyzed to
the (R)-phenylethylamine.The unconverted (S)-enantiomer can be racemized using
a palladium catalyst.
19.3.4.2
7-AminocephalosporanicAcid Formation by Amide Hydrolysis Catalyzed by Glutaryl
Amidase (E.C. 3.1.1.41)[66-691
The second step of the 7-aminocephalosporanicacid (7-ACA)process is the deami-

dation of glutaryl-7-ACA (Fig. 19-22), the first step is described in Sect. 19.3.2.2.
7-ACA is an intermediate for semi-synthetic cephalosporins. Hoechst Marion
Roussel uses the glutaryl amidase immobilized on a spherical carrier. Toyo Jozo and
Asahi Chemical immobilize the glutaryl amidase on porous styrene anion exchange
resin with subsequent cross-linking with 1% glutardialdehyde. The catalyst is
applied in a fixed bed reactor in a repetitive batch mode (70 cycles). Here, an
enzymatic process has replaced an existing chemical process for environmental
reasons (Fig. 19-23):
In the first step, the zinc salt of cephalosporin C is produced, followed by the
protection of the functional groups (NH2 and COOH) with trimethylchlorosilane.
1 = glutaryl-7-aminocephalosporanicacid
2 = 7-aminocephalosporanicacid (7-ACA)
E = glutaryl amidase, enzyme from Escherichia coli
Figure 19-22. Synthesis of 7-arninocephalosporanic acid (7-ACA)

(Asahi Chemical, Hoechst Marion Roussel, Toyo Jozo).
79.3 Examples I1437
COOH 0
ZnCPC J ++ 0H2 L
- H202
solventJTMSCl - NH,
/
HN-SI-
/
\
-si/
N
/o
*
0 phoy
CI 0
COOH 0 COOH 0
\
T c 0 "C hydrolysis
~ H o o c ~ c o o H
H2Np&oy
0
COOH 0
E l = D-aminnacid oxidase
E2 = glutaiyl amidase
Figure 19-23. Comparison o f chemical and biocatalytical route for the synthesis o f 7-ACA.
The imide chloride is synthesized in the subsequent step at 0 "C with phosphorous
pentachloride. Hydrolysis of this imide chloride yields 7-ACA. By replacement of
this synthesis with the biotransformation, the use of heavy-metal salts (ZnClz) and
chlorinated hydrocarbons as well as precautions for highly flammable compounds
can be circumvented. The off-gas quantities were reduced from 7.5 to 1.0 kg. Mother
liquors requiring incineration were reduced from 29 to 0.3 t. Residual zinc that was
recovered as Zn(NH4)P04is reduced from 1.8 to 0 t. The absolute costs of
environmental protection are reduced by 90% per tonne of 7-ACA. Asahi Chemical
and Toyo Jozo have produced 7-ACA since 1973 with a capacity of 90 t a-l and
Hoechst Marion Roussel since 1996 with a capacity of 200 t a-'.
79 Replacing Chemical Steps by Biotransformations
1438
I
1 2 3
1 = penicillin-G
2 = 6-amino penicillanic acid ( 6-APA)
3 = phenylaceticacid
E = penicillin amidase, enzyme from Escherichia coli
Figure 19-24. Synthesis of 6-amino penicillanic acid (multiple companies).
19.3.4.3
Penicillin C Hydrolysis by Penicillin Amidase from Escherichia coli (E.C. 3.5.1.11) [68-711
6-Amino penicillanic acid (6-APA)is used as the intermediate for manufacturing

semi-synthetic penicillins. Companies applying this technology (Fig. 19-24) include
Unifar, Turkey; Asahi Chemicals, Japan; Fujisawa Pharmaceutical Co., Japan; Gist-
Brocades/DSM, The Netherlands; Novo-Nordisk, Sweden; Pfizer, USA. The enzyme
is isolated and immobilized, often on Eupergit@C(Rohm, Germany). The produc-
tion is carried out in a repetitive batch mode. The immobilized enzyme is retained by
sieves. In case of the Eupergit@Cimmobilized amidase the residual activity is about
50 % of initial activity after 800 batch cycles. Therefore the hydrolysis time after 800
batch cycles increases from initially 60 min to 120 min. The space-time yield is 445 g
L-‘ d-*. Phenylacetic acid is removed by extraction and 6-APA can be crystallized.
Concentrating the “split” solution and/or the mother liquor of crystallization via
vacuum evaporation or reverse osmosis can increase the yield. The production plant
operates for 300 days per year with an average production of 12.8 batch cycles per day
(production campaigns of 800 cycles per campaign). Asahi Chemical utilizes a
penicillin amidase from Bacillus megaterium that is immobilized on aminated porous
polyacrylonitrile fibers. The production is carried out in a recirculation reactor
consisting of 18 parallel columns with immobilized enzyme. Each column has a
volume of 30 L. The circulation of the reaction solution is established with a flow rate
of 6 000 L h-’. One cycle time takes 3 h. The lifetime of each column is 360 cycles.
Purification of 6-APA is done by isoelectric precipitation at pH 4.2 with subsequent
filtration and washing with methanol.
7-Amino deacetoxy cephalosporanic acid (7-ADCA)is also produced by the same
technology.
Several chemical steps are replaced by a single enzyme reaction (Fig. 19-25).
Organic solvents, the use of low temperature (- 40 “C) and the need for absolutely
anhydrous conditions, which used to make the process difficult and expensive, are
no longer necessary in the enzymatic process.
19.3 €xurnp/es I
1439
pc15 1 -400c
n-butanol
“‘“P%
0
COOH
Figure 19-25. Chemical process for 6-APA.
19.3.4.4
Kinetic Resolution of a-Amino Acid Amides Catalyzed by Aminopeptidase from
Pseudomonas putida (E. C. 3.4.1.1 1) [72-751
Enantiomerically pure a-H-amino acids are intermediates in the synthesis of

antibiotics used for parenteral nutrition and for food and feed additives (see also
Chapter 12.2). Examples are D-phenylglycine and 4-hydroxyphenylalanine for semi-
synthetic j3-lactam antibiotics and L-phenylalaninefor the peptidic sweetener aspar-
tame. DSM used this process to produce also L-homophenylalanine, a potential
precursor molecule for several ACE-inhibitors.
The a-amino amides as substrates for this enantiospecific, biocatalytic amide
hydrolysis can be readily obtained from the appropriate aldehydes via the Strecker
synthesis (Fig. 19-26).
As whole cell catalyst, Pseudornonas putida, which accepts a wide range of
substrates, is applied. Subsequent to the biotransformation, benzaldehyde is added,
resulting in precipitation of the o-amide Schiff base, which can be easily isolated by
filtration. An acidification step leads to the D-amino acid. The L-amino acid can be
reused after racemization so that a theoretical yield of 100% D-amino acid is
possible.
The same process can be used for the synthesis of 100% of L-amino acids by
racemizing the Schiff base of the D-amide in a short time using small amounts of
base in organic solvents.
Using i n vivo protein engineering not only mutant strains of Pseudornonas putida
7 9 Replacing Chemical Steps by Biotransformations
1440
I 0 H,N CN
,AH
1
--zFT
+ NH, 2
1"- acetone
1) NH3
2) PhCHO D,L-3
3) OH- (pH 13)
i.
racemization 2) H,O'
4) H,O'
L-4
t = aldehyde
2 = amino nitrile
3 = a-amino acid amide
4 = a-amino acid methyl ester
5 = a-amino acid
6 = a-amino acid amide
7 = schiff base of a-amino acid amide
E = aminopeptidase, whole cells from Pseudomonas puhda
Figure 19-26. Production o f L-and D-a-amino acids by kinetic resolution o f a-amino

acids amides (DSM).
exhibiting L-amidase and also D-amidase but also amino acid amide racemase
activities were obtained. Using these mutants a convenient synthesis of a-H-amino
acids with 100% yield would be possible with one cell system. It is noteworthy that
only a-H-substrates can be used. By screening, a new biocatalyst of the strain
Mycobacterium neoaurum was found, which is capable of converting a-substituted
amino acid amides.
1 2 3 4
t racemization
> 99.5% ee
80% yield
1 = N-acetyl-D.L-methionine
2 = N-acetyl-D-methionine
3 = L-methionine
4 = acetic acid
E = aminoacylase, enzyme from AspergiNus niger
Figure 19-27. Biocatalytical production of L-rnethionine by kinetic resolution (Degussa)
19.3.4.5
Production of L-Methionine by Kinetic Resolution with Aminoacylase o f A s p e r g i h
oryzae (E.C. 3.5.1.14) [76-791
The N-acetyl-D,L-aminoacid precursors are conveniently accessible through either

acetylation of D,L-amino acids with acetyl chloride or acetic anhydride in a Schotten-
Baumann reaction or via amidocarbonylation[80].For the acylase reaction, Co2+as
metal effector is added to yield an increased operational stability of the enzyme. The
unconverted acetyl-D-methionineis racemized by acetic anhydride in alkali, and the
racemic acetyl-D,L-methionineis reused. The racemization can also be carried out in
a molten bath or by an acetyl amino acid racemase. Product recovery of L-methionine
is achieved by crystallization, because L-methionine is much less soluble than the
acetyl substrate. The production is carried out in a continuously operated stirred tank
reactor. A polyamide ultrafiltration membrane with a cutoff of 10 kDa retains the
enzyme, thus decoupling the residence times of catalyst and reactants. L-methionine
is produced with an ee > 99.5% and a yield of 80% with a capacity of > 300 t a-'. At
Degussa, several proteinogenic and non-proteinogenic amino acids are produced in
the same way e. g. L-alanine, L-phenylalanine, a-amino butyric acid, L-valine, L-
norvaline and L-homophenylalanine.
19.3.4.6
Production o f D-p-Hydroxyphenyl Clycine by Dynamic Resolution with Hydantoinase
from Bacillus breuis (E. C. 3.5.2.2) L8, 81-831
D-p-Hydroxyphenyl glycine is a key raw material for the semisynthetic penicillins

such as ampicillin and amoxycillin. It is also used in photographic developers.
Racemic hydantoins are synthesized starting from phenol derivatives, glyoxylic acid
and urea via the Mannich condensation (Fig. 19-28).The D-specific hydantoinase is
applied as immobilized whole cells in a batch reactor. The unreacted L-hydantoins
are readily racemized under the alkaline conditions (pH 8) of enzymatic hydrolysis,
yielding quantitative conversion. This process enables the stereospecific preparation
of various amino acids, such as L-tryptophane, r-phenylalanine, D-valine, D-alanine
1442
I I 9 Replacing Chemical Steps by Biotransformations
+ A H,N NH,
H d
L-1
H d
D-1
-
E
HO'
D-2
HO
1 = 5-(p-hydroxybenzyI)-hydantoin D-3
2 = D-N-carbamoylamino acid
3 = D-4-hydroxyphenylglycine
E = D-hydantoinase,whole cells from Bacillus brews
Figure 19-28. Synthesis o f o-amino acids (Kanegafuchi).
and D-methionine. Instead of chemical treatment with sodium nitrite, a carbamoy-

lase (EC 3.5.1.77) can also be applied to remove the carbamoyl group. Several other
companies have developed patented processes to produce D-hydroxyphenylglycine
(Ajinomoto,DSM, SNAM-Progetti,Recordati and others).
Here the biotransformation competes with the classical chemical route (Fig. 19-
29), which employs bromocamphorsulfonic acid (Br-CAS)as the resolving agent. In
both routes phenol is used as raw material since p-hydroxybenzaldehyde is too
expensive. The hydantoinase process for phenylglycines does not necessarily need
an extra racemization step since the hydantoin is racemized in situ at an alkaline pH.
Because of the dynamic resolution in the case of this biotransformation, higher
yields are reached.
19.3.4.7
Dynamic Resolution of a-Amino-E-caprolactam by the Action of Lactamase (E. C.
3.5.2.11) and Racemase (E.C. 5.1.1.15)[84* "1
Again a dynamic resolution is carried out, but this time the racemization is
introduced by an enzyme, a racemase from Achromobacter obae (Fig. 19-30). The
lactamase and racemase are applied as whole cells and are fortunately active at the
same pH, so that they can be used in one reactor. Reaction conditions enabling
I
79.3 Examp/es 1443
resolution with
Br-CAS
J
HO $”
\ /
c-
HNO,
E l = D-hydantoinase.whole cells from San//us brevis

E2E3 = D-hydantoinase/N-carbamoyl-D-amino acid hydrolase, whole cells, strain Pseudomonassp. contains both
enzymes
Figure 19-29. Comparison o f chemical and biocatalytical route for the synthesis
o f o-amino acids (Kanegafuchi).
D,L-1 D-1 L-2

> 99.5% ee
E2
1 = a-amino-E-caprolactam(ACLI
2 = lysine
E l = L-aminolactarn-hydrolase,whole cells from Cryptococcus laurentii
E2= amino-lactam-racemase,whole cells from Achromobacter obae
Figure 19-30. Synthesis of L-lysine (Toray Industries).
chemical racemization would reduce the enzyme stability. L-Lysine was produced
with an ee of 99.5 % at a capacity of 4000 t a-’. This process has been totally replaced
by highly effective fermentation methods.
1444
I
R’ fJqf + H21F> N /
COOH
R3 E
-R,H , R’Q-Yp&
0
OOH
D(-)-1 2 3
l a = phenylglycineamide (R’=H, R2=NH,) = PGA

1b = phenylglycinernethyleter(R’=H, RZ=OMe)= PGM
1c = hydroxyphenylglycineamide(R’=OH, R2=NH,) = HPGA
I d = hydroxyphenylglycinemethylester(R’=OH, RZ=OMe)= HPGM
2a = 7-amincdeacetoxycephalosporanicacid (&Me) = 7-ADCA
2b = 7-arn1ncdeaceioxymethyl-3-chlorocephalospnic acid (R3=CI)= 7-ACCA
3a = cefaclor (R’=H, R3=CI)
3b = cephalexin (R’=H, &Me)
3c = cefadroxil (R’=OH. &Me)
E = penicillin acylase
Figure 19-31. Synthesis o f p-lactam antibiotics (Chemferm).
19.3.4.8
Synthesis of B-Lactam Antibiotics Catalyzed by Penicillin Acylase (E. C. 3.5.1.1 1) ~86-8gl
The penicillin acylases do not accept charged amino groups. Therefore phenyl-
glycine itself cannot be used at a pH value at which the carboxyl function is
uncharged, because the amino group will then be charged.
To reach non-equilibrium concentrations of the product, the substrate must be
activated as an ester or amide (Fig. 19-31).By this means the amino group can be
partly uncharged at the optimal pH value of the enzyme. In biological systems, ATP
delivers the activation energy. Using the same synthetic pathway alternatively to
7-ADCA and 7-ACCA, 6-APA derivatives can also be synthesized.
The established chemical synthesis started from benzaldehyde and included the
fermentation of penicillin (Fig. 19-32).The process consists often steps with a waste
stream of 30-40 kg waste per kg product. The waste contains methylene chloride,
other solvents, silylating agents and many products from side-chain protection and
acylating promoters. In comparison, the chemoenzymatic route needs only six steps
including three biocatalytic ones. The biotransformations E l and E2 in Fig. 19-32
can be found in Sect. 19.3.4.3and 19.3.4.4.
19.3.4.9
Synthesis of hetidinone 8-Lactam Derivatives Catalyzed by Penicillin Acylase
(E.C. 3.5.1.11)[’Ot ”1
It was thought that the Pen G amidase would exhibit only a limited substrate
spectrum, since it does not hydrolyze the phenoxyacetyl side chain of penicillin V.
Nevertheless, Eli Lilly shows that the Pen G amidase acylates the amino function of
cis-3-amino-azetidinonewith the methyl ester of phenoxyacetic acid (Fig. 19-33).The
19.3 Examples I
1445
j
benzaldehyde Penicillium benzaldehyde Penicillium
J
~trecker
synthesis
D,L-phenylglycine
fermentation
penicillin G
Strecker
synthesis
J
penicillin G
fermentation
1 classical
resolution
D-( -)-phenylglycine
1 ring
enlargement
cephalosporin D,L-phenylglycine-
amide ester
1
cephalosporin
ring
enlargement
protection
Dane salt 7-ADCA

deacylation El
1
7-ADCA
deacylation
I
I
activation protection protection
D-(-)-phenylglycine-
mixed anhydride Protected7-ADCA protected 7-ADCA
amidd ester
coupling
deprotecting
cefalexin
-T coupling
cefalexin
E l = penicillin amidase
E2 = amino peptidase
E3 = penicillin acylase
Figure 19-32. C o m p a r i s o n of t h e chemical a n d biocatalytical synthesis of cefalexin.
1 = cis-3-amino-azetidinone
2 = phenoxy-aceticacid methyl ester
3 = B-lactam intermediate
4 = loracarbef
E = Pen G amidase, enzyme from Escherichiacolr
Figure 19-33. Synthesis of azetidione fi-lactam derivatives (Eli Lilly).

1446
I 79 Replacing Chemical Steps by Biotransformations
acylation occurs using methyl phenylacetate (MPA) or methyl phenoxyacetate

(MPOA)as the acylating agents. The penicillin amidase is immobilized on Eupergit
(Roehm GmbH, Germany).
The chemical resolution of the racemic azetidinone is only low yielding. The
(2R,3S)-azetidinoneis a key intermediate in the synthesis of the carbacephalosporin
antibiotic loracarbef.
19.3.4.10
EnantioselectiveSynthesis of an Aspartame Precursor with Thermolysin from Bacillus
proteolicus (E.C. 3.4.24.27) 931
Since the reaction (Fig. 19-34) is limited by the equilibrium the products have to be
removed from the reaction mixture to reach high yields. Therefore an excess of
racemic phenylalanine methylester (which is inert to the reaction) is added. The
carboxylic anion of the protected aspartame forms a poorly soluble adduct with D-
Phe-OCH3 that precipitates from the reaction mixture. The precipitate can be
removed easily by filtration. Final steps of the process are the separation of D-Phe-
ester, removal of protecting groups and racemization of the formed L-amino acid. a-
Aspartame is produced with > 99.9% and a worldwide capacity of - 10,000 t a-',
- 2,500 t 6' by enzymatic coupling.
The bacterial strain was found in the Rokko Hot Spring in central Japan.
Consequently it is very stable up to temperatures of GO "C.
The main problem in chemical synthesis coupling of Z-Asp anhydride with
COOCH , HOOCYCooH
HNXZ
D,L-l L-2
COOCH,
I I) HCI
1 1 ii)
1 ) H,, Pd/C, MeOH
\ RH,W + H O O C q A COOCH,
NHZ
aspartame
~~~ Figure 19-34. Biocata-
1 = phenylalanine methylester lytical synthesis of as-
2 = aspartic acid (protected)
3 = a-aspartame (protected) partame (HSC, Holland
E = thermolysin. enzyme from Bacillus proieolicus Sweetener Company).
79.3 Examples 11447
1 2 3
> 95% yield
1 = 2-cyanopyrazine
2 = pyrazine-2-carboxylicacid
3 = 5-hydroxypyrazine-2carboxylicacid
EVE2 = nitrilaselhydroxylase,whole cells, strain Agrobacteriumsp. contains both enzymes
Figure 19-35. Biocatalytical synthesis of 5-hydroxypyrazine-2-carboxylic acid (Lonza).
L-Phe-OCH3 is the by-product formation of P-aspartame. This isomer is of bitter

taste and has to be completely removed from the a-isomer. The advantages of the
enzymatic route are: (i) No p-isomer is produced, (ii) the enzyme is completely
stereoselective, so that racemic mixtures of the substrate or the appropiate enantio-
mer of the amino acid can be used, (iii)no racemization occurs during synthesis and
(iv) the reaction takes place in aqueous media under mild conditions.
19.3.4.11
Hydrolysis of Heterocyclic Nitrile by Nitrilase from Agrobacteriurn sp.
(E.C. 3.5.5.1) [94-961
5-Hydroxypyrazine-2-carboxylicacid (Fig. 19-35)is a versatile building block in the

synthesis of new antituberculous agents, e. g. 5-chloro-pyrazine-2-carboxylic acid
esters. The regioselectivehydroxylation of pyrazine-2-carboxylicacid is catalyzed by a
hydroxylase (E2,E. C. 1.5.1.13). This second enzyme is also in the applied suspended
whole cells from Agrobacteriurnsp. The biomass is separated by ultrafiltration (cutoff
10 kDa) after the biotransformation. 5-Hydroxypyrazine-2-carboxylic acid is precipi-
tated from the permeate by acidification with sulfuric acid to pH 2.5.
In contrast to the biotransformation, the chemical synthesis of 5-substituted
pyrazine-2-carboxylicacid leads to a mixture of 5- and 6-substituted pyrazine-
carboxylic acids and requires multiple steps.
19.3.5
Formation o f C - 0 Bonds by Lyases
19.3.5.1
Synthesis of Carnitine Catalyzed by Carnitine Dehydratase in Whole Cells
(E.C. 4.2.1.89) [', 46s 97-991
L-Carnitine is used in infant health, sport and geriatric nutrition. The biotransforma-
tion is catalyzed by carnitine dehydratase in whole cells (Fig. 19-36).(R)-carnitineis
produced with > 99.5% conversion of butyrobetaine and > 99.5% ee. The mutant
strain has blocked the L-carnitine dehydrogenase and excretes the accumulated
product. The purified enzyme could not be used for the biotransformation because
of its high instability. Apart from usual batch fermentations, continuous production
1448
Figure 19-36. Comparison of chemi-
,% cal and biocatalytical synthesis o f

carnitine (Lonza).
CI&COOEt
1
H, Ru-(S)-BINAP
Q”
tO
E
C, - ,A, - ,~lCOOC , , - , , . + N 3 e M
(R) 97% ee 1
Me3N\ 99.9% ee
H30t E
J > 99.5% yield
Q”
C
O
O
~, - , - , , ,N
+M
,e
( R)-2
1 = 4-butyro betaine
2 = carnitine
E = carnitine dehydratase, whole cells from Escherichia coli
is also feasible since the cells go into a “maintenance state” with high metabolic
activity and low growth rate. The cells can be recycled after separation from the
fermentation broth by filtration. A chemical resolution process with L-tartaric acid
that was developed at Lonza was no longer competitive with the biotechnological
route. A more attractive chemical route would be the Ru-BINAP catalyzed asym-
metric hydrogenation of 4-chloroacetoacetate (Fig. 19-36). Here an ee of 97% is
yielded.
19.3.6
Formation ofC-N Bonds by Lyases (E.C. 4)
19.3.6.1
Synthesis of L-Dopa Catalyzed by Tyrosine Phenol Lyase from Erwinia herbicola
(E.C. 4.1.99.2)
The product is applied for the treatment of Parkinsonism that is caused by a lack of L-
dopamine and its receptors in the brain. L-Dopamine is synthesized in organisms by
decarboxylation of ~-3,4-dihydroxyphenylalanine (L-dopa).Since L-dopamine cannot
pass the blood-brain barrier L-dopa is applied in combination with dopadecarbox-
ylase-inhibitors to avoid formation of L-dopamine outside the brain. Ajinomoto
produces L-dopa by this lyase-biotransformation with suspended whole cells in a fed
batch reactor on a scale of 250 t ax’. Much earlier, Monsanto has successfully scaled
up the chemical synthesis of L-dopa (Fig. 19-38).
1 2 L-3
1 = catechol
2 = pyruvic acid
3 =dopa
E = tyrosine phenol lyase. whole cells from Erwinia herbicola
Figure 19-37. Biocatalytical synthesis o f L-dopa (Ajinomoto).
HoucHo
HO
1
+ A~HN-COOH
Ac
2
\
1 =vanillin
2 = azlactone
3 = Z-enamide
3 = dopa
Figure 19-38. Chemical synthesis of L-dopa (Monsanto)
The enantioselectivehydrogenation of 3,4-dihydroxy-N-acetylamino cinnamic acid

is catalyzed by the cationic Rh-biphosphine complex DIPAMP, in which the
enantioselectivity is introduced by the chiral phosphine 1104*1051 . The hydrogenation
proceeds quantitatively with 94 % ee. The optically pure L-dopa is separated from the
catalyst by crystallization.
19.3.6.2
Synthesis o f 5-Cyano Valeramide by Nitrile Hydratase from Pseudomonas chlororaphis
823 (E.C. 4.2.1.84)['06. 1"'
5-Cyanovaleramideis used as intermediate for the synthesis of the DuPont herbicide

azafenidine (Fig. 19-39). The whole cells from Pseudornonas chlororuphis are im-
mobilized in calcium alginate beads. The biotransformation itself is catalyzed by a
nitrile hydratase that converts a nitrile into the corresponding amide by addition of
water. Nitrile hydratases belonging to the enzyme class of lyases (E. C. 4) are not be
1450
1 = adiponitrile
2 = 5-cyano-valerarnide
E = nitrile hydratase, whole cells from Pseudomonas chlororaphis
Figure 19-39. Comparison of chemical and biocatalytical synthesis of 5-cyano-valerarnide

(DuPont) .
confused with the nitrilases belonging to the class of hydrolases (E.C. 3) that
hydrolyze nitriles to the corresponding carbon acids. For strain selection it was
important that the cells did not show any amidase activity that would further
hydrolyze the amide to the carboxylic acid. The biotransformation is carried out in a
two-phase system with pure adiponitrile forming the organic phase. A reaction
temperature of 5 "C is chosen, since the solubility of the by-product adipodiamide is
only 37-42 mM in 1-1.5 M 5-cyanovaleramide.A batch reactor is preferred over a
fixed-bed reactor, because of the lower selectivity to 5-cyanovaleramide that was
observed and the possibility of precipitation of adipodiamide and plugging of the
column. Excess water is removed at the end of the reaction by distillation. The by-
product adipodiamide is precipitated by dissolution of the resulting oil in methanol
at z 65 "C. The raw product solution is directly transferred to the herbicide
synthesis.
By this method 13.6 tonnes have been produced in fifty-eight repetitive batch
cycles with 97 % conversion and 96 % selectivity. This biotransformation was chosen
over the chemical transformation because of the higher conversion and selectivity,
production of more product per catalyst weight (3 150 kg per kg dry cell weight), and
less waste. The catalyst consumption is 0.006 kg per kg product.
19.3.6.3
Synthesis of the Commodity Chemical Acrylamide Catalyzed by Nitrile Hydratase from
Rhodococcus rodochrous (E. C. 4.2.1.84) 12]
Acrylamide (Fig. 19-40) is an important commodity monomer used in coagulators,

soil conditioners and stock additives for paper treatment and paper sizing, and for
adhesives, paints and petroleum recovering agents.
Since acrylonitrile is the most poisonous of the nitriles, screening for micro-
organisms was conducted with low molecular weight nitriles instead.
Acrylamide is unstable and polymerizes easily; therefore the process is carried out
at a low temperature (5 "C). Although the cells, which are immobilized on poly-
acrylamide gel, and the contained enzyme are very stable towards acrylonitrile,the
starting material has to be fed continuously to the reaction mixture because of
inhibition effects at higher concentrations. The biotransformation is started with an
19.3 Examples 11451
E Figure 19-40. Biocatalytical

@CN __+ qNH2
synthesis of acrylamide (Nitto
+ H20 0 Chemical Industry).
1 2
-
1 = acrylonitrile
2 = acrylamide
E = nitrile hydratase,whole cells from Rhodococcuserythropolis
acrylonitrile concentration of 0.11 M and is stopped at an acrylamide concentration

of 5.6 M. The process is operated at a capacity of 30 000 t a-'.
This nitrile hydratase acts also on other nitriles with yields of 100%. The most
impressive example is the conversion of 3-cyanopyridine to nicotinamide. The
product concentration is about 1465 g L-l.This conversion (1.17 g L-' dry cell mass)
can be named "pseudocrystal enzymation", since at the start of the reaction the educt
is solid and with ongoing reaction it is solubilized.
The chemical synthesis uses copper salt as catalyst for the hydration of acryloni-
trile and has several disadvantages:
The rate of acrylamide formation is lower than that of acrylic acid formation.
The double bond of the starting material and the product causes the formation of
by-products such as ethylene, cyanohydrin and nitrilotrispropionamide.
Polymerization occurs.
Copper needs to be separated from the product (an extra step in the chemical
synthesis).
The biotransformation has the advantages that no recovering of unreacted nitrile is
necessary since the conversion is 100% and no copper catalyst removal is needed.
This is also the first case of a biocatalytic conversion of a bulk fiber monomer.
19.3.6.4
Synthesis o f Nicotinamide Catalyzed by Nitrile Hydratase from Rhodococcus
rodochrous (E.C. 4.2.1.84)* ' 4 i
Nicotinamide (vitamin B3) is used as a vitamin supplement for food and animal
feed. It is the same strain that is also used in the industrial production of acrylamide
(see Sect. 19.3.6.3). The biotransformation is carried out on a scale of 3000 t a-l
(Fig. 19-41).
In contrast to the chemical alkaline hydrolysis of 3-cyanopyridine with 4 % Ly-
product of nicotinic acid (96% yield) the biotransformation works with absolute
selectivity and no acid or base is required. The biotransformation (a continuous
process) is operated at low temperature and atmospheric pressure. In contrast to the
old synthesis route of nicotinamide at Lonza, the new one is environmentally
friendly and safe. There is only one organic solvent used throughout the whole
process in four highly selective continuous and catalqc reactions. The process
water, NH3 and HZare recycled.
1452
I
old route new route
NC
1
4
hydrogenation
I
+ NH,
H 2 N L N H 2
u
1 -NH,
cyclization
over zeolite
H (-"y
H
1 Pd-catalyzed
dehydrogenation
4 1 ammonoxidation
E = nitrile hydratase, whole cells from Rhodococcus eiythropolis

Figure 19-41. Comparison of chemical and biocatalytical synthesis
of nicotinarnide (Lonza).
19.3.7
Epimerase
19.3.7.1
Epimerization of Clucosamine Catalyzed by Epimerase from E. coli
(E.C. 5.1.3.8) [114-1161
N-Acetyl-D-mannosamineserves as the in situ generated substrate for the synthesis

of N-acetylneuraminicacid. Since N-acetyl-D-mannosamineis quite expensive it is
synthesized from N-acetyl-D-glucosamineby epimerization at C2. This biotransfor-
mation is integrated into the production of N-acetylneuraminicacid (Neu5Ac).
By application of N-acylglucosamine 2-epimerase it is possible to start with the
inexpensive N-acetyl-D-glucosamineinstead of N-acetyl-D-mannosamine (Fig. 19-
42). The epimerase is used for the in situ synthesis of N-acetyl-D-mannosamine
7 9.4 Some Misconceptions about lndustrial Biotransformations
I
,,A
1453
L H &
OH OH
NHAc
1 2
1 = N-acetyl-D-glucosamine
2 = N-acetyl-D-mannosamine
E = GlcNAc 2-epimerase, enzyme from Escherichia coli
Figure 19-42. Biocatalytical epimerization of glucosamine to

mannosamine (Marukin Shoyu).
(ManNAc).Since the equilibrium is on the side of the starting material, the reaction
is driven by the subsequent biotransformation of ManNAc together with pyruvate to
Neu5Ac.
The N-acylglucosamine2-epimerase is cloned from porcine kidney, transformed
and overexpressed in Escherichia coli. To reach maximal activitiy, ATP and Mg2+need
to be added. Since the whole synthesis is reversible, high GlcNAc concentrations are
used.
The chemical epimerization of GlcNAc is used by Glaxo. The equilibrium of the
chemical epimerization is on side of N-acetyl-D-glucosamine(G1cNAc:ManNAc =
4: 1).After neutralization and addition of isopropanol GlcNAc precipitates. In the
remaining solution a ratio of G1cNAc:ManNAc = 1: 1is reached. After evaporation to
dryness and extraction with methanol the ratio of G1cNAc:ManNAc is shifted to
1:4.
19.4
Some Misconceptions about Industrial Biotransformations
There are a lot of prejudices against biotransformations. The major ones are:
Biocatalysts are too expensive.
Biocatalysts only work under mild conditions.
The first prejudice that biocatalysts are too expensive is only partly true. If the cost
per mol or per unit weight is calculated they certainly are expensive. For example,
penicillin amidase costs $ 10 OOO/kg on a bulk scale. On the other hand the cost
contribution of penicillin amidase in the “splitting”of penicillin G is only $ l/kg of
product[117].In the case of L-aspartic acid production the cost contribution of
aspartase is even lower, $ O.l/kg. This demonstrates that it is not the absolute catalyst
cost but the cost contribution of the catalyst to the final product cost that has to be
considered and compared. This is also true for chemical catalysts;e. g., the bulk price
of BINAP is $ 4 0 OOO/kg[117].Important parameters influencing the cost contribu-
tion are the total turnover number (mol product/mol catalyst) and the turnover
frequency (mol product/mol catalyst and unit time).
The second prejudice, that biocatalysts only work in an aqueous phase with low
concentrations of starting material is also only partly true. The natural environment
1454
I Table 19-1. Highest concentrations applied in industrial biotransforrnations
EC enzyme substrate concentration medium
1.1.99.21 o-Sorbitol dehydrogenase 1-Amino-D-sorbitol 1.00 M Aqueous

(N-protected)
4.2.1.2 Fumarase Fumaric acid 1.00 M Aqueous
3.4.21.62 Subtilisin Phenylalanine isopropylester 1.20 M Aqueous
3.1.1.3 Lipase 1-Phenylethylamine 1.65 M MTBE
3.5.2.6 0-Lactamase y-Lactam 1.83 M Aqueous
4.2.1.84 Nitrile hydratase Adiponitrile 2.01 M Aqueous/
organic
4.3.1.1 L-Aspartase Fumaric acid 2.00 M Aqueous
4.1.1.12 Aspartate P-decarboxylase Aspartic acid 2.50 M Aqueous
3.1.1.25 Lactonase Pantolactone 2.69 M Aqueous
3.1.1.3 Lipase Palmitic acid 3.10 M 2-Propanol
3.1.1.3 Lipase Cyclopentenylester 4.16 M Aqueous/
organic
4.2.1.84 Nitrile hydratase Acrylonitrile 5.60 M Aqueous
(product)
4.3.1.5 L-Phenylalanineammonia- trans-Cinnamic acid 9.31 M Aqueous
lyase (NH3)
is in general the aqueous phase and ambient temperature. But the examples
described above demonstrate that biocatalysts can be also applied in emulsions or
even pure organic solvents (Table 19-1). Here, moreover, very high concentrations
are reached, e. g. in the case of acrylamide up to 5.6 M.
19.5
Outlook
Despite the progress biocatalysis has made in the last few years its potential is still
increasing. By improved screening methods new catalysts will be detected and made
available in large amounts by cloning and overexpression. Directed evolution will be
used to improve properties such as stability or selectivity[”’, . M etabolic engi-
neering will be used to analyze and remove bottlenecks in the metabolism or to
create novel biocatalysts[l2O1.
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20
Tabular Survey of Commercially Available Enzymes
Peter Rasor
Enzymes are catalysts. Nature has designed them to perform specific tasks necessary
for the survival of the organism producing the enzyme. The organic chemist tends to
name enzymes “biocatalysts”which means nothing more than catalysts of biological
origin.
These biocatalysts bring some confusion to the well-structured world of organic
chemistry:
the names are unfamiliar,
each enzyme has a variety of names which are all used making it as difficult as
distinguishing characters in a Russian novel (e.g. Penicillin G-amidase and
Penicillin acylase),
when it comes to microogranisms or plants, the origin of enzymes is described in
Latin (type face italic),
in order to add to the confusion, the names of microorganisms may change over
time, for example Pseudomonas cepacia is now Burkholderia cepacia, Candida
cylindracea is Candida rugosa,
even mammalian sources can be described differently - esterase from hog liver or
pig liver, but lipase from porcine pancreas (type face not italic).
For identifjmg synonyms or finding out the correct name of an enzyme, the Enzyme
Nomenclature Database (EC database) can be searched or downloaded under http://
www.expasy.ch/enzyme/.
If the chemist is still not confused and has mastered this hurdle, the manu-
facturers or suppliers introduce brand names for marketing reasons, and may even
change names once in a while. Additionally, not every supplier gives full information
on the origin of the biocatalyst and may use old names of microorganisms while
other suppliers already use new names.
Furthermore, the same biocatalyst by description may behave differently in a
specific reaction: for example, lipase from Candida rugosa from Amano (LipaseAY)
differs from Lipase MY or OF from Meito Sangyo with respect to activity and
stereoselectivitybecause it consists of a number of catalytically active species which
differ depending on the production strain used and thus, on the manufacturer.
1462
I 20 Tabular Survey ofCommercially Available Enzymes
Table 20-1. Abbreviation of most commonly used biocatalysts

Abbreviation Lipase from Abbreviation Lipase from
ANL Aspergillus niger PcamL Penicillium carnembertii
BCL (PCL) Burkholderia cepacia (formerly PFL Pseudomonasfluorescens
Pseudomonas cepacia)
CAL Candida antarctica PfragiL Pseudomonasjagi
CAL-A Candida antarctica, type A PPL Porcine pancreas
CAL-B Candida antarctica, type B ProqL Penicillium roquefortii
CLL Candida lipolytics PSL Pseudomonas sp.
CRL (CCL) Candida rugosa (formerly RML (MML) Rhizomucor miehei
C. cylindracea) (formerly Mucor miehei)
CVL Chromobacterium viscosum ROL Rhizopus oryzae (other names:
(identical to Pseudomonas RNL - Rhizopus niveus, RDL -
glumae) Rhizopus delemar, RJ L -
Rhizopusjavanicus)
GCL Geotrichum candidum TLL (HLL) Thermomyces lanuginosa
(formerly Humicola lanuginosa)
Abbreviation Esterase from Abbreviation Alcohol dehydrogenase from
PLE Pig liver YADH Yeast
TBADH Themoanaerobium brockii
HLADH Horse liver
Since the full enzyme name according to the EC nomenclature is rather long, the
most commonly used enzymes have gotten abbreviations. For esterases and lipases
there are certain rules: in most cases, the first (two or three) letters characterize the
source, the last the type of enzyme (E for esterase, L for lipases) (see Table 20-1).
Alcohol dehydrogenases are treated similarly.
All this may explain why many publications give only incomplete information on
the exact type of enzyme used in the work described and why many references to
enzymes are simply wrong. The author strongly recommends to provide at least the
following information:
Parameter Example 1 Example 2
Name of the product Lipase Type XI11 CHIRAZYME L-2, lyo

Description (if the name of Lipase from Pseudomonas Lipase from Candida a n t -
the product is a brand sp. arctica, type B
name or non-descriptive)
Formulation Powder Powder
Manufacturer Sigma Roche Diagnostics
Table20-2. Available screening sets
20 Tabular Survey ofCornrnercially Available Enzymes
I 1463
Enzyme type Company
Alcohol dehydrogenases ThermoGen, BioCatalytics

Esterases SC lipases Altus, Fluka, Roche, ThermoGen
Nitrilases BioCatalytics
Proteases Altus
Transaminases (aminotransferases) BioCatalytics
In the laboratory protocol, lot. no. and activity (incl. assay no. or assay conditions)
must be recorded as well in order to track variation in results because of lot to lot
inconsistency.
Every development of a biocatalytic reaction starts with a screening for the most
appropriate enzyme. Some companies offer screening sets (or kits) containing the
most commonly used enzymes (Table 20-2). Some Sets are single use (Altus,
ThermoGen) while others contain enough material to perform depending on the
scale 5-20 experiments (BioCatalytics, Fluka, Roche). These sets may include
enzymes available on industrial scale or on research scale only.
The following companies offer screening setlkits for quick enzyme selectiori
(Table 20-2). While some companies include only industrial scale enzymes, others
contain enzymes only available at lab quantities. Diversa Co. offers an enzyme
subscription program for lipases, esterases, nitrilases, cellulases, glycosidases,
phosphatases, and transaminases (aminotransferases).
Some enzymes of Novozymes A/S (formerly Novo Nordisk A/S) were widely
distributed on an experimental stage (SP nnn). Table 20-3 lists the most important
Table 20-3. List of experimental enzymes by Novozymes, current products names and suppliers
~
Old name Characterization Current brand name Availability
SP 361 Immobilized enzyme mixture discontinued

SP 409 from Rhodococcus sp. containing
nitrilase, nitril hydratase,esterase,
epoxide hydrolase and amidase
activity
S P 382 Immobilized lipase from Candida discontinued
antarctica, containing type A & B
SP 435 Immobilized lipase from Candida Novozym 435 Novo-Nordisk
antarctica, type B, rec. in Asper- CHIRAZYME L-2, Carrier 2 Roche Diagnostics
gillus oryzae
SP 523 Lipase powder from Thermomy- CHIRAZYME L-8, lyo Roche Diagnostics
ces lanuginosus (formerly Humi-
cola lanuginosa)
SP 524 Lipase powder from Rhizomucor CHIRAZYME L-9, lyo Roche Diagnostics
miehei, rec. in Aspergillus oryzae
SP 525 Lipase powder from Candida ant- CHIRAZYME L-2, lyo Roche Diagnostics
arctica, type B, rec. in Aspergillus
oryzae
S P 526 Lipase from Candida antarctica, CHIRAZYME L-5, lyo Roche Diagnostics
type A, rec. in Aspergillus oryzae
20 Tabular Survey ofComrnercially Available Enzymes
Table 20-4. Enzyme producers/suppliers and brief characterization
Company Adress Tel./Fax/Email/WWW Focus/Characterization’

Altus Altus Biologics lnc. Tel.: +1(G17) 299-2900 Manufacturer of stabilized
625 Putnam Avenue Fax: +1 (617) 299-2999 enzymes for use in indus-
Cambridge, MA 02139-4807 Email: info@altus.com trial, biocatalytical,diagnos-
USA http://www.altus.com tic, and medicinal applica-
tions. No enzyme produc-
tion itself. Biocatalytical
process development.
Amano Amano Pharmaceutical Co., Tel.: +81 (52) 211-3032 Specialtyenzyme producer
Ltd. Fax: +81 (52) 211-3054 for industrial, biocatalytical,
2-7, 1-chome,Nishiki http://www.amano-enzyme.co.jp diagnostic, and medicinal
Naka-ku, applications.
Nagoya, 460-8630
Japan
Asahi Diagnostics Division Tel.: +81 (3) 3259-5776 Specialityenzyme producer

Chemical Hibiya-Mitsui Building Fax: +81 (3) 325S-5741 for diagnostic and medici-
Industry Co. 1-2 Yurakucho 1-chome, Email: nal applications.
Chiyoda-ku shindan@ml.asahi-kasei.co.jp
Tokyo 100-8440 http://www.asahi-kasei.co.jp
lapan
Biocatalysts Biocatalysts Ltd Tel.: +44 (0) 1443843712 Producer and distributor of
Main Avenue, Treforest Fax: +44 (0) 1443 84 12 14 enzymes for use in indus-
lndustrial Estate Email: sales@biocats.com trial and diagnostic applica-
Pontypridd, Wales, http://www.biocatalysts.com tions.
CF37 5UD
United Kingdom
BioCataly- BioCatalytics Inc. Tel.: +1 (626) 229-0588 Biocatalytical process devel-

tics 39 Congress Street, Fax: +1 (626) 535-9465 opment. Experimental en-
Suite 303 Email info@biocatalytics.com zymes for biocatalysis. Lim-
Pasadena, CA 91105-3022 http://www.biocatalytics.com ited production capacity.
USA Distributor for Roche in
USA and Canada.
Biozyme USA and Canada: Tel.: +1 (858) 549-4484 or Specialityenzyme producer

Laboratories 9939 Hibert Street (800) 423-8199 for diagnostic and medici-
Inter- Suite 101 Fax: (858) 549-01 38 nal applications
national Ltd. San Diego, CA 92131-1029 Email: bioinfo@biozyme.com
USA
All other countries: Tel.: (+44)1495790678

Biozyme Laboratories Ltd Fax: (+44) 1495791780
Unit 6, Gilchrist Email: info@biozyme.co.uk
Thomas Estate http://www.biozyme.com/
Blaenavon, South Wales,
N P 4 9RL
United Kingdom
Calbiochem. Calbiochem-Novabiochem Tel.: +1 (858) 4509600 or Supplier of enzymes and

co., Corporation (800)8543417 biochemicals on research
CN Bio- 10394 Pacific Center Court Fax: +I (858) 45335 52 scale. Focus on life science,
sciences San Diego, CA 92121 Email: orders@calbiochem.com. not biocatalysis.
Mailing Address: technical@calbiochem.com
P.O. Box 12087 http: //www.calbiochem.com
La Jolla, CA 92039-2087 http://www.cnbi.com
USA
20 Tabular Survey ofComrnercially Available Enzymes I1465
Company Adress Tel./Fax/Ernail/WWW Focus/C haracterization’

Diversa Diversa Corporation Tel.: +1 (858) 526-5000 Discovery and development
4955 Directors Place Fax: +1 (858) 5 2 6 5 5 51 of industrial enzymes.
San Diego, CA 92121-1609 Email information@diversa.com No general biocatalyst
USA http://www.diversa.com portfolio.
DSM Gist- DSM Food Specialties Tel.: +31 (15) 2793474 Enzyme producer for
Brocades P. 0. Box 1 Fax: +31 (15) 2793540 industrial applications
2600 MA Delft http://www.dsm.nl/dfs/ (feed &food).
The Netherlands
Fluka see Sigma-AldrichFluka
Genencor Genencor International, Inc. Tel.: +1 (716) 256-5200 Enzyme producer for
200 Meridian Centre Blvd. Fax: +1 (716) 2566952 industrial applications
Rochester, NY 14618-3916 Email: ysmith@genencor.com
USA http://www.genencor.corn
Jiilich Juelich Enzyme Products Tel.: +49 (2461) 348188 Experimental enzymes for
Enzyme GmbH Fax: +49 (2461) 348186 biocatalysis. Limited
Products Karl-Heinz-Beckurts-Str. 13 E-mail juelichep@aol.com enzyme production
D-52428 Jiilich http://www.juelich-enzyme.com capacity.
Germany
Lee Lee Scientific Inc. Tel.: +1 (314) 968-1091 Specialty enzyme producer.
Scientific 2924 Mary Ave. Fax: +1 (314) 968-9851 Focus on life science and
St. Louis, MO 63144 Email: diagnostics. Some bio-
USA burtonlee@leescientific.com catalysts.
http://www.leescientific.corn/
Meito Fine Chemicals Dept. Tel.: +81 (3) 3242-1795 Producer and distributor of
Sangyo Co. Meito Sangyo Co. Ltd. Fax: +81 (3) 3242-1792 enzymes for use in indus-
Ltd. Sankeido Bldg., Email: jdt02625@niftyne.jp trial and diagnostic applica-
4 3 - 1 5 , Muromachi, tions.
Nihonhashi
Chuo-ku, Tokyo 103-0022
Japan
Novozyme Europe, Middle East & http://www.novozymes.com Largest enzyme producer
A/ s Africa: for industrial applications.
Novozymes France S. A. Distribution agreement
lmmeuble Challenge 92 Tel.: +33 146140746 with Roche for chiral
79, Avenue Frantois Arago Fax: +33 146140766 organic synthesis market.
92017 Nanterre Cedex,
France
Latin America: Tel.: +55 41641 1000

Novozymes Latin America Fax: +55 416431443
Limited
Rua professor Francisco
Ribeiro 683
CEP 83707-660 - Araucaria
- Parana
Brazil
20 Tabular Survey ofCommercially Available Enzymes
Company Adress Tel./Fax/Emailm Focus/Characterization’

USA:
Novozymes North America
Inc.
77 Perry Chapel Church
Road
Franklinton, N. C. 27525
Postal Address:
State Road 1003 Tel.: +19194943000
P.O. BOX 576 Fax: +19194943450
Franklinton, NC 27525
Asia Pacific, Hong Kong:

Novozymes Asia Pacific
Regional Office
7/F Chinachem Century
Tower Tel.: +852 25193380
178 Gloucester Road, Fax: +852 28 77 06 59
Wanchai
Recordati Via Matteo Civitali, I Tel.: +39 (02) 487871 Manufacturer of industrial
S.p.A. 20148 Milan http://www.recordati.it enzymes for beta-lactam an-
Italy tibiotics.
Roche Roche Diagnostics GmbH Tel.: +49 (621) 7598593 Specialityenzyme producer
Diagnostics Roche Molecular Bio- Fax: +49 (621) 7598986 for industrial, biocatalytical,
chemicals Email: ute.hill@roche.com diagnostic, and medicinal
Sandhofer Str. 116 http://indbio.roche.com applications. Broad range of
68298 Mannheim enzymes.
Germany
USA & Canada:

Refer to BioCatalytics Inc.
Seravac Seravac USA, Inc. Tel.: +1 (858) 679-4050 or Specialityenzyme producer

13220 Evening Creek Drive (800) 679-40 50 for diagnostic and medici-
San Diego, CA 92128 Fax: (858) 679-1438 nal applications.
USA Email: enzymes@seravac.com
http://www.seravac.com
Sigma- Sigma Co. Tel.: (314) 771-5765 Manufacturer and distribu-

Aldrich 3050 Spruce Street Fax: (314) 771-5757 tor of enzymes and bio-
Fluka (SAF) St. Louis, MO 63103 Email: sigma@sial.com chemicals on research
Mail: P. 0. Box 14508 http://www.sigma-aldrich.com scale.
St. Louis, MO 63178 Very broad range of
USA enzymes (Sigma and
Fluka). Limited range of
Fluka Chemical LLC. Tel.: +41 (81) 7552828 biocatalysts at Aldrich.
Industriestrasse 25 Fax: +41 (81) 756 5449 Within the group, Fluka has
CH-9471 Buchs EMail: fluka@sial.com the focus on biocatalysts on
Mail P. 0. Box 260 http://www.sigma-aldrich.com research scale. Production
CH-9471 Buchs of selected enzymes up to
Switzerland medium scale.
ThermoGen ThermoCen, Inc. Tel.: +1 (630) 783-4600 Enzyme discovery. Limited
2501 Davey Road Fax: +1 (630) 783-4909 enzyme production
Woolridge, IL 60517 info@thermogen.com capacity. Biocatalyhcal
USA http://www.thermogen.com process development.
20 Tabular Survey ofCommercially Available Enlymt!s

-
Company Adress Tel./Fax/Ernail/WWW Focus/Characterization I
Toyobo Co. Toyobo Co. Ltd. Tel.: +81 (3) 3660-4819 Speciality enzyme producer
Ltd. Biochemical Operations Fax: +81 (3) 366&4951 for diagnostic and medici-
Department EMail: nal applications.
17-9 Nihonbashi Koami-cho toshiro-ki!achi@bio.toyobo.co.jp
Chuo-ku http://www.toyobo.co.jp/e/
Tokyo 103-8530
Japan
Unitica Ltd. Medical Products Division Tel.: +81(6) 6281-5021 Specialty enzyme producer
Unitika Ltd. Fax: +81 (6) 6281-5256 for diagnostic and medici-
4-1-3, Kyutaro-machi, Email : medical@unitika.co.jp nal applications.
Chuo-ku, Osaka 541-8566 http://www.unitika.co.jp/
Japan home-e.htm
Wako Pure 1-2, Doshomachi 3-Chome, Tel.: +81 (6)6203-3741 Manufacturer and distribu-
Chemicals Chuo-Ku, Osaka 54&8605 Fax: +81(6) 6222-1203 tor of enzymes and bio-
Industries, Japan http://search.wako-chem.co.jp chemicals on research
Ltd. scale. Focus on life science,
not biocatalysis.
Worthing- Worthington Biochemical Tel.: +1(732)942-1660 Manufacturer and distribu-
ton Bioche- Corp. Fax: +1 (732) 942-9270 tor of enzymes and bio-
mica1 730 Vassar Ave http://www.worthington- chemicals. Focus on life
Lakewood, NI 08701 biochem.com/ science and diagnostics.
1 Industrial applications include detergents, feed and food, pulp & paper, etc.
enzymes and gives the current brand names, wherever possible. Some enzymes have
been discontinued at Novozymes but replacements are available from Roche
Diagnostics (CHIRAZYME product line).
Catalytic antibodies are not yet widely available. Aldrich is offering two aldolase
monoclonal antibodies.
The major enzyme producers and/or suppliers are listed and briefly characterized
in Table 20-4. The author is aware that the list of enzyme producers is not
complete.
The author has made the attempt to list enzymes that are commercially available
(Table 20-5)and thus can be used in biocatalysis. He knows that the list is incomplete
and therefore, the reader should not rely solely on this list but rather check the
suppliers listed in Table 20-4. Enzyme manufacturers also update their product
portfolio continuously, so this list probably needs updating before the book is even i n
print.
A special word is necessary with respect to the Sigma-Aldrich-Flukaconglomerate:
Fluka ha:; taken the lead in biocatalysis, while Sigma serves mostly the life science
market. Especially since the Sigma catalog is a book in itself, only enzymes from
Fluka are listed. The reader should be aware that the majority of enzymes is available
from Sigma as well, and with respect to enzymes not typically used in biocatalysis,
the portfolio may be even greater.
Explanations to Table 20-5:

The table is sorted by the EC number. In most cases the number is given in the
1468
respective chapter and can be used to find the enzyme in the table. If the EC no. is
not known, at least the general reaction of the enzyme class is given according to the
EC nomenclature.
Underneath the EC name, synonyms are given. The general reaction according EC
nomenclature is denoted too. Afterwards, the product (enzyme) names are listed, one
entry for each manufacturer per enzyme. If the product is sold under a brand name,
this name is listed too. In one enzyme class, the entries are sorted by origin.
The availability is characterized in three categories: lab, pilot and industrial scale.
It refers to the scale with respect to biocatalytical reactions. The author recognized
that this categorization is somewhat arbitrary and in some cases may not be correct
because the actual production scale is not generally known. Hopefully, though, it will
prove to be useful as a rough guide.
Enzyme producers are devoted to certain markets like food & feed, detergents,
diagnostics or research. Large enzyme producers such as Novozymes, Genencor or
DSM Gist-brocadesare categorized as “industrial”, specialty enzyme producers like
Amano, Asahi or Roche Diagnostics serve various markets and thus, scale varies
from pilot to industrial. Since the enzyme demand for diagnostics is much lower
than for biocatalysis, typical diagnostic enzymes are labeled as “pilot” although the
manufacturing process is certainly standardized and therefore, could be call “in-
dustrial” as well. Companies serving the life sciences market (e.g. Sigma-Aldrich
Fluka, Roche Diagnostics) have manufacturing capacities from small (“lab”)scale to
medium scale (here termed as “pilot”).It should also be recognized that the term
“pilot scale” in a context other than this table has a different meaning when
comparing for example Sigma, Roche Diagnostics, and Novozymes.
Table 20.5. Commercially available enzymes.
I 1469
Oxidoreductases. 1.1.1.-
Acting on the CH-OH group of donors.
With NADI+I or NADPf+) as accedor.
Alcohol Dehydrogenase Screening Kit; Origin: microorganism, rec. in E. coli
ThemoGen: ThermoCat Alcohol Dehydrogenase Kits Lab
Ketoreductase, broad-range; Origin: microorganism, rec. in E. coli
BioCatalytics: KRED- 1001 Lab
Ketoreductase, broad-range, Ongin microorganism, rec in E coli
BioCatalytics KRED-1002 Lab
Ketoreductase, broad-range, Ongin microorganism, rec in E cob
BioCatalytic s KRED- 1003 Lab
BioCatalytics: KRED-I004 Lab
BioCatalytics: KRED-1005 Lab
BioCatalytics: KRED-1006 Lab
Ketoreductase, broad-range; Ongin microorganism, rec in E coli
BioCatalytiLs KRED-1007 Lab
Ketoreductase, broad-range, Ongin. microorganism, rec. in E coli
BioCatalytiLs KRED-1008 Lab
Cholesterol Dehydrogenase, Ongin Nocardid sp
Amano: Ainano 5 [CHDH-51 Pilot
7-Hydroxysteroid Dehydrogenase, Ongin P\eudomonda sp.
Asahi Pilot
Alcohol dehydrogenase. 1.1.1.1

_I -
Aldehyde reductase
~ _ _ X ~ X *- I /_II
Alcohol Dehydrogenase, Ongin. Candida parapsilosis

~ * x x " % =
Julich Enzyrne Products Lab
Fluka Lab
Biocatalysts Sec ADH 300 Lab

Alcohol Dehydrogenase, Ongin Rhodococcus elythropolis
Julicb Enzyrne Products Lab
Alcohol Dehydrogenase, Ongin yeast
Biozyme Pilot
Fluka Pilot
Roche Diagnostics Alcohol Dehydrogenase (YADH), lyo Pilot
Alcohol Dehydrogenase Ohgin yeast
Roche Diagriostics Alcohol Dehydrogenase (YADH), susp Pilot
Alcohol Dehydrogenase, Ongin Zymomonas mobilis
Alcohol dehydrogenase (NADP+). 1.1.1.2

Aldehyde reductase (NADPH) An alcohol + NADP(+) = an aldehyde + NADPH
Alcohol Dehydrogenase, Ongin Lactobacillus kefir
Fluka PllOt
1470

Alcohol Dehydrogenase,Ongin Lactobacillus kefir
Julich Enzyme Products Lab
Alcohol Dehydrogenase:Ongin Thermoanaerobium brockii
nuka Pilot
Acetoin dehydrogenase. 1.1.1.5

Diacetyl reductase Acetoin + NAD(+) = diacetvl + NADH
Acetoin Dehydrogenase,Ongin Lactobacillus kefir
Fluka Lab
Diketone Reductase, Ongin Lactobacillus kefir
Glycerol dehydrogenase. 1.1.1.6

Glycerol + NAD(+) = glycerone + NADH
Glycerol Dehydrogenase,Ongin Bacillus megatenum
Asahi Pilot
Glycerol Dehydrogenase: Ongin Geotnchum candidum
Fluka Lab
Glycerol Dehydrogenase,Ongin Klebsiella pneumoniae (formerly Enterobacter aerogenes)
Roche Diagnostics Glycerol Dehydrogenase Lab
Glycerol Dehydrogenase (GIDH), Ongin nucroorganisms
Unitika Pilot
Glycerol-3-phosphate dehydrogenase (NAD+). 1.1.1.a

Sn-glycerol3-phosphate+ NAD(+) = glycerone phosphate +
L-iditol2-dehydrogenase.
ngin microorganism?
Pilot
Sorbitol Dehydrogenase,Ongin sheep liver
Fluka Lab
(S)-lactate + NAD(+) = pyruvate + NADH
Biozyme Pilot
L-Lactate Dehydrogenase,Ongin. bovine heart
Fluka Lab
L(+)-Lactate Dehydrogenase,Ongin hog muscle
Roche Diagnostics L(+)-Lact PllOt
L-Lactate dehydrogenase,
Biozyme Pilot
L(+)-Lactate Dehydrogenase,Origin pig muscle
Roche Diagnostics L(+)-Lactate Dehydrogenase (L-LDH) PllOt
L-Lactate dehydrogenase,0
Biozyme Pilot

I1471
L-Lactate dehydrogenase,Ongin rabbit muscle

Biozyme Pilot
L-Lactate Dehydrogenase, Ongin rabbit muscle
Fluka Lab
I I
Lactate Dehydrogenase,Ongin Staphylococcus sp

Amano Aniano 3 [LDH-31 Pilot
D-lactate dehydrogenase. 1.1.I .28

D-lacuc acid dehydrogenase D-lactic dehydrogenase (R)-lactate + NAD(+) = pyruvate + NADH
D-Lactate Dehydrogenase;Origin: Lactobacillus leichmanii
Fluka Lab
D(-)-Lactate Dehydrogenase;Ongin: Lactobacillus leichmannii
Roche Diagnostics: D(-)-Lactate Dehydrogenase (D-LDH) Pilot
D-Lactate Dehydrogenase;Origin: microorganisms
Toyobo Pilot
D-Lactate Dehydrogenase,Ongin nucroorganisms
Umtika D-Lactate Dehydrogenase (D-LDH) Pilot
B
3-hydroxybutyrate dehydrogenase. 1.I .I .30

D-beta-hydroxybutyrate dehydrogenase (R)-3-hydroxybutanoate + NAD(+) = acetoacetate + NADH
~ ycI~- ~~ "_
__^__x
_rllx, I__y- ~ _ x ~ c "- c_ y__ 1_1 xxx _x- .>^_ -* *-"* ~ I 1 -
3-Hydroxybutyrate Dehydrogenase
Asahi Pilot
D-3-HydrorybutyrateDehydrogenase;Origin: Pseudomonas sp.
Toyobo Pilot
3-Hydroxybutyrate Dehydrogenase;Origin: Rhodobacter sphaeroides (formerly Rhodopseudomonas sphaeroides)
Roche Diagnostics: 3-Hydroxybutyrate Dehydrogenase (3-HBDH), Grade I1 Lab
3-Hydroxybutyrate Dehydrogenase;Origin: Rhodopseudomonas spheroides
Fluka Lab
Malate dehydrogenase. 1.I .I .37

Malic dehydrogenase
Y l -~ _I c_ X r x x x
Malate Dehydrogenase,Ongin microorganisms

~ ~ 1_ _x L --
(S)-malate + NAD(+) = oxaloacetate + NADH
Tovoho Pilot
Malate Dehydrogenase,Ongin rmcroorganisms
Urukka Malate Dehydrogenase (MDH) Pilot
- 1 1 1
Malate dehydrogenase,Ongin pig heart

Biozyme Pilot
Malate Dehydrogenase,Ongin porcine heart
Fluka Lab
Amano. Ammo 3 [MDH-31 Pilot
lsocitrate Dehydrogenase,Ongin porcine heart

Fluka Lab
lsocitrate Dehydrogenase,Ongin. porcine heart
Ruka Lab
1472
I 20 Tabular Survey ofComrnercially Available Enzymes
Phosphogluconate dehydrogenase 1.I .I .44

(decarboxylating).
Phosphogluconic acid dehydrogenase 6-phosphogluconic 6-phospho-D-gluconate + NADP(+) = D-nbulose 5-phosphate +
dehydrogenase 6-phosphogluconic carboxylase 6PGD CO(2) +NADPH
6-PhosphogluconateDehydrogenase (6PGDH). Ongin Thermoactinomces intermedius

Unitika Pilot
6-PhosphogluconicDehydrogenase,Ongin Torula yeast
Fluka Lab
6-Phosphogluconic Dehydrogenase:Ongin yeaa
Fluka Lab
Glucose 1-dehydrogenase. 1.1.1.47

Beta-D-glucose + NAD(P)(+) = D-glucono-l,5-lactone + NAD(P)H
Fluka Pilot
Glucose Dehydrogenase,Ongin Bacillus sp
Amano Amano 2 [GLUCDH-2] Pilot
Glucose Dehydrogenase,Ongin. Cryptococcus un~guttulatus
Ash Pilot
Glucose Dehydrogenase,Ongin microorganisms
G6PD D-glucose 6-phosphate + NADP(+) = D-glucono-l,5-lactone

6-phosphate +NADPH
Glucose-6-Phosphate Dehydrogenase
Asahi Pilot
Glucose-6-Phosphate Dehydrogenase (GGPDH), Ongin Bacillus stearothermophilus
Unitika PllOt
Glucose-6-phosphate Dehydrogenase,Ongin baker's yeast
Fluka PllOt
Glucose-6-phosphatedehydrogenase,Ongin Leuconostoc mesenteroides
Biozyme Pilot
Glucose-6-phosphate Dehydrogenase,Ongm Leuconostoc mesenteroides
Fluka Pilot
Glucose-6-PhosphateDehydrogenase, Ongin Leuconostoc mesenteroides
Toyobo Pilot
Glucose-6-phosphate Dehydrogenase, Ongin Leuconostoc mesenteroides
Roche Diagnostics Glucose-6-phosphate Dehydrogenase (G6P-DH). susp PllOt
Glucose-6-phosphate Dehydrogenase,Ongin Leuconostoc mesenteroides, rec in E coli
Roche Diagnostics Glucose-6-phosphdte Dehydrogenase (G6P-DH). lyo PllOt
Glucose-6-phosphate Dehydrogenase,Ongin Torula yeast
Fluka Lab
Glucose-6-phosphatedehydrogenase,Origin yeast
Biozyme Pilot
Glucose-6-phosphate Dehydrogenase,Ongin yeast
Fluka Pilot
Glucose-6-phosphateDehydrogenase,Ongin yeast
Fluka Pilot
Glucose-6-phosphateDehydrogenase,Ongin yeast
Roche Diagnostics Glucose-6-phosphate Dehydrogenase (G6P-DH), lyo Pilot

I 1473
Glucose-6-Phosphate Dehydrogenase(GBPDH),Ongin Zymomonas mobilis

Unitika Pilot
3-alpha-hydroxysteroid dehydrogenase 1.1.1.50

(B-specific).
Hydroxyproc taglandin dehydrogenase 3-alpha-HSD Androsterone + NAD(P)(+) = 5-alpha-androstane-3,17-dione+
NAD(P)H.
3-Hydroxysteroid Dehydrogenase
Asahi Pilot
3-alpha-Hydroxysteroid Dehydrogenase(3alphaHSDH), Ongin mcroorganisms
Unitika Pilot
3-alpha-Hydroxysteroid Dehydrogenase, Ongin Pseudomonas testosteroni
Fluka Lab
Xanthine + NAD(+) + H(2)O = urat
Xanthine Olehydrogenase
Asahi Pilot
12-alpha-HydroxysteroidDehydrogenasePP ; Origin: Clostridium spec.
Jiilich Enzyme Products Lab
12-alpha-Hydroxysteroid Dehydrogenase, Ongin mcroorganisms
Asahi Pilot
Glucose oxidase. 1.1.3.4

Glucose oxyhydrase Beta-D-glucose oxygen 1-oxide-reductase Beta-D-glucose + O(2) = D-glucono-l,$-lactone+ H(2)0(2)
Glucose aerodehydrogenase D Glucose- 1-oxidase
y_x_ _ _ - c _ r _
Glucose Oxidase
> ~ x x x __ - I)
Seravac Industnal
Glucose oxidase, Ongin A\pergillus niger
Ammo Hyderase Industnal
I __ ~ __
Glucose oxidase,Ongin Aspergillus niger
Amano Hyderase L Industnal
I
Glucose oxidase, Ongin A5pergillus niger

Biozyme Pilot
Glucose Oxidase, Ongin Aspergillu$ niger
Fluka Industnal
Glucose oxidase, Ongin A\pergillus niger
Novozymes Gluzyme@ Industnal
Glucose Oxidase, Ongin Aspergillus niger overproducer
Roche Diagnostics Glucose Oxidase (GOD) Industnal
Amano Ammo 2 [GO-2] Pilot

Glucose Oxidase, Ongin A\pergillus sp
Amano Ainano LC [GOLC] Pilot
Glucose Oxidase, Ongin Aspergillus sp
Amano Arnano LD2 [GOLD-21 Pilot
Glucose Oxidase, Ongin Aspergillus sp
Toyobo Pilot
Glucose Oxidase, Ongin microorganism, rec in yeast
Roche Diagnostics Glucose Oxidase (GOD) Pilot
Glucose Oxidase, Ongin Penicillium sp
Biocatalysts Industrial
1474
Cholesterol oxidase. 1.1.3.6

Cholesterol-02 oxidoreductase Cholesterol + O(2) = cholest-4-en-3-one + H(2)0(2)
Asahi Pdot
Cholesterol Oxidase, Ongin Brevibactenum sterolicum, rec in rmcroorganiqm
Roche Diagnostics Cholesterol Oxidase Pilot
Cholesterol Oxidase, Ongin rmcroorganiams
Ammo Amano 6 [CHO-6] Pilot
Cholesterol Oxidase, Ongin mi~roorganisms
Asahi Pilot
Cholesterol Oxidase, Ongin rmuoorganisms
Toyobo Pilot
Cholesterol Oxidase, Ongin Nocardia erythropolis
Fluka PllOt
Cholesterol Oxidase, Ongin Pseudomonas sp
Amano Ammo 1 [CHO-I] Pdot
Cholesterol Oxidase, Ongin Pseudomonas sp
Ammo Amano 2 [CHO-2] Pilot
Cholesterol Oxidase: Ongin Pseudomonas sp
Fluka Pilot
Cholesterol Oxidase, Ongin Streptomyces cinnamomeus
Asahi Pilot
Galactose oxidase. 1.1.3.9

Beta-Galactose oxidase D-galactose + O(2) = D-galacto-hexodialose + H(2)0(2)
Galactose Dehydrogenase, Ongin Agrobactenum sp

Biocatalysts Pilot
Alcohol oxidase. 1.1.3.13

Methanol oxidase AOX A pnmary alcohol + O(2) = an aldehyde + H(2)0(2)
Alcohol Oxidase; Ongin Candida sp

As& PllOt
Alcohol oxidase, broad-range: Ongin microorganism, rec in E coli
BioCatalytics BRAO- 100I Ldb
Alcohol oxidase, Origin Pichia pastons
Biozyme Pilot
Alcohol Oxidase, Ongin Pichia pastons
Choline oxidase. 1.1.3.17

Choline + O(2) = betame aldehyde + H(2)0(2)
Choline Oxidase; Origin: Alcaligenes sp
Fluka Pilot
Choline Oxidase, Ongin Arthrobacter globiformis
Asahi Pilot
Glycerol-3-phosphate oxidase. 1.1.3.21

Sn-glycerol 3-phosphate + O(2) = glycerone phosphate + H(2)0(2)
L-GlycerophosphateOxidase
Asahi Pdot
Glycerol 3-phosphate Oxidase, Ongin Aerococcus vindans
Fluka Lab
20 Tabular Survey ofCornrnercially Available Enzyrncs
I 1475
L-GlycerophosphateOxidase; Origin: Aerococcus viridans

Asahi Pilot
L-Glycerol-3-phosphateOxidase; Origin: microorganism, rec. in E. coli
Roche Diagnostics: L-Glycerol-3-phosphate Oxidase (GPO), stabilized Pilot
L-alpha-GlycerophosphateOxidase; Origin: microorganisms
Toyobo Pilot
L-alpha-Glycerophosphate Oxidase; Origin: Pediococcus sp.
Toyobo Pilot
L-alpha-Glycerophosphate Oxidase; Ongin Streptococcus \p
Amano Arnano 2 [GPO-2] PllOt
wm&
Xanthine oxidase. 1.I
.3.22
Xanthine oxidoreductase Hypoxanthine oxidase Xanthine + H(2)O + O(2) = urate + H(2)0(2)
Hypoxanthiiie-xanthine oxidase Schardinger enzyme
Xanthine oxidase, Ongin buttemulk
Biozyme Pilot
Xanthine Oxidase, Ongin buttermilk
Fluka Pilot
Xanthine Oxidase, Ongin cow rmlk
Pilot
Fructose 5-dehydrogenase. 1.1.99.1 1

D-Fmctose dehydrogenase D-fructose + acceptor = 5-dehydro-D-fmcto\e + reduced acceptor
D-Fructose Dehydrogenase, Ongin Gluconobacter sp
Toyobo Pilot
Formate dehydrogenase. 1.2.1.2

~ -
_I”*
Formate nase;
Fluka Pilot
Formate Dehydrogenase; Origin: Candida boidinii
Formate Dehydrogenase, rec., Ongin: Candida boidinii, overexpressed in E. coli
Roche Diagnostics. Formate Dehydrogenase (FDH), rec Industrial
Formate Dehydrogenase rec., Ongin E coli
nuka Lab
Formate Dehydrogenase, Ongin microorganisms
Unitika Formate Dehydrogenase (FDH) Pilot
Formate Dehydrogenase, Ongin Pseudomonas sp
Fluka Lab
Formate Dehydrogenase, Ongin Pseudomonas sp
Fluka Lab
Formate Dehydrogenase, Ongin Xilana digitata (formerly Candida biodinii)
Roche Diagnostics Formate Dehydrogenase (FDH) Industnal
Formate Dehydrogenase, Origin yeast
Fluka Pilot
Aldehyde dehydrogenase (NAD(P)+). 1.2.1.5

An aldehyde + NAD(P)(+) + H(2)O = an acid + NAD(P)H
Aldehyde Dehydrogenase, Ongin baker’s yeast
Fluka Lab
1476
Aldehyde dehydrogenase, Ongin yeast
Biozyme Pilot
Aldehyde Dehydrogenase; Ongin yeast
Glyceraldehyde 3-phosphate dehydrogenase 1.2.1.12

(phosphorylating).
NAD-dependent glyceraldehyde-3-phosphatedehydrogenase D-glyceraldehyde 3-phosphate + phosphate + NAD(+) =
Tnosephosphate dehydrogenase GAPDH 3-phospho-D-glyceroyl phosphate + NADH
Glyceraldehyde-3-Phosphate Dehydrogenase (GapDH); Origin: Bacillus stearothermophilus

Unitika Pilot
Glyceraldehyde-3-phosphate dehydrogenase; Origin: rabbit muscle

Biozyme Pilot
Glyceraldehyde-3-phosphate Dehydrogenase, Ongin rabbit m u d e

Fluka Ldh
AD(+) + H(2)O = formate +
Formaldehyde Dehydrogenase, Ongin Pseudomonay putida

Fluka Lab
Formaldehyde Dehydrogenase, Ongin Pseudomonas sp
Toyoho Pilot
PyNvlL oxidase Pyruvate + phosphate + O(2)+ H(2)O = acetyl phosphate + CO(2)

+H(2)0(2)
Pyruvate Oxidase , On
Asahi Pilot
Pyruvate Oxidase, Ongin Lactobacillus plantarum, rec E coli
Pilot
Bilirubin oxidase. 1.3.3.5

Bilirubin + O(2)= biliverdin + H(2)O
Acyl-CoA oxidase. 1.3.3.6

Acyl-CoA + O(2)= trany-2.3-dehydroacyl-CoA + H(2)0(2)
Acyl-CoA Oxidase, Ongin Arthrobacter sp

Asahi Pilot
Acyl-CoA Oxidase, Ongin mcroorganism~
Amano Ammo 3 [ACO-3] Pilot
Alanine dehydrogenase.
NH(3) + NADH.
Alanine Dehydrogenase
Asahi Pilot
L-Alanine Dehydrogenase, Ongin Bacillus cereu5
Alanine Dehydrogenase, Ongin Bacillus ytearothermophilus
Unitika Alanine Dehydrogenase (AlaDH) Pilot
L-Alanine Dehydrogenase, Ongin Bacillus subtili\
Fluka Lab
20 Tabular Survey of Commercially Available Enzymes
I
-
1477
il!
Glutamate dehydrogenase (NAD(P)+). 1.4.1.3

Glutamic dehydrogenase. L-glutamate + H(2)O + NAD(P)(+) = 2-oxoglutarate + NH(3) +
NAD(P)H
Glutamate dehydrogenase; Origin: beef liver
Biozyme Pilot
Glutamate Dehydrogenase; Origin: bovine liver
Fluka Pilot
L-Glutamate Dehydrogenase; Origin: bovine liver
Roche Diagnostics: L-Glutamate Dehydrogenase (GlDH), lyo. Pilot
Glutamate Dehydrogenase; Origin: microorganisms
Toyobo Pilot
Glutamate Dehydrogenase, Ongin Proteus sp
Toyobo Pilot
Leucine dehydrogenase. 1.4.1.9

L-leucine + H(2)O + NAD(+) = 4-methyl-2-oxopentanoate + NH(3)
+ NADH
Leucine Dehydrogenase; Origin: Bacillus cereus
Biocatalysts Pilot
Leucine Dehydrogenase; Origin: Bacillus sp
Toyobo Pilot
Leucine Dehydrogenase, Ongin Bacillus stearothermophilus
Unitika Leucine Dehydrogenase (LeuDH) Pilot
L-phenylalanine + H(2)O + NAD(+) = phenylpymvate + NH(3) +

L I ~ ^ _ - --
I _x
Phenylala S
Unitika Phenylalanine Dehydrogenase (PheDH) Pilot
PhenylalanineDehydrogenase, Ongin Spororarcina sp
D-amino acid oxidase. 1.4.3.3

A D-amino acid + H(2)O + O(2) = a 2-0x0 acid + NH(3) +
H(2)0(2)
D-Amino Acid Oxidase; Origin: hog kidney
Fluka Lab
D-Amino Acid Oxidase; Origin: hog kidney
Fluka Lab
D-Amino Acid oxidase, Ongin porcine kidney
Bioryme PllOt
D-Amino Acid Oxidase, Ongin Tngonopsis vanabilir
Recordati DAAO Beads Industnal
D-Amino Acid Oxidase, carrier-fixed, Ongin: Tngonopsis vanabilis
Roche Diagnostics D-Armno Acid Oxidase (D-AOD), caner-fixed lndustnal
D-Amino acid Oxidase, immobilized, Ongin Tngonopsis vanabilis
Fluka Industnal
Amine oxidase (flavin-containing). 1.4.3.4

Monoamine oxidase Tyramine oxidase Tyraminase Amine RCH(2)NH(2) + H(2)O + O(2) = RCHO + NH(3) + H(2)0(2)
oxidase
Tyramine Oxidase; Origin: Arthrobacter sp.
Asahi Pilot
1478

ewmm*m*
Dihydrofolate reductase. 1.5.1.3
5,6,7,8-tetrahydrofolate+ NADP(+) = 7,8-dihydrofolate + NADPH
-_~ ~ ~ ---_
--~
Tetrahydrofolate dehydrogenaqe
~ - _.
" x Y I X X
Sarcosine Oxidase
Asahi Pilot
Sarcosine Oxidase, Ongin mcroorganisms
PllOt
With other acceptors. 1.5.99.
TMADh Tnmethylamine + H(2)O + acceptor = dimethylamine +

formaldehyde +reduced acceptor
x_11_____ lyl-"**- ~ -"--%% ~ ~ . ~ L ~ c I _ " ^-" _"__c ~
Trimethylamlne Dehydrogenase;Ongin Paracoccus spec
R
Glutathione reductase (NADPH). 1.6.4.2
NADPH + oxidized glutathione = NADP(+) + 2 glutathione
Glutathione Reductase, Origin baker's yeast
Fluka Lab
NADPH dehydrogenase. 1.6.99.1

NADPH diaphorase. NADPH + acceptor = NADP(+) + reduced acceptor
Diaphorase(NADPH), Ongin Bacillus megatenum
Asahi Pilot
Diaphorase I, Ongin Bacillu5 stearothermophilus
Unitika Pilot
Asahi PllOt
Uricase;Origin: Bacillus fastidiosus
Fluka Lab
Uricase;Origin: Bacillus sp
Toyobo Pilot
Uricase; Ongin pig liver
Biozyme Pilot
Dihydrolipoamide dehydrogenase. 1.8.1.4.

Lipoanude reductase (NADH) E3 component of alpha-ketoacid Dihydrohpoamide + NAD(+) = lipoamide + NADH
dehydrogenase complexes Lipoyl dehydrogenase Dihydrolipoyl
dehydrogenaqe
Diaphorase(NADH), Ongin Bacillus megatenum
Asahi Pilot
Diaphorase II, Ongin Bacillus stearothermophilus
Unitika Pilot
I 1479
Diaphorase,Ongin Clostndium kluyven

Fluka Pilot
~~~~- -
Unshiol oxidase
Laccase A,
~ -----ww-s*
_I__'
Ongin Agancus hispru5

_3 -* _....*"" ^-xx
4 henzenediol + O(2) = 4 henzosemiquinone + 2 H(2)O
*"m-_y_w-/-**__I u__- ***--_l___i--* --
- _ I - -
Laccase C, Ongin: Conolus versicolor
Laccase,Ongin: rec. nucroorganism
Novozymes. DeniLiteTM
*xj__ ---
ascorhate + O(2) = 2 dehydr
--* __I_
Asahi Pilot
Ascorbate Oxidase, Ongin Cucumber
Amano. Amano 2 [ASO-21 PllOt
-
Ascorbate oxidase, Ongin Cucurbita sp
Biozyme Pilot
Fiuka Pilot
Ascorbate Oxidase, Ongin nucroorganisms
Amano Aman
a
Oxidoreductases. 1.11 .-.-
Acting on a peroxide as acceptor (peroxidases).
Brornoperoxidase; Ongin Corallina officinalis
Fluka Lab
Catalase. 1.11.1.6
2 H(2)0(2) = O(2) + 2 H(2)O
- ) I ~ ~ ~ I I x I x x ~ ~ ~ . *%
m- -, y u ( I
^a
--
--
- *-m
-m
M
p
-----
Catalase
Biocatalysts CATALASE Industnal
Catalase
Seravac Industrial
Catalase; Origin: Aspergillus niger
Amano: Catalase NL "Amano" Industrial
Catalase, Ongin Aspergillus niger
Biozyme Pilot
Catalase, Ongin Aspergillus niger
Fluka Industrial
Novozyrnes: CatazymeB Industrial
Roche Diagnostics: Catalase, technical grade Industrial
Catalase; Origin: Aspergillus niger, rec.
Novozyrnes: TerminoxTM Ultra Industrial
Catalase, Ongin beef liver
Biozyme Pilot
-
Catalase, Ongin beef liver
Roche Diagnostics Industnal
1480
Catalase;Origin: bovine liver
Fluka Industrial
Catalase, immobilized on Euperglt C; Origin: bovine liver

Fluka Lab
Catalase;Origin: Corynebacterium glutamicum
Roche Diagnostics Industrial
Catalase, Ongin Micrococcus lysodeikticus

Fluka Lab
Catalase;Ongin nucroorganisms
Fluka Pilot
Catalase;Ongin microorgmsms
Toyobo Industnal
Peroxidase. 1.1 1 .I .7
Myeloperoxidase. Donor + H(2)0(2) = oxidized donor + 2 H(2)O
X I X X -_I x _ “ I I _ _ _ ~ ~ “I% 1 _ 1
Lactoperoxidase,Ongin bovine nulk

Biozyme Pilot
Lactoperoxidase,Ongin bovine milk
Fluka Pilot
Peroxidase, Ongin Copnnus cinereus
Novozymes. NovoLym 502 Industnal
Peroxidase, Ongin Copnnus cinereus
Novozymes NS18010 Industnal
Peroxidase, Ongin horse radish
Fluka Industnal
Peroxidase, Ongin horseradish
Amano Amano 2 [PO-21 Pilot
Ammo Amano 3 [PO-31 Pilot
Biocatalysts Industnal
Biozyme Pilot
Roche Diagnostics: Peroxidase (POD), Grade I lndustnal
Roche Diagnostics Peroxidase (POD), Grade I1 Pilot
Peroxidase , Ongin horseradirh
Seravac Industnal
Toyobo Pilot
Lab
Chloride peroxidase. 1.11.1.10

Chloroperoxidase 2 RH + 2 chlonde + H(2)0(2) = 2 RCI + 2 H(2)O
Chloroperoxidase,Ongin Caldanomyces fumago

Fluka Pilot

I
Chloroperoxidase,Ongin Leptoxyphium fumago
Lipoxygenase. 1.13.11.12
Lipoxidase Carotene oxidase Lipoperoxidase Linoleate + O(2) =
(9Z,IlE)-( 13S)-13-hydroperoxyoctadeca-9,1l-dienoate
LipoxygenaseII, Ongin. pea, rec in E coli

Biocatalysts Pilot
-_ - x
Lipoxygenase111, Ongin. pea, rec in E coli

Biocatalysts Pilot
Fluka Lab
Lipoxidase, Ongin soybeen
Biozvme Pilot
Lactate 2-monooxygenase. 1.13.12.4

Lactate oxidative decarboxylase. Lactate oxidase Lactate (S)-lactate + O(2) = acetate + CO(2) + H(2)O.
oxygenase.
Lactate Oxidase
Asahi Pilot
Lactate Oxidase, Ongin Pediococcus 5p
Fluka Pilot
Oxidoreductases. 1.14.13.-
Acting on paired donors with incorporation of
molecular oxygen.
With NADH or NADPH as one donor, and
incorporation of one atom of oxygen.
2-Tridecanone Monooxygenase, Ongin Pseudomonas cepacia
Fluka Lab
Cyclopentanone monooxygenase. 1.14.13.16

Cyclopentanone + NADPH + O(2) = 5-valerolactone + NADP(+) +
~ ~ "~ - ~ -- ~
~ x -~~ ~ y_"_1___ -- ~ ~-
- ~
_y
H(2)0
I___
ne Monooxygenase, Ongin P
Cyclohexanone monooxygenase. 1.14.13.22

Cyclohexanone oxygenase Cyclohexanone + NADPH + O(2) = 6-hexanolide + NADP(+) +
-*-- w.**-** x "-1 * x- ~ C" * " x
Cyclohexanone Monooxygenase, Ongin Acinetobacter sp

Fluka Lab
Cyclohexanone Monooxygenase; Origin: E. coli overproducer
Fluka Lab
Cyclohexanone Monooxygenase, Ongin Nocardia globemla
Fluka Lab
Cyclohexanone Monooxygenase, Ongin Xanthobacter sp
Fluka Lab
2-hydroxybiphenyl+ NADH + O(2) = 2,3-dihydroxybiphenyI+

NAD+ + H 2 0
monooxygenase,
Fluka Lab
1482

W#w
Camphor 5-monooxygenase. 1.14.15.1

Camphor 5-exo-methylene hydroxylase Cytochrome p450-cam. (+)-camphor + putidaredoxin + O(2) = (+)-exo-5-hydroxycamphor+
oxidizedputidaredoxin + H(2)O
(+)-Camphor Monooxygenase,Ongin Pseudomonas putida
Fluka Lab
Monophenol monooxygenase. 1.14.18.1

Tyrosinase Phenolase Monophenol oxidase Cresolase L-tyrosine + L-DOPA + O(2) = L-DOPA + DOPAquinone + H(2)O
Tyrosinase, Ongin mushroom

Fluka Lab
Fluka Lab
eroxide dismut 1.15.1.1

~ -
Superoxide Dismuta
*-- ** __ ---~ ~
Bacillus stearothennophilus
- i__L_ _i l Y %"
2 peroxide radical + 2 H(+) = O(2) + H(2)0(2)
I - * * * ~ 1_ -
Umtika Superoxide Dismutase (SOD) Pilot
Superoxide dismutase; Origin: bovine erythrocytes
Biozyme Pilot
Superoxide Dismutase;Origin: bovine erythrocytes
Fluka Pilot
Superoxide Dismutase; Origin: bovine erythrocytes
Roche Diagnostics: Superoxide Dismutase (SOD) Lab
Superoxide Dismutase, Ongin bovine liver
Fluka Pilot
Catechol 0-methyltransferase. 2.1.1.6

S-adenosyl-L-methionine + catechol = S-adenosyl-L-homocysteine
+guaiacol
Brenrkatechin-0-methyl-Transferase
Fluka Lab
Glycoaldehyde transferase Sedobeptulose 7-phosphate + D-glyceraldehyde 3-phosphate =

-___*_)_-_-I --_I- -_I__)__^- = C _ i - _ - ~~ I- _ Y 1 x Y _x x 11_^1 _^--
Transketolase,Ongin baker's yeast

FlUka Lab
I __
Transketolase ,Ongin E coli
Fluka Lab
Transketolase,Ongin. E coli K12 (rec )
Transaldolase. 2.2.1.2
Dihydroxyacetone transferase Glycerone transferase Sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate =
D-erythrose 4-phosphate + D-fructose 6-phosphate
"+ x -I ~ ~-
Fluka Lab
I
Transaldolase,Ongin E coli K12 (rec )

Julich EnLyme Products Lab
1143
Glucosamine-phosphateN-acetyltransferase. 2.3.1.4
Phosphoglucosamine transacetylase Phosphoglucosanune Acetyl-CoA + D-glucosmne 6-phosphate = CoA +
acetylase N-acetyl-D-glucosanune 6-phosphate
Phosphotransacetylase,Ongin Bacillus stearothennophilus
Urutlka Phosphotransacetylase (PTA) Lab
Camihne acetylase
Lab
Gamma-glutamyltranspeptldaseGlutamyl transpeptldase (5-L-glutamyl)-peptide + an anuno acid = pepude +

~ - -(__I-vw - *--s-e-
gamma-Glutamyltransferase. Ongin beef ludney

*
e
I -%- I~~-
-- I _ y I -
5-L-glutamyl-aminoacid
m I _ _ ~ l I I ~ _ I ~
__~ l~
_lm
Biozyme Pilot
gamma-Glutarnyl Transpeptidase, Ongin hog ludney
Fluka Lab
Muscle phosphorylase A and B Amylophosphorylase ((1,4)-alpha-D-glucosyl)(N)+ phosphate =

Polyphosphorylase
in rabbit musc e
- -x -**.' -
[ ( I ,4)-alpha-D-glucosyl] (N- I)+ alpha-D-glucose I-phosphate
-
p-
-L
--------x
l- -l__l_
Lab
UDP-glucose-fructose glucosyltransferase Sucrose-UDP UDP-glucose + D-fructose = UDP + sucrose

glucosyltransferase
--I- I_)_--**<-
Sucrose Synthase, Ongin nce grains

1,4-alpha-glucan branching enzyme. 2.4.1.18

Glycogen branchng enzyme Amyb(1.4 to 1,6)transglucosidase Formation of 1,6-glucosidic hnkages of glycogen
Branching enzyme Amylo-( 1,4-1,6)-transglycosylase
Transglucosldase, Ongin Aspergillus niger
Amano Transglucosidase L "Amano" lndustnal
Lactose synthase. 2.4.1.22

UDP-galactose-glucose galactosyltransferase N-acetyllactosamine UDP-galactose + D-glucose = UDP + lactose.
synthase
FlUka
Beta-N-acetylglucosamin yl-glycopeptide 2.4.1.38

beta-l,4-galactosyltransferase.
Glycoprotein 4-beta-galactosyltransferase Thyroid UDP-galactose + N-acety-beta-D-glucosanunylglycopeptide = UDP
galactosyltransferase UDP-galactose-glycoprotein +beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosmnylglycopeptide
-~
galactosyItransferase
------ - -- -- - - "
Beta-l,4-Galactosyltransferase,
Ijy
Ongin S )
1484
N-acetyllactosaminide 2.4.1 .I51

alpha-I ,3-galactosyltransferase.
Galactosyltransferase UDP-galactose +
beta-D-galactosyl-(1,4)-N-acetyl-D-glucosaminyl-R = UDP +
alpha-D-galactosyl-( 1,3)-beta-D-galactosyI-(1,4)-N-acetyl-D-glucos
Fluka Lab
alpha-l,3-Galactosyltransferase,Ongin E coli, rec
Inosine phosphorylase PNPase Punne nucleoside + phosphate = punne +

alpha-D-nbose 1-phosphate
_ x x x I xxI 1~ Y l l I - i
Purine-Nucleosidephosphorylase
Ash Pilot
Purine-NucleosidePhosphorylase,Ongin microorganisms
Transferring nitrogenous groups.

Transaminases (aminotransferases).
~ 1_7--1__1mm- - *a*-i -.,,- ~
Transaminase, branched-chain, L-specific, Ongin microorganism, rec in E coli

* I I
BioCatalytics AT-I02 Lab

Transaminase, broad-range, D-specific, Ongin rmcroorganism, rec In E coli
BioCatalytics AT-I03 Lab
Transaminase, broad-range, L-specific, Ongin rmcroorgannm, rec in E coli
Transaminase A Glutarmc--oxaloacehc transaminase L-aspartate + 2-oxoglutarate = oxaloacetate + L-glutamate

- -
Glutamic-aspartic transarmnase
- -., * *
Glutamicoxaloacetic transaminase,
< ^* ~
Ongin pig heart

_1
Biozyme Pilot
Glutamate-OxaloacetateTransaminase,Ongin pig heart (mitochondnal)
Roche Diagnostics Glutamate-OxaloaLetateTransaminase (GOT) Pilot
Glutamic-OxalaceticTransaminase, Ongin porcine heart
Glutarmc--pyruvic transaminase. Glutamc--alanine transarmnase. L-alanine + 2-oxoglutarate = pyruvate + L-glutamate

I_ I~ "~
Glutamate-PyruvateTransaminase, Ongin pig heart

Roche Diagnostics Glutamate-Pyruvate Transaminase (GIT) Pilot
Glutamic-pyruvictransaminase, Ongin pig heart
Biozyme Pilot
Glutamic-PyruvicTransaminase, Ongin porcine heart
exohnase type IV
Asahi Pilot
Hesperidinase,Ongin Penicillium decumbens
Amano Hespendinase "Amano" Conc Industrial
1143
Table 20.5. (cont.)
Hexokinase, Ongin Saccharomyces sp
Toyobo Pilot
-
Hexokinase, Ongin yeast
Biozyme Pilot
Hexokinase, Ongin yeast
Fluka Industnal
Hexokinase, Ongin. yeast
FlUka Industnal
f ___~ Xlmf ~ ~ - -- ~ -~~ c
stearothermophilus
Unitlka Glucokmase (GlcK) Pilot
Glucokinase, Ongin Zymomonas mobilis
-fructose 6-phosphate =
~---~-~~-~-~
Glycerokinase ATP glycerol 3-phosphotransferase
Glycerol Kinase
Y ~ x _ _ I x x __ __ I
ATP + glycerol = ADP + glycerol 3-phosphate
L r X -- I ~ I - - I_- _x
Asahi Pilot
Glycerol Kinase, Ongin. Arthrobacter sp
Ammo Amano 2 [GK-21 Pilot
_I
Glycerokinase, Ongin Bacillus stearothemophilus

Pilot
Roche Diagnostics Glycerokinase, sol Pilot

I _ I~ -
Glycerol Kinase, Ongin E coli
Fluka Pilot
I
Glycerol Kinase, Ongin rmcroorganisms

Toyobo PllOt
------
Phosphoenolpyruvate kmase Phosphoenol transphosphorylase
~ -*mix *-- - I *<--I
_i-__^-
~ - -- ~ i I-r ~ -- -*-=--- --
ATP + pyruvate = ADP + phosphoenolpyruvate
1 __<_( -- *-
PllOt
Biozyme Pilot
Pyruvate Kinase, Ongin rabbit muscle
Biozyme Pilot
Pyruvate Kinase, Ongin rabbit muscle
Fluka Lab
Pyruvate Kinase, Ongin Zymomonas mobilis
Unitika Pyruvate Kinase (PK) PllOt
1486

"%
Streptomycin 6-kinase. 2.7.1.72

ATP + streptomycin = ADP + streptomycin 6-phosphate
Streptidine kmase Streptomycin 6-phosphotransferase APH(6)
X " A x( c -- - _x
Acetate Kinase, Ongin Bacillus stearothermophilus

Unitika Acetate Kinase (AK) Pilot
Acetate Kinase, Ongin E coli
Phosphoglycerate kinase. 2.7.2.3

ATP + 3-phospho-D-glycerate = ADP + 3-phospho-D-glyceroyl
phosphate
Phosphoglycerate Kinase,Ongin Bacillus stearothermophilus

Umtika Phosphoglycerate Kinase (PGK) Pilot
Biozyme Pilot
Creatine Kinase; Origin: pig heart
Biozyme Pilot
Creatine Kinase; Origin: rabbit muscle
Biozyme Pilot
Creatine Phosphokinase, Ongin rabbit m u d e
Fluka Lab
Adenylate kinase. 2.7.4.3

Myokmase Adenylic kmase Adenylohnase ATP + AMP = ADP + ADP
Polyribonucleotide nucleotidyltransferase. 2.7.7.8

Polynucleotide phosphorylase. (RNA](N+I) + phosphate = (RNA)(N) + a nucleoside diphosphate
Polynucleotide Phosphorylase, Ongin Bacillus stearothermophilus
Unrtika Polynucleotide Phosphorylase (PNPase) Prlot
CMP-N-acetylneuraminic acid synthetase CMP-NeuNAc CTP + N-acylneurdrmnate = diphosphate +

synthetase CMP-bialate pyrophosphorylase CMP-sialate CMP-N-acylneurmnate
Acting on ester bonds.
rmcroorganisms
Roche Diagnostics CHIRAZYME Lipases & Esterases, Screening Set Industnal Enzymes 2 lndustnal
11437
Carboxylesterase. 3.1.1.1
Ah-esterase B-esterase. Monobutyrase Cocaine esterase A carboxylic ester + H(2)O = an alcohol + a carboxylic anion
Esterase basic kit
FlUka Lab
-
Esterase, Ongin Bacillus sp
Fluka Lab
. -
Esterase; Ongin Bacillus stearothermophilus
Fluka Lab
Esterase,Ongin Bacillus thermoglucosidasius
Fluka Lab
I
Esterase, Ongin Candida lipolytica

Fluka Lab
Esterase , Ongin. Candida rugosa
Alms Indusmal
_ _ _ I _- - -
Esterase, Ongin hog liver
Fluka Indusmal
Esterase, immobilized on Eupergim C, Ongin. hog liver
Fluka Pilot
- -
Esterase lsoenzyme 1, Ongin hog liver
Fluka Pilot
I _ _ _ - - I -
Esterase, Ongin horse liver
FIuka Lab
- _-__ - - - - - _- -_ - - -~
Esterase Screening Kit , Ongin microorganism, rec in E coli
ThemoGen Quickscreen Esterase IOts Lab
- - I _ _ _
Esterase, Ongin Mucor miehei

Fluka Lab
I -. -
I I I I - -_
Esterase; Ongin: pig liver
Julich Enzyme Products Esterase PL Lab
- ~ _ _ _ _ _ _ I
- ___- I -- ___ I - - _-_^ --
Esterase. immobilized, Ongin. pig liver
Roche Diagnostics CHIRAZYME E-I, c - f , lyo Industnal
~- I_ -- I I __ _ 111 _--_^ I __ _~
Pig Liver Esterase, Ongin. pig Liver
AltuS Industrial
~ ~ - ~ - l l _ l _ l l l l l l_-l___l " I _I____ __ I __I_ - __ I_I
Pig Liver Esterase, Ongin pig liver

Roche Diagnostics PLE, technical grade, susp Industnal
__l__-----l_-I___I1_I _-_- ___ - - ~ ~ I_ - - _ _ _ _ I _ _ ^ - I -
Esterase, Ongin pig liver, fraction 1
Roche Diagnoshcs CHIRAZYME E-I, lyo Industnal
~ ~ - l _ _ - l _ - _ _ I I _ _I I_ __ ---- -___ __I^ ~ -- l__l I -
Esterase,Ongin pig liver, fractlon 2
Roche Diagnostics CHIRAZYME E-2, lyo Industnal
l_l_l-l -- - -- ll_l ___ - -I_ I -- - -- ._ I
Esterase, Ongin Rluzopus arrhizus

Julich Enzyme Products Esterase EL9
~- I _ _ _ I I I I I x ~ - 1111 I _ _ _ _ - - _ Lab
___
Esterase, Ongin Rhodotorula pilimanae
Julich Enzyme Products Esterase EL5 Lab
--_^_ - - - - - I x -- I -
Esterase, Ongin Saccharomyces cerevisiae
Fluka Lab
~ --- __ _-- ll__l __ _ I I ~ -
Desacetylesterase, Ongin Therm sp ,rec in E coli
Recordati Desa-REC Indusmal
I - I x --__ I I I
Esterase, Ongin Thermoanaerobium broclai

FlUka Lab
1488
Table 20.5. (cont )
Triacylglycerol lipase. 3.1 .I .3

Lipase Tnglycende lipase Tnbutyrase
- c x _ _ -~ -
Lioase basic kit
Fluka Lab
Lipase extension kit
Fluka Lab
Monoglyceride Lipase
Asahi Pilot
Lipase, Ongin Achromobacter sp
Meito Sangyo. Lipase AL Industnal
Lipase, immobilized,Ongin Achromobacter sp
Meito Sangyo. Lipax ALCIALG Industnal
Lipase, Ongin Alcaligenes sp
Altus Industnal
Meito Sangyo Lipase PL Industndl

Lipase; Ongin Alcaligenes sp
Meito Sangyo' Lipase QLL lndustnal
Lipase, Ongin Alcaligenes sp
Meito Sangyo Lipase QLM Industnal
Lipase, immobilized, Ongin Alcaligenes sp
Meito Sangyo Lipase PLCPLG Industrial
Lipase, immobilized; Origin: Alcaligenes sp.
Meito Sangyo: Lipase QLCIQLG Industrial
Lipase, Ongin Alcaligines sp
Roche Diagnostics CHIRAZYME L-10, lyo Industnal
Lipase, Ongin Aspergillus mger
Altus lndustnal
Lipase, Ongin Aspergillus niger
Amano Lipase A "AmdnO" 6 lndustnal
Lipase; Origin: Aspergillus niger
Ammo : Lipase AS Industrial
Amano : Lipase DS Industrial
Fluka Industrial
Lipase;Origin: Aspergillus niger
Fluka Industrial
Lipase, immobilized in Sol-Gel-AK, Ongin Aspergillus niger
Fluka Lab
Lipase, Ongin Aspergillus oryzae
Fluka lndustnal
Meito Sangyo Lipase SL Industnal

Lipase , Ongin. Burkholdena ~p
Fluka PllOt
Lipase, Ongin Candida antarctica
Fluka Lab
Lipase A , Ongin Candida antarctica
Fluka Industrial

I 1489
Lipase, immobilized; Origin: Candida antarctica

Fluka Industrial
Lipase, immobilized in Sol-Gel-AK; Origin: Candida antarctica

Fhka Pilot
Lipase, immobilized in Sol-Gel-AK on sintered glass; Origin: Candida antarctica

Fluka Lab
Lipase, type A; Origin: Candida antarctica

AlNS Industrial
Lipase, type B; Ongin Candida antarctica

Alms Industnal
Lipase B, Ongin Candida antarctica, rec
Fluka Industnal
Ongin. Candida antarctica, type A
Lipase, Ongin: Candida antarctica, type A
Roche Diagnostics CHIRAZYME L-5, sol Industnal
Lipase, immobilized, Ongin Candida antarctica, type A
Roche Diagnostics CHIRAZYME L-5, c -f , lyo Industnal
Lipase; Ongin Candida antarctica, type A, rec in Aspergillus oryzae
Novozymes NovoCor AD Industnal
Lipase; Ongin Candida antarctica, type A, rec in Aspergillus oryzae
Novozymes NovozymB 868 Industnal
Lipase, Ongin Candida antarctica, type B
Roche Diagnostics CHIRAZYME L-2, lyo. Industnal
Lipase, Ongin Candida antarctica, type B
Roche Diagnostics. CHIRAZYME L-2, sol Industnal
Lipase, immobilized, Ongin Candida antarctica, type B
ME L-2, c.-f , C2, lyo (Novozym 435) Industnal
Cdndida antarctica, type B
Roche Diagnostics CHIRAZYME L-2, c -f , C3, lyo lndustnal
Lipase, immobilized, Ongin Candida antarctica, type B
Roche Diagnostics CHIRAZYME L-2, c -f , lyo Industnal
Lipase, Ongin Candida antarctica, type B, rec in Aqpergillus oryzae
Novozymes. Nocozym 525 L Industnal
Lipase, immobilized, Ongin Candida antarctica, type B, rec in Aspergillus oryzae
Lipase; Ongin. Candida cylindracae
Julich Enzyme Products. Lipase LEI 1 Lab
Lipase, Ongin Candida cylindracea
Biocatalyqts lndustnal
Fluka Industnal
Flub Industnal
Meito Sangyo Lipase MY Industnal

Meito Sangyo Lipase OF lndustnal
-
Meito Sangyo Lipase OF% InduStndl
_ _
1490

Lipase, immobilized: Ongin Candida cylindracea
Meito Sangyo Lipase OFC/OFG Industrial
- -
Lipase, immobilized in Sol-Gel-AK; Ongin Candida cylindracea
Fluka PllOt
Pilot
Industnal
Lipase; Ongin Candida rugosa
Altus Industnal
I
Lipase, Ongin Candida rugosa

Alms. ChiroCLEC-CR (dry) lndustnal
Lipase, Ongin. Candida rugosa
Alms ChiroCLEC-CR (slurry) lndustnal
-
Amano. Lipase AY "Amano" 30 lndustnal
Amano. Lipase AYS lndustnal
Lipase, Ongin Candida rugosa (formerly C cylindracea)
Roche Diagnostics: CHIRAZYME L-3, lyo Industnal
Lipase, purified, Ongin Candida rugosa (formerly C cylindracea)
Roche Diagnostics CHIRAZYME L-3, punfied, lyo Pilot
Lipase, purified, immobilized, Ongin: Candida rugosa (formerly C cylindracea)
Roche Diagnostics. CHIRAZYME L-3, punfied, c -f , C2, lyo Pilot
Lipase, Ongin Candida utihs
Fluka Lab
Lipase, Ongin Chromobactenum viscosum
Alms Industnal
Lipase, Ongin: Cromobactenum V ~ S C O S U ~
Asahi Pilot
Lipase, Ongin: Geotnchum candidum
Alms lndustnal
Lipase, Ongin. hog pancreas
Fluka Lab
Lipase, Ongin hog pancreas
Fluka 1ndus tnaI
-
Lipase, immobilized in Sol-Gel-AK, Ongin hog pancreas
Fluka Pilot
Pancreatin, Ongin. hog pancreas
Industnal
Roche Diagnostics CHIRAZYME L-8, sol Indu stnaI

Lipase 6 , covalently linked to carrier; Origin: microorganisms
Fluka Pilot
Lipoprotein Lipase, Ongin microorganisms
Amano Amano 6 [LPL-6] Pilot
Lipase, Ongin Mucorjavanicus
Alms Industnal
Lipase: Ongin. Mucor javanicus
Amano Lipase M "Amano" 10 Industnal
I
1491
Lipase, Ongin Mucorjavanicus

Fluka Industnal
-
Lipase, Ongin Mucor meihei
AltuS
I - - - - Industrial _I
Lipase, Ongin Mucor nuehei

Fluka Lab
- - -
Lipase, Ongin Mucor nuehei
Roche Diagnostics CHIRAZYME L-9, sol. Industnal

Lipase, immobilized: Ongin Mucor rmehei
Fluka Lipozymea, immobilized Industnal
Lipase, immobilized: Ongin: Mucor miehei
Roche Diagnostics CHIRAZYME L-9, c - f , C2. lyo Industrial
Lipase, immobilized, Ongin Mucor miebei
Roche Diagnoshcs CHIRAZYME L-9, c -f Industnal
Lipase, immobilized in Sol-Gel-AK, 0
Fluka Lab
Lipase, immobilized in Sol-Gel-AK on sintered glass, Ongin Mucor nuehei
Fluka Lab
Lipase, Ongin Mucor nuehei, rec
FlUka Industnal
Lipase, Ongin Penicillium camembertii
Ammo. Lipase G "Amano" 50 Industnal
Lipase: Ongin Penicillium roqueforti
Fluka Pilot
Lipase, Ongin Penicillium roquefortii
Amano Lipase R Indubtnal
-
Lipase; Ongin porcine pancreas
Alms Industnal
Lipase, Ongin porcine pancreas
Lipase, Ongin porcine pancreas
Roche Diagnoshcs. Lipase Pilot
- -
Lipase, Ongin Protein engineered in rec. Aspergillus
Novozymes Lipolase Ultra Industnal
- -I - - - - -
Lipase, Ongin Protein engineered in rec Aspergillus
Novozymes LipoPnme'M Industnal
I __
Lipase, Ongin Pseudomonas aeroginosa
Altus Industnal
Lipase, Ongin Pseudomonas cepacia
Altus Industnal
I _ - __ -
Lipase, Ongin. Pseudomonas cepacia
Altus CkoCLEC-PC (dry) lndustnal
- -
Altus ChiroCLEC-PC (sluny) Industrial
- I - _I _I - _-
Industnal
I -
Amano Lipase PS-C Industrial

- I - _ I - _I
1492
I 20 Tabular Survey ofCornrnercially Available Enzymes

Amano Lipase PS-D Industnal
Lipase, Ongin. Pseudomonas cepacia
Fluka Industnal
Lipase; Origin: Pseudomonas cepacia
Fluka Industrial
Lipase, immobilized in Sol-Gel-AK; Origin: Pseudomonas cepacia
Ruka Pilot
Lipase, immobilized in Sol-Gel-AK on sintered glass, Ongin Pseudomonas cepacia
Fluka Lab
Lipase, immobilized on Ceramic particles; Ongin Pseudomonas cepacia
Fluka Lab
Lipase, Ongin Pseudomonas fluorescens
Ammo LipaseAK Indurtnal
Lipase: Origin: Pseudomonas fluorescens
Fluka Pilot
Lipase, immobilized in Sol-Gel-AK, Ongin Pseudomonas fluorescens
Fluka Pilot
Lipase, immobilized in Sol-Gel-AK on sintered glass, Ongin Pseudomonas fluorescens
Fluka Lab
Lipase, immobilized on Eupergit C , Ongin Pseudomonas fluorescens
Fluka Lab
Lipase, Ongin. Pseudomonas sp
Roche Diagnostics CHIRAZYME L-6, lyo Industrial
Lipase, immobilized, Ongin Pseudomonas sp
Toyobo Industnal
Lipoprotein Lipase, Ongin Pseudomonas sp
Ammo Amano 3 [LPL-31 Pilot
Lipoprotein Lipase, Ongin Pseudomonas sp
Toyobo Pilot
Lipase, Ongin. Pseudomonas stutzen
Meito Sangyo Lipase TL Industrial
Lipase, Ongin Rhizomucor miehei
DSM Gist-brocades. Piccantase Industnal
Lipase, Ongin Rhizomucor nuehei
Fluka Indu\tnal
Lipase, Ongin Rhizomucor nuehei, rec in Aspergillus oryzae
Novozymes NovozymB 388 Indwtnal
Lipase, Ongin Rhizomucor nuehei, rec in Aspergillus oryzae
Novozymes PalataseB Industnal
Lipase, immobilized, 0 Rhizomucor miehei, rec in Aspergillus oryzae
Novozymes LipozymeB RM IM Induanal
Lipase, Ongin Rhizopus arrhizus
Fluka Industnal
Lipase, Ongin: Rhizopus delemar
Alms Industrial
Lipase, Origin Rhizopus delemar
Fluka Industnal
Altus Industnal
I 1493
Lipase;Origin: Rhizopus niveus

Amano: Newlase F Industrial
Lipase;Origin: Rhizopus niveus
Fluka Pilot
Lipase, Ongin Rhizopus niveus
Julich Enzyme Product? Lipase LE9 Lab
Lipase, Ongin Rhizopus oryLae
Altus Industnal
Lipase, Ongin Rhizopus oryzae
Amano Lipase F-APIS Industnal
Lipase; Origin: Rhizopus oryzae
Ammo : Lipase F-DS Industrial
Lipase, Ongin Rhizopus sp.
Meito Sangyo' Lipase UL
Lipase, Ongin Thermomyces lanuginosa
Altus Industnal
Lipase, Ongin Thermomyces lanuginosa, rec Aspergillus oryzae
Novozymes Novozym 677 BG Industnal
Lipase;Origin: Thermomyces lanuginosa rec. in Aspergillus oryzae
Novozymes: GreasexB Industrial
Lipase;Origin: Thermomyces lanuginosa rec. in Aspergillus oryzae
Novozymes: LipolaseB Industrial
Lipase, Ongin Thermomyces lanuginosa rec in Aspergillus oryzae
Novozymes. NovozymB 27007 lndustnal
Novozymes NovozymB 735 lndustnal
Lipase; Origin: Thermomyces lanuginosa rec. in Aspergillus oryzae
Novozymes: NovozymB 871 Industnal
Lipase; Origin: Thermomyces sp. (formerly Humicola sp.)
Roche Diagnostics: CHIRAZYME L-8, lyo. Industrial
Lipase; Origin: Thermus aquaticus
Fluka Lab
Lipase; Origin: Thermus flavus
Fluka Lab
Lipase; Origin: Thermus thermophilus
Fluka I.ah
Lipase, Ongin wheat germ
Fluka PIlOt
Phosp
Phosphatidylcholine 2-acylhydrolase Lecithinase A Phosphatidylcholine + H(2)O = 1-acylglycerophosphoholine + a
c_ -
Phosphatidase Phosphatidolipase
yI_xI_y^l_l~ ~ -
-i-____
-*" - _ f _
fattyacid anion
~ *-*
Phospholipase A2, Ongin. Aglustrodon halys
Fluka Lab
Phospholipase A2, Ongin bovine pancrea5
Fluka Pilot
Phospholipase A2, Ongin hog pancreas
Fluka Pilot
20 Tabular Survey ofCornmercially Available Enzymes
1494
PhospholipaseA2; Origin: porcine pancreas
Biocatalysts Pilot
PhospholipaseA2, Ongin porcine pancreas
Industnal
Acetylcholinesterase. 3.1.1.7
True cholinesterase Choline esterase I Cholinesterase Acetylcholine + H(2)O = choline +acetate
Acetylcholinesterase;Origin: bovine erythocytes
Biozyme Pilot
Acetylcholine Esterase, Ongin Electrophorus electncus
Fluka Lab
84
Cholinesterase. 3.1.1.8
Pseudocholinesterase Acylcholine acylhydrolase Butyrylcholine An acylcholine + H(2)O = choline + a carboxylic acid amon
esterase Non-specific cholinesterase
x- ~ l y _ _ _ ~ - -~
- x
_ X " ~ 1 Y xx Y ) i
Butyrylcholineesterase, Ongin horse serum

Biozyme Pilot
ButyrylcholineEsterase, Ongin horse semm
Fluka Lab
Pectinesterase. 3.1.1.11
Pectin methylesterae Pectm demethoxylase Pectin methoxylase Pectin + N H(2)O = N methanol + pectate
~ ~ - I _ _ _c
L _ I - I-_-I
~ -
__I-jli ~ _
_I ~ xI-_XI_ I x - ~ ~ x x -
Pectinesterase
Novozymes Cellubnx@ Industnal
Pectinesterase
Novozymes NovoclairTM FCE lndustnal
Pectinesterase
Novozymes Pectinex BE Industnal
-
Pectinesterase,Ongin Aspergillus niger
Amano Pectinase P lndustnal
Ammo Pectinase PL "Amano" Industnal

-
Pectin Esterase;Ongin orange peel
Fluka lndustnal
Pectinesterase,Ongin rec microorganism
Novozymes NovoshapeB Industnal
Pectinesterase,Ongin rec rmcroorganism
Novozymes Pectinex SMASH lndustnal
Sterol esterase. 3.1.1.13

Cholesterol esterase Cholesterol ester synthase Tnterpenol A steryl ester + H(2)O = a sterol + a fatty acid
esterase
Cholesterol Esterase
Asahi Pilot
Cholesterol Esterase, lyo., Ongin Candida rugosa (formerly C cylindracea)
Roche Diagnostics Cholesterol Esterase, lyo Pilot
I_ _I_
Cholesterol Esterase, sol., Ongin Candida rugosa (formerly C cylindracea)

Roche Diagnostics Cholesterol Esterase, sol. Pilot
111
Cholesterol Esterase, Ongin hog pancreas

Fluka Pilot
I 1495
Cholesterolesterase, Ongin pig pancreas

Biozyme Pilot
- I _ I -
Amano. Amano 2 [CHE-Z] PllOt

Cholesterol Esterase;Origin: Pseudomonas sp.
Amano: Amano 3 [CHE-3] Pilot
Cholesterol Esterase; Origin: Pseudomonas sp.
Asahi Pilot
Cholesterol Esterase, Ongin Pseudomonas sp
Toyobo Pilot
Tannase. 3.1.1.20
Digallate + H(2)O = 2 gallate
Cleanng factor lipase Diglycende lipase Diacylglycerol lipase Tnacylglycerol + H(2)O = diacylglycerol + a fatty acid anion
* w - ~ ~ - - - - - ~ ~ ~ e a ~ -m
-em* v ~ ~~ * *-!. _^
L xp p----xyxx
Lipoprotein Lipase,Ongin Chromobactenum viscosum

Fluka Pilot
I I I I I I I I I
LipoproteinLipase,Ongin Pseudomonas fluorescens

Pilot
-
Alkaline phosphatase. 3.1.3.1

Alkaline phosphomonoesterase Phosphomonoesterase An orthophosphonc monoester + H(2)O = an alcohol +phosphate
GIycerophosphatase
Phosphatase, alkaline
Seravac Pilot
Phosphatase, alkaline; Origin: Bacillus sp.
Biocatalysts Pilot
Phosphatase alkaline; Origin: bovine intestinal mucosa
Fluka Industrial
Phosphatase alkaline; Origin: calf intestinal mucosa
FlUka Industrial
Phosphatase alkaline, immobilizedon Agarose; Origin: calf intestinal mucosa
Fluka Industrial
Phosphatase alkaline, immobilizedon Agarose, Ongin calf intestinal mucosa
Fluka Industnal
__ - I I I I I _ I _ _ - - _-_-
Phosphatase, alkaline,Ongin calf intestine
Roche Diagnostics Phosphatase, alkaline, EIA Grade Industnal
I I I _ - I __ _ _ I I
Phosphatase, alkaline,
Biozyme Pilot
Phosphatase, alkaline, highly active; Origin: calf intestine, rec. in Pichia pastoris
Roche Diagnostics: Phosphatase, alkaline, EIA Grade, highly active Industrial
Phosphatase, alkaline, Ongin E coli
Fluka Lab
--- ~ _ _ _ -
Phosphatase, alkaline , Ongin Eschenchia coli
Asahi Pilot
I I _ I
Phosphatase, alkaline; Ongin microorganisms

Unitika Pilot
1496
I Table 20.5. (cont )
-sw~~u-~&<~~e
Acid phosphatase. 3.1.3.2
Acid phosphomonoesterase. Phosphomonoesterate An orthophosphonc monoester + H(2)O = an alcohol +phosphate
Glycerophosphatase
~ I " 1 c
ase, acid, otato

Roche Diagnostics Phosphatase, acid, grade I1 Pilot
Phosphatase, acid. Ongin potatoes
Fluka Pilot
Phytase Phytate 3-phosphatase Myo-inositol-hexaphosphate Myo-inositol hexalasphosphate + H(2)O =

*<_^__*we __- ___
3-phosphohydrolase
f^
Phytase; Ongin Aspergillus niger

I *_"" " - x L
D-myo-inositol1,2,4,5,6-pent&sphosphate + phosphate
- I x x x _ x
Amano Industnal
Phytase; Ongin Peniophora lycii, rec In Asp oryzae
Novozymes Bio-feed@Phytase Industnal
Lipphosphodiesterase I Lecithinase C Clostndium welchn A phosphatidylcholine + H(2)O = 1,2-diacylglycerol+

cholinephosphate
**
Asahi Pilot
Phospholipase C, Ongin. Bacillus cereus
Fluka Pilot
Phospholipase C, Ongin Clostndium perfnngens
Fluka Pilot
Glycerophosphorylcholine Phosphodiesterase, Ongin microorganisms

Asahi Pilot
Phospholipase D, Ongin Streptomyces chromofuscua
Asahi Pilot
Phospholipase D, Ongin Streptomyces chromofuscu?
Fluka Pilot
Phospholipase D, Ongin Streptomyces sp
Asahi PllOt
Pilot
Deoxyribonuclease 1. 3.1.21 .I
Pancreatic DNase DNase Thymonuclease Endonucleolytic cleavage to 5'-phosphodinucleotide
andS'-phosphooligonucleotldeend-products
_-" ~ x_xI -- - _j lil
ribonuclease I,Ongin bovine pancrea5
Guanylonbonuclease Aspergillus oryiae nbonuclease RNase N 1 Two-stage endonucleolytic cleavage to 3'-pho\phomononucleotides

RNase N2 and3'-pho?phoohgonucleotidesending in G-P with 2,3'-cychc
- -- I -
pho\phateintermediates.
Ribonuclease T1, Ongin Aspergillus oryzae

Fluka Lab
I
1497
mXX.I*
Pancreatic ribonuclease. 3.1.27.5

RNase. RNase I Rh'ase A. Pancreatic RNase Endonucleolytic cleavage to 3'-phosphomononucleotides and
3'-phosphooligonucleotldesendmg in C-P or U-P with
Ribonuclease,Ongin beef pancreas

_x "- -_ _l_l_l ----~-~
2',3'-cyclicphosphate intermediates
x I x I ~ ~ ~ ~ - x I I I x I __
Biozyme Pilot
RibonucleaseA, Ongin bovine pancreas
Fluka Pilot
__ - _I -
Ribonuclease,immobilized on EupergitC, Ongin bovine pancreas
Fluka Lab
Aspergillus nuclease S1. 3.1.30.1

Endonuclease S1 Single-stranded-nucleateendonuclease Endonucleolytlc cleavage to 5'-phosphomononucleotldeand
5'-phosphooligonucleotideend-products
- ~ - ~ -
Deoxynbonuclease S 1
~ __x_xa- -_ -m - - x I ~ __m --_ ~ ~ I - ~ ~ - ~ - ~ ~ -~ x y I ~
Amano Enzyme Rp-1 Industnal

Nuclease P1; Ongin Penicillium citnnum
Fluka Lab
i
Micrococcal endonuclease. Endonucleolytic cleavage to 3'-phosphomononucleotideand

3'-phosphooligonucleotideend-products
- ~ ~ - - ~ - - ~ = ~ ~ - -----
~ - ~ ~
I-_I___ -
~ -
"_I ~ ~ ~
" ~ " - ~ - - -
X _I
_ Y- ~ _ v y ~ ~ ~ I I F - ~ - * x x x I ~ l l x I I x l l
Nuclease micrococcal,Ongin Staphylococcus aureus

Fluka Lab
Alpha-amylase. 3.2.1 .I
1,4-alpha-D-glucanglucanohydrolase Glycogenase Endohydrolysis of 1,4-alpha-glucosidiclinkages in oligosacchandes
-=--* -*--
Amylase, Ongin Aspergillus niger
~ ------
and polysacchandes
xIIxuI*-I)*_^-
Ammo' Gluczyme NL4 2 Industnal

l_li - I I _ _
alpha-Amylase,Ongin Aspergillus oryzae
Fluka Industnal
-
Amylase, O n g n Aspergillus oryzae
Ammo Amylase DS Industnal
Amylase, Ongin Aspergillus oryzae
Ammo Biozyme F10 SD Industnal
- - _x
Amylase, Ongin Aspergillus oryzae

Ammo Biozyme S Conc Industnal
Taka-Diastase,Ongin Aspergillus oryzae
Fluka Pilot
alpha-Amylase,Origin Bacillus amyloliquefaciens
Fluka Industnal
alpha-Amylase,Ongin Bacillus lichenifomis
Fluka Industnal
alpha-Amylase,Ongin Bacillus suhhlis
Fluka Industnal
Amylase, Ongin Bacillus suhtilis
Amano Amylase A "Amano" Conc Induanal
alpha-Amylase;Origin: fungus
Novozymes: Fungarnylm Industrial
alpha-Amylase: Origin: hog pancreas
Fluka Lab
1498

Amylase; Origin: microbacterium
Amano : AMT "Amano" Industrial
Amylase; Origin: microorganisms
Novozymes: AquazymB
Amylase; Origin: microorganisms
Novozymes: BAN (Bacterial Amylase Nova) Industrial
alpha-Amylase; Origin: rec. microorganism
Novozymes: DuramylTM Industrial
Novozymes: Liquozyme@ Industrial
Novozymes: TermamylB Industrial
alpha-Amylase, Ongin rec nucroorganism
Novozymes Termamyl, Type LS Industnal
Industnal
Beta-amylase. 3.2.1.2
1,4-alpha-D-glucan maltohydrolase Saccharogen amylase Hydrolysis of 1,4-alpha-glucosidic linkages in polysacchandes so
Glycogenase asto remove successive maltose units from the non-reducing ends of
the chains
beta-Amylase, Ongin barley

Fluka Pilot
- -_I_ - --
beta-Amylase, Ongin. sweet potato
Fluka Pilot
Glucan 1,4-alpha-glucosidase. 3.2.1.3

Glucoamylase 1.4-alpha-D-glucan glucohydrolase Hydrolysis of tennmal 1,Clinked alpha-D-glucose residues
Amyloglucosidase Gamma-amylase successively from non-reducing ends of the chams with release
- "m-m--- "
A spergillus niger
Fluka Industnal
Amyloglucosidase; Ongin rec nucroorganism
Novozvmes. AMG Industnal
Cellulase. 3.2.1.4
Endoglucanase Endo- 1,4-beta-glucanase Carboxymethyl Endohydrolysis of 1.4-beta-D-glucosidic linkages in cellulose
celluldse
x _ ~ " - I _? ____ __-
Cellulase
NovoLymes Novozym@ 342 Industnal
- -
Cellulase, Ongin Aspergillus niger
Ammo Cellulase A "Amano" 3 Industnal
-
Cellulase; Ongin Aspergillus niger
Ammo Cellulase DS Industnal
Cellulase; Origin: Aspergillus niger
Fluka Industrial
Cellulase, Ongin fungus
Novozymes Celluzyme@ Industnal
Cellulase, Ongin. Hunucola insolens
Fluka Lab

I
1499
Cellulase, Ongin rec mcroorganism

Novozymes CarezymeB Industnal
- - I
Cellulase, Ongin rec mcroorganism

Novozymes: DeniMaxB Industnal
- _- _ _ I I I
Cellulase, Ongin Tnchoderma longibrachiatum

Fluka Industnal
_ I I I
Cellulase, Origin Tnchoderma reesei

Fluka Lab
Cellulase, Ongin: Tnchoderma vinde
Amano Cellulase T "Amano" 4 Industnal
hoderma vinde
Endo-l A-beta-glucanase Endo-l,3-beta-glucanase Laminannase Endohydrolysis of 1,3- or 1,CIinkages in beta-D-glucans when the
glucose residue whose reducing group i s involved in the linkage to
- . - - ~~~-------
~~-- y1I_ -_x_I
be hydrolysed i s itself substituted at C-3
beta-glucanase
Novozymes . CerefloB Industnal
beta-glucanase
Novozymes: FinizymB Industrial
beta-glucanase, heat-stable
Novozymes: UltrafloB Industrial
beta-Glucanase; Origin: Aspergillus niger
Fluka Industrial
beta-Glucanase, Ongin Bacillus subtilis
FlUka Industnal
Inulinase. 3.2.1.7
Inulase
--- _I_( _-i
Endohydrolysis of 2,l-beta-D-ftuctosidic linkages in inulin
-*( ---* li ~
e, Ongin Aspergillus niger
Endo-1,&beta-xylenase. 3.2.1.8
1,Cbeta-D-xylan xylanohydrolase Endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans
Xylanase
Novozymes : Pulpzyme'M HC Industrial
Xylanase; Origin: Aspergillus niger
Amano: Hemicellulase "Amano" 90 Industrial
Xylanase, Ongin Aspergillus niger
Ammo Hemcellulase "Amano" 90 Industnal
I_-- ____ - - - I
Xylanase, Ongin bactena

Fluka Industrial
I_ - I-I - - I _I - - I- - --
Xylanase, Ongin rec rmcroorganism
Novozymes PentopanTMMono Industnal
- - I I - - - -
Xylanase, Ongin rec mcroorganism
Novozymes Shearzyme'M Industnal
Xylanase; Origin: Tnchoderma viride
FlUka Lab
1500
Table 20.5. (cont ).
Dextranase. 3.2.1 .I 1
Alpha-l,6-glucan-6-glucanohydrolase
Dextranase
Novozymes Dextranase lndustnal
Dextranase,Ongin Chaetonuum erraticum)
Amano Dextranase L "Amano" Industnal
Dextranase,Ongin Paecilomyces lilacinus
Ruka Pilot
Chitinase. 3.2.1 .I 4
Chitodextnnase 1,4-beta-poly-N-acetylglucosamin1dase Hydrolysis of the 1.4-beta-linkages of N-acetyl-D-glucosanune
Poly-beta-glucosanunidase polymers of clutin
E_
Chitinase, Ongin. bean leaves

Julich Enzyme Products Chitinase BB Lab
Chitinase, Ongin Streptomyces gnseus
Ruka Pilot
Chitinase, Ongin sugar beet
Fluka Lab
Chitinase, Ongin sugar-beet leaves
Julich Enzyme Products: Chitinase ZR Lab
Pectin depolymerase Pectinase Random hydrolysis of 1,4-alpha-D-galactos1duroniclinkages in

pectate and other galacturonans
-
Fluka Industrial
Pectinase, Ongin mould
Fluka Industnal
Pectinase, Ongin Rhizopus sp
Murmdase Hydrolysis of the 1,4-heta-linkages between

N dcetyl-D-ghIcosanune and N-acetylmuranuc acid In peptidoglycan
aryotes cell walls
Lysozyme
Seravac Pilot
Lysozyme, Ongin chicken egg white
Biozyme Industnal
Lysozyme, Ongin hen egg white
Sialidase Neuraminldase N-acylneuraminate glycohydrolaie Hydrolysis of alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)-glycosidic

Alpha-neurammidase linkages of terminal sialic residues in oligosacchandes,
glycoprotein\, glycolipids, colominic acid and synthetic substrates
"_ c _
Neuraminidase,Ongin Clostndium perfnngens

Fluka Pilot
Neuraminidase,immobilized on Agarose4B, Ongin Clostndium perfnngens

Ruka Pilot
Neuraminidase,Ongin nucroorganisms
Unitika Pilot
Table 20.5. (cont.)
I 1501
Neuraminidase,Origin Streptococcus sp
Toyobo Pilot
-
Neuraminidase.Ongin Vibno cholerae
Fluka Lab
a*
Alpha-glucosidase.
Maltase Glucoinvertase. Glucosidosucrase Maltase-glucoamylase Hydrolysis of terminal, nowreducing 1,4-linked D-glucose residues
with release
XC--I-X_l%^*-Ym-M1 ** * --**-_ *',- " -*-- i r --c * -Ju*x-
alpha-Glucosidase,Ongin. Aspergillus niger

Fluka Industnal
alpha-Glucosidase,Ongin Bacillus stearothermopbilus
Urutlka alpha-Glucosidase Pilot
alpha-Glucosidase,Ongi
Toyobo Pilot
alpha-Glucosidase;Ongin yeast
Biozyme Pilot
alpha-Glucosidase,Ongin yeast
Fluka Lab
alpha-Glucosidase,Ongin yeast overproducer
Industnal
Beta-glucosidase. 3.2.1.21
Gentobiase Cellobiase Amygdalase Hydrolysis of temunal, non-reducing beta-D-glucose residues with
-- release of beta-D-glucose
Seravac Pilot
-
beta-Glucosidase,Ongin almonds
Fluka Pilot
beta-Glucosidase,Ongin sweet almond
Toyobo Pilot
-
beta-Glucosidase,Ongin sweet almonds
Alpha-galactosidase. 3.2.1.22
Melibiase Melibiose + H(2)O = galactose + glucose
alpha-Galactosidase;Ongin. rec microorganism
Novozymes Alpha-galTM Industnal
Beta-galactosidase. 3.2.1.23
Lactase. Hydrolysis of terminal. non-reducing beta-D-galactose residues in
beta-D-galactosides.
Lactase; Origin: Aspergillus oryzae
Amano: Lactase 14-DS Industrial
Lactase, Ongin Aspergillus oryzae
Amano Lactase F "Amano" Industnal
beta-Galactosidase,Ongin E coli
Fluka Pilot
I I
beta-Galactosidase,Ongin E coli overproducer

Roche Diagnostics beta-Galactosidase Industnal
beta-Galactosidase,Ongin Eschenchia coli
Toyobo Pilot
-_ I
beta-Galactosidase,Ongin Kluviromyces lactis

Recordati beta-Galactosidase, Lattasi beads Industnal
1502
I 20 Tabular Survey of Commercially Available Enzymes

beta-Galactosidase,Ongin Kluyveromyces fragilis
Fluka Pilot
Lactase, Ongin Kluyveromyces lactis
Novozymes Lactozym@ Industnal
-
beta-Galactosidase, Ongin nucroorganisms
Unitika beta-Galactosidase (beta-Gal) Pilot
Hydrolyw of terminal, non-reducing alpha-D-mannose residues in

_ _ I I _ X X _
alpha-Mannosldase
_ X " l " X " ~" - _ I _ ^ L I I _ ~ Y l / _ __ -- ~
alpha-D-mannosides
x *I_ x
Invertase Saccharase Hydrolysis of temunal non-reducing beta-D-fructofuranoside
-D-glucuronoside + H(2)O = an alcohol + D-glucuronate

i__ x_ ;
i - ; _ _ -
Seravac Pilot
beta-Glucuronidase: Ongin bovine liver
Fluka Lab
beta-Glucuronidase, Ongin E coli
Fluka Lab
-
beta-Glucuronidase, Ongin E coli K12
Fluka Lab
__ __
beta-Glucuronidase, Ongin: Helix pomatla
Hyaluronidase Random hydrolysis of 1.4-linkages between

N-acetyl-beta-D-glucosamine and D-glucuronate residues in
hyaluronate
x _rr x_ "* "__ ~ ^^ - 1 - I _- ~ * % * * 1
Hyaluronidase
Seravac Pilot
Hyaluronidase: Ongin bovine testes
Fluka Lab
Hyaluronidase, Ongin ovine or bovine teste5
Biozyme Pilot
Hyaluronidase, Ongin sheep testes
Fluka Lab
Hyaluronidase, Ongin Streptomyces hyalurolyticus
Fluka Lab
Hyaluronidase, Ongin Streptomyces sp.
Amano Amano 1 [HY-I] Pilot
Hyaluronidase, Ongin Streptomyces sp
Ammo Ammo 3 [HY-3] Pilot
I1503
w&8m
3.2.1.39
J
Glucan endo-l,3-beta-D-glucosidase.
(I->3)-beta-glucan endohydrolase Endo-l,3-beta-glucanase Hydrolysis of 1,3-beta-D-glucosidic linkages in 1,3-beta-D-glucans
hnannase
Pullulanase hllulan 6-glucanobydrolase. Limit dextnnase Starch-debrancbmg enzyme, hydrolyses ( l-6)-alpha-glucosidic

Debranclung enzyme linkages in pullulan and starch to form maltotnose
s
-s
-p*_
-_x -* a
-e
r
-
*
ia
--- *--
Pullulanase, Ongin Bacillus sp.
Amano Debranchngenzyme "Amano" 8 Industnal
I - _ _ __ _I I - - - - - 1-1 - I -
Pullulanase, Ongin Bacillus sp
Fluka Industnal
I I - _ _ I " I __
Pullulanase.Ongin rec. microorganism
Beta-hexosanunidase Hexosanunidase Hydrolysis of tenrunal non-reducing N-acetyl-D-hexosanune

N-acetyI-beta-glucosamimdase residues in N-acetyl-beta-D-bexosmnides
- - - -w
m* -b-
Fluka Lab
Agarase. 3.2.1.81
Hydrolysis of 1,3-beta-D-galactosidic linkages in agarose, giving the
teuamer as the predominant product
Agarase, Ongin: Pseudomonas atlantica
Fluka Lab
Thioglucosidase. 3.2.3.1
Myrosinase Sinignnase Sinigrase A thioglucoside + H(2)O = a tbiol + sugar
Myrosinase, Ongin Senapis alba (white mustard seed)
Biocatalysts Pilot
Epoxide hydrolase. 3.3.2.3

Epoxide hydratase Arene-oxide hydratase An epoxide + H(2)O = a glycol
Epoxide Hydrolase;Origin: Agrobacterium sp
Fluka Lab
Epoxide Hydrolase;Origin: Aspergillus niger
Fluka Lab
Epoxide Hydrolase;Origin: Rhodococcus rhodochrous
Flub Lab
Epoxide Hydrolase, Ongin Rbcdotorula glutinis
Fluka Lab
Hydrolases. 3.4%-
--
Acting on peptide bonds (peptide hydrolases).
m
-p
--*w
Protease, Ongin Aspergillus melleus

--mm -m*yL c^i-v_cx-.A~~~Iu--~-~*-- -- ---____l__M(_n-- _--
Amano. Protease DS lndustnal
Protease;Ongin. Aspergillus melleus
Amano Protease P "Amano" 6 Industnal
_
Alms Industnal
Protease; Origin: Aspergillus niger
Amano: Acid Protease A Industrial
1504
I 20 Tabular Suwey ofCommercially Available Enzymes

Protease, Origin Aspergillus niger
Ammo Acid Protease DS Industnal
Protease, Ongin Aspergillus niger
-
Protease , Ongin Aspergillus oryzae
Altus Industnal
Protease, Ongin Aspergillus oryzae
Altus Industnal
Altur Industnal
Ammo Protease A "Amano" 2G IndUStndl
-
Ammo Protease A-DS Industnal
I

Ammo Protease M "Amano" Industnal
Protease; Origin: Aspergillus oryzae
Proteinase 2A, Ongin Aspergillus oryzae
Huka Industnal
Protease, Ongin Aspergillus sp
Altus Industnal
Protease, neutral; Ongin Bacillus amyloliquefaciens
Novozymes NeutraseB Industnal
Protease, Ongin Bacillus lichenifomis
Novozymes Bio-FeedB Pro Indurtnal
Protease, Ongin Bacillus lichenifomis
Novozymes NovozymB FM Industnal
Protease, Endopeptidase ; Origin: Bacillus lichenifomis
Novozymes: AlcalaseB Industrial
Proteinase; Origin: Bacillus lichenifomis
Fluka Industrial
Protease, Ongin Bacillus sp
Altus Industnal
Protease, Ongin Bacillus sp
Novozymes NovoCor S Industnal
Protease, alkaline, Ongin Bacillus sp
Novozymes: EsperaseB Industnal
Proteinase, neutral; Origin: Bacillus sp.
Toyobo Industrial
Endoproteinase; Origin: Bacillus sp.. rec.
Fluka Pilot
Protease; Ongin Bacillus sp , rec
Novozymes Novo-ProTMD Industnal
Protease, Ongin Bacillus sp ,rec
Novozymes PyraseB Industnal
Protease; Origin: Bacillus stearothennophilus
Amano: Protease S "Amano" Industrial
Protease;Origin: Bacillus subtilis
Amano : Proleather FG-F Industrial
I1505

Ammo: Protease N "Amano" Industrial
Ammo: Protease NL "Ammo" Industrial
Protease; Origin: Bacillus subtilis
Proteinase; Origin: Bacillus subtilis
Fluka Industrial
Proteinase; Origin: Bacillus subtilis var. biotecus A
Fluka Industrial
Protease;Origin: Carica papaya L
Ammo : Papain W-40 Industrial
Protease, Proline-Specific Endopeptidase; Origin: Flavobacterium sp.
Toyobo Pilot
Protease, neutral to acidic; Origin: Fungus
Novozymes: NovoCor P Industrial
Protease, alkaline; Origin: microorganism, rec. in Bacillus sp
Novozymes: Savinase@ Industrial
Protease;Origin: microorganisms
DSM Gist-brocades: Fermizyme Industrial
Protease;Origin: Penicillum sp.
Altus Industrial
Protease; Origin: Protein engineered in rec. Bacillus
Novozymes: Kannase'M Industrial
Protease, alkaline; Origin: Protein engineered, rec. in Bacillus sp.
Novozymes: Everlase@ Industrial
Protease; Origin: Rhizomucor miehei, rec. in Aspergillus oryzae
DSM Gist-brocades: Optiren Industrial
Protease, neutral to acidic; Origin: Rhizomucor sp.
Novozymes: NovoCo~fBAB Industrial
Protease; Origin: Rhizopus niveus
Ammo : Acid Protease Industrial
Protease; Origin: Rhizopus oryzae
Ammo: Peptidase R Industrial
Pronase,Ongin Streptomyces gnseus
Fluka Industnal
Fluka lndustnal
Pronase nonspecific protease,Ongin. Streptomyces gnseus
Roche Diagnostics Pronase nonspecific protease Industnal
Protease, alkalophilic, Ongin Streptomyces sp
Toyobo Pilot
Cytosol anunopeptidase Leucine anunopeptidase Peptidase S Release of an N-terminal amino acid, Xaa-I-Xbb-, in which Xaa is
preferably Leu, but may be other amino acids including Pro
-- ~ _ Y X 1 I ~ ---_---
although not Arg or Lys, and Xhb may be Pro
x " ~ L I
Leucine Aminopeptidase, cytosol , Ongin hog ladney

Fluka Lab
Leucine arninopeptidase, Ongin pig ladney
Biozyme Pilot
1506

a ~~~~~~
Xaa-Pro dipeptidase. 3.4.13.9

X-Pro dipeptldase Proline dipeptidase Imdodipeptidase Hydrolysis of Xaa-I-Pro dipeptides, also acts on
Prolidase aminoacyl-hydroxyproline analogs No action on Pro-I-Pro
Prolidase,Ongin Lactococcus lactis
Fluka Lab
Prolidase, Ongin. pig ludney
Biozyme Pilot
Dipeptidyl-peptidase1. 3.4.14.1
Cathepsin C Cathepsin J Dipeptidyl mnopeptidase I Dipeptidyl Release of an N-terminal dipeptide, Xaa-Xbb-I-Xcc, except when
transferase. Xaa is Arg or Lys, or Xbb or Xcc is Pro
Cathepsin C, sol., Ongin bovine spleen
Roche Diagnostics. Cathepsin C, sol Industnal
a
Transferred entry: 3.4.1 6.5 and 3.4.16.6. 3.4.16.1
CarboxypeptidaseY; Origin: baker’s yeast
Fluka Lab
CarboxypeptidaseY, Ongin yeast
Roche Diagnostics Carboxypeptidase Y,Sequencing Grade Lab
CarboxypeptidaseA. 3.4.17.1
Carboxypolypeptidase Peptidyl-L-amino acid + H(2)O = peptide + L-amino acid
CarboxypeptidaseA
Seravac Pilot
CarboxypeptidaseA, Ongin bovine pancreas
Fluka Lab
Carboxypeptidase P Microsomal carboxypeptiddse Release of a C-temnal residue other than proline, by

-- -
-l____-
-v- x - x-- X
preferentialcleavage of
I - ~ - ~ ~ - P _ _ . I----
Pyroglutamyl-peptidase1. 3.4.19.3
5 oxoprolyl-peptidase Pyrrolidone-carboxylate peptidase 5-oxoprolyl-peptide + H(2)O = 5-oxoproline + peptide
Pyrrolidone carboxyl peptidase Pyroglutamyl aminopeptidase
Pyroglutamate Aminopeptidase, Ongin calf liver
Fluka Pilot
Hydrolases. 3.4.21.-
Acting on peptide bonds (peptlde hydrolases).
Serlne endopeptidases.
Endoproteinase Pro-C, Ongin. microorganism, rec in E coli
Fluka Lab
Chymotrypsin. 3.4.21.1
-
Chymotrypsin A Chymotrypsin B Alpha-chymotrypsin
yI--II--yx--~~-uI-
alpha-Chymotrypsln
I ~ x _ x _ - ~ ~ -
~ I ~” ”
__I --x*-i ~
Preferential cleavage Tyr-I-Xaa, Trp-I-Xaa, Phe-I-Xaa, Len-I-Xaa
_- -
~ I( li_ I-^*.,- - ~- nX”-we_
Seravac Pilot
alphaChymotrypsin. Ongin Bacillus Iicheniformis
Alms Industnal
alpha-Chymotrypsin; Origin: bovine pancreas
Fluka Pilot
I
1507
3.4.21.4
Preferentlal cleavage Arg-I-Xaa, Lys-I-Xaa.
-m-_
-'___)_- -x x _I*
Trypsin
Seravac Pilot
Trypsin
Seravac Pilot
Trypsin; Origin: bovine pancreas
Fluka Industrial
Trypsin; Origin: pig Pancreas
Biozyme Industrial
Trypsin ;Origin: porcine pancreas
Alms Industrial
Trypsin; Origin: porcine pancreas
Biocatalysts: Trypsin Industrial
Trypsin;Origin: porcine pancreas
Biocatalysts: Trypsin 250 Industrial
Trypsin; Origin: porcine pancreas
Biocatalysts: Trypsin 250 Industrial
Trypsin ; Origin: porcine pancreas
Novozymes: Crystalline Porcine Trypsin Industrial
Trypsin (Chrymotrypsinas minor constituent), Ongin porcine pancreas
Novozymes ITN (Pancreatic Trypsin Novo) Industnal
Thrombin. 3.4.21.5
Fibnnogenase Preferential cleavage Arg I-Gly, activates fibnnogen to fibnn
I_r
- ~~~-~ 1- _I _ x _ -1_1 _y ~~~ ~ -
andreledses fibnnopeptide A
-" - -
Thrombin, Ongin bovine plasma
Fluka Pilot
Enteropeptidase. 3.4.21.9
Enterokinase. Selectlve cleavage of 6-Lys-I-Ile-7bond in trypsinogen.
Enteropeptldase
Seravac Pilot
Glutamyl endopeptidase. 3.4.21.1 9

Staphylococcal senne proteinase V8 proteinase Protease V8 Preferential cleavage Asp-I-Xaa, Glu-I-Xaa
Endoproteinase Glu-C
EndoproteaseGIu-C; Origin: Endophrins
Alms Pilot
EndoproteinaseGIu-C, Ongin Staphylococcus aureus stram V8
Fluka Lab
EndoproteinaseGIu-C, Ongin Staphylococcus aureus strain V8
Roche Diagnostics. Endoproteinase Glu-C, Sequencing Grade Lab
Pancreatic elastase. 3.4.21.36

Pancreatopeptidase E Pancreatic elastase I Hydrolysis of proteins, including elastin Preferential
cleavage Ala-I-Xaa
Elastase;Origin: hog pancreas
Fluka Pilot
Elastase;Origin: pig pancreas
Biozvme
1508
Elastase, Ongin porcine pancreas
Altus Industnal
I Lysyl bond specific proteinase Preferential cleavage L y d X a a , includin

v_ ~ ~
Endoproteinase Lys-C, Ongin Lysobacter enzymogenes

Fluka Lab
Subtilisin. 3.4.21.62
Hydrolysis of proteins with brodd specificity for peptide bonds, and
a preference for a large uncharged residue in P1 Hydrolyses peptide
mdes
Pilot
Industnal
Bacillus Iichenifonnir
Industnal
Industnal
Altus PeptiCLEC-BL (slurry) Industnal

- _ _I
Subtilisin, Ongin Bacillus lichenifomis

Fluka Industnal
Roche Diagnostics CHIRAZYME P-1, lyo Industnal
Roche Diagnostics CHIRAZYME P-1, sol In dustn aI
Subtilisin Carlsberg , Ongin Bacillus licheniformis
Altus Industnal
Proteinase K. 3.4.21.64
Endopeptidase K Tntirachium alkaline proteinase Tntirachium Hydrolysis of keratin and of other proteins, with subtilisin-like
album proteinase K specificity Hydrolyses peptides amdes
Proteinase N, Ongin Bacillus subtilis
Ruka Industnal
Proteinase K, Ongin Tntirachium album
Fluka Industnal
Proteinase K, immobilized on EupergitC,Ongin Tntirachium album
Fluka Lab
Proteinase K, lyo., Ongin Tntirachium album
Roche Diagnostics Proteinase K, lyo. Industnal
Papaya peptidase 1 Hydrolysis of proteins with broad specificity for peptide bonds, with
preference for a residue beanng a large hydrophobic sidechain at the
Papain
Biocatalysts PROMOD 144L lndustnal
Papain, Ongin Canca papaya
Flukd Industnal
I1509
Papain, Ongin Canca papaya

Roche Diagnostics Papam Industnal
Papain, immobilizedon Eupergim C, Ongin Canca papaya
Fluka Lab
_-- " C x - _I___

Specificity sinular to that of papam
1 >_clwIc--c- - x-
Industnal
Clostndiopeptidase B Preferentlal cleavage Arg-I-Xaa, including Arg-I-Pro bond, hut not

~ ~ ~ ~ ~ -xIxxL
- - a
Clostripain, Ongin
Alms Industnal
Clostripain, Ongin Clostndium histolyticum
Fluka Pilot
_ _
Endoproteinase Arg-C, Ongin Clostndium histolyticum
se Arg-C, Sequencing Grade Lab
_ _ -
Fluka Pilot
- -
Endoproteinase Arg-C, Ongin rmce (suhmaxillary glands)
Bromelain Broad specificity for cleavage of proteins, but strong preference for
m___~ _1 - ~ x x I c ~ x-_- x _XX^ ~ _r xxx x _c - - x
_ " ~ _ _ I _ ~ x -_
Z-Arg-Arg-I-NHMec amongst small molecule substrates
1 x___x ~ ~ -_~ - - -~ -
x1_ x_Ix
Bromelain,Ongin. pinapple stem

Alms
Bromelain,Ongin pineapple stem
Fluka Pilot
Pepsin A. 3.4.23.1
Pepsin Preferentlal cleavage hydrophobic, preferably aromatic, residues in
PI and PI' positions Cleaves I-Phe-I-Val-2, 4-Gln-I-His-5,
^ _ I yII -- -
Pepsin, Ongin hog stomach
y f C I __ I x x
13-Glu-I-Ala-14, 1CAla-I-Leu-15, 15-Leu-I-Tyr-16,
_I" z x_ c I_ I- -~~~ ~~- _ ~ ) _ _ _ ~
Fluka Industnal
Pepsin, Ongin pig Ftomach mucosa
Biozyme Pilot
Pepsin ,Ongin porcine pancreas
Altus Industnal
Chymosin. 3.4.23.4
Rennin Broad specificity similar to that of pepsin A Clots nulk by
- -xl_*%". 1 x -- ^" -x
cleavageof a single bond in casein (kappa cham)
X I x - x "&* - _ I - _ ) _ j Y _"L
Chyrnosin,Ongin calf stomach

Altus Industnal
1510
Microbial collagenase. 3.4.24.3

Clostndium histolytlcum collagenase Clostndiopeptidase A Digestion of native collagen in the tnple helical region at Xaa-I-Gly
Collagenaye A Collagenase I bonds With synthetlc pepudes, a preference is shown for Gly at P3
and PI', Pro and Ala at P2 and P2': and hvdroxvmoline. Ala or Are
Collagenase; Origin: Clostridium histolyticum

Fluka Pilot
Collagenase; Origin: Clostridium histolyticum
Fluka Pilot
Collagenase: Origin: Clostridium sp.
Amano: Ammo 1 [CL-I] Pilot
Collagenase, Ongin Clostndium sp
Ammo Ammo s [CL-S] Pilot
Thermolysin; Ongin Bacillus thermoproteolyticus

Altus PeptCLEC-TR (dry) Industnal
Thermolysin; Origin: Bacillus thermoproteolyticus
AItus: PeptiCLEC-TR (slurry) Industrial
Thermolysin; Origin: Bacillus thermoproteolyticus
Fluka Pilot
Peptidyl-Asp rnetalloendopeptidase. 3.4.24.33

Endoproteinase Asp-N Cleavage of Xaa-I-Asp, Xaa-I-Glu and Xaa-I-cysteic acid bonds.
Endoproteinase Asp-N: Ongin Pseudomonas fragi (mutant)
Fluka Lab
-
EndoproteinaseAspN, Ongin Pseudomonas fragi (mutant)
Roche Diagnostics Endoproteinase Asp-N, Sequencing Grade Lab
Acting on carbon-nitrogen bonds, other than

peptide bonds.
Fluka Industnal
Glutaryl acylase, carrier-fixed,Ongin E coli overproducer
Roche Diagnostics Glutanl acvlase, camer-fixed (GI-Ac) Industrial
Glutaryl-7-ACA Acylase, Ongin microorganlsm, rec in E coh
Recordati GAA Beads Industrial
&S
L-asparagine + H(2)O = L-a5partate

" _ L 1 1 ""
_ I - _~~ ~ I -x
L-Asparaginase, Ongin E coli

Fluka Lab
Glutaminase. 3.5.1.2
L-glutanune + H(2)O = L-glutamate + NH(3)
_/_Lx” --~~-~ rxarw--*anr - - P l W * - - * - s a _
Acylanudase Acylase A monocarboxylic acid amde + H(2)O = a monocarboxylate t

_--_I_ ---_
l ~ l l ~ ~ -L_-l
iw_-i---m
Amidase, Ongin Pseudomonas aeruginosa, rec in E coli

)I- *-m-m- - _xn--m--m a _ - / _
NH(3)
_ _ _ l _ l ~ - ~ ~ _ _ l - i - _ I - ~ - _ ~ ~ ~ ~ ~ ~ ~
nukd Lab
Urea + H(2)O = CO(2) t 2 NH(3)

~ - ~ . ~ ~ ~ ~ ~ ~ ~ p -
--a---w
** ~ ” - ~ - - - ~ ~ ~ ~
Urease, Ongin jack bean
Fluka Industnal
-*--w-- 1 I-sl----a17--- -----*-
Urease, Ongin jack bean
Pilot
- - -
Roche Diagnostics Urease Industrial
Industnal
-
Toyobo
__I_x_
~ ~ I ___xl^ - _- 111 II I - I I_II - -- Pilot
__I_
Urease, immobilized on Eupergit C, Ongin jack bean

Fluka Lab
Penicillin amidase. 3.5.1.1 1

Perucillin acylase Penicillin + H(2)O = a fatty acid anion + 6-mnopenicillanate
-m
-m
--p
-- ---- ----*-me---* -*-* *
-
Penicillin Acylase, Ongin E coli

Alms Industnal
_- XI_- - -- _I I - I_ I - _ - -- - I __ I_I^ -_-__ -
Penicillin Acylase, Ongin E coh
Altus ChoCLEC-EC (dry) Industnal
_I___I_ __ _I ___- - _ - I - I^___ I_ I _I- 1_ I_ ~~ _-_
Penicillin Acylase, Ongin E coli
Altus ChoCLEC-EC (slurry) Industnal
_ _ _ I _ _ _ _ _ I __ I _ _ _ _ I _I_-- --___ I__ I I l _ l I I -_ --
Penicillin Amidase, Ongin E coli
FlUka
----- - I I I- ___ ~ - I - -___ Industnal
___ - - -_
Penicillin Amidase, immobilized on Eupergim C, E coli
Fluka Industnal
-_I_ --- __ I - _ - I _ 1 1 1 1111
Penicillin G Amidase, immobilized, Ongin E coli

lndustnal
FlUka
l _ l _ l I -_ I I - __ _I - I _-
Penicillin-G Acylase, Ongin E coli
Recordati PGA beads, Standard Industnal
- --^- - I_ _I -- I_ _ _ - I II ll_l - -
Penicillin-G Acylase, Ongin E coli
Recordati PGA beads, Superenzyme Industnal
I
_I__- l_l__l-
-I - -I _lll -- _I I 1-1- _ ^ - _ _ l l l
Penicillin G Amidase, Ongin E coli overproducer

Industnal
Aminoacylase. 3.5.1.14
Histozyme Hippuncase Benzanudase Dehydropeptidase I1 An N-acyl-L-anuno acid + H(2)O = a fatty acid amon + an L-anuno
--*“,- > _ _ _ _ ( _ J x xm
I^__c~~--mn_l
Acylase I, Ongin Aspergillus melleus

-*___I I__-a--v%- - -*
acid
Fluka Industnal
1512

Acylase I, immobilized on Eupergit C, Ongin Aspergillus sp
Fluka Pilot
Ammo. Acylase Industnal

Acylase I; Ongin hog ladney
Fluka Pilot
Acylase I, Ongin. pig kidney
Biozyme PllOt
Acylase, Ongin Streptomyces chartreusis
Fluka Lab
Acylase, Ongin Streptomyces gnseocarneus
Fluka Lab
Streptomyces hachijoensis
Fluka Lab
Acylase, Ongin Streptomyces toyocaensis
Fluka Lab
Acylase, Ongin Streptomyces zanmyceticus
Fluka Lab
Recordati Hyda-REC Industnal

D-Hydantoinase, Ongin Azuk beans
Fluka Lab
D-Hydantoinase 1, carrier-fixed,0 Bacillus thermoglucosidasius, rec in E coh
Roche Diagnostics D-Hydantoinase 1, camer fixed lndustnal
-
D-Hydantoinase, recombinant, immobilized,Ongin E coli
Beta-lactamase. 3.5.2.6
Penicillinase Ceohalosoonnase A beta-lactam + H(2\0 = a substituted beta-ammo acid
beta-Lactamase I: Origin: Bacillus cereus

Fluka Lab
beta-Lactamase, Ongin Enternbacter cloacae
Fluka Lab
Creatininase. 3.5.2.10
Creatinine armdohydrolase Creatinine + H(2)O = creatine
Creatininase, Ongin microorganisms
Asah Pilot
Arginase. 3.5.3.1
L-arginine + H(2)O = L-ornithine + urea
-~ -
Arginine amdinase Canavanase
”
Arginase
Biozyme Bovine liver PllOt
L-Arginase, Ongin bovine liver
Huka Lab
I 1513
P
Creatinase. 3.5.3.3
Creatine mdinohydrolase Creatine + H(2)O = sarcosine + urea
Creatinase, Ongin. Bacillus sp

Asahi Pilot
Creatinase, Ongin Flavobacterium sp
Fluka Pilot
Hydrolases. 3.5.4.-
peptide bonds.
In cyclic amidines.
Deaminase;Ongin. Aspergillus melleus
Adenosine deaminase. 3.5.4.4

Adenosine armnohydrolase. Adenosine + H(2)O = inosine + NH(3)
Adenosine Deaminase;Ongin: calf intestinal mucosa
Fluka Pilot
I ____
Adenosine Deaminase, Ongin calf intestine
Roche Diagnostics Adenosine Deanunase Industnal
Nitrilase. 3.5.5.1
A mtnle + H(2)O = a carboxylate + NH(3)
Nitrilase, broad-range;Origin: microorganism, rec. in E. coli
BioCatalytics: NIT-I01 Lab
Nitrilase, broad-range;Origin: microorganism, rec. in E. coli
BioCatalytics: NIT-I02 Lab
Nitrilase, broad-range, Ongin nucroorgamsm, rec in E coli
BiOCatdlytiCS' NIT- 103 Lab
Inorganic pyrophosphatase. 3.6.1.1

Inorganic diphospbahse. Pyropbosphate phosphohydrolase Diphosphate + H(2)O = 2 phosphate
Diphosphate phosphobydrolase
~ x I I I I ~ x I I a w x ~ ~ x I I x
- i - _ " ~ _ _
Pyrophosphatase,inorganic, Ongin bake

Fluka Lab
Pyrophosphatase,inorganic. Ongin E coli
Fluka
Alpha-carboxylase Pyruvic decarboxylase Alpha-ketoacid A 2-0x0 acid = an aldehyde + CO(2)

carboxylase
Pyruvate Decarboxylase,Ongin Zymomonas mobils, E coli (rec )
Julich Enzyme Products
Oxaloacetate decarboxylase. 4.1.1.3

Oxdlate beta-decarboxylase Oxaloacetate = pymvate + CO(2)
Oxalacetate Decarboxylase;Origin: Pseudomonas sp.
Fluka Lab
Oxaloacetate Decarboxylase;Origin: Pseudomonas sp.
Asahi Pilot
1514
Table 20.5. (cont.)
Acetolactate decarboxylase. 4.1.1.5

*-F*s7--M-
Acetolactate Decarboxylase, Ongin Bac Brevis in rec, Bac subtilis

Novozymes MaturexB Industnal
Phosphate + oxaloacetate = H(2)O + phosphoen

Phosphoenolpyruvate carboxylase, Ongin maze leaves
Biozvme Pilot
Phosphoenolpyruvate Carboxylase, Ongin maize leaves
Fluka Lab
- -__
Phosphoenolpyruvate Carboxylase, Ongin rmcroorganisms
Toyobo Pilot
alanine = phenethylamine +
%"-_ -_wm-w-I-x ~ _ a _ ( _ _ _ - ~ h _ _ --%cw- I * -L- -
L-Phenylalanine Decarboxylase, Ongin: Streptoco
Fluka Lab
Methionine decarboxylase. 4.1.1.57

L-methionine = 3-methylthiopropanane+ CO(2)
L-Methlonine decarboxylase; Ongin Streptomyces sp
Fluka Lab
Phosphodeoxynboaldolase Deoxynboaldolase 2-deoxy-D-nbose 5-phosphate = D-glyceraldehyde 3-phosphate

+acetaldehyde
*----- " L 1 a * --^_II/x_ _-
~ ~ " - ---
L-threonine = glycine + ace
*- I *-*
Fluka
__ I I -
Lab
Threonin Aldolase; Ongin Pseudomonas putida
Fluka Lab
Mandelonitrile lyase. 4.1.2.10

Hydroxynitnle lyase. (R)-oxynitnlase
(I?)-Oxynitrilase, Ongin bitter almonds ( h n u s amygdalus)
_- %-
Mandelonitnle = cyanide + benzaldehyde
~-- _-I ^ X -,-- x
Julich Enzvme Products I ah

11515
Table 20.5. (cont ).
R-Oxynitrilase rec., Ongin Pichia pastons
FlUka Pilot
Hydroxymandelonitrile lyase. 4.1.2.1 1

Hydroxynitnle lyase (S)-4-hydroxymandelonitnle
= cyanide + 4-hydroxyhenzaldehyde
(S)-Oxynitrilase, Ongin Sorghum bicolor or S vulgare

Juhch Enzyme Products Lab
Aldolase. Fructoye-1,6-bisphosphate tnosephosphate-lyase D-fructose 1,6-bisphosphate = glycerone phosphate +

---- D-glyceraldehyde 3-phosphate
--*----+*- --
Fluka Lab
Aldolase; Origin: rabbit muscle
Fluka Lab
Aldolase; Origin: rahhit muscle
Roche Diagnostics: Aldolase Lab
Aldolase; Origin: Staphylococcus aureus
Flub Lab
Aldolase ;Origin: Staphylococcus carnosus
Fluka Lab
Fructose 1,6-bisphosphateAldolase; Origin: Staphylococcus carnosus
Aldolase, Ongin Thermus aquaucus
Fluka Lab
6-phospho-2-dehydro-3-deoxygalactonate aldolase 2-dehydro-3-deoxy-D-galactonate

6-phosphate = pyruvate
6-phospho-2-keto-3-deoxygalactonatealdolase +D-glyceraldehyde 3-phosphate
2-0x0-3-deoxygalactonate6-phosphate aldolase.
------w.-vp*"w<"*- - x __ta-~- ~ II-I--Iyx -- - - - ~ ~ - - - ~ ~ - ~ -
v L I - ^ " I I I - ~ ~ x - x - - x L - ~
6-Phospho-2-dehydro-3-deoxygalactonatealdolase
Dihydroneopterin aldolase. 4.1.2.25

2-amino-4-hydroxy-6-(D-erythro-1,2,3-tnhydroxypropyl)-7,8-dihydr
optendine =
2-anuno-4-hydroxy-6-hydroxymethyl-7,8-dihydroptendine
+
DihydroneopterinAldolase
Fluka Lab
Industnal
N-Acetyl-neuraminicacid aldolase, Ongin E coli

Fluka Lab
NeuraminicAcid Aldolase, Ongin E coli K12
-
N-AcetylneuraminicAcid Aldolase, Ongin nucroorganisms
Toyobo Pilot
1516
I 20 Tabular Survey ofCammercially Available Enzymes

N-AcetylneuraminicAcid Aldolase, Ongin microorganisms
Unitika N-Acetylneuranunic Acid Aldolase (Nana-Ald) Pilot
Citntase Citndesmolase
Citrate Lyase, Ongin Enterohacter aerogenes
Fluka Lab
2-keto-4-hydroxyglutaratealdolase 2-0x0-4-hydroxyglutaate 4-hydroxy-2-oxoglutarate= pyruvate + glyoxylate
4-Hydroxy-2-oxoglutarate aldolase ,Ongin E coli

Fluka Lab
4-hydroxy-4-methyl-2-oxoglutarate aldolase. 4.1.3.17

= 2 pyruvate
4-hydroxy-4-methyl-2-oxoglutarate
L tryptophan + H(2)O -
Tryptophanase, Ongin microorganisms
PI101
Pilot
Carbonate dehydratase. 4.2.1.1

Carbonic dehydratase Carbonic anhydrase H(2)C0(3) = CO(2) + H(2)O
I X I - -
Carbonic anhydrase, Ongin Bhvine erythocytes

Biozyme Pilot
Carbonic Anhydrase,Ongin bovine erythrocyte?
Fluka Lab
Carbonic Anhydrase lsozyme II, Ongin bovine erythrocytes
Fluka
Chondroitin ABC lyase. 4.2.2.4

Chondroitinase Chondroitin ABC elinunase Elimlndtive degradation of polysacchandes contaming
1,4-beta-D-hexosamnyI and I ,3-heta-D-glucuronosyl or
1,3-alphd-L-iduronosyIlinkages lo disacchandes containing
droitinase ABC; Ongin Proteus vulgans
Pectin lyase. 4.2.2.10

hliminative deavage of pectin to give oligosaLchandes with
termindl 4-deoxy-6-methyl-alpha-D-galact-4-enuronosyl groups
Phenylalanine ammonia-lyase. 4.3.1.5

L-phenylalanine = trans-cinnamate + NH(3)
-
PhenylalanineDeaminase,Ongin Rhodotomla glutinis
Fluka Lab
I 1517
L-3-cyanoalanine synthase. 4.4.1.9

L-cysteine + cyanide = H(2)S + L-3-cyanoalanine
"I_x - _* ~
_1 ~ x c A
Ongin Bacillus stearothennophilus

Unitika Alanine Racemase (AlaR)
c Y " 1 ~~
Ammo Amano 2 [MUT-21 Pilot

Mutarotase,Ongin pig kidney
Triosephosphateisomerase, Ongin rabbit muscle

Biozyme Pilot
Xylose isomerase.
x % I ~ - -_
D-xylose = D-xylulose
* * *I
Glucose-6-phosphate isomerase. 5.3.1.9

Phosphoglucose isomerase Phosphohexo5e isomerase D-glucose 6-phosphate = D-fructose 6-phosphate
Phosphohexomutase Oxoisomerase
_-_ ___( _ - C I - i
PhosphoglucoseIsomerase,Ongi
Unitlka Phosphoglucose Isomerase (PGI) Pilot
_ _
PhosphoglucoseIsomerase. Ongin baker's yeast
S-S rearrangase Rearrangement of both intrachain and interchain diwlfide bonds

r - _ I ~ _% _"
er
Fluka Lab
I_
Protein disulfide isomerase,Ongin E coh
- ~ - _ _- _
I
ucomutase. Ongin
sphoglucose mutase
- q _c
rabbit muscle
Acyl-activating enzyme Acyl-CoA synthetase Fatty acid ATP + a long-chain carboxylic acid + CoA = AMP + diphosphate +
Acyl-CoA Synthetase,Ongin microorganisms

Asahi Pilot
1518
I 20 Tabular Survey ofComrnercially Available Enzymes

Acyl-CoA Synthetase,Ongin Pseudomonas sp
Ammo Ammo 2 [ACS-2] Pilot
Acyl-CoA Synthetase,Ongin Pseudomonas sp
Ammo Ammo3 IACS-31 Pilot
Acyl-coenzyme A Synthetase, Ongin Pseudomonas sp
Fluka Lab
Glutamate--ammonia ligase. 6.3.1.2

Glutamme synthetase ATP + L-glutamate + NH(3) = ADP + phosphate + L-glutamme
Glutamine Synthetase, Ongin Bacillus stearothermophilus

Urutka Glutamme Synthetase (GS) Lab
NAD(+) synthetase ATP + deamido-NAD(+) + NH(3) = AMP + diphosphate +
NAD Synthetase
Asahi Pilot
Urease (An-hydrolysing) Urea carboxylase (hydrolysing) ATP + urea + CO(2) = ADP + phosphate + urea-1-carboxylate
ATP--urea amidolyase Urea anudo-lyas
1Ix- -*-*m_-l --*--
Urea Amidolyase,Ongin. yeast

Toyobo Pilot
Index
a acetyl-CoA 963,1247
AADH 1048 acetylene carboxylatehydratase
- carbinolamine 1052 - malonic semialdehyde 690
- catalytic mechanism 1051 - propynoicacid 690
- protonated imine 1051 acetylene dicarboxylatehydratase
AAT - pyruvate 690
- directed evolution 877 N-acetyl-D-glucosamine1324,1452
- protein engineering 877 - 2-epimerase 1324
- site-directed mutagenesis 877 acetylhexosamidinase 636
abscisinic acid 1385 acetyllactosamine 615
ABTS 1113,1132,1175 N-acetyl-D-mannosamine 1324,1452
ACA acylase 1058 9-0-acetylneuraminate 945
7-ACA 1436 N-acetylneuraminate
ACE inhibitors 1056 - aldonase 944
- enalapril 874 - lyase 1324
acetaldehyde 343,478, 571, 574 - synthesis from N-acetyl-D-glucosamineand
acetate kinase 614,615, 902, 904,905,1246, pyruvate 1324
1319 N-acetylneuraminic acid 194,1452
acetates 397,458 - aldolase 194
acetic anhydride 473,545 acetylphosphate 615
acetoacetates 544 achiral 352
acetoin reductase 1129 - diol 344
acetone 342,352,478,1002 achromobacter 784
acetone cyanohydrin 544,565 - xylosoxidans 744
acetone powder 1002 acid phosphatase 918,919,921
acetonitrile 342, 545 acid-base conditions 281
acetophenone 995,1019 acidophilic xylose 1316
acetoxy ketone, 2- 565 acidovorax facilis 707
acetoxyethers 458 acids 544
60-acetoxyeudesmanone 1075 acinetobacter
acetoxyphosphonates 458 - calcoaceticus 1207, 1220, 1226, 1229, 1231
acetoxysulfides 458, 565 - calcoaceticus NCIMB 9871 1214,1220,1223
acetyl esterase 1378,1383 - calcoaceticus NCIMB 9871/TD 63 1225
acetyl kinase 904 - sp. 704
acetyl phosphate 614,902,904 - sp. TD 63 1222,1223,1226
N-acetyl-D,r-aminoacids 1441 AcK 907
- racemase 1308 aconitate hydratase
acetylamino glutarate 365 - cis-aconitate 688
acetylase 1483 - citrate 688
acetylcholine acetyl hydrolases 407 - isocitricacid 688
acetylcholine esterase 406,407 AcP 907
1520
I Index
acrylamide 173,1454 adenylate kinase 615,906, 907

- production 712 adipodiamide 1450
acrylonitrile 1454 adiponitrile 1454
actinomycetes 594 adsorption 170
activation 351 aerobacter 784
- energy 192 affinitychromatography 59
active site 146, 148, 337, 338, 346, 413 aging 1001
- model 352,398,407 agrarases 1503
- residues 146 agrobacterium 774
activity 342, 473, 571 - radiobacter 593, 777, 780, 781, 783
- of immobilized enzymes 177 - SP. 774,779-782
acyclic 427 - tumefaciens 705, 774
- diacetates 417 AIDS 814
- dicarboxylic acid esters 399 ajinomoto 1442, 1448
- ketones 996 alanine 1441
- monoesters 369 - as a component of cyclosponn A 1283
acyl - production form fumaate 1298
- activating enzyme 1517 - scanning 106
- donor 473,544 alanine dehydrogenase 1286
- transfer 820 - AlaDH 1049
acylamino acid racemase 1306 - characterization 1053
- gene cloning 1308 - isolation 1053
- microbial distribution 1307 - product synthesis 1053
- properties 1307 alanine racemase 1283,1517
- substrate specificity 1307 - from yeast schizosaccharomyces pombe 1283
acylases 351, 741, 746,747, 752, 753,757, - genecloning 1283
758,1441 - isozyme 1283
- ACA acylase 1058 - reaction mechanism 1284
- acylase1 1511 - stability 1284
- glutaryl acylase 1510 - thermolabile enzyme from a psychrophile
- penicillin acylase 180, 855, 1336, 1337, 1284
1341,1403,1405, 1407, 1444,1511 - thermostable enzyme from a thermophile
acylated 1284
- alcohols 545 alcalase 1346, 1379
- hemiacetal 558 alcaligenes 755
acylations 336, 342, 346, 348, 416, 473, 474, - faecalis 299, 744
478,486,525, 570-572 - SP. 752,754
- enantiosekctive 1435 - sp. hpase 443,458, 526
- reagents 343 - xylosoxidans 788, 789
acyl-CoA synthetase 1517 alcohol dehydrogenases 81,997, 1006, 1009,
acyknzyme 337, 338, 343, 398 1010,1014,1017,1018, 1029,1038,1231
- product 337 - yeast 996
acyloin condensation 962 alcohols 288, 342, 351, 352, 370, 383, 416,
acyloxy groups 343 442,458,473,486,525,526, 544, 545,1120,
adaptive walks 116 1122, 1127
additives l l G , 417, 556, 569, 570 alcoholysis 336,342, 343, 348, 416,459,473,
additivity 109, 118 486, 545, 554, 555,558,565
adenosine aldehyde dismutase activity 1196
- aminohydrolase 1513 aldehyde reductase 1010
- deaminase 1513 aldehydes 1194
S-adenosyl-L-homocysteine1290 aldofuranose 1317
- hydrolase 1290 aldol reactions 931
S-adenosyl-L-methionine 1290 aldolases 194, 618, 931, 946, 948, 950,953,
adenovirus endopeptidase 808 954,958,1321,1515
- N-acetylneuraminic acid aldolase 944,1515 amidohydolases 742,749,751,752,777,782,
- 2-dehydro-3-deoxyphosphogalactonate
aldo- 786,1303
lase 1515 - r-asparagin amidohydrolase 1510
- deoxyribose-phosphate aldolase 1514 - r-glutamine amidohydrolase 1511
- dihydroneopterin aldolase 1515 amines 569,1035,1037
- FDP aldolase 931-933,936,938,953,961 - kinetic resolution 1435
- fructose-biphosphate aldolase 1317, 1515 amino acids
- fucose 1-P aldolase 939 - a-amino acid 370,442, 1047, 1048
- 4-hydroxy-4-methyl-2-oxoglutarate aldolase - - amide 1439
1516 - D-aminoacid 1303
- 4-hydroxy-2-oxoglutarate aldolase 1516 - - N-acetyl-D,L-aminoacid 1441
- KDG aldolase 950 - - aminotransferase 1287, 1295
- KDO aldolase 946 - - donor 890
- KDPG aldolase 949 - -ester 405
- KHG aldolase 948 - - labeled with 13N by means of I3N-NH3
- rhamnose aldolase 939 1288
- TDP aldolase 942 - - oxidase trigonopsis variabilis 1426
- threonine aldolase 953, 1514 - - production of 1286
aldose 1-epimerase 1517 - - synthesis from 5-substituted D,L-hydan-
algorithms 139 toins through N-carbamoyl-L-aminoacids
alignment 141 1303
- score significance 144 - - synthesis with D-amino acid aminotrans-
aliphatic ferase 1296
- alcohols 1145 - - synthesis with formate dehydrogenase
- ketones 1019 1296
alitame 873 - - synthesis with glutamate dehydrogenase
alkaline phosphatases 897,908,918-921 1296
alkaline protease 1346 - - synthesis with glutamate racemase 1295,
alkenes 1085,1088,1089 1296
alkoxycarbonyl group 458 - - transferases 889
alkyl-alkyl 525 - L-amino acid 873
alkyl-aryl 525 - - 6-(L-aminoadipoyl)-L-cysteinyl-D-valine
akyl-enzyme 586 1308
alkylphenols 1173, 1189 - -derivative 405
allantoinases 764766, 793 - -ester 412
allenylic alcohols 525 - - homophenylalanine 874
2-allo-hydroxy-~-proline - - isotopically labeled 874
- substrate of proline racemase 1301 - - non-naturally occuring
D-allose - - D-penicillamine 874
- from D-fructose 1322 - - L-phosphinotrhicin 874
allyl alcohols 1004, 1142 - dehydrogenases 1047
allyl bromide 1004 - derivatives 558
allylic 525 - epimerase
- alcohols 458, 565, 1145 - - cofactor-independent 1293
- hydroxylation 1081 - esters 398,412
- isomerization by 1,3-shift 1282 - - ester derivatives 405
alzheimer’sdisease 744 - racemase
amidases 729,735,719,720,730-732,763, - - cofactor-independent 1293
765, 1336, 1436, 1511 - - pyridoxal S’-phosphate-dependent 1283
- penicillin amidase 1438, 1453,1511 - - with low substrate specificity 1289
- peptide amidase 197,1510 - receptor 1007
amidation - sequence similarity 584
- ofcarboxylic 716 - spimerase
amidinohydrolases 1512, 1513 - - pyridoxal S’-phosphate-dependent 1283
1522
I Index
amino alcohols 397,442, 1119 - - glutamate oxidase 882

a-amino butyric acid 1441 - aspartate aminotransferase 1484
a-amino-c-caprolactam 1442 - glutamic-pyruvictransaminase 875
- racemase - leucine aminotransferase 888
- - genecloning 1292 - mechanism pyridoxal phosphate 875
- - isolation and purification form achromo- - mechanism pyridoxamine phosphate inter-
bacter obae 1292 mediate 875
- - properties 1292 - mechanism Schiff-baselinkage 875
- - reaction mechanism 1292 8-aminovalerate 1301
2-4-amino-4-carboxybutyl-2-aziridine car- ammonia lyases 866
boxylic acid ammoniolysis 717
- inactivator of diaminopimelate epimerase - of carboxylic esters 716
1299 amoxycillin 1441
7-amino deacetoxy cephalosporanic acid (Z- ampicillin 1441
ADCA) 1438 amygdalase 1501
6-amino penicillanic acid (6-APA) 1438 amylases 1497
amino-2-thiazoline-4-carboxylate - a-amylase 315,654, 655, 1433, 1497
- hydrolase - P-amylase 656, 1498
- - chemical synthesis of D,r-cysteine 1302 - glucoamylase 315,656, 1498
- intermediate in chemical synthesis of D,L- amyloglucosidases 1433, 1498
cysteine 1301 amylopectin 315,654
- racemase amyloses 315,654
- - bacterial distribution 1301 anaerobic 1130
a-amino-6-valerolactam andida antarctica 413
- substrate of a-amino- c-caprolactam race- androstane 1390
mase 1292 androst-2-en-3,17-dione 1160
aminoacylases 194, 744, 747, 754, 755, 756 angiotensin 801,856
- aminoacylases 746 anhydrides 554,555
- D-aminoacylase 1306 anhydrous organic solvents 412
- L-aminoacylase anilines 1186
- - of pig ludney 1306 animal enzymes 46,48
- - substrates 1306 anisotropic 102
- - thermostable enyzme 1306 antagonist 1007
- spergillus olyzae 1441 anthracene 1100
aminoacyl-tRNA 822,842 antibiotics 729, 730, 735
4-aminobutyrate-2-ketoglutarate transaminase antibody
- broad-spectrum herbicide Basta 881 - anti-0-endorphin antibody 1400
- 7-~-phosphinothricin881 anticholesterol drugs 1423
aminocephalospoanic acid 1256,1426,1436 Ap4 hydrolases 923
aminocylase 1511 APG4 1002,1022
R-1-aminoethylphosphonicacid 1285 apoptosis 813
3-aminoglutarates 365 applied molecular evolution 96
aminolysis 599 aqueous media 259
aminopeptidases 720, 723,802,809, 1506 arabidopsis thaliana 744
- aminopeptidase N 1399 a-L-arabinofuranosidases671
- pseudomonas putida 1439 arabinogalactan 674
1-amino-D-sorbitol 1454 D-arabinOSeS 1323
6-amino-1-sorbitol 1425 archaea 313
6-amino-~-sorbose1425 arginases 46, 1512
aminotransferases 874,878, 1484 arginine 871
- alanine aminotransferase 1484 - amidinase 1512
- amino acid transferase 1287, 1295 - racemase 1290
- c-aminotransferase aromatic acids 1194
- - cephalosporin biosynthesis 882 aromatic cyanohydrin acetates 544
Index
arthrobacter 1158 asymmetric
- aurescens 778, 785, 786, 788, 789, 791 - dihydroxylation 580
- crystallopoietes 774, 778 - epoxidation 579
- SP. 725,791 - hydrolysis 473
- sp.lipase 459 - synthesis 335, 342,412,473
artificial - transformation 458
- redox coenzymes 1163 atenolo1 1087
- substrate 113 atomic absorption spectrometry 785
arylallylethers 1087 ATP 1245
2-aryloxy propionic acid 571 - regeneration
Asahi Chemicals 1436, 1438 - - by acetate kinase 1319
L-ascorbic acid 1427 - - systems 909
L-asparaginases 1510 ATPases 897
aspartame 831,834,838,852,856,866,859, autoregeneration 1129,1246
873,1337,1439 auxotrophy 119
- precursor 1446 axial 370
aspartases 866,867, 1325, 1453 axial-chiral 545
- aspartame 866 - diols 502
- for synthesis of D-alanine 1298 azafenidine 1449
- fumaric acid 866, 867 azasugars 934
L-aspartase 1454 azetidinone 1444
aspartate azide 1035
- aminotransferase 130,881 2-azidoaldehydes 934
- 0-decarboxylase 1454 azido-2-hydroxypropanal 934
- - fumaric acid 867 azidolysis 599
- - L-alanine 867 azure 1130
- - L-aspartic acid 867
- racemase b
- - bacterial distribution 1297 Bacillus 784, 1037
- - composite active site 1298 - acylamina acid racemase 1306
- - essential cysteine residues 1298 - brevis 766, 788,789
- - for synthesis of D-alanine 1298 - cereus 994
- - ofarchaea 1297 - circulans 774,779
- - properties 1298 - fastidiosus 765
- - purification from streptococcus therrnophi- - lichenijorrnis 1346
lus 1297 - megatenurn 582
- - reaction mechanism 1298 - sp. 774,777
- - substrate specificity 1298 - stearothemophillus 76,78, 81, 83,85, 123,
- synthesis with maleate cis-trans isomerase 743,747,774,775,777,779,788,789,1012,
and aspartase 1325 1013,1014,1028,1128
aspartic acid 866,1454 - subtilis 123,744, 1353
- maleicacid 866 - subtilis DY 1346
- peptidases 808,812 - themoglucosidius 748
- phosphinotricin 867 - therrnoproteolyticus 78, 1345
aspartoacylases 749 Baeyer-Villiger
aspergillus 746, 748 - monooxygenases 1212,1213,1234
- rnelleus 743 - oxidation 1065, 1202, 1204, 1210, 1219,
- niger 303, 588, 593, 1355 1222, 1226,1229, 1230
- niger I F 0 4415 1000 baker’s yeast 308,310, 1016,1020
- nigerlipase 418,428,443,474, 555, 559 balstobacter sp. 779,782, 789
- oryzae 743,746,752,754,758 BASF 1436
- oryzae aminoacylase 743 batch reactor 1450
astasia longu 1037 Bayer 1425
Astra-Zeneca 1422 beauveria bussiana 308, 588, 593, 1208
1524
I fndex
beef liver 1037 - using whole cells 33

benzaldehyde 1247 biphasic
- dehydrogenase 1247 - reaction systems 1184
benz[a]anthracenes 1100, 1101 - systems 1127,1151
benz[a]pyrene 1100 - - medium engineering with organic sol-
benzocycloalkenes 1080 vents 832,833
benzocyclobutenes 1080 bipyridine complexes 1130
benzoquinones 1130,1132 bisabolol 601
benzoyl formate 1247 BLAST 74, 144
- decarboxylase 1247 blastobacter sp. 771, 780, 788
benzoyl-phenyl alanine 557 bleomycine 1256
benzyl alcohols 545, 554, 1151, 1152, 1174 !3-blockers 1157
5-benzylhydantoin blocks 156
- substrate for hydantoin racemase 1304 Bower’s compound 602,603
benzylic branched chain
- alcohols 1145, 1171 - aminotransferase (BCAT)
- alkenes 1171 - - BCAT 878
benzylisoquinoline alkaloids 1259 - - selection system 878
a-benzyloxyketones 458 - - substrate specificity 878
6-benzyloxy-(3R,SS)-dihydroxy-hexanoicacid - a-keto acids
ethyl ester 1423 - - substrate specificity 1053
3-benzyloxy-1,3-propane478 BrCN-sepharose 359
bicyclic BRENDA 152,158
- diacetates 369 brevibacterium
- lactones 383 - R312 710
- monoesters 360 - sp. 753
bicyclo[3.1.O]cyclohexanes 486 Bristol-Myers Squibb 1423
bicyclo[3.3.0]octanoIs 545 broad-range
bifunctional enzyme 1309 - continuous production of L-p-fluorophenyl-
BINAP 1453 alanine 887
binaphthols 545 - transaminase
binding motifs 90 - - immobilization 886
biocatalysis 699, 736, 1419 - volumetric productivity 887
biocatalysts 67, 79, 90, 713 bromelain 1509
- effects on acid-base conditions 281 brominated cyclohexenol derivatives 545
- effects on enzyme form 260 4-bromo-1-butene 1088
- effects on residual water level 264 bromoketones 1025
- effects on solvent choice 276 bromoperoxidase 1142, 1264, 1267, 1479
- effects on temperature 274 bromotoluene 1100
- substrate concentrations 274 7-bromotryptophan 1271
bioconversion processes 127 burkholderia
biodegradation - cepacia 413, 1201
- alkenes 583 - SP. 719
- aromatics 583 - sp. lipase 503, 526
biofuel cells 1162 butanediol 3,487, 1154
bioinformatics 139 butanol 342,352, 370,1008,1009,1152
biological butanone 1009,1012
- complexity 120 butenol 545,1088
- pathways 153 butyl hydroperoxide 1143
- phosphorylating agents 899 butyl methyl ether 342, 413,473
biosensors 1130 butyrate 458
biostoning 667 butyrolactones 545
biotin 19
biotransformations 1419
index
C carboxypeptidases 197,802,809,853,1336,
Ca” 1004 1345,1352
cadaverine 1259 - carboxypeptidase A 10,11, 1506
caerulein 856 - carboxypeptidase P 1506
cakitonin 856 - carboxypeptidase Y 1506
calcium alginate bead 1433 carebastine 1160
caldariornycesfumago 1262 Carlsberg
camphor 1200 - subtilisin 1508
- (+)-camphor 1073 carnitine 1447
- &camphor 1066 - dehydratase 1447
candida - - 4-L-butyrobetaine 694
- albicans 310 carotenoid biosynthetic pathway 130
- antarctica 295, 296, 546, 716, 1383 carrageenan 571
- antarctica A lipase 428, 503, 546 carrier types 170
- antarctica B lipase 305, 425,435, 443,479, - inorganic 171
487, 503, 526,546, 555, 558, 559, 566 - organic-biopolymer 171
- antarctica lipase 294, 335, 347, 418,428, - organic-synthetic polymer 171
442,474,487, 559,566 - proteinaceous 171
- cylindracea 413,434,1377, 1389 - synthetic 171
- cylindracea lipase 300,425-428,442,443, (-)-carve01 1148
458,459,479,486,487, 503, 526, 545, 546, (-)-camone 1148
554,555,566 carvotanacetone 1072
- lipolytica lipase 487 caryophyllene 1096
- magnoliae 1000,1001, 1010, 1027 p-casomorphin 1398
- parapsilosis I F 0 0708 1000 caspases 808,812-814
- rugosa 413 cassette 123
- rugosa lipase 300, 347,418, 428, 435, 458, - mutagenesis 69, 105
459,474,479, 503, 526, 546,555 castanospermine 1386,1387
- sp.lipase 555 catalase 1145, 1428, 1479
candidate genes 159 catalysis by UDP-glucose pyrophosphorylase
canier activation methods 169 615
capillary electrophoresis 115 catalytic
caprolactamase 727 - activity 81,87, 89, 473
capsaicin 1174 - antibodies 840, 955, 1467
N-carbamoylamino acid amidohydrolase - residues 88
1303 - triad 585
- gene cloning 1304 catechin 1390
carbamoyl phosphate 905 catechol 1179,1186
carbamoylases 763,767,770,777,780,782, - methyltransferase 1482
786,787,790-792,795,1442 catecholase activity 1176
S-carbamoyl-L-cysteinehydrolase 1302 cathepsin 812, 1506
carbamyl kinase 905 - cathepsinc 1399
carbon dioxide 1038 cefalexin 1445
carbonate 335 cell extract
- dehydratase 1516 - acinetobacter calcoaceticus 1423
carbonyl reductase 993, 1010, 1016 cell-free transcription/translation system 121
carboxyl esterase 413 cellobiase 1501
carboxylase 73,79 cellobiohydrolases 663
- phosphoenolpyruvate carboxylase 1514 cellobiose 1379
- urea carboxylase 1518 cehlases 321, 663, 1498
carboxylester hydrolase 335 cellulose 321, 661
carboxylic acids 342, 351, 555, 558, 1154 - hydrolyzing enzymes 321
- esters 335,473, 554, 555 celhlosome 665
carboxymethyl thioester 848 cephalosporins 1341,1436,1512
1526
I lndex
ceramide glycanase 1398 chroman 1080

cetus process 1132 chromanone 1390
chain elongation 728 chromatography 49,757
chalcone 1390 chromobacterium viscosum lipase 418,426,
chaperonin GroESL 474,479,486
- solubilization of glutamate racemase 1293 chryseomonas luteola 594
characterization of immobilized biocatalysts chymopapain 1509
178 chymosin 1509
chelation affinity binding chymotrypsin 805, 832, 834,839,849,852,
- carbohydrate resin 166 854,855,857,1334,1336,1345,1377,1399
- diethylaminoethyl 166 - a-chymotrypsin 335, 336, 342, 347, 398,
- ionic adsorption 166 399,472,1337,1381,1394
chemferm 1444 - chymotrypsin-catalyzed 850,858
chemical - equilibrium-controlled synthesis 830
- modification 86, 335,407 - frozen aqueous systems 837
- mutagenesis 99,124 - medium engineering with organic solvents
- racemization 833
- - combination with enantioselective hydro- - prediction by S’subsite mapping 829, 830
ysis 1307 chymotrypsinogen 841,842
- resolution 1431 13-cineole 1073
chemoselectivity 706 cinnamic acid 1454
chiral cinnamyl alcohol 1152
- alcohols, primary 486 citraconase
- auxiliaries 397, 545 citraconate hydratase
- chromium carbonyl complex 502 - kitramalate 688
- glutarates 365 - 2-methylmaleate 688
- cis-glycols 1101 - (R)-2-methylmaleate dehydratase 688
- malonates 365 - 2-methylmalic acid 688
- monoacetates 370 citrate dehydratase
- monoesters 343, 346 - cis-aconitate 688
- synthesis 131 - citrate 688
chitin 325 - isocitric adic 688
- binding domain 820 citric acid derivatives 369
chitinases 326,1500 citronello1 1095
chlorinated hydrocarbons 342 classification 160
chlorine 673 - E.C: 809
chloroacetates 458,473 - of immobilization methods 165
a-chloroalanine cleavage of P-0-bonds 895
- production 1287 CLEC 342,413,473
l-chloro-l,2-epoxypropane, 1088 clostridium
a-chloroketoester 1025 - beijerinckii 1010
chloroketones 1025 - histolyticum 1335
4-chloro-3-oxobutanoate 993, 1000, 1001, 1026 - oroticum 765, 787-789
5-chloro-2-pentanone 1020, 1027 - uracilicum 780, 781
chloroperoxidases 581, 1143, 1145, 1262, clostripain 807, 808, 846, 854, 1509
1267, 1270,1273,1274,1480 - prediction by S’subsite mapping 829
chlorophenyl-acetate 565 dostripin 845
chlorotoluene 1100 CMP-N-acetylneuraminic acid 618
7-chlorotryptophan 1271 - derivatives 619
cholecystokinin 856 - synthetase 618,1486
cholesterol 1158 C-N bonds 1435
- esterases 347,413, 426, 434, 458,459, 1343 C-N ligase 859
choline esterase 148 C-0 bonds
chondroitinase 1516 - formation 1430
- hydrolytic cleavage 1430 correlation length 102
cobalamine 21 corresponding transition 338
cobalt tert-pyridine 1142 cortexolone 1079
cocaine derivatives 397 corticosteroids 1067
codon bias 104 corynebacterium 582,770
codon-levelmutagenesis 101 - sp. 1210
coenzymes 12,16, 335,991,1108 - strain 1033
- coenzyme A (CoA-SH) 18 - strain STlO 1010
- regeneration 992 corynesporium cassiicola 592
cofactor 12, 16, 1047 cosolvents 342,398,412,425
- consumption 238 cost contribution 1453
- regeneration 1048 coumarine 1390
- - FDH/formate 1054 counter-ions 281
- - glucose DH/glucose 1054 - loss 283
- specificity 82,89 coupled residue 102
COG database 157 covalent attachment 170
colchicoside 1383 - acylation 168
cold surface 269 - alkylation 168
cold-activeenzyme - conjugate addition 168
- alanine racemase 1284 - deactivation 168
collagenase 873,1335,1510 - imine formation 168
coUetotrichum gloeosporoides 307 creatinase 1513
colominic acid 1324 creatine amidinohydrolase 1513
colyophylization 571 creatininase 1512
comamonas creatinine amidohydrolase 1512
- acidovorans 719,725,780 cresolase 1482
- SP. 781 - activity 1176
- testosteroni 707, 752, 753 cross-linked
- testosteroni N I 1 700 - crystals 261
commercial - enzyme crystals 179,342,835
- biocatalysts 65 - - cross-linked enzyme aggregate (CLEA)
- enzymes 41 180
commodity chemical acrylamide 1450 - - lipases 179,180
comparative modeling 68,70, 73-75 - - penicillin acylase 179, 180
comparison of immobilization methods 170 - - proteases 179,180
conducting polymers 1113 - glutaraldehyde 179
conductive polymers 1186 cross-links 261,343
configured aldofuranose, 2R,3R cross-linking
- conversion by xylose isomerase 1317 - amorphous solid 175
confocal fluorescence coincidence analysis - high fructose corn syrup 175
(CFCS) 116 - whole cells 175
consensus crotonase 149
- concept 80,81 crotonobetainyl-CoAhydratase
- sequences 106,1098 - butyrobetaine 694
conservation patterns 145,146, 154 crotonylamino glutarate 365
continuous crown ether derivatives 544
-culture techniques 119 crude preparations 343
- flow membrane reactor 342 crystal structures 398, 413
- operated stirred tank reactor 1429, 1441 crystallization 64, 261, 757
- process 1451 cultivation 1003
control of water activity 269 cunninghamella echinulata 1031, 1229
conversion 196, 344, 346, 348 (+)-curdione 1074
coordination site 146 curvularia
core packing 127 - inaequalis 1270
1528
I lndex
- lunata 1222, 1226 - monoesters 360, 434

cyamide hydratase 702 cyclopentanol 1003, 1021
cyanidase 702,703 cyclopentanone monooxygenase 1214
5-cyano valeramide 1449 cyclopentenylester 1454
cyanoalanine synthase 1517 cylindrocarpon destructans 1225
cyanobacterium 994 cysteine 1302
cyanogenesis 975 - desulfhydrase
cyanohydrins 289,458,544, 565 - - degradation of cysteine by 1302
- acetates 458 - peptidases 805,812
- enantioselective synthesis 974 - - mechanism of catalysis 807
- follow-up chemistry 985 - synthesis from ~,~-2-amino-A~-thiazoline-
- ofaldehydes 979 4-carboxylate 1302
- ofketones 981 cytochrome
- of 3-phenoxybenzaldehyde 976 - cytochromeC 768
- preparations of optically active cyanohy- - cytochrome P-450 1066,1262
drins 974 - cytochrome P-450 monooxygenase 1263
- safe handling of cyanides 988
cyanopyridine 1451 d
cyclases 898, 1378 DAHP synthetase 947
cyclic dancus carota 1027
- dicarboxylic acids 426 Danvinian theory 95
- - diesters 360,427 databases 144,160
- diesters 365 DCIP 1146
- dimethanol derivatives 478 deacetoxycepharosporin C 1308
- diol diacetates 399 - hydroxylase 1309
- 1,2-diols 397 deactivation of the enzyme 343
- ketones 996 deacylation 337, 545
- monoacetates 370 deamidation 1436
- monoesters 365 deaminases 766, 1516
- secondary alcohols 545 - adenosin deaminase 1513
- secondary dialkanoates 425 - cyclodeaminase 871
cyclitols 936 - histidine deaminase 869
cycloalkanols 458, 545 - serine deaminase 871
cyclodextrins 316,659, 1007 - threonine deaminase 871
- glycosyltransferases 316, 659 deaminooxytocin 1334
cycloheptane derivatives 370 debranching enzyme 1503
cyclohexadiene carboxylates 383 decarboxylases
cyclohexanes 413,434 - !3-decarboxylase 877
- derivatives 349, 397 ~ acetolactate decarboxylase 1514
cyclohexanoid 370 - aspartate decarboxylase 867, 1454
- compounds 397 - benzoyl formate decarboxylase 1247
- dimethanol 486 - glutamate decarboxylase 1296,1297
- methylesters 360 - lysine decarboxylase 1514
cyclohexanol 545 - methionine decarboxylase 1514
cyclohexanone monooxygenase 1214,1216, - oxaloacetate decarboxylase 885, 1513
1263 - phenylalanine decarboxylase 1514
cyclohexene dicarboxylate 351 - pyruvate decarboxylase 1513
cyclohexenoid - tyrosinedecarboxylase 1514
- monoacetate 370 decarboxylation 877
- monoesters 351, 360, 398 I-decene 1086
cycloisomerase 1281 definition 163
cyclopentanoid 370, 398,434 degradation
- dimethanol 486 - enzymatic 725
- dimethanol diacetates 425 - ofaromatics 582
Degussa 1441 - hydroxybutyratedehydrogenase 1471
dehalogenase 149 - hydroxyisocaproatedehydrogenase 231,
dehalogenation 999 1015,1058
dehydratases 686,690-692,695 - hydroxysteroid dehydrogenase 84,85, 996,
- altronate dehydratase 690 1127,1160,1473
- arabionate dehydratase 690 - iditol2-dehydrogenase 1470
- captopril 694 - isocitrate dehydrogenase 1013, 1039
- carbapenems 694 - isopropylmalate dehydrogenase 1013
- carbonate dehydratase 1516 - intrasequential regeneration 1110
- L-carnitine 694 - lactate dehydrogenase 1012-1014,1036,
- citrate dehydratase 688-690 1109,1125,1470,1471
- diol dehydratase 582 - leucine dehydrogenase 1049,1052, 1053,
- glycerol dehydratase 692 1477
- (R)-2-hydroxyisobutyricacid 694 - maleate dehydrogenase 1471
- hydroxysteroid dehydratase 695 - rnandelate dehydrogenase 1310
- isopropylmalate dehydratasr 688 - mannitol dehydrogenase 1126
- mannonate dehydratase 690 - methanol dehydrogenase 1038,1147
- methylmalate dehydratase 088 - NADPH/ NAD(P)+)dehydrogenase 1111,
- serine dehydratase 871 1286, 1478
- tartaric acid dehydratase 6886 - - aldehyde dehydrogenase (NAD(P)+) 1475
- xylonate dehydratase 690 - - glutamate dehydrogenase (NAD(P)+)
dehydration 342 1477
dehydrogenases 25,26,288,754,766,896, - - isocitrate dehydrogenase (NADP') 1471
937,1108,1146,1454 - nicotinic acid dehydrogenase 1252
- acetoin dehydrogenase 14711 - other dehydrogenases 1127
- alanine dehydrogenase 1049,1053,1251, - phenylalanine dehydrogenase 1049,1054,
1283,1284,1286,1476,1517 1056,1477
- alcohol dehydrogenases 81,992,994,996, - phosphogluconate (decarboxylating)dehy-
997,1006,1009,1010,1014 1017,1018, drogenase 1472
1029,1038,1109,1120,1115,1120,1194, - polyol dehydrogenase 1126
1231,1469 - quinohemoprotein dehydrogenase 1147
- aldehyde dehydrogenase 1196 - quinoprotein dehydrogenase (QDH) 1146
- amino acid dehydrogenase 1047 - sorbitol dehydrogenase 1470
- benzaldehyde dehydrogenase 1247 - trimethylamine dehydrogenase 1478
- cellobiose dehydrogenase 1131 - triosephosphate dehydrogenase 1476
- dihydrolipoamide dehydrogenase 82.83, - xanthine dehydrogenase 1473
1478 dehydrogenation 1174
- dimethylamine dehydrogenase 1478 dehydroprogesterone 695
- enzyme-coupled regeneration 1110 dehydropyrirnidinases 767,774
- formaldehyde dehydrogenase 1476 deleterious mutations 102
- formate dehydrogenase 23 I , 993,1014, deletions 141,143
1048,113G,1184,1287,1295,1475 Delft Cleavage 732
- fructose dehydrogenase 1126,1475 delta sleep inducing peptide 856
- glucose dehydrogenase 99:1,994, 1037, dernethylation 1067
1126,1146,1148,1472 demonstration
- glucose phosphate dehydrogenase 993, - glucose isomerase 166
1126,1472 4-deoxy-4-fluorofructose1319
- glutamate dehydrogenase ?9,80, 1037, deoxy-fluoroglucose 1317, 1319
1054,1295 4'deoxy-4'-fluorosucrose 1319
- glyceraldehyde 3-phosphate (phosphorylat- 5-deoxy-~-fluoro-~-xylulose 1317
ing) dehydrogenase 1476 - production with xylose isomerase 1318
- glycerol dehydrogenase 992,1122,1470 deoxy-glucose 1317
- glycerol-%phosphatedehydrogenase 1124, deoxy-~-manno-2-octu~osonate 8-phosphate
1470 synthase 946
1530
I lndex
3-deoxy-~-manno-2-octulosonate aldolase 946 dihydrolipoamide dehydrogenase 82,83

1-deoxynojirimicine 1387 dihydroorotase 765,768
deoxyribonucleases 895,1496 dihydropyridines 434,1259
deoxyribose-5-phosphatealdolase (DERA) dihydropyrimidinases 765,771,774,775,
950,1321 1512
1-deoxy-5-thio-galactopyranose 935 dihydroxyacetonephosphate 1321
1-deoxy-5-thio-D-mannopyranose, 935 dihydroxyacid dehydratase 691
deoxy-thio sugars 935 dihydroxyphenylalanine 1448
depeptidyl peptidase 809 diisopropyl 544
depolymerases 1500 - ether 413,417,434,473, 545
deracemization 288,596,1136,1158,1251, - phosphofluoridate 805,816
1255,1256 diketene 544
derivatives 1100 diketocamphane monooxygenase 1216
descriptors 154 diketoester 1423
designer yeast 1241 diltiazem 252,1430
desoxynojirimycin 1150,1425 dilution series 119
detoxification 581 dimethyl sulfoxide 342,352,360,398,412
dextranase 1500 dimethylformamide 342,398,412
dextromethorphan 571 dimethylhexadiene 1093
DHAP 932,943 dinitrodibenzyl 1428
diacetates 346,350-352,369,370,407,417, diols 336,352,579,1120,1122,1129,1137,
478 1249
diacetyl reductases 1028,1129 - dehydratase 582
diacylated diols 346 dioxygenases 129,1100
dialkanoates 425 dioxygenation 1099
dialkyl dicarboxylate 370 dipeptidases 802,809, 1506
diaminase 1513 dipeptidyl
a-diamines 1259 - aminopeptidase 1506
diaminopimelate 1299 - peptidases 802
- epimerase - transferase 1506
- - essential cysteine residue 1299 dipropionate 473
- - evolutionary origin 1299 directed evolution 68,90,95,96,335,407,
- - genecloning 1299 1014,1099
- - mechanism-based inactivator 1299 - of hydantoin racemase 1305
- - properties 1299 disaccharides 1134,1142
- - tertiary structure 1300 dismutase 1482
- - topology of secondary structural element disparlure 600
1300 disulfide
diamond lattice model 996 - bond 89
diary1 ketones 1029 - bridge 89,90
dicarboxylic acids 352,1199 dithio
- diesters 351 - acetal derivative 427
- esters 370,383 - monoester 427
dichloroacetate 473 divergent evolution 1050
dichloroindophenol 1132 DMS 1439
dichloromethane 473,486 DMSO 597
dichlorophenacylchlorise 1025 DNA 124
Diels-Alderreaction 1177,1407 - ligases 895
diesterases 898,918,923-925 - polymerase 72,73,79,329,895
diesters 343,383,398,426 - shuffling 68,107
diethyl ether 398,342,473, 545,554 - shuppling 123
differentiation 343 - synthesizer 105
difluoroacetophenone 1023 DNases 918,922,923
digital imaging analysis 115 dodecadiene 1086
domains 149 - equilibrium-controlled synthesis 830
Index I””
r-dopa 1448 - prediction by S’subsite mapping 830
L-dopamine 1448 electrical
dried cells 37 - power 1185
drying agents 273 - wiring 1126
DSM 1440.1442 electrochemical
dual-action vasopeptidase inhibitor 882 - enzyme regeneration 1130
DuPont 1449 - methods 1098
dynamic - oxidation 1251
- kinetic resolution 351, 544, 558, 559, 565, - reduction 1145, 1199
1030,1441,1442 - regeneration 995, 1112, 1130
- programing 141, 144 electrode 1113, 1126, 1146, 1162
dynorphin 856 electron
- acceptors 1129, 1132
e - transfer 1130
E 348 eledoisin 856
- value 348, 349 Eli Lilly 1444
E.C. nomenclature 152 emulsion 342, 975, 1431
E.C.l.l.l.l 1422, 1423 - process 1184
E.C.1.1.99.21 1425 enantiocomplementary 416
E.C.I.I.X.X. 1424 enantioconvergent synthesis 598
E.C.1.11.1.6 1428 enantiodivergent synthesis 346
E.C.1.4.3.3 1426 enantiogenic 1079
E.C.1.5.1.13 1428, 1447 enantiomer
E.C.l.X.X.X 1427 - preference 347
E.C.3 1430, 1435 - recognition 473
E.C.3.1.1.3 1430 enantiomer-differentitating 344, 345, 348,
E.C.3.1.1.25 1433 352, 399,405,473,545,546, 566
E.C.3.1.1.41 1436 - acylation 486,487, 503, 525, 526, 544
E.C.3.2.1.1 1433 - alcoholysis 545, 554, 555, 557
E.C.3.2.1.3 1433 - hydrolysis 345, 352, 370, 371, 383, 384, 405,
E.C.3.4.1.11 1439 428,434,435,443,458,459,486,557
E.C.3.4.21.62 1431 - process 558
E.C.3.4.24.27 1446 enantiomeric
E.C.3.5.1.11 1438, 1444 - esters 343
E.C.3.5.1.14 1441 - monoesters 343, 344
E.C.3.5.1.77 1442 - ratio 348
E.C.3.5.2.2 1441 enantiomers 348
E.C.3.5.2.11 1442 enantioselective
E.C.3.5.5.1 1447 - acylation 571
E.C.4.1.99.2 1448 - hydrolysis 343
E.C.4.2.1.84 1449, 1450, 1451 enantioselectivity 103, 129, 131, 348, 370,
E.C.4.2.1.89 1447 1048
E.C.5.1.1.15 1442 - improvement 999
E.C.5.1.3.8 1452 enantiotopic
ebastine 1160 - acyloxy 343,344
EDP-GlcNAcpyrophosphorylase 616 - ester groups 336, 343,360, 365
ee value 344, 352 - groups 348, 349, 373,416,473
effect of temperature 204 - - recognition 346,417,425
effectors 26 - hydroxyl groups 336
EF-hand 146 - methoxycarbonyl groups 343
EGF 856 - selectivity 352
Eigen 96 enantiotopos 345
elastase 813, 1507 - differentiation 344
1532
I lndex
enantiotopos-differentiating 343, 344, 352, enzymatic

473,474,546, 566 - halogenation 1270
- acylation 479 - peptide synthesis 822
- alcoholysis 554, 555 - - chemically modified enzymes 835
- hydrolysis 351-353, 361, 366, 369, 399, - - fragment condensation 854
406,414,418,426 - - genetically engineered enzymes 835
encapsulation 170, 171 - - immobilized enzymes 834
- liposome encapsulation 174 - - in frozen aqueous systems 836
- microencapsulation 174 - - in solvent-free micro-aqueous systems
endoglucanase 321,663,1498,1499 838
endolinkers 1393 - - planning and process development 851
endomyces - - solid-to-solid peptide synthesis 839
- mugnusii 1020 - - solvent-modified enzymes 834
- resii 1020 - - stepwise chain elongation 853
endonucleases 129,895, 922,923 - - substrate engineering 839
endopeptidases 801,808-810 enzymes 158,261,281, 337, 342,343
- adenovirus endopeptidase 808 - activity 189, 190, 722
- endopeptidase K 1508 - availability 1468
- lysyl endopeptidase 810 - carriers, for 172
- serine endopeptidase 1506 - classification 991
endoproteinases - commercially available 1461
- Arg-C endoproteinase 1509 - committee 152
- Asp-N endoproteinase 1510 - consumption 238
- Glu/Asp-C endoproteinase - cross-linked 342
- - equilibrium-controlled synthesis 830 - crystals 343
- - prediction by S’subsite mapping 830 - database 152
- Glu-C endoproteinase 1507 - engineering 95
- Glu-C V8endoproteinase - experimental by Novozymes 1463
- - equilibrium-controlled synthesis 830 - families 148
- - prediction by S’subsite mapping 830 - flexibility 127
- Lys-C endoproteinase 1508 - form 260
- Pro-C endoproteinase 1506 - function 4
endoxylanase 670, 1499 - immobilization 193
enhancement 569 - isomerization 1282
enol - kinetics 23, 208
- acetates 458 - - catalytic 214
- esters 458 - - competitive inhibition 214
enolase G92 - - initial reaction rates 209
enolyl-CoA - - international unit 213
- hydratase - - Michaelis-Menten kinetics 210
- - enoylase 693 - - multiple enzyme systems 230
- - (R)-2-hydroxybutanoicacid 693 - - non-competitive inhibition 215
- hydratase/isomerase 88 - - ordered bi bi 220
- isomerase 149 - - ordered bi uni 221
enterobacter ugglomeruns 1149 - - ping pong bi bi 221
entrapment 170,171 - - randombibi 220
- alginate 173 - - random bi uni 220
- carrageenan 173, 174 - - resolution 558
- polyacid 173 - - reversibility 217
- polyacrylamide 173 - - substrate inhibition 227
- polyacrylate 173 - - theorell-chance 221
- polymer network 173,571 - - two-substrate reactions 218
- precipitation 173 - - uncompetitive inhibition 216
entropy calculation 117 - membrane reactor 237, 241
index
- metal combination 293 error
- nomenclature 21,1461,1465 - level 113
- quality requirements 32 - prone PCR 99, 105,131
- pH-optimum 191 - rates 100
- producers/supplicers 1464, 1468 - threshold 103
- productivity 1058 erythrose
- reactors 174 - substrate oftylose isomerase 1317
- - batchreactor 232 erjthrulose 1317
- - continuous stirred tank reactor 233 eschericha coli 68, 70,79, 82, 83, 123, 308, 311,
- - mass balances 234 775,777,782,791,1336
- - pflug flow reactor 233 essential 873
- - stirred tank reactor 232 ester 335, 336, 351, 545
- selectivity 190, 280 - formation 416
- specificity 280 esterases 158,677, 1487
- stability 190, 192 - acetyl esterase 1378, 1383
- structure 4 - acetylcholin esterase 406, 407, 1494
- substrate complex 337,850 - butyrylcholine esterase 1494
- suppliers 44,45 - carboxyl esterase 413,1487
- synonym 1468 - catalyzed hydrolyses 352
- temperature stability 191 - cholesterol esterase 347,413,426,434,458,
- tetrahedral intermediated 338 459, 1343,1494
- transition metal combination 294, 295 - cholin esterase 148, 1494
- typical abbrevitation 1462 - diesterase 898,918, 923-925
- use in organic solvents 31 - pectinesterase 1494
eosinophil peroxidase 1269 - porcine liver 335, 336, 342-344, 346,
epimerases 149, 1281, 1282,1299,1300 351-353,360,361,365,366,369-371,383,
- acetyl glucosamine epimerase 1324 384,398,407,425,472,571,572,840,1383
- aldose epimerase 1517 - rabbitserum 1378
- amino acid epimerase 1293 - resolution 983
- E. coli 1452 - sterol esterase 1494
- isopenicillin N epimerase 1308 esterification 336, 473, 555, 558
- tagatose epimerase 1322 esters 154, 338, 342, 546
epimerization 565, 1452 - of racemic alcohols 336,435,443,459
- of allo threonine 1290 - of racemic carboxylic acids 336
epoxidation 1067,1084 ethanol 352, 398,412, 545, 1152
epoxides 579, 592,1084 ethanolamine ammonia lyase 871
- aminolysis 599 ethene 1090
- azidolysis 599 ethers 342
- disubstituted 594 ethyl
- hydrolases 579 - acetate 342, 473, 478
- - isolation 582 - chloroacetate 1004
- - mechanism 584 - chlorohydroxybutanoate 1157
- - screening 587 - chlorooxobutanoate 1157
- -structure 584,585 - methoxy acetate 473, 1435
- isomerase 582 - octanoate 473
- monosubstituted 592 - thioacetate 458
- styrene oxide-type 593 - thiooctanoate 473
- trisubstituted 596 ethylene glycol 1137,1139
epoxy alcohols 442 eudesmanes 1075
epoxyindene 589 eugenol 1191
equilibration 287 Euglenales 1037
equilibrium 338, 341,748 eukaryotic
- conversion 194 - cell 1420
- of hydrolysis reactions 264 - enzymes 122
Eupergit 342,352, 571, 1438, 1446 flavin 991
evolution 148 - monooxygenase 1214
evolutional relationship - nucleotides 16
- between diaminopimelate epimerase and - reductase 1268
aspartate racemase 1299 jlavobacterium 785
- between diaminopimelate epimerase and - SP. 785,788,789
glutamate racemase 1299 flavocytochrome enzyme 1188
evolvable 97 flavoperoxidase 1143
exchange of water with the environment 269 fluorescence-activated cell sorter (FACS) 116
exoglucanase 321,663 fluorinated
exolinkers 1393 - acetophenones 1007
exonucleases 922, 923 - alcohols 1024
exopeptidases 802,808,809,853 - ketones 1005,1021, 1024
exosialidase 1500 - malonates 427
expanded bed absorption chromatography - toluenes 1100
1183 FMN 1111
experimental enzymes by novozymes 1463 - reductase 1111
expression 122, 791 focused mutagenesis 99,104,105
- pattern 160 folate coenzymes 20
extracellular enzymes 48 folding process 9
extraction of enzymes 47 formaldehyde 1038
extreme thermophiles 313 formate 1038, 1245
extremophiles 67 - dehydrogenase 231,993,1014, 1048, 1136,
1287,1295
f formation
FAD-containing monooxygenase 1263 - ofamides 716
FADHZ-dependent halogenase 1268,1275 - of carboxylic esters 472
family shuffling 100, 111 - of C-N bonds 699
farnesol 1093 - ofhydantoins 761
FAS 1027 - ofP-Obonds 895
FASTA 74,144 formylesters 1199
fatty acids 342, 1085, 1325 freeze-concentration effect 838
- synthase 1012 frontalin 602
FDH 1058 frozen aqueous systems 837
FDP aldolase 931-933,936,938,953,961 P-fructofuranosidase 1502
fed batch reactor 1448 fructose 1132
(+)-fenchone 1073 - bisphosphate 130,1319
fermantation 41 - - aldolase 1317
- ofenzymes 46 - - from glucose 1319
fermlipase 303 - dehydrogenase 1126
ferredoxin-NADP' 995 - phosphate 1318
ferricyanide 1146 - - kinase 1319
ferrocenes 1113, 1130, 1142, 1146 fucose aldolase 939
- alcohols 502 fucose analogue
fibrinogenase 1507 - modified at nonpolar terminus 1323
fingerprint 83 fucose isomerase 939,941
- motifs 82 - three-dimensional structure 1323
first-order rate constants 344 fucosidase 129, 636
fitness fucosylated oligosaccharide 1323
- information 117 fucosyltransferase 624
- landscape 97, 107 Fuji Chemical Industries 1433
fixed bed reactor 1433 Fujisawa Pharmaceutical Co. 1438
flap 348 fumarase
flavanone 1390 - aspartic acid 867
- malic acid 867 gluconobacter oxydans 1149
fumarate hydratase glucosamine 1452
- fumaricacid 687 glucose 1132,1433
- malic acid 687 - dehydrogenase 993,994,1037, 1126
fumaric acid 1454 - derivatives 1316
functional - isomerase 1313
- domain 149 - oxidase 1145
- space 106 - phosphate 615,1318
&sariurn s o h i pisi cultinase 418 - phosphate dehydrogenase 993,1126
fusion 149 - pyrophosphorylase 909
- sensors 1139
g glucosidases 654
galactose 1132, 1141 - a-glucosidase 315, 321,636,657, 663, 664,
a-galactosidases 635, 636, 1402, 1501 1501
galactosyltransferases 609, 6l!i, 619, 1484 - P-glucosidase 1501
galacturonic acid 673 - amyloglucosidase 1433, 1498
ganglioside G M 3 1398 - glucan glucosidase 1498, 1503
GAOX 1142 - thioglucosidase 1503
gap penalties 142,143 glucosyltransferases 632, 654
GC 115 glucuronidases 671, 1502
GDP-fucose 617 glutamate 1293
GDP-mannose 617 - ammonia ligase 1518
- pyrophosphorylase 617 - decarboxylase 1297
gel filtration 56, 57 - dehydrogenase 79,80, 1037,1054, 1295
generalized profiles 155 - oxidase 1297
genetic - production 1287,1289,1297,
- algorithms 98,102,112 - racemase 1295
- code 101,119 - - gene cloning 1293
- engineering 819 - - properties 1293
- methods 1010 - synthesis 1296
- modified organisms 45 - - catalytic 1294
genome sequences 1098 - - for poly-glutamate production 1294
genotype-phenotype coupling systems 120 - - from gram-positive bacteria 1294
gentobiase 1501 - - structure and mechanism 1294
geotrichurn candidurn 307, 3101,994, 1002, - - tertian structure 1294
1004,1006,1008,1018-1027,1156 - - thermostable enzyme from bacillus sub-
- glycerol dehydrogenase 992 tdis 1294
- lipase 418,479 glutaminase 25, 1511
geraniol 1069, 1152 glutamine synthetase 1518
geranylacetone 1093 glutamyl
germacrone 1074,1096 - endopeptidase 810
- epoxide 1074 - transferase 1483
gibbereliinic acid 1385 - transpeptidase 1483
Gist-Brocades/DSM 1438 glutaraldehyde 177
Glaxo 1453 glutarates 398
GlcNAc-phosphate 616 glutaric acid anhydride 558
GlcNAc-transferases 609 glutaryl-7-ACA 1427
Glu/Asp-specific endopeptidase (GSE)840 glutaryl amidase 1436
Glu4-oxytocin 1335 glutathione 1337, 1343
glucan glucosidase 1498, 1503 - peroxidase 1142
glucanase 1503 - reductase 82, 83, 1236
glucoamy1ases 315, 656, 1498 glycals 544
glucoinvertase 1501 glycanases 1398, 1499
glucokinase 1319, 1484 glyceraldehyde 1122
1536
I lndex
glycerol hanging drop method 72

- dehydatase hansenula anovnala 1022
- - PDO 692 Heck reaction 1407
- - polyols 692 helianthus annus 1385
- derivatives 571 helicase 1385
- diacetate 425 helicenediol 502
- kinase 907,908,909 helix 79
- monoacetate 417 - stabilization 127
glycidic acid methyl ester 1430 heme
glycidol 1147 - containing 1267
glycine-dependent aldolases 953 - type 1268
glycogenase 1497,1498 hemiacetals 558
glycohydrolases 1500 - hemithioacetals 291,292,565
glycolate oxidase 1137 hemiaminal 558
glycolic acid 1137 hepatitis B S antigen 856
glycol monoester enzyme 585 heptane 1084
glycopeptide 1349 heptanediol 478
glycoprotein 1483 heptyl methyl ether 1067
- remodeling 641 hesperidin 1383
glycosidases 633,1338 hesperidinase 1484
- transferase inhibitors 639 heterocyclic 1100
glycosides 637 - monoesters 360,370
glycosidic bonds 609 heterogeneous conditions 343
glycosyl transferases 611,612,619, 627-629, heterologous expression 1098
631,909,1394 heuea brasiliensis 976
- cyclodextrin glycosyl transferase 316,659 hexadecanoate 458
- inhibitors 639 I-hexadecene 1084,1087
glycosylation 611 hexadiene 1086,1093
glycosyl-enzyme intermediate 587 hexahydronaphthalenones 1081
glyoxal 1139 hexanal 1151
glyoxylic acid 1137 hexane 349,413,473,545
goat liver lipase 503 hexanediol 478
gold 157 hexanol 545,558,1151,1152
Golgi apparatus 609 hexoaminidases 1503
group preference 344 hexoketose I-phosphate 1317
growing cells 36 hexokinase 948
growth hormone releasing factor 856 hexylcyano complexes 1130
guanidinium chlorid 412 hexylchymotrypsin 835
guanidinophenyl ester 843-846,849,850 Hidden Markov model 143,144,154,156,160
l-guanyl-32,5-dimethylpyrazole841 high
guanylate kinase 906 - fructose corn symp 1433
- mutation rate 102,103
h - product concentrations 129
haemb 17 high-entropy positions 106
HAL 869 high-throughput 119,160
Haldane equation 218 - digital image analysis 130
halogenases 1268,1275 hinge movement 1050
halogenating enzymes 1267 histidinase 869
halogenations 1267,1271,1273,1277 histidine 869
halomethyl ketones 1025 - ammonia lyase 869
haloperoxidases 1262,1264,1267,1268,1271, - a-deaminase 869
1277 - nikkomycin 869
- perhydrolase 1267 - urocanoicacid 869
Hamming distance 118 HIV 814
- protease inhibitors 873, 1159 - bacillus brevis 1441
- retropepsin 814 hydratases 149,690,692,694
HLADH 994,1033,1196 - acetylene carboxylate hydratase 690
HMMs 158 - acetylene dicarboxylate hydratase 690
HOBT 1175 - aconitate hydratase 688, 689
Hoechst Marion Roussel 1427,1436 - captopril hydratase 694
Hoffmann La-Roche 1431 - carbapenems 694
Holland Sweetener Company 1446 - carnitine hydratase 694, 1447
hollow fiber ultrafiltration membrane 359 - citraconate hydratase 688
homo sapiens 743,744 - cyanamide hydratase 702
homoaconitate - dimethylmaleate hydratase 688
- hydratase 689 - enoyl-CoA hydratase 88,693
- homocitric acid 689 - fuconate hydratase 690
homoallylic 525 - - mannonate dehydratase 690
homology 142,145 - - phosphogluconate dehydratase 690
- domains 150 - fumarate hydratase 687
- modeling 70,73 - hydratase/dehydratase 690
homonojirimycin 934 - - farnesyl-CoA dehydratase 695
homophenylalanine 1439, 144-1 - - 3-hydroxybutyryl-CoAdehydratase 695
homopropargylic 525 - - 3-hydroxydecanoyl-ACP dehydratase 695
horse liver 1109, 1115 - - 3-hydroxypalmitoyl-ACPdehydratase 695
- alcohol dehydrogenases 992,994,996 - - isohexenylglutaconyl-CoAhydratase 695
- esterase 371, 383 - - itaconyl-CoA dehydratase 695
- peroxidase 122, 1034, 1262 - - lactoyl-CoA dehydratase 695
hot-spots 106 - - long-chain enoyl-CoA hydratase 695
HPLC 115 - - methylglucatonyl-Cothydratase 695
HSDH 1127 - hydratase/isomerase
Ht 31(493-515) peptide 856, 857 - - enoyl-CoA 88
human - (R)-2-hydroxyisobutyricacid 694
- immuno deficiency virus (HIV) 814 - maleate hydratase 688
- insulin 820,831,834,852, 558 - nitrile hydratase 173,686, 700-704, 707,
humicola 710,712,713,719,1449-1451,1454,
- lanuginosa lipase 435,487, 546 - oleate hydratase 695
- sp. lipase 443 - 2-oxopent-4-enoatehydratase 686
humulene 1096 hydrated
hyaluronidase 1502 - reverse micelles 263
hyaluronoglucosaminidase 1502 - salts 343
hybrid hydrations 383
- enzymes 151 hydride transfer 992
- in vitro-in vivo recombinatbm method 111 hydrindane 1081
hydantoins 761, 762, 767,785, 794, 1441 hydrocarbon monooxygenase 1263
- racemase hydrocarbons 342
- - bacterial distribution 130'3 hydrocyanation
- - genecloning 1304 - of carbonyl compounds 974
- - inactivation by hydantoin derivatives hydrogen
1305 - abstraction 1185
- - of hydantoin racemase 1305 - bonds 76,77,89,90,127
- - properties 1304 - bond chromatography 59
- - purification from Arthrobacter aurescens - peroxide 1129, 1143, 1428
DSM 3747 1304 - safe handling of cyanides 988
- - sulfur compounds 1305 hydrogenases 994
hydantoinases 306,762-765,767-775,777, hydrolases 22.81, 146, 335, 336, 344-346,
780, 784, 786, 787, 791, 792.,795, 1303, 351,407,525,581,686,771,919,1277,
1304,1512 1421,1430,1435,1486,1493
1538
I Index
- acetylcholine acetyl hydrolase 407 N-hydroxyglutamate 1294

- adenosine amino hydrolase 1513 hydroxyhistidine 1255
- adenosyl homocysteine hydrolase 1290 2-hydroxy-1-indanone 1159
- amidohydrolase 742,749, 751, 752,777, hydroxyisocaproate dehydrogenase 231,1015,
782,786,1302,1304,1510,1511 1058
- Ap4 hydrolase 923 hydroxyl groups 1122
- caprolactam hydrolase 1292 hydroxylases 1309,1447
- carboxylester hydrolase 335 - decane hydroxylase 1067
- catalyzed 545 hydroxylations 1066,1086,1170,1174,1176,
- cellobiohydrolase 663 1179,1186,1189, 1428
- creatine amidinohydrolase 1512, 1513 hydroxymethyl ketone 1016
- epoxide hydolase 579, 582, 584, 585, 1503 hydroxymethyl lactones 442
- fold 582 hydroxymethylisoxazolinebutyrates 442
- peptide hydrolase 1503, 1506 6-hydroxynicotinicacid 1254
- pullulan hydrolase 315, 316, 657-659 hydroxynitrilases 1515
- serine hydrolase 337 hydroxynitrile lyases
hydrolyases 686 - availability for organic synthesis 976
hydrolysis 335, 336, 338, 342, 343, 348-352, - catalyzed addition of hydrogen cyanide 976,
405,414,416,473,486,544, 545,555, 558, 978
565,699,705,721,723,748,753,1433,1447 - crystal structure of 975
- biocatalytic 716 - experimental techniques 981
- of amides 716,719,722 - immobilization 977
- of amino acid amides 720,741 - overexpression 976
- of carboxylic amides 719 - recombinant 977
- ofcyanide 702 - safe handling of cyanides 988
- of cyclic amides 727 - substrate acceptance 976
- ofdinitriles 705 - used for preparative application 975
- ofhydantoins 761 hydroxyphenyl glycine 1441
- of nitriles 699, 700, 703,708, 710, 716 - production by hydantoinase 1303
hydronaphthalenone 1081 hydroxyphenylalanine 1439
hydroperoxides 1034 2-hydroxy-~-proline,
hydrophilic 145 - substrate of proline racemase 1301
hydrophobic 79,145 5-hydroxypyrazine-2-carboxylicacid 1447
- core 145,149 hydroxysteroid dehydratase
- interaction 89, 90 - ergosterol 695
- interaction chromatography 54 - hydroprogesterone 695
- polymerXAD 1007 hydroxysteroid dehydrogenase 84,85,996
hydroquinones 1186 hyperthermophiles 67,79,313
hydroxy acids 1135,1136 hyperthemophilicarchaeon 73,79
- esters 405 hypohalogenic acid 1268
hydroxy butyric acid 1256 hysteresis 260, 266
hydroxy carboxylic acid esters 544,545,554,
1137 I
hydroxy ketones 544,1122 ibuprofen 604
hydroxy lactones 383 igase 822
2-hydroxyalkanes 1120 imidachlorid 1254
5-p-hydroxybenzylhydantoin imidases 772, 1336
- substrate for hydantoin racemase 1304 immiscible 342
hydroxybiphenyl 1179 immobilization 44, 342, 343, 352, 398, 571,
4-hydroxy-2-butanone 1155 786,1421
(R)-3-hydroxy-butyrate1422 immobilized 1431, 1435
3-hydroxy-2,4-dimethylglutarate 369 - cell 1433
6-hydroxydopamine 1259 - dye chromatography 61
hydroxyesters 545, 1031 - enzymes 189,261
index
imprinting 263 invertase 1502
improvement o f enantioselectivity 999 iodine 654
in silico screening 1098 iodoacetophenone 999
in situ iodoperoxidase 1267
- analyses of stereospecificityof hydrogen ion exchange
transfer 1286 - chromatography 49,757
- cofactor regeneration 626 - crystallization 757
- racemization 558 ion pairs 79
in vitro ionic
- assembly 105 - interaction 89, 90
- evolution 96 - liquids 206,412
- protein biosynthesis 1246 ionone 1071
- recombination 98 ion-pair networks 80
in vivo IPTG 69
- homologous DNA recombination 110 irreversible enzymatic
- oxidations 1190 - nonpeptidases 840
- recombination 124 - substrate engineering 840
- selection 113 - synthesis
inclusion bodies 69 - zymogens 841
- solubilization of glutamate racemase 1293 irreversible transformation 344, 345
1,2-indandiols 1159 isoamylase 657
indanols 545 isoatisene 1076
indigo 1181,1182 isobutylhydantoin
indinavir 589 - substrate for hydantoin racemase 1304
indole 1143,1181 isobutyrylhydantoin
- derivative 1271 - substrate for hydantoin racemase 1304
5-indolylmethylhydantoin isocitrate dehydrogenase 1013, 1039
- substrate for hydantoin racemase 1304 isoenzymes 351, 352
industrial isolation of enzymes 41
- enzymes 41 isoleucine 688
- reactor 119 isomerases 22,149, 1281, 1282, 1421
influence of pH 27 - &trans isomerase 1281, 1325
information theory 106 - classification of 1281
inhibition 351 - enoyl-CoA isomerase 149
inhibitors 26, 398, 998, 1004 - epoxide isomerase 582
inhibitory domains 151 - glucose isomerase 166, 1313, 1517
inorganic salts 569 - glucose-6-phosphate isomerase 1517
insertions 141, 143 - fucose isomerase 939,941, 1323
insulin (human) 856 - maleate isomerase 866, 1324
intact microbial cells 1201, L!47 - phosphoglucose isomerase 1318,1319,
integrated downstream processing 1420 1517
intein 642 - protein disulfide isomerase 1517
inter alcoholysis 545 - rhamnose isomerase 939, 941, 1321,
interfacial 1322
- activation 348 - sugar isomerase 1281
- deactivation 342 - triosephosphaste isomerase 1320,1517
intermolecular alcoholysis 54-5, 546 - tylose isomerase 1317
International BioSynthetics 3 427 - xyloseisornerase 1313-1318,1517
Internet 151 isomerization 661, 1281
INTERPRO 156 isooctane 554
intramolecular alcoholysis 545, 546 isopenicillin
inulinase 1499 - N epimerase 1308, 1309
invariant residues 145 - Nsynthetase 1308
inverse esters 843 isoporpylhydantoin
1540
I Index
- inactivator of hydantoin racemase 1305 - pyruvate kinase 614,902,907,909,921,

isoprenoid synthesis 965 1485
isopropanol 352 - streptomycin kinase 1486
isopropenyl kinetic 189
- acetate 342,473, 478 - assays 114
- alcohol 478 - constants 345
- esters 571 - enantiomer separation 486
isopropyl ester 360 - resolution 301, 351, 352, 370, 383, 398, 412,
(R)-isopropylideneglycericacid 1427 417, 544,565,571, 587,1118,1122,1129,
isopropylideneglycerol 1157, 1427 1136,1142,1427,1430,1431,1433,1439,
isopropylmalate dehydratase 688 1441
isopropylmalate dehydrogenase 1013 - - alcohols with cataylst 1, 296
isoquercitrin 1383 - - conventional 287,288,293
isoquinoline 1101 - - dynamic 287-289,296,298, 302,723
isovaleraldehyde 1153 - - enzymatic 287,293
- - of racemic cyanohydrin esters 983
k - - of racemic cyanohydrins 983
Kanegafuchi 1442,1443 - - simple 293
Kaneka process 776 kinetically controlled synthesis
kcat 88 - kinetics 827
- value 83 - prediction (of synthesis) by S' subsite map-
k c a t / h 85887 ping 827
- value 83 - synthesis 634
KDG aldolase 950 - - medium engineering with organic sol-
KDO vents 831
- aldolase 946 klebsiella
- 8-P synthetase 947 - pneumoniae 1031
- 8-phosphate phosphatase 918 - pneumoniae I F 0 3319 1000
KDPG aldolase 949 - temgena 719
KEGG 153 kloeckera
keto acids 1037, 1048, 1135 - magna 308,1031
- conversion to D-amino acid 1287 - saturnus 1028
a,a:keto diesters 398 KM 83
P-keto esters 383 - values 88
2-keto sugars 1132 Knowles' classification 896
ketopantoyl lactone 1000 kyotorphin 832,856
ketoreductase 1010, 1469
KHG aldolase 948 I
kinases 897,898 laccases 1129, 1130, 1162, 1170, 1174, 1479
- acetate kinase 614, 615, 902, 904, 905, lactam 1454
1246,1319,1486 - antibiotics 1439, 1444
- acetyl kinase 904 lactamase 1442,1454,1512
- adenylate kinase 615, 906, 907, 1486 lactase 1501
- carbamyl kinase 905 lactate 1256
- creatine kinase 1486 - dehydrogenase 1012-1014,1136
- fructose phosphat kinase 1319 - monoxygenase 1481
- glucokinase 1485 - oxidase 1256
- glycerol kinase 907-909, 1485 lactobacillus
- guanylate kinase 906 - brevis 1029
- hexokinase 948,1484 - delbrueckii bulgaricus 1015
- monophosphat kinase 615 - fermenturn 1027
- phosphoenolpyruvate kinase 1485 - kefir 994, 1017, 1027
- 6-phosphofmctokinase 1319,1485 - - alcohol dehydrogenase 992
- phosphoglycerate kinase 1486 - SP. 1014
lactococcus lactis 743 1353-1355,1368,1369,1377,1379,1381,
lactonase 1454 1383,1385,1388,1392
- fusarium oxyspoium 1433 - candida lipolytica lipase 487
lactones 335, 336, 338, 342, 371, 383,428, - candida rugosa lipase 300, 347, 418,428,
442,545,546,554,1120 435,458,459,474,479,503, 526,546,555
lactonization 383, 545 - candida sp. lipase 555
lactoperoxidase 1262,1269, 1480 - chrornobacterium viscosurn lipase 418, 426,
lactose 1379 474,479,486,1355,1379,1385,1388,1390
- synthase 1483 - fermlipase 303
laminarinase 1499 - geotrichum candidum lipase 418,479
landscape ruggedness 102, 1:16,118 - goat liver lipase 503
large scale 342 - humicola lipase 167,435,443,487,546
- experiments 359 - interesterification 167
- production 122, 351 - lipoprotein lipase 1492, 1495
- resolution 412 - mammalian lipase 413
lecithinase A 1493 - mucorjavanicus lipase 414,418,1352,1385
Leloir pathway 609, 611 - mucor meihei lipase 167, 413, 418,443,479,
lens esculenta 772 487,526,546,559, 566
leucine dehydrogenase 1049 - mucor sp. lipase 418,425,479
- characterization 1052 - penicillin lipase 546
- cloning overexpression 1052 - phospholipase 146,147,898, 1493, 1496
- isolation 1052 - porcine pancreas hpase 335, 346, 413, 414,
- kinetic parameters 1053 417,426,428,435,474,479,486,503, 526,
- substrate specificity 1052 546,555,559,1354,1355,1369,1377,1381,
leu-enkephalin 854456,1398 1390,1392
levomethorphan 571 - pseudodomonas aeruginosa lipase 347,435,
Lewis acids 624 443
LH-RH 856 - pseudomonas cepacia lipase 289,
lid 348 296298,347, 349, 413,414,418,428,435,
ligand database 153 442,443,458,459,474,479,487, 526,
hgases 22,895, 1518 544546,555,559, 566,1354,1369,1381,
- chain reaction 105 1388,1390,1391
- C-N- 804,859 - pseudodomonase chlororaphis 700,701, 707,
lignin peroxidase 1269 709-712
limited proteolysis 800, 814, 816, 817 - pseudomonasfluorescencelipase 294, 295,
limonene 600,1070,1092,1149 347,417,418, 425,428, 443,459, 474, 478,
R(+)-limonene 479,486,487, 503,526,544-546,555,559,
linum usitatissimurn 976 566,1368,1381,1385
lipases 167, 179, 180, 181, 2132, 289, 290, 292, - pseudomonas sp. 335,418,426,428,434,
296298,303,335,336,341-344, 370,407, 435,443,459, 474, 479, 503, 526, 546, 554,
413,414,417, 418, 425,426,428,434,435, 559,565,566
442,443,459, 473, 474,478, 479,486,487, - RB 001-05 lipase 1403
503, 525, 526, 544-546, 5584, 555, 558, 559, - resolution 983
565, 566, 569, 571,840, 1354, 1454, - rhizomucorjavanicuslipase 474
1488 - rhizomucor miehei lipase 413
- aspergillus niger lipase 13B9, 1385, 1390 - rhizomucor sp. lipase 418, 503
- burkholderia plantarii lipase 1435 - rhizopusjavanicus lipase 474, 1377
- candida antarctica lipase l67, 294, 305, 335, - rhizopus delemar lipase 418
347, 418, 425,428,442,4/.3,474, 479, 487, - rhizopus niveus lipase 1349
503, 526, 546, 555, 558, 559, 566, 1354, - rhizopus oryzae lipase 443
1381,1384,1388,1390 - serratia marcescens lipase 1430
- candida cylindracea lipase 300,425, - spergillus niger lipase 1379
426-428, 442, 443, 458,459,479, 486, 487, - triacylglycerol lipase 1488
503, 526, 545, 546, 554, 555, 566, - wheat germ lipase 1377, 1379, 1383
1542
I Index
lipotropin 1335 -- maleic acid 866

lipoxygenases 46,84, 1481 maltase 1501
liquefaction 661 maltobiose 1379
liquid fermentation 46 mammalian lipase 413
lithium chloride 571 mandelate
liver alcohol dehydrogenase (HLADH) 997 - dehydrogenase 1310
local alignment 150 - racemase
local fitness landscape 117 - - crystal structure 1311
log P 279 - - divalent metal ions 1310
- value 205 - - genecloning 1311
long-jump mutagenesis strategy 102 - - large-scale production 1310
Lonza 1428,1447,1448,1452 - - p(bromomethy1)mandelate 1311
loop flexibility 127 - - purification 1310
loracarbef 1445 - - reaction mechanism 1311
low solubility 1424 - - similarity to muconate lactonizing
low water enzyme 1311
- biocatalysts 260 mandelonitrile 975
- content 342, 343, 398 manganese peroxidase 1143
- media 259 manihot esculenta 976
luciferase 1198 mannitol 1427
lyases 22, 1421, 1447 - dehydrogenase 1126
- acetylneuraminatelyase 1324 mannosidase 636,1502
- ammonia lyase 866,868,870,1255 manufacturing cost 1431
- chondroitin ABC lyase 1516 Markov chain analysis 102
- citratelyase 1516 marmin 1094
- histidine ammonia lyase 869 Marukin Shoyu 1453
- hydrolyase 686 mass transfer effect 176
- hydroxymandelonitrile lyase 1515 mathematical 352
- hydroxynitrile lyase 975-978, 981, 988, - model 344,348
1514,1515 MDB 154
- mandelonitrile lyase 1514 mean-field theory 118
- pectin lyase 679, 1516 mechanisms 748
- phenylalanine ammonia lyase 1516 mediators 1113, 1139, 1141
- tyrosine phenol lyase 1448,1516 melibiase 1501
lyophilization 343 membrane 1421
lyophilized 478 - hollow-fiber 1431
- powder 260,413,473,478 - reactor 237, 1286
lyoprotection 263 menthols 1072
lysine 882, 1443 Merck Research Laboratories 1425
- aminogroups 343 MEROPS 154
- production mesaconate hydratase
- -total conversion of racemic a-amino-s-ca- - citramalate 688
prolactam 1292 - ethylmalic acid 688
- - with a-amino-c-caprolactamhydrolase - methylfumarate 688
1292 - methylmalate dehydratase 688
lysozyme 1500 - substituted malic acids 688
lysyl endopeptidase 810 mesophiles 313
metabolic
m - engineering 1288
maleate 688 - pathway 150
- dehydratase 688 metabolism 119
- hydratase 688 metal
- isomerase 1324 - chelate affinity chromatography 61
- - fumaric acid 866 - ions 146,584
metallopepidases 808,812,813 - kinetics 76
metalloproteases 147,148 microarrays 160
metalloproteins 154,743 microbacterium campoquemadoensis 1000,
Met-enkephalin 853,856,1335,1345 1001
methane 1089 microbial
- monooxygenases 1067 - enzymes 44,45,47
methanol 342,352,360,398,412, 545,1146 - lipase 336
- dehydrogenase 1038 micrococcus 784
methionine 1255,1305,1441,1442 microemulsion 263
- synthesis from (methylthioethy1)hydantoin microorganisms 299
1305 microtiter plate-based screening systems 115
methods of immboilization miglitol 1425
- covalent attachment 164 milbemycin 1082
- cross-linking 164 minimal screening requirement 116
- entrapment 164 minimum nucleotide sequence identity 110
- non-covalent adsorption 164 miscible 342
methoxy malonic acid dimethyl ester 554 - organic solvents 412
methoxy viologen 1038 misconception 1453
methoxycarbonyl phosphate 904 Mitsunobuy reaction 1407
methyl imidoesters 1086 model evaluation 75
methyl vinyl ketone 1004 modeling 73 ,
methyl viologen 995 modification 999
methylaspartase 868 - of the substrate 998
methylaspartate 868 modular enzymes 149
- mesaconicacid 868 modularity 151
methylaspartate ammonia lyase 868 modules 110
- chloroaspartic acid 868 molecular
- dialkylethylaspartates 868 - breeding 96
- ethylaspartic acid 868 - oxygen 1111,1129,1145
- methylaspartic acid 868 - sieves 273,554
methylbutanol 1152,1153 - traps 825
methylchrymotrypsin 835 monensin 1082,1083
methylcitrate dehydratase 689,690 monoacetates 350,352,370,398,407,416,
methyl-1,2-dimethoxysuccinate 369 417,425,473,478,486,525,544
methylesterases 677 monoalkanoates 427
methylgeranate 1091 monoalkyl glutarates 558
methylglucose 1317 monodechloroaminopyrrolinitrin-3-haloge-
methylhepteneone 1008,1020,1092,1096 nase 1268
methylhydantoinase 765 monoesters 344,346,351,352,398,442, 557
methyloxobutanoate 1000,1031 monofluoroacetophenone 1023
methylphenols 1189 monolactones 383
methylpropanol, 558 monomethyl 360
methylpropyl-butanedioicacid ethyl ester monooxygenases 581,582,1170,1176,1198,
1431 1214,1216,1262
methylthioethylhydantoin - Baeyer-Villingermonooxygenase 1212,
- substrate for hydantoin racemase 1304 1213,1234
methyltransferases 72,73,79, 1482 - camphor monooxygenase 1482
metoprolol 1087,1157 - cyclohexanone monooxygenase 1214,1216,
mevalonolactone 602,603 1263,1481
Mg” 1004 - cyclopentanone monooxygenase 1214,1481
Michaelis-Menten - cytochrome P-450 monooxygenase 130,
- complex 806,827 1160,1199,1263
- constant 24,748 - flavin monooxygenase 1181,1214
- equation 1005 - hydrocarbon monooxygenase 1263
1544
I lndex
hydroxybiphenyl monooxygenase 1179,

- mutator strain 131
1481 rnycobacterium 770
- lactate monooxygenase 1481 - neoaurum 725,726
- methan monooxygenase 1067 - tuberculosis 1241
- monophenol monooxygenase 1482 myeloperoxidase 1262, 1269
- progesterone monooxygenase 1482 myo-inositol derivatives 544
- tridecanone monooxygenase 1481 - hexakisphosphate phosphohydrolase 81
monophasic organic solvents 831, 834 myrcene 1073,1092
- medium engineering with organic solvents myrosinase 1503
832 myxococcusfihs 1268
monopropyl ester 360
monosaccharides 1133, 1142 n
monosubstituted hydantoins, 5 NAD hydrolase 639
- spontaneous racemization 1304 NAD' 82,83,84,1050
monoterpenes 1069 NADH 991,1047, 1290
Monsanto 1448 - dehydrogenases 1111
moraxella sp. 1009 - oxidases 1111, 1128
- alcohol dehydrogenase 992 - regeneration with formate dehydrogenase
MPEG 343, 571 1287
MPMS 1130 - stereospecifically deuterated 1286
mRNA-protein fusions 121 NAD(P)' 82-84, 1108
MSH 856 NAD(P)H 991, 1245
muconic acid 1248 - dependent oxygenase 1203
mucor naphthalene 1100
- circinelloides 307 naproxen 1080
- griseocyanus 308 naringine 1384
- heimalis 1020 natural
- javanicus 1020, 1349 - amino acid esters 412
- - dihydroxyacetone reductase 992 - diversity 106
- - lipase 414,418 - evolution 128
- miehei lipase 413, 418,443,479, 487, 526, nazlinin 1260
546, 559,566 nerol 1069
- racemosus 1025, 1026, 1031 nerolidol 1092, 1093
- racemosus kloeckera 307 nerylacetone 1093
- sp. lipase 418,425,479 NeuSAc 1452
multi neuraminidases 636, 1500
- catalytic enzymes 150 neuroligins 148
- domain proteins 149 neutral protease 78
- enzymesystem 1288 NHAA 1175
- substrate enzymes 150 nicked proteins 817
multiple nicotiana tabacurn 1037
- alignment 143,145 nicotinamide 1451
- generations of small libraries 116 - adenine dinucleotide (NAD') 82
muramidase 1500 - adenine dinucleotide phosphate (NADP')
murine epidermal growth factor 1335 82
mus musculus 744 - nucleotides 16
mutagenesis 71,123,124, 1067, 1098 nicotinic acids 1428, 1451
mutant nifknalol 597
- fitness distribution 118 nitration 1170, 1187
- library 99 - ofphenols 1187
- redundancy 99 nitrilases 700-704, 707, 711, 713, 1513
mutarotase 1517 - agrobacteriurn sp. 1447
mutases 897,925, 1281, 1517 - oxynitrilases 200, 975-978, 981, 982, 1514,
mutation rate 118, 119 1515
Index
nitriles 1447 0
- hydratases 173,686, 700-702,704,707, 0 2 1111
710,712,713,719,1454 ochobactrum anthropi 723-725
- - pseudomonas chlororaphis 1449 ocimene 1073
- - rhodococcus rodochrous 1450, 1451 octadiene 1086, 1088
nitro 1035 octane 1087
nitrobenzene 1187 - hydroxylase 1067
nitrogen 486 octanol 545
Nitto Chemical Industry 1451 octanone 1019
N-linked 609 octene 1084-1086
NMR spectroscopy 70,75 0-demethylation 1067
nocardia 582, 594 old yellow enzyme 1037
nonadienal 1196 oleate hydratase
non-additive 107 - linoleic acid 695
non-convalent adsorption - oleic acid 695
- affinitybinding 167 - pamitoleic acid 695
- chelation 167 oleic acid 695, 1436
- electrostatic binding 166 oligonucleotide
- ionic adsorption 165, 166 - cassette mutagenesis 105
- physical adsorption 165, lt17 - directed codon mutagenesis 125
non-heme oligopeptidases 802,810
- haloperoxidase 1267 oligosaccharides 622, 624, 627, 1142
- type 1268 0-linked 609
non-homologous recombination methods 110 omapatrilat 882
non-Leloir pathways 609 one base mechanism
non-native sugars 1142 - for racemization 1285
non-natural amino acids and ,esters 412, 873 one pot process 762,777
non-ribosomal peptide synthases 150,151 oninitrilase
norethisterone acetate 1079 - safe handling of cyanides 988
norlaudanosine 1259 operon 159
norvaline 1441 opposite
Novartis 1429 - enantiopreference 591,593
Novo-Nordisk 1438 - regioselectivity 598
N-oxides 1036 optimal mutation rate 103
N+ S acyl transfer optimalpH 81
- expressed protein ligation 821 optimization 190
nucleases 898,918, 922, 923, 1497 organic
- endonucleases 129,895,922,923 - biphasic system 831
- ribonucleases 825, 835, 856, 857, 859, 918, - cosolvent 335, 352,360, 370
923,924,1402,1496 - media 342
nucleophiles - phasebuffer 284
- non-natural 599 - polymers 343
nucleoside organic solvents 128, 203, 204, 259, 342, 346,
- monophosphate kinase 61 5 348, 351, 398, 413, 473, 474, 487, 503, 526,
- phosphorylase 638 545, 570-572, 1005
- phosphate regeneration system 901 ornithine 871
- triphosphates 614, 895 ornithine cyclodeaminase 871
nucleotidases 897 orthoester 558
nucleotidyl ortholog 157, 159
- cyclases 898 - search 157
- transferases 898 overexpression 1010
S-nucleotidyl-L-homocys teintt, ovoperoxidase 1269
- production 1291 oxaloacetate decarboxylase 885
- total conversion 1291 oxamesaconate hydratase
1546
I Index
- 4-carboxy-2-oxohexenedioatehydratase 690 oxoprolinases 765

oxazolinones 554,555,558 oxyanion
oxidases 26,1108,1170 - hole 807
- acyl-CoA oxidase 1476 - intermediates 338
- alcohol oxidase 1139,1141,1151,1170 oxygen 486
- amine oxidase 1256,1477 oxygenases 73,79,1203
- amino acid oxidase 1254,1477 - lipoxygenase 46,84,1481
- - galactose oxidase 1474 - monooxygenase 130,1214,1216,1263
- - glucose oxidase 1138,1162,1473 - NADH-dependent oxygenase 1203
- - glycerol-3-phosphateoxidase 1474 - toluate dioxygenase 1249
- - glycolate oxidase 1135 oxynitrilases 200,982,1514,1515
- - lactate oxidase 1481 - available for organic synthesis 976
- - trigonopsis variabilis 1426 - catalyzed addition of HCN to aldehydes
- ascorbate oxidase 1479 976
- bilirubin oxidase 1476 - catalyzed addition of HCN to ketones 978
- cholesterol oxidase 1142,1474 - catalyzed biotransformations in aqueous
- choline oxidase 1474 medium 981
- cytochrome c oxidase 1129 - catalyzed biotransformations in biphasic
- diamine oxidase 1259 medium 982
- glucose oxidase 1130,1145 - catalyzed biotransformations in organic me-
- glutamate oxidase 1297 dium 981
- glycerophosphate oxidase 1474 - crystal structure of 975
- glycolate oxidase 1137 - immobilization 977
- lactate oxidase 1256 - overexpression 976
- monoamine oxidase 1256 - recombinant 977
- NADH oxidases 1111,1128,1129 - substrate acceptance 976
- nucleoside oxidase 1138 - transhydrocyanation for HCN generation
- phenol oxidases 1176,1190 982
- pyranose oxidase 1131,1132 - used for preparative application 975
- pyruvate oxidase 1246,1476 oxypressin 1335
- sarcosine oxidase 1478 oxytocin 856
- tyramine oxidase 1477
- ureate oxidase 1478 P
- xanthine oxidase 1201,1475 P value
oxidations 1133,1134,1262,1264,1425,1427- prediction by S’subsite mapping 828
- alcohol 1425 P-450monooxygenase 130
- ofacids 1245 PAL 870
- ofalcohols 1108 - 3,Cdihydroxy-trans-caffeic acid 870
- ofaldehydes 1194 - dihydroxy-L-phenylalanineammonia lyase
- of C-N bonds 1250 palladium complexes 565
- ofdiols 1121 palmitic acid 1454
oxidative palmitoleic acid 1088
- coupling reactions 1185 pancreas lipase 487
- deamination 1426 pancreatic trypsin inhibitor 841
oxidoreductases 22,1108,1421,1422,1469, pantenoic acid 1433
1479,1481 pantolacetone 1433
- cresol oxidoreductase 1188 pantolactone 1454
- ethylphenol oxidoreductase 1189 pantoyl lactone 1000
oxime esters 342,473 papain 807,812,834,855, 1353,1508
oxirane acid esters 370 - equilibrium-controlled synthesis 830
oxobutyrates 1019 - prediction by S’subsite mapping 829,830
0x0-fatty acids 1199 papain-like endopeptidases of RNA virus 807
0x0-norleucine acetal 882 paptide antibiotics 823
oxoperoxidases 1269 paralog 157,158
Index
- search 157 - cell-surface peptidase 801
parental fitness 118 - clans 809,811
Parkinson's - classes of peptidases 805
- disease 761 - cysteine peptidase 805, 807, 812
- syndrome 744 - dipeptidyl-peptidase 809, 1506
partition 176 - endopeptidase 801,807-810,829,1508
- value 827 - enteropeptidase 1507
pathways 159, 160 - evolutionary classification 811
PCR 68-70,330 - families 809, 811
- mutagenesis 105 - glutamyl endopeptidase 1507
PDB 153 - inhibitors
pectic substances 673 - - active site-specific low-molecular-mass
pectinase 1500 inhibitors 816
pectinate 675 - - naturally occuring inhibitors 816
pectins 673 - leucyl aminopeptidase 1505
- depolymerase 1500 - lysyl endopeptidase 1508
PEG-modified enzymes 834 - metallopeptidase 808, 812, 813
penicillin 729-732, 735, 775, 1.441 - nomenclature 803
- acylase 855,1337, 1444 - oligopeptidase 802, 810
- amidase 1438, 1453 - peptidyl-Asp methalloendopeptidase 1510
- hydroylsis 1438 - peptidyl dipeptidase 802,809
- penicillin G 1341 - pyroglutamate aminopeptidase 1506
- - acylase 1336, 1337, 1341, 1403, 1405, - pyroglutamyl peptidase I 1506
1407 - serine 805,807, 810
- - cylase 1378 - structural probes of conformation of
- penicillin N soluble proteins 816
- - expandase 1308 - tripeptidyl peptidase 802,809
- - expandase/deacetoxycepharosporin C peptide
hydroxlase 1309 - amidase 197,1510
- penicillin V 1341 - nucleic acid 1398
penicillinase 1512 - synthesis 197,801
penicilliurn - - chemical peptide synthesis 818,858
- chrysogenum 307 - - coupling method 819
- roquefortelipase 546 - - expressed protein ligation (EPL) 820
- simplicissimum 1170 - - native chemical ligation 820
pentane 473 peptides 818
pentanediol 478 - bonds 800,803
pentanol 1008 - cleavingenzyrnes 801
pentanone 1012 - glycosylated 1338, 1349, 1352
PEP 902,904,907 - hydrolysis 801, 803
pepsin 1399, 1509 - lipidated 1338, 1352
peptidases 800,802,809,817,1505 - nucleopeptides 1349, 1352
- amino peptidase 720,723,802,809,1341, - phosphorylated 1338,1349,1352
1399 peptidyldipeptidases 802, 809
- aspartic peptidase 808, 81;! peptidyltransferase 804, 822
- ATP-dependent peptidase 815 peptidyl-tRNA 822, 842
- carboxypeptidase 10, 11, 197,802,809, 853, peracetic acid 1268
1336,1345,1352,1506 perhydralase 1267, 1277
- catalytic mechanism 805 permanent modification 344
- catalyzed modification 85;' permeabilized 37
- catalyzed peptide synthesis: 823 peroxidases 26,46, 581,1108, 1142,1170,
- - equilibrium-controlled synthesis 825, 826 1185,1262,1264,1267,1269,1352,1479,
- - general manipulations 824 1480
- - kinetically controlled synthesis 826 - bromo peroxidase 1142, 1264, 1267, 1479
1548
I Index
- chloride peroxidase 1480 deuterated 1290

-
- chloroperoxidase 581, 1143, 1145, 1262, isopropylester 1454
-
1267,1270,1273, 1274, 1480 - lyases 866
- eosinophil peroxidase 1269 - production 1287
- flavoperoxidase 1143 - synthesis with glutamate racemase 1296
- glutathione peroxidase 1142, 1480 phenylcatechol 1180
- haloperoxidase 1262, 1264, 1268, 1271, phenylethanol 565, 1152
1277 phenylethylamine 1435, 1454
- horseradish peroxidase 122, 1034,1186, phenylethylmethoxyamide 1436
1262 phenylglycidate
- iodoperoxidase 1267 - inactivator of mandelate racemase 1312
- lactoperoxidase 1262, 1269, 1480 phenylglycine 1439
- lignin peroxidase 1186, 1269 - deuterated 1290
- manganese peroxidase 1143 phenylmethanesulphonylfluoride 816
- ovoperoxidase 1269 phenyloxazolinone 473
- thyroid peroxidase 1269 phenylphenol 1180
- soybean peroxidase 1139, 1186 phenylpropanoic acid 1154
PFAM 156,158,160 phenylpyruvic acid 1255
Pfizer 1438 phenylthioethanol 1152
PH phosphatases 26, 897,918
- dependence 351 - acid phosphatase 918,919,921,1496
- effect 204 - alkaline phosphatase 897, 908, 918-921,
- memory 281 1495
- profiles 177 - phosphate phosphatase 918
- value 342 - pyrophosphat phosphatase 909,919,920,
phage display 68, 121 1513
pharmacological application 717 phosphatidylcholine 2-acylhydrolase 1493
phenanthrene 1100 phosphodiesterases 898,918,923-925,1496
phenanthroline complexes 1130 phosphoenolpyruvate 614,902
phenazine 1130,1146 - hydratase 692
phenolase 1482 phosphoesterase 158
phenols 1170,1179,1185,1186 phosphofructokinase 1319
phenothiazine 1130 phosphoglucomutase 925, 1517
phenoxy radicals 1170 phosphoglucosamine transacetylase 1483
phenoxypropionic acid 1191 phosphoglucose
phenyl benzyl oxazolinone 557 - isomerase 1319
phenyl ethanediol 1122 - - reaction mechanism 1318
phenyl propanol 1152 - - thermostable enzyme 1318
phenyl butyl acetate 352 ~ - X-ray structure 1318
phenylacetaldehyde 1154 - substituted 1319
- reductase 1033 phosphohydrolases 898,918, 919
phenylalanine 866, 870, 1255, 1287, 1441 phosphokinases 897
- ammonialyase 1454 phospholipases 146, 147,898, 1493, 1496
- - cinnamicacid 870 phosphomutases 897,925
- - coumaric acid 870 phosphopeptide 1349
- - DOPA 870 phosphoresters 896
- - tyrosine 870 phosphorous-containing racemic esters 383
- deaminase 1516 phosphoryl transfer 895,896
- dehydrogenase 1049 phosphorylases 612,638,897,901,923,1483
- - cloning 1056 phosphorylating
- - heterologous expression 1056 - agents 899,900,910
- - sequencing 1056 - enzymes 895
- - substrate specificity 1054 phosphorylations 896,897, 901,907, 918, 920,
- - synthesis of allysine ethylene acetal 1056 1246
Index
phosphotransacetylases 1483 porcine insulin 1337
phosphotransferases 897 position of chemical equilibrium 276
photochemical regeneration 1114 precipitant 72
photoelectrochemical 1039 precipitation 62
photosensitizers 1114 - by changing pH 63
photosynthetic microorganism 994, 1037 - by organic solvents 63
phthalyl amidase 1336 - by salting out 62
phylogenetic 155 - by water-soluble polymer 63
- relationships 158 preference 350
physical 171 prejudice 1453
phytase 81,1496 prenyl diphosphate synthase 85
pichia pastoris 1151 pre-screening 117
Pictet-Spengler reaction 1403 pressure 1002, 1009
pig liver 397 - swingreactor 777
- esterase 46, 335, 336, 342-344, 346, 351, PRINTS 156
353, 360,361, 365,366,369-371, 383,384, propanediol derivatives 478
398,407,425,472,571,572,840, 1377 PROCARD 156
pig pancreas 434 process
- lipase 46, 335, 346,413,414,417,418,426, - conditions 192
428,435,474,479,486,503, 526, 546, 555, - design 186
559 - optimization 186
pinene 1073 processing
piperidine 1080 - proteolytic processing 814
- derivatives 425 prochiral 416,473
PK 904,907 - acyclic 426
pK,value 81 - - dicarboxylic acid diesters 361
planar chiral - - diol diacetates 369,414
- [2.2]paracyclophane 458 - - diols 474
- racemic ester 370 - acylated diols 344, 370
planar chiralities 1033 - alcohols 571
plant - anhydrides 473
- cellcultures 1037 - cyclic
- enzymes 46,48 - - dicarboxylic acid esters 353, 399
P U P 352 - - diol diacetates 366, 406
plasma-atomicemission spectrometry 785 - - diol dialkanoates 418
plasmid 70, 71 - - diols in organic solvents 479
polyacrylamide gel 1450 - diacetates 407,473
polycyclic 1100 - dialkanoate 416
polygalacturonases 678, 1501) - diesters 554
- endopolygalacturonases 679 - diketones 458
- exopolygalacturonases 679 - diols 344, 346, 414, 473
polyghtamate 1294 - esters 408
polyketid 130 - glutarates 365
- synthases 150, 151 - glutaric anhydrides 557
polymerase chain reaction (I’CR) 98 - malonates 365, 398
polymerases 71, 72, 79, 329, 895 - substrates 343
polymer-modified NAD 1163 product
polynucleotide 923 - inhibition 344
- synthetases 898 - specificity 85
polyo1 1142 production of enzymes 41
- dehydrogenase 1126 productivity 185
polyphenolase 1176 profiles 143, 144, 154, 158, 160
polysaccharides 622 - method 142
pooling strategy 117 progesterone 1066, 1067
1550
I lndex
prokaryotic cell 1420 - 20s proteasome 815

proleather 1354 - 26Sproteasome 815
prolidase 1506 - PA700 regulatory complex 815
proline 871, 1256 protecting group 365
- competitive production 1301 protection
- racemase - of amino groups
- - free energy profile 1301 - - acetoxy-benzyloxycarbonyl 1338
- - properties 1301 - - Arg-OH 1334
- - purification 1301 - - benzoylphenylalanine 1334
- - reaction mechanism 1301 - - phenylacetylbenzyloxycarbonyl 1338
- - recombinant enzyme of clostridium stick - - phenylacetamide 1403, 1405
landii 1301 - - phenylacetic acid amides 1336, 1341,
- reductase 1301 1343
- rule 89 - - phthalyl imide 1343
prolyl - - pyroglutamyl amides 1341
- endopeptidase - - tetra-O-acetyl-galactopyranosyloxycarbo-
- - prediction by S’subsite mapping 829 nyl 1339
- oligopeptidase 810 - - tetra-0-acetyl-glucopyranosyloxycarbonyl
PROMISE 154 1339
promoter, T7 69 - of carboxy groups 1344
pronase 1505 - - allylesters 1353
propanediol - - benzyl esters 1346
- dehydratase 692 - - butyl esters 1345, 1350
- derivatives 417 - - bromoethyl esters 1348
propanol 545,994,1003, 1017, 1152, 1154 - - choline esters 1352
propargylic 525 - - cyclopentylesters 1353
- alcohols 1145 - - ethyl esters 1345, 1346
propene 1090 - - heptyl esters 1348
propenylphosphonate 1090 - - methoxyethoxyethylesters 1351
properties 171, 172 - - methoxyethyl esters 1351
- benefits 163 - - methyl esters 1345, 1346, 1350
- limitations 163, 164 - - nitrobenzyl esters 1348, 1350
- of immobilized biocatalysts 175 - - phenylhydrazide 1352
propionases 767, 770,777 - of hydroxy groups 1353
propionylphosphate 905 - - acetyl esters 1370,1371, 1375
propylene oxide 1134 - - butanoyl esters 1387
prosite 155, 156, 158, 160 - - butyrylesters 1375
prostaglandin 1101 - - octanoyl esters 1370
prosthetic - - pentanoyl esters 1370, 1371
- centers 154 - of thiol groups
- groups 584 - - phenylacetamidomethyl 1343
proteases 26,46, 78, 149, 801, 840, 847, 848, protein 4
852,1379, 1503 - aggregates 342
- alcaIase@ 1431 - comparison 140
- akaline protease 1346 - crystallization 68, 70, 75
- aspartic protease 326 - engineering 67, 76, 89, 90
- cysteine protease 326 - family 143
- endopeptidase 326 - peptidebonds 800
- exopeptidase 326 - scaffold 88
- heat-stable protease 327 - structure 73, 74
- metal protease 147, 148, 326 proteinases 801, 504, 1508
- serine protease 326, 398,407, 806 - endoproteinase 830,1506-1510
- subtilisin Carlsberg 1431 - inhibitor 816
proteasome proteolysis 804, 813, 816
proteolytic enzymes 154,800,801 - type I1 315,658
protoheme 1142 pulp 673
protonation state 281 purification 122
protopectin 675 - ofenzymes 49
protopectinases 676 purine-nucleoside phosphorylase 1484
protoporphyrin IX 1142 putidaredoxine 1199
pmnus amygdalus 976 putrescine 1259
pseudocrystal pyridoxal phosphate 19, 1283
- enzymation 1451 pyroglutamate aminopeptidase 1341
- process 1254 pyrophosphatases 909,919, 1513
pseudomonas 582,735,754,756, 784, 1158 pyrophosphate 920
- aemghosa 442,728,1205, '1241 pyrophosphokinases 898
- aemginosa lipase 347,435,443 pyrophosphorylases 612,615-618,909
- cepacia 289-298, 413 pyrrolecarboxylate
- cepacia lipase 347, 349,418,428,435, 442, - competitive inhibitor of proline racemase
443,458,459, 474, 479, 487, 503, 526, 1301
544-546,555,559,566 pyrrolidine 1080
- chlororaphis 707,709, 710, 712 pyrrolonitrin 1187
- chlororaphis B23 700, 711, '712 pyrroloquinohe quinone 991, 1038, 1141,
- desmolyticum 774 1145,1146
- fluorescens 291,413,719, 727, 774, 778, pyruvate 1245
1268 - kinase 614, 902, 907,909, 921
- fluorescens lipase 294,295, 347,414, 417, - oxidase 1246
418,425,428,443,459,474,478, 479, 486,
487, 503, 526, 546, 559, 566 9
- lipases 303 quasi-species theory 102, 103
- NCIMB9872 1232 quaternary
- putida 701,705,722,723, 770,775, 780, - carbon 442
782,788,789,1209,1210, 1227,1234 - structure 785
- putida ATCC 17453 1216,1224, 1229, 1232, quinazoline 1101
1238 quinoline 1101
- solanacearum 727 quinones 1176
- SP. 710, 718, 752,754, 774-, 778, 780, 782, - methide 1171, 1189
783,788,789,794,994 quinoxaline 1101
- sp. alcohol dehydrogenase 992
- sp. hpase 335,418,426,428,434,435,443, r
459,474,479,487, 503, 526, 546, 554, 559, racemases 758,763,765,766,771,791,792,
565,566 1281,1282
- Sp. NCIMB 9872 1207,1228 - achromobacter obae 1442
- sp. strain PED 1017 - alanine racemase 1284, 1285
- striata 774,778 - amino acid racemase 1283,1289, 1293,
- testosteroni 996 1306-1308,1440
PSI-BLAST 74 - amino caprolactam racemase 1292,
psicose - arginine racemase 1290
- conversion into allose 1322 - aspartate racemase 1297, 1298
psychrophiles 313 - glutamate racemase 1293, 1295, 1296
pulegone 1072 - hydantoin racemase 1303-1305
pullulan 657 - mandelate racemase 1310-1312
- hydrolases - p r o h racemase 1301
- - type1 315,659 racemates 336, 351, 383
- - type I1 316,659 - resolution 1251
- - type 111 659 - separation 412
pulldanases 1503 racemic
- type1 315,657 - acetates 384, 398, 473
1552
I Index
- acyclic alcohols 487, 503 - efficiency 108

-acylated alcohols 473 - familyshuffling 100
- alcohols 336, 416,473, 570-572 - singlegene 100
- alkanoate 416 Recordati 1442
- carboxylic acid esters 336, 371, 399, - process 776
428 recovery 342
- carboxylic acids 343,473 recursive PCR 105
- esters 408 red yeasts 584
- ketones 458 redox
racemization 287, 288, 290, 291,293, 294, - dyes 1130,1142
297,298, 301, 309,412, 544, 557, 558, 565, - mediators 1146
723,727,792,794,1439,1441 reductases
- by one-base mechanism 1285 - acetoin reductase 1129
- by two-base mechanism 1285 - aldehyde reductase 1010, 1033
RAMA 932,934-936 - diacetyl reductase 1028, 1129
random - dihydrofolte reductase 1478
- mutagenesis 68, 123, 1099 - dihydroxyacetone reductase 992
- point mutagenesis 99, 105 - diketone reductase 1470
- pointmutations 108 - flavin reductase 1268
randomized oligonucleotides 105 - FMNreductase 1111
random-priming method 108 - glutathion reductase 82, 83, 1236, 1478
Raney copper 711 - ketoreductase 1010, 1469
rare codons 69 - lipoamide NADH reductase 1478
rat liver imidase 1336 - oxidoreductase 22, 1108, 1188, 1189, 1421,
rate constants 344 1422, 1469, 1479, 1481
rational design 67 - phenylacetaldehyde reductase 1033
reactions - proline reductase 1301
- conditions 203 reductions 991
- consecutive 195 - enantioselective 1424
- engineering 185 - equivalents 1185
- kinetics 23, 189 - ketoester 1423
- mechanism 991 - ketone reduction 1422
- parallel 195 - of C=N bonds 1047
reactivity 417, 473, 569 - OfNAD' 1247
reactors reductive amination 1048
- batch reactor 232 - thermodynamic limitation 1050
- continuous stirred tank reactor 233 regeneration 1014, 1108
- fixed bed reactor 250 - coenzyme 992,1245
- flow reactor 233 - electrochemical 995
- fluidifizedbed 250 - enzyme 1126
- immobilized enzymes 250 - hydrogenases 994
- massbalance 234 - OfATP 902,909, 1246
- recirculation reactor 250, 1438 - OfNADH 1184,1247
- stirred tank reactor 232 - OfNADP' 1122
recognition 150 - of NAD(P)H 993
recombinant - of nucleoside triphosphates 901
- cells 112, 1011 - system 909
- DNA technology 819,820,855 regiospecificity 84, 85, 89
- enzymes 795 regular expression 155
- gene expression system 69 Reichstein-Grussner synthesis 1425
- genetechnology 68 relationship with water activity 267
- pig liver esterase 352 rennin 1509
- proteins 69 repetitive batch 1450
recombination 107 replacement of chloroalanine 966
replacing chemical steps by bicitransforma- - ribonuclease A 835,856,857,859,923,
tions 1402
- industrial application 1419 - ribonuclease T1 924, 1496
residence time 232 - ribonuclease T2 923,924
residual water level 264 ribonucleotide phosphohydrolase 919
resolution 351, 383, 565, 1255 ribosome 822,823
- of racemates - display 121
- - oxynitrilase as catalyst 982 ribozyme 804,823
resting cells 37, 777 ribulose 1,s-bisphosphatecarboxylase/oxyge-
retinal 1194 nase 73,79
retropepsin 812 Roehm GmbH 1446
reverse rubisco 72
- hydrolysis 346 rugged landscapes 119
- micelles 343, 833 ruthenium catalysts 565
- phase chromatography 58 rutin 1383
- transcriptases 895 rutinose 1379
rhamnose 673,1384
- aldolase 939 S
- isomerase 939,941 saccharase 1502
- - genecloning 1321 saccharification 661
- - immobilization 1322 saccharomycescerevisiae 124, 307, 794,1002,
- - metal-mediated hydride-shift mechanism 1009, 1022,1027,1241
1322 safety-catchlinker 1405
rhamnulose 1321 salicin 1383
rhizomucor salt hydrates 269
- jauanicus lipase 474 saltingout chromatography 62
- mieheilipase 413 santonin 1075
- sp.lipase 503 saturated salt solution 266
rhizopus saturation mutagenesis 104, 131
- arrhizus 1031 schizosaccharomycespombe 794
- delemarlipase 418 sdareol 1076
- niueus 1348 screen 101
- oryzaelipase 443 screening 104, 109, 112, 123, 1000
- sp.lipase 418 - sets 1463
rhodobacter sphaeroides 1034 SDS-PAGE 72
rhodococcus 582, 591, 594, 710 search engine 155
- butanica 704 secretory proteins 814
- equi 705,727,728 selection 112, 119, 123, 130
- erythropolis 728, 993, 1016, 1148 - methods 101
- rhodochrous 704-708 selective cleavage 728
- rhodochrous J l 701,709,712,713 selectivity 196, 344,417,473, 569,634
- SP. 700,701,705,710,1207 seleno subtilisin 835
- sp. N-774 711,712 selenomethionine
rhodosporidium toruloides 1029 - production 1287
rhodotorula 588 sequence
- glutinis 310, 592, 593, 1031 - alignment 74, 81-83, 85, 90, 106
- minuta I F 0 0920 1000 - analysis 140
- mucillaginosa 1025, 1026 - comparison 140,159
- pilimanae 1029 - region 143
- rubra 752 - similarity 140
riboflavin 1383 - space 97, 106
ribonucleases 825, 918 sequence-structure-functionrelationships 122
- ligase 895 sequential kinetic resolution 397
- polymerase 329 serine
1554
I Index
- deaminase 871 - poly(hydroxymethylsi1oxanes) 181

- - 2-oxobuturate 871 solid
- - pyruvate 871 - biocatalyst 260
- dehydatase 871 - fermentation 47
- hydrolase 337 - statebuffers 284
- peptidase 807 - support 343
- - catalytic mechanism 805 - - bio-beads 1399
- - clan 810 - - controlled pore glass 1343
- -family 810 - - kieselguhr-PDMA-resins 1402
- protease 398,407 - - MPEG-support 1402
- - catalytic mechanism 806 - - novasyn KA 1399
- residue 337 - - pepsyn K (polyacrylamide)resin 1398
serratia marcensens lipase 487 - - POE 6000 1406
sertraline 1025 - - SPOCC-resin 1399
serum albumin 359 - - TentaGel beads 1400
1,I-shift - - TentaGelS beads 1403
- by epimerase 1282 solid-phase
- by racemase 1282 - peptide synthesis 818,819,852
1,2-shift - synthesis 835
- by aldose-ketose isomerase 1282 solid-to-solidconversion 854
1,3-shift solketal 1147
- for allylic isomerization 1282 solubility 341
shikimic 1102 solubilized enzymes 263
sialic acid 1324 solubilizing protecting groups 832,852,854
- aldolase 618 - medium engineering with organic solvents
sialidase 1500 833
sialyltransferase (SiaT) 623 solvents 546
silapropanediol derivatives 478 - choice 276
silica 565 - engineering 417
- gel 359 - hydrophobicity 279
single - parameters 279
- gene 100 - polarity 277, 279
- turnover events 120 somatostatin 856
site Sonogashira reaction 1407
- entropies 106 sorbiton dehydrogenase 1454
- tolerances 120 sorbose derivatives
site-directed 71 - modified at position 5 1317
- mutagenesis 68,69,75, 81, 85, 87, 105, sorghum bicolor 976
335,407 soybean peroxidase 11 39
sitting drop method 72 space-timeyield 196
sliding window 845 specificity 103,129,1060
small-scale 342 sphingomyelin phosphodiesterase 1496
SMART 156 sphingomyelinase 1496
smart polymers 181 spiked oligonucleotides 105
SNAM-Progetti 1442 spimerases
- process 776 - amino acid spimerase 1283
sodium spirobicyclic amides 1081
- chloride 349 spontaneous mutations 124
- cyanide 1086 sporobolomyces
sol-gel - salmonicolor 1027, 1033
- gel entrapment 181, 343 - salmonicolor AKU4429 1010
- lipase 181 stability 177, 342, 571
- materials 413, 473 stabilization during purification 64
- poly(akylsi1oxanes) 181 staggered extension process 108
Index
stannylated derivatives 525 - - fragment condensation 845,846,848,
staphylococcus epidermidis 1015 851,852
starch 653, 1433 - - V8-protease-catalyzed 848
- processing enzymes 315 - models 346
statistical distribution of mutation 101 - modification 458
Stemmer method 108 - solvation 275
stemodine 1077 - specificity 68,84,89, 351
stereochemical control 997 subtiligase 835, 856
stereochemistry 992, 1127 subtilisin 86, 298, 335, 337, 342, 347,407,
stereoinversions 288, 1127, 1157, 1256 408,472,570,571,817,835,857,1346,
stereospecific aldol condensation 1317 1350,1354,1368, 1378,1379,1381,1383,
stereospecificity 84 1384,1399,1454, 1508
steroid synthesis 965 succinases 768,787
steroids 695, 1067, 1127, 1202 succinates 365
stop codon 102 succinic acid anhydride 473,486
storage of enzymes 64 sucrose 1379
Strecker reaction 725, 761 - derivatives 1317
streptavidin 1400 - isomerase 1281
streptidine kinase 1486 - nucleoside diphosphate 613
streptogrisin A 810 - synthase 1317, 1483
streptokinase 1486 - synthetase 625, 909
streptomyces 755, 756, 1022 suicide inhibitors 121
- coelicolor 744 sulcaton N-phenylcarbamate 1095
- griseus 298 sulfides 1034,1262-1266
- sp. 754 sulfoxidation 1263, 1264
- strain 1027 sulfoxides 1034, 1262, 1266
streptoverticillium 755 sulfur
string 159 - compounds 1004
structural - containing racemic esters 383
- genomics 140, 160 - functionalities 486, 1024
- tolerant protein 106 sulphenyl ketones 1024
structure 6 supercritical
- comparison 140,141 - carbon dioxide 1006
- units 110 - fluids 278,412
structure-function studies 108 - solvents 1006
styrene 1200 superfamily 1050
styrene superoxide dismutase 1482
- anion exchange resin 1436 surface 343
- oxide 588 - saltbridges 127
substance P 855-857 Suzuki reaction 1407
substituted hydantoin derivatives swinging arm 150
- precursors for amino acids 1303, 1306 SWISS-PROT 152
substitution matrix 141, 143 synechococcus sp. 994
substrate 101 synergism 666
- bindingenergy 76 synthesis 858
- concentrations 201, 274, 1005 - in frozen-aqueous systems 833
- mimetics 842, 843, 850, 851 synthetases 947
- - anionic substrate mimetics 847 - acyl-CoA synthetase 1517
- - cationic substrate mimetics 845 - CMP-Neu5Acsynthetase 618, 1486
- - hydrophobic substrate mimetics 849 - DAHP synthetase 947
- - kinetic model 844 - diphosphate synthetase 85
- - steady-state kinetic 843 - fatty acid synthetase 1012
- mimetic-mediated synthesis - glutamate synthetase 1293
- - clostripain-catalyzed coupling 846, 847 - glutamin synthetase 1518
1556
I Index
- isopenicillin synthetase 1308 therrnophiles 313

- KDO 8-P synthetase 947 therrnophilic organisms 157
- lactose synthetase 1483 thermostabile
- NAD(+) synthetase 1518 - DNA polymerase 71
- phosphate synthetase 946 - proteins 67
- polynucleotide synthetase 898 thermostability 78-81, 89,90, 127,473, 996,
- sucrose synthetase 625, 909, 1317, 1483 1120
- tryptophan synthetase 1290 thiacrown 571
- tyrosyl-tRNA synthetase 76, 77 ~ ethers 569
synthetic thiamine pyrophosphate 19
- applications 1222 thioesters 412,442, 565
- oligonucleotides 69 thioglucosidase 1503
thiol subtilisin 835
t thio-NAD 1147, 1163
o-tagatose 3-epimerase 1322 thionin 1130
Tanabe Siyaku 1430 three-dimensional
tannase 1495 - models 75
targeted random mutagenesis 125 - protein structures 67
tartaric acid 686 - structure 70, 75, 79, 81, 82, 87,89, 139
- dehydratase 686 threonine
tautomerase 1281 - aldolases 953, 1514
TBADH 1033,1196 - deaminase 871
TDP aldolase 942 thrombin 1400, 1507
temperature 28, 274, 1008 - inhibitors 873
template matching method, 3D 74 throughput 113
temporary 344 thymidine 1321
terpene 1092 thyroid
terpinolene 1072 - galactosyltransferase 1483
terpyridine complexes 1130 - peroxidase 1269
testosterone 1158 tiered screening 118
tetracyano-iron-l,l0-phenanthroline 1142 tight-bind inhibitor
tetracyanoquinodimethane (TCNQ) 1130 - of alanine racemase 1285
tetracyclic alcohols 545 tolerant 116
tetrahedral intermediates 806,807 toluate-l,2-dioxygenase 1249
tetrahydrofuran 342,473, 545 toluene 473, 545, 571, 1099
tetrahydropyran derivatives 425 toluic acid 1100
tetraphenylporphyrin 544 topaquinone 1259
tetrathiofulvalene (TTF) 1130 Toray Industries 1443
th. themophilus 124 total turnover number 238, 1453
thermal stability 157 Toyo Jozo 1436
thermitase 1347, 1349, 1353 TPL 870
themoactionomyces vulgaris 1347 Transacetylases 1483
therrnoanaerobacter ethanolicus 996,997, 1008, transaldolases 962, 1482
1010,1012 transaminases 874,886,1047,1484,1260
thermoanaerobium brockii 994,996,1010, - a-transaminases
1018,1109,1195,1231 - - 2-aminobutane served 880
- alcohol dehydrogenase 1120 - - enantiomerically pure arnines 880
themococcus - - isopropylamine transaminase 880
- kodakaraensis 79, 80 - - ketoglutarate transaminase 881
- kodakaraensis DODl 73 - - phenylethylamine transaminase 880
thermodynamic equilibrium 193 - - phenylisoproylamine transaminase 880
thermodynamics 748 - (R)-transaminase 877
thermolysin 78, 472, 813, 834, 1345, 1510 - (5’-transaminase 877
- bacillus proteolicus 1446 glutamate-pyruvate transaminase 1484
index
- ghtamic-oxaloacetic transam inase 1484 triethylamine 558,570
transamination 874 trifluoro esters 342
- decarboxylation spontaneous 884 trifluoro(2-thieny1)ethanone 1025
- equilibrium constant 884 trifluoromethyl ketones 1022
- oxaloacetate 884 triglycerides 417
transcriptases 895 trimethylsilylalanine
transcyanation 978 - synthesis from D,r-5-trimethylsilylmethyl-
transesterification 336,342,343,349,571, hydantoin 1305
574,717 trioctylamine 565
transferases 22,150,625, 1484 triosephosphate isomerase 1320
- acetylglucosaminyl-glycopeptidegalactosyl- tripeptidyl peptidases 802,809
transferase 1483 triphosphohydrolases 898
- N-acetyllactosaminidea-1,3-galactosyltran- Trusopt@ 1422
serase 1484 trypsin 802,831,8429345,849,852, 1334,
- acylneuraminate cytidylyltransferase 1486 1336,1345,1352
- amino acid transferase 889 - equilibrium-controlled synthesis 830
- aminotransferase 130,874,875,881,882, - prediction by S’subsite mapping 830
1484 trypsinogen 841
- dipeptidyl transferase 1506 tryptophan 1441
- fucosyltransferase 624 - halogenase 1268
- galactosyl transferase 615,619,1483,1484 - combination production from N-acetyl-D,r-
- glucosamine-phosphate N-acetyltransferase tryptophan 1307
1483 - production with tryptophan synthase 1290
- glucosidase transferase 639 tryptophanase 1516
- glycosyltransferases 316,611, 612, turnover frequency 1453
627-629,631,639,654,659 909,1394 two-base mechanism
- ghtamykransferase 609,1483 - for racemization 1285
- methyltransferases 1482 two-hybrid systems 68
- nucleotidyl transferase 898 two-liquid phase systems 1117,1118
- peptidyl transferase 804,822 two-phase
- polyribonuceotide nucleotidyltransferase - reactor 1431
1486 - systems 252,413,425,473
- sialyltransferase 623 tyrosinase 1176,1352,1482
- uridyltransferase 616 tyrosine 1176
transferrin receptor 148 - phenollyase 870
transformation 121 - - enuinia herbicola 1448
- protocols 112 - production 1287
transglucosidase 1483 - synthesis with glutamate racemase 1296
transhydrocyanation 978 P-tyrosinase 1516
- principle of 980 tyrosyl-tRNAsynthetase 76, 77
- procedureof 982
transhydrogenase 993 U
transition metal UBA 147
- combination 293 UDP 616
- complexes 365 - N-acetylgalactosamine 616
transition state analogs 120 - N-acetylmuramoyl-r-alanine
transketolase 960,1482 - - activator of glutamate racemase 1293
transpeptidases 1483 - N-acetylmuramoyl-r-alanyl-D-glutamate syn-
triad 338,413 thetase 1293
trichloroethyl - gaIactose 615
- acetate 570 - galactose pyrophosphorylase 615
- esters 342 - galactosylphosphate uridyltransferase
tricyclic alcohols 545 616
triethyl citrate 369 - glucose 615
1558
I Index
- glucose dehydrogenase 618 W

- glucose pyrophosphorylase 616,909 water
- glucuronic acid 618 - activity 205, 265
ulocladium atrum 582 - budget 271
ultrafiltration 49, 239, 342, 352, 1447 - concentration 267
UM-BBD 153 - control via vapor phase 272
uncoupling reaction 1181 - mimics 273
unfolding temperature 81 - filed porous supports 571
Unifar 1438 - immiscible solvents 338, 412
unsaturated fatty acid cis-trans isomerase - in-oil emulsions 121
- purification 1325 - miscible 338
unusual sugar derivative wheat germ lipase 1379
- production 1316 whole cell 1442, 1447
urea - achromobacter obae 1443
- amidolyase 1518 - achromobacter xylosoxidans 1428
- carboxylase 1518 - candida sorbophila 1424
urease 765, 1511 - catalyst 708
ureidopropionases 767, 770,777 - cluconobacter suboxydans 1425
ureidosuccinases 768,787 - crytococcus laurentii 1443
uricase 766,1478 - ertuinia herbicola 1448
uridine-4-diphosphoglucose dehydrogenase - neurospora crassa 1422
1126 - oxidation 1148
UTP 616 - process 1419
- pseudomonas chlororaphis 1449
Y - gseudomonasputida 1439
V8 protease 840,847,848,852 - zygosaccharomyces rouxii 1420
valencene 1096 wursters blue 1146
E-valerolactones 554
valine 1441 X
vanadium 1142,1267 XAD-7 resin 1420
- bromoperoxidase 1264 ranthobacter agilis 1336
vanillin 1174, 1191 X-ray
vanillylamine 1174, 1260 - crystal structure 347, 348
vanlev - crystallography 70
- 6-hydroxy-~-norleucine1054 - diffraction 70, 75
vasopressin 856 xylan 667
vic-diols 1119 - degrading enzymes 324
vinyl 478 xylanases 324
- acetates 342, 343, 346, 348, 473,478, 486, xylose 324,1132,1313
502,544, 545,565, 571 xylose isomerase
- butyrate 473 - by site-directed mutagenesis enhancement
- carboxylates 558 of thermostability 1316
- esters 342, 343, 571 - divalent metal cation 1314
- laurate 473 - from various microorganisms 1316
- palmitate 473 - gene cloning 1313
- propionate 473, 574 - immobilization 1316
vinylogous a-hydroxy carboxylic acids 442 - properties 1313
viologene 1113 - reaction mechanism 1314
vitamin B3 1451 - thermostable enzyme from thermophile
vitamin B6-dependent enzymes 875 1314
- three dimensional structure 1313
xylosidases 670
xylulose, 1313
Index
Y Z
YADH 1109 (-)-zeylena 1102
yarrowia Iipolytica 1002, 1009 zinc-fingers 146
yeast 125, 591, 1022, 1023, 1027, 1035, 1036, zoogloea ramigera 753
1109,1120 zopfiella karachiensis 582
- alcohol dehydrogenase 992 zygosaccharomycesrouxii 1007, 1010
yield 196 zymogens 801

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Edited by K.: Enzyme Catalysis in Organic Synthesis

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Enzyme Catalysis in Organic Synthesis

Edited by K. Drauz and H. Waldmann

B. Cornils, W. A. Herrmann (Eds.)

Applied Homogeneous Catalysis with

2000, ISBN 3-527-30434-7

6. Cornils, W. A. Herrmann, R. Schlogl, C.-H. Wong (Eds.)

Catalysis from A-Z

Chiral Catalysts Immobilization and Recycling

U. Th. Bornscheuer, R. J . Kazlauskas

Hydrolases in Organic Synthesis

R. A. Sheldon, H. van Bekkum

Fine Chemicals through Heterogeneous Catalysis

Second, Completely Revised and Enlarged Edition

Prof. Dr. Herbert Waldmann

British Library Cataloguingin-PublicationData

Die Deutsche Bibliothek - CIP Cataloguingin-

0 Wiley-VCH Verlag GmbH, Weinheim 2002

All rights reserved (including those of translation

Printed on acid-free paper.

Printed in the Federal Republic of Germany

Cover Gesine Schulte, Max-Planck-Institutfiir

syntheses. Biocatalysts should feature prominently in the repertoire of synthetic

Pasadena, January, 2002 Frances Arnold

Dortmund and Hanau, February 2002 Karlheinz Drauz, Herbert Waldmann

1.1 Enzymes as Catalysts 1

2 Production and I d a t i o n of Enzymes 41

2.4 Fermentation of Enzymes 46

3 Rational Design of Functional Proteins 67

3.1 Protein Engineering 67

4 Enzyme Engineering by Directed Evolution 95

5 Enzyme Bioinformatics 139

5.1 Introduction 139

6 Immobilization o f Enzymes 163

6.1 Introduction 163

6.3 Properties of Iminobilized Biocatalysts 175

7 Reaction Engineering for Enzyme-Catalyzed Biotransformations 185

7.1 Introduction 185

Conclusions and Outlook 253

8 Enzymic Conversions in Organic and Other Low-Water Media 259

8.1 Introduction 259

9 Enzymatic Kinetic Resolution 287

9.1 Introduction 287

10 Enzymes from Extreme Therrnophilic and HyperthermophilicArchaea and

11 Hydrolysis and Formation of C - 0 Bonds 335

11.2.3.2 Resolution of Racemic Epoxides 592

12 Hydrolysis and Formation of C-N Bonds 699

12.1 Hydrolysis of Nitriles 699

12.7 Transaminations 873

13 Formation and Cleavage of P - 0 Bonds 895

13.1 Introduction 895

14.1 Aldol Reactions 931

15 Reduction Reactions 991

15.1 Reduction of Ketones 991

16 Oxidation Reactions 1065

16.1 Oxygenation of C-H and C=C Bonds 1065

16.3.6.2 Monohydroxylationof (R)-2-PhenoxypropionicAcid and Similar

Andreas Schmid, thank Hollmann, and Bruno Buhler

17.1 Introduction 12.81