133

2.1.4 Enzymatic Carboxylation and Decarboxylation

R. Lewin, M. L. Thompson, and J. Micklefield

2.1.4.1 Biocatalytic Carboxylation

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Carboxylation reactions underpin many critical steps in the fundamental life cycles of or-
ganisms. For example, ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO),
thought to be the most abundant protein on Earth, catalyzes the carboxylation of ribulose
1,5-bisphosphate, the preliminary step in carbon fixation. Further examples of biocatalyt-
ic carboxylations can be found in many other biosynthetic and catabolic pathways, such
as de novo purine biosynthesis.[1] Those carboxylase enzymes involved in biodegradation
are thought to possess a broader substrate spectrum and, as such, could be of potential
interest for use in preparative-scale reactions.[2] In addition to the diversity of carboxyla-
tions which can be performed using these enzymes, the inherent regioselectivity of the
reactions will be explored in this chapter. Relatively few carboxylation reactions are per-
formed on an industrial scale; however, the Kolbe–Schmitt reaction is a widely used
chemical carboxylation process for the synthesis of salicylic acid and other aromatic hy-
droxycarboxylic acids. This reaction proceeds by heating alkali metal phenolates in car-
bon dioxide at high temperature and at high pressure (typically around 100 atm and
125 8C), and then treating the product with sulfuric acid. Carboxylation in the ortho posi-
tion results in the final product salicylic acid (1). Various salicylic acid derivatives are com-
mercially produced by this method for use as medicines, herbicides, and industrial prod-
ucts. However, the Kolbe–Schmitt reaction generates a large amount of byproducts,
which subsequently have to be separated (Scheme 1). Moreover, the high reaction temper-
ature and pressure have a negative impact on the environment.[3–5]

Scheme 1 Production of Salicylic Acid (and Byproducts) through Carboxylation via the
Kolbe–Schmitt Reaction in Traditional Nonenzymatic Synthesis

1. supercritical CO2
NaOH, 100 atm, 125 oC
OH 2. H2SO4

CO2H CO2H
OH OH OH OH
+ + +
CO2H HO2C HO2C
CO2H
1

The advantages conferred in biocatalytic carboxylation can be observed in the same reac-
tion catalyzed by the enzyme salicylic acid decarboxylase from Trichosporon moniliiforme,
which affords much greater regiocontrol in the production of salicylic acid from phenol,
thus highlighting a considerable benefit for commercial purposes (Scheme 2).[4]

for references see p 155

134 Biocatalysis 2.1 C—C Bond Formation

Scheme 2 Production of Salicylic Acid through an Enzymatic

Kolbe–Schmitt Reaction Using Salicylic Acid Decarboxylase from
Trichosporon moniliiforme[4]

CO2H
salicylic acid decarboxylase
OH OH
supercritical CO2

In addition to enhanced regioselectivity, this enzymatic Kolbe–Schmitt reaction avoids

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the requirement of high temperatures and pressures and the use of harsh reactants, a typ-
ical advantage shared with the use of biocatalysts over their nonenzymatic counterparts.
Whilst the fundamentals of enzymatic carboxylation have been studied extensively, only
recently have applications of carboxylase enzymes in biocatalysis and synthesis begun to
be explored,[6] with advances in protein engineering opening up a route toward tailoring
existing biocatalysts for specific reactions and substrates.[4] Examples to be described in
this chapter will serve to highlight the diversity and operational advantages of enzymatic
carboxylation with phenolic, aromatic, aliphatic, and heteroaromatic substrates, and the
potential scope for use of the corresponding enzymes in industry.

2.1.4.1.1 Regioselective Biocatalytic Carboxylation of Phenols

Proceeding through the Kolbe–Schmitt reaction, carboxylation of alkali metal phenox-

ides with carbon dioxide has provided a typical route toward carboxylated phenols for
more than a century. This has enabled the production of industrially important aromatic
hydroxy acids, such as salicylic acid, 4-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic
acid (p-aminosalicylic acid), and hydroxynaphthoic acids.[7] The large amounts of byprod-
ucts formed and the subsequent separation required to achieve pure carboxylated phe-
nols has led to the pursuit of more selective routes. Moderate yields (up to 60%) have
been obtained in the selective synthesis of 4-hydroxybenzoic and 4-hydroxy-3-methylben-
zoic acids from the corresponding phenols and carbon tetrachloride by using -cyclodex-
trin as a catalyst;[8] however, harsh reaction conditions, including the use of sodium hy-
droxide, elevated temperatures for extended periods, and the requirement of organic sol-
vents and copper powder precludes extensive scale-up for industrial purposes. This has
resulted in studies toward more environmentally benign conditions and a search for an
equivalent selective biocatalytic carboxylation of phenols. For instance, a biological route
toward 4-hydroxybenzoic acid has been identified in the initial steps of anaerobic phenol
metabolism (Scheme 3).[2,9]
 2.1.4 Enzymatic Carboxylation and Decarboxylation 135

Scheme 3 Carboxylation of Phenol in Thauera aromatica[2]

phenylphosphate phenylphosphate
synthase carboxylase

O− CO2 Pi
OH ATP AMP + Pi O
P O−
O

CO2H
further
metabolism
acetyl-CoA + CO2

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OH

Catabolism of phenol to acetyl-CoA in anaerobic bacteria involves the initial ATP depen-
dent carboxylation of phenol to give 4-hydroxybenzoate, in a process involving two en-
zymes that proceeds via a phenyl phosphate intermediate.[9,10] Firstly, phenylphosphate
synthase catalyzes the phosphorylation of phenol to give phenyl phosphate, which under-
goes subsequent carboxylation catalyzed by phenylphosphate carboxylase, resulting in
the formation of 4-hydroxybenzoate with the release of phosphate (Scheme 3).[9,11–13]
Later studies led to the isolation and characterization of a 4-hydroxybenzoate decarboxyl-
ase from Enterobacter cloacae P240. This enzyme, whilst its physiological function is to
carry out the decarboxylation of 4-hydroxybenzoate, is also capable of catalyzing the re-
verse carboxylation of phenol in the presence of 3 M potassium hydrogen carbonate to
generate 4-hydroxybenzoate with yields of up to 19% (Table 1, entry 1).[14] As well as their
role in the degradation of aromatic acids, microbial aromatic acid decarboxylases are also
able to provide carbon dioxide under carbon dioxide limiting conditions.[15]
Carboxylation reactions identified in biosynthetic pathways highlight a wealth of
possible enzymes to be used in biocatalytic processes for the carboxylation of other phe-
nol/aromatic derivatives. As with the example from Enterobacter cloacae mentioned above,
decarboxylase enzymes catalyzing the reverse carboxylation reaction provide an addi-
tional source of potential biocatalysts, and in the case of phenolic carboxylation are the
most commonly described. These may find application for replacement of chemical steps
in the synthesis of higher-value compounds, with the choice of carboxylation position de-
termining which particular regioselective enzyme should be used. A different regioselec-
tivity in phenol carboxylation is observed when using 2,3-dihydroxybenzoate decarboxyl-
ase from Aspergillus oryzae, which gives 2-hydroxybenzoate (salicylate) in 43% yield from
phenol in whole-cell reactions using a strain of E. coli that overproduces the A. oryzae de-
carboxylase (Table 1, entry 3).[6] Salicylic acid decarboxylase from Trichosporon moniliiforme
can also be used in the biocatalytic carboxylation of phenol to salicylic acid. In this case, E.
coli cells transformed with a plasmid containing the salicylic acid decarboxylase gene
from T. moniliiforme give salicylic acid in 27% yield in whole-cell reactions with phenol
and potassium hydrogen carbonate incubated at 30 8C for 9 hours (Table 1, entry 2).[4]
The carboxylase activity of the purified enzyme is dependent on potassium hydrogen car-
bonate concentration, with levels of 2.5 M and above resulting in highest product yields.
Optimal phenol substrate concentration and assay temperature for the purified enzyme
were determined to be 30 mM and 30 8C, respectively. Salicylic acid decarboxylase from T.
moniliiforme is also capable of carboxylating other phenols at the ortho-position under sim-
ilar reaction conditions. Carboxylation of 1-naphthol results in production of 1-hydroxy-2-
naphthoic acid with a yield of 62% (Table 1, entry 9),[6] and, interestingly, selective ortho-
carboxylation of 3-aminophenol yields the antituberculostatic agent 4-amino-2-hydroxy-
benzoic acid (p-aminosalicylic acid) with 80% conversion (Table 1, entry 10).[6] Salicylic

for references see p 155

136 Biocatalysis 2.1 C—C Bond Formation

acid decarboxylase has also been subjected to site-directed mutagenesis to obtain a higher
yielding p-aminosalicylic acid system.[16] Improved kinetic parameters for the carboxyla-
tion of 3-aminophenol are shown using the Tyr64Thr/Phe195Tyr (Y64T/F195Y) mutant,
which displays a 12-fold higher specific activity than the wild-type enzyme.

Table 1 Regioselective Biocatalytic Carboxylation of Phenolic Compounds[4–6,14,17,18]

HO2C
OH OH

R1 R1

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2

Entry Starting Organism/Enzyme Product Yield Ref

Material (%)

CO2H

[14]
1 Enterobacter cloacae 19

OH
OH

Trichosporon moniliiforme/salicylic [4]

2 27
acid decarboxylase CO2H
OH OH

Aspergillus oryzae/2,3-dihydroxy- [6]

3 43
benzoate decarboxylase CO2H
OH OH

CO2H

[5]
4 Pandoraea sp. 25
OH OH
OH OH

CO2H
Rhizobium sp./2,6-dihydroxyben- [6]
5 35
OH zoate decarboxylase OH
OH OH

OH OH

[17]
6 Agrobacterium tumefaciens 30
CO2H
OH OH

OH OH

[5]
7 Pandoraea sp. 48
CO2H
OH OH
 2.1.4 Enzymatic Carboxylation and Decarboxylation 137

Table 1 (cont.)
Entry Starting Organism/Enzyme Product Yield Ref
Material (%)

OH OH
Rhizobium radiobacter/resorcylic [18]
8 44
acid decarboxylase CO2H
OH OH

Trichosporon moniliiforme/salicylic [6]

9 62
acid decarboxylase CO2H

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OH OH

H2N H 2N
Trichosporon moniliiforme /salicylic [6]
10 80
acid decarboxylase CO2H
OH OH

Analogous to the carboxylation of phenol, the enzymatic carboxylation of catechol has

been uncovered in metabolic pathways of the sulfate-reducing bacterium Desulfobacterium
sp.,[19] and in the anaerobic denitrifying bacterium Thauera aromatica.[20] An initial phos-
phorylation of catechol, catalyzed by a phenylphosphate synthase, is followed by carbox-
ylation at the para-position to the phosphomonoester by a phenylphosphate carboxylase,
resulting in formation of 3,4-dihydroxybenzoic acid. Carboxylation of catechol by whole
cells of Pandoraea sp. 12B-2 has been developed as a preparative-scale biotransformation,
affording a yield of 25% of 2,3-dihydroxybenzoate (Table 1, entry 4).[5] The broad substrate
scope of this class of enzymes is highlighted by 2,6-dihydroxybenzoate decarboxylase
from Rhizobium sp., which in addition to the carboxylation of catechol (35% yield; Table
1, entry 5) will also carboxylate 3-aminophenol, 1-naphthol, 1,3-dihydroxybenzene, and
1,3-dihydroxy-5-pentylbenzene.[6] In addition to 2,6-dihydroxybenzoate decarboxylase
from Rhizobium sp., recent advances in the ortho-carboxylation of phenolic compounds
have identified additional benzoic acid decarboxylases to possess broad substrate scope,
with carboxylation of phenolic substrates containing alkyl, alkoxy, halo, and amino func-
tionalities.[21]
More extensive studies have been carried out into the carboxylation of 1,3-dihydroxy-
benzene (resorcinol).[5,22] For example, optimized culture and reaction conditions for
whole-cell biotransformations with Pandoraea sp. 12B-2 led to production of 1.43 M
(220 g • L–1) 2,6-dihydroxybenzoate (resorcylic acid) in the presence of 3 M potassium hy-
drogen carbonate, which corresponds to a 48% molar conversion of 1,3-dihydroxybenzene
(Table 1, entry 7). Carboxylation of 1,3-dihydroxybenzene by whole cells of Pandoraea sp.
can also be performed in supercritical carbon dioxide, from which 18 mM 2,6-dihydroxy-
benzoate is formed after 1 hour, and 30 mM product formation after 24 hours, corre-
sponding to 67% productivity compared to carrying out the carboxylation in the presence
of 3 M potassium hydrogen carbonate.[5] The use of supercritical carbon dioxide as a sol-
vent in such reactions is suggested to offer many environmental and practical benefits,
which are enhanced during scale-up of reactions for industrial purposes. Supercritical car-
bon dioxide is a benign, nontoxic, nonflammable, and inexpensive reaction medium
when compared to typical solvents used in organic synthesis, and offers the potential
for combining reaction and separation processes.[23] It is becoming increasingly used in
biocatalysis, and is particularly suited for performing the enzymatic carboxylation reac-
tions described in this chapter.[24] However, it should be noted that the high pressures re-
quired to generate supercritical carbon dioxide are energy intensive, and this needs to be

for references see p 155

138 Biocatalysis 2.1 C—C Bond Formation

considered if such processes are to be developed at scale. In summary, the growing num-
ber of enzymes available for performing the carboxylation of aromatic compounds and
their relatively broad substrate scope,[21] together with increasing studies into optimiza-
tion of product yield, highlights their potential application for larger-scale industrial bio-
transformations.

Hydroxybenzoic Acids 2 (Table 1); General Procedure for Whole-Cell Catalyzed Carboxyl-
ation of Phenolic Compounds:[5,6]
The source of whole-cell biocatalyst for performing the carboxylation of aromatic com-
pounds may vary between wild-type microorganisms enriched from cultures containing
the substrate of interest,[5] to recombinant E. coli cells overexpressing the enzyme that per-

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forms the carboxylation reaction.[6] In a typical procedure, lyophilized whole cells (gener-
ally 20–30 mg) were resuspended in 100 mM phosphate buffer (pH 5.5) and allowed to re-
hydrate for 30 min. Direct addition of the substrate, typically at a final concentration of
10–20 mM, preceded the addition of KHCO3 (3 M, 300 mg) as the source of CO2. Carboxyl-
ation reactions were performed in sealed glass vials to prevent leakage of CO2 gas. The
mixture was shaken at 30 8C and 120 rpm, and, after overnight incubation, the cells
were removed by centrifugation prior to purification by HPLC.

2.1.4.1.2 Biocatalytic Carboxylation of Hydroxystyrene Derivatives

In addition to arene carboxylation, phenolic acid decarboxylases have been found to carry
out the “reverse carboxylation” of hydroxystyrene derivatives, with carboxylation occur-
ring at the -carbon atom of the vinyl side chain.[6] Decarboxylases from both Lactobacillus
plantarum (PAD_Lp) and Bacillus amyloliquefaciens (PAD_Ba) have been shown to produce
cinnamic acid derivatives from styrene precursors.[6,25] It was noted by the authors that
this biotransformation does not have any direct counterpart in nonenzymatic catalysis;
the most similar nonenzymatic process being the nickel-catalyzed reductive carboxyla-
tion of styrenes, which forms 2-arylpropanoic acids rather than cinnamates.[26] Expression
of recombinant Lactobacillus plantarum and Bacillus amyloliquefaciens in E. coli has also en-
abled whole-cell biotransformations to be performed utilizing the same experimental
procedures as outlined previously for the carboxylation of phenolic compounds (Section
2.1.4.1.1). For instance, incubation of the resulting cells with the substrate 2-methoxy-4-
vinylphenol (3, R1 = H) at 30 8C for 24 hours results in conversions of 30 and 20% using Lac-
tobacillus plantarum and Bacillus amyloliquefaciens decarboxylases, respectively (Scheme 4).
However, the substrate scope of these enzymes toward carboxylation of other phenols
and styrene derivatives has been shown to be limited when compared to other phenolic
acid decarboxylases.[6]

Scheme 4 Enzymatic Carboxylation of Hydroxystyrene Derivatives Using Phenolic

Acid Decarboxylases from Lactobacillus plantarum and Bacillus amyloliquefaciens[6]

phenolic acid decarboxylase

R1 R1 CO2H
KHCO3 buffer

HO HO
OMe OMe
3
 2.1.4 Enzymatic Carboxylation and Decarboxylation 139

R1 Enzyme Conversion (%) Ref

[6]
H PAD_Lp 30
[6]
H PAD_Ba 20
[6]
OMe PAD_Lp 3
[6]
OMe PAD_Ba 5

2.1.4.1.3 Biocatalytic Carboxylation of Heteroaromatic Compounds

Owing to the abundance of carbon dioxide as a greenhouse gas, enzymatic carbon dioxide

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fixation reactions have gained importance for their potential as alternative biocatalytic
routes toward commercially applicable compounds for which the synthesis would typi-
cally require harsh reaction conditions and/or toxic reagents. In addition to the central
carbon dioxide fixation pathways in photosynthetic organisms, examples of enzymatic
carbon dioxide fixation can be found in the dehydrogenase-catalyzed reduction of carbon
dioxide to formic acid or methanol, and the reductive carbon dioxide fixation onto 2-oxo-
pentanedioate (2-oxoglutarate) and pyruvate by isocitrate or maleate dehydro-
genases.[27–29] Besides enzyme-catalyzed fixation of carbon dioxide onto aryl substrates
(see Section 2.1.4.1.1), heteroaromatic compounds, such as pyrrole and indole, can also
be carboxylated in the reverse reaction catalyzed by decarboxylase enzymes. Heteroaro-
matic compounds are synthesized for many applications, including those within the agro-
chemical and pharmaceutical industries. For example, many pyrrolecarboxylic acid deriv-
atives have been shown to possess pharmacological activity (Scheme 5).[30,31]

Scheme 5 Pyrrole-2-carboxylate Derivatives Which Have Found Application

in the Pharmaceutical Industry[30]

Cl
N
NH2
Bu
N
O
HO
O O O CO2H

NMe N

antiviral - active against herpes simplex virus angiotensin II blocker

CO2Et
O CO2H
H2N O
S
N
N

Ph
Cl
antiviral - against AIDS, HIV-1 IL-1 inhibitor

A nonoxidative aromatic acid decarboxylase has been isolated from Bacillus megaterium
PYR2910 and shown to convert pyrrole-2-carboxylate into pyrrole and carbon dioxide.[32]
In the reverse direction, this enzyme also catalyzes the carboxylation of pyrrole in the
presence of a carbon dioxide source (carbon dioxide or hydrogen carbonate), resulting in
yields of up to 80%.[33] Using purified pyrrole-2-carboxylate decarboxylase, carboxylation
of pyrrole is performed in tightly sealed vessels containing 100 mM potassium phosphate
buffer (pH 5.5), 140 mM ammonium acetate, 400 mM pyrrole, 20 mM dithiothreitol, and
100 U/mL enzyme. Whole-cell assays also proved viable for the carboxylation of pyrrole.

for references see p 155

140 Biocatalysis 2.1 C—C Bond Formation

Reactions in both cases were started with the addition of 3 M potassium hydrogen carbon-
ate, and stopped with sodium hydroxide prior to analysis by high-performance liquid
chromatography. Soluble carbon dioxide was determined to be the carboxylation-limiting
factor. Gaseous carbon dioxide and dry ice were also tested, though the optimal carbon
dioxide source proved to be potassium hydrogen carbonate, with formation of 82 mM pyr-
role-2-carboxylate from 100 mM pyrrole at saturating concentrations (2.5 M) of potassium
hydrogen carbonate. A lower Vmax of 47 mol • mg–1 • min–1 for the carboxylation of pyrrole
compared to 989 mol • mg–1 • min–1 for the corresponding decarboxylation reaction
shows that C—C bond formation is thermodynamically more difficult than C—C bond
cleavage.[33] Maximal rates of carbon dioxide fixation by the enzyme were achieved
using 300 mM of pyrrole and 100 U/mL of enzyme, which, in a batch reaction, resulted
in formation of 230 mM (25.5 g • L–1) pyrrole-2-carboxylate. This productivity could be fur-

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ther increased to 36.1 g • L–1 through feeding 150 mM pyrrole to a reaction 3 hours after an
initial addition of 250 mM substrate. An organic acid such as butanoic acid or propanoic
acid is required for enzymatic activity, and the saturation effect of such organic acids on
the catalysis suggests formation of an enzyme–acid complex.[32] In a proposed mecha-
nism, the organic carboxylate is suggested to deprotonate the pyrrole nitrogen atom,
thus facilitating electrophilic substitution at the C2 position, with the conjugate organic
acid protonating the pyrrole nitrogen upon rearomatization (Scheme 6). The postulated
role of the organic acid as cofactor in the reaction is supported by the finding that 1-meth-
ylpyrrole is not a substrate for enzymatic carboxylation.[30,33]

Scheme 6 Proposed Reaction Mechanism for Carboxylation of Pyrrole by Pyrrole-2-car-

boxylate Decarboxylase from Bacillus megaterium[30]

O−
O
•
H O
N O N
H
O
H
N
H H O−
− O
O O O

R1 R1

Enzymatic regioselective carboxylation of pyrrole derivatives would be particularly valu-

able in the preparation of pharmaceutical and agrochemical compounds. However, pyr-
role-2-carboxylate decarboxylase from Bacillus megaterium has been shown to be highly
specific for pyrrole, and is not reported to accept more diverse pyrrole derivatives,
which limits the use of the wild-type enzyme in the preparation of higher-value com-
pounds. The discovery of novel carbon dioxide fixing enzymes with a wider substrate
scope would be highly desirable. To this end it may be possible to engineer, through di-
rected-evolution approaches, decarboxylases with activity for alternative pyrrole sub-
strates. In addition to engineering existing biocatalysts, selective enrichment can be
used to discover new enzymes from nature with desirable decarboxylase/carboxylase ac-
tivities. For example, selective enrichment of microorganisms from soil samples using a
minimal medium supplemented with 50 g • L–1 of indole-3-carboxylic acid has resulted in
the identification of a bacterium Arthrobacter nicotianae that can degrade indole-3-carbox-
ylate. This subsequently led to characterization of a novel reversible indole-3-carboxylate
decarboxylase which is produced by A. nicotianae.[34] The deployment of this decarboxylase
in the reverse synthetic direction offers a more benign route toward carboxylated indole
compounds compared with nonenzymatic synthesis, which typically requires harsh and/
or deleterious reaction conditions (Scheme 7).[35]
 2.1.4 Enzymatic Carboxylation and Decarboxylation 141

Scheme 7 Base-Mediated Carboxylation of Indole Derivatives with Carbon Diox-

ide and Biocatalytic Carboxylation of Indole and 2-Methylindole by Indole-3-car-
boxylate Decarboxylase from Arthrobacter nicotianae[2,34,35]

R2 R2
R3 R3
t-BuOLi, CO2 (1 atm) CO2H
DMF, 100 oC, 24 h
R4 R4
N R1 R1
N
R5 H R5 H

CO2 CO2H
indole-3-carboxylate decarboxylase

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N R1 R1
N
H H
4 5
R1 = H, Me

In a similar approach to the carboxylation of pyrrole, reactions with indole are also per-
formed in tightly sealed vessels to prevent carbon dioxide leakage.
The procedures used in the discovery of these enzymes from A. nicotianae and B. meg-
aterium could be further applied in the future and, together with suitable screening meth-
ods, may allow the repertoire of regioselective enzymatic carboxylations to be expanded
for these heteroaromatic compounds.

1H-Indole-3-carboxylic Acid (5, R1 = H); Typical Procedure:[34]

A typical biotransformation contained indole (4, R1 = H; 40 mol), KHCO3 (6 mmol), and
dithiothreitol (20 mol) in potassium phosphate buffer (pH 6) together with whole-cell
mold mycelia expressing indole-3-carboxylate decarboxylase or the purified enzyme it-
self. The solubility of indole was increased by adding 8% (v/v) MeOH. The reaction was in-
cubated at 20 8C and stopped by the addition of NaOH prior to analysis using HPLC.

2.1.4.1.4 Biocatalytic Carboxylation of Epoxides

Nonenzymatic carboxylation of epoxides is utilized on an industrial scale for the synthe-

sis of cyclic carbonates, which find many applications including as solvents, fuel addi-
tives, herbicides, disinfectants, and in the preparation of cosmetic products.[36] The con-
version of epoxides into cyclic carbonate compounds with carbon dioxide typically re-
quires organic solvents and catalysts such as dialkyltin methoxides, alkali metal salts, or-
ganoantimony halides, or magnesium/aluminum mixed metal oxides. In addition to envi-
ronmental concerns regarding their disposal, the use of these metal catalysts leads to nu-
merous other problems such as poor solubility and difficult catalyst recycling.[37] The use
of supercritical carbon dioxide has enabled these reactions to be performed in dimethyl-
formamide without any catalyst, thus circumventing many of these issues.[37] Unlike the
chemically catalyzed routes, enzymatic carboxylation of epoxides has been shown to re-
sult in the formation of the corresponding -oxo acids,[2] and was first identified in the
metabolism of aliphatic epoxides, such as 2-methyloxirane (epoxypropane), which de-
rived from the corresponding alkenes as the main carbon source in Xanthobacter strain
Py2.[38] During epoxide degradation, coenzyme M (2-sulfanylethanesulfonate, 2-mercapto-
ethanesulfonate) attacks the highly reactive short-chain epoxide, to generate a 2-hydroxy-
alkyl sulfide catalyzed by epoxyalkane CoM transferase. Hydroxypropyl-CoM dehydro-
genase catalyzes oxidation of the alcohol to give a 2-oxoalkyl sulfide which serves as the
substrate for ketopropyl-CoM oxidoreductase/carboxylase (2-KPPC). An active-site cys-
teine residue of ketopropyl-CoM oxidoreductase/carboxylase subsequently attacks the

for references see p 155

142 Biocatalysis 2.1 C—C Bond Formation

sulfur atom of the sulfide, resulting in a covalent enzyme–CoM disulfide adduct with con-
comitant release of an enolate intermediate, which is carboxylated to form the product
-oxo acid (Scheme 8).[39,40] No practical applications of this pathway have been developed
to date, but it is suggested[2] that the -oxo acids (e.g., acetoacetate) could be further re-
duced in vivo to the corresponding -hydroxy acids before condensation to give polyhy-
droxyalkanoates (e.g., polyhydroxybutyrates, PHB), which are potentially valuable bio-
polymers.

Scheme 8 Biocatalytic Carboxylation in the Metabolism of 2-Methyloxirane by Xantho-

bacter Py2 Forming Acetoacetate[40]

O O

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S
HS O−
epoxyalkane O O hydroxypropyl-CoM
CoM transferase S dehydrogenase
O
S O−
OH
NAD+ NADH + H+

CO2
O O ketopropyl-CoM
S O− O O
S O− oxidoreductase/carboxylase
+ S
O O O HS O−

NADPH NADP+

2.1.4.1.5 Carboxylation of Aliphatic Compounds in Biosynthesis

Carboxylation reactions are endergonic (˜G > 0) and therefore thermodynamically unfav-
orable.[2] In the examples in the preceding sections, energetically favorable enzymatic de-
carboxylation reactions are driven in the reverse carboxylation direction through the use
of high concentrations of hydrogen carbonate or carbon dioxide in sealed vessels, so that
carbon dioxide cannot escape. Nature, on the other hand, typically employs cofactors in
coupled reactions to drive otherwise thermodynamically unfavorable carboxylation reac-
tions. For example, acetyl-CoA and propionyl-CoA carboxylase enzymes are adenosine tri-
phosphate (ATP) and biotin-dependent, with the coupled hydrolysis of adenosine triphos-
phate providing the thermodynamic driving force for substrate carboxylation, leading to
malonyl-CoA and methylmalonyl-CoA, respectively.[41]
In cell extracts of Rhodobacter sphaeroides, an enzyme has been found to catalyze the
adenosine triphosphate independent reductive carboxylation of the aliphatic enoyl-CoA
ester, crotonoyl-CoA (6), to generate ethylmalonyl-CoA.[42] The mechanism and stereo-
chemistry of crotonoyl-CoA carboxylase catalyzed reductive carboxylation and reduction
reactions have been determined by Alber and co-workers using the crotonoyl-CoA carbox-
ylase from R. sphaeroides.[43] Reductive carboxylation of Æ,-unsaturated acyl-CoA sub-
strates is initiated by transfer of the hydride from nicotinamide adenine dinucleotide
phosphate (NADPH) onto the -carbon to give a thioester enolate, and is followed by elec-
trophilic attack of carbon dioxide at the Æ-carbon position (Scheme 9). In both reactions
catalyzed by crotonoyl-CoA carboxylase, the pro-(4R) hydrogen of NADPH is transferred to
the Re face of the C3 position of crotonoyl-CoA.[44,45] In the case of crotonoyl-CoA carbox-
ylase, the reductive half reaction, in which NADPH is transformed to NADP+, is exergonic
and drives the unfavorable endergonic carboxylation half reaction.[44]
 2.1.4 Enzymatic Carboxylation and Decarboxylation 143

Scheme 9 Stereochemistry and Regioselectivity of Crotonoyl-CoA Car-

boxylase/Reductase during Reductive Carboxylation of (E)-Crotonoyl-CoA
to (2S)-Ethylmalonyl-CoA[44,45]

H O *H H O
3 CO2
CoA 2 CoA
1 S 3 1 S
2
H CO2−
H
(4R)-NADPH* NADP+
6

Crotonoyl-CoA carboxylase/reductase enzymes are widespread amongst actinomycetes

and proteobacteria, with analysis of genomes revealing increasing numbers of crotono-

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yl-CoA carboxylase genes clustered together with genes involved in polyketide and non-
ribosomal-peptide biosynthesis. The involvement of crotonoyl-CoA carboxylase enzymes
in polyketide biosynthetic gene clusters suggests that crotonoyl-CoA carboxylase homo-
logues may accept C2-malonyl-CoA substrate derivatives for polyketide assembly.[45] The
first crotonoyl-CoA carboxylase/reductase homologue was identified in the biosynthesis
of chloroethylmalonyl-CoA.[46] The chloroethylmalonyl-CoA pathway is unique to the ob-
ligate marine bacteria Salinispora tropica, in which precursors to this pathway were found
and linked to the hybrid polyketide synthase–nonribosomal-peptide synthetase cluster
generating functionalized ª-lactam--lactone bicyclic salinosporamides. The S. tropica bio-
synthetic gene cluster which produces this family of salinosporamide proteasome inhib-
itors contains the crotonoyl-CoA carboxylase homologue salG, for which the encoded pro-
tein catalyzes the reductive carboxylation of 4-chlorocrotonoyl-CoA (7) (Scheme 10).[45]
The salG gene was amplified from S. tropica genomic DNA and cloned into the
pHIS8 vector prior to transforming into E. coli BL2l(DE3). Overexpression and purification
of the salG chloroethylmalonyl-CoA carboxylase/reductase was performed, and the en-
zyme was incubated with various concentrations of crotonoyl-CoA and 4-chloro-
crotonoyl-CoA in 100 mM tris(hydroxymethyl)aminomethane buffer (pH 7.9) under satu-
rating levels of NADPH. Assays performed in the presence and absence of sodium hydro-
gen carbonate (80 mM) were used to determine the kinetic parameters for the reduction
and the reductive carboxylation reactions.[46] The apparent kinetic constants support a
preference for the chlorinated substrate, whereas the enzyme also accepts crotonoyl-
CoA with a sevenfold decrease in catalytic efficiency ( kcat/KM). Moreover, the turnover
( kcat) for the reductive carboxylation of chlorocrotonoyl-CoA is approximately 10-fold fast-
er than for the reductive reaction in the absence of hydrogen carbonate, which is consis-
tent with the kinetic properties of the known crotonoyl-CoA carboxylase from Rhodobact-
er sphaeroides.[46] Deletion of the salG gene results in no production of the chlorinated an-
alogue salinosporamide A, in which the C2 chloroethyl side chain was shown to be de-
rived from S-adenosylmethionine (SAM).[46,47]

for references see p 155

144 Biocatalysis 2.1 C—C Bond Formation

Scheme 10 Involvement of salG Crotonoyl-CoA Carboxylase/Reductase Homologue in the

Proposed Biosynthetic Pathway of the Polyketide Synthase Extender Unit Chloroethyl-
malonyl-CoA, Which Undergoes Further Transformations in the Production of Salinospor-
amide A[45,46]

N N
Cl−
H3N O N NH2 O N NH2
salL
S Cl
O
Me N N N N
O− HO OH HO OH
(S)-adenosyl-L-methionine 5-chloro-5'-deoxyadenosine

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O O O
CO2
CoA salG CoA
S S O−
NADPH

Cl Cl
7 4-chloroethylmalonyl-CoA

polyketide synthase
O
nonribosomal peptide synthetase NH H
OH
O
O
Cl
salinosporamide A

2.1.4.2 Biocatalytic Decarboxylation

Biocatalytic decarboxylation reactions are central to many metabolic pathways including,

but not limited to, key steps in the citric acid cycle,[48] catabolism of glucose,[49] amino acid
metabolism,[50] and fatty acid metabolism.[51] Unlike the carboxylation reactions described
in Section 2.1.4.1, decarboxylation is an energetically favorable process, which makes the
development of synthetically useful biotransformations more feasible. In the following
sections we describe several of the more promising decarboxylase enzymes that have
been developed for biocatalysis. A common theme in all of the following biotransforma-
tions is the decarboxylation of simple, achiral carboxylic acid substrates to give a carban-
ion intermediate that undergoes subsequent stereoselective C—C or C—H bond formation
to generate a more valuable homochiral building block.

2.1.4.2.1 Thiamine Diphosphate Dependent Decarboxylases

Thiamine diphosphate (ThDP; 8, also known as thiamine pyrophosphate; TPP) is a cofac-

tor in enzyme-catalyzed reactions that typically involve the formation and cleavage of
C—C bonds adjacent to carbonyl groups.[52,53] Thiamine diphosphate dependent enzymes
catalyze many important steps in metabolic pathways, including decarboxylation of
Æ-oxo acids. For example, pyruvate decarboxylase catalyzes the decarboxylation of pyru-
vate (10, R1 = Me) to give acetaldehyde (14, R1 = Me) (Scheme 11). Here, the catalytically ac-
tive thiamine diphosphate ylide (9), acts as a nucleophile attacking the ketone group of
pyruvate[54,55] to give the C2 Æ-lactyl–thiamine diphosphate intermediate 11. The lactyl–
thiamine diphosphate intermediate undergoes decarboxylation resulting in a C2 en-
amine/Æ-carbanion 12A/12B, which is protonated to give C2 Æ-hydroxyethylthiamine di-
phosphate 13, which breaks down to give acetaldehyde with regeneration of the thiamine
 2.1.4 Enzymatic Carboxylation and Decarboxylation 145

diphosphate ylide 9. The thiamine diphosphate dependent enzyme benzoylformate de-

carboxylase catalyzes the decarboxylation of benzoylformate (10, R1 = Ph) to give benzal-
dehyde by the same mechanism.[56]

Scheme 11 Thiamine Diphosphate and Its Catalytically Active Ylide Form Afford-
ing the Decarboxylation of Pyruvate to Acetaldehyde Catalyzed by Pyruvate De-
carboxylase, or Benzoylformate to Benzaldehyde Catalyzed by Benzoylformate
Decarboxylase

NH2
−O O
−O
N N
O P

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HO P O S N
O
8

O −O
R2
N Ar1
H R2 R1 O
S 10
14
9

N Ar1 Ar1
R1 N O
R1
S O
H S
O−
R2 R2 OH
13 11

H+ CO2

N Ar1 N Ar1
R1 R1
S OH S OH

R2 R2
12A 12B

NH2

O O
N O O OH
Ar1 = ; R1 = P P ; R2 = Me, Ph
O O

The potential for thiamine diphosphate dependent decarboxylases to catalyze ligations

has been known for many decades. First reported in 1921,[57] brewers yeast has been
used in the industrial manufacture of (R)-1-hydroxy-1-phenylpropan-2-one [15; (R)-phenyl-
acetylcarbinol], a precursor to (–)-ephedrine (16), since at least 1930 (Scheme 12).[58] Pro-
duction of 15 is achieved through the use of yeast containing pyruvate decarboxylase to
effect the condensation of pyruvate with benzaldehyde. In this transformation, the C2
Æ-carbanion of type 12 formed initially upon decarboxylation of C2 Æ-lactyl–thiamine di-
phosphate intermediate of type 11 attacks the Re face of the benzaldehyde carbonyl, re-
sulting in the R-configured alcohol 15.[55]

for references see p 155

146 Biocatalysis 2.1 C—C Bond Formation

Scheme 12 An Industrial Method for the Production of (–)-Ephedrine Utilizing Pyruvate

Decarboxylase[58]

−
O
O
O pyruvate OH MeNH2 OH
decarboxylase Pt, H2
Ph H Ph Ph
−CO2
O NHMe
15 16

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Traditionally, yeast has been used as a whole-cell biocatalyst for the formation of acyloins,
such as 15, from aldehydes. The high levels of pyruvate decarboxylase in the yeasts used
to catalyze the transformations had been noted[59] and studies by Crout and co-workers[60]
provided direct evidence to show that purified yeast pyruvate decarboxylase could pro-
duce acyloins from a range of aliphatic and aromatic aldehydes in the presence of pyru-
vate. Further studies[61] ligating pyruvate and a small range of halogenated benzaldehyde
derivatives using purified yeast pyruvate decarboxylase showed that the enzyme results
in the production of acyloins in higher optical purity and chemical yields than with whole
cells. The high enantioselectivity (up to 99% ee) displayed when purified pyruvate decar-
boxylase is used to perform these reactions on aromatic aldehydes was postulated to be
the result of the presence of a “vestigial” quinone binding site, possibly originating from
an ancestral pyruvate oxidase enzyme, which shares homology with pyruvate decarbox-
ylase.[61,62]
More recent work has broadened the scope for enzymatic carboligation reactions by
showing it is possible to achieve enantioselective C—C bond ligation using thiamine di-
phosphate dependent decarboxylases without the substrate actually having to undergo
the decarboxylation step. These transformations are more reminiscent of the nonenzy-
matic benzoin condensation, with enzyme-bound thiamine diphosphate ylide serving
the same function as cyanide. For example, benzoylformate decarboxylase catalyzes the
condensation of benzaldehyde with acetaldehyde to produce (S)-2-hydroxy-1-phenylpro-
pan-1-one (17) in 99% yield and 92% ee (Scheme 13).[63]

Scheme 13 Benzoylformate Decarboxylase Catalyzed Ligation of

Aldehydes[63]

benzoylformate decarboxylase O
O O
ThDP, Mg2+
+ Ph
Ph H H 99%
OH
17 92% ee

The stereoselectivity of this ligation is shown to increase with increased steric bulk on the
benzene ring; however, this is usually accompanied by a decrease in yield. A ligation of
benzaldehyde with itself results in an enantiomeric excess greater than 99%, but the
yield is less than 1%. More reasonable levels of conversion are displayed in the ligation
of meta-substituted benzaldehydes with acetaldehyde; 3-(methoxymethoxy)benzaldehyde
reacts with acetaldehyde to give the R-configured product in 88% yield and greater than
99% enantiomeric excess. Ligations of acetaldehyde can also be performed with furyl, thi-
enyl, pyridinyl, cyclohexyl, and cyclohexenyl aldehydes, showing that the scope of this
reaction is not just limited to benzaldehydes. Substrates containing more than one alde-
hyde group are also tolerated as benzoylformate decarboxylase can catalyze the ligation
of isophthalaldehyde (18) in a stereoselective addition with acetaldehyde to form
 2.1.4 Enzymatic Carboxylation and Decarboxylation 147

bis(Æ-hydroxy ketone) 19 with an enantiomeric excess greater than 99% (Scheme 14).[64]
Although benzoylformate decarboxylase requires aqueous media, with the addition of di-
methyl sulfoxide or cyclodextrin more hydrophobic substrates can be turned over by the
enzyme, potentially broadening the usability of these enzymes for organic synthesis.[65]

Scheme 14 Production of a Bis(Æ-hydroxy ketone) Using Benzoylformate

Decarboxylase[64]

O O

H benzoylformate decarboxylase
O ThDP, buffer
+ 2 OH
94%

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H
OH
O H O

18 19 >99% ee

Further studies have broadened the scope of ligations with thiamine diphosphate depen-
dent decarboxylases, showing that by using the previously little-known branched-chain
2-keto acid decarboxylase from Lactococcus lactis sup. Cremoris (KdcA), benzaldehyde
could be ligated to a wider range of aliphatic aldehydes (Scheme 15).[66] Aliphatic alde-
hydes may also be successfully ligated to one another, though with enantiomeric excesses
lower than those for ligations with benzaldehyde. Within the same work, the closely
related thiamine diphosphate dependent benzaldehyde lyase from Pseudomonas fluores-
cens biovar (which catalyzes the reverse acyloin reaction) was shown to catalyze the
same reactions with greater selectivity.[67]

Scheme 15 Ligations Performed by Thiamine Diphosphate Dependent

Decarboxylases[63,66,67]

O O O
enzyme 2
+ ∗ R
R1 H R2 H R1
OH

R1 R2 Enzymea ee (%) Config Yieldb (%) Ref

[63]
Ph Me BfD 92 S 99
[63]
Ph Ph BfD >99 R 0.4
[63]
Me Me BfD >99 S n.d.
[63]
2-furyl Me BfD 45 S 56
[63]
2-thienyl Me BfD 83 S 50
[63]
3-pyridyl Me BfD 87 S 65
[63]
Cy Me BfD 61 S 21
[63]
cyclohex-1-enyl Me BfD 94 S 50
[66]
Et Ph KdcA >98 R n.d.
[66]
Pr Ph KdcA 96.5 R n.d.
[66]
iBu Ph KdcA 88 R n.d.
[66]
cyclopropyl Ph KdcA 98 R n.d.
[67]
Me Me BfD 34 R >90
[67]
Et Et BfD 63 R >90

for references see p 155

148 Biocatalysis 2.1 C—C Bond Formation

R1 R2 Enzymea ee (%) Config Yieldb (%) Ref

[67]
Pr Pr BfD 80 R >90
[67]
Bu Bu BfD 65 R >90
[67]
iBu iBu BfD 85 R 60
a
BfD = benzoylformate decarboxylase; KdcA = 2-keto acid decar-
boxylase from Lactococcus lactis sup. cremoris.
b
n.d. = not determined.

(S)-2-Hydroxy-1-phenylpropan-1-one (17); Typical Procedure Using Benzoylformate De-

carboxylase:[63]

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CAUTION: Acetaldehyde and chloroform are toxic and potential carcinogens.
Benzaldehyde (10 mM), acetaldehyde (500 mM), and benzoylformate decarboxylase (puri-
fied with ThDP and Mg2+ present; 6.75 U) were incubated together at rt in 50 mM potassi-
um phosphate buffer (total volume 1.5 mL) at pH 7 for 20 h with no agitation. (S)-2-Hy-
droxy-1-phenylpropan-1-one (17) was extracted with CHCl3, and the organic layer was
dried (MgSO4); yield: 99%; 92% ee.

2.1.4.2.2 Arylmalonate Decarboxylases: Decarboxylation of Malonic Acids

Decarboxylation of malonic acids is a well-known route for the production of substituted

propanoic acids.[68–70] To produce a substituted propanoic acid from a propanoic ester via
enolate chemistry requires the use of a strong base, such as lithium diisopropylamide[71]
or sodium hydride,[72] to remove the Æ-proton. Removal of an Æ-proton from a malonate,
however, is much more facile, allowing the synthetic chemist to employ a weaker base,
such as ethoxide.[73] Saponification of the esters, followed by heating in acidic solution
produces the propanoic acid (Scheme 16).[74]

Scheme 16 Malonic Acid Synthesis of Propanoic Acids[68–74]

O O R2X O O
NaOEt NaOH
EtO OEt EtO OEt
R1 R2
R1

O O O
H3O+
R1
HO OH − CO2
OH
R1 R2 R2

Decarboxylation by just heating in acid, however, can only produce racemic products. A
number of methods do exist for performing this reaction enantioselectively, where the
proton is selectively delivered to one face of the intermediate enolate in a process
known as enantioselective decarboxylative protonation (EDP),[68] but these methods invar-
iably utilize either heavy metals or bulky chiral organocatalysts, and often result in prod-
ucts with low enantiomeric excess values.
Arylmalonate decarboxylases (AMDases) are decarboxylase enzymes which catalyze
the enantioselective decarboxylative protonation of Æ-aryl- and Æ-alkenylmalonic
acids.[75–78] Unlike the traditional synthetic methods outlined above, enantiomeric excess-
es for the R-configured propanoic acids produced are usually excellent[75,76,79–81] and are
generally only lowered by spontaneous background decarboxylation of the substrate.[76]
Chemical yields are also usually excellent,[76,77,79–81] and the reactions can be performed
in aqueous conditions at 37 8C and neutral pH.[79] These conditions are much less deleteri-
 2.1.4 Enzymatic Carboxylation and Decarboxylation 149

ous to other chemical functionalities possibly present in the substrate than other enantio-
selective decarboxylative protonation methods,[68] and also possess associated environ-
mental and economic benefits.
Arylmalonate decarboxylases are members of the aspartate/glutamate racemase su-
perfamily of enzymes,[75,82] which rely on general acid/base catalysis and do not require
cofactors.[79] Unlike thiamine diphosphate dependent decarboxylases, arylmalonate de-
carboxylases rely on the substituents of the substrate, along with a network of hydrogen
bonding in the enzyme active site to stabilize the anionic intermediates generated upon
decarboxylation. Following entry to the active site, the pro-(R) carboxylate of the malo-
nate substrate is lost, releasing carbon dioxide, which is suggested to be bound within a
hydrophobic pocket, the resulting electron density is then delocalized into the carboxy

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group and aryl or alkenyl substituent of a putative enediolate intermediate. This enediol-
ate is stabilized in the active site of the enzyme using a “dioxyanion hole” motif and also
through conjugation of the charge throughout the -system of the Æ-substituent of the
substrate. A cysteine residue then protonates this species on the Si face to give the R-con-
figured propanoic acid.[76]
Arylmalonate decarboxylases accept prochiral malonic acids that have two substitu-
ents in the Æ-position, provided that one of these substituents is small and the other sub-
stituent has a -system Æ to the carboxylates to assist in delocalization and thereby stabi-
lize the anionic intermediate resulting from decarboxylation.[76] Early work on arylmalo-
nate decarboxylases, which showed that malonates with an aryl group in the -position
were not turned over by arylmalonate decarboxylase,[79] suggested that the aryl group is
more than just a steric requirement for decarboxylation. Later work showed that malo-
nates with an alkenyl group in the Æ-position are also active substrates, indicating that it
is the -system of the substrate that is needed in the Æ-position, most likely to help stabi-
lize the postulated enediolate transition state.[76]
Modelling of 2-methyl-2-phenylmalonate into the active site of arylmalonate decar-
boxylase, based on a crystal structure obtained by Okrasa and co-workers with a phos-
phate inhibitor bound in the active site, showed the phenyl group could be held by the
enzyme in a position which would be coplanar to an enediolate generated by decarboxyl-
ation, ideally oriented to offer conjugative stabilization to the negative charge generated
by decarboxylation.[75] The Æ-phenyl group emerges from the active site in a solvent-acces-
sible channel, and consequently it can be replaced with groups ranging in size from a
vinyl group[76] to a napthyl group,[77] with a range of para-[77,80,83] and meta-substituted[80,83]
aryl groups accepted. ortho-Substituted aryl groups are generally not accepted as a result
of what are thought to be conformational constraints on these substrates as they attempt
to enter the active site.[83] The pro-(S) carboxylate group is held in the “dioxyanion hole”, a
hydrogen-bonding network comprising two adjacent oxyanion holes,[75] with the pro-(R)
carboxylate group held in a hydrophobic pocket. The second Æ-position substituent is
held in a restricted space, and so needs to be a moiety that is small in size (e.g., H, F, Me,
OH, NH2) (Scheme 17).[76,77,80,81]

for references see p 155

150 Biocatalysis 2.1 C—C Bond Formation

Scheme 17 Parameters for Arylmalonate Decarboxylase Catalyzed Transformations with

Arylmalonate Decarboxylase from Bordetella bronchiseptica[76,77,80,83]

R2 R2
arylmalonate decarboxylase
CO2H
R1 −CO2
R1 CO2H
CO2H
20 21

R1 R2 KM (mM)a kcat (s–1)a kcat/KM (s–1 • mM–1)a ee (%) Yielda (%) Ref
[76]
Ph H 10.7 316 29.6 – n.r.
[76]
Ph Me 26.9 279 10.4 99 n.r.

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[76]
Ph OH 30.2 101 3.3 99 n.r.
[76]
Ph NH2 n.r n.r. n.r. 33 n.r.
[80]
Ph F n.r n.r. n.r. 95 64
[77,83]
2-naphthyl Me 19.4 4321 223 99 n.r.
[77,83]
2-thienyl Me 12.5 200 16 95 97
[84]
2-thienyl OH 4.3 166 38.5 97 >99
[76]
CH=CH2 Me 7.8 42.8 5.5 99 n.r.
[76]
CH=CH2 OH 9.1 47.6 5.2 99 n.r.
[76]
CH=CH2 NH2 n.r. n.r. n.r. 66 n.r.
[76]
CH=CMe2 Me 3.5 5.2 1.5 99 n.r.
[76]
CH=CMe2 OH 7.3 20.7 2.8 76 n.r.
[76]
(E)-CH=CHEt Me 14.4 23.9 1.7 99 n.r.
a
n.r. = not reported.

Substrates can be screened for activity with arylmalonate decarboxylases using a 96-well
plate and a bromothymol blue based spectrophotometric assay by monitoring absorbance
at 620 nm.[85] To each well is added a 0.05% solution of bromothymol blue (180 L) in
10 mM MOPS (3-morpholinopropane-1-sulfonic acid) solution at pH 7.2, along with a
2 mg • mL–1 solution of the arylmalonate decarboxylase (10 L) and the substrate solution
(10 L; typically 0.25 M, pH 7.2). The plate is then incubated at 37 8C for 6 hours. Absorb-
ance at 620 nm is monitored, with active enzyme/substrate combinations displaying a sig-
nificant increase in absorbance over time. Alternatively, this assay can be performed
using whole cells on agar plates (1.5% agar) at pH 6 (so that the color change associated
with decarboxylation is apparent to the naked eye) using bromothymol blue (0.0025%)
and an appropriate substrate (0.36%), whereby colonies expressing an arylmalonate decar-
boxylase which turns over the substrate will show a noticeable blue color.
Arylmalonate decarboxylases can also catalyze aldol-like reactions between the pos-
tulated enediolate intermediate generated after decarboxylation and an aldehyde teth-
ered in the ortho-position of the phenyl ring (Scheme 18).[86] The decarboxylated species
is close enough in proximity to react with the adjacent aldehyde to produce a cyclic alco-
hol. Stereoselectivity in this transformation, however, has not been detected.
 2.1.4 Enzymatic Carboxylation and Decarboxylation 151

Scheme 18 Aldol Reaction Performed by Arylmalonate Decarboxylase[86]

O O
arylmalonate decarboxylase
H H
− CO2
CO2H

HO2C −O
O−

H+
OH

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CO2H

(R)-2-Phenylpropanoic acid (21, R1 = Ph; R2 = Me):[76]

Purified arylmalonate decarboxylase (3 mg • mL–1; 1 mL) was added to a 10 mM soln of
2-methyl-2-phenylmalonic acid (20, R1 = Ph; R2 = Me; 19 mL) and the mixture was left at
37 8C for 3.5 h, after which the enzyme was denatured and precipitated from the soln by
the addition of 1 M HCl (1 mL). After removal of the enzyme from the mixture by centrifu-
gation, the product was purified by reversed-phase HPLC; yield: 99%; 99% ee.

2.1.4.2.3 Acetolactate Decarboxylases

Acetolactate decarboxylases are decarboxylase enzymes derived from the lyase superfam-
ily that catalyze the decarboxylation of (S)-Æ-acetolactate [(S)-2-hydroxy-2-methyl-3-oxobu-
tanoic acid, (S)-22] to (R)-acetoin [(R)-3-hydroxybutan-2-one, (R)-23] (Scheme 19).[87–89]
Whilst thiamine diphosphate dependent decarboxylases and arylmalonate decarboxyl-
ases utilize either thiamine diphosphate or the substrate, respectively, to stabilize the
negative charge associated with decarboxylation, acetolactate decarboxylases use diva-
lent metal cations for this purpose. Although a crystal structure obtained of the enzyme
from Bacillus brevis showed an active-site zinc(II) ion,[89] studies have shown similar levels
of activity with other divalent cations, notably manganese(II) and cobalt(II).[90]

Scheme 19 Decarboxylation of (S)-Æ-Acetolactate by Aceto-

lactate Decarboxylase[87–89]

OH
OH acetolactate decarboxylase
OH
− CO2
O O O
(S)-22 (R)-23

Similarly to the mechanism for arylmalonate decarboxylases, acetolactate decarboxylases

catalyze the decarboxylation of natural (S)-acetolactate via an enantioselective decarbox-
ylation–protonation sequence, with the ketone and hydroxy groups held in a syn confor-
mation by chelation to a divalent metal cation, allowing the negative charge generated
upon decarboxylation to be delocalized into a planar enediol. This enediol is then stereo-
selectively protonated on the Si face by an active-site water molecule to give (R)-acetoin.[89]
Interestingly, the unnatural (R)-acetolactate [(R)-22] is thought to undergo a tertiary ketol
rearrangement catalyzed by acetolactate decarboxylase (Scheme 20) to give the (S)-aceto-
lactate enantiomer (S)-22, which is then turned over to (R)-acetoin [(R)-23].[89,91]

for references see p 155

152 Biocatalysis 2.1 C—C Bond Formation

Scheme 20 Tertiary Ketol Rearrangement of (R)-Æ-Acetolactate to (S)-Æ-Acetolactate by

Acetolactate Decarboxylase[89,91]

OH OH
OH acetolactate decarboxylase OH
OH
−CO2
O O O O O
(R)-22 (S)-22 (R)-23

Acetolactate decarboxylases have been exploited in the brewing industry to greatly speed
up the maturation process of beers. Acetolactate naturally decomposes to diacetyl (bu-
tane-2,3-dione), a compound which negatively affects the taste of the beer. This can be re-

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moved by allowing the beer to mature for upwards of two weeks. Alternatively, the addi-
tion of acetolactate decarboxylase can, in 24 hours, remove enough acetolactate to render
the concentration of diacetyl below the threshold of taste.[92]
Decarboxylation of acetolactate-like molecules has been demonstrated to be applica-
ble to a small range of Æ-hydroxy--oxo acids 24, producing the corresponding acyloins 25
in good yields and reasonable enantiomeric excesses, with those substrates most closely
related to acetolactate generally producing products in the best yields (Scheme 21).[58] All
of the substrates demonstrated to be turned over by the enzyme were introduced as race-
mates, showing that acetolactate decarboxylase is not only capable of decarboxylating
these substrates, but also effecting the tertiary ketol rearrangement, a process that
could be termed as deracemization with subsequent decarboxylation.

Scheme 21 Decarboxylations Catalyzed by Acetolactate

Decarboxylase[58]

O O O
acetolactate decarboxylase
OH
R2 OH − CO2 R2
1
R OH R1
24 25

R1 R2 ee (%) Yield (%) Ref

[58]
Me Me >96 70
[58]
Et Et 83 90
[58]
(CH2)3 82 90
[58]
(CH2)4 79 67
[58]
(CH2)5 83 90

2-Hydroxy Ketones 25; General Procedure:[58]

Owing to the inherent instability of -oxo acids, the substrates must be prepared immedi-
ately prior to enzymatic transformation from their ester-protected analogues. This may
be performed by alkaline hydrolysis, although this always results in some level of sponta-
neous decarboxylation.[58,93] An alternative approach is to use porcine liver esterase (PLE),
which will deprotect both enantiomers of the protected substrate.[91] Once produced, the
acid 24 (0.17 mmol in 1 mL) was added to a 0.01 mM preparation of acetolactate decarbox-
ylase from Klebsiella aerogenes [buffered to pH 6.2 with 2,2¢-(piperazine-1,4-diyl)diethane-
sulfonic acid (PIPES); 3.75 mL] and the mixture was incubated at 30 8C for 1.5 h.[94] The
crude mixture from the enzymatic reaction was eluted through an anion-exchange col-
umn with H2O, saturated with NaCl, and then extracted with Et2O continuously for 18 h,
followed by drying (MgSO4) and removal of solvent.[95]
 2.1.4 Enzymatic Carboxylation and Decarboxylation 153

2.1.4.2.4 A Postulated Malonic Semi-Aldehyde Decarboxylase

Discovered by screening microorganisms for the ability to utilize tropate as their sole
source of carbon, malonic semi-aldehyde decarboxylase is suggested to be an enzyme
that may exist within the KU1314 strain of the soil bacteria Rhodococcus sp.[96] Though
the enzyme itself has not been purified, the strain of Rhodococcus has been shown to be
capable of producing optically active 2-arylalkanoic acids from 3-hydroxy acids 26, with
the products being isolated as the methyl esters (R)-27 (Scheme 22). The stereochemistry
of the substrate is unimportant: both the R- and S-configured forms of acids 26 result in
the formation of R-configured products (R)-27.

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Scheme 22 Transformations of 3-Hydroxy Acids Catalyzed by
Rhodococcus sp.[96]

1. Rhodococcus sp.
R1 CO2H R1
2. TMSCHN2
OH
Ar1 Ar1 CO2Me
26 (R)-27

Ar1 R1 Time (d) ee (%) Yield (%) Ref

[96]
Ph Me 2 68 61
[96]
Ph Et 7 85 25
[96]
4-MeOC6H4 Me 2 74 61
[96]
4-ClC6H4 Me 5 0 75
[96]
2-naphthyl Me 6 60 60

A small series of experiments were carried out where a range of possible intermediates in
this transformation were incubated with the Rhodococcus strain to elucidate the nature of
this transformation. From these, it was hypothesized that the alcohol 28 is first oxidized
to an aldehyde 29, which is then decarboxylated by the postulated decarboxylase to give
aldehyde 30. This is then followed by oxidation of the aldehyde to give the alkanoic acid
31 (Scheme 23). Incubation of racemic aldehyde 30 or racemic acid 31 with the Rhodococ-
cus strain results in the recovery of the racemic ester after treatment with diazo(trimeth-
ylsilyl)methane/methanol, implying that the decarboxylase speculated to be responsible
for the production of aldehyde 30 operates in a stereospecific manner. However, without
the isolation and purification of the enzymes responsible for these transformations, noth-
ing can be said for certain about any of the steps in this transformation. Reaction times for
the transformation are very long and yields are relatively low, though this may be a result
of low levels of expression of the necessary enzymes under the reaction conditions, or
competing metabolism. Again, overexpression and purification of the enzymes involved
in the transformation would be very informative.

for references see p 155

154 Biocatalysis 2.1 C—C Bond Formation

Scheme 23 Postulated Route of Rhodococcus Catalyzed Transformation of 3-Hydroxy

Acids[96]

malonic semi-aldehyde
CO2H CO2H
decarboxylase
OH O
Ph Ph −CO2
H
28 29

O
Ph Ph CO2H
H

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30 31

Methyl 2-Arylalkanoates 27; General Procedure:[96]

CAUTION: Diazo(trimethylsilyl)methane and its solvolysis products are very toxic by inhalation.
All operations should be performed in a well-ventilated fume hood using appropriate safety pre-
cautions and procedures. Diazo(trimethylsilyl)methane is, however, a supposedly safer replace-
ment for diazomethane.
Cells of Rhodococcus sp. KU 1314, which were grown in the presence of tropate (0.2%), were
suspended in 0.1 M glycine buffer at pH 9, to which the hydroxy acid substrate 26 was
added to give a concentration of 10 mM. The mixture was shaken at 30 8C for 2–7 d, after
which it was acidified with HCl and extracted with iPr2O. The organic layers were washed
with brine, filtered, and dried (Na2SO4), and the solvent was removed under reduced pres-
sure. The crude residue was treated with MeOH and TMSCHN2, and the ester products 27
were purified by chromatography (silica gel).
 References 155

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