Trends in Preparative Scale Chemoenzymatic Transformations: Andrew Razgulin

Trends in Preparative Scale
Chemoenzymatic Transformations
Andrew Razgulin
Mecozzi Group
2/9/06
Would you like sugar with that?

COOCH3
+
NH2
COOH
HOOC
NHCbz
NHCbz
+
Thermolysin
O
from Bacillus Oproteolicus
H2N
COOMe
COOCH3
+
NH2
HOOC
CbzHN
H2N
N
H
COOCH3
Ph
O
HO
Ph
N
H
O
aspartame
COOMe
O
+
H2N
OH
O
Ph
N
H
COOMe
aspartame
Yuck!
Oyama, K; in: Chirality in Industry, John Wiley & Sons Ltd., 1992; Harad, T; Shinnanyo-shi, Y; Irino, S;
Kunisawa, Y; Oyama, K; Holland Sweetener Company, The Netherlands, EP 0768384, 1996.
Overview
General aspects of chemoenzymatic synthesis
Chemoenzymatic synthesis
History
Temperature, pH, and solvent effects
Enzyme recovery
Cofactor regeneration
Use of hydrolytic enzymes

Glycoside synthesis
Oxidation-Reductions
Industrial Case Studies

3
Early History of Enzymatic Transformations
BC Chymosin from cattle used in cheese production
1833 Active principle of malt (diastase) identified and application to

industrial art described (Payen and Persoz)
1894 Taka diastase produced commercially by surface culture (Takamine)
1897 Conversion of glucose to ethanol by cell-free yeast extract (Buchner)
1906 L-leucine from ester racemate (Warburg)
1911-1913 - Glucoside synthesis in high concentration ethanol and acetone

(Bourquelot, Bridel and Verdon)
1916 Immobilization on charcoal without loss of activity (Nelson and Griffin)
General aspects: Issues
need optimal pH
need optimal temperature
solvent requirements (water/organic)
General aspects: Immobilization
Stabilizes the enzyme in organic solvent

Eases recovery and reuse
Covalent
Epoxide-coated acrylamide beads

Glass beads treated with 3-aminopropyltriethoxysilane
Noncovalent
Ion exchange resin

Glass beads
6
General aspects: Cofactor regeneration
Cofactors too expensive for stoichiometric use
NADPH/NADP and NADH/NAD+ can be recycled in situ by the

glucose/glucose dehydrogenase system
ADP,CDP,GDP,UDP can be recycled in situ by pyruvate

kinase/phophoenol pyruvate system
Overview

History
Enzyme recovery
Hydrolytic enzymes
Enantioselectivity, (dynamic) kinetic resolution

Regioselectivity
Glycoside synthesis
Hydrolytic enzymes
Catalyze cleavage and formation of esters, amides,

epoxides in vivo
Lipases are most used
Stable in organic solvent

Accept non-natural substrates
Do not need cofactors
General enantioselectivity
Normal KR
50% yield
Et
O
protease
N
O
Et
O
Et + N
O
O
O
OH
Dynamic KR
In situ racemization
N
H
Et
96% ee, 43%
O
O
N
4 kg
1 weight eq Candida antarctica lipase B

F
methyl t-butyl ether

20mol% Et3N
N
H
Et
99% ee, 45%
F
O
N
H
OH
O
82%
Hu, S; Tat, D; Martinez, C; Yazbeck, D.R; Tao, J; Org. Lett. 2005, 7, 4329-4331; Limanto, J; Shafiee, A;
Devine, P.N; Upadhyay, V; Desmond, R.A; Foster, B.R; Gauthier Jr; Reamer, R.A;, Volante, R.P; J. Org.
Chem. 2005, 70, 2372-2375.
10
Kinetic Resolution: Overcoming the 50% mark

Resolve-and-trap!
CN
Pseudomonas cepacia
lipase on diatomite
OH
Cl
Cl
Cl
3 eq.
CN
Cl
Pseudomonas cepacia
lipase on diatomite
OH
O
Cl
Cl
C5H11
OAc
O
Cl
(S) 47%
(R) 45%
CN
PPh3, DEAD, HOAc
Cl
OAc
THF, 0 C, 24 h
lyophilized
Methylobacterium sp. FCC 031
(epoxide hydrolase)
O
buffer pH 7.8
18 h
OAc
O
Cl
(S) 80%, 94% ee
toluene, 35 C
100 mg
290 mg
CN
Cl
3 eq.
OH
OAc
toluene, 35 C, 6 h
500 mg
CN
C5H11
HO
HO
C5H11
(S) 82% yield, 82% ee
(R)
H+ cat.
dioxane/H2O/ 0 C, 30 min
Kamal, A; Chouhan, G; Tetrahedron: Asymmetry, 2005, 16, 2784-2789; Ueberbacher, B.J; Ospian, I;
Mayer, S.F; Faber, K; E. Eur. J. Org. Chem. 2005, 1266-1270.
11
Dynamic Kinetic Resolution: Classic example - hydantoins
L-specific
D-specific
O
OH
NH2
R
HN
HN
R
NH
O
L-Carbamoylase
O
R
ONH3+
O
L-Hydantoinase
NH
HN
O
D-Hydantoinase
HN
R
O
NH
OH
NH2
R
HN
pH > 8
D-Carbamoylase
O
L-amino acid
D-amino acid
ONH3+
Drauz, K; Waldman, H; (eds.) Enzyme Catalysis in Organic Synthesis. 2002, Wiley-VCH Verlag GmbH, Weinheim
12
Hydantoin feedstock
+
R-CHO
+
HCN
R'
(NH4)2CO3
COOH
NH2
+
OCN-
Strecker synthesis
D,L-hydantoin
H+
O
1. OH2. H2/cat
NH
HN
O
H+
R-CHO
+
1. Pd
2. H+
O
NH
HN
O
Amido
carbonylation
O
R-CHO +
H2N
NH2
CO
R'
COOEt
NCO
+
NH3
13
Traditional racemization strategies
Base catalyzed racemization
Acidic hydrogen at the stereocenter

O
Ph
OH
Candida antarctica lipase B

Amberlyt 15 ion exchange resin
hexanes
Ph
O
15 mg
O
O
90%, >91% ee
Dissociation/recombination
O
O
452 mg
CN
OH
Amberlite IRA-904
(OH- form)
toluene
OH
O

on Celite R-633
O
CN
toluene
OAc
CN
92%, 89% ee
Odman, P; Wessjohann, L.A; Bornscheuer, U.T; J. Org. Chem. 2005, 70, 9551-9555; Veum, L; Kanerva,
L.T; Halling, P.J; Maschmeyer, T; Hanefeld, U. Adv. Synth Catal. 2005, 347, 1015-1021.
14
New racemization strategies
Transition metal-based
Allows DKR of isolated secondary alcohols and
Ph
amines
Ph
Ph
Ph
Ph
CO Ru
CO Cl
X
M
R'
H
R'
Nu
+ M
R'
H
H
R'
X
R
H
H +
R'
R'
Nu
Pamies, O; Backvall, J.-E; Trends in Biotechnology, 2004, 22, 130-135
15
Resolution of secondary alcohols

OH
OH
R
OH
Ph
MeO
Ru cat.
OAc
OH
R
94%, >99% ee
R
OAc
Ph H
N
Ph
90%, >99% ee
Ph
CO Ru
CO Cl
OH
MeO
OAc
89%, 95% ee
Candida antartica lipase B
OH
OAc
KOtBu, Na2CO3, toluene, 25 C
86%, >99% ee
OAc
1.5 eq
OH
H OH
1 mmol
Lipase preference (R)
OAc
HO H
L
89%, 90.5% ee
Protease preference (S)
Choi, J.H; Kim, Y.H; Nam, S. H; Shin, T.S; Kim, M.-J; Park, J. Angew. Chem. Int. Ed., 2002, 41, 2373-2376
16
Resolution of secondary alcohols

OH
OOC5H9
F3C
Ph
Ph
F3C
Ph
Ph
Ph
CO Ru
CO Cl
CF3
OH
94%, 95% ee
CF3
OOC5H9
94%, 95% ee
Subtilisin Carlsberg
OH
OOC5H9
KOtBu, Na2CO3, THF, RT

OH
O
1.5 eq
OH
OOC5H9
H OH
S
1 mmol
80%, 99% ee
Lipase preference
67%, 95% ee
HO H
L
OOC5H9
96%, 95% ee
Protease preference
Boren, L; Martin-Matute, B; Xu, Y; Cordova, A; Backvall, J.-E; Chem. Eur. J. 2006, 12, 225-232.
17
Resolution of secondary amines

NH2
R
NH2
R
R
R
NH2
MeO
Ru cat.
R
CO Ru
CO
NH2
R
O
O
R H R
90%, 98% ee
R
R
Ru OC
OC
R = p-OMePh
NH2
NH2
NHAc
NHAc
95%, 99% ee
MeO
Candida antartica lipase B
NHAc
92%, 95% ee
Na2CO3, toluene, 90 C
NHAc
OAc
91%, 99% ee
F3C
0.5 mmol
F3C
Paetzold, J; Backvall, J.E; J. Am. Chem. Soc. 2005, 127, 17620-17621.
18
Resolution of aliphatic substrates

OH
OAc
N(iPr)2
Ph
Ph
O
OH
N(iPr)2
Ph
CO Ru
CO
O
O
Ph H Ph
N(iPr)2
93%, 98%
ee
N(iPr)2
65%, 98% ee
N(iPr)2
59%, 92% ee
N(iPr)2
90%, 80% ee
Ph
Ph
Ph
Ru OC
OC
OAc
O
Pseudomonas species lipase
OH
OAc
N(iPr)2
toluene, 70 C
OH
OAc
NC
N(iPr)2
3 eq
Cl
OAc
NC
OH
O
N(iPr)2
O
OAc
0.5 eq
O
OH
N(iPr)2
85%, 91% ee
0.1 mmol
Fransson, A-B.L; Boren, L; Pamies, O; Backvall, J.-E; J. Org. Chem. 2005, 70, 2582-2587.
19
Overview
History
Enzyme recovery
Hydrolytic enzymes
Glycoside synthesis
Enantioselectivity, (dynamic) kinetic resolution

Regioselectivity
Meso desymmetrization
Chemo-selectivity
Polymer synthesis
Peptide synthesis
20
Meso desymmetrization
O
BnN
NBn
H3CO2C
Pig liver esterase

0.1 M aqueous phosphate
0.1 M aqueous NaOH, pH 8
45 h, 30 C
O
BnN
CO2CH3
H3CO2C
NBn
CO2H
90 %
19.1 g
Candida rugosa lipase
hexane, MS 3
OH
HO
HO
OTBS
5 eq
OAc
OTBS
OAc
172 mg
OH
OH
94%, 97% ee
toluene, 30 C, 4h
O
O
O
O
OH
100%
Chen, F.-E; et al. Adv. Synth. Catal. 2005, 347, 549-554; Chenevert, R; et al. Tetrahedron: Asymmetry,
2003, 14, 2567-2571; Fernandez, V.G; et al. Bioorganic & Medicinal Chemistry, 2004, 12, 5443-5451.
21
Chemoselectivity
O
R
N
H2N
8 eq.
NH
O
MS 4
THF
O
NH2
R=H
R=Me
R=(E)-BrCH=CH2
NH
N
O HN
O
NH2
69 %
64%
69%
[L-RST-6]
O
O
H2N
O
100 uL
100 uL

1,4-dioxane, 30 C
N
H
90%
N
[L-RST-7]
Lavandera, I; Fernandez, S; Ferrero, M; Gotor, V; J. Org. Chem. 2004, 69, 1748-1751; Torre, O; GotorFernandez, V; Alfonso, I; Garcia-Alles, L.F; Gotor, V; Adv. Synth. Catal. 2005, 347, 1007-1014.
22
Chemoselectivity
O
2.5 eq
HO
OH
OH
O
C7H15
O
O
O
OH
350 mg
HO
C7H15
C7H15
O
90%
1 eq
O
OH

dichloromethane
4.01 g
C7H15
C7H15
6
C7H15

dichloromethane
4-dimethylaminopyridine
O
O
O
O
O
C7H15
6
90%
Halldorsson, A; Magnusson, C.D; Haraldsson, G.G; Tetrahedron, 2003, 59, 9101-9109
23
Polymerization
O
O
O
Candida antarctica lipase
9
1 mmol
CH3
*
isopropyl ether
60 C, 4h
1 mmol
* +
12 O
(R)-Enriched
(S)-Enriched
(unreacted)
76% ee, Mn=6000 Da

O
O
OH
O
8
1 mmol
Mn=600
1 mmol
OH
n
OH
70% yield

HO
Mw = 27000 Da
2 mmol
O
OH
OH OH
* O
2 mmol
O
Candida antarctica lipase
O
Bulk, 90 C
O *
n m
OH
80%, Mn = 18000 Da
Kumar, R; Chen, M.-H; Parmar, V.S; Samelson, L.A; Kumar, J; Nicolosi R; Yoganathan, S; Watterson, A.C; J.
Am. Chem. Soc., 2004, 126, 10640-10644; . Kobayashi, S; Uyama, H; ACS Symposium Series: Biocatalysis
in Polymer Science. 2003, 128-140
24
Short glycopeptide synthesis

HO
HO
OH OH
O
O
0.3 mmol Cbz Thr-Val-COOMe
OH
HO
HO
OH
O
OH
HO
HO
Ala-Tyr-COOH
1 mmol
Subtilisin BPN' 8397
48%
O
Cbz Thr-Val Ala-Tyr-COOH
water/DMF
Et3N pH=9
Boc Asp-Ala-Ser-COOMe
O
NH
+ Phe-Leu-CONH2
water/DMF
NHAc
0.1 mmol
Et3N pH=9
O
HO
0.02-0.04 mmol
OH
OH
O
HO
HO
OH
OH
Cbz-Ala-Ser-COOMe + HO
HO
0.1 mmol
OH OH
O
O
HO
Boc Asp-Ala-Ser Phe-Leu-CONH2

O
NH
NHAc
Gly-Ala-Asp-CONH2
O
NHAc
0.1 mmol
NH
53%

water/DMF
Et3N pH=9
Cbz-Ala-Ser Gly-Ala-Asp-CONH2
OH
HO
HO
O
NHAc
NH
45%
Wong, C.-H; Schuster, M; Wang, P; Sears, P; J. Am. Chem. Soc., 1993, 115, 5893-5901
25
Longer glycopeptide synthesis
Fmoc-Lys-Thr-Thr-Gln-Ala-Asn-Lys-His-Ile-Ile-Val-Ala-PAM
H-Gly-Gly-Ser-CONH2
1.3 mg
1.4 mg
Ac3GlcNAc
Subtilisin 8397 K256Y

DMF, N(EtOH)3, pH 7.8
37 C
Fmoc-Lys-Thr-Thr-Gln-Ala-Asn-Lys-His-Ile-Ile-Val-Ala-Gly-Gly-Ser
84%
Gly-Gly-Ser-CONH2
Ac3GlcNAc
Witte, K; Seitz, O; Wong, C.-H; J. Am. Chem. Soc., 1998, 120, 1979-1989.
26
Overview
History
Enzyme recovery
Hydrolytic enzymes
Glycoside synthesis
Glycosidases
Glycotransferases
27
How many steps?
OH OH
HO
NHAc
HO
AcHN
HO
AcHN
HO
OH
OH
HO
O
OH
O
COOH
OH
O
OH
OH
O
HO
O
OH
N3
O
COOH
28
Oligosaccharide Synthesis
Enzymes catalyze formation and cleavage of glycosidic linkages in

vivo
Glycosidases
Transferases
OH
HO
HO
O
OH
O
HO
OH
OH
HO O
HO
HO
OH
O
OH
O
HO
O
OH
OH
OH
29
Oligosaccharide Synthesis:
Glycosidases
Classified as hydrolases
Endo-acting hydrolyze inner linkages in a random manner releasing linear
and branched oligosaccharides
Exo-acting hydrolyze linkages from the non-reducing end of the chain
OH
OH
O
HO
OH
OH
O
HO
O
HO
O
OH HO
-amylase from Bacillus licheniformis
OH
OH
O
O
OH HO
O
O
OH
oligomers
OH
OHO
O
glucoamylase
from Aspergillus niger
OH
starch
OH
HO
HO
O
OH
OH
glucose
30
Glycosidases: Short oligosaccharides
HO
HO
OH
HO O
OH
OH
O
HO
N
5.5 mg
HO
HO
NHAc
H
N
Ala-Lys-Trp-Asn-Asn-Thr-Val-NH2
5.2 mg
endo--N-acetylglucosaminidase
buffer, pH 6.5
OH
HO
HO
OH
HO O
O
OH
O
HO
NHAc
82% yield
O
HO
O
NHAc
H
N
Ala-Lys-Trp-Asn-Asn-Thr-Val-NH2
Li, B; Zeng, Y; Hauser, S; Song, H; Wang, L.-X; J. Am. Chem. Soc. 2005, 127, 9692-9693.
31
Glycosidases: Complex structures
Li, H; Li, B; Song, H; Breydo, L; Baskakov, I.V; Wnag, L.-X.; J. Org. Chem. 2005, 70, 9990-9996.
32
Glycosidases:
Thermodynamic approach
HO
HO
HO
OH
HO O
OHHO
HO O
HO
HO
OH
OH O
O
HO
OH
HO
O
O
HO
HO
HO O
HO
HO
O
NHAc
NH O
AcNH-W-M-E-W-D-R-E-I-N-N-Y-T-S-L-I-H-S-L-I-E-E-S-Q-N-Q-Q-E-K-N-E-Q-E-L-L-CONH
2
HO O
11 %
[S-2]
O HO
7 mg
HO O
O
OH
+ OH
OO O
HO O
OH O
OHO
O
HO
O OOH
O
HO OO
HO
OHO
O HOH
HO
O
NH O
OH
O
HO
O
N AsnNHAc
NHAc
HO
HO
HO
O
O
HO
HO
NHAc
NHAc
O OOHHO HO
O
O
HO
HO
OH
OO
5 mg
HO HO
OH
HO
O
HO
OH
HO
HO O HO HO
HO
OO
Arthrobacter endo--N-acetylglucosaminidase
OH OH
OO
30% DMSO
OH
OH
HO
HO
pH 6
HO HO
HO O HO
(fromOsoy)
OH
OH
HO
HO
AcNH-W-M-E-W-D-R-E-I-N-N-Y-T-S-L-I-H-S-L-I-E-E-S-Q-N-Q-Q-E-K-N-E-Q-E-L-L-CONH2
Wang, L.-X; Song, H; Liu, S; Lu, H; Jiang, S; Ni, J; Li, H; ChemBioChem 2005, 6, 1068-1074.
33
Oligosaccharide Synthesis: Glycotransferases
Generate saccharide oligomers from activated mono-saccharides

Cofactors are required
Chen, X; Fang, J; Zhang, J; Liu, Z; Shao, J; Kowal, P; Andreana, P; Wang, P.J; J. Am. Chem. Soc. 2001,
123, 2081-2082.
34
Glycotransferases:
Stoichiometric use of cofactors
OH OH
OH
HO
O
HO
OH
NHTFA
O
OH
O
OH
13.6 mg
-(2,3)-Neu5Ac transferase
stoicheometric CMP-Neu5Ac
OH
HO
HO
AcHN
NaO2C
O
OH OH
O
HO
OH
HO
HO
AcHN
OH
OH
O
OH
-(1,4)-GalNAc transferase
UDP-GlcNAc 4-epimerase
stoicheometric UDP-GlcNAc
95%
O
HO
NaO2C
O
HO
O
HO
NHTFA
41.6 mg
OH
OH
HO
OH
88%
O
O
OH
O
OH
OH
O
HO
NHTFA
O
OH
O
OH
Jacques, S; Rich, J.R; Ling, C.-C; Bundle, D.R; Org. Biomol. Chem. 2006, 4, 142-154.
35
Glycotransferases:
Stoichiometric use of cofactors
OH OH
NaO3SO
OH
O HO
OH OH
O
O
OH
OH
O
HO
OH
NHAc
OH
20 mg
stoicheometric GDP-Fucose
FucT-III
MES pH 6.4
HO
62%
HO
O
OH OH
NaO3SO
OBn
OH
OH
O
OH
OH OH
O
O
NHAc
O
OH
OH
O
HO
O
OH
OBn
Blixt, O; Vasiliu, D; Allin, K; Jacobsen N; Warnock, D; Razi, N; Pulson, J.C; Bernatchez, S; Gilber, M;
Wakarchuk, W; Carbohydrate Research, 2005, 340, 1963-1972
36
In situ cofactor regeneration UDP-Gal Superbeads

Pyruvate
Galactose
ATP
GalK
PyK
PhosEnPyr
ADP
Gal-1-P
GalPUT
UDP-Gal
UDP-Glc
GalT
PPi
Glc-1-P
GlcPUT
Gal-OR
UTP
UDP
PyK
Pyruvate
Recombinant E. coli strains

overexpressing GalK, GalPUT
GlcPUT, PyK
R-OH
1. Fermentation
2. Lysation
3. Mix
4. Add to Ni+2 resin
PhosEnPyr
GalPUT
GlcPUT
GalT
GalK
PyK
123, 2081-2082.
37
UDP-Gal Superbeads in action
a: 1g LacOBn, 0.54g Gal, 123 mg ATP, 100 mg UDP, 73mg Glc-1-P

Others = 100 mg scale
123, 2081-2082.
38
2 steps!
OH OH
OH
HO
O
HO
OH
CMP-Neu5Ac
2.5 eq. CTP-Neu5Ac
OH
OH OH
HO
HO
AcHN
HO
HO
N3
O
OH
OH
O
HO
OH
O
OH
COOH
N3
N3
OH
OH
34% at 2.5 eq donor (2.4g)
COOH
UDP-GalNAc
1.8 eq. UDP-GlcNAc
UDP-GalNAc
4GalNac-transferase
GalNAc-epimerase
OH OH
HO
NHAc
HO
AcHN
HO
AcHN
HO
2.5 eq. CMP-Neu5Ac

(2-3/8)-sialyltransferase
CMP-Neu5Ac-synthetase
AcHN
OH
OH
HO
O
COOH
O
OH
OH
O
OH
OH
O
HO
COOH
O
OH
80-85% (0.5-0.6g)
Blixt, O; Vasiliu, D; Allin, K; Jacobsen N; Warnock, D; Razi, N; Pulson, J.C; Bernatchez, S; Gilber, M;
Wakarchuk, W; Carbohydrate Research, 2005, 340, 1963-1972
39
Overview

History
Enzyme recovery
Hydrolytic enzymes
Glycoside synthesis
40
Chemoenzymatic Synthesis: Redox
Catalyze oxidations and reductions in vivo

Cofactors
Whole-cell catalysis
Advantages
Disadvantages
41

ADH - recombinant from E. coli, purified by
denaturation of native proteins at 80 C
Thermus sp.
alcohol dehydrogenase
on Eupergit C
TN(TADH) 28000
TN(Ru) 233
14 g
Tris/phosphate/octanol
pH=7, 60 C
NADH
9% yield, 97% ee
NAD+
2.5 umol
N
CO2
OH
N
Rh
HCOOH
OH2
38 mmol
gradually to 0.01 mol%

Hollmann, F; Kleeb, A; Otto, K; Schmid, A; Tetrahedron: Asymmetry, 2005, 16, 3512-3519
42
BioCatalytics ketonereductase "kit"

Phosphate pH 6.7
OH
2.5% DMSO
CO2Et
EtO2C
O
EtO2C
R
13 mmol
R= -CH2iPr
-CH2Ph
d-gluconic acid
NADH
125 umol
NAD+
Glucose dehydrogenase
CO2Et
R
85-95% yield
Glucose
25 mmol
(a) Kambourakis, S; Rozzell, J.D; Adv. Synth. Catal. 2003, 345, 699-705. (b) Harrison, D.C; Biochem J.,
1932, 26, 1295-1299.
43

5-30g Saccharomyces cerevisiae
entrapped in calcium alginate pellets
O
R
6.6-36g glucose
citrate pH=4
OH
R
CO2Et
CO2Et
Br
Br
R=Ph
CH2Ph
CH2iPr
n-C4H9
40%, >99% ee
70%, 96% ee
74%, >99% ee
70%, >99% ee
2.2 mmol
O
O
O
104.6 mg
OH
C. floricola
IAM 13115
phosphate pH 7.5
glucose
HO
O
O
+
O
O
HO
(1:0.7)
68%, 94.4% ee
20%, 94.3% ee
Rodrigues J.A.R; Milagre, H.M.S; Milagre, C.D.F; Moran, P.J.S; Tetrahedron: Asymmetry, 2005, 16,
3099-3106. Fujieda, S; Tomita, M; Fuhshuku, K; Ohba, S; Nishiyama, S; Sugai, Adv. Synth. Catal.
2005, 347, 1099-1109.
44

E.coli [pQR239]
(cyclohexanone monooxygenase)
30 C, phosphate pH 7.5
O
O
OBn
Lewatit MP62
(anionic resin)
1.3 g
OBn
89%, 96% ee
E. coli
(aldose reductase YDR368w)
OEt
Cl
5 mmol
(gradually)
OH O
OEt
XAD-4 resin
(nonpolar resin)
phosphate pH 5.6
glucose
Cl
82%, 98% ee
Gutierrez, M.-C; Furstoss, R; Alphand, V; Adv. Synth. Catal. 2005, 347, 1051-1059. Fest, B,D; Stewart,
J.D; Tetrahedron: Asymmetry, 2005, 16, 3124-3127.
45
Overview

History
Enzyme recovery
Hydrolytic enzymes
Glycoside synthesis
46
Industrial case studies: Trusopt (Astra-Zeneca)

H+, MeOH
O
*
1. Py/TsCl
OH
COOCH3
(Commercially available biopolymer)

O
S
O O
O
HO
S
O O
S
S
3. H2O2, NaWO4
NHCH2CH3
S
S
O O
>99% de
>80% yield
S
1. 6M HCl
2. TFAA
OH
S
O O
O
2.
LiS
Neurosporia crassa
(alcohol dehydrogenase)
H3COOC
S
O O
Trusopt
LiAlH4
SO2NH2
OH
OH
S
O O
H2SO4
0C
S
O O
incomplete inversion
(a) Blacker, A.J; Holt, in: Chirality In Industry II, John Wiley&Sons, New York, 1997, 246-261. (b) Blacklock,
T.J; Sohar, P; Butcher, J.W; Lamanec, T; Grabowski, E.J.J; J. Org. Chem., 1993, 58, 1672-1679. (c) Holt,
R.A; Chimica Oggi, 1996, 9, 17-20. (d) Holt, R.A; Rigby, S.R; Zeneca Limited, 1996 US 5580764
47
Industrial case studies: Miglitol (Bayer)

CHO
OH
OH
NH2
OH
OH
reductive
amination
HO
Protection
HO
OH
CH2OH
OH
CH2OH
D-glucose
NHR
OH
OH
Gluconobacter oxidans
(D-sorbitol dehydrogenase)
NHR
OH
OH
HO
HO
O
CH2OH
OH
CH2OH
1-amino-D-sorbitol
6-amino-L-sorbose
90% yield
1. Deprotect
2. Reduce
OH
HO
HO
OH
H
N
OH
1-desoxynojirimycin
HO
HO
OH
OH
miglitol
(a) Kinast, G; Schedel, M; Bayer AG, DE 283 4122 A1, 1978. (b) Kinast, G; Schedel, M; Bayer AG,
DE 285 3573 A1, 1978. (c) Kinast, G; Schedel, M; Angew. Chem. 1981, 93, 799-800.
48
Industrial case studies:

Ro 31-9790 (Hoffmann La-Roche)
COOEt
H2N
COOtBu
COOH
COOBn
TfO
COOBn
t-BuOOC
t-BuOOC
COOtBu
COOBn
COOBn
H2N
COOH
COOBn
H2N
CONHMe
H
N
t-BuOOC
EtOOC
EtOOC
COOEt
COONa
CONHMe
O
O
HO N
H
H
N
O
CONHMe
Ro 31-9790
49
Industrial case studies:

Ro 31-9790 (Hoffmann La-Roche)
Subtilisin Carlsberg
from Bacillus sp.
+
EtOOC
EtOOC
COOEt
COOEt
EtOOC
COONa
87%
[IN-3]
NaOEt
Enzyme (9 mol%)
Up to 200 kg-scale reactions have been performed
Enzyme is highly stereoselective even at 20% substrate concentration.
(a) Wirz, B; Weisbrod, T; Estermann, H; Chimica Oggi. 1995, 14, 37-41. (b) Doswald, S; Estermann, H;
Kupfer, E; Stadler, H; Walther, W; Weisbrod, T; Wirz, B; Wostl, W; Bioorg. Med. Chem. 1994, 2, 403-410.
50
Industrial case studies: 7-ACA
(Asahi Chemical, Hoechst Marion Roussel, Toyo Jozo)

H
N
HOOC
NH2
O
COOH
Zn salt
D-aminoacid oxidase
solvent
O
TMSO
TMSCl
H
N
NHTMS
O
COOH
T<0C
H
N
HOOC
O
O
O
Cl
O
-CO2
S
H
N
HO
O
COOH
T<0C
O
COOH
PCl5
N
NHTMS
O
TMSO
O
COOH
hydrolysis
glutaryl amidase
H2N
O
S
N
HO
O
COOH
OH
O
7-aminocephalosporanic acid
51
Industrial case studies: 7-ACA
(Asahi Chemical, Hoechst Marion Roussel, Toyo Jozo)
Environmental impact reduction (Asahi)

Off-gas from 7.5 kg to 1.0 kg
Mother liquors incineration from 29 to 0.3 t
Zinc waste [Zn(NH4)PO4] from 1.8 to 0 t
Absolute costs of environmental protection reduced by 90%

per tonne 7-ACA
Asahi Chemical and Toyo Jozo = 90 t/a (since 1973)

Hoechst Marion Roussel = 200 t/a (since 1996)
(a) Christ, C; in: Ullmanns Encyclopedia of Industrial Chemistry, VCH Verlagsgesellschaft, Weinheim, 1995
(b) Verweij, J; de Vroom, E; Rec. Trav. Chem. Pays-Bas 1993, 112, 66-81. (c) Matsumoto, K; in: Industrial
Application of Immobilized Biocatalysts, Marcel Dekker, New York, 1993, 67-88. (d) Tramper, J; Biotechnol.
Bioeng. 1996, 52, 290-295.
52
Industrial case studies: 6-APA (multiple companies)
Amidase immobilized on resin

18 columns with enzyme V=30 L each
Space-time yield = 445 g/L*day
Lifetime of column = 360 cycles
Each cycle = 3 hrs
This works out to 12 tons of 6-APA
per 18 columns
H
N
O
O
Penicillin amidase
from E. coli
H2N
Penicillin-G
COOH
H2O, NH3
pH 8.0
30 C
OH
O
COOH
6-aminopenicillanic acid (6-APA)
[IN-4]
(a) Christ, C; in: Ullmanns Encyclopedia of Industrial Chemistry, VCH Verlagsgesellschaft, Weinheim, 1995
(b) Verweij, J; de Vroom, E; Rec. Trav. Chem. Pays-Bas 1993, 112, 66-81. (c) Matsumoto, K; in: Industrial
Application of Immobilized Biocatalysts, Marcel Dekker, New York, 1993, 67-88. (d) Tramper, J; Biotechnol.
Bioeng. 1996, 52, 290-295.
53
Summary
Demonstrated capability:
Resolve-and-trap sequence
Resolution of racemization-prone functionalities
(e.g. carboxyl, keto, cyano)
Resolution of secondary alcohols and amines
Desymmetrization
Chiral polymerization
Locale-specific glycopeptide ligation
Unprotected, regioselective oligosaccharide synthesis
Cofactor regeneration strategies
Whole-cell oxidations/reductions
On preparative scale!
54
Acknowledgements
Prof. Sandro Mecozzi

The Mecozzi group
Peter Anderson
Margherita de Bonis
Julee Byram
Jonathan Fast
Oana Martin
Claire Poppe
Jennifer Slaughter
Ratmir Derda
55

Trends in Preparative Scale Chemoenzymatic Transformations: Andrew Razgulin

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Trends in Preparative Scale

Would you like sugar with that?

General aspects of chemoenzymatic synthesis

Use of hydrolytic enzymes

Industrial Case Studies

Early History of Enzymatic Transformations

BC Chymosin from cattle used in cheese production

1833 Active principle of malt (diastase) identified and application to

1894 Taka diastase produced commercially by surface culture (Takamine)

1897 Conversion of glucose to ethanol by cell-free yeast extract (Buchner)

1906 L-leucine from ester racemate (Warburg)

1911-1913 - Glucoside synthesis in high concentration ethanol and acetone

1916 Immobilization on charcoal without loss of activity (Nelson and Griffin)

General aspects: Issues

need optimal temperature

solvent requirements (water/organic)

General aspects: Immobilization

Stabilizes the enzyme in organic solvent

Epoxide-coated acrylamide beads

Ion exchange resin

General aspects: Cofactor regeneration

Cofactors too expensive for stoichiometric use

NADPH/NADP and NADH/NAD+ can be recycled in situ by the

ADP,CDP,GDP,UDP can be recycled in situ by pyruvate

General aspects of chemoenzymatic synthesis

Enantioselectivity, (dynamic) kinetic resolution

Industrial Case Studies

Catalyze cleavage and formation of esters, amides,

Lipases are most used

Stable in organic solvent

96% ee, 43%

1 weight eq Candida antarctica lipase B

methyl t-butyl ether

99% ee, 45%

Kinetic Resolution: Overcoming the 50% mark

PPh3, DEAD, HOAc

(S) 80%, 94% ee

(S) 82% yield, 82% ee

Dynamic Kinetic Resolution: Classic example - hydantoins

Traditional racemization strategies

Base catalyzed racemization

Acidic hydrogen at the stereocenter

Candida antarctica lipase B

Candida antarctica lipase B

New racemization strategies

Pamies, O; Backvall, J.-E; Trends in Biotechnology, 2004, 22, 130-135

Resolution of secondary alcohols

Candida antartica lipase B

Lipase preference (R)

Protease preference (S)

Resolution of secondary alcohols

KOtBu, Na2CO3, THF, RT

Resolution of secondary amines

Candida antartica lipase B

Paetzold, J; Backvall, J.E; J. Am. Chem. Soc. 2005, 127, 17620-17621.

Resolution of aliphatic substrates

General aspects of chemoenzymatic synthesis

Enantioselectivity, (dynamic) kinetic resolution

Industrial Case Studies

Pig liver esterase

Candida antarctica lipase B

Candida antarctica lipase B