Reductions of cyclic β-keto esters by individual Saccharomyces cerevisiae dehydrogenases and a chemo-enzymatic route to (1R,2S)-2-methyl-1-cyclohexanol

Article abstract of DOI:10.1016/j.tetasy.2007.08.010

Twenty purified dehydrogenases cloned from bakers' yeast (Saccharomyces cerevisiae) and expressed as fusion proteins with glutathione (S)-transferase were tested for their ability to reduce three homologous cyclic β-keto esters. The majority of dehydrogenases reduced ethyl 2-oxo-cyclopentanecarboxylate, yielding a pair of diastereomeric alcohols with consistent (1R)-stereochemistry. Ethyl 2-oxo-cyclohexanecarboxylate reductions afforded only cis-alcohol enantiomers. Ethyl 2-oxo-cycloheptanecarboxylate was accepted by two enzymes in the collection, and both yielded mainly the cis-(1R,2S)-alcohol. Escherichia coli cells overexpressing the YDL124w gene were used in a dynamic kinetic resolution of ethyl 2-oxo-cyclohexanecarboxylate to produce the key intermediate in a chemo-enzymatic synthesis of (1R,2S)-2-methyl-1-cyclohexanol, an important chiral building block.

Full text of DOI:10.1016/j.tetasy.2007.08.010

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 18 (2007) 2133–2138
Reductions of cyclic b-keto esters by individual Saccharomyces
cerevisiae dehydrogenases and a chemo-enzymatic
route to (1R,2S)-2-methyl-1-cyclohexanol
Santosh Kumar Padhi,a, Iwona A. Kaluzna,a, ,à Didier Buisson,b,*
Robert Azeradb and Jon D. Stewarta,*
aDepartment of Chemistry, University of Florida, Gainesville, FL 32611, USA
bLaboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite´ Paris Descartes,
`
45 Rue des Saints-Peres, 75006 Paris, France
Received 1 June 2007; accepted 3 August 2007
Available online 25 September 2007
Abstract—Twenty purified dehydrogenases cloned from bakers’ yeast (Saccharomyces cerevisiae) and expressed as fusion proteins with
glutathione (S)-transferase were tested for their ability to reduce three homologous cyclic b-keto esters. The majority of dehydrogenases
reduced ethyl 2-oxo-cyclopentanecarboxylate, yielding a pair of diastereomeric alcohols with consistent (1R)-stereochemistry. Ethyl 2-
oxo-cyclohexanecarboxylate reductions afforded only cis-alcohol enantiomers. Ethyl 2-oxo-cycloheptanecarboxylate was accepted by
two enzymes in the collection, and both yielded mainly the cis-(1R,2S)-alcohol. Escherichia coli cells overexpressing the YDL124w gene
were used in a dynamic kinetic resolution of ethyl 2-oxo-cyclohexanecarboxylate to produce the key intermediate in a chemo-enzymatic
synthesis of (1R,2S)-2-methyl-1-cyclohexanol, an important chiral building block.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
trans-(1S,2S)-2
cis-(1R,2S)-3
OH
OH
Asymmetric reductions of substituted b-keto esters allow
simple access to densely-functionalized chiral building
blocks. Such reductions can be carried out by hydrogena-
tion in the presence of chiral transition metal catalysts1,2
or by using biological methods.3,4 In cases where the b-keto
ester contains an a-substituent, the biocatalytic strategy
can sometimes deliver higher stereoselectivities. This is par-
ticularly true for cyclic b-keto esters, and extensive micro-
bial screening programs have uncovered several whole-cell
biocatalysts that afford several stereoisomeric alcohols
from representative substrates (Scheme 1).5–9 Surprisingly,
whole cells of bakers’ yeast (Saccharomyces cerevisiae)
reduced substrates 1a–c with only modest stereoselectivi-
ties.6,8 Our prior experience suggested that this may result
from multiple dehydrogenases with overlapping substrate
1
1
2
CO2Et
2
CO2Et
n
n
O
[H]
1
2
CO2Et
n
1a n=1
b n=2
c n=3
OH
OH
1
1
2
2
CO2Et
CO2Et
n
n
trans-(1R,2R)-2
cis-(1S,2R)-3
Scheme 1.
*
Corresponding authors. Tel.: +33 1 42 86 21 70; fax: +33 1 42 86 83 87
(D.B.); tel.: +1 352 846 0743; fax: +1 352 846 2095 (J.D.S.); e-mail
addresses: didier.buisson@univ-paris5.fr; jds2@chem.ufl.edu
Both authors contributed equally to this work.
specificities but divergent stereopreferences.3,10,11 We have
therefore evaluated twenty individual, purified S. cerevisiae
dehydrogenases as catalysts for reducing 1a–c. These
enzymes were selected after displaying relatively broad sub-
strate specificities in earlier studies.
à Present address: BioCatalytics, Inc., 129 North Hill Avenue, Suite 103,
Pasadena, CA 91106, USA.
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.08.010

2134
S. K. Padhi et al. / Tetrahedron: Asymmetry 18 (2007) 2133–2138
Our current studies had two major goals. The first was to
compare dehydrogenase substrate- and stereopreferences
for structurally-related acyclic and cyclic b-keto esters.
These comparisons provide valuable information in pre-
dicting the likely outcomes of proposed reductions and also
shed light on how substrates interact with the active sites.
Second, we hoped to provide simple routes to the majority
of the stereoisomeric alcohol building blocks in enantiome-
rically pure form. Our prior experience has shown that if a
suitable dehydrogenase can be identified, Escherichia coli
cells that overproduce this protein can be used to supply
practical quantities of chiral alcohols for chemical synthe-
sis. In particular, we hoped to develop a chemo-enzymatic
route to homochiral cis-(1R,2S)-2-methylcyclohexanol.
(R)- and (S)-alcohols whereas the carboxyethyl substitu-
ent maintained a consistent configuration. Whether this
was due to two, flipped substrate binding orientations
being accessible in all the enzyme active sites, or whether
a single substrate binding mode permitted hydride addi-
tion from both the Si- and Re-faces could not be deter-
mined. Whole cells of commercial bakers’ yeast also
provided two diastereoisomeric alcohols from 1a,
although only one was also produced by our collection
of isolated dehydrogenases cis-(1R,2S)-3a. By contrast,
the trans-alcohol product was the enantiomer of that
produced by the isolated enzymes. This implies that at
least one additional dehydrogenase accepting 1a is missing
from our current collection.
Despite its simple structure, cis-(1R,2S)-2-methylcyclohex-
anol is difficult to prepare in enantiomerically pure form.
Several routes requiring kinetic resolutions involving ester
hydrolysis12–14 or synthesis,15 and enantioselective oxida-
tion16 or reduction17–19 have been reported. Unfortunately,
the enantioselectivities of these processes are modest, which
lessens their practical utility. In addition, separating the de-
sired alcohol from the structurally similar by-product is
undesirable on larger scales. The facile enolization of b-
keto esters allows carbonyl reductions to proceed via dy-
namic kinetic resolutions that convert 100% of the input
starting material to the desired product, thereby side-step-
ping the separation issue.
Reductions of cyclohexanone 1b were more complex. All of
the medium chain dehydrogenases and two of the four
short chain dehydrogenases failed to reduce 1b to a signif-
icant extent. In all cases where 1b was accepted as a
substrate, only cis-alcohols were formed; however, the
enantiomeric compositions of the product mixtures dif-
fered. Four enzymes (encoded by the YJR096w,
YDL124w, YGL185c, and YNL274c genes) produced
mainly cis-(1S,2R)-3b, although with a maximum of 90%
ee (YDL124w). By contrast, eight other enzymes produced
the opposite enantiomer in >98% de and >98% ee. This
behavior is reminiscent of the results obtained when the
same collection of dehydrogenases was used to reduce ethyl
2-chloropropionylacetate.11 It is not clear why this acyclic
substrate’s behavior should resemble that of 1b, which
lacks a polar group at the a-position. This further
underscores the advantages of screening individual
dehydrogenases to determine reaction outcomes directly,
rather than by relying on analogous substrates.
2. Results and discussion
Three representative cyclic b-keto esters 1a–c were tested as
substrates for our collection of twenty S. cerevisiae dehy-
drogenases and the results are collected in Table 1. All pro-
teins were expressed and purified as fusions with
glutathione S-transferase.3 NADPH served as cofactor
for all the dehydrogenases, which was supplied by enzy-
matic glucose-6-phosphate oxidation. Reactions were car-
ried out at 30 ꢁC in phosphate buffer (pH 7.0) and
reaction progress was determined by periodic GC analysis.
All four alcohol stereoisomers derived from 1a and 1b were
resolved by chiral-phase GC; products from 1c required
prior derivatization by trifluoroacetic anhydride for resolu-
tion by chiral-phase GC. The identities of all peaks were as-
signed using authentic standards available from our
previous studies.6,8 Since two numbering systems have
appeared in the literature, carbons are numbered unambi-
guously in Scheme 1.
Only two reductases in our collection accepted cyclohepta-
none 1c and the same alcohol stereoisomer predominated
in both cases [cis-(1R,2S)]. In addition to this major prod-
uct, aldose reductase Ypr1 also afforded a small quantity of
its enantiomer, while the short chain dehydrogenase en-
coded by the YGL039w gene produced a small quantity
of the trans-(1S,2S)-alcohol. The observation that rela-
tively few yeast dehydrogenases accept larger substrates
parallels our results from structurally diverse acyclic b-keto
esters, suggesting that simple sterics is the major contribu-
tor to this phenomenon.20
Our initially planned synthetic route to cis-(1R,2S)-2-meth-
ylcyclohexanol featured a kinetic resolution of racemic 2-
methylcyclohexanone by a yeast dehydrogenase. It was
abandoned after a screen of our enzyme collection failed
to uncover one with the necessary stereoselectivity for this
substrate. We therefore adopted the chemo-enzymatic
strategy outlined in Scheme 2, which was adapted from
our earlier work.6,8,21 While screening reactions were car-
ried out with purified fusion proteins and a cofactor regen-
eration system, preparative reductions of 1b used whole
E. coli cells that overexpressed the YDL124w protein under
non-growing conditions.22 NADPH was supplied by glu-
cose metabolism by intrinsic E. coli enzymes. Under non-
optimized conditions, ca. 3 g/L of 1b was reduced in
24 h. After product isolation by continuous extraction23
The majority of the yeast dehydrogenases examined re-
duced ethyl 2-oxo-cyclopentanecarboxylate 1a. Despite
the diversity in dehydrogenase sequences, the same two
alcohol products were always observed (trans-(1R,2R)-2a
and cis-(1R,2S)-3a). In no case did a yeast reductase
afford a single diastereomer, although YPR1 afforded
the best result (91% de; >98% ee, >98% ee). Our previous
experience with acyclic b-keto ester reductions suggested
that the configuration of the newly-created hydroxyl ste-
reocenter would be consistent in the alcohol products
from a given dehydrogenase.3,10,11 This was not the case
for 1a. Every yeast dehydrogenase afforded mixtures of

S. K. Padhi et al. / Tetrahedron: Asymmetry 18 (2007) 2133–2138
Table 1. Yeast dehydrogenase reductions of cyclic b-keto esters
2135
O
O
O
CO2Et
CO2Et
Entry
1
Yeast ORF
YJR096w
Gene name
CO2Et
a
—
—
—
2
3
4
YDL124w
YBR149w
YCR107w
—
—
—
—
ARA1
AAD3
5
6
YNL331c
YOR120w
AAD14
—
—
—
—
—
GCY1
7
8
YHR104w
YDR368w
GRE3
YPR1
N.D.b
—
9
YGL185c
—
—
—
10
11
YNL274c
YPL275w
GOR1
FDH2
—
—
—
—
12
13
14
YPL113c
YLR070c
YAL060w
—
—
—
—
XYL2
BDH1
—
—
—
—
15
16
YAL061w
YGL157w
—
—
—
—
—
—
17
18
19
YDR541c
YGL039w
YOL151w
—
—
—
—
GRE2
—
—
20
21
YMR226c
Yeast cells
—
—
—
—
c
c
d
Product compositions of reactions proceeding to >20% completion are indicated graphically using the patterns shown in Scheme 1. Yeast enzymes are
grouped by superfamily (aldose reductase, D-hydroxy acid dehydrogenase, medium chain dehydrogenase, short-chain dehydrogenase); results from using
commercial bakers’ yeast cells are included for comparison.
a Conversion <20% after 24 h.
b Not determined.
c Ref. 6.
d Ref. 8.
and purification by silica gel chromatography, cis-(1S,2R)-
3b was obtained in 80% yield. The stereochemical purity
was identical to that observed in the screening reaction,
demonstrating that the catalytic activities of any E. coli

2136
S. K. Padhi et al. / Tetrahedron: Asymmetry 18 (2007) 2133–2138
dehydrogenases were negligible under these reaction
conditions.
a concise synthesis of cis-(1R,2S)-6b, an important chiral
building block.
O
OH
Engineered
E. coli cells
4. Experimental
4.1. General procedures
Ca(BH4) 2
1
CO2Et
CO2Et
2
EtOH-THF, 0 °C
98% yield
NADP+
NADPH
>98% de, 80% ee
80% yield
cis-(1S,2R )- 3b
1b
All reagents were obtained from commercial suppliers and
1
used as received. H and 13C NMR spectra were recorded
SO2Cl
CH3
at 300 MHz using a Varian VXR instrument and refer-
enced to residual protonated solvent. FT-IR spectra were
recorded from thin films on a Bruker Vector 22 instrument.
Mass spectra were determined with an HP 5971 instrument
interfaced to an HP 5890 GC. Optical rotations were mea-
sured at room temperature using a Perkin–Elmer 241
polarimeter. GC analysis was carried out using
0.25 mm · 30 m DB-17 and 0.25 · 25 m Chirasil-Dex CB
columns for normal and chiral-phase separations, respec-
tively. Racemic standards were prepared by treating 1a–c
with excess NaBH4 in EtOH. While conditions allowing
adequate resolution of all four diastereomeric alcohols
from 1a,b using chiral-phase GC were identified, products
from 1c required prior conversion to trifluoroacetate deriv-
atives. Standard media and techniques for growth and
maintenance of E. coli were used and LB medium con-
tained 1% Bacto-Tryptone, 0.5% Bacto-Yeast Extract,
and 1% NaCl. GST-fusion proteins were isolated as de-
scribed previously and stored at À20 ꢁC in the presence
of 50% glycerol.3 E. coli strain BL21(DE3)(pIK8) produced
the GST-YDL124w fusion protein under control of a T7
promoter, as described previously.3 Glucose-6-phosphate
dehydrogenase (Sigma type XV from bakers’ yeast) was
used for NADPH regeneration. When required, glucose
concentrations were determined using Trinder reagent in
a commercially-available kit.
CH3
OH OH
OH OSO2Ar
OH
CH3
NaBH4
CH3
Et3N, DMAP
77% yield
EtOH, rt
53% yield
4b
5b
(1R,2S)- 6b
Scheme 2.
The remainder of the synthesis paralleled our earlier route.6
Complete carboethoxy reduction was accomplished by a
three-step procedure. Calcium borohydride reduction
afforded diol 4b in nearly quantitative yield. Reaction with
tosyl chloride occurred mainly at the primary alcohol,
although some bis-tosylate was formed. By contrast, mesi-
tyl sulfonyl chloride reacted with only the primary alcohol
to produce 5b. Many methods for reducing 5b to the target
were evaluated. Using optimized conditions (excess NaBH4
in EtOH at room temperature), the reaction proceeded
cleanly; the modest yield was caused by difficulties in isolat-
ing the volatile product. As expected, the enantiomeric pur-
ity of 6b (80% ee) matched that of the bio-reduction
product, indicating that no racemization had taken place
during the subsequent synthetic operations.
3. Conclusion
4.2. Screening yeast dehydrogenase fusion proteins
A collection of dehydrogenases from bakers’ yeast previ-
ously shown to reduce acyclic b-keto esters with high stereo-
selectivities also shows activity against representative cyclic
b-keto esters. Interestingly, the product mixtures from
yeast cell-mediated reductions of 1a–c do not match the
aggregate observed from twenty individual S. cerevisiae
dehydrogenases. This mismatch undoubtedly reflects the
absence of one or more participating yeast enzymes, but
the possibility that the natural expression levels of some en-
zymes in our collection may be very low should not be
overlooked. Individually expressing and isolating yeast
reductases eliminates low native expression levels, and is
a key advantage of the library approach.
Screening reaction mixtures contained NADP+ (0.15 mg,
0.20 lmol), glucose-6-phosphate (4.3 mg, 14 lmol), glu-
cose-6-phosphate dehydrogenase (5 lg) and a yeast dehy-
drogenase fusion protein (12–60 lg) in a total volume of
1.0 mL of 100 mM KPi (pH 7.0). Reactions were incubated
at 30 ꢁC for 24 h, then extracted with Et2O for GC analysis.
4.3. cis-(1S,2R) Ethyl 2-hydroxycyclohexanecarboxylate 3b
A single colony of BL21(DE3)(pIK8) was cultured over-
night in 50 mL of LB supplemented with 25 lg/mL kana-
mycin at 37 ꢁC. This preculture was diluted into 4 L of
the same medium in a New Brunswick M19 fermenter.
The culture was grown at 37 ꢁC with vigorous stirring
(700 rpm) and an air flow of 4 L/min until the O.D.600
reached 0.59, then it was cooled to 30 ꢁC and supplemented
with isopropylthio-b-D-galactoside (final concentration of
100 lM) and kept under these conditions for an additional
6 h. Cells were harvested by centrifugation at 6000g for
10 min at 4 ꢁC, then they were resuspended in 100 mL of
buffer (per liter: Na2HPO4, 12.8 g; KH2PO4, 3 g; NaCl,
0.50 g) and added to 900 mL of the same buffer in a B.
Braun Biostat B fermenter employing a 1 L vessel. Glucose
Despite their apparent structural similarities, the outcomes
of cyclic b-keto ester reductions often diverged from expec-
tations based on acyclic analogs. In particular, reductions
of cyclopentanone 1a proceed with a surprising lack of ste-
reoselectivity with respect to the hydroxyl configuration.
Reductions of cyclohexanone 1b and cycloheptanone 1c
were more in line with predictions from previous studies.
In both cases, cis-alcohols were the predominant (usually
sole) products. These observations could be put to use in

S. K. Padhi et al. / Tetrahedron: Asymmetry 18 (2007) 2133–2138
2137
was added to a final concentration of 4 g/L. The reduction
was carried out at 30 ꢁC at pH 7.0 (maintained with 3 M
KOH) with an air flow of 0.5 L/min and stirring set at
800 rpm. Ketone 1b (final quantity 20 mmol, 3.4 g) was
added in two 10 mmol portions at 0 and 2 h. The glucose
concentration was maintained at ca. 4 g/L by periodic
manual additions of a sterile 20% stock solution. After
24 h, the product was isolated by continuous extraction
with CH2Cl2, drying with MgSO4, and concentration under
reduced pressure followed by silica gel chromatography
(1:4 EtOAc/hexanes) to yield 2.3 g of 3b as a pale yellow
oil (80% yield). Chiral-phase GC analysis showed 80% ee.
Spectral data matched those reported previously.24
[a]D = À27.5 (c 0.6, CHCl3); lit.5 for enantiomer
[a]D = +20.0 (c 0.6, CHCl3).
CH2Cl2 (3 · 20 mL). The combined organic extracts were
washed with saturated NaHCO3 and brine, then dried with
MgSO4, and concentrated under reduced pressure. Pure 5b
was obtained after micro-distillation as a colorless oil
(60 mg, 53% yield). Spectral data matched those reported
previously.27 [a]D = À16.2 (c 2.12, CHCl3); lit.12 [a]D =
À8.3 (c 2.1, CHCl3).
Acknowledgments
Financial support from Pfizer, Inc. (S.K.P.), the Ruegamer
Foundation (I.A.K.), and the National Science Foundation
(CHE-0130315) is gratefully acknowledged.
4.4. (1R,2R)-2-(Hydroxymethyl)-cyclohexanol 4b
References
Sodium borohydride (0.48 g, 13 mmol) was added to a stir-
red solution of 3b (0.56 g, 3.2 mmol) and anhydrous CaCl2
(0.72 g, 6.5 mmol) in 9.5 mL of EtOH and 5.6 mL of THF
at 0 ꢁC. The reaction mixture was slowly warmed to rt over
16 h, then it was quenched with 1 M HCl and extracted
with Et2O (3 · 20 mL). The combined organic extracts
were washed with saturated NaHCO3 and brine, dried with
MgSO4, and concentrated under reduced pressure to afford
414 mg of 4b as a thick, colorless oil that required no fur-
ther purification (98% yield). 1H NMR data matched those
reported previously.25 [a]D = À32.1 (c 0.24, H2O); lit.26
[a]D = À36.0 (c 0.42, H2O).
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To a stirred solution of 4b (50 mg, 0.38 mmol) in CH2Cl2
(1.0 mL) was added Et3N (160 lL, 1.15 mmol) followed
by 2,4,6-trimethylbenzenesulfonyl chloride (92 mg,
0.42 mmol) and DMAP (5 mg, 0.04 mmol). After stirring
for 12 h at rt, additional 2,4,6-trimethylbenzenesulfonyl
chloride (92 mg, 0.42 mmol) and CH2Cl2 (0.50 mL) were
added, and stirring was continued for an additional 14 h.
The mixture was washed with 1 M HCl and saturated
NaHCO3, then dried with MgSO4, and concentrated under
reduced pressure. The crude product was purified by silica
chromatography (1:1 EtOAc/hexanes) to yield 93 mg of 5b
as a colorless oil (77% yield). 1H NMR (300 MHz, CDCl3)
d 1.16–1.86 (m, 9H), 2.31 (s, 3H), 2.63 (s, 6H), 3.77 (dd, 1H,
J = 9.6, 6.3 Hz), 3.98–4.05 (m, 2H), 6.98 (s, 2H) ppm. 13C
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65.4, 70.9, 130.5, 131.7, 139.8, 143.2 ppm. IR m 3549,
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1.0, CHCl3).
4.6. (1R,2S)-2-Methyl-cyclohexanol 6b
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