stereochemistry; these assignments were consistent with
those made by NMR in all cases.
and chromatographically purified to yield 3.7 g of syn-
3c (98% ee) in 89% isolated yield. We have used a similar
approach for reductions of 1d and 1e with equal success.22
Taken together, our results have demonstrated that
reductase enzymes uncovered by an analysis of the yeast
genome can deliver important chiral building blocks for
organic synthesis. At least two of the four possible
R-chloro-â-hydroxy ester diastereomers could be produced
in high optical purities in most cases. The major defi-
ciency in the present collection is a lack of stereoselective
reductases with D-specificities. Biocatalysts with these
properties might be identified by including enzymes from
additional organisms in our collection of fusion proteins,
and the increasing pace of the genome-sequencing project
bodes well for expanding the utility of our chemo-
enzymatic approach.
Comparing the outcomes of reactions using whole
baker’s yeast cells with those employing isolated yeast
reductases clearly demonstrates the utility of examining
individual biocatalysts (Table 1). Not only did the purified
yeast reductases deliver higher stereoselectivities in most
cases, they also produced diastereomers not observed in
reductions employing commercial yeast cells. This may
result from low expression of some reductases under the
physiological conditions prevailing in commercial baker’s
yeast, and this highlights an important advantage of
using isolated reductases, rather than relying on whole
yeast cells. Alternative methods to increase expression
levels of desirable reductases, such as adding specific
enzyme inhibitors, are more difficult to optimize and
control (for examples, see refs 9, 20, and 21). It should
also be noted that the screening reactions could be carried
out rapidly, and a complete data set was typically
obtained for each substrate within 48 h.
The smallest substrate, 1a, was accepted by all of the
yeast reductases examined, although the stereoselectivi-
ties of these reactions were relatively poor except for
YOR120w and YGL157w, which afforded syn- and anti-
2a as the major products, respectively. In all cases,
however, only L-alcohols were formed. This behavior
parallels our earlier observations from reactions in which
ethyl acetoacetate was used as a substrate for the same
collection of yeast fusion proteins.12 The behavior of
higher homologue 1b provides an interesting contrast.
In four cases, D-alcohols were the major products. This
is significant because D-alcohols are observed much less
commonly in biocatalytic reductions and enzymes that
deliver this enantioselectivity are correspondingly im-
portant. Six enzymes examined accepted 1d as a sub-
strate: four afforded only syn-3d while the remaining two
produced mainly syn-2d. Benzyl-substituted â-keto ester
1e was reduced by three enzymes, with very high
stereoselectivities in two cases.
Experimental Section
General Procedures. Standard media and techniques for
growth and maintenance of E. coli were used, and LB medium
contained 1% Bacto-Tryptone, 0.5% Bacto-Yeast Extract, and
1% NaCl. GST-fusion proteins were isolated as described previ-
ously.12 Glucose-6-phosphate dehydrogenase (Sigma type XV
from baker’s yeast) was used for NADPH regeneration. Ketones
1a-e were prepared by treating the corresponding â-keto esters
with sulfuryl chloride.14
NMR spectra were recorded with a 5 mm indirect detection
probe at 500 MHz for 1H and 125 MHz for 13C. Chemical shifts
are reported at 25 °C in ppm relative to TMS. Optical rotations
were measured from CHCl3 solutions at room temperature. GC
analyses were carried out with a 0.32 mm × 30 m DB-17 column
for nonchiral separations and a 0.25 mm × 25 m Chirasil-Dex
CB or a 0.25 × 25 m Chirasil-L-Val column for enantiomer
separations. GC samples were prepared by vortex mixing of 200
µL of the reaction mixture with an equal volume of Et2O and
then removing the organic layer for analysis. Racemic alcohols
were prepared from ketones 1a-e by reduction with NaBH4 and
GC conditions providing resolution of all products were used for
analyzing products from enzymatic reductions. In cases where
insufficient resolution was obtained, alcohol products were
acetylated prior to GC analysis.
General Procedure for Ketone Reductions Using Puri-
fied Yeast GST-Fusion Proteins. Reaction mixtures contained
NADP+ (0.20 µmoles, 0.15 mg), glucose 6-phosphate (14 µmoles,
4.3 mg), glucose 6-phosphate dehydrogenase (5 µg), R-chloro-â-
keto ester substrate (5 mM), and purified GST-fusion protein
(10-100 µL, containing 5-50 µg) in 1.0 mL of 100 mM KPi, pH
7.0. Reactions were incubated at 30 °C and sampled for GC
analysis periodically.
To determine relative and absolute configurations of alcohols,
bioconversions affording single products were scaled up 10- or
20-fold from the procedure described above. After nearly all of
the substrate had been consumed, the reaction mixture was
extracted with Et2O (3 × (5 × reaction volume)). The combined
organic extracts were washed with brine (1 volume) and water
(1 volume), dried with MgSO4, and concentrated in vacuo. If
required, the alcohol product was purified by flash column
chromatography prior to spectral analysis.
The studies summarized in Table 1 delineated the
subset of enzymes that might be useful in producing
specific R-chloro-â-hydroxy ester diastereomers. To show
that these enzymes could in fact be used for reactions on
preparatively useful scales, 1a was reduced by whole cells
of an E. coli strain overexpressing the YOR120w protein
in a 1 L laboratory-scale fermenter. Cells were grown in
rich medium under inducing conditions and then resus-
pended in phosphate buffer supplemented with glucose.
The reduction of 1a was carried out under aerobic
conditions (dissolved oxygen maintained at 75%), and the
pH was kept constant at 5.6. The ketone substrate was
added portionwise to a final concentration of 27 mM.
After reduction, the product was recovered by extraction
Optical rotation data for isolated R-chloro-â-hydroxy
esters: syn-2a (YOR120w), [R]D ) +11, c 2.0, lit.23 [R]D ) +12.4,
c 1 (CHCl3); syn-2b (YDR368w), [R]D ) +8.0, c 0.98; syn-3d
(YDL124w), [R]D ) -3.0, c 3.5, lit.3 [R]D ) -3.0, c 1.7 (CHCl3);
syn-2e (YDR368w), [R]D ) +24, c 0.7; anti-2a (YGL157w), [R]D
) +4.0, c 1.1; anti-2b (YGL157w), [R]D ) -0.17, c 0.25; anti-2c
(YGL157w), [R]D ) -9.8, c 1.7, lit.3 for enantiomer [R]D ) +8.5,
c 1 (CHCl3); anti-2e (YGL157w), [R]D ) -2.2, c 3.0.
(16) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Ta-
chibana, K. J. Org. Chem. 1999, 64, 866-876.
(17) Williamson, R. T.; Marquez, B. L.; Barrios Sosa, A. C.; Koehn,
F. E. Magn. Reson. Chem. 2003, 41, 379-385.
(18) Seco, J. M.; Quin˜oa´, E.; Riguera, R. Tetrahedron: Asymmetry
2001, 12, 2915-2925.
(19) Seco, J. M.; Quin˜oa´, E.; Riguera, R. Chem. Rev. 2004, 104, 17-
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(20) Nakamura, K.; Inoue, K.; Oshio, K.; Oka, S.; Ohno, A. Chem.
Lett. 1987, 679-682.
(21) Nakamura, K.; Kawai, Y.; Oka, S.; Ohno, A. Bull. Chem. Soc.
Jpn. 1989, 62, 875-879.
(22) Details of these conversions will be described elsewhere.
(23) Hamdani, M.; De Jeso, B.; Deleuze, H.; Saux, A.; Maillard, B.
Tetrahedron: Asymmetry 1993, 4, 1233-1236.
344 J. Org. Chem., Vol. 70, No. 1, 2005