Stereoselective reduction of alkyl 3-oxobutanoate by carbonyl reductase from Candida magnoliae

Article abstract of DOI:10.1016/S0957-4166(01)00279-8

The enantioselective reduction of alkyl 3-oxobutanoates by carbonyl reductase (S1) from Candida magnoliae was investigated. S1 reduced alkyl 4-halo-3-oxobutanoates to the corresponding enantiomerically pure (S)-3-hydroxy esters. Escherichia coli HB101 transformant co-overproducing the S1 and glucose dehydrogenase from Bacillus megaterium, produced optically pure alkyl 4-substituted-3-hydroxybutanoates in a two-phase water/organic solvent system.

Full text of DOI:10.1016/S0957-4166(01)00279-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 1713–1718
Stereoselective reduction of alkyl 3-oxobutanoate by carbonyl
reductase from Candida magnoliae
Yoshihiko Yasohara,a,* Noriyuki Kizaki,a Junzo Hasegawa,a Masaru Wada,b Michihiko Kataokac
and Sakayu Shimizuc
aFine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan
bDepartment of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Fukui 910-1195, Japan
cDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku,
Kyoto 606-8502, Japan
Received 1 May 2001; accepted 11 June 2001
Abstract—The enantioselective reduction of alkyl 3-oxobutanoates by carbonyl reductase (S1) from Candida magnoliae was
investigated. S1 reduced alkyl 4-halo-3-oxobutanoates to the corresponding enantiomerically pure (S)-3-hydroxy esters.
Escherichia coli HB101 transformant co-overproducing the S1 and glucose dehydrogenase from Bacillus megaterium, produced
optically pure alkyl 4-substituted-3-hydroxybutanoates in a two-phase water/organic solvent system. © 2001 Elsevier Science Ltd.
All rights reserved.
1. Introduction
(GDH) reduces NADP+ to NADPH. Herein, we report
on the substrate specificity and enantioselectivity of S1
and on applications for the synthesis of optically active
alkyl 3-hydroxybutanoates (Scheme 1).
The importance of optically active compounds has been
increasingly recognized in the pharmaceutical field.
Four optically active carbon compounds are promising
building blocks for asymmetric organic synthesis. Alkyl
(R)-3-hydroxybutanoate and alkyl (S)-4-halo-3-hy-
droxybutanoate are important synthetic intermediates
of various inhibitors of angiotensin-converting enzyme1
and HMG-CoA reductase,2,3 respectively. Alkyl (R)-4-
2. Results and discussion
Carbonyl reductase S1 reduces a- and b-ketoesters and
conjugated diketones but does not effect aldehyde
reduction.7 Aside from 1a, however, its selectivity for
the reduction of alkyl 3-oxobutanoate derivatives is
unknown. It is important to discern information about
the enantioselectivity of the enzyme for application in
the synthesis of optically active compounds and to gain
a greater understanding of the active center of the
enzyme. We investigated the relative substrate specific-
ity and the enantioselectivity of this enzyme for alkyl
4-substituted-3-oxobutanoate by using the purified
enzyme to eliminate the influences of other enzymes.
The results are summarized in Table 1. S1 was found to
reduce alkyl 4-halo-3-oxobutanoate 1a, 1b, 1c and 1d
halo-3-hydroxybutanoates are used for
L-cartinine
synthesis.4
Many studies have investigated the enantioselective
bioreductions of alkyl 3-oxobutanoates to the corre-
sponding optically active alkyl 3-hydroxybutanoates as
the most effective preparative method. However, in
order to accumulate a high yield of product this
method requires the regeneration of reduced nicoti-
namide coenzyme, which is an expensive factor.5 We
have made an Escherichia coli transformant that co-
produces carbonyl reductase S1 from Candida magno-
liae and glucose dehydrogenase from Bacillus
megaterium.6 The carbonyl reductase S1 reduces ethyl
4-chloro-3-oxobutanoate 1a to the corresponding (S)-
enantiomeric alcohol 2a. Glucose dehydrogenase
Scheme 1. Enzymatic enantioselective reduction of ethyl 4-
chloro-3-oxobutanoate 1a.
* Corresponding author. Fax: +81-794-45-2668; e-mail: Yoshihiko.
Yasohara@kaneka.co.jp
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00279-8

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Y. Yasohara et al. / Tetrahedron: Asymmetry 12 (2001) 1713–1718
Table 1. Enantioselective reduction of alkyl 3-oxobutanoate
lective reduction of 1k is a single isomer rather than
two isomers because of the inversion of the substrate.11–13
Purified enzymes from baker’s yeast12 or Geotrichum
candidum13 and recombinant enzyme from baker’s
yeast14 reduce 1k to give the syn product with high
diastereo- and enantioselectivities. In contrast, S1 gave
the anti product with low enantioselectivity. Cells of
Penicillium15 and Geotricum16 have been shown to give
anti-2k. The stereoselectivity of microbial reduction
changes depending on the cultivation conditions or cell
treatments.17–19 This occurs because a microorganism
has multiple reducing enzymes.20 In the reduction of 1l,
baker’s yeast reduction afforded equimolar amounts of
both diastereoisomers,21 but S1 gave the anti product
with 94% diastereomeric excess (d.e.) and low enan-
tioselectivity. Also, chemical reduction by sodium boro-
hydride gave the syn product with d.e. of 96%10 S1
catalyzed a unique stereoselective reduction for 1l. To
understand the structure of the active center of S1, it is
interesting to note that the stereoselectivity of S1 is
affected by the presence of a substituent at the a-posi-
tion of the substrate.
by carbonyl reductase S1
Entry
R1
R3
Relative
% e.e.
activity (%)
a
b
c
d
e
f
g
h
i
Cl
Br
I
Cl
N3
CH2CH3
CH2CH3
CH2CH3
(CH2)7CH3
CH2CH3
100
72
16
4
\99 S
\99 S
\99 S
\99 S
Nta
0
C6H5CH2O CH2CH3
HO
H
H
21
80
7
0
0.5
21 S
CH2CH3
CH2CH3
C(CH3)3
CH2CH3
\99 S
\99 R
Nt
j
CH3CH2
Nt
a Not tested.
A reduced form coenzyme regeneration system is neces-
sary for biological reduction catalyzed by dehydroge-
nase/reductase. Whole cells, such as baker’s yeast,
which contain both reduction and coenzyme regenera-
tion systems, are often used as the biological reduction
catalyst because of easy availability and handling. In
many cases, however, their weak coenzyme regenera-
tion system causes low product yields. We have made
an E. coli transformant that coproduces S1 and GDH.6
GDH reduces NAD(P)+ to NAD(P)H linked with glu-
cose oxidation.
and the 4-hydroxy derivative 1g to the optically pure
corresponding 3-hydroxy esters. The reactivity with
octyl ester 1d was low and the 4-benzyloxy derivative 1f
was reduced with low enantioselectivity. Poor enan-
tioselectivity in the baker’s yeast reduction of 1f has
also been reported.8 The reactivities in the reduction of
3-oxobutanoates were low, but the enantioselectivity
for the ethyl ester 1h was high. For the sake of synthe-
sizing optically active compounds, information on the
enantioselectivity of carbonyl reductase is more impor-
tant than that concerning reactivity.
The use of a two-phase aqueous/organic solvent system
is advantageous in an enzymatic reaction because the
problems of degradation of unstable substrates in the
aqueous phase and enzyme deactivation by a toxic
substrate or product are minimized by extraction into
an organic solvent.6,22 The enzymes responsible for the
reduction reaction are required to have tolerance for
the organic solvent used in the reaction, and S1 and
GDH are both known to be tolerant to n-butyl
acetate.6,22
Alkyl 2-substituted-3-oxobutanoates are interesting
substrates for enantioselective reduction because the
product of each contains two stereogenic centers.9,10
The stereoselectivities of the purified S1 for ethyl 2-sub-
stituted-3-oxobutanoates are summarized in Table 2. S1
gave anti-2k and anti-2l. The enantioselectivity for 1k
was high, but that for 1l was low. Biological reduction
of alkyl 2-substituted-3-oxobutanoates has been
reported.9–18,21 The product of the enzymatic enantiose-
Table 2. Reduction of ethyl 2-substituted-3-oxobutanoate by carbonyl reductase S1
Entry
R2
Relative activity (%)a
syn/anti (%)
% e.e.
syn-(2S,3R)
anti-(2S,3S)
k
l
CH3
Cl
14
11
22/78
3/97
70
55
99
15
a The activity of ethyl 4-chloro-3-oxobutanoate was taken as 100%.

Y. Yasohara et al. / Tetrahedron: Asymmetry 12 (2001) 1713–1718
1715
Scheme 2. Synthesis of optically active alkyl 3-hydroxybutanoate derivatives by Escherichia coli transformant in a water–organic
two-phase reaction.
Alkyl 4-substituent-3-oxobutanoates are relatively
unstable compounds, so we applied the two-phase reac-
tion system in order to prepare six optically active
corresponding 3-hydroxy esters (Scheme 2). A water
phase containing the E. coli cells, glucose, and a cata-
lytic amount of NADP+ was stirred with an organic
(n-butyl acetate) phase containing the substrate. The
pH of the reaction was maintained at 6.5. After 20
hours, the yields for 2a, 2b, 2g and 2h were over 90%,
but those for 2c and 2d were less than 10% and most of
the starting substrate was recovered in these latter two
cases. The course of the reduction of 1b with time is
shown in Fig. 1. The concentration of 2b in the organic
phase increased almost stoichiometrically, linking with
the concentration of consumed glucose and the volume
of NaOH to neutralize the formed gluconic acid. The
concentration of 2b reached 0.38 M in the n-butyl
acetate layer with the consumption of glucose and
sodium hydroxide. The turnover number of NADP+
was about 650. The amount of coenzyme was not
optimized for this reaction. After the reaction, the
product and the remaining substrate were easily
extracted to an organic phase with ethyl acetate and
were purified by column chromatography. The product
2g can be used to synthesize HMG-CoA reductase
inhibitors and other products.23,24
The coenzyme regeneration system by GDH produces
gluconic acid as a by-product. The oxidation of formate
to water and carbon dioxide by formate dehydrogenase,
coupled with NAD+ reduction, is a cleaner coenzyme
regeneration system. The NADH-dependent mutant of
S1 or the NADPH-dependent mutant of formate
dehydrogenase25 will allow a clean production system in
which the synthesis of optically active alcohols can be
realized. Biological enantioselective reduction has been
a conventional methodology, but it has entered a new
phase due to the application of a practical coenzyme
regeneration system and gene technology.
3. Conclusion
The enantioselective reduction of alkyl 3-oxobutanoate
by carbonyl reductase S1 from C. magnoliae was inves-
tigated. S1 reduced alkyl 4-halo-3-oxobutanoates to the
corresponding optically pure (S)-form 3-hydroxy esters.
It also reduced alkyl 2-substituted-3-oxobutanoates, but
the stereoselectivity was not necessarily high. E. coli
HB101 transformant co-overproducing the S1 and glu-
cose dehydrogenase from B. megaterium, produced
optically pure alkyl 4-substituted-3-hydroxybutanoates
in a water/organic solvent two-phase system. This is a
highly advantageous method for synthesizing homochi-
ral 3-hydroxybutanoate derivatives.
4. Experimental
4.1. General
Figure 1. Preparation of ethyl (S)-4-bromo-3-hydroxybu-
tanoate 2b by Escherichia coli transformant in a water–
organic two-phase reaction. Time-course plots are shown of
the enantioselective reduction of 1b. Reaction conditions are
described in the text. ꢀ, Concentration of glucose; ꢁ, con-
centration of 2b; ꢂ, volume of 5 M aqueous sodium hydrox-
ide to neutralize the formed gluconic acid.
Authentic ( )-alkyl 3-hydroxybutanoates were synthe-
sized by reducing the corresponding alkyl 3-oxobu-
tanoates with sodium borohydride. The reactions were

1716
Y. Yasohara et al. / Tetrahedron: Asymmetry 12 (2001) 1713–1718
followed by TLC on Kieselgel 60F254 TLC plates
(Merck, Germany). Detection was achieved by UV
inspection (254 nm). H NMR spectra were recorded in
mL) over 2 h. The reaction mixture was stirred at 25°C
for 15 h. After the reaction, nitrogen gas was bubbled
into the mixture to remove hydrogen bromide. Water
was added to the reaction mixture, and then the organic
layer was dried over anhydrous sodium sulfate. The
solvent was removed, and the residue was purified by
column chromatography on silica gel to give 1b. Yield
1
CDCl3 with an FT NMR, JM-400 spectrometer (400
MHz; Jeol, Japan). Chemical shifts are expressed in
parts per million (ppm), with trimethylsilane as the
internal standard. The spectra of all synthesized com-
pounds used in this study agree with the data found in
the literature. Optical rotation was carried out using a
digital polarimeter (SEPA-200, Hiroba, Japan). IR
spectra were recorded on an FTIR-8100M spectrometer
(Shimadzu, Japan). All other chemicals used were of
analytical grade and commercially available.
1
47%; H NMR l (ppm) 4.25 (2H, m), 4.10 (2H, s), 3.80
(2H, s), 1.30 (3H, t); 13C NMR: 13.9, 34.0, 46.0, 61.6,
166.4, 194.5; IR (neat) 2984, 1752, 1028, 565 cm−1.
4.5.2. Ethyl 4-iodo-3-oxobutanoate 1c. 1c was prepared
by a halogen exchange reaction of ethyl 4-chloro-3-
oxobutanoate 1a.27 The mixture, comprising sodium
iodide (13.7 g, 0.09 mol), 1a (10 g, 0.061 mol) and
methylethyketone (100 mL), was stirred under reflux
for 15 h. After the reaction, the solvent was removed
and the residue was extracted with ethyl acetate. The
residue was then purified by column chromatography
4.2. Microorganisms and cultivation
The carbonyl reductase S1-overproducing strain, E. coli
HB101/pNTS1, and the S1- and GDH-co-overproduc-
ing strain, E. coli HB101/pNTS1G, were used.6 2xYT
medium composed of 1.6% Bacto-tryptone, 1.0% yeast
extract and 0.5% NaCl, pH 7.0, was used. A test tube
containing 2 mL of 2xYT medium was inoculated with
an E. coli strain, which was then incubated at 37°C for
15 h by reciprocal shaking. An aliquot of this pre-cul-
ture broth (0.5 mL) was transferred to a 500-mL shak-
ing flask containing 100 mL 2xYT medium, and
cultivation was then performed at 37°C for 13 h by
reciprocal shaking.
1
on silica gel to give 1c. Yield 85%; H NMR l (ppm)
4.23 (2H, m), 3.44 (2H, s), 2.25 (2H, s), 1.30 (3H, t); 13
C
NMR: 6.2, 14.5, 45.9, 62.2, 167.2, 195.8; IR (neat)
2982, 1743, 1028, 544 cm−1.
4.5.3. Octyl 4-chloro-3-oxobutanoate 1d. 1d was pre-
pared by chlorination of diketene following esterifica-
tion by 1-octanol.28 Chlorine gas was bubbled into a
solution of diketene (16.8 g, 0.2 mol) in
dichloromethane (160 mL) at a temperature below
15°C. 1-Octanol (28.6 g, 0.22 mol) was added to the
mixture at a temperature below 10°C. The reaction
mixture was stirred at room temperature for 1 h and
washed with brine. After the solvent was removed, the
residue was purified by column chromatography on
4.3. Purification of carbonyl reductase S1
All purification procedures were performed at 0–4°C in
a 10 mM potassium phosphate buffer (pH 7.0) contain-
ing 2 mM 2-mercaptoethanol. The washed cells, iso-
lated from the culture broth of E. coli HB101/pNTS1,
were suspended in the buffer and disrupted by sonica-
tion. The supernatant obtained by centrifugation was
fractionated with solid ammonium sulfate. The precipi-
tate obtained at 45–70% saturation was collected by
centrifugation and dissolved in the buffer. This precipi-
tate was applied to a Blue Sepharose CL-6B column
that had been previously equilibrated with the same
buffer. The enzyme was eluted with a linear gradient of
NaCl (0–0.6 M in the buffer). The active fraction was
collected and used as the purified enzyme.
1
silica gel to give 1d. Yield 35%; H NMR l (ppm) 4.28
(2H, s), 4.16 (2H, m), 3.68 (2H, s), 1.65 (2H, m), 1.35
(10H, m), 0.88 (3H, m); 13C NMR: 14.0, 22.5, 25.7,
28.3, 29.0, 31.7, 42.3, 46.1, 48.0, 65.9, 166.5, 195.4; IR
(neat) 2856, 1751, 579 cm−1.
4.5.4. Ethyl 4-azide-3-oxobutanoate 1e. 1e was prepared
from reaction of ethyl 4-chloro-3-oxobutanoate 1a with
sodium azide.29 The mixture, comprising sodium azide
(9.5 g, 0.14 mol), 1a (20 g, 0.12 mol), acetone (150 mL)
and water (50 mL), was stirred under reflux for 5 h.
The solvent was removed and the residue was extracted
with ethyl acetate. After the solvent was removed, the
residue was purified by column chromatography on
4.4. Enzyme assay and kinetic measurement
1
Carbonyl reductase S1 activity was determined spec-
trophotometrically as follows. The assay mixture, com-
posed of 100 mM potassium phosphate buffer (pH 6.5),
0.1 mM NADPH and 1 mM substrate, and the reac-
tions at 30°C were monitored for decreases in
absorbance at 340 nm. One unit of S1 was defined as
the amount catalyzing the oxidation of 1 mmol
NADPH per minute.
silica gel to give 1e. Yield 78%; H NMR l (ppm) 4.30
(2H, m), 4.18 (2H, s), 3.55 (2H, s), 1.30 (3H, t); 13C
NMR: 13.9, 46.5, 57.5, 61.7, 166.2, 197.2; IR (neat)
2986, 2104, 1751, 1080 cm−1.
4.5.5. Ethyl 4-benzyloxy-3-oxobutanoate 1f. 1f was pre-
pared from 4-chloro-3-oxobutanoate 1a and benzyl
alcohol.30 Sodium hydride (3.6 g, 0.15 mol) (which had
previously been washed three times with pentane in a
nitrogen atmosphere) was suspended in tetrahydrofuran
(80 mL). Benzylalcohol (8.1 g, 0.075 mol) and 1a (12.3
g, 0.075 mol) were added to the suspension and stirred
at 40°C for 18 h. Tetrahydrofuran was removed in
vacuo and the residue was extracted with ethyl acetate.
The solvent was removed and the residue was purified
4.5. Preparation of alkyl 4-substituted-3-oxobutanoates
4.5.1. Ethyl 4-bromo-3-oxobutanoate 1b. 1b was pre-
pared by bromination at the 4-position of ethyl 3-
oxobutanoate 1h.26 Bromine (16 g, 0.1 mol) was added
to a solution of 1h (13 g, 0.1 mol) in chloroform (90

Y. Yasohara et al. / Tetrahedron: Asymmetry 12 (2001) 1713–1718
1717
by column chromatography on silica gel to give 1f.
80/100-mesh Chromosorb WAW DMCS (3.2 mmf×1.0
m) column (GL Science, Japan) and with a flame
ionization detector. The concentration of glucose was
measured with a glucose assay kit containing glucose
oxidase (Wako, Japan). After the reaction, the mixture
was extracted with 25 mL of ethyl acetate and the
organic layer was dried over anhydrous sodium sulfate.
The solvent was removed and the residue was purified
by column chromatography on silica gel to give alkyl
4-substituted-3-hydroxybutanoate.
1
Yield 60%; H NMR l (ppm) 7.50 (5H, m), 4.60 (2H,
s), 4.13 (2H, m), 3.54 (2H, s), 1.30 (3H, t); 13C NMR:
13.9, 45.9, 61.3, 73.3, 74.6, 127.6, 127.7, 128.0, 128.4,
136.8, 166.9, 201.6; IR (neat) 2984, 1751, 742, 700 cm−1.
4.5.6. Ethyl 4-hydroxy-3-oxobutanoate 1g. 1g was pre-
pared by hydrogenation of ethyl 4-benzyloxy-3-oxobu-
tanoate 1f.30 A mixture of 1f (4.5 g, 0.019 mol) in
methanol (50 mL) and 10% palladium on carbon (0.5 g)
was stirred under hydrogen at room temperature for 18
h. The catalyst was removed by filtration through
Celite™ (Celite, USA). The solvent was removed and
the residue was purified by column chromatography on
4.7.1. Ethyl (S)-4-chloro-3-hydroxybutanoate 2a. Oil.
Yield 95%; [h]2D5=−21.4 (c 7.0, CHCl3), lit. for (R)-2a
of 97% e.e. prepared by hydrogenation using (S)-
BINAP–Ru complex, [h]2D1=+20.9 (c 7.71, CHCl3);31
e.e. >99% (by HPLC of the 3,5-dinitrobenzoate ester
1
silica gel to give 1g. Yield 60%; H NMR l (ppm) 4.43
(2H, s), 4.20 (2H, m), 3.52 (2H, s), 3.20 (1H, s), 1.30
(3H, t); 13C NMR: 13.9, 45.2, 61.7, 68.4, 166.4, 202.5;
IR (neat) 3588, 2984, 1747 cm−1.
1
derivative); H NMR l (ppm) 4.20 (3H, m), 3.60 (2H,
d), 3.31 (1H, d), 2.65 (2H, d), 1.33 (3H, t); 13C NMR:
14.4, 38.8, 48.4, 61.3, 68.2, 172.1; IR (neat) 3450, 2984,
1736, 756 cm−1.
4.6. Enzymatic reaction and measurement of
enantioselectivity
4.7.2. Ethyl (S)-4-bromo-3-hydroxybutanoate 2b. Oil.
Yield 95%; [h]2D5=−9.1 (c 10.4, CHCl3), lit. for (S)-2b
derived from (S)-3-hydroxybutyrolactone, [h]2D0=−11 (c
1, EtOH);32 e.e.=99% (by HPLC of the 3,5-dinitroben-
The enantioselectivity of S1 for alkyl 3-oxobutanoate
was determined by analysis of the enantiomeric excess
of the 3,5-dinitrobenzoate ester derivative of the alkyl
3-hydroxybutanoate that was formed by the enzymatic
reaction. The reaction mixture, comprising 10 mg of
each substrate, 100 mg of NADPH, 0.1 mmol of potas-
sium phosphate buffer (pH 6.5) and 0.5 units of the S1
solution in a total volume of 1 mL, was stirred at 30°C
for 18 h. The formation of product was monitored by
TLC and the reaction mixture was extracted with tolu-
ene (0.5 mL). To the organic extract, 3,5-dinitrobenzoyl
chloride (20 mg) and pyridine were added (0.2 mL) and
the mixture was stirred for 1 h at room temperature.
After the reaction, HCl (6 M, 1 mL) was added and the
organic layer was applied to a preparative TLC. The
e.e. of the 3,5-dinitrobenzoate product was analyzed
with an HPLC on a Chiralpak AD (4.6 mmf×250 mm)
column (Daicel Chemicals, Japan). The retention times
of four isomers of ethyl 3-hydroxy-2-methylbutanoate
2k (2S,3S), (2R,3S), (2S,3R) and (2R,3R) were, respec-
tively, 18.4, 20.2, 24.0 and 25.6 min. The retention
times of the four analogous isomers of ethyl 2-chloro-3-
hydroxybutanoate 2l were 12.2, 14.9, 16.2 and 18.8
min, in that order.
1
zoate ester derivative); H NMR l (ppm) 4.25 (1H, m),
4.18 (3H, q), 3.51 (2H, m), 3.28 (1H, br), 2.75 (2H, m),
1.38 (3H, t); 13C NMR: 14.4, 37.6, 39.6, 61.3, 67.8,
172.0; IR (neat) 3456, 2982, 1736, 673 cm−1.
4.7.3. Ethyl (S)-4-iodo-3-hydroxybutanoate 2c. Oil.
Yield 10%; [h]2D5=−9.8 (c 11.7, CHCl3), lit. for (R)-2c
derived from (R)-3-hydroxybutyrolactone, [h]2D0=+10.2
(c 3.3, EtOH);33 e.e.=99% (by HPLC of the 3,5-dini-
1
trobenzoate ester derivative); H NMR l (ppm) 4.20
(2H, q), 4.00 (1H, m), 3.31 (3H, m), 2.65 (2H, d), 1.28
(3H, t); 13C NMR: 12.4, 14.4, 41.0, 61.3, 67.7, 172.0; IR
(neat) 3450, 2982, 1732, 633 cm−1.
4.7.4. Octyl (S)-4-chloro-3-hydroxybutanoate 2d. Oil.
Yield 7%; [h]2D5=−14.5 (c 6.1, CHCl3), lit. for (R)-2d
prepared by baker’s yeast reduction, [h]2D3=+12.6 (c
10.2, CHCl3);34 e.e. >99% (by HPLC of the 3,5-dinitor-
1
benzoate ester derivative); H NMR l (ppm) 4.26 (1H,
m), 4.12 (2H, t), 3.61 (2H, t), 3.17 (1H, d), 2.64 (2H,
m), 1.65 (2H, m), 1.30 (10H, m), 0.90 (3H, t); 13C
NMR: 14.3, 22.9, 26.1, 283.8, 29.4, 32.0, 38.7, 48.4,
65.5, 68.2, 172.2; IR (neat) 3450, 2928, 1732, 758 cm−1.
4.7. General procedures for the preparation of optically
active alkyl 4-substituted-3-hydroxybutanoates
4.7.5. Ethyl (S)-3,4-dihydroxybutanoate 2g. Oil. Yield
90%; [h]2D5=+6.0 (c 6.4, CHCl3), lit. for (S)-2g derived
from (S)-malic acid, [h]2D5=+6.3 (c 1.0, CDCl3);24 e.e.
>99% (by HPLC of the 3,5-dinitrobenzoate ester
For the synthesis of various optically active alkyl 3-
hydroxybutanoates, we used a transformant, producing
both S1 and GDH-regenerating NADPH, as a catalyst.
The reaction mixture, comprising 25 mL broth of E.
coli HB101/pNTS1G, 10 mmol of alkyl 3-oxobu-
tanoate, 10 mmol of glucose, 3.2 mg NADP+, 25 mg of
Triton X-100 and 25 mL of n-butyl acetate, was stirred
at 30°C for 20 h. The pH of the reaction mixture was
kept at 6.5 with aqueous 5 M sodium hydroxide. The
reaction was monitored by the titration of sodium
hydroxide. The concentrations of the product were
determined with a gas chromatograph (model 164,
Hitachi, Japan) equipped with a 10% PEG-20M on an
1
derivative); H NMR l (ppm) 4.20 (2H, q), 4.15 (1H,
s), 3.68 (1H, d), 3.55 (1H, d), 3.18 (1H, br), 2.55 (2H,
m), 1.30 (3H, t); 13C NMR: 14.3, 38.0, 61.2, 65.9, 68.9,
172.8; IR (neat) 3450, 2984, 1774 cm−1.
4.7.6. Ethyl (R)-3-hydroxybutanoate 2h. Oil. Yield 95%;
[h]2D5=−41.6 (c 7.1, CHCl3), lit. for (S)-2h of 85% e.e.
prepared by baker’s yeast reduction, [h]2D5=+37.2 (c 1.3,
CHCl3);35 e.e. >99% (by HPLC on the 3,5-dinitroben-
1
zoate ester derivative); H NMR l (ppm) 4.18 (2H, q),

1718
Y. Yasohara et al. / Tetrahedron: Asymmetry 12 (2001) 1713–1718
2.88 (1H, br), 2.45 (2H, m), 1.28 (3H, t), 1.19 (3H, d);
13C NMR: 14.3, 22.7, 43.1, 60.8, 64.4, 173.0; IR (neat)
3450, 2978, 1736, 1030 cm−1.
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