Highly Enantioselective Reduction of Carbonyl Compounds Using a Reductase Purified from Bakers' Yeast

Article abstract of DOI:10.1021/jo980165e

An NADPH-dependent reductase that shows reducing activity for 1-chloro-2-hexanone has been purified from bakers' yeast. SDS-PAGE and gel filtration suggested that the purified reductase is a monomeric enzyme with a molecular weight of ca. 37 kDa. Asymmetric reduction of several carbonyl compounds using the purified reductase has been carried out. 1-Chloro-2-hexanone, 1-acetoxy-2-heptanone, methyl acetoacetate, ethyl pyruvate, 1-chloro-2,4-pentanedione, and 2,4-hexanedione were reduced to the corresponding alcohols with high enantiomeric purities (>98% ee). The reductase showed high specificity constants (k_cat/K_m = 10³-10⁵ s^-1 M^-1) and relatively low Michaelis constants (K_m = 10^-4-10^-3 M) for all the substrates examined.

Full text of DOI:10.1021/jo980165e

4996
J . Org. Chem. 1998, 63, 4996-5000
High ly En a n tioselective Red u ction of Ca r bon yl Com p ou n d s Usin g
a Red u cta se P u r ified fr om Ba k er s’ Yea st
Tadashi Ema,* Yasushi Sugiyama, Minoru Fukumoto, Hiroyuki Moriya, J ing-Nan Cui,
Takashi Sakai, and Masanori Utaka*
Department of Applied Chemistry, Faculty of Engineering, Okayama University,
Tsushima, Okayama 700, J apan
Received J anuary 29, 1998
An NADPH-dependent reductase that shows reducing activity for 1-chloro-2-hexanone has been
purified from bakers’ yeast. SDS-PAGE and gel filtration suggested that the purified reductase
is a monomeric enzyme with a molecular weight of ca. 37 kDa. Asymmetric reduction of several
carbonyl compounds using the purified reductase has been carried out. 1-Chloro-2-hexanone,
1-acetoxy-2-heptanone, methyl acetoacetate, ethyl pyruvate, 1-chloro-2,4-pentanedione, and 2,4-
hexanedione were reduced to the corresponding alcohols with high enantiomeric purities (>98%
ee). The reductase showed high specificity constants (kcat/Km ) 103-105 s-1 M-1) and relatively
low Michaelis constants (Km ) 10-4-10-3 M) for all the substrates examined.
In tr od u ction
selectivity, as frequently observed in enzymatic reactions,
is one of the requirements for modern asymmetric
synthesis. The problem that the purification of an
enzyme is laborious can be eliminated, once the method
for producing the enzyme on a large scale using the
cloning technique has been established.8 Since many
enzymes whose substrate specificity is not strict are
currently known,1 it is an important task of synthetic
chemists to develop the potential ability of a purified
enzyme by applying a variety of multifunctional sub-
strates. In this paper, we report the highly enantio- and
regioselective reduction of several carbonyl compounds
using an NADPH-dependent reductase purified from BY.
Asymmetric synthesis with biocatalysts is becoming
more and more important because of its great potential
in environmentally benign organic syntheses. Among
many kinds of transformations with biocatalysts,1 asym-
metric reduction of carbonyl compounds with microor-
ganisms such as bakers’ yeast (BY, Saccharomyces
cerevisiae)2 is a traditional and useful method for the
preparation of optically active alcohols.3 Although the
BY whole-cell reduction is inexpensive and convenient,
stereoselectivity is often incomplete due to several oxi-
doreductases contained in the BY cells. On the other
hand, the enzymatic reduction using a purified reductase
usually shows high stereoselectivity.4-7 Complete stereo-
Resu lts
(1) (a) J ones, J . B. Tetrahedron 1986, 42, 3351. (b) Chen, C.-S.; Sih,
C. J . Angew. Chem., Int. Ed. Engl. 1989, 28, 695. (c) Klibanov, A. M.
Acc. Chem. Res. 1990, 23, 114. (d) Preparative Biotransformations:
Whole Cell and Isolated Enzymes in Organic Synthesis; Roberts, S.
M., Wiggins, K., Casy, G., Eds.; J ohn Wiley & Sons: Chichester, 1993.
(e) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic
Chemistry; Pergamon: Oxford, 1994. (f) Enzyme Catalysis in Organic
Synthesis; Drauz, K., Waldmann, H., Eds.; VCH: New York, 1994; Vol.
1. (g) Faber, K. Biotransformations in Organic Chemistry; Springer-
Verlag: Berlin, 1995.
(2) Abbreviations used in this paper: BSA, bovine serum albumin;
BY, bakers’ yeast; CFE, cell-free extract; GL6P, glucono-δ-lactone-6-
phosphate; G6P, glucose-6-phosphate; G6PDH, glucose-6-phosphate
dehydrogenase; MES, 2-(N-morpholino)ethanesulfonic acid; NADP+,
â-nicotinamide adenine dinucleotide phosphate; NADPH, reduced form
of NADP+; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis; TTN, total turnover number.
(3) (a) Servi, S. Synthesis 1990, 1. (b) Csuk, R.; Gla¨nzer, B. I. Chem.
Rev. 1991, 91, 49. (c) Utaka, M.; Sakai, T.; Tsuboi, S. J . Synth. Org.
Chem. J pn. 1991, 49, 647.
(4) (a) Kim, M.-J .; Whitesides, G. M. J . Am. Chem. Soc. 1988, 110,
2959. (b) Casy, G. Tetrahedron Lett. 1992, 33, 8159. In some cases,
the cell-free extracts can catalyze highly stereoselective reductions.
For example, see: (c) Keinan, E.; Seth, K. K.; Lamed, R. J . Am. Chem.
Soc. 1986, 108, 3474. (d) Bradshaw, C. W.; Hummel, W.; Wong, C.-H.
J . Org. Chem. 1992, 57, 1532.
P u r ifica tion of th e Red u cta se. We decided to purify
the enzyme that shows reducing activity toward 1-chloro-
2-hexanone (1), because the enantioselectivities in the
BY whole-cell reduction of a series of 1-chloro-2-al-
kanones were only moderate.9 The enzyme assay for the
cell-free extract (CFE) of BY using UV spectroscopy
revealed that the reductase requires NADPH as a
coenzyme. The reductase in the CFE was successfully
purified to homogeneity by hydrophobic chromatography
and ion-exchange chromatography. The detailed proce-
dure is described in the Experimental Section. After the
final chromatographic separation, only a single band (37
kDa) was detected by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) and by gel filtra-
tion, suggesting that the reductase is a monomeric
enzyme.10
Asym m etr ic Red u ction w ith th e P u r ified Red u c-
ta se. We carried out the enzymatic reduction of carbonyl
(5) (a) Shieh, W.-R.; Gopalan, A. S.; Sih, C. J . J . Am. Chem. Soc.
1985, 107, 2993. (b) Shieh, W.-R.; Sih, C. J . Tetrahedron: Asymmetry
1993, 4, 1259.
(6) (a) Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. J . Org.
Chem. 1991, 56, 4778. (b) Nakamura, K.; Kondo, S.; Kawai, Y.;
Nakajima, N.; Ohno, A. Biosci. Biotechnol. Biochem. 1994, 58, 2236.
(c) Nakamura, K.; Kondo, S.; Nakajima, N.; Ohno, A. Tetrahedron 1995,
51, 687.
(7) Ishihara, K.; Nakajima, N.; Tsuboi, S.; Utaka, M. Bull. Chem.
Soc. J pn. 1994, 67, 3314.
(8) (a) Casy, G.; Lee, T. V.; Lovell, H.; Nichols, B. J .; Sessions, R.
B.; Holbrook, J . J . J . Chem. Soc., Chem. Commun. 1992, 924. (b)
Sakowicz, R.; Gold, M.; J ones, J . B. J . Am. Chem. Soc. 1995, 117, 2387.
(9) Sakai, T.; Wada, K.; Murakami, T.; Kohra, K.; Imajo, N.; Ooga,
Y.; Tsuboi, S.; Takeda, A.; Utaka, M. Bull. Chem. Soc. J pn. 1992, 65,
631.
S0022-3263(98)00165-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/03/1998

Enantioselective Reduction of Carbonyl Compounds
J . Org. Chem., Vol. 63, No. 15, 1998 4997
Sch em e 1a
aged us, because any attempt to improve the enantiose-
lectivity in the BY whole-cell reduction of 1 has been
unsuccessful so far.9,14 The reductase exhibited several
interesting features as shown below.
Table 1 suggests that the purified reductase is one of
the enzymes contributing to Prelog’s rule, the empirical
rule proposed for the microbial reductions of carbonyl
compounds;15 the right-hand moiety of each structure is
bulkier than the left-hand moiety (see Table 1), and the
â face of the carbonyl group (the Si face in the cases of 1
and 5, and the Re face in the cases of the other
substrates) is attacked by NADPH. We suppose that the
right-hand moiety of the substrates is either accom-
modated in a binding pocket of the reductase by attrac-
tive interactions or disfavored by some steric repulsion.
In the case of the polyfunctional ketone 6, the steric
bulkiness and the polarity of the acetoxy group (the right-
hand moiety) are very similar to those of the acetyl group
(the left-hand moiety). Therefore, it is conceivable that
6 has two types of binding modes, with the Re or Si face
of the carbonyl group directed toward NADPH in some
ratio. Such ambiguous recognition of the enantioface
may be responsible for the exceptionally low enantiose-
lectivity for 6. Ketones 2-4, 6, and 7 were reduced to
the corresponding (S)-alcohols, whereas chlorine-contain-
ing substrates 1 and 5 were reduced to the corresponding
(R)-alcohols. This difference in the absolute configuration
may be convenient for organic synthesis, because deriva-
tives of both enantiomers of 1,2-diol can be prepared by
the choice of the R substituent (Cl or OAc).3c
a
For abbreviations, see ref 2.
compounds 1-7 using the purified reductase. To regen-
erate a catalytic amount of NADPH in situ, the glucose-
6-phosphate (G6P)/glucose-6-phosphate dehydrogenase
(G6PDH) system11 was employed (Scheme 1). It was
confirmed by UV spectrophotometric analysis that G6PDH
has little or no reducing activity for 1-7 in the presence
of NADPH. The results of the enzymatic reduction of
1-7 are summarized in Table 1, where the stereochem-
istry of the products is drawn in such a way that the â
(front) face of the carbonyl group in the substrates is
attacked by NADPH. The results of the whole-cell
reduction of 1-7 are also shown in Table 1 for compari-
son. The obtained alcohols are useful for organic syn-
thesis. For example, alcohols 9 and 13 are chiral building
blocks for prostaglandins12 and nonactin,13 respectively.
The reductase successfully reduced ketones 1-4 to the
corresponding alcohols 8-11 with high enantiomeric
purities (>98% ee). In the cases of 1-substituted-2,4-
pentanediones 5 and 6, the more hindered but more
activated carbonyl group was regioselectively reduced, in
contrast to 2,4-hexanedione (7) undergoing the reduction
at the less hindered carbonyl (acetyl) group. The absolute
configuration of alcohols 8 and 12 was found to be R,
which is opposite to the S-configuration of other alcohols.
Kin etic P a r a m eter s. We determined the kinetic
parameters of the reductase for 1-7 in order to evaluate
the capacity of the reductase as a catalyst from a
physicochemical viewpoint. The number of the kinetic
parameters so far determined and evaluated for enanti-
oselective enzymatic reactions toward nonnatural sub-
strates is limited.5-7 The initial rates v0 at different
substrate concentrations [S]0 were measured by monitor-
ing the decrease in the absorbance of NADPH at 340 nm.
The plot of v0 versus [S]0 afforded a typical saturation
curve. The apparent kcat and Km values were calculated
using the nonlinear least-squares method assuming the
Michaelis-Menten equation. The results are listed in
Table 2.
Another interesting observation is the regioselectivity
for â-diketones 5-7. It is known that â-diketones
undergo the BY reduction selectively at the less hindered
carbonyl group.16 The purified reductase has the ability
to completely recognize the steric difference between
methyl and ethyl groups of 7. This regiospecificity is
impressive when it is compared, for example, to the result
that the reduction of 7 with NaBH4 in water showed little
regioselectivity even at 0 °C. Furthermore, the regiose-
lectivity of the reductase was changed when 1-substituted-
2,4-pentanediones 5 and 6 were used (Table 1), indicating
that in this case the chemical reactivity of the functional
groups dominates over the steric effect.17
In the enzymatic reduction of 1, the isolated yield of 8
was initially very low under our standard reaction
conditions. By adding a small amount of bovine serum
albumin (BSA) to the reaction mixture according to the
report of Nakamura et al.,6a the yield was significantly
improved. The amino acid residues at the active sites of
the enzymes may be susceptible to functionalization by
the potential alkylating agent 1, which could have been
suppressed by BSA. In the enzymatic reduction of 5, a
large amount (51%) of furanone 15 was obtained, al-
though the enantiomeric purity of alcohol 12 was high.
Control experiments revealed that both the reductase and
Discu ssion
The reductase presented in this paper showed excellent
stereoselectivity toward several nonnatural substrates.
The enzymatic reduction showed higher stereoselectivity
than the whole-cell reduction. In particular, the high
enantioselectivity in the enzymatic reduction of 1 encour-
(10) The facts that the purified reductase showed high activity for
1-acetoxy-2-alkanones and that its molecular weight determined by
SDS-PAGE and gel filtration is ca. 37 kDa strongly suggest that it is
identical with the R-acetoxy ketone reductase (AcKR) previously
reported by us.7 We have suggested that AcKR is identical with
L-enzyme-2,7 which is one of the four â-keto ester reductases isolated
by Nakamura et al.6a
(11) Wong, C.-H.; Whitesides, G. M. J . Am. Chem. Soc. 1981, 103,
4890.
(12) Barry, J .; Kagan, H. B. Synthesis 1981, 453.
(14) Utaka, M.; Kitagawa, K. Unpublished results.
(15) Prelog, V. Pure Appl. Chem. 1964, 9, 119.
(16) (a) Ohta, H.; Ozaki, K.; Tsuchihashi, G. Agric. Biol. Chem. 1986,
50, 2499. (b) Ohta, H.; Ozaki, K.; Tsuchihashi, G. Chem. Lett. 1987,
2225. (c) Fauve, A.; Veschambre, H. J . Org. Chem. 1988, 53, 5215.
(17) Cui, J .-N.; Teraoka, R.; Ema, T.; Sakai, T.; Utaka, M. Tetra-
hedron Lett. 1997, 38, 3021.
(13) Kim, B. H.; Lee, J . Y. Tetrahedron Lett. 1992, 33, 2557.

4998 J . Org. Chem., Vol. 63, No. 15, 1998
Ema et al.
Ta ble 1. Asym m etr ic Red u ction of Ca r bon yl Com p ou n d s Usin g th e P u r ified Red u cta se a n d BY Wh ole Cells
a
Conditions: substrate (0.25 mmol), purified reductase (1.5 U with respect to 1), NADP+ (2.5 µmol), G6PDH (22 U), G6P (0.29 mmol),
b
10 mM phophate buffer (pH 7.0, 25 mL), 30 °C. Conditions: substrate (1.0 mmol), pressed bakers’ yeast (6 g), glucose (2 g), water (60
mL), 30 °C. c Isolated yield. BSA (2 mg) was added as an additive. e The values reported in ref 9 (with immobilized BY) are 69 (80).f The
d
g
h
values reported in ref 7 (with immobilized BY) are 51 (99). The values reported in ref 28 are 47 (91). BSA (2 mg) was added as an
additive. A large amount (51%) of furanone 15 was obtained. The values reported in ref 17 (with dry BY) are 53 (29, S). The values
reported in ref 24 (with dry BY) are 42 (82). The values reported in ref 16a are 48 (97).
i
j
k
Ta ble 2. Kin etic P a r a m eter s of th e P u r ified Red u cta sea
value is also lower than that observed for other enzymatic
reductions toward natural substrates (TTN ) 106-107)11
and is higher than that for the lipase-catalyzed enanti-
oselective transesterifications of secondary alcohols in an
organic solvent (TTN ) ca. 5000).21 The specificity
constants, kcat/Km, of the reductase (Table 2) amount to
substrate
kcat (s-1
)
Km (M)
kcat/Km
1
2
3
4
5
6
7
6.9 ( 0.5
(2.1 ( 0.5) × 10-3
(3.2 ( 0.2) × 10-4
(8.0 ( 0.9) × 10-4
(3.3 ( 0.9) × 10-4
(3.2 ( 0.4) × 10-3
(4.0 ( 0.5) × 10-3
(7.1 ( 1.4) × 10-3
3.3 × 103
1.3 × 105
1.3 × 104
3.6 × 103
1.1 × 103
3.8 × 103
1.4 × 103
(4.1 ( 0.1) × 10
(1.0 ( 0.04) × 10
1.2 ( 0.09
3.4 ( 0.2
the order of 103-105 s-1 M-1
. Despite the reactions
(1.5 ( 0.06) × 10
toward nonnatural substrates, the specificity constants
are high and are comparable to those of other enzymes
for natural substrates; recall that the rate constant for
the diffusion-controlled bimolecular association in water
is 109 s-1 M-1 and that the specificity constants of most
of the enzymatic reactions for natural substrates are in
(1.0 ( 0.1) × 10
Measurement conditions: reductase (2.8 × 10-8 or 6.3 × 10-8
M), NADPH (3.0 × 10-4 or 3.4 × 10-4 M), substrate concentration
range (substrate) ) 0.7-14 mM (1), 0.008-0.8 mM (2), 1.5-15
mM (3), 0.07-6 mM (4), 0.7-14 mM (5), 0.4-16 mM (6), 1.8-12
mM (7), 10 mM phosphate buffer (pH 7.0), 30 °C. The kinetic
parameters were calculated by means of the nonlinear least-
squares method applied to the Michaelis-Menten equation: v0 )
a
the range of 104-108 s-1 M-1 22
The results that the
.
reductase showed relatively low Km values for all the
substrates examined may suggest broad substrate speci-
ficity of the reductase, which is advantageous for organic
synthesis. The apparent binding energies approximately
calculated from the equation ∆G° ) RT ln Km range from
-3.0 to -4.8 kcal/mol. The ample binding energy of the
reductase for 1-7 may be used to recognize the enan-
tioface of the carbonyl group, because the differential
activation energy required for 98% ee can be calculated
to be -2.8 kcal/mol.23 These considerations confirm that
the reductase is an excellent catalyst for asymmetric
reduction.
k
cat[E]0[S]0/(Km + [S]0).
the buffer are responsible for the cyclization of 5.18
The total turnover number (TTN)1e for NADPH in the
enzymatic reduction is ca. 100 at 100% conversion, and
the TTN roughly estimated for the enzyme is ca. 29 000.19
The former value is lower than that reported for mem-
brane-reactor systems (TTN ) 103-105).20 The latter
(19) The weight of the reductase used in the preparative reduction
was ca. 0.32 mg as determined by the method of Bradford. Because
the molecular weight of the reductase is 37 000, the amount of
reductase used is 8.6 nmol.
(20) Nidetzky, B.; Haltrich, D.; Kulbe, K. D. Chemtech 1996, 31.
(21) Ema, T.; Maeno, S.; Takaya, Y.; Sakai, T.; Utaka, M. J . Org.
Chem. 1996, 61, 8610.
(22) Fersht, A. Enzyme Structure and Mechanism, 2nd ed.; Free-
man: New York, 1985.
(18) Control experiments were done without NADP(H), G6PDH, and
G6P in 10 mM phosphate buffer (pH 7.0). Conversions of 5 to 15 in
the presence and absence of the reductase (1.5 U with respect to 1)
after 4 h were 82% and 54%, respectively.

Enantioselective Reduction of Carbonyl Compounds
J . Org. Chem., Vol. 63, No. 15, 1998 4999
Ta ble 3. P u r ifica tion of th e Red u cta se
Exp er im en ta l Section
purification
step
total
total
specific
yield
Gen er a l. 1H NMR spectra were measured in CDCl3 at 500
or 200 MHz. Silica gel column chromatography was carried
out using Fuji Silysia BW-127 ZH (100-270 mesh), and TLC
was performed on Merck silica gel 60 F254. Butyl-Toyopearl
650M, Phenyl-Toyopearl 650M, and DEAE-Toyopearl 650M
were purchased from Tosoh Co. The ultrafiltration membrane
(10-kDa cutoff) and the seamless cellulose tubing for dialysis
were purchased from Advantec and Viskase Co., respectively.
G6PDH (from BY, lyophilized powder) and the molecular
weight markers for SDS-PAGE (lysozyme (14 kDa), â-lacto-
globulin (18 kDa), trypsinogen (24 kDa), ovalbumin (45 kDa),
albumin (66 kDa)) were purchased from Sigma. The pressed
BY, NADPH, NADP+, and G6P were purchased from Oriental
Yeast Co., Ltd. The substrates 1,9 2,7 5,17 6,24 and 725 were
prepared according to the literature. The reduction products
8,9 9,26 10,27 11,28 12,17 13,24 and 1416a were characterized
according to the literature.
En zym e Assa y. A chromatographic fraction (300 µL) was
added to a solution (2.4 mL) of 1-chloro-2-hexanone (1) (4.8
mM) and NADPH (0.33 mM) in 10 mM MES buffer (pH 6.0)
in a UV cuvette thermostated at 25 °C. After the solution was
quickly shaken, the reaction rate was measured by following
the decrease in the absorbance of NADPH at 340 nm as a
function of time. In this paper, 1.0 U of the enzymatic activity
is defined as the amount of enzyme that oxidizes 1.0 µmol of
NADPH/min at 25 °C under the reaction conditions indicated
above. The amount of proteins was determined by the method
of Bradford using BSA as the standard.29
Kin etic P a r a m eter s. An aliquot (200 or 500 µL) of the
purified reductase (0.37 µM) in 10 mM phosphate buffer (pH
7.0) was added to a solution (2.4 mL) of substrate and NADPH
(0.37 mM) in the phosphate buffer in a UV cuvette; the
substrate concentration range (substrate) ) 0.7-14 mM (1),
0.008-0.8 mM (2), 1.5-15 mM (3), 0.07-6 mM (4), 0.7-14
mM (5), 0.4-16 mM (6), and 1.8-12 mM (7). After the solution
was magnetically stirred for 30 s in a UV cell compartment
thermostated at 30 °C, the initial rate v0 was measured by
monitoring the decrease in the absorption at 340 nm as a
function of time. The initial rates v0 were measured at
different substrate concentrations [S]0. Plot of v0 versus [S]0
afforded the saturation curve. The apparent kcat and Km values
were calculated using the nonlinear least-squares method
assuming the Michaelis-Menten equation: v0 ) kcat[E]0[S]0/
(Km + [S]0).
P u r ifica tion of th e Red u cta se. Raw pressed BY (40 g)
suspended in 0.1 M MES buffer (pH 6.0, 40 mL) was homog-
enized by glass beads (0.5 mm in diameter, 60 mL) with a cell
mill (Vibrogen Co.) chilled with ice. This procedure was
repeated eight times (BY totally 320 g). The homogenate was
centrifuged at 12 000 rpm for 30 min at 4 °C. To the
supernatant CFE (360 mL) obtained by decantation was added
(NH4)2SO4 (52 g, 30%). After the solution was allowed to cool
in an ice bath for 45 min, the solution was centrifuged at
12 000 rpm for 30 min at 4 °C. All the purifications by column
chromatography that are described below were carried out at
4 °C. The supernatant solution was applied on a Butyl-
Toyopearl column (φ 4 × 12 cm) equilibrated with 10 mM MES
buffer (pH 6.0) containing 1 M (NH4)2SO4. The proteins were
eluted with the MES buffer using a stepwise gradient of
activitya (U) proteinsb (mg) activity (U/mg) (%)
CFE
35.6
23.2
15.3
12.5
3800
567
85.6
13.8
1.18
0.0094
0.041
0.18
0.91
2.9
100
65
43
35
10
butyl
phenyl
butyl
DEAE
3.47
a
Determined by the spectrophotometric enzyme assay method
described in the Experimental Section. One unit of enzymatic
activity is defined as the amount of enzyme that oxidized 1.0 µmol
b
of NADPH/min at 25 °C. Determined by the method of Brad-
ford.29
(NH4)2SO4 (from 1 to 0.6 M, 150 mL each). The elution pattern
of the proteins was examined by the absorbance at 280 nm.
The active fractions were detected by the enzyme assay method
described above. The active fractions (170 mL) eluted at 0.9-
0.8 M (NH4)2SO4 were then combined. After the concentration
of (NH4)2SO4 in the enzyme solution was adjusted to ca. 1.2
M by adding (NH4)2SO4, the enzyme solution was applied on
a Phenyl-Toyopearl column (φ 3 × 8 cm) equilibrated with 10
mM MES buffer (pH 6.0) containing 1.2 M (NH4)2SO4. The
proteins were eluted with the MES buffer using a stepwise
gradient of (NH4)2SO4 (from 1.2 to 0.8 M, 125 mL each). The
active fractions (160 mL) eluted at 0.9 M (NH4)2SO4 were then
combined. After the concentration of (NH4)2SO4 in the enzyme
solution was adjusted to ca. 1.0 M by adding (NH4)2SO4, the
enzyme solution was applied on a Butyl-Toyopearl column (φ
2.5 × 8 cm) equilibrated with 10 mM MES buffer (pH 6.0)
containing 1 M (NH4)2SO4. The proteins were eluted with the
MES buffer using a stepwise gradient of (NH4)2SO4 (from 1 to
0.6 M, 125 mL each). The active fractions (75 mL) eluted at
0.8 M (NH4)2SO4 were combined and concentrated to 20 mL
by ultrafiltration. After the solution was dialyzed against 10
mM phosphate buffer (pH 7.0, 250 mL × 4, 1000 mL × 2), the
solution was applied on a DEAE-Toyopearl column (φ 2.5 × 5
cm) equilibrated with the phosphate buffer. The proteins were
eluted with the phosphate buffer using a stepwise gradient of
NaCl (from 0 to 0.05 M, 100 mL each). The active fractions
(32 mL) eluted at 0.04 M NaCl were combined. Only a single
band was detected by SDS-PAGE (12.5%) around ca. 37 kDa
as estimated by using the molecular weight markers. These
results are summarized in Table 3. Gel filtration using a TSK-
GEL G3000 SW column (Tosoh Co.) (0.1 M phosphate buffer
(pH 7.0, 0.1 M Na2SO4), flow rate 0.5 mL/min, detection at
280 nm) showed a single peak at ca. 37 kDa as estimated by
using the molecular weight markers. The reductase thus
purified was used for the enzymatic reduction of carbonyl
compounds 1-7 and for the determination of the kinetic
parameters.
Gen er a l P r oced u r e for En zym a tic Red u ction . To a
solution of the purified reductase (1.5 U with respect to 1),
NADP+ (2.1 mg, 2.5 µmol), G6PDH (100 µg, 22 U), and G6P
(100 mg, 0.29 mmol) in 10 mM phosphate buffer (pH 7.0, 25
mL) was added the substrate (0.25 mmol). The mixture was
stirred with a magnetic stirrer in a water bath thermostated
at 30 °C for the appropriate reaction time. The progress of
the reaction was monitored by TLC. After the solution was
saturated with NaCl, the crude product was extracted with
ethyl acetate or ether (15 mL × 4). The combined organic
phase was dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel
column chromatography or distillation under reduced pressure
to give the product.
Gen er a l P r oced u r e for BY Wh ole-Cell Red u ction . To
bakers’ yeast (6.0 g) suspended in water (60 mL) was added
glucose (2.0 g). After the mixture was stirred with a magnetic
stirrer at 30 °C for 30 min, the substrate (1.0 mmol) was added
to the mixture. The reaction mixture was stirred at 30 °C for
the appropriate reaction time. After Celite (13 g) was added
to the reaction mixture, the mixture was filtered through
Celite. The Celite was washed with acetone (6 mL) and then
with ethyl acetate or ether (20 mL × 3). The filtrate was
(23) The ratio of the rate constants for parallel reactions is equal to
the ratio of the amounts of two products: E ) (kcat/Km)S/(kcat/Km)R
)
S/R. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J . J . Am. Chem.
Soc. 1982, 104, 7294. The above equation can be transformed to the
equation ∆∆G‡ ) -RT ln E ) -RT ln [(1 + ee(P))/(1 - ee(P))].
(24) Utaka, M.; Ito, H.; Mizumoto, T.; Tsuboi, S. Tetrahedron:
Asymmetry 1995, 6, 685.
(25) Adams, J . T.; Hauser, C. R. J . Am. Chem. Soc. 1944, 66, 1220.
(26) Ishihara, K.; Sakai, T.; Tsuboi, S.; Utaka, M. Tetrahedron Lett.
1994, 35, 4569.
(27) Utaka, M.; Watabu, H.; Higashi, H.; Sakai, T.; Tsuboi, S.; Torii,
S. J . Org. Chem. 1990, 55, 3917.
(28) Nakamura, K.; Inoue, K.; Ushio, K.; Oka, S.; Ohno, A. J . Org.
Chem. 1988, 53, 2589.
(29) Bradford, M. Anal. Biochem. 1976, 72, 248.

5000 J . Org. Chem., Vol. 63, No. 15, 1998
Ema et al.
saturated with NaCl. After the organic phase was separated,
the crude product was extracted with ethyl acetate or ether
(40 mL × 3). The combined organic phase was dried over
MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography or
distillation under reduced pressure to give the product.
Ack n ow led gm en t. We would like to thank Dr. T.
Tamura (Okayama University) for useful discussions
about purification of the enzyme and gel filtration
performance. We are grateful to the SC-NMR Labora-
tory of Okayama University for measurement of NMR
spectra. This work was financially supported by a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Science and Culture of J apan.
Deter m in a tion of En a n tiom er ic P u r ity a n d Absolu te
Con figu r a tion . The enantiomeric purities of alcohols 9 and
13 were determined by 1H NMR spectroscopy after the alcohols
were converted to the MTPA esters.30 The enantiomeric
purities of alcohols 8, 10 (acetate derivative), 11, 12, and 14
(acetate derivative) were determined by gas chromatography
with a CP-cyclodextrin-â-2,3,6-M-19 capillary column (Chrom-
pack Co.). The absolute configurations of alcohols 8-14 were
determined by comparison with the signs of the reported
specific rotations or with the retention time of authentic
samples by gas chromatography.
Su p p or tin g In for m a tion Ava ila ble: Spectroscopic data
for the optically active alcohols 8-14, copies of 1H NMR spectra
for 8-14, [S]0-v0 plots for 1-7, and results of SDS-PAGE
and gel filtration (14 pages). This material is contained in
libraries on microfiche, immediately follows this article in the
microfilm version of the journal, and can be ordered from the
ACS; see any current masthead page for ordering information.
(30) Dale, J . A.; Mosher, H. S. J . Am. Chem. Soc. 1973, 95, 512.
J O980165E