Asymmetric reduction of ketones by the acetone powder of Geotrichum candidum

Article abstract of DOI:10.1021/jo9812779

Aromatic ketones, β-keto esters, and simple aliphatic ketones were reduced with excellent selectivity to the corresponding (S)-alcohols by using the acetone powder of Geotrichum candidum. This method is superior in reactivity and stereoselectivity to reduction by the whole-cell. The experimental conditions for the reduction system such as ratio of the biocatalyst to the substrate, kinds of coenzymes, alcohol for coenzyme regeneration, and buffer, pH, and reaction temperature were investigated, and stability and preservability of the biocatalyst were also examined. This method is very convenient for the synthesis of optically pure alcohols on a gram scale.

Full text of DOI:10.1021/jo9812779

J . Org. Chem. 1998, 63, 8957-8964
8957
Asym m etr ic Red u ction of Keton es by th e Aceton e P ow d er of
Geotr ich u m ca n d id u m
K. Nakamura* and T. Matsuda
Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011 J apan
Received J une 30, 1998
Aromatic ketones, â-keto esters, and simple aliphatic ketones were reduced with excellent selectivity
to the corresponding (S)-alcohols by using the acetone powder of Geotrichum candidum. This
method is superior in reactivity and stereoselectivity to reduction by the whole-cell. The
experimental conditions for the reduction system such as ratio of the biocatalyst to the substrate,
kinds of coenzymes, alcohol for coenzyme regeneration, and buffer, pH, and reaction temperature
were investigated, and stability and preservability of the biocatalyst were also examined. This
method is very convenient for the synthesis of optically pure alcohols on a gram scale.
In tr od u ction
liver (HLADH),8 Thermoanaerobium brockii (TBADH),9
Thermoanaerobacter ethanolicus,10 Lactobacillus kefir,11
Pseudomonas sp.,12 Gluconobacter oxidans,13 Bacillus
stearothermophilus,14 Geotrichum candidum,15 et al. are
used to reduce a full range of carbonyl compounds.
Extensive research has been undertaken on the develop-
ment of enzymatic systems for the asymmetric reduction
of ketones, and many methods to control the stereochem-
istry of the reduction have been reported.16
The synthesis of enantiomerically pure compounds is
becoming increasingly important for research and devel-
opment in chemistry and biochemistry.1 Among chiral
compounds, enantiomerically pure alcohols are particu-
larly useful as building blocks for the synthesis of natural
products, pharmaceuticals, and agricultural chemicals
since a variety of methods have been developed to convert
the alcohol functionality to other useful functional groups
such as chloride,2 amine,3 azide,4 fluoride,5 etc. One of
the easiest methods for the preparation of optically active
alcohols is the asymmetric reduction of ketones by either
organometallic reagents with chiral ligands or biocata-
lysts such as alcohol dehydrogenases. Enzymatic reduc-
tions have attracted more and more attention due to the
high stereoselectivities.6 Currently, alcohol dehydroge-
nases from various sources such as baker’s yeast,7 horse
However, there is still a need to improve the enantio-
selectivity of biocatalytic reduction. Although the selec-
tivities of previously reported reduction systems were
relatively high (around 90-95% ee) to moderate, enan-
tiomerically pure compounds (>99% ee) could not be
obtained except for a few cases. For example, the selec-
tivity of the reduction of ketones by permeabilized cells
of Gluconobacter oxidans is around 95% for most of its
substrates(2-pentanone,2-hexanone,2-octanone,3-nonanone,
cyclohexyl methyl ketone), but a selectivity of 99% ee is
obtained only for the reduction of acetophenone and
methyl isopropyl ketone.13
(1) (a) Seebach, D.; Imwinkelried, R.; Weber, T. In Modern Synthetic
Methods 1986; Scheffold, R., Ed.; Springer-Verlag: Berlin, 1986; Vol.
4, pp 125-259. (b) Helmchen, G.; Karge, R.; Weetman, J . In Modern
Synthetic Methods 1986; Scheffold, R., Ed.; Springer-Verlag: Berlin,
1986; Vol. 4, pp 261-306. (c) Brown, H. C.; J adhav, P. K.; Singram, B.
In Modern Synthetic Methods 1986; Scheffold, R., Ed.; Springer-
Verlag: Berlin, 1986; Vol. 4, pp 307-356. (d) Santaniello, E., Ferra-
boschi, P., Grisenti, P., Manzocchi, A. Chem. Rev. 1992, 92, 1071-
1140. (e) Chiral Auxiliaries and Ligands in Asymmetric Synthesis; Sey-
den-Penn, J., Ed.; Wiley-Interscience: New York, 1995. (f) Stereoselective
Synthesis; Nograde, M., Ed.; VCH Publishers: New York, 1995.
(2) (a) Lewis, E. S.; Boozer, C. E. J . Am. Chem. Soc. 1952, 74, 308.
(b) Ward, A. M. Organic Syntheses; J ohn Wiley: New York, 1943;
Collect. Vol. 2, p 159. (c) Shoppee, C. W.; Coll, J . C. J . Chem. Soc. C
1970, 1124. (d) Carman, R. M.; Shaw, I. M. Aust. J . Chem. 1976, 29,
133.
(3) (a) Degerbeck, F.; Fransson, B.; Grehn, L.; Ragnarsson, U. J .
Chem. Soc., Perkin Trans. 1 1992, 245. (b) Corey, E. J .; Wei-guo, S.
Tetrahedron Lett. 1990, 31, 3833.
(4) Fabiano, E.; Golding, B. T.; Sadeghi, M. M. Synthesis 1987, 190.
(5) (a) Leroy, J .; Hebert, E.; Wakselman, C. J . Org. Chem. 1979,
44, 3406. (b) Hamman, S.; Barrelle, M.; Tetaz, F.; Beguin, C. G. J .
Fluorine Chem. 1987, 37, 85. (c) Kopecky, J .; Smejkal, J . Collect. Czech.
Chem. Commun. 1980, 45, 2971.
Moreover, the substrate specificity of most of the
systems is not wide enough to be beneficial for organic
(7) (a) MacLeod, R.; Prosser, H.; Fikentscher, L.; Lanyi, J .; Mosher,
H. S. Biochemistry 1964, 3, 838. (b) Servi, S. Synthesis 1990, 1.
(8) (a) Davies, J .; J ones, J . B. J . Am. Chem. Soc. 1979, 101, 5405.
(b) Haslegrave, J . A.; J ones, J . B. J . Am. Chem. Soc. 1982, 104, 4666.
(c) J ones, J . B.; Beck, J . F. Applications of Biochemical Systems in
Organic Chemistry; J ones, J . B., Sih, C. J ., Perlman, D., Eds.; J ohn
Wiley & Sons: New York, 1976; Part 1, pp 107-401.
(9) (a) Keinan, E.; Hafeli, E. K.; Seth, K. K.; Lamed, R. J . Am. Chem.
Soc. 1986, 108, 162. (b) Keinan, E.; Seth, K. K.; Lamed, R. J . Am.
Chem. Soc. 1986, 108, 3474.
(10) Zheng, C.; Pham, V. T.; Phillips, R. Catal. Today 1994, 22, 607.
(11) (a) Hummel, W. Appl. Microbiol. Biotechnol. 1990, 34, 15. (b)
Bradshaw, C. W.; Hummel, W.; Wong, C.-H. J . Org. Chem. 1992, 57,
1532.
(12) (a) Shen, G.-J .; Wang, Y.-F.; Bradshaw, C.; Wong, C.-H. J .
Chem. Soc., Chem. Commun. 1990, 677. (b) Bradshaw, C. W.; Fu, H.;
Shen, G.-J .; Wong, C.-H. J . Org. Chem. 1992, 57, 1526.
(6) (a) Servi, S. Microbial Reagents in Organic Synthesis, NATO ASI
Series C; Kluwer: Dordrecht, The Netherlands, 1992. (b) Faber, K.
Biotransformations in Organic Chemistry, 2nd ed.; Springer: Berlin,
1995. (c) Roberts, S. M.; Turner, N. J .; Willetts, A. J .; Turner, M. K.
Introduction to Biocatalysis Using Enzymes and Microorganisms;
Cambridge University Press: New York, 1995. (d) Azerad, R. Bull.
Soc. Chim. Fr. 1995, 132, 17. (e) Roberts, S. M.; Turner, N. J . J .
Biotechnol. 1992, 22, 227. (f) Turner, N. J . Nat. Prod. Rep. 1994, 11,
1. (g) Sih, C. J .; Chen, C.-S. Angew. Chem., Int. Ed. Engl. 1984, 23,
570. (h) Pratt, A. J . Chem. Brit. 1989, 282. (i) Klibanov, A. Acc. Chem.
Res. 1990, 23, 114.
(13) Adlercreutz, P. Enzyme Microb. Technol. 1991, 13, 9.
(14) Bortolini, O.; Fantin, G.; Fogagnolo, M.; Giovannini, P. P.;
Guerrini, A.; Medici, A. J . Org. Chem. 1997, 62, 1854.
(15) (a) Azerad, R.; Buisson, D. Microbial Reagents in Organic
Synthesis, NATO ASI Series C; Servi, S., Ed.; Kluwer Academic
Publishers: Dordrecht, The Netherlands, 1992; pp 421-440. (b)
Nakamura, K.; Yoneda, T.; Miyai, T.; Ushio, K.; Oka, S.; Ohno, A.
Tetrahedron Lett. 1988, 29, 2453. (c) Nakamura, K.; Takano, S.;
Terada, K.; Ohno, A. Chem. Lett. 1992, 951. (d) Nakamura, K.; Takano,
S.; Ohno, A. Tetrahedron Lett. 1993, 34, 6087. (e) Nakamura, K.; Inoue,
Y.; Ohno, A. Tetrahedron Lett. 1995, 36, 265.
10.1021/jo9812779 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/29/1998

8958 J . Org. Chem., Vol. 63, No. 24, 1998
Nakamura and Matsuda
Ta ble 1. Red u ction of 1a a by G. ca n d id u m
syntzhesis, some reasons being (1) the reactivity of
HLADH is high for cyclic ketones but not for acyclic
ketones,8c (2) the selectivity of TBADH is shown to be
excellent for the reduction of only aliphatic ketones,9 and
(3) when the substrate specificity with reduction by the
whole cell, such as baker’s yeast which is easy to
manipulate and commercially available, is very wide, the
selectivity is low.7b
reducing
agente
yield
(%)f
ee
catalyst
coenzymed
(%)f configg
An easily available and handy biocatalyst with very
high selectivity toward the reduction of any ketones,
either aromatic or aliphatic, under mild reaction condi-
tions would be of great value. Here, we report an
enzymatic reduction system by which both aromatic and
aliphatic ketones can be reduced with excellent stereo-
selectivity, resulting in the synthesis of secondary alco-
hols with a high yield and >99% ee. It should be
emphasized that the biocatalyst for the system is easy
to prepare and handle, the reaction proceeds under mild
conditions, and the product isolation is simple. Another
advantage of this system is that it is environmentally
friendly.
whole cellb
APG4c
APG4c
APG4c
APG4c
APG4c
APG4c
APG4c
APG4c
APG4c
none
none
none
none
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
cyclopentanol
cyclopentanol
52
0
1
28
R
none
NAD+
none
71
98
S
S
S
S
S
S
S
S
8
NAD+
NADH
NADP+
NADPH
NAD+
NADP+
89
89
88
83
97
86
>99
>99
>99
>99
>99
>99
Reaction conditions: 20 h at 30 °C at 130 rpm in water (3 mL)
for whole-cell reaction or in MES (2-(N-morpholino)ethanesulfonic
a
acid) buffer (pH 7.0, 0.1 M, 3 mL) for APG4 reactions. 0.08 mmol.
b
d
f
0.5 g wet weight. c 10 mg. 0.007 mmol. e 1.31 mmol. Deter-
g
mined by GC analyses. Determined by comparison of the GC
retention times with those of authentic samples.
A biocatalyst prepared from a dimorphic fungus, Geo-
trichum candidum IFO 4597, was employed for the
reduction of ketones owing to its high reactivity with
unnatural substrates and the simplicity of its growth. We
used the acetone powder17 of G. candidum (APG4), a
microbial dried-cell preparation dehydrated using ac-
etone, as a catalyst for the asymmetric reduction of
ketones for the first time and found that a number of
ketones from aromatic ketones, â-keto esters, and simple
aliphatic ketones are reduced to (S)-alcohols with >99%
ee.18 The properties and products of APG4, such as
coenzyme dependence, alcohols for coenzyme regenera-
tion, suitable buffers, optimum pH, optimum reaction
temperature, stability, and preservability were examined,
as well as the scope and limitations in regard to substrate
specificity.
Sch em e 1
ness of APG4 preparation is in striking contrast to the
tediousness associated with isolation of an enzyme in a
pure form.
B. Rea ction Con d ition s for Asym m etr ic Red u c-
tion of Acetop h en on e (1a ). The asymmetric reduction
of acetophenone (1a ) by G. candidum was investigated,
and the results are shown in Table 1. The reduction of
acetophenone (1a ) catalyzed by the whole cell resulted
in poor enantioselectivity (28% ee(R)). When the form
of the catalyst was changed from wet whole-cell to dried
powdered-cell (APG4), no reduction of 1a was observed,
which would indicate the loss of the necessary coen-
zyme(s) and/or coenzyme regeneration system(s) during
the treatment of the cells with acetone. Addition of
coenzyme, NAD+, did not have a significant effect on the
yield. Addition of 2-propanol resulted in only a small
increase in the yield, but a significant improvement in
the enantioselectivity was observed. Surprisingly, ad-
dition of both NAD+ and 2-propanol largely enhanced
both chemical yield and enantiomeric excess. Addition
of NADH, NADP+, or NADPH instead of NAD+ and
addition of cyclopentanol instead of 2-propanol also gave
enantiomerically pure alcohol in high yield.
Resu lts a n d Discu ssion
A. P r ep a r a tion s of th e En zym e (AP G4). The
crude alcohol dehydrogenase, the acetone powder of
Geotrichum candidum IFO 4597 (APG4), was prepared
as usual17 by treating wet cells with acetone. The
cultivation of G. candidum is very easy, requiring little
technique, and the preparation of the acetone powder is
also very simple (see Experimental Section). The simple-
(16) (a) Nakamura, K. Microbial Reagents in Organic Synthesis,
NATO ASI Series C; Servi, S., Ed.; Kluwer Academic Publishers:
Dordrecht, The Netherlands, 1992; pp 389-398. (b) Zhou, B.-n;
Gopalan, A. S.; VanMiddlesworth, F.; Shieh, W.-R.; Sih, C. J . J . Am.
Chem. Soc. 1983, 105, 5925. (c) Nakamura, K.; Kondo, S.; Nakajima,
N.; Ohno. A. Tetrahedron 1995, 51, 687. (d) Nakamura, K.; Miyai, T.;
Fukushima, K.; Kawai, Y.; Babu, B. R.; Ohno, A. Bull. Chem. Soc. J pn.
1990, 63, 1713. (e) Nakamura, K.; Kawai, Y.; Miyai, T.; Ohno, A.
Tetrahedron Lett. 1990, 31, 3631. (f) Nakamura, K.; Kawai, Y.;
Nakajima, N.; Ohno, A. J . Org. Chem. 1991, 56, 4778. (g) Nakamura,
K.; Kawai, Y.; Ohno, A. Tetrahedron Lett. 1991, 32, 2927. (h) Naka-
mura, K.; Kondo, S.; Kawai, Y.; Ohno, A. Tetrahedron: Asymmetry
1993, 4, 1253. (i) Nakamura, K.; Kondo, S.; Kawai, Y.; Ohno, A. Bull.
Chem. Soc. J pn. 1993, 66, 2738. (j) Nakamura, K.; Takano, S.; Ohno,
A. Tetrahedron Lett. 1993, 34, 6067. (k) Nakamura, K.; Kondo, S.;
Ohno, A. Bioorg. Med. Chem. 1994, 2, 433. (l) Fujisawa, T.; Itoh, T.;
Sata, T. Tetrahedron Lett. 1984 25, 5083.
(17) Goodhue, C. T.; Rosazza, J . P.; Peruzzotti, G. P. Manual of
Industrial Microbiology and Biotechnology; Demain, A. L., Solomon,
N. A., Eds.; American Society for Microbiology: Washington, DC, 1986;
pp 97-121.
(18) (a) Nakamura, K.; Kitano, K.; Matsuda, T.; Ohno, A. Tetrahe-
dron Lett. 1996, 37, 1629. (b) Nakamura, K.; Matsuda, T.; Itoh, T.;
Ohno, A. Tetrahedron Lett. 1996, 37, 5727.
The reaction mechanism is shown in Scheme 1. When
acetophenone (1a ) is reduced to (S)-1-phenylethanol ((S)-
1b), the reduced form of the coenzyme (NAD(P)H) is
oxidized to NAD(P)+, which is subjected to concomitant
recycling to its reduced form by a reducing agent, such
as 2-propanol or cyclopentanol. As the catalytic cycle is
effective only for the reduction to the (S)-alcohol and does
not involve any (R)-producing enzyme(s); the selectivity

Asymmetric Reduction of Ketones
J . Org. Chem., Vol. 63, No. 24, 1998 8959
Ta ble 2. Effect of Alcoh ols a s Red u cin g Agen ts on th e
Red u ction of 1a †
reducing agenta
yield (%)b
ee (%)b
configc
2-propanol
1-pentanol
2-pentanol
3-pentanol
cyclopentanol
cyclohexanol
ethanol
89
9
>99
94
>99
>99
>99
98
S
S
S
S
91
71
96
39
8
S
S
98
S†
Reaction conditions: 20 h at 30 °C at 130 rpm in MES buffer
(pH 7.0, 0.1 M, 3 mL): 1a , 0.08 mmol; APG4, 10 mg; NAD+, 0.007
mmol. 1.31 mmol. Determined by GC analyses. c Determined
by comparison of the GC retention times with those of authentic
samples.
a
b
F igu r e 1. Effect of biocatalyst/substrate ratio (B/S Ratio) on
the reduction of 1a . Reaction Conditions: 20 h at 30 °C at 130
rpm in MES buffer (pH 7.0, 0.1 M, 3 mL); 1a , 0.08 mmol;
NAD+, 0.007 mmol; 2-propanol, 1.31 mmol. Yields and ee were
determined by GC analyses, and ee was >99%(S) for any
amount of APG4 employed.
and cyclohexanone24 to regenerate the oxidized form of
coenzymes have been reported. For the APG4 reduction
system, the applicability of various alcohols as reducing
agents in the reduction of acetophenone (1a ) was inves-
tigated, and the results are shown in Table 2. Among
the alcohols tested for their abilities as a reducing agent,
2-alkanols (2-propanol and racemic 2-pentanol) and cy-
clopentanol exhibited excellent results for both yield and
ee.
of the APG4 reduction system consisting of APG4, NAD-
(P)H, and a reducing agent is excellent, whereas the
selectivity of the whole-cell reduction in which both (S)-
enzyme(s) and (R)-enzyme(s) catalyze the reaction is low
(28% ee(R)).
Ra tio of Bioca ta lyst (AP G4) to Su bstr a te (B/S).
The effect of the ratio of the biocatalyst (APG4) to the
substrate (B/S) on the yield of reduction of acetophenone
(1a ) was investigated; it was found that a 0.4/1 B/S ratio
was sufficient (Figure 1). One of the problems usually
encountered in biocatalytic processes in organic synthesis
is the requirement of an excessively high B/S ratios.6b,c,19
For example, in the baker’s yeast reduction of acetophe-
none (1a ), a B/S ratio of 360/1 (1800 g of baker’s yeast to
reduce 5 g of 1a ) is necessary to obtain a 23% yield of
(S)-1-phenylethanol ((S)-1b).7a When the whole cell of
G. candidum was used to reduce 1a , a B/S ratio of 50/1
is necessary.15e Moreover, the enantioselectivity of the
reduction was not affected by the amount of the catalyst
at all, and excellent selectivity (>99% ee) was obtained
regardless of the B/S ratio.
Coen zym es. Unlike biotransformations using the
whole cell, a coenzyme must be replaced in enzymatic
transformations. Using 2-propanol as a reducing agent,
the effectiveness of several nicotinamide coenzymes for
the reduction of acetophenone (1a ) was investigated. As
shown in Table 1, NAD+, NADH, NADP+, and NADPH
can be used to reduce 1a with high yield. The selectivity
of the reduction was hardly affected by the kind of
coenzymes and remained very high (ee >99%). Next, the
concentration effect of the NAD+ on the yield of (S)-1-
phenylethanol ((S)-1b) in the reduction of 1a was studied,
and it was found that only a small amount (0.025 mol
equivalence to the substrate) is necessary to maximize
the yield to 89%. The selectivity of the reduction also
remained >99% ee even when only 0.0009 mol equiva-
lence of NAD+ was employed in the reduction. The
concentration effects of NADH, NADP+, and NADPH
were also studied, and coenzyme concentration-yield
profiles similar to that for NAD+ were obtained.
Differences in effectiveness between cyclopentanol and
cyclohexanol can be explained on the basis of the higher
strain energy of cyclohexanone over that of cyclopen-
tanone. The facile reduction of cyclohexanone compared
with that of cyclopentanone was demonstrated in the
relative rate (23:1) of the sodium borohydride reduction.25
Cyclohexanone is easily reduced back to cyclohexanol
competing with the reduction of the main substrate (1a ),
but cyclopentanone is reduced much slower in the
undesired direction.
2-Alkanols from 2-propanol to 2-octanol and cyclopen-
tanol were employed for further investigation to examine
their concentration effects, which clarified that a large
excess of the reducing agents is necessary to maximize
the yield. The yield increased as the amount of the
reducing agent was increased and reached around 90%
when 15-20 equiv of the reducing agents to the substrate
were used. All 2-alkanols afford similar correlations in
the yield-mole equivalence relationship, whereas cyclo-
pentanol worked a little better than 2-alkanols. An
excess amount of the reducing agent is necessary to
obtain the product in high chemical yields since the
product, (S)-1-phenylethanol ((S) -1b), is oxidized back
to acetophenone (1a ) by the same enzyme(s) (see Scheme
1). Even with the excess amount of a reducing agent,
the yield of the reaction does not reach 100% since
increases in the yield stopped when the equilibrium
between the desired reaction (reduction of acetophenone
(1a )) and the undesired reverse reaction (oxidation of
1-phenylethanol (1b)) was reached. The undesired oxi-
(19) Sugai, T.; Katoh, O.; Ohta, H. Tetrahedron 1995, 51, 11987.
(20) Hirschbein, B. L.; Whitesides, G. M. J . Am. Chem. Soc. 1982,
104, 4458.
(21) (a) Vandecasteele, J .-P.; Lemal, J . Bull. Soc. Chim. Fr. 1980,
101. (b) Wong, C.-H.; Drueckhammer, D. G.; Sweers, H. M. J . Am.
Chem. Soc. 1985, 107, 4028.
(22) (a) Tischer, W.; Tiemeyer, W.; Simon, H. Biochimie 1980, 62,
331. (b) Wichmann, R.; Wandrey, C.; Bu¨ckmann, A. F.; Kula. M.-R.
Biotechnol. Bioeng. 1981, 23, 2789.
(23) J akovac, I. J .; Goodbrand, H. B.; Lok, K. P.; J ones, J . B. J . Am.
Chem. Soc. 1982, 104, 4659.
Red u cin g Agen ts. The reduction catalyzed by APG4
does not proceed without a reducing agent even in the
presence of a coenzyme as shown in Table 1. The use of
glucose-6-phosphate,11a,20 glucose,11a,21 formate,22 ethanol,8
2-propanol,9-12,15b,e 2-butanol,13 endo-bicyclo[3.2.0]hept-
2-en-6-ol,14 2-hexanol,15e and cyclopentanol15c,15d,15e to
regenerate the reduced form of coenzymes, and FMN23
(24) Nakamura, K.; Inoue, Y.; Ohno, A. Tetrahedron Lett. 1994, 35,
4375.
(25) Brown, H. C.; Ichikawa, K. Tetrahedron 1957, 1, 221.

8960 J . Org. Chem., Vol. 63, No. 24, 1998
Nakamura and Matsuda
dation of 1-phenylethanol (1b) through a different route
under aerobic conditions24,26 also prevents the yield from
becoming quantitative.
Although 2-alkanols from 2-propanol to 2-octanol and
cyclopentanol were effective for the reduction system,
from the synthetic point of view, the use of 2-propanol
as a reducing agent is most advantageous due to its high
volatility since a large amount of the remaining reducing
agent has to be separated from the product during the
workup.
original after heating at 40, 50, and 60 °C for 30 min,
respectively. However, most of the activity was lost with
heating at 70 °C. The selectivity of the reduction was
not influenced by incubation below 60 °C prior to the
reaction and was maintained at >99% ee.
Preservability of biocatalysts is one of the most im-
portant factors in judging applicability of catalysts to
organic synthesis. Accordingly, preservability of APG4
at 30 and -20 °C was investigated. Most of the activity
of APG4 was retained after 48 h at 30 °C, and 69% and
35% were retained after 10 days and 22 days at 30 °C in
air, respectively. Amazingly, the enzyme activities were
still preserved to a certain extent even after the storage
of APG4 for more than 45 days at 30 °C. APG4 can also
endure long-term preservations in a refrigerator; 69% of
the activity was preserved when the enzyme was stored
at -20 °C for more than 2 years without special care.
The enantioselectivity of the reduction remained very
high (>99%) even with the enzyme being stored for 45
days at 30 °C or stored for 2 years at -20 °C. This proved
that the observed stability and preservability of APG4
were remarkable.
As a result of the thorough examination of the reaction
conditions for APG4 reduction as well as the stability of
APG4, the yield of the reaction changes only with a
drastic change in the conditions, and the enantioselec-
tivity remains very high (>99% ee) regardless of the
reaction conditions. Similar experiments examining the
reaction conditions for the APG4 reduction of â-keto
esters were also conducted using ethyl 3-ketobutyrate or
methyl 3-ketobutyrate as substrates. The yield was a
little higher for the reduction of â-keto esters than that
of acetophenone (1a ), but analogous trends in the rela-
tionship between the yield and the conditions were
observed. Enantioselectivity was also high for the reduc-
tion of â-keto esters regardless of the reduction condi-
tions.
C. Syn th etic Ap p lica tion s. The present reduction
system has a great synthetic value due to the extremely
wide substrate specificity; aromatic ketones, â-keto es-
ters, and simple aliphatic ketones are reduced with
excellent selectivity to the corresponding (S)-alcohols.
The high enantioselectivity of the APG4 reduction
system was demonstrated by the reduction of acetophe-
none derivatives. Ortho-, meta-, or para-substituted
fluoro, chloro, bromo, methyl, methoxy, and trifluoro-
methyl acetophenone (2a -19a ) and pentafluoroacetophe-
none (20a ) were reduced by the APG4 reduction system,
and the results are listed in Table 3. An enantioselec-
tivity of more than 99% ee was obtained for the reduction
of all the acetophenone derivatives tested except that for
o-(trifluoromethyl)acetophenone (17a ) being 97% ee,
while the yield of the reduction depends on the position
of the substituent. Generally, the resulting alcohols were
obtained in quantitative yields when there was a sub-
stituent at the ortho position. The yield of the meta-
substituted acetophenone was slightly lower than that
of ortho-substituted acetophenone, and a para substitu-
ent decreased the yield. This trend is not affected by the
inductive effect of the substituent(s) since the fluoro or
chloro substituents have the same effect on the yield as
the methyl group. The plausible reason for the effect of
the position of the substituent on the yield is that the
yield is determined by the equilibrium between the
reduction and undesired oxidation as described in the
Reducing Agents section. It is known that this microbe
Kin etic P a r a m eter s. Using APG4 homogenized with
MES buffer (pH 7.0, 0.1 M), the initial rates of the
reduction of acetophenone (1a ) were measured as a
function of the substrate concentration. The apparent
kinetic parameters (Km and Vmax) were calculated via a
Lineweaver-Burk plot to be 1.7 mM and 0.0085 mM‚
min-1‚mg-1 of APG4 for Km and Vmax, respectively.
Bu ffer . The effect of the buffer used in the reduction
of acetophenone (1a ) by APG4 on the reaction rate, yield,
and ee was examined. It was found that various kinds
of buffers (MES (2-(N-morpholino)ethanesulfonic acid-
NaOH), KPB (potassium phosphate buffer), HEPES (4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid-NaOH),
MOPSO (â-hydroxy-4-morpholinepropanesulfonic acid-
NaOH), Tris (tris(hydroxymethyl)aminomethane-HCl),
and Im (imidazole-HCl)) at pH 7.0 can be used for this
reaction. Although the reaction rate depends slightly on
the kind of buffer used, little effect on the yield was
observed. Importantly, the enantioselectivity of the
reduction remained very high (>99% ee) regardless of the
kind of buffer. However, the buffer action must be
present for the reaction to proceed because the reaction
does not occur when ion exchanged water is used as a
solvent.
p H. The effect of pH on the reaction rate, yield, and
ee was also examined using MES buffer for pH between
4.5 and 7.7 and Tris buffer between 7.1 and 8.9. The
optimum pH for the reduction of acetophenone (1a ) by
APG4 is around 8.5, although it proceeds within a wide
pH range from 5.7 to higher than 8.9. The rate-limiting
step of this reduction system is supposed to be the
regeneration of the coenzyme from the oxidized form
(NAD+) to the reduced form (NADH) rather than the
reduction of the acetophenone (1a ) based on the optimum
pH being 8.5 since the reduction of NAD+ is faster in
basic conditions than in acidic conditions. Interestingly,
the reaction rate depends on the pH value, but the effect
of pH on the yield is very small between a pH value of
6.4 and 8.9. The enantioselectivity of the reduction is
not affected at all by the pH value between 5.7 and 8.9.
Rea ction Tem p er a tu r e. The optimum reaction tem-
perature was found to be 40 °C, whereas the reaction at
30 and 50 °C also proceeded smoothly, resulting in a high
yield. However, at 60 °C, the reaction rate dropped to
only one-third of the rate at the optimum reaction
temperature. The stereoselectivity of the reduction was
not influenced by the reaction temperature (>99% ee was
obtained at 30, 40, 50, and 60 °C).
Th er m osta bility. APG4 was incubated at various
temperatures for 30 min at pH 7.0 and the residual
activity was measured. APG4 was very stable at 30 °C
in MES buffer and did not lose any activity. The activity
of APG4 was retained at 94%, 82%, and 62% of the
(26) Nakamura, K.; Matsuda, T.; Ohno, A. Tetrahedron: Asymmetry
1996, 7, 3021.

Asymmetric Reduction of Ketones
J . Org. Chem., Vol. 63, No. 24, 1998 8961
Ta ble 3. Red u ction of Ar yl Meth yl Keton es by AP G4, NAD+, a n d 2-P r op a n ol
X
yield (%)a,b
ee (%)a
configc
H (1)
o-F (2)
89 (74)
>99 (94)
95 (90)
74 (60)
>99 (94)
95 (91)
62 (80)d
97 (89)
92 (85)
95 (66)
96 (73)
86 (83)
78 (70)
84 (85)d
90 (83)
29 (21)
6 (13)d
>99
>99
>99
>99
>99
99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
97
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
m-F (3)
p-F (4)
o-Cl (5)
m-Cl (6)
p-Cl (7)
o-Br (8)
m-Br (9)
p-Br (10)
o-Me (11)
m-Me (12)
p-Me (13)
o-MeO (14)
m-MeO (15)
p-MeO (16)
o-CF3 (17)
m-CF3 (18)
p-CF3 (19)
1′,2′,3′,4′,5′-F5 (20)
96 (83)
73 (85)
62 (80)
>99
>99
>99
a
b
Determined by GC analyses. Isolated yield in parentheses. See Experimental Section for the reaction conditions. c Determined as
d
described below. Higher isolated yield was obtained than GC yield with more enzyme or coenzyme, longer reaction time, or the reaction
under argon atmosphere. 1a (120 mg, 1.0 mmol) was converted to (S)-1b (90 mg). GC conditions: CPCD 110 °C; R, 9.0 min; S, 9.7 min;
[R]25 -55.1(c 1.63, CHCl3) (lit.33 [R]25 -57(c 5.12, CHCl3), S). 2a (1.11 g, 8.0 mmol) was converted to (S)-2b (1.06 g). GC conditions:
D
D
CPCD 110 °C; R, 8.6 min; S, 9.2 min; [R]25D -44.5 (c 0.782, MeOH). The absolute configuration of this compound was tentatively assigned
to be S. 3a (1.11 g, 8.0 mmol) was converted to (S)-3b (1.01 g). GC conditions: CPCD 110 °C; R, 10.3 min; S, 11.1 min; [R]25 -33.5 (c
D
1.435, MeOH). The absolute configuration of this compound was tentatively assigned to be S. 4a (360 mg, 2.6 mmol) was converted to
(S)-4b (219 mg). GC conditions: CPCD 115 °C; R, 10.2 min; S, 11.0 min; [R]25 -37.7 (c 0.931, MeOH) (lit.34 [R]31 -23.1 (c 3.12, MeOH),
D
D
70% ee(S)). 5a (365 mg, 2.4 mmol) was converted to (S)-5b (346 mg). GC conditions: CPCD 130 °C; R, 11.1 min; S, 12.8 min; [R]25 -62.7
D
(c 0.894, CHCl3) (lit.29e [R]D -56.5 (c 0.0463, CHCl3) 90% ee(S)). 6a (154 mg, 1.0 mmol) was converted to (S)-6b (143 mg). GC conditions:
CPCD 130 °C; R, 13.4 min; S, 14.2 min; [R]25 -43.5(c 1.08, CHCl3) (lit.35 [R]20 +36.7(c 1.0, CHCl3) 84.6% ee(R)). 7a (362 mg, 2.3 mmol)
D
D
was converted to (S)-7b (293 mg). GC conditions: CPCD 130 °C; R, 14.0 min; S, 15.0 min; [R]25 -49.0 (c 1.84, ether) (lit.29e [R]D -48.9
D
(c 0.0613, ether) 94% ee(S)). 8a (2.6 g, 13 mmol) was converted to (S)-8b (2.3 g). GC conditions: CPCD 145 °C; R, 10.5 min; S, 12.4 min;
[R]24 -54.6 (c 1.23, CHCl3) (lit.29e [R]D -50.5 (c 0.0305, CHCl3) 94% ee(S)). 9a (1.00 g, 5.0 mmol) was converted to (S)-9b (854 mg). GC
D
conditions: CPCD 145 °C; R, 18.5 min; S, 19.3 min); [R]26 -28.6 (c 1.78, EtOH). The absolute configuration was determined to be S by
D
debromination of 9b to (S)-1-phenylethanol (see Experimental Section). 10a (100 mg, 0.50 mmol) was converted to (S)-10b (67 mg). GC
conditions: CPCD 150 °C; R, 10.5 min; S, 10.9 min; [R]23 -37.9 (c 1.13, CHCl3) (lit.29e [R]D -37.5(c 0.0666 CHCl3) 96% ee(S)). 11a (362
D
mg, 2.7 mmol) was converted to (S)-11b (269 mg). GC conditions: CPCD 130 °C; R, 7.1 min; S, 7.8 min; [R]25D -64.3 (c 1.04, EtOH) (lit.29e
[R]D -58.6 (c 0.0665, EtOH) 95% ee(S)). 12a (309 mg, 2.3 mmol) was converted to (S)-12b (261 mg). GC conditions: CPCD 110 °C; R, 17.1
min, S: 18.3 min; [R]25 -39.8 (c 0.944, EtOH) (lit.38 [R]15 -41.9 (c 0.500, EtOH) >99.9% ee(S)). 13a (311 mg, 2.3 mmol) was converted
D
D
to (S)-13b. Yield: 70% (220 mg). GC conditions: CPCD 105 °C; R, 15.6 min; S, 17.2 min; [R]26 -43.5 (c 0.994, MeOH) (lit.34 [R]27 -22.3
D
D
(c 3.79, MeOH) 55% ee(S)). 14a (557 mg, 3.7 mmol) was converted to (S)-14b. Yield: 85% (477 mg); ee >99% (S) (determined by GC
analysis). GC conditions: CPCD 130 °C; S, 12.5 min; R, 13.4 min); [R]23 -63.0 (c 1.10, toluene) (lit.35 [R]20 +48.9 (c 1.1, toluene) 81.5%
D
D
ee(R)). 15a (352 mg, 2.3 mmol) was converted to (S)-15b (296 mg). GC conditions: CPCD 130 °C; R, 15.6 min; S, 16.4 min); [R]22 -34.9
D
(c 0.849, MeOH) (lit.36 [R]D +35 (c 1, MeOH) 97% ee(R)). 16a (704 mg, 4.7 mmol) was converted to (S)-16b (152 mg). GC conditions:
CPCD 130 °C; R, 14.5 min; S, 15.8 min); [R]22D -51.9 (c 0.718, CHCl3) (lit.29e [R]D -46.2 (c 0.0273, CHCl3) 87% ee(S)). 17a (2.0 g, 11 mmol)
was converted to (S)-17b (257 mg). GC conditions: CPCD 110 °C; R, 9.8 min; S, 10.8 min; [R]22D -45.5 (c 0.661, MeOH) (lit.34 [R]22D -23.0
(c 1.26, MeOH) 55% ee(S)). 18a (350 mg, 1.9 mmol) was converted to (S)-18b (294 mg). GC conditions: CPCD 120 °C; R, 8.2 min; S, 8.8
min; [R]22D -28.4 (c 1.26, MeOH) (lit.34 [R]24D -17.1(c 2.92, MeOH) 59% ee(S)). 19a (350 mg, 1.9 mmol) was converted to (S)-19b (299 mg).
GC conditions: CPCD 120 °C; R, 10.1 min; S, 11.1 min; [R]22 -28.1 (c 1.13, MeOH) (lit.29e [R]21 -10.8 (c 0.0578, MeOH) 61% ee(S)). 20a
D
D
(281 mg, 1.3 mmol) was converted to (S)-20b (227 mg). GC conditions: CPCD 120 °C; R, 5.1 min; S, 5.6 min; [R]22D -6.33 (c 1.01, pentane)
(lit.37 [R]D -8.1, (pentane), 97% ee(S)).
oxidizes para-substituted (S)-phenylethanol smoothly,
but ortho-substituted phenylethanol is not oxidized at
all27 meaning the yield of the reduction increases in the
order of para-, meta-, and ortho-substituted acetophe-
none.
The reduction on a gram scale proceeds smoothly
without any loss in ee, and the isolation of the product
alcohol with high yields was possible as illustrated in the
reduction of o-bromoacetophenone (8a , 2.6 g) to (S)-1-(o-
bromophenyl)ethanol ((S)-8b) giving an 89% yield (2.3
g, >99% ee). Importantly, any organic chemist will be
able to reproduce this result because the high selectivity
of this reduction system is very steady and independent
of small changes in the experimental conditions such as
scale of the reaction, pH, reaction temperature, etc.
Reduction by APG4 of several aromatic ketones having
different length alkyl chains demonstrated the scope and
limitations of the substrate specificity (Table 4). Phenyl
moiety of acetophenone can be replaced by a benzyl (21a )
or even by a 2-phenylethyl (22a ) group with slightly
better results of chemical yield than the reduction of
acetophenone (1a ) without a decrease in enantioselec-
tivity. However, when the methyl moiety of acetophe-
(27) Nakamura, K.; Inoue, Y.; Matsuda, T.; Ohno, A. Tetrahedron
Lett. 1995, 36, 6263.

8962 J . Org. Chem., Vol. 63, No. 24, 1998
Nakamura and Matsuda
Ta ble 4. Red u ction of Ar om a tic Keton es by AP G4,
NAD+, a n d 2-P r op a n ol
Ta ble 5. Red u ction of â-Keto Ester s by AP G4, NAD+,
a n d 2-P r op a n ol
a
Determined by GC analyses. The product alcohol was acety-
lated to determined ee by GC analysis for 30b, 32b, and 33b.
b
Isolated yield in parentheses. See Experimental Section for the
reaction conditions. c Determined as described below. 29b GC
conditions: G-TA 90 °C; S, 7.0 min; R, 7.5 min. 30a (136 mg, 1.0
mmol) was converted to (S)-30b (78 mg). GC conditions for ethyl
3-acetoxylbutanoate: G-TA, 85 °C; R, 19.2 min; S, 20.5 min; [R]25
D
+40.6 (c 1.09, CHCl3) (lit.41 [R]r.t.D +39.9 (c 1.8, CHCl3) 92% ee(S)).
31b GC conditions: DEX 90 °C; S, 12.5 min; R, 13.2 min. 32a
(172 mg, 1.0 mmol) was converted to (S)-32b (128 mg). GC
conditions for 2,2-dimethylpropyl 3-acetoxylbutanoate: DEX, 115
°C; S, 10.8 min; R, 11.4 min); [R]25 +31.1 (c 1.01, CHCl3) (lit.42
D
[R]25 +26 (c 1.0, CHCl3) 80% ee(S)). 33a (500 mg, 3.5 mmol) was
D
a
Determined by GC, HPLC, or NMR analyses. The product was
converted to (S)-33b (262 mg). GC conditions for ethyl 3-acetoxy-
b
acetylated to determine ee for 21b. Isolated yield in parentheses.
lpentanoate: G-TA, 110 °C; R, 8.7 min; S, 9.1 min; [R]26D +34.3 (c
See Experimental Section for the reaction conditions. c Determined
1.05, CHCl3) (lit.41 [R]r.t. +32.2 (c 5.1, CHCl3) 93% ee(S)).
D
d
as described below. Higher isolated yield was obtained than GC
yield with more enzyme or coenzyme, longer reaction time, or the
reaction under argon atmosphere. 21a (367 mg, 2.7 mmol) was
converted to (S)-21b (291 mg). GC conditions for 1-phenyl-2-propyl
results of the reduction of 3-ketobutyrates (29a -32a ) and
a 3-ketopentanoate (33a ) are shown in Table 5. 3-Ke-
tobutyrates of methyl (29a ), ethyl (30a ), tert-butyl (31a ),
or neopentyl (32a ) ester are reduced to (S)-hydroxyl
esters (29b-32b) with >99% ee quantitatively. The yield
and ee remained >99% regardless of the ester moiety.
Ethyl 3-ketopentanoate (33a ) can also be reduced to
S-hydroxyl ester (33b) with >99% ee in 72% yield. The
present reduction system is certainly suitable for the
reduction of 3-ketobutyrates and 3-ketopentanoates.
Efficient catalysts for the enantioselective reduction of
functionalized ketones have been reported,29 but the
asymmetric reduction of small aliphatic ketones remains
a major challenge in organic chemistry.9a Simple ali-
phatic ketones from 2-octanone to 2-undecanone (34a -
37a ), 6-methyl-5-heptene-2-one (38a ), and 5-chloro-2-
pentanone (39a ) were reduced by the APG4 system to
the corresponding (S)-2-alkanols (34b-39b) giving high
yields with 99% ee (Table 6). The present reduction
system is beneficial for the reduction of aliphatic ketones
over a nonenzymatic system by which, for example, in
the reduction of 2-octanone (34a ), 82% ee is the highest
to the best of our knowledge.30 Moreover, the reduction
of 2-octanone (34a ) gives only 24% ee for the product
alcohol with BINAL-H, the well-known “super” catalyst
acetate: CPCD 110 °C; S, 13.7 min; R, 14.4 min; [R]25 +41.7 (c
D
1.19. CHCl3) (lit.38 [R]15 +39.7 (c 0.515, CHCl3) >99.9% ee(S)).
D
22a (350 mg, 2.4 mmol) was converted to (S)-22b (338 mg). GC
conditions: CPCD 105 °C; S, 30.1 min; R, 31.6 min; [R]25 +21.0
D
(c 1.17, C6H6) (lit.38 [R]22 +22 (c 0.380, C6H6) >99.9% ee(S)). 23a
D
(345 mg, 2.6 mmol) was converted to (S)-23b (86 mg). GC
conditions: CPCD 110 °C; R, 15.5 min; S, 16.1 min; [R]25 -47.2
D
(c 0.643, CHCl3) (lit.29e [R]D -46.7 (c 0.0409, CHCl3, 95% ee(S)).
25a (1.00 g, 6.7 mmol) was converted to (S)-25b (113 mg). GC
conditions: G-TA 95 °C; R, 29.9 min; S, 31.2 min; [R]26 -49.1 (c
D
0.828, ether) (lit.29e [R]D -45.7 (c 0.0623, ether) 92% ee(S)). 27a
(2.0 g, 13 mmol) was converted to (R)-27b (99 mg). GC conditions:
DEX 120 °C; S, 16.2 min; R, 17.2 min; [R]25D -40.2 (c 1.02, acetone)
(lit.39 [R]D -34.4 (c 2, acetone) 76 ee(R)). 28a (1.01 g, 6.5 mmol)
was converted to (R)-28b (493 mg). HPLC conditions: OD 5:1
hexane-2-propanol, 0.5 mL/min; S, 15.6 min; R, 18.1 min; [R]25
D
-50.4 (c 1.78, cyclohexane) (lit.40 [R]25D -48.1 (c 1.73, cyclohexane)
100% ee(R)).
none is replaced by an ethyl (23a ), isopropyl (25a ), or
methoxymethyl (27a ) group, the yield dramatically de-
creases, although the enantioselectivity remained high
(>99% ee), and when the alkyl chain is elongated to a
propyl (24a ) or enlarged to a tert-butyl (26a ) group, the
reaction scarcely proceeds. R-Chloroacetophenone (28a )
was also reduced by the APG4 system, and R-chloro-1-
phenylethanol (28b) was obtained with 80% yield and
98% ee. This chiral alcohol can be converted to various
compounds via epoxide.28
(29) (a) Midland, M. M.; Kazubski, A. J . Org. Chem.. 1982, 47, 2495.
(b) Noyori, R.; Tomino, I.; Tanimoto, Y.; Nishizawa, M. J . Am. Chem.
Soc. 1984, 106, 6709. (c) de Vries, E. F.-J .; Brussee, J .; Kruse, C. G.;
van der Gen, A. Tetrahedron Asymmetry 1994, 5, 377. (d) Brown, H.
C.; Chandrasekharan, J .; Ramachandran, P. V. J . Am. Chem. Soc.
1988, 110, 1539. (e) Carter, M. B.; Schiøtt, B.; Gutie´rrez, A.; Buchwald,
S. L. J . Am. Chem. Soc. 1994, 116, 11667. (f) Ohkuma, T. Ooka, H.
Ikariya, T. Noyori, R. J . Am. Chem. Soc. 1995, 117, 10417. (g)
Pu¨ntener, K.; Schwink, L.; Knochel, P. Tetrahedron Lett. 1996, 37,
8165.
The versatility of the APG4 reduction system is further
exemplified by the â-keto esters as substrates. The
(28) (a) Calet, S.; Urso, F.; Alper, H. J . Am. Chem. Soc. 1989, 111,
931. (b) Brown, H. C.; Pai, G. G. J . Org. Chem. 1983, 48, 1784. (c)
Lucchi, O. D.; Buso, M.; Modena, G. Tetrahedron Lett. 1987, 28, 107.
(d) Atkins, R. K.; Frazier, J .; Moore, L. L.; Weigel, L. O. Tetrahedron
Lett. 1986, 27, 2451. (e) Niwa, M.; J iang, P.-F., Hirata, Y. Chem.
Pharm. Bull. 1987, 35, 108.
(30) Weissaman, S. A.; Ramachandran, P. V. Tetrahedron Lett. 1996,
37, 3791.

Asymmetric Reduction of Ketones
J . Org. Chem., Vol. 63, No. 24, 1998 8963
Ta ble 6. Red u ction of Alip h a tic Keton es by AP G4
Deter m in a tion of Absolu te Con figu r a tion s. The
absolute configuration of 1-(m-bromophenyl)ethanol (9b)
was determined by conversion to optically active 1-phen-
ylethanol (1b) by Pd/H2 and comparison of the sign of
optical rotation with that of the literature value.33 The
absolute configuration of 2-undecanol (37b) was deter-
mined by synthesizing the authentic (S)-37b from (S)-
propylene oxide.
Con clu sion s
A simple reagent for asymmetric reduction of various
kinds of ketones for use by organic chemists with a little
background in biochemistry or microbiology has been
created. The use of the acetone powder of the microbe
eliminates the laborious isolation of the enzyme and
problems of low selectivity associated with catalysis by
the whole cell. To investigate the substrate specificity,
a wide variety of ketones was subjected to the APG4
reduction system. The substrate specificity of the acetone
powder system is very wide, and excellent enantioselec-
tivity of the reduction was obtained for most of the wide
variety of substrates. Since the yield of the reduction is
also satisfactory, the present method is highly effective
for asymmetric synthesis of optically pure aromatic
alcohols, aliphatic alcohols, and hydroxy esters.
a
b
Determined by GC analyses. Isolated yield in parentheses.
See Experimental Section for the reaction conditions. c Determined
by GC analyses after acetylation of the product except for 38b.
Determined as described below. e Higher isolated yield was
d
obtained than GC yield with more enzyme or coenzyme, longer
reaction time, or the reaction under argon atmosphere. 34a (402
mg, 3.1 mmol) was converted to (S)-34b (252 mg). GC conditions
for 2-octyl acetate: CPCD, 100 °C; S, 7.0 min; R, 7.8 min; [R]25
D
+9.00 (c 1.23, CHCl3) (lit.9a [R]D +8.78 (CHCl3) 97% ee(S)). 35a
(399 mg, 2.8 mmol) was converted to (S)-35b (313 mg). GC
conditions for 2-nonyl acetate: CPCD 110 °C; S, 8.1 min; R, 8.7
min; [R]25 +8.66 (c 1.18, CHCl3) (lit.9a [R]D +7.96 (CHCl3) 98%
D
ee(S)). 36a (209 mg, 1.3 mmol) was converted to (S)-36b (103 mg).
GC conditions for 2-decyl acetate: CPCD 125 °C; S, 7.8 min; R,
8.4 min); [R]23 +10.3 (c 0.80, EtOH) (lit.43 [R]25 +4.22 (c 6.28,
Exp er im en ta l Section
D
D
EtOH) 83.0% ee(S)). 37a (450 mg, 2.6 mmol) was converted to (S)-
37b (276 mg). GC conditions for 2-undecyl acetate: CPCD 130 °C;
S, 10.7 min; R, 11.4 min); [R]21D +7.92 (c 0.75, EtOH). The absolute
configuration of this compound was determined to be S by
comparison of the optical rotation with that of the authentic
sample synthesized. 38a (397 mg, 3.2 mmol) was converted to (S)-
38b (306 mg). GC conditions: G-TA 85 °C; S, 7.3 min; R, 7.8 min;
[R]25D +12.0 (c 2.93, CHCl3) (lit.9a [R]D +10.76 (CHCl3) 99% ee(S)).
39a (612 mg, 5.1 mmol) was converted to (S)-39b (249 mg). GC
conditions for 5-chloro-2-pentyl acetate: DEX 110 °C; S, 5.3 min;
R, 6.2 min; [R]26D +14.9 (c 1.19, CHCl3) (lit.9a [R]D +15.58 (CHCl3)
98% ee(S)).
In str u m en ts. Gas chromatographic analyses were per-
formed using chiral GC-columns (Chiraldex G-TA; 40 or 30
m; He 2 mL/min (G-TA), CP-cyclodextrin-B-2,3,6-M-19; 25 m;
He 2 mL/min (CPCD), Chirasil-DEX CB; 25 m; He 2 mL/min
(DEX)). HPLC analyses were performed using Chiralcel OD
(0.46 cm × 25 cm (OD)).
P r ep a r a tion s of th e En zym e (AP G4). Cu ltiva tion of
Geotr ich u m ca n d id u m . Glycerol (30 g), yeast extract (10
g), polypeptone (5 g), KH2PO4 (11.18 g), and K2HPO4 (3.12 g)
were mixed with water and the volume was adjusted to 1.0 L
with water. A portion of the resulting solution (30 mL) was
placed in an 100-mL test tube, and the rest was placed in a
2-L Erlenmeyer flask, which were covered with silicone caps
and sterilized (121 °C, 20 min). The solution in the test tube
was inoculated with the stored microbe of G. candidum IFO
4597 and stirred for 24 h at 30 °C at 130 rpm. The resulting
mixture in the test tube was transferred to the flask and
stirred for 24 h at 30 °C at 130 rpm. The resulting mixture
was filtered to obtain the cells (18 g wet weight (wet wt)).
P r ep a r a tion of Aceton e P ow d er (AP G4). The cells of
G. candidum IFO 4597 (18 g wet wt) were mixed with cold
acetone (-20 °C, 150 mL), and the cells were collected by
filtration. The procedure was repeated five times, and then
the cells were dried under reduced pressure. The dried cells
(3.8 g) were obtained and used without further purification.
for hydrogenolysis.29b In contrast to these low selectivi-
ties, the reduction of 2-octanone (34a ) by this present
APG4 system affords (S)-alcohol (34b) with >99% ee.
This system can also reduce aliphatic ketones with
functional groups such as olefin or chloride giving a high
yield with 99% ee. For example, the reduction of 6-meth-
yl-5-heptene-2-one (38a ) by the APG4 system proceeded
smoothly and afforded (S)-(+)-sulcatol (39b), the ag-
gregation pheromone produced by males of Gnathotrichus
sulcatus,31 in one step. The reduction of 5-chloro-2-
pentanone (39a ) was also successful and afforded (S)-5-
chloro-2-pentanol (39b) giving high yields with 99% ee.
This chiral alcohol is an excellent bifunctional building
block that may be conveniently employed for synthesis
of natural products containing chiral carbinol centers.9b
For example, (S)-39b is used as a chiral building block
for the total synthesis of (+)-(S,S)-(cis-6-methyltetrahy-
dropyran-2-yl)acetic acid,9b a naturally occurring hetero-
cycle that has been isolated from the perfume material
civet, a glandular secretion of the civet cat (Viverra
civetta).32
(33) Kasai, M.; Froussios, C.; Ziffer, H. J . Org. Chem. 1983, 48, 459.
(34) Naemura, K.; Murata, M.; Tanaka, R.; Yano, M.; Hirose, K.;
Tobe, Y. Tetrahedron: Asymmetry 1996, 7, 3285.
(35) Hayashi, T.; Matsumoto, Y.; Ito, Y. Tetrahedron: Asymmetry
1991, 2, 601.
(36) Chen, C.-P.; Prasad, K.; Repic, O. Tetrahedron Lett. 1991, 32,
7175.
(37) Fukumasa, M.; Furuhashi, K.; Umezawa, J .; Takahashi, O.;
Hirai, T. Tetrahedron Lett. 1991, 32, 1059.
(38) Nakamura, K.; Kawasaki, M.; Ohno, A. Bull. Chem. Soc. J pn.
1996, 69, 1079.
(39) Ferraboschi, P.; Grisenti, P.; Manzocchi, A.; Santaniello, E.
Tetrahedron 1994, 50, 10539.
(40) Imuta, M.; Kawai, K.; Ziffer, H. J . Org. Chem. 1980, 45, 3352.
(41) Seebach, D.; Giovannini, F.; Lamatsch, B. Helv. Chim. Acta
1985, 68, 958.
(42) Tacke, R.; Linoh, H.; Stumpf, B.; Abraham, W.-R.; Kieslich, K.;
Ernst, L. Z. Naturforsch. 1983, 38b, 616.
(31) Byrne, K. J .; Swigar, A. A.; Silversteine, R. M.; Borden, J . H.;
Stokkink, E. J . Insect Physiol. 1974, 20, 1895.
(32) (a) Maurer, B.; Grieder, A.; Thommen, W. Helv. Chim. Acta
1979, 62, 44. (b) Maurer, B.; Thommen, W. Helv. Chim. Acta 1979,
62, 1096. (c) van Dorp, D. A.; Ward, J . P. Experientia 1981, 37, 917.
(43) Sonnet, P. E. J . Org. Chem. 1987, 52, 3477.

8964 J . Org. Chem., Vol. 63, No. 24, 1998
Nakamura and Matsuda
Wh ole-Cell Red u ction of Acetop h en on e (1a ). Acetophe-
none (1a ) (0.08 mmol) was added to a suspension of freshly
prepared whole cells (0.5 g wet wt) in water (3 mL) and shaken
at 130 rpm at 30 °C for 20 h. The resulting mixture was put
on Extrelut (Merck) and eluted with ether. The chemical yield
and ee of 1-phenylethanol (1b) were determined by GC
analysis of the ether extract to be 52% and 28%, respectively
(GC conditions: CPCD, 110 °C).
AP G4 Red u ction of Keton es. A ketone (0.08 mmol),
NAD+ (0.007 mmol), and 2-propanol (1.31 mmol) were added
to a suspension of APG4 (10 mg) in MES (2-(N-morpholino)-
ethanesulfonic acid) buffer (pH 7.0, 0.1 M, 3 mL). The mixture
was shaken at 130 rpm at 30 °C for 20 h, and the resulting
mixture was put on Extrelut and eluted with ether. Chemical
yield and ee of the product were determined by GC analysis,
(400 mg), and 5% palladium on carbon (45 mg) was stirred
overnight under an atmosphere of hydrogen at room temper-
ature. The catalyst was removed by filtration, and the filtrate
was neutralized with 1 N HCl. Brine was added to the
mixture, and the entire mixture was extracted with ether. The
combined extracts were washed with water, aqueous sodium
bicarbonate, and water and dried over Na2SO4, and the solvent
was evaporated. The residue was purified by silica gel column
chromatography (eluent 4:1 hexane-ethyl acetate) followed
by distillation with a Kugelrohr apparatus. The 1H NMR
spectrum of the product was identical with that of 1-phenyl-
ethanol. Ee of the product was determined to be 88% by GC
analysis (GC conditions: CPCD, 110 °C): yield 64% (53 mg);
[R]25 -46.8 (c 1.17, CHCl3) (lit.33 [R]25 -57 (c 5.12, CHCl3)
D
D
S).
HPLC analysis, or NMR analysis of the ether extract.
A
(S)-2-Un d eca n ol fr om (S)-P r op ylen e Oxid e. CuI (0.2
mmol, 40 mg) and Me2S (0.2 mL) were added to a cold (-20
°C) solution of n-octylmagnesium bromide (0.93 M in THF, 2.4
mL) under an atmosphere of argon. Then, a THF (2 mL)
solution of (S)-propene oxide (116 mg, 2 mmol) was added
dropwise to the mixture, and the mixture was stirred for 2 h
at the same temperature. The reaction was quenched by
aqueous NH4Cl, the mixture was acidified with 1 N HCl, and
the product was extracted with ether. The combined ether
extracts were washed with water, aqueous sodium bicarbonate,
and water and dried over anhydrous Na2SO4, and the solvent
was evaporated. The crude product was purified by silica gel
column chromatography (eluent 8:1 to 5:1 hexane-ethyl
acetate) followed by distillation with a Kugelrohr apparatus.
The NMR spectrum of the product was identical with that of
2-undecanol: yield 70% (240 mg); [R]25D +7.44 (c 1.27, EtOH).
The product was acetylated with acetyl chloride and pyridine
in CH2Cl2 to determine ee by GC analysis (GC conditions:
CPCD, 120 °C); ee 99%.
similar experiment was conducted using NADH, NADP+, and
NADPH as a coenzyme and cyclopentanol and 2-alkanols from
2-propanol to 2-octanol as a reducing agent.
Syn t h et ic Ap p lica t ion s. P r ep a r a t ion of 1-P h en yl-2-
p r op a n on e (21a ). This ketone was prepared through the
oxidation of 1-phenyl-2-propanol (22 mmol, 3.0 g) by stirring
in DMSO (300 mL), DCC (132 mmol, 25 g), and trifluoroacetic
acid (33 mmol, 3.8 g) under an atmosphere of argon at room
temperature for 4 days. Yield 39% (1.2 g); 1H NMR (CDCl3) δ
2.15 (s, 3H, CH3), 3.69 (s, 2H, CH2), 7.17-7.35 (m, 5H, Ph);
IR (neat) 700, 737, 1030, 1078, 1159, 1229, 1358, 1422, 1454,
1
1497, 1603, 1713, 2926, 3030, 3063 cm-
.
Gen er a l P r oced u r e for th e Red u ction of Keton es in
P r ep a r a tive Sca le. A ketone (1.6 mmol), 2-propanol (26
mmol, 2.0 mL), NAD+ (100 mg). and APG4 (200 mg) were
added to 60 mL of MES buffer (0.1 M, pH 7.0). The mixture
was stirred at 30 °C for 20 h at 130 rpm under an argon
atmosphere and filtered through Extrelut, which was washed
with ether. The filtrate was extracted with ether, and the
combined ether solution was washed with water, dried over
Na2SO4, and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography
(eluent 5:1 to 3:1 hexane-ethyl acetate) followed by distillation
with a Kugelrohr apparatus. The product was identified with
1H NMR, IR, and elemental analysis. Ee was determined by
GC analysis or HPLC analysis. An absolute configuration was
assigned on the basis of the literature values of optical rotation,
unless otherwise noted.
Ack n ow led gm en t. This work was supported by
Nagase Science and Technology Foundation and by a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Science, Sports, and Culture of J apan.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectral,
IR spectral, and elemental analysis data of chiral alcohols (1b-
23b, 25b, 27b, 28b, 30b, 32b-39b) (8 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.
Det er m in a t ion of Ab solu t e Con figu r a t ion s. (S)-1-
P h en yleth a n ol (1b) fr om Ch ir a l (m -Br om op h en yl)eth -
a n ol (9b) Obta in ed by AP G4 Red u ction of m -Br om o-
a cetop h en on e (9a ). Chiral m-(bromophenyl)ethanol (9b)
(137 mg, 0.68 mmol) obtained by APG4 reduction of m-
bromoacetophenone (9a ), ethanol (13 mL), sodium hydroxide
J O9812779