Stereoinversion of arylethanols by Geotrichum candidum

Article abstract of DOI:10.1016/S0957-4166(01)00554-7

Aromatic and aliphatic racemic alcohols were converted to the corresponding optically active alcohols in high yield with excellent enantioselectivities by the use of Geotrichum candidum IFO 5767.

Full text of DOI:10.1016/S0957-4166(01)00554-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 3147–3153
Stereoinversion of arylethanols by Geotrichum candidum
Kaoru Nakamura,a,* Mikio Fujiib and Yoshiteru Idab
aInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
bSchool of Pharmaceutical Science, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Received 9 November 2001; accepted 10 December 2001
Abstract—Aromatic and aliphatic racemic alcohols were converted to the corresponding optically active alcohols in high yield
with excellent enantioselectivities by the use of Geotrichum candidum IFO 5767. © 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
alcohols have been obtained in enantiopure form by
stereoinversion.
Homochiral secondary alcohols are increasingly recog-
nized as valuable chiral building blocks in organic
syntheses. In the synthesis of enantiomerically pure
alcohols, ketones or racemic alcohols are generally used
as starting materials. The reduction of prochiral
ketones by chemical or enzymatic methods can provide
optically active alcohols in quantitative yield with 100%
enantiomeric excess (e.e.). In contrast, the chemical
yield of enantiopure alcohols obtained by resolution of
racemic alcohols via selective oxidation or esterification
cannot exceed 50%. Lipase-catalyzed transesterification
of racemic alcohols is frequently used for the prepara-
tion of such compounds, but some problems still
remain; resolutions with excellent enantioselectivities
are rare and separation of the remaining substrate and
the product is often problematic. Therefore, the devel-
opment of a methodology to obtain enantiopure alco-
hols in high yield from the corresponding racemic
alcohols is highly desirable.
Practically, biocatalytic stereoinversion is divided into
two systems: a two-biocatalyst system11,12,15,16 and a
one-biocatalyst system.6–10,13,14,17–24 For example, two
biocatalysts, Alcaligenes bronchisepticus and Streptococ-
cus faecalis, were used for the stereoinversion of ( )-
mandelic
acid.12
A.
bronchisepticus
oxidized
(S)-mandelic acid selectively and the resulting ketone,
benzoylformate, was reduced by NADPH-dependent
benzoylformate reductase of S. faecalis to give (R)-
mandelic acid in 80% yield with >99% e.e. Stereoinver-
sion with one biocatalyst has also been reported;
(R)-3-pentyn-2-ol was obtained enantioselectively from
the corresponding racemic alcohol by Nocardia fusca.17–
19 Stereoinversion of arylethanols by Geotrichum
candidum20 and Sphingomonas pausimobilis21 have also
been reported.20,21 Plant cell cultures have also been
used as biocatalysts for the stereoinversion of racemic
alcohols.14,21–24 A cell culture of Catharanthus roseus
entrapped in calcium alginate has been employed for
the deracemization of pyridyl alcohols.14 Indeed, sev-
eral enantiopure alcohols have been prepared by
stereoinversion. However, the number of examples is
still somewhat limited and a new methodology for
enhancing the effectiveness of stereoinversion is needed.
Deracemization of racemic alcohols1–5 to the corre-
sponding enantiomerically pure alcohol permits 100%
conversion. Deracemization can be carried out either by
stereoinversion or dynamic kinetic resolution. In
stereoinversion, one enantiomer from the racemic sub-
strate is transformed into its mirror image by leaving
the other enantiomer unchanged. Stereoinversion of a
secondary alcohol catalyzed by a biocatalyst consists of
enantioselective oxidation of one enantiomer to the
corresponding ketone and sequential reduction to the
other enantiomer. Chiral diols,6–9 hydroxy acid
derivatives10–15 and aliphatic16–19 and aromatic20–23
Herein, we report the stereoinversion of 1-arylethanols
by G. candidum IFO 5767.25
2. Results and discussion
2.1. Stereoinversion of 1-phenylethanol
Firstly, we screened 18 microbes for their ability to
catalyze the stereoinversion of 1-phenylethanol 1a.
* Corresponding author. Tel./fax: 81-774-38-3201; e-mail: nakamura
@scl.kyoto-u.ac.jp
0957-4166/01/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00554-7

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K. Nakamura et al. / Tetrahedron: Asymmetry 12 (2001) 3147–3153
Microbes were cultivated in a glycerol medium as
described in the experimental section and the collected
cells (0.5 g wet weight) were added to a 0.03 mM
aqueous 1-phenylethanol solution (3 mL) and the reac-
tion mixture was shaken at 30°C for 1 day. The results
are summarized in Table 1. All microbes tested gave
high chemical yields except Geotrichum rectangulatum
and G. candidum ATCC34674. G. rectangulatum
afforded (R)-1a in 53% yield in 99% e.e., which shows
that (S)-1a was selectively oxidized to acetophenone 2a
but reduction of 2a to (R)-1a did not proceed. The
stereoinversion did not proceed with Aspergillus,
Mucor, and Trichosporon species; these microbes
afforded 1a in high yields with low enantioselectivities.
On the other hand, three microbes, Dipoduscus mag-
nusii IFO 4600, Endomyces geotrichum IFO 9541, and
G. candidum IFO 5767 gave (R)-1a in high yields with
high enantioselectivities.
(R)-1a. After 24 h, almost all of the (S)-1a presented at
the beginning was converted to (R)-1a, and the e.e.
reached 99%.
Although a detailed mechanism for the microbial
stereoinversion is not clear at present, useful informa-
tion about the reaction has been obtained. Firstly,
reduction of 2a with G. candidum IFO 5767 afforded
(R)-1a in low selectivities (15–63% e.e.) and the amount
of (R)-1a increased with prolonged reaction times.
These facts indicate that (S)-1a was oxidized to the
ketone 2a, which was converted into a mixture of
(R)-1a and (S)-1a, but e.e. of (R)-1a could not be
oxidized under the reaction conditions. Thus, one possi-
ble mechanism of stereoinversion is that oxidoreduction
between (S)-1a and 2a is reversible and the reduction of
2a is irreversible (Scheme 1).
Further information about the mechanism was
obtained from NMR experiments. When phenyl-
ethanol-1-d was used as the substrate, the deuterium
content in racemic 1a-1-d decreased to 50% after 24 h
of reaction. This reveals that the methine hydrogen of
(R)-1a produced by stereoinversion is not derived from
the hydrogen of (S)-1a. Thus, it can be reasonably
stated that at least two enzymes participate, one that
Among the microbes, G. candidum IFO 5767 gave the
best result (96% yield and 99% e.e.), and recovery of the
substrates 1a and 2a was quantitative. This indicated
that (S)-1a was not decomposed but isomerized to
(R)-1a. Fig. 1 shows the time course of stereoinversion
of 1a by G. candidum IFO 5767. Initially, (S)-1a was
oxidized to 2a, which was subsequently reduced to
Table 1. Screening of microbes for stereoinversion of 1a
Microbe
1-Phenylethanol
Yield (%)a
E.e. (%)a
Config.a
Aspergillus oryzae
Aspergillus niger
Aspergillus niger
Aspergillus niger
Aspergillus niger
Dipoduscas magnusii
Endomyces geotrichum
Galactomyces reessii
Geotrichum candidum
Geotrichum candidum
Geotrichum candidum
Geotrichum rectangulatumb
Mucor heimalis
IAM 2663
IAM 6661
IFO 9455
IFO 5374
IFO 4091
IFO 4600
IFO 9541
IFO 1112
IFO 4597
IFO 5767
ATCC 34674
100
100
100
100
100
78
98
90
88
96
66
53
96
97
97
99
97
97
0
0
0
0
0
90
73
13
17
97
98
99
2
R
R
R
R
R
R
R
R
R
R
R
R
R
IAM 6095
IAM 6087
IAM 6101
IAM 4324
IAM 12206
IAM 12246
Mucor Ja6anicus
Mucor Ja6anicus
Trichosporon capitatum
Trichosporon cutaneum
Trichosporon cutaneum
3
2
30
2
2
Reaction conditions: 24 h at 30°C at 130 rpm in water (3 mL); microbe (0.5 g wet wt.), 1a (0.03 M).
a Determined by GC analysis.
b 7% of phenol, which was produced by Baeyer–Villiger oxidation of acetophenone and subsequent hydrolysis, was detected.

K. Nakamura et al. / Tetrahedron: Asymmetry 12 (2001) 3147–3153
3149
Figure 1. Time course of stereoinversion of 1a. Reaction conditions: at 30°C/130 rpm; cells (0.5 g wet wt.), 1a (0.08 mmol), water
(3 mL)
OH
O
OH
G. candidum
G. candidum
(S)-1a
(R)-1a
2a
Scheme 1.
oxidizes (S)-1a and the other that reduces 2a into
(R)-1a, and these enzymes may use different coen-
zymes. The possibility of the participation of flavin
coenzyme is also conceivable. The former situation has
been reported in the stereoinversion of pentane diol,
where the first enzyme used NADH and the second
NADPH as coenzymes.6 Recently, we have found that
the reduction of methyl ketones by G. candidum IFO
4597 was conducted by two enzymes which exhibit
different stereoselectivities.26 One enzyme afforded (S)-
alcohols and the other gave (R)-alcohols. Since the
former enzyme could oxidize secondary alcohols but
the latter could not, we propose that the similar enzy-
matic system works in the stereoinversion by G. can-
didum IFO 5767. The ineffectiveness of G. candidum
IFO 4597 toward stereoinversion is thought to be due
to low activity of the (R)-producing enzyme in the
microbe.
2.3. Improvement of stereoinversion
To improve the effectiveness of stereoinversion by G.
candidum, three strategies are considered with respect to
the mechanism in Scheme 2; namely, inhibition of the
reduction of 2a back to (S)-1a, enhancement of reduc-
tion of 2a to (R)-1a, and activation of oxidation of
(S)-1a to 2a, respectively.
To inhibit the reduction of 2a to (S)-1a, several
inhibitors, such as methyl vinyl ketone, allyl alcohol,
and ethyl chloroacetate were used as additives.
Table 2. Stereoinversion of secondary alcohols by G. can-
didum IFO5767
Substrate
Yield (%)
E.e. (%)
Config.
1-(o-Methylphenyl)ethanol 1b
1-(m-Methylphenyl)ethanol 1c
1-(p-Methylphenyl)ethanol 1d
1-(o-Chlorophenyl)ethanol 1e
1-(m-Chlorophenyl)ethanol 1f
1-(p-Chlorophenyl)ethanol 1g
1-(p-Methoxypheny)ethanol 1h
1-(2%-Furyl)ethanol 1i
98
90
95
100
89
96
79
65
96
95
88
2
19
77
0
14
89
89
92
25
14
74
R
R
R
–
R
R
R
R
R
R
R
2.2. Stereoinversion of secondary alcohols
Several aryl alcohols were subjected to the microbial
reaction. The results are summarized in Table 2. para-
Substituted 1-phenylethanols 1d, 1g, 1h gave the corre-
sponding (R)-alcohols with high e.e. (>77%) and high
yields, but stereoinversion of meta- and ortho-substi-
tuted phenylethanols were less effective. Increasing the
length of the alkyl moiety of the phenylalkanol
decreased the reactivity and as such, the phenyl-
propanols 3a and 3b and phenylbutanol 3c were recov-
ered from the reactions in high yield.
1-Phenylpropan-1-ol 3a
1-Phenylpropan-2-ol 3b
4-Phenylbutan-2-ol 3c
Reaction conditions: 24 h at 30°C at 130 rpm in water (3 mL);
microbe (0.5 g wet wt.), and substrate (0.03 M).

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K. Nakamura et al. / Tetrahedron: Asymmetry 12 (2001) 3147–3153
O
HO D
D OH
HO D
HO H
G. candidum
(R)-1a-1d
(S)-1d-1d
50%
(R)-1a-1d
(R)-1a
2a
50%
50%
Scheme 2.
Recently, we have developed several methods for con-
trolling stereoselectivities of microbial reduction and
have found that the use of selective inhibitors is effec-
tive for controlling the stereoselectivities of microbial
reductions. For example, in the yeast reduction of
b-keto esters, addition of allyl alcohol27,28 and methyl
to oxidize the coenzyme NAD(P)H to NAD(P)+ and
thus allow subsequent acceleration of the oxidation of
the (S)-alcohol.
2.4. Stereoinversion of various secondary alcohols in
preparative scale
vinyl ketone28 afforded
D-hydroxy esters selectively. On
the other hand, the presence of ethyl chloroacetate27,30
and magnesium ions31 in the same biocatalytic system
Various chiral secondary alcohols were prepared via
stereoinversion of the corresponding racemates with G.
candidum IFO5767 under aerobic conditions. As shown
in Table 3, most para-substituted phenylethanols 1a–1l,
phenylpropanols 3a,3b, and pyridylethanols 1m, 1n, 1o
gave high e.e. (>95% e.e.) with high chemical yields.
The exception was p-methoxyphenylethanol 1h, which
gave relatively low yields. The reaction intermediate,
p-methoxyacetophenone, may not resist reduction
because of the strong electron-donating nature of its
methoxy moiety. Since the oxidation of (S)-1h was fast,
(S)-1h in the starting racemic 1h was consumed within
6 h and the e.e. of the remaining (R)-1h reached 100%.
Chiral pyridylethanols were obtained in high yields
with excellent e.e. Diol also reacted smoothly. Thus,
phenyl-1,2-ethanediol 3f exhibited excellent e.e. and
chemical yield. Aromatic ketones as well as hydroxy
esters 3g, 3h were reacted using this method, resulting
in chiral hydroxy esters with high enantioselectivities
(Scheme 3).
afforded
L-hydroxy esters selectively. When ethyl
chloroacetate was used as an additive in the stereo-
inversion, the e.e. of 1a decreased (97% e.e. was
observed without additive and 29% e.e. in the presence
of ethyl chloroacetate). In contrast, the e.e. of 1a
increased and its yield reduced by the addition of
methyl vinyl ketone (98% e.e., 54% yield) and allyl
alcohol (100% e.e., 91% yield). This is because ethyl
chloroacetate inhibits the (R)-alcohol-producing
enzyme and methyl vinyl ketone and allyl alcohol
inhibits the (S)-alcohol-producing enzyme.
To enhance selective reduction to the (R)-alcohol, sec-
ondary alcohols and sugar derivatives, such as glycerol
and glucose, were used as additives.29,32,33 However,
these additives unfortunately led to a decreased e.e. of
1a.
Finally, enhancement of the oxidation of (S)-1a to 2a
was attempted. Cyclohexanone was used since it is
known to increase the oxidation rate of (S)-alcohol by
G. candidum,34,35 however, this additive was not effec-
tive and the e.e. of (R)-1a was unsatisfactory (48% e.e.).
In addition, anaerobic conditions were used for the
stereoinversion. This is because anaerobic conditions
(for example, an argon atmosphere) are known to shift
the stereoselectivity of the reduction of ketones by G.
candidum toward the production of (S)-alcohols; the
stereoselectivity of the reduction under an aerobic con-
dition seems to facilitate the production of (R)-alco-
hols.36 In the experiment described above, a standard
test tube was used as the reaction vessel and the reac-
tion was conducted by shaking the test tube. A Sak-
aguchi flask is often used for cultivation of microbes
and is known to supply oxygen in the medium effec-
tively, which increases the growth rate of aerobic
microbes. Surprisingly, stereoinversion of 1a under aer-
obic conditions gave the best result; (S)-1a was
obtained in 96% yield with 99% e.e. A further increase
in the oxygen level inhibited the reaction and the reac-
tion under a pure oxygen atmosphere did not proceed.
We think that a suitable level of oxidation is required
3. Summary
Stereoinversion of racemic secondary alcohols by G.
candidum IFO 5767 was accomplished under aerobic
conditions, and broader substrate specificities were
observed. The racemic alcohols were converted to the
corresponding (R)-alcohols in excellent e.e. and chemi-
cal yields.
Previously, we have reported asymmetric reduction by
G. candidum IFO 4597 with the aid of a hydrophobic
polymer37 or water-absorbing polymer,35 which
afforded the corresponding (S)-alcohols with excellent
e.e. The acetone powder of G. candidum IFO 4597 also
afforded (S)-alcohols by the reduction of methyl or
trifluoromethyl ketones.26,32 Thus, both (S)- and (R)-
enantiomers, could be obtained with excellent enan-
tioselectivities and high yields by using G. candidum
species. Since the dehydrogenase from G. candidum IFO
5767 is active in an organic solvent35 and even in
supercritical carbon dioxide,38 and affords optically

K. Nakamura et al. / Tetrahedron: Asymmetry 12 (2001) 3147–3153
3151
Table 3. Stereoinversion of secondary alcohols by G. candidum IFO5767 under aerobic conditions
Substrate
Yield (isolated)c (%)
E.e. (%)
Config.
1-Phenylethanol 1a
96 (83)
99 (49)
99 (67)
94 (84)
93 (82)
65 (56)
95 (85)
96 (58)
98 (79)
99 (82)
99 (61)
96 (53)
100 (90)
100 (63)
87 (25)
99 (80)
65 (59)
100 (99)
80 (49)b
100 (69)
65 (26)
99 (48)
99
78
96
92
98
100
100
100
95
100
95
98
95
100
100
100
99
97
96
100
97
99
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
1-(m-Methylphenyl)ethanol 1c
1-(p-Methylphenyl)ethanol 1d
1-(p-Chlorophenyl)ethanol 1g
1-(p-Chlorophenyl)ethanola 1g
1-(p-Methoxyphenyl)ethanola 1h
1-(p-Fluorophenyl)ethanol 1j
1-(p-Fluorophenyl)ethanola 1j
1-(p-Bromophenyl)ethanol 1k
1-(p-Bromophenyl)ethanola 1k
1-(p-Trifluoromethylphenyl)ethanol 1l
1-(2%-Pyridyl)ethanola 1m
1-(3%-Pyridyl)ethanola 1n
1-(4%-Pyridyl)ethanol 1o
1-Phenylpropan-1-ola 3a
1-Phenylpropan-2-ola 3b
4-Phenylbutan-2-ola 3c
4-Phenoxypropan-2-ola 3d
1-Phenylthiopropan-2-ola 3e
Phenylethan-1,2-diol 3f
Methyl 3-hydroxybutanoate 3g
Methyl 3-hydroxypentanoate 3h
R
R
Conditions: substrate (600 mg) and G. candidum (30 g wet wt.) dissolved in water (180 mL).
a Substrate (200 mg) and G. candidum (20 g wet wt.) dissolved in water (100 mL).
b 28% of 1-phenylsulfenylpropan-2-ol with 0% d.e. which was oxidized on silica gel was obtained. The 1H NMR spectral data are in accord with
those of the authentic samples. 1a.42
c From 1a (600 mg, 4.92 mmol) to (R)-1a (499 mg, 4.09 mmol); [h]D +53.5 (c=1.13, CHCl3), (lit.)20 [h]D +58.7 (c=1.03, CHCl3), >99% e.e. (R).
1c: From 1c (623 mg, 4.58 mmol) to (R)-1c (303 mg, 2.22 mmol); [h]D +33.0 (c=1.00, ethanol), (lit.)39 [h]D +40.4 (c=0.53, ethanol), >99.9%
e.e. (R). 1d: From 1d (613 mg, 4.50 mmol) to (R)-1d (411 mg, 302 mmol); [h]D +51.5 (c=1.00, CHCl3), (lit.)39 [h]D +51.6 (c=1.0, CHCl3), 93.8%
e.e. (R). 1g: From 1g (603 mg, 3.87 mmol) to (R)-1g (500 mg, 321 mmol), conditions (a) from 1g (208 mg, 1.33 mmol) to (R)-1g (175 mg, 1.12
mmol); [h]D +44.8 (c=1.00, ether), (lit.)39 [h]D +51.3 (c=0.595, ether), >99.9% e.e. (R). 1h: From 1h (223 mg, 1.47 mmol) to (R)-1h (124 mg,
0.82 mmol); [h]D +51.2 (c=1.02, MeOH), (lit.)32 [h]D −51.9 (c=0.718, MeOH), >99% e.e. (S). 1j: From 1j (600 mg, 4.29 mmol) to (R)-1j (517
mg, 3.69 mmol), conditions (a) from 1j (205 mg, 1.46 mmol) to (R)-1j (120 mg, 0.86 mmol); [h]D +37.5 (c=1.01, MeOH), (lit.)32 [h]D −37.7
(c=0.931, MeOH), >99% e.e. (S). 1k: From 1k (610 mg, 3.03 mmol) to (R)-1k (487 mg, 2.42 mmol), conditions (a) from 1k (212 mg, 1.05 mmol)
to (R)-1k (177 mg, 0.88 mmol); [h]D +39.6 (c=1.24, CHCl3), (lit.)32 [h]D −37.9 (c=1.13, CHCl3), >99% e.e. (S). 1l: From 1l (607 mg, 3.19 mmol)
to (R)-1l (371 mg, 1.95 mmol); [h]D +28.7 (c=1.13, MeOH), (lit.)32 [h]D −28.4 (c=1.26, MeOH), >99% e.e. (S). 1m: From 1m (203 mg, 1.65
mmol) to (R)-1m (108 mg, 0.88 mmol); [h]D +24.5 (c=0.31, CHCl3), (lit.)40 [h]D +25.6 (c=1.27, CHCl3), >95% e.e. (R). 1n: From 1n (202 mg,
1.64 mmol) to (R)-1n (182 mg, 1.48 mmol); [h]D +51.5 (c=1.00, CHCl3), (lit.)40 [h]D +52.4 (c=1.21, CHCl3), >95% e.e. (R). 1o: From 1o (602
mg, 4.89 mmol) to (R)-1o (378 mg, 3.07 mmol); [h]D +44.9 (c=0.94, MeOH), (lit.)40 [h]D +42.5 (c=1.04, MeOH), >95% e.e. (R). 3a: From 3a
(204 mg, 1.50 mmol) to (R)-3a (51 mg, 0.38 mmol); [h]D −47.0 (c=1.00, CHCl3), (lit.)39 [h]D +39.7 (c=0.0409, CHCl3), 95% e.e. (S). 3b: from
3b (240 mg, 1.76 mmol) to (R)-3b (193 mg, 1.42 mmol); [h]D −38.7 (c=1.11, CHCl3), (lit.)39 [h]D +39.7 (c=0.515, CHCl3), >99.9% e.e. (S). 3c:
From 3c (230 mg, 3.03 mmol) (R)-3c (137 mg, 0.912 mmol); [h]D −21.1 (c=1.00, C6H6), (lit.)39 [h]D +22 (c=0.38, C6H6), >99.9% e.e. (S). 3d:
From 3d (244 mg, 1.57 mmol) to (R)-3d (242 mg, 1.57 mmol); [h]D +2.8 (c=0.58, EtOH), (lit.)41 [h]D −2.7 (c=1.80, EtOH), >99% e.e. (S). 3e:
From 3e (197 mg, 1.17 mmol) to (R)-3e (96 mg, 0.60 mmol); [h]D −8.8 (c=1.02, MeOH), (lit.)41 [h]D +8.20 (c=0.59, MeOH), 94% e.e. (S). 3f:
From 3f (600 mg, 4.35 mmol) to (S)-3f (411 mg, 2.99 mmol); [h]D +38.4 (c=1.31, ethanol) (lit.)6 [h]D +38.6 (c=1, ethanol), 99.9% e.e. (S). 3g:
From 3g (600 mg, 5.08 mmol) to (R)-3g (156 mg, 1.23 mmol); [h]D −41.9 (c=1.09, CHCl3), (lit.)32 [h]D +40.6 (c=1.09, CHCl3), >99% e.e. (S).
3h: From 3h (601 mg, 4.55 mmol) to (R)-3h (290 mg, 2.20 mmol); [h]D −34.8 (c=1.36, CHCl3), (lit.)32 [h]D +34.3 (c=1.05, CHCl3), >99% e.e.
(S).
active alcohols with excellent enantioselectivities, G.
candidum is highly recommended as a biocatalyst for
the synthesis of optically active secondary alcohols.
5. Experimental
The gas chromatographic analysis was performed using
a Shimadzu GC-14A gas chromatograph equipped with
a Shimadzu CR-6A Chromatopac equipped with 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
4. Conclusions
A range of chiral aromatic and aliphatic alcohols has
been produced from racemic mixtures by stereoinver-
sion using G. candidum IFO 5767. Further studies,
including the isolation of enzymes, are underway in our
laboratory.
1
mL/min (DEX-CB)). H NMR spectra were recorded
on a Varian VXR-200 spectrometer in CDCl3. Optical
rotation was measured with a Perkin–Elmer 241
polarimeter.

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K. Nakamura et al. / Tetrahedron: Asymmetry 12 (2001) 3147–3153
G. candidum
OH
R2
OH
R2
(R)-1,3
R1
R1
1,3
1; R1 = Ar, R2 = Me
1a-1h; Ar
1a; R = H, 1b; R = o-Me, 1c; R = m-Me, 1d; R = p-Me,
1e; R = o-Cl, 1f; R = m-Cl, 1g; R = p-Cl, 1h; R = p-MeO
=
R
1i; Ar = 2'-furyl, 1j; R = p-F, 1k; R = p-Br, 1l; R =p-CF3, 1m; Ar = 2'-pyridyl, 1n; Ar =
3'-pyridyl,1o; Ar = 4'-pyridyl,
OH
OH
OH
OH
OH
O
3a
3b
3c
3d
OH
OH
OH
S
OH
MeO2C
3g
MeO2C
3e
3f
3h
Scheme 3.
5.1. Materials
standard. Results are summarized in Tables 1 and 2.
GC conditions for determination of e.e. of alcohols
(column, retention time); 1a: CPCD 100°C, S 15.8 min,
R 14.6 min. 1b: CPCD 130°C, S 7.8 min, R 7.1 min. 1c:
CPD 120°C, S 13.7 min, R 13.3 min. 1d: CPCD 115°C,
S 12.1 min, R 11.2 min. 1e: CPCD 130°C, S 13.9 min,
R 13.1 min. 1f: CPCD 130°C, S 13.9 min, R 13.0 min.
1g: CPCD 130°C, S 14.3 min, R 13.3 min. 1h: CPCD,
130°C, S 17.5 min, R 16.8 min. 1j: DEX-CB 120°C, S
11.4 min, R 10.3 min. 1k: DEX-CB 150°C, S 12.7 min,
R 11.6 min. 1l: DEX-CB 120°C, S 16.3 min, R 13.5
min. 1m-Acetate: DEX-CB 100°C, S 6.9 min, R 6.3
min. 1n: DEX-CB 130°C, S 13.4 min, R 12.6 min. 1o:
DEX-CB 105°C, S 9.8 min, R 9.6 min. 3a: DEX-CB
120°C, S 14.2 min, R 13.7 min. 3b-Acetate: CPCD
105°C, S 15.8 min, R 16.8 min. 3c: G-TA 105°C, S 21.4
min, R 22.2 min. 3d-Acetate: CPCD 130°C, S 13.9 min,
R 14.4 min. 3e: G-TA 130°C, S 21.1 min, R 21.9 min.
3f-Diacetate: G-TA 115°C, S 56.1 min, R 58.3 min. 3g:
G-TA 85°C, S 11.9 min, R 13.1 min. 3h: DEX-CB
90°C, S 8.4 min, R 8.8 min.
Organic reagents were purchased from Nacalai Tesque,
Inc., Tokyo Chemical Industry Co., Ltd, Kanto Chem-
ical, Inc. or Wako Pure Chemical Industries, Ltd.
unless otherwise indicated. Racemic alcohols were
obtained by the reaction of the corresponding alde-
hydes with excess methylmagnesium bromide or by
reduction of the corresponding ketones with sodium
borohydride. 1-Phenylethanol-1-d was obtained by
reduction of acetophenone with lithium aluminium
duteride.
5.2. Cultivation of microbe
The microbe was grown in a complex medium consist-
ing of 3% (wt./vol.) glycerol, 1% (wt./vol.) yeast extract,
and 0.5% (wt./vol.) polypeptone. The medium, adjusted
to pH 6.2 using 0.1 M potassium phosphate buffer, was
placed in a Sakaguchi flask, sterilized (121°C, 20 min)
and inoculated with the preincubated culture. The culti-
vation was carried out for 24 h at 30°C with shaking.
5.4. Stereoinversion of 1-phenylethanol in the presence of
additives
5.3. General procedure of stereoinversion on small scales
The cells (0.5 g wet wt.), additive (0.5 mmol) and 1a
(0.08 mmol) were put into a 50 mL test tube equipped
with screw cap and containing water (3 mL), and
shaken at 130 rpm for 1 day at 30°C. The reaction
mixture was put on Extrelut (Merck) and eluted with
ether. The e.e. and chemical yield of the 1-
phenylethanol was determined by GC analysis of the
ether solution.
The cells (0.5 g wet wt.) and substrate (0.08 mmol) were
put into a 50 mL test tube equipped with screw cap and
containing water (3 mL) and shaken at 130 rpm for 1
day at 30°C. The reaction mixture was put on Extrelut
(Merck) and eluted with ether. The e.e. and chemical
yield of the products were determined by a GC analysis
of the ether extract using dodecane as an internal

K. Nakamura et al. / Tetrahedron: Asymmetry 12 (2001) 3147–3153
3153
5.5. Stereoinversion on preparative scale: (R)-1-
phenylethanol 1a
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Bioeng. 1995, 80, 247–250.
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3053.
17. Xie, S. X.; Ogawa, J.; Shimizu, S. Appl. Microbiol. Bio-
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Biochem. 1999, 63, 1721–1729.
20. Nakamura, K.; Inoue, Y.; Matsuda, T.; Ohno, A. Tetra-
hedron Lett. 1995, 36, 6263–6266.
21. Allan, G. R.; Carnell, A. J. J. Org. Chem. 2001, 66,
6495–6497.
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1995, 12, 2925–2928.
23. Takemoto, M.; Achiwa, K. Phytochemistry 1998, 49,
1627–1629.
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25. Previous communication: Ref. 20.
26. Matsuda, T.; Harada, T.; Nakajima, N.; Itoh, T.; Naka-
mura, K. J. Org. Chem. 2000, 65, 157–163.
27. Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. J.
Org. Chem. 1991, 56, 4778–4783.
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Chem. Lett. 1987, 679–682.
The cells (30 g wet wt.) and ( )-phenylethanol (600 mg,
4.92 mmol) were put into a Sakaguchi-flask containing
water (180 mL), and shaken at 130 rpm for 1 day at
30°C. The mixture was filtered and the filtrate and the
filtered microbe were extracted with ether separately.
The organic layers were collected, dried over anhydrous
magnesium sulfate, and concentrated in vacuo. The
residue was subjected to silica gel column chromatogra-
phy (hexane:ethyl acetate=8:1), followed by bulb-to-
bulb
distillation
(150°C/1.8
mmHg)
afforded
(R)-phenylethanol (499 mg, 4.09 mmol, 83%). The e.e.
and chemical yield of (R)-phenylethanol was deter-
mined by GC analysis (CP-Cyclodextrin-B-236-M-19,
0.25 mm×25 m, He: 2 mL/min, 100°C, retention times;
(S)-1-phenylethanol 15.8 min, (R)-1-phenylethanol 14.6
min) (96%, 99% e.e.); [h]D +53.5 (c=1.13, CHCl3),
1
(lit.)20 [h]D +58.7 (c=1.03, CHCl3), >99% e.e. (R); H
NMR (200 MHz, CDCl3) l 1.49 (d, 3H, J=6.8 Hz),
1.85 (s, 1H) 4.90 (q, 1H, J=6.8 Hz), 7.25–7.37 (5H,
J=6.4 Hz). Anal. calcd for C8H10O; C, 78.65; H, 8.25.
Found: C, 78.46; H, 8.40%.
Other alcohols were reacted with the same system, and
the results are listed in Table 3.
29. Nakamura, K.; Kawai, Y.; Oka, S.; Ohno, A. Bull. Chem.
Soc. Jpn. 1989, 62, 875–879.
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