Stereoselective oxidation and reduction by immobilized Geotrichum candidum in an organic solvent

Article abstract of DOI:10.1039/a900936a

Cells of the fungus, Geotrichwn candidum, were immobilized on a water-absorbing polymer and used for stereoselective oxidation and reduction in an organic solvent using cyclohexanone, cyclopentanol or alkan-2-ols as additive. Enantiomerically pure (.β)-1-arylethanols were obtained by the stereoselective oxidation of racemic 1-arylethanols, whereas enantiomerically pure (S)- 1-arylethanols were obtained by the reduction of the corresponding ketones, in contrast to reduction in water by the free cells in which (R)- or (S)- 1-arylethanols were produced in low ee. The reaction mechanism was investigated by measuring the partition of the substrates and products between the organic phase and aqueous phase in the polymer around which the cells were immobilized. Deuterated compounds were used to determine the role of the additives.

Full text of DOI:10.1039/a900936a

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Stereoselective oxidation and reduction by immobilized Geotrichum
candidum in an organic solvent
Kaoru Nakamura,* Yuko Inoue, Tomoko Matsuda and Ibuki Misawa
Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011 Japan
Received (in Cambridge) 3rd February 1999, Accepted 28th June 1999
Cells of the fungus, Geotrichum candidum, were immobilized on a water-absorbing polymer and used for stereo-
selective oxidation and reduction in an organic solvent using cyclohexanone, cyclopentanol or alkan-2-ols as additive.
Enantiomerically pure (R)-1-arylethanols were obtained by the stereoselective oxidation of racemic 1-arylethanols,
whereas enantiomerically pure (S)-1-arylethanols were obtained by the reduction of the corresponding ketones, in
contrast to reduction in water by the free cells in which (R)- or (S)-1-arylethanols were produced in low ee.
The reaction mechanism was investigated by measuring the partition of the substrates and products between the
organic phase and aqueous phase in the polymer around which the cells were immobilized. Deuterated compounds
were used to determine the role of the additives.
1-arylethanols, affording enantiomerically pure (R)-1-aryl-
ethanols. In the reduction of aryl methyl ketones, this system
produced (S)-1-arylethanol in excellent ee, in contrast to the
reduction in water in which the (R)- or (S)-1-arylethanol is pro-
duced in low ee. The mechanism of improvement in reactivity
and enantioselectivity by the use of the system was investigated.
Introduction
The growing interest in asymmetric synthesis has promoted a
great development in biotransformations in organic synthesis
and they have been used widely for the syntheses of chiral
compounds.1 The selectivity of reactions is usually high for
enzymatic reactions and for microbial reactions when the
microbe contains only the necessary enzyme(s). However, when
there are many enzymes in a microbe which transform a sub-
strate to products of different configurations, microbial trans-
formation does not afford a product of satisfactory optical
purity, and a new method for controlling the enantioselectivity
of microbial reactions is awaited. To date, several methods by
which to improve the selectivity of microbial reactions such as
screening of microorganisms,2 modification of substrates,3 and
modification of the reaction conditions4 have been developed.
Among these, the last is the most desirable since one can obtain
alcohols of excellent optical purity without modifying the kind
of microorganisms or substrates by controlling the activities of
desired and undesired enzymes. It would be convenient if the
reaction conditions could be modified to simplify the experi-
mental manipulation and to make the system suitable for large
scale productions, while concurrently controlling the stereo-
selectivity. For this purpose, the use of organic solvents5 and
immobilization of the microorganism are attractive approaches.
Dry or immobilized bakers’ yeast reduction in an organic
solvent has been developed. The reduction of various α-keto
esters with dry bakers’ yeast in benzene6 or immobilized yeast
in polyurethane in hexane7 affords the corresponding (R)-α-
hydroxy esters in high enantiomeric excess (ee), whereas that in
water gives the (S)-α-hydroxy esters. Similarly, β-keto esters are
reduced by dry bakers’ yeast in petroleum ether to the corre-
sponding (S)-β-hydroxy esters.8 Thus, stereochemical control
by the use of organic solvents and/or immobilization in the
bakers’ yeast reduction have been achieved.
Results and discussion
Oxidation
When racemic 1-phenylethanol (R/S)-1a was incubated with
resting free cells of G. candidum in water for 24 h, (S)-1a was
oxidized selectively to give the corresponding ketone (2a, 12%)
leaving (R)-1a of 17% ee (Table 1, entry 1). Although the oxid-
ation of (S)-1a but not (R)-1a took place, the reactivity of (S)-
1a was not strong, so that some (S)-1a remained after 24 h,
which lowered the ee of remaining (R)-1a. When the reaction
was conducted in a biphasic system of water and hexane, the
yield increased, giving (R)-1a of 43% ee (Table 1, entry 2), and
when the cell was immobilized on a water-absorbing polymer,
the oxidation in hexane proceeded more readily giving (R)-1a
of 86% ee (Table 1, entry 3). To further improve the system, an
additive was used. When cyclohexanone (2.5 equivalents with
respect to the substrate) was added, (R)-1a of 99% ee was ob-
tained (Table 1, entry 4). The ee enhancement of the remaining
alcohol is a reflection of the degree of conversion because (S)-
alcohol is selectively oxidized and when all of the (S)-alcohol is
converted to ketone, only (R)-alcohol which does not oxidize at
all remains in the reaction mixture.
An improvement in the ee of the remaining alcohol (R)-1a
was observed when hexane was employed, the cells were
immobilized, and cyclohexanone was added. The improvement
by the use of hexane is explained by the partition of the sub-
strates and products between the organic phase and aqueous
phase. The solubility of ketones in the aqueous layer is gener-
ally much lower than that of alcohols, and as shown in Scheme
1, the substrate-alcohol (R/S)-1a dissolves more easily in the
aqueous phase than ketone 2a: the local concentration of
(R/S)-1a around the cells is higher than that of 2a, which shifts
the equilibrium between the oxidation and reduction in the
direction more favorable for the oxidation of the alcohol (R/S)-
1a. (The process is reversible as for (S)-1a; the reduction of
ketones to (S)-1a is observed as detailed in the Reduction
We have found that Geotrichum candidum IFO 4597, a fungus
for which the cultivation is very simple, can reduce various
kinds of ketones with acceptable rates, although the stereo-
selectivity is not high. Here we report a new system which
improves the reactivity and stereoselectivity of G. candidum-
catalyzed transformations. The system consists of an organic
solvent, immobilized microbial cells, and additives such as
cyclohexanone, cyclopentanol and alkan-2-ols.5f–i The present
system increased the reactivity in the oxidation of racemic
J. Chem. Soc., Perkin Trans. 1, 1999, 2397–2402
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Table 1 Oxidation of 1a with G. candidum
Entry
Catalyst
Hexanea
Cyclohexanoneb
Yieldc (%)
ee of 1a (%)
1
2
3
4
Free cells in water
Free cells in water
Immobilized cellsd
Immobilized cellsd
—
ϩ
ϩ
ϩ
—
—
—
ϩ
12
29
44
51
17 (R)
43 (R)
86 (R)
99 (R)
Reaction conditions are described in the Experimental section. a 6 mL. b 0.2 mmol. c Yield of 2a. d The cell was immobilized on water–absorbing
polymer (BL-100).
Fig. 1 Picture of immobilized cells on the water-absorbing polymer.
The average diameter of the polymer after the absorption of water for
immobilization is 0.15 0.05 mm and the number of cells per polymer
particle is 50–100.
ation of other 1-arylethanols is also enhanced in the reaction
system with the immobilized cells on the water-absorbing
polymer in hexane in the presence of cyclohexanone as shown
Scheme 1
in Table 2. meta- and para-Substituted 1-arylethanols are
section.) Organic solvents more polar than hexane are in-
smoothly oxidized to leave (R)-1-arylethanol in high ee,
appropriate because the local concentration of not only 2a but
however, ortho-substituted alcohols are inert to the oxidation.
also (R/S)-1a around the cell is too low for the reaction to occur.
o,p-Dichlorophenylethanol 1j is not oxidized at all, even though
An improvement in the oxidation reactivity was also
it is para-substituted, due to the ortho-substitution. 1-Phenyl-
observed when the cells were immobilized on a water-absorbing
propan-2-ol (1k) and 4-phenylbutan-2-ol (1l) are oxidized
polymer. Immobilization was accomplished by adding the
smoothly, although 1-phenylpropan-1-ol (1m) and 1-phenyl-
water-absorbing polymer (BL-100) to a suspension of the cells
butan-1-ol (1n) are hardly oxidized, which means that the small
in water. Since the water is absorbed by the polymer instantly
substituent adjacent to the alcohol group must be a methyl.
and the cells are adsorbed on the polymer, the cells are spread
over a large polymer surface (Fig. 1), which increases the sur-
face area between the cells and hexane phase, and results in
Reduction
improvement in the reactivity.
G. candidum also catalyzes the reduction of ketones as well as
the oxidation of alcohols, although the reduction of 2a by the
free cells in water afforded only (R)-1a with 28% ee in 52% yield
(Table 3, entry 1). The reduction in hexane by the immobilized
cells barely proceeded (Table 3, entry 2), contrary to the oxid-
ation because of the unfavorable partition of 2a and 1a between
the organic and aqueous phases for the reduction. How-
ever, when an alcohol such as cyclopentanol or hexan-2-ol
was added to the reaction system, the reduction proceeded
smoothly to afford (S)-1a in excellent ee (Table 3, entry 3, 4).
Cyclopentanol or hexan-2-ol worked when they were used in
large excess. The yield increased as the amount of cyclo-
pentanol was increased and peaked at 6 mol equivalents with
respect to the substrate, whereas alkan-2-ols such as propan-2-
ol, butan-2-ol, pentan-2-ol, hexan-2-ol, heptan-2-ol and octan-
2-ol exerted saturation on the yield at 10 to 15 mol equivalents.
In addition, immobilization of the cells simplifies the workup
procedure; the reaction can be quenched easily by filtration of
the immobilized cells which is much more straightforward than
that of the free cells.
Addition of cyclohexanone also gave an improvement in the
reactivity. Among carbonyl compounds such as butyraldehyde,
isobutyraldehyde, octanol, benzaldehyde, chloroacetone, 4-
methylpentan-2-one, octan-2-one, cyclobutanone, cyclopentan-
one, cyclohexanone and 4-methylcyclohexanone tested for their
ability to promote oxidation, cyclohexanone and chloroacetone
were most effective (data not shown). The ee of the remaining
alcohol increases according to the amount of cyclohexanone
added and at least a 1.2 mol equivalent of cyclohexanone with
respect to the substrate is necessary to obtain >99% ee.
Various substrates were applied to the oxidation. The oxid-
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Table 2 Oxidation of various alcohols with the immobilized cell
Immobilized cells in hexane
with cyclohexanone
Free cells in water
Yielda (%)
Entry
Substrate
Eeb (%)
Yielda (%)
Eeb (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1-Phenylethanol (1a)
12
11
1
8
11
7
32
38
41
0
35
48
28
2
17 (R)
23 (R)
—
5 (R)
20 (R)
6 (R)
40 (R)
71 (R)
75 (R)
—
57 (R)
60 (R)
41 (R)
—
51
50
13
52
46
28
50
50
50
1
44
50
16
0
99 (R)
96 (R)
14 (R)
97 (R)
74 (R)
37 (R)
>99 (R)
97 (R)
99 (R)
—
76 (R)
97 (R)
17 (R)
—
1-(2-Furyl)ethanol (1b)
1-(o-Chlorophenyl)ethanol (1c)
1-(m-Chlorophenyl)ethanol (1d)
1-(p-Chlorophenyl)ethanol (1e)
1-(o-Methylphenyl)ethanol (1f)
1-(m-Methylphenyl)ethanol (1g)
1-(p-Methylphenyl)ethanol (1h)
1-(p-Methoxyphenyl)ethanol (1i)
1-(o,p-Dichlorophenyl)ethanol (1j)
1-Phenylpropan-2-ol (1k)
4-Phenylbutan-2-ol (1l)
1-Phenylpropan-1-ol (1m)
1-Phenylbutan-1-ol (1n)
Reaction conditions are described in the Experimental section. a Yield of ketones. b Ee of the remaining substrate. Preparative scale results: (R)-1a: ee
98%, [α]D ϩ53.0 (c 0.60, CHCl3) (lit.13 [α]D25 Ϫ57 (c 5.12, CHCl3) (S)). (R)-1k: ee 69%, [α]D Ϫ24.4 (c 0.97, CHCl3) (lit.12 [α]D14 ϩ39.7 (c 0.515, CHCl3),
>99.9% ee (S)).
Table 3 Reduction of 2a with G. candidum
Entry
Catalyst
Hexanec
Additive
Yield (%)
Ee (%)
1
2
3
4
5
Free cells in water
Immobilized cellsa
Immobilized cellsa
Immobilized cellsa
Immobilized cellsb
—
ϩ
ϩ
ϩ
ϩ
—
—
52
2
58
73
38
28 (R)
—
>99 (S)
>99 (S)
98 (S)
Cyclopentanold
Hexan-2-ole
Hexan-2-ole
Reaction conditions are described in the Experimental section. a The cells were immobilized on water absorbing polymer (BL-100). b The cells were
immobilized in calcium alginate. c 6 mL. d 0.5 mmol. e 1.0 mmol.
the yield and ee were the highest with hexane the same as for the
oxidation.
Aryl methyl ketones, 2b–2j, were also reduced smoothly by
the same system using 12 equivalents of hexan-2-ol to give the
corresponding (S)-alcohols in excellent ee (Table 4). The reduc-
tion of ortho-substituted acetophenones gives relatively better
yields than that of para-substituted acetophenones probably
because the reverse reaction, oxidation, does not proceed for
the ortho-substituted substrates. The better a substrate is for the
stereoselective oxidation, the worse it is for the reduction as for
the substituted acetophenone derivatives. However, a different
trend was observed for other substrates. Changing the phenyl
group in 2a to a benzyl (2k) or 2-phenylethyl (2l) group does not
interfere with the reduction, whereas the reaction is inhibited by
substitution of the methyl group in 1a with an ethyl or propyl
group (Table 4, entries 11–14); which is the same trend as for the
oxidation. A methyl group but not an ethyl or a propyl group is
suitable as the smaller group adjacent to the keto group for
both the reduction and the oxidation.
In the reduction of 2a by immobilized cells in hexane, alkan-
2-ols such as hexan-2-ol and octan-2-ol etc. and cyclopentanol
are effective additives. To clarify which stereoisomers of
alkan-2-ol to use as a reductant, optically active octan-2-ol was
prepared and used as an additive. As shown in Table 5, (S)-
octan-2-ol was more effective than the (R)-isomer. Although
the effect of (R)-octan-2-ol as an additive was small, addition
of (R)-octan-2-ol undoubtedly increased the yield up to
about 9% from 1%. To investigate the role of (R)-octan-2-ol, the
Fig. 2 Effect of immobilization on the reduction rate of 2a; ᭜ with
immobilization of the cells, ᮀ without immobilization of the cells.
The present immobilization method is more effective than
methods employing calcium alginate,9 a commonly used
immobilizing material, the reduction of 2a by the immobilized
cell in calcium alginate proceeded with low yield (38%) and 98%
ee (Table 3, entry 5).
Fig. 2 shows the time-course of the reduction in hexane.
Obviously, the reduction by the immobilized cells proceeded
more rapidly than that without immobilization. The reduction
with the immobilized cells is complete within 3 h. The initial
rate of reduction with the immobilized cells is calculated to be
28 mM hϪ1 per 1 g of cells.
Organic solvents other than hexane were also tested for in the
reduction by the immobilized cells with cyclopentanol and
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Table 4 Reduction of various ketones with immobilized cells
Immobilized cells in hexane
with hexan-2-ol
Free cells in water
Yield (%)
Entry
Substrate
Ee (%)
Yield (%)
Ee (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Acetophenone (2a)
52
6
46
22
49
4
50
10
14
37
47
31
14
7
28 (R)
1 (R)
>99 (R)
90 (R)
46 (R)
—
93 (R)
19 (R)
46 (R)
>99 (S)
88 (S)
60 (S)
19 (S)
>99
73
81
99
88
41
59
56
40
12
83
97
88
2
>99 (S)
99 (S)
99 (S)
>99 (S)
92 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
—
2-Acetylfuran (2b)
o-Chloroacetophenone (2c)
m-Chloroacetophenone (2d)
p-Chloroacetophenone (2e)
o-Methylacetophenone (2f)
m-Methylacetophenone (2g)
p-Methylacetophenone (2h)
p-Methoxyacetophenone (2i)
o,p-Dichloroacetophenone (2j)
1-Phenylpropan-2-one (2k)
Benzylacetone (2l)
Propiophenone (2m)
Butyrophenone (2n)
<1
—
The absolute configuration of the products was determined by comparison of the sign of the optical rotation with that reported previously or by
comparison of the GC retention times with those of authentic samples. (S)-1b: Isolated yield 63%, ee >99%, [α]D Ϫ54.7 (c 1.05, CHCl3) (lit.13 [α]D25 Ϫ57
(c 5.12, CHCl3), (S)). 1b: Product decomposed during purification. (S)- 1c: Isolated yield 88%, ee >99%, [α]D Ϫ62.8 (c 1.07, CHCl3) (lit.10 [α]D Ϫ56.5
(c 0.0463, CHCl3) 90% ee (S)). (S)-1d: Isolated yield 75%, ee >99%, [α]D Ϫ40.5 (c 1.19, CHCl3) (lit.11 [α]D20 ϩ36.7 (c 1.0, CHCl3) 84.6% ee (R)). (S)-1e:
Isolated yield 32%, ee 92%, [α]D Ϫ65.7 (c 1.15, CHCl3). (S)-1f: Isolated yield 25%, ee >99%, [α]D Ϫ58.9 (c 1.01, EtOH) (lit.10 [α]D Ϫ58.6 (c 0.0665,
EtOH) 95% ee (S)). (S)-1g: Isolated yield 43%, ee 99% [α]D Ϫ39.4 (c 1.05, EtOH) (lit.12 [α]D14 Ϫ41.9 (c 0.500, EtOH), >99.9%, ee (S)). (S)-1h: Isolated
yield 21%, ee 99%, [α]D Ϫ54.4 (c 1.02, CHCl3) (lit.14 [α]D ϩ39 (c 1, CHCl3), 76% ee (R)). (S)-1i: Isolated yield 10%, ee >99%, [α]D Ϫ23.5 (c 1.00,
CHCl3) (lit.10 [α]D Ϫ46.2 (c 0.0273, CHCl3) 87% ee (S)). (S)-1j: Isolated yield 71%, ee >99%, [α]D Ϫ58.7 (c 1.03, CHCl3); absolute configuration was
determined after dehalogenation of the product to 1a, [α]D Ϫ48.7 (c 1.13, CHCl3). (S)-1k: Isolated yield 65%, ee >99%, [α]D ϩ42.6 (c 1.01, CHCl3)
14
(lit.12 [α] ϩ39.7 (c 0.515, CHCl3), >99.9% ee (S)). (S)-1l: Isolated yield 71%, ee 98%, [α]D ϩ17.4 (c 1.80, CHCl3).
D’
Table 5 Effect of chirality of additives on the reduction of 2a with
immobilized cells in hexane
Table 6 Effect of deuterium content in 1a on the reduction of 2a with
immobilized cells in hexane
Additive
Yield (%)
Ee (%)
Additive
D in water (%)d
D in 1a (%)e
None
1
46
58
9
10
8
—
rac-[2-2H]Octan-2-ola
(S)-[2-2H]Octan-2-olb
(R)-[2-2H]Octan-2-olc
rac-Octan-2-ola
0
0
0
33
66
99
99
40
36
0
16
24
45
rac-Octan-2-ola
(S)-Octan-2-olb
(R)-Octan-2-olc
Tetrahydrofurand
1,2-Dimethoxyethanee
>99 (S)
>99 (S)
89 (S)
>99 (S)
>99 (S)
rac-Octan-2-ola
rac-Octan-2-ola
rac-Octan-2-ola
>99
a 0.4 mmol b 98% ee, 0.4 mmol 99.98% ee, 0.4 mmol. d 1.23 mmol.
c
e 0.96 mmol.
a 0.4 mmol. b 92% ee, 0.2 mmol. c >99% ee, 0.2 mmol. d Deuterium
content in the water used for immobilization of the cells. e Deuterium
content in 1a.
effect of other organic substances (tetrahydrofuran and 1,2-
dimethoxyethane) on reduction of 2a was studied. When these
additives were used, the yield of 1a increased to about 10%,
regardless of the kind and the amount, which indicates that the
yield of 1a increases as a result of the inhibition of the oxid-
ation of product, 1a. When (R)-octan-2-ol or these organic
compounds were used for the oxidation of 1a, they also
inhibited the oxidation (data not shown).
Further experiments clarified the function of the additives as
a reductant. Deuterated additives, [2-2H]octan-2-ol, and/or
deuterated water were employed for the reduction of 2a with
the immobilized cells to search for the source of the hydrogen
transferred to C1-H of 1-phenylethanol (S)-1a. As shown in
Table 6, the deuterium at CD-OH in the additive alcohol was
transferred onto the product except for the case of (R)-[2-2H]-
octan-2-ol.
Since the product was only partially deuterated in spite of
starting with completely deuterated additives, other sources
of the hydrogen in the C1 position were investigated. When
deuterated water was used in the immobilization step, the
C1 position of the product was deuterated. According to the
deuterium content of the water, the deuterium content of the
product was increased (Table 6). The hydrogen at CH-OH of
the product comes either from the additive (octan-2-ol) or
the solvent (water) since when both deuterated additive and
deuterated water were used for the reduction, the deuterium
content in the product at CH-OH was >99%. This suggests that
there are more than two routes for the reduction; (1) the
additive such as octan-2-ol reduces the NAD(P)ϩ which reduces
the main substrate directly, (2) initially the additive such as
octan-2-ol reduces the NAD(P)ϩ, then the NAD(P)H reduces
other cofactors like flavin, followed by exchange of the hydro-
gen on flavin etc. with one from water, and finally the reduction
of the main substrate occurs. To investigate the reaction
mechanisms further the enzymes reducing the substrates need
to be isolated.
Conclusion
Geotrichum candidum was immobilized on a water-absorbing
polymer and used for stereoselective oxidation and reduction in
hexane using cyclohexanone, cyclopentanol, and alkan-2-ols
as additives. Enantiomerically pure (R)-1-arylethanols were
obtained by the oxidation of racemic 1-arylethanols, whereas
enantiomerically pure (S)-1-arylethanols were obtained by the
reduction of the corresponding ketones. Using only (S)-
selective enzyme(s), the stereoselective synthesis of both enan-
tiomers with excellent ee was achieved because alcohol dehy-
drogenase(s) catalyze both oxidation and reduction. This sys-
tem is suitable for large scale synthesis because expensive
coenzymes are not necessary, and the use of the organic solvent
simplifies the work-up procedure.
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The system utilizing immobilized cells and hexane favors the
oxidation over the reduction reaction due to the partitioning of
the ketones and alcohols between the organic and aqueous
phases. However, by choosing the right additives the reactivity
can be controlled in both directions; cyclohexanone is suitable
for the oxidation of the main substrate, whereas alkan-2-ols
and cyclopentanol are suitable for the reduction. To elucidate
the role of the additive, deuterated alkan-2-ol was employed for
the reduction of the substrate and it was clarified that alkan-2-
ols act as a hydride source for the reduction.
Reduction of 2a–2n by the free cells in water
2a–2n was reduced by the free cells as described previously.4
Reduction of 2a–2n by the immobilized cells in hexane at GC
scale
To a suspension of the immobilized cells on BL-100 (4.0 g) in
hexane (6 mL), 2a–2n (0.08 mmol), hexan-2-ol (1.0 mmol) and
dodecane as an internal standard for GC analysis were added.
The resulting suspension was shaken at 130 rpm at 30 ЊC for
24 h. The organic layer was subjected to GC analysis to deter-
mine the yield and ee.
Experimental
General
Reduction of ketones 2a–2l by the immobilized cells in hexane at
1H NMR spectra were recorded at 200 MHz on a Varian VXR-
200 spectrometer in CDCl3. Capillary gas chromatograms were
recorded on a Shimadzu GC-9A gas chromatograph with
Shimadzu C-R6A Chromatopac. GC-MS spectra were
obtained with a JEOL JMS-DX300 spectrometer and analyzed
with a JMA-3500 spectrometer. Optical rotations were meas-
ured with a JASCO DIP-181 digital polarimeter and are given
preparative scale
To a suspension of the immobilized cells (80 g) in hexane (120
mL), 2a–2l (1.7 mmol) and hexan-2-ol (20 mmol) were added.
The resulting suspension was shaken at 130 rpm at 30 ЊC for
24 h. The suspension was filtered and the immobilized cells were
washed with ether. The combined organic layer was washed
with brine, dried over anhydrous magnesium sulfate and the
solvent was evaporated under reduced pressure. Hexan-2-ol was
removed from the residual oil under reduced pressure (60 ЊC/25
mmHg, (R)-hexan-2-ol exerted [α]D Ϫ1.72 (c 9.8, Et2O)). The
product was purified by silica gel column chromatography
(ethyl acetate–hexane, 1:5) and by distillation with a Kugelrohr
apparatus. The spectral data are in accord with those reported
previously.4
in units of 10Ϫ1 deg cm2 gϪ1
.
Commercially available reagents were purchased from
Nacalai Tesque Co., Ltd., Tokyo Kasei Co., Ltd. and Aldrich
Chemical Co., Ltd. unless otherwise indicated. Solvents and
purchased reagents were generally used without additional
purification unless otherwise indicated. BL-100 (water-
absorbing polymer) was kindly supplied by Osaka Yuki
Kagaku Kogyo Co., Ltd. Geotrichum candidum IFO 4597 was
cultivated as described previously.4
Determination of absolute configuration of (؊)-1-(2Ј,4Ј-
dichlorophenyl)ethanol; dehalogenation of (؊)-1j
Immobilization of the cells on the water-absorbing polymer,
BL-100
(Ϫ)-1-(2Ј,4Ј-Dichlorophenyl)ethanol obtained from the reduc-
tion of 2j by the immobilized cells was converted into 1-phenyl-
ethanol (1a) to compare the sign of optical rotation with that
reported in the literature.13 A mixture of (Ϫ)-1j (0.13 g, 0.68
mmol), ethanol (13 mL), sodium hydroxide (0.4 g) and 5%
palladium on carbon (45 mg) was stirred overnight under an
atmosphere of hydrogen at room temperature. The catalyst was
removed by filtration, and the filtrate was neutralized by 2 M
HCl and the solvent evaporated. Brine was added and the mix-
ture was extracted with ether. The ether solution was dried over
anhydrous magnesium sulfate and the solvent was evaporated.
The residue was distilled under reduced pressure to afford a
colorless oil (0.080 g). The NMR spectrum of the product was
identical to that of 1-phenylethanol and the product exerted
[α]D Ϫ48.6 (c 1.13, CHCl3), which corresponds to (S)-1a: yield
97%, ee 91%.
To a suspension of the resting cells (15 g wet wt) in water (90
mL), BL-100 (water-absorbing polymer, 15 g) was added and
the mixture was stirred with a spatula. The resulting powder
(120 g) was used for the reaction.
Oxidation of alcohols 1a–1n by the free cells in water
To a suspension of the cells (0.5 g) in water (3.0 mL), 1a–1n (0.08
mmol) was added and the mixture was shaken at 130 rpm at
30 ЊC for 24 h. The mixture was extracted with ether (3 × 2.0
mL), dodecane as an internal standard for GC analysis was
added, and the resulting ether solution was dried over
anhydrous magnesium sulfate and subjected to GC analysis to
determine the yield of 2a–2n and the ee of 1a–1n. The GC
conditions are as described previously.4
Oxidation of alcohols 1a–1n by the immobilized cells in hexane
with cyclohexanone at GC scale
Determination of the partition of 1a and 2a between hexane and
water absorbed in BL-100
To a suspension of the immobilized cells on BL-100 (4.0 g) in
hexane (6 mL), 1a–1n (0.08 mmol), cyclohexanone (0.2 mmol)
and dodecane as an internal standard for GC analysis were
added and the resulting suspension was shaken at 130 rpm and
30 ЊC for 24 h. The organic layer was subjected to GC analysis
to determine the yield of 2a–2n and ee of 1a–1n.
Water (3.0 mL) was absorbed on BL-100 (0.5 g) and the poly-
mer was placed in a test tube. A hexane solution (6.0 mL) of 1a
or 2a (0.08 mmol) and dodecane (4.30 mM) as an internal
standard was added to the test tube and the mixture was shaken
at 30 ЊC for 24 h. The hexane layer was subjected to GC analysis
to determine the partition of 1a or 2a between the hexane and
water phase.
Oxidation of alcohol 1a at preparative scale
To a suspension of the immobilized cells (120 g) in hexane
(180 mL), 1a (1.5 mmol) and cyclohexanone (0.30 mL) were
added and the resulting suspension was shaken at 130 rpm at
30 ЊC for 24 h. The resulting mixture was filtered and the
immobilized cell was washed with ether, then the combined
organic layer was evaporated to obtain the crude product. (R)-
1a was purified with silica gel column chromatography: 31%, ee
98%, [α]D ϩ53.0 (c 0.60, CHCl3) (lit.13 [α]D25 Ϫ57 (c 5.12, CHCl3)
(S)). The spectral data are in accord with those reported
previously.4
Reduction of 2a with immobilized cells on calcium alginate
Cells (2.0 g wet wt) were suspended in 12 mL of 1% sodium
alginate and the suspension was extruded slowly into a CaCl2
solution (0.05 M) through a syringe. The immobilized cells were
washed with distilled water and wiped with a Kimwipe. The
total weight of the immobilized cells was 7.3 g.
To a suspension of immobilized cells on calcium alginate (1.8
g) in hexane (6.0 mL), 2a (0.08 mmol), hexan-2-ol (1.0 mmol)
and dodecane as an internal standard for GC analysis were
J. Chem. Soc., Perkin Trans. 1, 1999, 2397–2402
2401

View Article Online
added. The resulting suspension was shaken at 130 rpm at 30 ЊC
for 24 h. The organic layer was subjected to GC analysis to
determine yield and ee.
24 h. The organic layer was washed with water and subjected to
GC analysis to determine yield and ee and to GC-MS analysis
(Thermon-3000 (Shincarbon A) 60–80 mesh, 5 mm × 1 m,
80–150 ЊC minϪ1) to determine the deuterium content of the
product.
Preparation of racemic and chiral [2-2H]octan-2-ol
(R/S)-[2-2H]Octan-2-ol. NaBD4 (7.9 mmol, 0.33 g) was
added to 50 mL of an ethanol solution of octan-2-one (15.8
mmol, 2.0 g) at 0 ЊC, and the mixture was stirred at rt for 4 h.
After neutralization with 1 M HCl, ethanol was removed under
reduced pressure, and the product was extracted with ether (30
mL × 3), washed with water, aqueous sodium hydrogen carb-
onate, and water, successively, and dried over anhydrous sodium
sulfate. Then, the ether was evaporated under reduced pressure,
and the residual oil was purified by distillation with a Kugelrohr
apparatus to give (R/S)-[2-2H]octan-2-ol (yield 89%, 1.84 g). 1H
NMR (CDCl3) δ 0.86 (t, 3H, CH3, J = 6.4 Hz), 1.15 (s, 3H,
CH3), 1.26–1.40 (m, 10H, CH2), 1.54 (s, 1H, OH); FTIR ν(NaCl
Acknowledgements
This work was supported by the Nagase Science and Technol-
ogy Foundation and by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science Sports and Culture of
Japan. The authors are grateful to Osaka Yuki Kagaku Kogyo
Co., Ltd. for providing BL-100 (water-absorbing polymer).
References
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neat) 2130 cmϪ1
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(S)-[2-2H]Octan-2-ol. Vinyl acetate (41 mmol, 3.6 g) was
added to a mixture of (R/S)-[2-2H]octan-2-ol (7.6 mmol, 1.0 g),
lipase (CAL SP 435, 0.2 g), and molecular sieves (0.5 g) in
benzene (50 mL), the resulting mixture was stirred for 3 h then
filtered through Extrelute, and the solvent was evaporated
under reduced pressure. The resulting (S)-octan-2-ol was separ-
ated from the (R)-2-octyl acetate by silica gel column chrom-
atography (eluent: hexane–ethyl acetate, 9:1). (S)-Octan-2-ol
was purified by distillation with a Kugelrohr apparatus, and the
ee was determined by GC analysis (yield 36%, ee 92%). The
spectral data are in accord with those for the racemic one.
2 M. Kataoka, S. Shimizu, Y. Doi, K. Sakamoto and H. Yamada,
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(R)-[2-2H]Octan-2-ol. The (R)-2-octyl acetate (4.3 mmol,
0.75 g, ee 93%) obtained above was hydrolyzed by stirring with
lithium aluminum hydride (3.4 mmol, 0.13 g) in ether (25 mL)
for 1 h, then the mixture was neutralized with 1 M HCl. The
organic layer was separated from the aqueous layer, and the
product was extracted with ether from the aqueous layer. The
combined organic portion was evaporated under reduced pres-
sure to obtain (R)-[2-2H]octan-2-ol, which was re-acetylated by
CAL and vinyl acetate and hydrolyzed as described above for
further enhancement of ee. After the second acetylation and
hydrolysis, the residual oil was purified by distillation with a
Kugelrohr apparatus (yield 23%, ee >99%). The spectral data
are in accord with those for the racemic one.
Reduction of 2a by deuterated additive, [2-2H]octan-2-ol
To a suspension of the immobilized cell on BL-100 (4.0 g) in
hexane (6 mL), 2a (0.08 mmol), [2-2H]octan-2-ol and dodecane
as an internal standard for GC analysis were added. The result-
ing suspension was shaken at 130 rpm and 30 ЊC for 24 h. The
organic layer was subjected to GC analysis to determine yield
and ee and to GC-MS analysis (Thermon-3000 (Shincarbon A)
60–80 mesh, 5 mm × 1 m, 80–150 ЊC minϪ1) to determine the
deuterium content of the product.
Reduction of 2a by immobilized cells with deuterated water
9 Y. Naoshima, J. Maeda, Y. Munakata, T. Nishiyama, M.
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For immobilization of the cells with 33% or 66% deuterated
water, to a suspension of cells (0.5 g wet wt) in deuterated water
(3 mL, D2O:H2O = 1:2 or 2:1), BL-100 (0.5 g) was added and
the mixture was stirred with a spatula. For immobilization of
the cells with >99% deuterated water, the cells (0.5 g wet wt)
were washed with deuterated water (6 mL × 4) and suspended
in deuterated water (3 mL), then BL-100 (0.5 g) was added
and the mixture was stirred with a spatula. To a suspension of
the immobilized cells on BL-100 (4.0 g) in hexane (6 mL), 2a
(0.08 mmol), octan-2-ol or [2-2H]octan-2-ol (0.4 mmol) and
dodecane as an internal standard for GC analysis were added.
The resulting suspension was shaken at 130 rpm at 30 ЊC for
13 M. Kasai, C. Frussios and H. Ziffer, J. Org. Chem., 1983, 48, 459.
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Paper 9/00936A
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J. Chem. Soc., Perkin Trans. 1, 1999, 2397–2402