Control of enantioselectivity in the baker's yeast asymmetric reduction of γ-chloro β-diketones to γ-chloro (S)-β-hydroxy ketones

Article abstract of DOI:10.1016/S0957-4166(98)00269-9

1-Chloro-2,4-alkanediones 1a-f prepared in one step were reduced using baker's yeast to afford 1-chloro-2-hydroxy-4-alkanones 2a-f regioselectively in S 29% to R 58% ee. Use of a small amount of organic solvents to dissolve the substrate enhanced the enantiomeric excess in favor of the S configuration. The function of the organic solvents was studied with reducing enzymes isolated from baker's yeast. Some polar solvents selectively inhibited the enzymes, while nonpolar solvents enhanced the concentration of substrate in water. Application of inhibitors with heat-treatment and organic solvents as additives enhanced the enantiomeric excesses of the products to S 66-96% ee.

Full text of DOI:10.1016/S0957-4166(98)00269-9

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 2681–2692
Control of enantioselectivity in the baker’s yeast asymmetric
reduction of γ-chloro β-diketones to γ-chloro (S)-β-hydroxy
ketones
Jing-Nan Cui, Tadashi Ema, Takashi Sakai and Masanori Utaka ∗
Department of Applied Chemistry, Faculty of Engineering, Okayama University, Tsushima-naka Okayama 700-8530, Japan
Received 8 June 1998; accepted 4 July 1998
Abstract
1-Chloro-2,4-alkanediones 1a–f prepared in one step were reduced using baker’s yeast to afford 1-chloro-2-
hydroxy-4-alkanones 2a–f regioselectively in S 29% to R 58% ee. Use of a small amount of organic solvents to
dissolve the substrate enhanced the enantiomeric excess in favor of the S configuration. The function of the organic
solvents was studied with reducing enzymes isolated from baker’s yeast. Some polar solvents selectively inhibited
the enzymes, while nonpolar solvents enhanced the concentration of substrate in water. Application of inhibitors
with heat-treatment and organic solvents as additives enhanced the enantiomeric excesses of the products to S
66–96% ee. © 1998 Elsevier Science Ltd. All rights reserved.
1. Introduction
Optically active β-hydroxy ketones are useful building blocks in organic synthesis. The chiral 1,3-
diol skeleton found in a variety of natural products such as antibiotics and pheromones1 can be obtained
from the chiral β-hydroxy ketones.2 Thus, synthesis of chiral β-hydroxy ketones has been investigated
intensively. For example, reduction of chiral β,δ-diketosulfoxides with DIBAL3 and aldol reactions
between ketones and aldehydes using a chiral ligand4 gave β-hydroxy ketones in high enantioselect-
ivities. On the other hand, in the enantioselective reduction of prochiral ketones by use of baker’s yeast,5
especially interesting is the fact that high regio- and enantioselectivities are achieved in the reduction of
β-diketones to afford β-hydroxy ketones.6 We have been investigating the preparation of functionalized
β-hydroxy ketones by use of baker’s yeast and reported that the baker’s yeast reduction of 1-acetoxy-2,4-
alkanediones afforded (S)-1-acetoxy-2-hydroxy-4-alkanones with high regio- and enantioselectivities.7
In connection with the work, we have also been studying the reduction of 1-chloro-2,4-alkanediones 1
∗
Corresponding author. E-mail: utaka@cc.okayama-u.ac.jp
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00269-9

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to 1-chloro-2-hydroxy-4-alkanones 2 to capitalize on the synthetic versatility of the chloro substituent as
reported in a preliminary paper.8 Herein we wish to report the scope of the asymmetric reduction of 1 to
(S)-2 using baker’s yeast (Scheme 1).
Scheme 1.
Reduction of β-keto esters using baker’s yeast cells was reported to be catalyzed by some enzymes
producing the (S)-enantiomers and by other enzymes producing the (R)-enantiomers,9 and thus the
enantioselectivity can be controlled by inhibiting either of those enzymes. Recent successes in the
stereochemical control of the baker’s yeast reduction of β-keto esters seemed to be promising10 and
prompted us to apply the control to attain high enantioselectivity in the reduction of our γ-chloro β-
diketones 1. Therefore, we tried to inhibit the reducing enzymes selectively by addition of inhibitors10a
and heat-treatment of the yeast,10b and found that these methods were also effective in enhancing the
enantiomeric excesses of products 2 in favor of the S configuration.
Incidentally, in the course of our work we noticed that a small amount of organic solvent, used to
dissolve the substrate, could enhance the enantioselectivity in favor of the (S)-enantiomer. Thus, the
effects of a small amount of organic solvent are also described in detail. Organic solvents so far have
been used as bulk solvents11 or substrate-dissolving solvents,12 but such a use as additives to control the
enantioselectivity has never been reported.
2. Results and discussion
2.1. Preparation of the substrates
γ-Chloro β-diketones 1 were prepared in 19–50% yield in one step by a condensation reaction between
ethyl chloroacetate and lithium enolates formed from methyl ketones and lithium diisopropylamide
(LDA). Two equivalents of the enolate were added to the ester in the reaction13 due to the acidity of
the produced γ-chloro β-diketone 1. Use of chloroacetyl chloride instead of the chloroacetate, gave only
about 10% of the desired product 1. We failed to obtain 1-chloro-4-phenyl-2,4-butanedione. The product
was found to be 5-phenyl-3-oxo-2,3-dihydrofuran. The phenyl group seems to enhance the enolization
of the carbonyl group at the C-4 position, leading to the formation of the conjugated ring structure.
2.2. Baker’s yeast reduction under conventional conditions
The regio- and enantioselectivities in the yeast reduction of γ-chloro β-diketones 1 were investigated
under conventional conditions. As shown in Table 1, the regioselectivity was perfect, as observed in the
reduction of 1-acetoxy-2,4-alkanediones using the yeast.7 The regioisomer having a hydroxy group at
the C-4 position was not detected, even in the reduction of 1a (R=CH3) having a rather easily reducible
methyl ketone functionality at the C-4. The enantioselectivity, however, varied from S 29% to R 58% ee

J.-N. Cui et al. / Tetrahedron: Asymmetry 9 (1998) 2681–2692
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Table 1
Reduction of 1-chloro-2,4-alkanediones 1 to 1-chloro-2-hydroxy-4-alkanones 2 with baker’s yeasta
depending on the alkyl chain length, in contrast to that for the reduction of 1-acetoxy-2,4-alkanediones
which mostly kept S 95–98% ee. The low enantioselectivity is similar to that for the reduction of 1-
chloro-2-alkanones varying from S 83% to R 65% ee.14 It seems that 1-chloro-2,4-alkanediones 1 are
reduced by the yeast as derivatives of 1-chloro-2-alkanones, not as those of 2,4-alkanediones which are
seemingly reduced as methyl ketones activated by the β-keto group. It has been reported that an isolated
carbon–carbon double bond cannot be reduced by the yeast.15 Similarly, the double bond remained intact
in the hydroxyl ketone 2f. The products 2d and 2f have similar ees and yields. The enzymes cannot
distinguish between the alkyl and alkenyl groups remote from the active site.
2.3. Effects of organic solvents as additives
Compound 1c (R=n-C3H7) was chosen as a probe to test various organic solvents in detail, because
it was reduced to a nearly racemic product of S 6% ee in the conventional yeast reduction. As shown
in Table 2, almost all of the organic solvents enhanced the enantiomeric excess in favor of the S
configuration considerably. The hydrophobic nonpolar solvents such as hexane, 2-methylpentane, and
octane were most effective, whilst the effects of cyclohexane, heptane, and benzene were smaller or
negligible. The polar solvents such as tetrahydrofuran, ethyl acetate, and diethyl ether were more effective
than the hydrophilic solvents such as acetone and alcohol.
To explain the mechanism by which the organic solvents used in small quantities caused significant
enhancement in the enantioselectivity, we suggest the following two factors. One is the action of organic
solvents as inhibitors. It is well-known that enzymes are denatured in aqueous–organic mixtures. In order
to estimate the highest concentration of organic solvent that allows the reduction, various concentrations
of hexane, tetrahydrofuran, ethyl acetate, and ethanol were tested. As shown in Fig. 1, we found that the
reaction took place at almost all concentrations of hexane, except neat hexane as a solvent. On the other
hand, no reaction took place in the region where concentrations of ethyl acetate, tetrahydrofuran, and
ethanol exceeded 5%, 12%, and 34%, respectively, indicating occurrence of the inhibition of enzymes or
metabolic pathway.
In order to clarify the function of the small amount of organic solvents, we isolated the reductases
from the cells of baker’s yeast using 1-chloro-2,4-heptanedione 1c as substrate. We found three dominant
competing enzymes. Among these, R-enzyme-1 and -2 catalyzed the reduction of 1c to (R)-2c with 94%

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Table 2
Effects of organic solvents as an additive on the reduction of 1-chloro-2,4-heptanedione 1c to 1-chloro-
2-hydroxy-4-heptanone 2c with baker’s yeasta
Fig. 1. Effects of the concentration of organic solvents on the reduction of 1c to 2c with baker’s yeast
and 44% ee,† respectively, and S-enzyme-1 afforded (S)-2c with 97% ee. The activity of the isolated
enzymes toward 1c was measured in the presence of 1.3% of organic solvent (Fig. 2). We found that
hexane did not affect the activity of the enzymes, while ethyl acetate, tetrahydrofuran, and diethyl ether
decreased the activities of R-enzyme-2 and S-enzyme-1 to about 20% and 50%, respectively. The effect
of ethanol was small.
The other factor is the enhanced concentration of substrate due to the solubilizing or dispersing power
of organic solvents. It is known that a variation of substrate concentration can shift the enantiomeric
excess in the yeast reductions16 because the variation should affect the kinetics of each enzyme. We
noticed that, without a small amount of organic solvent, it took more than 20 minutes to dissolve the
substrate completely into water under the reaction conditions in spite of the fact that about 90% of
†
The isolated R-enzyme-2 was purified further and the purified enzyme catalyzed the reduction of 1c to give 2c with R 98%
ee.

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2685
Fig. 2. Activity of the crude enzymes toward 1c in the presence of 1.3% organic solvent
the substrate was converted to the product within 30 minutes. Thus, the reduction proceeded in much
lower substrate concentrations than 13 mM as calculated (0.5 mmol of the substrate in 38 ml of water).
When the substrate, dissolved in a small amount of organic solvent, was added to the reaction mixture,
it is thought that the function of organic solvent was to change the state of the substrate in the aqueous
reaction mixture and make the reaction proceed in a substrate concentration near to 13 mM. This function
is essentially different from that of the organic solvents used in bulk, where the substrate is partitioned
between organic and aqueous layers and the enantioselectivity is affected by the diluted substrate
concentration in water. Therefore, the effect of the substrate concentration on the enantioselectivity was
tested in detail under conventional conditions. As shown in Fig. 3, the enantioselectivity increased toward
the (S)-enantiomer with an increase in the concentration of substrate. The results show that Km of the R-
enzyme is smaller than that of the S-enzyme in baker’s yeast and the enantioselectivity can be enhanced
in favor of the S configuration by increasing the substrate concentration.
Fig. 3. Effects of substrate concentration on the reduction of 1c to 2c with baker’s yeast
Thus, it can be concluded that the function of organic solvents is to enhance the effective concentration
of the substrate in water, while the function as enzyme inhibitors is conceivable for ethyl acetate, diethyl
ether, and tetrahydrofuran. The characterization of the reductases will be reported in due course.
2.4. Effects of inhibitor, heat-treatment, and organic solvent
We chose the substrate 1c (R=n-C3H7) to select the inhibitor and reduction conditions (Table 3). Thus
we found that allyl alcohol was better than methyl vinyl ketone as an S-enhancing inhibitor (entries 2
and 3) and the maximal S selectivity (89% ee) was obtained when the yeast was heat-treated at 50°C for
30 minutes with 67 mM allyl alcohol prior to the addition of the neat substrate (entry 12). An alternative
procedure of the heat-treatment followed by addition of allyl alcohol was found to be less effective (entry

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Table 3
Effects of inhibitor, heat-treatment, and organic solvent on the reduction of 1-chloro-2,4-heptanedione
1c to (S)-1-chloro-2-hydroxy-4-heptanone 2c with baker’s yeasta
13). The effect of a small amount of organic solvent was small under the conditions of heat-treatment
with allyl alcohol (entries 14 and 15), but we achieved S 94% ee by use of a mixture of hexane and diethyl
ether (entry 16).
It is worth noting that the yield was lowered by use of the inhibitor in excess (entry 8) and by heat-
treatment of the yeast over 30 minutes (entry 17). These results can be explained by suppression of the
yeast reduction, leaving side reactions such as a furanone formation to proceed further. A similar result
observed in the reduction using no organic solvent (entry 12) may also be attributed to slowness of the
yeast reduction due to lack of the solubilization of substrate described in the preceding section.
As shown in Table 4, we reduced the series of 1-chloro-2,4-alkanediones 1a–e to obtain the corres-
ponding (S)-β-hydroxy ketones 2a–e using the best method of entry 16 in Table 3. High enantiomeric
excesses of S 94–96% ee were realized for substrates 1a, 1b, and 1c, and inversions of the enantioselect-
ivity from R 39–58% ee to S 81–66% ee were brought about for substrates 1d and 1e as compared with
Table 1.
In summary, we have succeeded in controlling the enantioselectivity by the combined use of enzyme
inhibitor, heat treatment, and organic solvent as an additive. The organic solvent in the combined use

J.-N. Cui et al. / Tetrahedron: Asymmetry 9 (1998) 2681–2692
2687
Table 4
Control of the enantioselectivity using additives and heat-treatment in the baker’s yeast reduction of
1-chloro-2,4-alkanediones 1 to 1-chloro-2-hydroxy-4-alkanones 2a
may play a role in solubilizing the substrate rapidly rather than to inhibit the R-enzyme, enhancing the
substrate concentration in favor of the S selectivity.
3. Experimental
3.1. General
1H and 13C NMR spectra were measured in CDCl3 at 200 and 50 MHz, respectively. Column
chromatography was carried out using Nacalai silica gel 60 (70–230 mesh) or Fuji Silysia BW-127 ZH
(100–270 mesh), and thin layer chromatography (TLC) was performed on Merck silica gel 60 F254. Dry
baker’s yeast was purchased from Nissin Milling. Dry THF and benzene were prepared by distillation
from sodium. DEAE-Toyopearl 650M was purchased from Tosoh. The seamless cellulose tubing for
dialysis was purchased from Viskase. G6PDH (from baker’s yeast, lyophilized powder) was purchased
from Sigma. Pressed baker’s yeast, NADPH, NADP+, and G6P were purchased from Oriental Yeast.
3.2. Preparation of 1-chloro-2,4-alkanediones 1a–f
To a solution containing diisopropylamine (120 mmol) in 50 ml of THF under a nitrogen atmosphere
was added butyllithium (120 mmol) and the bright yellow reaction mixture was stirred at −10°C for
15 min. A methyl ketone (120 mmol) was added via syringe and the mixture was stirred at −30°C for
30 min. Ethyl chloroacetate (60 mmol) was added to the mixture at −50°C and stirring was continued
until the mixture was warmed to room temperature slowly. The mixture was neutralized (pH 4–5) with
10% HCl and extracted with ethyl acetate (3×50 ml). The combined organic layer was dried over
magnesium sulfate and concentrated under vacuum. The crude material was purified by chromatography
on a silica gel column using ethyl acetate:hexane (1:30) as eluent. The major fractions were combined
and concentrated to give the target molecule as a colorless oil.
3.2.1. 1-Chloro-2,4-pentanedione 1a
Yield 19%. IR (neat) 1716, 1609. Ratio of enol form:keto form in CDCl3 is 78:22. 1H NMR (CDCl3)
enol form: δ 2.12 (s, 3H, CH3), 4.02 (s, 2H, CH2Cl), 5.85 (s, 1H, CH_COH), 15.0 (bs, OH); keto form:
δ 2.28 (s, CH3), 3.79 (s, CH2CO), 4.17 (s, CH2Cl). 13C NMR (CDCl3) enol form: δ 24.5, 44.4, 98.2,

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188.1, 190.7; keto form: δ 30.8, 48.4, 54.3, 196.6, 201.2. Anal. calcd for C5H7O2Cl: C, 44.62; H, 5.24;
found: C, 44.55; H, 4.77.
3.2.2. 1-Chloro-2,4-hexanedione 1b
Yield 35%. IR (neat) 1717, 1604. Ratio of enol form:keto form in CDCl3 is 80:20. 1H NMR (CDCl3)
enol form: δ 1.16 (t, J=7.5 Hz, 3H, CH3), 2.39 (q, J=7.5 Hz, 2H, CH3CH2), 4.03 (s, 2H, CH2Cl), 5.84 (s,
1H, CH=COH), 15.0 (bs, OH); keto form: δ 1.09 (t, 3H, CH3), 2.57 (q, CH3CH2), 3.77 (s, COCH2CO),
4.18 (s, CH2Cl). 13C NMR (CDCl3) enol form: δ 9.4, 31.1, 44.4, 96.8, 187.3, 195.2; keto form: δ 7.3,
37.0, 48.5, 53.2, 196.7, 203.9. Anal. calcd for C6H9O2Cl: C, 48.48; H, 6.06; found: C, 48.51; H, 6.07.
3.2.3. 1-Chloro-2,4-heptanedione 1c
Yield 50%. IR (neat) 1717, 1603. Ratio of enol form:keto form in CDCl3 is 85:15. 1H NMR (CDCl3)
enol form: δ 0.96 (t, J=7.3 Hz, 3H, CH3), 1.58–1.76 (m, 2H, CH2CH2), 2.32 (t, J=7.4 Hz, 2H, CH2CO),
4.03 (s, 2H, CH2Cl), 5.83 (s, 1H, CH_COH), 15.0 (bs, OH); keto form: δ 2.53 (t, CH2CH2CO), 3.76 (s,
COCH2CO), 4.18 (s, CH2Cl). 13C NMR (CDCl3) enol form: δ 13.6, 19.1, 39.7, 44.5, 97.5, 188.2, 193.7;
keto form: δ 13.4, 16.8, 45.6, 48.5, 53.5, 196.8, 203.5. Anal. calcd for C7H11O2Cl: C, 51.69; H, 6.78;
found: C, 51.58; H, 6.78.
3.2.4. 1-Chloro-2,4-octanedione 1d
1
Yield 50%. IR (neat) 1717, 1603. Ratio of enol form:keto form in CDCl3 is 87:13. H NMR
(CDCl3) enol form: δ 0.93 (t, J=7.2 Hz, 3H, CH3), 1.28–1.45 (m, 2H, CH3CH2), 1.55–1.68 (m, 2H,
CH2CH2CH2), 2.34 (t, J=7.3 Hz, 2H, CH2CO), 4.03 (s, 2H, CH2Cl), 5.84 (s, 1H, CH_COH), 15.0 (bs,
OH); keto form: δ 2.54 (t, CH2CH2CO), 3.77 (s, COCH2CO), 4.18 (s, CH2Cl). 13C NMR (CDCl3) enol
form: δ 13.7, 22.2, 27.7, 37.6, 44.5, 97.4, 188.0, 194.1; keto form: δ 13.6, 22.0, 25.4, 43.4, 48.5, 53.5,
196.8, 203.6. Anal. calcd for C8H13O2Cl: C, 54.39; H, 7.37; found: C, 54.44; H, 7.36.
3.2.5. 1-Chloro-2,4-nonanedione 1e
1
Yield 38%. IR (neat) 1603. Ratio of enol form:keto form in CDCl3 is 79:21. H NMR (CDCl3) enol
form: δ 0.87–0.93 (m, 3H, CH3), 1.22–1.40 (m, 4H, CH3CH2CH2), 1.50–1.68 (m, 2H, CH2CH2CO),
2.34 (t, J=7.3 Hz, 2H, CH2CO), 4.03 (s, 2H, CH2Cl), 5.83 (s, 1H, CH_COH), 15.0 (bs, OH); keto form:
δ 2.51 (t, CH2CH2CO), 3.76 (s, COCH2CO), 4.18 (s, CH2Cl). 13C NMR (CDCl3) enol form: δ 13.8,
22.3, 25.3, 31.2, 37.8, 44.4, 97.4, 188.0, 194.0; keto form: δ 13.5, 22.4, 23.0, 31.0, 43.7, 48.5, 53.5,
196.8, 203.6. Anal. calcd for C9H15O2Cl: C, 56.69; H, 7.93; found: C, 56.94; H, 8.35.
3.2.6. 1-Chloro-7-octen-2,4-dione 1f
Yield 28%. IR (neat) 1717, 1605. Ratio of enol form:keto form in CDCl3 is 86:14. 1H NMR (CDCl3)
enol form: δ 2.35–2.51 (m, 4H, CH2CH2CO), 4.05 (s, 2H, CH2Cl), 4.99–5.21 (m, 2H, CH2_CH),
5.70–5.95 (m, 1H, CH2_CH), 5.86 (s, 1H, CH_COH), 15.0 (bs, OH); keto form: δ 2.67 (t, CH2CH2CO),
3.79 (s, COCH2CO), 4.19 (s, CH2Cl). 13C NMR (CDCl3) enol form: δ 29.9, 37.8, 44.9, 98.2, 116.3,
136.9, 188.2, 193.8; keto form: δ 27.8, 43.2, 48.9, 54.1, 116.3, 136.9, 197.2, 201.3. Anal. calcd for
C8H11O2Cl: C, 55.02; H, 6.35; found: C, 55.09; H, 6.60.
3.3. Conventional procedure for the baker’s yeast reduction of 1a–f
A mixture of dry baker’s yeast (2 g) and glucose (2 g) in 38 ml of boiled water was stirred at 30°C for
30 min. The substrate (0.5 mmol) was added to the mixture and the reaction was continued with stirring

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2689
at the same temperature. The progress of the reaction was monitored by GC. After the substrate was
consumed, Celite 545 was added to the mixture and the resulting suspension was filtered. The residual
Celite was washed with ethyl acetate and the combined filtrate was extracted with ethyl acetate. The
organic portion was dried over magnesium sulfate and concentrated under vacuum. The crude material
was purified by chromatography on a silica gel column using ethyl acetate:hexane (1:10) as eluent. The
major fractions were combined and concentrated to give the target molecule as a colorless oil. The ees
1
were determined by H NMR of the corresponding (R)-MTPA ester. The [α]D values were measured
using CHCl3 (c 1.9–2.3). The absolute configurations were determined by comparing the [α]D values of
dechlorinated derivatives with those of the corresponding 2-hydroxy-4-alkanones reported.6
3.3.1. 1-Chloro-2-hydroxy-4-pentanone 2a
1
IR (neat) 3350, 1720. H NMR (CDCl3) δ 2.22 (s, 3H, CH3), 2.77 (d, J=6.1 Hz, 2H, COCH2COH),
3.17 (br, 1H, OH), 3.58 (d, J=5.5 Hz, 2H, CH2Cl), 4.21–4.33 (m, 1H, CHOH). 13C NMR (CDCl3) δ
30.8, 46.7, 48.2, 67.4, 208.2. Anal. calcd for C5H9O2Cl: C, 43.96; H, 6.59; found: C, 43.81; H, 6.21.
3.3.2. 1-Chloro-2-hydroxy-4-hexanone 2b
1
IR (neat) 3385, 1710. H NMR (CDCl3) δ 1.08 (t, J=7.3 Hz, 3H, CH3), 2.50 (q, J=7.3 Hz, 2H,
CH3CH2), 2.74 (d, J=5.6 Hz, 2H, COCH2COH), 3.16 (d, J=4.6 Hz, 1H, OH), 3.58 (d, J=5.1 Hz, 2H,
CH2Cl), 4.21–4.33 (m, 1H, CHOH). 13C NMR (CDCl3) δ 7.46, 36.8, 45.4, 48.2, 67.6, 211.1. Anal. calcd
for C6H11O2Cl: C, 47.84; H, 7.31; found: C, 47.76; H, 7.23.
3.3.3. 1-Chloro-2-hydroxy-4-heptanone 2c
IR (neat) 3385, 1717. 1H NMR (CDCl3) δ 0.93 (t, J=7.3 Hz, 3H, CH3), 1.53–1.71 (m, 2H, CH3CH2),
2.45 (t, J=7.2 Hz, 2H, CH2CH2CO), 2.74 (d, J=5.8 Hz, 2H, COCH2COH), 3.19 (d, J=4.6 Hz, 1H, OH),
3.59 (d, J =5.7 Hz, 2H, CH2Cl), 4.21–4.33 (m, 1H, CHOH). 13C NMR (CDCl3) δ 13.6, 17.0, 45.5, 45.7,
48.2, 67.5, 210.8. Anal. calcd for C7H13O2Cl: C, 51.69; H, 6.78; found: C, 51.58; H, 6.78.
3.3.4. 1-Chloro-2-hydroxy-4-octanone 2d
IR (neat) 3414, 1708. 1H NMR (CDCl3) δ 0.90 (t, J=7.2 Hz, 3H, CH3), 1.23–1.40 (m, 2H,
CH3CH2), 1.49–1.65 (m, 2H, CH2CH2CO), 2.46 (t, J=7.2 Hz, 2H, CH2CH2CO), 2.73 (d, J=6.0 Hz,
2H, COCH2COH), 3.19 (br, OH), 3.57 (d, J=5.6 Hz, 2H, CH2Cl), 4.21–4.33 (m, 1H, CHOH). 13C NMR
(CDCl3) δ 13.8, 22.2, 25.6, 43.4, 45.7, 48.2, 67.6, 210.9. Anal. calcd for C8H15O2Cl: C, 53.78; H, 8.40;
found: C, 53.89; H, 8.28.
3.3.5. 1-Chloro-2-hydroxy-4-nonanone 2e
IR (neat) 3385, 1715. 1H NMR (CDCl3) δ 0.89 (t, J=6.8 Hz, 3H, CH3), 1.23–1.40 (m, 4H,
CH3CH2CH2), 1.49–1.65 (m, 2H, CH2CH2CO), 2.46 (t, J=7.3 Hz, 2H, CH2CH2CO), 2.73 (d, J=5.8
Hz, 2H, COCH2COH), 3.20 (br, OH), 3.58 (d, J=5.6 Hz, 2H, CH2Cl), 4.21–4.33 (m, 1H, CHOH). 13
C
NMR (CDCl3) δ 13.8, 22.4, 23.2, 31.2, 43.6, 45.7, 48.2, 67.6, 210.9. Anal. calcd for C9H17O2Cl: C,
56.10; H, 8.83; found: C, 55.87; H, 8.78.
3.3.6. 1-Chloro-2-hydroxy-7-octen-4-one 2f
1
IR (neat) 3421, 1711. H NMR (CDCl3) δ 2.34 (q, J=6.6 Hz, 2H, CHCH2CH2), 2.57 (t, J=7.0 Hz,
2H, CH2CH2CO), 2.74 (d, J=6.0 Hz, 2H, COCH2COH), 3.19 (br, OH), 3.58 (d, J=5.3 Hz, 2H, CH2Cl),
4.21–4.33 (m, 1H, CHOH), 4.97–5.09 (m, 2H, CH2_CH), 5.70–5.90 (m, 1H, CH2_CH). 13C NMR

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Table 5
Baker’s yeast reduction of 1c at various substrate concentrations
(CDCl3) δ 27.9, 43.1, 46.5, 48.8, 68.0, 116.1, 137.1, 210.1. Anal. calcd for C8H13O2Cl: C, 54.40; H,
7.42; found: C, 54.05; H, 7.79.
3.4. Baker’s yeast reduction of 1c at various substrate concentrations
A mixture of dry baker’s yeast (0.5–20 g) and glucose (2 g) in boiled water (9–150 ml) was stirred
at 30°C for 30 min. The substrate (0.074–1.18 mmol) was added to the mixture and the reaction was
continued with stirring at the same temperature for 1 h. The work-up procedure was essentially the same
as that described in the foregoing experiment. The results were listed in Table 5.
3.5. General procedure using heat-treated baker’s yeast with inhibitor and a small amount of organic
solvent
A mixture of dry baker’s yeast (2 g), water (38 ml), glucose (2 g) and allyl alcohol (2.5 mmol, 67 mM)
was stirred at 50°C for 30 min. After the mixture was cooled to 30°C, the substrate dissolved in 0.5 ml of
organic solvent was added and the reaction was continued with stirring at 30°C for 1–3 h. The work-up
procedure was essentially the same as that described in the foregoing experiment.
3.6. Preparation of MTPA esters from 2a–f
To a solution of 1-chloro-2-hydroxy-4-alkanones 2a–f (0.03 mmol) in benzene (1.5 ml) was added
(R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (0.09 mmol) and pyridine (0.1 ml). After
being stirred for 12 h at room temperature, the mixture was treated in the usual manner. The crude product
was purified by chromatography on silica gel to give the corresponding ester in 80–95% yield.
3.7. Isolation of γ-chloro β-diketone reductases
Raw pressed baker’s yeast (40 g) was suspended in 40 ml of 0.1 M MES buffer (pH 6.0). The suspended
cells were homogenized with 60 ml of glass beads (0.5 mm in diameter) using a Vibrogen cell mill chilled
with ice below 4°C. The homogenate obtained from 80 g of the pressed yeast was centrifuged at 12 000
rpm for 30 min at 4°C. The supernatant liquid (90 ml) was dialyzed overnight against 10 mM phosphate
buffer (pH 7.0). The dialyzate was applied to a column packed with DEAE-Toyopearl 650 equilibrated

J.-N. Cui et al. / Tetrahedron: Asymmetry 9 (1998) 2681–2692
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with the phosphate buffer. The column was eluted with the buffer and a nonadsorbed enzyme (crude
R-enzyme-1) was eluted. Then the elution was continued with a 0–0.5 M stepwise gradient concentration
of NaCl dissolved in the buffer. Crude R-enzyme-2 and S-enzyme-1 were eluted at 0.1 and 0.2 M NaCl,
respectively.
3.8. Enzyme assay
To a mixture of 1c (0.9 mM), organic solvent (35 µl, 1.3% v/v) and NADPH (0.26 mM) in 2.5 ml of
10 mM MES buffer (pH 6.0) was added the crude enzyme solution (0.2 ml). The rate of reaction was
determined spectrophotometrically at 30°C by following the decrease in absorbance of NADPH at 340
nm. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the oxidation of 1
µmol of NADPH per minute at 30°C under these conditions.
3.9. Enzymatic reduction
To the crude enzyme solution [R-enzyme-1 (70 ml), R-enzyme-2 (40 ml) or S-enzyme-1 (30 ml)] was
added NADP+ (3 µmol), D-glucose 6-phosphate disodium salt (G6P, 0.3 mmol), D-glucose 6-phosphate
dehydrogenase (G6PDH, 0.1 mg, 15 unit), and 1c (0.3 mmol). The mixture was stirred at 30°C for 3 h.
After work-up in the usual manner, R-enzyme-1 gave (R)-2c in 45% yield with 94% ee, R-enzyme-2 gave
(R)-2c in 34% yield with 44% ee, and S-enzyme-1 gave (S)-2c in 52% yield with 97% ee.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education,
Science and Culture of Japan. We are grateful to the SC-NMR Laboratory of Okayama University for the
measurement of NMR spectra.
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