Enzyme-catalyzed asymmetric synthesis of optically active (R)-and (S)-ethyl-4-phenyl-4-hydroxybutyrate with microbial cells

Article abstract of DOI:10.3109/10242422.2012.758116

In efforts to obtain carbonyl reductases with high activity and enantioselectivity, forty microorganisms belonging to different taxonomical groups were investigated for the ability to catalyze the enantioselective reduction of ethyl 4-phenyl- 4-oxobutyrat

Full text of DOI:10.3109/10242422.2012.758116

Biocatalysis and Biotransformation, 2013; 31(1): 66–70
SHORT PUBLICATION
Enzyme-catalyzed asymmetric synthesis of optically active (R)- and
(S)-ethyl -4-phenyl-4-hydroxybutyrate with microbial cells
SHIWEN XIA1, YONGZHENG CHEN2, ZHUO JUNRUI2 & HONGMEI XU2
1College of Bio-information, Chongqing University of Posts and Telecommunications, Chongqing, P. R. China and
2College of Pharmacy, Zunyi Medical University, Zunyi, P. R. China
Abstract
In efforts to obtain carbonyl reductases with high activity and enantioselectivity, forty microorganisms belonging to dif-
ferent taxonomical groups were investigated for the ability to catalyze the enantioselective reduction of ethyl 4-phenyl-
4-oxobutyrate (EPOB) to the corresponding optically active ethyl 4-phenyl-4-hydroxybutyrate (EPHB). Highly
enantioselective reduction of EPOB was achieved with Candida magnoliae CGMCC 2.1919 and Saccharomyces cerevisiae
CGMCC 2.399 giving the corresponding (R)-EPHB and (S)-EPHB in 99% ee, respectively. The highly enantioselective
bioreductions provide simple routes to optically active γ-hydroxyl acid esters that are useful pharmaceutical intermediates
and versatile chiral building blocks.
Keywords: Bioreduction, Candida magnoliae, Saccharomyces cerevisiae, Enantioselectivity
Introduction
derivatives with ruthenium-containing catalysts, and
Enantioselective reduction provides direct access to
chiral alcohols that are important building blocks for
the synthesis of fine chemicals and pharmaceuticals
(Matsuda et al. 2009). Optically active 4-aryl-
4-hydroxybutanoic acid derivatives are valuable inter-
mediates in the preparation of liquid crystalline
compounds, agrochemicals and pharmaceuticals
(Scheme 1), such as serotonin uptake inhibitors,
fluoxetine and platelet activating factor (PAF) antag-
onists (Corey et al. 1987, 1988).
The asymmetric synthesis of optically active
4-aryl-4-hydroxybutanoic acid derivatives has been
reported using chemical catalysts. For example,
Noyori et al. (1992) described the enantioselective
hydrogenation of γ-keto esters in the presence of
chiral ruthenium complexes. Corey and coworkers
reported the enantioselective synthesis of methyl
(R)-4-(3, 4-dimethoxyphenyl)-4-hydroxybutyrate in
98% yield and 95% ee using chiral oxazaborolidine
and borane reagents (Corey et al. 1987, 1988).
Militzer et al. (2005) further developed the stereo-
selective reduction of 4-aryl-4-oxobutanoic acid
a series of chiral 4-aryl-4-hydroxybutanoic acid
derivatives were obtained in 70–96% ee. In addition,
Ramachandran et al. (2002) developed effective
intramolecular asymmetric reduction of γ-keto acid
ester with β-chlorodiisopinocampheylborane to
produce (R)-4-phenyl-4-hydroxybutyrate in 94% ee.
Recently, Militzer and coworkers also designed and
developed a new chiral ruthenium catalyst, which
was used for asymmetric reduction of γ-keto acid
esters to their corresponding chiral hydroxyl acid
esters (Ramachandran et al. 2002).
In comparison with chemical catalysts, the use of
enzymes can provide benefits that cannot be obtained
with traditional chemical treatments. These include
mild reaction conditions, environmentally friendly
reduction in waste and reduced energy consump-
tion. Finding ideal biocatalysts is a critical part of
enzyme-catalyzed asymmetric synthesis. (Chen et al.
2007, 2008, 2009, 2010). To the best of our knowl-
edge, enzyme-catalyzed asymmetric reduction of
4-aryl-4-hydroxybutanoic acid esters has rarely been
reported. Manzocchi et al. (1987) have previously
Correspondence: Dr.Yongzheng Chen, Zunyi Medical University, College of Pharmacy, 201 Dalian Rd, Zunyi, Guizhou 563003, P. R. China. E-mail: yzchen@
zmc.edu.cn
(Received 20 June 2012; revised 11 September 2012; accepted 10 December 2012)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2013 Informa UK, Ltd.
DOI: 10.3109/10242422.2012.758116

Enantioselectivity Reduction 67
Scheme 1. Biocatalytic preparation and synthetic application of optically active (R) and (S)-EPHB.
reported that Baker’s yeast can be used as a biocata-
lyst for the reduction of EPOB with unspecified
enantioselectivity.
0.5% (w/v) beef extract, and 2% (w/v) peptone
solution (pH 6.8); and strains were maintained on
nutrient agar slants at 4°C. Erlenmeyer flasks of
250 mL containing 100 mL of the appropriate ster-
ilized cultivation medium were inoculated with the
test microorganism and incubated in an orbital
shaker (180 rpm) at 27°C. After 48 h of growth
(yeast, bacteria), the cells were harvested by cen-
trifugation and washed twice with cool physiological
saline (0.85%).
Materials and methods
General
All strains were obtained from our laboratory and
registered at the China General Microbiological
Culture Collection Center (CGMCC) and at our lab.
All other reagents were purchased from commercial
sources and were used without further purification.
1H NMR spectra were recorded on a Bruker-300
(300/75 MHz) spectrometer using CDCl3 as a sol-
vent and TMS as an internal standard. TLC was
performed on glass-backed silica plates. Column
chromatography was performed by using silica gel
(200–300 mesh) with ethyl acetate/petroleum ether
as an eluent. Enantiomeric excess was determined
by GC analysis using a Fuli GC9790 with a chiral
column (CP-Chirasil-DEX CB, Varian, USA) and
using a flame ionization detector, nitrogen was used
as the carrier gas at 1.5 mL/min, the split ratio was
1:50 (v/v), the injector and detector temperatures
were both set at 250°C, and the column temperature
was programmed as 80°C for 3 min ramped to
220°C at a rate of 3°C/min.The configuration of the
bioproduct 2 was assigned by using authentic sam-
ples of (R)-2 and (S)-2 as standards in chiral GC
analysis.
General procedure for enantioselective reduction
with resting cells of microorganisms
To a 50 mL Erlenmeyer shaking-flask were added
10 mL potassium phosphate buffer (0.1 M, pH 7.0),
1.0 g (wet weight) freshly harvested cells, 50 mg glu-
cose, and 10 mg EPOB, and the mixture was shaken
for 24 h at 30°C. After 24 h, ethyl acetate (30 mL)
was added to the reaction mixture, and the organic
layer was dried and analyzed by GC to determine
the yield and enantiomeric excesses.
Preparation of (R)-2 and (S)-2 with C. magnoliae
CGMCC 2.1919 and S. cerevisiae CGMCC 2.399,
respectively
To a 50 mL Erlenmeyer shaking-flask were added
a suspension of C. magnoliae CGMCC 2.1919 or
S. cerevisiae CGMCC 2.399 cells (5.0 g. wet weight)
in 50 mL of potassium phosphate buffer (0.1 M, pH
6.8), substrate EPOB (50 mg), 5% glucose (w/v),
and 10% ethanol (v/v), and the mixtures were shaken
for 24 h at 30°C. After the reaction was complete,
the mixture was centrifugated at 10,000 rpm for
10 min, the supernatant was then saturated with
sodium chloride, and extracted with ethyl acetate
(3ϫ30 mL). The combined organic layers were
General procedure for cell growth
Yeasts were grown in a medium containing 0.2%
(w/v) glucose, 2% (w/v) peptone, and 1% (w/v) yeast
extract solution (pH 6.8–7.0); bacteria were grown
in a medium containing 0.1% (w/v) sodium chloride,

68 S. Xia et al.
dried over anhydrous Na2SO4 and the solvent was
removed under vacuum. The chemical yield and ee
of the products were determined by GC analysis.
The products were purified by silica gel column
(S)-enantioselectivity was obtained using S.cerevisiae
CGMCC 2.399 as a biocatalyst.
Encouraged by these results, we investigated the
reaction process to monitor whether byproducts
were being formed and for process optimization.
Figure 1 shows the time course of bioreduction of
EPOB with resting cells of C. magnoliae CGMCC
2.1919.The reaction was very fast over the first 4 h,
(R)-EPHB with 12% yield and the ee of (R)-EPHB
was maintained over 99%. However, we found that
a lot of keto acid appeared in this whole reaction
process after 10 h. A 22% yield of (R)-EPHB with
78% residual 4-oxo-4-phenylbutanoic acid was
obtained after extending the reaction time to 24 h.
This indicates the presence of a competing ester
hydrolytic activity but also that 4-oxo-4-phenylbu-
tanoic acid is a poor substrate for the reductase and
EPHB is not a substrate for the hydrolase, as 4-
phenyl-4-hydroxybutyrate (PHB), the correspond-
ing hydroxy acid to EPHB was not detected in this
reaction process.
1
chromatography, and were identified by H NMR
analysis.The absolute configurations of the bioprod-
ucts (R)-EPHB and (S)-EPHB were assigned by
comparison of the retention time in GC analysis.
(R)-Ethyl-4-phenyl-4-hydroxybutyrate 2
1
Colorless oil (38% yield): H NMR (300 MHz,
CDCl3): δ7.25–7.35 (m, 5H, Ph), 4.68 (m, 1H,
CHOH), 4.11(q, 2H, Jϭ7.2 Hz, OCH2CH3), 2.40
(t, Jϭ7.8 Hz, 2H, -CH2CO), 2.36 (s, 1H, CHOH),
2.06 (q, 2H, Jϭ7.2 Hz, CHCH2), 1.25 (t, 3H, Jϭ7.2
Hz, CH3). 13C NMR (75 MHz, CDCl3): δ174, 144,
128, 127, 126, 74, 61, 34, 30, 14.
Results and discussion
Reaction conditions such as reaction time, pH,
glucose, and temperature were investigated for the
biotransformation by C.magnoliae CGMCC 2.1919.
These were shown to be pH 6.8–7.0, 30°C, and 5%
glucose. Glucose was particularly important for
cofactor regeneration in the biotransformation
process as a very low yield (Ͻ5.0%) was obtained
without glucose in the reaction system. As shown in
Figure 1, the highest yield was obtained when the
reaction time was extended to 24 h, (R)-EPHB
was obtained with 22% yield and Ͼ99% ee, and
extension of the reaction time had no effect on the
enantioselectivity.
Forty strains were screened for the enantioselective
reduction of EPOB to chiral EPHB, and the reaction
progress was monitored by GC analysis. As shown in
Table I. T.variabilis CGMCC 2.1570 and G.candidum
CGMCC 2.616 gave especially good enantioselectiv-
ity of (R)-EPHB (81%–85% ee) (entries 1–2). The
best results for the bioreduction were obtained with
S. cerevisiae CGMCC 109 and Candida magnoliae
CGMCC 2.1919, giving an ee for (R)-EPHB of
98% and 99%, respectively (entries 3–4). In
contrast, the reduction of EPOB with other strains
(S. cerevisiae CGMCC 2.396, S. cerevisiae CGMCC
2.25, T. cutaneum CGMCC 2.1795, and T. cutaneum
CGMCC 2.570; entries 5–8) gave (S)-EPHB with
moderate enantioselectivity of 53–77% ee.The highest
In order to increase the yield of product, a
cosolvent system was investigated in this bioreduc-
tion process. Usually, a small amount of an organic
Table I. Screening of microbial strains for the bioreduction of EPOB to EPHB.
Entrya
Strain
Time (h) Yield (%) ee (%) Configurationb
1
2
3
4
5
6
7
8
9
Trichosporon variabilis CGMCC 2.1570
Geotrichum candidum CGMCC 2.616
Saccharomyces cerevisiae CGMCC 2.109
Candida magnoliae CGMCC 2.1919
Trichosporon cutaneum CGMCC 2.25
Saccharomyces cerevisiae CGMCC 2.396
Trichosporon cutaneum CGMCC 2.1795
Trichosporon cutaneum CGMCC 2.570
Saccharomyces cerevisiae CGMCC 2.399
24
24
24
24
24
24
24
24
24
42
51
16
22
34
29
51
42
40
85
81
98
99
77
59
53
60
99
(R)
(R)
(R)
(R)
(S)
(S)
(S)
(S)
(S)
aThe reactions were run with 10 mg substrate EPOB in a 10-mL cell suspension (1 g fresh harvest wet
cells) of microorganism’s whole cells in 100 mM KH2PO4–K2HPO4 buffer (pH 6.8–7.0) containing
5% glucose (w/v) at 30°C and 160 rpm for 24 h.
bDetermined by GC analysis.

Enantioselectivity Reduction 69
Table II. Effect of cosolvents on the bioreduction of EPOB with
C. magnoliae CGMCC 2.1919.
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Time Yield
(h)
ee (%)
Entrya
Cosolvent (10% v/v)
None
Ethanol
Methanol
(%)b Configuration
1
2
3
4
5
6
7
24
24
24
24
24
24
24
22
38
21
19
19
26
10
99 (R)
99 (R)
91 (R)
89 (R)
97 (R)
99 (R)
98 (R)
Hexane
Methyl Tert-Butyl Ether
Dimethylformamide
Dimethyl Sulfoxide
aThe reactions were run with 10 mg substrate EPOB in a 10-mL
cell suspension (1 g fresh harvest wet cells) of microorganism’s
whole cells in 100 mM KH2PO4–K2HPO4 buffer (pH 6.8–7.0)
containing 5% glucose (w/v) and 10% cosolvent (v/v) at 30°C and
160 rpm for 24 h.
0
5
10
Time (h)
15
20
25
Figure 1.Time-course curve of bioreduction of EPOB in aqueous
phase with C. magnoliae CGMCC 2.1919 (¨-yield of (R)-EPHB,
·-ee of (R)-EPHB, ꢀ-Yield of EPOB, and ꢁ-yield of 4-oxo-
4-phenylbutanoic acid).
bDetermined by GC analysis.
with 40% yield. C. magnoliae CGMCC 2.1919 gave
the corresponding (R)-EPHB in 99% ee with 38%
yield under optimal reaction conditions. This pro-
vides a greener way and direct access to synthesize
optically active γ-hydroxy acid esters that are useful
pharmaceutical intermediates and versatile chiral
building blocks. Moreover, the resting cells of
C. magnoliae CGMCC 2.1919 and S. cerevisiae
CGMCC 2.399 are easily available in large quantity
and are easy to handle, thus being a routine biocata-
lyst for organic chemist.
solvent is used to ensure sufficient solubility of keto
ester substrates (Griebenow et al. 2001). We found
that cosolvents played an important role in the
bioreduction of EPOB to (R)-EPHB with Candida
magnoliae CGMCC 2.1919. From the results shown
in Table II, it can be noticed that addition of 10%
(v/v) ethanol or dimethylformamide increased the
yield but did not affect the enantioselectivity (entry
2 and entry 6). (R)-EPHB was obtained in 38% yield
and 99% ee in the presence of 10% ethanol as a
cosolvent. Addition of methyl tert-butyl ether, dime-
thyl sulfoxide, methanol, and hexane decreased both
the activity and enantioselectivity. (entries 3–5, 7). A
larger scale experiment was investigated under the
optimum conditions (24 h, pH 6.8, 5% (w/v) glucose,
10% (v/v) ethanol, 30°C); 5 g wet cells suspended in
50 mL potassium phosphate buffer converted 50 mg
EPOB to (R)-EPHB with 38% yield and 99% ee,
confirming that the cosolvent system (10% ethanol
v/v) was helpful for increasing the yield presumably
by increasing the solubility of substrates. Moreover,
40% yield and 99% ee (S)-EPHB was also obtained
under the optimum conditions (24 h, pH 6.8,
5% (w/v) glucose, 10% (v/v) ethanol, 30°C) with
S. cerevisiae CGMCC 2.399.
Acknowledgments
We are grateful for the financial support obtained
from The Education Department of Guizhou Prov-
ince, China (Project No. Qian Jiao Ke 2010046); the
Organization Department of the Provincial Commit-
tee of Guizhou Province (Project No. TZJF-2010-
063);and theTechnologicalActivities of Certification
for the Returned Oversea Students from the Depart-
ment of Human Resources and the Social Security
(No. 2011-1) and Natural Science Foundation Project
of CQ CSTC, Project No. 2009BB5286.
Declaration of interest: The authors report no
declarations of interest.The authors alone are respon-
sible for the content and writing of the paper.
Conclusion
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