Enantiocomplementary preparation of (S)- and (R)-mandelic acid derivatives via α-hydroxylation of 2-arylacetic acid derivatives and reduction of α-ketoester using microbial whole cells

Article abstract of DOI:10.1016/j.tetasy.2007.09.029

Forty one microorganisms belonging to different taxonomical groups were used to carry out the enantioselective reduction of methyl benzoylformate to afford the corresponding (R)-methyl mandelate, with moderate to high ee. In contrast, the monooxygenase en

Full text of DOI:10.1016/j.tetasy.2007.09.029

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Tetrahedron: Asymmetry 18 (2007) 2537–2540
Enantiocomplementary preparation of (S)- and (R)-mandelic acid
derivatives via a-hydroxylation of 2-arylacetic acid derivatives
and reduction of a-ketoester using microbial whole cells
Yongzheng Chen,a,b Jinggang Xu,a Xiaoying Xu,a Yu Xia,a Hui Lin,a
Shiwen Xiaa,* and Lixin Wanga
aKey Laboratory for Asymmetric Synthesis and Chirotechnology of Sichuan Province, Chengdu Institute of Organic Chemistry,
Chinese Academy of Sciences, Chengdu 610041, China
bGraduate School of Chinese Academy of Sciences, Beijing 100039, China
Received 13 August 2007; accepted 20 September 2007
Abstract—Forty one microorganisms belonging to different taxonomical groups were used to carry out the enantioselective reduction of
methyl benzoylformate to afford the corresponding (R)-methyl mandelate, with moderate to high ee. In contrast, the monooxygenase
enzyme in Helminthosporium sp. CIOC3.3316 catalyzed the hydroxylation of methyl 2-phenylacetate to (S)-methyl mandelate. This com-
bination of oxidation and reduction biotransformations thus provides a method for preparing the enantiomers of chiral a-hydroxy acid
derivatives.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The regio- and stereoselective hydroxylation of 2-phenyl-
acetic acid derivatives is the simplest route for preparing
optically active mandelic acid derivatives. However, regio-
and stereoselective hydroxylation on activated C–H
remains a challenge in synthetic chemistry.8 On the other
hand, hydroxylation reaction can be a useful tool for this
type of transformation.
The growing interest in asymmetric synthesis has promoted
a great development in biotransformations in organic
synthesis and has been widely used for the synthesis of
chiral compounds.1 Enantiomerically pure a-hydroxy acids
and their derivatives are important building blocks for the
synthesis of a wide variety of bioactive molecules and other
fine chemicals.2 Moreover, Mandelic acid and its deriva-
tives have been found to act as versatile intermediates for
pharmaceuticals and resolving agents in chiral resolution
processes.3 Due to the great interest in these compounds,
several methods have been developed to obtain the
enantiomerically pure form. These include the kinetic
resolution of racemic a-hydroxy acid ester,4 hydrolysis of
cyabohydrin,5 and reduction of prochiral a-ketoesters.6
However, methyl mandelate and its derivatives can hardly
be gained by C–H oxidation of their corresponding
prochiral aromatic carboxylic acid derivatives.7
Adam et al. reported the a-oxidation of long-chain carb-
oxylic acids to a useful optically active 2-hydroxy acids by
a oxidase of peas (Pisum sativum).9 Recently, Arnold et al.
reported the first enantioselective oxidation of 1 to (S)-2
with engineered cytochrome P450 BM-3;7b however, engi-
neered cytochrome P450 BM-3 shows low to moderate
yields and moderate to high ee (82–90%) on 2-phenylacetic
esters. Holland’s group reported the hydroxylation of long-
chain alkanenitriles (C6–C10) to give the corresponding (R)-
2-alkanols in low yields (8–19%) and high enantiomeric
purity (ee >95%) by Helminthosporium sp. NRRL4671.10
Herein, we report a microbial strain (Helminthosporium sp.
CIOC3.3316), which can hydroxylate methyl 2-phenyl-
acetate to (S)-methyl mandelate with high enantioselec-
tivity (92% ee). Moreover, several microorganisms, which
can reduce methyl benzoylformate to (R)-methyl mandel-
*
Corresponding author. Fax: +86 28 85211877; e-mail: swxia2008@
hotmail.com
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.09.029

2538
Y. Chen et al. / Tetrahedron: Asymmetry 18 (2007) 2537–2540
ate, have also been studied in this paper. This combination
of oxidation and reduction biotranformations thus pro-
vides a method for preparing the enantiomers of chiral a-
hydroxy acid derivatives (Scheme 1).
can be easily cultivated and stored, hence representing a
renewable source of the catalytic entity.
With the whole cells of a microbial strain (Helminthospo-
rium sp. CIOC3.3316) expressing high hydroxylase activity
as discovered in our group as the biocatalyst, a novel
microbial hydroxylation system will be constructed. Reac-
tion conditions, the interrelations of electronic and stereo
effects with regio- and enantioselectivity of the hydroxyl-
ation of aromatic and aliphatic carboxylic acid esters are
also currently under investigation.
OH
Hydroxylation
Reduction
COOCH3
COOCH3
COOCH3
(S)
OH
COOCH3
O
(R)
2.2. Reduction
Scheme 1. Enantioselective hydroxylation and reduction.
The results of the microbial screening for the reduction are
summarized in Table 2. In terms of reduction yields,
among the 41 microorganism strains evaluated, 10 of them
catalyzed the reduction of ketoester 3 to the corresponding
hydroxy acid ester 4 with high yields (>90%, entries 3–12);
six of which (Geotrichum candidum CIOC2.616, Geotrichum
candidum CIOC2.1062, Geotrichum candidum CIOC SY1,
Saccharomyces cerevisiae CIOC2.1396, Yarrowia lipolytica
CIOC2.1506) reduced ketoester 1 to the corresponding
(R)-mandelic acid ester with good enantioselectivities (90–
95% ee, entries 3–5, 7, 9, 11).
2. Results and discussion
2.1. Oxidation
From the results shown in Table 1, the Helminthosporium
sp. CIOC3.3316 performed an (S)-preference with high
enantioselectivity. Strains (entries 1, 3–5) showed little to
no activity. However, using Helminthosporium sp.
CIOC3.3316, 62% yield and 92% ee of methyl mandelate
were obtained (entry 2).
Table 2. Reduction of benzoylformate by whole microbial cells
O
OH
OCH3
OCH3
Table 1. Oxidation of methyl 2-phenylacetate by Helminthosporium sp.
whole microbial cells
O
O
OH
OCH3
OCH3
4
3
Helminthosporium sp.
(R)-methyl mandelate
O
O
Entry Microorganism
Yielda (%) eea (%)
1
2
1
2
3
4
5
6
7
8
9
Trichosporon cutaneum CIOC2.25
Geotrichum candidum CIOC2.1080
Geotrichum candidum CIOC2.616
Geotrichum candidum CIOC2.1062
Geotrichum candidum CIOC SY1
Saccharomyces cerevisiae CIOC2.399
Saccharomyces cerevisiae CIOC2.1396 93
Saccharomyces cerevisiae CIOC2.1090 93
Saccharomyces cerevisiae CIOC SY12 96
Saccharomyces cerevisiae CIOC SY13 93
84
69
92
97
92
97
92(R)
91(R)
95(R)
94(R)
94(R)
85(R)
90(R)
85(R)
90(R)
74(R)
95(R)
70(R)
(S)-methyl mandelate
Entry
Microorganism
Yield
eec
1
2
3
4
5
Helminthosporium sp. CIOC3.1033
Helminthosporium sp. CIOC3.3316
Helminthosporium sp. CIOC3.3319
Helminthosporium sp. CIOC3.3114
Helminthosporium sp. CIOC3.1669
0
—
92(S)
nd
—
nd
82a(63)b
<5%
0
<5%
a Determined by HPLC analysis.
b Isolated yield.
c Determined by HPLC.
10
11
12
Yarrowia lipolytica CIOC2.1506
Pichia farinosa CIOC2.1463
93
93
a Determined by HPLC.
Recently, Arnold et al. reported that the first enantioselec-
tive oxidation of 1 to (S)-2 (90% ee) with engineered cyto-
chrome P450 BM-3 (BM-3; isolated from Bacillus
megaterium) is capable of enantioselective hydroxylation
at the a-position of certain carboxylic and peptide
groups.7b Compared with what Arnold et al. reported, we
used a wild type strain (Helminthosporium sp. CIOC3.3316)
as a biocatalyst, and (S)-2 was prepared by using resting
whole cells with 92% ee. In addition, whole-cell fermenta-
tions offer a different approach to overcoming the cofactor
obstacle: living organisms provide natural recycling sys-
tems for all factors required.11 The tedious process of pro-
tein purification can be avoided, and applications are not
limited by possible enzyme instability. In many cases, cells
In terms of stereoselectivity, eight strains (Trichosporon
cutaneum CIOC2.25, Geotrichum candidum CIOC2.1080,
Geotrichum candidum CIOC2.616, Geotrichum candidum
CIOC 2.1062, Geotrichum candidum CIOC SY01, Saccha-
romyces cerevisiae CIOC2.1396, Yarrowia lipolytica
CIOC2.1506) follow ‘Prelog’s rule’ leading to the (R)-man-
delate while no one strain shows an anti-Prelog specificity
leading to the (S)-mandelate. These results are consistent
with ‘Prelog’s rule’. This simple model states that the
majority of dehydrogenases deliver the hydride ion to the
re-face of a prochiral ketoester. Geotrichum candidum
CIOC2.616 is selected as the best strain to further prepara-
tive synthesis of the (R)-methyl mandelate.

Y. Chen et al. / Tetrahedron: Asymmetry 18 (2007) 2537–2540
2539
3. Conclusion
and then extracted three times with ethyl acetate. Chemical
yields and ee of the product were determined by GC or
HPLC analysis.
In conclusion, the enantiomers of methyl mandelate were
obtained by reduction of methyl benzoylformate and oxi-
dation of methyl 2-phenylacetate. Several strains catalyzed
the enantioselective reduction of methyl benzoylformate
with high conversion and enantiomeric purity to give the
(R)-methyl mandelate. Moreover, it has been shown that
Helminthosporium sp. CIOC3.3316 is capable of hydroxyl-
ating methyl 2-phenylacetate to (S)-methyl mandelate.
Enantioselective hydroxylation in the a-position of carb-
oxylic acid ester provided a novel reaction type with micro-
bial whole cells, and it opens a new route to (S)-mandelic
acid derivates. More importantly, there have been signifi-
cant developments in the direct synthesis of (R) or (S)-
methyl mandelate using microbial cells under solvent free
conditions. As a result, it will contribute to the develop-
ment of green and sustainable synthetic processes.12
4.4. Isolation and characterization of the products
The biotransformation of methyl 2-phenylacetate and
methyl benzoylformate was carried out for 24 h on a
300 mg (2.0 mmol) and 328 mg (2.0 mmol) scale, respec-
tively. The product was extracted with ethyl acetate, dried
and concentrated. The crude of the reaction mixture was
purified by flash chromatography (25% EtoAc/petroleum
ether). This gave isolated yields of 209 mg (1.42 mmol)
and 268 mg (1.61 mmol), respectively (62–81% yield). This
concentrated sample was used for HPLC analysis to deter-
mine its enantiomeric purity. (S)-methyl mandelate:
20
½aꢀD ¼ þ130:2 (c 0.8, methanol), (R)-methyl mandelate:
20
1
½aꢀD ¼ ꢁ135:6 (c 1.0, methanol). The H NMR and 13C
NMR of methyl mandelate are as follows: 1H NMR
(CDCl3, 300 Hz), d 3.45 (s, 1H), 3.76 (s, 3H), 5.18 (s,
1H), and 7.37–7.40 (m, 5H). 13C NMR (CDCl3, 75Hz): d
174.0, 138.2, 128.5, 128.4, 126.6, 72.8, 53.0.
4. Experimental
4.1. General
4.5. Determination of ee of the product formed
Unless otherwise noted, reagents were purchased from
commercial suppliers and used without further purifica-
tion. Compound 1, compound 3, and rac-and (R)-methyl
mandelate were purchased from Aldrich Chemical Co.
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 eluent.
1H and 13C NMR spectra were recorded on a Brucker-
300 (300/75 MHz) spectrometer using CDCl3 as a solvent
and TMS as an internal standard.
The absolute configurations were assigned by chiral-phase
HPLC analysis using authentic (R)-2 or (S)-4 as a stan-
dard. The product from the biotransformation of methyl
2-phenylacetate and methyl benzoylformate was analyzed
on HPLC using a chiral column, chiralcel OD-H (u
0.46 cm · 25 cm) from Daicel, Japan. The mobile phase
used was hexane/isopropanol (90:10), 1 mL/min monitored
at 254 nm. The retention times for racemic methyl mandel-
ate were 11.53 min and 13.11 min.
4.2. Cultivation of microorganisms
Acknowledgment
Microorganisms screened were preserved in our labora-
tory. Yeasts were grown in a medium containing 0.2%
(w/v) glucose, 2% (w/v) peptone, and 1% (w/v) yeast ex-
tract solution (pH 6.8–7.0); bacteria were grown in a med-
ium containing 0.1% (w/v) sodium chloride, 0.5% (w/v)
beef extract, 2% (w/v) peptone solution (pH 6.8); fungi
were grown in a medium of potato with 2% (w/v) glucose.
Strains were maintained on nutrient agar slants at 4 ꢁC.
Erlenmeyer flasks (250 mL), each containing 100 mL of
the appropriate sterilized cultivation medium were inocu-
lated with the tested microorganism and incubated in an
orbital shaker (160 rpm) at 28 ꢁC. After 48 h of growth
(yeast, bacteria) and 72 h (fungi), respectively, the cells
were harvested by centrifugation and washed twice with
cool physiological saline (0.85%).
We are grateful for financial support from the National
Natural Science Foundation of China (No. 20672110).
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