Bioresolution production of (2R,3S)-Ethyl-3-phenylglycidate for chemoenzymatic synthesis of the taxol C-13 side chain by galactomyces geotrichum ZJUTZQ200, a new epoxide-hydrolase-producing strain

Article abstract of DOI:10.3390/molecules19068067

A newly isolated Galactomyces geotrichum ZJUTZQ200 strain containing an epoxide hydrolase was used to resolve racemic ethyl 3-phenylglycidate (rac-EPG) for producing (2R,3S)-ethyl-3-phenylglycidate ((2R,3S)-EPG). G. geotrichum ZJUTZQ200 was verified to be able to afford high enantioselectivity in whole cell catalyzed synthesis of this chiral phenylglycidate synthon. After the optimization of the enzymatic production and bioresolution conditions, (2R,3S)-EPG was afforded with high enantioselectivity (e.e.S > 99%, E > 49) after a 8 h reaction. The co-solvents, pH buffer solutions and substrate/cell ratio were found to have significant influences on the bioresolution properties of G. geotrichum ZJUTZQ200. Based on the bioresolution product (2R,3S)-EPG, taxol's side chain ethyl (2R,3S)-3-benzoylamino-2-hydroxy- 3-phenylpropionate was successfully synthesized by a chemoenzymatic route with high enantioselectivity (e.e.S > 95%).

Full text of DOI:10.3390/molecules19068067

Molecules 2014, 19, 8067-8079; doi:10.3390/molecules19068067
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Bioresolution Production of (2R,3S)-Ethyl-3-phenylglycidate for
Chemoenzymatic Synthesis of the Taxol C-13 Side Chain
by Galactomyces geotrichum ZJUTZQ200, a New
Epoxide-Hydrolase-Producing Strain
Chun Wei, Jinlong Ling, Honglei Shen and Qing Zhu *
College of Biological and Environmental Engineering, Zhejiang University of Technology,
Hangzhou 310014, China; E-Mails: springweispring@163.com (C.W.); jor_ling@163.com (J.L.);
shenhonglei-77@163.com (H.S.)
* Author to whom correspondence should be addressed; E-Mail: zhuq@zjut.edu.cn;
Tel./Fax: +86-571-8832-0781.
Received: 3 May 2014; in revised form: 6 June 2014 / Accepted: 11 June 2014 /
Published: 16 June 2014
Abstract: A newly isolated Galactomyces geotrichum ZJUTZQ200 strain containing an
epoxide hydrolase was used to resolve racemic ethyl 3-phenylglycidate (rac-EPG) for
producing (2R,3S)-ethyl-3-phenylglycidate ((2R,3S)-EPG). G. geotrichum ZJUTZQ200
was verified to be able to afford high enantioselectivity in whole cell catalyzed synthesis of
this chiral phenylglycidate synthon. After the optimization of the enzymatic production and
bioresolution conditions, (2R,3S)-EPG was afforded with high enantioselectivity (e.e.S > 99%,
E > 49) after a 8 h reaction. The co-solvents, pH buffer solutions and substrate/cell ratio
were found to have significant influences on the bioresolution properties of G. geotrichum
ZJUTZQ200. Based on the bioresolution product (2R,3S)-EPG, taxol’s side chain ethyl
(2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate was successfully synthesized by a
chemoenzymatic route with high enantioselectivity (e.e.S > 95%).
Keywords: ethyl 3-phenylglycidate; Galactomyces geotrichum; epoxide hydrolase;
synthesis; bioresolution

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1. Introduction
Optically pure EPG, an important pharmaceutical intermediate, has a wide variety of
applications. (2R,3S)-EPG is a key intermediate in the synthesis of taxol’s C-13 side chain ethyl
(2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate [1]. It is also used in the synthesis of the drug
reboxetine which is an inhibitor of the reuptake of norepinephrine [2]. In addition, (2S,3R)-EPG can be
applied to the synthesis of the nootropic drug clausenamide [3]. Analogues of EPG also have a wide
range of applications in drug synthesis. (2R,3S)-3-(4-methylphenyl)glycidic acid methyl ester is an
important intermediate in the synthesis of the cardiovascular drug diltiazem [4]. Moreoever,
(2S,3R)-benzylepoxy acid esters are important intermediates in the synthesis of a series of anti-cancer
drugs that act as aminopeptidase N inhibitors, such as bestatin, phebestin and probestin, etc. [5].
The preparation of chiral phenylglycidate synthons is mainly achieved by chemical synthesis or
enzymatic asymmetric hydrolysis [6]. Among the enzymatic methods, the use of lipase has been
extensively reported. Mithilesh et al. obtained (2R,3S)-EPG with e.e.S > 99% using Lecitase Ultra
immobilized by gelatin as biocatalyst [7]. Zheng et al. prepared (2R,3S)-3-phenylglycidic acid methyl
ester by hydrolyzing rac-3-phenylglycidic acid methyl ester using Pseudomonas putida cells as
biocatalyst [8]. After the optimization of fermentation and catalytic conditions, the enantiomeric
excess of the final product was greater than 99% with a yield of 48%. Manoucheh et al. obtained a
series of chiral epoxy esters including chiral EPG through selective hydrolysis of the corresponding
racemic compounds catalyzed by lipase from pig liver [9].
However, there are also several drawbacks in lipase-catalyzed asymmetric hydrolysis for producing
chiral phenylglycidates, which may hinder its industrial application. For example, the unstable epoxy
acid hydrolysis product may decarboxylate rapidly to the corresponding aldehyde [4], which affects the
stability of the reaction system, and even leads to irreversible degenerative inactivation of lipase.
Another enzymatic option is epoxide hydrolase (EH) [10], which has none of the above drawbacks
in the production of chiral phenylglycidates. In this field, EH-catalyzed hydrolysis of rac-EPG has
been reported using the whole cells of Pseudomonas sp., yielding (2R,3S)-EPG with 95% e.e.S
and 26% yield in 12 h from 0.2% (w/v) of the racemate [11]. Since there are few reports on the
preparation of chiral EPG by EH [11,12], the exploration of new microbial catalysts and the
improvement of EH enantioselectivity are thus necessary.
As a blockbuster anticancer drug, taxol represents one of the most clinically valuable natural
products known to mankind in the recent past [1,13]. The dominant method for the preparation of taxol
was the semisynthesis route by attaching a synthetic optically active C-13 side chain to natural
baccatin III derived from renewable yew leaves. Chemoenzymatic synthesis protocols have been
recognized as a very useful means to prepare optically active compounds [14,15]. Lipase-catalyzed
asymmetric hydrolysis routes have been widely reported in the chemoenzymatic synthesis of taxol’s
C-13 side chain [1]. In this work, we attempted to discover a new EH-producing microorganism that
can be used to prepare chiral (2R,3S)-EPG as a synthon for taxol’s C-13 side chain. As a result, we
obtained a newly isolated microorganism Galactomyces geotrichum ZJUTZQ200 to produce (2R,3S)-EPG
with excellent enantioselectivity by whole-cell catalysis (Scheme 1). Furthermore, (2R,3S)-EPG
prepared by EH was applied for the first time to the synthesis of the taxol C-13 side chain.

Molecules 2014, 19
Scheme 1. Enantioselective hydrolysis of rac-EPG by G. geotrichum ZJUTZQ200.
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2. Results and Discussion
2.1. Screening and Identification of a Strain Producing Enantioselective EH
We have successfully isolated more than 200 strains from soil samples using 3-phenylglycidol, an
analogue of rac-EPG, as the sole carbon source. The selective hydrolysis capabilities of rac-EPG were
checked by HPLC. More than 30 strains showed EH activity. Among them, ten strains could
preferentially hydrolyze (2S,3R)-EPG with high enantioselectivity leading to (2R,3S)-EPG configuration
retention. One of the fungi strains, ZJUTZQ200, afforded the highest e.e.S to produce (2R,3S)-EPG.
The ITS sequence analysis of the strain ZJUTZQ200 was carried out [16]. The sequence data had
been submitted to GenBank under the accession NO. KJ534595. A phylogenetic tree (Figure 1) was
further constructed, and this strain was closely clustered with Galactomyces geotrichum (GenBank
accession No. JF262197), having 99% sequence identity. Based on the results of phylogenetic analysis
and phenotypic tests, the isolated strain ZJUTZQ200 was designated as Galactomyces geotrichum
ZJUTZQ200 and deposited in the China Center for Type Culture Collection (CCTCC M 2013114).
Figure 1. The phylogenetic tree based on 18S-ITS rDNA sequences, constructed by the
neighbor-joining method, showing the relationship between the strain ZJUTZQ200 and the
representatives of some related taxa.
Galactomyces geotrichum(JF262183.1)
99
ZJUTZQ 200(KJ534595)
99
57
Galactomyces geotrichum(JF262197.1)
100
Geotrichum candidum(AB000652.1)
Galactomyces citri-aurantii(AB000664.1)
57
Galactomyces reessii(AB000646.1)
Geotrichum cucujoidarum(AY520175.1)
100
Geotrichum fermentans(AB000651.1)
Dipodascus aggregatus(AB000645.1)
89
100
Geotrichum klebahnii(AB000641.1)
73
Geotrichum carabidarum(AY520162.1)
100
Geotrichum histeridarum(AY520216.1)
0.01

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G. geotrichum was usually reported as a lipase producer. Its lipase also probably hydrolyzed EPG in
the bioresolution reaction, but our results indicated little EPG was hydrolyzed by the lipase in
G. geotrichum ZJUTZQ200 (data not shown). Hitherto, EH produced by G. geotrichum ZJUTZQ200
has never been reported.
2.2. Substrate Specificity of Chiral Resolution by G. geotrichum ZJUTZQ200
The structure of the substrate can significantly affect enzyme selectivity [17,18]. In order to identify
substrate specificity, we investigated the substrate scope of EH in G. geotrichum ZJUTZQ200. Nine
racemic epoxides, including five phenylglycidate compounds, were resolved by the whole cells of
G. geotrichum ZJUTZQ200. As seen in Table 1, G. geotrichum ZJUTZQ200 exhibited a relatively
high enantioselectivity (e.e.S > 95%) towards phenylglycidate compounds 1, 2, 3 and 4, suggesting that
it had potential in producing chiral phenylglycidate synthons. The enantioselectivity of G. geotrichum
ZJUTZQ200 towards compound 5 with a methoxy group substituent on the phenyl ring was
significantly lower than that of other phenylglycidate compounds. The epoxides 6, 7, 8 and 9 were
resolved with low enantioselectivity. The results indicated that chiral resolution by EH of
G. geotrichum ZJUTZQ200 was specific to EPG and its derivatives.
Table 1. Enantioselective hydrolysis of rac-EPG and its analogues by G. geotrichum ZJUTZQ200.
No.
Substrate
Reaction Time (h) a Retention Configuration e.e.S (%) b c (%)
E Value c
1
4
3
6
(2R,3S)
(2R,3S)
(2R,3S)
99.4
98.6
97.2
65
62.5
68
16
2
3
19
10
4
5
3
5
(2R,3S)
(2R,3S)
95.1
60.3
62.1
2.8
6
3
6
6
(2R,3S)
7.3
63.4
1
7
8
9
6
6
4
(2R,3S)
(2S,3R)
(2S,3R)
41.3
17.3
72.4
64.1
24.1
59
2
4
6
a
Racemic epoxide (5 mM, dissolved in DMSO) was hydrolyzed using wet mycelium of G. geotrichum
b
ZJUTZQ200 (1 g) in 10 mL KPB (100 mM, pH 7.0) at 30 °C, 200 rpm for several hours; The e.e.S was
calculated by the equation: e.e.S = (nR,S − nS,R)/(nR,S + nS,R), where nR,S and nS,R are the concentrations of the
(2R,3S)- and (2S,3R)-enantiomers, respectively; c The E value was enantiomeric selectivity which was calculated
by the following equation: E = ln[(1 − c)(1 − e.e.S)]/ln[(1 − c)(1 + e.e.S)], c is the conversion rate of epoxide.

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2.3. Optimization of Enzyme Production
In order to improve EH enzyme production, the culture conditions of G. geotrichum ZJUTZQ200,
such as carbon and nitrogen sources, metal ions, the initial pH and culture time, etc. were optimized by
single factor and orthogonal experiments. The optimal composition of enzyme production medium was
as follows (per liter, pH 5.0): glycerin 10 g, soybean flour 15 g, NaNO3 3 g, MgSO4 0.5 g, KH2PO4 1 g,
KCl 0.5 g. The biomass with EH was improved to 34.3 g/L from the initial 8.2 g/L.
2.4. Effect of Co-solvents on the Resolution of Rac-EPG
In enzymatic catalysis, a co-solvent can enhance the solubility of the substrate in water but also
make it easier to get into the mycelium [19–21]. The effect of different organic solvents as co-solvents
on the enantioselectivity was investigated at a concentration of 4‰ (v/v) (Figure 2). In comparison
with the control without a supplementing co-solvent, the addition of different co-solvents all increased
the e.e.S. Higher e.e.S and E values were obtained by the addition of DMSO, IPA and DMF. When
acetone or ethanol was added, the conversion rate increased greatly so that E value was significantly
decreased. In summary, DMSO as co-solvent afforded the best EH activity with the final e.e.S of
99.3% and E of 15. Therefore, DMSO was chosen as the optimal co-solvent.
Figure 2. Effects of co-solvents on the resolution of rac-EPG by G. geotrichum
ZJUTZQ200. Reaction conditions: 1.0 g wet mycelium, 0.01 mL rac-EPG, 10 mL KPB
(100 mM, pH 7.0), 30 °C, 200 rpm, 6 h.
1: Dimethyl sulfoxide (DMSO), 2: tetrahydrofuran (THF), 3: acetone , 4: isopropanol (IPA), 5: ethanol,
6: N,N-dimethylformamide (DMF).
2.5. Effect of pH Buffer Solutions on Resolution of rac-EPG
The spatial conformation of the enzyme can be significantly affected by the type and pH value of
the reaction buffer system [22], at the same time the dissociation situation of the substrate can also be
influenced to some extent [23,24], thus affecting the binding of the enzyme and the substrate.

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To investigate the effects of pH and buffer type on the EH activity and enantioselectivity, the e.e.S and
E values were examined in different buffer systems (citrate buffer (CB), KPB and Tris-HCl) with a pH
range from 3.6 to 9.0 at 30 °C (Figure 3). The spontaneous hydrolysis rate of rac-EPG in different
buffer systems was also examined. The results indicated that the enantioselectivity of the epoxide
hydrolase produced by G. geotrichum ZJUTZQ200 was very sensitive to pH, which was consistent
with Hellström’s report [25]. In acidic and alkaline buffer systems, the spontaneous hydrolysis rate
was high, while it was relatively low around neutral pH. In citrate buffer, the e.e.S increased with pH
and reached up to 94.7% at pH 6.4; meanwhile, the E value reached its maximum of 24 at pH 5.4. In
KPB of pH 7.2, the e.e.S and E all reached their maxima of 98.5% and 17; besides, the spontaneous
hydrolysis rate was in the lowest level (<10%). In Tris-HCl buffer, the e.e.S and E values were too low
in comparison with the other two buffer systems, and the spontaneous hydrolysis rate was too high. As
a result, KPB at pH 7.2 was chosen as the most favorable buffer solution.
Figure 3. Effects of buffer types and buffer pH on the resolution of rac-EPG by G. geotrichum
ZJUTZQ200. Reaction conditions: 1.0 g wet mycelium, 0.01 mL rac-EPG, 10 mL buffer,
30 °C, 200 rpm and 6 h.
2.6. Effect of Substrate/Cell Ratio on Resolution of rac-EPG
Due to the toxicity of the epoxide moiety to microbial cells and the transport resistance of
biocatalysts [26], the concentration of substrate and the amount of biocatalyst have great effects on the
enantioselectivity and reactivity of EH [27]. As shown in Table 2, when the substrate/cell ratio (%, w/w)
was less than 7%, the e.e.S of EPG could reach >99% after different bioconversion times. Under the
condition of 10% mass ratio, the e.e.S of EPG only reached 78.1%, even if the reaction time was
prolonged. E value increased with the mass ratio and reached its maximum value of 49 at the mass ratio
of 5%. The possible reason is that when the substrate concentration is low (EPG/mycelium < 3%),
the catalytic center of EH is not saturated, resulting in low catalytic efficiency manifested as a low

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E value. High substrate concentration (EPG/mycelium > 7%) may inhibit the activity of the enzyme’s
active center, meanwhile the hydrolysis product can change the pH value of the buffer leading to a
decrease of the EH activity, thus resulting in low enantiomeric selectivity (E value). High E value
and e.e.S were all targets of biocatalysis optimization. e.e.S above 99.5% was especially favorable
in the medicinal synthon preparation. Thus, we selected the mass ratio of 5% as an optimal
substrate/cell ratio.
Table 2. Effect of substrate/cell ratio on enantioselectivity of epoxide hydrolase in
G. geotrichum ZJUTZQ200. Reaction conditions: 1.0 g wet mycelium, 10 mL KPB (100 mM,
pH 7.2), 30 °C, 200 rpm.
Sub./Cell (%) Reaction Time (h) e.e.S (%)
c (%)
75
E Vaule
1
2
4
6
99.4
99.5
99.8
99.5
99.8
78.1
11
12
28
49
24
3
71.7
72.5
69
3
8
5
8
7
8
75.1
74.7
10
13
2.7. Synthesis of Taxol’s C-13 Side Chain
Taxol’s C-13 side chain is of notable importance in the drug’s powerful antileukemic and
tumor-inhibiting activity [28]. A new synthetic strategy towards taxol’s C-13 side chain ethyl
(2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate (13) is shown in Scheme 2. Firstly, based on
the optimal resolution process with G. geotrichum ZJUTZQ200, optically pure (2R,3S)-EPG
(e.e.S > 99%) was obtained in a yield of 37.1%. Secondly, (2R,3S)-EPG was reacted with sodium azide
in methanol to afford 11 in a yield of 95%. Subsequently, the reduction reaction with triphenylphosphine
in THF to afford 12, followed by acylation with benzoyl chloride to obtain the desired ethyl
(2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate (13, e.e.S > 95%) in a total yield of 33.8%. No
racemization was observed during the whole formation of taxol’s C-13 side chain.
Scheme 2. Preparation of taxol C-13 side chain from rac-EPG.
Reaction conditions: (a) G. geotrichum ZJUTZQ200, KPB; (b) NaN3, NH4Cl, methanol, 65 °C; (c)
triphenyl phosphine, THF, rt; (d) benzoyl chloride, dichloromethane, rt.

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3. Experimental Section
3.1. Materials
rac-EPG 1 and microbial media components were purchased from J&K Scientific Ltd. (Shanghai,
China). (2R,3S)-EPG was purchased from UHN Shanghai R&D Co., Ltd. (Shanghai, China). Other
chemicals used were of analytical reagent grade and purchased from various commercial sources.
3.2. Preparation of Derivatives of EPG
Phenylglycidates 2, 3, 4 and 5 were synthesized by Darzens reaction [29]. Briefly, methyl chloroacetate
(10 mmol) was dissolved in a solution of sodium methoxide (10 mmol) in dry methanol (10 mL) under ice
bath cooling and stirred for 5 min, followed by the addition of corresponding aldehyde (5 mmol). The
mixture was kept at 0 °C with stirring until completion and then the methanol was removed under
reduced pressure. Dichloromethane (40 mL) was added and then washed with saturated sodium
chloride solution (50 mL × 3), dried over anhydrous sodium sulfate and concentrated under vacuum
and finally purified by thin layer chromatography (TLC) to give the desired derivatives of EPG. TLC
was performed on F254 silica gel plates with the eluent petroleum ether/ethyl acetate (10:1, v/v). All
the synthesized substrates were confirmed by LC-MS and 1H-NMR. NMR spectra were recorded on a
Digital Avance 400 MHz spectrometer (Bruker) using tetramethylsilane as an internal standard.
Methyl 3-phenylglycidate (2): pale yellow liquid, yield 37%, 1H-NMR (CDCl3) δ 7.25 (s, 5H), 4.01 (s, 1H),
3.70 (d, J = 9.1 Hz, 3H), 3.42−3.39 (m, 1H). MS (ESI) m/z 178.0 (M)+.
Methyl 3-(2-methylphenyl) glycidate (3): pale yellow liquid, yield 42%, 1H-NMR (CDCl3) δ 7.05 (s, 2H),
6.72 (dd, J = 6.8, 1.9 Hz, 2H), 3.94 (s, 1H), 3.68–3.62 (m, 6H), 3.41 (s, 1H). MS (ESI) m/z 192.1 (M)+.
Methyl 3-(4-methylphenyl) glycidate (4): pale yellow liquid, yield 36%, 1H-NMR (CDCl3) δ 7.06 (s, 4H),
3.96 (d, J = 1.7 Hz, 1H), 3.96 (d, J = 1.7 Hz, 3H), 3.42 (s, 1H), 2.25 (s, 3H). MS (ESI) m/z 192.1 (M)+.
Methyl 3-(4-methoxyphenyl) glycidate (5): pale yellow liquid, yield 41%, 1H-NMR (CDCl3) δ 7.15–7.05
(m, 4H), 4.14 (s, 1H), 3.75 (s, 3H), 3.33 (s, 1H), 2.31 (s, 3H). MS (ESI) m/z 208.2 (M)+.
3.3. Screening of Strains and Enantioselective Hydrolysis to Rac-EPG
Soil samples were used for the screening of strains capable of utilizing 3-phenylglycidol as a sole
carbon source. Isolated strains were transferred to 50 mL basal enzyme production medium and
cultivated at 200 rpm, 30 °C for 48 h for bacteria, or 72 h for molds. After the cells were harvested, 1 g
wet cells were washed and suspended in 10 mL 0.1 M, pH 7.0 potassium phosphate buffer (KPB).
Cell suspensions were mixed with 50 μL substrate solution (EPG:DMSO = 2:8,V/V) and kept
at 200 rpm, 30 °C for 6 h reaction, then the optical purity of the remaining substrate was determined by
chiral HPLC. The screening medium contained (per liter, pH 6.5) 10 g soybean flour, 2 g NaNO3, 0.5 g
KCl, 0.5 g MgSO4·7H2O, 1 g K2HPO4·3H2O, and 0.1% (v/v) 3-phenylglycidol. Basal enzyme
production medium contained (per liter, pH 6.5) 10 g soybean flour, 2 g NaNO3, 0.5 g KCl, 0.5 g
MgSO4·7H2O, 1 g K2HPO4·3H2O, and 30 g sucrose.

Molecules 2014, 19
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3.4. Identification of the Strain ZJUTZQ200
The isolated strain was preliminarily identified by morphological and microscopic observation.
Furthermore, its 18S-internal transcribed spacer (ITS) regions of the rDNA were obtained through
gene sequencing. The 18S-ITS region sequence was deposited in the GenBank database. Related
sequences were obtained from GenBank database (National Center for Biotechnology Information)
using the BLAST system. The 18S-ITS regions determined were aligned with the reference sequences
obtained from GenBank databases using ClustalW ver.1.81 [30]. MEGA ver.5.1 was applied for the
calculation of evolutionary distance and finally a phylogenetic tree was constructed using the
neighbor-joining method [31,32].
3.5. Analytical Methods
The commercially available (2R,3S)-EPG was employed as a standard to identify the absolute
configuration. The absolute configurations of the other products were assigned by comparing the
measured specific rotations and retention times on chiral HPLC columns with the reported ones. EPG
and its analogues were detected by HPLC (Waters 1525) equipped with a Daicel Chiralpack AS-H
column (0.46 cm × 25 cm, 5 μm). The mobile phase was a mixture of n-hexane and isopropanol
(8:2, v/v) at a flow rate of 0.8 mL/min. The value of e.e.S was expressed as enantiomeric excess of the
remaining epoxide, which was calculated using the following equation: e.e.S = (nR,S − nS,R)/(nR,S + nS,R)
(nR,S and nS,R were the concentrations of the (2R,3S)- and (2S,3R)-enantiomer, respectively).
E value was enantiomeric ratio of epoxides, which was calculated using the following equation:
E = ln[(1 − c)(1 − e.e.S)]/ln[(1 − c)(1 + e.e.S)] (c, conversion rate) [33].
3.6. Synthesis of Taxol’s C-13 Side Chain
(2R,3S)-EPG (10): rac-EPG (1 mL) in DMSO (4 mL, 2:8, v/v) was dissolved in KPB (195 mL, 100 mM,
pH 7.2) using wet mycelium of G. geotrichum ZJUTZQ200 (20 g) as biocatalyst at 30 °C (oil bathing)
by stirring with a magnetic stir bar at 200 rpm. After 8 h biotransformation, the reaction solution was
centrifuged to remove the mycelium and then extracted by n-butanol (60 mL × 3). The combined
organic phase was concentrated under reduced pressure, followed by TLC purification with the
eluent petroleum ether/ethyl acetate (10:1, v/v). 0.36 g (2R,3S)-EPG (e.e.S > 99%) was obtained.
25
24
[α]D = −148.6 (c 2, CHCl3) {lit. [34] [α]D = −158.8 (c 1.06, CHCl3)}.
(2R,3S)-3-azido-2-hydroxy-benzenepropanoic acid ethyl ester (11): (2R,3S)-EPG (0.25 g, 1.3 mmol) was
dissolved in methanol (10 mL) in round-bottom flask followed by addition of sodium azide (0.25 g, 3 eq.)
and ammonium chloride (0.19 g, 3 eq.), then the mixture was stirred at 65 °C for 6 h. After
completion of the reaction, the solvent was removed under reduced pressure and redissolved
in ethyl acetate (30 mL). The organic phase was washed with saturated sodium chloride (2 × 30 mL)
and dried over anhydrous sodium sulfate and then concentrated under reduced pressure to give 0.3 g
(95% yield) of 11 as a pale yellow oil. 1H-NMR (CDCl3): δ 7.30 (s, 5H), 4.83 (s, 1H), 4.46 (s, 1H), 4.13 (s,
25
20
2H), 2.70 (s, 1H), 1.14 (s, 3H). MS (ESI) m/z 235.1 (M)+. [α]D = +141.1 (c 1.8, CHCl3) {lit. [35] [α]D
= +133.5 (c 2, CH2Cl2)}.

Molecules 2014, 19
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(2R,3S)-3-amino-2-hydroxy-3-phenylpropionic acid ethyl ester (12): Compound 11 (0.3 g, 1.3 mmol)
and triphenylphosphine (1 g, 3 eq.) were dissolved in tetrahydrofuran (30 mL) and then stirred at room
temperature for 6 h. The solvent was removed under reduced pressure and then purified by TLC
1
(eluent petroleum ether/ethyl acetate 2:1, v/v) to obtain 12 as pale yellow oil. H-NMR (CDCl3): δ
7.25–7.18 (m, 5H), 4.43 (s, 1H), 4.34 (s, 1H), 4.05–3.95 (m, 2H), 2.58 (s, 3H), 1.14 (s, 3H). MS (ESI)
25
25
m/z 209.0 (M)+. [α]D = −4 (c 1, CHCl3) {lit. [36] [α]D = −2.9 (c 0.26, CHCl3)}.
Ethyl (2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate (13): Triethylamine (0.72 g, 1.5 eq.) was
dissolved in a solution of 12 (0.1 g, 0.5 mmol) and benzoyl chloride (0.77 g, 1.5 eq.) in
dichloromethane (20 mL) and then stirred at room temperature for 4 h. The solvent was removed under
reduced pressure and then purified by TLC (eluent petroleum ether/ethyl acetate 2:1, v/v) to give 0.102 g
of 13 (e.e.S > 95%) as white solid with a total yield of 33.8%. 1H-NMR (CDCl3): δ 7.68 (d, J = 8.4 Hz,
1H), 7.37–7.14 (m, 10H), 5.51 (dd, J = 8.5, 3.5 Hz, 1H), 4.55 (d, J = 3.5 Hz, 1H), 4.07–3.95 (m, 2H),
25
3.41 (s, 1H), 1.12 (t, J = 7.1 Hz, 3H). MS (ESI) m/z 336.1 (M)+. [α]D = −11.4 (c 1, CHCl3) {lit. [37]
25
[α]D = –12 (c 2, CHCl3)}.
4. Conclusions
Bioresolution of rac-EPG is one of the important potential synthetic routes for industrial production
of enantiopure (2R,3S)-EPG. In this work, we obtained a new strain capable of resolving rac-EPG to
(2R,3S)-EPG with high enantioselectivity and identified it as G. geotrichum ZJUTZQ200. The use of
this strain in enantioselective synthesis of (2R,3S)-EPG has not been previously reported. The substrate
scope investigation results indicated G. geotrichum ZJUTZQ200 could afford high enantioselectivity in the
synthesis of the chiral phenylglycidate synthon, suggesting that it had good potential in industrial
applications. After the optimization of enzyme production and the whole cell catalysis process,
G. geotrichum ZJUTZQ200 could resolve rac-EPG to (2R,3S)-EPG with e.e.S of 99.5% and E value of
49, both of which were higher than those reported in the literature for (2R,3S)-EPG preparation by
enantioselective synthesis using whole cells containing EH [11,12]. Co-solvents, pH buffer solutions
and substrate/cell ratio significantly affected the enantioselectivity of G. geotrichum ZJUTZQ200.
Especially, the E value was improved to 49 from 17 at a substrate/cell ratio of 5%.
As a result, based on the optimized process of (2R,3S)-EPG synthesis by G. geotrichum
ZJUTZQ200, we established a new chemoenzymatic synthesis route to produce taxol’s C-13 side
chain, affording e.e.S of 95% and a total yield of 33.8%. In comparison with the chemoenzymatic
synthesis route based on lipase-catalyzed asymmetric hydrolysis, our scheme is a promising alternative
for the industrial preparation of taxol’s C-13 side chain due to EH’s advantages.
Acknowledgments
This work was supported by the National Natural Science Fund of China (No. 21272212) and the
Natural Science Fund of Zhejiang Province, China (No. LY12B02018, LY12B06010).

Molecules 2014, 19
8077
Author Contributions
C.W. participated in designing the study. H.S. performed the experiments and collected the data.
C.W. and J.L. analyzed the data and wrote the manuscript. Q.Z. directed the study.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the synthetic compounds are available in milligram amounts from
the authors.
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