Process Biochemistry 47 (2012) 1037–1041

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Synthesis of methyl (R)-3-(4-fluorophenyl)glutarate via enzymatic

desymmetrization of a prochiral diester
Weiming Liu a , Yi Hu a,b , Ling Jiang a,b , Bin Zou a , He Huang a,b,∗
a
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, PR China
b
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, Nanjing 210009, PR China

a r t i c l e i n f o a b s t r a c t

Article history: An efficient procedure for enzymatic desymmetrization of the prochiral dimethyl 3-(4-
Received 17 September 2011 fluorophenyl)glutarate (3-DFG) in an aqueous–organic phase was successfully developed to prepare
Received in revised form 13 January 2012 methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG). Novozym 435 was selected as a highly efficient
Accepted 17 January 2012
biocatalyst through lipase screening. The effects of various parameters in terms of co-solvent and its
Available online 4 February 2012
concentration, buffer pH, ionic strength and reaction temperature, on the reaction were investigated. It
was found that 0.2 M phosphate buffer (pH 8.0) containing 20% MTBE (v/v) was the optimum reaction
Keywords:
medium, and the optimum reaction temperature was 30 ◦ C. Under the optimized reaction conditions,
Enzymatic desymmetrization
Novozym 435
(R)-3-MFG was obtained in 95.6% ee value and 92.6% yield after 64 h when the concentration of 3-DFG
Co-solvent and Novozym 435 were 200 mmol/l and 20 g/l respectively. Furthermore, Novozym 435 showed an
Enantioselectivity excellent operational stability, retaining above 95% of the initial activity and enantioselectivity after 10
Methyl (R)-3-(4-fluorophenyl)glutarate cycles of reaction. The developed method has a potential to be used for efficient enzymatic production
of (R)-3-MFG.
© 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Optically pure methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-
MFG) is an attractive building block for the synthesis of a number of
pharmaceutically important compounds. The most representative
is (−)-paroxetine hydrochloride [1–3], a selective serotonin reup-
take inhibitor (SSRI) which was used in treatment of depression,
obsessive compulsive disorder, and panic disorder widely [4,5].
Two series of potent P2X7 receptor antagonists discovered by Roche
also can be obtained through an asymmetric synthetic route in
which (R)-3-MFG is an important precursor [6].
Although a vast amount of researches have been devoted to the
preparation of paroxetine [7–9], only a few routes dealing with
3-MFG are known. Until now, enzymatic or non-enzymatic desym-
metrization of prochiral compounds is the main method to obtain
enantiomerically pure 3-MFG, such as enantioselective hydroly-
sis of prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG) and
asymmetric alcoholysis of meso 3-(4-fluorophenyl)glutaric anhy-
dride (3-FGA) [1,6,10–12]. However, there are a limited number
of publications dealing with the enzymatic alcoholysis of meso
∗ Corresponding author at: College of Biotechnology and Pharmaceutical Engi-
neering, Nanjing University of Technology, Nanjing 210009, PR China.
Tel.: +86 25 83172094; fax: +86 25 83172094.
E-mail address: biotech@njut.edu.cn (H. Huang).
3-FGA [12], as the results obtained by enzymatic means are not
at the level reached using chemical transformations [6,10,11].
The enzymatic desymmetrization of prochiral diesters is a ver-
satile and powerful alternative for the synthesis of 3-substituted
glutaric acid monoesters [1,13–20]. López-García et al. [20] inves-
tigated the desymmetrization of 3-DFG by enzymatic aminolysis
and ammonolysis in organic solvent, however, yield was not so
satisfactory although high ee value can be obtained under the opti-
mized reaction conditions. Yu et al. employed porcine liver esterase
(PLE) for the enantioselective hydrolysis of 3-DFG to afford (S)-3-
MFG in 95% ee and 86% yield [1]. Nevertheless, inversion of the
stereochemistry was needed to provide the proper configuration,
as (S)-3-MFG was unwanted for the preparation of (−)-paroxetine
hydrochloride. In addition, PLE is difficult to be reused as it is a
free enzyme. Accordingly, we became interested in employing an
immobilized enzyme to prepare (R)-3-MFG via desymmetrization
of the prochiral diester 3-DFG.
The use of organic co-solvent will overcome the low solu-
bility of hydrophobic substrates in an aqueous medium. It has
been reported that the modification of a reaction medium by
adding organic co-solvent can improve the catalytic activity and
enantioselectivity of an enzyme [21–26]. In the present work,
we attempted to establish an efficient procedure to synthesis
of (R)-3-MFG via enzymatic desymmetrization of 3-DFG in an
aqueous–organic phase (Scheme 1). Effects of reaction parameters
such as co-solvent and its concentration, buffer pH, ionic strength

1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2012.01.014
1038 W. Liu et al. / Process Biochemistry 47 (2012) 1037–1041

F F Table 1
Enantioselective hydrolysis of 3-DFG using various lipases.

Enzyme Entry Lipase Yield (%) ee (%) Configuration

1 PPL 19.5 56.3 (S)

buffer/co-solvent 2 CRL 18.8 11.1 (S)
MeO2C CO2Me HO2C CO2Me 3 RNL 10.1 3.6 (R)
4 HLEa 19.2 26.4 (R)
5 Lipase AK 15.9 0 –
3-DFG (R)-3-MFG
6 Lipase PS 5.3 3.7 (S)
7 Lipase AYS 18.7 0 –
Scheme 1. Enzymatic enantioselective hydrolysis of 3-DFG to (R)-3-MFG in an
8 Lipozyme TLa 15.2 63.0 (S)
aqueous–organic phase.
9 Novozym 435a 53.9 91.8 (R)
a
Immobilized enzyme.
and reaction temperature on the activity and enantioselectivity
of immobilized enzyme were investigated. Moreover, operational
NMR (400 MHz, CDCl3 ): ı 2.58–2.79 (m, 4H), 3.59–3.64 (m, 4H), 6.96–7.00 (m, 2H),
stability of immobilized enzyme under the optimized reaction con-
7.17–7.20 (m, 2H). 13 C NMR (400 MHz, CDCl3 ): ı 37.35, 40.39, 40.59, 51.82, 115.62,
ditions was examined. 128.86, 138.01, 161.88, 172.01, 177.72. The absolute configuration of the product
was assigned by comparing with the results of Ostaszewski and co-workers [12].
2. Materials and methods

2.1. Materials 3. Results and discussion

Novozym 435 (lipase B from Candida antarctica, immobilized on a macroporous
acrylic resin, 10000 PLU/g) and Lipozyme TL (lipase from Thermomyces lanuginosus,
immobilized on silica granulation, 250 IUN/g) were purchased from Novozymes;
Porcine pancreas lipase (PPL, 400 U/mg), Candida rugosa lipase (CRL, 700 U/mg), Rhi-
zopus niveus lipase (RNL, 1.5 U/mg), esterase from hog liver (HLE, 0.2 U/mg) were
obtained from Sigma–Aldrich; Lipase AK (Pseudomonas fluorescens lipase, 20 U/mg),
Lipase PS (Pseudomonas cepacia lipase, 20 U/mg) and Lipase AYS (C. rugosa lipase,
20 U/mg) were kindly donated by Amano Pharmaceuticals. Isopropanol and n-
hexane were of HPLC grade. All other reagents were of analytical grade and used
without any further purification. 3-DFG (HPLC purity >99%) was prepared according
to a procedure previously described in Ref. [27].
2.2. Enantioselective hydrolysis of 3-DFG using different lipases
3-DFG (0.04 mmol) was dissolved in 0.1 M phosphate buffer (pH 8.0) contain-
ing 10% (v/v) acetonitrile as co-solvent and 10 mg lipase was added to 1 ml of this
solution. The mixtures were orbitally shaken at 30 ◦ C and 200 rpm for 24 h, then
stopped by adjusting the pH to ∼2.0 with 5 M HCl and extracted with ethyl acetate
(2× 2.5 ml), the extracts were dried over MgSO4 and analyzed by chiral high perfor-
mance liquid chromatography (HPLC).
3.1. Enzyme screening
Various lipases were firstly selected to catalyze the enantios-
elective hydrolysis of 3-DFG in phosphate buffer (0.1 M, pH 8.0)
containing 10% acetonitrile (v/v) as co-solvent. The results were
listed in Table 1. Of the 9 lipases employed, Novozym 435 showed
the best activity and enantioselectivity, with (R)-enantiomer being
preferentially formed, which has the correct configuration to com-
plete the synthesis of (−)-paroxetine. In the process catalyzed by
the other lipases, lower yields (<20%) and ee values were obtained.
It is noteworthy that PPL, CRL, lipase PS and Lipozyme TL possessed
(S)-stereochemical preference, which was opposite to that of RNL,
HLE and Novozym 435. Similar behaviors have been reported in
the enantioselective hydrolysis of other prochiral diesters using
different lipases [15,16,28]. Novozym 435 was consequently used
throughout the further studies.

2.3. General procedure for enantioselective hydrolysis of 3-DFG 3.2. Effect of reaction medium

The enzymatic hydrolysis was typically carried out by suspending 3-DFG
(0.2 mmol) and Novozym 435 (20 mg) in phosphate buffer or in mixtures with co-
solvents (10–20%, v/v). The mixtures were orbitally shaken at 20–60 ◦ C in a rotary
shaker (200 rpm). The reactions were stopped by adjusting the pH to ∼2.0 with 5 M
HCl and extracted with ethyl acetate (2× 2.5 ml), the extracts were dried over MgSO4
and analyzed by chiral HPLC.
2.4. Analytical methods
The determination of yield and enantiomeric excess (ee) of (R)-3-MFG was per-
formed by chiral HPLC analysis carried out on a HPLC system (DIONEX) equipped
with UV wavelength detector and autosampler, using a Chiralpak AY-H column
(250 mm × 4.6 mm, 0.5 ␮m) thermostated at a temperature of 30 ◦ C. The mobile
phase was a mixture of n-hexane, isopropanol and trifluoroacetic acid (90:10:0.1,
v/v/v) at a flow rate of 1.0 ml/min and UV detection was performed at 266 nm.
The retention time of 3-DFG, (S)-3-MFG and (R)-3-MFG were 15.5 min, 17.4 min
and 20.7 min, respectively. The yield of (R)-3-MFG was calculated as follows: yield
(%) = MP /MS , MP and MS are the final mole of (R)-3-MFG and the original mole of the
substrate, respectively. The ee values were obtained by the ratio of peak areas of
(S)-3-MFG (A1 ) and (R)-3-MFG (A2 ) as follows: ee (%) = (A2 − A1 )/(A2 + A1 ) × 100.
2.5. Production of methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG)
The production of (R)-3-MFG was carried out by suspending 3-DFG (1 g) and
Novozym 435 (0.4 g) in 20 ml reaction medium, which consisted of 16 ml phos-
phate buffer (0.2 M, pH 8.0) and 4 ml MTBE (20%, v/v). The mixture was orbitally
shaken at 30 ◦ C and 200 rpm. At the end of the reaction (∼60 h), the reaction mix-
ture was filtrated to eliminate the immobilized enzyme. The pH of the aqueous
solution was raised to 9 and the aqueous phase was extracted with ethyl acetate
(2× 20 ml) to remove unchanged 3-DFG. The aqueous phase was then acidified to
pH ∼2.0 with 5 M HCl and extracted with ethyl acetate (3× 30 ml). The collected
organic layers were dried over MgSO4 and concentrated under vacuum to yield (R)-
3-MFG as a white crystal (0.848 g, 89.7%, 94.8% ee). [˛]20 D = +6.8 (c = 1.5, CHCl3 ). 1 H
3.2.1. Choice of organic co-solvent
Since the prochiral substrate 3-DFG has a poor solubility in aque-
ous medium, organic solvent was used as co-solvent in order to
enhance the solubility of 3-DFG. However, the organic co-solvent
may influence the activity and enantioselectivity of Novozym 435.
The effect of various organic co-solvents on both the activity and
enantioselectivity of Novozym 435 was examined at a concen-
tration of 10% (v/v). As shown in Fig. 1, the use of hydrophobic
solvents (i-Pr2 O, MTBE) gave better results. Similar results were
also observed in the enzymatic hydrolysis of ␣-acetoxyamide [29].
The addition of MTBE profoundly enhanced the activity and enan-
tioselectivity of Novozym 435 compared to that in phosphate buffer
only. The lowest yield (4.4%) and ee value (73.7%) were obtained
when methanol was used, this may be attributed to that the addi-
tion of methanol will shift the reaction equilibrium to the reverse
direction as methanol is a byproduct of the hydrolysis of 3-DFG.
However, there seems to be no correlation between the activity
and enantioselectivity of Novozym 435 and the solvent parameters
such as log P, dielectric constant, dipole moment, the same as the
previous literature reported [30]. In the consequent experiments
MTBE was chosen as co-solvent for the enantioselective hydrolysis
of 3-DFG.
3.2.2. Effect of co-solvent concentration
Considering the concentration of a co-solvent also has profound
influence on the enzyme activity and enantioselectivity [22–25,31],
the influence of MTBE concentration on the yield and ee value of
 W. Liu et al. / Process Biochemistry 47 (2012) 1037–1041
Fig. 1. Effect of co-solvent (10%, v/v) on enantioselective hydrolysis of 3-DFG. Reac-
tion conditions: 20 mg Novozym 435, 0.2 mmol 3-DFG in 1 ml phosphate buffer
(0.1 M, pH 8.0), 200 rpm, 30 ◦ C, 24 h.
(R)-3-MFG at 24 h was investigated (Fig. 2). At a lower concentra-
tion (≤20%, v/v), the yield increased obviously with the addition
of MTBE, up to a yield of 69.1% at 20% (v/v) MTBE, probably due
to that all substrate can dissolve in MTBE until its volume ratio
reaches 20%. However, an excess of MTBE (>20%, v/v) resulted in
a loss of enzymatic activity. This may be attributed to the mass
action effect as further increase of MTBE concentration will dilute
the substrate concentration in the organic phase and increase the
enzyme concentration based on the aqueous phase. At the same
time, the influence of MTBE concentration on the ee value of (R)-3-
MFG was similar to that on the yield (Fig. 2). The highest ee value
was obtained when the MTBE concentration was 20% (v/v). The
results clearly indicated that 20% (v/v) of MTBE produced the best
effect on the activity and enantioselectivity of Novozym 435.
3.2.3. Effect of buffer pH
It is well known that both the activity and enantioselectivity
of enzymes are highly influenced by buffer pH [19,32] since the
conformation of an enzyme depends on its ionization state [33].
Fig. 2. Effect of MTBE concentration on enantioselective hydrolysis of 3-DFG. Reac-
tion conditions: 20 mg Novozym 435, 0.2 mmol 3-DFG in 1 ml phosphate buffer
(0.1 M, pH 8.0) containing MTBE, 200 rpm, 30 ◦ C, 24 h. Symbols: () yield; (䊉) ee
value.
1039
Fig. 3. Effect of buffer pH on enantioselective hydrolysis of 3-DFG. Reaction con-
ditions: 20 mg Novozym 435, 0.2 mmol 3-DFG in 1 ml phosphate buffer (0.1 M)
containing 20% (v/v) MTBE, 200 rpm, 30 ◦ C, 24 h. Citric acid–sodium citrate buffer,
4.0–5.0; sodium phosphate buffer, 6.0–8.0; glycine–sodium hydroxide buffer,
9.0–10.0. Symbols: () yield; (䊉) ee value.
The pH range of different buffered solutions from 4.0 to 10.0 was
selected to investigate the effect of buffer pH on the activity and
enantioselectivity of Novozym 435. As shown in Fig. 3, the activ-
ity of Novozym 435 exhibited high dependence on pH while the
enantioselectivity was less vulnerable to pH changes. As pH was
elevated from 4.0 to 8.0, the yield increased from 26.8% to 68.7%,
after which it decreased sharply as pH increased, and the yield
declined to 35.9% at pH 10.0, indicating that the enzymatic activity
was partially inhibited in acidic and strong basic solutions. How-
ever, ee value slightly increased with the pH range from 4.0 to 9.0,
and decreased to 91.0% as pH increased up to 10.0. According to the
data obtained in this experiment, pH 8.0 was the optimum for this
hydrolytic reaction.
3.2.4. Effect of ionic strength
Ionic strength is another variable parameter that may affect the
activity and enantioselectivity of enzymes [34,35]. Thus, the effect
of this variable was tested for a series of phosphate buffer con-
centrations between 0 and 1.0 M at pH 8.0 (Fig. 4). There was a
26.6% rise of yield with the increase of buffer concentration from
0 to 0.2 M, probably due to the lower enzyme activity in the acidic
solution when the buffer concentration was below 0.2 M. However,
further increase of buffer concentration gave a slight decrease of
Fig. 4. Effect of ionic strength on enantioselective hydrolysis of 3-DFG. Reaction
conditions: 20 mg Novozym 435, 0.2 mmol 3-DFG in 1 ml phosphate buffer (pH 8.0)
containing 20% (v/v) MTBE, 200 rpm, 30 ◦ C, 56 h. Symbols: () yield; (䊉) ee value.
1040 W. Liu et al. / Process Biochemistry 47 (2012) 1037–1041
Fig. 5. Effect of reaction temperature on enantioselective hydrolysis of 3-DFG. Reac-
tion conditions: 20 mg Novozym 435, 0.2 mmol 3-DFG in 1 ml phosphate buffer
(0.2 M, pH 8.0) containing 20% (v/v) MTBE, 200 rpm, 24 h. Symbols: () yield; (䊉) ee
value.
yield, demonstrating that Novozym 435 is stable even at higher
ionic strength buffer. An insignificant decrease of ee value was
observed while the buffer concentration ranged from 0.05 M to
1.0 M, namely, the influence of buffer concentration on the enan-
tioselectivity of Novyzym 435 could be neglected. Therefore, 0.2 M
phosphate buffer (pH 8.0) was established to be the optimum for
this hydrolytic reaction.
3.3. Effect of reaction temperature
In enzymatic reaction, temperature significantly influences the
activity, enantioselectivity and stability of a biocatalyst and the
thermodynamic equilibrium of a reaction as well [36,37]. Effect
of reaction temperature on enantioselective hydrolysis of 3-DFG
was investigated at different temperatures and the results were
displayed in Fig. 5. It was observed that with the rise of reac-
tion temperature from 20 ◦ C to 60 ◦ C, yield of (R)-3-MFG increased,
indicating that Novozym 435 showed a good thermostability. The
highest yield was obtained at 60 ◦ C, and the decrease of enan-
tioselectivity at the temperature exceeding 30 ◦ C was observed.
This may be due to the conformational change of the active site
of Novozym 435 caused by excessive high temperature. Similar
behaviors for the hydrolysis of diethyl 3-hydroxyglutarate using
Novozym 435 were reported [19]. However, low temperature
seems to be in favor for enhancement of the enantioselectivity of
Novozym 435 [38,39]. Thus, 30 ◦ C was selected as the optimum
temperature by considering a compromise between the yield and
ee value of (R)-3-MFG. Especially, enantioselectivity was prefer-
entially taken into accountant in the preparation of optical pure
compounds.
3.4. Operational stability of Novozym 435
We first monitored the time course of the enantioselec-
tive hydrolysis of 3-DFG catalyzed by Novozym 435 under the
optimized reactions. The results showed that 3-DFG was fully trans-
formed after 64 h and (R)-3-MFG was obtained with 95.6% ee and
92.6% yield. To further examine the potential of Novozym 435 for
the synthesis of (R)-3-MFG, its operational stability was investi-
gated. Novozym 435 was recovered by centrifugation after each
batch reaction (one batch for 64 h), followed by washing with MTBE
to remove substrate and product absorbed onto Novozym 435 and
then introduced into the fresh reactants. It was found that the rela-
tive activity and enantioselectivity of Novozym 435 still remained
above 95% after being reused for 10 cycles, indicating that Novozym
435 exhibited good operational stability and could be potentially
used in industrial production.
4. Conclusions
In this study, we have developed and optimized a new
enzymatic method for the synthesis of (R)-3-MFG through desym-
metrization of a prochiral substrate 3-DFG using a commercial
available immobilized lipase Novozym 435. A biphasic system
containing phosphate buffer (0.2 M, pH 8.0) and 20% MTBE (v/v)
has been established as the optimum reaction medium. Effects of
reaction temperature on the hydrolytic reaction and operational
stability of Novozym 435 were investigated in detail. Under the
optimized reaction conditions, (R)-3-MFG was obtained in 92.6%
yield and 95.6% ee after 64 h when the concentration of sub-
strate and Novozym 435 were 200 mmol/l and 20 g/l respectively.
Improved yield and approximate ee value were obtained in com-
parison with the previous study [1]. Moreover, Novozym 435 could
be reused for at least 10 cycles and showed an excellent opera-
tional stability, demonstrating that enzymatic desymmetrization
of prochiral substrates is a promising method for the preparation
of optically pure (R)-3-MFG.
Acknowledgements
This research was financially supported by the Key Project
of National Natural Science Foundation of China (Grant No.
20936002), the National Natural Science Foundation of China
for Young Scholars (Grant No. 20906049), the National Basic
Research Program of China (Grant No. 2011CB710800) and the
Hi-Tech Research and Development Program of China (Grant No.
2011AA02A209).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.procbio.2012.01.014.
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