Lipase catalyzed kinetic resolution for the production of (S)-3-[5-(4-fluoro-phenyl)-5-hydroxy-pentanoyl]-4-phenyl-oxazolidin-2-one: An intermediate for the synthesis of ezetimibe

Article abstract of DOI:10.1016/j.molcatb.2012.08.014

Efficient enzymatic methods were developed for the synthesis of (S)-3-[5-(4-fluoro-phenyl)-5-hydroxy-pentanoyl]-4-phenyl-oxazolidin-2-one, by transesterification of (RS)-3-[5-(4-fluorophenyl)-5-hydroxypentanoyl]-4(S)-4- phenyl-1,3-oxazolidin-2-one [(R,S)-FOP alcohol] and hydrolysis of (RS)-1-(4-fluorophenyl)-5-oxo-5-[(S)-2-oxo-4-phenyloxazolidin-3-yl] pentyl acetate [(R,S)-FOP acetate] using lipase as enzyme source. The synthesized S-diastereomer is an intermediate for the potent cholesterol absorption inhibitor, ezetimibe. Among various lipases tried, Candida rugosa lipase in diisopropyl ether was best for both the reactions. Vinyl acetate was found as suitable acyl donor in transesterification reaction. A higher amount of enzyme (500 mg) was required for the transesterification of 10 mM substrate; it may be due to the enzyme denaturation by acetaldehyde formed in the reaction. The ester hydrolysis reaction worked well, excellent conversion and de were obtained at 40 °C, pH 7. The 300 mg enzyme hydrolyzed 120 mg (R,S)-FOP acetate with 50% conversion and 99.5% de.

Full text of DOI:10.1016/j.molcatb.2012.08.014

Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 99–104
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Journal of Molecular Catalysis B: Enzymatic
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Lipase catalyzed kinetic resolution for the production of
(S)-3-[5-(4-fluoro-phenyl)-5-hydroxy-pentanoyl]-4-phenyl-oxazolidin-2-one:
An intermediate for the synthesis of ezetimibe
Amit Singh, Yogesh Goel, Amit Kumar Rai, U.C. Banerjee∗
Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar 160062, Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 June 2012
Received in revised form 22 August 2012
Accepted 22 August 2012
Available online 29 August 2012
Efficient enzymatic methods were developed for the synthesis of (S)-3-[5-(4-fluoro-phenyl)-5-
hydroxy-pentanoyl]-4-phenyl-oxazolidin-2-one, by transesterification of (RS)-3-[5-(4-fluorophenyl)-5-
hydroxypentanoyl]-4(S)-4-phenyl-1,3-oxazolidin-2-one [(R,S)-FOP alcohol] and hydrolysis of (RS)-1-
(4-fluorophenyl)-5-oxo-5-[(S)-2-oxo-4-phenyloxazolidin-3-yl] pentyl acetate [(R,S)-FOP acetate] using
lipase as enzyme source. The synthesized S-diastereomer is an intermediate for the potent cholesterol
absorption inhibitor, ezetimibe. Among various lipases tried, Candida rugosa lipase in diisopropyl ether
was best for both the reactions. Vinyl acetate was found as suitable acyl donor in transesterification reac-
tion. A higher amount of enzyme (500 mg) was required for the transesterification of 10 mM substrate;
it may be due to the enzyme denaturation by acetaldehyde formed in the reaction. The ester hydrolysis
reaction worked well, excellent conversion and de were obtained at 40 ◦C, pH 7. The 300 mg enzyme
hydrolyzed 120 mg (R,S)-FOP acetate with 50% conversion and 99.5% de.
Keywords:
Transesterification
Ester hydrolysis
Candida rugosa lipase
Ezetimibe
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
antagonizing Niemann-Pick C1-like protein 1 (NPC1L1) action
[11–13]. The synthesis of ezetimibe requires enantiomerically
Microbial enzymes are routinely used as green alternatives
for the synthesis of chiral pharmaceuticals with required enan-
tiomeric purity. Today, drug regulatory agencies prefer maximum
enantiomeric purity level in the drug substances to avoid varia-
tions in pharmacological responses elicited by racemates. Lipase
has been a preferred and popular choice as the biocatalyst, espe-
cially due to its ability to work at aqueous/organic interphase,
overcoming the solubility issue of hydrophobic compounds. Res-
olution of racemic alcohols using lipases has been the major tool
for the synthesis of chiral alcohols. Lipases can catalyze selec-
tive esterification or transesterification of the racemic alcohol to
yield an acylated alcohol, leaving other enantiomer unaffected
[1–6]. Alternatively, the racemic ester may be prepared first by
chemical means and then selectively hydrolyzed to alcohol by
lipase, leaving other ester unaffected [7–10]. Thus enantiopure
ester and alcohol may be recovered from the reaction mixture
by simple chromatographic methods, since, the ester formed and
alcohol left exhibit marked difference in physical and chemical
properties. Ezetimibe, the first marketed drug of ACAT (acyl-
CoA-cholesterol acyl transferase) inhibitor suppresses cholesterol
absorption, storage and transport from the intestine, mainly by
pure alcohol, 3-[5-(4-fluorophenyl)-5(S)-hydroxypentanoyl]-4(S)-
4-phenyl-1,3-oxazolidin-2-one [(S)-FOP alcohol], as crucial
a
intermediate. This is generally synthesized by the reduction of
the corresponding ketone, 1-(4-fluorophenyl)-5-(2-oxo-4-phenyl-
oxazolidin-3-yl)-pentane-1,5-dione [FOP dione] using various
chemical catalysts (Fig. 1). Fu et al. used Corey–Bakshi–Shibata
catalyst [(R)-MeCBS] with borane tetrahydrofuran complex (BTHF)
and obtained 92–96% de with <1% diol formation [14]. An improve-
ment of this process was provided by Bertrand et al. [15]
using (R)-MeCBS with borane diethylaniline complex (BDEA) as
the reducing agent. The de was up to 98% with <1% diol for-
mation. (−)-␤-Chloro diisopinocampheyl borane ((−)-DIP) and
(R)-diphenylprolinol have also been used as catalyst for this pur-
pose [16,17]. All the above mentioned catalysts produce diol
byproduct in the reduction process and the synthesized (S)-FOP
alcohol is not chirally pure (<98% de). Moreover, the boron used
in these catalysts is non degradable, may be hazardous for the
environment. Only one microbial catalyst (Schizosaccharomyces
octosporus ATCC 2479) has been reported for the reduction of FOP
dione with 33–34% product yield and 100% de [18]. There is no
report yet for the production of (S)-FOP alcohol using lipase as
catalyst.
The aim of this work was to develop the efficient enzymatic
method for the synthesis of pure (S)-FOP alcohol. Both, transesteri-
fication and hydrolysis reactions were tried for this purpose.
∗
Corresponding author. Tel.: +91 172 2214682/687x2142; fax: +91 172 2214692.
E-mail address: ucbanerjee@niper.ac.in (U.C. Banerjee).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.08.014

100
A. Singh et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 99–104
OH
O
O
O
OH
O
O
OH
N
N
O
O
N
F
F
F
O
F
FOP dione
(S)-FOP alcohol
Ezetimibe
Fig. 1. Reduction of FOP dione to (S)-FOP alcohol, intermediate of ezetimibe, by chemical catalysts.
4H). 13C NMR: ı 172.55, 163.32, 153.76, 140.21, 140.18, 139.39,
139.04, 129.24, 129.21, 128.92, 128.76, 115.31, 115.10, 73.28, 70.02,
57.56, 38.25, 35.17, 20.27.
2. Materials and methods
2.1. Chemicals
1-(4-Fluorophenyl)-5-(2-oxo-4-phenyl-oxazolidin-3-yl)-
pentane-1,5-dione (FOP dione) was a kind gift from Ind-Swift
Pharmaceutical Ltd. (Chandigarh, India). Lipases were purchased
from Sigma–Aldrich (Germany). Borontetrahydrofuran (BTHF) and
dimethylamino-pyridine (DMAP) were obtained from Aldrich Ltd
(Germany). Various solvents used for HPLC analysis were pur-
chased from J.T. Baker (Phillipsburg, NJ, USA), Rankem (Mumbai,
India) and Merck Ltd (Whitehouse Station, NJ, USA), and were
of HPLC grade. Solvents for synthesis and reaction were of the
highest analytical grade and were dried over molecular sieves
(3 A) before use. Buffer salts were obtained from Qualigens Inc.
(Mumbai, India).
2.3.2. Synthesis of (RS)-FOP acetate
(RS)-FOP alcohol (2 g, 5.6 mmol) was dissolved in DCM and reac-
tion mixture was cooled to 0 ◦C. Triethylamine (1.16 mL) was added
followed by acetic anhydride (0.53 mL) and DMAP (0.068 g). Reac-
tion was monitored for 4 h by TLC. The reaction was worked up by
diluting the reaction mixture with DCM, washing it with 1 N HCl
and at last with brine solution to remove DMAP. The product was
isolated and then purified using column chromatography on silica
gel (5:1 hexane:ethyl acetate), yielded 1.9 g (4.75 mmol, 95%). 1H
NMR: ı 7.32–7.39 (m, 3H), 7.25–7.30 (m, 4H), 6.97–7.01 (m, 2H),
5.65–5.68 (m, 1H), 5.37–5.41 (m, 1H), 4.67 (t, J = 8.1 Hz, 1H), 4.27
(dd, J = 7.9 Hz, 3.6 Hz, 1H), 2.92–2.98 (m, 2H), 2.04 (d, J = 2.44 Hz, 2H),
1.86–1.91 (m, 1H), 1.53–1.79 (m, 4H); 13C NMR (100 MHz, CDCl3):
ı 20.09, 21.05, 35.11, 57.56, 60.40, 70.02, 74.95, 115.23, 115.44,
125.91, 128.19, 128.27, 128.76, 129.21, 136.14, 139.05, 153.71,
161.09, 165.53, 170.26, 171.18, 172.15.
2.2. Analytical methods
FOP alcohol and ester formed (both by chemical and enzymatic
methods) were analyzed using chiral reversed phase column (Lux
5␮ Amylose, 4.6 mm × 250 mm, Phenomenex) in Shimadzu HPLC
system consisting of LC-10AT pump, SPD-10A UV–VIS detector.
The corresponding chiral alcohol and esters were eluted using
ACN:water (55:45) as mobile phase at a flow rate of 0.5 mL/min
and absorbance was measured at 215 nm with retention time 11.90
and 12.94 min for (S)- and (R)-alcohol, and retention time 16.80 and
17.92 min for (R)- and (S)-acetate ester, respectively.
Similarly, (R,S)-FOP butyrate/isobutyrate were synthesized by
esterification of (R,S)-FOP alcohol with butyric anhydride and
isobutyric anhydride, respectively.
2.3.3. Synthesis of FOP dione
Pyridinium chlorochromate (151 mg, 0.7 mmol) was dissolved
in DCM, placed on the magnetic stirrer and equipped with con-
denser. (R)-FOP alcohol (250 mg, 0.7 mmol) dissolved in DCM, was
added to pyridinium chlorochromate suspension with stirring and
heated to 40 ◦C. Progress of reaction was checked for 2 h by TLC (3:1
hexane:ethyl acetate). The reaction was worked up by dilution with
DCM and filtered through celite bed. The filtrate was washed with
1 M NaOH solution, followed by saturated salt solution and dried.
The product was purified using column chromatography on silica
gel (5:1 hexane:ethyl acetate), yielded 227 mg (0.64 mmol, 91%).
The structure was confirmed by 1H NMR (300 MHz) and 13C
NMR (100 MHz) using CDCl3 as solvent (Bruker DPX 300 spec-
trometer; Billerica, MA, USA). Chemical shifts were recorded in
parts per million, with tetramethylsilane as internal standard. Thin-
layer chromatography was performed using pre-coated silica gel
plates (Merck 60 F254; Merck, Darmstadt, Germany). Column chro-
matography was performed on Silica Gel 60 (0.040–0.063 mm,
230–400 mesh; Merck), employing hexane and ethyl acetate as
developing solvents.
2.3. Preparation of racemic substrates
2.4. Lipase-mediated transesterification of (R,S)-FOP alcohol
2.3.1. Synthesis of (RS)-FOP alcohol
Commercially available lipases from different sources were used
for the resolution of (R,S)-FOP alcohol. Each lipase was used in three
solvents (hexane, diisopropyl ether (DIPE) and toluene) separately
and vinyl acetate was used as acyl donor. The substrate (R,S)-FOP
alcohol had partial, moderate and good solubility in hexane, DIPE
and toluene, respectively. The reaction (5 mL) was set up with
3.6 mg substrate, 50 mg enzyme (free powder or immobilized on
solid support) and 5 mM vinyl acetate. The reaction contents were
stirred on magnetic stirrer (500 rpm, for lipases in powder form)
or rotatory shaker (250 rpm, for immobilized lipases) at 35 ◦C. The
samples (200 ␮L) from the organic layer of the reaction mixture
was taken out at 24 h interval for 5 days and dried on rotavapor.
FOP dione (5 g, 14 mmol) was dissolved in dry DCM (previously
dried by distillation with P2O5) at room temperature and then
THF:BORANE complex (13.95 mL) was added to reaction mixture
at 0–5 ◦C with stirring (300 rpm). The reaction was subjected to
nitrogen atmosphere to maintain the reducing condition. Reaction
was monitored for 6 h using TLC (3:1 hexane:ethyl acetate). Sol-
vent was evaporated and purified using column chromatography
on silica gel (5:1 hexane:ethyl acetate), yielded 3 g (8.4 mmol, 70%).
1H NMR: ı 7.32–7.42 (m, 4H), 7.23–7.28 (m, 3H), 6.99 (t, J = 8.1 Hz,
2H), 5.40 (dd, J = 8.6 Hz, 3.5 Hz, 1H), 4.59–4.77 (m, 2H), 4.26 (dd,
J = 8.0 Hz, 3.6 Hz, 1H), 2.89–3.00 (m, 2H), 2.17 (s, 1H), 1.52–2.04 (m,

A. Singh et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 99–104
101
It was re-dissolved in acetonitrile (1 mL), filtered through 0.22 ␮M
2.7.1. Temperature
filter and analyzed through HPLC system.
In order to determine the optimum temperature for the hydrol-
ysis of (R,S)-FOP acetate by Candida rugosa lipase, reactions were
carried out in 5 mL reaction volume having 10 mg substrate dis-
solved in 1.5 mL DIPE and 150 mg enzyme suspended in 3.5 mL
phosphate buffer (pH 7). The reaction contents were stirred on
magnetic stirrer (500 rpm) and incubated at various temperatures
ranging from 25 to 50 ◦C. The organic layer of the reaction mixture
was taken out at 24 h interval up to 5 days, dried on rotavapor and
analyzed by HPLC.
2.5. Optimization of transesterification reaction
Experiments were conducted to check the effect of acylating
agents, type of co-solvent and substrate and enzyme concentration
on the transesterification of (R,S)-FOP alcohol.
2.5.1. Acylating agent
Different acylating agents (vinyl acetate, vinyl butyrate, benzyl
acetate and acetic anhydride) were tried for the resolution of (R,S)-
FOP alcohol by C. antarctica and C. rugosa lipase. Toluene was used
as solvents with the C. antarctica lipase whereas DIPE was used with
C. rugosa lipase. Reactions (5 mL) were set up with 3.6 mg substrate
and 100 mg enzyme. The acylating agents (5 mM) were added, sep-
arately to each reaction. Samples (200 ␮L) were withdrawn at 24 h
interval for 5 days, dried in rotavapor and analyzed through HPLC
system.
2.7.2. Reaction pH
The substrate (10 mg) was dissolved in 1.5 mL organic solvent
and 150 mg enzyme suspended in 3.5 mL 0.1 M potassium phos-
phate buffer having a pH range from 5.8 to 8, was added. The
reaction contents were stirred on magnetic stirrer (500 rpm) at
40 ◦C. The samples (200 ␮L) from the organic layer of the reac-
tion mixture was taken out at 24 h interval up to 5 days, dried on
rotavapor and analyzed by HPLC.
2.7.3. Various (R,S)-FOP esters
2.5.2. Co-solvent
Butyric and isobutyric esters of racemic FOP-alcohol were also
tried. The hydrolysis reaction was set up with 150 mg enzyme,
10 mg esters in DIPE (1.5 mL) with 3.5 mL 0.1 M phosphate buffer,
pH 7 and incubated at 40 ◦C for 5 days. The samples (200 ␮L) of the
reaction mixture was taken out at 24 h interval for 5 days, dried on
Rotavapor and analyzed by HPLC.
The solubility of (R,S)-FOP alcohol in DIPE was increased by the
addition of various co-solvents such as tetrahydrofuran, toluene,
dioxane, DCM, DMF, etc. Reactions were set up with 3.6 mg sub-
strate, 100 mg C. rugosa lipase in DIPE and co-solvents (5–20%, v/v)
to make the total reaction volume 5 mL. The reaction was stirred on
magnetic stirrer (500 rpm) at 35 ◦C. The samples (200 ␮L) from the
organic layer of the reaction mixture was taken out at 24 h interval
for 5 days, dried over rotavapor and analyzed by HPLC.
2.7.4. Alcoholysis of the (R,S)-FOP acetate
Different alcohols (20 mM) were used in place of buffer for the
hydrolysis of (R,S)-FOP acetate (10 mg) by the C. rugosa lipase at
40 ◦C for 5 days. Samples (200 ␮L) were collected at 24 h interval
up to 5 days and dried on rotavapor. The reaction contents were
analyzed by HPLC.
2.5.3. Substrate and enzyme concentration
Reactions (5 mL) were set up containing various quantity (3.6,
9, 18 and 36 mg) of (R,S) FOP alcohol with 200 mg C. rugosa lipase
in DIPE as solvent and vinyl acetate as acylating agent. The reac-
tion mixture was stirred on magnetic stirrer (500 rpm) at 35 ◦C.
The samples (200 ␮L) from the organic layer of the reaction mixture
was taken out each day for 5 days, dried in rotavapor and analyzed
through HPLC system. Various enzyme concentrations were used
for the resolution of 18 mg (R,S)-FOP alcohol. Reactions (5 mL) were
set up containing 200–500 mg C. rugosa lipase in DIPE using vinyl
acetate as acyl donor and stirred on a magnetic stirrer (500 rpm) at
35 ◦C. The samples (200 ␮L) from the organic layer of the reaction
mixture was taken out each day up to 6 days, dried in rotavapor
and analyzed by HPLC.
2.7.5. Substrate and enzyme concentration
The (R,S)-FOP acetate at different quantity (10, 20, 40, 60,
90, 120, 150 and 180 mg) was hydrolyzed with varying enzyme
concentrations (150, 200, 250 and 300 mg) in order to achieve max-
imum conversion and de of the product formed. The reaction was
carried out in 5 mL DIPE:buffer (1.5:3.5 mL) at 40 ◦C, and stirred on
magnetic stirrer (500 rpm). The organic layer of the reaction mix-
ture was taken out at 24 h interval up to 5 days, dried on rotavapor
and analyzed by HPLC.
2.6. Lipase-mediated hydrolysis of (R,S)-FOP acetate
3. Results and discussion
Similar to the transesterification reaction, commercially avail-
able lipases from different sources were used in three solvents
[hexane, DIPE and toluene], for the hydrolysis of (R,S)-FOP acetate.
The reaction was set-up by dissolving the substrate, [(R,S)-FOP
acetate, 10 mg] in 1.5 mL organic solvent, separately and 50 mg
enzyme suspended in 3.5 mL phosphate buffer (pH 7) was added
to it. The reaction was stirred on magnetic stirrer (500 rpm, for
lipases in powder form) or rotatory shaker (250 rpm, for immobi-
lized lipases) at 35 ◦C. The samples (200 ␮L) from the organic layer
of the reaction mixture was taken out at 24 h interval up to 5 days
and dried on rotavapor. It was again dissolved in acetonitrile (1 mL),
filtered through 0.22 ␮M filter and analyzed by HPLC.
3.1. Transesterification of (R, S)-FOP alcohol
Various commercially available lipases from different sources
were tried for the resolution of (R,S)-FOP alcohol using differ-
ent solvents (heptanes, DIPE and toluene). Immobilized lipases on
various supports (Pseudomonas cepacia sol–gel, Candida antarctica
acrylic–resin and Lipozyme) and free lipases from porcine pancre-
atine and Mucor miehei showed no conversion. The lipases from
Candida cylindrica and Aspergillus niger showed resolution of (R,S)-
FOP alcohol in heptane only (Table 1), which was not desirable due
to the partial solubility of FOP alcohol in heptane. Only C. antarctica
lipase showed transesterification in polar solvent (toluene), how-
ever, the diastereomeric excess (de) was very less (69.28%). Various
other polar solvents such as di chloro methane, tetrahydrofuran,
dimethyl formamide and dioxane were used with C. antarctica
lipase, no improvement in de was observed. The C. rugosa lipase
in DIPE was found as best catalyst for this reaction, since 100% de
of the product was obtained (Table 1) and DIPE provided moderate
2.7. Optimization of ester hydrolysis
The various physico-chemical parameters such as temperature,
reaction pH, type of ester and substrate and enzyme concentration
on the ester hydrolysis were optimized.

102
A. Singh et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 99–104
Table 1
Screening of lipases from various commercial sources for the transesterification and hydrolysis of (R,S)-FOP alcohol and (R,S)-FOP acetate, respectively. (Substrate 2 mM,
enzyme 50 mg, reaction volume 5 mL.)
Source of lipases
Solvents
Heptane
Diisopropyl ether
Conversion (%)
Toluene
Conversion (%)
de (%)
de (%)
Conversion(%)
de (%)
T
H
T
H
T
H
T
H
T
H
T
H
Candida antarctica B
Candida rugosa
Candida cylindrica
Aspergillus niger
Mucor miehei
44.8
47.5
28.2
6.5
10.8
13.1
7.2
3.2
3.9
39
100
100
100
–
39.7
97.7
89.6
76.4
94.1
42.2
12.5
–
–
–
5.2
8.6
–
–
4.2
52.2
100
–
–
–
51.1
98.5
–
–
89.1
16.4
–
–
–
–
2.1
–
–
–
–
69.3
–
–
–
–
74.3
–
–
–
–
–
T, transesterification reaction; H, ester hydrolysis reaction; “–”, “not detectable”.
solubility of the substrate, (R,S)-FOP alcohol. All the used lipases
esterified the (R)-FOP alcohol to (R)-FOP acetate.
transesterification reaction, vinyl acetate is converted to vinyl alco-
hol which tautomerizes to acetaldehyde. The enzyme denaturation
during transesterification reaction using vinyl acetate as acylat-
ing agent [20,21], specially denaturation of C. rugosa lipase, is well
reported [22,23] in literature.
3.1.1. Effect of acylating agent
The effect of various acylating agents on the transesterification
of alcohol by lipases are well reported in the literature [19]. Various
acylating agents (vinyl acetate, vinyl butyrate, benzyl acetate and
acetic anhydride) were used for the transesterification of (R,S)-FOP
alcohol using C. antarctica and C. rugosa lipases. Toluene was used
as solvent for the C. antarctica lipase as an effort to increase both
the conversion and selectivity. The DIPE was used as reaction sol-
vent with the C. rugosa lipase to increase the conversion. However,
it was observed that in all the transesterification reactions, vinyl
acetate was the best acylating agent and no reaction was observed
with vinyl butyrate and acetic anhydride as the acylating agents.
Transesterification reaction was carried out with benzyl acetate as
acylating agent using C. antarctica lipase in toluene, but both the
conversion (21.2%) and de (54.8%) were lower compared to that
of carried out by vinyl acetate as acylating agent (35.9% conver-
sion and 58.4% de). Excellent selectivity (100%) and good conversion
(23.48%) was achieved with C. rugosa lipase with vinyl acetate using
DIPE as solvent. All the subsequent experiments were carried out
with C. rugosa lipase with DIPE as a solvent and vinyl acetate as
acylating agent.
3.2. Enantioselective hydrolysis of (R,S)-FOP acetate
The transesterification reaction for the resolution of (R,S)-FOP
alcohol resulted in low yield. Another approach, hydrolysis of
the corresponding ester (R,S)-FOP acetate to (S)-FOP alcohol, was
tried. All the lipases tried for the transesterification reaction were
also used for hydrolysis reaction. Here also, Pseudomonas cepacia
sol–gel, Porcine pancreatine and C. antarctica acrylic–resin lipases
did not show any conversion while lipase from M. miehei showed
very less conversion. More or less, similar pattern as seen in the
case of transesterification reaction, was observed in the hydrolysis
of (R,S)-FOP acetate. No conversion was shown by the immobi-
lized lipases and only C. antarctica free lipase showed conversion in
the polar (toluene) solvent with very less de. Lipases from Candida
cylindracea and A. niger showed very less conversion with lower
selectivity, when heptane was used as a solvent, however, there
was no conversion of the (R,S)-FOP acetate by Candida cylindracea
and A. niger lipases when the hydrolysis reactions were carried out
in DIPE and toluene as reaction medium. Good de was obtained
with C. rugosa lipase (98.54%) in DIPE as reaction medium with less
(8.61%) conversion (Table 1). Here also, all the enzymes preferen-
tially act on the (R) isomer i.e. hydrolyzed the (R)-FOP acetate to
(R)-FOP alcohol, thus followed the Kazlauskas rule [24].
3.1.2. Effect of co-solvent
Since, the substrate was moderately soluble in DIPE, various
polar solvents such as tetrahydrofuran, toluene, dioxane, DCM,
DMF, etc. were added as co-solvent (5–20%, v/v) in order to make
(R,S)-FOP alcohol more soluble in the reaction mixture. Marked
decrease in conversion was observed with the increased concen-
tration of all the co-solvents tried (data not shown). This may be
due to the detrimental effect of polar solvents on the C. rugosa lipase
activity.
Organic solvents with higher polarity such as tetrahydrofuran,
acetone, acetonitrile, pyridine, dichloromethane, 1,4-dioxane, etc.
3.1.3. Effect of substrate and enzyme concentration
The substrate concentration was increased from 3.6 to 36 mg
in the reaction mixture. Maximum conversion (48%) was achieved
with 3.6 mg substrate, the conversion decreased to 36 and 24%
at 18 and 36 mg substrate concentration, respectively. It may be
due to the inhibition of lipase activity at higher substrate con-
centrations. However, de remained unchanged with the increased
concentration of substrate. Resolution of 18 mg (R,S)-FOP alcohol
was tried with 200–500 mg C. rugosa lipase, complete conver-
sion (50%) with 100% de was achieved with 500 mg enzyme.
It was observed that at lower enzyme concentration (200 mg),
conversion had decreased to 33% (Fig. 2). The decrease in con-
version at lower enzyme concentration was probably due to the
denaturation of lipase by acetaldehyde formed during longer incu-
bation period (5–6 days), whereas, only 2–3 days were required
to achieve highest conversion at higher enzyme concentration. In
Fig. 2. Effect of enzyme concentration on the transesterification of (R,S)-FOP alcohol
by C. rugosa lipase (substrate 18 mg, DIPE 5 mL, vinyl acetate 25 mM). The bar and
line graphs represent conversion and de of the product formed, respectively.

A. Singh et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 99–104
103
Table 2
Effect of enzyme and substrate concentration on the resolution of (R,S)-FOP acetate
by C. rugosa lipase (solvent DIPE + water, 40 ◦C, pH 6.2, water content 70%).
Serial no
1
Enzyme used
(mg)
(R,S)-FOP acetate
(mg)
Conversion (%)
de (%)
150
200
250
300
10
20
40
40
60
50
51
36
50
51
36
51
37
32
50
35
30
99.2
98.6.
97.5
99.3
99.2
98.2
99.5
98.8
97.7
99.5
97.9
97.8
2
3
4
90
90
120
150
120
150
180
Fig. 3. Effect of temperature on the hydrolysis of (R,S)-FOP acetate by C. rugosa
lipase (substrate 10 mg, enzyme 150 mg, DIPE 1.5 mL, buffer 3.5 mL). The bar and
line graphs represent conversion and de of the product formed, respectively.
Table 3
Alcoholysis of (R,S)-FOP acetate by C. rugosa lipase. (Substrate 5 mM, enzyme 150 mg,
solvent DIPE, 40 ◦C.)
Serial no.
Alcohol
Conversion (%)
de (%)
Time (days)
were tried in place of DIPE for the hydrolysis of (R,S)-FOP acetate,
however, there was no conversion or very less conversion with low
de was obtained. Usually solvents with low log P values are more
hydrophilic and they reduce the catalytic activity of enzymes [25].
Heptane was not selected as reaction medium because of low sol-
ubility of the substrate, although conversion was higher (13.08%)
with comparable de (97.66%). Hence, DIPE was selected as reaction
medium. It may be mentioned that ester group imparted higher
solubility to the FOP acetate, making it well soluble in DIPE.
1
2
3
4
5
6
Methanol
Propanol
Butanol
Isobutyl alcohol
Hexanol
21.4
26.6
24.4
27.3
23.9
17.9
>95%
>95%
>95%
>95%
>95%
>95%
20
20
20
20
20
20
Octanol
3.3. Alcoholysis of the (R,S)-FOP acetate
Use of alcohols in place of water for the hydrolysis reaction is
well known to improve the selectivity of the reaction [26–28]. Var-
ious alcohols were tried for alcholysis of (R,S)-FOP acetate using
C. rugosa lipase. It was observed that rate of alcholysis of (R,S)-
FOP acetate was very less along with decreased stereoselectivity,
as compared to hydrolysis reaction (Table 3). Percentage conver-
sion was very less with lower de, moreover, all the reactions took a
longer incubation time (20 days) for conversion.
3.2.1. Effect of temperature and pH
The effect of temperature on the hydrolysis of (R,S)-FOP acetate
by C. rugosa lipase was studied at various temperatures ranging
from 25 to 50 ◦C. Increased conversion was observed at higher tem-
perature and it reached to maximum (50%) at 40 ◦C, and decreased
thereafter. However, the de values decreased slightly with the
increased conversion (Fig. 3). The (R)-FOP acetate was hydrolyzed
to (R)-FOP alcohol by C. rugosa lipase, leaving (S)-FOP acetate as
such which was the product of interest. The higher conversion
was also an important criterion of the reaction, where a slightly
decreased de may be compromised. Hence, 40 ◦C reaction tempera-
ture was selected for the hydrolysis of (R,S)-FOP acetate by C. rugosa
lipase. There was no variation of concentration when the pH of
reaction buffer was varied between 5.8 and 8 (data not shown).
4. Conclusion
For the synthesis of (S)-FOP alcohol, both transesterification
and hydrolysis reactions were studied. For both the reactions, C.
rugosa lipase in DIPE as solvent gave best results. The ester hydrol-
ysis reaction was more efficient as transesterification reaction
was associated with acetaldehyde-mediated enzyme denaturation.
Maximum ester hydrolysis was obtained at 40 ◦C (pH 7) with
300 mg enzyme and 120 mg (R,S)-FOP acetate as substrate. The
undesired (R)-FOP alcohol was oxidized to FOP dione using Pyri-
dinium chlorochromate, thus formed FOP dione may be recycled.
3.2.2. Effect of different (R,S)-FOP esters
The effect of ester groups on the hydrolysis reaction by C. rugosa
lipase was studied by replacing acetate with the butyric and isobu-
tyric esters. Lipase from C. rugosa could not selectively hydrolyze
the butyrate and isobutyrate esters of (R,S)-FOP alcohol, the conver-
sion was very high (95%), however, the de was totally lost (data not
shown). Hence, (R,S)-FOP acetate was the most suitable substrate
for the C. rugosa lipase.
Acknowledgements
AS, YG and AKR are grateful for the fellowships provided by CSIR
and DBT, Govt. of India, for carrying out this work.
3.2.3. Effect of substrate and enzyme concentration
References
After the optimization of all the necessary parameters for the
C. rugosa lipase-mediated hydrolysis reaction at 10 mg substrate
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represents the various concentrations of C. rugosa lipase and the
substrate resolved. It was found that 300 mg enzyme was suitable
for the resolution of 120 mg substrate completely. Preparative gram
scale reaction was setup with 1 g of (R,S)-FOP acetate and pure (S)-
ester was obtained with >99% de.
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