Exploration of the expeditious potential of Pseudomonas fluorescens lipase in the kinetic resolution of racemic intermediates and its validation through molecular docking

Article abstract of DOI:10.1002/chir.22771

A profoundly time-efficient chemoenzymatic method for the synthesis of (S)-3-(4-chlorophenoxy)propan-1,2-diol and (S)-1-chloro-3-(2,5-dichlorophenoxy)propan-2-ol, two important pharmaceutical intermediates, was successfully developed using Pseudomonas fluorescens lipase (PFL). Kinetic resolution was successfully achieved using vinyl acetate as acylating agent, toluene/hexane as solvent, and reaction temperature of 30°C giving high enantioselectivity and conversion. Under optimized condition, PFL demonstrated 50.2% conversion, enantiomeric excess of 95.0%, enantioselectivity (E?=?153) in an optimum time of 1?hour and 50.3% conversion, enantiomeric excess of 95.2%, enantioselectivity (E?=?161) in an optimum time of 3?hours, for the two racemic alcohols, respectively. Docking of the R- and S-enantiomers of the intermediates demonstrated stronger H-bond interaction between the hydroxyl group of the R-enantiomer and the key binding residues of the catalytic site of the lipase, while the S-enantiomer demonstrated lesser interaction. Thus, docking study complemented the experimental outcome that PFL preferentially acylated the R form of the intermediates. The present study demonstrates a cost-effective and expeditious biocatalytic process that can be applied in the enantiopure synthesis of pharmaceutical intermediates and drugs.

Full text of DOI:10.1002/chir.22771

Received: 13 July 2017
DOI: 10.1002/chir.22771
Revised: 28 August 2017
Accepted: 3 September 2017
R E G U L A R A R T I C L E
Exploration of the expeditious potential of Pseudomonas
fluorescens lipase in the kinetic resolution of racemic
intermediates and its validation through molecular docking
Surbhi Soni2 | Bharat P. Dwivedee1 | Vishnu K. Sharma3 | Gopal Patel1 | Uttam C. Banerjee1
1 Department of Pharmaceutical
Abstract
Technology (Biotechnology), National
A profoundly time‐efficient chemoenzymatic method for the synthesis of (S)‐3‐
Institute of Pharmaceutical Education and
Research, Punjab, India
2 Department of Biotechnology, National
(4‐chlorophenoxy)propan‐1,2‐diol and (S)‐1‐chloro‐3‐(2,5‐dichlorophenoxy)
propan‐2‐ol, two important pharmaceutical intermediates, was successfully
Institute of Pharmaceutical Education and
developed using Pseudomonas fluorescens lipase (PFL). Kinetic resolution was
Research, Punjab, India
3 Department of Pharmacoinformatics,
National Institute of Pharmaceutical
Education and Research, Punjab, India
successfully achieved using vinyl acetate as acylating agent, toluene/hexane
as solvent, and reaction temperature of 30°C giving high enantioselectivity
and conversion. Under optimized condition, PFL demonstrated 50.2%
conversion, enantiomeric excess of 95.0%, enantioselectivity (E = 153) in an
optimum time of 1 hour and 50.3% conversion, enantiomeric excess of 95.2%,
enantioselectivity (E = 161) in an optimum time of 3 hours, for the two racemic
alcohols, respectively. Docking of the R‐ and S‐enantiomers of the intermedi-
ates demonstrated stronger H‐bond interaction between the hydroxyl group of
the R‐enantiomer and the key binding residues of the catalytic site of the lipase,
while the S‐enantiomer demonstrated lesser interaction. Thus, docking study
complemented the experimental outcome that PFL preferentially acylated the
R form of the intermediates. The present study demonstrates a cost‐effective
and expeditious biocatalytic process that can be applied in the enantiopure syn-
thesis of pharmaceutical intermediates and drugs.
Correspondence
U.C. Banerjee, Department of
Pharmaceutical Technology
(Biotechnology), National Institute of
Pharmaceutical Education and Research
(NIPER), Sector 67, S.A.S.Nagar‐160062,
Punjab, India.
Email: ucbanerjee@niper.ac.in
KEYWORDS
biocatalysis, chemoenzymatic synthesis, enantiomeric excess, enantioselectivity, β‐aryloxyalcohols
1 | INTRODUCTION
drugs and drug intermediates have emerged.15 The stabil-
ity of lipases in organic solvents has further widened the
Biocatalysis has become a key strategy for the synthesis of
chiral drugs. The foremost reason for the wide application
of biocatalysis in synthetic reactions is the high degree of
selectivity along with ecological benefits.25 Lipases are the
most common enzymes applied in biocatalysis. Lipases
have achieved a distinguished position owing to their ver-
satile nature and easy availability. Over the years, several
lipase‐catalyzed processes for the synthesis of enantiopure
scope of their application, making lipase an excellent
biocatalyst for the synthesis of single stereoisomers of
racemic drugs.16 Among the lipases applied in biotrans-
formations, the lipase from Pseudomonas fluorescens
(PFL) is an interesting candidate. PFL displays specialities
in the kinetic resolution13 of racemic alcohols, acids, and
esters, as well as in the large‐scale synthesis of bulk prod-
ucts like vitamins4 and biodiesel.38 PFL has successfully
Chirality. 2017;1–10.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

2
SONI ET AL.
been applied in the enantiopure synthesis of intermedi-
ates of important beta blocker drugs like propanolol44,
atenolol2, and CNS drug intermediates like 3‐hydroxy-
methyl‐1‐tetralone tosylates6 antifungal N‐substituted
benzimidazole derivatives24 and has also been used at an
industrial scale for the synthesis of Carbovir, an antiviral
agent.26 Several additions to the biocatalytic toolbox of
PFL have been made by immobilization of PFL on various
matrices like nanoscaffolds, polymeric supports, and
hydrophobic sol‐gels.5,8
β‐Aryloxyalcohols are key intermediates in the synthe-
sis of many important drugs.30 It has been well established
that the desirable therapeutic activities of β‐aryloxyalcohols
reside mainly in the (S)‐enantiomers.27,42,43 The chemical
synthesis of enantiopure β‐aryloxyalcohols was per-
formed by the reaction of substituted phenols with
chiral epichlorhydrin or glycidol, to give epoxide inter-
mediates28,46 followed by the ring opening of these
epoxides.3,7 However, the use of chiral reagents makes
the process more cost intensive. Here we report an
extremely time‐efficient, economic, and environmen-
tally benign methodology for the synthesis of two
important pharmaceutical intermediates, which depict
the promising biocatalytic potential of PFL.
In the present study, two important racemic
β‐aryloxyalcohols, (RS)‐1‐chloro‐3‐(2,5‐dichlorophenoxy)
propan‐2‐ol [(RS)‐4], and (RS)‐3‐(4‐chlorophenoxy)pro-
pane‐1,2‐diol [(RS)‐8] were chemically synthesized and
subjected to lipase‐catalyzed kinetic resolution. Five
commercial lipase preparations were screened. The
enantiopure intermediates obtained by lipase‐catalyzed
kinetic resolution can be used for the enantiopure synthe-
sis of cloranolol (a nonselective β‐blocker)23,40 and
chlorphenesin (a muscle relaxant and antifungal).34 All
the reaction parameters were optimized using “one factor
at a time” approach. Finally, experimental observations
were validated by docking the compounds into the active
sites of the lipase.
2.1 | Analytical methods
Biocatalytic reactions were incubated in an incubator
shaker (Kuhner, Switzerland) at 200 rpm. H NMR and
1
13C NMR were obtained using Bruker DPX 400 (1H
400 MHz) in CDCl3. All the chemical shift values were
expressed in δ (ppm) units relative to tetramethylsilane
(TMS). HPLC (Shimadzu LC‐10AT pump, SPD‐10A UV‐
Vis detector) with Chiralcel® OD‐H column
(0.46 mm × 250 mm; 5 μm, Daicel Chemical Industries,
Japan) and Chiralcel® AD‐H column (0.45 mm × 250 mm;
5 μm, Daicel Chemical Industries, Japan) were used to
determine the conversion rate enantiomeric excess of
the substrates and products.
2.2 | Synthesis of (RS)‐1‐chloro‐3‐(2,5‐
dichlorophenoxy)propan‐2‐ol [(RS)‐4]
The intermediate of cloranolol was prepared via chemical
route using the following steps.
The starting racemic epoxide intermediate (RS)‐3 was
synthesized by dissolving 2,5‐dichlorophenol (1) (2.5 g,
15 mM, 1 eqv) in anhydrous acetonitrile followed by the
addition of K2CO3 (4.14 g, 15 mM, 2 eqv) at 80°C. After
stirring the reaction mixture for 30 minutes, (RS)‐
epichlorhydrin (RS)‐2 (1.8 mL, 15 mM, 1.5 eqv) was added
dropwise. The reaction mixture was extracted with ethyl
acetate after the completion of reaction in 30 hours, and
the filtrate was concentrated under vacuum. Recovered
product (RS)‐3 was subjected to the epoxide ring‐opening
reaction using acetyl chloride (1.1 mL, 10 mM, 1.5 eqv) in
dichloromethane : water (1:1) mixture. After the comple-
tion of reaction in 10 hours, the reaction mixture was
extracted with dichloromethane, dried using anhydrous
Na2SO4, and concentrated on a Rotavapor. The product
(RS)‐4 was purified by column chromatography using
hexane : ethyl acetate (94:6) as an eluent on silica gel.
(RS)‐4 was used as the substrate for lipase‐catalyzed
kinetic resolution (Scheme 1). Using similar method the
enantiopure intermediate of cloranolol, (R)‐4 was
synthesized.
(RS)‐3, light yellow liquid (96% yield), 1H NMR
(400 MHz; CdCl3) δ (ppm): 7.28 (dd, 1H, Ar–H), 7.25
(dd, 1H, Ar–H), 6.93 (m, 1H, Ar–H), 4.12 (m, 1H,
−CH2–), 3.98 (m, 1H, −CH2–), 3.40 (d, 1H, −CH–), 2.93
(dd,1H, oxirane), 2.83 (dd,1H, oxirane).13C NMR
(100 MHz, CDCl3): δ (ppm): 44.81, 53.88, 69.85, 114.67,
120.62, 122.81, 130.15, 135.21, 158.65.
2 | MATERIALS AND METHODS
2,5‐Dicholorophenol, 4‐chlorophenol, (RS)‐epichlorhydrin,
(S)‐epichlorhydrin, (RS)‐cyclopropylmethanol (glycidol),
(S)‐cyclopropylmethanol (glycidol), Candida antarctica
lipase (CALA and CALB), Candida rugosa lipase, and
P. fluorescens lipase were purchased from Sigma (St.
Louis, Missouri). Burkholderia cepacia lipase was pur-
chased from Fluka™. Anhydrous Na2SO4, K2CO3, TLC
plates, and HPLC grade solvents were purchased from
Merck (Germany). Silica gel (60‐120 mesh) for column
chromatography was obtained from SRL (India).
(RS)‐4, pale yellow liquid (95% yield), 1H NMR
(400 MHz; CdCl3) δ (ppm): 7.64 (dd, 1H, Ar–H), 7.25
(dd, 1H, Ar–H), 7.12 (dd, 1H, Ar–H), 4.11 (dd, 1H,
−CH), 4.05 (d, 2H, −CH2), 3.72 (d, 2H, −CH2). 13C
NMR (100 MHz, CDCl3): δ (ppm): 46.05, 69.67, 69.85,

SONI ET AL.
3
SCHEME 1 Kinetic resolution of
racemic alcohols using various lipases
114.67, 120.62, 122.81, 130.15, 135.21, 158.65. Optimized
HPLC method for the estimation of (RS)‐4 consisted of
Chiralcel OD‐H column (0.46‐mm diameter, 250‐mm
long, 5 μm, Chiralcel) with mobile phase Hexane : IPA
(90:10), flow rate of 1 mL/min and detected at 254 nm.
The retention times for the two enantiomers of (RS)‐4
were 9.0 and 10.5 minutes. The retention time for the
(R)‐enantiomer [(R)‐4] was 9.0 minutes. The retention
time for the (R)‐ester [(R)‐5] was 5.6 minutes (Supporting
Information S1‐S7).
2.4 | Synthesis of (RS)‐1‐chloro‐3‐(4‐
chlorophenoxy)propan‐2‐yl acetate (RS)‐9
The synthesis of the acetylated derivative of (RS)‐8 was
done using Ac2O at 4°C in the presence of pyridine.
Using similar method enantiopure‐acetylated derivative
of chlorphenesin, [(R)‐9] was also synthesized. The
optical purity of both the products was determined by
chiral HPLC.
1
(RS)‐9, a white solid (85% yield), H NMR (400 MHz;
CdCl3) δ (ppm): 7.32 (m, 2H, Ar–H), 6.95 (m, 2H, Ar–
H), 4.32 (m, 1H, −CH), 4.27 (m, 2H, −CH2), 4.12 (m,
2H, −CH2), 3.32 (m, 1H, −CH), 2.20 (s, 3H, −CH3). The
optimized HPLC method for the estimation of substrate
(RS)‐8 consisted of Chiralcel AD‐H column (0.46‐mm
diameter, 250‐mm long, 5 μm, Chiralcel) with mobile
phase Hexane : IPA (90:10) with a flow rate of 1 mL/
min and detected at 254 nm. The retention times for the
two enantiomers of (RS)‐8 were 12.7 and 14.0 minutes.
The retention time for (S)‐8 was 12.1 minutes. The reten-
tion time for the two enantiomers of (RS)‐9 were 18.7 and
22.0 minutes. The retention time for (R)‐ester (R)‐9 was
22.0 minutes (Supporting Information S10‐S15).
2.3 | Synthesis of (RS)‐3‐(4‐
chlorophenoxy)propane‐1,2‐diol [(RS)‐8]
Chlorphenesin was prepared via chemical route using the
following steps.
Chlorphenesin [(RS)‐8] was synthesized by dissolving
4‐chlorophenol (6) (2.6 g, 20 mM, 1 eqv) in anhydrous
acetonitrile followed by the addition of K2CO3 (5.2 g,
20 mM, 2 eqv). The reaction mixture was refluxed at
70°C with continuous stirring. After 30 minutes, (RS)‐
glycidol (RS)‐7 (2.2 g, 20 mM, 1.5 eqv) was added
dropwise. Upon completion of the reaction in 36 hours,
the reaction mixture was poured into a mixture of
water and dichloromethane, and the organic layer
was separated, filtered, dried using anhydrous Na2SO4,
and concentrated under vacuum. The concentrated
crude product was purified using silica gel (60‐120
mesh) column chromatography eluted with hex-
ane : ethyl acetate (96:4) affording (RS)‐8. Using similar
2.5 | Enzyme screening
(RS)‐4 and (RS)‐8 (0.02 mmol, 1 eqv) were subjected to
transesterification with vinyl acetate (acyl donor)
(100 μL) in toluene (900 μL) using commercial lipase
preparations. All the lipase preparations were individually
added and incubated in a shaker at 30°C (200 rpm). After
completion, the supernatant was separated and evapo-
rated under vacuum. Samples for HPLC analyses were
prepared using 2‐propanol, and enantiomeric excess and
ratio were determined.
method
enantiopure
chlorphenesin,
(S)‐8
was
synthesized.
1
(RS)‐8, a white solid (90% yield), H NMR (400 MHz;
CdCl3) δ (ppm): 7.26 (m, 2H, Ar–H), 6.95 (m, 2H, Ar–
H), 4.03 (m, 2H, –CH2), 3.96 (m, 2H, −CH2), 3.65 (m,
2H, −CH2), 3.55 (m, 1H, −CH). 13C NMR (100 MHz,
CDCl3): δ (ppm): 63.54, 69.44, 70.29, 76.71, 77.03, 77.34,
115.80, 126.24, 129.45, 157.01.
2.6 | Large‐scale synthesis of (S)‐4 and (S)‐8
Large‐scale synthesis of (S)‐4 and (S)‐8 was performed by
subjecting (RS)‐4 (511 mg, 2.0 mmol) and (RS)‐8 (405 mg,

4
SONI ET AL.
2.0 mmol), respectively, to transesterification with vinyl
acetate (acyl donor) (10 mL) in toluene (90 mL) using
P. fluorescens lipase. The reaction was incubated in a
shaker at 30°C (200 rpm). After completion, the superna-
tant was separated and evaporated under vacuum. The
solvent was evaporated, and crude of the reaction was
purified by column chromatography on silica gel (5‐20%
EtOAc/hexane), affording (S)‐4 [49% isolated yield and
>95% ee], (R)‐5 [48% isolated yield and >95% ee], and
(S)‐8 [49% isolated yield and >95% ee], (R)‐9 [48% isolated
yield and >95% ee].
incorporated OPLS_2005 force field. The grid for the pro-
tein was generated using the centroid of the bound ligand
as well as using selective active site residues mentioned in
the literature. The MOL files of ligand structures were
opened and prepared using LigPrep module of
Schrödinger software, and possible ionization states were
generated at neutral pH (7.0
0.5). Finally, prepared
ligands were docked in the active site of the lipase using
the standard precision method from the glide module of
Schrodinger software, and 30 poses per ligand were gener-
ated. The best docked conformations (poses) were
selected on the basis of G score and interactions.
2.7 | Optimization of process parameters
for the enantioselective transesterification
of (RS)‐4 and (RS)‐8
3 | RESULTS AND DISCUSSION
Lipases follow the well‐known “ping‐pong bi‐bi”
mechanism of enantioselective transesterification.10
Enantioselectivity is obtained because of the differential
interaction of R‐ and S‐enantiomers with catalytically
active site of the lipase.39,41 Lipases are well known for
the kinetic resolution of secondary alcohols with a stereo-
chemical preference toward the R‐enantiomer.20
Effect of each parameter influencing the lipase‐catalyzed
kinetic resolution was studied one at a time. Six organic
solvents with different log P values were used to study
the effect on the transesterification of (RS)‐4. The opti-
mum reaction time was determined by performing the
reaction and collecting the samples at various time inter-
vals. Various aspects of enzymatic acylation such as
enantioselectivity, conversion, and greenness have been
reported to be influenced by the acyl donors. The
enantioselectivity and rate of conversion of enzyme‐cat-
alyzed kinetic resolution was therefore studied using
various acyl donors. The reaction temperature also
influences the rate and enantioselectivity of the
transesterification reaction. Various enzyme concentra-
tions (15, 30, 45, 60, and 90 mg/mL) were used with a
fixed substrate concentration (20 mM). Substrate con-
centrations were also varied (10, 20, 30, 40, and
50 mM) to achieve its optimum concentration for the
reaction. Samples were analyzed for conversion and
enantioselectivity of the enzymes used.
3.1 | Lipase‐catalyzed transesterification
of (RS)‐4 and (RS)‐8
(RS)‐4 and (RS)‐8 were synthesized and subjected to
lipase‐catalyzed kinetic resolution (Scheme 1). Five differ-
ent commercially available lipases (Candida rugosa
[CRL], Burkholderia cepacia [BCL], P. fluorescens [PFL],
Candida antarctica [CALA], and Candida antarctica
[CALB]) were screened for the kinetic resolution of both
(RS)‐4 and (RS)‐8 in toluene using vinyl acetate as acyl
donor. No reaction was observed in the absence of lipase.
Among all the lipases screened, P. fluorescens lipase (PFL)
was the fastest enzyme to resolve both (RS)‐4 and (RS)‐8
into the corresponding (S)‐alcohol and (R)‐ester products,
with high enantioselectivity. Other lipases also performed
the enantioselective transesterification of (RS)‐4 and (RS)‐
8, but with poor conversion. Thus, PFL was chosen for
further reactions. The most interesting outcome of this
study was the rapid PFL‐catalyzed enantioselective con-
version of the racemates compared to other lipases
(Tables S1 and S2).
2.8 | Methodology for molecular docking
Glide module of Schrödinger software incorporated with
the maestro graphical user interface (GUI) was used for
molecular docking study. Initially, appropriate 3D crystal
structures for the lipase Pseudomonas sp. MIS38 (selected
on the basis of similarity with PFL) was retrieved from the
Protein Data Bank (PDB). The lipase was prepared using
protein preparation wizard incorporated in Schrodinger
software. The bond orders for the protein residues were
assigned, and hydrogen atoms, which were not assigned
for each amino acid in the 3D crystal structure, were
added. The disulfide bonds were created, and appropriate
crystal water molecules, which were part of active site,
were considered during molecular docking process.
Finally, lipase structure was minimized using
3.2 | Effect of solvents
Reaction medium plays an important role in deciding the
course of a biocatalytic reaction. Study of lipase catalysis
in organic solvents is very interesting and well
researched.22,45 Lipases are interfacial enzymes, and the
enzyme‐bound water (called as structural water, generally

SONI ET AL.
5
present in the form of a monolayer) plays a crucial role for
the enzyme structure and is necessary for their catalytic
activity.18 Hydrophilic solvents pose a higher tendency
to strip the tightly bound water decreasing the efficiency
of the lipase. Contrary to this, lipases exhibit good stability
in organic solvents, which have a lesser tendency to do so,
thus becoming a favorable reaction medium for lipase‐cat-
alyzed reactions.37 Many examples of improved lipase
activity and selectivity in organic solvents along with
other benefits like increased solubility of substrates have
been reported.19,22 In the same context, different organic
solvents, varying in log P values were examined for the
kinetic resolution of (RS)‐4 and (RS)‐8, with vinyl acetate
using PFL. In the case of (RS) 4, best result was obtained
in hexane depicting 50.7% conversion, 97.1% ees, 94.6%
eep, and enantiomeric ratio (E‐value) of 153 (Figure 1A).
Similarly, toluene gave maximum enantiomeric excess
and enantioselectivity for (RS)‐8 with 50.4% conversion,
96.3% ees, 95.0% eep, and enantiomeric ratio (E‐value) of
152 (Figure 2A).
3.3 | Type of acyl donors
Lipase‐catalyzed acyl transfer reactions involving normal
esters are generally reversible and yield low optical purity
of the product and substrate. The use of enol esters like
vinyl acetate or isopropenyl acetate shifts the reaction
equilibrium toward the product.33 These esters lead to
the liberation of unstable enols, which are rapidly
tautomerized to aldehydes and ketones, thereby making
the reaction irreversible in nature.9,29 However, the acet-
aldehyde formed during the reaction might act as an
alkylating agent and deactivate the lipase. This is not a
general case with all the lipases and the degree of deacti-
vation depends upon the nature of the lipase.11 Hence, the
type of acyl donor and interaction with the lipase is a piv-
otal determinant of reaction progression. Four different
acyl donors (BA, benzyl acetate; EA, ethyl acetate; IPA,
isopropenyl acetate; VA, vinyl acetate) were applied in
the reaction, among which vinyl acetate showed best
results in the kinetic resolution of both (RS)‐4 and (RS)‐
FIGURE 1 Effect of (A) solvents, (B) acyl donors [BA = benzyl acetate, EA = ethyl acetate, IPA = isopropenyl acetate, VA = vinyl acetate],
(C) reaction time, (D) substrate concentration, (E) enzyme concentration, and (F) temperature on PFL‐catalyzed kinetic resolution of [(RS)‐4]

6
SONI ET AL.
FIGURE 2 Effect of (A) solvents, (B) acyl donors, (C) reaction time, (D) substrate concentration, (E) enzyme concentration, and (F)
temperature on the PFL‐catalyzed kinetic resolution of [(RS)‐8]
8. The highest percentage conversion (50.1%), enantio-
meric excess (95.7% ees and 95.2% eep) and E‐value of
159 for PFL‐catalyzed kinetic resolution of (RS)‐4 in hex-
ane were obtained with vinyl acetate as an acyl donor
(Figure 1B). Similarly for (RS)‐8, highest percentage con-
version (50.6%), enantiomeric excess (97.7% ees and
95.3% eep), and E‐value of 185 were obtained in PFL‐cata-
lyzed kinetic resolution using vinyl acetate (Figure 2B).
for (RS)‐8, the optimum reaction time was 1 hour with
96.8% ees, 95.4% eep, 50.4% conversion, and E‐value 177
(Figure 2C). Other lipases also give moderate‐to‐good
results but in longer duration, making PFL the obvious
choice for this biocatalytic process. The prompt activity
of PFL could be due to better accommodation of a partic-
ular conformation of the substrate at the active site of
PFL. The molecular docking study in the later section is
a demonstrative evidence of such interactions. The swift
action of PFL with high degree of enantioselectivity and
rate of conversion makes it a very promising tool in such
reactions.
3.4 | Optimization of reaction time
The most important factor governing the utility of a bio-
catalytic process is reaction time. The industrial research
is focused on developing economically expedient pro-
cesses, yielding higher enantioselectivity in lesser time.
Gratifyingly, in the present study PFL catalyzed the
kinetic resolution of both (RS)‐4 and (RS)‐8 in a short
duration of 3 and 1 hour, respectively. In the reaction of
(RS)‐4, PFL took 3 hours to give 97.1% ees, 94.8% eep,
50.6% conversion, and E‐value 160 (Figure 1C). Likewise,
3.5 | Optimization of substrate and
enzyme concentration
Lipases are interfacial enzymes. It is a unique characteris-
tic of lipases. Several studies have revealed the effect of
the interfacial microenvironment in lipase catalysis. Thus,
it is important to determine the correlation of lipase

SONI ET AL.
7
activity with the concentration of enzymes and sub-
strates.31 The hydrophobic and electrostatic interactions
between the enzyme and substrate at the interface are
important with respect to the diffusion of substrates
toward the active site. At low interfacial concentration
of the substrate, lipase exhibits little activity. However,
once the substrate concentration reaches an optimum
value (exceeding its solubility limit), and the substrate
becomes available in the form of micelles, the reaction
rate increases.36 In the light of the aforementioned expla-
nations, we performed the reaction using a range of sub-
strate concentration. The substrate concentration was
varied between 1 and 30 mM. In case of (RS)‐4 the opti-
mal substrate concentration was 10 mM, at which PFL
displayed good conversion rate of 50.4% and E‐value of
161 along with the ees and eep values of 96.5% and
95.1%, respectively (Figure 1E). Similarly for (RS)‐8 the
optimal substrate concentration was 20 mM at which
the percentage conversion was 50.3, E‐value of 175 and
ees and eep were found to be 96.4% and 95.5%, respectively
(Figure 2E). The rate of an enzymatic reaction depends
upon the amount of the enzyme present in the reaction
medium, which in turn is an indicator of the number of
enzyme active sites of the enzyme. Generally, rate of the
reaction increases with the increase in enzyme concentra-
tion. However, at an optimum concentration of the
enzyme all the active sites of the enzyme are saturated,
and further increase in the enzyme concentration does
not improve the reaction rate. Thus, an optimum concen-
tration of enzyme is sought for high yield, less reaction
time, and economic viability of the process.21 The reac-
tions of (RS)‐4 and (RS)‐8 were performed using different
concentrations (100, 200, 300, 400, 600, and 800 IU/mL) of
PFL in hexane and toluene, respectively. At 400 IU/mL,
PFL showed highest conversion of 50.6%, E‐value of 141
and ees and eep were found to be 96.6% and 94.4%,
respectively for (RS)‐4 (Figure 1D). For (RS)‐8, while the
maximum conversion with 300 IU/mL PFL was 49.7%,
E‐value of 187 and ees and eep were found to be 95.0%
and 96.1%, respectively (Figure 2D).
stereochemistry of the reaction. While some studies sug-
gest that enantioselectivity increases upon lowering the
temperature, many other studies depict an increase in
enantioselectivity with the rise in temperature. Thus,
change in temperature has a variable effect on lipase‐
catalyzed reactions.32,35 The kinetic resolution of both
the racemates was performed at a range of temperature
(25, 30, 40, 50, and 60°C) to observe the effect on degree
of enantioselectivity and percentage conversion. In both
the reactions of (RS)‐4 and (RS)‐8, PFL catalyzed the con-
version with highest enantioselectivity at 30°C. The
increase in temperature showed a gradual demise in the
enantioselective resolution of the (R) and (S) forms of
the racemic alcohols. For (RS)‐4 the conversion at 30°C
was 50.2%, with 96.0% ees, 95.4% eep, and an E‐value of
167 after 3 hours of reaction (Figure 1F). For (RS)‐8 the
conversion at 30°C was 49.6%, with 94.0% ees, 95.5% eep,
and an E‐value of 156 after 1 hour of reaction (Figure 2
F; Supporting Information S9 and S17).
3.7 | Molecular docking study for the
validation of suitable biocatalyst
Molecular docking of substrates into the active site of the
enzyme gives an insight of the reaction mechanism. The
crystal structures of many lipases with small molecules
bound at the active site are well exploited in the study of
catalytic mechanism and enantioselectivity of biocatalytic
reactions.12,14 In our investigation of theoretical evidence
for the experimental work, we performed molecular
docking of the R‐ and S‐enantiomers of (RS)‐4 and (RS)‐
8, using Schrodinger's Maestro Glide 9.2 program. Wet
laboratory experiments revealed PFL as the most suitable
lipase for both (RS)‐4 and (RS)‐8. While searching the
PDB [www.rcsb.org], we discovered that the 3D crystal
structure information of PFL has not hitherto been
reported. A protein sequence annotated as lipase from
P. fluorescens (P41773 LIPB_PSEFL: 447 AA) was avail-
able in the UniProt database. BLAST analysis of this
sequence exhibited the highest percent identity (79%)
and query cover (99%) with Pseudomonas sp. MIS38
(PDB code 2Z8X).1 On the basis of the similarity score
Pseudomonas sp. MIS38 was used for docking of the 4
enantiomers. Crystal structure of the lipase displayed cat-
alytic triad composed of Ser207, Asp255, and His313 resi-
dues and the oxyanion hole containing the –NH groups of
the peptide backbone constituted by Tyr29, Gly59, and
Arg106. According to the well‐known “ping‐pong bi‐bi”
mechanism, the alcohol does not act on the apoenzyme
but on the acyl enzyme intermediate. Hydrogen bonds
between the carbonyl O atom of the acyl group and the
–NH groups of the oxyanion hole stabilize the acyl
enzyme complex. Further, a nucleophilic attack by the
3.6 | Optimization of reaction
temperature
The value of enantioselectivity (E‐value) in a lipase‐cata-
lyzed reaction depends upon the difference in the free
energy of activation (G) of the two enantiomers, which
in turn is related to the activation enthalpy (H) and the
activation entropy (S), as G = H − TS. The difference in
activation enthalpy of the enantiomers is due to the differ-
ence in the steric fit of the substituents into the respective
binding pockets of the lipase. If the values of H and TS are
balanced then temperature can modulate the

8
SONI ET AL.
alcohol leads to the formation of an ester.17 To explore
molecular docking for evaluation of binding differences
between the enantiomers, we prepared the acyl enzyme
intermediate by acylation of the hydroxyl group of serine
207. In case of (RS)‐4, the R‐enantiomer, ie, (R)‐4 showed
strong H‐bond interaction with Tyr29 and His 313 each
with a minimum distance of 1.9 Å (Figure 3A). The (S)‐
enantiomer showed lesser interactions needed for the
lipase‐catalyzed reaction (Figure 3B). The G score for the
R‐enantiomer was −5.67, while the S‐enantiomer had a
lower score of −5.24. It thus indicated that PFL catalyzed
the transesterification of both the enantiomers, with pref-
erence to the R form. However, this selectivity was time
dependent. Initially, PFL catalyzed the reaction very fast
in favor of transesterification of the R‐enantiomer. Maxi-
mum conversion of (RS)‐4 into the corresponding ester
(R)‐5 was achieved in 3 hours. The result obtained in
3 hours was C = 50.6%, eeS = 97.1%, eeP = 94.8%, and
E = 160. Beyond this period, lipase catalyzed nonselective
conversion of (RS)‐4, thereby losing the enantioselectivity.
Similarly, in the case of (RS)‐8, the R‐enantiomer, ie, (R)‐8
showed strong interactions with Tyr29 and Gly59 residue
each with an H‐bond distance of 1.9 and 2.6 Å (Figure 3
C). The S‐enantiomer showed lesser interaction
(Figure 3D). The G score for the R‐enantiomer was
−5.36, while the S‐enantiomer had a lower score of
−4.13. PFL catalyzed the maximum conversion of (RS)‐8
into (R)‐9 in a very short duration of 1 hour. The result
obtained in 1 hour was C = 50.4%, eeS = 96.8%, eeP = 95.4%,
and E = 177. Other lipases also performed the
enantioselective transesterification of (RS)‐4 and (RS)‐8,
but best results were obtained with PFL, the most inter-
esting feature being the least time taken by PFL compared
to other lipases.
3.8 | Synthesis of the enantiopure drugs
using the results of computational and
experimental studies
The results obtained from the initial screening of the bio-
catalyst and optimization of the reaction parameters were
incorporated in the synthesis of enantiopure cloranolol
and chlorphenesin. (S)‐4 was successfully synthesized by
the kinetic resolution of (RS)‐4 using P. fluorescens lipase
with C = 50.2%, E = 161, eep = 96.0%, and ees = 95.2%.
SCHEME 2 Synthesis of final drugs
FIGURE 3 Interaction of ligands (A) [(R)‐4], (B) [(S)‐4], (C) [(R)‐8], and (D) [(S)‐8] with Pseudomonas sp. MIS38 lipase

SONI ET AL.
9
3. Banoth L, Thakur NS, Bhaumik J, Banerjee UC. Biocatalytic
approach for the synthesis of enantiopure acebutolol as a β1‐
selective blocker. Chirality. 2015;27:382‐391.
(S)‐4 was further reacted with isopropyl amine (10) in
water to produce (S)‐cloranolol (89% yield).
Enantiopure chlorphenesin, (S)‐8 was obtained by the
kinetic resolution of (RS)‐8 using P. fluorescens lipase
(Scheme 2).
4. Bonrath W, Karge R, Netscher T. Lipase catalyzed transforma-
tions as key steps in the large scale preparation of vitamins.
J Mol Catal B: Enzym. 2002;19–20:67‐72.
5. Brem J, Turcu MC, Paizs C, et al. Immobilization to improve the
properties of Pseudomonas fluorescens lipase for the kinetic reso-
lution of 3‐aryl‐3‐hydroxy esters. Process Biochem. 2012;47:119‐
126.
4 | CONCLUSION
Our investigation was focused on the screening of lipases
for kinetic resolution of two useful racemates. Central to
this investigation was the observation that P. fluorescens
lipase catalyzed the kinetic resolution expeditiously tak-
ing only 3 and 1 hour for the two racemates, respectively.
Under optimized conditions, PFL exhibited selective acyl-
ation of the (R) enantiomer of (RS)‐4 [C = 50.2%,
ees = 96.0%, eep = 95.2%, and E = 161] and (RS)‐8
[C = 50.3%, ees = 96.2%, eep = 95.0%, E = 153]. To our
delight, molecular docking results displayed considerable
interaction between the lipase and R‐enantiomers of the
racemates, which was in agreement with experimental
results. The methodology of complementing biocatalytic
reactions with molecular docking proved to be a time‐effi-
cient and reliable process. The result of both studies taken
together warranties a broad application of P. fluorescens
lipase as a biocatalyst for the synthesis of enantiopure
intermediates.
6. Caro Y, Masaguer CF, Ravina E. Preparation of (R)‐(−)‐ and (S)‐
(+)‐3‐Hydroxymethyl‐1‐tetralone tosylates, key intermediates in
the synthesis of new CNS drugs via resolution of precursors. Tet-
rahedron Asymmetry. 2003;14:381‐387.
7. Dwivedee BP, Ghosh S, Bhaumik J, Banoth L, Banerjee UC.
Lipase‐catalyzed green synthesis of enantiopure atenolol. RSC
Adv. 2015;5:15850‐15860.
8. Dwivedee BP, Bhaumik J, Rai SK, Laha JK, Banerjee UC. Devel-
opment of nanobiocatalysts through the immobilization of
Pseudomonas fluorescens lipase for applications in efficient
kinetic resolution of racemic compounds. Bioresour Technol.
2017;239:464‐471.
9. Egri G, Baitz‐Gács E, Poppe L. Kinetic resolution of 2‐acylated‐
1,2‐diols by lipase‐catalyzed enantiomer selective acylation. Tet-
rahedron Asymmetry. 1996;7:1437‐1448.
10. Ema T. Mechanism of enantioselectivity of lipases and other syn-
thetically useful hydrolases. Curr Org Chem. 2004;8:1009‐1025.
11. Faber K, Riva S. Enzyme‐catalyzed irreversible acyl transfer.
Synthesis (Stuttg). 1992;10:895‐910.
12. Ferrario V, Ebert C, Knapic L, et al. Conformational changes of
lipases in aqueous media: a comparative computational study
ACKNOWLEDGMENTS
and
experimental
implications.
Adv
Synth
Catal.
S.S. would like to thank the Department of Science and
Technology, Government of India, for providing DST‐
INSPIRE fellowship. B.P.D. and V.K.S. acknowledge
NIPER, Mohali, for providing fellowship. G.P. acknowl-
edges the Department of Biotechnology, Government of
India, for providing DBT‐fellowship. Authors acknowl-
edge the Department of Pharmacoinformatics, National
Institute of Pharmaceutical Education and Research, S.
A.S. Nagar 160062, Punjab, India, for providing facilities
for docking study.
2011;353:2466‐2480.
13. Ferreira HV, Rocha LC, Severino RP, Porto ALM. Syntheses of
enantiopure aliphatic secondary alcohols and acetates by
bioresolution with lipase B from Candida antarctica. Molecules.
2012;17:8955‐8967.
14. García‐Urdiales E, Busto E, Ríos‐Lombardía N, Gotor‐Fernández
V, Gotor V. Computational study of the lipase‐mediated
desymmetrisation
Chembiochem. 2009;10:2875‐2883.
of
2‐substituted‐propane‐1,3‐diamines.
15. Gotor V. Biocatalysis applied to the preparation of pharmaceuti-
cals. Org Process Res Dev. 2002;6:420‐426.
16. Gotor‐Fernández V, Brieva R, Gotor V. Lipases: useful
biocatalysts for the preparation of pharmaceuticals. J Mol Catal
B: Enzym. 2006;40:111‐120.
ORCID
Uttam C. Banerjee http://orcid.org/0000-0002-7363-4042
17. Guieysse D, Cortés J, Puech‐Guenot S, et al. A structure‐con-
trolled investigation of lipase enantioselectivity by a path‐
planning approach. Chembiochem. 2008;9:1308‐1317.
REFERENCES
1. https://blast.ncbi.nlm.nih.gov/Blast.cgi.
18. Hari Krishna S, Karanth NG. Lipases and lipase‐catalyzed ester-
ification reactions in nonaqueous media. Cata Rev. 2002;44:161‐
4940.
2. Agustian J, Kamaruddin AH, Aboul‐Enein HY. Enantio‐conver-
sion and selectivity of racemic atenolol kinetic resolution using
free Pseudomonas fluorescence lipase (amano) conducted via
transesterification reaction. RSC Adv. 2016;6:26077‐26085.
19. Herbst D, Peper S, Niemeyer B. Enzyme catalysis in organic sol-
vents: influence of water content, solvent composition and

10
SONI ET AL.
temperature
on
candida
rugosa
lipase
catalyzed
36. Reis P, Holmberg K, Watzke H, Leser ME, Miller R. Lipases at
transesterification. J Biotechnol. 2012;162:398‐403.
interfaces:
a review. Adv Colloid Interface Sci. 2009;147–
148:237‐250.
20. Höhne M, Schätzle S, Jochens H, Robins K, Bornscheuer UT.
Rational assignment of key motifs for function guides in silico
enzyme identification. Nat Chem Bio. 2010;6:807‐813.
37. Salihu A, Alam MZ. Solvent tolerant lipases: a review. Process
Biochem. 2015;50:86‐96.
38. Salis A, Bhattacharyya MS, Monduzzi M, Solinas V. Role of the
support surface on the loading and the activity of Pseudomonas
fluorescens lipase used for biodiesel synthesis. J Mol Catal B:
Enzym. 2009;57:262‐269.
21. Janes LE, Kazlauskas RJ, Quick E. A fast spectrophotometric
method to measure the enantioselectivity of hydrolases. J Org
Chem. 1997;3:4560‐4561.
22. Klibanov AM. Improving enzymes by using them in organic sol-
39. Sawant G, Ghosh S, Banesh S, Bhaumik J, Banerjee UC. In‐silico
approach towards lipase mediated chemoenzymatic synthesis of
(S)‐ranolazine as an antianginal drug. RSC Adv. 2016;6:49150‐
49157.
vents. Nature. 2001;409:9‐11.
23. Kulcsár‐Gergely J. Action of beta‐adrenoceptor blockers on liver
function of rats. Arzneimittelforschung. 1989;39:782‐785.
24. Łukowska‐Chojnacka E, Staniszewska M, Bondaryk M, Maurin
JK, Bretner M. Lipase‐catalyzed kinetic resolution of novel anti-
fungal N‐substituted benzimidazole derivatives. Chirality.
2016;28:347‐354.
40. Syrenski AV, Tsyrlin VA. The effect of beta‐adrenoblockaders on
the elasticity of the vessels of the greater circulation. Eksp Klin
Farmakol. 1995;58:23‐24.
41. Thakur NS, Bhaumik J, Sooram B, Banoth L, Banerjee UC. Syn-
thesis of enantiopure drugs and drug intermediates using in
silico generated archetype biocatalyst: a case study using
alprenolol as a model drug. ChemistrySelect. 2016;1:871‐876.
25. Margolin AL. Enzymes in the synthesis of chiral drugs. Enzyme
Microb Technol. 1993;15:266‐280.
26. Muñoz Solano D, Hoyos P, Hernáiz MJ, Alcántara AR, Sánchez‐
Montero JM. Industrial biotransformations in the synthesis of
building blocks leading to enantiopure drugs. Bioresour Technol.
2012;115:196‐207.
42. Theil F, Ballschuh S, Kunath A, Schick H. Kinetic resolution of
rac‐3‐(2‐methylphenoxy)propane‐1,2‐diol (mephenesin) by
sequential lipase‐catalyzed transesterification. Tetrahedron
Asymmetry. 1991;2:1031‐1034.
27. Newman DJ. Natural product‐derived drugs based on β‐adrener-
gic agents and nucleosides. J Braz Chem Soc. 2016;27:1320‐1333.
43. Wang ZM, Zhang XL, Sharpless KB. Asymmetric
dihydroxylation of aryl allyl ethers. Tetrahedron Lett.
1993;34:2267‐2270.
28. Panchgalle SP, Gore RG, Chavan SP, Kalkote UR.
Organocatalytic enantioselective synthesis of β‐blockers: (S)‐Pro-
pranolol and (S)‐Naftopidil. Tetrahedron Asymmetry.
2009;20:1767‐1770.
44. Wang F, Zhang H, Wang J, et al. Ultrasound irradiation pro-
moted enzymatic transesterification of (R/S)‐1‐Chloro‐3‐(1‐
naphthyloxy)‐2‐propanol. Molecules. 2012;17:10864‐10874.
29. Paravidino M, Hanefeld U. Enzymatic acylation: assessing the
greenness of different acyl donors. Green Chem. 2011;13:2651‐
2657.
45. Zaks A, Klibanov AM. Enzymatic catalysis in nonaqueous sol-
vents. J Biol Chem. 1988;263:3194‐3201.
46. Zhang JY, Liu HM, Xu HW, Shan LH. Non‐enzymatic kinetic
resolution of β‐amino alcohols using C‐12 higher carbon sugar
as a chiral auxiliary. Tetrahedron Asymmetry. 2008;19:512‐517.
30. Pchelka BK, Loupy A, Plenkiewicz J, Petit A, Blanco L. Resolu-
tion of racemic 3‐aryloxy‐1‐nitrooxypropan‐2‐ols by lipase‐
catalyzed enantioselective acetylation. Tetrahedron Asymmetry.
2001;12:2109‐2119.
31. Petersen MTN, Fojan P, Petersen SB. How do lipases and ester-
SUPPORTING INFORMATION
ases work: the electrostatic contribution.
J
Biotechnol.
Additional Supporting Information may be found online
in the supporting information tab for this article.
2001;85:115‐147.
32. Phillips RS. Temperature modulation of the stereochemistry of
enzymatic catalysis: prospects for exploitation. Trends
Biotechnol. 1996;14:13‐16.
33. Puskas JE, Seo KS, Sen MY. Green polymer chemistry: precision
synthesis of novel multifunctional poly(ethylene glycol)s using
enzymatic catalysis. Eur Polym J. 2011;47:524‐534.
How to cite this article: Soni S, Dwivedee BP,
Sharma VK, Patel G, Banerjee UC. Exploration of
the expeditious potential of Pseudomonas
fluorescens lipase in the kinetic resolution of
racemic intermediates and its validation through
molecular docking. Chirality. 2017;1–10. https://
doi.org/10.1002/chir.22771
34. Quan J, Wu Q, Lin XF. Synthesis of polymeric prodrugs of
chlorphenesin with saccharide branches by chemo‐enzymatic
regioselective strategy. Polymer (Guildf). 2007;48:2595‐2604.
35. Reetz MT. Lipases as practical biocatalysts. Curr Opin Chem Biol.
2002;6:145‐150.