Enantioselective transesterification of (RS)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol using Pseudomonas aeruginosa lipases

Article abstract of DOI:10.1016/j.tetasy.2007.08.024

Lipases from the bacterial strain, Pseudomonas aeruginosa, isolated from the soil by enrichment techniques, are assessed for the enantioselective transesterification of (RS)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) to (R)-1-chloro-3-(3,4-dif

Full text of DOI:10.1016/j.tetasy.2007.08.024

Tetrahedron: Asymmetry 18 (2007) 2079–2085
Enantioselective transesterification of (RS)-1-chloro-3-(3,4-
difluorophenoxy)-2-propanol using Pseudomonas aeruginosa lipases
Manpreet Singh and Uttam Chand Banerjee*
Biocatalysis Laboratory, Department of Pharmaceutical Technology (Biotechnology), National Institute of
Pharmaceutical Education and Research, Sector 67, SAS Nagar, Punjab 160 062, India
Received 4 July 2007; accepted 21 August 2007
Abstract—Lipases from the bacterial strain, Pseudomonas aeruginosa, isolated from the soil by enrichment techniques, are assessed for
the enantioselective transesterification of (RS)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) to (R)-1-chloro-3-(3,4-difluoro-
phenoxy)-2-propanol, a key intermediate in the synthesis of the chiral drug (S)-Lubeluzole. The lipases produced by the organism yielded
the (S)-ester and the (R)-alcohol as the remaining substrate with an excellent yield (>49.9%) and almost complete enantioselectivity (ee
>99.9%) in the presence of vinyl butyrate as an acyl donor in an organic medium. In contrast, purified and expensive commercially avail-
able lipases of Candida rugosa and porcine pancreas achieved much lower conversion with enantioselectivities of 15% and 5%, respec-
tively. A well-mixed (ꢀ1000 rev minꢁ1) batch reactor having the aqueous phase finally dispersed in hexane was used in these studies. The
parameters of the transesterification reaction were optimized and the optimal concentrations of rac-CDPP and vinyl butyrate were found
to be 5 and 150 mM at 30 ꢀC. A preparative-scale reaction yielded the (S)-ester at 42% conversion and ee >99%.
ꢁ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
reductases, aminotransferases, aldolases, monooxygenases
and dioxygenases) with the ability to catalyze a large num-
ber of enantioselective reactions.4 Among the various bio-
catalysts, lipases (triacylglycerol acylhydrolases, E.C.
3.1.1.3) have provided chemists with the opportunity for
the development of novel chemical synthesis.5 The screen-
ing of microbial strains found in soil samples is the best
way to obtain microorganisms producing enzymes capable
of carrying out a specific chemical reaction. To obtain the
specific microorganism, soil samples are enriched with the
substrate itself or its easily available derivative as a sole
carbon source.6
Biocatalysts are increasingly being used to synthesize com-
plex molecules of industrial interest.1,2 The biggest role of
biocatalysis still remains in the pharmaceutical sector
where the exquisite regioselective and stereoselective prop-
erties of biocatalysts enable the difficult synthesis.3 In the
manufacturing of modern pharmaceutical drugs and in
the drug discovery process, chiral reagents and chiral build-
ing blocks play a pivotal role. For the synthesis of various
drugs and drug intermediates, among the various chiral
compounds, chiral alcohols represent a highly versatile
and attractive group of chiral building blocks. However,
the problem lies with the solubility of these chiral inter-
mediates, most of the time they are not miscible in the
aqueous phase. Organic synthesis employing the enzymes
as a catalyst for stereo-controlled reactions has grown
significantly as these are inexpensive and easy to operate
under mild conditions of temperatures and pH.
Lubeluzole [(S)-4-(2-benzothiazolylmethylamino)-a-((3,4-
difluorophenoxy)methyl)-1-piperidineethanol] is a novel
benzothiazole compound that has shown neuroprotective
activity in preclinical models of ischemic stroke.7 Lubeluz-
ole contains one stereocenter, and a retrosynthetic strategy
reveals that enantiopure (R)-1-chloro-3-(3,4-difluorophen-
oxy)-2-propanol and N-methyl-N-(4-piperidinyl)-2-benzo-
thiazolamine should be suitable building blocks for its
synthesis.8,9
Production of enantiomerically pure intermediates by the
enzymatic processes requires large-scale screening pro-
grams that reveal a number of enzymes (hydrolases, oxido-
The synthesis of (R)-1-chloro-3-(3,4-difluorophenoxy)-2-
propanol using commercially available (R)-epichloro-
hydrin and its reaction with 3,4-difluorophenol is
*
Corresponding author. Tel.: +91 (0) 172 2214682–87x2142; fax: +91 (0)
172 2214692; e-mail: ucbanerjee@niper.ac.in
0957-4166/$ - see front matter ꢁ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.08.024

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M. Singh, U. C. Banerjee / Tetrahedron: Asymmetry 18 (2007) 2079–2085
reported.8 The reaction is complete in 96 h with an average
conversion of 48%. The chemically synthesized enantio-
merically pure alcohol was then used for the convergent
synthesis of (S)-Lubeluzole. Overall yields ranged from
20% to 35% with a moderate enantiomeric excess of 94%,
which needs to be increased. There is precedent for
synthesis of the enantiomerically pure alcohol through
lipase catalyzed transesterification reaction, but on a very
small scale8 (<5 mM).
Enzyme activity
OD
2000
1500
1000
500
0
1.6
1.2
0.8
0.4
0
So far, there has been no report regarding the optimization
of reaction conditions for the enzymatic resolution of rac-
CDPP and its preparative-scale synthesis. Herein we report
for the first time the transesterification of rac-CDPP to
enantiopure (R)-alcohol using crude lipases from Pseudo-
monas aeruginosa MTCC 5113. The various parameters
for the transesterification reaction have been optimized
with the preparative-scale synthesis of (R)-1-chloro-3-
(3,4-difluorophenoxy)-2-propanol, for its subsequent use
in the production of anti-ischemic drug, Lubeluzole.
0
24
48
72
96
120
Time (h)
Figure 1. Time-course of growth and enzyme production by lipases from
P. aeruginosa.
2. Results and discussion
Conversion
ee
2.1. Screening of lipase producer
50
40
30
20
10
0
100
80
60
40
20
0
The screening was carried out as described in Section 4,
using 3,4-difluorophenol as the sole carbon source. We suc-
cessfully isolated 20 lipase-producing microorganisms. All
of these were tested for their enantioselective transesterifi-
cation capabilities on rac-CDPP. Among them, only one
strain gave moderate conversion (15%) with excellent (S)-
enantioselectivity (ee >99%). Morphological, biochemical
and physiological tests for the identification of the organ-
ism isolated were carried out in the Microbial Type Culture
Collection (MTCC) at the Institute of Microbial Technol-
ogy, Chandigarh. It was found that the isolated organism
was a bacterial strain, P. aeruginosa. All the taxonomic
studies of the organism indicated that it is the same organ-
ism (99% similarity), which was earlier isolated in our lab-
oratory and deposited in MTCC at the Institute of
Microbial Technology, Chandigarh, under the accession
number 5113.10 Previously, the same organism P. aerugin-
osa was used for the enantiospecific hydrolysis of methoxy-
phenyl glycidic acid methyl ester ( )-MPGM.11 To
further characterize the new isolate, the time-course of
the enzyme production and growth was monitored by cul-
tivating the organism in the production medium and per-
forming the enzyme assay as described in Section 4. As
seen in Figure 1, lipase activity started increasing after
24 h of incubation and continued up to 72 h, thereafter,
there was a fall in the enzyme activity.
0
10
20
30
40
50
60
Time (h)
Figure 2. Time-course of conversion and enantioselectivity of (RS)-1-
chloro-3-(3,4-difluorophenoxy)-2-propanol by crude lipase of P. aerugin-
osa at 30 ꢀC.
biotransformations. Therefore, all the subsequent reactions
were carried out up to 24 h.
2.3. Effect of temperature
The transesterification of rac-CDPP by lipase from P. aeru-
ginosa was examined at different temperatures ranging
from 20 to 60 ꢀC. Both above and below 30 ꢀC, a decrease
in conversion was observed. All the subsequent experi-
ments were carried out at 30 ꢀC (Table 1).
2.2. Time-course for transesterification
The time-course of the transesterification of rac-CDPP for
the production of (S)-1-chloro-3-(3,4-difluorophenoxy)-2-
butanoate is shown in Figure 2. The reaction was allowed
to proceed for 50 h and the conversion and ee were calcu-
lated. The conversion of alcohol to ester increased with
time, while the ee decreased as commonly observed in such
2.4. Effect of solvents
Keeping in mind the effect of organic solvents on lipase
activity as well as the specificity and insolubility of the sub-
strates in aqueous phase, various solvents with different

M. Singh, U. C. Banerjee / Tetrahedron: Asymmetry 18 (2007) 2079–2085
Table 1. Effect of temperature on the conversion of rac-CDPP
2081
Conversion
ee
Temperature (ꢀC)
Conversion (%)
20
15
10
5
120
100
80
60
40
20
0
20
30
40
50
60
28.57
40.05
39.45
37.01
35.02
logP (P is octanol–water partition coefficient for the sol-
vent) values were selected. Since solvents with high logP
values stabilize lipases compared to the solvents with low
logP values, six different types of solvents, that is, hexane,
heptane, toluene, benzene, tetrahydrofuran (THF), and
water-miscible solvent like acetonitrile (ACN), were as-
sessed for the lipase catalyzed transesterification reaction
(Fig. 3). Hexane was found to offer the maximum conver-
sion rate when compared to the others.
0
Conversion
ee
Figure 4. Effect of addition of DMSO (5%) in different organic solvents.
20
15
10
5
120
100
80
60
40
20
0
result in substrate inhibition. Therefore, the effect of sub-
strate concentration on the conversion rate is a very impor-
tant parameter, worthy of careful investigation. Several
concentrations of rac-CDPP (2–8 mM) were used for the
transesterification reaction at 30 ꢀC while keeping constant
amount of the following components in the reaction mix-
ture: vinyl butyrate (150 mM), hexane (25 ml), enzyme
(25 ml). The experiments were carried out in triplicate
and the samples were withdrawn after every 6 h up to
24 h. It is clear from Figure 5 that the final conversion
was highest when the initial concentration of rac-CDPP
was 5 mM. However, there was a decrease in conversion
rate beyond 5 mM, suggesting the toxicity of rac-CDPP
in higher concentration. Also, with the increase of rac-
CDPP concentration in the reaction mixture, there was a
decrease in the enantiomeric excess, indicating changing
0
Figure 3. Effect of solvent type on the transesterification reaction of rac-
CDPP by P. aeruginosa lipase at 30 ꢀC. [All reaction mixtures had 50%
(v/v) of organic solvent and the initial substrate concentrations were 5 mM
(rac-CDPP) and 150 mM (vinyl butyrate).]
2 mM
4 mM
5 mM
6 mM
8 mM
50
40
30
20
10
0
As reported by Goswami and Goswami in 2005, using di-
methyl sulfoxide (DMSO) as a co-solvent enhances the
enantioselectivity as well as the catalytic activity of li-
pase-mediated transesterification of chiral alcohols.12 In
view of this effect, the reactions were also evaluated using
DMSO (5%) as a co-solvent. This did not have any signif-
icant effect on the conversion rate when hexane, heptane,
benzene, and ACN were individually used as solvents. It
was indeed observed that when 5% DMSO was supple-
mented into THF, the conversion rate as well as the ee in-
creased (Fig. 4); however, the conversion rate was still
lower than that in hexane. Consequently, hexane was used
in all the subsequent experiments, in view of its superior
performance.
6
12
18
24
Time (h)
2.5. Effect of rac-CDPP concentration on transesterification
Figure 5. Effect of substrate concentration on the synthesis of (S)-butyric
acid 1-chloromethyl-2-(3,4-difluorophenoxy)-ethyl ester by lipases from P.
aeruginosa at 30 ꢀC.
The substrate concentration in the reaction medium influ-
ences sufficient expression of the enzyme activity, or may

2082
M. Singh, U. C. Banerjee / Tetrahedron: Asymmetry 18 (2007) 2079–2085
of the enzyme affinity for the (S)-enantiomer as compared
to its counterpart, that is, the (R)-enantiomer (Fig. 6).
butyrate. This is in contradiction with an earlier report,13
where the conversion rate was insensitive to vinyl ester
concentration.
Conversion
ee
2.7. Effect of various biocatalysts
60
50
40
30
20
10
0
100
80
60
40
20
0
Purified lipases from C. antarctica and porcine pancreatic
are known to be excellent catalysts for transesterification
reactions (Table 2); however, in the current study, C. ant-
arctica lipase showed very low conversion (12%) as well
as low enantioselectivity when compared to P. aeruginosa
lipase. Pancreatic lipase also showed almost no conversion
over 24 h. The higher activity of P. aeruginosa lipase is
probably due to its stability in the presence of acetalde-
hyde. It has been reported that acetaldehyde liberated by
the vinyl group of esters deactivates few enzymes through
the formation of a Schiff base with lysine residue.14–16
This may be the reason for the poor conversion rate and
enantioselectivity of C. antarctica and porcine pancreatic
lipase in the reaction mixture containing vinyl butyrate as
acyl donor.
0
2
4
6
8
10
12
Substrate concentration (mM)
Figure 6. Effect of substrate concentration on the conversion and
enantioselectivity of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol by
lipases from P. aeruginosa.
Table 2. Effect of lipases from different sources on the conversion of rac-
CDPP
2.6. Effect of vinyl butyrate concentration
Enzyme source
Conversion (%)
Candida rugosa
Porcine pancreatic
Pseudomonas aeruginosa MTCC 5113
15
5
>49
Figure 7 shows the effect of vinyl butyrate concentration on
the conversion of rac-CDPP. The concentration of vinyl
butyrate was varied from 100 to 200 mM while keeping
the constant concentrations of rac-CDPP (5 mM), hexane
(25 ml), and lipase solution (25 ml) in the reaction mixture.
It can be seen from Figure 7 that there was an increase in
the conversion rate with an increase in vinyl butyrate con-
centration up to 150 mM and it decreased with a further in-
crease of its concentration. This may be due to the decrease
in surface area for rac-CDPP molecules at the interface of
the reaction mixture at a higher concentration of vinyl
3. Conclusion
The biocatalytic transesterification of racemic alcohols
has been extensively explored and is now one of the best
methods for the preparation of enantiomerically pure
alcohols. However, the application of biocatalysts in
such a process has been hampered by the unavailability
of suitable biocatalysts. In our study, after a thor-
ough screening of 20 lipase producers, we used the best
one for the transesterification of rac-CDPP. This led
us to a bacterial strain (P. aeruginosa) that can catalyze
the stereoselective transesterification of rac-CDPP with
almost absolute enantioselectivity to (R)-1-chloro-3-(3,4-
difluorophenoxy)-2-propanol, a key intermediate of (S)-
Lubeluzole.
100 mM
125 mM
150 mM
175 mM
200 mM
60
50
40
30
20
10
0
The crude lipases from P. aeruginosa efficiently catalyzed
the enantioselective transesterification of rac-CDPP. Stud-
ies illustrated that a solvent, such as ACN, having a low
logP value decreases the reaction rate because such types
of solvents are hydrophilic in nature and tend to rip off
the water available for the enzyme activity. Also, DMSO
(mild base) when added in minute quantities into the reac-
tion mixture enhanced the conversion and enantioselectiv-
ity to a certain extent. Optimization of the substrate
concentration in the reaction revealed that with an increase
in substrate concentration, there is a substantial decrease in
both the conversion and the ee. This could be due to the
dead-end inhibition complex formation between lipases
and rac-CDPP or might be due to the presence of different
6
12
Time (h)
18
24
Figure 7. Effect of vinyl butyrate concentration on the transesterification
reaction of rac-CDPP by lipases from P. aeruginosa at 30 ꢀC.

M. Singh, U. C. Banerjee / Tetrahedron: Asymmetry 18 (2007) 2079–2085
2083
lipases in the crude mixture possessing opposite stereo-
chemical specificities and different affinities for rac-CDPP.
The reaction conditions were optimized so that rac-CDPP
was transesterified quantitatively and both the enantiomers
could be separated with ee >99% on a preparative scale
(conversion 42% and ee >99%). Thus, the lipases from P.
aeruginosa were used here for the first time on a preparative
scale and an efficient enzymatic resolution of rac-CDPP
was developed.
use of water in workup. The yield obtained was 95%.
Low melting white solid, Yield: 95%, 1H NMR
(300 MHz, CDCl3): d = 7.00–7.09 (m, 1H), 6.72–6.77 (m,
1H), 6.60–6.62 (d, J = 6.96 Hz, 1H), 4.19–4.23 (d,
J = 11.04 Hz, 1H), 3.81–3.85 (d, J = 10.98 Hz, 1H), 3.31
(s, 1H), 2.89 (s, 1H), 2.73 (s, 1H). 13C NMR (75 MHz,
CDCl3): d = 155.34, 166.52, 149.24, 147.33, 144.14,
177.83, 110.57, 104.89, 70.13, 50.42, 44.85. MS (ESI)
m/z = 186 (M+), 57 (100). On each occasion, the spectral
data (IR, NMR, and MS) of the prepared known com-
pounds were found to be identical with those reported in
the literature.
In conclusion, it can be said that the stereoselective transe-
sterification of rac-CDPP has been successfully carried out
with the lipase from a new bacterial strain, P. aeruginosa.
The result described here, in combination with the exist-
ing knowledge, makes the extracellular lipases from P. aeru-
ginosa very interesting biocatalysts for further investigation,
as well as for a variety of biotechnological applications.
Purification of lipases and cloning of the lipase gene in-
volved in the process and its application for the synthesis
of certain other chiral compounds are currently in progress
in our laboratory.
4.2.2. Synthesis of rac-CDPP from 2-(3,4-difluoro-phen-
oxymethyl)-oxirane. rac-CDPP was synthesized by dis-
solving 3,4-difluorophenoxy methyl oxirane (1.0 g,
5.3 mM), lithium chloride (0.4 g, 9.5 mM) in acetic acid
(1 ml) and THF (10 ml) at room temperature. The reaction
was carried out for 6 h and 90% yield of the product was
obtained. The reaction mixture was diluted with ethyl ace-
tate (15 ml), washed with water (5 ml), dried over Na2SO4,
and was concentrated under vacuum to afford (RS)-1-
chloro-3-(3,4-difluorophenoxy)-2-propanol.
4. Experimental
1H NMR (300 MHz, CDCl3): d = 7.02–7.11 (q, 1H), 6.70–
6.63 (m, 2H), 4.12–4.24 (m, 1H), 3.98–4.03 (m, 1H), 3.98–
4.03 (d, J = 5.106 Hz, 1H), 3.68–3.79 (m, 1H), 2.92 (br s,
1H). 13C NMR (75 MHz, CDCl3): d = 155.09, 152.620,
149.42, 147.52, 144.33. 118.37, 110.39, 104.92, 70.10,
46.33. MS (ESI) m/z = 222 (M+), 130 (100); spectroscopic
data were identical with an authentic sample.8
4.1. Chemicals and medium
3,4-Difluorophenol, epichlorohydrine, Triton X-100,
Tween-80, p-nitrophenyl palmitate and p-nitrophenol were
procured from Sigma–Aldrich Corporation (St. Louis,
MO, USA). Vinyl butyrate used in biological reactions
was purchased from Fluka. rac-CDPP was synthesized in
the laboratory and the structure was confirmed by 1H
13
and C NMR.17 Various components of growth media
4.3. Isolation of microorganism
were obtained from Himedia (Mumbai, India). Cultures
were grown in a medium containing peptone (0.5%), beef
extract (0.5%), yeast extract (0.1%), NaCl (0.5%) and agar
(1.5%). A minimal salt medium (MSM) used for the screen-
ing and growth of organisms under selection pressure
includes dihydrogen potassium phosphate (0.1%), di-
sodium orthophosphate (0.2%), ammonium chloride
(0.04%), magnesium chloride (0.04%) with 3,4-difluorophe-
nol as the sole carbon source. Agar plates were prepared by
supplementing MSM with 2 mM 3,4-difluorophenol. Soil
samples were collected from the different sites of an oil
industry in Punjab, India. Various solvents were of HPLC
grade and were obtained from Ranbaxy Fine Chemicals
Limited (New Delhi, India). Candida rugosa lipase was
obtained from Amano Pharmaceuticals Co. (Japan) and
porcine pancreas lipase was purchased from Sigma–
Aldrich Inc. (St. Louis, MO, USA).
Soil samples were collected from various places in and
around the oil industries in Punjab, India. A soil suspen-
sion was prepared by adding 10 ml of tap water to 1 g of
the soil sample. This was vortexed and 1 ml of its superna-
tant used as an inoculum in 100 ml MSM containing 1 mM
3,4-difluorophenol or 1 mM rac-CDPP as a sole carbon
source and incubated at 30 ꢀC in an orbital shaker
(200 rpm) for 4–5 days. A loop full of the broth was plated
onto selective 3,4-difluorophenol and rac-CDPP plates. All
positive isolates were transferred to nutrient agar plates
(0.5% peptone, 0.1% (v/v) yeast extract, 0.5% beef extract,
0.5% sodium chloride, pH 8). The enzyme production was
carried out using 1% (v/v) preculture (12 h) in 500 ml flask
containing 100 ml production medium with 2 mM 3,4-
difluorophenol or rac-CDPP as inducer. The production
was carried out at 30 ꢀC (200 rpm) for 24 h. Extracellular
broth containing lipase was harvested by centrifugation
(10,000g, 10 min) and enzyme activity and the enantioselec-
tivity for rac-CDPP transesterification were determined.
The reaction mixture was incubated at 30 ꢀC (ꢀ1000
rpm). Samples (1 ml) containing solvent layer were with-
drawn after every 3 h up to 48 h. The organic phase was
dried and removed under reduced pressure. Finally, the
conversion and ee of the (S)-1-chloro-3-(3,4-difluorophen-
oxy)-2-butanoate formed were determined using chiral
HPLC. The transesterification of rac-CDPP by P. aerugin-
osa is shown in Scheme 1.
4.2. Chemical synthesis of (RS)-1-chloro-3-(3,4-difluoro-
phenoxy)-2-propanol
4.2.1. Synthesis of 2-(3,4-difluoro-phenoxymethyl)-oxi-
rane. 3,4-Difluorophenol (1.0 g, 3.8 mM), epichlorohy-
drin (0.52 g, 5.7 mM) and potassium carbonate (1.05 g,
76 mM) or cesium carbonate were dissolved in acetonitrile
(10 ml). The reaction was stirred at reflux for 16 h, ex-
tracted, dried over Na2SO4, and concentrated in vacuo.
The reaction was carried out in acetonitrile to avoid the

2084
M. Singh, U. C. Banerjee / Tetrahedron: Asymmetry 18 (2007) 2079–2085
OH
O
Cl
F
F
1-Chloro-3-(3,4-difluoro-phenoxy)-propan-2-ol
vinyl butyrate
Lipase
O
H
OH
H
OCC3H7
Cl
F
F
O
Cl
O
F
F
+
(R)-1-Chloro-3-(3,4-difluoro-phenoxy)-propan-2-ol
(S)-Butyric acid 1-chloromethyl-2-
(3,4-difluoro-phenoxy)-ethyl ester
Scheme 1. Transesterification of (RS)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol to (S)-butyric acid 1-chloromethyl-2-(3,4-difluorophenoxy)-ethyl ester
by crude lipase from P. aeruginosa.
4.4. Enzyme production
spectrophotometrically at 410 nm against substrate blank.
One unit of enzyme activity was defined as the amount of
enzyme that liberated 1 lmol p-nitrophenol per minute un-
der the above specified assay condition.
A loop full of microorganisms maintained on nutrient agar
plates (0.5%, w/v peptone; 0.15%, w/v yeast extract; 0.5%,
w/v beef extract; 0.5%, w/v sodium chloride; 1.5%, w/v
agar; pH 8) was transferred to 20 ml sterilized (121 ꢀC,
20 min) nutrient broth (as above, without agar) to produce
the seed culture. A 1% (v/v) seed inoculum was used to
inoculate 100 ml nutrient broth in 500 ml shake flask and
incubated at 30 ꢀC (200 rpm, 96 h). Crude supernatant of
the fermentation broth was separated from the cells by cen-
trifugation (10,000g, 10 min). The supernatant was used as
crude lipase preparation.
4.6. Enantioselective transesterification of rac-CDPP
The transesterification reaction was carried out by extracel-
lular crude lipase in 100 ml capacity stoppered flask con-
taining substrate rac-CDPP and vinyl butyrate, that was
magnetically agitated (ꢀ1000 rpm) and held at the desired
constant temperature (30–60 ꢀC) in an incubator for 24 h
(Scheme 1). Samples (1 ml) were collected and processed,
as described above, to monitor the conversion and
enantioselectivity.
4.5. Enzyme assay
A modified form of the Winkler and Stuckmann method
was applied to the quantification of lipase activity in the
crude mixture of P. aeruginosa.18 The substrate (p-nitro-
phenyl palmitate) was dissolved in 2-propanol (3 mg/ml)
and an aqueous solution (9 ml) of gum arabic (0.11% w/
v) and Triton X-100 (0.44% w/v) was added into the sub-
strate solution. Intense agitation (vortex mixture) was used
to emulsify the mixture. This emulsion (0.9 ml) was mixed
with 1.5 ml tris–HCl buffer (50 mM, pH 8) and 0.5 ml
CaCl2 (75 mM). After 5 min of incubation, 100 ll of
appropriately diluted (in 50 mM tris–HCl buffer, pH 8.0)
enzyme solution was added. Incubation was continued
for a further 10 min. The reaction was stopped by putting
the test tubes in ice and the optical density was measured
4.7. Optimization of transesterification reaction
The effect of various solvents (hexane, heptane, toluene,
benzene, tetrahydrofuran, and water-miscible solvents such
as acetonitrile) on the transesterification of rac-CDPP was
evaluated. Unless otherwise specified, the total volume of
the solvent was always 50% of the total volume of the reac-
tion mixture. It has been reported that the addition of a
trace amount of mild base enhances the enantioselectivity
and catalytic activity in Pseudomonas lipase catalyzed
resolution of racemic alcohols.12 To evaluate the effect of
the co-solvent on the transesterification reaction, a cata-
lytic amount of denaturing organic DMSO was added into
each solvent in separate experiments.

M. Singh, U. C. Banerjee / Tetrahedron: Asymmetry 18 (2007) 2079–2085
2085
The optimum temperature was determined by incubating
the lipase with the substrate at different temperatures in
the range 20–60 ꢀC. To find out the effect of substrate con-
centration, varying amounts of rac-CDPP (1–10 mM) were
subjected to transesterification by P. aeruginosa. Finally, in
order to optimize the concentration of the acyl donor,
reactions were carried out by varying the concentrations
of vinyl butyrate (100–200 mM) and keeping the other
parameters constant. The samples were taken and analyzed
for transesterification efficiency and enantioselectivity.
a Shimadzu 10AVP Instrument equipped with a UV detec-
tor using a Chiracel ODH column (0.46 mm diam.,
250 mm long, 5 lm, Chiralcel). The mobile phase was hex-
ane–isopropyl alcohol at 95:05 (v/v) with a flow rate of
0.5 ml/min and detected at 220 nm. The retention times
of (S)- and (R)-1-chloro-3-(3,4-difluorophenoxy)-2-propa-
nol were 23 and 24.7 min, respectively, and the retention
time for the (S)-ester was 12.2 min in the Chiracel ODH
column. The ee was defined as the ratio of [R] ꢁ [S]/
[R] + [S] · 100%, where [R] and [S] are the concentrations
1
of the (R)- and (S)-enantiomers, respectively. H and 13C
4.8. Preparative-scale transesterification
NMR spectra were recorded on a Bruker Advance DPX
300 NMR spectrometer and optical rotations were mea-
sured on a Rudolph polarimeter (Rudolph Research Auto-
pol IV).
In order to scale-up the transesterification of rac-CDPP,
when the reaction was carried out at a preparative scale
with 3 g rac-CDPP, (S)-1-chloro-3-(3,4-difluorophenoxy)-
2-butanoate was obtained at a conversion of about 42%
with an ee >99%. This suggests that the crude lipase mix-
ture of P. aeruginosa is a versatile biocatalyst, able to
transesterify (RS)-1-chloro-3-(3,4-difluorophenoxy)-2-pro-
panol with excellent enantioselectivity. Also, the results
demonstrated promising prospects for the practical appli-
cation of P. aeruginosa in the production of (R)-1-chloro-
3-(3,4-difluorophenoxy)-2-propanol, a key intermediate in
the synthesis of anti-ischemic drug (S)-Lubeluzole.
Acknowledgments
We are grateful to Dr. A. K. Chakraborti for the helpful
discussion and for providing full access to his laboratory.
M.S. gratefully acknowledges the Department of Science
and Technology (DST), India, for providing fellowship.
This is NIPER Communication No. 402.
22
For the (S)-ester, ½aꢂD ¼ þ18:9 (c 1, CHCl3).Yellowish li-
quid, IR (Neat): 2965, 2934, 1743, 1609, 1518, 1462,
1435, 1327, 1262, 1216, 1204, 1162, 1101, 1043, 983, 963,
References
851, 786, 704 cmꢁ1
.
1H NMR (300 MHz, CDCl3):
d = 7.02–7.11 (m, 1H), 6.71–6.77 (m, 1H), 6.60–6.63 (m,
1H), 5.31–5.34 (m, 1H), 4.13 (s, 1H), 4.11 (s, 1H), 3.81–
3.85 (m, 1H), 3.73–3.78 (m, 1H), 2.33–2.38 (t,
J = 7.18 Hz, 2H), 1.62–1.73 (m, J = 7.16 Hz, 2H), 0.83–
0.87 (t, J = 7.07 Hz, 3H). 13C NMR (75 MHz, CDCl3):
d = 171.78, 153.53, 150.99, 146.08, 116.42, 108.86, 103.61,
69.50, 65.81, 41.3, 34.99, 17.37, 13.09. MS (MALDI-
TOF) m/z = 315.50 (M+), 242 (100).
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22
For the (R)-alcohol, ½aꢂD ¼ ꢁ1:9 (c 1.14, CHCl3). IR
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1211, 1162, 1116, 1037, 957, 840, 783 cmꢁ1 1H NMR
.
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2H), 3.68–3.79 (m, 1H), 3.13 (br s, 1H). 13C NMR
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149.2, 147.5, 147.4, 144.4, 144.2, 117.9, 117.7, 110.4,
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4.9. Effect of various biocatalysts
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Purified lipases from different sources (C. rugosa and Por-
cine pancreatic) were evaluated for the transesterification
of rac-CDPP with vinyl butyrate at 30 ꢀC. The results for
conversion and enantioselectivity were compared with the
crude lipases from P. aeruginosa.
4.10. Analytical methods
Conversion and the enantiomeric excess of the transesteri-
fication reaction were monitored by HPLC performed on