A new biocatalyst for the preparation of enantiomerically pure 2-arylpropanoic acids

Article abstract of DOI:10.1016/S0957-4166(01)00249-X

A new biocatalyst, a strain of Pseudomonas fluorescens MTCCB0015, is described, which produces ibuprofen, ketoprofen and flurbiprofen as enantiomerically pure (S)-2-arylpropanoic acids from their corresponding racemic esters. 2-Arylpropanoic acids are an important class of non-steroidal anti-inflammatory compounds, whose anti-inflammatory activity is mainly due to the (S)-enantiomer.

Full text of DOI:10.1016/S0957-4166(01)00249-X

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 1431–1434
A new biocatalyst for the preparation of enantiomerically pure
†
2
-arylpropanoic acids
‡
§
Ish Kumar, Kavita Manju and Ravinder S. Jolly*
Department of Chemistry, Institute of Microbial Technology, Sector 39, Chandigarh 160 036, India
Received 16 April 2001; accepted 25 May 2001
Abstract—A new biocatalyst, a strain of Pseudomonas fluorescens MTCCB0015, is described, which produces ibuprofen,
ketoprofen and flurbiprofen as enantiomerically pure (S)-2-arylpropanoic acids from their corresponding racemic esters.
2
-Arylpropanoic acids are an important class of non-steroidal anti-inflammatory compounds, whose anti-inflammatory activity is
mainly due to the (S)-enantiomer. © 2001 Elsevier Science Ltd. All rights reserved.
1
. Introduction
Biocatalyzed hydrolysis of the (±)-2-arylpropanoate
esters is an attractive method for the resolution of the
corresponding acids. Several lipases of microbial origin
have been used, but only the lipase of Candida cylin-
dracea gave the (S)-enantiomeric acid selectively. The
enantioselectivities obtained in the hydrolysis of various
2
-Arylpropanoic acids are an important class of non-
1
–3
steroidal anti-inflammatory compounds. Several rep-
resentatives of this class of compounds, such as
ibuprofen, ketoprofen, flurbiprofen, naproxen and
flunoxaprofen, have been introduced as pharmaceuti-
cals. (S)-Ibuprofen has been available since 1994, but
until recently, all except naproxen and flunoxaprofen
have been marketed and consumed as racemic mixtures.
The anti-inflammatory activity of this class of com-
pounds is mainly due to the active (S)-enantiomer. For
this reason, several investigations have been carried out
to obtain enantiomerically pure (S)-2-arylpropanoic
1
6
2
-arylpropanoic esters catalyzed by Candida cylindracea
33
have been described. While good enantioselectivity
was observed for the hydrolysis of the ester of ibupro-
fen with E values of 50, hydrolysis of 2-phenyl-
propanoic acid, ketoprofen and flurbiprofen proceeded
with poor enantioselectivity, with E values in the range
4
–10. Several methods, such as non-covalent enzyme
33 34
modification and enantioselective inhibition, have
been developed to improve the enantioselectivity of
Candida cylindracea enzyme. Recently, a Bacillus sub-
tilis strain has been described, which produced ibupro-
fen in about 96% e.e. from ibuprofen methyl ester. The
reduced e.e. was attributed to the presence of a non-
specific esterase in the organism. Cloning and over-
expression of the enzyme in the same organism
eliminated contaminant esterase activity and the e.e.
4
–12
acids. A number of stereoselective chemical
and
1
3–15
chemoenzymatic
methods have been described in
the last few years for the synthesis of 2-arylpropanoic
acids in non-racemic form. A resolution of (±)-2-aryl-
propanoic acids has been achieved by enzyme-catalyzed
enantioselective hydrolysis of the corresponding
racemic esters, amides
lective esterification or transesterification of (±)-2-aryl-
propanoic 5–3a2 cids with lipases has also been
1
6
17–19
20–24
and nitriles.
Enantiose-
35
2
was improved to >99%.
described.
Herein, we describe a new biocatalyst, a strain of
¶
Pseudomonas fluorescens MTCCB0015, that produced
ibuprofen, ketoprofen and flurbiprofen from their cor-
responding racemic esters in enantiomerically pure (S)-
configuration. The biocatalyzed hydrolysis reaction can
*
Corresponding author. Tel.: 91-172-690025; fax: 91-172-690585;
e-mail: jolly@imtech.res.in
Communication number 07/2001.
Present address: Postdoctoral Research Associate, SUNY, UpState
Medical University, Syracuse, NY 13210, USA.
Present address: Postdoctoral Research Associate, Department of
Chemistry, Colorado State University, Fort Collins, CO 80523,
USA.
†
‡
§
¶
Accession number: B0015, Microbial Type Culture Collection
(MTCC), Institute of Microbial Technology, Sector 39, Chandigarh
160 036, India.
0
957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00249-X

1
432
I. Kumar et al. / Tetrahedron: Asymmetry 12 (2001) 1431–1434
Table 1. Pseudomonas fluorescens MTCC0015-catalyzed hydrolysis of 2-arylpropanoic acids
O
COOR
COOR
COOR
COOR
F
1a-1c
2a-2c
3a-3c
4a-4c
a, R = H; b, R = CH ; c, R = CH CF
3
3
2
Entry
Ester
Conversion (%)
Time (h)
Produced (S)-acida
Recovered ester
Yieldb (%)
E.e. (%)
Yieldb (%)
1
2
3
4
5
6
7
8
9
1b
1b
2b
3b
4b
1c
1c
1c
2c
3c
4c
100
20
20
20
25
100
67
22
47
48
47
26
3
0
\99
\99
\99
\99
0
95
15
17
18
20
92
60
20
40
40
43
0
75
68
72
48
0
c
c
c
c
20
20
20
12
5
d
38
30
c
c
c
c
1
\99
\99
\99
\99
71
50
48
48
20
20
20
1
1
0
1
a
b
c
1
3
6
C
o
n
fi
g
u
r
a
t
i
o
n
a
n
d
e
.
e
.
w
e
r
e
d
e
t
e
r
m
i
n
e
d
b
y
t
h
e
H
N
M
R
m
e
t
h
o
d
o
f
F
u
l
w
o
o
d
e
t
a
l
.
Yield of the product isolated after column chromatography.
No (R)-acid could be detected.
d
E.e. of recovered ester, 74% by 1H NMR in the presence of Eu(hfc)3.
be carried out with whole cells or preferably with crude
lysate and required no further modification of organism
or enzyme to achieve high enantioselectivity.
0.5N aqueous HCl. The solvent was removed under
reduced pressure, the residue thus obtained was sub-
jected to flash chromatography over silica gel (ethyl
acetate/hexane; 1:4) to yield pure 1a and 1b. The e.e.
and the configuration of 1a were determined by follow-
ing a procedure proposed by Fulwood et al. 2-Phenyl-
propanoic acid 1a produced at 20% conversion was
found to be enantiomerically pure having (S)-
configuration.
36
2
. Results and discussion
To begin with, the biocatalyzed hydrolysis of the parent
compound methyl 2-phenylpropanoate 1b was
attempted. Initially whole cells of Pseudomonas fluores-
cens MTCCB0015 were used as biocatalyst. They were
able to hydrolyze 1b to the corresponding acid, but the
reaction was very slow. It was therefore decided to use
the cell-free extract as biocatalyst. The cell-free extract
was prepared by sonication of cells of Pseudomonas
fluorescens (2 g; wet mass) suspended in 5 mL of
phosphate buffer, pH 7.5, followed by removal of
debris by centrifugation at 16,000×g for 30 min. To this
cell-free extract 0.25 mmol of 1b was added and the
contents incubated at 37°C with constant shaking at
We next studied the biocatalyzed hydrolysis of ibupro-
fen methyl ester 2b using cell-free extract as biocatalyst.
The reaction was carried out under the reaction condi-
tions described above. In this case, the reaction was
slower compared to 1b; only 20% conversion occurred
in 20 hours at 37°C (entry 3, Table 1). Ibuprofen 2a
and unreacted ester 2b were separated by flash chro-
matography over silica gel (ethyl acetate/hexane; 1:4).
The ibuprofen so produced was found to be enan-
3
6
tiomerically pure and was assigned (S)-configuration.
200 rpm. The reaction was monitored by withdrawing
aliquots (100 mL) of reaction mixture, which were
acidified to pH 2.0 with 0.5N HCl and extracted with
ether. The ethereal layer was analyzed for the produc-
tion of 2-phenylpropanoic acid 1a by TLC. Under these
conditions, quantitative conversion of 1b to 1a occurred
in 20 h (entry 1, Table 1). The acid so produced was
found to be a racemic mixture. The reaction was
repeated under similar conditions, but stopped at about
Similarly, methyl esters of ketoprofen 3b (entry 4, Table
) and flurbiprofen 4b (entry 5, Table 1) on biocata-
1
lyzed hydrolysis under the reaction conditions described
above gave ketoprofen 3a and flurbiprofen 4a, respec-
tively. The rates of reaction were similar for both these
compounds; after 20 hours the conversion was found to
be 20 and 25%, respectively. The acids in both cases
were found to be enantiomerically pure and assigned
2
0% conversion (entry 2, Table 1). The reaction mixture
was extracted in ether after acidification to pH 2.0 with
36
(S)-configuration.

I. Kumar et al. / Tetrahedron: Asymmetry 12 (2001) 1431–1434
1433
Although the biocatalyst was able to enantioselectively
3.2. Preparation of cell-free extract
hydrolyze methyl esters of ibuprofen, ketoprofen and
flurbiprofen, the rates of the reaction were very low. It
has been reported that the conversion rate could be
considerably enhanced by the use of activated esters.
Thus, instead of methyl esters, biocatalyzed hydrolysis
A suspension of cells (2 g) in 50 mM phosphate buffer,
pH 7.50 (5 mL), was cooled in ice and then subjected to
sonication for 20 min at 4°C. The debris was removed
by centrifugation at 16,000×g for 0.5 h. The super-
natant was used as biocatalyst.
16
of 2,2,2-trifluoroethyl ester of 2-phenylpropanoic acid
1
c was studied under the reaction conditions described
above. As expected there was improvement in the con-
version rate (entries 6–8, Table 1). The reaction was
stopped at 22, 67 and 100% conversion to give the
3.3. General procedure for biocatalyzed hydrolysis
Ester (0.25 mmol) was suspended in cell-free extract
prepared above. The reaction mixture was incubated at
(
S)-acid in >99, 38 and 0% e.e., respectively.
3
7°C for 20 h, maintaining the pH at 7.50 by addition
Similarly, the biocatalyzed hydrolysis of 2,2,2-tri-
fluoroethyl esters of ibuprofen (2c; entry 9, Table 1),
ketoprofen 3c (entry 10, Table 1) and flurbiprofen 4c
of 0.1 M NaOH. The progress of the reaction was
monitored by TLC. It was then acidified to pH 2.0 with
0.5N HCl and extracted in ether. The ether layer was
washed with brine, dried (Na SO ) and evaporated
(
entry 11, Table 1) gave the corresponding profen and
unreacted ester. The conversion was found to be about
7–48% after 20 h under the reaction conditions
described above. The profen products were enantiomer-
2
4
under reduced pressure. The separation of acid and
4
unreacted ester was achieved by flash chromatography
1
over silica gel (ethyl acetate/hexane, 1:4). H NMR
3
6
ically pure and assigned (S)-configuration.
(CDCl ): l 1a, 1.51 (3H, d, J=7.2 Hz), 3.76 (1H, q,
3
J=7.2 Hz), 7.23–7.34 (m, 5H), 8.3 (1H, bs). 2a, 0.88
(6H, d, J=6.6 Hz), 1.49 (3H, d, J=7.3 Hz), 1.82 (1H,
m), 2.43 (2H, d, J=7.2 Hz), 3.70 (1H, q, J=7.3 Hz),
7.11 and 7.21 (each 2H, each d, J=8.1 Hz). 3a, 1.56
(3H, d, J=7.3 Hz), 3.82 (1H, q, J=7.3 Hz, H-2),
7.26–7.81 (9H, m). 4a, 1.56 (3H, d, J=7.0 Hz), 3.79
(1H, q, J=7.0 Hz), 7.13–7.54 (8H, m).
It is clear from the results described above that the rate
of the reaction and the enantioselectivity in this biocat-
alyzed hydrolysis is dependent on the bulk of the
substituent present in the phenyl ring of the 2-aryl-
propanoate. Substitution with a bulky group decreased
the rate of reaction but increased the selectivity. This is
further supported by our observations on the biocata-
lyzed hydrolysis of methyl esters of 2-(2-chlorophen-
oxy)propanoic acids 5, 2-(4-chlorophenoxy)propanoic
acid 6 and 2-(2,4-dichlorophenoxy)propanoic acid 7
using cell-free extract as biocatalyst. In all examples,
3.4. Determination of the e.e. of profens
1
The e.e. was determined by H NMR analysis of the
diastereoisomeric salt complex of the chiral acid with
1
00% conversion occurred in 16–20 hours to give corre-
(1R,2R)-1,2-diphenylenediamine as described by Ful-
36
sponding 2-aryloxypropanoic acid in racemic form.
Even at 15–20% conversion, acids produced from 5–7
were racemic.
wood et al. For samples of high optical purity, a
signal corresponding to the minor enantiomer could not
be detected. Validation of the method in these cases
was confirmed by addition of a small amount of race-
mate acid to the sample, which was again analyzed by
1
H NMR.
Acknowledgements
3
. Experimental
I.K. and K.M. thank CSIR, New Delhi for the award
of Senior Research Fellowship and Research Associate-
ship, respectively. We thank MTCC, Institute of Micro-
bial Technology for identification and storage of
culture of Pseudomonas fluorescens.
3
.1. Preparation of biocatalyst
A loopful of actively growing culture of Pseudomonas
fluorescens was inoculated into a 100 mL Erlenmeyer
flask containing 20 mL of sterile medium consisting of
(
per 1000 mL) peptone (10 g) and beef extract (5 g).
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