A practical enzymatic method for preparation of (S)-ketoprofen with a crude Candida rugosa lipase

Article abstract of DOI:10.1081/SCC-100106210

A simple and practical method for the preparation of (S)-Ketoprofen (2-(3-benzoylphenyl) propionic acid) has been developed through the hydrolysis of its chloroethyl ester catalyzed by a cheap and commercially available crude Candida rugosa lipase preparation under an extremely acidic condition, pH 2.5, and in the presence of Tween-80. As the reaction was carried out in a shake flask with baffles.

Full text of DOI:10.1081/SCC-100106210

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A PRACTICAL ENZYMATIC METHOD FOR PREPARATION
OF (S)-KETOPROFEN WITH A CRUDE CANDIDA RUGOSA
LIPASE
Hui-Yuan Wu a , Jian-He Xu b & You-Yan Liu c
a Laboratory of Applied Biocatalysis, State Key Laboratory of Bioreactor Engineering ,
East China University of Science and Technology , Shanghai, 200 237, P. R. China
b Laboratory of Applied Biocatalysis, State Key Laboratory of Bioreactor Engineering ,
East China University of Science and Technology , Shanghai, 200 237, P. R. China
c Department of Chemical Engineering , Guangxi University , Nanning, Guangxi, 530 004,
China
Published online: 22 Aug 2006.
To cite this article: Hui-Yuan Wu , Jian-He Xu & You-Yan Liu (2001) A PRACTICAL ENZYMATIC METHOD FOR PREPARATION
OF (S)-KETOPROFEN WITH A CRUDE CANDIDA RUGOSA LIPASE, Synthetic Communications: An International Journal for
Rapid Communication of Synthetic Organic Chemistry, 31:22, 3491-3496, DOI: 10.1081/SCC-100106210
To link to this article: http://dx.doi.org/10.1081/SCC-100106210
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SYNTHETIC COMMUNICATIONS, 31(22), 3491–3496 (2001)
A PRACTICAL ENZYMATIC
METHOD FOR PREPARATION
OF (S)-KETOPROFEN WITH A
CRUDE CANDIDA RUGOSA LIPASE
Hui-Yuan Wu, Jian-He Xu,*
y
andYou-Yan Liu
Laboratory of Applied Biocatalysis,
State Key Laboratory of Bioreactor
Engineering, East China University of
Science and Technology,
Shanghai 200 237, P. R. China
ABSTRACT
A simple and practical method for the preparation of
(S )-Ketoprofen (2-(3-benzoylphenyl) propionic acid) has
been developed through the hydrolysis of its chloroethyl
ester catalyzed by a cheap and commercially available crude
Candida rugosa lipase preparation under an extremely acidic
condition, pH 2.5, and in the presence of Tween-80. As
the reaction was carried out in a shake flask with baffles,
*Corresponding author. Fax: þ 86-21-64253904; E-mail: jianhexu@ecust.edu.cn
yCurrent address. Department of Chemical Engineering, Guangxi University,
Nanning 530 004, Guangxi, China.
3491
Copyright & 2001 by Marcel Dekker, Inc.
www.dekker.com

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3492
WU, XU, AND LIU
the degree of hydrolysis was enhanced from 31.5% to 42.2%.
A needle-like crystalline of (S )-Ketoprofen was obtained with
95.7% e.e. after simple separation and purification.
2-Arylpropionic acids represent an important group of non-steroidal
anti-inflammatory drugs (NSAIDs) whose anti-inflammatory activities
reside mainly in their (S)-enantiomers.1 Although a chemical method (via
diastereomeric crystallization) was traditionally used in optical resolution of
Naproxen (2-(6-methoxy-2-naphthyl) propionic acid), biocatalytic
approaches become more and more popular for kinetic resolution of enan-
tiomers due to the simplicity of processes and the high enantioselectivity of
enzymes,2 especially toward those substrates with complicated structures.
Among various enzyme preparations from commercial sources, only the
lipases from Candida rugosa (formerly Candida cylindracea) showed the
desired (S)-preference in the enantioselective hydrolysis of 2-arylpropionic
acid esters.3 However, the enantioselectivity of the crude Candida rugosa
lipase toward an ester of Ketoprofen (2-(3-benzoylphenyl) propionic acid)
was so low (E ¼ 4) that additional treatments such as purification and non-
covalent modification of the enzyme had to be adopted.4 Such a cumber-
some process is, therefore, rather difficult to be applied for the practical
resolution of Ketoprofen. Herein, several commercially available crude
lipases were tested as the bio-catalysts in enantioselective hydrolysis of the
chloroethyl ester of Ketoprofen (1) under various conditions (Scheme 1). As
a result, a novel and efficient enzymatic method for preparation of
((S )-Ketoprofen ((S )-2-(3-benzoylphenyl) propionic acid 2) was successfully
established, in which a crude Candida rugosa lipase (Lipase OF, from Meito
Sangyo Co., Japan), without any purification steps and pretreatments, was
directly used to prepare 2 from 1 with a commercially acceptable conversion
ratio and optical purity.
Scheme 1.

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(S)-KETOPROFEN
3493
Table 1. Enzymatic Resolution of Ketoprofen via Enantioselective Hydrolysis of
Its 2-Chloroethyl Ester Catalyzed by Different Candida rugosa Lipase Preparations
at Various pHsa
Entry
Enzymes
pH
Conv. (%)
e.e.b(%)
E c
1
Lipase OFd
7.0
7.0
7.0
4.0
4.0
4.0
2.5
2.5
2.5
13.0
20.9
9.5
20.5
24.5
8.5
29.2
8.7
10.4
4.5
23.6
12.0
26.9
60.0
27.6
77.6
86.9
89.4
1.1
1.7
1.3
1.9
4.8
1.8
13.2
15.5
19.8
2
3Type VII
Lipase MYd
e
4
5
6
7
8
9
Lipase OF
Lipase MY
Type VII
Lipase OF
Lipase MY
Type VII
aReactions were carried out on a shaker at 30ꢀC and 160 rpm for 120 h, with 1 mmol
substrate and 200 mg enzyme powder suspended in 10 ml of 0.1 M citrate/0.2 M
Na2HPO4 buffer at indicated pHs. bEnantiomeric excess of the acid produced.
cEnantiomeric ratio8: E = ln[1-c(1 + ee)]/ln[1-c(1 ꢁ ee)]. dFrom Meito Sangyo Co.,
e
Japan. From Sigma (L 1754).
EFFECT OF PH ON THE HYDROLYSIS OF
KETOPROFENATE CATALYZED BY DIFFERENT
LIPASE PREPARATIONS
At first, the effect of environmental pH on the three commercial
Candida rugosa crude lipases, Lipase OF, Lipase MY and Type VII was
examined. When the enzymatic hydrolysis of chloroethyl Ketoprofenate
catalyzed by these lipases was conducted at various pH values, it was
found that the reactions catalyzed by each of the tested lipases increased
in the enantioselectivity with the decrease of pH value from 7.0 to 2.5 (Table
1). But it was only Lipase OF that resulted in a remarkably enhanced con-
version of the ester from 13.0% to 29.2%. At pH 7.0 and 4.0, Lipase MY
afforded higher conversion and enantioselectivity than Lipase OF, but shar-
ply decreased in the conversion when pH was lowered to 2.5.
It was reported that lipases produced by Candida rugosa contained sev-
eral different isoenzyme components5 and some of them displayed different
preferences to the two enantiomers of the Ketoprofen ester.6 Therefore, the
results in Table 1 suggest that different commercial crude preparations of
Candida rugosa lipases might markedly distinguish in their components.

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WU, XU, AND LIU
Table 2. Enhanced Selectivity by Tween-80 in the Hydrolysis of 2-Chloroethyl
Ketoprofenate Catalyzed by Different Candida rugosa Lipases at Various pHsa
Entry
Enzymes
pH
Conv. (%)
e.e.b(%)
E c
1
Lipase OFd
7.0
7.0
7.0
4.0
4.0
4.0
2.5
2.5
2.5
69.9
10.6
10.0
29.0
8.9
8.5
33.7
4.5
16.5
54.2
50.1
41.9
1.9
3.6
3.2
2.9
2
3Type VII
Lipase MYd
e
4
5
6
7
8
9
Lipase OF
Lipase MY
Type VII
Lipase OF
Lipase MY
Type VII
86.2
14.7
86.314.7
95.0
92.6
62.2
27.2
32.8
4.9
93.8
aReactions were carried out on a shaker at 30ꢀC and 160 rpm for 24 h, with 1 mmol
substrate and 100 mg enzyme powder suspended in 10 ml 0.1 M citrate/0.2 M
Na2HPO4 buffer at indicated pHs with 2% of Tween-80. bEnantiomeric excess of
c
the acid produced. Enantiomeric ratio8: E = ln[1-c(1 + ee)]/ln[1-c(1 ꢁ ee)]. dFrom
e
Meito Sangyo Co., Japan. From Sigma (L1754).
EFFECT OF A NON-IONIC SURFACTANT ON THE
HYDROLYSIS OF KETOPROFENATE CATALYZED
BY DIFFERENT LIPASE PREPARATIONS
Surfactants are widely used in lipase-catalyzed reactions to increase
the lipid-water interfacial area and, thus to enhance the apparent rates of
these enzymatic reactions.7 When 2% (w/v) of Tween-80 was added into the
reaction system at various pHs, remarkably improved enantioselectivities
were observed for all the tested enzymes (Table 2). However, Tween-80
seemed more prone to inactivate Lipase MY and Type VII, since almost
all the reactions catalyzed by these two enzymes stopped at lower conver-
sions in the presence of Tween-80. But different results were observed in the
reactions using the crude Lipase OF. The enzyme seemed to be activated by
Tween-80. The conversion exceeded 50% at pH 7.0 after 24 h and even
reached 33.7% at pH 2.5, with the surprisingly high e.e. value of 95.0%,
which resulted in an E value of 60 times higher than that in the neutral
environment (pH 7.0).
The effect of Tween-80 concentration was further investigated in the
reaction system using Lipase OF at pH 2.5 (Figure 1). It was found that
both the conversion and the enantioselectivity were enhanced with the
increasing amount of Tween-80 up to 0.5%, but tended to be constant
when Tween-80 was higher than 0.5%.

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(S)-KETOPROFEN
3495
Figure 1. Effect of Tween-80 concentration on the yield and e.e. value of 2
produced by Lipase OF at pH 2.5.
EFFECT OF SUBSTRATE DISPERSION AND
MASS TRANSFER ON THE REACTION
It was also found that whether the reaction mixture was sufficiently
pre-emulsified or not affected the enantioselectivity notably. Among several
emulsification methods, mechanical homogenization resulted in a better
dispersed reaction system than the others, such as shaking or ultrasonication
(data not shown).
When a sufficiently homogenized mixture of 1 and 0.5% (w/v) of
Tween-80 in the buffer was used to prepare 2 on an amplified scale, the
conversion was still unsatisfactory. As the shake flasks with baffles were used
to enhance the mass transfer in the reaction system, the conversion of the
ester increased from 31.5% to 42.2%, without any decrease in the e.e. value.
In summary, the results above provide a novel method with both
commercially acceptable conversion ratio and e.e. value for preparation of
(S)-Ketoprofen, which was never achieved previously using crude enzymes.
General procedure for preparation of (S )-2-(3-benzoylphenyl) propionic
acid(2) from 1: To 200 ml of emulsion containing 6.3g (20 mmol) of 1
(which could be conveniently synthesized as previously described9), 1.0 g
of Tween-80 and 0.1 M citrate /0.2 M Na2HPO4 buffer at pH 2.5, which
was sufficiently pre-emulsified in a homogenizer at 10,000 rpm, was added
2.0 g of crude Candida rugosa lipase (Lipase OF, from Meito Sangyo Co.,
Japan). After reacting at 30ꢀC and 160 rpm for 24 h, the mixture was
adjusted to ca. pH 10.0 with 2N NaOH and then centrifuged to remove
most of the ester. The crude product was obtained as a precipitate after the
supernatant was acidified to pH 2.0, with 39.1% I.Y. and 95.5% e.e.. Then it
was eluted through a short silica gel column to remove the residual Tween-

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3496
WU, XU, AND LIU
80, ester and protein and afforded 2 (21.2% I.Y., 95.5% e.e.) as light yellow
powder. Then the product was recrystallized from NaHCO3/HAC, and the
structure of the crystalline solid was confirmed to be 2 (95.7% e.e.) by NMR
and chiral analysis on HPLC using a CHIRALCEL OJ column (Daicel
Chemical Industries Ltd., Japan).
2-(3-Benzoylphenyl) propionic acid(2):
C.Y., 42.2% 1H-NMR
(500 MHz, MeOH-d4), d/ppm: 7.77–7.48 (m, 9H, ArH); 3.81 (q, 1H,
J ¼ 7.16; -CH(CH3)COOH), 1.48 (d, 3H, J ¼ 7.17; -CH3); m.p. 74–78ꢀC
20
D
20
D
[lit10: 75–78ꢀC]; ½aŠ þ 45ꢀ (c ¼ 1.0, MeOH) [lit10: ½aŠ þ 47ꢀ (c ¼ 1.0,
MeOH)].
ACKNOWLEDGMENTS
This work was supported by National Natural Science Foundation of
China (Grant No. 29506043) and Commission of Science and Technology of
Shanghai Municipal Government (‘‘Scientific Phosphor’’ Program).
REFERENCES
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4. Wu, S.H.; Guo, Z.W.; Sih, C.J. J. Am. Chem. Soc. 1990, 112,
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5. Rua, M.L.; Diaz-Maurino, T.; Fernandez, V.M.; Otero, C.;
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Received in the Netherlands January 25, 2001

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