Dimethyl sulfoxide as a co-solvent dramatically enhances the enantioselectivity in lipase-catalysed resolutions of 2-phenoxypropionic acyl derivatives

Article abstract of DOI:10.1039/b100182p

We recently reported that the enantioselectivity for subtilisin-catalysed hydrolysis of ethyl 2-(4-substituted phenoxy)-propionates in aqueous buffer is found to be dramatically enhanced by addition of dimethyl sulfoxide (DMSO). In our present work, as one of the useful methods for improving the enzyme's enantioselectivity, this approach using DMSO is tested for both hydrolysis and transesterification catalysed by various lipases. For instance, for Candida rugosa lipase-catalysed hydrolysis in aqueous buffer containing DMSO, the optimum additive conditions (50-65 vol% DMSO) markedly enhance the enantioselectivity toward the substrates used, as compared with that for no-additive conditions, in spite of a decrease in the enzymatic activity. On the other hand, for Pseudomonas cepacia lipase-catalysed hydrolysis, the addition of DMSO to the reaction medium enhances the enantioselectivity with an increase in the enzymatic activity. Also, the DMSO effect on the enantioselectivity can apply to the lipase-catalysed transesterification in organic solvent. A mechanism for the DMSO-induced enhancement of the lipase's enantioselectivity is briefly discussed on the basis of the values of the initial rates obtained for each enantiomer of the substrate used.

Full text of DOI:10.1039/b100182p

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Dimethyl sulfoxide as a co-solvent dramatically enhances the
enantioselectivity in lipase-catalysed resolutions of 2-phenoxy-
propionic acyl derivatives
1
a
,b
Keiichi Watanabe and Shin-ichi Ueji*
a
The Graduate School of Science and Technology, Kobe University, Nada,
Kobe 657-8501, Japan
Division of Natural Environment and Bioorganic Chemistry, Faculty of Human Development
and Sciences, Kobe University, Nada, Kobe 657-8501, Japan. Fax: ϩ81-78-803-7761,
E-mail: ueji@kobe-u.ac.jp
b
Received (in Cambridge, UK) 2nd January 2001, Accepted 30th April 2001
First published as an Advance Article on the web 24th May 2001
We recently reported that the enantioselectivity for subtilisin-catalysed hydrolysis of ethyl 2-(4-substituted phenoxy)-
propionates in aqueous buffer is found to be dramatically enhanced by addition of dimethyl sulfoxide (DMSO).
In our present work, as one of the useful methods for improving the enzyme’s enantioselectivity, this approach
using DMSO is tested for both hydrolysis and transesterification catalysed by various lipases. For instance, for
Candida rugosa lipase-catalysed hydrolysis in aqueous buffer containing DMSO, the optimum additive conditions
(
50–65 vol% DMSO) markedly enhance the enantioselectivity toward the substrates used, as compared with that for
no-additive conditions, in spite of a decrease in the enzymatic activity. On the other hand, for Pseudomonas cepacia
lipase-catalysed hydrolysis, the addition of DMSO to the reaction medium enhances the enantioselectivity with
an increase in the enzymatic activity. Also, the DMSO effect on the enantioselectivity can apply to the lipase-
catalysed transesterification in organic solvent. A mechanism for the DMSO-induced enhancement of the lipase’s
enantioselectivity is briefly discussed on the basis of the values of the initial rates obtained for each enantiomer
of the substrate used.
which is due to the increase of the lipase’s flexibility arising
Introduction
6
from multiple hydrogen-bond formation with added water,
whereas it decreases the enantioselectivity for the subtilisin-
For the preparation of enantiomerically pure compounds
such as pharmaceutical and agricultural chemicals, the need
for new chemical transformations with high enantioselectivity
has increased, because their biological activity is usually con-
11
catalysed reaction. In particular, lipases are the most popular
enzymes in organic synthesis, because they display relatively
high enantioselectivity, possess broad substrate specificity, are
commercially available, and do not require expensive and
unstable coenzymes. For this reason, an applied study of the
enantioselectivity enhancement of lipases is highly desirable.
As part of our continuing interest in strategies for improving
enzymes’ enantioselectivity, we reported that the enantio-
selectivity for subtilisin-catalysed hydrolysis of ethyl 2-(4-
substituted phenoxy)propionates in aqueous buffer was
dramatically improved by the addition of dimethyl sulfoxide
1
fined to only one of the enantiomers. As one of the strategies
explored with this goal in mind, biocatalysis is recognized as a
useful method for the resolution of enantiomers under mild
reaction conditions, which are highly recommended from the
viewpoint of environmental factors. Thus, the use of enzymes
as enantioselective transformation catalysts is becoming increas-
ingly popular among chemists, and several reaction systems
and classes of enzymes have been successfully employed for this
2
12
synthetic purpose.
(DMSO). The high DMSO-induced enantioselectivity is
The high enantioselectivity of enzymes is always required for
effective resolution of racemates. Improvement of an enzyme’s
enantioselectivity has been achieved by variation of the reac-
related to a change in the conformation and/or the flexibility of
the subtilisin, arising from a partial decrease of its tertiary
structure caused by addition of DMSO to the reaction medium.
In addition, it is reported that the effect of DMSO showed
enantioselectivity enhancement for pig liver esterase-catalysed
3
4
tion conditions such as solvents, reaction temperature, or
7
5
6
even pressure, use of various additives, protein engineering,
8
13
modification of enzymes, or formation of ion-paired salts
with substrates. Among these approaches, the addition of
hydrolysis. In this paper, we report a new and a simple method
9
for markedly improving the enantioselectivity of the hydrolysis
of butyl 2-(4-substituted phenoxy)propionates catalysed by
various lipases in aqueous buffer containing DMSO. Also,
the DMSO effect on the enantioselectivity can apply to the
lipase-catalysed transesterification in organic solvents. This is
the first example of DMSO as a denaturing solvent of lipases
being used to enhance their enantioselectivity. Furthermore, the
mechanism of the high DMSO-induced enantioselectivity for
lipase-catalysed reactions will be briefly discussed on the basis
of the values of the initial rates obtained for each enantiomer
of the substrate used here.
a compound or a solvent to the reaction medium seems to
10
be advantageous for its simplicity. However, the selection of
a common additive for improving the enantioselectivity of a
variety of enzymes is lacking, because the additive used to
obtain a high enantioselectivity for one enzyme does not
always apply to the improvement of another enzyme’s enantio-
selectivity. For instance, it is well known that, for lipase-
catalysed reactions in organic solvents, a small amount of water
added to the medium improves the lipase’s enantioselectivity,
as compared with that for no-additive conditions, the origin of
1
386
J. Chem. Soc., Perkin Trans. 1, 2001, 1386–1390
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b100182p

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Table 1 Effects of addition of DMSO on the enantioselectivity for
lipase MY-catalysed hydrolysis of butyl 2-(4-substituted phenoxy)-
Results and discussion
Improvement of the enantioselectivity by addition of DMSO for
lipase-catalysed reactions
propionates 1 in aqueous buffer
DMSO
(vol%)
Time
(t/min)
Conversion
(%)
ee
(%)
For lipase-catalysed hydrolysis of butyl 2-(4-substituted
phenoxy)propionates 1 in aqueous buffer, we investigated the
behaviour of the enantioselectivity of Candida rugosa lipases
by addition of DMSO (Scheme 1). For these reactions, all the
lipases used catalysed preferentially the R enantiomer of
the substrates 1. 2-Phenoxypropionic acids are well-known
X
E-Value
1
a Et
0
30
40
20
20
25
39
38
42
39
42
41
37
<1
38
43
35
40
42
39
40
41
49
60
72
92
96
100
100
3.9
5.7
10
44
102
>200
>200
5
55
0
30
60
14
herbicides and also have other biological activities. Their R
6
6
7
0
5
0
0
0
5
0
0
0
5
0
90
enantiomers are known to be active in these cases.
720
720
20
20
60
300
12
30
360
7 days
1
1
b Cl
c H
22
17
95
100
15
44
1.8
1.6
65
>200
1.5
3.3
20
>200
3
5
6
3
5
6
84
100
Scheme
1
Lipase-catalysed hydrolysis of butyl 2-(4-substituted
phenoxy)propionates 1 in aqueous buffer with addition of DMSO.
Table 2 Effects of addition of DMSO on the enantioselectivity for
lipase AY-catalysed hydrolysis of butyl 2-(4-substituted phenoxy)-
propionates 1 in aqueous buffer
Table 1 summarizes the results of the variation of the enantio-
15
selectivity (E-value estimated from the known method ) at
ca. 40% conversion observed for the hydrolysis of butyl 2-(4-
substituted phenoxy)propionates 1 catalysed by lipase MY
originating from Candida rugosa in aqueous buffer with various
amounts of DMSO. As is seen in Table 1, when DMSO was
added to the reaction medium for lipase MY-catalysed
hydrolysis of 1a (X = Et), the enantioselectivity was found to be
dramatically enhanced, as compared with that for no-additive
conditions (E = 4). In particular, upon the addition of the
optimum amount of DMSO (60–65 vol%), lipase MY dis-
played excellent enantioselectivity (E > 200), thus resulting in
the complete resolution of the enantiomers of 1a. For the
hydrolysis of 1a, however, a steep decrease in the enzymatic
activity was produced by addition of an excess of DMSO
DMSO
(vol%)
Time
(t/min)
Conversion
(%)
ee
(%)
X
E-Value
1
1
a Et
0
0
12
12
15
24
60
80
20
12
90
7 days
10
20
1440
7 days
40
41
36
41
43
35
43
34
40
36
43
46
12
3
26
38
60
95
97
94
47
69
100
100
26
30
89
2.0
2.8
5.5
78
145
54
3.9
7.7
>200
>200
2.0
2.3
19
3
40
50
5
0
0
0
5
60
0
0
5
0
5
6
b Cl
3
5
(
70 vol%), probably because of the significant irreversible
1c H
3
5
6
deformation of lipase MY caused by DMSO. Then, the
enhancement effect induced by DMSO was tested using the
other substrates, 1b (X = Cl) and 1c (X = H), for lipase MY-
catalysed hydrolysis (Scheme 1). From the data summarized in
Table 1, the optimum additive conditions (60 vol% DMSO) also
produced excellent enantioselectivity for both these substrates.
Thus, the high DMSO-induced enantioselectivity is useful for
the enantioselective improvement of substrates bearing dif-
ferent substituents, although the enantioselectivity for lipase
MY-catalysed esterification of acids 2 in organic solvents was
affected by the nature of the substituents, such as their steric
of DMSO to the reaction medium, accompanying a decrease
in enzymatic activity with an increase in the amount of
DMSO added, as with lipase MY. Also, the optimum amount
of DMSO (55–60 vol%) to maximize the enantioselectivity for
lipase AY-catalysed hydrolysis is almost identical to that for
the lipase MY-catalysed one.
Furthermore, our approach based on the high DMSO-
induced high enantioselectivity was applied to the lipase-
catalysed transesterification of ethyl 2-(4-ethylphenoxy)-
propionate 3a with n-butyl alcohol in a dry organic solvent
(Scheme 2). Unfortunately, lipases except for Candida rugosa
16
and/or electronic effects.
Indeed, the effects of DMSO on the enantioselectivity
enhancement of lipase MY are consistent with results obtained
for subtilisin-catalyzed hydrolysis in aqueous buffer con-
taining DMSO, although the reaction rate as a measure of the
enzymatic activity for lipase MY-catalysed hydrolysis of the
substrates 1a–c decreased dramatically with an increase in
the amount of DMSO added to the medium (55–65 vol%)
(
Table 1). This observation concerning the enzymatic activity
of lipase MY can explain a decrease in the enzymatic activity
of the incorrectly binding S enantiomer (see also below, for
a discussion on the basis of the results of the initial rates for
each enantiomer).
Scheme 2 Lipase-catalysed transesterification of ethyl 2-(4-ethyl-
phenoxy)propionate 3a with n-butyl alcohol in isooctane with addition
of DMSO.
In order to investigate the effects of DMSO on the enantio-
selectivity of the hydrolysis catalysed by other lipases, lipase
AY originating from Candida rugosa was tested using the model
reaction (Scheme 1). Lipase AY is known to differ from lipase
MY in its catalytic activity, although both lipases were pro-
lipases did not catalyse the transesterification of 3a. Table 3
summarizes the variation in enantioselectivity (E-value) at
ca. 30–40% conversion caused by the addition of DMSO for
lipase MY- or AY-catalysed transesterifications in dry iso-
octane.† For both lipase-catalysed reactions, the addition of
4c,4f
duced from the same yeast (Candida rugosa).
From the
data summarised in Table 2, the enantioselectivity of lipase AY
for substrates 1a–c was dramatically improved by the addition
† Isooctane = 2,2,4-trimethylpentane.
J. Chem. Soc., Perkin Trans. 1, 2001, 1386–1390
1387

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Table 4 Effects of addition of DMSO on the enantioselectivity for
Pseudomonas cepacia lipase-catalysed hydrolysis of butyl 2-(4-ethyl-
phenoxy)propionate 1a in aqueous buffer
Table 3 Enantioselectivity of lipase MY- or AY-catalysed transesteri-
fications of ethyl 2-(4-ethylphenoxy)propionate 3a with n-butyl alcohol
in isooctane with addition of DMSO
DMSO
(vol%)
Time
(t/h)
Conversion
(%)
ee
(%)
DMSO
(vol%)
Time
(t/min)
Conversion
(%)
ee
(%)
Lipase
MY
E-Value
Lipase
AH
E-Value
0
150
120
90
39
43
37
38
38
41
43
39
36
33
37
7
41
41
39
38
41
39
61
79
88
89
85
62
37
51
57
70
58
61
64
69
70
71
67
57
6.0
16
26
30
21
0
6
6.5
8
17
24
24
6
6.5
8
17
24
24
37
40
40
38
2.2
<1
33
28
26
29
25
8.2
75
82
79
11
21
14
3
5
5
6
7
0
0
5
0
0
0
0
0
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.05
.10
.15
.20
.30
80
100
>200
120
900
90
75
70
63
70
70
270
210
170
120
130
190
6.4
2.8
4.2
5.0
7.9
5.2
4.3
7.0
8.7
8.7
9.0
7.9
5.2
PS
AY
85
83
83
91
89
19
15
14
31
23
3
5
5
6
7
.05
.10
.15
.20
.30
100
>200
AK
3
5
a small amount of DMSO enhanced their enantioselectivity, as
compared with those for no-additive conditions (Table 3).
In particular, upon the optimum additive conditions (0.15 vol%
DMSO for lipase MY, 0.3 vol% DMSO for lipase AY), both
these lipases displayed excellent selectivity (E > 200). Therefore,
our approach based on the high DMSO-induced enantio-
selectivity can be used as a valuable method for improving the
enantioselectivity for Candida rugosa lipase-catalysed trans-
esterification as well as hydrolysis. A drop in the E-value and
the enzymatic activity, however, was produced by the addition
of a slightly larger amount of DMSO (0.20 vol%) than the
optimum additive condition (0.15 vol%), probably because
even a small amount of DMSO may cause the deformation of
lipases due to the direct interaction between the lipase molecule
and DMSO, which results from the low solubility of DMSO in
isooctane.
55
60
7
0
Table 5 Lipase-catalysed hydrolysis of butyl 2-(4-ethylphenoxy)-
propionate 1a in aqueous buffer with addition of DMSO
DMSO
(vol%)
Time
(t/min)
Conver-
sion (%)
ee
(%)
Origin
E-Value
Candida lipolytica
0
60
30
30
30
30
30
60
60
60
30
30
30
4
0
0
12
3
1
9
7
1
62
4.3
3
5
0
0
0
Rhizopus niveus
Pig pancreas
9.0
1.2
30
5
0
0
0
0
0
30
50
33
86
2.0
14
In order to investigate the scope of the DMSO effect on the
enantioselectivity of lipases, which was independent of the
effect of the origin of the Candida rugosa lipases, Pseudomonas
cepacia lipases were submitted to the model reaction (Scheme
3
5
Rhizopus delemar
68
45
2
<1
<1
1.0
1.0
1
). Table 4 summarizes the variation in the enantioselectivity for
Pseudomonas cepacia lipase-catalysed hydrolysis of 1a (X = Et)
caused by the addition of DMSO from 0 to 70 vol%. For lipase
AH-catalysed hydrolysis, when DMSO was added to aqueous
buffer, the enantioselectivity was found to be largely enhanced,
as compared with that for no-additive conditions (Table 4).
In particular, upon the addition of 55 vol% DMSO, lipase
AH displayed the highest enantioselectivity (E = 30), which was
Effects of addition of DMSO on the initial rates of
each enantiomer of the substrate used for lipase-catalysed
reactions
For lipase-catalysed hydrolysis of butyl esters 1 in aqueous
buffer with addition of DMSO, we found two types of the
enantioselectivity enhancement of lipases caused by this
addition of DMSO, viz. high DMSO-induced enantioselectivity
with a decrease in the enzymatic activity for Candida rugosa
lipases, and high DMSO-induced enantioselectivity with an
increase in the enzymatic activity for Pseudomonas cepacia
lipases (Tables 1–4). In order to gain insight into the mechanism
of the enantioselectivity enhancement by the addition of
DMSO, we investigated the initial rates for each enantiomer of
1a for lipase MY (Candida rugosa)- or lipase AH (Pseudomonas
cepacia)-catalysed hydrolyses. The results of the initial rates
affected by the addition of DMSO are summarized in Table 6.
There was a marked difference between the behaviour of the
initial rate for lipase MY-catalysed hydrolysis and that for
the lipase AH-catalysed one. In the former hydrolysis, the initial
5
times as large as that for no-additive conditions (E = 6). Inter-
estingly, the DMSO caused an increase in the reaction rate of
the enzyme, although the enzymatic activity for Candida rugosa
lipase was decreased by the addition of DMSO. A drop in both
the E-value and the enzymatic activity, however, was produced
by the addition of a large amount of DMSO (60–70 vol%).
Furthermore, for lipase PS- or lipase AK-catalysed hydrolyses
of 1a, the addition of DMSO to the reaction medium slightly
enhanced their enantioselectivity (Table 4). Even an optimum
amount of DMSO (55–60 vol%) produced only a moderate
enantioselectivity (E = 8–9), which would not be sufficient for
an effective resolution of 1a.
Table 5 summarizes the results of the variation in the enantio-
selectivity for the hydrolysis of 1a catalysed by lipases except for
Candida rugosa or Pseudomonas cepacia lipases. Unfortunately,
Candida lipolytica, Rhizopus niveus, and Pig pancreas lipases
did not catalyse the model reaction, and the enantioselectivity
of Rhizopus delemar lipase was not improved by the addition of
DMSO at all. Thus, when lipases do not catalyse the hydrolysis
of 1a or do not show the relatively moderate enantioselectivity
over no-additive conditions, our approach based on the high
DMSO-induced enantioselectivity is not appropriate for an
improvement in the lipase’s enantioselectivity.
rate (V ) for the correctly binding R enantiomer remained
R
almost unchanged upon addition of 55 vol% DMSO (V = 2.5),
R
as compared with that for no-additive conditions (V = 2.9)
R
(Table 6). However, when 55 vol% DMSO was added to the
reaction medium, the hydrolysis of the incorrectly binding
S enantiomer resulted in the almost complete suppression of
its enzymatic activity (V ≈ 0), which was too low to obtain
S
a reproducible value. This observation may be explained by
1
388
J. Chem. Soc., Perkin Trans. 1, 2001, 1386–1390

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Table 6 Initial rates of lipases MY- or AH-catalysed hydrolyses of
each enantiomer of butyl 2-(4-ethylphenoxy)propionate 1a in aqueous
buffer with addition of DMSO
assuming that the accommodation of the S enantiomer into the
binding site of lipase MY and the stabilization of the complex
between the substrate and the lipase would be decelerated by a
change in the conformation and/or the flexibility of lipase MY
caused by DMSO. Also, the serious decrease of the initial rate
for the S enantiomer results in the extension of the reaction
time to reach ca. 40% conversion for lipase MY-catalysed
hydrolysis in 50–65 vol% DMSO (Table 1). Therefore, from the
large value of the quotient V /V = ∞, calculated from Table 6,
VR
VS
DMSO
(vol%)
Ϫ1
Ϫ1
Lipase
MY
(µmol h mg
)
V /VS
E-Value
R
0
55
0
2.9
2.5
0.12
0.35
0.89
≈0
0.049
0.024
3.3
3.9
102
6.0
R
S
the enantioselectivity enhancement by the addition of DMSO
55 vol%) for lipase MY-catalysed hydrolysis can be mainly
AH
2.4
15
5
5
30
(
ascribed to the serious deceleration of the initial rate for the
incorrectly binding S enantiomer, as compared with that for the
R enantiomer.
Furthermore, for lipase MY- and AY-catalysed transesterifi-
cations in organic solvents containing a small amount of
DMSO, we investigated the initial rates for each enantiomer of
Table 7 Initial rates of lipase MY- or AY-catalysed transesterific-
ations of each enantiomer of ethyl 2-(4-ethylphenoxy)propionate 3a
with n-butyl alcohol in isooctane with addition of DMSO
VR
(nmol h mg
VS
3
a (Table 7). As is seen in Table 7, for both lipase-catalysed
DMSO
(vol%)
Ϫ1
Ϫ1
transesterifications, the addition of DMSO to isooctane
decreased the initial rates for both enantiomers, as compared
with those for no-additive conditions. In particular, the increase
in the amount of DMSO (to 0.15 vol%) added for lipase
MY-catalysed transesterification dramatically decreased the
initial rate for the incorrectly binding S enantiomer. Thus,
the larger value of the quotient of the initial rates, V /V =
Lipase
MY
)
V /VS
R
0
621
357
66
9.4
674
0
0
0
0
.15
.20
0.53
0.02
0.2
AY
434
304
191
11
39
47
30
.15
6.5
6.3
R
S
0.20
6
74 calculated from Table 7, arising from the serious inhibition
of the initial rate for the S enantiomer, is found to be
responsible for the significant enhancement of the enantio-
selectivity for lipase MY. For lipase AY-catalysed trans-
esterification, the smaller decrease in the initial rate for the
incorrectly binding S enantiomer, as compared with that for
lipase MY-catalysed one, does not result in a marked enhance-
ment of enantioselectivity. Thus, the marked enhancement
of enantioselectivity for Candida rugosa lipase-catalysed
hydrolysis and transesterification is attributed to the almost
complete inhibition of the initial rate for the incorrectly binding
S enantiomer.
apply to the enantioselectivity enhancement for Candida rugosa
lipase-catalysed transesterification in an organic solvent, the
origin of which is almost the same as for the hydrolysis.
This is the first example of DMSO being used to markedly
improve the enantioselectivity, but via different mechanisms of
both Candida rugosa lipases and Pseudomonas cepacia lipases.
Our findings will form a valuable tool for the improvement
of enantioselectivity in various lipase-catalysed reactions in
aqueous or non-aqueous media.
In contrast, for lipase AH-catalysed hydrolysis, the addition
of 55 vol% DMSO was found to bring about an acceleration
Experimental
General
(
2.9-fold) of the initial rate for the R enantiomer, whereas that
for the S enantiomer was decelerated (0.49-fold), as compared
with those for no-additive conditions (Table 6). Thus, the high
DMSO-induced enantioselectivity of Pseudomonas cepacia
lipase AH can be characterized by the relative quotient
Materials
Lipase MY was supplied from Meito Sangyo Co., Ltd., Japan,
and lipases AY, AH, PS, AK, Candida lipolytica, Rhizopus
niveus, Pig pancreas, and Rhizopus delemar lipases were
supplied from Amano Pharmaceutical Co., Ltd., Japan, and
used without further purification. Phosphate buffer (pH 6.86)
and DMSO (analytical grade) were purchased from Wako
Pure Chemical Industries, Ltd., Japan. Racemic butyl 2-(4-
substituted phenoxy)propionates 1 were prepared by the
reaction of the appropriate 4-substituted phenol and ethyl
(
V /V )
/(V /V )
= 6.0, arising from the opposing
R
S
55 vol%
R
S
0 vol%
trends of the initial rates for each enantiomer.
Conclusions
The enantioselectivity for various lipase-catalysed hydrolyses
in aqueous buffer was significantly enhanced by the addition
of DMSO. Also, the addition of DMSO enhanced the enantio-
selectivity for Candida rugosa lipase-catalysed transesterifica-
tion in an organic solvent. In particular, the high DMSO-
induced enantioselectivity of Candida rugosa lipases was found
to be useful for dramatically improving the enantioselectivity,
as compared with that for no-additive conditions. The particu-
larly high DMSO-induced enantioselectivity for Candida rugosa
lipases and Pseudomonas cepacia lipases can be explained fol-
lowing a discussion of the results of the initial rates of each
enantiomer of the substrate used. For Candida rugosa lipases,
the serious deceleration in the initial rate for the incorrectly
binding S enantiomer upon addition of DMSO, as compared
with that for the correctly binding R enantiomer, is a main
factor in the significant enhancement of the enantioselectivity.
On the other hand, for Pseudomonas cepacia lipases, the high
DMSO-induced enantioselectivity was dependent on the
acceleration of the initial rate for the R enantiomer and the
deceleration of that for the S enantiomer, as compared with
those for no-additive conditions. Also, the DMSO effect can
2
-bromopropionate (Tokyo Kasei Kogyo Co., Ltd., Japan),
17
according to the known method, followed by hydrolysis of
the ethyl ester and lipase OF-catalysed esterification of the acid
with n-butyl alcohol.
Lipase-catalysed hydrolysis of butyl 2-(4-substituted phenoxy)-
propionates 1 in aqueous buffer with addition of DMSO
Racemic butyl 2-(4-substituted phenoxy)propionates 1 (0.05
mmol) were added to aqueous buffer containing DMSO (0–70
vol%) (2 ml), followed by ultrasonic dispersion (40000 Hz), and
then lipase (30 mg) was added. The reaction mixture was
Ϫ1
shaken (170 strokes min ) at 37 ЊC. At an appropriate time
interval, aliquots were withdrawn and the supernatant was
analysed by HPLC (using n-hexane–isopropyl alcohol 970 : 30
(v/v) as eluent with the addition of 0.1 vol% trifluoroacetic acid)
on a chiral column (Chiralcel OK or OJ, from Daicel Chemical
Industries Co. Ltd., Japan) to determine the conversion and the
enantiomeric excess (ee).
J. Chem. Soc., Perkin Trans. 1, 2001, 1386–1390
1389

View Article Online
Preparation of enantiomerically pure butyl 2-(4-ethylphenoxy)-
propionate 1a
Acknowledgement
We thank Meito Sangyo Co., Ltd., and Amano Pharmaceutical
Co. Ltd., for their generous gifts of lipases.
6g
According to our method, the R enantiomer of 2-(4-ethyl-
phenoxy)propionic acid 2a was obtained by repetition of the
enantiomeric esterification of 2-(4-ethylphenoxy)propionic
acid 2a (36 mmol) with n-butyl alcohol (108 mmol) in dry
diisopropyl ether (200 ml) with 0.75 vol% LiCl-containing
water by use of lipase MY (3 g), followed by hydrolysis of
the ester. The S enantiomer of 2a containing a small amount
of the R enantiomer was extracted from the reaction mixture,
and the S enantiomer of 2a was obtained by removal of the
R enantiomer by lipase MY-catalysed esterification. The value
of the ee for each enantiomer used in our work was more than
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1
390
J. Chem. Soc., Perkin Trans. 1, 2001, 1386–1390