A method to greatly improve the enantioselectivity of lipase-catalyzed hydrolysis using sodium dodecyl sulfate (SDS) as an additive

Article abstract of DOI:10.1016/j.tetasy.2005.10.003

The addition of sodium dodecyl sulfate (SDS) resulted in a dramatic improvement of the enantioselectivity of the lipase-catalyzed hydrolysis of racemic butyl 2-(4-substituted phenoxy)propanoates, racemic butyl 2-(4-isobutylphenyl)propanoate, and racemic butyl 2-(6-methoxy-2-naphthyl) propanoate in an aqueous buffer solution. An increase in the E value by up to two orders of magnitude was observed for some esters. As to the effects of SDS on the structure of a lipase, FT-IR and fluorescence measurements suggest some conformational change and/or an increase of the flexibility of the lipase, although the native secondary structure of the lipase is held even in the presence of 100 mM SDS. The origin of the enantioselectivity enhancement brought about by the addition of SDS is briefly discussed on the basis of the values of the initial rates obtained for each enantiomer of the substrate.

Full text of DOI:10.1016/j.tetasy.2005.10.003

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 3698–3702
A method to greatly improve the enantioselectivity of
lipase-catalyzed hydrolysis using sodium dodecyl
sulfate (SDS) as an additive
a
b
b
a
Shuichi Mori, Hiromi Yumoto, Rina Matsumi, Tomohiro Nishigaki,
a,b
a,b,
*
Yasuhito Ebara and Shin-ichi Ueji
a
Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan
b
Division of Natural Environment and Bioorganic Chemistry, Faculty of Human Development and Sciences,
Kobe University, Nada, Kobe 657-8501, Japan
Received 23August 2005; accepted 1 October 2005
Available online 10 November 2005
Abstract—The addition of sodium dodecyl sulfate (SDS) resulted in a dramatic improvement of the enantioselectivity of the lipase-
catalyzed hydrolysis of racemic butyl 2-(4-substituted phenoxy)propanoates, racemic butyl 2-(4-isobutylphenyl)propanoate, and
racemic butyl 2-(6-methoxy-2-naphthyl)propanoate in an aqueous buffer solution. An increase in the E value by up to two orders
of magnitude was observed for some esters. As to the effects of SDS on the structure of a lipase, FT-IR and fluorescence measure-
ments suggest some conformational change and/or an increase of the flexibility of the lipase, although the native secondary structure
of the lipase is held even in the presence of 100 mM SDS. The origin of the enantioselectivity enhancement brought about by the
addition of SDS is briefly discussed on the basis of the values of the initial rates obtained for each enantiomer of the substrate.
Ó 2005 Elsevier Ltd. All rights reserved.
1
. Introduction
A traditional approach for this problem is to screen
enzymes from other sources in the hope of finding a more
enantioselective one, although, this empirical method
can seem tedious to organic chemists. Herein, we used
sodium dodecyl sulfate (SDS), a denaturant, as an addi-
tive in the lipase-catalyzed hydrolysis of the racemic
esters 1–9. The addition of small amounts of SDS to
the reaction medium was anticipated to cause a large
flexibility in the lipase, yet retaining its function. This
strategy is based on our findings that the enantioselecti-
vity of lipase-catalyzed esterification was significantly
improved by addition of aqueous SDS directly to
Lipase-catalyzed transformations have been established
as an important tool in organic synthesis, because lipases
display broad substrate specificity, yet retain relative high
enantioselectivity not only in aqueous solution but also in
1
organic media. For this reason, organic chemists rou-
tinely use lipases as enantioselective catalysts in kinetic
resolutions of racemates to provide easy access to enantio-
merically pure compounds. These enzymatic reactions,
however, do not always offer satisfactory results with high
enantioselectivity toward non-natural substrates, such as
pharmaceuticals, agrochemicals, or other synthetic tar-
gets. The structures of these substrates are widely different
from those of the triacylglycerols of natural substrates for
native lipases, that is, in other words, the acceptance of
such non-natural substrates should force these enzymes
to alter their active site conformations. Furthermore,
the rigid enzymes seem to have difficultly in adjusting
their conformational states for favorable interactions
with substrates having undesirable structures.
2
suspended enzyme preparations in organic media. It is
certainly of value to see if this approach is valid even in
lipase-catalyzed hydrolysis in an aqueous buffer solution.
2. Results and discussion
2.1. Effects of SDS on the enantioselectivity of lipase-
catalyzed hydrolysis
*
Corresponding author. Fax: +81 78 8037761; e-mail: ueji@kobe-
u.ac.jp
Lipase MY from Candida rugosa catalyzes the enantio-
selective hydrolysis of racemic butyl 2-(4-substituted
0957-4166/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.10.003

S. Mori et al. / Tetrahedron: Asymmetry 16 (2005) 3698–3702
3699
O
CO Bu
O
CO2H
CO H
2
2
CO Bu
2
Hydrolysis
X
X
Lipase
SDS/Buffer
(S)-Preference
8
(R)-Preference
1
4
6
: X=Et, 2: X=H, 3: X=Me
: X=n-Pr, 5: X=t-Bu
: X=MeO, 7: X=CF3
CO H
2
CO Bu
2
MeO
MeO
(
S)-Preference
9
Scheme 1. Lipase-catalyzed hydrolysis of butyl 2-(4-substituted phenoxy)propanoates 1–7, butyl 2-(4-isobutylphenyl)propanoate 8, and butyl
-(6-methoxy-2-naphthyl)propanoate 9.
2
phenoxy)propanoates 1–7, racemic butyl 2-(4-isobutyl-
phenyl)propanoate 8 (butyl ester of ibuprofen) and race-
mic butyl 2-(6-methoxy-2-naphthyl)propanoate 9 (butyl
ester of naproxen) in an aqueous buffer solution
300
2
2
1
1
50
00
50
00
1
9
(
1
Scheme 1). Lipase MY favors the (R)-enantiomers of
–7 and the (S)-enantiomers of 8 and 9, however these
enantiomers of 1–9 have similar shapes, because of
changes in priorities of the substituents. Some products
of the hydrolysis of 1–7, (R)-2-aryloxypropanioc acids,
are well known herbicides and have other biological
E
3
activities. (S)-Naproxen and (S)-ibuprofen are also an
50
important class of non-steroidal anti-inflammatory
4
drugs. Unfortunately, the enantioselectivity observed
is low to moderate for these esters in an aqueous buffer
solution without any additive (Table 1), the E value of
which would not be sufficient for an effective resolution.
To improve the enantioselectivity, various concentra-
tions of SDS were tested as an additive in the lipase-cat-
alyzed hydrolysis using 1 and 9 (Fig. 1). As can be seen
in Figure 1, the addition of SDS significantly increases
the enantioselectivity toward both substrates. The opti-
mum concentration of SDS (100 mM) for 1, however,
is appreciably different from that (1 mM) for 9. One of
the possible explanations for this observation is that 9
associates with the active site of lipase differently to that
of 1, which contains an oxygen at the stereocenter. The
0
0.5
1
10 50 100 150 200
Concentration of SDS (mM)
Figure 1. Variation of the enantioselectivity of the lipase-catalyzed
hydrolysis of 1 and 9 by addition of SDS (0–200 mM).
scope of this enhancement effect of SDS on the enanti-
oselectivity was evaluated by submitting the other sub-
strates 2–8 to the lipase-catalyzed hydrolysis (Table 1).
Inspection of the data summarized in Table 1 reveals
that the remarkable enhancement of the E value brought
Table 1. Enhancement of the enantioselectivity by the addition of SDS for the lipase-catalyzed hydrolysis of 1–9 in an aqueous buffer solution
a
Ester
Additive
None
Time (min)
Conv. (%)
ee
p
(%)
E
1
8
18
5
330
5
10
20
20
15
20
7
10
4
5
1080
1080
660
720
36
45
37
29
40
4398
39
45
35
19
39
44
45
39
25
23>99
42
44
50
98
16
88
30
186
51
97
28
81
40
98
38
95
86
4
225
2
22
2
1
00 mM SDS
None
00 mM SDS
None
00 mM SDS
None
00 mM SDS
None
00 mM SDS
None
00 mM SDS
None
00 mM SDS
None
2
3
4
5
6
7
8
9
1
1
4
123
2
11
3
122
3
70
18
1
1
1
1
1
mM SDS
>500
92
98
None
49
254
1
mM SDS
a
Enantiomeric excess of the product.

3700
S. Mori et al. / Tetrahedron: Asymmetry 16 (2005) 3698–3702
Table 2. Effects of SDS on the enantioselectivity of the hydrolysis of 1
Table 3. Effects of SDS on the secondary structure of lipase MY
catalyzed by five different lipases
determined by FT-IR in the amide III spectral region
a
À1
Lipase Additive
Time (min) Conv. (%) % ee
E
Additive
None
Position (cm
)
Assignment
Area (%)
35
MY
OF
AY
PS
None
8
2.5
18
0.8
0.1
0.1
2
36
4361
45
38
40
50
6
4
1316
1301
1287
a
a
a
48
Random
Random
b
a
1
1
mM SDS
00 mM SDS
98
41
70
88
49
225
3
10
37
3
None
1273Random
1259
1
1
mM SDS
00 mM SDS
46
25
1244
1229
1315
1304
1290
1275
1260
1245
None
16
33
1
1
mM SDS
00 mM SDS
0.1
0.05
60
4394
43 09 7
43 34 3
40
10
39
70
13
100 mM SDS
a
a
None
1
1
mM SDS
00 mM SDS 125 h
30
65
26
65
87
55
7
2
7
Random
Random
Random
b
50
17
AH
None
90
90
47 h
1
1
mM SDS
00 mM SDS
42
5
26
4
1230
a
Enantiomeric excess of the product.
even in the presence of 100 mM SDS. On the other
hand, fluorescence spectra showed a marked decrease
of intensity due to the emission of Trp and/or Tyr resi-
dues and a somewhat red shift of the maximum wave-
length (Fig. 2), upon addition of SDS. The change of
the fluorescence spectra indicates that Trp and/or Tyr
residues in lipase MY are in a less hydrophobic environ-
ment when compared with those in the absence of SDS,
with some conformational change, thus resulting in an
increase of the flexibility of the lipase. This spectral
about by the action of SDS was observed for all the sub-
strates. An increase in the E value up to two orders of
magnitude was observed for some substrates examined.
Furthermore, to test the generality of this SDS effect, the
hydrolysis of 1 catalyzed by the other popular lipases
was also carried out in the presence of SDS (1 and
1
00 mM) (Table 2). As can be seen from the E value
listed in Table 2, this SDS effect is not limited to lipase
MY. Among the lipases used, lipase AY and lipase
OF from C. rugosa displayed almost the same enhance-
ment effect in the E value as that for lipase MY. Lipase
PS and lipase AH from Pseudomonas cepacia also mod-
erately increased the E value, although the optimum
concentration of SDS is different from that for lipase
MY. The mode of the enhancement of the enantioselec-
tivity and the optimum concentration of SDS seems to
depend on both the structure of the substrate and the
origin of lipase.
2
result is also supported by our previous ESR study in
which the flexibility of lipase in organic solvents is
affected by the addition of aqueous SDS.
5
4
(a)
3
2
(
b)
2.2. Effects of SDS on the structure of lipase and the
initial rate of each enantiomer of the substrate
(c)
1
First, a possible conformational change of lipase MY
caused by the addition of SDS was investigated on the
basis of the results of FT-IR and fluorescence spectra.
All the IR bands in the amide III region of lipase MY
in an aqueous buffer solution with and without the
SDS addition were assigned to individual secondary
structural elements (Table 3), according to the known
0
-1
2
50
300
350
Wavelength (nm)
400
450
5
method of Gaussian curve fitting. The a-helix content
Figure 2. Fluorescence spectra of lipase MY dissolved in an aqueous
buffer solution containing (a) 0 mM SDS, (b) 1 mM SDS, and (c)
00 mM SDS.
of 35% and the b-sheet of 16% (see Table 3) calculated
from the areas of the IR bands of lipase MY in an aque-
ous buffer solution agree with the 33% a-helix and 12%
b-sheet determined from its X-ray crystallographic
1
6
data. Furthermore, it was found by a comparison of
Next, to gain further insight into the origin of the
enantioselectivity enhancement triggered by the SDS
addition, we investigated the initial rate of reaction of
each enantiomer of 1 for lipase MY-catalyzed hydrolysis
(Table 4). Upon the addition of 100 mM SDS, the initial
rate (V ) of the correctly binding (R)-enantiomer was
accelerated, whereas that (V ) of the incorrectly binding
(S)-enantiomer was seriously decelerated, when com-
pared with no additive conditions. As can be seen from
the data in Table 3 that both IR spectra obtained in
the presence and absence of SDS had essentially the
same secondary structure composition. Additionally,
the far-UV CD spectra of lipase MY in an aqueous buf-
fer solution showed little change in the relative intensity
of the negative band at 222 nm due to the a-helix struc-
ture after the addition of SDS. These facts mean that the
native secondary structure of lipase MY is held steady
R
S

S. Mori et al. / Tetrahedron: Asymmetry 16 (2005) 3698–3702
3701
the larger value of the quotient of the initial rates (VR/
V ) listed in Table 4, the larger difference in the initial
S
4.2. Lipase-catalyzed hydrolysis, HPLC analysis, and
determination of the enantiomeric ratio
rates between the two enantiomers, arising from the
opposite direction of each initial rate, is responsible
for the significant enhancement of the enantioselectivity.
In other words, judging from the results so far obtained,
the enantioselectivity enhancement can be explained by
assuming that some conformational change and/or an
increase in the flexibility of lipase MY allow an easier
access to its active site for the correctly binding (R)-
enantiomer, but not for the incorrectly binding (S)-
enantiomer.
In a typical procedure, racemic butyl esters 1–9 (0.036
mmol) were added to an aqueous buffer solution
(2 mL), followed by the ultrasonic dispersion, and then
lipase (30 mg) was added. At frequent intervals, aliquots
were withdrawn and the supernatant analyzed by HPLC
(a Shimadzu LC-10A) on a Chiralcel OK or a Chiralcel
OD-H (Daicel Chemical Industries Co., Ltd, Japan)
using an appropriate proportion of hexane/2-propanol
as an eluent for each substrate. The enantiomers of the
butyl esters 1–9 and hydrolysis products, the corre-
sponding acids, were separated well enough for the
accurate determination of the conversion and the enan-
tiomeric excess (ee). The E value was calculated from the
enantiomeric excess for the acid produced, according to
Table 4. Effects of SDS on the initial rate of hydrolysis of each
enantiomer of 1
À1
À1
)
Additive
Initial rate (lmol h mg
V
R
/V
S
E
8
a
a
the literature. The results and conditions of HPLC
analysis are shown in Table 5.
V
R
V
S
None
13.4
44.5
1.72
0.22
8
200
4
225
1
00 mM SDS
The V and V
mers, respectively.
Table 5. HPLC characterization of the hydrolysis products
a
R
S
denote the initial rates of the (R)- and (S)-enantio-
Substrate
Column
Eluent
hexane/
-propanol)
Ret. time
(min)
b
(
2
a
R
S
3
. Conclusions
1
2
3
4
5
6
7
8
9
Chiralcel OK
Chiralcel OK
Chiralcel OK
Chiralcel OK
Chiralcel OK
Chiralcel OK
Chiralcel OK
Chiralcel OD-H
Chiralcel OD-H
98/2
98/2
98/2
98/2
98/2
98/2
98/2
99/1
94/6
12.0
15.4
14.2
10.2
8.5
37.4
9.2
19.1
8.8
15.8
18.8
19.2
14.1
11.2
56.9
12.5
22.5
10.1
The technique of SDS addition provides a useful method
to improve the enantioselectivity of the lipase-catalyzed
hydrolysis. This method is also appealing to organic
chemists, because of its simplicity. Furthermore, the
enantioselectivity enhancement brought about by SDS
addition was found to be dependent on the acceleration
of the initial rate of the correctly binding (R)-enantiomer
and the deceleration of that for the incorrectly binding
a
All the eluents contain 0.1 vol % trifluoroacetic acid.
Retention time of the hydrolysis product (the corresponding acid).
b
(
S)-enantiomer.
4.3. Measurement of the initial rate of each enantiomer
of 1
4. Experimental
4
.1. General
In a typical procedure, (R)- or (S)-1 (0.018 mmol) was
added to an aqueous buffer solution (1 mL), followed
by the ultrasonic dispersion after which lipase (15 mg)
was added. The reaction mixture was then shaken
Lipases MY and OF and lipases AY, PS, and AH were
generously provided by Meito Sangyo Co., Ltd, Japan,
and Amano Pharmaceutical Co., Ltd, Japan, respec-
tively. These lipases were used without further purifica-
tion for the hydrolysis. For the spectral measurements,
lipase MY was semi-purified by dialyzing and lyophiliz-
ing from crude material. The MALDI-TOF MS spec-
trum of the semi-purified lipase showed a single parent
peak, m/z 60.2 kD, which is consistent with the mole-
À1
(170 strokes min ) at 3 7°C. At appropriate times, ali-
quots were withdrawn and the supernatant analyzed
by HPLC on a chiral column to obtain the conversion.
Several data points were collected to determine the
initial rate. Each enantiomer used was prepared by the
9
lipase-catalyzed esterification, according to our method.
7
cular weight of C. rugosa lipase. Ibuprofen and naproxen
4.4. Preparation of racemic butyl esters 1–9
were purchased from Tokyo Kasei Kogyo Co., Ltd,
Japan. All other chemicals were from commercial
sources and of reagent grade. The FT-IR measurements
were performed on a BRUKER TENSOR 27 spectro-
Racemic butyl esters 1–9 were prepared by the esterifica-
tion of the corresponding acids with 1-butyl alcohol in
the presence of 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (EDCÆHCl). The correspond-
ing, 2-aryloxypropanioc acids, were also prepared
polarimeter. All the spectra were averaged over a total
À1
of 100 scans at 2 cm
resolution. The fluorescence
1
0
spectra were recorded on a Shimadzu RF-5300 PC
spectrofluorophotometer with an excitation wavelength
according to the known method.
1
1
of 280 nm and a bandwidth of 1.5 nm. The H NMR
Butyl 2-(4-ethylphenoxy)propanoate (RS)-1. H NMR
spectra were recorded on a Varian Unity instrument
(CDCl ): d 0.88 (3H, t, J = 7.4 Hz), 1.12 (3H, t,
J = 7.6 Hz), 1.30 (2H, m), 1.59 (5H, m), 2.58 (2H, q,
3
(
400 MHz) using CDCl as a solvent.
3

3702
S. Mori et al. / Tetrahedron: Asymmetry 16 (2005) 3698–3702
1
J = 7.6), 4.15 (2H, m), 4.72 (1H, q, J = 6.8 Hz), 6.80
Butyl 2-(6-methoxy-2-naphthyl)propanoate (RS)-9. H
(
2H, d, J = 8.8 Hz), 7.09 (2H, d, J = 8.4 Hz).
NMR (CDCl ): d 0.86 (3H, t, J = 7.4 Hz), 1.29 (2H,
3
m), 1.56 (5H, m), 3.84 (1H, q, J = 6.4 Hz), 3.91 (3H,
s), 4.08 (2H, m), 7.13(2H, m), 7.42 (1H, d,
J = 8.8 Hz), 7.69 (3H, m).
1
Butyl 2-phenoxypropanoate (RS)-2. H NMR (CDCl ):
3
d 0.88 (3H, t, J = 7.4 Hz), 1.29 (2H, m), 1.60 (5H, m),
4
.15 (2H, m), 4.76 (1H, q, J = 6.8 Hz), 6.87 (2H, m),
6
.97 (1H, m), 7.27 (2H, m).
Acknowledgments
1
Butyl 2-(4-methylphenoxy)propanoate (RS)-3. H NMR
(
(
CDCl ): d 0.89 (3H, t, J = 7.4 Hz), 1.31 (2H, m), 1.60
5H, m), 2.27 (3H, s), 4.15 (2H, m), 4.71 (1H, q,
The authors wish to thank Noriko Kitamura, Rieko
Tani, Emi Nakao, Takeshi Hamada, and Tomomi Mat-
suda for their help in some measurements of the E
values.
3
J = 6.4 Hz), 6.77 (2H, d, J = 8.8 Hz), 7.06 (2H, d,
J = 8.8 Hz).
1
Butyl 2-(4-n-propylphenoxy)propanoate (RS)-4.
H
NMR (CDCl ): d 0.90 (6H, m), 1.29 (2H, m), 1.59
3
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3
2
3
4
5
1
Butyl 2-(4-methoxyphenoxy)propanoate (RS)-6.
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3
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1
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3
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J = 9.0 Hz).
7
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1
Butyl 2-(4-isobutylphenyl)propanoate (RS)-8. H NMR
9
. Okamoto, T.; Ueji, S. Chem. Commun. 1999, 939.
(
CDCl ): d 0.90 (9H, m), 1.28 (2H, m), 1.52 (5H, m),
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1
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1
.84 (1H, m), 2.44 (2H, d, J = 7.2), 3.68 (1H, q,
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J = 8.0 Hz), 7.23(2H, d, J = 8.0 Hz).