A great improvement of the enantioselectivity of lipase-catalyzed hydrolysis and esterification using co-solvents as an additive

Article abstract of DOI:10.1246/bcsj.81.617

Addition of co-solvents such as tetrahydrofuran resulted in a great improvement of the enantioselectivity of lipase-catalyzed hydrolysis of butyl 2-(4-substituted phenoxy)propanoates in an aqueous buffer solution. On the other hand, lipase lyophilized from an aqueous solution containing the co-solvents catalyzed highly enantioselective esterification of 2-(4-substituted phenoxy)propionic acids, 2-(4-isobutylphenyl)propionic acid (ibuprofen), and 2-(6-methoxy-2-naph-thyl)propionic acid (naproxen) in an organic solvent. An increase in the E value up to two orders of magnitude was observed for some substrates. The origin of the enantioselectivity enhancement caused by the co-solvent addition was mainly attributed to a significant deceleration in the initial reaction rate for the incorrectly binding enantiomer, as compared with that for the correctly binding enantiomer. From the results of FT-1R, CD, and ESR spectra, the co-solvent addition was also found to bring about a partial destruction of the tertiary structure of lipase.

Full text of DOI:10.1246/bcsj.81.617

ꢀ 2008 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 81, No. 5, 617–622 (2008)
617
A Great Improvement of the Enantioselectivity of Lipase-Catalyzed
Hydrolysis and Esterification Using Co-Solvents as an Additive
Tomohiro Nishigaki,1 Yoshitaka Yasufuku,1 Sayuri Murakami,2
ꢀ2
Yasuhito Ebara,2 and Shin-ichi Ueji
1Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501
2Graduate School of Human Development and Environment, Kobe University, Nada, Kobe 657-8501
Received October 29, 2007; E-mail: ueji@kobe-u.ac.jp
Addition of co-solvents such as tetrahydrofuran resulted in a great improvement of the enantioselectivity of lipase-
catalyzed hydrolysis of butyl 2-(4-substituted phenoxy)propanoates in an aqueous buffer solution. On the other hand,
lipase lyophilized from an aqueous solution containing the co-solvents catalyzed highly enantioselective esterification
of 2-(4-substituted phenoxy)propionic acids, 2-(4-isobutylphenyl)propionic acid (ibuprofen), and 2-(6-methoxy-2-naph-
thyl)propionic acid (naproxen) in an organic solvent. An increase in the E value up to two orders of magnitude was ob-
served for some substrates. The origin of the enantioselectivity enhancement caused by the co-solvent addition was
mainly attributed to a significant deceleration in the initial reaction rate for the incorrectly binding enantiomer, as com-
pared with that for the correctly binding enantiomer. From the results of FT-IR, CD, and ESR spectra, the co-solvent
addition was also found to bring about a partial destruction of the tertiary structure of lipase.
Lipase-catalyzed transformations play an important role in
organic synthesis to fulfill the rapidly growing demand for
enantiomerically pure compounds such as pharmaceuticals,
agrochemicals, and natural products.1 Native lipases, however,
do not always show satisfying performance in terms of activity
and most importantly enantioselectivity, because the structures
of these synthetic targets are widely different from those of the
triacylglycerols of their natural substrates. Enantioselectivity is
one of the key features of lipases making them attractive for
biocatalysis. For this reason, an applied study of the enantiose-
lectivity enhancement of lipase is highly desirable.2 In general,
the enantioselectivity of lipase may be determined by a special
relationship between the substrate structure and the origin of
lipase, that is, the stereochemical environment of its active
site. A traditional approach for this problem is to screen a large
number of commercial lipases in the hope of finding a more
enantioselective one. This procedure, however, seems to be
somewhat tedious for organic chemists. Among several strat-
egies to improve the enantioselectivity of lipase-catalyzed
reactions, the method of additives seems to be attractive in
view of its simplicity of use. In fact, various types of additives
have been successfully applied to improve the outcome of the
reactions.3
Here, we wish to report that the enantioselectivity for lipase-
catalyzed hydrolysis is markedly improved by addition of co-
solvents such as tetrahydrofuran to an aqueous buffer solution
as the reaction medium (Scheme 1). On the other hand, lipase
lyophilized from an aqueous solution containing the co-sol-
vents also enhances the enantioselectivity of lipase-catalyzed
esterification in an organic solvent (Scheme 1). Furthermore,
the origin of the enhanced enantioselectivity of lipase caused
by the co-solvent addition will be discussed briefly on the basis
of the initial rate obtained for each enantiomer of the substrate
used and the structural change of lipase estimated from the
spectral results.
Results and Discussion
Effects of Co-Solvents on the Enantioselectivity of Li-
pase-Catalyzed Hydrolysis. The hydrolysis of butyl 2-(4-
substituted phenoxy)propanoates 1–7, butyl 2-(4-isobutylphen-
yl)propanoate 8 (butyl ester of ibuprofen), and butyl 2-(6-
methoxy-2-naphthyl)propanoate 9 (butyl ester of naproxen)
was catalyzed by Candida rugosa lipase Type VII in an aque-
ous buffer solution containing 0–30 vol % tetrahydrofuran
(THF) (Scheme 1). Lipase Type VII favors the R enantiomers
of 1–7 and the S enantiomers of 8 and 9, however, these enan-
tiomers have similar shapes, because of changes in priorities of
the substituents. Some products from the hydrolysis of 1–7, the
R enantiomer of 2-aryloxypropanoic acids, are well-known
herbicides and have other biological activities.4 Furthermore,
the S enantiomers of 8a and 9a belong to an important class
of non-steroidal anti-inflammatory drugs.5 Results of hydroly-
sis are listed in Table 1. The hydrolysis of these compounds
without addition of THF gave low enantioselectivities (E
values were less than 5), as was expected from the previously
reported hydrolysis of methyl 2-aryloxypropanoates with Can-
dida rugosa lipase (Table 1).6 On the contrary, the addition of
THF in the reaction mixture increased the enantioselectivities
of the hydrolysis (Table 1). Thus, the reaction proceeded enan-
tioselectively (E value > 100) when 20 vol % of THF was
added to substrates 1–7. For 8 and 9, the reaction rate was
too late to obtain a reliable E value.
In order to investigate the effect of co-solvent on the enan-
tioselectivity of the reaction, other co-solvents such as acetoni-
trile, acetone, and dioxane were tested using 1 as the substrate.
Figure 1 shows that all solvents tested had the ability to in-
Published on the web May 14, 2008; doi:10.1246/bcsj.81.617

618 Bull. Chem. Soc. Jpn. Vol. 81, No. 5 (2008)
Enhancement of Lipase’s Enantioselectivity
O
CO2H
O
CO2Bu
CO2Bu
CO2Bu
CO2H
Hydrolysis
X
X
Lipase, 37°C,
pH7 Buffer + Co-solvents
1-7
8
9
1a-7a
8a
9a
1, 1a: X = Et
2, 2a: X = H
3, 3a: X = Me
4, 4a: X = n-Bu
5, 5a: X = OMe
6, 6a: X = CF3
Esterification
CO2H
Lipase lyophilized with
co-solvents, 1-BuOH,
37°C, Isopropyl ether
MeO
MeO
7, 7a: X = Cl
Scheme 1. Lipase-catalyzed hydrolysis of 1–9 and esterification of 1a–9a.
Table 1. Enhancement of the Enantioselectivity of the Li-
pase Type VII-Catalyzed Hydrolysis of 1–7 by Addition
of THF (0–30 vol %) to pH 7 Buffer at 37 ꢁC
600
500
400
300
200
100
0
Substrate THF/vol % Time/min Convn./% ee/%
E
1
(X = Et)
0
10
15
20
25
30
0
10
15
20
25
30
0
10
15
20
25
30
0
10
15
20
25
30
0
10
15
20
25
30
0
10
15
20
25
30
0
10
15
20
25
30
6
25
70
42
43
42
44
19
4
47
41
40
8
36
48
79
3
4
15
220
1440
1440
35
100
540
1440
21600
1440
6
30
60
150
1440
1440
15
96 112
97
95
13
53
72
73
73
29
52
55
88
91
37
1
5
10
7
7
2
4
5
33
2
(X = H)
0
20
40
Co-solvents/vol%
Figure 1. Effects of co-solvents (THF: , acetonitrile:
acetone: , and dioxane: ) on the enantioselectivity of
the lipase Type VII-catalyzed hydrolysis of 1 in pH 7 buf-
fer at 37 ꢁC. Reaction times at the maximum E value are
220 min ( ), 1440 min ( ), 260 min ( ), and 1440 min
60
80
3
3
3
(X = Me)
37
42
45
37
16
2
37
46
31
23
7
,
97 138
99 171
80
35
27
68
92
90
86
56
67
86
9
3
2
(
), respectively.
4
(X = n-Bu)
50
crease enantioselectivity. Each of the co-solvents had its own
optimum concentration for the maximum E value toward 1.
An increase in the E value up to two orders of magnitude
was achieved by addition of these co-solvents (Figure 1). In
particular, acetone and dioxane gave an excellent E value
(E > 400), thus leading to almost complete resolution of the
enantiomers of 1. Several investigations have been undertaken
on the influence of the co-solvents on the enantioselectivity of
lipase-catalyzed reactions.7 Although many methods have
been reported to increase the enantioselectivity of lipase-cata-
lyzed reactions,2 the use of co-solvent in the reaction mixture
is probably the most convenient and useful method.
Furthermore, to test the generality of this co-solvent effect,
hydrolysis of 1 catalyzed by other readily available lipases was
also carried out in the presence of THF. Figure 2 shows the
variation of E values of different lipases as a function of con-
centration of THF. As can be seen from Figure 2, this co-sol-
vent effect is not limited to lipase Type VII. The E value ob-
served for lipases AY and OF from Candida rugosa was very
sensitive to the addition of THF, which was accompanied by
a marked improvement of their enantioselectivity, as well as
lipase Type VII. Lipase AH from Burkholderia cepacia also
180
1440
1440
1440
5
7
31
21
14
5
8
25
4
5
(X = OMe)
32
43
43
42
14
1
41
49
44
9
30
110
1440
1440
1440
8
97 129
98 105
81
38
35
54
88
87
30
30
81
81
94
96
—
10
3
3
5
18
9
6
(X = CF3)
55
195
8640
7200
1440
5
7
5
2
2
7
(X = Cl)
33
38
41
21
4
90
15
17
44
46
—
180
1440
1440
1440
0

T. Nishigaki et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 5 (2008)
619
Table 2. Enantioselectivity of the Lipase-Catalyzed Esteri-
fication of 1a in Isopropyl Ether at 37 ꢁC by Use of Lipase
Type VII Lyophilized with THF (0–70 vol %)
400
350
300
250
200
150
100
50
THF/vol %
Time/h
Convn./%
ee/%
E
0
10
15
20
25
30
40
50
60
70
30
30
30
30
30
30
48
48
48
48
42
38
38
38
38
34
40
39
39
41
55
73
80
87
91
95
85
89
88
82
5
10
15
24
35
67
23
30
27
17
0
0
10
20
30
THF/vol%
Table 3. Enhancement of the Enantioselectivity of the
Lipase-Catalyzed Esterification of 1a–9a in Isopropyl
Ether at 37 ꢁC by Use of Lipase Type VII Lyophilized
with THF (0 and 30 vol %)
Figure 2. Variation of the enantioselectivity of different
lipases (Type VII: , AY: , OF: , AH: , AK:
and PS: ) for the lipase-catalyzed hydrolysis of 1 by
,
addition of THF to pH 7 buffer at 37 ꢁC.
Substrate
THF/vol % Time/h Convn./% ee/%
E
moderately increased the E value while the corresponding in-
crease was small for lipase PS from Burkholderia cepacia and
lipase AK from Pseudomonas fluorescence (Figure 2).
Effects of Co-Solvents on the Enantioselectivity of Li-
pase-Catalyzed Esterification. It is certainly of value to
see if the co-solvent approach is valid in lipase-catalyzed ester-
ification in an organic solvent. First, lipase Type VII-catalyzed
esterification of 1a (Scheme 1) was carried out in isopropyl
ether containing THF as the co-solvent additive. In the pres-
ence of THF (10–30 vol %), however, the enantioselectivity
of the esterification of 1a decreased with an inhibition of the
lipase activity (data not shown). It is logical to suppose that
some of the essential waters, maintaining the lipase’s structure
and function, are stripped out by addition of water miscible
co-solvent, THF.
Next, lipase Type VII was lyophilized from aqueous-THF
(0–70 vol %) mixed solvents. Interestingly, this procedure in-
creased the enantioselectivities of the esterification of 1a in
isopropyl ether compared to those using un-treated lipase. This
treated lipase exhibited ‘‘solvent memory’’ as was observed in
the hydrolysis of esters catalyzed by the crude lipase treated
with organic solvents (Scheme 1 and Table 2).8 As is seen in
Table 2, the maximum E value (E ¼ 67) for 1a was obtained
around 30 vol % THF, the E value of which was about 13 times
as large as that for 0 vol % THF (E ¼ 5). Therefore, the lipase
Type VII lyophilized in the presence of 30 vol % THF was
subjected to esterification using the other substrates 2a–9a
(Scheme 1 and Table 3); the enantiopreferences for 1a–9a
are the same as those in the hydrolysis. Inspection of the data
summarized in Table 3 reveals that the remarkable enhance-
ment of the E value brought about by the action of THF was
observed for all the substrates studied here.
1a
(X = Et)
2a
(X = H)
3a
(X = Me)
4a
(X = n-Bu)
5a
(X = OMe)
6a
(X = CF3)
7a
(X = Cl)
8a
0
30
0
30
30
29
94
42
34
40
37
41
37
38
35
44
38
30
36
48
37
9
55
95
41
82
55
93
43
82
64
91
58
83
34
85
94
99
94
99
5
67
3
16
5
48
3
16
7
38
5
17
3
21
36
259
35
30
0
30
0
30
0
30
0
30
0
30
0
29
69
44.5
94
29
45
44.5
94
44.5
94
170
30
0
30
170
167
167
12
12
11
9a
189
marked enhancement of the E value, although this effect was
largest for THF.
Figure 4 represents the variation in the enantioselectivity of
the esterification of 1a catalyzed by various lipases lyophilized
in the presence of 30 vol % THF. The E value for lipases Type
VII, AY, and OF from Candida rugosa was found to be largely
improved by lyophilization with THF. For instance, the E val-
ue of lipase OF was raised from E ¼ 3 to E ¼ 355. In contrast
to this, the E value remained essentially unchanged for lipases
AH and PS from Burkholderia cepacia and lipase AK from
Pseudomonas fluorescence (Figure 4).
Effects of Co-Solvents on the Initial Rate of Each Enan-
tiomer of the Substrate and the Structure of Lipase. In or-
der to gain an insight into the origin of the enantioselectivity
enhancement caused by the co-solvent addition, we investigat-
ed the initial rate of each enantiomer of 1 for the hydrolysis
catalyzed by lipase Type VII in the presence of 20 vol %
THF (Table 4) and that of 1a for the esterification catalyzed
by lipase Type VII lyophilized in the presence of 30 vol %
Furthermore, the effect of the lyophilized lipase on the
enantioselectivity of the esterification of 1a was examined
using the other co-solvents and lipases, as in the case of the
hydrolysis. As is seen from the plot in Figure 3, lipase Type
VII lyophilized with various concentrations of the co-solvents
(THF, acetonitrile, acetone, and dioxane) also produced

620 Bull. Chem. Soc. Jpn. Vol. 81, No. 5 (2008)
Enhancement of Lipase’s Enantioselectivity
Table 4. Effects of THF on the Initial Rate of Lipase Type
VII-Catalyzed Hydrolysis and Esterification of Each
Enantiomer of 1 and 1a in pH 7 Buffer and Isopropyl
Ether, Respectively at 37 ꢁC
80
70
60
50
40
30
20
10
0
Reaction
Initial rate/mmol hꢂ1 mgꢂ1
THF/vol %
VR=VS E
(Substrate)
VR
VS
Hydrolysis
(1)
Esterification
(1a)
0
20
0
20.4
0.648
2.16
1.60
0.792
25.8
225 112
23.7
275
3
0.00288
0.0912
0.00580
5
67
30
0
20
40
60
80
Table 5. Effects of THF on the Secondary Structure of
Lipase Type VII Determined by FT-IR in the Amide I
Spectral Region
Co-solvents/vol%
Figure 3. Effects of lyophilization of lipase Type VII with
THF ( ), acetonitrile ( ), acetone ( ), and dioxane (
on the enantioselectivity of the lipase-catalyzed esterifica-
tion of 1a in isopropyl ether at 37 ꢁC. Reaction times at the
maximum E value are 30 h ( ), 25 h ( ), 25 h ( ), and
28.5 h ( ), respectively.
)
THF/vol %
0
Position/cmꢂ1
Assignment
Area/%
1621
1631
1640
1650
1659
1669
1679
1622
1631
1641
1650
1659
1669
1680
ꢀ-aggregates
ꢀ-sheet
irregular
14
16
17
ꢁ-helix
ꢁ-helix
33
400
ꢀ-turn
12
8
15
16
17
ꢀ-aggregates
ꢀ-aggregates
ꢀ-sheet
irregular
ꢁ-helix
350
300
250
200
150
100
50
THF 0 vol%
THF 30 vol%
20
33
ꢁ-helix
ꢀ-turn
ꢀ-aggregates
12
8
Table 6. Effects of Lyophilization with THF on the Secon-
dary Structure of Lipase Type VII Determined by FT-IR in
the Amide III Spectral Region
0
Type
VII
AY
OF
AH
AK
PS
THF/vol %
0
Position/cmꢂ1
Assignment
Area/%
13
Lipases
1229
1240
1250
1262
1276
1291
1305
1319
1230
1243
1257
1269
1280
1292
1306
1319
ꢀ-sheet
random
random
random
random
ꢁ-helix
ꢁ-helix
ꢁ-helix
ꢀ-sheet
random
random
random
random
ꢁ-helix
ꢁ-helix
ꢁ-helix
Figure 4. Variation of the enantioselectivity of lipases
Type VII, AY, OF, AH, AK, and PS lyophilized with
THF (0 and 30 vol %) for the lipase-catalyzed esterifica-
tion of 1a in isopropyl ether at 37 ꢁC.
51
THF (Table 4). As is seen from the data summarized in
Table 4, the initial rates of the R and S enantiomers for both
hydrolysis and esterification in the presence of THF were
decelerated, and its deceleration for the slower reacting (the
incorrectly binding) S enantiomer is much more serious than
that for the faster reacting (the correctly binding) R enantio-
mer. Thus, in the presence of THF, the larger value of the
quotient of the initial rates (VR=VS) listed in Table 4, arising
from the larger difference in the initial rates between the two
enantiomers, is responsible for the significant enhancement
of the enantioselectivity.
37
15
30
51
34
Then, a possible conformational change of lipase Type VII
caused by the addition of THF was investigated on the basis of
the results of FT-IR, CD, and ESR spectra. To estimate the
secondary structure of the lipase, FT-IR measurements in the
amide I region of lipase Type VII in D2O containing THF
and in the amide III region of lipase Type VII lyophilized in
the presence of THF were carried out. All the IR bands in
the amide I and III regions were assigned to individual secon-
dary structural elements (Tables 5 and 6), according to a
known method of Gaussian curve fitting.9 As is seen from

T. Nishigaki et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 5 (2008)
621
words, THF provides no direct effect on the conformational
flexibility of lipase.
12
10
8
THF 0 vol%
THF 10 vol%
THF 20 vol%
Conclusion
6
The technique of co-solvent addition provided a simple
and powerful tool to improve the enantioselectivity of lipase-
catalyzed hydrolysis. Also, the lipase lyophilized with the
co-solvent catalyzed nicely the esterification with high enan-
tioselectivity. The enantioselectivity enhancement observed
was found to be mainly attributed to the serious deceleration
in the initial rate for the incorrectly binding S enantiomer,
arising from a partial destruction of the tertiary structure
of lipase.
4
THF 30 vol%
2
0
-2
-4
250
260
270
280
290
300
Wavelength/nm
Experimental
Figure 5. CD spectra of lipase Type VII dissolved in water
containing THF (0, 10, 20, and 30 vol %).
Materials. Lipase OF and lipases AY, PS, AH, and AK were
generously provided by Meito Sangyo Co., Ltd. and Amano Phar-
maceutical Co., Ltd., respectively. Lipase Type VII was purchased
from Sigma Chemical. These lipases were used without further
purification for the hydrolysis and esterification. For the spectral
measurements, lipase Type VII was semi-purified by dialyzing
and lyophilizing from crude material. The MALDI-TOF-MS spec-
trum of the semi-purified lipase Type VII showed a single parent
peak, m=z 60.2 kDa, which is consistent with the molecular weight
of Candida rugosa lipase. Ibuprofen and naproxen were purchased
from Tokyo Kasei Kogyo Co., Ltd. All other chemicals were from
commercial sources and of reagent grade.
Preparation of Substrates. According to a known method,12
the substrates 1a–7a (racemic carboxylic acids) were prepared
from the corresponding 4-substituted phenols and ethyl 2-bromo-
propionate, followed by the hydrolysis of the esters. The products
were purified by recrystallization from hexane. The substrates 1–7
(racemic butyl esters) were prepared from the corresponding sub-
strates 1a–7a and 1-butanol. The R or S single enantiomer of 1a
(ca. 98% ee) was prepared according to our method.13
Lipase-Catalyzed Hydrolysis. In a typical procedure, the
substrates 1–9 (racemic butyl esters, 0.036 mmol) were added to
pH 7 phosphate buffer solution containing the co-solvent such
as tetrahydrofuran (2 mL), followed by the ultrasonic dispersion,
and lipase (10 mg) was added. The suspension was shaken (170
strokes minꢂ1) at 37 ꢁC until approximately 40% of the substrates
had reacted. After the reaction, the product and residual substrate
were extracted into hexane, and the filtered aliquot was used as the
sample solution.
THF 0 vol%
THF 30 vol%
Figure 6. ESR spectra of the spin-labeled lipase Type VII
lyophilized with THF (0 and 30 vol %).
the data listed in Tables 5 and 6, the contents of the ꢁ-helix
(33–37%) and ꢀ-sheet (13–16%) calculated from the areas
of the IR bands obtained are nearly identical to the X-ray crys-
tallographic data (33% ꢁ-helix and 12% ꢀ-sheet).10 Further-
more, it was found by comparison of the data in Tables 5
and 6 that both IR spectra in the presence and absence of
THF showed almost the same secondary structure composition
in the amide I and III regions. Thus, the native secondary
structure of lipase Type VII seems not to be changed by the
addition of THF.
On the other hand, the near-UV CD spectrum in water
showed a marked decrease of the relative intensity of the posi-
tive band in the range from 260 to 290 nm, corresponding
to the tertiary structure of lipase, upon addition of 0–
30 vol % THF (Figure 5). From this observation, one could
speculate that a partial destruction of the tertiary structure of
lipase Type VII produces a more compact conformation
or a local conformational change around its active site. In
fact, the incorrectly binding S enantiomer causes a significant
decrease in the initial rate, compared with the correctly bind-
ing R enantiomer (see also Table 4), probably because the
steric difficulty encountered by the S enantiomer would be-
come more serious in fitting into the more compact binding
pocket.
Lipase-Catalyzed Esterification.
In a typical procedure,
the substrates 1a–9a (racemic carboxylic acids, 0.36 mmol)
and 1-butanol (1.08 mmol) were dissolved in diisopropyl ether
(2 mL). To the solution, 1.2 vol % of water (24 mL) was added,
followed by ultrasonic dispersion, and lipase (30 mg) was
added. The suspension was shaken (170 strokes minꢂ1) at
37 ꢁC until approximately 40% of the substrates had reacted.
After the reaction, the filtered aliquot was used as the sample
solution.
Determination of the Enantiomeric Ratio (E Value). For
measurements of the enantiomeric excess (ee) of the product,
the sample solutions obtained from lipase-catalyzed hydrolysis
and esterification were analyzed by HPLC (Shimadzu LC-10A)
on a chiral column (CHIRALCEL OK or CHIRALCEL OD-H,
Daicel Chemical Industries Co., Ltd.) using a mixture of hexane,
2-propanol, and trifluoroacetic acid (970:30:1) as a mobile phase.
The enantiomeric ratio (E value) was calculated from the enantio-
In connection with a partial destruction of the tertiary
structure of lipase, the ESR spectrum of lipase Type VII with
a spin label was taken to estimate its conformational flexibility
(Figure 6). In the previous study, we showed that the enantio-
selectivity of lipase in organic solvents is mainly controlled by
its conformational flexibility estimated from the ESR spec-
trum.11 As judged from the ESR spectrum in Figure 6, contra-
ry to our expectations, the spectral lines were found to remain
unchanged even in the presence of 30 vol % THF. In other

622 Bull. Chem. Soc. Jpn. Vol. 81, No. 5 (2008)
Enhancement of Lipase’s Enantioselectivity
meric excess (ee), according to the literature.14
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Measurement of the Initial Rate for Lipase-Catalyzed
Hydrolysis of Each Enantiomer of 1. In a typical procedure,
(R)- or (S)-1 (0.018 mmol) was added to pH 7 phosphate buffer
solution containing the co-solvent (2 mL), followed by ultrasonic
dispersion, and lipase (5 mg) was added. The suspension was
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2 For a recent review, see: U. T. Bornscheuer, Curr. Opin.
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FT-IR Measurements of Lipase Type VII and Secondary
Structure Analysis. The FT-IR measurements were performed
at room temperature on a BRUKER TENSOR 27 spectrometer
purged with dry N2. All the spectra were averaged over a total
of 100 scans at 2 cmꢂ1 resolution. For the secondary structure
analysis of lipase, the IR spectra were taken as solutions of
lipase Type VII (50 mg mLꢂ1) in D2O containing 0, 30 vol %
THF (corresponding to the hydrolysis) and as KBr tablets
of lipase Type VII lyophilized in the presence of THF (corre-
sponding to the esterification). The spectral data obtained
were deconvoluted by using a BRUKER OPUS 5.0 curve-fitting
module.
CD Measurements of Lipase Type VII and Tertiary Struc-
ture Analysis. The CD measurements were performed at room
temperature on a JEOL MN-EX90A spectrometer. For the tertiary
structure analysis of lipase, the CD spectra of solutions of lipase
Type VII (4 mg mLꢂ1) in water containing 0–30 vol % THF were
measured.
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Measurements. The methionine residues on the surface of lipase
Type VII were spin-labeled with 4-(2-indoacetamido)-TEMPO
purchased from Aldrich Chemical, according to a procedure
reported by Morrisett and Broomfield.15 The spin-labeled lipase
Type VII lyophilized from aqueous solution containing 0 and
30 vol % THF was subjected to the ESR measurements in diiso-
propyl ether with 0.3 vol % water. All the ESR measurements
were carried out at room temperature on a BRUKER EMX081
spectrometer at X-band frequency.
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