Highly enantioselective lipase-catalyzed reactions at high temperatures up to 120°C in organic solvent

Article abstract of DOI:10.1016/j.tetasy.2003.09.040

Lipase-catalyzed kinetic resolutions of 1,1-diphenyl-2-propanol were performed at high temperatures up to 120°C. Burkholderia cepacia lipase immobilized on porous ceramic particles, lipase PS-C II (Amano Enzyme Inc.), gave an enantiopure product at 40-120

Full text of DOI:10.1016/j.tetasy.2003.09.040

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 3943–3947
Highly enantioselective lipase-catalyzed reactions at high
temperatures up to 120°C in organic solvent
Tadashi Ema,* Masafumi Kageyama, Toshinobu Korenaga and Takashi Sakai*
Department of Applied Chemistry, Faculty of Engineering, Okayama University, Tsushima, Okayama 700-8530, Japan
Received 8 September 2003; accepted 16 September 2003
Abstract—Lipase-catalyzed kinetic resolutions of 1,1-diphenyl-2-propanol were performed at high temperatures up to 120°C.
Burkholderia cepacia lipase immobilized on porous ceramic particles, lipase PS-C II (Amano Enzyme Inc.), gave an enantiopure
product at 40–120°C, with the highest conversion (39%) at 80–90°C. The mechanism of high enantioselectivity retained even at
120°C is also described briefly.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
Kogyo Co.), the activity and stability of the enzyme are
improved.9,10 We therefore examined the Toyonite-
immobilized lipase that is commercially available, lipase
PS-C II (Amano Enzyme Inc.), for the high-tempera-
ture biocatalysis. We used n-decane as solvent, having a
high boiling point (174°C), and a stainless autoclave as
a reaction vessel to suppress the evaporation of vinyl
acetate. The lipase-catalyzed kinetic resolutions of 1
were conducted with vinyl acetate (2 equiv.) at 30–
120°C for 3 h (Scheme 1). The results are listed in Table
1 and are plotted in Figure 1. The enantiomeric purities
(% ee) of 1 and 2 were determined by means of chiral
HPLC, and the E values were calculated according to
the literature.11
Potential capabilities of enzymes may be developed by
increasing or decreasing the reaction temperature.1 The
fact that the lipase-catalyzed transesterifications pro-
ceed even at −60°C is one of the most remarkable
examples of the temperature effect.2 Enantioselectivity
was increased dramatically with a decrease in the reac-
tion temperature.2 Several groups have demonstrated
that lipase- and esterase-catalyzed reactions can pro-
ceed at temperatures as high as 70–100°C.3–7 Klibanov
et al. have demonstrated that a dry lipase is extremely
thermostable and active at 100°C in organic media.3
Although enantioselectivity is high in some cases,6 the
number of highly enantioselective and efficient bio-
catalyses at high temperatures is limited. Previously, in
a series of mechanistic studies, we encountered several
substrates that showed little or no reactivity for lipases.
1,1-Diphenyl-2-propanol 1 is one of them.8 High tem-
peratures may force 1 to get over the high-energy
transition state. Here we report that the lipase-cata-
lyzed transesterifications of 1 proceeded at high temper-
atures up to 120°C in a completely enantioselective
manner.
Although the conversion was only 3% at 30°C after 3 h,
it increased with increasing the reaction temperature to
reach 39% at 80 and 90°C. Further increase in the
temperature resulted in lower conversions, and a small
amount of unknown byproduct was also detected. The
absolute configuration of alcohol 1 obtained in the
lipase-catalyzed reactions was determined to be (S) by
comparison of the sign of its specific rotation with the
2. Results and discussion
It is known that when a lipase is immobilized to a
porous ceramic support called Toyonite (Toyo Denka
* Corresponding author. Tel.: +81-86-251-8091; fax: +81-86-251-8092;
e-mail: ema@cc.okayama-u.ac.jp
Scheme 1.
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.09.040

3944
T. Ema et al. / Tetrahedron: Asymmetry 14 (2003) 3943–3947
Table 1. Enantioselective lipase-catalyzed kinetic resolu-
tions of 1 at high temperaturesa
tube with a rubber septum. As a result, the correspond-
ing ester was obtained in >98% ee and 8% yield.
Temp. (°C)
c (%)b
% Yieldc (% eed)
E valuee
Thermostability of the lipase in the Toyonite pores
(mean pore size: 60 nm) is much higher than that of the
same enzyme on Celite (lipase PS, Amano Enzyme Inc.)
because the latter showed 0% conversion for 1 at 100°C
for 3 h (Table 1). The thermostability of the lipase on
Toyonite was examined by the following two methods.
After the lipase was heat-treated at either 80, 100, 120,
130, or 140°C for 3 h in n-decane in an autoclave in the
absence of alcohol and vinyl acetate, the mixture was
filtered and dried in vacuo. In method A, it was used in
the kinetic resolution of 1-phenylethanol 3 in i-Pr2O at
30°C (Scheme 2), and in method B, it was used for the
measurements of the residual activity using Lipase Kit
S (Dainippon Pharmaceutical Co., Ltd.). The results
are listed in Table 2. In method A, enantiopure ester
(R)-4 was obtained in the kinetic resolution of 3 at
30°C using the lipase that had been heat-treated at 100,
120, and even 130°C, although the conversion
decreased gradually. In method B, although the resid-
ual activity was reduced with an increase in the heat-
treatment temperature, the activity (31%) remained
even after the heat treatment at 120°C for 3 h. The
activity was lost substantially after the heat treatment
at 140°C. These results indicate that although the lipase
on Toyonite is denatured gradually at elevated temper-
atures, it is very robust.
(R)-2
(S)-1
30
40
50
60
70
80
90
100
100f
110
120
120g
3
16
23
27
32
39
39
30
0
– (–)
– (–)
–
13 (>98)
17 (>98)
20 (>98)
27 (>98)
31 (>98)
33 (>98)
29 (>98)
– (–)
74 (16)
70 (27)
60 (39)
58 (46)
52 (58)
52 (59)
61 (42)
– (–)
>116
>129
>145
>156
>179
>181
>150
–
21
12
7
25 (>98)
13 (>98)
8 (>98)
62 (28)
71 (14)
78 (10)
>130
>114
>109
a Conditions: Lipase PS-C II (270 mg), 1 (0.82 mmol), vinyl acetate
(1.63 mmol), n-decane (5 mL), in an autoclave, 3 h.
b Conversion determined by 1H NMR for the reaction mixture.
c Isolated yield.
d Determined by HPLC (Chiralpak AD-H for 2, Chiralcel OJ-H for
1).
e Calculated from E=ln [1−c(1+ee(2))]/ln[1−c(1−ee(2))], where c=
ee(1)/(ee(1)+ee(2)).
f Lipase PS, in which Celite is used as a supporting material, was
used.
g Vinyl hexanoate was used as an acyl donor. The reaction was
conducted in a test tube.
Scheme 2.
The enantioselectivity for 1 was excellent even at high
temperatures. The transition-state model that we have
previously proposed (Fig. 2)8,14 suggests that the reac-
tivity of the unfavorable enantiomer, (S)-1, is com-
pletely suppressed by a severe steric repulsion between
the diphenylmethyl moiety of (S)-1 and the protein wall
and/or by a conformational strain which are caused in
the transition state, leading to the perfect enantiomeric
purity of (R)-2 even at 120°C. Thus, the configuration
of the alcohol and ester was predictable by the mecha-
nism-based enzymatic method.15 We have previously
observed no reactivity of 1 for lipase PS (on Celite) at
30°C,8 although the transition-state model predicts that
lipases will show activity for 1, having a small sub-
stituent (Me group) on one side. The crystal structure
of Burkholderia cepacia lipase (PDB entry 1OIL)16 sug-
gested a possibility that even (R)-1 undergoes a severe
steric repulsion between the diphenylmethyl moiety of
(R)-1 and a protein moiety, such as Tyr29, Leu287 and
Ile290, which are not shown in the transition-state
model. By elevating the temperature, the protein can
fluctuate to relieve the steric repulsion, and this fluctua-
tion may also have contributed to the rate acceleration
at elevated temperatures. The transition-state model
also predicts that both activity and enantioselectivity
for 5, which possesses bulky substituents on both sides,
will be very low (Fig. 2(b)), which were actually
Figure 1. Conversions plotted against the reaction tempera-
ture.
reported value.12 The obtained ester 2 was enantiomeri-
cally pure (>98% ee) in all cases measured (except
30°C). It should be noted that the enzymatic catalysis
proceeded completely enantioselectively even at 120°C,
a
sterilization temperature for microorganisms.
Although it is known that the enantioselectivity in
lipase-catalyzed reactions usually decreases with an
increase in temperature2,8 because of a high racemic
temperature,13 the enantioselectivities remained com-
plete at all the temperatures examined in this study. We
also examined vinyl hexanoate as an acyl donor
because its boiling point is higher than 120°C. In this
case, the reaction was conducted at 120°C in a glass test

T. Ema et al. / Tetrahedron: Asymmetry 14 (2003) 3943–3947
Table 2. Effect of heat-treatment on the catalytic activity of the lipase
3945
Heat-treatment temp. (°C)
Method Aa
(R)-4 (% ee)c
Method B
c (%)b
(S)-3 (% ee)c
Relative residual activity (%)d
80
52
50
38
8
99
>99
>99
>99
>99
>99
61
4
66
63
31
6
100
120
130
140
e
0
–
2
2
a Conditions: Lipase PS-C II (heat-treated, 100 mg), 3 (0.82 mmol), vinyl acetate (1.63 mmol), i-Pr2O (5 mL), 30°C, 1 h. The lipase heated at the
indicated temperature for 3 h was used.
b Conversion determined by 1H NMR for the reaction mixture.
c Determined by chiral GC.
d Measured with Lipase Kit S (Dainippon Pharmaceutical Co., Ltd.). The residual activity for the lipase which was treated at 30°C was used as
the standard.
e Not detected.
3. Conclusion
In summary, lipase PS-C II is a robust biocatalyst
useful for organic synthesis. Triggered by the concept
represented by the transition-state model, the capabili-
ties of this mesophilic enzyme have been explored,
finding that they are comparable to those of
(hyper)thermophilic enzymes.17 The lipase-catalyzed
reactions for 1 proceeded at 40–120°C to give enantio-
pure (R)-2, with the highest conversion at 80–90°C.
Since the lipase-catalyzed enantioselective transesterifi-
cations proceed at −60°C using the same enzyme as
reported previously,2,18 it follows that the single enzyme
can show the catalytic function at a very wide range of
temperatures from −60 to 120°C.
4. Experimental
4.1. General
Figure 2. (a) Transition-state model to rationalize the enan-
Silica gel column chromatography was performed using
tioselectivity in the lipase-catalyzed kinetic resolution of sec-
Fuji Silysia BW-127 ZH (100–270 mesh). Thin-layer
ondary alcohols. (i) The CꢀO bond of the substrate takes the
chromatography (TLC) was performed on Merck silica
gauche conformation with respect to the breaking CꢀO bond,
gel 60 F254. Lipase PS-C II and lipase PS were pur-
which is due to the stereoelectronic effect. (ii) The hydrogen
chased from Amano Enzyme Inc. The enantiomeric
atom attached to the stereocenter in the substrate is syn-ori-
purities of 1, 2, 5, and 6 were determined by HPLC
ented toward the carbonyl oxygen atom. Enantioselectivity is
using Chiralcel OJ-H (1), Chiralpak AD-H (2), Chiral-
explained by the conformational requirements and repulsive
cel OD-H (5), and Chiralpak AS-H (6) columns (Daicel
interactions and/or strains caused in the transition state. For
Chemical Industries), and that of 3 and 4 was deter-
details, see Ref. 14. (b) Alcohol 5 having bulky substituents
mined by capillary gas chromatography using a CP-
on both sides, whose reactivity is very low as predicted by the
cyclodextrin-b-2,3,6-M-19 column (Chrompack, ƒ 0.25
transition-state model.
1
mm×25 m). H NMR spectra were measured in CDCl3
at 500 MHz.
4.2. General procedure for lipase-catalyzed kinetic
observed. The 0% conversion was observed at 30°C
after 3 h. Even the bulky alcohol 5 was allowed to react
at a higher temperature but with a low E value. The
alcohol 5 was converted to the corresponding acetate
(R)-6 with 66% ee at 21% conversion (E=6) in the
reaction at 80°C for 3 h.
resolution
The relationship between the temperature of the solvent
in the reaction vessel (autoclave or test tube) and that
of an oil bath was checked in advance to set the
reaction temperature correctly. A mixture of alcohol

3946
T. Ema et al. / Tetrahedron: Asymmetry 14 (2003) 3943–3947
(0.82 mmol), lipase PS-C II (270 mg) and vinyl acetate
(150 mL, 140 mg, 1.63 mmol) in n-decane (5 mL) was
put in a stainless autoclave. The vessel was then placed
in a preheated oil bath. In the case of the reaction using
vinyl hexanoate, a mixture of 1 (173 mg, 0.82 mmol)
and lipase PS-C II (270 mg) in n-decane (5 mL) in a test
tube with a rubber septum was heated at 120°C for 15
min. To the mixture was added vinyl hexanoate (260
mL, 230 mg, 1.62 mmol) via syringe to start the reac-
tion. After being stirred for 3 h, the mixture was
cooled, filtered and concentrated in vacuo. The alcohol
and ester were separated by silica gel column
chromatography.
m, Inj. 250°C, Col. 100°C, Det. 220°C) for the determi-
nation of the enantiomeric purities of (R)-4 and (S)-3.8
In method B, a mixture of the recovered lipase (150 mg)
and water (5 mL) was stirred at room temperature for
30 min. After the mixture was filtered, the lipase activ-
ity in the filtrate was determined with Lipase Kit S
(Dainippon Pharmaceutical Co., Ltd.) according to the
manufacturer’s instructions. The residual activity for
the lipase which was treated at 30°C in the same way
was also determined and was used as the standard for
the relative residual activity.
Acknowledgements
4.3. Lipase-catalyzed kinetic resolution of 1
This work was supported by a Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan. We also
thank Venture Business Laboratory of Okayama Uni-
versity for financial support. We are grateful to the
SC-NMR Laboratory of Okayama University for the
measurement of NMR spectra.
(R)-2: 31% yield; >98% ee; [h]1D4=+44.2 (c 0.5, MeOH),
lit.12 [h]2D5=−41.3 (c 3.27, MeOH) for (S)-2 with 88%
1
ee; H NMR l 1.20 (d, J=6.5 Hz, 3H), 1.84 (s, 3H),
4.02 (d, J=9.5 Hz, 1H), 5.71–5.76 (m, 1H), 7.17–7.30
(m, 10H); HPLC: Chiralpak AD-H, hexane/i-PrOH=
50: 1, flow rate 0.5 mL/min, detection 254 nm, (S) 13.8
min, (R) 15.2 min. (S)-1: 52% yield; 58% ee; [h]1D8=
−28.6 (c 0.5, MeOH), lit.12 [h]2D5=+44 (c 0.55, MeOH)
for (R)-1 with 96.5% ee; 1H NMR l 1.20 (d, J=6.0 Hz,
3H), 1.56 (br s, 1H), 3.81 (d, J=9.0 Hz, 1H), 4.54–4.57
(m, 1H), 7.18–7.40 (m, 10H); HPLC: Chiralcel OJ-H,
hexane/i-PrOH=1: 1, flow rate 0.5 mL/min, detection
254 nm, (S) 22.6 min, (R) 28.0 min.
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