Control of enantioselectivity of lipase-catalyzed esterification in supercritical carbon dioxide by tuning the pressure and temperature

Article abstract of DOI:10.1016/S0957-4166(03)00431-2

The enantioselectivity of lipase-catalyzed esterification of 1-(p-chlorophenyl)-2,2,2-trifluoroethanol in supercritical carbon dioxide was controlled by tuning the pressure and temperature. The enantioselectivity was higher at low pressure and low temperature (E=60) than at high pressure and high temperature (E=10). At the density of 0.75 g/mL, a modified Eyring plot of ln E against 1/T was found to be linear, as expected from the theory on the effects of temperature on stereochemistry.

Full text of DOI:10.1016/S0957-4166(03)00431-2

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 2087–2091
Control of enantioselectivity of lipase-catalyzed esterification in
supercritical carbon dioxide by tuning the pressure and
temperature
Tomoko Matsuda,a,* Ryuzo Kanamaru,a Kazunori Watanabe,a Takashi Kamitanaka,a
Tadao Haradaa and Kaoru Nakamurab
aDepartment of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Otsu, Shiga 520-2194, Japan
bInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
Received 22 April 2003; accepted 16 May 2003
Abstract—The enantioselectivity of lipase-catalyzed esterification of 1-(p-chlorophenyl)-2,2,2-trifluoroethanol in supercritical
carbon dioxide was controlled by tuning the pressure and temperature. The enantioselectivity was higher at low pressure and low
temperature (E=60) than at high pressure and high temperature (E=10). At the density of 0.75 g/mL, a modified Eyring plot of
ln E against 1/T was found to be linear, as expected from the theory on the effects of temperature on stereochemistry. © 2003
Published by Elsevier Science Ltd.
1. Introduction
Our aim is to control the biocatalytic reaction in scCO2
by adjusting the pressure and temperature. Such attempts
to control enzymatic reactions have been done by using
supercritical fluoroform because its polarity changes
drasticallywithpressureandtemperature.1,2 Forexample,
Mori et al. demonstrated the reversible control of trans-
Supercritical carbon dioxide (scCO2, critical point:
31.0°C, 7.38 MPa (72.9 atm)) has recently attracted
attention as an environmentally friendly solvent for
extraction, chemical reactions and chromatography.1
Moreover, its properties, such as density, dielectric
constant, diffusivity, viscosity, solubility etc. can be tuned
by adjusting the pressure and temperature, which clearly
distinguishes this supercritical fluid from conventional
solvents. Therefore, with supercritical fluids, solvent
effects on the reaction can be examined without changing
the kind of solvent, and continuous change in a reaction
can be expected since the solvent properties can be
changed continuously by manipulating the pressure and
temperature.
glycosylation by a lipid-coated b-D
-galactosidase2a and
enantioselective esterification by a lipid-coated lipase.2b
Kamat et al. examined the enantioselectivity of Subtilisin
carlsberg and an Aspergillus protease2c,d in supercritical
fluoroform. However, only a few reports on the control
of biocatalytic reactions using scCO2 under various
pressures and temperatures have been reported.3 We have
examined the enantioselective acetylation of racemic 1-
(p-chlorophenyl)-2,2,2-trifluoroethanol (RS)-1 with li-
pases and vinyl acetate4 in scCO2 as shown in Scheme 1.5
Scheme 1.
* Corresponding author. Tel.: +81 77 543 7466; fax: +81 77 543 7483; e-mail: matsuda@rins.ryukoku.ac.jp
0957-4166/$ - see front matter © 2003 Published by Elsevier Science Ltd.
doi:10.1016/S0957-4166(03)00431-2

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T. Matsuda et al. / Tetrahedron: Asymmetry 14 (2003) 2087–2091
Table 1. Screening of lipases for enantioselective acetyla-
tion of (RS)-1 in supercritical carbon dioxide
The fluorinated compound was chosen as a model
substrate because enantiomerically pure fluorinated
alcohols have received much attention for the synthesis
of ferroelectric liquid crystals or bioactive compounds.6
We found that the enantioselectivity of a reaction using
the lipase Novozym can be controlled by adjusting the
pressure and temperature of scCO2. Moreover, at the
density of 0.75 g/mL (31°C at 9.5 MPa, 35°C at 11.2
MPa, 40°C at 13.2 MPa, 45°C at 15.3 MPa, 50°C at
17.5 MPa, 55°C at 19.6 MPa, and 60°C at 21.8 MPa),7
the modified Eyring plot of ln E8 against 1/T was found
to be linear, which correlates well with results predicted
by the theory of the effects of temperature on
enantiochemistry.9
Low pressure
conditions
High pressure
conditions
Lipase
LPL
(9.1 MPa)
(14.5 MPa)
Yield (%)
52
Ea
Yield (%)
38
Ea
12
16
(Pseudomonas
aeruginosa)
AY (Candida
rugosa)
AH (Pseudomonas
cepacia)
8
3
0
1b
2
0
0
2
–
29
PS-D
–
–
2. Results and discussion
2.1. Screening of lipases
(Pseudomonas
cepacia)
PS-C
(Pseudomonas
cepacia)
Lipozyme
(Rizomucor
miehei )
43
0
8
–
22
0
17
–
We screened various lipases for the enantioselective
acetylation of (RS)-1 with vinyl acetate4 in scCO2 at 9.1
and 14.5 MPa. Lipases tested were LPL (Pseudomonas
aeruginosa), AY (Candida rugosa), AH (Pseudomonas
cepacia), PS-D (Pseudomonas cepacia), PS-C (Pseu-
domonas cepacia), Lipozyme (Rizomucor miehei), and
Novozym (Candida antarctica). The results are listed in
Table 1. The (S)-enantiomer reacted faster than the
(R)-enantiomer, affording the (S)-acetate (S)-2 and the
remaining (R)-alcohol (R)-1 except when lipase AY
was used at 9.1 MPa. The highest enantioselectivity8
(E=38) was obtained using Novozym at 9.1 MPa.
Interestingly, the enantioselectivity was significantly
affected by the pressure.
Novozym
(Candida
antarctica)
25
38
24
23
Reaction conditions: 40°C, 4 h.
a The (S)-enantiomer reacted faster than (R)-enantiomer.
b In this case, the (R)-enantiomer reacted slightly faster than the
(S)-enantiomer.
2.3. Effect of pressure on the reaction using Novozym
at 55°C
2.2. Time course of the reaction using Novozym
The effect of pressure on the enantioselective acetyla-
tion of (RS)-1 with vinyl acetate in scCO2 by Novozym
was investigated at 55°C changing the reaction time
from 2 to 4 h.5 Below 8 MPa, the reaction hardly
proceeded, and the reaction above 19 MPa was not
examined. As shown in Figure 1(b), the E value
The enantioselective acetylation of (RS)-1 with vinyl
acetate in scCO2 by Novozym was investigated at 55°C
and 10 MPa.5 The time course of the reaction is shown
in Figure 1(a). The reaction yield of acetate 2 reached
approximately 50% at 5 h.
Figure 1. Acetylation of (RS)-1 in supercritical carbon dioxide catalyzed by lipase Novozym at 55°C (a) time course at 10 MPa,
(b) effect of pressure on enantioselectivity.

T. Matsuda et al. / Tetrahedron: Asymmetry 14 (2003) 2087–2091
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Figure 2. Effect of pressure on enantioselectivity of acetylation of (RS)-1 in supercritical carbon dioxide catalyzed by lipase
Novozym at 31°C (green), 40°C (magenta) and 60°C (blue).
2.4. Effect of temperature on the reaction using
Novozym
changed continuously from 50 to 10 when the pressure
was changed from 8 to 19 MPa, regardless of the
reaction time.
Effect of pressure on the enantioselective acetylation of
(RS)-1 with vinyl acetate in scCO2 by Novozym was
investigated at different temperatures. The results of the
experiments at 31, 40 and 60°C are shown in Figure 2.
As in the case at 55°C, the (S)-enantiomer reacted
faster than the (R)-enantiomer, affording (S)-2 and the
remaining (R)-1, and the E value changed continuously
according to the pressure. The cause of this change is
probably due to the change in density as in the case at
55°C. This explanation agrees with the following obser-
vations. At lower temperatures (31, 40°C), the E values
changed rapidly from high to low values within a small
range of pressure below 10 MPa. However, at higher
temperatures (55°C, 60°C), the E values changed gradu-
ally within a larger range of pressure below 14 MPa.
These changes correlate well with the change in density;
at low temperatures, the density changes rapidly from
0.2 to 0.6 g/mL below 10 MPa but at high tempera-
tures, it changes gradually over a large range of pres-
sure, as shown in Figure 3.7
The change in enantioselectivity of the reaction by the
pressure is indeed noteworthy, although the reason is
not clear at present. When the pressure of scCO2 was
changed, there was no significant change in the polarity
evaluated as dielectric const. (at 50°C; 1.12 at 8 MPa
and 1.29 at 11 MPa)10 and log P (at 50°C; 1.4 at 8 MPa
and 1.9 at 11 MPa).11 It is not clear if this small change
in polarity has a large effect on this reaction. On the
other hand, the density of scCO2 does change from 0.20
to 0.42 kg/L when the pressure is changed from 8 to 11
MPa at 55°C.7 Ikushima explained the high enantiose-
lectivity of lipase in a very limited pressure range at
304.1 K as resulting from the interaction between the
CO2 and enzyme molecules.3 We also propose that the
large change in density can significantly change the
interaction of CO2 and enzyme, causing formation of
carbamates from CO2 and the free amine groups on the
surface of the enzyme2e, CO2 adsorption on the enzyme
as reported in other proteins12 and/or CO2 incorpora-
tion in the substrate-binding pocket of the enzyme as
reported in the incorporation of organic molecule in
enzymes.13 These interactions may gradually change the
conformation of the enzyme in response to pressure,
resulting in a continuous change in enantioselectivity.
This conformational change is reversible because treat-
ment of the enzyme by scCO2 does not alter reactivities
or enantioselectivities.
However, when E values of the same density (at differ-
ent temperatures and different pressures) were com-
pared, E values were affected by the temperature. The
higher the temperature, the lower the enantioselectivity.
The enantioselectivity is determined not only by the
density but also by the temperature. In a reaction under
ambient conditions, the enantioselectivity in the kinetic
resolution is temperature-dependent and obeys the fol-
lowing modified Eyring equation.9,14

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T. Matsuda et al. / Tetrahedron: Asymmetry 14 (2003) 2087–2091
temperature or pressure. This contrasting result is prob-
ably due to the difference between fluoroform and CO2
in the magnitude of the change in dielectric constants
caused by the manipulation of pressure and tempera-
ture. In our case, both the density and the temperature
controlled the reaction, but in their case, the effect of
temperature on the reactions were probably negligible
compared to the effect of the dielectric constant.
3. Conclusions
The enantioselectivity of an environmentally benign
reaction was examined, and the continuous change in
enantioselectivity without changing the molecular struc-
ture of the solvent, which is not possible by simply
changing the organic solvent, was observed using
scCO2. The enantioselectivity of the reaction depends
not only on the density but also on the temperature,
and the Eyring plot was found to be linear. So we
believe this work to be helpful to further developments
in studying the origin of enantioselectivity and synthe-
sizing useful compounds while keeping harmony with
the natural environment.
Figure 3. Density versus pressure of CO2 at 31°C (green),
40°C (magenta) 55°C (black) and 60°C (blue).7
ln E=−(DDH"/R)(1/T)+(DDS"/R)
By enzymatic reactions performed at temperatures
ranging from 30 to −50°C, Sakai et al. showed the first
experimental evidence supporting the theory of the
effect of temperature on stereochemistry.14 However, to
the best of our knowledge, there are no experimental
examples for the scCO2 reaction. Here, we examined
whether the theory is applicable to the reaction in
scCO2. At the density of 0.75 g/mL (31°C at 9.5 MPa,
35°C at 11.2 MPa, 40°C at 13.2 MPa, 45°C at 15.3
MPa, 50°C at 17.5 MPa, 55°C at 19.6 MPa, and 60°C
at 21.8 MPa), ln E was plotted against 1/T. As shown
in Figure 4, the Eyring plot was found to be linear,
which indicates the conformational stability of the tran-
sition state at a temperature range from 31°C to 60°C
in scCO2 at 0.75 g/mL. This is the first example show-
ing that Eyring’s theory can be applied to the biocata-
lytic reaction in scCO2.
4. Experimental
4.1. General
Lipase LPL was kindly supplied by Toyobo, the lipases
AY, AH, PS-D and PS-C were kindly supplied by
Amano Enzymes Inc., and the lipases Lipozyme and
Novozym were kindly supplied by Novozymes. Chemi-
cals were purchased from Nacalai Tesque, Inc., Wako
Pure Chemical Industries, Ltd, and Aldrich Chemical
Co and used without further purification unless other-
wise indicated. Vinyl acetate was distilled and dried
over MS-4A before use. Gas chromatographic analyses
were performed using chiral GC-columns (Chrompack,
Chirasil-DEX CB: 25 m; He 2 mL/min) equipped on
the Shimadzu GC-14B with C-R7A plus.
The observation in the variation of the enantioselectiv-
ity by changing the temperature at the same density, i.e.
at the same dielectric constant,10 is in contrast to the
case for the transglycosylation by a lipid-coated b-D-
galactosidase2a or the enantioselective esterification by a
lipid-coated lipase2b in supercritical fluoroform by Mori
et al. In these reports, reactivities and selectivities were
controlled by the dielectric constant and not solely by
4.2. Experimental apparatus for scCO2 reactions
The apparatus consists of a CO2 gas cylinder, cooler
(−10°C), pump (Jasco PU-1580 pump), manometer
(Taiatsu Techno, Co., Osaka, 15 MP or 25 MPa),
stainless steel pressure-resistant vessel (Taiatsu Techno,
Co., Osaka, TVS-N2 type, 10 mL), stop valve
(Swagelok, SS3NBS4), oven and magnetic stir (Koike,
HE-16GA).
4.3. Enantioselective acetylation of (RS)-1 in scCO2
The lipase (Novozym, 25 mg), the racemic alcohol
(RS)-1 (2.5 mg, 0.012 mmol), vinyl acetate (0.025 mL,
0.27 mmol), and a magnetic stirrer bar were charged in
the vessel (the chemicals were placed in a glass tube to
prevent them from contacting the biocatalyst before
supercritical conditions were achieved). The vessel was
then warmed to 55°C, and CO2, preheated to 55°C, was
introduced at 9.3 MPa. The mixture was then stirred at
Figure 4. Effect of temperature on enantioselectivity of acetyl-
ation of (RS)-1 in supercritical carbon dioxide catalyzed by
lipase Novozym at 0.75 g/mL.

T. Matsuda et al. / Tetrahedron: Asymmetry 14 (2003) 2087–2091
2091
55°C for 3 h and the scCO2 was liquefied at −10°C;
subsequently, the gas pressure was released. The result-
ing residue was dissolved in ether, and the mixture put
on Extrelut and quickly eluted with ether. The ee values
of both the remaining alcohol 1 and the producing
acetate 2 were measured with a chiral GC-column
(130°C for 6 min followed by 10°C/min to 180°C) to
determine the yields and E values. The absolute
configurations were determined by comparing the GC
retention times with those of authentic samples.15 The
experiment was conducted using other lipases and at
different reaction times, temperatures or pressures.
5. Matsuda, T.; Kanamaru, R.; Watanabe, K.; Harada, T.;
Nakamura, K. Tetrahedron Lett. 2001, 42, 8319–8321.
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