Control on enantioselectivity with pressure for lipase-catalyzed esterification in supercritical carbon dioxide

Article abstract of DOI:10.1016/S0040-4039(01)01785-3

The enantioselectivity of lipase-catalyzed esterification of 1-(p-chlorophenyl)-2,2,2-trifluoroethanol in supercritical carbon dioxide changed continuously from E = 10 to 50 by decreasing the pressure at 55°C. The effect of the solvent on enantioselectivi

Full text of DOI:10.1016/S0040-4039(01)01785-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 8319–8321
Control on enantioselectivity with pressure for lipase-catalyzed
esterification in supercritical carbon dioxide
Tomoko Matsuda,a,* Ryuzo Kanamaru,a Kazunori Watanabe,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 19 July 2001; accepted 21 September 2001
Abstract—The enantioselectivity of lipase-catalyzed esterification of 1-(p-chlorophenyl)-2,2,2-trifluoroethanol in supercritical
carbon dioxide changed continuously from E=10 to 50 by decreasing the pressure at 55°C. The effect of the solvent on
enantioselectivity was examined without changing the kind of solvent. © 2001 Elsevier Science Ltd. All rights reserved.
Supercritical fluids are of continuing interest and
importance as tunable solvents and reaction media in
the chemical process industry.1 They are neither gas nor
liquid but can be compressed gradually from low to
high density. By adjusting the density, the properties of
these fluids can be customized and manipulated for a
particular process.1 Among many fluids, supercritical
carbon dioxide (scCO2) has the advantages of an envi-
ronmentally benign nature, non-flammability, low toxi-
city, high availability, and ambient critical temperature
(Tc=31.0°C).1 Several recent publications have demon-
strated the potential of scCO2 as an alternative reaction
medium to toxic organic solvents for synthetic transfor-
mations with transition metal catalysts.2,3 Our interest
is in the reaction by a natural catalyst, biocatalyst, for
synthesizing optically pure fluorinated alcohols that
have received much attention for the synthesis of fer-
roelectric liquid crystals or bioactive fluorinated com-
pounds.4–7 Mori et al.8,9 have demonstrated the benefit
of using scCO2 for biotransformations, for example
improved and controllable reaction rates. We examined
the enantioselective acetylation of racemic 1-(p-
chlorophenyl)-2,2,2-trifluoroethanol (R/S)-1 with the
lipase CAL (Novozym 435) and vinyl acetate10 in CO2,
affording (S)-acetate (S)-2 and remaining (R)-alcohol
(R)-1, as shown in Scheme 1. We found that the
enantioselectivity of the reaction can be tuned continu-
ously from E=10 to 50 by adjusting the pressure of
CO2 at 55°C. The effect of the solvent was examined
without changing the kind of solvent.
For the acetylation of (R/S)-1 with the lipase in CO2,
we used a high pressure-resistant stainless-steel vessel.11
In the vessel, the lipase (CAL, 25 mg), the racemic
alcohol (R/S)-1 (2.5 mg, 0.012 mmol), vinyl acetate
(0.025 mL, 0.27 mmol), and a magnetic stirrer bar were
charged (the chemicals were placed in a glass tube to
prevent them from contacting the biocatalyst before
achieving supercritical conditions). Then the vessel was
warmed to 55°C, and CO2, preheated to 55°C, was
introduced until a desired pressure (8–19 MPa, 1
MPa=9.87 atm) was reached. The mixture was then
Lipase CAL
OH
OH
OAc
OAc
+
F3C
F3C
F3C
CO2 (8 - 19 MPa), 55 oC
Cl
Cl
Cl
(R) - 1
(S) - 2
(R/S) - 1
Scheme 1.
Keywords: supercritical carbon dioxide; lipase; enantioselectivity; trifluoromethyl alcohol; esterification.
* Corresponding author. Fax: +81-77-543-7483; e-mail: matsuda@rins.ryukoku.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01785-3

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T. Matsuda et al. / Tetrahedron Letters 42 (2001) 8319–8321
Contrarily, the continuity of the enantioselectivity
change was not observed using conventional organic
solvents for the same reaction (the same enantiomer
(S)-1 reacted faster). As shown in Fig. 3, E values were
largely affected by the solvents used. However, it is
ambiguous whether the E values depended only on the
polarity17 of the solvent, because the polarity change is
inevitably accompanied by the change in the molecular
structure of the solvent (cyclic or acyclic). This result
agrees with the literature report that the molecular
structure of the solvent, besides its polarity, affects the
enantioselectivity of the reaction.18 On the other hand,
by using CO2, the solvent properties can be changed by
the manipulation of pressure, without changing the
kind of solvent.
stirred at 55°C for the desired time (2–4 h) and worked
up as described.11 The ee of both the remaining alcohol
1 and the producing acetate 2 were measured with a
chiral GC column (Chirasil-DEX CB; 25 m; He 2
mL/min, 130°C for 6 min followed by 10°C/min to
180°C) to determine the yields and E values, the ratio
of the specificity constants.12 The absolute configura-
tions were determined by comparing the GC retention
times with those of authentic samples.13
Many hydrolytic enzymes have been reported to show
activities in supercritical solvents,8,9,14–16 and, as
expected, (R/S)-1 was acetylated with the lipase CAL
in CO2 at 55°C under the pressure range of 8 MPa to
19 MPa. Below 8 MPa at 55°C, the reaction hardly
proceeded, and the reaction above 19 MPa was not
examined. The reaction yield of acetate 2 reached
around 50% in 5 h at 10 MPa (Fig. 1).
The tunable enantioselectivity of the reaction is indeed
noteworthy, but the cause is not clear at present. A
search of the literature uncovered several reports that
investigated the effect of solvent properties on enzyme
enantioselectivities.19–25 Kamat19 attributed the change
in the enantioselectivity of Subtilisin carlsberg and an
Aspergillus protease by manipulation of the pressure of
supercritical fluoroform to the change in the polarity of
the fluoroform.26 However, in our work, the change in
the pressure of CO2, rather than fluoroform, does not
cause a significant change in the polarity evaluated as
dielectric constant (at 50°C, 1.12 at 8 MPa and 1.29 at
11 MPa)27 and log P (at 50°C, 1.4 at 8 MPa and 1.9 at
11 MPa).28 It is not clear if this small change in polarity
has a large effect on this reaction. On the other hand,
the density of CO2 changes from 0.20 to 0.42 kg/L by
changing the pressure from 8 to 11 MPa at 55°C.29
Ikushima explained the high enantioselectivity of lipase
in a very limited pressure range at 304.1 K by the
interaction between CO2 and enzyme molecules.21–23
We also propose that the large change in density could
The effect of pressure on enantioselectivity was investi-
gated. As shown in Fig. 2, the E value changed contin-
uously from 50 to 10 when the pressure was changed
from 8 to 19 MPa, regardless of the reaction time.
Figure 1. Time course of acetylation catalyzed by lipase at 10
MPa.
Figure 3. Effect of organic solvent on enantiomeric ratio (E).
Reaction conditions: Substrate: 7.7 mmol, vinyl acetate: 88
mmol, lipase: 20 mg for reaction in hexane and cyclohexane,
,
50 mg for benzene and isopropyl ether, molecular sieves 4 A:
50 mg, organic solvent: 500 mL, 130 rpm, 55°C, 2 h for
hexane and cyclohexane, 2.5 h for benzene, 4 h for isopropyl
ether. The reaction was stopped at the conversion of 50% for
hexane, 47% for cyclohexane, 47% for benzene and 39% for
isopropyl ether.
Figure 2. Effect of pressure on enantiomeric ratio (E).

T. Matsuda et al. / Tetrahedron Letters 42 (2001) 8319–8321
8321
significantly change the interaction of CO2 and enzyme,
causing adsorption of CO2 on the enzyme as reported
in other proteins30 and/or incorporation of CO2 in the
substrate-binding pocket of the enzyme as reported in
the incorporation of organic molecule in the
enzymes.18,24 These interactions may change the confor-
mation of the enzyme gradually corresponding to the
pressure, resulting in a continuous change in enantiose-
lectivity. However, further investigation is necessary to
clarify the mechanism.
8. Mori, T.; Okahata, Y. Chem. Commun. 1998, 2215–2216.
9. Mori, T.; Kobayashi, A.; Okahata, Y. Chem. Lett. 1998,
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10. Wang, Y.-F.; Lalonde, J. J.; Momongan, M.; Bergbreiter,
D. E.; Wong, C.-H. J. Am. Chem. Soc. 1988, 110, 7200–
7205. Vinyl alcohol initially formed by the reaction is
usually converted to acetaldehyde spontaneously.
11. Matsuda, T.; Harada, T.; Nakamura, K. Chem. Commun.
2000, 1367–1368.
12. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J.
Am. Chem. Soc. 1982, 104, 7294–7299. The E value was
used to evaluate enantioselectivity. E=(VA/KA)/(VB/KB)
where VA, KA and VB, KB denote maximal velocities and
Michaelis constants of the fast- and slow-reacting enan-
tiomers, respectively.
13. Matsuda, T.; Harada, T.; Nakajima, N.; Itoh, T.; Naka-
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In conclusion, the enantioselectivity of an environmen-
tally benign reaction pairing the natural catalyst and
natural solvent was examined, and the continuous
change in enantioselectivity without changing the
molecular structure of the solvent, which is not possible
by simply changing the organic solvent, was observed
using CO2. We believe this reaction will be helpful not
only for studying the origin of enantioselectivity but
also for synthesis of useful compounds while keeping
harmony with the natural environment.
15. Cernia, E.; Palocci, C. In Methods in Enzymology; Abel-
son, J. N.; Simon, M. I., Eds. Lipases in supercritical
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Acknowledgements
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water) is usually used to understand the solvent effect of
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The authors greatly appreciate the helpful advice given
by Professor Okahata and Dr. Mori at the Tokyo
Institute of Technology. This work was supported by a
Grant-in-Aid from the Taisho Pharmaceutical Co.
Award in Synthetic Organic Chemistry, Japan, the
Sasakawa Scientific Research Grant from the Japan
Science Society, and a Grant-in-Aid for Encouragement
of Young Scientists (No. 12740400) and a Grant-in-Aid
for Scientific Research (C) (No. 11640602) from the
Ministry of Education, Science, Sports and Culture of
Japan.
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