The results are shown in Table 1. Cyclohexanol was obtained
successfully. The conversions to cyclohexanol in the first and
second trap were smaller than that of the third trap because some
of the product remained in the column. The conversions in the
third and fourth were constant, and that in the fifth decreased
slightly. This may be due to the change in the water content in
the water-absorbing polymer on which the cell was im-
mobilized. Viability of the cell was not examined, but this may
not effect enzyme activity.
Encouraged by this promising result, reduction of o-
fluoroacetophenone was conducted by the same method (Fig. 1
(b)), using cyclooctane as a GC internal standard. (S)-o-
Fluorophenylethanol was obtained successfully with a conver-
sion of 8%. The enantioselectivity was determined to be > 99%
ee by GC analysis using a chiral column (Chirasil-DEX CB; 25
m; He 2 mL min21, 130 °C). The absolute configuration was
determined by comparing the GC retention times with those of
the authentic samples. Similar results were obtained with
repeated-use of the biocatalyst.
The space-time yield of this flow system was compared with
that of the corresponding batch system.4 That of the flow system
(0.24 µmol min21) was almost twice as much as that of the
corresponding batch system (0.13 µmol min21) at 35 °C and 10
MPa using a pressure-resistant vessel (Taiatsu Techno Co.,
Osaka, TVS-N2 type, 10 mL) for the reduction of o-
fluoroacetophenone. Therefore, the flow-type reactor is more
efficient than the corresponding batch system.
In conclusion, the immobilized resting cell of G. candidum
was used in a semi-continuous flow system using scCO2 for the
first time. The alcohol dehydrogenase in the cell reduced the
cyclohexanone successfully, and when o-fluoroacetophenone
was used as a substrate, excellent enantioselectivity and a higher
space–time yield than the corresponding batch system were
obtained. The ability of this first biocatalytic-flow-reduction
process using scCO2 to solve the product-extraction problem in
the dehydrogenase reactions will be indispensable as a seminal
step toward further developments in the field.
The authors greatly appreciate the advice given by Professor
T. Ikariya of the Tokyo Institute of Technology. The authors are
grateful to Osaka Yuki Kagaku Kogyo Co., Ltd. for providing
BL-100 (water-absorbing polymer).
Notes and references
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Kiran, P. G. Debenedetti and C. J. Peters, Supercritical Fluids
Fundamentals and Applications, Kluwer Academic Publishers, Dor-
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and J. M. DeSimone, Chem. Rev., 1999, 99, 543.
2 A. J. Mesiano, E. J. Beckman and A. J. Russell, Chem. Rev., 1999, 99,
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and Y. Okahata, Chem. Commun., 1998, 2215; T. Mori, A. Kobayashi
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Yamada and T. Yano, Chem. Eng. Commun., 1986, 45, 207; H. Nakaya,
O. Miyawaki and K. Nakamura, Enzyme Microb. Technol., 2001, 28,
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3 M. G. Hitzler and M. Poliakoff, Chem. Commun., 1997, 1667.
4 T. Matsuda, T. Harada and K. Nakamura, Chem. Commun., 2000,
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5 J. L. Panza, A. J. Russell and E. J. Beckman, Tetrahedron, 2002, 58,
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6 The apparatus consists of a cooler (210 °C), pump (Jasco PU-1580
pump), manometer (Taiatsu Techno Co., Osaka, 15 MPa), six-port
sample injection valve for the HPLC (Rheodyne, 7725(i)), stainless steel
pressure-resistant vessel (Taiatsu Techno Co., Osaka, TVS-N2 type, 10
mL) and relief valve (Nupro Co., SS-4R3A-EP).
7 K. Nakamura, T. Matsuda and T. Harada, Chirality, 2002, 14, 703; K.
Nakamura, Y. Inoue, T. Matsuda and I. Misawa, J. Chem. Soc., Perkin
Trans. 1, 1999, 2397; K. Nakamura, Y. Inoue and A. Ohno, Tetrahedron
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