2
274
T. Matsuda et al. / Tetrahedron: Asymmetry 19 (2008) 2272–2275
Table 4
[IRA-96SB (Organo Co.)] was kindly supplied by Nacalai Tesque,
Reduction of various ketones by the immobilized cell in scCO
2
Inc., and was used after drying with vacuum pump. Polyallylamine
(PAS-880 (Nitto Boseki Co.)) was purchased from Nacalai Tesque,
Inc. and used without further purification. Other chemicals were
purchased from Nacalai Tesque, Inc., Wako Pure Chemical Indus-
tries, Ltd, and Aldrich Chemical Co. and used without further puri-
fication. Gas chromatographic analyses were performed using
chiral GC-columns (CP-Chirasil-Dex-CB (Varian, 0.32 mm Â
Substrate
Yield (%)
ee (%)
1
2
3
4
5
18
20
0
<1
12
4
>99 (S)
>99 (S)
—
—
>99 (S)
>99 (S)
>99 (S)
a
6
0
0
.25
l
mÂ50 m)
or
CP-cyclodextrin-B-2,3,6-M-19
(Varian,
7
8
.25 mm  0.25
l
m  25 m)) equipped on the Shimadzu GC-14A
Reaction conditions are described in the Section 4.
a
+
+
or GC-14B with C-R8A. The column temperatures and retention
time of the substrates and products for the reactions of 1–7 are
as follows, respectively. Compound 1: 120 °C, 8.3 min, (R)
NADP , 5 mg, was added instead of NAD , 5 mg.
the reaction. At 35 °C and 40 °C, the reaction proceeded and gave
similar results (35 °C: Yield 20%, ee >99%, 40 °C: Yield 27%, ee
12.0 min, (S) 12.6 min; 2: 135 °C, 4.8 min, (R) 7.6 min, (S)
7.9 min; 3: 130 °C, 6.5 min, (R) 10.0 min, (S) 10.6 min; 4: 140 °C,
6.4 min, (R) 10.5 min, (S) 11.4 min; 5: 140 °C, 18.7 min, (S)
26.3 min, (R) 26.8 min; 6: 135 °C, 3.1 min, (S) 14.4 min, (R)
14.7 min; and 7: 115 °C, 16.2 min, (S) 18.9 min, (R) 19.5 min.
>
99%). However, at 50 °C and 60 °C, the reaction did not proceed
at all. This is probably due to the low solubility of the substrate
at the low density of CO at higher temperatures.
Next, various ketones were reduced by the immobilized cell in
scCO , and excellent enantioselectivities were obtained, although
2
2
4
.2. Immobilization of the cell
the yields were poor, and it was necessary to improve this.
The feasibility of recycling was examined using o-fluoroaceto-
The whole cell of G. candidum was immobilized according to the
literature method as follows. To a solution of polyallylamine (PAS-
80, 2.9 g) in a potassium phosphate buffer (pH 7.0, 0.1 M, 5.8 g),
whole cell (6.5 g wet wt) was mixed, after which ion exchange
polymer (IRA-96SB, 48 mL) was mixed and dried for 2 h with a
fan and for 3 h under reduced pressure. The resulting immobilized
cell (14.1 g dry wt) was used for the reaction or stored at À30 °C.
phenone 2 as a substrate. After the reaction, the scCO
2
pressure
5
was decreased, and the product was extracted. Then, the catalyst
was kept under reduced pressure (50 mmHg) at rt for 30 min and
under 4 mmHg for 15 min to use it in the next cycle of reaction.
As shown in Table 5, it could be recycled up to two times, although
8
the yield decreased. This is the first report for the recycling of alco-
8
2
hol dehydrogenase used for reaction in scCO .
4
.3. Batch reaction in an aqueous solution
Table 5
Recycling of the immobilized cell for the reduction of 2 in scCO
2
NAD+ (5 mg) was suspended in an MES buffer (0.1 M, pH 7.0,
Run
Yield (%)
ee (%)
3.0 mL). The substrate (10 lL) and 2-propanol (1.3 mmol) were
added to the suspension, followed by the free cell (0.25 g) or
immobilized cell (0.50 g). The reaction mixture was stirred at
130 rpm for 5 h at 30 °C, extracted with ether (5 mL Â 4), and ana-
lyzed by GC using undecane as an internal standard. The absolute
configuration of the product was determined by comparison of the
GC retention times with those of authentic samples.
1
2
3
4
st
20
9
7
>99 (S)
>99 (S)
>99 (S)
—
nd
rd
th
0
Compound 2 (1
l
L) was converted at 35 °C, 10 MPa for 5 h in scCO
2
by immobilized
cell (0.5 g). After the extraction of the product, mixture was kept under reduced
pressure (50 mmHg) at rt for 30 min and under 4 mmHg for 15 min, and then to
carry out next reaction, MES buffer (0.75 mL), 2-propanol (0.65 mmol), NAD
+
4
.4. Flow reaction in aqueous solution
(
2
5 mg), and 2 (1 lL) were added followed by pressurization with CO .
The immobilized cell (2.5 g) was packed in an up-flow tubular
Furthermore, the immobilized cell could be used in the contin-
uous-flow reaction. This is the first complete flow system using
11
reactor (1/2 inch  10 mm  135 mm), and substrate solution
+
À5
(
1 (0.011 M), 2-propanol (12.72 M), and NAD (1.2 Â 10 M) in
9
alcohol dehydrogenase and scCO
. Conclusions
The whole cell of G. candidum was immobilized onto an ion ex-
2
.
an MES buffer (0.10 M, pH 7.0)) was pumped (JASCO, PU-2085)
at the rate of 0.15 mL/min. The system reached equilibrium after
3
3
h, and then the product was analyzed by GC using undecane as
an internal standard.
change resin with polyallylamine. The immobilization improved
the enantioselectivity and stability, as shown by the reaction con-
ducted in an MES buffer. The immobilization also enabled the con-
tinuous-flow reaction and recycling of the catalyst. The biocatalyst
4
.5. Experimental apparatus for scCO
2
reactions
The apparatus consists of a CO gas cylinder, pump (Jasco SCF-
2
Get), manometer (Taiatsu Techno, Co., Osaka, 15 MP an 25 MPa),
stainless steel pressure-resistant vessel (Taiatsu Techno, Co., Osa-
ka, TVS-N2 type, 10 mL), stop valve (Swagelok, SS3NBS4), water
bath, and magnetic stirrer (Koike, HE-16GA).
was then applied to the reaction in scCO
were reduced with high enantioselectivities. The recycling of the
catalysts accompanying the scCO depressurization and continu-
ous-flow reaction were possible for the first time for alcohol dehy-
drogenase using scCO
2
, and various ketones
2
2
.
4
.6. Reaction in scCO
2
4
4
. Experimental
.1. General
At first, NAD+ (5 mg) was added to the MES buffer (0.1 M, pH
.0, 0.75 mL) in a pressure-resistant vessel followed by the immo-
7
bilized cell (0.50 g). Substrate (0.008 mmol) and 2-propanol
0.65 mmol) were added to a small glass tube, and the glass tube
was placed in pressure-resistant vessel, so that the substrate did
not come into contact with the catalyst before adding CO . Then,
(
The whole cell of G. candidum NBRC 5767 was obtained by cul-
tivation according to the literature.10 The ion exchange polymer
2