9
72
T. Matsuda et al. / Tetrahedron: Asymmetry 13 (2002) 971–974
2
. Results and discussion
Table 1. Reduction of 2-pentanone 1a with the APG4 sys-
tem
2
.1. Reduction of 2-pentanone 1a
Entry Coenzyme Additive alcohols
Yield (%) E.e. (%)
a
(
mL)
First, 2-pentanone 1a was subjected to the reduction
with APG4, NAD , and 2-propanol in MES buffer.
+
+
1
2
3
4
5
NAD
2-Propanol (100)
2-Propanol (100)
Cyclopentanol (100) 88
Cyclopentanol (100) 86
88
85
\99
\99
\99
\99
\99
The yield and enantioselectivity of the resulting 2-pen-
tanol 1b was determined by GC analysis, and the
absolute configuration of 1b was determined by com-
parison of the GC retention times with those of the
authentic samples. It was found that the product was
obtained in 88% yield, with e.e. of >99% (Table 1, entry
+
NADP
+
NAD
+
NADP
NAD
+
2-Propanol (500)
97
The reaction conditions are described in Section 4.
a
Amount of an alcohol additive to reduce 10 mL (0.093 mmol) of 1a.
1). In this system, the enzyme clearly distinguished
between the Me and Pr groups. Other coenzyme and
alcohol additives were also tested to see if the high
enantioselectivities were steady, and >99% e.e. was
Table 2. Reduction of 2-butanone 2a with the APG4 sys-
tem
+
observed for any combination of coenzyme (NAD or
+
NADP ) and additive (2-propanol or cyclopentanol).
When the amount of 2-propanol was increased, the
yield increased without decreasing enantioselectivity.
The time course of the reduction shows that the yield of
Entry Coenzyme Additive alcohols
Yield (%) E.e. (%)
a
(mL)
+
2-pentanol was increased to 96% in 9 h.
1
2
3
4
5
6
NAD
2-Propanol (100)
2-Propanol (100)
Cyclopentanol (50)
Cyclopentanol (50)
Cyclopentanol (150) 79
2-Propanol (500) 73
85
85
88
89
77
76
84
83
93
94
+
NADP
NAD
NADP
+
2.2. Reduction of 2-butanone 2a
+
+
NAD
Next, 2-butanone 2a was reduced using the same sys-
tem to obtain (S)-2-butanol. The result is shown in
Table 2. Unfortunately, under the same conditions as
for the reduction of 2-pentanone 1a, the enantioselec-
tivity of the reduction was only moderate (77% e.e.,
Table 2, entry 1). This is probably due to the presence
of more than two competing enzymes with different
enantioselectivities ((R)-enzyme(s) and (S)-enzyme(s)).
Screening of the dried cells of other microorganisms
+
NAD
The reaction conditions are described in Section 4.
a
Amount of an additive alcohol to reduce 10 mL (0.11 mmol) of 2a.
6
). This improvement is probably due to the relatively
stronger inhibition of the (R)-enzyme(s) than that of
the (S)-enzyme(s) with the large amount of the additive
alcohols.
†
resulted in even lower e.e. The reaction conditions
were optimized using other coenzyme or additive alco-
+
+
hol. The use of NADP instead of NAD did not affect
the enantioselectivity (Table 2, entry 2). When
cyclopentanol was used instead of 2-propanol, an
improvement was observed (Table 2, entries 3 and 4).
When a larger amount of cyclopentanol or 2-propanol
was used, the enantioselectivity was greatly improved to
e.e.s of 93 and 94%, respectively (Table 2, entries 5 and
2
.3. Reduction of 3-hexanone 3a
A considerably challenging substrate for asymmetric
reduction, 3-hexanone 3a, was also reduced by the
APG4 system. Surprisingly, the enantioselectivity of the
reduction was 98%, as shown in Scheme 2. The GC
chromatograph of the propionate of the product 3b is
shown in Fig. 1. Thus, the system could clearly distin-
guish the Et and Pr groups.
†
Reduction of 2-butanone 2a (20 mL, 0.22 mmol) by the dried cell of
various organisms (10 mg) and four different combinations of a
+
coenzyme (5 mg) and an additive alcohol (100 mg), NAD and
+
2
-propanol (Condition A), NAD and cyclopentanol (Condition B),
2.4. Substrate specificity
+
+
NADP and 2-propanol (Condition C), NADP and cyclopentanol
Condition D), in MES buffer (pH 7.0, 0.1 M, 3 mL) at 30°C for 20
(
To examine the generality of these excellent enantiose-
lectivities, other aliphatic ketones were also reduced by
the APG4 system (Table 3). As expected, most of the
h gave the following results:
Endomyces magnusii IFO 4600: Conditions A 90% yield, 19% e.e./
Conditions B 66% yield, 57% e.e/Conditions C 85% yield, 40%
e.e./Conditions D 41% yield, 66% e.e.;
Endomyces geotrichum IFO 9541: Conditions A 87% yield, 42%
e.e./Conditions B 92% yield, 71% e.e./Conditions C 85% yield, 59%
e.e./Conditions D 89% yield, 78% e.e.;
Geotrichum candidum IFO 5767: Conditions A 86% yield, 46%
e.e./Conditions B 63% yield, 78% e.e./Conditions C 88% yield, 61%
e.e./Conditions D 92% yield, 83% e.e.;
O
OH
APG4(dried cell of G. candidum)
+
NAD , 2-propanol, MES buffer
Geotrichum candidum ATCC 34614: Conditions A 85% yield, 54%
e.e./Conditions B 91% yield, 80% e.e./Conditions C 86% yield, 52%
e.e./Conditions D 92% yield, 80% e.e.;
Galactomyces reessii IFO 1112: Conditions A 89% yield, 44% e.e./
Conditions B 92% yield, 74% e.e./Conditions C 86% yield, 52%
e.e./Conditions D 88% yield, 78% e.e.
3
a
3b
Yield 35%
ee 98%
Scheme 2.