Stereoselective reduction of 2-butenolides to chiral butanolides by reductases from cultured cells of Glycine max

Article abstract of DOI:10.1016/j.tetlet.2006.12.126

The stereoselective reduction of 2-butenolides by two reductases, p51 and p83, from cultured plant cells of Glycine max was investigated. The reduction of 2-methyl-2-butenolide by p51 reductase produced (R)-2-methylbutanolide, whereas the reduction by p83 reductase gave (S)-2-methylbutanolide. Both reductases reduced 3-methyl-2-butenolide to (R)-3-methylbutanolide. The reduction of 2,3-dimethyl-2-butenolide by p51 reductase gave (2R,3R)-2,3-dimethylbutanolide, whereas the reduction by p83 reductase produced (2S,3R)-2,3-dimethylbutanolide. The reduction of 4-alkyl-2-butenolides with these reductases was accompanied by resolution of chiral centers affording (R)-4-alkylbutanolides.

Full text of DOI:10.1016/j.tetlet.2006.12.126

Tetrahedron Letters 48 (2007) 1345–1347
Stereoselective reduction of 2-butenolides to chiral butanolides
by reductases from cultured cells of Glycine max
Kei Shimoda,a Naoji Kubota,a Toshifumi Hirata,b Yoko Kondoc and Hiroki Hamadac,*
aDepartment of Pharmacology and Therapeutics, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
bDepartment of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University,
1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan
cDepartment of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
Received 16 November 2006; revised 18 December 2006; accepted 21 December 2006
Available online 23 December 2006
Abstract—The stereoselective reduction of 2-butenolides by two reductases, p51 and p83, from cultured plant cells of Glycine max
was investigated. The reduction of 2-methyl-2-butenolide by p51 reductase produced (R)-2-methylbutanolide, whereas the reduction
by p83 reductase gave (S)-2-methylbutanolide. Both reductases reduced 3-methyl-2-butenolide to (R)-3-methylbutanolide. The
reduction of 2,3-dimethyl-2-butenolide by p51 reductase gave (2R,3R)-2,3-dimethylbutanolide, whereas the reduction by p83 reduc-
tase produced (2S,3R)-2,3-dimethylbutanolide. The reduction of 4-alkyl-2-butenolides with these reductases was accompanied by
resolution of chiral centers affording (R)-4-alkylbutanolides.
Ó 2007 Elsevier Ltd. All rights reserved.
Optically active c-lactones, butanolides, are versatile
chiral building blocks for drugs, natural products, and
ferroelectric liquid crystals.1–5 Many studies on the bio-
logical production of chiral butanolides by enantiomeric
resolution of racemic butanolides with microorganisms
have been reported so far.6–9 Because the maximal yield
of chiral products obtained in the resolution process is
theoretically 50%, the asymmetric induction process
which gives the chiral products directly may be more
advantageous. Asymmetric reduction of ketones and
C–C double bonds catalyzed by reductases from micro-
organisms and plants has been one of the most impor-
tant asymmetric induction processes.10–16 There have
been a few studies on the yeast-mediated reduction of
2-butenolides to give chiral butanolides.17,18 However,
little attention has been paid to the enzymatic reduction
of 2-butenolides with reductases. Furthermore, there are
no reports on the asymmetric reduction of 2,3-disubsti-
tuted 2-butenolides to chiral 2,3-disubstituted butanol-
ides and on the reductive resolution of 4-substituted
2-butenolides to give chiral 4-substituted butanolides
by a reductase, which contracts the stereocenter remote
from the reaction center. This Letter reports, for the first
time, the biological production of optically active but-
anolides including chiral 2,3-disubstituted butanolides
and 4-substituted butanolides by the stereoselective
reduction of butenolides with two reductases from cul-
tured plant cells of Glycine max.
2-Butenolide (0.1 mmol) was administered to a 300 mL
conical flask containing the suspension cultured cells
of G. max (50 g) and 100 mL of MSK-II medium,19
and the cultures were incubated at 25 °C for one day
on a rotary shaker (120 rpm). 2-Butenolide was reduced
to butanolide in >99% yield, showing that the cultured
G. max cells have high potential for the reduction of
2-butenolides.
Next, the reductases were purified from the cultured sus-
pension cells as follows. A crude enzyme fraction ex-
tracted from G. max with 50 mM Na–phosphate buffer
(pH 7.0) was subjected onto diethylaminoethyl-Toyo-
pearl column chromatography, which gave crude reduc-
tase fractions. Further purification by chromatography
on a AF-Red Toyopearl column and then a Sephadex
G-200 column gave homogeneous reductases as judged
by SDS-PAGE: p51 reductase was a dimer composed
of two identical 25.5 kDa subunits and p83 reductase
was a dimer composed of two identical 41.5 kDa sub-
units. The enzymatic reduction of substituted 2-butenol-
ides 1–5 with the isolated p51 reductase was examined.
The reaction was carried out at 36 °C for 5 or 12 h in
*
Corresponding author. Tel.: +81 86 256 9473; fax: +81 86 256
8468; e-mail: hamada@das.ous.ac.jp
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.12.126

1346
K. Shimoda et al. / Tetrahedron Letters 48 (2007) 1345–1347
a mixture consisting of 10 mL of 50 mM Na–phosphate
buffer (pH 7.0), the reductase (ca. 20 lg), 2 mM sub-
strate, and 5 mM NADPH. The products were identified
by comparison of their GLC, GC–MS, 1H and 13C
NMR data20 with those of authentic samples,1,21 and
the conversions of the products were determined by
GLC analyses. The absolute configuration of the prod-
ucts was determined by comparing the retention times
of the resulting butanolide in the chiral GLC on Rt-
bDEX with those of authentic chiral samples.1,21 The
enantiomeric purities of the products were determined
based on the peak areas of the corresponding enantio-
mers in the GLC on Rt-bDEX. The p51 reductase
reduced enantioselectively the C@C double bond of 2-
methylated substrate 1 to give (R)-butanolide 6a with
excellent optical purity (>99% ee) and yield (>99%) after
12 h incubation (Fig. 1). On the other hand, 3-methyl-
ated substrate 2 was reduced to (R)-butanolide 7 (99%
ee, 85% yield), suggesting that the steric hindrance of
3-methyl group reduced the yield of the product. The
2,3-dimethyl-2-butenolide (3) was reduced to (2R,3R)-
2,3-dimethylbutanolide (8a) with high enantiomeric ex-
cess (99% ee (99% de)) in 82% yield. Next, 4-substituted
2-butenolides 4 and 5 were subjected to the enzymatic
reduction with p51 reductase. After 5 h incubation, race-
mic 4-propyl-2-butenolide (4) was reduced to (R)-4-pro-
pylbutanolide (9) with 53% ee in 43% yield. This
suggests that reductive resolution of 4 occurred in the
p51 reductase, which contracts the stereocenter remote
from the reaction center.22 When 4-butyl-2-butenolide
(5) was used as substrate, (R)-4-butylbutanolide (10)
was obtained in 67% ee and 40% yield (see Table 1).
Substrates 1–5 were next subjected to the enzymatic
reduction by p83 reductase. The p83 reductase reduced
1 to (S)-butanolide 6b with 98% ee in >99% yield, show-
ing that the enantioselectivity at 2-position of 2-butenol-
ide in the reduction with p83 reductase was opposite to
that with p51 reductase. The substrate 2 was reduced to
(R)-product 7 (99% ee, 99% yield). The p83 reductase
reduced 2,3-dimethyl substrate 3 to (2S,3R)-product 8b
with 96% ee (98% de) in 90% yield. 4-Alkyl-2-butenol-
ides 4 and 5 were reduced to (R)-products 9 and 10,
enantiomeric purities of which were ees of 80% and 87%.
Thus, the enantioselective reduction of 2-butenolides
has been accomplished by two reductases from cultured
plant cells of G. max. It is worth noting that the enantio-
selective formation of each enantiomer of 2-substituted
butanolide has been achieved by selective use of these
enzymes, which are different in enantioselectivity.
Earlier, it has been reported that horse liver alcohol
dehydrogenase-catalyzed oxidation of cis-2,3-di-
methylbutane-1,4-diol gave (2S,3R)-2,3-dimethylbutanol-
ide with 100% ee in 15% yield.21 This Letter reports, for
the first time, the enantioselective reduction of 2,
3-dimethyl-2-butenolide to give both (2R,3R)- and
(2S,3R)-2,3-dimethylbutanolide with high optical purity
and yield, and the reductive resolution of 4-substituted
2-butenolides to afford chiral 4-substituted butanolides
R
1
R
2
p51 reductase from G. max
O
R
3
O
6a: R1=Me, R2,R3=H; 7: R2=Me, R1,R3=H;
8a: R1,R2=Me, R3=H; 9: R1,R2=H, R3=n-Pr;
10: R1,R2=H, R3=n-Bu
R
1
R
2
O
R
3
R
1
R
2
O
p83 reductase from G. max
1: R1=Me, R2,R3=H; 2: R2=Me, R1,R3=H;
3: R1,R2=Me, R3=H; 4: R1,R2=H, R3=n-Pr;
5: R1,R2=H, R3=n-Bu
O
R
3
O
6b: R1=Me, R2,R3=H; 7: R2=Me, R1,R3=H;
8b: R1,R2=Me, R3=H; 9: R1,R2=H, R3=n-Pr;
10: R1,R2=H, R3=n-Bu
Figure 1. Stereoselective reduction of 2-butenolides 1–5 by the reductases from cultured cells of G. max.
Table 1. Stereoselective reduction of 2-butenolides to chiral butanolides with reductases from cultured plant cells of G. max
Reductase
Substrates
Products
Reaction time (h)
Conversiona (%)
ee (%)
p51 reductase
1
2
3
4
5
6a
7
12
12
12
5
>99
85
82
43
40
>99
99
99
53
67
8a
9
10
5
p83 reductase
1
2
3
4
5
6b
7
12
12
12
5
>99
99
90
47
38
98
99
96
80
87
8b
9
10
5
a The conversions were expressed as the percentage of the product in the reaction mixture on the basis of GLC.

K. Shimoda et al. / Tetrahedron Letters 48 (2007) 1345–1347
1347
20. In order to obtain the products adequate for spectroscopic
analyses, the reaction was performed in a similar condition
to the standard assay system except that the scale was 10–
20-fold enlarged. Extraction from the reaction mixture
with ether followed by purification using column chroma-
tography on silica gel with pentane–ethyl acetate (95:5,
v/v) gave the products. Spectral data for the products.
Product 6a (obtained in the reduction of 1 with p51
reductase): HREIMS m/z 100.0252 [M]+; 1H NMR
(400 MHz, CDCl3) d 1.27 (3H, d, J = 7.2, CH3), 1.93
(1H, m, CH), 2.43–2.63 (2H, m, CH2), 4.17–4.37 (2H, m,
CH2); 13C NMR (100 MHz, CDCl3): d 14.7 (CH3), 30.3
(CH), 33.7 (CH2), 65.9 (CH2), 179.7 (C@O). Product 6b
(obtained in the reduction of 1 with p83 reductase):
HREIMS m/z 100.0255 [M]+; 1H and 13C NMR data were
entirely consistent with those for 6a. Product 7 (obtained
in the reduction of 2 with p51 reductase): HREIMS m/z
by reductases, which contracts the stereocenter remote
from the reaction center. Further studies on the whole
sequences of the reductases using molecular biological
techniques to clarify the role of the reductases in G.
max and to apply the expressed enzymes for large scale
synthesis of chiral butanolides are currently in progress.
References and notes
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1
100.0251 [M]+; H NMR (400 MHz, CDCl3) d 1.15 (3H,
d, J = 6.8, CH3), 2.14–2.22 (2H, m, CH2), 2.60–2.69 (1H,
m, CH), 4.09–4.28 (2H, m, CH2); 13C NMR (100 MHz,
CDCl3): d 17.7 (CH3), 36.5 (CH), 37.2 (CH2), 69.9 (CH2),
180.1 (C@O). Product 8a (obtained in the reduction of 3
with p51 reductase): HREIMS m/z 114.0681 [M]+; 1H
NMR (400 MHz, CDCl3) d 1.16 (3H, d, J = 6.2 Hz, CH3),
1.24 (3H, d, J = 6.4 Hz, CH3), 1.91–2.27 (2H, m, 2CH),
3.73 (1H, t, J = 9.0 Hz, Ha–CH2), 4.36 (1H, t, J = 9.0, Hb–
CH2); 13C NMR (100 MHz, CDCl3): d 13.2 (CH3), 15.8
(CH3), 38.7 (CH), 41.6 (CH), 72.2 (CH2), 180.0 (C@O).
Product 8b (obtained in the reduction of 3 with p83
reductase): HREIMS m/z 114.0680 [M]+; 1H NMR
(400 MHz, CDCl3) d 0.95 (3H, d, J = 6.8 Hz, CH3), 1.10
(3H, d, J = 6.8 Hz, CH3), 2.52–2.65 (2H, m, 2CH), 3.87
(1H, d, J = 9.0 Hz, Ha–CH2), 4.24 (1H, dd, J = 9.0,
5.6 Hz, Hb–CH2); 13C NMR (100 MHz, CDCl3): d 9.8
(CH3), 13.2 (CH3), 33.7(CH), 38.1 (CH), 73.1 (CH2), 179.5
(C@O). Product 9 (obtained in the reduction of 4 with p83
reductase): HREIMS m/z 128.0837 [M]+; 1H NMR
(400 MHz, CDCl3) d 1.05 (3H, t, J = 6.4 Hz, CH3),
1.28–1.74 (4H, m, 2CH2), 2.11–2.72 (4H, m, 2CH2), 4.03
(1H, m, OCH); 13C NMR (100 MHz, CDCl3): d 14.0
(CH3), 27.5 (CH2), 33.6 (CH2), 35.5 (CH2), 36.9 (CH2),
87.2 (CH), 176.6 (C@O). Product 10 (obtained in the
reduction of 5 with p83 reductase): HREIMS m/z
1
142.0998 [M]+; H NMR (400 MHz, CDCl3) d 0.98 (3H,
t, J = 6.4 Hz, CH3), 1.31–1.73 (6H, m, 3CH2), 2.10–2.75
(4H, m, 2CH2), 4.01 (1H, m, OCH); 13C NMR (100 MHz,
CDCl3): d 13.9 (CH3), 22.5 (CH2), 27.9 (CH2), 33.5 (CH2),
36.3 (CH2), 37.1 (CH2), 87.0 (CH), 176.5 (C@O).
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