Asymmetric reduction of 2-substituted 2-butenolides with reductase from Marchantia polymorpha

Article abstract of DOI:10.1016/j.tetasy.2004.11.006

A p68 reductase participating in the asymmetric reduction of the C-C double bond of 2-substituted 2-butenolides was isolated from Marchantia polymorpha. The enzyme reduced 2-substituted 2-butenolides to give (R)-butanolides, and the reduction of citraconic anhydride afforded (R)-methylsuccinic anhydride.

Full text of DOI:10.1016/j.tetasy.2004.11.006

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3827–3829
Asymmetric reduction of 2-substituted 2-butenolides with
reductase from Marchantia polymorpha
Kei Shimoda* and Naoji Kubota
Department of Pharmacology and Therapeutics, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
Received 11 October 2004; accepted 1 November 2004
Abstract—A p68 reductase participatingin the asymmetric reduction of the C–C double bond of 2-substituted 2-butenolides was
isolated from Marchantia polymorpha. The enzyme reduced 2-substituted 2-butenolides to give (R)-butanolides, and the reduction
of citraconic anhydride afforded (R)-methylsuccinic anhydride.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Optically active a-substituted c-lactones such as 2-
hydroxybutanolide (HBL) are versatile chiral building
blocks for drugs, natural products, and ferroelectric
liquid crystals.1–3 Lactonohydrolase from Fusarium
oxosporum has already been used for the industrial
production of chiral HBLs; enantiomeric resolution of
racemic HBL catalyzed by this enzyme gives (S)-HBL
and (R)-2,4-dihydroxybutanoic acid with high enantio-
meric purities.4 Because the maximum yield of chiral
products obtained in the resolution process is theoreti-
cally 50%, the asymmetric induction process which gives
the chiral products directly may be more advanta-
geous.5,6 A few studies on the yeast-mediated reduction
of 3-substituted 2-butenolides to 3-substituted (S)-but-
anolides, that is, b-chiral c-lactones, have been re-
ported.6 However, little attention has been paid to the
enzymatic production of a-chiral c-lactones such as
optically active HBLs by the reductases participating
in the hydrogenation of C–C double bonds. In the
course of developinga new asymmetric reduction, we
investigated the enantiofacially selective reduction of
2-substituted 2-butenolides and citraconic anhydride
by the reductase isolated from plant cell cultures of
the liverwort Marchantia polymorpha.
morpha7 at 25ꢁC under illumination (4000lux), 1 was
reduced to butanolide 6 with >99% conversion. To
examine the enzymatic reduction of 2-butenolides, the
reductase was purified from M. polymorpha. A crude
enzyme fraction extracted from M. polymorpha with
50mM Na-phosphate buffer (pH7.0) was subjected onto
diethylaminoethyl-Toyopearl column chromatography,
which gave a crude reductase fraction that reduces 1
to 6. Further purification by chromatography on a
Blue-Toyopearl column and then a Sephadex G-200
column gave homogeneous reductase as judged by
SDS-PAGE: the isolated p68 reductase was a dimer
composed of two identical 34kDa subunits.
Next, the enzymatic reduction of 2-butenolides with the
isolated p68 reductase was examined. The reaction was
carried out at 35ꢁC for 4 or 8h in a mixture consisting
of 10mL of 25mM Na-phosphate buffer (pH7.0), the
reductase (ca. 15lg), 2mM substrate, and 5mM
NADH. The products were identified by comparison
of their TLC, GLC, and GC–MS data with those of
authentic samples, and the conversions of the products
were determined by GLC analyses. The absolute config-
uration of the products was confirmed by comparingthe
The reduction system with cultured cells of M. polymor-
pha was preliminarily tested for its ability to reduce the
C–C double bond of 2-butenolide 1. After 1day incuba-
tion of 1 (30mg) with 50g of suspension cells of M. poly-
*
Correspondingauthor. Tel.: +81 97 586 5606; fax: +81 97 586
5619; e-mail: shimoda@med.oita-u.ac.jp
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.006

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K. Shimoda, N. Kubota / Tetrahedron: Asymmetry 15 (2004) 3827–3829
retention times of the resultingbutanolide or the corre-
spondingacetyl ester in the GLC with chiral stationary
phase with those of authentic optically active samples
alongwith determination of specific rotation of the
products.8,9 The enantiomeric purities of the products
were determined based on the peak areas of the corre-
spondingenantiomers or acetates in the GLC on Rt- b
DEX.8 As a result, it was found that the C–C double
bond of 1 was reduced to give 6 with >99% conversion
by the p68 reductase after 4h (Table 1), showingthat the
p68 reductase has potential for the reduction of 1. To
examine the enantioselectivity of the reduction at the
2-position of 2-butenolides by the p68 reductase, 2 and
3 were used as substrates. After 8h incubation, 2 was re-
duced to (R)-HBL 7 in >99% conversion and the chiral
GLC analysis of acetates of the product 7 showed that
the hydrogenation at the 2-position occurred with excel-
lent enantioselectivity (>99% ee) (Fig. 1b). This suggests
that the asymmetric reduction with the p68 reductase is
very useful for the practical preparation of (R)-HBL in
enantiomerically pure form. The reduction of 3 likewise
gave 8 of >99% ee in 99% conversion. On the other
hand, no reduction occurred in the case of 3-substituted
2-butenolide 4, indicatingthat the p68 reductase isolated
from M. polymorpha is a novel enzyme apparently dif-
ferent from the reductase in yeast with respect to the
substrate specificity. A challenging substrate for the
asymmetric reduction, citraconic anhydride 5, was re-
duced to give enantiomerically pure (R)-methylsuccinic
anhydride 9 (100% ee)10,11 which is a potentially useful
chiral buildingblock for organic and polymer synthe-
ses.11,12 These results demonstrate that the p68 reductase
from M. polymorpha is able to catalyze the enantioface-
discriminatinghydrogenation of the C–C double bond
of 2-substituted 2-butenolides to give (R)-butanolides
and capable of reducingcitraconic anhydride to igve
(R)-methylsuccinic anhydride.
Thus, a novel plant reductase which catalyzes the hydro-
genation of 2-substituted 2-butenolides has been isolated
from M. polymorpha. It should be emphasized that the
new asymmetric reduction of 2-substituted 2-buteno-
lides and citraconic anhydride usingthe reductase from
M. polymorpha as a biocatalyst is one of the most useful
methods for chiral generation; this method is useful for
practical preparation of 2-substituted (R)-butanolides,
such as (R)-HBL, and (R)-methylsuccinic anhydride in
enantiomerically pure forms.
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Table 1. Reduction of 2-butenolides and citraconic anhydride with the
p68 reductase from M. polymorpha
Sub-
strates
Products Reaction Conversion Ee
(%)a
Configuration
time (h)
(%)
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—
—
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7
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—
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a Percentage of the products in the reaction mixture on the basis of
GLC analyses.
Figure 1. Chiral GLC analyses of acetates of (a) racemic 2-hydroxybutanolide and (b) the product 7 obtained in the reduction of 2 for the
determination of enantioselectivity of enzymatic reduction.

K. Shimoda, N. Kubota / Tetrahedron: Asymmetry 15 (2004) 3827–3829
3829
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25
D
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25
D
25
D
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20
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25
rotation of 9; ½aŠ ¼ þ34:8 (c 0.28, MeOH) {lit.11
D
25
D
½aŠ ¼ ꢀ36:5 for (S)-enantiomer}. The enantiomeric
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