Asymmetric synthesis of a nitroalkane by the use of novel nitroalkene reductases from baker's yeast

Article abstract of DOI:10.1016/S0040-4039(01)00464-6

Two kinds of novel nitroalkene reductases were isolated from baker's yeast. Reduction of a trisubstituted nitroalkene by these reductases afforded the corresponding nitroalkane with excellent enantioselectivity, moderate diastereoselectivity, and in good yield.

Full text of DOI:10.1016/S0040-4039(01)00464-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 3367–3368
Asymmetric synthesis of a nitroalkane by the use of novel
nitroalkene reductases from baker’s yeast
Yasushi Kawai,* Yoshikazu Inaba, Motoko Hayashi and Norihiro Tokitoh
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
Received 19 February 2001; revised 15 March 2001; accepted 16 March 2001
Abstract—Two kinds of novel nitroalkene reductases were isolated from baker’s yeast. Reduction of a trisubstituted nitroalkene
by these reductases afforded the corresponding nitroalkane with excellent enantioselectivity, moderate diastereoselectivity, and in
good yield. © 2001 Elsevier Science Ltd. All rights reserved.
Chiral nitro compounds are important synthetic inter-
mediates, which can be easily converted into chiral
amines, and chiral amines are useful compounds in
organic synthesis. Asymmetric reduction of nitroalke-
nes with microbes affords chiral saturated nitro com-
advantage of the use of an isolated enzyme compared
with the whole cell reaction, in which the enzyme
catalyzes a reaction without any side reactions and
performs a reaction in excellent stereoselectivity.8–11
However, no investigation on the isolation of a
nitroalkene reductase has been reported. Herein, we
report the first purification and characterization of
novel nitroalkene reductases from the cells of baker’s
yeast, and the asymmetric reduction of a nitroalkene
with these reductases.
pounds.
The
baker’s
yeast
reduction
of
b,b-disubstituted nitroalkenes is known to afford the
(R)-nitroalkanes enantioselectively,1,2 while a,b-disub-
stituted nitroalkenes are transformed into almost
racemic saturated nitroalkanes under similar reduction
conditions.3–7 Recently, we have reported that the
reduction of trisubstituted nitroalkenes affords satu-
rated nitroalkanes with excellent enantioselectivities,
moderate diastereoselectivities and in good yield.7 On
the other hand, we have already demonstrated the
The reductases were isolated from a cell-free extract of
baker’s yeast (Oriental Yeast, Japan) by column chro-
matography such as anion exchange, hydrophobic and
gel-filtration, as the same method reported previ-
Table 1. Enzymatic reduction of (Z)-3-phenyl-2-nitro-2-butene (1) with YNAR-I and IIa
Enzyme
Coenzyme
Relative activity
d.e.b (%)
e.e.c (%)
e.e.d (%)
YNAR-I
YNAR-II
NADPH
NADH
NADPH
NADH
1.00
0.60
1.00
0.54
31
29
35
34
\98
\98
\98
\98
97
97
97
97
a Conditions: acetate buffer, 100 mM, pH 5.0.
b Excess of 2 over 3.
c e.e. of 2.
d e.e. of 3.
Keywords: enzymes and enzyme reactions; asymmetric reactions; nitro compounds.
* Corresponding author. Fax: +81-774-38-3209; e-mail: kawai@scl.kyoto-u.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00464-6

3368
Y. Kawai et al. / Tetrahedron Letters 42 (2001) 3367–3368
Table 2. Enzymatic reduction in D2Oa
these enzymes affords the product in excellent enan-
tioselectivity and modest diastereoselectivity, indicating
that stereoselective addition of hydride from NADPH
occurs at the b-position of the nitro group, followed by
less stereoselective protonation at the a-position.
The diastereoisomers thus obtained were readily separa-
ble from each other by column chromatography on
silica gel, allowing enantiomerically pure isomers of 2
and 3 to be obtained without difficulty. Nitroalkene
reductases, the new class of enzymes reported here, are
potentially useful as catalysts for obtaining chiral nitro
compounds in excellent stereoselectivity.
Deuterium content (%)
Enzyme
C-2
C-3
YNAR-I
YNAR-II
52
59
0
0
a The reaction was in more than 99% D2O.
Acknowledgements
ously.8,12,13 All purification steps were run at 4°C and
the enzymes were stored at −20°C. Activity was mea-
sured by employing (Z)-3-phenyl-2-nitro-2-butene (1)
as a substrate. Two enzymes, yeast nitroalkene reduc-
tase-I and -II (YNAR-I and -II), were isolated and the
stereoselectivities of the reduction are summarized in
Table 1. Both enzymes preferentially utilize NADPH as
the coenzyme, but NADH also serves as the coenzyme
to a certain extent. Although diastereoselectivities in the
reduction of (Z)-3-phenyl-2-nitro-2-butene with these
enzymes were moderate, enantioselectivities of these
enzymes were satisfactory (more than 98% e.e.), giving
(2R,3S)-3-phenyl-2-nitrobutane (2) in good yield. Com-
pared with the whole cell reaction, the diastereoselectiv-
ities were slightly improved by the use of an isolated
enzyme.7
This work was partially supported by a Grant-in-Aid
for Scientific Research No. 12680634 from the Ministry
of Education, Culture, Sports, Science and Technology
of Japan.
References
1. Ohta, H.; Ozaki, K.; Tsuchihashi, G. Chem. Lett. 1987,
191–192.
2. Ohta, H.; Kobayashi, N.; Ozaki, K. J. Org. Chem. 1989,
54, 1802–1804.
3. Mori, A.; Ishiyama, I.; Akita, H.; Suzuki, K.; Mitsuoka,
T.; Oishi, T. Chem. Pharm. Bull. 1990, 38, 3449–3451.
4. Takeshita, M.; Yoshida, S.; Kohno, Y. Heterocycles
1994, 37, 553–562.
5. Bak, R. R.; McAnda, A. F.; Smallridge, A. J.; Trewhella,
M. A. Aust. J. Chem. 1996, 49, 1257–1260.
6. McAnda, A. F.; Roberts, K. D.; Smallridge, A. J.; Ten,
A.; Trewhella, M. A. J. Chem. Soc., Perkin Trans. 1 1998,
501–504.
7. Kawai, Y.; Inaba, Y.; Tokitoh, N. Tetrahedron: Asymme-
try 2001, 12, 309–318.
8. Kawai, Y.; Hayashi, M.; Inaba, Y.; Saitou, K.; Ohno, A.
Tetrahedron Lett. 1998, 39, 5225–5228.
9. Kawai, Y.; Hida, K.; Dao, D. H.; Ohno, A. Tetrahedron
Lett. 1998, 39, 9219–9222.
10. Kawai, Y.; Hida, K.; Tsujimoto, M.; Kondo, S.; Kitano,
K.; Nakamura, K.; Ohno, A. Bull. Chem. Soc. Jpn. 1999,
72, 99–102.
11. Kawai, Y.; Hida, K.; Ohno, A. Bioorg. Chem. 1999, 27,
3–19.
12. Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. J.
Org. Chem. 1991, 56, 4778–4783.
13. Nakamura, K.; Kondo, S.; Kawai, Y.; Nakajima, N.;
McAnda et al. reported that the mechanism of the yeast
reduction of nitrostyrenes using deuterium labeling
experiments.6 They concluded that the yeast mediated
reduction of the nitroalkenes proceeds in two stages: a
reversible non-stereoselective protonation at the a-car-
bon followed by a stereoselective addition of a hydride
from a coenzyme at the b-carbon. Since they studied
the mechanism of the reduction using a whole cell
system, a detailed mechanism of the nitroalkene reduc-
tase remained unclear. To elucidate the hydrogenation
process with these enzymes, nitroalkene 1 was subjected
to the enzymatic reduction in D2O. The incorporation
1
of deuterium in the product was determined by H and
2H NMR spectrum analyses. The results summarized in
Table 2 reveal that the product 2 contains a deuterium
at the a-position of the nitro group (C-2). It has thus
been elucidated that the hydrogen incorporated into the
b-position of the nitro group by the enzymatic reduc-
tion is derived from NADPH and that into the a-posi-
tion comes from water or a certain amino acid residue
in the active site of the enzyme. The reduction with
Ohno, A. Biosci. Biotech. Biochem. 1994, 58, 2236–2240.
.