Asymmetric reduction of α,β-unsaturated carbonyl compounds with reductases from Nicotiana tabacum

Article abstract of DOI:10.1016/j.tetasy.2004.07.011

Two reductases, p44 and p90, isolated from Nicotiana tabacum, catalyzed the asymmetric reduction of α,β-unsaturated carbonyl compounds. The p44 reductase reduced α-alkylated enones to (R)-ketones, while the reduction using the p90 reductase gave the (S)-k

Full text of DOI:10.1016/j.tetasy.2004.07.011

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2443–2446
Asymmetric reduction of a,b-unsaturated carbonyl compounds
with reductases from Nicotiana tabacum
a
b
Kei Shimoda,a, Naoji Kubota and Hiroki Hamada
*
aDepartment of Pharmacology and Therapeutics, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
bDepartment of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
Received 1 July 2004; accepted 9 July 2004
Abstract—Two reductases, p44 and p90, isolated from Nicotiana tabacum, catalyzed the asymmetric reduction of a,b-unsaturated
carbonyl compounds. The p44 reductase reduced a-alkylated enones to (R)-ketones, while the reduction using the p90 reductase
gave the (S)-ketones. The p90 reductase reduced b-alkylated enones with excellent enantioselectivities to afford the (S)-ketones.
On the other hand, the hydrogenation of the C–C double bond of 2-methylmaleimide with both enzymes gave (R)-2-
methylsuccinimide.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Herein we report the asymmetric hydrogenation of the
C–C double bond of enones and 2-methylmaleimide
with the reductases isolated from N. tabacum. This
approach gives a new biocatalytic method for the
enzymatic production of enantiomerically pure b-
substituted ketones and 2-methylsuccinimide by the
reductases.
Optically active ketone derivatives are potentially useful
chiral building blocks for drugs and insecticides.1,2
Asymmetric reduction of enones with biocatalysts is
very attractive for the practical preparation of a-chiral
ketones because of the excellent enantioselectivities of
the reductases participating in the hydrogenation of
C–C double bonds.3–12 However, little attention has
been paid to the production of b-chiral ketones with
these reductases, because most reductases reduce the
C–C double bonds when the substrates have a hydrogen
atom at the b-position to the carbonyl group.13–21
2. Results and discussion
Two reductases, p44 and p90, were isolated from plant
cell cultures of N. tabacum in three steps involving col-
umn chromatographies. Reductions of enones 1–6 and
2-methylmaleimide 7 (see Fig. 1) with the p44 reductase
were performed at pH7.7 where the enzyme activity is
optimal. Enones 1 and 2, which have an alkyl substitu-
ent at the a-position to the carbonyl group, were re-
duced to give the corresponding (R)-ketones with very
high enantiomeric purities (ees of 100% and >99%), as
shown in Table 1. On the contrary, no reduction
occurred in the cases of b-alkylated substrates 3–6.
When N-phenyl-2-methylmaleimide 7, which was ex-
pected to be reacted as a a,b-unsaturated carbonyl com-
pound with either a- or b-methyl substituent, was used
as the substrate, the resulting N-phenyl-2-methylsuccin-
imide 14a had an (R)-configuration. The enantiomeric
excess of product 14a was 100% based on the peak anal-
ysis of methyl proton signal in the 1H NMR spectrum of
14a with Eu(hfc)3. This shows that the p44 reductase
Recently, we isolated two reductases from Nicotiana
tabacum; a 44kDa reductase (p44), which is capable of
reducing enones with a hydrogen atom at the b-position
to the carbonyl groupby the anti-addition of hydrogen
atoms, and a 90kDa reductase (p90), which is able to re-
duce b-methylated enones, catalyzing the syn-addition
of hydrogen atoms.10–12
On the other hand, it has been reported that the whole
cells of N. tabacum have the ability to catalyze the asym-
metric hydrogenation of the C–C double bond of
2-methylmaleimide to give (R)-2-methylsuccinimide
with high enantioselectivity.20
*
Corresponding author. 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.2004.07.011

2444
K. Shimoda et al. / Tetrahedron: Asymmetry 15 (2004) 2443–2446
(Table 2). Interestingly, b-alkylated enones 3–6 were re-
O
O
duced to give the corresponding (S)-ketones after 12h
incubation. The enantiomeric purities of the resulting ke-
tones 10b, 11b, 12b and 13b were ees of 100% and >99%,
suggesting that the asymmetric reduction of b-alkylated
enones with the p90 reductase is useful for the practical
preparation of b-substituted (S)-ketones in their enantio-
merically pure forms. The reduction of 7 gave 14a with
89% ee, indicating that 7 was accepted preferentially as
a b-alkylated substrate by the p90 reductase. These re-
sults demonstrate that the p90 reductase from N. taba-
cum is able to catalyze the asymmetric reduction of
both a- and b-alkylated enones to give (S)-ketones and
is capable of reducing 2-methylmaleimide to give (R)-2-
methylsuccinimide.
O
O
R1
R2
N
R
1: R1=Me, R2=H
2: R1=Et, R2=H
3: R1=H, R2=Me
4: R1=H, R2=Et
5: R=Me
6: R=Et
7
R2
N
R1
O
O
R1
O
O
R2
R3
R1
R2
R4
8a: R1,R3,R4=H, R2=Me 12a: R1=H, R2=Me
8b: R1=Me, R2,R3,R4=H 12b: R1=Me, R2=H
14a: R1=H, R2=Me
14b: R1=Me, R2=H
9a: R1,R3,R4=H, R2=Et
9b: R1=Et, R2,R3,R4=H
10a: R1,R2,R3=H, R4=Me
10b: R1,R2,R4=H, R3=Me
11a: R1,R2,R3=H, R4=Et
11b: R1,R2,R4=H, R3=Et
13a: R1=H, R2=Et
13b: R1=Et, R2=H
3. Conclusions
The asymmetric hydrogenation of the C–C double bond
of enones and 2-methylmaleimide has been accom-
plished by two reductases from N. tabacum. It is worth
noting that the enantioselective formation of each enan-
tiomer of the a-substituted ketones and b-substituted
(S)-ketones has been achieved by selective use of these
enzymes, which are different in substrate specificity
and enantioselectivity, and that the new asymmetric
hydrogenation of 2-methylmaleimide by the reductase
from N. tabacum produced (R)-2-methylsuccinimide in
an enantiomerically pure form.
Figure 1. Structures of a,b-unsaturated carbonyl compounds and
conversion products.
was able to recognize the 2-methylmaleimide derivative
as a substrate and that 7 was accepted preferentially as
a a-alkylated substrate by the p44 reductase. The results
obtained here demonstrate that the p44 reductase from
N. tabacum has the ability to reduce a-alkylated enones
to (R)-ketones and catalyze the enantioface-discriminat-
ing hydrogenation of 2-methylmaleimide to give (R)-2-
methylsuccinimide.
4. Experimental
4.1. General
Substrates 1–7 were next subjected to the reduction with
the p90 reductase at pH7.4 (optimum pH of the enzyme).
Enones 1 and 2 were reduced to give the corresponding
(S)-ketones, and the hydrogenation at the a-position
showed high enantioselectivities (ees of >99% and 98%)
Chemicals such as enones, N-phenyl-2-methylmale-
imide, (RS)-ketones and optically active ketones used
Table 1. Enantioselective reduction of a,b-unsaturated carbonyl compounds with the p44 reductase from N. tabacum
Substrates
Products
Reaction time (h)
Conversion (%)a
Ee (%)
Configuration
1
2
3
4
5
6
7
8a
9a
––
––
––
––
14
24
24
24
24
24
24
81
49
100
>99
––
R
R
––
––
––
––
––
––
R
––
––
––
––
––
100
a
12
>99
a The conversions are expressed as the percentage of the product in the reaction mixture on the basis of GLC.
Table 2. Enantioselective reduction of a,b-unsaturated carbonyl compounds with the p90 reductase from N. tabacum
Substrates
Products
Reaction time (h)
Conversion (%)a
Ee (%)
Configuration
1
8b
9b
24
24
12
12
12
12
86
44
>99
98
S
S
S
S
S
S
R
2
3
411b
10b
>99
95
100
100
100
>99
89
5
6
7
12b
13b
14
99
88
a
12
>99
a The conversions are expressed as the percentage of the product in the reaction mixture on the basis of GLC.

K. Shimoda et al. / Tetrahedron: Asymmetry 15 (2004) 2443–2446
2445
as substrates or authentic samples were purchased from
Aldrich Chemical Co. The suspension cells of N. taba-
cum were cultured in 500mL conical flasks containing
300mL of Murashige and SkoogÕs (MS) medium supple-
mented with 3% sucrose and 10mM 2,4-D under illumi-
nation (4000 lux) as reported previously.22 GLC
analyses were performed with FID and a capillary col-
umn of Rt-bDEX (0.25mm · 30m) using N2 as a carrier
gas (injector, 180ꢁC; detector, 180ꢁC; make up,
50mLminꢀ1). Optical rotation data were obtained on
a Jasco DIP-360 using a 4mL cuvette.
tones 8 and 9 in the GLC at the oven temperature of
100ꢁC were as follows: (S)- and (R)-8, 12.0 and
12.7min; (S)- and (R)-9, 15.8 and 16.6min.
The products obtained by the reduction of 3–6 with the
p90 reductase were identified as the corresponding ke-
1
tones by direct comparison of GLC, GC–MS and H
NMR with those of the authentic samples. The absolute
configuration of the resulting ketones were determined
by comparing the chiral GLC retention times with those
of authentic chiral ketones and the specific rotation of
the products {10b: CD [h]294 = ꢀ5653 (c 0.55, MeOH);
25
D
25
D
4.2. Enzyme preparation
½aŠ ¼ ꢀ147:0 (c 0.4, MeOH) {lit.24 ½aŠ ¼ þ154:8 for
(R)-enantiomer}; 11b: CD [h]294 = ꢀ6257 (c 0.36,
25
25
MeOH); ½aŠ ¼ ꢀ153:5 (c 0.3, MeOH); 12b: CD
Homogenates of cultured cells of N. tabacum in 100mM
Na-phosphate buffer (pH7.5) were centrifuged at
10,000g for 30min to give a cell-free extract, which
was then treated with ammonium sulfate (60–80% satd)
to give a crude enzyme preparation. Diethylaminoethyl-
Toyopearl column chromatography of the crude enzyme
preparation gave a good separation of the two different
reductases. Further purification by chromatography on
a hydroxylapatite column and then a Red-Toyopearl
column gave homogeneous reductases as judged by
SDS-PAGE: p44, dimeric form composed of two identi-
cal 22kDa subunits; p90, dimeric form composed of two
identical 45kDa subunits.10–12
D
[h]289 = ꢀ1877 (c 0.59, MeOH); ½aŠ ¼ ꢀ13:2 (c 0.4,
25
D
20
D
MeOH) {lit.25 ½aŠ ¼ þ12:6 for (R)-enantiomer}; 13b:
CD [h]288 = ꢀ2270 (c 0.50, MeOH); ½aŠ ¼ ꢀ19:9 (c
D
0.2, MeOH)}. The enantiomeric purities of the obtained
ketones were determined by the peak area of the corre-
sponding enantiomers in the GLC analyses of the prod-
ucts on Rt-bDEX at the oven temperature of 80ꢁC.
Retention times for ketones 10–13 in the GLC were as
follows: (R)- and (S)-10, 15.3 and 15.9min; (R)- and
(S)-11, 25.2 and 26.2min; (R)- and (S)-12, 25.7 and
26.6min; (R)- and (S)-13, 33.8 and 34.8min.
The products converted from 7 by the p44 and p90
reductases were identified as (R)-N-phenyl-2-methylsuc-
cinimide 14a {14a converted by the p44 reductase:
4.3. Reduction condition
25
22
Substrates 1–7 (10mg each) were administered to a mix-
ture of the p44 reductase (ca. 30lg) and 120mg of
NADH in 5mL of 50mM Na-phosphate buffer
(pH7.7) and incubated at 37ꢁC for 12 or 24h. The yields
of the products were determined by GLC analyses.
½aŠ ¼ þ7:3 (c 0.5, CHCl3) {lit.26 ½aŠ ¼ þ8}; 14a re-
D
D
25
D
duced by the p90 reductase: ½aŠ ¼ þ6:8 (c
0.4, CHCl3)}, respectively, by means of 1H and 13C
NMR. 1H NMR (CDCl3, 500MHz): d 1.46 (3H, d,
J = 7.1Hz, 2-Me), 3.04 (1H, ddq, J = 9.3, 4.6 and
7.3Hz, 2-H), 2.51 (1H, dd, J = 17.7 and 4.5Hz, 3-Ha),
3.10 (1H, dd, J = 17.6 and 9.3Hz, 3-Hb), 7.29 (2H, d,
J = 8.3Hz, o-H), 7.39 (1H, t, J = 7.4Hz, p-H), 7.47
(2H, t, J = 7.7Hz, m-H); 13C NMR (CDCl3,
125MHz): d 16.9 (Me), 34.9 (CH), 36.7 (CH2), 126.4
(o-C in Ph), 128.6 (p-C in Ph), 129.1 (m-C in Ph),
132.0 (N–C in Ph), 175.4 (C@O), 179.5 (C@O). The
intensities of the pair of methyl proton signals in the
1H NMR spectra of the reduction products in the pres-
ence of Eu(hfc)3 were used for the determination of the
enantiomeric excess of the resulting 14a as reported pre-
Reduction by the p90 reductase was performed under
similar conditions except that NADPH was used as
the coenzyme and that the pH of the reaction mixture
was 7.4.
In order to obtain the products adequate enough for
optical rotation analysis, the reaction was performed
under similar conditions to the standard assay system
except that the scale was 20–30 fold enlarged. Extraction
from the reaction mixture with ether followed by purifi-
cation using column chromatography on silica gel with
pentane–ethyl acetate (95:5, v/v) gave the products.
1
viously.20 Methyl proton signals of 14 in the H NMR
spectrum were revealed at d 2.56 (d, J = 7.0Hz, for
14a) and 2.64 (d, J = 7.0Hz, for 14b) in a CDCl3 solu-
tion of the product and Eu(hfc)3 (1:1 mol ratio). The
enantiomeric purity of 14a was confirmed by the peak
area of the corresponding enantiomers in the chiral
GLC analyses of the products on Rt-bDEX at the oven
temperature of 80ꢁC. Retention times for 14 in the GLC
were as follows: (R)- and (S)-14, 40.2 and 40.9min.
4.4. Product identification
The products obtained by the reduction of 1 and 2 with
the p44 and p90 reductases were identified as the corre-
sponding ketones by direct comparison of GLC, GC–
1
MS and H NMR with those of the authentic samples.
The absolute configuration and enantiomeric excesses
of the resulting ketones were determined by the specific
25
D
rotation of the products {8a: ½aŠ ¼ ꢀ112:4 (c 0.51,
25
Acknowledgements
25
D
25
D
CHCl3) {lit.23 ½aŠ ¼ ꢀ110:5}; 8b: ½aŠ ¼ þ104:3 (c
25
0.53, CHCl3); 9a: ½aŠ ¼ ꢀ125:7 (c 0.45, CHCl3); 9b:
This work was supported in part by a Grant-in-Aid for
Scientific Research (No. 16790014) from the Ministry of
Education, Culture, Sports, Science and Technology,
Japan.
D
½aŠ ¼ þ121:1 (c 0.58, CHCl3)} and the peak area of
D
the corresponding enantiomers in the GLC analyses of
the products on Rt-bDEX. Retention times for the ke-

2446
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