Asymmetric hydrogenation of the C-C double bond of enones with the reductases from Nicotiana tabacum

Article abstract of DOI:10.1246/cl.2000.850

Three enone reductases, p44, p74, and p90, from the cultured cells of Nicotiana tabacum catalyzed the asymmetric hydrogenation of the C-C double bond of enones. Reduction of 2-alkyl-2-cyclohexen-1-ones with the p44 reductase gave highly optically active (

Full text of DOI:10.1246/cl.2000.850

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50
Chemistry Letters 2000
Asymmetric Hydrogenation of the C-C Double Bond of Enones with the Reductases from
Nicotiana tabacum
Toshifumi Hirata,* Kei Shimoda, and Takayuki Gondai
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University,
1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526
(Received January 27, 2000; CL-000089)
Three enone reductases, p44, p74, and p90, from the cul-
tured cells of Nicotiana tabacum catalyzed the asymmetric
hydrogenation of the C-C double bond of enones. Reduction of
-alkyl-2-cyclohexen-1-ones with the p44 reductase gave high-
ly optically active (R)-2-alkylcyclohexanones, whereas reaction
with the p90 reductase gave (S)-2-alkylcyclohexanones. On the
other hand, reduction of 2-alkylidenecyclohexanones with the
p74 reductase gave (S)-2-alkylcyclohexanones.
C-C double bond were converted to the corresponding saturated
ketones, but 2-alkyl-2-cyclohexen-1-ones (3–5), which have an
endocyclic C-C double bond, remained unchanged, as shown in
Table 1. In the reduction of 2-alkylidenecyclohexanones (1 and
2), hydrogen atoms came from the re-face of their C-C double
bond to give (S)-2-methylcyclohexanone (6a) (97% ee) and (S)-
2
1
4
2-propylcyclohexanone (8a) (75% ee), respectively. On the
other hand, when the p90 and p44 reductases were used, enones
3–5 were converted to the corresponding saturated ketones, but
no reduction occurred for enones 1 and 2, as shown in Table 1.
In the reduction with the p90 reductase, a hydrogen atom was
stereoselectively added to C-2 from the re–re face of the C-C
double bond to give highly optically active (S)-2-alkylcyclo-
Previous studies on the asymmetric hydrogenation of the
C-C double bond of enones by plant cell cultures revealed that
there are two different types of enone reductase with respect to
substrate specificity; one is responsible for the reduction of the
endocyclic double bond and the other catalyzes the reduction of
1
7
hexanones, 6a–8a. On the contrary, reduction of enones 3–5
1
–3
the exocyclic double bond.
We recently isolated several
enone reductases from the cultured cells of N. tabacum partici-
pating in the reduction of the endocyclic C-C double bond of
enones. We reported that a 44 kDa enone reductase (p44) was
able to reduce only the C-C double bond bearing a hydrogen
4
atom at the β position to the carbonyl group, and that a 90 kDa
enone reductase (p90) was capable of reducing a broad range of
enones.5 We have now isolated a novel 74 kDa enone reduc-
tase (p74) from N. tabacum participating in the reduction of the
exocyclic C-C double bond of enones. In this paper, we
describe the enantioselective formation of chiral ketones by
reduction of the C-C double bond of enones with three enone
reductases from N. tabacum as biocatalysts.
,6
Three reductases, p44, p74 and p90, were isolated from the
7
cultured cells of N. tabacum by chromatographic separation.
The stereospecificity in the asymmetric reduction of enones
9
with these reductases was examined by using enones 1–5 as
substrates.10 In the case of the reduction with the p74 reduc-
tase, 2-alkylidenecyclohexanones (1 and 2) having an exocyclic
Copyright © 2000 The Chemical Society of Japan

Chemistry Letters 2000
851
with the p44 reductase gave highly optically pure (R)-2-alkyl-
µmol), Triton X-100 (0.1%), and enzyme preparation (5
µg) in 50 mM Tris-HCl buffer (pH 8.0) containing 1.0 mM
2-mercaptoethanol and 1.0 mM dithiothreitol was incubat-
1
8
cyclohexanones, 6b–8b. These results indicate that the stere-
ospecificity of the hydrogenation of the C-C double bond was
opposite between the p90 and p44 reductases.
o
ed at 35 C for 8 h. After incubation, the reaction mixture
Thus, it was demonstrated that the enone reductases from
N. tabacum were able to reduce enantiotropically the C-C dou-
ble bond of enones to afford optically active 2-alkylated
ketones. It is worth noting that each enantiomer of 2-alkylated
ketones can be synthesized by selective use of these enone
reductases from N. tabacum.
was subjected to GLC and GC-MS analyses, and then the
products were purified by column chromatography on sili-
ca gel. The absolute configurations and enantiomeric puri-
ties of the products were determined by circular dichroism
(CD) spectra and the peak area of the corresponding enan-
tiomers in the GLC analyses on CP cyclodextrin β 236M-
19 column.
The authors thank the Instrument Center for Chemical
Analysis of Hiroshima University for the measurements of H
NMR, GC–MS and CD spectra.
10 2-Alkylidenecyclohexanones (1 and 2) were prepared from
the α-alkylidene acetals of cyclohexanone according to the
1
1
1,12
+
1
reported procedure.
1: MS (EI) m/z 110 (M ); H
NMR (500 MHz; CDCl ) δ 6.01 (1H, t, J = 1.9 Hz,
3
References and Notes
>C=CH ), 6.12 (1H, t, J = 1.9 Hz, >C=CH ); 2: MS (EI)
2
2
+
1
1
T. Hirata, H. Hamada, T. Aoki, and T. Suga,
m/z 138 (M ), H NMR (CDCl ) δ 1.05 (3H, t, J = 7.6 Hz,
Me), 2.11 (2H, q, J = 7.6 Hz, –CH –Me), 6.61 (1H, tt, J =
3
Phytochemistry, 21, 2209 (1982).
2
2
3
T. Suga, H. Hamada, and T. Hirata, Chem. Lett., 1987, 471.
T. Suga, H. Hirata, H. Hamada, and S. Murakami,
Phytochemistry, 27, 1041 (1988).
T. Hirata, Y. X. Tang, K. Okano, and T. Suga,
Phytochemistry, 28, 3331 (1989).
T. Hirata, S. Izumi, K. Shimoda, and M. Hayashi, J. Chem.
Soc., Chem. Commun., 1993, 1426.
K. Shimoda, D. I. Ito, S. Izumi, and T. Hirata, J. Chem.
7.4 and 2.1 Hz, >C=CH–). 2-Alkyl-2-cyclohexen-1-ones
(3–5) were prepared from O-methoxybenzoic acid by
1
3
reductive alkylation according to the reported precedure.
+
1
4
5
6
7
3: MS (EI) m/z 110 (M ), H NMR (CDCl ) δ 1.77 (3H, q,
3
J = 1.5 Hz, Me), 6.74 (1H, tq, J = 4.3 and 1.5 Hz,
>C=CH–); 4: MS (EI) m/z 124 (M ), H NMR (CDCl ) δ
1.00 (3H, t, J = 7.5 Hz, Me), 2.20 (2H, qq, J = 7.5 and 1.5
+
1
3
Hz, –CH –Me), 6.69 (1H, tt, J = 4.1 and 1.5 Hz, >C=CH–);
2
+
1
Soc., Perkin Trans. 1, 1996, 355.
5: MS (EI) m/z 138 (M ), H NMR (CDCl ) δ 0.89 (3H, t,
J = 7.4 Hz, Me), 1.41 (2H, se, J = 7.5 Hz, –CH –Me), 2.15
3
1
The suspension cells of N. tabacum were cultivated at 25
2
o
8
C for 3 weeks in Murashige and Skoog’s medium on a
(2H, tq, J = 7.4 and 1.2 Hz, –CH –CH –Me), 6.69 (1H, t, J
2
2
rotary shaker (75 rpm). The cells (200 g) were homoge-
nized in 0.1 M Na–Pi buffer (pH 6.8) containing 10 mM 2-
mercaptoethanol and 5 mM dithiothreitol. After centrifu-
gation at 10000 g for 15 min, the supernatant was fraction-
ated by treatment with (NH ) SO (40 to 60% satn.). The
= 4.3 Hz, >C=CH–).
11 F. Huet, M. Pellet, and J. M. Conia, Tetrahedron Lett.,
1977, 3505.
12 K. Matsumoto, Y. Kawabata, J. Takahashi, Y. Fujita, and
M. Hatanaka, Chem. Lett., 1998, 283.
4
2
4
crude enzyme soln obtained was desalted and then applied
to a DEAE–Toyopearl column with a 0–0.5 M linear gradi-
ent of NaCl in 50 mM Tris-HCl buffer (pH 8.0) containing
13 D. F. Taber, J. Org. Chem., 41, 2649 (1976).
1
5
14 6a: [θ]288 +960 (c 0.25, MeOH) {lit. [θ]288 –987 for R
1
6
enantiomer}; 8a: [θ] +1860 (c 0.25, MeOH) {lit. [θ]288
2
88
1
.0 mM 2-mercaptoethanol and 1.0 mM dithiothreitol
+2480}.
(
buffer A) to give three enone reductase fractions, which
15 C. J. Cheer and C. Djerassi, Tetrahedron Lett., 1976, 3877.
16 A. I. Meyers, D. R. Williams, G. W. Erickson, S. White,
and M. Druelinger, J. Am. Chem. Soc., 193, 3081 (1981).
contained the 44, 74 and 90 kDa proteins, respectively.
Each of the fractions was further purified on a hydroxylap-
atite column with buffer A and a Red–Toyopearl column
with buffer A containing a 0–1.0 M linear gradient of
NaCl.
17 6a: [θ]288 +993 (c 0.3, MeOH); 7a: [θ] +2293 (c 0.1,
288
MeOH); 8a: [θ]288 +2351 (c 0.12, MeOH).
18 6b: [θ]288 –989 (c 0.2, MeOH); 7b: [θ] –2341 (c 0.1,
288
1
5
8
9
T. Murashige and F. Skoog, Physiol. Plant., 15, 473
MeOH) {lit. [θ]288 +2200 for S enantiomer}; 8b: [θ]288
–2483 (c 0.1, MeOH).
(1962).
The mixture (2 mL) of enone (100 µmol), NADPH (200