Diastereoselective reduction of β-keto carbonyl compounds by cultured plant cells

Article abstract of DOI:10.1016/j.tetlet.2006.01.005

The diastereoselective reduction of β-keto carbonyl compounds such as 2-benzamidomethyl-3-oxobutanoates and 2-methyl-2-(2-propenyl)cyclopentan-1,3- dione by cultured cells of higher plants was investigated. The reduction of the 2-benzamidomethyl-3-oxobuta

Full text of DOI:10.1016/j.tetlet.2006.01.005

Tetrahedron
Letters
Tetrahedron Letters 47 (2006) 1541–1544
Diastereoselective reduction of b-keto carbonyl compounds
by cultured plant cells
Kei Shimoda,a Naoji Kubota,a Hatsuyuki Hamadab and Hiroki Hamadac,*
aDepartment of Pharmacology and Therapeutics, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
bNational Institute of Fitness and Sports in Kanoya, 1 Shiromizu-cho, Kagoshima 891-2390, Japan
cDepartment of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
Received 14 November 2005; revised 27 December 2005; accepted 4 January 2006
Available online 23 January 2006
Abstract—The diastereoselective reduction of b-keto carbonyl compounds such as 2-benzamidomethyl-3-oxobutanoates and
2-methyl-2-(2-propenyl)cyclopentan-1,3-dione by cultured cells of higher plants was investigated. The reduction of the 2-benzami-
domethyl-3-oxobutanoates by Parthenocissus tricuspidata diastereoselectively produced the (2R,3S)-2-benzamidomethyl-3-hydroxy-
butanoates, whereas the reduction by Gossypium hirsutum gave the (2S,3S)-2-benzamidomethyl-3-hydroxybutanoates. The (2R,3S)/
(2S,3S) predominance in the reduction with Nicotiana tabacum, Glycine max, and Catharanthus roseus was reversed by the change in
the structure of the alkoxyl group in the substrate. On the other hand, the reduction of 2-methyl-2-(2-propenyl)cyclopentan-1,3-
dione by P. tricuspidata produced (2R,3S)-3-hydroxy-2-methyl-2-(2-propenyl)cyclopentan-1-one, whereas the reaction by N. taba-
cum, G. max, C. roseus, and G. hirsutum gave (2S,3S)-3-hydroxy-2-methyl-2-(2-propenyl)cyclopentan-1-one.
Ó 2006 Elsevier Ltd. All rights reserved.
Optically active b-hydroxy carbonyl compounds such as
2-substituted 3-hydroxybutanoates and 2,2-disubstituted
3-hydroxycycloalkan-1-ones are versatile chiral synthons
in the organic syntheses of biologically active com-
pounds, especially in drug syntheses; chiral 2-benzami-
domethyl-3-hydroxybutanoates are useful building
blocks for the b-lactam antibiotics such as penems and
carbapenems,1,2 and optically active 3-hydroxy-2-
methyl-2-(2-propenyl)cyclopentan-1-one is an important
chiral synthon for prostaglandins.3 Because the asym-
metric reduction of the carbonyl compounds with bio-
catalysts is a very attractive method for the practical
preparation of chiral alcohols, many studies on the bio-
logical reduction of 2-substituted 3-oxobutanoates and
2,2-disubstituted cyclopentan-1,3-diones by microorgan-
isms, which generally afford a mixture of (2R,3S)- and
(2S,3S)-isomers of 2-substituted 3-hydroxybutanoates,
and (2S,3S)-isomers of 2,2-disubstituted 3-hydroxy-
cyclopentan-1-ones, respectively, have already been
reported.4–17 However, little attention has been paid to
the reduction of the 2-substituted 3-oxobutanoates18
and the 2,2-disubstituted cyclopentan-1,3-diones by
cultured plant cells. Furthermore, a cell-mediated
process in which the 2-benzamidomethyl-3-oxobutano-
ate is reduced to essentially produce one diastereomer,
either the (2R,3S)- or (2S,3S)-isomer of the 2-benzami-
domethyl-3-hydroxybutanoate in enantiomerically pure
form, and the 2,2-disubstituted cyclopentan-1,3-
dione is transformed to (2R,3S)-isomer of 2,2-disubsti-
tuted 3-hydroxycyclopentan-1-one is still unavailable.
Herein, we report the asymmetric reduction of the
2-benzamidomethyl-3-oxobutanoates and 2-methyl-2-
(2-propenyl)cyclopentan-1,3-dione by cultured plant
cells of Parthenocissus tricuspidata, Nicotiana tabacum,
Glycine max, Catharanthus roseus, and Gossypium
hirsutum.
Just prior to use for this work, 50 g of cultured cells was
transplanted to a 300 mL conical flask containing
100 mL of freshly prepared medium (MS medium for
P. tricuspidata, N. tabacum, and G. max; SH medium
for C. roseus and G. hirsutum)19,20 containing 3%
sucrose and 10 mM 2,4-D and grown with continuous
shaking for 1 week at 25 °C. A total of 120 mg of each
substrate was administered to the 10 flasks (12 mg/flask,
none solvent) containing the suspension cultures and the
cultures were incubated at 25 °C for 3 days on a rotary
shaker (120 rpm). After incubation the cultures were
*
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 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.01.005

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K. Shimoda et al. / Tetrahedron Letters 47 (2006) 1541–1544
harvested and the medium was extracted with ethyl ace-
tate. The yields of the products were determined by
HPLC and GLC analyses. Purification of the ethyl ace-
tate extracts by column chromatography on silica gel
with pentane–ethyl acetate (95:5, v/v) gave the products.
The structures of the products were determined on the
basis of their HRFABMS, 1H and 13C NMR, H–H
COSY, and C–H COSY.21 The absolute configurations
of the products obtained in the reduction of 2-benzamido-
methyl-3-oxobutanoates were determined by comparing
the retention times of the corresponding (R)-(+)-a-
methoxy-a-(trifluoromethyl)phenylacetic acid (MTPA)
esters in the HPLC analyses on Deverosil 100-3 column
with those of the (R)-MTPA esters of authentic chiral
samples.22 The enantiomeric purities of the products
were determined based on the peak areas of the corre-
sponding (R)-MTPA esters in the HPLC. The absolute
configurations and the optical purities of the products
obtained in the reduction of 2-methyl-2-(2-propenyl)-
cyclopentan-1,3-dione were determined by the proce-
dure reported previously.7 All of the five plant strains
had a high potential for the reduction of methyl 2-benz-
amidomethyl-3-oxobutanoate (1) to give the corre-
sponding b-hydroxy ester 4 (Table 1). The reduction
with P. tricuspidata gave methyl (2R,3S)-2-benzamido-
methyl-3-hydroxybutanoate (4a) (100% de) with an
excellent enantioselectivity (>99% ee) in 97% yield. This
suggests that the reductive resolution via an enol inter-
mediate occurred in the P. tricuspidata cells.9 The
(2R,3S)-selectivity was also observed in the reduction
with N. tabacum, G. max, and C. roseus. The reduction
with these cultured cells proceeded with an excellent
enantioselectivity to give the corresponding (3S)-b-
hydroxy esters, for example, the reduction with N. taba-
cum afforded the (2R,3S)-isomer 4a (50% de) with >99%
ee and minor (2S,3S)-isomer 4b with 99% ee. On the
other hand, the reduction with G. hirsutum exhibited a
(2S,3S)-selectivity to give methyl (2S,3S)-2-benzamido-
methyl-3-hydroxybutanoate (4b) (84% de) with a high
enantioselectivity [(2R,3S)-isomer 4a: 98% ee; (2S,3S)-
isomer 4b: >99% ee] in 98% yield.
Next, ethyl 2-benzamidomethyl-3-oxobutanoate (2),
which contained an ethoxyl group, was subjected to
the same reduction system. The reduction with P. tricu-
spidata showed a (2R,3S)-selectivity to give ethyl
(2R,3S)-2-benzamidomethyl-3-hydroxybutanoate (5a)
(88% de) with an excellent enantioselectivity [(2R,3S)-
isomer 5a: >99% ee; (2S,3S)-isomer 5b: 98% ee] in
82% yield. Interestingly, the reduction with N. tabacum,
G. max, and C. roseus exerted a (2S,3S)-selectivity. The
reduction of 2 with G. hirsutum exhibited an excellent
(2S,3S)-selectivity to give ethyl (2S,3S)-2-benzamido-
methyl-3-hydroxybutanoate (5b) (100% de) with a high
enantioselectivity (>99% ee) and yield (97%).
These results demonstrate that the reduction of 2-benz-
amidomethyl-3-oxobutanoates by the cultured cells of
P. tricuspidata diastereoselectively gives the (2R,3S)-2-
benzamidomethyl-3-hydroxybutanoates, whereas the
reaction with G. hirsutum cells affords the (2S,3S)-2-
benzamidomethyl-3-hydroxybutanoates (Fig. 1). It is
worth noting that the (2R,3S)/(2S,3S) predominance
in the reduction with N. tabacum, G. max, and C. roseus
is reversed by a change in the structure of the alkoxyl
group in the substrate.
Further, 2-methyl-2-(2-propenyl)cyclopentan-1,3-dione
(3) was used as the substrate. The reduction by P. tricu-
spidata exerted a (2R,3S)-selectivity to produce (2R,3S)-
3-hydroxy-2-methyl-2-(2-propenyl)cyclopentan-1-one (6a)
(58% de) with an excellent enantioselectivity [(2R,3S)-
isomer 6a: 99% ee; (2S,3S)-isomer 6b: 95% ee] in 91%
yield (Fig. 2). On the other hand, a (2S,3S)-selectivity
was observed in the reduction with N. tabacum, G.
max, C. roseus, and G. hirsutum, for example, the reduc-
tion with G. hirsutum gave (2S,3S)-3-hydroxy-2-methyl-
2-(2-propenyl)cyclopentan-1-one (6b) with
a
high
(2S,3S)-selectivity (96% de) [(2R,3S)-isomer 6a: 78%
ee; (2S,3S)-isomer 6b: >99% ee]. These results suggest
that 3 is held and reduced by the reductases from each
cultured cells in the same manner as in the case of the
reduction of 2.
Table 1. Diastereoselective reduction of b-keto carbonyl compounds by the cultured plant cells
Substrates
Products
Cultured cells
Conversion (%)a
(2R,3S)/(2S,3S)
ee (%)
(2R,3S)
(2S,3S)
1
4a
P. tricuspidata
N. tabacum
G. max
97
95
>99
92
100/0
75/25
57/43
58/42
8/92
>99
>99
>99
>99
98
—
99
98
98
>99
C. roseus
G. hirsutum
4b
98
2
3
5a
5b
P. tricuspidata
N. tabacum
G. max
82
>99
>99
86
94/6
7/93
45/55
10/90
0/100
>99
91
99
99
—
98
>99
99
>99
>99
C. roseus
G. hirsutum
97
6a
6b
P. tricuspidata
N. tabacum
G. max
91
99
96
67
93
79/21
4/96
35/65
11/89
2/98
99
84
99
96
78
95
>99
99
99
>99
C. roseus
G. hirsutum
a The conversions were expressed as the percentage of the product in the reaction mixture on the basis of HPLC (4 and 5) and GLC (6).

K. Shimoda et al. / Tetrahedron Letters 47 (2006) 1541–1544
1543
OH
CO R
2
P. tricuspidata
O
NHCOPh
4a: R=Me, 100% de, >99% ee
5a: R=Et, 88% de, >99% ee
CO R
2
2
4
3
1
6
5
NHCOPh
OH
G. hirsutum
CO R
2
1: R=Me
2: R=Et
NHCOPh
4b: R=Me, 84% de, >99% ee
5b: R=Et, 100% de, >99% ee
Figure 1. Diastereoselective reduction of 2-benzamidomethyl-3-oxobutanoates 1 and 2 by the cultured cells of P. tricuspidata and G. hirsutum.
P. tricuspidata
HO
O
9
6
3
7
8
2
6a, 58% de, 99% ee
1
O
O
4
5
G. hirsutum
3
HO
O
6b, 96% de, >99% ee
Figure 2. Diastereoselective reduction of 2-methyl-2-(2-propenyl)cyclopentan-1,3-dione (3) by the cultured cells of P. tricuspidata and G. hirsutum.
2. Ohtake, H.; Imada, Y.; Murahashi, S. J. Org. Chem. 1999,
64, 3790.
3. Schick, H.; Welzel, H. P.; Schwarz, S.; Truckenbrodt, G.
J. Prakt. Chem. 1985, 327, 91.
4. Nakamura, K.; Higaki, M.; Ushio, K.; Oka, S.; Ohno, A.
Tetrahedron Lett. 1985, 26, 4213.
5. Ohta, H.; Ozaki, K.; Tuchihashi, G. Agric. Biol. Chem.
1986, 50, 2499.
6. Sakai, T.; Nakamura, T.; Fukuda, K.; Amano, E.; Utaka,
M.; Takeda, A. Bull. Chem. Soc. Jpn. 1986, 59, 3185.
7. Brooks, D. W.; Mazdiyasni, H.; Grothaus, P. G. J. Org.
Chem. 1987, 52, 3223.
8. Buisson, D.; Sanner, C.; Larcheveque, M.; Azerad, R.
Tetrahedron Lett. 1987, 28, 3939.
9. Nakamura, K.; Miyai, T.; Ushio, K.; Oka, S.; Ohno, A.
Bull. Chem. Soc. Jpn. 1988, 61, 2089.
10. Nakamura, K.; Miyai, T.; Nagar, A.; Oka, S.; Ohno, A.
Bull. Chem. Soc. Jpn. 1989, 62, 1179.
11. Nakamura, K.; Kawai, Y.; Ohno, A. Tetrahedron Lett.
1991, 32, 2927.
12. Nakamura, K.; Kawai, Y.; Miyai, T.; Honda, S.; Naka-
jima, N.; Ohno, A. Bull. Chem. Soc. Jpn. 1991, 64, 1467.
13. Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. J.
Org. Chem. 1991, 56, 4778.
14. Mori, K.; Takayama, S.; Yoshimura, S. Liebigs Ann.
Chem. 1993, 91.
15. Nakamura, K.; Takano, S.; Ohno, A. Tetrahedron Lett.
1993, 34, 6087.
Thus, the diastereoselective reduction of 2-benzamido-
methyl-3-oxobutanoates and 2-methyl-2-(2-propenyl)-
cyclopentan-1,3-dione has been accomplished by the
cultured plant cells of P. tricuspidata, N. tabacum, G.
max, C. roseus, and G. hirsutum. Recently, it has been
reported that the reduction of methyl 2-benzamido-
methyl-3-oxobutanoate with BINAP catalyst gave the
(2S,3R)-isomer of methyl 2-benzamidomethyl-3-
hydroxybutanoate (93% de, 99% ee).23–25 It should be
emphasized that the diastereoselective formation of each
of the (2R,3S)- and (2S,3S)-2-benzamidomethyl-3-
hydroxybutanoates in enantiomerically pure forms has
been achieved by the selective use of these plant cells.
On the other hand, it is worth noting that the (2R,3S)-
isomer of 2,2-disubstituted 3-hydroxycyclopentan-1-
one was preferentially produced by the cultured plant
cells of P. tricuspidata. Further investigations using the
enzyme preparation from these cultured plant cells are
now in progress.
Acknowledgments
The authors thank Dr. T. Miura of Takasago Perfumery
Co. Ltd. for helpful discussions.
16. Zhu, Y. Y.; Burnell, D. J. Tetrahedron: Asymmetry 1996,
7, 3295.
17. Nakamura, K.; Yamanaka, R.; Matsuda, T.; Harada, T.
Tetrahedron: Asymmetry 2003, 14, 2659.
References and notes
1. Murayama, T.; Kobayashi, T.; Miura, T. Tetrahedron
Lett. 1995, 36, 3703.
18. Nakamura, K.; Miyoshi, H.; Sugiyama, T.; Hamada, H.
Phytochemistry 1995, 40, 1419.

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K. Shimoda et al. / Tetrahedron Letters 47 (2006) 1541–1544
19. Murashige, T.; Skoog, F. Physiol. Plant. 1962, 15, 473.
20. Shenk, R. U.; Hildebrand, A. C. Can. J. Bot. 1979, 50,
199.
1.28 (3H, d, J = 6.4 Hz, H-4), 2.85 (1H, m, 2-H), 3.74
(1H, m, H-5a), 3.88–4.22 (4H, m, H-3, 5b, 10), 7.18 (1H, br
s, NH), 7.41 (2H, td, J = 6.4, 1.4 Hz, m-H), 7.47 (1H, td,
J = 7.2, 1.2 Hz, p-H), 7.76 (2H, dd, J = 6.4, 1.6 Hz, o-H);
13C NMR (100 MHz, CDCl3): d 14.1 (C-20), 20.9 (C-4),
38.8 (C-5), 51.0 (C-2), 60.9 (C-10), 67.1 (C-3), 126.9 (o-C in
Ph), 128.7 (m-C in Ph), 131.6 (p-C in Ph), 168.7 (C-6),
173.7 (C-1). Product 6a (obtained in the reduction of 3
with P. tricuspidata): HRFABMS m/z 155.1715 [M+H]+;
1H NMR (400 MHz, CDCl3) d 1.01 (3H, s, H-6), 1.68
(1H, br s, OH), 1.88–2.48 (6H, m, H-4, 5, 7), 4.23 (1H,
t, J = 6.2 Hz, H-3), 5.13 (2H, m, H-9), 5.77 (1H, m,
H-8); 13C NMR (100 MHz, CDCl3): d 15.2 (C-6), 27.4
(C-7), 35.0 (C-5), 39.9 (C-4), 53.2 (C-2), 75.6 (C-3),
118.6 (C-9), 133.5 (C-8), 220.0 (C-1). Product 6b (obtained
in the reduction of 3 with G. hirsutum): HRFABMS
m/z 155.1712 [M+H]+; 1H NMR (400 MHz, CDCl3) d
1.01 (3H, s, H-6), 1.77 (1H, d, J = 2.8 Hz, OH), 1.98–
2.51 (6H, m, H-4, 5, 7), 4.13 (1H, m, H-3), 5.16 (2H, m,
H-9), 5.88 (1H, m, H-8); 13C NMR (100 MHz, CDCl3):
d 19.9 (C-6), 27.8 (C-7), 34.2 (C-5), 35.6 (C-4), 53.2
(C-2), 77.3 (C-3), 118.2 (C-9), 134.6 (C-8), 220.5
(C-1).
21. Spectral data for the products; product 4a (obtained in the
reduction of 1 with P. tricuspidata): HRFABMS m/z
252.1933 [M+H]+; 1H NMR (400 MHz, CDCl3) d 1.24
(3H, d, J = 6.4 Hz, H-4), 2.63 (1H, m, 2-H), 3.62 (1H, m,
H-5a), 3.69 (3H, s, OCH3), 4.02 (2H, m, H-3, 5b), 4.53
(1H, d, J = 4.0 Hz, OH), 7.37 (2H, td, J = 6.4, 1.6 Hz, m-
H), 7.48 (1H, td, J = 7.2, 1.2 Hz, p-H), 7.76 (2H, dd,
J = 6.4, 1.2 Hz, o-H); 13C NMR (100 MHz, CDCl3): d
20.9 (C-4), 38.0 (C-5), 52.0 (OCH3), 52.6 (C-2), 65.7 (C-3),
127.3 (o-C in Ph), 128.6 (m-C in Ph), 131.7 (p-C in Ph),
168.7 (C-6), 174.1 (C-1). Product 4b (obtained in the
reduction of 1 with G. hirsutum): HRFABMS m/z
252.1928 [M+H]+; 1H NMR (400 MHz, CDCl3) d 1.26
(3H, d, J = 7.2 Hz, H-4), 2.63 (1H, m, 2-H), 3.57 (1H, m,
H-5a), 3.74 (3H, s, OCH3), 4.00 (1H, m, H-3), 4.12 (1H, m,
H-5b), 4.29 (1H, d, J = 4.0 Hz, OH), 7.04 (1H, br s, NH),
7.44 (2H, td, J = 7.2, 1.2 Hz, m-H), 7.48 (1H, td, J = 7.2,
1.2 Hz, p-H), 7.76 (2H, dd, J = 7.2, 1.2 Hz, o-H); 13C
NMR (100 MHz, CDCl3): d 20.6 (C-4), 37.7 (C-5), 51.9
(OCH3), 52.4 (C-2), 65.4 (C-3), 126.9 (o-C in Ph), 128.5
(m-C in Ph), 131.7 (p-C in Ph), 168.6 (C-6), 174.1 (C-1).
Product 5a (obtained in the reduction of 2 with P.
tricuspidata): HRFABMS m/z 266.2056 [M+H]+; 1H
NMR (400 MHz, CDCl3) d 1.27 (6H, m, H-4, 20), 2.60
(1H, m, 2-H), 3.61 (1H, m, H-5a), 4.06–4.21 (4H, m, H-3,
5b, 10), 4.36 (1H, d, J = 4.0 Hz, OH), 7.14 (1H, br s, NH),
7.42 (2H, td, J = 7.2, 1.2 Hz, m-H), 7.48 (1H, td, J = 7.2,
1.2 Hz, p-H), 7.77 (2H, dd, J = 7.2, 1.2 Hz, o-H); 13C
NMR (100 MHz, CDCl3): d 14.2 (C-20), 20.8 (C-4), 38.0
(C-5), 52.6 (C-2), 61.0 (C-10), 65.7 (C-3), 127.0 (o-C in Ph),
128.6 (m-C in Ph), 131.8 (p-C in Ph), 168.7 (C-6), 173.7 (C-
1). Product 5b (obtained in the reduction of 2 with
G. hirsutum): HRFABMS m/z 266.2104 [M+H]+; 1H
NMR (400 MHz, CDCl3) d 1.26 (3H, t, J = 7.2 Hz, H-20),
22. HPLC was carried out with Deverosil 100-3 (Nomura
Chemical Co. Ltd.) column [eluent: hexane–THF–MeOH
(1000:100:1); flow rate: 1 mL/min; detection: UV 254 nm].
Retention times for the (R)-MTPA esters of (2S,3R)-4,
(2R,3S)-4, (2R,3R)-4, (2S,3S)-4, (2S,3R)-5, (2R,3S)-5,
(2R,3R)-5, and (2S,3S)-5 in the HPLC were 25.1, 26.4,
31.9, 38.5, 26.7, 28.3, 34.4, and 40.1 min, respectively.
23. Noyori, R.; Ikeda, T.; Ohkuma, T.; Widhalm, M.;
Kitamura, M.; Takaya, H.; Akutagawa, S.; Sayo, N.;
Saito, T.; Taketomi, T.; Kumobayashi, H. J. Am. Chem.
Soc. 1989, 111, 9134.
24. Kitamura, M.; Tokunaga, M.; Noyori, R. J. Am. Chem.
Soc. 1993, 115, 144.
25. Noyori, R. Angew. Chem., Int. Ed. 2002, 41, 2008.