obtained in 76% yield with >99% ee (entry 1). This result was
surprising because the sterically demanding chlorine atom at
the ortho position might hinder the reaction and deteriorate the
enantioselectivity and because a-hydroxy ester 1, bearing the three
electron-withdrawing groups at the stereocenter, might be subject
to racemization. Indeed, the difficulty of asymmetric reduction of
2
can be inferred from the fact that Genet and co-workers gained
Scheme 2
(
R)-1 with 50% ee at most despite various attempts to increase the
enantioselectivity in the Ru-catalyzed asymmetric hydrogenation
of 2.
3
In summary, the present biotransformation provides an ef-
ficient and green method for the synthesis of methyl (R)-o-
chloromandelate ((R)-1). The hydride source is glucose, which is
cheap biomass, and the catalyst is E. coli, which can be multiplied
easily and inexpensively. The reaction is performed in an aqueous
solution under air. This is the first example of the direct asymmetric
synthesis of (R)-1 with >99% ee. Excellent productivity as high as
In view of the industrial utility of (R)-1 as a synthetic in-
termediate for clopidogrel, we next turned our attention to the
efficiency of this biotransformation. We tried to find the best
reaction temperature, at which the enzyme denaturation caused
by a large amount of substrate/product as well as temperature
is suppressed well, and at which the asymmetric reduction of
−
1
178 g L has been achieved. Because of the pharmaceutical value
2
proceeds smoothly, giving the highest productivity of (R)-1.
of the downstream product, clopidogrel, this bioprocess has good
potential for an industrial application.
This work was supported by a Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science
Table 1 outlines how we optimized the productivity by changing
the substrate concentration and the reaction temperature. When
◦
the reaction temperature was decreased by 5 C, the conversion
and isolated yield increased (entry 2), which prompted us to double
the substrate concentration. Even at the substrate concentration
of 0.6 M, the conversion reached 94% (entry 3). Therefore,
we further increased the substrate concentration up to 1.0 M,
which resulted in 90% conversion (entry 4). Finally, we further
lowered the reaction temperature (entries 5 and 6) to find the best
temperature giving the highest conversion at the same substrate
(
JSPS). We are grateful to the SC-NMR Laboratory of Okayama
University for the measurement of NMR spectra.
Notes and references
1
J. Grimley, Chem. Eng. News, 2006, Dec. 4th, pp. 17–28.
concentration. Thus, the whole-cell reduction of 1.0 M of 2 at
◦
2 A. Bousquet and A. Musolino, PCT Int. Appl., WO 9918110A1, Chem.
2
0 C gave 99% conversion and 1.78 g of isolated product (R)-
Abs., 1999, 130, 296510.
−
1
1
(entry 5), which corresponds to a productivity of 178 g L
3
4
5
J.-P. Genet, S. Juge, J.-A. Laffitte, C. Pinel and S. Mallart, PCT Int.
Appl., WO 9401390A1, Chem. Abs., 1994, 121, 156763.
Y. Sun, X. Wan, J. Wang, Q. Meng, H. Zhang, L. Jiang and Z. Zhang,
Org. Lett., 2005, 7, 5425–5427.
A. Glieder, R. Weis, W. Skranc, P. Poechlauer, I. Dreveny, S. Majer, M.
Wubbolts, H. Schwab and K. Gruber, Angew. Chem., Int. Ed., 2003,
42, 4815–4818.
(
weight of isolated product per litre of initial reaction volume).
Such a remarkable temperature effect on productivity was beyond
our expectation although other researchers had gained the highest
◦
productivity at 20 C in the whole-cell asymmetric reduction
12
of ethyl 4-chloroacetoacetate. Because only a few examples of
microbial reduction systems capable of giving productivity higher
6
7
8
9
L. M. van Langen, R. P. Selassa, F. van Rantwijk and R. A. Sheldon,
Org. Lett., 2005, 7, 327–329.
T. Ema, Y. Sugiyama, M. Fukumoto, H. Moriya, J.-N. Cui, T. Sakai
and M. Utaka, J. Org. Chem., 1998, 63, 4996–5000.
T. Ema, H. Moriya, T. Kofukuda, T. Ishida, K. Maehara, M. Utaka
and T. Sakai, J. Org. Chem., 2001, 66, 8682–8684.
−
1
13–18
than 100 g L have been reported,
the present whole-cell
reduction is quite promising. Moreover, the enantiomeric purities
of (R)-1 obtained under various conditions in Table 1 were
>99% ee in all cases.
T. Ema, H. Yagasaki, N. Okita, K. Nishikawa, T. Korenaga and T.
Sakai, Tetrahedron: Asymmetry, 2005, 16, 1075–1078.
Previously, we have obtained ethyl (R)-mandelate ((R)-3) with
10
9
2% ee using recombinant E. coli overproducing SCR. The fact
10 T. Ema, H. Yagasaki, N. Okita, M. Takeda and T. Sakai, Tetrahedron,
006, 62, 6143–6149.
2
that the (R)-enantiomer of 3 was obtained could not be explained
1
1 M. Ma, C. Li, L. Peng, F. Xie, X. Zhang and J. Wang, Tetrahedron
Lett., 2005, 46, 3927–3929.
8
well by the stereochemical trend observed for a series of products.
Before the attempt to reduce 2, therefore, we could not predict
how the enantioselectivity would change due to the structural
modifications in the analogous compound. To investigate the
factors responsible for the highly enantioselective production
of (R)-1, we determined the enantiomeric purities of 4 and 5
obtained by the whole-cell reduction of the corresponding ketones
12 H. Yamamoto, A. Matsuyama and Y. Kobayashi, Biosci., Biotechnol.,
Biochem., 2002, 66, 481–483.
1
3 N. Kizaki, Y. Yasohara, J. Hasegawa, M. Wada, M. Kataoka and S.
Shimizu, Appl. Microbiol. Biotechnol., 2001, 55, 590–595.
1
4 H. Gr o¨ ger, F. Chamouleau, N. Orologas, C. Rollmann, K. Drauz, W.
Hummel, A. Weckbecker and O. May, Angew. Chem., Int. Ed., 2006,
4
5, 5677–5681.
1
5 W. Kroutil, H. Mang, K. Edegger and K. Faber, Curr. Opin. Chem.
Biol., 2004, 8, 120–126.
◦
at 30 C. As a result, (R)-4 and (R)-5 were obtained in good
yields with 96 and 99% ee, respectively (Scheme 2). Clearly, the
presence of the chlorine atom and the replacement of the ethyl
group by the methyl group each contributed to the enhancement of
enantioselectivity, and the two modifications led to the production
of (R)-1 with >99% ee.
16 K. Nakamura, R. Yamanaka, T. Matsuda and T. Harada, Tetrahedron:
Asymmetry, 2003, 14, 2659–2681.
1
7 A. Liese, K. Seelbach and C. Wandrey, Industrial Biotransformations,
Wiley-VCH, Weinheim, 2000.
8 Biocatalysis in the Pharmaceutical and Biotechnology Industries, ed.
1
R. N. Patel, CRC, Florida, 2007.
1
176 | Org. Biomol. Chem., 2007, 5, 1175–1176
This journal is © The Royal Society of Chemistry 2007