Stereoselective, Biocatalytic Reductions of r-Chloro-β-keto Esters
SCHEME 1
Iwona A. Kaluzna, Brent D. Feske, Weerawut Wittayanan, Ion Ghiviriga, and Jon D. Stewart* Department of Chemistry, University of Florida, Gainesville, Florida 32611
[email protected] Received August 26, 2004
Eighteen known and putative reductases from baker’s yeast (Saccharomyces cerevisiae) were tested for the ability to reduce a series of R-chloro-β-keto esters. In nearly all cases, it was possible to produce at least two of the four possible R-chloro-β-hydroxy ester diastereomers with high optical purities. The utility of this approach was demonstrated by reducing ethyl 2-chloroacetoacetate to the corresponding syn(2R,3S)-alcohol on a multigram scale using whole cells of an Escherichia coli strain overexpressing a single yeast reductase identified from the screening studies.
Homochiral glycidic esters are versatile intermediates that can be converted into a variety of high-value products. Optically active glycidates can be prepared by a number of routes including asymmetric Darzens reactions, chiral alkene oxidation methodologies, and ring closure of homochiral R-halo-β-hydroxy esters (see ref 1 and references cited therein). We were particularly interested in the last strategy because asymmetric reductions of R-chloro-β-keto esters might afford each of the four possible glycidate precursors via dynamic kinetic resolution processes from single, inexpensive starting materials (Scheme 1). Here, we explore the potential of individual reductase enzymes from baker’s yeast (Saccharomyces cerevisiae) as solutions to the problem of obtaining homochiral glycidate precursors. Reductions of R-chloro-β-keto esters by whole cells of commercial baker’s yeast generally produce disappointing mixtures of alcohol diastereomers.2-5 Recent work has * To whom correspondence should be addressed. Phone: 352-8460743. Fax: 352-846-2095. (1) Imashiro, R.; Seki, M. J. Org. Chem. 2004, 69, 4216-4226. (2) Cabon, O.; Larcheveˆque, M.; Buisson, D.; Azerad, R. Tetrahedron Lett. 1992, 7337-7340. (3) Cabon, O.; Buisson, D.; Larcheveˆque, M.; Azerad, R. Tetrahedron: Asymmetry 1995, 6, 2199-2210. (4) Cabon, O.; Buisson, D.; Larcheveˆque, M.; Azerad, R. Tetrahedron: Asymmetry 1995, 6, 2211-2218. (5) Jo¨rg, G.; Bertau, M. ChemBioChem 2004, 4, 87-92.
revealed that the yeast genome encodes a large number of reductases,6 and it seemed likely that their simultaneous participation was mainly responsible for the modest stereoselectivities commonly observed in yeastmediated ketone reductions.7-9 In response, we have adapted a fusion protein strategy10 that allows the properties of yeast reductases to be assessed individually, so that enzymes yielding homochiral products can be uncovered.11,12 Moreover, after a reductase with the desired properties has been identified, whole Escherichia coli cells expressing the same protein can be employed for bioconversions on preparative scales using glucose fed-batch conditions.13 Cellular metabolic pathways supply NADPH and the whole cells display very high stereoselectivities because they express only a single yeast reductase. Results and Discussion A series of five R-chloro-β-keto esters 1a-e was used in this study (Scheme 1). Those not commercially available were synthesized from the corresponding β-keto esters by treatment with sulfuryl chloride.14 Eighteen yeast reductases were isolated as fusion proteins with glutathione S-transferase using previously described methods.12 The collection of enzymes included members of the aldose reductase, D-hydroxy acid dehydrogenase, medium-chain dehydrogenase, and short-chain dehydrogenase superfamilies. Each R-chloro-β-keto ester was tested as a substrate for each reductase in the presence (6) Stewart, J. D.; Rodriguez, S.; Kayser, M. M. In Enzyme Technologies for Pharmaceutical and Biotechnological Applications; Kirst, H. A., Yeh, W.-K., Zmijewski, M. J., Eds.; Marcel Dekker: New York, 2001; pp 175-208. (7) Shieh, W.-R.; Gopalin, A. S.; Sih, C. J. J. Am. Chem. Soc. 1985, 107, 2993-2994. (8) Shieh, W.-R.; Sih, C. J. Tetrahedron: Asymmetry 1993, 4, 12591269. (9) Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. J. Org. Chem. 1991, 56, 4778-4783. (10) Martzen, M. R.; McCraith, S. M.; Spinelli, S. L.; Torres, F. M.; Fields, S.; Grayhack, E. J.; Phizicky, E. M. Science 1999, 286, 11531155. (11) Kaluzna, I.; Andrew, A. A.; Bonilla, M.; Martzen, M. R.; Stewart, J. D. J. Mol. Catal. B: Enzymatic 2002, 17, 101-105. (12) Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J. Am. Chem. Soc. 2004, 126, 12832-12827. (13) Walton, A. Z.; Stewart, J. D. Biotechnol. Prog. 2004, 20, 403411. (14) Matsuki, K.; Sobukawa, M.; Kawai, A.; Inoue, H.; Takeda, M. Chem. Pharm. Bull. 1993, 41, 643-648. 10.1021/jo0484981 CCC: $30.25 © 2005 American Chemical Society
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J. Org. Chem. 2005, 70, 342-345
Published on Web 12/03/2004
TABLE 1. Biocatalytic Reductions of r-Chloro-β-keto Estersc
a
