Development of a Practical Biocatalytic Process for ( R)-2-Methylpentanol Owen W. Gooding,* Rama Voladri, Abigail Bautista, Thutam Hopkins, Gjalt Huisman, Stephan Jenne, Steven Ma, Emily C. Mundorff, and Megan M. Savile Codexis, Inc., 200 Penobscot DriVe, Redwood City, California 94063, U.S.A. Susan J. Truesdell and John W. Wong Biocatalysis Center of Emphasis, Chemical R&D, Pfizer Global Research and DeVelopment, Eastern Point Road, Groton, Connecticut 06340, U.S.A. Abstract: (R)-2-Methylpentanol is an important chiral intermediate for the synthesis of certain medicinally important compounds, natural products, and liquid crystals. Here we describe the development of a practical kinetic resolution utilizing an enantiospecific bio- catalytic reduction of racemic 2-methylvaleraldehyde. The process utilizes an evolved ketoreductase enzyme to selectively reduce the (R)-enantiomer of racemic 2-methylvaleraldehyde to the desired product with high volumetric productivity. A scaleable method for separating the desired product from the off-enantiomer of the starting material is also described. The process is cost-effective, green, and amenable to manufacturing scale. Introduction (R)-2-Methylpentanol (R-2-MP) is an important intermediate for organic synthesis that has been utilized for the production of certain pharmaceuticals 1 and liquid crystals. 2 Several different synthetic approaches to this material based on resolution or asymmetric syntheses have been previously described (Scheme 1). The first report utilized a resolution of racemic 2-MP through crystallization of the tosylate salt of L-valine-2-methylpentyl ester (Scheme 1a). 3 Although somewhat effective based on optical rotation, this procedure was low-yielding, and the enantiomeric excess was not determined. In 1985 Oppolzer described the first asymmetric synthesis via a diastereoselective ester-enolate alkylation of a chiral sultam (Scheme 1b). 4 This procedure was high-yielding but provided material with only modest enantiopurity. A year later Danishefsky utilized R-2- MP in the synthesis of the antibiotic Zincophorin. 5 In this work an Evans diastereoselective alkylation of a chiral oxazolidinone- derived imide enolate was employed (Scheme 1c). This alky- lation went with 8:1 diastereoselectivity, and enantiopure R-2- MP was obtained following separation of the diastereomers and reductive cleavage of the auxiliary. More recently, a lipase- catalyzed resolution of racemic 2-MP was described (Scheme 1d). 6 In this case high ee product could only be isolated in low yield due to relatively poor enantiospecificity of the enzyme. Application of microbial oxidation in a resolution reaction afforded the desired enantiomer with only 40% ee (Scheme 1e). 7 Although effective at small scale, the procedures outlined in Scheme 1 are all unsuitable for industry because each suffers from one or more of the following: high cost of starting materials, large number of processing steps, poor yields, poor selectivity, or difficult purification procedures. The first industrially viable synthesis of R-2-MP was developed at BASF in 2006 (Scheme 2). 8 In this hybrid biocatalytic-chemocatalytic process, the product was prepared by selective hydrogenation of the unsaturated aldehyde to the allylic alcohol followed by asymmetric hydrogenation of the double bond at 200 bar. Due to the relatively low enantiose- lectivity of the chemocatalyst (∼75% ee), an additional step was required to upgrade the product’s chiral purity. To that end, a lipase resolution was employed to increase the enantiopurity of the product to 98% ee. Despite the drawbacks of high pressure and the requirement for an extra step to upgrade the ee of the product, this process was effective as demonstrated by successful scale-up in a 3 m 3 reactor. 9 Enzymes that catalyze the reduction of ketones (or alde- hydes) to corresponding alcohols are known as ketoreductases (KRED). 10 The use of KREDs for organic synthesis has been growing rapidly because these biocatalysts can be highly enantioselective in the formation of chiral alcohols 11 and they are becoming more widely available. 12 With the advance of * Author for correspondence. E-mail: owen.gooding@codexis.com. (1) Evans, M. C.; Franklin, L. C.; Murtagh, L. M.; Nanninga, T. N.; Pearlman, B. A.; Saenz, J. E.; Willis, J. U.S. Patent Application 2,714,168A1, 2007. (2) Morita, K.; Hachiya, S.; Moriwaki, F.; Endo, H. U.S. Patent 5,281,685, 1994. (3) Jermyn, M. A. Aust. J. Chem. 1967, 20, 2283. (4) Oppolzer, W.; Dudfield, P.; Stevenson, T.; Godel, T. HelV. Chim. Acta 1985, 68, 212. (5) Zelle, R. E.; DeNinno, M. P.; Selnick, H. G.; Danishefsky, S. J. J. Org. Chem. 1986, 51, 5032. (6) Barth, S.; Effenberger, F. Tetrahedron: Asymmetry 1993, 4, 823. (7) Clark, D. S.; Geresh, S.; DiCosimo, R. Bioorg. Med. Chem. Lett. 1995, 5, 1383–1388. (8) Ja ¨kel, C.; Heydrich, G.; Sturmer, R.; Paciello, R. World Patent Application WO 2006/034812, 2006. (9) Ja ¨kel, C.; Paciello, R. Chem. ReV. 2006, 106, 2912–2942. (10) This enzyme type is sometimes referred to as alcohol dehydrogenase (ADH). (11) (a) Kosjek, B.; Nti-Gyabaah, J.; Telari, K.; Dunne, L.; Moore, J. C. Org. Process Res. DeV. 2008, 12, 584–588. (b) Lavandera, I.; Kern, A.; Ferreira-Silva, B.; Glieder, A.; de Wildeman, S.; Kroutil, W. J. Org. Chem. 2008, 73, 6003–6005. (c) Moore, J. C.; Pollard, D. J.; Kosjek, B.; Devine, P. N. Acc. Chem. Res. 2007, 40, 1421–1419. (12) A wide selection of KRED biocatalysts are commercially available from Sigma-Aldrich (Milwaukee, WI), IEP GmbH (Wiesbaden, Germany), Enzysource (Hangzhou, China), and Codexis (Redwood City, CA). Organic Process Research & Development 2010, 14, 119–126 10.1021/op9002246  2010 American Chemical Society Vol. 14, No. 1, 2010 / Organic Process Research & Development • 119 Published on Web 12/01/2009