Kinetic resolution of (±)-1-phenylethanol in [Bmim][PF 6 ] using high activity preparations of lipases Shweta Shah and Munishwar N. Gupta * Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India Received 20 September 2006; revised 16 November 2006; accepted 18 November 2006 Available online 22 November 2006 Abstract—Lipases from two different sources Candida rugosa (CRL) and Burkholderia cepacia (BCL) were formulated as enzyme precipitated and rinsed with organic solvents, organic solvent rinsed enzyme preparation, cross-linked enzyme aggregates (CLEAs) and protein coated micro-crystals (PCMCs). These various enzyme formulates were evaluated for the kinetic resolution of (±)-1- phenylethanol in ionic liquid [Bmim][PF 6 ] by transesterification with vinyl acetate. Of all the enzyme forms evaluated EPRP and PCMC in the case of CRL showed the best results with 26 % (E value = 153) and 53% (E value = 79) conversion, respectively, at 35 °C in 24 h. Carrying out this conversion with PCMC at lower temperature of 25 °C further improved the E value to 453 (with 44% conversion in 12 h). For BCL the acetone-rinsed enzyme preparation (AREP), CLEA and PCMC performed equally well with % conversion of 50 and 99 ee p (%) (E value >1000) in just 2 h, whereas, the free lipase gave only 8% conversion. Ó 2006 Elsevier Ltd. All rights reserved. The ionic liquids (ILs) are fast emerging as a useful reac- tion medium for enantioselective biocatalysis. 1 In many cases, especially with lipase-catalyzed transesterification reactions; higher reaction rates and greater enantioselec- tivity have been reported as compared to the corre- sponding performances in anhydrous organic media. 2 Most such studies have been carried out with pH-tuned lyophilized enzyme powders, either in free or immobi- lized forms. 2,3 In the last few years, it has become clear that structural changes during lyophilization are largely responsible for relatively low catalytic rates shown by enzymes in nearly anhydrous organic solvents. 4 Conse- quently, many enzyme formulations which ‘dry’ en- zymes for use in such media have been described. Such enzyme forms include propanol-rinsed enzyme prepara- tions (PREP), 5 cross-linked enzyme aggregates (CLEAs) 6 and protein coated micro-crystals (PCMCs). 7 PREP refers to propanol-rinsed immobilized enzymes, the corresponding free enzyme preparations rinsed with propanol (EPRP) also give higher reaction rates than pH-tuned lyophilized powders in low water-containing organic solvents. 8 Studies with such preparations in ILs have begin to be described. 1a The present work shows that these enzyme preparations, which have prov- en efficiency in organic solvents, work well in a water- immiscible IL, 1-butyl-3-methylimidazolium hexafluoro- phosphate ([Bmim][PF 6 ]). The reaction chosen is the kinetic resolution of (±)-1-phenylethanol (1) by transe- sterification with vinyl acetate (2) (Scheme 1); this reac- tion has been often chosen for evaluating the reaction rates and enantioselectivity with various lipases. 2,3 The Candida rugosa lipase (CRL) is reported to give worst result in [Bmim][PF 6 ] 3b (for kinetic resolution of (±)-1- phenylethanol). The lipase from Burkholderia cepacia (BCL) has been found to give very poor reaction rates in [Bmim][PF 6 ] for transesterification of (±)-1-phenyl- ethanol. 2b,c Hence these two enzymes were converted into above-mentioned formulations and these enzyme forms were evaluated for their comparative performance in the ionic liquid. The alcoholysis between (±)-1-phenylethanol (1 mmol) and vinyl acetate (1 mmol) was carried out in 1 ml ionic liquid (Acros Organics, USA; the purity of [Bmim][PF 6 ] as specified by the vendor was 99.6% (by HPLC) with water content of 0.05% v/v by Karl-Fischer) using differ- ent preparations of CRL/BCL (containing 10 mg of lip- ases) at a fixed temperature as mentioned in the legend with constant shaking at 250 rpm. CRL (lipase AYS ‘Amano’) and BCL (lipase PS ‘Amano’) were a kind gift from Amano, Nagoya, Japan. For analysis, 50 ll of the reaction mixture was extracted with 500 ll n-hexane/ 0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2006.11.057 Keywords: Cross-linked enzyme aggregates; Enantioselectivity; Enzy- me precipitated and rinsed with propanol; Ionic liquids; Lipases; Propanol-rinsed enzyme preparation; Protein coated microcrystals. * Corresponding author. Tel.: +91 11 2659 1503/91 11 2659 6568; fax: +91 11 2658 1073; e-mail: appliedbiocat@yahoo.co.in Bioorganic & Medicinal Chemistry Letters 17 (2007) 921–924