NOTES Kinetic Study on the Enzymatic Resolution of Homophenylalanine Ester Using Ionic Liquids Hua Zhao, Robert G. Luo, † and Sanjay V. Malhotra* Department of Chemistry and Environmental Science, New Jersey Institute of Technology, University Heights, Newark, New Jersey 07102 Two ionic liquids (ILs) were investigated as novel media for the enzymatic resolution of amino acid ester to obtain enantiomeric amino acid homophenylalanine. The effects of solvent nature, polarity, and concentration on the kinetic resolution were investi- gated. With change in solvent concentration, a systematic study shows that an improved enzyme activity can be obtained by adjusting these solvent parameters. Introduction R-Amino acids are important building blocks for many pharmaceutically and biologically important compounds. Therefore, development of new methodology to obtain optically pure R-amino acids has always been of interest and a challenge to chemists. Enzymatic resolution of amino acid derivatives is one of the simplest and most efficient methods of synthesizing enantiomerically en- riched amino acids. Traditionally, mixtures of organic solvents and water mixture have been used as reaction media for the kinetic resolution of amino acids. For example, the kinetic resolution of different amino acids by the enzyme alcalase has been studied in acetone- water (1), acetonitrile-water (1), ethanol-water (1), 1-propanol-water (1), tetrahydrofura--water (1), diox- ane-water, tert-butyl alcohol-water (2), 2-methyl-2- propanol-water (3), and DMF (4), etc. In all of these cases a mixture of water and organic solvent has been used. In recent years, ionic liquids have gained a lot of attention as green solvents in organic synthesis and other chemical processes. Ionic liquids with melting points at room temperature and below (as low as -96 °C) can now be produced, which is an important reason why ionic liquids are becoming a more attractive substitute for volatile and toxic organic solvents (5) and are considered environmentally friendly. Also, ionic liquids have many favorable properties, e.g., they are good solvents for a wide range of inorganic, organic, and polymeric materi- als, adjustable polarity, and catalytic effects, etc. There- fore, they have been investigated as reaction media in many organic and organometallic syntheses (6). More recently, ionic liquids have been used in the study of enzymatic systems, such as lipase-catalyzed kinetic resolution of 1-phenylethanol in ionic liquids (7); enzy- matic catalysis in the formation of Z-aspartame in ionic liquids (8); as catalysts in alcoholysis, ammoniolysis, and perhydrolysis reactions by lipase in ionic liquids 1-butyl- 3-methylimidazolium tetrafluoroborate or hexafluoro- phosphate (9); etc. Markedly enhanced enantioselectivity was achieved in ionic liquids for the lipase-catalyzed transesterifications (10). Recently we achieved the synthesis of homophenyla- lanine ester and piperazine-2-carboxylic acid ester and obtained single enantiomers through enzymatic resolu- tion (11, 12). With the foreseeable advantages and goal of finding a proper ionic medium to substitute organic solvents, we carried out a detailed kinetic study on the resolution of homophenylalanine ester. Herein we report the results of this study using ionic liquids [EtPy] + [BF 4 ] - and [EMIM] + [BF 4 ] - , which have proven to be promising medium for the enzymatic resolution of amino acids. Materials and Methods Materials. BL-alcalase is a commercially available endoproteinase of the serine type obtained from Bacillus licheniforms, the major component of which is subtilisin A (Subtilisin Carlsberg). BL-alcalase was produced by Novozymes, Inc. and distributed by Sigma-Aldrich as a brown liquid with a specific activity of 2.4 AU/g 3 for hydrolysis of dimethyl casein at 50 °C and pH 8.3. (According to Novozymes, one Anson Unit [AU] is the amount of enzyme that, under standard conditions, digests hemoglobin at an initial rate liberating per min an amount of tricholoroacetic acid (TCA)-soluble product that gives the same color of phenol reagents as 1 mequiv of tyrosine. Thus, 1 AU ) 1000 U, 1U ) 1 mmol of L-tyrosine methyl ester hydrolyzed per min.) The N-acetyl homophenylalanine ethyl ester was prepared through a three-step reaction strategy (11). All solvents have purity higher than 99%. Ionic liquids 1-ethyl-3-methylimidazo- lium tetrafluoroborate ([EMIM][BF 4 ]) and N-ethyl pyri- dinium tetrafluoroborate ([EtPy][BF 4 ]) were prepared by literature method (13) and dried at 60 °C under vacuum before use. Kinetic Resolution. General Method. Racemic amino acid ethyl ester (0.5 g) was dissolved in 80 mL of mixed * To whom correspondence should be addressed. Email: malhotra@adm.njit.edu. † Current address: Vector Process Development, GTI-Novartis, Gaithersburg, MD 20878. 1016 Biotechnol. Prog. 2003, 19, 1016-1018 10.1021/bp025721b CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 03/05/2003