The enantioselective resolution of (R,S)-3-n-butylphthalide can be achieved by Novozyme 435-catalyzed transesterification in microaqueous medium. We studied the influence of organic solvent and initial water activity on this resolution. Tetrahydrofuran markedly improved the conversion of (R,S)-3-n-butylphthalide, the enantiomeric excess of remaining 3-n-butylphthalide, and the enantiomeric ratio. The optimum solvents were mixtures (1.9 mL) of 50% (vol/vol) tetrahydrofuran and 50% (vol/vol) hexane. The optimal initial water activity was 0.62, and the optimal molar ratio of vinyl acetate to (RS)-3-n-butylphthalide (0.4:0.05 mmol) was 8:1 in a 2.0-mL reaction system containing the optimized solvents (1.9 mL). In addition, the optimal reaction time was 48 h. Under these conditions, enantiopure 3-n-butylphthalide with an optical purity of 97.2% enantiomeric excess and 49.5% yield could be obtained at 30 degrees C. Furthermore, the enantiomeric excess of product was over 98%. (C) 2008 Published by Elsevier Ltd.