Novozym 435-catalyzed regioselective acylation of 1-beta-D-arabinofuranosylcytosine (ara-C) with vinyl caprylate for the preparation of its 5'-O-acyl derivative has been performed in six co-solvent mixtures and three pure polar solvents for the first time. Novozym 435 displayed low activity towards 1-beta-D-arabinofuranosylcytosine in pure polar solvents, although those solvents can dissolve the nucleosides well. When a hexane-pyridine co-solvent system was adopted, both the initial rate and the substrate conversion were enhanced markedly.The most suitable co-solvent, initial water activity, reaction temperature and the molar ratio of vinyl caprylate to ara-C were hexane/pyridine (28/72, v/v), 0.03, 40 degrees C and 15, respectively. Under these conditions, the initial rate, the substrate conversion and the regioselectivity were as high as 99.0 mmol h(-1), 98% and >99%, respectively. The product of the Novozym 435-catalyzed reaction was characterized by C-13 NMR and confirmed to be 5'-O-octanoyl 1-beta-D-arabinofuranosylcytosine. (C) 2008 Elsevier B.V. All rights reserved.