Fruit fly (Drosophila melanogaster) deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non-natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity > 98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and room temperature for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraA-MP). Upon optimization of the reaction conditions (50 mM ammonium acetate, substrate/ATP ratio = 1:1.25, 2 mM MgCl 2 , 378C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (> 90%), whereas with the soluble enzyme lower conversions were achieved (78-87%). Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%).