Introduction of a bulky lipophilic acyl entity at the 2'-OH position of both 1-beta-D-arabinofuranosylthymine (araT) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), consistently resulted in a marked ( approximately 10-fold) increase in the inhibitory activity of these new arabinosyl nucleoside analogues for the mitochondrial thymidine kinase (TK-2)-catalysed conversion of 2 microM [methyl-(3)H]dThd to [methyl-(3)H]dTMP. The most potent derivatives were inhibitory to [methyl-(3)H]dThd phosphorylation by TK-2 within the lower micromolar concentration range. Substitution of the arabinosyl nucleoside derivatives with the acyl groups also dramatically increased the selectivity of these compounds. The inhibitory activity of araT and BVaraU to dThd phosphorylation by other related nucleoside kinases, including herpes simplex virus type 1 TK, varicella-zoster virus TK, and cytosolic TK-1, was completely annihilated upon 2'-O-acyl substitution (IC(50) > or = 1000 microM). Kinetic analysis revealed purely competitive inhibition of 2'-O-acyl-BVaraU against TK-2-catalysed thymidine phosphorylation (K(i)/K(m): 2.3). However, 2'-O-acyl-BVaraU was extremely poorly converted to the corresponding arabinosyl nucleoside 5'-monophosphate by TK-2 as revealed by [gamma-(32)P]phosphate transfer studies from [gamma-(32)P]ATP. Thus, the 2'-O-acyl derivatives of BVaraU did not behave as substrates, but rather as potent and highly selective inhibitors of TK-2. This is the first report on such a highly selective arabinosyl nucleoside inhibitor of mitochondrial TK-2, and opens perspectives for the rational design of selective mitochondrial TK-2 inhibitors.