Studies on the adenosine deaminase-catalyzed conversion of adenosine and nucleoside prodrugs by different capillary electrophoresis modes

Four kinds of fast and efficient capillary electrophoresis modes, i.e., immobilized enzymatic reactor (IER), electrophoretically mediated microanalysis (EMMA), capillary zone electrophoresis (CZE), and micellar electrokinetic chromatography (MEKC), were first developed to study the adenosine deaminase (ADA)-catalyzed conversion of adenosine and nucleoside prodrugs, which is critical for releasing prodrugs into the intracellular compartment for phosphorylation. The enzyme-activated prodrug approach is a strategy that has been successfully employed to improve physicochemical and pharmacokinetic properties of potential therapeutic agents, especially in the search for antiviral nucleoside analogues. Adenosine, amino-ddG, and amino-D4G could be converted by ADA to different extents under our experimental conditions. Steady-state parameters K_m, V_max, and k_cat were also determined. The substrate efficiencies (k_cat/K_m) of adenosine, amino-ddG, and amino-D4G were 0.19 ± 0.01, 0.047 ± 0.005, and 0.017 ± 0.010 μM⁻¹ s⁻¹, respectively. The enzymatic reaction could be performed at a nanoliter scale and all manipulation steps were combined into a fully automated assay in on-line modes, which opened the possibilities of high-throughput screening of large libraries of synthetic nucleoside analogues for biological activity and a relative mechanism study of nucleoside and its analogues.