High-throughput synthesis of HepDirect prodrugs of nucleoside monophosphates

A high-throughput phosphoramidite method for HepDirect prodrug synthesis was optimized on seven representative nucleosides, adenosine, inosine, guanosine, uridine, cytidine, AICA-riboside, and thymidine, each on a 5 mg scale. The variables optimized included (1) reaction time, (2) reaction temperature, (3) activating agent, (4) solvent, (5) purification method, and (6) stoichiometry. Preparative HPLC with mass-based fraction collection and yield determination from an ELSD standard curve enabled high-throughput. The optimized conditions for the representative nucleosides required 6 mol equiv of phosphoramidite to nucleoside and resulted in an average HPLC determined yield of 31 +/- 14% and HPLC purity of 93 +/- 3%.