Phosphoramidates 1 and 2 were synthesized by H-phosphonate methodology and subsequent oxidative amination with L-alanine methyl ester. The removal of the protecting groups at pH = 7.5 and 37 degrees C in the absence and presence of porcine liver esterase (PLE) or glutathione (GSH) was monitored by HPLC. The stability of phosphoramidate 1 was additionally studied at pH = 9 and 10. The reduction of the disulfide bond with glutathione from 2 triggers the removal of the protecting group by cyclization releasing quantitatively nucleoside 5 '-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]-phosphoramidate} (7) as the desired product. With 1, enzymatic deacetylation or acetyl migration from the sulfur atom to the adjacent hydrated oxo group followed by chemical cyclization produces 7. The S-S-bond-mediated dimerization (8) competes as a side reaction. Prolonged treatment, however, resulted in the conversion of the S-S dimer 8 into 7 that undergoes slow alanine methyl ester hydrolysis to form 10.