Regiochemical Analysis of the ProTide Activation Mechanism

ProTides are nucleotide analogues used for the treatment of specific viral infections. These compounds consist of a masked nucleotide that undergoes in vivo enzymatic and spontaneous chemical transformations to generate a free mononucleotide that is ultimately transformed to the pharmaceutically active triphosphorylated drug. The three FDA approved ProTides are composed of a phosphoramidate (P-N) core coupled with a nucleoside analogue, phenol, and an l-alanyl carboxylate ester. The previously proposed mechanism of activation postulates the existence of an unstable 5-membered mixed anhydride cyclic intermediate formed from the direct attack of the carboxylate group of the l-alanyl moiety with expulsion of phenol. The mixed anhydride cyclic intermediate is further postulated to undergo spontaneous hydrolysis to form a linear l-alanyl phosphoramidate product. In the proposed mechanism of activation, the 5-membered mixed anhydride intermediate has been detected previously using mass spectrometry, but the specific site of nucleophilic attack by water (P-O versus C-O) has not been determined. To further interrogate the mechanism for hydrolysis of the putative 5-membered cyclic intermediate formed during ProTide activation, the reaction was conducted in ¹⁸O-labeled water using a ProTide analogue that could be activated by carboxypeptidase Y. Mass spectrometry and ³¹P NMR spectroscopy were used to demonstrate that the hydrolysis of the mixed anhydride 5-membered intermediate occurs with exclusive attack at the phosphorus center.