Stereochemical Course of Phosphoanhydride Synthesis

In the synthesis of phosphoanhydride linkages in nucleoside polyphosphates such as ADP, ATP, and dinucleotide coenzymes according to the procedure introduced by Michelson (ref la), a nucleoside monophosphate is first activated by reaction with diphenyl phosphorochloridate in an inert solvent to produce a P¹-nucleoside P²-bis(phenyl) pyrophosphate. The latter reacts in pyridine with trialkylammonium salts of phosphoric acid, pyrophosphoric acid, or phosphoric esters to produce the desired phosphoanhydride and diphenyl phosphate. The mechanism by which the coupling of a p¹-nucleoside P²-bisphenyl pyrophosphate with phosphates occuss in pyridine has been investigated by analyzing the stereochemical course of the coupling reaction using R_p⁻[α-,¹⁸O]AMPS and S_p⁻[α-¹⁸O]AMPS as the nucleoside monophosphates. Configurational analysis of the coupling products showed that the reaction proceeded with epimerization at the chiral phosphorus. It was concluded that the most probable mechanism for the reaction involves nucleophilic covalent catalysis by pyridine.