Syntheses and characterizations of adenosine 5′-(α,β- N-methylimido)di and triphosphates and adenosine 5′-(β,γ- N-methylimido)triphosphate—Comparisons with their nonmethylated analogs

Abstract

N-Methylimidodiphosphate [PN(Me)P] has been synthesized for the first time by hydrolysis of N-methylimidodiphosphoryl tetrachloride. PN(Me)P was coupled with 5′-tosyladenosine to produce adenosine 5′-[α,β- N-methylimido]diphosphate [AMPN(Me)P] which in turn was treated with phosphocreatine in the creatine kinase reaction to generate adenosine 5′-[α,β- N-methylimido]triphosphate [AMPN(Me)PP]. Using the Michelson synthesis, PN(Me)P was coupled with adenosine 5′-monophosphate to produce adenylyl N-methylimidophosphate [AMP · PN(Me)P]. All three new purine nucleotide analogs were characterized by mass spectrometry and 31P NMR spectroscopy. Their enzyme kinetic parameters ( V max and K m values) were measured in the creatine kinase reaction and directly compared to similar kinetic parameters obtained for the corresponding nonmethylated analogs and ADP and ATP. AMPN(Me)P was shown to have a V max value ca. 40% of that of ADP although its K m value is ca. 100 times greater than that for ADP. AMPN(Me)PP was also a substrate, whereas AMP · PN(Me)P showed no detectable inhibitory properties.