Phosphorylation of Nucleotides with Inorganic Cyclo-Triphosphate

Abstract

Phosphorylation of nucleotides (nucleoside 3′- and 5′-monophosphates, and 2′-deoxynucleoside 5′-monophosphates) with inorganic sodium cyclo-triphosphate (P3m) was studied in aqueous solutions under various conditions (mixing ratio of P3m to nucleotides, pH, reaction temperature, and time). (I) Unprotected nucleoside 5′-monophosphates (5′-NMP’s) were easily phosphorylated at the cis-2′,3′-diol by P3m to form selectively nucleoside 2′,5′-bis(monophosphate) (2′,5′-NDP’s), nucleoside 3′,5′-bis(monophosphate) (3′,5′-NDP’s), and nucleoside 2′,3′-cyclic 5′-bis(monophosphate) (cNDP’s). (2) The phosphorylation of 5′-NMP’s was strongly dependent on mixing ratio, pH, reaction temperature, and time. Under conditions of high mixing ratios of P3m to 5′-NMP’s (5:1–10:1), high pH (12), and room temperature, 92–98% of 5′-NMP’s was converted into 3′,5′-NDP’s and 2′,5′-NDP’s in roughly equimolar quantities. (3) Small quantities (5–8%) of cNDP’s were formed at the initial stage of reaction of 5′-NMP’s with P3m but in the course of the reaction for a long period, cNDP’s were hydrolyzed to 2′,5′-NDP’s and 3′,5′-NDP’s. (4) Nucleoside 3′-monophosphates (3′-NMP’s) and 2′-deoxynucleoside 5′-monophosphates (dNMP’s) could not be phosphorylated by P3m, which indicates that the presence of hydroxyl groups at both 2′- and 3′-positions on nucleotides is indispensable for the phosphorylation of nucleotides with P3m. (5) The mechanism of the formation of 2′,5′-NDP’s, 3′,5′-NDP’s, and cNDP’s in the phosphorylation of 5′-NMP’s with P3m is discussed.