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Nucleosides or deoxynucleosides were converted to a number of phosphorylated nucleotide and deoxynucleotide derivatives by ammonium or alkali dihydrogen phosphates in formamide. Conversions were smaller and slower at room temperature and greater and faster at elevated temperatures. Nucleotides afforded product mixtures similar to those obtained for nucleosides under the same conditions, indicating the occurrence of transphosphorylation processes. Products of reaction at elevated temperatures were cyclic nucleotides, nucleoside monophosphates, nucleoside diphosphates and cyclic nucleotide phosphates. The relative amounts of products formed were quite temperature dependent. Cyclic nucleotides were found to be in greatest abudance for reactions run at 125° or above. Relative yields of 2', 3' and 5' nucleotides and 3' and 5' deoxynucleotides from several experiments are reported. 5'-Monophosphates were generally found to be present in larger quantities than 2' or 3' monophosphates. 2'-Deoxyadenosine showed a preference for phosphorylation at the 3' position. Conclusions reached from mechanistic studies are that the phosphorylations are a series of equilibrium reactions, with cyclic nucleotides being formed irreversibly.

A possible prebiotic phosphorylation method has been investigated in which formamide served as the reaction medium. Nucleotides and nucleotide derivatives were formed when nucleosides were allowed to react with different orthophosphate, hydrogen phosphate or dihydrogen phosphate salts or with different condensed phosphate salts. The reaction products obtained from the phosphorylation of adenosine were 2', 3' and 5'-AMPs, 2', 5' and 3', 5'-ADPs, and 2', 3'-cyclic AMP. The extent of phosphorylation in formamide exceeded 50% under favorable conditions after 15 days at 70°. The acidic dihydrogen phosphates and condensed hydrogen phosphates proved to be the best phosphorylating agents. The presence of water in the medium decreased the yield of nucleotide derivatives, but some phosphorylation of adenosine was detected using dihydrogen phosphate in formamide containing water. The phosphorylation reactions were also observed for deoxynucleosides. Little decomposition of the nucleosides was detected during the reaction time needed to form nucleotide derivatives. The facility with which phosphorylation takes place in formamide under very mild conditions may justify further studies both of prebiotic phosphorylation and synthetic phosphorylation using this solvent.

An objective of this work is to elucidate the mechanism of phosphorylation of nucleosides in amide solvents and in urea. A second objective is to assess the importance of phosphorylation and dephosphorylation of nucleotide derivatives in amide environments. Although the most complex amide studied here was N-methylacetamide, inferences are made on the importance of dephosphorylation for nucleotides in oligopeptide environments.

Simple aliphatic alcohols, deoxynucleosides and nucleosides undergo reaction with formamide yielding formate esters. Formate ester formation was observed to occur slowly at 100°C and more rapidly at 130°C. As expected, formate esters were hydrolyzed to the alcohol and formic acid upon heating in aqueous solution. It was proposed to study the possibility that formate esters are formed initially in amide solvents, followed by displacement of formate by dihydrogen phosphate ion to form monophosphate esters. Experiments are described which demonstrate the formation and hydrolysis of formate esters, as well as their lack of reaction with hydrogen phosphate ion. Formate esters are not intermediates in the phosphorylation of nucleosides in formamide. Their formation has been observed and such an esterification is a side reaction during the phosphorylation of nucleosides in formamide.