A general method for the chemical synthesis of gamma-32P-labeled or unlabeled nucleoside 5(')-triphosphates and thiamine triphosphate

Abstract

Several methods for the chemical synthesis of gamma-32P-labeled and unlabeled nucleoside 5(')-triphosphates and thiamine triphosphate (ThTP) have been described. They often proved unsatisfactory because of low yield, requirement for anhydrous solvents, procedures involving several steps or insufficient specific radioactivity of the labeled triphosphate. In the method described here, all these drawbacks are avoided. The synthesis of [gamma-32P]ThTP was carried out in one step, using 1,3-dicyclohexyl carbodiimide as condensing agent for thiamine diphosphate and phosphoric acid in a dimethyl sulfoxide/pyridine solvent mixture. Anhydrous solvents were not required and the yield reached 90%. After purification, [gamma-32P]ThTP had a specific radioactivity of 11Ci/mmol and was suitable for protein phosphorylation. The method can also be used for the synthesis of [gamma-32P]ATP of the desired specific radioactivity. It can easily be applied to the synthesis of unlabeled ThTP or ribo- and deoxyribonucleoside 5(')-triphosphates. In the latter case, inexpensive 5(')-monophosphate precursors can be used as reactants in a 20-fold excess of phosphoric acid. Deoxyribonucleoside 5(')-triphosphates were obtained in 6h with a yield of at least 70%. After purification, the nucleotides were found to be suitable substrates for Taq polymerase during polymerase chain reaction cycling. Our method can easily be scaled up for industrial synthesis of a variety of labeled and unlabeled triphosphoric derivatives from their mono- or diphosphate precursors.