An activator-free method for the synthesis of nucleotide 5′–5′ triphosphate mRNA capping agent

Abstract

The traditional synthesis of nucleotide 5′–5′ triphosphate linkages involves a “2 + 1” phosphate coupling mediated by a large excess of metal salts, where the salt removal is a labor and waste-intensive step. We have omitted the metal salts from this reaction by modulating the solubility and reactivity of the monophosphate coupling partner using a precise combination of the reaction solvent and the paired cation. This resulted in simplified downstream purification, where the amount of waste generated was reduced by over 60% throughout the whole process. We have demonstrated this synthetic approach on ARCA, an mRNA 5′-capping agent. Both the chemical purity of the product and its performance in mRNA was comparable to commercial samples.