Efficient and Improved Solution-Phase Synthesis of Modified RNA Dinucleotides: Versatile Synthons in Cap 1 mRNA Therapeutics

An efficient and improved chemical method for the gram-scale synthesis of RNA dinucleotides such as pA m pA, pA m pG, and pA m pU is described that utilizes phosphoramidite chemistry using a solution-phase strategy. The first step involves the coupling between 5′- O -MMT protected nucleoside-3′- O -phosphoramidite and protected nucleoside containing the free 5′-OH group in the presence of tetrazole, followed by the oxidation of phosphite triester using tert -butyl hydroperoxide to afford the corresponding protected N m pN. Then, the 5′- O -MMT is cleaved under 3% TCA/DCM conditions. Finally, the 5′-hydroxyl group is phosphorylated by the use of an activated bis(2-cyanoethyl)- N, N -diisopropyl phosphoramidite using tetrazole, followed by the oxidation of trivalent to pentavalent phosphorus using tert -butyl hydroperoxide and subsequent deprotection using ammonium hydroxide to afford the corresponding RNA dinucleotide, pN m pN, in good yields with high purity (>99.5%).