This article describes a simple, reliable, efficient, and improved solution-phase method for the gram-scale chemical synthesis of RNA dinucleotides such as pA_mpA, pA_mpG, and pA_mpU that utilizes phosphoramidite chemistry. The overall synthetic strategy involves three steps. The first step involves the coupling reaction between 5'-O-MMT protected nucleoside-3'-O-phosphoramidite and a protected nucleoside containing a free 5'-OH group in the presence of tetrazole, followed by the oxidation of phosphite triester using tert-butyl hy-droperoxide to give the corresponding protected N_mpN. Next, the 5'-O-MMT is cleaved under 3% trichloroacetic acid in dichloromethane 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 hy-droperoxide and subsequent deprotection using ammonium hydroxide to afford the corresponding RNA dinucleotide, pN_mpN, in good yields with high purity (>99.5%).
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