Nucleoside 3′-Phosphorofluoridates for P(V)-Based Oligonucleotide Synthesis

Oligonucleotides (ONs) are fundamental to modern life sciences and nucleic acid-based therapeutics. Although P(III)-based phosphoramidite chemistry remains industry standard for ON synthesis, its requirement for phosphorus oxidation every coupling cycle is operationally complex. Inspired by Michelson and Todd, who synthesized the first dinucleotide via a 3'-phosphorochloridate [P(V)-Cl] intermediate, we have developed a strategy for ON synthesis using nucleoside 3'-phosphorofluoridates [P(V)-F] as bench-stable building blocks. In the presence of silicon-based activators, the enhanced reactivity of the P(V)-F bond resulted in more efficient nucleotide coupling than conventional P(III)-based phosphoramidite chemistry. Furthermore, automated solid-phase synthesis of a 20-mer ON can be achieved on a standard DNA/RNA synthesizer. Our study demonstrates that P(V)-based phosphorofluoridate chemistry facilitates oxidation-free ON synthesis, providing a complementary approach to conventional P(III)-based phosphoramidite chemistry.