Scalable Synthesis and Biological Evaluation of 3′-Deoxy-3′,4′-didehydronucleoside Triphosphates

3'-Deoxy-3',4'-didehydronucleoside triphosphates (ddhNTPs) are antiviral metabolites produced by viperin and viperin-like enzymes, all of which are members of the radical SAM superfamily of enzymes. To date, synthetic access to ddhUTP, ddhGTP, and ddhATP has remained limited. We report a scalable synthesis of the complete ddhN nucleoside family using Bobbitt's salt-mediated oxidation of nucleoside orthoformates, including a chromatography-free route to ddhC on a gram scale. This methodology enabled the first synthesis and evaluation of ddhUTP, ddhGTP, and ddhATP as inhibitors of RNA-dependent RNA polymerases, with ddhATP confirmed to act as an obligate chain terminator through primer extension assays.