Facile Synthesis, Geometry, and 2′-Substituent-Dependent in Vivo Activity of 5′‑(E)- and 5′‑(Z)‑Vinylphosphonate-Modified siRNA Conjugates

(E)-Vinylphosphonate ((E)-VP), a metabolically stable phosphate mimic at the 5′-end of the antisense strand, enhances the in vivo potency of siRNA. Here we describe a straightforward synthetic approach to incorporate a nucleotide carrying a vinylphosphonate (VP) moiety at the 5′-end of oligonucleotides under standard solid-phase synthesis and deprotection conditions by utilizing pivaloyloxymethyl (POM) protected VP-nucleoside phosphoramidites. The POM protection enhances scope and scalability of 5′-VP-modified oligonucleotides and, in a broader sense, the synthesis of oligonucleotides modified with phosphonate moieties. Trivalent N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) comprising (E)-geometrical isomer of VP showed improved RISC loading with robust RNAi-mediated gene silencing in mice compared to the corresponding (Z)-isomer despite similar tissue accumulation. We also obtained structural insights into why bulkier 2′-ribosugar substitutions such as 2′-O-[2-(methylamino)-2-oxoethyl] are well tolerated only when combined with 5′-(E)-VP.