4′‑C‑Methoxy-2′-deoxy-2′-fluoro Modified Ribonucleotides Improve Metabolic Stability and Elicit Efficient RNAi-Mediated Gene Silencing

We designed novel 4′-modified 2′-deoxy-2′-fluorouridine (2′-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4′-C-methoxy (4′-OMe) as the alpha (C4′α) or beta (C4′β) epimers. The C4′α epimer was synthesized by a stereoselective route in six steps; however, both α and β epimers could be obtained by a nonstereoselective approach starting from 2′-F U. 1H NMR analysis and computational investigation of the α-epimer revealed that the 4′-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the β-epimer led to significant destabilization. Both 4′-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4′-OMe substituent and the vicinal 5′- and 3′-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs containing several C4′α-epimer monomers in the sense or antisense strands triggered RNAi-mediated gene silencing with efficiencies comparable to that of 2′-F U.