Synthesis and Structural Analysis of N3‐Methyluridine and 2'‐Alkoxy/Fluoro‐N3‐Methyluridine Nucleosides by Using NMR Spectroscopy, X‐Ray Crystallography, and Computational Methods.

Dual modifications (base and sugar) in siRNA have the potential to overcome off‐target effects and impart nuclease resistance. Here we present the synthesis and detailed structural analysis of N3‐methyluridine and 2'‐alkoxy/fluoro‐N3‐methyluridine modifications, the rationally designed modifications for oligonucleotide therapeutics. Using X‐ray crystallography and 1H NMR spectral data, it is clear that the C2'‐endo conformation of N3‐methyluridine switches to the C3'‐endo pucker state preferentially due to the introduction of electron‐withdrawing 2'‐alkoxy or fluoro substituents. DFT study confirms that the modifications preferentially acquire the C3'‐endo conformation. NBO analysis indicates that in the presence of the 2'‐alkoxy/fluoro substituents, the C3'‐endo conformation is stabilized due to nO4'→σ*C1'‐N1 anomeric as well as σC3'‐H→σ*C2'‐R and σC1'‐C2'→σ*C3'‐OH hyperconjugation effects. The W−C hydrogen bond energies of the modified nucleosides were calculated using the DFT and SAPT methods at the modified base pair regions, and it was observed that one of the hydrogen bonds gets disrupted due to the presence of the N3‐methyl group, causing significant decline in hydrogen bond energy. These detailed structural studies will help to expand the scope of these destabilizing modifications in the rational design of RNAi‐based therapeutics. [ABSTRACT FROM AUTHOR]