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

Abstract

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 N 3 -methyluridine and 2'-alkoxy/fluoro-N 3 -methyluridine modifications, the rationally designed modifications for oligonucleotide therapeutics. Using X-ray crystallography and 1 H NMR spectral data, it is clear that the C2'-endo conformation of N 3methyluridine 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 n O4' !σ* C1'-N1 anomeric as well as σ C3'-H !σ* C2'-R and σ C1'-C2' !σ* C3'-OH hyperconjugation effects. The WÀ 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 N 3 -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.

4′-C-Acetamidomethyl-2′-O-methoxyethyl Nucleic Acid Modifications Improve Thermal Stability, Nuclease Resistance, Potency, and hAgo2 Binding of Small Interfering RNAs

“…The sugar conformation of 4′- C -azidomethyl-2′- O -MOE ( 7a and 7b ), 2′- O -MOE ( 13 ), and 2′-F ( 19 ) was compared using 1 H NMR spectroscopy (Table S2). The population of S -conformer was calculated using S (%) = 10 × J H1′–H2′ . It is reported in the literature that 2′- O -Me, 2′-F, and 2′- O -MOE-modified nucleosides ( 13 , 16 , and 19 ) exhibited very less J H1′–H2′ (less than 4 Hz) .…”

“…The population of Sconformer was calculated using S (%) = 10 × J H1′−H2′ . 31 It is reported in the literature that 2′-O-Me, 2′-F, and 2′-O-MOEmodified nucleosides (13, 16, and 19) exhibited very less J H1′−H2′ (less than 4 Hz). 14 It indicated that the 2′-derivatives prefer C3′-endo (North) conformation in solution and calculated data also represent the same, whereas 4′-Cazidomethyl-2′-O-methoxyethyl thymidine 7a and uridine 7b nucleosides exhibited a higher value of J H1′−H2′ (6−7 Hz).…”

“…DCI (18 mg, 0.154 mmol) in dry acetonitrile (0.2 mL) and bis-amidite reagent (194 mg, 0.645 mmol) was added and stirred at room temperature for 9 h. Saturated NaHCO 3 (50 mL) solution was poured into the reaction mixture and extracted with DCM (3 × 30 mL). The organic layer was dried with Na 2 SO 4 , evaporated, and purified using column chromatography (60−70% EtOAc in hexane +2% NEt 3 ) to obtain compound 10a (75 mg, 66%) as a white solid: R f = 0.6 (100% EtOAc); mp 77−78 °C; 31…”

“…1H), 1.19−1.02 (m, 12H); 31 (20). 2′-Fluoro nucleoside 19 (250 mg, 1.01 mmol) was dissolved in dry pyridine.…”

2′-O-Alkyl-N3-Methyluridine Functionalized Passenger Strand Improves RNAi Activity by Modulating the Thermal Stability

“…Our laboratory has reported the synthesis of a series of 2′-alkoxy/fluoro- N 3 -methyluridine nucleosides including 2′-F-m 3 U, 2′-OMe-m 3 U, 2′-OEt-m 3 U, 2′-OPr-m 3 U, and 2′-OMOE-m 3 U. , The incorporation of these modified monomers at the central position in 12-mer and 14-mer oligonucleotides exhibited a substantial reduction in melting temperature (around 8–12 °C per modification). Moreover, nuclease resistance studies showed that 2′- O -alkyl-m 3 U modifications improved the half-life of oligonucleotides against 3′- and 5′- exonucleases as compared to 2′-fluoro and 2′-OMe modified oligonucleotides.…”