The synthesis of 6′S-Me-2′-O,4′-C-ethylenebridged 5-methyluridine (6′S-Me-ENA-T) was achieved using visible light-mediated stereoselective radical cyclization as a key step. This is the first example of a method for constructing a 2′,4′-bridged structure from a 4′-carbon radical intermediate. The 6′S-Me-ENA-T monomer was successfully incorporated into oligonucleotides, and their properties were examined. The oligonucleotides containing 6′S-Me-ENA-T exhibited a highly selective hybridization affinity toward single-stranded RNA and an excellent enzymatic stability, compared to the corresponding LNA-and ENA-modified oligonucleotides.

Novel 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) analogs with exocyclic methylene and (R)-methyl groups at the 6'position were synthesized. The obtained 6'-methylene-ENA and 6'R-Me-ENA were incorporated into oligonucleotides, and their properties were compared with those of the corresponding natural, ENA-, and 6'S-Me-ENA-modified oligonucleotides. The duplex-stabilizing ability of 6'-methylene-ENA and 6'R-Me-ENA with single-stranded RNA (ssRNA) was similar to that of ENA and 6'S-Me-ENA under both dilute and molecular crowding conditions. On the other hand, oligonucleotides containing 6'-methylene-ENA and 6'R-Me-ENA formed stable triplexes with double-stranded DNA (dsDNA) in comparison with those containing 6'S-Me-ENA. Their thermodynamic properties were also calculated to explore the influence of the substituents introduced at the 6'-position of ENA. Moreover, oligonucleotides with ENA analogs exhibited excellent resistance against nucleases. These results suggest that 6'-methylene-ENA and 6'R-Me-ENA are promising candidates for targeting ssRNA and dsDNA.