Development of Synthetic Routes to 2'-O,4'-C-Spirocyclopentylene-Bridged Nucleic Acids: Thymidine, Guanosine, and...

Modified nucleic acids are crucial for improving the efficacy and safety of antisense oligonucleotides (ASOs). We previously synthesized and evaluated 2'-O,4'-C-spirocyclopentylene-bridged nucleic acid (scpBNA2) bearing the pyrimidine nucleobases thymine (T) and 5-methylcytosine (^mC). Oligonucleotides incorporating scpBNA2 exhibited strong binding affinity toward complementary single-stranded RNA (ssRNA) and high resistance to nuclease degradation. These favorable properties enabled ASOs containing scpBNA2-T and scpBNA2-^mC in the wing regions to induce potent target RNA knockdown both in vitro and in vivo. Furthermore, replacing 2'-O,4'-C-methylene-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) with scpBNA2 significantly reduced hepatotoxicity, underscoring its potential as a promising nucleic acid analog for ASO design. In this study, we established efficient synthetic routes to scpBNA2 nucleosides bearing the purine nucleobases guanine (G) and adenine (A), in addition to the previously reported scpBNA2-T. The scpBNA2-T nucleoside was synthesized via 2',4'-lactonization followed by formation of a 2,2'-anhydropyrimidine intermediate. For scpBNA2-G and scpBNA2-A, we devised a robust strategy that combines late-stage transglycosylation-converting the thymine nucleobase into purines-with an iodocyclization reaction to construct the 2',4'-bridged framework. This concise approach provides access to scpBNA2 nucleosides and supports their broader applications in ASO development.