Synthesis and Properties of Nucleobase-Sugar Dual Modified Nucleic Acids: 2 ' -OMe-RNA and scpBNA Bearing a 5-Hydroxycytosine Nucleobase.

Naturally occurring 5-hydroxycytosine ( 5-OH Cyt), which is associated with DNA damage, was recently found to reduce the hepatotoxicity of antisense oligonucleotides (ASOs) without compromising its antisense activity when used as a replacement for cytosine (Cyt). Additionally, sugar-modified nucleic acids, such as 2'- O -methylribonucleic acid (2'-OMe-RNA) and 2'- O ,4'- C -spirocyclopropylene-bridged nucleic acid (scpBNA), have emerged as useful antisense materials. Herein, we aimed to combine these two advantages by designing dual modified nucleic acids 2'-OMe-RNA- 5-OH Cyt and scpBNA- 5-OH Cyt bearing the 5-OH Cyt nucleobase to develop efficient and safe ASOs. We describe the synthesis of 2'-OMe-RNA- 5-OH Cyt and scpBNA- 5-OH Cyt phosphoramidites and their incorporation into oligonucleotides (ONs). The duplex-forming ability and base discrimination properties of 2'-OMe-RNA- 5-OH Cyt- and scpBNA- 5-OH Cyt-modified ONs were similar to those of 2'-OMe-RNA-Cyt- and scpBNA- m Cyt-modified ONs, respectively. We also synthesized two 2'-OMe-RNA- 5-OH Cyt-modified ASOs, and one of the two was found to exhibit reduced hepatotoxicity while retaining target mRNA knockdown activity in in vivo experiments.