Synthesis of Sensitive RNAs Using Fluoride‐Cleavable Groups as Linkers and Amino‐Group Protection

A chemical method suitable for the synthesis of RNAs containing modifications such as N4‐acetylcytidine (ac4C) that are unstable under the nucleophilic conditions used by standard RNA synthesis methods is described. The method uses the 4‐((t‐butyldimethylsilyl)oxy)‐2‐methoxybutanoyl (SoM) group for the protection of exo‐amino groups of nucleobases and the 4‐((t‐butyldimethylsilyl)oxy)‐2‐((aminophosphaneyl)oxy)butanoyl (SoA) group as the linker for solid phase synthesis. RNA cleavage and amino deprotection are achieved using fluoride under the same conditions used for the removal of the 2′‐OH silyl protecting groups. Using this method, a wide range of electrophilic and base‐sensitive groups including those that play structural and regulatory roles in biological systems and those that are artificially designed for various purposes are expected to be able to be incorporated into any position of any RNA sequences. As a proof of concept, several RNAs containing the highly sensitive ac4C epitranscriptomic modification was synthesized and purified with RP HPLC. MALDI MS analysis indicated that the ac4C modification is completely stable under the fluoride deprotection conditions. The sensitive RNA synthesis method is expected to be able to overcome the long‐lasting obstacle of accessing various modified sensitive RNAs to projects in areas such as epitranscriptomics, molecular biology and the development of nucleic acid therapeutics.