Synthesis of N(4)-acetylated 3-methylcytidine phosphoramidites for RNA solid-phase synthesis

The growing interest in 3-methylcytidine (m(3)C) originates from the recent discoveries of m(3)C modified tRNAs in humans as well as its intensively debated occurrence in mRNA. Moreover, m(3)C formation can be catalyzed by RNA without the assistance of proteins as has been demonstrated for a naturally occurring riboswitch fold using the methylated form of its cognate ligand as cofactor. Additionally, new RNA sequencing methods have been developed to detect this modification in transcriptome-wide manner. For all these reasons, an increasing demand for synthetic m(3)C containing oligoribonucleotides is emerging. Their chemical synthesis relies on RNA solid-phase synthesis using phosphoramidite building blocks. Here, we describe a facile synthetic path towards N(4)-acetylated 2′-O-TBDMS- and 2′-O-TOM m(3)C phosphoramidites to provide an optimal toolbox for solid-phase synthesis of m(3)C containing RNA. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00706-022-02896-x.