Synthesis and Multiple Incorporations of 2′-O-Methyl-5-hydroxymethylcytidine, 5-Hydroxymethylcytidine and 5-Formylcytidine Monomers into RNA Oligonucleotides

The synthesis of 2′-O-methyl-5-hydroxymethylcytidine (hm5Cm), 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C) phosphoramidite monomers has been developed. Optimisation of mild post-synthetic deprotection conditions enabled the synthesis of RNA containing all four naturally occurring cytosine modifications (hm5Cm, hm5C, f5C plus 5-methylcytosine). Given the considerable interest in RNA modifications and epitranscriptomics, the availability of synthetic monomers and RNAs containing these modifications will be valuable for elucidating their biological function(s).We acknowledge support from the University of Cambridge and Cancer Research UK program. The Balasubramanian laboratory is supported by core funding from Cancer Research UK (C14303/A17197). S.B. is a Senior Investigator of the Wellcome Trust (grant no. 099232/z /12/z)