Chemistry of installing epitranscriptomic 5-modified cytidines in RNA oligomers.

Studies of 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C) and 5-carboxycytidine (ca5C) modifications as products of the 5-methylcytidine (m5C) oxidative demethylation pathway in cellular mRNAs constitute an important element of the new epitranscriptomic field of research. The dynamic process of m5C conversion and final turnover to the parent cytidine is considered a post-transcriptional layer of gene-expression regulation. However, the regulatory mechanism associated with epitranscriptomic cytidine modifications remains largely unknown. Therefore, oligonucleotides containing m5C oxidation products are of great value for the next generation of biochemical, biophysical, and structural studies on their function, metabolism, and contribution to human diseases. Herein, we summarize the synthetic strategies developed for the incorporation of hm5C, f5C and ca5C into RNA oligomers by phosphoramidite chemistry, including post-synthetic C5-cytidine functionalization and enzymatic methods.

Protection-Free, Two-step Synthesis of C5-C Functionalized Pyrimidine Nucleosides.

A simple, reliable, and efficient method for the gram-scale chemical synthesis of pyrimidine nucleosides functionalized with C5-carboxyl, nitrile, ester, amide, or amidine, starting from unprotected uridine and cytidine, is described. The protocol involves the synthesis of 5-trifluoromethyluridine and 5-trifluoromethylcytidine with Langlois reagent (CF3 SO2 Na) in the presence of tert-butyl hydroperoxide and subsequent transformation of the CF3 group to the C5-C 'carbon substituents' under alkaline conditions. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of 5-trifluoromethyluridine (5-CF3 U) and 5-trifluoromethylcytidine (5-CF3 C) Basic Protocol 2: Conversion of 5-CF3 U and 5-CF3 C to several C5-substituted ribonucleosides.