Total synthesis of all stereoisomers of C5- glyceryl-methyl-2′-deoxycytidine 5gmC and their occurrence in Chlamydomonas reinhardtii

C5-glyceryl-methyl-2′-deoxycytidine (5gmC) is a hypermodified DNA nucleoside found in Chlamydomonas reinhardtii. It is a newly discovered eukaryotic DNA modification thought to serve as an epigenetic marker to counteract DNA methylation in regulating photosynthesis. Natural 5gmC is formed by the 5mC-modifying enzyme (CMD1), which transfers a glyceryl moiety from vitamin C to the methyl group, generating two stereoisomeric derivatives. However, the existence of their side-chain epimers and their effects on the C. reinhardtii DNA remain unknown, and the total synthesis of the glyceryl family is unrealized. Our study presents the total synthesis of these nucleosides utilizing allylation-oxidation-amination-dihydroxylation reactions. All four 5gmC stereoisomers, including natural P1 and P2, are synthesized with an overall yield of 0.4% in 9 steps. This allows us to investigate their effects on C. reinhardtii growth and genomic disturbance. This synthesis provides a foundation for further analysis of the biological function of the 5gmC family.