Synthesis of 4-thiouridines with prodrug functionalization for RNA metabolic labeling

Metabolic labeling has emerged as a powerful tool to endow RNA with reactive handles allowing for subsequent chemical derivatization and processing. Recently, thiolated nucleosides, such as 4-thiouridine (4sU), have attracted great interest in metabolic labeling-based RNA sequencing approaches (TUC-seq, SLAM-seq, TimeLapse-seq) to study cellular RNA expression and decay dynamics. For these and other applications (e.g. PAR-CLIP), thus far only the naked nucleoside 4sU has been applied. Here we examined the concept of derivatizing 4sU into a 5'-monophosphate prodrug that would allow for cell permeation and potentially improve labeling efficiency by bypassing the rate-limiting first step of 5' phosphorylation of the nucleoside into the ultimately bioactive 4sU triphosphate (4sUTP). To this end, we developed robust synthetic routes towards diverse 4sU monophosphate prodrugs. Using metabolic labeling assays, we found that most of the newly introduced 4sU prodrugs were well tolerated by the cells. One derivative, the bis(4-acetyloxybenzyl) 5'-monophosphate of 4sU, was also efficiently incorporated into nascent RNA.