Mechanistic Insights Into the Oxidative Reactivity of 2‐Selenouridine in tRNA

ABSTRACT Selenium‐containing nucleosides, such as 2‐selenouridine, represent natural transfer RNA (tRNA) modifications that influence translation fidelity and cellular stress responses. In this study, we investigated the oxidative behavior of Se2U and its derivatives, including 5‐methylaminomethyl‐2‐selenouridine, both as free nucleosides and within a tRNA anticodon stem‐loop model. Electrochemical and spectroelectrochemical analyses revealed that Se2U exhibits greater redox reactivity than its sulfur analogue, 2‐thiouridine, undergoing rapid and reversible oxidation to diselenide species under anaerobic conditions. Chemical oxidation with hydrogen peroxide produced diselenides and deselenated products, while biologically relevant thiols, such as glutathione and dithiothreitol, efficiently restored the parent nucleosides. Oxidation of Se2U within RNA led to uridine and 4‐pyrimidinone riboside formation, potentially altering codon recognition, and enabled nucleophilic substitution at the C2 position both by glutathione, as a biologically relevant nucleophile, and by hexafluoroisopropanol, a nucleophilic buffer component used in our experiments, underscoring the general character of this transformation. These findings suggest that oxidation proceeds via transient acidic selenium species, which act as efficient leaving groups and may facilitate additional post‐transcriptional modifications under oxidative stress. Overall, this study provides mechanistic insight into Se2U oxidation and suggests that oxidative activation ‐ mediated tRNA modifications, could influence translation and cellular stress adaptation.