Influence of C5 Methylation on Cytidine Photohydration Revealed by Deep-Ultraviolet Transient Absorption Anisotropy Spectroscopy

C5 methylation enhances the photostability of monomeric cytidine (Cyd), but the molecular basis of this effect has remained unresolved. Here, we examine the photodynamics of Cyd and its C5-methylated analogue, 5-methylcytidine (5mCyd), using femtosecond deep-ultraviolet transient absorption anisotropy spectroscopy. Recent theoretical and spectroscopic studies have highlighted a metastable ground-state intermediate with a twisted C5═C6 double bond, which plays a key role in the photodynamics of pyrimidine nucleobases. In Cyd, our anisotropy-resolved measurements captured the sub-picosecond ¹ππ* → S₀ internal conversion, with no detectable signature of a long-lived ¹nπ* state. Instead, our results support the generation of a ground-state twisted conformer as the reactive intermediate for hydration. In 5mCyd, the yield of this intermediate is significantly reduced, indicating that C5 methylation suppresses its formation. The observed correlation between twisted conformer formation and the photostability toward hydration highlights how subtle chemical modifications can markedly alter photochemical activities.