Direct observation of triple-component fluorescence and triplet formation in epigenetic nucleoside...

2'-deoxy-5-carboxylcytidine (5cadCyd) is a naturally occurring epigenetic DNA nucleoside central to active DNA demethylation and cellular reprogramming, yet its UV-excited-state dynamics remain unresolved. Previous studies have led to conflicting assignments of excited states and relaxation pathways. Here, we combine femtosecond broadband time-resolved fluorescence, transient absorption spectroscopy, and theoretical calculations to delineate the radiative and nonradiative excited-state cascades of 5cadCyd in water. We reveal an unusual two-state-three-component fluorescence dynamics and an ultrafast bifurcation of the initially excited population into two competing relaxation channels. The dominant channel undergoes sub-picosecond internal conversion through an emissive ππ* state with a strongly distorted geometry, providing an efficient photo-stabilizing pathway. A second, less populated yet substantial channel proceeds through a carboxylate-derived nπ* state. Contrary to the prevailing view that nπ* states in pyrimidine nucleobases and their derivatives are optically dark, this state in 5cadCyd is weakly emissive and contributes appreciably to its fluorescence. More importantly, it serves as an efficient gateway for intersystem crossing through spin-orbit coupling, generating a long-lived triplet state with an 18.3 ps formation time. This triplet-forming pathway may enhance the susceptibility of 5cadCyd to photochemical reactions and oxidative photodamage. These results resolve earlier controversy and show that the C5-carboxylate group markedly reshapes the balance between photostability and photoreactivity relative to canonical pyrimidine nucleosides. More broadly, they establish 5cadCyd as a representative of an underrecognized class of nucleobase derivatives in which substituent-derived nπ* states contribute to fluorescence while mediating efficient triplet formation, suggesting an intrinsic epigenetic hotspot for photoinduced oxidative damage and genome instability.