Resolving solvent effects and intersystem crossing in 2′-deoxy-5-formylcytidine: The pivotal role of the emissive nπ* state revealed by femtosecond broadband fluorescence, transient absorption, and theoretical study

2'-deoxy-5-formylcytidine (5fodCyd) is a key epigenetic intermediate in DNA de-methylation and is implicated in cellular responses to oxidative stress, primarily due to its ultrafast intersystem crossing (ISC) upon UV excitation. Despite extensive studies, the precise ISC mechanism, the nature of the precursor state, and the influence of the solvent environment on 5fodCyd's excited-state dynamics remain contentious, particularly in biologically relevant aqueous solutions. Here, we combine femtosecond broadband time-resolved fluorescence, transient absorption spectroscopy, and theoretical calculations to comprehensively investigate the radiative and nonradiative pathways of 5fodCyd in water compared to aprotic solvents acetonitrile and tetrahydrofuran. Our results unequivocally demonstrate a solvent-independent ISC pathway mediated by the formyl carbonyl-based nfπ* state. Notably, we provide direct evidence that the nfπ* state, commonly considered optically dark, is in fact optically bright and contributes significantly to the fluorescence emission of 5fodCyd. This establishes 5fodCyd as the first nucleobase derivative to exhibit dual-state fluorescence from both ππ* and nfπ* states. Furthermore, we show that solvent-specific hydrogen bonding modulates the dynamics of the nfπ* state, offering a novel explanation for the distinct fluorescence behavior observed in protic vs aprotic environments. These findings elucidate the excited-state landscape of 5fodCyd, highlight the pivotal role of the nfπ* state, and emphasize the importance of solvent in shaping deactivation dynamics-not only for 5fodCyd but also for analogous nucleobase derivatives-laying the groundwork for understanding its function as an intrinsic DNA photosensitizer in oxidative stress and genomic instability.