Molecular tautomerism is ubiquitous in nature and plays a crucial role in regulating biological function. In nucleobases, for example, structural tautomerism not only influences base pairing and genetic coding in DNA but also modulates the molecular response to UV irradiation. The photostability of the nucleobases depends on efficient internal conversion and is highly sensitive to structural variations and micro-environmental effects. Among the pyrimidine bases, cytosine exhibits the greatest number of tautomeric forms, offering a rich landscape to explore diverse structural scenarios, while simultaneously posing significant experimental challenges. This study builds on that context by unveiling the gas-phase photophysics of 2-thiocytosine (2TC) from a unique tautomer perspective. Specifically, it elucidates the decay mechanism in the absence of a thiocarbonyl group but under the influence of chalcogen heavy atom substitution. In solution, 2TC exists in its thion form, whose photodynamics are characterized by efficient intersystem crossing to the triplet manifold. Electronic and structural factors associated with the thiocarbonyl group play a crucial role in suppressing internal conversion pathways to the ground state—pathways that are otherwise active in canonical cytosine. This ISC mechanism is tautomer specific and does not apply to the thiol form, which dominates in the gas phase. Time-resolved photoelectron spectroscopy of thiol-2TC reveals ultrafast internal conversion dynamics, alongside the emergence of a long-lived state with nanosecond lifetime. The latter distinguishes the photodynamics from its canonical counterpart, enol cytosine. Ab initio calculations provide detailed insights into the deactivation mechanism of thiol 2TC and clarify the differences on the effect of thionation on both tautomeric forms of cytosine. Finally, we discuss how protonation (and hydrogen bonding) can modulate intersystem crossing in thiobases, with broader implications to other thiocarbonyl-containing compounds.
