Distinct doorway states lead to triplet excited state generation in epigenetic RNA nucleosides

N ⁶-Hydroxymethyladenosine (hm⁶A) and N⁶-formyladenosine (f⁶A) are two important intermediates during the demethylation process of N⁶-methyladenosine (m⁶A), which has been proven to show epigenetic function in mRNA. However, there is no knowledge about how the chemical integrity and stability could be altered when these two nucleosides are exposed to ultraviolet (UV) radiation. Herein, we report the first study on excited state dynamics of hm⁶A and f⁶A in solutions by using femtosecond time-resolved spectroscopy and quantum chemistry calculations. Surprisingly, triplet excited species are clearly identified in both hm⁶A and f⁶A after UV excitation, which is in sharp contrast to the 10⁻³ level triplet yield of adenosine scaffolds. Moreover, the doorway states leading to triplet states are found to be an intramolecular charge transfer state and a lower-lying dark nπ* state in hm⁶A and f⁶A, respectively. These discoveries pave the way to further study their effects on RNA strands and provide insight for understanding RNA photochemistry.