Mechanistic Insights into the Excited-State Relaxation of Two Important Epigenetic RNA Nucleosides: N6-Hydroxymethyladenosine and N6-Formyladenosine

Abstract N6-Hydroxymethyladenosine (hm6A) and N6-formyladenosine (f6A) are two important epigenetic RNA nucleosides, which have shown important biological functions in the demethylation process of N6-methyladenosine (m6A). However, their excited-state decay mechanisms after ultraviolet (UV) radiation are unclear. Herein, we have investigated the excited-state properties and relaxation pathways of hm6A and f6A by using the CASPT2//CASSCF method. For hm6A, the photoexcited 1ππ* state first bifurcates to reach the 1ππ* minimum and 1ππ*CT minimum, which can either internally convert to the S0 state via the 1ππ*/S0 and 1ππ*CT/S0 conical intersections (CIs), or undergo intersystem crossing (ISC) to the 3ππ* state via the 1ππ*/3ππ* and 1ππ*CT/3ππ* singlet–triplet crossing points (STCPs), respectively. Finally, the populated 3ππ* state can further evolve into the nearby 3ππ*/S0 STCP to hop to the S0 state. Differently, for f6A, the photoexcited 1ππ* state first relaxes to its 1ππ* minimum, from which the system can internally convert to the 1nπ* state directly or to the S0 state via the nearby 1ππ*/S0 CI. Additionally, the 1ππ* → 3nπ* and 1nπ* → 3nπ* ISC processes can occur at the 1ππ* minimum and 1nπ* minimum, respectively, which are followed by ISC to the S0 state via the 3nπ*/S0 STCP or internal conversion (IC) to the 3ππ* state via the 3nπ*/3ππ* CI. Subsequently, the 3ππ* state deactivates to the S0 state via the 3ππ*/S0 and 3ππ*/S0-I STCPs. This work rationalizes the ultrafast excited-state relaxation and triplet formation of hm6A and f6A and could contribute to understanding the photophysical and photochemical mechanisms of similar epigenetic RNA nucleosides and their derivatives.