Ultrafast Excited State Decay Pathways in Epigenetic Deoxycytidine Derivatives

Epigenetic DNA modification of 2’-deoxycytidine (dC), 5-methyl-2’-deoxycytidine (mdC), together with its enzymatic oxidation products (5-hydroxymethyl-2’-deoxycytidine (hmdC), 5-formyl-2’-deoxycytidine (fdC), and 5-carboxyl-2’-deoxycytidine (cadC)), play a fundamental role in modulating gene expression and as such are forming a second layer of information in DNA [1,2].<br/><br/>Here we provide a unified description of the complex excited state (ES) decay pathways of the epigenetic cytidines through an extensive experimental and theoretical investigation. On the experimental side, we perform ultrafast transient absorption spectroscopy with sub-30-fs temporal resolution, to follow the rapid evolution of the photoexcited wavepacket, and broad spectral coverage in the 1.9-4.2 eV range to identify all major photoinduced signals, including the previously unexplored UV region. On the computational side, we employ a hybrid MS-CASPT2/MM scheme accounting for multi-reference dynamically correlated energies and gradients and considering explicitly the water solvent. We conducted the measurements by exciting the ππ* state with 4.35 eV pump pulse and tracking the response in a broad spectral window that allowed us to observe both photo-induced absorption and stimulated emission signals, that were crucial to correctly assign the spectra to the dynamics. The data has been fitted with global analysis and the evolution associated spectra have been compared with theoretically computed transitions.<br/><br/>In mdC, we find a 130 fs time constant for the motion of the wavepacket from the Franck-Condon point to the plateau. After spending 1 ps there, it further decays into the ππ* minimum, from which the conical intersection (CI) with the ground state is reached in 4.3 ps. For hmdC, the dynamics observed do not change much with respect to mdC. FdC shows entirely different picture. The emission signal is now very weak and covered by strong photo-induced absorption peaks. We again find the 130 fs time constant corresponding to wavepacket motion towards the plateau, and then 345 fs decay time for the minor pathway to the ground state through a CI. However, this time the dark nπ* state is populated at very early times and leads to triplet population through inter-system crossing reached in 2.1 ps. This large population trapped in the efficiently formed triplet could be the source of long living high energy state that can damage the DNA strand. Finally, in cadC, we find a 130 fs time constant that shows ballistic decay through a barrier-less CI to the ground state. Simultaneously we observe a rise of hot ground state population that decays in 960 fs.<br/><br/>In conclusion, our study provides a comprehensive picture of the ES dynamics of all four epigenetic dC nucleosides. By combining ultrafast transient absorption spectroscopy with CASPT2/MM computations, we have shown how the different chemical modifications dramatically affect the de-excitation pathways. The ultrafast ES decay along the S₁ potential energy surface in mdC and hmdC, as compared to the parent molecule, is slowed down due to an increased energy barrier to reach the CI compared to the canonical nucleoside. For fdC we identify the competition of intersystem crossing and internal conversion and a ballistic decay to the ground state for cadC. This work represents an important step towards understanding the intricate photophysics of epigenetic dC derivatives in the biologically relevant aqueous environment. Our results help to elucidate their role in the incidence of DNA photodamage, promoted by either longer singlet ES lifetimes or population of the triplet states, which may make the epigenetic derivatives more reactive.<br/><br/>[1] T. Carell et al. “Structure and Function of Noncanonical Nucleobases,” Angew. Chem. Int. Ed. 51, 7110-7131 (2012).<br/>[2] X. Wang et al. “Excited State Decay Pathways of 2’-Deoxy-5-methylcytidine and Deoxycytidine Revisited in Solution,” J. Phys. Chem. B 122, 7027-7037 (2018).