Abstract
Thiobases are modified nucleobases where an oxygen atom is replaced with a sulfur atom, a substitution that significantly alters their photophysics by causing a red shift in the absorption spectrum and reducing photostability. Unlike standard nucleobases, which relax to the ground state through ultrafast internal conversion, thiobases move to a long-lived triplet state with high quantum yield via ultrafast intersystem crossing. While this efficiency makes them excellent photosensitizers for photodynamic therapy, their use as prodrugs for diseases like leukemia and gliomas carries a risk of DNA damage from these long-lived triplet states. The HAPPY project (Understanding the pHotochemistry of sulfur-substituted dnA bases by advanced ultrafast sPectroscoPY for phototherapeutic applications) was established to overcome the challenges in detailing these relaxation processes, such as 100-fs timescales and the involvement of "dark" excited states. Through a collaborative effort between CNR-IFN, POLIMI, and UNIBO, the project combined advanced UV-visible transient absorption spectroscopy, time-resolved X-ray absorption spectroscopy (TR-XAS), and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to reveal the early stages of excited state relaxation in thiobases.
Results achieved
By the conclusion of the project in February 2026, the HAPPY project successfully achieved its three primary objectives: 1. Advanced Spectroscopic Infrastructure The project established a world-class experimental framework for investigating biomolecules. POLIMI optimized a UV-visible transient absorption (TA) spectroscopy setup achieving a temporal resolution of 10 fs and sensitivity down to a few microOD. CNR-IFN developed an innovative XUV source based on High-order Harmonic Generation (HHG) inside microfluidic chips, reaching a significant photon flux at the sulfur 2p edge (160 eV). Furthermore, the harmonic spectrum was extended into the "water window" (up to ~400 eV) using a near-IR OPA developed in collaboration with POLIMI. To deliver samples, the team commissioned multiple molecular sources, including an oven-based beam source for gas-phase molecules and a flat jet source for liquid-phase TR-XAS experiments. 2. Advanced Numerical Simulations UNIBO developed and deployed a sophisticated theoretical machinery within the COBRAMM/SHARC interface. This framework implements RASPT2-based trajectory surface hopping to model both internal conversion and intersystem crossing with unprecedented accuracy. By benchmarking optimal active spaces and basis sets, the unit enabled fully correlated simulations that quantitatively reproduce experimental spectral signals, providing a "molecular movie" of the photoinduced dynamics. 3. Ultrafast Photochemistry of DNA Bases and Thiobases The scientific investigation yielded high-impact results published in leading journals: • DNA Dynamics: Studies on the GT dinucleotide revealed how base stacking influences charge transfer states, while research on 2'-deoxyadenosine resolved longstanding ambiguities regarding its dark Sn states. • Thiobases & Photoswitches: The project characterized the photoisomerization of hemithioindigo (HTI) photoswitches using time-resolved XPS at the sulfur L-edge. • International Facilities: The team conducted several successful beamtimes at international facilities, including the European XFEL and FERMI ELETTRA, investigating the relaxation dynamics of uracil and the chiral dynamics of ergosterol. Long-term Impact and Sustainability A major outcome of the HAPPY project was the awarding of a €10M ERC Synergy Grant for the project "CONCERT". This new initiative, involving the project’s PIs, emerged directly from the scientific discussions and experimental infrastructure established during the HAPPY PRIN project, ensuring the long-term sustainability and expansion of this research program in Italy.Project details
Unibo Team Leader: Marco Garavelli
Unibo involved Department/s:
Dipartimento di Chimica Industriale "Toso Montanari"
Coordinator:
CNR - Consiglio Nazionale delle Ricerche(Italy)
Total Unibo Contribution: Euro (EUR) 70.466,00
Project Duration in months: 24
Start Date:
30/11/2023
End Date:
28/02/2026
