Structure and dynamics of photoexcited chromophore-radical systems: a light-induced EPR investigation

Pulsed Dipolar Electron Paramagnetic Resonance (EPR) techniques are powerful tools for obtaining structural information in macromolecules and biological systems labeled with paramagnetic probes, most commonly stable nitroxide radicals. In recent years, the transient triplet state of photoexcited chromophores has been proposed as an alternative spin probe, giving rise to a new class of techniques known as Light-induced Pulsed Dipolar Spectroscopies (LiPDS). These techniques rely on the interaction between a transient triplet state and a stable radical. The photoexcited triplet state is a paramagnetic species with non-Boltzmann electron spin polarization, a direct consequence of its formation mechanism, namely intersystem crossing (ISC). The hyperpolarization corresponds to larger population differences between the triplet spin sublevels, resulting in enhanced EPR signals. Furthermore, studies on peptide model systems have shown an increased nitroxide EPR signal: this is due to spin polarization transfer from the triplet state, if the interaction between the two paramagnetic centers falls in the weak-coupling regime. This effect can potentially further enhance the sensitivity and applicability of LiPDS techniques, which are typically used to investigate systems with inter-spin distances between 1.5 and 8 nm, which is the suitable range for weak electron-electron dipolar interaction. The first part of this thesis aims at the understanding of the mechanism involved in spin polarization transfer in a model peptide in the weakly-coupled regime. The system analyzed is a rigid α-helix peptide with the amino acid TOAC, which carries a nitroxide group, and the chromophore I₂BODIPY attached to the N-terminal position. Time-resolved EPR spectroscopy is employed to monitor the temporal evolution of the EPR signals of both the triplet state and the polarized radical. The experimental results reveal that radical polarization is long-lived, with its lifetime closely correlated to that of the triplet state. Field-modulation detection was implemented for time-resolved measurements to improve stability and sensitivity over extended timescales. These findings provide new insights into the mechanism of spin polarization transfer, clarifying the conditions required for it to happen, and suggest an additional polarization pathway, as the radical polarization shows a dependence on the microwave frequency. The second part of this thesis focuses on the implementation of a LiPDS experiment, the Refocused Laser-Induced Magnetic Dipole Spectroscopy (ReLaserIMD), carried out for the first time at Q-band frequency at the University of Padova. This work aims to provide future users with a practical guide to the experiment. The method was first applied to a standard model peptide to extract the distance distribution between a photoexcited chromophore triplet state and a nitroxide radical, and subsequently extended to a protein-peptide system. Particular emphasis was placed on optimizing the experimental setup and validating the technique, demonstrating its reliability and effectiveness for structural studies in the nanometer range.