Time-resolved EPR investigation on the hyperpolarization of radicals by photoexcited triplet states

The purpose of this thesis is to provide a methodology to improve the sensitivity of Pulsed Dipolar Electron Paramagnetic Resonance (EPR) techniques by taking advantage of the spin hyperpolarization phenomenon in order to accurately determine distances between spin labels in complex biological systems. Pulsed Dipolar Spectroscopy techniques allow for distance determination in macromolecules via the attachment of two spin labels, which are traditionally two nitroxide moieties. In recent years a number of alternative spin labels have been proposed in combination with orthogonal spin labeling, a strategy in which two types of spin labels with distinct spectroscopic properties are employed. This method allows to obtain more information from experimental data, especially if one of the probes is photo-switchable. Notably, a promising orthogonal pair of spin labels consists of the nitroxide radical and the photoexcited triplet state of a chromophore. Porphyrins, in particular, have been considered with interest due to their natural presence in biological structures. The use of triplet states as spin labels significantly enhances the sensitivity of Pulsed Dipolar Spectroscopy experiments due to the hyperpolarization of this paramagnetic state, consequence of the non-Boltzmann population of the triplet-state sublevels through intersystem crossing. As a further step, by transferring hyperpolarization to the nitroxide radicals, an additional sensitivity improvement is attainable. To investigate the hyperpolarization mechanism occurring between the triplet state and a radical species, which involves an electron spin polarization transfer via dipolar interactions in the weakly coupled regime, Time-Resolved EPR studies have been conducted using peptide-based model compounds. The evolution of the EPR signal in time and as a function of the temperature, which has been increased to reach the liquid phase transition, has been observed. The peptide chains allow a careful investigation of the effect of increasing the fixed and well-known distances between the radical and the triplet state. Additionally, the effect of changing the chromophore has been probed. In parallel, the isolated chromophores have been investigated to separate contributions from each part of the main framework. The ultimate goal of this thesis is to identify the conditions leading to the most efficient polarization transfer. The experiments provide valuable insight into the factors influencing the efficiency of the hyperpolarization mechanism mediated by dipolar interaction in the weakly coupled regime. Notably, the effect of the properties of each triplet state, its net polarization and the radical-triplet dipolar coupling are explored. The overlap of the weakly coupled regime with the 1 - 8 nm range in which Pulsed Dipolar Spectroscopy can be typically applied makes for a great tool for sensitivity enhancement in this type of experiments. Additionally, hyperpolarization mechanisms have the potential to be employed to increase sensitivity also in Nuclear Magnetic Resonance (NMR) and help face the challenge posed from small population differences between energy states.