Hyperpolarization of radicals to increase the sensitivity of photo-induced pulsed EPR techniques

The aim of this thesis is the improvement of Pulsed Dipolar Electron Paramagnetic Resonance (EPR) techniques by means of hyperpolarization in order to use them for accurate distance measurements between spin labels in biological systems, and more importantly in cell. Conventionally, distance measurements are performed between two nitroxide spin labels which have been artificially attached to the biological macromolecule. Recently, alternative spin labels have been proposed, together with a orthogonal spin labelling strategy based on the use of two e types of spin labels with different spectroscopic properties which allows to increase the information content of experimental data that can be obtained. It has been demonstrated that the photoexcited triplet state, localized on a porphyrin moiety, and the nitroxide radical constitute a promising orthogonal pair of spin labels. Hyperpolarization of the triplet state, resulting from a non-Boltzmann population of the triplet-state sublevels by intersystem crossing, is responsible for a significant increase in the sensitivity of the DEER experiment. Since hyperpolarization can be transferred to the nitroxide radicals, a further sensitivity improvement can be achieved. To study the hyperpolarization mechanism, based on electron spin polarization transfer by dipolar interactions in the weakly coupled regime, Time-Resolved EPR investigations have been carried out on peptide-based model compounds. The peptide chain allows to keep the radical and the photoexcited triplet at fixed well known distances. Different triplet-radical systems have been investigated: changing the peptide bridge lenght, the chromophore and also in a mixture without peptide bridge. The final aim is to identify the systems in which the most efficient polarization transfer is observed. The investigation has given some valuable insights into the hyperpolarization mechanism in the weakly-coupled dipolar interaction regime focusing on the interplay between radical-pair dipolar coupling and net polarization of the triplet state, as well as on their relevance. Working in the weakly coupled regime is important for the application of hyperpolarization in the increase of sensitivity of pulsed dipolar EPR spectroscopy, since distances are measured in the 1.-8 nm range. In general, the hyperpolarization mechanisms can be used to provide increased sensitivity for both EPR and NMR, as both magnetic spectroscopies usually suffer from small population differences between energy states.