Design, Synthesis and Optimization of Paramagnetic Tags for NMR Spectroscopy

Paramagnetic restraints have been used in biomolecular NMR for the last three decades in order to elucidate and refine structures but also to characterize protein/ligand interactions. Pseudo-Contact Shifts (PCS) are the most commonly used restraints, consisting in measuring a shift between a paramagnetic species and a diamagnetic reference, whose magnitude depends on the distance from the paramagnetic centre. A common technique to generate such restraints consists in the attachment of lanthanides ions to the protein via a Lanthanide-Binding-Tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. In this thesis, we first focused on the synthesis of cross-bridge azamacrocyles. After having discussed their synthesis and chelation-properties, we proposed a paramagnetic tag based on the cross-bridge cyclam for which we proposed several synthetic pathways. This tag is based on the thiol-ene reaction as its conjugation method, the efficiency and catalysis of which will be discussed. A protocol using UV coupled to a radical iniator was developed, allowing the fast and cysteine-selective paramagnetic tagging of our model protein GB1 T53C. In a second time, efforts were concentrated on the synthesis of enantiopure paramagnetic tags. To this purpose we proposed two ligands, which were synthesized, tagged to the model protein GB1 T53C, and characterized via NMR and FFC Relaxometry. Finally, we used the thiol-ene based paramagnetic tagging of tcPex14, a pharmaceutically relevant protein, as an application of paramagnetic NMR in drug discovery. Its crystal structure was refined, and the possibilities of characterizing the binding mode of small ligands using paramagnetic restraints were discussed.