The main subject of this thesis is the design and synthesis of paramagnetic molecules for protein studies with NMR and EPR spectroscopy. With the development of paraNMR and DEER experiments,... Show moreThe main subject of this thesis is the design and synthesis of paramagnetic molecules for protein studies with NMR and EPR spectroscopy. With the development of paraNMR and DEER experiments, synthetic paramagnetic centers are becoming popular. About half of the current paramagnetic probes were described in the past five years, reflecting this popularity. Several of them improved the stability and rigidity of the probes, mainly by introducing novel attachment groups forming thioether and triazole linkers.1–3 Such linkages help to extend the application of the probes to in-cell measurements.4,5 Some small probes were designed for 3d-block metal ions as well, but these probes show low metal ion binding affinity.6,7 The research described in this thesis contributed to the development of paramagnetic probes. Hereafter, the properties of these new probes are discussed, and some general conclusions and prospects are given. Show less
The majority of the work presented in this thesis involves the design and synthesis of paramagnetic NMR probes, including lanthanoids caged probes and spin labels. An overview of the development of... Show moreThe majority of the work presented in this thesis involves the design and synthesis of paramagnetic NMR probes, including lanthanoids caged probes and spin labels. An overview of the development of different types of lanthanoids caged probes is given. Among all of the reported lanthanoid probes, the caged lanthanoid NMR probe version 5 (CLaNP-5) shows the largest paramagnetic effects due to the fact that it is tagged to proteins via two-point attachment and its free complex presents a single conformation. Although CLaNP-5 is successfully applied to study proteins and protein complexes, the net charge of Ln CLaNP-5 complex and the weak disulfide linker are the drawbacks. A new paramagnetic probe, CLaNP-7, was synthesized, for which the net charge of lanthanoids complexes was reduced to +1 by introducing p-nitrophenol. The __-tensor of CLaNP-7 is pH-dependent when a histidine residue is located close to the attachment site. It is proposed that the pH dependence is due to the fact that the histidine forms a hydrogen bond with a water that acts as the ninth ligand of the lanthanoid. In order to enhance the stability of the tag linkers, two approaches, thioether and bioorthogonal reactions, were investigated. The results showed that a new thio-reactive CLaNP-9 was successfully attached to protein and the reaction product was stable in the reductive conditions. Moreover, inhibitor-based paramagnetic probes were also synthesized and the co-crystal structures of protein with inhibitor-based probes were also presented. Show less
