Design and synthesis of paramagnetic probes for structural biololgy

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 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.