Design and properties of lanthanoid chelating tags

The work presented in this thesis is centred around the magnetic anisotropy of lanthanoids and how it can be harnessed for the study of bio macromolecules. Following the introduction about paramagnetic NMR spectroscopy, lanthanoids and lanthanoid chelating tags are three individual chapters regarding the properties, design and synthesis of lanthanoid chelating tags.
The first chapter comprises a published study concerning the anisotropy of the magnetic susceptibility exhibited by lanthanoid (III) ions within lanthanoid chelating tags (LCTs). LCTs are widely used to induce pseudocontact shifts (PCSs) or residual dipolar couplings (RDCs) on bio macromolecules. The size of the observed PCSs or RDCs is dependent on the anisotropy of the magnetic susceptibility of the lanthanoid (III) ion. The anisotropy of the magnetic susceptibility can be described by the anisotropy parameters, which are for LCTs commonly determined from PCS observed on a conjugated protein. Because the PCS observed on the protein are inevitably reduced by motional averaging, the anisotropy parameters determined from them describe always only a fraction of the anisotropy of the magnetic susceptibility a lanthanoid ion exhibits within an LCT. This study presents for the first time the intrinsic anisotropy parameters for the full lanthanoid series determined from shifts observed on the LCT itself. The strongly shifted proton spectra could no longer be assigned using conventional 2D- NMR assignment strategies, due to the extremely short T2 times. Instead, the 1D proton spectra were fully assigned using extensive, site-specific 2H and 13C labelling in combination with combinatorial methods. The full assignments were used to determine the anisotropy parameters, which deliver an upper limit for future PCS applications relying on this coordination polyhedron as well as new insights into future LCT designs. Surprisingly, we observed an, at least at room temperature, unprecedented correlation between the oblate or prolate f-electron distribution of the lanthanoid and the orientation of the main magnetic axis. Furthermore, a comparison of different ligands revealed that the size of the anisotropy parameters depends on the interaction between the ligand and the lanthanoid ion.
In the second chapter, the focus lies on the development of a new single-arm LCT, which could provide predictable anisotropy parameters. For current single-arm LCTs the averaging of the observed PCS on a conjugated protein is not only dependent on the LCT but also on the tagging site. Therefore, the averaging of the PCS is different for each tagging site, requiring the determination of the anisotropy parameters in each case. It would greatly ease the application of PCS NMR spectroscopy if a single-arm LCT would provide predictable anisotropy parameters. A priori knowledge of the anisotropy parameters would allow the choice of the best-suited tagging site for a given application and facilitate the assignment of paramagnetic signals. Based on the insights gained from determining the intrinsic anisotropy parameters it was hypothesized that coaxiality between the rotation axis and z-axis of the tensor frame could provide predictable anisotropy parameters. The sought coaxiality can be achieved with a symmetric LCT that is tethered via a thioether to the protein. In order to develop a new LCT, two scaffolds were tested for their suitability as an LCT. The second scaffold provided a new symmetric LCT, which was successfully tethered to ubiquitin S57C. Determination of the anisotropy parameters and comparison to the estimated intrinsic anisotropy parameters showed that the PCS induced by the new LCT are barely affected by averaging. The current results were not sufficient to ascertain whether the new LCT is able to provide predictable anisotropy parameters but they hold great promise for further research.
The third chapter describes the synthesis of a new building block for DOTA-M8-based LCTs. DOTA-M8 provides excellent properties for the development of lanthanoid chelating tags but has the inherent problem that it is difficult to synthesize. The main challenge in the synthesis towards DOTA-M8 is the synthesis of M4-cyclen. In this chapter, a new cyclen building block is presented, which should be simpler to synthesise but still provides all the necessary properties for high performance LCTs. A convenient synthetic route towards the new building block was tested. The encountered obstacles revealed which changes would be necessary for the future success of the proposed synthetic strategy.