Ein neuer Weg zu asymmetrischen Klammern und Pinzetten mit Phosphoramiditen
Molecular diphosphate tweezers are able to bind to lysine and arginine residues on protein surfaces and are used as new enzyme inhibitors, blockers of pathological protein aggregation and for the disruption of membranes in enveloped viruses. Nevertheless, these powerful receptors still lack selectivity. Previous attempts to solve this problem in our group resulted in replacing one phosphate group by a neutral linker unit for the introduction of additional recognition elements. Since the neutral linkers generally lowered the tweezers` affinities towards basic amino acids and peptides, a new strategy which keeps both phosphate anions and allows installation of one recognition unit by “click” chemistry was established in this thesis. The introduction of the phosphate moiety containing the alkyne unit was easily achieved by following a classic phosphoramidite approach. After overcoming several difficulties in attaching the second phosphate moiety to the tweezers, the monobutynyl diphosphate tweezers 9 was obtained by first attaching a phosphoramidate to the monobutynyl cyanoethyl monophosphate tweezers 53, which was hydrolyzed under mild conditions afterwards. Binding studies with this new derivative 9 and peptide fragments derived from IAPP und PSMα3 showed moderate binging affinity if peptide aggregation and precipitation could be excluded, proving the new derivative as potent as the classic diphosphate tweezers. Based on this success story, the tweezers 9 was modified for biological applications. In cooperation with the Meyer group a lead structure for an inhibitor for the interaction between the p97 protein and its cofactor UBXD1 was designed by molecular modelling, based on the knowledge of the tweezers´ best binding position on the p97 N-domain surface determined in cooperation by the Bayer and Sanchez group. The resulting additional recognition unit was determined to be an α-helical peptide consisting of 15 amino acids with an azide connected to the peptide´s C-terminus. After establishing a successful synthesis strategy, the final peptide could not be obtained in sufficient quantities due to solubility problems. Even though the final inhibitor could not be synthesized, the small peptide already showed a competitive effect on the binding of UBXD1 to p97 at higher concentrations, which was shown in pulldown experiments by the Mayer group, giving a proof of concept. In another biological project in cooperation with the Ottmann group the lead structures for inhibitors of the protein-protein-interaction of 14-3-3 with ExoS and C-raf were determined resulting in the successful synthesis of a tweezer-ExoS hybrid molecule, which is currently crystalized and examined in the Ottmann group.