Reversible Cyclisierung von Peptiden – neue Synthesewege für Peptidantibiotika

Lasso peptides have a very unique structure which is not comparable with structurally simpler biopolymers found in nature. The formed cyclic structure can be described by the macroscopic form of the eponymous lasso and is synthesized in nature by enzymes. The synthesis of such structures is a major challenge in organic-synthetic chemistry and until now there is no synthetic procedure known which leads to the correct topological isomer. To reduce the energy barrier of this cyclization process and to analyze the ratio between the macrocycle and the chain in the chemical equilibrium we performed the cyclization in a reversible manner. This was achieved by the substitution of a peptide bond for an imine or oxime which transforms irreversible macrocyclization into a reversible process. In this work bioorthogonal synthetic methods for C-terminal and sidechain peptide aldehydes were developed. The cyclization tendency of these peptides as well as the spatial arrangement of the cyclic products, formed in chemical equilibrium, were analyzed in buffered solution by NMR-spectroscopy. By means of NMR-based molecular modelling the peptides were evaluated and the corresponding structures calculated. The cyclization tendency of C terminal peptide aldehydes was examined for the cyclic hexapeptide Segetalin A by NMR-spectroscopy. Instead of the expected head-to-tail cyclization the sidechain cyclization of the Trp-NH-indole was observed. Besides Segetalin A, several additional cyclic hexapeptides were cyclized reversibly and their structures characterized by NMR-spectroscopy. In contrast to the head-to-tail cyclization novel monomers for the head-to-sidechain cyclization had to be developed and synthesized in multi gram scale for the use in solid phase peptide synthesis. For the reversible cyclization of peptide aldehydes via the sidechain of glutamic acid two different synthetic routes were performed and analyzed. The first route which was focused on a precursor-based strategy involved the synthesis of the unnatural amino acid homoallyglycine. Derivatives of this amino acid were prepared and the oxidative cleavage of the terminal olefin to the corresponding aldehyde was investigated. The second synthesis route fashioned a sidechain dipeptide consisting of glutamic acid and a protected glycinal or glycinal precursor in the sidechain was performed. This significantly simplified the synthesis of the amino acid aldehydes, led to higher overall yields and a stable amino acid derivative which is synthesizable in a multi gram scale. The insertion of the synthetic amino acid in peptides was performed by manual and automated solid phase peptide synthesis. All sidechain peptide aldehydes which were synthesized in this way were analyzed by their cyclization tendency in buffered solution by NMR-spectroscopy. The establishment of the chemical equilibrium was achieved at pH 6.5 in buffered solution whereby a fast H/D exchange occurred. Because of the resulting signal broadening the quality of the measured NMR-spectra was too low for a complete assignment of the signals. By reductive amination of the reactive imines the equilibrium was shifted and the obtained macrocyclic peptides could be analyzed more easily. The analysis showed that the intramolecular interactions itself were not sufficient to form the correct lasso topology, only cyclic-branched peptides were isolated. Based on the insufficient pre-folding of the linear lasso peptide the C-terminal end was linked to the backbone by a disulfide bridge. The performed macrocyclization of the N-terminal ring should then lead to the correct folded lasso peptide. The isolation of the topologically correct peptide failed either. For the reversible cyclization of the N-terminal ring an amide bond was replaced by another disulfide bridge. By orthogonal protected cysteines which could be flexibly substituted in the primary sequence different disulfide patterns were synthesized and analyzed. The biological activity of the synthesized di- and tetrasulfides which showed the strongest similarity to the native Microcin J25 was investigated. Growth curves of gram-negative bacteria like Salmonella paratyphi, Shigella flexneri, enterohaemorrhagic Escherichia coli (EHEC) and Escherichia coli AS19 were measured in the presence of the synthetic peptides and their growth-inhibitory activity were characterized. In contrast to the native Microcin J25 the biological activity of the synthetic peptides was quite low.