Synthese und Untersuchung von Alanyl-PNA Oligomeren und deren Einfluß auf β-Faltblatt Strukturen

The activity and properties of proteins mainly depend on the three-dimensional arrangment of their secondary structure elements and their conformation. Conformational changes as well as protein-protein interactions, be influenced by molecular switches, contribute to changes in biological activity and the function of peptide.The molecular switches used are alanyl peptide nucleic acids (alanyl-PNA), alternating oligopeptides based on amino acid alanyl with nucleobases attached to the β-postition.In the present work we studied the interaction and so the stability of diffrent complementary alanyl-PNA oligomers with varying mismatches,lengths and buffers by using UV- and NMR-spectroscopy. Further on, alanyl-PNA was be incorporated in β-sheet forming peptides. In addition, we aimed to develop an efficient method to synthesize N-methylated amino acid derivatives from N-methyl-serinelactone 42 (Figure 61) and incorporate N-methylated nucleoamino acids in alanyl-PNAs. By replacing C-terminal helix of humane interleukine 8 (hIL-8) (amino acids 56-77) with an alanyl-PNA hexamer we wanted to switch from a helical-structured C terminus to nother stable secondary structure. It is very difficult to incorporate alanyl-PNA in peptides or proteins, so that we decided at end our study to performed the enzymatic ligation in cooperation with the Bordusa group.The first aim part of this work was to understand the interaction and so the stability of diffrent complementary alanyl-PNA oligomers with varying mismatches and buffers by using UV- and NMR-spectroscopy. The stability of duplex was investigated by varying the lengths of different alanyl-PNA oligomers. To avoid aggregation problems, we incorporated Boc-AlaT-OH or amino acids lysine and glutamine as a mismatch. The structure and the conformational changes of this sequences were investigated by temperature- dependent UV- and NMR-spectroscopy. We found that a modification increased/reduced the selfpairing. In order to increase the solubility of alanyl-PNA oligomers, amino acids lysine and glutamine were incorporated. NMR-spectroscopy revealed higher aggregation in pure G/C-alanyl-PNA than in the modified oligomers.To avoid aggregation the oligomere 68 with a central incorporated N-methylated nucleoamino acid was prepared. In addition, the complementary alanyl-PNA with charged N-alkyl chains was prepared to enable NMR-spectroscopy measurements blocking one aggregation side.The fourth goal of this work was to investigat interactions of alternating alanyl-PNA oligomeres as molecular switches. Based on the specific nucleobase recognition, the two complementary alanyl-PNA hexamers provide a stable double strand with linear topology. Considering this, one alanyl-PNA hexamer was inserted in a peptide or protein to stabilize a β sheet conformation. Alanyl-PNAs randomly brought together from a single strand. Two complementary strands are able to form duplex, resulting in the β-strand conformation. Next, alanyl-PNA was incorporated eigther in the model peptide or in proteins to function as a distrurbed β-sheet (PNA double strand). As sutch the duplex formed a switchable de-/stabilizing element of protein conformation.Further on, alanyl-PNA was incorporated in β-sheet forming peptides as well as in proteins in order to function either as a part of a β-sheet (PNA double strand) or a disturbed β-sheet (PNA single strand). The peptide/PNA sequence Ac Ser-Pro-Gly-Lys-Thr-Tyr-Thr-Glu-Val-Pro-Gly-Orn-Lys-Ile-Leu-Gln-Val-Gly-Pro-Gly-Gly-AlaC-AlaC-AlaG-AlaC-AlaG-AlaG-Lys-NH2 (85) was synthesized, but the recognition of the complementary alanyl-PNAs failed. This could be due to the sterical or electronical interactions between the nucleoamino acids (alanyl-PNA) and the amino acids of peptide. Becuse of this, and a shorter and less rigid peptide containing a weaker β-turn Ac-Orn-Lys-Ile-Leu-Gln-Val-Gly-Asn-Gly-Gly-AlaC-AlaC-AlaG-AlaC-AlaG-AlaG-Lys-NH2 (87) (Figure 60) was synthesized. A much more intensive influence was measured between complementary alanyl-PNA and peptide/ PNA sequence, confirming our hypotheses.As fifth part of this work, the C-terminal helix of humane interleukine 8 (hIL-8) (amino acids 56-77) was replaced with a peptide sequence that contained an alanyl-PNA hexamer with the intention to switch from a structured C terminus to a stable secondary structure. In the present approach the native chemival ligation was applied to obtain protein/alanyl-PNA-chimera that allows to switch the protein conformation and to modify the protein interaction by the base pairing. Interleukin 8 is the first example we already obtained the preliminary data from. The fragment hIL-8 (1-55) was expressed in E. coli by the IMPACT system and purified by the Beck-Sickinger group. A last aim, was an enzymatic ligation in coorperation with the Bordusa group. For this reason, it was necessary to synthesize alanyl-PNA oligomeres of different lenghts and N-terminal nucleo amino acids. The ligation step was done by using different sequences and clostripain as an enzyme (Figure 61 ). Bordusa ligation formed out to be a promising method to incorporate alanyl-PNA in peptides or proteins. The existence of 70 with SPPS (solid phase peptide synthesize), was made possible by extending it s sequence with two glycines. In conclusion, this work describes a novel method to synthesize N-methylated amino and nucleo amino acids and incorporated them in alanyl-PNA oligomers. We suggest that the Bordusa ligation methode is an efficient strategy to incorporate into alanyl-PNA in peptides and proteins. Furthermore, to optimize/prevent the switching attachement and aggregation problems a N-methylation over this peptide backbone to one or more central nucleo amino acids of the strand would be done.