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Isolated or in vitro generated oligomers of aggregation-prone peptides and proteins, which are associated with neurodegenerative diseases, are mixtures of several species with different sizes and morphologies. Therefore, determined neurotoxic properties can not be attributed to one specific oligomer. In this work a model system for the synthesis of a specific oligomer in aqueous solution was developed. The synthesis of such species with a given degree of oligomerization using classical and irreversible reactions is very time consuming and has a high material consumption. Therefore, reversible bond formation on templates, which control the degree of oligomerization, was used to obtain even large peptide oligomers with a much lesser effort. Homooligomers of the dihydroxylated thiazolidinelactam hot=Tap were used as templates and reacted with boronic acid modified peptides to synthesize boronic ester based Aβ(32-40)-oligomers in DMSO-d6 and PrP(106-118)-oligomers in aqueous solution. The synthesis of the hot=Tap homooligomers was successful up to the hexamer. A quantitative conversion to the Aβ-oligomers was achieved by using a three component system, which included a hot=Tap template, 2-formylphenylboronic acid and the unmodified Aβ(32-40) peptide. Besides the boronic ester, an imine was formed between the N-terminal amine of the peptide and the aldehyde of 2-formylphenylboronic acid. The high signal dispersion in the NMR spectra of the oligomers allowed a detailed structural analysis, whereby it was possible to generate a proposed structure of the boronic ester based Aβ(32-40)-trimer by using molecular dynamic simulations.
Subsequently, based on the results of the experiments in DMSO-d6 peptide oligomers with a given degree of oligomerization using boronic ester formation in aqueous solution at a physiological pH value of 7.4 were synthesized. Therefore, the water soluble and biological relevant part 106-118 of the prion protein was used. As suitable binding motif for the diols of the hot=Tap templates the benzoboroxole was identified and PrP oligomers up to the hexamer were synthesized with benzoboroxole modified PrP(106-118) peptides. The successful reaction in aqueous solution was confirmed by NMR and UV/Vis spectroscopy. Mrs Albus from the workgroup Dodel (Fachbereich Medizin, Philipps-Universität Marburg) performed Dot Blot experiments with these PrP(106-118) oligomers and the mouse anti prion protein antibody 3F4. These experiments revealed, that the oligomers with a spacer between the peptide sequence and the benzoboroxole can be used as mimetic or model for the early species of aggregation of the prion protein.
Additionally, the suitability of the hot=Tap as β-turn mimetic was investigated. Two cyclic hexapeptides were synthesized and analyzed by NMR spectroscopy and X-ray crystallography. In contrast to a typical β-turn mimetic the hot=Tap does not occupy the positions i+1 and i+2 of the turn. Furthermore, some Gramicidin S derivatives with a hot=Tap, replacing one D-Phe-Pro turn of the antimicrobial active Gramicidin S, were synthesized. NMR and CD spectroscopic investigations revealed a distorted structure of the synthesized derivatives in comparison to the native Gramicidin S. Nevertheless, by incorporation of ketals with an alkyl chain as protecting group for the diol of the hot=Tap comparable biological profiles like for the native compound were achieved. The determinations of the minimal inhibitory concentrations were measured by Mrs Dr. Berditsch from the workgroup Ulrich (Institut für Organische Chemie, Karlsruher Institut für Technologie). For the two highly hydrophobic compounds higher minimal inhibitory concentrations and an increased hemolytic activity in comparison to Gramicidin S were determined. However, one derivative has comparable minimal inhibitory concentrations against the tested bacteria strains and is significant lower cytotoxic compared to Gramicidin S and is therefore a suitable lead compound for further optimizations.