Analytik und Synthese von Pyrrol-Schwefelverbindungen der Gattung Allium sowie deren Verwendung in der Biosensorik

Mit über 850 Arten ist die Gattung Allium eine der bekanntesten Gattungen. Insbesondere in der Untergattung Melanocrommyum wurden zahlreiche interessante Verbindungen isoliert. So wurde in A. stipitatum Pyridinyl-N-oxide gefunden; Marasmin, hauptsächlich aus südafrikanischen Tulbaghia-Arten bekannt,...

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Bibliographic Details
Main Author: Rüger, Fabian
Contributors: Keusgen, Michael (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
Online Access:PDF Full Text
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Table of Contents: With over 850 species, the genus Allium is one of the most prominent genera. Especially in the subgenus Melanocrommyum, numerous exciting compounds have been isolated. For example, noteworthy concentrations of pyridinyl-N-oxides were found in A. stipitatum; marasmin, known chiefly from South African Tulbaghia species, was found in A. suworowii. The aim of this work was the characterization of a red pigment produced by some species of the Melanocrommyum subgenus, upon tissue damage from allinase reaction of pyrrole-2-cysteine sulfoxide. Although two publications describe the structure and potential substrates, because of prior results of the Keusgen group, doubts have arisen concerning the correctness of both structures. The second target was the synthesis of this compound, along with biosensor measurements, to determine the response upon treatment with oxidation agents and subsequent polymerization. The third component of the research, conducted during the Ph.D. thesis, was the development of novel surface modification procedures inspired by the genus Allium, or, more precisely, its several natural products like fructans and pyrrole compounds. Crystallization experiments showed that the previously published structures for the red pigment are not correct. It was confirmed that the chemical structure contains four pyrrole moieties forming two bipyrrole units connected by disulfide bridges. Thus, the substance is closely related to porphyrins but shows a distinct step-like structure. A simple one-pot synthesis for the target molecule, involving easily to prepare educts, was developed. Alkaline hydrolysis of 2-thiocyanato-pyrrole was used to create a biomimetic synthesis yielding pyrrolyl-2-thiosulfinato-pyrrole in low concentrations, finally leading to the red pigment after several condensation steps. A novel reaction concerning several 2-thiocyanato-pyrroles was discovered during the optimization of the red pigment synthesis. It was shown that after hydrolysis of three different thiocyanato-pyrroles in alcoholic solvents, the S-alkylated products and disulfides could be isolated in acceptable yields. This reaction poses an alternative approach to non-N-substituted pyrrole thioethers. For all substrates that were hydrolyzed in alkaline alcoholic solvents, carbonimido-thioates were produced. Inspired by the tendency of pyrrole compounds to polymerize, a novel surface modification, involving N-substituted-pyrrole/pyrrole copolymers was developed. Surface-Plasmon-Resonance-(SPR)-studies conducted with these surfaces did show a high degree of compatibility with existing protocols. EDC/NHS- activation and covalent immobilization of biomolecules were possible due to the versatility of this method. Another trait of the copolymers was the excellent binding affinity for polysaccharides like man-nan. Polar- (carbonic acid) and unipolar- modified copolymers allowed the binding of mannan: the subsequent immobilization and use of the fusion protein ConA-Sav, which was established in the Keusgen group, was one of the primary targets and showed good results. Regeneration of the surface using a complimentary monosaccharide (α-D-methyl-mannopyranoside) was unfortunately only partially possible. The second method for biosensing SPR-surfaces was developed using the natural affinity of bovine serum albumin (BSA) to gold substrates, which led to a gold/BSA/gold nanoparticle-sandwich-platform. Tests, using an antibody bound adsorptively to the surface of the nanoparticles, did show high performance. The measurements were reproducible concerning the binding rates of BSA and gold nanoparticles; the binding-event of anti-BSA showed a linear relationship of binding rate and concentration between 15 µg/mL and 30 µg/mL. Lastly, a polysaccharide-based surface modification was established using some broadly available polysaccharides: inulin, dextran, arabinogalactan, and pullulan. The saccharides were successfully carboxymethylated and modified with 4-mercapto aniline to allow simple modification of gold-SPR-chips. The binding rates of the different modified glycans were evaluated using the SPR-technique. They produced highly similar results, regardless of the average weight of the glycans, suggesting a horizontal instead of a vertical binding of such poly sugars. The layers did show a high affinity for the ConA-Sav fusion protein and allowed EDC/NHS procedure to immobilize e.g., BSA, and anti-BSA binding confirmed successful immobilization. Although this strategy using modified glycans did provide satisfying results; more research is ongoing to develop a spacer between the gold surface and the polysaccharides.