Dokument

Summary:
This thesis presents the synthesis of novel photoresponsive DNA-binding agents as well as their application for both visualization and control of biological processes. In addition, the implementation of an innovative course to transfer the interdisciplinary research culture of chemical biology to education is reported. This cumulative dissertation is mainly composed of four chapters, each of them dealing with either published or submitted articles under revision. An introduction, motivation and outlook are included, too. The first article is published in Angewandte Chemie International Edition, and describes the development of a new far-red fluorescent DNA binder, 6-TramTO-3, as an alternative chemical tool to the green fluorescent protein (GFP) for interaction studies of live multidrug-resistant pathogens and host cells. Thus, the derivatization of thiazole orange (TO) resulted in a new cyanine dye, 6-TramTO-3, whose spectroscopic properties were analyzed. DNA binding analysis by UV-vis, fluorescence and CD spectroscopy was performed to study the interaction mode upon addition to DNA. Initially, fluorescence confocal microscopy, FACS and growth curve analysis with live E. coli displayed an efficient staining of more than 90% and no effect on the bacterial viability. Importantly, 6-TramTO-3 overcomes common limitations of other frequently used fluorophores like ethidium bromide, carboxyfluorescein succinimidyl ester and SyTO-9, such as low staining efficiency and cytotoxicity without genetic manipulation. Encouraged by these unique properties, we tested 6-TramTO-3 with antibiotic-sensitive and multidrug-resistant Klebsiella pneumoniae strains. 6-TramTO-3 conferred both strains a highly specific red-fluorescent shift with 91% and 87% in the 652-671 nm red2 channel without observable cytotoxic effects. Next, we performed phagocytosis assays with human macrophages, which revealed a different strain behavior for the first time. Besides the publication, an European patent was filed (18174086.1 – 1011) and numerous collaborations with universities, research institutes and companies were initiated to apply 6-TramTO-3 in different contexts. The second article is published in Organic & Biomolecular Chemistry, and presents ortho-tetra-fluoroazobenzene derivatives as DNA intercalators for photocontrol of DNA and nucleosome binding upon visible-light irradiation. Its use is less harmful to biological systems and therefore, preferred over UV-light, which is needed for the most known nucleic acid targeting photoswitchable systems. Thus, the introduction of such moiety in the backbone of the minor groove binder netropsin added dynamic control and changed the recognition mode. UV-vis, HPLC and NMR analysis demonstrated the photoisomerization of the new compounds by visible light. DNA-binding analysis conducted by UV-vis, fluorescence and CD spectroscopy revealed an intercalating binding mode in the low nM-region with differences between isomers. Finally, the use of the new derivative as a light-controllable nucleosome binder was explored. The different binding affinities observed on free DNA were corroborated for the nucleosomal DNA. Interestingly, electromobility shift assays and dynamic light scattering experiments proved that when the compound is in its trans-form, it intercalated into the nucleosomal DNA, induced distortion, which further led to disruption of the nucleosomes. These findings will help to introduce photochemical control to pave the way to altering nucleosome-based processes by light. The third chapter of this thesis is a submitted manuscript to the journal Organic Letters under revision and deals with a novel synthetic approach to access pyrrole-imidazole (Py-Im) DNA binding polyamides based on 4-methyltrityl (Mtt) solid-phase peptide synthesis (SPPS). Commonly Py-Im polyamide hairpins are synthesized by Boc- and Fmoc- SPPS. New Mtt-protected pyrrole and imidazole building blocks were synthesized with higher yields than the Boc- and Fmoc-analogues. Their implementation could successfully be shown by the synthesis and study of a DNA-binding polyamide applying all Mtt-building blocks. Our methodology is orthogonal to Fmoc and will allow the implementation of base labile compounds and other sensitive groups in Py-Im polyamide chemistry affording novel polyamide conjugates. In a further attempt, it was already possible to use the new building blocks for the introduction of a photoswitchable compound into the polyamide-hairpin backbone, which will access the introduction of dynamic and spatial control in the level of sequence specific DNA binding in biological assays. The fourth chapter is a submitted manuscript to the Journal of Chemical Education under replied revision, in which a novel course for master students in chemistry and biology is introduced to fill the missing gap between cutting-edge interdisciplinary research and education of future scientists. In other words, our course aimed at bringing the authentic “big picture” of science by the combination of lectures, a discovery-based research laboratory and science communication. The students experienced the advantages of collaborative work, wrote a short article and were guided in the design and defense of their own research proposals in chemical biology. Evaluation of the course clearly proved that the course fosters cooperative research relationships between biology and chemistry students, provides a unique and genuine research experience and increased the students’ interest in interdisciplinary approaches and collaborative work. The course and assessment material was accessible to inspire other interdisciplinary fields

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