Bio21 - Theses

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    Exploring novel blue turn-on fluorescent probes for the direct detection of nitric oxide and free radicals in living cells
    Barzegaramiriolya, Mina ( 2017)
    Bacterial biofilms are causing considerable damage to different areas in industry such as food industry, oil industry and dentistry. Traditional methods to control biofilm formation and to treat the surfaces affected by these microorganisms has mostly focused on biocidal and antibacterial strategies. The drawbacks of these approaches is related to the development of tolerances that decrease effectiveness of chemicals apply to eradicate these microorganisms. The growth of biofilms therefore is linked to a significant adaptation by bacteria cells to control changes in their environment. In this regard, the development of efficient methods to control biofilms formation as well as their irreversible eradication from affected surfaces is an important area of scientific research. Bacterial biofilms at times undergo regulated and coordinated dispersal events, where sessile biofilm cells convert to free-swimming, planktonic bacteria. Nitric oxide (NO) is an important biochemical signalling molecule that has been linked to the inhibition of biofilm formation and activation of dispersal through the generation of nitrosative and oxidative stress. Therefore, the availability of methods that enable sensing and visualizing NO is critical to reveal details of the biological functioning of this molecule. Knowledge of these will provide important guidelines for the development of strategies to combat biofilm formation. In this thesis two different approaches for detecting NO and oxidative stress were explored, that are based on fluorescence measurements using coumarin as fluorophore. The first strategy explores “turn-on” fluorescence for direct detection of endogenously produced NO. A family of five blue fluorescent probes CB1-5 were designed and synthesized and the photophysical properties studied in detail. These probes feature a substituted 7-hydroxy coumarin chromophore coupled to 2-methyl-8-aminoquinoline, which act as tridentate ligand for Cu(II) and active site for monitoring NO using the replacement strategy. The UV-vis absorption and fluorescence emission characteristics of the probes are significantly influenced by the substitution pattern on the coumarin ring, as well as by solvent polarity and pH. Time-dependent Density Functional Theory (TD-DFT) calculations for CB4 and CB5 showed that the absorptions are due to π ® π* transitions localised on the coumarin system, with a small charge transfer contribution from the quinoline system at higher pH where the 7-hydroxycoumarin moiety is deprotonated. Complexation of the probes with Cu(II) leads to fluorescence quenching, which switches back on upon reaction with NO. In vitro studies revealed that the probes detect NO with high selectivity in nM concentrations and do not respond to other oxidizing species. In vivo studies for CB4 and CB5 showed that these probes enable detection of NO in living bacterial cells in multi-dye imaging experiments. Furthermore, CB5 also enables to detect NO in macrophages, where it is an important effector molecule in host defence against bacterial pathogens. Using confocal microscopy, it was shown that the probe can be trapped by the cells and reacts directly and specifically with NO, rendering it a promising tool for imaging NO in response to pharmacological agents that modulate its level, for example during bacterial infections. The second strategy explored in this work was the development of a profluorescent nitroxide probe, which can be utilized for detecting the formation of reactive nitrogen and oxygen intermediates and associated changes in redox states within microcolonies. Attachment of a nitroxide to a fluorophore leads to fluorescence quenching, which upon free radical scavenging, metabolism or redox processes, returns the molecule to its native fluorescent state. A large variety of synthetic approaches and procedures were explored to construct such structure, but unfortunately none of them were successful.