School of Chemistry - Theses
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ItemThe use of nitroxides in the control and understanding of biofilm formation on cultural materials( 2014)Considerable aesthetic and structural damage to culturally significant materials can be caused by the growth of biofilms and the production of harmful metabolites by microorganisms. Traditional methods to control biofilm formation and to treat biodeteriorated materials has focused on biocidal and antibacterial approaches. The pitfalls associated with such strategies involve the development of tolerance in addition to poor effectiveness due to the refractory nature of biofilms to exogenous physio-chemical pressures. The development of biofilms therefore represent a remarkable adaptation by microorganisms to manage changing environmental parameters. Pivotal to the continual maintenance of a community of microorganisms is the microbial cell-to-cell communication system, quorum sensing. Utilizing biochemical messenger molecules, quorum sensing allows the population to function in unison by initiating signal transduction cascades that culminate in population wide changes in gene expression. Key to this research are the quorum sensing controlled and synchronized dispersal events where sessile cells are transformed into the more biocide susceptible planktonic bacteria. Compounds which encourage cells to be planktonic rather than sessile through the manipulation of cell signalling therefore offers a novel technique to control biofilm formation and biodeterioration. Nitric oxide is one such signalling molecule that has been linked to the inhibition of biofilm formation and activation of dispersal through the generation of nitrosative and oxidative stress. Nitroxides have also shown to promote analogous stress conditions upon bacterial cells and are structurally similar to nitric oxide, both with a free radical that is delocalized between the nitrogen and oxygen atoms. However unlike the highly reactive nitric oxide, some nitroxides are persistent radicals as evidenced by their resistance to dimerization and disproportionation reactions. This thesis investigates the efficacy of nitroxides as anti-biofilm compounds that inhibit and disperse bacterial biofilms. Initial work was directed towards the construction of a nitroxide library. The approach adopted involved the design and synthesis of 22 nitroxide candidates (32, 35-58) with varying charges and hydrophilicities in order to target a range of intra- and extra-cellular compartments. A batch-culture technique was used to screen the biofilm modulatory activity of nitroxide candidates and this identified one nitroxide, 3-(dodecane-1-thiyl)-4-(hydroxymethyl)-2,2,5,5-tetramethyl-1-pyrrolinoxyl (53) that was able to significantly suppress biofilm formation and elicit dispersal events in both a model organism, P. aeruginosa, and on biofilms composed of organisms derived from cultural material. Furthermore using semi-solid agar motility assays, it was revealed that surface-associated cell motilities - twitching and swarming - closely linked to the biofilm phenotype, were exclusively enhanced by lead nitroxide 53, leaving the planktonic-specific swimming motility unaffected and suggesting that the 53-mediated biofilm modulation is linked to the hyperactivation of cell motility. In order to investigate the role of the free radical moiety in the biological activity of 53, the ethoxylamine derivative 83 was prepared and tested for biofilm modulation. Evaluation of its biological activity using a batch-culture assay revealed that the free radical moiety is critical in the efficacy of 53-mediated biofilm inhibition and dispersal and one can speculate that 53 may function via the same pathway as nitric oxide. To further elucidate the mechanism of 53-mediated dispersal and investigate the formation of reactive nitrogen and oxygen intermediates and associated changes in redox states within microcolonies that may be associated with a nitric oxide-mimetic dispersal mechanism, two cell permeable and biocompatible novel profluorescent nitroxide probes (125, 145) and their corresponding ethoxylamine derivatives (126, 146) were synthesized. Imaging of 125 within a biofilm showed that 53-mediated biofilm dispersal is not linked to the formation of free radical species or oxidative stress.