School of Chemistry - Theses
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ItemModel chemistry relevant to the molybdenum copper active site of CO dehydrogenase(University of Melbourne, 2007)
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ItemToward the synthesis and analysis of selenium-containing glucocorticoid prodrugs(University of Melbourne, 2007)
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ItemNMR studies of amyloid ab-peptide in membranes(University of Melbourne, 2006)
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ItemTowards large-scale fabrication of plasmonic nanomaterials by fluid-mediated forces( 2017)Nano-assembly promises powerful technological advances, yet there is a persistent need to make such assembly more economical before we see these benefits in daily life. Scalable and reliable nano-assembly of any kind has remained elusive to researchers worldwide. This thesis focuses on Capillary Force-assisted Assembly (CFA), particularly for plasmonic structures and materials. CFA assembles nanoparticles out of solution via liquid-mediated forces over a nano-template. It is significantly cheaper and faster than standard nano-fabrication routes. CFA-assembled optical plasmonic materials have not been demonstrated over sizes larger than square-micrometers. An early goal of this work is to demonstrate centimetre-squared assembly. An unexpected hindrance emerged in the reliable manufacture of large-area nano-templates. Despite this, scaled-up assembly has been demonstrated, producing an optical two-tone metamaterial from plasmonic nanorod pixels. Harnessing the CFA protocol is difficult. Experimenters rely heavily on the current CFA model to guide their efforts. This model is widely accepted yet has received little critical analysis. In pushing the envelope of CFA, the conventional CFA model is inadvertently put to the test, and it is found wanting. A much needed thorough review dismantles the conventional model from experimental and theoretical standpoints. An as-yet unmentioned type of capillary force is suggested to apply on nanoparticles in CFA, and its form is mathematically derived. A new framework for CFA is proposed. In this framework, contact line pinning on template cavities, and the subsequent local enhancement of convective flows, are the primary assembly drivers. This model draws well from the limited existing nano-wetting theory, and conforms well to high-level experimental expectations. However, for reliable and scalable CFA, core dynamics must be quantified and relevant experimental parameters ascertained. This is non-trivial, requiring knowledge of the time-resolved three-dimensional (3D) meniscus form during single CFA-assembly events. No techniques exist to non-invasively probe such dynamics. Therefore, a novel experimental technique is developed to rapidly and non-invasively profile a meniscus shape in 3D on the micro-scale. Meniscus dynamics are observed over templated cavities with millisecond resolution, showing that the meniscus pins and closes down over cavities, acting to clamp particles in place. Moreover, evaporative dynamics can be modelled on the 3D micro-meniscus profiles, paving the way for crucial thermodynamic analysis. Characterisation of the new model therefore begins by abundantly probing pinning dynamics on cavities. Surface tension is revealed to direct pinning dynamics down to the nano-scale; inertial/viscous forces become negligible. Interpolatable trends allow us to theoretically reconstruct meniscus forms under a range of experimental conditions at any moment during a single CFA assembly event. From this versatile CFA-meniscus model, and armed with the ability for thermodynamic analysis, convective flows during assembly events are estimated and validated against extant literature. The subsequent kinematic behaviour of a nanoparticle in a cavity’s vicinity during a CFA-assembly event is simulated under a variety of experimental conditions. This sheds valuable light on which experimental parameters are most crucial to optimise CFA dynamics, and why. This new and arguably successful model is called the pinned-convective model of nano-CFA.
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ItemExploring novel blue turn-on fluorescent probes for the direct detection of nitric oxide and free radicals in living cells( 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.
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ItemSynthesis and investigation of polyoxometalate-supported lanthanoid single-molecule magnets( 2017)The crystal field splitting of the literature lanthanoid single-molecule magnet (Ln-SMM) family Na9[Ln(W5O18)2] has been investigated using inelastic neutron scattering (INS). The experimental measurements have been complemented by complete active space self-consistent field (CASSCF) ab initio methods. Chapter 2 of this thesis is devoted to the preliminary study of the TbIII analogue alone. Given the novelty of INS applied to Ln-polyoxometalates (Ln-POMs), and the potential challenges posed by Ln-POM systems (i.e the neutron absorption of the lanthanoid and of tungsten atoms), the choice of the Tb analogue, which is not a SMM, was dictated by the fact that Tb has the lowest neutron absorption cross-section. It was not possible to rationalize the observed INS transitions using current models for the electronic structure of this system, so the INS spectra were interpreted based on crystal field CASSCF ab initio methods, the latter being an unprecedented theoretical approach for Ln-POMs. Ab initio calculations on the isolated polyanion [Tb(W5O18)2]9- (gas phase) allowed attribution of the complex INS spectra of Na9[Tb(W5O18)2] to the presence of two distinct polymorphs, with slightly different structural parameters. A simple magnetostructural model to correlate the INS signal of each polymorph with the structural parameters of the first coordination sphere of TbIII was proposed. In Chapter 3, based on the successful INS study of the TbIII analogue, the spectroscopic investigation was extended to the more challenging – in terms of neutron absorption cross-section - Nd, Ho and Er SMM analogues of the Na9[Ln(W5O18)2] family. Magnetic excitations were observed for all compounds investigated. The INS transitions and the magnetic properties were interpreted based on crystal field methods and the CASSCF ab initio method developed in Chapter 2 was extended to these compounds, including also the Dy analogue, showing that low-symmetry effects play an important role in defining the electronic properties of these systems. The electronic structures calculated ab initio outperform in many cases the current state-of-the-art theoretical models for the Na9[Ln(W5O18)2] family in terms of the ability to reproduce the energy of the low-lying crystal field levels as determined by INS. A model representing the distribution of electrostatic point charges associated to the atoms in the in the crystal lattice was introduced in the CASSCF calculations. This approach gave better agreement with the experimental data with respect to the “gas phase” methodology employed in Chapter 2. Chapter 4 presents the synthesis and characterisation of a new family of organic hybrid Ln-POMs. The compounds have been characterised by single crystal X-ray diffraction, X-ray photoelectron spectroscopy, elemental analysis, and FT-IR spectroscopy. Detailed direct (dc) and alternating current (ac) magnetic characterisation was performed, revealing slow relaxation of the magnetisation in the presence of an external dc magnetic field for one compound.
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ItemNew insights into the redox chemistry of protein thiols( 2017)Redox reactions play a crucial role in many biological processes. These include the role of Cu(II)/Cu(I) couples in enzyme reactions and that of the tripeptide glutathione (GSH) as a key redox buffer in cells via the GSSG/2GSH redox couple. Copper is an essential trace metal but, if redox balance is not properly maintained, excess or ‘free’ copper is toxic. Consequently, specific metabolic routes exist to safely maintain the copper balance within cells. A particular challenge in understanding molecular copper metabolism is purification of cupro-proteins in high purity and yield. A variety of affinity tags, such as the popular poly-histidine tag, have been developed to facilitate purification but they generally rely on expensive affinity resins and their presence may interfere with protein characterization. This work demonstrates that a poly-lysine tag at the C-terminus enables, for certain cupro-proteins, ready purification on large scales via cost-effective cation-exchange chromatography. Cleavage of the tag is normally not necessary since the poly-lysine tag is shown to have no detectable affinity for either Cu(I) or Cu(II) and imposes no interference to the copper binding properties of the target proteins. In contrast, the poly-histidine tag possesses a sub-picomolar affinity for Cu(I) and sub-nanomolar affinity for Cu(II) and may need to be removed for reliable characterization of the target proteins. The GSSG/2GSH couple partners protein thiols in reversible thiol-disulfide exchange reactions that act as redox switches and play important roles in cell signalling and redox homeostasis. Disruption of these processes induces oxidative stress that is linked directly to aging processes and to a range of conditions including cancer and neurodegenerative diseases. Glutaredoxins (Grxs) are a class of oxidoreductase enzymes that specifically catalyse GSH-dependent thiol-disulfide exchange reactions and play essential roles in the regulation and maintenance of redox balance in cells. They protect protein thiols from irreversible oxidation and regulate their cellular activities under a variety of conditions. However, the molecular basis and underlying mechanisms of Grx action remain elusive are controversial and uncertain in many situations. Grxs feature dithiol active sites and can shuttle rapidly between three oxidation states, namely, fully reduced dithiol Grx(SH)2, semi-oxidized mixed disulfide Grx(SH)(SSG) and fully oxidized disulfide Grx(SS). Each is characterized by a distinct standard reduction potential (E^o' ). E_(P(SS))^o' values for the redox couple Grx(SS)/Grx(SH)2 are available but a recent estimate differs by over 100 mV from literature values. No estimates are available for E_(P(SSG))^o' for the mixed disulfide couple Grx(SH)(SSG)/(Grx(SH)2+GSH). The work reported in this thesis determined both E_(P(SS))^o' and E_(P(SSG))^o' for two representative Grx1 enzymes, human hGrx1 and E. coli EcGrx1. The empirical approaches were verified rigorously to overcome the sensitivity of these redox-labile enzymes to experimental conditions. Classic ‘acid quenching’ is demonstrated to shift the thiol-disulfide redox equilibrium. The reduction potentials are in the range that favours dual catalytic functions for Grxs as either an oxidase at low [GSH] or a reductase at high [GSH]. These enzymes were demonstrated to catalyze glutathionylation and deglutathionylation of a target protein monothiol under conditions of low and high GSH concentration, respectively. The catalysis was demonstrated to proceed via a monothiol ping-pong mechanism relying on a single Cys residue only in the dithiol active site. Grxs also catalyze oxidation of a protein dithiol and reduction of a disulfide via conserved parallel monothiol and dithiol mechanisms. Consequently, Grxs are shown to be a class of versatile enzymes with diverse catalytic functions that are driven by specific interactions with GSH.
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ItemInhibitors of an essential M. tuberculosis cytochrome P450 enzyme CYP121( 2017)Tuberculosis (TB) is an infectious disease caused by a rod-shaped mycobacterium called Mycobacterium tuberculosis (Mtb). Since the discovery of streptomycin in the 1940s, several effective anti-TB drugs have been discovered and employed clinically. However, the battle against TB is ongoing with approximately one-third of world’s population estimated to be currently infected with Mtb, with an increased prevalence of multidrug resistant (MDR-TB) and extensively drug resistant (XDR-TB) tuberculosis observed over the last two decades. Thus, there is an on-going need to develop new antimicrobial drugs with novel modes of action. Genome sequencing of Mtb, completed in 1998, revealed the presence of 20 genes encoding cytochrome P450 enzymes (CYPs). One of these CYPs – CYP121A1 – has been shown to be essential for Mtb viability and catalyses a unique reaction involving carbon–carbon bond formation between two tyrosyl side-chains of the cyclodityrosine substrate. This suggests that it may be possible to develop selective inhibitors against CYP121A1 as potential novel anti-Mtb drug leads. During this project, a range of analogues of cyclodityrosine, the natural substrate of CYP121A1, have been synthesized and biochemically analyzed with the aim of defining the binding requirements for the enzyme active site and providing insights into the design of selective inhibitors against CYP121A1. Our analysis shows that modifications at positions 3 and 4 of the tyrosyl aromatic ring are well accommodated within the active site whereas substituents at position-2 are not tolerated. Further, substrate analogue with an iodine at position-3 on the tyrosyl aromatic ring exhibits a 100-fold greater binding affinity compared to the natural substrate. Antimicrobial assays of the substrate analogues including the 3-iodo analogue, show that they are not effective antimicrobial agents (MIC values > 100 μM).
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ItemUltrafast spectroscopy of nanostructures( 2017)This thesis presents studies of ultrafast laser spectroscopy of semiconductor and gold nanostructures, aiming to advance our understanding of, and consequently control, photoinduced charge carrier dynamics in nanostructures to further improve their performance in practical applications. Artificial nanostructures have drawn significant attention in applications such as optoelectronic devices, photo-catalysts, and solar cells. Compared to bulk materials, nanostructures provide unique optical properties, which more importantly can be directly and easily tailored through changing size or shapes of the structures, during their synthesis procedures. Photoinduced charge carrier dynamics in the nanostructures play an important role in the photon conversion processes. However, in contrast to the fast development of nanostructure-based devices, the mechanisms of these processes are still being experimentally unravelled. In this study, a range of ultrafast optical spectroscopy methods have been applied to investigate the carrier dynamics, with a focus on the electron transfer (ET) process. Semiconductor nanoparticles, or quantum dots (QDs), of core/shell heterostructures are promising for their good photostability and high photoluminescence quantum yields. The ET dynamics from the 1S$_\mathrm{e}$ electron state to adsorbed methyl viologen electron acceptors, in CdSe/CdS and CdSe/CdS/ZnS QDs, were studied using femtosecond transient absorption and time-resolved photoluminescence spectroscopy. By changing shell thickness or alloying the shell interface, significant modulation of the ET dynamics was observed. In CdSe/CdS QDs, the 1S$_\mathrm{e}$ ET dynamics exhibited a hole-coupled effect, which is ascribed to the Auger-assisted ET process. In CdSe/CdS/ZnS QDs, the formation of alloyed shell interfaces at elevated shelling temperatures reduced the shell potential barrier, leading to an observed greater ET rate. Photoinduced ET processes from gold nanorod and nanowire structures to TiO$_{2}$ were also investigated, using a visible pump-NIR probe transient absorption spectroscopy method. Partially embedded Au nanorods on a TiO$_{2}$ layer exhibited an enhanced but directional ET process. An Au nanowire grating supported on a TiO$_{2}$ layer structure underwent the plasmon-waveguide hybridisation mechanism. The ET dynamics from the split states showed a dependence on the light-matter coupling effect that can be varied with the Au grating period. In summary, this thesis shows the great ability of ultrafast optical spectroscopy to reveal photoinduced processes in nanostructures. Results indicate ways for rational design of nanostructure-based devices. A greater understanding in underlying physics leads to better control of the performance of these nano-systems in potential practical applications.
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ItemWater soluble fullerene and SPIO based nanoparticles by solvent exchange and polymer self-assembly methods( 2017)The synthesis of nanomaterials with tailored components and structures has drawn significant attention for industrial and research applications. Buckminsterfullerene (C60), metallofullerene and related materials have potential uses in varied fields such as MRI contrast agents, solar cells, bio-medicines and drug delivery systems. In particular, gadofullerene (Gd@C82) has drawn research attention as a new type of MRI contrast agent taking advantage of the Gd atom inside the fullerene cage. However, one of the main challenges of C60 or metallofullerene Gd@C82 for application in the biological sciences is their negligible solubility in aqueous solution due to their hydrophobic surfaces. In this research, different strategies have been investigated to explore water-soluble fullerene or related inorganic nanoparticles for potential MRI applications. A number of different strategies were investigated to design water-soluble fullerenes. In the second chapter, the solvent exchange technique was explored, which is a method for transferring C60 from an organic solvent into water under stirring or sonication. The use of tetrahydrofuran (THF) as a solvent to produce water dispersable fullerenes has been widely reported and extensively utilised. In this research, we have developed a related method of solvent exchange based on the use of N,N-dimethylformamide (DMF). The DMF approach results in the formation of fullerene nanoparticle agglomerates that are highly stable in phosphate buffered saline solution and also in water, while the THF agglomerates are only stable in water as previously reported. Moreover, we found that both of these approaches result in the significant degradation of the fullerene cage as shown by various techniques such as matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD), fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). This discovery was not previously reported in the literature. Our results have shown that the solvent exchange technique using THF results in partial oxidation and degradation of C60. In contrast, the DMF evaporative method results in greater oxidation and degradation of C60 but significantly enhanced colloidal stability in buffer solution. Another approach to modify fullerenes is through the use of polymers. RAFT polymerization is one of the widely used controlled living polymerization processes as it allows the synthesis of tailored polymer structures with controlled molecular weight and narrow molecular weight distribution. In our research, the parameters of RAFT polymerization of poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) were explored by the high throughput instrument, the Chemspeed platform, to explore the optimal polymerization conditions. The Chemspeed is a parallel synthesis platform with high-through output and automated robot synthesis instrument, which have been used to systematically investigate the parameters of different polymerizations and to discover new materials with accelerated efficiency. The polymers synthesised were pH responsive, meaning they changed structure with variation in pH. In particular, PDEAEMA polymers are attractive for its pH responsiveness with pKa ~ 7 which is between the pH values of healthy and cancerous tissues. The kinetics of RAFT polymerization of both PDEAEMA and PEGMA were investigated and homopolymers with different chain length were synthesized. These pre-synthesized PEGMA-b-PDEAEMA amphiphilic block copolymers have a great potential as a smart platform for both MRI contrast agent and drug delivery. In the forth chapter, the polymer/fullerene complex nanoparticles (PEGMA-b-PDEAEMA/C60 and PEGMA-b-PDEAEMA/Gd@C82) were successfully synthesized via a self-assembly method and then were characterized by dynamic light scattering (DLS), ultraviolet-visible (UV-vis) spectrophotometry, cryo transmission electron microscopy (cryo-TEM) and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The disassembly of the complex PEGMA-b-PDEAEMA/C60 nanoparticles could be triggered by lowering the pH in the solution which makes the nanoparticle as a smart platform with pH responsiveness. In addition, the PEGMA-b-PDEAEMA/Gd@C82 nanoparticles were also explored utilizing the self-assembly strategy developed for PEGMA-b-PDEAEMA/C60 nanoparticles. The particles demonstrated stable incorporation of Gd@C82. The MRI contrast behavior of these materials showed variation with the different length of block copolymers used. Our results indicate these materials are interesting as potential MRI contrast agents. In chapter five, this self-assembly synthetic strategy was applied to synthesize PEGMA-b-PDEAEMA/iron oxide nanoparticles as superparamagnetic iron oxide (SPIO) nanoparticles which have been widely used as MRI T2 contrast agents to improve the MRI imaging quality. The pH responsive PEGMA-b-PDEAEMA/Fe3O4 nanoparticles fabricated by the self-assembly method were characterized in detail by dynamic light scattering (DLS) and cryo transmission electron microscopy (cryo-TEM). Moreover, the pH responsive behavior of the complex PEGMA-b-PDEAEMA/Fe3O4 nanoparticles was also investigated via DLS and cryo-TEM. The complex PEGMA-b-PDEAEMA/Fe3O4 nanoparticles as a new MRI T2 contrast agent was also explored. The results indicated that the MRI response was tunable based on the polymer building block and also on the concentration of polymer used in the formulation. The simple and modular synthesis of water soluble C60, Gd@C82 and superparamagnetic iron oxide nanoparticles using pH responsive polymers provides a potential smart system for advanced MRI imaging, bio-medicines and drug delivery in the future.