School of Chemistry - Theses
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ItemDevelopment and characterisation of micro polymer inclusion beads (µPIBs) for the separation of rare earth elements( 2022)The global demand for rare earth elements (REEs) is predicted to significantly increase in the near future. This is due to expanding production of green technologies heavily dependent on the incorporation of REEs, such as electric vehicles (EVs) and wind turbines. However, REEs are currently supplied by mining REE minerals, which require high intensity industrial processing and releases REEs into the environment. As a result, our demand for REEs is causing a significant environmental impact. Therefore, there is a considerable interest in the development of more sustainable extraction technologies and their application to alternative sources of REEs, namely recycling from end-of-life (EOL) electronics. This thesis details the development of a new polymer-based extraction material, denominated as micro polymer inclusion beads (uPIBs), and its application to the separation of REEs. The thesis covers the development and optimisation of uPIBs composed of the base polymers poly(vinyl chloride) (PVC) or poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) which immobilise between their entangled chains the extractant di-(2-ethyl hexyl)phosphoric acid (D2EHPA). The uPIBs were fabricated via a recently developed phase inversion microfluidic method, which was modified to allow effective fabrication of the PVC and PVDF-HFP-based uPIBs containing up to 60 wt% D2EHPA. These modifications included acidification of the fabrication solutions to 0.1 M sulfuric acid, and increasing desolvation solution salinity from 5 to 15 wt% NaCl. The uPIBs demonstrated superior extraction performance over their polymer inclusion membrane (PIM) counterparts, attributed to the higher surface area exposed to the feed and receiving solutions. This thesis also covers the uPIBs characterisation via thermogravimetric analysis (TGA), which was used to confirm the uPIB’s D2EHPA content, using PIMs with equivalent composition as reference standards. Negligible D2EHPA leaching occurred from both the PVC and PVDF-HFP-based uPIBs under the fabrication conditions applied here. Characterisation of PVC-based uPIBs using an isothermal step highlighted the presence of Na+, extracted during the fabrication process, which was successfully removed through a 1 M sulfuric acid washing step. The removal of Na+ was found to significantly improve the extraction performance of the uPIBs and would not have been identified if not for the application of the isothermal TGA method developed here. The performance of the newly developed uPIBs (60 wt% D2EHPA, 40 wt% PVC) was characterised for the on-line separation of REEs by packing a column with uPIBs. La3+ and Gd3+ were initially used as model REE ions and could be separated by selective extraction or back-extraction. The column separation method was then applied successfully to the recovery of the REE ions Nd3+ and Dy3+ from EOL rare earth permanent magnet (REPM), which was digested in 2 M sulfuric acid. Upon decreasing the acidity of the digest to 0.03 M sulfuric acid, Nd3+ and Dy3+ were selectively extracted after the introduction of ascorbic acid at a 3:1 ratio with Fe to reduce Fe3+ to Fe2+. Nd3+ and Dy3+ could then be selectively back-extracted from the uPIB-column using 0.3 and 2.0 M sulfuric acid solutions, respectively. On the basis of the results presented in this thesis it can be concluded that the column separation method based on the newly developed uPIBs has shown promising potential as a future sustainable method for recycling REEs from digested EOL electronics.
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ItemSonosynthesis of Functional Micro/Nano-structures using Biomolecules( 2022)Malnutrition and access to affordable health services are some of the world’s most urgent problems. The development of nutrient and drug delivery systems by using sub-micro particles as carriers has attained much attention for improving the nutritional value of food and the efficacy of diagnostic/therapeutic treatments. Common methods for synthesizing biofunctional particles usually require many reagents and involve multiple steps. In this regard, a novel and advanced approach for material synthesis needs to be developed and investigated to address those limitations. Ultrasonic techniques have emerged as one-pot and eco-friendly methods for the synthesis of organic and inorganic materials. It has been found that acoustic cavitation derived from sound waves can induce emulsification of liquids to form microcapsules and promote chemical modifications of biomolecules. Protein-shelled microcapsules have been synthesized by using low-frequency ultrasound and reducing agents for food-based applications. However, the direct use of food ingredients for the microencapsulation of microcapsules without resorting to additional external agents was never explored and needs to be investigated further for food-based applications. On another note, drug carriers are usually prepared in nanoscale to enhance interaction with cell membranes for achieving efficient therapeutic treatment. Conventional strategies for preparing drug loaded nanoparticles require matrix materials as carriers, resulting in low drug loading capacity and safety issues. Therefore, synthesis of nanodrugs solely made of antibiotic molecules is a better method for development of drug delivery platforms. Many molecules bearing aromatic groups have been successfully sono-assembled into nanoparticles by high-frequency ultrasound, but they are mainly used as drug carriers. Transforming drug molecules into carrier-free nanodrug has not been widely investigated. As such, I intend to expand new research towards other drug molecules with aromatic moieties. In this regard, my Ph.D. project aims to sono-chemically synthesize various micro and nano structures from biomolecules by tuning the frequency/power of ultrasound without the usage of external reagents. The size of the obtained bio-functional structures is controllable, and their compositions are suitable for use in specific applications such as : i) nutrients delivery in food industries; ii) drug delivery for biomedical applications. The fundamental concepts of sono-chemistry for material synthesis, along with biomolecules (proteins, nutrients and antibiotics) based micro/nano structures and their applications are discussed in Chapter 1. Chapter 2 provides an overview of microencapsulation techniques for food industries and fabrication of nanoparticles for antibiotics delivery. In particular, methodology, formulating materials, current challenges, limitations and innovation are discussed. In Chapter 3, the materials, equipment and methodologies involved in the reactions used in this thesis are thoroughly described. Chapter 4 is the first chapter of result and discussion section. Microcapsules made of egg white protein (EWP), as commonly available biopolymers, were first conceptualized. Oil-soluble nutrients (Vitamin A, D and E) were encapsulated into EWP to form nutrients loaded proteinaceous microcapsules by employing 20 kHz ultrasound. This work primarily points out that high availability of free thiol groups in protein solution is crucial in forming stable microcapsules with robust shells, in order to protect micronutrients from degradation against detrimental effects. In Chapter 5, another two plant-based protein isolates extracted from soybean (SPI) and corn (CPI) were also formulated to form microcapsules. This study provided further insights into the structural, chemical and surface properties of proteins for efficient ultrasonic microencapsulation of micronutrients. A double emulsion technique was further developed to co-encapsulate both oil- (vitamin A and D) and water-soluble (vitamin B, C and minerals) micronutrients. In-vitro digestion study showed that the proteinaceous microcapsules enable sustained release of micronutrients, demonstrating their potential in food fortification applications. In Chapter 6, a sono-chemical strategy for transforming antibiotic doxycycline into carrier-free nanodrugs via high-frequency ultrasound (490 kHz) is reported. This study demonstrates that doxycycline undergoes hydroxylation and dimerization processes upon sonication in an aqueous solution to ultimately self-assemble into nanoparticles. The size of obtained particles could be finely controlled by tuning the applied ultrasonic powers. The nanodrugs exhibited antioxidant properties, along with antimicrobial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacterial strains. These results highlight the feasibility of the ultrasound-based approach for engineering carrier-free nanodrug with multiple controlled bio-functionalities. Chapter 7 provides an overall summary of the entirety of my PhD project as well as my conclusion and thoughts on it.
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ItemInvestigating the Mechanism of Cysteine Dioxygenase( 2022)Non-heme enzymes utilise reactive iron-oxygen intermediates in their respective mechanisms, which are key to their reactivity. Common intermediates postulated are Fe(III)-superoxide and Fe(IV)-oxo. Among non-heme enzymes, the most well-known mammalian thiol dioxygenase is cysteine dioxygenase (CDO). CDO is responsible for the oxidation of cysteine by adding molecular oxygen to the sulfur of cysteine to form a sulfinic acid known as cysteine sulfinic acid. Many studies have proposed which intermediate occur during CDO turnover, but the mechanism is still unknown. Chapter 1 provides a comprehensive review of the mechanisms of non-heme enzymes and the iron-oxygen intermediates involved, as well as a general introduction to thiol dioxygenases. Thiol dioxygenases exhibit high specificity with their respective substrates, and catalyse very similar reactions. However, due to the challenges faced by the lack of intense absorption and direct spectroscopic evidence of oxygen-bound intermediates, the mechanism of these enzymes remains unclear. Chapter 2 describes the purification of WT CDO and specific variants of CDO. It also describes all the different techniques performed in this work. In chapter 3, the different CDOs included C93G, Y157F and H155Q CDO were used to study the mechanism of disulfide formation, a side product of the CDO reaction. The amount of disulfide formed increases when a non-native substrate is used or substitutions of catalytically important residues are made. These effects were investgated through kinetic studies, 1H-NMR and mass spectrometry. In particular, the presence of the sulfenate, which is an intermediate in the formation of disulfide was probed during turnover. The identity and quantification of sulfenate supports formation of a Fe(IV)-oxo during turnover and that proper substrate chelation appears to be critical for S-oxygenation. In chapter 4, WT CDO, C93G and Y157F CDO were used to study reactivity upon substitution of iron with the non-native metal, Co(II). This was done in the hope of stabilising new reactive intermediates. A Co(III)-superoxide was characterised by EPR and UV-Vis for Y157F and C93G CDO . In contrast, a tyrosyl radical was observed in WT and C93G CDO. In the case of WT CDO this led to an increase in crosslink between C93 and Y157, suggesting that the Co(III)-superoxide undergoes rapid H atom abstraction from Y157. Chapter 5 describes the formation of cysteic acid (CA) as a second subsequent product of the CDO reaction at high pH (9.1). Product distribution of the reaction of C93G CDO pre-bound with cysteine and CSA were quantified by HPLC-mass spectrometry. The data shows that under these conditions, cysteine is oxidised initially to CSA and then further to CA. The final chapter re-evaluates the mechanism of CDO proposed by computational studies. The thesis provides one of the first protein-based evidence that support Fe(III)-superoxide and Fe(IV)-oxo intermediates are formed during the CDO catalytic mechanism. It also provides first experimental evidence that a sulfenate is indeed formed during catalytic turnover and that a reactive Co(III)-superoxide can be detected when iron is replaced by the non-native metal cobalt. Lastly, the products of the reaction of C93G CDO with cysteine and CSA at higher pH are presented and the implications of these results to the mechanism of CDO with its native substrate cysteine are discussed.
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ItemBiological Interactions of Responsive Polymeric Nanoparticles( 2022)Nanoparticles need to be capable of endosomal escape to facilitate the effective delivery of therapeutic cargo, particularly biological materials. While there are many mechanisms postulated, some of them remain poorly understood. In this study, pH-induced swelling and proton sponge mechanisms for endosomal escape have been investigated. In the first two projects, swellable pH-responsive nanoparticles were designed and synthesised through emulsion polymerisation. Nanoparticles exhibited pH responsive swelling when the pH decreased from pH 7.4 to lower than 6.2. The swelling pH and percentage were tuned by altering the type of crosslinker or the composition and amount of pH responsive monomers. Also, particles showed strong buffering capacity as well as the ability to associate with cells, however there was no clear evidence of endosomal escape using the calcein assay. The results have generated serious questions about the validity of both these mechanisms to explain the endosomal escape of nanoparticles. In the third project, covalently- and physically-doxorubicin (Dox)-loaded particles with the capability of pH-induced release were designed and studied. Loading capacity was tunable by introducing various level of pentafluorophenyl functional moieties into particles in covalently-Dox-loaded particles. However, physically-Dox-loaded particles showed higher cytotoxicity and lower IC50. These findings suggests that both covalently- and physically-Dox-loaded particles could be potential candidates as drug delivery systems. In the fourth project, camptothecin (CPT)-loaded disulfide particles were also designed and synthesised by the oxidation of thiol monomers. Hydrophobic CPT was loaded into particles covalently and was able to be released by thiol-disulfide exchange reaction in response to the redox environment of cells. Drug release was tuned by the change in particles compositions. Cytotoxicity of drug-loaded particles was confirmed compared to control particles (non-drug-loaded) by using Alamar blue viability assay on MCF-7 cells. The study on swellable pH-responsive polymeric nanoparticles indicates that more work needs to be done to understand the endosomal escape of cargo/particles through swelling and proton sponge effect mechanisms. The study on introducing novel methods to load drug into polymeric particles showed these particles have potential for the effective controlled release of hydrophilic/hydrophobic therapeutic cargo in physiological conditions in response to pH variation and redox increase.
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ItemTransport, bioaccumulation, and risk of per- and polyfluroalkyl substances (PFASs) in birds from south-east Australia( 2022)Over six million per- and polyfluoroalkyl substances (PFASs) have been identified with a broad range of uses, including commercial and industrial products such as aqueous-film-forming foam (AFFF), mist suppressant, food contact materials, cosmetics, and therapeutic drugs. Some classes of PFASs have been termed “forever chemicals” due to the strong carbon-fluorine bond that resists metabolism and degradation. To help inform key stakeholders of the potential risks involved in the exposure to these compounds the occurrence, transport, and fate of legacy and emerging PFASs within the Southeast Australian environment, this thesis aims to describe the exposure pathways and bioaccumulation in native avian species. Wastewater treatment plants (WWTPs) are a common source of PFASs to the environment and hourly variations in PFAS concentrations could have wide ranging implications for the estimation of discharge to the environment that determine risk-based human and environmental health policies. Optimised and novel sampling and analytical methodologies were developed to measure the concentrations of PFASs in biological matrices from avian species exposed to long-range and diffuse sources of PFASs in Australia, such as WWTPs. Exposure to PFASs derived from long-range transport in marine and freshwater species results in concentrations generally considered to be low risk. Conversely, a moderately impacted surface water body in Melbourne has revealed the relatively high exposure of PFASs, including novel substances, to a resident swan population that may serve as an important model for the distribution and bioaccumulation of the chemicals in Australian species for the first time. Due to the limited information regarding the occurrence, fate, and potential impact to avian species in Southeast Australia, monitoring and hypothesis-driven research is required to assess the risk of PFASs to animals and humans that may also be exposed to similar environments.
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ItemApplications and analysis of sulfoglycolysis pathways( 2022)Sulfoquinovose (SQ; 6-deoxy-6-sulfo-D-glucose) is an analogue of glucose with a 6-sulfonate group. It is a major species in the biogeochemical sulfur cycle and arises from the hydrolysis of various natural SQ glycosides, including sulfoquinovosyl diacylglycerol (SQDG), through the action of highly specific glycosidases termed sulfoquinovosidases (SQase). Five sulfoglycolysis pathways have been reported that enable the catabolism of SQ and all gene clusters encoding these pathways usually contain genes encoding SQases. Thus, SQases are considered a key gateway enzyme to allow SQ to enter every sulfoglycolysis pathway. These pathways also contain SQ importers with one system involving an SQ binding protein that captures SQ and delivers it to an ATP-binding cassette transporter. In Chapter 2, we report the first fluorogenic SQase substrate, 4-methylumbelliferyl alpha-D-sulfoquinovoside (MU-SQ), which we use to characterize several SQases, including a new SQase from Eubacterium rectale. Unexpectedly, compared to the established chromogenic substrate, p-nitrophenyl alpha-sulfoquinovoside (PNP-SQ), enzyme activity measured using MU-SQ was extremely low (rate constants 10^4-10^5-fold lower). We speculate that the difference arises because the bicyclic structure of MU is too bulky to easily bind to SQases as a result of their evolution to preferentially process substrates with slender aglycons like SQDG. While we initially considered developing new fluorogenic and histological substrates for the detection of SQase activity in complex natural samples, the poor SQase activity on MU-SQ led us to abort this aim as the proposed substrates included even bulkier aromatic systems. Chapter 3 presents a detailed sub-family classification for the Carbohydrate Active Enzyme family GH31, a grouping of which SQases are a member. In addition to hosting the SQases, family GH31 is one of the major glycoside hydrolase families, and more than 99% of its members remain uncharacterised. However, predictions of their activity are difficult as there are only 124 characterized enzymes that display fifteen different activities. We conducted a sub-family classification using sequence similarity networks (SSNs), hidden Markov models (HMMs) and the results were explored and validated using phylogenetic and structural analysis. Our classification created subfamilies with assigned members possessing usually just one activity, and provides improved predictive power for more than 75% of uncharacterized enzymes in the family. The new subfamily classification has been deployed to the CAZy database where is it undergoing internal assessment as part of their daily curation activities. Chapter 4 pursues an application of discoveries in the field of sulfoglycolysis for modern DNA biotechnology. A commonly used purification tag for heterologously-expressed proteins uses maltose-binding protein (MBP). However, purifications using MBP as a tag has a drawback: the crosslinked amylose column is degraded by amylases constitutively-expressed by the host. Therefore, we proposed a new expression and purification system using the SQ binding protein (SQBP) from Agrobacterium tumefaciens as an affinity tag. This chapter reports the synthesis of an ethylene-glycol-based linker equipped with SQ for conjugation to CNBr-activated Sepharose. Sepharose conjugated with SQ will, in future work, be used to explore the use of SQBP as an affinity purification tag for heterologously expressed proteins fused to SQBP. Collectively, this thesis provides new approaches to study SQases, new insights into how SQases sit within the broader sequence-based classification of sequence related glycosidases, and provides a potential new application for an SQ binding protein in recombinant protein purification. The study of sulfoglycolysis pathways continues to furnish new discoveries and potential new applications.
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ItemTotal Synthesis of Alkyl Citrate Natural Products via Cyclobutene Diesters( 2022)The alkyl citrate natural family of products all possess a common citric acid moiety with a lipophilic polyketide sidechain at the C2 position. Many members of the alkyl citrate natural products are potent squalene synthase (SSase) inhibitors along with many other biological properties. The main challenges of alkyl citrate synthesis are the construction of the contiguous asymmetric centres and introduction of the triacid oxidation state. For the synthesis of this family of compounds, the main aim was a synthesis of a common citrate fragment with the requisite stereochemistry and correct oxidation state.
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ItemSynthesis and antioxidant capacity of chalcogen-containing furanose derivatives( 2021)Cardiovascular diseases are known to be a leading cause of death around the globe. Oxidative damage of endothelium plays a crucial role in development and progression of hypertension, atherosclerosis and other pathological cardiovascular conditions. Therefore, antioxidants that can effectively remove reactive oxygen species, represent promising therapeutic approaches. Sulfur, selenium and tellurium compounds act as effective oxidant scavengers due to their fast reaction rates with typical biological oxidants. Previous studies have shown that a novel selenofuranose derivative, specifically 1,4-anhydro-seleno-D-talitol, can modulate oxidative damage of biomolecules in plasma and exhibits wound healing activity. It should be noted tissue repair is extremely important in the treatment of vascular pathologies. Little is known about furanose analogues of seleno-D-talitol. Their antioxidant capacity and biological action were not investigated while knowledge of those could provide guidance for the development of novel selenosugar based therapeutics. In this thesis, approaches to the synthesis of selenium and tellurium furanose derivatives are described. Selenium and tellurium sugars were prepared from a range of different starting materials such as monosaccharides, lactones and sugar alcohols. Selenide and telluride moieties were inserted into the sugar core using sodium selenide and sodium telluride prepared in situ. Final furanose analogues were synthesised in up to 8 steps with good yields. Unlike selenium containing furanose derivatives, most of their tellurium counterparts were unstable light sensitive compounds presumably due to the larger chalcogen atom causing strain in the sugar ring. Additionally, thio-D-talitol and D-talitol with oxygen in the sugar ring were prepared for comparison in antioxidant studies. Antioxidant activity of the synthesised compounds was determined in Chapter 3. Cyclic voltammetry and linear sweep voltammetry were used for investigation of oxidation of chalcogen-containing selenofuranose derivatives. It was shown that the primary oxidation of the sugars is an irreversible process which, presumably, occurs through oxygen transfer reaction. The oxidation potentials were determined and the half-wave potentials for electrochemical oxidation of the chalcogen-containing sugars were calculated. In Chapter 4 the in vitro vascular studies were carried out to investigate the effectiveness of the synthesised chalcogen-containing furanose derivatives, toward the prevention of endothelial damage in mouse aorta under oxidative stress conditions. The in vitro studies revealed that the stereo configuration of the sugar core significantly influences the biological activity of the chalcogen-containing furanose analogues of seleno-D-talitol. In the range of selenofuranose derivatives, seleno-D-talitol was shown to prevent endothelial dysfunction more effectively than its analogues. Furthermore, the structural isomer, hexose sugar selenogalactitol demonstrated different effects on mouse aorta than the selenopentoses, by presumably inhibiting endothelial nitric oxide synthase. As expected, thio-D-talitol exhibited a lower ability to protect the endothelium in mouse aorta from oxidative stress, than its selenium counterpart, seleno-D-talitol. Telluro-D-talitol appeared to be toxic to cells in mouse aorta.
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ItemTotal Synthesis of the Viridiofungins and Structural Elucidation of 8,13-Deoxynargenicin( 2022)A fourteen step total synthesis of alkyl citrate natural products viridiofungin A, B, A2 and NA808 and thirteen step synthesis of viridiofungin Z2 is described. The stereochemistry of the citrate core of these natural products is set up via a diastereoselective formal [2+2] cycloaddition followed by a HF-mediated rearrangement to afford a bicyclic lactone. A key step of this synthesis is a cross metathesis reaction of a hindered disubstituted alkene beta lactone with the alkene side chain, whereby a benzoquinone additive is used to suppress unwanted alkene isomerisation of the starting material. The next step in the synthetic route is the novel formation of an amide via the nucleophilic ring opening of a beta lactone with an amino acid ester. Deprotection afforded either viridiofungin A, B, A2 and NA808. Removal of the tert-butyl esters and concomitant hydrolysis of the beta lactone of viridiofungin Z2 tri-tert-butyl ester afforded viridiofungin Z2. The structural elucidation of NarN knockout metabolite and proposed biosynthetic precursor of antibiotic macrolide nargenicin A1, 8,13-deoxynargenicin, is described. A combination of 1H and 13C NMR spectroscopy, COSY, HSQC and HMBC 2D-NMR experiments were used to support the proposed structure of 8,13-deoxynargenicin. The relative stereochemistry of 8,13-deoxynargenicin was assigned from a combination of 1H-1H J coupling constants as well as 2D-NOESY NMR correlations.
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ItemTemperature Jump Spectroscopy Studies of Colloid Systems( 2022)Temperature jump spectroscopy is a powerful method that has been used since its development in the 1960s to measure kinetics in chemical systems. It has very rarely been applied to colloids however, despite these systems being inherently complex and well-suited for relaxation studies. In this thesis we demonstrate that temperature jump spectroscopy is a fantastic asset to probe structural and morphological changes in colloid systems. We focus on a class of thermoresponsive core-shell microgels that undergo different types of reversible phase transitions. The first of these is a volume phase transition, where the gel network in the shell swells and deswells in response to temperature. The second is a reversible aggregation transition, which can be prompted by temperature and occurs under suitably high electrolyte conditions. The dynamics of these phase transitions are still largely unexplored. We first develop a scattering model that determines the relationship between the visible-light scattering cross-section and the morphology of microgels as they undergo swelling and deswelling in response to temperature. This enables us to correlate optical density changes observed upon relaxation of the system to specific structural changes. We then use temperature jump spectroscopy to characterise the reaction pathway undertaken by microgels as they transition between different volume phase states. Finally, we characterise aggregation/dissociation dynamics, which occurs at long time-scales following the temperature jump under certain conditions. This kinetic data will allow us to better understand how dynamic processes can be harnessed in microgels, and applied to applications like nanoparticle self-assembly, sensing and drug delivery. The general procedures developed throughout also demonstrate how temperature jump spectroscopy can be extended to other complex colloid systems.