School of Chemistry - Theses
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ItemExploring a new generation of nitrification inhibitors( 2023-05)Intensive application of nitrogen-based (N-based) fertilisers has become a common practise to achieve high crop yields to feed the growing global population. However, a substantial fraction of the applied N is lost from the plant-soil system to the surrounding environment, via ammonia volatilisation, nitrate leaching, and denitrification. These N losses negatively impact the environment and lead to unwanted groundwater contamination, eutrophication of waterways, biodiversity loss, greenhouse gas emission and stratospheric ozone depletion. Addition of nitrification inhibitors (NIs) to fertilisers is an effective strategy to minimise N losses and improve overall N-use efficiency in agricultural soils. 3,4-Dimethylpyrazole phosphate (DMPP) is the most successful NI to date but has a highly variable efficacy. New compounds with reliable performance under different agricultural and environmental settings that will assist in better synchronising the fertiliser N supply and crop demand are needed. Therefore, this thesis uses a systematic approach to explore the stability of current and newly developed NIs through degradation studies, to gain a better understanding of how degradation impacts the inhibitory activity, which should assist in the development of more efficient NIs with reliable performance.
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ItemEnergy Transfer Systems for Light Harvesting( 2023-03)Luminescent solar concentrators (LSCs) are solar harvesting devices that employ luminescent materials embedded within a waveguide to collect solar energy. The waveguide is typically made of a low-cost material such as glass or plastic and is designed to capture sunlight and redirect it towards solar cells. The luminescent materials, also known as luminophores, absorb the sunlight and re-emit it at a longer wavelength, allowing it to be trapped within the waveguide and guided towards the edges, where solar cells can convert the light into electricity. However, their performance is associated with several drawbacks, including reabsorption, which refers to the primary emission of the luminophore in the waveguide being reabsorbed by other luminophores in the waveguide. To overcome this, various strategies have been explored such as using different types of luminophores, optimizing the concentration of luminophores, and improving the design of the waveguide. Foster energy transfer (FRET), which reduces luminophore reabsorption, is a crucial aspect for improving energy conversion and device efficiency in LSCs. FRET works on separating the absorption and emission spectra of the luminophores by controlling the intermolecular spacing between donor and emitter molecules. Implementing these strategies can significantly enhance the performance of LSCs and accelerate their practical applications for solar energy harvesting. This thesis aims to advance the understanding of different energy transfer strategies for light harvesting applications. To implement an effective FRET approach, the concentration of luminophores is a crucial factor. The donor luminophore concentration should be higher than that of the emitter to allow energy migration through several donors to reach the emitter. However, increasing the dye concentration often leads to dye aggregation, which can quench the dye's fluorescence properties. To mitigate dye aggregation, molecular insulation in the luminophores can be an effective approach. In this thesis, the molecular insulation strategy is applied to both the donor and emitter luminophores to suppress dye aggregation at high concentrations. Several sterically hindered groups have been employed for both the donor and emitter through the imide position of the luminophore, and their photophysical properties have been observed at various concentrations. The results demonstrate that these sterically hindered groups effectively reduce dye aggregation at high dye concentrations. Furthermore, incorporating luminophores in a polymer backbone enables efficient energy transfer from the donor to the emitter by controlling the distance between the luminophores. The polymer chain acts as a spacer between the luminophores, reducing dye aggregation and suppressing the reabsorption issue. To investigate this approach, a variety of linear polymers with incorporated luminophores were employed in this study, where the luminophores were covalently linked to the polymer chain. Optical properties were analysed in both solid and solution states, and it was determined that this strategy did not adversely affect the luminophore's optical characteristics. Moreover, it was discovered that the more sterically hindered donor efficiently transferred its energy to the emitter, effectively suppressing aggregation-caused quenching (ACQ) in comparison to the less sterically hindered donor molecule. The success of the FRET energy transfer technique in polymer chains has inspired the use of crosslinked luminophore embedded nanoparticles for light harvesting. This approach involves embedding the dye within the polymer particles, resulting in a high dye concentration in a small area and promoting efficient energy transfer. In this study, a series of donor-emitter luminophore embedded crosslinked nanoparticles were synthesized using various luminophore concentrations, and their photophysical properties were studied to examine FRET efficiency. The cross-linked polymer particles effectively reduced luminophore aggregation and reabsorption. These polymer nanoparticles were used to fabricate bulk LSC films, which also demonstrated effective energy transfer. The photophysical observations were subsequently utilized in Monte-Carlo simulations of a large-scale bulk LSC device. The simulation results indicated that the incorporation of cross-linked polymer particles had a significant effect in mitigating the reabsorption process of luminophores in the bulk LSC waveguide.
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ItemZwitterionic Donor-Bridge-Acceptor Solvatochromic Dyes( 2023-03)Push-pull molecules are widely used luminophores for applications in optoelectronics and photonics. They are discrete, functionally desymmetrised molecules, bearing donor and acceptor substituents as end-groups, with their delocalized pi-electron system responsible for nonlinear optical effects. Oligomeric systems with a donor phenol group and an acceptor pyridyl moiety separated by a conjugated para-phenylene chain or fluorene spacer were obtained by iterative Suzuki–Miyaura or Horner–Wadsworth–Emmons couplings. For synthesis of desymmetrised molecules, aryl or heteroaryl halides and aryl triflates are typically used as substrates in the former case. Due to moderate reactivity of aryl triflates and their high cost, aryl nonaflates have been proposed as a good alternative. So far, only few reports related to the application of aryl nonaflates in Suzuki–Miyaura coupling have been published, where aryl nonaflates have shown slightly higher reactivity and better yields compared to corresponding triflates. Zwitterionic forms of these donor-bridge-acceptor molecules were generated by consequent N-methylation and deprotonation reactions leading to large redshifts in absorbance maxima. UV-vis absorbance studies also revealed negative solvatochromic behaviour: a smooth bathochromic shift was observed with the decrease of the solvent polarity. Most of the examples have shown strong solvatochromic characteristics, where the magnitudes of these shifts in the studied polarity range were close or even greater to the one for Reichardt’s dye — one among the most solvatochromic organic dyes known. Additionally, systems with increased disparity between the donor and acceptor ends were synthesised and studied. It was achieved by introducing a stronger acceptor, which lead to molecules with more unusual electronic and structural properties. Among them there were species with a bulky donor phenolate moiety and an acceptor pyridinium group separated by a bridging unit of a different nature. One of the molecules incorporating a vinyl bridge and 2,4-dinitrobenzene acceptor group demonstrated the broadest and the most redshifted absorption profile in the N-arylated series. It also showed extraordinary behaviour in its 1H NMR spectrum as well as in its crystal structure compared to its analogues. Thus, it was found to be near the cyanine limit, which makes it to be a potential candidate for photorefractive materials.
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ItemElucidating Cysteine Dioxygenase substrate specificity using substrate analogues( 2023-02)Cysteine dioxygenase (CDO) is a mononuclear ferrous non-haem enzyme that catalyses the addition of molecular oxygen to the thiol of substrate L-cysteine to form L-cysteine sulfinic acid. Selective dioxygenation of L-cysteine is believed to be mediated through several binding requirements, namely the formation of a salt bridge between substrate carboxylate and Arg60 guanidium group, and S/N coordination of substrate thiol and amine to the active site iron. Indeed, CDO catalysis with many substrate analogues yields little to no sulfinate product, believed to be a consequence of improper binding. Presented here is an in-depth exploration into the binding, kinetics and product characterisation of off-pathway reactions between CDO and the substrate analogues 2-aminobenzenethiol, L-selenocysteine and L-homocysteine. These studies use the catalytically active CDO C93G variant, which lacks the ability to form a thioether crosslink between C93-Y157 as seen in wild-type CDO. As crosslink proportions vary in wild-type CDO, use of CDO C93G allows for easier experimental investigation due to it existing as a singular isoform. 2-ABT is an aromatic substrate analogue that lacks the carboxylate moiety to form a salt bridge with Arg60 within the CDO active site. Molecular docking of 2-ABT to CDO C93G displayed binding like that observed with native substrate L-Cys. However, Mossbauer spectroscopy revealed no such binding was occurring. Catalytic oxidation of 2-ABT by CDO C93G was still observed, forming the main product 2-ABT disulfide. These results highlight the necessity of substrate S/N coordination to the CDO iron centre for correct catalysis to follow. L-selenocysteine is structurally similar to L-cysteine, with replacement of thiol sulfur with selenium. Binding of L-selenocysteine to CDO C93G was characterised with UV/vis and Mossbauer spectroscopy, and exhibited tight five-coordinate binding to a high spin (S = 2) ferrous centre. CDO C93G was unable to catalyse the oxidation of L-SeCys. Instead, an off-pathway reaction occurred with the rapid oxidation of CDO C93G:Fe(II) to CDO C93G:Fe(III). These results suggest that the CDO iron centre is in tight redox control, with Se/N coordination modifying the reduction potential of iron allowing for its rapid oxidation. Compared to L-cysteine, homocysteine exhibits an extended thiol arm by one carbon unit. A four-coordinate CDO:homocysteine complex has previously been observed, with homocysteine exhibiting monodentate binding of thiolate to the iron centre. The studies shown here describe the first observation of a five-coordinate L-homocysteine-bound CDO, with L-homocysteine binding likely occurring with S/N ligation. Furthermore, an analysis of tetrahedral iron centres validated the previous assignment of the four-coordinate homocysteine-bound CDO. This proves that homocysteine can bind with variable coordination and that lack of CDO catalytic dioxygenation may result from steric hinderance of O2 binding to the iron centre. Overall, this thesis provides valuable insight into CDO substrate selectivity and why CDO is incapable of catalysing the dioxygenation of the aforementioned substrate analogues.
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ItemSynthesis of tyrosine-derived diketopiperazines and investigation of their inhibition of CYP121, an essential enzyme of Mycobacterium tuberculosis.( 2022-12)The growing incidence and degree of resistance exhibited by Mycobacterium tuberculosis (Mtb), the major causative agent of tuberculosis (TB), to currently available treatments is of great concern. With multi-drug resistant (MDR-TB) and extensively-drug resistant (XDR-TB) strains being reported, alternative chemotherapies are required to maintain viable approaches to treat TB. Treatments exploiting new therapeutic targets are a key avenue of exploration. In the pursuit of drug discovery, analysis of an organism’s genome is imperative to determine essential gene products. Genomic analysis has revealed the presence of 20 cytochrome P450 encoding genes, with subsequent knockout studies uncovering CYP121 and CYP128 as proteins essential to bacterial survival and growth in vitro. The only unique and essential Mtb CYP that has been functionally characterized is CYP121, known to catalyze the production of the secondary metabolite, mycocyclosin, from the cyclic dipeptide cyclo(L-tyrosinyl-L-tyrosine) (cYY). With a known substrate and product, the generation of substrate-like mimics and pursuit of transition state analogues are two possible avenues of inhibitor development. A series of cYY analogues were synthesized to investigate the effect of incorporating various P450 inhibitory functionalities, in the hope to develop selective Mtb inhibitors. Biochemical analysis shows that the incorporation of nitrogen containing heterocycles is tolerated by the enzyme. Numerous substrate analogues display potent binding to CYP121. Additionally, analogues of postulated transition states on the enzymatic reaction pathway to mycocyclosin were designed. The biaryl distance of these macrocycles was calculated to identify the most appropriate linker that closely mimics the calculated transition state distance.
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ItemFluorescence Based Sensors and Methods for the Study of Biomolecules in Protein Conformation Diseases( 2023-03)Understanding how cells maintain the functional proteome and respond to stress conditions is critical for deciphering molecular pathogenesis and developing treatments for conditions such as neurodegenerative diseases. Efforts towards finer quantification of cellular proteostasis machinery efficiency, phase transitions and local environment changes remain a priority. In this work, we look at three aspects of proteostasis, namely, phase separation in cationic and RNA rich biomolecules, protein conformation and condensates and environmental polarity and its roles in proteostasis. We developed a dye FRET-pair system for studying phase separated polycationic rich protein-RNA droplets. Characterisation of the dyes have been carried out in-vitro, with promising results, and future work will look at phase separated protein-RNA droplets in-vitro. Future experiments involving live cells will further validate the staining ability and feasibility of use for our dye FRET-pair. For protein conformation and condensates, we look to structural modifications on two series of dyes, one cationic, based on ASCP, and one neutral, based on NIAD-4, to attempt to improve cellular uptake and retention and dye performance, with the future goal of implementing improved dye system for monitoring protein condensates in cells. The ability to better understand the driving force of spatial discretization of biomolecules in the complex cellular matrix remains a challenging task. To monitor such phenomenon and environmental polarity, we report on the robust polarity sensitive solvatochromic probe, FLAM, in conjunction with spectral phasor analysis as a general method for studying environmental polarity in biological systems. We find that phase separated proteins of SFPQ have distinct polarity depending on the type of phase separation occurring, suggesting that polarity plays a role in the formation of phase separated condensates. When using FLAM in cells, distinct subcellular environmental polarity distribution but similar trend of changes is observed for cells under similar type of stressors. We further validate FLAM in zebrafish model, and this provides the groundwork for more demanding experiments to be carried out. Taken together, our method puts forth an exciting development in the tool set for the study of phase separation.
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ItemNo Preview AvailableSynthesis of Myxobacteria Metabolites and Analogues and Development of Metal Complexes for the Mukaiyama Hydrogen Atom Transfer Reaction( 2022)The spirangiens are a series of spiroketal containing polyketide natural products which are potently active against human and mouse cancer cell lines, and interleukin 8 expression. Oxidation at the C20 position occurs after polyketide synthesis is complete. Acyclic compounds lacking oxidation at the C20 position have been isolated, but never cyclic species; as such C20 deshydroxy spirangiens could be undiscovered natural products. This work saw the completion of the synthesis of a C20 deshydroxy derivative of spirangien M522, utilising a series of asymmetric aldol reactions to construct the polyketide backbone. A cross metathesis reaction followed by spirocyclisation forged the core, which was elaborated to by a Roush crotylation/Weix coupling sequence. Cell death, and cell growth inhibition assays in immortalised bone marrow derived macrophages (murine) and colon adenocarcinoma cells (murine), indicated that derivative retained its activity, and that C20 oxidation was not crucial for bioactivity. The Mukaiyama-Isayama hydration reaction hydrates a polar or non polar alkene to an alcohol via a C centred radical. Under Mn, Co or Fe catalysis, with phenylsilane in an alcoholic solvent, a metal hydride species forms which is able to facilitate hydrogen atom transfer (HAT) and generate the requisite radical. In the absence of oxygen alkene reduction occurs, and this also represents a major side product in the Mukaiyama hydration. In the presence of a Michael acceptor, the radical can undergo coupling, forging a new C-C bond. Three cis-beta octahedral complexes with a SALPN type ligand; Mn(SALPN)acac and Co(SALPN)acac were synthesised. These complexes were active in the Mukaiyama hydration of esters and ketones, and Fe(SALPN)acac successfully catalysed olefin coupling reactions. Compared to the control catalysts, the new complexes, showed superior reactivity (reduced side product formation, lower catalyst loading) or unusual reactivity (promotion of an acyloin rearrangement). Furthermore, the cobalt complex facilitated the synthesis of 7 acyloin natural products.
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ItemInvestigations into the metabolism and distribution of sulfoglycolytic bacteria( 2023)Sulfoquinovose (SQ) is an analogue of glucose (Glc) with a 6-sulfonate group in place of the 6-hydroxyl group. SQ is produced by most photosynthetic organisms and thus, SQ comprises a pool of organosulfur comparable in size to the amino acids cysteine and methionine and is a major compound in the biogeochemical sulfur cycle. ‘Sulfoglycolysis’ describes the catabolism of SQ, and to date, five pathways for sulfoglycolysis have been discovered. Two of these sulfoglycolytic pathways are discussed in this thesis: the sulfoglycolytic Embden-Meyerhof-Parnas (sulfo-EMP) pathway, discovered in Escherichia coli, named because of its resemblance to the glycolytic EMP pathway, and the sulfoglycolytic SQ monooxygenase (sulfo-SMO) pathway, of which Agrobacterium tumefaciens is a prototypical organism. The sulfo-EMP pathway results in catabolism of only three of the 6 carbons of the sugar, with a C3-sulfonate excreted from the cell. In contrast, the sulfo-SMO pathway, uniquely amongst sulfoglycolytic pathways, utilizes all 6 carbons of SQ, converting SQ to Glc. Thus, the sulfo-SMO pathway serves as an assimilatory ‘bolt-on’ to glycolysis, while the sulfo-EMP pathway replaces glycolysis and metabolises only half the sugar of glycolysis. We hypothesized that a switch from glycolysis to the sulfo-EMP pathway and a switch from glycolysis to the sulfo-SMO pathway will have substantially different impacts on cellular metabolism. Since the discovery of these pathways in the last decade, there has been some study on the distribution and variation of sulfo-EMP and sulfo-SMO gene clusters across the tree of life, and some speculations have been made as to the evolutionary origin of the sulfo-EMP and sulfo-SMO genes, but these questions have not been comprehensively investigated. In this thesis, the sulfo-EMP and sulfo-SMO pathways were investigated using metabolomics and bioinformatics. To support the metabolomics experiments, a more expeditious route for the synthesis of SQ on multigram scale was developed, and applied to the synthesis of (13C6)SQ and sulforhamnose (SR). The impact of a switch from growth on Glc and the EMP pathway to growth on SQ and the sulfo-EMP pathway on the metabolism of E. coli was investigated using comparative metabolite profiling and other metabolomics experiments. This showed that sulfoglycolytic E. coli use gluconeogenesis and accumulate Glc and intracellular glycogen, a situation that is reversed once cells exhaust SQ and revert to glycolytic metabolism. Comparative metabolite profiling was also used to investigate the impact of a switch from glycolysis to sulfoglycolysis by the sulfo-SMO pathway in A. tumefaciens, which revealed only minor changes in cellular metabolism. We developed a bioinformatics pipeline to systematically search for sulfo-EMP gene clusters, which enabled investigation of the distribution and variation of the sulfo-EMP gene cluster, as well as the evolutionary origins of the sulfo-EMP genes. This pipeline was also applied to study the distribution, variation and evolution of the sulfo-SMO pathway. Consistent with previous reports, we found that the sulfo-EMP pathway is almost limited to the Gammaproteobacteria, while the sulfo-SMO pathway is near-exclusive to the Alphaproteobacteria. We found evidence for an evolutionary relationship between the sulfo-SMO pathway and the alkanesulfonate utilization pathway of E. coli.
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ItemNo Preview AvailableBiochemical Characterisation of C. elegans Ferritins( 2022)Ferritin plays an important role in maintaining optimum cellular iron concentrations. Consisting of 24 protein subunits in a spherical architecture, it creates an interior cavity that stores iron atoms in the form of a ferric oxy-hydroxide. Extensive in-vitro enzymological studies have greatly assisted our understanding but it is not yet fully understood how iron is released from ferritin in-vivo. A clearer understanding could aid iron related disorders, including iron overload or deficiency. The use of the model organism Caenorhabditis elegans will allow genetic manipulation to further understand the mechanisms involved in iron metabolism within a whole organism. C. elegans expresses two types of ferritin, FTN-1 and FTN-2 which have not been biochemically characterised. The aim of this thesis is to establish the mechanism of iron storage for these proteins to develop strategies to control and manipulate the protein for in vivo and ex vivo studies. C. elegans ferritins have been expressed, purified, and characterised through a range of techniques. Both ferritins show ferroxidase activity but with L-type ferritin nucleation properties. Even with identical ferroxidase active sites, FTN-2 is shown to react ~10 times faster than FTN-1. Combined structural and stopped-flow spectroscopy studies revealed the source of the difference in rates to be iron transport to the ferroxidase site. Both FTN-1 and FTN-2 react initially to form a mu-1,2-peroxo diferric intermediate that absorbs at 595 nm. Structural comparison showed an asparagine residue (Asn106) near the ferroxidase active site, which is a valine in most other ferritins including human H ferritin. The mu-1,2-peroxo diferric intermediate of human H ferritin absorbs at 665 nm and replacement of Asn106 to valine in FTN-2 shows a similar absorption. This may explain the variation in wavelength of the peroxide-to-iron(III) charge transfer band observed for ferritin across taxa.
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ItemNo Preview AvailableInvestigating Photo-luminescent Materials with Advanced Micro-spectroscopic Methods( 2022)Microscopic and spectroscopic techniques are very practical tools for investigating details of energy relocation processes in solar cell materials. However, this is always challenging as energy migration processes occur on ultrafast timescales and are usually very complex as several processes could take place either simultaneously or sequentially. On the other hand, morphological variations in thin films lead to different charge separation behavior. This usually needs to be distinguished spatially at the nanoscale, which is beyond the resolution limit of conventional optical microscopy methods. To address the challenges mentioned above, this thesis focuses on the development of advanced microscopic and spectroscopic techniques, and exploring their applications in studying the morphology of thin-film materials of opto-electronic applications, including solar cells and organic light-emitting diodes (OLEDs). Novel and versatile approaches with high spatial and temporal resolutions, as well as polarisation sensitivity, have been explored to reveal the relationship between morphology and photo-physical properties of thin-film excitonic materials. First of all, the design and construction of two advanced techniques, a time-resolved fluorescence anisotropy imaging microscope (TR-FAI) and a super-resolution structured illumination microscope (SR-SIM), have been elucidated in detail. The TR-FAI system provides the ability to collect a time-resolved fluorescence image, a fluorescence anisotropy image and a polarised transmission image simultaneously over the same area. The home-built SR-SIM system has been applied to various samples, including standard fluorophores and excitonic materials, providing obviously enhanced spatial resolution compared to wide-field imaging. Moreover, the possibility of using evanescent wave (EW) excitation to probe interfacial dynamics of thin-film excitonic materials has been explored. Time-resolved fluorescence anisotropy measurements (TR-FAM) of Acridine-doped PAA thin films of various concentrations reveal complex time-dependent dynamics inside these films. Numerical simulations have been conducted using the COMSOL Multiphysics platform, suggesting that a proposed multi-layer approach for EW generation in materials of high refractive indices is not reliable. Finally, the molecular conformation and alignment of a polyfluorene-based polymer, PODPF, has been successfully altered by thermal annealing and physical stretching. The developed TR-FAI, together with multi-dimensional microspectroscopic techniques, have been applied to thin-films of PODPF and its derivatives, to monitor the morphological variations and the related photo-physical properties. These techniques have been shown to be very powerful for characterising the morphological variations and excitonic processes in thin films. In summary, this thesis demonstrates that SIM, EW, and TR-FAI are practical and powerful tools to investigate morphological variations and photo-induced processes in thin-film excitonic materials.