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  • (2019) Kuppusamy, Rajesh
    Thesis
    Antimicrobial resistance is a major worldwide threat to public health and there is an urgent need for the development of novel antibacterial agents. This research project focused on the development of short cationic peptidomimetics that employ 3'-amino-[1, 1 '-biphenyl)-3-carboxylic acid and anthranilic acid backbones segregated by hydrophobic and cationic groups. The biphenyl peptidomimetic compounds showed that simple diaminoethanes and their respective guanidine cationic groups were sufficient to mimic lysine and arginine amino acids of natural antimicrobial peptides. The biphenyl backbone was important for antibacterial activity and tryptophan was important for bacterial cell membrane permeability. The most active compound showed good minimum inhibitory concentrations (MIC) against S. aureus (15.6 μM) and E.coli (7.8 μM) but was inactive against P. aeruginosa strain PA01. Based on these results, anthranilamide derivatives with tryptophan and simple amine cationic groups were developed. The anthranilamide peptidomimetic compounds showed that the guanidine group was important for good antibacterial activity against S. aureus (3.9 μM), E.coli (15.6 μM), and these compounds had low cytotoxicity (>100 μM). Active compounds disrupted 75% of established S. aureus biofilms. Biphenyl could be used as an alternative to naphthoyl groups to give hydrophobic groups to the mimetics. Increasing the net charge by adding lysine decreased antibacterial activity compared to compounds containing simple amine groups but improved the compound's cytotoxicity.Various alkyl-substituted guanidine compounds were investigated. Increasing the lipophilicity (adding alkyl groups) at the guanidine residues decreased antibacterial activity. Increasing the cationicity increased antibacterial activity against P. aeruginosa. The most active compound showed broad-spectrum antibacterial activity of against S. aureus (2.0 μM), E.coli (7.8 μM), and P. aeruginosa (32.0 μM). The active compounds at 4.0-8.0 μM showed significant disruption (55-77%) of preformed S. aureus biofilms and one compound at 15.6 μM disrupted 45% of E.coli biofilms. Peptidomimetics are promising future antibiotics. These compounds can potentially circumvent current antimicrobial resistance that is generated when bacteria produce biofilms.

  • (2019) Du, Eric
    Thesis
    Biomimetic scaffolds have been an area of great interest in recent years for cell culture materials. Self-assembled peptide hydrogels are a type of biomimetic material that has excellent potential in this field owing to its ease of modification and unique physical characteristics. As many of nature’s peptides contain various biological motifs, it is easy to incorporate these motifs into a peptide gelator sequence. In addition, self-assembled peptide hydrogels are chemically well-defined, and the resulting fibrous networks are reversible due to the noncovalent interactions that mediate the self-assembly process. The encompassing aim of this Thesis is to monitor and control cell growth in and on multicomponent self-assembled peptide hydrogels. This was achieved by synthesising a small library of peptides containing biological motifs and using them in multicomponent systems to mimic the extracellular matrix which is a combination of many fibre types. The gels that were formed were characterised for the physical characteristics which revealed co-assembly and destructive interactions between different combinations of gels. A concentration study of the gelators also elucidated interesting concentration dependent traits. The hydrogels were also used in a series of in vitro experiments whereby factorial combinations (that is, all possible combinations of factors) were used to culture fibroblasts and human embryonic stem cells in 2D and 3D which revealed that cells not only survived, but on some surfaces were able to proliferate and attach. A novel chemiluminescent hydrogel was also synthesised to monitor oxidative stress in cells as an alternative to current assays to monitor cell behaviour. The gelator was shown to not only be able to respond to oxidative burst in T cells, but also be able to respond to the classic glucose oxidase and horse radish peroxidase reaction. This gel, therefore, holds good potential in multiple biomedical applications.

  • (2019) Lizarme Salas, Yuvixza
    Thesis
    Control molecular conformation via fluorination is a novel strategy for optimising the potency and selectivity of biological target binding. This project investigated selective fluorination strategies to control the conformations of diverse but structurally-related drug candidates. Two are quorum sensing (QS) inhibitor candidates, and the other two are Alzheimer’s disease (AD) drug candidates. QS controls several functions including antibiotic resistance, hence disrupting QS systems through inhibitors is a potential strategy for new antimicrobial therapies. In this project, six different fluorinated analogues of each QS inhibitor candidates were synthesised. The fluorination patterns were predicted to control the shape of the analogues to pinpoint the optimal conformation for QS inhibitory (QSI) activity. The conformation of these analogues was investigated, followed by docking studies to predict the best conformation for QSI activity, and finally, QSI activities were tested in bacteria and reveals that the activities of all analogues were improved compared to their respective lead compounds. AD is associated with low levels of the neurotransmitter acetylcholine (ACh). Therefore, drugs that mimic ACh and bind to its receptors are desired to treat AD. An example is xanomeline, which has been shown to improve cognitive function and reduce neurodegeneration, however, it has poor selectivity. It was hypothesised that fluorination may deliver a molecule with improved receptor selectivity through conformational control. Therefore, six fluorinated analogues were synthesised. It reveals that these analogues have different conformation. However, the binding affinities of all analogues to all receptor tested were very similar to that of xanomeline. Inhibition of acetylcholinesterase is another strategy to combat ACh deficit. Piperine has AChE inhibitory activity and possesses a rigid shape due to the presence of double bonds. However, the double bonds can undergo photoisomerisation. It was hypothesised that replacing the double bonds with fluorine atoms may avoid the photoisomerisation problem whilst maintaining inhibitory activity. Therefore, a difluorinated analogue that mimics the rigid shape of piperine was synthesised. It was revealed that this analogue is photostable and inhibitory activity is improved. Overall, this research has contributed to the field of medicinal chemistry by developing a novel strategy for controlling the conformations of four drug candidates.

  • (2019) Barnett, Christopher
    Thesis
    Three main subsets of the imidazol-2-ylidene scaffold have been synthesised and their electronic properties have been investigated. The respective [PdBr2(iPrBenz)(L)] complexes, where L = the imidazol-2-ylidene, were prepared in order to use the 13C NMR chemical shift of the benzimidazol-2-ylidene fragment NCN as a measure of σ-electron donating ability of the imidazol-2-ylidene. The respective selenone adducts were also prepared, with the 77Se NMR chemical shift used as a measure of the π-electron accepting ability of the imidazol-2-ylidenes. The subsets of NHCs consisted of imidazol-2-ylidene derivatives with systematically altered substituents. The first subset, discussed in Chapter Two, contained consistent N-methyl substituents with various electron withdrawing and donating groups at the 4,5-positions. This chapter describes the synthesis of the NHCs and NHC precursors, as well as the preparation of the palladium and selenium probes, and discusses the findings. The second group, discussed in Chapter Three contained consistent 4,5-diprotio groups and systematically altered N-alkyl groups chosen to vary in steric bulk (methyl, ethyl, iso-propyl, tert-butyl, and adamantyl). This chapter briefly discusses the preparation of the precursors, then the preparation of the palladium and selenium probe species and discusses the results. The third group, discussed in Chapter Four, contained consistent N-mesityl substituents with various electron donating and withdrawing groups at the 4,5-positions. This chapter discusses the preparation of the NHCs and NHC precursors, as well as the palladium and selenium probes, and the results obtained from the probes. With derivatives in these three sets, the previously reported extremes of both σ-electron donating and π-electron withdrawing abilities for the imidazol-2-ylidene class were extended. A fourth minor subset, discussed in Chapter Five, was investigated designed to compare sterically similar scaffolds with distinct electronic properties. 1,3-Dimesityl-4-methylimidazol-2-ylidene was compared to 1,4-dimesityl-3-methyl-1,2,3-triazol-5-ylidene, using the same [PdBr2(iPrBenz)(L)] complexes and selenium adducts, as well as complexes of [IrCl(CO)2(L)], and [AlH3(L)], where L = the ligand. The information obtained using the electronic character probes was used in conjunction with established electronic parameters of the substituents to examine correlations to allow prediction of the electronic character of related species. The performance of a selection of the analysed imidazol-2-ylidenes and the 1,2,3-triazol-5-ylidene was investigated in two organocatalytic reactions: the formation of a γ–butyrolactone from trans-cinnamaldehyde and para-bromobenzaldehyde, and the formation of a spiro-γ-butyrolactone from trans-cinnamaldehyde and 1,2-cyclohexanedione, and these results were rationalised through consideration of the quantified electronic characteristics.

  • (2019) Wang, Huixin
    Thesis
    Owing to the development of electrospray ionization (ESI), various ion dissociation techniques and software algorithms, mass spectrometry has become an indispensable tool for protein analysis. However, new approaches are needed to overcome some of the challenges in protein sequencing, including in the identification of post-translational modifications (PTMs), and in the analysis of protein complexes and protein-protein interaction networks. For PTM identification, commonly used database search algorithms for bottom-up proteomics often fail to identify peptides with unexpected PTMs due to their relatively low abundances and the absence of such modifications in the PTM databases. However, such unexpected PTMs can have important roles in biological functions. Here, we report a novel mass spectrometry method to identify the proteins targets of organophosphate (OP) insecticides in a non-targeted fashion. This method integrates a high-resolution twin-ion metabolite extraction program with Mascot database searching. Using this method, transmethylation was identified as a new reaction pathway for OP insecticides, in addition to the well-known phosphorylation modification that causes acute toxicity. Our results show that this method can be used for the reliable identification of unknown PTMs in complicated biomatrices which may ultimately benefit the discovery of protein biomarkers for a variety of conditions. For protein sequence identification, collision induced dissociation (CID) is the most widely used dissociation technique. In the CID of intact proteins, the fragmentation patterns of the protein ion depend strongly on the protein charge state. An optimal charge state can generate selective fragmentation resulting in a limited number of sequence ions in high abundances which can be useful for protein identification with high sensitivity. However, an accurate model to predict the fragmentation patterns of particular charge states is needed. Here, we report an approach to predict the specific cleavage sites of intact protein ions upon CID by use of an improved electrostatic model for calculating the proton configurations of protein ions at different charge states. The origin of highly selective cleavage sites in the CID of highly charged proteins ions is investigated using an improved electrostatic model, molecular dynamics and hybrid ONIOM simulations. The ONIOM results indicate that the protons located at low-basicity amino acid residues can dramatically reduce the reaction barrier to the cleavages at such amide bonds. The results from our electrostatic model suggest that unlike peptide ions at relatively high charge states, protons at low-basicity amino acid residue sites are electrostatically confined within a relatively narrow range of amino acid residues. Such confined protons can ‘trigger’ the fragmentation at the specific peptide bonds. Our model can potentially be used to predict the specific charge states that yield either specific sequence ions in high abundances, or fragment extensively for optimal protein sequence coverage. Recently, higher energy collision induced dissociation (HCD) has become available for the LTQ Orbitrap. There are some reports in the literature that demonstrate that HCD benefits the de novo sequencing of proteins and the identification of PTM sites. However, studies investigating the potential use of HCD for intact protein sequencing are relatively rare. Here, we systemically compared the performance of HCD and CID for intact protein analysis. Our results indicate that HCD yields significant performance gains compared to CID for obtaining high sequence coverage at relatively low charge states owing to higher ion trapping efficiencies and higher ion collision energies. The origin of the highly specific cleavages in the HCD of highly charged protein ions was investigated. We found that for HCD, highly specific fragmentation sites occur near the first sites that low-basicity amino acids are predicted to be protonated with increasing charge states, which is consistent with the mechanism for the formation of highly specific fragmentation of protein ions in CID. This result provides additional evidence that the fragmentation patterns of highly charged protein ions can be predicted to improve protein identification. Solution-phase labelling experiments have been increasingly used in combination with top-down proteomics to rapidly obtain structural information. However, the low charge state of protein ions formed from native solutions usually result in low sequence coverage which limits spatial resolution. Here, we demonstrate that theta-capillary nanoelectrospray ionization can be used to form protein ions with the highest known charge densities to date from native-like solutions by use of alkyl cyclic carbonate “supercharging” additives. It is anticipated this approach will be particularly promising for top-down hydrogen-deuterium exchange mass spectrometry to obtain significantly more protein structural information in native solutions than by use of more conventional approaches.

  • (2019) Zhao, Manchen
    Thesis
    Understanding protein-protein interactions is important in the fields of biochemistry, molecular biology, clinical diagnostics and drug discovery. However, conventional study assays require many analytes on the biointerface to give a single averaged output. As a single signal constitutes the measurement, the main challenge is non-specific adsorption of other species, leading to the sophisticated design of the biointerface to repel non-specific species adsorption with molecular level control. Hence, such knowledge, like how protein-protein interactions behave on the biointerface and how well the biointerface works at a single-molecule level, is valuable to exploit. The goal of this thesis is to establish a novel biosensing platform to realize surface characterisation and quantitative monitoring of molecular interactions simultaneously. By employing super-resolution techniques, like single molecule localization microscopy (SMLM), individual molecules can be localized with a spatial resolution of 20 nm, allowing a greater density of single molecules being detected simultaneously. The strategy to achieve this goal is described here. Firstly, a low background surface for SMLM, poly-L-lysine grafted polyethylene glycol surface, was developed that limited non-specific protein adsorption even after many modification steps, satisfying the criteria to study bioaffinity reactions with SMLM. Secondly, with BSA and anti-BSA as the study model, dual-colour SMLM with a further physical analysis enabled to quantitatively monitor many antigen/antibody interactions and distinguish specific binding events from non-specific adsorption. Furthermore, the low proportion of specific binding events on the surfaces led to a study and comparison that revealed some factors that possibly affected the performance of biointerfaces with different interfacial designs or imaging parameters. Finally, as an important aspect of protein-protein interactions, protein stoichiometry could be also obtained from SMLM imaging, showing hemagglutinin (HA) tagged mEos2 was monomer and HA-mEos2-Folden was assembled into trimer when they were pulled down from cell lysates. These studies illustrated SMLM, an imaging tool as well as a quantitative analytical platform, to characterise surfaces and study molecular interactions simultaneously, to provide guidance for the design of new biointerfaces and even study protein stoichiometry, by determining one cluster of detected events acquired in SMLM from one single molecule or several closely spaced molecules.

  • (2019) Pardehkhorram, Raheleh
    Thesis
    Surface-enhanced Raman spectroscopy (SERS) represents a powerful analytical technique with a great promise for ultrasensitive bio analysis and detection. SERS tags are the new generation of nanoprobe offering remarkable features such as high signal uniformity, great physical and optical robustness and excellent multiplexing capability for indirect identification of the targets. Nevertheless, there are still plenty of opportunities and challenges to improve the performance of such assays. In this regard, anisotropic gold nanoparticles are potential candidates as they can support much stronger signal enhancement than those of commonly used sphere nanoparticles. Gold nanorod (NRs) in particular is a classic example of anisotropic nanostructure possessing unique properties including strong plasmonic effect, optical tunability as well as the capability for preferential surface modification. The motivation of this thesis is to develop a high-performance SERS tag for NIR detection of food-borne pathogenic bacteria. At the first step gold NRs with a similar optical absorption as the NIR laser, which is excitation source for cheap and portable Raman instruments, were prepared in order to achieve strong enhancement effect. At the second Chapter, as-synthesized gold NRs were rationally designed on the surface to provide the tags with the optimum signal enhancement and performance. In this regard, Raman reporter molecules were selectively attached to the NRs tips, where the local electromagnetic field is effectively concentrated. The side-body of encoded NRs was subsequently modified by attaching a mixed layer of two carboxyl terminated ligands aiming to improve colloidal stability as well as the coupling efficiency of the targeting moiety (bacteria antibody). In the last Chapter, performance of the tag was investigated for identification of Salmonella as one of the most common cause of food-related hospitalizations and deaths. In addition, the selective design of the tag was employed in preparation of two other tip-encoded NRs tags with distinctive Raman signatures. The multiplexing capability of the developed probes was examined for simultaneous detection of Salmonella typhimurium and other pathogenic bacteria including Staphylococcus aureus and E. coli O157:H7. Moreover, the specificity of the tags toward the target bacteria was also evaluated in food matrices prepared from chicken meat and leafy vegetables.

  • (2019) Cao, Cheng
    Thesis
    In this thesis, the main objective is to understand the relationship between the physicochemical properties and the biological activities of nanoparticles based on the self-assembled block glycopolymers and the zwitterionic poly(2-methacryloyloxyethyl phosphorycholine) (MPC) block copolymers. In addition to the physicochemical properties of nanoparticles such as size, shape, and surface chemistry, drug effects on the nanoparticles’ structure and function are investigated. Due to the interaction between the hydrophobic drugs and hydrophilic polymers, hydrophobic drugs might unexpectedly locate in the hydrophilic environment (hydrophilic shell) which can not only induce morphological transitions, but also influence the internal structure of nanoparticles, such as hydration and grafting density. These factors affect the cellular uptake in vitro. Generally, higher hydration of nanoparticles’ shells is correlated with an enhanced cellular uptake in MCF-7 cells, MDA-MB-231 cells, and RAW 264.7 cells due to the bioactive group more efficiently contacting to cells in a water environment. Moreover, nanoparticles with higher grafting density polymers suggest a lower cellular uptake due to their increased ability to adsorb protein, and the adsorbed protein not only influences the internal structure change (e.g. thickness of shell and core radius) but also induces an overall shape transition from sphere to oblate. All these factors may lead to lower cellular uptake. Additionally, higher grafting density does not afford an enhanced cellular uptake when the nanoparticles do not adsorb proteins, such as the zwitterionic poly(2-methacryloyloxyethyl phosphorycholine) (MPC) nanoparticles. Small angle X-ray scattering (SAXS) and small angle neutron scattering (SANS), with the help of DLS and TEM, play important roles in not only characterizing the size and shape, but also providing information on the internal structure of nanoparticles (e.g. hydration, aggregation number, and grafting density). In this thesis, small angle scattering (SAS) techniques are employed to investigate the relationship between the physicochemical properties of nanoparticles and their biological activities for better drug delivery vehicles.

  • (2019) Luis, Ena
    Thesis
    Supramolecular chemistry and visible-light photoredox catalysis are two research areas which are inspired by biological processes. This thesis presents strategies for bridging these two areas, accompanied by studies within each field that will benefit future developments of this research. The focus of this study is photoactive ruthenium(II) polypyridyl complexes and their roles as photoredox catalysts and in supramolecular structures. Chapter 2 introduces a method to characterise model ruthenium(II) complexes by NMR spectroscopy. This method was used to distinguish between chemically similar but spatially different proton environments on common precursors. This technique is important for accurately characterising more complex supramolecular systems built upon these complexes, which are described in Chapter 3. These complexes were extended into linear metalloligands designed to directly incorporate photoactivity into larger structures. The combination of these metalloligands with Zn(II) and Fe(II) ions leads to heterometallic tetrahedral supramolecular cages. Like the component metalloligands, the self-assembled cages retain their photoactivity. Chapter 4 examines the photocatalysed cycloaddition of trans-anethole, which was studied in depth using NMR spectroscopy with in situ irradiation. The concentration of oxygen affected the rate of reaction, but the final product concentration was governed by the equilibrium distribution. Equilibria are not discussed in photocatalytic reactions in the literature, and could be relevant but overlooked in many reported mechanisms. Chapter 5 describes the study of photostable heteroleptic complexes as photocatalysts. The photophysical and redox properties of five complexes were characterised in acetonitrile and water. The heteroleptic complexes were photostable in the presence of excess chloride, but their excited state potentials precluded the comparison in a reaction mixture. Chapter 6 focuses on the encapsulation of complexes in cucurbit[10]uril as supramolecular photocatalysts. The properties of the complex can be altered by encapsulation, and the host-guest system can bind additional substrates. Quenching of the photocatalyst’s excited state can occur within the internal cavity, which is a requirement for photocatalysis within this confined space. This strategy opens a door for a photoactive supramolecular catalyst capable of using visible light and a confined space to control the reaction outcome.

  • (2019) Elton, Timothy
    Thesis
    Multi-electron reduction of substrates is an ongoing challenge in catalysis, and Organic Hydride Donor (OHO) ligands tethered to metals could unlock new reaction pathways to realise this goal. This Thesis describes the synthesis of bio-inspired ligands containing a phosphine donor substituted by NADH/NAD• mimics. Transition metal complexes containing these ligands were produced, and their reactivity investigated. The prospect of an intermolecular reaction between an OHO and metal-bound formate was explored. Chapter 1 introduces the research. Chapter 2 describes dihydropyridine OHDs as possible reagents for W transfer to the formate ligand of the complex [Fe(dmpe)H(OCHO)], which is produced from CO, insertion to an Fe-H bond. Experiments revealed the hypothesised W transfer does not occur. It was also shown that 1,2-dihydropyridine is unable to transfer H- to CO, in THF. The syntheses of novel phosphine ligands P1-P3 bearing a nicotinamidinium (P1, P3), nicotinamide (P2) and OHO dihydronicotinamide (P1 b) functional group are described in Chapter 3. Ligand P1 could be converted to P1 b using borohydride. The syntheses of gold, rhodium and platinum complexes bearing the ligands P1 and P3 is discussed in Chapter 4. The complexes were incompatible with reducing agents including P1 b and inappropriate as synergistic OHO-metal complexes. The antimicrobial activity of these complexes was investigated, with [AuCl(P3)]CI and its thioglycosylated derivative, [Au(Tl3DGT)(P3)]CI showing modest antimicrobial activity. This activity is likely due to gold present in the structures - ligand P3 may simply serve to transport the gold into the cell. Chapter 5 describes the syntheses of ruthenium p--cymene complexes from P1-P3. [Ru(p-cymene)Cl2(P1 b)] was investigated as a H- transfer agent, reducing hexachloroacetone with concomitant production of [Ru(p-cymene)Cl2(P1 )]. Reactivity studies showed that it is likely the [Ru(p-cymene)Cl2(P3b)] is produced by treating [Ru(p-cymene)Cl,(P3)] with HCOONa. Investigation of the anti-carcinogenic activity of [Ru(p-cymene)Cl2(P3)]CI showed a significant anti-proliferative effect against breast cancer cell lines. Chapter 6 details the synthesis of three (propanedithiolato)diiron carbonyl complexes of ligands P1-P3, and an investigation of possible electrocatalytic activity. Each of the dimers displayed complicated electrochemistry with no useful electrocatalysis observed. Chapter 7 summarises the knowledge gained during this PhD research, and provides suggestions for future progress.