School of Chemistry - Theses
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ItemOxidative and chemical modification of amino acids by nitrogen dioxide, ozone and the reactive paracetamol metabolite NAPQI( 2016)Oxidative damage has been implicated in a wide range of diseases including cardiovascular disease, diabetes, asthma, cancer and atherosclerosis. While this damage is typically caused by ROS or RNS generated in vivo, oxidative damage is also mediated by environmental and secondary oxidants such as NO2•, O3 and reactive drug metabolites. This thesis explores the fundamental reactivity of environmental and secondary oxidants towards model amino acids and peptides. According to the WHO, environmental pollution represents the single greatest environmental risk to human health. Exposure of NO2• and O3, common polluting gases, has been linked to the development of asthma, bronchitis, heart disease, stroke, cancer and COPD. While this link is clear, the precise molecular mechanism through which NO2• and O3 cause these adverse health effects is largely unknown. The first section of this thesis explores the reactivity of NO2•, O3 and NO3• towards model biomolecules. N-Acetyl and C-terminal methyl ester protected amino acids and peptides were treated with NO2•, O3 and NO3•. NO3• was generated in situ from the reaction of NO2• and O3 or from the UV irradiation of CAN. These model biomolecules are irreversibly damaged as a result of exposure. The reaction of NO3• generated from the UV irradiation of CAN yields β-nitrate esters from aromatic amino acids, while NO3• generated in situ generally yields nitration products. In the case of tryptophan, pyrroloindoline and nitrosopyrroloindoline products were obtained. Exposure of NO2• to phenylalanine, glycine, alanine and valine containing peptides was found to lead to an unprecedented modification, rearrangement and ultimate excision of amino acids in a peptide chain. The thesis proceeds to examine the fundamental reactivity of a secondary oxidant, the reactive paracetamol metabolite NAPQI. Paracetamol is one of the most widely used analgesic drugs in the world and overdose represents a significant burden on the health system. NAPQI, generated in high concentrations in the liver by CYP450 enzymes, is known to form protein adducts, which have been linked to the development of liver toxicity. The focus of many studies has been on the role of cysteine – paracetamol adducts, formed from the reaction of NAPQI with cysteine residues. In this work, the reaction of NAPQI with a range of amino acids (Cys, Tyr, Trp, His, Lys, Arg, Met, Gln, Glu, Ser and Val) was examined and it was found that NAPQI forms adducts with Cys, Tyr, Trp and Met. Novel paracetamol – amino acid adducts were isolated and characterised by spectroscopic methods. The final part of this thesis explores the reaction of aromatic amino acids and peptides with CAN under UV irradiation. This method was utilised to form β- substituted amino acids with high diastereoselectivity in a single reaction step. Method development was performed in an effort to improve the yield of the β- nitrate ester products. This included 1H NMR based reaction screening of N- terminal protecting groups, work-up procedure and reaction conditions. From these experiments, it was found that ideal reaction conditions included N-acetyl protection, evaporation in vacuo, an excess of CAN and dilute solution concentrations.
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ItemTowards valence tautomerism in polynuclear complexes( 2012)The synthesis, structural and physical characterisation of families of dinuclear cobalt complexes with the bridging bis-o-dioxolene ligands 3,3,3',3'-tetramethyl-1,1'-spirobis(indane-5,5',6,6'-tetrol) (spiroH4) and 3,3'-bis-2-hydroxy-1,4-naphthoquinone (bhnqH2) as well as dinuclear nickel complexes with N1,N2-bis(2-((E)-(3,5-di-tert-butyl-2-hydroxyphenylimino)methyl)phenyl)oxalamide (H4Ltert) and N1,N2-bis(2-((E)-(2-hydroxy-3,5-dimethylphenylimino)methyl)phenyl)oxalamide (H4Lmethyl) were undertaken, with an emphasis on investigating potential valence tautomeric (VT) behaviour. A new family of dinuclear cobalt complexes incorporating deprotonated spiroH4 and ancillary ligands derived from tris(2-pyridylmethyl)amine (TPA) was investigated. Systematically increasing the number of methyl groups at the 6-position of the pyridine arms of the ancillary ligand was shown to fine tune the charge distribution within each complex. Pure samples of [Co2(spiro)(TPA)2]2+ (12+), [Co2(spiro)(MeTPA)2]2+ (22+), [Co2(spiro)(Me2TPA)2]2+ (32+), [Co2(spiro)(Me3TPA)2]2+ (42+), [Co2(spiro)(TPA)2]3+ (53+), [Co2(spiro)(TPA)2]4+ (64+) have been isolated. In the solid state, crystallographic analysis, variable temperature magnetic susceptibility and X-ray absorption spectroscopy (XAS) data revealed 12+, 42+ and 64+ exist in the LS-CoIII-(spirocat-cat)4--LS-CoIII, HS-CoII-(spiroSQ-SQ)2--HS-CoII and LS-CoIII-(spiroSQ-SQ)4--LS-CoIII redox states, respectively, invariant of temperature (2-360 K). In solution, electrochemistry, electronic absorption and EPR spectroscopy analysis are consistent with the above assigned redox states, and also confirmed the presence of another redox state of LS-CoIII-(spiroSQ-cat)3--LS-CoIII in complex 53+. Due to spiroconjugation, electronic communication is observed across the spirocyclic carbon atom in the spiro ligand, which gives rise to weak antiferromagnetic coupling between the two halves of (spiroSQ-SQ)2- in 64+ and electron delocalisation across the ligand for (spiroSQ-cat)3- in 53+. The only complex to exhibit a temperature dependent charge distribution is 32+. Variable temperature magnetic susceptibility and XAS data are consistent with two transitions in the solid state occurring at around 100 and 300 K, whereas UV-visible absorption spectroscopy indicated that two transitions occur at 200 and 250 K in butyronitrile. Therefore, complex 32+ undergoes two thermally-induced VT transitions between LS-CoIII-(spirocat-cat)4--LS-CoIII and HS-CoII-(spiroSQ-cat)3--LS-CoIII and then to HS-CoII-(spiroSQ-SQ)2--HS-CoII. Evidence of a photo-induced VT transition was observed in the solid state at 10 K. Analogous to the spiro complexes, three new dinuclear complexes [Co2(bhnq)(MenTPA)2]2+ (n = 1 for 72+; 2 for 82+; 3 for 92+) with the deprotonated form of bhnqH2 were isolated. In the solid and solution states all three of these complexes existed in the HS-CoII-(bhnqQ-Q)2--HS-CoII redox state independent of temperature (2-300 K). Increasing the number of methyl substituents on the TPA ligand favours the stabilisation of HS-Co(II). An increase in distortion from octahedral coordination around each cobalt centre was correlated with the decrease in room temperature magnetic susceptibility due to quenching of the orbital contribution. A search for potential dinuclear nickel VT systems afforded two square-planar and two octahedral complexes: [Ni2(Ltert)] (10), [Ni2(Lmethyl)] (11), [Ni2(Ltert)(py)4] (12) and [Ni2(Lmethyl)(py)4] (13), where the metal is Ni(II). None of these complexes displayed evidence of a VT transition. Chemical (with (NH4)2Ce(NO3)6) and electrochemical oxidation of 10 led to the decomposition of the complex, giving N1,N2-bis(2(5,7-di-tert-butylbenzo[d]oxazol-2-yl)phenyl)oxalamide (14).
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ItemNovel fluorescent angiotensin AT1 receptor antagonists( 2012)Hypertension is a severe condition that affects many people worldwide. The pathophysiology of hypertension is unknown. Sartans (selective AT1 receptor antagonists) are known to be the most direct and widely used class of antihypertensive drugs that block the vasoconstrictive hormone angiotensin II from binding at its AT1 receptor. Since the discovery of Losartan (2), many drugs are clinically used today. Sartans display a diverse pattern of antagonism however there is some debate as to why this occurs. Some believe it’s due to the antagonist induced changes in the AT1 receptor conformation, others believe it’s due to the levels of cell surface receptor expression and internalisation of the antagonist-receptor complex. Binding of fluorescent sartans to AT1 receptors, will enable the investigation of AT1 receptor localisation. This will provide an insight of the molecular pharmacology of AT1 receptors in cell and tissue systems and in turn will provide a better understanding of the physiological mechanisms involved with the disease. With the aim of preparing fluorescent sartans for use in the study of the molecular pharmacology and cellular localisation of AT1 receptors in cell-based systems, initial work was thus directed towards the synthesis of a series of analogues 103 - 114 of Fonsartan (14), in order to explore the effect of heteroatom substitution and substituent size on the AT1 receptor binding ability of the sartans. The approach involved direct coupling of the common bromo-biphenyl scaffold 32 with several novel aryl-thio substituted imidazoles 90 - 95. Also prepared were coumarin analogues 151 and 152 of Fonsartan, which required synthesis of the novel imidazole 149. In addition, the coumarin analogue 176 of Losartan (2) was also synthesised. Pharmacological testing revealed that compounds 103 – 108, 109 – 114, 151 and 152 were potent AT1 receptor antagonists. The novel AT1 receptor antagonists synthesised in this project that have shown strong inhibition activity and possess fluorescence emission properties suitable for in vitro cellular imaging were sartans 106, 112 - 114, 151 and 152. The novel fluorescent angiotensin AT1 receptor antagonists 113, 114 and 176 showed weak inhibition activity. Results from the in vitro cellular imaging, using Chinese hamster ovary (CHO) cells stably expressing the rat AT1a receptor, revealed that the novel fluorescent sartans 106, 112 - 114, 151 and 152 were too lipophilic to observe the localisation of AT1 receptors in CHO cells. The synthesis of less lipid soluble selective AT1 receptor antagonists with appropriate fluorescence emissions useful for biological investigations may prove as useful tools for the investigation of cellular localisation and trafficking of the AT1 receptors both in vitro and in vivo.
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ItemSynthesis and biological analysis of novel Fluorine-18 positron emission tomography (PET) imaging agents for hypoxic tissues in tumours( 2012)The significance of imaging hypoxia with the PET ligand [18F]FMISO (1.1) has been demonstrated in a variety of cancers. However, as a result of the slow kinetics of [18F]FMISO (1.1), a 2 h delay between tracer administration and patient scanning is required. Labelled chloroethyl sulfoxides have shown faster kinetics and higher contrast than [18F]FMISO (1.1) in a rat model of ischemic stroke. However, these nitrogen mustard analogues are unsuitable for routine production and use in humans. Development of new PET imaging agents was achieved by attempting structural modifications of parent sulfoxides. Five novel radiotracers [18F]1.18, [18F]1.20, [18F]1.21, [18F]1.23 and [18F]1.25 representing various of structural modifications of the parent sulfoxide-based imaging agents were synthesized. These modifications included the introduction of the propargyl group, acetic ester group and trizole-ethyl group to the parent sulfoxides. Radiotracers [18F]1.18, [18F]1.20, [18F]1.21, [18F]1.23 and [18F]1.25 were successfully prepared (with RCY of 2.5%, 15%, 28%, 2.5%, and 3.5% respectively) from the corresponding precursor molecules 1.17, 1.19, 1.22 and 1.24 either via 2-[18F]fluoroethyl azide click chemistry or conventional nucleophilic displacement of a chloride leaving group. Radiotracers [18F]1.20 and [18F]1.21 were synthesized from a single precursor compound 1.19, utilizing both radiolabelling methods. In-vitro and in-vivo evaluation of radiotracers [18F]1.18, [18F]1.20, [18F]1.21, [18F]1.23 and [18F]1.25 were accomplished. Only radiotracer [18F]1.25 was stable in both mice and rat S9 liver fraction during phase I aerobic metabolism studies. Phase I aerobic metabolism studies of tracer [18F]1.20 and [18F]1.21 showed that both tracers were stable in mice S9 liver fraction but undergo metabolism in rat S9 liver fraction, with biological half-lives of 32.39 min and 43.32 min respectively. While, tracer [18F]1.23 underwent minor phase I metabolism in mice and rat S9 liver fractions. Imaging studies using an SK-RC-52 tumour model in BALB/c nude mice have revealed that [18F]1.23 is the most promising hypoxia radiotracer in the series. This tracer shown high t/m ratio of 3.3 in large sized tumours with a pO2 value less than 5 mmHg. Thus, [18F]1.23 has emerged as an exciting new lead compound for further development. Radiotracers [18F]1.18 and [18F]1.20 are also retained in hypoxic tissues at highly hypoxic condition and mildly hypoxic conditions respectively, showing different hypoxia selectivity. [18F]1.18 and [18F]1.20 has emerged as a promising new lead structure for further development of sulfoxide-based hypoxia imaging agents. In particular the mechanism of uptake needs to be elucidated and changes to the chemical structure need to be made in order to reduce metabolism and improve radiotracer kinetics. Whereas [18F]1.21 and [18F]1.25 are cleared from those tumours at a rate similar to that of muscle tissue, showing great promise in future development of hypoxic radiotracer. Investigation of new PET imaging agent with diazobenzene as a bioreducible functional group, replacing the sulfoxide moiety was achieved and radiotracer [18F]1.27 was synthesized from precursor 1.26 in RCY 6.6%. Radiotracer [18F]1.27 underwent metabolism in faster rate in rat liver fraction in comparison to mice liver fraction, with the biological half-life of [18F]1.27 being 118.5 min for rat S9 liver fraction, while the biological half-life in mice S9 liver fraction was calculated to pass the 2 h incubation period (139 min). The imaging studies of [18F]1.27 shows some promises as a platform for new development of hypoxia radiotracer. However, structure modification of this aniline yellow derivative needs to be undertaken to improve this derivative further towards hypoxia imaging agents.
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ItemConfined conjugation MEH-PPV derivatives: synthesis, photophysics and energy transfer( 2012)Conjugated polymers based on poly(2-methoxy-5-(2’-ethylhexyloxy)-1,4- phenylenevinylene) (MEH-PPV) have received considerable attention owing to their interesting luminescent and semi-conducting properties. The performance of optoelectronic devices based on conjugated polymers, such as organic light-emitting diodes (OLEDs) and organic photovoltaic devices (OPVs), is predicated by an understanding of energy transfer processes occurring within and between polymer chains. Despite the large body of knowledge accumulated over the last twenty years, there remains a need to develop an energy transfer model capable of describing the observed fluorescence properties of MEH-PPV. The purpose of this thesis is to explore the photophysics and energy transfer dynamics of well-defined MEH-PPV oligomers and pendant polymers. These materials allow for the establishment of structure-property relationships, and serve as models for understanding the fluorescence properties of MEH-PPV. A series of MEH-PPV oligomers, ranging from 3 to 5 repeat units, was prepared using Horner-Wadsworth-Emmons chemistry from readily synthesised precursors. The oligomers displayed the expected red-shift in absorption and emission maxima as conjugation length increased. Fluorescence quantum yields and fluorescence lifetimes decreased in the order trimer > tetramer > pentamer, owing to increases in the rates of both radiative and non-radiative processes. The non-mirror image relationship between absorption and emission spectra, also observed in MEH-PPV, was investigated using a number of techniques. X-ray crystallography on n-hexyl derivatives, synthesised via a similar route to the MEH-PPV oligomers, showed that the oligomers adopted twisted helical conformations in the ground state, with dihedral angles of ~7° measured for the MEH-PPV trimer. MM2 calculations on a methoxy substituted trimer indicated that a family of conformers was likely to exist in room temperature solution. Temperature dependent absorption and fluorescence measurements provided additional evidence for the presence of non-isoenergetic torsional isomers in the ground state. The rate of conformational relaxation in the excited state was measured using ultrafast transient absorption spectroscopy on the MEH-PPV tetramer and pentamer, with relaxation times of 4 and 5 ps obtained, respectively. Luminescence measurements in neat films and in PMMA matrices demonstrated aggregation occurred in the solid state. Pendant polymers consisting of short MEH-PPV segments (from 3 to 5 units) attached to a non-conjugated poly(styrene) backbone were prepared using a post-polymerisation functionalisation approach. This method involved RAFT polymerisation of vinylbenzyl chloride, followed by a series of post-polymerisation modifications. The final polymers possessed narrow polydispersities (1.08 for each), and molecular weights ranging from 10100 to 17500 g/mol. The absorption and fluorescence profiles of the polymers redshifted as side-chain conjugation length increased. The non-mirror image relationship between absorption and emission spectra was attributed to absorption by a population of side-chain torsional isomers, followed by geometry relaxation to more planar structures prior to emission. Fluorescence decays were modelled using a double-exponential function, although this most likely approximated more complex decay behaviour. Thin film measurements provided evidence for aggregation in the series, with red-shifting of the [0,0] band observed in the neat film emission spectra. Energy transfer in the series was investigated using fluorescence polarisation measurements, and was found to be highly efficient. A Förster resonance energy transfer model was applied for describing energy transfer dynamics between adjacent chromophores, giving rates in the order of 1012 s-1. Pendant polymers labelled with low band-gap acceptors were also investigated. A statistical donor-acceptor polymer consisting of 15 trimer donor chromophores for every pentamer acceptor was prepared by a post-polymerisation functionalisation approach. Photophysical measurements indicated that energy transfer occurred with an average efficiency of 85%. A pendant polymer bearing a terminal perylene diimide (PDI) end-group was also prepared. This synthesis required the use of a PDIfunctionalised RAFT agent, followed by post-polymerisation functionalisation to attach the tetramer donor chromophores. This system was found to undergo electron transfer following selective excitation of the tetramer donors. The relative HOMO/LUMO levels were determined for the model poly(tetramer), and found to be conducive for electron transfer to PDI. The synthesis and solution photophysics of a water-soluble polymer were also explored. Water solubility was conferred by functionalisation of each pendant trimer chromophore with a PEG-3 group. This polymer adopts an aggregate structure in aqueous solution and intermolecular energy transfer between this polymer and an encapsulated MEH-PPV pentamer acceptor was demonstrated
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ItemMolecular characterization of copper tolerance and resistance proteins and enzymes in gram-negative bacteria( 2011)Copper is an essential trace element required by all living organisms. However, it is potentially toxic when in excess or in its “free” form due to its redox activity and highly competitive binding affinities. Specific metabolic pathways exist to safeguard its transport within the cells. A disruption at any point in these pathways in humans is manifested in several fatal diseases (eg. Wilson, Menkes) and contributes to symptoms of neurodegenerative diseases such as Alzheimer. Bacterial cells protect themselves from toxic environmental copper by expressing resistance defense, similar to antibiotic/drug resistance. The cue system in E. coli is part of the chromosomally encoded copper homeostatic system that enables the survival of bacterial cells at micromolar copper concentrations. This system involves two proteins: CopA, a P-type ATPase which removes excess CuI ions from the cytoplasm to the periplasm where they are oxidised by CueO to less toxic CuII ions. CueO is a multicopper oxidase (MCO). MCOs are a large family of enzymes capable of coupling four one-electron oxidation steps of substrates to the four-electron reduction of one molecule of dioxygen to water. Their active sites feature at least four copper atoms which are traditionally classified into three categories designated T1, T2 and binuclear T3 Cu sites. The T1 site catalyses substrate oxidation while a trinuclear cluster T2/T3 site catalyses the dioxygen reduction. However, CueO is unusual: it displays an extra methionine rich -helix insert which covers the T1 site and which accommodates a labile CuII binding site. This work demonstrates that this labile binding site is actually a specific CuI substrate docking/oxidation (CuI-SDO) site. As a cuprous oxidase, this site must be empty to allow catalytic turnover of CuI. As a phenol oxidase, this site must be occupied by a CuII ion to act as an electron-transfer mediator between the buried T1 site and substrates which are not able to dock directly at the specific CuI-SDO site. Using novel approaches, the affinities of this site for both CuI and CuII have been determined. The combined data provide compelling evidence that CueO is a cuprous oxidase in vivo and not a phenol oxidase. The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions, including copper. The resistance to copper is attributed primarily to the presence of a large plasmid pMOL30 that includes a cop cluster composing of 21 genes. Expression of the three soluble periplasmic proteins CopK, CopC and CopA is highly induced by the presence of copper indicating their important roles in the copper resistance. This work has expressed and characterised these three proteins. CopK is unique to this system and exhibits intriguing copper binding chemistry. It is a weakly associated dimer that has little affinity for CuII (KD > 10-6 M), but can¬ bind CuI with modest affinity (KD = 2 x 10-11 M¬). However, CuI binding induces structural change that leads to dimer dissociation and generation of a high affinity CuII binding site (KD = 3 x 10-12 M) and the high affinity CuII binding in turn enhances the CuI¬ binding affinity by a factor of 102. Such strong binding cooperativity between CuI and CuII is unprecedented in copper binding proteins. The molecular basis for this unusual property has been explored and uncovered via biochemical studies (including site-specific mutagenesis) and structural approaches (including X-ray crystallography and NMR spectroscopy). CopC shares high sequence similarity with two well-characterised protein homologs, CopC from Pseudomonas syringae and PcoC from E. coli. All potential protein ligands for CuI and CuII are also highly conserved among these three proteins. The latter two proteins bind CuI and CuII specifically at two separate sites. Indeed, CopC from C. metallidurans expressed and isolated in this work features similar CuI and CuII binding chemistries. CopA has not been isolated and studied previously. Its primary sequence derived from the copA gene predicts it to be a MCO and to contain a Met-rich sequence motif in a position equivalent to that in CueO that accommodates the specific CuI-SDO site. Therefore, CopA is assumed to take a similar role as a cuprous oxidase in vivo. However, preliminary studies in this work indicate that CopA is not only a robust cuprous oxidase, but also a robust phenol oxidase. The former function is buffer-dependent, but in contrast to CueO, the reaction buffers have only minimum effect on the phenol oxidase activity of CopA. Preliminary data suggest that, relative to the enzyme functions of CueO, the new enzyme properties of CopA are arisen from the fact that CopA may feature a CuI-SDO site that can bind either CuI or CuII with higher affinities than those of the equivalent site in CueO. This work also demonstrates that CopA catalyses air-oxidation of CuICuII-CopC efficiently, but not of CuICuII-CopK. Although these two proteins bind CuI with similar affinities, their CuI binding sites are quite different in aspects such as solvent-exposure, ligand number and ligand composition. All these differences are likely to affect their specific interactions with the CopA enzyme and emphasise that specific molecular recognition and interactions are important in copper homeostasis in biology.