School of Chemistry - Theses
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ItemNMR studies of amyloid ab-peptide in membranes(University of Melbourne, 2006)
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ItemThe Evaluation of Oxorhenium(V) and Oxotechnetium(V) Complexes for the Diagnosis of Alzheimer’s Disease( 2020)Alzheimer’s disease (AD) is the most common neurodegenerative condition and is characterised by the presence of insoluble deposits within the brain that primarily consist of aggregated forms of the amyloid-beta (AB) peptide. The role that AB plays in the development and progression of AD remains uncertain. Accurate diagnostic information for suspected AD patients is imperative for the development of patient care plans, potential therapeutics and may aid in further understanding of AD pathology. The development of radiotracers that can bind to AB is of great interest as they can allow estimation of the plaque burden in patients that may have AD. Such compounds must bind specifically to AB plaques and be able to cross the blood brain barrier (BBB). Technetium-99m is the most commonly utilised radionuclide for single-photon-emission computed tomography (SPECT) imaging. This is attributed to its widely applicable half-life of six hours and its availability from benchtop generators. Lipophilic, charge-neutral technetium complexes have been shown to cross the BBB and this sets a precedent for the development of diagnostic amyloid-targeting complexes using the technetium-99m radionuclide. As there are no stable isotopes of technetium, its group seven congener rhenium is utilised for exploratory synthesis and characterisation. Ligands of a pyridyl-N3S donor set and their corresponding oxorhenium(V) complexes have been synthesised and characterised as models for the potential to incorporate amyloid-targeting groups directly into rhenium and technetium complexes. The small, charge-neutral complexes [ReOL30] and [ReOL31] were analysed by X-ray crystallography and the ligand H2L30-Trt was successfully radiolabelled with technetium-99m under mild conditions using a kit-based approach. A series of tetradentate N3S ligands that directly incorporate a styrylpyridyl amyloid-targeting group have been characterised and the corresponding oxorhenium(V) complexes show excellent binding to AB plaques on human brain tissue. The ligands H2L34-Trt and H2L35-Trt were both radiolabelled with technetium-99m under mild conditions and were shown to be suitably lipophilic for BBB permeability. The biological properties of the two technetium-99m complexes were examined by biodistribution experiments in wild-type mice. The styrylpyridyl complexes [ReOL34-36] confirm that the direct incorporation of amyloid targeting groups to metal complexes is a viable strategy in the design of radiotracers for assisting in the diagnosis of Alzheimer’s disease. Further work is required to improve the BBB permeability of this class of compounds. The design of technetium complexes with very high specificity for diagnostic targets is of great interest in the diagnosis of AD as well as other diseases. A bidentate pyridyl thiosemicarbazide ligand that includes a 1,2,4,5-tetrazine group was synthesised to form 2:1 complexes with the oxorhenium(V) and oxotechnetium(V) cores. The tetrazine groups of the rhenium complex [ReO(HL38)2]+ were shown to rapidly react with a simple transcyclooctene by an inverse electron demand Diels-Alder reaction under mild conditions. The corresponding technetium 99m complex [99mTc][TcO(HL38)2]+ was also synthesised. There exist many diagnostic and therapeutic applications for the tetrazine-containing complex [ReO(HL38)2]+ in bioorthogonal reactions for excellent pathological selectivity.
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ItemCopper-based radiopharmaceuticals for diagnostic imaging of Alzheimer’s disease( 2017)Alzheimer’s disease (AD) is characterised by the presence of extracellular cerebral plaques, comprised mostly of the aggregated amyloid-beta (Abeta) peptide, and intraneuronal neurofibrillary tangles consisting of hyperphosphorylated tubulin associating unit (tau). A radiotracer that can bind specifically to either of these deposits would allow estimation of the burden of these deposits in suspected AD patients and assist in diagnosis and in the development of disease modifying therapies. A potential tracer for either Abeta or tau must be sufficiently chemically stable in vivo and able to cross through the blood-brain barrier (BBB). Copper has a range of useful isotopes for diagnostic applications. In particular, the positron (beta+) emitting isotopes 61Cu (beta+, t1/2 = 3.4 h), 62Cu (beta+, t1/2 = 9.8 min) and 64Cu (beta+, t1/2 = 12.7 h) are able to be integrated into essentially planar, lipophilic complexes and are able to be visualised by positron emission tomography (PET). Copper complexes based on the hybrid thiosemicarbazone-pyridylhydrazone (TPH) ligands form lipophilic, and a sufficiently stable coordination environment for Cu(II) while allowing functionalisation of the pyridyl group to include a targeting motif that can be tailored to the desired target. A series of complexes and their corresponding ligands have been synthesised and characterised that target Abeta using the N,N-dimethylaminostyrylpyridine targeting group ([CuL7,9-13]) and a novel pyridyl-4-vinylpyridyl core ([CuL14]. Both targeting groups show good binding to amyloid-plaques in human brain tissue. The pyridyl-4-vinylpyridyl-based [64Cu][CuL14] complex could be formed in high radiochemical purity under mild conditions and is a promising candidate for pre-clinical biodistribution studies to assess blood-brain barrier (BBB) permeability. The TPH framework has the potential to be modified to integrate a range of potential targeting groups for Abeta. A family of TPH complexes ([CuL15-22]) and corresponding ligands containing the benzofuran motif as a potential Aβ plaque targeting group have been synthesised and characterised. The most promising of these candidates is [CuL20], which interacts with both fibrillar and plaque forms of Abeta and has been shown greater selectively than the N,N-dimethylaminostyrypyridyl-based complex [CuL7] using laser-ablation inductively coupled plasma mass spectrometry. Initial biodistribution studies indicate that [64Cu][CuL20] is able to cross the BBB in appreciable amounts (1.54 ± 0.60% Injected Dose/gram of tissue (ID/g) at 2 minutes post injection). The development of radiotracers specific to aggregated tau are highly desirable from a diagnostic perspective. Tau deposition is thought to more closely follow the progression of symptoms in AD. To develop TPH complexes targeted to tau, a series of complexes ([CuL23-26]) and their corresponding ligands featuring the pyridylquinoline motif were synthesised and characterised. It was found that selectivity for tau could be modulated by the introduction of more sterically bulky substituents on the periphery of the TPH chelate. The most promising of these candidates [CuL26] has been selected for further evaluation to determine BBB permeability and investigate any off-target binding.
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ItemMolecular characterisation of the amyloid precursor protein: a key biomolecule in Alzheimer's disease( 2017)The amyloid precursor protein (APP) is strongly implicated in Alzheimer’s disease pathogenesis due to the observed aggregation of its Aβ sequence in the brain of AD patients. Despite being a molecule of intense interest, a sound molecular understanding of the normal functions of APP remains elusive. This work provides molecular insights into two ostensibly significant interactions involving the extracellular domain of APP, namely: (i) copper binding; and (ii) heparin-induced homo-dimerization. Biological copper is available in two oxidation states: Cu(I) and Cu(II). Well-characterised spectroscopic probes for Cu(I) binding studies are available, but reliable probes for Cu(II) binding studies are lacking. This work has developed a new method for in vitro characterisation of Cu(II) binding affinities. This involved the design and characterisation of four fluorescent peptide-based probes which respond to the binding of paramagnetic Cu(II) ions. Each displays a different affinity for the metal and together they are capable of detecting Cu(II) binding from micromolar to femtomolar range. This allows quantification of the Cu(II) binding affinities of individual protein targets on a unified scale. These probes facilitated studies of copper binding in the APP ectodomain. Biological studies performed over the last two decades have suggested that APP has a role in maintaining cellular copper homeostasis and it is commonly cited as a copper-binding protein. Several putative copper binding sites in the extracellular domain have previously been identified using structural techniques (NMR, X-ray crystallography). However, the chemistry of these has never been systematically studied under uniform conditions and the preferred metal coordination site is unknown. This work has characterised the thermodynamics of Cu(I) and Cu(II) binding in the APP ectodomain. The preferred binding site for both metal oxidation states is a (His)4 centre of picomolar affinity in the α-helical E2 domain. Interestingly, copper coordination leads to conformational changes in this flexible subdomain which could be significant in a cellular context. Homo- and hetero-dimeric interactions between APP and its ‘amyloid precursor-like protein’ homologues (APLP1 and APLP2) have been observed in many previous in vivo studies. These interactions are promoted by the binding of heparins (glycosaminoglycan chains abundant in the extracellular space). The dimers are thought to play a critical role in the normal functions of this protein family including trans-cellular adhesion and synaptogenesis. Detailed structural studies of these dimeric assemblies have not previously been performed. This is largely due to the size and heterogeneity of these complexes which present challenges for high-resolution techniques such as NMR and X-ray crystallography. However, homo-dimeric structures have been solved for two isolated extracellular domains (E1 and E2) which each contain heparin-binding regions. In this work, a chemical cross-linking mass spectrometry approach is used to provide structural information about the dimeric assembly of the complete secreted ectodomain sAPPα. The results offer direct proof of a parallel dimeric interaction between two sAPPα monomers. Additionally, this provides a means to evaluate E1 and E2 homo-dimeric models within the context of the overall ectodomain structure. This preliminary study provides a basis for further investigation of homo- and hetero-dimeric interactions involving APP homologues.
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ItemMembrane interactions of the Alzheimer’s Aβ42 peptide and pore-forming protein equinatoxin II in atomistic detail( 2015)Cytolytic properties of membrane active peptides and proteins (MAPS), in particular the 42-residue isoform of the amyloid beta peptide (Aβ42) from Alzheimer’s disease and the potent cytolysin Equinatoxin II (EqtII) from the sea anemone Actinia equina, are fundamentally dependent on their interactions with membrane interfaces. Atomsitic details of these interactions, however, cannot be identified by current experimental approaches, but may be infrerred from additional molecular dynamics (MD) simulation methods. In Chapter 2, high-resolution NMR of Aβ42 is enabled by development of a high-yielding biosynthetic protocol for producing uniformly 15N and 13C15N labelled material in excess of 10 mg/L of culture media. The final HPLC-purified product was stable for long periods of spectral acquisition by solution state NMR, and further characterised by thioflavin T assays, circular dichroism, electrospray mass spectrometry and dynamic light scattering. Chapter 3 details solid-state NMR investigations, complemented by thioflavin T assays and circular dichrosim, to characterise perturbations to model phospholipid membranes comprising cholesterol, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylserine, ganglioside GM1 or brain total lipid extract. Perturbations to headgroup and tail regions of model bilayers were modulated by lipid composition and by addition of Cu2+. The theory and applicability of MD simulation for studying protein-lipid interactions are discussed Chapter 4, in addition to analytical code developed to detect highly-specific cation-π complexes, hydrogen bonds and hydrophobic interactions. While MD applications for studying Aβ42-membrane interactions remain limited due to availability of data, structural and mechanistics aspects of EqtII are described by experimental research. In Chapter 5, the phosphocholine binding site of EqtII was mapped by solution NMR using uniformly 15N-labelled EqtII with dodecylphosphocholine (DPC). Subsequent docking of EqtII onto a DPC micelle, followed by all-atom MD simulation, identified several lipid-binding pockets stabilized by cation-π, hydrogen-bonding and hydrophobic interactions. Additional simulation with an N-acetyl sphingomyelin (SM) micelle suggested well-documented SM specificity might occur via hydrogen bonding to unique 3-OH and 2-NH functional groups by several conserved residues at the POC binding site and proximate to the hinge of the N-terminal helix that detaches upon pore formation. By combining NMR and MD results, atomistic details of membrane active peptides and proteins can be revealed.
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ItemTechnetium complexes for diagnostic imaging of amyloid-beta plaques to assist in the diagnosis of Alzheimer's disease( 2014)The pathological hallmarks of Alzheimer’s disease (AD) include extracellular plaques, primarily composed of aggregated Aβ peptide, and intracellular neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein. As cerebral Aβ plaque formation can precede the clinical symptoms of AD by decades, in vivo detection of Aβ plaques could assist in accurate diagnosis. The nuclear imaging modality of Single Photon Emission Computed Tomography (SPECT) is a widely available technique that uses the most popular radionuclide in nuclear imaging, technetium-99m. A probe that incorporates 99mTc and allows in vivo evaluation of Aβ plaque burden would be of great clinical utility. A series of tridentate ligands are presented that coordinate to the fac-[M(CO)3]+ core (M = Re/⁹⁹ᵐTc). A pendant group based on the trans-stilbene motif was attached via a small linker to facilitate binding of complexes to Aβ aggregates. Both neutral and monocationic complexes were synthesised by using ligands containing a tertiary amine donor combined with carboxylate and pyridyl or two pyridyl donating groups respectively. Binding of fac-[Re(CO)3Lx] complexes to Aβ plaques was assessed using human brain tissue. Binding to Aβ plaques only occurred for complexes that had a dimethylamino substituent connected to the trans-stilbene pendant group. The tridentate ligands coordinate efficiently at the no carrier added level with selected ⁹⁹ᵐTc complexes used for further animal model experiments. Bidentate 1,4-substituted pyridyl-1,2,3-triazole ligands for coordination to fac-[⁹⁹ᵐTc(CO)3]+ were also investigated. A cyclic peptide (cyclic-RGDfK) served as a model for incorporation of other biomolecules in the future. The type of linker for peptide conjugation affected ligand coordination. Ligands functionalised with an isothiocyanato group via a polyether chain formed the desired ⁹⁹ᵐTc complexes in high radiochemical purity. As a bidentate ligand is unable to exchange with all three water ligands of [⁹⁹ᵐTc(CO)3(H2O)3]+, exchange of the final water ligand was accomplished with pyridine, forming a monodentate ligand in a [2+1] ligand complex. A series of tetradentate ligands were synthesised for the formation of neutral complexes by triple deprotonation upon coordination to [MO]3+ (M = Re/⁹⁹ᵐTc). Synthesis and evaluation of rhenium complexes with ligands containing either a N4 or N3O donor atom set led to the selection of a diamido ligand containing the N3O donor atom set for further development. Incorporation of a styrylpyridine functional group enabled the complex to bind to Aβ plaques and also supplied a pyridyl donor group for coordination to the metal. Further development saw the synthesis and preliminary evaluation of the corresponding [⁹⁹ᵐTcO]3+ complex.
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ItemMetal complexes for diagnostic imaging of cerebral perfusion and amyloid-beta plaques in Alzheimer's disease( 2012)Both Ga-68 and Cu-64 radioisotopes have properties suitable for positron emission tomography (PET) diagnostic imaging but their use is hindered by the lack of suitable chelators. Alzheimer’s Disease (AD) is the most common form of irreversible dementia, characterized by the cerebral deposition of (i) insoluble extracellular amyloid-beta plaques, (ii) intracellular neurofbrillary tangles and (iii) overall brain atrophy. Currently, there is no cure for the progressive neurodegenerative disorder but development in AD diagnostic imaging agents can increase the accuracy of clinical diagnosis and assist in identifying patients in the “high risk group” for potential therapeutic interventions. Two approaches were taken to incorporate the Ga-68 and Cu-64 radioisotopes into potential AD diagnostic radiotracers: development of (1) cerebral perfusion radiotracers and (2) amyloid-beta plaques targeting bifunctional chelators and complexes. Inspired by the Tc amine oxime cerebral perfusion radiotracers 99mTc(V)O(PnAO) and 99mTc(V)O(d,l-HMPAO), the triamine trioxime TAMOX ligand was synthesized. The coordination behaviour of TAMOX in presence of Cu(II), Ga(III), Ni(II) and Co(III) was investigated by NMR studies, mass spectrometry, UV-Vis absorption spectroscopy and single crystal X-ray diffraction studies. The robust bis(thiosemicarbazone) ligand systems were known to form stable Cu(II)N2S2 complexes that are well characterized and tested in biodistribution studies. New bifunctional bis(thiosemicarbazone) ligands, with the diacetyl-bis(N4-methylthiosemicarbazone) framework were functionalized with amyloid-beta plaques targeting functional groups. Cu(II)L(2-4,6-8) complexes were successfully synthesized and characterized by ESI-mass spectrometry, reverse-phase HPLC, elemental analyses, cyclic voltammetry, UV-Vis absorption spectroscopy and fluorescence emission spectroscopy. Cu(II)L(2-4,6-8) were found to have binding affinities for amyloid-beta fibrils in vitro. CuL2 and CuL7 were also capable of binding to amyloid-beta plaques on AD human brain tissues. 64Cu(II)L2 and 64Cu(II)L7 were prepared in high radiochemical purities. Preliminary animal microPET carried out using 64Cu(II)L2 revealed significant findings: (i) the 64Cu(II)L2 complex is BBB permeable and (ii) had significantly higher brain uptake (p= 0.005) in APP/PS1 AD transgenic mice when compared with wildtype mice (percentage of injected dose per gram of animal weight, %ID/g= 2.5% ± 0.6 vs 1.7% ± 0.6, respectively).