School of Chemistry - Theses
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ItemBiomaterial porous networks of hydroxyapatite and titanium dioxide( 2014)A gap in a bone that exceeds 2.5 times the bone radius is termed a critical size bone defect and will not heal naturally. The defect needs to be filled with a synthetic bone substitute (a biomaterial scaffold). \(\textit{In situ}\) delivery of medicinal drugs may assist with treating a bone defect, but current drug delivery vehicles (DDVs) are neither able to controllably release drug molecules nor allow for targeted delivery. Porous networks of either hydroxyapatite (HA) or anatase titanium dioxide could be used as biomaterial scaffolds or DDVs. Using sol-gel chemistry and a templating technique, the preparation of such networks, with potential as materials for biomedical applications, forms the research objective. Polyurethane sponge (PU), polyethylenimine-modified polyurethane sponge, polyurethane-agarose gel composite sponge (AG) and natural sea sponge were used as templates for open-celled, porous HA networks. Two concentrations of sol-gel precursor solutions were employed; the higher concentration obscured the template structure in the final network. The choice of template, multiple sol-gel coating, and the rate of temperature increase when removing the template by calcination led to evolution of the HA fibre surface. PU-templated and AG-templated HA networks were contrasted against each other, with the agarose gel component influencing results. All final networks were HA, but other calcium species were present as well. As an initial alternative to the HA networks, titanium dioxide networks were templated on PU sponge, but these lacked both high surface area and mesoporosity. Next, a Type I collagen gel was employed as a template for anatase titanium dioxide networks composed of mesoporous fibres. A standard method for titanium dioxide network preparation is firstly described, where selective solvothermal treatment preceded calcination. This is followed by modified preparations exploring the morphological transition from the collagen to titanium dioxide network structures, and solvothermal fluids containing varying solvent ratios or ammonia. The collagen fibres were 50-100 nm thick, while the titanium dioxide fibres had walls up to 300 nm thick but retained the collagen structure. Compared to networks that only underwent calcination, solvothermal treatment altered the fibre morphology and enhanced the textural properties (surface area, mesopore diameter and total pore volume). Three titanium dioxide networks, previously templated on collagen gel, and spanning a large surface area range were studied for possible biomedical applications. Biomineralisation took place in a simulated body fluid. Apatite grew on each network indicating in vitro bioactivity, but surface area may affect sustained biomineralisation. Ibuprofen drug delivery was monitored by two methods, with a loading of 58.9 mg/g achieved on the highest surface area network. The drug release was modelled as a sustained diffusion mechanism. Ibuprofen could be stored in mesopores or adsorb to the titanium dioxide fibre surface. Overall, HA and titanium dioxide porous networks were fabricated by sol-gel chemistry and templating. In general, morphology and textural properties were influenced by the choice of template, precursor concentrations and processing conditions, including the rate of heating, calcination time and solvothermal treatment. The collagen-templated titanium dioxide networks have potential as materials for biomedical applications.
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ItemPhotoisomerisation action spectroscopy( 2014)A custom ion mobility mass spectrometer has been designed and built to investigate the photoisomerisation of molecular ions in the gas phase. The instrument differs from existing ion mobility mass spectrometers in that there is provision for optical access to the drift region to allow photo-excitation of the drifting ions as they drift. Photoisomerisation manifests as a change in the ions’ drift mobility, altering the time they take to drift through the instrument. By monitoring changes in the arrival time distribution as a function of laser wavelength it is possible to to collect photoisomerisation action spectra. The instrument has been used to probe the photoisomerisation of selected cationic carbocyanine dyes. Comparison of the dyes’ laser-off and laser-on arrival time distributions reveals that these dyes have a multitude of isomers, that can be interconverted using laser light. The peak absorptions of these dyes are shown to be blue-shifted relative to their absorption spectra in solution by 30-50nm. This work demonstrates that ion mobility can form the basis of a new spectroscopic technique for molecular ions that are not responsive to existing methods.
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ItemControlling the microbial deterioration of cultural materials: Investigations into potential free radical, nitric oxide-based treatments( 2014)The biodeterioration of cultural heritage objects can cause adverse changes to the physical and chemical properties of cultural materials. This can lead to a reduction in the integrity of materials and changes to the aesthetic qualities of objects that can have a negative impact on their long-term preservation and may ultimately result in the loss/damage/alteration of unique objects of cultural significance. The biodeterioration of cultural heritage caused by microorganisms is a consequence of the successful establishment and activities communities of microorganism contained within the protective environment of a biofilm. Treatment methods aimed at reducing biofilm formation and enhancing biofilm dispersal can potentially reduce bacterial activity associated with bio-decay. The free-radical molecule nitric oxide (NO•) has been shown to possess both anti-biofilm properties. The approach to and design of investigations that underpin this thesis aimed to assess the potential and suitability of NO• as an intervention material in the treatment of cultural heritage objects. These investigations into NO• based treatments have examined the effects of NO• on biofilm formation, biofilm dispersal and cell motility in test populations that have included a model microorganism Pseudomonas. aeruginosa PAO1 as well as microorganisms representative of those found in association with cultural materials (CMO). The NO• based treatments studied include the nitric oxide donor (Z)- 1-[N-(2-aminoethyl)-N-(2 ammonioethyl) amino]diazen-1-ium- 1,2- diolate (DETA/NO•) and potassium nitrate (KNO3) as a source of nitrate in the bacterial generation of bioactivated NO•. These investigations demonstrate that treatment effects are concentration dependent; are evident at sub-lethal concentrations and are influenced by the duration of a NO• based treatment. Fluorescence based imaging techniques, using confocal laser scanning microscopy (CLSM), have been used to confirm the presence of bioactivated NO• in cells, as well as the properties of biofilms to which NO• based treatments have been administered. The results indicate that NO• based treatments have a dose dependant, non-toxic, anti-biofilm effect on the test populations studied.
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ItemInvestigating the structure and dynamics of DNA with fluorescence and computational techniques( 2014)Nucleic acids, such as DNA, play an essential role in all known forms of life; however, despite their fundamental importance, there is still a significant lack of understanding surrounding their functional behaviour. This thesis explores the structure and dynamics of DNA by employing methods based on fluorescence and through the use of computational calculations. Time-resolved fluorescence experiments have been performed on dinucleotides containing 2-aminopurine (2AP) in various alcohol-water mixtures. 2AP, a fluorescent analogue of the nucleobase adenine, has been used extensively to investigate nucleic acids because of its ability to be incorporated into their structures with minimal perturbation and its high sensitivity to its local environment. Direct solvent effects on 2AP were established through measurements on the free fluorophore. Analysis of the complex fluorescence decays associated with the dinucleotides was challenging but has provided insight into their conformational dynamics. Solvent polarity was found to play a significant role in determining both photophysical and conformational properties in these systems. The complicated fluorescence decay of 2AP in nucleic acids highlights the need for accurate and unbiased analysis methods. Various time-resolved fluorescence analysis methods, including iterative reconvolution and the exponential series method, have been investigated with real and simulated data to obtain an overview of their benefits and limitations. The main outcome of the evaluation is that no single method is preferred in all situations and there is likely to be value in using a combination when there is ambiguity in the interpretation of the results. Regardless of the analysis technique used, the parameterised description of the observed fluorescence decay is meaningless if the underlying physical model is unrealistic. The advance of computational methods has provided a new means to rigorously test the viability of proposed models. Calculations have been performed at the M06-2X/6-31+G(d) level of theory to investigate the stability of 2AP-containing dinucleotides in conformations similar to those observed in the double-helical structure of DNA. The results help to explain the similarity of the time-resolved fluorescence behaviour of 2AP in dinucleotide and DNA systems but also bring to light subtle differences that could perhaps account for experimental discrepancies. The recent emergence of advanced optical microscopy techniques has offered the prospect of being able to directly visualise nucleic acid structure at the nanoscale but, unfortunately, limitations of existing labelling methods have hindered delivery of this potential. To address this issue, a novel strategy has been used to introduce reversible fluorescence photoswitching into DNA at high label density. Photophysical studies have implicated aggregation and energy-transfer as possible quenching mechanisms in this system, which could be detrimental to its future application. The reliability of fluorescence photoswitching was investigated at ensemble and single-molecule level and by performing optical lock-in detection imaging. These developments lay the foundations for improved and sequence-specific super-resolution microscopy of DNA, which could offer new insights into the 3D nanoscale structure of this remarkable biopolymer. In summary, the work presented in this thesis outlines important observations and developments that have been made in the study of the structure and dynamics of nucleic acids.
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ItemUltrasonic formation of stable food emulsions for the delivery of nutrients( 2014)Growing market trend for the consumption of healthy food has led to production of foods rich in bioactives. This thesis reports on the outcomes of an investigation into the formation of stable emulsions of a bioactive, flaxseed oil, in two complex liquid food matrices, namely, skim milk and carrot juice by employing low frequency high intensity ultrasound, particularly in the absence of external food additives, pre-emulsification procedures and external heat and/or pressure. Initially, the study focussed at analysing the process-induced changes to skim milk at sono-emulsification experimental conditions. Sonication of skim milk for up to 8 min (maximum emulsification time) caused minor modifications to about less than 20% of total whey proteins. The modifications to whey proteins is also an advantage since they can act as emulsifiers to stabilise ultrasonically generated flaxseed oil emulsion droplets. The minimal change noticed at short sonication time indicates the safety of the technique. The changes to whey proteins resulted in a decrease in the turbidity of milk but did not affect the viscosity of milk. The casein micelles showed stability against disruption even until 60 min of sonication, except for the very minor changes in their sizes. This work is published in Innovative Food Science and Emerging Technologies and presented in Chapter 3. The feasibility of making finer and stable 7% flaxseed oil-skim milk emulsions was explored at different power levels ranging from 88 to 176 W and processing time from 1 to 8 min. Highly stable emulsions were obtained at 3 min & 176 W and 5 min & 132 W in the absence of external food additives. The emulsion stability test reported a shelf life of 9 days at 4±2ºC against phase separation of emulsions. The efficiency of sono-emulsification was checked up to oil loading of 21%. The factors responsible for emulsion stability were identified to be partially denatured whey proteins and the optimum power-process time combinations employed in the process. The study also drew comparisons between conventional and sono-emulsification processes. High shear mixing and pressure homogenization processes were compared against ultrasound at equivalent energy densities. On comparison, it was observed that at similar energy densities, high shear process of 12.5 min at 70 W & 17500 rpm and pressure homogenization at 200 bar (5 pass) & 655 W did not produce finer and stable emulsions as observed with 5 min of ultrasound process at 176 W. This work is published in Food Hydrocolloids and presented in Chapter 4. After characterization of emulsions, the functionality studies of 7% flaxseed oil-skim milk emulsions were conducted primarily to understand the nature/characteristics of downstreamed products, viz., beverage emulsions (ready-to-drink) and yoghurts (lactic acid gel). Results showed that sonication process did not induce oxidation in emulsions and did not change their viscosity until 9 days of storage at 4±2ºC. Also, the acid gels obtained from milk emulsions showed improved functionalities such as higher gel strength, reduced syneresis, higher storage modulus and reduced gelation time against controls. The evidences obtained suggested that the newly formed fat globular surface (by sonication) stabilized by denatured whey proteins played a major role in functionality, mainly by interacting with other whey proteins and caseins during gelation and formation of network. All the observations helped at arriving at a complete mechanism of gelation of those sono-emulsions. This work is published in Ultrasonics Sonochemistry and presented in Chapter 5. Finally, the feasibility of sonication to disperse flaxseed oil in carrot juice was explored. Stable emulsions of 1% bioactives were obtained at 4 min & 176 W and the size of emulsions were 0.6 μm until 8 days of storage at 4±2ºC. The pectins and/or proteins of carrot juice were responsible for emulsion stability, either by electrostatic repulsion or steric stabilization. The emulsions showed high negative zetapotential values of above -30 mV until 9 days of storage at 4±2ºC. The results of oxidative stability of emulsions study suggested the need for inert atmospheric conditions while processing. It showed that carotenoids exhibited pro-oxidant behaviour mainly because of its concentration, presence of oxygen rich atmosphere and formation of few process-induced free radicals during sonication. The study also looked at delivering flaxseed oil in an aqueous mixture of milk and carrot juice system. The sonication of carrot juice under proposed emulsification conditions also led to many advantages in the juices, such as improvement in cloud stability, increased carotenoid content, reduction in pectin methyl esterase activity, absence of non-enzymatic browning, etc. This work is submitted in Food and Bioprocess Technology and presented in Chapter 6.
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ItemQuantum dots for biological applications( 2014)The use of Quantum Dots (QDs) as an alternative to fluorescent organic dyes for the labelling of biological molecules has attracted a significant amount of interest in recent years. Due to their unique spectral and physical properties, QDs promise to overcome some of the limitations of organic dyes, such as broad emission profiles and low photobleaching thresholds. In this thesis, a flexible platform for the use of QDs in biology is presented. CdSe/ZnS core-shell QDs are rendered water-soluble and biocompatible through a polymer encapsulation technique that ensures colloidal and optical stability, low non-specific binding and that allows the nanocrystal surface to be equipped with a tunable amount of various functional groups (e.g. azides). The conjugation to biomolecules is achieved through the use of bifunctional linkers able to react via strain-promoted click-chemistry with the azide groups present on the QDs. The elements of this conjugation strategy are extremely versatile and can be used in a range of systems. As a proof-of-concept, the protein transferrin is conjugated to QDs and followed during its internalisation path in cell. Next, QDs are conjugated to antibodies and a QD-based ELISA assay is developed. Finally, the concepts are applied to a novel approach for the delivery of photodynamic therapy.
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ItemDevelopment of highly sensitive and selective microfluidic paper-based analytical devices (µPADs) for environmental monitoring( 2014)The increase in world population and the level of industrialisation of many developed and developing countries have affected the quality of the world’s environment significantly since last century. High demand for raw materials and agricultural products has resulted in the release of increasing amounts of mining toxins, fertilisers and biocides. Electronic wastes contain large quantities of hazardous materials, including heavy metals, and their disposal presents huge environmental challenges to all developed and most developing countries. These problems highlight the necessity for low-cost, portable and accurate chemical analysers as indispensable monitoring and analysis tools for environmental protection activities. The objective of the work described in this thesis was to develop microfluidic paper-based analytical devices (µPADs) for the detection of reactive phosphate, ammonia, nitrate/nitrite and copper to meet the increasing need for rapid, sensitive and selective analysis of nutrients in drinking, natural, and industrial and domestic wastewaters. Interest in the determination of reactive phosphate in aquatic systems stems from the realisation in the late-1960s to mid-1970s that phosphorus was a critical nutrient that promoting eutrophication in aquatic systems. A simple µPAD was prepared and evaluated for the determination of reactive phosphate based on the phosphomolybdenum reaction. Hydrophilic reagent zones were defined by printing filter paper with a hydrophobic paper-sizing agent using an inkjet printer. Under optimal conditions, the µPAD is characterised by a working range of 0.1–10 mg L-1 P, and limits of detection (LOD) and quantitation (LOQ) of 0.05 and 0.16 mg L-1 P, respectively. There was no significant difference between the results obtained using the µPAD and reference methods. A simple and effective µPAD for the determination of ammonia in wastewaters was based on gas-diffusion separation combined with colourimetric detection. The sample was spotted onto the µPAD and ammonium ion present in the sample reacted with sodium hydroxide to form molecular ammonia, which diffused through a hydrophobic microporous membrane into an adjacent paper zone containing an acid-base indicator (3-nitrophenol or bromothymol blue). The resultant change in indicator colour was monitored using a desktop scanner and the Red, Green and Blue (RGB) signal was used for quantification. Under optimal conditions, the µPAD gave LOD of 0.8 and 1.8 mg L-1 N and repeatability, expressed as relative standard deviation (RSD) (n ≥ 10), of 3.1% and 3.7% at 20 mg L-1 N for 3-nitrophenol and bromothymol blue, respectively. The small dimensions, minimal reagent consumption, low cost and simplicity of application make the proposed µPAD an attractive option for on-site wastewater process and discharge monitoring. A low-cost disposable colourimetric µPAD was developed for the determination of nitrite and nitrate by the Griess reaction. First, nitrate is reduced to nitrite in the µPAD’s hydrophilic channel using immobilised zinc microparticles. Under optimal conditions, the LOD and LOQ for nitrite are 1.0 and 7.8 µM respectively, while the corresponding values for nitrate are 19 and 48 µM respectively. This µPAD is suitable for on-site measurement of environmental and drinking waters. A µPAD based on a polymer inclusion membrane (PIM) was developed for the selective measurement of Cu(II) in drinking and mine-tailing waters. The PIM consists of di(2-ethlyhexyl) phosphoric acid as the carrier, dioctyl phthalate as a plasticiser, poly(vinyl chloride) as the base polymer and 1-(2’-pyridylazo)-2-naphthol as the colourimetric reagent. Under optimal conditions, the device is characterised by LOD and LOQ of 0.06 and 0.21 mg L-1 Cu(II), respectively. This µPAD was stable for more than 5 months under ambient conditions.
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ItemThe use of nitroxides in the control and understanding of biofilm formation on cultural materials( 2014)Considerable aesthetic and structural damage to culturally significant materials can be caused by the growth of biofilms and the production of harmful metabolites by microorganisms. Traditional methods to control biofilm formation and to treat biodeteriorated materials has focused on biocidal and antibacterial approaches. The pitfalls associated with such strategies involve the development of tolerance in addition to poor effectiveness due to the refractory nature of biofilms to exogenous physio-chemical pressures. The development of biofilms therefore represent a remarkable adaptation by microorganisms to manage changing environmental parameters. Pivotal to the continual maintenance of a community of microorganisms is the microbial cell-to-cell communication system, quorum sensing. Utilizing biochemical messenger molecules, quorum sensing allows the population to function in unison by initiating signal transduction cascades that culminate in population wide changes in gene expression. Key to this research are the quorum sensing controlled and synchronized dispersal events where sessile cells are transformed into the more biocide susceptible planktonic bacteria. Compounds which encourage cells to be planktonic rather than sessile through the manipulation of cell signalling therefore offers a novel technique to control biofilm formation and biodeterioration. Nitric oxide is one such signalling molecule that has been linked to the inhibition of biofilm formation and activation of dispersal through the generation of nitrosative and oxidative stress. Nitroxides have also shown to promote analogous stress conditions upon bacterial cells and are structurally similar to nitric oxide, both with a free radical that is delocalized between the nitrogen and oxygen atoms. However unlike the highly reactive nitric oxide, some nitroxides are persistent radicals as evidenced by their resistance to dimerization and disproportionation reactions. This thesis investigates the efficacy of nitroxides as anti-biofilm compounds that inhibit and disperse bacterial biofilms. Initial work was directed towards the construction of a nitroxide library. The approach adopted involved the design and synthesis of 22 nitroxide candidates (32, 35-58) with varying charges and hydrophilicities in order to target a range of intra- and extra-cellular compartments. A batch-culture technique was used to screen the biofilm modulatory activity of nitroxide candidates and this identified one nitroxide, 3-(dodecane-1-thiyl)-4-(hydroxymethyl)-2,2,5,5-tetramethyl-1-pyrrolinoxyl (53) that was able to significantly suppress biofilm formation and elicit dispersal events in both a model organism, P. aeruginosa, and on biofilms composed of organisms derived from cultural material. Furthermore using semi-solid agar motility assays, it was revealed that surface-associated cell motilities - twitching and swarming - closely linked to the biofilm phenotype, were exclusively enhanced by lead nitroxide 53, leaving the planktonic-specific swimming motility unaffected and suggesting that the 53-mediated biofilm modulation is linked to the hyperactivation of cell motility. In order to investigate the role of the free radical moiety in the biological activity of 53, the ethoxylamine derivative 83 was prepared and tested for biofilm modulation. Evaluation of its biological activity using a batch-culture assay revealed that the free radical moiety is critical in the efficacy of 53-mediated biofilm inhibition and dispersal and one can speculate that 53 may function via the same pathway as nitric oxide. To further elucidate the mechanism of 53-mediated dispersal and investigate the formation of reactive nitrogen and oxygen intermediates and associated changes in redox states within microcolonies that may be associated with a nitric oxide-mimetic dispersal mechanism, two cell permeable and biocompatible novel profluorescent nitroxide probes (125, 145) and their corresponding ethoxylamine derivatives (126, 146) were synthesized. Imaging of 125 within a biofilm showed that 53-mediated biofilm dispersal is not linked to the formation of free radical species or oxidative stress.
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ItemHuman Relaxin-2: design, synthesis and development of novel RXFP1 agonists and antagonists( 2014)The ten members of the human insulin-relaxin superfamily comprise insulin, IGFs 1 and 2, relaxin-1, 2 and 3, and INSL3, 4, 5 and 6. Members of this superfamily share a common tertiary structure, but each has unique physiological functions. This thesis focuses on human relaxin-2 (relaxin) and efforts towards the design, synthesis and development of novel agonists and antagonists of its cognate receptor. The six cysteines found within the A- and B-chain of human relaxin forms characteristic disulfide bonds, which are a structural feature shared by this superfamily. One disulfide bond is held within the A-chain and another two between the A- and B-chains. These cysteines are conserved amongst these peptide hormones. Relaxin, a 53 amino acid peptide, is the major stored and circulatory form found within the human body. Although able to bind to two relaxin family peptide receptors, class 1 (RXFP1) and 2 (RXFP2), its cognate receptor is RXFP1. The relaxin-RXFP1 interaction is regulated in two modes. Both binding of the ligand to the receptor and its downstream biochemical activation is activated by this interaction. The primary interaction mode involves interaction of the binding cassette within the relaxin B-chain (the RXXXRXXI binding motif) with the leucine rich repeats of RXFP1. A secondary interaction involves the relaxin A-chain with the RXFP1 exoloops, hence, activating the receptor and consequently the downstream signalling cascade. The focus of this thesis was the primary binding interaction between relaxin and RXFP1. Relaxin passed Phase III clinical trials for the treatment of acute heart failure in late 2012 and, if it enters the clinic, will be the first novel therapy in more than 20 years. Despite its high specificity and excellent safety profile with no major adverse effects reported so far, like many other native peptides, it has poor pharmacokinetic properties. It is not orally available and survives in the blood only for a very short period (half-life ca. 10 minutes). Chapter 3 focuses on a short-term goal of this thesis which is to improve the pharmacokinetic properties of this peptide hormone using established approaches. Chemical modifications, specifically by oligomerisation and PEGylation, are employed to slow down enzymatic degradation. Dimerisations were carried out as preliminary studies to ensure the RXFP1 binding and activation properties of the analogues were retained. Relaxin dimers were formed with the via a disulfide bond and by click chemistry techniques. Both dimers were found to bind and activate RXFP1 similar to native relaxin. The PEGylated relaxin dimer was shown to be significantly more stable in serum than native relaxin and the disulfide bond-dimerised relaxin. Numerous studies have shown that relaxin is expressed by tumors in mammary, endometrial, thyroid and prostate cells and can act in both autocrine and paracrine manner on RXFP1 receptors in these cells. In human males, relaxin is exclusively produced in prostate secretory epithelial cells and, correspondingly, from prostate cancer cells. There is clear evidence that progression of aggressive prostate cancer can be stimulated by relaxin and/or RXFP1 overexpression. A significant decrease in tumor size and fewer metastases are reported for siRNA targeting of RXFP1 expression in the PC-3 xenograft model. These observations highlight the significant potential for the inhibition of relaxin physiological actions via RXFP1 antagonists to block prostate cancer growth and metastasis. Detailed studies in Chapter 4 focused on the design and development of RXFP1 antagonists specific to the primary relaxin-RXFP1 binding site via the RXXXRXXI binding motif. These two residues, Arg13 and Arg17 together with IleB20 within relaxin B-chain, were found to be essential for binding to and activating RXFP1 receptor. However, when these Arg were mutated to Lys, the analogue exhibited antagonistic properties both in vitro and in vivo by interfering with relaxin-induced signalling and, hence, impairment of prostate tumour growth by reduced affinity to RXFP1. The results from this chapter highlight efforts to increase the affinity of chemical analogues with RXFP1 whilst retaining antagonistic propensity. In particular, the more isosterically similar homo-Arg (HR) to Arg analogue, H2: BR13.17HR, showed improved RXFP1 affinity compared to the current antagonist, H2: BR13.17K whilst being a full antagonist in primary LNCaP cells. Relaxin also has other non-reproductive functions including cardioprotective and anti-fibrotic roles. To develop a highly specific next generation peptide drugs based on relaxin, three main concerns need to be addressed: large size (53 amino acids), complex structure (three disulfide bonds) and short half-life in blood. Simpler relaxin analogues need to be developed that are easier to prepare and modify, highly selective for RXFP1 and able to retain their activity for an extended therapeutic time-frame in patients. Chapter 5 addressed the effects of elimination of the A-chain intradisulfide bond on relaxin interaction with RXFP1. Various truncations were carried out within the A- and B-chains to elucidate the highest affinity RXFP1 analogue. H2: A(C10.15S) at nanomolar concentrations was found to activate RXFP1, which demonstrated that the intramolecular disulfide bond is not necessary for RXFP1 interaction and activation but instead maintained the overall 3D structure of relaxin. Development of these new analogues highlighted that the chemical syntheses of the complex, two chain and two- or three disulfide bond, relaxin peptides as an extremely laborious process. Assembly of the two chains is notoriously challenging due to the poor solubility of the B-chain and post-synthesis handling and purification of the peptide often results in low yields. From knowledge of the binding motifs of relaxin with RXFP1, efforts to develop RXFP1-specific single chains were explored in Chapter 6. The relaxin B-chain has most, if not all, of the amino acids responsible for RXFP1 binding and activation. The single chain peptide, B7-33, was shown to activate primary cells in vitro like relaxin, albeit with lower affinity. Importantly, B7-33 was shown also to mimic the anti-fibrotic actions of relaxin in a mouse model of acute heart failure disease. Together, the results of this thesis have identified novel relaxin agonists and antagonist that possess high affinity RXFP1 binding and potency. These analogues promise to be important lead analogues for further identifying the structural mechanism of relaxins actions in vivo and for development as potential therapeutic agents.
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ItemStudies towards the synthesis of rhizopodin( 2014)The asymmetric synthesis of the C1 – C18 fragment of rhizopodin is described. Initial work focused on the construction of the C9 – C18 fragment utilising the addition of the anion derived from 2-methyloxazole 195 to an aldehyde (235) to construct the C15 – C16 bond. The synthesis of iodide fragment 259 began with known aldehyde 244 and utilised a stereoselective novel acetylide addition to install the C3 stereocentre as well as a regioselective hydrostannylation of the terminal alkyne followed by tin-iodide exchange to install the vinyl iodide. Attempted construction of the C7 – C8 bond by intramolecular Heck coupling lead to the preferential formation of 6E, 8Z macrocycle 262 over the desired 6E, 8E 261 whilst an intermolecular Heck coupling between alkene 240 and iodide 259 resulted in the formation of a mixture of 6E, 8E (263) and 6E, 8Z (264) dienes with a slight preference towards the desired 6E, 8E (263). Due to the low selectivity, an alternative method for the construction of the C6 – C9 diene was then utilised. Suzuki coupling between boronate 311 and iodide 259 installed the diene (310), which was then esterified with acid 316. Installation of the azide by a Mitsunobu reaction (318) allowed for the application of an O, N-acyl shift to provide hydroxy amide 319 that was subjected to oxidation and cyclodehydration to give oxazole (320). This completed the synthesis of the C1 – C18 fragment of rhizopodin with NMR spectral analysis showing good correlations with literature values for the relevant fragments in rhizopodin (4) itself.