Protein modification is an essential tool in Chemical Biology, allowing a functional biomolecule to be equipped with a small molecule tag or label. However, as proteins are constructed from a limited palette of around 20 canonical amino acids, achieving selective modification can be problematic. Previously reported methods for protein modification will be discussed in Chapter 1; these often rely on alteration of the protein sequence to introduce a uniquely reactive (often non-canonical) amino acid which may then be covalently modified in a bioorthogonal manner. An alternative approach is to identify a uniquely reactive site within the native protein sequence, such as the protein N-terminus or the reactive side chain of an amino acid with low frequency, and modify this using selective chemistry. In this project, modification of a native sequence protein was achieved by targeting a low abundance residue, tyrosine (Tyr), in a selective manner. Tyr was identified as the ideal candidate as it displays only ~3% frequency in the proteome and, due to its electron-rich aryl ring, it can be selectively modified by electrophilic aromatic substitution. Using a diazonium salt as the tuned electrophile, modification results in formation of an azobenzene motif which may be orthogonally cleaved under mild reducing conditions. The resulting cleavage product bears an o-aminophenol modification on the Tyr side chain, which can then be conjugated to a fluorescent label using established chemistry. This system has been developed on a solid-phase platform to give further control over the extent of modification achieved. In Chapter 2, the component parts of this method are developed through reactions performed in-solution on small molecule substrates. In Chapter 3, this work is then moved onto a solid-phase resin in order to 'catch-and-release' small molecule and peptide substrates. Finally in Chapter 4, the resin-based catch-and-release system is optimised for use in protein modification, and analysis of the modification site is explored.

Glycoproteins have diverse and essential roles within biological systems. They are formed by enzymatic addition of saccharides to proteins during, or shortly after, translation. However, saccharides can also react with proteins non-enzymatically, a process termed glycation, which can cause impaired function and improper folding. Glycated proteins further react to form advanced glycation end-products, which have been implicated in the pathogenesis and progress of many diseases. Due to this pathological effect, glycation has been studied as a potential biomarker of these diseases. Photoaffinity labelling is a technique that is used to investigate the structure, and presence, of biological molecules; a precedent exists for its use in the study of carbohydrates in biological systems. Chapter 1 outlines the background of this thesis exploring previous studies of glycation, its effects, and methods used in recognition and photoaffinity labelling. Chapter 2 details the design and synthesis of a novel photoaffinity probe, and the optimisation of this synthesis. The target molecule was successfully produced and simpler alternatives to the initial synthetic route with similar yields are discussed. In Chapter 3 the use of the photoaffinity probe is studied. Labelling trials were performed on three proteins: human serum albumin (HSA), macrophage migration inhibitory factor (MIF), and casein. Mass spectrometry showed that the experiments with both HSA and MIF were successful, while the procedure appeared to lead to degradation of casein. Additionally, our work into developing techniques for identifying labelled samples is detailed. A diol-doped electrophoresis gel was not successful created, however, staining protein samples in polyacrylamide gel electrophoresis with curcumin showed promise. Chapter 4 explores the electrochemistry of the photoaffinity probe and details the use of the probe in functionalising a fluorine doped tin oxide (FTO) glass electrode. Cyclic voltammograms of Alizarin Red S (ARS), obtained using a treated electrode, suggest that surface functionalisation was successful.

A Research Report submitted to the School of Animal, Plant and Environmental Sciences, Faculty of Science, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Masters in Science (course work / research report). 28 October 2015 / Eco-labels are voluntary policy tools for promoting environmental justice. Eco-labels have the potential to achieve environmental justice when they are environmentally reliable, and when they help consumers to be aware of, understand and feel motivated to purchase eco-labels. This study analysed the current situation of eco-labels in South Africa. There are a number of generic eco-labelling terms that have come into use on eco-labelled products. The history of the environmental problems that led to the use of these generic terms, along with the accreditation of such eco-labels was considered. The six most commonly found terms considered are “Dolphin Safe”, “Badger Friendly Honey”, “Genetically Modified Organism (GMO)-free”, “Natural”, “Free-range” and “Organic”. The environmental reliability of the most common eco-labels found in local supermarkets was analysed. Overall, eco-labels scored well on environmental soundness and transparency but lacked in communication with consumers. The majority of the eco-labels were third-party certified and accredited by ISEAL (International Social and Environmental Accreditation and labelling) and IFOAM (International Federation of Organic Agriculture Movements). There are a variety of eco-labelled products for consumers to choose from in major supermarkets but the ratio of eco-labelled to regular food products is low, and the availability of eco-labelled products is not consistent. A questionnaire was used to assess the awareness, understanding and perception of consumers regarding eco-labels. The results were compared to a similar study on Swedish consumers to determine whether South African consumer perception regarding eco-labels differed greatly from first-world perceptions, as South African consumers have not had as much exposure to eco-labelled products. Consumer awareness and understanding varied significantly between local and international eco-labels. Generally, the number of consumers that have fully adopted eco-labels is low. However, South African consumers were found to have a pro-environmental attitude; many respondents felt that they would be willing to purchase eco-labelled products if they knew more about the eco-label. Improved stakeholder engagement among eco-labelling initiatives, supermarkets and consumers is required to foster better consumer knowledge of eco-labels and promote environmental justice locally.

This thesis describes the study of the three-dimensional structure and dynamics of the hepatitis B virus (HBV) encapsidation signal, epsilon, by means of nuclear magnetic resonance (NMR) and mutational data. HBV replicates by reverse transcription of an RNA pregenome into the viral DNA genome, which becomes enclosed in viral particles (encapsidation). Epsilon is a stem-loop structure within the RNA pregenome and both the primary sequence and secondary structure of epsilon are strongly conserved, in agreement with its essential function of propagating HBV. Epsilon is therefore a potential target for drug design. Studying the structure of epsilon requires development of new methods in the field of structural biology, as it is such a large RNA. Knowing the structure of epsilon will help to better understand the encapsidation mechanism and priming step of reverse transcription. This will help us in the search for antiviral drugs that block epsilon and prevent the viral reverse transcriptase from binding. NMR spectroscopy is a method that provides detailed structural and dynamical data in solution under natural conditions. However, the size of the molecules that can be studied with NMR is limited. NMR spectra become more and more difficult to interpret as the size of the molecule increases. To circumvent this problem, large RNA molecules can be divided into smaller parts and only the parts essential for NMR studies are selected. The information obtained from these smaller fragments can then be used to determine the structure of the larger molecule. Furthermore, a new method of enzymatically synthesizing nucleoside triphosphates with isotopes suitable for NMR has made it possible to specifically label the RNA molecules. Using this method it is possible to derive highly detailed molecular structures of RNA up to a size of 150 nucleotides. The method of selective isotope labelling was applied to different parts of HBV epsilon. Three RNA fragments of 27 (apical loop), 36 (internal bulge) and 61 (whole epsilon) nucleotides (nt) were synthesized in the unlabelled form. The 27-nt and 36-nt RNAs were also synthesized with (13C, 15N, 1', 3', 4', 5', 5"-2H5)-labelled uridines. The 61-nt sequence was (13C, 15N)-guanidine labelled. This labelling allowed unambiguous assignment of otherwise inaccessible parameters. The unlabelled and labelled RNA sequences provided the necessary data for structure derivation of the whole epsilon. The apical loop of epsilon forms a pseudo-triloop motif. There is only one conformation of the loop that fulfils all the restraints, including experimental chemical shifts. However, the loop adopts several structures that fulfil the experimental distance, torsion angle and residual dipolar coupling restraints. This may reflect true flexibility. Indeed, relaxation studies on the unlabelled and labelled 27-nt sequences show that the residues that show multiple conformations are flexible. This can be an important feature for the recognition and subsequent binding of epsilon to the viral polymerase. The information gained on the HBV encapsidation signal is useful in our understanding of the initiation of replication of the virus. This can in turn contribute to the search for drugs against HBV.

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Currently, nutritional labelling is difficult to interpret and time-consuming to read. This is a major problem as many consumers are overweight and resort to eating readymade meals and snacks. These are likely to be energy-dense food and beverages that are high in fat, sugar and artificial preservatives. Simplifying nutritional labels could help stem rising obesity rates. Front-of-pack labels are a tool to help overcome this problem by providing consumers with understandable, visible information to aid them into making healthier food choices. This study expands on past research by evaluating 7 separate pre-existing, proposed and fictitious front-of-pack nutritional labels. It includes Information, Image or a combination of both Information and Image based labels. Plus No label, which is a control variable to determine the effectiveness of each label. The nutritional labels were placed on a chicken salad sandwich which was kept consistent for all 14 manipulations. The nutritional components were altered to reflect either an Unhealthy or Healthy sandwich. The design of this experiment is a 2 (nutritional level: Healthy, Unhealthy) X7 (labelling system: Traffic Light, Star, Running, Walking, Third Party, Daily Intake, Caloric, None) between subjects design. The results provide evidence of the urgent need to communicate nutritional information more effectively. Images, simplicity, colour and reliability, are determining label elements that influence consumption behaviour. The results from this study help to understand behaviours associated to labels. This study draws differences between those who partake in health behaviours and those who do not. This information could help to trigger support for a new, more effective front-of-pack labelling system to be put in place globally to guide consumers in making healthier food choices.

Polymer surfaces were modified by exposure to a silent discharge plasma, by exposure to UV radiation and by chemical functionalisation. Additionally, the silent discharge treatment of alkali halide disks was investigated. Employing XPS and IR, the silent discharge treatment of poly (phenylmethylsilane) and poly (cyclohexylmethylsilane) thin films was found to result in the formation of a carbonaceous SiO(_x) layer. Oxidation occurred faster and to a larger degree in the case of the aromatic polysilane. A XPS study of the UV irradiation of poly (phenyhnethylsilane) thin films in the presence of CCI(_4) vapour revealed the formation of a chlorinated silicon species. The analysis of aged samples showed that this initially formed product was unstable in moist air. The silent discharge treatment of alkali halide disks (KCI, KBr, KI) was studied in ambient air, as well as in dried and humidified gases (artificial air. He, N(_2), O(_2)). IR and XPS were used as analytical methods, hi most cases nitrate and halogenate were formed upon treatment in air. Depending on the reaction conditions treated KI disks sometimes showed the presence of nitrite as an additional or as the main product. In oxygen atmospheres halogenate was formed as the exclusive product. Treatments in the remaining atmospheres did not lead to product formation. The presence of water vapour in the feed gas increased the amount of product. Changes in the IR spectra of the nitrate species upon storage in a desiccator and after exposure to heat were found and monitored. Pentafluoropropionic anhydride was tested for its suitability as a vapour phase labelling reagent for hydroxyl groups on polymer surfaces. Derivatised films were analysed by XPS and IR. Using Polyvinyl alcohol as a model polymer the reaction proceeded fast and quantitative. Moreover, the cross-reaction with a number of polymers containing functionalities other than hydroxyl was studied. The reaction with nylon 6,6 was investigated m detail. The vacuum photodegradation of polyethersulfone upon irradiation with the full and a selected part of a Hg (Xe) lamp spectrum was studied. The volatile products were identified with in-situ quadrupole mass spectrometry. Monitoring the intensities of some products in subsequent irradiation phases provided evidence for a crosslinking process. In samples irradiated with the complete lamp spectrum crosslinkmg occurred faster. Additionally, the XP spectra of the corresponding samples revealed a stronger modification which became most obvious in the presence of a reduced sulfur species.

This thesis is divided into two parts and covers biosynthetic studies on two secondary metabolites, tropic acid in Part I and piliformic acid, in Part II.(S)-Tropic acid is the acid moiety of the alkaloids hyoscyamine and scopolamine, which are produced by a number of plants of the Solanacae family. An intriguing rearrangement of the L-phenylalanine side chain gives rise to the isopropanoid (S)-tropic acid skeleton. The detailed nature of the rearrangement has however remained elusive despite continued interest over the years. In chapter two the identification of intermediates between L-phenylalanine and (S)-tropic acid is discussed, which has placed (R)-D-phenyllactic acid as an immediate precursor. The stereochemical features of the rearrangement are described in chapter 3 and finally in chapter 4 a mechanism for the rearrangement is proposed. This is based on information obtained from the incorporation of various isotopically labelled precursors to tropic acid into two of the minor alkaloids, 3a-2'-hydroxyacetoxytropane and 3a- phenylacetoxytropane. This work was carried out in collaboration with Dr Richard Robins at the AFRC Institute of Food Research in Norwich. Piliformic acid is elaborated by the slow growing fungus Poronia piliformis. The incorporation of a number of isotopically labelled substrates into piliformic acid has revealed a mixed biosynthetic origin, comprising C(_8) and C(_3) fragments. These have been shown to be of acetogenic and citric acid cycle origins respectively. The C(_8) fragment has been further demonstrated to be a degradation product of a longer chain fatty acid. The mode of coupling of the two fragments has been investigated and suggests the intermediacy of a novel a-carboxyoctanoate. A pathway for the assembly of piliformic acid, involving a 1,3-hydrogen shift, is proposed, consistent with the above findings. These results are the subject of chapter 6.

Molecular imaging involves the development of probes which can specifically label a certain object in the body at cellular or subcellular level. This thesis consists of three parts, each involving the development of novel labelling methods for viruses or virus-like particles with specific applications. Virus-like particles (VLP) derived from the E. coli bacteriophage Qβ are widely employed as a nano-carrier for drugs and vaccines, but a powerful method for tracing its circulation without affecting its structure is yet to be developed. In the first part of the thesis, the electrophilic fluorine source ¹⁹F-Selectfluor^TM was employed for introducing single fluorine atoms on Qβ VLPs. For the 'tag-and-modify' approach, site-selective electrophilic C-F bond formation was achieved on the dehydroalanine (Dha) amino acid tag of VLPs under aqueous conditions. Chemoselective electrophilic aromatic fluorination on tyrosine residues were also achieved using the same reagent by manipulating the amino acid sequence. Similar results were observed in conditions required for ¹⁸F-Selectfluor™ reaction, indicating the potential of this technique for positron emission tomography (PET) imaging. In addition, there is a lack of in situ technique for tracking the functional status of Qβ VLPs and hence the release of cargos. In the second part of the thesis, a simple way to monitor the disassembly of ¹⁹F-labelled Qβ VLPs by ¹⁹F NMR spectrosocpy is reported. Analysis of resonances, using experiments under a range of conditions, allowed determination not only of the intact particle but also the disassembled multimeric species and even smaller peptides upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems. In the third part of the thesis, a new type of rhodamine B fluorescent dye functionalised with a 2-imino-2-methoxyethyl (IME) group is reported. The amidine linkage formed between the IME group and lysine residue retains the pKaH of the original side chain, which cannot be achieved using commercially available conjugating dyes. This in turn minimises the change in net charge hence virus infectivity following virus labelling. By employing adenovirus (AV) as an example, the IME dye was shown to be a better choice in retaining virus infectivity compared to dyes linked with other coupling groups. In addition, preliminary experiments on dengue virus with the synthesised dyes were also performed.

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