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.

New NMR Tools for Impurity Analysis was written by Jane Power and submitted for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences at the University of Manchester, on 31st March 2016.NMR spectroscopy is rich in structural information and is a widely used technique for structure elucidation and characterization of organic molecules; however, for impurity analysis it is not generally the tool of choice. While 1H NMR is quite sensitive, due to its narrow chemical shift range (0 - 10 ppm) and the high abundance of hydrogen atoms in most drugs, its resolution is often poor, with much signal overlap. Therefore, impurity signals, especially for chemically cognate species, are frequently obscured. 19F NMR on the other hand offers extremely high resolution for pharmaceutical applications. It exhibits far wider chemical shift ranges (± 300 ppm) than 1H NMR, and typical fluorinated drugs, of which there are many on the market, have only one or two fluorine atoms. In view of this, 19F NMR is being considered as an alternative for low-level impurity analysis and quantification, using a chosen example drug, rosuvastatin. Before 19F NMR can be effectively used for such analysis, the significant technical problem of pulse imperfections, such as sensitivity to B1 inhomogeneity and resonance-offset effects, has to be overcome. At present, due to the limited power of the radiofrequency amplifiers, only a fraction of the very wide frequency ranges encountered with nuclei such as fluorine can be excited uniformly at any one time. In this thesis, some of the limitations imposed by pulse imperfections are addressed and overcome. Two new pulse sequences are developed and presented, CHORUS and CHORUS Oneshot, which use tailored, ultra-broadband swept-frequency chirp pulses to achieve uniform constant amplitude and constant phase excitation and refocusing over very wide bandwidths (approximately 250 kHz), with no undue B1 sensitivity and no significant loss in sensitivity. CHORUS, for use in quantitative NMR, is demonstrated to give accuracies better than 0.1%. CHORUS Oneshot, a diffusion-ordered spectroscopic technique, exploits the exquisite sensitivity of the 19F chemical shift to its local environment, giving excellent resolution, which allows for accurate discrimination between diffusion coefficients with high dynamic range and over very wide bandwidths. Sulfur hexafluoride (SF6) is investigated and shown to be a suitable reference material for use in 19F NMR. The bandshape of the fluorine signal and its satellites is simple, without complex splitting patterns, and therefore good for reference deconvolution; in addition, it is sufficiently soluble in the solvent of choice, DMSO-d6.To demonstrate the functionality of the CHORUS sequences for low-level impurity analysis, 470 MHz 1H decoupled 19F spectra were acquired on a 500 MHz Bruker system, using a degraded sample of rosuvastatin, to reveal two low-level impurities. Using a standard Varian probe with a single high frequency channel, simultaneous 1H irradiation and 19F acquisition was made possible by time-sharing. Simultaneous 19F{1H} and 19F{13C} double decoupling was then performed using degraded and fresh samples of rosuvastatin, to reveal three low-level impurities (in the degraded sample) and low-level 1H and 13C modulation artefacts.

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