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Proteins are biopolymers constructed from 20 canonical amino acids which, while limited in number, work together to carry out an extensive variety of functions essential to life. Genetic code expansion allows for the site-specific incorporation of non-canonical amino acids with novel functions not found in nature, creating proteins with unique properties that can be applied to tasks that are otherwise unachievable. Photocaged amino acids contain a bulky 'caging' group conjugated to the side chain of a canonical amino acid. This caging group can render the protein inactive by blocking its active site, but can be rapidly removed by illumination with 365 nm light, restoring the canonical amino acid and allowing for the photoactivation of the protein. Photocaged amino acids have been previously used in Caenorhabditis elegans to develop tools for controlling gene expression, apoptosis, and protein-protein interactions. In this thesis, I give a general introduction on genetic code expansion and the use of photocaged amino acids, as well as the use of C. elegans as a model organism. I then explore the use of photocaged amino acids in developing optogenetic tools in C. elegans. The main focus of this thesis is the development of a photocaged FLP recombinase which can drive gene expression with high spatiotemporal control. I show that this can be done in two ways, by photocaging either the catalytic lysine residue, or the catalytic tyrosine residue. In both cases, expression of a target gene is inhibited by the use of an FRT-flanked transcriptional terminator, which can be excised by the photoactivated FLP to drive gene expression with single-cell resolution. This system provides a valuable tool for the study of functions of cells that could not be targeted by other methods. We demonstrate this by using photocaged FLP to drive expression of a channelrhodopsin in the PVC neurons, which do not have their own cell-specific promoter, allowing us to study the contributions of the PVCs in locomotion and sleep. I also discuss the use of photocaged amino acids in the generation of genetargeted random mutagenesis tools, an area of genetic screening that is so far underutilised in C. elegans. These tools are designed to be targeted to a specific gene of interest and generate a random set of mutations in the area, which would facilitate the study of that gene in reverse genetics experiments.

Judicious addition of photolabile caging groups (PCGs) to protect biologically im- portant molecules, has enabled the development of many powerful chemical tools for the study of biological processes. These tools have the potential to be activated in a cellular setting by irradiation with light of appropriate wavelengths, restoring functionality, with excellent spatial and temporal control. This D. Phil. dissertation highlights two biologically relevant examples where PCGs can be applied: (i) 4,5-dimethoxynitrobenzyl (DMNB) caged derivatives of GSH and ESG, were synthesised and uncaging of the DMNB group at 350 nm to reveal the free α-carboxylic acid of the glycine residue was demonstrated. These molecules have the potential to probe the binding mode within the KefC KTN binding domain, a ligand-gated K+ efflux system, critical for bacterial response to electrophilic assault. The molecules are currently with collaborators awaiting further evaluation. (ii) The wavelength-dependent application of PCGs toward the study of protein post- translational modifications (PTMs) was developed. PTMs modulate protein function and have a ubiquitous role in a diverse range of cellular functions. A wavelength selective sequential pair of PCGs was developed and demonstrated in a tripeptide, using the diethylamino coumarin (DEACM), which was cleaved at 420 nm, and the DMNB caging group, which was subsequently cleaved at 350 nm. A chromatically orthogonal pair of PCGs, with possible applications in both organic synthesis and biological systems, incorporating the BODIPY-based and DEACM caging groups, which could be cleaved at 530 nm and 420 nm, respectively, was also developed. Photolysis was conducted on a tripeptide and hexapeptide and an in vitro application was demonstrated where when caged, the peptide was not susceptible to peptidolysis when incubated with enzymes. Following irradiation at 420 nm, the DEACM group was uncaged and the peptide underwent peptidolysis in the presence of Endoproteinase AspN, whereas following irradiation at 530 nm to uncage the BODIPY group, the peptide underwent peptidolysis in the presence of Endoproteinase LysC. These results not only provide conclusive evidence of the ability of this pair of caging groups to act in a wavelength orthogonal sense but also demonstrates the potential applicability of the pair to biochemical studies. This pair of PCGs represents the first example of an orthogonal pair of caging groups where both PCGs could be cleaved, at wavelengths longer than 400 nm, in a sequence independent manner.

Caged compounds are biologically active molecules that are rendered inert by masking an important functionality with a photolabile protecting, ‘caging’, group. The caging group can be removed by irradiation with light to reveal the active compound with restored pharmacological activity with high spatial and temporal control. This technology provides an ideal tool for the study of many chemical, physiological and biological systems. This DPhil dissertation highlights several projects in which caging technology has been employed to address biological problems and questions. The first example of spatially controlled mitochondrial inactivation is reported - a tool for the study of the role of mitochondria in Ca2+ signalling. Caged TRPV1 agonists and antagonists have been developed to probe TRPV1, specifically the location of the agonist-binding site. T cell activation has been controlled with light as a tool to gain insight into the adaptive immune response. Caged sodium channel blockers have been investigated. Wavelength-orthogonal photolysis in a neuronal system has been demonstrated using the neurotransmitters glutamate and GABA - this represents a significant advancement in caging technology. This dissertation also includes investigations into the development of novel caging groups.

O-Sulfation is an important chemical code existing widely in nature, participating in a variety of biological activities including immune response, hemostasis, hormone regulation, cell signaling, and viral invasion. The heterogeneous nature, high polarity with negative charge, and the chemical lability of the sulfate modification have created significant challenges in the synthesis and structure-function studies of O-sulfated biomolecules. It is therefore highly desirable to achieve caging and selective release of the O-sulfated biomolecules. Inspired by sulfur (VI) fluoride exchange reaction, our group developed a series of general approaches to caged O-sulfated biomolecules and their selective deprotection. First, an O-sulfation strategy is developed by coupling aromatic fluorosulfate with silylated target molecules. Scalable synthesis was demonstrated on monosaccharides, disaccharides, amino acid, and steroid. Selective hydrolytic and hydrogenolytic removal of the aryl masking groups yielded the corresponding O-sulfated products in excellent yields. Furthermore, a complete knowledge gap was noted in biocompatible caging of sulfate. With the rational design and systematic optimizations, we discovered that fluorosulfotyrosine in peptides and proteins was an ideal precursor for sulfotyrosine (sY), which can be efficiently converted into the anionic active form by hydroxamic acid activators under physiologically relevant conditions. Photocaging the hydroxamic acid activators further allowed for light-controlled activation of functional sulfopeptides. This system featuring fast kinetics, high selectivity, excellent robustness, and on-demand release provides a valuable tool for probing functional roles of sulfation in the peptides and proteins.

Laboratory mice housed in modern vivariums are chronically cold-stressed. Cold-stressed mice exhibit compensatory non-shivering thermogenesis that drives a significant increase in total energy expenditure. In this manuscript, we will describe the visualization andquantification of non-shivering thermogenesis with thermography as a metric of murine cold-stress (Chapter 2). Utilizing thermography to measure cold-stress within the vivarium, we will show mice housed in individually ventilated caging, a popular and important housing system,exhibit significantly greater non-shivering thermogenesis than mice housed in static cages. Xenograft tumors implanted in mice housed in individually ventilated cages have blunted growth and metabolism compared to static cages. The experimental variability between housingsystems can be significantly, but incompletely, mitigated by the addition of shelters that allow mice to maintain heated micro-climates (Chapter 3). Housing dependent variations in experimental results is likely a confounder throughout murine dependent research. We designed and validated novel ventilated caging designed to minimize the chilling effects of individually ventilated cages while preserving the benefits of ventilated caging (Chapter 4).