The aim of this thesis was to rationally design, construct and test novel protocells equipped with primitive cytoskeletons, which could be produced by integration of simple chemical systems. This was achieved by the non-covalent assembly of a functionalised amino acid, N-fluorenylmethyloxycarbonyl-tyrosine (FMOC-Tyr), to yield highly structured nanofilament morphologies within the aqueous interiors of micron-sized, membrane-bounded compartments. Initially. the formation of the non-covalently assembled FMOC-Tyr nanofilaments was studied in isolation. Significantly, at a critical filament concentration and appropriate pH, the formation of supramolecular hydrogels was ascertained. Moreover, by substituting FMOC-Tyr with the hydrophilic analogue N-fluorenylmethyloxycarbonyl- tyrosine-( O)-phosphate (FMOC-Tyr-POH), cleavage of the phosphate group by alkaline phosphatase or cerium oxide nanopal1icles resulted in the formation of gel networks with tuneable (PH or temperature) gel-sol and sol-gel transitions. After the experimental design parameters for hydrogel formation were determined, the FMOC-Tyr nanofilaments were grown within two contrasting model compartments: phospholipid vesicles and inorganic colloidosomes. Significantly, FMOC-Tyr supramolecular hydrogel-containing vesicles showed enhanced resistance to mechanical deformation, osmotic pressure and increased structural persistence. Moreover, primitive cytoskeletal function was further demonstrated via heat-induced structural deformations of vesicles, where morphological transformations were driven by disassembly of the internalised nanofilaments. Growth of FMOC-Tyr cytoskeletal-like filaments within inorganic colloidosomes resulted in silica membrane-directed nanofilament growth from the interior of the inorganic surface towards the centre of the microcapsule. In this instance, it was possible to demonstrate that temperature-dependent filament assembly and disassembly could be used to modulate the interior viscosities of colloidosomes, which in tum regulated intra-protocellular enzyme kinetics. The work presented in this thesis describes the first examples of protocells equipped with functional artificial cytoskeletons comprising self-assembled synthetic building blocks. The incorporation of these bio-inspired scaffolds within protocells not only provides the structural persistence that is central in all living cells, but also a mechanism to re-structure interior environments and control compartment shape.

The research contained herein is concerned with morpholinone based peptide synthesis incorporating N- and C-terminus chain extension. Chapter 1 firstly considers the bio-significance of peptides, the conformation of the peptide bond, local restriction of peptide chains and the reactivity of peptide bonds. Three general problems in peptide synthesis including chemoselectivity, racemization and yield are discussed. Subsequently, several important coupling systems such as dicyclohexylcarbodiimide, acid chlorides, acid fluorides, acid azides, phosphonium salts and uronium reagents are considered. The process of solid phase peptide synthesis is described and the total synthesis of a cyclic heptapeptide, hymenamide C, is given as an example of a successful solid phase synthesis. A recently developed protein synthesis methodology that employs a chemical ligation strategy is also presented. Synthetic strategies for the construction of small cyclic peptides are also described. Various methods for the aminolysis of lactones and N-debenzylation of amides are reviewed. Finally, previous morpholinone-based amino acid and peptide analogue syntheses are reviewed. The first part of the "Results and Discussion" chapter concentrates on the N-acylation of the 3-alkylsubstituted morpholinone templates. It was found that Fmoc acid chlorides are very powerful coupling reagents for the N-terminus extension of the hindered secondary amines. In the second part of this chapter, the investigation of the C-terminus extensions of the 3-alkylsubstituted morpholinone dipeptide derivatives is described, wherein dimethyl aluminium amides were found to be the ideal lactone aminolysis reagents. The third part of the chapter focuses on the synthesis and C- terminus extension of the five membered oxazolidinone dipeptide derivatives. Next, several amide N-debenzylation methods were tried leading ultimately to the successful synthesis of tri-L-alanine and L-D-L-alanine using lithium in liquid ammonia in the presence of t-butanol at -78°C. The synthesis of cyclic peptides based on 3-methyl morpholinone was attempted but was unsuccessful and this led us to construct the potentially more reactive thiamorpholinone templates. A universal three step synthesis of thiamorpholinones was developed and the expected high electrophilicity of these compounds was observed during the reduction process. A brief investigation of the thiamorpholinone N,C-termini extensions was carried out. It was found that the proline derived thiamorpholinone could be aminolyzed by a secondary amine under very mild conditions. The N-terminus extension of thiamorpholinones was also achieved using the method developed in the morpholinone series in good yields. However, the C-terminus extension of these dipeptide derivatives could not be observed under mild conditions. Suggestions for future C-terminus extension of thiamorpholinone dipeptide derivatives and a new pathway to the synthesis of non-proteinogenic amino acid residue containing thiamorpholinones are made.

Tachykinins are a family of neuropeptides characterised by the conserved C-terminal motif FXGLM-NH2, where X represents a hydrophobic amino acid. Substance P (SP) a member of the tachykinin family has recently been shown to stimulate platelet aggregation and a SP-like immunoreactivity has been demonstrated in platelets and shown to be released upon platelet activation, suggesting that SP may act as a secondary platelet agonist. In recent years a gene encoding new members of the tachykinin family has been identified named TTAC4, which unlike the classical tachykinins is predominantly expressed in the periphery, with high expression in the megakaryocytic cell line HEL. The predicted products of the human TAC4 gene, endokinins A and B share high homology with SP and display similar binding characteristics as SP for the neurokinin-1 (NKl) receptor, which is present on the platelet surface. The high sequence homology between endokinins A and B and SP renders them indistinguishable using SP-imunoassays raising the possibility that platelets may be a source of endokinins. The purpose of this study was to assess the roles of peripheral tachykinins in regulating platelet function.

Naturally occurring macromolecules such as proteins and DNA adopt very specific conformations and three dimensional arrangements which allow them to perform their sophisticated functions in nature. Unnatural oligomerscan also adopt well defined conformations and these have been termed 'foldamers'. This project is concerned with the design and synthesis of β-peptide foldamers which are assembled from nucleoside derived β -amino acids. It was hoped that the combination of the inherent helix folding properties of peptides and the associated characteristics of nucleosides would allow us to create novel foldamers with specific recognition properties. The structure of the β -amino acids involves conversion of the 3'-hydroxyl group of the nucleoside sugar into an amino group (with retention of configuration) and oxidation of the 5'-hydroxymethylene group to a carboxylic acid. Beginning with the natural nucleosides thymidine and 2'-deoxyadenosine, syntheses of 4 different nucleoside β -amino acid monomers was accomplished. The syntheses were based around the use of 3'-azido-2'-deoxynucleosides as key synthetic intermediates and also required optimisation of protecting groups to prevent side reactions associated with cleavage of the glycosidic bond. In general, the preferred protecting group strategy involved the 3'-amino group being either protected with a fluorenylmethoxycarbonyl (Fmoc) group or being masked as an azide. The benzhydryl group also proved useful in the protection of the carboxylic acid function for couplings in solution. The synthesis of the β -peptides was investigated by both solid-phase peptide synthesis and standard couplings in solution. A range of peptide dimers, trimers and a tetramer were prepared, but the isolated yields of the final products were low due to difficulties with purification.

Chemoselective ligation between two peptides can be used to produce synthetic peptides and proteins that cannot easily be synthesised as single peptides by solid phase peptide synthesis. Chemoselective reaction between an aldehyde and hydrazine or hydroxylamine, masking the aldehyde as a 1, 2-amino alcohol, has been investigated as a method of sequential ligation that will allow synthesis of individual peptides in high yield and purity, enabling more than two peptides to be ligated. Protected amino acids were used as precursors for the synthesis of 1, 2-amino alcohol derivatives that could be used in solid phase peptide synthesis for the production of peptides bearing a C-terminal 1, 2-amino alcohol, as a masked aldehyde for use in ligation reactions. Limitations of this method led to alternative investigations, using peptides bearing an N-terminal serine as a masked aldehyde, and a C-terminal hydrazide. Bovine pancreatic ribonuclease A was chosen as an example protein for sequential ligation and using this method, peptides were synthesised in high yield and purity for use in ligation reactions. Trial ligation reactions with short test peptides were performed successfully, however problems were experienced during ligations using peptide fragments of ribunuclease due to involvement of the cysteine side chains. Methods to overcome these unwanted reactions resulted in insoluble peptide fragments. Computer modelling using molecular dynamics simulations has been used to investigate the effect of replacing native peptide bonds in ribonuclease on the structure of the protein. The method of molecular dynamics simulation was validated through comparison of the structure of a mutant of ribonuclease from experimental NMR data to the structure produced after a molecular dynamics simulation. Results of the modelling simulations suggest that replacement of native peptide bonds with the chemoselective bond formed through reaction of an aldehyde and hydrazine will have only minor implications for the structure of ribonuclease, and therefore should only have a small impact on enzyme activity .

The pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are neuropeptides belong to the VIP-glucagon growth hormone releasing factor-secretin superfamily. A broad spectrum of physiological effects can be elicited by PACAP and VIP via PAC₁, VPAC₁ and VPAC₂ receptors. The activation of PAC₁ receptor is PACAP-selective with relatively low affinity for VIP. The splice variants of human PACi (hPAC₁) receptor containing full N terminus and either the hopl (hPAC₁₋hop₁) or null (hPAC₁₋null) form of the third intercellular loop (ic3) had been demonstrated to mediate cyclic AMP and inositol phosphates production by PACAP38 stimulation (Lutz et aL, 2006). This present study created novel cell lines stably expressing either hPAC₁₋hop₁ or hPAC₁₋null receptors that were fused with green fluorescent protein (GFP) at the C terminus.

Reactive Oxygen Species (ROS) produced during normal aerobic metabolism, if not promptly removed by the detoxification mechanisms of the cells, can easily react with cell components, especially lipids, producing secondary cytotoxic molecules called reactive carbonyl species (RCSs). RCSs exhibit significant chemical reactivity and can cause protein modification and dysfunction. One of the most important ReSs is 4-hydroxinonenal (HNE), an unsaturated aldehyde that has been strongly linked to Alzheimer'S disease (AD). A new series of dipeptide histidyl hydrazide analogues of carnosine was prepared, with the aim of producing molecules with enhanced HNE scavenging activity. These compounds were demonstrated to scavenge HNE and to protect SH-SY5Y cells from HNE-induced tOxlcity and were superior in action to carnosine. The synthesis of an analogue of the best compound of the series containing caffeic acid, resulted in the generation of a new molecule exhibiting complete retention of HNE-scavenging activity and also possessing free Radical Scavenging Activity (RSA). Mitochondria are the major sites of high levels of oxidative stress and the targeting of a reactive carbonyl scavenger directly to these organelles would result in extinguishing the primary source of RCS, thus arresting any consequent cellular damage. In the present work, the possibility of specifying the cellular localization of histidyl hydrazide was investigated using previously described mitochondria penetrating peptides (MPPs) and new examples of such sequences I modified to be more resistant to protease degradation. The ligation of histidyl hydrazide to a previously reported MPP was successful in targeting the former to the mitochondria of HeLa cells. Finally. a particular chemical ligation approach was investigated for the development of a system of linking a common peptide vector and a variety of cargo molecules of choice through facile in situ coupling, without requiring the de novo synthesis of a new chemical conjugate in each instance.

Amphibian skin is a rich source of biologically-active compounds that are assumed to have diverse physiological and defence functions It has been known for a long time that the granular glands of amphibian skin produce these biologically-active compounds The glands are controlled by sympathetic nerves, and discharge their contents onto the dorsal surface of the animal in response to a variety of stimuli. The compounds secreted by the glands are thought to play various roles, either in the regulation of physiological functions of the skin, or in defence against predators or microorganisms Amphibian skin secretions contain four major categories of biochemical components which are: biogenic amines, steroids, alkaloids and peptides/proteins. It has been demonstrated that among the secretion components, peptides account for a major percentage. Many hundreds of bioactive peptides have been identified in the skin secretions of species from many families of frogs. They can be classified into two major classes: -- antimicrobial peptides and pharmacological peptides such as retroactive peptides, vasoactive peptides and anticancer peptides. Bioactive peptides are superior to many chemical drugs in several ways. For example, antimicrobial peptides have fewer side effects, have good thermal stability, a broad-spectrum antimicrobial action and, because of their non-specific antibacterial mechanism, are less inclined to induce drug resistance. To date, antimicrobial peptides, trypsin inhibitory peptides, bradykinins, tachykinins, caeruleins, litorins, opioids and tryptophyllins, have been discovered in the skin secretions of frogs from the genus, Phyllomedusa. However, species from other genera of phyllomedusine frogs remain relatively unstudied and thus this project aimed to more systematically-identify novel bioactive peptides from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor , that is the only species within its genus. The focus of the research was to "shotgun" clone the skin peptide precursor -encoding cDNAs, to deduce the peptide structures prior to confirmation by sequencing and to evaluate their bioactivities following chemical synthesis. In this thesis, twelve skin peptides are described from the skin secretion of Pachymedusa dacnicolor. Chapter 3 describes seven of these peptides which were discovered through the ~shotgun " cloning approach. In Chapter 4, five of these peptides were synthesized and subjected to functional assays. In Chapter 5, four bradykinin-related peptides are described and characterized and Chapter 7 describes the isolation of a novel Kazal-type trypsin inhibitor peptide.

Theanine is a unique non-protein amino acid in tea and contributes to many health benefits. However, the progress towards understanding the synthesis and metabolism of theanine in tea has been slow. The main objective of this work was to identify genes involved in theanine metabolism in tea. A deeper understanding of theanine metabolism in tea should facilitate the natural enhancement of theanine levels in shoots.

The synthesis of a range of amino acid-based ionic liquids (AAILs) each containing a 1-alkyl-3-methylimidazolium cation have been completed. The synthesis of these materials was carried out by a three step approach via the dialkylimidazolium hydroxide. New synthetic procedures and analytical methods are described. Each of the AAIL samples have been fully characterised by traditional analytical techniques including multi- nuclear NMR, MS and polarimetry. The stability of the AAILs under ultra high vacuum (UHV) has been investigated; furthermore stability during X- ray beam irradiation has also been studied. XPS studies of each of the AAILs have been completed and the stoichiometry of each sample confirmed. A modified C is fitting protocol was developed, to include contributions from additional C, Nand 0 in the anion, this protocol was subsequently used to investigate the electronic environment of each element present. The measured BEs of the Ncation is peak of AAILs were all close to each other, within experimental error, indicating that the R group of the amino acid-based anion has very little effect on cation-anion interaction. The cation-anion interaction in AAILs was compared to that observed in non-functionalised ILs and was shown to be similar to that of halide-based dialkylimidazolium ionic liquids. The effect of sample exposure under X-ray irradiation for prolonged periods of time was studied, the magnitude of damage was quantified by comparison of N is XP spectra, supported by peak fitting with three components. The nature of the damage and its dependence on the chemical structure of the AAIL was established. The maximum exposure time (tmax,5%) at which point each sample was stable to X-ray irradiation was determined, this was used to develop longer scan sequences, particularly in the case of angle resolved, ARXPS, experiments. ARXPS of amino acid-based ionic liquids is reported for the first time. ARXPS of four AAILs of the general type [CsC1Im][AA], with differing amino acid-based anions, all showed that the alkyl chain is preferentially oriented above the imidazolium cation and sticking out towards the vacuum. The anion was shown to sit slightly above the imidazolium cation, but below the alkyl rich surface.