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Peptide nucleic acids (PNAs) were used as encoding tags to enable the analysis of peptide libraries by PNA/DNA hybridisation onto DNA microarrays. This allowed entire peptide libraries to be organised and sorted in a two dimensional format whereby all library members could be interrogated and analysed on a one-byone basis. In this thesis, PNA-encoded peptide libraries, generated by split-and-mix library synthesis, were screened for a variety of functions. Peptide sequences identified from the screening of a PNA-encoded library were analysed in detail as the first specific substrates for chymopapain. A new PNAencoded library consisting of D-amino acids was synthesised and screened with a number of proteases in attempts to identify novel/unusual substrates. PNA-encoded libraries were also used in the screening of peptide libraries for other activities. Thus substrates for catalyst-free Hüisgen cycloaddition were identified following the reaction between an alkyne modified peptide library and azidofluorescein, while cell-penetrating peptides were identified by hybridization of an internalized encoded library onto a DNA microarray.

The steady-state and time-resolved fluorescence of 2-aminopurine (2AP) have been used to monitor base dynamics and base stacking interactions in DNA single strands and dinucleotides, and to investigate the interactions between DNA and a polymerase, Pfu-Pol. A new class of base analogues has also been investigated using a combination of experiment and quantum chemical computation. In recent years, 2AP has been widely used as a fluorescent probe to study conformational changes and inter-bases interactions in duplex DNA, but the conformational behaviour of DNA in single strands has been far less investigated. In the present work, six 2AP–labelled single strands have been studied by steady-state and time-resolved fluorescence measurements. Single strands were found to show similar conformational heterogeneity (manifested by 4-exponential fluorescence decays) to duplex DNA, but highly stacked conformations, in which 2AP is rapidly quenched by inter-base charge transfer, are less populated in single strands, whereas imperfectly stacked (weakly quenched) conformations are more highly populated. The effect of base pairing in constraining base mobility is evident. To further investigate the influence of base stacking interaction on DNA conformation and the mechanism of inter-base quenching of 2AP, the time-resolved fluorescence of 2AP-containing dinucleotides was measured. The fluorescence decay of 2AP-containing dinucleotides in PBS buffer at room temperature is also multiexponential and the shortest lifetime varies with the identity of the natural base partner, in a manner consistent with quenching by inter-base electron transfer. When the dinucleotides are frozen to 77 K, the quenching of 2AP is almost eliminated, demonstrating the importance of thermal fluctuations of the bases in facilitating inter-base quenching at room temperature. In the frozen dinucleotides, an additional decay component with a lifetime significantly longer than unquenched 2AP is also observed, suggesting the formation of a new, delocalised, inter-base excited-state. Archaeal family-B DNA polymerases bind tightly to uracil and stall replication when they encounter this base in template strands, four bases ahead of the primer-template junction. If the polymerase progresses further towards the uracil, the 3′-5′ proof reading exonuclease becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and the introduction of mutations into the genome. Time-resolved fluorescence of 2AP has been used to investigate the role played by unwinding of primer-templates in this mechanism. 2AP-labelled primer-templates (2AP positioned next to the terminal 3′ base of the primer strand), with a misincorpated uracil at the +2 position (U+2) or +4 position (U+4) from the replication fork in the complementary template strand, were investigated in complex with the polymerase Pfu-Pol. For the U+2 primer-template, the fluorescence decay parameters show clear evidence for a decrease in the amount of double-stranded DNA on polymerase binding, manifested by marked weakening of inter-base stacking and a large transfer of population from highly stacked to poorly stacked conformations. In contrast, for the U+4 primer-template only a small perturbation to inter-base stacking is seen, together with the persistence of a high population of strongly stacked states. A new class of base analogues with selenium replacing oxygen at the 4 position of thymine and the 6 position of guanine has been investigated experimentally and computationally. These base analogues are interesting because they show a large shift (>80 nm) in their absorption spectrum compared with the natural bases, taking their absorption into the visible region, with minimal change in molecular structure. The potential of two examples of these analogues, 4-selenium thymine-3’-phosphate and 6-selenium-2’-deoxyguanosine-3-phosphate as luminescent probes has been investigated. However, they prove to have very low emission quantum yields for both fluorescence and phosphorescence. The effect of selenium-substitution on the structural and photophysical properties of the bases has been studied by various ab initio computational methods. It has been found that replacement of oxygen by selenium does not affect the ground state structure but changes the structure of the first excited-state from buckled to nearly planar. The shift in the absorption spectrum on introduction of selenium is successfully predicted by the calculations; the red-shifted absorption band of selenium-substituted thymine is due to a new electronic transition that is not present in the natural base, whereas that of selenium-substituted guanine is from red-shifting of a guanine-like transition.

The three-dimensional structure of a protein, formed as a result of amino-acid sequences folding into compact domains, is regarded as a key factor in its biological function. How and why proteins fold into specific topologies, remain the key focus of scientific research in the field of biophysics. By stripping down complex reactions down to the most basic elements, biophysicists aim to develop simplified models for biological phenomena such as antibody discrimination, viral fusion or self-assembly. Focusing on small model peptide systems, rather than the full proteins from which they were derived, was hoped to result in accurate structural measurements and provide a more transparent comparison between simulation and experiment. The aim of this research was therefore to investigate how accurate these models were when compared against experiment. Furthermore, while breaking down the complex biological phenomena into simple models, there was also a conscious effort to ensure that the models were representative of real biological systems, and a major focus was therefore aimed at determining whether any meaningful biomedical insight may be extrapolated from such models. Peptides found in hormones (human chorionic gonadotropin, luteinizing hormone), viruses (HIV) and amyloid diseases (transthyretin) were selected in order to probe a variety of questions in relation to the aforementioned biological phenomena. Namely, how the primary sequence influenced the three-dimensional structure (and thus its biological function), how its environment could influence such a confirmation, and how these systems aggregated. This doctoral study has made use of a combination of computer simulations and experimental techniques to investigate a selection of biologically relevant peptides; utilising classical atomistic molecular dynamics (MD) simulations to characterise the free-energy landscapes of the chosen peptides, and compare these findings with the secondary structure content predicted by spectroscopic methods such as circular dichroism and infrared spectroscopy. The peptide systems studied within, were found to be characterised by rugged free-energy landscapes unlike their protein counterparts (defined by singular, deep minima). Furthermore, these landscapes were found to be highly plastic and sensitive to changes in the local environment.

The incorporation of a propargyl group to a natural product target allows for a streamlined approach to the investigation of structure activity relationships (SARs) and target identification in forward chemical genetics programmes using a ‘click’-based approach. To this end, an efficient synthesis of O-propargylated tyrosine derivatives was designed, and these have been used in the construction of peptide motifs both (a) derived from phage display libraries and (b) found in natural products. The L-tyrosine derivative Y* (compound I, X=H, R=H) was incorporated into a peptide sequence, PTTIYY, which is known to prevent the inhibition of p53 by the AG-2 protein. Y* has been included as both the terminal and the internal tyrosine in the peptide sequence. ELISA assays were carried out to determine how the binding of PTTIYY* and PTTIY*Y to AG-2 compared to that of the un-marked PTTIYY sequence. The results of these assays allowed new conclusions to be drawn regarding the important binding features of the peptide and possible sites for further optimisation of the AG-2 binding properties of this peptide through ‘click’ functionalisation of the modified tyrosine. The binding of the peptides incorporating Y* was also assessed using MCF-7 breast cancer cell lysate, known to contain the AG-2 protein. These results confirmed those seen for the purified AG-2 ELISA. The related bromo-D-tyrosine derivative (compound I, X=Br, R=Me) has been prepared and employed towards the synthesis of a bisebromoamide derivative. Bisebromoamide is a newly discovered polypeptide, and a promising anti-cancer agent. The bisebromoamide derivative contains a thiazole unit (Tzl), two N-methylated amino acids, and an oxopropyl pyrrolidine (Opp) moiety, which is unique to bisebromoamide in natural products. The activity of this bisebromoamide derivative will be investigated via ‘click’-based affinity chromatography using a new supported linker recently developed within the Hulme group.

Molecularly imprinted polymers (MIPs) are a novel, promising family of porous materials with potential applications ranging from separations, chemical sensing and catalysis to drug delivery and artificial immunoassays. The unique feature of these materials is their biomimetic molecular recognition functionality. Molecular recognition is the biological phenomenon of specific, selective and strong association between a substrate and a ligand. In man made MIPs this functionality is implemented via templated synthesis protocol. MIPs are synthesized in the presence of additional template molecules which form complexes with functional monomers in the pre‐polymerization mixture. After polymerization, the template is removed, leaving cavities in the structure which are complementary in shape and interaction patterns to the template molecules. These cavities act as mimics of biological receptors and are able to recognize and rebind template molecules. Although the imprinting concept is simple in principle, synthesis of MIPs with precisely controlled characteristics and performance remains a challenging task. Composition, polymerization conditions, template removal process and application conditions all affect the properties of MIPs. The material is affected at different scales, but crucially at the microscopic level, the number, fidelity and accessibility of binding sites are dependent on all the factors mentioned. The full potential of these materials can only be achieved if researchers can control and optimize the properties of MIPs through detailed understanding of adsorption and molecular recognition processes in these materials. The objective of this work is to, using computer simulations and statistical mechanics; develop a fundamental description of MIP formation and function, and to link morphological features of the model materials to their molecular recognition capabilities. In general, molecular simulations employed in this study should allow easier and more efficient exploration of a vast number of factors influencing the behaviour of MIPs. At the heart of the approach developed in this thesis is a computational strategy that imitates all the stages of MIP formation and function. First, the model simulates the pre‐polymerization mixture, allowing the formation of template‐functional monomer complexes. (This stage is implemented via canonical Monte Carlo simulation). Complexes can have different structures, depending on the chemical nature of template and functional monomer; therefore complexes can have a range of association constants. The distribution of template‐functional monomer complexes also translates into a distribution of binding sites of different specificity after template removal. In the second stage of the process, adsorption simulations (grand canonical Monte Carlo) are performed for a variety of model MIPs prepared to assess the role of various processing conditions such as composition, density and binding sites degeneration. This strategy was first applied to a simplified description of MIP species in order to identify the minimal model capable of molecular recognition and thus shed the light on the very nature of this phenomenon. In the developed model, the molecular species are constructed from hard spheres, featuring small interaction sites on their surfaces. The bond between two interaction sites has the strength and topological features of a typical hydrogen bond. The model exhibits molecular recognition, being able to preferentially adsorb template molecules. The associations between template and functional monomers were analyzed and classified to describe the distribution of binding sites and their heterogeneity. Using this model, several experimental trends typically observed in MIP studies could be explained, such as maximum in the selectivity as a function of monomer concentration. Using this model, we were also able to explore hypothetical, alternative protocols for MIP synthesis in order to improve their performance. These include the use of alternative templates and the post‐synthetic surface modifications of MIPs. The general strategy to modelling MIP, employed in this thesis, was then applied to a more detailed description of MIPs with realistic force field potentials for all the species involved. This more elaborate model is simulated with a combination of molecular dynamics (MD) and Monte Carlo techniques. This detailed model provided a wealth of information on various types of complexes observed in the pre‐polymerization mixture. Specifically, it revealed the relative weight of different interactions in the complex and their role in the binding energy of adsorbates. These simulations also provided the comparison of the relative contribution of different types of interactions (van der Waals, Coulombic) involved in a molecular recognition process. We believe the insights gained in this work will contribute to the development of rational MIP design strategies. In the discussion of the results of the thesis we speculate on how these models can be further developed in order to generate quantitative predictions and what type of systems it would be interesting and important to investigate in the future.

Merrilactone A (1) was isolated in only 0.004% yield from the methanol extracts of the pericarps of Illicium merrillianum. Structural elucidation of Merrilactone A revealed a compact, cage-like pentacyclic architecture of high molecular complexity, featuring seven stereocentres, five of which as contiguous fully substituted carbon atoms, two γ-lactones and a central oxetane ring. Merrilactone A also exhibits an important neurotrophic activity, significantly promoting neurite outgrowth in the primary cultures of foetal rat cortical neurons at very low concentrations. Structurally, merrilactone A is related to anislactones A and B, a pair of epimeric sesquiterpene dilactones discovered ten years earlier by Kouno and co-workers from the related Illicium anisatum plant. Fukuyama has shown that anislactone B can be converted into merrilactone A using a simple three step sequence, suggesting that the anislactones may be biogenetic precursors to merrilactone A. Described in this thesis are our research efforts directed towards developing a conceptually novel synthetic route enabling regiodivergent total synthesis of both anislactone A / B and merrilactone A. Our synthetic route (around 22 steps) features several key reactions, which include a [2+2] photo-cycloaddition reaction, Tiffeneau-Demjanov ring expansion and titanium(III) mediated radical cyclization.

Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) constitute important sources of genetic variation which provide insight into disease aetiology and idiosyncratic differences in drug response. The analysis of such genetic variation relies upon the generation of allele-specific products, typically by enzymatic extension or the hybridization of allele-specific DNA probes. Herein, a distinct enzyme-free, dynamic chemistry-based method of producing allele-specific products for genotyping was developed. The approach was initially demonstrated in model systems using synthetic DNA, which was used as a template in a base-filling reductive amination reaction on a PNA backbone. The templated dynamic reaction between a free secondary amine at a ‘blank’ position on the PNA strand and four aldehyde-modified nucleobases drove selective formation of the ‘correct’ iminium intermediate according to Watson-Crick base-pairing rules. In a blind trial, the method was extended to genotype twelve cystic fibrosis patients for two mutations (one SNP and one indel) linked to this disease. Enzyme-free dynamic chemistry thus permitted successful genotyping in both singleplex and duplex formats, demonstrating the application of dynamic chemistry as a distinct method of allelediscrimination with certain advantages over those reported previously. The application of this method as a tool for the discovery of non-natural nucleobases with improved properties for antisense and genotyping applications was also investigated. Furthermore, progress was made towards the use of dynamic chemistry as a means of full nucleic acid sequence analysis, through the templated sequence-selective extension of PNA probes by reductive amination.

Virtual Sreening (VS) is an in silico technique for drug discovery. An overview of VS methods is given and is seen to be approachable from two sides: structure based and ligand based. Structure based virtual screening uses explicit knowledge of the target receptor to suggest candidate receptor-ligand complexes. Ligand based virtual screening can infer required characteristics of binders from known ligands. A consideration for all virtual screening techniques is the amount of computing time required to arrive at a solution. For this reason, techniques of high performance computing have been applied to both the structural and ligand based approaches. A proven structure based virtual screening code LIDAEUS (Ligand Discovery At Edinburgh University) has been ported and parallelised to a massively parallel computing platform, the University of Edinburgh’s IBM Bluegene/l, consisting of 2,048 processor cores. A challenge in achieving scaling to such a large number of processors required implementation of a minimal communication parallel sort algorithm. Parallel efficiencies achieved within this parallelisation exceeded 99%, confirming that a near optimum strategy has been followed and capacity for running the code on a greater number of processors exists. This implementation of the program has been successfully used with a number of protein targets. The development of a new ligand based virtual screening code has been completed. The program UFSRAT (Ultra Fast Shape Recognition with Atom Types) takes the features of known binders and suggests molecules which will be able to make similar interactions. This similarity method is both fast (1 million molecules per hour per processor) and independent of input orientation. Along with UFSRAT, some other methods (VolRAT and UFSRGraph) based on UFSRAT have been developed, addressing different approaches to ligand based virtual screening. UFSRAT as an approach to discovering novel protein-ligand complexes has been validated with the discovery of a number of inhibitors for 11β-Hydroxysteroid Dehydrogenase type 1 and FK binding protein 12.

Proliferating cell nuclear antigen (PCNA) is an essential protein in the cell. It is involved in transcription and many types of DNA repair and replication. Homologues of this protein are found in all orders of life. The high level of conservation and essential nature of PCNA infers that it may be a potential drug target for anti-caner drugs in humans and also a potential anti-parasitic target. X-ray structures of PCNA from Homo sapiens (Hs), Schizosaccharomyces pombe (Sp) and Leishmania major (Lm) are now available and can be used as a template for structure based drug design. In this work PCNA from these three species have been prepared in milligram quantities for biochemical and biophysical studies. The previously unknown structure of LmPCNA has been solved in an uncomplexed form and also complexed with a dodecapeptide to a resolution of 3.0Å. A comparison of PCNA structures and their peptide complexes for the three species identifies structural differences which may be relevant in analysing thermodynamic contributions of binding. All eukaryotic PCNA molecules exist as ring shaped trimers which form around DNA. In this work the oligomeric state of LmPCNA has been determined to be hexameric both in solution and in the crystal. It has also been hypothesised that HsPCNA is hexameric however these would seem to form hexamers in which the trimeric rings associate “back-to-back” while LmPCNA trimers would seem to associate “face-to-face”. The binding affinities for these three PCNAs have been determined with a selection of peptides derived from the Hs p21 protein. This work has shown, using a selection of different techniques including Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC) and Dynamic Scanning Fluorimetry (DSF); that HsPCNA and SpPCNA have similar affinities for a 12mer peptide (Kd of ~1μM) however LmPCNA shows significantly weaker interactions (Kd of ~10μM). This is most likely due to divergence in the sequence and structure of LmPCNA. A systematic investigation by SPR on the effect of peptide linker length on binding has been carried out using a series of synthesised peptides with different lengths of chemical spacer. The series of streptavidin immobilised peptides show that longer spacers are required for the recovery of the PCNA peptide binding affinity. The results presented in this work indicate that a linker length of at least 20Å is required for measurable protein binding activity. This interaction is improved with longer peptide spacers.

Chapter One introduces the principle of alkoxide and phosphine oxide as ligands for lanthanides and electropositive metals, ligand self-recognition, stereoselective polymerisation of lactide, fixation of CO2 and finally copolymerisation of CO2 and epoxide. Chapter Two shows the synthesis of the proligands rac-HLR (a racemic phosphine oxide-alkoxide, A, where R = tBu, Ph or C6H3-Me-3,5) and explores the resolution into diastereomeric RRR- and SSS-M(LR)3 to afford C3–symmetric M(LR)3 complexes, B (where M = Sc, Lu, Y, In, Bi or La). It also demonstrates that the process is under thermodynamic control and driven by ligand self-recognition via the synthesis of bis(LR) adducts (LR)2MX, C, (where M = Y or In and X = N(SiMe3)2 or OC6H3-tBu2-2,6) and mono(LR) adducts (LR)MX2, D (where M = Al or In and X = N(SiMe3)2, CH2SiMe3 or Me). Finally, it outlines the fixation of CO2 into an indiumamide bond. Chapter Three contains a detailed investigation of the potential of the MIII complexes as initiators for the stereoselective polymerisation of lactide, - caprolactone, glycolide and copolymerisation of lactide and -caprolactone, lactide and glycolide and CO2 and epoxide. Chapter Four investigates the use of rac-HLtBu in the resolution into diastereomeric RR- and SS-M(LtBu)2 complexes, E (where M = Ca, Zn or Sn), and of rac-HLPh into [M(LR)2]2 complexes, F (where M = Mg, Co or Sn and R = Ph or C6H3-Me-3,5) and mono-(LtBu) adducts (LtBu)MgX, G (X = N(SiMe3)2 or OC6H3-tBu2-2,6). It also describes the synthesis of protonated MII complexes (HLR)MCl2, H (where M = Mg, Zn or Sn and R = tBu or Ph). Finally, it details the polymerisation of lactide and its copolymerisation with glycolide using MII complexes as initiators. Chapter Five gives full experimental details and analytical data for the herein described novel compounds.

Protein kinases represent one of the largest families of enzymes. Due to their crucial roles in many cellular processes, protein kinases have become one of the major drug targets for pharmaceutical companies. In order to be able to determine selective substrates, elucidate pathway functionality and design potential kinase inhibitors, it is important to determine kinase substrate specificity. A 10,000-member split and mix peptide nucleic acid (PNA)-encoded peptide library was used to establish the substrate specificity of three different protein tyrosine kinases, using a DNA microarray and a Cy3-fluorescently labelled secondary anti-phosphotyrosine antibody. This approach was proven to be a powerful tool as several peptides (“hits”) were rapidly identified as good substrates for the three kinases. Split and mix combinatorial libraries can generate large peptide libraries consisting of potentially millions of compounds, but the main disadvantage is the deconvolution process required to identify active individual peptides from mixtures. The aim of the second part of the project was to build a PNA-encoded positional scanning library of FRET-based tetrapeptides to carry out microarray-based proteolytic profiling. As proof-of-principle, a 625-member library was first synthesised on solid phase using a split and mix methodology, and successfully used to profile three different proteases. The concept was then extended to the synthesis of a larger positional scanning library containing 50,625 different tetrapeptides encoded for by just 60 PNA tags.

A new family of nucleotides has been designed, synthesised and evaluated for DNA sequencing by synthesis. Each of the nucleotide analogues had a free 3’-OH group and a fluorophore attached to the base through a small peptide linkage that was designed to stop multiple base additions. Analysis by HPLC and MALDI-TOF proved the modified nucleotides were incorporated into a growing DNA strand by a DNA polymerase and that the peptide linker was cleaved with high efficiency after incubation of the extended primer with a protease. Enzymatically cleavable nucleotides were successfully applied to chip-based sequencing by synthesis cycles. The fluorescent emission revealed the identity of the incorporated nucleotide and the removal of the fluorophore by a protease ensured the detection of the next base in the following cycle. Additionally, a practical microwave-mediated solid-phase protocol has been developed for the synthesis of cyanine dyes spanning the visible and the near infrared spectrum to allow the preparation of a series of fluorescent nucleotides for the design of a four colour DNA sequencing technology.

I. Synthesis of pyroglutamic acid derivatives via double Michael reactions of alkynones Pyroglutamic acids and their derivatives are common structural units of widespread chemical significance and they have been heavily utilised as building blocks for asymmetric synthesis. A new method for the synthesis of highly functionalised pyroglutamic acid derivatives, which consists in a Double Michael addition route that utilises amidetethered carbon diacids and aromatic alkynones as substrates, is here described. The reaction proceeds with good levels of trans-diastereoselectivity, provided that substoichiometric quantities of Mg(OTf)2 or Ni(acac)2 are employed. II. Michael additions combined with Friedel-Crafts cyclisations A domino Michael/Friedel-Crafts alkyation of aryl ethers with propargyl ketones was developed as a continuation of our double Michael additions. The reaction, here described, proceeds in good yield over two steps, when the aryl ethers and alkynones are treated with substoichiometric quantities of Yb(OTf)3 and heated to 100 oC in a microwave oven.

Inspired by the motor protein kinesin, an ambitious and unprecedented mimic is proposed – a synthetic molecular motor that can walk. This thesis aims to explain the basic principles which define such walking molecules, with reference to both natural and synthetic systems. In light of these tenets, the rational design of the proposed synthetic kinesin analogue will then be expounded. The putative design envisages the use of a series of stimuli-induced binding events to cause a “walker unit” to process along a polypyridyl track in a unidirectional, hand-over-hand fashion. The chemistry behind the stepping mechanisms of both feet of the walker unit will be discussed in detail, along with a complete description of the synthesis of the track and walker unit to date. The future challenges and potential applications of the proposed system will be addressed.

This thesis describes a study of the aggregational behaviour of egg yolk lecithin (EYL), a natural lecithin, and bile salt mixtures especially with respect to an increase of the ionic strength of the solvent. Mixtures of two amphiphiles with very different spontaneous curvature as EYL lecithin and bile salt form mixed micelles and vesicles in aqueous solution. Their properties have been well-studied under physiological conditions, i.e. 150 mM electrolyte concentration and pH 7- 8, while other conditions are still hardly explored. Upon increasing ionic strength the formed structures and the transitional pathways (micelles, coexistence of micelles and vesicles, and vesicles) change the generated structures completely from those observed under physiological conditions. We quantitatively determined these structures formed in a broad range of electrolyte concentrations with various scattering techniques, x-ray, light and neutron scattering and calorimetry. With calorimetry, phase diagrams in the EYL and bile salt concentration phase plane were determined at various ionic strength ranging from physiological salt concentration to up to 1000 mM. Additionally a new electrochemical approach using functionalised electrodes, i.e. sensitive and selective to bile salt, and thus to control the bile salt concentration in solution (concentrations below the critical micellar concentration (cmc)) was attempted, since bile salt removal or injection drives the micelle-to-vesicle or the vesicle-to-micelle transition, respectively, of the mixed aggregational system of EYL/bile salt. Although this control was not achieved within the framework of this thesis, promising results show directions for future experiments.

Effective methods for the selective passage of therapeutic molecules or probes through biological barriers are essential for the study of cellular functions. Cell penetrating cationic peptoids, based on a lysine like unit, were efficiently synthesised a combination of solid phase synthesis strategies and microwave heating.  A linear 7mer peptoid was thus prepared by direct solid phase synthesis and conjugated to both a peptide hormone (α- Melanocyte Stimulating Hormone, a 13mer peptide) and to peptide nucleic acids (9mer, 12mer and 15mer prepared using Dde/Mmt protected monomers) targeting tyrosinase mRNA. These conjugates were able to efficiently penetrate different cell lines (average uptake > 90%), and even more, to penetrate human skin, with the heptapeptoid carrier being able to deliver a 12mer PNA deep inside the dermis stratus. Antisense DNA oligomer (19mer, targeting tyrosinase mRNA) conjugated to the heptapeptoid carrier by a disulfide linkage was able to down-regulate tyrosinase expression, showing the potential of the carrier in delivering DNA into cells. Delivery of macromolecules such as protein and plasmid DNA were attempted. Heptapeptoids functionalised with a tetradentate Ni2+ ligand were tested for the delivery of hexahistidine (His6) tagged GFP protein into cells, while 3 generations of a novel peptoid dendrimer were prepared and tested for the cellular delivery of DNA.

The twin questions of how and why protein molecules fold into the specific topologies which enable them to fulfill their biological function have been the subject of continuous scientific investigation since the early twentieth century. Interactions between biological macromolecules and water are obviously crucial to both folding and function but attempts to gain understanding are impeded by the size and complexity of these systems. A useful approach is to consider much simpler model systems which capture some essential element of real biological systems but are experimentally and theoretically tractable. N-methylacetamide (NMA) is a minimal model of the peptide linkage which forms the backbone of protein molecules. Its behaviour in aqueous solution therefore captures the important competition between peptide - peptide and peptide - water hydrogen bonds which arises in protein hydration. In this thesis aqueous NMA solutions are studied across the full concentration range using classical molecular dynamics simulation. This gives access to the complete spectrum of behaviour between the two important limiting cases of dilute NMA in water and, conversely, dilute water in NMA. Water is now known to be an active player in biological interactions and the simple system studied here displays significant disruption of the structure and dynamics of pure water with the addition of only a small proportion of peptide groups. At dilute NMA concentrations water molecules continue to form system-size hydrogen bonded networks. Water molecules appear to optimise their local tetrahedral order by forming hydrogen bonds with a combination of NMA and water neighbours, rather than solely with members of their own species. NMA molecules hydrogen bond through the amide and carbonyl groups to form linear and branched chains in both the pure liquid and in the aqueous solutions. In the NMA rich region water molecules preferentially donate both hydrogens to chain-end or midchain carbonyl oxygens, forming bridges between NMA chains which resemble buried water configurations found in protein cavities. These bridge structures are thought to contribute to the observed slowing of the system dynamics at these concentrations. The investigation of dynamics by classical simulation is complemented by a quasielastic neutron scattering study of NMA in its liquid and aqueous phases.

A scalable synthesis of core-1 disaccharide (β-galactose(1-3)α-N-acetylgalactosamine), O-linked to threonine was accomplished. This building block was then incorporated into a peptide using solid phase peptide synthesis. Additionally, a novel neo-glycopeptide linkage was created using the Cu(l)-catalysed modification of Huisgen cycloaddition (click chemistry) between glycosyl azides and peptides modified with acetylenes moieties. This linkage can be formed either on solid support or in aqueous solution. Moreover, we investigated the synthesis on solid phase of a tripeptide scaffold (Ac-L-Cys-L-Lys-L-Cys-NH2) that displays the N-acetyllactosamine through the thiol groups of the two cysteine residues as a potential 'oligosaccaride mimic'. This structure can be linked to a peptide via the glycosyl azide of N-acetylglucosamine appended to the amino group of the central lysine residue. Therefore, two such oligosaccharide mimics were incorporated into an erythropoietin (EPO) derived peptide fragment of 69% yield using our novel neo-glycopeptide linkage in a buffered solution. To demonstrate the tripeptide scaffold displays carbohydrates correctly, and be recognised in a biological environment, the EPO peptide fragment bearing two oligosaccharide mimics was joined to a Green Fluorescent Protein C-terminal thioester using native chemical ligation. Afterwards, the protein-carbohydrate interaction was observed between this glycoprotein mimetic and lectin Ricinnus communis RCA120 immobilised on agarose beads.

Homogeneous, structurally defined glycoproteins can be assembled by coupling synthetic glycopeptides to synthetic or bacterially-derived protein fragments using the native chemical ligation (NCL) reaction. A limitation of NCL is the requirement for an Nterminal cysteine residue in one of the peptide fragments. One method for cysteine-free peptide ligation utilizes thiol acyl transfer auxiliaries which effect ligation and can then be removed under mild conditions. We developed new, rapid routes to 1-phenyl-2-mercaptoethyl and 2-mercaptobenzyl auxiliaries. The key steps involved: 1) introduction of a suitably protected thiol to auxiliary precursors; 2) direct reductive amination of the auxiliary aldehyde or ketone to afford the auxiliary-amine, which can be conjugated to a peptide via the “sub-monomer” approach, or the auxiliary-amino acid “cassette” for use in conventional solid phase peptide synthesis. Overall yields are 53-83 %. A glycopeptide was then assembled via auxiliary-mediated ligation at a Gly-Gly junction, which was complete within 48 hours. Thioester- and auxiliary-peptides were assembled to investigate the scope and limitations of auxiliary-mediated ligation for non Gly-Gly junctions. The 1-phenyl-2-mercaptoethyl auxiliary effected ligation at Ala-Gly, Lys-Gly and Gly-Ala junctions in 24-70 % yield, whereas the 2-mercaptobenzyl auxiliary effected ligation at a Gly-Ala junction in 42 % yield. Excess thiol was found to inhibit ligation, indicating a change in rate-determining step relative to cysteine ligation.

The problems caused by Phytophthora medecaginis; together with the isolation, biological activity and biosynthesis of zwittermicin A (which shows potential as an inhibitor of this disease) are reviewed. Studies towards the synthesis of the aminopolyol antibiotic zwittermicin A I will be described. (Fig. 6438) Chapter 2 outlines the approaches investigated towards the synthesis of the nitrogen-rich fragment II which include: enzymatic separation of racemic 2,3-diaminopropionic acid using a D-amino acid oxidase; and synthetic routes based on a Hofmann rearrangement of N-protected asparagine. Chapter 3 describes studies towards the synthesis of aminopolyol species IV from threonine-derived ketone V and the serine-derived aldehyde VI. Methodology for the “matched” lithium-mediated aldol reaction between these two components was developed, together with directed reductions of the resultant aldol adducts to give either syn or anti diol relationships. The future application of this methodology to the total synthesis of zwittermicin A is discussed in Chapter 4. Chapter 5 presents a preliminary study of high pressure conditions for enantioselective organocatalytic aldol reactions.

Structure of bicyclic pseudo octapeptides containing a sequence arginine-glycine-aspartic acid (RGD peptides) was investigated by high-resolution NMR spectroscopy. The compounds, synthesized by General Electric Healthcare, are used as contract agents in imaging of cancer. Structures were generated using different protocols in vacuum or explicit solvent. The best results were obtained through the analysis of a series of NOESY spectra acquired using varying mixing times. Full relaxation matrix analysis in explicit solvent was performed on these data yielding a consistent set of structure. The polyethylene glycol moiety and the metal binding site did not show any signs of tertiary structure. The distance restraints for structure calculations were supplemented by dihedral restraints provided by the analysis of vicinal coupling constants. Towards this end, an extensive set of coupling constants related to dihedral angles of amino acids was obtained. Various experiments, including HMBC, HSQC and HETLOC, were evaluated yielding a basic set of experiments suitable for the measurement of coupling constants of peptides with natural abundance of isotopes. The structures of the studied peptides were found to be similar and to adopting a reverse γ-turn centered on the aspartic acid of the RGD motif. Despite the bicycling nature of these peptides the compounds showed signs of flexibility, which was more pronounced in H2O. Side chains of the compounds are flexible as implied from the analysis of the distributions of cl angles. Structures were determined in two solvents, H2O and DMSO. It was found that the water based structures are more extended, while the DMSO based structure are more compact. DMSO structure of one peptide was compared with a published NMR structure of a similar peptide and a good agreement was found for the common RGD loop. The water based structures are similar to a published X-ray structure of a RGD peptide in complex with αvβ3.

Over the last three decades high-pressure X-ray diffraction techniques have been widely utilised to perform structural studies in many areas of research. For example, physicists make use of these experimental techniques to investigate metals, conductor and semi-conductor compounds among others, whereas geochemists apply them to study the conditions deep within the Earth’s interior. Furthermore, pressure studies have reached an important status in chemistry, biology and planetary science, and have proved to be a new notable tool to study the structure of a variety of small molecule compounds, from inorganic (e.g. rock salt) to organic (e.g. urea) and biological molecules (e.g. amino acids). The main reason for this is the necessity to obtain a better understanding of different processes which take place at extreme conditions of pressure, such as the existence of life in the deep ocean (e.g. extremophiles) or the finding of amino acids, such as cysteine, in space. Small molecules, such as glycine and glutathione, may play important roles in these biological processes and therefore a good knowledge of their structural features could be essential to explain how they happen. The work described here focuses on one of the principal features of intermolecular bonding in small organic and biological molecules, namely the hydrogen bond. Although the hydrogen bond has been studied for almost a century, this important directional intermolecular interaction is still one of the most highly investigated topics in chemistry and biology due to the many aspects of its nature which are still unknown or not well understood. The hydrogen bond is directly involved with the crucial biochemical processes of amino acids, peptides, proteins and even DNA and RNA and therefore, in short, it is essential for life. Its importance goes beyond biomolecules and organic molecules, since the hydrogen bond is also present in inorganic compounds, such as clusters containing different types of ligands and metals, e.g. manganese and iron, and consequently it is implicated in many catalytic pathways. In this work, we have combined experimental and computational techniques to investigate the effect of pressure on the crystal structures of amino acids and small organic molecules, and in particular on the crystal packing of cyclopropylamine, α-glycine, L-α-aspartic acid and L-α-glutamine. The experimental study of these structures was aimed to determine the principal structural changes as a function of pressure. The computational study was carried out to investigate the energetics of the crystal structures: i.e., the sublimation, lattice and proton transfer energies, as well as the energies of the individual hydrogen bonds, in order to relate the structural changes observed by experiment. A new computational method was initially tested on the simplest naturally occurring amino acid, α-glycine, which had been previously studied experimentally at high pressure by our research group. Experimental results support the existence of a new polymorph of cyclopropylamine (Phase II), which was found at 1.2 GPa. This new molecular structure crystallises in the orthorhombic space group Pbca, with 8 molecules in the unit cell and one in the asymmetric unit. The molecular packing is formed by zig-zag chains of molecules linked via a short N-H…N hydrogen bond, with unusually only one of the hydrogen atoms of the NH2 group of the cyclopropylamine molecule involved in the hydrogen bonding network. A computational study was applied to the two polymorphs of cyclopropylamine (Phase I and Phase II), in order to investigate the energetics of these systems. It was found that the energies of the hydrogen bonds present in the Phase I and Phase II crystal structures of cyclopropylamine have very similar energies, falling in the range of 1 to 4 kcal mol-1 (6 to 16 kJ mol-1). Experimental results on amino acids showed that the effect of pressure (from 0 to approximately 6 GPa) did not cause significant changes in the crystal structures of L-α-aspartic acid and L-α-glutamine, other than a decrease in the lengths of the various hydrogen bonds and intermolecular contacts. Additionally, ab initio calculations carried out on α-glycine, L-α-aspartic acid and L-α-glutamine indicated that the hydrogen bonds present in the crystal structure of amino acids exhibit very different energies, falling over a much wider range of 1 to 30 kcal mol-1 (∼4 to 120 kJ mol-1), compared to that found for small organic molecules. This finding does not agree with what was expected according to previous hydrogen bonding classifications, and we attribute this to the zwitterionic nature of the amino acid molecules which is present in the solid state. Finally, it was found that the energies of the hydrogen bonds found in the amino acids investigated correlates with the experimental compressibility studies, so that the hydrogen bond exhibiting the lowest energy is also the weakest, and most easily compressed.

Chemical glycopeptide synthesis requires access to gram quantities of glyco-amino acid building blocks. An efficient and fast synthetic route has been developed for such building blocks with high yields using microwave-assisted Kochetkov amination as a key reaction. The resulting glyco-amino acid building blocks were successfully incorporated into target glycopeptides by Fmoc-solid phase peptide synthesis (SPPS) and were characterized by LC-MS and HRMS(FAB). The glycopeptides were used to investigate the effect of glycosylation on enzymatic cleavage of peptide bonds. At first, enzyme-cleavable linkers for solid-phase peptide and glycopeptide synthesis were developed. Commonly used hydroxymethyphenoxy linker (Wang Linker) in solid phase peptide synthesis was surprisingly susceptible to efficient cleavage to serine protease chymotrypsin. In order to investigate this observation further a broad range of amino acid residues were coupled to PEGA1900 resin using the Wang linker and subsequent treatment with chymotrypsin gave cleavage of the amino acids of the resin in quantitative yields. A similar strategy was also applied to a glyco-tripeptide and again chymotrypsin hydrolysis gave glycopeptide in quantitative yield. It was also established that not only the acid-labile linker (HMPA) but also base labile (HMBA) and unreactive ester (HOA) linkers could be cleaved using chymotrypsin. The solid supported peptide and glycopeptides were then used to study the effect of glycosylation on proteolytic hydrolysis of peptides with two different proteases such as chymotrypsin and thermolysin. Our hydrolysis results in combination with those of others show that protection from hydrolysis is highly dependent on the individual protease and also on the position of the glycosylation site in respect to the scissile bond.

We have characterised two biologically important protein complexes. Firstly, we have captured and analysed a complex of the biotin protein ligase (BPL) and the biotinylation domain (BCCPΔ67) of acetyl-CoA carboxylase (ACC) from the hyperthermophile Aquifex aeolicus. The genes encoding both BPL and BCCPΔ67 were overexpressed in E. coli and purified to homogeneity. Isolation of milligram amounts of both recombinant proteins allowed us to perform kinetic analysis of the BPL enzyme using steady-state techniques. Furthermore, a chemically crosslinked complex of BPL and BCCPΔ67 was isolated and a comprehensive mutational study has identified a salt bridge between the two proteins which is important for heterodimerisation. The role of a conserved ‘thumb-like’ motif in BCCPΔ67 was also investigated by mutagenesis. Our results suggest an interaction between the biotin moiety and the ‘thumb’ reduces the propensity of BPL and holo-BCCPΔ67 to heterodimerise. In a separate project we have investigate the relationship between the tertiary structure and biological activity of a novel β-defensin related peptide (Defrl). Defensins are cationic antimicrobial peptides which have a characteristic six cysteine motif and are important components of the innate immune system Defr1 is a polymorphism of mouse β-defensin 8 and contains only 5 cysteines. Against a panel of pathogens, we found that oxidized synthetic Defr1 had significantly higher activity than its reduced form, and the oxidized and reduced forms of its six-cysteine containing analogue (Defrl Y5C). Using non-denaturing gel electrophoresis and high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) we observed Defrl and Defrl Y5C dimers.

The electropolymerisation of a range of 5-substituted indoles has been shown previously to result in the formation of redox-active films. The oxidation potentials of both the indole monomers and their polymers can be altered by substitution of the monomer with different electron-withdrawing groups. Both the indole monomers and polymers also have high fluorescence quantum yields. However the fluorescence wavelengths of the indole polymers are not tuneable by substitution. In this thesis the work on the electropolymerisation of indole(s) is extended to two monomers which have larger conjugated systems than the indoles; indolo{3,2,1-jk}carbazole (IC) and pyrrolo{3,2,1-jk} carbazole (PC). A combination of computational, electrochemical and spectroscopic techniques have been employed to investigate both these monomers and their polymers. Computational confidence tests have been performed on a range of small organic molecules to assess methods of calculating oxidation potentials, radical cation spin density distributions and excited states using the software Gaussian 98. Calculations have predicted the oxidation potentials of such molecules to within 0.19 V of their experimental values. These calculated oxidation potentials are systematically underestimated. Gaussian 98 has been used to calculate the radical cation spin density distribution of indole and has been found to reproduce the results of previous calculations performed using the software DMol3.  Configuration interaction singles (CIS) calculations incorrectly predicted the energetic ordering of the two lowest singlet excited states of indole and carbazole. However CIS correctly predicted the dipole moment of the ILa state to be larger than the dipole moment of the ILb state of indole. The oxidation potentials of IC and PC have been measured experimentally and calculated. IC and PC have both been electropolymerised to form redox-active polymers. IC forms a conducting film which has been shown to contain two different dimers by mass spectrometry and NMR. Although successful in predicting the ring positions at which indole(s) link during electropolymerisation, radical cation spin density calculations have been shown to be unsuccessful in predicting the linkage sites for IC. The mechanism of electropolymerisation of IC has been shown to be similar to the mechanism of electropolymerisation of indole(s) with initial film formation occurring in solution, followed by linkage of IC radical cations which are adsorbed on the electrode surface. The diffusion coefficient has been measured from Koutecky-Levich plots as 5.9 x 10-5 cm2 s-1 (±2.0 x 10-3). The rate of film formation for IC has been shown to be approximately 0.8 times that of indole-5-carboxylic acid and 0.7 times that of 5-cyanoindole. Steady-state fluorescence spectroscopy in solution at room temperature has shown both the IC and PC monomers and their polymers to be highly fluorescent. There is no significant difference between the equilibrium geometries and potential energy surfaces of the IC monomer in the ground and first singlet excited states. However such a difference does not exist between the ground and first singlet excited states of the PC monomer. The electropolymerisation of each monomer produces a number of emitting species which have more extensive p-electron delocalisation than the monomers. The emission from the IC dimer peaks at 408 nm. IC electropolymerisation has been shown to proceed in a similar manner to indole electropolymerisation, with the first step the formation of dimer followed by the linkage of dimer units to form longer polymer species. The IC polymer has been found to be fluorescent in the solid state.

Serine and cysteine proteases are potently inhibited by peptide aldehydes. Compared with the plethora of methods available for solution synthesis of peptide aldehydes, there are relatively few methods for the solid phase synthesis of peptide aldehydes. The development of novel linkers for the solid phase synthesis of peptide aldehydes is reported (Scheme 1). When X = N, the aldehyde 4 can be cleaved in mild acid with 97% enantiomeric excess (e.e.). When X = O, base hydrolysis results in aldehyde 4 cleavage with complete epimerisation of the α-stereocentre. The use of Mucor meihei lipase and penicillin acylase generates the aldehyde 4 in 19 and 31% yield respectively, with complete retention of chiral integrity (>99%). The utility of linker 1 was demonstrated by the solid phase synthesis of a tetrapeptide aldehyde.

Two approaches for the preparation of some novel functionalised polymers were investigated using a combination of nitrile oxide chemistry and Ring opening Metathesis Polymerisation (ROMP). In the first case, homopolymers of norbornene (NBE) and norbornadiene (NBD) were synthesised and then modified by 1,3-dipolar cycloaddition to nitrile oxides (RCNO) to the poly(di)enes. These reactions were studied at varying molar ratios of the various nitrile oxides (R = Ph, CO2Et, CO2Me), which were generated in situ by the thermal dehydrochlorination of the corresponding hydroximoyl chlorides. The second method was via the ROMP of novel isoxazolino norbornenes. Thus, exo-3-phenyl-4,7-methano-3a,4,7,7a-tetrahydrobenzisoxazole and exo-3-oxa-4-aza-ethyl-tricyclo[5.2.1]dec-4-ene-5-carboxylate, were synthesised via the cycloaddition to NBD of the corresponding nitrile oxides, and were subsequently polymerised using {RuCl2(=CHPh)[P6H11)3]2] 1 and {RuCl2(=CHPh)[P(C6H11)3][IH2Mes]} 2. Extensive use has been made of monomeric alkenes as model compounds both to test the synthetic pathways and to assist in establishing the structure of products from the equivalent polymer reactions, for this purpose trans-5-decene and cyclopentene were used. The Isoxazolino norbornenes were polymerised with control of molecular weight (Mw and Mn) by reacting exo-3-oxa-4-aza-ethyl-tricyclo[5.2.1]dec-4-ene-5-carboxylic with varying ratios of hex-1-ene as a chain transfer agent (CTA). Thus, [CTA]/[Monomer] of 0.00, 0.5, 0.10, 0.15 and 0.25 yielded oligomers with an average degree of polymerisation av DP, of 554, 157, 16, 8 and 4 (determined by GPC and NMR end group analysis). A sample of an isoxazolino functionalised oligomer (n = 16) was converted into its hydrogenated analogue (83%) by treatment with p-toluenesulfonhydrazide. Having established that isoxazolino-norbornenes were suitable monomers for ROMP, the feasibility of using the analogues isoxazolino compounds was examined. exo-4-Methyl-5exo-phenyl-3-oxa-4-aza-tricyclo[5.2.1.02,6oxo]dec-8-ene (70%), was prepared by the cycloaddition of N-methyl-C-phenyl nitrone to NBD. The analogous exo, endo cycloadduct from the cycloaddition of C,N-diphenylnitrone to NBD was also synthesised (32%) and the isoxazolidines polymerised using the initiators 1 and 2. The polymerisation of the N-methyl-C-phenyl and the C-N-diphenyl isoxazolidines with 1 yielded polymers with an av DP of 251 and 234, whereas with 2 the chain length increased to n = 490 and 683 respectively. An increase in polydispersity index (PDI) of 1.84 to 2.37 and 1.54 to 2.09 for was observed on going from 1 to 2.

Enantiomerically pure β-branched-α-amino acids are important synthetic targets and a challenging group of compounds to prepare. A series of diastereoisomers of β-methylphenylalanine analogues have been prepared in enantiomerically pure form using a combination of chemo-and bio-catalysis. Starting from L-threonine methyl ester and proceeding via the vinyl halide with Suzuki chemistry, a range of β,β-disubstituted didehydroamino acids were obtained as their (Z)-isomers. Asymmetric hydrogenation of these alkenes, using either the [Rh(R,R)-Et-DuPhos(COD)BF4 or [Rh(S,S)-Et-DuPhos(COD)]BF4 catalyst, followed by hydrolysis, yielded two of the four possible sets of diastereoisomers of the β-branched amino acid. Subsequent stereoinversion, using a stereoselective amino acid oxidase in combination with a non-selective reducing agent, furnished the remaining two sets of diastereomers.

It is possible for some chemicals to achieve an effect within a system other than that of the intended/desired result for which they were employed. One such side effect can be the perturbation of an organism’s endocrine system. Chemicals capable of this are collectively known as “endocrine disrupting chemicals” (EDCs). The presence of these EDCs in the environment has the potential to cause adverse effects in wildlife and humans. This investigation focuses on two EDCs, nonylphenol (NP) and nonylphenol polyethoxylates (NPEOx), and their occurrence in various environmental matrices. NPEOx is a surfactant used in a variety of applications including detergents and pesticides. NP is used in the production of certain types of plastic and is also found during the biodegradation of NPEOx. Methods for the extraction (e.g. liquid/liquid, solid phase, Soxhtherm and Accelerated Solvent Extraction (ASE)) and analysis (i.e. GC-MS and LC-MS) of these chemicals from waste water treatment works (WWTW) waters, industrial effluents, river waters, sludges and sediments were developed and applied. A study of six WWTW was conducted to investigate concentrations of NP (1.1-29.3 mg/l) and NPEOx (1.6-28.4 mg/l) in effluents within each works. It was found that several, but not all, WWTW effluents contained detectable concentrations. An investigation of the bottom sediments of two contrasting freshwater lochs, Tay and Leven, showed that neither NP nor NPEOx was detectable in bottom sediments from Loch Tay, whereas Loch Leven bottom sediments revealed detectable concentrations of NP (8-631 mg/kg) and NPEOx (87-925 mg/kg). Other matrices, including single-amended soils (239-586 mg/kg NP and 123-343 mg/kg NPEOx), WWTW sludges (86-3843 mg/kg NP and <0.45-<10.8 mg/kg NPEOx ), and river waters (1.1-5.4 mg/l NP and 0.6-1.6 mg/l NPEOx, also had detectable concentrations in some of the samples. Improvement of NP and NPEOx removal processes by the water industry, during water purification, is needed as contamination by waste water effluents introduces NP into the fresh water environment at concentrations above the recommended safe level of 1.0 mg/l (Environmental Agency for England and Wales). No recommended limits have been set for NPEOx  at this juncture.

Two unique features of FT-ICR mass spectrometry are high mass resolution and mass accuracy. These features have been exploited in a novel approach to the study of metal exchange reactions in metallo-proteins using stable isotope labelling. The system chosen, Zn4-SmtA, is a cyanobacterial metallothionein (MT) Deconvoluted ESI-FT-ICR spectra for Zn4-FT-ICR spectra for Zn4-Smt containing Zn isotopes in natural abundance as well as enriched with 93% 67Zn show that the Zn4 cluster of SmtA, in contrast to the structurally-analogous cluster of mammalian MT, contains a kinetically-inert Zn site, a feature which can be related to its secondary and tertiary structure, and which is of potential importance to its biological function. The main body of this work is a systematic study of the dissociation, via ECD, of doubly protonated peptides, such as luteinising hormone releasing hormone (LHRH), bradykinin and bombesin. The most common fragments reported in the literature occur from cleavage of the N-C12 backbone bond, yielding c’ (N-terminal) and z’ (C-terminal) fragments. One striking observation in the spectra of the peptides studied is the presence of prominent radical c fragments (labelled herein c’) and even-electron z fragments (labelled z’). A study of the correlation between the relative abundance of each of the fragment ions as a function of the amino acid sequence for each of the peptides is shown. These experimental studies have been complemented molecular mechanics modelling, using the Amber force field, to correlate with putative gas-phase structures for these doubly protonated peptides. Here, a hypothesis is put forward that the relative ECD fragmentation pattern is related to the gas-phase structure of the peptide ion prior to electron capture, and in particular that hydrogen bonding of the protonated site to the backbone enhances backbone cleavage at that site (referred to as ‘ECD/structure correlation’). The relative ECD fragmentation pattern for bombesin and four LHRH variants are compared to their modelled gas-phase structures. A detailed study of the LHRH variants with sequence pEHWSYGLRPG-OH {1} and pEHWSYGLRPG-NH2 {2}, and bombesin {3}, showed good agreement between the relative ECD fragment abundances and predicted Amber structures for {1} and {3}, but not for {2}. This may imply that the postulated model is not a full description of the ECD process.

The chemical synthesis of the 184aa anti-angiogenic peptide, endostatin, was undertaken. Preparation via the stepwise of two large fragments approximately 90aa in length was attempted but was unsuccessful. A method that would allow the efficient, sequential coupling of several small fragments was required. To this end, the segment coupling of minimally protected fragments via transfer active ester condensation (TAEC), a technique recently developed within this research group, was investigated. The fragments for synthesis were provisionally selected based on hydrophobicity and potential coupling sites. These peptide fragments were then optimised for stepwise solid phase peptide synthesis (SPPS), using the Fmoc strategy. Peptides containing two types of C-terminal functionality - hydrazides and semicarbazides - were prepared. An alternative strategy for the synthesis of the Wang resin-based hydrazide linker, first proposed by Wang and Merrifield in 1969, was developed to facilitate this process. Peptide fragments of up to 20aa in length were successfully coupled using TAEC. A novel approach to the protection of arginine side chains was also investigated. This target was based on the dibenzocycloheptenyl system, a species which was originally deigned for use as a linker in the preparation of peptide amides. Recent work by Noda involved a derivative of this linker, which has proven to be significantly more acid labile than current arginine protection. Concurrent work on an improved design led to an alternative system - the dimethoxy-suberyl compound - being proposed. The synthesis of the target molecule was achieved in seven steps; key steps involved a Perkin reaction and an intramolecular Friedel Krafts acylation. Coupling to arginine was undertaken but proved unsuccessful.

A number of groups have been investigated as potential protecting groups for the thiol functionality of cysteine residues during Solid Phase Peptide Synthesis. Of these the 4-picolyl system has been found to be successful. A short, efficient synthesis to the protected amino acid and mild cleavage conditions have been developed. This protected amino acid has been applied in the synthesis of a number of interesting cysteine-containing peptide targets. Deglycosylated human erythropoietin (dhEPO) has been synthesised by SPPS, employing the picolyl group for cysteine protection. Purification of this material has been achieved by gel filtration. The synthetic material has been characterised by amino acid analysis, SDS-PAGE, iso-electric focusing and N-terminal sequencing. Attempts have been made to synthesise deglycosylated human erythropoietin via a fragment coupling technique. All fragments were successfully synthesised and purified and model ligation studies carried out.

Flavocytochrome c3 is a fumarate reductase expressed by the marine bacterium Shewanella frigidimarina during anaerobic growth in the presence of fumarate. This soluble periplasmic enzyme is composed of three domains; a flavin domain containing non-covalently bound FAD, a cytochrome domain and a clamp domain. The active site is located in the flavin domain at the interface with the clamp domain. The enzyme catalyses the reduction of fumarate to succinate with a kcat of 509 ± 15 s-1 and Km of 25 ± 2mM at pH 7.2, 25°C. Reduction of fumarate requires hydride transfer from the FAD and protonation by an active site acid. Residues implicated in catalysis have been studied using site directed mutagenesis. Substitution of Arg402 by alanine leads to complete loss of activity whereas neither of the two active site histidines (His504 and His365) is essential for catalysis. The H365A:H504A double mutant enzyme was found to have a kcat of 0.84 ± 0.05 s-1 at pH 7.2. Substitution of Arg402 by lysine, histidine or tyrosine led to a fall in kcat to 0.55 s-1, 0.091 s-1 and 0.05 s-1 respectively. Substrate specificity and inhibition studies have been carried out to probe the active site structure. Fcc3 was unable to catalyse the reduction of alternative enoates to fumarate. However, oxaloacetate, methylsuccinate and 3-nitropropinoate were found to be inhibitors of succinate oxidation with Ki values of 5.3 mM, 1.7 mM, and 0.5 mM respectively.

This thesis documents the design and synthesis of chiral amino alcohol monomers 161, 162, 167, chiral oxazolidine monomer 168 and chiral oxazolidinone monomers 203 and 204. The synthetic strategy relies on the key Suzuki coupling of aryl triflates derived from tyrosine with p-vinylphenylboronic acid where the resultant vinyl monomers are further modified to provide the desired functionality. (Fig. 15997a) Model gel and macroporous-type suspension copolymerisations of styrene, divinylbenzene and vinylbenzylchloride are investigated to allow modification of the experimental conditions to produce chloromethylpolystyrene beads 3 in optimum yield, size, shape and uniformity. (Fig. 15997b) The chiral amino alcohol 167 and oxazolidinone 204 monomers are subsequently polymerised utilising the previously optimised suspension protocol. Characterisation of the resultant gel-type polymers 217 and 218 confirms that the functionalised monomers are successfully incorporated with the desired loading. This versatile approach to the synthesis of chiral moiety is polymer-bound thus affording complete control. Finally, the utility of the polymer-bound amino alcohol 217 as a catalyst in the addition of dialkylzinc reagents to aldehydes and the application of the supported auxiliary 218 in asymmetric alkylation and aldol reactions are evaluated.

RS-a-Amino acids bearing a b- or g-substituent have been prepared using standard methods for a-amino acid synthesis. The deracemisation of RS-a-amino acids, using D-amino acid oxidase (DAAO) from Trigonopsis variabilis and hydride reducing agents, has been investigated. The enzyme has been shown to tolerate the b- and g-substituted a-amino acids as substrates. The use of NaCNBH3 for the deracemisation of acyclic a-amino acids has been demonstrated for the first time. Screening of a random library of organisms was undertaken to identify novel oxidase enzymes for use in the deracemisation reaction. The screen revealed five organisms that were able to oxidase L-phenylalanine to phenylpyruvic acid, indicating possible L-amino acid oxidase activity. In the screening for monoamine oxidase the assay methods employed lacked the sensitivity to identify conversion of benzylamine to benzaldehyde. For the first time the cyclic oxidation-reduction sequence used for the deracemisation of RS-a-amino acids has been extended to encompass the deracemisation of racemic amines. The deracemisation of RS-a-methylbenzylamine, using an enantioselective monoamine oxidase and ammonia:borane as reducing agent, has been shown to proceed with moderate yields and high selectivity.

This thesis compares methods of producing protein for structural analysis by NMR spectroscopy and describes the structural characterisation of the N-terminal CP-module of membrane cofactor protein (MCP-1). CP-modules are autonomously folding domains composed of approximately sixty amino acids. They are defined by a consensus sequence including four cysteines, and have been identified in over forty different proteins. The proteins discussed in this thesis, MCP and factor H, are members of the regulators of complement activation family. Each comprises a contiguous array of CP-modules which serve as the binding sites for complement components and various other ligands. The structures of CP-modules have been determined previously by NMR and X-ray crystallography. Whilst individual modules are structurally similar, the nature of the interface between modules is highly variable. The high-resolution structure determination of MCP-1 forms part of an on-going study to determine the structure of the extracellular portion of MCP. MCP-1 is of particular interest as it comprises part of the measles virus receptor. It is anticipated that understanding the structural characteristics of this module in solution may assist in determining the nature of the interaction with the virus. Synthetic and recombinant methods have been assessed for the production of CP-modules in sufficient yield and purity for structure determination by NMR. Two CP-modules, MCP-1 and the fifteenth module of factor H (fH~15), were synthesised using standard Fmoc methodology. fH~15 was used as a model since the 3D-solution structure of this stable and soluble module has been determined by NMR. The purification and renaturation strategy utilised a number of chromatographic techniques and was monitored by mass spectrometry and NMR. However, it was not possible to purify the synthetic modules to homogeneity in sufficient yield for structural analysis.

This thesis documents the development of serine derived aldehyde 73 and its application to natural product synthesis. (Fig. 9978A) Highly efficient syntheses of the biologically important natural products DAB1 35 and nectrisine 36 from aldehyde 73 are discussed. The synthesis of these molecules made use of highly efficient asymmetric boron mediated syn aldol reaction to generate a common intermediate, lactam 114. (Fig. 9978B) A alternate synthesis of lactam 114, making use of the Sharpless asymmetric dihydroxylation reaction is also discussed, along with an extension of the aldol reaction methodology which allowed the development of syntheses directed toward the C4Me analogue of 35 and the C3Me analogue of 36. Finally, the utility of aldehyde 73 towards the synthesis of the potential hydantocidin analogues 176 and 181 and the lactacystin analogue 183 was investigated (Fig. 9978C).

TiIV compounds have pronounced anticancer, antiviral and antibacterial activities, and titanocene dichloride (TDC) is currently on phase II clinical trials as an anticancer drug. However, the biological chemistry and mechanisms of action of TiIV are poorly understood. Proteins and nucleic acids are expected to be biological targets for TiIV. Human transferrin (hTF) is a bilobal serum glycoprotein (80 kDa) which transports FeIII to cells via receptor-mediated endocytosis. A structurally similar periplasmic iron binding protein (FBP, 34 kDa) is present in some pathogenic bacteria and is required for virulence. In this thesis, the aqueous coordination chemistry of TiIV with the phenolate ligand N,N'-ethylene-bis-(o-hydroxyphenylglycine)(H4ehpg) was investigated as a model for Ti-hTF (or FBP) interactions. TiIV forms 7-coordinae monomer (rac) and dimer (meso) complexes with H4ehpg (rac + meso) with novel stereo-selectivity. 1H and 31P NMR studies show that TDC binds selectivity to H4ehpg at neutral pH, but preferentially to adenosine triphosphate (APT) at pH values below 5; TiIV transfers from TiIV-ehpg to ATP at acidic pH values. The interactions of TDC with hTF and that of Ti2-hTF with ATP have characteristics which could allow transferrin to act as a mediator for titanium delivery to tumour cells. TDC reacts rapidly with apo-hTF under extra-cellular conditions and binds in the specific FeIII sites with release of the Cp and Cl ligands. TiIV is readily released from Ti2-hTF at endosomal pH (ca 5.0) and in the presence of ATP. Ti2hTF competes effectively for cell uptake of 59Fe-hTF into BeWo cancer cells. TDC binds strongly to the phosphate group of nucleotides in aqueous solution and TiIV binds to the phosphodiester groups of nucleotides in the less polar solvent N,N-dimethylformamide. This behaviour contrasts with that of the anticancer drug cisplatin which binds mainly to N-sites of nucleobases, and may account for the intracellular localisation behaviour of TiIV drugs.

Reactions of isolated carbonyl groups in ethene/propene-carbon monoxide alternating copolymers were accomplished by acid-catalysed condensation with alkene-1,2-diols (ethane-, propane-, butane- and hexane-1,2-diols) affording alkene-dioxolane copolymers. Oximation with hydroxylamine, O- methylhydroxylamine and O-benzylhydroxylamine resulted in complete conversion of the carbonyl groups to their oxime derivatives. In addition, the conversions of polyketoximes, derived from ethene/propene-carbon monoxide copolymers, to 1-acteoxyiminotrimethylene and 1-benzoxyimino-trimethylene polymers were achieved by treatment with acetic anhydride and benzoyl chloride, respectively. Reaction of 1,4-dione units in ethene/propene-carbon monoxide alternating copolymers has been investigated. Paal-Knorr reactions with aniline, benzylamine and glycine ethyl ester yielded poly(alkenepyrrole) derivatives. The conversion of the carbonyl groups in ethene/propene-carbon monoxide alternating copolymers to methoximes allowed directed a-substitution reactions to be carried out on the polymer backbone. Treatment with n-butyllithium followed by addition of electrophiles (e.g. iodomethane, 1-iodopropane, 1-bromobutane, benzyl bromide and allyl bromide) afforded the polymer products in which 20-38% of the a-syn methylene hydrogens were substituted. The syn selectivity of the lithiation process was established b treatment of the polymethoxime with BuLi/[O-2H]methanol and confirmed by the presence of deuterium signal in the 2H-NMR spectrum corresponding to those observed for the model compounds. Beckmann rearrangement reactions of model compounds [2.5-bis(tosyloxyimino)hexane, 2,5-bis(mesyloxyimino)hexane and 2,5-bis(hydroxyimino)hexane] were investigated and resulted in rearrangement products: N,N'-ethanediyl-bis-acetamide, N-acetyl-b-alaninemonomethylamide and N,N'-dimethylsuccinamide.

There is much interest in the synthesis of unnatural, enantiomerically pure amino acids, including dehydroamino acids and b,b-disubstituted amino acids. Described here is a method of synthesising dehydroamino acids via oxazolones derived from a modified version of the Erlenmeyer reaction. (Fig. 13661A) Rh-DuPHOS and Rh-BPE hydrogenation of these dehydroamino acids led to the synthesis of single products in high d.e. and e.e. Synthesis of such oxazolones on solid phase would provide a method for on-resin amino acid modification, allowing for greater flexibility in the combinatorial synthesis of peptides. Linkage via a silyl linker would allow mild and selective cleavage, while use of an acid functionalised linker would allow inverse (N(r)C) peptide synthesis. Attempts to synthesise I are described, along with a method of oxazolone formation which avoids the use of acid, and is therefore compatible with silyl linkers (Fig. 13661B).

Flavocytochrome b2 from Saccharomyces cerevisiae is an L-lactate:cytochrome c oxidoreductase. The crystal structure of this homotetrameric enzyme has been solved to 2.4 A (Xia and Mathews, 1990), and many aspects of its function have been investigated by the construction and kinetic analysis of site-directed mutant forms. From inspection of the crystal structure, Arg289 can be seen to interact with Arg376 which is a key residue for formation of the Michaelis complex. In addition Arg289 also forms a hydrogen bond with a water molecule which is itself hydrogen-bonded to a heme propionate. Substitution of this arginine by lysine results in an enzyme with altered kinetic properties. The Arg289(r)Lys mutant flavocytochrome b2 (R289K-b2) is shown to have a decreased kcat for ferricyanide-dependent L-lactate dehydrogenation (33.0 ± 3.9 vs. 400 ± 10 s-1 for the wild-type enzyme) with an increased value of kcat for L-lactate (1.40 ± 0.20 mM vs. 0.49 ± 0.05 mM for the wild-type enzyme). With cytochrome c as electron acceptor, the kcat for L-lactate dehydrogenation by R289K-b2 is also decreased by an order of magnitude relative to wild-type enzyme. Pre-steady state kinetics confirm that the rate constant for FMN reduction by L-lactate is significantly decreased (19.6 ± 1.7 s-1 cf. 604 ± 60 s-1 in the wild-type enzyme), leading to the conclusion that substitution of Arg289 with lysine has caused FMN reduction to become the rate-determining step of the catalytic cycle. This is contrary to the situation for the wild-type enzyme, where transfer of an electron from FMN semiquinone to the heme is rate limiting (Daff et al., 1996a). It is thought that this altered behaviour may be due to structural changes at the active site which affect substrate binding and dehydrogenation. Ligand-binding properties of R289K-b2 were investigated by a series of inhibition experiments which showed significant changes from wild-type behaviour. This was particularly evident with pyruvate, the product of the reaction which displayed a different type of inhibition in R289K-b2. The crystal structure of R289K-b2 was solved to 2.75 A resolution, and this revealed changes in the position of some active site residues, in particular Arg376, a residue which is important in substrate binding.

Methodology for the synthesis of peptide C-terminal aldehydes has been investigated. Modification of a linker system, based on a terabenzosuberyl construct, has been demonstrated to be suitable for the synthesis of peptide C-terminal semicarbazones. The route has been fully optimised to yield a series of peptide C-terminal semicarbazones and the corresponding peptide aldehydes. The chemical synthesis of deglycosylated human interferon-gamma (143 residues) has been carried out. The purification of this protein has been investigated and a short purification protocol developed which is sufficiently general to allow application to other similar protein systems. Purification and characterisation of the synthetic interferon-gamma molecule has been completed and folding of the molecule attempted.

In recent years, much effort has been directed towards the development of new strategies for the efficient synthesis of libraries of peptidomimetics as potential therapeutic leads. The solid phase inverse synthesis of peptides(N(r)C) has only received a fraction of the attention given to the classical (C(r)N) solid phase assembly of peptides due to oxazolone-induced epimerisation. However, rather than trying to prevent its formation we have demonstrated how the peptide-derived 5(4H)-oxazolone can be used effectively as an ideal precursor to a range of peptidomimetics. (Fig. 9971) The development of a suitable strategy for the solid phase inverse synthesis of peptides is described and investigated as a valuable route into C-terminally modified peptides. This methodology is then applied to the construction of the polymer-supported 5(4H)-oxazolone II and various transformations thereof have been examined, often with solution phase comparisons.

A glycoprotein can exist as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties. As a result of this 'microheterogeneity' there is a need for the synthesis of homogeneously glycosylated proteins for analytical purposes. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine has been developed (Figure 10919). Through site-directed mutagenesis, whereby the natural asparagine glycosylation sites can be exchanged for cysteine, it was possible to selectively glycosylated proteins at predetermined sites. This provided a general method for the synthesis of homogeneously glycosylated proteins. We chose the glycoprotein hormone erythropoietin as a model system since the N-glycans at residues 24, 38 and 83 are essential for in vivo biological activity. Using a modified recombinant erythropoietin gene (prepared in Chapter 2) we optimized protein expression, and over-expressed and purified His₁₀-WThEPO, His₁₀-Asn24Cys, His₁₀-Asn38Cys, His₁₀-Asn83Cys and His₁₀-Asn38/83 CyshEPO's in yields of 13mgL^-1 from E. coli. This allowed us to probe the structure of rhEPO using techniques such as NMR spectroscopy and electrospray MS (Chapter 3). Having access to larger quantities of EPO, we were also able to develop on-line LC-ESI-MS methods, which allowed us to monitor protein glycosylation reactions with glycosyl β-N-iodoacetamides and map the position of the glycosylation site. The synthesis of relevant iodoacetamides was attempted and simple glycosyl iodoacetamides and ¹³C labeled iodoacetamides were prepared and successfully employed as probes for protein glycosylation.

The dynamic kinetic resolution of 2-phenyl-4-subsittuted-5(4H)-oxazolones 89a-g has been investigated as a method for the synthesis of optically active α-amino acid derivatives. The effects of lipase, [either Novozyme (Candida antarctica lipase B), or Lipozyme (Rhizomucor miehei lipase)], solvent, nucleophile, and the addition of external triethylamine to the reaction is described. When R¹ = Ph, an 88% yield and 98% enantiomeric excess (e.e.) of α-amino acid ester 96a was obtained with Novozyme^® in acetonitrile as solvent. The synthesis of novel 5(4H)-oxazolones 129a-e, which are identified as key intermediates in the synthesis of a series of matrix metalloproteinase inhibitors 94, is described. Application of the lipase catalysed dynamic kinetic resolution conditions to 129a-e, afforded high yields (96%) and diastereomeric excesses (d.e.'s), (86%) of the resulting pseudodipeptides (2R,2'S)-127a-e and 130-132, by careful selection of the reaction conditions.(Figs. 1382A and 1382B).

Flavocytochrome c3 is a respiratory fumarate reductase produced by the marine organism Shewanella frigidimarina. It is a soluble enzyme located in the periplasm. The enzyme is composed of three domains; a flavin-binding domain, a cytochrome domain and a clamp domain. The flavin domain (residues 108-364 and 503-571) contains a non-covalently bound flavin adenine dinucleotide (FAD) as the redox cofactor. The smaller cytochrome domain (residues 1-107) encapsulates four c-type haems which are organised in a dog-leg arrangement. The enzyme catalyses the conversion of fumarate to succinate with a kcat of 509 ± 15 s-1 and Km of 25 ± 2 mM at pH 7.2, 25°C, which corresponds to a catalytic efficiency of 2.1 x 107 M-1s-1. The reverse reaction, succinate oxidation, proceeds less efficiently with a kcat of 0.7 ± 0.02 s-1 and a Km of 0.8 ± 0.1 mM. This results in a catalytic efficiency of 933 M-1 s-1 at pH 8.5. The efficiency of succinate oxidation is a factor of 2 x 104 less than for fumarate reduction confirming that flavocytochrome c3 is a unidirectional fumarate reductase. Flavocytochrome c3 differs in this respect from the wider family of fumarate reductases which are freely reversible. The effect of pH on both fumarate reduction and succinate oxidation has been studied. The results yield a pKa of 7.32 ± 0.5 for fumarate reduction and 8.6 ± 0.1 for succinate oxidation, indicative of the presence of an essential histidine at the active site of the enzyme. A programme of site-directed mutagenesis was undertaken with a view to probing the catalytic mechanism. Kinetic analysis of mutated forms of flavocytochrome c3, in conjunction with the recently determined crystal structure, has allowed the proposal of a mechanism for the reaction. Substitution of the active site histidines (365 and 504) by alanine resulted in dramatic changes in the pH profile for fumarate reduction. The overall rate of reaction was lowered 100-fold in each case. Active-site arginines 402 and 381 were also studied. The substitution of arginine 402 by alanine results in a complete loss of fumarate reductase activity, leading to the assignment of this residue as the essential active site base. Arginine 381, on the other hand, is likely to be involved in a more peripheral role, possibly in stabilising the transition state structure. Aspartate 195 was also studied but the substitution of this to alanine was found to have no effect on the catalytic parameters.

A previously unknown protein produced by an mRNA mutation has recently been identified in Alzheimer's patients. The protein, designated UbN, is composed of the first 75 residues of ubiquitin followed by an unrelated sequence of 20 residues. The synthesis of this 10.7kDa entity was carried out, as well as its purification which was based on a novel affinity support in conjunction with the tetrabenzofluoroene moiety. In vitro testing established how the protein could compromise the ubiquitin-dependent proteolytic system, hence exerting a toxic effect in Alzheimer's patients. The synthesis of an analogue of ubiquitin (76 residues, 8.5kDa), which contained the unnatural amino acid 2S, 4S-5-fluoroleucine in place of leucine at the 50 and 67 positions, has been carried out. A short purification and folding protocol was developed, and comparison of the analogue with the native structure was undertaken. A possible application of the analogue in the study of protein folding using 19F nmr has been examined.

The increased application of combinatorial synthesis as a valuable tool in the drug discovery process has led to the recent escalation in research into solid phase synthesis techniques. The use of multiple parallel solid phase synthesis has been described as an efficient method of lead compound optimisation. This method is explored for the generation of analogues of a substituted acrylic acid, identified from random screening, in order to increase receptor affinity. The optimised solution phase synthesis of the lead compound via a Horner-Wadsworth-Emmons reaction is reported. The synthetic route is then described for the solid phase synthesis of a simple analogue. Attempts have been made towards the multiple parallel solid phase syntheses of a range of substrates. Alternative methods for facile product purification as alternatives to covalent binding to a polymeric resin have recently been researched. The affinity of tetrabenzo [a, c,g,i]fluorene (Tbf) for charcoal has already been described as a useful method for the purification of peptides and proteins, and this property has been harnessed, for the first time, for application to general organic synthesis. An efficient solution-solid phase synthesis of the quinolone antibacterial, Ciprofloxacin, is described, in which charcoal was used to purify Tbf-bound intermediates in a pseudo solid phase purification step.

The following work documents three studies undertaken using solid phase synthesis techniques. Interaction of the zinc metalloprotease, endothelin converting enzyme-1(ECE-1), with its peptidic natural substrate big endothelum-1 has been investigated via an SAR study, using solid phase peptide synthesis (SPPS). Truncated forms of the substrate had been previously reported to inhibit ECE-1, this was confirmed however the big ET-1 analogues were shown to be substrates for the enzyme. A short study of the substrate specificity of ECE-1 was carried out. The synthesis of vast libraries of peptides using combinational synthesis has been used to accelerate the drug discovery process. Purification of these mixtures has not been previously attempted. 17-Tetrabenzo [a,c,g,i]fluoroeneyhethoxycarbonyl (Tbfmcc) developed for use with single peptides of proteins, has been used to achieve facile purification of five peptide libraries synthesised using SP. The methodology was fully optimised for the efficient separation of the desired library members from all impurities by exploiting the affinity of TBfmoc for carbon. A potential small molecule inhibitor of the zinc metalloenzyme, farnesyl transferase (FTase), was designed. The efficient solution phase synthesis of this novel structure is reported its adaptation to solid phase synthesis is described, with the view to using multiple parallel synthesis techniques to synthesise a range of analogous.

Morphine 1 is metabolised in the liver into its active form, morphine-6-glucuronide (M6G) 2. When M6G 2 is administered directly to patients it has greater analgesic potency than morphine 1 and this stimulated interest in the synthesis of M6G 2 as a novel analgesic. We have examined several synthetic routes to the precursor morphine-6-glucoside 4 with selective oxidation to M6G 2 as the final step. (Fig. 10917A). The coupling of a glucose residue to morphine 1 was investigated using both chemical and enzymatic approaches. However, selective oxidation at the C6' position of morphine-6-glucoside 4 did not yield the desired metabolite M6G 2. The replacement of the glucose reside in scheme 1 by galactose and arabinose gave morphine-6-glucoside analogues which are potentially an alternative source of analgesics. (Fig. 10917B).