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The incorporation of polyfluorinated regions into carbohydrates has been proposed as a strategy to improve the typically low protein–carbohydrate affinity. The creation of a hydrophobic region, while maintaining a potential for attractive dipolar interactions (mediated by the polarised C–F bond) may increase binding affinity. This thesis describes the synthesis of previously reported 2,3-dideoxy-2,2,3,3-tetrafluorogalactopyranose and 2,3-dideoxytetrafluoroglucopyranose. Further optimisations have been carried out to provide highly efficient and enantioselective routes to both compounds. The synthesis of the novel 2,3-dideoxy-2,2,3,3-tetrafluorogalactofuranose via a modification to the pyranose route has been successfully achieved and optimised. The large scale synthesis of these compounds has enabled their further derivatisation. This has included the development of phosphorylation methodology for 2,2,3,3-tetrafluorogalactose in both pyranose and furanose isomers. The synthesis of the first disaccharides to contain a tetrafluoroethylene moiety has been accomplished, providing tetrafluorinated analogues of known bioactive disaccharides. Studies have also been carried out towards different glycosylation methodologies of 2,2,3,3-tetrafluorinated sugars, enabling access to glycosylation by both electrophilic and nucleophilic reagents. Detailed conformational analysis has been implemented, with the results displaying high similarities between 2,2,3,3-tetrafluorogalactopyranosides and natural non-fluorinated carbohydrates.

The introduction of electron capture dissociation mass spectrometry in 1998 has provided a unique technique for the analysis of peptides and proteins, especially for the identification and localisation of posttranslational modifications. Despite many successes debate continues on the radical-based mechanism of ECD. This thesis explores ECD behaviour in a wide range of PTMs with the intention of furthering our knowledge of the radical-based mechanism. Studies were undertaken on the effect of 3-nitrotyrosine, which is an electron withdrawing group, on ECD. The presence of nitration dramatically decreases peptide backbone sequence coverage but results in the presence of abundant small neutral losses. The key finding is the insight provided into the hierarchy of the various proposed ECD mechanisms. ECD of cysteine bound modifications is shown to result in the fragmentation of the sulfur-modification bond and backbone sequence coverage is highly diminished when analysing S-nitrosopeptides. ECD behaviour of hydrogen-deficient radical peptides is highly dependent on gas-phase peptide structure, with electron capture typically resulting in an increase in charge-reduced precursor intensity. Comparisons of the intermolecular phospho-guanidinium bond strengths between phospho-serine, -threonine and -tyrosine were undertaken. ECD of these complexes results in the retention of the noncovalent bond allowing backbone sequence coverage.

The first part of this thesis describes the synthesis, solution studies and biological evaluation of 2-keto-3-deoxy-ulosonic acids. A synthetic route was developed for 2-keto- 3-deoxy-gluconate (o-KDG) and 2-keto-3-deoxy-galactonate (D-KDGal) that provided the targets via concise four step routes from naturally occurring sugar substrates. These routes make use of Horner-Wadsworth-Emmons reactions between the anion of ethyl 2- ((terl-butyldimethylsi lyl)oxy)-2-(dimethoxy-phosphoryl) acetate 1 with enantiopure sugarderived aldehydes to afford silyl-enol et hers that cou d be globally deprotected to give the target 2-keto-3-deoxy-ulosonic acids in high purity (Scheme I). The effect of temperature on the isomeric composition of these C6-sugars was studied and they were then supplied as substrates for the directed evolution of a stereochemically promiscuous aldolase from Sulfolobus solfataricus, to develop mutant aldolases with high diastereoselectivity for the aldol reaction of D-glyceraldehyde 6 and pyruvate 7 to exclusively afford either D-KDG or D-KDGal. The synthetic methodology was then applied to the synthesis of enantiopure 2-keto-3- deoxy-D-xylonale (D-KDX) and 2-keto-3-deoxY-l-arabonate (l -KDA) and these C5-sugars were fully characterised by 1H and DC NMR spectroscopy for the first time and had their enantiopurity confirmed by optical rotatory dispersion analysis. The kinetic parameters for D-KDX and L-KDA using KOG aldolase were determined using a modified thiobarbituric aCid assay, with good catalytic efficiencies being found for each. enantiomer (0.45 and 0.53 s•1mM•1). This gives a more complete understanding of the metabolism of S. solfataricus confirming that the archaeon uses the same KDG aldolase for the catabolism of not only the diastereotopic C6-sugars D-KDG and D-KDGal but also for the ' enantiomeric CS-sugars D-KDX and L-KDA.

Iminosugars are polyhydroxylated alkaloids, and can be generally defined as sugar mimetics in which the endocyclic oxygen atom has been replaced with a basic nitrogen. A common affect of this atomic substitution is to bestow these compounds with the ability to inhibit various sugarprocessing enzymes; most significantly the glycosidases (glycoside hydrolases) which areintimately involved in a huge array of biological functions. Compounds which inhibit these enzymes concordantly possess much potential as medicinal agents for the treatment of a variety of diseases. Several iminosugars have already achieved market approval as drugs, and many more are promising candidates in the late stages of clinical development. As such there remains considerable interest in this class of compound, both in terms of the exploration of novel iminosugar structures, as well as the continual development of more efficient general methodology for their synthesis. The densely-packed functionality and stereochemical information present in iminosugars makes them challenging targets for asymmetric chemical synthesis, whereas carbohydrates are clearly very attractive as chiral-pool starting materials for this purpose. Indeed, the majority of the most successful syntheses of iminosugars use the latter approach, and such is the focus of this thesis. Chapter 1 presents a relatively brief introduction to iminosugars, including their types of structure, natural occurrence and biological mode of action. The rationale behind their use as therapeutic agents for the treatment of some significant disease targets is also discussed. Chapter 2 is concerned with the preparation of a number of novel polyhydroxylated azetidines, and their evaluation as glycosidase inhibitors. Such compounds represent an almost entirely neglected class of iminosugars within the literature. An overview of natural and synthetic products incorporating an azetidine motif is given, as well as a brief review of preparative methods and known azetidine iminosugars. A highly efficient and flexible method for the key azetidine ring formation is demonstrated by the cyclisations of 3,5-di-O-triflates of pentoses and hexoses, and of a 2,4-di-O-triflate of glucose, with various primary amines. In this manner, many azetidine triols and tetrols were prepared in good yield. Furthermore, this process is readily adaptable to the installation of added functionality to the azetidine scaffold, as demonstrated by the preparation of 1-acetamido analogues. The initial biological screening of these compounds showed a promising array of glycosidase inhibition, including that of selective inhibition of fungal enzymes. Chapter 3 describes a strategy with which to prepare all sixteen stereoisomers of a known piperidine iminosugar, alpha-homonojirimycin (alpha-HNJ), in a highly divergent manner from just four of the possible thirty-two 6-azidoheptitols using traditional chemical synthesis in tandem with biotechnological transformations. One half of the execution of this strategy is described in this thesis. Two 6-azidoheptitols were prepared from D-mannose, thereby providing access to four 6-azidoketoheptoses through a combination of microbial oxidation and enzymatic epimerisation. Catalytic hydrogenation of these 6-azidoketoheptoses furnished four diastereomeric mixtures of 2,6-iminoheptitols, with varying degrees of stereoselectivity. Purification of these mixtures allowed six 2,6-iminoheptitols to be isolated, two of which have never previously been tested for glycosidase inhibition. Significantly, one of them was found to be a potent and highly selectiveinhibitor of alpha-galactosidases, and may therefore be of interest in the treatment of Fabry disease.

Carbohydrate-carbohydrate interactions (CCIs) have been shown to be an important interaction in molecular recognition. These interactions present characteristics such as the synergistic effect with other interactions e.g. protein-protein interactions, specificity, polyvalency and in some cases requirement of divalent cations. CCIs remain insufficiently documented due to the weakness of such an interaction, which is difficult to probe by classical techniques and study at the molecular level in a monovalent system has not been performed. In order to study the CCI, a peptide based on alanine and lysine has been designed. Carbohydrates have been ligated to this peptide and changes in a-helix and random coil conformations are examined using CD spectroscopy. This system was shown to function as a reporter for CCls through changes in the conformations of the peptide. A second system that will be employed to study eCls is utilising the thiol-thioester exchange reaction. This involves a reaction between a carbohydrate with a thioester linkage and a carbohydrate linked to a thiol moiety. The resulting equilibrium is to be probed using HPLC. Synthetic routes have been developed in order to obtain the desired thiols and thioester. First studies showed that a CHO-π interaction can be quantified using this system. The carbohydrates to be attached are: Lex, sLex, LeY. The synthesis of Lex has been improved within the Gallagher group. A new synthesis of LeY was performed using an armed/disarmed strategy. Meanwhile, studies towards the synthesis of sLex were carried out.

In this thesis four projects related to bioactive carbohydrates are described. The first project is about the extraction of iminosugars from Hyacinthoides non-scripta. This is the first time that extraction from English bluebell seeds has been described. Efficient extraction and isolation methods are reported. Another project discusses the development of a total synthetic carbohydrate conjugate vaccine candidate against Streptococcus pneumoniae type 14 using Gold nanoparticles as carrier. The synthetic pathway of the introduction of a linker for conjugation, and the deprotection of the tetrasaccharide corresponding to the repeating unit of the Streptococcus pneumoniae type 14 capsular polysaccharide is described. The biological results of the developed vaccine candidate are briefly discussed. In the third project, attempts to synthesise regioselectively sulfated disaccharides to be used in binding studies with FedF adhesin of E. coli are described. In this section, an improved high-yielding method based on the Heyns rearrangement for the synthesis of N-acetyl lactosamine (LacNAc) is also reported. In the fourth part, conjugation of the Lewis b hexasaccharide to be used for studies of Lewis b blood group antigen binding adhesin is reported.

In this project, the structures of exopolysaccharides (EPS) produced by bacterial strains were characterised. The current techniques utilised for structural elucidation were also investigated. The structure of the novel EPS isolated from the fermentation of the lactic acid bacteria(LAB) strain, Lactobacillus helveticus Rosyjski, has been characterised. The strain of LAB was grown on skimmed milk supplemented with glucose; the subsequent EPS produced was isolated using established protocols. The 1H NMR spectrum identified the presence of five anomeric monosaccharide signals corresponding to the existence of a pentasaccharide repeating unit oligosaccharide. HP-SEC-MALLS analysis revealed the EPS has a weight average molecular weight of less than 1.4 x106 g mol-1. A combination of GC-MS and HPAEC-PAD analysis confirmed that the structure was composed of D-glucose, D-galactose and D-N-acetyl mannosamine in a molar ratio of 2:2:1. Linkage analysis of the EPS, by GCMS and 2D-NMR experiments showed that the repeating unit contains two terminal, one dilinked and two tri-linked monosaccharides. All of the data obtained allowed for the elucidation of the structure of the EPS produced by Lactobacillus helveticus Rosyjski. The current techniques used for the determination of the monomers and linkages present in EPS structures were investigated. Monomer analysis was studied by using the previously characterised EPS, Lactobacillus acidophilus 5e2 as a model. A variety of acids were used to catalyse the hydrolysis of the polysaccharide. The monosaccharides liberated from the EPS were analysed by HPAEC-PAD. It was determined that hydrolysis with TFA was the simplest technique to employ whilst also providing reliable results. Linkage analysis was investigated by the production of a number of disaccharide-derived model linkage standard compounds. This resulted in the creation of a number of terminally and di-linked linkage standards which can be used as model reference compounds when characterising previously unidentified EPS. The bacterial strain Bifidobacterium animalis subsp. lactis A1dOxR produces EPS. Initial inspection of the 1H NMR spectrum however displayed a complex anomeric region with many overlapping signals. Analysis by HP-SEC-MALLS revealed multiple peaks, further adding to the evidence of the presence of more than one EPS in the recovered ‘crude’ sample. The crude sample was subjected to dialysis and a fraction (over 100,000 Da) was recovered and denoted as high molecular weight (HMW) EPS. Examination of the 1H NMR spectrum from HMW EPS indicated a hexasaccharide repeating unit oligosaccharide, whilst HPEAC-PAD and GC-MS analysis confirmed that the structure was composed of Lrhamnose, D-galactose and D-glucose in a molar ratio of 3:2:1. Further analysis determined that one of the galactose monosaccharides was present in the furanose form as appose to the more commonly observed pyranose configuration. Linkage analysis of the EPS, by GCMS and 2D-NMR experiments, showed that the repeating unit contains one terminal, four dilinked and one tri-linked monosaccharide. All of the data obtained allowed for the elucidation of the structure of the HMW EPS from by Bifidobacterium animalis subsp. lactis A1dOxR. Solubilising EPSs has been a constant challenge, however, it was hoped with the advent of ionic liquids (IL) this issue could be solved. Ultimately, dissolution of EPS in ionic liquids though proved to be unsuccessful, so attention was turned to combining derivatisation and dissolution, as a method for solubilising polysaccharides. Derivatisation of a number of model systems of di- and polysaccharides were explored. By studying both 1D- and 2D-NMR coupled with GC-MS analysis it has demonstrated that polysaccharides such as cellulose along with a number of common disaccharides can be successfully dissolved and modified in ionic liquids.

This thesis describes a new method for the synthesis of pseudodisaccharides containing a carbasugar analogue attached to a "true" sugar. The methodology is based on a Diels-Alder cycloaddition of vinyl sugars and appropriately substituted pyran-2-ones, followed by chemical manipulation of the resulting cycloadducts. The thesis also describes the synthesis of inhibitors of Golgi α-mannosidase II and glucokinase. The first chapter is a comprehensive survey of the reported synthetic routes to pseudodisaccharides from the literature. The results and discussions are presented in chapter 2. This chapter starts by discussion of the preparation of vinyl sugars and pyran-2-ones and the regio- and stereoselectivity of their cycloadditions. This is followed by reporting the chemical manipulations of these cycloadducts and the synthesis of a pseudodisaccharide. Cycloadducts are shown to lose carbon dioxide at elevated temperatures to afford dihydrobenzenes. The loss of the bridging carbon dioxide from the cycloadducts is experimentally and computationally investigated. The resulting dihydrobenzenes are shown to also be useful as precursors in the synthesis of pseudodisaccharides. The chemical manipulation of these dihydrobenzenes is used towards the synthesis of a pseudodisaccharide. The third and fourth chapters focus on the synthesis of new inhibitors of Golgi α-mannosidase II and glucokinase respectively. A range of 6-aminoglucose and mannose derivatives were prepared and tested for the inhibition of Jack bean α-mannosidase, but were found to lack any inhibition. Similarly, a range of 6-triazologlucose derivatives were prepared but were found to lack any cytotoxicity. The fifth chapter contains the details of the preparation, experimental procedures and spectroscopic characterisation of the synthesised chemical compounds. Rate calculations are reported in Appendix I and the X-ray crystallographic data are presented in the Appendix II.

Higher carbon chain sugars have gained increased interest recently; they are important building blocks of natural and unnatural products with biological properties. The synthesis of these higher sugar skeletons is commonly known to be achieved with the Wittig methodology which exploits phosphorus ylide chemistry. This method has been successfully used for the synthesis of the higher carbon sugars. The aim of this project was to synthesise ß,ß'-dioxo sugar-derived phosphorus ylides, a new class of ylides, as versatile intermediates to valuable higher carbon sugar derivatives and carbohydrate mimics. Model reactions were initially conducted; tetrahydro-2-furoic acid and tetrahydro-2H-pyran-4-carboxylic acid, compounds which are structurally similar to the precursor sugars, were identified as suitable model compounds. These compounds were converted to acyl chlorides and then converted to ß,ß'-dioxo phosphorus ylides precursors by acylation. The methodology proved successful and 8 examples were isolated. However, low yields were obtained due to the inevitable formation of triphenylphosphine oxide. The method was then extended to sugar derivatives, prepared using standard protecting group chemistry. It was found that acylation could be achieved using the simple acyl chloride route or peptide coupling methodology for sugar derivatives which were acid sensitive. ß,ß'-dioxo sugar-derived phosphorus ylides (16 examples) were successfully isolated in low yields. The oxidation and thermal reactivity of the ß,ß'-dioxo ylides were studied. Oxidation resulted in the successful synthesis of vicinal tricarbonyls, isolated as a mixture with the gem-diols (hydrates). The thermal decomposition of the ylides gave alkynes in moderate yields.

Singly, doubly and triply deprotonated monosaccharide and disaccharide molecules of the Heparin family of Glycosaminoglycans (GAGs) have been studied in the gas phase to investigate their geometric structures and intrinsic stabilities. Low energy Collision Induced Dissociation (CID) in a quadrupole ion trap was used to investigate the relative stabilities of the systems along with high energy (50-150 keV) Mass Induced Kinetic Energy (MIKE) experiments. The related alkali metal cation complexes were also studied for comparison. The detailed effects of the cations Li+, Na+ and K+ on the stabilities and low energy fragmentation pathways of these highly sulphated isomeric sugars have been determined, allowing suitable ionic fragments to be selected to assist in future analytical studies. High-energy MIKE experiments have provided information rich mass spectra which allow the locations of the biologically crucial sulphate groups to be unambiguously determined and are suggested as an alternative to Electron Detachment Dissociation (EDD). The conformational space available to the prototypical monosaccharide residue Iduronic Acid has been investigated using a combined molecular mechanics and quantum chemical approach. Merck Molecular Force Field 94’ has been utilized to generate candidate structures for higher level B3LYP, PW91PW91, M05-2X and MP2 calculations using the 6-31++G** basis set. This method was used to explore the preferred low energy structures of this highly flexible sulphated sugar around the 1C4, 4C1, 2S0 and 1S5 sugar ring conformations and to assess the suitability of the various methods for studying this class of compounds. This work showed that M05-2X method compared very favourably to the MP2 results, and that it provided improved relative energies over B3LYP. Additionally, the results presented demonstrated the need to study the three dimensional space available to sub conformers when assessing the relative energies of ring conformations. Finally, the CID technique was also used to investigate the Potential Energy Surface of two multiply charged inorganic anions. The Dichromate dianion Cr2O72- was seen to fragment via electron loss and ionic fragmentation. Density Functional Theory was used to determine that the relative barrier height for electron loss was lower than that for ionic fragmentation. The gas phase stability of the Re2X82- (X = Cl, Br) and Re2XnY8-n2- (X = Cl, Y=Br, n=1-3) metal-metal bond complexes were then studied. The ab initio calculations performed for Re2Cl82- and Re2Br82- indicated that Re2Cl82- was intrinsically stable in the gas-phase, while for Re2Br82-, loss of Br- was an exothermic process. However, gas-phase Re2Br82- was rendered metastable due to the presence of the repulsive coulomb barrier on the fragmentation potential energy surface. The intrinsic gas-phase stability displayed by the dirhenium complexes studied indicated that they could be investigated using more quantitative CID measurements to obtain accurate metal-ligand binding energies.

Glucosyl and galactosyl bearing methacrylates were synthesised using a combination of well known carbohydrate chemistry and ultrasonic chemistry. Polymerisation of these monomers by aqueous reversible addition-fragmentation chain transfer polymerisation yielded glycosylated polymers in high yield with a high degree of control over molecular weight and narrow polydispersity. Polymers of varying levels of glycosylation could be achieved through addition of a comonomer. Block copolymers were synthesised through sequential monomer addition. Binding studies of these polymers to Ricinus communis agglutinin 120 by both isothermal titration calorimetry and surface plasmon resonance found a high degree of molecular weight dependence on avidity. Studies also suggest that the increase in avidity through multivalency is due to both simultaneous binding to multiple receptors and the increased probability of binding due to the high local concentration of ligands. Polymers synthesised have been used in the synthesis of glycosylated gold nano-particles. Some degree of control over nanoparticle size was achieved through adjustment of the gold:polymer ratio but was found to be limited. Agglutination assays show the particles display specific lectin binding but in vitro cell studies were unable to give conclusive information regarding the uptake of particles into cells.

Branched chain sugars display a varied and valuable range of biological activities. This thesis concerns the synthesis of 3,5-di-C-methyl-D-glucose, a potential inhibitor of glycogen phosphorylase (GP), and therefore a proposed therapeutic agent for type 2 diabetes. Chapter 1 looks at the occurrence of branched sugars in the natural world and current therapies for type 2 diabetes. Inhibition of GP is explored, and the molecular modelling studies which led to the design of the project target. Chapter 1 also looks into the development of new foodstuffs, the chemistry and biochemistry of imino sugars and branched hydroxy proline analogues. In Chapter 2, a range of different approaches to 3,5-di-C-methyl-D-glucose are investigated. Most of the initial investigations were carried out on the L-enantiomer, a readily available test system deriving from 2-C-methyl-D-ribono lactone. 2-C-Methyl-D-ribono lactone is synthesised rapidly from D-glucose in a one-pot reaction; as the key starting material for this work, the scalability of this process was investigated. One of the attempted syntheses of di-C-methyl glucose lead to the development of a route towards 3,5-di-C-methyl fructose, a novel dibranched ketose sugar. It was envisaged that through an enzymatic transformation, it might be possible to produce 3,5-di-C-methyl glucose stereoselectively. Synthesis of both enantiomers of 3,5-di-C-methyl glucose and mannose are reported, alongside results of GPb inhibition studies. Analysis of the preferred ring size of a range of di-C-methyl branched sugars and sugar lactones generated in this work is also presented. Chapter 3 explores the chemistry of 2,4-di-C-methyl-L-arabinono lactone, a key intermediate in the synthesis of 3,5-di-C-methyl-L-glucose. From this lactone a novel deoxy sugar, 2-deoxy-2,4-di-C-methyl-L-arabinono lactone, was generated. Routes towards a selection of imino sugars were explored, resulting in the synthesis of a methyl branched isofagomine analogue. A substituted aziridine was synthesised, from which a route to a di-C-methyl branched piperidine was proposed, and a pyrrolidine. Also presented is a synthesis of a dihydroxy di-C-methyl branched proline analogue. Detailed NMR analysis of several of the sugars generated in this work was carried out by Dr M. Wormald, of the University of Oxford Biochemistry department. The results of these investigations are presented in the Appendix. Throughout this work, the presence of quaternary centres has posed a problem with the assignment of relative configuration. As a result, this work has been greatly supported by X-ray crystallography, and the structures shown herein were wholly generated by me. Several other crystals were run during the course of this work, not all pertaining to these projects, and are provided in the CD appendix.

A new metal-based method to synthesize C(1)-substituted glycals has been developed. Starting with the obtained substituted C-glycals, the possibility of a stereocontrolled addition (via azidoselenation) to form β-C-substituted-2-aminoglycosides has been investigated. Instead of forming the expected C-glycosyl derivatives of 2-amino sugars, an inseparable mixture of diastereomers, which were derived from the corresponding C(1)-allylated glycals, has been obtained.

Co-crystallising sugars by the supersaturation process was investigated using sucrose and lactose as the matrix sugars. The components to be cocrystallised with either sucrose or lactose were a variety of mono- and disaccharides along with sweeteners such as saccharin. Analysis of the materials yielded from the supersaturation process was done primarily by differential scanning Calorimetry (DSC) and powder x-ray diffraction (PXRD). DSC analysis allowed information to be obtained on the thermal behaviour of the various co-crystalline materials, whilst PXRD permitted information on the structural aspects of the materials to be gained. These two forms of analysis were complimentary to each other, each revealed unique characteristics of the co-crystalline materials. To unambiguously differentiate the difference between a material that is co-crystalline and one that is not, physical blends of the sugars to be cocrystallised were analysed by DSC and PXRD. This approach allowed for identification of all the components in the mixture, more importantly, this identification was achievable at all levels of the second component in the mixture. Co-crystallising either sucrose or lactose, with various sugars, yielded solely co-crystalline material up to a certain level of the added sugar. Once this level has been reached, two distinct phases appear in both DSC and PXRD analysis. A co-crystallised and a phase relating to the added sugar can be observed. The formation of a potentially co-crystalline material appears to result from a direct inclusion of the added sugar into the matrix sugar. DSC analysis of the co-crystallised material revealed thermal behaviour that is suggestive of a doping of the matrix sugar by the added component. PXRD analysis did provide some data to further this argument, axis elongation for co-crystallised material is suggestive of a sopping of the main phase. However, determination of the unit cell volumes did not yield conclusive evidence to help prove this hypothesis though. This behaviour in both forms of analysis was generally proportional to the quantity of the second component that has become included into the matrix sugar. The formation of solely co-crystalline materials appears to rely on the structural similarity between the matrix sugar and the component to be included. A higher degree of similarity is reflected by a high level of inclusion of the added component. Co-crystallisation appears to rely on a degree of intermolecular sugar-sugar recognition. The inclusion of a second component is not solely down to structural similarities between materials however. It appears that there are kinetic factors potentially involved to. Varying the method of co-crystallisation allowed for higher, or lower, amounts of various second components to be included within a matrix sugar. The appearance of co-crystalline materials may be due to the inclusion of the second component in an amorphous state. Analysis by Autosorb of various co-crystalline materials has dispelled this idea. All co-crystalline materials behaved in a manner that was indicative of a crystalline material. During the course of the work with sucrose, it was noted that a unique melting point was observed. From previous work, this unique phase was thought to be a hydrated form of sucrose. Further analysis on this material has allowed for further postulation on its formation and on new methods of its synthesis.

We have developed a novel chemoenzymatic strategy to GDP-azidodeoxymannoses which differs from the natural substrates of mannosyltransferases of the Leloir type due to the presence of an azide group instead of hydroxyl functionality on the mannopyranose ring. We first chemically synthesized 2-, 3-, 4- and 6-azidodeoxy-α-D-mannose-1-phosphates, and then tested them as substrates for a recombinant GDP-Mannose Pyrophosphorylase from Salmonela enterica produced as polyhistidine-tagged fusion protein in E. coli. This enzymatic step yielded all the corresponding GDP-azidodeoxymannoses in 41%, 52%, 55% and 63% yields respectively, which were characterized by high resolution MS, 1H- 13C and 31P NMR. Furthermore, we have optimized a high throughput colourimetric screen (via malachite green dye) which allows for indirect monitoring of this enzymatic reaction, and could be applied in future studies of enzymatic directed evolution to improve the efficiency of unnatural products formation. The GDP-azidodeoxymannoses so formed were tested as substrates for the soluble fragment of an α-1,2-Mannosyltransferase (Kre2p/Mnt1p) from S. cerevisiae which we produced in E. coli. Our preliminary data demonstrated that GDP-4-azidodeoxymannose was the most efficient donor substrate when transferred to a mannose α-methyl glycoside acceptor, as monitored by electrospray ionization mass spectrometry, and we foresee that the other isomers may find similar applications on further enzymes. Consequently, GDP-azidodeoxymannoses may serve as valuable tools for glycobiological studies using bioorthogonal azide chemistry.

The Fmoc synthetic strategy was employed to synthesise two identical combinatorial peptide libraries on a hydrophilic PEG-PS resin. One library was appended with boronic acid moieties at two positionally-fixed locations. Successful inclusion of the boronic acid units was confirmed using a novel UV fluorescent colorimetric assay employing carminic acid as the dye compound. A study of the effect had by the resin-bound peptides bearing boronic acid groups on the binding characteristics of vancomycin, a medically relevant antibiotic glycoprotein, was conducted. In all, 132 library compounds were tested for their binding affinity with vancomycin, via immobilisation of the glycopeptide onto the solid support through hydrogen bonding or complexation with the boronic acid moieties. Subsequent cleavage via acidolysis afforded vancomycin containing solutions which were quantified by growth inhibition of methicillin susceptible Staphylococcus aureus. Comparison of the diameters of the resultant zones of inhibition and those produced by vancomycin of known concentrations afforded a means of calculating the vancomycin concentration of the cleavage solutions, and thereby determining the binding affinity of vancomycin to each peptide sequence. Five peptide sequences and twenty one of the peptidyl-boronic acid sequences showed zones of inhibition, demonstrating their reversible affinity for vancomycin. Three peptide sequences showed zones of inhibition in both libraries. The presence of boronic acid was therefore shown to impart, enhance, detract and remove the affinity of vancomycin to a range of resin-bound peptide sequences.

The work within this thesis describes the optimisation of the synthesis of a series of [l,4]-morpholines, [l,4]-oxazepanes and disaccharide mimics prepared by reductive amination of carbohydrate-derived dialdehydes with primary amines. Also described are the investigations into synthesis of heptanoside motifs via nitroaldol reactions of carbohydrate-derived dialdehydes and Beckmann rearrangements of carbohydrate oximes. Chapter 1 provides an introduction to the involvement of carbohydrates within biological pathways, with particular interest in monosaccharide; importance and the methodologies used in the synthesis of heptanoside motifs. Chapter 2 provides an overview of oxidative cleavage, reductive amination and nitroaldol reactions with particular emphasis on the application of these reactions to carbohydrate substrates.

In modern society people are concerned about their state of health and if they do unfortunately become ill they want the best possible treatment to be made available to them. In order to satisfy these demands new treatments have to be developed. This can be a long and expensive process. Before any new treatment can come to market it has to be proved to be both clinically effective and economically cost-effective. With limited health care resources the cost-effectiveness of treatments is becoming ever more relevant. In order to show whether a treatment is clinically effective a clinical trial is carried out and this is now usually accompanied by an economic evaluation, so that the cost effectiveness of the treatment can be assessed. When a clinical trial aimed at preventing clinical events is analysed, a time-to-first event analysis is often performed together with a cost-effectiveness analysis. These analyses do not always make the best use of the large amounts of patient information recorded during the clinical trial. Using the randomised controlled trial (RCT) the Impact Of Nicorandil in Angina (IONA) as an exemplar, ways in which the clinical and economic evaluations of clinical trials can be expanded are explored. There are three main parts of this thesis. Firstly, following a more detailed introduction in Chapter 1, in Chapters 2 and 3 the IONA Study is introduced and the main clinical results of the study are given. Secondly, in Chapters 4, 5 and 6 the fact that patients could suffer more than one clinical endpoint is considered. The models that can be used to incorporate the recurrent events are introduced and then applied to the data from the IONA Study. Following on from this, through the simulation of recurrent event data, the performance of the models under different known conditions is assessed. Thirdly, in Chapters 7 and 8 an introduction to health economics is given and following this the main results of the economic evaluation of the IONA Study are presented. Areas in which the results of the economic evaluation can be expanded are then investigated. Finally, in Chapter 9 there is a discussion of the work as a whole and areas where there would be the possibility of further work.

Protein-carbohydrate interactions are at the basis of cell-cell and cell-external agent interactions and therefore play a major role in the biological events in the body. However, protein-carbohydrate interactions are characterized by low binding constants and the scientific community is searching for methods for increasing the rate of binding of carbohydrates to proteins. One proposed method for increasing the binding in protein-carbohydrate interactions is the fluorination of carbohydrates. Pioneering work of DiMagno, who synthesized a racemic hexafluorinated hexose and tested its capacity of transmembrane transport, has shown that the hexafluorinated hexose crosses the erythrocyte membrane ten fold faster than glucose. It was postulated that this is because of the enhanced affinity in the protein-carbohydrate interactions, instilled by the increased hydrophobicity of the carbohydrate. This thesis describes the successful enantioselective synthesis of 2,3-dideoxy-2,2,3,3-tetrafluoro -galactose and 2,3-dideoxy-2,2,3,3-tetrafluoro-glucose and a preliminary investigation of the glycosylation of 2,3-dideoxy-2,2,3,3-tetrafluoro-galactose. A tetrafluorinated building block approach was considered for the synthesis of 2,3-dideoxy-2,2,3,3-tetrafluoro-galactose and 2,3-dideoxy-2,2,3,3-tetrafluoro-glucose. Key steps in the synthesis of galactose and glucose were the β- and α-SAD reactions for the formation of the respective diols and also for introducing the enantioselectivity in the synthesis. The pentose rings were formed by the anionic cyclization reactions, methodology which was previously developed in our group. The glycosylation was achieved via an anomeric alkylation strategy.

Chitin is an abundant polysaccharide that is mainly found in the exoskeleton of arthropods (insect, crustaceans, etc.). It is chemically and structurally similar to cellulose but chitin has an acetamido group in the second position: Chitosan, the main derivative ofchitin, is deacetylated leaving the amide groups uncovered and liable to be protonated in acid environments. These together with other features provide chitin and chitosan with a range of distinctive functional properties that including structural qualities, adsorption capacity, reactivity, biocompatibility, bioactivity and biodegradability. Such chitin and chitosan properties have been the origin of increasingly interest and oftheir application in diverse fields. In order to improve the availability ofthe functional groups ofchitin and chitosan in the solid stat~ it is proposed the generation ofporous materials with large surface area. The proposal is remove the liquid phase of chitin and chitosan physical gels using C02 critical point drying to minimize the disruption of the formed microstructure. The analysis of the diverse solution characteristics and their relationships with' the features ofthe gel and porous structures produced provide basic information that could allow the design offunction specific structures. Two types ofchitin solutions were obtained using dimethylacetamide containing 5% LiCI and cold «4°C) 10% NaOH as solvents. Chitosan was dissolved in diluted acetic acid and in formaldehyde sodium bisulfite (FSB) solution. Homogeneously deacetylated chitosan was obtained and dissolved in water. The basic characterisation ofthe solutions indicate that the molecular interaction ofthe polysaccharide with each solvent is distinctive. Every solution type were cast and physical gels were generated by diffusion of gelating agent fumes. Additionally, gels of alkali chitin (chitin in 10% NaOH) were produced by thermal treatment. Once washed, a portion of each type ofgel was equilibrated in water and other part in acetone; except the homogeneous chitosan gels that were equilibrated only in acetone. The water gels were freeze dried and the acetone was removed by C02 critical point drying to get porous materials. The porous structure of such materials were analyzed by scanning electron microscopy, crystallinity, chemical compositionand BET gas adsorption. The freeze dried materials have porous structure morphology similar to what has been previously reported. On the other hand, most ofthe critical point dried porous structures are in the mesoporous range and have large specific surface areas (323-887 m2/g). Apparently, for this tYpe ofmaterials the molecular interactions occurring in the solution could be related with the morphology of their respective porous structure

This thesis describes the synthesis of 2-C-branched carbohydrate derivatives from ketohexoses and some useful synthetic applications thereof. Chapter 1 encompasses a general introduction to branched-chain sugars, with the emphasis upon 2C- hydroxymethyl and -methyl branched sugars. Strategies for their synthesis are discussed, both from nucleophilic and electrophilic carbohydrate derivatives. Some synthetic applications of carbohydrates and consequently branched carbohydrates are highlighted, with a view to the derivatisation of a proposed chiron from the Kiliani ascension of L-sorbose, 2-C-hydroxymethyl2,3: 5,6-di-O-isopropylidene-L-gulono,1,4-lactone. In chapter 2, the Kiliani ascension of L-sorbose and two subsequent complementary di-O-isopropylidene protections of the branched-chain lactones generated (thermodynamic and kinetic) are discussed. Further studies into the same transformations of three other ketohexoses, D-fructose, D-tagatose and D-psicose are then discussed. Further useful synthetic derivatives of the three major isomeric L-sorbose-derived branched di-protected lactones: selective deprotections and cleavage of C5-C6 bonds. One major area wh!ch utilizes carbohydrate building blocks is iminosugar synthesis. Iminosugars are the major compound class of interest in the realm of glycosidase inhibition, and also have other therapeutic properties, thus making them desirable synthetic targets. The application of 2-C- . branched sugar derivatives in the synthesis of novel iminosugars is proposed. Chapter 4 describes the syntheses of eight novel 2-C- hydroxymethyl and -methyl branched iminosugars from the major thermodynamic product (2-C-hydroxymethyl-2,3 :5,6-di-O-isopropylidene-L-gulono-l ,4-lactone) obtained from the Kiliani ascension of L-sorbose, as described in chapter 2. The syntheses of two additional novel iminosugars from isomeric 2,3:5,6-di-O-isopropylidene branched derivatives is described. Some results of inhibitory activity follow the synthetic results. The last two chapters provide a general introduction to macrocycles, and some synthetic investigations into macrocycles based on open-chain sugar amino acids (SAAs). SAA-derived macrocycles are structural hybrids of macrocyclic peptides and cyclodextrins. Strategies for the synthesis of large rings and previous work on D-galactose-derived-SAA macrocycles within the group is described with a view to the extension of this work towards novel asymmetric macrocycles with functionalisable rings. Chapter 6 reports synthetic investigations into three related families of macrocycles, each based on an open-chain galacto SAA. The pure galacto SAA macrocycles are revisited and two novel families of macrocycles are born, one containing proteinogenic amino acids and the other incorporating branched-chain SAAs. The branched-chain SAA itself is derived from 2-C-hydroxymethyl-2,3 :5,6-di-O-isopropylidene-D-mannono-l ,4-lactone, the major protected Kiliani lactone derived from D-fructose. Two major improvements to the original methodology for macrocycle formation are reported. Some structural analysis of macrocylic compounds through NMR and CD spectroscopy.

The efficient synthesis of oligosaccharides is becoming increasingly important as their roles in biological recognition and signalling mechanisms are exploited as potential therapeutic agents. Carbohydrate processing enzymes are desirable catalysts for creating oligosaccharides as the use of multi-step chemical syntheses are avoided; however, they do not always meet exact experimental requirements.

The aim of this project is to develop and test methodologies for conformational studies of free and bound GAGs. Gas phase conformations of heparin-derived oligosaccharides were studied by using Ion Mobility Mass Spectrometry (IMMS) and molecular modelling. Gas phase conformations were modelled using the AMBER force field. Their theoretical collision cross-sections were compared with the IMMS data and a good agreement was obtained. The gas phase conformations of heparin oligosaccharides were more compact than those observed in solution or crystal structures of heparin-protein complexes. This was attributed to the effects of sodium cations interacting with the negatively charged sulphate and carboxyl groups of oligosaccharides. Adiabatic maps of dihedral angles vs. potential energy of disaccharide fragments of the tetrasaccharides were calculated in the absence of the sodium cations. New NMR methods for the measurement of scalar and dipolar 1H-1H coupling constants and 13C-13C coupling constants in natural abundance 13C samples were developed. Performance of these methods was tested extensively. Solution conformation of the fully sulphated heparin-derived tetrasaccharide was studied by NMR spectroscopy. 1H-1H scalar coupling constants were used to characterize the dynamic equilibria of flexible monosaccharide rings. 1H-1H and 1H-13C RDCs were used in the study of the conformation of the glycosidic linkages. RDC-refined structures were obtained from molecular dynamics with explicit water and sodium cations. Interactions of the heparin-derived fully sulphated tetrasaccharide with factor H modules, fH~19, 20 and fH~7, were studied using AUTODOCK. Conformation of a spin-labelled heparin-derived fully sulphated disaccharide was studied by NMR and molecular modelling.

It was proposed that a phase separated system might be utilised to deliver a concentrated polysaccharide mucosal protective coating in gastro oesophageal reflux disease (GORD). In this context the phase behaviour of xanthan gum in combination with sodium alginate and other polymers was studied. Above a threshold concentration of alginate, aqueous mixtures of xanthan exhibited phase separation, resulting in loss of normal viscoelastic properties and the formation of a low viscosity system. The shape of the phase diagram showed behaviour typical of a segregative system, with the continuous phase composed exclusively of alginate and the disperse phase being rich in xanthan gum. Increasing alginate molecular weight reduced the threshold concentration for separation, as predicted by the Flory-Huggins theory, but changes in alginate mannuronate:guluronate ratio had no effect. Increasing ionic strength elevated the threshold concentration. Xanthan separation was elicited by other aqueous anionic polyelectrolytes, but not neutral water soluble polymers. Scleroglucan, another rigid-rod polysaccharide, was investigated as an alternative to xanthan but did not show similar separation behaviour, suggesting that the charge on the xanthan molecule is a necessary prerequisite. Reversal of phase separation by dilution across the phase boundary provided increases in viscosity. A 1% xanthan:2% alginate mixture doubled in viscosity whereas if diluted with simulated gastric fluid a seven-fold increase was seen, as a result of conversion to an alginic acid gel. This offers a mechanism for producing the desired viscosity barrier. Low viscosity polyelectrolytes, with concentrations close to the phase boundary yielded the greatest viscosity increases. In the phase separated system, the disperse phase exhibited an unusual strand-like morphology whose birefringence suggests a liquid crystalline structure. The variable size of the strands was explained in terms of kinetics of xanthan molecular aggregation in media of different viscosity.

The aim of this project was to investigate ways of chemically modifying cellulose using ionic liquids. Cellulose is a readily available, naturally occurring, polymeric carbohydrate which is ideal for chemical modification owing to the high density of its functional groups. However, it is insoluble in all common organic solvents. Therefore, interest has recently turned to the use of a novel class of solvents, ionic liquids, which have been found to dissolve cellulose. Initially, studies were undertaken into the acylation of simple carbohydrates using a Lewis acidic choline based ionic liquid, (ZnCl 2)2ChCl. It was found that the yields obtained were comparable to those achieved when traditional solvents and catalysts have been used. It was also found that when only one equivalent of acylating agent was used, all of the carbohydrate hydroxyl groups were acylated. This was thought to be unique to this ionic liquid system. The acetylation of cellulose was also studied in (ZnCl2)2ChCl. Here, it was discovered that the ionic liquid alters the morphology of cellulose by wetting or dissolution thus enabling a range of acetylated products to be produced. Research into the cationic modification of cellulose in an ionic liquid based on chlorcholine chloride (ClChCl) and urea was also undertaken. In this case, it was found that the ionic liquid could act as both the reagent and solvent. It was shown using this system, that all the available (surface) hydroxyl groups on cellulose were modified and that the material produced was significantly more hydrophilic than unmodified cellulose. Finally, preliminary investigations were untaken into other potential surface modifications of cellulose in ionic liquids.

The aim of the research to be undertaken is the synthesis of hetero-annulated glucose derivatives. In particular the synthesis of five-, six-, and seven- and eight-membered N-containing fused heterocycles is desired, with the nitrogen attached at the C-2 and C-3 position of the sugar ring. Emphasis is placed upon using the sugar ring as a scaffold to enable diastereoelective synthesis. A variety of synthetic techniques are to be employed to achieve ring-closure, especially reductive lamination, ring-closing metathesis and photochemical methods of annulation. (Fig. 9206A).;Such compounds could be useful as precursors to alkaloid synthesis due to the huge range of methods for the conversion of pyranosides to other ring systems. In addition, previous work has shown that such compounds have an inhibitory effect on glycoside processing enzymes. The pyrollidines synthesised have been tested as Glycosidase inhibitors, and some have been shown to be weak specific beta-Galactosidase inhibitors.;Using similar techniques as those employed to produce the lactam above, 2,O- and 3,O-fused lactones were also produced, with dimerised by-products also isolated. Intramolecular photocyclisation was also employed to produce complex multicyclic fused carbocyles. (Fig. 9206B).;Synthesis of N-linked amino sugar dimers was also achieved, and initial attempts at deprotection made. (Fig. 9206C).