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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 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.

1,3-Nucleophilic addition reactions of thiols and amines with pyranosylnitrile oxides have been employed in the synthesis of C-glycosides. The nitrile oxides were generated by base-induced dehydrochlorination of the corresponding hydroximoyl chlorides 106 and 107. D-Glucose derived hydroximoyl chloride 107 was prepared in four steps from D-glucose and employed in collaborative work toward the synthesis of glucosinolate analogue. Reaction of alkyl and aryl thiols with D-xylose nitrile oxide 151 afforded a series of desulfoisoglucosinolates in 55-76% yields. Reactions of allyl and aryl amines with the D-xylose and D-glucose derived nitrile oxides 151 and 115 under basic conditions afforded the corresponding Z-amidoximes. For example, (Z)-N-benzyl-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)formamide oxime (137) was prepared from 107 and benzylamine in 88% yield. The addition of amino acid derived nucleophiles was also investigated Reaction of the D-xylose and D-glucose nitrile oxides 115 with L-cysteine derivatives 147 under basic conditions afforded thiohydroximates 148 and 150 in high yield. Reaction of the D-xylose nitrile oxide 151 with glycine, L-leucine and L-phenylalanine esters initially afforded amidoximes 152, 155 and 161. Cyclisation of 152, 155 and 161 to form 1,2,4-oxadiazin-6-ones occurred on exposure to silica or prolonged standing. Employing L-proline benzyl ester as the nucleophile led to the formation of predominantly oxadiazinone 163. Attempts to synthesis pyranosyl-1,2,4-oxathiazin-6-ones were not successful 6-Amino-6-deoxy-1,2,3,4-di-O-isopropylidene-α-D-galactopyranose hydrochloride was prepared from D-galactose in 4 steps, and reaction with D-xylose and D-glucose derived nitrile oxides 151 and 115 afforded (1→6) amidoxime linked pseudo-disaccharides 178 (81%) and 181 (75%) D-Xylose and D-glucose amines 182 and 183 were prepared from the parent aldoses in 5 steps. Reaction of these amines with D-xylose and D-glucose nitrile oxides 151 and 115 yielded a series of (1→1) amidoxime linked pseudo-disaccharides (41-49% yields).

The sugar-derived alkenes were prepared using three methods; in the first hex-5-enofuranoses were generated in good yields from the corresponding 5,6-dimesylates using Tipson-Cohen conditions; the second employed an aprotic Bamford-Stevens reaction to give the 1-methylene sugar from the analogous pyranosyl tosylhydrazone. The final approach gave a series of 1-methylene sugars in moderate to good yields (50-82%) by the methylation of sugar lactones with dimethyl titanocene (Petasis Reagent).

Our research has been based on the biosynthetic pathway to N-linked glycoproteins, species which are comprised of an oligosaccharide moiety attached to the side-chain nitrogen atom of an asparagines residue in a peptide. In order to effectively study the steps involved in the biosynthesis of lipid-linked oligosaccharides and their subsequent transfer to a nascent protein in cotranslational modification processes, it is essential to have a source of pure dolichols in quantities sufficient to allow the synthesis of both biosynthetic intermediates and synthetic analogues in the pathways responsible for the biogenesis of glycoproteins. Since supplies of naturally occurring dolichols are scarce and they are difficult to synthesise, a number of lipid substrates were prepared as synthetic analogues of naturally occurring dolichyl phosphate. (Fig. 6302A) The synthesis and biological testing of lipids 1 and 2 are reported. The result of a study of the fluorescence of lipid 1 is also presented. Work towards the preparation of lipid 3 is detailed. Many of the enzymes involved in the biosynthesis of N-linked glycoproteins have already been isolated and characterised, however, some of those enzymes involved in the early stages of N-glycan biosynthesis remain unidentified. In order to gain more information about the unknown mannosyltransferases that are active in the early stages of N-glycan biosynthesis, a phytanyl-linked monosaccharide (glucosamine), 4, and disaccharide (chitobiose), 5, were prepared.  (Fig. 6302B) The syntheses of chitobiose phosphate from commercially available chitobiose octaacetate is reported. Details of the preparation of N-acetyl glucosaminyl phosphate from commercially available N-acetyl glucosamine are pretend. The lipid moiety, phytanyl phosphate, was derived from commercially available phytol. The manner in which phytanyl phosphate was coupled to both the monosaccharide phosphate and the disaccharide phosphoryl species is detailed and results of subsequent biological testing are reported. Finally, work towards the preparation of a fluorescently-labelled asparagine-linked chitobiosyl substrate, 6, is reported.

Three methods for the generation of pyranosyl nitrile oxide have been utilised: firstly isocyanate-induced dehydration of pyranosylnitromethanes (the Mukaiyama approach); secondly hypochlorite-mediated oxidation of pyranosyl aldoximes, and finally base-catalysed dehydrohalogenation of pyranosyl hydroxyimoyl chlorides. The pyranosyl aldoximes derived from D-mannose, D-glucose, D-xylose and L-fucose were synthesised via stannate (II) mediated reduction of the corresponding pyranosylnitromethane (80-90%), whilst direct chlorination of the aldoximes gave the hydroxyimoyl chlorides (80-95%). The efficiency of nitrile oxide generation by all three methods was illustrated by the high levels of 3,4-(dipyranosyl)-1,2,5-oxadiazole-2-oxides (furoxans) obtained by dimerisation on generation in the absence of a dipolarophile. For example, 3,4-(2,3,4,6-tetra-O-acetyl-β-D-mannopyranosyl)-1,2,5-oxadiazole-2-oxide was prepared from 2,3,4,6-tetra-O-acetyl-β-D-mannopyranosylnitromethane in a 90% yield. The D-glucose, D-xylose and L-fucose derived analogues were synthesised in high yields (79-96%) utilising all three methods. Where compatible, the Mukaiyama approach gave cycloadducts in good yields (>75%) (e.g. on reaction with methylenecyclohexane and styrene). However for low boiling or thermally unstable dipolarophiles or for dipolarophiles with functional groups incompatible groups incompatible with the isocyanate dehydrating agent (e.g. dimethyl acetylenedicarboxylate, allyl alcohol), cycloadditions were carried out successfully using pyranosyl aldoximes and pyranosyl hydroximoyl chlorides as precursors. The cycloaddition reactions with achiral alkenes proceeded with low levels of diastereoselectivity (d.e. < 10%), in contrast to the cycloaddition of chiral alkenes where significant p-facial selectivity was observed (d.e. 40-80%). The chemistry of the dipyranosyl furoxans has also been investigated. Hydrogenation of 3,4-(2,3,4-tri-O-acetyl-β-D-xylopyranosyl)-furoxan in the presence of Raney nickel yielded the corresponding dipyranosyl-1,2-dioxime in 77% yield. Preliminary studies of the chemistry of this class of dioxime have been carried out, for example dehydration to the 1,2,5-oxadiazole (furazan) and re-oxidation to the dipyranosyl furoxan. Attempts at complexation of these novel chiral ligands with nickel show promising results.

The orthogonally protected trisaccharide benzyl 4-O-[4'-O-(2",3",4",6"-tetra-O-acetyl-b-D-glucopyranosyl)-2',3'-di-O-allyl-6'-O-benzyl-a-D-glucopyranosyl]-2,3,6-tri-O-allyl-b-D-glucopyranoside X has been synthesised from maltose and 1,2,3,4,6-pentaO-acetyl-b-D-glucopyranoside in 11 steps with an average yield of 76% and a scale of one gram. Allyl ether protection could not be removed successfully so another target was designed. The orthogonally protected trisaccharide pent-4-enyl 2,3,6-tri-O-benzyl-4-O-{2',3'-di-O-benzyl-4'-O-(2",3", 4", 6"-tetra-O-acetyl-b-D-glucopyranosyl)-6'-O-pivaloyl-a-D-glucopyranosyl}-b-D-glucopyranoside X was synthesised on a scale of 2.6g from maltose and 1,2,3,4,6-pentaO-acetyl-b-D-glucopyranoside in only 12 steps with an average yield of 86%. (Fig. 16083) This trisaccharide was sequentially deprotected in order to generate six partially protected analogues. Each of these analogues was oxidised selectivity using the catalytic oxoammonium radical 2,2,6,6-tetramethylpiperidinoxy (TEMPO) and the oxidant(s) sodium chlorite and/or sodium hypochlorite. The TEMPO oxidation was developed in both aqueous and biphasic conditions in order to enable the oxidation of substrates with hydrophilic, hydrophobic and intermediate polarities. Several oxidation protocols were developed and the reactions proceeded on mmolar and mmolar scales and could be monitored by HPLC. Six oxidised trisaccharides were synthesised including mono, di and tri-uronate species.

Oligosaccharides were isolated directly from tomato fruit tissue; a series of homo-oligogalacturonides with a DP of 2-6 was identified in red-ripe and over-ripe fruit. Two glucose disaccharides, identified as gentiobiose and nigerose, were isolated from both mature green (MG) and red-ripe fruit. Gentiobiose was able to accelerate the initiation of ripening when vacuum infiltrated into MG tomato fruit, particularly when co-infiltrated with nigerose. The possible origin of these disaccharides is discussed. The potential role of organic acids (ascorbate, citrate and oxalate) in fruit softening was evaluated in vitro on MG tomato fruit alcohol-insoluble residue (AIR). The hydroxyl radical (•OH), produced via a Fenton reaction in which ascorbate acts as a pro-oxidant, released pectin into solution in the presence of oxygen. •OH production in vivo may be localised in the PCW by the positioning of metal ions such as Cu2+ on pectins or glycoproteins. Probes used to detect ·OH in the tomato fruit apoplast produced inconclusive results but suggested that this active oxygen species is present during ripening. The incubation of tomato fruit AIR with naturally occurring chelators such as citrate and oxalate also led to solubilisation of uronate-containing material. The alteration of the PCW during ripening is likely to be the result of a number of processes. These processes may occur throughout the PCW or may occur in specific areas but their contribution to the softening process should not be over-looked in favour of processes only involving hydrolytic enzymes, as this could lead to an over-simplified view fruit ripening.

The novel exo-enzyme cleavable linker N-1-(4-(3-aminopropylcarbamoyl)-phenyl)ethylsulfanyl)-methyl)-1-phenylacetamide (1) was successfully synthesised and its potential for use in solid phase carbohydrate chemistry was investigated. Sugar substrates were synthesised in solution and attached to solid support via exo-linker 1, which is also cleavable under mildly acidic conditions. (Fig. 9963)

A route to C-glycosides and C-disaccharides with functionalised linkages has been investigated. This convergent strategy involves cycloaddition of selected alkenes and sugar derived nitrile oxide moieties. The resultant 2-isoxazolines can be modified and subsequently ring opened to afford compounds with carbonyl or amino containing linkages. The alkenes selected for study were two w-unsaturated monosaccharide derivatives: 5,6-dideoxy-1,2-O-isopropylidene-a-D-xylo-hex-5-enofuranose and methyl 5,6-dideoxy-2,3-O-isopropylidene-a-D-lyxo-hex-5-enofuranoside. Styrene, oct-1-ene and methylenecyclohexane were also studied as models. Four sugar based nitrile oxides were investigated in cycloadditions to these alkenes, based on D-glucose, D-galactose, D-mannose and L-fucose. Three complementary cycloaddition procedures were developed and evaluated: the dehydration of 2,6-anhydro-1-deoxy-1-nitroalditols, the hypochlorite-mediated oxidative dehydrogenation of pyranosyl oximes, and the dehydrochlorination of a pyranosyl hydroximoyl chloride. The oximes and hydroximoyl chloride compounds were synthesised from the corresponding pyranosylnitromethanes. Reactions involving the sugar oximes typically resulted in variable yields of the by-product furazan N-oxide (furoxan) in addition to the cycloadducts, depending on the dipolarophile reactivity. In contrast, uniformly high yields of cycloadducts (70-90%) were obtained from the nitromethyl compounds and hydroximoyl chloride with a range of dipolarophiles; little or no nitrile oxide dimerisation was observed in these reactions.

Novel linker N-({[4-(3-aminopropylcarbamoyl)phenyl]ethysulphanyl}methyl)-2-phenylacetamide 87 has been developed which is compatible with a range of reactions (e.g. base oxidation, alkylation), can be cleaved under mild conditions and has the ability to release alcohols and amines. An efficient synthesis of the linker has been devised and attachment to a variety of solid supports (Tentagel, polystyrene) has been achieved. (Fig. 9966A). As a general building block 2-acetamido-3, 4-di-O-acetyl-2-deoxy-N-(fluoren-9-yl-methoxycarbonyl)-b-D-glucopyranosylamine 106 was synthesised in gram quantities from N-acetylglucosamine in 8 steps. The saccharide was linked through the 6-hydroxyl group onto carboxy-Tentagel via linker 87 in excellent yield and extension of the glycoside amino group provides a route towards the synthesis of glycopeptides. (Fig. 9966B). As an alternative, sugar building block 2-acetamido-2-deoxy-N-(benzyloxycarbonyl)-b-D-glucopyranosylamine 129 was synthesised from N-acetylglucosamine in 5 steps. The saccharide was attached onto carboxy-Tentagel via linker 87 and has been used in initial studies into the esterification reactions to generate a small library of compounds. (Fig. 9966C).

This work describes the development of a protecting group for sulfate monoesters in carbohydrates. After initial trial studies, the trifluoroethyl ester was selected as a suitable protecting group. The ester was formed from the sulfate by treatment with trifluoro diazoethane. This protection method is shown to be compatible with other common protecting groups used in carbohydrate chemistry. The protecting group is proven to be stable to the manipulations of many other protecting groups. The utility of monosaccharides incorporating protected sulfate esters in synthesis has been examined. It has been proven that they are useful and versatile building blocks in the synthesis of more complex structures. Selective cleavage of the trifluoroethyl ester was achieved with potassium tert-butoxide. The mechanism for this deprotection reaction has been studied and this investigation has led to the advancement of alternative deprotection methods.

This thesis is concerned with the structural studies of Brucine and some adducts. The use of brucine as a resolving agent is well known, but the use of the brucine a co-crystallising agent for molecules which crystallise with difficult or not all is unknown. Carbohydrates are notorious for their difficult crystallisations if they can be crystallised at all. One aim of this thesis, explained in Chapter 1, is to investigate this use of brucine by crystallographic means. The experimental methods by which this was achieved are described in Chapter 2. Chapter 3 describes the simplest of the structures; the brucine hydrates. This comprises three brucine structures; the anhydrous, di-hydrate and tetra-hydrate. The description of these structures by means of crystallography, thermal analysis and powder diffraction gives a solid basis for further discussion. In particular, anhydrous brucine taken as a 'groundstate' is important in this aspect. Chapters 4 and 5 discuss the co-crystallisation of brucine with uronates and nucleic acids. These classes of molecules are treated as precursors of carbohydrates and show the tendency of brucine to form ribbons in crystalls. They crystallise readily with brucine. The conformation of each acid is compared to conformations found in other crystal structures of these acids and found to be related. Chapter 6 describes the dicarboxylic acid series (oxalic acid to glutaric acid) and derivatives pyruvic, lactic and oxamic acid. These acids were chosen to co-crystallise with brucine to investigate the effect of slight changes between them and on structural methods. Brucine is shown again to favour crystallisation in ribbons, although with a few notable exceptions. In Chapter 7 the same dicarboxylic acids are crystallised with strychnine to make a comparison between brucine and strychnine co-crystallisations. Here the predominant strychnine packing is a bilayer, but crystallisations with uronates and nucleic acids are not favourable.

The improved synthesis of two methyl glycosides, viz methyl 4-O-methyl-α-D-glucopyranoside and methyl 4-O-(α-D-glucopyranosyl)-α-D-glucopyranoside (methyl α-maltoside) from readily available starting materials is described for use as model compounds for oxidative studies of the industrially important polysaccharide amylose. Attempts to acetylate methyl-α-D-glucoside regioselectivity proved fruitless, whilst regioselective pivaloylation was more successful, albeit in yields of poor synthetic utility. In a variation to this approach, the glucoside, is synthesized via selective tri-O-benzoylation of methyl-α-D-glucoside with the regiospecificity of the reaction being a function of the relative strengths of intramolecular hydrogen bound rings, which are governed by the conditions under which the reaction is performed. In the second step, methylation of the free hydroxy group can only be achieved under pressure with silver oxide as catalyst. The target compound is obtained in the final step by conventional cleavage of the ester functions. Initial attempts to synthesise methyl-α-maltoside were convergent, being conducted with variants of the Koenigs-Knorr methodology and using reactions of previously prepared haloglucosides with variously protected 4-hydroxymethyl-α-D-glucosides. No combination of catalysts could be found to activate either of the substrates, or in an alternative strategy, mediate the reaction between a halomaltoside and an alkoxy nucleophile. The target maltoside is formed eventually via the selective anomeric deprotection of per-O-acetylated maltose, followed by stereospecific methylation in a manner similar to that described previously. It is found that in this penultimate step an intramolecular silver alkoxy - esteric salt inhibits mutarotation, thus allowing the predominant formation of the α-anomer. Further deacetylation gives rise to methyl-α-maltoside in 63% yield and in only three steps.

Bacterial biofilms are formed when bacteria in a liquid environment adhere to a surface, multiply to form microcolonies and synthesis a protective glycocalyx composed mainly of hydrated exopolysaccharide (EPS). Bacterial biofilms form in natural, medical and industrial environments and are usually highly heterogeneous. Biofilms can be single or multi-species and their characteristics are dictated by the environment in which they develop. The biofilm bacteria analysed were isolated from a factory environment and all were members of the Enterobacteriaceae. The composition of their extracellular polysaccharides was examined using standard biochemical assays, HPLC and paper chromatography. Most were anionic due to uronic acids, except for the EPS of two strains, 53b and Ent (both Enterobacter agglomerans), which contained only neutral sugars and were highly insoluble. Periodate oxidation revealed a high degree of 1→3 linkages. Bacteriophages which possessed polysaccharide depolymerase enzymes specific for the EPS of strains 53b and Ent were isolated from sewage from a number of sources. The glycanase of one phage (SF153b) was highly specific, had optima over a wide range of temperature and pH, and activity was increased by Ca²⁺ ions. Degradation of 53b and Ent EPS by the depolymerase produced oligosaccharide repeat units. HPLC and size exclusion chromatography gave an estimation of DP for 53b and Ent repeat units of 8-9 and 7-8 respectively. Polysaccharide samples derived from 53b biofilm and planktonic cells were both degraded by the same phage glycanase suggesting that biofilm formation does not stimulate production of a biofilm specific EPS.

A convergent route, based on nitrile oxide/isoxazoline chemistry to carbon-linked disaccharides (C-disaccharides) and C-glycosides with functionalised linkages has been investigated. The sequence involves three steps. Firstly, the cycloaddition of a sugar-derived nitrile oxide to a terminal alkene provides a 3,5-disubstituted 2-isoxazoline. Next, the substituents attached to the isoxazoline may be modified, and finally the heterocycle subjected to either hydrogenolysis or reductive cleavage to afford a C-glycoside with a carbonyl or an amino functionalised linkage. Four terminal alkenes were selected; two ω-unsaturated monosaccharide derivatives, methyl 5, 6-dideoxy-2,3-0-isopropylidene-α-D-lyxo-hex-5-enofuranoside (93) and 3-O-benzyl-5, 6-dideoxy-2, 3-O-isopropylidene-α-D-xylo-hex-5-enofuranose (84), and two non-carbohydrate model alkenes, methylenecyclohexane and styrene. The in situ Mukaiyama dehydration of the peracetylated 2,6-anhydro-1-deoxy-1-nitroalditol derivatives from nitromethane and D-xylose, D-galactose and D-mannose provided the source of nitrile oxides (79), (89) and (90) respectively. Cycloadditions of the xylopyranosyl and galactopyranosyl nitrile oxides (79) and (89) to alkenes (93) and (84) proceeded regiospecifically and with a high degree of π-facial selectivity (40-64% d.e.). In each case the major adduct was found to possess R-configuration at the newly formed chiral centre C-5, corresponding to an erythro relationship between it and the adjacent C-4 position of the attached furanose unit. The structure of the isoxazoline formed from xylopyranosyl nitrile oxide (79) and D-mannose derived alkene (93) was established by X-ray crystallography. The observed selectivies may be rationalised in terms of the "inside alkoxy effect" proposed by Houk and the "homoallylic" modification of De Micheli et al.

Nitrile oxide-isoxazoline methodology has been employed in a novel convergent approach towards 2-ulosonic acid analogues, in particular those of 3-deoxy-D-arabino-2-heptulosonic acid (DAH) and 3-deoxy-D-manno-octulosonic acid (KDO). It involves regio- and diastereoselective [3+2] cycloaddition of a nitrile oxide to carbohydrate alk-1-enes containing four, five or six carbons, yielding 2-isoxazolines. Subsequent deprotection followed by reductive hydrolytic cleavage affords the 2-ulosonic acid analogues. The four carbon alkene 1,2-dideoxy-3,4-0-cyclohexylidene-D-glycero-1-enitol was chosen as a model alkene on which to establish the methodology which could then be applied directly to the two target classes of compounds. Cycloadditions were performed using four nitrile oxides: ethoxycarbonylformonitrile oxide, benzonitrile oxide, acetonitrile oxide and (diethoxyphosphoryl)acetonitrile oxide. These proceeded in 40-88% yield with moderate π-facial selectivity (54-65% dc) in favour of Erythro adducts, with the major product in each case possessing S-configuration at the new asymmetric centre, C-5. This selectivity can be rationalised in terms of the 'inside alkoxy effect' proposed by Houk et al, and the 'homoallylic' modification offered by De Micheli et al. In an attempt to control selectivity in the nitrile oxide cycloaddition reactions, a dispiroketal protecting group was utilised. However, the increase in steric bulk and presence of a six-membered ring as the protecting group incorporating the diol, rather than the five-membered ring, does not appear to influence π-facial selectivity. Cycloadditions were also performed using the five carbon alkenes 1,2-dideoxy-3,5-0-ethylidene-D-erythro-pent-1-enitol and 1,2-dideoxy-3,5-0-benzylidene-D-threo-pent-1-enitol. With the former alkene these proceeded in 40-80% yield but with minimal π-facial selectivity (4-6% DE). However, changing the homoallylic substituent from axial to equatorial in the later alkene significantly increased the selectivity (to 62-64% de). In both instances the major product was shown by X-ray crystallography to possess S-configuration at the new chiral centre (C-5) corresponding to a threo relationship about the C-5/C-6 bond. The corresponding acetate derivatives also yielded threo adducts preferentially.

The composition of gellan gum elaborated by wild type Sphingomonas paucimobilis was studied. Gas chromatographic methods of analysis were compared. Analysis of neutral sugars was carried out by Saemen hydrolysis and Stones method. Stones method was preferred due to its simplicity and the production of comparable results to those obtained following Saemen hydrolysis. The neutral sugars of gellan gum were analysed by GC/MS following preparation of per-methylated alditol acetates (PMAAs). Two methods were used for making alditol acetates, the Hakamori and sodium hydroxide (NaOH) methods. Although the NaOH method was simpler and faster to carry out, the results were not representative of the true composition of gellan gum. The Hakamori method revealed the presence of 33%, 1,4 rha, 30% 1,3 glc and 31% 1,4 glc in agreement with the sugar compositions of O'Neill et al. (1983) and Jansson et al. (1983) who elucidated the structure of gellan gum simultaneously. To determine the glucuronic acid content of gellan gum, samples were carboxy-reduced prior to analysis. Two methods were compared, the lithium aluminium deuteride and tetrahydrofuran (THF) methods. The THF method revealed the presence of 24%, 1, 4 rha, 24% 1,3 glc 26% 1,4 glc and 25% 1,4, 6 glc (corresponding to glcA). Physiological studies were carried out with twenty mutant strains of S. paucimobilis in batch culture. The chemical composition, viscosity, gelation and rheological properties of EPS produced from many of these strains were further investigated. Some of the strains elaborated polysaccharide, named fine precipitate, which differed chemically from gellan gum. Rheological measurements were carried out on several EPS samples using a mechanical rheometer. Large differences in gel strength were detected.

Gum arabic was exposed to varying degrees of ionising gamma irradiation. The result confirms the lability of certain sugar and amino acid residues in the gum structure. A chemical deproteination was carried out on a gum arabic solution using butan-1-o1, and confirms previous results which indicate that the protein content of gum arabic cannot be totally isolated from the polysaccharide structure. Mild sequential Smith degradations were carried out on gum arabic, these degradations were less structurally destructive than previous periodate oxidations. Gum arabic was also fractionated by its ability to emulsify limonene oil and associate with its molecular surface. All the previous degradative studies give valuable structural information to elucidate the gum's heterogeneous branched nature. An analytical study was carried out on ten gum arabic samples of Sudanese and Nigerian origin, collected between 1950-1989. The gum's polysaccharide and amino acid composition were fully characterised and their emulsification stability determined. There are convincing indications that gum arabic has remained remarkably constant in chemical nature over the past 40 years. Seven gum exudates of the genus Albizia, four Acacia gum exudates and six Combretum gum exudates, all of which are not permitted as food additives, were analysed for their polysaccharide and amino acid composition. Food manufacturers require analytical data to characterise non-permitted gums to prevent their use as adulterants to substitute for gum arabic.

A route to carbon-linked disaccharides (two monosaccharide units linked by a carbon bridge rather than a glycosidic oxygen) employing nitrile oxide/isoxazoline chemistry has been investigated. This convergent approach is based on cycloaddition of sugar-derived alkene and nitrile oxide fragments, followed by ring cleavage and functional group manipulation of the resulting 2-isoxazoline. Careful selection of the nitrile oxide and alkene fragments defines much of the stereochemistry of the products. Two ω-unsaturated hexofuranoses were chosen for study: 3-O-benzyl-5,6-dideoxy-1,2-O-isopropylidene-α-D-xylo-hex-5-enofuranose (37) derived from D-glucose and methyl 5,6-dideoxy-2,3-O-isopropylidene-α-D-lyxo-hex-5-enofuranoside (137), which has the opposite configuration to (37) at C_2, derived from D-mannose. Four nitrile oxides were employed in cycloadditions to these alkenes: ethoxycarbonylformonitrile oxide (36) was used as a model 1,3-dipole to probe the π-facial selectivity of cycloadditions to alkene (137), and three pyranose 1-nitrile oxides (122), (146) and (150), derived from D-xylose, D-arabinose and D-galactose respectively. Additions to alkene (37) proceeded with a high degree of π-facial selectivity (56-82% d.e.) in favour of the 2-isoxazoline with erythro configuration. Similar selectivity was noted in additions to alkene (137). This selectivity can be explained in terms of the 'inside alkoxy effect' proposed by Houk and modified to include the 'homoallylic effect' put forward by De Micheli. Reductive hydrolytic cleavage of the 2-isoxazolines to release the latent β-hydroxy ketone functionality was carried out by both Pd/C and Ra-Ni catalysed hydrogenolysis. In the Pd/C case significant loss of the 3-O-benzyl protecting group was noted, however, Ra-Ni hydrogenolysis afforded the required β-hydroxy ketones (1→6)-carbonyl-linked C-disaccharides) in moderate yield (43-56%) along with a minor product identified as an epimeric mixture of the corresponding γ-amino alcohols ((1→6)-aminomethylene-linked C-disaccharides). Finally, reduction of the β-hydroxy ketone functionality and subsequent removal of the isopropylidene protecting groups afforded the dipyranose (1→6)-hydroxymethylene-linked C-disaccharides.

Studies presented in this Thesis fall into four distinct groups: 1. Analytical characterizations have been made of the gum exudates from: (a) Eight species of the Series Gummiferae and Vulgares and three highly proteinaceous species of the Series Juliflorae of the genus Acacia; (b) Six species from the genus Leucaena which are chemically very close to gum arabic; (c) Nine specimens of gum obtained from Combretum nigricans growing in different locations; (d) Six A.senegal gum samples from Uganda and a further six 'gum arabic' samples from different regions; (e) Seventeen species of gum exudates from 15 different genera such as Cassia grandis, Cercidium praecox, Parkia nitida, Prosopis chilensis, Sesbania sesban, Atalaya hemiglauca, Sclerocarya birrea, Pseudocedrela kotschyi, Senna nicaraguensis, etc. 2. A study of some of the structural features of Combretum nigricans gum by Smith-degradation showed that uronic acid and rhamnose groups occur in internal locations within the overall structure, and not as end-groups as is the case in gum arabic (A.senegal). 3. Amino acid data and ¹³C NMR spectra are presented for various fractions of A.senegal gum and for some highly proteinaceous Acacia gums (e.g. A.difficilis, A.eriopoda, A.tumida). The effect of enzymes on these gums is reported. 4. ¹³C NMR spectra for thirty different gum exudates are presented to show the characteristic 'fingerprint' patterns given by their polysaccharide structures. Information concerning their component monosaccharides, anomeric configurations, and linkages between the various sugar residues is given by spectrum analysis. This provides a most sensitive way to identify botanical species based on the total structure of exuded gums. The arabinose form (pyranose or furanose) and the various linkage configurations (α or β) in the gum structures are important and directly affect the physico-chemical behaviour of gum exudates. Acacia gums in which rhamnose is absent (or nearly absent) generally have a large proportion of β-L-arabinopyranose (A.seyal, A.sieberana, A.arabica gums etc.).

Starch is one of the most important natural products. It finds many industrial uses and applications, and, in different forms, is used throughout the world as a staple item of the human diet. Consequently, the structure and properties of starch have deservedly formed the subject of extensive investigations both by chemists and bio-chemists. Because of the inherent difficulties in the many problems which arose, and the continual need for development of new techniques, progress prior to 1925 was slow and uncertain. However, the subsequent elucidation of the precise structural configurations of the simpler sugars e.g. glucose, maltose and cellobiose, gave a firm foundation for future work, and steady progress ensued. One of the most important advances was the realisation of the apparent inhomogeneity of starch; this was followed in 1941 by the first quantitative fractionation into simpler components. As a result, the highly polymeric nature and main structural features of the molecule are now well estab¬ lished. In general, starches can be separated into two distinct entities, both of which are high polymers of α-D-glucopyranose units joined by α-1:4-glycosidic linkages: (a) Amylose - a mixture of very long, essentially inbranched, chains of these units. (b) Arnylopectin - a mixture of much shorter, highly branched chains of these units, the inter-chain, or branching, linkages being essentially α-l;6. Yet many problems still remain. Some are concerned with the clarification of controversial issues on which the present experimental evidence, as reported by various workers, is either at variance or considered to be inconclusive; the majority, however, are concerned with the fundamentally important details of fine structure and the molecular size and shape of the starch components. It is now accepted that there is little prospect of progress on these particular problems unless full use is made by the carbohydrate chemist of physical and enzymic methods to supplement purely chemical approaches. When the researches reported in this thesis were begun, it was apparent that, before the established physical techniques used in polymer chemistry for the determination of molecular size and shape could be profitably applied, work on the following topics was an essential preliminary in order that the materials to be studied could be adequately and accurately characterized:- (1) The development of a differential potentiometric iodine titration technique for the determination of the % amylose in starches and their fractionated components, and for the possible characterization of other a-1:4-glucosans. It was desirable that the apparatus should be considerably more sensitive and accurate than any of those previously reported, yet also be capable of simple operation as a routine analytical method. (2) A study of the oxidation of starches by the periodate ion in view of the low quoted percentage accuracy of the technique, and the then existing lack of agreement in the literature on the following points; (a) conditions for the quantitative estimation of the formic acid released, (b) the length of time required for complete oxidation, and the possibility of over-oxidation, (c) the presence of intact glucose residues after oxidation, suggesting that inter-chain linkages other than 1:6 existed in amylopectin. (3) A study of the effect of the presence of contaminating protein on results obtained by use of the potentiometric iodine titration and periodate oxidation techniques. (4) The preparation, and purification, of starches from several botanical sources which had not previously been studied, so that the use at any stage of industrial samples of unknown origin, treatment, and history could be avoided. By preparation of the starches under the mildest possible conditions, so as to minimise degradation, these same samples could later be used for physico-chemical studies of molecular size and shape. The opportunity was taken, after the extraction of each starch, to apply a graded series of extractions to the residual plant material; the fractions isolated were analysed for protein, ash, and uronic acid content, and the polysaccharide content was investigated by chromatographic analyses of the sugars liberated on hydrolysis.