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The application of nitrile oxide/isoxazoline chemistry towards the synthesis of higher sugars (monosaccharides containing seven or more contiguous carbon atoms) has been investigated. This convergent approach, involving the cycloaddition of sugar derived alkene and nitrile oxide fragments and subsequent manipulation of the resulting isoxazoline, has much of the product stereochemistry preselected. Three ω-unsaturated monosaccharides were chosen for study: the D-glucose-derived six carbon unit (<i>94</i>), its C-3 epimer (<i>102</i>) and a seven carbon alkene, (<i>97</i>), prepared from D-galactose. Benzonitrile oxide was employed in preliminary studies as a model 1,3-dipole to probe the π-facial selectivity of cycloaddition to these alkenes. Cycloadditions with the carbohydrate derived nitrile oxides (<i>117</i>), (<i>121</i>) and (<i>125</i>) were subsequently examined. For alkenes (<i>94</i>) and (<i>97</i>) a high degree of stereoselectivity (typically <i>ca</i> 85:15) favouring the formation of the <i>erythro</i> 2-isoxazoline was obtained. This observation can be rationalised in terms of the 'inside alkoxy effect' proposed by Houk in which the allylic oxygen occupies the <i>inside</i> position in the transition state. D-<i>Ribo</i>-alkene (<i>102</i>) proved to be an exception; cycloaddition with ethoxycarbonylformonitrile oxide (<i>117</i>) occurred with negligible stereoselectivity (51:49). This result demonstrates the role played by the homoallylic oxygen in determining the stereoselectivity. Pd/C mediated reductive hydrolytic cleavage of the 2-isoxazolines unmasked the β-hydroxy ketone functionality of the deoxy-ulose derivatives. Subsequent reduction furnished a pair of separable diastereomeric 1,3-diols, whose stereochemistry was determined by examination of the ^1H n.m.r. spectra of the corresponding isopropylidene ketals. A series of deoxy -octose, -nonose, -decose, -dodecose and -tridecose monosaccharides have been thus prepared. Finally, other aspects of isoxazoline chemistry have been investigated. The reduction of isoxazoline (<i>123</i>) by lithium aluminium hydride afforded the <i>syn</i>-γ-amino alcohol (<i>176</i>) as the only isolated product. <i>Trans</i> α-enones (<i>174</i>) and (<i>175</i>) were synthesised stereospecifically by dehydration of the corresponding β-hydroxy ketones.

Mucins fonn the polymer matrix of mucous gels and give them their unique lubricating and protective properties. Most mucous secretions contain a heterogeneous population of mucins. The identification and quantitation of mucin phenotypes obtained from mucous secretions is a problem due to the density of protein glycosylation in these molecules. With the detennination of large portions or in some cases the complete primary sequence of a number of mucins. we now have precise protein compositional data. In particular it is becoming clear from the sequences of the heavily glycosylated domains that they contain distinctive ratios of the amino acids Ser, Thr and Pro. There is also evidence to suggest that mucin molecules have a unique pattern of glycosylation. These criteria could be used as a means for biochemically identifying mucins purified from mucous gels. The aims of this study were three-fold. Firstly to provide an approach for assessing the heterogeneity of mucin mixtures. Secondly to provide criteria by which the different mucins may be identified. Thirdly to establish methods for measuring their mass contribution to the mixture. The benefits of attaching mucins to PVDF membrane supports in order to simplify chemical analyses are described. j3-elimination, aqueous hydrazinolysis and anhydrous hydrazinolysis methods for the removal of O-linked glycan chains and fluorophore labelling and PAGE techniques for obtaining a chemical signature of the glycan chains are discussed. A strategy is presented for isolating, purifying and analysing mucins. using the previously uncharacterised high molecular weight salivary mucin MG 1 as a model. Intact mucins were extracted in 4 M GuHCI and separated using gel filtration chromatography and isopycnic density gradient centrifugation. The mucins were reduced and thereafter fractionated using ion exchange chromatography. Mucins were analysed using agarose gel electrophoresis and Western blotting. A broad and heterogeneous distribution of salivary mucin subunits was detected by both separation methods. Subunit fractions were trypsin digested, generating large glycopeptides and small peptide fragments, which were separated by gel chromatography. Amino acid analysis of the large glycopeptides from subunit fractions across the Mono Q yielded distinctive and identical Ser:Thr:Pro:Ala ratios. A chemical "fingerprint" of the small tryptic fragments was obtained by a combination of reverse phase chromatography and MALDI-TOF MS analysis. The composition and peptide mapping data were almost identical to that obtained from the MUCSB mucin from respiratory tract mucus. These data indicated that the MG 1 mucin in saliva is composed almost entirely of the MUCSB mucin. It was concluded that the physical heterogeneity observed must have been a result of variable glycosylation. Application of this strategy was used to show that MUCSB is the major large secreted mucin in nonnal cervical mucous secretions and many pathological respiratory mucous secretions. It was concluded that the approaches outlined above may prove valuable in three respects:- the assessment of mucin heterogeneity. the identification of their underlying polypeptide and the characterisation of the glycan chains from mucins.

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This thesis describes studies on the difficulties and solutions involved with the separation and analysis of heparin and heparan sulfate (HS) saccharides. Their repeating uronic acid and glucosamine nature means that they can only be distinguished or separated via their sulfates and acetates, making them a significant challenge using the high performance liquid chromatography (HPLC) techniques available. More comprehensive knowledge of the limited methods available, as well as new methods, is essential for optimal results. Size exclusion chromatography (SEC) separation of glycosaminoglycans (GAG) oligosaccharide mixtures is commonly used but the products have not previously been fully characterised, and studies here aimed at filling in some gaps in understanding of this complex process. Mass spectrometry is particularly useful for this task, as it can provide detailed molecular masses and structural information. Mass spectrometry analysis of SEC separated oligosaccharides showed that although these mixtures were separated via their hydrodynamic volume as expected, complex separations were observed. Increasingly poor resolution and overlapping of saccharide sizes was evident, particularly for hexasaccharides and larger, and indicated that previous suppositions about the nature of "size-defined" saccharides produced by SEC are unsound. Mass spectrometry also led to an unexpected identification of much higher proportions of odd-numbered oligosaccharides than previously described. High performance liquid chromatography (HPLC) techniques are commonly used in the glycosaminoglycan (GAG) field, but still have limitations, making the isolation of homogenous glycosaminoglycan (GAG) oligosaccharides of bioactive size (typically dp6-8+) an ambitious task. Here the development and application of porous graphitised carbon (PGC) chromatography columns was explored for preparative, analytical and micro- separations of heparin/HS saccharides based on a complex interplay of size, shape and charge. Graphitised carbon displays an additional advantage over most high performance liquid chromatography techniques used in the separation and analysis of heparin and heparan sulfate (HS) as it is compatible with mass spectrometry. Compatibility with electrospray mass spectrometry (ESI-MS) was confirmed, permitting development of an in- line method for the separation and characterisation of complex mixtures and pure saccharide species. Heterogeneous saccharide pools can be analysed rapidly, providing structural insights into multiple saccharide components, and detailed structural / information can be obtained on pure saccharide species. Finally, this thesis described studies used existing and novel high performance liquid chromatography techniques to separate and isolate single glycosaminoglycan oligosaccharides with the goal of sequencing these structures using electrospray mass spectrometry. Heparin and heparan sulfate (HS) disaccharide standards were initially studied by characterising internal fragmentation patterns unique to their sulfation patterns. This information was then used to explore sequencing of larger pure GAG oligosaccharides isolated from tissue HS. Compatibility of derived oligosaccharide sequences with disaccharide compositional analysis was confirmed. Verification that this electrospray mass spectrometry approach could sequence accurately was obtained by blind testing of chemically synthesised heparin / heparan sulfate oligosaccharides with known structures validated by different methods 'from other labs. The initial data suggest that the method has potential for sequencing of pure oligosaccharides, and by combining this method in future with bioactivity assays should assist in unravelling the structure-activity relationships of heparin / heparan sulfate saccharides.