Towards the Structural Characterisation of Sulfated Polysaccharides from Red Seaweeds and Animal Tissues

Sulfated polysaccharides are structurally diverse biomolecules with broad applications across the medical, pharmaceutical, and food industries. They are used in drug delivery systems, wound dressings, anticoagulants, tissue engineering scaffolds, and as stabilisers or thickeners in food and cosmetic formulations. The functional properties of these complex biopolymers are closely linked to structural features including molecular weight, monosaccharide composition, glycosidic linkages, and sulfation patterns. However, this structural complexity presents significant challenges for detailed structural characterisation, requiring the use of advanced, complementary analytical techniques specifically suited to their complex structures. Research described in this thesis focused on the detailed structural characterisation of two naturally occurring sulfated polysaccharides, carrageenan and heparan sulfate, with the specific aims of: 1. Advancing our understanding of carrageenan structures from the Gigartinaceae family, and 2. Developing chemical strategies for preparing and analysing structurally informative disaccharides from heparan sulfate. Carrageenans are sulfated polysaccharides produced by red seaweeds. In this research, carrageenans from two species of Rhodoglossum were extracted and characterised: Rhodoglossum sp., collected in New Zealand, and R. gigartinoides, collected in Australia. Both species belong to the Gigartinaceae family and are closely related, as indicated by rbcL sequence data. Carrageenans from the gametophytic (haploid) and tetrasporophytic (diploid) life stages of both species were characterised using a combination of chemical derivatisation, chromatography, and spectroscopy techniques. Similar to other members of the Gigartinaceae, the different life stages of these species were found to produce different types of carrageenans. The gametophytes of both Rhodoglossum species were shown to contain κ/ι-hybrid type carrageenans. The tetrasporophytes contained λ-type carrageenans which included π, ξ, and a modified ξ-type carrageenan having a higher sulfate content. Carrageenans from tetrasporophytic plants of R. sp. and R. gigartinoides differed in the proportion of ξ and modified ξ-type carrageenans. These carrageenans have similar structures to those of other closely related endemic New Zealand species of the Gigartinaceae, supporting the use of carrageenan structures as chemotaxonomic markers.  In continuation of the investigation into sulfated polysaccharides, chemical strategies were explored for generating disaccharides from heparan sulfate, a glycosaminoglycan found in animal tissues. The first approach involved butanolysis, which preserves uronic acid epimers but results in the loss of sulfate and acetyl groups. UPLC–MS analysis revealed a complex mixture of monomers, disaccharides, and longer oligosaccharides. The disaccharides generated through this approach were subsequently characterised.  The second approach adapted a methylation methodology, commonly used for plant polysaccharides, to facilitate the formation of disaccharides from which information on sulfation pattern could be inferred. This strategy involved carboxyl reduction, permethylation, acid-catalysed cleavage, and subsequent acetylation to produce derivatives amenable to GC–MS analysis. Hydrolysis of carboxyl-reduced, permethylated HS using TFA was not suitable for the intended purpose. In contrast, methanolysis yielded disaccharides as the major products, however; not in the proportions expected. The results demonstrated that the application of this methodology to glycosaminoglycans is feasible, but the complexity of the chemical processing involved necessitates further work before it can be used as a diagnostic analytical tool.