Alginates are a class of natural unbranched linear polysaccharides, and consist of the monomers β-D-mannuronic acid and α-L-guluronic acid. The inherent physical properties, relative ease of modification, wide availability and good biocompatibility of alginates have gained a great deal of attention with regards to therapeutic applications. Heparin is a highly negatively charged linear glycosaminoglycan that is widely used as an anticoagulant. The presence of carboxyl groups and several sulfate groups gives heparin its negative charge, while iduronic acid moieties confer a high degree of flexibility to the polysaccharide by being able to assume different stable conformations. Heparin shows diversity in molecular weight, monomer sequence and modification pattern, resulting in a vast range of biological effects. When administered therapeutically, this can in cause an unpredictable dose response and potentially severe adverse effects in certain patients. The main objective of this study was to create a structural analog of heparin exhibiting a more regular structure and distribution, through chemical sulfation of alginate using chlorosulfonic acid. Other important aims were to characterize the analog in terms of structure, distribution and sulfation degree, and assess protein binding and anticoagulating properties of the sulfated alginates in comparison with heparin and the unmodified alginate templates. Sulfation was performed using chlorosulfonic acid in formamide on a polymannuronic acid (poly-M) and a polyalternating alginate with a guluronic acid fraction of FG = 0.46 (poly-MG), introduced through enzymatic epimerization. FTIR, elemental analysis with HR-ICP-MS and carbon NMR were employed to detect the attached sulfate groups on the alginate. The average molecular weights and the mass distributions of the alginate samples were studied using SEC-MALLS. Elemental analysis was used to estimate the sulfation degrees of the alginates, and 13C NMR was employed to study substitution patterns, provide additional DS estimates and assess sample purity. The protein binding properties of the sulfated alginates were evaluated by studying their ability to release hepatocyte growth factor and osteoprotegerin bound to myeloma cells. Anticoagulating properties were studied by measuring prolongation of plasma coagulation time as a result of sulfated alginate supplementation. The alginates were successfully sulfated and exhibited different degrees of sulfation obtained by varying the chlorosulfonic acid concentration used (1 - 10 %), as estimated by elemental analysis. The poly-MG alginate showed increased solubility during the sulfation reaction, resulting in a higher estimated DS at lower chlorosulfonic acid concentrations compared with poly-M. No apparent degradation of the alginates as a result of the sulfation was observed, although preliminary acid hydrolysis resulted in a molecular weight disparity between poly-M and poly-MG samples. Analysis of carbon NMR spectra allowed characterization of novel peaks and secondary DS estimations for the sulfated poly-M samples, while the complexity of the sulfated poly-MG spectra prevented confident characterization of the structures. Sulfation resulted in a profound improvement of the protein binding properties of the alginates, and showed prolongation of the plasma coagulation time at high treatment concentrations.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:ntnu-16802 |
| Date | January 2012 |
| Creators | Arlov, Øystein |
| Publisher | Norges teknisk-naturvitenskapelige universitet, Institutt for bioteknologi, Institutt for bioteknologi |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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