Defense Date

2014

Document Type

Thesis

Degree Name

Master of Pharmaceutical Sciences

Department

Pharmaceutical Sciences

First Advisor

Umesh Desai

Abstract

Glycosaminoglycans (GAGs) are a family of linear bio-polysaccharides, which are heterogeneously modified with negatively charged sulfate and carboxylate groups. They are located on every cell surface, extracellular matrix or intracellular space in the body. GAGs are composed of alternating units of an amino sugars (glucosamine or galactosamine) and hexuronic acid/hexose (iduronic acid, glucoronic acid/ or galactose), which are linked by glycosidic bonds with different geometries. In recent years, GAGs have attracted considerable interest. GAGs play vital roles in fundamental biological processes, such as hemostasis, angiogenesis, cell signaling, growth and differentiation. Thus, GAGs contribute to a number of diseases such as thrombosis, cancer, inflammation, osteoarthritis and degenerative diseases. One of the most studied GAGs is heparin, which is widely used as an anticoagulant and is also implicated in other biological processes. Despite its extensive clinical use, heparin continues to suffer from major problems, such as life threatening hemorrhagic complications and heparin-induced thrombocytopenia. These activities originate from the large number of glycan sequences generated during its biosynthesis. Many different enzymes act in a non-template fashion to produce heparin chains with various chain lengths, sulfation and acetylation patterns. Their inherent heterogeneity, complexity and highly anionic nature have seriously limited the development of tools for rapid identification of sequences critical for many biological activities. A RPIP-UPLC MS protocol was developed to separate and characterize structures of heparin oligosaccharides prepared through enzymatic cleavage process and chemoenzymatic synthesis. In designing such protocol, several UPLC and MS parameters were considered. An efficient separation of each oligosaccharide mixture was achieved with different ion-pairing reagents. Yet, the structural elucidation of the multiple chromatographic peaks was hindered by the heterogeneity inherent in these mixtures even with supposedly defined standards. In order to elucidate the structures of these complex molecules, we utilized a strategy, which exploits the ease with which sulfate groups can be fragmented during MS ionization. A systematic increment of the MS cone voltage was employed starting from a minimum dissociation voltage, where the intact molecule is preserved, to a high enough dissociation voltage, where the only core oligosaccharide backbone was retained. This allowed identifying the number of sulfate groups and the core structures. However, positions of substituents were difficult to pinpoint because of the phenomenal number of possibilities. Such analysis would require tandem MS/MS–based approaches.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-19-2014

Available for download on Friday, August 16, 2024

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