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Molecular Level Characterization of Heparin Structure


Heparin is unbranched anionic polysaccharide that is part of the glycosaminoglycan family. Although its structural microheterogeneity allows heparin to mediate a variety of biological processes by binding to over 400 different proteins, it also makes structural characterization challenging. This dissertation focuses on the separation and isolation of unique heparin oligosaccharides and their structure elucidation using nuclear magnetic resonance (NMR) and mass spectrometry. The impact of sequence on secondary structure is evaluated through detection of specific intramolecular hydrogen bonds.

Separation of the low-molecular-weight heparin enoxaparin was accomplished using size-exclusion chromatography (SEC) followed by strong anion-exchange high-performance liquid chromatography (SAX-HPLC). Typically, size-similar SEC fractions are combined prior to SAX-HPLC separation. However, screening individual SEC fractions using their sulfamate 1H and 15N chemical shifts allowed for SAX-HPLC resolution of the SEC fractions containing 3-O-sulfated oligosaccharides and led to the isolation and characterization of a unique 3-O-sulfated heparin tetrasaccharide.

The effect of structure, particularly glucosamine 3-O-sulfation, on hydroxyl proton hydrogen bonding was explored. Experimental conditions were optimized for the NMR detection and identification of heparin hydroxyl protons. Solvent exchange rates were determined using exchange spectroscopy (EXSY). Structurally specific differences in the hydroxyl proton exchange rates of the hydroxyl protons were explored for Arixtra, a synthetic pentasaccharide that mimics the Antithrombin-III binding sequence, and for isolated oligosaccharides that do not contain a 3-O-sulfated glucosamine. Transverse relaxation rates (R2) measured for the Arixtra hydroxyl protons using the Carr-Purcell-Meiboom-Gill experiment are compared with EXSY solvent exchange rates. Because R2 values are sensitive to factors other than exchange, good correlations with the EXSY exchange rates were not uniformly obtained.

Experimental conditions are reported allowing the 1H NMR observation of the amine protons of glucosamine. Exchange properties of amine protons in monosaccharides differing only by 3-O-sulfation are compared. Amine group acidity (pKa) was measured to probe participation of the amine group in a salt bridge. Although no evidence of a salt bridge between the amine and sulfate groups was found in the 3-O-sulfated glucosamine (GlcN3S) monosaccharide, these experiments demonstrate the feasibility of probing for structurally important salt bridges in longer oligosaccharides.

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