UC Riverside

The focus of this dissertation is on the development of improved analytical methods for the characterization of anionic carbohydrate biopolymers. Our goal is to extract important information from complex mixtures of heterogeneous polysaccharides by characterizing their substituent oligosaccharides in terms of monosaccharide composition and primary and secondary structure. This work focuses on the application of two major analytical platforms: spectroscopy and chromatography.

The development of new nuclear magnetic resonance spectroscopy (NMR) tools for the characterization of the biologically active carbohydrates heparin and heparan sulfate (HS) is presented in Chapters 2-4. These biopolymers are members of a special class of nitrogen containing polysaccharides called glycosaminoglycans (GAGs). Our discovery of experimental parameters for detection of the 1H and 15N resonances of the sulfamate (NHSO3-) groups of the N-sulfoglucosamine residues of heparin and HS was an important breakthrough and demonstrated the sensitivity of these chemical shifts to local structure. Evaluation of the exchange kinetics of the sulfamate group protons with the bulk aqueous solvent allowed us to explore the relationship between primary and secondary structure in heparin oligosaccharides and led to our identification of the first solution state hydrogen bond between a sulfamate group NH proton and the adjacent 3-O-sulfate moiety of the heparin drug Arixtra (Fonduparinux sodium). Generation of a 1H and 15N chemical shift library for a series of heparin-derived oligosaccharides and chemically modified heparins allowed the assignment of the [1H,15N] HSQC correlations in spectra measured for unfractionated and low-molecular-weight heparins (LMWH) as well as commercial and human-derived 15N-enriched HS isolated from cell culture.

The analysis of LMWH samples by reversed-phase ion-pairing ultraperformance liquid chromatography with detection by mass spectrometry (RPIP-UPLC-MS) is described in Chapter 5. This method utilizes volatile amphiphilic ion-pairing reagents to retain anionic oligosaccharides on a hydrophobic stationary phase while facilitating detection with the information rich method of mass spectrometry. With this platform we were able to separate individual components of complex LMWH mixtures providing a fingerprint of different drug preparations. The potential of this approach for quality assurance applications was illustrated by the comparison of the relative abundances of individual oligosaccharides highlighting slight differences between LMWH samples.