Glycosaminoglycans (GAGs) mediate a variety of biological processes through their interactions with proteins. To advance our understanding of these interactions, it is necessary to explore the structural characteristics and physico-chemical properties of GAGs. The major focus of the research presented in this dissertation is the structural analysis of GAG oligosaccharides with emphasis on the chemical modification and isolation of oligosaccharides with unique structures. Reactions for the solvolytic de-N-sulfation, chemoselective N-acetylation, and reduction of reducing end residues with sodium borohydride were employed to obtain oligosaccharides containing amide and free amine moieties with unique structural elements. The impact of oligosaccharide primary structure on elements of secondary structure in aqueous solution was explored using 1H NMR.

The role of rare, but biologically important 3-O-sulfated glucosamine residues in heparan sulfate related oligosaccharides was examined. Possible salt bridge formation was probed through the measurement of amino proton temperature coefficients, amino proton solvent exchange rates, and comparison of the carboxylate and amino group pKa values of de-N-sulfated Arixtra (dNSA). Reduced amino proton temperature coefficients, accelerated exchange rates measured by exchange spectroscopy (EXSY), and altered pKa values of amino and carboxylate groups provided evidence that the dNSA 3-O-sulfated glucosamine amino group is likely involved in salt bridge with the neighboring 3-O-sulfo group and possibly with carboxylate group of the adjacent iduronic acid residue. This hypothesis was supported by the results of molecular dynamics simulations.

The effect of oligosaccharide structure on the solvent exchange behavior of GAG oligosaccharide amide group protons was evaluated through comparison of temperature coefficients, activation energy barriers, and pH titrations of oligosaccharides containing amide groups. A library of 20 oligosaccharides differing in size, glycosidic linkage, saccharide type, and sulfation position and extent, allowed the examination of the effect of charge repulsion by nearby negatively charged sulfate and carboxylate groups on the hydroxyl ions responsible for catalyzing amide proton exchange. This charge repulsion effect calls into question the validity of using exchange rates and activation energies for detection of hydrogen bonds without support from temperature coefficients measured for labile protons.

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.

The goal of this dissertation is to develop improved analytical methods for the separation and characterization of ionic analytes while gaining insights into their underlying mechanism. The work focused reversed-phase ion-pair high-performance liquid chromatography (RPIP-HPLC) and capillary isotachophoresis (cITP) separations of the highly charged molecules, heparin and heparan sulfate. Furthermore, this work will explore methods to improve the sensitivity of NMR, with a focus on both microcoil and microslot NMR probes.

RPIP-HPLC is an important method for the separation of ionic solutes using lipophilic ions, referred to as ion-pairing reagents (IPR), as mobile phase modifiers to aid in their retention on a hydrophobic stationary phase. However, the fine details of the RPIP separation mechanism are still being debated. The described work investigates the role that competition between ion-pairing reagents plays in the separation of structural isomers of heparin and heparan sulfate (HS) disaccharides, and how factors such as IPR concentration and buffer pH affect the resolution of their anomeric forms.

NMR spectroscopy can yield a vast amount of structural information about a compound without destroying the sample. However, NMR is often limited by poor sensitivity and for samples containing more than one compound of interest, the spectra

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obtained can quickly become convoluted, complicating interpretation. To solve these problems we use the pre-concentration/separation method cITP coupled with microcoil NMR detection. The first part of this work uses doxepin as a model compound to study both the type and strength of interactions that occur during cITP. This work focuses on the binding interactions of doxepin with the buffer modifier Beta-cyclodextrin and the role that the counterion acetate plays in the cITP process.

The insights gained in the doxepin cITP study were then used to develop an improved cITP-NMR method for the analysis of heparin and HS derived oligosaccharides, focusing on designing a buffer system that reduces the effects of current induced resonance broadening that occurs when cITP is coupled to microcoil NMR. Finally, this work demonstrates the coupling of cITP with a new microslot NMR probe design which eliminates the deleterious affects of the magnetic field produced by the separation.

The goal of this dissertation was to advance plant metabolomics through optimization of biological experimental design, sampling and sample preparation, data acquisition and pre-processing, and multivariable data analysis. The analytical platform most employed for comparative metabonomics was nuclear magnetic resonance (NMR). Liquid-chromatography (LC) coupled to NMR and mass spectrometry (MS) extended metabolic profile coverage from primary into secondary metabolic pathways. Comparative profiling of tissue extracts by LC-MS and complementary analyses by NMR were performed to establish metabolite identity and quantify responses to low-oxygen stress using the model Angiosperm, Arabidopsis thaliana.

Angiosperms, or flowering plants, constitute the most diverse and numerous group of land plants, and the most commercially important, producing an estimated 200,000 small molecule metabolites through conserved primary metabolism and divergent secondary pathways. No single metabolic profiling experiment has been able to quantify metabolite abundance over the relevant dynamic range (spanning seven orders of magnitude) and across the necessary structural diversity (chemical space coverage exceeds all known synthetic molecules). Challenges in plant metabolic profiling also arise through inherent limitations in methods (availability of diverse solid-phases, solvents, pure standards), instrument performance (reproducibility, robustness, speed, sensitivity), and data management (pre-processing, modeling, data basing and sharing).

The field of plant metabolic profiling has grown steadily over the past two decades, although it is still considered an emerging technique. Comparison of methods for sample preparation provided a foundation for one-dimensional NMR-based comparative metabonomics. Signal overlap was alleviated by liquid-liquid extraction (LLE), solid-phase extraction (SPE), and liquid chromatography (LC). A combined approach involving ultraviolet-visible absorption spectroscopy (UV-Vis), NMR, exact mass MS, and tandem MS (MS/MS) was used for dereplication.

Results are presented illustrating the ability of one-dimensional NMR combined with multivariable data analysis (MVDA) to quantify responses of a model plant to a biological problem of fundamental and practical relevance: cellular respiration under oxygen-limited conditions. The suitability of LC-NMR and LC-MS for dereplication is highlighted. Profiling of secondary metabolites by UPLC-MS combined with MVDA was undertaken for comparison with NMR-based comparative metabonomics. Efforts toward quantitative targeted analyses, hurdles and solutions, are highlighted.

The colon has long been considered as a waste-packing organ, but has been gaining importance with the discovery of the complex gut microbiome which is located primarily in the colon. The gut microbiome is comprised of more than 800 species of bacteria that help harvest energy, synthesize vitamins, and shape the immune system. Disturbances in the gut microbiome have been implicated in many diseases including allergies, autism, colorectal cancer, Crohn’s disease, diabetes, and obesity, making it critically important for human health. While the focus of most researchers has been solely on the bacterial component of the gut, little is understood about the mechanism of metabolic communication between the gut microbiome and its human host.

In this work, nuclear magnetic resonance, ion chromatography, and gas chromatography-mass spectrometry are used to identify and quantify major metabolites in extracts of fecal material isolated from the rat ileum, cecum, proximal, mid, and distal colon. These metabolites include short chain fatty acids that are the preferred energy source for colonocyte, amino acids, polyamines, bile acids, choline metabolites, phenolic compounds, and inorganic ions. This study provides information on the luminal metabolome, its stratification along the colon, and the assortment of metabolites available for absorptive transporters within each segment.

Metabolic profiling of along different segments of the colon provides knowledge about the metabolites available in the stool for transport across the epithelial tissue into the bloodstream or for further transformation by the gut microbiome. In this work, use of the Ussing chamber provides evidence of the transport behavior of metabolites including short-chain fatty acids, amino acids, vitamins, and choline derivatives across excised colonic epithelial tissue. These in vitro experiments utilize two chambers separated by epithelial tissue representing both the lumen and serosal sides of the colon. 1H-NMR analysis of these chambers has shown that over a dozen metabolites including SCFAs, amino acids, and choline derivatives are involved in transport.

Due to rising salinity of freshwater systems and the lack of information about contaminants in saltwater lakes, there is a need for new methods to identify stressors and their effects in saline environments. We propose the use of the saltwater aquatic crustacean, Artemia franciscana, as a model organism for environmental metabolomics analysis of stressors for saltwater ecosystems. Artemia is an ideal indicator species because it is well-studied, have short life-cycles, and are robust. Their hemolymph has a high concentration of small molecule metabolites in an open circulatory system that is susceptible to environmental conditions and amenable to metabolomics analysis. Environmental metabolomics methods use analytical techniques, such as nuclear magnetic resonance (NMR) and mass spectrometry (MS), with chemometric analysis to characterize the interactions of an organism with its environment. When exposed to a stressor, these methods are used to identify biomarkers of exposure and assess the biochemical pathways that are impacted.

The small molecule metabolite profile of Artemia was characterized using 1D and 2D NMR and GC-MS and 43 metabolites were identified. Environmental metabolomics methods were developed using untargeted 1H NMR and GC-MS analysis coupled with high-content imaging to assess Artemia exposed to established and emerging stressors, which included temperature stress, Roundup® herbicide, and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) flame retardant. Targeted metabolomics analysis with LC-MS/MS was introduced to study the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), 118 metabolites were identified with this method. Multivariate and univariate statistical analysis was used to identify biomarkers of exposure for each stressor and pathway analysis identified biochemical pathways that were likely affected. Although each stressor had a unique effect, it was determined that sugars, which are important for energy pathways such as glycolysis, and osmolytes, such as betaine and gadusol, play an important role in Artemia stress response.

The goal of this dissertation is to develop and apply NMR methods for the analysis of heparin and its impurities. As a result of the intentional contamination of heparin with oversulfated chondroitin sulfate (OSCS), significant efforts have been placed on the development of analytical methods to evaluate heparin purity. To advance the suite of analytical methods available for heparin analysis, HPLC-NMR and diffusion NMR methods were developed for its characterization. Our HPLC-NMR method focuses on the WAX separation of intact glycosaminoglycans (GAGs) coupled to on-line UV and NMR spectroscopy for detection and identification of heparin contaminants. The weak anion exchange (WAX) method developed allows for the timely separation of heparin and its impurities, OSCS, dermatan sulfate, and chondroitin sulfate A. In addition to the study of heparin impurities using WAX-HPLC, the characterization of intact and digested heparin samples was investigated using diffusion NMR. Because the diffusion behavior of the intact biopolymers can be dominated by viscosity and molecular crowding effects even in dilute GAG solutions, NMR diffusion results must only be interpreted qualitatively. These limitations can be largely overcome by digesting heparin to low molecular weight oligosaccharides, significantly improving the resolution of heparin and its biopolymer impurities.

The second part of this dissertation is on the characterization of heparin-derived oligosaccharides. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, extensive or partial digestion of the full length polysaccharides is a common practice. Size-exclusion chromatography was used to separate digested heparin oligosaccharides into size-based fractions while SAX-HPLC was used to further purify individual oligosaccharides for NMR characterization. Although, NMR spectroscopy is a powerful tool for structure determination, it trails other analytical techniques in sensitivity. Microcoil NMR is an inexpensive method for enhancing the mass sensitivity of NMR allowing structure elucidation with 5-10 μg of isolated material. Results are presented showing the utility of microcoil NMR for the structure characterization of microgram amounts of heparin-derived tetrasaccharides, reducing the NMR sample requirements by 2-fold when compared to analysis in a Shigemi tube.

In Chapter 6, an IMPACT-HNMBC experiment was used in the detection of ^{15N in amino sugars through long-range couplings to carbon-bound protons of the sugar ring. Comparison of the ge-HMBC, IMPACT-HMBC and a modified IMPACT-HNMBC pulse sequence revealed improved sensitivity, resolution and reduced F1 noise with the IMPACT-HNMBC experiment.}

Wastewater is a complex mixture of natural and anthropogenic pollutants including inorganic and organic species. The use of disinfection treatments such as chlorine is necessary to attenuate pollutants encountered in wastewater prior to its discharge into the environment. In recent years, the presence of pharmaceuticals and personal care products (PPCPs) in wastewater has received increasing attention due to the concern that these compounds may disrupt the normal endocrine function of aquatic biota. Less attention has been paid to the potential for PPCP transformation into new, chemically unique compounds via disinfection treatments. This dissertation focuses on the identification and quantification of halogenated pharmaceutical analogs in wastewater effluent generated by disinfection treatment with chlorine. The goal of this research is twofold: 1. Identify chlorination transformation products (TPs) of selected PPCPs, synthesize and purify standards (with and without deuterium labeling) for each unique TP and characterize the structure and purity of the TP standards; 2. Use these TP standards to develop targeted high performance liquid chromatography hyphenated to mass spectrometry (HPLC-MS) methods for the analysis of the selected PPCPs and their TPs and accurately quantify each analyte in solid phase extracts (SPE) of several wastewater effluents from Southern California.

This dissertation initially focuses on the chlorination TPs of the lipid regulator gemfibrozil and their occurrence in advanced primary wastewater effluent. Standards of gemfibrozil's TPs were synthesized, purified by HPLC and characterized by nuclear magnetic resonance (NMR) spectroscopy and accurate mass MS. This research is extended to identify and quantify the chlorination TPs of the analgesic salicylic acid, the non-steroidal anti-inflammatory (NSAID) diclofenac and the plasticizer bisphenol A in secondary wastewater effluent. Standards for TPs of the additional compounds were synthesized, isolated and characterized by the same methods as used for the gemfibrozil TPs. The dissertation culminates by expanding the selected PPCPs for secondary and tertiary wastewater analysis to include the surfactants octylphenol and nonylphenol, the NSAID naproxen, in addition to the synthesis of deuterated analogs of each PPCP and TP. Several of the identified TPs were quantified for the first time at ng/L levels in several of the analyzed wastewaters.