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The synthesis of glycoconjugates has facilitated a wide variety of techniques for the detailed study of carbohydrates and their interactions in biological systems. However, when only small amounts of the isolated oligosaccharide are available, multistep synthetic approaches are not possible. This thesis explores new synthetic methods for the preparation of glycoconjugates without protecting group manipulations. A new glycosidation method was developed which introduces N-glycopyranosylsulfonohydrazides as glycosyl donors for the protecting group-free synthesis of O-glycosides, glycosyl azides and oxazolines. The glycosyl donors were synthesized in a single chemical step by condensing p-toluenesulfonylhydrazide with the corresponding mono- and disaccharides. The N-glycopyranosylsulfonohydrazides were activated with NBS and subsequently glycosylated with the desired alcohol or transformed to the oxazoline or glycosyl azide. Recent advances in chemoselective ligation methods for the functionalization of unprotected carbohydrates have provided new routes towards complex glycoconjugates. Despite the wide use of those chemoselective methods, the properties of these linkages have not been thoroughly investigated. Characterization of a series of glycoconjugates formed by chemoselective ligation of xylose, glucose and N-acetylglucosamine with either an acyl hydrazide, a p-toluenesulfonylhydrazide or an N-methylhydroxylamine were carried out to gain further insight into the optimal conditions for the formation and the stability of these useful conjugates. Their apparent association constants (9-74 M-1) at pD 4.5, as well as rate constants for hydrolysis were determined at pH 4.0, 5.0 and 6.0. The half-lives of the conjugates varied between 1 h and 300 days. All the compounds were increasingly stable as the pH approached neutrality. Finally, selective chemical modification of a glycosaminoglycan chondroitin sulfate was attempted at the non-reducing end by utilizing the Δ4-uronic acid functional group formed upon cleavage of the glycosaminoglycan with a bacterial lyase enzyme. The captodative double bond of the unique Δ4-uronic acid functionality was unreactive towards Michael addition, even if the carboxylate was methylated. Trials towards radical addition using thiyl radicals were unsuccessful, although a synthesized model phenyl Δ4-uronic acid monosaccharide was successfully functionalized under the same conditions.

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D.Phil. (Chemistry) / Please refer to full text to view abstract

Wong Ka-kei. / Thesis submitted in: December 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 123-139). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.i / ACKNOWNLEDGEMENT --- p.ii / ABSTRACT (ENGLISH VERSION) --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / LIST OF TABLES --- p.ix / ABBREVIATIONS --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Edible mushrooms --- p.1 / Chapter 1.1.1 --- Classification and terminology --- p.1 / Chapter 1.1.2 --- Mode of nutrition --- p.3 / Chapter 1.1.3 --- World consumption --- p.3 / Chapter 1.1.4 --- Nutritional values of edible mushroom --- p.6 / Chapter 1.1.5 --- Medicinal values of mushrooms --- p.7 / Chapter 1.2 --- Mushroom mycelium --- p.11 / Chapter 1.2.1 --- Uses and applications --- p.11 / Chapter 1.2.2 --- Submerged fermentation (SmF) --- p.12 / Chapter 1.2.3 --- Factors affecting the growth of mycelium in submerged fermentation --- p.14 / Chapter 1.2.3.1 --- Nutritional requirements - Carbon sources --- p.14 / Chapter 1.2.3.2 --- Nutritional requirements ´ؤ Nitrogen sources --- p.16 / Chapter 1.2.3.3 --- Nutritional requirements ´ؤ Minerals --- p.16 / Chapter 1.2.3.4 --- Environmental factors ´ؤ Temperature --- p.17 / Chapter 1.2.3.5 --- Environmental factors - Aeration --- p.17 / Chapter 1.2.3.6 --- Environmental factors - Agitation --- p.18 / Chapter 1.2.4 --- Optimization of growth of mycelium and production of EPS --- p.18 / Chapter 1.3 --- Mushroom polysaccharides --- p.21 / Chapter 1.3.1 --- Biologically active mushroom polysaccharides --- p.21 / Chapter 1.3.2 --- Chemical structures of mushroom polysaccharides --- p.21 / Chapter 1.3.2.1 --- β-glucans --- p.23 / Chapter 1.3.2.2 --- α-glucans --- p.25 / Chapter 1.3.2.3 --- Mannans --- p.26 / Chapter 1.3.2.4 --- Protein-bound polysaccharides --- p.26 / Chapter 1.3.2.5 --- Other heteroglycans --- p.28 / Chapter 1.4 --- Mushrooms under investigation --- p.28 / Chapter 1.4.1 --- Pleurotus tuber-regium (Fr.) Sing. (PTR) --- p.28 / Chapter 1.4.2 --- Agrocybe cylindracea (AC) --- p.30 / Chapter 1.4.3 --- Grifola frondosa (GF) --- p.31 / Chapter 1.5 --- Objectives and experimental design --- p.32 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.35 / Chapter 2.1 --- Source of mushroom mycelium --- p.35 / Chapter 2.2 --- Effect of different carbon sources on submerged fermentation --- p.37 / Chapter 2.2.1 --- Production of mycelium by submerged fermentation using 250 mL and 1L shake-flasks --- p.37 / Chapter 2.2.2 --- Scale-up production of mycelium of PTR using fermentor --- p.39 / Chapter 2.2.3 --- Concentration of dissolved oxygen in 250 mL and 1L shake-flasks. --- p.39 / Chapter 2.3 --- Isolation and fractionation of mushroom polysaccharides --- p.40 / Chapter 2.3.1 --- Isolation of exo-polysaccharides (EPS) from culture medium by ethanol precipitation --- p.40 / Chapter 2.3.2 --- Isolation of EPS from culture medium by ultra-filtration --- p.40 / Chapter 2.3.3 --- Hot water extraction of PTR mycelium --- p.41 / Chapter 2.3.4 --- Fractionation of HWE by fractional ethanol precipitation --- p.41 / Chapter 2.4 --- Chemical composition of HWE and EPS --- p.42 / Chapter 2.4.1 --- Phenol-sulphuric acid method --- p.42 / Chapter 2.4.2 --- Modified Lowry method --- p.43 / Chapter 2.4.3 --- Monosaccharide composition analysis of HWE and EPS --- p.43 / Chapter 2.4.3.1 --- Acid depolymerization --- p.43 / Chapter 2.4.3.2 --- Neutral sugar derivatization --- p.44 / Chapter 2.4.3.3 --- Determination of neutral sugar composition by gas chromatography (GC) --- p.45 / Chapter 2.4.3.4 --- Uronic acid content --- p.46 / Chapter 2.5 --- Structural studies of HWE and EPS --- p.47 / Chapter 2.5.1 --- High Pressure Liquid Chromatography (HPLC) --- p.47 / Chapter 2.5.2 --- Methylation study and gas chromatography- mass spectrometry (GC-MS) --- p.48 / Chapter 2.5.2.1 --- Preparation of dry dimethyl sulfoxide (DMSO) --- p.48 / Chapter 2.5.2.2 --- Preparation of methylsulfinyl methyl sodium (CH3SOCH2-Na+) --- p.48 / Chapter 2.5.2.3 --- Methylation --- p.49 / Chapter 2.5.2.4 --- Extraction of methylated polysaccharide --- p.49 / Chapter 2.5.2.5 --- Acid depolymerization and preparation of aditol acetate derivatives --- p.50 / Chapter 2.5.2.6 --- Determination of partially methylated alditol acetates (PMAAs) by gas chromatography-mass spectrometry (GC-MS) --- p.50 / Chapter CHAPTER 3 --- RESULTS AND DISCUSSION --- p.51 / Chapter 3.1 --- "Production of mycelium and EPS of PTR, AC and GF by submerged fermentation in 250 mL shake-flask with liquid medium containing different carbon sources" --- p.51 / Chapter 3.1.1 --- "Mycelial biomass production of PTR, AC and GF" --- p.51 / Chapter 3.1.2 --- "Production of EPS of PTR, AC and GF" --- p.57 / Chapter 3.1.3 --- "Characterization of EPS of PTR, AC and GF" --- p.62 / Chapter 3.1.3.1 --- Carbohydrate and protein content --- p.62 / Chapter 3.1.3.2 --- Monosaccharide composition --- p.67 / Chapter 3.1.4 --- Summary --- p.72 / Chapter 3.2 --- "Production of mycelium, EPS of PTR by submerged fermentation in 1L shake-flask and 8L fermentor with liquid medium containing different carbon sources" --- p.75 / Chapter 3.2.1 --- Mycelial production of PTR --- p.75 / Chapter 3.2.2 --- EPS Production of PTR --- p.80 / Chapter 3.2.3 --- Chemical characteristics of EPS of PTR --- p.83 / Chapter 3.2.3.1 --- Carbohydrate and protein content --- p.83 / Chapter 3.2.3.2 --- Monosaccharide composition --- p.85 / Chapter 3.2.4 --- Structural characteristics of EPS of PTR --- p.87 / Chapter 3.2.4.1 --- Molecular weight of EPS of PTR by HPLC --- p.87 / Chapter 3.2.4.2 --- Glycosyl linkages of EPS of PTR by GC-MS of PMAA --- p.90 / Chapter 3.2.5 --- Summary --- p.93 / Chapter 3.3 --- Hot water extraction of mycelium of PTR from the scale-up submerged fermentation in 1L shake-flask and 8L fermentor with liquid medium containing different carbon sources --- p.95 / Chapter 3.3.1 --- Yield of hot water extract (HWE) of mycelium of PTR --- p.95 / Chapter 3.3.2 --- Chemical characteristics of HWE of PTR --- p.101 / Chapter 3.3.2.1 --- Carbohydrate and protein content --- p.101 / Chapter 3.3.2.2 --- Monosaccharide composition --- p.104 / Chapter 3.3.3 --- Structural characteristics of HWE of PTR --- p.112 / Chapter 3.3.3.1 --- Molecular weight of HWE of PTR by HPLC --- p.112 / Chapter 3.3.3.2 --- Glycosyl linkages of HWE of PTR by GC-MS ofPMAA --- p.116 / Chapter 3.3.4 --- Summary --- p.119 / Chapter CHAPTER 4 --- CONCLUSIONS AND FUTURE WORKS --- p.120 / Chapter 4.1 --- Conclusions --- p.120 / Chapter 4.2 --- Future works --- p.121 / REFERENCES --- p.123

An atmospheric pressure argon glow discharge is shown to detect trace levels of carbohydrates in aqueous flowing systems, using either of two glow discharge solution interface configurations. The first configuration consists of an oscillating glow discharge sustained between a flowing aqueous cathode and platinum anode. Picomole and micromolar mass and concentration detection limits, respectively, are obtained for sucrose in an aqueous flow injection system when monitoring discharge oscillation frequency or discharge current. The second configuration consists of a non-oscillating glow discharge sustained between metallic electrodes near the flowing output of a high performance liquid chromatography system. A conductivity detector detects the acidic product formed when each carbohydrate elutes and is exposed to the glow discharge. This detector yields femtomole and nanomolar mass and concentration detection limits, respectively, for a variety of carbohydrates and competes with the best of the commercially available liquid chromatography carbohydrate detectors. An increase in the discharge electrode spacing or reduction in the liquid flow rate increases detector sensitivity, since the discharge area and solution exposure time are increased, respectively. The aqueous carbohydrate products formed from exposure to the glow discharge are similar to those formed from exposure to high energy radiation. Acid, hydrogen peroxide, and an absorbing species all form in amounts proportional to carbohydrate concentration and glow discharge exposure time, with yields approximating those encountered when using high energy radiation. / Graduation date: 1997