A highly sulphated, methylated polysaccharide, aeodan, isolated from the red seaweed Aeodes orbitosa was shown to contain galactose, 2-̲̲O-methyl-D-galactose, 4-O̲-methyl-Lgalactose, 6-O̲-methyl-D-galactose, xylose, and glycerol. The polysaccharide was desulphated with methanolic hydrogen chloride. Periodate oxidation of aeodan and desulphated aeodan, followed by reduction and hydrolysis, revealed the presence of 1,4- and 1,3-linked galactose residues and 1,3-linked 6-O̲-methy l-D-galactose residues in aeodan. Treatment of aeodan with sodium hydroxide revealed that the majority of the ester sulphate groups were alkali stable. Methylation of desulphated aeodan revealed that the polysaccharide was composed entirely of 1,3 and 1,4 links. Methylation of aeodan revealed the presence of 1,3- and 1,4- linked units, 1,3-linked galactose-2-sulphate, and 1,3-linked galactose-2, 6-disulphate units in the polysaccharide. Partial hydrolysis of aeodan resulted in the isolation and characterisation of 3-O̲-D-galactopyranosyl-D-galactose and 4-O̲-ß-D-galactopyranosyl- D-galactose. A sulphated, methylated polysaccharide, phyllymenan, isolated from the red seaweed Phyllymenia cornea was shown to contain galactose, 2-O̲-methyl-D-galactose, 4-O̲-methyl L- galactose , 6-O̲-methyl -D-galactose, and xylose. The polysaccharide was completely desulphated with methanolic hydrogen chloride. Periodate oxidation of phyllymenan before and after desulphation revealed that removal of the sulphate ester groups had not produced any new adjacent hydroxyl groups. Alkali treatment of phyllymenan revealed that the ester sulphate groups were alkali stable. Methylation studies on phyllymenan revealed the presence of 1,3- and 1,4-linked units, 1,3-linked galactose-2-sulphate, and 1,3-linked galactose- 2,6-disulphate units in the polysaccharide. Partial hydrolysis of phyllymenan revealed the presence or 4-O-̲ß- D-Dgalactopyranosyl- D-galactosc, 4-O-̲ß-D-galactopyranosyl -2-0- methyl-D-galactose, a galactosylgalactose composed of D and L-galactose, and adjacent 6-O̲-methyl- and 2-O̲-methyl-D- galactose units in the polysaccharide.

Hot-water extraction of Anatheca dentata, a red seaweed belonging to the family Solieriaceae, yielded a mixture of polysaccharides. Fractionation of this mixture with Cetavlon gave a glucomannan as minor component and a highly sulphated major component, which gave D- and L-galactose, D-xylose and small amounts of 3-0 (underscore)-methylgalactose, pyruvic acid and uronic acid on hydrolysis. All subsequent investigations were carried out on the sulphated major component. The sulphate was not labile to alkali, but was removed with methanolic hydrogen chloride. Periodate oxidation of the polysaccharide before and after desulphation indicated that new a-glycol groups were formed during desulphation. All the xylose units in the polymer were cleaved by periodate and this, together with the fact that the major xylose product from methylation analysis of the desulphated polymer was the 2,3, 4-tri-0 (underscore)-methyl derivative, indicated that the xylose occurs as a non-reducing end-group. Methylation of the desulphated polysaccharide revealed the presence of 1,4- and 1,3- linked D- galactose and 1,4- linked L-galactose units in the polymer. D-Glucuronic acid occurred as non-reducing end-groups. Summary, p. 1.

The research presented in this thesis forms part of an on-going collaborative programme concerned with the determination of the chemical structures of the surface antigens of bacteria belonging to genera within the family Enterobacteriaceae. Bacteria of this family are opportunistic pathogens and are frequently responsible for serious infections in animals and man. Surface antigens produced by virulent strains are largely polysaccharides and occur as lipopolysaccharides (the O-antigens) and capsular polysaccharides (the K-antigens) respectively. The extracellular polysaccharide antigens expressed by strains of the species Escherichia coli are of considerable . interest due to their effect on immunological processes and the relationship which exists between their chemical structure and virulence. To date, some seventy-four K-antigens have been distinguished serologically within the species E. coli and structures have been determined for most of these. The K-antigens of E. coli are structurally diverse and exhibit serological cross-reactivity with other pathogenic bacteria. The structures of five previously unstudied E. coli K-antigens, viz. those produced by serotypes 020:K1 01 :H-, 08:K45:H9, 08:K50:H-, 0101 :K1 03:H-, and 08:K43:H11, are presented in this thesis. A variety of chemical techniques has been employed in the structural analysis, and these are discussed. Two-dimensional NMR spectroscopic techniques proved invaluable for the structural elucidation of these complex carbohydrates, and high-field NMR spectroscopy alone was used in the analysis of the K43 antigen. Structural analysis of the K1 03 antigen was facilitated by specific enzymatic degradation, using a bacteriophage-borne endoglycanase. The K45 antigen was found to contain the unusual sugar 3-acetamido-3,6-dideoxygalactopyranose, while the K50 and K103 antigens join a minority group of polysaccharides which contain pyruvate as their only acidic component.

2, 4-Di-O-methyl-L-erythrose is thought to occur in the hydrolysis and periodate oxidation products of polysaccharides. This sugar has now been synthesized from methyl-L-arabofuranoside by tosylation, methylation, detosylation and periodate oxidation. The free sugar was obtained as a sirup and was characterized by the preparation of a crystalline phenylhydrazone and a crystalline p nitrobenzoate. / Science, Faculty of / Chemistry, Department of / Graduate

Part I - The work described in this thesis was part of a project to test the possible selective toxicity of some of the secondary alkyl homologues of dinitro-ortho-cresol. The 2-and 4-isobutyl-and isoamylphenols were prepared by an unequivocal synthesis by the method of Klages. Part A describes a Grignard reaction carried out on 2-and 4-methoxybenzaldehyde with isopropylbromide and isobutylbromide respectively. The four carbinols prepared were characterised whenever possible by methoxyl determinations and formation of phenylurethane derivatives, which were analysed for methoxyl and nitrogen values. Dehydration of the carbinols was achieved by refluxing the alcohol with sulphuric acid (20% by weight). The substituted ethylenes were characterised by methoxyl determinations and by formation of nitrosyl chloride derivatives which, were analysed for methoxyl and nitrogen values. The substituted ethylenes were hydrogenated under pressure to give the alkyl anisoles which were characterised by methoxyl determinations and formation of sulphonamide derivatives. The derivatives were analysed for methoxyl and nitrogen values. The alkyl anisoles were demethylated by refluxing with pyridine hydrobromide at 200°C. for five hours. The alkylphenols were characterised by carbon and hydrogen analyses and by formation of 3,5-dinitrobenzoate derivatives. The derivatives were analysed for their nitrogen content. Part B reports the Fries rearrangement of phenylisobutyrate. Isobutyryl chloride was prepared from isobutyric acid. Part of the acid chloride was reacted with phenol to give phenylisobutyrate. A Fries rearrangement of the ester in the absence of solvent at 140°C. gave 2-and 4-hydroxyisobutyrophenone in almost equimolar portions. A low temperature Fries rearrangement of the ester in nitrobenzene solvent gave the 4-hydroxyisobutyrophenone isomer only. A Friedel and Crafts reaction of phenol with isobutyryl chloride using two molar quantities of aluminium chloride in a nitrobenzene solvent gave the two hydroxyisobutyrophenone isomers. The two isomeric hydroxyisobutyrophenones were methylated by using sodium hydroxide and dimethyl sulphate to give 2-and 4-methoxyisobutyrophenone. A Friedel and Crafts reaction of anisole with isobutyryl chloride in carbon disulphide gave 2-and 4-methoxyisobutyrophenone. Oxidation of the two isomeric methoxyisobutyrophenones with potassium permanganate and sodium carbonate gave ortho-and para-methoxybenzoic acid, thus proving the orientation of the substituents. Part II - Mesquite gum, lemon gum, sapote gum A and sapote gum B were analysed quantitatively for their sugar content by a refined step-by-step hydrolytic technique. Standard curves of optical density versus concentration in the submicro scale were experimentally drawn from readings taken on a Beckman D U spectrophotometer on sugar solutions of L-arabinose, D-galactose, L-rhamnose, D-ribose and D-xylose, using a phenol-sulphuric acid method developed by Smith. Synthetic mixtures of L-arabinose, D-galactose, L-rhamnose and D-xylose were separated quantitatively by using paper partition chromatography techniques and were analysed by the above method. Procedure and techniques were thus standardised, Mesquite gum was hydrolysed into its components by N sulphuric acid; the composition of the gum was found to be D-galactose 56.8%, L-arabinose 30.0%, the remaining part representing uronic acid residues. Lemon gum was hydrolysed under controlled acid conditions into the components L-arabinose 22.35% and D-galactose 54.94%, the remaining portion representing uronic acid residues. Similarly sapote gum A was found to be composed of L-arabinose 25.45%, D-xylose 41.62% and uronic acid residues. Lastly sapote gum B was found to be divisible in two portions: a water soluble fraction composed of L-arabinose 25.10%, D-galactose 46.25%, L-rhamnose 6.80%, D-xylose 5.28%, the remaining unaccounted portion representing the uronic acid residues, and a water-insoluble, alkali-soluble fraction composed of L-arabinose 16.27%, D-galactose 25.14%, D-xylose 10.25% and uronic acids. / Science, Faculty of / Chemistry, Department of / Graduate

Aqueous dispersions of xanthan and locust bean gum, in combination, show a synergistic increase in viscosity. At sufficiently high concentrations, firm gels are formed, although neither component forms true gels when alone. The actual molecular processes resulting in this phenomenon are still incompletely understood and Theological studies can provide some clues to the mechanism of the interaction. Rheological properties of the polysaccharide blend were therefore investigated. Moisture, ash and inorganic elements and protein content as well as the intrinsic viscosity of the individual polysaccharides were determined. Dynamic viscoelastic properties of dispersions prepared from the polysaccharide blend were evaluated in four solvent treatments capable of disrupting weak intermolecular bonds. The effect of polysaccharide concentration, temperature, ionic strength, pH, ratio of mixing of the two gums and urea concentration on steady shear rheological properties were evaluated in a fractional factorial experiment. More detailed studies were carried out on the effects of temperature at two levels of concentration and ionic strength. Solvent treatments significantly affected the viscoelastic properties of xanthan-locust bean gum solutions. At 20°C and under the conditions used, dipole interactions appeared to be the primary force responsible for stabilizing the xanthan-1ocust bean gum interacting system. Hydrogen bonding and hydrophobic interactions seemed to play less important roles. Under conditions of low ionic strength and increased temperature, the interactions were lost and polysaccharide solution behavior passed from that of a viscoelastic solid to that of a viscoelastic liquid. At 60°C and at high ionic strengths, hydrophobic interactions may become important in the stabilization of the three dimensional gel network. Temperature effects on steady shear viscosity of xanthan-locust bean gum solutions were dependent on the concentration and ionic strength of the system. In general, steady flow properties were comparable to the reported behavior of xanthan solutions and so reflected the weakness of the interaction (dipole interactions) between the two polysaccharides. / Land and Food Systems, Faculty of / Graduate

Seventy eight serologically distinct strains of Klebsiella bacteria are known to exist. The capsular polysaccharide surrounding the bacterial cell of these pathogenic Enterobacteria is of immunological significance. Structures of the capsular polysaccharides of nearly sixty seven strains of Klebsiella have been established, and each one found to be unique. The structures of the K antigens from Klebsiella, serotypes K67 and K80 are presented as a contribution to the continuing program of elucidation of the chemical structures of these antigens in an attempt to explain their immunological responses. Chemical methods of structural elucidation were employed and the following two structures were obtained. [formula omitted] The polysaccharide from K67 was unique among the Klebsiella K antigens in having a four-plus-two-plus-one repeating-unit (indicating a branch on a side chain), while K80 was unique as it was the first instance that a pyruvic acetal was found on a terminal rhamnose unit. The importance of bacteriophage-borne enzymes in the generation of single repeating-units containing labile substituents is demonstrated. Klebsiella K44 polysaccharide was degraded using a crude solution of Φ44 bacteriophage. The oligosaccharides obtained were crucial in the determination of the position of the 0-acetate group. In the case of the polysaccharide from Klebsiella K26, the degradation performed using a crude solution of Φ26 bacteriophage resulted in the isolation of a single repeating-unit containing a pyruvic acetal together with an oligosaccharide corresponding to a single repeating-unit devoid of its terminal pyruvate containing sugar. The structures of these compounds which are as follows, were useful in further confirmation of the structures of the original polysaccharides. [formula omitted] Escherichia coli. another pathogenic Enterobacteria possessing immunologically significant K antigens, has been found to contain capsular polysaccharides bearing a strong resemblance to those of Klebsiella. Recently it was discovered that some strains of E. coli contained K antigens comprising amino sugars. A preliminary study on the chemical behaviour of amino sugars, and chemical methods of structure elucidation of such polysaccharides have been included in an appendix as this has been a new area of research in this laboratory. An appendix containing compilations of the cross-reactions, known structures, and chemotypes of the Klebsiella K antigens has also been included. / Science, Faculty of / Chemistry, Department of / Graduate

Polysaccharides are abundant in Chinese medicines (CMs) and play important roles in these materials' bioactivities. But at present, the polysaccharides are seldom incorporated into the quality assessment system of CMs. One of the big bottlenecks encountered is the difficulties in quality analysis due to their large molecular mass, diverse and complex chemical structure. Hereby, in current study, evidence and improved analysis approaches related to polysaccharides quality are proposed by employing several CMs as objectives. Firstly, Lingzhi (Ganoderma lucidum and G. sinense) was used to provide evidence of the importance of polysaccharides quality assessment in CMs. The results showed that the polysaccharides from two species shared the same structural features in terms of mono-/oligo-saccharide profiles, molecular size, sugar linkages, and IR/NMR spectra. And these polysaccharides showed no obvious difference in antitumor/immunomodulating activities and mechanism related to the activation of some proteins via TLR-4 related signaling pathway and gut-microbiota modulatory effects. Following that, to solve the problems existed in qualitative and quantitative analysis of polysaccharide in CMs, a fluorescence-labeled oligosaccharide-marker approach was raised accordingly based on LC-DAD-qTOF -MS analysis. Taking Danggui Buxue Tang (DBT) as an example, the result showed that 1) selected oligosaccharide marker generated by mild hydrolyzation and ABEE-labeling could powerfully differentiate individual herb polysaccharides. 2) good linearity was established between the identified oligosaccharide-markers and individual polysaccharides (r≥0.99). 3) the established method had satisfactory repeatability (RSD≤8.4%), recovery (≥80%) and sensitivity in polysaccharide quantitation in DBT. Finally, a polysaccharide quality analysis was conducted to dynamically trace the absorption, dynamic distribution and degradation of orally-dosed DOP (a marker polysaccharide from Dendrobium officinale) in mice and in vitro using near-infrared fluorescence imaging and chromatographic analysis. The results indicate that DOP was not absorbed and digested but were quickly degraded to short-chain fatty acids in the large intestine where gut microbiota existed. The interaction could be associated with DOP's suppression of 4T1 tumor growth in mice. In conclusion, by these studies, the importance of bioactive polysaccharides in quality assessment of CMs was confirmed. The mentioned predicaments in quality analysis of polysaccharides were greatly improved whether in CMs or complex biological matrix in vivo and in vitro. All the approaches proposed will contribute to other polysaccharides or polysaccharide-dominated CMs' analysis.

Numerous of studies have reported that polysaccharides have many bioactivities including immune system modulation, anti-oxidative and anti-tumor activities. Because herbal materials are rich in polysaccharides, some of the herbs like Dendrobium offcinale, its polysaccharide marker (DOP) has been applied in the quality analysis of polysaccharides in D. officinale by high performance gel permeation chromatography (HPGPC). However, such polysaccharide marker is not presented in every herb, and DOP also has limitation in monitoring herb formula. In addition, even though the same herbal materials and the same extraction methods were used, different studies gave out different conclusion regarding the structure of polysaccharides. Therefore, in the current study, D. officinale was used as a case study to demonstrate the application of ABEE-labeled oligosaccharides approach on structure elucidation and quantification of polysaccharides in herbs and herb formulae. First, the elucidation of polysaccharide structure remains challenging due to the lack of accurate analytical methods to determine the sequence and nature of glycosidic linkages. Oligosaccharide fragments from hydrolysis of polysaccharides are believed to provide accurate structure information, however, they are hard-to-separate and hard-to-detect. In the proposed method, the oligosaccharides generated from partial acid hydrolysis of DOP were labeled with p-aminobenzoic ethyl ester (ABEE), which made them separable and detectable by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS-DAD). Subsequently, nine ABEE-labeled oligosaccharide fragments (dimer to decamer) were isolated and identified by MS sequencing and 2D-NMR, and were confirmed by methylation analysis. The results indicated that the backbone should be β-D-1,4-linked Manp chain instead of mixed mannose and glucose chain. Second, two ABEE-labeled oligosaccharides namely, Te-Man-ABEE and Pen-Man-ABEE, were selected as chemical markers in the quantification of DOP in D. officinale and D.officinale (DO) products due to their high specificity in herb formula. The linear relationship between the content of these two markers and the content of DOP was successfully established. The linear relationship was further transformed to that between peak area of chemical markers and DOP content so that chemical markers were not necessary to be isolated for analysis. This linear relationship was systemically validated in terms of repeatability, precision and accuracy. The results showed that these two oligosaccharide markers presented a good linear relationship with DOP (R2 ≥ 0.997) in the range of 0.68-16.02 µg and also demonstrated satisfactory repeatability (RSD < 7.0%), and recovery (91.41% - 118.30%) in real sample determination. There was no significant difference between the results given by the two chemical markers as the RSD values were not more than 7.0%. While concerning the results given by the oligosaccharide-markers and the previously-published polysaccharide marker, the RSD value was not more than 6.4%. In conclusion, this approach provided an efficient and reliable method to obtain accurate structure information of polysaccharides and quantify specific polysaccharide in herb formula. it is believed that ABEE-labeled oligosaccharides approach can be also applied in the analysis of other saccharide-dominant herbal materials which in turns helps to find out the bioactivity mechanism of polysaccharides.

Includes bibliographical references. / The structures of some non-cellulosic β D-Glycans from three plant sources have been investigated and each was found to be characterised by linked D-pyranosyl a main chain consisting of β -(1-44)- sugars. The polysaccharides were, however, different in structural features in a manner apparently related to their respective locations within the organs of the plants concerned. The polysaccharides were isolated and purified using standard fractionation methods including chromatographic techniques and selective precipitation methods. Structural information was obtained by employing techniques such as methylation analysis (involving use of gas liquid chromatography mass spectrometry), optical rotation measurements, mass spectrometry and n.m.r. spectroscopy on the original polysaccharides and on degraded products obtained by methods such as acid- or enzyme-catalysed hydrolysis and Smith degradation.