Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1963 S16 / Master of Science

Cellooligosaccharides are the reaction intermediates produced during the hydrolysis of cellulose to glucose. Hence, they have the same chemical structure as cellulose, just shorter chain lengths. Cellooligosaccharides up to DP eight are soluble in water. The soluble cellooligosaccharides can be used "as is" in the food industry as non-digestible oligosaccharides and in the laboratory as representative substrates for cellulolytic enzymes. The soluble cellooligosaccharides may also be chemically modified for use in the laboratory, in this case serving as affinity ligands, reporter groups, or model substrates. A number of methods are available for the separation of cellooligosaccharides differing only with respect to DP. This type of separation is relevant to both laboratory and industrial applications. A new approach to the chromatographic separation of cellooligosacchandes is presented in this thesis. It is shown that cellulose stationary phases, in conjunction with ethanol-water mobile phases, may be used for cellooligosaccharide fractionation. The system appears to behave as an affinity/partition system, with retention times increasing as the DP of the cellooligosaccharides increase. The feasibility of using such a chromatographic system for the "clean-up" of cellooligosaccharide mixtures is demonstrated. The relative merit of different chromatographic approaches putatively used for the fractionation of cellooligosaccharides was determined. Affinity-, adsorption-, ion-mediated- and molecular exclusion-approaches were tested. Adsorption chromatography, using a charcoal-celite stationary phase, was the most generally applicable method for the preparation of near gram quantities of pure cellooligosaccharides. Cellulose-based affinity/partition chromatography was found to be the least time consuming and most economical method for the preparation of cellotetraose and cellopentaose. Studies using chemically modified cellooligosaccharides are typically limited to derivatives whose aglycone group is conjugated to the reducing end of the sugar. This is because the chemistry involved in modifying the reducing end is typically much easier than that involved in selectively modifying other sites on the oligosaccharides. A portion of the studies presented herein was aimed at exploring approaches for the modification of the non-reducing end of cellooligosaccharides. Methyl 6-O-p-nitrobenzoyl-β-D-glucoside was synthesized by reacting methyl 4,6-O-p-nitrobenzylidine-β-Dglucoside with N-bromosuccinimide. This method has potential as a general method for the modification of the reducing terminus of oligosaccharides, including, cellooligosaccharides. / Graduation date: 2003

Many protein-linked oligosaccharides are believed to impart biological specificities to the molecules. The knowledge of detailed structural characteristics of oligosaccharides is essential for understanding their functions. In order to develop methodology for characterization of oligosaccharides of glycoproteins, and to compare glycosylation patterns of different immunoglobulins, oligosaccharides of IgM from two cell lines, MOPC 104E and PC 700, were analyzed. Homogeneous preparations of glycopeptides carrying individual glycosylation sites of the heavy chain were obtained from the two IgM's. The oligosaccharides of these glycopeptides were prepared by hydrazinolysis, and fractionated by HPLC under conditions that resolve oligosaccharides by charge and size, and by affinity chromatography on Concavalin A-Sepharose. Structures of some of these oligosaccharides were determined by 400 MHz NMR spectroscopy. HPLC fractionation by charge resolved oligosaccharides with zero, one, two, and three sialic acids. As indicated by HPLC analyses, oligosaccharides at all the glycosylation sites of both the IgM's were highly heterogeneous. A comparative study on oligosaccharides prepared by peptide-N-glycosidase F digestion of glycopeptides showed a similar degree of heterogeneity. Therefore, it was concluded that the observed heterogeneity of oligosaccharides was not an artefact caused by hydrazinolysis. Major differences between the glycosylation patterns of the two IgM's were evident from analyses of the oligosaccharides by both chromatographic techniques and NMR spectroscopy. MOPC IgM contained a high proportion of sialylated oligosaccharides when compared to PC IgM. Furthermore, the major oligosaccharide structures of MOPC IgM were of triantennary type whereas PC IgM contained biantennary oligosaccharides as its major species. In both the IgM's, a decreased trend of oligosaccharide processing was observed from the N-terminus to the C-terminus.

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