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

The stereospecific synthesis of the Glc 3 Man N-glycan tetrasaccharide via an iterative tethering and allyl-mediated intramolecular glycosylation strategy is reported.

No description available.

This thesis describes investigations into the use of electrochemical oxidation as a method for the activation of glycosyl donors, and in particular the application of this technique to the selective activation of one electrochemically active glycoside over another. The synthesis of twenty eight electrochemically active monosaccharide donors, including thio-, seleno- and O-glycosides with varying protecting group patterns and anomeric substituents is described, together with the synthesis of three electrochemically active monosaccharides with the potential to act as both glycosyl donors and glycosyl acceptors. The electrochemical analysis of these monosaccharides is reported and gives a detailed insight into the effect of various factors on the oxidation potentials of the monosaccharide donors and allows some general conclusions to be drawn. In addition the analysis of six monosaccharides by cyclic voltammetry at scan rates of up to 25 000 Vs^-1 allows their homogenous kinetics to be outrun and formal oxidation potentials obtained. Investigations into selective electrochemical glycosylations are reported, and the applicability of the analytical electrochemical studies to synthetic electrochemical reactions is demonstrated. Selective glycosylations are possible with selenoglycoside donors and either thio- or O-glycoside acceptors to give disaccharides. However the selective activation of selenoglycosides over thioglycosides is shown to be complicated by some underlying pathway for indiscriminate activation of both donor and acceptor. In contrast the use of an O-glycoside donor experiences no such problems. More detailed work on the underlying problems experienced with the thioglycoside acceptor was conducted, and the results are reported here. Investigations into electrochemical activation of the disaccharides are discussed, and the thioglycoside is shown to be easily activated to give a trisaccharide. At the time of writing this is believed to be the only electrochemically mediated trisaccharide synthesis reported in the literature. The O-glycoside however is shown to be inactive under the electrochemical oxidation conditions employed.

No description available.

Oligosaccharides are important biomolecules that are targets and also components of many medical treatments, including treatments for cancer, HIV, and inflammation. While the demand for medically-relevant oligosaccharides is increasing, these compounds have proven difficult to synthesize. Whole-cell oligosaccharide synthesis is a promising method that requires relatively inexpensive substrates and can complete the synthesis in just one step. However, whole-cell oligosaccharide synthesis employing common microorganisms like E. coli have been plagued by low yields. This dissertation investigates an alternative microorganism for oligosaccharide production: Agrobacterium sp. ATCC 31749. This Agrobacterium strain produces high levels of curdlan polysaccharide, demonstrating its natural ability to produce the sugar nucleotide precursor for oligosaccharide production. The two main objectives of this dissertation are 1) to develop biocatalysts for oligosaccharide synthesis by engineering ATCC 31749 and 2) to determine what factors affect poly- and oligosaccharide production in this Agrobacterium strain. ATCC 31749 was engineered to produce two oligosaccharides of medical importance: N-acetyllactosamine and galactose-α 1,3-lactose. Oligosaccharide production in the biocatalyst was further improved with additional metabolic engineering. Substrate uptake was increased through expression of a lactose permease, and availability of the sugar nucleotide substrate improved with gene knockout of the curdlan synthase gene. Both of these engineering efforts led to increased oligosaccharide synthesis in the Agrobacterium biocatalyst. Overall, the engineered Agrobacterium strains synthesized gram-scale quantities of the oligosaccharide products in just one step and requiring only a few inexpensive substrates and cofactors. Additional improvement of the oligosaccharide-producing biocatalysts required further investigation of the factors influencing poly- and oligosaccharide production in ATCC 31749. In this dissertation, several environmental and intracellular factors are identified that affect both oligosaccharide and curdlan production. Sucrose was the preferred carbon source for oligosaccharide synthesis, and the addition of citrate to the synthesis reaction led to significant improvement in oligosaccharide production. To identify the genetic factors and possible mechanisms regulating curdlan production, the genome of ATCC 31749 was sequenced. The genome sequence was utilized for transcriptome analysis of ATCC 31749. In the transcriptome analysis, genes significantly up- and down-regulated during curdlan production were identified. Subsequent gene knockout experiments showed several factors to be important for curdlan synthesis, namely the nitrogen signaling cascade, polyphosphate, and the GTP-derived second messengers (p)ppGpp and c-di-GMP. In addition to the development of biocatalysts for oligosaccharide production, this investigation provides insight into the complex mechanisms regulating exopolysaccharide synthesis.

Mycobacterial infection is a major health concern. High drug resistivity of the mycobacterium is due to its multi-layered, thick hydrophobic waxy cell wall components, consisting of cross-linked peptidoglycan (PG), mycolyl arabinogalactan (mAG) and lipoarabinomannan (LAM) polysaccharides. These polysaccharides are composed of arabinose and galactose in the furanose form and mannose in the pyranose form. The high waxy hydrophobic components of the mycobacterial cell wall acts as a barrier for most hydrophilic antibacterial agents. Enzymes responsible for the biosynthesis of polysaccharides of mAG and LAM are arabinosyl transferase (AraT), galactosyl transferase (GlfT) and mannosyl transferase (ManT). In the absence of furanoside derivatives of D-arabinose and D-galactose in mammalian systems, inhibitors based on these sugars arise an interest. Upon realizing structural characteristics of cell wall polysaccharides, the chemical syntheses of such polysaccharides were reported. Biological studies of synthetic arabinomannan and arabinogalactan oligosaccharides were performed, in order to identify their effects in enzymatic, as well as, mycobacterial growth assays. Chapter 1 of the thesis describes the structural features of mAG and LAM polysaccharides. Chemical synthesis of oligosaccharides related to mycobacterial cell wall components and their effects of mycobacterial growth and enzymatic assays are discussed. In my research program, synthesis and studies of oligosaccharides pertaining to mycobacterial cell wall components were undertaken. Monovalent and bivalent glycolipids 1 and 2 (Figure 1), containing arabinofuranoside trisaccharide as the sugar head group, were synthesized and their effects on the growth of M. smegmatis strain were studied. In the presence of arabinan glycolipids, retardation of the growth of M. smegmatis was observed and the inhibitory activity was found to be specific with glycolipids containing arabinofuranoside head groups. Glycolipid with maltosyl sugar and arabinofuranoside trisaccharide without lipid chains, did not affect the mycobacterial growth. Continuing the effort, tri- and tetrasaccharide of arabinomannan glycolipids were synthesized and their effects in the mycobacterial growth were studied. It was found that 3 was inhibiting the growth of the mycobacterium, whereas in the case of 4, inhibition was found to be less when compared to 3. Relative inhibitions of mycobacterial growth by synthetic glycolipids 1-4, at a concentration 200 µg/mL, were found to be in a varying degrees, ranging from 16 % in the case of 4 and 65 % in the case of 3. Figure 1. Molecular structures of arabinan and arabinomannan oligosaccharides 1-7. Following mycobacterial growth inhibition studies, surface plasmon resonance studies of synthetic oligosaccharides were performed, in order to identify their interactions with mycobacterial cell lysates. Amine tethered glycosides 5-7 (Figure 1) were synthesized and immobilized onto SPR sensor chip through amine coupling methodology. From SPR studies, it was found that the binding affinity was higher with cell lysates from motile strains than non-motile strain. Among various arabinomannans, glycoside 5, presenting two mannose units showed higher affinity than 6 and 7, having no or one mannose unit, respectively. Chapter 2 of the thesis provides details of synthesis, biological and biophysical studies of arabinan and arabinomannan glycolipids. Continuing the synthesis and studies with arabinose oligosaccharides, a linear tetra-, hexa and octasaccharide glycolipids, containing α-(1→5) linkages (10-12), as well as, a branched heptasaccharide containing α-(1→2) and α-(1→5) linkages (14) between the arabinofuranoside units (Figure 2) were synthesized. In addition to glycolipids, oligosaccharides without alkyl chains (8, 9 and 13) were also prepared. Synthesis was performed using trichloroacetimidate and Figure 2. Molecular structures of linear and branched arabinan derivatives 8-14. thioglycosides as glycosyl donors. Synthesis of linear oligosaccharide derivatives 8-12 was achieved by iterative glycosylation and deprotection strategies. Branched heptasaccharide derivatives 13 and 14 were synthesized by using block glycosylation method, wherein two fold excess of arabinose disaccharide was reacted with a suitably protected arabinose trisaccharide. Upon synthesis, molecular modeling studies were performed to identify the conformational behavior of arabinan glycolipids. Conformational studies were performed in three steps, namely, (i) dihedral scan (ii) conformational search and (iii) molecular dynamics. Dihedral scan was performed to assess favorable torsion angles at each glycosidic linkage with respect to overall conformation of the molecule. Monte-Carlo conformational search was performed to obtain the lowest energy structure of arabinan glycolipids. Relative orientations of lipidic portions and sugar portions were identified for linear and branched arabinan glycolipids. The least energy conformations of 10, 11, 12 and 14 are shown in Figure 3. In the case of linear molecules 10, 11 and 12, alkyl chains and arabinofuranoside portion did not phase segregate, whereas in the case of branched glycolipid 14, the alkyl chains were observed to move away from the sugar moieties. Molecular dynamic calculations were performed for the lowest energy structure, in order to evaluate the torsion angles in the trajectory. Following the synthesis and conformational analysis of the arabinan glycolipids, surface plasmon resonance studies were performed to assess their interactions with a host protein, namely, pulmonary surfactant protein-A (SP-A). For the interaction studies, SP-A was immobilized on to the CM-5 sensor chip using amine coupling method. Varying concentrations of arabinan glycolipids 10, 11, 12 and 14 and oligosaccharides 8, 9 and 13 were used as analytes. Responses from the surface of SP-A were subtracted from that of ethanolamine to eliminate the non-specific interactions. Primary sensorgrams were fitted using 1:1 Langmuir model to obtain the kinetic parameters of the interactions. Specificities and relative binding affinities of arabinan oligosaccharides interacting with SP-A are presented in Table 1. The affinities between Figure 3. Lowest energy structures of glycolipids 10, 11, 12 and 14 derived from molecular modeling studies. arabinan oligosaccharides and SP-A were found in the range of 4.9-47x103 M-1. Among the series, branched arabinan oligosaccharides 13 and 14 showed higher Ka values than the linear arabinan glycolipids. The association rate constants (kon) were generally higher for the oligosaccharides without lipidic chain, whereas, the dissociation rate constants (koff) were slower with oligosaccharides having lipidic chains. Faster kon was also associated with a faster koff for oligosaccharides without the lipidic chains. For the glycolipids, a relatively slower koff was found to be the trend. In the case of branched heptasaccharide derivatives, glycolipid 14 showed higher binding constant than heptasaccharide with a thiocresyl group at the reducing end 13. Chapter 3 of the thesis presents the synthesis, conformational analysis and SPR studies of linear and branched arabinan glycolipids. Table 1. Kinetic parameters of the interactions between arabinose derivatives 8-14 and SP-A. Compound kon (M-1s-1) kd (s-1) (104) Ka (M-1) (10-3) χ2 12 3.9 7.91 4.9 8.3 11 1.5 3.98 3.77 2.9 10 0.384 0.22 17.5 6.7 14 27.3 5.79 47.2 4.5 8 11.3 6.14 18.4 2.3 9 23.3 11.6 20.1 2.4 13 53.6 17.9 29.9 5.4 Upon assessing the biophysical studies of the α-arabinofuranoside glycolipids, an effort was undertaken to prepare glycolipids containing β-arabinofuranoside linkages and to study their conformational and biophysical properties. Arabinan glycolipids 15 and 16 (Figure 4), containing β-(1→2), β-(1→3) and β-(1→5) linkages between furanoside units were synthesized to compare the properties with the corresponding synthetic α-arabinan glycolipids. Incorporation of β-arabinofuranoside linkages in 15 and 16 was achieved using low temperature activation of silyl substituted glycosyl donor 17 (Figure 4), with NIS and AgOTf. The configurations in 15 and 16 were confirmed through 1H-1H COSY, 1H-13C HMQC NMR techniques. During the synthesis of 15 and 16, stereoselective incorporation of two β-Araf linkages on a single furanoside unit was achieved for the first time. Conformational studies of 15 and 16 were conducted similar to α-arabinan glycolipids, as above, to identify most favorable conformations of inter-ring, as well as, overall conformation of the molecule. The interactions between the SP-A and β-arabinofuranoside glycolipids 15 and 16 were also assessed with the aid of SPR technique. The analysis showed that the affinities of glycolipids 15 and 16 to SP-A were found to be relatively lower when compared to α-arabinofuranoside glycolipids. Synthesis and studies of β-arabinofuranoside glycolipids are described in chapter 4 of the thesis. Figure 4. Molecular structures of β-arabinofuranoside glycolipids 15 and 16. In summary, the present thesis describes synthesis, conformational and biophysical studies of synthetic arabinan and arabinomannan glycolipids. Monovalent and bivalent arabinan, tri- and tetrasaccharide arabinomannan glycolipids were synthesized and their effects in the mycobacterial growth were studied. It was found that arabinan and arabinomannan glycolipids inhibited the growth of the mycobacterium. The inhibitory activity is specific with the arabinan and arabinomannan glycolipids and the glycolipids with higher arabinose composition were found to be better inhibitors for mycobacterial growth. The interactions of mycobacterial cell lysates with arabinomannan compounds were evaluated through SPR technique. Linear tetra-, hexa-, octa- and branched heptasaccahride arabinan glycolipids containing α-Araf linkages between furanoside units were synthesized. Molecular modeling studies of arabinan glycolipids were performed, in order to identify their lowest energy conformations. Biophysical studies of linear and branched arabinan glycolipids were conducted to assess their interactions with pulmonary surfactant protein-A (SP-A) through surface plasmon resonance technique. Syntheses, conformational and biophysical studies were extended further to β-arabinofuranoside glycolipids. Overall, the thesis provides synthesis, conformational, biological and biophysical studies of a series of lipoarabinomannan oligosaccharides. The results provide a possibility to evolve newer types of glycolipids that can act as inhibitors of mycobacterial growth. (For structural formula pl see the hard copy)

No description available.