Thesis (M.S.)--University of Toledo, 2007. / Typescript. "Submitted as partial fulfillments of the requirements for the Master of Science Degree in Chemistry." "A thesis entitled"--at head of title. Bibliography: leaves 56-65.

Thesis (Ph.D.)--Tufts University, 2001. / Adviser: Marc d'Alarcao. Submitted to the Dept. of Chemistry. Includes bibliographical references (leaves 210-218). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (M.E.)--University of Waikato, 2009. / Title from PDF cover (viewed Apr. 20, 2010). Includes bibliographical references (p. 36-46)

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains xviii, 145 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--Illinois State University, 2005. / Title from title page screen, viewed September 27, 2006. Dissertation Committee: Wade Nichols (chair), Jon Friesen, Craig Gatto, Laura Vogel, Brian Wilkinson. Includes bibliographical references (leaves 87-93) and abstract. Also available in print.

The conformation and dynamics of sialyl Lewisx and related oligosaccharides were investigated using high resolution nuclear magnetic resonance measurements, and molecular dynamics calculations. In order to increase the number of structural parameters for inclusion in the molecular modelling simulations, the oligosacchaiide sialyl Lewisx was chemo-enzymatically synthesised to a high degree of carbon-13 enrichment (< 99%). The incorporation of labelling allows editing of standard homonuclear NMR experiments by the 13C chemical shift, thus overcoming the spectral overlap which plagues carbohydrate NMR. Three dimensional heteronuclear NOESY/ROESY-HSQC experiments have allowed the unambiguous assignment and quantitation of NOEs/ROEs in a number of oligosaccharides including evidence that the anti-conformer is populated in aqueous solution for the Gal 1- 4GlcNAc linkage in sialyl 2,3-N-acetyllactosamine trisaccharide. Additional structural information was gained from the measurement of trans-glycosidic three bond carbon- carbon coupling constants. A Karplus relationship was derived for the C-O-C-C fragment allowing the back-calculation of 3Jcocc values from molecular dynamics simulation for comparison to experimental data. Additional distance restraints were derived from NOEs to exchangeable protons in low temperature studies using a mixed solvent system of H2O/acetone-d6. The increase in the number of distance restraints, have allowed the use time-averaged molecular dynamics simulations, and from these it is shown that NOEs to certain hydroxyls in sialyl Lewisx can only be explained if these hydroxyls are constrained within hydrogen bonds.

The three-dimensional conformations of various oligosaccharides have been investigated using high-resolution nuclear magnetic resonance measurements and molecular dynamics calculations. A fundamental problem with such studies is the lack of structural restraints across the glyosidic linkage as well as the short-range nature of these restraints. In this thesis this problem has been addressed by developing new techniques that increase the total number of structural parameters for inclusion in the molecular modelling simulations. The measurement of inter-glycosidic heteronuclear NOEs is described and four 1H- NOEs were measurable in a model disaccharide with appropriate 13C and 2H enrichment. NMR studies were also carried out with a series of oligosaccharides dissolved in a dilute liquid crystalline medium. This resulted in a degree of molecular alignment for the oligosaccharides that in turn allowed the measurement of dipole-dipole coupling constants. These were incorporated in dynamic simulated annealing calculations in order to verify their usefulness in structural calculations. The three-dimensional structure and dynamics of Gal?1-4Glc were investigated using the additional restraints mentioned above, as well as 13C-13C trans-glycosidic long-range coupling constants and 1H-1H NOEs. It was shown that the conformation cannot be represented by a single structure, but is best represented by a dynamic model. Other techniques developed in this thesis include the measurement of three-bond scalar coupling constants to probe the existence of inter-glycosidic hydrogen bonding, and the direct measurement of bond angles using relaxation via cross-correlated dipolar couplings. Finally the bound-state conformation of 13C-enriched Sialyl Lewis-x in association with E-selectin was investigated using a three-dimensional nuclear Overhauser effect 13C-1H heteronuclear single quantum correlation experiment.

Inulin hydrolysis can occur as a result of the action of exoinulinases and endoinulinases acting alone or synergistically. Exoinulinases cleave the non-reducing β-(2, I) end of inulin releasing fructose while endoinulinases act on the internal linkages randomly to release inulotrioses (F₃), inulotetraoses (F₄) and inulopentaoses (F₅) as major products. Fructosyltransferases act by cleaving a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the short chain fructooligosaccharide. The production of high yields of oligosaccharides of specific chain length from simple raw materials such as inulin and sucrose is a challenge. Oligosaccharides of chain length up to degree of polymerisation (DP) 5 and fructose were produced using preparations of three commercial microbial enzymes. Production of these novel oligosaccharides was achieved by employing response surface methodology (RSM) with central composite experimental design (CCD) for optimising product yield. Using a crude Novozyme 960 endoinulinase preparation isolated from Aspergillus niger, the following conditions gave a high inulooligosaccharide (lOS) yield, temperature (60 ºC), 150 g/L inulin concentration, 48 h incubation; pH 6.0 and enzyme dosage of 60 U/ml. Under these conditions, inulotrioses (70.3 mM), inulotetraoses (38.8 mM), and inulopentaoses, (3.5 mM) were produced. Response surface regression predicted similar product levels under similar conditions. The crude endoinulinase was purified through a three step purification procedure with a yield of 1.11 % and 3.5 fold purification. The molecular weight of this endoinulinase was estimated to be 68 .1 kDa by SDS-PAGE and its endoinulinase nature was confirmed by native PAGE. The purified endoinulinase was more efficient in production of lOS than the crude endoinulinase preparation. The purified endoinulinase demonstrated a high affinity for the inulin substrate (Km[subscript] 3.53 mM, Vmax[subscript] 666.67 μmol/min/ml). Pectinex Ultra SP-L, a commercial crude enzyme preparation isolated from Aspergillus aculeatus is a cocktail of several enzymes including a fructosyltransferase. The crude enzyme showed both transfructosylation and hydrolytic activity in 200 to 600 g/L sucrose. The main fructooligosaccharides produced from sucrose were l-kestose (GF₂), nystose (GF₃) and fructofuranosyl nystose (GF₄). After the first RSM, with the coded independent variables of temperature, incubation time, pH and sucrose concentration, the highest levels of GF₂, was 68.61 mM, under sucrose concentration 600 g/L, temperature 60°C, enzyme dosage 20 U/ml , pH 5.6, after 4 h incubation. A sucrose concentration of 400 g/L favoured the synthesis of high levels of GF₃ and GF₄. In the second RSM the maximal yields of GF₂, GF₃ and GF₄ were 152.07 mM, 131.38 mM and 43.99 mM respectively. A purified fructosyltransferase did not synthesise GF₄. Ammonium ions were demonstrated to enhance the yield of FOS. A mixture of glucose and fructose was used as substrate for FOS synthesis and no FOS were formed. Glucose was shown to be an end product inhibitor of the fructosyltransferase and therefore hinders the formation of high FOS yield. Fructozyme, isolated from Aspergillus ficuum is a mixture of exo and endoinulinases with the former being predominant was used for fructose production from inulin hydrolysis. The exoinulinase was purified to electrophoretic homogeneity by a three step purification procedure. The molecular weight of the enzyme was estimated to be 53 kDa with a 2 I % yield and 4.2-fold. Response surface regression was used to predict the maximum fructose levels achievable under the combinations of temperature, enzyme dosage and incubation time. A reaction time (48 h), enzyme dosage (100 U/ml) and inulin concentration (150 g/l) at pH 5.0 at 50°C gave higher fructose levels (106.6 mg/ml) using crude exoinulinase as compared to 98.43 mg/ml using the purified exoinulinase. These findings indicate that higher levels of fructose require longer incubation periods and higher inulin substrate concentrations with higher enzyme dosage. The crude exoinulinase preparation gave fairly higher levels of fructose than the purified exoinulinase and this is due to the presence of other hydrolytic enzymes in the crude preparation. The conditions established by RSM and CCO were adequate in producing high yield of oligosaccharides and fructose and can therefore be applied for their industrial production since they are in high demand due to their health benefits as prebiotics.

Fructooligosaccharides (FOS) are short-chain fructans with a terminal glucose moiety and are found naturally in many plant species. Besides their wide use as an alternative sweetener in food and beverage industry, FOS have shown great potential as neutraceuticals against diabetes, colon cancer and bowel disease. The uses of FOS are dependent on the degree of polymerisation that they exhibit. β-fructofuranosidase (FFase) and fructosyltransferase (FTase) enzymes are capable of synthesing FOS from carbohydrate raw materials such as chicory and sugar beet. The aim of this study was to investigate the synthesis of FOS of a pre-defined chain length, from sucrose, by the enzyme FopAp; a β-fructofuranosidase from Aspergillus niger. ATCC 20611. The crude enzyme FopAp was successfully purified, with a yield of 78.20 %, by ammonium sulphate precipitation and anion exchange chromatography. Two protein fractions, named FA and FB were shown to exhibit FFase activity. SDS PAGE analysis revealed two proteins with molecular weights of 112 kDa and 78 kDa, which were identified as a FFase and a hydrolase. Temperature and pH optima of 20 ºC and 9, respectively, were observed for the transfructosylation activity in the FFase. The purified FFase exhibited a half life of 1.5 hrs under optimal conditions. Substrate kinetic studies indicated a high hydrolytic activity at low sucrose concentrations, with Vmax and Km of 1.25 μmol/ml/min and 3.28 mM, respectively. Analysis by response surface methodology identified temperature and pH to be significant factors for the production of kestose and nystose, at a 95 % level of confidence. These findings were confirmed by neural networks constructed to identify optimal conditions of FOS synthesis.FOS synthesis was found to be optimal between pH 6 and pH 9 at 25 ºC. The factor of reaction time was found to be insignificant within the selected experimental constraints, for both FOS species. The findings of this investigation are very important as the foundations of a commercially viable synthetic process for the production of FOS.

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.