The Design, Synthesis, Biological Activity Assay Of β-(1â†'3)-D-Oligoglucoside Derivatives, And The Construction Of Oligosaccharide Microarray

Glycobiology is a discipline which studies on the oligosaccharide structure, biosynthesis, and biological function of glycoconjugates. Drug design of structure-assisted and mechanism-based is a newer strategy of drug design, and can open up a new method for glycobiology. The glycoarray can be extensively used to study on carbohydrates, such as the throughput analysis of carbohydrate-protein interactions, genomics of drug, and so on. It is a necessary tool for post-genomic era. This thesis mainly studies on the design, synthesis, biological activity assay of β-(1→3)-D-oligoglucoside derivatives, and the construction of oligosaccharide microarray. The main results are as follows: The β-(1 →3)-D-glucan from Saccharomyces cerevisiae was extracted by the alkaline-acid method. The paper chromatography and UV analysis showed that the product extracted by alkaline-acid method was chemically pure, i.e. it contained no other carbohydrates and proteins, which was further confirmed by FTIR spectrum. The hydrolysis mechanism was analyzed. It was considered that the alkaline-acid method was ideal for extracting β-(1→3)-D-glucan from Saccharomyces cerevisiae. Fluorophore-assisted carbohydrate electrophoresis (FACE) is a simple and inexpensive method for separating the carbohydrates. The (oligo)saccharides were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and the reductive amination reactions were essentially complete after approximately 16h under the given experimental conditions. Saccharide-ANTS adducts were then separated from one another by electrophoresis on a 32% CACR, 2.4% CBIS polyacrylamide gel at alkaline pH. This technique doesn't require sophisticated instrumentation and highly trained personnel. It indicates that the linear relationship between band fluorescence intensity and carbohydrate concentration in the range of 5 to 100 pM is used to calculate relative abundance. At the same time, no one chain length is derivatized more readily than any other chain length. β-(1→3)-D-Oligoglucosides of various lengths of Saccharomyces cerevisiae glucan were abtained by using acidic hydrolysis [c(CF3COOH)=2.0mol/L], and they were tested by FACE, confirming their degree of polymerization from 1 to 7. According to the action mechanism of β-(1→3)-D-glucanase, the epoxyalkyl β-(1→3)-D-oligoglucoside mixtures were designed and synthesized successively by acetylation, glycosidation, oxidation, and deacetylation ofβ-(1→3)-D-oligoglucosides. Thereinto, a sample was analysed by ESI-MS. In epoxyalkyl β-(1→3)-D-oligoglucosides-binding β-(1→3)-D-glucanase assay, we found that the β-(1→3)-D-glucanase was obviously inactivated by epoxyalkyl β-(1→3)-D-oligoglucosides. Moreover, the shorter the chain length of epoxyalkyl introduced, the stronger the anti-enzyme ability will be. The scavenging ability on superoxide anion, immunological activities (phagocytosis of peritoneal macrophages, superoxide anion production activity, and lymphocyte proliferation), and inducting phytoalexins of anβ-(1→3)-D-oligoglucoside mixture and its 3,4-epoxybutyl derivative were investigated. The main results are as follows: ①β-(1→3)-D-Oligoglucoside mixture and its 3,4-epoxybutyl derivative both with 0.1 μg/mL had a little scavenging ability towards superoxide anion. ②β-(1→3)-D-Oligoglucoside mixture and its 3,4-epoxybutyl derivative both with 200 μg/mL enhanced the phagocytosis of peritoneal macrophages. ③β-(1→3)-D-Oligoglucoside mixture and its 3,4-epoxybutyl derivative both with 20 μg/mL stimulated superoxide production. ④β-(1→3)-D-Oligoglucoside mixture and its 3,4-epoxybutyl derivative both with 200 μg/mL accelerated the lymphocyte proliferation. ⑤β-(1→3)-D-Oligoglucoside mixture and its 3,4-epoxybutyl derivative can induct phytoalexins. Moreover, the 3,4-epoxybutyl derivative could be kept for a longer time than β-(1→3)-D-oligoglucoside mixture, which indicated 3,4-epoxybutyl derivative is much more stable than β-(1→3)-D-oligoglucoside mixture. A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions was demonstrated using QDs as a fluorescence label coupled with protein. 1, 3-Dipolar cycloaddition between azide and alkyne was exploited to attach α-D-glucopyranoside to a C14 hydrocarbon chain that noncovalently binds to the microtiter well surface, and the product formation was detected by both ESI-MS and QD (or FITC)-conjugated lectin binding. It indicated that the peak intensity of the fluorescence emission was proportional to the initial Con A concentration of in the range of 2×10-3 μmol/L～2×10-2 mmol/L with a detection limit at least 100 times lower than that of the FITC-based method.