【作者】 黄刚良；

【导师】 刘曼西；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2005， 博士

【摘要】 糖生物学的研究领域主要包括糖缀合物糖链的化学合成与结构分析、糖缀合物糖链的生物合成、糖缀合物糖链在复杂生物系统中的功能,以及糖缀合物糖链操作技术。结构辅助并基于机理进行有目的药物设计,仍是当前药物设计的主流,它可以为糖生物学开辟一个新的领域。而糖芯片可以广泛地用于研究糖类,例如高通量分析糖-蛋白的相互作用、药物基因组学等,是后基因组时代必不可少的一种研究工具。本文的主要工作是研究了β-(1→3)-D-葡聚寡糖衍生物的设计合成、生物活性测定和寡糖芯片的构建。完成的工作主要包括以下几个方面: 采用酸碱法来提取啤酒酵母中的β-(1→3)-D-葡聚糖。纸色谱和紫外光谱分析表明,所得产物为高纯度的β-(1→3)-D-葡聚糖,此结论由傅立叶红外(FTIR)光谱得到进一步的证实。这表明酸碱法是从啤酒酵母中提取β-(1→3)-D-葡聚糖的理想途径。荧光辅助糖电泳(FACE)是一种快捷、花费少的分离糖类方法。寡糖首先经8-氨基萘基-1,3,6-三磺酸(ANTS)胺化还原衍生,此反应过程在所给实验条件下需要16h。然后,ANTS 衍生的寡糖在由32%丙烯酰胺-2.4%双丙烯酰胺组成的碱性分离胶上电泳从而得以分离。此方法无需专门的仪器和操作熟练的技术人员。实验结果表明,当糖的浓度范围在5～100pM 之间时,带的荧光强度和糖的浓度之间成线性关系,并且没有哪个链长比其它的链长更加容易衍生化。用2.0mol/L 的CF3COOH 来水解酵母葡聚糖,得到的寡糖混合物经FACE 分析,结果证实得到了聚合度为1-7 的β-(1→3)-D-葡聚寡糖。根据β-(1→3)-D-葡聚糖酶的作用机理,设计出环氧烷基-β-(1→3)-D-葡聚寡糖。然后经过乙酰化、糖苷化、氧化和去乙酰化这四步来合成它。用ESI-MS 分析了所得到的一产物。酶活性分析表明,在β-(1→3)-D-葡聚寡糖分子中引入环氧烷基后,的确能够提高其抗酶水解能力,并且所引入环氧烷基的链长越短,其抗酶水解能力越强。测定了β-(1→3)-D-葡聚寡糖及其3,4-环氧丁基衍生物对超氧阴离子自由基的清除作用、对动物免疫调节的影响、对单核细胞产生超氧阴离子的影响、对Con A 诱更多还原

【Abstract】 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.更多还原