合成硫酸化N-乙醯乳糖胺及其衍生物以用於研究半乳糖凝集素-8的結合專一性

Synthesis of N-Acetyllactosamine Sulfates and Analogues to Examine the Binding Specificity of Galectin-8

陳立涵(Li-Han Chen)

指導教授 ： 林俊宏

共同指導教授 ： 羅禮強(Lee-Chiang Lo)

國立臺灣大學/理學院/化學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01927

摘要

半乳糖凝集素家族(galectins)是一群可以辨識β-半乳糖苷(β
-galactosides)單元的蛋白家族。它們在調控生物的生理活性上扮演了重要角色。像是半乳糖凝集素-8(galectin-8, Gal-8)可以調控B細胞的分化與影響大腸癌細胞的轉移。在近幾年相關研究中開始以分子層級探討半乳糖凝集素對醣分子辨識的關係，用以解釋不同的半乳糖凝集素具有不同的醣辨識專一性(carbohydrate recognition specificity)與生物功能。
本實驗室之前已開發出硫酸化N-乙醯乳糖胺的合成方法，本論文則著重於合成一系列的Gal-β1,3/4-GlcNAc分子，在Gal的3號與6號羥基以及GlcNAc的6號羥基修飾上硫酸根基團。並且成功針對數種關鍵步驟改善產率，像是：醣基化反應(glycosylation)與硫酸化反應(sulfation)，以大量生產硫酸化雙醣分子用於進一步的研究。
製備完成的目標分子會以偏極化螢光(fluorescence polarization)的方式進行定量分析以評估醣分子與Gal-8的結合情形。在初步的分析結果中顯示將硫酸根基團修飾在N-乙醯乳糖胺雙醣分子Gal的3號羥基以及GlcNAc的6號羥基可以有效增進Gal-8對於醣分子的結合。亦可看出Gal-8的兩種醣辨識區具有不同的專一性。未來可應用硫酸化醣分子對生物特性的影響進行更深入的探討，像是研究硫酸化醣分子對細胞附著(adhesion)與細胞分化(differentiation)所造成的影響。

關鍵字

β-半乳糖苷半乳糖凝集素 ； 硫酸化 ； 偏極化螢光 ； N-乙醯乳糖胺

並列摘要

Galectins represent a family of animal lectins that bindβ-galactosides. They play an essential role in numerous physiological activities. Among 12 human galectins, galectin-8 was shown to serve as a key regulator in the immune responses of B-cell and affects the migration of human colon cancer cell. As a matter of fact, the sugar-binding specificity of each galectin is different, explaining the molecular basis of the functional differences corresponding to the biological activity.
Our lab previously developed an efficient method for preparing the sulfated N-acetyllactosamines and their analogues. My thesis is focused on the synthesis of Gal-β1, 3/4-GlcNAc sulfates with a sulfate group at either O3/6 of Gal or O6 of GlcNAc,as well as a methoxy group installed in the reducing terminus. Several reactions have been examined in detail to improve the yields, such as glycosylation and sulfation. We also scaled up the syntheses in order to obtain a sufficient quantity of the final products for further applications.
Finally the target disaccharides were measured for the binding affinity with galectin-8 based on fluorescence anisotropy. In the result of assay, we found galectin-8 had much higher affinity for 6-O-sulfated or 3’-O-sulfated N-acetyllactosamine disaccharides than for non-sulfated analogues. Also, the binding specificity of the two carbohydrate recognition domain are different. In the future, the sulfated disaccharides will be applied to the investigation of the biological function further.

並列關鍵字

β-galactoside ； galectins ； sulfation ； fluorescence anisotropy ； N-Acetyllactosamine

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