針對醣苷水解酶分子工具合成之探討

Synthetic effort on the development of molecular tools for glycosidases

陳函郁(Han-Yu Chen)

指導教授 ： 羅禮強

國立臺灣大學/理學院/化學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202101274

摘要

近年來小分子探針工具的開發在化學生物學相關領域研究中的重要性日益增加，本論文以α-甘露醣苷酶與α-岩藻醣苷酶為對象，對其啟動式分子工具進行系統性合成策略的探討。本研究嘗試採用模組化的合成策略，以含有羥基苯甲醇/疊氮雙官能基的捕捉單元前驅物為核心架構，將複雜的合成步驟分為三個主要階段來發展具雙酬載特性的分子工具：(一)合成出全乙醯化醣苷（甘露醣與岩藻醣）連接到捕捉單元/標示部前驅物的重要中間體；（二）將第一酬載引入上述中間體的苯甲基位置；（三）去除醣苷保護基，再應用點擊化學引入第二酬載，完成下列目標化合物之合成。目標一：我們先於核心架構裡第一酬載位置引入氟原子，生成單氟甲基衍生物的捕捉機制前驅物，再藉由點擊反應在疊氮基位置分別引入螢光的發報基團（BODIPY/Cy5），完成4個α-甘露醣苷酶的活性探針分子。此探針分子庫將用於探討連接段長度以及不同螢光團對標示效能的影響。目標二：我們在捕捉機制前驅物雙酬載的位置分別引入淬滅團（BHQ2）及螢光團（Cy5）之組合，利用螢光共振轉移（FRET）的放光機制，來發展醣苷酶啟動式螢光活性探針分子。針對α-甘露醣苷酶，共完成2個此類活性探針分子，將應用於細胞顯影，來比較並驗證其效能。目標三：我們以α-岩藻醣作為辨識端，也設計了兩種探針。其一為搭配氟離去基/螢光團（BODIPY），其二為搭配淬滅團/螢光團（Dabcyl/BODIPY）之組合。我們後續也將進行一階段標示實驗，來比較此兩種螢光探針在蛋白標示上的效能。

關鍵字

分子探針 ； 雙酬載 ； 醣苷水解酶 ； 螢光共振能量轉移 ； 點擊反應

並列摘要

The utilization of molecular tools based on small organic molecules to tackle biological processes is increasingly gaining importance in chemical biology. In this paper, benzyl alcohol azide is used as the core framework to develop dual-payload molecular tools. The focus of discussion is systematic synthesis strategies of glycosidase-initiated molecular tools by taking α-mannosidase and α-fucosidase as research objects. It is mainly divided into three stages: (1) Synthesize of important intermediates with acetylated-mannosides and fucosides connected to the precursor capturing unit and azido labeling tag; (2) Introduce the first payload to the hydroxyl side of molecular tools precursor; (3) Remove the protection and utilize click chemistry to load the second payload to the molecular framework to complete the synthesis of the target compound.Our first goal is introducing fluorine atoms into the core structure as the first payload, generating activated molecules with monofluoromethyl derivatives as the capture mechanism. As for the part of the reporting group, an azide group is used, where fluorophores (BODIPY Cy5) are introduced respectively by click reaction to attain the purpose of capturing and labeling specific protein. We complete four kinds of activated probes of α-mannosidase. This library of probe molecules will be used to explore the length of the linker and the influence of different fluorophores on the labeling performance.Second, to develop glycosidase-activated functional molecules, we introduce quencher (BHQ2) and fluorophore (Cy5) as the double payload pair and adopt fluorescence resonance transfer (FRET) mechanism. Cell imaging and efficacy are involved in the comparison with these two molecular probes.Finally, we mainly use α-fucose as the recognition unit, and design two probes, one is a fluorine-containing active probe, and the other is combination of a quencher (Dabcyl) and a fluorophore (BODIPY). We also perform a one-stage labeling method to compare the performance of these two fluorescent probes on protein labeling.

並列關鍵字

glycosidases ； dual-payload ； FRET ； activity probe ； click chemistry

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