臺灣博碩士論文加值系統

生物分子的硫酸酯化程度與其生物活性有密切關連性，而硫酸酯水解酵素活性的調控在此過程扮演著重要的角色，因此為藥物開發的對象，本論文即是針對芳基硫酸酯水解酶此一重要的酵素族群來開發活性標示分子。活性標示分子的結構上包含有辨識端、捕捉機制、連接橋段與發報端。基於芳香基硫酸酯水解酶可以水解簡單的芳香基硫酸酯的特性，因此本研究在活性標示分子的辨識端以及捕捉機制部分是以酪胺酸為基本架構，來合成出二種不同系列的標示分子。這二系列標示分子的主要差異在於其標示機制，第一系列是源自於自殺性受質的概念，利用水解後的中間體會生成高反應性的quinone methide，來達到標示的效果；第二系列則是屬於親核性試劑型，採用環狀胺基磺酸衍生物做為辨識端及捕捉機制，預期在其進入酵素的受質結合區後能與酵素催化中心形成共價鍵結。發報端的部份則設計為生物素或具備疊氮基團，可分別應用於行一階段及二階段的標示反應，以增加活性標示分子應用的靈活度。後續的研究將探討這二系列標示分子的專一性以及他們在複雜蛋白質系統中的標示效能。

Sulfatases play a key role in regulating the sulfation status of many physiologically important biomolecules, and have become a target for drug development. In this thesis we report the design and synthesis of activity-based probes for this important enzyme family. An activity-based probe consists of four structural components; a recognition head, a trapping device, a linker, and a reporter group. Since most arylsulfatases could hydrolyze simple aryl sulfates therefore we attempted to develop two series of probes utilizing tyrosine derivatives as the core structure for the recognition heads and the trapping devices. These two series of probes differ in their mechanism of action. The first series exploits the concept of suicide substrate. When the probe is hydrolyzed by the target sulfatase, it will release an intermediate that undergoes elimination to form a reactive quinone methide which in turn reacts with nearby nucleophiles. The second series belongs to an electrophilic reagent which utilizes a cyclic sulfamate moiety serving both as the recognition unit and the trapping device. It is expected to form a covalent linkage with the target enzyme at the active site. A biotin and an azido group were respectively used as the reporter group for the one-stage and two-stage labeling protocols. Their labeling performance is currently under investigation.

謝誌…………………………………………………………………I
中文摘要……………………………………………………………II
英文摘要……………………………………………………………III
目錄…………………………………………………………………IV
化學符號縮寫………………………………………………………VIII
第一章 緒論…………………………………………………………1
1.1 前言……………………………………………………………1
1.2 研究對象………………………………………………………2
1.2.1 Aryl Sulfatase的簡介…………………………………2
1.2.2 Aryl Sulfatase的水解機制……………………………4
1.3 活性標示分子…………………………………………………6
1.3.1 活性標示分子之設計………………………………………8
1.3.2目標分子之設計……………………………………………14
第二章 結果與討論…………………………………………………16
2.1 化合物1之合成………………………………………………16
2.1.1 化合物1的合成策略……………………………………16
2.1.2 化合物1的合成方法……………………………………19
2.2 化合物2之合成………………………………………………30
2.2.1 化合物2的合成策略……………………………………30
2.2.2 化合物2的合成方法……………………………………32
2.3 化合物3之合成………………………………………………41
2.3.1 化合物3合成策略………………………………………41
2.3.2 化合物3的合成方法……………………………………43
2.4 化合物4、5之合成…………………………………………45
2.4.1 化合物4、5合成策略…………………………………45
2.4.2 化合物4、5的合成方法………………………………48
第三章 實驗部分…………………………………………………53
3.1 一般敘述……………………………………………………53
3.2 有機合成實驗步驟及光譜數據……………………………55
參考文獻……………………………………………………………73
附錄 化合物之核磁共振光譜圖…………………………………78

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