羅禮強Lo, Lee-Chiang臺灣大學:化學研究所陳昭凱Chen, Chao-KaiChao-KaiChen2010-06-302018-07-102010-06-302018-07-102009U0001-1006200914293300http://ntur.lib.ntu.edu.tw//handle/246246/187571目前已知蛋白質酪胺酸磷酸酯水解酵素（Protein Tyrosine Phosphatases, PTPs）參與調控許多細胞間訊號傳導的速率和過程，PTPs的活性表現與許多疾病間都具有關聯性，如癌症、神經失調和糖尿病等。本篇論文針對PTPs發展不同用途的活性標示分子來更加了解它在生物體內特定的動態表現。我們設計並合成出四個活性標示分子，具備了相同的磷酸化酪胺酸類似物作為對PTPs有專一性的辨識端，和連接橋連接潛在的捕捉基團—2-氟甲基-苯氧基基團和不同的發報端或親和標記用來觀察標示結果或純化蛋白質。首先，由酪胺酸開始建構對PTPs的辨識端，接著利用三甘醇衍生得到一端為疊氮基或帶有保護基的胺基作為連接橋，再與辨識端藕合得到具有疊氮基的活性標示分子前驅物，和其他活性標示分子的共同中間體，進一步連接上生物素、螢光團及具雙硫鍵的發報端，最後去除所有磷酸根的丙烯基保護基即完成四個活性標示分子的建構。未來將陸續完成這一系列活性標示分子的標示實驗並進一步和過去的成果比較及評估其發展潛力。A number of protein tyrosine phosphatases (PTPs) are linked to diseases, ranging from cancers to neurological disorders and diabetes. It was believed that PTPs were involved in controlling the rate and duration of the signal response. The main goal in this thesis is developing different uses of activity-based probes for profiling PTPs in dynamic systems. We designed and synthesized four activity-based probes that carry the same phosphotyrosine analog as a PTP-specific recognition head and a linker that connects the latent trapping device, 2-fluoromethylphenoxyl group, with different reporter groups or affinity tags for visualization and purification. First, a recognition head for PTPs was constructed from tyrosine and linkers with one side carrying an azido group or protected amino group were derived from triethylene glycol. Afterward a precursor of azido-based probe and common intermediates of probes were obtained by coupling the recognition head and linkers. Further, connecting reporter groups of biotin, fluorophore and disulfide-containing linker to the intermediates, and then removing all the allyl protecting groups of phosphates to afford the four probes. All the labeling performance and versatile applications for these probes will be compared to our previous work and evaluated in the future.口試委員會審定書誌……………………………………………………………………………….…..i文縮寫………………………………………………………………………….….ii文摘要…………………………………………………………………………….iv文摘要……………………………………………………………………………..v一章 緒論………………………………………………………………………....1.1 前言…………………………….……….………………………………….1.2 活性導向的蛋白質標記技術……..………………..……………...………2.2.1 依照不同反應機制的活性標示分子分類………………………....2.2.2 連接橋…………………………………………………..…………..8.2.3 發報端/親和標記的系統和偵測的策略..........................................9.2.4 可切斷的連接橋系統…………………...………………..…...…..12.3 研究對象………..……..…………….....………………………………….16.3.1 PTPs的作用機制…………………………………………………...16.4 目標分子的設計……….……………………………...………………......19二章 結果與討論………………………………………………………………..21.1 目標分子的逆合成分析………...…..…………………………….………21.2 辨識端的合成………………..……………………………………………24.3 連接橋的合成……………..………………………………………………31.4 活性標示分子前驅物的合成…………..…………………………………33.5 磷酸的去保護基反應………………………………………………….….38.5.1 發報端為生物素或螢光團的去保護基反應………………………39.5.2 發報端為疊氮基的去保護基反應…………………………………41.5.3 活性標示分子含雙硫鍵的去保護基反應…………………………43.6 目標分子4的新合成策略和方法………………..………………..……..45.7 活性標示分子之生物活性測試……………………………………….….48.7.1 ECL western blotting的原理及應用……………………….………48.7.2 針對PTP1B的敏感度比較………………………………..………50.7.3 針對各種酵素的專一性比較……………………….......…………51.7.4 針對各種磷酸酯水解酵素的選擇性比較……………...…………52.8 結論…………………………………………………………………….….54三章 實驗部份……………………………………………………………...……55.1 一般敘述..…………………………………………………………………55.2 有機合成實驗步驟及光譜數據………………………………………..…57考文獻………………………...……………………………………………..……78錄 化合物之核磁共振光譜圖...……..…………….…………………………….877840395 bytesapplication/pdfen-US蛋白質酪胺酸磷酸酯水解酵素活性標示分子生物素螢光團疊氮基雙硫鍵Protein tyrosine phosphatases (PTPs)activity-based probes2-fluoromethylphenoxyl groupbiotinfluorophoreazido groupdisulfide[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/187571/1/ntu-98-R95223056-1.pdf