臺灣博碩士論文加值系統

　　蛋白質磷酸化與去磷酸化是細胞中調控蛋白質活性的重要機制之一，因此參與此過程的兩大酵素家族，包括蛋白酪胺酸磷酸酶(Protein Tyrosine Phosphatases, PTPs)與蛋白酪胺酸激酶(Protein Tyrosine Kinases, PTKs)，在細胞的訊息傳遞、生長分化與代謝的調控上扮演了重要角色。失調的酵素活性常會造成人類疾病，例如腫瘤與癌症等。過去的文獻對於PTKs家族著墨較多，對於重要性不亞於PTKs的PTPs家族則相對較少。在所有PTPs蛋白家族中，它們的活性區位中都包含一段共同的胺基酸序列：HC(X)5R(S/T)，被稱為PTP的signature motif，其中半胱胺酸的親核性反應主導了PTPs催化磷酸水解的進行。本論文的研究即是利用此一結構特性，來發展針對PTPs蛋白家族具有專一性的活性探針，希望能應用於偵測細胞中PTPs的活性。
　　在活性探針最關鍵的反應端部分，我們設計了具有ａ,ｂ-不飽和結構的酪胺酸磷酸酯類似物，並採用組合式化學的概念，總共合成四個化學探針分子。這些探針分子除了反應端的差異外，皆有相同的生物素發報端以及四乙二醇的連結橋段。我們後續則評測這些探針分子能否被PTPs所辨識，進而利用其中的ａ,ｂ-不飽和結構來和PTPs活性位中的半胱胺酸發生麥可加成反應(Michael addition)，形成不可逆的共價加成產物。它們在與PTPs (SHP2、TCPTP、VHR)進行標識實驗的結果顯示，其中兩個探針能和PTPs產生共價作用，並在對PTPs家族酵素有專一性及選擇性。因此，本論文第二階段採取前述兩個能和PTPs作用探針分子的架構，但改用FQCA螢光團來取代生物素發報端，合成出兩個帶有螢光的探針。標記實驗結果證實螢光探針可提高偵測靈敏度。
　　螢光探針未來可應用在探針分子於PTPs上修飾位置的鑑定，藉由PTPs標識→胰蛋白酶消化→HPLC分離與螢光偵測→質譜分析的過程來確認。

Protein Tyrosine Phosphatases (PTPs) and Protein Tyrosine Kinases (PTKs) are two enzyme families which control protein tyrosine dephosphorylation and phosphorylation reactions. Reversible tyrosine phosphorylation plays critical roles in regulating cellular activities such as growth, differentiation, metabolism and signal transduction. Thus, aberrant activities of these enzymes have been implicated in many human diseases including tumors and cancer. Among the PTP superfamily, they share a conserved signature motif, HC(X)5R(S/T), in their active site. The nucleophilic cysteine dominates the catalytic mechanism of dephosphorylation process.

In this work, we focused on this structural feature and developed specific active site probes for PTPs in the hope that they could be used to profile intracellular PTP activities. We have designed and synthesized four chemical probes for PTP enzyme family. Those probes carry the same tetraethylene glycol linker and biotin reporter but differ in their reactive groups which consist of ,-unsaturated structure as phosphotyrosine mimetics. Later we investigated the reactivity between our probes and PTPs to see if they can covalently modify of active site cysteine through Michael addition. Labeling results showed that two of our chemical probes form covalent adducts with the PTPs tested (SHP2, TCPTP and VHR) and the probes are specific toward PTPs.
　　In second part we synthesized two fluorescent probes based on the previous PTPs reactive structure but used FQCA fluorophore as reporter. The fluorescent probes provided higher detection sensitivity and they could further be used to examine the labeling site of our probes on PTPs by processes of protein labeling→ digested by trypsin→ HPLC separation and fluorescence detection→ Mass analysis.

摘要 i
Abstract ii
目錄 iv
圖目錄 vi
表目錄 ix
反應式目錄 x
第一章 緒論 1
1.1 PTPs的分類 2
1.1.1 具酪胺酸專一性的PTPs 3
1.1.2 雙重專一性的PTPs 4
1.2. PTPs的催化機制 5
1.3. PTPs與疾病 6
1.3.1 PTP1B與胰島素訊息傳遞途徑及肥胖症 6
1.3.2 SHP2與努南式症候群 6
1.3.3 PTPs與免疫疾病 7
1.3.4 PTPs與癌症及腫瘤 7
1.4. 針對PTPs的活性探針(Activity-Based Probes, ABPs) 8
1.4.1 可產生Quinone methide的探針 9
1.4.2 Bromobenzyl phosphonate 11
1.4.3 Aryl vinyl sulfone and sulfonate 13
1.5. 實驗目的 14
1.5.1 以麥可受體為基礎發展新型仿磷酸酪胺酸單元 14
1.5.2 針對PTPs的活性探針分子設計 15
第二章 結果與討論 17
2.1. 探針2a-2d之逆合成分析 17
2.2. 合成分析及討論 18
2.2.1 連接橋－化合物6之合成 18
2.2.2 共同的中間體－化合物3a/3b之合成 18
2.2.3 探針2a/2b之合成 21
2.2.4 探針2c/2d之合成 21
2.3. 標記實驗結果 24
2.3.1 探針2a-2d與PTPs的反應性 24
2.3.2 探針2a為PTPs的活性位探針 30
2.3.3 探針2a對PTPs家族的選擇性 30
2.4. 發展以2a/2b為基礎的螢光探針 33
2.5. 合成分析與討論 36
2.5.1 FQCA螢光試劑－化合物13之合成 36
2.5.2 FQCA與胺基酸生成異吲哚產物之條件優化 40
2.5.3 螢光團－化合物11之合成 45
2.5.4 探針9a/9b之合成 48
2.5.5 探針9a/9b對PTPs的敏感度測試 49
2.6. 結論 51
第三章 實驗部分 52
3.1. 一般敘述 52
3.2. 有機合成實驗步驟及光譜數據 53
參考文獻 80
附錄 86

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