小分子感測對於基礎生物學的研究十分重要，因此許多研究致力於發展可於細胞内直接偵測小分子的方法。目前，利用螢光共振能量轉移 (Förster resonance energy transfer, FRET) 機制的感測蛋白為偵測活體細胞內小分子的主要方法，但其應用仍有許多限制如 : 螢光基團更換不易及需要使用基因工程等。本論文發展一種基於立體障礙調節的新型生物素化半合成蛋白，其原理為利用小分子與配體之間的競爭，開啟生物素衍生物與鏈黴親和素之間的專一性結合，進而測得螢光訊號。在沒有小分子分析物的情況下，此半合成蛋白會形成關閉狀態，使生物素辨識端因立體障礙無法與鏈黴親和素結合，而在加入小分子分析物後，呈現開啟形式，進一步使生物素辨識端暴露，並與鏈黴親和素結合，達到快速偵測小分子的目的。基於這個設計，我們相信這樣的變構半合成蛋白設計會成為基礎生物學研究和醫學診斷上重要的工具。

Small molecule sensing is very important to the understanding of biological processes. Currently, protein sensors based on Förster resonance energy transfer (FRET) have been employed as the main method for detecting small molecules in living cells. However, these FRET-based proteins cannot be applied on native cells and are restricted with limited choice of fluorophores. In this thesis, we introduce a new affinity-switchable biotinylated semisynthetic protein in which binding of the small metabolite target with the protein triggers interaction of streptavidin-biotin. In the absence of a small molecule analyte, the semisynthetic protein forms a closed state where the biotin is positioned in close proximity to the protein, imposing a large steric hindrance to preclude binding with streptavidin. In the presence of a small molecule target, the steric hindrance is removed as a result of open conformation, thereby exposing the biotin for streptavidin binding. We believe that this allosterically controlled semisynthetic protein design will become a powerful tool for fundamental biological research and medical diagnosis.

摘要.................................. I
Abstract ............................ II
謝誌................................. III
目錄.................................. V
第一章、緒論........................... 1
1-1 小分子的介紹 ...................... 1
1-1.1 生物體內的小分子 ................ 1
1-1.2 動物用藥 ........................ 2
1-1.3 磺胺類藥物 ...................... 3
1-2 小分子的檢測方法 .................. 5
1-2.1 質譜分析法 ...................... 5
1-2.2 酵素結合免疫吸附分析法 ........... 7
第二章、文獻回顧....................... 9
2-1 自然界中的小分子感測 ............... 9
2-1.1 G蛋白偶合受體 .................. 10
2-1.2 乳糖操縱子 ..................... 12
2-1.3 天然變構蛋白之應用 .............. 13
2-2 半合成蛋白 ....................... 15
第三章、探針設計...................... 18
3-1 探針設計構想 ..................... 18
3-2 探針合成設計與策略 ............... 21
3-2.1 蛋白及配體選擇 ................. 21
3-2.2 探針結構設計 .................. 22
第四章、實驗結果與討論................. 25
4-1 探針A、B、C合成與實驗結果 ......... 25
4-1.1 化合物13之測試 ................. 25
4-1.2 探針A之合成..................... 26
4-1.3 探針B之合成..................... 27
4-1.4 探針C之合成..................... 28
4-1.5 探針A、B、C之實驗結果 ........... 29
4-1.6 化合物36之測試 ................. 30
4-1.7 探針A、B、C之探討 .............. 31
4-2 探針D之合成與實驗結果 ............. 33
4-2.1 探針D之合成 .................... 33
4-2.2 探針D之實驗結果 ................ 34
第五章、實驗結論....................... 36
第六章、實驗部分....................... 37
6-1 實驗器材與藥品 .................... 37
6-2 細胞影像實驗 ...................... 39
6-2.1 細胞之準備 ...................... 39
6-2.2 探針之MCF-7細胞影像 ............. 39
6-3 有機合成與光譜資料 ................ 40
第七章、參考資料....................... 76
附錄.................................. 80

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