芳香磺醯氟化物化學探針之開發及其在蛋白質體學上的應用

Abstract

研究蛋白質體(proteome)中數目龐大的蛋白質之功能與結構，是很具挑戰性的工作，而各式化學探針的發展則提供了高效能的工具，以便能快速的在複雜蛋白質系統中發現標的，進而進行目標蛋白質的分析與鑑定。本論文主要是發展以芳香磺醯氟基作為捕捉基團的化學探針，運用組合式化學的概念，將芳香磺醯氟基與由胺基酸或鹼基所形成的辨識端連結，再搭配不同位向的鍵結方式，來合成出化學探針分子庫。探針分子的作用原理，是利用辨識端來進入特定的蛋白質族群的受質結合區，使探針分子的磺醯氟基能與其附近的蛋白質上的親核基作用形成共價鍵，以達捕捉及標示特定蛋白質的目的。 在本研究中，探針分子辨識端的部分共採用三種胺基酸：離氨酸(Lysine)、麩氨酸(Glutamic acid)、苯丙胺酸(Phenylalanine)以及三種鹼基：腺嘌呤(adenine)、胸腺嘧啶 (Thymine)、鳥嘌呤(Guanine)；發報基團的部分則是採用疊氮基(azido)，在完成蛋白質的捕捉反應後，將與含有炔基(alkyne)的螢光團進行1,3-偶極環加成(1,3-dipolar cycloaddition)，如此將可利用螢光來顯示和定量被標示的蛋白質。對於所合成出的探針分子庫，將用於探討其對特定蛋白質的標示專一性並觀察在複雜蛋白質系統中的標示效能。此外，也將嘗試將此二階段標示法應用於細胞內以驗證其性能。

The presence of a large variety of proteins within cells makes it a challenge to analyze function and structure for individual proteins. This problem would be simplified if the analysis could be confined to certain functionally related protein families with the assistance of target-selective chemical probes. In this thesis we exploited the concept of combinatorial chemistry by utilizing arene sulfonyl fluoride as the reactive group in conjunction with an appropriate recognition unit to construct structurally diverse probe libraries. The reactive sulfonyl fluoride is expected to undergo a nucleophilic attack with a suitable residue near the active site of the target proteins. The recognition unit covers three kinds of amino acids (lysine, glutamic acid, and phenylalanine) and two kinds of nucleobases (adenine and thymine). Probes all carry an azido tail that could undergo 1,3-dipolar cycloaddition with the terminal alkyne of a fluorescent reporter group. The probe libraries will be subjected to labeling experiments with various protein extracts in order to validate the two-stage labeling protocol and to evaluate their labeling performance.