活性標示分子之設計與合成及其性能改進之研究

Design and synthesis of activity-based probes as well as efforts in performance improvement

周子超(Tzyy-Chao Chou)

指導教授 ： 羅禮強

國立臺灣大學/理學院/化學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.10323

摘要

本論文主要分為兩部分，第一部分乃著重在新型高專一性、高產率且可應用於生理條件 (水相溶液、pH 值介於 6~8 與常溫常壓) 下之特殊化學反應 (Bioorthogonal Chemistry) 的開發。藉由磺酸疊氮官能基可與三苯磷化氫衍生物生成穩定的 Iminophosphorane 鍵結或與硫化酸生成天然的醯胺鍵結等特殊反應，我們開發出以乙二醇衍生物為主架構之磺酸疊氮基連接橋，並提供羧酸及胺類兩種官能基作為連接時可選擇的作用單元，且連帶合成一系列含有相對應官能基之發報端。藉由長時間 31P-NMR 實驗證明有機溶液中磺酸疊氮基與三苯磷化氫衍生物的反應速率較 Staudinger Ligation 為快，並由 ESI-MS 進一步確認此反應於水溶液中的可能。由初步結論得知，此反應具有高產率 (> 90%)、速率快 (< 0.5 h) 與適用於水相溶液等特點，並已成功地在牛血清蛋白表面進行兩階段標示實驗。此外，我們也在合成硫化酸發報端的過程裡建立了一套操作簡單且高產率的反應途徑，且依此得以對文獻中尚未進行詳細結構鑑定的生物素硫化酸作解析，更進一步擴展整體研究的面向。
第二部分則是針對蛋白質酪胺酸磷酸酶和醣苷酶作用之活性探針分子庫的設計與合成，並嘗試將其連接於固相載體上藉以比較其與溶液相中表現性質的差異。我們以 Bromobenzylphosphonate (BBP) 作為與蛋白質酪胺酸磷酸酶活化區進行親核性加成的反應基團，並建立起含有此特殊單元之非天然胺基酸的合成系統，且開發出以其作為仿酪胺酸磷酸酯的相關衍生物。除了以生物素作為發報端的活性探針之外，我們也成功的合成出含有疊氮官能基的對應化合物，並搭配具有末端炔的多種螢光團與 TBTA 配位基，建立一套以 Click Chemistry 進行兩階段標示的化學工具。同時也以對照實驗說明此非天然胺基酸適用於固相胜肽合成的可行性，並嘗試建構以苯丙胺酸為起始物且步驟較少的另一合成路徑，使得大量製備相關抑制劑分子庫的想法得以實現。以醣苷酶為作用對象之活性探針則以一系列與目標蛋白作用後可釋出醌化甲基活化體的化合物為主，並可藉由醣類辨識單元的調整達到針對特定蛋白進行標示的目的。我們也成功合成出具有單/雙氟甲基衍生物作為捕捉機制的活性標示分子，並連接上生物素與螢光團作為親和性純化與偵測訊號的依據。此系統的主要概念在於引入含有疊氮官能基的連接橋，除了直接以兩階段標示偵測目標蛋白的訊號之外，還可藉由 Staudinger Reduction 轉為相對應的胺類官能基並與所需的多種化合物做連接，也使得單一路徑獲得多種活性探針分子成為可能。

關鍵字

硫化酸 ； 活性探針 ； 蛋白質酪胺酸磷酸 ； 酶醣苷酶

並列摘要

This thesis is divided into two parts. In first part, we attempt to develop a new Bioorthogonal Chemistry – a reaction with basic characters such as high specificity between two counterparts, high isolated yield and can be applied to physiological conditions (aqueous medium, pH between 6 to 8, atmospheric pressure and room temperature). We develop an ethyleneglycol-based linker with sulfonylazide group, which provides carboxylic acid or amino functional groups as alternative choice for amide bond condensation. Due to the unique chemical reactions between sulfonylazide and triphenylphosphine or thioacid, we also synthesize a series of corresponding reporter molecules. By the result of time course 31P-NMR experiments, it’s hard not to mention that the reaction rate of sulfonylazide and triphenylphosphine derivative is faster than Staudinger Ligation in organic solvents. ESI-MS experiments further confirm the feasibility of this reaction in aqueous solution. The preliminary data shows that this chemical reaction can be performed in aqueous medium with high yield (> 90%) and high reaction rate (< 0.5 h), should have great potential in the field of selective labeling of target protein, cell imaging or constructing biochips. In addition, we also establish a practical and high yield synthetic pathway to obtain biotin thioacid, and performed a detailed spectral characterization of the compound.
The second part puts emphasis on the design and synthesis of activity- based probes library for protein tyrosine phosphatase and glycosidase, and the different performance of immobilized probes on solid supports. We make use of bromobenzylphosphonate (BBP) as the reactive moiety for nucleophilic addition with the active site of protein tyrosine phosphotase, and develop a promising system to obtain the non-natural amino acid containing this unit. With this material in hand, we further serve it as phosphoryltyrosyl mimetic group to synthesis a series of related derivatives. In addition to the activity-based probes with biotin as reporter group, we also successfully construct the corresponding compounds with azide functional groups. With the help of a variety of fluorescent groups with terminal alkyne and TBTA ligand, we establish a chemical tool fot two-stage labeling purpose by means of Click Chemistry. Besides, the control experiments show the feasibility of our non-amino acid for solid phase peptide synthesis, and a more practical synthetic pathway from phenylalanine as the starting material make the preparation of related inhibitors library can be achieved. The activity-based probes for glycosidase are based on a series of quinine methide-releasing compounds, which selective label their target enzymes with the sugar recognition unit. We also successfully synthesize various molecules with (mono/di-fluoromethyl)benzyl trapping devices, and further attach to biotin or fluorophore for affinity purification and detection. The main concept of this sunthetic route is the introduction of a linker containing azide functional group, which can serve as the source of readout signal by two-stage labeling experiments, but also convert to the corresponding amine by Staudinger reduction for connection with other compounds, and makes it possible to acquire a variety of activity-based probes in a single pathway.

並列關鍵字

Bioorthogonal Chemistry ； Thioaicd ； Activity-Based Probes ； Protein Tyrosine Phosphotase ； Glycosidase

參考文獻

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