蛋白質微陣列晶片在蛋白質-蛋白質間交互作用的研究中是快速且經濟的高通量篩選平台，此方法已廣泛應用於蛋白質體學的研究中。本論文合成具親核性胺基側鏈之環辛炔以及一系列亞硼酸-苯基磺醯基化學探針，並將其應用在微陣列晶片表面修飾。此亞硼酸-苯基磺醯基化學修飾化之晶片表面可將目標蛋白質溶液與晶片表面進行接合。在利用其亞硼酸官能基與醣蛋白之醣基上的雙醇進行反應而形成亞硼酸酯。藉由鄰近效應，蛋白質表面具親核性的胺基酸殘基可與化學探針的苯基磺醯基進行 SN2 親核性取代反應。即可將蛋白質以位向專一性共價鍵方式固定化於晶片表面。然後利用丙三醇溶液對亞硼酸酯進行可逆之競爭反應，使醣蛋白之醣基恢復其原生結構。最後以修飾上螢光基團的凝集素 ( 與醣蛋白醣基具有高度特異性之結合蛋白 )，來探討蛋白質-蛋白質交互作用。此方法利用勻相反應環境，能有效提升亞硼酸-苯基磺醯基式化學探針在晶片表面的修飾效率，是一個新穎的位向專一性共價鍵式醣蛋白固定化化學策略。

Protein microarray is a high-throughput screening platform in the study of protein-protein interaction. However, the orientation and structure integrity of immobilized proteins can considerably affect the corresponding biological activity. To date this end, amino-cyclooctyne and a series of boronic acid (BA)-tosyl chemical probes have been synthesized, and used in the fabrication of glycoprotein microarray. Accordingly, strain-promoted azide-alkyne cycloaddition under homogeneous condition have been shown to improve the efficiency of BA-tosyl chemical probe modification on microarray surface. BA-tosyl functionalized surface can conduct the anchoring of glycoproteins. The boronic acid component recognizes the diols of glycan to form boronate diester ring. The resulting proximity effect can trigger a SN2 reaction by nucleophilic residues of protein to the tosyl group of the chemical probe, thereby immobilizes the protein of interest on microarray surface. In this research, a highly efficient strategy for the immobilization of glycoproteins is proposed and then applied in the study of glycoprotein-protein interactions.

論文審定書........................................................................................................................i
誌謝...................................................................................................................................ii
論文提要..................................................................................................................iii
中文摘要..........................................................................................................................iv
英文摘要...........................................................................................................................v
目次..................................................................................................................................vi
圖目錄............................................................................................................................viii
流程目錄..........................................................................................................................ix
表目錄..............................................................................................................................xi
光譜目錄.........................................................................................................................xii
縮寫表..........................................................................................................................xviii
第一章 緒論.....................................................................................................................1
　　第一節 研究背景.....................................................................................................1
　　　　1.1.1 微陣列晶片簡介.....................................................................................1
　　　　1.1.2 蛋白質微陣列的種類.............................................................................2
　　　　1.1.3 蛋白質固定化策略.................................................................................4
　　　　1.1.4 位向專一性共價鍵式蛋白質固定化法.................................................7
　　　　1.1.5 張力促進疊氮-炔環加成反應..............................................................12
　　第二節 研究動機及實驗設計...............................................................................14
第二章 實驗結果與討論...............................................................................................20
　　第一節 具有環張力炔類分子之合成...................................................................20
　　　　2.1.1 具胺基側鏈環辛炔片段之合成...........................................................21
　　　　2.1.2 具胺基側鏈環辛炔之合成...................................................................23
　　第二節 亞硼酸-苯基磺醯基化學探針之合成......................................................27
　　　　2.2.1 短鏈錨固官能基之合成.......................................................................29
　　　　2.2.2 中鏈錨固官能基之合成.......................................................................29
　　　　2.2.3 長鏈錨固官能基之合成.......................................................................30
　　　　2.2.4 亞硼酸-短鏈-苯基磺醯基-疊氮基分子之合成...................................31
　　　　2.2.5 亞硼酸-中鏈-苯基磺醯基-疊氮基分子之合成...................................34
　　第三節 亞硼酸-苯基磺醯基化學探針分子於醣蛋白微陣列晶片之應用..........37
　　　　2.3.1 張力促進疊氮-炔環加成反應之反應性測試......................................38
　　　　2.3.2 阻抗劑降低背景值干擾之比較...........................................................39
　　　　2.3.3 不同長度亞硼酸-苯基磺醯基化學探針對醣蛋白晶片效能之比較..40
　　第四節 利用二維核磁共振光譜圖進行一維氫譜及碳譜之核磁共振光譜標定
　　　　　 ...................................................................................................................43
第三章 結論...................................................................................................................49
第四章 引用文獻...........................................................................................................50
第五章 實驗步驟與光譜數據.......................................................................................55
　　第一節 儀器設備與藥品材料...............................................................................55
　　第二節 合成步驟與光譜數據...............................................................................57
　　第三節 醣蛋白晶片之製備.................................................................................103
　　　　5.3.1 晶片表面修飾環辛炔之方法.............................................................103
　　　　5.3.2 張力促進疊氮-炔環加成反應之反應性測試....................................103
　　　　5.3.3 阻抗劑之比較實驗.............................................................................104
　　　　5.3.4 晶片表面修飾化學探針之方法.........................................................104
　　　　5.3.5 晶片表面固定化醣蛋白之方法.........................................................105
　　　　5.3.6 以修飾螢光標記之凝集素標定醣蛋白之分析.................................105
第六章 光譜資料.........................................................................................................107

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