臺灣博碩士論文加值系統

　　近期有許多研究團隊發展親和型探針 (affinity-based probes, AfBPs) 的相關技術，第一個成功運用於蛋白質研究的 AfBPs，是由 Cravatt 以及 Yao 教授團隊各自發表的金屬蛋白酶探針。AfBPs 與蛋白質上的特定位置結合後，再經由共價鍵的生成，以達到對特定蛋白質的標記。AfBPs 的研究工作蓬勃發展，讓我們明瞭設計與合成標記不同生物分子的探針，為勢在必行的工作。
　　胚胎細胞的遷移過程對於生物體的形態發生與體型之建立相當重要。本實驗室先前研究指出，類固醇荷爾蒙－孕烯醇酮 (pregnenolone) 可維持微管的穩定，進而有效地促使細胞的遷徙。因此我們推測存在某種蛋白質，能與孕烯醇酮結合並調控微管的形成，我們稱此為孕烯醇酮結合蛋白質 (pregnenolone binding proteins, PBPs)。為了驗證此推論，我們設計與合成以孕烯醇酮為辨識基團、二苯丙酮為光反應基團以及生物素為序號標籤的光反應型探針 (P5C7b-O-NBPN)，並期待能運用此探針來鑑定與純化 PBPs。
　　除了蛋白質的研究之外，我們也對去氧核醣核酸 (DNA) 特殊的二級結構之相關研究深感興趣。在細胞分裂期間，DNA 尾端的端粒序列，能保護基因組的不穩定以及避免細胞衰老。許多腫瘤細胞中有過度表現的端粒酶，可維持端粒序列的動態平衡，使端粒長度不會因細胞分裂而縮短，此為腫瘤細胞不死的重要因素之一。所幸富含鳥嘌呤的端粒序列，在特定的環境下傾向於形成鳥嘌呤四股結構 (G-quardruplex)，此特殊的結構可抑制端粒酶延長端粒序列。近年來咔唑衍生物 BMVC 在文獻中被報導可作為四方結構 (G-quartet) 的穩定劑以及螢光探針。有鑑於此，我們設計與合成一系列以 BMVC 骨架為辨識端的芥子氣衍生物 (BMVC-CnM; n = 2, 3, 6)，以嘗試發展成為端粒導向的 DNA 烷化劑。先前我們已利用多種光譜學以及凝膠電泳實驗，來探討 BMVC-CnM 與四股結構之間的結合辨識。在本論文中，我們藉由螢光共振能量轉移 (FRET) 以及 DNA 足跡 (footprinting) 等分析方法，對 BMVC-CnM 與四股結構間的結合模式做更深入的研究。此外，端粒酶抑制性的測試以及細胞顯影等實驗結果，顯示出 BMVC- CnM 有潛力發展成為抗癌藥物。

　　Affinity-based probes (AfBPs) have been developed recently by several research groups. Both of Cravatt and Yao groups independently reported the first AfBPs for metalloproteases. AfBPs achieves labelling by binding at a specific site of a protein followed by a non-specific covalent bond-forming event. The seminal works of AfBPs research enlighten us to design and synthesize probes for targeting different biomolecules of interest.
　　Embryonic cell movement is essential for morphogenesis and establishment of body shape. In the previous research reported by our group, showed the steroid hormone, pregnenolone, can preserve the abundance of microtubules, and thus effectively promotes the cell movement. In light of this study, we speculate that there might be pregnenolone binding proteins (PBPs), which can bind pregnenolone and further control the formation of microtubules. To verify our assumption, the photoreactive probes (P5C7b-O-NBPN) composed of a pregnenolone as the PBPs binding group, benzophenone as the photo cross-linker and a biotin as the reporter were designed and synthesized. It is hoped that by using this set of probes, PBPs could be identified and further purified prospectively.
　　On the other hand, telomeres are specialized DNA ends providing protection against genomic instability and cell senescence during cell division. The homeostasis of telomere is maintained by telomerase, which is selectively expressed in most tumors, and hence has a crucial role in cellular immortalization.38(?) The G-rich sequence of human telomeric DNA has a strong propensity to form the DNA G-quadruplex secondary structure, which can inhibit the activity of telomerase.53(?) Recently, 3,6-bis(1-methyl-4-vinylpyridium) carbazole diiodide (BMVC) was reported as a G-quartet stabilizer and a fluorescence probe. Preserving the key structural features of BMVC, a series of BMVC-mustard conjugates (BMVC-CnM; n = 2, 3, 6) were designed and synthesized in our group in attempts to develop telomere-directed DNA alkylating agents. The recognition between BMVC-CnM and G-quarduplex were analyzed and confirmed by various spectroscopic tools and DNA-PAGE studies previously. In this thesis, FRET analysis and DNA footprinting were applied to investigate the binding modes of BMVC-CnM with G-quarduplex. Furthermore, the TRAP-assay and cell images experimental results indicate BMVC-CnM have great potentials to be developed as anti-tumor agents.

目錄

目錄 i
圖目錄 iii
表目錄 viii
中文摘要 ix
英文摘要 xi

緒論 1
第一章 孕烯醇酮的生理作用以及設計合成孕烯醇酮衍生物的 AfBP 5
1-1 孕烯醇酮的簡介以及其生理作用 5
1-2 孕烯醇酮穩定微管生成以及與微管協助蛋白質之作用 6
1-3 孕烯醇酮對斑馬魚胚胎細胞發育的影響 10
1-4 AfBP 技術用於膽固醇結合蛋白質的研究 13
1-5 孕烯醇酮衍生物的 AfBP 之分子設計概念 14
1-6 P5C7β-O- NBPN 的逆合成分析與合成介紹 16
第二章 以 DNA 為標的之化學癌症治療方法的回顧以及發展 27
2-1 蛋白質標的與標靶藥物設計 27
2-2 去氧核醣核酸標的與標靶藥物設計 28
2-2.1 代碼解讀分子 (Code-reading molecules) 30
2-3 端粒的介紹 34
2-3.1 獨特的結構標的：鳥嘌呤四股結構的發現與鑑定 37
2-3.2 潛在抑制端粒酶的抗癌藥物：鳥嘌呤四股結構穩定劑 44
2-3.3 結合鳥嘌呤四股結構穩定劑的DNA 烷化劑 48
第三章 以 BMVC 為端粒導向的芥子氣衍生物 (BMVC-CnM) 應用於烷化鳥嘌呤四股結構的研究 53
3-1 分子設計概念 53
3-1.1 BMVC-CnM 與端粒序列作用後的光物理性 53
3-1.2 BMVC-CnM 與端粒序列之反應產物的非變性及變性聚丙烯醯胺凝膠電泳分析結果 56
3-2 BMVC-CnM 與端粒序列作用的機制 59
3-2.1 BMVC-CnM 與具螢光標記的端粒片段作用後的 FRET 螢光光譜分析 60
3-2.2 定序不同 DNA 序列的烷基化產物及其紫外光/可見光、螢光光譜分析 62
3-3 BMVC-CnM 對端粒酶的抑制性 72
3-4 BMVC-CnM 之細胞顯影 76
3-5 總結 78
實驗部分 80
一、一般敘述 80
二、實驗步驟及光譜數據 83
三、DNA 反應以及凝膠電泳分析 92
參考文獻 95
附錄 106

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