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研究生: 劉宛瑄
LIU, WAN-HSUAN
論文名稱: 結核病藥物開發:設計合成麥芽糖轉移酶抑制劑
Drug Discovery for Anti-Tuberculosis: Design and Synthesis of Maltosetransferase Inhibitors
指導教授: 謝俊結
Shie, Jiun-Jie
林文偉
Lin, Wen-Wei
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2019
畢業學年度: 107
語文別: 中文
論文頁數: 111
中文關鍵詞: 麥芽糖轉移酶麥芽糖轉移酶抑制劑高通量篩選細菌影像測試CuAACsulfonamido-oxine-based fluorescent
英文關鍵詞: maltose transferase, maltose transferase inhibitor, high throughput screening, bacterial imaging test, CuAAC, sulfonamido-oxine-based fluorescent
DOI URL: http://doi.org/10.6345/NTNU201900977
論文種類: 學術論文
相關次數: 點閱:114下載:0
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  • 結核病(Tuberculosis ,簡稱TB)是一種全球普遍性流行疾病,全球多重抗藥性結核桿菌(MDR)TB的例子日益嚴重增加,這說明需要開發一種治療結核病的新藥。參與細菌細胞壁合成α-D-葡聚醣 (α-D-glucan)成份的結核分枝桿菌麥芽糖轉移酶(Mtb GlgE)是近年來發現為結核桿菌生長的特定必需酶,適合用於設計抑制結核菌生長的目標酵素。在本論文中,我們合理設計了一系列模擬過渡態的類似物作為GlgE酶抑制劑,透過用氮原子取代醣苷 (glycosides) 的內環氧原子,將iminosugars和pseudo-醣苷上含有氮雜環官能基,作為與另一種醣苷相連的結構支架,希望藉由酶活性中心與分子所產生的電荷作用力用於抑制GlgE酶。此外,我們設計了螢光基質用於檢測GlgE酶活性指標,以磺酰氨基氧化物為螢光基團的sulfonamido-oxine-based fluorescent maltose-1-phosphates (Sox-M1P)作為主體基質,當發光團結合Mg2+,並在GlgE水解Sox-M1P後,藉由螯合增強螢光(CHEF)反應機制,以螢光強度變化達到高靈敏快速檢測的效果。另一方面,在本論文中,我們也描述了利用炔基修飾的單醣衍生物(GlcNAl,ManNAl,GalNAl和Fucyne)作為探針,藉由醣代謝生成途徑標記細菌壁中的醣共軛體 (glycoconjugates)策略。搭配一價銅離子催化疊氮-炔正交性化學反應偶合炔基官能化螢光探針的策略,希望以螢光顯影的方式檢測和區分不同菌株細胞壁表面醣共軛體,達到肉眼快速檢測的目的。

    Tuberculosis (TB) is an epidemic disease and the growing burden of multidrug-resistant (MDR) TB worldwide underlines the need to develop a new drug to treat the infectious disease. Mycobacterium tuberculosis maltosyltransferase (Mtb GlgE) involved in α-D-glucan is an essential enzyme only for tuberculosis growth. We designed a series of transition-state mimic analogues as inhibitors against GlgE by replacing the endocyclic oxygen atom of glycosides with a nitrogen atom, the iminosugars and fusing membered nitrogen heterocyclic ring into the pseudo-glycoside as the structural scaffold that is linked to another glycoside for inhibiting GlgE. In addition, we designed a fluorescence dye, sulfonamido-oxine-based fluorescent maltose-1-phosphates (Sox-M1P), the chromophore binds Mg2+ and undergoes chelation-enhanced fluorescence (CHEF) upon hydrolysis of Sox-M1P by GlgE. On the other hand, we describe a metabolic oligosaccharide engineering (MOE) strategy for exploiting sugar metabolic pathways to label glycocojugates in bacteria with alkyne-modified sugar substrates (GlcNAl, ManNAl, GalNAl and Fucyne). Subsequent CuAAC [Cu(I) catalyzed Azide-Alkyne Cycloaddition] reaction with azide-functionalized probes enabled fluorescent detection and visualization of bacteria.

    摘要 i Abstract ii 誌謝 iii 目錄 iv 圖目錄 vi 表目錄 viii 第一章 緒論 1 1.1 前言 1 1.2 腸道細菌抗生素與其細胞壁 3 1.3 生物正交化學反應 (Bioorthogonal Chemical Reaction) 5 1.4 結核病與診斷方式 8 1.5 結核病藥物 10 1.6 麥芽糖轉移酶 12 1.7高通量篩選(high throughput screening) 15 第二章麥芽糖轉移酶GlgE抑制劑及高通量篩選設計 16 2.1 麥芽糖轉移酶GlgE抑制劑 16 2.2麥芽糖轉移酶GlgE抑制劑設計 18 2.3芽醣轉移酶GlgE抑制劑高通量篩選設計 19 第三章實驗結果與討論 20 3.1麥芽糖轉移酶GlgE抑制劑合成 20 3.1.1前驅物內醯胺lactam (7) 與thiolactam (8) 合成 21 3.1.2麥芽糖轉移酶GlgE抑制劑(14)合成 22 3.1.3麥芽糖轉移酶GlgE抑制劑(15)合成 22 3.1.4麥芽糖轉移酶GlgE抑制劑(16)合成 22 3.1.5麥芽糖轉移酶GlgE抑制劑(17 )合成 23 3.1.6麥芽糖轉移酶GlgE抑制劑(18)合成 23 3.1.7麥芽糖轉移酶GlgE抑制劑(19)合成 23 3.2麥芽糖轉移酶GlgE活性指標螢光基質(Sox-M1P)合成 24 3.3細菌影像測試 25 第四章結論 35 第五章實驗部分 37 4.1 實驗儀器 37 4.1.1核磁共振光譜儀(Nuclear Magnetic Resonance Spectroscopy, NMR) 37 4.1.2高解析質譜儀 37 4.1.3螢光顯微鏡 38 4.1.4流式細胞儀 38 4.2 實驗藥品 38 4.3 實驗菌種 38 4.4 細菌培養條件與螢光測試條件 39 4.5合成步驟與光譜資料 40 參考文獻 62 附錄 66

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