在現在這個時代，抗生素是人類用來對抗細菌感染的重要利器。然而，濫用抗生素的情況下，導致許多具抗藥性的菌種相繼產生，開發新穎的抗生素成為刻不容緩的目標。細菌細胞壁的主要成分為&;#32957;聚糖，其主要功用為維持細菌細胞形狀來抵抗外界滲透壓差，對細菌之生存是相當重要的。細菌細胞壁的生合成路徑主要是透過轉胜&;#32957;&;#37238;和轉醣&;#37238;催化下所進行，由於轉醣&;#37238;裸露在細胞膜外側，藥物不需進入細胞膜內即可對酵素進行抑制，因此被科學家們認為以此為目標開發新型的抗生素是相當具有潛力的。
我們所設計之轉醣&;#37238;抑制劑，主要概念是以模擬lipid II為單體，在轉醣化過程中所形成的過渡態之電荷和結構，搭配天然轉醣&;#37238;抑制劑—moenomycin作為結構的參考，設計出一系列具有潛力的轉醣&;#37238;抑制劑。我們以moenomycin脂質區特徵基團作為基礎骨架，以短鏈(C8)之phosphoglycerate衍生物來模擬lipid II的雙磷酸脂質鏈，透過適當長度的連接鏈，連接模擬lipid II多醣部分骨架之芳香環結構。 合成出所設計之一系列轉醣&;#37238;抑制劑之後，我們透過轉醣&;#37238;活性分析及最低抑菌濃度檢測來驗證所設計之化合物是否具抑菌能力。在這幾個合成之phosphoglycerate衍生物中，苯環上帶有能產生氫鍵的官能基之化合物，對金黃色葡萄球菌的最低抑菌濃度為100 μM，但是在相同濃度下對轉醣&;#37238;卻不具有抑制性。綜合所有本實驗室所設計之轉醣&;#37238;抑制劑，我們認為保留磷酸鹽（phosphate）結構是必要的，之後再利用還原胺化方式引入能產生氫鍵作用力的取代基（如OH基）之苯環結構，同時保留苯環與氨基酸殘基之作用力，還能產生額外氫鍵吸引力，期望能提抑制劑之抑制活性，開發出更有潛力的轉醣&;#37238;抑制劑。

In this day and age, antibiotics are used to treat people for bacterial infections. However, abuse of antibiotics also leads to the appearance of multi-drug resistant bacteria. There is an urgent need to develop new agents that are active against multi-drug resistant bacteria. The main constituent of bacteria cell wall is peptidoglycan, which is responsible for a defined cell shape to allow bacteria to live in a variable internal osmotic environment. Transpeptidase (TPase) and transglycosylase (TGase) are two major enzymes which can catalyze the synthesis of bacteria cell wall. TGase is located on the external surface of bacterical cell membrane, so that it is unnecessary for inhibitors to enter cytoplasm. We consider TGase as a promising antibiotic target due to its essential function and ready accessibility.
We have designed and synthesized some potential transglycosylase inhibitors with an aryl moiety bearing several hydrogen-bonding groups to mimic the saccharide part of lipid II, the nature substrate of TGase. In addition, a long chain phosphoglycerate was used to mimic the long chain pyrophosphate part of lipid II. These inhibitors are designed to block the process of transglycosylation by mimicking the transition state in lipid II polymerization. Some potential transglycosylase inhibitors were synthesized and subjected to the HPLC-based TGase fluorescence assay and MIC assay. Among these derivatives of phosphoglycerate with the aryl moiety bearing hydrogen-bonding groups showed the best MIC value of 100 μM against Staphylococcus aureus. However, it showed no inhibition against TGase at same concentration. Among all of the transglycosylase inhibitors designed in our laboratory, we considered that it is necessary to retain phosphate structure and introduce benzene structure bearing hydrogen-bonding groups (e.g. OH). We hope that through the interaction between benzene ring and amide side chain and further hydrogen bonding might improve the inhibitory activity to act as efficient TGase inhibitors.
&;#8195;

目錄
謝誌……......................................................................................................I
中文摘要.................................................................................................III
英文摘要..................................................................................................V
目錄.......................................................................................................VII
圖目錄....................................................................................................IX
表目錄...................................................................................................XII
流程目錄...............................................................................................XIII
簡稱用語對照表.........................................................................................................XIV
第一章　緒論 1
1.1 細菌細胞壁之重要性與生合成路徑…………………………………………….1
1.2 革蘭氏陽性菌與革蘭氏陰性菌………………………………………………….2
1.3 青黴素結合蛋白 4
1.4 轉醣化機制探討 7
1.5 轉醣&;#37238;活性分析法 ……………………………………………………………...11
1.5.1 放射標誌分析法……………………………………………………...11
1.5.2 高效能液相層析分析法……………………………………………. .11
1.5.3 表面電漿共振分析法………………………………………………. .13
1.5.4 螢光異向性分析……………………………………………………. .14
1.5.5 螢光共振能量轉移…………………………………………………. .15
1.6 抗生素的介紹與發展 17
1.7 轉醣&;#37238;抑制劑的發展 19
第二章　結果與討論 28
2.1 轉醣&;#37238;抑制劑的設計概念 28
2.2 各轉醣&;#37238;抑制劑之合成方法 32
2.2.1 含脂質鏈之醇類片段的合成………………………………………...32
2.2.2 含適當長度連接鏈之膦酸(phosphonic acid)片段的合成…………. .33
2.2.3 耦合反應………………………………………………………….......34
2.2.4 含benzylamino基團化合物之合成…………………………….…...34
2.3 各潛在轉醣&;#37238;抑制劑之活性分析 40
2.3.1 轉醣&;#37238;活性檢測……………………………………………………...41
2.3.2 最低抑菌濃度檢測…………………………………………………...45
2.4 結論 48
第三章　實驗部分 51
3.1 General Part 51
3.2 General procedures of HPLC-based TGase fluorescence assay 52
3.3 General procedures of MIC assay 53
3.4 Synthetic procedures and characterization of compounds 53
參考文獻 75
附錄: 化合物之NMR光譜.. 82






圖目錄
圖一、細菌細胞壁的生合成 2
圖二、革蘭氏陽性菌與革蘭氏陰性菌概要圖 3
圖三、Moenomycin與Staphylococcus aureus PBP2鍵結之錯合物結構圖
………………………………………………………………………………………………………………….5
圖四、Neryl-moenomycin A與Aquifex aeolicus PBP1A鍵結之錯合物
結構圖 6
圖五、大腸桿菌(E. coli) PBP1b和moenomycin之錯合物共結晶結構圖 7
圖六、轉醣&;#37238;催化下，lipid II聚合之反應機構 8
圖七、Lipid II聚合之轉醣化機制，S1 、S2分別為glycosyl acceptor和
donor site 9
圖八、聚醣生長鏈(growing glycan chain)兩種可能延伸方式 10
圖九、利用[14C]Gal來分析lipid II受質於轉醣化過程扮演之角色 11
圖十、以螢光lipid II衍生物，配合HPLC分析轉醣&;#37238;活性的方法 12
圖十一、Lipid II-Dansyl-C20之設計概念 13
圖十二、Moenomycin A之衍生物 (Moe-NH2) 14
圖十三、運用F-Moe螢光異向性分析法篩選轉醣&;#37238;抑制劑 15
圖十四、利用螢光共振能量轉移法進行轉醣&;#37238;活性分析示意圖 16
圖十五、萬古黴素與突變前後細菌之lipid II作用力 18
圖十六、抑制細菌細胞壁合成之抗生素 19
圖十七、轉醣&;#37238;抑制劑nisin與moenomycin A之結構 20
圖十八、Sofia研究團隊發表之轉醣&;#37238;抑制劑結構 22
圖十九、Walker與Vederas研究團隊設計之轉醣&;#37238;抑制劑 23
圖二十、不同長度脂質鏈之moenomycin對抑菌效果之影響 24
圖二十一、中研院基因體中心高通量分子庫篩選出的轉醣&;#37238;抑制劑 25
圖二十二、鄭偉杰研究團隊設計之轉醣&;#37238;抑制劑 26
圖二十三、本實驗室所設計之轉醣&;#37238;抑制劑 27
圖二十四、Lipid II經轉醣化過程中所形成之過渡態和設計概念 28
圖二十五、本實驗室張容華所合成之轉醣&;#37238;抑制劑 29
圖二十六、Moenomycin在轉醣&;#37238;活性區可能具重要作用力的
胺基酸殘基 31
圖二十七、本實驗室鄭&;#32137;蓉所合成之轉醣&;#37238;抑制劑 31
圖二十八、引入苯環結構之phosphoglycerate衍生物的合成設計 32
圖二十九、本實驗室設計含苯環之轉醣&;#37238;抑制劑結構 35
圖三十、嘗試直接利用化合物38和2,3,4-trihydroxybenzaldehyde
進行還原胺化反應 37
圖三十一、透過OsO4對雙鍵進行氧化 37
圖三十二、嘗試利用Pd(PPh3)4去除烯丙基 38
圖三十三、設計之轉醣&;#37238;抑制劑結構 41
圖三十四、以HPLC分析轉醣&;#37238;活性 41
圖三十五、化合物38和53 (100 μΜ)之轉醣&;#37238;活性檢測結果 42
圖三十六、化合物46、47和50 (200 μΜ)之轉醣&;#37238;活性檢測結果 43
圖三十七、化合物54、55 和57 (500、200 μΜ)之轉醣&;#37238;活性檢測結果 43
圖三十八、化合物48和56 (100 μΜ)之轉醣&;#37238;活性檢測結果……………………..44
圖三十九、欲合成之帶有苯環骨架抑制劑的結構 45
圖四十、化合物38、46、47和50之最低抑菌濃度檢測結果 46
圖四十一、化合物53–55和57之最低抑菌濃度檢測結果 46
圖四十二、化合物48和56之最低抑菌濃度檢測結果……………………………….47
圖四十三、本實驗室所設計之轉醣&;#37238;抑制劑 49














表目錄
表一、　　革蘭氏陽性菌與革蘭氏陰性菌基本比較........................................ 4
表二、　　合成之轉醣&;#37238;抑制劑TGase MIC assay結果整理……………….49






















流程目錄
流程一、　　化合物33之合成方法............................................................................. 33
流程二、　　化合物36之合成方法............................................................................. 34
流程三、　 化合物38之合成方法............................................................................. 34
流程四、　　利用還原胺化合成化合物46、47…………………………………….36
流程五、　　化合物50之合成方法............................................................................ 38
流程六、　　化合物48之合成方法............................................................................. 39
流程七、　　化合物53–57之合成方法....................................................................... 40

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