臺灣博碩士論文加值系統

自從青黴素被發現以後，數十年來抗生素一直被用來對抗細菌感染所造成的疾病。然而廣泛使用抗生素的情況下，許多具抗藥性的菌種相繼產生，引起人與動物嚴重的用藥問題，因此發展新穎的抗生素成為刻不容緩的議題。細菌細胞壁主要是由肽聚醣所構成，其可提供胞壁的堅韌與剛性來保持菌體形狀來抵抗外界滲透壓差，對細菌之生存是相當重要的。肽聚醣生合成路徑中的獨特轉醣酶(TGase)由於位在細胞膜外側，藥物不需進入細胞膜內側與其作用，較不受其他因素干擾，且胜肽鏈的多醣體骨架在眾多細菌突變種中依然相同，不易產生突變，因而日漸成為抗菌劑研究的關注方向。
設計轉醣酶抑制劑的概念，主要是模擬以lipid II為單體經轉醣酶催化時，聚合成一多醣鏈狀的過渡態；當其行聚合反應時，經由電子轉移之化學過程中，會形成oxonium過渡態，因此我們設想可仿照此過渡態，設計新穎的轉醣酶抑制劑。我們利用特殊的醣–DANA, GlcNAc或是帶有能產生氫鍵官能基團的苯環結構來模擬lipid II多醣部分的骨架結構；另外，用脂質鏈(C8)malonate或是天然轉醣酶抑制劑—moenomycin結構中之脂質鏈phosphoglycerate來仿照過渡態中脂質鏈雙磷酸的部分。此外，我們也從脂質鏈phosphoglycerate的磷酸基團上延伸不同長度的胺鹽，預期用來找出多醣部分和脂質鏈雙磷酸間之最佳距離。

成功合成出一系列具有潛力成為轉醣酶抑制劑之化合物後，我們將其進行轉醣酶活性以及最低抑菌濃度之檢測。篩選結果顯示，在所有我們設計之轉醣酶抑制劑中，化合物59之轉醣酶活性抑制效果和抑菌效果都是最好的，其可以在100 μM下完全抑制住轉醣酶之活性，此外其最低抑菌濃度為100 μM；而化合物60的轉醣酶活性和抑菌效果也相當不錯，其在100 μM下有80%抑制轉醣酶之活性。
OOHOOPOOOHH2N59Bestinhibition(100%inhibitionat100uM)OOEtOOPOOOH6080%inhibitionat100uMH2N
另外，我們發現到從脂質鏈phosphoglycerate的磷酸基團上延伸不同長度的胺基鹽這一系列之化合物，在100 μM下幾乎都有些許抑制轉醣酶之活性；可惜當我們引入用來模擬lipid II多醣部分的骨架結構之DANA, GlcNAc或是帶有能產生氫鍵官能基團的苯環結構後，發現到轉醣酶抑制效果皆消失，推測是由於原先用來模擬轉醣化部分正價性質之胺基鹽結構被無正電性質之triazole或醯胺鍵取代所導致。因此從轉醣酶活性檢測結果中，我們發現脂質鏈phosphoglycerate可成功取代lipid II中脂質鏈雙磷酸的部分，但脂質鏈malonate則無法達到此效果。依據活性的化合物結構特徵來看，轉醣酶抑制劑之設計結構除了應含有脂質鏈與磷酸團基之特性，且由磷酸根延伸胺基鹽之正價性質是相當重要的；未來將這些具活性的結構衍生修飾，期望能發展出更具轉醣酶抑制效果的抑制劑。

The discovery of penicillin antibiotics has a great impact on the treatment of infections caused by bacteria. However, the emergence of drug resistance has become a serious threat to human health. There is an urgent need to develop new agents that are active against resistant bacteria. The major constituent of bacterial cell wall is the peptidoglycan, which allows bacteria to live in a variable internal osmotic environment. Transglycosylase (TGase) is located on the extracellular surface of the bacterial cell membrane, where it catalyzes the polymerization of lipid II to establish the bacterial cell wall. Thus, TGase can be a potential target for development of new antibiotics.
We designed and synthesized some potential transglycosylase inhibitors using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA), N-acetylglucosamine (GlcNAc) or benzene ring bearing several hydrogen-bonding groups to mimic the saccharide part of lipid II, the natural substrate of TGase. On the other hand, long chain malonate or phosphoglycerate, a characteristic motif of moenomycin, were used to mimic the long chain pyrophosphate part. We also elongated different lengths of terminal amine from phosphoglycerate in order to determine the effect of the distance from phosphoglycerate to the saccharide part of lipid II. These kinds of inhibitors are expected to block the process of transglycosylation by mimicking the transition state in lipid II polymerization.

Some potential transglycosylase (TGase) inhibitors were synthesized and subjected to HPLC-based TGase fluorescence assay and MIC assay. The results have revealed that compound 59 is the best inhibitor having complete inhibition of TGase at 100 μM, among the designed compounds based on the transition-state of lipid II polymerization. Compound 60 has 80% inhibition at 100 μM.
OOHOOPOOOHH2N59Bestinhibition(100%inhibitionat100uM)OOEtOOPOOOH6080%inhibitionat100uMH2N
Most derivatives of phosphoglycerate bearing a terminal amino group showed some inhibition against TGase at 100 μM according to the HPLC-based fluorescence assay; however, the inhibitory activity diminished after connection with DANA, GlcNAc or benzene ring bearing several hydrogen-bonding groups. Though the part of long chain pyrophosphate could be replaced by the phosphoglycerate, a characteristic motif of moenomycin in TGase binding, it cannot be replaced by malonates. Phosphoglycerate coupled with the terminal amine might play an important role in TGase inhibition. Modification of these active compounds may eventually lead to more efficient inhibitors of TGase.

謝誌………………………………………………………………… I
中文摘要……………………………………………………………III.
英文摘要……………………………………………………………V
目錄…………………………………………………………………VII
圖目錄………………………………………………………………X
表目錄………………………………………………………………XI.
流程目錄……………………………………………………………XII
簡稱用語對照表……………………………………………………XIII
第一章 緒論…………………………………………………………1
1.1細菌細胞壁之生合成機制與重要性…………………………….1
1.2抗生素的介紹與抗藥性…………………………………………4
1.3青黴素結合蛋白與轉醣酶構造…………………………………6
1.4轉醣化機制………………………………………………………8
1.5轉醣酶抑制劑的發展……………………………………………11
1.6轉醣酶活性分析法………………………………………………19
1.6.1 放射標誌分析法……………………………………………19
1.6.2 高效能液相層析分析法……………………………………19.
1.6.3 表面電槳共振分析法………………………………………22
1.6.4 螢光異向性分析法…………………………………………23
1.7過渡態模擬抑制劑……………………………………………24
第二章 結果與討論………………………………………………26
2.1轉醣酶抑制劑的設計概念……………………………………26
2.2各轉醣酶抑制劑之設計來源及合成方法……………………28
2.2.1 DANA四號位置修飾脂質鏈pyrophosphate………………28.
2.2.2 DANA四號位置修飾脂質鏈malonate衍生物………………39
2.2.3脂質鏈phosphoglycerate衍生物…………………………45
2.2.4 DANA四號位置修飾脂質鏈phosphoglycerate衍生物……54
2.2.5其他脂質鏈phosphoglycerate衍生物………………………62
2.2.5.1 以GlcNAc模擬多醣部分骨架結構…………………………62
2.2.5.2 以苯環結構模擬多醣部分骨架結構………………………64
2.3各合成之潛在轉醣酶抑制劑活性分析…………………………66
2.3.1轉醣酶活性檢測………………………………………………68
2.3.2最低抑菌濃度之檢測…………………………………………72
2.4結論………………………………………………………………74
第三章 實驗部份……………………………………………………77
3.1 General part…………………………………………………78
3.2 General procedures of HPLC-based TGase fluorescence assay……79
3.3 General procedures of MIC assay……………………79
3.4 Synthetic procedures and characterization of compounds…………………………………………………………80
參考文獻……………………………………………………………139.
附錄：1H, 13C, 31P NMR光譜……………………………………146

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