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標題: | 以磷酸基甘油酸酯衍生物為架構來設計及合成細菌轉醣酶的抑制劑 Structure-based design and synthesis of phosphoglycerate–lipid derivatives as the inhibitors against bacterial transglycosylases |
作者: | Jui-Yin Yu 俞瑞胤 |
指導教授: | 方俊民(Jim-Min Fang) |
關鍵字: | 轉醣?,抑制劑,抗生素,磷酸基甘油酸酯,聯苯, transglycosylase,inhibitor,antibiotic,moenomycin,phosphoglycerate,biphenyl, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 在治療細菌的感染上,抗生素的使用扮演舉足輕重的角色,然而面對具有多重抗藥性的細菌威脅,必須不斷探索新穎的抗生素及新的藥物作用標靶來對抗。轉醣酶與轉胜肽酶是兩個參與細菌細胞壁生合成的重要酵素。藉由這兩種酵素的催化,單體lipid II可以進行聚合而生成網狀聚合物“肽聚醣”。肽聚醣的生成可以幫助細菌對抗內部所產生的滲透壓,進而保持細菌的形狀與完整性。除了像β-內醯胺類抗生素等針對轉胜肽酶的抑制劑,由於轉醣酶在細胞壁生合成中扮演不可或缺的角色及具有藥物易達性,使得以轉醣酶為目標而開發的新型抗生素是極具吸引力的,而且轉醣酶裸露在細菌細胞膜外側,使得藥物在與酵素結合的過程中不會遇上阻礙。
依據天然抑制劑moenomycin的結構為基礎,我們設計並合成出一系列phosphorylpropanoate衍生物作為具有潛力的轉醣酶抑制劑。設計的抑制劑具有一些帶有不同取代基的芳香烴基團、一個比phosphoglycerate更穩定並連接有一段由triazole為架構之長直脂鏈的phosphorylpropanoate基團,以及一段用來連結上述兩基團的高絲胺酸、胺基或醯胺基連接鏈。期望這些抑制劑可以用來模擬單體lipid II在進行轉醣化過程中所形成過渡態之電荷與結構。 在成功合成出一系列具有潛力的轉醣酶抑制劑後,我們透過高效液相層析–轉醣酶活性分析法及最低抑菌濃度檢測來確認所合成之抑制劑是否具有良好的抑菌效果。根據活性測試的結果,phosphorylpropanoate化合物具有長直脂鏈(例如63a、63c及63e)或聯苯基鏈(63g)的衍生物顯示比具有膽固醇鏈、含支鏈之脂鏈和不飽和脂鏈的衍生物擁有更高的轉醣酶抑制活性,然而這些phosphorylpropanoate化合物卻喪失了抗菌活性。在所有合成的phosphorylpropanoate衍生物中,帶有聯苯基團及胺基連接鏈的化合物(例如94及123)具有較顯著的轉醣酶抑制活性,推論是由於化合物與轉醣酶活性中心產生特定π系統間的作用力。此外化合物94可有效抑制金黃色葡萄球菌,並擁有最低抑菌濃度值為6.3 μM。若將氟取代基引入聯苯基團上(例如112e及112f)可更加提升化合物的抗菌活性。然而若在聯苯基團上3號或5號位置引入R3取代基將會使聯苯的立體構形改變,進而降低與轉醣酶活性中心之間所產生特定π系統間的作用力。相較於化合物94,雙碳鏈長的化合物118預期其正電胺離子將更接近轉醣化過渡態中所形成之電荷的位置,而具有更好的轉醣酶抑制活性。此外,具有三級胺基化合物121及123則具有提升此正電胺離子的生成能力,而活性測試結果也顯示出這類化合物均具有比化合物94更高的轉醣酶抑制活性。若將連接鏈換成較為剛性的piperidine(例如127及128),則維持一樣的轉醣酶抑制活性。 除了利用轉醣酶分子模擬的方式,我們也希望用光親和性標示法來確立聯苯基phosphorylpropanoate衍生物與轉醣酶活性中心的結合區域。此外,綜合本文設計的所有轉醣酶抑制劑,顯示其phosphorylpropanoate基團、聯苯基團及能產生正價性質之胺基連接鏈是提供與轉醣酶活性中心之間作用力的重要區域,這些結構上的重要特性將是一個基礎去開發出具有更佳效果的轉醣酶抑制劑和抗菌劑。 The use of antibiotics has a great impact on the treatment of bacterial infections. However, emergence of multi-drug resistant bacteria requires discovery of new antibacterial targets and drugs. Transglycosylase (TGase) and transpeptidase (TPase) are two important enzymes involved in bacteria cell wall biosynthesis. By catalysis of TGase and TPase, the substrate lipid Ⅱ is polymerized to form the netlike polymer peptidoglycan, which can resist internal osmotic pressure to maintain the cell shape and integrity. In addition to TPase inhibitors (e.g. β-lactam antibiotics), TGase has become an attractive target for development of new antibiotics because of its indispensable function and accessibility. As TGase is located on the external surface of bacterial membrane, the inhibitor would bind to TGase without any obstacle. Based on the structure of moenomycin, we designed and synthesized some potential TGase inhibitors of phosphorylpropanoate derivatives that contain the aromatic moieties with different substituents, a phosphorylpropanoate moiety, which is expected to increase the stability of phosphoglycerate, bearing a triazole-based straight lipid chain, and two above-mentioned moieties are connected by homoserine, amide or amine linkers. These inhibitors were expected to mimic the oxonium transition state during the lipid II transglycosylation. The potential TGase inhibitors were synthesized and subjected to HPLC-based TGase fluorescence assay and MIC measurement. The phosphorylpropanoate compounds bearing straight lipids (e.g. 63a, 63c and 63e) or biphenyl group (63g) displayed higher TGase inhibitory activity than the analogous compounds having cholestery, branched or unsaturated lipids. However, these phosphorylpropanoate compounds lost antibacterial activity. Among all the synthesized phosphorylpropanoate derivatives, compounds bearing biphenyl moiety and amine linker (e.g. 94 and 123) showed appreciable TGase inhibition, presumably due to the particular π–π interaction in TGase active site. In addition, compound 94 effectively suppressed the growth of S. aureus with MIC value of 6.3 μM. Introduction of fluorine atoms to biphenyl moiety (e.g. 112e and 112f) did increase antibacterial activity. However, the conformation of biphenyl group may vary by the (R3)n substituents at 3- or 5-positions, and thus may not retain the presumed π–π interaction in the TGase active site. Compound 118 bearing a two-carbon linker, which was expected to place the aminium ion at a closer position to mimic the oxonium ion in the transition state of transglycosylation, had better TGase inhibitory activity than compound 94. Compounds 121 and 123 containing tertiary amine moiety, which could enhance the formation of positively charged aminium ion, also showed higher TGase inhibitory activity than compound 94. Replacing the flexible linker with rigid piperidine (e.g. 127 and 128) retain the TGase inhibitory activity. In addition to molecular docking experiments, the photoaffinity labeling strategy could be used to identify the exact amino acid residues of TGase for binding with phosphorylpropanoate derivatives. Among all of the TGase inhibitors designed in this study, the phosphorylpropanoate, amine linkage and biphenyl group appear to be important moieties to provide the necessary interactions with the active site of TGase. These important elements will serve as a foundation to explore more effective TGase inhibitors and antibacterial agents. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77767 |
DOI: | 10.6342/NTU201801845 |
全文授權: | 有償授權 |
電子全文公開日期: | 2028-12-31 |
顯示於系所單位: | 化學系 |
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