合成Lipid II及其衍生物以研究細胞壁合成中的轉醣化過程

Abstract

自從八十幾年前弗萊明發現青黴素以後，抗生素一直被用來對抗細菌感染所造成的疾病。然而，即使是萬古黴素，人類用來對抗細菌的最後一樣武器，也因細菌的突變而產生抗藥性。因此，發展新一代的抗生素以對抗細菌是當今全民健康的重大課題。 轉醣酶(TG)是一個極具發展價值以研發新一代抗生素的重要目標。因為它裸露在細胞膜的外側，藥物不需進入細菌細胞內側，較不易受到其他干擾。它的功能為催化lipid II聚合而形成細菌的細胞壁。 研究TG的困難處有幾點，其中最難以解決的是缺乏lipid II的來源。從細菌的細胞利用分離而獲得研究所需的量，是困難且步驟繁雜的。我們利用化學合成的方法合成了lipid II以及只含有四個Z-form脂肪鏈的lipid II衍生物，試著解決這個問題。 之後，我們更進一步的將所合成的lipid II和其衍生物利用化學方法連結螢光團(dansyl，fluorescein和naphthalene)。再與Clostridium difficile的penicillin-binding protein 1b (PBP1b)混合反應，以HPLC和螢光偵測器以研究它們和TG之間的相互作用關係。由實驗的結果可知，因Fluorescein-LII的胜肽鏈上所含的fluorescein較Dansyl分子結構大了許多，造成TG無法辨識。但是，Dansyl-LII, D-C20-LII, and FRET-LII則仍為TG的受質。因此，我們可於D-C20-LII與PBP1b的反應中加入內標準品，以定量分析的方法初步篩選TG抑制劑。

Since the discovery of penicillin by Fleming more than 80 years ago, antibiotics have been needed to treat infections caused by bacteria. However, even vancomycin, a potent antibiotic as the final weapon against bacterial infections, has encountered the problem of drug resistance. The development of new antibiotic to treat resistant bacterial infections is an important issue for public health. Transglycosylase (TG) is a potential target for development of new antibiotics. It 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. One of the difficulties in studying TG is the lack of lipid II, though it can be isolated from bacterial membrane in little amount by tedious procedure. In this thesis, I report a chemical synthesis that provides an alternative method to obtain substantial amounts of lipid II and its analogues containing four cis-isoprene units. Furthermore, the natural lipid II and its analogues were elaborated with fluorescence probes, for example, dansyl, fluorescein, and naphthyl via chemical methods. These fluorescent substrates were used to study the transglycosylation in cell wall formation. The reactivity between lipid II analogs and PBP1b were moitored by HPLC using fluorescence detector. Based on the analysis, fluorescein conjugated with Fluorescein-LII is too bulky to be recognized by TG, but Dansyl-LII, D-C20-LII, and FRET-LII are still the substrates of TG. By normalization with internal standard, we may screen the inhibitors based on the decrease of D-C20-LII monitored by HPLC.