設計與合成香豆素偵測器應用於細菌GlgE與轉醣酶活性測試

Design and Synthesis of Coumarin Sensors for Bacterial TGase and GlgE Enzymatic Assays

李薇立(Wei-Li Lee)

指導教授 ： 方俊民

國立臺灣大學/理學院/化學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601018

摘要

為促進抗生素的發展，發展精確和簡易的測定法以篩選出適合的藥物是至關重要的。我們致力於發展可應用於細菌性轉醣酶(TGase)與肺結核桿菌(TB) Glg酶抑制劑測定的分子探針設計。轉醣酶是負責建構細菌細胞壁糖苷鍵的酶。轉醣酶催化lipid II分子的聚合，並在反應中釋放酯質磷酸(C55PP)離子，而形成聚醣鏈。GlgE催化麥芽糖-1-磷酸(M1P)的聚合反應，並在反應中釋放磷酸離子(Pi)，而形成α-1,4-葡聚醣(α-1,4-glucan)。為了測量轉醣酶和GlgE酶的活性，分子探針須能選擇性地結合C55PP和Pi。
我們設計的分子探針包含一個香豆素感測單位與一個Zn/Cu2+–di(pyridylmethyl)amine (DPA)辨識單位。在DPA吡啶的二號位置，我們修飾上不同的烷基提供立體障礙，阻擋lipld II 與M1P分子。探針中感測與辨識單位的電子是共軛的，以分子內電荷轉移(internal charge transfer) (ICT)機制偵測待測物，此種感測方式較為敏感且受溶劑影響較小。
在所有合成的分子中，兩個以銅為螯合中心的coumarin-DPA–Cu2+ 探針6 與 11 可以在HEPES緩衝液中，選擇性地偵測磷酸與二磷酸分子，而不與其它陰離子反應。其中，探針11 在微莫耳濃度下，可與磷酸及二磷酸進行定量實驗，在HEPES緩衝液中的結合常數分別為1.5 × 105 M–1與3.1 × 105 M–1。因此，探針11有潛力應用於GlgE與TGase活性測試中。

關鍵字

螢光 ； 探針 ； 香豆素 ； 蛋白?活性測試 ； 轉醣? ； GlgE

並列摘要

To facilitate the development of antibiotics, a precise and facile assay for test of potential drug candidates is required. Here we focus on design of the molecular probes that can be applied to discover the inhibitors of bacterial transglycosylase (TGase) and the GlgE enzyme in Mycobacterium tuberculosis (TB). TGase is an enzyme responsible for formation of the glycosidic linkages in bacteria cell wall. TGase catalyzes polymerization of lipid II along with release of lipid diphosphate (C55PP) ions. GlgE catalyzes the linkage of maltose 1-phosphate (M1P) to α-1,4-glucan by releasing a unit of phosphate (Pi) ion. To measure the activity of TGase and GlgE enzymes, the detection probes must be able to selectively bind C55PP and Pi.
Our designed probes contain a sensing motif of coumarin and a binding motif of Zn/Cu2+–di(pyridylmethyl)amine (DPA). The 2-position of pyridines on DPA is modified with alkyl substituent (R) to enhance the ligand-metal bonding and to provide steric effect to block undesirable binding with lipid II and M1P. Our probes use an internal charge transfer (ICT) mechanism to sense substrates, and are expected to be more sensitive to substrate and less susceptible to solvents.
Among the synthesized probes, two coumarin-DPA–Cu2+ probes 6 and 11 acted as selective fluorescent chemosensors for Pi and PPi over other anions in water and buffer solution. In particular, 11 could undergo quantitative analysis with Pi and PPi at micromolar scale with association constant 1.5 × 105 M–1 and 3.1 × 105 M–1 in HEPES buffer for Pi and PPi, respectively. Probe 11 is potentially applied in both TGase and GlgE assays.

並列關鍵字

Fluorescence ； probe ； coumarin ； enzymatic assay ； TGase ； GlgE

參考文獻

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