設計及合成針對磷酸酯偵測的化學感測器

Design and Synthesis of Chemosensors to Detect Phospholipids

陳冠宏(Kuan-Hung Chen)

指導教授 ： 方俊民

國立臺灣大學/理學院/化學研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.02958

摘要

第一部份: 利用化學感測組合偵測磷酸酯
在本實驗室先前的研究中，已設計能針對磷酸根離子偵測的螢光感測器。此類磷酸根受體具有類似四面體的構形，與磷酸根離子形成化學計量比為1:1的氫鍵錯合物。對於如GPP類的磷酸酯偵測，我們則對受體分子修飾了長碳鏈以增加受體與磷酸酯上長碳鏈間的疏水性作用力，並保留著芘分子基團，以利於螢光訊號的讀取。
在本實驗中，我們利用了化學感測組合系統作為偵測方法，並以香豆素類分子做為此系統中的指示劑分子。當指示劑與受體結合時，因香豆素指示劑與芘分子間作用力，螢光光譜可在波長340 nm光源的激發下，產生螢光共振能量轉移 (FRET)的訊號。由於GPP與受體間的高結合能力，因此GPP的加入可置換出指示劑分子。當香豆素指示劑分子離開受體分子後，便減弱了螢光共振轉移的現象。因此，利用此方法可容易的偵測磷酸酯，並且經由滴定過程中螢光訊號的改變，而決定受體與GPP間的結合常數。
第二部份: 合成BPA–Zn類錯合物以用以選擇性結合LysoPA
我們合成了具有剛性的苯環分子以及BPA–Zn類錯合物的受體分子，此類受體在與磷酸離子結合時，每個鋅離子可與BPA以及磷酸根上的氧原子形成四配位的形式，由於鋅離子可擁有五配位的錯合，因此鋅離子上的空軌域可與額外的配位基錯合。根據此概念，每個在BPA–Zn類錯合物上的鋅離子可與LysoPA上的磷酸與羥基共同作用，而提高與LysoPA的結合能力。我們利用了化學感測組合系統，搭配使用香豆素類衍生物作為指示劑分子，可測定出與LysoPA以及PA間的結合常數。如同我們所預期，受體分子與LysoPA的結合能力比PA高約四倍左右。
第三部份: 利用磷酸酯誘導蒽分子聚集及激發雙體的形成:應用於磷酸酯的偵測
有別於其他磷酸衍生物，受體60與水解磷酸酯LysoPA在水溶液中結合後，可因磷酸酯濃度的不同，形成聚集現象，並經由照光而產生激發雙體的放光。此聚集現象，主要憑藉於磷酸酯上所具有疏水性質的長碳鏈。因聚集作用而在基態便形成雙體分子或簇團的蒽分子，在受到光激發後，便可放出激發雙體的放光。由吸收及激發滴定光譜所展現出的光譜變寬與紅位移變化，證明了水溶液中蒽分子的聚集，並反映出可由此作用而導致類似微小晶體的形成。利用觀測螢光半衰期的改變，也可解釋我們以上所推測的螢光偵測機制。當受體60與磷酸酯結合所造成激發雙體與單體分子在放光上的變化，提供了一種對於LysoPA更有利的偵測方法。此外，藉由受體60在結合能力上，可選擇性地辨識單酯基焦磷酸與二酯基焦磷酸的特性，上述方法或許也可應用於醣轉移酵素的活性偵測，進而在抑制細菌細胞壁增生的研究上，提供一套有效篩選抑制劑的新方法。

關鍵字

磷酸 ； 感測器 ； 水解磷酸酯

並列摘要

Part 1: Detection of Phospholipids Base on Chemosensing Ensemble
We have previously designed a fluorescence receptor for sensing of phosphate ions. This kind of receptor is established by incorporating four additional amido groups onto pyridine 2,6-biscarboxamide to provide a pseudo-tetrahedral cleft and multiple hydrogen bondings to hold phosphates in a 1:1 complexation stoichiometry. For sensing phospholipids, such as geranyl pyrophosphate (GPP), a modified receptor was synthesized by incorporation of long hydrocarbon chains to increase hydrophobic interaction with the lipid moiety of phospholipids, and two pyrene units were kept for the fluorescence readout.
In this study, a chemosensing ensemble system was applied by using coumarin derivatives as the indicator. Upon photoirradiation at 340 nm, a fluorescence resonance energy transfer (FRET) would occur due to the interaction of the coumarin with the pyrene unit. Because of high binding affinity of the receptor with GPP, addition of GPP would cause displacement of the coumarin indicator. Once the coumarin indicator was extruded out of the receptor cleft, the FRET would be diminished. Thus, phospholipid is readily detected by this method, and the binding constant is determined by the fluorescence changes during the titration.
Part 2: Synthesis of BPA–Zn Complex for Selective Binding with LysoPA.
A bis(2-pyridylmethyl) amine–Zn complex [Ph–(BPA–Zn)2, 48] is designed to contain a rigid benzene unit and dinuclei of Zn2+ ions. On binding with a phosphate ion, each zinc ion is four-coordinated with BPA and one oxygen of phosphate. Because a Zn2+ ion can have coordination number of five, the residual vacant orbital on the Ph–BPA–Zn–phosphate is available to incorporate an additional ligand. Based on this concept, both Zn2+ ions of BPA–Zn can bind synergistically to the hydroxyl group and the phosphate moiety of lysophosphatidic acid (LysoPA). The binding constants for LysoPA and phosphatidic acid (PA) are determined in a chemosensing ensemble system using coumarin derivative as the indicator. As our anticipation, the binding of Ph–(BPA–Zn)2 receptor with LysoPA is about 4–fold stronger than that with PA.
Part 3: Phospholipid-Induced Aggregation and Anthracene Excimer Formation: Application to sensing phospholipids.
Receptor 60 displays an unusual and selective fluorescence response to LysoPA in aqueous buffer solutions through a concentration-dependent formation of aggregates and fluorescent anthracene excimers. The aggregation behavior relies on the hydrophobic aliphatic chains in LysoPA, and the excimer emission results from excitation of the ground-state pre-associated anthracene dimer or clusters. The aggregate formation is consistent with the broadening and red-shift of their absorption and excitation titration spectra. The elevation of absorption might reflect the formation of microcrystal-like aggregates in the buffer solution. The proposed fluroescence sensing mechanism can also account for the observed changes in the fluorescence decay time. The anthracene excimer vs monomer emission would allow a more favorable ratiometric detection of LysoPA. Furthermore, this method may be used to detect the activity of transglycosylase based on the selective binding of receptor 60 with pyrophosphate mono-ester over the corresponding di-ester. It may serve as a new tool for screening the inhibitors against formation of bacterial cell wall.

並列關鍵字

phosphate ； sensor ； Lysophosphatidic acid

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