- 學位論文
以誘導聚集之螢光模式偵測磷酸脂
Detection of Phospholipids by Aggregation–based Fluorescence Sensing Mode
摘要
第一部分: 磷酸脂誘導產生激發雙體放光偵測系統之應用－Lipid II 轉醣酶 轉醣酶是細菌之細胞壁生合成途徑中重要的酵素。由於細胞壁包覆在細菌最外層，除了決定細菌之細胞形狀，並具有保護細菌的作用，因此轉醣酶被視為開發新型抑制劑的目標之一。然而對於抑制劑的開發，建立具良好選擇性、靈敏性且篩選通量高，甚至可即時偵測的感測器檢測系統是必須的。 我們致力於利用受體 2 與具有長碳鏈部分的磷酸脂作用後，因長碳鏈之疏水性質而誘發聚集現象並使得受體 2 產生激發雙體放光的特性，以提供一套有效偵測轉醣酶活性的新檢測方法。此設計的關鍵在於受體 2 對單酯基磷酸/焦磷酸與其相對應的二酯基焦磷酸在結合能力上的選擇性，為了對此性質有更深入的了解，我們設計合成替代的受質 S-P 12、 SPP-GlcNAc 17 和 FPP-GlcNAc 18，並且檢測其與受體 2 的結合能力。所設計的化合物皆具有磷酸根與長碳鏈，因此合成及滴定實驗過程中，需要克服無水環境、純化方法及溶解度的問題。而滴定實驗結果顯示，隨著碳鏈長度的增加 (C15 vs C45) 提供更強的疏水性質，使得引發顯著的激發雙體放光現象；並且受到醣類性質的影響，使得所設計的二酯基焦磷酸 (如化合物 17 和 18) 及其相對應的單酯基磷酸/焦磷酸 (如化合物 12) 對受體 2 有相當的結合能力，說明了在此實驗系統中，受體 2 與具有醣基團的受質產生特殊的結合模式。而在所選擇的 Lipid II 轉醣酶系統中，存在具雙醣、胜肽鏈及其聚合形式的複雜結構分子，因此仍有機會將此套系統應用於檢測轉醣酶活性。 第二部分: 磷酸脂誘導產生激發雙體放光偵測系統之應用－磷脂酶 D 甘油磷酸脂廣泛地存在於微生物、植物及動物中，是構成生物膜的主要成分之一， 並且參與在許多細胞代謝、訊息傳遞機制中。然而目前廣為使用於檢測磷脂酶 (phospholipases) 活性的方法，缺點是需要另外使用它種酵素以間接的方式檢測。 我們選擇了被證實不易與受體 2 有結合作用的磷脂膽鹼 (phosphatidylcholine, PC) 做為磷脂酶 D 的受質，而其產物之一的磷脂酸 (phosphatidic acid, PA) 亦被證實與受體 2 結合後會產生激發雙體現象。為了符合往後進行酵素實驗時的環境，改變偵測系統溶液之 pH 值及添加界面活性劑 (Triton X-100)。由螢光滴定光譜的變化，得知 pH 值的調動，不會影響結合模式；而 Triton X-100 的量在小於 0.02% 時，仍然可見到有激發雙體的產生，但當存在有 0.04% 比例時，受到 TritonX-100 性質的影響，使得磷脂質產生聚集的現象不足以使產生激發雙體放光；並如同我們所預期的，受體 2 對 PC 及 PA 之間有良好的結合選擇性。
關鍵字
磷酸脂並列摘要
Part 1. Phospholipid-induced aggregation fluorescence sensing mode: application to Lipid II transglycosylases The bacterial transglycosylases (TGase) are the enzymes that synthesize the peptidoglycan layers surrounding bacterial cell membranes. TGase are promising targets for development of antibacterial agents. Therefore, it is desirable to explore a TGase activity assay that is highly sensitive and amenable to real-time screening of inhibitors. In this study, we use receptor 2, which displays an phospholipid-induced aggregation fluorescence sensing mode, to detect phospholipids as a new tool for screening the activity of TGase. The aggregation behavior is due to the hydrophobic chain in phospholipids, and the fluorescence emission results from excitation of the dimer or clusters form of anthracene. The selective binding of receptor 2 with phosphate/pyrophosphate monoester over the corresponding phosphate diester is the key point of this study. We thus synthesize S-P 12, SPP-GlcNAc 17 and FPP-GlcNAc 18, and compare their binding behavior with receptor 2. There are a lot of problems to be overcome in the synthesis of the designed compounds, for example, control of anhydrous conditions, application of proper purification methods, and solubility in proper solvent. On the basis of fluorescence and UV-vis titration studies, we prove that the formation of the 480 nm emission band (excimer emission) is associated with the behavior of its aliphatic chains. Surprisingly, receptor 2 can strongly bind to not only phosphate monoesters (e.g. 12) but also to phosphate diesters (e.g. 17 and 18), which attribute to the distinct binding interaction between receptor 2 and the saccharide moiety in this assay system. In transglycosylation of bacterial cell wall formation, the structure of natural substrate (Lipid II) consists of disaccharide, pentapeptide and even the oligo/polymeric products, so it still has chance to apply this sensing system to detect the process of transglycosylation. Part 2. Phospholipid-induced aggregation fluorescence sensing mode: application to phospholipase D Glycerophospholipids are ubiquitous in nature as key components of the biomembrane of cells or organelles. Phospholipids are also linked to many fundamental physiological processes such as bioenergetics, cellular rec¬ognition, and signal transduction across the cell membrane. However, the most wildly used method for sensitive measurement of phospholipase activity is based on the enzyme-coupled assay mode, which is an indect method. Phophatidylcholine (PC) is one of substrates of phospholipase D (PLD) that is known to exhibit weak binding with receptor 2. On the other hand, phosphatidic acid (PA), a product of PC by PLD-catalyzed hydrolysis, is proved to bind with receptor 2 to induce the excimer emission. Therefore, the phospholipid-induced aggregation fluorescence sensing mode may be used to detect the activity of PLD. To accommodate the conditions for assay in PLD, the titration studies of PA with receptor 2 was performed at pH 8.0, and in the presence of proper amount of Triton X-100. The fluorescence titration results imply the binding mode of receptor 2 with PA is not affected in weakly basic conditions with less than 0.02% Triton X-100. The receptor 2 still forms the excimer due to the phospholipid-induced aggregation. To our expectation, receptor 2 binds selectivity with PA over PC.