運用分子感測器檢測細菌轉醣酶及麥芽糖基轉移酶的活性
第一部分、開發以三聯吡啶感測器以及二階散射作為檢測細菌轉醣酶活性之方法
第二部分、合成銅(II)–二吡咯胺香豆素感測器運用於麥芽糖基轉移酶的活性測試

第一部分、開發以三聯吡啶感測器以及二階散射作為檢測細菌轉醣酶活性之方法長碳鏈萜類焦磷酸，尤其是十一萜類焦磷酸(UPP)在自然界中可擔任酯質定錨的功能以催化O-以及N-醣基化的反應。以往在檢測轉醣酶(例如:青黴素結合蛋白轉醣酶(PBP-TGase)、轉多醣酶(PglB))的活性常依賴以同位素或是修飾螢光基團的方式作分析，然而此類方法並無法在一般實驗室進行，或需花費大量時間製備螢光修飾的受質。因此在此研究中，我們首先建立以三聯吡啶–鋅螢光感測器(Tpy-Zn, 23)作為選擇性檢測長碳鏈焦磷酸單酯(UPP類似物)的方法，而不感應焦磷酸雙酯(Lipid II類似物)。此方法可成功運用於HEPES緩衝溶液中，並對應出明顯的螢光差異。此研究也經由在不同極性溶劑中的溶致變色(solvatochromism)之分析實驗來推斷出可能的檢測機構。此外我們也嘗試在三聯吡啶系統中鑲入炔取代基，並探討其在螢光性質上的變化。在此篇研究中我們也發現焦磷酸單酯(法尼基、茄尼基衍伸物)在含有氯化鈣的HEPES緩衝溶液中有著顯著的二階散射現象(SOS)，而焦磷酸雙酯(lipid II類似物)以及肽聚醣單體(PGM)對二階散射不靈敏。最終我們合成出天然物lipid II，並成功的以三聯吡啶感測器以及二階散射效應來分析轉醣酶反應，以及檢測抑制劑moenomycin A的對青黴素結合蛋白的半抑制濃度(IC50)。第二部分、合成銅(II)–二吡咯胺香豆素感測器運用於麥芽糖基轉移酶的活性測試麥芽糖基轉移酶(GlgE)在結核桿菌中扮演著催化麥芽糖-1-磷酸(M1P)的聚合反應以形成α-葡聚糖(α-glucan)，並在反應中離去一當量的磷酸根離子(Pi)。由於GlgE是結核桿菌的必要蛋白質且不存在於人體中，因此被認為是一個有潛力用來對抗結核桿菌的標的。考慮到目前使用MESG檢測磷酸根離子的方法，需經由酵素反應所帶來的限制，因此開發出穩定且靈敏的檢測方法來探討GlgE的活性是必要的。在此研究中我們利用銅(II)–二吡咯胺香豆素(化合物108)以內部電荷轉移(ICT)為基礎的螢光開啟式感測器，在HEPES緩衝溶液中檢測磷酸根離子。我們也另外合成麥芽糖-1-磷酸來證實感測器108在水相中對受質的選擇性。在實際的酵素實驗中我們成功地運用此方法去檢測麥芽糖基轉移酶的活性，此終點測定的螢光檢測有潛力運用於篩選GlgE抑制劑，並使用於抗結核病藥物的開發。

關鍵字

焦磷酸酯；化學感測器；細菌轉醣酶；麥芽糖基轉移酶

並列摘要

Part 1. Probing Bacterial Transglycosylation Using Terpyridine-Derived Sensors and Secondary-Order Scattering Effect Long-chain isoprenoid pyrophosphates, especially undecaprenyl pyrophosphate (UPP), can serve as the lipid anchor to promote O- and N-transglycosylation of lipid II and oligosaccharides, respectively. The previous methods for monitoring transglycosylase (i.e., PBP-TGase and PglB) mostly rely on incorporation of radioactive isotope or fluorophore. However, those methods are hampered by their unavailability in common laboratory and the extra work to prepare fluorophore-modified substrates. In this study, we developed a fluorometric method using terpyridine zinc complex (Tpy-Zn, 23) for selective sensing long-chain lipid pyrophosphate monoesters (UPP analogs), but not lipid pyrophosphate diesters (lipid II analogs). The sensing properties can be applied in HEPES buffer to show strong fluorescence responses. The purposed sensing mechanism was also studied through solvatochromism experiment in different solvent polarities. In addition, we also introduced the alkynyl substituents to pyridine core of Tpy-Zn to examine their sensing properties toward substrates. Furthermore, farnesyl, solanesyl and undecaprenyl pyrophosphates form colloidal solutions in HEPES buffer containing CaCl2 to show a remarkable secondary-order scattering effect (SOS), while lipid II and peptidoglycan monomer are soluble without SOS. The fluorometric and SOS assays were successfully applied to probe the PBP-TGase reaction, and determined the inhibitory activity (IC50) of moenomycin A. Part 2. A Copper(II)–dipicolylamine–coumarin Sensor for Maltosyltransferase Assay Maltosyltransferase enzyme (GlgE) catalyzed the polymerization of maltose-1-phosphate (M1P) to linear α-glucan in Mycobacterium tuberculosis (Mtb) with releasing a unit of phosphate ion (Pi). GlgE is essential for the survival of Mtb but absent in human body, and is validated as an anti-tuberculosis (anti-TB) target. To overcome the limitation of using the MESG-based phosphate assay to screen the enzyme activity, a thermal-stable and sensitive method to detect the Pi released in GlgE reaction is essential. In this study, a Cu(II)−[di(2-methylpyridyl)methylamino]coumarin fluorescence turn-on sensor 108 using intramolecular charge transfer (ICT) as the sensing mechanism is designed to detect phosphate ion in HEPES buffer. We also synthesized M1P as maltosyltransferase substrate and proved its insensitivity to sensor in aqueous media. In a real enzymatic reaction, the protocol is modified and successfully applied to probe the GlgE catalyzed maltosyltransfer reaction by fluorescence enhancement. This end-point fluorescence turn-on assay may facilitate the screening of GlgE inhibitors for discovery of new anti-TB drugs.

並列關鍵字

lipid pyrophosphate ； chemosensor ； transglycosylase ； maltosyltransferase

參考文獻

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