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

Abstract

Part 1. Probing Bacterial Transglycosylation Using Terpyridine-Derived Sensors and Secondary-Order Scattering Effect\n 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.\n\nPart 2. A Copper(II)–dipicolylamine–coumarin Sensor for Maltosyltransferase Assay\n 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.