| Summary: | 碩士 === 臺灣大學 === 化學研究所 === 98 === 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.
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