Abstract：
A fluorescence-quenching-based assay system was constructed to determine the hydrolytic activity of endo-β-N-acetylglucosaminidases (ENGases) interacting with hybrid-type N-glycans. This was achieved using a dual-labeled fluorescent probe with a nonasaccharide structure. We produced the nonasaccharide skeleton by the stepwise glycosylation of the galactose residue on a galactosyl chitobiose derivative. Next, we introduced azido and acetoxy groups into the nonasaccharide derivative in a stepwise manner, which led to stereochemistry inversion at both the C-4 and C-2 hydroxy groups on its galactose residue. The protecting groups of the resulting nonasaccharide derivative were removed, and the derivative was labeled with an N-methylanthraniloyl group to obtain a reporter dye and a 2,4-dinitrophenyl group as a quenching molecule to obtain target probe 1. The use of this probe along with a microplate reader enabled a facile evaluation of the hydrolytic activities of ENGases Endo-H, Endo-M, Endo-F3, Endo-S, and Endo-CC. Furthermore, this probe could also assist in the search for novel ENGases that are specific to hybrid-type N-glycans.
摘要：
构建了基于荧光猝灭的测定系统，以确定与杂合型N-聚糖相互作用的内切β-N-乙酰氨基葡萄糖苷酶（ENGases）的水解活性。这是使用具有非糖结构的双标记荧光探针实现的。我们通过逐步糖基化半乳糖基壳二糖衍生物上的半乳糖残基来产生非糖骨架。接下来,我们将叠氮基和乙酰氧基逐步引入九糖衍生物中，导致其半乳糖残基上C-4和C-2羟基的立体化学反转。除去所得九糖衍生物的保护基，用N-甲基邻氨基苯并酰基标记该衍生物以获得报告染料和2,4-二硝基苯基作为猝灭分子以获得目标探针1。该探针与酶标仪一起使用可以轻松评估ENGasesEndo-H的水解活性，Endo-M,Endo-F3,Endo-S,和Endo-CC。此外，该探针还可以帮助寻找对杂合型N-聚糖具有特异性的新型ENGase.
