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.

构建了基于荧光淬灭的测定系统，以确定内切-β- N-乙酰氨基葡萄糖苷酶（ENGase）与混合型N-聚糖相互作用的水解活性。这是使用具有九糖结构的双标记荧光探针实现的。我们通过半乳糖基壳二糖衍生物上的半乳糖残基的逐步糖基化产生了九糖骨架。接下来，我们逐步将叠氮基和乙酰氧基引入到九糖衍生物中，导致其半乳糖残基上的C-4和C-2羟基发生立体化学反转。将得到的九糖衍生物的保护基除去，用N-甲基邻氨基苯甲酰基标记该衍生物，得到报告色素，用2，4-二硝基苯基标记作为猝灭分子，得到目标探针1 。使用该探针与酶标仪可以轻松评估 ENGases Endo-H、Endo-M、Endo-F3、Endo-S 和 Endo-CC 的水解活性。此外，该探针还可以帮助寻找针对混合型N -聚糖的新型 ENGase。