Graduate School of Science and Technology, Gunma University, 1-5-1, Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
Graduate School of Science and Technology, Gunma University, 1-5-1, Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
Bioorg Med Chem. 2024 Feb 15;100:117612. doi: 10.1016/j.bmc.2024.117612. Epub 2024 Jan 27.
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.
构建了基于荧光猝灭的分析系统，用于测定与杂合型 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。该系统使用带有九糖结构的双标记荧光探针来实现。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生成九糖骨架。然后，我们逐步在九糖衍生物中引入叠氮基和乙酰氧基，导致其半乳糖残基上的 C-4 和 C-2 羟基的立体化学反转。所得九糖衍生物的保护基团被去除，并用 N-甲基邻氨基苯甲酸基标记该衍生物，得到报告染料和 2,4-二硝基苯作为猝灭分子，得到目标探针 1。使用该探针和微孔板读取器可以轻松评估 ENGases Endo-H、Endo-M、Endo-F3、Endo-S 和 Endo-CC 的水解活性。此外，该探针还可以协助寻找对杂合型 N-聚糖具有特异性的新型 ENGases。
