Fluorogenic probes for assessing endo-β- N -acetylglucosaminidase activity on complex-type N -glycans

Endo-β-N-acetylglucosaminidases (ENGases) cleave the N-glycan core from glycoproteins and are important tools for structural analysis of glycans. Currently, only a few ENGases can specifically cleave the complex-type N-glycans, highlighting the need for discovering novel ENGases for glycoengineering applications. Nevertheless, conventional techniques for ENGase analysis are limited by low throughput and a lack of real-time detection. Herein, we describe the development of a fluorescence quenching assay to measure the hydrolytic activity of ENGases against fucosylated and afucosylated biantennary complex-type N-glycans. Two chemically synthesized probes, MG2FD (1) and MG2D (2)—a decasaccharide and a nonasaccharide, respectively—were labeled with an N-methylanthraniloyl group (fluorophore) and a 2,4-dinitrophenyl group (quencher). These probes were used to evaluate commercially available ENGases: Endo-M, Endo-CC, Endo-F3, Endo-H, and Endo-S. Endo-M and Endo-CC selectively cleaved probe 2 but not probe 1; Endo-F3 cleaved only probe 1; however, Endo-H and Endo-S showed no detectable activity. These findings align with known substrate specificities, validating the assay as a rapid and reliable method for assessing ENGase activity, profiling substrate specificity, and identifying novel ENGases targeting complex-type glycans.