{Reference Type}: Journal Article
{Title}: Synthesis of a fluorescent probe for measuring the activity of endo-β-N-acetylglucosaminidases recognizing hybrid-type N-glycans.
{Author}: Ishii N;Inoue S;Sano K;Takahashi S;Matsuo I;
{Journal}: Bioorg Med Chem
{Volume}: 100
{Issue}: 0
{Year}: 2024 Feb 15
{Factor}: 3.461
{DOI}: 10.1016/j.bmc.2024.117612
{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.