J Pharm Biomed Anal. 2026 Apr 3;277:117496. doi: 10.1016/j.jpba.2026.117496. Online ahead of print.
ABSTRACT
The Endoglycosidase CC N180H mutant (Endo-CC N180H) is extensively utilized in the synthesis of homogeneous glycopeptides and glycoconjugates due to its high transglycosylation activity. However, its potential in analytical glycomics remains underexplored. In this study, various structural acceptors were synthesized using N-acetylglucosamine (GlcNAc) as a substrate, and their transglycosylation reactions catalyzed by Endo-CC N180H were investigated. Modifications at the C6 position of GlcNAc completely abolished transglycosylation activity, while alterations at the C1 position were well tolerated. Among these acceptors, d0-Bn-β-GlcNAc exhibited the highest transglycosylation efficiency, with optimal reaction conditions identified as 150 mU/mL Endo-CC N180H at 45 °C for 36 h. Leveraging these conditions, d5-Bn-β-GlcNAc was further synthesized, enabling the development of a stable isotope-labeled chemoenzymatic strategy for N-glycan relative quantification. Using sialylated glycopeptides as a model, the method achieved excellent linearity over a 1:10-10:1 molar ratio range (R2 = 0.9991), high precision (CV = 0.22-6.24%), and a limit of detection of 50 fmol. Moreover, the strategy was successfully applied to the profiling and relative quantification of high-mannose N-glycans in ribonuclease B. Collectively, this work expands the analytical utility of Endo-CC N180H and establishes a robust and sensitive platform for quantitative glycomics for relative quantification of selected N-glycan species in glycomics research.
PMID:41946240 | DOI:10.1016/j.jpba.2026.117496