Glycoprotein N-linked oligosaccharides in the endoplasmic reticulum function as tags to regulate glycoprotein folding, sorting, secretion and degradation. Since the N-glycan structure of a glycoprotein should reflect the folding state, N-glycan processing may be affected by the aglycone state. In this study, we examined the influence of aglycone structures on N-glycan processing using synthetic substrates. We prepared (Glc(1))Man(9)GlcNAc(2) linked to hydrophobic BODIPY-dye with a systematic series of different linker lengths. With these compounds, glucose transfer, glucose trimming and mannose trimming reactions of an endoplasmic reticulum fraction were examined. The results showed that substrates with shorter linkers between the N-glycan and hydrophobic patch had higher activities for both the glucose transfer and the mannose trimming reactions. In contrast, the glucose trimming reaction showed lower activity when substrates had shorter linkers. Thus, the reactivity for N-linked oligosaccharide processing of glycoproteins in the endoplasmic reticulum might be tunable by the aglycone structure, e.g., protein portion of glycoproteins. (C) 2016 Elsevier Ltd. All rights reserved.