Optimizing Phosphate Couplings for Dolichyl Diphosphochitobiose to Enable Protein N-Glycosylation Studies

Herein we report an optimized synthesis for dolichyl diphosphochitobiose (GlcNAc₂-PP-Dol₂₅), a probe useful for biochemical and structural studies of protein N-glycosylation in eukaryotic cells. We improved three phosphate coupling steps in terms of yields and reaction times, namely chitobiose phosphorylation, dolichol phosphorylation, and phosphate-phosphate coupling, by adjusting reagents and conditions. We also developed an efficient preparative reverse-phase HPLC purification protocol followed by ion exchange step to obtain the pure product as a stable sodium salt. These optimized procedures ensure a reliable supply of GlcNAc₂-PP-Dol₂₅ for enzymatic studies.