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

Herein we report an optimized synthesis for dolichyl diphosphochitobiose (GlcNAc(2)-PP-Dol(25)), 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 GlcNAc(2)-PP-Dol(25) for enzymatic studies.

Automated summary

This study presents an optimized synthesis method for a probe used in studying protein N-glycosylation in eukaryotic cells. The yields and reaction times of three phosphate coupling steps were improved by adjusting reagents and conditions. An efficient purification protocol was also developed to obtain the pure product.