Human milk oligosaccharides (HMOs) have highly diverse and branched structures that present a significant challenge for chemical synthesis. Here we show that masking the amino group in glucosamine with a p -nitrobenzyloxycarbonyl ( p NZ) protecting group enhances coupling and deprotection efficiency during automated glycan assembly (AGA) of homogeneous HMOs, including the lacto- N -tetraose (LNT), lacto- N -neotetraose (LNnT), lacto- N -fucopentaose (LNFP), lacto- N -difuco-hexaose (LNDFH), and lacto-N-neohexaose (LNnH) series. Deprotection strategies are developed to achieve excellent purity of linear and branched HMO structures. The end-to-end tractability of p NZ-protected oligosaccharides underscores the robustness of this approach, while three orthogonal deprotection pathways offer synthetic versatility for HMO compounds.
