p-nitrobenzyloxycarbonyl protective group as key to automated glycan assembly of neutral human milk oligosaccharides

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 (pNZ) 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 pNZ-protected oligosaccharides underscores the robustness of this approach, while three orthogonal deprotection pathways offer synthetic versatility for HMO compounds.