Versatile Strategy for the Chemoenzymatic Synthesis of Branched Human Milk Oligosaccharides Containing the Lacto‐N‐Biose Motif

Abstract Human milk oligosaccharides (HMOs) exhibit prebiotic, antimicrobial, and immunomodulatory properties and confer significant benefits to infants. Branched HMOs are constructed through diverse glycosidic linkages and prominently feature the lacto‐ N ‐biose (LNB, Gal‐β1,3‐GlcNAc) motif with fucose and/or sialic acid modifications, displaying structural complexity that surpasses that of N ‐ and O ‐glycans. However, synthesizing comprehensive libraries of branched HMO is challenging due to this complexity. Although a few systematic synthetic strategies have emerged, many of them rely on labor‐intensive chemical methodologies or exploit the substrate specificity of human N ‐acetylglucosaminyltransferase 2 (hGCNT2). In this study, we capitalized on the substrate promiscuities of hGCNT2 and bacterial glycosyltransferases (GTs) to construct a universal tetrasaccharide core in a highly efficient manner. This core was systematically and flexibly extended to generate diverse branched HMOs utilizing the promiscuity of bacterial GTs coupled with N ‐trifluoroacetyl glucosamine (GlcNTFA), which facilitated sugar chain elongation. The GlcNTFA residues were subsequently converted into various N ‐modified glucosamines through straightforward chemical manipulations to modulate the activities of additional GTs during glycan extension. These masked amino groups were ultimately reverted to N ‐acetyl groups, facilitating the synthesis of a broad range of asymmetric and multiantennary HMOs featuring LNB moieties, including many previously inaccessible structures.