Poly‐N‐acetyllactosamine (poly‐LacNAc) is ubiquitously expressed on cell surface glycoconjugates, serving as the backbone of complex glycans and an extended scaffold that presents diverse glycan epitopes. The branching of poly‐LacNAc, where internal galactose (Gal) residues have β1‐6 linked N‐acetylglucosamine (GlcNAc) attached, forms the blood group I‐antigen, which is closely associated with various physiological and pathological processes including cancer progression. However, the underlying mechanisms remain unclear as many of the I‐antigen sequences are undefined and inaccessible. In this study, we developed a highly efficient orthogonal‐group‐controlled approach to access site‐selectively I‐branched poly‐LacNAc chains. The approach relies on three orthogonal protecting groups, each of them “caps” one internal Gal residue of poly‐LacNAc. These groups can be readily “decapped” by specific enzymes or chemical reduction to expose desired sites for GCNT2‐catalyzed I‐branching. This approach enabled the rapid preparation of a diverse library of 41 linear and branched poly‐LacNAc glycans from a single precursor. Glycan microarray analysis using these complex glycans revealed unique recognitions of I‐branches by lectins, anti‐I mAbs, and galectins. Surprisingly, oxidized forms of linear poly‐LacNAc strongly bound to several glycan‐binding proteins (GBPs). These findings help to bridge the gap in recognition of I‐branching and open new avenues for therapeutic development by targeting galectins.
