In the present work, bacterial glycosyltransferases are utilized to construct ganglioside glycans in a convergent approach via a sugar‒nucleotide regeneration system and one‐pot multienzyme reactions. Starting from β‐lactoside enables the diversification of both the glycan moieties and the linkages in the lower α‐arm and upper β‐arm. Overall, a comprehensive panel of 24 natural a‐series (GM3, GM2, GM1a, GD1a, GT1a, and fucosyl‐GM1), b‐series (GD3, GD2, GD1b, GT1b, and GQ1b), c‐series (GT3, GT2, GT1c, GQ1c, and GP1c), α‐series (GM1α, GD1aα, and GT1aα), and o‐series (GA2, GA1, GM1b, GalNAc‐GM1b, and GD1c) ganglioside glycans are prepared, which are suitable for biological studies and further applications. Moreover, a microarray is constructed with these synthesized ganglioside glycans to investigate their binding specificity with recombinant Fc‐fused Siglec‐7 and Siglec‐9, which are immune checkpoint‐like glycan recognition proteins on natural killer cells. The microarray binding results reveal that GD3 and GT1aα are specific ligands for Siglec‐7 and Siglec‐9, respectively, and this discovery can lead to the identification of appropriate ligands for investigating the roles of these Siglecs in immunomodulation.

The excellent molecular recognition capabilities of monoclonal antibodies (mAbs) have opened up exciting opportunities for biotherapeutic discovery. Taking advantage of the full potential of this tool necessitates affinity ligands capable of conjugating directly with small molecules to a defined degree of biorthogonality, especially when modifying natural Abs. Herein, a bioorthogonal boronate‐affinity‐based Ab ligand featuring a 4‐(dimethylamino)pyridine and an S‐aryl thioester to label full‐length Abs is reported. The photoactivatable linker in the acyl donor facilitated purification of azide‐labelled Ab (N₃‐Ab) was quantitatively cleaved upon brief exposure to UV light while retaining the original Ab activity. Click reactions enabled the precise addition of biotin, a fluorophore, and a pharmacological agent to the purified N₃‐Abs. The resulting immunoconjugate showed selectivity against targeted cells. Bioorthogonal traceless design and reagentless purification allow this strategy to be a powerful tool to engineer native antibodies amenable to therapeutic intervention.