General and Robust Chemoenzymatic Method for Glycan-Mediated Site-Specific Labeling and Conjugation of Antibodies: Facile Synthesis of Homogeneous Antibody–Drug Conjugates

of serotype M49 (Endo-S2) exhibited remarkable activity in transferring the functionalized disaccharides carrying site-selectively modified azide, biotin, or fluorescent tags to antibodies without hydrolyzing the resulting transglycosylation products. This discovery, together with the excellent Fc deglycosylation activity of Endo-S2 on recombinant antibodies, allowed direct labeling and functionalization of antibodies in a one-pot manner without the need of intermediate and enzyme separation. The site-specific introduction of varied numbers of azide groups enabled a highly efficient synthesis of homogeneous antibody-drug conjugates (ADCs) with a precise control of the drug-to-antibody ratio (DAR) ranging from 2 to 12 via a copper-free strain-promoted click reaction. Cell viability assays showed that ADCs with higher DARs were more potent in killing antigen-overexpressed cells than the ADCs with lower DARs. This new method is expected to find applications not only for antibody-drug conjugation but also for cell labeling, imaging, and diagnosis.