Efficient Preparation of Homogenous Antibody Conjugates via Glycosite‐Specific Transglycosylation Enabled by Readily Available Glycosyl Donors

Site‐specific antibody conjugation through glycoengineering offers a promising route to generate homogeneous glycosite‐specific antibody‒drug conjugates (gsADCs) with improved therapeutic indices. Dozens of gsADCs are advancing from preclinical studies to clinical trials. However, current methods involve either multiple enzymes or lengthy preparation of substrates. Herein, we report a novel and synthetically streamlined platform utilizing LacNAc‐derived 4,6‐acetal glycosyl donors for glycosite‐specific transglycosylation mediated by a single enzyme. These glycosyl donors can be synthesized in as few as two steps, representing a major advancement in synthetic accessibility compared to previously reported glycosyl donors, which often require more than 15 steps. Computational analysis showed that the acetal ring restricts conformation, directing donor 7 to a π–π‐stabilized groove of the enzyme. Donor 7 , along with a positive control, was evaluated in the context of gsADCs, consistently demonstrating potent and selective cytotoxicity toward HER2‐positive cancer cells, while sparing HER2‐negative cells. Furthermore, donor 7 was successfully adapted to generate glycosite‐specific degrader‐antibody conjugates (gsDACs), highlighting its broad utility. Additional studies revealed that donor 7 produces antibodies with markedly enhanced resistance to Endo S2 mediated hydrolysis. Together, these findings establish a practical and broadly applicable platform for glycosite‐specific antibody conjugation, paving the way for next‐generation antibody‐based therapeutics.