N -Azidoacetylglucosamine (GlcNAz)-Derived Sugar Oxazolines as a New Class of Substrates for Chemoenzymatic Site-Specific Antibody Bioconjugation

Endoglycosidase-catalyzed Fc glycan remodeling using glycan oxazolines as substrates has become a general method to produce homogeneous antibody glycoforms and site-specific antibody–drug conjugates (ADCs). While endoglycosidases generally tolerate structural modifications at nonreducing terminal sugar moieties, the reducing-terminal GlcNAc oxazoline has been regarded as an essential and largely immutable structural motif for enzyme recognition. In this work, we sought to better understand substrate recognition by endoglycosidases and to develop simpler and more efficient strategies for synthesizing site-specific ADCs. We have performed site-selective modifications on the GlcNAc oxazoline moiety concurrent with structure–activity relationship (SAR) studies. We found that while the endoglycosidase Endo-S2 does not tolerate modifications at the C-3 or C-6 positions, the enzyme can tolerate certain modifications at the methyl group of the oxazoline portion, allowing the introduction of azide and halogen atoms at this site. In contrast to the conventional method of introducing tags to the nonreducing terminal glycans through ether bonds, which requires tedious protection–deprotection steps, this new strategy enables the one-step introduction of a tag (e.g., azide) to the reducing-terminal glucosamine moiety while leaving free hydroxyl groups on other positions intact. This approach significantly enhances the efficiency of the ADC preparation. These findings open a new avenue to antibody tagging and bioconjugation with a class of much simpler disaccharide substrates. In addition, we found that a second sugar moiety β-1,4-linked to the GlcNAc oxazoline also played an important role, with the mannose moiety being the most efficient for enzymatic transglycosylation. A structural modeling analysis indicated that there was a cavity in the enzyme pocket that permits certain modifications at the methyl group. This new method was successfully used to produce site-specific ADCs. Cell-based assays showed that the resulting ADCs exhibited potent cell killing of cancer cells that overexpressed the corresponding antigen.