Site-Selective Bioconjugations of Distinct Methionine Residues in a Protein via Reactivity-Based Reagent Control

The reaction of thioethers in biological macromolecules with oxaziridines enables their functionalization with moieties having utility in both basic and applied science. Exploitation of that reaction has advantages over common bioconjugations due to the relatively low abundance and weak reactivity of thioethers. Herein, we report a novel and improved route to 1-ureal piperidine,3-aryl oxaziridines that enables the preparation of bioconjugation reagents that are either electronically activated or deactivated. We leveraged the differential reactivities of the oxaziridine reagents and engineered methionine residues in distinct environments within a therapeutically relevant antibody fragment (Fab) to effect site-selective bioconjugations. We capitalized on this novel means of kinetic resolution to effect dual functionalization of a HER2-targeted Fab with distinct fluorophores for imaging receptor internalization. This work broadens the scope of what is possible in bioconjugations of thioethers and demonstrates that site-selective bioconjugations of the same residue at more than one site in a protein can be realized via reactivity-based reagent control.