Bioorthogonal click and release: A general, rapid, chemically revertible bioconjugation strategy employing enamine N-oxides

A chemically revertible bioconjugation strategy featuring a new bioorthogonal dissociative reaction employing enamine N-oxides is described. The reaction is rapid, complete, directional, and traceless and displays a broad substrate scope. Reaction rates for the cleavage of fluorophores from proteins are on the order of 82 M⁻¹s⁻¹, and the reaction is relatively insensitive to common aqueous buffers and pHs between 4 and 10. Diboron reagents with bidentate and tridentate ligands also effectively reduce the enamine N-oxide to induce dissociation and compound release. This reaction can be paired with the corresponding bioorthogonal hydroamination reaction to afford an integrated system of bioorthogonal click and release via an enamine N-oxide linchpin with a minimal footprint. The tandem associative and dissociative reactions are useful for the transient attachment of proteins and small molecules with access to a discrete, isolable intermediate. We demonstrate the effectiveness of this revertible transformation on cells by using chemically cleavable antibody-drug conjugates.