Title: Bioorthogonal click and release: A general, rapid, reversible bioconjugation strategy employing enamine N-oxides
Abstract: A functionally reversible bioconjugation strategy employing a new bioorthogonal dissociative reaction has been designed using enamine N-oxides. The reaction is rapid, complete, directional, traceless, and displays broad substrate scope. Reaction rates for cleavage of small molecules from proteins are fast, and the reaction is insensitive to common aqueous buffers, pHs between 4 and 10, complex tissue homogenates, and cells. 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 between N,N-dialkylhydroxylamines and strained or push-pull-activated alkynes 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 traceless and transient attachment of proteins and small molecules with access to a discrete, isolable intermediate. The effectiveness of this reversible transformation is demonstrated through several applications.