Hydrazonyl Sultones as Stable Tautomers of Highly Reactive Nitrile Imines for Fast Bioorthogonal Ligation Reaction

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Here we report the design and synthesis of a new class of bioorthogonal reagents called hydrazonyl sultones (HS) that serve as stable tautomers of highly reactive nitrile imines (NI). Compared to the photogenerated NI, HS display a broad range of aqueous stability and tunable reactivity in a 1,3-dipolar cycloaddition reaction, depending on substituents, sultone ring structure, and solvent conditions. DFT calculations have provided vital insights into the HS → NI tautomerism, including a base-mediated anionic tautomerization pathway and a small activation barrier. Comparative kinetic analysis of tetrazole vs HS-mediated cycloadditions reveals that a tiny fraction of the reactive NI (∼15 ppm) is present in the tautomeric mixture, underpinning the extraordinary stability of the six-membered HS. We further demonstrate the utilities of HS in selective modification of bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN)-lysine-containing nanobodies in phosphate buffered saline and fluorescent labeling of a BCN-lysine-encoded transmembrane glucagon receptor on live cells.