Systematic Structural Tuning Yields Hydrazonyl Sultones for Faster Bioorthogonal Protein Modification

We report the synthesis of a series of hydrazonyl sultones (HS) containing an ortho-CF3 group, a five- or six-membered sultone ring, and a varying N-aryl substituent, and characterization of their aqueous stability and reactivity toward bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) in a 1,3-dipolar cycloaddition reaction. To avoid the purification of highly polar intermediates, we employed two protecting groups in our synthetic schemes. Most HS were obtained in moderate to good yields with optimized reaction conditions. The X-ray structure analysis of two HS revealed that the partially negative-charged fluorine atoms in CF3 electrostatically shield the electrophilic nitrile imine (NI) center from a nucleophilic attack, underpinning their extraordinary aqueous stability. In addition, the N-aryl substituents further modulate HS reactivity and stability, with the electron-rich six-membered HS displaying excellent aqueous stability and increased cycloaddition reactivity. The utility of these improved HS reagents was demonstrated through fast and selective modification of a BCNK-encoded nanobody with second-order rate constants as high as 1488 M-1 s-1 in phosphate-buffered saline--ethanol (9:1), representing the fastest HSB--CN ligation reported in the literature.