Ultrafast 4‐Azidopyridinium‐Based Staudinger (UApS) Ligation for Bioorthogonal Labeling and Imaging

ABSTRACT Fast click reactions that operate efficiently in aqueous buffers are highly desirable across many areas of chemistry, yet remain scarce. Herein, we report an u ltrafast 4‐ a zido p yridinium‐based S taudinger (UApS) ligation that proceeds with large bimolecular reaction rate constants (10 3 –10 4 M −1 s −1 ) under catalyst‐free, physiologically relevant conditions. We introduced a new class of substituted 4‐azido‐1‐alkylpyridinium reagents that are both water‐stable and exceptionally reactive toward triarylphosphines, yielding phosphazide adducts that release nitrogen gas and convert into iminophosphoranes. These products were structurally validated by single‐crystal x‐ray diffraction studies and possess good aqueous stability. Mechanistic and computational investigations elucidated the UApS ligation pathway and its fast kinetics arising from the substituted 4‐azidopyridinium scaffolds. We demonstrate that the UApS ligation enables efficient protein labeling and cell‐surface imaging at low reagent concentrations. In combination with the established tetrazine ligation, we demonstrate that the UApS ligation facilitates dual labeling of distinct cellular compartments in living cells. Moreover, the UApS ligation is suitable for live‐cell stimulated emission depletion (STED) super‐resolution imaging of filopodia. Overall, the UApS ligation should extend fast click chemical space, providing a new addition to the bioconjugation toolkit.