Beyond traditional strain-promoted azide–alkyne cycloadditions by achieving orthogonality and rapid kinetics with fluoroalkyl azides

Strain-promoted azide–alkyne cycloaddition (SPAAC) is a cornerstone of bioorthogonal chemistry, offering metal-free and biocompatible ligation for applications ranging from bioconjugation to live-cell imaging. However, its relatively slow kinetics and limited selectivity hinder the simultaneous labelling of multiple targets. Here, we report on a systematic study of fluoroalkyl azides as SPAAC reagents that display enhanced reactivity with electron-rich cyclooctynes, while showing significantly reduced reactivity with electron-deficient dipolarophiles. Kinetic measurements revealed over 100-fold rate differences depending on the azide–alkyne pair, enabling orthogonal bioconjugation in both purified proteins and living cells. Quantum chemical calculations support the feasibility and qualitative trends of these SPAAC reactions while highlighting the limitations of simple frontier-orbital descriptors and inverse-electron-demand arguments. Fluorescently labelled fluoroalkyl and alkyl azide probes demonstrate the selective labeling of modified antibody trastuzumab and protein concanavalin A in vitro and the selective labelling of organelles in living cells. This dual-selectivity strategy enables orthogonal SPAAC labeling. Strain-promoted azide–alkyne cycloaddition (SPAAC) is crucial for bioorthogonal chemistry but suffers from slow kinetics and limited selectivity, complicating multi-target labeling. Here, the authors introduce fluoroalkyl azide-based SPAAC reagents, achieving enhanced reactivity and selectivity, enabling orthogonal bioconjugation in proteins and living cells.