Beyond Traditional SPAAC: 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 novel 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. Experimental and computational data support the rate trends and mechanistic rationale based on electronic complementarity. Fluorescently labelled fluoroalkyl and alkyl azide probes demonstrate the selective labeling of modified antibodies and lectin protein in vitro and the selective labelling of organelles in living cells. This dual-selectivity strategy enables orthogonal SPAAC labeling.