We present the concept, synthesis, and kinetic characterization of PYRROC as the first functionalized cycloalkyne which cannot form isomers in the reaction with azides. In aqueous buffer, PYRROC displays unprecedented rate accelerations in SPAAC of three to four orders of magnitude, leading to rate constants exceeding 400 M(-1) s(-1).

Chlorhexidine and alexidine have long been used as oral disinfectants by humans. Both compounds inhibit protein–protein interactions mediated by the anti‐apoptotic protein Bcl‐xL at physiologically relevant concentrations and induce apoptosis in a series of tumor cell lines derived from the tongue and pharynx (see picture). Inhibition of protein–protein interactions is a potential mode of action of drugs in current human use.

Strain-promoted azide-alkyne cycloadditions (SPAAC) have proven extremely useful for labeling of biomolecules, but typically produce isomeric mixtures. This is not appropriate for the formation of bioactive molecules in living cells. Here, the first use of SPAAC for the isomer-free synthesis of a bioactive molecule is reported both in vitro and inside cultured cells. We developed the symmetrical cyclooctyne SYPCO and used it for the generation of a chemically uniform triazole inhibitor of protein-protein interactions mediated by Bcl-x via isomer-free SPAAC (iSPAAC). Tumor cells treated with the reactants of the iSPAAC reaction contained higher concentrations of triazole, and displayed higher apoptosis levels, than cells treated with pre-synthesized triazole. We envision iSPAAC as a broadly applicable method for modulating intracellular targets with organic molecules with molecular weights prohibitively large for cellular uptake, via smaller and thus more cell-permeable components.

High‐affinity inhibitors of large protein–protein interactions often have a high molecular weight, which compromises their cell permeability and oral bioavailability. We recently presented isomer‐free, strain‐promoted azide‐alkyne cycloaddition (iSPAAC) as a method by which to generate large, chemically uniform bioactive molecules inside living cells from two smaller components with higher cell permeability. Here, we present the synthesis of Fmoc‐protected azacyclonon‐5‐yne (Fmoc‐ACN) as the first cyclononyne suitable for iSPAAC. ACN facilitated the structure‐guided development of a single‐digit micromolar triazole inhibitor of the protein–protein interaction domain of the antiapoptotic protein Bcl‐xL. Inhibitor formation in aqueous buffer at 37 °C, templated by the target protein Bcl‐xL, proceeded 2800 times faster than the reaction between Fmoc‐ACN and benzyl azide under standard conditions in acetonitrile. Our data demonstrate the utility of cyclononynes for iSPAAC and their potential for achieving vastly accelerated templated reactions in aqueous environments.

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