Templated Generation of a Bcl‐x(L) Inhibitor by Isomer‐Free SPAAC Based on Azacyclonon‐5‐yne

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‐x(L). Inhibitor formation in aqueous buffer at 37 °C, templated by the target protein Bcl‐x(L), 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.