Templated Generation of a Bcl-xL 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.