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-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.

iSPAAC: Isomer-Free Generation of a Bcl-xL -Inhibitor in Living Cells.

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-xL 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.

PYRROC: the first functionalized cycloalkyne that facilitates isomer-free generation of organic molecules by SPAAC.

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).

Synthesis and biochemical evaluation of highly enantiomerically pure (R,R)- and (S,S)-alexidine.

Alexidine is in everyday human use as oral disinfectant and contact lens disinfectant. It is used as a mixture of stereoisomers. Since all of alexidine's known biological targets are chiral, the biological activity of any of its chiral stereoisomers could be significantly higher than that of the mixture of stereoisomers. This makes a synthetic methodology for obtaining the individual enantiomers of the chiral diastereoisomer highly desirable. Here, we describe the first synthesis of both enantiomers of alexidine in high enantiomeric purity, and demonstrate their activity against the protein-protein interaction between the anti-apoptotic protein Bcl-xL and the pro-apoptotic protein Bak.