Cyclopropenones for Metabolic Targeting and Sequential Bioorthogonal Labeling

Cyclopropenones are attractive motifs for bioorthogonal chemistry, owing to their small size and unique modes of reactivity. Unfortunately, the fastest-reacting cyclopropenones are insufficiently stable for routine intracellular use. Here we report cyclopropenones with improved stability that maintain robust reactivity with bioorthogonal phosphines. Functionalized cyclopropenones were synthesized and their lifetimes in aqueous media and cellular environments were analyzed. The most robust cyclopropenones were further treated with a panel of phosphine probes, and reaction rates were measured. Two of the phosphine scaffolds afforded ∼100-fold rate enhancements compared to previously reported reagents. Importantly, the stabilized cyclopropenones were suitable for recombinant protein production via genetic code expansion. The products of the cyclopropenone ligation were also amenable to traceless Staudinger ligations, setting the stage for tandem labeling experiments.