Angew. Chem. Int. Ed. Engl., published online 3 May 2012, doi:10.1002/anie.201108181

First-generation azide-alkyne cycloaddition reactions required high doses of copper, the toxicity of which limited cellular applications. Next-generation reactions have thus focused on decreasing the copper required by altering metal ligands or by using ring strain to drive the reaction. Uttamapinant et al. now present a third strategy: raising the effective copper concentration by merging a copper chelator with the substrate azide. To test this idea, the authors synthesized substrates containing pyridine rings with nitrogen atoms positioned next to the reactive azide; these substrates showed 40–80% conversion to product in 30 minutes, whereas control phenyl-modified substrates showed no conversion in that time. Adding the known copper ligand THPTA increased conversion rates, but not as much as chelation, as a pyridine-modified substrate without THPTA was equally or more effective than a phenyl-modified substrate with THPTA. To extend this reaction to cells, the authors used their PRIME strategy to specifically link azides onto protein fusions prior to cycloaddition. Rates of protein labeling mirrored in vitro results, with chelation increasing yields by 2.7- to 25-fold. The combined use of chelation and secondary copper ligands allowed the use of copper at sufficiently low concentrations that proteins could be labeled without disrupting neuron cultures known to be highly sensitive to toxic substances, suggesting that this strategy will find immediate utility in chemical biology research.