Fast, Cell-Compatible Click Chemistry with Copper-Chelating Azides for Biomolecelar Labeling

摘要

The copper-catalyzed azide-alkyne cycloaddition, or CuAAC, has been used extensively for the conjugation, immobilization, and purification of biomolecules. Despite excellent reaction kinetics, high specificity, and bioorthogon-ality, CuAAC has been used to a far lesser extent in the cellular context because of toxicity caused by the Cu~I-mediated generation of reactive oxygen species (ROS) from O2. One way to address this problem is to remove the Cu~I requirement, by using alkynes activated by ring strain. However, even the fastest of the strained cyclooctynes react with azides more than tenfold slower than terminal alkynes in the presence of Cu~I (k_(obs) ≈ 1M~(-1) s~(-1) for (aza)dibenzocyclo octynel compared to k_(obs) ≈ 10-100~(-1) s~(-1) per 10-100 μm CuV Cu" for CuAAC). A second approach to improve cell compatibility is to use water-soluble ligands such as tris-(hydroxypropyltriazolylmethyl)amine (THPTA), bis[(tert-butyltriazoyl)methyl]-[(2-carboxymethyltriazoyl)methyl]-amine (BTTAA), or bis(L-histidine) for Cu~I. These ligands both accelerate the cycloaddition reaction and act as sacrificial reductants, helping to protect cells and biomole-cules from ROS.