We report that azides capable of copper-chelation undergo much faster “Click chemistry” (copper-accelerated azide-alkyne cycloaddition, or CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allowed us to perform site-specific protein labeling on the surface of living cells with only 10–40 µM CuI/II and much higher signal than could be obtained using the best previously-reported live-cell compatible CuAAC labeling conditions. Detection sensitivity was also increased for CuAAC detection of alkyne-modified proteins and RNA labeled by metabolic feeding.

The copper-catalyzed azide-alkyne cycloaddition, or CuAAC, has been used extensively for the conjugation, immobilization, and purification of biomolecules. [1] Despite excellent reaction kinetics, high specificity, and bioorthogonality, CuAAC has been used to a far lesser extent in the cellular context because of toxicity caused by the Cu Imediated generation of reactive oxygen species (ROS) from O 2 . [2] One way to address this problem is to remove the Cu I requirement, by using alkynes activated by ring strain. [3][4][5] However, even the fastest of the strained cyclooctynes [6] react with azides more than tenfold slower than terminal alkynes in the presence of Cu I (k obs % 1m À1 s À1 for (aza)dibenzocyclo octyne [6] compared to k obs % 10-100 m À1 s À1 per 10-100 mm Cu I / Cu II for CuAAC [7] ). A second approach to improve cell compatibility is to use water-soluble ligands such as tris-(hydroxypropyltriazolylmethyl)amine (THPTA), [8] bis[(tertbutyltriazoyl)methyl]-[(2-carboxymethyltriazoyl)methyl]amine (BTTAA), [9] or bis(l-histidine) [10] for Cu I . These ligands both accelerate the cycloaddition reaction and act as sacrificial reductants, helping to protect cells and biomolecules from ROS. [8] Here we explore a third approach to improve the cell compatibility and performance of CuAAC. In general, decreasing the copper concentration lowers the toxicity of CuAAC to cells, but this is accompanied by a large decrease in reaction kinetics. [9] We reasoned that it might be possible to compensate for this decrease by using an azide reaction partner that contains an internal copper-chelating moiety (Figure 1 A), which would raise the effective copper concentration at the reaction site. This concept has been explored for azide-alkyne reactions in organic solvents, with Cu II rather than Cu I species, and at very high copper (10 mm) and reactant (200-400 mm) concentrations, [11,12] but never before under conditions relevant to biomolecular labeling. The goal of our study was to examine the effect of substrate chelation assistance on CuAAC kinetics and biocompatibility.The rate-determining step of CuAAC is postulated to be the formation of the metallacycle from the Cu I acetylide and the organic azide. [15] We decided to test whether an organic azide containing an internal Cu I ligand could accelerate formation of the metallacycle and hence the overall rate of the CuAAC reaction. We prepared two azides with proximal pyridine nitrogen atoms to chelate the Cu I ion (picolyl azides 2 and 4), as well as their nonchelating carbocyclic analogues, 1 and 3 ( Figure 2).CuAAC reaction timecourses were measured using 7ethynylcoumarin, a fluorogenic alkyne whose quantum yield (QY) increases from 1 % to 25 % upon reaction with azides [4] (Figure 2 A). Assays were first performed with 10 mm CuSO 4 in the absence of Cu I ligands. Reaction timecourses are shown in Figure S1 (see Supporting Information) and values for percent conversion into product after 10 and 30 minutes are given in Figure 2 B. Whereas the conventional azides 1 a...

This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: PRobe Incorporation Mediated by Enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase site-specifically attaches a picolyl azide derivative to a 13-amino acid recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing picolyl azide are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize picolyl azide, perform PRIME labeling, and achieve CuAAC derivatization of picolyl azide on live cells, fixed cells, and purified proteins. Reagent preparations, including synthesis of picolyl azide probes and expression of lipoic acid ligase, take 12 d, while the procedure to perform site-specific picolyl azide ligation and CuAAC on cells or on purified proteins takes 40 min-3 h.

3,4‐Disubstituted and 3,4,5‐trisubstituted isoxazoles have been formed from alkynes and nitrile oxides in a ruthenium(II)‐catalyzed process (see scheme; cod=cycloocta‐l,5‐diene, Cp*=C5Me5). These reactions are experimentally simple, proceed at room temperature, and produce isoxazoles with excellent regioselectivity in high yield.

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