Fast, Cell‐Compatible Click Chemistry with Copper‐Chelating Azides for Biomolecular Labeling

Abstract

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...

Tissue-based metabolic labeling of polysialic acids in living primary hippocampal neurons

“…In other words, the Ac 4 ManNAz feeding was performed at 0 d in vitro (DIV), and the CuAAC reaction was performed at 2 DIV. For the CuAAC reaction, a recently developed ligand, 2-[4-{{bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl}-1H-1,2,3-triazol-1-yl]acetic acid (36)(37)(38), was used to minimize the cytotoxicity of Cu(I) ions and fasten the coupling reaction (39). After a 5-min reaction at 4°C, the intense red fluorescence was observed at the entire cellular surface, including neuronal cell bodies (somas) and neurites ( Fig.…”

Highly accelerated inverse electron-demand cycloaddition of electron-deficient azides with aliphatic cyclooctynes

“…The latter mode of reactivity enabled the development of highly accelerated SPAAC by means of introduction of electron-withdrawing substituents on the aromatic azide, thereby leading to unprecedented reaction rates (42 M À 1 s À 1 ), up to 30-fold faster than 'traditional' SPAAC. In this respect, use of IED SPAAC also compares favourably with the so-called supersensitive copper-catalysed click chemistry, which can be accelerated only 4-6 times by including picolyl azides instead of regular benzyl azides 34,35 . It must be noted that several research groups reported on a sizable effect of electronegative substituents on aryl azides in cycloaddition with alkenes already 50 years ago 23,36,37 .…”

Selective chemical protein modification

“…Indeed, these have allowed the labelling of living systems including the labelling of glycans in developing zebrafish embryos 85 . Moreover, an alternative approach has recently been reported by Uttamapinant et al Rather than reducing the toxicity of the catalyst, they used chelating azides, leading to a significant reduction in the required metal loading; this too allowed cell-compatible labelling 86 . Despite these developments, the use of CuAAC for intracellular modification in live cells is still to be reported and it is worth considering that eukaryotic cells are likely to offer additional unique challenges, which may be highly dependent on cell type.…”