Labeling Live Cells by Copper-Catalyzed Alkyne−Azide Click Chemistry

Hong, Vu; Steinmetz, Nicole F.; Manchester, Marianne; Finn, M. G.

doi:10.1021/bc100272z

Cited by 361 publications

(363 citation statements)

References 46 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

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

Kang

Joo

Choi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The posttranslational modification of neural cell-adhesion molecule (NCAM) with polysialic acid (PSA) and the spatiotemporal distribution of PSA-NCAM play an important role in the neuronal development. In this work, we developed a tissue-based strategy for metabolically incorporating an unnatural monosaccharide, peracetylated N-azidoacetyl-D-mannosamine, in the sialic acid biochemical pathway to present N-azidoacetyl sialic acid to PSA-NCAM. Although significant neurotoxicity was observed in the conventional metabolic labeling that used the dissociated neuron cells, neurotoxicity disappeared in this modified strategy, allowing for investigation of the temporal and spatial distributions of PSA in the primary hippocampal neurons. PSA-NCAM was synthesized and recycled continuously during neuronal development, and the two-color labeling showed that newly synthesized PSANCAMs were transported and inserted mainly to the growing neurites and not significantly to the cell body. This report suggests a reliable and cytocompatible method for in vitro analysis of glycans complementary to the conventional cell-based metabolic labeling for chemical glycobiology.

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Section: Resultsmentioning

confidence: 99%

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

Kang

Joo

Choi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Perhaps at odds with a notion of general toxicity is the fact that several essential proteins in organisms utilize copper, leading in some cases to a relatively high cellular content 83 . For example, in yeast, estimates of over 10 5 atoms per cell have been made with a relatively low associated toxicity as judged by MIC 50 of 40.7 mM 84 .…”

Section: Review Nature Communications | Doi: 101038/ncomms5740mentioning

confidence: 99%

“…Lack of toxicity is attributed to a highly conserved biological system for maintaining Cu(I) bound to a series of carriers, preventing the release of free copper ions that could generate reactive oxygen species. Therefore, ligands such as THPTA 83 , BTTES 85 and histidine 82 that have been used to generate Cu(I) complexes and that maintain and stabilize the metal oxidation state while also negating the potential toxicity of exogenous reductants seem a logical approach. Indeed, these have allowed the labelling of living systems including the labelling of glycans in developing zebrafish embryos 85 .…”

Section: Review Nature Communications | Doi: 101038/ncomms5740mentioning

confidence: 99%

Selective chemical protein modification

2014

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“…The latter click reaction involves copper(I) catalyzed coupling of an azide and terminal alkyne to generate a stable triazole (2). Until recently, the necessity of the copper catalyst precluded use of this reaction in biological systems due to toxicity concerns (3,4). Bertozzi and others elegantly solved this problem by developing several new ring strained dienophile derivatives that do not require catalysts (5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

Reactive polymer enables efficient in vivo bioorthogonal chemistry

Devaraj

Thurber

Keliher

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

173

187

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There has been intense interest in the development of selective bioorthogonal reactions or "click" chemistry that can proceed in live animals. Until now however, most reactions still require vast surpluses of reactants because of steep temporal and spatial concentration gradients. Using computational modeling and design of pharmacokinetically optimized reactants, we have developed a predictable method for efficient in vivo click reactions. Specifically, we show that polymer modified tetrazines (PMT) are a key enabler for in vivo bioorthogonal chemistry based on the very fast and catalyst-free [4 þ 2] tetrazine/trans-cyclooctene cycloaddition. Using fluorescent PMT for cellular resolution and 18 F labeled PMT for whole animal imaging, we show that cancer cell epitopes can be easily reacted in vivo. This generic strategy should help guide the design of future chemistries and find widespread use for different in vivo bioorthogonal applications, particularly in the biomedical sciences.in vivo chemistry | pharmacokinetics | PET imaging | intravital microscopy | pretargeting T he ability to perform selective chemistries in living systems such as single cells, 3D cultures, invertebrates, or mammals would have far reaching applications in tracking biomolecules, designing new therapeutic approaches, and in visualizing medically relevant biomarkers. To date, only a few practical bioorthogonal reactions have been reported, the most popular being the Staudinger ligation and the [3 þ 2] cycloaddition "click" reaction between azides and alkynes (1, 2). The latter click reaction involves copper(I) catalyzed coupling of an azide and terminal alkyne to generate a stable triazole (2). Until recently, the necessity of the copper catalyst precluded use of this reaction in biological systems due to toxicity concerns (3, 4). Bertozzi and others elegantly solved this problem by developing several new ring strained dienophile derivatives that do not require catalysts (5-9). However, many of these derivatives have low water solubility, require complex multistep synthesis, and possess suboptimal kinetics. Our search for alternative rapid, selective, and chemically accessible coupling reactions without need for a catalyst led us and others to investigate the [4 þ 2] inverse Diels-Alder cycloaddition (10-13). We realized that this set of chemistries is more uniquely suited to biological applications and may indeed represent a universal platform technology ( Fig. 1 A and B). Specifically we and others have shown that the cycloaddition between tetrazine (Tz) and trans-cyclooctene (TCO) can proceed orders of magnitude faster than previously studied azide and alkyne click reactions and importantly does not require the action of a catalyst. This reaction has also been adapted for single cell imaging using newer fluorogenic Tz probes (14).Although initial work focused on in vitro labeling there has been a surge of recent work applying this reaction for various in vivo applications (15-17). Despite the above progress, our results with initial TCO...

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Labeling Live Cells by Copper-Catalyzed Alkyne−Azide Click Chemistry

Cited by 361 publications

References 46 publications

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

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

Selective chemical protein modification

Reactive polymer enables efficient in vivo bioorthogonal chemistry

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