A Chemoselective Rapid Azo-Coupling Reaction (CRACR) for Unclickable Bioconjugation

Addy, Partha Sarathi; Erickson, Sarah B.; Italia, James S.; Chatterjee, Abhishek

doi:10.1021/jacs.7b05125

Cited by 88 publications

(93 citation statements)

References 52 publications

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“…This had previously been reported by both our group and others to facilitate release of aryl diazonium for protein labeling via increasing the basicity of the compound. [9] We were relieved to find that the presence of a fluorophore in conjugation with the triazabutadiene did not prevent the photochemistry. Once more we utilized BSA as a model protein to show labeling could be attained at pH 7 by irradiation at 350 nm (Figure 1c).…”

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confidence: 99%

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Coumarin Triazabutadienes for Fluorescent Labeling of Proteins

et al. 2018

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The use of small-molecule fluorophores to label proteins with minimal perturbation in response to an external stimulus is a powerful tool to probe chemical and biochemical environments. Herein we describe first the use of a coumarin modified triazabutadiene that can deliver aryl diazonium ions to fluorescently label proteins via tyrosine selective modification. The labeling can be triggered by low pH induced liberation of the diazonium species, making the fluorophore specifically useful in labeling biochemical surroundings such as those found within the late endosome. Additionally, we show that a variety of coumarin triazabutadienes may also be prone to releasing their diazonium cargo via irradiation with UV light.

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confidence: 99%

“…This result is consistent with previous work on using light to release aryl diazonium ions from triazabutadienes. [9] …”

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Coumarin Triazabutadienes for Fluorescent Labeling of Proteins

et al. 2018

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“…Indeed, we have recently developed an oxidative coupling reaction that enables selective attachment of various aromatic amines to the 5HTP residue . Additionally, we have shown that various aryldiazonium ions react rapidly with the 5HTP residue, leading to the development of a chemoselectively rapid azo‐coupling reaction (CRACR) for site‐specific protein labeling …”

Section: Figurementioning

confidence: 76%

“…Although we have previously kinetically characterized the azo‐coupling reaction between free 5HTP and 4CDZ, it is presumable that the 5‐hydroxyindole residue on a protein may react somewhat differently. The quenchergenic aspect of CRACR described herein should enable facile measurement of the kinetics of this labeling reaction on sfGFP, by monitoring the concomitant decrease in its fluorescence.…”

Section: Figurementioning

confidence: 99%

A “Quenchergenic” Chemoselective Protein Labeling Strategy

et al. 2019

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Dynamic changes in protein structure can be monitored by using a fluorescent probe and a dark quencher. This approach is contingent upon the ability to precisely introduce a fluorophore/quencher pair into two specific sites of a protein of interest. Despite recent advances, there is continued demand for new and convenient approaches to site‐selectively label proteins with such optical probes. We have recently developed a chemoselectively rapid azo‐coupling reaction (CRACR) for site‐specific protein labeling; it relies on rapid coupling between a genetically encoded 5‐hydroxytryptophan residue and various aromatic diazonium ions. Herein, it is reported that the product of this conjugation reaction, a highly chromophoric biarylazo group, is a potent fluorescence quencher. The absorption properties of this azo product can be tuned by systematically altering the structure of the aryldiazonium species. A particular “quenchergenic” aryldiazonium has been identified that, upon conjugation, efficiently quenches the fluorescence of green fluorescent protein, which is a widely used genetically encoded fluorescent probe that can be terminally attached to target proteins. This fluorophore/quencher pair was used to evaluate the protein‐labeling kinetics of CRACR, as well as to monitor the proteolysis of a fusion protein.

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“…Indeed, we have recently demonstrated that 5HTP undergoes azo-coupling reaction significantly faster than canonical amino acids (e.g., tyrosine), providing a basis for chemoselective protein labeling. [6] 5-hydroxyindoles such as serotonins are also known to be prone to oxidation under relatively mild conditions, producing oligomeric products. [7] This provides an opportunity to develop new chemoselective protein modification strategies targeted to 5HTP through oxidative coupling reactions (Figure 1).…”

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An Oxidative Bioconjugation Strategy Targeted to a Genetically Encoded 5‐Hydroxytryptophan

2018

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Approaches that enable the chemoselective, covalent modification of proteins in a site-specific manner have emerged as a powerful technology for a wide range of applications. The electron-rich unnatural amino acid 5-hydroxytryptophan was recently genetically encoded in both Escherichia coli and eukaryotes, thereby allowing its site-specific incorporation into virtually any recombinant protein. Herein, we report the chemoselective conjugation of various aromatic amines to full-length proteins under mild, oxidative conditions that target this site-specifically incorporated 5-hydroxytryptophan residue.

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A Chemoselective Rapid Azo-Coupling Reaction (CRACR) for Unclickable Bioconjugation

Cited by 88 publications

References 52 publications

Coumarin Triazabutadienes for Fluorescent Labeling of Proteins

Coumarin Triazabutadienes for Fluorescent Labeling of Proteins

A “Quenchergenic” Chemoselective Protein Labeling Strategy

An Oxidative Bioconjugation Strategy Targeted to a Genetically Encoded 5‐Hydroxytryptophan

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