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. 2018 Dec 18;19(24):2550-2552.
doi: 10.1002/cbic.201800599. Epub 2018 Nov 15.

Coumarin Triazabutadienes for Fluorescent Labeling of Proteins

Mehrdad Shadmehr  1 , Garrett J Davis  1 , Bereketab T Mehari  1 , Stephanie M Jensen  1 , John C Jewett  1
Affiliations

Coumarin Triazabutadienes for Fluorescent Labeling of Proteins

Mehrdad Shadmehr et al. Chembiochem. .

Abstract

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 the use of a coumarin-modified triazabutadiene that can deliver aryl diazonium ions to fluorescently label proteins by tyrosine-selective modification. The labeling can be triggered by low-pH-induced liberation of the diazonium species, thus making the fluorophore especially useful in labeling biochemical surroundings such as those found within the late endosome. Additionally, we show that a variety of coumarin triazabutadienes might also be prone to releasing their diazonium cargo after irradiation with UV light.

Keywords: electrophilic substitution; fluorescent probes; protecting groups; protein modifications; substituent effects.

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Figures

Figure 1.
Figure 1.
A. Coumarin triazabutadiene 2 was synthesized from the corresponding aryl azide, 3. B. and C. are scans from SDS-PAGE gels. Fluor. = fluorescence scan; Coom. = coomassie blue staining. B. Bovine serum albumin (BSA) labeled with 2 (0.2 mM) in 100 mM PBS buffer with increasing pH (4, 5, 6, 7 and 8) for 12 hours. At far right, resorcinol is added to trap diazonium ion intermediate and reduces fluorescence signal. The samples were then run on a gel and analyzed. C. Samples of BSA in 100 mM PBS (pH 7) are irradiated with 350 nm light for varying times. At far right, resorcinol is added to trap diazonium ion intermediate and reduces fluorescence signal. The samples were then run on a gel and analyzed.
Figure 2.
Figure 2.
A. A series of triazabutadienes was synthesized and evaluated for their ability to label proteins. B., C. and D. are scans from SDS-PAGE gels. Fluor. = fluorescence scan; Coom. = coomassie blue staining. B. Minimal difference was observed when bovine serum albumin (BSA) was treated with compounds 2, 5-7 at pH 5 for 2 hours. The samples were quenched with resorcinol and then run on a gel and analyzed. C. BSA was incubated with 2, 5-7 in pH 7 PBS buffer and UV irradiation for either 0 or 15 minutes by incubation for an additional 45 minutes in the absence of light. The samples were then run on a gel and analyzed. D. A sample of E. coli lysate was incubated with 6 in Tris-HCl buffer pH 8.8 for 40 minutes in the presence or absence of UV light followed by incubation for an additional 2 hours in the absence of light. The samples were then run on a gel and analyzed.
Scheme 1.
Scheme 1.
A. Aryl diazonium ions can react with proteins and generate a fluorescent product that is minimally perturbing. B. Coumarin diazonium 1 reacted with p-cresol to form a fluorescent azobenzene.
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