A red-emission fluorescence probe based on 1,4-addition reaction mechanism for the detection of biothiols in vitro and in vivo
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In this work, a new fluorescence probe (DC) with a donor-π-acceptor (D-π-A) structure was designed and synthesized for the detection of three kinds of biothiols (Cys, Hcy and GSH) in live cells and organisms. DC displayed an intense red-emission centered at 625 nm. In the presence of biothiols, the nucleophilic addition reaction between the C=C double bond of DC and the sulfhydryl group (-SH) of biothiols occurred, resulting in obvious fluorescence quenching responses. DC exhibited high selectivity towards biothiols over other common bioactive species with low detection limits (0.26, 0.43, and 0.44 µM for Cys, Hcy and GSH, respectively). In addition, DC displayed a rapid response to biothiols within 4 min. The applications of DC in biothiols detection and imaging were then successfully demonstrated for the real-time monitoring of endogenous and exogenous biothiols in live cells and live animals.Keywords:
Nucleophilic Addition
Acceptor
Biothiols, including homocysteine (Hcy), cysteine (Cys) and reduced glutathione (GSH), play various roles in physiological and pathological processes. Because these biothiols possess similar in structures, it is difficult to discriminate Hcy, Cys and GSH from one another. In this work, a novel fluorescent probe, 4-BrCP, based on coumarin as a fluorophore to discriminate GSH from Hcy and Cys was rationally designed and synthesized in three steps with good total yield. The benzothiazol ring played the role of a fluorescent emission wavelength adjuster, while the ester part served as a fluorescent quencher and reactive sites with Hcy, Cys and GSH. The results of responsive experiment indicated that the probe could discriminate GSH from Hcy and Cys distinctly and exhibited a relatively high fluorescence quantum yield (0.84) and low detection limit (9.8 nM). In addition, the probe 4-BrCP also showed good stability and low toxicity. The reaction mechanism of 4-BrCP with GSH was speculated on according to the LC-MS data. Most importantly, because of the length of the skeletons of Hcy, Cys and GSH, different dynamic fluorescent phenomena were observed. Furthermore, imaging experiments suggested that the probe could be used to monitor GSH in living cells and organisms.
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Michael reaction
2-Mercaptoethanol
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ABSTRACT Electronic excitation energy transfer (EET) between molecules of polymethine dyes bound to human serum albumin (HSA) has been established and studied by absorption and fluorescence spectroscopy as well as by fluorescence decay measurements. In this system, excitation of the donor dye molecule leads to fluorescence of the acceptor dye molecule, both bound to HSA, with donor fluorescence quenching by the acceptor. The short distance between the donor and the acceptor (25‐28 Å) revealed from the Förster model of EET as well as some spectroscopic data show that both molecules are probably located in the same binding domain of HSA. The role of HSA is to bring donor and acceptor molecules together to a distance adequate to achieve EET as well as to increase the donor and acceptor fluorescence quantum yields. Efficient quenching of the intrinsic HSA fluorescence by some polymethine dyes (oxonols) is observed. The experimental results fit well a model for the formation of a weakly fluorescent dye‐HSA complex; the quencher in this complex should be located in the immediate vicinity of the HSA fluorophore group (Trp 214 ).
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Spiropyran
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Three fluorescent probes TP1–3 for thiols were rationally designed and synthesized to distinguish cysteine (Cys) from glutathione (GSH)/homocysteine (Hcy). TP1–3 are almost non-fluorescent and colorless 4-nitro-1,8-naphthalimide derivatives. Upon the substitution of nitro by Cys, TP1–3 were transformed into weakly fluorescent green-emitting 4-amino analogs via highly fluorescent blue-emitting thioether intermediates. The three-channel signaling capability allows discrimination between Cys and GSH/Hcy. The fluorescence intensity at 498 nm was linearly proportional to GSH concentration in the range of 0–20 μM, and the detection limit was 5 × 10−8 mol L−1. A good linear relationship between A446/A350 and Cys concentration was found in the range of 0–70 μM, and the detection limit was 2 × 10−7 mol L−1. Moreover, TP3 was used for living cell imaging as well as for detecting mercapto-containing proteins.
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A fluorescent probe (N-(4-methyl-2-oxo-2H-chromen-7-yl)-2,4-dinitrobenzenesulfonamide), which exhibits high selectivity to glutathione and cysteine among amino acids including sulphur-containing methionine and metal ions, was synthesized. The experiments demonstrate that the fluorescent probe is a reliable and specific probe for glutathione and cysteine in living cells.
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ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTChanges of selectivity in the reactions of substituted 4-nitrobenzyl sulfonates with nucleophilic reagentsPaul Dietze and William P. JencksCite this: J. Am. Chem. Soc. 1989, 111, 15, 5880–5886Publication Date (Print):July 1, 1989Publication History Published online1 May 2002Published inissue 1 July 1989https://pubs.acs.org/doi/10.1021/ja00197a057https://doi.org/10.1021/ja00197a057research-articleACS PublicationsRequest reuse permissionsArticle Views584Altmetric-Citations11LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
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ENERGY TRANSFER AND FLUORESCENCE QUENCHING IN COMPLEXES OF POLYMETHINE DYES WITH HUMAN SERUM ALBUMIN
Electronic excitation energy transfer (EET) between molecules of polymethine dyes bound to human serum albumin (HSA) has been established and studied by absorption and fluorescence spectroscopy as well as by fluorescence decay measurements. In this system, excitation of the donor dye molecule leads to fluorescence of the acceptor dye molecule, both bound to HSA, with donor fluorescence quenching by the acceptor. The short distance between the donor and the acceptor (25-28 A) revealed from the Forster model of EET as well as some spectroscopic data show that both molecules are probably located in the same binding domain of HSA. The role of HSA is to bring donor and acceptor molecules together to a distance adequate to achieve EET as well as to increase the donor and acceptor fluorescence quantum yields. Efficient quenching of the intrinsic HSA fluorescence by some polymethine dyes (oxonols) is observed. The experimental results fit well a model for the formation of a weakly fluorescent dye-HSA complex; the quencher in this complex should be located in the immediate vicinity of the HSA fluorophore group (Trp(214)).
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Human serum albumin
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