Electrophilic aromatic substituted luciferins as bioluminescent probes for glutathione S-transferase assays

Zhou, Wenhui; Shultz, John; Murphy, Nancy; Hawkins, Erika M.; Bernad, Laurent; Good, Troy; Moothart, Leonard; Frackman, Susan; Klaubert, Dieter H.; Bulleit, Robert F.; Wood, Keith V.

doi:10.1039/b610682j

Cited by 60 publications

(37 citation statements)

References 25 publications

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“…The lower pK a of the thiol leaving group usually results in higher reaction rates. 25 Since the thiol leaving group of 1 has a much lower pKa value than 5 (the thiol pK a values of GSH and 4-nitrothiophenol are 8.5 26 and 4.67 20a , respectively), the alkyl thioester 5 produced by thiol-thioester exchange is rather unreactive, thus almost no further NCL reaction between 5 and Cys being observed. Another plausible explanation is that phenyl thioesters react faster when compared to the alkyl thioesters under NCL conditions.…”

Section: Resultsmentioning

confidence: 99%

A dual emission fluorescent probe enables simultaneous detection of glutathione and cysteine/homocysteine

et al. 2014

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Many studies have shown that glutathione (GSH) and cysteine (Cys) / homocysteine (Hcy) levels are interrelated in biological systems. To unravel the complicated biomedical mechanisms by which GSH and Cys/Hcy are involved in various disease states, probes that display distinct signals in response to GSH and Cys/Hcy are highly desirable. In this work, we report a rhodol thioester (1) that responds to GSH and Cys/Hcy with distinct fluorescence emissions in neutral media. Probe 1 reacts with Cys/Hcy to form the corresponding deconjugated spirolactam via a tandem native chemical ligation (NCL) reaction. This intramolecular spirocyclization leads to the “quinone – phenol” transduction of rhodol dyes, and an excited-state intramolecular proton transfer (ESIPT) process between the phenolic hydroxyl proton and the aromatic nitrogen in the benzothiazole unit occurs upon photoexcitation, thus affording 2-(2’-hydroxyphenyl) benzothiazole (HBT) emission (454 nm). In the case of the tripeptide GSH, only transthioesterification takes place removing the intramolecular photo-induced electron transfer (PET) process caused by the electron deficient 4-nitrobenzene moiety giving rise to a large fluorescence enhancement at the rhodol emission band (587 nm). The simultaneous detection of GSH and Cys/Hcy is attributed to the significantly different rates of intramolecular S,N-acyl shift of their corresponding thioester adducts derived from 1. The utility of probe 1 has been demonstrated in various biological systems including serum and cells.

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Section: Resultsmentioning

confidence: 99%

A dual emission fluorescent probe enables simultaneous detection of glutathione and cysteine/homocysteine

et al. 2014

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“…Our results indicate that the AL scaffold is appropriate for a range of functional luminophores and represents a useful alternative substrate to luciferin. Keywords: aminoluciferin · bioluminescent probes · biosensors · firefly luciferase · luminescence dase, [12] phosphatase, [13] glutathione S-transferase, [14] and lactamase [15] activity, have also been reported as highly sensitive indicators for target molecules. But, because the design principle is based only on using luciferin 6'-ether or 6'-ester derivatives, which have no emission prior to hydrolysis, the range of available chemical modifications is restricted and this approach is applicable to only a limited range of target molecules.…”

Section: Introductionmentioning

confidence: 99%

Aminoluciferins as Functional Bioluminogenic Substrates of Firefly Luciferase

Takakura

Kojima

Urano

et al. 2011

Chemistry An Asian Journal

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Firefly luciferase is widely used as a reporter gene in assays to study gene expression, gene delivery, and so on because of its extremely high signal-to-noise ratio. The availability of a range of bioluminogenic substrates would greatly extend the applicability of the luciferin-luciferase system. Herein, we describe a design concept for functional bioluminogenic substrates based on the aminoluciferin (AL) scaffold, together with a convenient, high-yield method for synthesizing N-alkylated ALs. We confirmed the usefulness of ALs as bioluminogenic substrates by synthesizing three probes. The first was a conjugate of AL with glutamate, Glu-AL. When Glu-AL, the first membrane-impermeable bioluminogenic substrate of luciferases, was applied to cells transfected with luciferase, luminescence was not observed; that is, by using Glu-AL, we can distinguish between intracellular and extracellular events. The second was Cy5-AL, which consisted of Cy5, a near-infrared (NIR) cyanine fluorescent dye, and AL, and emitted NIR light. When Cy5-AL reacted with luciferase, luminescence derived from Cy5 was observed as a result of bioluminescence resonance energy transfer (BRET) from AL to Cy5. The NIR emission wavelength would allow a signal to be observed from deeper tissues in bioluminescence in vivo imaging. The third was biotin-DEVD-AL (DEVD = the amino acid sequence Asp-Glu-Val-Asp), which employed a caspase-3 substrate peptide as a switch to control the accessibility of the substrate to luciferase, and could detect the activity of caspase-3 in a time-dependent manner. This generalized design strategy should be applicable to other proteases. Our results indicate that the AL scaffold is appropriate for a range of functional luminophores and represents a useful alternative substrate to luciferin.

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“…Deethylation of 7 with trimethyliodosilane, followed by treatment with toluidine gave 8 in a yield of 75 %. The cyclization of 8 with D ‐cysteine under basic conditions8b, c gave the desired luciferin trimethyl lock, 1 .…”

Section: Methodsmentioning

confidence: 99%

Self‐Cleavable Bioluminogenic Luciferin Phosphates as Alkaline Phosphatase Reporters

Zhou¹,

Andrews²,

Liu³

et al. 2008

ChemBioChem

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Electrophilic aromatic substituted luciferins as bioluminescent probes for glutathione S-transferase assays

Abstract: New highly sensitive latent bioluminescent luciferin substrates were designed and synthesized for monitoring mammalian glutathione S-transferase (GST) and Schistosoma japonicum enzyme activities.

Cited by 60 publications

References 25 publications

A dual emission fluorescent probe enables simultaneous detection of glutathione and cysteine/homocysteine

A dual emission fluorescent probe enables simultaneous detection of glutathione and cysteine/homocysteine

Aminoluciferins as Functional Bioluminogenic Substrates of Firefly Luciferase

Self‐Cleavable Bioluminogenic Luciferin Phosphates as Alkaline Phosphatase Reporters

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