1,5-Diazabicyclo[3.3.0]octadienediones (9,10-dioxabimanes). Strongly fluorescent syn isomers

Kosower, Edward M.; Pazhenchevsky, Barak; Hershkowitz, Eli

doi:10.1021/ja00488a050

Cited by 98 publications

(48 citation statements)

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“…The synthesis of the bromobimanes is described elsewhere (1). mBBr and bBBr were dissolved in acetonitrile and the 50 mM stock solutions were stored in the dark; the solutions are stable for at least 2 months.…”

Section: Methodsmentioning

confidence: 99%

“…These bimane labels provide a class of labeling agents that may have wide applicability in biological materials. syn-9,10-Dioxabimanes, a new class of compounds, are highly fluorescent (1). Exceptions to this generalization are the essentially nonfluorescent bromoderivatives, illustrated by a monobromobimane (mBBr) (I), a dibromobimane (bBBr) (II), and a monobromotrimethylammoniobimane (qBBr) (III).…”

mentioning

confidence: 99%

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Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Kosower¹,

Kosower²,

Newton³

et al. 1979

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

238

142

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The bimane fluorescent labels, monobromobimane, dibromobimane, and monobromotrimethylammoniobimane, are derivatives of syn-9,10-dioxabimane:1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione. They efficiently label hemoglobin (reactive thiol groups), membrane proteins, and glutathione of normal human red cells under physiological conditions. Monobromobimane and dibromobimane are effective on intact cells while red cell membranes may be impermeable to the positively charged monobromotrimethylammoniobimane, the latter being effective only on lysed cells. These bimane labels provide a class of labeling agents that may have wide applicability in biological materials. syn-9,10-Dioxabimanes, a new class of compounds, are highly fluorescent (1). Exceptions to this generalization are the essentially nonfluorescent bromoderivatives, illustrated by a monobromobimane (mBBr) (I), a dibromobimane (bBBr) (II), and a monobromotrimethylammoniobimane (qBBr) (III). We have now found that these three bromobimanes are highly efficient labeling agents for cells, proteins, and small molecules like glutathione, giving rise to highly fluorescent derivatives. The fluorescent derivatives are very stable in air and under irradiation. The labeling procedures are simple and can be carried out rapidly under physiological conditions. We shall describe in the present article the use of these reagents on normal human red cells. MATERIALS AND METHODSBromobimanes. The synthesis of the bromobimanes is described elsewhere (1). mBBr and bBBr were dissolved in acetonitrile and the 50 mM stock solutions were stored in the dark; the solutions are stable for at least 2 months. The salt, qBBr, was dissolved in aqueous buffer (pH 7.4) shortly before use; the 1-4 mM solutions have a half-life at 220C of approximately 4 hr.Red Cells. Human blood, anticoagulated with heparin, was obtained from normal individuals. The buffy coat was removed after centrifugation; the red cells were washed twice with 135 mM NaCI/10 mM phosphate buffer, pH 7.4, and resuspended in the same buffer to a packed cell volume of 5-10%. Ghosts were obtained from the cells by the procedure of Steck and Kant (2). Hemoglobin solutions were prepared by the centrifugation of lysed red cells at 22,000 X g for 30 min, followed by dialysis of the membrane-free supernatants against 10 mM phosphate buffer, pH 7.4.Reactions of Bromobimanes with Intact Cells, Lysates, and Hemoglobin Solutions. The reagents were added in a ratio of 5-20 ,umol of reagent per ml of packed red cells (1-4 moles of reagent per mole of hemoglobin). Usually 10-20 Aul of the stock solution was used per ml of cell suspension of 5-10% packed cell volume. The reagent solution was placed in a dry test tube and the-sample (cell suspension, hemoglobin solution) was added with rapid mixing. A stock solution (50 mM) of the mBBr reagent could be diluted with buffer to 1-2 mM before it was mixed with a sample. The bBBr compound, which is less soluble in water, was not diluted. qBBr was dissolved in buffer immediately before use...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Kosower¹,

Kosower²,

Newton³

et al. 1979

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

238

142

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“…The fluorogenic bromobimanes 14–17 are popular as analytical reagents for identifying cellular thiols and metabolites. Chromogenic vinylic sulfones, 18 dye-maleimides, 19 and cyanine-type linker electrophiles 20 have been recently described, all of them highly reactive and selective.…”

Section: Introductionmentioning

confidence: 99%

Thiol-Selective Fluorogenic Probes for Labeling and Release

2009

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Thiol alkylation is a powerful technique for the labeling of proteins. We report a new class of highly reactive, selective, and fluorogenic probes for thiols in aqueous solution at neutral pH, based on the 7-oxanorbornadiene (OND) framework. The maleate moiety in 7-oxabicyclo[2.2.1]hept-2,5-diene-2,3-dicarboxylic acid esters serves as both a tunable electrophile and an intramolecular quencher of an attached dansyl fluorophore. Thiols have been found to add with high rates (second-order rate constants of 40–200 M−1s−1) to give adducts that exhibit enhancements of fluorescence intensity up to 180-fold. The resulting adducts are also versatile with respect to cleavage (release) reactions by two different mechanisms. First, retro-Diels–Alder fragmentation occurs with half lives from days to weeks at room temperature, and an epoxide derivative is also reported that is incapable of cycloreversion cleavage. Second, monoamide OND derivatives undergo rapid closure to succinimide derivatives upon thiol addition, providing a thiol-triggered mechanism for immediate alcohol release. Peptides and proteins containing free thiol groups were labeled with OND electrophiles with high chemoselectivity. Since the system is so easily assembled from readily accessible modules, various functional groups can be added to OND linkers to allow the attachment of other molecules of interest.

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“…Significantly, these compounds are generally of good photostability and some of them are water soluble. The isomer anti-(R 2 ,R 1 ) bimane is only weakly fluorescent [6,7]. The Coumarin laser dyes presently used for the spectral region from 440 to 540 nm have rather poor photochemical stability.…”

Section: Syn-bimanes As Laser Dyesmentioning

confidence: 99%