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Fluorescent metal ion indicators with large stokes shift

a technology of metal ions and fluorescence, applied in the field of compositions and methods for the detection and isolation of metal ions, can solve the problems of limiting the effectiveness of metal ions and having a deleterious

Inactive Publication Date: 2006-02-02
MOLECULAR PROBES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0044] The present invention also provides kits for binding, detecting, quantitating, monitoring and otherwise analyzing metal ions wherein the kit comprises at least one compound according to the present invention and instructions for use thereof. In a further embodiment, the kit comprises one or

Problems solved by technology

Metal ions can also have a deleterious effect when present in excess of bodily requirements or capacity to excrete.
In addition, due to their relatively small size, they are readily sequestered non-selectively in intracellular vesicles, further limiting their effectiveness.

Method used

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  • Fluorescent metal ion indicators with large stokes shift
  • Fluorescent metal ion indicators with large stokes shift
  • Fluorescent metal ion indicators with large stokes shift

Examples

Experimental program
Comparison scheme
Effect test

example 1

Synthesis of 5-(Aminomethyl) BAPTA

[0183] To a stirred suspension of 5-formyl BAPTA 1 (5.60 g, 10 mmol) in EtOH (100 mL) was added a solution of hydroxylamine hydrochloride (1.40 g, 20 mmol) in H2O (5 mL) followed by 3N NaOAc (5 mL, 15 mmol). The mixture was stirred at 60° C. for 4 h, cooled to room temperature and evaporated. Water (200 mL) was added to the residue, the product filtered and washed with water (10×50 mL), dried in air, then in vacuo to give oxime 2, 5.37 g (91%) as an off-white solid. The product 2 does not require purification to use in the next step.

[0184] To a stirred solution of oxime 2 (4.71 g, 8 mmol) in acetic acid (80 mL), powdered Zn (2.62 g, 40 mmol) was added in one portion. The mixture was stirred for 6 h, diluted with CHCl3 (300 mL) and filtered from inorganic material. The filtrate was evaporated and the residue loaded onto a SiO2 column (3×30 cm bed, made in 5% MeOH and 1% AcOH in CHCl3). The product was eluted with a gradient of 5-20% MeOH in CHCl3 w...

example 2

Synthesis of 5-(Dansylaminomethyl) BAPTA derivatives

[0185] To a solution of amine 3 (160 mg, 0.28 mmol) in pyridine (3 mL), dry powdered dansyl chloride (98 mg, 0.36 mmol) was added in three portions within 5 min. The mixture was stirred for 2 h, then evaporated. The solid residue was dissolved in CHCl3 (100 mL), washed with H2O (100 mL), 1% AcOH (2×100 mL), and sat. NaCl (100 mL). Chloroform was evaporated and the residue was purified by preparative TLC on silica gel using 3% MeOH in CHCl3 as eluant to give 5-(dansylaminomethyl) BAPTA, tetramethyl ester 4, 60 mg (27%) as a white powder.

[0186] A mixture of tetramethyl ester 4 (60 mg, 0.074 mmol), MeOH (1 mL), dioxane (1 mL), H2O (1 mL), and 1N KOH (0.75 mL, 0.75 mmoL) was stirred for 16 h, then 0.2 N HCl added to achieve pH 9.5, and the mixture was evaporated. The residue was purified by column chromatography on Sephadex LH-20 (3.5×50 cm bed, made in H2O) using H2O as eluant to give 5-(dansylaminomethyl) BAPTA, tetrapotassium salt...

example 3

Synthesis of 5-(N-(5′-fluoresceinyl)aminomethyl) derivatives

[0188] To a stirred solution of amine 3 (115 mg, 0.2 mmol) and DIEA (0.17 mL, 1.0 mmol) in CH2Cl2 (5 mL) was added a solution of acyl chloride 8, prepared from the acid 7 (149 mg, 0.3 mmol) and oxalyl chloride (0.1 mL, 1.2 mmol). The mixture was stirred 16 h, then diluted with CHCl3 (100 mL), and washed with 1% AcOH (2×20 mL), sat. NaCl, filtered and evaporated. The residue was purified by preparative TLC on silica gel using 5% MeOH in CHCl3 as eluant to give tetramethyl ester 9, 71 mg (34%) as an off-white solid.

[0189] A mixture of tetramethyl ester 9 (50 mg, 0.047 mmol), MeOH (2 mL), dioxane (2 mL), and 1N KOH (0.5 mL, 0.5 mmoL) was stirred for 16 h, then 0.2 N HCl added to pH 9.0, and the mixture was evaporated. The residue was purified by column chromatography on Sephadex LH-20 (6×70 cm bed, made in H2O) using H2O as eluant to give hexapotassium salt 10, 39 mg (72%) as a yellow solid (after lyophilization).

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Abstract

The present invention provides fluorogenic compounds for the detection of target metal ions wherein the compounds exhibit a Stokes shift greater than 50 nm and the detectable signal is modulated by photoinduced electron transfer (PET). The present compounds consist of three functional elements, the ion sensing moiety (chelating moiety), the reporter moiety (fluorophore or fluorescent protein) and spacer or linker between the sensing and reporter moieties of the present compound that allows for PET upon binding of a metal ion and excitation by an appropriate wavelength.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Ser. No. 60 / 591,750, filed Jul. 27, 2004, which disclosure is herein incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to novel compositions and methods for the detection and isolation of metal ions, including physiological concentrations of calcium. The invention has applications in the fields of cell biology, neurology, immunology and proteomics. BACKGROUND OF THE INVENTION [0003] Metal ions play an important role in biological systems. Cells utilize metal ions for a wide variety of functions, such as regulating enzyme activity, protein structure, cellular signaling, as catalysts, as templates for polymer formation and as regulatory elements for gene transcription. Metal ions can also have a deleterious effect when present in excess of bodily requirements or capacity to excrete. A large number of natural and synthetic materials are known to selectively or non...

Claims

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Application Information

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IPC IPC(8): G01N33/20
CPCC07C229/18C07D221/14C07D265/38C07D311/82C09B5/60C09B5/62G01N33/20C09K11/06C09K2211/1014G01N33/84C07D209/14C07D311/90C07D413/04C09B11/06G01N2015/1006C07C311/44C07K14/195C07K14/43595C09B11/08C09B19/00C09B19/005C09B57/08G01N2458/00
Inventor GEE, KYLE RICHARDMARTIN, VLADIMIR
Owner MOLECULAR PROBES
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