Cysteine fluorescence probe adopting coumarin as fluorophore and application of cysteine fluorescence probe

A fluorescent probe, cysteine ​​technology, applied in the field of cysteine ​​fluorescent probe

Inactive Publication Date: 2014-05-07
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the search found that Chinese patents such as [201110439870], [201210586667], and [201210441256] reported that they can select active thiols in organisms, but there are few patents that specifically select cysteine

Method used

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  • Cysteine fluorescence probe adopting coumarin as fluorophore and application of cysteine fluorescence probe
  • Cysteine fluorescence probe adopting coumarin as fluorophore and application of cysteine fluorescence probe
  • Cysteine fluorescence probe adopting coumarin as fluorophore and application of cysteine fluorescence probe

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Experimental program
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Effect test

Embodiment 1

[0023] 3-Acetyl-7-hydroxycoumarin is obtained by reacting 2,4-dihydroxybenzaldehyde with ethyl acetoacetate, and then reacted with acryloyl chloride to obtain 2-(3-acetyl)coumarinyl acrylate. The generated 2-(3-acetyl)coumarin-based acrylate can undergo Mike addition cyclization reaction with cysteine ​​to release fluorescent 3-acetyl-7-hydroxycoumarin, thereby realizing detection .

[0024] Above-mentioned reaction process reaction formula is as follows:

[0025]

[0026] Add 2,4-dihydroxybenzaldehyde (1, 20mmol), ethyl acetoacetate (24mmol) and 1mL piperidine into 30mL absolute ethanol, heat to reflux, react for 3 hours and cool to room temperature, a green solid precipitates , filtered with suction, washed with ethanol three times, and the crude product was recrystallized with ethanol to obtain light green solid 2, melting point: 237°C, yield 91%.

[0027] Infrared measurement (KBr, cm -1 ): 3482(O-H), 3050(Ar-H), 2928(=CH), 1678(C=O), 1620(C=C), 1211(C-O-C).

[0028...

Embodiment 2

[0037] Add 60μM of Cys,Hcy,GSH,arginine,aspartic acid,glutamicacid,glycine,histidine,lysine,proline,threonine,tryptophan,tyrosine,KNO 3 , Ca(NO 3 ) 2 ,NaNO 3 ,Mg(NO 3 ) 2 , Zn(NO 3 ) 2 ,Fe(NO 3 ) 3 ,H 2 o 2 And the aqueous solution of glucose, carry out UV-visible spectrophotometry and fluorescence spectrophotometry test after 3 hours, show that probe 2-(3-acetyl) coumarin base acrylate has good selectivity to cysteine , the control before and after adding cysteine ​​showed a strong fluorescence enhancement effect. See figure 1 ,2.

Embodiment 3

[0039] Intracellular fluorescence imaging test:

[0040] The two groups of A and B are the control group, and the control group A: under the condition of 37 ℃, HeLa cells are cultured for 5 hours in the cell culture medium added with 20.0 μM 2-(3-acetyl)coumarin-based acrylate; Group B: HeLa cells were soaked in cell culture medium containing 20mM N-ethylmaleimide for 2 hours, washed three times with PBS buffer solution, and then added 20.0μM 2-(3-acetyl)coumarin Incubate in acrylated cell culture medium for 5 hours. Fluorescence imaging shows the penetration of 2-(3-acetyl)coumarinyl acrylate into the cells. In the control group A cells showed strong fluorescence. Control group B showed no fluorescence. See Figure 4 .

[0041] Fluorescence test in bovine serum:

[0042] At 37°C, 1 μM of 2-(3-acetyl)coumarinyl acrylate was added to different concentrations of bovine serum solutions (0, 0.01, 0.05, 0.2, 0.5, 1) and incubated for 20 minutes. The fluorescence intensity wa...

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Abstract

The invention discloses a cysteine fluorescence probe adopting coumarin as a fluorophore. The cysteine fluorescence probe is 2-(3-acetyl) coumarin-based acrylate and has the chemical structural formula shown as a formula (I) described in the specification. The 2-(3-acetyl) coumarin-based acrylate disclosed by the invention can act with cysteine selectively, becomes light yellow from colorless, has a fluorescence enhancing effect, can be distinguished by the naked eyes, and also can be analyzed by ultraviolet absorption and fluorospectrophotometry. The cysteine fluorescence probe disclosed by the invention has the advantages that trace high-sensitivity detection to the cysteine can be realized and the application value is important.

Description

technical field [0001] The present invention relates to a fluorescent probe with coumarin as the fluorescent parent, in particular to a cysteine ​​fluorescent probe with 2-(3-acetyl) coumarinyl acrylate as the parent and its application; The invention belongs to the field of small organic molecule fluorescent probes. Background technique [0002] Coumarin has a large molar extinction coefficient and quantum yield, and is a very important green fluorescent dye. After modification, it can be used in luminescent materials, chemical sensors and labeling biomolecules. In recent years, the use of fluorescence enhancement effect to detect cysteine ​​in living cells has been continuously developed. [0003] The content of cysteine ​​in the cell is 30-200μM, which is a very important component in the cell. It is the precursor of glutathione, coenzyme A, taurine, and the source of iron-sulfur sulfides. At the same time, it also has many important biochemical functions, such as bioca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D311/16G01N21/64
Inventor 赵宝祥苗俊英戴溪吴清华
Owner SHANDONG UNIV
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