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ESIPT (excited state intramolecular proton transfer) type fluorescent probe for biological mercaptan detection and application

A fluorescent probe, thiol technology, applied in fluorescence/phosphorescence, luminescent materials, organic chemistry, etc., to achieve the effects of simple synthesis steps, good stability, and strong anti-interference ability

Active Publication Date: 2018-01-19
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the use of it to detect biothiols

Method used

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  • ESIPT (excited state intramolecular proton transfer) type fluorescent probe for biological mercaptan detection and application
  • ESIPT (excited state intramolecular proton transfer) type fluorescent probe for biological mercaptan detection and application
  • ESIPT (excited state intramolecular proton transfer) type fluorescent probe for biological mercaptan detection and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of ESIPT-type fluorescent probes for biothiol detection

[0045] The ESIPT fluorescent probe is ethyl 2-(4-(2,4-dinitrophenylsulfonyloxy)-3-formylphenyl)-4-methylthiazole-5-carboxylate (NL- S). 2-(4-(2,4- Dinitrophenylsulfonyloxy)-3-formylphenyl)-4-methylthiazole-5-carboxylic acid ethyl ester, the generated 2-(4-(2,4-dinitrophenylsulfonyl Acyloxy)-3-formylphenyl)-4-methylthiazole-5-carboxylic acid ethyl ester (NL) can react with biothiol to remove 2,4-dinitrophenylsulfonyloxy Releases the fluorescent ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-2-carboxylate, followed by 2-(3-formyl-4-hydroxyphenyl)-4- Ethyl methylthiazole-2-carboxylate continues to react with cysteine ​​(Cys) and homocysteine ​​(Hcy) to form a Schiff base, respectively to obtain 2-(5-(5-(B Oxycarbonyl)thiazol-2-yl)-2-hydroxyphenyl)-1,3-thiadioxane-4-carboxylic acid and 2-(5-(5-(ethoxycarbonyl)thiazol-2-yl )-2-hydroxyphenyl)thiazolidine-4-carboxylic acid, so as to achieve the det...

Embodiment 2

[0056] For the detection of biothiols in solution systems

[0057] For the detection of biothiols in 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) / dimethylsulfoxide (DMSO) buffer, use 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) / dimethyl Fluorescent probe working solution with a concentration of 10 μM was prepared with sulfoxide (DMSO) buffer and ESIPT-type fluorescent probe; the specific operation process was as follows:

[0058] Use a pipette gun to add 2-(4-(2,4-dinitrophenylsulfonyloxy)-3-formylphenyl)-4-methylthiazole-5-carboxylic acid ethyl ester to 3 mL In the solution of HEPES / DMSO buffer (pH=7.4, v / v=2:8, 10μM), add 200μM cysteine ​​(Cys), homocysteine ​​(Hcy), glutathione Peptide (GSH), proline, aspartic acid, tryptophan, arginine, tyrosine, histidine, gluten glutamic acid, lysine, threonine, glycine, potassium nitrate (KNO 3 ), calcium nitrate (Ca(NO 3 ) 2 ), sodium nitrate (NaNO 3 ), magnesium nitrate (Mg(NO 3 ) 2 ), copper nitrate (Cu(NO 3 ) ...

Embodiment 3

[0060] Intracellular fluorescence imaging detection

[0061] For the detection of biothiols in human tumor cells HeLa cells, use 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) / dimethyl sulfoxide (DMSO) buffer and ESIPT fluorescent probe to prepare a concentration of 20 μM Fluorescent probe working solution; the specific operation process is as follows:

[0062]Take A, B, C, D four groups, A group: HeLa cells; B group: at 37 ℃, HeLa cells with 20μM probe 2-(4-(2,4-dinitrophenylsulfonyloxy )-3-formylphenyl)-4-methylthiazole-5-carboxylate ethyl ester treatment for 0.5 hours; Panel C: HeLa cells with 1mM N-ethylmaleimide (NEM) in cell culture medium Incubate for 2 hours, wash three times with PBS buffer, and then wash with 20 μM 2-(4-(2,4-dinitrophenylsulfonyloxy)-3-formylphenyl)-4-methylthiazole- Ethyl 5-carboxylate solution was incubated for 0.5 hours; in group D, HeLa cells were incubated with 1 mM NEM in cell culture medium for 2 hours, washed three times with PBS buffe...

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PUM

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Abstract

The invention discloses an ESIPT (excited state intramolecular proton transfer) type fluorescent probe for biological mercaptan detection. The ESIPT type fluorescent probe is 2-(4-(2,4-dinitrophenyl sulfonyloxy)-3-formylphenyl)-4-methylthiazole-5-ethyl carboxylate, wherein 2,4-dinitrophenyl sulfonyl serves as a recognition group, and 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-2-ethyl formate serves as an information reporter group. A preparation method of the ESIPT type fluorescent probe is simple, the fluorescent probe can enter cells simply and rapidly and specifically bound with biological mercaptan in the cells, so that the fluorescent probe has an obvious fluorescence enhancement effect, can realize distinguishing by naked eyes, has high anti-interference capacity on common biological molecules, has quite efficient selectivity and can perform analysis through ultraviolet absorption and fluorescence spectrophotometry. The ESIPT type fluorescent probe is good in stability, can bestored and used for a long time, is applicable to growing environments of various living cells, can realize high-sensitivity detection of trace biological mercaptan in the cells, can be applied to cell and living imaging and has quite important application value.

Description

technical field [0001] The invention belongs to the field of organic small molecule fluorescent probes, and relates to a fluorescent probe using phenylthiazole as a fluorescent matrix, in particular to 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole- A fluorescent probe of biothiol with ethyl 2-formate as the fluorescent matrix and its application. Background technique [0002] Cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH) are three very important thiol amino acids in cells, also known as biothiols. The intracellular content of cysteine ​​(Cys) is 30-200 μM, homocysteine ​​(Hcy) is 5.0-13.9 μM and glutathione (GSH) is 1-10 mM. They play key roles in the molecular and physiological processes of biological systems. Examples include maintaining aminothiol homeostasis, biocatalysis, metal binding, post-translational modification, and detoxification of xenobiotics. These biotins have received much research attention recently. Changes in aminothiol levels have bee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D277/32C09K11/06G01N21/64
Inventor 王毅朱美庆蒋而康那日松刘佳花日茂吴祥为范世锁王振滕婧
Owner ANHUI AGRICULTURAL UNIVERSITY
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