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Synthesis and application of fluorescent reagent for selectively detecting cysteine based on aggregation-induced emission principle

A cysteine ​​and fluorescent reagent technology, applied in the fields of luminescent materials, fluorescence/phosphorescence, nitro compound preparation, etc., can solve the problems of low detection signal-to-noise ratio, fluorescence quenching, limiting the practical application of Cys fluorescent probes, etc. To achieve the effect of simple synthesis steps, high selectivity, and overcoming the easy bleaching of dye molecules

Active Publication Date: 2017-06-30
GANNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the structures of Cys, Hcy, and GSH are very similar, and the reactivity of the sulfhydryl group contained is equivalent, there are still relatively few fluorescent probes that can specifically distinguish Cys from Hcy, and GSH, and these few fluorescent probes Most of the fluorescent molecules used in the probe are prone to fluorescence quenching at high concentrations, that is, aggregation caused quenching (ACQ).
This phenomenon forces researchers to use only dilute solutions during the detection process, leading to low detection signal-to-noise ratios and limiting the practical application of these Cys fluorescent probes.

Method used

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  • Synthesis and application of fluorescent reagent for selectively detecting cysteine based on aggregation-induced emission principle
  • Synthesis and application of fluorescent reagent for selectively detecting cysteine based on aggregation-induced emission principle
  • Synthesis and application of fluorescent reagent for selectively detecting cysteine based on aggregation-induced emission principle

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Experimental program
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Effect test

Embodiment 1

[0024] Target compound TPENNO 2 Synthesis

[0025]

[0026] (1) Synthesis of Compound 1: Add benzophenone (1.8g, 10mmol) and zinc powder (5.2g, 80mmol) to tetrahydrofuran, cool to -20°C for half an hour while stirring, and then slowly drop in tetrachloride Titanium (4.4mL, 40mmol), continue to stir at -20°C for half an hour, then slowly rise to room temperature, then heat to reflux overnight, cool, filter, concentrate and separate and purify by column chromatography to obtain a compound with aggregation-induced luminescent properties 1. The yield is 72%.

[0027] (2) Synthesis of Compound 2: Compound 1 (2.64g, 8mmol), acetic acid (1.9mL, 32mmol) were added to dichloromethane, and after cooling to -20°C, concentrated nitric acid (65%, 1.6mL, 24mmol ) was added to the reaction liquid, and after stirring at -20°C for 15 min, cold water was added for liquid extraction, the organic phase was washed three times with water, dried and concentrated, and the residue was recrystalli...

Embodiment 2

[0031] Cys selective recognition detection

[0032] (1) Determination of Cys response time: take 50 μL compound TPENNO 2 DMSO solution (1mM) in 2mL PBS buffer solution, adding 200μM Cys, the mixture was incubated at 37°C for different times, and the fluorescence intensity of the mixture solution was measured as a function of the incubation time. The results showed that after 30min, the fluorescence intensity basically reached saturation.

[0033] (2) Cys fluorescence titration test: take 50 μL compound TPENNO 2 DMSO solution (1mM) in 2mL PBS buffer solution, and then add different amounts of Cys in PBS solution, after the mixed solution was incubated at 37°C for 30min, the fluorescence emission spectrum of the solution after adding different Cys was measured (E x =341nm), the fluorescence intensity of the mixed solution showed a good linear relationship in the concentration range of 60 to 100μM Cys (such as figure 1 shown).

[0034] (3) Compound TPENNO 2 Selectivity test ...

Embodiment 3

[0036] Fluorescence imaging of sulfhydryl compounds in human breast cancer (HeLa) cells: HeLa cells were inoculated in DEME medium containing 10% fetal bovine serum after resuscitation, at 37°C, 5% CO 2 , 100% saturated humidity incubator. Then cultured on 18mm coverslip for 24h, ready to use.

[0037] HeLa cells were respectively immersed in 4 μM compound TPENNO 2 culture medium at 37°C, 5% CO 2 1. After incubating in an incubator with 100% saturated humidity for 20 minutes, pour out the medium, and wash the cells 3 times with fresh medium. Observed under a laser confocal fluorescence microscope, and photographed in bright field and dark field (such as image 3 shown). The results showed that the target compound TPENNO 2 Green fluorescent signal appears in HeLa cells, and under the same conditions, the medium containing 0.5mM Cys was first added to the HeLa cell culture dish and incubated for 24h, and then the target compound TPENNO was added 2 4μM medium was incubated...

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Abstract

The invention discloses a fluorescent reagent for selectively detecting cysteine based on an aggregation-induced emission principle, a synthesis method and application thereof. Benzophenone is used as an initial raw material for synthesizing a target compound TPENNO2. The detection research of the target compound TPENNO2 to sulfydryl-contained amino acids (such as the cysteine, homocysteine and glutathione) is developed, and the target compound TPENNO2 is discovered to have the good sensitivity and the high selectivity to the cysteine, and the interference to the homocysteine and the glutathione is small. Compared with the prior art, the fluorescent reagent has the advantages of simple synthesis, easily obtained raw materials, high target compound fluorescence quantum yield, and strong photobleaching resistant capacity. The defect of traditional fluorochrome that cannot be detected in the high concentration is avoided, and the target compound TPENNO2 is successfully used for the fluorescence imaging of a sulfhydryl compound in cells. The target compound TPENNO2 has the large application prospect in the aspects of selectively detecting the cysteine and detecting the content of the sulfhydryl compound in the cells.

Description

technical field [0001] The invention belongs to the field of biochemical materials, and relates to a fluorescent reagent for selective detection of cysteine ​​based on the principle of aggregation-induced luminescence, a synthesis method and an application. Background technique [0002] Cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH) are the three most common thiol amino acids in the human body and are the main active substances in cells. Among them, Cys is the only sulfhydryl amino acid among the 20 basic amino acids that constitute proteins, and has important physiological functions. The sulfhydryl groups in the two Cys residues can maintain the three-dimensional structure of the protein by forming disulfide bonds. The redox properties of sulfhydryl groups in Cys directly determine the biological activity of proteins and oligopeptides, making them have anti-aging properties, can remove free radicals in organisms, and play a vital role in the conversion and ab...

Claims

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Application Information

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IPC IPC(8): C07C303/38C07C311/21C09K11/06G01N21/64
CPCC07C1/2074C07C201/08C07C209/36C07C303/38C07C311/21C09K11/06C09K2211/1014G01N21/6428G01N2021/6443C07C211/45C07C205/06C07C15/52
Inventor 王建国姜国玉朱温平吴勇权范小林
Owner GANNAN NORMAL UNIV
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