Long wavelength emitting fluorescence probe for specifically detecting cysteine in living cells and preparation method and application thereof

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

Active Publication Date: 2019-12-13
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cysteine, homocysteine ​​and glutathione have similar structure and reactivity properties, they are present in almost all mammalian cells, therefore, cysteine, homocysteine ​​and glutathione Selective discrimination of glycides remains an important challenge in the field

Method used

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  • Long wavelength emitting fluorescence probe for specifically detecting cysteine in living cells and preparation method and application thereof
  • Long wavelength emitting fluorescence probe for specifically detecting cysteine in living cells and preparation method and application thereof
  • Long wavelength emitting fluorescence probe for specifically detecting cysteine in living cells and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preparation of compound 1: N-diethylaminophenol (1.8 g, 10.9 mmol) and phthalic anhydride (1.8 g, 12.2 mmol) were reacted in 200 mL of benzene at 130 °C for 12 hours to obtain White solid, then white solid (0.626 g, 2 mmol) was added to 10 mL of methanesulfonic acid containing p-hydroxyacetophenone (0.272 g, 2 mmol), reacted at 90 ° C for 8 hours, cooled and added to 200 mL of ice Water, add 10 mL perchloric acid, extract with dichloromethane (50 mL × 3), dry and rotary evaporate to dryness, use dichloromethane and glacial acetic acid mixed solvent (volume ratio: 10:1) to separate by column chromatography Compound 1 was obtained with a yield of 33%. Synthetic route such as figure 1 shown.

Embodiment 2

[0034] Example 2: Preparation of fluorescent probes for detecting biothiols in water-soluble environments according to the present invention: Compound 1 (600 mg, 1.45 mmol) was mixed with acryloyl chloride (468 μL, 5.8 mmol), triethylamine (804 μL, 5.8 mmol) in 30 mL redistilled dichloromethane, stirred in an ice bath for 1 hour, then returned to room temperature and stirred for 16 hours. Diluted with 30 mL of dichloromethane, washed with water (50 mL × 3), dried and spun to dryness, and separated by column chromatography with a mixed solvent of dichloromethane and methanol (volume ratio 30:1) to obtain the target compound PA-A , Yield: 50%.

Embodiment 3

[0035] Example 3: The titration experiment of pH=7.4 fluorescent probe and cysteine: In the PBS buffer solution with pH=7.4, add the fluorescent probe with an initial concentration of 2 mM, so that the concentration of the fluorescent probe in the solution is 10 μM. Then, different amounts of biothiols with an initial concentration of 20 mM were added sequentially, so that the concentrations of biothiols in the solution were 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM, 20 μM, 24 μM, 28 μM, 32 μM, 36 μM, 40 μM, 45 μM, 50 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM, 200 μM, 250 μM, 300 μM, 500 μM, without adding cysteine ​​as a control, let stand for 0.5 hours to fully react cysteine ​​and fluorescent probe. The absorption and fluorescence spectra of cysteine ​​under different concentrations were measured by absorption spectrometer and fluorescence spectrometer respectively. The excitation wavelength of the fluorescence spectrum was 570 nm, the emission...

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Abstract

The invention belongs to the technical field of fluorescence probes, and provides a long wavelength emitting fluorescence probe for specifically detecting cysteine in living cells and a preparation method and an application thereof. The molecular formula of the fluorescence probe is C29H26NO5+, and the name of the probe compound is 2-(4-(acryloyloxy)phenyl)-4-(2-carboxyphenyl)-7-(diethylamino)benzopyran, abbreviated as PA-A. A fluorescent probe for specifically recognizing cysteine, is constructed with anthocyanin derivatives as fluorophore and acrylate as recognition unit. Michael addition-pyrolysis reaction of cysteine and probe is used for detecting with high selectivity. The invention belongs to the technical field of organic small molecule fluorescent probes, the probe has obvious color change and good water solubility, and can efficiently identify cysteine in water solubility environment, organic environment and cell environment. The method of the invention has the advantages ofsimple operation, high sensitivity, good selectivity and stable properties, and can be stored and used for a long time.

Description

technical field [0001] The invention belongs to the technical field of fluorescent probes, in particular to a long-wavelength emission fluorescent probe for specifically detecting cysteine ​​in living cells and its preparation method and application, capable of highly selective and sensitive recognition of cysteine , responds in aqueous, organic and cellular environments. Background technique [0002] In recent years, amino acids containing sulfhydryl groups have attracted much attention because of their crucial roles in regulating physiological and pathological processes, including cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH). Among them, cysteine ​​is involved in protein synthesis, detoxification and metabolism, and is an important biothiol, which is related to many diseases. For example, a lack of cysteine ​​can lead to slow growth, edema, lethargy, liver damage, muscle and fat damage, and skin lesions in children. Excess cysteine ​​is also an important...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D311/60C09K11/06G01N21/64
CPCC07D311/60C09K11/06G01N21/6428C09K2211/1088
Inventor 李亚平乔柳琪蔡建华吕鑫
Owner SHANXI UNIV
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