阅读说明：本技术 一种特异性检测活细胞中的半胱氨酸的长波长发射荧光探针及其制备方法和应用 (Long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells and preparation method and application thereof ) 是由 李亚平 乔柳琪 蔡建华 吕鑫 于 2019-08-27 设计创作，主要内容包括：本发明属于荧光探针技术领域,提供了一种特异性检测活细胞中的半胱氨酸的长波长发射荧光探针及其制备方法和应用,荧光探针分子式为C<Sub>29</Sub>H<Sub>26</Sub>NO<Sub>5</Sub><Sup>+</Sup>,该探针化合物名称为2-(4-(丙烯酰氧基)苯基)-4-(2-羧基苯基)-7-(二乙基氨基)苯并吡喃,简称PA-A,以花色素衍生物为荧光团,以丙烯酸酯作为识别单元构建的特异性识别半胱氨酸的荧光探针。利用半胱氨酸与探针的迈克尔加成-裂解反应进行高选择性检测。本发明属有机小分子荧光探针技术领域,探针颜色变化明显,水溶性好,能在水溶性环境、有机环境以及细胞环境中高效识别半胱氨酸。本发明方法操作简单、灵敏度高、选择性好,性质稳定,可以长期保存使用。(The invention belongs to the technical field of fluorescent probes, and provides a long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells, and a preparation method and application thereof, wherein the molecular formula of the fluorescent probe is C 29 H 26 NO 5 + 3 the 3 probe 3 compound 3 is 3 named 3 as 32 3- 3 ( 3 4 3- 3 ( 3 acryloyloxy 3) 3 phenyl 3) 3- 3 4 3- 3 ( 32 3- 3 carboxyphenyl 3) 3- 3 7 3- 3 ( 3 diethylamino 3) 3 benzopyran 3, 3 PA 3- 3 A 3 for 3 short 3, 3 and 3 the 3 fluorescent 3 probe 3 which 3 is 3 constructed 3 by 3 taking 3 an 3 anthocyanidin 3 derivative 3 as 3 a 3 fluorophore 3 and 3 taking 3 acrylate 3 as 3 a 3 recognition 3 unit 3 and 3 can 3 specifically 3 recognize 3 cysteine 3. 3 The Michael addition-cleavage reaction of cysteine and a probe is utilized for high-selectivity detection. The invention belongs to the technical field of organic small molecule fluorescent probes, and the probe has obvious color change, good water solubility and can be arranged in a water-soluble ringEfficient cysteine recognition in environmental, organic and cellular environments. The method has the advantages of simple operation, high sensitivity, good selectivity and stable property, and can be stored and used for a long time.)

1. a long wavelength emitting fluorescent probe for specifically detecting cysteine in a living cell, comprising: the molecular formula of the fluorescent probe is C₂₉H₂₆NO₅ ⁺ 3 the 3 name 3 of 3 the 3 probe 3 compound 3 is 32 3- 3 ( 3 4 3- 3 ( 3 acryloyloxy 3) 3 phenyl 3) 3- 3 4 3- 3 ( 32 3- 3 carboxyphenyl 3) 3- 3 7 3- 3 ( 3 diethylamino 3) 3 benzopyran 3, 3 PA 3- 3 A 3 for 3 short 3, 3 and 3 the 3 structural 3 formula 3 of 3 the 3 fluorescent 3 probe 3 is 3 as 3 follows 3: 3。

2. A method for preparing a long wavelength-emitting fluorescent probe for specifically detecting cysteine in a living cell according to claim 1, characterized in that: the method comprises the following steps:

(1) dissolving m-N-diethylaminophenol and phthalic anhydride in benzene, and reacting at 130 ℃ for 12 h to obtain a white solid;

(2) Adding the white solid obtained in the step (1) and p-hydroxyacetophenone into a methanesulfonic acid solution, reacting for 8 h at 90 ℃, treating the obtained solid with ice water and perchloric acid, extracting with dichloromethane, and finally performing column chromatography separation to obtain a compound 1, wherein the structural formula is as follows:；

(3) 3 reacting 3 the 3 compound 3 1 3 with 3 acryloyl 3 chloride 3 in 3 alkaline 3 environment 3 triethylamine 3 for 3 16 3 h 3 at 3 normal 3 temperature 3, 3 and 3 separating 3 and 3 purifying 3 to 3 obtain 3 the 3 fluorescent 3 probe 3 compound 3 PA 3- 3 A 3. 3

3. The method of preparing a long wavelength-emitting fluorescent probe specifically detecting cysteine in a living cell according to claim 2, characterized in that: the molar ratio of the m-N-diethylaminophenol to the phthalic anhydride in the step (1) is 1: 1; every millimole of m-diethylaminoaminophenol was dissolved in 10-70 mL of benzene.

4. The method of preparing a long wavelength-emitting fluorescent probe specifically detecting cysteine in a living cell according to claim 2, characterized in that: the molar ratio of the white solid to the p-hydroxyacetophenone in the step (2) is 1: 1-1.5; the dosage of the methanesulfonic acid is 5-10 mL per millimole of p-hydroxyacetophenone; the dosage of the ice water is 50-100mL of ice water for each millimole of p-hydroxyacetophenone; the perchloric acid is used in an amount of 3 to 5 mL per millimole of p-hydroxyacetophenone.

5. The method of preparing a long wavelength-emitting fluorescent probe specifically detecting cysteine in a living cell according to claim 2, characterized in that: in the step (3), the molar ratio of the compound 1 to the acryloyl chloride is 1: 1-4; the molar ratio of triethylamine to the compound 1 in the alkaline environment is 1: 1-4.

6. The method of preparing a long wavelength-emitting fluorescent probe specifically detecting cysteine in a living cell according to claim 2, characterized in that: the specific method for separating and purifying in the step (3) comprises the following steps: and (3) diluting the solution after the reaction is finished with dichloromethane, washing with water for three times, drying, performing rotary evaporation to remove the solvent, dissolving the solid with dichloromethane, and performing column chromatography separation by using a mixed solvent of dichloromethane and methanol to obtain the fluorescent probe compound.

7. Use of a long wavelength emitting fluorescent probe for the specific detection of cysteine in living cells according to claim 1, characterized in that: the fluorescent probe is applied to detection of cysteine in a water-soluble environment, an organic environment or a cell tissue environment.

Technical Field

The invention belongs to the technical field of fluorescent probes, and particularly relates to a long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells, a preparation method and application thereof, which can sensitively identify high selectivity of cysteine and respond to water solubility, organic environment and cell environment.

Background

In recent years, thiol-containing amino acids have attracted much attention because they play a crucial role in the regulation of physiological and pathological processes, including cysteine (Cys), homocysteine (Hcy) and Glutathione (GSH). Among them, cysteine is involved in protein synthesis, detoxification and metabolism, is an important biological thiol, and is involved in many diseases. For example, cysteine deficiency can lead to slow growth, edema, lethargy, liver damage, muscle and fat damage, skin lesions in children. Excess cysteine is also a significant cause of cardiovascular disease and alzheimer's disease.

Electrochemical analysis, mass spectrometry, and High Performance Liquid Chromatography (HPLC) can be used to detect cysteine. However, these methods are complicated to operate and require expensive equipment. Compared with the prior art, the fluorescence detection method has the advantages of simple operation, high sensitivity, good selectivity and real-time monitoring. In addition, the fluorescent probe can image cysteine in living cells by a non-invasive confocal method, and provides possibility for further application of the fluorescent probe in the future. However, cysteine, homocysteine and glutathione have similar structural and response properties and are present in almost all mammalian cells, and therefore, selective differentiation of cysteine, homocysteine and glutathione remains a significant challenge in this field.

Currently, many small molecule fluorescent probes for selective detection of cysteine have been based on specific chemical reactions, such as michael addition, cyclization with aldehydes, disulfide exchange and cleavage reactions, and the like. In 2011, Strongin et al constructed benzothiazole derivatives for the first time using acryloyl as a reactive site for specific recognition of cysteine. Since then, fluorescent probes based on this design concept have been widely developed. After reaction of the acryloyl group with cysteine, the acrylate unit of the probe is cleaved by cysteine to release the fluorescent signal unit, and highly selective detection of cysteine is achieved by fluorescent signal enhancement.

Disclosure of Invention

The invention provides a long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells, a preparation method and application thereof, a novel fluorescent dye is synthesized, the cysteine can be specifically identified by the novel probe generated after the novel fluorescent dye is combined with an acryloyl group at an identification site, and the novel probe is simple and convenient in synthesis method, strong in operability, good in selectivity, high in sensitivity and capable of being identified by naked eyes.

The invention is realized by the following technical scheme: a long wavelength emission fluorescent probe for specifically detecting cysteine in living cells, the molecular formula of the fluorescent probe is C₂₉H₂₆NO₅ ⁺ 3 the 3 name 3 of 3 the 3 probe 3 compound 3 is 32 3- 3 ( 3 4 3- 3 ( 3 acryloyloxy 3) 3 phenyl 3) 3- 3 4 3- 3 ( 32 3- 3 carboxyphenyl 3) 3- 3 7 3- 3 ( 3 diethylamino 3) 3 benzopyran 3, 3 PA 3- 3 A 3 for 3 short 3, 3 and 3 the 3 structural 3 formula 3 of 3 the 3 fluorescent 3 probe 3 is 3 as 3 follows 3: 3。

The method for preparing the long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells comprises the following steps:

(1) Dissolving m-N-diethylaminophenol and phthalic anhydride in benzene, and reacting at 130 ℃ for 12 h to obtain a white solid;

(2) Adding the white solid obtained in the step (1) and p-hydroxyacetophenone into a methanesulfonic acid solution, reacting for 8 h at 90 ℃, treating the obtained solid with ice water and perchloric acid, extracting with dichloromethane, and finally performing column chromatography separation to obtain a compound 1, wherein the structural formula is as follows:；

(3) 3 reacting 3 the 3 compound 3 1 3 with 3 acryloyl 3 chloride 3 in 3 alkaline 3 environment 3 triethylamine 3 for 3 16 3 h 3 at 3 normal 3 temperature 3, 3 and 3 separating 3 and 3 purifying 3 to 3 obtain 3 the 3 fluorescent 3 probe 3 compound 3 PA 3- 3 A 3. 3

The molar ratio of the m-N-diethylaminophenol to the phthalic anhydride in the step (1) is 1: 1; every millimole of m-diethylaminoaminophenol was dissolved in 10-70 mL of benzene.

The molar ratio of the white solid to the p-hydroxyacetophenone in the step (2) is 1: 1-1.5; the dosage of the methanesulfonic acid is 5-10 mL per millimole of p-hydroxyacetophenone; the dosage of the ice water is 50-100mL of ice water for each millimole of p-hydroxyacetophenone; the perchloric acid is used in an amount of 3 to 5 mL per millimole of p-hydroxyacetophenone.

In the step (3), the molar ratio of the compound 1 to the acryloyl chloride is 1: 1-4; the molar ratio of triethylamine to the compound 1 in the alkaline environment is 1: 1-4.

the specific method for separating and purifying in the step (3) comprises the following steps: and (3) diluting the solution after the reaction is finished with dichloromethane, washing with water for three times, drying, performing rotary evaporation to remove the solvent, dissolving the solid with dichloromethane, and performing column chromatography separation by using a mixed solvent of dichloromethane and methanol to obtain the fluorescent probe compound.

Use of a long wavelength emitting fluorescent probe for the specific detection of cysteine in living cells for the detection of cysteine in a water soluble environment, an organic environment or a cellular tissue environment.

After the fluorescent probe reacts with cysteine, the color of the solution is changed from light pink to light purple, and the color change is obvious.

The invention also provides a preparation method of the cysteine fluorescent probe, which is synthesized by stirring the corresponding anthocyanidin derivative corresponding to the cysteine fluorescent probe and acryloyl chloride in a dichloromethane solution for 16 h at room temperature.

the synthetic route of the cysteine fluorescent probe is shown in figure 1.

The fluorescent probe can act with cysteine to generate the change of a fluorescence spectrum, thereby realizing the quantitative detection of the cysteine.

The cysteine fluorescent probe disclosed by the invention respectively acts with homocysteine, glutathione, arginine, aspartic acid, histidine, lysine, glutamic acid, tryptophan, threonine, serine, alanine, tyrosine, sulfide ion, hypochlorite ion, hydrosulfite, sulfite ion, chloride ion, carbonate ion, bromide ion, acetate ion, thiosulfate radical, sulfate radical, magnesium ion, lead ion, copper ion, barium ion, manganese ion, zinc ion and other substances, so that the fluorescence spectrum of the probe cannot be obviously changed; after cysteine was added to these solutions, the fluorescence spectra of the solutions changed significantly and closely to the changes produced by the direct addition of cysteine, indicating that the reactants in the experiment had no effect on the detection of cysteine.

The reaction mechanism of the fluorescent probe is clarified through the nuclear magnetic hydrogen spectrum titration reaction, and the double bond peak disappears gradually after the reaction of the acrylate of the probe and the cysteine, thereby proving that the reaction does occur.

the fluorescent probes of the present invention successfully imaged cysteine in living cells.

Drawings

FIG. 1 is a schematic diagram of the synthesis of the fluorescent probe for detecting biological thiols in an aqueous environment according to the invention;

FIG. 2 is the change of fluorescence intensity of the fluorescent probe under the condition of different concentrations of cysteine in the environment of PBS buffer solution with pH =7.4 in example 3; wherein the lowest curve is a fluorescence intensity curve when no biological thiol is added, the concentrations of the biological thiols in the curves sequentially increase from bottom to top, and the fluorescence curve of the biological thiol is when the concentration of the uppermost curve is 500 mu M (50 equivalent);

FIG. 3 is a fluorescence curve of fluorescence intensity of the fluorescent probe at 575 nm with respect to the ratio of bio-thiol to probe at pH =7.4 in example 3 of the present invention, and it can be seen that the saturation equivalent of cysteine reacted with the fluorescent probe is 15 equivalents (150. mu.M);

FIG. 4 is a linear relationship of cysteine in a concentration range of 0 to 36 μ M when reacted with a fluorescent probe at pH =7.4 in example 3 of the present invention;

FIG. 5 is a graph showing the change of fluorescence intensity with time of the reaction between the fluorescent probe and cysteine at pH =7.4 in example 3 of the present invention, and it can be seen that the optimal reaction time of the fluorescent probe and cysteine is 30 min;

FIG. 6 shows the change of fluorescence intensity at 575 nm between a fluorescent probe and cysteine at pH = 2-12 in example 3 of the present invention;

FIG. 7 shows the reaction of fluorescent probes with different equivalents of cysteine, homocysteine and glutathione;

FIGS. 8(a), 8(b) are graphs comparing the change in fluorescence intensity of fluorescent probes after the addition of different amino acids and ions, respectively, under the same conditions in example 5 of the present invention;

FIG. 9 is a nuclear magnetic titration reaction hydrogen spectrum of the probe in the deuterated reagent and cysteine in example 6 of the present invention, wherein the double bond hydrogen disappears after the reaction, which indicates that the probe actually reacts with cysteine;

FIG. 10 is an image of cysteine in cells imaged by the probe of example 7 of the present invention, which is a confocal image of the red channel, white channel and overlay when the probe is added, the thiol depleting reagent maleimide (NEM) and the probe are added, and the NEM and the probe are added and cysteine is added, wherein: a is an image added with the probe, B is an image added with the NEM and the probe, C is an image when the NEM, the probe and the added cysteine are added, 1 is a red light confocal image, 2 is a white light channel confocal image, and 3 is a confocal image when the NEM, the probe and the added cysteine are superposed.

Detailed Description