阅读说明：本技术 基于苯硒醚基团专一响应Cys的荧光探针、制备方法及应用 (Fluorescent probe based on phenylselenol group specific response Cys, preparation method and application ) 是由 张健 赵伟利 陶远芳 王楠楠 王瀚 岳金磊 苏慧慧 于 2020-06-17 设计创作，主要内容包括：本发明提供了一种基于苯硒醚基团专一响应Cys的荧光探针、制备方法及应用。本探针选择BODIPY染料为荧光团,苯硒醚为识别团。检测机制是Cys同苯硒醚发生亲核取代-重排反应,引起荧光信号变化。上述探针通过紫外和荧光光谱仪检测Cys且不受其他氨基酸和活性氧类的干扰,该检测过程简便、快速、灵敏,检测限为33 nM。更重要的是该探针可以检测细胞中Cys,在生物监测领域具有良好应用前景。(The invention provides a phenylselenol group-based specific response Cys fluorescent probe, a preparation method and application. The probe selects BODIPY dye as a fluorophore and phenylselene as a recognition group. The detection mechanism is that Cys and phenylselenol undergo nucleophilic substitution-rearrangement reaction to cause fluorescence signal change. The probe detects Cys through an ultraviolet spectrometer and a fluorescence spectrometer without interference of other amino acids and active oxygen, the detection process is simple, convenient, rapid and sensitive, and the detection limit is 33 nM. More importantly, the probe can detect Cys in cells and has good application prospect in the field of biological monitoring.)

1. A fluorescent probe specifically responding to Cys based on a phenylselenol group is characterized in that the structural formula of the probe is as follows:

2. the method for preparing the phenylselenol group-specific response Cys-based fluorescent probe according to claim 1, which comprises the following steps:

（1）N₂under the protection of ice water bath, dissolving acetone-based p-toluenesulfonate oxime ester and p-methoxystyryl methyl ketone in a mixed solvent, reacting for 4 hours at 50 ℃, quenching with ice water, extracting with dichloromethane, and separating by column chromatography to obtain a compound 1;

(2) dissolving the compound 1 prepared in the step (1) in tetrahydrofuran, adding N, N-diisopropylethylamine and triphosgene or thiophosgene, stirring at room temperature, then removing the solvent under reduced pressure, and performing column chromatography separation to obtain a compound 2;

(3) dissolving the compound 2 obtained in the step (2) in dichloromethane, adding phosphorus oxychloride for reacting overnight, adding alkali and boron trifluoride diethyl etherate for complexing, and performing column chromatography separation to obtain a compound 3;

(4) dissolving diphenyl diselenide and lithium aluminum hydride in ethanol for reduction, adding a compound 3 for reaction at room temperature, then quenching, decompressing and removing the solvent, and carrying out column chromatography separation on the residue to obtain the probe BDP-Se-3.

3. The method for preparing the phenylselenol group-specific response Cys-based fluorescent probe according to claim 1, wherein the structural formula of the compound 1 in the step (1) is as follows:。

4. the method for preparing the phenylselenol group-specific response Cys-based fluorescent probe according to claim 1, wherein the structural formula of the compound 2 in the step (2) is as follows:

5. the method for preparing the phenylselenol group-specific response Cys-based fluorescent probe according to claim 1, wherein the structural formula of the compound 3 in the step (3) is as follows:。

6. the method for preparing the phenylselenol group-specific response Cys-based fluorescent probe according to claim 1, wherein the fluorescent probe comprises: the mixed solvent in the step (1) is obtained by adding sodium hydride into a tetrahydrofuran solvent and mixing, wherein the acetone-based p-toluenesulfonate oxime ester and the p-methoxystyryl methyl ketone have an equivalent weight of 1: the ratio of (2-3) is dissolved in the mixed solvent.

7. The method for preparing the phenylselenol group-specific response Cys-based fluorescent probe according to claim 1, wherein the fluorescent probe comprises: in the step (2), N-diisopropylethylamine and triphosgene or thiophosgene are added according to the ratio of equivalent weight of 1: (1-1.5) and stirring at room temperature for 1-1.5 h.

8. The method for preparing the phenylselenol group-specific response Cys-based fluorescent probe according to claim 1, wherein the fluorescent probe comprises: the alkali in the step (3) is N, N-diisopropylethylamine or triethylamine, and the ratio of the alkali to boron trifluoride ethyl ether is 6: (8-10.5) adding, and complexing for 4-6h at room temperature.

9. The method for preparing the phenylselenol group-specific response Cys-based fluorescent probe according to claim 1, wherein the fluorescent probe comprises: in the step (4), the diphenyl diselenide and the lithium aluminum hydride are mixed and dissolved according to the proportion of 1:2, 2-2.5 equivalents of compound 3 are added, and the reaction is carried out for 1-1.5h at room temperature.

10. The use of a probe prepared by the preparation method according to any one of claims 2-9 for the specific detection of Cys.

Technical Field

The invention relates to a fluorescent probe, in particular to a fluorescent probe specifically responding to Cys based on a phenylselenol group, a preparation method and application.

Background

The biological sulfide plays an important role in aspects such as metabolism of animals, plants and microscopic life bodies, food processing and the like. Small molecule thiols are part of biogenic sulfides which include: cysteine (Cys), homocysteine (Hcy), and Glutathione (GSH). Abnormal concentrations of the three thiols can lead to a variety of diseases, such as liver damage, cardiovascular disease, alzheimer's disease, cancer, and the like. Cysteine (Cys) is a proteinogenic, semi-essential amino acid in animals that stabilizes protein structure by providing disulfide bonds in the folded protein, increases protein rigidity, and regulates protein function. The precursor for Cys synthesis is Hcy, methionine is converted to homocysteine, which is then combined with serine to form the asymmetric thioether cysteine Hcy combined with serine to form cystathionine under the action of cystathionine b-synthase (CBS), and then cleaved to Cys under the action of cystathionine g-lyase (CSE). It is also a precursor amino acid for the synthesis of GSH, taurine and acetyl-coa. In addition, Cys can be obtained from foods, and Cys is found in high protein foods, including meat, eggs, dairy products, and the like. And Cys has been assigned as a flavoring agent by the Joint experts committee for cereal and agriculture organization (JECFA), therefore, the specific detection of Cys is very important.

However, due to the structural similarity of thiols themselves, both have the same active site thiol and amine groups, especially Cys and Hcy differ only by one methylene group. Achieving three distinctions remains a significant challenge. The phenylselenol group is a group easy to leave, is easy to be attacked by a nucleophilic reagent and generates a substitution reaction. The reports of the strong nucleophilicity of sulfydryl by using the phenylselene as a detection group on active sulfides are very few, and particularly, a probe for specifically recognizing Cys has not been reported. Red fluorescent probes are well known for their long emission wavelength and strong tissue penetration. Therefore, the development of a red fluorescent probe based on the phenylselenol for detecting the group specificity response animal cell Cys is of great significance.

Disclosure of Invention

The invention provides a fluorescent probe based on phenylselenol group specific response Cys, a preparation method and application thereof.

The technical scheme for realizing the invention is as follows:

a fluorescent probe specifically responding to Cys based on a phenylselenol group has a structural formula as follows:

。

the preparation method of the fluorescent probe specifically responding to Cys based on the phenylselenol group comprises the following steps:

（1）N₂under the protection of ice water bath, adding sodium hydride into tetrahydrofuran solvent to be mixed to obtain mixed solvent, and mixing acetone-based p-toluenesulfonate oxime ester with p-methoxystyryl methyl ketone according to the equivalent weight of 1: dissolving the mixture in a ratio of 2-3 in a mixed solvent, reacting for 4 hours at 50 ℃, quenching with ice water, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 1;

the structural formula of compound 1 is as follows:；

(2) dissolving the compound 1 prepared in the step (1) in tetrahydrofuran, and mixing N, N-diisopropylethylamine and triphosgene or thiophosgene according to an equivalent weight of 1: (1-1.5), stirring for 1-1.5h at 25 ℃, then removing the solvent under reduced pressure, and performing column chromatography separation to obtain a compound 2;

the structural formula of compound 2 is as follows:

，

(3) dissolving the compound 2 obtained in the step (2) in dichloromethane, adding 1.5 equivalents of phosphorus oxychloride to react overnight, and then adding alkali and boron trifluoride diethyl etherate according to an equivalent ratio of 6: (8-10.5) adding the mixture into the system for complexing, complexing for 4-6 hours at 25 ℃, and performing column chromatography separation to obtain a compound 3;

the structural formula of compound 3 is as follows:

，

(4) diphenyl diselenide and lithium aluminum hydride in an equivalent weight of 1: dissolving the mixture 2 in ethanol for reduction, adding 2-2.5 equivalents of compound 3, reacting at room temperature for 1-1.5 hours, quenching, decompressing, removing the solvent, and performing column chromatography separation on residues to obtain the probe BDP-Se-3.

The probe prepared by the preparation method is applied to the specific detection of Cys.

The synthetic route of the fluorescent probe is as follows:

the invention is based on a fluorescent probe which takes phenylselenol as a response group and is used for specifically detecting Cys, and the probe can judge the reaction time and concentration dependency relationship between the probe and Cys by utilizing ultraviolet and fluorescence spectra in solution test; according to the test observation of selectivity and anti-interference capability, the probe can specifically detect Cys, does not react with active oxide and has strong anti-interference capability; and the probe has strong pH stability and small cytotoxicity. The purpose of Cys detection in HeLa cells can be achieved through a confocal fluorescence microscope.

The invention has the beneficial effects that:

(1) the fluorescent probe for specifically detecting Cys by using the phenylselenol as the response group has the advantages of simple synthetic method and convenient operation;

(2) the detection method can realize Cys specific detection, and is not interfered by other amino acids and active oxides;

(3) the invention has obvious detection signals and is a near-infrared-like fluorescence enhanced fluorescent probe.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a nuclear magnetic hydrogen spectrum of a fluorescent probe BDP-Se-3;

FIG. 2 is a nuclear magnetic carbon spectrum of a fluorescent probe BDP-Se-3;

FIG. 3 shows the time-dependent UV change of the interaction of fluorescent probe BDP-Se-3 with Cys;

FIG. 4 shows the time fluorescence change of the interaction of fluorescent probe BDP-Se-3 with Cys;

FIG. 5 shows the titration experiment fluorescence change for Cys concentration measured by fluorescent probe BDP-Se-3;

FIG. 6 is a linear fit of the maximum fluorescence emission wavelength 610nm to Cys concentration;

FIG. 7 shows the fluorescence selectivity of common amino acids for detecting Cys by probe BDP-Se-3;

FIG. 8 shows the fluorescence interference of common amino acids on probe BDP-Se-3 to detect Cys;

FIG. 9 is a graph showing the maximum fluorescence intensity changes of the fluorescent probe BDP-Se-3 and the probe plus Cys in buffer solutions with different pH values;

FIG. 10 is a probe BDP-Se-3 detecting Cys cytotoxicity;

FIG. 11 is an image of Cys HeLa cell detected by fluorescent probe BDP-Se-3.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.