Fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate as well as preparation method, use method and application of fluorescent probe

Abstract

The invention provides a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate as well as a preparation method, a use method and application of the fluorescent probe, and relates to the field of analytical chemistry. The structural formula of the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate is # imgabs0. The fluorescent probe provided by the invention is good in selectivity, high in sensitivity and strong in anti-interference performance, can be used for directly and simultaneously quantifying Cys, Hcy, GSH and ATP in an environment, and can be used for simultaneously detecting tumor markers such as cysteine, homocysteine, glutathione and adenosine triphosphate. Meanwhile, Cys, Hcy, GSH and ATP in biological systems such as fluorescence imaging tumor cells are distinguished.

Description

Technical Field

[0001] The present application relates to the field of analytical chemistry, and in particular to a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, and a preparation method, use method and application thereof. Background Art

[0002] Cysteine ​​(Cys), homocysteine ​​(Hcy) and reduced glutathione (GSH) are common biothiols in organisms and play important roles in various physiological and pathological processes. Among them, Cys is the precursor of GSH and is involved in many aspects of protein synthesis, detoxification and metabolism. Hcy has been identified as an independent risk factor for many diseases. A slight increase in Hcy (more than 15 μM) can lead to severe hyperhomocysteinemia. As the most abundant intracellular thiol, GSH plays a central role in combating oxidative stress and maintaining redox homeostasis. Abnormal levels of these three biosulfurs are associated with various diseases, such as cancer, AIDS, cardiovascular disease, liver damage and neurodegenerative diseases (Anal. Chim. Acta., 2019, 1074, 123–130). Adenosine triphosphate (ATP) is considered to be the "currency molecular unit" of energy transfer within living cells; it transports chemical energy within cells for metabolism. Basic activities of life such as protein transport activities, regulation of ion channels and activation of signal cascades involve the production or consumption of ATP. Cells, tissues, and organ systems maintain a constant cellular ATP concentration in a wide range of uses, which is called ATP homeostasis. For example, a lack of ATP levels may represent disease states such as ischemia, cancer, Parkinson's disease, and hypoglycemia (Chem. Commun., 2012, 48, 3206–3208). The above four substances Cys, Hcy, GSH, and ATP have very complex roles, levels, localization, distribution, and biological functions in tumor cells. Therefore, the simultaneous detection of Cys, Hcy, GSH, and ATP is of great significance in the pathological analysis and early diagnosis of tumors.

[0003] In recent years, fluorescent probes have attracted extensive attention due to their good selectivity, high sensitivity, simple operation, and ability to monitor and visualize biological molecules in living cells. So far, some fluorescent probes have been reported for the selective detection of Cys / Hcy / GSH / ATP, and some probes have been reported for the simultaneous detection of two of the three, but no fluorescent probe has achieved the simultaneous differential detection of Cys, Hcy, GSH, and ATP, which cannot meet the application requirements of simultaneous differential detection and imaging of endogenous Cys, Hcy, GSH, and ATP in biological systems such as tumor cells. Summary of the invention

[0004] The purpose of the present application is to provide a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, and its preparation method, use method and application, so as to solve the above problems.

[0005] To achieve the above objectives, this application adopts the following technical solutions:

[0006] A fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, the structural formula of which is:

[0007]

[0008] The present application also provides a method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, comprising:

[0009] 4-((4-(butylthio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and 2-(benzo[d]thiazol-2-yl)acetonitrile are added to anhydrous ethanol to carry out a first reaction. After the reaction is complete, the reaction solution is concentrated and purified by column chromatography to obtain a red solid product;

[0010] Under protective atmosphere conditions, (E)-4-((3-(2-(benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and 4-dimethylaminopyridine are added to anhydrous dichloromethane to carry out a second reaction, and then 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthine]-3-one and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are added to carry out a third reaction. After the reaction is complete, water and dichloromethane are added to the reaction solution, the liquids are separated, the organic layer is concentrated, and the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate is obtained by column chromatography.

[0011] Preferably, the temperature of the first reaction is 50-90°C and the time is 12-24h.

[0012] Preferably, the temperature of the second reaction is room temperature, and the reaction time is 10-30 min.

[0013] Preferably, the temperature of the third reaction is room temperature, and the reaction time is 4-12 hours.

[0014] Preferably, the first reaction, the second reaction and the third reaction are all carried out under stirring.

[0015] Preferably, the protective atmosphere comprises argon.

[0016] The present application also provides a method for using the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, comprising:

[0017] Dissolving the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate in a mixture of an organic phase and an aqueous phase, and performing detection under specific wavelength conditions after the reaction;

[0018] Among them, after reacting with cysteine, it emits 455nm blue fluorescence at an excitation wavelength of 375nm; after reacting with homocysteine, it emits 555nm red fluorescence at an excitation wavelength of 493nm; after reacting with glutathione, it emits 529nm green fluorescence at an excitation wavelength of 455nm; after reacting with adenosine triphosphate, it emits 587nm pink fluorescence at an excitation wavelength of 520nm.

[0019] Preferably, when detecting cysteine, homocysteine, and glutathione, the organic phase is dimethyl sulfoxide, the aqueous phase is a phosphate buffer solution with a pH of 7.4 and an aqueous solution of the analyte;

[0020] When detecting adenosine triphosphate, the organic phase is methanol, and the aqueous phase is a phosphate buffer solution with a pH of 7.4 and an aqueous solution of the analyte.

[0021] The present application also provides an application of the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, which is used for quantitatively detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate.

[0022] Compared with the prior art, the beneficial effects of this application include:

[0023] The fluorescent probe provided by the present application for simultaneously distinguishing and detecting the tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate has multiple active reaction sites. Based on different reaction mechanisms, it can realize the simultaneous distinguishing and detecting of Cys (λex / λem=375 / 455nm), Hcy (λex / λem=493 / 555nm), GSH (λex / λem=455 / 529nm) and ATP (λex / λem=520 / 587nm). It has good selectivity, high sensitivity and strong anti-interference, and can be used to directly and simultaneously quantify Cys, Hcy, GSH and ATP in the environment, and simultaneously distinguish Cys, Hcy, GSH and ATP in biological systems such as fluorescent imaging tumor cells.

[0024] The present application provides a method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, and the process is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0026] Figure 1 It is the change of fluorescence emission intensity with Cys concentration in the quantitative detection of Cys by fluorescent probe;

[0027] Figure 2 It is the change of fluorescence emission intensity with Hcy concentration in the quantitative detection of Hcy by fluorescent probe;

[0028] Figure 3 It is the change of fluorescence emission intensity with GSH concentration in the quantitative detection of GSH by fluorescent probe;

[0029] Figure 4 It is the change of fluorescence emission intensity with ATP concentration in the quantitative detection of ATP by fluorescent probe;

[0030] Figure 5 This is an image of a fluorescent probe that simultaneously differentiates and images endogenous Cys, Hcy, GSH, and ATP in Hela tumor cells. DETAILED DESCRIPTION

[0031] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0032] Example 1

[0033] This embodiment provides a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, and the preparation method thereof is as follows:

[0034] 1. Synthesis of (E)-4-((3-(2-(Benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid:

[0035] 4-((4-(Butylthio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid (200.0 mg, 510.9 μmol) and 2-(benzo[d]thiazol-2-yl)acetonitrile (89.0 mg, 510.9 μmol) were added to 10 mL of anhydrous ethanol and stirred at 50 degrees Celsius for 24 hours. After the reaction was complete, the reaction solution was concentrated and purified by column chromatography to obtain 205.0 mg of a red solid product with a yield of 73.3%.

[0036] 2. Synthetic probe (E)-4-((3-(2-(Benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)-N-(2-((2',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthine]-2-yl)ethyl)amino]ethyl)butyramide:

[0037] Under argon protection, (E)-4-((3-(2-(benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid (96.0 mg, 175.3 μmol) and 4-dimethylaminopyridine (2.1 mg, 17.53 μmol) were added to 10 mL of anhydrous dichloromethane and stirred at room temperature for 20 minutes. Then, 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthine]-3-one (92.5 mg, 175.3 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (50.4 mg, 262.9 μmol) were added to the reaction solution at once, and the reaction was continued to stir under argon protection for 6 hours. After the reaction was completed as monitored by TLC, appropriate amounts of dichloromethane and water were added, the layers were separated, the organic layer was washed with water three times, and concentrated to obtain a crude product, which was purified by column chromatography to obtain 100.0 mg of a red solid with a yield of 53.9%.

[0038] The reaction equation is as follows:

[0039]

[0040] Applications of the above fluorescent probes for simultaneous detection of Cys, Hcy, GSH and ATP:

[0041] Experiment on the spectral properties of fluorescent probes for detecting Cys, Hcy, GSH and ATP: The probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution with a concentration of 1 mM, and aqueous solutions of Cys, Hcy, GSH and ATP with a concentration of 1 mM were prepared respectively. The specific test method for detecting Cys, Hcy, GSH and ATP is as follows: 20 μL of probe solution (1 mM), 980 μL / 580 μL of analytical grade DMSO / MeOH, the required amount of 1 mM Cys / Hcy / GSH / ATP aqueous solution and the required amount of PBS buffer aqueous solution were placed in a 2 mL sample tube. For all tests, the final volume ratio of the organic phase and the aqueous phase was kept at 7:3 / 5:5 (the total volume of each test sample was 2 mL). After shaking at room temperature for 20 / 30 minutes, the fluorescence emission spectra of the probes for detecting Cys, Hcy, GSH and ATP were measured at excitation wavelengths of 375 nm, 493 nm, 455 nm and 520 nm, respectively. Other test operations were similar to the above steps. The probe has low detection limits and high sensitivity for detecting Cys, Hcy, GSH and ATP.

[0042] Figure 1 It is the variation of the fluorescence emission intensity with the Cys concentration in the quantitative detection of Cys by the fluorescent probe of the present invention, the abscissa is the wavelength, and the ordinate is the fluorescence intensity. Figure 2 This is the variation of the fluorescence emission intensity with the Hcy concentration in the quantitative detection of Hcy by the fluorescent probe of the present invention. The horizontal axis is the wavelength and the vertical axis is the fluorescence intensity. Figure 3 This is the variation of the fluorescence emission intensity with the GSH concentration in the quantitative detection of GSH by the fluorescent probe of the present invention. The horizontal axis is the wavelength, and the vertical axis is the fluorescence intensity. Figure 4 This is the change of the fluorescence emission intensity with the ATP concentration in the quantitative detection of ATP by the fluorescent probe of the present invention. The horizontal axis is the wavelength and the vertical axis is the fluorescence intensity.

[0043] Application of simultaneous differentiation of endogenous Cys, Hcy, GSH and ATP in Hela tumor cells:

[0044] Hela cells were subcultured into a cell culture dish for confocal imaging. After culturing under standard growth conditions for 24 hours, an appropriate amount of probe (5 μM) was added and continued to be cultured under standard growth conditions for 30 minutes. Then, the cells were imaged under a confocal fluorescence microscope. Fluorescence imaging was performed using blue, red, green, and red fluorescence channels. Figure 5 It can be seen that the fluorescent probe of the present invention successfully realizes the simultaneous differentiation of endogenous Cys, Hcy, GSH and ATP in cells, and has great application value in the fields of biochemistry, analytical detection and environmental science.

[0045] The synthesis of the fluorescent probe provided in the present application and the method for using it in the simultaneous distinguishing detection of tumor markers Cys, Hcy, GSH and ATP have the advantages of high efficiency, simplicity, low cost and high accuracy, and can realize the simultaneous distinguishing detection and imaging analysis of Cys, Hcy, GSH and ATP in environmental and biological samples.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, characterized in that: Its structural formula is:

2. A method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, characterized in that: include: 4-((4-(butylthio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and 2-(benzo[d]thiazol-2-yl)acetonitrile are added to anhydrous ethanol to carry out a first reaction. After the reaction is complete, the reaction solution is concentrated and purified by column chromatography to obtain a red solid product; Under protective atmosphere conditions, (E)-4-((3-(2-(benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and 4-dimethylaminopyridine are added to anhydrous dichloromethane to carry out a second reaction, and then 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthine]-3-one and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are added to carry out a third reaction. After the reaction is complete, water and dichloromethane are added to the reaction solution, the liquids are separated, the organic layer is concentrated, and the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate is obtained by column chromatography.

3. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate according to claim 2, characterized in that: The temperature of the first reaction is 50-90° C. and the time is 12-24 hours.

4. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate according to claim 2, characterized in that: The temperature of the second reaction is room temperature, and the reaction time is 10-30 minutes.

5. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate according to claim 2, characterized in that: The temperature of the third reaction is room temperature, and the reaction time is 4-12 hours.

6. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate according to claim 2, characterized in that: The first reaction, the second reaction and the third reaction are all carried out under stirring.

7. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate according to any one of claims 2 to 6, characterized in that: The protective atmosphere includes argon.

8. A method for using the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate as claimed in claim 1, characterized in that: include: Dissolving the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate in a mixture of an organic phase and an aqueous phase, and performing detection under specific wavelength conditions after the reaction; Among them, after reacting with cysteine, it emits 455nm blue fluorescence at an excitation wavelength of 375nm; after reacting with homocysteine, it emits 555nm red fluorescence at an excitation wavelength of 493nm; after reacting with glutathione, it emits 529nm green fluorescence at an excitation wavelength of 455nm; after reacting with adenosine triphosphate, it emits 587nm pink fluorescence at an excitation wavelength of 520nm.

9. The method for using the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate according to claim 8, characterized in that: When detecting cysteine, homocysteine, and glutathione, the organic phase is dimethyl sulfoxide, and the aqueous phase is a phosphate buffer solution with a pH of 7.4 and an aqueous solution of the analyte; When detecting adenosine triphosphate, the organic phase is methanol, and the aqueous phase is a phosphate buffer solution with a pH of 7.4 and an aqueous solution of the analyte.

10. Use of the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate as claimed in claim 1, characterized in that: Used for quantitative detection of tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate.

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