Fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine and mercury ions in tumor cells as well as preparation method, use method and application of fluorescent probe

Abstract

The invention provides a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine and mercury ions in tumor cells 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 cysteine, homocysteine and mercury ions in tumor cells is as follows: # imgabs0, and the probe does not have obvious fluorescence, so that specific analytes can be detected by specific excitation and fluorescence emission signals, and cysteine, homocysteine and mercury ions can be simultaneously distinguished and detected; the catalyst has no obvious response to other common amino acids, active oxygen and active nitrogen species, and has good selectivity.

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 cysteine, homocysteine ​​and mercury ions in tumor cells, and a preparation method, a use method and an application thereof. Background Art

[0002] Cysteine ​​(Cys) is a small amino acid containing a sulfhydryl group and plays a vital role in many physiological and pathological processes. Excessive Cys is associated with diseases such as rheumatoid arthritis, cancer, Parkinson's disease and Alzheimer's disease. On the other hand, Cys deficiency has been reported to lead to slowed growth, edema, liver damage, skin damage and weakness (J. Am. Chem. Soc. 2017, 139, 8, 3181–3185). Hcy, as a substrate for the synthesis of Cys and GSH, has different physiological functions from Cys / GSH, which can cause cellular oxidative damage (Chem. Commun., 2017, 53, 13168-13171). Elevated Hcy levels can lead to neurodegenerative diseases, cancer, Alzheimer's disease and cardiovascular diseases (Anal. Chem. 1999, 71, 20, 4527–4533). Mercury, as a common toxic heavy metal, can easily be biomagnified and enriched through the food chain and eventually enter the human body. Mercury ion (Hg 2+ ) has a strong affinity for enzymes and thiols in proteins, which can cause cellular dysfunction. Long-term exposure to high levels of mercury can cause irreversible damage to the nervous system, endocrine system, immune system, and digestive system (ACS Omega 2020, 5, 18176-18184). Therefore, it is important to monitor Cys, Hcy, and Hg in living systems. 2+ There is a great demand for new methods with high sensitivity and selectivity.

[0003] In recent years, fluorescent probes have attracted extensive attention due to their good selectivity, high sensitivity, ease of operation, and ability to monitor and visualize biomolecules in living cells. So far, some fluorescent probes have been reported for the selective detection of Cys / Hcy / Hg 2+ Some probes have been reported for the simultaneous detection of two of the three, but no fluorescent probe has been able to simultaneously distinguish and detect Cys, Hcy, and Hg. 2+ , which cannot meet the requirements of simultaneously distinguishing endogenous Cys, Hcy and exogenous Hg in biological systems such as cells. 2+ application requirements. Summary of the invention

[0004] The purpose of the present application is to provide a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells, and a preparation method, a use method and an application thereof, so as to solve the above-mentioned problems.

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

[0006] A fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells, the structural formula of which is as follows:

[0007]

[0008] The present application also provides a method for preparing the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells, 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 and heated to carry out a first reaction. After the reaction is complete, the reaction solution is concentrated and then purified by column chromatography to obtain a red solid product;

[0010] Under a protective atmosphere, (E)-4-((3-(2-(benzo[d]thiazol-2-yl)-2-cyano)(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-amino-3'-(diethylamino)-6'-(piperazine-1-yl)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 then column chromatography is performed to purify the solution to obtain the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells.

[0011] Preferably, the temperature of the first reaction is 50-90°C, and the reaction 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 cysteine, homocysteine ​​and mercury ions in tumor cells, comprising:

[0017] The fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells is dissolved in a mixture of an organic phase and an aqueous phase, and after the reaction, detection is performed under a specific wavelength condition;

[0018] Among them, after reacting with cysteine, it emits strong blue fluorescence of 456nm under an excitation wavelength of 360nm; after reacting with homocysteine, it emits strong green fluorescence of 558nm under an excitation wavelength of 500nm; after reacting with mercury ions, it emits strong red fluorescence of 580nm under an excitation wavelength of 530nm.

[0019] Preferably, 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 target analyte;

[0020] The volume ratio of the organic phase to the aqueous phase is 6:4-4:6.

[0021] The present application also provides an application of the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells, which is used to simultaneously distinguish and detect cysteine, homocysteine ​​and mercury ions in tumor cells.

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

[0023] The fluorescent probe provided in the present application for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells has multiple active reaction sites. Based on different reaction mechanisms, it can realize simultaneous distinguishing and detecting Cys (λex / λem=360 / 456nm), Hcy (λex / λem=500 / 558nm) and Hg2+ (λex / λem=530 / 580nm). The probe itself has no obvious fluorescence, so it can realize specific excitation and fluorescence emission signal detection of specific analytes. It has no obvious response to other common amino acids, reactive oxygen and reactive nitrogen species. It has good selectivity, high sensitivity and strong anti-interference, and can be used to directly and simultaneously quantify Cys, Hcy and Hg in the environment. 2+ , simultaneously distinguishing Cys, Hcy and Hg in biological systems such as fluorescent imaging of tumor cells 2+ .

[0024] The present application provides a method for preparing a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells, 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 As a fluorescent probe for quantitative detection of Hg 2+ The fluorescence emission intensity increases with the Hg 2+ Changes in concentration;

[0029] Figure 4 Simultaneous differentiation and imaging of endogenous Cys, Hcy and Hg in HeLa cells using fluorescent probes 2+ picture. DETAILED DESCRIPTION

[0030] 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.

[0031] Example

[0032] This embodiment provides a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells, and the preparation method thereof is as follows:

[0033] 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

[0034] 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%.

[0035] 2. Synthetic probe (E)-3-(7-((4-(4-(2-amino-3-oxo-3'-(pentan-3-yl)spiro[isoindoline-1,9'-xanthine]-6'-yl)piperazin-1-yl)-4-oxobutyl)(ethyl)amino)-4-(butylthio)-2-oxo-2H-chromen-3-yl)-2-(benzo[d]thiazol-2-yl)acrylonitrile

[0036] 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 (80.0 mg, 146.1 μmol) and 4-dimethylaminopyridine (1.8 mg, 14.61 μmol) were added to 7 mL of anhydrous dichloromethane and stirred at room temperature for 20 minutes. Then, 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one (68.6 mg, 146.1 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (42.0 mg, 219.1 μmol) were added to the reaction solution at one time, 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 55.0 mg of a red solid with a yield of 37.7%.

[0037] The reaction equation is as follows:

[0038]

[0039] Fluorescent probes for detection of Cys, Hcy and Hg 2+ Experiment on the spectral properties of:

[0040] The probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution with a concentration of 1 mM, and Cys, Hcy and Hg were prepared with a concentration of 1 mM respectively. 2+ Detection of Cys, Hcy and Hg 2+ The specific test method is: take 20 μL of probe solution (1mM), 980 μL of analytical grade DMSO, the required amount of 1mM Cys / Hcy / Hg 2+ The aqueous solution and the required amount of PBS buffered aqueous solution were placed in a 2 mL sample tube. The final volume ratio of the organic phase to the aqueous phase was kept at 6:4 / 4:6 for all tests (the total volume of each test sample was 2 mL). After shaking at room temperature for 20 / 30 minutes, the probes were measured at excitation wavelengths of 360 nm, 500 nm, and 530 nm to detect Cys / Hcy / Hg. 2+The fluorescence emission spectrum of the probe is similar to the above steps. 2+ The detection limit is low and the sensitivity is high.

[0041] Figure 1 It is the change of fluorescence emission intensity with Cys concentration in the quantitative detection of Cys by fluorescent probe. The horizontal axis is wavelength and the vertical axis is fluorescence intensity.

[0042] Figure 2 It is the change of fluorescence emission intensity with Hcy concentration in the quantitative detection of Hcy by fluorescent probe. The horizontal axis is wavelength and the vertical axis is fluorescence intensity.

[0043] Figure 3 It is the change of fluorescence emission intensity with Hg2+ concentration in the quantitative detection of Hg2+ by fluorescent probe. The horizontal axis is wavelength and the vertical axis is fluorescence intensity.

[0044] Simultaneous differentiation of endogenous Cys, Hcy, and Hg in HeLa cells 2+ Application:

[0045] Tumor HeLa cells were passaged into a cell culture dish for confocal imaging. After culturing for 24 hours under standard growth conditions, an appropriate amount of probe (5 μM) was added and continued to be cultured for 30 minutes under standard growth conditions. Then, the cells were imaged under a confocal fluorescence microscope, and fluorescence imaging was performed using blue, green, and red fluorescence channels. Figure 4 It can be seen that the fluorescent probe of the present application successfully achieves the endogenous Cys, Hcy and exogenous Hg in tumor cells. 2+ It can also distinguish fluorescence imaging analysis, which has great application value in biochemistry, analytical testing and environmental science.

[0046] Synthesis of fluorescent probes provided in this application and simultaneous detection of Cys, Hcy and Hg 2+ The application method has the advantages of high efficiency, simplicity, low cost and high accuracy, and can achieve the detection of Cys, Hcy and Hg in environmental and biological samples. 2+ Simultaneous differentiation detection and imaging analysis.

[0047] 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 cysteine, homocysteine ​​and mercury ions in tumor cells, characterized in that: Its structural formula is as follows:

2. A method for preparing a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells according to claim 1, 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 and heated to carry out a first reaction. After the reaction is complete, the reaction solution is concentrated and then purified by column chromatography to obtain a red solid product; Under a protective atmosphere, (E)-4-((3-(2-(benzo[d]thiazol-2-yl)-2-cyano)(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-amino-3'-(diethylamino)-6'-(piperazine-1-yl)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 then column chromatography is performed to purify the solution to obtain the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells.

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

4. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells 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 cysteine, homocysteine ​​and mercury ions in tumor cells 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 cysteine, homocysteine ​​and mercury ions in tumor cells 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 cysteine, homocysteine ​​and mercury ions in tumor cells 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 cysteine, homocysteine ​​and mercury ions in tumor cells according to claim 1, characterized in that: include: Dissolving the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells 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 456nm blue fluorescence at an excitation wavelength of 360nm; after reacting with homocysteine, it emits 558nm green fluorescence at an excitation wavelength of 500nm; after reacting with mercury ions, it emits 580nm red fluorescence at an excitation wavelength of 530nm.

9. The method for using the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells according to claim 8, characterized in that: 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 a target analyte; The volume ratio of the organic phase to the aqueous phase is 6:4-4:

6.

10. Use of the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and mercury ions in tumor cells according to claim 1, characterized in that: Used to simultaneously differentiate and detect cysteine, homocysteine ​​and mercury ions in tumor cells.

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