Fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine and superoxide anions as well as preparation method and application of fluorescent probe

Abstract

The invention provides a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine and superoxide anions as well as a preparation method and application of the fluorescent probe, and relates to the technical field of analytical chemistry. The structural formula of the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine and superoxide anions is # imgabs0, the fluorescent probe has a plurality of active reaction sites, Cys, Hcy and O2 <.-> are detected through different reaction strategies, different fluorescent substances are generated, and Cys, Hcy and O2 <.-> can be simultaneously distinguished and detected. The probe is good in selectivity, high in sensitivity and strong in anti-interference performance, can be used for directly and simultaneously quantifying Cys, Hcy and O2 <.-> in an environment, and can also be used for simultaneously distinguishing Cys, Hcy and O2 <.-> in biological systems such as imaging cells and the like.

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 superoxide anions, and a preparation method and use thereof. Background Art

[0002] Cys, as a thiol with a relatively abundant content in cells, plays an important role in maintaining the redox of cells, but the excess or deficiency of Cys can lead to slow growth, cardiovascular and liver damage and other diseases (Chemical Society Reviews, 2015, 44 (17): 6143-6160.). In addition, unlike the physiological function of Cys, Hcy, as a substrate for synthesizing Cys, may cause oxidative damage to cells. Excessive Hcy content can also cause diseases such as folic acid and VB12 deficiency and Alzheimer's disease. Increased Hcy levels can lead to severe oxidative stress, which in turn induces cell apoptosis and tissue damage, and is closely related to diseases such as cardiovascular disease, cancer, neurodegenerative diseases and pregnancy syndrome (New England Journal of Medicine, 1997, 337 (4): 230-237.). Superoxide anion (O 2. - ) is the main ROS, mainly from the mitochondrial electron transport chain and other enzyme systems. 2. - The abnormal increase of superoxide anions can lead to oxidative stress, metabolic disorders and organelle damage. Excessive accumulation of superoxide anions can further cause DNA damage in cells, trigger inflammation, and increase the occurrence of cancer (Angewandte Chemie International Edition, 2020, 59(38): 16698-16704.). The above three active substances Cys, Hcy and O 2. - The role, level, location, distribution and biological function of cells are very complex. Real-time monitoring of Cys, Hcy and O 2. - The dynamic changes and mutual influences of life are of great significance to the study of the automatic regulation mechanism of living organisms.

[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. 2. - Some probes have been reported for the simultaneous detection of two of the three, but no fluorescent probe has been achieved to simultaneously distinguish and detect Cys, Hcy, and O 2. -, which cannot meet the requirements of simultaneously distinguishing and imaging endogenous Cys, Hcy and O 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 superoxide anion, as well as a preparation method and use 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 superoxide anion, the structural formula of which is:

[0007]

[0008] The present application also provides a method for preparing the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion, 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 the reaction solution is concentrated after a first stirring reaction, and a first separation and purification is performed to obtain a red solid product (E)-4-((3-(2-(benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid;

[0010] 5-(4-(tert-butyloxycarbonyl)piperazine-1-carbonyl)-1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide and 4-formyl-2,3-dihydro-1H-xanthen-6-yl trifluoromethanesulfonic acid are added to anhydrous ethanol, and the reaction solution is concentrated after a second stirring reaction, and a second separation and purification is performed to obtain a blue solid product (E)-1-ethyl-3,3-dimethyl-5-(4-pivaloylpiperazine-1-carbonyl)-2-(2-(6-((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indole-1-iodide;

[0011] The (E)-4-((3-(2-(benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid is dissolved in anhydrous dichloromethane, 4-dimethylaminopyridine is added, and after the third stirring, the (E)-1-ethyl-3,3-dimethyl-5-(4-pivaloylpiperazine-1-carbonyl)-2-(2-(6-((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indole-1-iodide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added, and after the fourth stirring reaction, the third separation and purification is performed to obtain the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion.

[0012] Preferably, the first separation and purification, the second separation and purification, and the third separation and purification are each independently performed by column chromatography.

[0013] Preferably, the temperature of the first stirring reaction and the second stirring reaction are both 50°C.

[0014] Preferably, the temperatures of the third stirring reaction and the fourth stirring reaction are both room temperature.

[0015] Preferably, the third stirring time is 10-15 min.

[0016] Preferably, the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion is a blue solid.

[0017] The present application also provides a use of the fluorescent probe for distinguishing and detecting cysteine, homocysteine ​​and superoxide anions, which is used for simultaneously quantifying and / or distinguishing cysteine, homocysteine ​​and superoxide anions.

[0018] Preferably, the cysteine, the homocysteine ​​and the superoxide anion are present in the environment or in a biological system.

[0019] Preferably, the environment comprises an aquatic environment;

[0020] The biological system includes cells.

[0021] Compared with the prior art, the beneficial effects of this application include:

[0022] The fluorescent probe provided in the present application has multiple active reaction sites, and reacts to cysteine ​​Cys, homocysteine ​​Hcy, and superoxide anion O through different reaction strategies. 2. -Different fluorescent substances can be generated for detection, which can realize the simultaneous detection of Cys (λex / λem=360 / 456nm), Hcy (λex / λem=480 / 561nm) and O 2. - (λex / λem=650 / 721nm). This probe has good selectivity, high sensitivity and strong anti-interference, and can be used to directly and simultaneously quantify Cys, Hcy and O in the environment. 2. - , and can also simultaneously distinguish Cys, Hcy, and O in biological systems such as imaging cells 2. - .

[0023] The fluorescent probe was dissolved in dimethyl sulfoxide (DMSO), and the probe was dissolved in an organic phase and an aqueous phase (6:4, v / v) solution. After reacting with Cys at 37 degrees Celsius for 20 minutes, it emitted a strong blue fluorescence of 456 nm at an excitation wavelength of 380 nm; after reacting with Hcy at 37 degrees Celsius for 20 minutes, it emitted a strong red fluorescence of 561 nm at an excitation wavelength of 480 nm; the probe was dissolved in an organic phase and an aqueous phase (4:6, v / v) solution, and reacted with O 2. - After reacting at 37 degrees Celsius for 30 minutes, strong blue fluorescence of 721nm is emitted under an excitation wavelength of 650nm; the probe itself has no obvious fluorescence, so specific excitation and fluorescence emission signals are used to detect specific analytes. It has no obvious response to amino acids such as Asp, Pro, Glu, Tyr, Ser, Val, Ala, Leu, Thr, Dop, sulfur-containing derivatives and other active oxygen substances, and has good selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 A curve showing the change of the fluorescence emission intensity of the fluorescent probe provided in the present application with respect to the Cys concentration when quantitatively detecting Cys;

[0026] Figure 2 A curve showing the change of fluorescence emission intensity of the fluorescent probe provided in the present application with respect to Hcy concentration when quantitatively detecting Hcy;

[0027] Figure 3 The fluorescent probe provided in this application is used for quantitative detection of O 2. - The fluorescence emission intensity increases with the O 2. -Changes in concentration;

[0028] Figure 4 The fluorescent probe provided in this application simultaneously distinguishes endogenous Cys, Hcy and O in HELA cells 2. - . DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1

[0031] This embodiment provides a fluorescent probe, and its reaction equation is as follows:

[0032]

[0033]

[0034] The specific preparation method is as follows:

[0035] 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

[0036] (1.04 g, 2.66 mmol) 4-((4-(butylthio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and (462.83 mg, 2.66 mmol) 2-(benzo[d]thiazol-2-yl)acetonitrile 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 1.02 g of a red solid product with a yield of 73.3%.

[0037] 2. Synthesis of (E)-1-ethyl-3,3-dimethyl-5-(4-pivaloylpiperazine-1-carbonyl)-2-(2-(6-((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indole-1-iodine salt

[0038] (1.02 g, 1.93 mmol) 5-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide and (696.77 mg, 1.93 mmol) 4-formyl-2,3-dihydro-1H-xanthen-6-yltrifluoromethanesulfonic acid 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 1.06 g of a blue solid product with a yield of 64.2%.

[0039] 3. Synthesis of fluorescent probes

[0040] (312.00 mg, 569.67 μmol) (E)-4-((3-(2-(Benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butanoic acid was dissolved in 6 mL of anhydrous DCM, 4-dimethylaminopyridine (DMAP) (34.80 mg, 284.83 μmol) was added, and the mixture was stirred at room temperature for 10-15 minutes. Then, (486.35 g, 56 9.67 μmol) (E)-1-ethyl-3,3-dimethyl-5-(4-pivaloylpiperazine-1-carbonyl)-2-(2-(6-((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indole-1-iodide and (132.66 mg, 854.50 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were stirred at room temperature for 2 hours. After the reaction was complete, the crude product was purified by column chromatography to obtain 298.12 mg of a blue solid, namely the fluorescent probe, with a yield of 40.28%.

[0041] Experimental Example 1

[0042] Fluorescent probes can simultaneously distinguish and detect Cys, Hcy and O 2. - Application:

[0043] Detection of Cys, Hcy and O 2. - Spectral property experiment: The probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe stock solution with a concentration of 1 mM, and Cys, Hcy and O were prepared with a concentration of 10 mM respectively. 2. - Unless otherwise specified, the final volume ratio of organic phase to aqueous phase was 5:5 for all tests. 2. -The specific test method is as follows. Here, Cys is taken as an example: the ratio of organic phase to water phase is 6:4 for the test: 20 μL of probe solution (1 mM), 1180 μL of analytical grade DMSO, 20 μL of 10 mM Cys aqueous solution and 780 μL of PBS buffer aqueous solution are placed in a 2 mL sample tube (the total volume of each test sample is 2 mL). After shaking at 37 degrees Celsius for 20-30 minutes, the fluorescence emission spectrum of the probe detection of Cys can be measured at an excitation wavelength of 360 nm. Other test operations are similar to the above steps. The probe molecule can distinguish and detect Cys, Hcy and O by using different excitation wavelengths and fluorescence emission signals. 2. - , with high sensitivity and low detection limit, is very suitable for endogenous Cys, Hcy and O in living cells 2. - Imaging and analysis.

[0044] Figure 1 The curve of the change of the fluorescence emission intensity of the fluorescent probe provided in the present application with the Cys concentration when quantitatively detecting Cys; the abscissa is the wavelength and the ordinate is the fluorescence intensity;

[0045] Figure 2 The curve of the change of fluorescence emission intensity of the fluorescent probe provided in the present application with the Hcy concentration when quantitatively detecting Hcy; the abscissa is the wavelength and the ordinate is the fluorescence intensity;

[0046] Figure 3 The fluorescent probe provided in this application is used for quantitative detection of O 2. - The fluorescence emission intensity increases with the 2. - Concentration change curve; the horizontal axis is wavelength and the vertical axis is fluorescence intensity.

[0047] Experimental Example 2

[0048] Simultaneous differentiation of endogenous Cys, Hcy, and O in HeLa cells 2. - Application:

[0049] HeLa cells were passaged into confocal dish cell culture medium and cultured under standard growth conditions for 24 hours. Then, an appropriate amount of probe (5 μM) was added and cultured under standard growth conditions for another 30 minutes. Then, the cells were photographed under a confocal fluorescence microscope and fluorescence imaging was performed using blue, red, and pink fluorescence channels, respectively. 2. - ,from Figure 4 It can be seen that the multi-detection fluorescent probe of the present invention successfully realizes the detection of endogenous and exogenous Cys, Hcy and O in cells.2. - The multi-channel fluorescence imaging analysis is of great application value in biochemistry, analytical detection and other fields.

[0050] Figure 4 The fluorescent probe provided in this application simultaneously distinguishes endogenous Cys, Hcy and O in HELA cells 2. - .

[0051] The present invention provides a fluorescent probe for simultaneous detection of Cys, Hcy and O 2. - The application method has the advantages of high efficiency, simplicity, low cost and high accuracy, and can realize the detection of Cys, Hcy and O in environmental and biological samples. 2. - Simultaneous differentiation detection and imaging analysis.

[0052] 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 superoxide anion, characterized in that: Its structural formula is:

2. A method for preparing a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion 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 the reaction solution is concentrated after a first stirring reaction, and a first separation and purification is performed to obtain a red solid product (E)-4-((3-(2-(benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid; 5-(4-(tert-butyloxycarbonyl)piperazine-1-carbonyl)-1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide and 4-formyl-2,3-dihydro-1H-xanthen-6-yl trifluoromethanesulfonic acid are added to anhydrous ethanol, and the reaction solution is concentrated after a second stirring reaction, and a second separation and purification is performed to obtain a blue solid product (E)-1-ethyl-3,3-dimethyl-5-(4-pivaloylpiperazine-1-carbonyl)-2-(2-(6-((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indole-1-iodide; The (E)-4-((3-(2-(benzo[d]thiazol-2-yl)-2-cyano)-4-(butylthio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid is dissolved in anhydrous dichloromethane, 4-dimethylaminopyridine is added, and after the third stirring, the (E)-1-ethyl-3,3-dimethyl-5-(4-pivaloylpiperazine-1-carbonyl)-2-(2-(6-((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indole-1-iodide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added, and after the fourth stirring reaction, the third separation and purification is performed to obtain the fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion.

3. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion according to claim 2, characterized in that: The first separation and purification, the second separation and purification, and the third separation and purification are each independently performed by column chromatography.

4. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion according to claim 2, characterized in that: The temperatures of the first stirring reaction and the second stirring reaction are both 45-55°C.

5. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion according to claim 2, characterized in that: The temperatures of the third stirring reaction and the fourth stirring reaction are both room temperature.

6. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion according to claim 2, characterized in that: The third stirring time is 10-15 min.

7. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion according to any one of claims 2 to 6, characterized in that: The fluorescent probe for simultaneously distinguishing and detecting cysteine, homocysteine ​​and superoxide anion is a blue solid.

8. Use of the fluorescent probe for distinguishing and detecting cysteine, homocysteine ​​and superoxide anion according to claim 1, characterized in that: For simultaneous quantification and / or differentiation of cysteine, homocysteine, and superoxide anion.

9. The use of the fluorescent probe for distinguishing and detecting cysteine, homocysteine ​​and superoxide anion according to claim 8, characterized in that: The cysteine, the homocysteine, and the superoxide anion exist in the environment or in a biological system.

10. The use of the fluorescent probe for distinguishing and detecting cysteine, homocysteine ​​and superoxide anion according to claim 9, characterized in that: The environment includes an aquatic environment; The biological system includes cells.

Images