A fluorescent probe and its preparation method and application

Abstract

The invention discloses a fluorescent probe for simultaneously detecting hypochlorite and peroxynitrite anions, and a preparation method and application thereof. The invention adopts xanthene fluorophore as a fluorescent matrix and prepares the fluorescent probe by an organic synthesis method. The probe can achieve high selectivity for hypochlorite and peroxynitrite anions, is not interfered by other active oxygen and active nitrogen species, has high-sensitivity detection and the like, can be used to detect the content of hypochlorite and peroxynitrite anions in solutions and cells, and has broad application prospects in the field of active small molecule detection.

Description

Technical Field

[0001] The invention belongs to the technical field of organic small molecule fluorescent probes, and specifically relates to a fluorescent probe, in particular to a fluorescent probe for simultaneously detecting hypochlorite and peroxynitrite anions, and a preparation method and application thereof. Background Art

[0002] Hypochlorite (OCl-) is an important class of reactive oxygen species (ROS) that plays a leading role in a variety of physiological and pathological processes. Hypochlorite (OCl-) is generated by the reaction of hydrogen peroxide and chloride ions catalyzed by myeloperoxidase produced by endogenous pathways. Dysregulation of hypochlorite (OCl-) is associated with many potentially fatal diseases, such as cardiovascular disease, neuronal degeneration, diabetes, and cancer. Peroxynitrite anion (ONOO-) is generated by the diffusion-controlled coupling reaction of superoxide radical anion and NO. It is an important reactive nitrogen species (RNS) that often reacts with proteins, DNA, RNA, etc. in living systems.

[0003] Peroxynitrite anion (ONOO-) is produced by non-enzymatic reactions of superoxide anion and nitric oxide in living systems. Excessive ONOO- may cause damage to cells. Because ONOO- has strong oxidative and nucleophilic properties, it can react with many substances in cells, including proteins, DNA, lipids, etc., eventually leading to cell necrosis and apoptosis.

[0004] Hypochlorous acid (HOCl) and peroxynitrite anion (ONOO-) are the two most common and important active substances, which are related to various diseases. However, the reported probes only identify OCl- or ONOO-, ignoring that the occurrence and development of diseases such as tumors are the result of the interaction of multiple superoxides in cells, and cannot reflect the real-time and dynamic quantitative changes of ClO- and ONOO- levels in cells. In addition, the accuracy of repeated and step-by-step tests is low and the systematic error is high. Many of their functions have not been revealed due to the lack of methods for simultaneous detection of OCl- and ONOO-.

[0005] Fluorescent probes have the advantages of good selectivity, high sensitivity, simple and fast operation, and little damage to the detected object. They have been widely used in the detection of metal cations, anions, and active small molecules in the environment and biological systems. The development and utilization of fluorescent probe molecules is an interdisciplinary field of chemistry, biology, medicine, agriculture and other sciences. Fluorescent probe technology is an analytical method for studying the research object at the molecular level. Fluorescent probes have been widely used in the detection of heavy metals, bioactive small molecules and other substances in the environment and organisms. Summary of the invention

[0006] In order to solve the above technical problems, the inventors designed, synthesized, characterized and applied a fluorescent probe based on an "AND" logic gate, thereby completing the present invention. The probe selectively produces a large fluorescence enhancement only in the presence of ONOO- and OCl-, and has good water solubility, fast response, good selectivity and high sensitivity.

[0007] In one aspect, the present invention provides a fluorescent molecular probe for simultaneously identifying hypochlorite and peroxynitrite anions, wherein the structural formula of the molecular probe is shown in Formula 1:

[0008]

[0009] Among them, R1 and R2 are alkyl groups, and R1 and R2 are the same or different; R3 is oxygen, sulfur, selenium or tellurium; R4 is sulfur, selenium or tellurium, R5 is an alkyl chain, an aromatic group or an olefin chain, and R6 is a carboxyl group, a sulfonic acid group or a phosphoric acid group.

[0010] Furthermore, the number of carbon atoms in the alkyl chain is ≤20; the number of carbon atoms in the olefin chain is ≤20;

[0011] Preferably, the number of carbon atoms in the alkyl chain is ≤5; the number of carbon atoms in the olefin chain is ≤5;

[0012] In another aspect, the present invention also provides a method for synthesizing the fluorescent molecular probe, and the synthesis route is as follows:

[0013]

[0014] The specific steps include:

[0015] Step 1: Compound b and compound a react in a solvent at room temperature or under heating, pour into ice water after the reaction, add ion exchange reagent, and filter to obtain compound c;

[0016] Step 2: Add compound c, compound d and an organic base into an anhydrous solvent and react at room temperature; pour into water, extract and purify to obtain compound e;

[0017] Step 3: Add compound e and compound f into a solvent, add an organic base, react at room temperature, remove the solvent under reduced pressure, and purify to obtain probe g.

[0018] Preferably, in the first step, compound a and compound b are reacted at room temperature or under heating for 2-12 hours, the heating reaction temperature is 20-90° C., the ion exchange reagent is perchloric acid or potassium hexafluorophosphate, and the molar ratio of compound a to compound b is 1:(1-5).

[0019] Preferably, in the second step, the molar ratio of compound c to compound d is 1:(1-5); the reaction is carried out at room temperature for 2-48 hours, the organic base is triethylamine or diisopropylethylamine, the extractant is dichloromethane, petroleum ether, ethyl acetate or acetone; the solvent is N,N-dimethylformamide, dichloromethane, anhydrous ethanol, acetonitrile; the purification is silica gel column chromatography purification, the mobile phase used is dichloromethane and methanol in a volume ratio of 1:10-1:50, and then the product compound e is obtained by direct filtration with a yield of 20-95%; the reaction temperature is room temperature.

[0020] Preferably, in the third step, the molar ratio of compound e to compound f is 1:(1-5); the solvent is N,N-dimethylformamide, dichloromethane, acetonitrile; the reaction is carried out at room temperature for 2-48 hours; the organic base is triethylamine or diisopropylethylamine; the purification is silica gel column chromatography purification, and the mobile phase used is dichloromethane and methanol in a volume ratio of 1:10-1:50, and then the product compound g is obtained by direct filtration, with a yield of 20-95%; the reaction temperature is room temperature.

[0021] In a third aspect, the present invention provides the use of the above fluorescent molecular probe in detecting hypochlorite and peroxynitrite anions.

[0022] Furthermore, the detection is a quantitative detection.

[0023] Furthermore, the application is the detection of hypochlorite and peroxynitrite ions in cells or solutions.

[0024] Furthermore, the application is carried out under physiological conditions of pH=5-9, preferably pH=7.4.

[0025] The present invention has been experimentally verified that, in a PBS buffer system with a pH of 7.4, when the excitation wavelength is set to 530 nm, and when hypochlorite and peroxynitrite anions are added simultaneously, the fluorescence at 563 nm is significantly enhanced with the increase of time.

[0026] The fluorescent probe g synthesized by the present invention reacts with hypochlorite and peroxynitrite anions, resulting in the breaking of CN bonds and the release of fluorophores, thereby generating fluorescence with a maximum emission peak of 563 nm.

[0027] Beneficial Effects

[0028] The fluorescent probe g of the present invention is a fluorescent probe for simultaneously detecting hypochlorite and peroxynitrite anions, has the advantages of fast response time, high sensitivity, good specificity, etc., can eliminate the interference of various active ions, and has broad application prospects in the field of biomolecule detection.

[0029] In order to solve the above technical problems, the present invention provides a fluorescent probe for simultaneously detecting hypochlorite and peroxynitrite anions, and a synthesis method and application thereof. The fluorescent molecular probe is suitable for simultaneous detection and imaging of hypochlorite and peroxynitrite anions in mitochondria; the synthesis method has simple process, low cost and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the synthesis route of the fluorescent probe P34 synthesized in Example 2 of the present invention.

[0031] Figure 2 This is the H-NMR spectrum of the fluorescent probe P34 synthesized in Example 2 of the present invention.

[0032] Figure 3 This is a fluorescence spectrum diagram showing the fluorescence intensity of the fluorescent probe P34 synthesized in Example 2 of the present invention after hypochlorite is pre-added as a function of the concentration of peroxynitrite anions.

[0033] Figure 4 This is a fluorescence change diagram of the fluorescence intensity of the fluorescent probe P34 synthesized in Example 2 of the present invention in the presence of hypochlorite and peroxynitrite anions over time.

[0034] Figure 5 This is a fluorescence change diagram of the fluorescence intensity of the fluorescent probe P34 synthesized in Example 2 of the present invention at different pH values ​​in the presence of hypochlorite and peroxynitrite anions.

[0035] Figure 6 It is the fluorescence selectivity and competitiveness of the fluorescent probe P34 synthesized in Example 2 of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described in detail below in conjunction with specific embodiments and drawings; the reagents used in the present invention can be purchased unless otherwise specified.

[0037] Embodiment 1:

[0038] A method for preparing a fluorescent probe for detecting hypochlorite and peroxynitrite anions, the synthetic route is as follows:

[0039]

[0040] The specific steps include:

[0041] Step 1: Dissolve compound 1 (10 mmol) and compound 2 (10.5 mmol) in 30 ml of methanesulfonic acid and react at room temperature for 6 hours. Pour into ice water, add 3 mL of perchloric acid, and filter to obtain red solid compound 3;

[0042] Step 2: Add compound 3 (3 mmol) and compound 4 (3 mmol) and triethylamine (0.1 mL) to anhydrous N,N-dimethyldiamide and react at room temperature for 8 hours. Pour into 150 ml of water, extract with dichloromethane, dry with anhydrous sodium sulfate, and separate by column chromatography on a silica gel column (dichloromethane: methanol = 50:1) to obtain a red solid compound 4;

[0043] Step 3: Add compound 3 (3 mmol) and diphenyl diselenide (4 mmol) to 20 ml of dichloromethane, add 30 μl of diisopropylethylamine, react at room temperature for 4 hours, remove the solvent under reduced pressure, and separate by column chromatography using silica gel column chromatography (dichloromethane: methanol = 20:1) to obtain a red solid compound, which is probe P32.

[0044] Example 2

[0045] A method for preparing a fluorescent probe for detecting hypochlorite and peroxynitrite anions, the synthetic route is as follows:

[0046]

[0047] The specific reaction steps are as follows:

[0048] Step 1: Dissolve compound 1 (10 mmol) and compound 2 (10.5 mmol) in 30 ml of methanesulfonic acid and react at room temperature for 6 hours. Pour into ice water, add 3 mL of perchloric acid, and filter to obtain red solid compound 3;

[0049] Step 2: Add compound 3 (3 mmol) and compound 4 (3 mmol) and triethylamine (0.1 mL) to anhydrous N,N-dimethyldiamide and react at room temperature for 8 hours. Pour into 150 ml of water, extract with dichloromethane, dry with anhydrous sodium sulfate, and separate by column chromatography on a silica gel column (dichloromethane: methanol = 50:1) to obtain a red solid compound 4;

[0050] Step 3: Add compound 3 (3 mmol) and propanethiol (4 mmol) to 20 ml of dichloromethane, add 20 μl of triethylamine, react at room temperature for 4 hours, remove the solvent under reduced pressure, and separate by column chromatography using silica gel column chromatography (dichloromethane: methanol = 30:1) to obtain a red solid compound, which is probe P34.

[0051] The structure of the final product P34 synthesized in Example 2 of the present invention was confirmed by H NMR spectrum; its H NMR spectrum is as follows Figure 2 shown.

[0052] Experimental Example 1: Fluorescence Performance Test

[0053] Fluorescence intensity changes after adding different concentrations of peroxynitrite anions in the presence of hypochlorite

[0054] The fluorescent probe P34 synthesized in Example 2 of the present invention was prepared into a mother solution with a final concentration of 1 mM; 100 μM hypochlorite solution was added in advance, and then 0-100 μM peroxynitrite anions of different concentrations were added respectively. After half an hour of reaction, fluorescence detection was performed (excitation 500 nm, maximum emission 563 nm); the changes in fluorescence intensity after adding different concentrations of peroxynitrite anions are shown in FIG. Figure 3 As shown; it can be seen from the figure that as the concentration of peroxynitrite anion increases, the fluorescence intensity of the probe at 563nm continues to increase.

[0055] Response time of fluorescent probe P34

[0056] The fluorescent probe P34 in Example 2 of the present invention was prepared into a mother solution with a final concentration of 1 mM; after adding a certain concentration of hypochlorite and peroxynitrite anion to the mother solution, the fluorescence intensity changed as follows: Figure 4 As shown in the figure, when the excitation wavelength is 500nm, the fluorescence intensity of the probe at 563nm changes significantly and reaches the maximum value within 40 seconds.

[0057] Fluorescence intensity changes of P34 in recognition of hypochlorite and peroxynitrite anions at different pH

[0058] The PBS solution with a pH value of 4-10 was divided into three groups. The pH value of each group was 4-10. 3.0 ml of each pH value was taken into an analysis bottle. A certain amount of probe was added to each bottle to make the final concentration 10 μM. The first group was the probe alone (10 μM), and the second group was the probe (10 μM) + hypochlorite (100 μM) + peroxynitrite anion (100 μM). After half an hour, when the excitation wavelength was 500 nm, the change in fluorescence intensity of the probe at 563 nm was plotted, as shown in FIG. Figure 5 As shown, the probe is suitable for detecting hypochlorite and peroxynitrite anions under physiological conditions.

[0059] Selectivity and competitiveness of fluorescent probe P34 ion

[0060] Selectivity and competitiveness studies of probe P34 (10 μM) at different excitation wavelengths.

[0061] The PBS solution with pH 7.4 was divided into two groups, each with pH 7.4. 3.0 ml was taken into analysis bottles. A certain amount of probe was added to each bottle to make the final concentration 10 μM. After adding the single probe (10 μM) to the first group, other ions or small molecules numbered 1-25 (1.F- ,2.Cl - ,3.Br - ,4.SO4 2- ,5.S2O8 2- ,6.HS-,7.HSO3 - ,8.HCO3 - ,9.CO3 2- ,10.O2 .- ,11..OH,12.H2O2,13.NO,14.NO2 - ,15.NO3 - ,16.ONOO - ,17.ClO - ,18.PO4 3- ,19.K + ,20.Na + ,21.Ca 2+ ,22.Ac - ,23.GSH,24.Cys,25.Hcy; Cys concentration is 500μM, and other concentrations are 100μM).

[0062] The second group added a single probe (100 μM), and then added other ions or small molecules numbered 1-26 (1.F - ,2.Cl - ,3.Br - ,4.SO4 2- ,5.S2O8 2- ,6.HS-,7.HSO3 - ,8.HCO3 - ,9.CO3 2- ,10.O2 .- ,11..OH,12.H2O2,13.NO,14.NO2 - ,15.NO3 - ,16.ONOO - ,17.ClO - ,18.PO4 3- ,19.K + ,20.Na + ,21.Ca 2+ ,22.Ac - ,23.GSH,24.Cys,25.Hcy,26.Probe; Cys concentration is 500μM, other concentrations are 100μM), then add peroxynitrite anion (100μM), half an hour later, when the excitation wavelength is 500nm, take the change of the fluorescence intensity of the probe at 563nm and plot it, as shown in Figure 6 As shown, the probe has good selectivity.34 Only when hypochlorite and peroxynitrite anions exist simultaneously will there be obvious fluorescence changes, indicating that the probe can only undergo fluorescence changes when hypochlorite and peroxynitrite anions exist simultaneously.

[0063] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in this field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A fluorescent molecular probe for simultaneously identifying hypochlorite and peroxynitrite anions, wherein the structural formula of the molecular probe is shown in general formula 1: ； in, R1 and R2 are alkyl groups, and R1 and R2 are the same or different; R3 is oxygen or sulfur; R4 is sulfur, R5 is an alkyl group, and R6 is a carboxyl group, a sulfonic acid group or a phosphoric acid group, and the carbon number of the alkyl group is ≤5.

2. The method for synthesizing the fluorescent molecular probe according to claim 1, wherein the synthetic route is as follows: ； The specific steps include: Step 1: Compound b and compound a react in a solvent at room temperature or under heating, pour into ice water after the reaction, add ion exchange reagent, and filter to obtain compound c; Step 2: Add compound c, compound d and an organic base into an anhydrous solvent and react at room temperature; pour into water, extract and purify to obtain compound e; Step 3: Add compound e and compound f into a solvent, add an organic base, react at room temperature, remove the solvent under reduced pressure, and purify to obtain Formula 1.

3. The synthesis method according to claim 2, wherein In the first step, at least one of the following must be met: 1) Compound a and compound b are reacted at room temperature or under heating for 2-12 hours; 2) The heating reaction temperature is 20-90°C; 3) The ion exchange reagent is perchloric acid or potassium hexafluorophosphate; 4) The molar ratio of compound a to compound b is 1:(1-5).

4. The synthesis method according to claim 2, wherein In the second step, at least one of the following must be met: 1) The molar ratio of compound c to compound d is 1:(1-5); 2) React at room temperature for 2-48 hours; 3) The organic base is triethylamine or diisopropylethylamine; 4) The extractant is dichloromethane, petroleum ether, ethyl acetate or acetone; 5) The solvent is N,N-dimethylformamide, dichloromethane, anhydrous ethanol, acetonitrile; 6) The purification is performed by silica gel column chromatography, and the mobile phase used is dichloromethane and methanol in a volume ratio of 1:10-1:

50.

5. The synthesis method according to claim 2, wherein In the third step, at least one of the following must be met: 1) The molar ratio of compound e to compound f is 1:(1-5); 2) The solvent is N,N-dimethylformamide, dichloromethane, acetonitrile; 3) React at room temperature for 2-48 hours; 4) The organic base is triethylamine or diisopropylethylamine; 5) The purification is performed by silica gel column chromatography, and the mobile phase used is dichloromethane and methanol in a volume ratio of 1:10-1:

50.

6. Use of the fluorescent molecular probe according to claim 1 in the preparation of a fluorescent probe for detecting hypochlorite and peroxynitrite anions.

7. The use according to claim 6, wherein: The detection is a quantitative detection.

8. The use according to claim 6, wherein the use is the detection of hypochlorite and peroxynitrite ions in cells or solutions.

9. The use according to claim 6, which is carried out under physiological conditions of pH 5-9.

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