A fluorescent probe for detecting hypochlorous acid, its preparation method and usage method

Abstract

This invention discloses a fluorescent probe for detecting hypochlorous acid, its preparation method, and its application method. The invention utilizes 7-diethylaminocoumarin to construct a classic intramolecular charge transfer (PeT) system. The probe itself is almost non-fluorescent due to the PeT effect of the Se atom. However, in the presence of hypochlorous acid, the hypochlorous acid reacts with the selenylphenyl portion of the probe molecule through oxidation to form a selenium-oxygen double bond, thereby blocking the PeT process, and the probe molecule emits strong green fluorescence. The 7-diethylaminocoumarin-based "on" hypochlorous acid probe provided by this invention exhibits excellent response to hypochlorous acid solutions, enabling sensitive and quantitative detection of trace amounts of hypochlorous acid in samples. It has advantages such as simple operation, low cost, sensitive response, and ease of promotion and application.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to 4-trifluoromethyl-7-diethylamino-8-selenylphenylcoumarin used as a hypochlorous acid fluorescent probe, its preparation method and usage method. Background Technology

[0002] Hypochlorous acid (HClO), as one of the most important reactive oxygen species (ROS) in biological systems, is catalyzed by myeloperoxidase (MPO) under physiological conditions to form chloride ions (Cl-). - Hypochlorous acid (HClO) is a double-edged sword in biological processes. On the one hand, HClO can protect human health by killing pathogens and bacteria that invade the immune system; on the other hand, excessive HClO can cause excessive oxidation of biomolecules (such as proteins, nucleic acids, lipids, and enzymes), leading to tissue damage, inflammation, and a range of diseases such as cardiovascular disease, rheumatoid arthritis, liver ischemia-reperfusion injury, and even cancer. Given the special importance of hypochlorous acid in biology and medicine, exploring a highly selective, highly sensitive, and easily detectable method for endogenous hypochlorous acid has become a hot topic in the current biomedical field. There are many methods for detecting hypochlorous acid, such as the starch-potassium iodide method, the N,N-diethyl-p-phenylenediamine colorimetric method, chemiluminescence, spectrophotometry, and fluorescence analysis.

[0003] Among these detection methods, fluorescent probe technology has outstanding advantages such as good selectivity, high sensitivity, low detection limit, simple operation and low cytotoxicity, making it widely used in chemical and biological analysis.

[0004] Numerous hypochlorous acid fluorescent probes have been reported to date, most of which use electron-rich groups such as S, Se, Te, double bonds, hydrazine, and hydroxy acids as HClO / ClO. - The recognition site. The partially positively charged (+) Cl atoms in hypochlorous acid readily react with the aforementioned electron-rich (-) groups to form chlorides. Alternatively, they can be hydrolyzed or eliminated through the strong oxidizing properties of hypochlorous acid to induce fluorescence changes in the fluorophore.

[0005] Reactive hypochlorous acid fluorescent probes (see review Baruah M., Kwon HY, Cho H., Chang Y.T., Samanta AA Photoinduced Electron Transfer-Based Hypochlorite-Specific Fluorescent Probe for Selective Imaging of Proinflammatory M1 in a Rheumatoid Arthritis Model. Anal. Chem., 2023, 95(8):4147-54.) have been developed, including the oxidation of sulfur-containing groups to sulfoxides, the conversion of dithioacetal protecting groups to carbonyl groups, and the oxidation of tellurium to tellurium-oxygen double bonds, which have become commonly used selective recognition mechanisms for hypochlorous acid fluorescent probes. However, these reported probes still suffer from drawbacks such as poor fluorophore stability or difficulty in synthesis, which hinders their accurate assessment of the dynamic levels of hypochlorous acid in complex systems. In addition, the detection limits of current hypochlorous acid fluorescent probes are relatively high, making it difficult to quantitatively detect trace or even extremely trace amounts of hypochlorous acid in samples, thus limiting their application in biological systems. Summary of the Invention

[0006] To overcome the aforementioned deficiencies in the prior art, this invention proposes a fluorescence-activated fluorescent probe (Cou440) for the quantitative detection of hypochlorous acid. This invention enables the quantitative detection of trace amounts of hypochlorous acid in samples.

[0007] The core of this invention lies in constructing a classic photoinduced electron transfer (ICT) system using 7-diethylaminocoumarin. The probe itself exhibits a good PeT effect with almost no fluorescence. However, in the presence of hypochlorous acid, the hypochlorous acid reacts with the selenylphenyl portion on the probe molecule to form a selenium-oxygen double bond, thereby blocking the PeT effect of the molecule. The probe molecule then emits strong green fluorescence. Through the above scheme, an "on" type fluorescence response is obtained, achieving highly sensitive and specific detection of hypochlorous acid.

[0008] The hypochlorous acid fluorescent probe described in this invention is named Cou440, and its structural formula is shown in formula (I):

[0009]

[0010] The preparation method of the above fluorescent probe is as follows: a certain amount of 4-trifluoromethyl-7-diethylaminocoumarin (1), diphenyldiselenoether (2) and [bis(trifluoroacetoxy)iodide]benzene (3) are dissolved in dichloromethane, reacted for a certain time, dried by rotary evaporation and purified by column chromatography to obtain compound 4-trifluoromethyl-7-diethylamino-8-selenophenylcoumarin (4), namely: Cou440;

[0011] Preferably, the molar ratio of 4-trifluoromethyl-7-diethylaminocoumarin (1), diphenyldiselenoether (2) and [bis(trifluoroacetoxy)iodo]benzene (3) is 1:1.2:1.

[0012] Preferably, the molar-volume ratio of 4-trifluoromethyl-7-diethylaminocoumarin to dichloromethane is 1:2.27 to 5.56.

[0013] Preferably, the reaction temperature is 25°C and the reaction time is 0.5 hours.

[0014] The reaction formula for preparing the above probe is as follows:

[0015]

[0016] The usage method of the above-mentioned hypochlorous acid fluorescent probe is as follows:

[0017] Step 1: Add the same concentration of the compound shown in formula (I) to phosphate buffer solutions (10 mM, pH = 7.4) of hypochlorous acid at different concentrations to prepare at least 5 standard solutions containing the compound shown in formula (I) with different hypochlorous acid contents.

[0018] The concentration of the compound represented by formula (I) in the standard solution shown is 10 μM;

[0019] The hypochlorous acid content in the standard solutions shown ranges from 0.0 μM to 55.0 μM.

[0020] Step 2: Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 395 nm. Plot the hypochlorous acid concentration on the x-axis and Ig on the y-axis. 497 Establish a standard curve with the vertical axis as the ordinate;

[0021] I 497 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 497 nm;

[0022] Step 3: Add the compound shown in formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; measure its fluorescence emission spectrum under excitation light with an excitation wavelength of 395 nm, and calculate the hypochlorous acid content of the sample to be tested based on the standard curve.

[0023] This invention has the following characteristics:

[0024] 1) The fluorescent probe provided by this invention is a yellow solid powder with a stable structure.

[0025] 2) The fluorescent probe provided by this invention has a solution that is sensitive to the concentration of hypochlorous acid. As the concentration of hypochlorous acid increases, the fluorescence of its aqueous solution under ultraviolet light changes from no fluorescence to bright green.

[0026] 3) The fluorescent probe provided by this invention has an emission wavelength of 497nm, which is a fluorescence "on" response, and the fluorescence intensity changes significantly before and after the reaction (about 25 times). This can greatly eliminate the influence of differences in detection conditions on the results and improve the sensitivity of detection.

[0027] 4) The fluorescent probe provided by this invention has a linear relationship with the concentration of hypochlorous acid and can be used for the accurate measurement of hypochlorous acid concentration.

[0028] The "open" hypochlorous acid probe based on 7-diethylaminocoumarin dye provided by this invention has a good response to hypochlorous acid solution, enabling sensitive quantitative detection of hypochlorous acid in samples. It has the advantages of simple operation, low cost, sensitive response, and easy promotion and application. Attached Figure Description

[0029] Figure 1 : The proton NMR spectrum of the fluorescent probe Cou440.

[0030] Figure 2 The color response of the fluorescent probe Cou440 to hypochlorous acid.

[0031] Figure 3 UV response curve of fluorescent probe Cou440 in hypochlorous acid in phosphate buffer solution, where the probe concentration is 10.0 μM.

[0032] Figure 4 The fluorescence titration curve of the fluorescent probe Cou440 in hypochlorous acid in phosphate buffer solution, where the excitation wavelength was 395 nm and the probe concentration was 10.0 μM.

[0033] Figure 5 The fluorescence response of the fluorescent probe Cou440 to common reactive oxidized small molecules is shown in the figure. The excitation wavelength is 395 nm, the probe concentration is 10.0 μM, and the analyte concentration is 100.0 μM. Detailed Implementation

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] The compound numbers in the examples correspond to the numbers in the compounds described above.

[0037] Example 1: Synthesis of compound Cou440.

[0038] Synthesis of compound 4-trifluoromethyl-7-diethylamino-8-selenophenylcoumarin (4).

[0039] Take a 50 mL single-necked flask and accurately weigh 250 mg (0.88 mmol) of 4-trifluoromethyl-7-diethylaminocoumarin (1). Dissolve it in 2 mL of dichloromethane, then add diphenyldiselenoether (2) (328 mg, 1.05 mmol) and [bis(trifluoroacetoxy)iodide]benzene (3) (377 mg, 0.88 mmol) sequentially. React at room temperature for 0.5 hours. After the reaction is complete, evaporate the dichloromethane under reduced pressure. The crude product is then eluted by silica gel column chromatography with petroleum ether:dichloromethane = 5:1 to give a yellow solid Cou440 (251 mg, 65%).

[0040] 1 H NMR(400MHz,Chloroform-d)δ7.61(dt,J=9.0,2.0Hz,1H),7.39(dd,J=6.6,3.0Hz,2H),7.21(p, J=3.8Hz,3H),7.04(d,J=8.9Hz,1H),6.58(s,1H),3.34(q,J=7.0Hz,4H),1.03(t,J=7.0Hz,6H), such as Figure 1 The image shows the proton NMR spectrum of the fluorescent probe Cou440.

[0041] Example 2: Color response of compound Cou440 to hypochlorous acid.

[0042] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cou440 for detecting hypochlorous acid described in this invention. Measure 50 μL of this stock solution and add it dropwise to a phosphate buffer solution of hypochlorous acid of a certain concentration, then dilute to 5 mL with the corresponding phosphate buffer solution, so that the probe concentration in the test solution is 10.0 μM and the hypochlorous acid concentration is 35.0 μM, for color response testing. Figure 2 As shown, after adding hypochlorous acid solution, the fluorescence of the solution changed from no fluorescence to bright green fluorescence, indicating that the probe Cou440 has an intuitive color response to hypochlorous acid.

[0043] Example 3: Ultraviolet titration of compound Cou440 with different concentrations of hypochlorous acid.

[0044] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cou440 for detecting hypochlorous acid described in this invention. Take 50 μL of this stock solution and add it dropwise to phosphate buffer solutions of different concentrations of hypochlorous acid, then dilute to 5 mL with the corresponding phosphate buffer solution, so that the probe concentration in the test solution is 10.0 μM and the hypochlorous acid concentration is 35.0 μM. Perform absorption spectroscopy measurements. Obtain the UV absorption curve of the system. Figure 3 As shown, the absorbance at 395 nm decreases with the addition of hypochlorous acid.

[0045] Example 4: Fluorescent titration detection of compound Cou440 with different concentrations of hypochlorous acid.

[0046] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cou440 for detecting hypochlorous acid described in this invention. Measure 50 μL of this stock solution and add it dropwise to phosphate buffer solutions of different concentrations of hypochlorous acid, then dilute to 5 mL with the corresponding phosphate buffer solution. This ensures that the probe concentration in the test solution is 10.0 μM and the hypochlorous acid concentration is 0-55.0 μM for fluorescence detection (λex = 395 nm, λem = 497 nm). Measure the fluorescence intensity in each system and establish a standard curve comparing fluorescence intensity with hypochlorous acid concentration. Figure 4 As shown, the fluorescence intensity at 497 nm gradually increases with increasing hypochlorous acid concentration, reaching its maximum when the hypochlorous acid concentration reaches 35.0 μM. Furthermore, at low concentrations, the fluorescence intensity at 497 nm (Ig) is relatively low. 497 There was a good linear relationship between the concentrations of α and β and hypochlorous acid (0-35.0 μM) (R0). 2 =0.992).

[0047] Example 5: Selectivity experiment of different common reactive oxygen species on compound Cou440.

[0048] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cou440 for detecting hypochlorous acid as described in this invention. Prepare 10 mM solutions of various small reactive oxygen species to be tested as backup. Measure 50 μL of this stock solution and add it dropwise to the phosphate buffer solutions of different small molecule targets, and then dilute to 5 mL with the corresponding phosphate buffer solutions, so that the concentration of the probe in the test solution is 10.0 μM and the concentration of the small molecule target is 100.0 μM for fluorescence detection (λex = 395 nm, λem = 497 nm). Calculate the fluorescence intensity in each system and establish a fluorescence intensity (IL) indicator. 497 A bar chart showing the relationship between the analytes and the various test objects. (e.g.) Figure 5 As shown, apart from hypochlorous acid, other common reactive oxygen species have almost no effect on the fluorescence of probe Cou440.

[0049] Example 6: UV selectivity experiment of different common reactive oxygen species to compound Cou440.

[0050] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cou440 for detecting hypochlorous acid as described in this invention. Prepare 10 mM solutions of various different reactive oxygen species (ROS) molecules for testing. Measure 50 μL of this stock solution and add it dropwise to phosphate buffer solutions of different ROS molecules, and then dilute to 5 mL with the corresponding phosphate buffer solutions to achieve a probe concentration of 10.0 μM and an ROS concentration of 100.0 μM in the test solution. Perform absorption spectroscopy measurements. Obtain the UV absorption curves for each system and establish a standard curve for absorbance versus different ROS molecules. Except for hypochlorous acid, other common ROS molecules have almost no effect on the absorbance of probe Cou440.

Claims

1. A fluorescent probe for detecting hypochlorous acid, characterized in that: Its molecular formula C 20 H 18 F3NO2Se, abbreviated as Cou440, has the structural formula (I). 。 2. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that, The synthesis steps are as follows: 4-Trifluoromethyl-7-diethylaminocoumarin (1), diphenyldiselenoether (2) and [bis(trifluoroacetoxy)iodide]benzene (3) were dissolved in dichloromethane and reacted for a period of time to obtain 4-trifluoromethyl-7-diethylamino-8-selenylphenylcoumarin, namely: Cou440.

3. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The molar ratio of 4-trifluoromethyl-7-diethylaminocoumarin (1), diphenyldiselenoether (2) and [bis(trifluoroacetoxy)iodo]benzene (3) is 1:1.2:

1.

4. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The molar-volume ratio of 4-trifluoromethyl-7-diethylaminocoumarin (1) to dichloromethane is 1:(2.27-5.56).

5. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The reaction temperature was 25℃ and the reaction time was 0.5 hours.

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