A ratiometric fluorescent probe for detecting hypochlorous acid and its preparation method and use method

Abstract

The present invention discloses a ratiometric fluorescent probe for detecting hypochlorous acid, and its preparation and use methods. The probe obtained by the present invention itself has a good ICT effect and exhibits a weak light orange fluorescence. However, when a small amount of hypochlorous acid is present, hypochlorous acid reacts with thioketal to generate acetyl groups, at which point the orange fluorescence weakens. When the concentration of hypochlorous acid in the system further increases, hypochlorous acid can undergo an oxidation reaction with the sulfur atom on the probe molecule phenothiazine to generate sulfoxide, further blocking the ICT effect of the molecule. The probe molecule emits a strong cyan-blue fluorescence. Through the above-mentioned step-by-step oxidation design, a broader and more sensitive "ratio" type fluorescence response to hypochlorous acid is obtained, achieving highly sensitive and specific detection of hypochlorous acid. The present invention can achieve sensitive and quantitative detection of trace amounts of hypochlorous acid in a sample, and has the advantages of simple operation, low cost, sensitive response, and easy promotion and application.

Description

Technical Field

[0001] The present invention belongs to the field of organic small molecule fluorescent probes, and specifically relates to 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazine-2(11H)-one used as a hypochlorous acid fluorescent probe, and a preparation method and a use method thereof. Background Art

[0002] Hypochlorous acid (HClO) is one of the important endogenous reactive oxygen species (ROS) in organisms. It is mainly generated by the reaction of hydrogen peroxide (H2O2) and chloride ions (Cl2) induced by the heme enzyme myeloperoxidase (MPO) on the mitochondrial membrane of neutrophils to reduce nicotinamide adenine dinucleotide phosphate (NADPH). - ) reaction, and the process of producing hypochlorous acid is also called MPO-H2O2-Cl - Hypochlorous acid plays an important role in the innate immune system of living organisms. When neutrophils engulf foreign pathogens, they will pass through MPO-H2O2-Cl - The system produces hypochlorous acid to kill pathogens. Under normal circumstances, the generation and clearance of ROS in organisms is a dynamic equilibrium process. Excessive ROS are cleared by intracellular antioxidants such as glutathione peroxidase, superoxide dismutase, and vitamin E. However, when a large number of pathogens invade or the body suffers a major trauma, the immune system produces excessive hypochlorous acid, which not only kills pathogens but also causes damage to biological macromolecules, including lipid peroxidation, oxidative damage to DNA and RNA, oxidation of monosaccharides, and oxidation of proteins, leading to inflammation and a series of diseases. Therefore, monitoring the level of hypochlorous acid in the body helps us better understand the immune process and physiological and pathological mechanisms, which is of great significance.

[0003] Fluorescence detection has attracted widespread attention from researchers due to its excellent detection sensitivity and selectivity, and its ability to achieve real-time, online detection of samples to be tested.

[0004] Currently developed small-molecule fluorescent probes for hypochlorous acid detection are primarily designed based on hypochlorous acid's selective oxidation, deprotection, and oxidative cleavage reactions. In the presence of hypochlorous acid, the detection groups in the probe molecules react specifically with the acid, altering the molecule's inherent PET or ICT effects and causing changes in the probe's fluorescence properties, thereby enabling specific recognition of hypochlorous acid.

[0005] However, reaction-based fluorescent probes for hypochlorous acid (reviewed by Nahyun Kwon, Yahui Chen, Xiaoqiang Chen, Myung Hwa Kim, Juyoung Yoon, Dyes and Pigments, 2022, 200, 110132)—including oxidation of sulfur-containing groups to sulfoxides, conversion of dithioacetal protecting groups to carbonyls, and selective conversion of oximes or hydrazones to aldehydes or carboxylic acids—have become common selective recognition mechanisms for hypochlorous acid fluorescent probes. However, these reported probes still suffer from poor fluorophore stability, difficult synthesis, or short detection wavelengths, hindering their ability to accurately assess the dynamic levels of hypochlorous acid in complex systems. Furthermore, the relatively high detection limits of current hypochlorous acid fluorescent probes make it difficult to quantitatively detect trace or even extremely trace amounts of hypochlorous acid in samples, limiting their application in vivo. Summary of the Invention

[0006] To overcome the above-mentioned drawbacks of the prior art, the present invention proposes a ratiometric fluorescent probe (EMDP) for quantitative detection of hypochlorous acid. The present invention can be used to quantitatively detect trace amounts of hypochlorous acid in samples.

[0007] The core of this invention lies in constructing a classic intramolecular charge transfer (ICT) system using 3-acetyl-11-methylpyrazolo[2,3-b]phenothiazin-2(11H)-one. The probe itself exhibits a strong ICT effect, resulting in a weak, light-orange fluorescence. However, in the presence of a small amount of hypochlorous acid, the hypochlorous acid reacts with the thioketal to form an acetyl group, which weakens the orange fluorescence. As the concentration of hypochlorous acid in the system increases, the hypochlorous acid oxidizes the sulfur atom on the phenothiazine molecule to form a sulfoxide, further blocking the ICT effect. The probe molecule then emits strong cyan-blue fluorescence. This step-by-step oxidation design achieves a broader, more sensitive "ratiometric" fluorescence response to hypochlorous acid, enabling highly sensitive and specific detection of hypochlorous acid.

[0008] The hypochlorous acid fluorescent probe of the present invention is named EMDP, and its structural formula is shown in formula (I):

[0009]

[0010] The preparation method of the fluorescent probe is as follows: a certain amount of 3-acetyl-11-methylpyrazolo[2,3-b]phenothiazine-2(11H)-one (1), 1,2-ethanedithiol (2) and boron trifluoride etherate (3) are dissolved in dichloromethane, reacted for a certain time, and then dried and washed with water to obtain the compound 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazine-2(11H)-one (4), namely: EMDP.

[0011] The preparation reaction formula of the above probe is as follows:

[0012]

[0013] The method for using the above hypochlorous acid fluorescent probe is as follows:

[0014] Step 1: Add the same concentration of the compound represented by formula (I) to phosphate buffer solutions (10 mM, pH = 7.4) containing hypochlorous acid at different concentrations to prepare at least 8 standard solutions containing the compound represented by formula (I) at different hypochlorous acid contents;

[0015] The concentration of the compound represented by formula (I) in the standard solution shown is 1 nM to 10 μM;

[0016] The content of hypochlorous acid in the standard solutions shown is 0.1 nM to 1 mM;

[0017] Step 2: Measure the fluorescence emission spectrum of the standard solution respectively, with the excitation wavelength being 425 nm, the hypochlorous acid concentration being the horizontal axis, and the I being the 495 / I 566 As the vertical axis, establish a standard curve;

[0018] I 495 / I 566 It represents the ratio of the fluorescence emission intensity of the standard solution at wavelengths of 495 nm and 566 nm;

[0019] Step 3: Add the compound represented by formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound represented by formula (I) in the standard solution; measure its fluorescence emission spectrum under excitation light with an excitation wavelength of 425 nm, that is, calculate the hypochlorous acid content of the sample to be tested based on the standard curve.

[0020] The present invention has the following characteristics:

[0021] 1) The fluorescent probe provided by the present invention is a yellow solid powder with a stable structure.

[0022] 2) The fluorescent probe provided by the present invention has a solution that is sensitive to the concentration of hypochlorous acid. As the concentration of hypochlorous acid increases, the fluorescence of the aqueous solution observed under ultraviolet light changes from orange-yellow fluorescence to bright cyan-blue fluorescence.

[0023] 3) The fluorescent probe provided by the present invention has an emission wavelength of 566 nm. After reacting with hypochlorous acid, the emission wavelength decreases to 495 nm. This is a fluorescence "ratio" response, which can effectively overcome the influence of external factors such as probe concentration, environmental conditions, instrument and voltage stability during detection, thereby improving the sensitivity of detection.

[0024] 4) The fluorescent probe provided by the present invention has a linear relationship with the concentration of hypochlorous acid and can be used to accurately measure the concentration of hypochlorous acid.

[0025] The "ratio" type hypochlorous acid probe based on 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazine-2(11H)-one dye provided by the present invention has a good response to hypochlorous acid solution, can realize sensitive quantitative detection of hypochlorous acid in samples, and has the advantages of simple operation, low cost, sensitive response, and easy promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : H NMR spectrum of fluorescent probe EMDP.

[0027] Figure 2 : Color response diagram of fluorescent probe EMDP to hypochlorous acid phosphate buffer solution.

[0028] Figure 3 : Fluorescence response of fluorescent probe EMDP to hypochlorous acid phosphate buffer solution.

[0029] Figure 4 : UV titration curve of fluorescent probe EMDP with hypochlorous acid in phosphate buffer solution, where the probe concentration is 10.0 μM.

[0030] Figure 5 : Fluorescence titration curve of fluorescent probe EMDP with hypochlorous acid in phosphate buffer solution, where the excitation wavelength is 425 nm and the probe concentration is 10.0 μM.

[0031] Figure 6 : Fluorescence response diagram of the fluorescent probe EMDP to common active oxidative small molecules, where the excitation wavelength is 425nm, the probe concentration is 10.0 μM, and the analyte concentration is 100.0 μM. DETAILED DESCRIPTION

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples were obtained from commercial sources.

[0034] The compound numbers in the examples correspond to the compound numbers described above.

[0035] Example 1. Synthesis of compound EMDP.

[0036] Synthesis of compound 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazin-2(11H)-one (4).

[0037] 0.2 g of 3-acetyl-11-methylpyrazolo[2,3-b]phenothiazin-2(11H)-one (1) (618.49 mmol) and 77.81 μL of 1,2-ethanedithiol (2) (927.74 mmol) were dissolved in 10 mL of dichloromethane, and 391.88 μL of boron trifluoride etherate (3) (3.09 mol) was added. The mixture was reacted at room temperature for 8 hours, and then dried and washed with water to obtain 0.211 g of compound 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazin-2(11H)-one (4) with a yield of 85%.

[0038] 1 H NMR (400 MHz, Chloroform-d) δ 7.83 (d, J = 8.2 Hz, 1H), 7.56 –7.46 (m, 4H), 7.44 (d, J = 1.1 Hz, 1H), 7.37 (dq, J = 6.1, 2.1, 1.5 Hz, 7H), 6.96 (t, J = 7.7 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.10 (s, 1H), 2.61 (s,3H), 2.34 (s, 3H).

[0039] Example 2: Synthesis of compound EMDP.

[0040] Synthesis of compound 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazin-2(11H)-one (4).

[0041] 0.2 g of 3-acetyl-11-methylpyrazolo[2,3-b]phenothiazin-2(11H)-one (1) (618.49 mmol) and 77.81 μL of 1,2-ethanedithiol (2) (927.74 mmol) were dissolved in 1 mL of dichloromethane, and 391.88 μL of boron trifluoride etherate (3) (3.09 mol) was added. The mixture was reacted at 2°C for 22 hours, and then the mixture was dried and washed with water to obtain 0.206 g of compound 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazin-2(11H)-one (4) with a yield of 82%.

[0042] Example 3: Synthesis of compound EMDP.

[0043] Synthesis of compound 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazin-2(11H)-one (4).

[0044] 0.2 g of 3-acetyl-11-methylpyrazolo[2,3-b]phenothiazin-2(11H)-one (1) (618.49 mmol) and 77.81 μL of 1,2-ethanedithiol (2) (927.74 mmol) were dissolved in 90 mL of dichloromethane, and 391.88 μL of boron trifluoride etherate (3) (3.09 mol) was added. The mixture was reacted at 40°C for 2 hours, and the mixture was dried and washed with water to obtain 0.203 g of compound 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazin-2(11H)-one (4) with a yield of 81%.

[0045] Example 4: Color response of compound EMDP to hypochlorous acid.

[0046] Prepare a 1 mM dimethylformamide stock solution of the fluorescent probe EMDP for detecting hypochlorous acid described in the present invention. Measure 50 μL of this stock solution and add it dropwise to a phosphate buffer solution containing a certain concentration of hypochlorous acid. Then, dilute the volume to 5 mL with the corresponding phosphate buffer solution. This allows the probe concentration in the test solution to be 10.0 μM and the hypochlorous acid concentration to be 80.0 μM for color response testing. Figure 2 and 3 As shown in the figure, after adding hypochlorous acid solution, the color of the solution was observed to change from light yellow to colorless by naked eyes, and the fluorescence of the solution also changed from orange-yellow fluorescence to bright cyan-blue fluorescence, indicating that the probe EMDP has an intuitive color response to hypochlorous acid.

[0047] Example 5: UV titration detection of compound EMDP using hypochlorous acid at different concentrations.

[0048] Prepare a 1 mM dimethylformamide mother liquor solution of the fluorescent probe EMDP for detecting hypochlorous acid described in the present invention for use. Measure 50 μL of this mother liquor and add it dropwise to phosphate buffer solutions of different hypochlorous acid concentrations, and dilute it 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 0-80.0 μM for absorption spectrum testing. Obtain the ultraviolet absorption curve in each system and establish a standard curve of absorbance and hypochlorous acid concentration. Figure 4 As shown in Figure 2, with the increase of hypochlorous acid concentration, the absorbance at 566 nm gradually decreased (A 566 ), while the absorbance at 495 nm continued to increase (A 495 ), and the ratio of the two is A 495 / A 566There was a good linear relationship (R 2 =0.990).

[0049] Example 6. Fluorescence titration detection of compound EMDP using hypochlorous acid at different concentrations.

[0050] Prepare a 1 mM dimethylformamide mother solution of the fluorescent probe EMDP for detecting hypochlorous acid described in the present invention for use. Measure 50 μL of this mother solution and add it dropwise to phosphate buffer solutions of different hypochlorous acid concentrations, and dilute it 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 0-80.0 μM for fluorescence detection (λex = 495 nm, λem = 566 nm). Obtain the fluorescence intensity in each system and establish a standard curve of fluorescence intensity and hypochlorous acid concentration. Figure 5 As shown in the figure, with the increase of hypochlorous acid concentration, the fluorescence intensity ratio of the system (I 566 / I 495 ) gradually increased. When the concentration of hypochlorous acid reached 80.0 μM, the fluorescence intensity ratio of the reaction system (I 565 / I 494 ) reaches its maximum value. In addition, at low concentrations, the fluorescence intensity at 566 nm (I 566 ) and the concentration of hypochlorous acid (0-60 μM) showed a good linear relationship (R 2 = 0.990).

[0051] Example 7: Selectivity of compound EMDP for different common reactive oxygen species.

[0052] Prepare a 1 mM dimethylformamide mother solution of the fluorescent probe EMDP for detecting hypochlorous acid described in the present invention for standby use. Prepare 10 mM solutions of various reactive oxygen species to be tested for standby use. Measure 50 μL of this mother solution and add it dropwise to the phosphate buffer solutions of different small molecules to be tested, and dilute it to 5 mL with the corresponding phosphate buffer solution so that the concentration of the probe in the test solution is 10.0 μM and the concentration of the small molecule to be tested is 100.0 μM for fluorescence detection (λex = 495 nm, λem = 566 nm). Obtain the fluorescence intensity in each system and establish a fluorescence intensity ratio (I 495 / I 566 ) and the relationship between each analyte. Figure 6 As shown, other common reactive oxygen species small molecules have little effect on the fluorescence of the probe EMDP.

Claims

1. A fluorescent probe for detecting hypochlorous acid, characterized in that: Its molecular formula C 20 H 17 NO2S3, referred to as EMDP, has the structural formula (I); 2. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, wherein: The synthesis steps are as follows: 3-Acetyl-11-methylpyrazolo[2,3-b]phenothiazine-2(11H)-one, 1,2-ethanedithiol and boron trifluoride etherate were dissolved in dichloromethane and reacted for a period of time to obtain 11-methyl-3-(2-methyl-1,3-dithiolan-2-yl)pyrone[2,3-b]phenothiazine-2(11H)-one, namely: EMDP. The preparation reaction formula of the probe is as follows:

3. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, wherein: The molar ratio of 3-acetyl-11-methylpyrazolo[2,3-b]phenothiazine-2(11H)-one, 1,2-ethanedithiol and boron trifluoride etherate is 1:1.5:5; the reaction temperature is 0-40°C, and the reaction time is 1-24 hours.

4. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, wherein: i) the molar ratio of 3-acetyl-11-methylpyrazolo[2,3-b]phenothiazine-2(11H)-one, 1,2-ethanedithiol and boron trifluoride etherate is 1:1.5:5; the reaction temperature is 25° C., and the reaction time is 8 hours.

5. A method for using the fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that: Adding the compound of formula (I) according to claim 1 at the same concentration to phosphate buffer solutions containing hypochlorous acid at different concentrations to prepare at least five standard solutions containing the compound of formula (I) at different hypochlorous acid contents; The concentration of the compound represented by formula (I) in the standard solution is 1 nM to 10 μM; The content of hypochlorous acid in the standard solution shown is 0.1nM to 1mM; The fluorescence emission spectra of the standard solutions were measured respectively, with the excitation wavelength being 425 nm, the hypochlorous acid concentration being the abscissa, and the I being the ordinate. 495 / I 566 As the vertical axis, establish a standard curve; I 494 / I 565 It represents the ratio of the fluorescence emission intensity of the standard solution at wavelengths of 495 nm and 566 nm; Adding the compound represented by formula (I) to the sample to be tested, controlling the concentration thereof to be equal to the concentration of the compound represented by formula (I) in the standard solution; The fluorescence emission spectrum of the sample under the excitation light having an excitation wavelength of 425 nm is measured, and the hypochlorous acid content of the sample to be tested is calculated based on the standard curve.

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