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

Abstract

This invention discloses a reversible fluorescent probe for detecting hypochlorous acid, its preparation method, and its usage. The invention utilizes 7-diethylaminocoumarin to construct a classic intramolecular charge transfer system. The probe itself exhibits excellent ICT (Intramolecular Charge Transfer) effect and strong green fluorescence. However, in the presence of hypochlorous acid, the hypochlorous acid reacts with the selenomethyl group on the probe molecule to form a selenium-oxygen double bond, transforming the linking group from an electron-donating group to an electron-withdrawing group, thus weakening the ICT effect and quenching the fluorescence of the probe molecule. Simultaneously, by adding glutathione to the hypochlorous acid, the fluorescence emission becomes reversible. Glutathione reacts with the selenomethyl group on the probe molecule to form a selenium-oxygen double bond, restoring the linking group from an electron-withdrawing group to an electron-donating group, enhancing the ICT effect, and significantly increasing the fluorescence of the probe molecule. This invention exhibits excellent response to hypochlorous acid solution.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to 3-selenomethyl-4-methyl-7-diethylaminocoumarin used as a reversible fluorescent probe for hypochlorous acid, and its preparation and usage methods. Background Technology

[0002] Hypochlorous acid (HOCl) is an important reactive oxygen species in cells, produced by the catalysis of hydrogen peroxide and chloride ions by myeloperoxidase. HOCl plays a vital physiological role in organisms, participating in numerous physiological and pathological processes, effectively resisting the invasion of microbial pathogens and stimulating the body's acquired immune response. However, excessive expression of HOCl can damage tissues and organs, leading to various diseases such as rheumatoid arthritis, cardiovascular disease, and even cancer. Therefore, the detection of HOCl concentration and the study of its physiological functions have received widespread attention. However, due to the low concentration and short duration of HOCl in living organisms, its detection and research have always been challenging. The low concentration requires probe molecules to possess not only high selectivity but also very high sensitivity, while the short duration necessitates a rapid response between the probe molecules and HOCl.

[0003] Traditional methods for detecting HOCl mainly include iodometric methods, colorimetric methods, electrochemical methods, and spectrophotometric methods. However, most of these methods suffer from drawbacks such as complex operation, expensive equipment, and time-consuming pretreatment. In contrast, fluorescence methods, as a non-invasive detection technique, have attracted widespread attention from researchers due to their numerous advantages over traditional detection techniques (such as high sensitivity, good selectivity, rapid response, in-situ detection, and real-time monitoring).

[0004] Researchers have designed and developed various fluorescent probes for the detection of hypochlorous acid based on the strong oxidizing properties of HOCl. The partially positively charged (+) chlorine atoms in hypochlorous acid readily react with the aforementioned electron-rich (-) groups to form chlorides. Alternatively, the strong oxidizing properties of hypochlorous acid can be used to hydrolyze or eliminate these groups, inducing fluorescence changes in the fluorophore.

[0005] However, reactive hypochlorous acid fluorescent probes (see review: Geng Y., Wang Z., Zhou J., et al., Recent progress in the development of fluorescent probes for imaging pathological oxidative stress[J]. Chemical Society Reviews, 2023, 52(11), 3873-3926.) have become commonly used selective recognition mechanisms for hypochlorous acid fluorescent probes, 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. However, many probes still suffer from drawbacks such as lengthy synthesis steps, low quantum yield, slow response, and small Stokes shift, which limit their biological applications. Summary of the Invention

[0006] To overcome the aforementioned deficiencies in existing technologies, this invention proposes a reversible fluorescent probe (Cse325) for the quantitative detection of hypochlorous acid. This probe, after fluorescence quenching by hypochlorous acid oxidation, can be restored by glutathione reduction. This invention enables the dynamic detection of trace amounts of hypochlorous acid in samples.

[0007] The core of this invention lies in constructing a classic intramolecular charge transfer (ICT) system using 7-diethylaminocoumarin. The probe itself exhibits a strong ICT effect and intense green fluorescence. However, in the presence of hypochlorous acid, the hypochlorous acid reacts with the selenomethyl group on the probe molecule to form a selenium-oxygen double bond, transforming the linking group from an electron-donating group to an electron-withdrawing group. This weakens the ICT effect and quenches the fluorescence of the probe molecule. Simultaneously, by adding glutathione to the hypochlorous acid, the fluorescence emission becomes reversible. Glutathione reacts with the selenomethyl group on the probe molecule to form a selenium-oxygen double bond, restoring the linking group from an electron-withdrawing group to an electron-donating group. This enhances the ICT effect and significantly strengthens the fluorescence of the probe molecule, achieving highly sensitive and specific detection of both hypochlorous acid and glutathione.

[0008] This invention comprises compounds 1-6, with the structural formula shown in formula (I):

[0009]

[0010] The reversible fluorescent probe described in this invention is named Cse325, i.e., compound 1, and its structural formula is shown in formula (II):

[0011]

[0012] The preparation method of the above fluorescent probe is as follows: a certain amount of 4-methyl-7-diethylaminocoumarin (1a), dimethyl diselenoether (1b) and [bis(trifluoroacetoxy)iodide]benzene (1c) are dissolved in dichloromethane, reacted for a certain time, dried by rotary evaporation and purified by column chromatography to obtain compound 3-selenomethyl-4-methyl-7-diethylaminocoumarin (1), namely: Cse325.

[0013] The reaction formula for preparing the above probe is as follows:

[0014]

[0015] Compounds 2-6 were prepared using the same method as probe Cse325.

[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 (II) to phosphate buffer solutions (10mM, pH=7.4) of hypochlorous acid of different concentrations to prepare at least 5 standard solutions containing the compound shown in formula (II) with different hypochlorous acid contents.

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

[0019] The hypochlorous acid content in the standard solutions shown ranges from 0 μM to 100 μM.

[0020] Step 2: Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 400 nm. Plot the hypochlorous acid concentration on the x-axis and Ig on the y-axis. 486 Establish a standard curve with the vertical axis as the ordinate;

[0021] I 486 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 486 nm;

[0022] Step 3: Add the compound shown in formula (II) to the sample to be tested, and control its concentration to be equal to the concentration of the compound shown in formula (II) in the standard solution; measure its fluorescence emission spectrum under excitation light with an excitation wavelength of 400 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 bright green to weak fluorescence.

[0026] 3) The fluorescent probe provided by this invention has an emission wavelength of 486nm, is a fluorescent "reversible" response, and has a large change in fluorescence intensity before and after the reaction (about 5 times), which 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 redox hypochlorous acid probe based on 7-diethylaminocoumarin dye provided by this invention has a good response to hypochlorous acid solution, enabling sensitive dynamic 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 Cse325.

[0030] Figure 2 The color response of the fluorescent probe Cse325 to hypochlorous acid.

[0031] Figure 3 UV titration curve of fluorescent probe Cse325 in hypochlorous acid in phosphate buffer solution, where the probe concentration is 10.0 μM.

[0032] Figure 4 Fluorescence titration curve of fluorescent probe Cse325 in hypochlorous acid in phosphate buffer solution, where the excitation wavelength is 400 nm and the probe concentration is 10.0 μM.

[0033] Figure 5 The fluorescence response of the fluorescent probe Cse325 to common reactive oxidized small molecules is shown in the figure, where the excitation wavelength is 400 nm, the probe concentration is 10.0 μM, and the analyte concentration is 20.0 μM.

[0034] Figure 6 The fluorescent probe Cse325 undergoes a three-cycle reaction in phosphate buffer solution: hypochlorous acid-glutathione-hypochlorous acid, with an excitation wavelength of 400 nm and a probe concentration of 10.0 μM. Detailed Implementation

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] The compound numbers in the examples correspond to the numbers in the compounds described above.

[0038] Example 1: Synthesis of compound Cse325.

[0039] Synthesis of compound 3-selenomethyl-4-methyl-7-diethylaminocoumarin (1).

[0040] Take a 50 mL single-necked flask and accurately weigh 4-methyl-7-diethylaminocoumarin (1a) (400 mg). Dissolve it in 5 mL of dichloromethane, then add dimethyl diselenyl ether (1b) (390.18 mg, 1.2 eq) and [bis(trifluoroacetoxy)iodide]benzene (1c) (743.71 mg, 1 eq) sequentially. React at room temperature for 0.5 hours. After the reaction is complete, evaporate the solvent under reduced pressure. The crude product is then subjected to silica gel column chromatography with petroleum ether:ethyl acetate = 20:1 as the eluent to obtain a yellow solid Cse325 (300 mg, 53%).

[0041] 1 H NMR(400MHz,Chloroform-d)δ7.45(d,J=9.0Hz,1H),6.61(dd,J=9.1,2.6Hz,1H),6.51( d,J=2.6Hz,1H),3.44(q,J=7.1Hz,4H),2.70(s,3H),2.33(s,3H),1.24(t,J=7.1Hz,6H).

[0042] Example 2: Synthesis of compounds 2-6.

[0043] Synthesis of the same compound Cse325.

[0044] Compound 2: 1 H NMR(400MHz,Chloroform-d)δ7.49(d,J=9.1Hz,1H),7.40(d,J=7.4Hz,2H),7.22(q,J=8.9,7.6Hz,3H),6.6 3(dd,J=9.1,2.6Hz,1H),6.52(d,J=2.5Hz,1H),3.46(q,J=7.1Hz,4H),2.68(s,3H),1.25(t,J=7.1Hz,6H).

[0045] Compound 3: 1 H NMR(400MHz,Chloroform-d)δ7.34(d,J=9.0Hz,1H),7.24–7.10(m,5H),6.58(dd,J=9.1,2.6Hz,1 H), 6.51 (d, J = 2.6Hz, 1H), 4.15 (s, 2H), 3.43 (q, J = 7.1Hz, 4H), 2.32 (s, 3H), 1.23 (t, J = 7.1Hz, 7H).

[0046] Compound 4: 1 H NMR (400MHz, Chloroform-d) δ7.06 (s, 1H), 3.29 (dt, J=8.3, 5.5Hz, 4H), 2.92 (t, J= 6.5Hz,2H),2.82(t,J=6.4Hz,2H),2.67(s,3H),2.30(s,3H),2.00(h,J=5.8Hz,4H).

[0047] Compound 5: 1 H NMR(400MHz,Chloroform-d)δ7.41–7.36(m,2H),7.25–7.17(m,3H),7.09(s,1H),3.31(dt,J=8.1,5. 6Hz, 4H), 2.93 (t, J=6.5Hz, 2H), 2.82 (t, J=6.3Hz, 2H), 2.65 (s, 3H), 2.01 (qd, J=7.8, 6.9, 2.5Hz, 4H).

[0048] Compound 6: 1 H NMR(400MHz,Chloroform-d)δ7.30–7.20(m,5H),7.02(s,1H),4.21(s,2H),3.36(q,J=5.6 Hz, 4H), 3.01 (t, J = 6.5Hz, 2H), 2.85 (t, J = 6.3Hz, 2H), 2.37 (s, 3H), 2.08 (h, J = 6.0Hz, 4H).

[0049] Example 3: Color response of compound Cse325 to hypochlorous acid.

[0050] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cse325 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 15.0 μM, for color response testing. Figure 2 As shown, after adding hypochlorous acid solution, the fluorescence of the solution changed from bright green fluorescence to no fluorescence, indicating that the probe Cse325 has an intuitive color response to hypochlorous acid.

[0051] Example 4: Ultraviolet titration detection of compound Cse325 with different concentrations of hypochlorous acid.

[0052] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cse325 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 15.0 μM. Perform absorption spectroscopy measurements. Obtain the UV absorption curve of the system. Figure 3 As shown, with the addition of hypochlorous acid, the absorption peak position shifted from 400 nm to 413 nm.

[0053] Example 5: Fluorescent titration detection of compound Cse325 with different concentrations of hypochlorous acid.

[0054] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cse325 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. This ensures that the probe concentration in the test solution is 10.0 μM and the hypochlorous acid concentration is 0-22.0 μM for fluorescence detection (λex = 400 nm, λem = 486 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 486 nm gradually decreases with increasing hypochlorous acid concentration, reaching its minimum when the hypochlorous acid concentration reaches 15.0 μM. Furthermore, at low concentrations, the fluorescence intensity at 486 nm (Ig) is significantly lower. 486 There was a good linear relationship between the concentrations of α and β and hypochlorous acid (0-15.0 μM) (R0). 2 =0.994).

[0055] Example 6: Fluorescence selectivity experiment of different common reactive oxygen species on compound Cse325.

[0056] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cse325 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 15.0 μM for fluorescence detection (λex = 400 nm, λem = 486 nm). Calculate the fluorescence intensity in each system and establish a fluorescence intensity (IL) indicator. 486 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 Cse325.

[0057] Example 7: Cyclic response of compound Cse325 to hypochlorous acid-glutathione

[0058] Prepare a 1 mM ethanol stock solution of the fluorescent probe Cse325 for detecting hypochlorous acid described in this invention. Prepare a 10 mM hypochlorous acid solution. Prepare a 25 mM glutathione solution. 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 15.0 μM. Perform absorption spectroscopy testing. Obtain the fluorescence emission spectrum of the system. Figure 6 As shown, probe Cse325 can achieve a three-cycle redox cycle of hypochlorous acid-glutathione-hypochlorous acid in phosphate buffer solution, proving the reversibility of probe Cse325.

Claims

1. A reversible fluorescent probe for detecting hypochlorous acid, characterized in that: Its molecular formula C 15 H 19 NO2Se, abbreviated as Cse325, has the structural formula (I); 2. The method for preparing a reversible fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that, The synthesis steps are as follows: 4-Methyl-7-diethylaminocoumarin, dimethyl diselenyl ether, and [bis(trifluoroacetoxy)iodide]benzene were dissolved in dichloromethane and reacted for a period of time to obtain 3-selenomethyl-4-methyl-7-diethylaminocoumarin, namely: Cse325.

3. The method for preparing a reversible fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The molar ratio of 4-methyl-7-diethylaminocoumarin, dimethyl diselenyl ether, and [bis(trifluoroacetoxy)iodide]benzene is 1:1.2:1; the amount of dichloromethane is 5 mL; the reaction temperature is 25 °C, and the reaction time is 0.5 h.

4. A method for using a reversible fluorescent probe for detecting hypochlorous acid; characterized in that: 1) Add the same concentration of the compound shown in formula (I) to phosphate buffer solutions of hypochlorous acid of different concentrations to prepare at least 5 standard solutions containing the compound shown in formula (I) with different hypochlorous acid contents. The concentration of the compound represented by formula (I) in the standard solution shown is 10 μM; The hypochlorous acid content in the standard solution shown ranges from 0.0 nM to 100 μM. 2) Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 400 nm. Plot the hypochlorous acid concentration on the x-axis and Ig on the y-axis. 486 Establish a standard curve with the vertical axis as the ordinate; I 486 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 486 nm; 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; Measurement The fluorescence emission spectrum of the sample under excitation light with an excitation wavelength of 400 nm is used to calculate the hypochlorous acid content of the sample based on the standard curve.

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