Nitric oxide fluorescent probe as well as preparation method and use method thereof

Abstract

The invention discloses a fluorescent probe for detecting nitric oxide as well as a preparation method and a use method of the fluorescent probe. According to the probe, isophorone is used as a fluorescent mother group, substituted aniline is used as an NO recognition group, and the concentration level of NO in a water phase can be detected with high selectivity and high sensitivity. The probe itself presents red fluorescence, in the presence of nitric oxide, nitric oxide and amido on probe molecules are subjected to a nitrosation-deamination reaction to generate a corresponding denitrification product, the ICT effect of the probe molecules is inhibited, the emission wavelength of the probe is blue-shifted, and yellow fluorescence is emitted. According to the scheme, the probe obtains'ratio 'type fluorescence response to NO, and high-sensitivity and specific detection of nitric oxide is realized.

Description

Technical Field

[0001] The invention belongs to the field of organic small molecule fluorescent probes, and specifically relates to a preparation method and a use method of (E)-2-(3-(4-amino-3-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile used as a nitric oxide fluorescent probe. Background Art

[0002] Nitric oxide (NO) is a ubiquitous messenger molecule in biological systems, endogenously produced from NADPH, O2 and L-arginine under the action of three NO synthases (eNOS, nNOS and iNOS). It has a variety of physiological functions, including signal transduction, vasodilation and smooth muscle relaxation, and actively participates in immune-mediated pathological processes, playing important physiological functions in the cardiovascular system, reproductive system, central and peripheral nervous system. Dysregulation of NO will lead to a variety of diseases, including cancer, inflammation, endothelial dysfunction and neurodegeneration. Therefore, the development of specific detection methods for NO levels in the human body is crucial to understanding and studying its related physiological and pathological processes.

[0003] Fluorescence detection has attracted extensive attention from researchers due to its advantages such as strong operability, low cost, good biocompatibility and the ability to achieve real-time online detection in vivo.

[0004] In the past decade, researchers have developed a variety of NO fluorescent probes based on transition metal complexes and o-phenylenediamine recognition groups. However, due to poor biocompatibility, transition metal complex probes are rarely used in the study of NO in organisms; the N-acyl triazole product generated by the reaction of o-phenylenediamine-rhodamine lactam-based NO fluorescent probes with NO has a slow hydrolysis reaction and is easily attacked by biological thiols such as Cys, resulting in fluorescence quenching. In recent years, researchers have successively developed many new NO probes, such as amino nitrosation type, deamination type, Se ether type and Hans ester type. The development of these probes has solved the problems of biocompatibility, reactivity and selectivity to NO to a certain extent.

[0005] However, based on reactive nitric oxide fluorescent probes (see review Chenqian Ye, Shufang Lin, Jinyi Li, Peng Meng, Luqiang Huang, Daliang Li, Comprehensive insights into fluorescent probes for the determination nitric oxide for diseases diagnosis, Bioorganic Chemistry, 2024, 150, 107505), including o-phenylenediamines, Hans esters, amino nitrosation and other NO response modes, although they can specifically detect the concentration level of NO, most of the probes still have problems such as poor stability of fluorescent probes, complicated and difficult synthesis process, and insufficient sensitivity due to the single detection mode, which limits their further application in vivo. Summary of the invention

[0006] In order to overcome the above defects of the prior art, the present invention proposes a ratiometric fluorescent probe (BNOH) for detecting nitric oxide. The present invention can be used to quantitatively detect the content of nitric oxide in a sample.

[0007] Compared with traditional fluorescent dyes, dicyanoisophorone fluorescent dyes have the characteristics of red emission and large Stokes shift, and are easy to synthesize. They have been widely used in the field of fluorescent probes in recent years. Therefore, based on the isophorone fluorescent parent group, we constructed a classic intramolecular charge transfer (ICT) fluorescent probe. Due to its ICT effect, the probe itself exhibits red fluorescence, but when nitric oxide is present, nitric oxide undergoes a reduction deamination reaction with the amine group on the probe molecule, thereby inhibiting the ICT effect of the molecule, causing the probe molecule to emit yellow fluorescence. Through the above scheme, a "ratio" type fluorescence response is obtained, achieving highly sensitive and specific detection of nitric oxide.

[0008] The nitric oxide fluorescent probe of the present invention is named (E)-2-(3-(4-amino-3-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile, 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 (1), stannous chloride dihydrate (2) and hydrochloric acid (3) are dissolved in anhydrous ethanol and reacted at a certain temperature for a period of time. After the reaction is complete, the mixture is dried and subjected to column chromatography to obtain a black-red solid compound (E)-2-(3-(4-amino-3-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (4), namely: BNOH.

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

[0012]

[0013] The method of using the above-mentioned nitric oxide fluorescent probe is as follows:

[0014] Step 1: adding the compound of formula (I) at the same concentration to nitric oxide solutions of different concentrations to prepare at least 5 standard solutions containing the compound of formula (I) with different nitric oxide contents;

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

[0016] The content of nitric oxide in the standard solution shown is 10nM to 100μM;

[0017] Step 2: Determine the fluorescence emission spectrum of the standard solution, with an excitation wavelength of 415 nm, the concentration of nitric oxide as the horizontal axis, and I 522 and I 656 As the ordinate, establish a standard curve;

[0018] I 522 represents the peak intensity value of fluorescence emission of the standard solution at a wavelength of 522 nm; 656 represents the peak intensity value of fluorescence emission of the standard solution at a wavelength of 656 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 415 nm, that is, calculate the nitric oxide 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 black-red solid powder with good optical stability.

[0022] 2) The fluorescent probe provided by the present invention has a solution that is sensitive to the concentration of nitric oxide. As the concentration of nitric oxide increases, the fluorescence of the aqueous solution changes from red to yellow when observed under a fluorescent lamp.

[0023] 3) The fluorescent probe provided by the present invention has an emission wavelength at 656 nm. After adding nitric oxide, a new emission peak appears at 522 nm, which is a "ratio" type response. It can greatly eliminate the influence of detection environment differences on the results during detection and improve the sensitivity of detection.

[0024] 4) The fluorescent probe provided by the present invention has a linear relationship with the concentration of nitric oxide and can be used for accurate measurement of nitric oxide.

[0025] The "ratio" type nitric oxide probe based on isophorone dye provided by the present invention has a good response to nitric oxide solution, can realize sensitive quantitative detection of nitric oxide in the sample, and has the advantages of simple operation, low cost, sensitive response, easy promotion and application, etc. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] Figure 2 : Color response diagram of fluorescent probe BNOH to nitric oxide solution under visible light.

[0028] Figure 3 : Color response diagram of fluorescent probe BNOH to nitric oxide solution under fluorescent light.

[0029] Figure 4 : UV titration curve of fluorescent probe BNOH against nitric oxide in solution, where the probe concentration is 10.0μM.

[0030] Figure 5 : Fluorescence titration curve of fluorescent probe BNOH to nitric oxide in solution, where the excitation wavelength is 415nm and the probe concentration is 10.0μM.

[0031] Figure 6 : Fluorescence response diagram of fluorescent probe BNOH to common small molecules, where the excitation wavelength is 415nm, the probe concentration is 10.0μM, and the analyte concentration is 100.0μM. 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 numbers in the above compounds.

[0035] Example 1. Synthesis of compound BNOH.

[0036] Synthesis of compound (E)-2-(3-(4-amino-3-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (4).

[0037] 500 mg (E)-2-(3-(3-hydroxy-4-nitrophenyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (1) (1.49 mmol), 1.01 g stannous chloride dihydrate (2) (4.47 mmol) were dissolved in 10 mL of anhydrous ethanol, and 46 μL of hydrochloric acid (3) (1.49 mmol) was added. After reacting at 78°C for 12 hours, 137 mg of compound (E)-2-(3-(4-amino-3-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (4) was obtained, namely: BNOH, with a yield of 30%. The NMR spectrum is shown as follows Figure 1 shown.

[0038] 1 H NMR (400MHz, CDCl3-d6) δ7.02–6.93(m,3H),6.81–6.70(m,3H),2.60(s,2H),2.46(s,2H),1.09(s,6H).

[0039] Example 2. Synthesis of compound BNOH.

[0040] 250 mg of (E)-2-(3-(3-hydroxy-4-nitrophenyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (1) (0.75 mmol) and 1.01 g of stannous chloride dihydrate (2) (4.47 mmol) were dissolved in 90 mL of anhydrous ethanol, and 46 μL of hydrochloric acid (3) (1.49 mmol) was added. After reacting at 35° C. for 2 hours, column chromatography was performed to obtain 120 mg of compound (E)-2-(3-(4-amino-3-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (4).

[0041] Example 3. Synthesis of compound BNOH.

[0042] 62.5 mg of (E)-2-(3-(3-hydroxy-4-nitrophenyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (1) (0.19 mmol) and 1.01 g of stannous chloride dihydrate (2) (4.47 mmol) were dissolved in 50 mL of anhydrous ethanol, and 46 μL of hydrochloric acid (3) (1.49 mmol) was added. After reacting at 60° C. for 23 hours, 110 mg of compound (E)-2-(3-(4-amino-3-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (4) was obtained by spin column chromatography.

[0043] Example 4: Color response of compound BNOH to nitric oxide.

[0044] Prepare a 1mM dimethyl sulfoxide (DMSO) test stock solution of the fluorescent probe BNOH for detecting nitric oxide of the present invention for standby use. Take 50μL of this stock solution and drop it into a solution of ethanol:PBS=1:1 with a certain concentration of nitric oxide, and dilute it to 5mL with the corresponding ethanol:PBS=1:1 solution, so that the concentration of the probe in the test solution is 10.0μM and the concentration of nitric oxide is 100.0μM for color response test. Figure 2 and 3 As shown, after adding nitric oxide solution, the color of the solution was observed to change from red to light yellow by naked eyes, and the fluorescence of the solution also changed from red to yellow fluorescence, indicating that the probe BNOH has an intuitive colorimetric and fluorescence response to nitric oxide.

[0045] Example 5: UV titration detection of compound BNOH with different concentrations of nitric oxide.

[0046] Prepare a 1mM dimethyl sulfoxide (DMSO) test mother solution of the fluorescent probe BNOH for detecting nitric oxide described in the present invention for standby use. Take 50μL of this mother solution and drop it into a certain concentration of nitric oxide in ethanol:PBS=1:1 solution, and dilute it to 5mL with the corresponding ethanol:PBS=1:1 solution, so that the concentration of the probe in the test solution is 10.0μM, and the concentration of nitric oxide is 0-200.0μM for absorption spectrum test. Obtain the ultraviolet absorption curve in each system, and establish the standard curve of absorbance and nitric oxide concentration. Figure 4 As shown, with the increase of nitric oxide concentration, the absorbance at 482 nm gradually decreased, and the absorbance at 400 nm gradually increased. When the nitric oxide concentration was 100.0 μM, the absorbance in the reaction system reached a equilibrium state.

[0047] Example 6: Fluorescence titration detection of compound BNOH by different concentrations of nitric oxide.

[0048] Prepare a 1mM dimethyl sulfoxide (DMSO) test mother solution of the fluorescent probe BNOH for detecting nitric oxide described in the present invention for later use. Take 50μL of this mother solution and add it dropwise to a certain concentration of nitric oxide in an ethanol:PBS=1:1 solution, and dilute it to 5mL with the corresponding ethanol:PBS=1:1 solution, so that the concentration of the probe in the test solution is 10.0μM and the concentration of nitric oxide is 0-200.0μM for fluorescence detection. (λex=415nm,λem1=522nm,λem2=656nm). Obtain the fluorescence intensity in each system and establish a standard curve of fluorescence intensity and nitric oxide concentration. Figure 5As shown in the figure, with the increase of nitric oxide concentration, the fluorescence intensity of the system at 656nm gradually decreases, and the fluorescence intensity at 522nm gradually increases. When the concentration of nitric oxide reaches 100.0μM, the fluorescence intensity in the reaction system reaches a balance state.

[0049] Example 7. Selectivity of compound BNOH for different common reactive oxygen small molecules.

[0050] Prepare a 1mM dimethyl sulfoxide (DMSO) test mother solution of the fluorescent probe BNOH for detecting nitric oxide described in the present invention for standby use. Prepare 10mM solutions of various reactive oxygen species to be tested for standby use. Take 50μL of this mother solution and add them dropwise to 1:1 ethanol:PBS solutions of different small molecules to be tested, and make the volume to 5mL with the corresponding 1:1 ethanol:PBS solution, so that the concentration of the probe in the test solution is 10.0μM, and the concentration of the small molecule to be tested is 100.0μM for fluorescence detection (λex=415nm, λem1=522nm, λem2=656nm). Obtain the fluorescence intensity in each system and establish the fluorescence intensity (I 522 / I 656 ) and each analyte. Figure 6 As shown, other common small molecules have little effect on the fluorescence of probe BNOH.

Claims

1. A fluorescent probe for detecting nitric oxide, characterized in that: Its molecular formula C 19 H 19 N3O, referred to as BNOH, has a structural formula of formula (I); 2. The method for preparing a fluorescent probe for detecting nitric oxide according to claim 1, characterized in that: The synthesis steps are as follows: (E)-2-(3-(3-hydroxy-4-nitrophenyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile, stannous chloride dihydrate and hydrochloric acid are dissolved in anhydrous ethanol, and after a period of reaction, (E)-2-(3-(4-amino-3-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile, namely: BNOH, is obtained.

3. A method for using a fluorescent probe for detecting nitric oxide; characterized in that: 1) adding the compound of formula (I) at the same concentration to acetonitrile solutions containing nitric oxide at different concentrations to prepare at least 5 standard solutions containing the compound of formula (I) at different nitric oxide contents; The concentration of the compound represented by formula (I) in the standard solution shown is 10 μM; The content of nitric oxide in the standard solution shown is 10 μM to 100 μM; 2) respectively measuring the fluorescence emission spectrum of the standard solution, with the excitation wavelength being 415 nm, the concentration of nitric oxide as the horizontal axis, and the fluorescence emission spectrum of the standard solution as the horizontal axis. 522 and I 656 As the ordinate, establish a standard curve; I 522 represents the peak intensity value of fluorescence emission of the standard solution at a wavelength of 522 nm; I 656 represents the peak intensity value of fluorescence emission of the standard solution at a wavelength of 656 nm; 3) adding the compound of formula (I) to the sample to be tested, and controlling the concentration thereof to be equal to the concentration of the compound of formula (I) in the standard solution; The fluorescence emission spectrum of the sample under the excitation light with an excitation wavelength of 415 nm is measured, that is, the nitric oxide content of the sample to be tested is calculated according to the standard curve.

4. The method for preparing a fluorescent probe for detecting nitric oxide according to claim 1, characterized in that: In the method i), the molar ratio of (E)-2-(3-(3-hydroxy-4-nitrophenyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (1), stannous chloride dihydrate (2) and hydrochloric acid (3) is 0.1 to 1:3:1, and the molar volume ratio of (E)-2-(3-(3-hydroxy-4-nitrophenyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile to anhydrous ethanol is 1:6 to 250; the reaction temperature is 30 to 80 degrees, and the reaction time is 1 to 24 hours.

5. The method for preparing a fluorescent probe for detecting nitric oxide according to claim 1, characterized in that: i) (E)-2-(3-(3-hydroxy-4-nitrophenyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (1), stannous chloride dihydrate (2), hydrochloric acid (3) and the molar ratio is 1:3:1.5; anhydrous ethanol is 8 ml; the reaction temperature is 78 degrees, and the reaction time is 12 hours.

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