A fluorescent probe for detecting nitric oxide and its preparation method and use method

Abstract

The present invention discloses a fluorescent probe for detecting nitric oxide, and a preparation method and a method for using the same. Isophorone, as an excellent fluorescent mother group, has its unique D‑π‑A conjugated system. Under light excitation, an intramolecular charge transfer process from an electron donor to an electron acceptor occurs, and it has excellent fluorescence emission in the near-infrared region, a large Stokes shift caused by ultrafast intramolecular charge transfer, and excellent performance such as two-photon absorption. Therefore, based on the isophorone fluorescent mother group, we constructed a classic intramolecular charge transfer system. Due to its ICT effect, the probe itself exhibits red fluorescence, but in the presence of nitric oxide, nitric oxide reacts with the amine group on the probe molecule, thereby suppressing the ICT effect of the molecule and 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.

Description

Technical Field

[0001] The present invention belongs to the field of organic small molecule fluorescent probes, and specifically relates to a preparation method and a use method of (E)-(4-((4-(2-(3-(dicyanomethylidene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine used as a nitric oxide fluorescent probe. Background Art

[0002] Nitric oxide (NO) is a ubiquitous messenger molecule in biological systems. It is primarily produced through the conversion of endogenous L-arginine to L-citrulline, catalyzed by three NO synthases (eNOS, nNOS, and iNOS). It has diverse physiological functions, including regulating vascular tone, inhibiting platelet aggregation, and neurotransmission, and actively participates in immune-mediated pathological processes. It plays a vital role in the cardiovascular, immune, and nervous systems. The physiological effects of NO are highly dependent on its concentration. Inadequate NO concentrations can promote tumor growth and lead to thrombosis and immune dysfunction. Excessive NO concentrations can cause oxidative stress, cell damage, and vascular and neurotoxicity. Therefore, developing methods to specifically measure NO concentrations in the human body is crucial for understanding its physiological and pathological processes.

[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] Over the past decade, a large number of fluorescent NO probes have been developed, including transition metal complex-based and o-phenylenediamine-based NO probes. Transition metal complex-based NO fluorescent probes have rarely been used to study NO in vivo due to their poor biocompatibility. The o-phenylenediamine-based NO fluorescent probe was the earliest developed and most widely used. Its response mechanism involves the reaction of o-phenylenediamine with NO in the presence of oxygen to form a benzotriazole derivative, thereby inhibiting the photoinduced electron transfer (PeT) process. However, o-phenylenediamine-based fluorescent probes can also be interfered with by DHA / AA / MGO, resulting in fluorescence signal changes. In recent years, a number of new reaction-based NO probes have been developed, including o-phenylenediamine-rhodamine lactam-based, monoamino-based, Se-ether-forming, and Hans ester-based aromatization-based NO probes. These probes have improved fluorescent probes and, to a certain extent, addressed issues such as biocompatibility and selectivity for NO.

[0005] However, 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) have been developed to detect NO, including o-phenylenediamines, Hans esters, nitrogen nitrosation, and other methods. However, these reported probes still have defects such as poor fluorophore stability, difficult synthesis, and single-wavelength detection that makes the detection susceptible to interference from the body's autofluorescence, which will limit their application in vivo. Summary of the Invention

[0006] To overcome the above-mentioned defects in the prior art, the present invention proposes a ratiometric fluorescent probe (BNPPh3) for quantitative detection of nitric oxide. The present invention can be used to quantitatively detect nitric oxide in samples.

[0007] Isophorone, as an excellent fluorophore, possesses a unique D-π-A conjugated system. Upon photoexcitation, it undergoes intramolecular charge transfer from an electron donor to an electron acceptor, resulting in excellent properties such as excellent fluorescence emission in the near-infrared region, a large Stokes shift caused by ultrafast intramolecular charge transfer (ICT), and two-photon absorption. Therefore, a classic intramolecular charge transfer (ICT) system was constructed based on the isophorone fluorophore. The ICT effect gives the probe its inherent red fluorescence. However, in the presence of nitric oxide, the nitric oxide reacts with the amine groups on the probe molecule, suppressing the ICT effect and resulting in yellow fluorescence emission. This scheme achieves a ratiometric fluorescence response, enabling highly sensitive and specific detection of nitric oxide.

[0008] The nitric oxide fluorescent probe of the present invention is named (E)-(4-((4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine, and its structural formula is shown in Formula (I):

[0009] The fluorescent probe is prepared as follows: a certain amount of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile, (4-bromobutyl)triphenylphosphine bromide, sodium iodide, and N,N-diisopropylethylamine are dissolved in N,N-dimethylformamide and reacted at a certain temperature for a period of time. After the reaction is complete, the product is dried and purified by column chromatography to obtain a dark red solid compound (E)-(4-((4-(2-(3-(dicyanomethylidene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine, namely BNPPh3.

[0010] The preparation reaction formula of the above probe is as follows:

[0011]

[0012] Preferably, in said i), the molar ratio of (E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (1), (4-bromobutyl)triphenylphosphine bromide (2), sodium iodide (3) and N,N-diisopropylethylamine (4) is 0.1-1:1:1:3.5, and the amount-to-volume ratio of sodium iodide to N,N-dimethylformamide is 1:1-25; the reaction temperature is 30-120 degrees, and the reaction time is 1-24 hours.

[0013] Preferably, the molar ratio of i) (E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (1), (4-bromobutyl)triphenylphosphine bromide (2), sodium iodide (3) and N,N-diisopropylethylamine (4) is 1:1:1:3.5; the amount-to-volume ratio of sodium iodide to N,N-dimethylformamide is 1:5; the reaction temperature is 120 degrees, and the reaction time is 5 hours.

[0014] The method for using the above-mentioned nitric oxide fluorescent probe is as follows:

[0015] Step 1: adding the compound of formula (I) at the same concentration to nitric oxide solutions of different concentrations to prepare at least five standard solutions containing the compound of formula (I) with different nitric oxide contents;

[0016] The concentration of the compound represented by formula (I) in the standard solution shown is 10 μM;

[0017] The nitric oxide content in the standard solutions shown is 10 nM ~100 μM;

[0018] Step 2: Measure the fluorescence emission spectrum of the standard solution respectively, with the excitation wavelength being 402 nm, the concentration of nitric oxide as the horizontal axis, and the concentration of nitric oxide as the horizontal axis. 547 and I656 As the vertical axis, establish a standard curve;

[0019] I 547 represents the peak fluorescence emission intensity value of the standard solution at a wavelength of 547 nm; 656 represents the peak fluorescence emission intensity value of the standard solution at a wavelength of 656 nm;

[0020] 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 402 nm, that is, calculate the nitric oxide content of the sample to be tested based on the standard curve.

[0021] The present invention has the following characteristics:

[0022] 1) The fluorescent probe provided by the present invention is a black-red solid powder with good optical stability.

[0023] 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.

[0024] 3) The fluorescent probe provided by the present invention has an emission wavelength at 656 nm. After the addition of nitric oxide, a new emission peak appears at 547 nm, which is a "ratio" response. It can greatly eliminate the influence of differences in detection conditions on the results during detection and improve the sensitivity of detection.

[0025] 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.

[0026] The isophorone dye-based "ratio" nitric oxide probe provided by the present invention has a good response to nitric oxide solution, can realize sensitive quantitative detection of nitric oxide in samples, and has the advantages of simple operation, low cost, sensitive response, and easy promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : H NMR spectrum of fluorescent probe BNPPh3.

[0028] Figure 2 : Color response diagram of fluorescent probe BNPPh3 to nitric oxide solution under visible light.

[0029] Figure 3 : Color response diagram of fluorescent probe BNPPh3 to nitric oxide solution under fluorescent light.

[0030] Figure 4: UV titration curve of fluorescent probe BNPPh3 against nitric oxide in solution 1, where the probe concentration is 10.0 μM.

[0031] Figure 5 : Fluorescence titration curve of fluorescent probe BNPPh3 against nitric oxide in solution, where the excitation wavelength is 402 nm and the probe concentration is 10.0 μM.

[0032] Figure 6 : Fluorescence response diagram of fluorescent probe BNPPh3 to common reactive oxygen species, where the excitation wavelength is 402nm, the probe concentration is 10.0 μM, and the analyte concentration is 100.0 μM. DETAILED DESCRIPTION

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples were obtained from commercial sources.

[0035] The compound numbers in the examples correspond to the compound numbers described above.

[0036] Example 1. Synthesis of compound BNPPh3.

[0037] Synthesis of compound (E)-(4-((4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine (5).

[0038] 500 mg (E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (1) (1.73 mmol), 825 mg (4-bromobutyl)triphenylphosphine bromide (2) (2.07 mmol) and 284 mg sodium iodide (3) were dissolved in 10 mL N,N-dimethylformamide, and then 1 mL N,N-diisopropylethylamine (4) (6.05 mmol) was added and reacted at 120°C for 5 hours. The mixture was then spin-dried and column chromatography was performed to obtain 273 mg of compound (E)-(4-((4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine (5), namely: BNPPh3, with a yield of 27%. The NMR spectrum is shown in FIG. Figure 1 shown.

[0039] 1H NMR (400 MHz, CDCl3-d6) δ 7.85 – 7.79 (m, 11H), 7.75 – 7.68 (m,7H), 7.32 (s, 1H), 7.02 (d, J = 15.9 Hz, 1H), 6.77 – 6.70 (m, 4H), 3.71 (td,J = 12.8, 7.7 Hz, 2H), 3.31 (t, J = 6.1 Hz, 2H), 2.58 (s, 2H), 2.46 (s, 2H),2.08 (q, J = 6.7 Hz, 2H), 1.86 (p, J = 7.8 Hz, 2H), 1.08 (s, 6H).

[0040] Example 2. Synthesis of compound BNPPh3.

[0041] Synthesis of compound (E)-(4-((4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine (5).

[0042] 300 mg of (E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (1) (1.03 mmol), 825 mg of (4-bromobutyl)triphenylphosphine bromide (2) (2.07 mmol) and 284 mg of sodium iodide (3) were dissolved in 10 mL of N,N-dimethylformamide, and then 1 mL of N,N-diisopropylethylamine (4) (6.05 mmol) was added and reacted at 60 °C for 22 hours. The reaction was then performed by spin column chromatography to obtain 273 mg of compound (E)-(4-((4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine (5), namely: BNPPh3, with a yield of 25%.

[0043] Example 3. Synthesis of compound BNPPh3.

[0044] Synthesis of compound (E)-(4-((4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine (5).

[0045] 60 mg of (E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (1) (0.2 mmol), 825 mg of (4-bromobutyl)triphenylphosphine bromide (2) (2.07 mmol) and 284 mg of sodium iodide (3) were dissolved in 10 mL of N,N-dimethylformamide, and 1 mL of N,N-diisopropylethylamine (4) (6.05 mmol) was added. After the mixture was reacted at 35°C for 1.5 hours, 273 mg of compound (E)-(4-((4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine (5) was obtained, namely: BNPPh3, with a yield of 26%.

[0046] Example 4: Color response of compound BNPPh3 to nitric oxide.

[0047] Prepare a 1 mM dimethyl sulfoxide (DMSO) stock solution of the fluorescent probe BNPPh3 for detecting nitric oxide described in the present invention. Measure 50 μL of this stock solution and add it dropwise to a solution of ethanol and PBS (1:1) containing a certain concentration of nitric oxide. Then, dilute the volume to 5 mL with the corresponding solution so that the concentration of the probe in the test solution is 10.0 μM and the concentration of nitric oxide is 100.0 μM for color response testing. 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 BNPPh3 has an intuitive colorimetric response to nitric oxide.

[0048] Example 5. UV titration detection of compound BNPPh3 with different concentrations of nitric oxide.

[0049] Prepare a 1 mM dimethyl sulfoxide (DMSO) test mother solution of the fluorescent probe BNPPh3 for detecting nitric oxide described in the present invention. Measure 50 μL of this mother solution and add it dropwise to a solution of ethanol:PBS=1:1 with a certain concentration of nitric oxide, and dilute it to 5 mL with the corresponding solution so that the concentration of the probe in the test solution is 10.0 μM and the concentration of nitric oxide is 0-100.0 μM for absorption spectrum test. Obtain the ultraviolet absorption curve in each system and establish a standard curve of absorbance and nitric oxide concentration. Figure 4 As shown in Figure 2, with the increase of nitric oxide concentration, the absorbance at 510 nm gradually decreased, the absorbance at 402 nm gradually increased, and A 402 / A 510 There was a good linear relationship between the concentration of nitric oxide (0-40.0 μM).

[0050] Example 6. Fluorescence titration detection of compound BNPPh3 by different concentrations of nitric oxide.

[0051] Prepare a 1 mM dimethyl sulfoxide (DMSO) test stock solution of the fluorescent probe BNPPh3 for detecting nitric oxide described in the present invention. Measure 50 μL of this stock solution and add it dropwise to a solution of ethanol:PBS=1:1 with a certain concentration of nitric oxide, and dilute it to 5 mL with the corresponding solution so that the concentration of the probe in the test solution is 10.0 μM and the concentration of nitric oxide is 0-100.0 μM for fluorescence detection. (λex = 402 nm, λem = 547 nm). Obtain the fluorescence intensity in each system and establish a standard curve of fluorescence intensity and nitric oxide concentration. Figure 5 As shown in Figure 2, with the increase of nitric oxide concentration, the fluorescence intensity of the system at 656 nm gradually decreased, and the fluorescence intensity at 547 nm gradually increased. When the nitric oxide concentration reached 100.0 μM, the fluorescence intensity of the reaction system reached equilibrium. In addition, at lower concentrations, I 547 / I 656 There was a good linear relationship between the concentration of nitric oxide (0-40.0 μM) and the 2 = 0.97).

[0052] Example 7. Selectivity of compound BNPPh3 for different common reactive oxygen species.

[0053] Prepare a test mother solution of dimethyl sulfoxide (DMSO) with a concentration of 1 mM for the fluorescent probe BNPPh3 for detecting nitric oxide of the present invention. Prepare solutions of various reactive oxygen species to be tested with a concentration of 10 mM as standby. Measure 50 μL of this mother solution and add it dropwise to a 1:1 solution of ethanol:PBS of different small molecules to be tested, and dilute it to 5 mL with the corresponding 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 = 402 nm, λem = 547 nm). Obtain the fluorescence intensity in each system and establish the fluorescence intensity (I 547 / I 656 ) and each analyte. Figure 6 As shown, other common reactive oxygen species to be detected have almost no effect on the fluorescence of the probe BNPPh3.

Claims

1. A fluorescent probe for detecting nitric oxide, characterized in that: The structural formula is formula (I); 2. The method for preparing a fluorescent probe for detecting nitric oxide according to claim 1, wherein: The synthesis steps are as follows: (E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile, (4-bromobutyl)triphenylphosphine bromide, sodium iodide, and N,N-diisopropylethylamine are dissolved in a certain amount of N,N-dimethylformamide. After a period of reaction, (E)-(4-((4-(2-(3-(dicyanomethylidene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)amino)butyl)triphenylphosphine is obtained. The preparation reaction formula of the probe is as follows:

3. The method for preparing a fluorescent probe for detecting nitric oxide according to claim 2, wherein: In the step i), the molar ratio of (E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile, (4-bromobutyl)triphenylphosphonium bromide, sodium iodide and N,N-diisopropylethylamine is 0.1 to 1:1:1:3.5, and the amount-to-volume ratio of sodium iodide to N,N-dimethylformamide is 1:1 to 25. The reaction temperature is 30 to 120 degrees Celsius, and the reaction time is 1 to 24 hours.

4. The method for preparing a fluorescent probe for detecting nitric oxide according to claim 2, wherein: i) The molar ratio of (E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile, (4-bromobutyl)triphenylphosphonium bromide, sodium iodide and N,N-diisopropylethylamine is 1:1:1:3.5; the amount-to-volume ratio of sodium iodide to N,N-dimethylformamide is 1:5; the reaction temperature is 120 degrees, and the reaction time is 5 hours.

5. The method for using a fluorescent probe for detecting nitric oxide according to claim 1, 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 five 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 nitric oxide content in the standard solutions shown is 10 nM to 100 μM; 2) respectively measuring the fluorescence emission spectrum of the standard solution, with the excitation wavelength being 402 nm, the concentration of nitric oxide as the horizontal axis, and the concentration of nitric oxide as the horizontal axis. 547 and I 656 As the vertical axis, establish a standard curve; I 547 represents the peak fluorescence emission intensity value of the standard solution at a wavelength of 547 nm; I 656 represents the peak fluorescence emission intensity value of the standard solution at a wavelength of 656 nm; 3) adding the compound of formula (I) to the sample to be tested, controlling its concentration 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 402 nm is measured, and the nitric oxide content of the sample to be tested is calculated based on the standard curve.

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