A ratiometric fluorescent probe for detecting peroxynitrite and its preparation method and use method

Abstract

The application discloses a ratio fluorescent probe for detecting peroxynitrite and a preparation method and use method thereof. The application uses (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-en-1-alkyl malonitrile to construct a classic intramolecular charge transfer system. When peroxynitrite exists, the peroxynitrite and an amide part on the probe molecule are oxidized to break and generate a strong electron donor-amino, and then the ICT effect of the molecule is enhanced, the emission wavelength is red-shifted, and the probe molecule emits strong red fluorescence. The ratio peroxynitrite probe of the dicyanisophorone dye provided by the application has good response performance to a peroxynitrite solution, can realize sensitive, rapid and quantitative detection of trace peroxynitrite in a sample, and has the advantages of simple operation, low cost, good water solubility and sensitive response.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of organic small molecule fluorescent probes, and particularly relates to (E)-N-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl-vinyl)phenyl)-2,2,2-trifluoroacetamide as a fluorescent probe for peroxynitrite and a preparation method and use method thereof. BACKGROUND

[0002] Peroxynitrite (ONOO - ) is a typical endogenous reactive nitrogen species, which is usually generated in situ in vivo by the reaction of highly reactive nitric oxide (·NO) radical and superoxide (O2 ·- ). It has high oxidizing and nitration activity. Based on these characteristics, it shows a two-way effect. (i) Positive: peroxynitrite is an essential physiological activator and signal molecule in vivo. Under normal physiological conditions, ONOO - as a signal molecule helps to regulate the dynamic redox homeostasis of the organism and resist the invasion of exogenous microorganisms. It can also regulate redox-sensitive transcription factors in cells and affect the expression of subsequent genes. (ii) Negative: when the level of ONOO - exceeds a reasonable value, the life system may not be able to react and utilize these excess ONOO - , causing the redox state of the cell to become a peroxidation state. This state induces the oxidation of biological molecules, leading to damage to cells and organelles, and ultimately inducing severe inflammation and disease. Therefore, ONOO - is considered to be a pathogenic factor for many human diseases, such as Alzheimer's disease, cardiovascular disease, metabolic disease, inflammation, diabetes, cancer, etc. Therefore, developing a reliable method to monitor the level of ONOO - in vivo in real time is of great significance to better understand the related pathophysiological role of ONOO - .

[0003] Fluorescence detection method has attracted widespread attention from researchers due to its excellent detection sensitivity and selectivity, and the ability to realize real-time and visual detection of the sample to be measured.

[0004] The small molecule fluorescent probes currently developed for detecting peroxynitrite are mainly designed based on the selective oxidative elimination reaction and oxidative cleavage reaction of peroxynitrite. In the presence of peroxynitrite, the detection group in the probe molecule reacts specifically with peroxynitrite, changes the original PeT or ICT effect of the molecule, and causes the fluorescence properties of the probe molecule to change, thereby realizing the specific recognition of peroxynitrite.

[0005] However, the reaction-based peroxynitrite fluorescent probe (see review, Q. Ma, S. Xu, O. Z. Zhai, K. Wang, X. Liu, H. Xiao, S. Zhuo, Y. Liu, Chem. Eur. J. 2022, 28, e202200828.) including the oxidation of phenylboronic acid or phenylboronic ester to phenolic compounds, selective oxidation and hydrolysis of oxime or hydrazone, etc. has become a commonly used selective recognition mechanism for peroxynitrite fluorescent probe. The ratio-based peroxynitrite fluorescent probe (see review, M. J. Afshari, X. Cheng, G. Duan, R. Duan, S. Wu, J. Zeng, Z. Gu, M. Gao, ACS Nano, 2023, 17, 7109.) including fluorescent probes based on oxidized phenylboronic acid or phenylboronic ester, fluorescent probes based on C=C / C=N bond oxidative cleavage, fluorescent probes based on hydrazine oxidation, fluorescent probes based on benzopyridine oxidation, and fluorescent probes based on diphenyl phosphate hydrolysis. However, these reported probes still have the disadvantages of poor fluorophore stability, difficult synthesis, short detection wavelength, and inability to be used to visualize the fluctuation of peroxynitrite in real-time systems, which are not conducive to accurate evaluation of the dynamic level of peroxynitrite in complex systems. SUMMARY

[0006] In order to overcome the above-mentioned defects in the prior art, the present application proposes a ratio-type fluorescent probe (DNTF) for quantitative detection of peroxynitrite. The present application can be used for quantitative detection of trace amounts of peroxynitrite in a sample.

[0007] The core of the present application is to use (E)-2-(3-formyl-4-hydroxy styryl)-5,5-dimethylcyclohexyl-2-en-1-ylidene malonitrile to construct a classic intramolecular charge transfer (ICT) system. The probe itself has good ICT effect and weak yellow fluorescence, but when peroxynitrite is present, peroxynitrite reacts with the amide part on the probe molecule to break and generate amino groups, thereby enhancing the ICT effect of the molecule, and the probe molecule emits strong red fluorescence. Through the above scheme, a "ratio" type fluorescent response is obtained, realizing high sensitivity and specificity detection of peroxynitrite.

[0008] The peroxynitrite fluorescent probe described in the present application is named DNTF, and the structural formula is shown as formula (I):

[0009]

[0010] The preparation method of the fluorescent probe is as follows: a certain amount of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-en-1-ylidene malonitrile (1) is mixed with trifluoroacetic anhydride (2), reacted for a certain time, and then filtered to obtain compound (E)-N-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-ethenyl)phenyl)-2,2,2-trifluoroacetamide, (3), namely: DNTF.

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

[0012]

[0013] As preferred, the molar ratio of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-en-1-ylidene malonitrile to trifluoroacetic anhydride is 1:5-30; the reaction temperature is 0-50°C, and the reaction time is 1-24 hours.

[0014] As preferred, the molar ratio of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-en-1-ylidene malonitrile to trifluoroacetic anhydride is 1:10.

[0015] As preferred, the reaction temperature is 40°C.

[0016] As preferred, the reaction time is 8 hours.

[0017] The use method of the peroxynitrite fluorescent probe is as follows:

[0018] Step 1: the same concentration of compound represented by formula (I) is added to phosphate buffer solutions (10 mM, pH = 7.4) of different concentrations of peroxynitrite, and at least 5 standard solutions containing compound represented by formula (I) with different peroxynitrite contents are configured;

[0019] The concentration of compound represented by formula (I) in the standard solution is 1 nM-10 μM;

[0020] The content of peroxynitrite in the standard solution is 0.1 nM-1 mM;

[0021] Step 2: the fluorescence emission spectrum of the standard solution is measured respectively, the excitation wavelength is 429 nm, the peroxynitrite concentration is taken as the abscissa, and I 611 / I 554 is taken as the ordinate, and a standard curve is established;

[0022] I 611 / I 554the ratio of the fluorescence emission intensity of the standard solution at a wavelength of 611 nm to 554 nm;

[0023] Step 3: adding the compound shown in formula (I) into the sample to be tested, controlling the concentration of the compound shown in formula (I) to be equal to the concentration of the compound shown in formula (I) in the standard solution; measuring the fluorescence emission spectrum under the excitation light with an excitation wavelength of 429 nm, i.e. calculating the peroxynitrite content of the sample to be tested according to the standard curve.

[0024] The present application has the following characteristics:

[0025] 1) The fluorescence probe provided by the present application is an orange solid powder, which is stable in structure.

[0026] 2) The fluorescence probe provided by the present application is sensitive to the concentration of peroxynitrite in solution, and as the concentration of peroxynitrite increases, the fluorescence of its aqueous solution observed under ultraviolet light changes from weak orange-yellow fluorescence to bright red fluorescence.

[0027] 3) The fluorescence probe provided by the present application has an emission wavelength of 554 nm, and after reacting with peroxynitrite, the emission wavelength red shifts to 611 nm, which is a fluorescence "ratio" type response, which can effectively overcome the influence of external factors such as probe concentration, environmental conditions, instruments and voltage stability during detection, and improve the sensitivity of detection.

[0028] 4) The fluorescence probe provided by the present application has a linear relationship with the concentration of peroxynitrite, and can be used for accurate measurement of the concentration of peroxynitrite.

[0029] The "ratio" type peroxynitrite probe based on dicyanoisophorone dye provided by the present application has a specific response to peroxynitrite solution, can realize sensitive and quantitative detection of peroxynitrite in a sample, and has the advantages of simple operation, low cost, sensitive response, good water solubility, easy popularization and application, etc. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 : Nuclear magnetic resonance hydrogen spectrum of the fluorescence probe DNTF.

[0031] Figure 2 : Color response diagram of the fluorescence probe DNTF to peroxynitrite phosphate buffer solution.

[0032] Figure 3 : Fluorescence response diagram of the fluorescence probe DNTF to peroxynitrite phosphate buffer solution.

[0033] Figure 4 : Ultraviolet absorption spectra of the fluorescence probe DNTF in phosphate buffer solution before and after reaction with peroxynitrite, wherein the concentration of the probe is 10.0 μM and the concentration of peroxynitrite is 10.0 μM.

[0034] Figure 5 Figure 9 shows the fluorescence titration curve of fluorescent probe DNTF in phosphate buffer solution, wherein the excitation wavelength is 429 nm, and the concentration of the probe is 10.0 μM.

[0035] Figure 6 Figure 10 shows the fluorescence response of fluorescent probe DNTF to common active oxygen small molecules and reducing molecules, wherein the excitation wavelength is 429 nm, the concentration of the probe is 10.0 μM, and the concentration of the test substance is 100.0 μM. DETAILED DESCRIPTION

[0036] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0037] The materials, reagents, etc. used in the following examples are obtained from commercial channels unless otherwise specified.

[0038] The compound numbers in the examples correspond to the numbers in the above compounds.

[0039] Example 1, synthesis of compound DNTF.

[0040] Synthesis of compounds.

[0041] 1.0 g of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-en-1-ylidene malonitrile (1) (3.46 mmol) and 5 mL of trifluoroacetic anhydride (2) (35.97 mmol) were added to a 50 mL two-necked flask, mixed thoroughly, and reacted at 40°C for 8 hours. After filtration, the filter residue was washed with an appropriate amount of anhydrous ethanol to obtain 0.84 g of compound (E)-N-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1- vinyl)phenyl)-2,2,2-trifluoroacetamide (3) with a yield of 63%.

[0042] 1 H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.74 (s, 4H), 7.40 (d, J = 16.1 Hz, 1H), 7.27 (d, J = 16.1 Hz, 1H), 6.88 (s, 1H), 2.61 (s, 2H), 2.54 (s, 2H), 1.02 (s, 6H); as Figure 1 indicated;

[0043] Example 2, color response of compound DNTF to peroxynitrite.

[0044] Prepare a 0.1 mM dimethylformamide stock solution of the fluorescent probe DNTF for detecting peroxynitrite described in this invention. Take 1 mL of this stock solution, dilute it with 8.9 mL of phosphate buffer, then add a certain concentration of peroxynitrite solution, and finally adjust the volume to 10 mL with phosphate buffer, so that the probe concentration and peroxynitrite concentration in the test solution are both 10.0 μM. Then, perform color response testing. Figure 2 and 3 As shown, after adding peroxynitrite solution, the color of the solution changed from colorless to pale yellow, and the fluorescence of the solution also changed from a weak pale yellow fluorescence to a brighter red fluorescence, indicating that the probe DNTF has a direct color response to peroxynitrite.

[0045] Example 3: Ultraviolet titration detection of compound DNTF by different concentrations of peroxynitrite.

[0046] Prepare a 0.1 mM dimethylformamide stock solution of the fluorescent probe DNTF for detecting peroxynitrite described in this invention. Take 1 mL of this stock solution, dilute it with 8.9 mL of phosphate buffer, add a certain concentration of peroxynitrite solution, and finally adjust the volume to 10 mL with phosphate buffer, so that the probe concentration in the test solution is 10.0 μM, and the peroxynitrite concentration is 0 and 10.0 μM, respectively, for absorption spectroscopy testing. Figure 4 As shown, after adding a certain amount of peroxynitrite, the ultraviolet absorption peak shifted from 403 nm to 429 nm.

[0047] Example 4: Fluorescent titration detection of compound DNTF by different concentrations of peroxynitrite.

[0048] Prepare a 0.1 mM dimethylformamide stock solution of the fluorescent probe DNTF for detecting peroxynitrite described in this invention. Take 1 mL of this stock solution, dilute it with 8.9 mL of phosphate buffer, add a certain concentration of peroxynitrite solution, and finally adjust the volume to 10 mL with phosphate buffer, so that the probe concentration in the test solution is 10.0 μM and the peroxynitrite concentration is 0-20.0 μM for fluorescence detection (λex = 429 nm). Calculate the fluorescence intensity in each system and establish a fluorescence intensity ratio (IF). 611 / I 554 ( ) and the standard curve of peroxynitrite concentration. For example Figure 5 As shown, with the increase of peroxynitrite concentration, the fluorescence intensity ratio (I) of the system decreases. 611 / I 554 As the concentration of peroxynitrite gradually increases, when the concentration reaches 10.0 μM, the fluorescence intensity ratio (I0.0) of the reaction system increases. 611 / I 554) reaches a maximum.

[0049] Example 5, selectivity of compound DNTF to different common active oxygen small molecules and reducing molecules.

[0050] Prepare a dimethyl sulfoxide mother liquor solution of the fluorescent probe DNTF for detecting peroxynitrite according to the present application with a concentration of 0.1 mM. Prepare a solution of various different small molecules to be tested with a concentration of 10 mM as standby. Take 1 mL of the mother liquor, dilute with 8.9 mL of phosphate buffer solution, then add a certain concentration of the small molecule to be tested, and finally dilute to 10 mL with 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 10.0 μM for fluorescence detection (λex= 429 nm). The fluorescence spectra of each system are shown in Figure 1, and common small molecules to be tested have little effect on the fluorescence of the probe DNTF. Figure 6

[0051] Example 6, synthesis of compound (E)-N-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1- ylidene)phenyl)-2,2,2-trifluoroacetamide.

[0052] Put 1.0 g of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (3.46 mmol) and 14 mL of trifluoroacetic anhydride (100.71 mmol) into a 50 mL two-necked flask, mix well, and react at 50°C for 23 hours. After filtration, wash the filter residue with an appropriate amount of anhydrous ethanol to obtain 0.80 g of compound (E)-N-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1- ylidene)phenyl)-2,2,2-trifluoroacetamide, with a yield of 60%.

[0053] Example 7, synthesis of compound (E)-N-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1- ylidene)phenyl)-2,2,2-trifluoroacetamide.

[0054] Put 1.0 g of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (3.46 mmol) and 14 mL of trifluoroacetic anhydride (100.71 mmol) into a 50 mL two-necked flask, mix well, and react at 50°C for 23 hours. After filtration, wash the filter residue with an appropriate amount of anhydrous ethanol to obtain 0.80 g of compound (E)-N-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1- ylidene)phenyl)-2,2,2-trifluoroacetamide, with a yield of 60%.​

Claims

1. A fluorescent probe for detecting peroxynitrite, characterized in that: Its molecular formula C 21 H 18 F3N3O, abbreviated as DNTF, has the structural formula (I); 2. The method for preparing a fluorescent probe for detecting peroxynitrite according to claim 1, characterized in that, The synthesis steps are as follows: (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-en-1-ylmalonidonitrile was mixed with trifluoroacetic anhydride and reacted for a period of time to obtain (E)-N-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-vinyl)phenyl)-2,2,2-trifluoroacetamide, i.e., DNTF.

3. The method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2, characterized in that: The molar ratio of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-ene-1-ylidene malononitrile to trifluoroacetic anhydride is 1:5 to 30; the reaction temperature is 0 to 50°C; and the reaction time is 1 to 24 hours.

4. The method for preparing a fluorescent probe for detecting peroxynitrite according to claim 3, characterized in that: The molar ratio of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-ene-1-ylidene malononitrile to trifluoroacetic anhydride is 1:

10.

5. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, characterized in that: The reaction temperature is 40°C.

6. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, characterized in that: The reaction time is 8 hours.

7. A method for using a fluorescent probe for detecting peroxynitrite; characterized in that: 1) Prepare at least five standard solutions containing the compound of formula (I) with different concentrations of peroxynitrite by adding the same concentration of the compound shown in formula (I) to phosphate buffer solutions containing different concentrations of peroxynitrite; the structural formula of formula (I) is: The concentration of the compound represented by formula (I) in the standard solution shown is 1 nM to 10 μM; The peroxynitrite content in the standard solution shown is 0.1 nM to 1 mM; 2) Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 429 nm. Plot the peroxynitrite concentration on the x-axis and I on the y-axis. 611 / I 554 Establish a standard curve with the vertical axis as the ordinate; I 611 / I 554 This represents the ratio of the fluorescence emission intensity of the standard solution at wavelengths of 611 nm and 554 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 429 nm is used to calculate the peroxynitrite content of the sample based on the standard curve.

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