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

Abstract

The present invention discloses a fluorescent probe for detecting peroxynitrite and a preparation method and a method for using the same. The present invention utilizes (E)-2-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile to construct a classic intramolecular charge transfer (ICT) system. The probe itself has a good ICT effect and almost no fluorescence occurs, but when peroxynitrite is present, peroxynitrite and the imine part on the probe molecule undergo an oxidative hydrolysis reaction and break to generate an aldehyde group, thereby blocking the ICT effect of the molecule, and the probe molecule emits a strong red fluorescence. The open-type peroxynitrite probe of the dicyanoisophorone dye provided by the present invention has a good response to a peroxynitrite solution, can realize sensitive and quantitative detection of trace peroxynitrite 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 2-amino-3-(((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohexenyl)vinyl)-2-hydroxybenzylidene)amino)maleonitrile used as a peroxynitrite fluorescent probe, and a preparation method and a use method thereof. Background Art

[0002] Peroxynitrite (ONOO - ) is a typical endogenous reactive nitrogen species, usually composed of highly reactive free radicals nitric oxide (·NO) and superoxide (O2 ·- ) is produced in situ in the body and has high oxidation and nitrification activity. Based on these characteristics, it exhibits a bidirectional effect: (i) Positive: Peroxynitrite is an essential physiological activator and signaling molecule in the body. Under normal physiological conditions, ONOO - As a signaling molecule, it regulates a series of physiological homeostasis. - It helps regulate the dynamic redox homeostasis of organisms and resists 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 ONOO - When levels exceed reasonable values, living systems may not be able to respond to and utilize the excess ONOO. - , causing the redox state of the cell to change to a pro-oxidative state. This state induces oxidation of biomolecules, leading to damage to organelles and subcellular organelles, and ultimately inducing severe inflammation and diseases such as cardiovascular disease, neurodegenerative diseases, Alzheimer's disease, and even cancer. Therefore, it is necessary to develop a rapid, sensitive, and selective detection method for ONOO - This method helps us better study ONOO - Its dynamic role in biochemistry also contributes to the early diagnosis and treatment of diseases and is of great significance.

[0003] Fluorescence detection has attracted extensive 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] The small molecule fluorescent probes currently developed for the detection of peroxynitrite are mainly designed based on the selective oxidation reaction, deprotection reaction and oxidative cleavage reaction of peroxynitrite. When peroxynitrite is present, the detection group in the probe molecule reacts specifically with peroxynitrite, changing the original PET or ICT effect of the molecule, resulting in changes in the fluorescent properties of the probe molecule, thereby achieving specific recognition of peroxynitrite.

[0005] However, reaction-based peroxynitrite fluorescent probes (see review, Q.Ma, S.Xu, Z.Zhai, K.Wang, X.Liu, H.Xiao, S.Zhuo, Y.Liu, Chem.Eur.J.2022,28,e202200828.), including oxidation of phenylboronic acid or phenylboronic acid esters to phenolic compounds, selective oxidation and hydrolysis of oximes or hydrazones, etc., have become the commonly used selective recognition mechanisms of peroxynitrite fluorescent probes. However, these reported probes still have the disadvantages of poor fluorophore stability, difficulty in synthesis, short detection wavelength, or inability to be used to visualize the fluctuation of peroxynitrite in real-time systems, which is not conducive to the accurate assessment of the dynamic level of peroxynitrite in complex systems. Summary of the invention

[0006] In order to overcome the above defects in the prior art, the present invention proposes a fluorescence-on type fluorescent probe (DCI-OH-DAMN) for quantitatively detecting peroxynitrite. The present invention can be used to quantitatively detect extremely small amounts of peroxynitrite in a sample.

[0007] The core of the present invention is to use (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile to construct a classic intramolecular charge transfer (ICT) system. The probe itself has a good ICT effect and almost no fluorescence occurs, but when peroxynitrite is present, the peroxynitrite and the imine part on the probe molecule undergo an oxidative hydrolysis reaction to break and generate an aldehyde group, thereby blocking the ICT effect of the molecule, and the probe molecule emits a strong red fluorescence. Through the above scheme, an "on" type fluorescence response is obtained, and highly sensitive and specific detection of peroxynitrite is achieved.

[0008] The peroxynitrite fluorescent probe of the present invention is named DCI-OH-DAMN, and its molecular formula is C 24 H 20 N6O, the structural formula is shown in formula (I):

[0009]

[0010] The preparation method of the above fluorescent probe is as follows: a certain amount of (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile, diaminomaleonitrile and glacial acetic acid are dissolved in anhydrous ethanol, reacted for a certain time, and then filtered to obtain the compound 2-amino-3-(((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohexenyl)vinyl)-2-hydroxybenzylidene)amino)maleonitrile, namely: DCI-OH-DAMN.

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

[0012]

[0013] Preferably, the molar ratio of (E)-2-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile, diaminomaleonitrile and glacial acetic acid is 1:1-2:3-5; the mass volume ratio of (E)-2-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile and anhydrous ethanol is 1:10-200; the reaction temperature is 0-50°C, and the reaction time is 1-24 hours.

[0014] Preferably, the molar ratio of the (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile, diaminomaleonitrile and glacial acetic acid is 1:1:3.

[0015] Preferably, the mass volume ratio of the (E)-2-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile to anhydrous ethanol is 100.

[0016] Preferably, the reaction temperature is 50°C.

[0017] Preferably, the reaction time is 24 hours.

[0018] The method of using the above peroxynitrite fluorescent probe is as follows:

[0019] Step 1: adding the compound of formula (I) at the same concentration to phosphate buffer solutions (10 mM, pH=7.4) containing peroxynitrite at different concentrations to prepare at least 5 standard solutions containing the compound of formula (I) at different peroxynitrite contents;

[0020] The concentration of the compound represented by formula (I) in the standard solution is 1 nM to 10 μM;

[0021] The content of peroxynitrite in the standard solution shown is 0.1nM to 1mM;

[0022] Step 2: Determine the fluorescence emission spectrum of the standard solution, with an excitation wavelength of 532 nm, peroxynitrite concentration as the horizontal axis, and I 653 As the ordinate, establish a standard curve;

[0023] I 653 represents the peak intensity value of fluorescence emission of the standard solution at a wavelength of 653 nm;

[0024] 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 532 nm, that is, calculate the peroxynitrite content of the sample to be tested according to the standard curve.

[0025] The present invention has the following characteristics:

[0026] 1) The fluorescent probe provided by the present invention is a brown solid powder with a stable structure.

[0027] 2) The fluorescent probe provided by the present invention has a solution that is sensitive to the concentration of peroxynitrite. As the concentration of peroxynitrite increases, the fluorescence of the aqueous solution observed under ultraviolet light changes from weak fluorescence to bright red fluorescence.

[0028] 3) The fluorescent probe provided by the present invention has an emission wavelength of 653nm, which is a fluorescence "on" type response, and the fluorescence intensity changes greatly before and after the reaction (about 30 times), which can greatly eliminate the influence of differences in detection conditions on the results during detection and improve the sensitivity of detection.

[0029] 4) The fluorescent probe provided by the present invention has a linear relationship with the concentration of peroxynitrite and can be used for accurate measurement of the concentration of peroxynitrite.

[0030] The "on" type peroxynitrite probe based on dicyanoisophorone dye provided by the present invention has a good response to peroxynitrite solution, can realize sensitive quantitative detection of peroxynitrite in a sample, and has the advantages of simple operation, low cost, sensitive response, easy promotion and application, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : H NMR spectrum of fluorescent probe DCI-OH-DAMN.

[0032] Figure 2 : Color response diagram of fluorescent probe DCI-OH-DAMN to peroxynitrite phosphate buffer solution.

[0033] Figure 3 : Fluorescence response of fluorescent probe DCI-OH-DAMN to peroxynitrite phosphate buffer solution.

[0034] Figure 4 : UV absorption spectra of fluorescent probe DCI-OH-DAMN before and after reaction with peroxynitrite in phosphate buffer solution, where the probe concentration is 10.0 μM and the peroxynitrite concentration is 20.0 μM.

[0035] Figure 5: Fluorescence titration curve of peroxynitrite of fluorescent probe DCI-OH-DAMN in phosphate buffer solution, where the excitation wavelength is 532nm and the probe concentration is 10.0μM.

[0036] Figure 6 :The fluorescence response diagram of the fluorescent probe DCI-OH-DAMN to common reactive oxygen small molecules, halogen ions, anions and metal ions, where the excitation wavelength is 532nm, the probe concentration is 10.0μM, and the analyte concentration is 100.0μM. DETAILED DESCRIPTION

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples were obtained from commercial sources.

[0039] The compound numbers in the examples correspond to the numbers in the above compounds.

[0040] Example 1. Synthesis of compound DCI-OH-DAMN.

[0041] Synthesis of the compound 2-amino-3-(((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohexenyl)vinyl)-2-hydroxybenzylidene)amino)maleonitrile.

[0042] 5.0 g of (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenepropanonitrile (1) (15.70 mmol) and 1.7 g of diaminomaleonitrile (2) (15.70 mmol) were dissolved in 500 mL of anhydrous ethanol, and 2.70 mL of glacial acetic acid (3) (47.11 mmol) was added. The mixture was reacted at 50° C. for 24 hours. After filtration, 3.1 g of compound 2-amino-3-(((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohexenyl)vinyl)-2-hydroxybenzylidene)amino)maleonitrile (4) was obtained with a yield of 48%.

[0043] 1H NMR (400MHz, DMSO-d6) δ10.96(s,1H),8.58(s,1H),8.43(d,J=2.2Hz,1H),8.01(s,2H),7.69(dd,J=8.6,2.3Hz,1H),7.3 3(d,J=16.1Hz,1H),7.23(d,J=16.1Hz,1H),6.99(d,J=8.6Hz,1H),6.85(s,1H),2.63(s,2H),2.55(s,2H),1.03(s,6H); such as Figure 1 As shown;

[0044] Example 2: Color response of compound DCI-OH-DAMN to peroxynitrite.

[0045] Prepare a 0.1 mM dimethyl sulfoxide mother solution of the fluorescent probe DCI-OH-DAMN for detecting peroxynitrite of the present invention for standby use. Take 1 mL of this mother solution, dilute it with 8.9 mL of phosphate buffer solution, then add a certain concentration of peroxynitrite solution, and finally dilute it 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 peroxynitrite is 10.0 μM for color response test. Figure 2 and 3 As shown, after adding the peroxynitrite solution, the color of the solution was observed to change from light yellow to light red by the naked eye, and the fluorescence of the solution also changed from weak fluorescence to bright red fluorescence, indicating that the probe DCI-OH-DAMN has an intuitive color response to peroxynitrite.

[0046] Example 3: UV titration detection of compound DCI-OH-DAMN with different concentrations of peroxynitrite.

[0047] Prepare a 0.1 mM dimethyl sulfoxide mother solution of the fluorescent probe DCI-OH-DAMN for detecting peroxynitrite of the present invention for standby use. Take 1 mL of this mother solution, dilute it with 8.9 mL of phosphate buffer solution, then add a certain concentration of peroxynitrite solution, and finally dilute it 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 peroxynitrite is 0 and 20.0 μM respectively for absorption spectrum test. Figure 4 As shown, after adding a certain amount of peroxynitrite, the ultraviolet absorption peak red-shifts from 413nm to 505nm.

[0048] Example 4: Fluorescence titration detection of compound DCI-OH-DAMN by peroxynitrite at different concentrations.

[0049] Prepare a 0.1 mM dimethyl sulfoxide mother liquor solution of the fluorescent probe DCI-OH-DAMN for detecting peroxynitrite described in the present invention for later use. Take 1 mL of this mother liquor, dilute it with 8.9 mL of phosphate buffer solution, then add a certain concentration of peroxynitrite solution, and finally make the volume 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 peroxynitrite is 0-30.0 μM for fluorescence detection (λex=532 nm). Obtain the fluorescence intensity in each system, and establish a standard curve of fluorescence intensity and peroxynitrite concentration. Figure 5 As shown, with the increase of peroxynitrite concentration, the fluorescence intensity of the system at 653nm gradually increased. When the peroxynitrite concentration reached 20.0μM, the fluorescence intensity of the reaction system reached the maximum value.

[0050] Example 5. Selectivity of the compound DCI-OH-DAMN to different common reactive oxygen small molecules, halogen ions, anions and metal ions.

[0051] Prepare a 0.1mM dimethyl sulfoxide mother solution of the fluorescent probe DCI-OH-DAMN for detecting peroxynitrite described in the present invention for standby use. Prepare solutions of various small molecules to be tested with a concentration of 10mM for standby use. Take 1mL of this mother solution, dilute it with 8.9mL of phosphate buffer solution, then add a certain concentration of the test substance solution, and finally dilute it to 10mL with phosphate buffer 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 20.0μM for fluorescence detection (λex=532nm). The fluorescence spectra in each system are obtained as follows: Figure 6 As shown, common small molecules have little effect on the fluorescence of the probe DCI-OH-DAMN.

[0052] Example 6. Synthesis of the compound 2-amino-3-(((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohexenyl)vinyl)-2-hydroxybenzylidene)amino)maleonitrile.

[0053] 5.0 g of (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile (15.70 mmol) and 2.55 g of diaminomaleonitrile (23.55 mmol) were dissolved in 100 mL of anhydrous ethanol, and 3.60 mL of glacial acetic acid (62.80 mmol) were added. The mixture was reacted at 50° C. for 24 hours. After filtering, the filter residue was washed with anhydrous ethanol to obtain 2.7 g of compound 2-amino-3-(((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohexenyl)vinyl)-2-hydroxybenzylidene)amino)maleonitrile with a yield of 42%.

[0054] Example 7. Synthesis of the compound 2-amino-3-(((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohexenyl)vinyl)-2-hydroxybenzylidene)amino)maleonitrile.

[0055] 5.0 g of (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile (15.70 mmol) and 3.4 g of diaminomaleonitrile (31.40 mmol) were dissolved in 800 mL of anhydrous ethanol, and 4.50 mL of glacial acetic acid (78.50 mmol) was added. The mixture was reacted at 50° C. for 24 hours. After filtering, the filter residue was washed with anhydrous ethanol to obtain 2.9 g of compound 2-amino-3-(((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohexenyl)vinyl)-2-hydroxybenzylidene)amino)maleonitrile with a yield of 45%.

Claims

1. A fluorescent probe for detecting peroxynitrite, characterized in that: Its molecular formula C 24 H 20 N6O, referred to as DCI-OH-DAMN, has a structural formula of 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-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile, diaminomaleonitrile and glacial acetic acid are dissolved in anhydrous ethanol and reacted for a period of time to obtain 2-amino-3-(((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohexenyl)vinyl)-2-hydroxybenzylidene)amino)maleonitrile, namely: DCI-OH-DAMN.

3. The method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2, characterized in that: The molar ratio of the (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile, diaminomaleonitrile and glacial acetic acid is 1:1-2:1-5; the mass volume ratio of the (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile and anhydrous ethanol is 1:20-200 g / mL; the reaction temperature is 0-50° C., and the reaction time is 1-24 hours.

4. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, characterized in that: The molar ratio of the (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile, diaminomaleonitrile and glacial acetic acid is 1:1:

3.

5. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, characterized in that: The mass volume ratio of the (E)-2-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohexyl-2-ene-1-ylidenemalononitrile to anhydrous ethanol is 1:100 g / mL.

6. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, characterized in that: The reaction temperature is 50°C.

7. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, characterized in that: The reaction time is 24 hours.

8. The method for using a fluorescent probe for detecting peroxynitrite according to claim 1, characterized in that: 1) adding the same concentration of the compound represented by formula (I) to phosphate buffer solutions containing peroxynitrite at different concentrations to prepare at least 5 standard solutions containing the compound represented by formula (I) at different peroxynitrite contents; The concentration of the compound represented by formula (I) in the standard solution is 1 nM to 10 μM; The content of peroxynitrite in the standard solution shown is 0.1nM to 1mM; 2) respectively measuring the fluorescence emission spectrum of the standard solution, with the excitation wavelength being 532 nm, the peroxynitrite concentration being the abscissa and the I being the 653 As the ordinate, establish a standard curve; I 653 represents the peak intensity value of fluorescence emission of the standard solution at a wavelength of 653 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 532 nm is measured, that is, the peroxynitrite content of the sample to be tested is calculated according to the standard curve.

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