A nitric oxide fluorescent probe and its preparation and application methods
By designing a ratiometric fluorescent probe based on isophorone fluorescent parent groups, the biocompatibility and stability issues of existing NO fluorescent probes in vivo applications have been resolved, achieving highly sensitive and specific detection of nitric oxide, suitable for accurate measurement of nitric oxide.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2026-04-03
AI Technical Summary
The application of existing NO fluorescent probes in organisms is limited by problems such as poor biocompatibility, insufficient stability, cumbersome synthesis process, and insufficient detection sensitivity.
A ratiometric fluorescent probe (E)-2-(3-(4-amino-3-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile based on isophorone fluorescent parent groups was designed. Through intramolecular charge transfer effect, the red to yellow fluorescence response is achieved, which can be used for high sensitivity and specificity detection of nitric oxide.
It achieves highly sensitive and specific detection of nitric oxide, has good optical stability and is easy to operate, is suitable for accurate measurement of nitric oxide, and reduces the impact of differences in detection environment on the results.
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Figure CN119874566B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to a method for preparing and using (E)-2-(3-(4-amino-3-hydroxystyryl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile as a nitric oxide fluorescent probe. Background Technology
[0002] Nitric oxide (NO) is a ubiquitous messenger molecule in biological systems, endogenously produced from NADPH, O2, and L-arginine by three NO synthases (eNOS, nNOS, and iNOS). It possesses diverse physiological functions, including signal transduction, vasodilation, and smooth muscle relaxation. It also actively participates in immune-mediated pathological processes, playing crucial roles in the cardiovascular, reproductive, central, and peripheral nervous systems. NO dysregulation can lead to various diseases, including cancer, inflammation, endothelial dysfunction, and neurodegeneration. Therefore, developing specific methods for detecting NO levels in the human body is essential for understanding and studying its associated physiological and pathological processes.
[0003] Fluorescence detection has attracted widespread attention from researchers due to its advantages such as high operability, low cost, good biocompatibility, and ability to achieve real-time online detection in vivo.
[0004] Over the past decade, researchers have developed various NO fluorescent probes based on transition metal complexes and o-phenylenediamine recognition groups. However, poor biocompatibility has limited the application of transition metal complex probes in NO research in organisms. NO fluorescent probes based on the o-phenylenediamine-rhodamine lactam type react with NO to form an N-acyltriazole product, which exhibits slow hydrolysis and is easily quenched by biothiols such as Cys. In recent years, researchers have developed many novel NO probes, such as aminonitrosyl, deamination, Se ether, and hans ester types. These probes have addressed issues related to biocompatibility, reactivity, and selectivity with NO to some extent.
[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), including NO response modes such as o-phenylenediamine, hesperidin, and aminonitrosation, although capable of specifically detecting NO concentration levels, still suffer from problems such as poor fluorescent probe stability, cumbersome and difficult synthesis processes, and insufficient sensitivity due to a single detection mode, which limit their further application in organisms. Summary of the Invention
[0006] To overcome the aforementioned deficiencies of existing technologies, this invention proposes a ratiometric fluorescent probe (BNOH) for detecting nitric oxide. This invention allows for the quantitative detection of nitric oxide content in samples.
[0007] Compared to traditional fluorescent dyes, cyanoisophorone fluorescent dyes exhibit red emission and a large Stokes shift, and are easy to synthesize, leading to their widespread application 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. However, in the presence of nitric oxide, nitric oxide undergoes a reductive deamination reaction with the amino groups on the probe molecule, thereby suppressing the ICT effect and causing the probe molecule to emit yellow fluorescence. Through this scheme, a ratiometric fluorescence response was obtained, achieving highly sensitive and specific detection of nitric oxide.
[0008] The nitric oxide fluorescent probe of this invention is named (E)-2-(3-(4-amino-3-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile, and its structural formula is shown in formula (I):
[0009]
[0010] The preparation method of the above 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 solution is evaporated to dryness and then subjected to column chromatography to obtain a blackish-red solid compound (E)-2-(3-(4-amino-3-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malononitrile (4), namely: BNOH.
[0011] The reaction formula for preparing the above probe is as follows:
[0012]
[0013] The usage method of the above-mentioned nitric oxide fluorescent probe is as follows:
[0014] Step 1: Add the same concentration of the compound shown in formula (I) to nitric oxide solutions of different concentrations to prepare at least 5 standard solutions containing the compound shown in 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 nitric oxide content in the standard solution shown is 10 nM to 100 μM;
[0017] Step 2: Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 415 nm. Plot the nitric oxide concentration on the x-axis and Ig on the y-axis. 522 and I 656 Establish a standard curve with the vertical axis as the ordinate;
[0018] I 522 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 522 nm; I 656 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 656 nm;
[0019] Step 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; measure its fluorescence emission spectrum under excitation light with an excitation wavelength of 415 nm, and calculate the nitric oxide content of the sample to be tested based on the standard curve.
[0020] This invention has the following characteristics:
[0021] 1) The fluorescent probe provided by this invention is a blackish-red solid powder with good optical stability.
[0022] 2) The fluorescent probe provided by this invention has a solution that is sensitive to the concentration of nitric oxide. As the concentration of nitric oxide increases, the fluorescence of its aqueous solution changes from red to yellow under a fluorescent lamp.
[0023] 3) The fluorescent probe provided by this invention has an emission wavelength of 656nm. After the addition of nitric oxide, a new emission peak appears at 522nm, which is a "ratio" type response. This can greatly eliminate the influence of differences in the detection environment on the results and improve the sensitivity of the detection.
[0024] 4) The fluorescent probe provided by this invention has a linear relationship with the concentration of nitric oxide and can be used for accurate measurement of nitric oxide.
[0025] The isophorone dye-based ratio-type nitric oxide probe provided by this invention has a good response to nitric oxide solution, enabling sensitive quantitative detection of nitric oxide in samples. It has the advantages of simple operation, low cost, sensitive response, and easy promotion and application. Attached Figure Description
[0026] Figure 1 : The proton NMR spectrum of the fluorescent probe BNOH.
[0027] Figure 2 Color response of the fluorescent probe BNOH to nitric oxide solution under visible light.
[0028] Figure 3 Color response of the fluorescent probe BNOH to nitric oxide solution under fluorescent light.
[0029] Figure 4 UV titration curve of nitric oxide by fluorescent probe BNOH in solution, where the probe concentration is 10.0 μM.
[0030] Figure 5 The fluorescence titration curve of the fluorescent probe BNOH on nitric oxide in solution, with an excitation wavelength of 415 nm and a probe concentration of 10.0 μM.
[0031] Figure 6 Fluorescence response of the fluorescent probe BNOH to common small molecules, with an excitation wavelength of 415 nm, a probe concentration of 10.0 μM, and an analyte concentration of 100.0 μM. Detailed Implementation
[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0034] The compound numbers in the examples correspond to the numbers in the compounds described above.
[0035] Example 1: Synthesis of compound BNOH.
[0036] Synthesis of compound (E)-2-(3-(4-amino-3-hydroxystyryl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile (4).
[0037] 500 mg of (E)-2-(3-(3-hydroxy-4-nitrophenyl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonitrile (1) (1.49 mmol) and 1.01 g of stannous chloride dihydrate (2) (4.47 mmol) were dissolved in 10 mL of anhydrous ethanol, and then 46 μL of hydrochloric acid (3) (1.49 mmol) was added. After reacting at 78 °C for 12 hours, rotary cyclohexane chromatography was used to obtain 137 mg of compound (E)-2-(3-(4-amino-3-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonitrile (4), i.e., BNOH, with a yield of 30%. The NMR spectrum is shown below. Figure 1 As 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-en-1-yl)malonitrile (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 then 46 μL of hydrochloric acid (3) (1.49 mmol) were added. After reacting at 35 °C for 2 hours, the mixture was dried by rotary column chromatography to obtain 120 mg of compound (E)-2-(3-(4-amino-3-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile (4).
[0041] Example 3: Synthesis of compound BNOH.
[0042] 62.5 mg (E)-2-(3-(3-hydroxy-4-nitrophenyl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile (1) (0.19 mmol) and 1.01 g stannous chloride dihydrate (2) (4.47 mmol) were dissolved in 50 mL of anhydrous ethanol, and then 46 μL of hydrochloric acid (3) (1.49 mmol) were added. After reacting at 60 °C for 23 hours, the mixture was dried by rotary column chromatography to obtain 110 mg of compound (E)-2-(3-(4-amino-3-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile (4).
[0043] Example 4: Color response of compound BNOH to nitric oxide.
[0044] Prepare a 1 mM dimethyl sulfoxide (DMSO) test stock solution of the fluorescent probe BNOH for detecting nitric oxide described in this invention. Measure 50 μL of this stock solution and add it dropwise to a 1:1 ethanol:PBS solution of a certain concentration of nitric oxide, and then dilute to 5 mL with the corresponding ethanol:PBS 1:1 solution, so that the probe concentration in the test solution is 10.0 μM and the nitric oxide concentration is 100.0 μM. Then proceed with the color response test. Figure 2 and 3 As shown, after adding nitric oxide solution, the color of the solution changed from red to light yellow, and the fluorescence of the solution also changed from red to yellow, indicating that the probe BNOH has a direct colorimetric and fluorescence response to nitric oxide.
[0045] Example 5: Ultraviolet titration detection of compound BNOH with different concentrations of nitric oxide.
[0046] Prepare a 1 mM dimethyl sulfoxide (DMSO) test stock solution of the fluorescent probe BNOH for detecting nitric oxide described in this invention. Measure 50 μL of this stock solution and add it dropwise to a 1:1 ethanol:PBS solution of a certain concentration of nitric oxide, and then dilute to 5 mL with the corresponding ethanol:PBS 1:1 solution, so that the probe concentration in the test solution is 10.0 μM and the nitric oxide concentration is 0-200.0 μM. Perform absorption spectroscopy measurements. Obtain the UV absorption curves for each system and establish a standard curve of absorbance versus nitric oxide concentration. Figure 4 As shown, with the increase of nitric oxide concentration, the absorbance at 482 nm gradually decreases and the absorbance at 400 nm gradually increases. When the nitric oxide concentration is 100.0 μM, the absorbance in the reaction system reaches equilibrium.
[0047] Example 6: Fluorescent titration detection of compound BNOH with different concentrations of nitric oxide.
[0048] Prepare a 1 mM dimethyl sulfoxide (DMSO) test stock solution of the fluorescent probe BNOH for detecting nitric oxide described in this invention. Measure 50 μL of this stock solution and add it dropwise to a 1:1 ethanol:PBS solution of a certain concentration of nitric oxide, and then dilute to 5 mL with the corresponding ethanol:PBS 1:1 solution, so that the probe concentration in the test solution is 10.0 μM and the nitric oxide concentration is 0-200.0 μM for fluorescence detection (λex = 415 nm, λem1 = 522 nm, λem2 = 656 nm). Obtain the fluorescence intensity in each system and establish a standard curve of fluorescence intensity versus nitric oxide concentration. Figure 5As shown, with increasing nitric oxide concentration, the fluorescence intensity at 656 nm gradually decreases, while the fluorescence intensity at 522 nm gradually increases. When the nitric oxide concentration reaches 100.0 μM, the fluorescence intensity in the reaction system reaches equilibrium.
[0049] Example 7: Selectivity of compound BNOH for different common reactive oxygen species.
[0050] Prepare a 1 mM test stock solution of dimethyl sulfoxide (DMSO) for detecting nitric oxide (BNOH) as described in this invention. Prepare 10 mM solutions of various small reactive oxygen species (ROS) for testing. Measure 50 μL of this stock solution and add it dropwise to ethanol:PBS (1:1) solutions of different small molecule analytes, and then dilute to 5 mL with the corresponding ethanol:PBS (1:1) solutions to achieve a probe concentration of 10.0 μM and an analyte concentration of 100.0 μM for fluorescence detection (λex = 415 nm, λem1 = 522 nm, λem2 = 656 nm). Calculate the fluorescence intensity in each system and establish a fluorescence intensity (IL) measurement method. 522 / I 656 A bar chart showing the relationship between the analytes and the various test objects. (e.g.) Figure 6 As shown, other common small molecules to be tested have almost no effect on the fluorescence of the probe BNOH.
Claims
1. A fluorescent probe for detecting nitric oxide, characterized in that: Its molecular formula C 19 H 19 N3O, abbreviated as BNOH, has the structural 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-nitrostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile, stannous chloride dihydrate, and hydrochloric acid were dissolved in anhydrous ethanol and reacted for a period of time to obtain (E)-2-(3-(4-amino-3-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile, i.e., BNOH.
3. A method for using a fluorescent probe for detecting nitric oxide; characterized in that: 1) Add the same concentration of the compound shown in formula (I) to acetonitrile solutions of different concentrations of nitric oxide to prepare at least 5 standard solutions containing the compound shown in formula (I) with 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 ranges from 10 μM to 100 μM. 2) Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 415 nm. Plot the nitric oxide concentration on the x-axis and Ig on the y-axis. 522 and I 656 Establish a standard curve with the vertical axis as the ordinate; I 522 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 522 nm; I 656 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 656 nm; 3) Add the compound of formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound of formula (I) in the standard solution; Measurement Its fluorescence emission spectrum under excitation light with an excitation wavelength of 415 nm, that is, the nitric oxide content of the sample to be tested can be calculated based on the standard curve; The method of use described is for non-disease diagnostic purposes.
4. The method for preparing a fluorescent probe for detecting nitric oxide according to claim 2, characterized in that: The molar ratio of (E)-2-(3-(3-hydroxy-4-nitrostyryl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile, stannous chloride dihydrate, and hydrochloric acid is 0.1~1:3:1; the molar volume ratio of (E)-2-(3-(3-hydroxy-4-nitrostyryl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile to anhydrous ethanol is 1:6~250; the reaction temperature is 30~80 degrees Celsius, and the reaction time is 1~24 hours.
5. The method for preparing a fluorescent probe for detecting nitric oxide according to claim 2, characterized in that: (E)-2-(3-(3-hydroxy-4-nitrostyryl)-5,5-dimethylcyclohex-2-ene-1-yl)malonadionitrile, stannous chloride dihydrate, and hydrochloric acid were used in a molar ratio of 1:3:1.5; 8 mL of anhydrous ethanol was used; the reaction temperature was 78 degrees Celsius, and the reaction time was 12 hours.