Liver-targeted peroxynitrite fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a liver-targeted peroxynitrite fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of chemical and biological materials. The invention aims to solve the problem that the relationship between ONOO- and a certain specific disease and the pathogenesis of ONOO- cannot be studied deeply due to the lack of tissue or organtargeting of most probes. The liver-targeted peroxynitrite fluorescent probe disclosed by the invention contains a liver-targeted unit ursodeoxycholic acid; the preparation method of the fluorescent probe comprises the following steps: dissolving a compound 4-bromo-1,8-naphthalene dicarboxylic anhydride in dioxane, adding n-propyl amine, concentrating the reaction solution, and carrying out columnchromatography separation to obtain a compound NA-1; dissolving in 2-methoxy ethanol, adding hydrazine hydrate and sodium bicarbonate, and carrying out column chromatography separation to obtain a compound NA-2; dissolving the compound NA-2 and ursodeoxycholic acid in N,N-dimethyl formamide, then adding 1-hydroxyl benzotriazole, 1-ethyl-(3dimethylaminopropyl) carbodiimide hydrochloride and N,N-diisopropyl ethyl amine, and carrying out column chromatography on the crude product to obtain the target compound NA-3.

Description

technical field

[0001] The invention belongs to the technical field of chemical and biological materials, and in particular relates to a liver-targeted peroxynitrite fluorescent probe, a preparation method and an application. Background technique

[0002] Peroxynitrite (ONOO - ) as an important active oxygen molecule in the organism, participates in important physiological and pathological processes in the organism, mainly composed of nitric oxide (NO) and superoxide free radicals (O 2 .- )synthesis. Highly reactive ONOO - On the one hand, it can react with many biomolecules such as proteins, lipids and NDA, and on the other hand, it can cause oxidative stress toxicity to cells. However, the abnormal concentration of ONOO - Known as a key pathogenic factor in many diseases, such as neurodegenerative diseases, ischemia-reperfusion diseases, inflammation and cancer, etc. Therefore, the establishment of an efficient, highly selective ONOO - Detection technology is very i...

Examples

Embodiment 1

[0036] A liver-targeted peroxynitrite fluorescent probe

[0037] (1) Synthesis of compound NA-1: Compound 4-bromo-1,8-naphthalic anhydride (5g, 18.05mmol) was dissolved in 150mL of dioxane, stirred until clear, and n-propylamine (1.12g, 19.85 mmol), replaced with nitrogen, and reacted at 110° C. for 8 hours. TLC showed that the reaction was complete, the solvent was spin-dried, and the crude product was separated by column chromatography (petroleum ether: ethyl acetate = 40:1-10:1) to obtain 4 g of a yellow solid. 1 H NMR (400MHz, DMSO-d 6 )δ8.55-8.50 (m, 2H), 8.31 (d, J = 8.0Hz, 1H), 8.31 (d, J = 8.0Hz, 1H), 8.20 (d, J = 8.0Hz, 1H), 7.99 ( t,J=12.0Hz,1H), 4.01(t,J=16.0Hz,2H), 1.69-1.67(m,2H), 0.95(t,J=12.0Hz,3H); 13 C NMR (100MHz, DMSO-d 6 )δ163.1, 132.9, 131.9, 131.7, 131.2, 130.0, 129.5, 128.5, 123.0, 122.2. HRMS: [M+H + ] 318.0131, Calculated: 318.0124;

[0038] (2) Compound NA-1 (2g, 6.29mmol) was dissolved in 50mL of 2-methoxyethanol, 85% hydrazine hydrate (1mL, 12...

Embodiment 2

[0041] Fluorescent probes for different concentrations of ONOO - identification

[0042] Dissolve the fluorescent probe obtained in Example 1 with dimethyl sulfoxide, prepare the probe mother solution, and add different equivalents of ONOO - The solution was incubated at 37° C. for 30 min, diluted with phosphate buffer solution to a concentration of 10 μM to be tested, and then measured by fluorescence spectrum. Such as Image 6 As shown, the probe was able to respond to 0-14 μM ONOO - Response, and has a good linear relationship at 550nm, the linear regression equation is y=7.48x+0.24, R 2 = 0.9933.

Embodiment 3

[0044] Fluorescent probes are affected by the pH environment

[0045] Dissolve the fluorescent probe obtained in Example 1 with dimethyl sulfoxide, prepare the probe mother solution, and add 10 equivalents of ONOO - , after incubating at 37°C for 30min, dilute it to the test concentration of 10μM with phosphate buffer solution with different pH values, and measure its fluorescence spectrum (λ ex =450nm); according to its fluorescence intensity, evaluate the impact of different pH environments on the fluorescence intensity of the fluorescent probe, the results are as follows Figure 7 shown. The pH values ​​are 4, 5, 6, 7, 8, 9, respectively. Fluorescent probes with ONOO - The fluorescence intensity before and after the treatment was not affected by the pH environment.

technical field

[0001] The invention belongs to the technical field of chemical and biological materials, and in particular relates to a liver-targeted peroxynitrite fluorescent probe, a preparation method and an application. Background technique

[0002] Peroxynitrite (ONOO - ) as an important active oxygen molecule in the organism, participates in important physiological and pathological processes in the organism, mainly composed of nitric oxide (NO) and superoxide free radicals (O 2 .- )synthesis. Highly reactive ONOO - On the one hand, it can react with many biomolecules such as proteins, lipids and NDA, and on the other hand, it can cause oxidative stress toxicity to cells. However, the abnormal concentration of ONOO - Known as a key pathogenic factor in many diseases, such as neurodegenerative diseases, ischemia-reperfusion diseases, inflammation and cancer, etc. Therefore, the establishment of an efficient, highly selective ONOO - Detection technology is very i...

Examples

Embodiment 1

[0036] A liver-targeted peroxynitrite fluorescent probe

[0037] (1) Synthesis of compound NA-1: Compound 4-bromo-1,8-naphthalic anhydride (5g, 18.05mmol) was dissolved in 150mL of dioxane, stirred until clear, and n-propylamine (1.12g, 19.85 mmol), replaced with nitrogen, and reacted at 110° C. for 8 hours. TLC showed that the reaction was complete, the solvent was spin-dried, and the crude product was separated by column chromatography (petroleum ether: ethyl acetate = 40:1-10:1) to obtain 4 g of a yellow solid. 1 H NMR (400MHz, DMSO-d 6 )δ8.55-8.50 (m, 2H), 8.31 (d, J = 8.0Hz, 1H), 8.31 (d, J = 8.0Hz, 1H), 8.20 (d, J = 8.0Hz, 1H), 7.99 ( t,J=12.0Hz,1H), 4.01(t,J=16.0Hz,2H), 1.69-1.67(m,2H), 0.95(t,J=12.0Hz,3H); 13 C NMR (100MHz, DMSO-d 6 )δ163.1, 132.9, 131.9, 131.7, 131.2, 130.0, 129.5, 128.5, 123.0, 122.2. HRMS: [M+H + ] 318.0131, Calculated: 318.0124;

[0038] (2) Compound NA-1 (2g, 6.29mmol) was dissolved in 50mL of 2-methoxyethanol, 85% hydrazine hydrate (1mL, 12...

Embodiment 2

[0041] Fluorescent probes for different concentrations of ONOO - identification

[0042] Dissolve the fluorescent probe obtained in Example 1 with dimethyl sulfoxide, prepare the probe mother solution, and add different equivalents of ONOO - The solution was incubated at 37° C. for 30 min, diluted with phosphate buffer solution to a concentration of 10 μM to be tested, and then measured by fluorescence spectrum. Such as Image 6 As shown, the probe was able to respond to 0-14 μM ONOO - Response, and has a good linear relationship at 550nm, the linear regression equation is y=7.48x+0.24, R 2 = 0.9933.

Embodiment 3

[0044] Fluorescent probes are affected by the pH environment

[0045] Dissolve the fluorescent probe obtained in Example 1 with dimethyl sulfoxide, prepare the probe mother solution, and add 10 equivalents of ONOO - , after incubating at 37°C for 30min, dilute it to the test concentration of 10μM with phosphate buffer solution with different pH values, and measure its fluorescence spectrum (λ ex =450nm); according to its fluorescence intensity, evaluate the impact of different pH environments on the fluorescence intensity of the fluorescent probe, the results are as follows Figure 7 shown. The pH values ​​are 4, 5, 6, 7, 8, 9, respectively. Fluorescent probes with ONOO - The fluorescence intensity before and after the treatment was not affected by the pH environment.

Images