A single-shot detection of ONOO - Near-infrared ratiometric fluorescent probe and preparation method and application thereof

Abstract

The invention discloses a method for detecting peroxynitrite (ONOO) by single excitation. ‑ Near-infrared ratiometric fluorescent probe and its preparation method and application. The structural formula of the fluorescent probe is shown in Formula I below, and is named ER-MPTC (R=H) and ER-DMPTC (R=CH3). The fluorescent probe of the present invention is a reactive probe, and the fluorescent probe uses the oxidative cleavage of the carbon-carbon double bond as ONOO ‑ The fluorescent probe of the present invention showed in vitro detection of ONOO ‑ The fluorescent probe of the present invention has good ratio response, excellent selectivity and high sensitivity. At the same time, it has good photostability, is stable in a wide pH range and has an optimal response signal in the physiological pH range, which can meet the requirements of ONOO in biological tissue samples. ‑ testing requirements.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent probe technology. More specifically, it relates to a single excitation detection method for ONOO - Near-infrared ratiometric fluorescent probe and its preparation method and application. Background Art

[0002] Peroxynitrite anion ONOO - It is a member of the reactive oxygen family and can be produced by the rapid reaction of superoxide anion radicals and nitric oxide radicals. It has strong oxidizing and nucleophilic properties and can react with a variety of biologically active species such as proteins and nucleic acids. - It profoundly affects the cellular oxidative stress, inflammation, and immune response, and can trigger cell death through various signaling pathways. Under physiological conditions, ONOO - It plays an important role in the signal transduction and gene expression of normal cells. - Overproduction of ONOO is also thought to be associated with many diseases and pathological conditions, including drug-induced liver toxicity, stroke, ischemia-reperfusion injury, neurodegenerative diseases, inflammation, and cancer. Therefore, studying ONOO in disease-related processes is of great significance. - Changes in ONOO - It is of great significance to promote a deeper understanding of the etiology of diseases and their treatment.

[0003] In recent decades, fluorescence imaging based on molecular probes has become a popular method for studying ONOO. - It is an excellent choice for biological and pathological processes. Compared with other imaging methods, fluorescence imaging has outstanding advantages in biological sample imaging, such as high sensitivity, high target selectivity, good biosafety, non-invasive visualization, and high spatiotemporal resolution. In recent years, many methods have been developed for detecting ONOO. - There are many fluorescent probes, but most of these probes only show the turn-on fluorescence signal in a single fluorescence channel, and their performance will be affected by the type of instrument used, the internal microenvironment of the cell, the local concentration of the probe, and photobleaching. In contrast, ratiometric fluorescent probes can adjust two emission signals at the same time and have self-calibration capabilities, which can greatly eliminate the above interference and make the response signal more stable; at the same time, single-excitation ratiometric fluorescent probes can avoid signal errors caused by intensity fluctuations of different excitation light sources. Summary of the invention

[0004] One object of the present invention is to provide a method for detecting peroxynitrite (ONOO) with a single excitation wavelength. - The near-infrared ratiometric fluorescent probes ER-MPTC and ER-DMPTC are used to detect ONOO -It has good ratio response, excellent selectivity and high sensitivity, as well as good photostability. It is stable in a wide pH range and has the best response signal in the physiological pH range, which can meet the detection requirements of ONOO- in biological tissue samples.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned detection of ONOO - Preparation method of near-infrared ratiometric fluorescent probe.

[0006] In order to achieve the first object, the present invention discloses a method for detecting peroxynitrite (ONOO) by single excitation. - A near-infrared ratiometric fluorescent probe, the structural formula of which is shown in Formula I. When R is H, it is named ER-MPTC, and when R is CH3, it is named ER-DMPTC;

[0007]

[0008] Wherein, the R is selected from H or CH3.

[0009] In order to achieve the above second purpose, the present invention adopts the following technical solutions:

[0010] The present invention discloses a method for preparing the above-mentioned near-infrared ratiometric fluorescent probe, comprising the following steps:

[0011]

[0012] (1) Compound 1, triethylamine and solvent were mixed at 0-4°C, compound 2 was added, stirred for 1-2 hours, returned to room temperature, and stirred for 6-12 hours; after the reaction was completed, the mixture was concentrated, separated and purified by silica gel column chromatography to obtain compound 3;

[0013] (2) Under an inert atmosphere, a catalyst, Molecular sieve and solvent, heating under reflux for 6-10h; after the reaction is completed, concentrating, separating and purifying by silica gel column chromatography to obtain ER-MPTC or ER-DMPTC.

[0014] Furthermore, in step (1), the molar ratio of compound 1, compound 2 and triethylamine is 0.5-1:1.5:1.5.

[0015] Furthermore, the solvent is selected from dichloromethane and / or acetonitrile.

[0016] Furthermore, in step (2), the molar ratio of compound 3 to compound 4 is 1:1-4.

[0017] Furthermore, the catalyst is piperidine.

[0018] Furthermore, in step (2), the molar volume ratio of the compound 3 to the catalyst is 1 mmoL:(10-50) μL.

[0019] Further, in step (2), the compound 3 and the The molar mass ratio of the molecular sieve is 1mmoL:(1-4)g.

[0020] Furthermore, in step (1), the volume ratio of dichloromethane to methanol used as the eluent for silica gel column chromatography is 10-100:1.

[0021] Furthermore, in step (2), the volume ratio of dichloromethane to ethyl acetate used as the eluent for silica gel column chromatography is 10-100:1.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention discloses a single excitation detection method for ONOO - Near-infrared ratiometric fluorescent probe and its preparation method and application. The fluorescent probe of the present invention is a reactive probe, which uses the oxidative cleavage of carbon-carbon double bonds as ONOO - A new ONOO - Near infrared ratio probe, which has the following advantages:

[0024] 1. The ER-MPTC and ER-DMPTC near-infrared ratiometric fluorescent probes provided by the present invention are used to detect ONOO - It can show obvious fluorescence changes and is similar to ONOO - The concentration showed a good ratio change trend. With the addition of ONOO - As the concentration increases, the emission peak intensity at 540nm gradually increases, while the emission peak intensity at 680nm gradually decreases, showing a ratio change.

[0025] 2. The ER-MPTC and ER-DMPTC near-infrared ratiometric fluorescent probes provided by the present invention for ONOO - The detection has excellent selectivity and can detect related interfering substances (Ca 2+ , Cu 2+ , Fe 3+ Mg 2+ , K + 、Zn 2+ 、NO2 - 、NO3 - , Cys, GSH, NO, H2O2, t-BuOOH, O2 -. , ·OH, ROO· and ClO -) added to ER-MPTC or ER-DMPTC solution had no significant effect on the fluorescence of ER-MPTC or ER-DMPTC. - After addition, the ratio of probe fluorescence intensity changed significantly.

[0026] 3. The ER-MPTC and ER-DMPTC near-infrared ratiometric fluorescent probes provided by the present invention have good photostability. After continuous irradiation with a 150W xenon lamp for 90 minutes, the fluorescence intensity of the probe did not change significantly.

[0027] 4. The ER-MPTC and ER-DMPTC near-infrared ratiometric fluorescent probes provided by the present invention are stable in a wide pH range and have an optimal response signal in the physiological pH range, which can meet the requirements of ONOO in biological tissue samples. - testing requirements.

[0028] 5. The ER-MPTC and ER-DMPTC near-infrared ratiometric fluorescent probes provided by the present invention have good biocompatibility and can realize ONOO in vivo. - Detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0030] Figure 1 The compound 3 prepared in Example 1 of the present invention is shown 1 HNMR spectrum.

[0031] Figure 2 The ER-MPTC near-infrared fluorescent probe prepared in Example 1 of the present invention is shown. 1 HNMR spectrum.

[0032] Figure 3 The ER-DMPTC near-infrared fluorescent probe prepared in Example 1 of the present invention is shown. 1 HNMR spectrum.

[0033] Figure 4 The results show that the ER-MPTC prepared in Example 2 of the present invention has an effect on the ONOO in 1xPBS (pH 8.2, 3mM CTAAB) solution. - Response fluorescence change curve.

[0034] Figure 5 The results show that the ER-DMPTC prepared in Example 2 of the present invention has an effect on the ONOO in 1xPBS (pH 8.2, 3 mM CTAAB) solution. - Response fluorescence change curve.

[0035] Figure 6The results show that the ER-DMPTC prepared in Example 2 of the present invention has different concentrations of ONOO - The fluorescence ratio response curve was fitted.

[0036] Figure 7 The selectivity test results of ER-DMPTC prepared in Example 2 of the present invention are shown.

[0037] Figure 8 The light stability test results of ER-DMPTC prepared in Example 3 of the present invention are shown.

[0038] Fig. 9 The pH stability test results of ER-DMPTC prepared in Example 3 of the present invention are shown. DETAILED DESCRIPTION

[0039] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0040] Unless otherwise specified, the raw materials used in the present invention can be obtained commercially. Any range recorded in the present invention includes the end value and any numerical value between the end values ​​and any sub-range formed by the end value or any value between the end values.

[0041] Example 1

[0042] Synthesis of compound 3

[0043] At 0°C, compound 1 (251.3 mg, 1 mmol), acid-binding agent triethylamine (303.6 mg, 3 mmol) and solvent dichloromethane 20 mL were added to the reaction container, and after stirring evenly, compound 2 (785.2 mg, 3 mmol) was added. Stirring was maintained at 0°C for 2 hours, and after returning to room temperature, stirring was continued for 12 hours. After the reaction was completed by thin layer chromatography, the solvent was concentrated, and the product was separated and purified by silica gel column chromatography, and the eluent was dichloromethane and methanol (100 / 1, v / v). A light yellow solid product, namely compound 3 (217.4 mg, 46%), was obtained. 1 HNMR spectrum Figure 1 shown.

[0044] Synthesis of ER-MPTC

[0045] Compound 3 (238.3 mg, 0.5 mmol) and compound 4 (108.6 mg, 0.5 mmol) were added to the reaction vessel, and under nitrogen protection, catalyst piperidine (5 μL), solvent anhydrous acetonitrile 20 mL and Molecular sieve 0.5g, heated under reflux reaction for 10h. After the reaction was completed by thin layer chromatography, the solvent was concentrated, separated and purified by silica gel column chromatography, and the eluent was dichloromethane and ethyl acetate (100 / 1, v / v), and a black solid product, ER-MPTC (118.3mg, 35%), was collected. 1 HNMR spectrum Figure 2 shown.

[0046] Synthesis of ER-DMPTC

[0047] Compound 3 (238.3 mg, 0.5 mmol) and compound 5 (115.7 mg, 0.5 mmol) were added to the reaction vessel, and under nitrogen protection, 5 μL of catalyst piperidine, 20 mL of solvent anhydrous acetonitrile and Molecular sieve 0.5g, heated under reflux reaction for 10h. After the reaction was completed by thin layer chromatography, the solvent was concentrated, and the mixture was separated and purified by silica gel column chromatography. The eluent was dichloromethane and ethyl acetate (100 / 1, v / v), and a black solid product, ER-DMPTC (100.0mg, 29%), was collected. 1 HNMR spectrum Figure 3 shown.

[0048]

[0049] Preparation method of probe stock solution

[0050] Preparation of 1 mM ER-MPTC or ER-DMPTC probe stock solution: Weigh an appropriate amount of sample on an analytical balance, pipette a corresponding volume of dimethyl sulfoxide (DMSO) to dissolve the sample, prepare a probe concentration of 1 mM, and store it in a refrigerator at 4°C. The following examples prepare probe stock solutions in the same manner.

[0051] Embodiment 2:

[0052] Synthesis of compound 3

[0053] At 0°C, compound 1 (502.6 mg, 2 mmol), acid-binding agent triethylamine (303.6 mg, 3 mmol) and solvent dichloromethane 20 mL were added to the reaction container, and after stirring evenly, compound 2 (785.2 mg, 3 mmol) was added. Stirring was maintained at 0°C for 1 hour, and after returning to room temperature, stirring was continued for 6 hours; after the reaction was observed to be completed by thin layer chromatography, the solvent was concentrated, and the product was separated and purified by silica gel column chromatography, and the eluent was dichloromethane and methanol (10 / 1, v / v), and a light yellow solid product, i.e., compound 3, was collected.

[0054] Synthesis of ER-MPTC

[0055] Compound 3 (238.3 mg, 0.5 mmol) and compound 4 (434.4 mg, 2 mmol) were added to the reaction vessel, and under nitrogen protection, 15 μL of catalyst piperidine, 20 mL of solvent anhydrous acetonitrile and Molecular sieve 1g, heated under reflux reaction for 8h. After the reaction was completed by thin layer chromatography, the solvent was concentrated, separated and purified by silica gel column chromatography, the eluent was dichloromethane and ethyl acetate (10 / 1, v / v), and a black solid product, namely ER-MPTC, was collected.

[0056] Synthesis of ER-DMPTC

[0057] Compound 3 (238.3 mg, 0.5 mmol) and compound 5 (462.8 mg, 2 mmol) were added to the reaction vessel, and under nitrogen protection, 15 μL of catalyst piperidine, 20 mL of solvent anhydrous acetonitrile and Molecular sieve 1g, heated under reflux reaction for 8h. After the reaction was completed by thin layer chromatography, the solvent was concentrated, and the product was separated and purified by silica gel column chromatography, with dichloromethane and ethyl acetate (10 / 1, v / v) as the eluent, and a black solid product, namely ER-DMPTC, was obtained.

[0058]

[0059] Probe response ONOO - Determination of fluorescence emission spectrum changes

[0060] Take 10 μL of ER-MPTC fluorescent probe stock solution, dilute it with 1xPBS (pH 8.2, 3 mM CTAAB) solution, prepare 12 groups in parallel, and add ONOO - The solution to be tested was such that each probe solution was 10 μM, and ONOO - The concentrations of the test solutions were 0-200 μM (0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 140 μM, 160 μM, 180 μM and 200 μM, respectively). Incubate at room temperature in the dark for 12 hours. After sufficient reaction, the fluorescence spectra of parallel samples were tested in a 10 mm cuvette. The changes in the fluorescence emission spectrum are shown in Figure 4 .

[0061] Take 10 μL of ER-DMPTC fluorescent probe mother solution, dilute it with 1xPBS (pH 8.2, 3 mM CTAAB) solution, prepare 21 groups in parallel, and add ONOO - The solution to be tested was such that each probe solution was 10 μM, and ONOO -The concentrations of the test solutions were 0-200 μM (0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM and 200 μM). Incubate at room temperature in the dark for 12 hours. After sufficient reaction, test the fluorescence spectra of parallel samples in a 10 mm cuvette. See the changes in the fluorescence emission spectrum. Figure 5 .

[0062] from Figure 4 and 5 As can be seen, the fluorescent probes ER-MPTC and ER-DMPTC have similar responses to ONOO-. - With the addition of , the fluorescence of the probe at 680nm gradually decreased, while the fluorescence at 540nm gradually increased. In subsequent experiments, ER-DMPTC was selected as a representative substance for testing.

[0063] Probes for different concentrations of ONOO - The fitting curve of the fluorescence ratio response is shown in Figure 6 . Probe ER-DMPTC for 0-200 μM MONOO - Has good linear ratio response.

[0064] Probe detection of ONOO - Selective determination

[0065] Take 10 μL fluorescent probe mother solution, dilute it with 1xPBS (pH 8.2, 3 mM CTAAB) solution, prepare 17 groups in parallel, add 500 μM related interfering substances, including Ca 2+ , Cu 2+ , Fe 3+ Mg 2+ , K + 、Zn 2+ 、NO2 - 、NO3 - , Cys, GSH, NO, H2O2, t-BuOOH, O2 -. , ·OH, ROO· and ClO - , so that each probe solution is 10 μM, incubate at room temperature in the dark for 12 h, and after sufficient reaction, test the fluorescence spectrum in a 10 mm cuvette. The test results are shown in Figure 7 As can be seen from the figure, the fluorescent probe is sensitive to ONOO - Has good selectivity.

[0066] Embodiment 3:

[0067] Synthesis of compound 3

[0068] At 0°C, compound 1 (377.0 mg, 1.5 mmol), acid-binding agent triethylamine (303.6 mg, 3 mmol) and solvent dichloromethane 20 mL were added to the reaction vessel, and after stirring evenly, compound 2 (785.2 mg, 3 mmol) was added. Stirring was maintained at 4°C for 2 h, and after returning to room temperature, stirring was continued for 12 h. After the reaction was completed by thin layer chromatography, the solvent was concentrated, and the product was separated and purified by silica gel column chromatography, with dichloromethane and methanol (50 / 1, v / v) as the eluent, and a light yellow solid product, i.e., compound 3, was obtained.

[0069] Synthesis of ER-MPTC

[0070] Compound 3 (238.3 mg, 0.5 mmol) and compound 4 (217.2 mg, 1 mmol) were added to the reaction vessel, and under nitrogen protection, 25 μL of catalyst piperidine, 20 mL of solvent anhydrous acetonitrile and Molecular sieve 2g, heated under reflux for 6h. After the reaction was completed by thin layer chromatography, the solvent was concentrated, and the product was separated and purified by silica gel column chromatography with dichloromethane and ethyl acetate (50 / 1, v / v) as the eluent, and a black solid product, namely ER-MPTC, was obtained.

[0071] Synthesis of ER-DMPTC

[0072] Compound 3 (238.3 mg, 0.5 mmol) and compound 5 (231.4 mg, 1 mmol) were added to the reaction vessel, and under nitrogen protection, 25 μL of catalyst piperidine, 20 mL of solvent anhydrous acetonitrile and Molecular sieve 2g, heated under reflux for 6h. After the reaction was completed by thin layer chromatography, the solvent was concentrated, and the product was separated and purified by silica gel column chromatography with dichloromethane and ethyl acetate (50 / 1, v / v) as the eluent, and a black solid product, ER-DMPTC, was obtained.

[0073]

[0074] Fluorescent probe photostability assay

[0075] Take 10 μL of fluorescent probe stock solution, dilute it to 10 μM with DMSO, place it under a 150W xenon lamp for 90 minutes, and collect the fluorescence spectrum of the solution every 1 minute. The test results are shown in Figure 8 As can be seen from the figure, the fluorescent probe has good photostability.

[0076] pH stability of fluorescent probe and its effect on ONOO at different pH values - Response test

[0077] Adjust the 1xPBS (3mM CTAB) solution to pH values ​​of 3.16, 4.06, 5.13, 5.98, 6.60, 7.12, 7.80, 8.29, 9.29, 10.03 and 11.20. Take 10μL fluorescent probe stock solution, dilute it to 10μM with PBS solution of corresponding pH value, and stand it at room temperature in the dark for 12h. Take another set of parallel samples and add 200μM MONOO - , incubate at room temperature in the dark for 12 hours. After sufficient reaction, test the fluorescence spectrum of the sample in a 10 mm cuvette. The test results are shown in Fig. 9 As can be seen from the figure, the fluorescent probe is stable in a wide pH range and has an optimal response signal in the physiological pH range, which can meet the requirements of ONOO in biological tissue samples. - testing requirements.

[0078] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A single excitation detection method for peroxynitrite (ONOO) - A near-infrared ratiometric fluorescent probe, characterized in that The structural formula of the near-infrared ratiometric fluorescent probe is shown in Formula I: Wherein, R is selected from H or CH3.

2. A method for preparing a near-infrared ratiometric fluorescent probe as claimed in claim 1, characterized in that: The steps include: (1) Compound 1, triethylamine and solvent were mixed at 0-4°C, compound 2 was added, stirred for 1-2 hours, returned to room temperature, and stirred for 6-12 hours; after the reaction was completed, the mixture was concentrated, separated and purified by silica gel column chromatography to obtain compound 3; (2) Under an inert atmosphere, a catalyst, Molecular sieve and solvent, heat to reflux for reaction for 6-10h; after the reaction is completed, concentrate, separate and purify by silica gel column chromatography to obtain the product.

3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of compound 1, compound 2 and triethylamine is 0.5-1:1.5:1.

5.

4. The preparation method according to claim 2, characterized in that: The solvent is selected from dichloromethane and / or acetonitrile.

5. The preparation method according to claim 2, characterized in that: In step (2), the molar ratio of compound 3 to compound 4 is 1:1-4.

6. The preparation method according to claim 2, characterized in that: The catalyst is piperidine.

7. The preparation method according to claim 2, characterized in that: In step (2), the molar volume ratio of the compound 3 to the catalyst is 1 mmoL:(10-50) μL.

8. The preparation method according to claim 2, characterized in that: In step (2), the compound 3 and the The molar mass ratio of the molecular sieve is 1mmoL:(1-4)g.

9. The preparation method according to claim 2, characterized in that: In step (1), the volume ratio of dichloromethane to methanol used as the eluent for silica gel column chromatography is 10-100:

1.

10. The preparation method according to claim 2, characterized in that: In step (2), the volume ratio of dichloromethane to ethyl acetate used as the eluent for silica gel column chromatography is 10-100:1.

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