Ratio-type fluorescent probe for specifically responding to ONOO <-> based on triphenylamine dye as well as preparation method and application of ratio-type fluorescent probe

Abstract

The invention discloses a ratio type fluorescent probe for specifically responding to ONOO <-> based on triphenylamine dye as well as a preparation method and application of the ratio type fluorescent probe, and belongs to the technical field of fluorescent probes. The invention discloses a ratio type fluorescent probe for specifically responding to ONOO <-> based on triphenylamine dye. The chemical formula of the ratio type fluorescent probe is (C38H38BN2O2) < + > Br <->. The fluorescent probe detects ONOO <-> through ultraviolet and fluorescence spectrometers and is not interfered by active nitrogen and active oxygen, the detection process is simple, convenient, rapid and sensitive, and the detection limit is 0.87 nM. More importantly, the probe can detect ONOO <-> in cells and has a good application prospect in the field of biological monitoring.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and more specifically to a method for specifically responding to ONOO based on triphenylamine dyes. - Ratio-type fluorescent probe and preparation method and application thereof. Background Art

[0002] As a kind of reactive oxygen species (ROS), peroxynitrite ion (ONOO - ) plays an irreplaceable role in biological processes. - Imbalanced levels will oxidize nucleic acids and proteins in cells, causing cellular metabolic disorders. This is closely related to many diseases, such as diabetes, cardiovascular disease, neurodegenerative diseases, cancer, etc. In recent years, researchers have proven that ROS is closely related to cancer treatment. Therefore, ROS can be used as a biological target for detecting anti-cancer treatment. However, there is still a lack of detection methods for ONOO - analysis tools.

[0003] So far, ONOO - The main methods include iodine titration, colorimetry, chromatography, fluorescent probe method, electrochemical method, etc. Among them, small molecule fluorescent probes have attracted great attention from researchers due to their advantages such as non-invasiveness, real-time evaluation and simple operation. So far, researchers have reported many methods for detecting ONOO - Although many detection groups for ONOO have been reported - A fluorescent probe that can detect ONOO in the process of oxidative stress very quickly - Fluorescent probes that can detect level fluctuations are still rare. - The development of new ratiometric fluorescent probes with high specificity and the realization of cell imaging is still urgent.

[0004] Triphenylamine has the advantages of low raw material cost, simple synthesis, multiple modifiable sites, high fluorescence quantum yield and good photostability. More importantly, triphenylamine fluorescent dyes have relatively small molecular weight, good biocompatibility, easy penetration of living cells, and are very suitable for biological fluorescence imaging, which has attracted widespread attention. Based on this, the preparation of triphenylamine fluorescent dyes that specifically respond to ONOO in biological cells is a new method for preparing triphenylamine fluorescent dyes. - The ratiometric fluorescent probe is of great significance.

[0005] Therefore, a triphenylamine-based dye specifically responds to ONOO - Ratio-type fluorescent probes and preparation methods and applications thereof are issues that urgently need to be addressed by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a triphenylamine dye-specific response ONOO - The ratiometric fluorescent probe and its preparation method and application are convenient to operate, the raw materials are easily available, and the intracellular ONOO - Specificity detection.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A triphenylamine dye-specific ONOO - A ratiometric fluorescent probe, wherein the chemical formula of the fluorescent probe is: (C 38 H 38 BN2O2) + Br - , named NBONOO, with the structural formula:

[0009]

[0010] Further, the triphenylamine dye specifically responds to ONOO - The preparation method of the ratiometric fluorescent probe is as follows:

[0011] (1) 4-Diphenylaminobenzaldehyde and 2-methylpyridine were added to anhydrous ethanol under the catalytic action of piperidine, and stirred at 25°C to 35°C for 5 to 6 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using dichloromethane / petroleum ether as the mobile phase to obtain compound 1;

[0012] The 4-diphenylaminobenzaldehyde, 2-methylpyridine, piperidine and anhydrous ethanol are mixed in a molar ratio of 1: (1-1.5): 0.1: 500;

[0013] The volume ratio of dichloromethane / petroleum ether is 12:1;

[0014] (2) Compound 1, 4-bromomethylphenylboronic acid pinacol ester prepared in step (1) was refluxed with acetonitrile at 81° C. to 82° C. and stirred for 5 to 6 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by neutral alumina column chromatography using dichloromethane / methanol as the mobile phase to obtain a fluorescent probe;

[0015] The compound 1, 4-bromomethylphenylboronic acid pinacol ester and acetonitrile are added in a molar ratio of 1: (1-2): 300;

[0016] The volume ratio of dichloromethane / methanol is 10:1.

[0017] Furthermore, the structural formula of the compound 1 in step (1) is:

[0018]

[0019] Furthermore, the fluorescent probe or the fluorescent probe prepared by the preparation method is used to prepare a fluorescent probe specifically for detecting ONOO - Application of reagents.

[0020] Furthermore, the fluorescent probe is used to specifically detect ONOO in the prepared cells. - Application of reagents.

[0021] Furthermore, the cells are MCF-7 cells.

[0022] Furthermore, a method for detecting ONOO - A reagent or kit comprising the fluorescent probe or the fluorescent probe prepared by the preparation method.

[0023] Further, the kit is used to detect ONOO - Application in.

[0024] The fluorescent probe uses triphenylamine derivatives as the fluorophore and phenylboronic acid ester as the recognition group. The detection mechanism is ONOO - After the oxidation reaction with phenylboronic acid ester, 1,6-elimination occurs, releasing the fluorophore and causing the fluorescence signal to change. The fluorescent probe detects ONOO by UV and fluorescence spectrometer. - The detection process is simple, rapid and sensitive, and the detection limit is 0.87nM. More importantly, the probe can detect ONOO in cells. - , which has good application prospects in the field of biological monitoring.

[0025] The present invention is based on triphenylamine dye for specific detection of ONOO - The ratiometric fluorescent probe can determine the concentration dependence of the probe by using UV and fluorescence spectra in solution testing; the probe can specifically detect ONOO through selectivity and anti-interference ability tests. - , does not react with active oxygen species and has strong anti-interference ability; the probe has strong pH stability and low cytotoxicity. It can also be used to detect ONOO in MCF-7 cells through fluorescence imaging technology. - Purpose of testing.

[0026] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for preparing a triphenylamine dye-specific ONOO - The ratio-type fluorescent probe and its preparation method and application are simple in synthesis and easy in operation; the detection method can realize ONOO -Specific detection without interference from other active oxygen species; the detection signal is obvious, and it is a ratiometric fluorescent probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The accompanying drawing is a synthetic route of the fluorescent probe of the present invention;

[0029] Figure 2 The accompanying drawing is a nuclear magnetic hydrogen spectrum of the fluorescent probe NBONOO of the present invention;

[0030] Figure 3 The accompanying drawing is a nuclear magnetic carbon spectrum of the fluorescent probe NBONOO of the present invention;

[0031] Figure 4 The attached figure shows the fluorescent probes NBONOO and ONOO of the present invention. - Fluorescence changes over time

[0032] Figure 5 The attached figure shows the fluorescent probe NBONOO of the present invention for measuring ONOO - Absorption changes in concentration titration experiments;

[0033] Figure 6 The attached figure shows the fluorescent probe NBONOO of the present invention for measuring ONOO - Fluorescence changes in concentration titration experiments;

[0034] Figure 7 The accompanying drawings are the present invention I 510 / I 665 Fluorescence intensity ratio and ONOO - The concentration linear fitting diagram of

[0035] Figure 8 The attached figure shows the fluorescent probe NBONOO of the present invention detecting ONOO - Fluorescence selectivity;

[0036] Fig. 9 The attached figure shows the fluorescent probe NBONOO of the present invention detecting ONOO - Fluorescence interference;

[0037] Fig.10 The attached figure shows the fluorescent probe NBONOO and probe plus ONOO of the present invention. -Maximum fluorescence intensity changes in different pH buffer solutions;

[0038] Fig.11 The accompanying drawing shows the detection of ONOO by the probe HBTC1 of the present invention. - MCF-7 cell toxicity;

[0039] Fig.12 The accompanying drawings show the detection of exogenous and endogenous ONOO by the probe HBTC1 of the present invention. - MCF-7 cell imaging. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1

[0042] Preparation steps of fluorescent probe (synthesis route see Figure 1 )as follows:

[0043] (1) Preparation of Compound 1

[0044]

[0045] 4-Diphenylaminobenzaldehyde and 2-methylpyridine were added to anhydrous ethanol under the catalytic action of piperidine, and stirred at 25°C for 6 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using dichloromethane / petroleum ether as a mobile phase to obtain compound 1;

[0046] 4-Diphenylaminobenzaldehyde, 2-methylpyridine, piperidine and anhydrous ethanol are mixed in a molar ratio of 1:1:0.1:500; the volume ratio of dichloromethane / petroleum ether is 12:1.

[0047] (2) Preparation of fluorescent probe NBONOO

[0048]

[0049] The compound 1, 4-bromomethylphenylboronic acid pinacol ester prepared in step (1) was refluxed with acetonitrile at 81° C. for 6 h, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by neutral alumina column chromatography using dichloromethane / methanol as the mobile phase to obtain the fluorescent probe NBONOO.

[0050] Compound 1, 4-bromomethylphenylboronic acid pinacol ester and acetonitrile were added in a molar ratio of 1:1:300; the volume ratio of dichloromethane / methanol was 10:1.

[0051] The H NMR spectrum of the fluorescent probe NBONOO is shown in Figure 2 ; The NMR carbon spectrum of the fluorescent probe NBONOO is shown in Figure 3 .

[0052] 1 H NMRδ(500MHz,DMSO-d6)δ9.08-9.05(m,1H),8.57-8.52(m,2H),7.97-7.92(m,1H) ),7.85(d,J=15.8Hz,1H),7.69-7.66(m,2H),7.60-7.57(m,2H),7.40(d,J=2.0H z,1H),7.39(d,J=1.1Hz,2H),7.37(d,J=1.9Hz,1H),7.30(d,J=7.8Hz,2H),7.20 -7.16(m,2H),7.14-7.12(m,4H),6.92-6.89(m,2H),6.10(s,2H),1.26(s,12H).

[0053] 13 C NMR (126MHz, DMSO) δ153.05,151.52,150.40,146.50,146.06,145.14,145 .01,144.66,144.52,143.60,140.85,139.82,137.93,135.60,135.00,130 .61,130.35,130.02,129.77,129.33,127.80,127.17,126.72,126.68,126 .07,125.20,124.50,123.92,120.67,114.52,84.32,25.43,25.12,25.05.

[0054] Example 2

[0055] The preparation steps of the fluorescent probe are as follows:

[0056] (1) Preparation of Compound 1

[0057] 4-Diphenylaminobenzaldehyde and 2-methylpyridine were added to anhydrous ethanol under the catalytic action of piperidine, and stirred at 30°C for 5.5 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using dichloromethane / petroleum ether as mobile phase to obtain compound 1;

[0058] 4-Diphenylaminobenzaldehyde, 2-methylpyridine, piperidine and anhydrous ethanol are mixed in a molar ratio of 1:1.2:0.1:500; the volume ratio of dichloromethane / petroleum ether is 12:1.

[0059] (2) Preparation of probe NBONOO

[0060] The compound 1, 4-bromomethylphenylboronic acid pinacol ester prepared in step (1) was refluxed with acetonitrile at 82° C. for 5.5 h, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by neutral alumina column chromatography using dichloromethane / methanol as the mobile phase to obtain the fluorescent probe NBONOO.

[0061] Compound 1, 4-bromomethylphenylboronic acid pinacol ester and acetonitrile were added in a molar ratio of 1:1.5:300; the volume ratio of dichloromethane / methanol was 10:1.

[0062] Example 3

[0063] The preparation steps of the fluorescent probe are as follows:

[0064] (1) Preparation of Compound 1

[0065] 4-Diphenylaminobenzaldehyde and 2-methylpyridine were added to anhydrous ethanol under the catalytic action of piperidine, stirred at 35°C for 5 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using dichloromethane / petroleum ether as a mobile phase to obtain compound 1;

[0066] 4-Diphenylaminobenzaldehyde, 2-methylpyridine, piperidine and anhydrous ethanol are mixed in a molar ratio of 1:1.5:0.1:500; the volume ratio of dichloromethane / petroleum ether is 12:1.

[0067] (2) Preparation of probe NBONOO

[0068] The compound 1, 4-bromomethylphenylboronic acid pinacol ester prepared in step (1) was refluxed with acetonitrile at 81° C. for 5 h, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by neutral alumina column chromatography using dichloromethane / methanol as the mobile phase to obtain the fluorescent probe NBONOO.

[0069] Compound 1, 4-bromomethylphenylboronic acid pinacol ester and acetonitrile were added in a molar ratio of 1:2:300; the volume ratio of dichloromethane / methanol was 10:1.

[0070] Test example

[0071] Performance test of the fluorescent probe prepared in Example 1

[0072] (1) Detection of ONOO in solution- Reaction time test of fluorescent probe NBONOO

[0073] Prepare a 1 mM NBONOO fluorescent probe stock solution in acetonitrile;

[0074] Fluorescent probe NBONOO (10 μM) and ONOO - (50 μM) was reacted in a solution system of acetonitrile / PBS buffer (1:99, v / v, 10 mm, pH 7.4). The results are shown in Figure 4 , join ONOO - Then, the fluorescent probes NBONOO and ONOO - The response time is super fast (about 3 minutes).

[0075] (2) Detection of ONOO in solution - Concentration titration test of fluorescent probe NBONOO and concentration linear relationship

[0076] The spectral performance of the probe NBONOO (10 μM) and different concentrations of HClO (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 μM) were tested in acetonitrile / PBS (1:99, v / v, 10 mm, pH 7.4) at 37°C. - As the concentration of the probe increases gradually, the absorption of the probe at 465nm gradually increases; the fluorescence peak at 665nm also gradually weakens, and the fluorescence peak at 515nm also gradually increases. 665 / I 515 The fluorescence intensity ratio of ONOO - The concentration reached a maximum of 50 μM and the enhancement was about 79 times (probe NBONOO (10 μM) in I 665 / I 515 The fluorescence intensity ratio is 0.11268, probe NBONOO (10 μM) + ONOO - (50μM) in I 665 / I 515 The fluorescence intensity ratio is 8.72009) (see Figure 5 and Figure 6 ).

[0077] ONOO - The concentration is the horizontal axis, and the probe NBONOO is in I 510 / I 665 The fluorescence intensity at the position is taken as the ordinate, and a graph is drawn and a linear fit is performed to obtain the linear regression equation of the probe: Y = 0.1825X +

[0078] 0.0825, linear correlation coefficient R2 = 0.9926 and the detection limit was calculated to be 0.75 (see Figure 7 ).

[0079] (3) Interference and anti-interference ion experiments

[0080] In different fluorescence cuvettes, 4 mL DMSO / PBS buffer (1:99, v / v, 10 mm, pH 7.4) and 10 μL fluorescent probe stock solution were added, respectively. The results are shown in Figure 8 (Blank only adds probe), when the probe NBONOO is added with active nitrogen and active oxide (ONOO - 、H2O2、O2 - , 1 O2、O t Bu, OH, HClO, TBHP, HS - 、SO3 2- , Hcy, Cys, GSH) and other analytical species (100 μM), the probe NBONOO can be used to detect ONOO - Perform specific identification and 510 / I 665 The fluorescence intensity ratio is significantly enhanced, so the probe can specifically respond to ONOO - When NBONOO (10 μM) was added to the above analytes and then 50 μM NBONOO was added for 100 seconds, it can be seen that NBONOO can still specifically detect ONOO in a complex solution system when it coexists with various analytes. - , and the fluorescence intensity is significantly enhanced. Experiments have shown that NBONOO can respond to ONOO - No interference from other substances (see Fig. 9 ).

[0081] (4) pH responsiveness experiment

[0082] PBS buffer with pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 was prepared for NBONOO (10 μM) and NBONOO and ONOO - The changes in fluorescence intensity after the reaction of 50 μM were studied.

[0083] The results are as follows Fig.10 As shown in the figure, the fluorescence intensity of the probe remained basically unchanged in solutions with a pH value of 4.0-10.0; - After, and at a pH value of 4.0-10.0, its I 510 / I 665The fluorescence intensity ratio remains basically unchanged. The experiment proves that the probe NBONOO can adapt to the pH environment in the body.

[0084] (5) MTT cytotoxicity assay

[0085] At 37°C, DMEM medium, and 5% CO2, an MTT experiment was performed in a 96-well plate to evaluate the cytotoxicity of the probe NBONOO (0-20 μM). Different concentrations of the probe BTHCl were used to treat different MCF-7 cells for three parallel experiments and continued to be cultured for 24 hours, then washed 3 times with PBS, 0.5 mg / L of MTT was added and continued to be incubated for 4 hours, the medium was aspirated, and 100 μL of DMSO was added (about 15 minutes). Each experiment was performed in triplicate. The ultraviolet absorption at 490 nm was measured using an ELISA instrument, and the cell viability was calculated based on the following equation: Cytotoxicity (%) = (average absorbance of the experimental group / absorbance of the control group)

[0086] ×100%.

[0087] The results are as follows Fig.11 As shown, the cell survival rate can be as high as over 90% at low concentrations, and the probe has almost no cytotoxicity.

[0088] (6) Detection of ONOO - Fluorescent probe for ONOO in MCF-7 cells - Detection performance test

[0089] Before cell imaging, adherent cells were detached with 0.2% (w / v) trypsin-EDTA solution and dispersed into cell culture medium. The cell suspension was then transferred to a confocal dish and allowed to grow on the bottom of the dish. -The cells were divided into 4 groups for experiments. The first group (NBONOO): cells were incubated with NBONOO (10 μM, 0.5 h) only, washed with PBS three times, and then confocal imaging was performed; the second group (SNI-1): cells were incubated with SNI-1 (200 μM, 0.5 h), then incubated with NBONOO (10 μM, 0.5 h), washed with PBS three times, and then confocal imaging was performed; the third group (LPS / IFN-γ): cells were incubated with SNI-1 (200 μM, 0.5 h), and then incubated with LPS (1 μg / mL) and IFN-γ (50 ng / mL) for 12 h, then incubated with NBONOO (10 μM, 0.5 h), washed 3 times with PBS, and then confocal imaging was performed; the fourth group (LPS / IFN-γ+UA): cells were incubated with SNI-1 (200 μM, 0.5 h), then incubated with LPS (1 μg / mL) and IFN-γ (50 ng / mL) for 12 h, then incubated with UA (200 μM), 0.5 h, and then incubated with NBONOO (10 μM, 0.5 h) and washed 3 times with PBS, and then confocal imaging was performed. SNI-1 is ONOO - LPS and IFN-γ are ONOO - Stimulant; UA is ONOO - of the remover.

[0090] The results are as follows Fig.12 As shown, red fluorescence was observed in cells after the probe was first incubated, and then with the addition of ONOO - As the concentration of ONOO increases, the red fluorescence in the cell gradually weakens and the green fluorescence gradually increases. This result also shows that the fluorescent probe of the present invention has the ability to detect ONOO in cells. - The red fluorescence is the intrinsic fluorescence of the probe, and the green fluorescence is the fluorescence after the probe reacts.

[0091] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A triphenylamine dye-specific ONOO - A ratiometric fluorescent probe, characterized in that The structural formula of the fluorescent probe is:

2. A triphenylamine dye-specific ONOO according to claim 1 - A method for preparing a ratiometric fluorescent probe, characterized in that: The specific steps are as follows: (1) 4-Diphenylaminobenzaldehyde and 2-methylpyridine were added to anhydrous ethanol under the catalytic action of piperidine, and stirred at 25°C to 35°C for 5 to 6 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using dichloromethane / petroleum ether as the mobile phase to obtain compound 1; The 4-diphenylaminobenzaldehyde, 2-methylpyridine, piperidine and anhydrous ethanol are mixed in a molar ratio of 1: (1-1.5): 0.1: 500; The volume ratio of dichloromethane / petroleum ether is 12:1; (2) Compound 1, 4-bromomethylphenylboronic acid pinacol ester prepared in step (1) was refluxed with acetonitrile at 81° C. to 82° C. and stirred for 5 to 6 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by neutral alumina column chromatography using dichloromethane / methanol as the mobile phase to obtain a fluorescent probe; The compound 1, 4-bromomethylphenylboronic acid pinacol ester and acetonitrile are added in a molar ratio of 1: (1-2): 300; The volume ratio of dichloromethane / methanol is 10:

1.

3. A method according to claim 2 for specifically responding to ONOO based on triphenylamine dye - A method for preparing a ratiometric fluorescent probe, characterized in that: The structural formula of compound 1 in step (1) is:

4. The fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 3 is used to prepare a fluorescent probe specifically for detecting ONOO - Application of reagents.

5. The use according to claim 4, characterized in that: The fluorescent probe is used to specifically detect ONOO in the preparation of cells. - Application of reagents.

6. The use according to claim 5, characterized in that: The cells are MCF-7 cells.

7. A method for detecting ONOO - A reagent or kit, characterized in that The invention comprises the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 3.

8. The kit according to claim 7 is used to detect ONOO - Application in.

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