Multifunctional near-infrared fluorescent probe based on carbazole-phenylboronic acid ester as well as preparation method and application of multifunctional near-infrared fluorescent probe

Abstract

The invention discloses a multifunctional near-infrared fluorescent probe based on carbazole-phenylboronic acid ester and a preparation method and application of the multifunctional near-infrared fluorescent probe, the molecular formula of the multifunctional near-infrared fluorescent probe is (C40H42BN2O2) + Br <->, the multifunctional near-infrared fluorescent probe is named CNB-2, and the structural formula of the multifunctional near-infrared fluorescent probe is as follows: # imgabs0 #. The multifunctional near-infrared fluorescent probe can detect polarity, viscosity and ONOO <-> at the same time, and the detection process is simple, convenient, rapid and sensitive. More importantly, the probe can be used for detecting polarity, viscosity and ONOO <-> change in cells, and has a good application prospect in the fields of biological monitoring, medical diagnosis, clinical medicine and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a multifunctional near-infrared fluorescent probe based on carbazole-phenyl borate, and a preparation method and application thereof. Background Art

[0002] In recent years, with the rapid development of chemical sensors, fluorescent probes have received more and more attention, and their research in many fields such as chemistry, biology, medicine and environment has become more and more in-depth. Fluorescent probe technology has been widely used in the detection of various ions due to its advantages of good selectivity, high sensitivity, simplicity and rapidity, and no need for expensive instruments. The relationship between ultraviolet or fluorescence properties and ion concentration can be used to quantitatively or qualitatively analyze ions, thereby realizing the detection of ions more quickly and conveniently. The high sensitivity, high selectivity and non-invasiveness of fluorescent probes are very suitable for real-time or in-situ detection of ions.

[0003] Viscosity is a key parameter of the cellular microenvironment and is involved in numerous cellular processes such as membrane fusion, metabolite diffusion, protein aggregation, signal transduction, etc. Abnormal deviations in cellular viscosity levels can interfere with many cellular functions and induce diseases, including Alzheimer's disease, diabetes, atherosclerosis, and hypertension. Therefore, it is very important to accurately monitor changes in viscosity to reveal its chemical and biological roles in relevant diseases.

[0004] In addition to intracellular viscosity, polarity is also an important environmental factor that plays an important role in the metabolic process of organisms, especially at the cellular level. Changes in intracellular polarity play a key role in embryonic development, postnatal development, and cell homeostasis (including directional cell movement, cell proliferation, cell survival / apoptosis, and cell differentiation). More importantly, the occurrence of many diseases, such as diabetes and depression, often manifests as changes in the polarity of the body, so it is very important to monitor changes in intracellular polarity. Changes in intracellular polarity, especially changes in subcellular polarity, are associated with many pathologies. For example, the development of cirrhosis is closely related to changes in mitochondrial polarity. The process of fusion of lysosomes with autophagosome membranes may lead to changes in intralysosomal polarity. Therefore, real-time and accurate detection of subcellular structural polarity is crucial for related diseases caused by polarity changes.

[0005] Peroxynitrite (ONOO - ) is a typical representative of reactive nitrogen species (RNS), which is usually generated by the free radical nitric oxide and superoxide anion through a diffusion-controlled reaction in vivo and can act as an endogenous oxidant under different physiological and pathological conditions. In addition, ONOO has been reported to - It can participate in the metabolic process of cells through the oxidation of DNA, lipids and proteins, and ONOO -Excessive production of ONOO can cause a variety of diseases, such as inflammation, cardiovascular disease, Alzheimer's disease, neurodegenerative diseases, autoimmune diseases, cancer and diabetes, which seriously endanger the safety of humans and various living organisms. Therefore, a simple and sensitive method for detecting ONOO is established. - It is very necessary.

[0006] Carbazole has the advantages of low raw material cost, simple synthesis, multiple modifiable sites, high fluorescence quantum yield and good photostability. More importantly, carbazole 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, dyes based on carbazole groups are prepared for detecting polarity, viscosity and ONOO - The near-infrared fluorescent probe is of great significance. Summary of the invention

[0007] The present invention provides a multifunctional near-infrared fluorescent probe based on carbazole-phenyl borate and a preparation method and application thereof. The fluorescent probe preparation method of the present invention is easy to operate, the raw materials are easily available, and the intracellular polarity, viscosity and ONOO can be achieved. - Multifunctional detection.

[0008] The multifunctional near-infrared fluorescent probe based on carbazole-phenyl borate of the present invention has the molecular formula (C 40 H 42 BN 2 O 2 ) + Br - , named CNB-2, the structural formula is as follows:

[0009] .

[0010] The preparation method of the multifunctional near-infrared fluorescent probe based on carbazole-phenyl borate of the present invention comprises the following steps:

[0011] Step 1: Preparation of intermediate CN-2

[0012] 200 mg (0.80 mmol) of 9-butyl-9H-carbazole-3-carboxaldehyde and 171 mg (1.19 mmol) of 4-methylquinoline were placed in a 25 mL round-bottom flask containing 8 mL of DMF, and 2 drops of methanesulfonic acid were added. The mixture was stirred under reflux at 150 °C for 3 h. After cooling to room temperature, the reactant was poured into a saturated NaCl solution and filtered to obtain a red crude product. The intermediate CN-2 (120 mg, 40%) was purified by column chromatography (PE / EA = 4 / 1, V / V) as a yellow solid.

[0013] Step 2: Preparation of target product CNB-2

[0014] 200 mg (0.53 mmol) of intermediate CN-2 and 237 mg (0.80 mmol) of 4-bromomethylphenylboronic acid pinacol ester were weighed and placed in a 100 mL three-necked flask and filled with nitrogen. 20 mL of acetonitrile was added under anaerobic conditions. The mixture was refluxed and stirred at 80 °C for 8 h. A dark red solid was precipitated and the crude product was filtered off. The crude product was washed with anhydrous acetonitrile to obtain 165 mg of a dark red solid (165 mg, 52%), which was the target product.

[0015] The synthetic route of the present invention is as follows:

[0016] .

[0017] The invention discloses an application of a multifunctional near-infrared fluorescent probe based on carbazole-phenyl borate in preparing a polarity detection reagent.

[0018] The invention discloses an application of a multifunctional near-infrared fluorescent probe based on carbazole-phenyl borate in preparing a viscosity detection reagent.

[0019] The multifunctional near-infrared fluorescent probe based on carbazole-phenyl borate is used in the preparation of ONOO - Application in detection reagents.

[0020] The fluorescent probe of the present invention selects carbazole derivatives as fluorophores and phenylboronic acid ester as recognition groups. The detection mechanism is that phenylboronic acid ester is reacted with ONOO - After oxidation, an elimination reaction occurs, releasing the fluorophore and causing a change in the fluorescence signal. The fluorescent probe detects ONOO by UV and fluorescence spectrometer. - The fluorescent probe can also be used to detect changes in polarity and viscosity, and the detection process is simple, rapid and sensitive. More importantly, the probe can simultaneously detect polarity, viscosity and ONOO in cells. - , has good application prospects in the fields of biological monitoring, medical diagnosis and clinical medicine.

[0021] The present invention is based on carbazole dyes for detecting polarity, viscosity and ONOO - The near-infrared fluorescent probe can be used to detect polarity and viscosity by using fluorescence spectroscopy in solution testing; the probe response to ONOO can be determined by using UV and fluorescence spectroscopy. - The probe can specifically detect ONOO through selectivity and anti-interference ability tests. - , does not react with other active oxygen species and has strong anti-interference ability; the probe has strong pH stability and low cytotoxicity. Fluorescence imaging technology can also be used to measure the polarity, viscosity and ONOO in Hep G2 cells.- Purpose of testing.

[0022] Compared with the prior art, the present invention discloses a dye based on carbazole-phenyl borate for detecting polarity, viscosity and ONOO - The near-infrared fluorescent probe has a simple synthesis method and is easy to operate. The detection method can realize the detection of polarity, viscosity and ONOO - Multifunctional detection; the detection signal is obvious, and it is a near-infrared fluorescent probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 This is the synthetic route of the fluorescent probe of the present invention.

[0025] Figure 2 This is the H NMR spectrum of the fluorescent probe CNB-2 of the present invention.

[0026] Figure 3 This is the nuclear magnetic carbon spectrum of the fluorescent probe CNB-2 of the present invention.

[0027] Figure 4 This is the fluorescence spectrum of the fluorescent probe CNB-2 of the present invention for detecting polarity.

[0028] Figure 5 This is the fluorescence spectrum of the fluorescent probe CNB-2 of the present invention for detecting viscosity.

[0029] Figure 6 The fluorescent probe CNB-2 and ONOO - Fluorescence changes over time.

[0030] Figure 7 The fluorescent probe CNB-2 of the present invention is used to measure ONOO - Absorption changes in concentration titration experiments.

[0031] Figure 8 The fluorescent probe CNB-2 of the present invention is used to measure ONOO - Fluorescence changes in concentration titration experiments.

[0032] Fig. 9 For the present invention I 520 Fluorescence intensity and ONOO - Linear fitting diagram of the concentration.

[0033] Fig.10 The fluorescent probe CNB-2 of the present invention detects ONOO - Fluorescence selectivity.

[0034] Fig.11 The fluorescent probe CNB-2 of the present invention detects ONOO - fluorescence interference.

[0035] Fig.12 The fluorescent probe CNB-2 and probe plus ONOO - Changes in maximum fluorescence intensity in buffer solutions with different pH values.

[0036] Fig.13 The probe CNB-2 of the present invention detects ONOO - Toxicity to Hep G2 cells.

[0037] Fig.14 This is an imaging picture of Hep G2 cells using the probe CNB-2 of the present invention to detect polarity.

[0038] Fig.15 This is an image of Hep G2 cells using the probe CNB-2 of the present invention to detect viscosity.

[0039] Fig.16 The probe CNB-2 of the present invention detects endogenous ONOO - Hep G2 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: Preparation of fluorescent probe

[0042]

[0043] 1. Synthesis and characterization of intermediate CN-2

[0044] 200 mg (0.80 mmol) of 9-butyl-9H-carbazole-3-carboxaldehyde and 171 mg (1.19 mmol) of 4-methylquinoline were placed in a 25 mL round-bottom flask containing 8 mL of DMF, and 2 drops of methanesulfonic acid were added. The mixture was refluxed and stirred at 150 °C for 3 h. After cooling to room temperature, the reactants were poured into a saturated NaCl solution and filtered to obtain a red crude product, which was purified by column chromatography (PE / EA = 4 / 1, V / V) to obtain a yellow solid (120 mg, 40%).

[0045] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.89 (d, J = 4.7 Hz, 1H), 8.69 (s, 1H), 8.63 (d, J = 8.4 Hz, 1H), 8.25 (d, J = 7.7 Hz, 1H), 8.17-8.02 (m, 2H), 7.96(d, J = 8.6 Hz, 1H), 7.90 (d, J = 4.7 Hz, 1H), 7.84-7.76 (m, 2H), 7.67 (dt, J= 15.6, 7.9 Hz, 3H), 7.49 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 7.4 Hz, 1H), 4.44(t, J = 7.1 Hz, 2H), 1.78 (p, J = 7.2 Hz, 2H), 1.32 (h, J = 7.4 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). HRMS (m / z) calcd for C 27 H 24 N 2 [M + H] + : 377.1985, found:377.2002.

[0046] 2. Synthesis and characterization of fluorescent probe CNB-2

[0047] Weigh 200 mg (0.53 mmol) CN-2 and 237 mg (0.80 mmol) 4-bromomethylphenylboronic acid pinacol ester into a 100 mL three-necked flask and fill it with nitrogen. Add 20 mL acetonitrile under anaerobic conditions. Stir under reflux for 8 h. A dark red solid precipitates. Filter to obtain a crude product. Wash the crude product with anhydrous acetonitrile to obtain 165 mg of a dark red solid (165 mg, 52%).

[0048] 1H NMR (400 MHz, DMSO-d 6 ) δ 9.53 (d, J = 6.6 Hz, 1H), 9.17 (d, J = 8.7Hz, 1H), 8.92 (d, J = 1.6 Hz, 1H), 8.64 (d, J = 6.7 Hz, 1H), 8.54 (d, J =15.7 Hz, 1H), 8.42 (d, J = 15.8 Hz, 1H), 8.28 (dd, J = 11.3, 8.3 Hz, 2H), 8.15 (t, J = 8.3 Hz, 2H), 8.01 (t, J = 7.7 Hz, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.70 (t, J = 7.9 Hz, 3H), 7.55 (t, J = 7.7 Hz, 1H), 7.40-7.29 (m, 3H), 6.29(s, 2H), 4.49 (t, J = 7.0 Hz, 2H), 1.80 (p, J = 7.1 Hz, 2H), 1.27 (s, 14H),0.91 (t, J = 7.4 Hz, 3H). 13 C NMR (151 MHz, DMSO-d 6 ) δ 154.60, 148.24, 146.48,142.46, 138.52, 138.26, 135.57, 135.53, 129.42, 128.22, 127.17, 126.87,126.77, 122.67, 122.57, 121.04, 120.38, 119.89, 116.73, 110.57, 84.30, 59.47,42.81, 31.22, 25.10, 20.23, 14.21. HRMS (m / z) cald for C 40 H 42 BN 2 O 2 + [M] + :593.3305, found: 593.3312.

[0049] The H NMR spectrum of fluorescent probe CNB-2 is shown in Figure 2 ; The NMR spectrum of fluorescent probe CNB-2 is shown in Figure 3 .

[0050] Example 2: Polarity response test of fluorescent probe CNB-2

[0051] The spectral performance of probe CNB-2 (10 μM) was tested in 2 mL solutions of 1,4-dioxane and water at different volume ratios. Figure 4 As shown in the figure, when the proportion of water in the mixed solution of 1,4-dioxane and water increases from 0% to 100%, the polarity of the solution gradually increases. At an excitation wavelength of 499 nm, the emission wavelength increases with the increase of polarity, the emission wavelength red-shifts from 630 nm to 660 nm, and the fluorescence quenching degree reaches 95%. This shows that the probe CNB-2 is a good polarity response probe.

[0052] Example 3: Viscosity response test of fluorescent probe CNB-2

[0053] The spectral performance of probe CNB-2 (10 μM) was tested in solutions of 2 mL glycerol and water with different volume ratios. Figure 5 It can be seen that at an excitation wavelength of 499 nm, as the glycerol content increases, the emission wavelength of the probe CNB-2 blue-shifts with the increase in the viscosity of the test system, and the emission wavelength blue-shifts from 660 nm to 627 nm; at the same time, the fluorescence intensity gradually increases with the increase in the viscosity of the test system, and the fluorescence intensity increases by about 111 times. The experimental results show that the probe CNB-2 responds well to viscosity and can be used as a viscosity probe.

[0054] Example 4: Detection of ONOO in solution - Reaction time test of fluorescent probe CNB-2

[0055] Fluorescent probe CNB-2 (10 μM) and ONOO - (50 μM) was reacted in a solution system of ethanol / water (3:7, v / v, pH = 7.4). The results are shown in Figure 6 , join ONOO - Afterwards, the fluorescent probes CNB-2 and ONOO - The response time is super fast (about 3 minutes).

[0056] Example 5: Detection of ONOO in solution - Concentration titration test of fluorescent probe CNB-2 and concentration linear relationship

[0057] The probe CNB-2 (10 μM) was tested in ethanol / water (3:7, v / v, pH = 7.4) with different concentrations of ONOO - (0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM) spectral properties. It was found that with the increase of ONOO- As the concentration of the probe increases gradually, an absorption peak appears at 383 nm and the intensity gradually increases; the fluorescence peak at 650 nm also gradually weakens, and the fluorescence peak at 520 nm gradually increases. 520 Fluorescence intensity in ONOO - The concentration reached a maximum of 18 μM and the enhancement was about 11 times (see Figure 7 and Figure 8 ). - The concentration is the horizontal axis, and the probe CNB-2 is in I 520 The fluorescence intensity at the position is taken as the ordinate, and a graph is drawn and linear fitting is performed to obtain the linear regression equation of the probe: Y = 75.98X + 45.55, and the linear correlation coefficient R 2 = 0.9943, and the detection limit was calculated to be 51.3 nM (see Fig. 9 ).

[0058] Example 6: Selectivity and anti-interference ion experiments

[0059] In different fluorescence cuvettes, 2 mL of ethanol / water (3:7, v / v, pH = 7.4) and 10 μM fluorescent probe stock solution were added. When active nitrogen and active oxide (ONOO - , ClO - , H 2 O 2 , •OH, Na + , K + , Ca 2+ Mg 2+ , Cu 2+ Cr 3+ , Fe 2+ , Cl - Br - ,I - , HSO 4 - , S 2 O 3 2- 、SO 3 2- 、NO 2 - 、SCN - ions and Cys, Hcy, GSH small molecules) and other analytical species (50 μM), the probe CNB-2 can detect ONOO - Perform specific identification and 520 The fluorescence intensity is significantly enhanced, so the probe can specifically respond to ONOO - (See Fig.10:The left side of the illustration is CNB-2, and the right side is CNB-2 + ONOO - ). When CNB-2 (10 μM) was added to the above analytes and then 50 μM ONOO - After 3 minutes of reaction, it can be seen that CNB-2 can still specifically detect ONOO in a complex solution system when coexisting with various analytes. - , and the fluorescence intensity was significantly enhanced. Experiments have shown that CNB-2 can respond to ONOO - No interference from other substances (see Fig.11 ).

[0060] Example 7: pH responsiveness experiment

[0061] PBS buffer with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 was prepared for CNB-2 (10 μM) and CNB-2 and ONOO - The changes in fluorescence intensity after the reaction of 50 μM were studied. Fig.12 As shown in the figure, the fluorescence intensity of probe CNB-2 remained basically unchanged in solutions with a pH value of 3.0-9.0; - After that, under the condition of pH value of 3.0-9.0, its I 520 The fluorescence intensity ratio remained basically unchanged. The experiment proved that the probe CNB-2 can adapt to the pH environment in the body.

[0062] Example 8: Hep G2 cell toxicity experiment

[0063] Before confocal imaging of Hep G2 cells, the standard MTT assay was used to evaluate the cytotoxic effect of probe CNB-2 on live Hep G2 cells. Fig.13 As shown in Figure 2, even when Hep G2 cells were co-incubated with 25 μM CNB-2 for 24 h, the cell viability remained above 80%. This indicates that the probe CNB-2 has low cytotoxicity and can be used for biological imaging.

[0064] Example 9: Testing the performance of fluorescent probes for detecting polarity in Hep G2 cells

[0065] Before cell imaging, adherent cells were detached with 0.2% (w / v) trypsin-EDTA solution and dispersed into cell culture medium, and then the cell suspension was transferred to the confocal dish and allowed to grow on the bottom of the dish. In order to study the ability of probe CNB-2 to detect cell polarity, Hep G2 cells were co-cultured with CNB-2 and DMSO, and fluorescence images were recorded at different times, such as Fig.14As shown. It can be observed that after adding DMSO, the fluorescence intensity of the red channel was significantly weakened after 15 min, and after 30 min, the fluorescence intensity of the red channel was weakened again. After about 1 hour, the fluorescence of the red channel was almost invisible. The decrease in fluorescence intensity in the red channel confirmed that CNB-2 can effectively detect the increase in DMSO-induced mitochondrial polarity in a short time. This shows that the probe CNB-2 can be used as a new polarity probe to detect polarity changes in cells.

[0066] Example 10: Testing the performance of the fluorescent probe for detecting viscosity on the viscosity in Hep G2 cells

[0067] Before cell imaging, adherent cells were separated with 0.2% (w / v) trypsin-EDTA solution and dispersed into cell culture medium, and then the cell suspension was transferred to the confocal dish to allow it to grow on the bottom of the dish. In order to explore whether CNB-2 can be used as a new fluorescent probe for detecting viscosity changes in cells, Hep G2 cells were first cultured with nystatin for 30 minutes, then the probe CNB-2 was added and incubated for 30 minutes, and then fluorescence imaging was performed. Fig.15 As shown, compared with cells treated with CNB-2 alone, the fluorescence intensity of the red channel of Hep G2 cells co-cultured with CNB-2 and nystatin for 30 minutes was significantly enhanced. This indicates that the probe CNB-2 can be used as a new viscosity probe to detect viscosity changes in cells.

[0068] Example 11: Detection of ONOO - Fluorescent probe for ONOO in Hep G2 cells - Detection performance test

[0069] 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 (CNB-2): cells were incubated with CNB-2 (5 μM, 30 min) only, washed three times with PBS, and then subjected to confocal imaging; the second group (CNB-2+UA): cells were first incubated with UA (200 μM, 30 min), then incubated with CNB-2 (5 μM, 30 min), washed three times with PBS, and then subjected to confocal imaging; the third group (CNB-2+LPS): cells were incubated with LPS (1 μg / mL, 30 min), then incubated with CNB-2 (5 μM, 30 min), washed three times with PBS, and then subjected to confocal imaging; the fourth group (CNB-2+LPS+UA): cells were incubated with LPS (1 μg / mL, 30 min), then incubated with UA (200 μM, 30 min), and then incubated with CNB-2 (5 μM, 30 min), washed three times with PBS, and then subjected to confocal imaging. LPS for ONOO - Release agent; UA is ONOO - Scavenger.

[0070] The results are as follows Fig.16 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 yellow 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 yellow fluorescence is the fluorescence after the probe reacts.

[0071] 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 multifunctional near-infrared fluorescent probe based on carbazole-phenylboronic acid ester, characterized in that: The molecular formula of the multifunctional near-infrared fluorescent probe is (C 40 H 42 BN2O2) + Br - , named CNB-2, and its structural formula is as follows: 。 2. The method for preparing the multifunctional near-infrared fluorescent probe based on carbazole-phenylboronic acid ester according to claim 1, characterized in that The steps include: Step 1: Preparation of intermediate CN-2 9-Butyl-9H-carbazole-3-carboxaldehyde and 4-methylquinoline were added to DMF, and methylsulfonic acid was added, and the mixture was heated to reflux and stirred for reaction; after the reaction was completed, the mixture was cooled to room temperature, and the reactants were poured into a saturated NaCl solution, and filtered to obtain a red crude product, which was purified by column chromatography to obtain the intermediate CN-2; Step 2: Preparation of target product CNB-2 The intermediate CN-2 and 4-bromomethylphenylboronic acid pinacol ester were added to a reactor, nitrogen was introduced, acetonitrile was added under anaerobic conditions, the temperature was raised to reflux and stirred for reaction, a dark red solid was precipitated, a crude product was obtained by suction filtration, and the crude product was washed with anhydrous acetonitrile to obtain the target product; The synthetic route is as follows: 。 3. The multifunctional near-infrared fluorescent probe according to claim 1 is used in the preparation of polarity detection reagents and / or viscosity detection reagents and / or ONOO - Application in detection reagents.

4. The use according to claim 3, characterized in that: The detection reagent is in I 520 The fluorescence intensity at ONOO - The concentration is linear.

5. The use according to claim 3 or 4, characterized in that: The pH value range of the detection system is 3.0-9.

0.

6. The use according to claim 3, characterized in that: The detection system is a cell system or a solution system.

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