Near-infrared fluorescent probe DCI-TSO based on ICT mechanism as well as preparation method and application of near-infrared fluorescent probe DCI-TSO

Abstract

The invention belongs to the technical field of hydrogen sulfide and peroxynitroso ion detection, and particularly discloses a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism and a preparation method and application thereof, and the preparation method comprises the following steps: S1, synthesizing a compound 1; s2, synthesizing a compound 2; s3, synthesizing a compound 3: dissolving the compound 1 obtained in S1 and the compound 2 obtained in S2, and dropwise adding 1, 8-diazabicyclo [5.4. 0]-7-undecene for reaction; s4, synthesizing a compound 4; and S5, synthesizing a probe DCI-TSO: dissolving the compound 3 obtained in S3 and the compound 4 obtained in S4, dropwise adding sodium methoxide, and carrying out normal-temperature stirring reaction in a protective atmosphere. According to the near-infrared fluorescent probe DCI-TSO based on the ICT mechanism and the preparation method and application thereof, H2S and ONOO <-> are detected through two channels, and the near-infrared fluorescent probe DCI-TSO has good optical properties and biocompatibility and has great commercial value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hydrogen sulfide and peroxynitrite ion detection, in particular to a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism, a preparation method and application thereof. BACKGROUND

[0002] Hydrogen sulfide (H2S) is considered the third key gas transmitter after nitric oxide and carbon monoxide, and has been proven to exist in various tissues and organs of mammals and participate in various physiological and pathological processes, and can freely cross the cell membrane without relying on transport proteins and membrane receptors. The physiological concentration of H2S in mammalian blood is between 30-100 mM, and the physiological concentration level in the brain is between 50-160 mM. Clinical studies have shown that H2S mainly exists in the form of HS - in mammals at normal physiological levels, and maintains a dynamic balance, playing a very important role in relaxing smooth muscle, regulating neuronal transmission, regulating insulin release and anti-inflammation.

[0003] Peroxynitrite (ONOO - ) is one of the important reactive oxygen species (ROS) and reactive nitrogen species (RNS), and plays an important role in physiological and pathological processes, especially in a series of oxidative stress-related diseases. It has strong oxidizing and nucleophilic properties on many bioactive molecules, and overexpressed ONOO - can oxidize multiple targets or produce free radicals with high reactivity, leading to structural modification and dysfunction of proteins, nucleic acids and lipids, and producing severe cytotoxicity, which is closely related to various diseases such as Alzheimer's disease, Parkinson's disease, cardiovascular disease and diabetes.

[0004] The concentrations of H2S and ONOO - in biological systems are closely related to health. Therefore, it is very necessary to develop efficient and convenient tools for detecting H2S and ONOO - in biological systems. Traditional techniques such as colorimetry, gas chromatography and electrochemical voltammetry cannot achieve in-situ real-time detection and are not suitable for non-invasive in-situ detection and real-time imaging of living cells. In recent years, fluorescent probe technology has been greatly developed due to its unique advantages in real-time imaging, short response time, high sensitivity and low biological toxicity.

[0005] In the prior art, researchers have developed numerous fluorescent probes for detecting H2S and ONOO - respectively, but there are problems of complex operation, and fluorescent probes based on the same probe and different recognition sites for simultaneously detecting H2S and ONOO - have rarely been reported so far. SUMMARY

[0006] The application aims to provide a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism, and a preparation method and application thereof - , and has good optical properties and biocompatibility, and has great commercial application value.

[0007] To achieve the above-mentioned purpose, the application provides a preparation method of a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism, and the following steps are included.

[0008] S1, synthesizing compound 1:

[0009] According to a proportion, 4-hydroxybenzaldehyde and 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate are dissolved in anhydrous acetonitrile, and then stirred under the catalysis of triethylamine; after the reaction is completed, extraction, evaporation and purification are performed, and the compound 1 is obtained;

[0010] S2, synthesizing compound 2:

[0011] According to a proportion, 4-nitrophenol and imidazole are dissolved in anhydrous DMF, and then tert-butyldimethylchlorosilane is added under a protective atmosphere; the reaction is stirred at room temperature, and after the reaction is completed, extraction, evaporation and purification are performed, and the compound 2 is obtained;

[0012] S3, synthesizing compound 3:

[0013] According to a proportion, the compound 1 obtained in S1 and the compound 2 obtained in S2 are dissolved in anhydrous acetonitrile, and then 1,8-diazabicyclo[5.4.0]-7-undecene is added dropwise after stirring; the reaction is stirred at room temperature, and after the reaction is completed, extraction, drying, removal of the solvent and purification are performed, and the compound 3 is obtained;

[0014] S4, synthesizing compound 4:

[0015] According to a proportion, isofuroline and malondialdehyde are dissolved in ethanol, and then refluxed under the catalysis of piperidine; after the reaction is completed, evaporation and purification are performed, and the compound 4 is obtained;

[0016] S5, synthesizing the probe DCI-TSO:

[0017] According to a proportion, the compound 3 obtained in S3 and the compound 4 obtained in S4 are dissolved in anhydrous methanol, and then sodium methoxide is added dropwise; the reaction is stirred at room temperature under a protective atmosphere overnight, and then the organic layer is purified, and the probe DCI-TSO is obtained.

[0018] Preferably, in S1, the equivalent ratio of the 4-hydroxybenzaldehyde, the triethylamine, the anhydrous acetonitrile and the 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate is 1:1.5:10:1.5;

[0019] The temperature of the stirring reaction is 0-5℃, and the reaction time is 4h.

[0020] Preferably, in S1, the extraction is performed using water, saturated brine, and ethyl acetate, and the volume ratio of the saturated brine to the ethyl acetate is 3:1.

[0021] The evaporation is performed using a rotary evaporator under reduced pressure or a freeze dryer.

[0022] The purification is performed using silica gel column chromatography, and the volume ratio of the eluent is PE:EtOAc=50:1-20:1.

[0023] Preferably, in S2, the equivalent ratio of the 4-nitrophenol to the imidazole to the anhydrous DMF to the tert-butyl dimethylchlorosilane is 1:1.5:15:1.3.

[0024] The extraction is performed using diethyl ether and water, and the purification is performed using silica gel column chromatography, and the volume ratio of the eluent is PE:DCM=50:1.

[0025] Preferably, in S3, the equivalent ratio of the compound 1 to the compound 2 to the anhydrous acetonitrile to the 1,8-diazabicyclo[5.4.0]-7-undecene is 1:1:10:0.25.

[0026] The extraction is performed using ethyl acetate and water.

[0027] The drying is performed using anhydrous sodium sulfate.

[0028] The removal of the solvent is performed using a reduced pressure desolvation method, and the reduced pressure condition is 0.1 MPa.

[0029] The purification is performed using silica gel column chromatography, and the volume ratio of the eluent is PE:EtOAc=30:1.

[0030] Preferably, in S4, the equivalent ratio of the isophorone to the malononitrile to the ethanol to the piperidine is 1:2:71:0.01.

[0031] The reflux reaction condition is 80-85℃ for 10-15 hours.

[0032] Preferably, in S5, the equivalent ratio of the compound 3 to the compound 4 to the anhydrous methanol to the sodium methoxide is 1:1:10:1.

[0033] The stirring reaction condition is 20-25℃ for 10-12h.

[0034] The purification is performed using column chromatography, and the volume ratio of the eluent is PE:EtOAc=20:1.

[0035] This invention also provides a near-infrared fluorescent probe DCI-TSO based on the ICT mechanism, the structural formula of which is as follows:

[0036]

[0037] This invention also provides a near-infrared fluorescent probe DCI-TSO based on the ICT mechanism for the simultaneous detection of H2S and ONOO in cells. - For the application of content detection, the steps are as follows:

[0038] The probe DCI-TSO, the test solution, and PBS solution were mixed and incubated. H2S and ONOO were qualitatively or quantitatively analyzed by observing changes in solution color or detecting changes in fluorescence signal using a fluorescence detection instrument. - content.

[0039] This invention also provides a near-infrared fluorescent probe DCI-TSO based on the ICT mechanism for the simultaneous detection of H2S and ONOO during preparation. - Applications in reagents.

[0040] Therefore, the present invention employs the above-mentioned near-infrared fluorescent probe DCI-TSO based on the ICT mechanism, its preparation method, and its application, with the following beneficial effects:

[0041] (1) The probe DCI-TSO provided by this invention has obvious ultraviolet absorption at 390 nm in its initial state, and its absorption spectrum red-shifts to 435 nm after responding with H2S; and with ONOO - After the response, the absorption intensity at 390 nm of the probe decreased significantly, while the absorption intensity at 505 nm increased significantly, thus indicating a connection with H2S or ONOO. - After the response, the probe structure changes, and the absorption wavelength of the probe redshifts.

[0042] (2) The probe DCI-TSO provided by this invention responds to H2S or ONOO - Rapid and highly sensitive, reaching peak fluorescence intensity in 60 seconds; the limit of detection for H2S is as low as 132.4 nM; ONOO - The lowest detection limit is 97.2 nM, which can meet the requirements for rapidly changing H2S and ONOO in vivo. - The need for real-time in-situ horizontal monitoring.

[0043] (3) The probe DCI-TSO provided by this invention has good selectivity and high specificity, and can accurately identify H2S or ONOO in complex biological systems. - This can effectively avoid interference from other biomolecules, active substances, and the background, ensuring the repeatability, accuracy, and reliability of the test results.

[0044] (4) The probe DCI-TSO provided by the application is a dual-response fluorescent probe, one probe molecule can detect two active substances at the same time, the detection method is simple, the detection can be completed without interference in a complex environment, the probe has excellent biocompatibility, excellent fluorescence imaging can be realized in living cells, and no obvious toxic side effects are observed.

[0045] (5) The preparation method provided by the application can successfully synthesize the probe DCI-TSO, the synthesis method is simple and efficient, raw materials are cheap and easy to obtain, is conducive to large-scale industrial production, and has a broad market application prospect.

[0046] The technical solutions of the application will be further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the probe DCI-TSO prepared in Example 1 of the application;

[0048] Figure 2 is the nuclear magnetic resonance carbon spectrum of the probe DCI-TSO prepared in Example 1 of the application;

[0049] Figure 3 is the high-resolution mass spectrum of the probe DCI-TSO prepared in Example 1 of the application;

[0050] Figure 4 is the result of the probe DCI-TSO prepared in Example 1 of the application before and after response to H2S and ONOO - , wherein (A) is an ultraviolet absorption spectrum, and (B) is a fluorescence emission spectrum;

[0051] Figure 5 is the result of the probe DCI-TSO prepared in Example 1 of the application, wherein (A) is the fluorescence emission spectrum of the probe DCI-TSO and H2S concentration titration, (B) is the linear relationship of the probe DCI-TSO and H2S concentration titration, (C) is the fluorescence emission spectrum of the probe DCI-TSO and ONOO - concentration titration, and (D) is the linear relationship of the probe DCI-TSO and ONOO - concentration titration;

[0052] Figure 6Time-dependent and pH stability determination results of probe DCI-TSO prepared in Example 1 of the present application, wherein (A) is the time stability of probe DCI-TSO (10.0 μM) before and after response to H2S (20 μM), (B) is the fluorescence stability of probe DCI-TSO (10.0 μM) after response to blank and H2S (20 μM) at different pH values, (C) is the time stability of probe DCI-TSO (10.0 μM) before and after response to ONOO - (10.0 μM), (D) is the fluorescence stability of probe DCI-TSO (10.0 μM) after response to blank and ONOO - (10 μM) at different pH values;

[0053] Figure 7 Selectivity and competition determination results of probe DCI-TSO prepared in Example 1 of the present application, wherein (A) is the fluorescence intensity (λ ex = 430 nm) of probe DCI-TSO (10.0 μM) after response to 28 kinds of ions respectively, (B) is the fluorescence intensity of probe after response to H2S and then adding other ions, (C) is the fluorescence intensity (λ ex = 500 nm) of probe DCI-TSO (10.0 μM) after response to 28 kinds of ions respectively, (D) is the fluorescence intensity of probe after response to ONOO - and then adding other ions, (E) is the photos of probe DCI-TSO after response to different ions under daylight lamp, (F) is the photos of probe DCI-TSO after response to different ions under ultraviolet lamp;

[0054] Figure 8 Experimental results of HepG2 cell toxicity of probe DCI-TSO prepared in Example 1 of the present application under different concentrations;

[0055] Figure 9 Confocal fluorescence imaging maps of probe DCI-TSO prepared in Example 1 of the present application on HepG2 cells under different concentrations, wherein (A) is the confocal fluorescence imaging map of DCI-TSO (10.0 μM) under the action of different concentrations of H2S (0.0 μM, 5.0 μM, 10.0 μM and 15.0 μM), (B) is the confocal fluorescence imaging map of DCI-TSO (10.0 μM) under the action of different concentrations of ONOO - , (C) is the confocal fluorescence imaging map of DCI-TSO (10.0 μM) under the action of H2S and ONOO - under different excitation wavelengths. DETAILED DESCRIPTION

[0056] The technical solutions of the present application are further described below through the drawings and examples.

[0057] Unless otherwise defined, the technical and scientific terms used in the present application shall have the meanings that are commonly understood by a person of ordinary skill in the art to which this application belongs.

[0058] The instruments and reagents used in the present application are obtained through commercial channels; the method steps not specifically described are the conventional technical means in the art.

[0059] Example 1

[0060] A near-infrared fluorescent probe DCI-TSO based on an ICT mechanism, the synthesis line is as follows:

[0061]

[0062] Wherein (a) is Et3N, ACN, 0℃-4℃, 4h, 69.3%, (b) is Imidazole, TBS-Cl, DMF, r.t., overnight, 87.9%, (c) is DBU, ACN, r.t., 6h, 52.5%, (d) is Piperidine, EtOH, 85℃, 12h, 66.0%, (e) is NaOMe, MeOH, r.t., overnight, 64.3%.

[0063] The preparation method is as follows:

[0064] (1) Synthesis of compound 1

[0065] 4-hydroxybenzaldehyde (200.00 mg, 1.64 mmol, 1.0 equiv) was dissolved in 10 mL of anhydrous acetonitrile, triethylamine (342.00 μL, 2.46 mmol, 1.5 equiv) was added dropwise at 0℃, and the reaction was stirred for 10 min. After adding 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate (807.52 mg, 2.46 mmol, 1.5 equiv), the reaction was stirred at room temperature for 4 h. After the reaction was completed, water was added to the reaction liquid, and 10.0 mL of ethyl acetate was used for extraction. The organic phases were combined, and the organic phase was washed with water and 30.0 mL of saturated brine. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and rotary evaporated under reduced pressure. The white solid compound was obtained by silica gel column chromatography (eluent volume ratio PE:EtOAc = 50:1-20:1) (260.80 mg, yield 69.3%, Rf= 0.65, PE:EtOAc = 4:1). f

[0066] (2) Synthesis of compound 2

[0067] ​In a 25.0 mL round-bottom flask, 4-nitrophenol (500.00 mg, 3.60 mmol, 1.0 equiv) and imidazole (367.63 mg, 5.40 mmol, 1.5 equiv) were added and dissolved with 15.0 mL of anhydrous DMF, and then tert-butyldimethylsilyl chloride (705.37 mg, 4.68 mmol, 1.3 equiv) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was diluted with ether, washed with water three times to remove DMF, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure using a rotary evaporator. The residue was purified by column chromatography on silica gel (PE:DCM = 50:1) to obtain 802.10 mg of a white solid (yield 87.9%, Rf= 0.62, PE:EtOAc = 10:1). f

[0068] (3) Synthesis of compound 3

[0069] In a 25.0 mL round-bottom flask, compound 1 (210.0 mg, 1.12 mmol, 1.0 equiv) and compound 2 (283.78 mg, 1.12 mmol, 1.0 equiv) were dissolved in 10.0 mL of anhydrous acetonitrile, and then 1,8-diazabicyclo[5.4.0]-7-undecene (41.80 μL, 0.28 mmol, 0.25 equiv) was added dropwise. The reaction was stirred at room temperature for 6 h. After the reaction was completed, the reaction solution was diluted with ethyl acetate, extracted with water three times, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure using a rotary evaporator. The residue was purified by column chromatography on silica gel (PE:EtOAc = 30:1) to obtain 190.00 mg of a white solid (yield 52.5%, Rf= 0.38, PE:EtOAc = 4:1). f

[0070] (4) Synthesis of compound 4

[0071] In a 250.0 mL round-bottom flask, isophorone (5.00 g, 36.18 mmol, 1.0 equiv) and malononitrile (5.00 g, 75.69 mmol, 2.0 equiv) were weighed, dissolved in 150.0 mL of ethanol, and then piperidine (112.0 mg, 0.362 mmol, 0.01 equiv) was added and heated to 85°C for reflux for 12 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure using a rotary evaporator, and then purified by column chromatography on silica gel (eluent: PE) to obtain 4.45 g of a white solid (yield 66.0%, Rf= 0.23, PE:EtOAc = 10:1). f

[0072] (5) Synthesis of probe DCI-TSO

[0073] ​​​Compound 4 (109.47 mg, 0.59 mmol, 1.0 equiv) and compound 3 (190.0 mg, 0.59 mmol, 1.0 equiv) were weighed into a 25.0 mL round-bottom flask, dissolved with 10.0 mL of anhydrous methanol, and then sodium methoxide (31.75 mg, 0.59 mmol, 1.0 equiv) was added. The reaction was carried out at room temperature overnight. After the reaction was completed, the rotary evaporator was directly used to spin dry under reduced pressure, and column chromatography (PE:EtOAc = 20:1) was used for purification to obtain 186.40 mg of a yellow solid (yield 64.3%, R f = 0.32, PE:EtOAc = 6:1).

[0074] The nuclear magnetic resonance hydrogen spectrum data of the probe DCI-TSO are as follows:

[0075] 1 HNMR (400 MHz, DMSO-d6) δ 8.31-8.24 (m, 2H), 8.15-8.10 (m, 2H), 7.94 (m, J = 9.1 Hz, 2H), 7.67 (d, J = 16.3 Hz, 1H), 7.39-7.35 (m, 2H), 7.01 (s, 1H), 6.93 (m, J = 2.3 Hz, 1H), 2.65 (s, 2H), 2.55 (s, 2H), 1.03 (s, 6H).

[0076] The nuclear magnetic resonance carbon spectrum data of the probe DCI-TSO are as follows:

[0077] 13 CNMR (101 MHz, DMSO-d6) δ 170.60, 164.39, 155.02, 153.58, 146.60, 143.15, 140.06, 134.98, 134.41, 133.51, 129.38, 129.14, 126.67, 126.34, 125.29, 123.93, 116.26, 78.67, 42.70, 38.52, 32.17, 27.88.

[0078] The nuclear magnetic resonance hydrogen spectrum of the probe DCI-TSO is as shown in Figure 1 The nuclear magnetic resonance carbon spectrum of the probe DCI-TSO is as shown in Figure 2 The high-resolution mass spectrum of the probe DCI-TSO is as shown in Figure 3 The successful synthesis of the probe DCI-TSO is indicated.

[0079] Test test

[0080] The performance test of the probe DCI-TSO prepared in Example 1 is as follows:

[0081] (1) Spectral test

[0082] 1) Spectral measurement conditions:

[0083] The probe DCI-TSO was dissolved in DMSO to prepare a standard solution with a concentration of 1.0 mmol / L. 100.0 μL of the probe standard solution was added to a colorimetric tube, followed by 100.0 μL of the ion solution to be measured, 1.0 mL of DMSO, and 1.0 mL of PBS buffer solution (pH = 7.4). Deionized water was added to make up to 5.0 mL, and the mixture was shaken well. The ultraviolet absorption spectrum and fluorescence emission spectrum of the probe before and after the response to ions were measured using an ultraviolet absorption spectrometer and a fluorescence spectrophotometer. When scanning the system responding to H2S, the conditions were λ ex = 430 nm, slits: 5 / 10 nm, 700 V; and when scanning the system responding to ONOO - , the conditions were λ ex = 500 nm, slits: 10 / 10 nm, 700 V.

[0084] The results are as follows:

[0085] As shown in Figure 4 (A), the probe DCI-TSO has a clear ultraviolet absorption at 390 nm, and after responding to H2S, the absorption spectrum of the probe is red-shifted to 435 nm. After responding to ONOO - , the absorption intensity of the probe at 390 nm is obviously decreased, and the absorption intensity at 505 nm is obviously increased, so that it is judged that the structure of the probe changes after responding to H2S or ONOO - , and the absorption wavelength of the probe is red-shifted. Then, the excitation and emission spectra of the probe DCI-TSO before and after responding to H2S and ONOO - , respectively, were scanned, as shown in Figure 4 (B), the probe DCI-TSO has no fluorescence emission at 570 nm and 655 nm under the excitation wavelength of 430 nm and 500 nm, respectively. After adding H2S and ONOO - , respectively, the fluorescence intensity of the probe at 570 nm and 655 nm is obviously enhanced, and it can be clearly observed that there are different excitation and emission wavelengths, and they do not interfere with each other when responding to H2S and ONOO - , respectively. The probe can be used to detect H2S and ONOO - , respectively.

[0086] 2) Effect of concentration on fluorescence intensity

[0087] Firstly, the relationship between H2S concentration and probe fluorescence intensity was explored, and the same solvent system as above was used, i.e. 100.0 μL of DCI-TSO probe solution with a concentration of 1.0 mmol / L was added, and 1.0 mL of DMSO, 1.0 mL of PBS buffer solution (pH = 7.4), and deionized water were added to make the total volume 5.0 mL. The fluorescence intensity of the probe after responding to ions with different concentrations was scanned. The relationship between ONOO - concentration and the fluorescence intensity of the probe DCI-TSO was explored in the same way as above, and ONOO - concentration gradient was 0-90.0 μM, with 19 groups.

[0088] The results are as follows:

[0089] As shown in Figure 5 (A), under excitation at 430 nm, the fluorescence intensity of the probe DCI-TSO at 570 nm gradually increased with the increase of H2S concentration. Figure 5 (B) is a linear relationship diagram of the concentration titration fluorescence intensity of the probe DCI-TSO and H2S. The H2S concentration had a good linearity between 0.0-70.0 μM (Y = 29.5112X + 62.8619, the correlation coefficient R 2 reached 0.9989), and the minimum detection limit was 132.4 nM. Then the relationship between the probe and ONOO - concentration was explored, as shown in Figure 5 (C), the concentration titration fluorescence spectrum of the probe and ONOO - showed that under excitation at 500 nm, the fluorescence intensity of the probe at 655 nm gradually increased in proportion to the ONOO - concentration. Figure 5 (D) is a linear relationship between the fluorescence intensity of the probe and the ONOO - concentration. A good linearity was shown within the linear range of 0.0-65.0 μM, the correlation coefficient R 2 was 0.9988, and the minimum detection limit was 97.2 nM. It can be seen that the probe DCI-TSO can be used to detect the concentration changes of H2S and ONOO - .

[0090] 3) Time dependence and pH stability determination of the probe

[0091] Time dependence: 100.0 μL of DCI-TSO probe standard solution was added to a colorimetric tube, and 100.0 μL of responding ions, 1.0 mL of DMSO, 1.0 mL of PBS buffer solution, and deionized water were added to make the total volume 5.0 mL. After shaking, the fluorescence spectrum before and after adding ions was determined.

[0092] pH stability study: according to the above system, the colorimetric tube was added with the probe DCI-TSO and the responding ions, then DMSO was added, and then PBS buffer solution with different pH values was added, the pH range was 2-13, and each pH unit was measured once, and the fluorescence spectrum at different pH values was measured.

[0093] The results are as follows:

[0094] As shown in Figure 6 (A) and Figure 6 (C), the probe itself does not show fluorescence intensity without the addition of response ions under the excitation of 430 nm and 500 nm wavelengths. After the response with H2S, the fluorescence signal is obviously enhanced compared with that before the addition, and the fluorescence signal quickly tends to be balanced (within 1 min). Similarly, after the addition of ONOO - , the probe can be observed to have obvious fluorescence signal, quickly reach the maximum absorption intensity, and maintain stable fluorescence intensity within 30 min. Thus, it can be shown that the probe DCI-TSO can quickly identify the concentration changes of H2S and ONOO - , and has excellent light stability.

[0095] Subsequently, it was evaluated whether DCI-TSO can achieve detection of H2S and ONOO - in the pH 2-12 range, as shown in Figure 6 (B) and Figure 6 (D), the probe DCI-TSO itself has almost no fluorescence production under the condition of pH 2-12, and is not sensitive to the pH environment. Subsequently, the fluorescence intensity change of the probe DCI-TSO before and after the addition of ions H2S and ONOO - was detected under different pH conditions, and the probe DCI-TSO has stable responsiveness to H2S in the pH=5-9 range, and has good fluorescence response to ONOO - in the pH=4-8 range. The above shows that the probe DCI-TSO can achieve detection of H2S and ONOO - under physiological conditions.

[0096] 4) Determination of selectivity and competition of the probe

[0097] Determination of selectivity of the probe: 100.0 μL of the DCI-TSO probe standard solution was added to the colorimetric tube, 100.0 μL of the prepared common active oxygen, active sulfur, metal ions were added, respectively, 1.0 mL of DMSO and 1.0 mL of PBS buffer solution were added, and deionized water was added to 5.0 mL, and then shaken, and the fluorescence spectrum was measured.

[0098] The competitive determination of the probe: 100.0 μL of the DCI-TSO probe standard solution was added to a colorimetric tube, followed by 100.0 μL of the responding ion, 1.0 mL of DMSO and 1.0 mL of PBS buffer solution, and deionized water was added to 5.0 mL, and then it was shaken uniformly, and the fluorescence spectrum was measured.

[0099] The results are as follows:

[0100] As shown in Figure 7 (A), after the probe DCI-TSO was added to various ions, the fluorescence spectrum of the solution was scanned, and after H2S was added, under the excitation of 430 nm wavelength, the probe exhibited a strong fluorescence signal at 570 nm compared with other ions. However, after other active ions were added, no fluorescence signal was exhibited, and after ONOO - was added, the probe also did not exhibit obvious fluorescence intensity at 570 nm, but exhibited weak fluorescence intensity at 650 nm, which did not interfere with the detection of H2S, indicating that the probe had good selectivity for H2S. Subsequently, competitive research was carried out, as shown in Figure 7 (B), after the probe was added to H2S, other interfering ions were added, and it was found that the fluorescence intensity did not change obviously. The above results show that the probe DCI-TSO has good selectivity and anti-interference ability for H2S under the excitation of 430 nm wavelength.

[0101] As shown in Figure 7 (C), after the DCI-TSO probe was added to ONOO - , under the excitation of 500 nm wavelength, the fluorescence signal of the probe at 655 nm was obviously enhanced. After other active ions were reacted with the probe, the fluorescence signal of the probe did not change. In the competitive research, as shown in Figure 7 (D), after the probe was reacted with ONOO - , other active interfering ions were added, and the fluorescence intensity of the probe was basically not affected. In summary, it is shown that the probe DCI-TSO exhibits good specific selectivity and anti-interference ability for H2S and ONOO - , and the detection of H2S and ONOO - does not interfere with each other under different excitation wavelengths.

[0102] In the process of studying the selectivity, it was also found that the detection of H2S and ONOO - by the probe DCI-TSO can be realized by naked eye observation. After the probe was added to the ion to be detected, it was observed under the daylight lamp and the 365 nm ultraviolet lamp, respectively, as shown in Figure 7 (E) and Figure 7(F) shown, under daylight lamp, the probe is colorless and transparent after adding other ions, the solution becomes yellow after H2S ion response, the solution becomes pink after ONOO - - ion response; under UV lamp, other ions emit blue fluorescence, H2S ion solution emits orange-yellow fluorescence, and ONOO - ion solution emits pink fluorescence, indicating that the probe DCI-TSO can realize naked-eye detection of H2S and ONOO

[0103] (2) Biocompatibility test

[0104] MTT method was used to evaluate the cytotoxicity of the probe DCI-TSO. HepG2 cell suspension was inoculated into 96-well plate culture medium, and cultured in a 5% CO2, 37°C incubator for 24 h, then standard concentration of DCI-TSO probe solution (0.0 μM, 0.5 μM, 1.0 μM, 2.0 μM, 5.0 μM, 10.0 μM, 15.0 μM) was added accurately, and incubated in the incubator for 24 h. After incubation, 100.0 μL of MTT solution (0.50 mg / mL) was added to each well, and incubated for 4 h. 100.0 μL of supernatant was aspirated, 100.0 μL of DMSO was added, and the tin paper was wrapped and shaken in a shaker for 15 min. The absorbance of the solution at 490 nm was recorded using a microplate reader.

[0105] The results are as follows:

[0106] As shown in Figure 8 , even at a probe concentration of 15 μM, the survival rate of cells can still reach more than 85%.

[0107] (3) Application example 1 prepared probe DCI-TSO to detect intracellular peroxynitrite ion

[0108] The specific process is as follows:

[0109] ​The incubated HepG2 cell suspension was inoculated into a confocal special dish, washed with PBS solution three times, and an appropriate amount of culture medium was added. The dish was placed in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere. After incubation, the cells were incubated with 10 μM probe DCI-TSO in tin foil under light protection at 37°C in a 5% CO2 incubator for 20 minutes. After completion, the cells were washed three times with PBS buffer and divided into three groups. In the first group, the probe was incubated with different concentrations of H2S in the incubator under light protection for 20 minutes. The H2S concentrations were 0.0 μM, 5.0 μM, 10.0 μM, and 15.0 μM, respectively. After incubation, the cells were washed three times with PBS buffer and subjected to confocal imaging. The second group was the same as the first group, and 0.0 μM, 5.0 μM, 10.0 μM, and 15.0 μM of ONOO - were added, respectively. After 20 minutes of incubation in the incubator under light protection, the cells were washed with PBS and subjected to confocal imaging. In the third group, the cells were divided into three groups: a blank group with only the probe, and two groups with the addition of 10.0 μM H2S and ONOO - , respectively. After incubation, the cells were washed with PBS and subjected to confocal imaging. The H2S group was imaged under the orange channel, and the ONOO - group was imaged under the red channel.

[0110] The results are as follows:

[0111] As shown in Figure 9 (A), first, the probe DCI-TSO with a concentration of 10.0 μM was co-incubated with the cells, and then H2S and ONOO - with concentrations of 0.0 μM, 5.0 μM, 10.0 μM, and 15.0 μM, respectively, were incubated with the cells for 20 minutes. After co-incubation, confocal imaging was performed. In the blank group without ions, there was almost no fluorescence in the orange channel. After incubation with different concentrations of H2S, obvious fluorescence was observed in the orange channel, and the fluorescence intensity gradually increased with increasing H2S concentration. Then, imaging experiments were performed on ONOO - , as shown in Figure 9 (B). Similarly, after co-incubation of the probe with ONOO - , there was almost no fluorescence signal in the red channel when the cells were incubated with only the probe. When incubated with different concentrations of ONOO - , the fluorescence intensity in the red channel increased with increasing ONOO - concentration. This indicates that the probe DCI-TSO can detect H2S and ONOO - in living cells and achieve visual imaging.

[0112] Finally, H2S and ONOO -Meanwhile, the fluorescence imaging of the probe DCI-TSO in living cells was explored. As shown in Figure 9(C), after incubation of the probe with cells, the probe was co-incubated with H2S and ONOO - , and fluorescence imaging was performed under orange and red channels, respectively. No fluorescence was observed in the blank group, and yellow fluorescence was observed under the orange channel when H2S and ONOO - coexisted, while red fluorescence was observed under the red channel. - The results showed that different fluorescence signals could be observed under different channels when H2S and ONOO - coexisted, and H2S and ONOO - could be detected and imaged in living cells, respectively, and the two fluorescence signals did not interfere with each other.

[0113] Therefore, the present application adopts the above-mentioned near-infrared fluorescent probe DCI-TSO based on the ICT mechanism, and the preparation method and application thereof, and the dual-channel detection of H2S and ONOO - has good optical properties and biocompatibility, and has great commercial application value.

[0114] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not

[0115] to limit them, although the present application has been described in detail with reference to the preferred embodiments, the

[0116] person skilled in the art should understand: its still can be on the technical scheme of the present application

[0117] modification or equivalent replacement, and these modifications or equivalent replacements also cannot make the modified technical

[0118] solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing an ICT mechanism-based near-infrared fluorescent probe DCI-TSO, characterized in that, The method comprises the following steps: S1, synthesizing compound 1: According to the proportion, 4-hydroxybenzaldehyde and 1-(fluorosulfonyl)-2, 3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate are dissolved in anhydrous acetonitrile, and then stirred under the catalysis of triethylamine. After the reaction is completed, extraction, evaporation and purification are performed to obtain compound 1. S2, synthesizing compound 2: According to the proportion, 4-nitrophenol and imidazole are dissolved in anhydrous DMF, and then tert-butyl dimethyl chlorosilane is added under a protective atmosphere. After stirring at room temperature, extraction, evaporation and purification are performed to obtain compound 2. S3, synthesizing compound 3: According to the proportion, compound 1 obtained in S1 and compound 2 obtained in S2 are dissolved in anhydrous acetonitrile, and then 1, 8-diazabicyclo[5.4.0]-7-undecene is added dropwise. After stirring at room temperature, extraction, drying, removal of solvent and purification are performed to obtain compound 3. S4, synthesizing compound 4: According to the proportion, isoforone and malononitrile are dissolved in ethanol, and then refluxed under the catalysis of piperidine. After evaporation and purification, compound 4 is obtained. S5, synthesizing probe DCI-TSO: According to the proportion, compound 3 obtained in S3 and compound 4 obtained in S4 are dissolved in anhydrous methanol, and then sodium methoxide is added dropwise. After stirring at room temperature overnight under a protective atmosphere, the organic layer is purified to obtain probe DCI-TSO.

2. The preparation method of the near-infrared fluorescent probe DCI-TSO based on the ICT mechanism according to claim 1, characterized in that, In S1, the equivalent ratio of the 4-hydroxybenzaldehyde, the triethylamine, the anhydrous acetonitrile and the 1-(fluorosulfonyl)-2, 3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate is 1:1.5:10:1.5; The stirring reaction is performed at a temperature of 0-5°C for 4 hours.

3. The method according to claim 1, wherein, In S1, the extraction is performed by using water, saturated brine and ethyl acetate, and the volume ratio of the saturated brine to the ethyl acetate is 3:1; The evaporation is performed by using a rotary evaporator or a freeze dryer; The purification is performed by using a silica gel column, and the volume ratio of the eluent is PE:EtOAc=50:1-20:

1.

4. The method according to claim 1, wherein, In S2, the equivalent ratio of the 4-nitrophenol, the imidazole, the anhydrous DMF and the tert-butyl dimethyl chlorosilane is 1:1.5:15:1.3; The extraction is performed by using ether and water, and the purification is performed by using a silica gel column, and the volume ratio of the eluent is PE:DCM=50:

1.

5. The method according to claim 1, wherein, In S3, the equivalent ratio of the compound 1, the compound 2, the anhydrous acetonitrile and the 1, 8-diazabicyclo[5.4.0]-7-undecene is 1:1:10:0.25; The extraction is performed by using ethyl acetate and water; The drying is performed by using anhydrous sodium sulfate; The removal of solvent is performed by using a reduced-pressure desolventizing method; The purification is performed by using a silica gel column, and the volume ratio of the eluent is PE:EtOAc=30:

1.

6. The method for preparing a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism according to claim 1, characterized in that, In S4, the equivalent ratio of the isoforone, the malononitrile, the ethanol and the piperidine is 1:2:71:0.01; The reflux reaction is performed at a temperature of 80-85°C for 10-15 hours.

7. The method according to claim 1, wherein, In S5, the equivalent ratio of the compound 3: the compound 4: the anhydrous methanol: the sodium methoxide is 1:1:10:1; The stirring reaction is carried out at 20-25℃ for 10-12h. The purification is carried out by column chromatography, and the volume ratio of the eluent is PE:EtOAc=20:

1.

8. The preparation method of the ICT mechanism-based near-infrared fluorescent probe DCI-TSO according to any one of claims 1-7, wherein the probe DCI-TSO is prepared by the method. The structure of the probe DCI-TSO is as follows:

9. A probe DCI-TSO for simultaneous detection of H2S and ONOO in cells as claimed in claim 8. - for use, characterized in that, The detection steps are as follows: The probe DCI-TSO, the sample to be tested and the PBS solution are mixed and incubated, the color change of the solution or the change of the fluorescence signal is detected by a fluorescence detection instrument, and H2S and ONOO are qualitatively or quantitatively analyzed - content.

10. Use of a probe DCI-TSO according to claim 8 for the preparation of a reagent for the simultaneous detection of H2S and ONOO - -

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