Near-infrared fluorescent probe based on ICT mechanism, preparation method and application thereof

Abstract

The application belongs to the technical field of hydrogen sulfide and peroxynitrite ion detection, and specifically discloses a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism, a preparation method and application, and comprises the following steps: S1, synthesizing compound 1; S2, synthesizing compound 2; S3, synthesizing compound 3: dissolving compound 1 obtained in S1 and compound 2 obtained in S2, and adding dropwise 1,8-diazabicyclo[5.4.0]-7-undecene reaction; S4, synthesizing compound 4; S5, synthesizing probe DCI-TSO: dissolving compound 3 obtained in S3 and compound 4 obtained in S4, and adding dropwise sodium methoxide under stirring reaction at room temperature in a protective atmosphere. The application adopts the above-mentioned near-infrared fluorescent probe DCI-TSO based on an ICT mechanism, a preparation method and application, and can detect H2S and ONOO ‑ with good optical properties and biocompatibility, and has great commercial value.

Description

Technical Field

[0001] This invention relates to the field of hydrogen sulfide and peroxynitroso ion detection technology, and in particular to a near-infrared fluorescent probe DCI-TSO based on the ICT mechanism, its preparation method, and its application. Background Technology

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

[0003] peroxynitrite (ONOO) - ONOO is an important reactive oxygen species (ROS) and reactive nitrogen species (RNS), playing a crucial role in physiological and pathological processes, particularly in a range of diseases associated with oxidative stress. It exhibits strong oxidizing and nucleophilic properties towards many bioactive molecules, and overexpression of ONOO... - It can oxidize multiple targets or generate highly reactive free radicals, leading to structural modifications and functional disorders of proteins, nucleic acids and lipids, and producing severe cytotoxicity. It is closely related to a variety of diseases such as Alzheimer's disease, Parkinson's disease, cardiovascular disease and diabetes.

[0004] H2S and ONOO in biological systems - Concentration is closely related to health. Therefore, developing efficient and convenient detection systems for H2S and ONOO is crucial. - Such tools are essential. 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 live cells. In recent years, fluorescent probe technology has seen tremendous development due to its unique advantages in real-time imaging, short response time, high sensitivity, and low biotoxicity.

[0005] In existing technologies, researchers have developed numerous methods for detecting H2S and ONOO. - Fluorescent probes are available, but they present operational challenges, such as the simultaneous detection of H2S and ONOO at different recognition sites using the same probe. - Fluorescent probes have been rarely reported to date. Summary of the Invention

[0006] The purpose of this invention is to provide a near-infrared fluorescent probe DCI-TSO based on the ICT mechanism, its preparation method, and its application for dual-channel detection of H2S and ONOO. - It also possesses excellent optical properties and biocompatibility, making it highly valuable for commercial applications.

[0007] To achieve the above objectives, this invention provides a method for preparing a near-infrared fluorescent probe DCI-TSO based on the ICT mechanism, comprising the following steps:

[0008] S1, Synthetic compound 1:

[0009] According to the proportion, 4-hydroxybenzaldehyde and 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-onium trifluoromethanesulfonate were dissolved in anhydrous acetonitrile and then stirred under the catalysis of triethylamine. After the reaction was completed, the mixture was extracted, evaporated to dryness and purified to obtain compound 1.

[0010] S2, Synthetic compound 2:

[0011] 4-Nitrophenol and imidazole were dissolved in anhydrous DMF in proportion, and tert-butyldimethylchlorosilane was added under a protective atmosphere. The mixture was stirred at room temperature and reacted. After the reaction was completed, the mixture was extracted, evaporated to dryness and purified to obtain compound 2.

[0012] S3, Synthetic Compound 3:

[0013] According to the proportion, compound 1 obtained from S1 and compound 2 obtained from S2 were dissolved in anhydrous acetonitrile, and 1,8-diazabicyclo[5.4.0]-7-undecene was added dropwise after stirring. The reaction was stirred at room temperature. After the reaction was completed, the mixture was extracted, dried, the solvent was removed, and the mixture was purified to obtain compound 3.

[0014] S4, Synthetic Compound 4:

[0015] Isoflurone and malondiol were dissolved in ethanol in proportion, and the mixture was refluxed under piperidine catalysis. After the reaction was completed, the mixture was evaporated to dryness and purified to obtain compound 4.

[0016] S5, Synthesis probe DCI-TSO:

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

[0018] Preferably, in S1, the equivalent ratio of 4-hydroxybenzaldehyde: triethylamine: anhydrous acetonitrile: 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazolium-3-onium 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 carried out using water, saturated saline solution, and ethyl acetate, wherein the volume ratio of the saturated saline solution to the ethyl acetate is 3:1.

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

[0022] The purification was performed using silica gel column chromatography, with an eluent volume ratio of PE:EtOAc of 50:1 to 20:1.

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

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

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

[0026] The extraction was performed using ethyl acetate and water;

[0027] The drying process uses anhydrous sodium sulfate.

[0028] The solvent removal is performed using a reduced pressure solvent removal method, with a reduced pressure of 0.1 MPa.

[0029] The purification was performed using a silica gel column, with an eluent volume ratio of PE:EtOAc = 30:1.

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

[0031] The reflux reaction conditions are 80–85°C for 10–15 hours.

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

[0033] The stirring reaction conditions are 20–25°C for 10–12 hours;

[0034] The purification was performed using column chromatography with an eluent volume ratio of 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 present invention is a dual-response fluorescent probe. One probe molecule can detect two active substances at the same time. The detection method is simple and can be completed without interference in complex environments. It has excellent biocompatibility, can achieve excellent fluorescence imaging in living cells, and no obvious toxic side effects have been observed.

[0045] (5) The preparation method provided by the present invention can successfully synthesize the probe DCI-TSO. The synthesis method is simple and efficient, and the raw materials are inexpensive and readily available, which is conducive to large-scale industrial production and has broad market application prospects.

[0046] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

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

[0048] Figure 2 This is the carbon NMR spectrum of the probe DCI-TSO prepared in Example 1 of this invention;

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

[0050] Figure 4 The probe DCI-TSO prepared in Example 1 of this invention is combined with H2S and ONOO. - The results before and after the response are shown in the figure, where (A) is the ultraviolet absorption spectrum and (B) is the fluorescence emission spectrum;

[0051] Figure 5 These are the results of the effect of probe DCI-TSO concentration on fluorescence intensity prepared in Example 1 of this invention, where (A) is the fluorescence emission spectrum of probe DCI-TSO titrated with H2S concentration, (B) is the linear relationship of probe DCI-TSO titrated with H2S concentration, and (C) is the fluorescence emission spectrum of probe DCI-TSO titrated with H2S concentration. - Concentration titration fluorescence emission spectrum, (D) is the fluorescence emission spectrum of probe DCI-TSO and ONOO. - Linearity of concentration titration;

[0052] Figure 6The results show the time dependence and pH stability of the probe DCI-TSO prepared in Example 1 of this invention. (A) shows the time stability of probe DCI-TSO (10.0 μM) before and after the response to H2S (20 μM); (B) shows the fluorescence stability of probe DCI-TSO (10.0 μM) after the response to blank and H2S (20 μM) at different pH values; and (C) shows the fluorescence stability of probe DCI-TSO (10.0 μM) after the response to ONOO. - (10.0 μM) Time stability before and after response, (D) shows the difference between probe DCI-TSO (10.0 μM) and blank at different pH values, and ONOO. - (10 μM) fluorescence stability after response;

[0053] Figure 7 These are the results of the selectivity and competitiveness determination of the probe DCI-TSO prepared in Example 1 of this invention, where (A) represents the fluorescence intensity (λ) of the probe DCI-TSO (10.0 μM) after responding to 28 ions. ex =430nm), (B) is the fluorescence intensity of the probe after reacting with H2S and adding other ions, and (C) is the fluorescence intensity of the probe DCI-TSO (10.0μM) after reacting with 28 kinds of ions (λ). ex =500nm), (D) is the probe and ONOO - The fluorescence intensity of the probe DCI-TSO after the reaction with other ions is shown in (E) under fluorescent light and (F) under ultraviolet light.

[0054] Figure 8 These are the experimental results of the cytotoxicity of the probe DCI-TSO prepared in Example 1 of this invention on HepG2 cells at different concentrations;

[0055] Figure 9 These are confocal fluorescence images of HepG2 cells at different concentrations of the probe DCI-TSO prepared in Example 1 of this invention. (A) is a confocal fluorescence image of DCI-TSO (10.0 μM) on cells reacting with different concentrations of H2S (0.0 μM, 5.0 μM, 10.0 μM, and 15.0 μM). (B) is a confocal fluorescence image of cells reacting with different concentrations of ONOO. - Confocal fluorescence imaging of cells under the action of DCI-TSO (10.0 μM), (C) shows cells with H2S and ONOO added at different excitation wavelengths. - Confocal fluorescence imaging of DCI-TSO (10.0 μM) under the influence of DCI-TSO. Detailed Implementation

[0056] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0057] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0058] The instruments, equipment, reagents and materials used in this invention are all obtained through commercial means; the methods and steps not described in detail are all conventional techniques in the field.

[0059] Example 1

[0060] A near-infrared fluorescent probe, DCI-TSO, based on the ICT mechanism, is synthesized via the following route:

[0061]

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

[0063] Its 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 °C, and the mixture was stirred for 10 min. Then, 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazolium-3-onium trifluoromethanesulfonate (807.52 mg, 2.46 mmol, 1.5 equiv) was added, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with 10.0 mL of ethyl acetate. The organic phases were combined, washed with water and 30.0 mL of saturated brine, and then combined again. The mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure using a rotary evaporator. The solution was then purified by silica gel column chromatography (eluent volume ratio of PE:EtOAc = 50:1 to 20:1) to give 260.80 mg of a white solid compound (yield 69.3%). f =0.65, PE:EtOAc = 4:1).

[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 in 15.0 mL of anhydrous DMF. Then, tert-butyldimethylchlorosilane (705.37 mg, 4.68 mmol, 1.3 equiv) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with diethyl ether, washed three times with water to remove DMF, and the organic phase was collected. The phase was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure using a rotary evaporator, and purified by silica gel column chromatography (PE:DCM = 50:1) to give 802.10 mg of a white solid (yield 87.9%). f =0.62, PE:EtOAc=10:1).

[0068] (3) Synthesis of compound 3

[0069] 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 in a 25.0 mL round-bottom flask and stirred until homogeneous. 1,8-diazabicyclo[5.4.0]-7-undecene (41.80 μL, 0.28 mmol, 0.25 equiv) was added dropwise, and the mixture was stirred at room temperature for 6 h. After the reaction was complete, ethyl acetate was added, and the mixture was extracted three times with water. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure (0.1 MPa) using a rotary evaporator. The resulting solution was purified by column chromatography (PE:EtOAc = 30:1) to give 190.00 mg of a white solid (yield 52.5%). f =0.38, PE:EtOAc = 4:1).

[0070] (4) Synthesis of compound 4

[0071] Isophorone (5.00 g, 36.18 mmol, 1.0 equiv) and malononitrile (5.00 g, 75.69 mmol, 2.0 equiv) were weighed into a 250.0 mL round-bottom flask, dissolved completely in 150.0 mL of ethanol, and then piperidine (112.0 mg, 0.362 mmol, 0.01 equiv) was added. The mixture was heated to 85 °C and refluxed for 12 h. After the reaction was complete, the mixture was evaporated to dryness under reduced pressure using a rotary evaporator, and purified by silica gel column chromatography (eluent: PE) to give 4.45 g of a white solid compound (yield: 66.0%, R). f =0.23, PE:EtOAc=10:1).

[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 completely in 10.0 mL of anhydrous methanol, and then sodium methoxide (31.75 mg, 0.59 mmol, 1.0 equiv) was added. The mixture was reacted overnight at room temperature. After the reaction was complete, the mixture was directly evaporated to dryness under reduced pressure using a rotary evaporator, and purified by column chromatography (PE:EtOAc = 20:1) to give 186.40 mg of a yellow solid (yield 64.3%). f =0.32, PE:EtOAc=6:1).

[0074] The proton NMR spectrum data of the probe DCI-TSO are as follows:

[0075] 1 HNMR (400MHz, DMSO-d6) δ8.31-8.24(m,2H),8.15-8.10(m,2H),7.94(m,J=9.1Hz,2H),7.67(d,J=16.3 Hz, 1H), 7.39-7.35 (m, 2H), 7.01 (s, 1H), 6.93 (m, J = 2.3Hz, 1H), 2.65 (s, 2H), 2.55 (s, 2H), 1.03 (s, 6H).

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

[0077] 13 CNMR(101MHz,DMSO-d6)δ170.60,164.39,155.02,153.58,146.60,143.15,140.06,134.98,134.41,1 33.51,129.38,129.14,126.67,126.34,125.29,123.93,116.26,78.67,42.70,38.52,32.17,27.88.

[0078] The proton NMR spectrum of the probe DCI-TSO is shown below. Figure 1 As shown, the carbon NMR spectrum is as follows: Figure 2 As shown, the high-resolution mass spectrum is as follows: Figure 3 As shown, this indicates that the probe DCI-TSO was successfully synthesized.

[0079] Test

[0080] The performance of the probe DCI-TSO prepared in Example 1 was tested, as follows:

[0081] (1) Spectral testing

[0082] 1) Spectroscopic measurement conditions:

[0083] The probe DCI-TSO was dissolved in DMSO to prepare a 1.0 mmol / L standard solution. 100.0 μL of the probe standard solution was added to a colorimetric tube, followed by 100.0 μL of the analyte solution, 1.0 mL of DMSO, and 1.0 mL of PBS buffer (pH = 7.4). Deionized water was added to bring the volume to 5.0 mL, and the mixture was shaken well. The UV absorption and fluorescence emission spectra of the probe before and after the ion response were measured using a UV absorbance spectrophotometer and a fluorescence spectrophotometer. The scanning conditions for the H₂S response system were λ. ex =430nm, slits: 5 / 10nm, 700V; scanning with ONOO - The system time condition for the response is λ. ex =500nm, slits:10 / 10nm, 700V.

[0084] The results are as follows:

[0085] like Figure 4 As shown in (A), the probe DCI-TSO exhibits significant UV absorption at 390 nm, and its absorption spectrum red-shifts to 435 nm after responding to 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. - Following the response, the probe structure changed, and the probe's absorption wavelength red-shifted. Subsequently, the probe DCI-TSO was scanned in reactions with H2S and ONOO. - Excitation and emission spectra before and after the response, such as Figure 4 As shown in (B), the probe DCI-TSO showed no fluorescence emission at excitation wavelengths of 430 nm and 500 nm, and at wavelengths of 570 nm and 655 nm, respectively. However, when H2S and ONOO were added, fluorescence emission was observed. - After the response, the fluorescence intensity of the probe was significantly enhanced at wavelengths of 570 nm and 655 nm, and different excitation and emission wavelengths were clearly observed in response to H2S and ONOO, respectively. - They do not interfere with each other. The probes can be used to detect H2S and ONOO separately. - .

[0086] 2) Effect of concentration on fluorescence intensity

[0087] First, the relationship between H2S concentration and probe fluorescence intensity was investigated. Using the same solvent system as described above, 100.0 μL of 1.0 mmol / L DCI-TSO probe solution was added, followed by the addition of H2S ion solution in gradients from 0 to 100.0 μM, 1.0 mL of DMSO, and 1.0 mL of PBS buffer solution (pH = 7.4). The volume was then adjusted to 5.0 mL with deionized water. The fluorescence intensity of the probe in response to different concentrations of ions was then scanned. The investigation of ONOO... - The relationship between concentration and probe DCI-TSO fluorescence intensity is the same as described above, except that ONOO is added. - The concentration gradient ranged from 0 to 90.0 μM, with a total of 19 groups.

[0088] The results are as follows:

[0089] like Figure 5 As shown in (A), under excitation at a wavelength of 430 nm, the fluorescence intensity of the probe DCI-TSO gradually increases at 570 nm as the H2S concentration increases. Figure 5 (B) shows the linear relationship between the concentration titration fluorescence intensity of the probe DCI-TSO and H2S. The H2S concentration exhibits good linearity in the range of 0.0–70.0 μM (Y = 29.5112X + 62.8619, correlation coefficient R0). 2 The detection limit was 0.9989, and the detection limit was 132.4 nM. The interaction between the probe and ONOO was then investigated. - The relationship between concentrations, such as Figure 5 As shown in (C), the probe is connected to ONOO. - The concentration titration fluorescence spectrum showed that, under excitation at a wavelength of 500 nm, the fluorescence intensity of the probe gradually increased at 655 nm, similar to that of ONOO. - It is directly proportional to the concentration. Figure 5 (D) represents the probe fluorescence intensity and ONOO - The linear relationship between concentration and concentration showed good linearity in the range of 0.0–65.0 μM, with a correlation coefficient R0. 2 The limit of detection (LOD) is 0.9988, and the limit of detection (LOD) is 97.2 nM. Therefore, the DCI-TSO probe can be used to detect H2S and ONOO. - The concentration change.

[0090] 3) Determination of probe time dependence and pH stability

[0091] Time dependence: Add 100.0 μL of DCI-TSO probe standard solution to the colorimetric tube, then add 100.0 μL of response ion, 1.0 mL of DMSO, and 1.0 mL of PBS buffer solution. Make up the volume to 5.0 mL with deionized water, shake well, and measure the fluorescence spectra before and after the addition of ions.

[0092] pH stability study: Following the above system, the probe DCI-TSO and the responding ions were added to the colorimetric tube, followed by DMSO, and then PBS buffer at different pH values, ranging from 2 to 13. The fluorescence spectrum was measured once for each pH unit.

[0093] The results are as follows:

[0094] like Figure 6 (A) and Figure 6 As shown in (C), when the probe is excited at wavelengths of 430 nm and 500 nm without the addition of response ions, the probe DCI-TSO itself does not exhibit fluorescence intensity. However, upon response to H2S, the fluorescence signal is significantly enhanced compared to before the addition, and the fluorescence signal rapidly reaches equilibrium (within 1 min). Similarly, the probe shows increased fluorescence intensity upon the addition of ONOO. - Subsequently, a significant fluorescence signal was observed, rapidly reaching maximum absorption intensity and remaining stable for 30 minutes. This demonstrates that the probe DCI-TSO can rapidly identify H2S and ONOO. - It exhibits excellent photostability and resistance to concentration changes.

[0095] Subsequently, it was evaluated whether DCI-TSO could effectively control H2S and ONOO within the pH range of 2–12. - The detection, such as Figure 6 (B) and Figure 6 As shown in (D), the probe DCI-TSO itself exhibits almost no fluorescence under pH conditions ranging from 2 to 12, indicating that it is not sensitive to pH. Subsequently, the effects of adding H₂S and ONOO₂ ions on the probe DCI-TSO were investigated under different pH conditions. - The fluorescence intensity changes before and after showed that the probe DCI-TSO exhibited a stable response to H2S within the pH range of 5–9, and a response to ONOO within the pH range of 4–8. - It exhibits a good fluorescence response. This indicates that the probe DCI-TSO can achieve H2S and ONOO under physiological conditions. - The detection.

[0096] 4) Determination of probe selectivity and competitiveness

[0097] Selectivity determination of the probe: Add 100.0 μL of DCI-TSO probe standard solution to a colorimetric tube, then add 100.0 μL of prepared common reactive oxygen species, reactive sulfur species, and metal ions respectively, then add 1.0 mL of DMSO and 1.0 mL of PBS buffer solution, and dilute to 5.0 mL with deionized water. Shake well and measure the fluorescence spectrum respectively.

[0098] Probe competition determination: Add 100.0 μL of DCI-TSO probe standard solution to the colorimetric tube, then add 100.0 μL of the responding ion, then add other non-responding ions of the same concentration, and finally add 1.0 mL of DMSO and 1.0 mL of PBS buffer solution. Make up the volume to 5.0 mL with deionized water, shake well, and measure the fluorescence spectrum.

[0099] The results are as follows:

[0100] like Figure 7 As shown in (A), fluorescence spectroscopy was performed on the DCI-TSO probe solution after the addition of various ions. With the addition of H₂S, under excitation at 430 nm, the probe exhibited a strong fluorescence signal at 570 nm compared to other ions. However, the addition of other active ions did not produce a fluorescence signal, and the addition of ONOO₂ did not produce a fluorescence signal. - Subsequently, the probe showed no significant 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 has good selectivity for H2S. Competitive studies were then conducted, such as... Figure 7 As shown in (B), after adding H2S to the probe, the addition of other interfering ions did not result in a significant change in fluorescence intensity. These results indicate that the DCI-TSO probe exhibits good selectivity and anti-interference ability towards H2S under excitation at 430 nm wavelength.

[0101] like Figure 7 As shown in (C), the DCI-TSO probe is added to ONOO. - Subsequently, under excitation at a wavelength of 500 nm, the fluorescence signal of the probe at 655 nm was significantly enhanced. After other active ions reacted with the probe individually, no change in the probe's fluorescence signal was observed. In competitive studies, such as... Figure 7 As shown in (D), the probe is connected to ONOO. - After the response, the fluorescence intensity of other active interfering ion probes was essentially unaffected. In summary, this indicates that the probe DCI-TSO is effective against H2S and ONOO. - It exhibits good specificity and anti-interference ability, and at different excitation wavelengths, it is effective against H2S and ONOO. - The two detection methods do not interfere with each other.

[0102] During the study of selectivity, the probe DCI-TSO was also found to be effective against H2S and ONOO. - All detection methods are observable with the naked eye. After adding the analyte ion to the probe, observations are performed under fluorescent light and a 365nm ultraviolet light, respectively. Figure 7 (E) and Figure 7As shown in (F), under fluorescent light, it can be observed that the probe is colorless and transparent after the addition of other ions, turns the solution yellow after reacting with H2S ions, and turns yellow after reacting with ONOO. - After the ion response, the solution turns pink; under ultraviolet light, other ions emit blue fluorescence, while the H2S ion solution emits orange-yellow fluorescence. - The ionic solution emitted pink fluorescence, indicating that the probe DCI-TSO can achieve naked-eye detection of H2S and ONOO. - The detection.

[0103] (2) Biocompatibility testing

[0104] The cytotoxicity of the probe DCI-TSO was assessed using the MTT assay. HepG2 cell suspensions were seeded into 96-well plates and cultured for 24 h in a 5% CO2 incubator at 37°C. Then, precisely measured concentrations 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) were added, and the plates were incubated for another 24 h. After incubation, 100.0 μL of MTT solution (0.50 mg / mL) was added to each well, and the plates were incubated for another 4 h. 100.0 μL of the supernatant was then aspirated, and 100.0 μL of DMSO was added. The plates were wrapped in aluminum foil and shaken for 15 min. The absorbance at 490 nm was recorded using a microplate reader.

[0105] The results are as follows:

[0106] like Figure 8 As shown, even at a probe concentration of 15 μM, the cell survival rate can still reach over 85%.

[0107] (3) Detection of intracellular peroxynitrite ions using the probe DCI-TSO prepared in Example 1

[0108] Specifically as follows:

[0109] The incubated HepG2 cell suspension was seeded into a confocal microscopy dish, washed three times with PBS, and an appropriate amount of culture medium was added. The dish was then incubated at 37°C with 5% CO2 for 24 hours until cell adhesion. After incubation, the cells were incubated with 10 μM DCI-TSO probe (covered with foil and kept in the dark) at 37°C with 5% CO2 for 20 minutes. Afterward, the cells were washed three times with PBS buffer and divided into three groups. The first group was incubated with different concentrations of H2S (0.0 μM, 5.0 μM, 10.0 μM, and 15.0 μM) for 20 minutes in the dark. After incubation, the cells were washed three times with PBS buffer and then subjected to confocal imaging. The second group was the same as the first group, but with the addition of 0.0 μM, 5.0 μM, 10.0 μM, and 15.0 μM ONOO. - After incubation in the dark for 20 min, the cells were washed with PBS and subjected to confocal imaging. The third group was divided into three subgroups: a blank group with only the probe, a group receiving both 10.0 μM H2S and ONOO2, and a group receiving both. - After incubation, the two groups were washed with PBS and subjected to confocal imaging. The H2S group was imaged in the orange channel. - Imaged in the red channel.

[0110] The results are as follows:

[0111] like Figure 9 As shown in (A), the cells were first co-incubated with a 10.0 μM probe DCI-TSO, followed by the introduction of H2S and ONOO at concentrations of 0.0 μM, 5.0 μM, 10.0 μM, and 15.0 μM, respectively. - After incubating with cells separately for 20 min, co-incubation was performed, followed by confocal imaging. In the blank group (without added ions), the orange channel showed almost no fluorescence. After incubation with different concentrations of H2S, significant fluorescence was observed in the orange channel, and the fluorescence intensity gradually increased with increasing H2S concentration. Next, ONOO... - Imaging experiments were conducted, such as Figure 9 As shown in (B), similarly, the probe is connected to ONOO. - After co-incubation, when cells were incubated only with the probe, there was almost no fluorescence signal in the red channel, compared to different concentrations of ONOO. - During incubation, the fluorescence intensity in the red channel increased with ONOO - The effect is enhanced with increasing concentration. This indicates that the probe DCI-TSO can detect H2S and ONOO in living cells. - Furthermore, it can achieve visual imaging.

[0112] Finally, regarding H2S and ONOO -The fluorescence imaging of the probe DCI-TSO in live cells was investigated in the presence of both. As shown in Figure 9(C), after incubating the probe with cells, the probe was then reacted with H2S and ONOO. - Co-incubation was performed, and fluorescence imaging was conducted in the orange and red channels respectively. No fluorescence was observed in the blank group. (H2S and ONOO) - Simultaneously, yellow fluorescence was observed in the orange channel, while red fluorescence was observed in the orange channel. The results indicate that in H2S and ONOO... - When both H2S and ONOO are present, different fluorescence signals can be observed in different channels, enabling the realization of H2S and ONOO. - The fluorescence signals were detected and imaged separately in living cells, and the two fluorescence signals did not interfere with each other.

[0113] Therefore, this invention employs the aforementioned near-infrared fluorescent probe DCI-TSO based on the ICT mechanism, its preparation method, and its application for dual-channel detection of H2S and ONOO. - It also possesses excellent optical properties and biocompatibility, making it highly valuable for commercial applications.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to be construed as...

[0115] Although the invention has been described in detail with reference to preferred embodiments, this invention is not intended to limit it.

[0116] Those skilled in the art should understand that they can still make modifications to the technical solutions of this invention.

[0117] Modifications or equivalent substitutions are made, but these modifications or equivalent substitutions cannot improve the modified technology.

[0118] The proposed solution deviates from the spirit and scope of the technical solution of this invention.

Claims

1. A method for preparing a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism, characterized in that, Includes the following steps: S1, Synthetic compound 1: 4-hydroxybenzaldehyde and 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-onium trifluoromethanesulfonate were dissolved in anhydrous acetonitrile according to a certain ratio, and then stirred and reacted under the catalysis of triethylamine. After the reaction was completed, the mixture was extracted, evaporated to dryness, and purified to obtain compound 1. ; S2, Synthetic compound 2: 4-Nitrophenol and imidazole were dissolved in anhydrous DMF according to the specified ratio. Then, tert-butyldimethylchlorosilane was added under a protective atmosphere, and the mixture was stirred at room temperature. After the reaction was completed, the mixture was extracted, evaporated to dryness, and purified to obtain compound 2. ; S3, Synthetic Compound 3: Compound 1 obtained in S1 and compound 2 obtained in S2 were dissolved in anhydrous acetonitrile according to the specified ratio. After stirring, 1,8-diazabicyclo[5.4.0]-7-undecene was added dropwise. The reaction was stirred at room temperature. After the reaction was completed, the mixture was extracted, dried, and the solvent was removed. The mixture was then purified to obtain compound 3. ; S4, Synthetic Compound 4: Isoflurone and malondioxonine were dissolved in ethanol according to the specified ratio, and the mixture was refluxed under piperidine catalysis. After the reaction was completed, the mixture was evaporated to dryness and purified to obtain compound 4. ; S5, Synthesis probe DCI-TSO: Compound 3 obtained in S3 and compound 4 obtained in S4 were dissolved in anhydrous methanol according to the specified ratio, and sodium methoxide was added dropwise. The mixture was stirred overnight at room temperature under a protective atmosphere. The organic layer was purified to obtain the probe DCI-TSO. .

2. The method for preparing a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism according to claim 1, characterized in that, In S1, the equivalent ratio of 4-hydroxybenzaldehyde, triethylamine, anhydrous acetonitrile, and 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazolium-3-onium trifluoromethanesulfonate is 1:1.5:10:1.

5. The temperature of the stirring reaction is 0~5℃, and the reaction time is 4h.

3. The method for preparing a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism according to claim 1, characterized in that, In S1, the extraction is carried out using water, saturated saline solution, and ethyl acetate, wherein the volume ratio of saturated saline solution to ethyl acetate is 3:

1. The evaporation process is performed using a rotary evaporator under reduced pressure or a freeze dryer. The purification was performed using silica gel column chromatography, with an eluent volume ratio of PE:EtOAc = 50:1 to 20:

1.

4. The method for preparing a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism according to claim 1, characterized in that, In S2, the equivalent ratio of 4-nitrophenol: imidazole: anhydrous DMF: tert-butyldimethylchlorosilane is 1:1.5:15:1.3; The extraction was performed using ether and water, and the purification was performed using silica gel column chromatography with an eluent volume ratio of PE:DCM = 50:

1.

5. The method for preparing a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism according to claim 1, characterized in that, In S3, the equivalent ratio of compound 1: compound 2: anhydrous acetonitrile: 1,8-diazabicyclo[5.4.0]-7-undecene is 1:1:10:0.25; The extraction was performed using ethyl acetate and water; The drying process uses anhydrous sodium sulfate. The solvent removal method is a reduced pressure solvent removal method; The purification was performed using a silica gel column, with an eluent volume ratio of 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 equivalence ratio of isophorone:malononitrile:ethanol:piperidine is 1:2:71:0.01; The reflux reaction conditions are 80~85℃ for 10~15 hours.

7. The method for preparing a near-infrared fluorescent probe DCI-TSO based on an ICT mechanism according to claim 1, characterized in that, In S5, the equivalent ratio of compound 3: compound 4: anhydrous methanol: sodium methoxide is 1:1:10:1; The stirring reaction conditions are 20~25℃, 10~12h; The purification was performed using column chromatography with an eluent volume ratio of PE:EtOAc = 20:

1.

8. A probe DCI-TSO prepared by the method for preparing a near-infrared fluorescent probe DCI-TSO based on the ICT mechanism as described in any one of claims 1-7.

9. A probe DCI-TSO as described in claim 8 for the simultaneous detection of H2S and ONOO in cells for non-diagnostic purposes. - The application of content is characterized by, The testing steps are as follows: 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.

10. A probe DCI-TSO as described in claim 8 for the simultaneous detection of H2S and ONOO during preparation. - Applications of reagents.

Images