A near-infrared fluorescent probe and a preparation method and application thereof

Abstract

This invention presents a novel near-infrared fluorescent probe, TPA-NI-NO2, prepared using triphenylamine-naphthalimide as the fluorescent group and 2,4-dinitrophenyl ether as the recognition group. This fluorescence-on type probe is used for the detection of 4-methoxythiophenol (MTP) and exhibits good selectivity and anti-interference properties. Under qualitative detection of MTP, bright orange fluorescence with a rapid response is observed. Under quantitative detection, the fluorescence emission intensity at 607 nm after excitation at 470 nm shows a significant linear correlation with the MTP concentration, with a detection limit reaching 80 nM. This probe can also be used for detection at environmental pH values ​​from 2 to 12 and for intracellular detection, showing broad development prospects in environmental and biological sensing applications.

Description

Technical Field

[0001] This invention relates to the fields of detection and materials, specifically to a near-infrared fluorescent probe, its preparation method, and its applications. Background Technology

[0002] Thiophenols are widely used in the preparation of pesticides, pharmaceuticals, and various industrial products. However, thiophenol compounds are highly toxic, and long-term exposure can cause serious damage to the central nervous system and other nervous systems, including shortness of breath, muscle weakness, hind limb paralysis, coma, and even death. The presence of thiophenol compounds in water and soil also damages natural habitats. It is listed as one of the priority pollutants. Animal model studies have shown that thiophenol compounds are toxic to aquatic organisms and animals, with a median lethal concentration (LC50) in fish. 50 The median lethal dose (LD50) in mice was 0.01–0.4 mM. 50 The concentration was 46.2 mg / kg. Therefore, monitoring trace amounts of thiophene compounds in the environment and organisms is essential.

[0003] Traditional methods for detecting benzenethiols include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and UV-VIS spectroscopy. However, these methods suffer from drawbacks such as high cost and complex sample pretreatment. Fluorescent probes offer high specificity and sensitivity for the detection of target analytes and are easy to operate. The research group of Lin (Lin, W., Long, L., Tan, W. A highly sensitive fluorescent probe for detection of benzenethiols in environmental samples and living cells. Chem. Commun., 2010, 46, 1503–1505.) formed a fluorescent probe by linking hydroxycoumarin dye and 2,4-dinitrophenyl groups together via ether bonds. When detecting benzenethiols, nucleophilic aromatic substitution breaks the ether bonds of the probe, releasing fluorescence. Most fluorescent probes use 2,4-dinitrophenyl (DNP) as a quencher, binding with fluorophores such as naphthalimide, nitrobenzofuran, coumarin, and BODIPY to form fluorescent probes. Mechanistically, they detect thiophenol compounds through intramolecular charge transfer (ICT) or photoinduced electron transfer (PET) mechanisms. However, currently reported probes only exhibit fluorescence response in the visible light region, have small stoke shifts, and mostly require large amounts of organic co-solvents (≥30%) and have slow fluorescence responses. Furthermore, fluorescence imaging is easily interfered with by endogenous molecular background fluorescence. Additionally, some probes are significantly affected by solvent and pH conditions, preventing the detection of thiophenol compounds in real-world environments and biological samples. Therefore, from a practical application perspective, designing and synthesizing near-infrared fluorescent probes with large stoke shifts, rapid responses, and significant color changes is essential. Summary of the Invention

[0004] The present invention aims to provide a near-infrared fluorescent probe, its preparation method and application. Compared with fluorescence quenching probes, the probe of the present invention is a fluorescence-on type, which has better specificity, selectivity, anti-interference and higher sensitivity, and is more conducive to the detection of thiophenol compounds in the near-infrared region.

[0005] The thiophenol compounds described in this invention include thiophenol and substituted thiophenols. A substituted thiophenol is a compound having at least one substituent at positions 2-5 of the benzene ring of the thiophenol. The substituent is selected from alkyl, alkoxy, halogen, hydroxyl, amino, amide, or sulfonyloxy groups that can be further substituted or are unsubstituted. Specific examples of substituted thiophenols include, but are not limited to, p-methylthiophenol, m-methylthiophenol, m-ethylthiophenol, 4-ethylthiophenol, 3-chlorothiophenol, 2,3-dichlorothiophenol, 4-fluorothiophenol, 2,4-difluorothiophenol, 3-bromothiophenol, 3-aminothiophenol, 2,4-dimethylthiophenol, 4-methoxythiophenol, m-methoxythiophenol, m-ethoxythiophenol, p-hydroxythiophenol, 2-amino-5-methoxythiophenol, p-chloro-o-aminothiophenol, 4-acetaminothiophenol, and 4-methylsulfonyloxythiophenol.

[0006] During the research, the inventors unexpectedly discovered that the near-infrared fluorescent probe of the present invention exhibits high specificity, rapid response, and high sensitivity for the detection of 4-methoxythiophenol (MTP), and that the detection range is applicable to a wider pH environment and can be used for detection in live cells.

[0007] The present invention is achieved through the following technical solutions.

[0008] This invention provides a near-infrared fluorescent probe, characterized in that it is a compound represented by formula (I):

[0009] .

[0010] In this invention, the near-infrared fluorescent probe is also referred to as "TPA-NI-NO2".

[0011] This invention also provides a method for preparing the near-infrared fluorescent probe, comprising the following steps: under a protective atmosphere, heating compound TPA-NI-OH with 2,4-dinitrofluorobenzene and triethylamine under reflux until the reaction is complete, to obtain the near-infrared fluorescent probe.

[0012] .

[0013] The protective atmosphere employs conventional practices in the art, such as nitrogen protection or inert gas protection.

[0014] Preferably, the molar ratio of TPA-NI-OH to 2,4-dinitrofluorobenzene and triethylamine is 1:1-1.5:2-4.

[0015] Preferably, the solvent for the reaction is selected from N,N-dimethylformamide or tetrahydrofuran.

[0016] Preferably, the reflux reaction time is 6-8 hours.

[0017] After the reaction was completed, the reaction solution was cooled to room temperature, extracted with water and dichloromethane, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by column chromatography (petroleum ether PE: ethyl acetate EA = 3:1, v / v) to obtain TPA-NI-NO2, an orange solid.

[0018] Further, the compound TPA-NI-OH is prepared by the following method: reacting compound NI-OH with triphenylamine 4-borate, tetra(triphenylphosphine)palladium and potassium carbonate under reflux in an organic solvent.

[0019] .

[0020] Preferably, the molar ratio of NI-OH to triphenylamine 4-borate, tetra(triphenylphosphine)palladium and potassium carbonate is 1:1.1-1.4:0.03-0.10:5-12.

[0021] Preferably, the organic solvent is selected from N,N-dimethylformamide or tetrahydrofuran.

[0022] Preferably, the reflux reaction time is 6-12 hours.

[0023] After the reaction was complete, the reaction solution was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by column chromatography (dichloromethane DCM) to give TPA-NI-OH, which was an orange solid.

[0024] Further, the compound NI-OH was prepared by the following method: 4-bromo-1,8-naphthenic anhydride was dissolved in acetic acid, 3-aminophenol was added, the temperature was raised to 118°C, and the mixture was refluxed and stirred for 5 hours. After the reaction was completed, the reaction solution was cooled to precipitate a solid, which was then filtered, washed with water, and dried to obtain the compound NI-OH, which was a brown solid.

[0025]

[0026] After screening, the detection solvent for the near-infrared fluorescent probe TPA-NI-NO2 of the present invention is a mixed solution of HEPES buffer solution (10 mM, pH=7.4) and dimethyl sulfoxide (DMSO), containing 10-20% (v / v) DMSO; preferably, it contains 10% (v / v) DMSO. In the present invention, "detection solvent A" refers to a detection solvent containing 90% HEPES buffer solution (10 mM, pH=7.4) and 10% DMSO.

[0027] The advantages of the detection solvent of this invention are: it does not use a large amount of organic solvent; under 470nm excitation conditions, the fluorescence emission intensity of the probe detecting 4-methoxythiophenol (MTP) is twice that of the fluorescence emission intensity of the single probe when the component ratio of the detection solvent is within the range; the detection solvent does not contain components that can dissolve cell membranes or have toxic effects on cells, such as CTAB, making it suitable for intracellular detection.

[0028] The working concentration range of the near-infrared fluorescent probe TPA-NI-NO2 in the detection solvent is 10-30 μM, preferably 10 μM.

[0029] The pH range of the working environment for the near-infrared fluorescent probe TPA-NI-NO2 of this invention is 2-12, preferably 2-8.

[0030] The near-infrared fluorescent probe TPA-NI-NO2 of this invention and the sample to be tested were dispersed in a detection solvent and tested using a UV-Vis spectrophotometer and fluorescence spectroscopy. The probe showed the following properties:

[0031] (1) The probe has two absorption peaks at 308 nm and 446 nm.

[0032] (2) Under the excitation condition of 470 nm, the probe itself has no obvious fluorescence brightness.

[0033] (3) The test sample containing 4-methoxythiophenol (MTP) was added to the probe and observed under a 365 nm UV lamp. The probe responded in about 20 minutes, changing from weak fluorescence to bright orange fluorescence, indicating that the probe is a fluorescence-on type and can achieve a rapid response to MTP.

[0034] (4) Add other substances (Br) to the probe - CO3 2- SO3 2- SO4 2- HSO3 - NO3 - Na + Mg 2+ K + L-cysteine ​​(L-cys), glutathione (GSH), ClO - H2S o -CH3O-C6H4SH、 o -CH3-C6H4SH, C6H5S, o The fluorescence emission intensity of the sample to be tested (-CH3CH2-C6H4SH) did not change significantly under the excitation condition of 470 nm.

[0035] (5) When the test sample containing 4-methoxythiophenol (MTP) was added to the probe and the fluorescence intensity at 607 nm was monitored over time under 470 nm excitation, the fluorescence intensity increased within 10 min and reached saturation within 60 min, with strong orange fluorescence emission at 607 nm. Even in the presence of other interfering substances, the probe's recognition of MTP was not affected, and the fluorescence emission intensity remained at a high level. This indicates that the probe is a fluorescence-on type, with a rapid response to MTP in the near-infrared region, and has good fluorescence specificity, selectivity, and anti-interference ability.

[0036] (6) Under the excitation condition of 470nm, the change in the fluorescence signal of the probe itself is negligible, indicating that the probe itself has good stability over time.

[0037] Therefore, this invention provides a visual fluorescence detection method for 4-methoxybenzylthiophenol, comprising the following steps: dispersing a near-infrared fluorescent probe TPA-NI-NO2 and a sample containing 4-methoxybenzylthiophenol in a detection solvent; under 365 nm ultraviolet light, if the probe exhibits fluorescence activation (i.e., a bright orange fluorescence is observed), it indicates the presence of 4-methoxybenzylthiophenol in the sample. Preferably, the detection solvent is a mixture of HEPES buffer solution (10 mM, pH=7.4) and dimethyl sulfoxide, containing 10-20% (v / v) dimethyl sulfoxide. This visual fluorescence detection method belongs to the category of qualitative detection methods.

[0038] Therefore, the present invention also provides a fluorescence detection method for 4-methoxybenzylthiophenol, comprising the following steps: dispersing a near-infrared fluorescent probe TPA-NI-NO2 and a test sample containing 4-methoxybenzylthiophenol in a detection solvent, and measuring the fluorescence emission intensity at 607 nm under 470 nm excitation conditions. Preferably, the detection solvent is a mixed solution of HEPES buffer solution (10 mM, pH=7.4) and dimethyl sulfoxide, wherein the solvent contains 10-20% (v / v) dimethyl sulfoxide; more preferably, it contains 10% (v / v) dimethyl sulfoxide. Preferably, the working concentration range of the near-infrared fluorescent probe TPA-NI-NO2 is 10-30 μM.

[0039] Specifically, this invention provides a quantitative fluorescence detection method for 4-methoxybenzylthiophenol, comprising the following steps: dispersing a near-infrared fluorescent probe TPA-NI-NO2 and a sample containing 4-methoxybenzylthiophenol in a detection solvent; measuring the fluorescence emission intensity at 607 nm under 470 nm excitation conditions; and performing quantitative detection by establishing a linear correlation between the concentration of 4-methoxybenzylthiophenol and the fluorescence emission intensity. Preferably, the detection solvent is a mixture of HEPES buffer solution (10 mM, pH=7.4) and dimethyl sulfoxide (DMSO), containing 10-20% (v / v) DMSO; more preferably, it contains 10% (v / v) DMSO. Preferably, the working concentration range of the near-infrared fluorescent probe TPA-NI-NO2 is 10-30 μM. Preferably, the linear regression equation for the quantitative detection is: Y = 15923.80548X - 274837.92549, R² = 0.99539, where the unit for the concentration of 4-methoxybenzylthiophenol on the x-axis is μM. The limit of detection for 4-methoxybenzylthiophenol in the quantitative detection can reach 80 nM.

[0040] This invention also provides a method for detecting intracellular 4-methoxybenzylthiophenol using fluorescence, comprising the following steps: culturing test cells in a solution containing the near-infrared fluorescent probe TPA-NI-NO2, adding 4-methoxybenzylthiophenol during the culture, and then observing fluorescence emission at 490-800 nm under 470 nm excitation conditions. If the probe exhibits fluorescence activation, i.e., orange fluorescence is observed, it indicates the presence of 4-methoxybenzylthiophenol in the test cells. Preferably, the working concentration of the solution containing the near-infrared fluorescent probe TPA-NI-NO2 is 2.5-10 μM.

[0041] Based on the aforementioned properties of the near-infrared fluorescent probe TPA-NI-NO2 of this invention, and the method for detecting 4-methoxybenzylthiophenol (MTP) using the near-infrared fluorescent probe TPA-NI-NO2, the fluorescent probe TPA-NI-NO2 of this invention can be used for near-infrared detection of MTP, for rapid and specific detection of MTP, for qualitative and quantitative detection of MTP, for intracellular MTP detection, or for preparing reagents, test strips, or kits for MTP detection. It possesses advantages such as high specificity and selectivity, strong anti-interference ability, rapid recognition, high sensitivity, accurate detection results, and wide applicability. The pH value of the application environment for the near-infrared fluorescent probe TPA-NI-NO2 in MTP detection can be 2-12.

[0042] The working principle of the near-infrared fluorescent probe TPA-NI-NO2 of this invention is as follows: The triphenylamine-naphthalimide (TPA-NI-OH) moiety serves as the fluorophore, forming a push-pull electron system with the 2,4-dinitrophenyl ether moiety. The 2,4-dinitrophenyl ether moiety quenches the probe's fluorescence through photoinduced electron transfer (PET) effect, resulting in weak fluorescence in TPA-NI-NO2. Upon addition of 4-methoxythiophenol (MTP), due to its strong nucleophilicity, a nucleophilic reaction occurs with the probe molecule, leading to the breakage of the bond between triphenylamine-naphthalimide and the 2,4-dinitrophenoxy group, generating the fluorescent product TPA-NI-OH. The system then releases a significantly enhanced fluorescence signal, thereby achieving effective detection of MTP.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] (1) Based on nucleophilic reaction and the near-infrared fluorescence "off-on" effect of the probe, this invention establishes a novel near-infrared fluorescent probe TPA-NI-NO2, which is an on-type probe with good water solubility and anti-interference properties. When applied to the detection of 4-methoxythiophenol (MTP), it shows high selectivity and high sensitivity.

[0045] (2) This invention uses a fluorescence visualization method for qualitative analysis of MTP. The near-infrared fluorescent probe TPA-NI-NO2 responds in about 20 minutes, and bright orange fluorescence is observed under 365 nm ultraviolet light. This invention also uses a fluorescence method for quantitative detection of MTP. Under 470 nm excitation conditions, the sample concentration is significantly linearly correlated with the fluorescence emission intensity at 607 nm of the probe. The detection results are accurate, and the lowest detection limit is as low as 80 nM.

[0046] (3) The near-infrared fluorescent probe TPA-NI-NO2 of the present invention has a large stoke shift, which can effectively avoid background interference during fluorescence measurement. After detecting MTP, bright near-infrared fluorescence can be observed and the response is fast. It is less affected by other interfering substances and can detect MTP in an environment with a pH value of 2 to 12.

[0047] (4) The near-infrared fluorescent probe TPA-NI-NO2 of the present invention is easy to prepare, the detection process is simple, it has low toxicity to cells, and it can rapidly detect MTP in cell samples. It has good development and application prospects in the environmental and biological fields. Attached Figure Description

[0048] Figure 1 The selection of the solvent for the near-infrared fluorescent probe TPA-NI-NO2 detection in Example 2 is shown in the fluorescence emission diagram under 470 nm excitation conditions.

[0049] Figure 2The image shows the UV-Vis absorption spectrum of the near-infrared fluorescent probe TPA-NI-NO2 in Example 3.

[0050] Figure 3 The fluorescence spectrum of the near-infrared fluorescent probe TPA-NI-NO2 recognizing 4-methoxybenzylthiophenol (MTP) is shown in Example 3.

[0051] Figure 4 The fluorescence spectra of the near-infrared fluorescent probe TPA-NI-NO2 used in Example 4 to identify different substances are shown.

[0052] Figure 5 This is a bar chart showing the fluorescence emission intensity of the near-infrared fluorescent probe TPA-NI-NO2 for different substances in Example 4.

[0053] Figure 6 The bar chart for Example 5 shows the anti-interference ability of the near-infrared fluorescent probe TPA-NI-NO2 to recognize 4-methoxybenzylthiophenol (MTP).

[0054] Figure 7 The response time diagram for the recognition of 4-methoxybenzylthiophenol (MTP) by the near-infrared fluorescent probe TPA-NI-NO2 in Example 6 is shown.

[0055] Figure 8 The image shows the fluorescence emission intensity of the near-infrared fluorescent probe TPA-NI-NO2 in Example 7 at different pH values ​​in response to 4-methoxybenzylthiophenol (MTP).

[0056] Figure 9 The fluorescence spectra of the near-infrared fluorescent probe TPA-NI-NO2 in Example 8 for recognizing different concentrations of 4-methoxythiophenol (MTP).

[0057] Figure 10 The working curve for quantitative detection of 4-methoxybenzylthiophenol (MTP) by the near-infrared fluorescent probe TPA-NI-NO2 in Example 9 is shown.

[0058] Figure 11 A schematic diagram of the mechanism by which the near-infrared fluorescent probe TPA-NI-NO2 recognizes 4-methoxybenzylthiophenol (MTP).

[0059] Figure 12 High-performance liquid chromatography (HPLC) chromatogram for the mechanism verification of the near-infrared fluorescent probe TPA-NI-NO2 recognizing 4-methoxybenzylthiophenol (MTP) in Example 10.

[0060] Figure 13 The figure shows the results of the near-infrared fluorescent probe TPA-NI-NO2 cytotoxicity experiment on HeLa cells in Example 11.

[0061] Figure 14This is a fluorescence imaging image of the near-infrared fluorescent probe TPA-NI-NO2 recognizing 4-methoxybenzylthiophenol (MTP) in HeLa cells, as shown in Example 12. Detailed Implementation

[0062] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0063] The chemical reagents and solvents used in the preparation of the near-infrared fluorescent probe TPA-NI-NO2 of this invention were all purchased from Tansun Reagent Company, and the metal ions were all purchased from Aladdin Reagent Company. Fluorescent probe confirmation and performance testing were performed using a Bruker 400 MHz nuclear magnetic resonance spectrometer (DMSO-d6 solvent, TMS as internal standard). High-resolution mass spectrometry data were recorded using a Thermo Q-Exactive HR-MS mass spectrometer. UV-Vis absorption spectra were measured using a Varian Cary-300 UV-Vis spectrometer. Fluorescence spectra were recorded using an Edinburgh FS-5 fluorescence spectrometer. Confocal imaging was performed using an LSM700 confocal microscope.

[0064] Example 1: Preparation of near-infrared fluorescent probe TPA-NI-NO2

[0065]

[0066] 1. Preparation of Ni-OH

[0067] 4-Bromo-1,8-naphthoic anhydride (1.00 g, 3.61 mmol) and 3-aminophenol (0.47 g, 4.33 mmol) were dissolved in 10 mL of acetic acid at room temperature. The solution was gradually heated to 118 °C under nitrogen protection to obtain a clear brown solution. The solution was refluxed and stirred for 5 hours. After the reaction was completed, the reaction solution was cooled and a solid precipitated. The solid was filtered, and the filter residue was washed with an appropriate amount of water (20 mL). The residue was dried to obtain the final brown solid compound (1.26 g, yield 94.8%).

[0068] 2. Preparation of TPA-NI-OH

[0069] Compound NI-OH (600 mg, 1.63 mmol), triphenylamine 4-borate (518.3 mg, 1.79 mmol), and tetra-(triphenylphosphine)palladium (94.2 mg, 0.0815 mmol) were dissolved sequentially in tetrahydrofuran (15 mL) and K₂CO₃ solution (2.24 g, 16.23 mmol / 5 mL water), and the mixture was heated under reflux for 8 hours. After the reaction was completed, the reaction solution was cooled, and the crude product was obtained by extraction. The crude product was purified by column chromatography using dichloromethane (DCM) as the chromatographic solvent. The chromatographic solvent was collected and rotary evaporated to give the orange pure product TPA-NI-OH (700 mg, yield 80.65%).

[0070] Structural characterization of TPA-NI-OH

[0071] Nuclear magnetic resonance (NMR) measurement: 1 H NMR (400 MHz, DMSO-d6) δ ppm: 9.67 (s, 1H), 8.57 -8.51 (m, 2H), 8.45 (d, J = 8.5 Hz, 1H), 7.95 - 7.88 (m, 1H), 7.84 (d, J = 7.6Hz, 1H), 7.52 (d, J = 8.5 Hz, 2H), 7.39 (t, J = 7.9 Hz, 4H), 7.31 (t, J = 8.0Hz, 1H), 7.15 (dd, J = 16.1, 7.7 Hz, 8H), 6.87 (d, J = 7.5 Hz, 1H), 6.80 (d,J = 6.9 Hz, 2H).

[0072] 13 C NMR (100 MHz, DMSO-d6) δ ppm: 164.06, 163.84, 158.30, 148.22,147.26, 131.94, 131.49, 130.91, 130.23, 129.88, 125.24, 124.27, 122.48.

[0073] 3. Preparation of near-infrared fluorescent probe TPA-NI-NO2

[0074] In a three-necked flask, TPA-NI-OH (600 mg, 1.13 mmol), 2,4-dinitrofluorobenzene (314.5 mg, 1.69 mmol), and triethylamine (544 mg, 3.38 mmol) were dissolved in 15 mL of N,N-dimethylformamide (DMF). The mixture was heated to reflux for 7 hours under nitrogen protection, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was cooled to room temperature, water was added, and the mixture was extracted with dichloromethane. The solution was dried over anhydrous Na₂SO₄ and concentrated by rotary evaporation. The crude product was purified by column chromatography (petroleum ether PE: ethyl acetate EA = 3:1, v / v) to give an orange solid (381 mg, yield 48.4%).

[0075] Structural characterization of the near-infrared fluorescent probe TPA-NI-NO2

[0076] Nuclear magnetic resonance (NMR) measurement: 1 H NMR (400 MHz, DMSO-d6) δ 8.92 (d, J = 2.8 Hz, 1H), 8.58- 8.54 (m, 3H), 8.46 (d, J = 8.5 Hz, 1H), 7.94 - 7.90 (m, 1H), 7.86 (d, J = 7.6Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.44 - 7.37 (m, 7H), 7.30 (d, J = 9.3 Hz, 1H), 7.15 (dd, J = 11.4, 4.5 Hz, 8H). 13C NMR (101 MHz, DMSO-d6) δ 164.02, 163.79, 154.98, 154.40, 148.28, 147.26, 146.53, 142.19, 140.10,138.38, 133.07, 131.82, 131.46, 131.29, 131.26, 131.00, 130.21, 129.84,129.06, 128.25, 127.81, 127.61, 125.24, 124.30, 123.30, 122.42, 122.38,121.70, 121.63, 120.48, 119.98, 40.63, 40.42, 40.21, 40.00, 39.79, 39.58, 39.38.

[0077] High-resolution mass spectrometry (HRMS) determination: HRMS (ESI): calcd. for C 42 H 26 N4O7: 698.1835; found: 698.18885.

[0078] Example 2: Selection of detection solvent for near-infrared fluorescent probe TPA-NI-NO2

[0079] Prepare HEPES buffer (10mM, pH=7.4) for later use.

[0080] Prepare a 1 mM stock solution of the near-infrared fluorescent probe TPA-NI-NO2 (hereinafter referred to as "probe stock solution") using DMSO for later use.

[0081] Eleven clean quartz cuvettes were filled with HEPES buffer and DMSO in different proportions to prepare 2 mL of detection solvent. The volume ratios of HEPES buffer to DMSO were 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10, respectively. Then, 20 μL of probe stock solution was added to each cuvette to bring the working probe concentration to 10 μM. Finally, 4-methoxythiophenol (MTP) was added to each cuvette to bring the MTP concentration to 20 μM. Fluorescence spectroscopy was used to investigate the individual probe concentration in these 11 different detection solvents under 470 nm excitation conditions. Figure 1 (gray bar) and probe identification of MTP ( Figure 1 The fluorescence emission of the black column (as shown in the results) is as follows: Figure 1 As shown.

[0082] Figure 1The results showed that the fluorescence emission intensity of the individual probes varied but was weak in 11 different detection solvents. After the probes recognized MTP, the fluorescence emission intensity doubled in detection solvents with HEPES buffer to DMSO volume ratios of 10:0, 9:1, and 8:2, with the best effect observed at 9:1.

[0083] Therefore, a suitable detection solvent for the near-infrared fluorescent probe TPA-NI-NO2 is a mixed solution of HEPES buffer (10 mM, pH=7.4) and dimethyl sulfoxide (DMSO), containing 10-20% (v / v) DMSO; preferably, it contains 10% (v / v) DMSO. In this invention, "detection solvent A" refers to a detection solvent containing 90% HEPES buffer (10 mM, pH=7.4) and 10% DMSO.

[0084] Example 3: Fluorescence spectral response of near-infrared fluorescent probe TPA-NI-NO2 to recognize 4-methoxythiophenol (MTP)

[0085] Add 2 mL of detection solvent A to a clean quartz cuvette, then add 20 μL of the probe stock solution from Example 2 to a working concentration of 10 μM. Next, add 4-methoxythiophenol (MTP) to a concentration of 20 μM. Test using a UV-Vis spectrophotometer and a fluorescence spectrometer.

[0086] like Figure 2 As shown, when the probe was tested on a UV-Vis spectrophotometer, two absorption peaks were observed at 308 nm and 446 nm.

[0087] like Figure 3 As shown, when the probe alone is tested on a fluorescence spectrometer, it exhibits very weak orange fluorescence emission in the near-infrared region due to the quenching effect of 2,4-dinitrophenyl ether. However, after the probe recognizes MTP, it emits a distinct orange fluorescence at 607 nm under a 470 nm excitation condition, and the fluorescence intensity increases significantly.

[0088] Example 4: Selective detection of 4-methoxythiophenol (MTP) by the near-infrared fluorescent probe TPA-NI-NO2

[0089] Add 2 mL of detection solvent A to each clean quartz cuvette, and then add 20 μL of the probe stock solution from Example 2 until the working concentration of the probe is 10 μM. Next, add 20 μM of the following substance: Br - CO3 2- SO3 2- SO4 2- HSO3 - NO3- , ClO - Na + Mg 2+ , K + , L-cys, GSH, H2S, o -CH3O-C6H4SH, o -CH3-C6H4SH,C6H5S, o The selectivity of the probe to 4-methoxybenzylthiophenol (MTP) was investigated using fluorescence spectroscopy under excitation conditions of 470 nm.

[0090] like Figure 4 The near-infrared fluorescent probe TPA-NI-NO2 is shown in the fluorescence spectra of different substances, as follows: Figure 5 The bar chart showing the fluorescence emission intensity of the near-infrared fluorescent probe TPA-NI-NO2 against different substances reveals that the probe alone exhibits no significant fluorescence emission. With the addition of MTP, the probe displays a strong orange fluorescence emission intensity at 607 nm. However, the addition of other interfering substances (Br...)... - CO3 2- SO3 2- SO4 2- HSO3 - NO3 - , ClO - Na + Mg 2+ , K + L-cys, GSH, H2S o -CH3O-C6H4SH, o -CH3-C6H4SH, C6H5S, o When -CH3CH2-C6H4SH is used, the fluorescence emission intensity of the probe does not change significantly compared to the fluorescence emission intensity of the probe alone.

[0091] The above experimental results show that the near-infrared fluorescent probe TPA-NI-NO2 for 4-methoxybenzylthiophenol (MTP) has good fluorescence specificity and selectivity in detection solvent A.

[0092] Example 5: Determination of the anti-interference ability of the near-infrared fluorescent probe TPA-NI-NO2 to recognize 4-methoxybenzylthiophenol (MTP)

[0093] Add 2 mL of detection solvent A to each clean quartz cuvette, and then add 20 μL of the probe stock solution from Example 2 until the working concentration of the probe is 10 μM. Then add one other interfering substance (blank, Br) to each. -CO3 2- SO3 2- SO4 2- HSO3 - NO3 - , ClO - Na + Mg 2+ , K + , L-cys, GSH, H2S, o -CH3O-C6H4SH, o -CH3-C6H4SH, C6H5S, o The concentrations of 4-methoxybenzylthiophenol (MTP) and other interfering substances were 20 μM in each quartz cuvette. The concentration of MTP in each quartz cuvette was also 20 μM. Fluorescence spectroscopy was used to detect whether the probe's recognition of MTP was interfered with by the aforementioned interfering substances under excitation conditions of 470 nm. The resulting fluorescence emission histogram is shown below. Figure 6 As shown, black bars indicate the addition of other interfering substances to the probe, while gray bars indicate the addition of other interfering substances and MTP to the probe.

[0094] Figure 6 The results showed that, in the presence of other interfering substances, the near-infrared fluorescent probe TPA-NI-NO2 was not affected by other interfering substances in the detection solvent A for the recognition of 4-methoxybenzylthiophenol (MTP), and the fluorescence emission intensity remained at a high level, demonstrating good anti-interference ability.

[0095] Example 6: Response time of near-infrared fluorescent probe TPA-NI-NO2 to 4-methoxythiophenol (MTP)

[0096] Add 2 mL of detection solvent A to a clean quartz cuvette, followed by 20 μL of the probe stock solution from Example 2 to a working concentration of 10 μM. Then add 4-methoxythiophenol (MTP) to a concentration of 20 μM. The response time of the probe to MTP was then investigated using a fluorescence spectrometer under 470 nm excitation. The time-dependent fluorescence generated by the interaction between the probe and MTP was studied by monitoring the change in fluorescence intensity at 607 nm over time. Figure 7 As shown, fluorescence enhancement occurs within 10 minutes and saturation occurs within 60 minutes, demonstrating that the near-infrared fluorescent probe TPA-NI-NO2 detects MTP in a fluorescence enhancement (on-mode) that increases with time, eventually resulting in a bright orange fluorescence. Furthermore, the fluorescence signal change of the probe itself within the same time interval is negligible, indicating that the probe itself is stable over time.

[0097] Example 7: Testing the response of the near-infrared fluorescent probe TPA-NI-NO2 to 4-methoxythiophenol (MTP) at different pH values.

[0098] Prepare HEPES buffer solutions (10 mM) with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0099] Next, detection solvents with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 were prepared. Specifically, for example, the detection solvent with pH=2 was prepared by mixing 90% (v / v) HEPES buffer (10mM, pH=2) and 10% (v / v) DMSO; detection solvents with other pH values ​​were obtained by a similar method.

[0100] 2 mL of detection solvent with different pH values ​​(2-12) and 20 μL of probe stock solution from Example 2 were added to 11 clean cuvettes to make the working concentration of the probe 10 μM. The stability of the probe in detection solvents with different pH values ​​was first investigated. As shown in Figure 8, the fluorescence intensity of the probe itself remained basically unchanged in a wide pH range of 2-12, indicating that the probe is very stable.

[0101] Next, 4-methoxythiophenol (MTP) was added to each of the 11 cuvettes until the MTP concentration reached 20 μM. Then, the effect of the probe on the MTP response in detection solvents at different pH values ​​was investigated using a fluorescence spectrometer under 470 nm excitation conditions. Figure 8 As shown, significant fluorescence enhancement was observed in the pH range of 2 to 12 after the probe was added to MTP.

[0102] The results showed that the near-infrared fluorescent probe TPA-NI-NO2 can detect 4-methoxythiophenol (MTP) in a wide range of pH environments.

[0103] Example 8: Near-infrared fluorescent probe TPA-NI-NO2 identifies different concentrations of 4-methoxythiophenol (MTP).

[0104] Add 2 mL of detection solvent A and 20 μL of the probe stock solution from Example 2 to a clean quartz cuvette until the working concentration of the probe is 10 μM. Prepare a 0.02 M 4-methoxythiophenol (MTP) solution with anhydrous ethanol, and gradually add the 4-methoxythiophenol (MTP) solution to the quartz cuvette until the MTP concentration is 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. Detect the fluorescence emission intensity at 607 nm using a fluorescence spectrometer under 470 nm excitation conditions. Figure 9 As shown.

[0105] Figure 9 The results showed that as the concentration of MTP increased (0, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM), the orange fluorescence intensity at 607 nm gradually increased. Therefore, it is believed that the near-infrared fluorescent probe TPA-NI-NO2 can be used for quantitative detection of MTP.

[0106] Example 9: Investigation of the detection limit of the near-infrared fluorescent probe TPA-NI-NO2 for recognizing 4-methoxybenzylthiophenol (MTP)

[0107] The operation of Example 9 is the same as that of Example 8.

[0108] Fluorescence emission intensity was measured on a fluorescence spectrometer using 470 nm as the excitation wavelength. The concentration of 4-methoxythiophenol (MTP) was plotted on the x-axis, and the fluorescence emission intensity at 607 nm on the y-axis. A working curve was obtained by establishing a linear correlation between MTP concentration and fluorescence emission intensity. The linear regression equation was: Y = 15923.80548X - 274837.92549, R0 2 =0.99539; Y represents the fluorescence emission intensity at 607 nm; X represents the concentration of 4-methoxybenzylthiophenol in μM. Therefore, the detection limit of the near-infrared fluorescent probe TPA-NI-NO2 for 4-methoxybenzylthiophenol (MTP) is calculated (LOD=3σ / k) to be 80 nM. Figure 10 As shown.

[0109] The results showed that the near-infrared fluorescent probe TPA-NI-NO2 had high sensitivity for the quantitative detection of 4-methoxythiophenol (MTP). The MTP concentration was significantly linearly correlated with the fluorescence emission intensity at 607 nm, and the limit of detection was 80 nM.

[0110] Example 10: Mechanistic Study of Detection of 4-Methoxythiophenol (MTP) by Near-Infrared Fluorescent Probe TPA-NI-NO2

[0111] The near-infrared fluorescent probe TPA-NI-NO2 of this invention contains a quenching and recognition component, 2,4-dinitrophenyl, which is linked to naphthalimide-triphenylamine via an ether bond. The photoinduced electron transfer (PET) behavior of the triphenylamine-naphthalimide to the 2,4-dinitrophenyl ether moiety results in almost no fluorescence in the probe. 4-Methoxythiophenol (MTP) can be nucleophilically substituted, causing the ether bond of the probe to break and electron rearrangement to restore the bright orange fluorescence of the TPA-NI-OH fluorophore. Figure 11 ).

[0112] The reaction process was further investigated using high-performance liquid chromatography, such as... Figure 12 As shown in the report, liquid chromatography analysis of individual near-infrared fluorescent probes TPA-NI-NO2, TPA-NI-OH, and MTP molecules revealed that TPA-NI-NO2 had a retention time of 11.8 min and a single chromatographic peak at the chromatogram. After the addition of MTP, a new peak appeared at 8.4 min, which coincided with the chromatogram of compound TPA-NI-OH. Figure 1 The results demonstrate that the near-infrared fluorescent probe TPA-NI-NO2 reacts with MTP, breaking the ether bond to produce TPA-NI-OH. Therefore, the liquid chromatography results indicate that MTP can induce the probe to transform into TPA-NI-OH, i.e., a nucleophilic addition reaction occurs.

[0113] Example 11 Near-infrared fluorescent probe TPA-NI-NO2 cytotoxicity assay

[0114] HeLa cells were seeded in 96-well plates in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin. A 1 mM probe stock solution was prepared using DMSO, and different concentrations of the probe (working concentrations of 0, 1.25, 2.5, 5, and 10 μM) were added to the HeLa cells and cultured for 24 hours. Cell viability was then measured using the CCK-8 assay. The results showed that even at higher probe concentrations (10 μM), cell viability remained above 90%. Figure 13 This indicates that the near-infrared fluorescent probe TPA-NI-NO2 is safe, low in toxicity, and can be used for intracellular fluorescence imaging, showing great potential for biological applications.

[0115] Example 12 Application of near-infrared fluorescent probe TPA-NI-NO2 in HeLa cell imaging

[0116] HeLa cells were seeded in 96-well plates in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin. HeLa cells were washed three times with HEPES buffer, pre-cultured at 37°C with a probe (working concentration up to 10 μM) for 30 minutes, then treated with 4-methoxythiophenol (MTP) (MTP concentration up to 20 μM) and cultured for another 60 minutes. Fluorescence was observed using a fluorescence spectrometer, with excitation at 470 nm and emission collected at 490–800 nm. Confocal imaging was performed using a confocal microscope (LSM700). Figure 14 As shown.

[0117] Figure 14The results showed that no obvious fluorescence signal was detected in cells after preculturing them with the near-infrared fluorescent probe TPA-NI-NO2 alone. However, after pretreating cells with TPA-NI-NO2, followed by treatment with MTP and continued culturing, orange fluorescence was observed in the cells. This indicates that the near-infrared fluorescent probe TPA-NI-NO2 can be used to detect 4-methoxybenzylthiophenol (MTP) in cells.

Claims

1. A near-infrared fluorescent probe, characterized in that, It is the compound represented by Formula I.

2. The method for preparing the near-infrared fluorescent probe according to claim 1, comprising the following steps: Step (1) involves refluxing compound NI-OH with triphenylamine 4-borate, tetra(triphenylphosphine)palladium, and potassium carbonate in an organic solvent. The compound TPA-NI-OH was prepared. Step (2): Under a protective atmosphere, the compound TPA-NI-OH obtained in step (1) is heated under reflux with 2,4-dinitrofluorobenzene and triethylamine until the reaction is complete. The near-infrared fluorescent probe was prepared.

3. The preparation method according to claim 2, characterized in that, The molar ratio of NI-OH to 4-boronic acid triphenylamine, tetra(triphenylphosphine)palladium and potassium carbonate in step (1) is 1:1.1-1.4:0.03-0.10:5-12, and the molar ratio of TPA-NI-OH to 2,4-dinitrofluorobenzene and triethylamine in step (2) is 1:1-1.5:2-4.

4. The application of the near-infrared fluorescent probe according to claim 1 or the near-infrared fluorescent probe prepared by the preparation method according to claim 2 or 3 in the preparation of reagents, test strips and kits for detecting 4-methoxythiophenol.

5. The application according to claim 4, characterized in that, The pH value of the application environment is 2-12.

6. A non-diagnostic visual fluorescence detection method for 4-methoxythiophenol, comprising the following steps: The near-infrared fluorescent probe of claim 1 is dispersed in a detection solvent with a test sample containing 4-methoxybenzylthiophenol. If the probe exhibits fluorescence activation under 365 nm ultraviolet light, i.e., a bright orange fluorescence is observed, it indicates that 4-methoxybenzylthiophenol is present in the test sample.

7. A non-diagnostic quantitative fluorescence detection method for 4-methoxythiophenol, comprising the following steps: The near-infrared fluorescent probe of claim 1 and the test sample containing 4-methoxybenzylthiophenol were dispersed in a detection solvent. The fluorescence emission intensity at 607 nm was measured under 470 nm excitation conditions. Quantitative detection was performed by the linear correlation between the concentration of 4-methoxybenzylthiophenol and the fluorescence emission intensity.

8. The non-diagnostic visual fluorescence detection method according to claim 6 or the non-diagnostic quantitative fluorescence detection method according to claim 7, characterized in that, The detection solvent is a mixture of 10 mM HEPES buffer solution with pH=7.4 and dimethyl sulfoxide, with a volume concentration of dimethyl sulfoxide of 10-20% in the mixture; the working concentration of the near-infrared fluorescent probe is 10-30 μM.

9. The non-diagnostic quantitative fluorescence detection method according to claim 7, characterized in that, The linear regression equation for quantitative detection is: Y = 15923.80548X - 274837.92549, R0 2 =0.99539; Y represents the fluorescence emission intensity at 607 nm; X represents the concentration of 4-methoxythiophenol in μM.

10. A non-diagnostic, visual fluorescent detection method for 4-methoxythiophenol in cells, comprising the following steps: The cells to be tested were cultured with a near-infrared fluorescent probe solution with a working concentration of 2.5-10 μM, and 4-methoxybenzylthiophenol was added during the culture. After culture, the fluorescence emission at 490-800 nm was observed under the excitation condition of 470 nm. If the probe showed fluorescence activation, i.e., orange fluorescence was observed, it indicated that 4-methoxybenzylthiophenol was present in the cells to be tested.

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