Water-soluble near-infrared aggregation-induced emission coumarin-quinolyl viscosity fluorescent probe and application thereof

Abstract

The invention discloses a water-soluble near-infrared aggregation-induced emission coumarin-quinolyl viscosity fluorescent probe and application thereof, and the water-soluble near-infrared aggregation-induced emission coumarin-quinolyl viscosity fluorescent probe is realized by the following method: carrying out Knoevenagel condensation reaction on a 7-(N, N '-diphenylamino)-coumarin-3-formaldehyde compound and 1, 2-dimethyl quinoline iodized salt as raw materials under the catalytic action of p-toluenesulfonic acid and piperidine to synthesize the water-soluble near-infrared aggregation-induced emission coumarin-quinolyl viscosity fluorescent probe. The chemical structural formula is shown as a formula (III). The preparation method has the advantages of simple reaction steps, mild reaction conditions and high yield. The fluorescent probe emits fluorescence in a near-infrared region, has the advantages of large Stokes shift, low background interference, small light damage to biological samples and the like, has aggregation / viscosity-induced fluorescence enhancement capability, and can be used for detecting viscosity changes in solutions and cells. And an effective research tool is provided for researching physiological activities under different viscosities in cells.

Description

Technical Field

[0001] The invention belongs to the fields of organic compound synthesis, functional probes and fine chemicals, and specifically relates to a water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe and an application thereof. Background Art

[0002] The use of fluorescent probes to identify and visualize living systems is of vital importance in clinical diagnosis and real-time monitoring of human vital signs. Important parameters in living systems include pH, polarity, and viscosity, all of which affect various physiological functions of cells. Among them, viscosity is one of the most important microenvironmental parameters in living systems, affecting the transport process of various nutrients and decomposition metabolites in living bodies. At the organelle level, abnormalities in lysosomal or mitochondrial viscosity often herald the occurrence of various diseases and dysfunctions, such as fibrosis, inflammation, immune diseases, and tumors.

[0003] Given the complexity and dynamic nature of living systems, ideal fluorescent dyes for living systems should minimize background noise and amplify signals when specific targets are detected. However, traditional fluorescent dyes are usually composed of fluorophores with large π-π conjugated systems (e.g., coumarin, quinoline), and the fused ring structures often lead to water-soluble crosstalk and are more susceptible to J-aggregation or H-aggregation, where strong π-π stacking can lead to red-shift or blue-shift of the spectrum, and also lead to aggregation-induced quenching (ACQ) effect, which often reduces or even eliminates the signal. The aggregation-induced emission (AIE) effect is the opposite of the ACQ effect. AIE luminogens (AIEgens) usually have obvious twisted propeller-like conformations or rotatable structures. These structures restrict intramolecular motion and reduce intermolecular π-π stacking in the aggregated state. This strategy regulates energy dissipation in the excited state and ultimately emits brighter fluorescence signals. A notable feature of AIEgens is that their fluorescence intensity is significantly positively correlated with the degree of aggregation, making them suitable for viscosity measurement. It is well known that viscous media can cause restricted intramolecular motion (RIM) and polar solvents can induce aggregation of hydrophobic solutes. Both effects activate the restricted intramolecular rotation / vibration (RIR / RIV) process of AIEgens, thereby blocking their nonradiative decay channels and turning on their fluorescence emission in aggregated systems. The viscosity and polarity increase with increasing glycerol volume, enabling viscosity quantification.

[0004] In the past decade, chemists have witnessed great progress in the use of coumarin-based fluorescent dyes for multifunctional imaging. However, most coumarin-based fluorescent dyes (especially natural coumarins and their derivatives) absorb or emit fluorescence signals in the visible light region. These molecules have poor penetration depth, which greatly limits their imaging applications in biological tissues or in vivo. Near-infrared (NIR) fluorescent probes have advantages over conventional dyes with shorter emission wavelengths (e.g., low tissue light damage, strong biocompatibility, and high imaging resolution). Researchers have mainly improved the photophysical properties (UV-visible absorption and fluorescence emission spectra, molar extinction coefficient, fluorescence quantum efficiency, and Stokes shift, etc.) and bioactivity (water solubility, targeting, and physiological toxicity, etc.) of coumarins by regulating the donor-π-acceptor (D-π-A) effect, extending the π-π conjugated system, and enhancing rigidity and planarity. However, it is difficult to have optimal performance in all these aspects with only a single fluorescent group, so it cannot be widely used. In order to obtain ideal NIR photophysical properties, integrating two fluorophores into one skeleton is a better regulatory strategy. The quinoline ring system is one of the most promising N-heterocyclic nuclei and is present in various natural products, especially alkaloids. The quinoline group with cations has a great influence on the overall spectral and biological properties of the luminophore, such as photostability, water solubility, emission efficiency, biological properties, etc. Due to the difference in electrochemical potential across the inner membrane of mitochondria or lysosomes, cationic compounds can accumulate in specific organelles according to the Nernst equation. In view of the advantages of quinoline and coumarin fluorescence, the introduction of combinatorial chemistry can further accelerate the efficient synthesis of candidate dyes and promote the further development of coumarin-quinoline binary compounds as near-infrared viscosity fluorescent probes.

[0005] The present invention synthesizes a water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe in one step through a Knoevenagel condensation reaction. The poor water solubility and biocompatibility caused by the coumarin and quinoline fused ring structure are greatly improved by the introduction of cations. The triphenylamine group with a twisted propeller-shaped conformation enhances the aggregation / viscosity-induced fluorescence enhancement ability of the compound. The coumarin-quinoline (D-π-A) structure causes strong intramolecular charge transfer to red-shift the spectrum to the near-infrared region. At the same time, the distortion of the macromolecular structure of the strong ground state-excited state causes the Stokes shift to be significantly increased. The near-infrared fluorescent probe has strong specificity and sensitivity to viscosity, is expected to give an accurate signal when an abnormal condition occurs in a living body, and senses the occurrence of a disease as soon as possible, and has potential application prospects in the prevention, diagnosis and treatment of diseases. Summary of the invention

[0006] Purpose of the invention: In view of the deficiencies in the prior art, the purpose of the present invention is to provide a water-soluble near-infrared aggregation-induced emission coumarin-quinoline-based viscosity fluorescent probe and its application.

[0007] Technical solution: In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0008] The present invention provides a water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe and its application, characterized in that the probe has a structural formula shown in the following formula (III):

[0009]

[0010] A water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe and its application, comprising the following steps:

[0011] Under anhydrous conditions, 7-(N,N′-diphenylamino)-coumarin-3-carboxaldehyde (I) and 1,2-dimethylquinoline iodide (II) are added to a dry toluene solution, and then p-toluenesulfonic acid (PTSA) and piperidine are added, stirred and heated, the temperature is controlled at 50-80°C, the reaction time is 2-5 hours, the reaction is terminated, cooled to room temperature, diluted with dichloromethane, extracted, washed with water, separated, dried over anhydrous sodium sulfate, and after removing the organic solvent, a silica gel column is used for chromatography separation and purification to obtain a water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (III). The specific chemical reaction formula is as follows:

[0012]

[0013] In the above step (1), the molar ratio of 7-(N,N′-diphenylamino)-coumarin-3-carboxaldehyde (I) to 1,2-dimethylquinoline iodide (II) is 1:1.0-2.0.

[0014] In the above step (1), the volume ratio of toluene and piperidine added is 50:1.

[0015] In the above step (1), the catalyst is p-toluenesulfonic acid (PTSA) and piperidine.

[0016] Beneficial effects of the present invention

[0017] Compared with the prior art, the water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe and its application of the present invention have the following advantages: (1) the preparation method is simple and easy, with fewer synthesis steps, simple reaction conditions and higher yield; (2) the maximum fluorescence emission wavelength of the fluorescent probe is 688 nm, and the Stokes shift is 168 nm, which can effectively enhance the imaging sensitivity; (3) as the aggregation degree of the fluorescent probe increases, J-aggregation phenomenon will occur, and the fluorescence intensity at 688 nm will gradually increase, which can effectively avoid background interference; (4) as the viscosity of the system increases, the fluorescence intensity of the fluorescent probe (III) will gradually increase, and there is a good linear relationship between the fluorescence intensity and the logarithm of the viscosity of the system (log(F 660 )=0.3868*log(η)+1.5817,R 2 =0.9959), which can realize the quantitative detection of viscosity; (5) It has good cell permeability and biocompatibility and can be used for fluorescence imaging of cells with different viscosities. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the UV-visible absorption spectra of water-soluble near-infrared aggregation-induced emission coumarin-quinoline-based viscosity fluorescent probe (III) solution (10 μM) in chloroform / methanol solutions with different ratios;

[0019] Figure 2 The fluorescence emission spectra of water-soluble near-infrared aggregation-induced emission coumarin-quinoline-based viscosity fluorescent probe (III) solution (10 μM) in chloroform / methanol solutions with different ratios. Excitation wavelength λ ex =520nm;

[0020] Figure 3 The fluorescence emission spectra of water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe (III) solution (10 μM) in different ratios of glycerol / PBS aqueous solution; the excitation wavelength λ ex =520nm;

[0021] Figure 4 It is a linear relationship between the logarithm of the fluorescence intensity of the water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (III) solution (10 μM) at 660 nm and the logarithm of the system viscosity; the excitation wavelength λ ex =520nm;

[0022] Figure 5Figure 2 is a confocal fluorescence image of HCT-116 cells under different treatment conditions. Control cells, monensin, nystatin, and LPS-induced HCT-116 cells were incubated in the presence of a water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (III) solution (10 μM) to detect cell viscosity. The excitation wavelength λ ex =520nm; DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with the specific drawings.

[0024] use 1 H-NMR, 13 C-NMR and ESI-HRMS characterized and confirmed the structure of water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe (III). The spectral properties were determined by UV-Vis spectrum and fluorescence emission spectrum; the instruments used for detection were: Bruker ARX 400 nuclear magnetic resonance instrument (deuterated dimethyl sulfoxide as solvent), German Leica SP8 laser confocal microscope, NeXion-300X inductively coupled plasma mass spectrometer, Shimadzu UV-3100 ultraviolet-visible spectrophotometer, American LS-55 fluorescence spectrophotometer (the slits were all 2.5 mm).

[0025] Example 1 Preparation of water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (III)

[0026] Under anhydrous conditions, a dry round-bottom flask equipped with a Dean-Stark apparatus was used to dissolve 7-(N, N′-diphenylamino)-coumarin-3-carboxaldehyde (170 mg, 0.5 mmol), 1,2-dimethylquinoline iodide (171 mg, 0.6 mmol) and p-toluenesulfonic acid (26 mg, 0.15 mmol) in 25 mL of toluene and 0.5 mL of piperidine and heated to 60°C. The reaction was detected by TLC. After 3 hours of reaction, the 7-(N, N′-diphenylamino)-coumarin-3-carboxaldehyde compound completely disappeared and the reaction was stopped. The mixture was cooled to room temperature, diluted with dichloromethane, washed with water, separated, dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure. The residue was separated and purified by silica gel column chromatography, and the eluent was 100% dichloromethane to obtain a reddish brown solid product, a water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (III) (72 mg, 30%). 1H NMR (400MHz, DMSO-d6, ppm) δ = 9.07 (d, J = 8.8Hz, 1H), 8.62-8.49 (m, 3H), 8.41-8.34 (m, 1H), 8.26-8.15 (m, 2H), 8.07-7.91 (m, 2H), 7.67 (d, J=8.8Hz, 1H), 7.50 (t, J=7.6Hz, 4H), 7.33 (dd, J=9.6, 8.4Hz, 6H), 6.80 (dd, J=8.8, 2.4Hz, 1H), 6.54 (d, J=2.0Hz, 1H), 4.50 (s, 3H). 13 C NMR (100MHz, DMSO-d6, ppm) δ = 159.709, 156.506, 155.852, 153.425, 146.697, 145.216, 144.644, 141.746, 139.678, 135.491, 131.445, 130 .742, 130.585, 129.497, 128.290, 127.409, 126.928, 121.437, 120.666, 119.743, 117.062, 115.769, 112.302, 103.267, 55.395.ESI-HRMS calculated forC 33 H 25 N2O2 + :481.1911; found: 481.2620( Figure 1 ).

[0027] Example 2 Preparation of water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (III)

[0028] Similar to Example 1, the difference is that in this example, 7-(N,N′-diphenylamino)-coumarin-3-carboxaldehyde and 1,2-dimethylquinoline iodide are reacted in a molar ratio of 1:1.5; the reaction temperature is controlled at 70°C, the reaction time is 4 hours, and the yield is 55%.

[0029] Example 3 Preparation of water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (III)

[0030] Similar to Example 1, the difference is that in this example, 7-(N,N′-diphenylamino)-coumarin-3-carboxaldehyde is reacted with 1,2-dimethylquinoline iodide and p-toluenesulfonic acid in a molar ratio of 1:2:0.25; the reaction temperature is controlled at 60°C, the reaction time is 5 hours, and the yield is 47%.

[0031] Example 4 Changes in UV-Vis absorption spectra of water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (10 μM) in chloroform / methanol solutions with different ratios

[0032] The probe (III) prepared in Example 1 was dissolved in a methanol solution to prepare a 3 mmol / L stock solution. The stock solution (3 mM) of the probe (III) was added to a chloroform / methanol mixed solution (4 mL) of different volume ratios to obtain 10 μM probe (III) solutions of different chloroform / methanol ratios, and their UV-visible absorption spectra were measured. As the proportion of the chloroform solution increased, the absorbance at 520 nm gradually decreased, and the absorbance at 566 nm gradually increased, indicating that the probe (III) had J-aggregation ability in the chloroform / methanol solvent; Figure 1 The figure shows the UV-visible absorption spectra of water-soluble near-infrared aggregation-induced emission coumarin-quinoline-based viscosity fluorescent probe (III) (10 μM) in chloroform / methanol solutions with different ratios.

[0033] Example 5 Fluorescence emission spectrum changes of water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline viscosity fluorescent probe (10 μM) in chloroform / methanol solutions with different ratios

[0034] The 10 μM probe (III) solution in different chloroform / methanol ratios prepared in Example 4 was taken and its fluorescence emission spectrum was measured. As the ratio of the chloroform solution increased, the fluorescence intensity at 688 nm gradually increased, indicating that the probe (III) has aggregation-induced fluorescence enhancement ability in the chloroform / methanol solvent; Figure 2 The fluorescence emission spectra of the water-soluble near-infrared aggregation-induced emission coumarin-quinoline-based viscosity fluorescent probe (III) (10 μM) in chloroform / methanol solutions with different ratios are shown.

[0035] Example 6 Fluorescence emission spectra of water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline viscosity fluorescent probe (III) (10 μM) in glycerol / PBS aqueous solutions with different ratios

[0036] The probe (III) prepared in Example 1 was dissolved in a dimethyl sulfoxide solution to prepare a 3 mmol / L stock solution. The stock solution (3 mM) of the probe (III) was added to glycerol / PBS aqueous solution (4 mL) of different volume ratios to obtain 10 μM probe (III) solutions of different glycerol / PBS aqueous solution ratios, and the fluorescence emission spectra thereof were measured; as the ratio of the glycerol solution increased, the fluorescence at 660 nm gradually increased, indicating that the probe (III) had the ability of viscosity-induced fluorescence enhancement in the glycerol / PBS aqueous solution. Figure 3It is the fluorescence emission spectra of water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe (III) (10 μM) in glycerol / PBS aqueous solutions with different ratios.

[0037] Example 7 Determination of the linear range of fluorescence intensity of water-soluble near-infrared aggregation-induced emission coumarin-quinoline-based viscosity fluorescent probe (III) (10 μM) with viscosity change

[0038] By analyzing the relationship between the fluorescence intensity of the water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe (III) at 660 nm and the logarithm of the system viscosity at different viscosities (λ ex =520nm), and the response ability of the probe to viscosity was investigated; it showed that the probe (III) had a good linear relationship within a certain viscosity range, and the linear relationship was log(F 660 )=0.3868*log(η)+1.5817,F 660 represents the fluorescence intensity of probe (III) at 660 nm, η represents the viscosity of the probe (III) solution system; the square of the linear correlation coefficient is R 2 =0.9959, Figure 4 It is a linear relationship diagram between the fluorescence intensity of the water-soluble near-infrared aggregation-induced emission coumarin-quinoline-based viscosity fluorescent probe (III) (10 μM) at 660 nm and the logarithm of the system viscosity.

[0039] Example 8 Confocal fluorescence imaging of HCT-116 cells incubated in the presence of a water-soluble near-infrared aggregation-induced emission coumarin-quinoline-based viscosity fluorescent probe (III) (10 μM) under different viscosity conditions

[0040] Human colon cancer cells (HCT-116) were cultured in DMEM medium containing D-glucose and fetal bovine serum. The cells were divided into four groups: negative control group, Monensin group, Nystatin group and LPS group. The negative control group, Monensin group, Nystatin group and LPS group were treated with normal saline, 10ng / mL Monensin, Nystatin and LPS, respectively. After treatment, the cells in all groups were incubated with 10μM probe (III) for 30 minutes and then washed 3 times with 1.0mL PBS aqueous solution. Finally, the cells were examined using a high-resolution fluorescence confocal microscope, and the confocal fluorescence imaging was analyzed using Leica application software. The fluorescent dye entered the HCT-116 cells and was clearly imaged, with good biocompatibility and cell permeability. Confocal fluorescence imaging showed that after different drug induction, the cell morphology changed, the viscosity increased, and the fluorescence signal increased. Figure 5These are confocal fluorescence images of HCT-116 cells under different treatment conditions.

Claims

1. A water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe (III), characterized in that: Its structural formula is shown in the following formula (III):

2. The water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe (iii) according to claim 1, characterized in that: The method is achieved by the following steps: under anhydrous conditions, 7-(N,N′-diphenylamino)-coumarin-3-carboxaldehyde (I) and 1,2-dimethylquinoline iodide (II) are added to a dry toluene solution, p-toluenesulfonic acid (PTSA) and piperidine are added, stirred and heated, the temperature is controlled at 50-80° C., the reaction time is 2-5 hours, the reaction is stopped, cooled to room temperature, extracted with dichloromethane, separated, dried with anhydrous sodium sulfate, after removing the organic solvent, chromatographically separated and purified using a silica gel column, and a water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (III) is obtained. The chemical reaction formula of the preparation process is as follows:

3. The method for preparing a water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe according to claim 2, characterized in that: The molar ratio of 7-(N,N-diphenylamino)-coumarin-3-carboxaldehyde (I) to 1,2-dimethylquinoline iodide (II) is 1 mol:1.0-2.0 mol.

4. The method for preparing a water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe according to claim 2, characterized in that: The volume to amount ratio of toluene, piperidine and 7-(N,N′-diphenylamino)-coumarin-3-carboxaldehyde (I) is 50 mL:1 mL:1 mmol.

5. The water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline-based viscosity fluorescent probe (III) according to claim 1 has aggregation-induced fluorescence enhancement capability in chloroform / methanol solution.

6. The water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe (iii) according to claim 1 has the ability of viscosity-induced fluorescence enhancement in a glycerol / PBS aqueous solution.

7. Use of the water-soluble near-infrared aggregation-induced emission coumarin-quinoline-based viscosity fluorescent probe (III) according to claim 1 in viscosity detection.

8. The use according to claim 7, characterized in that: The fluorescent probe can detect the viscosity changes of HCT-116 cells induced by monensin, nystatin and lipopolysaccharide (LPS).

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