A water-soluble near-infrared aggregation-induced luminescence coumarin-quinolinyl viscosity fluorescent probe and its application

Abstract

This invention discloses a water-soluble near-infrared aggregation-induced fluorescence coumarin-quinoline-based viscosity fluorescent probe and its applications. It is synthesized via a Knoevenagel condensation reaction using 7-(N,N′-diphenylamino)-coumarin-3-carboxaldehyde and 1,2-dimethylquinoline iodide as raw materials, catalyzed by p-toluenesulfonic acid and piperidine. Its chemical structure is shown in formula (III). This preparation method features simple reaction steps, mild reaction conditions, and high yield. The fluorescent probe emits fluorescence in the near-infrared region, exhibiting advantages such as a large Stokes shift, low background interference, and minimal photodamage to biological samples. It possesses aggregation / viscosity-induced fluorescence enhancement capabilities and can be used to detect viscosity changes in solutions and cells. This provides an effective research tool for studying physiological activities in cells at different viscosities.

Description

Technical Field

[0001] This 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 emission coumarin-quinolinyl viscosity fluorescent probe and its application. Background Art

[0002] The use of fluorescent probes to identify and visualize living systems is crucial for clinical diagnosis and real-time monitoring of human vital signs. Important parameters within living systems include pH, polarity, and viscosity, all of which influence various cellular physiological functions. Viscosity is one of the most important microenvironmental parameters within living systems, affecting the transport of various nutrients and catabolites. At the organelle level, abnormalities in lysosomal or mitochondrial viscosity often foreshadow various diseases and functional disorders, such as fibrosis, inflammation, immune diseases, and tumors.

[0003] Given the complexity and dynamic nature of living systems, ideal fluorescent dyes for use in living systems should minimize background noise and amplify the signal when a specific target is detected. However, traditional fluorescent dyes typically consist of fluorophores with large π-π conjugated systems (e.g., coumarin, quinoline). Fused ring structures often lead to water solubility crossover and are prone to J- or H-aggregation. Strong π-π stacking can cause spectral redshift or blueshift, and also aggregation-induced quenching (ACQ) effects, often resulting in signal reduction or even disappearance. Aggregation-induced emission (AIE) is the opposite of ACQ. AIE luminescent organisms (AIEgens) typically have a pronounced twisted propeller-like conformation or rotatable structure. These structures restrict intramolecular motion and reduce intermolecular π-π stacking in the aggregated state. This strategy modulates energy dissipation in the excited state, ultimately emitting a brighter fluorescence signal. A significant characteristic of AIEgens is that their fluorescence intensity is significantly positively correlated with the degree of aggregation, making them suitable for viscosity measurements. It is well known that viscous media cause restricted intramolecular motion (RIM), and polar solvents induce the aggregation of hydrophobic solutes. Both of these effects activate the intramolecular rotational / vibrational confinement (RIR / RIV) processes of AIEgens, thereby blocking their nonradiative decay channels and opening their fluorescence emission in aggregated systems. Viscosity and polarity increase with increasing glycerol volume, enabling viscosity quantification.

[0004] Over the past decade, chemists have witnessed tremendous progress in the application of coumarin-based fluorescent dyes to multifunctional imaging. However, most coumarin-based fluorescent dyes (especially natural coumarins and their derivatives) absorb or emit fluorescence signals in the visible light region. The poor penetration depth of these molecules greatly limits their imaging applications in biological tissues or within the body. Near-infrared (NIR) fluorescent probes are superior to conventional dyes with shorter emission wavelengths (e.g., low tissue light damage, strong biocompatibility, and high imaging resolution). Researchers have primarily focused on improving the photophysical properties (UV-Vis absorption and fluorescence emission spectra, molar extinction coefficient, fluorescence quantum efficiency, and Stokes shift, etc.) and biological activities (water solubility, targeting, and physiological toxicity, etc.) of coumarins by modulating the donor-π-acceptor (D-π-A) effect, extending the π-π conjugated system, and enhancing rigidity and planarity. However, it is difficult to achieve optimal performance in all these aspects using only a single fluorophore, thus limiting its widespread application. To obtain ideal NIR photophysical properties, integrating two fluorophores into a single framework is a better regulatory strategy. The quinoline ring system is one of the most promising N-heterocyclic nuclei, found in various natural products, especially alkaloids. The cation-containing quinoline group significantly influences the overall spectral and biological properties of the luminescent group, such as photostability, water solubility, emission efficiency, and biological characteristics. Due to differences in electrochemical potential across mitochondrial or lysosomal membranes, cationic compounds can accumulate in specific organelles according to the Nernst equation. Given the advantages of quinoline and coumarin fluorescence, the introduction of combinatorial chemistry can further accelerate the efficient synthesis of candidate dyes, promoting further research on near-infrared viscosity fluorescent probes of coumarin-quinoline binary compounds.

[0005] This invention synthesizes a water-soluble, near-infrared aggregation-induced emission (AIE) coumarin-quinoline-based viscosity-fluorescent probe in one step via a Knoevenagel condensation reaction. The introduction of cations significantly improves the poor water solubility and biocompatibility issues caused by the fused-ring structures of coumarin and quinoline. The triphenylamine group with a twisted propeller-like conformation enhances the compound's AIE / viscosity-induced fluorescence enhancement. The coumarin-quinoline (D-π-A) structure leads to strong intramolecular charge transfer, redshifting the spectrum to the near-infrared region. Simultaneously, the strong distortion of the macromolecular structure between the ground and excited states results in a significant increase in the Stokes shift. This near-infrared fluorescent probe exhibits strong specificity and sensitivity to viscosity, and is expected to provide precise signals when abnormal conditions occur in vivo, enabling rapid detection of disease occurrence. It holds potential application prospects in disease prevention, diagnosis, and treatment. Summary of the Invention

[0006] Purpose of the invention: To address the shortcomings of existing technologies, the purpose of this invention is to provide a water-soluble near-infrared aggregation-induced emission coumarin-quinolinyl viscosity fluorescent probe and its applications.

[0007] Technical solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0008] This invention relates to a water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscositoid fluorescent probe and its application, characterized in that the probe has the structural formula shown in formula (III):

[0009]

[0010] A water-soluble near-infrared aggregation-induced emission coumarin-quinolinyl viscosity fluorescent probe and its application are described below:

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

[0012]

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

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

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

[0016] Beneficial effects of the present invention

[0017] Compared with the prior art, the advantages of the water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe of the present invention and its application are as follows: (1) The preparation method is simple and easy, with few synthesis steps, simple reaction conditions, and high 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 degree of aggregation increases, the fluorescent probe will exhibit J-aggregation phenomenon, and the fluorescence intensity at 688 nm gradually increases, which can effectively avoid background interference; (4) As the viscosity of the system increases, the fluorescence intensity of the fluorescent probe (III) gradually increases, and there is a good linear relationship between the fluorescence intensity and the logarithm of the system viscosity (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. Attached Figure Description

[0018] Figure 1 The UV-Vis absorption spectra of a water-soluble near-infrared aggregation-induced emission coumarin-quinolinyl viscosity fluorescent probe (III) solution (10 μM) in chloroform / methanol solutions of different ratios are shown.

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

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

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

[0022] Figure 5These are confocal fluorescence images of HCT-116 cells under different treatment conditions. Control cells, monensin, nystatin, and lipopolysaccharide (LPS)-induced HCT-116 cells were incubated in the presence of a water-soluble near-infrared aggregation-induced luminescence coumarin-quinolinyl viscosity fluorescent probe (III) solution (10 μM) to detect cell viscosity. The excitation wavelength was λ. ex =520nm; Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings.

[0024] use 1 H-NMR, 13 The structure of the water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe (III) was characterized and confirmed by C-NMR and ESI-HRMS. Spectroscopic properties were determined using UV-Vis spectroscopy and fluorescence emission spectroscopy. The instruments used for the analysis were: a Bruker ARX 400 NMR spectrometer (deuterated dimethyl sulfoxide as solvent), a Leica SP8 laser confocal microscope, a NeXion-300X inductively coupled plasma mass spectrometer, a Shimadzu UV-3100 UV-Vis spectrophotometer, and an LS-55 fluorescence spectrophotometer (all with a slit width of 2.5 mm).

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

[0026] Under anhydrous conditions, in a dry round-bottom flask equipped with a Dean-Stark apparatus, 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) were dissolved in 25 mL of toluene and 0.5 mL of piperidine, and heated to 60 °C. The reaction was monitored 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 purified by silica gel column chromatography using 100% dichloromethane as the eluent to obtain a reddish-brown solid product, a water-soluble near-infrared aggregation-induced luminescence coumarin-quinoline 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 a water-soluble near-infrared aggregation-induced luminescence coumarin-quinolinyl viscosity fluorescent probe (III)

[0028] Similar to Example 1, except that in this example, 7-(N,N′-diphenylamino)-coumarin-3-carboxaldehyde reacts with 1,2-dimethylquinoline iodide at 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 a water-soluble near-infrared aggregation-induced luminescence coumarin-quinolinyl viscosity fluorescent probe (III)

[0030] Similar to Example 1, except that in this example, 7-(N,N′-diphenylamino)-coumarin-3-carboxaldehyde reacts 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: UV-Vis absorption spectra of a water-soluble near-infrared aggregation-induced luminescence coumarin-quinolinyl viscosity fluorescent probe (10 μM) in chloroform / methanol solutions of 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 probe (III) was added to chloroform / methanol mixed solutions (4 mL) of different volume ratios to obtain 10 μM probe (III) solutions with different chloroform / methanol ratios, and their UV-Vis absorption spectra were measured. With increasing chloroform solution ratio, the absorbance at 520 nm gradually decreased, while the absorbance at 566 nm gradually increased, indicating that probe (III) possesses J-aggregation ability in chloroform / methanol solvent. Figure 1 The UV-Vis absorption spectra of the water-soluble near-infrared aggregation-induced emission coumarin-quinolinyl viscosity fluorescent probe (III) (10 μM) in chloroform / methanol solutions of different ratios are shown.

[0033] Example 5: Fluorescence emission spectra of a water-soluble near-infrared aggregation-induced luminescence coumarin-quinolinyl viscosity fluorescent probe (10 μM) in chloroform / methanol solutions of different ratios.

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

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

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

[0037] Example 7: Determination of the linear range of fluorescence intensity as a function of viscosity for a water-soluble near-infrared aggregation-induced luminescence coumarin-quinolinyl viscosity fluorescent probe (III) (10 μM).

[0038] The relationship between the fluorescence intensity at 660 nm of the water-soluble near-infrared aggregation-induced emission coumarin-quinolinyl viscosity fluorescent probe (III) and the logarithm of the system viscosity was analyzed. ex =520nm), the probe's response to viscosity was examined; it was shown that probe (III) exhibits good linearity within a certain viscosity range, with the linearity being log(F) 660 F = 0.3868 * log(η) + 1.5817 660 The fluorescence intensity of probe (III) at 660 nm is represented by η, and the viscosity of the probe (III) solution system is represented by η; the square of the linear correlation coefficient is R. 2 =0.9959, Figure 4 This is a graph showing the linear relationship between the fluorescence intensity of the water-soluble near-infrared aggregation-induced emission coumarin-quinolinyl 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 with water-soluble near-infrared aggregation-induced luminescence coumarin-quinolinyl 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. Cells were divided into four groups: negative control, Monensin, Nystatin, and LPS. The negative control, Monensin, Nystatin, and LPS groups were treated with physiological saline, 10 ng / mL Monensin, 10 ng / mL Nystatin, and 10 μM LPS, respectively. After treatment, all cells were incubated with 10 μM probe (III) for 30 minutes, followed by washing three times with 1.0 mL PBS. Finally, high-resolution fluorescence confocal microscopy was used to examine the cells, and Leica software was used for image analysis of the confocal fluorescence imaging. The fluorescent dyes penetrated the HCT-116 cells clearly, demonstrating good biocompatibility and cell permeability. Confocal fluorescence imaging showed changes in cell morphology, increased viscosity, and increased fluorescence signal after induction by different drugs. 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-quinolinyl viscosity fluorescent probe (III), characterized in that, Its structural formula is shown in equation (III) below:

2. The preparation method of the water-soluble near-infrared aggregation-induced emission coumarin-quinolinyl viscosity fluorescent probe (III) according to claim 1, characterized in that, The process is achieved through 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, followed by the addition of p-toluenesulfonic acid (PTSA) and piperidine. The mixture is stirred and heated at 50-80°C for 2-5 hours. The reaction is then stopped, cooled to room temperature, extracted and separated with dichloromethane, dried over anhydrous sodium sulfate to remove the organic solvent, and purified by silica gel column chromatography to obtain a water-soluble near-infrared aggregation-induced emission coumarin-quinoline viscosity fluorescent probe (III). The chemical reaction formula for this preparation process is as follows:

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

5. The application of the water-soluble near-infrared aggregation-induced emission coumarin-quinolinyl viscosity fluorescent probe (III) according to claim 1 in the preparation of viscosity detection reagents.

6. The application according to claim 5, characterized in that, The viscosity was measured as changes in the viscosity of HCT-116 cells induced by Monensin, Nystatin, or lipopolysaccharide (LPS).

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