Viscosity fluorescent probe based on ESIPT mechanism and preparation method and application thereof
By designing a viscosity fluorescent probe based on the ESIPT mechanism, and utilizing intramolecular proton transfer and specific groups, the problems of low sensitivity and poor biocompatibility of existing probes are solved, achieving high-sensitivity viscosity detection and simple preparation, which is suitable for monitoring viscosity changes in human liver cancer cells and zebrafish.
Patent Information
- Application Number
- CN202511069300.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-11-11
AI Technical Summary
Existing fluorescent probes have low sensitivity when detecting microenvironment viscosity, making it difficult to distinguish minute viscosity changes in biological systems. They also have poor biocompatibility and complex preparation processes, which are not conducive to large-scale applications.
A viscosity-fluorescent probe based on the ESIPT mechanism was designed. The molecular structure includes a rigid planar large conjugated system, a proton donor group, and a proton acceptor group. By introducing a cyanopyridine salt, high intramolecular proton transfer efficiency is ensured. The fluorescence properties are highly sensitive to changes in microenvironment viscosity, and the preparation method is simple.
It achieves highly sensitive viscosity detection, has good biocompatibility, and can be used to detect viscosity changes in human liver cancer cells and zebrafish. It also features a simple preparation process.
Smart Images
Figure CN120923491A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescence sensing and bioimaging technology, specifically relating to a viscosity fluorescent probe based on the ESIPT mechanism, its preparation method, and its application. Background Technology
[0002] Microenvironment viscosity, as a key physical parameter in biological systems, plays a crucial regulatory role in a series of physiological processes, including intracellular substance transport, signal transduction, and enzymatic reactions. Numerous studies have shown that abnormal changes in intracellular viscosity are often closely related to the occurrence and development of various diseases, such as cancer, Alzheimer's disease, and cardiovascular diseases.
[0003] In tumor cells, intracellular viscosity typically increases significantly due to metabolic abnormalities and cytoskeleton remodeling. This viscosity change can serve as a potential biomarker for tumor diagnosis and disease monitoring. Therefore, achieving accurate, real-time, and in-situ detection of the viscosity of the microenvironment within biological systems is of great significance for a deeper understanding of the laws governing life activities, revealing the pathogenesis of diseases, and developing novel diagnostic technologies.
[0004] Currently, methods for detecting microenvironment viscosity mainly include rotational viscometry, dynamic light scattering, and fluorescence polarization. However, most of these methods have certain limitations: rotational viscometry requires sample preparation and cannot achieve in-situ detection; dynamic light scattering is limited by detection sensitivity and spatial resolution, making it difficult to apply to viscosity analysis at the single-cell level; and while traditional fluorescence polarization has a certain degree of real-time capability, the Stokes displacement of the fluorescent probe is small, making it susceptible to excitation light interference, and its sensitivity to viscosity changes needs improvement.
[0005] Cell viscosity is an important indicator of cellular health. It affects intracellular substance transport, signal transduction, interactions between macromolecules such as proteins, nucleic acids, and polysaccharides, and the diffusion of various reactive metabolites (such as reactive oxygen species (ROS) and reactive nitrogen species (RNS)) within the cell. Viscosity controls the rate of substance transfer between different substances in life processes and the transfer of substances between multiphase liquids.
[0006] Fluorescent probe technology has attracted widespread attention in the detection of microenvironments in biological systems due to its advantages such as ease of operation, high sensitivity, and good spatiotemporal resolution. Among them, fluorescent probes based on the excited-state intramolecular proton transfer (ESIPT) mechanism have become a research hotspot in recent years due to their unique properties such as large Stokes shift, tunable fluorescence emission wavelength, and good photostability.
[0007] Although some fluorescent probes based on the ESIPT mechanism have been reported for viscosity detection, existing probes still have some shortcomings: some probes have low viscosity response sensitivity, making it difficult to distinguish small viscosity changes in biological systems; some probes have poor biocompatibility, affecting the accuracy of detection results; in addition, the preparation process of some probes is complex and the synthesis steps are cumbersome, which is not conducive to their large-scale application and promotion. Summary of the Invention
[0008] The purpose of this invention is to provide a viscosity fluorescent probe based on the ESIPT mechanism, its preparation method and application, so as to solve the problems mentioned in the background art.
[0009] To achieve the above objectives, the present invention provides the following technical solution:
[0010] A viscosity-fluorescent probe based on the ESIPT mechanism has the following molecular structure: a rigid planar large conjugated system as the fluorophore, a proton donor group (hydroxyl-OH), and a proton acceptor group (a nitrogen heterocycle). This structural feature ensures efficient intramolecular proton transfer in the excited state. The molecular structure is as follows:
[0011]
[0012] A method for preparing a viscosity fluorescent probe based on the ESIPT mechanism includes the following steps.
[0013] Synthesis of S1 and Compound 1:
[0014] Take a 50 mL single-necked round-bottom flask, weigh 2.0 g of 3-methylsalicylaldehyde (14.7 mmol) and 1.6 g of 2-aminobenzylthiophenol (12.8 mmol) and add them to the flask, then add 20 mL of dry N,N-dimethylformamide to the flask.
[0015] Stir at room temperature for 5 minutes, weigh sodium metabisulfite (3.2 g, 16.8 mmol) and add it to the reaction system, and connect a spherical reflux condenser to the mouth of the reaction flask;
[0016] Subsequently, the reaction temperature was raised to 160°C using a constant temperature heating device, and the solution began to reflux. The reaction was carried out under these conditions for 2 hours. Then, the heating device was turned off, and the mixture was allowed to cool naturally to room temperature. The reaction solution was then slowly added to a small beaker containing 20 mL of distilled water, and solids gradually precipitated out.
[0017] Continue stirring for 30 minutes, filter, wash three times with 20 mL of distilled water, and dry to obtain a white solid compound 1;
[0018] Synthesis of S2 and Compound 2:
[0019] Weigh compound 1 (1.4 g, 5.81 mmol) using a balance and add it to a 50 mL round-bottom flask. Then weigh hexamethylenetetramine (1.8 g, 12.86 mmol) and add it to the reaction flask as well.
[0020] Add a magnetic stir bar, and slowly add 20 mL of trifluoroacetic acid into the reaction flask using a syringe. Stir for 5 minutes at room temperature until the reactants are dissolved.
[0021] Connect a spherical condenser to the mouth of the reaction flask and pass cooling water through it. Then heat the reaction to 75°C using a constant temperature heating device. The solution will exhibit reflux. Under these conditions, react for 36 hours. When the reaction is complete, turn off the heating device and cool to room temperature.
[0022] Prepare a dilute potassium hydroxide solution and adjust the pH of the reaction system to neutral.
[0023] As the pH of the solution changed, solids continuously precipitated out. The mixture was stirred for another 30 minutes, filtered, and dried under vacuum. The dried solids were dissolved in dichloromethane and purified by column chromatography to obtain a pale yellow solid compound 2.
[0024] Synthesis of S3 and Compound 3:
[0025] Compound 2 (100 mg, 0.37 mmol) and 4-pyridine acetonitrile (53 mg, 0.45 mmol) were added to a 25 mL round-bottom flask, and 10 mL of acetonitrile was used as the reaction solvent using a syringe.
[0026] Stir at room temperature for a while, add 2 drops of piperidine to the reaction system, and the solution color will change immediately; connect a spherical reflux condenser to the mouth of the reaction flask and pass reflux water through it, then use a constant temperature heating device to raise the temperature of the reaction solution to 80°C, and react under these conditions for 24 hours;
[0027] After the reaction was complete, the heating device was turned off and the mixture was allowed to cool naturally to room temperature, where a solid precipitated out. The solid was then filtered and washed with acetonitrile to obtain a pale yellow compound 3.
[0028] Synthesis of S4 and Compound 4:
[0029] Compound 3 (97 mg, 0.26 mmol) was added to a 25 mL reaction flask, followed by 10 mL of acetonitrile.
[0030] Stir at room temperature for 5 min, add iodomethane (0.1 mL, 1.6 mmol) using a micropipette, then attach a spherical condenser to the mouth of the reaction flask and connect it to cooling water;
[0031] The reaction was refluxed for 24 hours. After the reaction was completed, the reaction system was cooled to room temperature, and a solid precipitated out. The solid was filtered, washed three times with 5 mL of acetonitrile, and dried under vacuum to obtain a yellow solid compound 4, which was then used to obtain the product.
[0032] Preferably, the compound 1 prepared in S1 is 1.98 g with a yield of 64.3%, the compound 2 prepared in S2 is 917 mg with a yield of 58.7%, the compound 3 prepared in S3 is 61.8 mg with a yield of 45.3%, and the compound 4 prepared in S4 is 64.6 mg with a yield of 48.6%.
[0033] Applications of ESIPT-based viscosity fluorescent probes in detecting microenvironment viscosity and viscosity changes in biological systems, including cells and zebrafish.
[0034] Compared with the prior art, the beneficial effects of the present invention are:
[0035] The structural features of this invention ensure that the molecule can undergo efficient intramolecular proton transfer in the excited state. In addition, the probe molecule is designed by introducing a specific electron-withdrawing group—cyanopyridine salt—so that its fluorescence properties are highly sensitive to changes in the viscosity of the microenvironment.
[0036] The ESIPT mechanism viscosity fluorescent probe of this invention has advantages such as high sensitivity, good biocompatibility and simple preparation method, and can be used to detect viscosity changes in human hepatocellular carcinoma cells HepG2 and zebrafish. Attached Figure Description
[0037] Figure 1 This is a molecular structure diagram of the present invention;
[0038] Figure 2 This is a schematic diagram of the specific synthetic route structure of the present invention;
[0039] Figure 3 Compound 4 of the present invention 1 H NMR spectrum;
[0040] Figure 4 The fluorescence emission spectra of the ESIPT mechanism viscosity fluorescent probe of the present invention in different viscosity environments are shown.
[0041] Figure 5 The following are fluorescence emission spectra of the viscosity fluorescent probe of the ESIPT mechanism of the present invention in different solvents;
[0042] Figure 6 This is a cytotoxicity diagram of the ESIPT mechanism viscosity fluorescent probe of the present invention;
[0043] Figure 7This is a graph showing the viscosity changes in cells detected by the ESIPT mechanism of the present invention using a viscosity fluorescent probe.
[0044] Figure 8 This diagram illustrates the viscosity changes in zebrafish monitored by the ESIPT mechanism of this invention using a viscosity fluorescent probe. Detailed Implementation
[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.
[0046] Example 1:
[0047] The molecular structure of a viscosity fluorescent probe based on the ESIPT mechanism is as follows;
[0048]
[0049] The method for preparing the molecular formula includes the following steps:
[0050] The specific synthesis steps are as follows:
[0051] Synthesis of Compound 1: A 50 mL single-necked round-bottom flask was used. 3-Methylsalicylaldehyde (2.0 g, 14.7 mmol) and 2-aminothiophenol (1.6 g, 12.8 mmol) were weighed and added to the flask, followed by 20 mL of dried N,N-dimethylformamide. The mixture was stirred at room temperature for 5 min. Sodium metabisulfite (3.2 g, 16.8 mmol) was then added to the reaction system, and a spherical reflux condenser was connected to the mouth of the flask. The reaction temperature was then raised to 160 °C using a constant-temperature heating device, and the solution began to reflux. The reaction was carried out under these conditions for 2 h. Afterward, the heating device was turned off, and the mixture was allowed to cool naturally to room temperature. The reaction solution was slowly added to a small beaker containing 20 mL of distilled water, and a solid gradually precipitated. The mixture was stirred continuously for 30 min, filtered, washed three times with 20 mL of distilled water, and dried to obtain a white solid, Compound 1 (1.98 g, yield 64.3%).
[0052] Synthesis of Compound 2: Compound 1 (1.4 g, 5.81 mmol) was weighed into a 50 mL round-bottom flask using a balance, followed by hexamethylenetetramine (1.8 g, 12.86 mmol). A magnetic stir bar was added, and 20 mL of trifluoroacetic acid was slowly added to the flask using a syringe. The mixture was stirred for 5 min at room temperature until the reactants dissolved. A spherical condenser was connected to the mouth of the flask and cooled with cooling water. The reaction was then heated to 75 °C using a constant-temperature heating device until reflux occurred. The reaction was carried out for 36 h under these conditions until complete. The heating device was then turned off, and the mixture was cooled to room temperature. A dilute potassium hydroxide solution was prepared, and the pH of the reaction system was adjusted to neutral. As the pH of the solution changed, solid precipitated continuously. The mixture was stirred for another 30 min, filtered, and dried under vacuum. The dried solid was dissolved in dichloromethane, and the crude product was purified by column chromatography to obtain a pale yellow solid, Compound 2 (917 mg, yield 58.7%).
[0053] Synthesis of Compound 3: Compound 2 (100 mg, 0.37 mmol) and 4-pyridineacetonitrile (53 mg, 0.45 mmol) were added to a 25 mL round-bottom flask, and 10 mL of acetonitrile was used as the reaction solvent using a syringe. The mixture was stirred at room temperature for a short time, and then 2 drops of piperidine were added to the reaction system, causing a color change in the solution. A spherical reflux condenser was connected to the mouth of the flask and reflux water was introduced. The reaction solution was then heated to 80 °C using a constant temperature heating device and reacted under these conditions for 24 h. After the reaction was completed, the heating device was turned off, and the mixture was allowed to cool naturally to room temperature, resulting in the precipitation of a solid. The solid was filtered and washed with acetonitrile to obtain a pale yellow compound 3 (61.8 mg, yield 45.3%).
[0054] Synthesis of compound 4: Compound 3 (97 mg, 0.26 mmol) was added to a 25 mL reaction flask, followed by 10 mL of acetonitrile. The mixture was stirred at room temperature for 5 min, and iodomethane (0.1 mL, 1.6 mmol) was added using a micropipette. A spherical condenser was then attached to the mouth of the reaction flask and connected to cooling water. The mixture was heated under reflux for 24 h. After the reaction was complete, the reaction system was cooled to room temperature, and a solid precipitated. The precipitate was filtered, washed three times with 5 mL of acetonitrile, and dried under vacuum to give a yellow solid, compound 4 (64.6 mg, yield 48.6%).
[0055] The NMR data of the above compounds are as follows: 1H NMR (400MHz, DMSO-d6, ppm): δ 13.22 (s, 1H), 8.95 (d, J = 6.8Hz, 2H), 8.54 (s, 1H), 8.44 (d, J = 1.7Hz, 1H), 8.24 (d, J = 6.9Hz, 2H), 8.16 (d, J = 7.9Hz, 1H), 8.01 (d, J = 8.6Hz, 2H), 7.54 (t, J = 7.2Hz, 1H), 7.45 (t, J = 7.7Hz, 1H), 4.31 (s, 3H), 2.28 (s, 3H).
[0056] Application Example 1
[0057] Fluorescence changes of ESIPT-based viscosity fluorescent probes in different viscous environments
[0058] like Figure 4 As shown, with 526 nm as the excitation wavelength, the fluorescence emission intensity of the ESIPT mechanism-based fluorescence probe (compound 4) continuously increases at 589 nm in different viscosities in vitro as the proportion of glycerol (G) in the PBS (P) system increases (system viscosity increases). When the solvent is pure glycerol, the fluorescence intensity of the probe at 589 nm reaches its maximum value, which is 50 times higher than that in pure PBS, indicating that the probe has a good fluorescence response to viscosity.
[0059] Application Example 2
[0060] Fluorescence changes of ESIPT-mechanically-based viscosity fluorescent probes in different solvents
[0061] To investigate the influence of solvent environment on the optical properties of viscosity-fluorescent probes, this study tested the fluorescence emission spectra of an ESIPT-mechanism viscosity-fluorescent probe (compound 4) in a series of common organic solvents. The results are as follows: Figure 5 As shown, the fluorescence emission intensity of this probe exhibits a significant solvent dependence. Particularly noteworthy is the significant enhancement of fluorescence emission intensity in high-viscosity glycerol, with intensity values far exceeding those observed in other low-viscosity solvents (such as PBS, ethanol, methanol, acetonitrile, DMSO, DMF, water, and dichloromethane). This striking contrast clearly demonstrates that the ESIPT mechanism's viscosity-fluorescent probe is highly sensitive to solvent viscosity, thus exhibiting significant potential as a highly efficient viscosity sensing tool.
[0062] Application Example 3
[0063] The toxic effects of ESIPT-mediated viscosity fluorescent probes on HepG2 cells
[0064] This study evaluated the cytotoxicity of a viscosity-sensitive fluorescent probe based on the ESIPT mechanism against HepG2 cells using an MTT assay. Results are as follows: Figure 6 As shown, within the concentration range of 0 to 20 μM, the survival rate of HepG2 cells treated with this probe was higher than 90%, indicating that the probe has extremely low toxicity to cells within this concentration range, thus confirming its excellent biocompatibility.
[0065] Application Example 4
[0066] Viscosity fluorescent probes based on the ESIPT mechanism are used to detect intracellular viscosity changes.
[0067] Nystatin increases intracellular viscosity by disrupting intracellular ion balance and inducing mitochondrial dysfunction. Therefore, this experiment used nystatin as an inducer of intracellular viscosity in HepG2 cells and set up different concentrations to observe changes in the cellular adhesive environment. The results are as follows: Figure 6 As shown, the intensity of red fluorescence in the cells significantly increased with the increase of Nystatin treatment concentration, indicating that the fluorescent probe can effectively monitor the dynamic changes in intracellular viscosity.
[0068] Application Example 5
[0069] ESIPT-based viscosity fluorescent probes were used to measure viscosity changes in zebrafish.
[0070] To verify the properties of the ESIPT mechanism-based viscous fluorescent probe in vivo, this study used zebrafish for experiments. The results are as follows: Figure 7 As shown, with the increase of Nystatin treatment concentration, the red fluorescence signal in zebrafish showed a significant and dose-dependent enhancement trend, confirming that the probe can sensitively sense and visualize viscosity changes in live zebrafish.
[0071] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.
[0072] 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 limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A viscosity-fluorescent probe based on the ESIPT mechanism, characterized in that, The general molecular structure formula is: a rigid planar large conjugated system as the fluorophore, a proton donor group (hydroxyl -OH) and a proton acceptor group (nitrogen heterocycle). This structural feature ensures efficient intramolecular proton transfer in the excited state. The molecular structure formula is:
2. The method for preparing a viscosity fluorescent probe based on the ESIPT mechanism according to claim 1, characterized in that, Includes the following steps; Synthesis of S1 and Compound 1: Take a 50 mL single-necked round-bottom flask, weigh 2.0 g of 3-methylsalicylaldehyde (14.7 mmol) and 1.6 g of 2-aminobenzylthiophenol (12.8 mmol) and add them to the flask, then add 20 mL of dry N,N-dimethylformamide to the flask. Stir at room temperature for 5 minutes, weigh sodium metabisulfite (3.2 g, 16.8 mmol) and add it to the reaction system, and connect a spherical reflux condenser to the mouth of the reaction flask; Subsequently, the reaction temperature was raised to 160°C using a constant temperature heating device, and the solution began to reflux. The reaction was carried out under these conditions for 2 hours. Then, the heating device was turned off, and the mixture was allowed to cool naturally to room temperature. The reaction solution was then slowly added to a small beaker containing 20 mL of distilled water, and solids gradually precipitated out. Continue stirring for 30 minutes, filter, wash three times with 20 mL of distilled water, and dry to obtain a white solid compound 1; Synthesis of S2 and Compound 2: Weigh compound 1 (1.4 g, 5.81 mmol) using a balance and add it to a 50 mL round-bottom flask. Then weigh hexamethylenetetramine (1.8 g, 12.86 mmol) and add it to the reaction flask as well. Add a magnetic stir bar, and slowly add 20 mL of trifluoroacetic acid into the reaction flask using a syringe. Stir for 5 minutes at room temperature until the reactants are dissolved. Connect a spherical condenser to the mouth of the reaction flask and pass cooling water through it. Then heat the reaction to 75°C using a constant temperature heating device. The solution will exhibit reflux. Under these conditions, react for 36 hours. When the reaction is complete, turn off the heating device and cool to room temperature. Prepare a dilute potassium hydroxide solution and adjust the pH of the reaction system to neutral. As the pH of the solution changed, solids continuously precipitated out. The mixture was stirred for another 30 minutes, filtered, and dried under vacuum. The dried solids were dissolved in dichloromethane and purified by column chromatography to obtain a pale yellow solid compound 2. Synthesis of S3 and Compound 3: Compound 2 (100 mg, 0.37 mmol) and 4-pyridineacetonitrile (53 mg, 0.45 mmol) were added to a 25 mL round-bottom flask, and 10 mL of acetonitrile was used as the reaction solvent using a syringe. Stir at room temperature for a while, add 2 drops of piperidine to the reaction system, and the solution color will change immediately; connect a spherical reflux condenser to the mouth of the reaction flask and pass reflux water through it, then use a constant temperature heating device to raise the temperature of the reaction solution to 80°C, and react under these conditions for 24 hours; After the reaction was complete, the heating device was turned off and the mixture was allowed to cool naturally to room temperature, where a solid precipitated out. The solid was then filtered and washed with acetonitrile to obtain a pale yellow compound 3. Synthesis of S4 and Compound 4: Compound 3 (97 mg, 0.26 mmol) was added to a 25 mL reaction flask, followed by 10 mL of acetonitrile. Stir at room temperature for 5 min, add iodomethane (0.1 mL, 1.6 mmol) using a micropipette, then attach a spherical condenser to the mouth of the reaction flask and connect it to cooling water; The reaction was refluxed for 24 hours. After the reaction was completed, the reaction system was cooled to room temperature, and a solid precipitated out. The solid was filtered, washed three times with 5 mL of acetonitrile, and dried under vacuum to obtain a yellow solid compound 4, which was then used to obtain the product.
3. The method for preparing a viscosity fluorescent probe based on the ESIPT mechanism according to claim 2, characterized in that: Compound 1 prepared in S1 was 1.98 g with a yield of 64.3%; Compound 2 prepared in S2 was 917 mg with a yield of 58.7%; Compound 3 prepared in S3 was 61.8 mg with a yield of 45.3%; and Compound 4 prepared in S4 was 64.6 mg with a yield of 48.6%.
4. The application of the ESIPT-based viscosity fluorescent probe according to claim 1 in detecting microenvironment viscosity and viscosity changes in biological systems, including cells and zebrafish.