Viscosity-sensitive fluorescent probe, and preparation method and use method thereof

Abstract

The application discloses a viscosity-sensitive fluorescent probe and a preparation method and use method thereof. The probe is (E)-2-(3-(2-(1H-imidazole-5-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (DCI-V7). The probe is based on a twisted intramolecular charge transfer (TICT) mechanism, has weak fluorescence in a low-viscosity environment, and has significantly enhanced fluorescence in a high-viscosity environment due to hindered molecular rotation, and presents an "on" response. The probe has simple synthesis steps, high sensitivity, good selectivity and a large Stokes shift to viscosity, and can realize quantitative detection of sample viscosity through a linear relationship between fluorescence intensity and viscosity, and has application prospects in biological microenvironment analysis and pathological mechanism research.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to a method for preparing and using (E)-2-(3-(2-(1H-imidazol-5-yl)vinyl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile as a viscosity fluorescent probe. Background Technology

[0002] Intracellular viscosity, as a key parameter of the microenvironment, significantly influences various dynamic processes, including signal transduction, macromolecular interactions, metabolic processes, and apoptosis. Cells regulate viscosity to maintain the stability of their complex internal systems. As part of stress response and homeostasis regulation mechanisms, abnormal fluctuations in viscosity not only disrupt the dynamic equilibrium of small molecules within the cell that depend on viscosity diffusion but also induce disturbances in other microenvironmental factors (such as pH and polarity). More seriously, significant viscosity abnormalities can lead to organ dysfunction, thereby triggering various diseases such as inflammation, fatty liver, Alzheimer's disease, and Parkinson's syndrome. Therefore, developing a highly sensitive, non-invasive imaging tool is of paramount scientific and clinical value for deeply elucidating the mechanisms of viscosity-related diseases, clarifying the function of viscosity in biology and pathology, and achieving real-time in-situ imaging of intracellular viscosity.

[0003] To date, the main tools available for viscosity measurement include falling ball viscometers, rotational viscometers, and capillary viscometers. These traditional instruments provide reliable viscosity data at the macroscopic scale and are widely used in materials science and industry. However, these methods are inherently macroscopic measurements and are difficult to implement in-situ, real-time monitoring of the microscopic environment, such as cells and subcellular organelles. With the deepening of life science research, it has become increasingly clear that intracellular viscosity, as a key parameter of the microenvironment, plays a crucial role in dynamic processes such as signal transduction, substance transport, metabolic regulation, and even apoptosis. Therefore, developing imaging tools capable of real-time, in-situ monitoring of microviscosity changes in living cells has become an important research direction in the field of biomedical imaging (see review: MM Sreejaya, Vineeth M Pillai, Ayesha A, Maanas Baby, Manoranjan Bera, and Moumita Gangopadhyay, Mechanistic analysis of viscosity-sensitive fluorescent probes for applications in diabetes detection. J. Mater. Chem. B, 2024, 12, 2917-2937). https: / / doi.org / 10.1039 / D3TB02697C .). Summary of the Invention

[0004] The present invention aims to provide an "on" type viscosity fluorescent probe based on the twisted intramolecular charge transfer (TICT) mechanism, which has a large Stokes shift, high sensitivity and good selectivity.

[0005] In a first aspect, the present invention provides a fluorescent probe for detecting viscosity, which is (E)-2-(3-(2-(1H-imidazol-5-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-yl)malonadionitrile (abbreviated as DCI-V7), with the structure shown in formula (I):

[0006]

[0007] Preferably, the fluorescent probe is an orange solid powder.

[0008] The working principle of the probe in this invention is as follows: the compound has a donor-π-acceptor (D-π-A) structure, wherein isophorone-malononitrile is the acceptor, imidazole is the donor, and vinyl group is the π-bridge. In a low-viscosity environment, the molecule can rotate freely in the excited state, rapidly dissipating energy through nonradiative relaxation (twisted intramolecular charge transfer, TICT), resulting in weak fluorescence. When the ambient viscosity increases, molecular rotation is hindered, the TICT process is suppressed, the nonradiative pathway is reduced, and the fluorescence is significantly enhanced, thereby achieving "on" detection of viscosity.

[0009] In a second aspect, the present invention provides a method for preparing the fluorescent probe, the synthetic route of which is as follows:

[0010]

[0011] The process includes the following steps: Under nitrogen protection, 2-(3,5,5-trimethylcyclohex-2-en-1-yl)malononitrile, 4-imidazolium carboxaldehyde and piperidine are dissolved in acetonitrile and reacted at 0-50°C for 1-24 hours. An orange solid is precipitated during the reaction, and the fluorescent probe DCI-V7 is obtained after separation and purification.

[0012] Preferably, the molar ratio of 2-(3,5,5-trimethylcyclohexyl-2-ene-1-yl)malonitrile, 4-imidazolium formaldehyde, and piperidine is 1:1 to 1.2:0.2 to 1; the amount of acetonitrile used is 1 to 10 mL per 0.54 mmol of 2-(3,5,5-trimethylcyclohexyl-2-ene-1-yl)malonitrile.

[0013] More preferably, the molar ratio of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile, 4-imidazolium carboxaldehyde, and piperidine is 1:1.2:0.2; the amount of acetonitrile used is 3 mL per 0.54 mmol of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile; the reaction temperature is 50°C, and the reaction time is 4 hours.

[0014] A third aspect of the present invention provides a method for using the fluorescent probe to detect sample viscosity for non-diagnostic purposes, thereby achieving quantitative detection by establishing a standard curve of fluorescence intensity versus viscosity.

[0015] The usage method of the above-mentioned viscosity fluorescent probe is as follows:

[0016] Step 1: Add the same concentration of the compound shown in formula (I) to methanol / glycerol solutions of different contents to prepare at least 10 standard solutions containing the compound shown in formula (I) with different viscosity gradients;

[0017] The concentration of the compound represented by formula (I) in the standard solution shown is 10 μM;

[0018] The standard solutions shown contain 0% to 100% glycerol (viscosity 0.6 cP to 1091.2 cP).

[0019] Step 2: Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 415 nm. Plot log(η) as the abscissa and log(I) as the ordinate. 535 Establish a standard curve with y as the ordinate;

[0020] η represents the logarithm of the viscosity value in the standard solution; I 535 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 535 nm;

[0021] Step 3: Add the compound shown in formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; measure its fluorescence emission spectrum under excitation light with an excitation wavelength of 415 nm, and calculate the viscosity of the sample to be tested based on the standard curve.

[0022] Preferably, the standard solutions with different viscosity gradients include glycerol / methanol mixed solvents with glycerol volume fractions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

[0023] Preferably, as the viscosity of the test system increases, the fluorescence emission intensity of the fluorescent probe at 535 nm increases.

[0024] This invention has the following characteristics:

[0025] 1) The fluorescent probe provided by this invention is an orange solid powder with good optical stability.

[0026] 2) The fluorescent probe provided by this invention has a solution that is sensitive to viscosity. As the viscosity of glycerol increases, the fluorescence of the methanol / glycerol solution under a fluorescent lamp changes from no fluorescence to yellow fluorescence.

[0027] 3) The fluorescent probe provided by this invention has an absorption wavelength at 415 nm and an emission wavelength at 535 nm. It has a large Stokes shift, and as the viscosity increases, the emission peak at 535 nm gradually increases. It is an "off-on" type response and can sensitively respond to changes in viscosity.

[0028] 4) The fluorescent probe provided by this invention has a linear relationship with viscosity and can be used for accurate viscosity measurement.

[0029] The isophorone dye-based "off-on" type viscosity probe provided by this invention has a good response to viscosity, enabling sensitive quantitative detection of viscosity within a sample. It has the advantages of simple operation, low cost, sensitive response, and ease of promotion and application. Attached Figure Description

[0030] Figure 1 1H NMR spectrum of fluorescent probe DCI-V7

[0031] Figure 2 Photographs of the color response of the fluorescent probe DCI-V7 (10 μM) in methanol (low viscosity) and glycerol (high viscosity) under visible light.

[0032] Figure 3 Photographs of the fluorescence response of the fluorescent probe DCI-V7 (10 μM) in methanol and glycerol under UV light.

[0033] Figure 4 UV-Vis absorption spectra of fluorescent probe DCI-V7 (10 μM) in methanol / glycerol mixed solvents with different glycerol contents (0%–100%).

[0034] Figure 5 Fluorescence emission spectra (λ) of fluorescent probe DCI-V7 (10 μM) in methanol / glycerol mixed solvents with different glycerol contents (0%–100%) ex = 415 nm).

[0035] Figure 6 : Fluorescence response bar chart of fluorescent probe DCI-V7 (10 μM) to common small molecule interfering agents (50 μM) (solvent: glycerol / methanol mixture with fixed viscosity, λ ex = 415 nm, λem = 535 nm). Detailed Implementation

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] The compound numbers in the examples correspond to the numbers in the compounds described above.

[0039] Example 1: Synthesis of fluorescent probe DCI-V7.

[0040] Under a nitrogen atmosphere, 100 mg (0.54 mmol) of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonadionitrile and 62 mg (0.64 mmol) of 4-imidazolium carbaldehyde were placed in a two-necked flask. The solid was dissolved in 3 mL of anhydrous acetonitrile, and then 11 μL (0.11 mmol) of piperidine was added. The mixture was heated to 50 °C and stirred for 4 hours, during which an orange solid gradually precipitated. After the reaction was complete, the mixture was filtered, washed with cold acetonitrile, and dried to give 83 mg of the orange solid product DCI-V7, with a yield of 55%.

[0041] like Figure 1 As shown, 1 H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H), 7.79 (s, 1H),7.52 (s, 1H), 7.23 (d, J = 15.8 Hz, 1H), 7.08 (d, J = 16.1 Hz, 1H), 6.67 (s,1H), 2.59 (s, 2H), 1.01 (s, 6H).

[0042] Example 2: Color and fluorescence response of compound DCI-V7 to viscosity.

[0043] Prepare a 1 mM DCI-V7 dimethyl sulfoxide (DMSO) stock solution. Add 50 μL of this stock solution to 5 mL of pure methanol (low viscosity) and 5 mL of pure glycerol (high viscosity), respectively, to achieve a final probe concentration of 10 μM. Figure 2 As shown, under visible light, methanol solution appears pale yellow, while glycerol solution appears yellow. Figure 3 As shown, under ultraviolet light, the methanol solution showed almost no fluorescence, while the glycerol solution emitted a bright yellow fluorescence. This indicates that the probe has a direct fluorescent response to viscosity.

[0044] Example 3: UV absorption spectra of DCI-V7 at different viscosities.

[0045] A series of methanol / glycerol mixed solvents with different volume ratios were prepared (glycerol content 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, corresponding viscosities 0.6 cP to 1091.2 cP). DCI-V7 stock solution was added to each solution to a final concentration of 10 μM. The UV-Vis absorption spectra of each solution were measured. Figure 4 As shown, with the increase of glycerol content (viscosity), the maximum absorption wavelength gradually redshifts from 405 nm to 419 nm.

[0046] Example 4: Fluorescence emission spectra of DCI-V7 at different viscosities.

[0047] Prepare a series of solutions identical to those in Example 3, and measure the fluorescence emission spectra at an excitation wavelength of 415 nm. Figure 5 As shown, the fluorescence intensity at 535 nm gradually increases with increasing viscosity, exhibiting a typical "on" response. Plotting log(viscosity) as the x-axis and log(I) as the y-axis... 535 Plotting y = R on the ordinate yields a good linear relationship. 2 (>0.98), which can be used to quantitatively detect the viscosity of unknown samples.

[0048] Example 5: Selectivity of probe DCI-V7 for common interfering substances.

[0049] Prepare a glycerol / methanol mixed solvent (9:1 volume ratio, for a high-viscosity environment), and add DCI-V7 stock solution to a final concentration of 10 μM. Then add the following interfering substances (final concentration 50 μM): glutathione, cysteine, homocysteine, H₂O₂, HClO, NO, and ONOO₂. - Zn 2+ Cu 2+ Ca 2+ The fluorescence intensity of each solution was measured (λex = 415 nm, λem = 535 nm). For example... Figure 6 As shown, compared with the blank control, the above-mentioned interfering substances have no significant effect on the fluorescence intensity of the probe, indicating that the probe has excellent selectivity for viscosity.

[0050] Example 2: Synthesis of fluorescent probe DCI-V7

[0051] Under a nitrogen atmosphere, 80 mg (0.43 mmol) of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile and 50 mg (0.52 mmol) of 4-imidazolium carbide were placed in a two-necked flask, and 2.5 mL of anhydrous acetonitrile was added to disperse and dissolve them. Then, 9 μL (0.09 mmol) of piperidine was added. The reaction system was heated to 45 °C and stirred for 6 hours. During the reaction, an orange solid gradually precipitated. After the reaction was completed, the mixture was cooled to room temperature, the solid was collected by filtration, washed 2–3 times with cold acetonitrile, and dried under vacuum to give 64 mg of the orange solid product DCI-V7, with a yield of 53%.

[0052] The product was confirmed by 1H NMR spectroscopy, and its characteristic peaks were consistent with those of the product obtained in Example 1, indicating that the target fluorescent probe DCI-V7 could be prepared in this example.

[0053] Example 3: Synthesis of fluorescent probe DCI-V7

[0054] Under a nitrogen atmosphere, 150 mg (0.81 mmol) of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonadionitrile and 86 mg (0.90 mmol) of 4-imidazolium carbide were placed in a two-necked flask, followed by the addition of 8 mL of anhydrous acetonitrile and then 40 μL (0.40 mmol) of piperidine. After stirring at room temperature for 1 hour, the temperature was increased to 50 °C and the reaction was continued for 3 hours, resulting in the precipitation of an orange solid. After the reaction was completed, the mixture was cooled, filtered, washed with cold acetonitrile, and dried to give 132 mg of the orange solid product DCI-V7, with a yield of 58%.

[0055] The product was confirmed by 1H NMR spectroscopy, and its characteristic peaks were consistent with those of the product obtained in Example 1, indicating that the target fluorescent probe DCI-V7 could be prepared in this example.

Claims

1. A viscosity-sensitive fluorescent probe, characterized in that, The fluorescent probe is (E)-2-(3-(2-(1H-imidazol-5-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile, with the molecular formula C2. 16 H 16 N4, with a structure as shown in equation (I), namely: DCI-V7: 。 2. The fluorescent probe according to claim 1, characterized in that, The fluorescent probe is an orange solid powder.

3. A method for preparing a fluorescent probe according to claim 1 or 2, characterized in that, Includes the following steps: Under nitrogen protection, 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile, 4-imidazolium formaldehyde, and piperidine were dissolved in acetonitrile and reacted at 0–50 °C for 1–24 hours. An orange solid precipitated during the reaction, and after separation and purification, the (E)-2-(3-(2-(1H-imidazol-5-yl)vinyl)-5,5-dimethylcyclohexyl-2-en-1-yl)malononitrile was obtained.

4. The preparation method according to claim 3, characterized in that, The molar ratio of 2-(3,5,5-trimethylcyclohexyl-2-ene-1-yl)malonitrile, 4-imidazolium carbide, and piperidine is 1:1 to 1.2:0.2 to 1; the amount of acetonitrile used is 1 to 10 mL per 0.54 mmol of 2-(3,5,5-trimethylcyclohexyl-2-ene-1-yl)malonitrile.

5. The preparation method according to claim 4, characterized in that, The molar ratio of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile, 4-imidazolium carbaldehyde, and piperidine is 1:1.2:0.2; the amount of acetonitrile used is 3 mL per 0.54 mmol of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile; the reaction temperature is 50℃, and the reaction time is 4 hours.

6. A method for detecting sample viscosity for non-diagnostic purposes using the fluorescent probe according to claim 1 or 2, characterized in that, Includes the following steps: (a) Prepare a series of glycerol / methanol mixed solvents with different viscosities, and add the same concentration of the compound shown in formula (I) to each solvent to obtain at least 5 standard solutions with different viscosity gradients; The concentration of the compound represented by formula (I) in the standard solution is 10 μM; (b) Measure the fluorescence emission spectrum of the standard solution with an excitation wavelength of 415 nm. Establish a standard curve with the logarithm of viscosity value η log(η) as the abscissa and the logarithm of fluorescence emission peak intensity I535 at 535 nm wavelength log(I535) as the ordinate. (c) Add the compound of formula (I) to the sample to be tested, so that the concentration of the compound of formula (I) in the sample to be tested is equal to the concentration of the compound of formula (I) in the standard solution, measure the fluorescence emission spectrum of the sample to be tested at an excitation wavelength of 415 nm, and calculate the viscosity of the sample to be tested according to the standard curve.

7. The method according to claim 6, characterized in that, The volume fraction of glycerol in the glycerol / methanol mixed solvent is 0% to 100%, corresponding to a viscosity of 0.6 cP to 1091.2 cP.

8. The method according to claim 6 or 7, characterized in that, The standard solutions with different viscosity gradients include glycerol / methanol mixed solvents with glycerol volume fractions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

9. The method according to any one of claims 6 to 8, characterized in that, As the viscosity of the test system increases, the fluorescence emission intensity of the fluorescent probe at 535 nm increases.

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