Preparation method and application of multi-channel fluorescent probe

Abstract

The application discloses a preparation method and application of a multi-channel fluorescent probe, and discloses a preparation method and application of a multi-channel fluorescent probe.The structure of the multi-channel fluorescent probe is shown as formula (I), and the chemical name of the compound of formula (I) is (E)-4-(4-(dimethylamino)styryl)-1-(4-((diphenylphosphinoyl)benzyl)quinoline.The multi-channel fluorescent probe has the performance of simultaneously detecting viscosity, polarity and peroxynitrite.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a preparation method and application of a multi-channel fluorescent probe. Background Art

[0002] Cells are the fundamental structural and functional units of living organisms and play a vital role in maintaining the normal functioning of life. Their physiological activities are regulated by their corresponding biomolecules and the cellular microenvironment, including reactive oxygen species (ROS) levels, viscosity, and polarity. Microenvironmental homeostasis is crucial for normal cellular function, and changes in this microenvironment are often associated with cellular dysfunction and can even lead to disease. Therefore, detecting and measuring these parameters in living cells is of great significance to biological science and biomedical research.

[0003] Redox balance is crucial for maintaining normal cellular function and regulating signal transduction. Reactive oxygen species (ROS) are a class of important active substances produced when mitochondria provide energy for biological systems. They possess strong oxidative properties and exist in various forms, playing a crucial role in maintaining cellular function, transmitting signals, and regulating physiological processes. Among them, peroxynitrite (ONOO-) is a key ROS species. Excessive accumulation of ONOO- can cause biomembrane damage and cellular dysfunction, leading to various pathological processes such as inflammation, acute kidney injury, cardiovascular disease, cancer, depression, and neurodegenerative diseases.

[0004] Viscosity is a key parameter in the cellular microenvironment, playing a crucial role in intracellular interactions such as signal transduction and the diffusion of various substances. Abnormal cell viscosity is often closely associated with cellular pathophysiological states. Polarity, in addition to establishing and maintaining cellular homeostasis, is also a component of the cellular microenvironment. Changes in cell polarity occur when cells engage in processes such as differentiation, migration, and growth, leading to changes in spatial arrangement and protein composition. Therefore, abnormal changes in cell polarity may indicate cellular disorders or even the onset of disease. Currently, reports on fluorescent probes primarily focus on detecting a single parameter. While the simultaneous use of multiple single-parameter probes can address the need for multi-parameter detection, cells exist in a dynamic equilibrium, where each parameter is constantly changing and interacting with each other. Therefore, the simultaneous use of multiple probes still presents challenges such as dye overlap, increased toxicity, and analytical errors caused by background variations. More importantly, it is difficult to track changes in multiple parameters in real time. Therefore, the development of multi-channel fluorescent probes that simultaneously detect multiple parameters is crucial for analyzing changes in the cellular microenvironment. Therefore, a method for preparing and applying multi-channel fluorescent probes is needed. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of a multi-channel fluorescent probe.

[0006] The present application is realized by the following technical solutions:

[0007] The technical scheme adopted by the present application is:

[0008] A multi-channel fluorescent probe, the chemical name of the fluorescent probe is (E)-4-(4-(dimethylamino) styryl)-1-(4-((diphenylphosphoryl) benzyl) quinoline; the structure is shown in formula I:

[0009] ;

[0010] Further, the method comprises the following steps:

[0011] (a) preparing compound (II): mixing p-dimethylaminobenzaldehyde and 4-methylquinoline according to a molar ratio of 1.1:1, dissolving in a DMF solution, adding toluenesulfonic acid dropwise to form an ene reaction, the reaction temperature is 150 DEG C, the reaction time is 12h, and column chromatography purification is carried out to obtain compound (II) solid shown in formula (II):

[0012] ;

[0013] (b) preparing compound (III): 4-hydroxybenzyl alcohol is mixed and dissolved in anhydrous tetrahydrofuran solution to obtain a reaction solution, and phosphinic chloride is added dropwise to carry out catalytic reaction, the 4-hydroxybenzyl alcohol and the phosphinic chloride are added dropwise according to a molar ratio of 1:1, the dissolution ratio of the 4-hydroxybenzyl alcohol and the phosphinic chloride to the anhydrous tetrahydrofuran solution is 24.03 mg / ml; and the reaction solution is stirred at room temperature for 24h, the solvent is evaporated under reduced pressure, and column chromatography purification is carried out to obtain compound (IV) ;

[0014] The volume ratio of the triethylamine to the reaction solution is 1:40; compound (IV) is generated by reaction in acetonitrile,

[0015] Compound (IV) is dissolved in dichloromethane according to a mol ratio of 1:1:1 with carbon tetrabromide and triphenylphosphine, the reaction solution is stirred at room temperature for 12h, saturated sodium bicarbonate solution is added to the above reaction solution, dichloromethane extraction is carried out, and column chromatography purification is carried out on the organic phase to obtain compound (III) solid shown in formula (III):

[0016] ;

[0017] (c) mixing compound (II) and compound (III) according to a mol ratio of 1:1, dissolving in acetonitrile solution, the dissolution ratio of the compound (II) and the compound (III) to the acetonitrile solution is 0.06 mmol / mL, heating refluxing for 12h, and column chromatography purification is carried out to obtain compound (I) solid shown in formula (I).

[0018] Furthermore, in step a, column chromatography with petroleum ether / ethyl acetate 5:1 was used to obtain compound (II) as a solid.

[0019] Furthermore, in step c, column chromatography using dichloromethane / methanol 20:1 was performed to obtain compound (I) as a solid.

[0020] Furthermore, when performing the extraction operation in step b, the organic phase needs to be washed multiple times with saturated saline solution and saturated sodium bicarbonate solution.

[0021] Furthermore, when saturated sodium bicarbonate solution is used to quench the reaction in step b, the saturated sodium bicarbonate solution needs to be added in small amounts and multiple times to ensure that the mixed solution does not overflow until no more gas is produced.

[0022] A multi-channel fluorescent probe is used to detect viscosity or solvent polarity in the presence of peroxynitrous acid.

[0023] Compared with the prior art, the present invention has the beneficial effect of providing a multi-channel fluorescent probe capable of simultaneously detecting viscosity, polarity and peroxynitrous acid.

[0024] Compared with the prior art, the present invention adopts the above technical solution, and its biggest feature is:

[0025] Compared with the prior art, the present invention has the following effects:

[0026] The present invention adopts a cold expression system to prepare full-length PA and full-length LF to obtain highly active LT toxin, and the vaccine potency after immunization is evaluated by adding the LT toxin into the post-immunization serum and inhibiting the LT toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the fluorescence emission spectra of compound (I) under the optimal wavelength of 568 nm excitation in water / glycerol systems with different ratios;

[0028] Among them, the vertical axis represents the fluorescence intensity and the horizontal axis represents the wavelength;

[0029] Figure 2 Schematic diagram of the fluorescence emission spectrum of compound (I) before and after the addition of peroxynitrous acid under excitation at an optimal wavelength of 400 nm;

[0030] Among them, the vertical axis represents the fluorescence intensity and the horizontal axis represents the wavelength;

[0031] Figure 3 Schematic diagram of the fluorescence emission spectra of compound (I) after adding peroxynitrous acid in solvents of different polarities and excited at the optimal wavelength of 405 nm;

[0032] The ordinate represents the normalized fluorescence intensity, and the abscissa represents the wavelength. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further illustrated below in conjunction with embodiments and comparative examples, but they should not be construed as limiting the present invention:

[0034] The overall synthetic route of compound (I) of the present invention is as follows:

[0035]

[0036] The structural formula of compound (I) provided herein contains an N,N-dimethyl group that can rotate freely, resulting in twisted intramolecular charge transfer (TICT), leading to fluorescence quenching of compound (I). Once in a high-viscosity environment, the restricted intramolecular rotation weakens the TICT effect, causing compound (I)'s fluorescence to increase, providing a recognition site that responds to viscosity. The diphenylphosphoryl group specifically recognizes peroxynitrous acid, leading to the disappearance of the cation and a cascade reaction to form compound (II), which weakens the intramolecular charge transfer (ICT) effect and causes a blue shift in the fluorescence spectrum. Furthermore, compound (II) molecules themselves can form a charge-separated state in their excited state, making them excellent polarity-sensitive probes. Therefore, the present invention further provides the use of the compound of formula (I) as a multi-channel fluorescent probe for the simultaneous detection of viscosity, polarity, and peroxynitrous acid.

[0037] Example 1 Compound (IV)

[0038] Multi-channel fluorescent probe: 4-Hydroxybenzyl alcohol (248 mg, 2 mmol) and triethylamine (1 mL) were dissolved in 30 mL of anhydrous tetrahydrofuran. Phosphinyl chloride (473 mg, 2 mol) was slowly added dropwise to the solution, and the solution was stirred at room temperature overnight. The resulting solution was quenched with 10 mL of water, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography to yield compound IV (454 mg, 70%) as a white solid.

[0039] The detection parameters are:

[0040] 1H NMR (500 MHz, DMSO-d6) δ 7.94 – 7.86 (m, 4H), 7.65 – 7.58 (m, 2H), 7.54 (td, J = 7.4, 3.7 Hz, 4H), 7.26 – 7.20 (m, 4H), 5.14 (t, J = 5.5 Hz,1H), 4.40 (d, J = 5.1 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ 149.85, 149.79,139.40, 133.19, 133.17, 132.05, 131.96, 131.83, 130.75, 129.43, 129.32,128.32, 120.67, 120.63, 62.70.

[0041] Multi-channel fluorescent probe Example 2 Compound (III)

[0042] Compound IV (324 mg, 1 mmol), carbon tetrabromide (331 mg, 1 mmol), and triphenylphosphine (262 mg, 1 mmol) were dissolved in dichloromethane (20 mL). The solution was stirred at room temperature for 12 hours. Saturated sodium bicarbonate solution (5 mL) was added to the reaction solution. Extraction with dichloromethane was performed, and the organic phase was purified by column chromatography to yield compound III (246 mg, 64%) as a white solid.

[0043] The detection parameters are:

[0044] 1 H NMR (500 MHz, DMSO-d6) δ 7.91 (m, J = 13.0, 7.7 Hz, 4H), 7.63 (m,J = 6.8 Hz, 2H), 7.56 (m, J = 10.9, 5.4 Hz, 4H), 7.40 (d, J = 8.6 Hz, 2H),7.26 (d, J = 7.9 Hz, 2H), 4.64 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ 150.97,150.91, 134.95, 133.32, 133.29, 132.01, 131.93, 131.64, 131.39, 130.55,129.49, 129.38, 121.25, 121.22, 34.21.

[0045] Example 3 Compound (II)

[0046] 4-Methylquinoline (429 mg, 3 mmol) and p-dimethylaminobenzaldehyde (492 mg, 3.3 mmol) were dissolved in 10 mL of DMF. Six drops of methanesulfonic acid were added, and the reaction was heated to 150°C for 12 hours. The mixture was added to ice water, extracted with dichloromethane, and dried by rotary evaporation. Purification by column chromatography afforded an orange-yellow solid (548 mg, 67%).

[0047] Example 4 Compound (I)

[0048] Compound III (129 mg, 0.33 mmol) and compound II (92 mg, 0.33 mmol) were dissolved in 6 mL of acetonitrile and heated under reflux for 12 hours. The solvent was evaporated to dryness, and the product was purified by column chromatography to obtain a purple solid I (140 mg, 64%).

[0049] The detection parameters are:

[0050] 1 H NMR(500 MHz, DMSO-d6) δ 9.31 (d, J = 6.8 Hz, 1H), 9.03 (dd, J =8.9, 1.4 Hz, 1H), 8.43 (d, J = 6.8 Hz, 1H), 8.29 – 8.21 (m, 2H), 8.08 – 8.02(m, 2H), 7.91 – 7.85 (m, 7H), 7.64 – 7.59 (m, 2H), 7.56 – 7.51 (m, 4H), 7.36– 7.32 (m, 2H), 7.30 – 7.26 (m, 2H), 6.86 – 6.80 (m, 2H), 6.10 (s, 2H), 3.08(s, 6H).

[0051] Example 5 Response of Compound (I) to Viscosity

[0052] The experiment for testing the viscosity response performance of the compound (I) of the present invention is as follows:

[0053] To examine the viscosity response of compound (I), different volume ratios (0% to 100%) of glycerol and water were mixed to form solvents of varying viscosities. 10 μL of 1 mM compound (I) was added to each solvent. After mixing, absorption spectra were measured, and fluorescence spectra were measured at an excitation wavelength of 568 nm.

[0054] During the fluorescence spectrum test, compound (I) was placed in a quartz cuvette with light passing through all four sides, the slit width was set to 20 nm, and the voltage was set to 800 V at level 13.

[0055] According to the above experimental method, the fluorescence intensity of compound (I) was tested to increase with the increase of viscosity, and within a certain viscosity range, the logarithm of the fluorescence intensity had a good linear relationship with the logarithm of the viscosity. Figure 1 The fluorescence emission spectra of compound (I) under the optimal wavelength of 568 nm excitation in water / glycerol systems with different ratios.

[0056] Example 6 Response of Compound (I) to Peroxynitrous Acid

[0057] The experiment for testing the response performance of the compound (I) of the present invention to peroxynitrous acid is as follows:

[0058] In the experiment, to detect the response of compound (I) to peroxynitrous acid, peroxynitrous acid was added to 2 ml of compound (I) (10 μM) solution, 10 mM PBS buffer (pH 7.4, containing 20% ​​DMSO, v / v) was added, and after mixing, the absorption spectrum was measured, and the fluorescence spectrum was measured at an excitation wavelength of 400 nm.

[0059] During the fluorescence spectrum test, compound (I) was placed in a quartz cuvette with light passing through all four sides, the slit width was set to 10 nm, and the voltage was set to 800 V at level 13.

[0060] According to the above experimental method, the fluorescence intensity of compound (I) was significantly increased after adding peroxynitrous acid, and increased by nearly 31 times. Figure 2 The fluorescence emission spectra of compound (I) before and after the addition of peroxynitrous acid are excited at an optimal wavelength of 400 nm.

[0061] Example 7 Response of Compound (II) to Polarity

[0062] The experiment for testing the polarity response performance of compound (II) of the present invention is as follows:

[0063] To examine the polarity response of compound (II), 1 mM compound (I) was diluted with solvents of varying polarity to a final concentration of 10 μM (ET = 2.9 to 10.2) and peroxynitrous acid was added. Fluorescence spectra were measured at an excitation wavelength of 405 nm.

[0064] During the fluorescence spectrum test, compound (I) was placed in a quartz cuvette with light passing through all four sides, the slit width was set to 20 nm, and the voltage was set to 700 V at level 11.

[0065] According to the above experimental method, the test showed that compound (I) exhibited different emission wavelengths in different solvents after adding peroxynitrous acid, and the polarity parameter had a good linear relationship with the optimal emission wavelength. Figure 3The fluorescence emission spectra of compound (I) after adding peroxynitrous acid in solvents of different polarities and excited with the optimal wavelength of 405 nm.

[0066] Various spectroscopic experiments showed that compound (I) and its compound (II) have good application potential in monitoring viscosity, peroxynitrite, and polarity at the cellular level, and provide a good reference for the molecular design of multi-channel fluorescent probes.

[0067] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, and they are all covered by the scope of protection of the present invention.

Claims

1. A multi-channel fluorescent probe, characterized in that: The chemical name of the fluorescent probe is (E)-4-(4-(dimethylamino)phenylvinyl)-1-(4-((diphenylphosphino)phenylmethyl)quinoline; the structure is shown in Formula I: 。 2. A method for preparing a multi-channel fluorescent probe according to claim 1, characterized in that: The steps include: (a) Preparation of Compound (II): p-Dimethylaminobenzaldehyde and 4-methylquinoline were mixed at a molar ratio of 1.1:1 and dissolved in a DMF solution. Toluenesulfonic acid was added dropwise to allow olefin formation to occur at a temperature of 150°C for 12 h. The solid compound (II) was purified by column chromatography to obtain: ； (b) Preparation of compound (III): 4-hydroxybenzyl alcohol and triethylamine were mixed and dissolved in anhydrous tetrahydrofuran solution to obtain a reaction solution, and phosphinoyl chloride was added dropwise to carry out a catalytic reaction. The 4-hydroxybenzyl alcohol and phosphinoyl chloride were added dropwise at a molar ratio of 1:1, and the dissolution ratio of the 4-hydroxybenzyl alcohol and phosphinoyl chloride to the anhydrous tetrahydrofuran solution was 24.03 mg / ml. The reaction solution was stirred at room temperature for 24 hours, and the solvent was evaporated under reduced pressure. The solution was purified by column chromatography to obtain compound (IV). ； The volume ratio of the triethylamine to the reaction solution is 1:40; the reaction is carried out in acetonitrile to generate compound (IV), Compound (IV), carbon tetrabromide and triphenylphosphine were dissolved in dichloromethane at a mol ratio of 1:1:

1. The reaction solution was stirred at room temperature for 12 hours. Saturated sodium bicarbonate solution was added to the reaction solution and extracted with dichloromethane. The organic phase was purified by column chromatography to obtain a solid compound (III) as shown in formula (III): ； (c) Compound (II) and compound (III) were mixed at a mol ratio of 1:1 and dissolved in an acetonitrile solution, wherein the solubility ratio of compound (II) and compound (III) to the acetonitrile solution was 0.06 mmol / mL. The mixture was heated under reflux for 12 hours and purified by column chromatography to obtain a solid compound (I) represented by formula (I).

3. The preparation method according to claim 2, wherein: In step a, column chromatography was performed using petroleum ether / ethyl acetate 5:1 to obtain compound (II) as a solid.

4. The preparation method according to claim 2, wherein: In step c, column chromatography using dichloromethane / methanol 20:1 was used to obtain compound (I) as a solid.

5. The preparation method according to claim 2, wherein: When performing the extraction operation in step b, the organic phase needs to be washed multiple times with saturated brine and saturated sodium bicarbonate solution.

6. The preparation method according to claim 2, wherein: When quenching the reaction with a saturated sodium bicarbonate solution in step b, the saturated sodium bicarbonate solution needs to be added in small amounts and multiple times to ensure that the mixed solution does not overflow until no more gas is produced.

7. Use of the multi-channel fluorescent probe according to claim 1 for detecting viscosity or detecting polarity of a solvent in the presence of peroxynitrous acid.

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