Preparation method and application of multi-channel fluorescence probe for identifying viscosity, polarity and ONOO <->

Abstract

The invention discloses a preparation method and application of a multi-channel fluorescent probe for identifying viscosity, polarity and ONOO <->, and the structure of the multi-channel fluorescent probe for identifying viscosity, polarity and ONOO <-> is as shown in formula (I), the chemical name of the compound in the formula (I) is (E)-4-(4-(dimethylamino) styryl)-1-(4-(((trifluoromethyl) sulfonyl) oxy) benzyl) quinoline-1-ammonium bromide. The multi-channel fluorescence probe for identifying viscosity, polarity and ONOO <->, disclosed by the invention, has the performance of simultaneously detecting viscosity, polarity and ONOO <->.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a preparation method and application of a multi-channel fluorescent probe for identifying viscosity, polarity and ONOO-. Background Art

[0002] The physiological activities of all cells are regulated by the corresponding biomolecular microenvironment. Key physicochemical properties, such as pH, viscosity, polarity, temperature, electron potential, and mechanical force, play an integral role in this regulation. For example, pH significantly affects cell proliferation, endocytosis, and ion transporters require appropriate pH and temperature ranges to perform their normal functions. Rapid changes in neuronal membrane potential are essential for normal brain function, while mechanical forces are essential in maintaining cell shape, cell growth, and migration. Microenvironmental homeostasis is essential for the normal function of cells, and changes in the microenvironment are often associated with cell dysfunction and occasionally lead to disease. Therefore, the detection and measurement of these parameters in living cells are of great significance to biological science and biomedical research.

[0003] Peroxynitrite (ONOO-) is an endogenous reactive oxygen species (ROS) produced in living organisms that plays an important role in signal transduction and has antimicrobial and antibacterial activities. However, due to its high oxidative and nitrating properties, ONOO- also often causes damage to many biomacromolecules in cells, including DNA and proteins. Abnormally high levels of ONOO- are believed to be associated with many diseases, such as neurodegenerative diseases, cardiovascular diseases, and inflammatory diseases, and even cancer. However, due to the lack of effective methods to visualize ONOO- in living systems, many biological pathogenic effects of ONOO- are still not well understood. Therefore, the establishment of highly selective and sensitive detection methods with high biocompatibility and site specificity is of great significance for elucidating the biological functions of ONOO-.

[0004] The intracellular microenvironment includes factors such as viscosity and polarity, which play a vital role in maintaining redox balance and controlling the diffusion of active substances. Abnormalities in these factors can lead to cell damage. Viscosity is a key parameter in the cellular microenvironment and is indispensable in various physiological processes at the cellular level, including enzyme catalysis, signal transduction, protein aggregation, and molecular diffusion. Polarity, as another important microenvironment parameter, plays a key role in cell differentiation, proliferation, immune system regulation, and other related physiological processes. Abnormalities in intracellular viscosity and polarity values ​​can reduce the activity of membrane-bound proteins and lead to various diseases, including fatty liver, Alzheimer's disease, inflammation, and diabetes. Accurate monitoring of changes in polarity and viscosity, especially within biological systems, is of vital importance for understanding the pathological implications of diseases, understanding physiological processes, and achieving early diagnosis.

[0005] So far, many fluorescent probes have been developed for detecting viscosity, polarity or ONOO- separately, but single-channel probes cannot track and compare these three changes in real time. Various parameters in cells are in dynamic balance, and various parameters change dynamically and affect each other. Therefore, it is of great significance to track the real-time changes of multiple parameters at the same time. Therefore, it is urgent to develop a multi-channel fluorescent probe for detecting multiple parameters. Therefore, a preparation method and application of a multi-channel fluorescent probe for identifying viscosity, polarity and ONOO- are needed. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation method and application of a multi-channel fluorescent probe for identifying viscosity, polarity and ONOO-.

[0007] The present invention is achieved through the following technical solutions:

[0008] The technical solution adopted by the present invention is:

[0009] A multi-channel fluorescent probe for identifying viscosity, polarity and ONOO-, the chemical name of the fluorescent probe is (E)-4-(4-(dimethylamino)phenylvinyl)-1-(4-(((trifluoromethyl)sulfonyl)oxy)benzyl)quinolin-1-ammonium bromide, and the structure is shown in Formula I:

[0010]

[0011] A method for preparing a multi-channel fluorescent probe for identifying viscosity, polarity and ONOO-, comprising the following steps:

[0012] (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, p-toluenesulfonic acid was added dropwise to cause an olefin reaction at a temperature of 150° C. for 12 h, and purified by column chromatography to obtain a solid compound (II) as shown in formula (II):

[0013]

[0014] (b) Preparation of compound (III): p-hydroxyphenol and trifluoromethanesulfonic anhydride were mixed in a molar ratio of 1.1:1 and added to pyridine, the mass-volume ratio of p-hydroxyphenol to pyridine was 0.1211 g / mL, and the mass-volume ratio of trifluoromethanesulfonic anhydride to pyridine was 0.2821 g / mL, under argon protection, reacted at 0°C for 10 minutes, reacted at room temperature for 5 hours, diluted with DCM, pyridine was removed with CuSO4 solution, the organic phase was washed with water and saturated sodium chloride, excess solvent was removed by rotary evaporation, and purified by column chromatography to obtain compound (V);

[0015] Compound V was dissolved in ethanol and then added dropwise to ethanol containing NaBH4, wherein the molar ratio of compound V to NaBH4 was 1.3:1. After reacting for 1 h at room temperature in an ice bath, dilute hydrochloric acid was added to quench the reaction, the mixture was spin-dried, water was added, and the mixture was extracted with DCM, dried over anhydrous Na2SO4, and the solvent was evaporated to obtain compound (IV);

[0016] Compound (IV) and PBr3 were dissolved in dichloromethane at a molar ratio of 1:1.3, the ratio of compound IV to dichloromethane was 25.6 mg / mL, and the ratio of PBr3 to dichloromethane was 35.1 mg / mL. The reaction solution was stirred at room temperature for 2 h. Saturated sodium bicarbonate solution was added to the reaction solution and then extracted with dichloromethane. The organic phase was purified by column chromatography to obtain a solid compound (III) as shown in formula (III):

[0017]

[0018] (c) Compound (II) and compound (III) are mixed in a molar ratio of 1:1 and dissolved in an acetonitrile solution, wherein the dissolution ratio of compound (II) and compound (III) to the acetonitrile solution is 23.2 mg / mL, and the mixture is heated under reflux for 12 hours, and purified by column chromatography to obtain a solid compound (I) as shown in formula (I).

[0019] Furthermore, in step a, a solid compound (II) is obtained by column chromatography using petroleum ether / ethyl acetate 5:1.

[0020] Furthermore, in step c, column chromatography with dichloromethane / methanol 20:1 is used to obtain compound (I) as a solid.

[0021] 5. The preparation method according to claim 2, characterized in that: when performing the extraction operation in step b, the organic phase needs to be washed multiple times with saturated salt water and saturated sodium bicarbonate solution.

[0022] 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 gas is produced.

[0023] The multi-channel fluorescent probe for identifying viscosity, polarity and ONOO- is used for detecting viscosity, polarity and peroxynitrite fluorescent probe.

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

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

[0026] The multi-channel fluorescent probe for identifying viscosity, polarity and ONOO- of the present invention has the performance of simultaneously detecting viscosity, polarity and peroxynitrite. The multi-channel fluorescent probe for identifying viscosity, polarity and ONOO- of the present invention has the performance of simultaneously detecting viscosity, polarity and peroxynitrite. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] The ordinate represents the fluorescence intensity, and the abscissa represents the wavelength;

[0029] Figure 2 The fluorescence emission spectrum of compound (I) before and after the addition of peroxynitrous acid under the optimal wavelength of 405 nm excitation;

[0030] The ordinate represents the fluorescence intensity, and the abscissa represents the wavelength;

[0031] Figure 3 The fluorescence emission spectra of compound (I) in different polar solvents after adding peroxynitrous acid and excited by 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 scheme of the present invention is further described 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 CC single bonds on both sides of the double bond in the structural formula of the compound (I) provided by the present invention can rotate freely, that is, twisted intramolecular charge transfer (Twisted intramolecular Charge Transfer, TICT) occurs, resulting in fluorescence quenching of the compound (I). Once in a high viscosity environment, the intramolecular rotation is restricted, which will weaken the TICT effect, so that the fluorescence of the compound (I) increases, providing a recognition site for responding to viscosity. The trifluoromethanesulfonic acid group can specifically recognize ONOO-, resulting in the disappearance of the cation to produce a tandem reaction to generate the compound (II), so that the intramolecular charge transfer (ICT) effect is weakened, resulting in a blue shift in the fluorescence spectrum. In addition, the compound (II) molecule itself can form a charge separation state in the excited state, and is a good polarity sensitive probe. Therefore, the present invention further provides the use of the compound of formula (I) as a multi-channel fluorescent probe for simultaneously detecting viscosity, polarity and peroxynitrous acid.

[0037] The technical solution of the present invention is described below with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0038] Example 1 Compound (V)

[0039] Add p-hydroxyphenol (484.5 mg, 4.4 mmol) and trifluoromethanesulfonic anhydride (1128.5 mg, 4 mmol) to 4 ml of pyridine, protect with argon, react at 0°C for 10 minutes, react at room temperature for 5 hours, dilute with DCM, remove pyridine with CuSO4 solution, wash the organic phase with water and saturated sodium chloride, remove excess solvent with rotary evaporation, and obtain compound (V) (711 mg, 70%) by column chromatography.

[0040] Example 2 Preparation of Compound (III) and Compound (IV)

[0041] Compound V (495 mg, 1.95 mmol) was dissolved in 5 ml of ethanol, and added dropwise to 5 ml of ethanol containing NaBH4 (56.7 mg, 1.5 mml). The mixture was placed in an ice bath, and the addition was continued for 1 h. The reaction was allowed to react at room temperature for 1 h. 10% dilute hydrochloric acid was added to quench the reaction. The mixture was dried by rotary evaporation, and water was added. The mixture was extracted with DCM, and dried over anhydrous Na2SO4. The solvent was evaporated to obtain compound IV (486 mg, 97%).

[0042] Compound IV (256 mg, 1 mmol) and PBr3 (351 mg, 1.3 mmol) were dissolved in dichloromethane (10 mL) and reacted at room temperature for 2 h under argon protection. The mixture was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, and purified by column chromatography to obtain compound (III) (207 mg, 65%).

[0043] Example 3 Compound (II)

[0044] 4-Methylquinoline (429 mg, 3 mmol) and p-dimethylaminobenzaldehyde (492 mg, 3.3 mmol) were dissolved in 10 mL DMF, 6 drops of p-toluenesulfonic acid were added, and the reaction was heated to 150°C for 12 hours. Added into ice water, extracted with dichloromethane, dried by spin drying, and purified by column chromatography to obtain an orange-yellow solid (548 mg, 67%).

[0045] Example 4 Compound (I)

[0046] Compound (III) (125 mg, 0.39 mmol) and compound (II) (107 mg, 0.39 mmol) were dissolved in 10 mL of acetonitrile solution and heated under reflux for 12 hours. The solvent was evaporated to dryness and purified by column chromatography to obtain a purple solid (I) (135 mg, 58%).

[0047] 1 H NMR (500MHz, DMSO) δ9.36(d,J=6.8Hz,1H),9.07(dd,J=8.8,1.3Hz,1H),8.48(d,J=6.8Hz,1H),8.31(d,J=15.5Hz,1H),8. 25(d,J=8.9Hz,1H),8.13–8.04(m,2H),7.94–7.89(m,3H),7.54(s,4H),6.85(d,J=9.0Hz,2H),6.25(s,2H),3.09(s,6H).

[0048] Example 5 Response of Compound (I) to Viscosity

[0049] The experiment for testing the viscosity response performance of the compound (I) of the present invention is as follows:

[0050] In the experiment, in order to detect the response of compound (I) to viscosity, glycerol and water were mixed at different volume ratios (0% to 90%) to form solvents with different viscosities. 10 μL of 2 mM compound (I) was added to each solvent, mixed evenly, and the absorption spectrum was measured, and the fluorescence spectrum was measured at an excitation wavelength of 568 nm.

[0051] In the fluorescence spectrum test, compound (I) was placed in a quartz cuvette with four sides transparent to light, the slit width was set to 20 nm, and the voltage was 900 V at 15 levels.

[0052] According to the above experimental method, the fluorescence intensity of compound (I) is tested to increase with the increase of viscosity, and within a certain viscosity range, the logarithm of the fluorescence intensity has 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.

[0053] Example 6 Response of Compound (I) to ONOO-

[0054] The experiment for testing the ONOO-responsive performance of the compound (I) of the present invention is as follows:

[0055] In the experiment, in order to detect the response of compound (I) to ONOO-, ONOO- was added to 2 ml of compound (I) (10 μM) solution, the solvent system was PBS buffer containing 30% DMSO (concentration of 0.01 M, pH = 7.4), after mixing evenly, the absorption spectrum was measured, and the fluorescence spectrum was measured at an excitation wavelength of 405 nm.

[0056] In the fluorescence spectrum test, compound (I) was placed in a quartz cuvette with four sides transparent to light, the slit width was set to 10 nm, and the voltage was 750 V at 12 levels.

[0057] According to the above experimental method, the fluorescence intensity of compound (I) was significantly improved after adding ONOO-, and the increase was nearly 223 times. Figure 2 The fluorescence emission spectra of compound (I) before and after the addition of ONOO- are excited by the optimal wavelength of 405 nm.

[0058] Example 7 Response of Compound (II) to Polarity

[0059] The experiment for testing the polarity response performance of the compound (II) of the present invention is as follows:

[0060] In the experiment, in order to detect the response of compound (II) to polarity, 2 mM compound (I) was diluted with solvents of different polarity to a final concentration of 10 μM (ET=36-63.1), and ONOO- (final concentration of 20 μM) was added. The fluorescence spectrum was measured at an excitation wavelength of 405 nm.

[0061] During the fluorescence spectrum test, compound (I) was placed in a quartz cuvette with four sides through which light could pass, the slit width was set to 20 nm, and the voltage was set to 750 V at 12 levels.

[0062] According to the above experimental method, it was tested that compound (I) exhibited different emission wavelengths in different solvents after adding ONOO-, and the polarity parameter had a good linear relationship with the optimal emission wavelength. Figure 3 The fluorescence emission spectra of compound (I) in solvents of different polarities after adding ONOO- under excitation with the optimal wavelength of 405 nm.

[0063] Various spectral experiments show that compound (I) and compound (II) thereof have good application potential in monitoring viscosity, ONOO- and polarity at the cellular level. And it provides a good reference for the molecular design of multi-channel fluorescent probes. The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field within the technical scope disclosed by the present invention, according to the technical solution and its inventive concept of the present invention, is equivalently replaced or changed, which is covered within the protection scope of the present invention.

Claims

1. A multi-channel fluorescent probe for identifying viscosity, polarity and ONOO-, characterized in that: The chemical name of the fluorescent probe is (E)-4-(4-(dimethylamino)phenylvinyl)-1-(4-(((trifluoromethyl)sulfonyl)oxy)benzyl)quinolin-1-ammonium bromide, and the structure is shown in Formula I:

2. A method for preparing a multi-channel fluorescent probe for recognizing viscosity, polarity and ONOO- as claimed in 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, and toluenesulfonic acid was added dropwise to cause an olefin reaction at a temperature of 150° C. for 12 h. The solid compound (II) was purified by column chromatography to obtain the following formula (II): (b) Preparation of compound (III): p-hydroxyphenol and trifluoromethanesulfonic anhydride were mixed in a molar ratio of 1.1:1 and added to pyridine, the mass-volume ratio of p-hydroxyphenol to pyridine was 0.1211 g / mL, and the mass-volume ratio of trifluoromethanesulfonic anhydride to pyridine was 0.2821 g / mL, under argon protection, reacted at 0°C for 10 minutes, reacted at room temperature for 5 hours, diluted with DCM, pyridine was removed with CuSO4 solution, the organic phase was washed with water and saturated sodium chloride, excess solvent was removed by rotary evaporation, and purified by column chromatography to obtain compound (V); Compound V was dissolved in ethanol and then added dropwise to ethanol containing NaBH4, wherein the molar ratio of compound V to NaBH4 was 1.3:

1. The mixture was reacted in an ice bath and at room temperature for 1 h. After that, dilute hydrochloric acid was added to quench the reaction. The mixture was spin-dried and water was added. The mixture was extracted with DCM, dried over anhydrous Na2SO4, and the solvent was evaporated to obtain compound (IV); Compound (IV) and PBr3 were dissolved in dichloromethane at a molar ratio of 1:1.3, the ratio of compound IV to dichloromethane was 25.6 mg / mL, and the ratio of PBr3 to dichloromethane was 35.1 mg / mL. The reaction solution was stirred at room temperature for 2 h. Saturated sodium bicarbonate solution was added to the reaction solution and then 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) are mixed in a molar ratio of 1:1 and dissolved in an acetonitrile solution, wherein the dissolution ratio of compound (II) and compound (III) to the acetonitrile solution is 23.2 mg / mL, and the mixture is heated under reflux for 12 hours, and purified by column chromatography to obtain a solid compound (I) as shown in formula (I).

3. The preparation method according to claim 2, characterized in that: In step a, column chromatography with petroleum ether / ethyl acetate 5:1 was used to obtain compound (II) as a solid.

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

6. The preparation method according to claim 2, characterized in that: 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 gas is produced.

7. An application of the multi-channel fluorescent probe for identifying viscosity, polarity and ONOO- as claimed in claim 1 for detecting viscosity, polarity and peroxynitrite fluorescent probe.

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