Water-soluble monophenyl ring fluorescent dye, and preparation method and application thereof

Abstract

The application provides a water-soluble single benzene ring fluorescent dye and a preparation method and application thereof. The single benzene ring fluorescent dye has a structure shown in formula I, and the preparation method comprises the following steps: adding tetrafluoro-p-xylylene cyanide into an organic solvent, stirring and dissolving, adding an alkali, and performing reaction to obtain the water-soluble single benzene ring fluorescent dye. The organic small molecule fluorescent dye has excellent water solubility, and fluorescence quenching does not occur in water. The fluorescent dye has a rigid conjugated structure, the fluorescent dye 1 has a quantum yield of 0.83 in an aqueous solution, and the fluorescent dye 2 can reach a quantum yield of 0.97 in an aqueous solution. In addition, the fluorescent dye has excellent thermal stability and light stability, provides a possibility for further detection, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a water-soluble monobenzene ring fluorescent dye, its preparation method, and its application, belonging to the field of organic fluorescent dyes. Background Technology

[0002] In recent years, organic fluorescent dyes have been widely used in fields such as bioimaging, fluorescent labeling, anti-counterfeiting, and environmental monitoring. Organic fluorescent dyes generally have advantages such as simple synthesis, absence of heavy metals, and ease of modification; however, they often exhibit poor water solubility or significant fluorescence quenching in water. Furthermore, fluorescent dyes need to possess properties such as bright luminescence, high quantum yield, resistance to photobleaching, and a large Stokes shift.

[0003] Traditional organic fluorescent dyes are mostly based on fluorescein, coumarin, anthocyanin, and rhodamine backbones, and generally have good lipid solubility. In specific applications, they need to be modified to increase their water solubility. Furthermore, in order to obtain fluorescent dyes with large Stokes shifts and long fluorescence emission wavelengths, it is necessary to increase the conjugated structure of the dye system and introduce strong electron-donating and electron-withdrawing groups. This results in an increase in the conjugated structure and molecular volume of the dye molecule, an increase in the coplanarity and rigidity of the fluorescent dye molecule, and a decrease in solubility. This makes the fluorescent dyes prone to fluorescence quenching at high concentrations or in the solid state, which seriously limits the development of dyes.

[0004] Currently, most traditional organic fluorescent dyes still suffer from several problems, such as short excitation and emission wavelengths, low brightness, poor water solubility, and poor stability. Therefore, developing a novel, water-soluble organic fluorescent dye with a simple structure and excellent fluorescence performance is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a water-soluble monophenyl ring fluorescent dye, its preparation method, and its applications. The monophenyl ring fluorescent dye of this invention is water-soluble, exhibiting excellent solubility in water, and requires no organic additives. The fluorescent dye is designed based on a monophenyl ring D-π-A conjugated luminescent structure, with the luminescence region in the visible light region. The fluorescent dye exhibits excellent photophysical properties, displaying bright luminescence in both water and organic solvents, extremely high quantum yield, and good thermal and photostable stability.

[0006] This invention is achieved through the following technical solution:

[0007] A water-soluble monobenzene ring fluorescent dye having the structure shown in Formula I:

[0008]

[0009] In Formula I, the substituent R is fluorine or hydroxyl.

[0010] According to the present invention, the water-soluble monobenzene ring fluorescent dye has the structure shown in formula I-1 or I-2 as follows:

[0011]

[0012] According to the present invention, the preparation method of the above-mentioned water-soluble monobenzene ring fluorescent dye includes the following steps:

[0013] Tetrafluoroterephthalonitrile was added to an organic solvent, stirred and dissolved, and then an alkali was added to carry out the reaction, resulting in a water-soluble monobenzene ring fluorescent dye.

[0014] According to a preferred embodiment of the present invention, the organic solvent is N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), dichloromethane, or acetonitrile; the volume ratio of the organic solvent to the mass ratio of tetrafluoroterephthalonitrile is 10-100 mL: 1 g.

[0015] According to a preferred embodiment of the present invention, the base is an organic base or an inorganic base; the organic base is triethylamine, pyridine, 4-dimethylaminopyridine, or N,N-diisopropylethylamine; the inorganic base is potassium carbonate or cesium carbonate; and the molar ratio of the base to tetrafluoroterephthalonitrile is 0.1-10:1.

[0016] According to a preferred embodiment of the present invention, the reaction temperature is 60-90°C; and the reaction time is 0.5-10 hours.

[0017] According to the present invention, the generation of monohydroxy-substituted and dihydroxy-substituted fluorescent dye products can be controlled by controlling the reaction time and the type of base, wherein the source of the hydroxyl group may be moisture contained in the solvent or moisture in the air; preferably, the base is 4-dimethylaminopyridine, and when the reaction time is 6-10 h, a water-soluble monophenyl ring fluorescent dye in Formula I with hydroxyl substituent R can be obtained, i.e., the fluorescent dye shown in Formula I-2; when the reaction time is 0.5-6 h, a water-soluble monophenyl ring fluorescent dye in Formula I with fluorine substituent R can be obtained, i.e., the fluorescent dye shown in Formula I-1.

[0018] According to a preferred embodiment of the present invention, the post-treatment steps of the reaction solution obtained from the reaction are as follows: after the reaction is completed, dichloromethane and water are added to the obtained reaction solution for extraction; the lower organic phase is washed with water 3-5 times, and then the obtained organic phase is dried with anhydrous sodium sulfate to remove the solvent, thereby obtaining a crude product. The crude product is purified by column chromatography and dried under vacuum to obtain the final product.

[0019] According to a preferred embodiment of the present invention, the eluent for the column chromatography is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol in the mixed solution is 10:0 to 9:1.

[0020] The fluorescent dye of the present invention has a single benzene ring skeleton, which is a type of fluorophore with "D-π-A" properties, wherein -CN and -F are electron-withdrawing groups (acceptors) and -OH is an electron-donating group (donor). Through electron conjugation of the single benzene ring, the fluorescent dye has fluorescent properties.

[0021] According to the present invention, the above-mentioned water-soluble monobenzene ring fluorescent dyes are used in bioimaging, fluorescent labeling, anti-counterfeiting and environmental monitoring.

[0022] The technical features and beneficial effects of this invention are as follows:

[0023] 1. The fluorescent dye of the present invention adopts a novel monobenzene ring D-π-A conjugated luminescent skeleton, which emits bright light, has a simple structure, and a small molecular weight. Unlike other small organic molecule fluorescent dyes, the fluorescent dye of the present invention is a novel organic fluorescent dye with good solubility in most common organic solvents and water. It does not undergo fluorescence quenching in water and has excellent water solubility and lipid solubility, thus expanding its application field.

[0024] 2. The monobenzene ring organic fluorescent dye of the present invention has a rigid monobenzene ring structure, which is a rigid conjugated structure, thereby restricting molecular vibration and weakening vibrational relaxation. It has bright luminescence and high fluorescence quantum yield. Experiments have shown that the fluorescent dye of the present invention has an extremely high quantum yield in water, which can reach 97%, and at the same time has excellent photostability and thermal stability.

[0025] 3. The method for preparing the monobenzene ring fluorescent dye of the present invention is simple, and the product can be obtained in one pot and one step without further modification. The raw materials are safe, the preparation is simple, the cost is low, and it is easy to promote and utilize. Attached Figure Description

[0026] Figure 1 The fluorescent dye I-1 obtained in Example 1 19 F NMR spectrum.

[0027] Figure 2 The mass spectrum of fluorescent dye I-1 obtained in Example 1 is shown.

[0028] Figure 3 The fluorescent dye I-1 obtained in Example 1 13 C10 NMR spectrum.

[0029] Figure 4 The fluorescent dye I-2 obtained in Example 219 F NMR spectrum.

[0030] Figure 5 The mass spectrum of fluorescent dye I-2 obtained in Example 2 is shown.

[0031] Figure 6 The fluorescent dye I-2 obtained in Example 2 13 C10 NMR spectrum.

[0032] Figure 7 The fluorescence spectra of fluorescent dye I-1 obtained in Example 1 are shown in different organic solvents and water; in the figure, Ace is acetone, CH3CN is acetonitrile, CH3OH is methanol, DCE is 1,2-dichloroethane, DCM is dichloromethane, DMF is N,N-dimethylformamide, DMSO is dimethyl sulfoxide, DOX is 1,4-dioxane, EA is ethyl acetate, EtOH is ethanol, H2O is water, TCM is chloroform, THF is tetrahydrofuran, and Tol is toluene.

[0033] Figure 8 The fluorescence spectra of fluorescent dye I-2 obtained in Example 2 are shown in different organic solvents and water; in the figure, Ace is acetone, CH3CN is acetonitrile, CH3OH is methanol, DCE is 1,2-dichloroethane, DCM is dichloromethane, DMF is N,N-dimethylformamide, DMSO is dimethyl sulfoxide, EtOH is ethanol, H2O is water, and TCM is trichloromethane.

[0034] Figure 9 The image shows the quantum yield of fluorescent dye I-1 obtained in Example 1 in water.

[0035] Figure 10 This is a fluorescence lifetime image of the fluorescent dye I-1 obtained in Example 1 in water.

[0036] Figure 11 The image shows the quantum yield of fluorescent dye I-2 obtained in Example 2 in water.

[0037] Figure 12 The image shows the fluorescence lifetime of fluorescent dye I-2 obtained in Example 2 in water.

[0038] Figure 13 The fluorescence intensity change of fluorescent dye I-1 obtained in Example 1 after a heating-cooling cycle.

[0039] Figure 14 The fluorescence intensity change of fluorescent dye I-2 obtained in Example 2 after a heating-cooling cycle.

[0040] Figure 15 The fluorescence intensity change of fluorescent dye I-1 obtained in Example 1 after 1 hour of illumination.

[0041] Figure 16 The fluorescence intensity changes of fluorescent dye I-1 obtained in Example 1 were continuously tested 50 times without interruption.

[0042] Figure 17 The fluorescence intensity change of fluorescent dye I-2 obtained in Example 2 after 1 hour of illumination.

[0043] Figure 18 The fluorescence intensity changes of fluorescent dye I-2 obtained in Example 2 were continuously tested 50 times without interruption. Detailed Implementation

[0044] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0045] The solvent DMF used in the examples was analytical grade and had not undergone further processing. It is available from Tianjin Fuyu Fine Chemical Co., Ltd.

[0046] Example 1

[0047] A method for preparing a water-soluble monobenzene ring fluorescent dye includes the following steps:

[0048] 400 mg of tetrafluoroterephthalonitrile was added to a round-bottom flask containing 10 mL of DMF and sonicated until fully dissolved. 1.45 g of triethylamine was added, and the mixture was heated to 80 °C and reacted at 80 °C for 4 hours. The reaction was monitored by TLC until complete. After the reaction was complete, 50 mL of dichloromethane and 100 mL of water were added to the reaction system for extraction. The lower organic phase was collected and washed three times with water (100 mL × 3). The obtained organic phase was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated and purified by column chromatography. The eluent was a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol was 10:0 to 9:1). After removing the solvent by rotary evaporation, the product was dried under vacuum at 60 °C for 5 hours to obtain a water-soluble monobenzene ring fluorescent dye, namely fluorescent dye I-1, with a yield of 80%.

[0049]

[0050] The fluorescent dye I-1 obtained in this embodiment in deuterated chloroform 19 F NMR spectrum as shown Figure 1 As shown, by Figure 1As can be seen, three different chemical shifts of F were identified, located at -130.44, -130.76, and -137.47 ppm, with an integrated area ratio of 1:1:1. This means that there are three F atoms in the compound with the same proportion but in different chemical environments, indicating that an aromatic nucleophilic substitution reaction has occurred, and one F atom on tetrafluoroterephthalonitrile has been substituted. Furthermore, the mass spectra of the fluorescent dye I-1 obtained in this example in negative ion mode are as follows: Figure 2 As shown. The theoretical simulated molecular weight of the structure shown by fluorescent dye I-1 is 198.0041. In negative ion mode, it is composed of... Figure 2 A nucleus-to-mass ratio of 196.9966 (MH) can be found. - The molecular ion peak. The fluorescent dye I-1 obtained in this example in deuterated chloroform... 13 The C NMR spectrum is as follows Figure 3 As shown, 13 C NMR (101 MHz, Chloroform-d) δ 143.22, 143.06, 123.85, 117.66, 107.24, 105.92, 105.88, 97.10. Based on the fluorescence of dye I-1 19 F NMR, 13 Analysis by C NMR and mass spectrometry confirmed the presence of fluorescent dye I-1.

[0051] Example 2

[0052] A method for preparing a water-soluble monobenzene ring fluorescent dye includes the following steps:

[0053] 400 mg of tetrafluoroterephthalonitrile was added to a round-bottom flask containing 10 mL of DMF and sonicated until fully dissolved. 500 mg of 4-dimethylaminopyridine was added, and the mixture was heated to 80 °C and reacted at 80 °C for 8 hours. The reaction was monitored by TLC until complete. After the reaction was completed, 50 mL of dichloromethane and 100 mL of water were added to the reaction system for extraction. The lower organic phase was collected and washed three times with water (100 mL × 3). The obtained organic phase was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated and purified by column chromatography. The eluent was a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol was 10:0 to 9:1). After removing the solvent by rotary evaporation, the product was dried under vacuum at 60 °C for 5 hours to obtain a water-soluble monobenzene ring fluorescent dye, namely fluorescent dye I-2, with a yield of 30%.

[0054]

[0055] The fluorescent dye I-2 obtained in this embodiment in deuterated dimethyl sulfoxide 19 F NMR spectrum as shown Figure 4 As shown, by Figure 4 As can be seen, only one chemical shift of F was identified, located at -147.10 ppm. This means that there is only one chemical environment for F in the compound, indicating that an aromatic nucleophilic substitution reaction occurred, and two F atoms on the tetrafluoroterephthalonitrile were substituted. Furthermore, the mass spectra of the fluorescent dye I-2 obtained in this example in negative ion mode are as follows: Figure 5 As shown, the theoretical simulated molecular weight of the structure of fluorescent dye I-2 is 196.0084. In negative ion mode, it is composed of... Figure 5 A nucleus-to-mass ratio of 194.9887 (MH) can be found. - The molecular ion peak. The fluorescent dye I-2 obtained in this example in deuterated dimethyl sulfoxide... 13 The C NMR spectrum is as follows Figure 6 As shown, 13 C NMR (101MHz, DMSO-d6) δ 164.12, 156.77, 149.83, 143.22, 142.40, 108.70, 108.03, 103.31. Based on 19 F NMR, 13 Analysis by C NMR and mass spectrometry confirmed the presence of the fluorescent dye I-2.

[0056] In this embodiment, the required reaction time and the choice of the base are crucial for the preparation of fluorescent dye I-2 and have a significant impact on the result of the aromatic nucleophilic substitution reaction. When the reaction time is less than 6 hours, TLC monitoring shows that the main product is fluorescent dye I-1. When the reaction time is greater than 6 hours, the formation of fluorescent dye I-2 is detected, but the yield is low. Regarding the choice of base, only the addition of 4-dimethylaminopyridine yields a certain amount of fluorescent dye I-2; with further increase in reaction time, fluorescent dye I-2 is gradually formed.

[0057] Comparative Example 1

[0058] A method for preparing a water-soluble monophenyl ring fluorescent dye is described in Example 2, except that 500 mg of potassium carbonate is used to replace 500 mg of 4-dimethylaminopyridine to obtain water-soluble monophenyl ring fluorescent dye I-1, with a separation yield of up to 80%. By TLC monitoring, the formation of fluorescent dye I-2 is almost undetectable, and the yield is extremely low, so it cannot be separated and purified by column chromatography to obtain fluorescent dye I-2 with a certain yield.

[0059] Experimental Example 1

[0060] 1. Solvation effect

[0061] The solvation effects of monobenzene ring fluorescent dye I-1 and fluorescent dye I-2 were determined. The concentration of the fluorescent dyes was 100 μmol / L. The fluorescence spectra of fluorescent dye I-1 in different solvents are shown in the figure. Figure 7 The excitation wavelength was 370 nm. The fluorescence spectra of fluorescent dye I-2 in different solvents are shown in the figure. Figure 8 The excitation wavelength is 400 nm. Fluorescent dyes exhibit good solubility in most organic solvents, especially water. With increasing solvent polarity, the fluorescence emission wavelength exhibits a redshift. Fluorescent dye I-1 displays bright blue fluorescence in both organic solvents and water, with emission wavelengths distributed in the visible light region, ranging from 380 nm to 600 nm, while exhibiting weaker blue fluorescence in solids. Fluorescent dye I-2 displays bright green fluorescence in both organic solvents and water, with emission wavelengths ranging from 400 nm to 650 nm. Furthermore, under the same conditions, fluorescent dye I-1 exhibits the strongest fluorescence intensity in water, emitting bright blue fluorescence. Similarly, fluorescent dye I-2 also exhibits the strongest fluorescence intensity in water, emitting bright green fluorescence.

[0062] 2. Quantum yield and lifetime testing:

[0063] Fluorescent dyes I-1 and I-2 were dissolved in PBS buffer at a concentration of 10 μmol / L, and quantum yield and lifetime were tested.

[0064] The emission peak of the fluorescent dye I-1 is located at 440 nm, and its quantum yield can reach 83%. Figure 9 The fluorescence lifetime is 14.3 ns. Figure 10 The emission peak of the fluorescent dye I-2 is located at 480 nm, and the quantum yield can reach 97%. Figure 11 The fluorescence lifetime is 6.0 ns. Figure 12 ).

[0065] 3. Thermal stability test:

[0066] Fluorescent dyes I-1 and I-2 were dissolved in water, each at a concentration of 100 μmol / L. Temperature-dependent fluorescence spectroscopy was performed on the aqueous solutions of the fluorescent dyes. The temperature was increased from 30℃ to 80℃ and then decreased back to 30℃, constituting one temperature cycle. The temperature gradient was 10℃. The change in fluorescence intensity of fluorescent dye I-1 at 440 nm was monitored. Figure 13 ), monitoring the fluorescence intensity change of fluorescent dye I-2 at 480 nm ( Figure 14 ).Depend on Figure 13-14 As can be seen, after a temperature cycling test, the fluorescence emission can be completely restored to its original intensity, indicating that fluorescent dyes I-1 and I-2 have excellent thermal stability.

[0067] 4. Light stability test:

[0068] Fluorescent dye I-1 was dissolved in water at a concentration of 100 μmol / L. Photostable properties were tested under 365 nm ultraviolet light, including irradiation time and number of irradiations. The aqueous solution was irradiated for 60 minutes, and the fluorescence intensity at 440 nm was monitored every 10 minutes. The results are shown below. Figure 15 ,Depend on Figure 15 It can be seen that the fluorescence intensity in water decreases to some extent over time, but remains essentially flat, ultimately maintaining 78% of the original fluorescence intensity. Furthermore, after multiple uninterrupted excitation fluorescence tests on the aqueous solution, the fluorescence intensity maintained 86% of the original intensity after 50 uninterrupted tests. Figure 16 This indicates that fluorescent dye I-1 has good photostability in water.

[0069] Similarly, fluorescent dye I-2 was dissolved in water at a concentration of 100 μmol / L. The photostability of this aqueous solution was tested under the same conditions as that of fluorescent dye I-1. After 60 minutes of light irradiation, the results showed that fluorescent dye I-2 retained 92% of its original fluorescence intensity after 1 hour of light irradiation. Figure 17 Meanwhile, after 50 uninterrupted fluorescence tests, the fluorescent dye I-2 maintained 97% of its original fluorescence intensity. Figure 18 The test results show that fluorescent dye I-2 has extremely high photostability, allowing it to withstand prolonged irradiation and undergo multiple fluorescence tests, thus providing possibilities for further detection.

Claims

1. A water-soluble monobenzene ring fluorescent dye, characterized in that, It has the structure shown in Equation I: ； In Formula I, the substituent R is fluorine or hydroxyl.

2. The method for preparing the water-soluble monobenzene ring fluorescent dye according to claim 1, comprising the following steps: Tetrafluoroterephthalonitrile was added to an organic solvent, stirred and dissolved, and then an alkali was added to carry out the reaction, resulting in a water-soluble monobenzene ring fluorescent dye.

3. The method for preparing a water-soluble monobenzene ring fluorescent dye according to claim 2, characterized in that, The organic solvent is N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, or acetonitrile; the volume ratio of the organic solvent to the mass ratio of tetrafluoroterephthalonitrile is 10-100 mL: 1 g.

4. The method for preparing a water-soluble monobenzene ring fluorescent dye according to claim 2, characterized in that, The base is an organic or inorganic base; the organic base is triethylamine, pyridine, 4-dimethylaminopyridine, or N,N-diisopropylethylamine; the inorganic base is potassium carbonate or cesium carbonate; the molar ratio of the base to tetrafluoroterephthalonitrile is 0.1-10:

1.

5. The method for preparing a water-soluble monobenzene ring fluorescent dye according to claim 2, characterized in that, The reaction temperature is 60-90℃; the reaction time is 0.5-10 hours.

6. The method for preparing a water-soluble monobenzene ring fluorescent dye according to claim 2, characterized in that, When the base is 4-dimethylaminopyridine, a water-soluble monobenzene ring fluorescent dye with hydroxyl substituent R in Formula I is obtained when the reaction time is 6-10 h; when the reaction time is 0.5-6 h, a water-soluble monobenzene ring fluorescent dye with fluorine substituent R in Formula I is obtained.

7. The method for preparing a water-soluble monobenzene ring fluorescent dye according to claim 2, characterized in that, The post-processing steps of the reaction solution are as follows: After the reaction is completed, dichloromethane and water are added to the reaction solution for extraction; the lower organic phase is washed with water 3-5 times, and then the organic phase is dried with anhydrous sodium sulfate to remove the solvent and obtain the crude product. The crude product is purified by column chromatography and dried under vacuum to obtain the product.

8. The method for preparing a water-soluble monobenzene ring fluorescent dye according to claim 7, characterized in that, The eluent for the column chromatography is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol in the mixed solution is 10:0 to 9:

1.

9. The application of the water-soluble monobenzene ring fluorescent dye of claim 1 in anti-counterfeiting and environmental detection.

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