CN115677554A - 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission, and preparation method and application thereof - Google Patents
3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of fluorescence sensors and discloses 3,4-disulfide maleimide derivative fluorescent molecules based on aggregation-induced emission and a preparation method and application thereof. The invention has the beneficial effects that: the fluorescent molecular probe has stronger fluorescence intensity, and the higher the concentration is, the stronger the fluorescence emission is, has significant AIE characteristics when probes are used with H 2 Quenching of fluorescence intensity after S contact to achieve H 2 Examination of SThe ACQ effect is effectively avoided; and in detecting H 2 The S is not easily interfered by other potential interferents, has high sensitivity, strong selectivity and small cytotoxicity, and is applied to the field of hydrogen evolution (H) 2 The S detection has important application value.
Description
Technical Field
The invention relates to the technical field of fluorescence sensors, in particular to 3,4-disulfide maleimide derivative fluorescent molecules based on aggregation-induced emission and a preparation method and application thereof.
Background
Hydrogen sulfide (H) 2 S) is a common toxic gas molecule with the smell of the rotten eggs, and researches show that H in organisms 2 S is a third type of endogenous ' gas signal molecule ' of organisms except CO and NO, and the abnormal expression of the concentration of the S is closely related to the occurrence and development of a plurality of diseases, such as Huntington ' S disease, alzheimer disease, diabetes, liver cirrhosis and the like. Thus, for H 2 Detection of S, in particular H in vivo 2 The detection of S is of great significance. At present, related to H 2 The detection method of S mainly comprises spectrophotometry, gas chromatography, electrochemistry and polarography. Although these techniques have many advantages, their disadvantages are not negligible, such as high cost, complicated sample processing, and the need to destroy the integrity of the sample during sample preparation, thereby causing H in the organism 2 The detection application of S is greatly limited.
In recent years, the small-molecule fluorescent probe has the advantages of high sensitivity, good selectivity, high spatial resolution, good biocompatibility, suitability for in-situ detection and the like, and is H 2 S and other bioactive molecules provide an effective way to detect. However, when the conventional fluorophore is in a high concentration Aggregation state, the intermolecular interaction is enhanced, which causes energy to be dissipated by non-radiative energy transition, so that an Aggregation induced Quenching (ACQ) effect appears, and the luminescence property is affected. Due to the hydrophobic characteristic of the conventional fluorescent probe, the probe can be still aggregated in a hydrophobic cavity of some macromolecules such as protein and the like even in a dilute solution stateExcessive aggregation causes local probe concentration increase and results in fluorescence quenching, which may interfere with the detection result, greatly limiting the application of fluorescent probes.
In contrast to ACQ dyes, aggregation Induced Emission (AIE) fluorescent dyes have a rotatable structure or a twisted propeller-shaped conformation, and can emit light with high efficiency due to the blocking of intramolecular movement caused by aggregation, especially when the concentration of a solution is higher, the fluorescence emission thereof is stronger. Therefore, AIE fluorescent probes are an ideal candidate for detecting active species in organisms. For example, the Chinese patent publication with publication number CN108641711A discloses a H 2 The method comprises the following steps of (1) synthesizing a target product by taking 2-azido-9-fluorenone, salicylaldehyde and salicylaldehyde monohydrazone as raw materials through two different ways, wherein the target compound can respond to trace substances of a human body, namely hydrogen sulfide; however, this technique does not mention how toxic the probe is to cells in the organism, and the selectivity and sensitivity in the detection process are still to be further improved.
Disclosure of Invention
The technical problem to be solved by the invention is how to provide a 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission and a preparation method thereof, and the prepared fluorescent molecule is applied to H 2 The S detection has the advantages of good selectivity, high sensitivity and low cytotoxicity.
The invention solves the technical problems through the following technical means:
the invention provides a 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission, which has the following molecular structural formula:
has the advantages that: the 3,4-dithioether maleimide derivative fluorescent molecule is used as a probe, on one hand, the probe has strong fluorescence intensity, and fluorescence emission is carried out when the concentration of a probe compound is higherThe stronger the probe compound, the more pronounced AIE properties are exhibited when the probe compound is reacted with H 2 After S contact, the fluorescence intensity of the probe is completely quenched, thereby realizing H 2 The detection of S effectively avoids the ACQ effect; on the other hand, the fluorescent molecule pair H 2 S identification shows good selectivity and sensitivity, and H detection is carried out 2 The S is not easily interfered by other potential interferents such as anions, cations, biomolecules and other common active substances in the process of H 2 S detection has the advantages of high sensitivity and high selectivity.
In addition, the 3,4-dithioether maleimide derivative fluorescent molecule has strong cell permeability and low cytotoxicity, and can realize H in vivo by using the fluorescent molecule as a probe 2 Detection of S, H in biomedicine and Living systems 2 S detection has extremely important application value.
The invention provides a preparation method of 3,4-dithioether maleimide derivative fluorescent molecules based on aggregation-induced emission, which comprises the following steps: adding anhydrous sodium acetate into a 4- (3,4-dichloromaleimide) benzaldehyde solution, uniformly stirring, then dropwise adding a 3,5-bis (trifluoromethyl) thiophenol solution under the protection of inert gas, removing an organic solvent under reduced pressure after stirring reaction is finished, and separating and purifying to obtain a yellow solid 3,4-dithioether maleimide derivative fluorescent molecule.
Preferably, the molar ratio of 4- (3,4-dichloromaleinamide) benzaldehyde to 3,5-bis (trifluoromethyl) thiophenol is 1:1 to 1:10.
preferably, the molar ratio of the 4- (3,4-dichloromaleimide) benzaldehyde to anhydrous sodium acetate is 10:1 to 1:10.
preferably, the 4- (3,4-dichloromaleinamide) benzaldehyde and the 3,5-bis (trifluoromethyl) thiophenol are both dissolved by using an organic solvent, wherein the organic solvent is at least one of tetrahydrofuran, dichloromethane, chloroform and acetone.
Preferably, the stirring temperature is 0-50 ℃ and the stirring time is 8-48 h.
Preferably, the separation and purification adopts column chromatography for purification.
Preferably, the reaction equation of the preparation process of the 3,4-dithioether maleimide derivative fluorescent molecule is as follows:
preferably, the preparation method of the 4- (3,4-dichloromaleimido) benzaldehyde comprises the following steps: dissolving aminobenzaldehyde and 3,4-dichloromaleic anhydride in glacial acetic acid, refluxing for 12-48 h at 80-150 ℃, cooling to room temperature, filtering and collecting yellow precipitate, washing the precipitate by using an organic solvent precooled at-4-0 ℃, and removing the organic solvent by vacuum drying to obtain yellow solid 4- (3,4-dichloromaleic amide) benzaldehyde.
Preferably, the molar ratio of the p-aminobenzaldehyde to the 3,4-dichloromaleic anhydride is 1.0:1.0 to 3.0.
Preferably, the organic solvent is at least one of methanol and dichloromethane.
Preferably, the preparation method of the paraaminobenzaldehyde comprises the following steps: dissolving 4-acetamino benzaldehyde in methanol, adding sodium hydroxide water solution, refluxing at 60-65 deg.c for 1-5 hr, cooling to room temperature, extracting with chlorine dioxide, collecting organic phase, washing, drying, decompression eliminating organic solvent, separating and purifying to obtain yellowish solid p-aminobenzaldehyde.
Preferably, the molar ratio of 4-acetamidobenzaldehyde to sodium hydroxide is 1.0:3.0 to 5.0.
Preferably, the crude product of the p-aminobenzaldehyde is separated and purified by adopting a medium-pressure chromatographic column, and the volume ratio of petroleum ether of 2:1: dichloromethane mixed solution is used as eluent.
Preferably, the preparation method of the 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission comprises the following specific steps:
(1) Preparing p-aminobenzaldehyde: dissolving 4-acetamidobenzaldehyde in methanol, and adding an aqueous solution of sodium hydroxide to obtain a molar ratio of 4-acetamidobenzaldehyde to sodium hydroxide of 1.0:3.0 to 5.0; carrying out reflux reaction on the mixed solution at the temperature of between 60 and 65 ℃ for 1 to 5 hours, cooling the mixed solution to room temperature, extracting the mixed solution by using chlorine dioxide, collecting an organic phase, washing and drying the organic phase, removing the organic solvent under reduced pressure, and separating and purifying a crude product by using a medium-pressure chromatographic column to obtain a light yellow solid p-aminobenzaldehyde;
(2) Preparation of 4- (3,4-dichloromaleimido) benzaldehyde: dissolving the p-aminobenzaldehyde and 3,4-dichloromaleic anhydride in glacial acetic acid, wherein the molar ratio of the p-aminobenzaldehyde to 3,4-dichloromaleic anhydride is 1.0:1.0 to 3.0; carrying out reflux reaction on the mixed system at the temperature of 80-150 ℃ for 12-48 h, cooling to room temperature, filtering and collecting yellow precipitate, washing the precipitate by using precooled methanol at the temperature of-4-0 ℃ and dichloromethane, and drying in vacuum to remove an organic solvent to obtain yellow solid 4- (3,4-dichloromaleimide) benzaldehyde;
(3) Preparation of 3,4-dithioethermaleimide derivative fluorescent molecule: anhydrous sodium acetate was added to an organic solution of 4- (3,4-dichloromaleamide) benzaldehyde at a molar ratio of 4- (3,4-dichloromaleamide) benzaldehyde to anhydrous sodium acetate of 10:1 to 1:10, after stirring uniformly, adding 3,5-bis (trifluoromethyl) thiophenol solution dropwise under the protection of inert gas, so that the molar ratio of 4- (3,4-dichloromaleamide) benzaldehyde to 3,5-bis (trifluoromethyl) thiophenol is 1:1 to 1:10; and after the dropwise addition, stirring the mixed system at the temperature of 0-50 ℃ for 8-48 h, decompressing after the reaction is finished, removing the organic solvent, and separating and purifying to obtain the yellow solid 3,4-disulfide maleimide derivative fluorescent molecule.
Preferably, the reaction equation of the preparation process of the 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission is as follows:
the third aspect of the invention provides a method for detecting H in preparation of 3,4-dithioether maleimide derivative fluorescent molecules based on aggregation-induced emission 2 Application in S fluorescent probe.
Has the advantages that: this applicationPlease show that the 3,4-dithioether maleimide derivative based on aggregation-induced emission has excellent fluorescent molecular luminescence property and can be used as sensitive 'on-off' type H 2 The S fluorescent probe has extremely important application value in the fields of biosensors, fluorescent probes, fluorescent imaging, organic light-emitting devices and the like.
Preferably, the 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission is used as H 2 The application of the S fluorescent probe in cell imaging.
Has the beneficial effects that: the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission is applied to cell imaging as an 'on-off' type fluorescent probe, and H in biomedicine and a life system can be efficiently and accurately realized 2 And S, detecting.
The invention has the advantages that:
1. the 3,4-dithioether maleimide derivative fluorescent molecule is used as a probe, on one hand, the probe has strong fluorescence intensity, the fluorescence emission is stronger when the concentration of a probe compound is higher, the AIE characteristic is obvious, and the probe compound and H are combined to form the fluorescent molecule 2 After S contact, the fluorescence intensity of the probe is completely quenched, thereby realizing H 2 The detection of S effectively avoids the ACQ effect; on the other hand, the fluorescent molecule pair H 2 S identification shows good selectivity and sensitivity, and H detection is carried out 2 The S is not easily interfered by other potential interferents such as anions, cations, biomolecules and other common active substances in the process of H 2 S detection has the advantages of high sensitivity and high selectivity; in addition, the 3,4-dithioethermaleimide derivative fluorescent molecule has strong cell permeability and low cytotoxicity, and can realize H in vivo by being used as a probe 2 Detection of S, H in biomedicine and Living systems 2 S detection has extremely important application value;
2. the preparation method is simple, and the prepared 3,4-disulfide maleimide derivative based on aggregation-induced emission has excellent fluorescent molecular luminescence performance and can be used as sensitive 'on-off' type H 2 The S fluorescent probe has extremely important application value in the fields of biosensors, fluorescent probes, fluorescent imaging, organic light-emitting devices and the like;
3. the 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission is applied to cell imaging as an 'on-off' type fluorescent probe, and H in biomedicine and a life system can be efficiently and accurately realized 2 And S, detecting.
Drawings
FIG. 1 is a fluorescence emission spectrum of 3,4-dithioether maleimide derivative fluorescent molecules in different volume fractions of water based on aggregation-induced emission in example 4 of the present invention.
FIG. 2 is the fluorescence emission spectra of the fluorescent molecules of 3,4-dithioethermaleimide derivative based on aggregation-induced emission in PBS/DMSO solution with different concentrations of NaHS in example 5 of the present invention.
FIG. 3 is a linear graph of the fluorescence intensity of the fluorescent molecules of 3,4-dithioether maleimide derivative in PBS/DMSO solution with different concentrations of NaHS based on aggregation-induced emission in example 5 of the present invention.
FIG. 4 is a bar graph showing the change in KJ fluorescence intensity of compound before and after adding NaHS to PBS/DMSO solution of 3,4-dithioether maleimide derivative fluorescent molecules based on aggregation-induced emission in example 6 of the present invention in the presence of other interferents.
FIG. 5 is a cytotoxicity plot of 3,4-dithioethermaleimide derivative fluorescent molecules based on aggregation-induced emission in example 7 of the present invention.
FIG. 6 shows the application of 3,4-dithioethermaleimide derivative fluorescent molecules based on aggregation-induced emission in Hela cells in example 8 of the present invention 2 Fluorescence imaging of S.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.
Example 1
In a first aspect, this embodiment provides a 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission, having the following molecular structural formula:
the second aspect of the present invention provides a method for preparing 3,4-dithioethermaleimide derivative fluorescent molecules based on aggregation-induced emission, comprising the following steps:
(1) Preparing p-aminobenzaldehyde: in a 250mL round-bottom flask, 4-acetylaminobenzaldehyde (3.5 g,21.5 mmol) was dissolved in 140mL of methanol, and then 50mL of an aqueous solution of sodium hydroxide (3.43g, 85.7 mmol) was added to the above solution; refluxing the mixed solution at 65 ℃ for 2h, cooling to room temperature after the reaction is finished, adding 100mL of water into the system, extracting with dichloromethane (50mL x 4), collecting an organic phase, washing with a saturated saline solution, drying with anhydrous sodium sulfate, and removing the organic solvent under vacuum condition; then, a medium-pressure chromatographic column is adopted, and petroleum ether and dichloromethane in the volume ratio of 2:1 are used as eluent to separate and purify the crude product, so that a light yellow solid product is obtained. The prepared product is subjected to nuclear magnetic hydrogen spectrum characterization, and the result is as follows:
1 H NMR(400MHz,CDCl 3 ,δppm):9.67(s,1H),7.61(d,J＝8.6Hz,2H),6.75(d,J＝8.6Hz,2H),5.71(s,2H)。
the obtained product is p-aminobenzaldehyde and the yield is 75%.
(2) Preparation of 4- (3,4-dichloromaleimido) benzaldehyde: dissolving the prepared p-aminobenzaldehyde (1g, 8.26mmol) and 3,4-dichloromaleic anhydride (1.37g, 8.26mmol) in 30mL of glacial acetic acid, carrying out reflux reaction on the mixed system at 120 ℃ for 12h, cooling to room temperature after the reaction is finished, filtering by using a Buchner funnel, collecting yellow precipitates, washing the precipitates by using precooled methanol and dichloromethane in a refrigerator at-4 ℃, and carrying out vacuum drying to remove an organic solvent to obtain a yellow solid product. The prepared product was subjected to nuclear magnetic hydrogen spectrum characterization, and the results were as follows:
1 H NMR(400MHz,CDCl 3 ,δppm):10.02(s,1H),7.97(d,J＝8.4Hz,2H),7.59ppm(d,J＝8.4Hz,2H)。
the product was 4- (3,4-dichloromaleimido) benzaldehyde and the yield was 85%.
(3) Preparation of 3,4-dithioethermaleimide derivative fluorescent molecule: at room temperature, adding anhydrous sodium acetate solid (205mg, 2.5 mmol) into 10mL of anhydrous tetrahydrofuran solution dissolved with a compound 4- (3,4-dichloromaleimido) benzaldehyde (269mg, 1mmol), stirring for 10 minutes, slowly dropwise adding 1mL of tetrahydrofuran solution containing 2.2mmol3, 5-bis (trifluoromethyl) thiophenol into the solution under the protection of nitrogen, reacting the mixed system at room temperature for 12 hours after dropwise adding, removing the organic solvent under reduced pressure under vacuum after the reaction is finished, and separating and purifying to obtain yellow solid. Respectively performing nuclear magnetic hydrogen spectrum and carbon spectrum characterization on the prepared product, wherein the results are as follows:
1 H NMR(400MHz,d 6 -DMSO,δppm):10.07(s,1H),8.12(d,J＝8.0Hz,2H),7.94(s,2H),7.76(s,4H),7.71ppm(d,J＝8.0Hz,2H)。
13 C NMR(600MHz,d 6 -DMSO,δppm):192.8，166.3，137.0，135.48，132.86，131.38，131.0，130.8，130.7，130.2，126.7，124.5，121.8，121.6。HR-MS(ESI)：m/z＝689.0003。
the obtained product was 3,4-dithioethermaleimide derivative fluorescent molecule (expressed by KJ) in 80% yield.
The reaction equation of the preparation process is as follows:
example 2
The embodiment provides a preparation method of 3,4-dithioether maleimide derivative fluorescent molecules based on aggregation-induced emission, which comprises the following steps:
(1) Preparing p-aminobenzaldehyde: in a 250mL round-bottom flask, 4-acetamidobenzaldehyde (3.5g, 21.5 mmol) was dissolved in 140mL of methanol, and then 50mL of an aqueous solution of sodium hydroxide (2.57g, 64.5 mmol) was added to the above solution; refluxing the mixed solution at 63 ℃ for 1h, cooling to room temperature after the reaction is finished, adding 100mL of water into the system, extracting with dichloromethane (50mL x 4), collecting an organic phase, washing with a saturated saline solution, drying with anhydrous sodium sulfate, and removing the organic solvent under vacuum condition; then, a medium-pressure chromatographic column is adopted, petroleum ether and dichloromethane in a volume ratio of 2:1 are used as eluent to separate and purify the crude product, and a light yellow solid product, namely the p-aminobenzaldehyde, is obtained, wherein the yield is 64%.
(2) Preparation of 4- (3,4-dichloromaleimido) benzaldehyde: dissolving the prepared p-aminobenzaldehyde (1g, 8.26mmol) and 3,4-dichloromaleic anhydride (2.74g, 16.5 mmol) in 30mL of glacial acetic acid, carrying out reflux reaction on the mixed system at 80 ℃ for 48h, cooling to room temperature after the reaction is finished, filtering by using a Buchner funnel, collecting yellow precipitates, washing the precipitates by using methanol and dichloromethane precooled in a refrigerator at-4 ℃, and carrying out vacuum drying to remove an organic solvent to obtain a yellow solid product, namely 4- (3,4-dichloromaleic amide) benzaldehyde, wherein the yield is 70%.
(3) Preparation of 3,4-dithioethermaleimide derivative fluorescent molecule: at room temperature, anhydrous sodium acetate solid (205mg, 2.5 mmol) is added into 10mL anhydrous tetrahydrofuran solution dissolved with compound 4- (3,4-dichloromaleimido) benzaldehyde (269mg, 1mmol), after stirring for 10 minutes, 1mL tetrahydrofuran solution containing 5mmol of 3,5-bis (trifluoromethyl) thiophenol is slowly dripped into the solution under the protection of nitrogen, after the dripping is finished, the mixed system reacts for 48 hours at 10 ℃, after the reaction is finished, the organic solvent is removed under the reduced pressure under the vacuum condition, and the yellow solid product 3,4-disulfide maleimide derivative fluorescent molecule (expressed by KJ) is obtained through separation and purification, wherein the yield is 78%.
The reaction equation of the preparation process is as follows:
example 3
The embodiment provides a preparation method of 3,4-dithioether maleimide derivative fluorescent molecules based on aggregation-induced emission, which comprises the following steps:
(1) Preparing p-aminobenzaldehyde: in a 250mL round-bottom flask, 4-acetamidobenzaldehyde (3.5 g,21.5 mmol) was dissolved in 140mL methanol and then 50mL aqueous sodium hydroxide (4.28g, 107.5 mmol) was added to the solution; refluxing the mixed solution at 60 ℃ for 5 hours, cooling to room temperature after the reaction is finished, adding 100mL of water into the system, extracting with dichloromethane (50mL x 4), collecting an organic phase, washing with a saturated saline solution, drying with anhydrous sodium sulfate, and removing the organic solvent under vacuum condition; then, the crude product is separated and purified by adopting a medium-pressure chromatographic column and taking petroleum ether and dichloromethane with the volume ratio of 2:1 as eluent, and a light yellow solid product, namely the p-aminobenzaldehyde, is obtained, wherein the yield is 72%.
(2) Preparation of 4- (3,4-dichloromaleimido) benzaldehyde: p-aminobenzaldehyde (1g, 8.26mmol) prepared above and 3,4-dichloromaleic anhydride (4.11g, 24.8mmoll) were dissolved in 30mL of glacial acetic acid, and the mixture system was refluxed at 150 ℃ for 25 hours, cooled to room temperature after the reaction was completed, filtered with a buchner funnel and collected a yellow precipitate, and the precipitate was washed with methanol and dichloromethane precooled in a refrigerator at-4 ℃, and vacuum-dried to remove the organic solvent, to obtain 4- (3,4-dichloromaleic amide) benzaldehyde as a yellow solid product with a yield of 80%.
(3) Preparation of 3,4-dithioethermaleimide derivative fluorescent molecule: at room temperature, anhydrous sodium acetate solid (205mg, 2.5 mmol) is added into 10mL anhydrous tetrahydrofuran solution dissolved with compound 4- (3,4-dichloromaleimido) benzaldehyde (269mg, 1mmol), after stirring for 10 minutes, 1mL tetrahydrofuran solution containing 10mmol of 3,5-bis (trifluoromethyl) thiophenol is slowly dripped into the solution under the protection of nitrogen, after the dripping is finished, the mixed system reacts for 8 hours at 50 ℃, after the reaction is finished, the organic solvent is removed under reduced pressure under vacuum condition, and the yellow solid product 3,4-disulfide maleimide derivative fluorescent molecule (expressed by KJ) is obtained through separation and purification, wherein the yield is 75%.
The reaction equation of the preparation process is as follows:
example 4
In this example, AIE characteristics of 3,4-dithioethermaleimide derivative fluorescent molecules prepared as above were studied, and the specific method included: 11 centrifuge tubes were prepared, 3ml of 3,4-dithioether maleimide derivative fluorescent molecules (KJ) prepared in example 1, with a concentration of 10 μ M, were taken by pipette and added to different centrifuge tubes, respectively, and then DMSO and deionized water were added, respectively, so that the volume fractions of deionized water to DMSO in each centrifuge tube were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, respectively, and after mixing uniformly, fluorescence emission spectra were measured in a fluorescence emission spectrometer with a fluorescence excitation wavelength of 425nm and a slit of 10/10nm, respectively, and the results are shown in fig. 1.
As can be seen from fig. 1, when the volume fraction of water is less than 50%, compound KJ hardly fluoresces because DMSO is a benign solvent for KJ, and when its volume fraction is large, KJ is in a state of low concentration in the test solution. After the volume fraction of water reaches 50%, the fluorescence intensity of the compound KJ is also obviously enhanced along with the gradual increase of the volume fraction of water, particularly, the fluorescence intensity of the compound KJ is strongest when the volume fraction of water reaches 80% -90%, which indicates that the compound KJ has obvious AIE characteristics, because when the volume fraction of water is higher, water is used as a poor solvent of KJ, KJ is in a highly aggregated state in DMSO, the rotation of thiol bonds is inhibited, the conjugate plane of the structure is increased, and the fluorescence intensity is also enhanced; whereas when the volume of water is higher than 90%, the fluorescence intensity is slightly reduced due to the precipitation of molecular aggregates, but compound KJ still has significant AIE properties.
Example 5
The embodiment provides an application of 3,4-dithioether maleimide derivative fluorescent molecules based on aggregation-induced emission, in particular to KJ to H 2 Fluorescence response detection of S, hydrolysis of NaHS in solution to generate H 2 S, and H 2 The concentration of S is positively correlated with the concentration of NaHS, and H with different concentrations can be obtained by adding NaHS with different concentrations into the KJ solution 2 And (5) preparing an S solution. The specific method comprises the following steps: PBS, DMSO as 9:1 to give a PBS/DMSO mixture, and 3,4-dithioethermaleimide derivative fluorescent molecules prepared in example 1 were dissolved in the PBS/DMSO mixture to give a KJ solution at a concentration of 10. Mu.M. Adding the KJ solution into different cuvettes, dripping different amounts of NaHS solution into the cuvettes to ensure that the NaHS concentration in the solution is respectively 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 25 mu M, fully shaking and mixing the solution at room temperature, and respectively carrying out fluorescence spectrum scanning in a fluorescence emission spectrometer with the fluorescence excitation wavelength of 425nm and the slit of 10/10nm, wherein the fluorescence spectrum of the NaHS with different concentrations is shown in figure 2.
As can be seen from fig. 2, compound KJ itself has significant fluorescence intensity when NaHS concentration in solution is 0 μ M; with the continuous increase of the NaHS concentration, the fluorescence intensity of the compound KJ at 540nm is obviously quenched; when the concentration of NaHS reached 25. Mu.M, the fluorescence was almost completely quenched. The fluorescence of the KJ solution is quenched from yellow after the NaHS is continuously added. This indicates that the compound KJ is present against H 2 S exhibits an "on-off" type fluorescent response.
For the above experiments, the NaHS concentrations were 0, 2, 4, 6, 8, 10, 12, 14, 16, 18. Mu.M and correspond to a fluorescence at 540nmLinear relationship of intensity As shown in FIG. 3, the linear fit results in the equation y =1915.2364-88.0318x (R) 2 = 0.998), the detection limit was calculated by the formula LOD =3 σ/k, where the value of k is the slope, the value of σ is the background noise, and the detection limit was calculated to be 75nM.
Example 6
The embodiment provides application of 3,4-dithioether maleimide derivative fluorescent molecules based on aggregation-induced emission, and particularly provides application of the fluorescent molecules in exploring KJ to H through competitive experiments of other interfering substances 2 The specific detection method of S comprises the following steps: PBS, DMSO as 9:1 to give a PBS/DMSO mixture, and 3,4-dithioethermaleimide derivative fluorescent molecules prepared in example 1 were dissolved in the PBS/DMSO mixture to give a KJ solution at a concentration of 10. Mu.M. Adding the KJ solution into different cuvettes, and adding other potential interferents such as anion solution (F) - 、Cl - 、Br - 、I - 、PO 4 3- 、NO 2 - 、NO 3 - 、HCO 3 - 、CO 3 2- 、HSO 3 - 、SO 3 2- 、HSO 4 - 、SO 4 2- 、A C O - ) Cationic solution (Na) + 、K + 、Mg 2+ 、Zn 2+ 、Ca 2+ ) A biomolecule (. OH. A. C.), 1 O 2 、H 2 O 2 、ONOO - 、TBHP、Vc、GSH、Cys、H 2 S), after fully shaking and mixing at room temperature, respectively carrying out fluorescence spectrum scanning in a fluorescence emission spectrometer with the fluorescence excitation wavelength of 425nm and the slit of 10/10 nm; then, a NaHS solution was added at a concentration of 25. Mu.M, and the fluorescence spectrum was scanned again. The bar graph of the change in KJ fluorescence intensity of the compound before and after addition of NaHS under the condition of co-existence of interferents is shown in FIG. 4.
As can be seen from fig. 4, when a potential interfering substance was added to the KJ solution, it hardly caused a significant change in fluorescence; whereas addition of NaHS to KJ solution containing different potential interferents almost completely quenched the fluorescence, indicating H 2 S InductionThe fluorescent response of (a) is not affected by other interferents. Thus, the 3,4-dithioethermaleimide derivative fluorescent molecule KJ based on aggregation-induced emission was directed against H in various competitive analytes 2 S detection has excellent selectivity and interference resistance.
Example 7
The embodiment provides an application of 3,4-dithioether maleimide derivative fluorescent molecules based on aggregation-induced emission, and the KJ prepared in the embodiment 1 is subjected to cytotoxicity research through an MTT (3- (4,5-dimethylthiazole-2) -2,5 diphenyl tetrazole bromide) colorimetric method, and the specific method comprises the following steps: the 3,4-dithioether maleimide derivative fluorescent molecules prepared in example 1 were dissolved in DMSO to obtain KJ mother solutions, and then different amounts of KJ mother solutions were added to the cell culture solution to obtain fluorescent molecule KJ (0, 20, 40, 60, 80, 100 μ M) culture solutions with different concentration gradients for use. Inoculating the human squamous uterine carcinoma cells SiHa in logarithmic growth phase into a 96-well microplate to a cell inoculation density of 5000 cells/well, placing at 37 ℃,5% CO 2 After the cells adhere to the wall for 16h, the old culture medium is discarded, and fluorescent molecule KJ (0, 20, 40, 60, 80, 100 mu M) culture solutions with different concentration gradients are respectively added, and untreated cells are used as a control group, and 6 parallel holes are simultaneously arranged. After the culture is continued for 24 hours, removing the culture solution containing the fluorescent molecules KJ, then adding 20 mu L of MTT with the concentration of 5.0mg/mL into each well, placing the well in an incubator for 4 hours, removing old solution, adding 150 mu L of DMSO, placing the well in a shaking table, fully shaking for 10min, detecting the absorbance of each well at 490nm on an enzyme-linked immunosorbent assay detector, and calculating the cell survival rate of each experimental group according to a cell survival rate calculation formula as follows:
SR＝(A/A 0 )*100％
wherein SR is cell survival rate, A is absorbance value of experimental group, A is 0 Absorbance values for the blank control.
The results of SiHa cell viability at different concentrations of compound KJ are shown in figure 5. As can be seen from fig. 5, cell survival decreased with the increase of the concentration of compound KJ, but the cell survival rate remained at 80% or more even at the concentration of KJ as high as 100 μ M, and thus it was revealed that compound KJ had very low cytotoxicity.
Example 8
The embodiment provides application of 3,4-dithioether maleimide derivative fluorescent molecules based on aggregation-induced emission, and particularly relates to application of the fluorescent molecules in H in SiHa cells 2 The fluorescence imaging of S comprises the following specific steps: the 3,4-dithioethermaleimide derivative fluorescent molecule prepared in example 1 was prepared in cell culture broth to give a KJ solution with a concentration of 10 μ M. Adding it to previously cultured SiHa cells, at 37 deg.C, 5% 2 Incubating in a cell incubator for 30min, washing the cells three times with cold PBS, and removing the KJ solution which does not enter the cells; incubation was then continued for 30min with 30 μ M NaHS solution, washed twice with cold PBS, and immediately followed by intracellular fluorescence imaging using confocal fluorescence microscopy. The wavelength of the excitation light is 405nm, the receiving wavelength is 420-600 nm, and KJ is obtained and used for H in SiHa cells 2 The fluorescence image of S is shown in FIG. 6.
In FIG. 6, the two images on the left side are fluorescence images, the two images on the right side are bright field images of cells, and the middle image corresponds to the Merge image of the left and right images in the same row; the three images in the upper row are fluorescence images of SiHa cells when the NaHS solution is not added, and the three images in the lower row are fluorescence images of SiHa cells after the NaHS solution is added. It can be seen that cells treated with compound KJ alone have a clear yellow fluorescence, whereas when H was added 2 After S, yellow fluorescence in cells is obviously quenched, which shows that the 3,4-dithioether maleimide derivative fluorescent molecule KJ based on aggregation-induced emission has good cell membrane permeability; meanwhile, the fluorescent molecule KJ can detect H in living cells 2 S。
The implementation principle of the application is as follows: the 3,4-dithioether maleimide derivative fluorescent molecule is used as a probe, on one hand, the probe has strong fluorescence intensity, the fluorescence emission is stronger when the concentration of a probe compound is higher, the AIE characteristic is obvious, and the probe compound and H are combined to form the fluorescent molecule 2 Complete quenching of the fluorescence intensity of the probe occurs after S contact, therebyRealization of H 2 The detection of S effectively avoids the ACQ effect; on the other hand, the fluorescent molecule pair H 2 S identification shows good selectivity and sensitivity, and H detection is carried out 2 The S is not easily interfered by other potential interferents such as anions, cations, biomolecules and other common active substances in the process of H 2 S detection has the advantages of high sensitivity and high selectivity; in addition, the 3,4-dithioether maleimide derivative fluorescent molecule has strong cell permeability and low cytotoxicity, and can realize H in vivo by using the fluorescent molecule as a probe 2 Detection of S, H in biomedical and Life systems 2 S detection has extremely important application value.
The preparation method is simple, and the prepared 3,4-disulfide maleimide derivative based on aggregation-induced emission has excellent fluorescent molecule luminescent performance and can be used as sensitive 'on-off' type H 2 The S fluorescent probe has extremely important application value in the fields of biosensors, fluorescent probes, fluorescent imaging, organic light-emitting devices and the like.
The 3,4-disulfide maleimide derivative fluorescent molecule based on aggregation-induced emission is applied to cell imaging as an 'on-off' type fluorescent probe, and H in biomedicine and a life system can be efficiently and accurately realized 2 And S, detecting.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission is characterized in that: has the following molecular structural formula:
2. the method of claim 1 for preparing 3,4-dithioethermaleimide derivative fluorescent molecules based on aggregation-induced emission, wherein: the method comprises the following steps: adding anhydrous sodium acetate into a 4- (3,4-dichloromaleimide) benzaldehyde solution, uniformly stirring, then dropwise adding a 3,5-bis (trifluoromethyl) thiophenol solution under the protection of inert gas, removing an organic solvent under reduced pressure after stirring reaction is finished, and separating and purifying to obtain a yellow solid 3,4-dithioether maleimide derivative fluorescent molecule.
3. The method for preparing 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation induced emission as claimed in claim 2, wherein: the molar ratio of the 4- (3,4-dichloromaleimido) benzaldehyde to the 3,5-bis (trifluoromethyl) thiophenol is 1:1 to 1:10.
4. the method for preparing 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation induced emission as claimed in claim 2, wherein: the molar ratio of the 4- (3,4-dichloromaleimide) benzaldehyde to anhydrous sodium acetate is 10:1 to 1:10.
5. the method for preparing 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation induced emission as claimed in claim 2, wherein: the 4- (3,4-dichloromaleinamide) benzaldehyde and 3,5-bis (trifluoromethyl) thiophenol are both dissolved by an organic solvent, and the organic solvent is at least one of tetrahydrofuran, dichloromethane, chloroform and acetone.
6. The method for preparing 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation induced emission as claimed in claim 2, wherein: the reaction temperature is 0-50 ℃, and the reaction time is 8-48 h.
7. The method for preparing 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation induced emission as claimed in claim 2, wherein: and (4) separating and purifying by adopting a column chromatography.
8. The method for preparing 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation induced emission as claimed in claim 2, wherein: the reaction equation of the preparation process of the 3,4-dithioether maleimide derivative fluorescent molecule is as follows:
9. the method for detecting H in the preparation of the 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission according to claim 1 2 Application in S fluorescent probe.
10. The use of 3,4-dithioethermaleimide derivative fluorescent molecules based on aggregation-induced emission as claimed in claim 9, wherein: the 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission is used as H 2 The application of the S fluorescent probe in cell imaging.
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