Design, synthesis and bactericidal application of an aggregation-induced luminescence probe based on a bioorthogonal reaction metabolic anchoring strategy for live bacteria

Abstract

The present invention belongs to the technical field of photosensitizers, and in particular to a kind of aggregation-induced luminescence probe design, synthesis and sterilization application based on the metabolic anchoring live bacteria strategy of bioorthogonal reaction.The aggregation-induced luminescence compound of the present invention (aggregation-induced luminescence probe) is typical D π π A structure, using triphenylamine structural unit as the electron donor spacer group in molecular system, to prevent π π from piling up, using thiophene ring and carbon-carbon double bond as electron donor and π bridge, nitro unit as electron acceptor, the strong push-pull effect of overall formation electron, finally, also connected with the important synthetic raw material D-amino acid (D Ala) of bacterial cell wall.The aggregation-induced luminescence compound prepared by the present invention has long fluorescence lifetime, and fluorescence intensity is large, and a large amount of reactive oxygen can be produced after illumination to effectively kill live bacteria, and can be absorbed and utilized by bacteria by metabolism, to achieve the difference detection of live and dead bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of photosensitizers. More specifically, it relates to the design, synthesis and bactericidal application of an aggregation-induced luminescence probe based on a bioorthogonal reaction metabolic anchoring strategy for live bacteria. Background Art

[0002] Microorganisms are ubiquitous in our daily lives, playing both beneficial and detrimental roles in human survival, production, and development. Once food is contaminated by spoilage bacteria, they use them as nutrients and multiply in large numbers, ultimately degrading the food's sensory qualities and losing its nutritional value. Ingesting food contaminated by pathogenic bacteria can cause symptoms ranging from abdominal pain and diarrhea to life-threatening consequences. Currently, a wide variety of sterilization technologies exist. In addition to traditional thermal and ultraviolet sterilization, new technologies are constantly emerging, such as ultrasonic sterilization, plasma sterilization, electromagnetic pulse sterilization, irradiation sterilization, and high-pressure sterilization. However, each of these sterilization technologies has limitations: thermal sterilization can affect food quality and reduce nutritional value; ultraviolet sterilization can be harmful to skin and eyes; ultrasonic sterilization cannot be used on solid foods; improper irradiation sterilization can easily lead to food safety issues; and plasma and electromagnetic pulse sterilization require specialized equipment.

[0003] Anti-microbial photodynamic technology (APDT) uses specific photosensitizers (PS) to selectively accumulate in target tissues (such as microorganisms). The absorbed light energy triggers a series of photochemical reactions, generating large amounts of reactive oxygen species (ROS). These ROS, in turn, disrupt the normal physiological functions of microorganisms, causing cell damage and necrosis while sparing the properties of surrounding cells and tissues. While stable concentrations of ROS act as messengers to regulate physiological processes, excessive amounts of ROS are highly effective, broad-spectrum, and non-polluting gases that damage microbial morphology, cell membranes, nucleic acids, and proteins, triggering a variety of cell death mechanisms without causing bacterial tolerance. APDT was initially used primarily in the medical field for the treatment of inflammation and the inhibition and killing of cancer cells. With the advancement of research, APDT has gradually entered the food industry, finding application as an antimicrobial and bactericidal method. For safety reasons, APDT is primarily used in the food industry for preservation and freshness preservation through the mediation of natural photosensitizers such as curcumin, chlorophyll, and riboflavin. However, most natural photosensitizers suffer from aggregation-induced quenching (ACQ), which leads to a decrease in the production of reactive oxygen species and affects their bactericidal effect.

[0004] In addition to sterilization, timely monitoring and prevention are also very important steps in food preservation and freshness preservation. However, common microbial detection methods have many limitations. For example, the plate culture method has cumbersome and time-consuming operation steps, molecular biology detection technology has high requirements for operators and the environment, and the difficulty in preparing microbial antibodies leads to low specificity of immunoassays. In recent years, due to the advantages of fluorescence detection technology such as high selectivity, high sensitivity, low dosage, fast response speed, and low cost, more and more researchers have focused on how to develop new fluorescent sensors. However, the quenching phenomenon caused by aggregation in most fluorescent materials greatly limits their use, resulting in low detection signals, poor sensitivity, and easy photobleaching.

[0005] Aggregate induced emission (AIE) molecules can effectively solve ACQ. They do not emit fluorescence or emit very weak fluorescence in dilute solutions or at low concentrations, but release photons to emit bright light in aggregated states or at high concentrations, thus providing lower background and more reliable signals in biological and chemical detection. In addition, AIE has excellent photodynamic properties. After being irradiated with light of a specific wavelength, it absorbs photon energy and reacts with surrounding oxygen or related molecules to generate reactive oxygen species, which can accurately damage and kill biological macromolecules, cells, bacteria, etc. AIE materials can not only produce stable fluorescence signals, but also produce reactive oxygen species. Therefore, AIE-type photosensitizers (AIE-PS) have shown good detection-killing application prospects in the medical field. However, most AIE-PS mainly bind to bacteria through electrostatic interactions and hydrophobic interactions, and cannot distinguish between live and dead bacteria. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and provide an aggregation-induced emission compound.

[0007] The object of the present invention is to provide a method for preparing the aggregation-induced emission compound.

[0008] Another object of the present invention is to provide the use of the aggregation-induced luminescence compound in preparing a photosensitizer or in distinguishing between the living and dead states of bacteria.

[0009] Another object of the present invention is to provide use of the aggregation-induced emission compound in preparing a fungicide or in sterilization.

[0010] Another object of the present invention is to provide a use of the aggregation-induced emission compound in preparing a cell wall-specific staining reagent.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides an aggregation-induced emission compound, the structural formula of which is shown in formula (I):

[0013]

[0014] The aggregation-induced luminescence compound of the present invention has a typical D-π-A structure. The triphenylamine structural unit serves as an electron donor spacer in the molecular system to prevent π-π stacking. The thiophene ring and carbon-carbon double bond serve as electron donors and π bridges, and the nitro unit serves as an electron acceptor. The overall structure forms a strong push-pull interaction for electrons. Finally, D-amino acid (D-Ala) is an important raw material for the synthesis of bacterial cell walls. Bacteria can absorb and utilize it through metabolism, enabling the differential detection of live and dead bacteria. Furthermore, the D-π-A structured aggregation-induced luminescence compound of the present invention can generate a large amount of reactive oxygen species upon illumination, effectively killing live bacteria.

[0015] The metabolic labeling of the present invention, based on bioorthogonal reactions, integrates bacterial nutrients (D-Ala) with specific signals into bacteria through bacterial metabolic uptake. This is a relatively effective live bacteria targeting strategy. After the bacteria take up the aggregation-induced luminescence compound containing D-Ala for cell wall synthesis, the aggregation-induced luminescence compound is enriched on the bacterial cell wall to produce fluorescence, ultimately achieving the detection and killing of live bacteria.

[0016] Preferably, the present invention also protects pharmaceutically acceptable salts, prodrugs, hydrates, solvates, stereoisomers, polymorphs, tautomers or isotopic compounds of the aggregation-induced emission compound.

[0017] Furthermore, the present invention provides a method for preparing the aggregation-induced emission compound, and the synthesis route is as follows:

[0018]

[0019] The following steps are involved:

[0020] S1.4-Triphenylamine borate and 5-bromo-2-thiophenecarboxaldehyde undergo condensation reaction to obtain compound 1;

[0021] S2. Compound 1 is subjected to a condensation reaction with methyl 2-methyl-5-nitrobenzoate to obtain compound 2;

[0022] S3. Compound 2 is subjected to amidation reaction with Boc-D-2,3-diaminopropionic acid to obtain compound 3;

[0023] S4. The Boc group of compound 3 is removed to obtain the aggregation-induced emission compound represented by formula (I).

[0024] Preferably, the steps include:

[0025] S1.4-Triphenylamine borate, 5-bromo-2-thiophenecarboxaldehyde and palladium catalyst are subjected to condensation reaction under an inert gas atmosphere and post-processed to obtain compound 1;

[0026] S2. Compound 1, methyl 2-methyl-5-nitrobenzoate and an alkaline reagent are subjected to a condensation reaction and post-processing to obtain compound 2;

[0027] S3. Compound 2 is subjected to amidation reaction with Boc-D-2,3-diaminopropionic acid and a catalyst under an inert gas atmosphere, and post-processed to obtain compound 3;

[0028] S4. Compound 3 reacts with trifluoroacetic acid to remove the Boc group, and then post-processes to obtain the aggregation-induced emission compound represented by formula (I).

[0029] Preferably, in step S1, the temperature of the condensation reaction is 55-65°C.

[0030] Preferably, in step S2, the temperature of the condensation reaction is 75-85°C.

[0031] Preferably, in step S3 and step S4, the reaction temperature is 25-30°C.

[0032] Preferably, in step S3, the catalyst is one or both of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0033] Preferably, in step S1, the palladium catalyst is a zero-valent palladium catalyst conventionally used in the art for condensation reactions; more specifically, the zero-valent palladium catalyst includes but is not limited to tetrakis(triphenylphosphine)palladium, etc.

[0034] Preferably, in step S2, the alkaline reagent is an alkaline reagent commonly used in the art for condensation reactions; more specifically, the alkaline reagent includes but is not limited to anhydrous potassium carbonate, etc.

[0035] In steps S1 to S4, the post-treatment is a separation and purification process, which can be any compound separation and purification method in the field of organic synthesis, for example, one or more combinations of column chromatography, solvent extraction, and distillation.

[0036] For reference, the present invention provides a specific post-processing purification method for steps S1 to S4 as follows:

[0037] In step S1, the post-treatment is extraction, washing, separation and drying of the organic layer, and column chromatography; preferably, the eluent of the column chromatography is petroleum ether / dichloromethane with a volume ratio of 1:2.5-3.5;

[0038] In step S2, the post-treatment is extraction, removing the solvent by rotary evaporation of the organic layer, and column chromatography; preferably, the eluent of the column chromatography is petroleum ether / ethyl acetate with a volume ratio of 2.5 to 3.5:1;

[0039] In step S3, the post-treatment is extraction, removing the solvent by rotary evaporation of the organic layer, and column chromatography; preferably, the eluent of the column chromatography is petroleum ether / ethyl acetate / triethylamine in a volume ratio of 20 to 30:1:1;

[0040] In step S4, the post-treatment is extraction, removing the solvent by rotary evaporation of the organic layer, and column chromatography; preferably, the eluent of the column chromatography is petroleum ether / ethyl acetate / formic acid in a volume ratio of 75 to 85:40:1.

[0041] Furthermore, the present invention protects the use of the aggregation-induced luminescence compound in preparing a photosensitizer or in distinguishing between the living and dead states of bacteria.

[0042] The aggregation-induced luminescence compound of the present invention can be metabolized and absorbed by living bacteria for cell wall synthesis, and then enriched on the cell wall to produce fluorescence, thereby "lighting up" the bacteria, while no fluorescence is produced for dead bacteria.

[0043] In addition, the present invention also protects the use of the aggregation-induced emission compound in preparing a fungicide or in sterilization.

[0044] The aggregation-induced luminescence compound of the present invention exerts a current bactericidal effect under light conditions, and the bacterial survival rate is 0% at a concentration of 50 μM.

[0045] Furthermore, the present invention protects the use of the aggregation-induced emission compound in the preparation of a cell wall-specific staining reagent.

[0046] The present invention has the following beneficial effects:

[0047] First, the aggregation-induced luminescence compound of the present invention has a typical D-π-A structure. Triphenylamine acts as an electron donor and spacer to prevent π-π stacking, thiophene and the carbon-carbon double bond act as electron donors and π bridges, and the nitro unit acts as an electron acceptor. Overall, this D-π-A aggregation-induced luminescence compound generates a large amount of reactive oxygen species upon illumination, effectively killing live bacteria, making it a potent sterilant.

[0048] Secondly, the present invention abandons the use of electrostatic interactions to target bacteria. Instead, by linking D-amino acids (D-Ala) to the molecular structure, bacteria can metabolize and absorb them, enabling the differential detection of live and dead bacteria. Furthermore, the aggregation-induced luminescence compound prepared by the present invention exhibits a long fluorescence lifetime and high fluorescence intensity. After being metabolized and absorbed by living bacteria for cell wall synthesis, it accumulates on the cell wall, producing fluorescence and "illuminating" the bacteria.

[0049] Finally, the aggregation-induced luminescence compound described in the present invention, as a photosensitizer, overcomes the disadvantage of fluorescence quenching of traditional photosensitizers at high concentrations. It does not emit fluorescence or emits very weak fluorescence at lower concentrations, but releases photons to emit bright light in the aggregated state or at higher concentrations, thereby providing lower background and more reliable signals in biological and chemical detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the H NMR spectrum of the AIE photosensitizer.

[0051] Figure 2 The fluorescence emission diagrams of the AIE photosensitizers in Example 1 and Comparative Examples 1 to 3 in different DMSO / water systems are shown. Figure 2 Figure A is the fluorescence emission diagram of the AIE photosensitizer in Example 1 in different ratios of DMSO / water systems. Figure 2 Figure B is the fluorescence emission diagram of the AIE photosensitizer in comparative example 1 in different ratios of DMSO / water systems. Figure 2 Figure C is the fluorescence emission diagram of the AIE photosensitizer in comparative example 2 in different ratios of DMSO / water systems. Figure 2 Figure D is the fluorescence emission diagram of the comparative example 3AIE photosensitizer in different ratios of DMSO / water systems.

[0052] Figure 3 The ROS characterization diagram of AIE photosensitizers in Example 1 and Comparative Examples 1 to 3. Figure 3 Figure A is the fluorescence spectrum of DCFH-Da probe under light conditions. Figure 3 Figure B is the fluorescence spectrum of the DCFH-Da probe after adding the AIE photosensitizer of Example 1 under light conditions. Figure 3 Figure C shows the fluorescence intensity change of the DCFH-Da probe after adding the AIE photosensitizer of Example 1 under light conditions. Figure 3 Figure D shows the fluorescence intensity change of the DCFH-Da probe after adding the AIE photosensitizer of Comparative Example 1 under light conditions. Figure 3 Figure E shows the fluorescence intensity change of the DCFH-Da probe after adding the AIE photosensitizer of Comparative Example 2 under light conditions. Figure 3Figure F shows the change in fluorescence intensity of the DCFH-Da probe after adding the AIE photosensitizer of comparative example 3 under light conditions.

[0053] Figure 4 This is a characterization diagram of the stability of AIE photosensitizers. Figure 4 Figure A is the UV absorption graph of DMSO under light conditions. Figure 4 Figure B is the UV absorption diagram of AIE photosensitizer under light conditions. Figure 4 Figure C shows the change in ultraviolet absorption of AIE photosensitizer under light conditions.

[0054] Figure 5 This is a characterization diagram of the bactericidal effect of AIE photosensitizer. Figure 5 Figure A shows the growth of culture dishes with different concentrations of AIE photosensitizer under light and dark conditions. Figure 5 Figure B is a statistical diagram of the number of colony survival data in culture dishes under different treatment conditions; Figure 5 Figure C is a statistical graph of bacterial survival rate data of AIE photosensitizer under different treatment conditions.

[0055] Figure 6 These are the confocal laser scanning microscope fluorescence and bright field images of AIE photosensitizer and Lactobacillus acidophilus at different incubation times. DETAILED DESCRIPTION

[0056] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0057] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0058] Example 1

[0059] 1. Synthesis of triphenylamine-thiophene fragment

[0060] 4-Triphenylamine borate (722 mg, 2.5 mmol) and 5-bromo-2-thiophenecarboxaldehyde (363 mg, 1.9 mmol) were mixed and dissolved in a tetrahydrofuran / water mixture (v / v=60 / 20). 121 mg, i.e. 0.1 mmol, of tetrakis(triphenylphosphine)palladium was added, and the mixture was reacted at 60° C. in an N2 atmosphere for 12 hours. After completion of the reaction, the reaction mixture was cooled to ambient temperature, extracted three times with ethyl acetate and washed with water, and then the ethyl acetate layer was separated and dried over sodium sulfate to evaporate the ethyl acetate solvent. The crude product solid was then separated and purified by column chromatography using petroleum ether / dichloromethane (1 / 3, v / v) as eluent to give a yellow solid, i.e., Compound 1.

[0061]

[0062] 2. Nitro electron-withdrawing group connection

[0063] First, prepare 2-methyl-5-nitrobenzoic acid methyl ester: 2-methyl-5-nitrobenzoic acid (15 g, 27.6 mmol) is dissolved in 50 mL of methanol, and thionyl chloride (6 mL, 99 mmol) is added dropwise below 0° C., with stirring while adding dropwise. After the addition is completed, place in a constant temperature oil bath, stir at 65° C. for 6 hours, reflux with a condenser, and monitor whether the reaction is complete by thin layer silica gel chromatography. The developing solvent system is petroleum ether / ethyl acetate (volume ratio, 3 / 1). After the reaction is complete, the solvent is spin-dried by vacuum rotary evaporation, methanol is added to fully dissolve it, and then rotary evaporation is continued to form a solid. The thionyl chloride in the system is fully removed twice, and the remaining white solid is freeze-dried with ultra-low temperature freeze drying to obtain 2-methyl-5-nitrobenzoic acid methyl ester for standby use.

[0064] Compound 1 was connected with methyl 2-methyl-5-nitrobenzoate: 355 mg (1.0 mmol) of compound 1 was then added to 1.0 mmol (209.6 mg) of methyl 2-methyl-5-nitrobenzoate, dissolved in 5 mL of dehydrated dimethyl sulfoxide, and 0.275 g (2.02 mmol) of anhydrous potassium carbonate. The mixture was placed in an eggplant-shaped flask and stirred overnight in a constant temperature oil bath at 80°C. The next day, the reaction flask was transferred to room temperature and cooled. The reaction was monitored for completion by thin-layer silica gel chromatography using a developing solvent of petroleum ether: ethyl acetate: formic acid (volume ratio, 80:40:1). After the reaction was complete, the reaction system was transferred to a separatory funnel and extracted with ethyl acetate. The product was dissolved in the ethyl acetate layer. All the ethyl acetate was removed by rotary evaporation to remove the solvent, and the resulting solid crude product was separated and purified by column chromatography with petroleum ether: ethyl acetate: formic acid (volume ratio, 80:40:1) as eluent. The resulting product was freeze-dried and weighed to obtain the intermediate product triphenylamine-thiophene-nitro product, namely compound 2.

[0065]

[0066] 3. Metabolic molecule D-Ala connection

[0067] 0.212 g of N-hydroxysuccinimide NHS (2.3 mmol) and 0.884 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC (4.6 mmol) were weighed and added to 30 mL of a DMF solution containing 1 mmol of compound 2. The mixture was stirred at room temperature under nitrogen for 6 hours. 0.224 g (1.1 mmol) of Boc-D-Dap-OH was added and stirred at room temperature for another 24 hours. The reaction system was then transferred to a separatory funnel and extracted with ethyl acetate. The product dissolved in the ethyl acetate layer. The ethyl acetate was removed by rotary evaporation, and the resulting crude solid product was isolated and purified by column chromatography using a developing solvent of petroleum ether:ethyl acetate:triethylamine (volume ratio, 25:1:1). Finally, the product with the NHBoc group, compound 3, was dried by rotary evaporation.

[0068]

[0069] 4. Obtaining the final product AIE photosensitizer

[0070] Take 0.39mmol of compound 3, dissolve it with 4mL of dichloromethane solution, add 4mL of trifluoroacetic acid dropwise at 0℃, stir at room temperature for 4 hours to remove the Boc group. Then, add saturated NaHCO3 solution dropwise until the mixture solution no longer produces bubbles. The reaction system is then transferred to a separatory funnel and extracted with ethyl acetate, and the product is dissolved in the ethyl acetate layer. Take the ethyl acetate layer, remove the solvent from all ethyl acetate by rotary evaporation, and the resulting solid crude product is separated and purified by column chromatography, and the developing solvent is petroleum ether: ethyl acetate: formic acid (volume ratio, 80:40:1). Finally, rotary evaporation and drying give the final product, an aggregation-induced emission compound of the structure shown in formula (I), i.e., an AIE photosensitizer.

[0071]

[0072] The H NMR spectrum of AIE photosensitizer is shown in Figure 1 The specific NMR data are as follows:

[0073] 1H NMR(600MHz,Chloroform-d)δ8.75(d,J=2.5Hz,1H),8.24(ddd,J=8.2,5.5,2.5Hz,1H),7.78(d,J=3.9Hz,1H),7.53(d,J=8.6Hz,2H),7.43(t,J=7.0Hz ,1H),7.30(d,J=1.9Hz,1H),7.27(d,J=1.9Hz,1H),7.14–7.06(m,13H),3.5 8–3.55(m,1H),3.50–3.43(m,2H),1.96(dddd,J=9.9,5.9,4.1,1.5Hz,2H).

[0074] Comparative Example 1

[0075] Compared with Example 1, the difference is that 2-methyl-5-nitrobenzoic acid methyl ester in step 2 is replaced by 2-methyl-5-trifluoromethylbenzoic acid methyl ester, and the remaining experimental operations and experimental steps are consistent with those in Example 1.

[0076] Comparative Example 2

[0077] Compared with Example 1, the difference is that 2-methyl-5-nitrobenzoic acid methyl ester in step 2 is replaced by 2-methyl-5-cyanobenzoic acid methyl ester, and the remaining experimental operations and experimental steps are consistent with those in Example 1.

[0078] Comparative Example 3

[0079] Compared with Example 1, the difference is that 2-methyl-5-nitrobenzoic acid methyl ester in step 2 is replaced by 2-methyl-1,5-dimethyl isophthalate, and the remaining experimental operations and experimental steps are consistent with those in Example 1.

[0080] Example 2: Verification of Aggregation-Induced Emission Properties

[0081] In order to explore the aggregation-induced emission properties of the AIE photosensitizer prepared by the present invention, its fluorescence emission spectrum in H2O / DMSO mixed solutions with different volume fractions was measured. The DMSO content was set to 1%, 10%, 30%, 50%, 70%, 90%, 99%, and 100%, respectively. The AIE photosensitizers prepared in Example 1 and Comparative Examples 1 to 3 (dissolved in DMSO) were diluted to 50 μM with DMSO, and then primary water was added to a specific DMSO volume fraction according to different mixing ratios, and then placed in a black ELISA plate for fluorescence measurement.

[0082] The experimental results are as follows Figure 2As shown in Figure A, the AIE photosensitizer prepared in Example 1 reaches a peak fluorescence value at 575 nm with an excitation wavelength of 410 nm. In 100% DMSO, the fluorescence value is 9,030,439 au, while in 1% DMSO, the fluorescence value is only 672,795 au, a 1,085-fold difference in fluorescence values, which is typical of aggregation-induced emission.

[0083] The experimental results of the AIE photosensitizer prepared in Comparative Example 1 are as follows: Figure 2 As shown in Figure B, with an excitation wavelength of 375 nm, the fluorescence value reaches a peak at 530 nm. In 100% DMSO, the fluorescence value is 163,184,128 au, while in 1% DMSO, the fluorescence value is only 150,344 au, a difference of 13.42 times, which is significantly lower than that in Example 1.

[0084] The experimental results of the AIE photosensitizer prepared in Comparative Example 2 are as follows: Figure 2 As shown in Figure C, with an excitation wavelength of 380 nm, the fluorescence value reaches a peak at 535 nm. In 100% DMSO, the fluorescence value is 38524552 au, while in 1% DMSO, the fluorescence value is only 131935 au, a difference of 292 times, which is significantly lower than that in Example 1.

[0085] The experimental results of the AIE photosensitizer prepared in Comparative Example 3 are as follows: Figure 2 As shown in Figure D, with an excitation wavelength of 360 nm, the fluorescence value reaches a peak at 470 nm. In 100% DMSO, the fluorescence value is 59,928,448 au, while in 1% DMSO, the fluorescence value is only 393,093 au, a difference of 152 times, which is significantly lower than that in Example 1.

[0086] By comparing Example 1 of the present invention with Comparative Examples 1-3, it can be found that by replacing the electron-withdrawing groups on the molecular structure, the photosensitizer shows a phenomenon of gradually weakening fluorescence intensity, and the AIE properties are also weakened to varying degrees.

[0087] Example 3 Determination of active oxygen generation capacity

[0088] 10 μM of the AIE photosensitizer prepared in Example 1 (dissolved in DMSO) was added to an aqueous solution containing 40 μM DCFH-Da (2',7'-dichlorofluorescein diacetate). 2 After the solution was irradiated with white light, its fluorescence signal in the range of 515-700 nm was monitored every 5 min with an excitation wavelength of 488 nm and a slit of 5 nm. The change in fluorescence intensity at 525 nm was recorded to indicate the total ROS production rate of the AIE photosensitizer.

[0089] The experimental results are as follows Figure 3 As shown in Figures A and B, no AIE photosensitizer was added to the blank group, and only the DCFH-Da probe was illuminated. As the illumination time increased, the fluorescence value did not show significant changes ( Figure 3 Figure A); After adding the AIE photosensitizer prepared in Example 1 to the experimental group, the fluorescence value gradually increased with the increase of illumination time. Figure 3 As shown in Figure C, within 55 minutes of illumination, the fluorescence intensity of the AIE group gradually increased and reached a peak at the 55th minute, which was 175 times the initial (0 min) fluorescence intensity, indicating that the AIE photosensitizer prepared in Example 1 has a strong ability to produce active oxygen.

[0090] After adding the AIE photosensitizer prepared in Comparative Example 1, the experimental results are as follows Figure 3 As shown in Figure D, after the nitro group in the photosensitizer molecule was replaced with a trifluoromethyl group, the fluorescence intensity of the AIE group of the photosensitizer gradually increased within 55 minutes of illumination, and reached a peak at the 55th minute, which was 98 times the initial (0 min) fluorescence intensity, that is, its ability to produce reactive oxygen species was weaker than that in Example 1.

[0091] After adding the AIE photosensitizer prepared in Comparative Example 2, the experimental results are as follows Figure 3 As shown in Figure E, after the nitro group in the photosensitizer molecule was replaced with a cyano group, the fluorescence intensity of the AIE group of the photosensitizer gradually increased within 55 minutes of illumination, and reached a peak at the 55th minute, which was 160 times the initial (0 min) fluorescence intensity, that is, its ability to produce active oxygen was weaker than that in Example 1.

[0092] After adding the AIE photosensitizer prepared in Comparative Example 3, the experimental results are as follows Figure 3 As shown in Figure F, after the nitro group in the photosensitizer molecule was replaced with a carboxyl group, the fluorescence intensity of the AIE group of the photosensitizer gradually increased within 55 minutes of illumination, and reached a peak at the 50th minute, which was 13 times the initial (0 min) fluorescence intensity, that is, its ability to produce reactive oxygen species was weaker than that in Example 1.

[0093] Comparing Example 1 of the present invention with Comparative Examples 1-3, it can be found that by replacing the electron-withdrawing groups on the molecular structure, the ability of the photosensitizer to produce active oxygen is weakened to varying degrees, which ultimately leads to a significant decrease in its bactericidal ability.

[0094] Example 4 Fluorescence stability determination

[0095] Use white light (20mW / cm 2 ) irradiated aliquots (300 μL) of each AIE photosensitizer solution (50 μM, dissolved in DMSO) for 60 min in triplicate, 100 μL each, and were detected by BioTek SynergyTM The UV-visible absorption spectrum of the AIE photosensitizer was measured every 5 minutes using a multi-mode microplate reader. The control group consisted of a DMSO solution without the AIE photosensitizer. The maximum absorption intensity (Amax) was determined, and the photostability of the AIE photosensitizer was expressed as follows:

[0096] (Amax-Amax(control)) / (Amax(0min)-Amax(control))

[0097] Wherein, Amax represents the maximum absorbance of the AIE photosensitizer at a certain time within 0 to 60 min; Amax(0 min) represents the maximum absorbance of the AIE photosensitizer at 0 min; Amax(control) represents the maximum absorbance of the control group at the same time as Amax.

[0098] like Figure 4 In Figures A and B, both the blank group and the experimental group have absorbance values ​​that are stable within a certain range after 60 minutes of illumination, indicating that the AIE photosensitizer has good stability. Figure 4 Figure C further determines the stability of the AIE photosensitizer by the absorbance ratio. The fluorescence ratio varies between 94% and 101%, indicating that the AIE photosensitizer prepared in Example 1 of the present invention has good fluorescence stability and will not be weakened by light.

[0099] Example 5 Bactericidal Ability Determination

[0100] MRS medium: MRS broth dry powder medium, Guangdong Huankai Microbiology Technology Co., Ltd., product number 027312.

[0101] Take a small amount of glycerol-frozen Lactobacillus acidophilus culture and add it to MRS medium. Incubate at 37℃ for 1-2 days and measure OD 600 Confirm the initial bacterial concentration (the bacterial concentration when the OD value is 1 is about 2×10 9 CFU / mL).

[0102] Take 1mL of bacterial solution and centrifuge to remove the supernatant, then resuspend the bacteria with sterile PBS, repeat the washing twice and dilute the bacterial solution to 10 7 CFU / mL, set aside.

[0103] 10 μL of AIE photosensitizers of different concentrations (2 μM, 10 μM, 50 μM) were added to 1 mL of diluted bacterial solution to obtain mixed bacterial solutions. The mixed bacterial solutions were incubated in a 37°C constant temperature shaker at 220 rpm in the dark for 30 minutes. The mixed bacterial solutions were then exposed to white light (20 mW / cm 2) for 40 minutes. A light blank control group (10 μL of PBS was added to 1 mL of diluted bacterial solution, and all other procedures were the same) was set up. A dark blank control group (10 μL of PBS was added to 1 mL of diluted bacterial solution, incubated in the dark, and then in the dark for 40 minutes) and a dark photosensitizer group (after incubation in the dark, not exposed to white light, and then in the dark for 40 minutes, and all other procedures were the same) were set up. Each experiment was performed in triplicate.

[0104] After the above white light irradiation or light protection is completed, the mixed bacterial solution is mixed and 100 μL of the mixed bacterial solution is aspirated and diluted 10 times in PBS, and then graded diluted 5 times (from the mixed bacterial solution concentration of about 10 7 CFU / mL starts dilution, each dilution is 10 times, and the final dilution factor is about 10 2 After thorough mixing, 100 μL of the mixed bacterial solution was evenly spread on the MRS medium and cultured in a 37°C constant temperature incubator for 48 h.

[0105] The experimental results are as follows Figure 5 As shown, Figure 5 The small figures numbered a, b, and c in Figure A show the bacterial growth under light conditions at 2μM, 10μM, and 50μM AIE photosensitizers, respectively. It can be seen from the figures that adding only 2μM AIE photosensitizer has played a good role in inhibiting bacterial growth, and complete sterilization is achieved at 50μM.

[0106] Figure 5 Panels d, e, and f in Figure A represent bacterial growth in the dark at 2μM, 10μM, and 50μM AIE photosensitizers. Clearly, without light to provide energy to the AIE photosensitizer, it cannot generate reactive oxygen species to kill bacteria, resulting in the growth of numerous colonies in all three concentration gradients.

[0107] Figure 5 The panels g and h in Figure A represent bacterial growth in culture dishes without the addition of an AIE photosensitizer, under light and dark conditions, respectively. The two blank groups exhibited strong colony growth, indicating that the lighting conditions were appropriate and did not affect bacterial growth.

[0108] In order to intuitively reflect the sterilization effect, the plate counting method was used to count the number of bacterial colonies ( Figure 5 Figure B). Under light conditions, the number of colonies on the plate treated with three concentrations of AIE photosensitizer was 5.3×10 7 CFU / mL, 1.3×10 7 CFU / mL, 0 CFU / mL; under dark conditions, the number of colonies on the plate treated with three concentrations of AIE photosensitizer was 9.2×10 7CFU / mL, 6.5×10 7 CFU / mL, 7.6×10 7 CFU / mL; the colony counts on the dark blank group and the light blank group were 8.2×10 7 CFU / mL, 7.8×10 7 CFU / mL. AIE photosensitizer can inhibit nearly half of the bacteria at a low concentration of 2μM, and can completely kill the bacteria at 50μM. Figure 5 In Figure C, the bacterial survival rate of 2 μM AIE photosensitizer is 66%, the bacterial survival rate of 10 μM AIE photosensitizer is 20%, and the bacterial survival rate of 50 μM AIE photosensitizer is 0%. This shows that the bactericidal effect of this AIE photosensitizer is very obvious.

[0109] Example 6 Bacterial Imaging Identification

[0110] Take 1mL of Lactobacillus acidophilus liquid (1.2×10 9 The supernatant was removed by centrifugation at 100 μL (0.175 μM) per well (0.175 μM CFU / mL). The bacteria were then resuspended in sterile PBS. 10 μL of AIE photosensitizer (50 μM) was then added to the bacterial suspension to form a suspension. After incubation at 37°C in the dark for 0, 20, 40, and 60 minutes, 10 μL of the suspension was transferred to a glass slide, quickly covered with a coverslip, and imaged on a confocal laser scanning microscope.

[0111] The experimental results are as follows Figure 6 shown. Figure 6 Figures A to D in the figure are fluorescence images of AIE photosensitizer and Lactobacillus acidophilus; Figure 6 Figures E to H in the figure are bright field images of the AIE photosensitizer and Lactobacillus acidophilus. At 0 minutes, the field of view of the fluorescence image is dark, but combined with the bright field image, it can be seen that Lactobacillus acidophilus is present in the field of view, but it does not emit light. This shows that the AIE photosensitizer is not bound by the bacteria at this time, so it cannot produce fluorescence. At the 20th minute, the bacteria in the fluorescence field of view began to show a faint yellow fluorescence, and it is worth noting that the fluorescence mainly comes from the bacterial cytoplasm, and the fluorescence on the cell wall is lighter. This shows that after 20 minutes of incubation, the bacteria began to absorb the AIE photosensitizer into the body. As the incubation time increased to 40 minutes, the bacterial cell wall was clearly illuminated, and the fluorescence of the cytoplasm decreased, indicating that the bacteria had utilized and integrated the AIE photosensitizer into the cell wall, and the AIE photosensitizer produced strong fluorescence due to large-scale aggregation. Finally, when the incubation time was 60 minutes, a large number of bacteria in the fluorescence field of view were illuminated, so this probe has good bacterial imaging capabilities.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An aggregation-induced emission compound, characterized in that The structural formula is shown in formula (I):

2. The method for preparing the aggregation-induced emission compound according to claim 1, characterized in that: The synthetic route is as follows: The following steps are involved: S1.4-Triphenylamine borate and 5-bromo-2-thiophenecarboxaldehyde undergo condensation reaction to obtain compound 1; S2. Compound 1 is subjected to a condensation reaction with methyl 2-methyl-5-nitrobenzoate to obtain compound 2; S3. Compound 2 is subjected to amidation reaction with Boc-D-2,3-diaminopropionic acid to obtain compound 3; S4. The Boc group of compound 3 is removed to obtain the aggregation-induced emission compound represented by formula (I).

3. The preparation method according to claim 2, characterized in that The following steps are involved: S1.4-Triphenylamine borate, 5-bromo-2-thiophenecarboxaldehyde and palladium catalyst are subjected to condensation reaction under an inert gas atmosphere and post-processed to obtain compound 1; S2. Compound 1, methyl 2-methyl-5-nitrobenzoate and an alkaline reagent are subjected to a condensation reaction and post-processing to obtain compound 2; S3. Compound 2 is subjected to amidation reaction with Boc-D-2,3-diaminopropionic acid and a catalyst under an inert gas atmosphere, and post-processed to obtain compound 3; S4. Compound 3 reacts with trifluoroacetic acid to remove the Boc group, and then post-processes to obtain the aggregation-induced emission compound represented by formula (I).

4. The preparation method according to claim 3, characterized in that In step S1, the temperature of the condensation reaction is 55-65°C.

5. The preparation method according to claim 3, characterized in that: In step S2, the temperature of the condensation reaction is 75-85°C.

6. The preparation method according to claim 3, characterized in that: In step S3 and step S4, the reaction temperature is 25-30°C.

7. The preparation method according to claim 3, characterized in that: In step S3, the catalyst is one or both of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

8. Use of the aggregation-induced emission compound according to claim 1 in preparing a photosensitizer or in distinguishing between the living and dead states of bacteria.

9. Use of the aggregation-induced emission compound according to claim 1 in the preparation of a fungicide.

10. Use of the aggregation-induced emission compound according to claim 1 in preparing a cell wall-specific staining reagent.

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