Design, synthesis and sterilization application of aggregation-induced emission probe based on biological orthogonal reaction metabolism anchoring viable bacteria strategy

Abstract

The invention belongs to the technical field of photosensitizers, and particularly relates to design, synthesis and sterilization application of an aggregation-induced emission probe based on a biological orthogonal reaction metabolism anchoring viable bacterium strategy. The aggregation-induced emission compound (aggregation-induced emission probe) is of a typical D-pi-A structure, takes a triphenylamine structural unit as an electron donor spacer group in a molecular system to prevent pi-pi accumulation, and takes a thiophene ring and a carbon-carbon double bond as electron donors and a pi bridge and a nitro unit as electron acceptors to synthesize the aggregation-induced emission probe. And finally, an important synthetic raw material D-type amino acid (D-Ala) of a bacterial cell wall is also connected. The prepared aggregation-induced emission compound is long in fluorescence lifetime and high in fluorescence intensity, a large amount of active oxygen can be generated after illumination to effectively kill viable bacteria, the active oxygen can be absorbed and utilized by bacteria through metabolism, and differential detection of the viable bacteria and dead bacteria is achieved.

Description

Technical Field

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

[0002] Microorganisms are everywhere in our daily lives, and they play a positive or negative role in the survival, production, and development of human society. Once food is contaminated by spoilage bacteria, the spoilage bacteria will multiply it as a nutrient, eventually causing the sensory degradation of the food and loss of nutritional value. Once humans accidentally eat food contaminated by pathogenic bacteria, they may suffer from abdominal pain and diarrhea, or even life-threatening conditions. At present, there are many sterilization technologies. In addition to traditional thermal sterilization and ultraviolet sterilization, new sterilization technologies are constantly emerging, such as ultrasonic sterilization technology, plasma sterilization technology, electromagnetic pulse sterilization technology, irradiation sterilization technology, high-pressure sterilization technology, etc. However, these sterilization technologies all have their limitations to varying degrees: thermal sterilization will affect food quality and reduce nutritional value, ultraviolet sterilization will damage the skin and eyes, ultrasonic sterilization cannot be used for solid food, improper irradiation sterilization operation may easily cause food safety problems, and plasma and electromagnetic pulse sterilization technologies require professional equipment.

[0003] Anti-microbial photodynamic technology (APDT) refers to a new technology that uses specific photosensitizers (PS) to selectively enrich in target tissues (such as microorganisms), absorb light energy to undergo a series of photochemical reactions, and produce a large amount of reactive oxygen species (ROS), thereby affecting the normal physiological functions of microorganisms, causing cell damage and necrosis, but not affecting the characteristics of surrounding cells and tissues. A stable concentration of ROS can serve as a messenger to regulate physiological processes, while excessive ROS is a highly efficient, broad-spectrum, and non-secondary-pollution gas that can destroy the morphological structure, cell membrane, nucleic acid, and protein of microorganisms, trigger a variety of death mechanisms, and is not easy to cause bacterial tolerance. APDT was initially mainly used in the medical field and can be used to treat inflammation, inhibit and kill cancer cells, etc. With the continuous advancement of research, APDT has gradually entered the food field and has been used as an antibacterial and sterilization method. For safety reasons, the food field mainly uses APDT mediated by natural photosensitizers such as curcumin, chlorophyll, and riboflavin for preservation and freshness. However, most natural photosensitizers suffer from aggregation-induced quenching (ACQ), which results in a decrease in the amount of reactive oxygen species produced, affecting their bactericidal effect.

[0004] In addition to sterilization, timely monitoring and prevention are also very important links in food 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, small dosage, fast response speed, and low cost, more and more researchers have focused on how to develop new fluorescent sensors. However, the aggregation of most fluorescent materials leads to quenching, which 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 light 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) show 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 shortcomings 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 dead and alive states of bacteria.

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

[0010] Another object of the present invention is to provide the 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 is a typical D-π-A structure, with a triphenylamine structural unit as an electron donor spacer group in the molecular system to prevent π-π stacking, a thiophene ring and a carbon-carbon double bond as electron donors and π bridges, and a nitro unit as an electron acceptor, forming a strong push-pull effect of electrons as a whole, and finally, D-amino acid (D-Ala) is an important synthetic raw material for bacterial cell walls, which can be absorbed and utilized by bacteria through metabolism, and can achieve the detection of live and dead bacteria. At the same time, the aggregation-induced luminescence compound of the D-π-A structure of the present invention can generate a large amount of active oxygen after illumination, and effectively kill live bacteria.

[0015] The metabolic labeling of the present invention based on bioorthogonal reaction integrates bacterial nutrients (D-Ala) with specific signals into bacteria through bacterial metabolic uptake, which 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, thereby ultimately realizing 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-boric acid triphenylamine 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 an 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-boric acid triphenylamine, 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 condensation reaction and post-treatment 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, followed by post-treatment to obtain compound 3;

[0028] S4. Compound 3 reacts with trifluoroacetic acid to remove the Boc group, and then post-processes to obtain an 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 and the like.

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

[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 in a volume ratio of 1:2.5-3.5;

[0038] In step S2, the post-treatment is extraction, taking the organic layer to remove the solvent by rotary evaporation, 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, taking the organic layer to remove the solvent by rotary evaporation, 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 dead and alive 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 is used as an electron donor and spacer group to prevent π-π stacking, thiophene and carbon-carbon double bonds are used as electron donors and π bridges, and the nitro unit is used as an electron acceptor, forming a strong push-pull effect of electrons as a whole. This aggregation-induced luminescence compound with a D-π-A structure can generate a large amount of active oxygen to effectively kill live bacteria after being irradiated with light, and is a powerful sterilizing agent.

[0048] Secondly, the present invention abandons the direction of using electrostatic interaction to target bacteria. By connecting D-amino acids (D-Ala) to the molecular structure, bacteria can absorb and utilize them through metabolism, thereby realizing the differential detection of live and dead bacteria. In addition, the aggregation-induced luminescence compound prepared by the present invention has a long fluorescence lifetime and high fluorescence intensity. After being absorbed by live bacteria for cell wall synthesis, it is enriched on the cell wall to produce fluorescence, which "lights up" the bacteria.

[0049] Finally, the aggregation-induced luminescence compound of the present invention is used as a photosensitizer to overcome 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 an aggregated state or at a higher concentration. Therefore, it can provide 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 ratios of 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 AIE photosensitizer of comparative example 3 in DMSO / water systems with different ratios.

[0052] Figure 3 The ROS characterization diagram of AIE photosensitizers in Example 1 and Comparative Examples 1 to 3. Figure 3 Figure A in the figure is the fluorescence spectrum of the DCFH-Da probe under illumination 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 in the figure 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 in the figure is a graph showing the fluorescence intensity change of the DCFH-Da probe after adding the AIE photosensitizer of Comparative Example 1 under illumination conditions. Figure 3 Figure E in the figure 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 illumination conditions.

[0053] Figure 4 This is a characterization diagram of the stability of AIE photosensitizer. Figure 4 Figure A is the UV absorption graph of DMSO under light conditions. Figure 4 Figure B is the UV absorption diagram of the AIE photosensitizer under light conditions. Figure 4 Figure C shows the change in ultraviolet absorption of the 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 the culture dish 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 Confocal laser scanning microscopy fluorescence and bright field images of AIE photosensitizer and Lactobacillus acidophilus at different incubation times. DETAILED DESCRIPTION

[0056] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. 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 are commercially available.

[0058] Example 1

[0059] 1. Synthesis of triphenylamine-thiophene fragment

[0060] 4-boric acid triphenylamine (722mg, 2.5mmol) and 5-bromo-2-thiophenecarboxaldehyde (363mg, 1.9mmol) were mixed and dissolved in a tetrahydrofuran / water mixture (v / v=60 / 20). 121mg, i.e. 0.1mmol, of tetrakis(triphenylphosphine)palladium was added, and the mixture was reacted at 60°C in a N2 atmosphere for 12 hours. After the reaction was completed, 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 with sodium sulfate to evaporate the ethyl acetate solvent. The crude product solid was separated and purified by column chromatography, using petroleum ether / dichloromethane (1 / 3, v / v) as an eluent to finally obtain a yellow solid, i.e., compound 1.

[0061]

[0062] 2. Nitro electron-withdrawing group connection

[0063] First, prepare methyl 2-methyl-5-nitrobenzoate: 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 in an environment below 0° C., with stirring while adding dropwise, and placed in a constant temperature oil bath pot, stirred at 65° C. for 6 hours, accompanied by reflux with a condenser, and thin layer silica gel chromatography is used to monitor whether the reaction is complete, and the developing solvent system is petroleum ether / ethyl acetate (volume ratio, 3 / 1). After the reaction is complete, the solvent is evaporated under reduced pressure to dryness, methanol is added to fully dissolve, and then the solvent is evaporated to form a solid, and the thionyl chloride in the system is fully removed twice, and the remaining white solid is freeze-dried by ultra-low temperature freeze drying to obtain methyl 2-methyl-5-nitrobenzoate for standby use.

[0064] Compound 1 was connected with methyl 2-methyl-5-nitrobenzoate: Then 355 mg, i.e. 1.0 mmol, of compound 1 was taken, 1.0 mmol, i.e. 209.6 mg, of methyl 2-methyl-5-nitrobenzoate was added, dissolved in 5 mL of dehydrated dimethyl sulfoxide, and 0.275 g (2.02 mmol) of anhydrous potassium carbonate was added, placed in an eggplant-shaped bottle, and stirred overnight in a constant temperature oil bath at 80 ° C. The next day, the reaction bottle was transferred to room temperature for cooling, and the reaction was monitored by thin layer silica gel chromatography to see if it was complete. The developing solvent was 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, and 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. The eluent was petroleum ether: ethyl acetate: formic acid (volume ratio, 80:40:1). 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] 0.212g N-hydroxysuccinimide NHS (2.3mmol) and 0.884g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC (4.6mmol) were added to 30mL DMF solution containing 1mmol of compound 2, stirred at room temperature under nitrogen protection for 6 hours, 0.224g, i.e. 1.1mmol of Boc-D-Dap-OH was added, and stirring was continued at room temperature for 24 hours, and then the reaction system was transferred to a separatory funnel and extracted with ethyl acetate. The product was dissolved in the ethyl acetate layer. All ethyl acetate was removed by rotary evaporation, and the obtained solid crude product was separated and purified by column chromatography, and the developing solvent was petroleum ether: ethyl acetate: triethylamine (volume ratio, 25:1:1). Finally, the product with NHBoc group, i.e. compound 3, was obtained by rotary evaporation and drying.

[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°C, 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. Then transfer the reaction system to a separatory funnel, extract 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 luminescent 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 luminescence 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 content of DMSO was set to 1%, 10%, 30%, 50%, 70%, 90%, 99%, and 100%, respectively. The AIE photosensitizer (dissolved in DMSO) prepared in Example 1 and Comparative Examples 1 to 3 was 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 uses 410 nm as the excitation wavelength, and its fluorescence value reaches a peak at 575 nm. In 100% DMSO, the fluorescence value is 9030439 au, while the fluorescence value of 1% DMSO is only 672795 au, which is a 1085-fold difference between the two fluorescence values, which is a typical aggregation-induced emission phenomenon.

[0083] The experimental results of the AIE photosensitizer prepared in Comparative Example 1 are as follows Figure 2 As shown in Figure B, with 375 nm as the excitation wavelength, the fluorescence value at 530 nm reaches a peak. In 100% DMSO, the fluorescence value is 163184128 au, while the fluorescence value of 1% DMSO is only 150344 au, which is 13.42 times different from that of Example 1, which is significantly lower.

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

[0085] The experimental results of the AIE photosensitizer prepared in Comparative Example 3 are as follows Figure 2 As shown in Figure D, with 360 nm as the excitation wavelength, the fluorescence value at 470 nm reaches a peak. In 100% DMSO, the fluorescence value is 59928448 au, while the fluorescence value of 1% DMSO is only 393093 au, which is 152 times different from that of Example 1, which is significantly lower.

[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. The excitation wavelength was 488 nm, the slit was set to 5 nm, and the change in fluorescence intensity at 525 nm was recorded to indicate the total ROS yield 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 a significant change ( Figure 3 A in the experimental group); after adding the AIE photosensitizer prepared in Example 1, the fluorescence value gradually increases 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 is replaced by a trifluoromethyl group, the fluorescence intensity of the AIE group of the photosensitizer gradually increases within 55 minutes of illumination time, and reaches a peak value at the 55th minute, which is 98 times the initial (0 min) fluorescence intensity, that is, its ability to produce active oxygen is 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 is replaced by a cyano group, the fluorescence intensity of the AIE group of the photosensitizer gradually increases within 55 minutes of illumination time, and reaches a peak value at the 55th minute, which is 160 times the initial (0 min) fluorescence intensity, that is, its ability to produce active oxygen is 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 is replaced by a carboxyl group, the fluorescence intensity of the AIE group of the photosensitizer gradually increases within 55 minutes of illumination time, and reaches a peak value at the 50th minute, which is 13 times the initial (0min) fluorescence intensity, that is, its ability to produce active oxygen is weaker than that in Example 1.

[0093] 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 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 Aliquots (300 μL) of each AIE photosensitizer solution (50 μM, dissolved in DMSO) were irradiated for 60 min in triplicate, with 100 μL per group, and the images were taken by BioTek SynergyTM The UV-visible absorption spectrum of the AIE photosensitizer was measured every 5 minutes by a multi-mode microplate reader. The control group was a DMSO solution without the AIE photosensitizer. The maximum absorption intensity (Amax) was determined, and the photostability of the AIE photosensitizer was expressed by the following formula:

[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(0min) 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 Microbiological Technology Co., Ltd., catalog 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 1 mL of bacterial solution and centrifuge to remove the supernatant. Then resuspend the bacteria in sterile PBS. Repeat the washing twice and dilute the bacterial solution to 10 7 CFU / mL, for future use.

[0103] 10 μL of AIE photosensitizers of different concentrations (2 μM, 10 μM, and 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 min and then exposed to white light (20 mW / cm 2) for 40 minutes. At the same time, a light blank control group (10 μL PBS was added to 1 mL of diluted bacterial solution, and the rest of the operations were the same), a dark blank control group (10 μL 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, still in the dark for 40 minutes, and the rest of the operations were the same), and each group of experiments was performed in parallel 3 times.

[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 gradiently diluted 5 times (from the mixed bacterial solution concentration of about 10 7 CFU / mL, dilute 10 times each time, and the final dilution factor is about 10 2 After fully mixing, 100 μL of the mixed bacterial solution was evenly spread on the MRS medium and placed 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 The small figures numbered d, e, and f in Figure A represent the growth of bacteria under dark conditions at 2μM, 10μM, and 50μM AIE photosensitizers. Obviously, since there is no light to provide energy to the AIE photosensitizer, it is unable to produce reactive oxygen species to kill bacteria, so a large number of colonies grow in the three concentration gradient culture dishes.

[0107] Figure 5 The small figures numbered g and h in Figure A represent the bacterial growth in the culture dish under light and dark conditions without adding AIE photosensitizer. The colony growth of the two blank groups is good, indicating that the lighting conditions are suitable and will not cause additional effects on bacterial growth.

[0108] In order to intuitively reflect the bactericidal 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, 0CFU / 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 of 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 2μM AIE photosensitizer is 66%, the bacterial survival rate of 10μM AIE photosensitizer is 20%, and the bacterial survival rate of 50μM AIE photosensitizer is 0%. This shows that the bactericidal effect of the AIE photosensitizer is very obvious.

[0109] Example 6 Bacterial imaging identification

[0110] Take 1mL of Lactobacillus acidophilus solution (1.2×10 9 CFU / mL) was centrifuged to remove the supernatant, and then the bacteria were resuspended in sterile PBS, and then 10 μL of AIE photosensitizer (50 μM) was added to the bacterial solution 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 placed on 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 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 contained in the field of view, but it does not emit light. This shows that the AIE photosensitizer is not bound by 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 take in the AIE photosensitizer into the body. As the incubation time increased to 40 minutes, the bacterial cell wall was clearly lit up, and the fluorescence of the cytoplasm decreased, indicating that the bacteria had utilized and integrated the AIE photosensitizer into the cell wall at this time, 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 lit up, 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 equivalent replacement methods and are included in the protection scope 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-boric acid triphenylamine 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 an 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-boric acid triphenylamine, 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 condensation reaction and post-treatment 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, followed by post-treatment to obtain compound 3; S4. Compound 3 reacts with trifluoroacetic acid to remove the Boc group, and then post-processes to obtain an 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 luminescence compound according to claim 1 in preparing a photosensitizer or in distinguishing between the dead and alive states of bacteria.

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

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

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