Synthesis and antibacterial application of thiophene D-pi-A type organic photosensitizer based on molecular modification

Abstract

The invention discloses synthesis and antibacterial application of a thiophene D-II-A type organic photosensitizer based on molecular modification, and a compound TPA-SFCN with excellent performance is synthesized by improving the molecular structure of a natural photosensitizer 2, 2 ': 5', 2 ''-trithiophene-5-formaldehyde. In the invention, the thiophene compound is used as an efficient electron transfer pi bridge, and functional groups of electron donor triphenylamine (TPA) and electron acceptor cyano (CN) are successfully introduced through ordered molecular engineering modification. The modification is realized through a simple and efficient chemical reaction, so that the target compound shows a remarkable red shift phenomenon in the aspect of spectral characteristics, including simultaneous red shift of an ultraviolet-visible absorption spectrum and a fluorescence emission spectrum. In addition, compared with an initial compound, the TPA-SFCN shows remarkably improved singlet oxygen generation efficiency and bactericidal ability, so that the TPA-SFCN shows wide application prospects and important development value in the field of organic photosensitive therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of photosensitizers, and specifically to the synthesis and antibacterial application of a molecularly modified thiophene-based D-Π-A type organic photosensitizer. Background Art

[0002] Photodynamic therapy is an emerging non-invasive treatment method that has been widely used in the clinical field in recent years. Its mechanism of action mainly involves the excitation of photosensitizers under specific light irradiation conditions to generate singlet oxygen, thereby achieving the selective elimination of diseased cells or pathogenic microorganisms. With its significant advantages of spatio-temporal selectivity, low toxicity, and minimally invasive nature, it has gradually become an important means for treating various diseases such as cancer, skin diseases, ophthalmic diseases, and bacterial infections. Compared with traditional treatment methods (such as surgery, chemotherapy, and radiotherapy), it exhibits unique advantages, including reducing damage to normal tissues, avoiding the induction of drug resistance, and repeatable treatment.

[0003] Reactive oxygen species (ROS) are a class of highly oxidative molecules that can effectively cause the death of diseased tissues or pathogens through mechanisms such as inducing apoptosis, destroying cell structures, damaging blood vessels, and stimulating immune responses. The generation of ROS depends on the excitation of specific wavelength light irradiation and has a short action distance and survival period. Therefore, photodynamic therapy can specifically damage diseased tissues or microorganisms in the region of specific wavelength light irradiation, significantly reducing side effects on normal tissues and improving the treatment accuracy. From the perspective of the photochemical reaction mechanism, the generation of ROS can be achieved through two pathways: energy transfer (type II mechanism) and electron transfer (type I mechanism). In the type II mechanism, the photosensitizer undergoes energy transfer with oxygen molecules to generate singlet oxygen ( 1 O 2 ). In the type I mechanism, the triplet excited state photosensitizer interacts with neighboring molecules to generate free radical intermediates, which further react with water and oxygen to produce reactive oxygen species such as hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 ·- ), and hydroxyl radical (·OH). These two mechanisms usually coexist in the PDT process and show a competitive relationship, and their activity ratios are regulated by factors such as the characteristics of the photosensitizer, solvent polarity, photosensitizer concentration, oxygen concentration, and action substrates. Research shows that in a system with a higher oxygen concentration, the type II pathway dominates; when the oxygen concentration decreases, the type I pathway becomes the main reaction path. However, due to the lack of a systematic type I photosensitizer design strategy and theoretical guidance, current photosensitizer research and development mainly focus on the study of the type II PDT process.

[0004] The natural photosensitizer 2,2':5',2”-terthiophene-5-carbaldehyde exists in phototoxic nematicides and antibiotics such as marigolds. The phototoxic nematicides and antibiotics in marigolds have strong resistance to many strains. As a natural photosensitive compound, 2,2':5',2”-terthiophene-5-carbaldehyde can generate singlet oxygen under aerobic excitation and exhibits excellent ultraviolet-enhanced antibacterial properties, thus attracting extensive attention and application. However, its characteristic that the ultraviolet absorption band is close to the ultraviolet region significantly restricts its practical application.

[0005] To address this limitation, this study achieved a red shift of the ultraviolet absorption band through a molecular modification strategy. The D-Π-A structured organic photosensitizer 2-((5”-(4-(diphenylamino)phenyl)-[2,2':5',2”-terthiophen]-5-yl)methylene)malononitrile was successfully synthesized and its application in the field of photosensitizing antibacterial was verified. By comparing its properties with those of 2,2':5',2”-terthiophene-5-carbaldehyde, it showed a fluorescence and absorption red shift and exhibited better photosensitizing antibacterial activity. Summary of the Invention

[0006] The object of the present invention is, based on the deficiencies of natural thiophene photosensitizers, to synthesize a D-Π-A organic photosensitizer and its application in the field of photosensitizing antibacterial on the basis of molecular modification. The technical solution adopted by the present invention is as follows:

[0007] To achieve the purpose of preparing a compound with a specific wavelength of thiophene, based on molecular modification to make the fluorescence and ultraviolet absorption of the compound red shift, triphenylamine groups and cyano groups are introduced on both sides of the compound to form a D-Π-A structure, and an explanation is given from a molecular perspective.

[0008] To achieve and verify the above technical solution, based on 2,2':5',2”-terthiophene-5-carbaldehyde, a D-Π-A type organic photosensitizer TPA-SFCN is synthesized. The TPA-SFCN is 2-((5”-(4-(diphenylamino)phenyl)-[2,2':5',2”-terthiophen]-5-yl)methylene)malononitrile, and the structural formula of the TPA-SFCN is:

[0009]

[0010] The TPA-SFCN is prepared by the following method: 51.9 mg of TPA-SFQ is placed in a two-necked flask. Under a nitrogen atmosphere, 10 mL of ultra-dry n-propanol, 0.1 mL of malononitrile, and 0.2 mL of pyridine are gradually added using a syringe. The reaction mixture is refluxed overnight at 80 °C. After the reaction is completed and cooled to room temperature, the solvent is removed by vacuum concentration, and the product is purified by silica gel column chromatography using petroleum ether / ethyl acetate (V / V = 10 / 1) as the eluent. The obtained product is TPA-SFCN.

[0011] The TPA-SFQ is prepared by the following method: 71.0 mg of Br-SFQ, 86.7 mg of 4-(diphenylamino)phenylboronic acid, and 5% equivalent (7.2 mg) of 1,1'-bis(diphenylphosphino)ferrocene dichloropalladium(II) are placed in a two-necked flask. Under a nitrogen atmosphere, 10 mL of toluene is added using a syringe to dissolve them, and finally 2 mL of 2 mg·L -1 potassium carbonate solution is added. The reaction mixture is refluxed overnight at 110 °C. After the reaction is completed and cooled to room temperature, the system is extracted with ethyl acetate, and the organic phase is washed successively with brine and deionized water. The organic phase is dried over anhydrous sodium sulfate and then the solvent is removed by vacuum concentration. The crude product is purified by silica gel column chromatography using petroleum ether / ethyl acetate (V / V = 10 / 1) as the eluent. The obtained product is TPA-SFQ.

[0012] The Br-SFQ is prepared by the following method: 138 mg of [2,2':5',2”-terthiophene]-5-carbaldehyde and 106.2 mg of N-bromosuccinimide are placed in a two-necked flask and dissolved in N,N-dimethylformamide. The reaction mixture is stirred overnight at room temperature under the protection of an inert gas (nitrogen) atmosphere and in the dark. After the reaction is completed, the mixture is extracted with dichloromethane and deionized water. The organic phase is dried over anhydrous sodium sulfate and then the solvent is removed by vacuum concentration. The obtained product is Br-SFQ.

[0013] Application of the thiophene-based D-Π-A type organic photosensitizer in in vitro sterilization.

[0014] The pathogenic bacteria are S. aureus, E. coli, and P. aeruginosa.

[0015] The beneficial effects obtained by the present invention are as follows:

[0016] (1) In the present invention, triphenylamine is used as an electron donor, and the thiophene group is used as a Π bridge to be connected to the malononitrile group with strong electron-withdrawing ability.

[0017] (2) The D-Π-A type organic photosensitizer provided in this experiment can generate a large amount of singlet oxygen under a xenon lamp and has a better production effect than the widely studied 2,2':5',2”-terthiophene-5-carbaldehyde.

[0018] (3) This experiment is based on the idea of molecular modification and successfully redshifts the ultraviolet absorption and fluorescence from SFQ to TPA-SFCN, providing ideas for compound design.

[0019] (4) It has a high bactericidal rate against three common pathogenic bacteria in life (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa), and the minimum bactericidal concentration reaches 6.6×10 -6 mol·L -1 .

[0020] In summary, the present invention provides an idea of molecular modification, which can design compounds according to requirements to make the ultraviolet absorption and fluorescence redshift. A thiophene-based D-Π-A organic photosensitizer compound obtained by molecular modification on the basis of SFQ. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the structural formula of the thiophene-based D-Π-A type organic photosensitizer of the present invention;

[0022] Figure 2 is the molecular modification route diagram of the compound;

[0023] Figure 3 is the redshift of ultraviolet absorption during compound modification;

[0024] Figure 4 is the redshift of fluorescence during compound modification;

[0025] Figure 5 is the change of LUMO-HOMO of the molecular calculation compound;

[0026] Figure 6 is the electrostatic formula and orbital change of the molecular calculation compound;

[0027] Figure 7 is the ABDA degradation diagram of the original compound SFQ;

[0028] Figure 8 is the ABDA degradation diagram of the modified product TPA-SFCN;

[0029] Figure 9 is the ABDA normalized degradation diagram;

[0030] Figure 10 is the antibacterial schematic diagram of TPA-SFCN;

[0031] Figure 11 is the evaluation of the antibacterial effect of TPA-SFCN by a live / dead staining kit. DETAILED DESCRIPTION OF THE INVENTION

[0032] To illustrate the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. The following are the implementation examples of the present invention, which are not intended to limit the present invention. Any modifications, substitutions, improvements, etc. made on the basis of the present invention are included within the protection scope of the present invention.

[0033] Example 1

[0034] This example is an application example for preparing TPA-SFCN based on the molecular modification process. The specific steps are as follows:

[0035] Preparation of Br-SFQ:

[0036] Take 138 mg of 2,2':5',2”-terthiophene-5-carbaldehyde and 106.2 mg of N-bromosuccinimide in a two-necked flask, dissolve them in N,N-dimethylformamide, and stir the reaction mixture overnight at room temperature under the protection of an inert gas, nitrogen, and in the dark. After the reaction is completed, the mixture is extracted with dichloromethane and deionized water. The organic phase is dried over anhydrous sodium sulfate and then concentrated in vacuo to remove the solvent. What is obtained is Br-SFQ.

[0037] Preparation of TPA-SFQ:

[0038] Take 71.0 mg of Br-SFQ, 86.7 mg of 4-(diphenylamino)phenylboronic acid, and 5% equivalent of 7.2 mg of 1,1'-bis(diphenylphosphino)ferrocene dichloropalladium(II) in a two-necked flask. Add 10 mL of toluene to dissolve it with a syringe under a nitrogen atmosphere, and finally add 2 mL of 2 mg·L -1 potassium carbonate solution. The reaction mixture is refluxed overnight at 110 °C. After the reaction is completed and cooled to room temperature, the system is extracted with ethyl acetate, and the organic phase is washed successively with brine and deionized water. The organic phase is dried over anhydrous sodium sulfate and then concentrated in vacuo to remove the solvent. The crude product is purified by silica gel column chromatography, and eluted with petroleum ether / ethyl acetate (V / V = 10 / 1) as the eluent. What is obtained is TPA-SFQ.

[0039] Preparation of TPA-SFCN:

[0040] Take 51.9 mg of TPA-SFQ in a two-necked flask, and gradually add 10 mL of ultradry n-propanol, 0.1 mL of malononitrile, and 0.2 mL of pyridine with a syringe under a nitrogen atmosphere. The reaction mixture is refluxed overnight at 80 °C. After the reaction is completed and cooled to room temperature, the solvent is removed by vacuum concentration, and it is purified by silica gel column chromatography with petroleum ether / ethyl acetate (V / V = 10 / 1) as the eluent. What is obtained is TPA-SFCN.

[0041] Example 2

[0042] This example is to verify the correctness of the red-shift of fluorescence and ultraviolet absorption of three compounds, namely SFQ, TPA-SFQ, and TPA-SFCN.

[0043] Weigh a certain amount of the target compound and prepare a stock solution with a concentration of 1×10 -4 M in tetrahydrofuran (THF). Subsequently, using THF as a diluent, further dilute the stock solution to a working solution with a concentration of 1×10 -5 M. Use a UV-visible spectrophotometer (model: UV-1901PC, produced by Shanghai Lengguang Technology Co., Ltd.) and a fluorescence photometer (model: F97PRO, produced by Shanghai Lengguang Technology Co., Ltd.) to measure the ultraviolet and fluorescence spectra respectively. The ultraviolet-visible absorption spectra and fluorescence spectra of three compounds, SFQ, TPA-SFQ, and TPA-SFCN, were measured in the experiment. Figure 3 and Figure 4 The results show that with the optimization of the molecular structure, the ultraviolet-visible absorption band shows a gradual red-shift trend: from the maximum absorption peak of SFQ at 390 nm, through 440 nm of TPA-SFQ, and finally red-shifting to 520 nm of TPA-SFCN. At the same time, the fluorescence emission peak also shifts from 480 nm of SFQ, through 600 nm of TPA-SFQ, and finally red-shifts to 720 nm of TPA-SDCN. This reflects the effectiveness of molecular design.

[0044] Example 3

[0045] This example is to verify the rationality of the design and the explanation of the red-shift of fluorescence and ultraviolet absorption from the perspective of molecular calculation by combining three compounds, SFQ, TPA-SFQ, and TPA-SFCN.

[0046] The electronic structures of the three compounds were analyzed by theoretical calculation. Using Gaussian 09W software and the density functional theory (DFT) method, the distributions of the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) of each compound were calculated. As Figure 5As shown, the electron cloud distribution characteristics of the three compounds are extremely similar: when the molecule is in the LUMO state, the electron cloud is mainly concentrated on the thiophene ring; while in the HOMO state, the electron cloud is mainly distributed on the triphenylamine (TPA) structure. Comparing the three compounds, TPA-SFQ and TPA-SFCN exhibit better LUMO-HOMO separation characteristics. The calculation results show that the LUMO-HOMO energy gaps (ΔE) of SFQ, TPA-SFQ, and TPA-SFCN are 3.86 eV, 3.09 eV, and 2.27 eV, respectively. The gradually decreasing trend of the energy gap is highly consistent with the red-shift phenomena of the ultraviolet-visible absorption spectrum and the fluorescence emission spectrum. A smaller energy gap indicates a more significant red-shift of the absorption spectrum and the emission spectrum, which is beneficial to improving the utilization efficiency of long-wavelength light such as visible light by the compound.

[0047] Example 4

[0048] This example is another application example for verifying the rationality of the design and the explanation of the red-shift of fluorescence and ultraviolet absorption.

[0049] Further analyze the distribution of the molecular surface electrostatic potential (ESP), Figure 6 showing that the electron density in the TPA-SFCN molecule is mainly concentrated in the cyano region. This molecule presents a typical D-π-A structure. Calculate the energy gap difference (ΔE 1 ) between the lowest singlet excited state (S 1 ) and the lowest triplet excited state (T st ) by the time-dependent DFT (TD-DFT) method. The results show that the ΔE st of SFQ, TPA-SFQ, and TPA-SFCN are 1.02 eV, 0.50 eV, and 0.32 eV, respectively, and the ΔE st gradually decreases. This LUMO-HOMO separation not only promotes the reduction of ΔE st , but also leads to the red-shift of the absorption spectrum and the fluorescence spectrum, further verifying the scientificity and rationality of the compound design in this study.

[0050] Example 5

[0051] This example is to verify the singlet oxygen generation ability of TPA-SFCN and compare it with that of the pre-modified molecule SFQ.

[0052] Using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) as a specific chemical probe for singlet oxygen ( 1 O 2 ), the reactive oxygen species generation abilities of the two photosensitizers (SFQ and TPA-SFCN) were quantitatively analyzed.

[0053] The specific experimental protocol is as follows: First, accurately weigh ABDA (2.1 mg) and add it to deionized water (2.0 mL). Dissolve it thoroughly by ultrasonic treatment to prepare an ABDA stock solution with a concentration of 2.5×10-3 M, and store it for later use under dark and low-temperature conditions. Subsequently, prepare the photosensitizers to be studied into test solutions with a concentration of 10 μM (the solvent is a methanol / water mixed solution with a volume ratio of 15:85). During the experiment, take 80.0 μL of the ABDA stock solution and add it to 4.0 mL of the test solution, mix well, and then irradiate it uniformly (using a xenon lamp as the light source). To ensure the comparability of the experimental results, strictly control the irradiation time to 20.0 min. During the spectral analysis process, continuously monitor the change in the absorbance value of the solution at 378 nm. By normalizing the absorbance value of ABDA at this characteristic wavelength, calculate the ABDA degradation efficiency of different photosensitizers within the same irradiation time, and then quantitatively evaluate the influence degree of their singlet oxygen generation ability. As shown in the figure, in the case of adding SFQ, the absorbance of ABDA decreases significantly. Specifically, compared with the SFQ treatment group ( Figure 7 ), the TPA-SFCN treatment group ( Figure 8 ) shows a faster decreasing trend in absorbance. By normalizing the degradation curve ( Figure 9 ), it is calculated that the ABDA degradation rate of TPA-SFCN is significantly higher than that of SFQ, further verifying its superior 1 O 2 generation ability.

[0054] Example 6

[0055] This example is an example for evaluating antibacterial activity.

[0056] Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa) are taken out from the -80°C ultra-low temperature freezing storage conditions respectively, inoculated into LB broth medium, placed in a 37°C constant temperature shaking incubator, with the condition set to a vibration rate of 120 r / min, and cultured for 12 - 16 hours until the bacteria are activated. The activated bacterial culture solutions are diluted to OD 600 = 0.01 respectively using LB liquid medium to prepare standard bacterial suspension solutions. Mix 200 μL of the bacterial suspension solution with 20 μL of different concentrations of the compound respectively, place them in a 96-well plate, and incubate at 37°C for 1 hour. Subsequently, the light irradiation group is treated with light irradiation using a xenon lamp (CEL-TCX250) for 30 minutes, while the dark treatment group is incubated in the dark for the same time. Spread each group of mixed solutions evenly on the surface of LB agar solid medium by the dilution coating method, and then place them in a 37°C constant temperature incubator for 12 hours. After the culture is completed, take pictures of the colonies on each group of culture media.

[0057] As Figure 10 shown, the growth of bacteria under different treatments was observed through LB agar. The experimental results showed that under dark conditions, regardless of whether TPA-SFCN was added or not, all three bacteria could grow normally, and the colony distribution was uniform, indicating that the photosensitizer itself had no direct killing effect on bacteria. However, under light conditions, the bacteria in the drug-added group were completely eliminated, and almost no colonies could be seen, showing a significant photodynamic antibacterial effect. Specifically, the bactericidal efficiency of TPA-SFCN against S. aureus, E. coli, and P. aeruginosa reached 99.9% under light illumination, indicating its excellent antibacterial performance.

[0058] Example 7

[0059] This example is another evaluation example of antibacterial activity.

[0060] To further verify the photodynamic antibacterial effect, the LIVE / DEAD staining method was used to treat the three bacteria. Specifically, the bacteria before and after treatment were grouped, and the bacteria were stained by LIVE / DEAD staining and observed under a fluorescence microscope.

[0061] The experimental results showed that in the light-illuminated and drug-added group, the bacterial cells showed red fluorescence, indicating that the cells had lost their activity, and the killing of bacteria was successfully achieved ( Figure 11 ). This result was verified with the bactericidal efficiency data, further confirming the effectiveness of the photosensitizer.

Claims

1. A thiophene D-II-A type organic photosensitizer, characterized in that: It has the following structural formula:

2. The method for preparing the thiophene D-II-A type organic photosensitizer according to claim 1, characterized in that: The following steps are involved: (1) Preparation of intermediate Br-SFQ 138 mg of [2,2':5',2"-thiophene]-5-carboxaldehyde and 106.2 mg of N-bromosuccinimide were dissolved in N,N-dimethylformamide, and the reaction mixture was stirred overnight under an inert gas atmosphere at room temperature and away from light; after the reaction, the mixture was extracted with dichloromethane and deionized water, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo to remove the solvent to obtain an orange-yellow solid Br-SFQ; (2) Preparation of intermediate TPA-SFQ Take 71.0 mg of Br-SFQ, 86.7 mg of 4-(diphenylamino)phenylboronic acid and 7.2 mg of 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) dichloride and add 10 mL of toluene to dissolve them under nitrogen atmosphere. Finally, add 2 mL of 2 mg·L -1 Potassium carbonate solution; the reaction mixture was refluxed at 110°C overnight; after the reaction was completed and cooled to room temperature, the system was extracted with ethyl acetate, and the organic phase was washed with brine and deionized water in turn, the organic phase was dried over anhydrous sodium sulfate and then concentrated in vacuo to remove the solvent; the crude product was purified by silica gel column chromatography and eluted to obtain red solid TPA-SFQ; (3) Preparation of TPA-SFCN: 51.9 mg of TPA-SFQ was gradually added with 10 mL of super dry n-propanol, 0.1 mL of malononitrile and 0.2 mL of pyridine using a syringe under a nitrogen atmosphere; the reaction mixture was refluxed at 80°C overnight, and after the reaction was completed and cooled to room temperature, the solvent was removed by vacuum concentration, and the product was purified by silica gel column chromatography and eluted to obtain a black solid, namely TPA-SFCN.

3. The method for preparing the thiophene D-II-A type organic photosensitizer according to claim 2, characterized in that: The elution in step (2) is performed using petroleum ether:ethyl acetate in a volume ratio of 10:1 as the eluent.

4. The method for preparing the thiophene D-II-A type organic photosensitizer according to claim 2, characterized in that: The elution in step (3) is performed using petroleum ether:ethyl acetate in a volume ratio of 10:1 as the eluent.

5. The method for preparing the thiophene D-II-A type organic photosensitizer according to claim 2, characterized in that: The inert gas in step (1) is nitrogen.

6. Use of the thiophene D-II-A type organic photosensitizer according to claim 1 in in vitro sterilization.

7. The use of the thiophene D-II-A type organic photosensitizer in in vitro sterilization according to claim 6, characterized in that: The pathogenic bacteria are S. aureus, E. coli, and P. aeruginosa.

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