AIE imitated antibacterial peptide material as well as preparation method and application thereof

Abstract

The invention discloses an AIE imitated antibacterial peptide material and a preparation method and application thereof, and belongs to the technical field of polymer materials, the AIE imitated antibacterial peptide material takes an alkyl chain as a main chain, the position of a cation primitive is regulated, and AIE primitives with different D-A structures are combined as side chains, so that the AIE imitated antibacterial peptide material is prepared. Through the D-A structure in the AIE element, the molecular can be used for the performance regulation and control of generating ROS or photo-thermal through illumination response, so that the growth and proliferation of microorganisms can be inhibited, and the damage to a biofilm is further inhibited. The AIE imitated antibacterial peptide material is good in biocompatibility and has a wide application prospect in the field of antibacterial agents.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polymer materials, in particular to an AIE mimetic antibacterial peptide material and a preparation method and application thereof. BACKGROUND

[0002] The problem of microbial resistance to conventional antibiotics is one of the biggest challenges in the field of biomedicine, and the emergence of drug-resistant bacteria has seriously affected the efficacy of conventional antibiotics, increasing the difficulty of disease cure. Antibacterial peptides can also have antibacterial effects on drug-resistant strains due to their broad-spectrum antibacterial properties, making them important candidate materials for the development of new antibacterial drugs. However, the inherent limitations of antibacterial peptides, such as low stability, susceptibility to proteolysis, complex and time-consuming synthesis, and high production costs, limit their application. To overcome the limitations of natural antibacterial peptides, researchers have developed a variety of mimetic antibacterial peptides. Molecules with cationic motifs as a kind of mimetic antibacterial peptide have high antibacterial activity and a broad antibacterial spectrum. They usually have a lipophilic part and a positive charge part, which helps to recognize and inhibit drug-resistant bacteria. The positive charge on the cationic motif allows it to adhere to the negatively charged bacterial membrane surface, and the lipophilic part inserts into the lipid bilayer, ultimately destroying the cell membrane and causing the bacteria to die or enter the bacterial interior to disturb and inhibit the bacteria.

[0003] It is known that phototherapy is an emerging method for treating microbial infections, with obvious advantages such as high spatial and temporal resolution, precise spatiotemporal controllability, and non-invasiveness, including photodynamic therapy (PDT) and photothermal therapy (PTT). PDT involves energy transfer processes of molecular excited states, and light-excited molecular energy transfer produces reactive oxygen species (ROS), while PTT converts light energy into heat energy through non-radiative transitions of molecular excited states, both of which can inhibit or kill microorganisms to achieve therapeutic purposes. Compared with antibacterial drugs such as antibiotics, PDT and PTT can greatly reduce the risk of drug resistance, so phototherapy has broad application prospects in the treatment of infections.

[0004] In addition, the localization and diagnosis of microbial infection foci are also crucial in the treatment of infectious diseases. Fluorescence imaging (FLI) is a method with high sensitivity and fast response. However, the spatial resolution of FLI is limited. Photoacoustic imaging (PAI) is an imaging technique that combines the advantages of optics and acoustics. After the absorption of light energy by tissues, thermal-elastic expansion occurs, and then ultrasonic waves are emitted. The instrument reconstructs the image by detecting the ultrasonic waves. PAI has the advantage of high spatial resolution, but it has the disadvantage of low sensitivity. Therefore, combining the advantages of FLI and PAI will make the imaging of microbial infection foci clearer and more conducive to the tracking and treatment of the foci. Therefore, the development of a multi-modal diagnosis and treatment system that combines FLI, PAI, PDT, and PTT will help improve the efficacy of infectious diseases. Aggregation-induced emission (AIE) materials are the first choice for building a multi-modal diagnosis and treatment system due to their characteristics of emitting more light when aggregated, good light stability, and difficulty in forming strong intermolecular π-π interactions. In addition, AIE materials can be designed to have excellent photosensitivity and photothermal conversion efficiency by cleverly regulating the donor-acceptor (D-A) structure motif, which further broadens the potential applications of materials in FLI, PAI, PDT, PTT, and other aspects of infectious disease diagnosis and treatment.

[0005] Developing new antibacterial drugs is an important strategy to combat microbial infectious diseases, aiming to break through the existing drug resistance barrier and improve clinical efficacy. The Chinese patent document with publication number CN120209206A discloses a light-responsive-essential oil synergistic antibacterial polymer. The invention prepares CMA monomers by esterification of citronellol (CT), copolymerizes them with glycidyl methacrylate (GMA) monomers to prepare polymer PG9C2, and then introduces TBO to obtain antibacterial polymer PG9C2T1. The improvement of its antibacterial activity is due to the fact that the polymer improves the liposolubility of TBO, enhances its interaction with bacterial cells, improves its accumulation in cells, enables it to penetrate the cell membrane and play a role inside the cell, improves the utilization efficiency of ROS, and at the same time improves the water solubility of essential oil to kill bacteria by destroying the bacterial membrane. The Chinese patent document with publication number CN113929892A discloses a high-efficiency cationic antibacterial polymer containing metal. The metal cationic polymer is composed of a macromolecular polyester backbone and side groups. The macromolecular polyester backbone is produced by the polycondensation of trimethylolpropane allyl ether and diacyl chloride, and the side groups are dimethylaminopyridine amine-metal complexes. The dimethylaminopyridine amine-metal complexes can have strong coordination with the phosphatidylserine on the surface of bacterial membranes, so the metal cationic polymer can kill a variety of bacteria at a lower concentration.

[0006] However, there are few reports of antibacterial peptide mimics with AIE properties and multi-modal diagnosis and treatment in the prior art. SUMMARY

[0007] In order to solve the above-mentioned problems existing in the prior art, the present application provides an AIE imitated antibacterial peptide material, which has good biocompatibility and inhibitory effect on various microorganisms, especially can inhibit the growth and proliferation of drug-resistant bacteria such as MRSA and their biofilm.

[0008] The specific technical solutions adopted are as follows:

[0009] An AIE imitated antibacterial peptide material, the structural formula of which is shown in any one of formula I, formula II or formula III:

[0010]

[0011] In formula I, 6 >= m >= 1;

[0012] In formula I, formula II and formula III, n is an integer of 5-20 (both of the two repeating segment numbers in formula III are the same n);

[0013] R has an electron-donating group and an electron-withdrawing group, each independently selected from one of the following structures, wherein * represents the connecting position;

[0014]

[0015] The present application takes an alkyl chain as the main chain, adjusts the position of the cationic group, and combines different D-A structure AIE groups as side chains, so that the performance regulation of the molecule for generating ROS or photothermal effect under light response is realized through the D-A structure in the AIE group, and an AIE imitated antibacterial peptide with ROS generation or photothermal conversion ability is obtained. The AIE imitated antibacterial peptide can better interact and combine with the surface of microorganisms through the cationic group, can inhibit the growth and proliferation of microorganisms, and further inhibit and destroy the biofilm.

[0016] The present application also provides a preparation method of the AIE imitated antibacterial peptide material, which specifically comprises the following steps:

[0017] (1) Under the protection of inert gas, Suzuki reaction is carried out by using aryl boronic acid and aldehyde-substituted bromoheteroarene, the obtained nitrobenzaldehyde derivative is reacted with format reagent acetylene magnesium bromide under the protection of inert gas, and the obtained product is treated with manganese dioxide to obtain a first intermediate AIE activated alkyne;

[0018] (2) The first intermediate AIE activated alkyne is reacted with bis(2-bromoethyl)amine hydrobromide to obtain a second intermediate, and the second intermediate is polymerized with a bis-tertiary amine compound under the protection of inert gas to obtain the AIE imitated antibacterial peptide shown in formula I;

[0019] (3) reacting the first intermediate AIE-activated alkyne with 3-bromoprop-1-amine hydrobromide to obtain a third intermediate, and subjecting the first polymerization monomer to radical polymerization to obtain a polymerization product, and reacting the polymerization product with the third intermediate to obtain the AIE mimetic antibacterial peptide shown in formula II;

[0020] (4) subjecting the second polymerization monomer to radical polymerization to obtain a polymerization product, and reacting the polymerization product with the first intermediate AIE-activated alkyne to obtain the AIE mimetic antibacterial peptide shown in formula III.

[0021] In step (1), a catalyst and an auxiliary agent are further added in the Suzuki reaction; the catalyst is preferably Pd(PPh3)4, and the auxiliary agent is preferably potassium carbonate.

[0022] Preferably, in step (1), the Suzuki reaction is carried out at 70-80℃ for 16-48h, and the reaction of the nitrobenzaldehyde derivative with the Grignard reagent ethynylmagnesium bromide is carried out at 0-40℃ for 2-12h.

[0023] Alternatively, in step (1), the nitrobenzaldehyde acetylene derivative obtained is oxidized by manganese dioxide under the conditions of 25-40℃ for 2-4h.

[0024] Specifically, the first intermediate AIE-activated alkyne includes ketone alkyne, ester alkyne or amide alkyne, and is preferably ketone alkyne.

[0025] Preferably, in step (2), the reaction of the first intermediate AIE-activated alkyne with bis(2-bromoethyl)amine hydrobromide is carried out at 25-60℃ for 2-48h, and the polymerization of the second intermediate and the bis-tertiary amine compound is carried out at 70-120℃ for 16-72h.

[0026] Specifically, the first polymerization monomer is N-[2-(dimethylamino)ethyl]acrylamide, and the second polymerization monomer is N-ethylmethacrylamide and N-[2-(dimethylamino)ethyl]acrylamide bromoethane salt.

[0027] Preferably, the polymerization monomer is subjected to radical polymerization under the action of an initiator, and the initiator is azobisisobutyronitrile (AIBN), and the first polymerization monomer or the second polymerization monomer is subjected to radical polymerization under the conditions of 40-120℃ for 24-72h.

[0028] Preferably, in step (3), the reaction of the first intermediate AIE-activated alkyne with 3-bromoprop-1-amine hydrobromide is carried out at 25-60℃ for 2-48h, and the reaction of the polymerization product with the third intermediate is carried out at 70-120℃ for 16-72h.

[0029] Preferably, in step (4), the reaction conditions of the polymerization product and the first intermediate AIE activated alkyne are 25-60 DEG C, 4-48h.

[0030] By regulating the position of the cationic unit of the product AIE antimicrobial peptide, the solubility, active oxygen (ROS) production / photothermal conversion performance, antibacterial effect, etc. can be regulated.

[0031] The application also provides an antibacterial agent, wherein the antibacterial agent component comprises the AIE antimicrobial peptide.

[0032] The AIE antimicrobial peptide has good biocompatibility, and as an antibacterial photosensitizer, it has inhibitory effect on a variety of bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus MRSA, Escherichia coli, carbapenem-resistant multi-drug-resistant Escherichia coli CREc, and Candida albicans, etc.).

[0033] Research has found that the AIE antimicrobial peptide not only has inhibitory effect on planktonic bacteria, but also has inhibitory effect on biofilm, and phototherapy can simultaneously improve the antibacterial effect.

[0034] The antibacterial agent can be used alone or in combination with light treatment. The AIE antimicrobial peptide can be used to generate ROS / heat under light, realize energy conversion, and realize inhibition of microorganisms at low concentration.

[0035] Further preferably, the light treatment conditions are: light density 20-200 mW / cm 2 , irradiation time 5-40 min.

[0036] Compared with the prior art, the application has the following beneficial effects:

[0037] (1) The application synthesizes an AIE antimicrobial peptide with an alkyl chain as the main chain, a cationic unit position that can be regulated, and an AIE unit and a photosensitive / photothermal agent unit introduced in the side chain through Suzuki reaction, coupling reaction, and free radical polymerization reaction, so as to obtain an AIE antimicrobial peptide with ROS production or photothermal conversion capability.

[0038] (2) The AIE antimicrobial peptide provided by the application has good biocompatibility, little or no damage to normal cells, and as a photosensitive / photothermal agent, it has inhibitory effect on a variety of microorganisms, especially can inhibit the growth and proliferation of drug-resistant bacteria such as MRSA and their biofilm. DETAILED DESCRIPTION

[0039] Figure 1 A schematic diagram of the AIE antimicrobial peptide prepared in the examples.

[0040] Figure 2(A) is the AIE mimicking antibacterial peptide molecular structure, (B) is the absorption and emission spectrum, (C) is the fluorescence quantum yield, (D) is the Zeta potential, (E) is the ROS ratio curve.

[0041] Figure 3 (A) is the OMPA-TT, (B) is the OMHA-TT, (C) is the OSMA-TT, (D) is the OSEA-TT.

[0042] Figure 4 (A) is the OSEA-TT plate picture after acting on MRSA and the inhibition rate statistics, (B) is the SEM picture of OSEA-TT after acting on MRSA.

[0043] Figure 5 (A) is the schematic diagram of OSEA-TT curing mouse subcutaneous abscess assisted by white light, (B) is the picture of mouse subcutaneous abscess treatment process, (C) is the skin smear plate inhibition rate statistics data of mouse subcutaneous abscess after treatment in different groups, (D) is the skin smear plate picture of mouse subcutaneous abscess after treatment in different groups. DETAILED DESCRIPTION

[0044] In order to make the objects, features and advantages of the present application more apparent, the following will be described in detail through specific embodiments. In the following description, a lot of specific details are set forth in order to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the spirit of the present application, so the present application is not limited to the specific embodiments disclosed below. The technical features of each embodiment of the present application can be combined accordingly without mutual conflict.

[0045] The operation methods in the following examples without specific conditions are usually according to the conventional conditions, or according to the conditions suggested by the manufacturers. The contents not described in detail in the specification belong to the prior art known by those skilled in the art. The experimental materials used in the following examples can be purchased from the conventional biochemical reagent company, unless otherwise specified.

[0046] The preparation schematic diagram of AIE mimicking antibacterial peptide is shown in Figure 1 .

[0047] The synthesis of the first intermediate AIE-activated alkyne used in the following examples is shown below:

[0048]

[0049] To 5-bromothiophene-2-carboxaldehyde (2.19 g, 11.44 mmol), 4-bis(4- methoxyphenyl)aminophenylboronic acid (5.00 g, 14.30 mmol), potassium carbonate (3.95 g, 28.60 mmol) and Pd(PPh3)4(0.79 g, 0.68 mmol), these mixtures were added to a double necked round bottom flask with a condenser and sealed, then vacuumed and purged with nitrogen three times. Then THF (40 mL) and deionized water (14.3 mL) were added to the double necked flask and stirred at 80 °C under nitrogen atmosphere for 24 hours. After that, cooled to room temperature, the reaction mixture was concentrated, the solid was dissolved, DCM and saturated NaCl solution were added to the system and extracted three times, then the organic layer was dried over anhydrous Na2SO4, concentrated by rotary evaporation, then silica gel powder was added to the sample, the crude product was purified by column chromatography using DCM:PE (1:1, v / v) as eluent. Orange-yellow solid (nitrobenzaldehyde derivative) was obtained.

[0050] The orange-yellow solid (1.50 g, 3.60 mmol) synthesized in the previous step was placed in an oven-dried double-necked round-bottom flask, then vacuumed and purged with nitrogen three times, and then super-dry THF (40 mL) was added to the double-necked round-bottom flask. The flask was placed in an ice-water bath, and 21.6 mL of 0.5 M ethynyl magnesium bromide THF solution was added dropwise. After returning to room temperature, the reaction was continued to stir for 4 hours under a nitrogen atmosphere. The reaction solution was quenched with saturated aqueous ammonium chloride solution (40 mL), and the resulting mixed solution was extracted with DCM three times. Then the organic layer was dried over anhydrous Na2SO4, concentrated by rotary evaporation, then silica gel powder was added to the sample, and the crude product was purified by column chromatography using DCM:PE (3:1, v / v) as eluent to obtain an orange-red solid.

[0051] The orange-red solid (1.50 g, 3.42 mmol) synthesized in the previous step was dissolved in 40 mL of DCM, and then manganese dioxide (4.00 g, 45.98 mmol) was slowly added while stirring. The reaction was monitored by thin layer chromatography and was complete after about 2 hours of stirring at 35 °C. The remaining manganese dioxide was removed by filtration using a sintered glass funnel packed with silica gel, and the organic layer was concentrated by rotary evaporation. Silica gel powder was then added to the sample, and the crude product was purified by column chromatography using DCM:PE (1:1, v / v) as eluent to obtain the first intermediate AIE-activated alkyne as an orange-red solid with a yield of 84% (1.33 g).

[0052] The first intermediate AIE-activated alkyne has the following NMR hydrogen / carbon spectral data:

[0053] 1 H NMR (500 MHz, CDC13) δ 7.94 (d, J = 4.1 Hz, 1H), 7.60 (d, J = 2.1 Hz, 2H), 7.50 (d, J = 4.1 Hz, 1H), 7.10-7.07 (m, 4H), 6.97-6.93 (m, 4H), 6.72 (d, J = 7.0 Hz, 2H), 4.97 (s, 1H), 3.74 (s, 6H).

[0054] 13 C NMR (125 MHz, CDC13) δ 167.74, 156.41, 154.95, 149.85, 140.00, 138.87, 127.49, 127.25, 123.39, 122.76, 117.69, 115.04, 83.47, 79.75, 55.18. Synthesis of AIE mimetic antimicrobial peptide OMPA-TT

[0055] The structural formula of AIE mimetic antimicrobial peptide OMPA-TT and OMHA-TT are shown below, the R group of OMPA-TT and OMHA-TT is different, X of OMPA-TT is (CH2) 1.5 , and X of OMHA-TT is (CH2)3;

[0056]

[0057] The specific synthesis steps are as follows:

[0058]

[0059] Put bis(2-bromoethyl)amine hydrobromide (0.42 g, 1.37 mmol), triethylamine (0.14 g, 1.37 mmol) and DCM (2 mL) into a 10 mL double-necked round-bottom flask, stir at 35°C for 0.5 hours, then add the first intermediate AIE activated alkyne (5-(dimethoxytriphenylamine)thiophene-2-carbonyl alkyne) (0.40 g, 0.91 mmol) to the flask and continue to stir for 2 hours. After rotary evaporation, add silica gel powder to the sample, and purify the crude product by column chromatography using DCM:methanol (40:1, v / v) as the eluent to obtain a red solid, which is the second intermediate shown in the formula above, with a yield of 51% (0.31 g).

[0060] The nuclear magnetic hydrogen / carbon spectrum data of the second intermediate are as follows:

[0061] 1H NMR (500 MHz, C2D6OS) δ 8.32 (d, J = 4.2 Hz, 1H), 7.70-7.60 (m, 4H), 7.14-7.09 (m, 5H), 6.98-6.93 (m, 4H), 6.78-6.73 (m, 2H), 3.76 (s, 4H), 3.66 (q, J = 7.1 Hz, 6H), 3.12 (d, J = 4.2 Hz, 2H).

[0062] 13 C NMR (125 MHz, C2D6OS) δ 177.80, 155.90, 154.76, 149.31, 143.42, 139.86, 138.39, 137.18, 126.96, 126.75, 124.70, 122.82, 117.24, 114.55, 54.70, 53.62.

[0063]

[0064] In a 25 mL polymerization tube, the second intermediate (40.00 mg, 0.60 mmol) was added, then the nitrogen was pumped in and out for three times, DMF (0.5 mL) was added into the polymerization tube, then tetramethylethylenediamine (6.95 mg, 0.60 mmol) dissolved in 0.5 mL DMF was added dropwise slowly under the stirring environment of room temperature and nitrogen atmosphere, it took 0.5 hours to drop, then the reaction was continued to be heated to 80 °C and stirred for 24 hours. After the reaction was completed, the reaction solution was gradually dropped into 100 mL DCM: n-hexane (1:9, v / v) under rapid stirring, after standing, filtration, washing with DCM: n-hexane (1:9, v / v), and natural air drying, the orange red gel-like AIE mimetic antimicrobial peptide OMPA-TT was obtained, the yield was 93% (0.04 g). The M w was 6870, was 1.56, and the molecular weight test result showed that the repeat unit 15 > n > 5, n was an integer.

[0065] The infrared spectrum and nuclear magnetic hydrogen spectrum data of the AIE mimetic antimicrobial peptide OMPA-TT were as follows:

[0066] FT-IR (KBr disk), v (cm -1 ): 3008, 2940, 1644, 1624, 1462, 1321, 1280, 1230, 954.

[0067] 1H NMR (500 MHz, C2D6OS) δ 9.59 (s, 1H), 8.94 (s, 2H), 7.71 (s, 1H), 7.62 (d, J = 12.5 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.9 Hz, 4H), 6.94 (d, J = 9.0 Hz, 4H), 6.76 (d, J = 8.8 Hz, 2H), 5.57 (s, 1H), 4.10 (s, 18H), 2.79 (s, 8H), 2.00 (d, J = 16.2, 4H), 0.85 (t, J = 6.9 Hz, 2H).

[0068] The reaction raw material was changed from tetramethyl ethylenediamine to tetramethyl hexanediamine, and other raw materials and operation steps were consistent with the synthesis method of AIE antimicrobial peptide OMPA-TT. Orange-red gel AIE antimicrobial peptide OMHA-TT was obtained, and the yield was 71% (0.03 g). The molecular weight of AIE antimicrobial peptide OMHA-TT was 8330, w 1.55, and the molecular weight test result showed that the repeating unit 15 > n > 5, n was an integer.

[0069] The infrared spectrum and nuclear magnetic hydrogen spectrum data of AIE antimicrobial peptide OMHA-TT were as follows:

[0070] FT-IR (KBr disk), v (cm -1 ): 3017, 2943, 1626, 1600, 1465, 1323, 1238, 1227, 953.

[0071] 1 H NMR (500 MHz, C2D6OS) δ 9.42 (s, 4H), 7.72-7.50 (m, 2H), 7.10-6.88 (m, 4H), 6.76 (d, J = 7.0 Hz, 1H), 4.04 (s, 8H), 3.77 (s, 4H), 3.10 (d, J = 8.5 Hz, 2H), 3.08-2.94 (m, 12H), 2.63 (s, 1H), 2.07-1.93 (m, 2H), 1.71-1.50 (m, 12H), 0.85 (t, J = 6.9 Hz, 2H).

[0072] Example 2 Synthesis of AIE antimicrobial peptide

[0073] The structural formula of AIE antimicrobial peptide OSMA-TT was as follows:

[0074]

[0075] The synthesis steps of AIE antimicrobial peptide OSMA-TT were as follows:​

[0076]

[0077] Into a 10 mL two-necked round bottom flask, 3-bromoprop-1-amine hydrobromide (0.30 g, 1.37 mmol), triethylamine (0.14 g, 1.37 mmol) and DCM (2 mL) were placed and stirred at 35 °C for 0.5 h. Then the first intermediate AIE-activated alkyne (5-(dimethoxytriphenylamino)thiophene-2-carbonyl alkyne) (0.40 g, 0.91 mmol) was added and the reaction was stirred for 2 h. After concentration by rotary evaporation, silica gel powder was added to the sample and the crude product was purified by column chromatography using DCM:methanol (40:1, v / v) as eluent to give the third intermediate as a red solid in 79% yield (0.40 g).

[0078] The NMR data of the third intermediate are as follows:

[0079] 1 H NMR (500 MHz, C2D6OS) δ 8.32 (s, 1H), 7.69 (d, J = 13.5 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 4.1 Hz, 1H), 7.12 (d, J = 9.2 Hz, 5H), 6.96 (d, J = 8.9 Hz, 4H), 6.75 (d, J = 8.9 Hz, 2H), 3.76 (s, 4H), 3.67 (d, J = 7.3 Hz, 6H).

[0080] 13 C NMR (125 MHz, C2D6OS) δ 177.80, 155.90, 154.76, 149.31, 143.42, 139.87, 137.19, 126.96, 126.75, 124.70, 122.81, 122.47, 117.40, 114.55, 54.69, 53.62, 44.98, 25.73.

[0081]

[0082] AIBN (12.10 mg, 0.12 nmol) purified by recrystallization was added to a 25 mL polymerization tube, which was sealed and then evacuated and refilled with nitrogen three times. Ethanol (1 mL) and N-[2-(dimethylamino)ethyl]acrylamide (0.28 g, 2.00 mmol) were added, and the reaction was stirred at 70 °C for 24 h under a nitrogen atmosphere. After the reaction was completed, 1 mL of ethanol was added to the polymerization tube to dilute the solution, and the solution was gradually dropped into 100 mL of rapidly stirred n-hexane. After standing, the product was filtered, washed with n-hexane, and air-dried. A yellowish transparent gel, the fourth intermediate, was obtained in 92% yield (0.13 g).

[0083] The nuclear magnetic hydrogen / carbon spectrum data of the fourth intermediate are as follows:

[0084] 1 H NMR (500 MHz, C2D6OS) δ 8.14 (s, 1H), 3.44 (q, J = 7.0 Hz, 2H), 3.38 (s, 2H), 2.15 (s, 10H).

[0085] 13 C NMR (125 MHz, C2D6OS) δ 173.12, 56.71, 55.41, 44.69, 44.21, 35.94, 34.80, 17.95.

[0086]

[0087] The third intermediate (0.23 g, 0.56 mmol) and the fourth intermediate (30.00 mg, 0.28 mmol) were added together to a two-necked flask equipped with a condenser, and THF (3 mL) was added. The reaction was stirred at 70 °C for 72 h. After the reaction was completed, the solution in the flask was concentrated, and the solution was gradually dropped into 100 mL of rapidly stirred DCM:n-hexane (1:9, v / v). After standing, the product was filtered, washed with DCM:n-hexane (1:9, v / v) until no 5-(dimethoxytriphenylammoniumyl)thiophene-2-carbonyl alkyne was present in the system, and air-dried. An orange-yellow gel, AIE mimicking antimicrobial peptide OSMA-TT, was obtained in 42% yield (0.05 g). The M w was 7930, was 1.46, and the molecular weight test results indicated that the repeating unit 15 > n > 5, where n is an integer.

[0088] The infrared spectrum and the nuclear magnetic hydrogen spectrum data of AIE mimicking antimicrobial peptide OSMA-TT are as follows:

[0089] FT-IR (KBr disk), v (cm -1): 2967, 2917, 1624, 1603, 1539, 1506, 1324, 1280, 1235, 1030.

[0090] 1 H NMR (400 MHz, C2D6OS) δ 7.71 (d, J = 4.0 Hz, 2H), 7.52 (d, J = 8.7 Hz, 4H), 7.33 (d, J = 3.9 Hz, 2H), 6.75 (d, J = 8.7 Hz, 4H), 5.82 (d, J = 12.5 Hz, 2H), 3.75 (s, 12H), 1.99 (d, J = 13.2 Hz, 4H).

[0091] Synthesis of AIE mimetic antibacterial peptide

[0092] The structural formula of AIE mimetic antibacterial peptide OSEA-TT is as follows:

[0093]

[0094] The synthesis steps of AIE mimetic antibacterial peptide OSEA-TT are as follows:

[0095]

[0096] In a 25 mL polymerization tube, recrystallized azobisisobutyronitrile AIBN (12.10 mg, 0.12 nmol) was added and sealed, then evacuated and filled with nitrogen three times, and then 1 mL of ethanol, N-ethyl methacrylamide (0.10 g, 1.00 mmol) and N-[2-(dimethylamino)ethyl] acrylamide bromoethane salt (0.25 g, 1.00 mmol) were added, and then heated to 70°C under nitrogen atmosphere and stirred for 24 hours. After the reaction was completed, 1 mL of ethanol was added to dilute the polymerization tube, and the solution was gradually dropped into 100 mL of rapidly stirred n-hexane, and after standing, it was filtered, washed with n-hexane, and naturally air-dried. A milky white transparent gel was obtained, which was the fifth intermediate shown in the formula above, with a yield of 72% (0.14 g).

[0097] The nuclear magnetic resonance hydrogen / carbon spectrum data of the fifth intermediate are as follows:

[0098] 1H NMR (500 MHz, C2D6OS) δ 8.22 (t, J = 5.8 Hz, 1H), 4.84 (d, J = 29.5 Hz, 2H), 3.44 (q, J = 6.7 Hz, 7H), 3.27 (t, J = 6.9 Hz, 3H), 3.10 (s, 9H), 2.60 (t, J = 7.3 Hz, 2H), 2.40 (q, J = 7.2 Hz, 2H), 2.24 (t, J = 7.4 Hz, 2H), 1.25 (t, J = 6.3 Hz, 5H), 0.94 (t, J = 7.2 Hz, 3H).

[0099] 13 C NMR (125 MHz, C2D6OS) δ 171.41, 59.87, 59.35, 58.22, 55.40, 49.40, 48.08, 45.77, 32.44, 27.96, 25.94, 19.97, 17.95, 10.81, 7.49, 7.16.

[0100]

[0101] The fifth intermediate (0.30 g, 1.37 mmol), the first intermediate AIE activated alkyne (5-(dimethoxytriphenylamine)thiophene-2-carbonyl alkyne) (0.14 g, 1.37 mmol), ethanol (0.5 mL) and DCM (2 mL) were put into a 10 mL double-necked round-bottom flask, stirred at 35 °C for 4 hours, and then 5-(dimethoxytriphenylamine)thiophene-2-carbonyl alkyne (0.40 g, 0.91 mmol) was added to continue the stirring reaction for 24 hours. After the reaction was completed, the solution in the flask was concentrated, and the solution was gradually dropped into 100 mL of rapidly stirred DCM: n-hexane (1:9, v / v), and after standing, it was filtered, washed with DCM: n-hexane (1:9, v / v) until no 5-(dimethoxytriphenylamine)thiophene-2-carbonyl alkyne was present in the system, and then naturally air-dried. An orange-red gel-like AIE antimicrobial peptide OSEA-TT was obtained with a yield of 98% (0.06 g). The M w was 8580, was 1.42, and the molecular weight test results showed that the repeating unit 15 > n > 5, n being an integer.

[0102] The infrared spectrum and nuclear magnetic hydrogen spectrum data of the AIE antimicrobial peptide OSEA-TT were as follows:

[0103] FT-IR (KBr disk), v (cm -1 ): 2955, 2920, 1665, 1624, 1542, 1503, 1321, 1280, 1233, 1030.

[0104] 1 H NMR (500 MHz, C2D6OS) δ 8.19 (s, 1H), 7.52 (d, J = 7.0 Hz, 2H), 7.32 (d, J = 32.3 Hz, 1H), 7.06 (d, J = 3.0 Hz, 3H), 6.95 (d, J = 7.9 Hz, 3H), 6.76 (d, J = 8.8 Hz, 1H), 3.76 (s, 6H), 3.21 - 2.96 (m, 19H), 1.99 (d, J = 16.1 Hz, 2H), 1.27 (d, J = 40.7 Hz, 19H).

[0105] Sample analysis

[0106] The methicillin-resistant Staphylococcus aureus MRSA used in the experiment was purchased.

[0107] 1. The test method for the inhibition rate of planktonic bacteria is:

[0108] (1) Broth dilution method for antibacterial analysis:

[0109] Pour 10 mL of fresh LB medium into a 50 mL centrifuge tube, and inject 10 μL of bacterial solution into the corresponding centrifuge tube. Place it in a shaker and incubate overnight (37°C, 1700 r / min). Wash the bacterial solution with PBS for 3 times, and prepare mixed solutions of different concentrations with the sample to be tested. Dilute the bacterial solution to 5.0 x 10 5 individuals, shake for 10 seconds, and place the 96-well plate in a microplate reader to test the original absorbance at 600 nm. Then, place the 96-well plate in a shaker and incubate for 16 hours (37°C, 1700 r / min). If light is required, place the plate under a white light or laser after incubation for 1 hour, and then continue to incubate in the shaker for 15 hours (37°C, 1700 r / min). After incubation, place the plate in a microplate reader, shake for 10 seconds to disperse the bacteria, and test the absorbance at 600 nm to calculate the inhibition rate.

[0110] (2) Plate dilution method for antibacterial experiment:

[0111] Take the MRSA bacterial solution and wash it with PBS three times. Test the OD 600 = 0.5 using a microplate reader. Mix 80 μL of the sample with 20 μL of the diluted bacterial solution, and incubate at 37°C with shaking at 1700 rpm for 30 minutes. For the white light irradiation group, place the bacterial suspension under white light. For the control group, place it at room temperature for the corresponding time. Dilute the bacterial suspension with PBS to 1 x 10 4The diluted bacterial solution (100 μL) was dropped into an agar plate, and the bacteria were evenly dispersed using a spreader. The agar plate with the bacteria was incubated in a 37°C incubator until obvious colonies were formed, and the colonies of the bacteria were counted.

[0112] 2. The method for testing the biofilm inhibition rate is as follows:

[0113] In a 50 mL centrifuge tube, 10 mL of fresh LB medium was poured, 10 μL of bacterial solution was taken and injected into the corresponding centrifuge tube, and the centrifuge tube was placed on a shaker for overnight culture (37°C, 1700 r / min). The bacterial solution was washed with PBS for 3 times, and mixed with the sample to be tested to prepare a mixed solution with different concentrations. The bacterial solution was diluted to 1.0×10 6 / mL, and then 100 μL of the mixed solution was taken and added to a 96-well plate, and the number of bacteria in each well was 50,000. The plate was placed in an incubator for culture. After the biofilm was formed, the culture solution was replaced with 100 μL of the sample with different concentrations prepared with PBS, and the plate was incubated in a 37°C incubator for 1 hour. The biofilm solution was irradiated with white light in the white light irradiation group, and the control group was placed at room temperature for the corresponding time. The biofilm solution was mixed and diluted 1×10 4 times with PBS. The diluted bacterial solution (100 μL) was dropped into an agar plate, and the bacteria were evenly dispersed using a spreader. The agar plate with the bacteria was incubated in a 37°C incubator until obvious colonies were formed, and the colonies of the bacteria were counted.

[0114] 3. Live and dead bacteria imaging:

[0115] (1) Planktonic bacteria imaging:

[0116] The bacterial solution was washed with PBS for 3 times, and was placed in an EP tube. The bacterial solution was mixed with the sample to be tested to prepare a mixed solution, and the bacterial solution was diluted to 1.0×10 9 / mL. 2 μg mL -1 of DMAO and 6 μg mL -1 of PI were added, and the EP tube was placed on a shaker for culture (37°C, 1700 r / min) for a certain time. The EP tube was washed with PBS again. 3-5 μL of the solution in the EP tube was taken and pressed on a carrier wave sheet to prepare a glass sheet, which was observed under a confocal microscope and photographed.

[0117] (2) Biofilm imaging:

[0118] The biofilm in the confocal dish was washed with PBS for 3 times. The sample solution to be tested was added to the biofilm, and 2 μg mL -1 of DMAO and 6 μg mL -1PI, incubate in incubator (37℃) for a certain time, then wash with PBS. Put the confocal dish under the confocal microscope to observe the imaging and take pictures. Use different excitation and emission combinations for each dye to image the bacteria: DMAO (live cells show green), excitation wavelength is 488 nm, fluorescence emission range is 490-520 nm, showing green fluorescence; PI (dead cells show red), excitation wavelength is 543 nm, fluorescence emission range is 600-650 nm, showing red fluorescence.

[0119] 4. Scanning electron microscope sample preparation:

[0120] AIE antimicrobial peptide analogue and MRSA biofilm were co-cultured in a 37℃ incubator for 1 hour. Then, the light group was irradiated with a white light lamp, and the non-light group was incubated at room temperature for the corresponding time as a reference. Then wash with PBS three times, then fix with 2.5% glutaraldehyde overnight. Wash with PBS, then dehydrate with 10, 20, 40, 60, 70, 80, 90, 95 and 100% ethanol solution respectively, 10 minutes for each concentration. After mixing each sample, 2-4 μL was dropped on a silicon wafer, naturally air-dried, then pasted on conductive glue, placed on the sample stage, and sprayed with gold twice. SEM was used for observation.

[0121] 5. Cell survival rate determination:

[0122] L929 cells were dispersed in complete culture medium and inoculated in a 96-well plate at 8×10 3 cells / well in a 5% CO2 incubator at 37℃ overnight until the cells adhered. Then, fresh culture medium containing different concentrations of AIE antimicrobial peptide analogue was used to replace the old culture medium, and the light group was irradiated with a white light lamp after 1 hour. The non-light group was used as a reference. Continue to culture for 24 hours, then remove the original culture medium, leave a row of cells with only DMEM medium as a background, and add CCK8 DMEM culture medium to the other wells for 2.5 hours. The absorbance of each well at 450 nm was measured by a microplate reader, and then the relative activity of the cells was calculated.

[0123] 6. The method for testing the effect of antibacterial drugs in animal experiments is:

[0124] By subcutaneously injecting bacterial solution MRSA (125 μL, 1×10 8 / animal) into the leg, an abscess was formed after 1 day, and OSEA-TT (100 μL, 800 μM) was injected into the abscess. After 1 hour, the wound site was irradiated with a white light lamp (100 mW / cm 2 , 30 min). The PBS and OSEA-TT groups required light irradiation, while one group did not require light irradiation.

[0125] Experimental results

[0126] The molecular structures of different AIE antimicrobial peptide mimics synthesized in the examples are shown in (A) of FIG. 1, and the absorption and emission spectra, fluorescence quantum yield, and Zeta potential are shown in (B)-(D) of FIG. 1, respectively. Figure 2 Figure 2 The ROS production efficiency of AIE antimicrobial peptide mimics and commercial photosensitizer Rose Bengal (RB) was detected using the fluorescent probe activated 2',7'-dichlorofluorescein diacetate (DCFH-DA), and the results are shown in (E) of FIG. 2. Figure 2

[0127] The inhibition rate of different AIE antimicrobial peptide mimics on MRSA biofilm under dark and light conditions is shown in (A)-(D) of FIG. 3, and the results show that AIE antimicrobial peptide mimics can significantly inhibit the growth and proliferation of methicillin-resistant Staphylococcus aureus (MRSA) at a lower concentration under light conditions, and the effect of AIE antimicrobial peptide mimic OSEA-TT is the best. Figure 3

[0128] The inhibition of AIE antimicrobial peptide mimic OSEA-TT on MRSA biofilm under dark and light conditions is shown in (A) and (B) of FIG. 4, and the results show that AIE antimicrobial peptide mimics can significantly inhibit MRSA biofilm at a lower concentration under light conditions. Figure 4

[0129] The treatment of AIE antimicrobial peptide mimic OSEA-TT on mouse subcutaneous abscess under dark and light conditions is shown in (A)-(D) of FIG. 5, and it is shown that OSEA-TT assisted by white light treatment of mouse subcutaneous abscess has good effect. Figure 5

[0130] The above examples have been described in detail, and it should be understood that the above examples are only specific embodiments of the present application and are not intended to limit the present application. Any modification, supplement or similar replacement within the principle range of the present application should be included in the protection scope of the present application.​​​​​

Claims

1. An AIE mimicking antibacterial peptide material, characterized in that, The structural formula is shown in any one of Formula I, Formula II or Formula III: In Formula I, 6≥m≥1; In Formula I, Formula II and Formula III, n is an integer of 5-20; The electron-donating group and the electron-withdrawing group in R are each independently selected from one of the following structures, wherein * represents the connecting position; 2. The method for preparing an AIE mimicking antibacterial peptide material according to claim 1, characterized in that, The method comprises the following steps: (1) Under inert gas protection, Suzuki reaction is carried out by using aryl boronic acid and aldehyde group-substituted bromo heteroarene, and then the obtained nitrobenzaldehyde derivative is reacted with format reagent acetylene magnesium bromide under inert gas protection, and after the obtained product is treated by manganese dioxide, the first intermediate AIE activated alkyne is obtained; (2) The first intermediate AIE activated alkyne is reacted with bis(2-bromoethyl)amine hydrobromide to obtain a second intermediate, and then polymerization reaction is carried out under inert gas protection by using the second intermediate and a bis-tertiary amine compound to obtain the AIE antimicrobial peptide mimetic shown in Formula I; (3) The first intermediate AIE activated alkyne is reacted with 3-bromoprop-1-amine hydrobromide to obtain a third intermediate, radical polymerization is carried out by using the first polymerization monomer, and then the obtained polymerization product is reacted with the third intermediate to obtain the AIE antimicrobial peptide mimetic shown in Formula II; (4) Radical polymerization is carried out by using the second polymerization monomer to obtain a polymerization product, and then the polymerization product is reacted with the first intermediate AIE activated alkyne to obtain the AIE antimicrobial peptide mimetic shown in Formula III.

3. The method for preparing an AIE mimicking antibacterial peptide material according to claim 2, characterized in that, In step (1), the condition of Suzuki reaction is 70-80℃, 16-48h; and the condition of reaction of the nitrobenzaldehyde derivative and the format reagent acetylene magnesium bromide is 0-40℃, 2-12h.

4. The method for preparing the AIE mimicking antibacterial peptide material according to claim 2, characterized in that, In step (2), the reaction condition of the first intermediate AIE activated alkyne and bis(2-bromoethyl)amine hydrobromide is 25-60℃, 2-48h; the bis-tertiary amine compound includes tetramethylethylenediamine or tetramethylhexanediamine; and the polymerization reaction condition of the second intermediate and the bis-tertiary amine compound is 70-120℃, 16-72h.

5. The method for preparing the AIE mimicking antibacterial peptide material according to claim 2, characterized in that, The first polymerization monomer is N-[2-(dimethylamino)ethyl]acrylamide, the second polymerization monomer is N-ethylmethacrylamide and N-[2-(dimethylamino)ethyl]acrylamide bromoethane salt, and the condition of radical polymerization of the first polymerization monomer or the second polymerization monomer is 40-120℃, 24-72h.

6. The method for preparing an AIE mimicking antibacterial peptide material according to claim 2, characterized in that, In step (3), the reaction condition of the first intermediate AIE activated alkyne and 3-bromoprop-1-amine hydrobromide is 25-60℃, 2-48h; and the reaction condition of the polymerization product and the third intermediate is 70-120℃, 16-72h.

7. The method for preparing an AIE mimicking antibacterial peptide material according to claim 2, characterized in that, In step (4), the reaction condition of the polymerization product and the first intermediate AIE activated alkyne is 25-60℃, 4-48h.

8. An antibacterial agent, characterized by, The component comprises the AIE antimicrobial peptide mimetic material of claim 1.

9. The antimicrobial agent of claim 8, wherein, The object of the antibacterial agent includes S.aureus, MRSA, E.coli, CREc and C.albicans.

10. The antimicrobial agent of claim 8, wherein, The antibacterial agent is used alone or in combination with light treatment.

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