A conformation-locked melittin Anoplin derivative and its preparation and use

Abstract

The present invention discloses a conformation-locked bee venom peptide Anoplin derivative and its preparation and use, belonging to the technical field of pharmaceutical chemistry. It is represented by general formula (I) or (II), wherein n is an integer of 0 to 5, m is an integer of 0 to 5, n+m=5 or 2, and each AA is derived from consecutive amino acids in bee venom peptide Anoplin. The preparation method of the Anoplin derivative is simple, and its antibacterial activity is better than that of the uncyclized cysteine-substituted Anoplin antibacterial peptide analogs, and better than the parent peptide Anoplin. Among them, Ano‑13, Ano‑14, Ano‑17 and Ano‑18 have the best antibacterial activity against Staphylococcus aureus and Escherichia coli, which greatly improves the antibacterial activity of the parent peptide Anoplin.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemistry, and in particular to a conformation-locked melittin Anoplin derivative and a preparation method and application thereof. Background Art

[0002] The discovery and use of antibiotics is one of the greatest medical advances of the 20th century. However, due to the extensive use of antibiotics, bacterial resistance has become an increasingly serious global public health crisis. It is estimated that by 2050, drug-resistant infections may claim 10 million lives each year and impose a cumulative burden of $100 trillion on the global economy. The development of new antibacterial drugs for multidrug-resistant bacteria, including drug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, Enterococci, Staphylococcus aureus, etc., will become one of the important directions of innovative drug research and development in my country in the future.

[0003] Antimicrobial peptides (AMPs) are a class of small molecule active proteins widely found in organisms to resist exogenous pathogens. They have the advantages of broad-spectrum antimicrobial activity, thermal stability and wide sources. Different from the bactericidal mechanism of traditional antibiotics, antimicrobial peptides act on the cell membrane to form transmembrane ion channels on the membrane, thereby inducing the leakage of cell contents to produce antimicrobial effects. This bactericidal mechanism is not easy to produce drug resistance, and has a high efficiency and broad-spectrum killing effect on both Gram-negative and Gram-positive bacteria. It has great potential in the development of new antimicrobial drugs for multi-drug resistant bacteria. Anoplin is an amphipathic α-helical antimicrobial peptide isolated from wasp venom. It consists of 10 amino acids with the sequence of GLLKRIKTLL-NH2. It has broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, and also has multiple activities such as antifungal, antitumor and anti-malarial parasites. Due to its simple structure and low hemolytic activity, Anoplin has great potential as a new type of antibiotic and has high clinical application value. However, studies have found that Anoplin still has defects such as insufficient antibacterial activity and inability to resist protease hydrolysis, which limits its clinical application. Summary of the invention

[0004] The purpose of the present invention is to provide a conformation-locked melittin Anoplin derivative and its preparation and use, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a class of Anoplin derivatives containing perfluoroaromatic conformational locking modification, represented by general formula (I) or (II),

[0007] , ;

[0008] Wherein, n is an integer of 0 to 5, m is an integer of 0 to 5, n+m = 5 or 2, and each AA is derived from consecutive amino acids in Anoplin.

[0009] The second technical solution of the present invention is a method for preparing the Anoplin derivative of melittin, comprising the following steps: the intermediate peptide is subjected to a nucleophilic substitution reaction under the action of a base by chemical synthesis with perfluorobenzene or decafluorobiphenyl as a bridging group to achieve locking of the α-helical structure, thereby obtaining the Anoplin derivative of melittin.

[0010] The third technical solution of the present invention is the use of the Anoplin derivative in the preparation of drugs for preventing and treating bacterial infections.

[0011] A fourth technical solution of the present invention is the use of the Anoplin derivative in the preparation of an antibacterial agent.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] The invention provides a class of perfluoroaromatic conformationally locked modified bee venom peptide Anoplin derivatives. The preparation method of the Anoplin derivatives is simple, and the antibacterial activity thereof is superior to that of uncyclized cysteine-substituted Anoplin antibacterial peptide analogs and the parent peptide Anoplin. Among them, Ano-13, Ano-14, Ano-17 and Ano-18 have the best antibacterial activity against Staphylococcus aureus and Escherichia coli, and greatly improve the antibacterial activity of the parent peptide Anoplin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 is the mass spectrum of Ano-10;

[0016] Figure 2 is the mass spectrum of Ano-11;

[0017] Figure 3 is the mass spectrum of Ano-12;

[0018] Figure 4 is the mass spectrum of Ano-13;

[0019] Figure 5 is the mass spectrum of Ano-14;

[0020] Figure 6 is the mass spectrum of Ano-15;

[0021] Figure 7 is the mass spectrum of Ano-16;

[0022] Figure 8 is the mass spectrum of Ano-17;

[0023] Fig. 9 This is the mass spectrum of Ano-18. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0030] The chemical structure of Anoplin is as follows:

[0031] .

[0032] The modified intermediate peptide Ano1 of the present invention is obtained by replacing the first Gly glycine and the fifth Arg arginine sites of the natural bee venom peptide Anoplin with cysteine, thereby obtaining a bee venom peptide Anoplin derivative containing cysteine, which is named Ano-1 and has a structural formula of Cys-Leu-Leu-Lys-Cys-Ile-Lys-Thr-Leu-Leu-NH2.

[0033] The modified intermediate peptide Ano2 of the present invention is obtained by replacing the 2nd Leu leucine and the 6th Ile isoleucine sites of the natural bee venom peptide Anoplin with cysteine, thereby obtaining a bee venom peptide Anoplin derivative containing cysteine, which is named Ano-2 and has a structural formula of Gly-Cys-Leu-Lys-Arg-Cys-Lys-Thr-Leu-Leu-NH2.

[0034] The modified intermediate peptide Ano3 of the present invention is obtained by replacing the 3rd Leu leucine and the 7th Lysine sites of the natural bee venom peptide Anoplin with cysteine, thereby obtaining a bee venom peptide Anoplin derivative containing cysteine, which is named Ano-3 and has a structural formula of Gly-Leu-Cys-Lys-Arg-Ile-Cys-Thr-Leu-Leu-NH2.

[0035] The modified intermediate peptide Ano4 of the present invention is obtained by replacing the 4th Lys and 8th Thr sites of the natural bee venom peptide Anoplin with cysteine, thereby obtaining a bee venom peptide Anoplin derivative containing cysteine, which is named Ano-4 and has a structural formula of Gly-Leu-Leu-Cys-Arg-Ile-Lys-Cys-Leu-Leu-NH2.

[0036] The modified intermediate peptide Ano5 of the present invention is obtained by replacing the 5th Arg arginine and the 9th Leu leucine of the natural bee venom peptide Anoplin with cysteine, thereby obtaining a bee venom peptide Anoplin derivative containing cysteine, which is named Ano-5 and has a structural formula of Gly-Leu-Leu-Lys-Cys-Ile-Lys-Thr-Cys-Leu-NH2.

[0037] The modified intermediate peptide Ano6 of the present invention is obtained by replacing the 6th Ile isoleucine and the 10th Leu leucine of the natural bee venom peptide Anoplin with cysteine, thereby obtaining a bee venom peptide Anoplin derivative containing cysteine, which is named Ano-6 and has a structural formula of Gly-Leu-Leu-Lys-Arg-Cys-Lys-Thr-Leu-Cys-NH2.

[0038] The modified intermediate peptide Ano7 of the present invention is obtained by replacing the first Gly glycine and the eighth Thr threonine sites of the natural bee venom peptide Anoplin with cysteine, thereby obtaining a bee venom peptide Anoplin derivative containing cysteine, which is named Ano-7 and has a structural formula of Cys-Leu-Leu-Lys-Arg-Ile-Lys-Cys-Leu-Leu-NH2.

[0039] The modified intermediate peptide Ano8 of the present invention is obtained by replacing the 2nd Leu leucine and the 9th Leu leucine sites of the natural bee venom peptide Anoplin with cysteine, thereby obtaining a bee venom peptide Anoplin derivative containing cysteine, which is named Ano-8 and has a structural formula of Gly-Cys-Leu-Lys-Arg-Ile-Lys-Thr-Cys-Leu-NH2.

[0040] The modified intermediate peptide Ano9 of the present invention is obtained by replacing the 3rd Leu leucine and the 10th Leu leucine sites of the natural bee venom peptide Anoplin with cysteine, thereby obtaining a bee venom peptide Anoplin derivative containing cysteine, which is named Ano-9 and has a structural formula of Gly-Leu-Cys-Lys-Arg-Ile-Lys-Thr-Leu-Cys-NH2.

[0041] Among them, Ano-1~Ano-9 contained in the present invention were all purchased from Anhui Peptide Biotechnology Co., Ltd.

[0042] The present invention provides a class of Anoplin derivatives containing perfluoroaromatic conformational locking modifications, represented by the general formula (I) or (II),

[0043] , ;

[0044] Wherein, n is an integer of 0 to 5, m is an integer of 0 to 5, n+m = 5 or 2, and each AA is derived from consecutive amino acids in Anoplin.

[0045] In some specific embodiments, the Anoplin derivative comprises:

[0046] , , , , , , , ,and .

[0047] The embodiment of the present invention also provides a method for preparing the Anoplin derivative of melittin, comprising the following steps: the intermediate peptide is subjected to a nucleophilic substitution reaction under the action of a base by chemical synthesis with hexafluorobenzene or decafluorobiphenyl as a bridging group to achieve locking of the α-helical structure, thereby obtaining the Anoplin derivative of melittin.

[0048] In some specific embodiments, the steps include:

[0049] (1) Add hexafluorobenzene or decafluorobiphenyl and tris(hydroxymethylaminomethane) to the intermediate peptide, mix vigorously at 1500 rpm for 30 seconds, react at room temperature, and freeze-dry after the reaction to obtain a crude product;

[0050] (2) Purifying the crude product to obtain the Anoplin derivative.

[0051] In some specific embodiments, the molar ratio of the intermediate peptide, hexafluorobenzene or decafluorobiphenyl to tris(hydroxymethyl)aminomethane is 1:(2-30):10.

[0052] In some specific embodiments, the concentration of the hexafluorobenzene or decafluorobiphenyl DMF solution is 1:50 mM to 1:200 mM.

[0053] In some specific embodiments, in the DMF solution of tris(hydroxymethylaminomethane), the molar ratio of tris(hydroxymethylaminomethane) to DMF is 1:25 mM to 1:100 mM.

[0054] In some specific embodiments, the room temperature reaction conditions are: standing at room temperature and rotating speed of 1000 rpm for 4.5 to 5 hours.

[0055] In some specific embodiments, the purification is RP-HPLC purification, mobile phase A is 0.1% TFA / water, mobile phase B is 0.1% TFA / acetonitrile, and linear gradient elution is performed.

[0056] The intermediate peptide is selected from any one of Ano-1 to Ano-9, and the structural formulas of Ano-1 to Ano-9 are as follows:

[0057] .

[0058] The embodiment of the present invention also provides the use of the Anoplin derivative in preparing a drug for preventing and treating bacterial infection.

[0059] In some specific embodiments, the bacteria include Staphylococcus aureus and / or Escherichia coli.

[0060] The embodiment of the present invention also provides the use of the Anoplin derivative in the preparation of an antibacterial agent.

[0061] Example 1

[0062] In the preparation of the conformation-locked antimicrobial peptide derivative Ano10-18 of the present invention, all reagents used in the examples are commercially available analytically pure, and all solvents in the preparative liquid phase are chromatographically pure.

[0063] Table 1 Amino acid sequences of natural antimicrobial peptide Anoplin derivatives Ano-1 to Ano-18 of the present invention

[0064]

[0065] Among them, Ano-1~Ano-9 were purchased from Anhui Zhuanpeptide Biotechnology Co., Ltd.

[0066] The following is a specific description of the preparation of perfluoroarene conformationally locked Anoplin derivatives Ano-10~Ano-18 as an example:

[0067] Preparation of Ano-10

[0068] Accurately weigh the calculated amount of Ano-1 (7.5 µmol, 8.59 mg) and place it in a 5 mL reaction bottle, add 2 mL of 100 mM hexafluorobenzene DMF solution (25 equiv., 187.5 µmol) and 1.5 mL of 50 mM tris(hydroxymethyl)aminomethane DMF solution (10 equiv., 75 µmol). Mix vigorously at 1500 rpm for 30 seconds in a constant temperature mixer and place at 1000 rpm for 4.5 hours at room temperature. After the reaction is completed, the reaction solution is transferred to a glass culture dish and placed in a -80 °C refrigerator, and then placed in a freeze dryer. The freeze-drying process lasts about 12-16 hours, and the peptide sample is completely freeze-dried to obtain a dry crude peptide. Subsequently, it was purified by RP-HPLC (using mobile phase A as 0.1% TFA / water, mobile phase B as 0.1% TFA / acetonitrile, linear gradient elution), and the target peak effluent was collected, lyophilized, and identified by mass spectrometry to obtain Ano-10 (6.9 mg), see Figure 1 .

[0069] The synthetic route is as follows:

[0070] .

[0071] Preparation of Ano-11

[0072] Accurately weigh the calculated amount of Ano-2 (7.5 µmol, 8.49 mg) and place it in a 5 mL reaction bottle, add 2 mL 100 mM hexafluorobenzene DMF solution (25 equiv., 187.5 µmol) and 1.5 mL 50 mM tris(hydroxymethyl)aminomethane DMF solution (10 equiv., 75 µmol). Mix vigorously at 1500 rpm for 30 seconds in a constant temperature mixer and place at 1000 rpm for 4.5 hours at room temperature. After the reaction is completed, the reaction solution is transferred to a glass culture dish and placed in a -80 ° C refrigerator, and then placed in a freeze dryer. The freeze-drying process lasts about 12-16 hours, and the peptide sample is completely freeze-dried to obtain a dry crude peptide. Subsequently, it was purified by RP-HPLC (using mobile phase A as 0.1% TFA / water, mobile phase B as 0.1% TFA / acetonitrile, linear gradient elution), and the target peak effluent was collected, lyophilized, and identified by mass spectrometry to obtain Ano-11 (5.7 mg), see Figure 2 .

[0073] The synthetic route is as follows:

[0074] .

[0075] Preparation of Ano-12

[0076] Accurately weigh the calculated amount of Ano-3 (7.5 µmol, 8.38 mg) and place it in a 5 mL reaction bottle, add 2 mL 100 mM hexafluorobenzene DMF solution (25 equiv., 187.5 µmol) and 1.5 mL 50 mM tris(hydroxymethyl)aminomethane DMF solution (10 equiv., 75 µmol). Mix vigorously at 1500 rpm for 30 seconds in a constant temperature mixer and place at 1000 rpm for 4.5 hours at room temperature. After the reaction is completed, the reaction solution is transferred to a glass culture dish and placed in a -80 °C refrigerator, and then placed in a freeze dryer. The freeze-drying process lasts about 12-16 hours, and the peptide sample is completely freeze-dried to obtain a dry crude peptide. Subsequently, it was purified by RP-HPLC (using mobile phase A as 0.1% TFA / water, mobile phase B as 0.1% TFA / acetonitrile, linear gradient elution), and the target peak effluent was collected, lyophilized, and identified by mass spectrometry to obtain Ano-12 (6.0 mg), see Figure 3 .

[0077] The synthetic route is as follows:

[0078] .

[0079] Preparation of Ano-13

[0080] Accurately weigh the calculated amount of Ano-4 (7.5 µmol, 8.47 mg) and place it in a 5 mL reaction bottle, add 2 mL 100 mM hexafluorobenzene DMF solution (25 equiv., 187.5 µmol) and 1.5 mL 50 mM tris(hydroxymethyl)aminomethane DMF solution (10 equiv., 75 µmol). Mix vigorously at 1500 rpm for 30 seconds in a constant temperature mixer and place at 1000 rpm for 4.5 hours at room temperature. After the reaction is completed, the reaction solution is transferred to a glass culture dish and placed in a -80 °C refrigerator, and then placed in a freeze dryer. The freeze-drying process lasts about 12-16 hours, and the peptide sample is completely freeze-dried to obtain a dry crude peptide. Subsequently, it was purified by RP-HPLC (using mobile phase A as 0.1% TFA / water, mobile phase B as 0.1% TFA / acetonitrile, linear gradient elution), and the target peak effluent was collected, lyophilized, and identified by mass spectrometry to obtain Ano-13 (6.1 mg), see Figure 4 .

[0081] The synthetic route is as follows:

[0082] .

[0083] Preparation of Ano-14

[0084] Accurately weigh the calculated amount of Ano-5 (7.5 µmol, 8.17 mg) and place it in a 5 mL reaction bottle. Add 2 mL of 100 mM hexafluorobenzene DMF solution (25 equiv., 187.5 µmol) and 1.5 mL of 50 mM tris(hydroxymethyl)aminomethane DMF solution (10 equiv., 75 µmol). Mix vigorously at 1500 rpm for 30 seconds in a constant temperature mixer and place at 1000 rpm for 4.5 hours at room temperature. After the reaction is completed, transfer the reaction solution to a glass culture dish and freeze it in a -80 °C refrigerator, and then place it in a freeze dryer. The freeze-drying process lasts for about 12-16 hours, and the peptide sample is completely freeze-dried to obtain a dry crude peptide. Subsequently, it was purified by RP-HPLC (using mobile phase A as 0.1% TFA / water, mobile phase B as 0.1% TFA / acetonitrile, linear gradient elution), and the target peak effluent was collected, lyophilized, and identified by mass spectrometry to obtain Ano-14 (5.8 mg), see Figure 5 .

[0085] The synthetic route is as follows:

[0086] .

[0087] Preparation of Ano-15

[0088] Accurately weigh the calculated amount of Ano-6 (7.5 µmol, 8.49 mg) and place it in a 5 mL reaction bottle, add 2 mL 100 mM hexafluorobenzene DMF solution (25 equiv., 187.5 µmol) and 1.5 mL 50 mM tris(hydroxymethyl)aminomethane DMF solution (10 equiv., 75 µmol). Mix vigorously at 1500 rpm for 30 seconds in a constant temperature mixer and place at 1000 rpm for 4.5 hours at room temperature. After the reaction is completed, the reaction solution is transferred to a glass culture dish and placed in a -80 °C refrigerator, and then placed in a freeze dryer. The freeze-drying process lasts about 12-16 hours, and the peptide sample is completely freeze-dried to obtain a dry crude peptide. Subsequently, it was purified by RP-HPLC (using mobile phase A as 0.1% TFA / water, mobile phase B as 0.1% TFA / acetonitrile, linear gradient elution), and the target peak effluent was collected, lyophilized, and identified by mass spectrometry to obtain Ano-15 (6.7 mg), see Figure 6 .

[0089] The synthetic route is as follows:

[0090] .

[0091] Preparation of Ano-16

[0092] Accurately weigh the calculated amount of Ano-7 (7.5 µmol, 9.00 mg) and dissolve it in 3 mL of DMF solution in a 5 mL reaction bottle. Add 0.15 mL of 100 mM decafluorobiphenyl DMF solution (2 equiv., 15 µmol) and 1.5 mL of 50 mM tris(hydroxymethyl)aminomethane DMF solution (10 equiv., 75 µmol). Mix vigorously at 1500 rpm for 30 seconds in a thermostatic mixer and place at 1000 rpm for 5 h at room temperature. After the reaction is completed, transfer the reaction solution to a glass culture dish and freeze it in a -80 °C refrigerator, and then place it in a freeze dryer. The freeze-drying process lasts for about 12-16 hours, and the peptide sample is completely freeze-dried to obtain a dry crude peptide. Subsequently, it was purified by RP-HPLC (using mobile phase A as 0.1% TFA / water, mobile phase B as 0.1% TFA / acetonitrile, linear gradient elution), and the target peak effluent was collected, lyophilized, and identified by mass spectrometry to obtain Ano-16 (5.7 mg), see Figure 7 .

[0093] The synthetic route is as follows:

[0094] .

[0095] Preparation of Ano-17

[0096] Accurately weigh the calculated amount of Ano-8 (7.5 µmol, 8.49 mg) and dissolve it in 3 mL of DMF solution in a 5 mL reaction bottle. Add 0.15 mL of 100 mM decafluorobiphenyl DMF solution (2 equiv., 15 µmol) and 1.5 mL of 50 mM tris(hydroxymethyl)aminomethane DMF solution (10 equiv., 75 µmol). Mix vigorously at 1500 rpm for 30 seconds in a thermostatic mixer and place at 1000 rpm for 5 h at room temperature. After the reaction is completed, transfer the reaction solution to a glass culture dish and freeze it in a -80 °C refrigerator, and then place it in a freeze dryer. The freeze-drying process lasts for about 12-16 hours, and the peptide sample is completely freeze-dried to obtain a dry crude peptide. Subsequently, it was purified by RP-HPLC (using mobile phase A as 0.1% TFA / water, mobile phase B as 0.1% TFA / acetonitrile, linear gradient elution), and the target peak effluent was collected, lyophilized, and identified by mass spectrometry to obtain Ano-17 (4.6 mg), see Figure 8 .

[0097] The synthetic route is as follows:

[0098] .

[0099] Preparation of Ano-18

[0100] Accurately weigh the calculated amount of Ano-9 (7.5 µmol, 8.49 mg) and dissolve it in 3 mL of DMF solution in a 5 mL reaction bottle. Add 0.15 mL of 100 mM decafluorobiphenyl DMF solution (2 equiv., 15 µmol) and 1.5 mL of 50 mM tris(hydroxymethyl)aminomethane DMF solution (10 equiv., 75 µmol). Mix vigorously at 1500 rpm for 30 seconds in a thermostatic mixer and place at 1000 rpm for 5 h at room temperature. After the reaction is completed, transfer the reaction solution to a glass culture dish and freeze it in a -80 °C refrigerator, and then place it in a freeze dryer. The freeze-drying process lasts for about 12-16 h, and the peptide sample is completely freeze-dried to obtain a dry crude peptide. Subsequently, it was purified by RP-HPLC (using mobile phase A as 0.1% TFA / water, mobile phase B as 0.1% TFA / acetonitrile, linear gradient elution), and the target peak effluent was collected, lyophilized, and identified by mass spectrometry to obtain Ano-18 (4.6 mg), see Fig. 9 .

[0101] The synthetic route is as follows:

[0102] .

[0103] Example 2

[0104] 1. The high-resolution mass spectrometry data of the antimicrobial peptide Anoplin derivatives Ano-1~18 of the present invention are shown in Table 2.

[0105] Table 2 High-resolution mass spectrometry data of natural antimicrobial peptide Anoplin derivatives Ano-1-18 of the present invention

[0106]

[0107] 2. In vitro antibacterial experiment of Anoplin derivatives modified with perfluoroaromatic conformation locking of the present invention

[0108] The common two-fold dilution method was used to determine the minimum inhibitory concentration of the drug, i.e., the MIC value. First, 4 to 5 colonies of the standard strains of Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 were picked from the test bacteria plate and inoculated into 3 to 5 mL of MH liquid culture medium. The culture was shaken at 37°C for 12 to 24 hours to keep it in the logarithmic growth phase. After determining the concentration of the bacterial solution, it was diluted with MH broth to 1×10 6 CFU / mL. The peptide solution and Anoplin with a concentration of 512 μM were diluted into 5 to 8 concentrations with MH broth, and the dilution concentrations were: 256 μM, 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, and 2 μM. They were added to a 96-well plate, and 100 μL of the diluted bacterial solution was added to each well. Finally, the final drug concentration in each well was 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, and 1 μM. There were 3 parallel wells for each concentration. Anoplin was used as the positive control, and the blank culture medium was used as the negative control. Finally, the turbidity was observed with the naked eye after incubation in a 37 ℃ constant temperature incubator for 18 h. The minimum concentration at which the bacterial growth was clearly inhibited was the MIC value. The parallel experiment was repeated more than three times. The results are shown in Table 3.

[0109] Table 3 Minimum inhibitory concentration against standard strains

[0110]

[0111] The results in Table 3 show that the antibacterial activity of the Anoplin antimicrobial peptide analogs modified with perfluoroaromatic hydrocarbons is better than that of the uncyclized Anoplin antimicrobial peptide analogs containing cysteine ​​substitutions, and the antibacterial activity of the Anoplin antimicrobial peptide analogs modified with perfluoroaromatic hydrocarbons is better than that of the parent peptide Anoplin. Among them, Ano-13, Ano-14, Ano-17 and Ano-18 have the best antibacterial activity against Staphylococcus aureus and Escherichia coli, which greatly improves the antibacterial activity of the parent peptide Anoplin.

[0112] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A perfluoroarene conformationally locked modified melittin Anoplin derivative, characterized in that: Its structure is as shown in any of the following:

2. The method for preparing the Anoplin derivative according to claim 1, characterized in that: The following steps are involved: The intermediate peptide is synthesized by chemical method, with hexafluorobenzene or decafluorobiphenyl as a bridging group, and undergoes a nucleophilic substitution reaction under the action of a base to achieve locking of the α-helical structure, thereby obtaining the bee venom peptide Anoplin derivative.

3. The preparation method according to claim 2, characterized in that: The following steps are involved: (1) Add hexafluorobenzene or decafluorobiphenyl and tris(hydroxymethylaminomethane) to the intermediate peptide, mix vigorously at 1500 rpm for 30 seconds, react at room temperature, and freeze-dry after the reaction to obtain a crude product; (2) Purifying the crude product to obtain the Anoplin derivative.

4. The preparation method according to claim 3, characterized in that: The molar ratio of the intermediate peptide, hexafluorobenzene or decafluorobiphenyl and tris(hydroxymethyl)aminomethane is 1:(2-30):

10.

5. The preparation method according to claim 3, characterized in that: The room temperature reaction conditions are: standing at room temperature and rotating speed of 1000 rpm for 4.5 to 5 h.

6. The preparation method according to claim 3, characterized in that: The purification was performed by RP-HPLC purification, with mobile phase A being 0.1% TFA / water, mobile phase B being 0.1% TFA / acetonitrile, and linear gradient elution.

7. The preparation method according to claim 3, characterized in that: The intermediate peptide is selected from any one of the following: 。 8. The use of the Anoplin derivative of claim 1 in the preparation of a drug for preventing and treating bacterial infection, characterized in that: The bacteria include Escherichia coli and Staphylococcus aureus.

9. The use of the Anoplin derivative of melittin in the preparation of an antibacterial agent according to claim 1, characterized in that: The bacteria include Escherichia coli and Staphylococcus aureus.

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