Modified antibacterial peptide and antibacterial peptide-antibiotic conjugate prepared from same

Abstract

The invention provides a modified antibacterial peptide and an antibacterial peptide-antibiotic conjugate prepared from the modified antibacterial peptide, and belongs to the technical field of biochemical drugs. The modified antibacterial peptide is obtained by connecting an alkyl chain with hydroxyl into an antibacterial peptide through an amido bond. According to the modified antibacterial peptide provided by the invention, the antibacterial property of the antibacterial peptide is enhanced by introducing an alkyl chain with a hydroxyl group and a specific length into the antibacterial peptide, and meanwhile, a coupling site capable of forming an easy-to-break chemical bond with an antibiotic drug is provided. According to the modified antibacterial peptide, a chemical bond (ester bond) with hydrolytic ability is formed between a coupling site and an antibiotic drug, and then coupling is carried out, so that the antibacterial peptide-antibiotic conjugate is obtained. The conjugate also has an antibacterial effect, and the antibiotic drug can be released through hydrolysis to achieve the antibacterial effect. In addition, the conjugate can also reduce the drug resistance risk of antibiotics and broaden the antibacterial spectrum. The invention provides a better treatment mode for bacterial infection, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical drugs, and in particular relates to a modified antimicrobial peptide and an antimicrobial peptide-antibiotic conjugate prepared therefrom. Background Art

[0002] Bacterial infections pose a serious threat to people's lives and health. Antibiotics are famous for their excellent bactericidal effects. However, the abuse of traditional antibiotics in the animal husbandry and pharmaceutical industries has led to the rapid development of drug resistance. The current discovery of antibiotics is becoming increasingly difficult. Most of the antibiotics approved between 2014 and 2018 were based on discoveries made 30 years ago. Antibiotics are increasingly showing signs of decline.

[0003] Natural antimicrobial peptides are widely distributed in organisms. They contain cationic amino acids and hydrophobic amino acids and have antimicrobial activity. Some antimicrobial peptides also have anticancer and antiparasitic effects. Antimicrobial peptides themselves have positive charges and can contact negatively charged bacterial membranes under electrostatic forces. Antimicrobial peptides are usually irregular conformations in solution. After contacting bacterial membranes, most of them will undergo conformational transformation to form secondary structures of α-helix or β-sheet. After antimicrobial peptides bind to bacterial membranes, they undergo structural transformation and rely on hydrophilic and hydrophobic abilities to interact with bacterial membranes, gradually achieving membrane lysis or membrane penetration to exert antimicrobial ability. Antimicrobial peptides have unique antimicrobial mechanisms of membrane destruction and immune regulation, are not easy to induce bacterial resistance, and have a wide range of antimicrobial activity against Gram-positive and Gram-negative bacteria. Therefore, antimicrobial peptides can be used as candidates for the next generation of antimicrobial drugs. In recent years, there have been more and more literature, patents, and clinical trial projects on antimicrobial peptides.

[0004] At the same time, the conjugation of antimicrobial peptides with traditional antibiotics has attracted widespread attention. As a new antimicrobial strategy, antimicrobial peptide-antibiotic conjugates have many advantages, such as enhancing antimicrobial activity, reducing the risk of drug resistance, and broadening the antimicrobial spectrum. When preparing antimicrobial peptide-antibiotic conjugates, the selection of the conjugation site of the antimicrobial peptide is crucial. Different conjugation sites may lead to changes in the synergistic effect between the antimicrobial peptide and the antibiotic. At the same time, some antimicrobial peptides themselves lack conjugation sites that can form easily broken chemical bonds after connection, resulting in the inability to release antibiotic drugs in the conjugate, affecting the antibacterial effect. Therefore, they need to be functionalized to improve them. However, the introduction of functional groups may reduce or even lose the antibacterial effect of antimicrobial peptides. How to appropriately functionalize antimicrobial peptides so that they can be coupled with antibiotic drugs through easily broken chemical bonds without sacrificing their own antibacterial effects, and obtain antimicrobial peptide-antibiotic conjugates with certain antibacterial effects, requires further study. Summary of the invention

[0005] The object of the present invention is to provide a modified antimicrobial peptide and an antimicrobial peptide-antibiotic conjugate prepared therefrom.

[0006] The present invention provides a modified antimicrobial peptide, which is obtained by linking an alkyl chain with a hydroxyl group to the antimicrobial peptide via an amide bond; the structure of the alkyl chain with a hydroxyl group is n is an integer from 10 to 16.

[0007] Furthermore, n is 12.

[0008] Furthermore, the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.1.

[0009] Furthermore, the structure of the modified antimicrobial peptide is:

[0010]

[0011] The present invention also provides a method for preparing the aforementioned modified antimicrobial peptide, wherein the preparation method is a solid phase synthesis method;

[0012] Preferably,

[0013] The preparation method comprises the following steps:

[0014] (1) Swelling the Rink resin and then deprotecting it;

[0015] (2) using a solid phase synthesis method, the target amino acid, HBTU and DIEA are dissolved in a solvent in sequence and reacted with the deprotected Rink resin to obtain a resin grafted with Fmoc-KFKWPW;

[0016] (3) The amino acid in step (2) is replaced with terminal hydroxyl dodecanoic acid, and the terminal hydroxyl dodecanoic acid is connected to the resin in the same manner to obtain HO-C 12 -KFKWPW resin;

[0017] (4) using a cleavage system containing trifluoroacetic acid to cleave the resin, filtering the obtained cleavage solution, precipitating it with anhydrous ether, and purifying the precipitate to obtain the product.

[0018] Further,

[0019] In step (1), the solvent used for swelling is dichloromethane;

[0020] And / or, in step (1), the solvent used for the deprotection is a mixed solution of tetramethylpiperidine and N',N-dimethylformamide;

[0021] And / or, in step (2), the equivalent ratio of the target amino acid, HBTU and DIEA is 1:(0.1-1):(1-3);

[0022] And / or, in step (2), the solvent is N,N-dimethylformamide;

[0023] And / or, in step (2), the reaction temperature is 25 to 30° C., and the reaction time is 1 to 5 h;

[0024] And / or, in step (4), the trifluoroacetic acid-containing cracking system is a mixed solution consisting of 95% by volume of trifluoroacetic acid, 2.5% by volume of water and 2.5% by volume of triisopropylsilane;

[0025] And / or, in step (4), the purification method is purification using high performance liquid chromatography;

[0026] Preferably,

[0027] In step (1), the volume ratio of tetramethylpiperidine to N',N-dimethylformamide is 1:4;

[0028] And / or, in step (2), the equivalent ratio of the target amino acid, HBTU and DIEA is 1:0.9:2.

[0029] The present invention also provides an antimicrobial peptide-antibiotic conjugate, which is obtained by connecting the aforementioned modified antimicrobial peptide and the antibiotic drug through a chemical bond with hydrolysis ability;

[0030] Preferably, the chemical bond having hydrolysis ability is an ester bond;

[0031] More preferably, the antibiotic drug is ciprofloxacin.

[0032] Furthermore, the structure of the antimicrobial peptide-antibiotic conjugate is:

[0033]

[0034] The present invention also provides a method for preparing the aforementioned antimicrobial peptide-antibiotic conjugate, which comprises the following steps:

[0035] 1) In a solvent, ciprofloxacin and di-tert-butyl dicarbonate react to obtain Boc-protected ciprofloxacin;

[0036] 2) After the Boc-protected ciprofloxacin, catalyst, and water absorbent are dissolved in a solvent, the aforementioned HO-C 12 -KFKWPW resin reaction;

[0037] 3) After the reaction is completed, the resin is cut using a cracking system containing trifluoroacetic acid, the cleavage solution obtained is filtered, and anhydrous ether is used for precipitation, and the precipitate is purified to obtain;

[0038] Preferably,

[0039] In step 1), the solvent is a mixed solution of NaOH aqueous solution and dioxane;

[0040] And / or, in step 1), the mass ratio of ciprofloxacin to di-tert-butyl dicarbonate is 1:(1-5);

[0041] And / or, in step 1), the reaction temperature is 25-30°C and the reaction time is 20-30h;

[0042] And / or, in step 2), the solvent is DMF;

[0043] And / or, in step 2), the mass ratio of the Boc-protected ciprofloxacin to the catalyst is 9:(2-5);

[0044] and / or, in step 2), the mass volume ratio of the Boc-protected ciprofloxacin and the water absorbent is 0.9 g: (1-5) mL;

[0045] And / or, in step 2), the Boc-protected ciprofloxacin and HO-C 12 -The mass ratio of KFKWPW resin is 0.9:(1-2);

[0046] And / or, in step 2), the reaction temperature is 80-100° C., and the reaction time is 20-30 h;

[0047] And / or, in step 3), the trifluoroacetic acid-containing cracking system is a mixed solution consisting of 95% by volume of trifluoroacetic acid, 2.5% by volume of water and 2.5% by volume of triisopropylsilane;

[0048] And / or, in step 3), the purification method is purification using high performance liquid chromatography;

[0049] More preferably,

[0050] In step 2), the catalyst is tetrakis dimethylaminopyridine;

[0051] And / or, in step 2), the water absorbent is 1,3-diisopropylcarbodiimide.

[0052] The present invention also provides the use of the aforementioned modified antimicrobial peptide or the aforementioned antimicrobial peptide-antibiotic conjugate in the preparation of antimicrobial drugs.

[0053] Antimicrobial peptide HB1345(C 10 -KFKWPW) is a lipopeptide compound with broad-spectrum antibacterial activity, showing good antibacterial effect against Propionibacterium acnes. Although the antimicrobial peptide HB1345 can be conjugated with antibiotic drugs, it lacks a conjugation group that can form an easily breakable chemical bond with the antibiotic, resulting in the inability to release the antibiotic drug in the conjugate and exert its antibacterial effect.

[0054] The present invention introduces an alkyl chain (OH-C) with a hydroxyl group into the antimicrobial peptide HB1345.12 alkyl chain) to replace the C 10 Alkyl chain is used to realize its functionalization. It not only effectively improves the antibacterial effect of antimicrobial peptides, but also facilitates the coupling of antimicrobial peptides and antibiotic drugs through easily breakable chemical bonds. The present invention uses ester bonds to couple antimicrobial peptides and antibiotic drugs, and antibiotic drugs can be hydrolyzed and released through ester bonds to exert antibacterial effects.

[0055] In the present invention, in addition to using an ester bond to link the hydrophobic alkyl chain and the antibiotic, using a hydrolyzable chemical bond such as a carbonate bond to link the two should also fall within the scope of this solution.

[0056] The present invention has achieved the following beneficial effects:

[0057] The present invention provides a modified antimicrobial peptide, which enhances the antimicrobial performance of the antimicrobial peptide by introducing an alkyl chain of a specific length with a hydroxyl group into the antimicrobial peptide, and provides a coupling site that can form an easily breakable chemical bond with an antibiotic drug. The modified antimicrobial peptide forms a chemical bond (ester bond) with hydrolysis ability between the coupling site and the antibiotic drug, and then couples to obtain an antimicrobial peptide-antibiotic conjugate. The conjugate also has an antimicrobial effect, and the antibiotic drug can be released by hydrolysis to exert the antimicrobial effect. In addition, the conjugate can also reduce the risk of antibiotic resistance and broaden the antimicrobial spectrum. The present invention provides a better treatment for bacterial infection, and at the same time, the present invention provides an example of coupling antibiotics to antimicrobial peptides, which has good application prospects.

[0058] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0059] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Modified antimicrobial peptide HO-C 12 -The molecular structure, HPLC and LC-Mass spectra of KFKWPW.

[0061] Figure 2 Reaction route for Boc protection of ciprofloxacin.

[0062] Figure 3 HPLC and LC-Mass spectra of Boc-protected ciprofloxacin (Boc-CIP).

[0063] Figure 4 The antibiotic ciprofloxacin and the antimicrobial peptide HO-C 12 -KFKWPW coupling molecule CIP-C 12 -Molecular structure formula of KFKWPW.

[0064] Figure 5 The antibiotic ciprofloxacin and the antimicrobial peptide HO-C 12 -KFKWPW coupling molecule CIP-C 12 -HPLC and LC-Mass spectra of KFKWPW.

[0065] Figure 6 Modified antimicrobial peptide HO-C 10 -The molecular structure, HPLC and LC-Mass spectra of KFKWPW.

[0066] Figure 7 Modified antimicrobial peptide HO-C 14 -The molecular structure, HPLC and LC-Mass spectra of KFKWPW.

[0067] Figure 8 Modified antimicrobial peptide HO-C 16 -The molecular structure, HPLC and LC-Mass spectra of KFKWPW.

[0068] Fig. 9 The antibacterial test results of each modified antimicrobial peptide against Escherichia coli and Staphylococcus aureus: HO-C10, HO-C12, HO-C14 and HO-C16 represent HO-C 10 -KFKWPW, HO-C 12 -KFKWPW, HO-C 14 -KFKWPW and HO-C 16 -KFKWPW.

[0069] Fig.10 The results of the antibacterial experiments of different antimicrobial peptides against Escherichia coli and Staphylococcus aureus at a concentration of 128 μg / mL are shown in Table 1. 12 -KFKWPW compared, ns indicates no significant difference, ** indicates p < 0.01, **** indicates p < 0.0001.

[0070] Fig.11 The antimicrobial peptide-antibiotic conjugate (CIP-C 12 -KFKWPW)'s antibacterial test results. DETAILED DESCRIPTION

[0071] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0072] Example 1. Preparation of the antimicrobial peptide-antibiotic conjugate of the present invention

[0073] 1. Preparation of modified antimicrobial peptide HB1345

[0074] The peptide sequence KFKWPW (SEQ ID NO. 1) was obtained on Rink resin by solid phase synthesis, and then the C with a hydroxyl group at the end was connected by solid phase synthesis. 12 The hydrophobic tail chain of carbon chain length was obtained to obtain the modified antimicrobial peptide (HO-C 12 -KFKWPW), to achieve functionalization of the antimicrobial peptide. The specific preparation method of the modified antimicrobial peptide comprises the following steps:

[0075] Using a standard solid phase synthesis method, 1.14 g of Rink resin with a substitution degree of 0.416 mmol / g was swollen with dichloromethane (DCM) for 15 minutes, and then deprotected in 25 mL of a solvent of tetramethylpiperidine (HTMP): N', N-dimethylformamide (DMF) = 1:4 (volume ratio) for 15 minutes. 1 mmol of Fmoc-L-tryptophan (Fmoc-W-OH) was mixed with benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU) and N, N-diisopropylethylamine (DIEA) and dissolved in 30 ml of N, N-dimethylformamide (DMF), with an equivalent ratio of Fmoc-W-OH: HBTU: DIEA = 1: 0.9: 2. Then 40 ml of the mixed solution was added to the deprotected Rink resin and reacted at 25°C for 2 hours to obtain a resin with Fmoc-W attached. The above deprotection and reaction steps are then repeated to sequentially access different amino acids to obtain a resin accessed with Fmoc-KFKWPW.

[0076] Then, the above deprotection and reaction steps are repeated to replace the amino acid with terminal hydroxyl dodecanoic acid, thereby connecting the terminal hydroxyl dodecanoic acid to the resin to obtain HO-C 12 -KFKWPW resin. After the reaction was completed, 10 ml of a mixed solution of 95% trifluoroacetic acid (TFA), 2.5% water (H2O) and 2.5% triisopropylsilane (TIS) was used as a cleavage system to cut the resin, the cleavage solution was filtered and precipitated with anhydrous ether, and the precipitate was purified by high performance liquid chromatography (HPLC) to obtain the modified antimicrobial peptide HO-C 12 -KFKWPW. Modified antimicrobial peptide HO-C 12 The molecular structure of -KFKWPW and the characterization results of HPLC and LC-Mass are as follows Figure 1 shown.

[0077] 2. Esterification conjugation of antibiotics and antimicrobial peptides

[0078] The antibiotic molecule selected in this example is ciprofloxacin (CIP), which has extremely poor solubility in common DCM and DMF solvents.

[0079] 1g of ciprofloxacin was dissolved in a mixed solution of 10mL of 1M NaOH aqueous solution and 10mL of dioxane, and then 1g of di-tert-butyl dicarbonate was added to react at room temperature of 25°C. After 24 hours, the reaction was completed and a powdery solid was automatically precipitated. The Boc-protected ciprofloxacin solid, i.e., Boc-CIP, was obtained by filtration. The reaction route for Boc protection of ciprofloxacin is as follows: Figure 2 The HPLC spectrum and LC-Mass spectrum of Boc-protected ciprofloxacin (Boc-CIP) are shown in Figure 3 shown.

[0080] Boc-CIP molecules and HO-C 12 -KFKWPW resin cannot react in conventional esterification systems. The present invention has been improved through multiple experiments. 0.9g Boc-CIP, 0.2g catalyst tetradimethylaminopyridine (DMAP), and 1mL water absorbent 1,3-diisopropylcarbodiimide (DIC) are dissolved in 10ml DMF solvent, and HO-C 12 -KFKWPW resin 1.9g, and reacted it at 80°C for 24 hours. After the reaction was completed, a mixed solution of 95% trifluoroacetic acid (TFA), 2.5% water (H2O) and 2.5% triisopropylsilane (TIS) was used as a cleavage system to cut the resin, and the cleavage solution was filtered and precipitated with anhydrous ether. The precipitate was purified by high performance liquid chromatography (HPLC) to obtain the antimicrobial peptide-antibiotic conjugate CIP-C 12 -KFKWPW. HPLC was used to characterize the molecular purity, and LC-Mass was used to characterize the correctness of the synthesis. 12 -The molecular structure of KFKWPW is as follows Figure 4 As shown, CIP-C 12 -HPLC and LC-Mass characterization results of KFKWPW are as follows Figure 5 The results show that CIP-C 12 -KFKWPW was prepared successfully.

[0081] Comparative Example 1: Preparation of antimicrobial peptide HB1345

[0082] Using the standard solid phase synthesis method, 1.14g of Rink resin with a substitution degree of 0.416mmol / g was swollen with dichloromethane (DCM) for 15 minutes, and then deprotected in 25ml solvent of tetramethylpiperidine (HTMP): N', N-dimethylformamide (DMF) = 1:4 (volume ratio) for 15 minutes. 1mmol of Fmoc-L-tryptophan (Fmoc-W-OH) was mixed with benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA) and dissolved in 30ml N,N-dimethylformamide (DMF), with an equivalent ratio of Fmoc-W-OH: HBTU: DIEA = 1:0.9:2. Then 40ml of the mixed solution was added to the deprotected Rink resin and reacted at 25℃ for 2h to obtain a resin with Fmoc-W attached. The above deprotection and reaction steps are then repeated to sequentially access different amino acids to obtain a resin accessed with Fmoc-KFKWPW.

[0083] Then repeat the above deprotection and reaction steps to replace the amino acid with decanoic acid (C 10 ), so that C 10 Alkyl groups are linked to the resin to give C 10 -KFKWPW resin. After the reaction was completed, the resin was cleaved with a mixed solution of 10 ml 95% trifluoroacetic acid (TFA), 2.5% water (H2O) and 2.5% triisopropylsilane (TIS) as a cleavage system, the cleavage solution was filtered and precipitated with anhydrous ether, and the precipitate was purified by high performance liquid chromatography (HPLC) to obtain the antimicrobial peptide HB1345 (C 10 -KFKWPW).

[0084] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0085] Experimental Example 1: Functionalization Screening of Antimicrobial Peptides

[0086] According to the method described in Example 1, the polypeptide sequence KFKWPW was obtained on Rink resin and then connected to different carbon chain lengths (C 10 , C 12 , C 14 , C 16 ) to regulate the hydrophilicity and hydrophobicity of the peptide and obtain a series of modified antimicrobial peptides (HO-C 10 -KFKWPW, HO-C 12 -KFKWPW, HO-C 14 -KFKWPW and HO-C 16 -KFKWPW). The acids used are hydroxy-terminated deca-acid, hydroxy-terminated dodecanoic acid, hydroxy-terminated tetradecanoic acid and hydroxy-terminated hexadecanoic acid.10 -KFKWPW, HO-C 14 -KFKWPW and HO-C 16 The molecular structure of -KFKWPW and the characterization results of HPLC and LC-Mass are as follows Figures 6 to 8 shown.

[0087] Through the antibacterial experiment screening on Escherichia coli and Staphylococcus aureus, we found functionalized modified antimicrobial peptides with excellent effects. The specific experimental method of the antibacterial experiment is as follows:

[0088] The refrigerated Staphylococcus aureus and Escherichia coli were cultured in liquid culture medium for 24 hours (incubator at 37°C). The ratio was 100 μL bacteria + 900 μL liquid culture medium; the activated bacteria were approximately 10 8 CFU / mL. Dilute the activated bacteria to 10 5 After 10 μL of the solution was added, 190 μL of the solution was placed in a 96-well plate, and 10 μL of the antimicrobial peptide aqueous solution samples of different concentrations were added. Three independent experiments were performed for each sample. The 96-well plate was placed in a 37°C incubator for 24 h, and then the OD was measured with a microplate reader. 600 The survival rate of bacteria was calculated.

[0089] Calculation formula:

[0090] Bacterial survival rate = (OD 600测量样品 -OD 600阴性对照 ) / (OD 600阳性对照 -OD 600阴性对照 )×100%

[0091] The negative control was pure culture medium without bacterial solution, and the positive control was bacterial solution without antimicrobial peptide.

[0092] Staphylococcus aureus and Escherichia coli were cultured for 24 hours, and the antibacterial results of each antimicrobial peptide at different concentrations were obtained by absorbance measurement ( Fig. 9 ) and the antibacterial results of each antimicrobial peptide at a concentration of 128 μg / mL ( Fig.10 ). The experimental results show that the modified antimicrobial peptide HO-C 12 -KFKWPW has the best antibacterial effect, significantly better than other modified antimicrobial peptides with different chain lengths. 12 -KFKWPW has an antibacterial effect on Staphylococcus aureus that is even better than the antimicrobial peptide HB1345. 12 The antibacterial properties of antimicrobial peptides can be improved by modifying the carbon chain.

[0093] Experimental Example 2: Study on the antibacterial properties of antimicrobial peptide-antibiotic conjugates

[0094] The antibacterial efficacy of the antimicrobial peptide-antibiotic conjugate was verified by an antibacterial experiment in which Escherichia coli and Staphylococcus aureus were incubated with the antimicrobial peptide-antibiotic conjugate for 24 hours.

[0095] The refrigerated Staphylococcus aureus and Escherichia coli were cultured in liquid culture medium for 24 hours (incubator at 37°C). The ratio was 100 μL bacteria + 900 μL liquid culture medium; the activated bacteria were approximately 10 8 CFU / mL. Dilute the activated bacteria to 10 5 CFU / mL (10 μL bacteria + 10 mL liquid culture medium), 190 μL was placed in a 96-well plate, and different concentrations of CIP-C 12 -10 μL of KFKWPW aqueous solution sample, three independent experiments were performed for each sample, the 96-well plate was placed in a 37°C incubator for 24 h, and then the OD was measured with a microplate reader 600 The survival rate of bacteria was calculated.

[0096] Calculation formula:

[0097] Bacterial survival rate = (OD 600测量样品 -OD 600阴性对照 ) / (OD 600阳性对照 -OD 600阴性对照 )×100%

[0098] The negative control was pure culture medium without bacterial solution, and the positive control was bacterial solution without antimicrobial peptide.

[0099] Antimicrobial peptide-antibiotic conjugates (CIP-C 12 -KFKWPW) using ester bonds to carry out the antibiotic ciprofloxacin and antimicrobial peptide HO-C 12 -KFKWPW link, which is conducive to the hydrolysis and release of antibiotics. CIP-C 12 -KFKWPW has high bactericidal ability at low concentrations such as 128, 64, and 32 μg / ml ( Fig.11 ). The CIP-C prepared by the present invention 12 -KFKWPW can reduce the risk of antibiotic resistance and broaden the antibacterial spectrum while having bactericidal ability.

[0100] In summary, the present invention provides a modified antimicrobial peptide, which enhances the antimicrobial properties of the antimicrobial peptide by introducing an alkyl chain of a specific length with a hydroxyl group into the antimicrobial peptide, and provides a coupling site that can form an easily breakable chemical bond with an antibiotic drug. The modified antimicrobial peptide forms a chemical bond (ester bond) with hydrolysis ability between the coupling site and the antibiotic drug, and then couples to obtain an antimicrobial peptide-antibiotic conjugate. The conjugate also has an antimicrobial effect, and the antibiotic drug can exert its antimicrobial effect through hydrolysis and release. In addition, the conjugate can also reduce the risk of antibiotic resistance and broaden the antimicrobial spectrum. The present invention provides a better treatment for bacterial infections, and the present invention provides an example of coupling antibiotics to antimicrobial peptides, which has good application prospects.

Claims

1. A modified antimicrobial peptide, characterized in that: It is obtained by linking an alkyl chain with a hydroxyl group to an antimicrobial peptide via an amide bond; the structure of the alkyl chain with a hydroxyl group is n is an integer from 10 to 16.

2. The modified antimicrobial peptide according to claim 1, characterized in that: n is 12.

3. The modified antimicrobial peptide according to claim 1 or 2, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

1.

4. The modified antimicrobial peptide according to claim 3, characterized in that: The structure of the modified antimicrobial peptide is:

5. The method for preparing the modified antimicrobial peptide according to claim 4, characterized in that: The preparation method is a solid phase synthesis method; Preferably, The preparation method comprises the following steps: (1) Swelling the Rink resin and then deprotecting it; (2) using a solid phase synthesis method, the target amino acid, HBTU and DIEA are dissolved in a solvent in sequence and reacted with the deprotected Rink resin to obtain a resin grafted with Fmoc-KFKWPW; (3) The amino acid in step (2) is replaced with terminal hydroxyl dodecanoic acid, and the terminal hydroxyl dodecanoic acid is connected to the resin in the same manner to obtain HO-C 12 -KFKWPW resin; (4) using a cleavage system containing trifluoroacetic acid to cleave the resin, filtering the obtained cleavage solution, precipitating it with anhydrous ether, and purifying the precipitate to obtain the product.

6. The preparation method according to claim 5, characterized in that: In step (1), the solvent used for swelling is dichloromethane; And / or, in step (1), the solvent used for the deprotection is a mixed solution of tetramethylpiperidine and N',N-dimethylformamide; And / or, in step (2), the equivalent ratio of the target amino acid, HBTU and DIEA is 1:(0.1-1):(1-3); And / or, in step (2), the solvent is N,N-dimethylformamide; And / or, in step (2), the reaction temperature is 25 to 30° C., and the reaction time is 1 to 5 h; And / or, in step (4), the trifluoroacetic acid-containing cracking system is a mixed solution consisting of 95% by volume of trifluoroacetic acid, 2.5% by volume of water and 2.5% by volume of triisopropylsilane; And / or, in step (4), the purification method is purification using high performance liquid chromatography; Preferably, In step (1), the volume ratio of tetramethylpiperidine to N',N-dimethylformamide is 1:4; And / or, in step (2), the equivalent ratio of the target amino acid, HBTU and DIEA is 1:0.9:

2.

7. An antimicrobial peptide-antibiotic conjugate, characterized in that: It is obtained by connecting the modified antimicrobial peptide described in any one of claims 1 to 4 with an antibiotic drug through a chemical bond with hydrolysis ability; Preferably, the chemical bond having hydrolysis ability is an ester bond; More preferably, the antibiotic drug is ciprofloxacin.

8. The antimicrobial peptide-antibiotic conjugate according to claim 7, characterized in that: The structure of the antimicrobial peptide-antibiotic conjugate is:

9. The method for preparing the antimicrobial peptide-antibiotic conjugate according to claim 8, characterized in that: It includes the following steps: 1) In a solvent, ciprofloxacin and di-tert-butyl dicarbonate react to obtain Boc-protected ciprofloxacin; 2) After the Boc-protected ciprofloxacin, catalyst and water absorbent are dissolved in a solvent, the HO-C 12 -KFKWPW resin reaction; 3) After the reaction is completed, the resin is cut using a cracking system containing trifluoroacetic acid, the cleavage solution obtained is filtered, and anhydrous ether is used for precipitation, and the precipitate is purified to obtain; Preferably, In step 1), the solvent is a mixed solution of NaOH aqueous solution and dioxane; And / or, in step 1), the mass ratio of ciprofloxacin to di-tert-butyl dicarbonate is 1:(1-5); And / or, in step 1), the reaction temperature is 25-30°C and the reaction time is 20-30h; And / or, in step 2), the solvent is DMF; And / or, in step 2), the mass ratio of the Boc-protected ciprofloxacin to the catalyst is 9:(2-5); And / or, in step 2), the mass volume ratio of the Boc-protected ciprofloxacin and the water absorbent is 0.9 g: (1-5) mL; And / or, in step 2), the Boc-protected ciprofloxacin and HO-C 12 -The mass ratio of KFKWPW resin is 0.9:(1-2); And / or, in step 2), the reaction temperature is 80-100° C., and the reaction time is 20-30 h; And / or, in step 3), the trifluoroacetic acid-containing cracking system is a mixed solution consisting of 95% by volume of trifluoroacetic acid, 2.5% by volume of water and 2.5% by volume of triisopropylsilane; And / or, in step 3), the purification method is purification using high performance liquid chromatography; More preferably, In step 2), the catalyst is tetrakis dimethylaminopyridine; And / or, in step 2), the water absorbent is 1,3-diisopropylcarbodiimide.

10. Use of the modified antimicrobial peptide according to any one of claims 1 to 4 or the antimicrobial peptide-antibiotic conjugate according to claim 7 or 8 in the preparation of antimicrobial drugs.

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