CN117143196A - Alpha-helical polypeptide antibiotic bound by full-hydrocarbon side chain, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an α-helical polypeptide antibiotic bound with all hydrocarbon side chains and its preparation method and application. The polypeptide antibiotic uses the linear peptide shown in SEQ ID NO: 1 as a template. First, the linear polypeptide antibiotic is obtained through the Fmoc solid-phase synthesis method; then, on the basis of retaining the key amino acid residues, (S) is used at a specific position. ‑2‑(4‑pentenyl)alanine replaces the original amino acid, and the olefin metathesis reaction is carried out under the catalysis of Grubbs I reagent, and the i-th and i+4-th positions of the linear polypeptide antibiotic are stapled and cyclized. Obtain the target peptide antibiotic. The present invention can inhibit the growth and reproduction of Gram-negative bacteria and Gram-positive bacteria, has low hemolytic toxicity to red blood cells and cytotoxicity to embryonic fibroblasts, has high serum stability, has anti-inflammatory activity, and promotes the recovery of mice. The healing of bacterially infected skin wounds is expected to replace traditional antibiotics in the treatment of bacterial infection-related diseases.

Description

A kind of α-helical polypeptide antibiotic bound with full hydrocarbon side chain and preparation method thereof application

Technical field

The invention belongs to the field of biomedicine, and specifically relates to an α-helical polypeptide antibiotic bound with full hydrocarbon side chains and its preparation method and application.

Background technique

Bacterial infection refers to the process in which pathogenic bacteria invade host tissues or organs and cause varying degrees of pathological reactions in tissues and organs through massive growth and reproduction and the release of toxic substances (toxins). Bacteria can infect any tissue and organ of the human body, and can even invade the blood circulation system, causing acute systemic infection and developing into sepsis or sepsis. Currently, antibiotics remain the mainstay of treatment for bacterial infections. It generally exerts its antibacterial effect by affecting the metabolic process of bacteria, such as inhibiting the synthesis of bacterial cell walls, proteins and DNA. Bacteria will use corresponding methods, such as changing cell wall components to reduce the binding sites of antibiotics, using efflux pumps to exclude antibiotics from the cell, and producing specific active enzymes to degrade antibiotics and resist their antibacterial effects, producing Resistance. From an evolutionary perspective, the development of bacterial resistance is an inevitable process of natural selection, and the number of existing antibiotics that can effectively treat drug-resistant bacteria will continue to decrease. However, the process of developing new antibiotics through high-throughput screening and synthesis using natural products and chemical libraries is extremely slow and will not be enough to meet the challenges posed by drug-resistant bacteria that are increasing rapidly around the world. Therefore, antimicrobial peptides, natural “antibacterial” weapons that exist abundantly in nature, have attracted increasing attention.

Antimicrobial peptides are generally composed of 20-60 cationic amino acids and hydrophobic amino acids in a certain sequence. They mainly exert their antibacterial effects by destroying the integrity of the bacterial cell membrane structure and have broad-spectrum antibacterial activity. Among them, LL-37 is the only antibacterial peptide found in the human body so far. It consists of 37 amino acids forming an amphipathic α-helical structure. The positively charged LL-37 is first adsorbed on the surface of the negatively charged bacterial membrane through electrostatic interaction, and then inserts its hydrophobic structure into the bacterial phospholipid membrane to destroy the fluidity of the phospholipid membrane. At the same time, it is continuously enriched in the phospholipid membrane to form Ion channels leak bacterial contents, causing bacterial death. Therefore, resistance is less likely to develop compared to traditional antibiotics. However, in terms of clinical translation and application, LL-37 antimicrobial peptide still faces the following problems: (1) The number of amino acids in the peptide chain is large, and solid-phase synthesis is expensive; (2) It is easily degraded by proteases and has poor stability in vivo; (3) Selection It has low toxicity and high hemolytic toxicity and normal cell toxicity.

Relevant studies have shown that using an all-carbon skeleton to form a side chain cyclic structure can stabilize the active conformation of α-helical polypeptides and improve its cell membrane penetration and enzyme stability. At the same time, the change in charge caused by side chain cyclization can also improve its selective toxicity. Therefore, the full hydrocarbon side chain binding strategy is expected to be the most direct and effective method to overcome the limitations of clinical application of LL-37 antimicrobial peptide. There are no relevant research reports yet.

Contents of the invention

The technical problem to be solved by the present invention is to provide an α-helical polypeptide antibiotic bound with all hydrocarbon side chains and its preparation method and application, so as to solve the problems faced by LL-37 antimicrobial peptide in clinical translation and application.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides an α-helical polypeptide antibiotic bound with all hydrocarbon side chains. The polypeptide sequence is the shortest sequence with antibacterial activity among the LL-37 antimicrobial peptides, including positions 18 to 29. 12 amino acid residues between. Using the polypeptide sequence as a peptide chain template, replace the i-th and i+4-th amino acids with (S)2(4-pentenyl)alanine (S5) to perform full hydrocarbon side chain binding;

Preferably, the fully hydrocarbon side chain bound α-helical polypeptide antibiotic is selected from one of the following:

a) Using Ac-KRIVQRIKDFLR-NH ₂ as the peptide chain template, 3 _I and 7 _I are replaced by S ₅ and cyclized;

b) Using Ac-KRIVQRIKDFLR-NH ₂ as the peptide chain template, 5 _Q and 9 _D are replaced by S ₅ and cyclized;

c) Using Ac-KRIVQRIKDFLR-NH ₂ as the peptide chain template, 7 _I and 11 _L are replaced by S ₅ and cyclized;

The structural formula of the fully hydrocarbon side chain bound α-helical polypeptide antibiotic provided by the present invention is as follows:

KR(I ₃ , I ₇ )

KR(Q ₅ ,D ₉ )

KR(I ₇ ,L ₁₁ )

In a second aspect, the present invention provides a method for preparing an α-helical polypeptide antibiotic bound with all hydrocarbon side chains, including the following steps:

Step 1): Use a deprotection reagent to remove the Fmoc protecting group on the amino resin;

Step 2): Use a condensation reagent to couple the carboxyl group of the Fmoc-protected amino acid with the exposed amino group on the Rink Amide MBHA resin;

Step 3): Use a deprotection reagent to remove the Fmoc protecting group on the amino acid;

Step 4): Repeat the coupling-deprotection operations described in steps 2) and 3) to synthesize a peptide chain according to the amino acid sequence, wherein the amino acids at the i and i+4 cyclization sites are replaced with S5 respectively;

Step 5): Use an acetylation reagent to modify the deprotected amino group of the last amino acid on the peptide chain;

Step 6): Use a cyclization reagent to catalyze the olefin metathesis reaction of the S5 side chain at position i and i+4;

Step 7): Use a cleavage reagent to cut the peptide chain from the resin, and use glacial ether to precipitate to obtain a crude polypeptide antibiotic;

Step 8): Use high performance liquid chromatography to separate and purify the crude peptide antibiotic.

As a preferred example of the present invention, during the solid-phase synthesis in step 1), the loading amount of the resin is 0.3 mmol/g.

As another preferred example of the present invention, the deprotecting reagent in step 1) is a mixed solution of piperidine and DMF, with a ratio of 1:4 (v/v).

As another preferred example of the present invention, deprotection in step 1) is performed using a deprotecting reagent for 5 minutes and then again for 5 minutes; the reaction temperature for removing the Fmoc group is 20 to 30°C, more preferably 25°C.

As another preferred example of the present invention, the condensation agent used in step 2) is a DIC-Oxyme condensation system, the activator is DIC, and DMF is used as the solvent.

More preferably, the ratio of amino acids, Oxyme, DIC and DMF in step 2) is 1:1:1:6 (mol/mol/mol/mL).

As another preferred example of the present invention, the temperature of the coupling reaction in step 2) is 50-60°C, more preferably 55°C; the coupling reaction time is 20-30 min, more preferably 20 min.

As another preferred example of the present invention, the reaction time of the first amino acid after S ₅ is 1 hour, and the reaction is repeated once under the same conditions before proceeding to the next step.

As another preferred example of the present invention, in step 5), the acetylation reagent used is a mixed solution of acetic anhydride, DIEA and DMF, and the feeding ratio is 1:1:8 (v/v/v).

As another preferred example of the present invention, the acetylation in step 5) uses resin to react in an acetylation reagent for 20 minutes; the reaction temperature is 20-30°C, more preferably 25°C.

As another preferred example of the present invention, the cyclizing agent in step 6) is a solution of Grubbs I reagent and DCE, and the feeding ratio is resin loading capacity: Grubbs I reagent: DCE = 0.3:58:6 (mmol/mg/ mL).

As another preferred example of the present invention, the cyclization in step 6) involves shaking the resin in the cyclization reagent twice for 2 hours each time; the reaction temperature is 20-30°C, more preferably 25°C.

As another preferred example of the present invention, in step 7), the cutting reagent is a mixed solution of TIPS, H ₂ O and TFA, with a volume ratio of 2.5:2.5:95; the volume to mass ratio of the cutting reagent to resin is 1:50(mL/mg).

As another preferred example of the present invention, in step 7), the cutting temperature is 20-30°C, more preferably 25°C; the cutting time is 4 hours.

As another preferred example of the present invention, the purification method adopted in the present invention is reverse-phase high-performance liquid chromatography, using SHIMADZU (LC-6A) reverse-phase high-performance liquid chromatography (RP-HPLC), C18 column (Daisogel, 20× 250mm), the flow rate is 10mL/min. Mobile phase buffer A is acetonitrile plus 0.1% TFA, and mobile phase buffer B is water plus 0.1% TFA. Starting from 10% buffer A, linear gradient elution was performed to 75% buffer A within 50 minutes to obtain the target peptide antibiotic.

In a third aspect, the present invention provides an application of an α-helical polypeptide antibiotic bound with all hydrocarbon side chains.

Preferably, the present invention provides the use of the polypeptide antibiotic in the preparation of medicaments for inhibiting the growth and reproduction of Gram-positive and -negative bacteria.

Preferably, the present invention provides the use of the polypeptide antibiotic in the preparation of medicaments for treating infectious diseases caused by Gram-positive or/and Gram-negative bacteria.

Preferably, the present invention provides the use of the polypeptide antibiotic in the preparation of medicaments for anti-inflammation and promoting wound healing.

Compared with the existing LL-37 antimicrobial peptide, the polypeptide antibiotic provided by the present invention has the following beneficial effects:

(1) The polypeptide antibiotic contains fewer amino acid sequences, the preparation method is simple and easy, and the product can be obtained with high yield and high purity;

(2) The polypeptide antibiotic can more significantly inhibit the growth and reproduction of Gram-negative bacteria and Gram-positive bacteria;

(3) The polypeptide antibiotic has lower hemolytic toxicity to red blood cells and lower cytotoxicity to embryonic fibroblasts;

(4) The peptide antibiotic has higher serum stability;

(5) The polypeptide antibiotic has more significant anti-inflammatory activity and can more significantly promote the healing of bacterially infected skin wounds in mice.

Description of the drawings

Figure 1 is a schematic sequence diagram of the polypeptide antibiotic prepared in Example 1;

Figure 2 is a synthesis route diagram of Example 1;

Figures 3-7 are high performance liquid chromatograms and mass spectra of the polypeptide antibiotics prepared in Example 1;

Figure 8 is a summary table of the minimum inhibitory concentrations of the polypeptide antibiotics prepared in Example 1;

Figure 9 is a hemolytic toxicity diagram of the polypeptide antibiotic prepared in Example 1;

Figure 10 is a cytotoxicity diagram of the polypeptide antibiotic prepared in Example 1;

Figure 11 is a serum stability chart of the polypeptide antibiotic prepared in Example 1;

Figure 12 is a graph of the anti-inflammatory activity of the polypeptide antibiotic prepared in Example 1;

Figure 13 is an experimental diagram of the polypeptide antibiotic prepared in Example 1 promoting bacterial infection wound healing in mice.

Detailed ways

In order to make the present invention more obvious and understandable, preferred embodiments are described in detail below along with the accompanying drawings.

In the following examples, the abbreviations involved are explained as follows:

Fmoc: fluorenylmethoxycarbonyl

DCM: dichloromethane

DCE: 1,2-dichloroethane

DMF: N,N-dimethylformamide

Oxyme: Ethyl Cyanoglyoxylate-2-Oxime

DIEA: N,N-diisopropylethylamine

DIC: N,N-diisopropylcarbodiimide

S5: (S)-2-Amino-2-methyl-4-pentanoic acid

TFA: trifluoroacetic acid

TIPS: Triisopropylsilane

GrubbsⅠ: phenylmethylenebis(tricyclohexylphosphonium)ruthenium dichloride

%: Unless otherwise stated in the present invention, it refers to mass percentage.

The sources of experimental materials involved are as follows:

Amino acids and Rink Amide MBHA resin were purchased from Shanghai Gill Biochemical Co., Ltd.; N,N-diisopropylcarbodiimide (DIC), Ethyl Cyanoglyoxylate-2-Oxime, trifluoroacetic acid (TFA), and acetonitrile (chromatographically pure) were purchased from From Beijing Bailingwei Technology Co., Ltd.; N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF), anhydrous ether, dichloromethane (DCM), 1,2-di Ethyl chloride (DCE), piperidine, and phenol were all of analytical grade and purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.

Example 1

The invention provides an α-helical polypeptide antibiotic bound with all hydrocarbon side chains, and its preparation method is as follows:

1. Synthesis of polypeptide antibiotics (as shown in Figure 2)

(1) Preparation of compound 1

Add 500 mg of amino resin (loading amount: 0.30 mmol/g) into the solid-phase synthesis reaction tube, soak in DCM for 20 minutes to fully swell the resin, and drain it for use.

Add 20% piperidine-DMF solution until the resin is completely submerged, shake for 5 min × 2 at 25°C to remove Fmoc from the resin, and wash the resin three times each with DCM and DMF.

(2) Preparation of compound 2

Mix the first amino acid in the sequence (1mmol), Oxyme (142mg, 1mmol) and DIC (155μL, 1mmol) in 6mL DMF, add to the resin and shake at 60°C for 20min (S ₅ followed by an amino acid reaction for 1h, repeat Reaction once), wash the resin with DCM and DMF three times each.

(3) Preparation of compound 3

Repeat steps (1) and (2), and mix Fmoc amino acid (1mmol), Oxyme (142mg) and DIC (155μL) in 6mL DMF according to the polypeptide sequence, add it to the resin, and shake at 60°C for 20min. Repeat deprotection→condensation→deprotection until all amino acids are connected. After the last amino acid is deprotected, 6 mL of acetic anhydride:DIEA:DMF (1:1:8) mixture is added, shaken at 25°C for 20 minutes, and the resin is washed three times each with DCM and DMF.

(4) Preparation of compound 4

Add DCE solution (6 mL) of Grubbs I (58 mg) reagent, and react with shaking at 25°C twice for 2 hours each time. After the reaction is completed, the resin is washed three times each with DCM, DMF, and anhydrous ether, and the resin is dried under vacuum.

(5) Preparation of target compounds

Wash the resin and drain it, add 10 mL of TIPS:H ₂ O:TFA = 2.5:2.5:95 (V/V/V), shake at room temperature for 4 hours, filter, wash the resin with a little TFA, and collect the filtrate. Blow away excess TFA by bubbling argon, pour in ice-cold ether to precipitate and centrifuge, discard the supernatant, continue washing and centrifuging with ice-cold ether three times, and blow-dry with argon to obtain crude staple peptide.

2. Purification of target peptide antibiotics

The crude peptide was dissolved in acetonitrile and water and purified by preparative RP-HPLC. The separation conditions are as follows:

Instrument: SHIMADZU (LC-6A) reversed-phase high performance liquid chromatography (RP-HPLC)

Chromatographic column: C18 column (Daisogel, 20×250mm)

Mobile phase: Mobile phase A is an aqueous solution with a volume fraction of 0.1% TFA, and mobile phase B is an acetonitrile solution with a volume fraction of 0.1% TFA;

Steps and parameters: Start with 10% buffer A and proceed to 75% buffer A within 50 minutes for linear gradient elution. The flow rate is 10mL/min, the injection volume is 5mL, and the detection wavelength is 214nm.

Example 2: Product identification and purity analysis

The product obtained in step 2 of Example 1 was subjected to purity analysis by HPLC and molecular weight identification of the product by ESI-MS. ESI-MS was measured on a Shimadzu LCMS-8040 mass spectrometer. Scanning mode: positive ion; determination time: 0-2min; determination range: 400-2000; data collection time: 2min; total flow rate: 0.2mL/min; buffer A is water plus 0.1% formic acid, buffer B is acetonitrile Add 0.1% formic acid and maintain 80% buffer B within 2 min for compound analysis. It was determined that the peak time of the main peak of the crude product was consistent, and the purity of the stapled peptide prepared by this method was >98%. The HPLC and ESI-MS mass spectrometer analysis results are shown in Figure 3-5.

Example 3: Determination of antibacterial activity of polypeptide antibiotics according to the invention

The minimum inhibitory concentration (MIC) of stapled peptides was determined using the microbial dilution method according to the Clinical and Laboratory Standards Institute (CLSI) method. Prepare a bacterial suspension of 10 ⁶ CFU/mL in MH-II broth, prepare peptide solutions of different concentrations in phosphate buffer saline (PBS), and then mix 50 μL of the peptide solution with 50 μL of the bacterial suspension in a 96-well plate , incubate the plate at room temperature of 37°C for 24 hours. 10 μL of AlamarBlue cell viability reagent (Thermo Fisher Scientific, UK) was added to each well and the plates were incubated for an additional 2 h at 37°C. The MIC was recorded as the lowest peptide concentration required to completely inhibit bacterial growth. Assays were performed in three independent experiments. It can be seen from the results that the polypeptide antibiotics KR (I ₃ , I ₇ ), KR (Q ₅ , D ₉ ) and KR (I ₇ , L ₁₁ ) of the present invention can significantly inhibit the growth of Gram-positive bacteria and Gram-negative bacteria. Growth and reproduction, among which KR (Q ₅ , D ₉ ) has the best effect. The results are shown in Table 6.

Example 4: Determination of hemolytic toxicity of the polypeptide antibiotic prepared in Example 1

The peptide solution with a concentration of 256 μg/mL was serially diluted in a 96-well plate and then mixed with an equal volume of 4% (v/v) rabbit red blood cell suspension. The plate was incubated at 37°C for 1 hour, and then centrifuged at 1000 rpm for 10 minutes to separate the supernatant. The amount of released hemoglobin was determined using a microplate reader (BioTek, USA). Measure the absorbance at a wavelength of 570 nm and calculate the hemolysis percentage as: Hemolysis (%) = (Abspeptide – Absblank) / (Abscontrol – Absblank), where Absblank and Abscontrol are the samples treated with PBS and 0.1% Triton X-100, respectively. Absorbance. It can be seen from the results that the polypeptide antibiotics KR (I ₃ , I ₇ ), KR (Q ₅ , D ₉ ) and KR (I ₇ , L ₁₁ ) of the present invention have lower hemolytic toxicity than LL-37. The results are shown in the figure 7 shown.

Example 5: Determination of cytotoxicity of the polypeptide antibiotic prepared in Example 1

NIH 3T3 cells were seeded in a 96-well plate at a density of 1.5 × ¹⁰ cells/well and incubated at 37°C for 24 hours. Cells were then treated with peptide solutions of different concentrations in fresh DMEM and incubated for 24 hours, then 10 μL of CCK-8 was added and incubated for another 1 hour. The absorbance was measured at a wavelength of 450 nm using a Cytation 5 microplate reader (BioTek, USA), and the percentage of cell viability was calculated as follows: Cell viability (%) = (Abspeptide-Absblank)/(Abscontrol-Absblank) × 100%, where Abscontrol is in PBS The absorbance of the treated cells, Absblank is the absorbance of the cell-free medium. Fit the data using nonlinear regression (curve fitting) in Graphpad Prism to obtain _IC50 values. Determinations were performed in three independent experiments, and standard errors (SD) are shown in the histograms. It can be seen from the results that the polypeptide antibiotics KR (I ₃ , I ₇ ), KR (Q ₅ , D ₉ ) and KR (I ₇ , L ₁₁ ) of the present invention have lower cytotoxicity than LL-37. The results are shown in the figure As shown in 8.

Example 6: Determination of serological stability of the polypeptide antibiotic prepared in Example 1

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect the serum stability of stapled peptides. Prepare a 2 mg/mL peptide solution in pH 7.4 PBS and incubate with human serum at a final volume ratio of 1:4 at 37 °C. Sampling was performed at different time intervals, and 50 μL of the mixture was added to 50 μL of ethanol to terminate enzymatic hydrolysis. After centrifugation at 10,000 rpm for 10 min at 4°C, the supernatant was collected and analyzed using HPLC with a wavelength of 220 nm. Record the integrated peak area of the intact peptide and calculate the percentage of remaining peptide based on the integrated area as follows: Remaining peptide (%) = Peak area of remaining peptide/Peak area of intact peptide × 100% in three independent experiments Determinations were performed and standard errors (SD) are shown in histograms. It can be seen from the results that the polypeptide antibiotics KR (I ₃ , I ₇ ), KR (Q ₅ , D ₉ ) and KR (I ₇ , L ₁₁ ) of the present invention have higher serum stability than LL-37. The results are as follows As shown in Figure 9.

Example 7: Determination of anti-inflammatory activity of the polypeptide antibiotic prepared in Example 1

RAW264.7 cells were seeded in a 48-well plate at a density of ⁴ × 10 cells/well and incubated at 37 °C for 24 h. Cells were then treated with different concentrations of peptides in fresh DMEM and incubated for 2 h. Add 2 μL of LPS (10 μg/mL) and incubate for an additional 6 hours. After centrifugation at 1809 rpm for 10 min, the cell supernatants were collected, and the production levels of TNF-α and IL-6 were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits (Lianke, China) according to the manufacturer's instructions. Measurements were performed in three independent experiments, and standard errors (SD) are shown in the histograms. It can be seen from the results that the polypeptide antibiotic KR (Q ₅ , D ₉ ) of the present invention has higher anti-inflammatory activity than LL-37, and can more significantly reduce the production of TNF-α and IL-6. The results are shown in Figure 10 Show.

Example 8: Experiment on how the polypeptide antibiotic prepared in Example 1 promotes bacterial infection wound healing in mice

Animal experiments were approved by the Ethics Committee of Shanghai University (ECSHU). Adult female BALB/c mice (6–8 weeks, 15–20 g) were purchased from Jiangsu Huachuang Cigna Pharmaceutical Technology Co., Ltd. (Jiangsu, China) and housed in standard plastic rodent cages under artificially controlled conditions ( 25 ℃, humidity 50%-70%, 12 hours light/dark cycle). All mice were anesthetized with an intraperitoneal injection of 4% chloral hydrate at a dose of 10 mL/kg, and then shaved with a razor and rinsed with 75% ethanol. A full-thickness wound of 8 mm diameter was then prepared on the back of the mouse using a biopsy punch and infected with 20 μL E. coli suspension with a density of 2.5 × 10 ⁸ CFU/mL in pH 7.4 PBS. Infected wounds are covered with sterile gauze and secured with an elastic adhesive bandage. After 2 days of continuous infection, the mice were divided into 3 groups, with 10 mice in each group, treated with 20 μL PBS, 20 μL LL-37 solution (12.8 mg/mL, 100×MIC) and 20 μL KR (Q ₅ , D ₉ ) solution (0.8 mg/mL). ,100×MIC). The treatment was repeated on days 3, 7, and 10, and the wound area of the mice was recorded on days -2, 0, 3, 5, 7, 10, 12, and 14. It can be seen from the results that the polypeptide antibiotic KR (Q ₅ , D ₉ ) of the present invention promotes the healing of bacterially infected skin wounds in mice more significantly than LL-37. The results are shown in Figure 11.

The above examples show that the present invention successfully prepared a stapled peptide based on the linear template peptide Ac-KRIVQRIKDFLR-NH ₂ , and proved that the polypeptide antibiotic of the present invention can significantly inhibit the growth of Gram-negative bacteria and Gram-positive bacteria. Growth and reproduction, low hemolytic toxicity to red blood cells and cytotoxicity to embryonic fibroblasts, high serum stability, and certain anti-inflammatory activity. It can significantly promote the healing of bacterially infected skin wounds in mice and has good applications. prospect.

Claims (10)

1. An α-helical polypeptide antibiotic bound with all hydrocarbon side chains, characterized in that the polypeptide antibiotic is selected from one of the following: a) Using Ac-KRIVQRIKDFLR-NH ₂ as the peptide chain template, 3I and 7I are replaced by S5 and cyclized; b) Using Ac-KRIVQRIKDFLR-NH ₂ as the peptide chain template, 5Q and 9D are replaced by S5 and cyclized; c) Using Ac-KRIVQRIKDFLR-NH ₂ as the peptide chain template, 7I and 11L are replaced by S5 and cyclized. 2. The preparation method of α-helical polypeptide antibiotic according to claim 1, characterized in that it includes the following steps: Step 1): Use deprotection reagent to remove the Fmoc protecting group on Rink Amide MBHA resin; Step 2): Use a condensation reagent to couple the carboxyl group of the Fmoc-protected amino acid with the exposed amino group on the resin; Step 3): Use a deprotection reagent to remove the Fmoc protecting group on the amino acid; Step 4): Repeat the coupling-deprotection operations described in steps 2) and 3) to synthesize a peptide chain according to the amino acid sequence, wherein the amino acids at the i and i+4 cyclization sites are replaced with S5 respectively; Step 5): Use an acetylation reagent to modify the deprotected amino group of the last amino acid on the peptide chain; Step 6): Use a cyclization reagent to catalyze the olefin metathesis reaction of the S5 side chain at position i and i+4; Step 7): Use a cleavage reagent to cut the peptide chain from the resin, and use glacial ether to precipitate to obtain a crude polypeptide antibiotic; Step 8): Use high performance liquid chromatography to separate and purify the crude peptide antibiotic. 3. The preparation method according to claim 2, wherein the deprotecting reagent in step 1) is a solution in which piperidine and DMF are mixed in a volume ratio of 1:4, and its feeding ratio relative to the amino resin is 20: 1mL/mmol. 4. The preparation method according to claim 2, wherein the condensation reagent in step 2) is a mixed solution of amino acids, Oxyme, DIC and DMF, and its feeding ratio relative to the amino resin is 24:1mL/ mmol; among them, the ratio of amino acid, Oxyme, DIC, and DMF is 1mol:1mol:1mol:6mL. 5. The preparation method according to claim 2, wherein the acetylation reagent in step 5) is a solution in which acetic anhydride, DIEA and DMF are mixed in a volume ratio of 1:1:8, relative to the amino resin The feeding ratio is 20:1mL/mmol. 6. The preparation method according to claim 2, wherein the cyclization reagent in step 6) is a DCE solution of Grubbs I reagent with a concentration of 29mg/3mL, and its feeding ratio relative to the amino resin is 20: 1mL/mmol. 7. The preparation method according to claim 2, wherein the cutting reagent in step 7) is a mixed solution of TIPS, H ₂ O and TFA in a volume ratio of 2.5:2.5:95, relative to the amino resin. The feeding ratio is 20:1mL/mmol. 8. The use of the α-helical polypeptide antibiotic according to claim 1 in the preparation of drugs for inhibiting the growth and reproduction of Gram-positive and -negative bacteria. 9. The use of the α-helical polypeptide antibiotic according to claim 1 in the preparation of drugs for the treatment of infectious diseases caused by Gram-positive or/and Gram-negative bacteria. 10. Use of the α-helical polypeptide antibiotic according to claim 1 in the preparation of drugs for anti-inflammation and promoting wound healing.