Low-toxicity high-stability cyclic lipid antibacterial peptide and application thereof

Abstract

The invention discloses a low-toxicity high-stability cyclic lipid antibacterial peptide and application thereof. The antibacterial peptide is prepared by the following steps: firstly, designing an antibacterial peptide template Cn-D '-(M)-DY-cyclo [KXXWZZE], then replacing Cn in the antibacterial peptide template with octanoic acid, decanoic acid and dodecanoic acid, adding Dab or W between Dab and DY, replacing 2 and 3 positive charge amino acids in a ring with arginine or lysine analogues, and replacing 5 and 6 hydrophobic amino acids with DF or DL. Wherein X is equal to R, K, Dab and Orn; z = DW, DF and DL; n is equal to 8, 10 and 12; an in-vitro antibacterial experiment, a hemolysis experiment, a salt ion stability experiment and an enzymolysis stability experiment show that the antibacterial peptide has the advantages of broad-spectrum antibacterial activity, low toxicity and high stability. Induced drug resistance experiments show that the antibacterial peptide has the characteristic that drug resistance is not easy to generate. In-vivo acute toxicity experiments show that the antibacterial peptide has high in-vivo safety and has a good application prospect in preparation of clinical antibacterial drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and relates to a class of low-toxic and high-stability cyclic lipid antimicrobial peptides. The invention also relates to the application of the antimicrobial peptides in the preparation of clinical antimicrobial drugs. Background Art

[0002] Since the "golden age" of antibiotic discovery (1940-1960), the pace of research into antibiotics has slowed significantly, with only 30 new compounds entering clinical use in the past 20 years (Antibiotics in the clinical pipeline in 2015). Due to the lack of new treatments, Gram-negative bacteria have become increasingly resistant to current first-line antibiotics in recent years, and an antibiotic crisis is approaching. Most recently approved antibiotics are directed against Gram-positive bacteria, which are more complicated to treat. Gram-negative bacteria have two membranes, and the outer membrane (OM) contains lipopolysaccharide (LPS) molecules that prevent the entry of many antibiotics, so the discovery of new antibiotics to combat these pathogens is urgent.

[0003] Antimicrobial peptides, also known as host defense peptides, are an important component of the innate immune system. They have significant activity against bacteria, fungi, viruses and parasites (Medicinal Research Reviews 2019; 39: 831–859). The antibacterial mechanism of antimicrobial peptides is different from that of existing antibiotics. They are a class of small molecule peptides that inhibit microbial invasion and have immunomodulatory effects (Biochemical Pharmacology 133 (2017) 117–138). They are characterized by their small size (7-100 amino acids) and molecular weight generally less than 10kD. Most antimicrobial peptides have common characteristics, such as net positive charge and amphipathic structure. The positive charge carried by cationic antimicrobial peptides electrostatically interacts with the negatively charged bacterial surface structure and adsorbs together. The amphipathic structure of the peptide has obvious hydrophilic and hydrophobic regions, which allows the antimicrobial peptide to insert into the hydrophobic core of the bacterial cell membrane, destroying the integrity of the cell membrane and leading to cell death (Journal of Molecular Biology (2019) 431, 3547–3567). Due to its unique cell membrane breaking mechanism, which changes the permeability of bacterial membranes, bacteria are not likely to acquire resistance to antimicrobial peptides (International Journal of Molecular Sciences 2019, 20, 3954). Antimicrobial peptides act preferentially at the membrane level, which may destroy various biochemical processes of pathogens and activate the host's immune system. Moreover, antimicrobial peptides can not only be used alone to kill microorganisms, but can also be combined with antibiotics to achieve more effective synergistic effects (Expert Opinion on Biological Therapy 2017, 20, 1471-2598).

[0004] However, natural antimicrobial peptides have many disadvantages, such as low antibacterial activity, high toxicity to mammalian cells, and poor stability in salt ion and protease environments. Among them, antimicrobial peptides are easily degraded by various proteases and cannot exert biological activity in vivo, which is the main factor hindering the clinical application of AMPs. In the past few years, many chemical modifications have been proposed to reduce the hydrolysis of antimicrobial peptides on proteins (Acta Biomaterialia 153 (2022) 540–556). Jia et al. introduced halogens into Jelleine-I, and the halogenation treatment increased its in vitro antibacterial activity, anti-biofilm activity and in vivo antibacterial effect by 1-8 times, and the proteolytic stability was increased by 10-100 times (Peptides 112 (2019) 56–66). Qiu et al. used D-amino acids and non-natural amino acids instead of L-amino acids to synthesize antimicrobial peptides, making them resistant to proteolysis (Journal of. Peptide Science 2017; 23 (5): 392-402.). Main chain cyclization is often considered to be a method to impart stability to polypeptides. The cyclic antimicrobial peptide ZY4 designed by Mwangi et al. (Proc Natl Acad Sci US A. 2019; 116(52): 26516-26522.) increases antimicrobial activity and selectivity through disulfide bond cyclization and exhibits high stability in vivo. Lipidation and glycosylation are also an option to improve serum stability and activity and reduce rapid clearance through the kidneys and liver (Biochim Biophys Acta Biomembr. 2020; 1862(8): 183195.). Zhong et al. obtained a potent and highly stable antimicrobial drug by combining fatty acids at the amino termini of some D-amino acid-substituted analogs and dimerizing them (European Journal of Medicinal Chemistry 182(2019)111636).

[0005] Lipopeptides are a class of microbial secondary metabolites that have diverse biological effects. Some lipopeptides have been shown to have antibacterial, antiviral, antifungal, antitumor and immunomodulatory effects (BioMed Research International 2015: 473050.). Lipopeptides are composed of hydrophilic peptides and hydrophobic fatty acid acyl chains (Biochim Biophys Acta.

[0006] 2012;1818(3):566-573.). The number of amino acids is generally between 7 and 25, while the length of fatty acids is generally between 13 and 17 carbons. Currently, most lipopeptides are found in Bacillus and Paenibacillus (Medicinal Research Review. 2016;36(1):4-31.). Studies have shown that lipopeptides can effectively inhibit pathogenic strains (Antimicrob Agents Chemother. 2012;56(3):1458-1465.), and they have the ability to neutralize lipopolysaccharide (LPS) (Huang and Yousef 2014b). Some lipopeptide drugs have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA), including daptomycin and echinocandins (caspofungin, micafungin and anidulafungin) (Clin Ther.

[0007] 2009; 31(7): 1595-1603.). In addition, the synergistic effect of colistin (polymyxin E) in inhibiting multidrug-resistant (MDR) Acinetobacter baumannii has been reviewed (Infectious Disease Therapy. 2014; 3(2): 69-81.). Various studies on cyclolipopeptides have shown that this structure has great research potential.

[0008] Based on the above, the applicant has designed a seven-membered cyclic antimicrobial peptide template from scratch. However, although antimicrobial peptides (AMPs) have obvious potential as new antimicrobial drugs, AMPs have poor activity, cytotoxicity, poor stability and high large-scale production costs as antimicrobial drugs, which affect their clinical application (Acta Biomaterialia. 2014, Vol. 10 (No. 1): 244-257.). As mentioned above, existing studies have shown that strategies such as adjusting charge, changing hydrophobicity, replacing D-type amino acids, introducing non-natural amino acids, and lipidation (Scientific Reports. 2016: 27258) can effectively improve the antimicrobial activity and stability of antimicrobial peptides and reduce toxicity. Therefore, based on the template designed by the applicant, ideal cyclic lipid antimicrobial peptides can be obtained by transformation through different strategies. Summary of the invention

[0009] The purpose of the present invention is to provide a class of cyclic lipid antimicrobial peptides with low toxicity and high stability.

[0010] Another object of the present invention is to provide the use of the above antimicrobial peptide in the preparation of clinical antimicrobial drugs.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] (I) Design of a class of low-toxic and high-stability cyclic lipid antimicrobial peptides

[0013] The present invention provides a low-toxic and high-stability cyclic lipid antimicrobial peptide, which is based on the polymyxin B sequence as a framework to design an antimicrobial peptide template C n -D'-(M)- D Y-cyclo[KXXWZZE], and then the template was subjected to N-terminal fatty acid modification, tail chain amino acid replacement and ring amino acid replacement; its general structural formula is:

[0014] C8-D'- D Y--cyclo[KXXW D L D LE], recorded as: X4X5;

[0015] or: C n -D'- D Y--cyclo[KD'D'W D L D LE], recorded as: C n -Dab2;

[0016] Or: C8-D'- D Y--cyclo[KD'D'WZZE], denoted as: Dab2-Z2;

[0017] Or: C8-D'-M- D Y--cyclo[KD'D'W D L D LE], denoted as: M-Dab2;

[0018] or: C 10 -D'- D Y--cyclo[KD'D'WZZE], recorded as: C 10 -D2-Z2;

[0019] or: C 10 -D'-M- D Y--cyclo[KD'D'W D L D LE], recorded as: C 10 -M-Dab2;

[0020] Or: C8-D'-D'-W- D Y--cyclo[KD'D'WZZE], written as: DW-D2-Z2;

[0021] Where, X=R,K,D',Orn;D'=Dab;M=D',W;Z= D W, D F, D L; n = 8, 10, 12;

[0022] Specifically, the low-toxicity and high-stability cyclic lipid antimicrobial peptides described in the present invention have a structure

[0023] The structure is as follows:

[0024] C8-D'- D Y--cyclo[KXXW D L D LE], denoted as: X4X5; where X = R, K, D', Orn;

[0025] Or: C8-D'- D Y--cyclo[K D'D'W D L D LE], denoted as: Dab4Dab5;

[0026] or: C n -D'- D Y--cyclo[KD'D'W D L D LE], recorded as: C n -Dab2; wherein n=8, 10, 12;

[0027] or: C 10 -D'- D Y--cyclo[KD'D'W D L D LE], recorded as: C 10 -Dab2;

[0028] or: C 12 -D'- D Y--cyclo[KD'D'W D L D LE], recorded as: C 12 -Dab2;

[0029] Or: C8-D'- D Y--cyclo[KD'D'WZZE], denoted as: Dab2-Z2; where Z = D W, D F, D L;

[0030] Or: C8-D'- D Y--cyclo[KD'D'W D WD WE], recorded as: Dab2- D W2;

[0031] Or: C8-D'- D Y--cyclo[KD'D'W D F D FE], recorded as: Dab2- D F2;

[0032] Or: C8-D'-M- D Y--cyclo[KD'D'W D L D LE], denoted as: M-Dab2; where M = D', W;

[0033] Or: C8-D'-W- D Y--cyclo[KD'D'W D L D LE], denoted as: W-Dab2;

[0034] Or: C8-D'-D'- D Y--cyclo[KD'D'W D L D LE], denoted as: Dab-Dab2;

[0035] or: C 10 -D'- D Y--cyclo[KD'D'WZZE], recorded as: C 10 -D2-Z2; where Z= D W, D F, D L;

[0036] or: C 10 -D'- D Y--cyclo[KD'D'W D F D FE], denoted as: C 10 -D2- D F2;

[0037] or: C 10 -D'-M- D Y--cyclo[KD'D'W D L D LE], recorded as: C 10 -M-Dab2; wherein, M = D', W;

[0038] or: C 10 -D'-W- D Y--cyclo[KD'D'WD L D LE], recorded as: C 10 -W-Dab2;

[0039] or: C 10 -D'-D'- D Y--cyclo[KD'D'W D L D LE], recorded as: C 10 -Dab-Dab2;

[0040] Or: C8-D'-D'-W- D Y--cyclo[KD'D'WZZE], recorded as: DW-D2-Z2; where Z = D F, D L

[0041] Or: C8-D'-D'-W- D Y--cyclo[KD'D'W D F D FE], recorded as: DW-D2- D F2;

[0042] Or: C8-D'-D'-W- D Y--cyclo[KD'D'W D F D LE], recorded as: DW-D2- D F D L;

[0043] Or: C8-D'-D'-W- D Y--cyclo[KD'D'W D L D LE], recorded as: DW-D2- D L2;

[0044] Preferably, the low-toxic and high-stability cyclic lipid antimicrobial peptides of the present invention are Dab4Dab5, C 10 -Dab2, C 12 -Dab2, Dab2- D W2, Dab2- D F2, W-Dab2, Dab-Dab2, C 10 -D2- D F2, C 10 -W-Dab2, C 10 -Dab-Dab2, DW-D2- D F2, DW-D2- D F D L or DW-D2-D More preferably, the cyclic lipid antimicrobial peptide with low toxicity and high stability of the present invention is W-Dab2, Dab-Dab2, C 10 -Dab-Dab2 or DW-D2- D L2; Most preferably, the cyclic lipid antimicrobial peptides with low toxicity and high stability of the invention are Dab-Dab2 and DW-D2- D L2.

[0045] (II) Synthesis of low-toxic and high-stability cyclic lipid antimicrobial peptides

[0046] The low-toxicity and high-stability cyclic lipid antimicrobial peptide of the present invention is synthesized by using Rink-MBHA Resin as a raw material and a classical polypeptide Fmoc solid phase synthesis method. In the polypeptide synthesis process, HOBT / HBTU is used as a condensing agent for amino acid coupling, and a ninhydrin colorimetric method is used to detect secondary amines. The polypeptide connected with MBHA resin is obtained by coupling in sequence according to the polypeptide sequence. The cyclization method after the linear sequence synthesis is divided into two steps: ① the removal of the side chain protecting group: the Dde protecting group of the lysine side chain at position 1 in the ring is 2% hydrazine hydrate; the Alloc protecting group of glutamic acid at position 7 in the ring is phenylsilane, tetrakis(triphenylphosphine)palladium and dichloromethane; ② the amide bond cyclization method uses HOBT / TBTU as a condensing agent for reaction, and the polypeptide is obtained after cutting and HPLC purification.

[0047] (III) Application of low-toxic and high-stability cyclic lipid antimicrobial peptides

[0048] 1. In vitro antibacterial experiment

[0049] The minimum inhibitory concentration (MIC) of antimicrobial peptide analogs against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae was determined by the classic micro-serial two-fold dilution method. The antibiotic Polymyxin B was used as a positive control. The results are shown in Table 1.

[0050] Table 1 Minimum inhibitory concentration of the antimicrobial peptides of the present invention against common standard strains

[0051]

[0052]

[0053] The results in Table 1 show that polymyxin B has a strong inhibitory effect on Gram-negative bacteria. The cyclic lipid antimicrobial peptides designed in the present invention have strong antibacterial activity against common experimental strains, and have a strong inhibitory effect on both Gram-positive bacteria and Gram-negative bacteria, showing broad-spectrum antibacterial activity. Most of the analogs have slightly stronger inhibitory activity against Gram-positive bacteria than polymyxin B.

[0054] 2. Hemolytic activity test

[0055] In order to investigate the toxicity of the antimicrobial peptide synthesized in the present invention to normal mammalian cells, the applicant measured the hemolysis of the antimicrobial peptide after incubation with mouse red blood cells for 1 hour. Fig.14 , Fig.15 .

[0056] Fig.14 , Fig.15 The results show that the cyclic lipid antimicrobial peptide Dab2- D W2, Dab2- D F2, W-Dab2, Dab-Dab2, C 10 -Dab2, C 10 -Dab-Dab2, DW-D2- D F D L, DW-D2- D L2 all had low hemolytic toxicity, among which W-Dab2, Dab-Dab2 and DW-D2- D The hemolysis rate of L2 at the highest detection concentration (256 μM) was less than 10%, and the hemolysis rates of the other antimicrobial peptides at 256 μM were higher than 10%, but their hemolysis concentrations were much higher than the effective concentrations for exerting antimicrobial activity. In general, the antimicrobial peptide analogs synthesized in the present invention have low hemolytic toxicity and high safety.

[0057] 3. Salt ion stability experiment

[0058] Physiological salts interfere with the electrostatic interaction between antimicrobial peptides and bacterial membranes, thereby affecting the antimicrobial activity of antimicrobial peptides. In order to explore the effect of the type and amount of positive charge on the stability of antimicrobial peptide salt ions, the applicant determined the preferred antimicrobial peptide DW-D2- D MIC values ​​of L2, Dab-Dab2 and polymyxin B against Staphylococcus aureus and Escherichia coli in the presence of salt ion solutions with different concentrations.

[0059] Table 2 MIC of the antimicrobial peptides of the present invention against Staphylococcus aureus in salt ion solution

[0060]

[0061]

[0062] Table 3 MIC of the antimicrobial peptides of the present invention against Escherichia coli in salt ion solution

[0063]

[0064] As shown in Table 2 and Table 3, polymyxin B and the preferred antimicrobial peptide DW-D2- DL2 and Dab-Dab2 maintained good antibacterial activity against Staphylococcus aureus and Escherichia coli in salt ion solutions of different concentrations, with the MIC unchanged or increased by 1-4 times, and DW-D2- D The stability of L2 is slightly better than that of Dab-Dab2.

[0065] 4. Enzymatic stability

[0066] In order to evaluate the resistance of the preferred antimicrobial peptides of the present invention to enzymes, the antimicrobial peptides of the present invention were incubated with trypsin or chymotrypsin at different concentrations for 6 h, inactivated at 60°C for 15 min, and then the inhibitory ability of the antimicrobial peptides on the growth of Staphylococcus aureus and Escherichia coli was detected. The final concentration of the antimicrobial peptides was 2×MIC. The results are shown in Figure 16-19 .

[0067] Figure 16-19 The results showed that the control substance polymyxin B was hardly affected by trypsin and chymotrypsin; after incubation with trypsin for 6 hours, the antimicrobial peptide DW-D2- D L2 and Dab-Dab2 can still completely inhibit the growth of the two bacteria and have very high enzymatic stability; after incubation with chymotrypsin for 6 hours, the antimicrobial peptide Dab-Dab2 of the present invention maintains the inhibitory activity against the two bacteria at the highest test concentration, and the antimicrobial peptide DW-D2- D L2 lost its inhibitory ability against both bacteria after incubation with the highest concentration of 2000 μg / mL chymotrypsin, but maintained its ideal inhibitory ability after incubation with chymotrypsin at concentrations of 200 μg / mL and below. The chymotrypsin hydrolysis stability of Dab-Dab2 was higher than that of DW-D2- D L2.

[0068] 5. Induced resistance experiment

[0069] Compared with traditional antibiotics, the most prominent advantage of antimicrobial peptides is that they are difficult to develop drug resistance. This is due to their membrane dissolution mechanism, which makes it difficult for bacteria to change the cell membrane components and thus develop drug resistance. Therefore, in order to investigate whether the antimicrobial peptides of the present invention have the characteristic of being difficult to develop drug resistance, the preferred cyclic lipid antimicrobial peptide DW-D2- D The drug resistance induced by L2 and Dab-Dab2 after continuous action with Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 for 20 days was determined, with antibiotics Gentamycin and Polymyxin B as controls. The results are shown in Fig. 20 , Fig.21 .

[0070] Generally speaking, it is normal for MIC to fluctuate within the range of 1-4 times. If the MIC increases by more than 4 times, it indicates that antibiotic resistance has developed.

[0071] like Fig. 20 The results showed that the traditional antibiotic gentamicin had already caused drug resistance in Staphylococcus aureus ATCC 25923 after 6 days of continuous action, and the MIC of gentamicin increased by 4096 times after 20 days of continuous action; Fig.21 The results showed that the traditional antibiotic gentamicin had already produced drug resistance to Pseudomonas aeruginosa ATCC 27853 after 4 days of continuous action, and the MIC of gentamicin increased by 512 times after 20 days of continuous action. D After 20 days of continuous action, neither of the two bacteria developed resistance, and the MIC fluctuated only within a 1-4-fold range. Polymyxin B did not develop resistance after 20 days of continuous action with Staphylococcus aureus ATCC 25923, and the MIC increased 8 times after 20 days of continuous action with Pseudomonas aeruginosa ATCC27853. D L2, Dab-Dab2 and polypeptide antibiotic polymyxin B have obvious advantages in terms of drug resistance compared with traditional antibiotics.

[0072] 6. Acute toxicity test in mice

[0073] The experimental mice were BALB / C mice, male, 18-22 g, and fed according to the Lanzhou University Laboratory Animal Ethics Management Regulations.

[0074] Acute toxicity median lethal dose LD 50 Preliminary experiment: 3 mice in each group were injected intraperitoneally with cyclic lipid peptide DW-D2- D L2, Dab-Dab2, observe the mortality within 7 days, use Polymyxin B as a control, explore the minimum dose that causes 100% mortality and the maximum dose that causes 0% mortality, and determine the upper and lower limits of the dose.

[0075] Formal test: DW-D2- D LD of L2 50 The dose range is 30-130 mg / kg, and the LD of Dab-Dab2 50 The dosage range is 30-100 mg / kg, and the dosage range of the control drug Polymyxin B is 15-23.78 mg / kg. Therefore, the applicant has determined the dosage range of the control drug Polymyxin B according to DW-D2- DThe common ratio of the grouping of L2 was calculated to be 1.443, and it was divided into 5 groups, namely 30, 43.29, 62.47, 90.14, and 130 mg / kg. The common ratio of the grouping of Dab-Dab2 was 1.493, and it was divided into 4 groups, namely 30, 44.8, 66.9, and 100 mg / kg. The common ratio of the grouping of the control drug Polymyxin B was 1.259, and it was divided into 3 groups, namely 15, 18.89, and 23.78 mg / kg, and the subsequent formal experiments were carried out separately. According to the grouping, each group of mice was intraperitoneally injected with the corresponding dose of DW-D2- D L2, Dab-Dab2 or Polymyxin B, observe the mortality within 7 days and calculate LD 50 The results are shown in Table 4.

[0076] Table 4 LD of antimicrobial peptides and Polymyxin B in BALB / C mice 50 .

[0077] name <![CDATA[LD 50 (mg / kg)]]> <![CDATA[LD 50 Confidence interval (mg / kg)]]> <h2 style=";text-align:left;direction:ltr"><![CDATA[DW-D2- <h2 style=";text-align:left;direction:ltr"> D <h2 style=";text-align:left;direction:ltr"> L2]]><h2 style=";text-align:left;direction:ltr"> 62.52 48.128～81.283 <![CDATA[Dab-Dab2]]> 49.4 36.53～66.788 Polymyxin B 18.9 16.11～22.18

[0078] From the results in Table 4, it can be seen that the cyclic lipid antimicrobial peptide DW-D2- D The in vivo toxicity of L2 and Dab-Dab2 is 2.6 to 3.3 times lower than that of Polymyxin B. The two antimicrobial peptides have higher in vivo safety compared with Polymyxin B.

[0079] In summary, in vitro antibacterial experiments, hemolysis experiments, salt ion stability experiments, and enzymatic stability experiments show that the antimicrobial peptides of the present invention have the advantages of broad-spectrum antimicrobial activity, low toxicity, and high stability. Induced resistance experiments show that the antimicrobial peptides of the present invention are not easy to develop drug resistance. In vivo acute toxicity experiments show that the antimicrobial peptides of the present invention have high in vivo safety. Therefore, it has a good application prospect in the preparation of clinical antimicrobial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1-13 The mass spectrum of the cyclic lipid antimicrobial peptide of the present invention, wherein 1-13 correspond to the peptides: Dab4Dab5, C 10 -Dab2, C 12 -Dab2, Dab2- D W2, Dab2- D F2, W-Dab2, Dab-Dab2, C 10 -D2- D F2, C 10 -W-Dab2, C 10 -Dab-Dab2, DW-D2- D F2, DW-D2-D F D L and DW-D2- D L2;

[0081] Fig.14 The hemolytic activity results of the cyclic lipid antimicrobial peptide 1-7 of the present invention on mouse erythrocytes after incubation with erythrocytes for 1 hour;

[0082] Fig.15 The hemolytic activity results of the cyclic lipid antimicrobial peptide 8-13 of the present invention on mouse erythrocytes after incubation with erythrocytes for 1 hour;

[0083] Fig.16 The antibacterial activity of the cyclic lipid antimicrobial peptide of the present invention against Staphylococcus aureus after incubation with trypsin for 6 hours;

[0084] Fig.17 The antibacterial activity of the cyclic lipid antimicrobial peptide of the present invention against Escherichia coli after incubation with trypsin for 6 hours;

[0085] Fig.18 The antibacterial activity of the cyclic lipid antimicrobial peptide of the present invention against Staphylococcus aureus after incubation with chymotrypsin for 6 hours;

[0086] Fig.19 The antibacterial activity of the cyclic lipid antimicrobial peptide of the present invention against Escherichia coli after incubation with chymotrypsin for 6 hours;

[0087] Fig. 20 The antibacterial activity of the cyclic lipid antibiotic of the present invention against Staphylococcus aureus S.aureus ATCC 25923 for 20 consecutive days;

[0088] Fig.21 The antibacterial activity of the cyclic lipid antibiotic of the present invention against Pseudomonas aeruginosa ATCC 27853 was continuously exerted for 20 days. DETAILED DESCRIPTION

[0089] The following is a specific example of the low-toxicity and high-stability cyclic lipid antimicrobial peptide DW-D2- D L2, Dab-Dab2, Dab4Dab5, C 10 -Dab2, C 12 -Dab2, Dab2- D W2, Dab2- D F2, W-Dab2, C 10 -D2- D F2, C 10 -W-Dab2, C 10 -D-Dab2, DW-D2- D F2, DW-D2- D FD The synthesis of L is further described.

[0090] Example 1: Antimicrobial peptide DW-D2- D Synthesis of L2

[0091] (1) Activation of resin

[0092] Weigh 0.625 g of MBHA resin (0.48 mmol / g), put it at the bottom of the solid phase synthesizer, add DCM to swell for 30 min, drain, wash with DMF 3 times, drain, and identify the resin with ninhydrin colorimetry. If the resin is colorless, it can be used normally.

[0093] (2)Fmoc-DW-D2- D Synthesis of L2-MBHA

[0094] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0095] According to the above method, the following reactions are condensed in sequence: Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU and DIEA are the same as above, to obtain Fmoc-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0096] After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, a 3-fold excess (0.6 mmol) of CH3(CH2)6COOH was added to the synthesizer for condensation reaction for 1 hour, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C8-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp-D-Leu-D-Leu-Glu(Alloc)-resin.

[0097] (3) Removal of Dde protecting group

[0098] Add 2% hydrazine hydrate (2 μL / 100 Ml DMF) to the synthesizer, stir at room temperature for 3 min × 3 times, wash with DMF for 2 min × 3 times, and identify the resin by ninhydrin colorimetry. If the resin is dark purple, it indicates that the Dde protecting group is successfully removed, and C8-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin with the Dde protecting group removed is obtained.

[0099] (4) Removal of Alloc protecting group

[0100] First, the solvent system was replaced with DCM, and the product was washed with DCM for 2 min × 3 times. 58 mg of tetrakistriphenylphosphine palladium and 246 μL of phenylsilane were dissolved in DCM and added to the synthesizer. The product was stirred at room temperature for 3 h, and then washed with DCM for 2 min × 3 times and DMF for 2 min × 3 times to obtain AllocC8-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu-resin.

[0101] (5) Cyclization reaction

[0102] A 3-fold excess of condensing agents HOBT and TBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 6 hours under argon protection. After the reaction time expired, C8-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-cyclo-[Lys-Dab(Boc)-Dab(Boc)-Trp(Boc)-Trp-D-Leu-D-Leu-Glu] was obtained.

[0103] (6) Peptide cleavage

[0104] The obtained C8-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-cyclo-[Lys-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu] was washed 3 times with a DMF solution containing 20% ​​piperidine and then dried. After washing 3 times with DMF, the resin was dried and identified by the ninhydrin colorimetric method. If the resin was dark blue-purple, it indicated that the Fmoc protecting group had been removed. After the resin was washed 3 times with DCM and methanol alternately, it was pumped with a vacuum pump for 1 hour. After the resin was dried into dry particles, 10 mL of a cleavage reagent (TFA:Tris:water=9.5:0.25:0.25 (v:v:v)) was added to react for 3 hours, stirring slowly every 20 minutes. After the reaction time was reached, the cleavage reagent was collected, extracted with ether, and freeze-dried.

[0105] (7) Peptide purification

[0106] The freeze-dried peptide was purified by RP-HPLC. The RP-HPLC purification conditions were as follows: mobile phase A: 0.05% TFA / water, mobile phase B: 0.05% TFA / acetonitrile, linear gradient elution was used, the target peak effluent was collected, and freeze-dried to obtain the antimicrobial peptide DW-D2- D L2, its mass spectrum is as follows Figure 1 shown.

[0107] DW-D2- D The theoretical calculation result of the molecular weight of L2 is 1543.9027, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0108] Example 2: Synthesis of antimicrobial peptide Dab-Dab2

[0109] (1) Activation of resin

[0110] Same as Example 1.

[0111] (2) Synthesis of Fmoc-Dab-Dab2-MBHA

[0112] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0113] According to the above method, the following reactions are condensed in sequence: Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU and DIEA are the same as above, to obtain Fmoc-Dab(Boc)-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0114] After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, a 3-fold excess (0.6 mmol) of CH3(CH2)6COOH was added to the synthesizer for condensation reaction for 1 hour, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C8-Dab(Boc)-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0115] (3) Removal of Dde protecting group: Same as Example 1.

[0116] (4) Removal of Alloc protecting group: Same as Example 1.

[0117] (5) Cyclization reaction: Same as Example 1.

[0118] (6) Polypeptide cleavage: Same as Example 1.

[0119] (7) Peptide purification: The antimicrobial peptide Dab-Dab2 was obtained in the same manner as in Example 1. Its mass spectrum is shown in Figure 2 shown.

[0120] The theoretical calculation result of the molecular weight of Dab-Dab2 is 1357.8234, which is consistent with the result of mass spectrometry identification, proving that the structure of the antimicrobial peptide is correct.

[0121] Example 3: Synthesis of antimicrobial peptide Dab4Dab5

[0122] (1) Activation of resin

[0123] Same as Example 1.

[0124] (2) Synthesis of Fmoc-Dab4Dab5-MBHA

[0125] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0126] According to the above method, the following reactions were condensed in sequence: Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU, and DIEA were the same as above, to obtain Fmoc-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0127] After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, a 3-fold excess (0.6 mmol) of CH3(CH2)6COOH was added to the synthesizer for condensation reaction for 1 hour, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C8-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0128] (3) Removal of Dde protecting group: Same as Example 1.

[0129] (4) Removal of Alloc protecting group: Same as Example 1.

[0130] (5) Cyclization reaction: Same as Example 1.

[0131] (6) Polypeptide cleavage: Same as Example 1.

[0132] (7) Peptide purification: The antimicrobial peptide Dab4Dab5 was obtained in the same manner as in Example 1. Its mass spectrum is shown in Figure 3 shown.

[0133] The theoretical calculation result of the molecular weight of Dab4Dab5 is 1257.7598, which is consistent with the mass spectrometry identification result, proving that the structure of the antimicrobial peptide is correct.

[0134] Example 4: Antimicrobial Peptide C 10 -Synthesis of Dab2

[0135] (1) Activation of resin

[0136] Same as Example 1.

[0137] (2) Fmoc-C 10 Synthesis of -Dab2-MBHA

[0138] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0139] According to the above method, Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH are condensed in sequence, wherein the amounts of amino acid, HOBT, HBTU and DIEA are the same as above, to obtain

[0140] Fmoc-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-re sin.

[0141] After removing the terminal Fmoc protecting group with DMF solution containing 20% ​​piperidine, 3 times excess (0.6 mmol) CH3(CH2)8COOH was added to the synthesizer for condensation reaction for 1 h, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C 10 -Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0142] (3) Removal of Dde protecting group: Same as Example 1.

[0143] (4) Removal of Alloc protecting group: Same as Example 1.

[0144] (5) Cyclization reaction: Same as Example 1.

[0145] (6) Polypeptide cleavage: Same as Example 1.

[0146] (7) Peptide purification: As in Example 1, antimicrobial peptide C was obtained. 10 -Dab2. Its mass spectrum is as follows Figure 4 shown.

[0147] C 10 -The theoretical calculation result of the molecular weight of Dab2 is 1286.7911, which is consistent with the mass spectrometry identification result, proving that the structure of the antimicrobial peptide is correct.

[0148] Example 5: Antimicrobial Peptide C 12 -Synthesis of Dab2

[0149] (1) Activation of resin

[0150] Same as Example 1.

[0151] (2) Fmoc-C 12 Synthesis of -Dab2-MBHA

[0152] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0153] According to the above method, the following reactions were condensed in sequence: Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU, and DIEA were the same as above, to obtain Fmoc-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0154] After removing the terminal Fmoc protecting group with DMF solution containing 20% ​​piperidine, 3 times excess (0.6 mmol) of CH3(CH2) 10 COOH was added to the synthesizer for condensation reaction for 1 h, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C 12 -Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0155] (3) Removal of Dde protecting group: Same as Example 1.

[0156] (4) Removal of Alloc protecting group: Same as Example 1.

[0157] (5) Cyclization reaction: Same as Example 1.

[0158] (6) Polypeptide cleavage: Same as Example 1.

[0159] (7) Peptide purification: As in Example 1, antimicrobial peptide C was obtained. 12 -Dab2. Its mass spectrum is as follows Figure 5 shown.

[0160] C 12 -The theoretical calculation result of the molecular weight of Dab2 is 1313.8224, which is consistent with the mass spectrometry identification result, proving that the structure of the antimicrobial peptide is correct.

[0161] Example 6: Antimicrobial peptide Dab2- D Synthesis of W2

[0162] (1) Activation of resin

[0163] Same as Example 1.

[0164] (2) Fmoc-Dab2-D Synthesis of W2-MBHA

[0165] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0166] According to the above method, the following condensations are performed in sequence: Fmoc-D-Trp(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU and DIEA are the same as above, to obtain Fmoc-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Trp(Boc)-D-Trp(Boc)-Glu(Alloc)-resin.

[0167] After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, a 3-fold excess (0.6 mmol) of CH3(CH2)6COOH was added to the synthesizer for condensation reaction for 1 hour, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C8-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Trp(Boc)-D-Trp(Boc)-Glu(Alloc)-resin.

[0168] (3) Removal of Dde protecting group: Same as Example 1.

[0169] (4) Removal of Alloc protecting group: Same as Example 1.

[0170] (5) Cyclization reaction: Same as Example 1.

[0171] (6) Polypeptide cleavage: Same as Example 1.

[0172] (7) Peptide purification: The antimicrobial peptide Dab2- DW2. Its mass spectrum is as follows Figure 6 shown.

[0173] Dab2- D The theoretical calculation result of the molecular weight of W2 is 1403.7503, which is consistent with the mass spectrometry identification result, proving that the structure of the antimicrobial peptide is correct.

[0174] Example 7: Antimicrobial peptide Dab2- D Synthesis of F2

[0175] (1) Activation of resin

[0176] Same as Example 1.

[0177] (2) Fmoc-Dab2- D Synthesis of F2-MBHA

[0178] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0179] According to the above method, the following reactions were condensed in sequence: Fmoc-D-Phe-OH, Fmoc-D-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU, and DIEA were the same as above, to obtain Fmoc-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Phe-D-Phe-Glu(Alloc)-resin.

[0180] After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, a 3-fold excess (0.6 mmol) of CH3(CH2)6COOH was added to the synthesizer for condensation reaction for 1 hour, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C8-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Phe-D-Phe-Glu(Alloc)-resin.

[0181] (3) Removal of Dde protecting group: Same as Example 1.

[0182] (4) Removal of Alloc protecting group: Same as Example 1.

[0183] (5) Cyclization reaction: Same as Example 1.

[0184] (6) Polypeptide cleavage: Same as Example 1.

[0185] (7) Peptide purification: The antimicrobial peptide Dab2- D F2. Its mass spectrum is as follows Figure 7 shown.

[0186] Dab2- D The theoretical calculation result of the molecular weight of F2 is 1325.7285, which is consistent with the result of mass spectrometry identification, proving that the structure of the antimicrobial peptide is correct.

[0187] Example 8: Synthesis of antimicrobial peptide W-Dab2

[0188] (1) Activation of resin

[0189] Same as Example 1.

[0190] (2) Synthesis of Fmoc-W-Dab2-MBHA

[0191] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0192] According to the above method, the following reactions are condensed in sequence: Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU, and DIEA are the same as above, to obtain Fmoc-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0193] After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, a 3-fold excess (0.6 mmol) of CH3(CH2)6COOH was added to the synthesizer for condensation reaction for 1 hour, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C8-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0194] (3) Removal of Dde protecting group: Same as Example 1.

[0195] (4) Removal of Alloc protecting group: Same as Example 1.

[0196] (5) Cyclization reaction: Same as Example 1.

[0197] (6) Polypeptide cleavage: Same as Example 1.

[0198] (7) Peptide purification: The antimicrobial peptide W-Dab2 was obtained in the same manner as in Example 1. Its mass spectrum is shown in Figure 8 shown.

[0199] The theoretical calculation result of the molecular weight of W-Dab2 is 1443.8391, which is consistent with the result of mass spectrometry identification, proving that the structure of the antimicrobial peptide is correct.

[0200] Example 9: Antimicrobial Peptide C 10 -D2- D Synthesis of F2

[0201] (1) Activation of resin

[0202] Same as Example 1.

[0203] (2) Fmoc-C 10-D2- D Synthesis of F2-MBHA

[0204] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0205] According to the above method, the following reactions were condensed in sequence: Fmoc-D-Phe-OH, Fmoc-D-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU, and DIEA were the same as above, to obtain Fmoc-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Phe-D-Phe-Glu(Alloc)-resin.

[0206] After removing the terminal Fmoc protecting group with DMF solution containing 20% ​​piperidine, 3 times excess (0.6 mmol) CH3(CH2)8COOH was added to the synthesizer for condensation reaction for 1 h, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C 10 -Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Phe-D-Phe-Glu(Alloc)-resin.

[0207] (3) Removal of Dde protecting group: Same as Example 1.

[0208] (4) Removal of Alloc protecting group: Same as Example 1.

[0209] (5) Cyclization reaction: Same as Example 1.

[0210] (6) Polypeptide cleavage: Same as Example 1.

[0211] (7) Peptide purification: As in Example 1, antimicrobial peptide C was obtained. 10 -D2- DF2. Its mass spectrum is as follows Fig. 9 shown.

[0212] C 10 -D2- D The theoretical calculation result of the molecular weight of F2 is 1353.7598, which is consistent with the mass spectrometry identification result, proving that the structure of the antimicrobial peptide is correct.

[0213] Example 10: Antimicrobial Peptide C 10 Synthesis of -W-Dab2

[0214] (1) Activation of resin

[0215] Same as Example 1.

[0216] (2) Fmoc-C 10 Synthesis of -W-Dab2-MBHA

[0217] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0218] According to the above method, the following reactions are condensed in sequence: Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU, and DIEA are the same as above, to obtain Fmoc-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0219] After removing the terminal Fmoc protecting group with DMF solution containing 20% ​​piperidine, 3 times excess (0.6 mmol) CH3(CH2)8COOH was added to the synthesizer for condensation reaction for 1 h, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C 10-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0220] (3) Removal of Dde protecting group: Same as Example 1.

[0221] (4) Removal of Alloc protecting group: Same as Example 1.

[0222] (5) Cyclization reaction: Same as Example 1.

[0223] (6) Polypeptide cleavage: Same as Example 1.

[0224] (7) Peptide purification: As in Example 1, antimicrobial peptide C was obtained. 10 -W-Dab2. Its mass spectrum is as follows Fig.10 shown.

[0225] C 10 The theoretical calculation result of the molecular weight of -W-Dab2 is 1471.8704, which is consistent with the mass spectrometry identification result, proving that the structure of the antimicrobial peptide is correct.

[0226] Example 11: Antimicrobial Peptide C 10 Synthesis of -D-Dab2

[0227] (1) Activation of resin

[0228] Same as Example 1.

[0229] (2) Fmoc-C 10 Synthesis of -D-Dab2-MBHA

[0230] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0231] According to the above method, the following reactions are condensed in sequence: Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU and DIEA are the same as above, to obtain Fmoc-Dab(Boc)-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0232] After removing the terminal Fmoc protecting group with DMF solution containing 20% ​​piperidine, 3 times excess (0.6 mmol) CH3(CH2)8COOH was added to the synthesizer for condensation reaction for 1 h, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C 10 -Dab(Boc)-Dab(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Leu-D-Leu-Glu(Alloc)-resin.

[0233] (3) Removal of Dde protecting group: Same as Example 1.

[0234] (4) Removal of Alloc protecting group: Same as Example 1.

[0235] (5) Cyclization reaction: Same as Example 1.

[0236] (6) Polypeptide cleavage: Same as Example 1.

[0237] (7) Peptide purification: As in Example 1, antimicrobial peptide C was obtained. 10 -D-Dab2. Its mass spectrum is as follows Fig.11 shown.

[0238] C 10 The theoretical calculation result of the molecular weight of -D-Dab2 is 1385.8755, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0239] Example 12: Antimicrobial peptide DW-D2- D Synthesis of F2

[0240] (1) Activation of resin

[0241] Same as Example 1.

[0242] (2)Fmoc-DW-D2- D Synthesis of F2-MBHA

[0243] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0244] According to the above method, the following reactions are condensed in sequence: Fmoc-D-Phe-OH, Fmoc-D-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU and DIEA are the same as above, to obtain Fmoc-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Phe-D-Phe-Glu(Alloc)-resin.

[0245] After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, a 3-fold excess (0.6 mmol) of CH3(CH2)6COOH was added to the synthesizer for condensation reaction for 1 hour, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C8-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Phe-D-Phe-Glu(Alloc)-resin.

[0246] (3) Removal of Dde protecting group: Same as Example 1.

[0247] (4) Removal of Alloc protecting group: Same as Example 1.

[0248] (5) Cyclization reaction: Same as Example 1.

[0249] (6) Polypeptide cleavage: Same as Example 1.

[0250] (7) Peptide purification: As in Example 1, the antimicrobial peptide DW-D2- D F2. Its mass spectrum is as follows Fig.12 shown.

[0251] DW-D2- D The theoretical calculation result of the molecular weight of F2 is 1611.8714, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0252] Example 13: Antimicrobial peptide DW-D2- D F D Synthesis of L

[0253] (1) Activation of resin: Same as Example 1.

[0254] (2)Fmoc-DW-D2- D F D Synthesis of L-MBHA

[0255] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, and then drained. After washing with DMF three times, the resin was drained and identified by the ninhydrin colorimetric method. If it was dark blue-purple, it indicated that the Fmoc protecting group had been removed. A 3-fold excess of Glu, HOBT, HBTU and a 6-fold excess of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred at room temperature for 1 hour under argon protection. After the reaction time was reached, the resin was identified by the ninhydrin colorimetric method. If the resin was colorless and transparent, it indicated that the Glu condensation was successful, and Fmoc-Glu(Alloc)-MBHA was obtained.

[0256] According to the above method, the following reactions are condensed in sequence: Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dab(Boc)-OH, and Fmoc-Dab(Boc)-OH, wherein the amounts of amino acid, HOBT, HBTU and DIEA are the same as above, to obtain Fmoc-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Phe-D-Leu-Glu(Alloc)-resin.

[0257] After removing the terminal Fmoc protecting group with a DMF solution containing 20% ​​piperidine, a 3-fold excess (0.6 mmol) of CH3(CH2)6COOH was added to the synthesizer for condensation reaction for 1 hour, wherein the amounts of HBTU, HOBT and DIEA were the same as above, to obtain C8-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Thr(tBu)-Lys(Dde)-Dab(Boc)-Dab(Boc)-Trp(Boc)-D-Phe-D-Leu-Glu(Alloc)-resin.

[0258] (3) Removal of Dde protecting group: Same as Example 1.

[0259] (4) Removal of Alloc protecting group: Same as Example 1.

[0260] (5) Cyclization reaction: Same as Example 1.

[0261] (6) Polypeptide cleavage: Same as Example 1.

[0262] (7) Peptide purification: The same as in Example 1, the antimicrobial peptide DW-D2- D F D L. Its mass spectrum is as follows Fig.13 shown.

[0263] DW-D2- D F D The theoretical calculation result of the molecular weight of L is 1577.8871, which is consistent with the result of mass spectrometry identification, proving that the structure of the antimicrobial peptide is correct.

Claims

1. A low-toxic and high-stability cyclic lipid antimicrobial peptide, characterized in that: The cyclic lipid antimicrobial peptide is first designed as an antimicrobial peptide template C n -D'-(M)- D Y-cyclo[KXXWZZE], and then the template was subjected to N-terminal fatty acid modification, tail chain amino acid replacement and ring amino acid replacement; its general structural formula is: C8-D'- D Y--cyclo[KXXW D L D LE], recorded as: X4X5; or: C n -D'- D Y--cyclo[KD'D'W D L D LE], recorded as: C n -Dab2; Or: C8-D'- D Y--cyclo[KD'D'WZZE], denoted as: Dab2-Z2; Or: C8-D'-M- D Y--cyclo[KD'D'W D L D LE], denoted as: M-Dab2; or: C 10 -D'- D Y--cyclo[KD'D'WZZE], recorded as: C 10 -D2-Z2; or: C 10 -D'-M- D Y--cyclo[KD'D'W D L D LE], recorded as: C 10 -M-Dab2; Or: C8-D'-D'-W- D Y--cyclo[KD'D'WZZE], written as: DW-D2-Z2; Where, X=R,K,D',Orn;D'=Dab;M=D',W;Z= D W, D F, D L; n = 8, 10, 12; The N-terminal fatty acid modification refers to the addition of C n Replaced with octanoic acid, decanoic acid and dodecanoic acid to adjust its hydrophobicity; the tail chain amino acid replacement refers to the replacement of Dab and D Dab or W is added between Y; the amino acid replacement in the ring refers to the replacement of the positively charged amino acids at positions 2 and 3 in the ring by arginine or lysine analogs, and the hydrophobic amino acids at positions 5 and 6 by D F or D L replacement.

2. A low-toxicity and high-stability cyclic lipid antimicrobial peptide according to claim 1, characterized in that: The cyclic lipid antimicrobial peptides are Dab4Dab5, C 10 -Dab2, C 12 -Dab2, Dab2- D W2, Dab2- D F2, W-Dab2, Dab-Dab2, C 10 -D2- D F2, C 10 -W-Dab2, C 10 -D-Dab2, DW-D2- D F2, DW-D2- D F D L or DW-D2- D L2.

3. A low-toxicity and high-stability cyclic lipid antimicrobial peptide according to claim 2, characterized in that: The low-toxicity, high-stability and broad-spectrum antimicrobial peptides are W-Dab2, Dab-Dab2, C 10 -D-Dab2 or DW-D2- D L2.

4. A low-toxicity and high-stability cyclic lipid antimicrobial peptide according to claim 3, characterized in that: The low-toxicity, high-stability, broad-spectrum antimicrobial peptide is DW-D2- D L2 or Dab-Dab2.

5. Use of a low-toxic and high-stability cyclic lipid antimicrobial peptide as described in any one of claims 1 to 4 in the preparation of clinical antimicrobial drugs.

6. The use of a low-toxic and high-stability cyclic lipid antimicrobial peptide according to claim 5 in the preparation of clinical antimicrobial drugs, characterized in that: The bacteria inhibited by the antibacterial drug are Gram-positive bacteria or Gram-negative bacteria.

7. The use of a low-toxicity and high-stability cyclic lipid antimicrobial peptide according to claim 6 in the preparation of clinical antimicrobial drugs, characterized in that: The Gram-positive bacteria are Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis or Enterococcus faecalis, and the Gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Acinetobacter baumannii.

8. The use of a low-toxic and high-stability cyclic lipid antimicrobial peptide according to claim 5 in the preparation of clinical antimicrobial drugs, characterized in that: The cyclic lipid antimicrobial peptide is used in combination with Gentamycin to reduce the generation of Gentamycin resistance.

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