Derivative lipopeptides based on lipocyclic peptide laterocidine synthesis and applications thereof

Abstract

This invention belongs to the field of biochemistry, specifically relating to a derived lipopeptide synthesized from the lipocyclic peptide Laterocidine. This derived lipopeptide is obtained by replacing the amino acid at position 1, 6, or 9 of the linear Laterocidine peptide chain obtained after ring-opening of Laterocidine. The amino acids replacing positions 1 and 6 are positively charged, while the amino acid replacing position 9 carries a phenyl, thiol, or hydroxyphenyl group. The derived lipopeptide of this invention exhibits good antibacterial effects against Gram-negative bacteria, low cytotoxicity, and excellent stability in salt ion solutions. It can be used in antibacterial drugs or compositions against Gram-negative bacteria, and also in feed additives, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a derived lipopeptide synthesized based on the lipocyclic peptide Laterocidine and its applications. Background Technology

[0002] Antimicrobial resistance has become a global public health problem, and curbing it is urgent. In recent years, the rapid spread of Gram-negative bacteria in human and animal clinical practice has led to a dwindling supply of clinically available antibiotics. The emergence of multidrug-resistant Gram-negative bacteria, in particular, has resulted in a near-complete lack of effective treatments, causing a series of food safety and public health security issues.

[0003] Lipocyclic peptides, a class of polypeptide bioactive substances widely found in the biological world, are core defense molecules of the innate immune system. Their unique antibacterial mechanisms enable them to exhibit significant inhibitory effects not only against common pathogens but also remarkable bactericidal activity against multidrug-resistant strains. Of particular note is that lipocyclic peptides achieve their bactericidal activity through membrane permeation, making them extremely difficult to induce bacterial resistance. Laterocidine, due to its unique cyclic structure, exhibits highly efficient activity against Gram-negative bacteria and has become a key research focus. However, in-depth research has revealed significant limitations in the application of natural laterocidine: its cyclic structure is easily hydrolyzed and its significant salt ion sensitivity limits its application value. Therefore, developing laterocidine-derived lipopeptides with low hemolytic activity and high stability using structural modification strategies is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a derived lipopeptide based on the synthesis of the lipocyclic peptide Laterocidine, so as to solve the problem of poor in vivo and in vitro stability of the natural lipocyclic peptide Laterocidine.

[0005] To achieve the above objectives, the present invention provides a derivative lipopeptide synthesized based on the lipocyclic peptide Laterocidine. The derivative lipopeptide is obtained by replacing the amino acid at position 1, position 6, or position 9 in the linear Laterocidine peptide chain obtained after ring opening of the lipocyclic peptide Laterocidine. The amino acids replacing the amino acids at positions 1 and 6 are positively charged, and the amino acid replacing the amino acid at position 9 is phenyl, mercapto, or hydroxyphenyl.

[0006] The working principle and beneficial effects of this scheme are as follows: the linear laterocidine peptide chain obtained after ring opening of the lipocyclic peptide laterocidine is C9H. 17The derived lipopeptides, O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly, obtained by replacing the negatively charged amino acids in the linear latexidine peptide chain with positively charged amino acids, exhibit superior antibacterial activity compared to linear latexidine. The derived lipopeptide obtained by replacing glycine at position 6 with 2,4-diaminobutyric acid (2–8 μg / mL) showed the best antibacterial effect. Furthermore, the hemolytic activity of the derived lipopeptides was not significantly increased compared to the natural lipocyclic peptide latexidine, indicating lower cytotoxicity and broad application prospects. In addition, the derived lipopeptides showed good stability in ionic salt solutions, with the derived lipopeptide obtained by replacing glycine at position 6 with 2,4-diaminobutyric acid exhibiting the best stability in ionic salt solutions.

[0007] Optionally, the structure of the derived lipopeptide is as follows: C9H 17 O-(D-Orn)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Dab)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Arg)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Lys)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Orn-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Dab-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Arg-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Lys-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Phe-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Cys-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Tyr-Ile-Asn-Gly-Gly.

[0008] This invention also provides the application of the derived lipopeptides synthesized from the lipocyclic peptide laterocidine described above in the preparation of antibacterial drugs or antibacterial compositions. Based on the good antibacterial effect of the derived lipopeptides in this method, they can be used as antibacterial drugs.

[0009] Alternatively, the bacteria may be Gram-negative.

[0010] Optionally, Gram-negative bacteria include Escherichia coli, Salmonella, and Pseudomonas aeruginosa.

[0011] The present invention also provides the application of the above-mentioned derivative lipopeptides based on the synthesis of the lipocyclic peptide Laterocidine in the preparation of feed additives. Attached Figure Description

[0012] Figure 1 The above are the RP-HPLC and mass spectrometry analysis results of linear Laterocidine in Example 1 of this invention. Figure 2 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 1 in Example 2 of this invention. Figure 3 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 2 in Example 3 of this invention. Figure 4 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 3 in Example 4 of this invention. Figure 5 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 4 in Example 5 of this invention. Figure 6 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 5 in Example 6 of this invention. Figure 7 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 6 in Example 7 of this invention. Figure 8 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 7 in Example 8 of this invention. Figure 9 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 8 in Example 9 of this invention. Figure 10 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 9 in Example 10 of this invention. Figure 11 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 10 in Example 11 of this invention. Figure 12 The above are the RP-HPLC and mass spectrometry analysis results of the derived lipopeptide 11 in Example 12 of this invention. Figure 13 This is a graph showing the hemolytic evaluation results of the derived lipopeptide in Experimental Example 2 of this invention. Detailed Implementation

[0013] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description. The following description is intended to disclose the present invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and those skilled in the art can conceive of other obvious modifications. The basic principles of the invention defined in the following description can be applied to other embodiments, modifications, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the invention. In this invention, unless otherwise specified, the raw materials and equipment used are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0014] The experimental materials used in this invention: The natural lipocyclic peptide Laterocidine was isolated and purified from Bacillus fermentation broth in our laboratory (referring to the preparation method described in Chinese Invention Patent Application No. 2024102352564, entitled "A Bacillus belyssus and its Production Method and Application in the Preparation of Anti-Enterobacterial Tridecapeptide"), with a purity >95%. The amino acids and fatty acids required for the synthesis of the derived lipopeptide, trypsin, pepsin, Triton X-100, etc., were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Other conventional reagents were either imported and repackaged or domestically produced analytical grade. The sources of the antibacterial experimental strains are shown in Table 1.

[0015] Table 1. Strains and their sources ; Example 1 Synthesis of Linear Laterocidine Weigh 3.0 g of Wang Resin resin and place it in a peptide synthesis tube. Add 10 mL of N,N-dimethylformamide (DMF) to swell for 30 min. After the resin swelling is complete, filter to remove the DMF from the peptide synthesis tube. Add 10 mL of 20% (v / v) piperidine solution (prepared with DMF) to remove the Fmoc protecting groups of the amino groups on the resin. Calculate the molar amount of active groups on the resin based on the resin loading and weight. Weigh 3.0 g of Fmoc-Gly(Boc)-OH and 3.0 g of 1-hydroxybenzotriazole (HOBt) into a dry beaker, add 10 mL of DMF to dissolve, then transfer 20 mL of N,N'-diisopropylcarbodiimide (DIC) into a beaker, activate, and then transfer to the peptide synthesis tube. Couple the amino acids at room temperature for 2 h. Based on the linear laterocidine peptide chain C9H... 17The O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly sequence continues to extend the peptide chain from the C-terminus to the N-terminus, with isononanoic acid as the final component coupled to the N-terminal D-serine (D-Ser). The resin was washed alternately with 10 mL of DMF and 10 mL of anhydrous methanol, then vacuum filtered and dried. The resin was then transferred to a round-bottom flask, and 50 mL of cleavage reagent (trifluoroacetic acid (TFA):triisopropylsilane (Tis):water in a volume ratio of 95:2.5:2.5) was added. The mixture was magnetically stirred at room temperature in the dark for 3–4 h to complete the cleavage process. The peptide solution in the round-bottom flask was filtered into an Erlenmeyer flask using an hourglass. 20 mL of ice-cold diethyl ether was added, and the mixture was allowed to stand at -20°C for 24 h to precipitate the peptide. The precipitate was then transferred to a centrifuge tube, centrifuged to collect the precipitate, and washed three times thoroughly with 20 mL of diethyl ether. Distilled water was added to dissolve the precipitate, and the mixture was freeze-dried to obtain the crude peptide product. The crude peptide product was purified using preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain a pure peptide, which was linear latexidine. Purity was determined by HPLC, and molecular weight was identified by mass spectrometry. The RP-HPLC and mass spectrometry chromatograms of linear latexidine are shown below. Figure 1 .

[0016] Example 2 Synthesis of Derivative Lipopeptide 1 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the D-serine (D-Ser) at position 1 in the linear latexidine peptide chain was replaced with D-ornithine (D-Orn). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Orn)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 1, has the RP-HPLC and mass spectrometry chromatograms of Derived Lipopeptide 1 as shown in [reference needed]. Figure 2 .

[0017] Example 3 Synthesis of Derivative Lipopeptide 2 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the D-serine residue (D-Ser) at position 1 in the linear latexidine peptide chain was replaced with D-2,4-diaminobutyric acid (D-Dab). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Dab)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 2, has RP-HPLC and mass spectrometry chromatograms for Derived Lipopeptide 2 as shown in [reference needed]. Figure 3 .

[0018] Example 4 Synthesis of Derivative Lipopeptide 3 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the D-serine (D-Ser) at position 1 in the linear latexidine peptide chain was replaced with D-arginine (D-Arg). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Arg)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 3, has the RP-HPLC and mass spectrometry chromatograms of Derived Lipopeptide 3 as shown in [reference needed]. Figure 4 .

[0019] Example 5 Synthesis of Derived Lipopeptide 4 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the D-serine (D-Ser) at position 1 in the linear latexidine peptide chain was replaced with a D-lysine (D-Lys). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Lys)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 4, has the RP-HPLC and mass spectrometry chromatograms of Derived Lipopeptide 4 shown in [reference needed]. Figure 5 .

[0020] Example 6 Synthesis of Derived Lipopeptide 5 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the glycine (Gly) at position 6 of the linear latexidine peptide chain was replaced with ornithine (Orn). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Orn-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 5, has RP-HPLC and mass spectrometry chromatograms for Derived Lipopeptide 5 as shown in [reference needed]. Figure 6 .

[0021] Example 7 Synthesis of Derived Lipopeptide 6 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the glycine (Gly) at position 6 in the linear latexidine peptide chain was replaced with 2,4-diaminobutyric acid (Dab). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Dab-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 6, has the RP-HPLC and mass spectrometry chromatograms of Derived Lipopeptide 6 shown in [reference needed]. Figure 7 .

[0022] Example 8 Synthesis of Derivative Lipopeptide 7 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the glycine (Gly) at position 6 of the linear latexidine peptide chain was replaced with arginine (Arg). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Arg-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 7, has RP-HPLC and mass spectrometry chromatograms for Derived Lipopeptide 7 as shown in [reference needed]. Figure 8 .

[0023] Example 9 Synthesis of Derivative Lipopeptide 8 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the glycine (Gly) at position 6 of the linear latexidine peptide chain was replaced with lysine (Lys). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Lys-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 8, has RP-HPLC and mass spectrometry chromatograms for Derived Lipopeptide 8 as shown in [reference needed]. Figure 9 .

[0024] Example 10 Synthesis of Derivative Lipopeptide 9 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the threonine (Thr) at position 9 in the linear latexidine peptide chain was replaced with phenylalanine (Phe). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Phe-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 9, has RP-HPLC and mass spectrometry chromatograms for Derived Lipopeptide 9 as shown in [reference needed]. Figure 10 .

[0025] Example 11 Synthesis of Derivative Lipopeptide 10 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the threonine (Thr) at position 9 in the linear latexidine peptide chain was replaced with cysteine ​​(Cys). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Cys-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 10, has RP-HPLC and mass spectrometry chromatograms for Derived Lipopeptide 10 as shown in [reference needed]. Figure 11 .

[0026] Example 12 Synthesis of Derivative Lipopeptide 11 In this embodiment, the derived lipopeptide was synthesized according to the method described in Example 1, except that the 9th threonine (Thr) in the linear latexidine peptide chain was replaced with a tyrosine (Tyr). That is, the peptide chain of the derived lipopeptide in this embodiment is C9H. 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Tyr-Ile-Asn-Gly-Gly, denoted as Derived Lipopeptide 11, has the RP-HPLC and mass spectrometry chromatograms of Derived Lipopeptide 11 shown below. Figure 12 .

[0027] Depend on Figures 1-12 It can be seen that the calculated molecular weight and mass spectrometry identification results of the linear Laterocidine synthesized in Examples 1-12 and the derived lipopeptides 1-11 are consistent, proving that the lipopeptide structure is correct. After purification, the purity of all of them is >98%.

[0028] Example 1: Evaluation of the antibacterial activity of derived lipopeptides A broth-based microdilution method was used, and the procedure was performed according to the CLSI Guidelines for Antimicrobial Susceptibility Testing. *Escherichia coli* CVCC 1515, *Escherichia coli* CVCC 1557, *Escherichia coli* CVCC 1569, *Salmonella pullorum* CVCC 534, *Salmonella choleraesuis* CVCC 79500, and *Pseudomonas aeruginosa* ATCC 27853 were cultured in broth and grown to the logarithmic growth phase. The bacterial suspensions were then diluted with broth to a concentration of 10. 5 ~10 6CFU / mL. Natural lipocyclic peptides Laterocidine, linear Laterocidine, and derived lipopeptide 1-11 were dissolved separately in sterile broth to obtain a stock solution with a concentration of 512 μg / mL. Test concentrations were prepared by two-fold dilution. 50 μL of each of the sequentially two-fold diluted natural lipocyclic peptides Laterocidine, linear Laterocidine, and derived lipopeptide solutions, along with 50 μL of indicator bacterial dilution, were added to sterile 96-well plates, with three replicates for each concentration. The plates were incubated at 37°C for 18 h, and the clarity of the solution was observed visually. If indicator bacteria grew, the solution would become turbid. The concentrations of Laterocidine, linear Laterocidine, and derived lipopeptide corresponding to the clear wells at the boundary between turbidity and clarity were defined as the minimum inhibitory concentrations (MICs). Sterile MH broth was used as a negative control, and polymyxin was used as a positive control. The results are shown in Table 2.

[0029] Table 2. MIC values ​​of natural lipocyclic peptides Laterocidine, linear Laterocidine, and derived lipopeptides ; Table 2 shows that polymyxins exhibit significant antibacterial activity against Gram-negative bacteria, with MIC values ​​ranging from 2 to 4 μg / mL. The natural lipocyclic peptide laterocidine has an MIC of 4 μg / mL against Gram-negative bacteria, showing activity comparable to polymyxins. However, this cyclic structure is easily hydrolyzed and opens to form linear laterocidine, leading to a significant decrease in its antibacterial activity (MIC range increases to 2–16 μg / mL), indicating that the cyclic structure is crucial for maintaining its antibacterial activity. To improve the antibacterial activity of linear laterocidine, through rational design, site-specific substitutions were made at positions 1, 6, and 9 of its peptide chain, yielding a series of lipopeptide derivatives (derived lipopeptides 1-11). Activity assays showed that introducing positively charged D-ornithine, D-2,4-diaminobutyric acid, D-arginine, or D-lysine at position 1 slightly enhanced the antibacterial activity of the corresponding derived lipopeptides (lipopeptides 1-4) (MIC 2–16 μg / mL). In contrast, replacing glycine at position 6 with positively charged 2,4-diaminobutyric acid significantly enhanced the antibacterial effect of the corresponding derived lipopeptide (derived lipopeptide 6), with a MIC value (MIC 4–8 μg / mL) comparable to that of the natural lipocyclic peptide Laterocidine. However, replacing position 9 with phenylalanine (phenyl), cysteine ​​(thiol), or tyrosine (hydroxyphenyl) significantly decreased the antibacterial activity (MIC 4–32 μg / mL). These results indicate that derived lipopeptide 6 exhibits the best antibacterial activity, comparable to that of the natural lipocyclic peptide. Furthermore, derived lipopeptide 2 also showed superior antibacterial activity compared to linear Laterocidine, demonstrating potential for further development.

[0030] Example 2: Evaluation of the hemolytic properties of derived lipopeptides Red blood cells were isolated from pig blood and washed 4–5 times with phosphate-buffered saline (PBS, pH 7.4). The washed red blood cells were dispersed in PBS to prepare a 0.25% (v / v) red blood cell suspension. Natural lipocyclic peptides Laterocidine, linear Laterocidine, and derived lipopeptide 1-11 were dissolved in PBS and diluted to 512 μg / mL stock solutions, which were then diluted two-fold with PBS to obtain working solutions. Equal volumes of red blood cell suspension and different concentrations of natural lipocyclic peptides Laterocidine, linear Laterocidine, and derived lipopeptide working solutions were added to 96-well plates. Triton X-100 solution (1%, v / v 20 μL) was used as a positive control, and the PBS solution of red blood cells served as a negative control. The 96-well plates were incubated at 37°C for 1 h, then centrifuged at 3000 r / min and 4°C for 10 min. The supernatant was carefully transferred to new wells, and the absorbance was measured at 490 nm. Hemolysis rate is calculated using the following formula: .

[0031] All experiments were repeated three times, and the results were as follows: Figure 13 As shown in Table 3.

[0032] Table 3. Hemolysis rate of lipocyclic peptides and their derivatives ; pass Figure 3 As shown in Table 3, the natural lipocyclic peptide Laterocidine itself exhibits low hemolytic activity. However, increasing the positive charge of the peptide typically enhances its interaction with the erythrocyte membrane, potentially increasing hemolytic activity. Therefore, the hemolytic activity of linear Laterocidine and derived lipopeptide 1-11 was evaluated. The results showed that within the concentration range of 256–1 g / mL, the hemolytic rate of natural Laterocidine was 0–0.67%, that of linear Laterocidine was 0–0.75%, and that of derived lipopeptide 1-11 was 0–1.61%, all below 5%. These results indicate that increasing the charge of linear Laterocidine does not significantly increase hemolytic activity, suggesting that the derived lipopeptide, after substitution with positively charged amino acids, has lower cytotoxicity and broad application prospects.

[0033] Experiment Example 3: Stability of Derived Lipopeptides in Solutions with Different Salt Ions The stability of derived lipopeptide 1-11 against Escherichia coli CVCC1569 was evaluated in the presence of different salt ions (150 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 4 μM CuCl2 and 4 μM FeCl3) by measuring the MIC value. The results are shown in Table 4.

[0034] Table 4. MIC values ​​of natural lipocyclic peptides Laterocidine, linear Laterocidine, and derived lipopeptides in salt ion solutions. ; Physiological salts interfere with the electrostatic interaction between peptides and bacterial membranes, thus affecting the antibacterial activity of the peptides. Table 4 shows that, compared to the control group with a MIC value of 4 μg / mL, the MIC values ​​of the natural lipocyclic peptide Laterocidine in different salt ion solutions ranged from 16 to 512 μg / mL, indicating that salt ions affect the antibacterial activity of Laterocidine. The antibacterial activity of linear Laterocidine against *E. coli* did not change significantly compared to the natural lipocyclic peptide Laterocidine. To enhance the antibacterial activity of linear Laterocidine in different salt ion environments, we rationally designed and performed site-specific substitutions of the 1st, 6th, and 9th amino acids of its peptide chain, obtaining a series of lipopeptide derivatives (derived lipopeptides 1-11). Activity assays showed that introducing positively charged D-ornithine, D-2,4-diaminobutyric acid, D-arginine, or D-lysine at position 1 only maintained moderate antibacterial activity (MIC of 8–256 μg / mL) in the corresponding derivatives (derived lipopeptides 1–4). In contrast, replacing position 6 with positively charged ornithine, 2,4-diaminobutyric acid, arginine, or lysine significantly improved the antibacterial activity of the corresponding derived lipopeptides. In particular, 2,4-diaminobutyric acid substitution resulted in the best antibacterial effect in the derivative (derived lipopeptide 6), with an MIC of 4–16 μg / mL. Substituting position 9 with phenylalanine (phenyl), cysteine ​​(thiol), or tyrosine (hydroxyphenyl) did not significantly alter the antibacterial activity. These results indicate that derived lipopeptide 6 exhibits the best antibacterial activity in different salt ion solutions and has potential for further development. This molecule shows promise as an antibiotic alternative added to livestock feed and may also be developed into a drug for treating Gram-negative bacterial infections, demonstrating broad application prospects.

[0035] The above are merely embodiments of the present invention. The invention is not limited to the fields covered by these embodiments. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can improve and implement this solution based on the guidance provided in this application and their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness or practicality of the invention. The scope of protection claimed in this application should be determined by the content of its claims. The specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A derivative lipopeptide synthesized based on the lipocyclic peptide laterocidine, characterized in that: The derived lipopeptide is obtained by replacing the amino acid at position 1, position 6, or position 9 in the linear latexidine peptide chain obtained by opening the lipocyclic peptide latexidine. The amino acids that replace the amino acids at positions 1 and 6 are positively charged, and the amino acid that replaces the amino acid at position 9 is phenyl, mercapto, or hydroxyphenyl.

2. The derived lipopeptide synthesized based on the lipocyclic peptide laterocidine according to claim 1, characterized in that: The structure of the derived lipopeptide is as follows: C9H 17 O-(D-Orn)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; or, C9H 17 O-(D-Dab)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Arg)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; or, C9H 17 O-(D-Lys)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Orn-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Dab-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Arg-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Lys-(D-Orn)-Trp-Thr-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Phe-Ile-Asn-Gly-Gly; or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Cys-Ile-Asn-Gly-Gly; Or, C9H 17 O-(D-Ser)-(D-Tyr)-(D-Trp)-(D-Orn)-Orn-Gly-(D-Orn)-Trp-Tyr-Ile-Asn-Gly-Gly。 3. The use of the derived lipopeptide synthesized from the lipocyclic peptide Laterocidine as described in claim 1 or 2 in the preparation of antibacterial drugs or antibacterial compositions.

4. The application according to claim 3, characterized in that: The bacteria are Gram-negative.

5. The application according to claim 4, characterized in that: Gram-negative bacteria include Escherichia coli, Salmonella, and Pseudomonas aeruginosa.

6. The application of the derived lipopeptide synthesized based on the lipocyclic peptide Laterocidine as described in claim 1 or 2 in the preparation of feed additives.

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