Cyclic peptide as well as preparation method and application thereof

Abstract

The invention provides a cyclic peptide as well as a preparation method and application thereof. The cyclic peptide has an amino acid sequence # imgabs0 # represented by formula (1) (wherein X is a Lys residue modified with a fatty acid molecule, and a line connecting Arg and Lys represents an amide bond. The cyclic peptide prepared by the invention has wide antifungal activity and antibacterial activity on drug-resistant bacteria; meanwhile, the biotoxicity and the protease degradation resistance are high, and the in-vivo antibacterial effect of the cyclopeptide can be prolonged. The cyclic peptide can be applied to preparation of anti-bacterial and anti-fungal infection drugs, and can be used as an excellent substitute drug or an auxiliary drug of existing antibiotics. The method for preparing the cyclic peptide is simple, raw materials are easy to obtain, and industrial production can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and in particular to a cyclic peptide and a preparation method and application thereof. Background Art

[0002] Antibiotics are the most effective means of treating infectious diseases. However, the long-term use and abuse of antibiotics have led to increasingly serious drug resistance in major pathogenic microorganisms, causing pathogenic drug-resistant bacteria to become a major threat to human health. With the lack of development of new antibiotics, the emergence of multidrug-resistant bacteria, and the restrictions on the use of antibiotics in many fields, it is urgent to find safe, effective, and non-resistance-producing antibacterial drugs. Antimicrobial cyclic peptides have become important candidate molecules for combating drug-resistant bacteria due to their core advantages such as structural stability, broad-spectrum high efficiency, low toxicity, resistance to drug resistance, and modifiability. They have broad application prospects in the fields of medicine, agriculture, and industry. Therefore, the discovery of cyclic peptides that have antibacterial activity against both pathogenic non-resistant and resistant bacteria and fungi is of great significance to the research and development of new antibiotics. Summary of the Invention

[0003] The purpose of the present invention is to provide a cyclic peptide and its preparation method and application in response to the above-mentioned deficiencies in the prior art.

[0004] To achieve the above object, the present invention adopts the following technical solutions: The first object of the present invention is to provide a cyclic peptide having an amino acid sequence represented by formula (I), , Here, X is a Lys residue modified with a fatty acid molecule, and the line connecting Arg and Lys represents an amide bond.

[0005] Furthermore, the fatty acid molecule is any one of lauric acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid and eicosanoic acid.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned cyclic peptide, comprising the following steps: S1. Solid-phase chemical synthesis of linear peptides The Fmoc solid-phase peptide synthesis method was used, with dichlororesin as the carrier, to synthesize fully protected linear peptides according to the amino acid sequence, in which Fmoc-Lys(Mtt)-OH was used at the 7th position of Lys; S2. Fatty acid modification The fully protected linear peptide obtained in step S1 was used to remove the Mtt protecting group of the Lys side chain using a DCM solution containing 1% TFA by volume. Fatty acids, HOBT, HCTU, and DIEA were dissolved and mixed in DMF, and then condensed with the linear peptide without the Mtt protecting group to obtain a fatty acid-modified linear peptide resin. S3, polypeptide cyclization The fatty acid-modified linear peptide resin obtained in step S2 is cleaved by a cleavage reagent to obtain a fully protected linear peptide, which is then concentrated to a viscous state using a rotary evaporator, dissolved in water, and then freeze-dried using a freeze dryer. The linear peptide, HOBT, and DIC are then reacted in a DMF liquid phase to cyclize the fully protected cyclic peptide, which is then deprotected using trifluoroacetic acid and precipitated with diethyl ether to obtain a crude cyclic peptide. S4. Purification of cyclic peptides The crude cyclic peptide in step S3 was purified by high performance liquid chromatography to remove impurity peptides, and then freeze-dried to obtain pure cyclic peptide.

[0007] Furthermore, in step S1, a dichloro resin with a substitution value of 0.89-1.25 mmol / g was selected, and 2-4 mM HOBT, 2-4 mM HCTU, and 2-6 mM Fmoc-Lys(Boc)-OH were added to DMF containing 5% N-methylmorpholine for dissolution and activation for coupling reaction.

[0008] Furthermore, the fatty acid was coupled to the linear peptide by dissolving and mixing a system of 2~4 mM fatty acid, 2~4 mM HOBT, 2~4 mM HCTU and 3~6 mL DIEA, and then condensing with the linear peptide without the side chain Mtt protecting group for 2~4 hours.

[0009] Furthermore, the peptide was cyclized using 2-4 mM HOBT and 2-4 mM DIC in a DMF liquid phase to obtain a fully protected cyclic peptide.

[0010] Furthermore, the cyclic peptide was purified by preparative reverse-phase high performance liquid chromatography. The chromatographic column was an XB-C18 reverse-phase column, and ultrapure water and acetonitrile were used as mobile phases. The detection wavelengths were 215 nm and 280 nm. The flow rate was 3 mL / min. The mobile phase A was acetonitrile containing 0.1% TFA, and the mobile phase B was ultrapure water containing 0.1% TFA. The elution method was 15%-45% phase A, 85%-55% phase B, and the elution time was 30 min.

[0011] The third object of the present invention is to provide the use of the above cyclic peptide in the preparation of antibiotic drugs.

[0012] The fourth object of the present invention is to provide a medicine, the effective active ingredient of which includes the above-mentioned cyclic peptide.

[0013] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: The antimicrobial cyclic peptide prepared by the present invention has broad antifungal activity and antibacterial activity against drug-resistant bacteria. It also has low biotoxicity and high resistance to protease degradation, which can prolong the antimicrobial effect of the peptide in vivo and further reduce the development of drug resistance by inhibiting biofilm formation. It can be used in the preparation of antibacterial and antifungal drugs as an excellent alternative or adjuvant to existing antibiotics. Furthermore, the method for preparing the cyclic peptide of the present invention is simple, and the raw materials are readily available, enabling industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a high performance liquid chromatogram of the cyclic peptide Ra-CycloAMP-12-K7-C16 prepared in Example 1; Figure 2 This is the mass spectrum of the cyclic peptide Ra-CycloAMP-12-K7-C16 prepared in Example 1; Figure 3 This is the mass spectrum of the cyclic peptide Ra-CycloAMP-12-K11-C16 prepared in Comparative Example 1; Figure 4 This is the hemolytic activity graph of the cyclic peptide Ra-CycloAMP-12-K7-C16; Figure 5 This is the cytotoxicity graph of the cyclic peptide Ra-CycloAMP-12-K7-C16; Figure 6 This is a diagram showing the inhibition of Malassezia biofilm formation by cyclic peptide Ra-CycloAMP-12-K7-C16; Figure 7 This is a scanning electron micrograph of Malassezia treated with cyclic peptide Ra-CycloAMP-12-K7-C16. DETAILED DESCRIPTION

[0015] To make the purpose, technical solutions and advantages of the present invention clearer, the specific embodiments of the present invention are described in further detail below in conjunction with specific examples and accompanying drawings. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0016] The cyclic peptide provided by the present invention has an amino acid sequence represented by formula (1): Arg-Trp-Trp-Arg-Phe-Phe-Lys-Phe-Trp-Trp-Lys-Lys, and is obtained by forming an amide bond between the amino group and the carboxyl group of the first and last amino acids of the amino acid sequence to form a ring, wherein the seventh Lys residue is modified with a fatty acid molecule. The fatty acid molecule is selected from any one of dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, and eicosanoic acid.

[0017] Example 1 Preparation of cyclic peptide modified with hexadecanoic acid at position 7 Lys.

[0018] Step S1, solid phase chemical synthesis of linear peptides (1) Weigh 4 mM dichlororesin with a substitution value of 1.05 mmol / g, add dichloromethane (DCM), react for 5 min, and drain. Add 2 mM Fmoc-Lys(Boc)-OH amino acid and 3 mL N,N-diisopropylethylamine (DIEA), react for 15 min, drain, add 5 mL of an organic solution containing dichloromethane (80%), methanol (15%), and N,N-diisopropylethylamine (5%), react for 20 min, drain, and wash with DMF to obtain a blocked resin containing Fmoc-Lys(Boc).

[0019] (2) For single amino acid reaction, 20% piperidine was added to remove the FMOC protecting group on the resin. 4 mM HOBT, 4 mM HCTU (peptide condensing agent), and 2 mM Fmoc-Lys (Boc)-OH were weighed and added to DMF containing 5% N-methylmorpholine for dissolution and activation. After activation, the resin was added to the deprotected and cleaned resin for amino acid coupling reaction. The coupling time was 1 h. The resin was drained and washed with DMF.

[0020] (3) Repeat the above step (2) and sequentially add Fmoc-Lys(Boc)-OH, Fmoc-Trp-OH, Fmoc-Trp-OH, Fmoc-Phe-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Trp-OH, Fmoc-Trp-OH, and Fmoc-Arg (Pbf)-OH to obtain a fully protected linear peptide resin.

[0021] Step S2, fatty acid modification The fully protected linear peptide resin obtained above was used to remove the Mtt protecting group of the side chain Lys of amino acid 7 using a DCM solution containing 1% TFA by volume. 2mM hexadecanoic acid, 4mM HOBT, 4mM HCTU and 3mL DIEA were weighed and dissolved and mixed respectively, and condensed with the linear peptide without the side chain Mtt protecting group for 2h. The resin was drained and washed to obtain the fatty acid-modified fully protected linear peptide resin.

[0022] Step S3, polypeptide cyclization The fatty acid-modified fully protected linear peptide resin obtained above was cleaved with a cleavage reagent (dichloromethane (80%), trifluoroethanol (TFEA) (20%)) for 20 minutes to obtain a fully protected linear peptide, which was concentrated to a viscous state using a rotary evaporator, dissolved in water, and then freeze-dried using a freeze dryer. The linear peptide, 4mM HOBT (1-hydroxybenzotriazole), and 4mM DIC (diisopropylcarbodiimide) were then reacted in a DMF liquid phase to cyclize the fully protected cyclic peptide. Finally, the peptide was deprotected using 6mL of cleavage buffer (2% anisole, 3% thioanisole, 95% trifluoroacetic acid) and precipitated with ether to obtain the crude cyclic peptide.

[0023] Step S4, purification of cyclic peptide The crude cyclic peptide obtained above was separated and purified using preparative reverse-phase high-performance liquid chromatography (RP-HPLC, XB-C18 reverse-phase column) using ultrapure water and acetonitrile as mobile phases. Detection wavelengths were 215 nm and 280 nm, and the flow rate was 10 mL / min. Mobile phase A consisted of acetonitrile containing 0.1% TFA, and phase B consisted of ultrapure water containing 0.1% TFA. The elution ratio was 15%-45% phase A, 85%-55% phase B, and the elution time was 40 min. The eluted peak was collected, and molecular weight was determined by mass spectrometry. Purity was analyzed by analytical reverse-phase high-performance liquid chromatography on a Waters 2615 column using a C18, 5 μm, 4.6 mm × 250 mm column. Ultrapure water and acetonitrile were used as mobile phases. Detection wavelengths were 215 nm and 280 nm, and the flow rate was 1 mL / min. Mobile phase A consisted of acetonitrile containing 0.1% TFA, and phase B consisted of ultrapure water containing 0.1% TFA. The elution ratio was 15%-45% phase A, 85%-55% phase B, and the elution time was 30 min. The peak eluting at 14.2 min was collected and identified as the target cyclic peptide, designated Ra-CycloAMP-12-K7-C16.

[0024] The structure of the cyclic peptide Ra-CycloAMP-12 is shown in Formula II:

[0025] Formula II like Figure 1 and Figure 2 As shown in the figure, the chromatogram and mass spectrum of the prepared Ra-CycloAMP-12-K7-C16 were obtained. The elution peak at 14.2 min was collected in the preparative chromatography and was identified by mass spectrometry as the target cyclic peptide Ra-CycloAMP-12-K7-C16, which was consistent with the theoretical molecular weight.

[0026] Example 2 Preparation of cyclic peptides modified with dodecanoic acid at position 7 Lys.

[0027] The preparation process was the same as that in Example 1, except that in step S2, dodecanoic acid was selected as the fatty acid for the coupling reaction; in step S4, the elution peak at 10.5 min was collected, which was the target cyclic peptide named Ra-CycloAMP-12-K7-C12.

[0028] Example 3 Preparation of cyclic peptide modified with tetradecanoic acid at position 7 Lys.

[0029] The preparation process was the same as that in Example 1, except that in step S2, myristic acid was selected as the fatty acid for the coupling reaction, and in step S4, the elution peak at 11.6 min was collected, which was the target cyclic peptide named Ra-CycloAMP-12-K7-C14.

[0030] Example 4 Preparation of cyclic peptide modified with octadecanoic acid at position 7 Lys.

[0031] The preparation process was the same as that in Example 1, except that in step S2, octadecanoic acid was selected as the fatty acid for the coupling reaction, and in step S4, the elution peak at 15.5 min was collected, which was the target cyclic peptide named Ra-CycloAMP-12-K7-C18.

[0032] Example 5 Preparation of cyclic peptides modified with eicosanoic acid at position 7.

[0033] The preparation process was the same as that in Example 1, except that in step S2, eicosanoic acid was selected as the fatty acid for the coupling reaction, and in step S4, the elution peak at 16.1 min was collected, which was the target cyclic peptide named Ra-CycloAMP-12-K7-C20.

[0034] Example 6 Preparation of cyclic peptides without modification of Lys at position 7.

[0035] The preparation process was the same as that in Example 1, except that in step S2, the fatty acid coupling reaction was not performed, and in step S4, the elution peak at 10.1 min was collected, which was the target cyclic peptide named Ra-CycloAMP-12.

[0036] The different fatty acid coupled cyclic peptides prepared in Examples 1-5 were compared and analyzed. The structures are shown in Table 1. The purity of each different fatty acid coupled cyclic peptide was above 95%.

[0037] Table 1.

[0038] Comparative Example 1 Preparation of cyclic peptide modified with hexadecanoic acid at position 11 Lys. Step S1, solid phase chemical synthesis of linear peptides (1) Weigh 4 mM dichlororesin with a substitution value of 1.05 mmol / g, add dichloromethane (DCM), react for 5 min, and drain. Add 2 mM Fmoc-Lys(Boc)-OH amino acid and 3 mL N,N-diisopropylethylamine (DIEA), react for 15 min, drain, add 5 mL of an organic solution containing dichloromethane (80%), methanol (15%), and N,N-diisopropylethylamine (5%), react for 20 min, drain, and wash with DMF to obtain a blocked resin containing Fmoc-Lys(Boc).

[0039] (2) For single amino acid reaction, 20% piperidine was added to remove the FMOC protecting group on the resin. 4 mM HOBT, 4 mM HCTU, and 2 mM Fmoc-Lys (Boc)-OH were weighed and added to DMF containing 5% N-methylmorpholine for dissolution and activation. After activation, the resin that had been deprotected and cleaned was added to carry out amino acid coupling reaction. The coupling time was 1 h. The resin was then drained and washed with DMF.

[0040] (3) Repeat the above step (2) and sequentially add Fmoc-Lys(Mtt)-OH, Fmoc-Trp-OH, Fmoc-Trp-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Trp-OH, Fmoc-Trp-OH, and Fmoc-Arg (Pbf)-OH to obtain a fully protected linear peptide resin.

[0041] Step S2, fatty acid modification The fully protected linear peptide resin obtained above was used to remove the Mtt protecting group of the side chain Lys of amino acid at position 11 using a DCM solution containing 1% TFA by volume. 2mM hexadecanoic acid, 4mM HOBT, 4mM HCTU and 3mL DIEA were weighed and dissolved and mixed respectively, and condensed with the linear peptide without the side chain Mtt protecting group for 2h. The resin was drained and washed to obtain the fatty acid-modified fully protected linear peptide resin.

[0042] Step S3, polypeptide cyclization The fatty acid-modified fully protected linear peptide resin obtained above was cleaved with a cleavage reagent (dichloromethane (80%), trifluoroethanol (TFEA) (20%)) for 20 minutes to obtain a fully protected linear peptide, which was concentrated to a viscous state using a rotary evaporator, dissolved in water, and then freeze-dried using a freeze dryer. The linear peptide, 4mM HOBT, and 4mM DIC were then reacted in a DMF liquid phase to cyclize the fully protected cyclic peptide. Finally, the peptide was deprotected using 6mL of lysis buffer (2% anisole, 3% thioanisole, 95% trifluoroacetic acid) and precipitated with ether to obtain a crude cyclic peptide.

[0043] Step S4, purification of cyclic peptide The crude cyclic peptide obtained above was separated and purified using preparative reverse-phase high-performance liquid chromatography (RP-HPLC, XB-C18 reverse-phase column) using ultrapure water and acetonitrile as mobile phases. Detection wavelengths were 215 nm and 280 nm, and the flow rate was 10 mL / min. Mobile phase A consisted of acetonitrile containing 0.1% TFA, and phase B consisted of ultrapure water containing 0.1% TFA. The elution ratio was 15%-45% phase A, 85%-55% phase B, and the elution time was 40 min. The eluted peak was collected, and molecular weight was determined by mass spectrometry. Purity was analyzed by analytical reverse-phase high-performance liquid chromatography on a Waters 2615 using a C18, 5 μm, 4.6 mm x 250 mm column. Ultrapure water and acetonitrile were used as mobile phases. Detection wavelengths were 215 nm and 280 nm, and the flow rate was 1 mL / min. Mobile phase A consisted of acetonitrile containing 0.1% TFA, and phase B consisted of ultrapure water containing 0.1% TFA. The elution ratio was 15%-45% phase A, 85%-55% phase B, and the elution time was 30 min.

[0044] like Figure 2 As shown in FIG, the mass spectrum of the prepared Ra-CycloAMP-12-K11-C16 was obtained. The elution peak at 14.5 min was collected in the preparative chromatography and was identified by mass spectrometry as the desired hexadecanoic acid-modified antibacterial cyclic peptide Ra-CycloAMP-12-K11-C16 with a purity of 96.2%. The final yield of 4 mM synthesis amount after freeze-drying was 10.4%.

[0045] In order to better illustrate that the cyclic peptide of the present invention has better antibacterial activity, good stability and safety, the applicant conducted the following research: (1) Verification of the antibacterial activity of antibacterial cyclic peptides.

[0046] Bacterial culture: Escherichia coli, Bacillus subtilis, Candida albicans, Staphylococcus aureus and Streptococcus mutans were diluted to 1×10 5 CFU / mL concentration, dilute Propionibacterium acnes, Malassezia, and Trichophyton rubrum to 1×10 6 The concentration of CFU / mL.

[0047] Preparation of 96-well microplates: The fatty acid-modified antimicrobial cyclic peptide Ra-CycloAMP-12 prepared in Example 1 and Comparative Example 1 was used to verify antimicrobial activity. The peptide concentration was 2-256 μg / mL. Physiological saline was used as a negative control, and clindamycin was used as a positive control. A bacterial suspension was taken during the logarithmic growth phase, and Propionibacterium acnes was diluted to a final concentration of 1×10 6 CFU / mL, dilute other strains to 1×10 5 CFU / mL. 100 μL of bacterial suspension was added to each well. After 96 hours of static culture for P. acnes and 17-20 hours for other strains, the absorbance of the bacterial suspension at 600 nm was measured using a microplate reader. The MIC value is the lowest drug concentration observed without any signs of bacterial growth. The MIC value was calculated as the average of the sample concentrations in the well with no detectable bacterial growth and the adjacent wells. The test results were obtained by repeating 4 to 6 independent experiments. The results are shown in Table 2.

[0048] Table 2. Minimum inhibitory concentrations of antimicrobial cyclic peptides against different strains

[0049] As can be seen from Table 2, Ra-CycloAMP-12 was modified with hexadecanoic acid at position 7 of Lys and then cyclized to obtain an antibacterial cyclic peptide. The minimum inhibitory concentration was determined and the broad-spectrum antibacterial activity was significantly improved compared with other fatty acid modifications.

[0050] (2) Verification of the toxicity of antimicrobial cyclic peptides.

[0051] (2.1) Verification of hemolytic activity of antimicrobial cyclic peptides 1 mg of the hexadecanoic acid-modified antimicrobial cyclic peptide Ra-CycloAMP-12-K7-C16 prepared in Example 1 was weighed and dissolved in 100 μL ddH2O to prepare a 10 mg / mL stock solution. 0.1% Triton X100 was then prepared and 4 mL of fresh blood was extracted and transferred to an anticoagulant tube. The blood was transferred from the anticoagulant tube to a 1.5 mL EP tube using a 1 mL pipette, centrifuged at 8000 rpm for 3 min, and then centrifuged three times to discard the supernatant and remove the plasma. 200 μL of the washed blood cells were taken out of the EP tube and diluted in 5 mL PBS, and gently mixed. 200 μL of the diluted blood cells were added to the EP tube with 2-256 μg / mL antimicrobial cyclic peptide, PBS was used as a negative control, and 0.1% Triton X 100 was used as a positive control, and both were gently mixed. The prepared EP tube was placed in a 37°C constant temperature incubator and incubated for 30 min. The EP tube was removed and centrifuged at 12000 rpm for 3 min. min, the supernatant was added to a 96-well plate, 100 μL per well, and each group was repeated 3 times; the hemolytic activity of the antimicrobial cyclic peptide was detected at 490 nm on an enzyme-labeled instrument. Figure 4 It can be seen that the antibacterial cyclic peptide obtained by modification of Lys fatty acid at position 7 and first cyclization has no obvious hemolytic activity at high concentrations.

[0052] (2.2) Cytotoxicity Verification of Antimicrobial Cyclic Peptides The cytotoxicity of antimicrobial cyclic peptides on NIH-3T3 cells was measured using CCK-8 assay. 4 / well) were seeded in a 96-well plate and grown overnight, and then the complete medium was replaced with fresh medium containing 2-256μg / mL Ra-CycloAMP-12-K7-C16, leaving a group untreated as the control group. For NIH-3T3 cells, they were further incubated at 37°C, 5% CO2 for 8 hours. After 8 hours, the lights were turned off and placed in a dark environment. 10μL / well CCK-8 was added to each well in a sterile operating table, and after incubation at 37°C for 2 days, the absorbance was measured at 450nm. The cytotoxicity of the peptide was estimated by comparing the cell inhibition rate of cells treated with the fusion peptide with that of untreated cells (the inhibition rate of the untreated control was set to 0%). Figure 5 It can be seen that the antibacterial cyclic peptide obtained by modification of Lys fatty acid at position 7 and first cyclization has no obvious toxicity to normal NIH-3T3 cells.

[0053] (3) Verification of protease stability of antibacterial cyclic peptides.

[0054] 2.5 mg / mL of different proteases were mixed with 256 μg / mL of the hexadecanoic acid-modified antimicrobial cyclic peptide prepared in Example 1 and incubated at 37°C for 1 hour. The minimum inhibitory concentration (MIC) of the antimicrobial cyclic peptide against P. acnes ATCC6919 was then determined using the microdilution method described in Example 2. The experiment was repeated three times in duplicate. The results are shown in Table 3.

[0055] Table 3. Protease stability of antimicrobial cyclic peptides

[0056] As can be seen from Table 3, the antimicrobial cyclic peptide still retained its original antibacterial activity after being treated with 2.5 mg / mL trypsin and pepsin, and had good resistance to protease degradation.

[0057] (4) Inhibition experiment of Ra-CycloAMP-12-K7-C16 on Malassezia biofilm.

[0058] Take 100 μL of Malassezia in the logarithmic growth phase and place it in a 96-well microtiter plate. Add the antibacterial substance Ra-CycloAMP-12-K7-C16 to make the peptide concentration 16 μg / mL. Use an equal volume of sterile water as a control group. After incubation at 30°C for 24 hours, add 0.1% crystal violet to stain for 30 minutes, wash three times with PBS buffer, wash off the excess dye, and take pictures. Figure 6 As shown in the results, Ra-CycloAMP-12-K7-C16 has obvious biofilm inhibitory activity and can reduce the development of drug resistance in Malassezia.

[0059] (5) Study on the mechanism of action of Ra-CycloAMP-12-K7-C16.

[0060] The M. furfur suspension (10 6 CFU / mL) was inoculated in a culture medium containing a polypeptide concentration of 16 μg / mL and inoculated with an untreated control group. After incubation at 37°C for 48 hours, the fungi were collected by centrifugation, washed with PBS, and fixed with electron microscopy fixative for 12 hours at 4°C. After washing with PBS, the samples were dehydrated through a series of ethanol solutions (30, 50, 70, 80, 90, 95 and 100%), treated with a mixture of ethanol and isoamyl acetate (v / v=1 / 1), and then treated with pure isoamyl acetate. After treatment, a small amount of solution was fixed on the sample pile for spraying gold and observed under a scanning electron microscope. As Figure 7As shown, the control group showed intact oval cells with clear cell surfaces, plump and dense protoplasts, and some budding. In contrast, after treatment with Ra-CycloAMP-12-K7-C16, the cells showed irregular shapes, shrinkage, depressions, and varying degrees of surface holes and damage, with leakage of cell contents.

[0061] The above embodiments and features of the embodiments may be combined with each other unless they conflict. The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A cyclic peptide, characterized in that The cyclic peptide has an amino acid sequence represented by formula (I), ， Here, X is a Lys residue modified with a fatty acid molecule, and the line connecting Arg and Lys represents an amide bond.

2. A cyclic peptide according to claim 1, characterized in that The fatty acid molecule includes any one of lauric acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid and eicosanoic acid.

3. A method for preparing the cyclic peptide according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Solid-phase chemical synthesis The Fmoc solid-phase peptide synthesis method was used, with dichlororesin as the carrier, to synthesize fully protected linear peptides according to the amino acid sequence, in which the 7th Lys was synthesized using Fmoc-Lys(Mtt)-OH; S2. Fatty acid modification The fully protected linear peptide obtained in step S1 was used to remove the Mtt protecting group of the Lys side chain using a DCM solution containing 1% TFA by volume. Fatty acids, HOBT, HCTU, and DIEA were dissolved and mixed in DMF, and then condensed with the linear peptide without the Mtt protecting group to obtain a fatty acid-modified linear peptide resin. S3, polypeptide cyclization The fatty acid-modified linear peptide resin obtained in step S2 is cleaved by a cleavage reagent to obtain a fully protected linear peptide, which is then concentrated to a viscous state using a rotary evaporator, dissolved in water, and then freeze-dried using a freeze dryer. The linear peptide, HOBT, and DIC are then reacted in a DMF liquid phase to cyclize the fully protected cyclic peptide, which is then deprotected using trifluoroacetic acid and precipitated with diethyl ether to obtain a crude cyclic peptide. S4. Purification of cyclic peptides The crude cyclic peptide of step S3 was purified by preparative chromatography to remove impurity peptides, and then freeze-dried to obtain pure cyclic peptide.

4. The preparation method according to claim 3, wherein In step S1, a dichlororesin with a substitution value of 0.89-1.25 mmol / g is selected. For the synthesis of amino acids, 2-4 mM HOBT, 2-4 mM HCTU, and 2-6 mM Fmoc-Lys(Boc)-OH are added to DMF containing 5% N-methylmorpholine for dissolution and activation to perform a coupling reaction.

5. The preparation method according to claim 3, wherein In step S2, the fatty acid is coupled to the linear peptide by dissolving and mixing a system of 2-4 mM fatty acid, 2-4 mM HOBT, 2-4 mM HCTU, and 3-6 mL DIEA, and then condensing with the linear peptide without the side chain Mtt protecting group for 2-4 hours.

6. The preparation method according to claim 3, wherein In step S3, the polypeptide is cyclized by reacting 2-4 mM HOBT and 2-4 mM DIC in a DMF liquid phase to obtain a fully protected cyclic peptide.

7. The preparation method according to claim 3, characterized in that In step S4, the cyclic peptide was purified by preparative high performance liquid chromatography using an XB-C18 column and gradient elution. The eluent A was acetonitrile containing 0.1% TFA, and the eluent B was ultrapure water containing 0.1% TFA. The elution program was 15%-45% phase A, 85%-55% phase B, and the elution time was 30 min.

8. Use of the cyclic peptide according to any one of claims 1 to 2 in the preparation of antibacterial and antifungal infection drugs.

9. A drug, characterized in that The effective active ingredient of the drug includes the cyclic peptide according to any one of claims 1 to 2.

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