CN116178519A

CN116178519A - Melittin glycopeptide and preparation method and application thereof

Info

Publication number: CN116178519A
Application number: CN202211213491.9A
Authority: CN
Inventors: 苏春丽; 黎容; 梁冰馨; 廖洪利; 李林吉; 唐铭; 廖秀飞; 陆黎; 李春苗; 汪磊; 李庆雯
Original assignee: Chengdu Medical College
Current assignee: Chengdu Medical College
Priority date: 2022-08-09
Filing date: 2022-09-30
Publication date: 2023-05-30

Abstract

The invention provides a melittin glycopeptide and a preparation method and application thereof, belonging to the field of polypeptide medicaments. The melittin glycopeptide of the invention adopts Ac-GIGAVLKVLTTGLPALISWIKRKRQQ-NH ₂ Arginine at position 22 and/or 24 is modified by glycosylation as a peptide chain. The melittin glycopeptides are successfully prepared, and the hemolysis effect of the melittin glycopeptides is greatly reduced compared with that of the melittin glycopeptides, so that the problem that the melittin in the prior art has strong hemolysis is effectively solved, and the clinical application is facilitated. Meanwhile, the antibacterial activity and the activity of inhibiting tumor cells of the partial melittin glycopeptide prepared by the method are obviously superior to those of melittin, and the preparation method has good application prospect in the research and development fields of antibacterial drugs and antitumor drugs.

Description

Melittin glycopeptide and preparation method and application thereof

Technical Field

The invention belongs to the field of polypeptide medicaments, and in particular relates to a melittin glycopeptide and a preparation method and application thereof.

Background

Melittin (melittin) is a main component of bee venom with pharmacological action and biological activity, and has antibacterial, antiviral, antiparasitic, and antitumor effects. Researches show that melittin has the advantages of good thermal stability, low immunogenicity, difficult anaphylactic reaction generation, better sterilization efficiency than that of conventional antibiotics and the like, and has great potential medicinal value. However, the clinical application of melittin is greatly limited due to the existence of strong hemolysis.

Protein glycosylation is one of the most common and complex Post-translational modifications of proteins (Post-translational modifications, PTMs) found in all areas of life, plays a key role in regulating the biological complexity of eukaryotes, and has become one of the hot-spot directions in current chemical and biological research. The glycosylation of polypeptide molecules can change the physical and pharmacological properties of the original peptide, is hopeful to improve the treatment effect of the polypeptide drug and improves the defects of the polypeptide drug in clinical use.

In glycosylation modification of polypeptide, arginine-glycosylation modification is a brand-new glycosylation modification method, and researches show that part of antibacterial peptide modified by the method is important for germ colonization and pathological effect generation. At present, no literature report on the glycosylation modification of melittin to obtain melittin glycopeptide is known.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art when melittin is clinically applied, and provides melittin glycopeptide and a preparation method and application thereof.

The invention provides a melittin glycopeptide which is prepared by Ac-GIGAVLKVLTTGLPALISWIKRKRQQ-NH ₂ Arginine at position 22 and/or 24 is modified by glycosylation as a peptide chain.

Further, the arginine at position 22 and/or 24 is modified by galactose, N-acetylglucosamine, rhamnose, xylose, glucose, ribose, maltose, or lactose glycosylation.

Further, the melittin glycopeptide is a compound represented by formula I:

wherein R is ₁ 、R ₂ Independently selected from none or

And R is ₁ And R is ₂ At least one is selected from->

R ₃ Selected from the group consisting of

Further, the melittin glycopeptide is a compound represented by formula II:

wherein R is ₃ As indicated previously;

alternatively, the melittin glycopeptide is a compound of formula III:

wherein R is ₃ As indicated previously;

alternatively, the melittin glycopeptide is a compound of formula IV:

wherein R is ₃ As previously indicated.

Further, the melittin glycopeptide is one of the following compounds:

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the invention also provides a preparation method of the melittin glycopeptide, which comprises the following steps:

(1) Synthesizing peptide chains shown as SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4 on the resin by adopting a polypeptide solid-phase synthesis method, and performing deprotection and acetylation on the last amino acid to obtain peptide-containing resin;

(2) Under the action of a deprotection agent, removing a protective group DDe of ornithine in a peptide chain shown as SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4 to expose side chain amino;

(3) In the presence of alkali, carrying out guanylation reaction on the peptide-containing resin containing the exposed side chain amino groups obtained in the step (2) and silver nitrate in a solvent, and connecting the sugar ligand to a peptide chain;

(4) Removing acetyl groups on the sugar ligands;

(5) And cutting off peptide chains from carrier resin, and purifying to obtain the corresponding melittin glycopeptides.

Further, the method comprises the steps of,

in the step (1), the polypeptide solid-phase synthesis method is Fmoc solid-phase synthesis method;

and/or, in the step (2), the method for removing the protecting group DDe includes the steps of: in a solvent, the peptide-containing resin reacts with hydrazine hydrate;

and/or in the step (3), the mass ratio of the peptide-containing resin containing the exposed side chain amino group, the silver nitrate and the sugar ligand is (1-10): (1-10): (1-10);

and/or, in the step (3), the solvent is DMF;

and/or, in step (3), the base is triethylamine;

and/or, in step (3), the reaction temperature is room temperature; and/or the reaction time is 6-12 h;

and/or, in step (4), the method for removing acetyl groups on the sugar ligand comprises the following steps: removing acetyl groups on the sugar ligands with 5% hydrazine hydrate in a solvent;

and/or, in the step (5), the solution used for cutting the peptide chain from the carrier resin is TIPS, H ₂ Mixed solution of O, phenol and TFA, TIPS, H ₂ The volume ratio of O, phenol and TFA is 2:5:5:88; the volume-mass ratio of the solution to the peptide is 1:20mL/g;

and/or, in step (5), the purification method is reverse-phase high performance liquid chromatography;

preferably, the method comprises the steps of,

in the step (1), in the Fmoc solid-phase synthesis method, the condensing agent is DIC-Oxyme condensation system, the activating agent is DIC, and NMP is used as solvent; and/or the deprotection reagent is a mixed solution of Oxyme, piperidine and DMF in a ratio of 7.1:100:400 (g/ml/ml);

and/or, in the step (1), when the last amino acid is acetylated after deprotection, the acetylating agent is a mixed solution of DIEA, acetic anhydride and DMF, and the feeding ratio is 1:1:8 (v/v);

and/or in the step (2), the deprotection agent is a DMF solution of 2% hydrazine hydrate, and the feeding ratio is the resin loading amount, namely NH ₂ NH ₂ Reagent DMF=0.1:0.2:10 (mmol/ml/ml);

and/or, in the step (2), the solvent is DMF when the protecting group DDe is removed;

and/or, in the step (4), the solvent is DMF when acetyl groups on the sugar ligand are removed.

Further, the sugar ligand structure is as follows:

the invention also provides application of the melittin glycopeptide in preparation of antibacterial drugs;

preferably, the medicine is an anti-staphylococcus aureus, streptococcus pyogenes, escherichia coli and salmonella.

The invention also provides application of the melittin glycopeptide in preparing antitumor drugs;

preferably, the medicament is a medicament for the prevention and/or treatment of colorectal cancer.

The invention also provides a medicine, which is a preparation prepared by taking the melittin glycopeptide as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Compared with the prior art, the invention has the advantages that:

1. the invention designs and synthesizes a melittin glycopeptide, and experiments prove that part of melittin glycopeptides can improve antibacterial activity and inhibit tumor cells, and meanwhile, most of melittin glycopeptides can reduce hemolytic side effects, thus having potential application value in antibacterial and antitumor treatment.

2. The invention takes amino resin as a carrier and adopts template Melittin (Melittin): ac-GIGAVLKVLTTGLPALISWIKRKRQQ-NH ₂ The amino acid sequence is synthesized into peptide chain in DIC-oxide condensation system through Fmoc solid phase synthesis method. Wherein ornithine is substituted at the position of arginine (R) to synthesize a long peptide fragment of the primary sequence. Removal of ornithine side chain protecting groups by treatment with 2% hydrazine hydrate in DMF followed by triethylamine and AgNO ₃ Under the action of the above-mentioned components, the sugar ligand is connected on the peptide chain of the resin, the DMF solution containing 5% hydrazine hydrate is used for removing acetyl group from the sugar ligand, and finally the target melittin glycopeptide is obtained by cutting off from the resin. The obtained compound is purified and subjected to characterization analysis by adopting HPLC, MS and other spectrums. The method is simple and easy to implement, and the purity of the obtained melittin glycopeptide is more than 95%.

In conclusion, the melittin glycopeptides are successfully prepared, and the hemolysis effect of the melittin glycopeptides is greatly reduced compared with that of the melittin, so that the problem that the melittin in the prior art has strong hemolysis is effectively solved, and the clinical application is facilitated. Meanwhile, the antibacterial activity and the activity of inhibiting tumor cells of the partial melittin glycopeptide prepared by the method are obviously superior to those of melittin, and the preparation method has good application prospect in the research and development fields of antibacterial drugs and antitumor drugs.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is an HPLC chart of MT-1 a.

FIG. 2 is a mass spectrum of MT-1 a.

FIG. 3 is an HPLC chart of MT-2 a.

FIG. 4 is a mass spectrum of MT-2 a.

FIG. 5 is an HPLC chart of MT-3 a.

FIG. 6 is a mass spectrum of MT-3 a.

FIG. 7 is a high performance liquid chromatography of MT-1 b.

FIG. 8 is a mass spectrum of MT-1 b.

FIG. 9 is an HPLC chart of MT-2 b.

FIG. 10 is a mass spectrum of MT-2 b.

FIG. 11 is an HPLC chart of MT-3 b.

FIG. 12 is a mass spectrum of MT-3 b.

FIG. 13 is a high performance liquid chromatography of MT-1 c.

FIG. 14 is a mass spectrum of MT-1 c.

FIG. 15 is an HPLC chart of MT-2 c.

FIG. 16 is a mass spectrum of MT-2 c.

FIG. 17 is an HPLC chart of MT-3 c.

FIG. 18 is a mass spectrum of MT-3 c.

FIG. 19 is a high performance liquid chromatography of MT-1 d.

FIG. 20 is a mass spectrum of MT-1 d.

FIG. 21 is an HPLC chart of MT-2 d.

FIG. 22 is a mass spectrum of MT-2 d.

FIG. 23 is an HPLC chart of MT-3 d.

FIG. 24 is a mass spectrum of MT-3 d.

FIG. 25 is an HPLC chart of MT-1 e.

FIG. 26 is a mass spectrum of MT-1 e.

FIG. 27 is an HPLC chart of MT-2 e.

FIG. 28 is a mass spectrum of MT-2 e.

FIG. 29 is an HPLC chart of MT-3 e.

FIG. 30 is a mass spectrum of MT-3 e.

FIG. 31 is a high performance liquid chromatography of MT-1 f.

FIG. 32 is a mass spectrum of MT-1 f.

FIG. 33 is an HPLC chart of MT-2 f.

FIG. 34 is a mass spectrum of MT-2 f.

FIG. 35 is an HPLC chart of MT-3 f.

FIG. 36 is a mass spectrum of MT-3 f.

FIG. 37 is a high performance liquid chromatography of MT-1 g.

FIG. 38 is a mass spectrum of MT-1 g.

FIG. 39 is an HPLC chart of MT-2 g.

FIG. 40 is a mass spectrum of MT-2 g.

FIG. 41 is an HPLC chart of MT-3 g.

FIG. 42 is a mass spectrum of MT-3 g.

FIG. 43 is a high performance liquid chromatography for MT-1 h.

FIG. 44 is a mass spectrum of MT-1 h.

FIG. 45 is an HPLC chart of MT-2 h.

FIG. 46 is a mass spectrum of MT-2 h.

FIG. 47 is an HPLC chart of MT-3 h.

FIG. 48 is a mass spectrum of MT-3 h.

FIG. 49 shows the hemolysis of rabbit erythrocytes by each melittin glycopeptide.

FIG. 50 shows the results of cytotoxicity assays of each melittin glycopeptide against human colorectal cancer HCT-116.

Detailed Description

The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

The 8 sugar ligands used in the present invention are prepared according to the documents Pan M, li S, li X, et al Synthesis of and specific antibody generation for glycopeptides with arginine N-GlcNAcNAcNAcation [ J ]. Angew Chem Int Ed Engl,2014,53 (52): 14517-14521 and Ye W, Y ule L, wei C, et al Total synthesis of TRADD death domain with arginine N-GlcNAcylation by hydrazide-based native chemical ligation [ J ]. Chinese Chemical Letters,2020,31 (1): 107-110. The 8 sugar ligands had the following structure:

wherein the galactose sugar ligand and the glucose ligand are isomers of each other; xylose ligands and ribose ligands are isomers of each other; maltose ligands and lactose ligands are isomers of each other.

The invention is based on the template Melittin (Melittin):

Ac-GIGAVLKVLTTGLPALISWIKRKRQQ-NH ₂ (SEQ ID NO. 1) amino acid sequence design and synthesis of 24 melittin glycopeptides. The specific sequence structure of each melittin glycopeptide is shown as follows:

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wherein MT-1 a-3 a and MT-1 e-3 e are isomers with each other; MT-1 d-3 d and MT-1 f-3 f are isomers with each other; MT-1 g-3 g and MT-1 h-3 h are isomers of each other.

The abbreviations involved in the following examples are explained as follows: fmoc: fluorenylmethoxycarbonyl; DCM: dichloromethane; DMF: n, N-dimethylformamide; oxyme: 2-oxime cyanoacetic acid ethyl ester; DIC: n, N-diisopropylcarbodiimide; NMP: n-methylpyrrolidone; TFA: trifluoroacetic acid; tigs: triisopropylsilane.

The sources of the related part of experimental materials are as follows: amino acids and amino resins were purchased from Shanghai Jier Biochemical Co., ltd; n-methylpyrrolidone (NMP), N-Diisopropylcarbodiimide (DIC), ethyl 2-oxime cyanoacetate (Oxyme), trifluoroacetic acid (TFA), acetonitrile (chromatographic purity) were purchased from Hengyu-auspicious glass laboratory apparatus manager in GaoXin; n, N-Dimethylformamide (DMF), anhydrous diethyl ether, dichloromethane (DCM), piperidine and phenol are all analytically pure and purchased from Hengyu-auspicious glass laboratory instruments and engineering division in Gao Xin district.

EXAMPLE 1 preparation of melittin glycopeptide MT-1a of the present invention

1. Synthesis of substrate peptide of melittin glycopeptides

The substrate peptides (compound 1, compound 2 and compound 3) of melittin glycopeptides have the following structures:

in the present invention

Resin, i.e., solid phase synthetic support, is represented.

(1) Preparation of Compound 1

526mg (sample loading of amino resin (Rink Amide resin) was taken out and the sample was 0.38 mmol. G ^-1 ) Adding the mixture into a solid phase synthesis reaction tube, soaking the mixture in DCM for 20min to fully swell the resin, and pumping the resin for later use.

20% piperidine-DMF solution (0.1M 2-oxime ethyl cyanoacetate) was added until the resin was completely submerged, shaking was performed at 25℃for 5min X2 to remove Fmoc from the resin, and the resin was washed 5, 3 times with DMF, DCM, DMF in sequence. The first amino acid in the sequence (1 mmol), oxyme (142 mg,1 mmol) and DIC (155.0 μl,1 mmol) were dissolved in 6ml N-methylpyrrolidone (NMP), added to the resin and shaken for 20min at 60 ℃ and the resin washed 5, 3 times sequentially with DMF, DCM, DMF. Repeating the step of deprotecting and coupling amino acids according to the polypeptide sequence: ac-GIGAVLKVALTTGLPALISWIKORnQQ-NH ₂ Fmoc amino acid (1 mmol), oxyme (142 mg) and DIC (155. Mu.l) were sequentially miscible in 6ml NMP, added to the resin and shaken for 20min at 60℃and the deprotection, condensation and deprotection were repeated until all amino acid ligation was completed. After deprotection of the last amino acid, DIEA was added: acetic anhydride: 10ml of DMF (volume ratio=1:1:1) mixture was shaken at 25℃for 20min, the resin was washed 5, 3 times sequentially with DMF, DCM, DMF, and the resin was washed with anhydrous diethyl ether and dried under vacuum to give Compound 1.

(2) Preparation of Compound 2

According to the polypeptide sequence: ac-GIGAVLKVALTTGLPALISWIKORnKRQQ-NH ₂ The method is the same as that of the preparation of the compound 1, and the compound 2 is obtained.

(3) Preparation of Compound 3

According to the polypeptide sequence: ac-GIGAVLKVALTTGLPALISWIKORnKORnQQ-NH ₂ The method is the same as that of the preparation of the compound 1, and the compound 3 is obtained.

2. Removal of ornithine protecting group DDe from melittin glycopeptide substrate peptide

Orn is abbreviated as Orn in the substrate peptide chain, and the structure is shown as follows:

removing ornithine protecting group DDe on the substrate peptide to prepare compound 4, compound 5 and compound 6. The structures of compound 4, compound 5 and compound 6 are as follows:

(1) Preparation of Compound 4

To 526mg of the dried peptide-containing resin (Compound 1), a DMF solution (10 ml) containing 2% hydrazine hydrate was added, the reaction was allowed to proceed with shaking three times at 25℃for 6min each, and after completion of the reaction, the resin was washed 3 times with DCM, DMF and anhydrous diethyl ether, respectively, and the resin was dried under vacuum to give Compound 4.

(2) Preparation of Compound 5

The raw material is peptide-containing resin (compound 2), and the method is the same as that of the preparation of the compound 4 to obtain the compound 5.

(3) Preparation of Compound 6

The raw material is peptide-containing resin (compound 3), and the method is the same as that of the preparation of the compound 4 to obtain the compound 6.

3. Preparation of melittin glycopeptide MT-1a

(1) Preparation of crude MT-1a

263.2mg of peptide-containing resin (compound 4) is placed in a 50ml centrifuge tube, 102mg of silver nitrate, 278 μl of triethylamine, 400mg of galactose sugar ligand and 10ml of DMF are added, the mixture is subjected to shaking reaction at room temperature for 12h, and after completion, the resin is washed 5, 5 and 3 times by DMF, meOH, DCM in sequence, and the resin is dried by vacuum pumping to obtain the compound 7.

After the above steps are completed, 263.2mg of resin (compound 7) is mixed with DMF solution (10 ml) containing 5% hydrazine hydrate, and the mixture is oscillated at room temperature for reaction for 10 hours, acetyl groups on the sugar ligand are removed, and after completion, DMF and H are sequentially used ₂ O, meOH, DCM the resin was washed 5, 5 times and dried under vacuum to give compound 8.

TIPS was added to compound 8: h ₂ O: phenol: tfa=2:5:5:88 (V/V) 10mL, shaking for 4h at normal temperature, filtering, washing the resin with a little TFA, and collecting the filtrate. Pouring the glacial ethyl ether into the filtrate, precipitating and centrifuging, discarding the supernatant, repeatedly washing and centrifuging with the glacial ethyl ether for three times, and drying with argon to obtain the MT-1a crude product.

(2) Purification of MT-1a

The crude MT-1a was dissolved in acetonitrile and water (50% +50%, v/v) and purified by preparative RP-HPLC. The separation conditions were as follows:

instrument: pre-HPLC SD-1VARIAN high performance liquid chromatograph;

chromatographic column: YMC-Pack ODS-AQ (250×20mml.D, S-10 μm,12 nm);

mobile phase: mobile phase a was an aqueous solution with a volume fraction of 0.1% tfa, and mobile phase B was an acetonitrile solution with a volume fraction of 0.1% tfa;

the steps and parameters are as follows: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 48 to 75 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

Finally preparing MT-1a purified product.

(3) Identification and structural analysis of products

The MT-1a prepared above was identified by HPLC and subjected to structural analysis by HR-Q-TOF-MS (high resolution matrix assisted laser desorption ionization time-of-flight mass spectrometry). The chromatographic mobile phase is acetonitrile and water. Flow ofThe mobile phase A is an aqueous solution with the volume fraction of 0.1 percent TFA, the mobile phase B is an acetonitrile solution with the volume fraction of 0.1 percent TFA, and the gradient elution is carried out (0 to 5min, 5% of the mobile phase B, 5 to 30min, 5 to 95% of the mobile phase B); the flow rate is 1.0 mL-min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The detection wavelengths are 214nm and 254nm, and the sample injection amount is 20 μl. The measured peak time is consistent with the peak time of the main peak of the crude product, and the purity of the melittin glycopeptide prepared by the method>98% (fig. 1). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 2.

EXAMPLE 2 preparation of melittin glycopeptide MT-2a of the present invention

263.2mg of peptide-containing resin (compound 5) and 400mg of galactose sugar ligand are taken as raw materials, and MT-2a purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 68 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-2a purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 3). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 4.

EXAMPLE 3 preparation of melittin glycopeptide MT-3a of the present invention

263.2mg of peptide-containing resin (compound 6) and 800mg of galactose sugar ligand are taken as raw materials, and MT-3a purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 68 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-3a purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 5). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 6.

EXAMPLE 4 preparation of melittin glycopeptide MT-1b of the present invention

263.2mg of peptide-containing resin (compound 4) and 410mg of N-acetylglucosamine ligand are taken as raw materials, and MT-1b purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 40 to 70 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-1b purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 7). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 8.

EXAMPLE 5 preparation of melittin glycopeptide MT-2b of the present invention

263.2mg of peptide-containing resin (compound 5) and 410mg of N-acetylglucosamine ligand are taken as raw materials, and MT-2b purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 40 to 70 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-2b purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 9). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 10.

EXAMPLE 6 preparation of melittin glycopeptide MT-3b of the present invention

263.2mg of peptide-containing resin (compound 6) and 820mg of N-acetylglucosamine ligand are taken as raw materials, and MT-3b purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 68 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-3b purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 11). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 12.

EXAMPLE 7 preparation of melittin glycopeptide MT-1c of the present invention

263.2mg of peptide-containing resin (compound 4) and 377mg of rhamnose ligand are taken as raw materials, and MT-1c purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: 40% of mobile phase B is eluted for 0-5 min, and 40% -60% of mobile phase B is eluted for 5-80 min; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-1c purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 13). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 14.

EXAMPLE 8 preparation of melittin glycopeptide MT-2c of the present invention

263.2mg of peptide-containing resin (compound 5) and 377mg of rhamnose ligand are taken as raw materials, and MT-2c purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 55 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-2c purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 15). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 16.

EXAMPLE 9 preparation of melittin glycopeptide MT-3c of the present invention

263.2mg of peptide-containing resin (compound 6) and 754mg of rhamnose ligand are taken as raw materials, and MT-3c purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 37 percent for 0 to 5 minutes and eluting the mobile phase B with the concentration of 37 to 60 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-3c purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 17). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 18.

EXAMPLE 10 preparation of melittin glycopeptide MT-1d of the present invention

263.2mg of peptide-containing resin (compound 4) and 368mg of xylose ligand are used as raw materials, and MT-1d purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: 40% of mobile phase B is eluted for 0-5 min, and 40% -70% of mobile phase B is eluted for 5-80 min; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-1d purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 19). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 20.

EXAMPLE 11 preparation of melittin glycopeptide MT-2d of the present invention

263.2mg of peptide-containing resin (compound 5) and 338mg of xylose ligand are used as raw materials, and MT-2d purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: 40% of mobile phase B is eluted for 0-5 min, and 40% -70% of mobile phase B is eluted for 5-80 min; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-2d purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 21). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 22.

EXAMPLE 12 preparation of melittin glycopeptide MT-3d of the present invention

263.2mg of peptide-containing resin (compound 6) and 736mg of xylose ligand are used as raw materials, and MT-3d purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 68 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-3d purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 23). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 24.

EXAMPLE 13 preparation of melittin glycopeptide MT-1e of the present invention

263.2mg of peptide-containing resin (compound 4) and 400mg of glucose ligand are taken as raw materials, and MT-1e purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 65 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-1e purified product was identified and structurally analyzed according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 25). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 26.

EXAMPLE 14 preparation of melittin glycopeptide MT-2e of the present invention

263.2mg of peptide-containing resin (compound 5) and 400mg of glucose ligand are taken as raw materials, and MT-2e purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 55 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-2e purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 27). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 28.

EXAMPLE 15 preparation of melittin glycopeptide MT-3e of the present invention

263.2mg of peptide-containing resin (compound 6) and 800mg of glucose ligand are taken as raw materials, and MT-3e purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 35 percent for 0 to 5 minutes and eluting the mobile phase B with the concentration of 35 to 50 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-3e purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 29). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 30.

EXAMPLE 16 preparation of melittin glycopeptide MT-1f of the present invention

263.2mg of peptide-containing resin (compound 4) and 368mg of ribose sugar ligand are taken as raw materials, and MT-1f purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with 36-60% for 0-5 min and the mobile phase B with 36-60% for 5-80 min; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-1f purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 31). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 32.

EXAMPLE 17 preparation of melittin glycopeptide MT-2f of the present invention

263.2mg of peptide-containing resin (compound 5) and 368mg of ribose sugar ligand are taken as raw materials, and MT-2f purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: 40% of mobile phase B is eluted for 0-5 min, and 40% -65% of mobile phase B is eluted for 5-80 min; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-2f purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 33). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 34.

EXAMPLE 18 preparation of melittin glycopeptide MT-3f of the present invention

263.2mg of peptide-containing resin (compound 6) and 736mg of ribose sugar ligand are taken as raw materials, and MT-3f purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 35 percent for 0 to 5 minutes and eluting the mobile phase B with the concentration of 35 to 50 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-3f purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 35). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 36.

EXAMPLE 19 preparation of melittin glycopeptide MT-1g according to the present invention

263.2mg of peptide-containing resin (compound 4) and 593mg of maltose ligand are taken as raw materials, and MT-1g of purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 50 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

MT-1g purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of melittin glycopeptide prepared by this method was >98% (FIG. 37). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 38.

EXAMPLE 20 preparation of melittin glycopeptide MT-2g according to the present invention

263.2mg of peptide-containing resin (compound 5) and 593mg of maltose ligand are taken as raw materials, and MT-2g of purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 55 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

MT-2g purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of melittin glycopeptide prepared by this method was >98% (FIG. 39). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 40.

EXAMPLE 21 preparation of melittin glycopeptide MT-3g according to the invention

263.2mg of peptide-containing resin (compound 6) and 800mg of maltose ligand are used as raw materials, and MT-3g of purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 60 percent for 5 to 60 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The purified MT-3g was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the melittin glycopeptide prepared by this method was of poor purity (FIG. 41). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 42.

EXAMPLE 22 preparation of melittin glycopeptide MT-1h of the present invention

263.2mg of peptide-containing resin (compound 4) and 593mg of lactose sugar ligand are taken as raw materials, and MT-1h purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 60 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-1h purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 43). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 44.

EXAMPLE 23 preparation of melittin glycopeptide MT-2h of the present invention

263.2mg of peptide-containing resin (compound 5) and 593mg of lactose sugar ligand are taken as raw materials, and MT-2h purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 38 percent for 0 to 5 minutes and the mobile phase B with the concentration of 38 to 60 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-2h purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 45). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 46.

EXAMPLE 24 preparation of melittin glycopeptide MT-3h of the present invention

263.2mg of peptide-containing resin (compound 6) and 800mg of lactose sugar ligand are taken as raw materials, and MT-3h purified product is prepared and purified according to the preparation method of melittin glycopeptide MT-1 a. Purification steps and parameters: eluting the mobile phase B with the concentration of 35 percent for 0 to 5 minutes and eluting the mobile phase B with the concentration of 35 to 50 percent for 5 to 80 minutes; the flow rate is 15ml/min, the sample injection amount is 5ml, and the detection wavelength is 214nm.

The MT-3h purified product was identified and analyzed for structure according to the method of example 1, and the peak time was determined to be consistent with the main peak of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% (FIG. 47). The results of analysis by HR-ESI-MS mass spectrometry are shown in FIG. 48.

The beneficial effects of the present invention are demonstrated by specific test examples below.

Test example 1, antibacterial Activity test

Preparing an antibacterial drug storage solution: the target compound was prepared with physiological saline to 1024. Mu.g/ml for use.

Dilution of antibacterial drug: the stock of antimicrobial drug was diluted in a 96-well plate with M-H broth (the concentration range of the drug-containing broth series should cover the sensitive and drug-resistant break points of the drug, and the MIC range of the quality control bacteria).

Inoculating bacterial liquid: first, a 0.5 turbidity of the bacteria suspension was prepared and the above bacteria suspension was subjected to 1:100 dilution (concentration about 10 ⁶ CFU/ml) was added to the above-mentioned drug-containing broth 96-well plates by taking 100. Mu.l of the diluted bacterial suspensions, respectively. Mixing, diluting the antibacterial agent concentration by 1:2, and the final bacterial liquid degree is about 5×10 ⁶ CFU/ml。

Control wells were set: growth control: 100 μl of broth without antibacterial agent and 100ul of diluted bacteria solution; negative control: broth 200.mu.1 with only antibacterial agent and broth 200.mu.1 without antibacterial agent.

Checking the purity of the inoculated bacteria: an inoculating loop of the diluted bacterial solution was streaked and inoculated on a blood agar plate, and the inoculated product was cultured at 37℃in a constant temperature incubator to examine the purity of the inoculated product.

Incubation: the inoculated 96-well plate containing the bacteria drug mixture is placed in a constant temperature incubator for 16-20 hours at 37 ℃.

Check control wells: bacterial purity was first observed and the growth of the plate bacteria was checked to determine if it was contaminated. The growth control tube was then observed for bacterial growth (should growth). The negative control should be grown aseptically; checking whether MIC values of the quality control strain are in the corresponding quality control range.

Interpretation of the results: the highest dilution of the drug without macroscopic bacteria growth test tube or hole is the antibacterial endpoint hole, and the drug concentration is MIC value. Based on the MIC measurement values, the CISI related file MIC interpretation criteria are consulted to determine whether the final result is drug resistance (R), sensitivity (S) or mediator (I).

The results are shown in Table 1, and it is found that the partial melittin glycopeptides of the present invention can enhance the activity of melittin against bacteria, wherein the MIC values of MT-1a, MT-3a, MT-2c for Puzza Streptococcus are doubled; the MIC value of MT-2d and MT-2h for E.coli is doubled. The antibacterial activity of the melittin glycopeptides is obviously improved compared with that of the melittin.

TABLE 1 antibacterial Activity results Table (MIC value μg/mL)

Note that: N/A indicates undetected.

Test example 2 hemolysis test

Preparation of erythrocyte suspensions: taking rabbit blood with good growth state, placing in a pretreated centrifuge tube coated with heparin, centrifuging at 2500r/min for 10min, discarding upper plasma, washing precipitate with equal volume of PBS solution until the upper solution is clear, re-suspending blood cell liquid with PBS buffer, and mixing to obtain final concentration of 2%.

Dilution of sample concentration: the melittin glycopeptides were diluted with PBS to give working solutions at concentrations of 100, 50, 25, 12.5, 6.25, 3.125. Mu.g/ml.

Clean 1.5ml centrifuge tubes were numbered, from one to six, 500. Mu.l of the previously treated cell suspension was added to each tube, and an equal volume of PBS was used as a negative control, and the positive control was 1% Triton X-100. And sequentially adding the prepared working solution into a test tube. Water bath at 37deg.C for 1h at 2500r/min for 10min, centrifuging to obtain upper layer, and measuring OD ₅₄₀ Hemolysis curves were plotted and median hemolysis values (HC were fitted by software Graphpad ₅₀ ) The hemolysis rate and the median hemolysis value (HC were calculated ₅₀ )。

Hemolysis ratio (%) = [ (OD) _{Sample of} -OD _{Negative of} )/(OD _{Positive and negative} -OD _{Negative of} )]×100％

TABLE 2 HC of melittin glycopeptides of each group on rabbit erythrocytes ₅₀ Value of (. Mu.g/ml)

Name of the name	HC ₅₀ (μg/ml)	Name of the name	HC ₅₀ (μg/ml)
				MT-1a	42.51	MT-1e	37.24
MT-2a	51.97	MT-2e	55.33
				MT-3a	>100	MT-3e	53.11
MT-1b	>100	MT-1f	33.08
				MT-2b	69.3	MT-2f	55.11
MT-3b	>100	MT-3f	124.4
				MT-1c	38.91	MT-1g	55.24
MT-2c	45.29	MT-2g	54
				MT-3c	93.49	MT-3g	N/A
MT-1d	27.96	MT-1h	33.74
				MT-2d	27.88	MT-2h	36.5
MT-3d	26.09	MT-3h	>100

Note that: N/A indicates undetected, melittin HC ₅₀ The value was 18.47. Mu.g/mL.

The results of the hemolysis experiments are shown in Table 2 and FIG. 49, and the results of the experiments show that the side effects of the hemolysis of melittin glycopeptides are lower than those of the original peptides, wherein the hemolysis of MT-3a, MT-1b, MT-3b and MT-3h is obviously reduced.

Test example 3 cytotoxicity test

Cytotoxicity assays were performed using CCK-8, using human colorectal cancer cells (HCT-116), and three replicates were used.

Cell culture: cell suspensions were prepared, cell counts, seeded into 96-well plates: according to the number of suitable plating cells (about. Gtoreq.5X 10) ³ Per well), about 100 μl of cell suspension per well, the same sample was replicated 3 times.

Adding the liquid medicine: the 96-well plate with the cells laid is cultured in an incubator at 37 ℃ for 24 hours, and then fresh culture medium containing polypeptide (5 gradients from 100 mu M) is added for half dilution for further incubation for 48 hours.

CCK-8 solution (0.1X) was added: incubating at 37 ℃ for 1-4h. Optical Density (OD) was measured at a wavelength of 450nm using a microplate reader.

Cell viability (%) = [ (OD experimental well-OD blank well)/(OD control well-OD blank well) ]x100% cytotoxicity the results of the experiments are shown in fig. 50, and the results of the experiments indicate that the compounds MT-3d, MT-1h, MT-2h have cytotoxicity to human colorectal cancer stronger than that of melittin pro peptide. The melittin glycopeptides have high toxicity to human colorectal cancer cells and good effect of inhibiting tumor cell growth.

Claims

1. A melittin glycopeptide, characterized by: the melittin glycopeptide is Ac-GIGAVLKVLTTGLPALISWIKRKRQQ-NH ₂ Arginine at position 22 and/or 24 is modified by glycosylation as a peptide chain.

2. The melittin glycopeptide of claim 1, wherein: the arginine at position 22 and/or 24 is modified by galactose, N-acetylglucosamine, rhamnose, xylose, glucose, ribose, maltose, or lactose glycosylation.

3. The melittin glycopeptide of claim 1 or 2, wherein: the melittin glycopeptide is a compound shown in a formula I:

wherein R is ₁ 、R ₂ Independently selected from none or

And R is ₁ And R is ₂ At least one is selected from->

R ₃ Selected from the group consisting of

4. The melittin glycopeptide of claim 3, wherein: the melittin glycopeptide is a compound shown in a formula II:

wherein R is ₃ As shown in claim 3;

alternatively, the melittin glycopeptide is a compound of formula III:

wherein R is ₃ As shown in claim 3;

alternatively, the melittin glycopeptide is a compound of formula IV:

wherein R is ₃ As shown in claim 3.

5. The melittin glycopeptide of claim 4, wherein: the melittin glycopeptide is one of the following compounds:

/>

/>

6. the method for producing melittin glycopeptide according to any one of claims 1 to 5, wherein: it comprises the following steps:

(4) Removing acetyl groups on the sugar ligands;

7. The method of manufacturing according to claim 6, wherein:

and/or, in the step (3), the solvent is DMF;

and/or, in step (3), the base is triethylamine;

and/or, in the step (5), the solution used for cutting the peptide chain from the carrier resin is TIPS, H ₂ Mixing O, phenol and TFAMixing solution, TIPS, H ₂ The volume ratio of O, phenol and TFA is 2:5:5:88; the volume-mass ratio of the solution to the peptide is 1:20mL/g;

preferably, the method comprises the steps of,

8. Use of a melittin glycopeptide of any one of claims 1 to 5 in the manufacture of an antibacterial medicament;

9. Use of a melittin glycopeptide according to any one of claims 1 to 5 for the manufacture of an anti-tumour medicament;

10. A medicament, characterized in that: the melittin glycopeptide of any one of claims 1-5 is used as an active ingredient, and pharmaceutically acceptable auxiliary materials or auxiliary ingredients are added to prepare the preparation.