A kind of bee venom peptide glycopeptide and its preparation method and use

Abstract

The present invention provides a melittin glycopeptide and a preparation method and use thereof, belonging to the field of polypeptide drugs. The melittin glycopeptide described in the present invention has Ac‑GIGAVLKVLTTGLPALISWIKRKRQQ‑NH2 as a peptide chain, and the arginine at the 22nd and / or 24th positions is glycosylated. The present invention successfully prepares a class of melittin glycopeptides, and proves that the hemolytic effect of this class of melittin glycopeptides is greatly reduced compared to melittin, effectively solving the problem of strong hemolysis of melittin in the prior art, which is beneficial to clinical application. At the same time, the antibacterial activity and tumor cell inhibition activity of some melittin glycopeptides prepared by the present invention are significantly better than those of melittin, and have good application prospects in the research and development of preparation of antibacterial drugs and anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the field of polypeptide drugs, and specifically relates to a melittin glycopeptide and a preparation method and use thereof. Background Art

[0002] Melittin is the main component of bee venom that exerts pharmacological and biological activities, and has antibacterial, antiviral, antiparasitic, and antitumor effects. Studies have shown that melittin has the advantages of good thermal stability, low immunogenicity, low allergic reaction, and better bactericidal efficiency than conventional antibiotics, and has great potential medicinal value. However, due to its strong hemolytic property, the current clinical application of melittin is greatly limited.

[0003] Protein glycosylation is one of the most common and complex post-translational modifications (PTMs) of proteins in all areas of life. It plays a key role in regulating the biological complexity of eukaryotic organisms and has become one of the hot topics in current chemical and biological research. Glycosylation of peptide molecules can change the physical and pharmacological properties of the original peptide, which is expected to improve the therapeutic effect of peptide drugs and improve the defects of peptide drugs in clinical use.

[0004] Among the glycosylation modifications of peptides, arginine-glycosylation modification is a new glycosylation modification method. Studies have shown that some antimicrobial peptides modified by this method are essential for the colonization of pathogens and the production of pathological effects. At present, there are no literature reports on the glycosylation modification of bee venom peptide to obtain bee venom peptide glycopeptide. Summary of the invention

[0005] The purpose of the present invention is to provide a melittin glycopeptide and a preparation method and use thereof in view of the shortcomings of melittin in the prior art in clinical application.

[0006] The invention provides a melittin glycopeptide. The melittin glycopeptide has Ac-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 as a peptide chain, and the arginine at position 22 and / or position 24 is modified by glycosylation.

[0007] Furthermore, the arginine at position 22 and / or arginine at position 24 is glycosylated with galactose, N-acetylglucosamine, rhamnose, xylose, glucose, ribose, maltose or lactose.

[0008] Furthermore, the melittin glycopeptide is a compound represented by formula I:

[0009]

[0010] Wherein, R1 and R2 are independently selected from none or and at least one of R1 and R2 is selected from

[0011] R3 is selected from

[0012] Furthermore, the melittin glycopeptide is a compound represented by formula II:

[0013]

[0014] Among them, R3 is as shown above;

[0015] Alternatively, the melittin glycopeptide is a compound represented by formula III:

[0016]

[0017] Among them, R3 is as shown above;

[0018] Alternatively, the melittin glycopeptide is a compound represented by formula IV:

[0019]

[0020] Among them, R3 is as shown above.

[0021] Furthermore, the melittin glycopeptide is one of the following compounds:

[0022]

[0023]

[0024]

[0025] The present invention also provides a method for preparing the aforementioned melittin glycopeptide, which comprises the following steps:

[0026] (1) synthesizing the peptide chain represented by SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 on a resin by a peptide solid phase synthesis method, and acetylating the last amino acid after deprotection to obtain a peptide-containing resin;

[0027] (2) under the action of a deprotecting agent, removing the protecting group DDe of ornithine in the peptide chain represented by SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4 to expose the side chain amino group;

[0028] (3) in the presence of a base, reacting the peptide-containing resin containing exposed side chain amino groups obtained in step (2) with silver nitrate and a sugar ligand in a solvent to carry out a guanidation reaction, thereby connecting the sugar ligand to the peptide chain;

[0029] (4) Removal of acetyl groups from sugar ligands;

[0030] (5) The peptide chain is cut off from the carrier resin and purified to obtain the corresponding bee venom peptide glycopeptide.

[0031] Further,

[0032] In step (1), the polypeptide solid phase synthesis method is Fmoc solid phase synthesis method;

[0033] And / or, in step (2), the method for removing the protecting group DDe comprises the following steps: reacting the peptide-containing resin with hydrazine hydrate in a solvent;

[0034] and / or, in step (3), the mass ratio of the peptide-containing resin containing exposed side chain amino groups, silver nitrate, and sugar ligand is (1-10):(1-10):(1-10);

[0035] And / or, in step (3), the solvent is DMF;

[0036] And / or, in step (3), the base is triethylamine;

[0037] And / or, in step (3), the reaction temperature is room temperature; and / or, the reaction time is 6 to 12 hours;

[0038] And / or, in step (4), the method for removing the acetyl group on the sugar ligand comprises the following steps: removing the acetyl group on the sugar ligand using 5% hydrazine hydrate in a solvent;

[0039] And / or, in step (5), the solution used to cut the peptide chain from the carrier resin is a mixed solution of TIPS, H2O, phenol and TFA, the volume ratio of TIPS, H2O, phenol and TFA is 2:5:5:88; the volume mass ratio of the solution to the peptide is 1:20 mL / g;

[0040] And / or, in step (5), the purification method is reverse phase high performance liquid chromatography;

[0041] Preferably,

[0042] In step (1), in the Fmoc solid phase synthesis method, the condensing agent used is a DIC-Oxyme condensation system, the activating agent is DIC, and NMP is used as the solvent; and / or, the deprotecting reagent is a mixed solution of Oxyme, piperidine and DMF, and the ratio is 7.1:100:400 (g / ml / ml);

[0043] And / or, in step (1), when the last amino acid is deprotected and then acetylated, the acetylation reagent is a mixture of DIEA, acetic anhydride and DMF, and the feed ratio is 1:1:8 (v / v);

[0044] And / or, in step (2), the deprotecting agent is a 2% hydrazine hydrate solution in DMF, and the feed ratio is resin loading: NH2NH2 reagent: DMF = 0.1: 0.2: 10 (mmol / ml / ml);

[0045] And / or, in step (2), when removing the protecting group DDe, the solvent is DMF;

[0046] And / or, in step (4), when removing the acetyl group on the sugar ligand, the solvent is DMF.

[0047] Furthermore, the sugar ligand structure is as follows:

[0048]

[0049] The present invention also provides the use of the aforementioned melittin glycopeptide in the preparation of antibacterial drugs;

[0050] Preferably, the drug is a drug against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Salmonella.

[0051] The present invention also provides the use of the aforementioned melittin glycopeptide in the preparation of anti-tumor drugs;

[0052] Preferably, the drug is a drug for preventing and / or treating colorectal cancer.

[0053] The present invention also provides a medicine, which is a preparation prepared by taking the aforementioned bee venom peptide glycopeptide as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0054] Compared with the prior art, the advantages of the present invention are:

[0055] 1. The present invention designs and synthesizes a class of bee venom peptide glycopeptides. Experiments have confirmed that some bee venom peptide glycopeptides can improve antibacterial activity and inhibit tumor cells. At the same time, most bee venom peptide glycopeptides can reduce hemolytic side effects and have potential application value in antibacterial and anti-tumor treatment.

[0056] 2. The present invention uses amino resin as a carrier, and synthesizes a peptide chain in a DIC-Oxime condensation system according to the template melittin: Ac-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 amino acid sequence through Fmoc solid phase synthesis. Wherein, ornithine is substituted at the position of arginine (R) to synthesize a long peptide fragment of the primary sequence. The side chain protecting group of ornithine is removed by treatment with a DMF solution containing 2% hydrazine hydrate, and then the sugar ligand is connected to the peptide chain on the resin under the action of triethylamine and AgNO3, and the acetyl group on the sugar ligand is removed by treatment with a DMF solution containing 5% hydrazine hydrate, and finally the target melittin glycopeptide is cut off from the resin. After the obtained compound is purified, HPLC and MS spectra are used for characterization and analysis. The method is simple and easy, and the purity of the obtained melittin glycopeptide is greater than 95%.

[0057] In summary, the present invention successfully prepared a class of bee venom peptide glycopeptides, and proved that the hemolytic effect of this class of bee venom peptide glycopeptides is greatly reduced compared with bee venom peptide, which effectively solves the problem of strong hemolysis of bee venom peptide in the prior art, and is conducive to clinical application. At the same time, the antibacterial activity and tumor cell inhibition activity of some bee venom peptide glycopeptides prepared by the present invention are significantly better than those of bee venom peptide, and have good application prospects in the research and development of preparation of antibacterial drugs and anti-tumor drugs.

[0058] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0059] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the HPLC spectrum of MT-1a.

[0061] Figure 2 This is the mass spectrum of MT-1a.

[0062] Figure 3 This is the HPLC spectrum of MT-2a.

[0063] Figure 4 This is the mass spectrum of MT-2a.

[0064] Figure 5 This is the HPLC spectrum of MT-3a.

[0065] Figure 6This is the mass spectrum of MT-3a.

[0066] Figure 7 This is the HPLC spectrum of MT-1b.

[0067] Figure 8 This is the mass spectrum of MT-1b.

[0068] Fig. 9 The HPLC spectrum of MT-2b.

[0069] Fig.10 This is the mass spectrum of MT-2b.

[0070] Fig.11 This is the HPLC spectrum of MT-3b.

[0071] Fig.12 This is the mass spectrum of MT-3b.

[0072] Fig.13 This is the HPLC spectrum of MT-1c.

[0073] Fig.14 This is the mass spectrum of MT-1c.

[0074] Fig.15 This is the HPLC spectrum of MT-2c.

[0075] Fig.16 This is the mass spectrum of MT-2c.

[0076] Fig.17 The HPLC spectrum of MT-3c.

[0077] Fig.18 This is the mass spectrum of MT-3c.

[0078] Fig.19 This is the HPLC spectrum of MT-1d.

[0079] Fig. 20 This is the mass spectrum of MT-1d.

[0080] Fig.21 This is the HPLC spectrum of MT-2d.

[0081] Fig. 22 This is the mass spectrum of MT-2d.

[0082] Fig.23 This is the HPLC spectrum of MT-3d.

[0083] Fig.24 This is the mass spectrum of MT-3d.

[0084] Fig.25 This is the HPLC spectrum of MT-1e.

[0085] Fig.26 This is the mass spectrum of MT-1e.

[0086] Fig. 27 This is the HPLC spectrum of MT-2e.

[0087] Fig.28 This is the mass spectrum of MT-2e.

[0088] Fig.29 The HPLC spectrum of MT-3e.

[0089] Fig.30 This is the mass spectrum of MT-3e.

[0090] Fig.31 This is the HPLC spectrum of MT-1f.

[0091] Fig.32 This is the mass spectrum of MT-1f.

[0092] Fig.33 This is the HPLC spectrum of MT-2f.

[0093] Fig.34 This is the mass spectrum of MT-2f.

[0094] Fig.35 The HPLC spectrum of MT-3f.

[0095] Fig.36 This is the mass spectrum of MT-3f.

[0096] Fig.37 This is the HPLC spectrum of MT-1g.

[0097] Fig.38 This is the mass spectrum of MT-1g.

[0098] Fig.39 This is the HPLC spectrum of MT-2g.

[0099] Fig.40 This is the mass spectrum of MT-2g.

[0100] Fig.41 This is the HPLC spectrum of MT-3g.

[0101] Fig.42 This is the mass spectrum of MT-3g.

[0102] Fig.43 This is the HPLC spectrum of MT-1h.

[0103] Fig.44 This is the mass spectrum of MT-1h.

[0104] Fig.45 This is the HPLC spectrum of MT-2h.

[0105] Fig.46 This is the mass spectrum of MT-2h.

[0106] Fig.47 This is the HPLC spectrum of MT-3h.

[0107] Fig.48 is the mass spectrum of MT-3h.

[0108] Fig.49 The hemolytic effect of each bee venom glycopeptide on rabbit erythrocytes.

[0109] Fig.50 The results of the cytotoxicity experiments of various bee venom glycopeptides on human colorectal cancer HCT-116. DETAILED DESCRIPTION

[0110] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0111] The 8 sugar ligands used in the present invention are prepared according to the literature Pan M, Li S, Li X, et al. Synthesis of and specific antibody generation for glycopeptides with arginine N-GlcNAcylation [J]. Angew Chem Int Ed Engl, 2014, 53 (52): 14517-14521 and Ye W, YuleL, 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 structures of the 8 sugar ligands are as follows:

[0112]

[0113] Among them, galactose ligand and glucose ligand are isomers of each other; xylose ligand and ribose ligand are isomers of each other; maltose ligand and lactose ligand are isomers of each other.

[0114] The present invention is based on the template melittin:

[0115] Ac-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 (SEQ ID NO.1) amino acid sequence was used to design and synthesize 24 melittin glycopeptides. The specific sequence structure of each melittin glycopeptide is shown below:

[0116]

[0117]

[0118]

[0119] Among them, MT-1a~3a and MT-1e~3e are isomers of each other; MT-1d~3d and MT-1f~3f are isomers of each other; MT-1g~3g and MT-1h~3h are isomers of each other.

[0120] The abbreviations involved in the following examples are explained as follows: Fmoc: fluorenylmethoxycarbonyl; DCM: dichloromethane; DMF: N,N-dimethylformamide; Oxyme: ethyl 2-oximecyanoacetate; DIC: N,N-diisopropylcarbodiimide; NMP: N-methylpyrrolidone; TFA: trifluoroacetic acid; TIPs: triisopropylsilane.

[0121] The sources of some experimental materials involved are as follows: amino acids and amino resins were purchased from Shanghai Jier Biochemical Co., Ltd.; N-methylpyrrolidone (NMP), N,N-diisopropylcarbodiimide (DIC), ethyl 2-oximecyanoacetate (Oxyme), trifluoroacetic acid (TFA), and acetonitrile (chromatographic grade) were purchased from Hengyuxiang Chemical Glass Experimental Instrument Business Department in the High-tech Zone; N,N-dimethylformamide (DMF), anhydrous ether, dichloromethane (DCM), piperidine, and phenol were all of analytical grade and purchased from Hengyuxiang Chemical Glass Experimental Instrument Business Department in the High-tech Zone.

[0122] Example 1. Preparation of Melittin Glycopeptide MT-1a of the Present Invention

[0123] 1. Synthesis of substrate peptide of bee venom peptide glycopeptide

[0124] The structures of the substrate peptides of bee venom glycopeptide (compound 1, compound 2 and compound 3) are as follows:

[0125]

[0126]

[0127]

[0128] In the present invention Resin refers to the support for solid phase synthesis.

[0129] (1) Preparation of Compound 1

[0130] Take 526 mg of amino resin (Rink Amide resin) (the loading amount is 0.38 mmol g -1 ) was added into a solid phase synthesis reaction tube, soaked in DCM for 20 min to allow the resin to fully swell, and then drained for later use.

[0131] Add 20% piperidine-DMF solution (0.1M ethyl 2-oximecyanoacetate) until the resin is completely submerged, shake at 25°C for 5 min×2 to remove Fmoc on the resin, and wash the resin with DMF, DCM, and DMF 5, 5, and 3 times in sequence. Dissolve the first amino acid in the sequence (1 mmol), Oxyme (142 mg, 1 mmol) and DIC (155.0 μL, 1 mmol) in 6 ml N-methylpyrrolidone (NMP), add to the resin, shake at 60°C for 20 min, and wash the resin with DMF, DCM, and DMF 5, 5, and 3 times in sequence. Repeat the deprotection and coupling amino acid steps. According to the peptide sequence: Ac-GIGAVLKVLTTGLPALISWIKRKOrnQQ-NH2, Fmoc amino acid (1mmol), Oxyme (142mg) and DIC (155μl) were mixed and dissolved in 6ml NMP, added to the resin, and shaken at 60°C for 20min, and deprotection → condensation → deprotection were repeated until all amino acids were connected. After the last amino acid was deprotected, 10ml of DIEA: acetic anhydride: DMF (volume ratio = 1:1:1) was added and shaken at 25°C for 20min. The resin was washed with DMF, DCM, and DMF 5, 5, and 3 times in sequence, and then washed with anhydrous ether, and the resin was vacuum dried to obtain compound 1.

[0132] (2) Preparation of Compound 2

[0133] According to the polypeptide sequence: Ac-GIGAVLKVLTTGLPALISWIKOrnKRQQ-NH2, the method was the same as that of compound 1 to obtain compound 2.

[0134] (3) Preparation of Compound 3

[0135] According to the polypeptide sequence: Ac-GIGAVLKVLTTGLPALISWIKOrnKOrnQQ-NH2, compound 3 was obtained by the same preparation method as compound 1.

[0136] 2. Removal of the protecting group DDe of ornithine on the substrate peptide of bee venom peptide

[0137] Ornithine in the substrate peptide chain is abbreviated as Orn, and the structure is shown below:

[0138]

[0139] The protecting group DDe of ornithine on the substrate peptide was removed to prepare compound 4, compound 5 and compound 6. The structures of compound 4, compound 5 and compound 6 are as follows:

[0140]

[0141]

[0142]

[0143] (1) Preparation of Compound 4

[0144] To 526 mg of dried peptide-containing resin (Compound 1), a DMF solution (10 ml) containing 2% hydrazine hydrate was added and the mixture was shaken at 25°C for three times, each time for 6 min. After the reaction was completed, the resin was washed with DCM, DMF and anhydrous ether for three times each, and the resin was vacuum dried to obtain Compound 4.

[0145] (2) Preparation of Compound 5

[0146] The starting material is a peptide-containing resin (Compound 2), and the method is the same as that of Compound 4 to obtain Compound 5.

[0147] (3) Preparation of Compound 6

[0148] The starting material is a peptide-containing resin (compound 3), and the method is the same as that of compound 4 to obtain compound 6.

[0149] 3. Preparation of Melittin Glycopeptide MT-1a

[0150] (1) Preparation of crude MT-1a

[0151] 263.2 mg of peptide-containing resin (compound 4) was placed in a 50 ml centrifuge tube, and 102 mg of silver nitrate, 278 μl of triethylamine, 400 mg of galactose ligand and 10 ml of DMF were added. The reaction was shaken at room temperature for 12 h. After completion, the resin was washed with DMF, MeOH and DCM for 5, 5 and 3 times respectively. The resin was vacuum dried to obtain compound 7.

[0152]

[0153] After completing the above steps, mix 263.2 mg of resin (compound 7) with a DMF solution (10 ml) containing 5% hydrazine hydrate and shake the reaction at room temperature for 10 hours to remove the acetyl group on the sugar ligand. After completion, wash the resin with DMF, H2O, MeOH, and DCM 5, 5, 5, and 5 times in sequence. Vacuum dry the resin to obtain compound 8.

[0154]

[0155] Add 10 mL of TIPS:H2O:phenol:TFA=2:5:5:88 (V / V / V / V) to compound 8, shake at room temperature for 4 h, filter, wash the resin with a little TFA, and collect the filtrate. Pour icy ether into the filtrate for precipitation and centrifugation, discard the supernatant, continue to wash with icy ether and centrifuge three times, and blow dry with argon to obtain the crude MT-1a.

[0156] (2) Purification of MT-1a

[0157] 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:

[0158] Instrument: Pre-HPLC SD-1VARIAN high performance liquid chromatograph;

[0159] Chromatographic column: YMC-Pack ODS-AQ (250×20mml.D, S-10μm, 12nm);

[0160] Mobile phase: Mobile phase A is an aqueous solution with a volume fraction of 0.1% TFA, and mobile phase B is an acetonitrile solution with a volume fraction of 0.1% TFA;

[0161] Steps and parameters: 38% mobile phase B elution for 0-5 min, 48%-75% mobile phase B elution for 5-80 min; flow rate is 15 ml / min, injection volume is 5 ml, and detection wavelength is 214 nm.

[0162] Finally, the purified MT-1a product was prepared.

[0163] (3) Product identification and structural analysis

[0164] The MT-1a prepared above was identified by HPLC and structurally analyzed by HR-Q-TOF-MS (high resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometry). The chromatographic mobile phases were acetonitrile and water. 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, with gradient elution (0-5 min, mobile phase B: 5%; 5-30 min, mobile phase B: 5%-95%); flow rate 1.0 mL min -1 ; Detection wavelength 214nm and 254nm, injection volume 20μl. The peak time was consistent with the main peak of the crude product, and the purity of the bee venom peptide prepared by this method was >98% ( Figure 1 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Figure 2 shown.

[0165] Example 2: Preparation of Melittin Glycopeptide MT-2a of the Present Invention

[0166] Using 263.2 mg of peptide-containing resin (Compound 5) and 400 mg of galactose ligand as raw materials, the purified product of MT-2a was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 68% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0167] The purified MT-2a product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Figure 3 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Figure 4 shown.

[0168] Example 3: Preparation of Melittin Glycopeptide MT-3a of the Present Invention

[0169] Using 263.2 mg of peptide-containing resin (Compound 6) and 800 mg of galactose ligand as raw materials, the MT-3a purified product was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 68% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0170] The purified MT-3a product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Figure 5 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Figure 6 shown.

[0171] Example 4: Preparation of Melittin Glycopeptide MT-1b of the Present Invention

[0172] Using 263.2 mg of peptide-containing resin (compound 4) and 410 mg of N-acetylglucosamine sugar ligand as raw materials, the MT-1b purified product was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 40% to 70% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0173] The purified MT-1b product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Figure 7 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Figure 8 shown.

[0174] Example 5: Preparation of Melittin Glycopeptide MT-2b of the Present Invention

[0175] Using 263.2 mg of peptide-containing resin (Compound 5) and 410 mg of N-acetylglucosamine sugar ligand as raw materials, the purified product of MT-2b was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 40% to 70% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0176] The purified MT-2b product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig. 9 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.10 shown.

[0177] Example 6: Preparation of Melittin Glycopeptide MT-3b of the Present Invention

[0178] Using 263.2 mg of peptide-containing resin (Compound 6) and 820 mg of N-acetylglucosamine sugar ligand as raw materials, the purified product of MT-3b was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 68% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0179] The purified MT-3b product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.11 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.12 shown.

[0180] Example 7: Preparation of Melittin Glycopeptide MT-1c of the Present Invention

[0181] Using 263.2 mg of peptide-containing resin (compound 4) and 377 mg of rhamnose glycoligand as raw materials, the MT-1c purified product was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 40% mobile phase B elution for 0 to 5 min, 40% to 60% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0182] The purified MT-1c product was identified and structurally analyzed according to the method of Example 1. The peak time of the main peak was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.13 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.14 shown.

[0183] Example 8: Preparation of Melittin Glycopeptide MT-2c of the Present Invention

[0184] Using 263.2 mg of peptide-containing resin (Compound 5) and 377 mg of rhamnose ligand as raw materials, the purified product of MT-2c was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 55% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0185] The purified MT-2c product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.15 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.16 shown.

[0186] Example 9: Preparation of Melittin Glycopeptide MT-3c of the Present Invention

[0187] Using 263.2 mg of peptide-containing resin (Compound 6) and 754 mg of rhamnose ligand as raw materials, the purified product of MT-3c was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 37% mobile phase B elution for 0 to 5 min, 37% to 60% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0188] The purified MT-3c product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.17 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.18 shown.

[0189] Example 10: Preparation of Melittin Glycopeptide MT-1d of the Present Invention

[0190] Using 263.2 mg of peptide-containing resin (compound 4) and 368 mg of xylose ligand as raw materials, the purified product MT-1d was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 40% mobile phase B elution for 0 to 5 min, 40% to 70% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0191] The purified MT-1d product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.19 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig. 20 shown.

[0192] Example 11. Preparation of Melittin Glycopeptide MT-2d of the Present Invention

[0193] Using 263.2 mg of peptide-containing resin (Compound 5) and 338 mg of xylose ligand as raw materials, the purified product MT-2d was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 40% mobile phase B elution for 0 to 5 min, 40% to 70% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0194] The purified MT-2d product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.21 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig. 22 shown.

[0195] Example 12: Preparation of Melittin Glycopeptide MT-3d of the Present Invention

[0196] Using 263.2 mg of peptide-containing resin (Compound 6) and 736 mg of xylose ligand as raw materials, the purified product of MT-3d was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 68% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0197] The purified MT-3d product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.23 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.24 shown.

[0198] Example 13: Preparation of Melittin Glycopeptide MT-1e of the Present Invention

[0199] Using 263.2 mg of peptide-containing resin (compound 4) and 400 mg of glucose ligand as raw materials, the MT-1e purified product was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 65% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0200] The purified MT-1e product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.25 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.26 shown.

[0201] Example 14: Preparation of Melittin Glycopeptide MT-2e of the Present Invention

[0202] Using 263.2 mg of peptide-containing resin (Compound 5) and 400 mg of glucose ligand as raw materials, the purified product of MT-2e was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 55% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0203] The purified MT-2e product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig. 27 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.28 shown.

[0204] Example 15. Preparation of Melittin Glycopeptide MT-3e of the Present Invention

[0205] Using 263.2 mg of peptide-containing resin (Compound 6) and 800 mg of glucose ligand as raw materials, the purified product of MT-3e was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 35% mobile phase B elution for 0 to 5 min, 35% to 50% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0206] The purified MT-3e product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.29 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.30 shown.

[0207] Example 16. Preparation of Melittin Glycopeptide MT-1f of the Present Invention

[0208] Using 263.2 mg of peptide-containing resin (compound 4) and 368 mg of ribose ligand as raw materials, the MT-1f purified product was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 36% mobile phase B elution for 0 to 5 min, 36% to 60% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0209] The purified MT-1f product was identified and structurally analyzed according to the method of Example 1. The peak time of the main peak was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.31 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.32 shown.

[0210] Example 17: Preparation of Melittin Glycopeptide MT-2f of the Present Invention

[0211] Using 263.2 mg of peptide-containing resin (Compound 5) and 368 mg of ribose ligand as raw materials, the purified product of MT-2f was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 40% mobile phase B elution for 0 to 5 min, 40% to 65% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0212] The purified MT-2f product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.33 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.34 shown.

[0213] Example 18: Preparation of Melittin Glycopeptide MT-3f of the Present Invention

[0214] Using 263.2 mg of peptide-containing resin (Compound 6) and 736 mg of ribose ligand as raw materials, the purified product of MT-3f was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 35% mobile phase B elution for 0 to 5 min, 35% to 50% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0215] The purified MT-3f product was identified and structurally analyzed according to the method of Example 1. The peak time of the main peak was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.35 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.36 shown.

[0216] Example 19: Preparation of Melittin Glycopeptide MT-1g of the Present Invention

[0217] Using 263.2 mg of peptide-containing resin (compound 4) and 593 mg of maltose ligand as raw materials, the purified product MT-1g was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 50% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0218] The purified MT-1g product was identified and structurally analyzed according to the method of Example 1. The peak time of the main peak was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.37 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.38 shown.

[0219] Example 20: Preparation of Melittin Glycopeptide MT-2g of the Present Invention

[0220] Using 263.2 mg of peptide-containing resin (Compound 5) and 593 mg of maltose ligand as raw materials, MT-2 g of purified product was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 55% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0221] The purified MT-2g product was identified and structurally analyzed according to the method of Example 1. The peak time of the main peak was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.39 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.40 shown.

[0222] Example 21. Preparation of Melittin Glycopeptide MT-3g of the Present Invention

[0223] Using 263.2 mg of peptide-containing resin (Compound 6) and 800 mg of maltose ligand as raw materials, MT-3g purified product was prepared and purified according to the preparation method of melittin glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 60% mobile phase B elution for 5 to 60 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0224] The purified MT-3g product was identified and structurally analyzed according to the method of Example 1. The peak time of the main peak was consistent with that of the crude product. The purity of the bee venom peptide glycopeptide prepared by this method was poor ( Fig.41 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.42 shown.

[0225] Example 22: Preparation of Melittin Glycopeptide MT-1h of the Present Invention

[0226] Using 263.2 mg of peptide-containing resin (compound 4) and 593 mg of lactose ligand as raw materials, the MT-1h purified product was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 60% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0227] The purified MT-1h product was identified and structurally analyzed according to the method of Example 1. The peak time of the main peak was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.43 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.44 shown.

[0228] Example 23. Preparation of Melittin Glycopeptide MT-2h of the Present Invention

[0229] Using 263.2 mg of peptide-containing resin (Compound 5) and 593 mg of lactose ligand as raw materials, the purified product of MT-2h was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 38% mobile phase B elution for 0 to 5 min, 38% to 60% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0230] The purified MT-2h product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.45 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.46 shown.

[0231] Example 24: Preparation of Melittin Glycopeptide MT-3h of the Present Invention

[0232] Using 263.2 mg of peptide-containing resin (Compound 6) and 800 mg of lactose ligand as raw materials, the purified product of MT-3h was prepared and purified according to the preparation method of bee venom glycopeptide MT-1a. The purification steps and parameters were as follows: 35% mobile phase B elution for 0 to 5 min, 35% to 50% mobile phase B elution for 5 to 80 min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0233] The purified MT-3h product was identified and structurally analyzed according to the method of Example 1. The peak time was consistent with that of the crude product, and the purity of the melittin glycopeptide prepared by this method was >98% ( Fig.47 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Fig.48 shown.

[0234] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0235] Test Example 1: Antibacterial Activity Test

[0236] Preparation of antimicrobial drug stock solution: Prepare the target compound with physiological saline to 1024 μg / ml for later use.

[0237] Dilution of antibiotics: dilute the stock solution of antibiotics in multiples with MH broth in a 96-well plate (the concentration range of the drug-containing broth series should cover the sensitive and resistant breakpoints of the drug, as well as the MIC range of the quality control bacteria).

[0238] Inoculation: First prepare a 0.5 McFarland turbidity bacterial suspension, then dilute the suspension 1:100 (concentration of about 10 6 CFU / ml), and 100 μl of the diluted bacterial suspension was added to the above-mentioned 96-well plate containing the drug broth. Mix well, and the concentration of the antibiotic is diluted 1:2. The final bacterial solution density is about 5×10 6 CFU / ml.

[0239] Set up control wells: Growth control: 100 μl of broth without antibiotics and 100 ul of diluted bacterial solution; Negative control: 200 μl of broth with only antibiotics added and 200 μl of broth without antibiotics.

[0240] Purity check of inoculum: Take an inoculation loop and streak the diluted bacterial solution onto a blood agar plate, then place the plate in a constant temperature incubator at 37°C to check the purity of the inoculum.

[0241] Incubation: The inoculated 96-well plate containing the bacterial-drug mixture was placed in a constant temperature incubator at 37°C for 16 to 20 hours.

[0242] Check the control wells: first observe the bacterial purity and test the bacterial growth on the plate to determine whether it is contaminated. Then observe the bacterial growth in the growth control tube (it should grow). The negative control tube should have no sterile growth; check whether the MIC value of the quality control strain is within the corresponding quality control range.

[0243] Interpretation of results: The highest drug dilution in the test tube or well with no visible bacterial growth is the endpoint well, and its drug concentration is the MIC value. According to the measured MIC value, refer to the CISI document MIC interpretation standard to determine whether the final result is resistant (R), sensitive (S) or intermediate (I).

[0244] The results are shown in Table 1, and it is found that some of the melittin glycopeptides of the present invention can enhance the antibacterial activity of melittin, among which the MIC values ​​of MT-1a, MT-3a, and MT-2c against Puzza Streptococcus are reduced by one time; the MIC values ​​of MT-2d and MT-2h against E. coli are reduced by one time. This indicates that the antibacterial activity of these melittin glycopeptides is significantly improved compared with melittin.

[0245] Table 1. Antibacterial activity results (MIC value μg / mL)

[0246]

[0247]

[0248] Note: N / A means not tested.

[0249] Test Example 2: Hemolysis Test

[0250] Preparation of red blood cell suspension: Take the blood of a rabbit in good growth condition, place it in a pre-treated heparin-coated centrifuge tube, centrifuge at 2500r / min for 10min, discard the upper plasma, wash the precipitate with an equal volume of PBS solution until the upper solution is clear, resuspend the blood cell solution with PBS buffer, mix well, and make the final concentration of 2%.

[0251] Dilution of sample concentration: dilute bee venom glycopeptide with PBS to prepare working solutions with concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 μg / ml.

[0252] Take clean 1.5ml centrifuge tubes and number them from one to six. Add 500μl of previously treated cell suspension to each tube. Set an equal volume of PBS as the negative control group and 1% TritonX-100 as the positive control group. Add the above prepared working solutions to the test tubes in turn. Incubate in a 37℃ water bath for 1h, 2500r / min, 10min, centrifuge and take the upper layer to measure OD 540 The hemolysis curve was drawn and the half hemolysis value (HC) was fitted by Graphpad software. 50 ), calculate the hemolysis rate and half hemolysis value (HC 50 ).

[0253] Hemolysis rate (%) = [(OD 样品 -OD 阴性 ) / (OD 阳性 -OD 阴性 )]×100%

[0254] Table 2. Effect of each group of bee venom peptides on HC of rabbit erythrocytes 50 (μg / ml) value

[0255] name <![CDATA[HC 50 (μg / ml)]]> name <![CDATA[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

[0256] Note: N / A means not tested, melittin original peptide HC 50 The value is 18.47μg / mL.

[0257] The results of the hemolysis test are shown in Table 2 and Fig.49 As shown, the experimental results show that the hemolytic side effects of bee venom peptide glycopeptide are lower than those of the original peptide, among which the hemolytic properties of MT-3a, MT-1b, MT-3b, and MT-3h are significantly reduced.

[0258] Test Example 3: Cytotoxicity Test

[0259] The CCK-8 method was used for cytotoxicity test, human colorectal cancer cells (HCT-116) were selected, and three parallel experiments were set up.

[0260] Cell culture: Prepare cell suspension, count cells, and inoculate into 96-well plates: according to the appropriate number of cells to be plated (approximately ≥5×10 3 Each well contained about 100 μl of cell suspension, and the same sample was repeated three times.

[0261] Addition of drug solution: The 96-well plate with cells was cultured in a 37°C incubator. After incubation for 24 hours, fresh culture medium containing the polypeptide (the concentration was diluted in half starting from 100 μM, and 5 gradients were made) was added and incubated for another 48 hours.

[0262] Add CCK-8 solution (0.1X): Incubate at 37°C for 1-4 h. Measure the optical density (OD) at 450 nm using a microplate reader.

[0263] Cell survival rate (%) = [(OD experimental well - OD blank well) / (OD control well - OD blank well)] × 100% Cytotoxicity test results are as follows Fig.50 As shown, the experimental results show that the toxicity of compounds MT-3d, MT-1h, and MT-2h to human colorectal cancer cells is stronger than that of melittin original peptide. This shows that these melittin glycopeptides are highly toxic to human colorectal cancer cells and have a good effect in inhibiting the growth of tumor cells.

[0264] In summary, the present invention successfully prepared a class of bee venom peptide glycopeptides, and proved that the hemolytic effect of this class of bee venom peptide glycopeptides is greatly reduced compared with bee venom peptide, which effectively solves the problem of strong hemolysis of bee venom peptide in the prior art, and is conducive to clinical application. At the same time, the antibacterial activity and tumor cell inhibition activity of some bee venom peptide glycopeptides prepared by the present invention are significantly better than those of bee venom peptide, and have good application prospects in the research and development of preparation of antibacterial drugs and anti-tumor drugs.

Claims

1. A melittin glycopeptide, characterized in that: The melittin glycopeptide is one of the following compounds:

2. The method for preparing the melittin glycopeptide according to claim 1, characterized in that: It includes the following steps: (1) synthesizing the peptide chain represented by SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 on a resin by a peptide solid phase synthesis method, and acetylating the last amino acid after deprotection to obtain a peptide-containing resin; (2) under the action of a deprotecting agent, removing the protecting group DDe of ornithine in the peptide chain represented by SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4 to expose the side chain amino group; (3) in the presence of a base, reacting the peptide-containing resin containing exposed side chain amino groups obtained in step (2) with silver nitrate and a sugar ligand in a solvent to carry out a guanidation reaction, thereby connecting the sugar ligand to the peptide chain; (4) Removal of acetyl groups from sugar ligands; (5) cutting the peptide chain from the carrier resin and purifying it to obtain the corresponding bee venom peptide glycopeptide; The sugar ligand structure is as follows:

3. The preparation method according to claim 2, characterized in that: In step (1), the polypeptide solid phase synthesis method is Fmoc solid phase synthesis method; And / or, in step (2), the method for removing the protecting group DDe comprises the following steps: reacting the peptide-containing resin with hydrazine hydrate in a solvent; and / or, in step (3), the mass ratio of the peptide-containing resin containing exposed side chain amino groups, silver nitrate, and sugar ligand is (1-10):(1-10):(1-10); And / or, in 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 to 12 hours; And / or, in step (4), the method for removing the acetyl group on the sugar ligand comprises the following steps: removing the acetyl group on the sugar ligand using 5% hydrazine hydrate in a solvent; And / or, in step (5), the solution used to cut the peptide chain from the carrier resin is a mixed solution of TIPS, H2O, phenol and TFA, the volume ratio of TIPS, H2O, phenol and TFA is 2:5:5:88; the volume mass ratio of the solution to the peptide is 1:20 mL / g; And / or, in step (5), the purification method is reverse phase high performance liquid chromatography.

4. The preparation method according to claim 3, characterized in that: In step (1), in the Fmoc solid phase synthesis method, the condensing agent used is a DIC-Oxyme condensation system, the activating agent is DIC, and NMP is used as the solvent; and / or, the deprotecting reagent is a mixed solution of Oxyme, piperidine and DMF, and the ratio is 7.1:100:400 (g / ml / ml); And / or, in step (1), when the last amino acid is deprotected and then acetylated, the acetylation reagent is a mixture of DIEA, acetic anhydride and DMF, and the feed ratio is 1:1:8 (v / v); And / or, in step (2), the deprotecting agent is a 2% hydrazine hydrate solution in DMF, and the feed ratio is resin loading: NH2NH2 reagent: DMF = 0.1: 0.2: 10 (mmol / ml / ml); And / or, in step (2), when removing the protecting group DDe, the solvent is DMF; And / or, in step (4), when removing the acetyl group on the sugar ligand, the solvent is DMF.

5. Use of the melittin glycopeptide according to claim 1 in the preparation of drugs against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Salmonella.

6. Use of the melittin glycopeptide according to claim 1 in the preparation of a drug for treating colorectal cancer.

7. A drug, characterized in that: The preparation is prepared by taking the bee venom peptide glycopeptide as claimed in claim 1 as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

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