Peanut endogenous peptide Hypogin derivative and application thereof

Abstract

The invention belongs to the field of medicinal chemistry, and particularly relates to a peanut endogenous polypeptide Hypogin derivative and application thereof. The method comprises the following steps: firstly, carrying out structure simplification on a Hypogin endogenous peptide, searching an active fragment, and designing and synthesizing a series I of Hypogin derivatives; a Hypogin derivative with good activity in the series I is selected to be combined with cinnamic acid, and a series II compound is designed and synthesized. Research finds that the II-1 compound can well inhibit growth of sclerotium rolfsii mycelia, and also finds that I-12 affects a signal channel of sclerotium rolfsii PI3K, and potential targets are GPCR, RTK and PI3K of sclerotium rolfsii. Cytotoxicology tests show that II-1 has low human LO2 hepatotoxicity and conforms to the development direction of green bactericides. The Hypogin derivative disclosed by the invention has bacteriostatic activity, can be used as a basis for development of small peptide bacteriostatic agents, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry and relates to peanut endogenous peptides. Background Art

[0002] Peanut is one of the most important oil crops in the world and plays an important role in global oil production. In recent years, peanut white rot caused by Sclerotium rolfsii Sacc. has caused serious damage, severely restricted peanut production, and caused huge economic losses to the peanut industry. At present, the prevention and control of peanut diseases mainly relies on chemical control, but this method has problems such as pathogen resistance, pesticide residues and environmental pollution. Nowadays, people's awareness of their own health and environmental protection has gradually increased, and the choice of pesticides has also changed. They are increasingly inclined to choose green pesticides with less residues, safe for humans and animals, and less environmental pollution.

[0003] In recent years, peptides have become a hot topic in the field of plant protection due to their wide range of raw material sources, excellent activity and ideal environmental compatibility. They are widely used in insecticides, antibacterial, weed control, immune activation and plant growth regulation. The development of antimicrobial peptides for peanut white rot is in line with the development direction of green pesticides. Many experts at home and abroad have found that peanut peptides have a variety of good functional activities, such as anti-oxidation, free radical scavenging, antibacterial and antifungal, and immune enhancement. Chen Guitang et al. found that peanut peptides have strong antioxidant effects (Chen Guitang, Zhao Liyan, Cong Tao, et al. Preparation of peanut peptides and their antioxidant effects on oxidative damage model mice [J]. Food Science, 2007, (03): 324-327.). Zhang Yuhao et al. showed through experiments that peanut short peptides with a molecular weight of less than 1 ku can significantly inhibit the activity of angiotensin converting enzyme (Zhang Yuhao, Ma Liang, Wang Qiang. Study on the antihypertensive activity of peanut short peptides [J]. Food Science, 2008, (06): 399-403.). Chen Tong et al. found that the peptide Hypogin (HYP) produced by the hydrolysis of neutral protease A has an inhibitory effect on Escherichia coli and Fusarium graminearum, can inhibit the growth of fungi, and can inhibit the reverse transcription of HIV (Chen Tong, Wang Changqing, Bai Yunyun, et al. Study on the antibacterial effect of enzymatic peptides from peanut cake [J]. Agricultural Products Processing, 2015, (07): 23-25.).

[0004] With the development of science and technology, more methods will be applied to the preparation and research and development of antimicrobial peptides, and the application fields will continue to expand, including food preservation, agricultural development, medicine, cosmetics and other fields. Patent CN202411350080.3 discloses a peanut cysteine-rich peptide and its encoding gene, antibacterial agent and application. A new antimicrobial peptide family member was identified and extracted from the peanut transcriptome library and named peanut cysteine-rich peptide. The cysteine-rich peptide prepared by solid-phase chemical synthesis method exhibited broad-spectrum antibacterial activity, and its polypeptide fragments showed strong inhibitory and killing ability against a variety of common pathogens and crop molds. Peanut antimicrobial peptides are expected to become an important natural antibacterial substance in the future, bringing more benefits to human health and life, and their research and application will continue to deepen and develop. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a peanut endogenous peptide Hypogin derivative and its application.

[0006] The technical solution of the present invention is achieved in this way:

[0007] In one aspect, the present invention provides a peanut endogenous peptide Hypogin derivative, wherein the Hypogin derivative is selected from any of the following situations:

[0008] (1) tetrapeptide or pentapeptide fragments constituting the Hypogin polypeptide;

[0009] (2) A polypeptide-carboxylic acid compound having a structure similar to that of the compound in case (1) formed by carboxylation.

[0010] Preferably, the Hypogin derivative in the above situation (1) is one or more of KSPYY-NH2, QKKTE-NH2, NPQAQ-NH2, RQLQS-NH2, DDQEP-NH2, AKLK-NH2, PAKLK-NH2, SDDQE-NH2, QRQLQ-NH2, ENPQA-NH2, YQKKT-NH2 and KSPY-NH2.

[0011] Preferably, the carboxylic acid in the above situation (2) is cinnamic acid.

[0012] Preferably, the polypeptide-carboxylic acid compound in the above situation (2) is cinnamic acid-KSPY-NH2 or cinnamic acid-RQLQ-NH2.

[0013] In a second aspect, the above-mentioned peanut endogenous peptide Hypogin derivative is used in the preparation of drugs for preventing and treating white rot.

[0014] Preferably, the white rot is caused by infection with Sclerotium solani.

[0015] In a third aspect, the present invention also provides a drug, the active ingredients of which contain one or more of the above-mentioned peanut endogenous peptide Hypogin derivatives.

[0016] Preferably, the above-mentioned medicine further contains auxiliary materials that make the medicine present a corresponding dosage form.

[0017] Preferably, the above-mentioned agent type is any one of powder, wettable powder, soluble powder, granule, aqueous emulsion, microemulsion, water, suspension, dispersible oil suspension, microcapsule and emulsifiable concentrate.

[0018] Fourthly, the above-mentioned drug is used as a drug for preventing and treating peanut white rot.

[0019] The present invention has the following beneficial effects:

[0020] 1. The present invention obtains a highly active Hypogin polypeptide fragment by designing and optimizing the structure of the peanut endogenous polypeptide Hypogin, and further adopts an active substructure splicing method to introduce highly active natural carboxylic acids to improve the compound to obtain a polypeptide-carboxylic acid compound. The in vitro activity of the improved compound is better than that of the lead compound. The compound of the present invention is safe, efficient, environmentally friendly, and easy to synthesize, and has potential development and application prospects.

[0021] 2. The Hypogin derivatives series I and II provided by the present invention both exhibit antibacterial activity against Sclerotium sclerotiorum, among which compounds I-12 and II-1 have the best antibacterial effects, with antibacterial rates of 56.61% and 50.20%, respectively. The present invention selected II-1 for non-target human hepatocyte LO2 toxicity test, and the results showed that the Hypogin derivatives have extremely low cytotoxicity. The Hypogin derivatives provided by the present invention are new green fungicide candidate compounds, and their structural characteristics are small peptide fragments containing 4-5 amino acids, which are low or non-toxic to humans and the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 For compound I-1 1 H NMR (400M) spectrum.

[0024] Figure 2 This is the high resolution mass spectrum (HRMS) of compound I-1.

[0025] Figure 3 For compound II-1 1 H NMR (400M) spectrum.

[0026] Figure 4 This is the high resolution mass spectrum (HRMS) of compound II-1.

[0027] Figure 5 The I-12 compounds had antibacterial activity against Sclerotium sclerotiorum.

[0028] Figure 6 The II-1 compound has an antibacterial activity against Sclerotium sclerotiorum.

[0029] Figure 7 The effects of three polypeptides on the relative expression levels of phosphatidylinositol 3-kinase (PI3K) mRNA in Sclerotium microspore were aligned.

[0030] Figure 8 The molecular docking diagram of I-12 compound; (a) is rhodopsin from Pseudomonas aeruginosa, (b) is yeast RET; (c) is the molecular docking of yeast PI3K.

[0031] Fig. 9 The toxicity of II-1 compound and thiothiocarbamide to human LO2 cells. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0034] The present invention uses Hypogin as a lead compound, designs and optimizes the structure of the peanut endogenous polypeptide Hypogin, and uses a polypeptide solid phase synthesis method to obtain a highly active Hypogin polypeptide fragment. Further, an active substructure splicing method is used to introduce highly active natural carboxylic acids to improve the compound to obtain a polypeptide-carboxylic acid compound, and the in vitro activity of the improved compound is better than that of the lead compound.

[0035] The present invention provides peanut endogenous polypeptide Hypogin and its derivatives. The structure of the Hypogin derivative series I is shown below:

[0036]

[0037]

[0038] The structure of the Hypogin derivative series II is shown below:

[0039]

[0040] The mass spectrometry data of series I and series II are shown in Table 1:

[0041] Table 1 Mass spectral data of Hypogin derivatives series I and II

[0042]

[0043] The polypeptide compounds of the present application are synthesized by using the method of polypeptide solid phase synthesis, and further improved:

[0044] 1. The usage ratio (volume ratio) of the deprotecting agent 1,8-diazabicycloundec-7-ene (DBU) was optimized to 20%; 2. The post-treatment was simplified, trifluoroacetic acid was blown off by N2 gas, and most of the solvent was evaporated by a rotary evaporator to avoid TFA damaging the respiratory tract of the experimental operators.

[0045] Example 1

[0046] This example is a method for preparing compound I-1:

[0047] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of dichloromethane (DCM), shake for 30 minutes, add 15 mL of 20% (V / V) 1,8-diazabicycloundec-7-ene (DBU) in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0048] 2. Add 3 times the molar equivalent of Fmoc-L-Tyr-OH, then add 6 times the molar equivalent of diisopropylethylamine (DIEA), add 15 mL of dimethylformamide (DMF) to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0049] 3. Add 3 times molar equivalent of Fmoc-L-Tyr-OH, then add 6 times molar equivalent of DIEA, add 15 mL of dimethylformamide (DMF) to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0050] 4. Add 3 times molar equivalent of Fmoc-L-Pro-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0051] 5. Add 3 times molar equivalent of Fmoc-L-Ser-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 2 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0052] 6. Add 3 times molar equivalent of Fmoc-L-Lys-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0053] 7. Prepare cutting solution: trifluoroacetic acid (TFA) 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0054] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0055] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-1.

[0056] Example 2

[0057] This example is a method for preparing compound I-2:

[0058] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0059] 2. Add 3 times molar equivalent of Fmoc-L-Glu-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0060] 3. Add 3 times molar equivalent of Fmoc-L-Thr-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0061] 4. Add 3 times molar equivalent of Fmoc-L-Lys-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0062] 5. Add 3 times molar equivalent of Fmoc-L-Lys-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0063] 6. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0064] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0065] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0066] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-2.

[0067] Example 3

[0068] This example is a method for preparing compound I-3:

[0069] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0070] 2. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0071] 3. Add 3 times molar equivalent of Fmoc-L-Ala-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 2 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0072] 4. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0073] 5. Add 3 times molar equivalent of Fmoc-L-Pro-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0074] 6. Add 3 times molar equivalent of Fmoc-L-Asn-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0075] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0076] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0077] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-3.

[0078] Example 4

[0079] This example is a method for preparing compound I-4:

[0080] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0081] 2. Add 3 times molar equivalent of Fmoc-L-Ser-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 2 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0082] 3. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0083] 4. Add 3 times molar equivalent of Fmoc-L-Leu-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0084] 5. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0085] 6. Add 3 times the molar equivalent of Fmoc-L-Arg-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0086] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0087] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0088] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-4.

[0089] Example 5

[0090] This example is a method for preparing compound I-5:

[0091] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0092] 2. Add 3 times molar equivalent of Fmoc-L-Pro-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0093] 3. Add 3 times molar equivalent of Fmoc-L-Glu-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0094] 4. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0095] 5. Add 3 times molar equivalent of Fmoc-L-Asp-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0096] 6. Add 3 times molar equivalent of Fmoc-L-Asp-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0097] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0098] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0099] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-5.

[0100] Example 6

[0101] This example is a method for preparing compound I-6:

[0102] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0103] 2. Add 3 times the molar equivalent of Fmoc-L-Lys-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0104] 3. Add 3 times molar equivalent of Fmoc-L-Leu-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0105] 4. Add 3 times the molar equivalent of Fmoc-L-Lys-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0106] 5. Add 3 times molar equivalent of Fmoc-L-Ala-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 2 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0107] 6. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0108] 7. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0109] 8. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-6.

[0110] Example 7

[0111] This example is a method for preparing compound I-7:

[0112] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0113] 2. Add 3 times the molar equivalent of Fmoc-L-Lys-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0114] 3. Add 3 times molar equivalent of Fmoc-L-Leu-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0115] 4. Add 3 times molar equivalent of Fmoc-L-Lys-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0116] 5. Add 3 times molar equivalent of Fmoc-L-Ala-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 2 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0117] 6. Add 3 times molar equivalent of Fmoc-L-Pro-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0118] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0119] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0120] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-7.

[0121] Example 8

[0122] This example is a method for preparing compound I-8:

[0123] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0124] 2. Add 3 times molar equivalent of Fmoc-L-Glu-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0125] 3. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0126] 4. Add 3 times molar equivalent of Fmoc-L-Asp-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0127] 5. Add 3 times molar equivalent of Fmoc-L-Asp-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0128] 6. Add 3 times molar equivalent of Fmoc-L-Ser-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0129] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0130] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0131] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-8.

[0132] Example 9

[0133] This example is a method for preparing compound I-9:

[0134] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0135] 2. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0136] 3. Add 3 times molar equivalent of Fmoc-L-Leu-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0137] 4. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0138] 5. Add 3 times molar equivalent of Fmoc-L-Arg-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0139] 6. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0140] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0141] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0142] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-9.

[0143] Example 10

[0144] This example is a method for preparing compound I-10:

[0145] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0146] 2. Add 3 times the molar equivalent of Fmoc-L-Ala-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 2 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0147] 3. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0148] 4. Add 3 times molar equivalent of Fmoc-L-Pro-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0149] 5. Add 3 times molar equivalent of Fmoc-L-Asn-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0150] 6. Add 3 times molar equivalent of Fmoc-L-Glu-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0151] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0152] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0153] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-10.

[0154] Embodiment 11

[0155] This example is a method for preparing compound I-11:

[0156] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0157] 2. Add 3 times the molar equivalent of Fmoc-L-Thr-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0158] 3. Add 3 times the molar equivalent of Fmoc-L-Lys-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0159] 4. Add 3 times the molar equivalent of Fmoc-L-Lys-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0160] 5. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0161] 6. Add 3 times molar equivalent of Fmoc-L-Tyr-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0162] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0163] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0164] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-11.

[0165] Example 12

[0166] This example is a method for preparing compound I-12:

[0167] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0168] 2. Add 3 times the molar equivalent of Fmoc-L-Tyr-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0169] 3. Add 3 times molar equivalent of Fmoc-L-Pro-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0170] 4. Add 3 times molar equivalent of Fmoc-L-Ser-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0171] 5. Add 3 times molar equivalent of Fmoc-L-Lys-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 2 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0172] 6. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0173] 7. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0174] 8. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-12.

[0175] Example 13

[0176] This example is a method for preparing compound II-1:

[0177] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0178] 2. Add 3 times the molar equivalent of Fmoc-L-Tyr-OH, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0179] 3. Add 3 times molar equivalent of Fmoc-L-Pro-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0180] 4. Add 3 times molar equivalent of Fmoc-L-Ser-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0181] 5. Add 3 times molar equivalent of Fmoc-L-Lys-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 2 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0182] 6. Add 3 times the molar equivalent of cinnamic acid, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0183] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0184] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0185] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product II-1.

[0186] Embodiment 14

[0187] This example is a method for preparing compound II-2:

[0188] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution for deprotection for 20 minutes, add 10 mL of DCM to wash three times, and filter out the solvent DCM through a vacuum filtration device.

[0189] 2. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0190] 3. Add 3 times molar equivalent of Fmoc-L-Leu-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0191] 4. Add 3 times molar equivalent of Fmoc-L-Gln-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0192] 5. Add 3 times molar equivalent of Fmoc-L-Arg-OH, then add 6 times molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 3 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0193] 6. Add 3 times the molar equivalent of cinnamic acid, then add 6 times the molar equivalent of DIEA, add 15 mL of DMF to dissolve, and shake for 4 hours. After the reaction is completed, wash with DMF and DCM alternately for 3 times. Add 15 mL of 20% (V / V) DBU dichloromethane solution for deprotection for 20 minutes, wash with DMF and DCM alternately for 3 times, and monitor the reaction with Kaiser's reagent.

[0194] 7. Prepare cutting solution: TFA 90.0%; water 10.0%. Put the resin containing the crude product into a 50 mL flask and cut for 2.5 hours, then add 20 mL of 90.0% TFA aqueous solution.

[0195] 8. After the lysate was evaporated to remove the solvent using a rotary evaporator, it was washed six times with 15 mL of ether and then dried in a vacuum drying oven to obtain a crude peptide sequence.

[0196] 9. Use semi-preparative liquid chromatography to separate and purify the crude peptide, and finally freeze-dry the purified solution to obtain the target product II-2.

[0197] Application Example 1

[0198] Virulence assay of hypogin derivatives against the growth of whole Sclerotium mycelium:

[0199] The antibacterial activity of series I and series II against peanut white rot fungus was tested according to the mycelium growth rate method. First, the test strain Sclerotium uniformis was activated until the mycelium reached two-thirds of the culture dish before inoculation; the Hypogin derivative was taken and dissolved by adding 0.1% DMSO and 0.1% Tween 80, and sterile water was added to prepare a 4g / L mother solution. The final concentration of the drug-containing culture medium of the series I compound was 400mg / L, and the final concentration of the drug-containing culture medium of the series II compound was 200mg / L. After preparation, they were poured into a sterile 90mm disposable culture dish. A PDA plate containing 0.1% DMSO and 0.1% Tween-80 was used as a control. The activated 5mm bacterial cake was inoculated into the drug-containing PDA culture medium, and the culture dish was placed in a 28℃ incubator for dark culture. After 2 days, the diameter (mm) of the colony of each treatment was measured twice using the cross method, and the average value was taken to represent the size of the bacterial circle. Each treatment was repeated 3 times, and the test was performed twice. The calculation formula for the mycelium growth inhibition rate is as follows:

[0200] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - 5 mm) × 100%

[0201] The test results are shown in Table 2:

[0202] Table 2 In vitro inhibition of mycelial growth of Sclerotium truncatum by series I at a concentration of 400 mg / L

[0203]

[0204] Note: The data in the table are mean ± standard deviation. Different letters indicate significant differences among different treatments (Waller-Duncan, P<0.05).

[0205] From the results in Table 2, it can be seen that at a concentration of 400 mg / L, some of the I series compounds have inhibitory activity on the mycelial growth of Sclerotium sclerotium. Among them, the I-12 compound (structure KSPY-NH2) has the most obvious inhibitory effect on the growth of Sclerotium sclerotium, with an inhibition rate of 56.61%. Its antibacterial activity is as follows Figure 5 As shown; the second is compound I-4 (structure is RQLQS-NH2), whose antibacterial rate is 41.28%.

[0206] Table 3 In vitro inhibition of mycelial growth of Sclerotium truncatum by series II at a concentration of 200 mg / L

[0207]

[0208] As shown in Table 3, the antibacterial rate of compound II-1 modified with I-12 as the lead compound is 50.20% at a concentration of 200 mg / L. Figure 6 As shown; the antibacterial rate of compound II-2 is 27.93%, therefore, II-1 can be used as a secondary lead for further structural optimization.

[0209] Application Example 2:

[0210] Mechanism of action of target compound:

[0211] Table 4 PI3K content determined by enzyme-linked immunosorbent assay (pmol / L, n=4)

[0212]

[0213] Note: The values ​​are mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences.

[0214] The effects of two peptides (Table 4) at a concentration of 800 mg / L on the expression of PI3K gene in Sclerotium truncatum hyphae were tested ( Figure 7 ).Depend on Figure 7 It can be seen that after treatment with I-4 and I-12, the relative expression of PI3K gene mRNA decreased. Enzyme-linked immunosorbent assay found that after treatment with I-12, the PI3K content in the mycelium was significantly reduced (Table 4). Molecular docking experiments showed that I-12 had good docking activity with fungal GPCR, RTK and PI3K ( Figure 8 ). This indicates that the potential targets of HYP derivative I-12 are GPCR, RTK and PI3K of Sclerotium uniformis.

[0215] Application Example 3

[0216] Cytotoxicity experiment of Hypogin derivative II-1:

[0217] LO2 cells with good growth status were digested and single cell suspension was prepared. The cell density was adjusted to 1×10 5 / mL, inoculated into 96-well plates, 100μL per well, after culturing the cells for 24h, switch to serum-free high-glucose DMEM medium and continue culturing for 24h to starve the cells and stop the division activity, then discard the serum-free medium, add the corresponding drug to each well and incubate for 24h, after the drug action time is over, discard the medium containing the drug, add 100μL of fresh serum-free high-glucose DMEM medium and 20μL of MTT to each well, continue culturing in an incubator for 4h, carefully remove the supernatant, add 100μL DMSO to each well to dissolve the crystals, oscillate on a micro-oscillator for 10min to fully dissolve the crystals, and detect the OD value of each well at 492nm with an enzyme labeled analyzer.

[0218] The formula for calculating cytotoxicity is: Cell mortality rate = (1-sample absorbance / blank group absorbance) × 100%

[0219] Depend on Fig. 9 The results show that the II-1 compound has low toxicity to normal human liver cells LO2 cells. After being treated with 400 mg / L of the II-1 compound, the cell death rate of the LO2 cells is only 2.89%; however, at the same concentration, the mortality rate of the LO2 cells treated with the commercial fungicide thiophanate-methyl has reached 97.33%. This shows that the Hypogin derivatives of the present invention have low or no toxicity, are harmless to humans, animals and the environment, conform to the trend of sustainable development of green agriculture, and have good development and application prospects.

[0220] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A derivative of the endogenous peanut peptide Hypogin, characterized in that: The Hypogin derivative is selected from any one of the following: (1) Tetra-peptide or penta-peptide fragments constituting the Hypogin polypeptide; (2) A polypeptide-carboxylic acid compound having a structure similar to that of the compound in situation (1) formed by carboxylation.

2. The peanut endogenous peptide Hypogin derivative according to claim 1, characterized in that: The hypogin derivative in the case (1) is one or more of KSPYY-NH2, QKKTE-NH2, NPQAQ-NH2, RQLQS-NH2, DDQEP-NH2, AKLK-NH2, PAKLK-NH2, SDDQE-NH2, QRQLQ-NH2, ENPQA-NH2, YQKKT-NH2 and KSPY-NH2.

3. The peanut endogenous peptide Hypogin derivative according to claim 2, characterized in that: In the case (2), the carboxylic acid is cinnamic acid.

4. The peanut endogenous peptide Hypogin derivative according to claim 3, characterized in that: The polypeptide-carboxylic acid compound in the situation (2) is cinnamic acid-KSPY-NH2 or cinnamic acid-RQLQ-NH2.

5. Use of the peanut endogenous peptide Hypogin derivative according to any one of claims 1 to 4 in the preparation of a drug for preventing and treating white rot.

6. The use according to claim 5, characterized in that: The white rot is caused by infection with Sclerotium solani.

7. A drug, characterized in that: The active ingredients of the drug contain one or more of the peanut endogenous peptide Hypogin derivatives described in any one of claims 1-4.

8. The drug according to claim 7, characterized in that: The drug also contains auxiliary materials that make the drug into a corresponding dosage form.

9. The drug according to claim 8, characterized in that: The drug type is any one of powder, wettable powder, soluble powder, granule, aqueous emulsion, microemulsion, water, suspension, dispersible oil suspension, microcapsule and emulsifiable concentrate.

10. Use of the drug according to any one of claims 7 to 9 as a drug for preventing and treating peanut white rot.

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