A staple peptide that can enhance broad-spectrum antibacterial activity, its preparation method and application

Abstract

This invention relates to a staple peptide with enhanced broad-spectrum antibacterial activity, its preparation method, and its applications. Using an amino resin as a carrier, the peptide chain is synthesized in a DIC-Oxime condensation system according to the template Chem-KVL:Ac-KVLGRLVKVLGRLV-NH2 amino acid sequence via Fmoc solid-phase synthesis. During synthesis, while retaining key amino acid residues, S5 is substituted at specific positions to replace the original amino acid. The linear peptide is then cyclized in a dichloroethane solution with Grubbs I reagent to obtain the target staple peptide. The method of this invention is simple, easy to implement, and yields high purity and high efficiency. Further experiments have confirmed that the staple peptide of this invention exhibits significant broad-spectrum antibacterial activity, including activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. In the pharmaceutical field, it can serve as an excellent alternative to antibiotics and has potential application value in in vitro and in vivo antibacterial therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of polypeptide drugs, in particular to a stapled peptide with improved broad-spectrum antibacterial activity and a preparation method and application thereof. BACKGROUND

[0002] Bacteria are widely present in human and nature. In the case of invasion of various pathogenic bacteria into human body and low collective resistance, bacterial infectious diseases may occur. For example, acute gastroenteritis, pneumonia, folliculitis, and the like, and in severe cases, gangrenous deep abscesses and the like may be infected. In order to ensure human health and improve the quality of life, a large number of antibacterial drugs have been developed and applied to the clinical treatment of bacterial infectious diseases. Among them, antibacterial peptides are widely used in clinical treatment. Antibacterial peptides are a kind of basic polypeptide substances found and isolated from insects, higher plants, mammals, bacteria, fungi and other organisms, which have antibacterial activity induced. They have broad-spectrum and high-efficiency bactericidal activity on bacteria. However, most of the antibacterial peptides have the problems of poor stability, strong drug resistance and the like. At present, solving the clinical use difficulty of antibacterial peptides and improving the in-vivo stability have become one of the research hotspots.

[0003] Existing studies have shown that the clinical use of some antibacterial peptides can be improved and side effects can be reduced by structural modification, gene fusion and the like. One of the modification methods is to introduce two unnatural amino acids containing α-methyl and α-alkenyl during the solid-phase synthesis of the peptide chain, and to cyclize the stable α-helix structure conformation of the all-carbon scaffold under the catalysis of the cyclization agent, thereby synthesizing the stapled peptide. The stapled polypeptide after modification significantly increases the affinity with the target and also significantly improves the cell permeability. The stapled modification technology can not only make the antibacterial peptide more stable to in-vivo proteases, but also can improve the activity and increase the clinical use. At present, the stapled technology has been a strategy that can effectively improve the activity, increase the protein stability and stabilize the polypeptide chain structure, which has been widely applied in drug chemistry for disease intervention. However, there are still problems such as drug resistance and side effects in clinical treatment. The application of all-carbon scaffold to form side chain ring structure to modify the activity conformation of α-helix peptide, i.e. stapled peptide, is the most direct and effective method to overcome these difficulties.

[0004] CN113651874A, published on November 16, 2021, discloses a stapling peptide with inhibitory effect on the growth and reproduction of Candida, and a preparation method and application thereof. The peptide chain is synthesized by Fmoc solid-phase synthesis method in a DIC-oxime condensation system according to the straight-chain peptide template Aurein1.2: Ac-GLFDIIKKIAESF-NH2 amino acid sequence, and on the basis of retaining key amino acid residues, S5 is used to replace the original amino acid at a specific position, and the straight-chain peptide is connected to the resin. After olefin metathesis reaction and ring closure in Grubbs I reagent dichloroethane solution, the target stapling peptide is obtained by cutting off the resin. However, the present inventors have noticed from the numerous information and literature reports in the prior art that human chemotatic protein, known for its antibacterial activity, is an inactive precursor protein with 163 amino acid residues, which can be widely expressed in various epithelial cells, and plays an important role in the chemotaxis of immune cells, the differentiation and metabolic function of adipocytes, and glucose metabolism function. In human chemotatic protein, a short peptide with 7 residues after tandem repeat sequence (Chem-KVL) was found to have strong broad-spectrum antibacterial activity, which has the highest density of hydrophobic residues and positively charged residues in the entire protein.

[0005] In the present patent, the tandem repeat short peptide (Chem-KVL) is designed to be stapled and modified to make its structure more stable. The i and i+4 position amino acids are replaced by S5 at the key residue position of the peptide chain, and the stapling peptide with stable structure is obtained after ring closure. However, there is no report on the stapling peptide with broad-spectrum antibacterial activity prepared by the present application and its preparation method and application. SUMMARY

[0006] The first object of the present application is to provide a stapling peptide and its preparation method and application.

[0007] The second object of the present application is to provide the use of the stapling peptide.

[0008] The third object of the present application is to provide a preparation method of the stapling peptide.

[0009] To achieve the above-mentioned first object, the technical solution adopted by the present application is:

[0010] A stapling peptide selected from one of the following:

[0011] a) Ac-KVLGRLVKVLGRLV-NH2 as a peptide chain template, wherein 10L and 14V are replaced by S5 and ring-closed;

[0012] b) Using Ac-KVLGRLVKVLGRLV-NH2 as a peptide template, where 9V and 13L are replaced by S5 and cyclized;

[0013] c) Using Ac-KVLGRLVKVLGRLV-NH2 as a peptide template, where 7V and 11G are replaced by S5 and cyclized;

[0014] d) Using Ac-KVLGRLVKVLGRLV-NH2 as a peptide template, where 6L and 10L are replaced by S5 and cyclized;

[0015] e) Using Ac-KVLGRLVKVLGRLV-NH2 as a peptide template, where 5R and 9V are replaced by S5 and cyclized;

[0016] f) Using Ac-KVLGRLVKVLGRLV-NH2 as a peptide template, where 3L and 7V are replaced by S5 and cyclized.

[0017] g) Using Ac-KVLGRLVKVLGRLV-NH2 as a peptide template, where 2V and 6L are replaced by S5 and cyclized.

[0018] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:

[0019] The application of the staple peptide in the preparation of antibacterial drugs.

[0020] The staple peptide is used in the preparation of preparations that inhibit harmful bacteria such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0021] To achieve the third objective mentioned above, the technical solution adopted by the present invention is as follows:

[0022] The method for preparing the staple peptide includes the following steps:

[0023] (1) The first amino acid at the C-terminus is coupled to the solid support under the action of the condensing agent;

[0024] (2) Use a deprotection reagent to remove the Fmoc protecting group on the amino acid;

[0025] (3) The next amino acid is linked under the action of a condensing agent;

[0026] (4) Repeat the deprotection-coupling operation to synthesize peptide chains according to the amino acid sequence; wherein, the looping sites are replaced by S5 for amino acids at positions i and i+4, respectively.

[0027] (5) The last amino acid is deprotected and then acetylated;

[0028] (6) Under the action of the cyclizing agent, the S5 amino acids at positions i and i+4 undergo an olefin metathesis reaction, cyclizing the peptide chain;

[0029] (7) Use a cleavage reagent to cut the peptide chain from the vector and purify it to obtain the corresponding staple peptide.

[0030] As a preferred example, the purification method used was reversed-phase high-performance liquid chromatography (RP-HPLC), with the following conditions: chromatographic column: YMC-Pack ODS-AQ column; mobile phase: mobile phase A was 0.1% TFA / water, mobile phase B was 0.1% TFA / acetonitrile; gradient elution program: elution with 32% B for 0–5 min, 32% B–56% B for 5–60 min; flow rate was 20 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

[0031] As another preferred embodiment of the present invention, the condensing agent used in step (1) is a DIC-Oxyme condensation system, the activator is DIC, and NMP is used as the solvent. The ratio of the amino acid, Oxyme, DIC and NMP is 1:1:1:6 (mol / mol / mol / ml) or 1:0.9:0.9:6 (mol / mol / mol / ml).

[0032] As another preferred embodiment of the present invention, in step (1) during solid-phase synthesis, the resin loading amount is 0.49 mmol / g.

[0033] As another preferred embodiment of the present invention, the temperature of the coupling reaction in step (1) is 50-60°C, more preferably 55°C; the time of the coupling reaction is 20-30 min, more preferably 20 min.

[0034] As another preferred embodiment of the present invention, in step (2), the deprotection agent is a mixed solution of Oxyme, piperidine and DMF in a ratio of 71:1:4 (m / v / v).

[0035] As another preferred embodiment of the present invention, in step (2), the Fmoc protection is removed by applying a protective reagent for 5 minutes and then applying it again for 5 minutes; the reaction temperature for removing the Fmoc group is 20-30°C, more preferably 25°C.

[0036] As another preferred embodiment of the present invention, the reaction time of the first amino acid after S5 is 1 hour, and the reaction is repeated once under the same conditions before proceeding to the next step.

[0037] As another preferred embodiment of the present invention, in step (5), the acetylation reagent used is a mixture of pyridine and acetic anhydride, with a feeding ratio of 1:1 (v / v).

[0038] As another preferred embodiment of the present invention, in step (5), the acetylation is carried out by reacting the resin in the acetylation reagent for 20 minutes; the reaction temperature is 20-30°C, more preferably 25°C.

[0039] As another preferred embodiment of the present invention, the cyclizing agent in step (6) is a solution of Grubbs I reagent in dichloroethane, and the feeding ratio is resin loading amount: Grubbs I reagent: dichloroethane = 0.3:58:6 (mmol / mg / ml).

[0040] As another preferred embodiment of the present invention, the cyclization in step (6) involves shaking the resin twice in the cyclization reagent for 2 hours each time; the reaction temperature is 20-30°C, more preferably 25°C.

[0041] As another preferred embodiment of the present invention, in step (7), the cleavage reagent is a mixed solution of TIPS, H2O, phenol and TFA in a volume ratio of 2:5:5:88; the volume-to-mass ratio of the cleavage reagent to the linear peptide is 1:10 mL / mg.

[0042] As another preferred embodiment of the present invention, in step (7), the cutting temperature is 20-30°C, more preferably 25°C; and the cutting time is 4 hours.

[0043] The advantages of this invention are:

[0044] 1. Based on extensive research experience, the inventors of this application recognized that the staple peptide of Chem-KVL may have higher antibacterial activity. Experiments have confirmed that it can significantly improve the inhibitory activity against Escherichia coli and Staphylococcus aureus, and has potential application value in the treatment of clinical bacterial infections and related diseases.

[0045] 2. This invention uses amino resin as a carrier, following the template Chem-KVL:

[0046] The Ac-KVLGRLVKVLGRLV-NH2 amino acid sequence was synthesized into a peptide chain via Fmoc solid-phase synthesis in a DIC-Oxime condensation system. During the synthesis, key amino acid residues were retained, and S5 was substituted at specific positions to link the linear peptide to resin. The linear peptide underwent olefin metathesis in a dichloroethane solution with Grubbs I reagent, followed by cyclization and cleavage from the resin to obtain the target staple peptide. The resulting compound was purified and characterized by HPLC and MS. This method is simple and easy to perform, yielding a staple peptide with a purity greater than 98% and a high yield. Attached Figure Description

[0047] Appendix Figure 1 This is a schematic diagram of the staple peptide of the present invention.

[0048] Appendix Figure 2 This is a synthetic route diagram for the staple peptide of this invention.

[0049] Appendix Figures 3-4High-performance liquid chromatogram of the purified target compound.

[0050] Appendix Figure 5 The mass spectrum of the purified compound Chem-KVL; Chem-KVL: HR-Q-TOF-MS m / z calcd for C74H139N23O15 1591.0720; found [M+H] + =1592.0916, [M+2H] 2+ =796.0541,[M+3H]3+=531.0494.

[0051] Appendix Figure 6 The mass spectrum of the purified compound SCL-1; SCL-1: HR-Q-TOF-MS m / z calcd for C77H141N23O151629.1210; found [M+H] + =1630.1064,[M+2H] 2+ =815.0547, [M+3H] 3+ =543.7047.

[0052] Appendix Figure 7 The mass spectrum of the purified compound SCL-2; SCL-2: HR-Q-TOF-MS m / z calcd for C77H141N23O151629.1210; found [M+2H] 2+ =815.0576,[M+3H] 3+ =543.7087.

[0053] Appendix Figure 8 The mass spectrum of the purified compound SCL-3; SCL-3: HR-Q-TOF-MS m / z calcd for C81H149N23O15 1685.2290; found [M+2H] 2+ =843.5945, [M+3H] 3+ =562.7212.

[0054] Appendix Figure 9 The mass spectrum of the purified compound SCL-4; SCL-4: HR-Q-TOF-MS m / z calcd for C76H139N23O151615.0940; found [M+H] + =1616.0992,[M+2H] 2+ =808.0532,[M+3H]3+=539.0575.

[0055] AppendixFigure 10 The mass spectrum of the purified compound SCL-5; SCL-5: HR-Q-TOF-MS m / z calcd for C77H140N20O15 1585.0920; found [M+H] + =1586.0966,[M+2H] 2+ =793.5519,[M+3H] 3+ =529.7158.

[0056] Appendix Figure 11 The mass spectrum of the purified compound SCL-6; SCL-6: HR-Q-TOF-MS m / z calcd for C77H141N23O15 1629.1210; found [M+H] + =1630.1160,[M+2H] 2+ =815.0624, [M+3H] 3+ =544.0552.

[0057] Appendix Figure 12 The mass spectrum of the purified compound SCL-7; SCL-7: HR-Q-TOF-MS m / z calcd for C77H141N23O15 1629.1210; found [M+H] + =1630.1160,[M+2H] 2+ =815.0621,[M+3H] 3+ =544.0563. Detailed Implementation

[0058] This invention designs and synthesizes seven staple peptides according to the amino acid sequence of the template Chem-KVL: Ac-KVLGRLVKVLGRLV-NH2 (SEQ ID NO: 1). Each staple peptide is as follows: Figure 1 As shown.

[0059] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0060] The abbreviations used in the following examples are explained below:

[0061] Fmoc: Fluorenylmethoxycarbonyl; DCM: Dichloromethane; DCE: Dichloroethane; DMF: N,N-Dimethylformamide; Oxyme: Ethyl Cyanoglyoxylate-2-Oxime; DIC: N,N-Diisopropylcarbodiimide; NMP: N-Methylpyrrolidone; S5: 2-amino-2-methylhept-6-enoic acid; TFA: Trifluoroacetic acid; TIPs: Triisopropylsilane; GrubbsⅠ: Phenylenemethylenebis(tricyclohexylphosphine)ruthenium dichloride.

[0062] The experimental materials used in the examples were sourced from the following sources:

[0063] Amino acids and amino resins were purchased from Shanghai Jier Biochemical Co., Ltd.; N-methylpyrrolidone (NMP), N,N-diisopropylcarbodiimide (DIC), Ethyl Cyanoglyoxylate-2-Oxime, trifluoroacetic acid (TFA), and acetonitrile (chromatographic grade) were purchased from Beijing Bailingwei Technology Co., Ltd.; N,N-dimethylformamide (DMF), anhydrous diethyl ether, dichloromethane (DCM), dichloroethane (DCE), piperidine, and phenol were all analytical grade and purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.

[0064] Example 1: Preparation of improved Chem-KVL staple peptide

[0065] 1. Synthesis of staple peptide

[0066] The synthesis of staple peptide is shown in the appendix. Figure 2 As shown.

[0067] (1) Preparation of compound 1

[0068] Take 204 mg of amino resin (sample loading capacity: 0.49 mmol·g). -1 Add the resin to the solid-phase synthesis reaction tube, soak it in DCM for 20 minutes to allow the resin to fully swell, and then dry it for later use.

[0069] Add 20% piperidine-DMF solution (0.1M Oxyme) until the resin is completely submerged, shake at 25°C for 5 min × 2 to remove Fmoc from the resin, and wash the resin with DCM and DMF 3 times each.

[0070] (2) Preparation of compound 2

[0071] The first amino acid in the sequence (0.5 mmol), Oxyme (71 mg, 0.5 mmol) and DIC (77.5 μL, 0.5 mmol) were mixed in 6 ml of NMP and added to the resin. The mixture was shaken at 60 °C for 20 min (the reaction of the amino acid after S5 was 1 h, and the reaction was repeated once). The resin was washed three times each with DCM and DMF.

[0072] (3) Preparation of compound 3

[0073] Repeat steps (1) and (2). According to the polypeptide sequence, Fmoc amino acids (0.5 mmol), Oxyme (71 mg), and DIC (77.5 μl) are dissolved in 6 mL of NMP and added to the resin. The mixture is shaken at 60 °C for 20 min. The process of deprotection → condensation → deprotection is repeated until all amino acids are linked. After the last amino acid is deprotected, 6 mL of a pyridine:acetic anhydride (1:1) mixture is added and the mixture is shaken at 25 °C for 20 min. The resin is then washed three times each with DCM, DMF, and anhydrous diethyl ether, and finally dried under vacuum.

[0074] (4) Preparation of compound 4

[0075] After the resin is completely dry, add 6 mL of dichloroethane solution containing Grubbs I (58 mg) reagent, and shake the reaction twice at 25 °C for 2 h each time. After the reaction is complete, wash the resin three times each with DCM, DMF and anhydrous diethyl ether, and then dry the resin under vacuum.

[0076] (5) Preparation of the target compound

[0077] Wash and dry the resin, add 10 mL of TIPS:phenol H2O:TFA = 2:5:5:88 (V / V / V), shake at room temperature for 4 hours, filter, wash the resin with a small amount of TFA, and collect the filtrate. Bubble away excess TFA with argon gas, pour in ice-cold ether, centrifuge, discard the supernatant, and continue washing and centrifuging with ice-cold ether three times. Dry with argon gas to obtain crude stapling peptide.

[0078] 2. Purification of the target binding peptide

[0079] The crude peptide was dissolved in acetonitrile and water, and then purified by preparative RP-HPLC. The separation conditions were as follows:

[0080] Instrument: Pre-HPLC SD-1VARIAN high-performance liquid chromatograph;

[0081] Column: YMC-Pack ODS-AQ (250×20mm l.D, S-5μm, 12nm);

[0082] Mobile phase: Mobile phase A is an aqueous solution of 0.1% TFA by volume, and mobile phase B is an acetonitrile solution of 0.1% TFA by volume;

[0083] Procedure and parameters: Elution with 32% B for 0–5 min, 32% B–56% B for 5–60 min; flow rate of 20 ml / min, injection volume of 5 mL, detection wavelength of 214 nm.

[0084] Identification and structural analysis of the product in Example 2

[0085] The product obtained in step 2 of Example 1 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 mobile phase was acetonitrile and water. Mobile phase A was an aqueous solution of 0.1% TFA (v / v), and mobile phase B was an acetonitrile solution of 0.1% TFA (v / v). Gradient elution was used (0–5 min, mobile phase B: 5%; 5–30 min, mobile phase B: 5%–65%). The flow rate was 15.0 mL / min. -1 The detection wavelengths were 214 nm and 254 nm, and the injection volume was 20 μl. The peak elution time was consistent with that of the crude product, and the purity of the staple peptide prepared by this method was >98% (see [link to product details]). Figures 3-4 ) The results of analysis by HR-ESI-MS mass spectrometry are as follows Figures 5-12 As shown.

[0086] Example 3: Experiment on inhibition of Gram-positive and Gram-negative bacteria

[0087] Antibacterial activity test: Antibacterial activity was determined by the MIC value of the peptide. *S. aureus* ATCC 25923, *P. aeruginosa* ATCC 27853, *E. coli* ATCC 25922, and *A. baumannii* ATCC 17978 strains were inoculated onto TSA plates and incubated upside down at 37°C for 18-24 hours. A suitable number of pure colonies were then picked from the plates and added to 3 mL of CAMHB medium. After thorough mixing, 60 μL of the 0.5 McF value bacterial suspension was added to CAMHB medium to a final volume of 9 mL (i.e., a 150-fold dilution). After thorough mixing, 1×10⁻⁶ cells were obtained. 6 One colony forming unit (CFU) / mL of bacterial suspension to be inoculated. Except for the blank control wells, each well was inoculated with 50 μL of the test bacterial suspension. 50 μL of CAMHB medium was added to the blank control wells. The final concentrations of each polypeptide were 64, 32, 16, 8, 4, and 2 μg / mL, and the bacterial concentration was 5 × 10⁻⁶. 5 CFU / mL. The bacterial suspension to be tested was inoculated within 15 min and incubated at 37°C for 18 h. The MIC value was recorded the next day.

[0088] The results are shown in Table 1, indicating that the staple peptides prepared in this invention can improve the antibacterial activity of the original peptides, with SCL-4 and SCL-7 showing the most significant effects.

[0089] Table 1 shows the experimental results of antibacterial activity.

[0090]

[0091] The above examples demonstrate that the present invention successfully prepared a Chem-KVL-based staple peptide, and proved that the staple peptide can significantly inhibit the growth and reproduction of harmful bacteria, showing good application prospects.

[0092] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A stapling peptide, characterized in that, The stapled peptide is selected from one of the following: a) Ac-KVLGRLVKVLGRLV-NH2 as a template for the peptide chain, only the 10 L and 14 V substitution with S5 and cyclization; b) using Ac-KVLGRLVKVLGRLV-NH2 as a template for the peptide chain, only the 9 V and 13 L substitution with S5 and cyclization; c) Ac-KVLGRLVKVLGRLV-NH2 as a peptide chain template, only 6 L and 10 L substitution with S5 and cyclization; d) Ac-KVLGRLVKVLGRLV-NH2 as a peptide chain template, only the 5 R and 9 V substitution with S5 and ring closure; e) Ac-KVLGRLVKVLGRLV-NH2 as a template for the peptide chain, only 3 L and 7 V substitution with S5 and cyclization; f) Ac-KVLGRLVKVLGRLV-NH2 as a peptide chain template, only 2 V and 6 L substitution with S5 and ring closure; The S5 is 2-amino-2-methylhept-6-enoic acid.

2. Use of the stapled peptide of claim 1 in the preparation of a medicament for inhibiting E. coli, S. aureus, P. aeruginosa.

3. The method of claim 1, wherein the stapling peptide is prepared by, The preparation comprises the following steps: (1) coupling the first amino acid at the C-terminal with a solid phase carrier under the action of a condensing agent; (2) removing the Fmoc protecting group on the amino acid using a deprotection reagent; (3) connecting the next amino acid under the action of a condensing agent; (4) repeating the deprotection-coupling operation to synthesize the peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S5 at positions i and i+4, respectively; (5) acetylating after deprotection of the last amino acid; (6) allowing the S5 amino acids at positions i and i+4 to undergo olefin metathesis under the action of a cyclization agent to cyclize the peptide chain; (7) using a cleavage reagent to cut the peptide chain from the carrier, and purifying to obtain the corresponding stapled peptide.

4. The production method according to claim 3, characterized by, In step (7), the purification method is reverse phase high performance liquid chromatography, and the conditions are as follows: column: YMC-Pack ODS-AQ column; mobile phase: mobile phase A is 0.1% TFA / water, and mobile phase B is 0.1% TFA / acetonitrile; gradient elution program: 32% B elution for 0-5 min, 32% B to 56% B for 5-60 min; flow rate is 20 ml / min, injection volume is 5 ml, and detection wavelength is 214 nm.

5. The preparation method according to claim 3, characterized in that, In step (1), the condensing agent is a DIC-Oxyme condensing system, the activating agent is DIC, and NMP is used as the solvent, and the ratio of the amino acid, Oxyme, DIC, and NMP is 1:1:1:6 (mol / mol / mol / ml) or 1:0.9:0.9:6 (mol / mol / mol / ml).

6. The preparation method according to claim 3, characterized in that, In step (2), the deprotection reagent is a mixed solution of Oxyme, piperidine, and DMF, and the ratio is 71:1:4 (m / v / v).

7. The preparation method according to claim 3, characterized in that, In step (5), the acetylating reagent is a mixed solution of pyridine and acetic anhydride, and the feeding ratio is 1:1 (v / v).

8. The preparation method according to claim 3, characterized in that, In step (6), the cyclization agent is a dichloroethane solution of Grubbs I reagent, and the feeding ratio is resin sample amount: Grubbs I reagent: dichloroethane = 0.1:58:6 (mmol / mg / ml).

9. The preparation method according to claim 3, characterized in that, In step (7), the cleavage reagent is a mixed solution of TIPS, H2O, phenol, and TFA, and the volume ratio is 2:5:5:88, and the volume-to-mass ratio of the cleavage reagent to the linear peptide is 1:10 mL / mg.

Images