WO2017190619A1 - 一种能抑制金葡菌毒素的化学合成环七修饰肽及其应用 - Google Patents

一种能抑制金葡菌毒素的化学合成环七修饰肽及其应用 Download PDF

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WO2017190619A1
WO2017190619A1 PCT/CN2017/082106 CN2017082106W WO2017190619A1 WO 2017190619 A1 WO2017190619 A1 WO 2017190619A1 CN 2017082106 W CN2017082106 W CN 2017082106W WO 2017190619 A1 WO2017190619 A1 WO 2017190619A1
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peptide
isomer
trt
natural
residue
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PCT/CN2017/082106
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French (fr)
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邵宁生
杨光
王良友
丁红梅
高亚萍
李少华
李慧
李洁
董洁
夏文晖
梁小平
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重程投资管理（上海）有限公司
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Priority to CN201780021188.XA priority Critical patent/CN109071604B/zh
Priority to US16/097,964 priority patent/US10905735B2/en
Publication of WO2017190619A1 publication Critical patent/WO2017190619A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

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  • the present invention relates to a chemically synthesized cyclic seven-modified peptide capable of inhibiting staphylococcal toxin and uses thereof.
  • the chemically synthesized peptide is capable of specifically inhibiting the production of toxin of S. aureus by binding to the autocrine RNAIII activator of S. aureus, and the present invention also relates to the use of the chemically synthesized peptide in the field of medicine.
  • Staphylococcus aureus is a common type of Gram-positive pathogen, which is the main microorganism causing fatal diseases such as burns and war wounds, pneumonia, endocarditis, sepsis, toxic shock.
  • Staphylococcus aureus is a common type of Gram-positive pathogen, which is the main microorganism causing fatal diseases such as burns and war wounds, pneumonia, endocarditis, sepsis, toxic shock.
  • the treatment of Staphylococcus aureus is often used in combination with antibiotics, but the effect is not satisfactory. Because Staphylococcus aureus is highly susceptible to drug resistance and there is no good solution, many commonly used antibiotics are ineffective. Controlling Staphylococcus aureus infection is one of the problems that need to be solved in clinical medicine.
  • RNAIII activates gene transcription of virulence factors and regulates translation of virulence factors by base complementation. In the early logarithm of bacterial growth, the level of RNAIII is low, but the level of RNAIII in the late logarithm is increased by 40-fold. The level of RNAIII is regulated by the protein secreted by Staphylococcus aureus and regulated by RANIII activator protein (RAP).
  • the factor RAP is also known as the Staphylococcus aureus virulence stimulating factor. Staphylococcus aureus continues to secrete RAP, and the activation of virulence factors is only activated after RAP reaches a certain concentration. Staphylococcus aureus that is not produced by RAP does not itself cause disease.
  • Balaban et al. published a study in the journal Science, which showed that antibodies prepared by RAP immunized animals can effectively protect mice from S. aureus (Balaban N, et al. Autoinducer of virulence as a target). For vaccine and therapy against Staphylococcus aureus. Science, 1998, 280(17): 438-440).
  • the present inventors used the phage display technology to select a small 7-peptide MRK (CQHWWHWYC) which can specifically bind to the RAP molecule and inhibit its activity from the random loop 7 peptide library, see CN0315020.5 and other preliminary work.
  • CQHWWHWYC small 7-peptide MRK
  • the inventors found that the small peptide has a better inhibitory effect on the production of staphylococcal toxin.
  • the inventors found that the small peptide has a major defect - insoluble in water or physiologically. The solution is only soluble in organic solvents (such as DMSO), thus seriously affecting its biological activity, and it is not clinically applicable.
  • the present invention relates to a chemical modification of a cyclic heptapeptide which belongs to a small molecule peptide.
  • the main object of the present invention is to improve water solubility and maintain biological activity.
  • the present inventors chemically engineered the small peptide, and the specific modified sequence is: CH 3 -(CH 2 )mXG-(CQHWWHWYC)-(R)nY.
  • MRG modified chemically synthesized peptide
  • MRG modified chemically synthesized peptide
  • the compounds of the present invention are completely new, both from the source and the structure, and no literature has been reported.
  • the object of the present invention is to provide a novel chemical synthesis cyclic seven-modified peptide capable of inhibiting staphylococcal toxin and a preparation method thereof, which can specifically inhibit the production of toxin of Staphylococcus aureus.
  • Another object of the present invention is to provide an application of the chemically synthesized peptide in the field of medicine.
  • the compound of the present invention is a small molecule polypeptide analog (hereinafter also referred to as "the peptide described", “cyclic seven modified peptide”), and has the following general chemical structural formula:
  • n 0-20, preferably 3-17, more preferably 6-14, most preferably 8-12;
  • n 1-10, preferably 1-7, more preferably 2-4, most preferably 3;
  • Y is selected from OH or NH 2 ;
  • G represents a natural L-type glycine residue or a D-form isomer thereof
  • C represents a natural L-type cysteine residue or a D-form isomer thereof
  • Q represents a natural L-type glutamine residue or a D-form isomer thereof
  • H represents a histidine residue or D-isomer
  • W represents a natural L-type tryptophan residue or a D-isomer thereof
  • Y represents a natural L-type tyrosine residue or a D-isomer thereof
  • R represents a natural The L-type arginine residue or its D-isomer
  • the two cysteines represented by C are linked by a disulfide bond.
  • the peptide specifically binds to the S. aureus virulence stimulating factor RAP and inhibits the production of staphylococcal toxin.
  • the peptide is obtained by chemical synthesis. In other words, it is obtained by chemical synthesis.
  • G, C, Q, H, W, Y, R independently represent a group: G represents a natural L-type glycine residue or a D-form isomer thereof; C represents a natural L-type cysteine residue Or a D-isomer thereof; Q represents a natural L-type glutamine residue or a D-isomer thereof; H represents a histidine residue or a D-isomer thereof; W represents a natural L a -type tryptophan residue or a D-isomer thereof; Y represents a natural L-type tyrosine residue or a D-isomer thereof; R represents a natural L-type arginine residue or D thereof - isomer.
  • the two cysteines of the nine amino acid sequences C-Q-H-W-W-H-W-Y-C are linked by a disulfide bond.
  • a solid phase synthesis method for preparing the peptide comprising the steps of:
  • Step 1 The conventional protected amino acid is coupled one by one with the substitution ratio of Rink Amide-AM Resin as sub-0.45-0.55 mmol/g.
  • Step 2 using a solid phase polypeptide stepwise condensation method, under the action of condensation test 1-hydroxybenzotriazole (HOBT) and N, N'-diisopropylcarbodiimide (DIC), according to the peptide sequence from C From the end to the N-terminus, the Fmoc-protected amino acids are coupled one by one to obtain:
  • HOBT 1-hydroxybenzotriazole
  • DIC N, N'-diisopropylcarbodiimide
  • Step 3 using the condensation reagent benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) Coupling the dodecanoic acid to obtain the sequence:
  • HBTU benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • DIPEA N,N-diisopropylethylamine
  • Step 4 mixing the conventional cleavage reagent with the linear peptide resin obtained in the third step to generate a cleavage reaction, thereby removing the Rink Amide-AM Resin and the MRG side chain protecting group, and then passing through sedimentation, centrifugation, Washing and drying to obtain a MRG linear crude peptide;
  • Step 5 After step 4, the dried linear crude peptide is ground to a powder form, and the MRG linear crude peptide is dissolved in a mixed solution of pure water and acetonitrile (2:1) to a concentration of 1 mmol/500 ml, thereby obtaining a linear crude peptide solution. ;
  • Step 6 Adding dilute ammonia water to the MRG linear crude peptide solution to adjust the pH of the MRG linear crude peptide solution to 7.1-7.3, and adjusting the temperature to about 30 degrees Celsius to carry out the cyclization experiment, and the cyclization time is 20- After 40 minutes, the cyclization was determined by high performance liquid chromatography (HPLC). After the cyclization was completed, acetic acid was added to adjust the pH to acidic conditions to terminate the cyclization reaction.
  • HPLC high performance liquid chromatography
  • Step 7 The crude product of the target product in the step 6 is separated and purified by a C 18 reversed-phase high performance liquid chromatography column, and then lyophilized to obtain a high-purity target product.
  • the invention also relates to the use of the peptide for the preparation of a medicament for the infection of S. aureus.
  • the chemically synthesized peptide having the above structure is capable of specifically binding to the Staphylococcus aureus virulence stimulating factor RAP and inhibiting its activity.
  • the invention relates to the peptides which increase the solubility of "cycloheptapeptide" (C-Q-H-W-W-H-W-Y-C).
  • the main reason for the antibiotic resistance of traditional antibiotic treatment is that the bacteria produce an enzyme that decomposes the effective group in the antibiotic under the pressure of survival after administration.
  • the present invention utilizes a polypeptide compound which specifically inhibits RAP activity to establish a treatment for Staphylococcus aureus infection, and finds a new way for the treatment of a common, multiple and fatal disease which has been plagued by clinical drug-resistant Staphylococcus aureus infection.
  • the invention has important significance for developing a novel small molecule polypeptide drug against Staphylococcus aureus infection, and has wide application value and broad market prospect.
  • Figure 1 is a HPLC purity identification map of chemically synthesized cyclic heptapeptide MRK
  • Figure 2 is a mass spectrometric identification map of chemically synthesized cyclic heptapeptide MRK
  • Figure 3 is a HPLC purity identification map of chemically synthesized cyclic seven-modified peptide MRG
  • Figure 4 is a mass spectrometric identification map of chemically synthesized cyclic seven-modified peptide MRG
  • Figure 5 is a therapeutic effect of chemically synthesized peptides on bacteremia in mice caused by Staphylococcus aureus infection.
  • Step 1 Solid phase synthesis of raw materials
  • Step two solid phase synthesis method
  • Step 1 Synthesis of 2mmol MRG Select Rink Amide-AM Resin
  • the degree of substitution is 0.45mmol/g.
  • this method removes the Fmoc protecting group generally twice, each time 5min and 15min respectively to ensure complete deprotection, after the deprotection is completed, the sputum test reagent is used:
  • test reagent is added with two drops of 105 degrees Celsius for 3 minutes to observe the color. If it is blue, the protective solution can be removed. If the color is not developed, the deprotection is not complete and the cause is removed again. After deprotection, DMF can be washed six times to put Fmoc-Arg(pbf)-OH and condensation test 1-hydroxybenzotriazole (HOBT) and N,N'-diisopropylcarbodiimide (DIC). Coupling reaction (coupling sequence from the C-terminus to the N-terminus according to the peptide sequence, coupling Fmoc-protected amino acids one by one), reaction time 2-3 h.
  • HOBT 1-hydroxybenzotriazole
  • DIC N,N'-diisopropylcarbodiimide
  • the ninhydrin color reaction reaction is still used to detect whether the reaction is complete, that is, a small amount of resin and a detection tube are added.
  • Each of the above three test solutions is added dropwise two drops of 105 degrees Celsius for 3 minutes to observe the color if it is colorless.
  • the reaction is complete, otherwise the reaction is not completely required to be charged or repeated until the reaction is complete.
  • the amino acid in the peptide sequence is coupled once by the method described above to obtain:
  • Step 3 In step 2, the peptide sequence is coupled to Gly.
  • the last amino acid, dodecanoic acid (monthly silicic acid), needs to be coupled.
  • the coupling washing should be noted in the experiment.
  • the deprotection and detection methods are the same except that the coupling reagent used by the inventors is benzotriazole-N,N,N',N'-four.
  • the reaction time of methyl urea hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) was about 1 h. After the completion of the reaction, the ninhydrin coloring method was used to detect whether the reaction was complete.
  • Step 4 Take the liquid-repellent peptide resin in the third step and weigh 8.2 g.
  • First, prepare 100 ml of the conventional lysing reagent according to the ratio of TFA: thioanisole: EDT: anisole 90:5:3:2.
  • 8.2g needs to remove about 70ml of lysing reagent from 100ml for cleavage reaction, the reaction time is about 2-3h.
  • the resin is filtered to obtain about 60ml of filtrate.
  • V water : V acetonitrile 2:1
  • Step 6 Add the diluted dilute ammonia water to the linear crude peptide solution described in the fifth step to make the pH value of the solution between 7.1 and 7.3 and then slowly add about 10 times of 30% hydrogen peroxide to the solution.
  • the amount of hydrogen peroxide added is 1 mmol, 0.3 ml of 30% hydrogen peroxide (usually diluted and then slowly added).
  • the temperature is generally controlled at 30 ° C to carry out the cyclization reaction.
  • HPLC analysis and trace detection are required. Whether the cyclization is complete or not, whether or not cyclization is judged by the displacement of the linear peptide and the cyclized peptide mainly detected by HPLC analysis.
  • the cyclization time is generally about 20-40 minutes.
  • the acetic acid solution is added in time to adjust the pH to 4.5 to terminate the cyclization.
  • Step 7 The crude peptide of interest after cyclization in HPLC in step 6 is separated and purified by C 18 reversed-phase high performance liquid chromatography column, and then lyophilized to obtain a fine product.
  • the main steps are as follows:
  • the crude crude peptide solution after cyclization was filtered through a 0.45 ⁇ m microporous filter passed through a integrity test. After the filtration is completed, the solution is adjusted to a suitable acidic pH to prepare.
  • Step 8 Freeze and dry the concentrated liquid obtained in the previous step to obtain a white powder, that is, obtain the finished product.
  • the concentrated sample solution is pre-frozen, and the sample is placed on the separator in the freeze-drying box for pre-freezing, and the temperature of the product is lowered to below -40 ° C for about 120 min.
  • the electric heating was set to 10 ° C and the deviation time was 500 min.
  • the electric heating was set to 35 ° C and the deviation time was 420 min.
  • Step 9 Purification and structural determination of chemically synthesized cyclic seven-modified peptide MRG
  • the small peptide obtained by chemical synthesis was identified by HPLC to have a purity greater than 95% (Fig. 3); the molecular weight identified by mass spectrometry was 2055.08 (1028.54 x 2-2) (Fig. 4).
  • Example 3 Comparison of water solubility between chemically synthesized cyclic heptapeptide MRK and cyclic seven modified peptide MRG
  • the modified cycloheptapeptide of the present invention has a solubility in water for injection or physiological saline of more than 1 mg/ml. It is indicated that the cyclic seven modified peptide MRG is far more water soluble than the cyclic heptapeptide MRK.
  • the cycloheptapeptide MRK itself has a solubility in water for injection or physiological saline of less than 0.1 mg/ml, and a larger amount of the cyclic seven-modified peptide MRG, specifically 10 mg, is mixed with 1 ml of water for injection or physiological saline, all Dissolved, stood still without precipitation, and the results of multiple tests were consistent.
  • the solubility of the cyclic seven modified peptide MRG in water for injection or physiological saline exceeds 1.0%, completely solving the technical problem that the procyclic heptapeptide MRK is insoluble in water.
  • Example 4 Inhibition of staphylococcal toxin production by chemical synthesis of cycloheptapeptide MRK and cyclic seven modified peptide MRG
  • Staphylococcus aureus 04018 strain Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences; blood plate (fresh blood agar plate) purchased from Beijing Aoboxing Biotechnology Co., Ltd., BHI plate home; Imported BHI medium (BactoTM Brain Heart Infusin) was purchased from BD Company of the United States; DMEM medium was purchased from CIBCO Company of the United States; imported fetal bovine serum was purchased from PAN BIOTECH Company of the United States; 0.22 ⁇ m membrane was purchased from PALL Company of the United States; MDBK cells were obtained from the cells of this laboratory.
  • the experimental results show that the chemically synthesized cyclic seven-modified peptide MRG prepared in the examples of the present invention can be well dissolved in physiological saline; the chemically synthesized peptide can inhibit the production of the staphylococcal toxin, and the MDBK cells caused by the staphylococcal toxin
  • the inhibition of proliferation has obvious protective effect and a certain dose-effect relationship.
  • the 5 ⁇ g/ml concentration of small peptide inhibited the production of staphylococcal toxins more than the positive control TP protein, but the chemical synthesis of the cyclic heptapeptide MRK was insoluble in water, and the suspension was added to the Staphylococcus aureus medium to inhibit the staphylococcal toxin.
  • Table 1 is an observation of the inhibitory effect of chemically synthesized cyclic heptapeptide MRK and cyclic seven modified peptide MRG on the production of staphylococcal toxin.). This difference in bacteriostatic activity is believed by the inventors to be a result of a combination of an improvement in solubility and a change in the structure of the modified cyclic heptapeptide, which is not caused by a simple change in solubility.
  • Example 5 Therapeutic effect of chemically synthesized cyclic seven-modified peptide MRG on bacteremia induced by Staphylococcus aureus infection in mice
  • Staphylococcus aureus Newman strain Institute of Basic Medical Sciences, Academy of Military Medical Sciences
  • blood plate fresh blood agar plate
  • BHI plate home purchased from Beijing Aoboxing Biotechnology Co., Ltd., BHI plate home
  • imported BHI medium Bactetrachloride
  • pentobarbital was purchased from Sigma, USA
  • desktop centrifuge EPPDORF, Germany
  • other experimental consumables such as centrifuge tubes, graduated straws and disposable syringes came from the laboratory.
  • a Newman monoclonal strain with a hemolysis circle was picked, inoculated into 3 mL of BHI medium, and shaken at 37 rpm overnight at 37 rpm.
  • Anesthetic (1% pentobarbital) BALB/c mice (female, 8 weeks) (200 ⁇ L/head) were injected intraperitoneally. Newman bacterial solution (OD600 0.2, 100 ⁇ L/head) was injected into the posterior venous plexus.
  • peptides 0.5 mg/mL, 200 ⁇ L/cell
  • a control group sterile water, 200 ⁇ L/cell
  • the experimental results show that the chemically synthesized cyclic seven-modified peptide MRG prepared in the examples of the present invention can reduce the death caused by Staphylococcus aureus-induced bacteremia in mice at a dose of 5 mg/kg body weight, and the control group for sterile water injection. Compared with the survival rate, the biosynthesis of the cyclic heptapeptide MRK also inhibited the infection of S. aureus in vivo (Fig. 5).

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Abstract

提供一种能抑制金葡菌毒素的化学合成环七修饰肽及其应用。该化学合成肽能够通过与金葡菌自分泌的RNAIII激活蛋白结合特异抑制金葡菌的毒素产生，在该环七肽的基础上，将小肽进行化学改造，具体改造后序列通式为：CH3-(CH2)m-X-G-(C-Q-H-W-W-H-W-Y-C)-(R)n-Y。结果表明，该修饰肽能极好地溶于水，而且具有良好的抗金葡菌毒素活性。

Description

一种能抑制金葡菌毒素的化学合成环七修饰肽及其应用 技术领域
本发明涉及一种能抑制金葡菌毒素的化学合成环七修饰肽及其应用。该化学合成肽能够通过与金葡菌自分泌的RNAIII激活蛋白结合特异抑制金葡菌的毒素产生，本发明还涉及此化学合成肽在医药领域中的应用。
背景技术
金黄色葡萄球菌(金葡菌)是一类常见的革兰氏阳性致病菌，是引起烧伤及战伤感染、肺炎、心内膜炎、败血症、中毒性休克等致命性疾病的主要微生物之一。每年仅医院内感染金葡菌的人数就超过数百万。目前临床上对金葡菌的治疗多采用联合使用抗生素的办法，但是效果并不理想。由于金葡菌极易产生耐药性且无好的解决方法，常用的许多抗生素对之无效，控制金葡菌感染是临床医学殛待解决的问题之一。
金葡菌的主要致病物质是毒素，包括溶血毒素、杀白细胞素、肠毒素等。最新研究表明，金葡菌这些毒力因子的合成是受一种可调节RNA分子，及RNAIII控制的。RNAIII激活毒力因子的基因转录，通过碱基互补调节毒力因子的翻译。在细菌生长的对数早期其RNAIII水平低，但到对数晚期RNAIII水平会增加40倍，而RNAIII的水平是由金葡菌自身分泌的蛋白，RANIII激活蛋白(RNA III activating protein，RAP)调节的，故因子RAP又称为金葡菌毒力刺激因子。金葡菌持续分泌RAP，在RAP达到一定浓度后才有激活毒力因子产生的作用。没有RAP产生的金葡菌本身并不致病。1998年Balaban等在“Science”杂志发表研究结果表明，用他们制备的RAP免疫动物，其抗体可以有效地保护小鼠免受金葡菌的感染(Balaban N,et al.Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus.Science,1998,280(17):438-440)。
本发明人前期采用噬菌体展示技术从随机环7肽库中筛选出能特异与RAP分子结合并抑制其活性的小7肽MRK(CQHWWHWYC)，见CN0315020.5等前期工作。体内外实验证明，该小肽虽然有较好的抑制金葡菌毒素产生的作用。但本发明人在随后的研究中，发现该小肽存在重大的缺陷——不溶于水或生理学上 的溶液，只溶于有机溶剂(如DMSO)，因而严重影响其生物活性的发挥，更无法在临床上应用。为此，这成为困扰本发明人的重大问题，以至于研究及开发工作无法推进。本发明人经过反复的筛选实验，尝试各种结构改造的方法，包括引入亲水性基团如PEG等、利用纳米技术手段进行纳米化、甚至采用脂质体或胶束载药等，但这些方法收效甚微，甚至大大影响环七肽的活性和稳定性。另外，近年来，在酶、蛋白质及多肽药物创制的技术领域，有了一些关于化学修饰的报道，例如改进稳定性、长效性(2012-2013生物化学与分子生物学学科发展报告，中国科学技术出版社，2014.04)，但采用何种化学分子修饰因功能和结构不同仍然是扑朔迷离的，需要视具体情况决定。
本发明涉及是环七肽的化学修饰，属于小分子肽。本发明的主要目的在于改善水溶性并保持生物活性。本发明人在该环七肽的基础上，将小肽进行化学改造，具体改造后序列通式为:CH3-(CH2)m-X-G-(C-Q-H-W-W-H-W-Y-C)-(R)n-Y。结果表明，改造后的化学合成肽(缩写为MRG)与原环七肽相比，不但能极好地溶于水，而且具有良好的抗金葡菌毒素活性，解决了上述技术问题。术语解释：“环七肽”指代(C-Q-H-W-W-H-W-Y-C)的组成，缩写为MRK。
本发明的化合物无论从来源还是从结构上完全是新的，未见任何文献报道。
发明内容
本发明的目的是涉及一种能抑制金葡菌毒素的新颖化学合成环七修饰肽及其制备方法，该化学合成肽能特异抑制金葡菌的毒素产生。
本发明的另一目的是提供该化学合成肽在医药领域中的应用。
本发明的化合物是小分子多肽类似物(以下也称“所述的肽”、“环七修饰肽”)，具有以下通用化学结构式：
CH3-(CH2)m-X-G-(C-Q-H-W-W-H-W-Y-C)-(R)n-Y；
其中，
m＝0-20、优选3-17、更优选6-14、最优选8-12；
X选自CONH、NHCO、O或S，优选X＝CONH；
n＝1-10、优选1-7、更优选2-4、最优选3；
Y选自OH或NH2；
G代表天然L-型甘氨酸残基或其D-型异构体；
C代表天然L-型半胱氨酸残基或其D-型异构体，Q代表天然L-型谷氨酰胺残基或其D-型异构体，H代表组氨酸残基或其D-型异构体，W代表天然L-型色氨酸残基或其D-型异构体，Y代表天然L-型酪氨酸残基或其D-型异构体，R代表天然L-型精氨酸残基或其D-型异构体，C表示的两个半胱氨酸通过二硫键相连。
所述的肽能与金葡菌毒力刺激因子RAP特异结合并抑制金葡菌毒素产生。
所述的肽通过化学合成得到。换言之，其是以化学合成方法获得。
其中，CH3-(CH2)m-代表烷酰基的烷基部分，优选为CH3(CH2)10-(m＝10)，其与X＝CONH连接时，构成十二烷酰修饰G。
G、C、Q、H、W、Y、R独立地代表如下基团：G代表天然L-型甘氨酸残基或其D-型异构体；C代表天然L-型半胱氨酸残基或其D-型异构体；Q代表天然L-型谷氨酰胺残基或其D-型异构体；H代表组氨酸残基或其D-型异构体基；W代表天然L-型色氨酸残基或其D-型异构体；Y代表天然L-型酪氨酸残基或其D-型异构体；R代表天然L-型精氨酸残基或其D-型异构体。
其中的9个氨基酸序列C-Q-H-W-W-H-W-Y-C的两个半胱氨酸通过二硫键相连。
制备所述的肽的固相合成方法，包括以下步骤：
步骤一、以Rink Amide-AM Resin替代度为sub＝0.45～0.55mmol/g为起始载体逐个偶联常规保护氨基酸。
步骤二、采用固相多肽逐步缩合法，在缩合试1-羟基苯并***(HOBT)和N,N'-二异丙基碳二亚胺(DIC)的作用下，根据肽序从C端至N端，逐个偶联Fmoc保护的氨基酸得到:
Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Rink Amide-AM Resin
步骤三、利用缩合试剂苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)和N,N-二异丙基乙胺(DIPEA)的作用下偶联十二烷酸得到序列:
CH3(CH2)10CO-Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Rink Amide-AM Resin
步骤四、将常规的裂解试剂与步骤三获得的线性肽树脂混合发生裂解反应，从而去除Rink Amide-AM Resin和MRG侧链保护基，再依次通过沉降、离心、 洗涤、干燥获得MRG线性粗肽；
步骤五、将步骤四获得干燥后的线性粗肽研磨至成粉末状，用纯水和乙腈(2:1)的混合溶液溶解MRG线性粗肽至浓度为1mmol/500ml，从而获得线性粗肽溶液；
步骤六、向所述MRG线性粗肽溶液中滴加稀氨水调节MRG线性粗肽溶液pH至7.1-7.3，并将温度调至30摄氏度左右即可搅拌进行环化实验，环化时间为20-40min经过高效液相色谱HPLC分析检测环化是否完全，待环化完全后加入醋酸调PH至酸性条件进行终止环化反应；
步骤七、将步骤六中目的产物粗品溶液经过C18反相高效液相色谱柱分离纯化，旋蒸冻干后，制得高纯度的目的产物。
本发明还涉及所述的肽在制备抗金葡菌感染药物中的应用。
具有上述结构的化学合成肽能够与金葡菌毒力刺激因子RAP特异结合，并抑制其活性。
本发明特别涉及所述的肽提高“环七肽”(C-Q-H-W-W-H-W-Y-C)的溶解性。
传统抗生素治疗产生抗药性的原因主要为用药后细菌在生存压力下产生分解抗生素中有效基团的诱导酶。本发明利用特异抑制RAP活性的多肽化合物建立的治疗金葡菌感染的方案，为一直困扰临床的抗药性金葡菌感染这一常见、多发且有致命性的疾病的治疗找到新的出路。
本发明对研制新型抗金葡菌感染的小分子多肽药物具有重要意义，并具有广泛的应用价值及广阔的市场前景。
附图说明
图1是化学合成环七肽MRK的HPLC纯度鉴定图谱
图2是化学合成环七肽MRK的质谱鉴定图谱
图3是化学合成环七修饰肽MRG的HPLC纯度鉴定图谱
图4是化学合成环七修饰肽MRG的质谱鉴定图谱
图5是化学合成肽对金葡菌感染所致小鼠菌血症的治疗作用
具体实施方式
下面结合实施例对本发明作进一步详细说明。
实施例1.化学合成环七肽MRK固相合成的制备
1、合成肽序列：CQHWWHWHWYC(首尾成环，分子量1346.5)
2、合成步骤：
步骤一、固相合成所需原料
H-Cys(Trt)-2-Chlorotrityl Resin(上海吉尔生化)，Fmoc-Tyr(tBu)-OH,Fmoc-Trp-OH,Fmoc-His(Trt)-OH,Fmoc-Gln-OH，Fmoc-Cys(Trt)-OH，DCC，HOBt，TFA，EDT，m-Cresol
步骤二、固相合成方法
标准Fmoc-AA-OH/DCC/HOBt法。
以1.1g H-Cys(Trt)-2-Chlorotrityl Resin(0.6mmol)为载体，氨基酸和缩合剂均过量3倍，合成完毕得2.218g
Cys(Trt)-Gln-His(Trt)-Trp-Trp-His(Trt)-Trp-Tyr(tBu)-Cys(Trt)-2-Chlorotrityl Resin
步骤三、肽树脂裂解
0.5gCys(Trt)-Gln-His(Trt)-Trp-Trp-His(Trt)-Trp-Tyr(tBu)-Cys(Trt)-2-Chlorotrityl Resin，0.1ml m-Cresol，0.3ml EDT，7.5ml TFA，0℃反应90min，得210mg粗品线性肽。
步骤四、线性肽氧化
200mg粗品线性肽溶于2000ml水中，用NaHCO3调pH 7-8，缓慢滴加K3Fe(CN)6(2mg/ml)溶液至反应液呈浅黄色，继续反应1h。用C18柱进行固相萃取，80％乙腈水溶液洗脱，得环肽76.7mg(纯度大于95％)。
步骤五、纯化与结构确证
以C18柱进行RP－HPLC纯化，可得纯度大于99％的产品(图1)。用质谱和氨基酸组成分析进行结构确证，分子量1346.5(图2)。
实施例2.化学合成环七修饰肽MRG固相合成的制备
1、合成肽序列：CH3(CH2)10CO-G-(CQHwWHWYC)-RRR-NH2(分子量2055.08)
2、合成步骤：
步骤一、合成2mmolMRG选取Rink Amide-AM Resin替代度为0.45mmol/g根据公式称取裸树脂的量＝合成的摩尔量/裸树脂的替代度即需要称取的裸树脂Rink Amide-AM Resin的量＝2mmol/0.45mmol/g＝4.5g,将称量好的裸树脂投入到反应柱 中用N,N-二甲基甲酰胺和二氯甲烷(DMF/DCM＝2/1)的混合溶液进行溶胀约30min,抽掉溶胀液后用N,N-二甲基甲酰胺(DMF)作为洗涤溶液洗涤3遍。
步骤二、将洗涤好的树脂采用去保护溶液脱除其保护基Fmoc，具体操作首先配置20％的脱保护液(V哌啶/VDMF＝20％)加入到步骤一中溶胀好的裸树脂中鼓氮气反应，此方法去除Fmoc保护基一般为两次，每次分别为5min和15min确保去保护彻底，脱保护完毕后采用茚检试剂：
(a)5％茚三酮的无水乙醇溶液(w/v)
(b)苯酚：无水乙醇溶液(4:1，w/v)
(c)吡啶
每个检测试剂滴加二滴105摄氏度条件下加热3min观察其颜色，若显蓝色可以抽掉保护液，若不显色说明脱保护不彻底立刻查明原因重新脱除。去保护完毕后DMF洗涤六遍即可投入Fmoc-Arg(pbf)-OH和缩合试1-羟基苯并***(HOBT)及N,N'-二异丙基碳二亚胺(DIC)进行偶联反应(偶联顺序根据肽序从C端至N端，逐个偶联Fmoc保护的氨基酸)，反应时间2-3h。反应完毕后仍用茚三酮显色反应实验来检测反应是否完全即取少量树脂与检测管中将上述三种检测液每个溶液滴加二滴105摄氏度下加热3min观察其颜色若无色说明反应完全，反之说明反应不完全需补投料或是重复投料至反应完全。按上述所述方法以此类推将肽序中的氨基酸一次偶联完毕得到：
Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Rink Amide-AM Resin
步骤三、在步骤二中肽序偶联至Gly在脱完保护基的情况下需偶联最后一个氨基酸即十二烷酸(月硅酸)，这时在实验中应注意的是偶联洗涤去保护及检测方法都相同的情况下不同的是偶联十二烷酸(月硅酸)本发明人所采用的缩合试剂为苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)和N,N-二异丙基乙胺(DIPEA)反应时间约1h。反应完毕后采用上述同样的方法茚三酮显色法检测反应是否完全，在反应完全的情况下DMF洗涤树脂3-4遍，甲醇(MeOH)收缩树脂两遍，每遍收缩时间为10min，再将树脂真空抽干至流沙状即可得到目的肽的线性肽树脂：
CH3(CH2)10CO-Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-RinkAmide -AM Resin
步骤四、取出步骤三中的流沙状肽树脂称量得到8.2g,首先按TFA:苯甲硫醚：EDT:苯甲醚＝90:5:3:2比例配制100ml的常规裂解试剂摇匀后按每1g肽树脂加8-10ml的裂解试剂进行裂解，则8.2g需要从100ml中取出约70ml裂解试剂进行裂解反应，反应时间约2-3h.反应完毕后滤除树脂得到滤液约60ml(在过滤中有部分损失)按沉降比例1:6(滤液：无水***)的比例将滤液缓慢加入到无水***中放置30min使之充分沉降再依次通过离心、洗涤、干燥获得线性粗肽：CH3(CH2)10CO-Gly-Cys-Gln-His-D-Trp-Trp-His-Trp-Tyr-Cys-Arg-Arg-Arg-NH2,干燥后称量粗肽约3.6g。
步骤五、将步骤四中获得干燥后的线性粗肽研磨至成粉末状，用纯水和乙腈的混合溶液(V水:V乙腈＝2:1)溶解MRG的线性粗肽至浓度为1mmol/500ml，从而获得的线性粗肽溶解液共1000ml,取出小样HPLC分析定位出峰时间。
步骤六、向步骤五中所述线性粗肽溶液中滴加稀释后的稀氨水使溶液的pH值7.1-7.3之间再缓慢的像溶液中滴加稀释约10倍左右的30％的双氧水，加入双氧水的量按1mmol滴加0.3ml30％的双氧水(一般都需稀释后再缓慢加入)，加入双氧水后温度一般控制在30摄氏度即可进行环化反应，在环化过程中需HPLC分析跟踪检测环化是否完全，环化是否在进行主要根据HPLC分析检测的线性肽与环化肽的位移情况来判断。环化时间一般约20-40min即可完成，环化实验完成后需及时加入醋酸溶液调pH至4.5终止环化。得到目的物的粗品即：
CH3(CH2)10CO-Gly-Cyclo(Cys-Gln-His-D-Trp-Trp-His-Trp-Tyr-Cys)-Arg-Arg-Arg-NH2
步骤七、将步骤六中HPLC分析环化完全后的目的肽粗品经过C18反相高效液相色谱柱分离纯化，旋蒸冻干后，制得精品。主要步骤如下：
将环化粗滤后的粗肽溶液经过通过完整性试验的0.45μm微孔滤膜进行过滤。过滤完成后将溶液调至纯化制备适宜的酸性pH后方可进行制备。
制备型液相色谱分离纯化采用分析仪器为DIONEX U3000高效液相色谱仪，分析柱C18、5μm、
Figure PCTCN2017082106-appb-000001
4.6*250mm，分析条件流动相：A相：1％0TFA，B相：乙腈，制备仪器采用创新5cm制备型高效液相色谱仪，制备色谱柱C18、10μm、
Figure PCTCN2017082106-appb-000002
150*250mm反相硅胶高压制备层析柱。
将上述过滤后的粗品溶液使用制备型HPLC的制备泵上样，5cm的层析柱进 行进样，流动相：A相：TFA(取1mL的TFA加入1000mL水中)B相：100％乙腈；
检测波长：λ＝230nm；柱温：室温；用干净的三角烧瓶收集样品，收集的样品纯度大于98％单杂小于1％的纯化溶液为合格品否则为不合格样品重复上面步骤，将溶液全部处理完，得到合格与不合格的分类纯化后溶液，再重复上述步骤将不合格样品进行二次纯化尽量得到更多的合格样品。然后进行旋蒸浓缩
步骤八、将上步分装好的浓缩液冷冻干燥得白色粉末，即得成品。首先将浓缩好的样品溶液先预冻即将样品置于冻干箱内隔板上进行预冻，制品温度下降至-40℃以下，维持约120min。
2)升华干燥：电加热设置为0℃，偏差时间1min,维持约40min。
电加热设置为10℃，偏差时间500min。
电加热设置为35℃，偏差时间420min。
3)解吸附：温度升至33℃左右，保持约240min。
步骤九、化学合成环七修饰肽MRG的纯度鉴定及结构确定
将化学合成制备获得小肽经HPLC鉴定，纯度大于95％(图3)；经质谱鉴定分子量为2055.08(1028.54×2-2)(图4)。
实施例3：化学合成环七肽MRK及环七修饰肽MRG水溶性比较
分别称取3份化学合成环七肽MRK，每份1mg置1ml离心管中，每管加入1ml注射用水、生理盐水及二甲基亚砜(DMSO)，观察溶解情况，结果表明化学合成环七肽MRK不溶于水或生理盐水，只溶于DMSO。通过计算，环七肽MRK本身在注射用水或生理盐水中的溶解度小于0.1mg/ml。同样方法检测环七修饰肽MRG，结果表明，环七修饰肽MRG能完全溶于水、生理盐水以及DMSO。通过计算，本发明修饰后的环七肽在注射用水或生理盐水中的溶解度大于1mg/ml。说明环七修饰肽MRG水溶性远远优于环七肽MRK。
为了进一步比较化学合成环七肽MRK及环七修饰肽MRG的水溶性差异。本发明人对合成工艺进行放大，并逐渐由小试向中试规模的生产过渡，使得制备工艺条件更加稳定，并将制得的环七修饰肽MRG再次验证水溶性。结果表明，环七肽MRK本身在注射用水或生理盐水中的溶解度小于0.1mg/ml，而将更大量的环七修饰肽MRG，具体是10mg的量与1ml注射用水或生理盐水混合，全部 溶解，静置无沉淀析出，多次试验的结果一致。换算百分比浓度，环七修饰肽MRG在注射用水或生理盐水的溶解度超过1.0％，完全解决了原环七肽MRK难溶于水的技术难题。
实施例4：化学合成环七肽MRK及环七修饰肽MRG对金葡菌毒素产生的抑制作用
1、实验试剂、耗材及仪器
化学合成环七肽MRK(CQHWWHWHWYC)及化学合成环七修饰肽MRG(CH3(CH2)10CO-G-(CQHwWHWYC)-RRR-NH2)均由苏州中科天马工程有限公司化学合成，纯度大于95％。金葡菌04018菌株：军事医学科学院基础医学研究所生物化学与分子生物学研究室保存；血平板(新鲜血琼脂培养板)购自北京澳博星生物技术有限责任公司、BHI平板本室自制；进口BHI培养基(BactoTM Brain Heart Infusin)购自美国BD公司；DMEM培养基购自美国CIBCO公司；进口胎牛血清购自美国PAN BIOTECH公司；0.22μm膜购自美国PALL公司；MDBK细胞由本室细胞库保存；台式离心机:德国EPPDORF公司；酶联仪：来自美国MICROPLATE公司；细胞培养瓶购自美国CORNING公司；离心管、刻度吸管、毛细滴管及一次性注射器等其它实验耗材来自本室。
2、实验方法
金葡菌毒素产生抑制水平测定方法参照文献(Yang G,et al.A novel peptide screened by phage display can mimic TRAP antigen epitope against Staphylococcus aureus infections.J Biol.Chem.2005,280:27431-27435)，具体如下：
1)取-70℃冰箱中冻存的金葡菌菌株04018，划线至血平板或者BHI平板内；37℃培养箱中培养16h后，观察板内长出单克隆，放置4℃冰箱中备用；
2)8小时后在超净台内随机挑取平板内的单克隆菌落，接入5ml BHI培养基试管中，共挑取2管；37℃摇床，200rpm振摇，16h后收菌，将2管菌液均匀混和，备用；
3)化学合成肽MRK和MRG及阳性对照品(金葡菌毒素抑制蛋白TP)分别用生理盐水溶解，稀释成不同浓度，加入细菌BHI培养基中，使小肽终浓度分别为0，1.5，5，15，50，150，500μg/ml。溶剂对照组为等量生理盐水；阴性对照组为金葡菌BHI培养基；另设正常细胞对照组。
4)将不同浓度的化学合成肽与金葡菌04018共孵育，培养6h后收菌，将细菌悬液按照编号分别取700μl加入至EP管中，离心，8000rpm,5min,取上清，100℃煮沸7min后，14000rpm离心10min；取10μl上清加入预先接种1×105/mlMDBK细胞的96孔细胞培养板内；继续培养18-20h后，加入MTT液(5mg/ml)5μl/孔，3h后加入10％SDS+0.01M HCL液100μl/孔终止。37℃继续孵箱培养20h后，酶联仪595nm处检测并读数。
3、实验结果与结论
实验结果表明，本发明实施例制备的化学合成环七修饰肽MRG，能很好溶于生理盐水；该化学合成肽能够较好地抑制金葡菌毒素产生，对金葡菌毒素引起的MDBK细胞的增殖抑制具有明显保护作用，呈一定的剂量-效应关系。5μg/ml浓度小肽抑制金葡菌毒素产生效果大于阳性对照TP蛋白的作用,但是化学合成环七肽MRK不溶于水，其混悬液加入金葡菌培养基中不能抑制金葡菌毒素的产生(下表1是化学合成环七肽MRK及环七修饰肽MRG对金葡菌毒素产生的抑制作用观察。)。这种抑菌活性上的差异，本发明人认为是溶解性的改进和修饰后的环七肽结构上的变化共同导致的结果，并非简单的溶解性能的改变导致。
表1、化学合成肽对金葡菌毒素产生的抑制作用
Figure PCTCN2017082106-appb-000003
实施例5：化学合成环七修饰肽MRG对金葡菌感染所致小鼠菌血症的治疗作用
1、实验试剂、耗材及仪器
化学合成环七修饰肽MRG(CH3(CH2)10CO-G-(CQHwWHWYC)-RRR-NH2)由苏州中科天马工程有限公司化学合成，纯度大于95％。金葡菌Newman菌株：军事医学科学院基础医学研究所保存；血平板(新鲜血琼脂培养板)购自北京澳博星生物技术有限责任公司、BHI平板本室自制；进口BHI培养基(BactoTM Brain Heart Infusin)购自美国BD公司；戊巴比妥纳购自美国Sigma公司；台式离心机:德国EPPDORF公司；离心管、刻度吸管及一次性注射器等其它实验耗材来自本室。
2、实验方法
1)金黄色葡萄球菌Newman菌液制备
挑取有溶血圈的Newman单克隆菌株，接种于3mL BHI培养基，220rpm，37℃摇过夜。
取过夜菌1mL，5000rpm，2min离心，去上清。
加入20mL无菌PBS重悬，5000rpm，5min离心，去上清。
用5mL无菌PBS重悬细菌，稀释至OD600＝0.2。
2)BALB/c小鼠菌血症模型建立和多肽活性测定
腹腔注射麻醉剂(1％戊巴比妥纳)BALB/c小鼠(雌性，8周)(200μL/只)。眼球后静脉丛注射Newman菌液(OD600＝0.2，100μL/只)。
感染30分钟后，腹腔注射多肽(0.5mg/mL，200μL/只)，共6只，对照组(无菌水，200μL/只)，共7只。
记录一周内小鼠时间，绘制存活曲线
3、实验结果与结论
实验结果表明，本发明实施例制备的化学合成环七修饰肽MRG，在剂量5mg/kg体重下能很好减少金葡菌所致小鼠菌血症引起的死亡，与无菌注射用水对照组相比生存率大幅度提高；表明化学合成环七肽MRK在体内也有良好的抑制金葡菌感染作用(图5)。

Claims (8)

  1. 一种肽，具有以下通用化学结构式：
    CH3-(CH2)m-X-G-(C-Q-H-W-W-H-W-Y-C)-(R)n-Y；
    其中，
    m＝0-20、优选3-17、更优选6-14、最优选8-12；
    X选自CONH、NHCO、O或S，优选X＝CONH；
    n＝1-10、优选1-7、更优选2-4、最优选3；
    Y选自OH或NH2；
    G代表天然L-型甘氨酸残基或其D-型异构体；
    C代表天然L-型半胱氨酸残基或其D-型异构体，Q代表天然L-型谷氨酰胺残基或其D-型异构体，H代表组氨酸残基或其D-型异构体，W代表天然L-型色氨酸残基或其D-型异构体，Y代表天然L-型酪氨酸残基或其D-型异构体，R代表天然L-型精氨酸残基或其D-型异构体，C表示的两个半胱氨酸通过二硫键相连。
  2. 如权利要求1的肽，其特征在于，其能与金葡菌毒力刺激因子RAP特异结合并抑制金葡菌毒素产生；该肽化合物通过化学合成得到。
  3. 如权利要求1的肽，其特征在于，CH3-(CH2)m-代表烷酰基的烷基部分，优选为CH3(CH2)10-(m＝10)，其与X＝CONH连接时，构成十二烷酰修饰G。
  4. 如权利要求1的肽，其特征在于，G、C、Q、H、W、Y、R独立地代表如下基团：G代表天然L-型甘氨酸残基或其D-型异构体；C代表天然L-型半胱氨酸残基或其D-型异构体；Q代表天然L-型谷氨酰胺残基或其D-型异构体；H代表组氨酸残基或其D-型异构体基；W代表天然L-型色氨酸残基或其D-型异构体；Y代表天然L-型酪氨酸残基或其D-型异构体；R代表天然L-型精氨酸残基或其D-型异构体。
  5. 如权利要求1的肽，其特征在于，其中的9肽(9个氨基酸序列C-Q-H-W-W-H-W-Y-C)的两个半胱氨酸通过二硫键相连。
  6. 如权利要求1至5中的任意一项所述的肽，其特征在于，其是以化学合成方法获得。
  7. 权利要求1至5中的任意一项所述的肽的固相合成制备方法，其特征在于，包括以下步骤：
    步骤一、以Rink Amide-AM Resin替代度为sub＝0.45～0.55mmol/g为起始载体逐个偶联常规保护氨基酸。
    步骤二、采用固相多肽逐步缩合法，在缩合试1-羟基苯并***(HOBT)和N,N'-二异丙基碳二亚胺(DIC)的作用下，根据肽序从C端至N端，逐个偶联Fmoc保护的氨基酸得到:
    Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Rink Amide-AM Resin
    步骤三、利用缩合试剂苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)和N,N-二异丙基乙胺(DIPEA)的作用下偶联十二烷酸得到序列:
    CH3(CH2)10CO-Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Rink Amide-AM Resin
    步骤四、将常规的裂解试剂与步骤三获得的线性肽树脂混合发生裂解反应，从而去除Rink Amide-AM Resin和MRG侧链保护基，再依次通过沉降、离心、洗涤、干燥获得MRG线性粗肽；
    步骤五、将步骤四获得干燥后的线性粗肽研磨至成粉末状，用纯水和乙腈(2:1)的混合溶液溶解MRG线性粗肽至浓度为1mmol/500ml，从而获得线性粗肽溶液；
    步骤六、向所述MRG线性粗肽溶液中滴加稀氨水调节MRG线性粗肽溶液pH至7.1-7.3，并将温度调至30摄氏度左右即可搅拌进行环化实验，环化时间为20-40min经过高效液相色谱HPLC分析检测环化是否完全，待环化完全后加入醋酸调PH至酸性条件进行终止环化反应；
    步骤七、将步骤六中目的产物粗品溶液经过C18反相高效液相色谱柱分离纯化，旋蒸冻干后，制得高纯度的目的产物。
  8. 权利要求1至6中的任意一项所述的肽在制备抗金葡菌感染药物中的应用。
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