阅读说明：本技术 阳离子桥联订书针肽及其应用 (Cationic bridged staple peptides and uses thereof ) 是由 张金强 夏学锋 李红 胡宇晨 于 2019-09-29 设计创作，主要内容包括：本发明公开了一种阳离子桥联订书针肽,所述订书针肽为具有10到20个氨基酸残基的环状多肽,具有以下序列及结构特征：多肽序列可以P<Sub>n</Sub>-K<Sub>c</Sub>-P<Sub>m</Sub>-K<Sub>c</Sub>-P<Sub>i</Sub>为通式；其中,P<Sub>n</Sub>,P<Sub>m</Sub>,及P<Sub>i</Sub>是具有“n”“m”“i”个氨基酸残基的多肽片段,n,i均为自然数,m为3或6；其中K表示赖氨酸,c表示两个赖氨酸侧链氨基之间形成环状结构。本发明提供此类阳离子桥联订书针肽的制备方法及其应用。本发明创新性的在两亲性阳离子抗菌肽的亲水性表面一侧形成环状结构,制备了一类订书针抗菌肽。该类抗菌肽具有以下优点：具有广谱抗菌活性,尤其对临床耐药细菌具有显著抑制效果；结构简单,易于合成；生物稳定性好；可以应用于人或动物感染性疾病或肿瘤的预防和治疗。(The invention discloses a cationic bridged staple peptide, which is a cyclic polypeptide with 10 to 20 amino acid residues and has the following sequence and structural characteristics: the polypeptide sequence may be P n ‑K c ‑P m ‑K c ‑P i Is shown in the general formula (II); wherein, P n ，P m And P i Is a polypeptide fragment with 'n','m' and 'i' amino acid residues, wherein n and i are natural numbers, and m is 3 or 6; wherein K represents lysine, and c represents a cyclic structure formed between two lysine side chain amino groups. The present invention provides a process for the preparation of such cationic bridged staple peptidesPreparation method and application thereof. According to the invention, a ring structure is innovatively formed on one side of the hydrophilic surface of the amphiphilic cationic antibacterial peptide, so that the staple antibacterial peptide is prepared. The antibacterial peptide has the following advantages: has broad-spectrum antibacterial activity and especially has obvious inhibition effect on clinical drug-resistant bacteria; the structure is simple and the synthesis is easy; the biological stability is good; can be applied to the prevention and treatment of infectious diseases or tumors of human beings or animals.)

1. Cationic bridged staple peptides, characterized in that the staple peptides are cyclic polypeptides having 10 to 20 amino acid residues and have the following sequence:

P_n-K_c-P_m-K_c-P_i；

wherein, P_n，P_mAnd P_iIs a polypeptide fragment with n, m and i unspecified amino acid residues, wherein n and i are natural numbers, and m is 3 or 6; wherein K represents lysine, and c represents a cyclic structure formed between two lysine side chain amino groups.

2. The cationic bridged staple peptide of claim 1, characterized in that: the staple peptide consists of at least two lysines and other L-type amino acid residues, and at least two polypeptide fragments consisting of 3 or 6 amino acid residues are separated between the two lysines, and the derivatives are all L-type amino acids and conform to the following sequences:

P_n-K_c-P_m-K_c-P_i

wherein K is selected from the group consisting of lysine or a derivative thereof;

the amino acids at the other positions are L-type amino acids or derivatives thereof.

3. The cationic bridged staple peptide of claim 2, characterized in that: the structure of the staple peptide is as follows:

wherein, K is lysine, and the amino groups at the tail ends of the two lysine side chains are connected with an alkylating reagent through nucleophilic substitution reaction; the R group is selected from alkyl, aryl, heteroaryl, aryl-substituted alkyl, heteroaryl-substituted alkyl or cycloalkyl-substituted alkyl.

4. The cationic bridged staple peptide of claim 3, characterized in that: the R group is 1, 2-dimethylene benzene, 1, 3-dimethylene benzene, 1, 4-dimethylene benzene or trans-1, 4-dimethylene-2-butene.

5. A process for the preparation of the cationic-bridged staple peptide of any one of claims 1 to 4, comprising the steps of:

1) preparing oligopeptide solid phase resin: loading a target oligopeptide on Rink-AM resin by a standard solid-phase synthesis method;

2) preparation of compound 2: loading the side chain protected lysine on a resin by standard solid phase synthesis methods to generate compound 3;

3) preparation of compound 3: carrying out nucleophilic substitution reaction on the compound 2 and a corresponding alkylating reagent to obtain a compound 3;

4) preparation of compound 4: deprotecting the compound 3 to obtain a compound 4;

5) preparation of compound 5: compound 4 is cleaved from the resin to yield compound 5.

6. Cationic bridged staple peptides for use in the preparation of a medicament against microbial infections or tumors in humans or animals, characterized in that the cationic bridged staple peptides according to any one of claims 1 to 4 are used as the staple peptide derivative.

Technical Field

The invention designs a cationic bridged staple peptide, and also relates to preparation and application of the polypeptides, belonging to the field of biomedicine.

Background

There are many documents that show that cyclic peptides are useful for enhancing the stability and increasing the biological activity of polypeptides. There are three main methods for forming cyclic peptides: the side chain is cross-linked with the side chain, head-to-tail, and the side chain is connected with the tail end. Wherein the amino acid side chains and the side chains are mutually connected to form the staple peptide. The formation of the staple peptide helps to stabilize the secondary conformation of the polypeptide and improve the binding capacity of the polypeptide to the receptor. Current methods of forming staple peptides are primarily through olefin metathesis reactions, with cysteine disulfide linkages forming thioethers or amino groups of amino acid side chains coupled with carboxyl groups to form staple peptides. However, the existing methods can only form a ring on the hydrophobic side of the polypeptide, and how to construct the staple peptide on the hydrophilic side is still a blank. For polypeptides, especially antibacterial peptides, charge and amphiphilicity have important influence on the activity of the polypeptides, and how to introduce a staple structure under the condition of keeping the original amphiphilicity of the polypeptides is extremely important.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a cation bridging staple peptide which is not easy to degrade by protease, and simultaneously provides a preparation method of the cation bridging staple peptide and application of the cation bridging staple peptide. The amphiphilic cationic antibacterial peptide staple peptide has a remarkable antibacterial effect, particularly has a good inhibition effect on clinical drug-resistant bacteria, and has the advantages of simple structure, convenience in artificial synthesis, wide antibacterial spectrum and the like. The antibacterial peptide has the remarkable characteristics of resistance to degradation by serum protease and long retention time of antibacterial activity. Can be used for preventing and treating infectious diseases of human or animals.

In order to solve the technical problems, the invention adopts the following technical scheme:

a cationic bridged staple peptide having the sequence:

P_n-K_c-P_m-K_c-P_i

wherein, P_n，P_mAnd P_iIs a polypeptide fragment with 'n','m' and 'i' amino acid residues, wherein n and i are natural numbers, and m is 3 or 6; wherein K represents lysine, c represents a ring structure formed between terminal amino groups of two lysine side chains, and the total length of the polypeptide sequence is 10-20 amino acid residues. The invention obtains the staple peptides with the general formula by a novel, green and efficient method, and activity tests prove that the method effectively enhances the stability and the bioactivity of the polypeptide and has potential medicinal value.

In a preferred embodiment of the present invention, the staple peptide comprises a polypeptide sequence consisting of at least two lysine residues and other L-type amino acid residues, wherein the two lysines are separated by 3 or 6 amino acid residues, and the derivatives are all L-type amino acids and correspond to one of the following sequences:

P_n-K_c-P_m-K_c-P_i

wherein K is selected from the group consisting of lysine or a derivative thereof;

the amino acids at the other positions are L-type amino acids or derivatives thereof.

In another preferred embodiment of the present invention, the structure of the staple peptide is as follows:

wherein, K is lysine, and the amino groups at the tail ends of the two lysine side chains are connected with an alkylating reagent through nucleophilic substitution reaction; the R group is selected from alkyl, aryl, heteroaryl, aryl-substituted alkyl, heteroaryl-substituted alkyl or cycloalkyl-substituted alkyl.

As a further preferred embodiment of the invention, the R group is 1, 2-dimethylene benzene, 1, 3-dimethylene benzene, 1, 4-dimethylene benzene or trans 1, 4-dimethylene-2-butene.

A method for preparing a cationic-bridged staple peptide, the method comprising the steps of:

1) preparing oligopeptide solid phase resin: loading a target oligopeptide on Rink-AM resin by a standard solid-phase synthesis method;

2) preparation of compound 2: loading the side chain protected lysine on resin by standard solid phase synthesis method to generate compound 2;

3) preparation of compound 3: carrying out nucleophilic substitution reaction on the compound 2 and a corresponding alkylating reagent to obtain a compound 3;

4) preparation of compound 4: deprotecting the compound 3 to obtain a compound 4;

5) preparation of compound 5: compound 4 is cleaved from the resin to yield compound 5.

The staple peptide can be used for preparing medicaments for resisting tumors or microbial infection of human or animals.

Compared with the prior art, the invention has the technical effects that:

1. the invention designs and synthesizes the cation-bridged antibacterial peptide with the staple structure by taking the linear antibacterial peptide as a template, enhances the stability of the polypeptide while enhancing the antibacterial activity, and effectively prolongs the action time.

2. The obtained antibacterial peptide with the staple structure has a remarkable antibacterial effect, particularly has a good inhibition effect on clinical drug-resistant bacteria, and compared with other antibacterial peptides, the antibacterial peptide with the staple structure has the advantages of simple structure, convenience in artificial synthesis, wide antibacterial spectrum and the like, particularly has stronger capability of resisting hydrolysis of serum protease, and can keep the activity of microorganisms for a longer time; the antibacterial peptide has the remarkable characteristics of resistance to protease degradation and long antibacterial activity retention time; can be used for preventing and treating infectious diseases of human or animals.

3. The obtained antibacterial peptide with the staple structure consists of L-type amino acid residues, resists the degradation of serum protease, and has obvious difference with linear peptide in the aspect of antimicrobial activity; the staple antibacterial peptide has remarkable capability of inhibiting the growth of microorganisms, and particularly has good inhibition effect on clinical pressure-resistant bacteria.

4. The invention keeps the charge and the amphipathy unchanged while forming the staple peptide, is beneficial to reducing the influence of structural change on the biological activity of the polypeptide and has larger potential drug application value.

Drawings

FIG. 1 is a flow diagram of the preparation of cationic bridged staple peptides;

FIG. 2 is a graph showing the results of a stability test of the staple peptide in 25% serum;

FIG. 3 is a graph of the sterilization efficiency of LH 11516 ug/mL;

FIG. 4 is a graph of the sterilization efficiency of LH 06532 ug/mL;

FIG. 5 is a graph showing the antitumor effect of LH 115.

Detailed Description

The invention is described in further detail below with reference to the figures and the detailed description.

The full name or corresponding Chinese name of a part of the material is as follows:

DIEA N, N-diisopropylethylamine

DMF: n, N-dimethylformamide

THF: tetrahydrofuran (THF)

TBAH tetrabutylammonium hydroxide

DBU 1, 8-diazabicyclo [5.4.0] undec-7-ene example 1: preparation of antibiotic peptide for stitching needle

The preparation route is as follows, as shown in figure 1:

1. preparing oligopeptide solid phase resin: Rink-AM resin (loading 0.35mmol/g, initial loading 200mg per peptide), Fmoc-Val-OH was loaded on Rink-AM resin by standard solid phase synthesis methods.

2. Preparation of side chain o-nitrobenzenesulfonyl protected lysine: Fmoc-Lys (Boc) -OH 4.68g is weighed and placed in a 250mL round-bottom flask, and then solvent CH is measured₂Cl₂60mL and 20mL trifluoroacetic acid (TFA) were added to a round bottom flask with 10mL CH₂Cl₂The TFA measuring cylinder was measured and the rinsing solution was transferred to a round bottom flask and stirred for 1 h. To the resulting intermediate Fmoc-Lys-OH & TFA, H was added₂Tetrahydrofuran (THF): 1 (55 mL each), N-ethyldiisopropylamine (PH) to 9, o-nitrobenzenesulfonyl chloride (2.1 g), stirring at room temperature for 2h, extracting, and separating by column chromatography to obtain the amino acid with higher purity.

3. Preparation of the antimicrobial peptide linear peptide: amino acids are sequentially coupled to resin from a carbon end according to a sequence by a standard solid phase synthesis method (lysine with a side chain protected by o-nitrobenzenesulfonyl is inserted when the second amino acid and the sixth amino acid are coupled), and after each coupling is finished, the resin is washed 3 times by DMF, methanol and dichloromethane, so that the linear peptide antibacterial peptide LH091a is finally obtained.

4. Preparation of alkylated polypeptide chains: the linear antimicrobial peptide synthesized by standard solid phase synthesis method is swelled in DCM for 10min, added with THF 4mL and TBAH (6eq) and shaken for 10min, then added with alkylating reagent 1, 4-dibromobutene (6eq), shaken for 24h on a shaking table and washed with DMF, methanol and dichloromethane for 3 times respectively to obtain LH091 b.

5. Preparation of deprotected polypeptide chains: the alkylated staple peptide was swollen in DCM for 10min, 2-mercaptoethanol (5eq), DBU (10eq) was added, shaken on a shaker for 12h and washed 3 times with DMF, methanol, dichloromethane in turn to give LH091 c.

6. Preparation of the final product of the staple peptide: adding a cleavage reagent (TFA: TIS: H) to the deprotected staple peptide solid phase tube₂O95: 2.5: 2.5), cutting overnight on a shaker. Collecting the cutting solution, air drying, adding cold diethyl ether for precipitation, centrifuging, discarding diethyl ether, adding water for dissolution, freeze-drying, and semi-preparing, separating and purifying to obtain LH 090. Yield 18.41%, purity>95％。

7. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into the 5 th and 9 th amino acids, and the other treatments are the same as LH090 to obtain a compound LH 065. The yield is 16.63 percent, and the purity is more than 95 percent.

8. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into 6 th and 10 th amino acids, and other treatments are the same as LH090 to obtain the compound LH 092. The yield is 19.07 percent, and the purity is more than 95 percent.

9. The site of the lysine with the side chain modified by o-nitrobenzenesulfonyl is changed into the 10 th and 14 th amino acids, and the other treatments are the same as LH090 to obtain the compound LH 091. The yield is 15.97%, and the purity is more than 95%.

10. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into 13 th and 17 th amino acids, and other treatments are the same as LH090 to obtain a compound LH 089. The yield is 13.79 percent, and the purity is more than 95 percent.

11. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into the 2 nd and 9 th amino acids, and other treatments are the same as LH090 to obtain the compound LH 109. The yield is 10.68 percent, and the purity is more than 95 percent.

12. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into 6 th and 13 th amino acids, and other treatments are the same as LH090 to obtain the compound LH 110. The yield is 9.5%, and the purity is more than 95%.

13. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into the 9 th and 16 th amino acids, and the other treatments are the same as LH090 to obtain the compound LH 124. The yield is 13.6 percent, and the purity is more than 95 percent.

14. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into the 10 th and 17 th amino acids, and the other treatments are the same as LH090 to obtain a compound LH 111. The yield is 9.96 percent, and the purity is more than 95 percent.

15. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into 9 th and 13 th amino acids, and the other treatments are the same as LH090 to obtain a compound LH 123. The yield is 14.61%, and the purity is more than 95%.

16. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into the 5 th and 9 th amino acids, the alkylating reagent is changed into 1, 2-dibromomethylbenzene, and the other treatments are the same as LH090 to obtain a compound LH 115. The yield is 16.01 percent, and the purity is more than 95 percent.

17. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into the 5 th and 9 th amino acids, the alkylating reagent is changed into 1, 3-dibromomethylbenzene, and other treatments are the same as LH090 to obtain a compound LH 116. The yield is 12.57%, and the purity is more than 95%.

18. The site of lysine with side chain modified by o-nitrobenzenesulfonyl is changed into the 5 th and 9 th amino acids, the alkylating reagent is changed into 1, 4-dibromomethylbenzene, and other treatments are the same as LH090 to obtain a compound LH 117. The yield is 13.96 percent, and the purity is more than 95 percent.