阅读说明：本技术 一组含n-甲基化氨基酸及n端脂肪酸修饰的抗菌肽类似物及其合成方法和应用 (group of antibacterial peptide analogues containing N-methylated amino acid and N-terminal fatty acid modification, and synthetic method and application thereof ) 是由 倪京满 王锐 刘天琪 王一杰 于 2019-09-04 设计创作，主要内容包括：本发明公开了一组含N-甲基化氨基酸及N端脂肪酸修饰的抗菌肽类似物,是在天然抗菌肽Anoplin上引入多个N-甲基化氨基酸,在结构的N端引入脂肪酸碳链修饰,经多肽切割和纯化获得。通过对抗菌肽体外抑菌和酶解稳定性实验,表明本发明合成的抗菌肽类似物随脂肪酸碳链的增长抗菌活性增加,且活性优于母肽Anoplin；同时在高浓度酶环境中仍能保持抗菌活性,这表明本发明合成的抗菌肽类似物在胰蛋白酶和糜蛋白酶环境中具有良好的稳定性。因此,该含N-甲基化氨基酸及N端脂肪酸修饰的抗菌肽类似物在制备临床抗菌药物方面具有很好的应用前景。(the invention discloses a group of antibacterial peptide analogues containing N-methylated amino acids and N-terminal fatty acid modification, which are obtained by introducing a plurality of N-methylated amino acids into natural antibacterial peptide Anoplin, introducing fatty acid carbon chain modification into the N terminal of the structure, and performing polypeptide cutting and purification. Experiments on in-vitro bacteriostasis and enzymolysis stability of the antibacterial peptide show that the antibacterial activity of the synthesized antibacterial peptide analogue is increased along with the growth of a fatty acid carbon chain, and the activity of the synthesized antibacterial peptide analogue is superior to that of the parent peptide Anoplin; meanwhile, the antibacterial peptide analogue can still maintain antibacterial activity in a high-concentration enzyme environment, which shows that the antibacterial peptide analogue synthesized by the invention has good stability in trypsin and chymotrypsin environments. Therefore, the antibacterial peptide analogue containing N-methylated amino acid and N-terminal fatty acid modification has good application prospect in the aspect of preparing clinical antibacterial drugs.)

1. A group of antibacterial peptide analogues containing N-methylated amino acid and N-terminal fatty acid modification has the following structure:

Cn-Gly-Leu-(N-Me-Leu)-Lys-Arg-(N-Me-Ile)-Lys-Thr-Leu-Leu-NH₂Labeled as Cn-M3.6;

Cn-Gly-Leu-Leu-(N-Me-Lys)-Arg-Ile-(N-Me-Lys)-Thr-Leu-Leu-NH₂labeled as Cn-M4.7;

Cn-Gly-Leu-Leu-Lys-(N-Me-Arg)-Ile-(N-Me-Lys)-Thr-Leu-Leu-NH₂Labeled as Cn-M5.7;

Wherein n = 8-14.

2. the use of an analog of an antimicrobial peptide comprising an N-methylated amino acid and an N-terminal fatty acid modification of claim 1 as an active ingredient in the preparation of an antimicrobial medicament.

3. The method of synthesizing an analog of an antimicrobial peptide comprising an N-methylated amino acid and an N-terminal fatty acid modification of claim 1, wherein: respectively introducing N-methylated amino acids into natural antimicrobial peptide Anoplin, respectively introducing fatty acid carbon chain modification into the N end of the structure, and performing polypeptide cutting and purification to obtain the antimicrobial peptide analogue containing the N-methylated amino acids and the N-end fatty acid modification.

4. a method of synthesizing an analog of an antimicrobial peptide containing an N-methylated amino acid and an N-terminal fatty acid modification of claim 3, wherein: respectively introducing N-methylated amino acids into natural antimicrobial peptide Anoplin, wherein the introduction sites are 3 and 6 sites, respectively, then respectively introducing fatty acid carbon chain modification into the N end of the structure, and obtaining the antimicrobial peptide analogue Cn-M3.6 containing the N-methylated amino acids and the N end fatty acid modification through polypeptide cutting and purification.

5. A method of synthesizing an analog of an antimicrobial peptide containing an N-methylated amino acid and an N-terminal fatty acid modification of claim 3, wherein: respectively introducing N-methylated amino acids into natural antimicrobial peptide Anoplin, wherein the introduction sites are 4 and 7 sites, respectively, then respectively introducing fatty acid carbon chain modification into the N end of the structure, and obtaining the antimicrobial peptide analogue Cn-M4.7 containing the N-methylated amino acids and the N end fatty acid modification through polypeptide cutting and purification.

6. A method of synthesizing an analog of an antimicrobial peptide containing an N-methylated amino acid and an N-terminal fatty acid modification of claim 3, wherein: respectively introducing N-methylated amino acids on natural antimicrobial peptide Anoplin, wherein the introduction sites are 5 and 7 sites, respectively, then respectively introducing fatty acid carbon chain modification at the N end of the structure, and obtaining the antimicrobial peptide analogue Cn-M5.7 containing the N-methylated amino acids and the N-end fatty acid modification through polypeptide cutting and purification.

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a group of antibacterial peptide analogs containing N-methylated amino acid and N-terminal fatty acid modification, and a synthesis method and application thereof, which are mainly used for preparing clinical antibacterial drugs.

background

Antibiotics are widely used as first-line drugs for treating infectious diseases, saving countless lives. However, the abuse of antibiotics finally leads to the emergence of antibiotic-resistant bacteria, and the continuous evolution of multi-drug-resistant bacteria threatens the health and safety of human beings, so that the development of new antibacterial drugs is urgently needed to solve the problem. Antimicrobial peptides (AMPs), also known as host defense peptides, are important components of the innate immune system, are widely distributed in nature, and have various biological activities such as antibacterial, antifungal, antitumor, antiviral, antiparasitic, immunomodulating, and the like. Different from the traditional antibiotics mainly acting on specific targets of bacteria, the unique nonspecific membrane destruction mechanism of the antibacterial peptide enables the antibacterial peptide to effectively resist multi-drug resistant bacteria, is not easy to induce the generation of drug resistance, and can be used as an antibiotic substitute with development potential.

The extraction process of the natural antibacterial peptide is complex, and the natural antibacterial peptide is easily degraded by protease due to the fact that the natural antibacterial peptide is mostly L-shaped amino acid in composition, and the stability is poor, so that the further clinical application of the natural antibacterial peptide is limited. The N-methylated amino acid substitution is a modification method widely applied to various bioactive peptides, and the introduction of the N-methylated amino acid can effectively improve the stability of the polypeptide and enhance the capability of resisting protease hydrolysis. Blake et al first applied N-methylated amino acids in the synthesis of [2-N-Methylphenylalanine ] -alpha-ACTH to increase resistance to exopeptidase degradation (International Journal of Peptide and protein research, 1972,4: 343-345.). In addition, the bioactive natural product containing N-methylated amino acid also has obvious therapeutic effect, and the cyclosporine is cyclic peptide containing 7N-methylated amino acids, has immunosuppressive effect and also has good oral bioavailability. It has been reported that the half-life of the antibacterial peptide oncocin in the serum of 25% mice is prolonged by replacing Arg at the C-terminal cleavage site of the sequence of the antibacterial peptide oncocin with N-Me-Arg, and the original antibacterial activity is retained (International journal of antibacterial Agents,2011,37 (2)). The introduction of single or multiple N-methylated amino acids in the R1 backbone of a polypeptide significantly improves the stability of the polypeptide against degradation by serum proteins (Journal of Biological Chemistry,2009,284 (14)).

polymyxin B is a polypeptide antibiotic effective against gram-negative bacteria, and its antibacterial activity is almost lost after removal of its fatty acid moiety, indicating that linking fatty acids in the antibacterial peptide sequence improves the antibacterial activity of the antibacterial peptide, facilitating the interaction of the antibacterial peptide with the bacterial cell membrane. It has been found that by introducing fatty acid chains of different lengths into the polypeptide dendrimer G2KL, the antimicrobial activity is increased in the form of "U", and the optimal antimicrobial activity is obtained after C10/C12 is linked, wherein TNS18 has broad-spectrum antimicrobial activity against multidrug-resistant bacteria and MRSA, and the plasma half-life is extended (Journal of the American chemical Society,2018,140 (1)).

Disclosure of Invention

it is an object of the present invention to provide a group of analogs of antimicrobial peptides containing N-methylated amino acids and modifications of the N-terminal fatty acid.

the invention also aims to provide a synthesis method of a group of antibacterial peptide analogues containing N-methylated amino acid and N-terminal fatty acid modification.

The invention also aims to provide an application of a group of antibacterial peptide analogues containing N-methylated amino acid and N-terminal fatty acid modification in preparation of antibacterial drugs.

in order to achieve the purpose, the invention adopts the technical scheme that:

The structure and the synthesis method of a group of antibacterial peptide analogues containing N-methylated amino acid and N-terminal fatty acid modification:

The invention relates to an antibacterial peptide analogue containing N-methylated amino acid and N-terminal fatty acid modification with high antibacterial activity and high enzymolysis stability, which is characterized in that N-methylated amino acid modification is respectively carried out on Leu at the 3 position, Arg at the 5 position, Ile at the 6 position, Lys at the 4 position and Lys at the 7 position of natural antibacterial peptide Anoplin, and fatty acid carbon chain (C8-C14) modification with different lengths is carried out on the screened analogue with higher stability at the N terminal thereof, and the specific steps are as follows:

respectively introducing N-methylated amino acids on natural antimicrobial peptide Anoplin, wherein the introduction sites are respectively 3 and 6, respectively introducing fatty acid carbon chain modification at the N end of the structure, and performing polypeptide cutting and purification to obtain an antimicrobial peptide analogue containing the N-methylated amino acids and the N-end fatty acid modification, wherein the antimicrobial peptide analogue is named as Cn-M3.6 and has the structural formula: Cn-Gly-Leu- (N-Me-Leu) -Lys-Arg- (N-Me-Ile) -Lys-Thr-Leu-Leu-NH₂wherein n = 8-14.

in the natural antibacterial peptide Anoplrespectively introducing N-methylated amino acids into the in, wherein the introduction sites are 4 and 7, respectively, then respectively introducing fatty acid carbon chain modification into the N end of the structure, and performing polypeptide cutting and purification to obtain the antibacterial peptide analogue containing the N-methylated amino acids and the N-end fatty acid modification, wherein the name of the analogue is Cn-M4.7, and the structural formula of the analogue is as follows: Cn-Gly-Leu-Leu- (N-Me-Lys) -Arg-Ile- (N-Me-Lys) -Thr-Leu-Leu-NH₂wherein n = 8-14.

Respectively introducing N-methylated amino acids on natural antimicrobial peptide Anoplin, wherein the introduction sites are 5 and 7, respectively, then respectively introducing fatty acid carbon chain modification at the N end of the structure, and obtaining the antimicrobial peptide analogue containing the N-methylated amino acids and the N-end fatty acid modification through polypeptide cutting and purification, wherein the name is Cn-M5.7, and the structural formula is as follows: Cn-Gly-Leu-Leu-Lys- (N-Me-Arg) -Ile- (N-Me-Lys) -Thr-Leu-Leu-NH₂wherein n = 8-14.

The invention relates to a synthesis method of an antibacterial peptide analogue containing N-methylated amino acid and N-terminal fatty acid modification, which adopts a polypeptide solid phase synthesis method, uses HOAt/HATU to replace HOBt/HBTU as a condensing agent to couple amino acid and N-methylated amino acid in the polypeptide synthesis process, adopts a chloranil method to detect secondary amine, synthesizes a polypeptide containing N-methylated amino acid and connected with MBHA resin, respectively connects fatty acid carbon chains (C8-C14) with different lengths at the N tail end of the polypeptide for modification, and obtains the antibacterial peptide analogue containing N-methylated amino acid and N-terminal fatty acid modification after cutting and purifying the polypeptide.

Secondly, the application of the antibacterial peptide analogue containing N-methylated amino acid and N-terminal fatty acid modification as an active ingredient in the preparation of antibacterial drugs, namely an antibacterial peptide in-vitro bacteriostasis experiment:

The Minimum Inhibitory Concentration (MIC) value of the medicine to different gram-positive bacteria and gram-negative bacteria is determined by adopting a common trace continuous two-fold dilution method. Results three or more replicates were performed. The results are shown in Table 1.

the results in Table 1 show that the antibacterial activity of the antibacterial peptide analogue modified by N-terminal esterification is increased along with the growth of a fatty acid carbon chain, wherein C12-M3.6/M4.7/M5.7 and C14-M3.6/M4.7/M5.7 have the best antibacterial activity, and the activity is better than that of the parent peptide Anoplin.

Thirdly, enzymolysis stability experiment:

to examine the stability of the synthetic antimicrobial peptide analogs of the present invention in the enzymatic environment, antimicrobial activity of P.aeruginosa was determined by incubating the parent peptide Anoplin and antimicrobial peptide Cn-M3.6/M4.7/M5.7 (n = 8-14) with varying concentrations of trypsin and chymotrypsin, respectively, for 6h (see FIGS. 1-2).

The results show that the parent peptide Anoplin loses antibacterial activity in the presence of low concentrations of trypsin and chymotrypsin, while the antibacterial peptide analogue containing N-methylated amino acids and N-terminal fatty acid modifications can still maintain antibacterial activity in high concentrations of enzyme. The antibacterial peptide analogue synthesized by the invention has good stability in the environments of trypsin and chymotrypsin.

Drawings

FIG. 1 is a graph showing the inhibition and killing ability of antimicrobial peptide against Pseudomonas aeruginosa after incubation for 6h with trypsin at different concentrations;

FIG. 2 is a graph showing the inhibition and killing ability of antimicrobial peptide on Pseudomonas aeruginosa after incubation for 6h with chymotrypsin of various concentrations;

FIG. 3 is a mass spectrum of C8-M3.6;

FIG. 4 is a mass spectrum of C8-M4.7;

FIG. 5 is a mass spectrum of C8-M5.7;

FIG. 6 is a mass spectrum of C10-M3.6;

FIG. 7 is a mass spectrum of C10-M4.7;

FIG. 8 is a mass spectrum of C10-M5.7;

FIG. 9 is a mass spectrum of C12-M3.6;

FIG. 10 is a mass spectrum of C12-M4.7;

FIG. 11 is a mass spectrum of C12-M5.7;

FIG. 12 is a mass spectrum of C14-M3.6;

FIG. 13 is a mass spectrum of C14-M4.7;

FIG. 14 is a mass spectrum of C14-M5.7.

Detailed Description

the structure and synthesis of the analogs of the peptides of the invention containing N-methylated amino acids and N-terminal fatty acid modifications are further illustrated by the following specific examples.