CN110563802B - Group of antibacterial peptide analogues containing N-methylated amino acid and N-terminal fatty acid modification, and synthetic method and application thereof - Google Patents
Group of antibacterial peptide analogues containing N-methylated amino acid and N-terminal fatty acid modification, and synthetic method and application thereof Download PDFInfo
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Abstract
The invention discloses a group of antibacterial peptide analogs 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.
Description
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, and immunoregulatory effects. 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 C-terminal cleavage site of the oncocin sequence of the antibacterial peptide is prolonged by N-Me-Arg and the original antibacterial activity is retained in 25% of mouse serum (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 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 a "U-shape", and the C10/C12 linkage has the best antimicrobial activity, among which TNS18 has broad-spectrum antimicrobial activity against multidrug-resistant bacteria and MRSA, and has an extended plasma half-life (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:
1. a group of antibacterial peptide analogues containing N-methylated amino acid and N-terminal fatty acid modification has the structure and a synthesis method:
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 (C10-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-NH 2 Wherein n =8-14.
Respectively introducing N-methylated amino acids on natural antimicrobial peptide Anoplin, wherein the introduction sites are 4 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-M4.7, and the structural formula is as follows: cn-Gly-Leu-Leu- (N-Me-Lys) -Arg-Ile-(N-Me-Lys)-Thr-Leu-Leu-NH 2 Wherein n =8-14.
Respectively introducing N-methylated amino acids on natural antimicrobial peptide Anoplin, wherein the introduction sites are respectively 5 and 7, 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 Cn-M5.7, and the structural formula of the antimicrobial peptide analogue is as follows: cn-Gly-Leu-Leu-Lys- (N-Me-Arg) -Ile- (N-Me-Lys) -Thr-Leu-Leu-NH 2 Wherein 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.
2. 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 increase of the carbon chain of the fatty acid, 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.
3. 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.
Example 1 Synthesis of antimicrobial peptide Cn-M3.6
(1) Activation and pretreatment of resins
Accurately weighing MBHA resin (0.43 mmol/g) in calculated amount, adding into polypeptide solid phase synthesizer, adding DCM, stirring for 30min, and draining. After washing with DMF, the resin is colorless and transparent as identified by ninhydrin color development, which indicates that the resin is normal and can be used.
(2) Synthesis of Cn-M3.6-MBHA
a. Coupling of Leu: the swelled resin is washed by 20% piperidine/DMF solution, drained and washed by redistilling DMF, and the indeno resin is bluish purple, which shows that Fmoc protective group is removed and amino acid can be connected. Weighing 3 times of excessive amino acid and 3 times of excessive HOBt and HBTU, dissolving by redistilled DMF, adding 6 times of excessive DIEA to start reaction, adding into a synthesizer, reacting and stirring for 1h under the protection of argon, draining after the reaction time is up, washing by redistilled DMF, and obtaining the Fmoc-Leu-MBHA if the indene detection resin is colorless and transparent, which indicates that the reaction is finished and Leu is successfully coupled;
b. coupling of Leu: the method is the same as the step a, and Fmoc-Leu-Leu-MBHA is obtained;
c. coupling of Thr: the method is the same as the step a, and Fmoc-Thr-Leu-Leu-MBHA is obtained;
d. coupling of Lys: the method is the same as the step a, and Fmoc-Lys-Thr-Leu-Leu-MBHA is obtained;
e. coupling of N-Me-Ile: the method is the same as the step a, and Fmoc- (N-Me-Ile) -Lys-Thr-Leu-Leu-MBHA is obtained;
f. coupling of Arg: removing Fmoc protection from 20% piperidine/DMF solution for 1h, and verifying whether the Fmoc protection group is removed by a chloranil method: if the Fmoc protecting group is colorless, the Fmoc protecting group is not removed, and the steps are repeated; in the case of blue color, the Fmoc protecting group removal was successful. Weighing 3 times of excessive amino acid, 3 times of excessive HOAt and HATU and 6 times of excessive DIEA, adding into a synthesizer, reacting and stirring for 2h under the protection of argon, draining after the reaction time is up, washing by using redistilled DMF, detecting by using a chloranil method, and repeating the steps if the resin is blue, which indicates that the coupling is incomplete; if the resin is colorless, arg is successfully coupled to obtain Fmoc-Arg- (N-Me-Ile) -Lys-Thr-Leu-Leu-MBHA;
g. coupling of Lys: the method is the same as the step a, and Fmoc-Lys-Arg- (N-Me-Ile) -Lys-Thr-Leu-Leu-MBHA is obtained;
h. coupling of N-Me-Leu: the method is the same as the step a, and Fmoc- (N-Me-Leu) -Lys-Arg- (N-Me-Ile) -Lys-Thr-Leu-Leu-MBHA is obtained;
i. coupling of Leu: the same procedure as step f to obtain Fmoc-Leu- (N-Me-Leu) -Lys-Arg- (N-Me-Ile) -Lys-Thr-Leu-Leu-MBHA;
j. coupling of Gly: the method is the same as the step a, obtaining Fmoc-Gly-Leu- (N-Me-Leu) -Lys-Arg- (N-Me-Ile) -Lys-Thr-Leu-Leu-MBHA;
k. acylation of the N-terminus: removing Fmoc protection from 20% piperidine/DMF solution, washing with DMF, and detecting with indene to be bluish purple to obtain Gly-Leu- (N-Me-Leu) -Lys-Arg- (N-Me-Ile) -Lys-Thr-Leu-Leu-MBHA. Respectively mixing fatty acid, HOBt, HBTU, DIEA and DMF uniformly, reacting and stirring for 1.5h under the protection of argon, draining, washing with DMF, and detecting with indene to obtain Cn-Gly-Leu- (N-Me-Leu) -Lys-Arg- (N-Me-Ile) -Lys-Thr-Leu-Leu-MBHA, wherein N =8-14;
(3) Polypeptide cleavage
The Cn-Gly-Leu- (N-Me-Leu) -Lys-Arg- (N-Me-Ile) -Lys-Thr-Leu-Leu-MBHA (N = 8-14) obtained in the above process was washed with redistilled DCM and redistilled methanol in sequence, sealed with a plug and then completely drained until the resin was totally powdery. Cleavage was performed by adding a cleavage reagent (TFA: tris: water = 9.5.
(4) Polypeptide purification
RP-HPLC purification: mobile phase a 0.1% tfa/water, mobile phase B0.1% tfa/acetonitrile, linear gradient elution, collection of target peak effluent, lyophilization, mass spectrometric identification, see figures 3, 6, 9 and 12.
Example 2 Synthesis of antimicrobial peptide Cn-M4.7
(1) Activation and pretreatment of resins
The procedure is as in example 1.
(2) Synthesis of Cn-M4.7-MBHA
a. Coupling of Leu: the swelled resin is washed by 20% piperidine/DMF solution, drained and washed by redistilling DMF, and the indenylated resin is bluish purple, which shows that Fmoc protective groups are removed and amino acid can be connected. Weighing 3 times of excessive amino acid and 3 times of excessive HOBt and HBTU, dissolving by redistilled DMF, adding 6 times of excessive DIEA to start reaction, adding into a synthesizer, reacting and stirring for 1h under the protection of argon, draining after the reaction time is up, washing by redistilled DMF, and obtaining the Fmoc-Leu-MBHA if the indene detection resin is colorless and transparent, which indicates that the reaction is finished and Leu is successfully coupled;
b. coupling of Leu: the method is the same as the step a, and Fmoc-Leu-Leu-MBHA is obtained;
c. coupling of Thr: the method is the same as the step a, and Fmoc-Thr-Leu-Leu-MBHA is obtained;
d. coupling of N-Me-Lys: the method is the same as the step a, and Fmoc- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
e. coupling of Ile: removing Fmoc protection from 20% piperidine/DMF solution for 1h, and verifying whether the Fmoc protection group is removed by a chloranil method: if the Fmoc protecting group is colorless, the Fmoc protecting group is not removed, and the steps are repeated; in the case of blue color, the Fmoc protecting group removal was successful. Weighing 3 times of excessive amino acid, 3 times of excessive HOAt and HATU and 6 times of excessive DIEA, adding into a synthesizer, reacting and stirring for 2h under the protection of argon, draining after the reaction time is up, washing by using redistilled DMF, detecting by using a chloranil method, and repeating the steps if the resin is blue, which indicates that the coupling is incomplete; if the resin is colorless, successfully coupling Ile to obtain Fmoc-Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA;
f. coupling of Arg: the method is the same as the step e, and Fmoc-Arg-Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
g. coupling of N-Me-Lys: the method is the same as the step e, and Fmoc- (N-Me-Lys) -Arg-Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
h. coupling of Leu: the method is the same as the step e, and Fmoc-Leu- (N-Me-Lys) -Arg-Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
i. coupling of Leu: the method is the same as the step a, and Fmoc-Leu-Leu- (N-Me-Lys) -Arg-Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
j. coupling of Gly: the method is the same as the step a, and Fmoc-Gly-Leu-Leu- (N-Me-Lys) -Arg-Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
k. acylation of the N-terminus: removing Fmoc protection from 20% piperidine/DMF solution, washing with DMF, and detecting with indene to be bluish purple to obtain Gly-Leu-Leu- (N-Me-Lys) -Arg-Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA. Respectively mixing fatty acid, HOBt, HBTU, DIEA and DMF uniformly, reacting and stirring for 1.5h under the protection of argon, draining, washing with DMF, and detecting with indene to obtain Cn-Gly-Leu-Leu- (N-Me-Lys) -Arg-Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA, wherein N =8-14;
(3) Polypeptide cleavage
The procedure is as in example 1.
(4) Polypeptide purification
The procedure is as in example 1, mass spectrometric identification, see FIGS. 4, 7, 10 and 13.
Example 3 Synthesis of antimicrobial peptide Cn-M5.7
(1) Activation and pretreatment of resins
The same as in example 1.
(2) Synthesis of Cn-M5.7-MBHA
a. Coupling of Leu: the swelled resin is washed by 20% piperidine/DMF solution, drained and washed by redistilling DMF, and the indenylated resin is bluish purple, which shows that Fmoc protective groups are removed and amino acid can be connected. Weighing 3 times of excessive amino acid and 3 times of excessive HOBt and HBTU, dissolving by redistilled DMF, adding 6 times of excessive DIEA to start reaction, adding into a synthesizer, reacting and stirring for 1h under the protection of argon, draining after the reaction time is up, washing by redistilled DMF, and obtaining Fmoc-Leu-MBHA (methyl methacrylate-ethyl methacrylate) if the indene detection resin is colorless and transparent, which indicates that the reaction is finished and Leu is successfully coupled;
b. coupling of Leu: the method is the same as the step a, and Fmoc-Leu-Leu-MBHA is obtained;
c. coupling of Thr: the method is the same as the step a, and Fmoc-Thr-Leu-Leu-MBHA is obtained;
d. coupling of N-Me-Lys: the method is the same as the step a, and Fmoc- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
e. coupling of Ile: removing Fmoc protection from 20% piperidine/DMF solution for 1h, and verifying whether the Fmoc protection group is removed by a chloranil method: if the Fmoc protecting group is colorless, the Fmoc protecting group is not removed, and the steps are repeated; in the case of blue color, the Fmoc protecting group removal was successful. Weighing 3 times of excessive amino acid, 3 times of excessive HOAt and HATU and 6 times of excessive DIEA, adding into a synthesizer, reacting and stirring for 2h under the protection of argon, draining after the reaction time is up, washing by using redistilled DMF, detecting by using a chloranil method, and repeating the steps if the resin is blue, which indicates that the coupling is incomplete; if the resin is colorless, successfully coupling Ile to obtain Fmoc-Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA;
f. coupling of N-Me-Arg: the method is the same as the step e, and Fmoc- (N-Me-Arg) -Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
g. coupling of Lys: the method is the same as the step e, and Fmoc-Lys- (N-Me-Arg) -Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
h. coupling of Leu: the method is the same as the step a, and Fmoc-Leu-Lys- (N-Me-Arg) -Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
i. coupling of Leu: the method is the same as the step a, and Fmoc-Leu-Leu-Lys- (N-Me-Arg) -Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
j. coupling of Gly: the method is the same as the step a, and Fmoc-Gly-Leu-Leu-Lys- (N-Me-Arg) -Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA is obtained;
k. acylation of the N-terminus: removing Fmoc protection from 20% piperidine/DMF solution, washing with DMF, and detecting with indene to be bluish purple to obtain Gly-Leu-Leu-Lys- (N-Me-Arg) -Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA. Respectively mixing fatty acid, HOBt, HBTU, DIEA and DMF uniformly, reacting and stirring for 1.5h under the protection of argon gas, draining, washing with DMF, and detecting with indene to obtain Cn-Gly-Leu-Leu-Lys- (N-Me-Arg) -Ile- (N-Me-Lys) -Thr-Leu-Leu-MBHA, wherein N =8-14;
(3) Cleavage of polypeptides
The procedure is as in example 1.
(4) Polypeptide purification
The procedure is as in example 1, mass spectrometric identification, see FIGS. 5, 8, 11 and 14.
Claims (6)
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-NH 2 labeled as Cn-M3.6;
Cn-Gly-Leu-Leu-(N-Me-Lys)-Arg-Ile-(N-Me-Lys)-Thr-Leu-Leu-NH 2 labeled as Cn-M4.7;
Cn-Gly-Leu-Leu-Lys-(N-Me-Arg)-Ile-(N-Me-Lys)-Thr-Leu-Leu-NH 2 labeled as Cn-M5.7;
wherein n =10-14.
2. The use of an N-methylated amino acid and N-terminal fatty acid modified antibacterial peptide analog of claim 1 as an active ingredient in the preparation of antibacterial drugs comprising anti-Staphylococcus aureus, anti-Bacillus subtilis, anti-Escherichia coli, anti-Klebsiella pneumoniae, and anti-Pseudomonas aeruginosa drugs.
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 respectively 3 and 6, 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 Cn-M3.6 containing the N-methylated amino acids and the N-end fatty acid modification.
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.
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| CN111875707B (en) * | 2020-05-20 | 2022-05-24 | 华南理工大学 | Fusion polypeptide and preparation method and application thereof |
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