CN110054664B - Side chain fatty acid modified antimicrobial peptide analogue containing D-type amino acid and its synthesis and application - Google Patents

Abstract

The present invention designs and synthesizes a side chain fatty acid modified antimicrobial peptide analogue containing D-type amino acids, which uses the linear amphipathic α-helical natural antimicrobial peptide Anoplin as a template, and replaces D-type amino acids to enhance enzymatic stability and antibacterial effect after replacement. Combining the strategy of D-type amino acid side chain fatty acid modification to enhance the activity of anti-drug-resistant bacteria, a new class of D-type amino acid side chain fatty acid-modified antimicrobial peptide analogs Ano‑D4, 7‑4C _n and Ano‑D4 obtained D4,7-7C _n , n=4-16. Antibacterial experiments in vitro, PI staining flow cytometry experiments and enzymolysis stability experiments all show that the side chain fatty acid modified antimicrobial peptide analogs containing D-type amino acids of the present invention have strong anti-drug-resistant bacteria activity and high enzymolysis stability ; Compared with traditional antibiotics, the novel antimicrobial peptide analog obtained in the present invention has a good application prospect in the development of clinical antibacterial drugs.

Description

Side chain fatty acid-modified antimicrobial peptide analogs containing D-amino acids and their synthesis and application

Technical Field

The present invention relates to the field of biochemical technology, and relates to a class of D-amino acid side chain fatty acid modified antimicrobial peptide analogs with a completely new structure, and the synthesis and application thereof, and in particular to a class of D-amino acid side chain fatty acid modified antimicrobial peptide analogs with strong anti-resistant bacteria activity and high enzymatic stability, and the synthesis and application thereof.

Background Art

In recent years, due to the abuse of traditional antibiotics in clinical use, drug-resistant strains, i.e., "super bacteria", have continued to emerge, which are resistant to most or all available antibiotics (Health Econ. 1996 May-Jun; 5(3): 217-26). Polymyxin B and polymyxin E (also known as colistin) were widely used as cationic peptide antibacterials in the 1960s, but due to serious toxicity problems, their clinical application was greatly reduced in the 1970s. Although the use of these two antibacterial drugs has revived with the emergence of multidrug-resistant Gram-negative bacteria that became popular in the 1970s, becoming the last antibiotic, unfortunately, polymyxin-resistant "super bacteria" have also appeared one after another (Expert Rev Anti Infect Ther. 2012 Aug; 10(8): 917-34; Biomed Res Int. 2015; 2015: 679109). Undoubtedly, the development of a new class of antibiotics has become crucial (Lancet Infect Dis. 2013 Dec; 13(12): 1057-98). As a new type of antibiotic with great potential, antimicrobial peptides (AMPs), especially cationic antimicrobial peptides, have attracted great attention due to their broad-spectrum antimicrobial activity and rapid bactericidal ability (Chembiochem. 2015 Jan 19; 16(2): 242-53). AMPs are usually produced by a variety of biological organisms, including bacteria, fungi, plants, insects, amphibians, crustaceans, fish and mammals (Clin Microbiol Rev. 2006 Jul; 19(3): 491-511). Most importantly, compared with traditional antibiotics, bacteria are less likely to develop resistance to antimicrobial peptides that do not have specific targets. The mode of action of antimicrobial peptides usually involves nonspecific interactions with the bacterial cytoplasmic membrane, which causes the antimicrobial peptides to accumulate in the bacterial membrane, increase the permeability of the membrane and lose its barrier function, ultimately leading to leakage of bacterial contents and death (Eur. J. Biochem. 2001, 268, 5589-5600; Nat Rev Microbiol. 2005 Mar; 3(3): 238-50).

However, although AMPs are expected to defeat "superbugs" compared with traditional antibiotics, as ideal antibacterial drugs, poor antibacterial activity, host cell toxicity, intolerance to physiological conditions, sensitivity to enzymatic degradation, and high manufacturing costs due to complex designs limit the clinical application of AMPs. A large number of studies have shown that the introduction of D-amino acids can effectively avoid protease degradation and improve the enzymatic stability of antimicrobial peptides (Sci Rep. 2017 Jul 31; 7 (1): 6953; Chem Biol Drug Des. 2006 Feb; 67 (2): 162-73), but the introduction of D-amino acids usually leads to a decrease in their antibacterial activity. Fatty acids, as an important component of biological cell membrane phospholipids, have high hydrophobicity. Introducing them into antimicrobial peptides is beneficial to increase the hydrophobicity of antimicrobial peptides and enhance their affinity for bacterial cell membranes, thereby enhancing their antimicrobial activity. Fatty acids can also reduce the degradation of proteases, enhance the enzymatic stability of antimicrobial peptides, and prolong the in vivo action time of antimicrobial peptides (Biochem J, 2005, 385 (Pt 1): 135-43; Biophys Chem, 2015, 199: 25-33).

Summary of the invention

One of the purposes of the present invention is to provide a new type of antimicrobial peptide analogs containing D-amino acids and side chain fatty acids modified with strong anti-resistant bacteria activity and high enzymatic stability.

The second object of the present invention is to provide the application of the above antimicrobial peptide analogs in the development of clinical antimicrobial drugs.

The third object of the present invention is to provide a method for synthesizing the above-mentioned antimicrobial peptide analogs.

(I) Antimicrobial peptide analogs modified with side chain fatty acids containing D-amino acids

The side chain fatty acid modified antimicrobial peptide analogs containing D-amino acids of the present invention are prepared by introducing a D-type special non-natural amino acid Fmoc-D-Lys(Mtt)-OH with a side chain protecting group Mtt into the 4th or 7th position of the analog Ano-D4,7, which is a partial D-amino acid replacement analog of the parent peptide Anoplin, and then removing the side chain protecting group, and modifying the side chain of the D-type special non-natural amino acid with fatty acids (C _n , n=4-16) of different lengths to obtain the side chain fatty acid modified antimicrobial peptide analogs containing D-amino acids of novel structure Ano-D4,7-4C _n , Ano-D4,7-7C _n , n=4-16.

The structural formulas are as follows:

Gly-Leu-Leu-D-Lys(C _n )-Arg-Ile-D-Lys-Thr-Leu-Leu-NH2

Where n = 4-16, named Ano-D4,7-4C _n ;

Gly-Leu-Leu-D-Lys-Arg-Ile-D-Lys(C _n )-Thr-Leu-Leu-NH2

Where n = 4-16, named Ano-D4,7-7C _n ;

The method for synthesizing the antimicrobial peptide analogs modified by side chain fatty acids containing D-amino acids of the present invention comprises the following process steps:

1. Synthesis of Ano-D4,7-4C _n

Dissolve Fmoc-Leu-OH, HOBT, HBTU and DIEA in DMF and mix them evenly, and carry out condensation reaction with MBHA resin from which the Fmoc protecting group has been removed to obtain Fmoc-Leu-resin; carry out condensation reaction of amino acids Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Arg(pbf)-OH, Fmoc-D-Lys(Mtt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH and Fmoc-Gly-OH in sequence to obtain Fmoc-Gly-Leu-Leu-D-Lys(Mtt)-Arg-Ile-D-Lys-Thr-Leu-Leu-resin, namely Fmoc-Ano-D4,7-4(Mtt)-resin;

The Fmoc-Ano-D4,7-4(Mtt)-resin obtained above is used to remove the side chain Mtt protecting group with a DCM solution containing 1% TFA by volume to obtain Ano-D4,7-4(NH ₂ )-resin; fatty acid, HOBT, HBTU and DIEA are respectively dissolved and mixed in DMF, and condensed with Ano-D4,7-4(NH ₂ )-resin to obtain Ano-D4,7-4C _n -resin; Ano-D4,7-4C _n -resin is cut and purified to obtain antimicrobial peptide analogs Ano-D4,7-4C _n , where n=4-16.

2. Synthesis of Ano-D4,7-7C _n

Dissolve Fmoc-Leu-OH, HOBT, HBTU and DIEA in DMF and mix them evenly, and carry out condensation reaction with MBHA resin from which the Fmoc protecting group has been removed to obtain Fmoc-Leu-resin; carry out condensation reaction of amino acids Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Lys(Mtt)-OH, Fmoc-Ile-OH, Fmoc-Arg(pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH and Fmoc-Gly-OH in sequence to obtain Fmoc-Gly-Leu-Leu-D-Lys-Arg-Ile-D-Lys(Mtt)-Thr-Leu-Leu-resin, namely Fmoc-Ano-D4,7-7(Mtt)resin;

The Fmoc-Ano-D4,7-7(Mtt)-resin obtained above is used to remove the side chain Mtt protecting group with a DCM solution containing 1% TFA by volume to obtain Ano-D4,7-7(NH ₂ )-resin; fatty acid, HOBT, HBTU and DIEA are respectively dissolved and mixed in DMF, and condensed with Ano-D4,7-7(NH ₂ )-resin to obtain Ano-D4,7-7C _n -resin; Ano-D4,7-7C _n -resin is cut and purified to obtain antimicrobial peptide analogs Ano-D4,7-7C _n , where n=4-16.

The concentrations of the above-mentioned amino acids, fatty acids, HOBT, HBTU and DIEA in DMF are 20-100 mg/mL, 20-100 mg/mL, 10-40 mg/mL, 20-100 mg/mL and 20-60 mg/mL, respectively; the molar mass ratios of the amino acids, fatty acids, HOBT, HBTU and the MBHA resin without the Fmoc protecting group are all 6:1-3:1, and the molar mass ratio of DIEA to the MBHA resin without the Fmoc protecting group is 6:1-12:1.

The cleavage reagent is a mixed solution of TFA, triisopropylsilane and water in a volume ratio of 9.5:0.25:0.25.

The purification process is as follows: the crude peptide is first freeze-dried to obtain freeze-dried powder, and then RP-HPLC separation is performed; the RP-HPLC purification conditions are: mobile phase A: 0.05% TFA aqueous solution, mobile phase B: 0.05% TFA acetonitrile solution; linear gradient elution, and the effluent of the main absorption peak is collected.

According to mass spectrometry identification, the method of the present invention successfully synthesized novel antimicrobial peptide analogs Ano-D4,7-4C _n and Ano-D4,7-7C _n containing D-amino acids and modified by side chain fatty acids, where n=4-16.

(II) Study on the in vitro activity of antimicrobial peptide analogs modified with side chain fatty acids containing D-amino acids

1. Antibacterial experiment

The minimum inhibitory concentration, i.e., MIC value, of the above antimicrobial peptide analogs was determined by the classic two-fold dilution method. The experimental strains used included standard normal strains: E. coli ATCC 25922, P. aeruginos ATCC 27853, K. pneumoniae ATCC 700603, S. aureus ATCC 25923, B. subtilis ATCC 23857, S. epidermidis ATCC 12228 and clinically isolated multidrug-resistant strains: A. baumannii 9828, A. baumannii 9840, P. aeruginosa 1240, P. aeruginosa 1190, E. coli 8500, E. coli 8040, S. aureus 4800, S. aureus 5200. The specific experimental method is as follows: the experimental bacteria cultured in MH medium overnight to the logarithmic growth phase were diluted into a bacterial suspension of 1×10 ⁶ CFU/mL; the antimicrobial peptide analogs were dissolved in sterile water, and a series of peptide solutions with different concentrations of 1-128 μmol/L were prepared by the two-fold dilution method, mixed with the above bacterial suspension in equal volumes, incubated at 37°C in a 96-well culture plate for 18-24 hours, and observed. The minimum concentration at which no obvious bacterial growth was visible to the naked eye was the minimum inhibitory concentration (MIC); the antibiotics Erythromycin, Kanamycin, and Penicillin were used as positive control drugs; the above experiment was repeated three times in parallel, and the results are shown in Tables 1 and 2.

Table 1 Minimum inhibitory concentration against standard normal strains

Table 2 Minimum inhibitory concentration against multidrug-resistant strains

The results in Table 1 show that the side chain fatty acid modified antimicrobial peptide analogs Ano-D4,7-4C _n and Ano-D4,7-7C _n containing D-amino acids have good antibacterial activity against standard normal bacterial strains, and their antibacterial activity is significantly improved compared with the parent peptide Anoplin, and is partially superior to traditional antibiotics; the results in Table 2 show that the side chain fatty acid modified antimicrobial peptide analogs Ano-D4,7-4C _n and Ano-D4,7-7C _n containing D-amino acids have strong antibacterial activity against clinically isolated multidrug-resistant bacterial strains, and their antibacterial activity is significantly improved compared with the parent peptide Anoplin, and is superior to traditional antibiotics; with the increase of fatty acid chain length, the antibacterial activity of antimicrobial peptide analogs against standard strains and multidrug-resistant bacteria is significantly enhanced, but when the fatty acid chain length increases to a certain extent, the antibacterial activity no longer increases.

2. Flow cytometry experiment

The standard strain of Escherichia coli (ATCC 25922) was used for PI staining flow cytometry to detect the destructive effect of antimicrobial peptide analogs on bacterial membranes. The specific experimental method is as follows: Escherichia coli cultured to the logarithmic phase was diluted to 10×10 ⁸ CFU/mL, washed with PBS (10mM, pH 7.4) and resuspended in half volume to obtain a bacterial suspension; the antimicrobial peptide analog was dissolved in PBS at a concentration of 8×MIC, mixed with an equal volume of the above bacterial suspension, incubated at 37°C for 2h, stained with iodinated pyridinium (PI) in the dark for 15min, and then washed with PBS to remove excess dye, and the PI fluorescence uptake ability was detected by flow cytometry, and the results are shown in Figure 9.

The results in Figure 9 show that the antimicrobial peptide analogs Ano-D4,7-4C _n and Ano-D4,7-7C _n modified with side chain fatty acids containing D-amino acids have good bacterial cell membrane destruction ability; as the length of the side chain fatty acid increases, the antimicrobial peptide analogs' ability to destroy bacterial cell membranes is significantly enhanced, which can explain the significant enhancement of the above-mentioned antibacterial activity.

3. Enzymatic stability test

The peptide solution was incubated with trypsin solutions of different concentrations (1 mg/mL, 0.5 mg/mL, 0.2 mg/mL, 0.1 mg/mL) at 37°C for 1 h and 6 h; after inactivation at 60°C for 15 min, the minimum inhibitory concentration (MIC) of E. coli ATCC 25922 in different trypsin solutions was determined in the same manner as the above-mentioned minimum inhibitory concentration determination method. The results are shown in Table 3.

Table 3 Minimum inhibitory concentration of trypsin against E.coli ATCC 25922 at different concentrations

Control: Minimum inhibitory concentration against E.coli ATCC 25922 in the absence of trypsin

The results in Table 3 show that the parent peptide Anoplin lost its antibacterial activity in the environment of different concentrations of trypsin and showed low stability; however, the antimicrobial peptide analogs Ano-D4,7-4C _n and Ano-D4,7-7C _n modified with side chain fatty acids containing D-amino acids did not lose their antibacterial activity in the environment of different concentrations of trypsin, and the minimum inhibitory concentration MIC only changed slightly, and still showed good antibacterial activity, indicating that the antimicrobial peptide analogs modified with side chain fatty acids containing D-amino acids have higher stability in the environment of trypsin, and their stability is significantly better than that of the parent peptide.

In summary, the present invention uses the linear amphipathic α-helical natural antimicrobial peptide Anoplin as a template, combines the strategy of D-amino acid substitution to enhance enzymatic stability and fatty acid modification of the replaced D-amino acid side chain to enhance anti-resistant bacteria activity, and obtains a new type of D-amino acid side chain fatty acid modified antimicrobial peptide analog. The results of in vitro biological activity studies show that the D-amino acid side chain fatty acid modified antimicrobial peptide analog has strong antimicrobial activity against resistant bacteria and high enzymatic stability, and has good application prospects in the development of clinical antimicrobial drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a mass spectrum of the antimicrobial peptide analog Ano-D4,7-4C ₄ of the present invention;

FIG2 is a mass spectrum of the antimicrobial peptide analog Ano-D4,7-4C ₈ of the present invention;

FIG3 is a mass spectrum of the antimicrobial peptide analog Ano-D4,7-4C ₁₂ of the present invention;

FIG4 is a mass spectrum of the antimicrobial peptide analog Ano-D4,7-4C ₁₆ of the present invention;

FIG5 is a mass spectrum of the antimicrobial peptide analog Ano-D4,7-7C ₄ of the present invention;

FIG6 is a mass spectrum of the antimicrobial peptide analog Ano-D4,7-7C ₈ of the present invention;

FIG7 is a mass spectrum of the antimicrobial peptide analog Ano-D4,7-7C ₁₂ of the present invention;

FIG8 is a mass spectrum of the antimicrobial peptide analog Ano-D4,7-7C ₁₆ of the present invention;

FIG9 is a graph showing the experimental results of PI staining flow cytometry of the antimicrobial peptide analogs of the present invention; in the figure, from left to right and from top to bottom, are the control group, Anoplin group, Ano-D4,7-4C ₈ group, Ano-D4,7-4C ₁₂ group, Ano-D4,7-7C ₈ group and Ano-D4,7-7C ₁₂ group.

DETAILED DESCRIPTION

The synthesis method of the antimicrobial peptide analogs modified with side chain fatty acids containing D-amino acids of the present invention is further described below through specific examples.

Example 1: Synthesis of Ano-D4,7-4C ₄

(1) Activation and pretreatment of resin

0.47 g of MBHA resin (0.43 mmol/g) was accurately weighed and placed in a peptide solid phase synthesizer. After swelling with DCM solution for 30 min, the resin was tested by ninhydrin colorimetry and it was colorless and transparent, indicating that the resin was normal.

(2) Synthesis of Fmoc-Ano-D4,7-4(Mtt)-resin

The above-mentioned normal MBHA resin was subjected to DMF solution containing 20% piperidine by volume to remove the Fmoc protecting group. The resin was tested by ninhydrin colorimetry and it turned blue-purple, indicating that the protecting group had been removed. Fmoc-Leu-OH (212 mg), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were added to 5-10 mL The mixture was dissolved in DMF and mixed evenly, added to the synthesizer and mixed with the MBHA resin from which the Fmoc protecting group had been removed, and the condensation reaction was carried out for 1 hour. The resin was colorless and transparent when tested by the ninhydrin colorimetric method, indicating that the condensation reaction was successful to obtain Fmoc-Leu-resin. The method was the same as above, and the subsequent amino acids were condensed in sequence: Fmoc-Leu-OH (212 mg), Fmoc-Thr(tBu)-OH (239 mg), Fmoc-D-Lys(Boc)-OH (281 mg), Fmoc-Ile-OH (212 mg), Fmoc-Arg(pbf)-OH (390 mg), Fmoc-D -Lys(Mtt)-OH (376 mg), Fmoc-Leu-OH (212 mg), Fmoc-Leu-OH (212 mg), Fmoc-Gly-OH (238 mg), the amounts of HOBT, HBTU and DIEA are the same as above, wherein the condensation reaction time of Fmoc-D-Lys(Mtt)-OH is 1.5 h, and the others are 1 h, to obtain Fmoc-Gly-Leu-Leu-D-Lys(Mtt)-Arg-Ile-D-Lys-Thr-Leu-Leu-resin, i.e., Fmoc-Ano-D4,7-4(Mtt)-resin;

(3) Synthesis of Ano-D4,7-4C ₄ -resin

The side chain Mtt protecting group of the Fmoc-Ano-D4,7-4(Mtt)-resin was removed by using a DCM solution containing 1% TFA by volume. The resin was blue-purple when tested by ninhydrin colorimetry, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-4(NH ₂ )-resin. Butyric anhydride (C _n , n=4; 0.87 mL), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were dissolved and mixed in 5-10 mL DMF, respectively, and added to a synthesizer and mixed with the Ano-D4,7-4(NH ₂ )-resin from which the side chain Mtt protecting group had been removed. The condensation reaction was carried out for 1.5 h. The resin was colorless and transparent when tested by ninhydrin colorimetry, indicating that the condensation reaction was complete, thereby obtaining Ano-D4,7-4C ₄ -resin; using DMF solution containing 20% piperidine by volume, the N-terminal Fmoc protecting group was removed, and the resin was tested by ninhydrin colorimetry, and it turned blue-purple, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-4C ₄ -resin.

(4) Peptide cleavage

Ano-D4,7-4C ₄ -resin was cleaved with a mixed solution of TFA, triisopropylsilane and water in a volume ratio of 9.5:0.25:0.25 as a cleavage reagent, extracted with ice ether and water, and freeze-dried to obtain a crude peptide lyophilized powder;

(5) Peptide purification

The crude peptide freeze-dried powder obtained by the freeze-drying was separated and purified by RP-HPLC, and the effluent was collected and freeze-dried again. Ano-D4,7-4C ₄ was identified by mass spectrometry, and the molecular weight was 1223Da. The mass spectrum is shown in Figure 1; wherein, the RP-HPLC purification conditions are: mobile phase A: 0.05% TFA/water; mobile phase B: 0.05% TFA/acetonitrile; linear gradient elution, and the effluent of the main absorption peak was collected.

Example 2: Synthesis of Ano-D4,7-4C ₈

(1) Activation and pretreatment of resin

Same as Example 1.

(2) Synthesis of Fmoc-Ano-D4,7-4(Mtt)-resin

Same as Example 1.

(3) Synthesis of Ano-D4,7-4C ₈ -resin

The Fmoc-Ano-D4,7-4(Mtt)-resin obtained above was used to remove the side chain Mtt protecting group with a DCM solution containing 1% TFA by volume. The resin was blue-purple when tested by ninhydrin colorimetry, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-4(NH ₂ )-resin; octanoic anhydride (C _n , n=8; 1.53 mL), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were dissolved and mixed in 5-10 mL DMF, respectively, and added to a synthesizer and mixed with the Ano-D4,7-4(NH ₂ )-resin from which the side chain Mtt protecting group had been removed, and the condensation reaction was carried out for 1.5 h; the resin was colorless and transparent when tested by ninhydrin colorimetry, indicating that the condensation reaction was complete, thereby obtaining Ano-D4,7-4C ₈ -resin; using DMF solution containing 20% piperidine by volume, the N-terminal Fmoc protecting group was removed, and the resin was blue-purple when tested by ninhydrin colorimetric method, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-4C ₈ -resin.

(4) Peptide cleavage

Same as Example 1.

(5) Peptide purification

As in Example 1, Ano-D4,7-4C ₈ was obtained by mass spectrometry identification, with a molecular weight of 1279 Da. The mass spectrum is shown in FIG2 .

Example 3: Synthesis of Ano-D4,7-4C ₁₂

(1) Activation and pretreatment of resin

Same as Example 1.

(2) Synthesis of Fmoc-Ano-D4,7-resin

Same as Example 1.

(3) Synthesis of Ano-D4,7-4C ₁₂ -resin

The Fmoc-Ano-D4,7-4(Mtt)-resin obtained above was used to remove the side chain Mtt protecting group with DCM solution containing 1% TFA by volume. The resin was blue-purple when tested by ninhydrin colorimetry, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-4(NH ₂ )-resin; dodecanoic acid (C _n , n=12; 240 mg), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were dissolved and mixed in 5-10 mL DMF respectively, and added to the synthesizer and mixed with the Ano-D4,7-4(NH ₂ )-resin from which the side chain Mtt protecting group had been removed, and the condensation reaction was carried out for 1.5 h; the resin was colorless and transparent when tested by ninhydrin colorimetry, indicating that the condensation reaction was complete, thereby obtaining Ano-D4,7-4C ₁₂ -resin; using DMF solution containing 20% piperidine by volume, the N-terminal Fmoc protecting group was removed, and the resin was tested by ninhydrin colorimetric method, and it turned blue-purple, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-4C ₁₂ -resin.

(4) Peptide cleavage

Same as Example 1.

(5) Peptide purification

As in Example 1, Ano-D4,7-4C ₁₂ was identified by mass spectrometry, and its molecular weight was 1335 Da. The mass spectrum is shown in FIG3 .

Example 4: Synthesis of Ano-D4,7-4C ₁₆

(1) Activation and pretreatment of resin

Same as Example 1.

(2) Synthesis of Fmoc-Ano-D4,7-resin

Same as Example 1.

(3) Synthesis of Ano-D4,7-4C ₁₆ -resin

The Fmoc-Ano-D4,7-4(Mtt)-resin obtained above was used to remove the side chain Mtt protecting group with DCM solution containing 1% TFA by volume. The resin was blue-purple when tested by ninhydrin colorimetry, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-4(NH ₂ )-resin; hexadecanoic acid (C _n , n=16; 307 mg), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were dissolved and mixed in 5-10 mL DMF respectively, and added to the synthesizer and mixed with the Ano-D4,7-4(NH ₂ )-resin from which the side chain Mtt protecting group had been removed, and the condensation reaction was carried out for 1.5 h; the resin was colorless and transparent when tested by ninhydrin colorimetry, indicating that the condensation reaction was complete, thereby obtaining Ano-D4,7-4C ₁₆ -resin; using DMF solution containing 20% piperidine by volume, the N-terminal Fmoc protecting group was removed, and the resin was blue-purple when tested by ninhydrin colorimetric method, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-4C ₁₆ -resin.

(4) Peptide cleavage

Same as Example 1.

(5) Peptide purification

As in Example 1, Ano-D4,7-4C ₁₆ was identified by mass spectrometry, and its molecular weight was 1391 Da. The mass spectrum is shown in FIG4 .

Example 5: Synthesis of Ano-D4,7-7C ₄

(1) Activation and pretreatment of resin

Same as Example 1.

(2) Synthesis of Fmoc-Ano-D4,7-7(Mtt)-resin

The above-mentioned normal MBHA resin was subjected to DMF solution containing 20% piperidine by volume to remove the Fmoc protecting group. The resin was tested by ninhydrin colorimetry and it turned blue-purple, indicating that the protecting group had been removed. Fmoc-Leu-OH (212 mg), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were added to 5-10 mL The product was dissolved in DMF and mixed evenly, added to the synthesizer and mixed with the MBHA resin from which the Fmoc protecting group was removed, and the condensation reaction was carried out for 1 hour. The resin was colorless and transparent when tested by the ninhydrin colorimetric method, indicating that the condensation reaction was successful to obtain Fmoc-Leu-resin. The method was the same as above, and the subsequent amino acids were condensed in sequence: Fmoc-Leu-OH (212 mg), Fmoc-Thr(tBu)-OH (239 mg), Fmoc-D-Lys(Mtt)-OH (376 mg), Fmoc-Ile-OH (212 mg), Fmoc-Arg(pbf)-OH (390 mg), Fmoc-D -Lys(Boc)-OH (281 mg), Fmoc-Leu-OH (212 mg), Fmoc-Leu-OH (212 mg), Fmoc-Gly-OH (238 mg), the amounts of HOBT, HBTU and DIEA are the same as above, wherein the condensation reaction time of Fmoc-D-Lys(Mtt)-OH is 1.5 h, and the others are 1 h, to obtain Fmoc-Gly-Leu-Leu-D-Lys-Arg-Ile-D-Lys(Mtt)-Thr-Leu-Leu-resin, i.e., Fmoc-Ano-D4,7-4(Mtt)-resin;

(3) Synthesis of Ano-D4,7-7C ₄ -resin

The Fmoc-Ano-D4,7-7(Mtt)-resin obtained above was used to remove the side chain Mtt protecting group with a DCM solution containing 1% TFA by volume. The resin was blue-purple when tested by ninhydrin colorimetry, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-7(NH ₂ )-resin; butyric anhydride (C _n , n=4; 0.87 mL), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were dissolved and mixed in 5-10 mL DMF, respectively, and added to a synthesizer and mixed with the Ano-D4,7-7(NH ₂ )-resin from which the side chain Mtt protecting group had been removed, and the condensation reaction was carried out for 1.5 h; the resin was colorless and transparent when tested by ninhydrin colorimetry, indicating that the condensation reaction was complete, thereby obtaining Ano-D4,7-7C ₄ -resin; using DMF solution containing 20% piperidine by volume, the N-terminal Fmoc protecting group was removed, and the resin was tested by ninhydrin colorimetry, and it turned blue-purple, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-7C ₄ -resin.

(4) Peptide cleavage

Same as Example 1.

(5) Peptide purification

As in Example 1, Ano-D4,7-7C ₄ was identified by mass spectrometry, with a molecular weight of 1223 Da. The mass spectrum is shown in FIG5 .

Example 6: Synthesis of Ano-D4,7-7C ₈

(1) Activation and pretreatment of resin

Same as Example 1.

(2) Synthesis of Fmoc-Ano-D4,7-7(Mtt)-resin

Same as Example 5.

(3) Synthesis of Ano-D4,7-7C ₈ -resin

The Fmoc-Ano-D4,7-7(Mtt)-resin obtained above was used to remove the side chain Mtt protecting group with DCM solution containing 1% TFA by volume. The resin was blue-purple when tested by ninhydrin colorimetry, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-7(NH ₂ )-resin; octanoic anhydride (C _n , n=8; 1.53 mL), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were dissolved and mixed in 5-10 mL DMF respectively, and added to the synthesizer and mixed with the Ano-D4,7-7(NH ₂ )-resin from which the side chain Mtt protecting group had been removed, and the condensation reaction was carried out for 1.5 h; the resin was colorless and transparent when tested by ninhydrin colorimetry, indicating that the condensation reaction was complete, thereby obtaining Ano-D4,7-7C ₈ -resin; using DMF solution containing 20% piperidine by volume, the N-terminal Fmoc protecting group was removed, and the resin was blue-purple when tested by ninhydrin colorimetric method, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-7C ₈ -resin.

(4) Peptide cleavage

Same as Example 1.

(5) Peptide purification

As in Example 1, Ano-D4,7-7C ₈ was identified by mass spectrometry, and its molecular weight was 1279 Da. The mass spectrum is shown in FIG6 .

Example 7: Synthesis of Ano-D4,7-7C ₁₂

(1) Activation and pretreatment of resin

Same as Example 1.

(2) Synthesis of Fmoc-Ano-D4,7-7(Mtt)-resin

Same as Example 5.

(3) Synthesis of Ano-D4,7-7C ₁₂ -resin

The Fmoc-Ano-D4,7-7(Mtt)-resin obtained above was used to remove the side chain Mtt protecting group with DCM solution containing 1% TFA by volume. The resin was blue-purple when tested by ninhydrin colorimetry, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-7(NH ₂ )-resin; dodecanoic acid (C _n , n=12; 240 mg), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were dissolved and mixed in 5-10 mL DMF respectively, and added to the synthesizer and mixed with the Ano-D4,7-7(NH ₂ )-resin from which the side chain Mtt protecting group had been removed, and the condensation reaction was carried out for 1.5 h; the resin was colorless and transparent when tested by ninhydrin colorimetry, indicating that the condensation reaction was complete, thereby obtaining Ano-D4,7-7C ₁₂ -resin; using DMF solution containing 20% piperidine by volume, the N-terminal Fmoc protecting group was removed, and the resin was tested by ninhydrin colorimetry, and it turned blue-purple, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-7C ₁₂ -resin.

(4) Peptide cleavage

Same as Example 1.

(5) Peptide purification

As in Example 1, Ano-D4,7-7C ₁₂ was identified by mass spectrometry, and its molecular weight was 1335 Da. The mass spectrum is shown in FIG7 .

Example 8: Synthesis of Ano-D4,7-7C ₁₆

(1) Activation and pretreatment of resin

Same as Example 1.

(2) Synthesis of Fmoc-Ano-D4,7-7(Mtt)-resin

Same as Example 5.

(3) Synthesis of Ano-D4,7-7C ₁₆ -resin

The Fmoc-Ano-D4,7-7(Mtt)-resin obtained above was used to remove the side chain Mtt protecting group with a DCM solution containing 1% TFA by volume. The resin was blue-purple when tested by ninhydrin colorimetry, indicating that the protecting group had been removed, thereby obtaining Ano-D4,7-7(NH ₂ )-resin; hexadecanoic acid (C _n , n=16; 307 mg), HOBT (81 mg), HBTU (228 mg), and DIEA (0.2 mL) were dissolved and mixed in 5-10 mL DMF, respectively, and added to a synthesizer and mixed with the Ano-D4,7-7(NH ₂ )-resin from which the side chain Mtt protecting group had been removed, and the condensation reaction was carried out for 1.5 h; the resin was colorless and transparent when tested by ninhydrin colorimetry, indicating that the condensation reaction was complete, thereby obtaining Ano-D4,7-7C ₁₆ -resin; using DMF solution containing 20% piperidine by volume, the N-terminal Fmoc protecting group was removed, and the resin was tested by ninhydrin colorimetry. The resin was blue-purple, indicating that the protecting group had been removed, and Ano-D4,7-7C ₁₆ -resin was obtained.

(4) Peptide cleavage

Same as Example 1.

(5) Peptide purification

As in Example 1, Ano-D4,7-7C ₁₆ was identified by mass spectrometry, with a molecular weight of 1335 Da. The mass spectrum is shown in FIG8 .

Claims (9)

1. A class of side chain fatty acid modified antimicrobial peptide analogs containing D-type amino acids, characterized in that the structural formula of the antimicrobial peptide analogs is: Gly-Leu-Leu-D-Lys(C _n )-Arg-Ile-D-Lys-Thr-Leu-Leu-NH ₂ Ano-D4,7-4C _n , C _n is octanoic acid, dodecanoic acid or hexadecanoic acid; or Gly-Leu-Leu-D-Lys-Arg-Ile-D-Lys(C _n )-Thr-Leu-Leu-NH ₂ Ano-D4,7-7C _n , C _n is octanoic acid, dodecanoic acid or hexadecanoic acid. 2. The application of the side chain fatty acid modified antimicrobial peptide analog containing D-amino acid as claimed in claim 1 in the preparation of clinical antibacterial drugs. 3. the synthetic method of the side chain fatty acid modification antimicrobial peptide analog containing D-type amino acid as claimed in claim 1, is characterized in that: Using the classic solid-phase synthesis method, the D-type special unnatural amino acid Fmoc-D-Lys(Mtt)-OH with the side chain protection group Mtt was introduced into Anoplin's partial D-type amino acid replacement analog Ano-D4, 4 of the 7 sequence or 7, synthesize Ano-D4,7-4(Mtt)-resin or Ano-D4,7-7(Mtt)-resin; The side chains are modified with fatty acids of different lengths; finally, after cleavage and purification, the side chain fatty acid modified antibacterial peptide analogues Ano-D4,7-4C _n or Ano-D4,7-7C _n containing D-type amino acids are obtained, wherein, C _n is octanoic acid, dodecanoic acid or hexadecanoic acid. 4. the synthetic method of the side chain fatty acid modification antimicrobial peptide analog containing D-type amino acid as claimed in claim 3, is characterized in that, the synthetic method of described antibacterial peptide analog is specifically: (1) Synthesis of Ano-D4,7-4C _n Dissolve and mix Fmoc-Leu-OH, HOBT, HBTU, and DIEA in DMF, and perform condensation reaction with MBHA resin without Fmoc protecting group to obtain Fmoc-Leu-resin; sequentially condense amino acid Fmoc-Leu- OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Arg(pbf)-OH, Fmoc-D-Lys(Mtt)-OH, Fmoc- Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, get Fmoc-Gly-Leu-Leu-D-Lys(Mtt)-Arg-Ile-D-Lys-Thr-Leu-Leu-resin, which is Fmoc-Ano-D4,7-4(Mtt)-resin; Use the Fmoc-Ano-D4,7-4(Mtt)-resin obtained above to remove the side chain Mtt protecting group with a DCM solution containing 1% TFA by volume to obtain Fmoc-Ano-D4,7-4(NH ₂ )-resin; respectively dissolve and mix fatty acid, HOBT, HBTU and DIEA in DMF, and perform condensation reaction with Fmoc-Ano-D4,7-4(NH ₂ )-resin to obtain Fmoc-Ano-D4,7- 4C _n -resin; remove the Fmoc-protecting group from Fmoc-Ano-D4,7-4Cn-resin, cut and purify to obtain the antimicrobial peptide analog Ano-D4,7-4C _n , C _n is octanoic acid, dodecane acid or hexadecanoic acid; (2) Synthesis of Ano-D4,7-7C _n Dissolve and mix Fmoc-Leu-OH, HOBT, HBTU, and DIEA in DMF, and perform condensation reaction with MBHA resin without Fmoc protecting group to obtain Fmoc-Leu-resin; sequentially condense amino acid Fmoc-Leu- OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Lys(Mtt)-OH, Fmoc-Ile-OH, Fmoc-Arg(pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc- Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, get Fmoc-Gly-Leu-Leu-D-Lys-Arg-Ile-D-Lys(Mtt)-Thr-Leu-Leu-resin, which is Fmoc-Ano-D4,7-7(Mtt)-resin; Use the Fmoc-Ano-D4,7-7(Mtt)-resin obtained above to remove the side chain Mtt protecting group with a DCM solution containing 1% TFA by volume fraction to obtain Fmoc-Ano-D4,7-7(NH ₂ )-resin; respectively dissolve and mix fatty acid, HOBT, HBTU and DIEA in DMF, and perform condensation reaction with Fmoc-Ano-D4,7-7(NH ₂ )-resin to obtain Fmoc-Ano-D4,7- 7C _n -resin; Fmoc-Ano-D4,7-7C _n -resin was cut and purified after removing the Fmoc-protecting group to obtain the antimicrobial peptide analog Ano-D4,7-7C _n , C _n is octanoic acid, dodecanoic acid alkanoic acid or hexadecanoic acid. 5. the synthetic method of the side chain fatty acid modification antimicrobial peptide analog containing D-type amino acid as claimed in claim 4, is characterized in that, the concentration of described each amino acid, each fatty acid, HOBT, HBTU and DIEA in DMF is respectively 20-100mg/mL, 20-100mg/mL, 10-40mg/mL, 20-100mg/mL, 20-60mg/mL. 6. as claimed in claim 4 or 5, the synthetic method of the side chain fatty acid modification antimicrobial peptide analog containing D-type amino acid is characterized in that, described each amino acid, each fatty acid, HOBT, HBTU and slough Fmoc protecting group The molar mass ratio of MBHA resin is 6:1-3:1, and the molar mass ratio of DIEA to MBHA resin without Fmoc protecting group is 6:1-12:1. 7. the synthetic method of the side chain fatty acid modification antimicrobial peptide analog containing D-type amino acid as described in any one of claim 3-5, it is characterized in that, described cleavage reagent is TFA, triisopropylsilane and water and A mixed solution formed with a volume ratio of 9.5:0.25:0.25. 8. the synthetic method of the side chain fatty acid modification antimicrobial peptide analog containing D-type amino acid as described in any one of claim 3-5, it is characterized in that, described purification process is, after freeze-drying, carry out RP-HPLC Separation and purification; RP-HPLC purification conditions are: mobile phase A: 0.05% TFA in water, mobile phase B: 0.05% TFA in acetonitrile; linear gradient elution, and collect the effluent of the main absorption peak. 9. the synthetic method of the side chain fatty acid modification antimicrobial peptide analogue that contains D-type amino acid as claimed in claim 6, is characterized in that, described purification process is, after the thick peptide that obtains is freeze-dried, carries out RP-HPLC separation Purification; RP-HPLC purification conditions are, mobile phase A: 0.05% TFA in water, mobile phase B: 0.05% TFA in acetonitrile; linear gradient elution, collect the effluent of the main absorption peak.

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