CN117186181A - Hydroxy phosphorylated antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and high stability and application thereof - Google Patents
Hydroxy phosphorylated antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and high stability and application thereof Download PDFInfo
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Abstract
The invention discloses a hydroxy phosphorylated antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and high stability and application thereof. The hydroxy phosphorylated antibacterial peptide is obtained by adjusting the hydrophobic moment of the antibacterial peptide A10 and phosphorylating the antibacterial peptide after amino acid substitution. The antibacterial experiment, the hemolysis experiment, the serum stability experiment, the drug resistance induction experiment and the acute toxicity experiment show that the phosphorylated antibacterial peptide has broad-spectrum antibacterial activity, low hemolytic toxicity, high serum stability and no drug resistance induction, and the combination of the phosphorylated antibacterial peptide and the Rifampin or the Kanamycin can reduce the generation of the drug resistance of the Rifampin or the Kanamycin; the phosphorylated antibacterial peptide can also be used for treating bacterial pneumonia induced by klebsiella pneumoniae in mice, so that the phosphorylated antibacterial peptide has good application prospect in preparing clinical antibacterial medicaments and is hopeful to become a candidate medicament of novel antibiotics.
Description
Technical Field
The invention belongs to the technical field of biochemistry, relates to a hydroxy-phosphorylated antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and high stability, and simultaneously relates to application of the hydroxy-phosphorylated antibacterial peptide as an antibacterial drug.
Background
Since penicillin discovery, antibiotics have played an important role in the prevention and treatment of infectious diseases in humans, bioscience research, agriculture, animal husbandry, and food industry. Unfortunately, abuse of various antibiotics has led to an increase in bacterial resistance at a striking rate, posing a serious threat to the health of people worldwide. Infections caused by antibiotic-resistant bacteria lose lives for at least 70 tens of thousands of people each year, and 1000 tens of thousands of people are expected to die each year in 2050, with a global economy losing $ 100 trillion due to productivity loss. According to The report from The center of disease prevention control, more than about 280 million people in The united states are infected with multi-drug resistant bacteria annually, resulting in at least 3.5 thousands of people dying from such infection and causing a medical expenditure of $200 billion (The Lancet,2020,396 (10257):1050-1053.).
Bacteria continuously increase the drug resistance to antibiotics through various drug resistance mechanisms, while antibacterial peptides exert antibacterial activity through non-target specific membrane dissolving mechanisms, so that microbial drug resistance is seldom caused, and the antibacterial peptide is a promising antibacterial agent (Expert Opinion on Biological Therapy,2017,17 (6): 663-676). Most antimicrobial peptides are cationic antimicrobial peptides, which have an amphiphilic structure that rapidly migrates to the site of infection after microbial infection, increasing the permeability of bacterial cell membranes, disrupting the homeostasis of the cell membranes, leading to bacterial cell lysis and release of the cell contents, thereby exerting antimicrobial activity (Biosafety and Health,2022,4 (2): 17.). The antibacterial peptide has wide sources, multiple varieties, wide antibacterial spectrum, high sterilization speed and difficult generation of resistance mutation of target strains, becomes a hotspot for research and development in the fields of domestic and foreign human and animal medicine, food (feed) science, immunology, nutrition, aquaculture and the like, and has wide application prospect. However, the use of antimicrobial peptides still faces challenges such as weak activity compared to antibiotics, showing some cytotoxicity and/or hemolysis, poor stability, high sensitivity to protease degradation, high mass production costs, etc. (Advances in Experimental Medicine and Biology,2019,1117: 175-214).
At present, the improvement of the antibacterial peptide is mainly pursued to achieve the aims of reducing toxicity, improving antibacterial activity, improving stability and the like. The modification means mainly comprise residue substitution, truncation, construction of hybrid peptide and the like. Strategies such as D-type amino acid substitution, halogenation treatment, disulfide bond cyclization, introduction of unnatural amino acids, coupling with organic metals, pegylation prodrugs, lipidation, glycosylation and the like effectively improve the antibacterial activity and stability of the antibacterial peptide and reduce toxicity. Therefore, molecular design, optimization of expression systems, structural transformation and modification of antibacterial peptides based on their physical and chemical properties are the primary problems in the development stage, and are also the main factors controlling the overall mass production.
The increase in toxicity of antibacterial peptides is generally associated with high hydrophobicity, high charge density and secondary structure (International journal of antimicrobial agents,2008,32 (2): 130-138.). Protein phosphorylation/dephosphorylation is the most common reversible modification, the most abundant and most studied post-translational modification (PTM). The phosphorylation modification is used for designing the antibacterial peptide, so that the toxicity of the antibacterial peptide can be reduced, and the stability is improved.
Disclosure of Invention
It is an object of the present invention to provide a novel hydroxy phosphorylated antibacterial peptide having broad-spectrum antibacterial activity, low toxicity and high stability.
The second purpose of the invention is to provide the application of the phosphorylated antibacterial peptide in preparing clinical antibacterial drugs.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
1. design of hydroxy-phosphorylated antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and high stability
The invention relates to a hydroxy phosphorylating antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and high stability, which is obtained by taking alpha helical peptide A10 as a base, carrying out hydrophobic moment adjustment and amino acid substitution, and then phosphorylating, and is denoted as W 3 BipY 8 -P, the amino acid sequence of which is as follows:
Gly-Leu-Bip-Lys-Arg-Leu-Lys-Tyr(H 2 PO 3 )-Leu-Leu-NH 2 。
wherein the amino acid sequence of the alpha helical peptide A10 is as follows:
Gly-Leu-Leu-Lys-Arg-Trp-Lys-Thr-Leu-Leu-NH 2 ；
the hydrophobic moment adjustment means that the 6 th tryptophan and the 3 rd leucine in the alpha helical peptide A10 peptide chain are exchanged under the premise of keeping strict amphipathy so as to reduce the hydrophobic moment, and the polypeptide is marked as W after the hydrophobic moment adjustment 3 The amino acid sequence is as follows:
Gly-Leu-Trp-Lys-Arg-Leu-Lys-Thr-Leu-Leu-NH 2 ；
the amino acid substitution refers to the substitution of W 3 Tryptophan at position 3 in peptide chain is replaced by unnatural amino acid 4' -biphenylalanine, threonine at position 8 is replaced by tyrosine, and the polypeptide is marked as W after amino acid replacement 3 BipY 8 The amino acid sequence is as follows:
Gly-Leu-Bip-Lys-Arg-Leu-Lys-Tyr-Leu-Leu-NH 2 。
2. synthesis of hydroxy-phosphorylated antibacterial peptides with broad-spectrum antibacterial activity, low toxicity and high stability
The invention relates to a hydroxy phosphorylating antibacterial peptide W 3 BipY 8 -P is synthesized by classical polypeptide Fmoc solid phase synthesis, in particular:
amino acid coupling is carried out by taking Rink-MBHA Resin as a raw material, taking HOBt/HBTU as a condensing agent in the polypeptide synthesis process, detecting secondary amine by adopting an ninhydrin chromogenic method, sequentially coupling according to a polypeptide sequence to obtain a polypeptide connected with MBHA Resin, and obtaining the novel phosphorylated antibacterial peptide after polypeptide cleavage and HPLC purification.
3. Application of hydroxy phosphorylated antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and high stability in preparation of clinical antibacterial drugs
1. In vitro bacteriostasis experiment
Determination of phosphorylated antibacterial peptide W by classical trace continuous double dilution method 3 BipY 8 P and its non-phosphorylated form W 3 BipY 8 Minimal inhibitory concentration against gram-positive bacteria (Staphylococcus aureus, bacillus subtilis) and gram-negative bacteria (Escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii)Degree. The experiments were repeated 3 times in parallel with the antibiotics Rifampin, polymyxin B, vancomycin and Ciprofloxacin as positive controls, and the results are shown in tables 1 and 2.
TABLE 1 minimum inhibitory concentration of the antibacterial peptides of the invention on gram-positive bacterial strains
TABLE 2 minimum inhibitory concentration of the antibacterial peptides of the invention against gram-negative bacterial strains
The results in tables 1 and 2 show that the hydroxy-phosphorylated antibacterial peptide W 3 BipY 8 P and its non-phosphorylated form W 3 BipY 8 Has strong inhibition effect on gram-positive bacteria represented by staphylococcus aureus and bacillus subtilis, and gram-negative bacteria represented by escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and acinetobacter baumannii, and has broad-spectrum antibacterial activity, and better effect on the gram-negative bacteria than Rifampicin and Vancomycin.
2. Hemolytic Activity assay
To examine the toxicity of the hydroxyphosphorylated antibacterial peptide synthesized by the present invention to normal mammalian cells, the hydroxyphosphorylated antibacterial peptide W 3 BipY 8 P and its non-phosphorylated form W 3 BipY 8 The hemolysis after incubation with mouse erythrocytes for 1h was determined and the results are shown in fig. 4.
FIG. 4 shows the results of W 3 BipY 8 Is 27.58 mu M, and the novel phosphorylated antibacterial peptide W 3 BipY 8 The 10% hemolytic concentration of P was much greater than the maximum test concentration of 256. Mu.M, showing no hemolytic toxicity. The result shows that the novel phosphorylated antibacterial peptide synthesized by the invention greatly reduces the hemolytic toxicity, and compared with the non-phosphorylated parent peptide, the novel phosphorylated antibacterial peptide has the advantages of 38 times reduction of the hemolytic toxicity and safer administration.
3. Serum stability test
In order to examine the stability of the novel phosphorylated antibacterial peptide synthesized by the present invention in serum, the phosphorylated antibacterial peptide W was monitored by RP-HPLC, respectively 3 BipY 8 P and its non-phosphorylated form W 3 BipY 8 Degradation at various time points when incubated with mouse serum at 37 ℃ was measured for serum half-life and the results are shown in figure 5.
FIG. 5 shows the results of W 3 BipY 8 Is 81.65min, and the novel phosphorylated antibacterial peptide W 3 BipY 8 The serum half-life of P was 510.80min, and the serum half-life of the phosphorylated antibacterial peptide was about 6.3 times that of the non-phosphorylated parent peptide. The results further demonstrate that the novel phosphorylated antibacterial peptide synthesized by the invention is more stable in mouse serum than parent peptide, and is not easily degraded by serum to lose antibacterial activity.
4. Experiment for inducing drug resistance
To examine whether the novel phosphorylated antibacterial peptide synthesized by the present invention is resistant to the phosphorylated antibacterial peptide W 3 BipY 8 P and its non-phosphorylated form W 3 BipY 8 The induced drug resistance after 20 days of continuous action with klebsiella pneumoniae ATCC700603 was measured, and the results are shown in fig. 6, with antibiotic Rifampin, kanamycin and Polymyxin B as controls.
FIG. 6 shows the results of phosphorylated antibacterial peptide W 3 BipY 8 P and its non-phosphorylated form W 3 BipY 8 After 20 days of continuous action with klebsiella pneumoniae ATCC700603, no drug resistance is generated, rifampin and Kanamycin quickly generate drug resistance, the MIC is improved by 128 times after 20 days, and the MIC of Polymyxin B is improved by 64 times after 20 days. Notably, phosphorylated antibacterial peptide W 3 BipY 8 P and its non-phosphorylated form W 3 BipY 8 The mixture with Rifampin or Kanamycin (1:1 mix) did not develop resistance. The results show that the novel phosphorylated antibacterial peptide synthesized by the invention has no induced drug resistance and can reduce the drug resistance of antibiotics.
To investigate whether Klebsiella pneumoniae is resistant to Rifampin and Kanamycin and would result in cross-over of the antibacterial peptideCross-drug resistance, and the phosphorylated antibacterial peptide W is measured by classical trace continuous double dilution method 3 BipY 8 P and its non-phosphorylated form W 3 BipY 8 The minimum inhibitory concentration of klebsiella pneumoniae after 20 days of continuous action on Rifampin and Kanamycin is used as a control. Experiments were repeated 3 times in parallel. The results are shown in Table 3.
TABLE 3 minimum inhibitory concentration of klebsiella pneumoniae after 20 days of continuous action on Rifampin or Kanamycin
Table 3 shows that the results of Rifampin or Kanamycin induced resistance of Klebsiella pneumoniae against phosphorylated antibacterial peptide W 3 BipY 8 -P and non-phosphorylated parent peptide W 3 BipY 8 None of them has cross drug resistance.
In conclusion, phosphorylated antibacterial peptide W 3 BipY 8 P has no induced drug resistance, and can reduce the generation of drug resistance of Rifampin or Kanamycin after being combined with Rifampin or Kanamycin.
5. Acute toxicity test in mice
The experimental mice are BALB/C mice, male, 18-22g, and are fed according to the ethical management method of experimental animals in Lanzhou university.
Acute toxicity half-lethal dose LD 50 Pre-experiment: 3 non-phosphorylated parent peptide W dissolved in physiological saline for intraperitoneal injection 3 BipY 8 Phosphorylated antibacterial peptide W 3 BipY 8 -P, observed for mortality within 7 days, with Polymyxin B as control, the minimum dose that caused 100% mortality and the maximum dose that caused 0% mortality were explored, and the upper and lower dose limits were determined.
Formal test: we measured W 3 BipY 8 LD of (2) 50 The dosage range is 40-130mg/kg, W 3 BipY 8 LD of P 50 The dosage range is 220-350mg/kg, and the dosage range of the control drug Polymyxin B is 21.2-30mg/kg. Thus we are based on W 3 BipY 8 The dose ranges of (2) were calculated to have a public ratio of 1.4812 and were divided into 4 groups of 40, 59.25, 87.76, 130mg/kg, respectively. W (W) 3 BipY 8 The public ratio of the P groups is 1.1674, which is divided into 4 groups of 220, 256.83, 299.82, 350mg/kg, respectively. The comparative drug Polymyxin B was classified into 3 groups of 21.2, 25 and 30mg/kg at a public ratio of 1.19, and the following main test was carried out. According to the grouping, the corresponding doses of W are injected into the abdominal cavity of each group of mice once 3 BipY 8 、W 3 BipY 8 P or Polymyxin B, death was observed within 7 days, and LD was calculated 50 And taking the heart, liver, spleen, lung and kidney of the dead mice as pathological sections, and observing the toxicity of the medicine to viscera. The results are shown in Table 4 and FIG. 7.
TABLE 4 LD of antibacterial peptides and Polymyxin B against BALB/C mice 50
From the results of Table 4, it can be seen that the phosphorylated antibacterial peptide W 3 BipY 8 In vivo toxicity of P compared to non-phosphorylated parent peptide W 3 BipY 8 4.2 times lower, 11 times lower than Polymyxin B, and lower toxicity. From the HE stained sections of the viscera of FIG. 7, polymyxin B was shown to be toxic to various tissues to various degrees, with bleeding and inflammatory cell infiltration, especially severe toxicity to the lungs and kidneys, renal cell swelling, severe alveolar contraction, pulmonary congestion, massive inflammatory cell infiltration, mice were likely to die from choking hypoxia, and Polymyxin B-killed mice were all rapidly dying within 2 hours. Non-phosphorylated parent peptide W 3 BipY 8 Has serious toxicity to liver and kidney, liver hemorrhage, inflammatory cell infiltration, and a large amount of scattered red blood cells can be seen in the liver and kidney; renal hemorrhage, edema; has slight toxicity to lung, and has small amount of red blood cells in alveoli. Phosphorylated antibacterial peptide W at half lethal dose 3 BipY 8 P is not obvious in toxicity to heart, lung and spleen, and is slightly toxic to liver and kidney. Phosphorylated antibacterial peptide W 3 BipY 8 The lethal dose of P is much greater than that of the non-phosphorylated parent peptide W 3 BipY 8 And Polymyxin B has higher in vivo safety.
6. Phosphorylated antibacterial peptide W 3 BipY 8 P treatment of mouse Klebsiella pneumoniae-induced bacterial pneumonia experiments
Male BALB/C mice, 18-22g, were fed according to the laboratory animal ethics management method at the university of Lanzhou. 154 BALB/C mice were randomly divided into a blank group (without infection treatment), a model group (without any treatment), W 3 BipY 8 Group (5 mg/kg), W 3 BipY 8 Group P (5.31 mg/kg) and Polymyxin B (2.46 mg/kg), 35 bacteria per group, 14 blanks. After intraperitoneal injection of pentobarbital (10 mg/mL) at a dose of 60mg/kg, klebsiella pneumoniae ATCC700603 (25. Mu.L, 3X 10) was administered to the pulmonary bronchus of each mouse with an atomizing needle under laryngoscope support 10 CFU/mL) to establish a model of pulmonary infection in mice. The corresponding dose of the drug was administered by intraperitoneal injection for the first time 4 hours after the administration group, and each infected mouse was intraperitoneally injected with the corresponding dose of the drug or sterile physiological saline every morning and evening within three days after the infection. Each experimental group was sacrificed by 5 mice per day, right lung homogenates were taken in sterile physiological saline, serial dilutions were made, and bacterial loads were counted on MH agar plates, each group of lung bacterial counts were recorded per day, and observations were made for 7 consecutive days. The left lung was fixed with 10% formaldehyde, paraffin embedded and hematoxylin-eosin stained. The results are shown in FIGS. 8 and 9.
FIG. 8 shows that the model group treated with physiological saline had a mortality rate of 30% in the first 3 days and no death of mice in the latter 4 days, but bacterial load remained high, and reached-5.7X10 at the 7 th day of modeling 5 Bacterial load of CFU/g. Accept W 3 BipY 8 、W 3 BipY 8 P and Polymyxin B treated mice showed improved outcome after bacterial infection, with one mouse dying on day 1 only, W 3 BipY 8 Group sum W 3 BipY 8 Group P no mice died within 7 days. W (W) 3 BipY 8 And W is 3 BipY 8 The P administration group reaches the level of being cleared by immune response on the 4 th day and completely kills the bacteria in the lungs of the mice on the 6 th day, thereby achieving better treatment of the miceAction of bacterial infection in the lungs. Cure rate ratio of Polymyxin B W 3 BipY 8 And W is 3 BipY 8 Slow P, reaching a level that can be cleared by immune response on day 6 and completely killing the mice lung bacteria on day 7. At any time point, there was a significant difference in bacterial load between the dosing group and the model group, demonstrating that the novel phosphorylated antibacterial peptide bacterial infection had significant therapeutic effects. From the lung tissue sections of fig. 9 we found that the lung tissue of the model group and the polymyxin B treated group had developed severe inflammation, with massive inflammatory cell infiltration, alveolar contraction and bacterial load significantly higher than the other groups. Phosphorylated antibacterial peptide W 3 BipY 8 -P and non-phosphorylated parent peptide W 3 BipY 8 After treatment, the alveoli are effectively expanded, the alveoli aggregation is improved, and simultaneously inflammatory response and bacterial load are obviously reduced.
In conclusion, the invention introduces the phosphorylation strategy into the design of the antibacterial peptide, the negative charge carried by the phosphorylated amino acid can neutralize partial positive charge of the antibacterial peptide, and meanwhile, the hydrophilicity of the phosphorylated amino acid can be greatly enhanced, and the hydrophobicity of the antibacterial peptide can be integrally weakened, so that the toxicity of the antibacterial peptide is reduced. In addition, the original secondary structure of the antibacterial peptide is destroyed after phosphorylation, and the electrostatic interaction of positively charged amino groups and negatively charged phosphate groups leads to helical conformation distortion, so that the toxicity of the amphiphilic peptide is weakened. In addition, phosphorylation can further increase the steric hindrance of the antibacterial peptide contacted with the protease, slow down the enzymolysis of the protease and increase the stability of the antibacterial peptide.
In-vitro bacteriostasis experiments, hemolytic activity experiments, serum stability experiments, drug resistance induction experiments and acute toxicity experiments show that the hydroxy phosphorylated antibacterial peptide has broad-spectrum antibacterial activity, low toxicity, high serum stability and no drug resistance induction, and the generation of the drug resistance of the Rifampin or the Kanamycin can be reduced by combining the hydroxy phosphorylated antibacterial peptide with the Rifampin or the Kanamycin. The hydroxy phosphorylated antibacterial peptide can also be used for treating bacterial pneumonia induced by mouse klebsiella pneumoniae, so that the hydroxy phosphorylated antibacterial peptide has good application prospect in the aspect of preparing clinical antibacterial medicines.
Drawings
FIG. 1 is W 3 Mass spectrum of (3);
FIG. 2 is W 3 BipY 8 Mass spectrum of (3);
FIG. 3 is a phosphorylated peptide W 3 BipY 8 -mass spectrum of P;
FIG. 4 is a phosphorylated peptide W 3 BipY 8 -P and non-phosphorylated parent peptide W 3 BipY 8 Hemolytic activity results on erythrocytes after incubation with mouse erythrocytes for 1 h;
FIG. 5 is a phosphorylated peptide W 3 BipY 8 -P and non-phosphorylated parent peptide W 3 BipY 8 Serum stability results after co-incubation with mouse serum;
FIG. 6 is a phosphorylated peptide W 3 BipY 8 -P and non-phosphorylated parent peptide W 3 BipY 8 Induced drug resistance results after 20 days of continuous action with klebsiella pneumoniae ATCC 700603;
FIG. 7 is a phosphorylated peptide W 3 BipY 8 -P and non-phosphorylated parent peptide W 3 BipY 8 And a graph of pathological examination results after acute toxic death of the mice caused by Polymyxin B;
FIG. 8 is a phosphorylated peptide W 3 BipY 8 -P and non-phosphorylated parent peptide W 3 BipY 8 And Polymyxin B treatment results of bacterial pneumonia in mice.
FIG. 9 is a phosphorylated peptide W 3 BipY 8 P, non-phosphorylated parent peptide W 3 BipY 8 Results of pulmonary histopathological examination of Polymyxin B in mice treated for bacterial pneumonia.
Detailed Description
The synthesis of novel hydroxyphosphorylated antimicrobial peptides of the present invention having broad spectrum antimicrobial activity, low toxicity and high stability is further illustrated by the following examples.
Example 1: antibacterial peptide W 3 Is synthesized by (a)
(1) Resin activation and pretreatment
0.5g of Fmoc-NH was weighed out 2 MBHA resin (0.42 mmol/g), added to a polypeptide solid phase synthesizer, swelled with DCM for 30min, washed with DMF and identified by ninhydrin chromogenic method, if notThe color indicated that the resin was normal.
(2)Fmoc-W 3 Synthesis of MBHA
Washing the swelled resin with DMF solution containing 20% piperidine to remove Fmoc protecting group, and obtaining the indene detection resin with bluish violet color. 3 times of Leu, 3 times of HOBt and HBTU and 6 times of DIEA are dissolved by redistilled DMF and added into a synthesizer to be stirred for 1h, and after the reaction is carried out for a period of time, the indene detection resin is colorless and transparent to indicate successful condensation, and Fmoc-Leu-MBHA is obtained.
Condensing Leu, leu, thr, lys, leu, arg, lys, trp, leu and Gly in sequence according to the method to obtain Fmoc-Gly-Leu-Trp-Lys-Arg-Leu-Lys-Thr-Leu-Leu-MBHA.
(3) Polypeptide cleavage
The Fmoc-Gly-Leu-Trp-Lys-Arg-Leu-Lys-Thr-Leu-Leu-MBHA obtained was purified. After removal of the Fmoc protecting group by washing with a solution of 20% piperidine in DMF, the resin was thoroughly drained by washing with DCM and methanol in sequence. 10mL of cleavage reagent (TFA: tris: water=9.5:0.25:0.25 (v: v)) was added and reacted for 3 hours, extracted with diethyl ether and lyophilized.
(4) Polypeptide purification
RP-HPLC purification conditions were mobile phase A:0.1% TFA/acetonitrile, mobile phase B:0.1% TFA/water, eluting with a linear gradient, collecting the target peak effluent, and lyophilizing.
The mass spectrum is shown in figure 1. The molecular weight theoretical calculation result is 1226.55, which is consistent with the mass spectrum identification result, and the antibacterial peptide structure is proved to be correct.
Example 2: antibacterial peptide W 3 BipY 8 Is synthesized by (a)
(1) Resin activation and pretreatment
As in example 1.
(2)Fmoc-W 3 BipY 8 Synthesis of MBHA
Washing the swelled resin with DMF solution containing 20% piperidine to remove Fmoc protecting group, and obtaining the indene detection resin with bluish violet color. 3 times of Leu, 3 times of HOBt and HBTU and 6 times of DIEA are dissolved by redistilled DMF and added into a synthesizer to be stirred for 1h, and after the reaction is carried out for a period of time, the indene detection resin is colorless and transparent to indicate successful condensation, and Fmoc-Leu-MBHA is obtained.
Condensing Leu, leu, tyr, lys, leu, arg, lys, bip, leu and Gly sequentially according to the method to obtain Fmoc-Gly-Leu-Bip-Lys-Arg-Leu-Lys-Tyr-Leu-Leu-MBHA.
(3) Polypeptide cleavage
As in example 1.
(4) Polypeptide purification
As in example 1.
The mass spectrum is shown in figure 2. The molecular weight theoretical calculation result is 1325.62, which is consistent with the mass spectrum identification result, and the antibacterial peptide structure is proved to be correct.
Example 3: antibacterial peptide W 3 BipY 8 Synthesis of P
(1) Resin activation and pretreatment
As in example 1.
(2)Fmoc-W 3 BipY 8 Synthesis of P-MBHA
Washing the swelled resin with DMF solution containing 20% piperidine to remove Fmoc protecting group, and obtaining the indene detection resin with bluish violet color. 3 times of Leu, 3 times of HOBt and HBTU and 6 times of DIEA are dissolved by redistilled DMF and added into a synthesizer to be stirred for 1h, and after the reaction is carried out for a period of time, the indene detection resin is colorless and transparent to indicate successful condensation, and Fmoc-Leu-MBHA is obtained.
Sequentially condensing Leu, leu, tyr (H) 2 PO 3 ) Lys, leu, arg, lys, bip, leu, gly to Fmoc-Gly-Leu-Bip-Lys-Arg-Leu-Lys-Tyr (H) 2 PO 3 )-Leu-Leu-MBHA。
(3) Polypeptide cleavage
As in example 1.
(4) Polypeptide purification
As in example 1.
The mass spectrum is shown in figure 3, the molecular weight theoretical calculation result is 1405.60, and the mass spectrum identification result is consistent with that of the phosphorylated antibacterial peptide, so that the structure is correct.
Claims (4)
1. A hydroxy-phosphorylated antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and high stability, which is characterized in thatBased on alpha helical peptide A10, is subjected to hydrophobic moment adjustment and amino acid substitution, and is then phosphorylated to obtain, denoted as W 3 BipY 8 -P, the amino acid sequence of which is as follows:
Gly-Leu-Bip-Lys-Arg-Leu-Lys-Tyr(H 2 PO 3 )-Leu-Leu-NH 2 。
wherein the amino acid sequence of the alpha helical peptide A10 is as follows:
Gly-Leu-Leu-Lys-Arg-Trp-Lys-Thr-Leu-Leu-NH 2 ；
the hydrophobic moment adjustment means that the 6 th tryptophan and the 3 rd leucine positions in the alpha helical peptide A10 peptide chain are interchanged to reduce the hydrophobic moment, and the polypeptide is marked as W after the hydrophobic moment adjustment 3 The amino acid sequence is as follows:
Gly-Leu-Trp-Lys-Arg-Leu-Lys-Thr-Leu-Leu-NH 2 ；
the amino acid substitution refers to the substitution of W 3 Tryptophan at position 3 in peptide chain is replaced by unnatural amino acid 4' -biphenylalanine, threonine at position 8 is replaced by tyrosine, and the polypeptide is marked as W after amino acid replacement 3 BipY 8 The amino acid sequence is as follows:
Gly-Leu-Bip-Lys-Arg-Leu-Lys-Tyr-Leu-Leu-NH 2 。
2. the use of a hydroxy phosphorylated antibacterial peptide of claim 1 having broad-spectrum antibacterial activity, low toxicity and high stability for the preparation of a clinical antibacterial drug.
3. The use of phosphorylated antibacterial peptide of claim 2 having broad-spectrum antibacterial activity, low toxicity and high stability for the preparation of clinical antibacterial drugs, wherein the hydroxy phosphorylated antibacterial peptide W is 3 BipY 8 P is used in combination with Rifampin or Kanamycin to reduce the development of Rifampin or Kanamycin resistance.
4. The use of a phosphorylated antibacterial peptide having broad-spectrum antibacterial activity, low toxicity and high stability according to claim 1 for the preparation of a medicament for the treatment of bacterial pneumonia induced by klebsiella pneumoniae in mice.
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