N-terminal lauric acid modified full-D-type ultrashort lipopeptide with high stability and gram-positive bacterium resisting activity and application of N-terminal lauric acid modified full-D-type ultrashort lipopeptide

Abstract

The invention discloses a full D-type ultrashort lipopeptide Lip 1 (C12-rrw-NH2) which is modified by N-terminal lauric acid and has high stability and gram-positive bacterium resisting activity, the sequence of the full D-type ultrashort lipopeptide Lip 1 is composed of two D-type arginine (D-Arg) and one D-type tryptophan (D-Trp), and the N-terminal of the full D-type ultrashort lipopeptide Lip 1 is modified by lauric acid. An in-vitro bacteriostasis experiment, a hemolysis experiment and a stability experiment show that Lip 1 has excellent antibacterial activity on gram-positive bacteria, is relatively low in hemolysis toxicity and has relatively high stability in protease, serum and salt ion environments. In addition, animal experiments show that Lip 1 has good in-vivo safety and antibacterial activity. Therefore, the designed and synthesized ultrashort lipopeptide Lip 1 has a good application prospect in preparation of gram-positive bacterium resistant polypeptide drugs, especially oral antibacterial drugs, due to high stability and efficient gram-positive bacterium resistant activity.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical technology and relates to an N-terminal lauric acid-modified all-D-type ultra-short lipopeptide Lip 1 (C 12 -rrw-NH2), the present invention also relates to the use of the ultrashort lipopeptide in the preparation of clinical anti-Gram-positive bacteria drugs. Background Art

[0002] Since the discovery of the first antibiotic, penicillin, antibiotics have played an extremely important role in protecting human health and curing bacterial infections. However, with the abuse of antibiotics, bacteria are developing drug resistance at an alarming rate, which not only poses a great threat to human life and health, but also brings huge economic impacts (Science. 2016Aug 26; 353(6302): 874-5). In particular, the growing drug resistance of Gram-positive bacteria has led to many difficulties in clinical antibacterial treatment. For example, since the discovery of methicillin-resistant Staphylococcus aureus (MRSA), its prevalence has continued to expand and has become one of the most common pathogens in today's society. Compared with ordinary Staphylococcus aureus infected people, MRSA infected people have a higher incidence and mortality rate of diseases such as acute renal failure, bacteremia, and necrotizing pneumonia, and the treatment time is longer, the treatment is more difficult, and the medical expenses required are higher. Therefore, the development of new anti-Gram-positive bacteria drugs is imminent.

[0003] Antimicrobial peptides (AMPs) are small molecule active peptides with advantages such as broad-spectrum antimicrobial activity and rapid bactericidal ability. At the same time, antimicrobial peptides mainly target bacterial cell membranes, destroy their integrity, and ultimately kill bacteria. This unique membrane destruction mechanism makes it difficult for bacteria to develop resistance to antimicrobial peptides (Lancet Infect Dis. 2020 Sep; 20 (9): e216-e230). At present, most of the existing antimicrobial peptides come from nature, including insects, animals, microorganisms and other organic organisms, and only a small part is artificially synthesized. However, natural antimicrobial peptides have long and complex sequence structures, poor stability, and high toxicity, which greatly limits their development and application.

[0004] Ultrashort lipopeptides are usually composed of a few amino acids or amino acids and hydrophobic groups. They have hydrophobic residues (tails) and hydrophilic segments (heads), showing obvious amphiphilic structural characteristics, and thus showing ideal antibacterial potential. At the same time, because its antibacterial mechanism is similar to that of antimicrobial peptides, it is not easy to develop drug resistance (Molecules. 2020 Jan 8; 25(2): 257), and has been widely studied in recent years. For example, Emilia Sikorska et al. reported four ultrashort lipopeptides C 16 -RRRR-NH2, C 14 -RRRR-NH2, C 12 -RRRR-NH2 and C 16 -PRRR-NH2 has antibacterial effect on Gram-positive bacteria (Biochim Biophys Acta Biomembr. 2018Nov; 1860(11): 2242-2251); Two palmitoylated lipopeptides and two D-lysine-substituted peptides C reported by Anindyasundar Adak et al. 16 -YKK, C 16 -WKK, C 16 -Ykk and C 16 -Wkk not only has good cell compatibility with fibroblasts, but also has antibacterial activity against both Gram-negative and Gram-positive bacteria (Biomacromolecules. 2024Feb12; 25(2): 1205-1213). The applicant has also conducted relevant research work in the early stage and made some progress. However, the ultra-short lipopeptides currently studied are mostly composed of L-type amino acids, and still have limitations such as poor stability and narrow application range. Summary of the invention

[0005] The purpose of the present invention is to provide an N-terminal lauric acid-modified all-D-type ultrashort lipopeptide with high efficiency stability and high efficiency anti-Gram-positive bacteria activity.

[0006] Another object of the present invention is to provide the use of the all-D ultrashort lipopeptide in the preparation of clinical anti-Gram-positive bacteria drugs.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] 1. Structural design of all-D ultra-short lipopeptide Lip 1

[0009] In order to obtain ultra-short lipopeptides with high stability and high anti-Gram-positive bacteria activity, the present invention, based on the applicant's previous research work, designed and synthesized ultra-short lipopeptides Lip 1, Lip 2 and Lip 3 containing different numbers of D-amino acids. Then, this series of ultra-short lipopeptides were subjected to antibacterial activity, hemolytic toxicity and stability experiments, and compared with the ultra-short lipopeptide C12-R2 (C 12 -RRW-NH2) were compared, and finally the all-D-type ultra-short lipopeptide Lip 1 with high antibacterial activity against Gram-positive bacteria, low hemolytic toxicity and high stability was screened. The ultra-short lipopeptide Lip 1 includes two hydrophilic D-arginines (D-Arg) and one hydrophobic D-tryptophan (D-Trp), and its N-terminus is modified with lauric acid.

[0010] The above series of ultrashort lipopeptides of the present invention have the following structural formula:

[0011]

[0012] The above series of ultrashort lipopeptides were prepared by classical solid phase synthesis method.

[0013] 2. Application of all-D ultra-short lipopeptide Lip 1 in the preparation of clinical antibacterial drugs

[0014] 1. In vitro antibacterial test

[0015] The experimental strains selected included: Gram-positive bacteria (S. aureus ATCC 25923, S. aureus ATCC29213, MRSA ATCC 33591, S. epidermidis ATCC 12228). The experimental bacteria grown to the mid-logarithmic phase were diluted to 1×10 6 CFU / mL bacterial suspension. The ultrashort lipopeptide was added to MH medium and prepared into a series of peptide solutions of different concentrations by two-fold dilution method, and then mixed with an equal volume of bacterial suspension in a 96-well plate, incubated at 37°C for 18-24h, and observed. The minimum concentration without obvious bacterial growth was the minimum inhibitory concentration (MIC) of the ultrashort lipopeptide; antibiotics Pobmyxin B and Gentamicin were used as positive controls; the experiment was repeated three times in parallel. The results are shown in Table 1.

[0016] Table 1 Minimum inhibitory concentration of the ultrashort lipopeptide of the present invention against standard strains

[0017]

[0018] The results in Table 1 show that the ultrashort lipopeptide of the present invention has strong antibacterial activity against common Gram-positive experimental strains, and the antibacterial activity is better than that of the antibiotics Pobmyxin B and Gentamicin.

[0019] 2. Hemolysis test

[0020] Take fresh blood from healthy mice, centrifuge at 1000g for 10min (4℃), collect red blood cells, wash with PBS 3 times, and prepare a red blood cell suspension with a concentration of 8%. Add peptide solutions of different concentrations and equal volumes of red blood cell suspension to a 96-well plate and incubate at 37℃ for 1h; after centrifugation at 1200g for 15min, transfer the supernatant to a new 96-well plate, and detect its absorbance at 490nm with an enzyme reader; PBS blank solution is used as a negative control, and 1% Triton X-100 is used as a positive control. The hemolysis rate is calculated according to the following formula:

[0021] Hemolysis rate (%) = [(OD 490nm(peptides) -OD 490nm(negative control) ) / (OD 490nm(positive control) -OD 490nm(negative control) )]×100%

[0022] Figure 5 The results showed that the ultrashort lipopeptides of the present invention had low hemolytic toxicity, among which C12-R2 and Lip3 showed hemolytic toxicity at 128 μM, and Lip 1 and Lip 2 showed hemolytic toxicity at 64 μM. However, the hemolytic concentration of all ultrashort lipopeptides was much greater than the effective concentration for exerting antibacterial activity.

[0023] 3. In vitro stability test

[0024] (1) Enzymatic stability

[0025] The experimental strains used were Gram-positive bacteria (MRSA ATCC 33591). The experimental bacteria grown to the mid-logarithmic phase were diluted to 1×10 6 CFU / mL bacterial suspension. Use MH broth medium to prepare trypsin, chymotrypsin and pepsin solutions with a concentration of 1 mg / mL, and use MH medium containing different proteases to prepare a series of ultra-short lipopeptide solutions with enzyme solutions by double dilution method, incubate them at 37°C for 6 hours, then inactivate them at 60°C for 15 minutes, add them to a 96-well plate and mix with an equal volume of bacterial suspension, incubate them at 37°C for 18-24 hours to observe their MIC. The results are as follows:

[0026] Table 2 Minimum inhibitory concentration of the ultrashort lipopeptide of the present invention against standard strains in an enzyme environment

[0027]

[0028] The results in Table 2 show that Lip 1 can still maintain high antibacterial activity in the presence of trypsin, chymotrypsin and pepsin, which indicates that it has excellent enzymatic stability. The enzymatic stability to pepsin provides the possibility for its development into an oral antimicrobial peptide.

[0029] (2) Serum sensitivity

[0030] The experimental strains used were Gram-positive bacteria (MRSA ATCC 33591). The experimental bacteria grown to the mid-logarithmic phase were diluted to 1×10 6 CFU / mL bacterial suspension. Mouse serum and fetal bovine serum (FBS) were added to MH medium to prepare a medium containing 10% serum, and a series of ultra-short lipopeptide solutions of different concentrations were prepared with the medium, incubated at 37°C for 6 hours, then inactivated at 60°C for 15 minutes, added to a 96-well plate and mixed with an equal volume of bacterial suspension, incubated at 37°C for 18-24 hours, and the MIC was observed and the MIC change factor was calculated.

[0031] Figure 6 The results showed that the MIC of this series of ultra-short lipopeptides increased in the serum environment. After treatment with 10% serum, the MIC of all peptides increased by 8 times; while after treatment with 10% FBS, only the MIC of Lip 1 increased by 8 times, while the others only increased by 4 times. Combining all the results, it was found that the stability of this series of ultra-short lipopeptides in FBS was better than that in mouse serum, but overall the stability of these four peptides in serum was not much different.

[0032] (3) Salt ion sensitivity

[0033] The experimental strains used were Gram-positive bacteria (MRSA ATCC 33591). The experimental bacteria grown to the mid-logarithmic phase were diluted to 1×10 6 CFU / mL bacterial suspension. Prepare MH medium containing 150mM NaCl and 1mM MgCl2, and use this medium to prepare a series of ultra-short lipopeptide solutions of different concentrations, add them to a 96-well plate and mix with an equal volume of bacterial suspension, incubate at 37°C for 18-24h, observe MIC, and calculate its MIC change multiple.

[0034] Figure 7 The results showed that after treatment with 150 mM NaCl and 1 mM MgCl2, the MIC of ultra-short lipopeptides changed little, only Lip 3 increased by more than 2 times, and the increases of other peptides were less than 2 times, which indicated that this series of ultra-short lipopeptides were all sensitive to MgCl2. 2+ and Na+ Has good resistance.

[0035] 4. In vivo safety experiments

[0036] Twenty female and male Balb / c mice weighing 18-22g SPF were selected and randomly divided into five groups, each group including four male mice and four female mice. Polymyxin B (20mg / kg) and Lip 1 (20mg / kg, 40mg / kg, 80mg / kg) were intraperitoneally injected into each group of mice. Normal saline was used as the control group. The state changes and survival rate of the mice were recorded within 8 days.

[0037] The results are as follows Figure 8 As shown, 20mg / kg and 40mg / kg of Lip 1 had less effect on mice and the survival rate of mice was 100%, while 80mg / kg of Lip 1 and 20mg / kg of polymyxin B had greater effect on mice, and the survival rate of mice in the 80mg / kg Lip 1 group was 70%, while the survival rate of mice in the 20mg / kg polymyxin B group was only 60%. In summary, compared with polymyxin B, Lip 1 has less effect on mice and is safer in vivo.

[0038] 5. In vivo antibacterial activity test

[0039] Twenty-four 18-22g SPF Balb / c male mice were randomly divided into three groups, with 8 mice in each group. Each group of mice was intraperitoneally injected with 2×10 8 CFU / mL of MRSA ATCC 33591 was used to establish an acute peritonitis infection model. Treatment was performed 1 hour after infection. Lip 1 (10 mg / kg) and polymyxin B (10 mg / kg) were injected intraperitoneally into the mice for treatment. Normal saline was used as the model group. 6 hours after infection, the mice were euthanized, and the liver, spleen, kidney and lung of the mice were removed by aseptic operation, homogenized, and evenly spread on MH agar plates. They were incubated at 37°C for 18-24 hours, and the bacteria were counted after removal to determine the bacterial load of the organs.

[0040] Fig. 9 The bacterial counts in the liver, spleen, kidneys and lungs of mice were displayed. The results of the model group showed that a systemic bacterial infection model had been established. After treatment with Lip 1 (10 mg / kg), the bacterial counts in each test organ of the mice were significantly reduced, and its antibacterial activity was equivalent to that of the same dose of polymyxin B.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. Polypeptide antibacterial drugs are easily hydrolyzed by proteins and have poor stability, which is still an important problem that needs to be solved in the development of antibacterial drugs. The novel ultrashort lipopeptide Lip 1 synthesized by the present invention is composed of 3 D-amino acids, including 2 hydrophilic D-arginines (D-Arg) and 1 hydrophobic D-tryptophan (D-Trp), and its N-terminus contains lauric acid modification. The use of all D-amino acids in the entire peptide sequence makes Lip 1 highly stable, which is in sharp contrast to its all-L-type amino acid sequence C12-R2. C12-R2 has good antibacterial activity and low cytotoxicity, but poor stability; Lip 1 retains the advantages of C12-R2 while also having high stability. The high stability presented by Lip 1 also makes it possible for its application in vivo and its development into oral antibacterial peptide drugs. The excellent performance of Lip 1 in treating systemic MRSA infection in mice also proves this point, broadening the application range of antibacterial peptide drugs.

[0043] 2. As an ultra-short lipopeptide containing only three amino acids, Lip 1 has a novel design strategy, simple structure, low preparation cost, and is easy to mass produce. While ensuring the stability of the peptide, Lip 1 also takes into account the balance between antibacterial activity and toxicity, which makes Lip 1 have high stability while also having excellent antibacterial activity against Gram-positive bacteria, low hemolytic toxicity, good in vivo safety and in vivo antibacterial activity. In general, Lip 1 is able to ensure its medicinal value while also having many advantages, all of which make Lip 1 have extremely high application value and development prospects in future clinical antibacterial treatments.

[0044] 3. The ultra-short lipopeptide of the present invention is modified with lauric acid at the N-terminus, which can not only enhance the hydrophobicity of the ultra-short lipopeptide, but also facilitate the interaction between the ultra-short lipopeptide and the bacterial membrane, thereby improving the antibacterial activity. In addition, the applicant's previous studies have found that the antibacterial activity of the antibacterial lipopeptide is improved in a "U"-shaped relationship with the fatty acid chain length. When the fatty acid chain length is 4 to 12 carbons, the increase in hydrophobicity significantly reduces its MIC value to a minimum threshold, but when the fatty acid chain length exceeds 12 carbons, due to excessive hydrophobicity, its toxicity increases, and the antibacterial activity also decreases with the increase in the MIC value. Therefore, the ultra-short lipopeptide of the present invention chooses to use lauric acid for modification, which can most effectively improve the antibacterial activity of the antibacterial peptide, and the appropriate fatty acid chain length will not increase its toxicity. Lauric acid plays a key role in the antibacterial potential and selectivity of lipopeptides. In addition, lauric acid modification can act as a hydrophobic amino acid in the peptide chain, compensate for the hydrophobic volume, shorten the peptide chain length, and facilitate the design and synthesis of ultra-short lipopeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is the mass spectrum of the ultrashort lipopeptide Lip 1 of the present invention;

[0046] Figure 2 is the mass spectrum of the ultrashort lipopeptide Lip 2 of the present invention;

[0047] Figure 3 is the mass spectrum of the ultrashort lipopeptide Lip 3 of the present invention;

[0048] Figure 4 The ultrashort lipopeptide C of the present invention 12 -Mass spectrum of R2;

[0049] Figure 5 This is a diagram showing the results of a hemolytic experiment of the ultrashort lipopeptide of the present invention;

[0050] Figure 6 This is a diagram showing the results of a serum stability experiment of the ultrashort lipopeptide of the present invention;

[0051] Figure 7 This is a diagram showing the experimental results of the salt ion stability of the ultrashort lipopeptide of the present invention;

[0052] Figure 8 This is a graph showing the results of an in vivo safety mouse survival rate experiment of the ultrashort lipopeptide of the present invention;

[0053] Fig. 9 This is a graph showing the results of a bacterial count experiment in the liver, kidney, spleen and lung of mice after systemic infection treated with the ultrashort lipopeptide of the present invention. DETAILED DESCRIPTION

[0054] The following is a detailed description of the synthesis method of the all-D-type ultrashort lipopeptide Lip 1 with high stability and high anti-Gram-positive bacteria activity, as well as its in vitro antibacterial activity, hemolytic toxicity and in vitro stability of the present invention in conjunction with the accompanying drawings and specific examples.

[0055] Example 1: Synthesis of Lip 1 and its in vitro antibacterial activity, hemolytic toxicity and in vitro stability studies

[0056] (1) Synthesis of Lip 1

[0057] ① Activation and pretreatment of resin

[0058] 0.43 g of MBHA resin (substitution value 0.46 mmol / g) was accurately weighed and placed in a synthesizer. After swelling with DCM solution for 30 min, the resin was tested by ninhydrin colorimetric method. The result showed that the resin was colorless and transparent, indicating that the resin was normal.

[0059] ②Synthesis of Lip 1

[0060] 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 Fmoc protecting group had been removed. Fmoc-D-Trp(Boc)-OH (237 mg), HOBT (61 mg), HBTU (171 mg), and DIEA (0.15 ml) were added to 8 ml The product was dissolved and mixed in DMF, 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, and Fmoc-D-Trp-resin was obtained. The method was the same as above, and the subsequent amino acids were condensed in sequence: Fmoc-D-Arg(pbf)-OH (292 mg), Fmoc-D-Arg(pbf)-OH (292 mg), wherein the amounts of HOBT, HBTU and DIEA were the same as above, and Fmoc-(D-Arg)-(D-Arg)-(D-Trp)-resin, i.e., Fmoc-rrw-resin, was obtained. The same operation was carried out by condensation reaction to connect lauric acid (181 mg), except that HBTU was replaced by HATU (171 mg) when connecting fatty acids. After the fatty acids were connected, no deprotection was required, and the peptide was directly cleaved after treatment with dichloromethane and methanol.

[0061] ③ Peptide cleavage

[0062] The C was cleaved using a mixed solution of TFA, triisopropylsilane and water in a volume ratio of 9.5:0.25:0.25. 12 -rrw-resin was cut, extracted with ice ether and water, and freeze-dried to obtain a crude peptide freeze-dried powder;

[0063] ④Peptide purification

[0064] The crude peptide freeze-dried powder obtained above was separated and purified by RP-HPLC, wherein the RP-HPLC purification conditions were as follows: mobile phase A: 0.05% TFA / water; mobile phase B: 0.05% TFA / acetonitrile; linear gradient elution, collecting the effluent of the main absorption peak, and then freeze-drying, and mass spectrometry identification to obtain Lip 1, the molecular formula of which was C 35 H 58 N 10 O5, molecular weight 698.5Da, mass spectrum see Figure 1 The structural formula is as follows:

[0065]

[0066] (2) Study on the antibacterial activity of Lip 1 in vitro

[0067] The experimental strains used were Gram-positive bacteria (S. aureus ATCC 25923, S. aureus ATCC29213, MRSA ATCC 33591, S. epidermidis ATCC 12228). The bacteria grown to the logarithmic phase were diluted to 1×10 6 CFU / mL bacterial suspension. Lip 1 was added to MH medium and prepared into a series of peptide solutions with different concentrations by two-fold dilution method, and then mixed with an equal volume of bacterial suspension in a 96-well plate, incubated at 37°C for 18-24h and observed. The minimum concentration with no obvious bacterial growth was the minimum inhibitory concentration (MIC) of Lip 1.

[0068] The results are shown in Table 1. Lip 1 has strong antibacterial activity against common Gram-positive experimental strains.

[0069] (3) Study on the hemolytic toxicity of Lip 1

[0070] Take healthy fresh mouse blood, centrifuge at 1000g for 10min (4℃), collect red blood cells, wash with PBS 3 times, and prepare a red blood cell suspension with a concentration of 8%. Add Lip 1 solution and an equal volume of red blood cell suspension to a 96-well plate and incubate at 37℃ for 1h; after centrifugation at 1200g for 15min, transfer the supernatant to a new 96-well plate and detect its absorbance at 490nm with an enzyme marker; PBS blank solution is used as a negative control, and 1% Triton X-100 is used as a positive control. The hemolysis rate is calculated according to the following formula:

[0071] Hemolysis rate (%) = [(OD 490nm(peptides) -OD 490nm(negative control) ) / (OD 490nm(positive control) -OD 490nm(negative control) )]×100%

[0072] The results are as follows Figure 5 Lip 1 showed hemolytic toxicity at 64 μmol / L, but its hemolytic concentration was much higher than the effective concentration for antibacterial activity.

[0073] (4) In vitro stability study of Lip 1

[0074] 1) Enzymatic stability

[0075] The experimental strains used were Gram-positive bacteria (MRSA ATCC 33591). The experimental bacteria grown to the mid-logarithmic phase were diluted to 1×10 6CFU / mL of bacterial suspension. MH broth medium was used to prepare trypsin, chymotrypsin and pepsin solutions with a concentration of 1 mg / mL. A series of Lip 1 solutions with different concentrations were prepared by two-fold dilution using MH medium containing different proteases. The solutions were incubated at 37°C for 6 hours, then inactivated at 60°C for 15 minutes, added to a 96-well plate and mixed with an equal volume of bacterial suspension, and incubated at 37°C for 18-24 hours to observe their MIC.

[0076] The results are shown in Table 2. The MIC of Lip 1 did not change in trypsin and chymotrypsin, but increased by 1 times in pepsin.

[0077] 2) Serum sensitivity

[0078] The experimental strains used were Gram-positive bacteria (MRSA ATCC 33591). The experimental bacteria grown to the mid-logarithmic phase were diluted to 1×10 6 CFU / mL bacterial suspension. Mouse serum and fetal bovine serum (FBS) were added to MH medium to prepare a medium containing 10% serum, and a series of Lip 1 solutions of different concentrations were prepared with the medium, incubated at 37°C for 6 hours, then inactivated at 60°C for 15 minutes, added to a 96-well plate and mixed with an equal volume of bacterial suspension, incubated at 37°C for 18-24 hours, and the MIC was observed and the MIC change factor was calculated.

[0079] The results are as follows Figure 6 , the MIC of Lip 1 in 10% serum and 10% FBS increased by 8-fold.

[0080] 3) Salt ion sensitivity

[0081] The experimental strains used were Gram-positive bacteria (MRSA ATCC 33591). The experimental bacteria grown to the mid-logarithmic phase were diluted to 1×10 6 CFU / mL bacterial suspension. Prepare MH medium containing 150mM NaCl and 1mM MgCl2, and use this medium to prepare a series of Lip 1 solutions of different concentrations, add them to a 96-well plate and mix with an equal volume of bacterial suspension, incubate at 37°C for 18-24h, observe MIC, and calculate its MIC change multiple.

[0082] The results are as follows Figure 7 , the MIC changes of Lip 1 were less than 2-fold in the environments of 150 mM NaCl and 1 mM MgCl2.

[0083] (5) In vivo safety study of Lip 1

[0084] Twenty female and male Balb / c mice weighing 18-22g SPF were selected and randomly divided into five groups, each group including four male mice and four female mice. Polymyxin B (20mg / kg) and Lip 1 (20mg / kg, 40mg / kg, 80mg / kg) were intraperitoneally injected into each group of mice. Normal saline was used as the control group. The state changes and survival rate of the mice were recorded within 8 days.

[0085] The results are as follows Figure 8 , 20mg / kg and 40mg / kg of Lip 1 had less effect on mice and the survival rate of mice was 100%, 80mg / kg of Lip 1 and 20mg / kg of polymyxin B had greater effect on mice, and the survival rate of mice in the 80mg / kg Lip 1 group was 70%, while the survival rate of mice in the 20mg / kg polymyxin B group was only 60%. In summary, compared with polymyxin B, Lip 1 has less effect on mice and is safer in vivo.

[0086] (6) In vivo antibacterial activity of Lip 1

[0087] Twenty-four 18-22g SPF Balb / c male mice were randomly divided into three groups, with 8 mice in each group. Each group of mice was intraperitoneally injected with 2×10 8 CFU / mL of MRSA ATCC 33591 was used to establish an acute peritonitis infection model. Treatment was performed 1 hour after infection. Lip 1 (10 mg / kg) and polymyxin B (10 mg / kg) were injected intraperitoneally into the mice for treatment. Normal saline was used as the model group. 6 hours after infection, the mice were euthanized, and the liver, spleen, kidney and lung of the mice were removed by aseptic operation, homogenized, and evenly spread on MH agar plates. They were incubated at 37°C for 18-24 hours, and the bacteria were counted after removal to determine the bacterial load of the organs.

[0088] Fig. 9 The bacterial counts in the liver, spleen, kidneys and lungs of mice were displayed. The results of the model group showed that a systemic bacterial infection model had been established. After treatment with Lip 1 (10 mg / kg), the bacterial counts in each test organ of the mice were significantly reduced, and its antibacterial activity was equivalent to that of the same dose of polymyxin B.

[0089] Example 2: Synthesis of Lip 2

[0090] (1) Synthesis of Lip 2

[0091] ① Activation and pretreatment of resin

[0092] Same as step (1) of Example 1;

[0093] ②Synthesis of Lip 2

[0094] 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 Fmoc protecting group had been removed. Fmoc-L-Trp(Boc)-OH (237 mg), HOBT (61 mg), HBTU (171 mg), and DIEA (0.15 ml) were added to 8 ml The product was dissolved and mixed in DMF, 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, and Fmoc-L-Trp-resin was obtained. The method was the same as above, and the subsequent amino acids were condensed in sequence: Fmoc-L-Arg(pbf)-OH (292 mg), Fmoc-D-Arg(pbf)-OH (292 mg), wherein the amounts of HOBT, HBTU and DIEA were the same as above, and Fmoc-(D-Arg)-(L-Arg)-(L-Trp)-resin, i.e., Fmoc-rRW-resin, was obtained. The same operation was carried out by condensation reaction to connect lauric acid (181 mg), except that HBTU was replaced by HATU (171 mg) when connecting fatty acids. After the fatty acids were connected, no deprotection was required, and the peptide was directly cleaved after treatment with dichloromethane and methanol.

[0095] ③ Peptide cleavage

[0096] Same as step (1) of Example 1 (3);

[0097] ④Peptide purification

[0098] The same as step (1) of Example 1, Lip 2 was obtained by mass spectrometry, and the molecular formula was C 35 H 58 N 10 O5, molecular weight 698.5Da, mass spectrum see Figure 2 The structural formula is as follows:

[0099]

[0100] (2) Study on the antibacterial activity of Lip 2 in vitro

[0101] Same as step (2) in Example 1;

[0102] The results are shown in Table 1. Lip 2 has strong antibacterial activity against common Gram-positive experimental strains.

[0103] (3) Study on the hemolytic toxicity of Lip 2

[0104] Same as step (3) of Example 1;

[0105] The results are as follows Figure 5 Lip 2 showed hemolytic toxicity at 128 μmol / L, but its hemolytic concentration was much greater than the effective concentration for antibacterial activity.

[0106] (4) In vitro stability study of Lip 2

[0107] 1) Enzymatic stability

[0108] Same as step (4) 1) in Example 1;

[0109] The results are shown in Table 2. The MIC of Lip 2 increased 16-fold in trypsin, 8-fold in chymotrypsin, and 4-fold in pepsin.

[0110] 2) Serum sensitivity

[0111] Same as step (4) 2) in Example 1;

[0112] The results are as follows Figure 6 , the MIC of Lip 2 in 10% serum increased 8-fold, and the MIC in 10% FBS increased 4-fold.

[0113] 3) Salt ion sensitivity

[0114] Same as step (4) 3) in Example 1;

[0115] The results are as follows Figure 7 , the MIC changes of Lip 2 were less than 2-fold in the environments of 150 mM NaCl and 1 mM MgCl2.

[0116] Example 3: Synthesis of Lip 3

[0117] (1) Synthesis of Lip 3

[0118] ① Activation and pretreatment of resin

[0119] Same as step (1) of Example 1;

[0120] ②Synthesis of Lip 2

[0121] 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 Fmoc protecting group had been removed. Fmoc-L-Trp(Boc)-OH (237 mg), HOBT (61 mg), HBTU (171 mg), and DIEA (0.15 ml) were added to 8 ml The product was dissolved and mixed in DMF, 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, and Fmoc-L-Trp-resin was obtained. The method was the same as above, and the subsequent amino acids were condensed in sequence: Fmoc-D-Arg(pbf)-OH (292 mg), Fmoc-D-Arg(pbf)-OH (292 mg), wherein the amounts of HOBT, HBTU and DIEA were the same as above, and Fmoc-(D-Arg)-(D-Arg)-(L-Trp)-resin, i.e., Fmoc-rrW-resin, was obtained. The same operation was carried out to connect lauric acid (181 mg) through the condensation reaction, except that HBTU was replaced by HATU (171 mg) when connecting fatty acids. After the fatty acids were connected, no deprotection was required, and the peptide was directly cleaved after treatment with dichloromethane and methanol.

[0122] ③ Peptide cleavage

[0123] Same as step (1) of Example 1 (3);

[0124] ④Peptide purification

[0125] The same as step (1) of Example 1, Lip 3 was obtained by mass spectrometry, and the molecular formula was C 35 H 58 N 10 O5, molecular weight 698.5Da, mass spectrum see Figure 3 The structural formula is as follows:

[0126]

[0127] (2) Study on the antibacterial activity of Lip 3 in vitro

[0128] Same as step (2) in Example 1;

[0129] The results are shown in Table 1. Lip 3 has strong antibacterial activity against common Gram-positive experimental strains.

[0130] (3) Study on the hemolytic toxicity of Lip 3

[0131] Same as step (3) of Example 1;

[0132] The results are as follows Figure 5Lip 3 showed hemolytic toxicity at 128 μmol / L, but its hemolytic concentration was much greater than the effective concentration for antibacterial activity.

[0133] (4) In vitro stability study of Lip 3

[0134] 1) Enzymatic stability

[0135] Same as step (4) 1) in Example 1;

[0136] The results are shown in Table 2. The MIC of Lip 2 increased 8-fold in trypsin, 16-fold in chymotrypsin, and 2-fold in pepsin.

[0137] 2) Serum sensitivity

[0138] Same as step (4) 2) in Example 1;

[0139] The results are as follows Figure 6 The MIC of Lip 3 in both 10% serum and 10% FBS increased by 8 times.

[0140] 3) Salt ion sensitivity

[0141] Same as step (4) 3) in Example 1;

[0142] The results are as follows Figure 7 , the MIC changes of Lip 3 were less than 3 times in the environments of 150 mM NaCl and 1 mM MgCl2.

[0143] Example 4: Synthesis of C12-R2

[0144] (1) Synthesis of C12-R2

[0145] ① Activation and pretreatment of resin

[0146] Same as step (1) of Example 1;

[0147] ②Synthesis of Lip 2

[0148] 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 Fmoc protecting group had been removed. Fmoc-L-Trp(Boc)-OH (237 mg), HOBT (61 mg), HBTU (171 mg), and DIEA (0.15 ml) were added to 8 ml The product was dissolved and mixed in DMF, 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-L-Trp-resin. The method was the same as above, and the subsequent amino acids were condensed in sequence: Fmoc-L-Arg(pbf)-OH (292 mg), Fmoc-L-Arg(pbf)-OH (292 mg), wherein the amounts of HOBT, HBTU and DIEA were the same as above, to obtain Fmoc-(L-Arg)-(L-Arg)-(L-Trp)-resin, i.e., Fmoc-RRW-resin. The same operation was carried out to connect lauric acid (181 mg) through the condensation reaction, except that HBTU was replaced by HATU (171 mg) when connecting fatty acids. After the fatty acids were connected, no deprotection was required, and the peptide was cleaved directly after treatment with dichloromethane and methanol.

[0149] ③ Peptide cleavage

[0150] Same as step (1) of Example 1 (3);

[0151] ④Peptide purification

[0152] The same as step (1) of Example 1, C12-R2 was identified by mass spectrometry, and the molecular formula was C 35 H 58 N 10 O5, molecular weight 698.5Da, mass spectrum see Figure 4 The structural formula is as follows:

[0153]

[0154] (2) Study on the in vitro antibacterial activity of C12-R2

[0155] Same as step (2) in Example 1;

[0156] The results are shown in Table 1. C12-R2 has strong antibacterial activity against common Gram-positive experimental strains.

[0157] (3) Study on the hemolytic toxicity of C12-R2

[0158] Same as step (3) of Example 1;

[0159] The results are as follows Figure 5, C12-R2 showed hemolytic toxicity at 128 μmol / L, but its hemolytic concentration was much greater than the effective concentration for antibacterial activity.

[0160] (4) In vitro stability study of C12-R2

[0161] 1) Enzymatic stability

[0162] Same as step (4) 1) in Example 1;

[0163] The results are shown in Table 2. The MIC of C12-R2 increased more than 32-fold in trypsin and chymotrypsin, and increased 8-fold in pepsin.

[0164] 2) Serum sensitivity

[0165] Same as step (4) 2) in Example 1;

[0166] The results are as follows Figure 6 , the MIC of C12-R2 in 10% serum increased 8-fold, and the MIC in 10% FBS increased 4-fold.

[0167] 3) Salt ion sensitivity

[0168] Same as step (4) 3) in Example 1;

[0169] The results are as follows Figure 7 , the MIC changes of C12-R2 were less than 2-fold in the environments of 150 mM NaCl and 1 mM MgCl2.

Claims

1. An all-D-type ultra-short lipopeptide modified with N-terminal lauric acid and having high stability and anti-Gram-positive bacterial activity, characterized in that: The ultrashort lipopeptide is composed of two D-arginines (D-Arg) and one D-tryptophan (D-Trp), and the N-terminus is modified by lauric acid; its sequence is: 12 -rrw-NH2, labeled as Lip 1; its structural formula is:

2. Use of the all-D-type ultrashort lipopeptide modified with N-terminal lauric acid having high stability and anti-Gram-positive bacteria activity as claimed in claim 1 in the preparation of anti-Gram-positive bacteria drugs.

3. Use of the N-terminal lauric acid-modified all-D-type ultrashort lipopeptide with high stability and anti-Gram-positive bacteria activity as claimed in claim 2 in the preparation of anti-Gram-positive bacteria drugs, characterized in that: The anti-Gram-positive bacteria drug is an oral anti-Gram-positive bacteria drug.

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