A quaternary ammonium salt-modified antibacterial peptidomimetic structure and its preparation method and application

Abstract

The present invention provides a quaternary ammonium salt-modified antimicrobial peptidomimetic structure and a preparation method and application thereof, and belongs to the technical field of antimicrobial materials. The present invention conjugates a quaternary ammonium salt with cationicity and several corresponding polypeptide fragments to prepare a series of novel quaternary ammonium salt-modified antimicrobial peptidomimetic structures. Compared with monoquaternary ammonium salts, the antimicrobial performance of the obtained antimicrobial peptidomimetic is significantly improved, and it exhibits excellent antimicrobial activity against a variety of Gram-positive bacteria and Gram-negative bacteria including clinical drug-resistant bacteria. At the same time, it has good stability, low toxicity, simple preparation process, and broad-spectrum antimicrobial properties. The series of antimicrobial peptidomimetic provided by the present invention have good application prospects in the field of medical antimicrobial materials, especially for the inhibition of drug-resistant bacteria, and can be used as antimicrobial materials or as drugs for antibacterial infections.

Description

Technical Field

[0001] The invention belongs to the technical field of medical antibacterial materials, relates to a novel quaternary ammonium salt small molecule antibacterial agent derivative structure, and specifically relates to a quaternary ammonium salt modified antibacterial peptidomimetic structure and a preparation method and application thereof. Background Art

[0002] Pathogenic bacterial infection has become one of the major issues threatening global public health. According to reports, if no timely action is taken, the number of deaths caused by pathogenic bacterial infection worldwide may increase to 10 million per year by 2050, which will bring great challenges to human health. Therefore, materials with antibacterial and bactericidal functions are gaining more and more attention, and research on antimicrobial agents is being carried out continuously.

[0003] Since Fleming discovered the first antibiotic, penicillin, in 1928, antibiotics have been considered the most effective solution to combat biofilm-related bacterial infections. However, in recent years, the widespread use and abuse of antibiotics has led to serious bacterial resistance problems. The antibacterial mechanism of antibiotics is mainly aimed at specific targets of bacteria. This mechanism has its own inherent limitations. Microorganisms can develop resistance through various resistance mechanisms such as gene mutations. The widespread use and abuse of antibiotics will accelerate the development of microbial resistance and even acquire multi-drug resistance.

[0004] According to a report in 2015, 700,000 people die each year worldwide due to antimicrobial resistance, and this number is still rising. In the United States alone, multidrug-resistant Staphylococcus aureus (MRSA) causes more than 100,000 life-threatening infections each year, and in some European countries, the prevalence of MRSA in blood cultures has exceeded 50%. Therefore, exploring new and efficient strategies for treating bacterial infectious diseases and developing new antimicrobial agents to replace antibiotics have become important issues that need to be addressed urgently.

[0005] There are a large number of antimicrobial agents, and there are many varieties. There are huge differences in their sources, structures, antimicrobial mechanisms, and many other aspects. Generally speaking, antimicrobial agents are divided into the following categories: inorganic antimicrobial agents, organic antimicrobial agents, natural antimicrobial agents, and composite antimicrobial agents. The antimicrobial process of antimicrobial agents is a complex process involving multiple disciplines. The antimicrobial agents themselves have different structural characteristics and different bactericidal mechanisms. There are also many factors that affect the antimicrobial effect.

[0006] Quaternary ammonium salts are a class of widely used strong cationic surfactants with antibacterial properties. They are compounds formed by replacing all four hydrogen atoms in ammonium ions with hydrocarbon groups. The quaternary ammonium group is a cationic functional group with a positive charge, which can destroy the integrity of the bacterial cell membrane structure, causing the outflow of cytoplasm and other components in the bacterial cells, thereby causing cell death and achieving an antibacterial effect. As a commonly used organic small molecule antibacterial agent, quaternary ammonium salts have the advantages of fast sterilization speed and not easy to induce bacterial resistance.

[0007] According to the structural characteristics of the four hydrocarbon groups of quaternary ammonium salts and the amount of quaternary ammonium nitrogen, quaternary ammonium salts can be divided into the following three categories: monoquaternary ammonium salts, diquaternary ammonium salts, and polyquaternary ammonium salts. The antibacterial effect of quaternary ammonium salts is mainly affected by the length of the N-alkyl chain and the content of the quaternary ammonium salt. Increasing the length of the N-alkyl chain can increase the hydrophobic interaction with the phospholipid bilayer of the cell wall, thereby increasing the antibacterial activity of quaternary ammonium compounds. However, studies have shown that the effect of the length of the hydrophobic alkyl chain on antibacterial properties varies in different systems.

[0008] The length of the substituted alkyl chain of cationic antimicrobial agents is closely related to the antimicrobial activity. Generally speaking, the longer the substituted alkyl chain, the better its hydrophobicity, the more intense the hydrophobic reaction with the bacterial cell membrane, and the faster the bacteria are killed. However, studies have also found that not all antimicrobial agents conform to this rule. For some compounds, there is an optimal length range for the relationship between the substituted alkyl chain and the antimicrobial activity. When the length exceeds the maximum value of this range, the antimicrobial ability decreases. For example, Chen et al. [1] A macromolecular dendritic antibacterial agent reported had the highest antibacterial activity when the side chain length was 10 C alkyl chain, followed by 8 and 12 C alkyl chains, and the lowest antibacterial activity when the length was 14 and 16 C.

[0009] In recent years, there are many quaternary ammonium salt antibacterial agents on the market, such as dodecyl dimethyl benzyl ammonium chloride (Chlorhexidine). Generally speaking, long alkyl chain quaternary ammonium salts have lipophilic and hydrophobic long carbon chains and hydrophilic ammonium ions. After quaternary ammonium salts bind to the surface of bacteria, they change the permeability of the cell membrane, causing the bacteria to rupture and kill the bacteria. However, recent studies have shown that [2] It was found that with the extensive use of quaternary ammonium antimicrobial agents, bacteria's tolerance to monoquaternary ammonium salts continued to increase, resulting in a decrease in the product's bactericidal ability, limiting its use.

[0010] Diquaternary ammonium salts are also called Gemini quaternary ammonium salts, which have two N + In 1991, Menger et al. [3] The first synthesis of a rigid group connecting two N +The surfactant with an ionic head group is named Gemini. Its structure generally consists of three parts: the first is the hydrophobic group, which is usually a long-chain fatty alkane, located on both sides of the molecule. The two chains can be the same or different. At the same time, functional groups such as benzyl and ester groups can be introduced into the hydrophobic chain; the second is the hydrophilic group, which is usually two N + ions; the third part is the linking group, which is located in the middle of the hydrophilic group. The types of linking groups are also varied, which can be rigid structures or flexible structures. In addition to connecting two N + In addition to the role of the group, its nature and position also have a great influence on the physical and chemical properties of Gemini quaternary ammonium salts.

[0011] Studies have shown that diquaternary ammonium salts can interfere with the synthesis of nucleic acids, thereby further interfering with the synthesis of proteins. + With at least two long hydrophobic chains, the charge strength and molecular polarity of the quaternary ammonium salt are much greater than those of the monoquaternary ammonium salt, making it easier to adsorb on the surface of bacteria through electrostatic action. Multiple long hydrophobic chains are also more likely to embed into the bacteria and destroy their integrity. Therefore, the bactericidal performance of diquaternary ammonium salt is stronger than that of monoquaternary ammonium salt.

[0012] In 2013, Guo Shengnan et al. [4] The bactericidal activity of cationic Gemini amphiphilic molecules against Escherichia coli and Staphylococcus aureus was studied. The results showed that this type of compound had good bactericidal activity, with the bactericidal concentration range of 4-512 mg / mL. It was also found that changes in the length of the connecting chain did not lead to an increase in the corresponding antibacterial activity.

[0013] Dong Le et al. [5] A series of Gemini quaternary ammonium surfactants (mnm) were synthesized by the ring method. It was found that the linking group and hydrophobic group of Gemini quaternary ammonium salts have a great influence on their physicochemical properties.

[0014] Although there have been many studies on the antibacterial activity of small molecule quaternary ammonium salt antimicrobial agents, traditional small molecule quaternary ammonium salt antimicrobial agents have the disadvantages of poor stability, high volatility, difficulty in processing, penetration into human skin, and easy loss to cause secondary pollution, which also affect the performance of their antibacterial activity. On the other hand, quaternary ammonium salts have the problem of high cytotoxicity, and their cytotoxicity increases with the growth of the carbon chain. This disadvantage limits the application of quaternary ammonium salts in biology.

[0015] Studies have shown that the general rule of toxicity and antibacterial activity of small molecule quaternary ammonium salt antimicrobial agents with the change of quaternary ammonium salt structure is: the toxicity of the same quaternary ammonium salt with short alkyl chain is greater than that of the long alkyl chain; when the alkyl chain length is the same, the toxicity of the benzyl group is less than that of the methyl group; the toxicity of the monoalkyl group is less than that of the methyl group, and the toxicity of the monoalkyl group is greater than that of the dialkyl group. As the alkyl chain grows, the antibacterial ability increases; but when it reaches a certain length, the antibacterial ability decreases.

[0016] Although researchers have made some progress in discovering new antimicrobial agents, the continued increase in drug-resistant bacteria and the continued decline in the number of newly approved antibiotics make the search for new antimicrobial agents a pressing need in the field of medical and pharmaceutical research.

[0017] Therefore, how to design a series of new small molecule quaternary ammonium antimicrobial agents with high antibacterial efficiency and good biosafety to solve the problems of high cytotoxicity, difficult processing and poor stability of small molecule quaternary ammonium antimicrobial agents, and further improve the antibacterial activity and biocompatibility of quaternary ammonium salts, has become a technical problem that needs to be solved urgently.

[0018] [1]Chen CZ, Beck-Tan NC, Dhurjati P., et al. Quaternary ammonium functionalized poly(propylene imine) dendrimers as effective antimicrobials: Structure-activity studies[J]. Biomacromolecules, 2000, 1(3):473-480.

[0019] [2]ALEXANDRA MB, MARTA F G. Polymeric materials with antimicrobialactivity[J]. Progress In Polymer Science, 2012, 37(2): 281-339.

[0020] [3]Menger F M. Gemini-surfactants: synthesis and properties[J]. Journal of the American Chemical Society, 1991, 113(4):1451-1452.

[0021] [4] Guo Shengnan. Preparation and performance study of antibacterial polymers containing glycine ester-type Gemini surfactants [D]. Wuhan, Hubei University, 2013, 1-90.

[0022] [5] Dong Le, Ge Xiujuan, Gao Wenchao, Li Xing, Wei Wenlong, Chang Honghong. Synthesis and properties of mnm-type Gemini quaternary ammonium salt surfactants[J]. Surfactant Chemical Industry, 2018, 48(09): 495-499. Summary of the invention

[0023] The present invention is to solve the above technical problems, thereby providing a quaternary ammonium salt modified antimicrobial peptidomimetic structure and its preparation method and application. The technical purpose of the present invention is mainly to provide a series of quaternary ammonium salt modified antimicrobial peptidomimetic structures with high antimicrobial activity, good biocompatibility and safety, and its preparation method and application, to solve the problems of existing small molecule quaternary ammonium salt antimicrobial agents, such as high cytotoxicity, low safety, difficult processing, poor stability, and the antimicrobial activity and biocompatibility of the antimicrobial agents need to be further improved.

[0024] In order to achieve the above-mentioned purpose, the present invention adopts the following series of technical solutions.

[0025] In a first aspect, the present invention provides an antimicrobial peptidomimetic structure modified with a quaternary ammonium salt, wherein the antimicrobial peptidomimetic is obtained by conjugating various quaternary ammonium salts with different hydrophobic alkyl chain lengths with polypeptide fragments;

[0026] The quaternary ammonium salt has a structure shown in the following formula:

[0027]

[0028] Wherein, R1, R2 and R3 are independently selected from one of C1-C20 straight chain alkyl or branched chain alkyl, and at least one of R1, R2 and R3 is selected from C4-C20 straight chain alkyl or branched chain alkyl; R is halogen; A group is (CH2) a , a=1-6; the B group is CH2 or -NHCO-; the D group is NH2 or COOH; the m is an integer of 1-6; the n=1, 2 or 3, when n=2 or 3, the chain lengths of the A groups may be the same or different;

[0029] The polypeptide fragment has the following two structures:

[0030] (X-NH-CO-Z) b (Y) or (X-NH-CO-Z) b ;

[0031] Wherein, X, Y and Z represent amino acid residues, X is an L-tryptophan residue, a phenylalanine residue or an L-leucine residue, Z is an L-alanine residue or an L-lysine residue; b is an integer of 2 to 5; and Y is an L-tyrosine residue or a phosphorylated L-tyrosine residue.

[0032] Furthermore, the antimicrobial peptidomimetic of the present invention has a structure shown in the following formula <Ⅱ> or formula <Ⅲ>:

[0033]

[0034] Specifically, the antimicrobial peptidomimetic structures provided by the present invention include the following formulas: <xiii>Several options are shown:

[0035]

[0036]

[0037]

[0038]

[0039] Wherein, the R is selected from a C1-C20 straight-chain alkyl group or a branched-chain alkyl group.

[0040] Among them, it is more preferred that the antimicrobial peptidomimetic has the structure shown in Formula <Ⅳ>, Formula <VII>, Formula <IX> and Formula <XI>. The antimicrobial peptidomimetic with these structures has the best antibacterial effect.

[0041] It is confirmed by the examples of the present invention that the above-mentioned series of antimicrobial peptidomimetic structures obtained by the present invention have high antimicrobial activity and biocompatibility against a variety of clinical drug-resistant bacteria and sensitive bacteria including Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), and the antimicrobial peptidomimetic structures provided by the present invention are easy to process and prepare, not easy to volatilize, have good stability, low toxicity and good safety, which well solves the problems of poor stability, difficult processing, high toxicity and low antimicrobial activity of small molecule quaternary ammonium salt antimicrobial agents.

[0042] Compared with quaternary ammonium salts alone, the antibacterial properties of the series of antibacterial peptoids obtained by the present invention are significantly improved, and they exhibit excellent antibacterial activity against a variety of Gram-positive bacteria and Gram-negative bacteria including clinical drug-resistant bacteria. The specific antibacterial activity is shown in the examples.

[0043] Due to the different structural characteristics of antibacterial agents, the sterilization mechanisms they bring about are also different, and there are many factors that affect the antibacterial effect. The present invention studies the structure-performance relationship of the obtained antibacterial peptidomimetic through in vitro antibacterial experiments, hemolysis experiments and MTT experiments, obtains an antibacterial peptidomimetic structure with good potential application value, and studies its antibacterial mechanism against Staphylococcus aureus through sterilization kinetics, cell membrane depolarization experiments, inner membrane permeability experiments and scanning electron microscopy (SEM) observations. Bacterial resistance experiments show that a series of quaternary ammonium salt derivative compounds obtained by the present invention have good drug resistance.

[0044] Since antimicrobial peptides have always shown excellent and broad-spectrum antimicrobial activity, and are expected to solve the bacterial resistance problem currently faced by humans, the inventors have chosen to use a method of combining polypeptides with quaternary ammonium salts to obtain antimicrobial peptoids to solve the problem of quaternary ammonium salts. The inventors have conducted experimental studies on a series of polypeptide fragments, and found that the method of obtaining antimicrobial peptoids by conjugating polypeptides to quaternary ammonium salts is not able to easily solve the above-mentioned technical problems of the present invention. As shown in the comparative examples of the present invention, when various polypeptide fragments in the comparative examples are selected to prepare antimicrobial peptoids, the antimicrobial activity of quaternary ammonium salts cannot be significantly improved.

[0045] At the same time, antimicrobial peptides face the following problems in clinical application: (1) Antimicrobial peptides have toxic side effects on normal cells; most antimicrobial peptides achieve their antimicrobial function by destroying bacterial cell membranes. This physical destruction mode is not selective and will inevitably cause certain damage to normal mammalian cells; in addition, most antimicrobial peptides have broad-spectrum antimicrobial properties, and some normal and beneficial bacteria in the human body may be indiscriminately attacked, causing dysbiosis. Therefore, current research on antimicrobial peptides mainly focuses on improving their toxic side effects; (2) They have low metabolic stability in the body and are easily affected by metabolic factors. They are easily affected by various enzymes, pH, salt, serum and various ions, resulting in a significant reduction in antimicrobial activity or loss of antimicrobial activity; (3) The manufacturing cost is expensive and difficult to achieve industrialization.

[0046] In order to solve the problems existing in small molecule quaternary ammonium salt antibacterial agents and the above-mentioned problems existing in antimicrobial peptides, the present inventors have conducted a lot of research on polypeptide fragments combined with quaternary ammonium salts, and finally found that when the above-mentioned polypeptide fragments of the present invention are selected to construct antimicrobial peptoids, the MIC experimental results show that with the increase of alkyl chain length, the antibacterial activity of quaternary ammonium salts is significantly increased.

[0047] As shown in the examples of the present invention, the simple polypeptide fragments (WA)3, (FA)3 and (LA)3 did not show obvious antibacterial activity at the test concentration, while QA8C and (WK)3 showed weak antibacterial activity. When the polypeptide fragments of the present invention were selected and combined with quaternary ammonium salts, the antibacterial activity of the obtained antibacterial peptoids was significantly increased, indicating that there is a certain synergistic effect between the polypeptide fragments selected by the present invention and the quaternary ammonium salts, making it more conducive to exerting efficient antibacterial activity.

[0048] In a second aspect, the present invention provides a method for preparing the above-mentioned quaternary ammonium salt-modified antimicrobial peptidomimetic, which comprises first synthesizing the required quaternary ammonium salt structure, then obtaining the corresponding polypeptide fragment by solid phase synthesis, and conjugating the quaternary ammonium salt with the polypeptide by amidation reaction to obtain the quaternary ammonium salt-modified antimicrobial peptidomimetic.

[0049] Specifically, the preparation method of the quaternary ammonium salt comprises the following steps:

[0050] (1) mixing an N,N'-dimethyl substituted raw material having different hydrophobic alkyl chain lengths with an excess of a halogenated hydrocarbon, adding a catalyst to react at elevated temperature under the action of an organic solvent, and obtaining a quaternized product;

[0051] (2) reacting the obtained quaternary ammonium product with excess 1,3-propylenediamine, and purifying the product to obtain the quaternary ammonium salt.

[0052] Alternatively, the N,N'-dimethyl substituted raw material is mixed with an excess of halogenated hydrocarbon, and a catalyst is added under the action of an organic solvent to directly carry out a temperature-raising reaction to obtain the quaternary ammonium salt.

[0053] The above two methods can both prepare the quaternary ammonium salt of the present invention and be used for the subsequent synthesis of antimicrobial peptidomimetics.

[0054] Furthermore, the method for preparing the antimicrobial peptidomimetic of the present invention comprises: synthesizing a polypeptide fragment and conjugating the polypeptide with a quaternary ammonium salt under the action of a condensing agent.

[0055] Specifically, the method for preparing the antimicrobial peptidomimetic of the present invention comprises the following steps:

[0056] (1) Using the standard Fmoc protection strategy solid phase peptide synthesis method, 2-chlorotrityl chloride resin is used as a carrier, HBTU, HOBt or DIEA is used as a condensation reagent, and the Fmoc protection strategy solid phase peptide synthesis extending from the C-terminus to the N-terminus obtains a Boc-protected peptide fragment;

[0057] (2) Conjugation of quaternary ammonium salts with polypeptide fragments

[0058] The polypeptide synthesized in step (1) is dissolved in DCM or DMF, mixed with a quaternary ammonium salt, and a condensing agent DCC or NHS or TEA is added under ice bath, and the reaction is carried out at room temperature for 48 hours. The obtained product is washed and precipitated to obtain a light yellow solid, which is dissolved in DCM or TFA, the Boc protecting group is removed under ice bath conditions, precipitated with ice ether, and frozen to obtain a crude antibacterial peptidomimetic product.

[0059] Furthermore, the crude antimicrobial peptidomimetic product of the present invention is also subjected to the following purification treatment: using preparative high performance liquid chromatography and reverse phase preparative liquid column, the crude product is dissolved in water / acetonitrile, the mobile phase ratio is: 0.1% TFA+H2O / acetonitrile, gradient elution, the organic phase concentration is increased from 30% to 70% within 15 minutes, and the flow rate is 1min / mL.

[0060] The preparation method provided by the present invention can stably obtain the antibacterial polypeptide, and the preparation method is simple and the process is mature, which can well solve the problems of difficult processing, easy volatility and poor stability of small molecule quaternary ammonium salt antibacterial agents.

[0061] In a third aspect, the present invention provides an application of the above-mentioned quaternary ammonium salt-modified antimicrobial peptidomimetic, which is to use the antimicrobial peptidomimetic for preparing medical anti-infection materials, and to use the antimicrobial peptidomimetic for preparing drugs for bacterial infections to achieve antibacterial effects on common bacteria or drug-resistant bacteria.

[0062] The beneficial effects of the present invention are as follows:

[0063] (1) The present invention provides a series of quaternary ammonium salt-modified antimicrobial peptoids with high antibacterial activity, good biocompatibility and safety, and their preparation methods and applications. The preparation method is simple and well solves the problems of difficult processing, poor stability and high cytotoxicity of existing small molecule quaternary ammonium salt antibacterial agents.

[0064] (2) Compared with quaternary ammonium salts alone, the antibacterial properties of the series of antibacterial peptoids provided by the present invention are significantly improved, have broad-spectrum antibacterial properties, and will not produce drug resistance;

[0065] (3) The series of antibacterial peptidomimetics provided by the present invention have good antibacterial effects on a variety of clinical drug-resistant bacteria, and they show excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP). They also have good antibacterial activity against carbapenem-resistant Acinetobacter baumannii (CRAB), extended-spectrum β-lactamase Escherichia coli (ESBL E.coli), etc. It has great application prospects in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The results of the hemolytic activity test of the antimicrobial peptidomimetic;

[0067] Figure 2 The cytotoxicity test results of the antimicrobial peptidomimetics;

[0068] Figure 3 The bactericidal kinetics of antimicrobial peptidomimetics;

[0069] Figure 4 The results of the drug resistance test of the antimicrobial peptidomimetic. DETAILED DESCRIPTION

[0070] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not intended to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention content still belong to the protection scope of the present invention.

[0071] The present invention provides a series of quaternary ammonium salt structures and corresponding antimicrobial peptidomimetic structures, which are as follows:

[0072] The present invention provides a series of quaternary ammonium salt structures, the general structural formula of which is shown in formula <Ⅰ>:

[0073]

[0074] Wherein, R1, R2 and R3 are independently selected from one of C1-C20 straight chain alkyl or branched chain alkyl, and at least one of R1, R2 and R3 is selected from C4-C20 straight chain alkyl or branched chain alkyl; R group is halogen; A group is (CH2) a , a=1~6; B group is CH2 or -NH-CO-; m is an integer from 1 to 6, n=1, 2 or 3. When n=2 or 3, the chain lengths of A groups may be the same or different; D group is NH2 or COOH.

[0075] The present invention also provides a series of polypeptide fragment structures, which have two structural formulas as shown below:

[0076] (X-NH-CO-Z) b (Y) or (X-NH-CO-Z) b ;

[0077] The above structural formula is represented as follows: Some polypeptide fragments in the present invention may have a Y group, while other polypeptide fragments do not have a Y group, see the examples for details.

[0078] Wherein, X, Y and Z represent amino acid residues respectively, X is L-tryptophan residue, phenylalanine residue or L-leucine residue, Z is L-alanine residue or L-lysine residue; b is an integer of 2 to 5, and Y is L-tyrosine residue or phosphorylated tyrosine residue.

[0079] The antimicrobial peptidomimetic structure of the present invention is shown in the following formula <Ⅱ> or formula <Ⅲ>:

[0080]

[0081] The following examples list several typical antimicrobial peptidomimetic structures and preparation methods of the present invention.

[0082] In the following embodiments, the synthesis routes of Gemini quaternary ammonium salts with different hydrophobic alkyl chain lengths are shown in the following reaction formula (I):

[0083]

[0084] The synthetic route of aminobutyric acid-derived quaternary ammonium salt (QAs-COOH) is shown in the following reaction formula (II):

[0085]

[0086] The synthetic route of the quaternary ammonium salt modified antimicrobial peptoid is shown in the following reaction formula (III) or reaction formula (IV):

[0087]

[0088] In the above reaction formula, the black sphere represents: resin for polypeptide synthesis (2-chlorotrityl chloride resin is selected in the embodiment, other types of resins can also be used).

[0089] The main experimental raw materials involved in the embodiment are as follows:

[0090] 1-Bromobutane, 1-bromooctane, 1,3-propylenediamine, N,N-diisopropylethylamine (DIEA), N,N'-diisopropylcarbodiimide (DCC), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBT), amoxicillin, ciprofloxacin, vancomycin hydrochloride, and glucose were all from Aladdin Reagent Company, China; various amino acids and 2-chlorotrityl resin (CTC) were from Anpai Biotech Co., Ltd.; sodium heparin, Mueller-Hinton medium, LB broth medium, propidium iodide (PI), DiBAC4(3) cell membrane potential fluorescent probe, and 2,3,5-triphenyltetrazolium chloride (TTC) were all from Beijing Solebao Technology Co., Ltd.; and all standard strains were from Nanjing Lezhen Biological Co., Ltd.

[0091] Example 1

[0092] (I) Synthesis of Gemini quaternary ammonium salt

[0093] (1) Add N,N,N,N-tetramethyllysine ethyl ester to a three-necked flask, then add an excess of 1-bromobutane and an appropriate amount of isopropanol as a solvent, and then add a small amount of NaI as a catalyst, stir and reflux in an oil bath at 84°C, and monitor the progress of the reaction by TLC. After the reaction is completed, most of the solvent is removed by rotary evaporation to obtain a yellow viscous oil, and a petroleum ether / ether mixture is added for precipitation and washing. After repeated washing, the supernatant is discarded and evaporated to dryness to obtain a light yellow oily product, which is recorded as: LG4 (R = C4H9), and its structural formula is as follows:

[0094]

[0095] (2) The quaternized product LG4 prepared above was added to a three-necked flask, and an excess of 1,3-propylenediamine (10 times) was added under nitrogen protection. After all the 1,3-propylenediamine was dissolved, the mixture was stirred and refluxed in an oil bath at 70°C for reaction, and the degree of reaction was monitored by TLC. After the reaction was completed, most of the unreacted 1,3-propylenediamine was removed by rotary evaporation, and a small amount of ethyl acetate was added and precipitated and washed with a mixture of petroleum ether / ether. After repeated multiple times, the supernatant was poured out and evaporated to dryness to obtain the Gemini quaternary ammonium salt product, which was recorded as: GQA4C (R = C4H9), and its structural formula is as follows:

[0096]

[0097] The theoretical molecular value of GQA4C is: 532.45, and the actual value measured by mass spectrometry is: ((M 2+ -2Br) / 2z): 186.20.

[0098] (II) Synthesis of polypeptide fragments

[0099] The peptide sequence was synthesized using the standard Fmoc protection strategy solid phase peptide synthesis (SPPS) method, with 0.984 mmol / g 2-chlorotrityl chloride (2-CTC) resin as a carrier, HBTU, HOBt or DIEA as a condensation reagent, and an Fmoc protection strategy solid phase peptide synthesis method extending from the C-terminus to the N-terminus. The basic operation is as follows:

[0100] (1) Loading of the first amino acid: Take 1g of 2-CTC resin in a solid phase reactor, add 10mL DCM to swell for 30min, and wash three times with DMF, MeOH, and DCM. Weigh 4equiv Fmoc-AA-OH and 12equiv DIEA, dissolve them in 10mL DCM and add them to the reactor, and stir with nitrogen for 2h at room temperature. Remove excess raw materials and condensing agent by suction filtration, and wash three times with 15mL DCM. Add MeOH / DIEA / DCM (v / v / v, 80:15:5) to cap for 0.5-1h to block the unreacted active sites on the resin, and wash three times with 15mL DCM;

[0101] (2) Removal of Fmoc protecting group: Add 10 mL of deprotection reagent piperidine / DMF (v / v, 25:75), react at room temperature for 25 min, wash three times with 15 mL of DMF, filter and take a small amount of resin and add ninhydrin / ethanol for detection (110°C, 3 min). If the resin is blue, it means that the amino group is exposed and the next step can be carried out;

[0102] (3) Condensation of amino acids: Weigh 3 equiv of Fmoc-AA-OH, 3 equiv of HBTU or HOBt or DIEA, dissolve them in 20 mL of DCM and add them to the reactor. Stir under nitrogen for 2 h at room temperature. Remove excess raw materials and condensing agent by suction filtration, wash three times with 15 mL of DCM, take a small amount of resin and add ninhydrin / ethanol for detection (110°C, 3 min). If the resin is colorless, the reaction is complete and the next step can be performed. When condensing to the last amino acid, a Boc-protected amino acid is usually used;

[0103] (4) Peptide cleavage: After the last amino acid is condensed, wash with 10 mL of DMF, DCM and MeOH three times, filter and dry with nitrogen. Weigh the dried resin and place it in an eggplant-shaped bottle. Add cleavage solution TFA:H2O (v / v 98:2) at a ratio of 10 mL of cleavage solution per gram of resin under ice bath conditions. After reacting for 2 hours, collect the filtrate by suction and concentrate it by rotary evaporation at low temperature. Then add a small amount of DCM and rotary evaporation again to remove the residual TFA. Then add a large amount of ice ether for precipitation. Repeat this process many times to obtain the Boc-protected peptide fragment.

[0104] According to the above method, tryptophan and alanine are condensed to obtain a polypeptide fragment, which is denoted as (WA)3 and has the following structural formula:

[0105]

[0106] (III) Conjugation of Gemini quaternary ammonium salts with polypeptide fragments

[0107] The above synthesized polypeptide fragment (WA) 3 was dissolved in DCM / DMF, 1.1 equiv Gemini quaternary ammonium salt was added, condensation agent DCC / NHS / TEA was added under ice bath, and the reaction was carried out at room temperature for 48 hours. The reaction progress was detected by TLC. After the reaction was completed, the precipitate was removed by suction filtration, and the filtrate was washed three times with 1M dilute hydrochloric acid, saturated sodium chloride solution and deionized water respectively. The organic phase was dried over anhydrous sodium sulfate overnight, filtered and concentrated, and a large amount of ice ether was added for precipitation. The precipitate was centrifuged and washed again with ice ether, and this was repeated three times to obtain a light yellow powder solid. It was dissolved in DCM / TFA, the Boc protecting group was removed under ice bath conditions, and after 4 hours of reaction, a large amount of ice ether was added to precipitate after rotary evaporation concentration, and the precipitate was poured into a centrifuge tube for centrifugal precipitation, the supernatant was poured out, the precipitate was washed with ice ether, and centrifuged, and repeated three times. The precipitate was taken and frozen to obtain a crude antimicrobial peptidomimetic product. The crude product was purified by preparative high performance liquid chromatography and reverse phase preparative liquid column. The product was dissolved in water / acetonitrile. The mobile phase ratio was 0.1% TFA+H2O / acetonitrile. The gradient elution was performed. The organic phase concentration increased from 30% to 70% within 15 minutes. The flow rate was 1 min / mL.

[0108] The antimicrobial peptidomimetic was prepared according to the above method and was recorded as: (WA)3GQA4C, and its structural formula is as follows:

[0109]

[0110] The actual value of the antimicrobial peptidomimetic (WA) 3GQA4C is: 572.10 ([M2+] / 2Z), and the theoretical value is: 571.87 ([M2+] / 2Z).

[0111] Example 2

[0112] (I) Synthesis of Gemini quaternary ammonium salt

[0113] (1) Add N,N,N,N-tetramethyl lysine ethyl ester to a three-necked flask, then add an excess of 1-bromohexane and an appropriate amount of isopropanol as solvent, and then add a small amount of NaI as a catalyst, stir and reflux in an oil bath at 84°C, and monitor the progress of the reaction by TLC. After the reaction is completed, most of the solvent is removed by rotary evaporation to obtain a yellow viscous oil, and a petroleum ether / ether mixture is added for precipitation and washing. After repeated washing, the supernatant is discarded and evaporated to dryness to obtain a light yellow oily product, which is recorded as: LG6 (R = C8H 17 ), whose structural formula is as follows:

[0114]

[0115] (2) The quaternized product LG6 prepared above was added to a three-necked flask, and an excess of 1,3-propylenediamine (10 times) was added under nitrogen protection. After all the 1,3-propylenediamine was dissolved, the mixture was stirred and refluxed in an oil bath at 70°C for reaction, and the degree of reaction was monitored by TLC. After the reaction was completed, most of the unreacted 1,3-propylenediamine was removed by rotary evaporation, and a small amount of ethyl acetate was added and precipitated and washed with a mixture of petroleum ether / ether. After repeated multiple times, the supernatant was discarded and evaporated to dryness to obtain the Gemini quaternary ammonium salt product, which was recorded as: GQA6C (R = C6H 13 ), whose structural formula is as follows:

[0116]

[0117] The theoretical molecular value of GQA6C is: 588.75, and the actual value measured by mass spectrometry is: ((M 2+ -2Br) / 2z): 214.27.

[0118] (II) Synthesis of polypeptide fragments

[0119] The polypeptide fragment (WA) 3 was prepared according to the polypeptide sequence synthesis method of Example 1;

[0120] (III) Conjugation of Gemini quaternary ammonium salts with polypeptide fragments

[0121] The antimicrobial peptidomimetic was prepared according to the method of Example 1 and was recorded as: (WA)3GQA6C, and its structural formula is as follows:

[0122]

[0123] The actual value of antimicrobial peptide (WA) 3GQA6C is: 600.70 ([M 2+ ] / 2Z), the theoretical value is: 600.40([M 2+ ] / 2Z).

[0124] Example 3

[0125] (I) Synthesis of Gemini quaternary ammonium salt

[0126] (1) Add N,N,N,N-tetramethyl lysine ethyl ester to a three-necked flask, then add an excess of 1-bromooctane and an appropriate amount of isopropanol as solvent, and then add a small amount of NaI as a catalyst, stir and reflux in an oil bath at 84°C, and monitor the progress of the reaction by TLC. After the reaction is completed, most of the solvent is removed by rotary evaporation to obtain a yellow viscous oil, and a petroleum ether / ether mixture is added for precipitation and washing. After repeated washing, the supernatant is discarded and evaporated to dryness to obtain a light yellow oily product, which is recorded as: LG8 (R = C8H 17 ), whose structural formula is as follows:

[0127]

[0128] (2) The quaternized product LG8 prepared above was added to a three-necked flask, and an excess of 1,3-propylenediamine (10 times) was added under nitrogen protection. After all the 1,3-propylenediamine was dissolved, the mixture was stirred and refluxed in an oil bath at 70°C for reaction, and the degree of reaction was monitored by TLC. After the reaction was completed, most of the unreacted 1,3-propylenediamine was removed by rotary evaporation, and a small amount of ethyl acetate was added and precipitated and washed with a mixture of petroleum ether / ether. After repeated multiple times, the supernatant was discarded and evaporated to dryness to obtain the Gemini quaternary ammonium salt product, which was recorded as: GQA8C (R = C8H 17 ), whose structural formula is as follows:

[0129]

[0130] The theoretical molecular value of GQA8C is: 644.65, and the actual value measured by mass spectrometry is: ((M 2+ -2Br) / 2z): 242.27.

[0131] (II) Synthesis of polypeptide fragments

[0132] The polypeptide fragment (WA) 3 was prepared according to the polypeptide sequence synthesis method of Example 1;

[0133] (III) Conjugation of Gemini quaternary ammonium salts with polypeptide fragments

[0134] The antimicrobial peptidomimetic was prepared according to the method of Example 1, and was recorded as: (WA)3GQA8C, and its structural formula is as follows:

[0135]

[0136] The actual value of the antimicrobial peptidomimetic (WA) 3GQA8C is: 427.70 ([M2++H] / 3Z), and the theoretical value is: 427.40 ([M2++H] / 3Z).

[0137] Example 4

[0138] (i) preparing Gemini quaternary ammonium salt LG8 according to the method of Example 2;

[0139] (ii) According to the method of Example 1, phenylalanine and alanine were condensed to obtain a polypeptide fragment, denoted as: (FA)3, whose structural formula is as follows:

[0140]

[0141] (III) According to the method of Example 1, the Gemini quaternary ammonium salt LG8 was conjugated with the polypeptide fragment (FA) 3 to obtain an antimicrobial peptoid, which is denoted as: (FA) 3GQA8C, and its structural formula is as follows:

[0142]

[0143] The actual value of the antimicrobial peptidomimetic (FA) 3GQA8C is: 569.65 ([M2+] / 2Z), and the theoretical value is: 569.41 ([M2+] / 2Z).

[0144] Example 5

[0145] (i) preparing Gemini quaternary ammonium salt LG8 according to the method of Example 3;

[0146] (ii) According to the method of Example 1, leucine and alanine were condensed to obtain a polypeptide fragment, denoted as: (LA)3, whose structural formula is as follows:

[0147]

[0148] (III) Conjugation of Gemini quaternary ammonium salts with polypeptide fragments

[0149] The antimicrobial peptidomimetic was prepared according to the method of Example 1, and was recorded as: (LA)3GQA8C, and its structural formula is as follows:

[0150]

[0151] The actual value of the antimicrobial peptidomimetic (LA) 3GQA8C is: 518.70 ([M2+] / 2Z), and the theoretical value is: 518.44 ([M2+] / 2Z).

[0152] Example 6

[0153] The quaternary ammonium salt and the polypeptide fragment were synthesized according to the method of Example 1, and the quaternary ammonium salt and the tyrosine polypeptide fragment were conjugated to obtain an antimicrobial peptidomimetic, which was recorded as: (WK)3YQA8C, and its structural formula is as follows:

[0154]

[0155] Example 7

[0156] The quaternary ammonium salt and the polypeptide fragment were synthesized according to the method of Example 1, and the quaternary ammonium salt and the phosphorylated tyrosine polypeptide fragment were conjugated to obtain an antimicrobial peptidomimetic, which was recorded as: (WK)3YpQA8C, and its structural formula is as follows:

[0157]

[0158] Example 8

[0159] The quaternary ammonium salt and the polypeptide fragment were synthesized according to the method of Example 1, and the quaternary ammonium salt and the tyrosine polypeptide fragment were conjugated to obtain an antimicrobial peptidomimetic, which was recorded as: (WA)3YGQA8C, and its structural formula is as follows:

[0160]

[0161] Example 9

[0162] The quaternary ammonium salt and the polypeptide fragment were synthesized according to the method of Example 1, and the quaternary ammonium salt phosphorylated tyrosine polypeptide fragment was conjugated to obtain an antimicrobial peptidomimetic, which was recorded as: (WA)3YpGQA8C, and its structural formula is as follows:

[0163]

[0164] Comparative Example 1

[0165] Referring to the method of Example 1, WKWKWK was selected as the polypeptide fragment and combined with quaternary ammonium ions to prepare an antimicrobial peptoid, which was recorded as: WKWKWK-GQA8C, and its structural formula is as follows:

[0166]

[0167] Comparative Example 2

[0168] Referring to the method of Example 1, RKVRGGG was selected as the polypeptide fragment, and combined with quaternary ammonium ions to prepare an antimicrobial peptoid, which was recorded as: RKVRGGG-QA8C, and its structural formula is as follows:

[0169]

[0170] Comparative Example 3

[0171] Referring to the method of Example 1, RWKGGG was selected as the polypeptide fragment, and combined with quaternary ammonium ions to prepare an antimicrobial peptoid, which was recorded as: RWKGGG-QA8C, and its structural formula is as follows:

[0172]

[0173] Comparative Example 4

[0174] Referring to the method of Example 1, RKVRGGG was selected as the polypeptide fragment, and combined with quaternary ammonium ions to prepare an antimicrobial peptoid, which was recorded as: RKVRGGG-QA8C, and its structural formula is as follows:

[0175]

[0176] Comparative Example 5

[0177] Referring to the method of Example 1, RWKGGG was selected as the polypeptide fragment and combined with the quaternary ammonium salt to prepare an antimicrobial peptoid, which was recorded as: RWKGGG-QA8C, and its structural formula is as follows:

[0178]

[0179] Comparative Example 6

[0180] Referring to the method of Example 1, WGWGWG was selected as the polypeptide fragment and combined with a quaternary ammonium salt to prepare an antimicrobial peptoid, which was recorded as: WGWGWG-QA8C, and its structural formula is as follows:

[0181]

[0182] Experimental Example 1

[0183] The antibacterial activity and hemolytic activity of the antimicrobial peptoids obtained in the examples and comparative examples were tested as follows:

[0184] 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) test of antimicrobial peptidomimetics

[0185] The MIC value of the sample was tested by the microbroth dilution method. The test strains were Gram-positive bacteria (such as Staphylococcus aureus S.aureus, ATCC6538) and Gram-negative bacteria (such as Escherichia coli E.coli, ATCC25922). The specific strains are shown in Table 1-5. The stored working strain was inoculated into LB broth medium, cultured at 37°C overnight, collected by centrifugation and redispersed into MH broth medium, and the bacterial solution was diluted to 10 6 CFU / mL and set aside. Take a 96-well plate, add 100 μL of sterile liquid culture medium to each well, then add 100 μL of sample solution, dilute stepwise by two-fold dilution method, and obtain antimicrobial peptide concentration range of 500 to 0.244 μg / mL, and finally add 100 μL of diluted bacterial solution (total volume 200 μL). Culture in a 37°C incubator for 16-20 hours, add only MH culture medium as a negative control, add MH culture medium and bacterial solution (without sample) as a positive control, make three to five parallel samples for each sample, and repeat three times at different times. Observe with the naked eye to find the first well that does not become turbid as the minimum inhibitory concentration, add 5 μL of 0.5% triphenyltetrazolium chloride (TTC) and place at 37°C for 15 minutes, and observe that the first well that does not turn red is the minimum inhibitory concentration.

[0186] 2. Evaluation of sterilization time dynamics

[0187] The stock working strain was inoculated into LB broth medium, cultured at 37°C overnight, collected by centrifugation and redispersed into MH broth medium. The bacterial solution was diluted to 10 6 CFU / mL, set aside, dilute the sample to 4MIC with MH medium, add the diluted bacterial solution and mix quickly, place in a 37℃ incubator, and take samples at a series of time points at 0h, 10min, 30min, 1h, 2h, 4h and 8h, take 100μL of samples each time for the experimental group, apply the original solution or dilute the plate, apply the blank control dilute plate, and count the bacteria by plate spreading method. After culturing at 37℃ for 24 hours, count the number of colonies. Convert the number of colonies in the control group to 100%, convert the number of colonies in the experimental group to the survival percentage, and draw the time-killing curve.

[0188] 3. Hemolytic activity test of antimicrobial peptidomimetics

[0189] Take 10mL of fresh blood from rats and place it in a beaker soaked with a small amount of sodium heparin. Use a glass rod to stir continuously to remove fibrinogen, blood clots, etc. Add 0.9% saline for washing, centrifuge at 2500rpm for 5min, discard the supernatant, and repeat the washing until the supernatant no longer appears red after centrifugation. Take 2mL of red blood cells from the lower layer and add 48mL of saline to dilute it into a 4% red blood cell suspension for later use. Dilute the sample to a series of concentrations. Add 500μL of 4% red blood cell suspension and 500μL of sample solution to a 48-well plate, and make three replicates for each sample. The negative control group contains 500μL of 0.9% saline and 500μL of 4% red blood cell suspension, and the positive control group contains 500μL of 0.5% TritanX-100 and 500μL of red blood cell suspension. All samples were incubated in an incubator at 37°C for 3h. Then all samples were centrifuged at 4000rpm for 5min. Take 100 μL of the supernatant and place it in another 96-well plate. Use an ELISA reader to measure its absorbance at 576 nm, and use the following formula to calculate the hemolysis rate of the sample on red blood cells:

[0190]

[0191] 4. Antimicrobial peptidomimetic resistance experiment

[0192] The strains tested in the drug resistance experiment were Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The stored working strains were inoculated into LB broth medium, cultured at 37°C overnight, collected by centrifugation and redispersed into HEPES working solution. The bacterial solution was diluted to 10 7 CFU / mL and set aside. After testing the MIC of the sample according to the method above, take the bacterial solution at 0.5×MIC value and dilute it 100 times and add it to MH medium as the working strain, and then continue to test the sample MIC value, repeating 14 generations. Plot the number of generations of resistance test and the increase multiple of MIC to obtain the resistance curve.

[0193] 5. Cytotoxicity of antimicrobial peptidomimetics

[0194] The toxicity of antimicrobial peptides to cells was detected by MTT assay. 5 The cells were plated at a density of 1000 μg / well in a 96-well culture plate, incubated in a constant temperature incubator under 5% CO2 and 37°C for 24 hours, and then the sample solution dissolved in DMEM medium (containing serum) was added. After continued cultivation for 24 hours and 72 hours, 20 μL MTT solution (5 mg / mL) was added to each well, and the culture was continued for 4 hours. The supernatant was discarded, and 150 μL DMSO was added to each well. After 15 minutes, the absorbance at 495 nm was measured with an enzyme reader.

[0195] (II) Experimental results

[0196] 1. Antimicrobial activity of antimicrobial peptoids

[0197] (1) The antibacterial activity test results of the antimicrobial peptoids, quaternary ammonium salts and polypeptide fragments obtained in the examples are shown in Table 1 below, and the antibacterial activity test results of the antimicrobial peptoids obtained in the control examples are shown in Table 2 below:

[0198] Table 1

[0199]

[0200]

[0201] Table 2

[0202]

[0203] As shown in Tables 1 and 2, the in vitro antibacterial activity of the synthesized antimicrobial peptidomimetics against Gram-positive bacteria Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli) was evaluated. Commercially available antibiotics amoxicillin, vancomycin, and ciprofloxacin were used as standard drugs in the evaluation of antibacterial activity. The minimum inhibitory concentration (MIC) is an important indicator for measuring the antibacterial activity of antibacterial substances. It is the lowest drug concentration that can inhibit bacterial growth after 18-24 hours of in vitro culture. MIC is used as an indicator to characterize in vitro antibacterial activity.

[0204] As can be seen from Table 1, for the Gram-positive bacteria S. aureus, the MIC values ​​of the antimicrobial peptoids (WA)3GQA4C, (WA)3GQA6C, (WA)3GQA8C, (FA)3GQA8C, (LA)3GQA8C, (WK)3QA8C, (WK)3YQA8C, (WK)3YQA8C, (WK)3YQA8C, (WK)3YpQA8C, (WA)3YQA8C and (WA)3YpQA8C were between 1-31.25 μg / mL. For the Gram-negative bacteria E. coli, (WA)3GQA4C showed no antibacterial activity at the highest concentration tested, and the MIC values ​​of (WA)3GQA6C, (WA)3GQA8C, (FA)3GQA8C, (LA)3GQA8C, (WK)3QA8C, (WK)3YQA8C, (WK)3YQA8C, (WK)3YpQA8C, (WA)3YQA8C and (WA)3YpQA8C were between 7.8-62.5 μg / mL. Compared with the single GQAs and the simple polypeptide fragments (WA)3, (FA)3, (LA)3, (WK)3, (WA)3Y, (WA)3Yp, (WK)3Y and (WK)3Yp, the antimicrobial peptidomimetics obtained after conjugation of polypeptide fragments have significantly improved antimicrobial activity against both Gram-positive and Gram-negative bacteria. This may be because in addition to the alkyl chain of the quaternary ammonium salt that can penetrate the bacterial plasma membrane and cause certain membrane damage, the hydrophobic part of the polypeptide fragment can also be inserted into the lipid bilayer of the bacterial cell membrane, causing it to produce a stronger membrane damage effect. The effect on Gram-positive bacteria is better than that on Gram-negative bacteria, which is caused by the different structures of their cell walls and cell membranes. Gram-positive bacteria are surrounded by only one layer of cell membrane, while Gram-negative bacteria have a double-layer membrane structure, which is divided into an outer membrane and an inner membrane. In addition, Gram-negative bacteria usually contain more neutral lipid phosphatidylethanolamine (PE) than Gram-positive bacteria, while Gram-positive bacteria contain more anionic phosphatidylglycerol (PG). Therefore, the negative charge of the positive bacterial plasma membrane is higher than that of negative bacteria, and the interaction with cationic antimicrobial peptides is stronger.

[0205] As can be seen from Table 2, the antibacterial activity of the antimicrobial peptoids obtained by combining the polypeptide fragments in Comparative Examples 1-6 with quaternary ammonium salts is generally low, and the antibacterial activity of the quaternary ammonium salts is not significantly improved, and some even reduce the antibacterial activity of the antimicrobial peptoids.

[0206] Comparing the minimum inhibitory concentrations of (WA)3GQA4C, (WA)3GQA6C, and (WA)3GQA8C, it can be seen that with the increase in alkyl chain length, the antibacterial properties of (WA)3GQA8C against S. aureus and E. coli are significantly better than those of (WA)3GQA4C. This may be because (WA)3GQA8C has a stronger overall hydrophobicity, which enhances the interaction with the hydrophobic region of the lipid bilayer to disrupt the bilayer structure and enhance the permeability of the membrane, and there is a certain positive correlation between hydrophobicity and antibacterial properties within a certain range. On the other hand, compared with the short alkyl chain of (WA)3GQA4C, the long alkyl chain of (WA)3GQA8C is conducive to piercing the bacterial cell wall and destroying the cell membrane, causing the contents to leak out, and ultimately leading to bacterial death.

[0207] In order to study the effect of different hydrophobic amino acids on the antimicrobial activity of antimicrobial peptidomimetics, (FA)3GQA8C and (LA)3GQA8C were synthesized by replacing tryptophan (W) in (WA)3GQA8C with phenylalanine (F) and leucine (L) under the condition of ensuring the same alkyl chain length of quaternary ammonium salt, and the ClogP values ​​of the compounds were calculated by ChemBioDraw software. The ClogP value represents the oil-water partition coefficient of organic compounds, also known as the hydrophobic constant. The larger the value, the more hydrophobic it is. The ClogP values ​​of (WA)3GQA8C, (FA)3GQA8C and (LA)3GQA8C are 0.125, 0.155 and 0.272, respectively, that is, under the condition of ensuring the same alkyl chain length of quaternary ammonium salt, the hydrophobicity of the three increases in turn. However, as can be seen from Table 1, their antibacterial activities decrease in turn, which indicates that hydrophobicity is not the main factor affecting the antibacterial activity at this time. From the MIC results, we can see that when the hydrophobic aromatic tryptophan is replaced by the hydrophobic fatty leucine, the MIC values ​​for positive bacteria and negative bacteria increase by 10 times and 5 times, respectively, and the antibacterial performance decreases significantly. This may be because compared with the aliphatic hydrophobic side chain, the aromatic hydrophobic side chain can use the larger aromatic ring to pierce the bacterial wall (membrane) of the bacteria, causing the cytoplasm to flow out and causing the bacterial death.

[0208] When tryptophan was replaced with phenylalanine, which is also a hydrophobic aromatic amino acid, the antibacterial activity was also reduced, as shown by a 5-fold and 2.6-fold increase in the MIC values ​​for positive and negative bacteria, respectively. This may be because there is a negatively charged π-electron cloud above and below the indole ring of the tryptophan side chain, which allows it to interact with both the positively charged amino acid side chain and aminocholine in the lipid bilayer. Therefore, it is speculated that in the presence of tryptophan, the positively charged quaternary ammonium salt can be protected in a highly hydrophobic environment, making it easier to penetrate the lipid bilayer. In addition, the indole side chain of tryptophan can destroy the hydrophobicity of the lipid acyl chain, resulting in the further insertion of the antimicrobial peptoid into the lipid bilayer. Due to the cation-π electron cloud effect, the peptide-membrane interaction is promoted, improving the antimicrobial activity of the antimicrobial peptoid.

[0209] (2) Antibacterial activity testing of various antimicrobial peptoids against various strains

[0210] In order to verify whether the antimicrobial peptoids obtained in the present invention have broad-spectrum antimicrobial activity, antimicrobial activity tests of various strains were carried out, and the selected strains are shown in Tables 3 to 5 below.

[0211] The test results for sensitive bacteria are shown in Tables 3 and 4 below:

[0212] Table 3

[0213]

[0214] Table 4

[0215]

[0216]

[0217] The test results for multiple drug-resistant bacteria in clinical practice are shown in Table 5 below:

[0218] Table 5

[0219]

[0220]

[0221] 2. Hemolytic activity of antimicrobial peptidomimetics

[0222] As an antimicrobial agent, hemolytic activity is an important indicator for evaluating its biocompatibility. Antimicrobial agents with high hemolytic activity will have limited biological applications. The half hemolytic value (HC50) is calculated by regression software SPSS. Figure 1 shown.

[0223] from Figure 1 It can be seen that (WA)3GQA4C (HC50>500μg / mL) showed lower hemolytic activity than (WA)3GQA8C (HC50: ~450μg / mL), indicating that increasing hydrophobicity will increase hemolytic activity while improving antibacterial activity. (FA)3GQA8C (HC50: ~337μg / mL) has a slightly higher hydrophobicity than (WA)3GQA8C, so its hemolysis is also slightly higher than (FA)3GQA8C. However, although (LA)3GQA8C has the highest hydrophobicity among the three, its HC50 value is >500μg / mL, which may be due to the fact that the interaction between the side chain aliphatic hydrophobic amino acids and the cell membrane is weaker than that between the aromatic hydrophobic amino acids and the cell membrane. The hemolysis rate of all the above antimicrobial peptides is less than 5% when the concentration does not exceed 62.5μg / mL. The results of erythrocyte hemolysis showed that some antimicrobial peptoids had low toxicity to mammalian erythrocytes. Considering the antimicrobial properties and hemolytic activity, it was believed that the (WA)3GQA8C antimicrobial peptoid had potential application value.

[0224] 3. Cytotoxicity of antimicrobial peptidomimetics

[0225] In order to characterize the biosafety of the obtained antimicrobial peptoids, L929 (mouse fibroblasts) were selected to test the cytotoxicity of the obtained antimicrobial peptoids. Figure 2 As shown in the figure, when the concentration was 100 μg / mL, the cell survival rates of (WA)3GQA4C, (WA)3GQA8C, (FA)3GQA8C and (LA)3GQA8C co-cultured cells were all over 80%, with no obvious cytotoxicity. When the concentration reached 1 mg / mL, the cell survival rates of (WA)3GQA4C and (WA)3GQA8C were 95.7% and 79.4%, respectively, with no obvious cytotoxicity. However, the cell survival rates of (FA)3GQA8C and (LA)3GQA8C were only 53.5% and 65.9%, showing certain toxicity. The MIC values ​​of (WA)3GQA4C, (WA)3GQA8C, (FA)3GQA8C and (LA)3GQA8C against S. aureus did not exceed 31.25 μg / mL, and the MIC values ​​against E. coli did not exceed 62.5 μg / mL. Therefore, within the MIC value range of the antimicrobial peptidomimetics, there was no obvious cytotoxicity.

[0226] 4. Bactericidal time dynamics of antimicrobial peptoids

[0227] The bactericidal ability and speed of (WA)3GQA8C against S. aureus and E. coli at different concentrations were measured. The concentrations of (WA)3GQA8C were 1×MIC, 2×MIC, 4×MIC and 8×MIC. The bactericidal time kinetic results showed that the bactericidal activity of the antimicrobial peptoids was concentration-dependent. The higher the concentration, the stronger and faster the bactericidal effect. Figure 3 As shown. (WA)3GQA8C showed rapid bactericidal performance against Staphylococcus aureus. When the concentration was 4×MIC and 8×MIC, it could significantly reduce the number of S. aureus (killing more than 99% of the bacteria) within a short time of 10 minutes, and more than 99.9% of S. aureus could be killed within 1 hour. At the concentrations of 1×MIC and 2×MIC, all bacteria could be killed within 4 hours. The bactericidal ability of the antimicrobial peptidomimetic against E. coli was slightly inferior to that of S. aureus. When the concentration was 4×MIC and 8×MIC, about 90% of the bacteria could be killed within a short time of 10 minutes, and the bactericidal rate could reach 99.9% after 2 hours. At the concentrations of 1×MIC and 2×MIC, more than 99% of the bacteria could be killed after 4 hours, and all bacteria could be killed after 8 hours at all tested concentrations. Rapid bactericidal performance is crucial to prevent the spread of bacterial infections, shorten treatment time, and reduce the probability of drug resistance. On the other hand, the rapid bactericidal performance reveals that the antibacterial mechanism of this type of antimicrobial peptoids may not be similar to antibiotics acting on a specific target (such as protein), but acting on the cell membrane and achieving the bactericidal effect by destroying the integrity of the cell membrane.

[0228] 5. Resistance of antimicrobial peptidomimetics

[0229] The tendency of bacteria to develop resistance can be assessed by continuously exposing them to sublethal concentrations of antimicrobial agents. (WA)3GQA8C was used as a representative for resistance studies. S. aureus and E. coli were serially subcultured in the presence of sublethal concentrations of (WA)3GQA8C and control antibiotics vancomycin and ciprofloxacin, and new MIC values ​​were determined each time. The definition of antimicrobial resistance is that the MIC value increases by more than 4 times during serial subculture. Figure 4 It can be seen that when the continuous passage reached the 12th generation, (WA)3GQA8C began to show a tendency to drug resistance, and its MIC value became 4 times the original. Traditionally, vancomycin is used as the "last line of defense" for positive bacteria treatment. It began to show drug resistance when S. aureus was passaged to the 7th generation. For E. coli, (WA)3GQA8C began to show drug resistance at the 10th generation, and the final MIC value became 8 times the original. The above results show that for the treatment of S. aureus, (WA)3GQA8C may have certain advantages over conventional antimicrobial drugs and is not easy to induce bacterial resistance.< / xiii>

Claims

1. A quaternary ammonium salt-modified antimicrobial peptidomimetic, characterized in that: The antimicrobial peptoid is obtained by conjugating a quaternary ammonium salt with a polypeptide fragment; The antimicrobial peptidomimetic has a structure as shown in any one of the following formulas <Ⅰ> to <Ⅸ>:

2. A method for preparing the quaternary ammonium salt-modified antimicrobial peptidomimetic according to claim 1, characterized in that: The method comprises subjecting the quaternary ammonium salt to an amidation reaction with a polypeptide fragment to obtain the antimicrobial peptoid modified with the quaternary ammonium salt.

3. The preparation method according to claim 2, characterized in that: The preparation method of the quaternary ammonium salt comprises: mixing an N,N'-dimethyl substituted raw material with an excess of halogenated hydrocarbon, adding a catalyst to carry out a temperature-raising reaction under the action of an organic solvent to obtain a quaternary ammonium product; and then reacting the obtained quaternary ammonium product with an excess of 1,3-propylenediamine, and purifying the mixture to obtain the quaternary ammonium salt.

4. The preparation method according to claim 2, characterized in that: The preparation method of the quaternary ammonium salt comprises: mixing an N,N'-dimethyl substituted raw material with an excess of halogenated hydrocarbon, adding a catalyst under the action of an organic solvent, and directly performing a temperature-raising reaction to obtain the quaternary ammonium salt.

5. The preparation method according to claim 2, characterized in that: The preparation method of the antimicrobial peptoid comprises: synthesizing polypeptide fragments and conjugating the polypeptide fragments with quaternary ammonium salts under the action of a condensing agent.

6. The preparation method according to claim 5, characterized in that: The preparation method of the antimicrobial peptoid comprises the following steps: (1) Using the standard Fmoc protection strategy solid phase peptide synthesis method, 2-chlorotrityl chloride resin is used as a carrier, HBTU, HOBt or DIEA is used as a condensation reagent, and the Fmoc protection strategy solid phase peptide synthesis extending from the C-terminus to the N-terminus obtains a Boc protected peptide fragment; (2) Conjugation of quaternary ammonium salts with polypeptide fragments The polypeptide fragment synthesized in step (1) is dissolved in DCM or DMF, mixed with a quaternary ammonium salt, and a condensing agent DCC or a condensing agent NHS or a condensing agent TEA is added under ice bath, and the reaction is carried out at room temperature for 48 hours. The obtained product is washed and precipitated to obtain a light yellow solid, which is dissolved in DCM or TFA, the Boc protecting group is removed under ice bath conditions, ice ether is precipitated, and the antibacterial peptoid is obtained.

7. The preparation method according to claim 6, characterized in that: The antimicrobial peptidomimetic further comprises the following treatment: dissolving the product obtained in step (2) in water or acetonitrile, and purifying the product by using preparative high performance liquid chromatography and reverse phase preparative liquid column, with the mobile phase ratio of 0.1% TFA+H2O / acetonitrile, gradient elution, the organic phase concentration increasing from 30% to 70% within 15 minutes, and the flow rate of 1 min / mL.

8. Use of the quaternary ammonium salt-modified antimicrobial peptidomimetic as claimed in claim 1 or the quaternary ammonium salt-modified antimicrobial peptidomimetic prepared by the method according to any one of claims 2 to 7 in preparing medical antimicrobial materials for the surface of medical catheters or devices, characterized in that: When the antimicrobial peptidomimetic is a compound represented by formula <Ⅰ>, the targeted strains are S. aureus, B. subtilis, E. faecalis, P. mirabilis, clinical isolate MRSA; When the antimicrobial peptidomimetic is a compound represented by formula <VI> to <IX>, the targeted strains are E. coli and S. aureus; When the antimicrobial peptidomimetic is a compound represented by formula <Ⅱ> to formula <Ⅴ>, the targeted strains are E. coli, S. aureus, B. subtilis, E. faecalis, P. aeruginosa, P. mirabilis, clinical isolate MRSA, clinical isolate A. baumannii, clinical isolate VRE, clinical isolate PRSP, and clinical isolate S. pneumoniae.

9. Use of the quaternary ammonium salt-modified antimicrobial peptidomimetic as claimed in claim 1 or the quaternary ammonium salt-modified antimicrobial peptidomimetic prepared by the method according to any one of claims 2 to 7 in the preparation of drugs for bacterial infection, characterized in that: When the antimicrobial peptidomimetic is a compound represented by formula <Ⅰ>, the targeted strains are S. aureus, B. subtilis, E. faecalis, P. mirabilis, clinical isolate MRSA; When the antimicrobial peptidomimetic is a compound represented by formula <VI> to <IX>, the targeted strains are E. coli and S. aureus; When the antimicrobial peptidomimetic is a compound represented by formula <Ⅱ> to formula <Ⅴ>, the targeted strains are E. coli, S. aureus, B. subtilis, E. faecalis, P. aeruginosa, P. mirabilis, clinical isolate MRSA, clinical isolate A. baumannii, clinical isolate VRE, clinical isolate PRSP, and clinical isolate S. pneumoniae.

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