CN115286692A - Antibacterial peptide mimetic structure modified by quaternary ammonium salt, preparation method and application thereof - Google Patents

Abstract

The invention provides a quaternary ammonium salt-modified antibacterial peptidomimetic structure and a preparation method and application thereof, belonging to the technical field of antibacterial materials. In the present invention, cationic quaternary ammonium salts are conjugated with several corresponding polypeptide fragments to prepare a series of novel quaternary ammonium salt-modified antibacterial peptidomimetic structures. Compared with monoquaternary ammonium salts, the antibacterial properties of the obtained antibacterial peptoids have been significantly improved, and they have excellent antibacterial activities against a variety of Gram-positive 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 antibacterial properties. The series of antibacterial peptidomimetics provided by the invention have good application prospects in the field of medical antibacterial materials, especially for the inhibition of drug-resistant bacteria, and can be used as antibacterial materials and antibacterial infection drugs.

Description

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

technical field

The invention belongs to the technical field of medical antibacterial materials, and relates to a novel structure of a quaternary ammonium salt small-molecule antibacterial agent derivative, in particular to a quaternary ammonium salt-modified antibacterial peptidomimetic structure and its preparation method and application.

Background technique

Pathogenic bacterial infection has become one of the major issues threatening global public health. According to the report, if no timely action is taken, by 2050, the number of deaths caused by pathogenic bacterial infection in the world may increase to 10 million each year, This will bring great challenges to human health. Therefore, materials with antibacterial and bactericidal functions have attracted more and more attention, and research on antibacterial agents has been continuously carried out.

Since Fleming discovered the first antibiotic, penicillin, in 1928, antibiotics have been considered the most effective solution to combat bacterial infections associated with biofilms. 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 the specific target of bacteria. This mechanism has its own inherent limitations. Microorganisms can develop drug resistance through multiple drug resistance mechanisms such as gene mutations. The widespread use and abuse of antibiotics will accelerate microbial resistance. and even acquire multidrug resistance.

According to a 2015 report, 700,000 people worldwide die each year from antimicrobial resistance, and the number is rising. Multidrug-resistant Staphylococcus aureus (MRSA) is responsible for more than 100,000 life-threatening infections each year in the United States alone, and the prevalence of MRSA in blood cultures has exceeded 50% in some European countries. Therefore, exploring effective new strategies for treating bacterial infectious diseases and developing new antibacterial agents to replace antibiotics have become important problems that need to be solved urgently.

There are a large number and varieties of antibacterial agents, and there are huge differences in many aspects such as their sources, structures, and antibacterial mechanisms. Generally speaking, antibacterial agents are divided into the following categories: inorganic antibacterial agents, organic antibacterial agents, natural antibacterial agents and compound antibacterial agents. The antibacterial process of antibacterial agents is a complex process involving multiple disciplines. Antibacterial agents have different structural characteristics and bactericidal mechanisms. There are also various factors that affect the antibacterial effect.

Quaternary ammonium salts are a class of strong cationic surfactants that are widely used and have antibacterial properties. They are compounds in which four hydrogen atoms in the ammonium ion are replaced by hydrocarbon groups. The quaternary ammonium group is a cationic functional group with a positive charge. It can destroy the integrity of the bacterial cell membrane structure, causing the cytoplasm and other components in the bacterial cell to flow out, thereby causing cell death and achieving antibacterial effect. Quaternary ammonium salt, as a commonly used organic small molecule antibacterial agent, has the advantages of fast bactericidal speed and not easy to induce bacterial resistance.

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: single quaternary ammonium salts, double quaternary 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 quaternary ammonium salts. Increasing the length of the N-alkyl chain can improve the hydrophobic interaction with the phospholipid bilayer of the cell wall, thereby increasing the amount of quaternary ammonium compounds. antibacterial activity. However, studies have shown that in different systems, the length of the hydrophobic alkyl chain has different effects on the antibacterial properties.

The length of the substituted alkyl chain of cationic antibacterial agents is closely related to the antibacterial 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 bacterial cell will be killed. However, studies have also found that not all antibacterial agents conform to this rule. Some compounds have an optimal length range for the relationship between substituted alkyl chains and antibacterial activity. When the length exceeds the maximum value of this range, the antibacterial ability will decrease instead. As reported by Chen et al. ^[1] , a macromolecular dendritic antibacterial agent, when the side chain length is 10 C alkyl chains, the antibacterial activity is the highest, followed by 8 and 12 C alkyl chains, and 14 and 16 C lengths, the antibacterial activity was the lowest.

In recent years, there are many kinds of quaternary ammonium antibacterial agents on the market, such as dodecyldimethylbenzyl ammonium chloride (geeramine) and so on. 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 and cause the bacteria to rupture, thereby killing the bacteria. However, a recent study ^[2] found that with the extensive use of quaternary ammonium salt antibacterial agents, the bacteria's tolerance to monoquaternary ammonium salts has been continuously enhanced, resulting in a decline in the bactericidal ability of the product, which limits its use.

Double quaternary ammonium salt is also called Gemini (Jimi Qi) type quaternary ammonium salt, that is, a quaternary ammonium salt with two N ⁺ . In 1991, Menger et al. ^[3] took the lead in synthesizing a surfactant with two N ⁺ ion head groups connected by a rigid group, named Gemini. Its structure generally consists of three parts: one is a hydrophobic group, usually a long-chain aliphatic alkane, which is located on both sides of the molecule. The two chains can be the same or different. At the same time, benzyl and ester groups can also be introduced into the hydrophobic chain. and other functional groups; the second is the hydrophilic group, usually two N ⁺ ions; the third part is the linking group, located in the middle of the hydrophilic group, and the types of linking groups are also various, which can be rigid structures, It can also be a flexible structure. In addition to the function of connecting two N ⁺ groups, the nature and position of the linking group also have a great influence on the physical and chemical properties of the Gemini quaternary ammonium salt.

Studies have shown that diquaternary ammonium salts can interfere with the synthesis of nucleic acids, thereby further interfering with protein synthesis, and diquaternary ammonium salts have two N ⁺ and at least two long hydrophobic chains, and their molecular charge strength and molecular polarity are far greater Compared with single quaternary ammonium salt, it is easier to adsorb on the surface of bacteria through electrostatic interaction, and multiple hydrophobic long chains are also easier to embed inside bacteria, destroying their integrity. Therefore, the bactericidal performance of the double quaternary ammonium salt is stronger than that of the single quaternary ammonium salt.

In 2013, Guo Shengnan et al ^[4] studied the bactericidal activity of cationic Gemini amphiphilic molecules against Escherichia coli and Staphylococcus aureus. It was found that the change of linking chain length did not lead to corresponding enhancement of antibacterial activity.

Dong Le et al. ^[5] synthesized a series of Gemini-type quaternary ammonium salt surfactants (mnm) by the ring method, and found that the linking group and hydrophobic group of Gemini-type quaternary ammonium salt have a great influence on their physical and chemical properties.

Although there have been many studies on the antibacterial activity of small-molecule quaternary ammonium salt antibacterial agents, traditional small-molecule quaternary ammonium salt antibacterial agents have poor stability, are volatile, are not easy to process, will penetrate into human skin, and are easily lost. Disadvantages such as secondary pollution have also affected the performance of its antibacterial activity. On the other hand, quaternary ammonium salts have the problem of high cytotoxicity, and their cytotoxicity increases with the growth of carbon chains, which limits the application of quaternary ammonium salts in biology.

Existing studies have shown that the toxicity and antibacterial properties of small-molecule quaternary ammonium salt antibacterial agents vary with the general law of quaternary ammonium salt structure: the toxicity of similar quaternary ammonium salts with short alkyl chains is greater than that with long alkyl chains; When the base chain length is the same, the toxicity of the benzyl group is less than that of the methyl group; the toxicity of the mono-alkyl group is less than that of the methyl group, and the toxicity of the mono-alkyl group is greater than that of the di-alkyl group. With the growth of the alkyl chain, the antibacterial ability is enhanced; but to a certain length, the antibacterial ability decreases instead.

Although researchers have made some progress in discovering new antibacterial agents, the continuous increase of drug-resistant bacteria and the continuous decline in the number of newly approved antibiotics make the search for new antibacterial agents an urgent need in the field of medical and drug research.

Therefore, how to design a series of novel small-molecule quaternary ammonium salt antibacterial agents with high-efficiency antibacterial and good biological safety, to solve the problems of large cytotoxicity, difficult processing, poor stability, and further Improving the antibacterial activity and biocompatibility of quaternary ammonium salts has become an urgent technical problem to be solved.

[1] Chen C.Z., Beck-Tan N.C., Dhurjati P., et al. Quaternary ammoniumfunctionalized poly(propylene imine) dendrimers as effective antimicrobials: Structure-activity studies[J]. Biomacromolecules, 2000, 1(3):473-480 .

[2] ALEXANDRA M B, MARTA F G. Polymeric materials with antimicrobial activity [J]. Progress In Polymer Science, 2012, 37(2): 281-339.

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

[4] Guo Shengnan. Preparation and performance research of antibacterial polymer containing glycine ester type Gemini surfactant [D]. Wuhan, Hubei University, 2013, 1-90.

[5] Dong Le, Ge Xiujuan, Gao Wenchao, Li Xing, Wei Wenlong, Chang Honghong. Synthesis and performance research of m-n-m type Gemini quaternary ammonium salt surfactant [J]. Daily Chemical Industry, 2018, 48(09): 495-499.

Contents of the invention

The present invention aims to solve the above technical problems, thereby providing a quaternary ammonium salt-modified antibacterial 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 antibacterial peptidomimetic structures with high-efficiency antibacterial activity and good biocompatibility and safety, as well as their preparation methods and applications, so as to solve the problem of existing small-molecule quaternary ammonium salts. Antibacterial agents have the problems of high cytotoxicity, low safety, difficult processing, and poor stability, and the antibacterial activity and biocompatibility of antibacterial agents need to be further improved.

In order to achieve the above object, the present invention adopts the following series of technical solutions.

In the first aspect, the present invention provides a quaternary ammonium salt-modified antibacterial peptoid structure, and the antibacterial peptoid is obtained by conjugating various quaternary ammonium salts with different hydrophobic alkyl chain lengths to polypeptide fragments;

The quaternary ammonium salt has a structure shown in the following formula <I>:

Wherein, the R ₁ , R ₂ and R ₃ are independently selected from one of C1-C20 straight-chain alkyl or branched-chain alkyl, and at least one of R ₁ , R ₂ and R ₃ is selected from C4 ~C20 straight-chain or branched-chain alkyl; the R is halogen; the A group is (CH ₂ ) _a , a=1~6; the B group is CH ₂ or -NHCO-; The D group is NH ₂ or COOH; the m is an integer of 1 to 6; the n=1, 2 or 3, when n=2 or 3, the chain length of the A group can be the same or can be different;

The polypeptide fragment has the following two structures:

(X-NH-CO-Z) _b (Y) or (X-NH-CO-Z) _b ;

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

Further, the antibacterial peptidomimetic of the present invention has the structure shown in the following formula <II> or formula <III>:

Specifically, the structure of the antibacterial peptidomimetic provided by the present invention includes several schemes as shown in the following formula <IV> to formula <XIII>:

Wherein, the R is selected from C1-C20 straight chain alkyl or branched chain alkyl.

Among them, more preferably, the antibacterial peptidomimetic has structures as shown in formula <IV>, formula <VII>, formula <IX> and formula <XI>. Antibacterial peptidomimetics having these structures have the best antibacterial effect.

Confirmed by the examples of the present invention, the above-mentioned series of antibacterial peptidomimetic structures obtained by the present invention are effective against clinical multiple drug-resistant bacteria and sensitive bacteria including Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli). Both have high-efficiency antibacterial activity and biocompatibility, and the antibacterial peptidomimetic structure provided by the invention is easy to process and prepare, is not volatile, has good stability, low toxicity, and good safety, which is a good solution to the problem of small molecule quaternary ammonium salt antibacterial agents. The problems of poor stability, difficult processing, high toxicity and low antibacterial activity exist.

Compared with a single quaternary ammonium salt, the antibacterial performance of a series of antibacterial peptidomimetics obtained by the present invention has been significantly improved, and it is effective against a variety of Gram-positive bacteria and Gram-negative bacteria including clinical drug-resistant bacteria. Show excellent antibacterial activity, specific antibacterial activity sees embodiment.

Due to the different structural characteristics of antibacterial agents, the bactericidal mechanisms brought about are also different, and there are many factors affecting the antibacterial effect. The present invention has studied the structure-performance relationship of the obtained antibacterial peptidomimetic through in vitro antibacterial experiments, hemolysis experiments and MTT experiments. Obtained an antibacterial peptidomimetic structure with good potential application value, and studied its antibacterial mechanism against Staphylococcus aureus through bactericidal kinetics, cell membrane depolarization experiments, inner membrane permeability experiments, and scanning electron microscope (SEM) observations , Bacterial drug resistance experiments show that a series of quaternary ammonium salt derivative compounds obtained in the present invention have good drug resistance.

Since antimicrobial peptides have always exhibited excellent and broad-spectrum antibacterial activity, it is hopeful to solve the problem of bacterial drug resistance that humans are currently facing. The problem with ammonium salts. The inventors conducted experimental research on a series of polypeptide fragments, and found that the method of obtaining antibacterial peptidomimetics by conjugating polypeptides with quaternary ammonium salts cannot 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 antibacterial peptoids, the antibacterial activity against quaternary ammonium salts cannot be significantly improved.

At the same time, antimicrobial peptides face the following problems in clinical application: (1) antimicrobial peptides have toxic and side effects on normal cells; most antimicrobial peptides achieve antibacterial function by destroying bacterial cell membranes, and this physical destruction mode is not selective. It will inevitably cause some damage to normal mammalian cells; in addition, most antimicrobial peptides have broad-spectrum antibacterial properties, and some normal and beneficial flora in the human body may be attacked indiscriminately, resulting in flora imbalance. (2) Low metabolic stability in the body, easily affected by metabolic factors, easily affected by various enzymes, pH, salt, serum and various ions, resulting in significantly reduced antibacterial activity or Loss of antibacterial activity; (3) expensive manufacturing costs, difficult to achieve industrialization.

In order to solve the existing problems of small molecule quaternary ammonium salt antibacterial agents and solve the above-mentioned problems of antimicrobial peptides, the inventors have conducted a lot of research on polypeptide fragments combined with quaternary ammonium salts, and finally found that when the above-mentioned polypeptides of the present invention are selected When the antibacterial peptidomimetic was constructed from the fragment, the MIC test results showed that the antibacterial activity of the quaternary ammonium salt was significantly increased with the increase of the alkyl chain length.

As shown in the examples of the present invention, for simple polypeptide fragments (WA) 3, (FA) 3 and (LA) 3, no obvious antibacterial activity was shown at the test concentration, while QA8C and (WK) 3 showed weak Antibacterial activity, and when the polypeptide fragment of the present invention is combined with the quaternary ammonium salt, the antibacterial activity of the obtained antibacterial peptidomimetic increases significantly, indicating that there is a certain synergy between the polypeptide fragment selected in the present invention and the quaternary ammonium salt, making it more conducive to exerting high-efficiency antibacterial activity.

In the second aspect, the present invention provides a method for preparing the above-mentioned quaternary ammonium salt-modified antibacterial peptidomimetic, which is to first synthesize the required quaternary ammonium salt structure, and then obtain the corresponding polypeptide fragment by solid-phase synthesis, and utilize amidation reaction The quaternary ammonium salt is conjugated with the polypeptide to obtain the antibacterial peptoid modified by the quaternary ammonium salt.

Concrete, the preparation method of described quaternary ammonium salt comprises the following steps:

(1) Mix N,N'-dimethyl substituent raw materials with different hydrophobic alkyl chain lengths with excess halogenated hydrocarbons, and add a catalyst under the action of an organic solvent to perform a temperature-raising reaction to obtain a quaternized product;

(2) reacting the obtained quaternized ammonium product with excess 1,3-propanediamine, and purifying to obtain the quaternary ammonium salt.

Alternatively, the quaternary ammonium salt is obtained by mixing N,N'-dimethyl substituent raw materials with excess halogenated hydrocarbons, adding a catalyst under the action of an organic solvent, and directly performing a temperature-raising reaction.

Both of the above two methods can prepare the quaternary ammonium salt described in the present invention and be used for the subsequent synthesis of antibacterial peptoids.

Further, the preparation method of the antibacterial peptidomimetic of the present invention includes: synthesizing polypeptide fragments and conjugating the polypeptide with a quaternary ammonium salt under the action of a condensing agent.

Specifically, the preparation method of the antibacterial peptidomimetic of the present invention comprises the following steps:

(1) Adopt the standard Fmoc protection strategy solid-phase peptide synthesis method, use 2-chlorotrityl chloride resin as the carrier, HBTU, HOBt or DIEA as the condensation reagent, and extend the Fmoc protection strategy solid phase from the C-terminus to the N-terminus Polypeptide synthesis to obtain Boc-protected polypeptide fragments;

(2) Conjugation of quaternary ammonium salts and polypeptide fragments

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

Further, the crude antibacterial peptidomimetic product of the present invention is also purified as follows: using preparative high-performance liquid chromatography and reverse-phase preparative liquid phase column, the crude product is dissolved in water/acetonitrile, mobile phase ratio: 0.1% TFA +H ₂ O/acetonitrile, gradient elution, the concentration of the organic phase increased from 30% to 70% within 15 min, and the flow rate was 1 min/mL.

The above preparation method provided by the present invention can stably obtain the antibacterial polypeptide. The preparation method is simple and the process is mature, and can well solve the problems of small molecule quaternary ammonium salt antibacterial agents that are not easy to process, easy to volatilize, and have poor stability.

In a third aspect, the present invention provides the application of the above-mentioned quaternary ammonium salt-modified antibacterial peptidomimetic, which is to use the antibacterial peptidomimetic to prepare medical anti-infection materials, and to use the antibacterial peptidomimetic to prepare drugs against bacterial infections, To achieve antibacterial against common bacteria or drug-resistant bacteria.

The beneficial effects of the present invention are as follows:

(1) The present invention provides a series of quaternary ammonium salt-modified antibacterial peptidomimetics with high antibacterial activity, good biocompatibility and safety and its preparation method and application. Molecular quaternary ammonium antibacterial agents have the problems of difficult processing, poor stability and high cytotoxicity;

(2) Compared with a single quaternary ammonium salt, a series of antibacterial peptoids provided by the present invention have significantly improved antibacterial properties, have broad-spectrum antibacterial properties, and will not produce drug resistance;

(3) A series of antibacterial peptidomimetics provided by the present invention have good antibacterial effects on a variety of clinical drug-resistant bacteria. Enterococcus (Vancomycin-Resistant Enterococcus, VRE) and penicillin-resistant Streptococcus pneumoniae (Penicillin resistant Streptococcus pneumoniae, PRSP) showed excellent antibacterial activity. It is also effective against carbapenem-resistant Acinetobacter baumannii (CRAB), Extended-Spectrum β-Lactamases Escherichia coli (ESBL E.coli), etc. Good antibacterial activity. It has great application prospects in the field of biomedicine.

Description of drawings

Fig. 1 is the hemolytic activity test result of antibacterial peptidomimetic;

Fig. 2 is the cytotoxicity test result of antibacterial peptidomimetic;

Fig. 3 is the bactericidal kinetics of antibacterial peptidomimetic;

Figure 4 is the results of drug resistance testing of antimicrobial peptoids.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be specifically described below in conjunction with examples. It should be pointed out that the following examples 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 content of the invention still belong to the protection scope of the present invention.

The present invention provides a series of quaternary ammonium salt structures and corresponding antibacterial peptidomimetic structures, specifically as follows:

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

Among them, R ₁ , R ₂ and R ₃ are independently selected from one of C1-C20 straight-chain alkyl or branched-chain alkyl, and at least one of R ₁ , R ₂ and R ₃ is selected from C4-C20 straight chain alkyl or branched chain alkyl; R group is halogen; A group is (CH ₂ ) _a , a=1~6; B group is CH ₂ or -NH-CO-; m is 1~ An integer of 6, n=1, 2 or 3, when n=2 or 3, the chain lengths of the A groups can be the same or different; the D groups are NH ₂ or COOH.

The present invention also provides a series of polypeptide fragment structures, which have the following two structural formulas:

(X-NH-CO-Z) _b (Y) or (X-NH-CO-Z) _b ;

The above structural formula is expressed as: 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.

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

The structure of the antibacterial peptidomimetic in the present invention is shown in the following formula <II> or formula <III>:

The following examples list several typical antibacterial peptidomimetic structures and their preparation methods in the present invention.

In the following examples, the synthetic route of Gemini quaternary ammonium salts with different hydrophobic alkyl chain lengths is shown in the following reaction formula (1):

The synthetic route of aminobutyric acid derivative quaternary ammonium salt (QAs-COOH) is shown in following reaction formula (two):

The synthetic route of the quaternary ammonium salt modified antibacterial peptidomimetic is shown in the following reaction formula (3) or reaction formula (4):

In the above reaction formula, the black ball represents: resin for polypeptide synthesis (2-chlorotrityl chloride resin is selected in the embodiment, other resins are also acceptable).

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

1-bromobutane, 1-bromooctane, 1,3-propanediamine, N,N-diisopropylethylamine (DIEA), N,N'-diisopropylcarbodiethylene Amine (DCC), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBT), amoxicillin , ciprofloxacin, vancomycin hydrochloride, and glucose were all from China Aladdin Reagent Company; various amino acids and 2-chlorotrityl resin (CTC) were from Anpaibo Biotechnology Company; heparin sodium, Mueller-Hinton culture Base, LB broth medium, propidium iodide (PI), DiBAC4(3) cell membrane potential fluorescent probe, 2,3,5-triphenyl tetrazolium chloride (TTC) were all from Beijing Suo Lai Bao Technology Co., Ltd.; all standard strains were from Nanjing Lediagnostic Biotechnology Company.

Example 1

(1) Synthesis of Gemini quaternary ammonium salt

(1) Add N,N,N,N-tetramethyllysine ethyl ester into the three-necked flask, then add excess 1-bromo-n-butane and appropriate amount of isopropanol as solvent, then add a small amount of NaI as Catalyst, stirring and reflux reaction under the condition of 84°C oil bath, TLC monitors the progress of the reaction. After the reaction was completed, most of the solvent was removed by rotary evaporation to obtain a yellow viscous oil, which was precipitated and washed by adding petroleum ether/diethyl ether mixture. After repeated washings, the supernatant was discarded, and after evaporation to dryness, a light yellow oily product was obtained, which was recorded as: LG4 (R=C ₄ H ₉ ), its structural formula is as follows:

(2) Add the quaternized ammonium product LG4 prepared above into a three-necked flask, add excess 1,3-propanediamine (10 times) under nitrogen protection conditions, dissolve all of them, and stir and reflux in an oil bath at 70°C The reaction was monitored by TLC. After the reaction is completed, remove most of the unreacted 1,3-propylenediamine by rotary evaporation, add a small amount of ethyl acetate and precipitate and wash with petroleum ether/ether mixture, pour off the supernatant after repeated several times, and evaporate to dryness to obtain Gemini quarter The ammonium salt product is denoted as: GQA4C (R=C ₄ H ₉ ), and its structural formula is as follows:

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

(2) Synthesis of polypeptide fragments

The synthesis of the peptide sequence adopts the standard Fmoc protection strategy solid-phase peptide synthesis method (SPPS), with 0.984mmol/g 2-chlorotrityl chloride (2-CTC) resin as the carrier, HBTU, HOBt or DIEA as the condensation reagent , the Fmoc protection strategy solid-phase peptide synthesis method extended from the C-terminus to the N-terminus, its basic operation is as follows:

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

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

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

(4) Polypeptide cleavage: After the last amino acid condensation is completed, wash with 10 mL DMF, DCM and MeOH three times respectively, and dry with nitrogen after suction filtration. The dried resin was weighed, placed in an eggplant-shaped bottle, and the lysate TFA:H ₂ O (v/v 98:2) was added at a ratio of 10 mL of lysate per gram of resin in an ice bath. After reacting for 2 hours, the filtrate was collected by suction filtration and concentrated by rotary evaporation at low temperature, then a small amount of DCM was added to rotary evaporation again to take away the residual TFA, and then a large amount of glacial ether was added for precipitation. After repeated several times, the Boc protected polypeptide fragment was obtained.

According to the above method, tryptophan and alanine are condensed to obtain polypeptide fragments, which are denoted as (WA)3, and its structural formula is as follows:

(3) Conjugation of Gemini quaternary ammonium salt to polypeptide fragments

Dissolve the above synthesized polypeptide fragment (WA) 3 in DCM/DMF, add 1.1 equiv Gemini quaternary ammonium salt, add condensing agent DCC/NHS/TEA under ice bath, react at room temperature for 48 hours, and detect the progress of the reaction 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, and the organic phase was dried overnight with anhydrous sodium sulfate. After filtration, the filtrate was concentrated, and a large amount of glacial ether was added for precipitation. The precipitate was collected by centrifugation and washed with glacial diethyl ether again, and this was repeated three times to obtain a light yellow powder solid. Dissolve it in DCM/TFA, remove the Boc protecting group under ice bath conditions, react for 4 hours, add a large amount of glacial ether to precipitate, pour it into a centrifuge tube, and centrifuge to precipitate, pour out the supernatant, The precipitate was washed with glacial ether, centrifuged and repeated three times. Take the precipitate and freeze it to get the crude product of antibacterial peptidomimetic. The crude product is purified by preparative high performance liquid chromatography and reversed phase preparative liquid column. The product is dissolved in water/acetonitrile, mobile phase ratio: 0.1% TFA+H ₂ O/ Acetonitrile, gradient elution, the concentration of the organic phase increased from 30% to 70% within 15 minutes, and the flow rate was 1min/mL.

According to the above-mentioned method, the antibacterial peptidomimetic is prepared, which is denoted as: (WA)3GQA4C, and its structural formula is as follows:

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

Example 2

(1) Synthesis of Gemini quaternary ammonium salt

(1) Add N,N,N,N-tetramethyllysine ethyl ester into the three-necked flask, then add excess 1-bromo-n-hexane and appropriate amount of isopropanol as solvent, and then add a small amount of NaI as catalyst , stirred and refluxed in an oil bath at 84°C, and monitored the progress of the reaction by TLC. After the reaction was completed, most of the solvent was removed by rotary evaporation to obtain a yellow viscous oil, which was precipitated and washed by adding petroleum ether/diethyl ether mixture. After repeated washings, the supernatant was discarded, and after evaporation to dryness, a light yellow oily product was obtained, which was recorded as: LG6 (R=C ₈ H ₁₇ ), its structural formula is as follows:

(2) Add the quaternized ammonium product LG6 prepared above into a three-neck flask, add excess 1,3-propanediamine (10 times) under nitrogen protection, dissolve all of it, and stir and reflux in an oil bath at 70°C The reaction was monitored by TLC. After the reaction is completed, remove most of the unreacted 1,3-propylenediamine by rotary evaporation, add a small amount of ethyl acetate and precipitate and wash with petroleum ether/ether mixture, pour off the supernatant after repeated several times, and evaporate to dryness to obtain Gemini quarter The ammonium salt product is denoted as: GQA6C (R=C ₆ H ₁₃ ), and its structural formula is as follows:

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

(2) Synthesis of polypeptide fragments

The polypeptide fragment (WA) 3 was prepared according to the polypeptide sequence synthesis method in Example 1;

(3) Conjugation of Gemini quaternary ammonium salt to polypeptide fragments

According to the method of Example 1, the antibacterial peptidomimetic is prepared, which is denoted as: (WA)3GQA6C, and its structural formula is as follows:

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

Example 3

(1) Synthesis of Gemini quaternary ammonium salt

(1) Add N,N,N,N-tetramethyllysine ethyl ester into the three-necked flask, then add excess 1-bromo-n-octane and appropriate amount of isopropanol as solvent, and then add a small amount of NaI as Catalyst, stirring and reflux reaction under the condition of 84°C oil bath, TLC monitors the progress of the reaction. After the reaction was completed, most of the solvent was removed by rotary evaporation to obtain a yellow viscous oil, which was precipitated and washed by adding petroleum ether/diethyl ether mixture. After repeated washings, the supernatant was discarded, and after evaporation to dryness, a light yellow oily product was obtained, which was recorded as: LG8 (R=C ₈ H ₁₇ ), its structural formula is as follows:

(2) Add the quaternized ammonium product LG8 prepared above into a three-neck flask, add excess 1,3-propanediamine (10 times) under nitrogen protection conditions, dissolve it completely, and stir and reflux in an oil bath at 70°C The reaction was monitored by TLC. After the reaction is completed, remove most of the unreacted 1,3-propylenediamine by rotary evaporation, add a small amount of ethyl acetate and precipitate and wash with petroleum ether/ether mixture, pour off the supernatant after repeated several times, and evaporate to dryness to obtain Gemini quarter The ammonium salt product is denoted as: GQA8C (R=C ₈ H ₁₇ ), and its structural formula is as follows:

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

(2) Synthesis of polypeptide fragments

The polypeptide fragment (WA) 3 was prepared according to the polypeptide sequence synthesis method in Example 1;

(3) Conjugation of Gemini quaternary ammonium salt to polypeptide fragments

According to the method of Example 1, the antibacterial peptidomimetic is prepared, which is denoted as: (WA)3GQA8C, and its structural formula is as follows:

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

Example 4

(1) prepare Gemini quaternary ammonium salt LG8 according to the method of embodiment 2;

(2) According to the method of Example 1, condensing phenylalanine and alanine to obtain polypeptide fragment fragments, denoted as: (FA) 3, its structural formula is as follows:

(3) According to the method of Example 1, the Gemini quaternary ammonium salt LG8 is conjugated with the polypeptide fragment (FA) 3 to obtain an antibacterial peptidomimetic, which is denoted as: (FA) 3GQA8C, and its structural formula is as follows:

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

Example 5

(1) prepare Gemini quaternary ammonium salt LG8 according to the method of embodiment 3;

(2) According to the method of Example 1, the polypeptide fragment fragment is obtained by condensing leucine and alanine, which is denoted as: (LA) 3, and its structural formula is as follows:

(3) Conjugation of Gemini quaternary ammonium salt to polypeptide fragments

According to the method of Example 1, the antibacterial peptidomimetic is prepared, which is denoted as: (LA)3GQA8C, and its structural formula is as follows:

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

Example 6

According to the method of Example 1, the quaternary ammonium salt and the polypeptide fragment were synthesized, and the quaternary ammonium salt and the tyrosine polypeptide fragment were conjugated to obtain an antibacterial peptoid, which was recorded as: (WK)3YQA8C, and its structural formula was as follows:

Example 7

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 antibacterial peptidomimetic, denoted as: (WK)3YpQA8C, and its structural formula is as follows:

Example 8

According to the method of Example 1, the quaternary ammonium salt and the polypeptide fragment were synthesized, and the quaternary ammonium salt and the tyrosine polypeptide fragment were conjugated to obtain an antibacterial peptoid, which was recorded as: (WA)3YGQA8C, and its structural formula was as follows:

Example 9

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 antibacterial peptidomimetic, denoted as: (WA)3YpGQA8C, and its structural formula is as follows:

Comparative example 1

With reference to the method of Example 1, WKWKWK was selected as the polypeptide fragment, combined with quaternary ammonium salt ions, to prepare an antibacterial peptidomimetic, denoted as: WKWKWK-GQA8C, and its structural formula is as follows:

Comparative example 2

Referring to the method of Example 1, RKVRGGG was selected as the polypeptide fragment, and combined with quaternary ammonium salt ions to prepare an antibacterial peptidomimetic, which was recorded as: RKVRGGG-QA8C, and its structural formula was as follows:

Comparative example 3

Referring to the method in Example 1, RWKGGG was selected as the polypeptide fragment, combined with quaternary ammonium salt ions, to prepare an antibacterial peptidomimetic, denoted as: RWKGGG-QA8C, and its structural formula is as follows:

Comparative example 4

Referring to the method of Example 1, RKVRGGG was selected as the polypeptide fragment, and combined with quaternary ammonium salt ions to prepare an antibacterial peptidomimetic, which was recorded as: RKVRGGG-QA8C, and its structural formula was as follows:

Comparative example 5

Referring to the method of Example 1, RWKGGG was selected as a polypeptide fragment, combined with a quaternary ammonium salt, to prepare an antibacterial peptidomimetic, denoted as: RWKGGG-QA8C, and its structural formula is as follows:

Comparative example 6

Referring to the method of Example 1, WGWGWG was selected as the polypeptide fragment, combined with a quaternary ammonium salt, to prepare an antibacterial peptidomimetic, denoted as: WGWGWG-QA8C, and its structural formula is as follows:

Experimental example 1

Performances such as antibacterial activity and hemolytic activity are tested to embodiment and comparative example gained antibacterial peptidomimetic activity, method is as follows:

1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) test of antibacterial peptoids

The micro broth dilution method was used to test the MIC value of the samples. The tested strains are 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 strains were inoculated in LB broth medium, cultivated overnight at 37°C, collected by centrifugation and redispersed into MH broth medium, and diluted to 10 ⁶ CFU/mL before use. Take a 96-well plate, add 100 μL of sterile liquid medium to each well, then add 100 μL of sample solution, and gradually dilute with the double dilution method to obtain an antimicrobial peptide concentration ranging from 500 to 0.244 μg/mL, and finally add 100 μL of the diluted bacterial solution (total volume 200 μL). Cultivate in a 37°C incubator for 16-20 hours. Add only MH medium as a negative control, add MH medium and bacterial solution (without samples) as a positive control, and make three to five parallel samples for each sample. The time is repeated three times. Visually observe the first well that does not become turbid as the minimum inhibitory concentration, add 5 μL of 0.5% triphenyltetrazolium chloride (TTC) and place it at 37°C for 15 minutes, and observe the first well that does not change red is the lowest Inhibitory concentration.

2. Kinetic evaluation of sterilization time

The stored working strains were inoculated in LB broth, cultured overnight at 37°C, collected by centrifugation and redispersed into MH broth. Dilute the bacterial solution to 10 ⁶ CFU/mL before use. Dilute the sample to 4MIC with MH medium, add the diluted bacterial solution and mix quickly, put it in a 37°C incubator and cultivate it at 0h, 10min, Samples were taken at a series of time points of 30min, 1h, 2h, 4h and 8h. The experimental group took 100 μL of samples each time, and carried out stock solution coating or dilution coating, blank control dilution coating, and plate coating method for bacterial counting. After culturing at 37°C for 24 hours, the number of colonies was counted. Convert the number of colonies in the control group to 100%, and convert the number of colonies in the experimental group into survival percentages, and draw a time-killing curve.

3. Hemolytic Activity Test of Antibacterial Peptoids

Take 10 mL of fresh blood from rats, put it in a beaker soaked with a small amount of sodium heparin, and stir continuously with a glass rod to remove fibrinogen, blood clots, etc. Add 0.9% physiological saline for washing, centrifuge at 2500 rpm for 5 min, discard the supernatant, and repeat the washing until the supernatant no longer appears red after centrifugation. Take 2 mL of the lower layer of red blood cells and add 48 mL of normal saline to dilute it into a 4% red blood cell suspension for later use. Samples were diluted to a range of concentrations. 500 μL of 4% erythrocyte suspension and 500 μL of sample solution were added to a 48-well plate, and three replicate holes were made for each sample. The negative control group contained 500 μL 0.9% normal saline and 500 μL 4% erythrocyte suspension, and the positive control group contained 500 μL 0.5% TritanX-100 and 500 μL erythrocyte suspension. All samples were incubated in a 37°C incubator for 3h. All samples were then centrifuged at 4000 rpm for 5 min. Take 100 μL of the supernatant in another 96-well plate, measure its absorbance at 576 nm with a microplate reader, and use the following formula to calculate the hemolysis rate of the sample on red blood cells:

4. Drug resistance experiment of antibacterial peptoids

The strains tested in the drug resistance experiment were Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli). The stored working strains were inoculated in LB broth medium, cultivated overnight at 37°C, collected by centrifugation and collected. Redisperse into HEPES working solution. And the bacterial solution was diluted to 10 ⁷ CFU/mL before use. After testing the MIC of the sample according to the above method, take the bacterial solution at 0.5×MIC value and dilute it 100 times and add it to the MH medium as the working strain. Then continue to test the MIC value of the sample and repeat for 14 generations. The drug resistance test algebra and the MIC increase multiple were plotted to obtain the drug resistance curve.

5. Cytotoxicity of antimicrobial peptoids

The toxicity of antimicrobial peptides to cells was detected by the MTT method. L929 cells were spread in 96-well culture plates at a density of 10 ⁵ cells/well, incubated in a constant temperature incubator for 24 hours under 5% CO ₂ and 37°C culture environment, and then added The sample solution dissolved in DMEM medium (containing serum) in advance, after continuing to culture for 24h and 72h, add 20μL of MTT solution (5mg/mL) to each well, continue to culture for 4h, discard the supernatant, add 150μL of DDMSO to each well for 15min The absorbance at 495 nm was measured with a microplate reader.

(2) Experimental results

1. Antibacterial activity of antibacterial peptoids

(1) The antibacterial activity test results of embodiment gained antibacterial peptoids, quaternary ammonium salts and polypeptide fragments are shown in Table 1 below, and the antibacterial activity test results of control example gained antibacterial peptidomimetics are shown in Table 2 below:

Table 1

Table 2

As shown in Table 1 and Table 2, the in vitro antibacterial activity of the synthesized antimicrobial peptoids 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 index to measure the antibacterial activity of antibacterial substances, and it is the lowest drug concentration that can inhibit the growth of bacteria after 18-24 hours of in vitro culture. The in vitro antibacterial activity was characterized by MIC.

As can be seen from Table 1, for Gram-positive bacteria S. The MIC values of 3QA8C, (WK)3YQA8C, (WK)3YQA8C, (WK)3YpQA8C, (WA)3YQA8C and (WA)3YpQA8C were between 1-31.25μg/mL. For Gram-negative bacteria E.coli, (WA)3GQA4C showed no antibacterial activity at the highest concentration tested, (WA)3GQA6C, (WA)3GQA8C, (FA)3GQA8C, (LA)3GQA8C, ( The MIC values of WK)3QA8C, (WK)3YQA8C, (WK)3YQA8C, (WK)3YpQA8C, (WA)3YQA8C and (WA)3YpQA8C were between 7.8-62.5μg/mL. Compared with individual GQAs and pure polypeptide fragments (WA)3, (FA)3, (LA)3, (WK)3, (WA)3Y, (WA)3Yp, (WK)3Y and (WK)3Yp , the antibacterial peptidomimetic obtained after conjugating polypeptide fragments has significantly improved antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria. This may be because in addition to the alkyl chain of the quaternary ammonium salt that can penetrate into the bacterial plasma membrane and cause certain membrane damage, the hydrophobic part of the polypeptide fragment can also insert into the lipid bilayer of the bacterial cell membrane, making it produce Stronger membrane disruptive action. The effect on Gram-positive bacteria is better than that of Gram-negative bacteria, which is due to their different cell wall and cell membrane structures. Gram-positive bacteria are surrounded by only one layer of cell membrane, while Gram-negative bacteria It has 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), so positive bacteria The negative charge of the membrane will be higher than that of the negative bacteria, and the effect of the cationic antimicrobial peptide will be stronger.

It can be seen from Table 2 that the antibacterial activity of the obtained antibacterial peptidomimetics is generally low when the polypeptide fragments in Comparative Examples 1-6 are combined with quaternary ammonium salts, and the antibacterial activity of quaternary ammonium salts is not significantly improved, and some even Reduced antibacterial activity of antimicrobial peptoids.

Comparing the minimum inhibitory concentrations of (WA)3GQA4C, (WA)3GQA6C and (WA)3GQA8C, it can be seen that with the increase of the alkyl chain length, (WA)3GQA8C has obvious antibacterial properties against S.aureus and E.coli It is better than (WA)3GQA4C, which may be due to the stronger overall hydrophobicity of (WA)3GQA8C, which enhances the interaction with the hydrophobic region of the lipid bilayer to disturb the bilayer structure and enhance the permeability of the membrane, and in a certain range There is also a certain positive correlation between internal hydrophobicity and antibacterial properties. On the other hand, it may be that compared with the short alkyl chain of (WA)3GQA4C, the long alkyl chain of (WA)3GQA8C is conducive to puncturing the bacterial cell wall and destroying the cell membrane, resulting in the leakage of the contents, and eventually leading to the death of the bacteria.

In order to study the effect of different types of hydrophobic amino acids on the antibacterial activity of antibacterial peptidomimetics, under the condition that the alkyl chain length of the quaternary ammonium salt was consistent, tryptophan (W) in (WA)3GQA8C was replaced with phenylalanine (F) and leucine Amino acid (L) was substituted to synthesize (FA)3GQA8C and (LA)3GQA8C. At the same time, the ClogP value of the compound was calculated by ChemBioDraw software. The ClogP value represents the oil-water partition coefficient of the organic compound, also known as the hydrophobic constant. Hydrophobic. The ClogP values of (WA)3GQA8C, (FA)3GQA8C and (LA)3GQA8C were 0.125, 0.155 and 0.272, respectively, that is, under the condition of ensuring the same chain length of the quaternary ammonium salts, the hydrophobicity of the three increases in turn. However, it can be seen from Table 1 that their antibacterial activity decreased sequentially, which shows that hydrophobicity is not the main factor affecting antibacterial activity at this time. It can be seen from the MIC results that when the hydrophobic aromatic tryptophan is replaced by hydrophobic fatty leucine, the MIC values for positive bacteria and negative bacteria are increased by 10 times and 5 times, respectively, and the antibacterial performance is significantly reduced, which may be Because compared with aliphatic hydrophobic side chains, aromatic hydrophobic side chains can use larger aromatic rings to pierce the bacterial wall (membrane) of bacteria, causing cytoplasmic efflux, resulting in bacterial death.

When tryptophan was replaced with phenylalanine, which is also a hydrophobic aromatic amino acid, the antibacterial activity was also reduced, and the MIC values against positive and negative bacteria were increased by 5 times and 2.6 times, respectively. This may be because there is a negatively charged π-electron cloud above and below the indole ring of the tryptophan side chain, so that it can not only interact with the positively charged amino acid side chain with a cationic-π interaction, but also interact with the lipid bilayer Aminocholine works. Therefore, it is speculated that in the presence of tryptophan, positively charged quaternary ammonium salts can be protected under highly hydrophobic environmental conditions, so that they can more easily penetrate the lipid bilayer. In addition, the indole side chain of tryptophan can disrupt the hydrophobicity of lipid acyl chains, leading to further insertion of antimicrobial peptidomimetics into lipid bilayers. Due to the cation-π electron cloud interaction, the peptide-membrane interaction is promoted and the antibacterial activity of the antibacterial peptidomimetic is enhanced.

(2) Antibacterial activity test of various antibacterial peptoids against various strains

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

The test results for sensitive bacteria are shown in Table 3 and Table 4 below:

table 3

Table 4

The test results for clinical multi-drug resistant bacteria are shown in Table 5 below:

table 5

2. Hemolytic activity of antibacterial peptoids

As an antibacterial agent, hemolytic activity is an important index to evaluate its biocompatibility, and the biological application of antibacterial agents with high hemolytic activity will be limited. Half hemolysis value (HC50) was calculated by software SPSS regression. The hemolytic activity test results of antimicrobial peptoids are shown in Figure 1.

It can be seen from Figure 1 that (WA)3GQA4C (HC50>500μg/mL) showed lower hemolytic activity compared with (WA)3GQA8C (HC50: ~450μg/mL), indicating that increasing hydrophobicity can improve antibacterial activity It also increases hemolytic activity. (FA)3GQA8C (HC50: ~337 μg/mL) is slightly more hydrophobic than (WA)3GQA8C, so its hemolysis is also slightly higher than (FA)3GQA8C. However, although the hydrophobicity of (LA)3GQA8C is the highest among the three, its HC50 value is >500 μg/mL, which may be due to the weaker interaction between the side chain aliphatic hydrophobic amino acid and the cell membrane than that between the aromatic hydrophobic amino acid and the cell membrane. between the role. The hemolysis rate of all the above antimicrobial peptides is less than 5% when the concentration is not more than 62.5 μg/mL. The results of erythrocyte hemolysis showed that some antibacterial peptoids were less toxic to mammalian erythrocytes. Considering the antibacterial performance and hemolytic activity, (WA)3GQA8C antibacterial peptoids had potential application value.

3. Cytotoxicity of antimicrobial peptoids

In order to characterize the biological safety of the obtained antibacterial peptoids, L929 (mouse fibroblasts) were selected to test the cytotoxicity of the obtained antibacterial peptoids. As shown in Figure 2, when the concentration was 100 μg/mL, the survival rate of cells co-cultured with (WA)3GQA4C, (WA)3GQA8C, (FA)3GQA8C and (LA)3GQA8C were all over 80%, without 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, without obvious cytotoxicity. The survival rate of (FA)3GQA8C and (LA)3GQA8C cells was only 53.5% and 65.9%, showing certain toxicity. However, the MIC values of (WA)3GQA4C, (WA)3GQA8C, (FA)3GQA8C and (LA)3GQA8C to S.aureus were not more than 31.25 μg/mL, and the MIC values to E.coli were not more than 62.5 μg/mL , so within the range of MIC values of antimicrobial peptides, there is no obvious cytotoxicity.

4. Bactericidal time kinetics of antibacterial peptoids

Take (WA)3GQA8C as a representative to measure the bactericidal ability and speed of S.aureus and E.coli at different concentrations. (WA) 3GQA8C concentrations were 1×MIC, 2×MIC, 4×MIC and 8×MIC. The bactericidal time kinetics results showed that the bactericidal activity of antimicrobial peptoids was concentration-dependent, and the higher the concentration, the stronger and faster the bactericidal effect, as shown in Figure 3. (WA) 3GQA8C showed rapid bactericidal performance against Aureus aureus. When the concentration was 4×MIC and 8×MIC, it could significantly reduce the number of S.aureus (kill more than 99% of the bacteria) in a short time of 10 minutes, 1 It can kill more than 99.9% of S.aureus within hours. And at the concentration of 1×MIC and 2×MIC, all bacteria can be killed within 4 hours. The bactericidal ability of antimicrobial peptoids on E.coli is slightly inferior to that of S.aureus. When the concentration is 4×MIC and 8×MIC, it can kill about 90% of the bacteria in a short time of 10 minutes, and the bactericidal rate after 2 hours Up to 99.9%. Concentrations of 1×MIC and 2×MIC also killed more than 99% of bacteria after 4 hours, and all bacteria were killed after 8 hours at all concentrations tested. Rapid bactericidal properties are critical to prevent the spread of bacterial infections, shorten treatment time and reduce the probability of drug resistance development. On the other hand, the rapid bactericidal performance reveals that the antibacterial mechanism of this type of antibacterial peptidomimetic may not be similar to that of antibiotics acting on a specific target (such as a protein), but acting on the cell membrane to achieve sterilization by destroying the integrity of the cell membrane Effect.

5. Drug resistance of antimicrobial peptoids

The propensity of bacteria to develop resistance can be assessed by continuously exposing bacteria to sublethal concentrations of antimicrobial agents. (WA)3GQA8C as a representative for drug resistance research. In the presence of sublethal concentrations of (WA)3GQA8C and control antibiotics vancomycin and ciprofloxacin, S.aureus and E.coli were continuously passaged, and each new MIC value was determined. The general definition of antimicrobial resistance is that the MIC value increases by more than 4 times during the continuous subculture process. It can be seen from Figure 4 that (WA)3GQA8C began to show drug resistance when it was continuously subcultured to the 12th generation, and its MIC value became 4 times that of the original. Traditionally, vancomycin was treated as a positive bacteria. "The last line of defense", it began to show drug resistance when S.aureus was subcultured to the 7th generation. For E.coli, (WA)3GQA8C began to show drug resistance at the 10th generation, and finally the MIC value changed. 8 times the original. The above results indicated that for S.aureus treatment, (WA)3GQA8C may have certain advantages compared with conventional antibacterial drugs, and it is not easy to induce bacterial resistance.

Claims (10)

1. A quaternary ammonium salt modified antibacterial peptoid, characterized in that, the antibacterial peptoid is obtained by conjugating a quaternary ammonium salt and a polypeptide fragment; The quaternary ammonium salt has a structure shown in the following formula <I>:

In formula <I>, the R ₁ , R ₂ and R ₃ are independently selected from one of C1-C20 straight-chain alkyl or branched-chain alkyl, and at least one of R ₁ , R ₂ and R ₃ A linear or branched alkyl group selected from C4-C20; the R group is halogen; the A group is (CH ₂ ) _a , a=1-6; the B group is CH ₂ or -NHCO-; the D group is NH ₂ or COOH; the m is an integer of 1 to 6; the n=1, 2 or 3, when n=2 or 3, the chain of the A group The length can be the same or different; The polypeptide fragment has the following two structures: (X-NH-CO-Z) _b (Y) or (X-NH-CO-Z) _b ; Wherein, X, Y and Z represent amino acid residues respectively, and said X is L-tryptophan residue, phenylalanine residue or L-leucine residue, and said Z is L-alanine residue base or L-lysine residue; b is an integer of 2 to 5; said Y is L-tyrosine residue or phosphorylated tyrosine residue. 2. The antibacterial peptoid modified by quaternary ammonium salt according to claim 1, characterized in that, the antibacterial peptoid has a structure shown in the following formula <II> or formula <III>:

3. The antibacterial peptidomimetic modified with quaternary ammonium salt according to claim 1 or 2, characterized in that, the antibacterial peptidomimetic has a structure shown in any one of the following formula <IV>～formula <XIII>:

Wherein, the R is selected from C1-C20 straight-chain alkyl or branched-chain alkyl; Preferably, the antibacterial peptidomimetic has structures as shown in formula <IV>, formula <VII>, formula <IX> and formula <XI>. 4. a preparation method of the antibacterial peptoid modified by quaternary ammonium salt as described in any one of claim 1-3, is characterized in that, is to carry out amidation reaction with quaternary ammonium salt and polypeptide segment, obtains described quaternary ammonium salt Ammonium salt modified antimicrobial peptidomimetic. 5. the preparation method according to claim 4, is characterized in that, the preparation method of described quaternary ammonium salt comprises the following two modes: (1) with N,N'-dimethyl substituent raw material and excessive halogenated Hydrocarbons are mixed, and under the action of an organic solvent, a catalyst is added for a temperature-raising reaction to obtain a quaternized product; then the obtained quaternized product is reacted with excess 1,3-propylenediamine, and purified to obtain the quaternized ammonium salt or, (2) mix N,N'-dimethyl substituted raw materials with excess halogenated hydrocarbons, add a catalyst under the action of an organic solvent, and directly carry out a temperature-raising reaction to obtain the quaternary ammonium salt. 6. The preparation method according to claim 4, characterized in that the preparation method of the antibacterial peptidomimetic comprises: synthesizing polypeptide fragments and conjugating the polypeptide fragments with quaternary ammonium salts under the action of a condensing agent. 7. preparation method according to claim 6, is characterized in that, the preparation method of described antibacterial peptidomimetic comprises the following steps: (1) Adopt the standard Fmoc protection strategy solid-phase peptide synthesis method, use 2-chlorotrityl chloride resin as the carrier, use HBTU, HOBt or DIEA as the condensation reagent, and extend the Fmoc protection strategy from the C-terminus to the N-terminus. Phase polypeptide synthesis to obtain Boc-protected polypeptide fragments; (2) Conjugation of quaternary ammonium salts and polypeptide fragments Dissolve the polypeptide fragment synthesized in step (1) in DCM or DMF, mix with quaternary ammonium salt, add condensing agent DCC or condensing agent NHS or condensing agent TEA under ice bath, react at room temperature for 48 hours, and the obtained product is washed and precipitated to obtain The light yellow solid is dissolved in DCM or TFA, the Boc protecting group is removed under ice-bath conditions, the ice ether precipitates, and the antibacterial peptidomimetic is obtained by cryopreservation. 8. The preparation method according to claim 7, characterized in that, the antibacterial peptidomimetic also includes the following treatment: the product obtained in step (2) is dissolved in water or acetonitrile, and is prepared by preparative high performance liquid chromatography and reverse phase Prepare a liquid phase column, mobile phase ratio: 0.1% TFA+H ₂ O/acetonitrile, gradient elution, the concentration of the organic phase rises from 30% to 70% within 15 minutes, and the flow rate is 1min/mL for purification. 9. an antibacterial peptidomimetic modified with quaternary ammonium salt as described in any one of claims 1-3 or an application of the antibacterial peptoid modified with quaternary ammonium salt as described in any one of claims 4-8, wherein It is characterized in that the application includes using the antibacterial peptoid to prepare medical antibacterial materials, and the medical antibacterial material includes using the antibacterial peptoid to prepare antibacterial coatings on the surface of medical catheters or instruments. 10. An antibacterial peptidomimetic modified with a quaternary ammonium salt as described in any one of claims 1-3 or an application of an antibacterial peptidomimetic modified with a quaternary ammonium salt as described in any one of claims 4-8, wherein It is characterized in that the application also includes using the antibacterial peptidomimetic to prepare medicines against bacterial infections.

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