CN114522180A

CN114522180A - Use of polypeptide polymers or peptide mimetics in the breeding industry

Info

Publication number: CN114522180A
Application number: CN202011322890.XA
Authority: CN
Inventors: 刘润辉; 马鹏程
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2022-05-24
Also published as: WO2022105837A1

Abstract

The invention relates to the use of polypeptide polymers or peptide mimetics in the breeding industry as antibacterial agents, as antibiotic substitutes for the prevention and treatment of diseases in animals. The polypeptide polymer or the polypeptide mimic has high-efficiency antibacterial activity and good biocompatibility on pathogenic bacteria in the breeding industry, is not easy to induce bacteria to generate drug resistance and cross drug resistance, can reduce the risk of microbial infection of animals in the breeding industry, and has the effects of disease prevention and treatment.

Description

Use of polypeptide polymers or peptide mimetics in the breeding industry

Technical Field

The invention relates to a polypeptide polymer or a polypeptide mimic used as an antibacterial agent in the breeding industry, such as aquatic products, livestock breeding and the like, and is used in the field of disease prevention and treatment of animals.

Background

To improve the economic efficiency of the aquatic, livestock and veterinary industries, the FDA in the united states officially approved the use of antibiotics in feed in 1950, after which the use of antibiotics became more and more widespread. However, many antibiotics are slowly degraded, and a large amount of non-degradable antibiotics are remained in the environment and the cultured organisms by measures of adding antibiotics into the feed or directly adding antibiotics into the growth environment, and even remained in the final cultured products (such as antibiotic residues in pork), so that the drug resistance pressure of microorganisms and the generation of drug-resistant microorganisms are stimulated, and more safety problems such as the generation of bacterial drug resistance in the environment are caused. For decades, the abuse of antibiotics in humans, animals and agriculture has caused medical emergencies and enormous economic impact. Various countries around the world have successively introduced multiple policies for coping with drug resistance problems. As early as 1 month in 2006, the european union announced that antibiotics were prohibited from being added to the feed, and subsequently, countries such as the united states and japan also continued to set up relevant policies. In China, the large countries for aquaculture and livestock breeding as well as the large countries for production and use of veterinary antibiotics, in order to control the problem of bacterial drug resistance caused by abuse of antibiotics, the' action plan for restraining bacterial drug resistance (2016-. Currently, the Ministry of agriculture has planned to realize 'comprehensive resistance forbidding' at the feed end and 'resistance reduction' and 'resistance limitation' at the culture end in 2020.

However, due to the severe dependence of modern high-density breeding on the need for antibiotics, there is a great need to develop antibacterial agents that can replace traditional antibiotics. In the face of the emergence of "general banning" policies, the search for antibiotic alternatives in the breeding industry for the prevention and treatment of animal diseases has been compelling and requires that antimicrobial agents meet the following requirements: 1) has effective antibacterial activity, preferably broad-spectrum antibacterial activity and is still effective against drug-resistant bacteria; 2) the drug resistance is not easy to generate, and the generation of the drug resistance of microorganisms in the environment is reduced; 3) good biocompatibility and no obvious influence and harm on the cultured objects.

In the alternative scheme of the current research, the Chinese herbal medicine contains certain antibacterial ingredients, and can reduce the colonization of bacteria in the animal body and regulate intestinal flora when being used as a feed additive, but the effective ingredients of the Chinese herbal medicine are complex, the raw materials are difficult to obtain, and the antibacterial activity is low, so that the consideration is still needed in the aspects of medication safety and use effect. The microbial ecological agent is a biological agent prepared from normal microbes which are beneficial to a host through a special process, and can maintain microbial ecological balance and regulate the immune function in an animal body, so that the immunity of the animal in the breeding industry is improved, and the microbial infection is reduced. The host defense peptide has the advantages of broad-spectrum antibacterial activity and difficult generation of drug resistance, is a potential novel antibacterial drug research object, but has complicated preparation steps and high cost, and is greatly limited in application in the breeding industry. Currently, there is no antimicrobial agent for use in the aquaculture world that fully satisfies the above needs.

Therefore, the development of an antibiotic substitute with high-efficiency antibacterial activity, good biocompatibility, simple preparation process and low cost for the breeding industry is urgently needed in the field.

Disclosure of Invention

The invention aims to provide an antibiotic substitute for the breeding industry.

The present invention provides the use of a polypeptide polymer or peptidomimetic as an antimicrobial agent in aquaculture for killing or inhibiting the growth of pathogenic bacteria; or for the preparation of a medicament for the treatment of bacterial infections in animals in the breeding industry.

The polypeptide polymer or the polypeptide mimic of the present invention is not susceptible to microbial resistance during use. During the process of continuously stimulating the bacteria for 24 days, the minimum inhibitory concentration of the polypeptide polymer or the polypeptide mimic to the bacteria is not increased, and the drug resistance of the bacteria is not easily caused.

In another preferred example, the aquaculture is aquaculture, poultry farming, livestock farming or domesticated animal farming.

In another preferred embodiment, the aquaculture is fish farming.

In another preferred embodiment, the fish is selected from: zebrafish, tilapia, turbot, herring, grass carp, silver carp, bighead carp, rainbow trout, catfish, carp, crucian carp, weever, hilsa herring, yellow croaker, bream, mandarin fish, salmon, eel and grouper.

In another preferred example, the polypeptide polymer or the polypeptide mimic has no influence on the life activity of adult zebra fish when the content of the polypeptide polymer or the polypeptide mimic in the culture water body is not more than 50 mg/L.

In another preferred example, the polypeptide polymer or the polypeptide mimic can reduce the risk of microbial infection of zebra fish and improve the survival rate when being used in the culture water body at the concentration of 5-50 mg/L.

In another preferred example, after the zebra fish suffers from systemic bacterial infection, 5-50mg/kg of the polypeptide polymer or the polypeptide mimic is injected into the zebra fish body to kill bacteria in the body and improve the survival rate of the zebra fish.

In another preferred example, the pathogenic bacteria is one or a combination of more than two of staphylococcus aureus, streptococcus suis, nocardia asteroides, streptococcus agalactiae, escherichia coli, vibrio anguillarum, vibrio alginolyticus, vibrio parahaemolyticus, vibrio harveyi, vibrio cholerae, mannheimia haemolytica, salmonella pullorum, salmonella typhimurium, salmonella choleraesuis, vibrio fluvialis, aeromonas hydrophila, edwardsiella tarda, pseudomonas fluorescens and salmonella ducks.

In another preferred example, the pathogenic bacteria is one or a combination of more than two of staphylococcus aureus, escherichia coli, vibrio anguillarum, vibrio parahaemolyticus, vibrio alginolyticus, vibrio cholerae, vibrio harveyi, salmonella typhimurium, salmonella pullorum, mannheimia haemolytica, salmonella choleraesuis and streptococcus suis.

In another preferred embodiment, the polypeptide polymer or polypeptide mimetic is one or more than two homopolymers, copolymers or multipolymers selected from the group consisting of: alpha-amino acid, beta-amino acid, gamma-amino acid and oxazoline.

In another preferred embodiment, the polypeptide polymer or polypeptide mimetic is an oxazoline polymer, an α -amino acid polymer, an α/β -amino acid polymer, a γ -amino acid polymer, or a β -amino acid polymer.

In another preferred embodiment, the polypeptide polymer is an alpha-amino acid polymer, an alpha/beta-amino acid polymer, a gamma-amino acid polymer, or a beta-amino acid polymer.

In another preferred embodiment, the peptidomimetic is an oxazoline polymer.

In another preferred embodiment, the binary copolymer is a random copolymer or a block copolymer.

In another preferred embodiment, the polypeptide polymer or peptidomimetic is a homopolymer, and the side chains of its repeating units all carry positively charged groups.

In another preferred embodiment, the starting material may be a natural amino acid or an unnatural amino acid.

In another preferred embodiment, the α -amino acid polymer and the α/β -amino acid polymer are degraded to amino acids and short peptides having no antibacterial activity in the presence of enzymes in the natural environment and organisms.

The alpha-amino acid polymer and the alpha/beta-amino acid polymer in the antibacterial polymer can be degraded in the natural environment and organisms in which proteases such as trypsin, chymotrypsin, protease XXIII and the like exist, and degraded products no longer have antibacterial activity, so that the generation of drug resistance can be avoided.

In another preferred embodiment, the polypeptide polymer or peptidomimetic is a homopolymer, copolymer or multipolymer of the following A, A ', B, B', C, C ', C ", D, D', E, E ', F, F' and G structures, the total number of repeating units being a positive integer from 5 to 5000:

wherein r is each independently at

occurrence

0, 1, 2, 3, 4 or 5; r' is independently at each

occurrence

1, 2 or 3; r "is independently at each

occurrence

0, 1, 2 or 3;

in the formula₁、R₂、R₃、R₄、R₅And R₆Each independently at each occurrence is selected from the group consisting of: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylhydroxy, C1-C6 alkoxy, C1-C6 alkylsulfonyl, C1-C6 alkylguanidino, C1-C6 alkyl ester group, thio C1-C6 alkyl ester group, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, C6-C12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group, C1-C6 alkyl-C6-C592 cycloalkyl, C6-C12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group12 aryl, amino and

p1 is a protecting group, each occurrence independently selected from the group consisting of: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), phthaloyl (Pht), acetyl (Ac), trifluoroacetyl (Tfa), benzyl (Bn), triphenylmethyl (Tr);

p2 is independently selected from the following groups at each occurrence: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

each occurrence of X is independently selected from the following groups: none, hydrogen, amino, guanidino, hydroxyl, carboxyl, amido, sulfhydryl, methylthio, alkenyl, alkynyl, ester (-COO-), C6-C12 aryl or 5-12 membered heterocyclyl;

each L, at occurrence, is independently selected from: -CHR'₁-, -CO-, -COO-, -S (═ O) 2-; q is an integer of 0 to 6;

R'₁each occurrence is independently selected from the group consisting of substituted or unsubstituted: hydrogen, amino, C1-C15 alkyl, C1-C15 alkylamino, C1-C15 alkylhydroxy, C1-C15 alkylaldehyde, C1-C15 alkyl ester, thio-C1-C15 alkyl ester, C6-C15 aryl, C2-C15 alkenyl, C2-C15 alkynyl, -Rc-COO-Rc ", -Rc-CO-Rc", -Rc-O-Rc' -, -Rc-S-Rc ", 5-15 membered heteroaryl, 5-12 membered heterocyclic group;

x is selected from the following groups: none, hydrogen, amino, guanidino, hydroxyl, carboxyl, amido, mercapto, methylthio, alkenyl, alkynyl, ester, aryl, or 5-12 membered heterocyclyl;

ra and Rb are each independently at the occurrence selected from the group consisting of substituted or unsubstituted: -absent, hydrogen, C1-C15 alkyl, C1-C15 alkylamino, C1-C15 alkylhydroxy, C1-C15 alkylaldehyde, C1-C15 alkylsulfonyl, C2-C15 alkenyl, C2-C15 alkynyl, -Rc-COO-Rc ", -Rc-CO-Rc", -Rc-O-Rc-, -Rc-S-Rc ", C3-C12 cycloalkyl, C4-C12 cycloalkenyl, 5-12 membered heterocyclyl, C6-C12 aryl, 5-12 membered heteroaryl;

rc is independently at each occurrence selected from the group consisting of substituted or unsubstituted: C1-C15 alkylene, C2-C15 alkenylene, C2-C15 alkynylene, C3-C12 cycloalkylene, C4-C12 cycloalkenylene, 3-12 membered heterocyclylene, C6-C12 arylene, 5-12 membered heteroarylene;

rc "is each independently at each occurrence selected from the group consisting of substituted or unsubstituted: C1-C15 alkyl, C1-C15 alkylamino, C2-C15 alkenyl, C2-C15 alkynyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, 3-12 member heterocyclyl, C6-C12 aryl, 5-12 member heteroaryl;

each of the above substituents independently means being substituted with one or more substituents selected from: halogen, hydroxy, amino, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C8 cycloalkyl, C6-C12 aryl, 5-12 membered heteroaryl, or 3-12 membered heterocyclyl.

In another preferred embodiment, the amino acids represented by structures A, A ', B, B ', C, C ', C ', D, D ', E, E ', F, F ' and G are in the L configuration, the D configuration, or a mixture of both the D and L configurations.

In another preferred embodiment, the total number of repeating units is from 5 to 100, preferably from 5 to 50.

In another preferred embodiment, R₁、R₂、R₃、R₄、R₅And R₆Each independently at each occurrence is selected from the group consisting of: hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkylhydroxy, C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylguanidino, C1-C4 alkylcarboxylate, thio C1-C4 alkylcarboxylate, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, phenyl, naphthyl, 5-6 membered heteroaryl, 5-6 membered heterocyclyl, C1-C4 alkyl-C6 aryl, amino and

in another preferred embodiment, each occurrence of X is independently selected from the group consisting of: none, hydrogen, amino, guanidino, hydroxyl, carboxyl, amido, sulfhydryl, methylthio, alkenyl, alkynyl, ester (-COO-), phenyl or 5-6 membered heterocyclyl.

In another preferred embodiment, each L, at the occurrence, is independently selected from: -CH₂-、-CO-、-COO-。

In another preferred embodiment, q is 0, 1, 2, 3, 4 or 5. When q is 0, (L) q is absent.

In another preferred embodiment, Ra and Rb, where present, are each independently selected from the group consisting of substituted or unsubstituted: absent, hydrogen, C1-C6 alkyl, C1-C6 alkylamino, C1-C6 alkylhydroxy, C1-C6 alkylaldehyde, C1-C6 alkylsulfonyl, C2-C6 alkenyl, C2-C6 alkynyl, -Rc-COO-Rc ", -Rc-CO-Rc", -Rc-O-Rc-, -Rc-S-Rc ", C3-C6 cycloalkyl, C4-C6 cycloalkenyl, 5-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl.

In another preferred embodiment, the salt of the polypeptide polymer or peptidomimetic is a hydrochloride, bromate, trifluoroacetate, phosphate, lithium, sodium, potassium salt.

The polypeptide polymer or the polypeptide mimetic also includes the derivative of the polypeptide polymer or the polypeptide mimetic, wherein the derivative means that the amino group of the side chain of the polymer is changed into other functional groups such as guanidyl and the like, or the amino group contained in the side chain is connected with other molecules through chemical reaction, such as drug molecules, fluorescent small molecules, protecting groups and the like; the polymer is chemically modified at its ends, such as by attachment of fluorescent molecules, or drug molecules.

For structure E

The side chain structures may be attached at different positions, and the side chain substitution may be at position 2, or position 3 of the beta, gamma amino acid, for structure C

And vice versa.

In the invention, when the polypeptide polymer or the polypeptide mimic is a binary or multi-polymer, the repeating unit is selected from two or more of the following groups, and the side chain of one type of repeating unit has a positively charged group,

the substituents are as defined above.

In another preferred embodiment, the polypeptide polymer or polypeptide mimetic is selected from the group consisting of:

wherein n is the total number of repeating units of the polypeptide polymer or the polypeptide mimetic, and x and y are the ratios of the respective components, respectively. n is a positive integer of 5-5000; a is a positive integer of 0 to 100. X is more than 0% and less than or equal to 100%, y is more than or equal to 0% and less than or equal to 100%, and x + y is equal to 100%;

R_zeach independently at each occurrence is selected from the group consisting of: any one of halogen, carboxyl, active ester group, acyl chloride, alkylene oxide, sulfhydryl-SH, C2-C15 alkylene group, C2-C15 alkynyl, azide, maleimide, ortho-dithiopyridyl (OPSS), cyclodextrin and adamantane;

R_seach independently at the occurrence is hydrogen or

R_tEach occurrence is independently selected from the group consisting of C1-C15 alkyl, C2-C15 alkenyl, C2-C15 alkynyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, 3-12 member heterocyclyl, C6-C12 aryl, 5-12 member heteroaryl, C1-C15 alkyl ester;

R_wat each occurrence independently of the others

Or

Or

Wherein

Is a joint; r' "are each independently at the

occurrence

1, 2 or 3; r₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃And R₁₄Each independently at each occurrence is selected from the group consisting of: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylhydroxy, C1-C6 alkoxy, C1-C6 alkylsulfonyl, C1-C6 alkylguanidino, C1-C6 alkyl ester, thio C1-C6 alkyl ester, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, C6-C12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, C1-C6 alkyl-C6-C12 aryl, amino and

Q₁are protecting groups, each independently at the occurrence, selected from the group consisting of: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), phthaloyl (Pht), acetyl (Ac), trifluoroacetyl (Tfa), benzyl (Bn), triphenylmethyl (Tr);

Q₂each independently at occurrence is selected from the group consisting of: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

x and y are calculated as the number of repeat units of the corresponding component divided by the total number of repeat units of the polypeptide polymer or polypeptide mimetic.

In another preferred embodiment, the amino acid in the above structural unit is in L configuration, D-configuration, or a mixture of D configuration and L configuration.

In another preferred embodiment, the structural anion is selected from: cl^-、Br^-、CF₃COO^-、H₂PO₄ ^-、HPO₄ ^2-、PO₄ ^3-。

In another preferred embodiment, the arrangement of the two repeating units in the polymer structure is random, alternating or block, preferably random.

In another preferred embodiment, n is 5 to 100, preferably 5 to 50.

In another preferred example, x: y is 0.01: 0.99 to 0.99:0.01, preferably 0.05:0.95 to 0.95:0.05, even 1:9 to 9: 1.

in the formula, n is a positive integer of 5-5000; a is a positive integer of 0 to 100;

x is more than 0% and less than or equal to 100%, y is more than or equal to 0% and less than or equal to 100%, and x + y is equal to 100%;

R_zeach independently at each occurrence is selected from the group consisting of: halogen, carboxyl, active ester group, acyl chloride, alkylene oxide, sulfhydryl, C2-C15 alkylene group, C2-C15 alkynyl, azide, maleimide, ortho-dithiopyridyl (OPSS), cyclodextrin and adamantane.

In another preferred embodiment, the anion of the above structure is selected from: cl^-、Br^-、CF₃COO^-、H₂PO₄ ^-、HPO₄ ^2-、PO₄ ^3-。

In another preferred embodiment, n is 5 to 100, preferably 5 to 50.

The polypeptide polymer or the polypeptide mimic has good biocompatibility and has no obvious hemolytic activity on human red blood cells, mouse red blood cells and the like; has no obvious cytotoxicity to mammalian cells such as mouse embryonic fibroblast, African monkey kidney cells, human umbilical vein endothelial cells, canine kidney cells, arterial smooth muscle cells and the like.

The polypeptide polymer or the polypeptide mimic has no obvious hemolytic activity on human red blood cells and mouse red blood cells;

the polypeptide polymer or the polypeptide mimic has no obvious cytotoxicity on mouse embryonic fibroblasts, African monkey kidney cells, human umbilical vein endothelial cells, canine kidney cells, arterial smooth muscle cells and other mammalian cells.

The polypeptide polymer or the polypeptide mimic can be used as a substitute of antibiotics in the breeding industry, and can be widely applied to the field of disease prevention and treatment of bred animals.

The mass use of antibiotics in the breeding industry is easy to promote the generation of drug resistance of microorganisms, and most of antibiotic substitutes reported in the breeding industry at present are limited by factors such as insufficient antibacterial activity of the antibiotics, high production process requirement, incapability of mass production and the like.

The main advantages of the invention are:

1. the polypeptide polymer or the polypeptide mimic used by the invention relies on the existing polymerization method, can overcome harsh polymerization conditions such as strict dehydration and the like, can complete a large amount of polymerization under an open condition, and has the potential of realizing industrial mass production.

2. The polypeptide polymer or the polypeptide mimic is not easy to promote the microorganisms to generate drug resistance and cross drug resistance, and the alpha-amino acid polymer and the alpha/beta-amino acid polymer can be degraded in the natural environment and in the presence of enzymes in organisms, so that the possibility of drug resistance is further reduced. Meanwhile, the residues in aquatic products and meat are reduced, the food safety is ensured, and the health of people and livestock is guaranteed.

3. The polypeptide polymer or the polypeptide mimic has high-efficiency antibacterial activity and good biocompatibility on pathogenic bacteria in aquaculture, livestock breeding and other breeding industries, can improve the survival rate of zebra fish infected by bacteria, plays a role in preventing and curing diseases of animals in the breeding industries, and can replace antibiotics in the breeding industries.

Drawings

FIG. 1 is a graph showing the results of the resistance test of an α -amino acid polymer, wherein a is the results of the resistance test of the α -amino acid polymer and norfloxacin against Staphylococcus aureus; b is the test result of the drug resistance of the alpha-amino acid polymer and ampicillin to escherichia coli.

FIG. 2 is a diagram showing the degradation performance characterization results of an alpha-amino acid polymer, wherein a is a nuclear magnetic spectrum of the alpha-amino acid polymer at different time points under the action of protease XXIII; b is a minimum inhibitory concentration graph of degradation products of the alpha-amino acid polymer at different time points.

FIG. 3 is a graph showing the results of acute toxicity tests of alpha-amino acid polymers on adult zebrafish.

FIG. 4 is a graph showing the results of a therapeutic test of an α -amino acid polymer for infection in zebrafish.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions (e.g.as described in Sambrook et al, molecular cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Preparation of alpha-amino acid polymers

Example 1: preparation of random alpha-amino acid copolymer by using N-epsilon-tert-butyloxycarbonyl-L-lysine-N-carboxyanhydride and L-glutamic acid-5-benzyl ester-N-carboxyanhydride initiated by lithium hexamethyldisilazide (LiHMDS)

Weighing N-epsilon-tert-butyloxycarbonyl-L-lysine-N-carboxyanhydride and L-glutamic acid-5-benzyl ester-N-carboxyanhydride in a glove box protected by nitrogen, and taking dried tetrahydrofuran as a solvent. 1.8mL of N-epsilon-t-butoxycarbonyl-L-lysine-N-carboxyanhydride (0.2M) and 0.2mL of L-glutamic acid-5-benzyl ester-N-carboxyanhydride (0.2M) were mixed and stirred with a magnet. Initiator hexamethyldisilazane lithium salt was weighed to prepare a solution (0.5M), 160. mu.L of the solution was quickly added with the initiator, reacted at room temperature for 5 minutes, taken out from the glove box and quenched with 1 drop of benzoic acid. Petroleum ether (45mL) was added to each reaction solution, and after a white flocculent precipitate was precipitated, the precipitate was collected by centrifugation, dissolved in tetrahydrofuran (1mL), and precipitated with petroleum ether. After repeating this three times, a polymer having a protective group at the side chain amino group was obtained, and the molecular weight Mn of the polymer was 4400 and the molecular weight distribution PDI was 1.21 as determined by Gel Permeation Chromatography (GPC). After trifluoroacetic acid (2mL) was added to the protected polymer, the protecting group was removed by shaking for 2 hours, and most of the trifluoroacetic acid (CF) was purged₃COOH), adding glacial methyl tert-butyl ether (50mL) to precipitate a white precipitate, centrifuging, collecting, dissolving with methanol (1mL), precipitating with glacial ethyl ether (50mL), repeating the steps for three times, pumping out the residual solvent with an oil pump, dissolving samples with ultrapure water (5mL) respectively, and finally freeze-drying to obtain the deprotected random alpha-amino acid copolymer (yield 81%).

Example 2: preparation of random alpha-amino acid copolymer by using hexamethyldisilazane based lithium salt to initiate N-epsilon-tert-butyloxycarbonyl-L-lysine-N-carboxyanhydride and O-tert-butyl-L-serine-N-carboxyanhydride

Experimental procedure as in example 1, except that 1.8mL of N-epsilon-t-butoxycarbonyl-L-lysine-N-carboxyanhydride (0.2M) and 0.2mL of L-glutamic acid-5-benzyl ester-N-carboxyanhydride (0.2M) were changed to 1.6mL of N-epsilon-t-butoxycarbonyl-L-lysine-N-carboxyanhydride (0.2M) and 0.4mL of O-t-butyl-L-serine-N-carboxyanhydride (0.2M), the reaction was completed within 5 minutes. The molecular weight Mn of the polymer having the side chain amino group-based protecting group was 4200 and the molecular weight distribution PDI was 1.14 as determined by Gel Permeation Chromatography (GPC). After final deprotection, a random alpha-amino acid copolymer was obtained (yield 78%).

Example 3: preparation of block alpha-amino acid copolymer by using N-epsilon-tert-butyloxycarbonyl-L-lysine-N-carboxyanhydride and L-glutamic acid-5-benzyl ester-N-carboxyanhydride initiated by lithium hexamethyldisilazide

Experimental procedure as in example 1, except that 0.1mL of LiHMDS solution (0.4M) was added to 1mL of N- ε -tert-butoxycarbonyl-L-lysine-N-carboxyanhydride (0.2M) until the monomer reaction was complete, 1mL of the second block monomer L-glutamic acid-5-benzyl ester-N-carboxyanhydride (0.2M) was added, and the reaction was complete within 10 minutes. The molecular weight Mn of the polymer having the side chain amino group-based protecting group was 4400 and the molecular weight distribution PDI was 1.24 as determined by Gel Permeation Chromatography (GPC). Finally, a block alpha-amino acid copolymer (yield 75.5%) was obtained after deprotection

Example 4: under the open condition, hexamethyldisilazane-based amino lithium salt initiates N-epsilon-tert-butyloxycarbonyl-L-lysine-N-carboxyanhydride to prepare alpha-amino acid homopolymer

Experimental procedure as in example 1, except that the entire polymerization was carried out in an open atmosphere, and 20mL of N-. epsilon. -t-butyloxycarbonyl-L-lysine-N-carboxyanhydride (0.2M) and 1mL of lithium hexamethyldisilazane as an initiator (0.8M) were added to the reaction monomers, the reaction was completed within 5 minutes. The molecular weight Mn of the polymer having the side chain amino group-based protecting group was 4600 and the molecular weight distribution PDI was 1.22 by Gel Permeation Chromatography (GPC). After final deprotection, a-amino acid homopolymer was obtained (yield 68%).

Preparation of beta-amino acid polymers

Example 5: preparation of random beta-amino acid copolymer by co-initiating beta-lactam monomer MM and beta-lactam monomer CH with p-tert-butylbenzoyl chloride and lithium hexamethyldisilazide

Beta-lactam monomer MM and beta-lactam monomer CH were weighed in a nitrogen-protected glove box, and 1.2mL of MM (0.2M) and 0.8mL of CH (0.2M) were mixed in a reaction flask with dried tetrahydrofuran as a solvent, followed by stirring with a magnet. P-tert-butylbenzoyl chloride (0.2M) and lithium hexamethyldisilazide (0.5M) were prepared as coinitiators, and 100uL of each was added into the reaction flask rapidly. After 4 hours reaction at room temperature, it was removed from the glove box and quenched by adding 1 drop of methanol. Petroleum ether (45mL) was added to each reaction solution, and after a white flocculent precipitate was precipitated, the precipitate was collected by centrifugation, dissolved in tetrahydrofuran (1mL), and precipitated with petroleum ether. After repeating the above steps three times, the polymer with the side chain amino group having the protecting group is obtained. The molecular weight Mn of the polymer was identified by Gel Permeation Chromatography (GPC) as 3800 and the molecular weight distribution PDI as 1.21. Adding trifluoroacetic acid (2mL) into the polymer with the protection, shaking for 2 hours to remove the protection group, blowing off most of the trifluoroacetic acid, adding glacial methyl tert-butyl ether (50mL) to precipitate a white precipitate, centrifuging, collecting, dissolving with methanol (1mL), and removing the protecting groupPrecipitating with glacial ethyl ether (50mL), repeating the above steps for three times, pumping off the residual solvent with an oil pump, dissolving the sample with ultrapure water (5mL), and lyophilizing to obtain deprotected random beta-amino acid copolymer¹H NMR) showed a Degree of Polymerization (DP) of 20 with a yield of 80%.

Example 6: preparation of random beta-amino acid copolymer by CO-initiating beta-lactam monomer MM and beta-lactam monomer CO by p-tert-butylbenzoyl chloride and lithium hexamethyldisilazide

Experimental procedure as in example 5, except that 1.2mL of MM (0.2M) and 0.8mL of CH (0.2M) were changed to 1.2mL of MM (0.2M) and 0.8mL of CO (0.2M). The molecular weight Mn of the polymer having a protective group in the side chain was identified by Gel Permeation Chromatography (GPC) as 3900, and the molecular weight distribution PDI was identified as 1.19. After final deprotection a random beta-amino acid copolymer was obtained (yield 79%, from¹H NMR characterization gave DP ═ 20).

Example 7: preparation of random beta-amino acid copolymer by co-initiating N-tert-butyloxycarbonyl-beta-lactam-DL-lysine and beta-lactam monomer HG by 2- (tritylthio) acetic acid-N-succinimide ester and lithium hexamethyldisilazide

Experimental procedure as in example 5, except that 100. mu.L of p-tert-butylbenzoyl chloride (0.2M) was changed to 100. mu.L of 2- (tritylthio) acetic acid-N-succinimidyl ester (0.2M), 1.2mL of MM (0.2M) and 0.8mL of CH (0.2M) were changed to 1.4mL of benzyl N-chloroformate-beta-lactam-L-lysine (0.2M) and 0.6mL of beta-lactam monomer HG (0.2M), and dried N, N-dimethylformamide was used as a solvent. The molecular weight Mn of the polymer having a protective group in the side chain was 3800 and the molecular weight distribution PDI was 1.18 as identified by Gel Permeation Chromatography (GPC). After final deprotection, a random β -amino acid copolymer was obtained (yield 82%).

Preparation of alpha/beta-amino acid polymers

Example 8: p-tert-butylbenzylamine initiated N-epsilon-tert-butoxycarbonyl-L-lysine-N-carboxyanhydride and DL-beta-glycine N-carboxythiocarbonylic anhydride (beta)³HG NTA) preparation of random alpha/beta-amino acid copolymers

In a nitrogen-protected glove box, N-epsilon-tert-butoxycarbonyl-L-lysine-N-carboxyanhydride and DL-beta-glycine N-carboxythiocarbonyl cyclic anhydride were weighed, and 1.2mL of N-epsilon-tert-butoxycarbonyl-L-lysine-N-carboxyanhydride (0.2M) and 0.8mL of DL-beta-glycine N-carboxythiocarbonyl cyclic anhydride (0.2M) were mixed in a reaction flask with dry N, N-dimethylformamide as a solvent, followed by stirring with a magnet. Weighing initiator p-tert-butylbenzylamine and preparing into solution (0.2M), quickly adding 100 mu L into a reaction bottle, stirring the reaction in a glove box at room temperature for 3 days, taking out the reaction solution from the glove box, adding cold petroleum ether (45mL), centrifugally collecting after white flocculent precipitate is separated out, dissolving with tetrahydrofuran (1mL), precipitating with cold petroleum ether, and repeating the steps for three times to obtain the polymer with the side chain amino group protection group. The molecular weight Mn of the polymer having a protective group in the side chain was 3300 and the molecular weight distribution PDI was 1.14, as determined by Gel Permeation Chromatography (GPC). Then trifluoroacetic acid (2mL) is added into the polymer, the polymer is shaken for 2 hours to remove the protecting group, most of the trifluoroacetic acid is blown off, glacial methyl tert-butyl ether (50mL) is added to separate out white precipitate, the white precipitate is centrifugally collected, then methanol (1mL) is used for dissolving, the glacial ethyl ether (50mL) is used for precipitating, after the steps are repeated for three times, the residual solvent is pumped by an oil pump, then ultrapure water (5mL) is used for dissolving samples, and finally freeze-drying is carried out to obtain the random alpha/beta-amino acid copolymer after deprotection (the yield is 72.5 percent)¹Characterization by H NMR revealed that DP 20)

Example 9: initiation of N-epsilon-tert-butylbenzylamine-L-lysine-N-carboxyanhydride and beta^2,3-cyclohexyl N-carboxythiocarbonylic cyclic anhydride (. beta.)^2,3Preparation of-CH NTA)Preparation of random alpha/beta-amino acid copolymers

Experimental procedure as in example 8, except that 1mL of N-epsilon-t-butoxycarbonyl-L-lysine-N-carboxyanhydride (0.2M) and 1mL of DL-beta-glycine N-carboxythiocarbonylic anhydride (0.2M) were changed to 0.8mL of N-epsilon-t-butoxycarbonyl-L-lysine-N-carboxyanhydride (0.2M) and 1.2mL of beta-carboxyanhydride (0.2M)^2,3Cyclohexyl N-carboxythiocarbonyl cyclic anhydride (0.2M) using dry tetrahydrofuran as solvent. The reaction was complete after 4 days. The molecular weight Mn of the polymer with the protective group at the side chain was 3300 and the molecular weight distribution PDI was 1.14 as identified by Gel Permeation Chromatography (GPC). After final deprotection a random alpha/beta-amino acid copolymer was obtained (yield 75%, from¹Characterization by H NMR revealed that DP 20)

Preparation of oxazoline polymers

Example 10: preparation of oxazoline copolymer by using trifluoromethyl sulfonic acid methyl ester to initiate N-epsilon-tert-butyloxycarbonyl-2- (aminomethyl) oxazoline and 2- (tert-butyl) oxazoline

Weighing N-epsilon-tert-butoxycarbonyl-2- (aminomethyl) oxazoline and 2- (tert-butyl) oxazoline in a nitrogen-protected glove box, and taking dry N, N-dimethylacetamide as a solvent. 1.2mL of N-epsilon-t-butoxycarbonyl-2- (aminomethyl) oxazoline (0.2M) and 0.8mL of 2- (t-butyl) oxazoline (0.2M) were mixed in a reaction flask, and then stirred with a magnet. Weighing initiator methyl trifluoromethanesulfonate to prepare a solution (0.2M), quickly adding 0.1mL of initiator methyl trifluoromethanesulfonate into a reaction bottle, stirring the reaction at 120 ℃ for 6 hours, cooling to room temperature, adding cold petroleum ether (45mL), centrifugally collecting after white flocculent precipitate is separated out, dissolving with tetrahydrofuran (1mL), precipitating with cold petroleum ether, and repeating the steps for three times to obtain the polymer with the side chain amino group having the protective group. The polymerization was identified by Gel Permeation Chromatography (GPC)The molecular weight Mn of the compound is 3400, and the molecular weight distribution PDI is 1.23. Then trifluoroacetic acid (2mL) is added into the polymer, the polymer is shaken for 2 hours to remove the protecting group, most of the trifluoroacetic acid is blown off, glacial methyl tert-butyl ether (50mL) is added to separate out white precipitate, the white precipitate is centrifugally collected, the white precipitate is dissolved by methanol (1mL) and precipitated by glacial ethyl ether (50mL), after the steps are repeated for three times, the residual solvent is pumped by an oil pump, the sample is dissolved by ultrapure water (5mL), and finally the oxazoline copolymer after deprotection is obtained by freeze-drying (the yield is 75 percent)¹Characterization by H NMR revealed that DP 19)

Example 11: preparation of oxazoline copolymer by using trifluoromethyl sulfonic acid methyl ester to initiate N-epsilon-tert-butyloxycarbonyl-2- (aminomethyl) oxazoline and 2- (p-tert-butylbenzyl) oxazoline

The experimental procedure is the same as in example 10 except that 1.2mL of N-epsilon-tert-butoxycarbonyl-2- (aminomethyl) oxazoline (0.2M) and 0.8mL of 2- (tert-butyl) oxazoline (0.2M) are replaced with 1mL of 2- (p-tert-butylbenzyl) oxazoline (0.2M) and 1mL of 2- (p-tert-butylbenzyl) oxazoline (0.2M). The molecular weight Mn of the polymer having the side chain amino group-based protecting group was 4200 and the molecular weight distribution PDI was 1.22 as determined by Gel Permeation Chromatography (GPC). After final deprotection the oxazoline copolymer was obtained (yield 77.5%; from¹Characterization by H NMR revealed that DP 20)

Example 12: antimicrobial Activity testing of polypeptide polymers or peptidomimetics

The antibacterial activity tests were carried out with the selected antibacterial agents being the α -amino acid polymers of examples 1 to 4, the β -amino acid polymers of examples 5 to 7, the α/β -amino acid polymers of examples 8 to 9, the oxazoline polymers of examples 10 to 11, and the commercial antibiotic thiamphenicol used in the breeding industry. The selected strains include Staphylococcus aureus, Escherichia coli, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Salmonella typhimurium, Salmonella pullorum, Mannheimia haemolyticus, Salmonella choleraesuis, and Streptococcus suis. These strains are all from the American type culture CollectionThe center (ATCC), the China center for Industrial microorganism culture Collection (CICC), the China Center for General Microorganism Culture Collection (CGMCC) and the China center for veterinary microorganism culture Collection (CVCC). The antibacterial activity was demonstrated as the Minimum Inhibitory Concentration (MIC), and the following test method was mainly used. Firstly, culturing the bacteria in a proper culture medium of each bacterium in a shaking table with proper growth temperature of the corresponding strain for 10 hours, transferring the bacteria to a centrifuge to centrifuge at the rotation speed of 4000rpm for 5 minutes after the bacteria grow to the mature period, pouring off the supernatant, dispersing the bacteria at the bottom by using a small amount of test culture medium, measuring the OD value on a microplate reader, diluting the bacteria liquid to 2 multiplied by 10 according to the OD value⁵CFU/mL is ready for use. Adding 10 mu L of polymer with the concentration of 4mg/mL to be detected in the first row of a 96-well plate, adding 90 mu L of culture medium, uniformly mixing, taking 50 mu L of the polymer, diluting from the row B to the row H step by step, adding 50 mu L of bacterial liquid into each hole, and taking the culture medium as a negative control and the bacterial liquid as a positive control. Then the strain is put into an incubator with the appropriate growth temperature for 9 hours, and then the 96-well plate is placed on a microplate reader to read by using the wavelength of 600 nm. Finally, the bacterial growth rate is expressed as (OD)^{Polymer and method of making same}-OD^{Blank space})/(OD^{Control of}-OD^{Blank space}) X 100% calculated, two replicates per sample in the antimicrobial activity test, and three replicates were run. The obtained MICs are shown in the table 1, and the alpha-amino acid polymer, the beta-amino acid polymer, the alpha/beta-amino acid polymer and the oxazoline polymer all show high-efficiency antibacterial activity to pathogenic bacteria in cultivation, and the MICs of partial strains are even better than those of commercial antibiotics.

TABLE 1 antibacterial Activity of alpha-amino acid polymers (MIC, μ g/mL)

TABLE 2 antibacterial Activity of beta-amino acid polymers (MIC, μ g/mL)

TABLE 3 antibacterial Activity of alpha/beta-amino acid polymers (MIC, μ g/mL)

TABLE 4 antibacterial Activity of oxazoline polymers (MIC, μ g/mL)

Example 13: drug resistance testing of polypeptide polymers or peptidomimetics

The alpha-amino acid polymer of example 2 and commercial antibiotics were used to stimulate the gram-positive bacteria staphylococcus aureus (a in fig. 1) and the gram-negative bacteria escherichia coli (b in fig. 1) for resistance testing. The following test methods are mainly used to test the growth of resistance of bacteria to antibacterial agents. And (3) adding 0.2 mu L of bacterial liquid in the bacterial maturation period into 1mL of culture medium, adding an antibacterial agent, controlling the stimulation concentration to be 1/2MIC value, culturing for 24 hours in a shaking table at 37 ℃, testing the Minimum Inhibitory Concentration (MIC) of the bacteria after repeating the steps for four days, determining a new MIC value, and continuing to test according to the operation method. Repeating the process for four days, and measuring the final MIC value 24 days after the antibacterial agent is stimulated, and determining the drug resistance growth of the bacteria. Tests have found that the MIC of both bacteria continuously stimulated with the polymer does not increase significantly after 24 days, while the MIC of bacteria continuously stimulated with the antibiotics norfloxacin and ampicillin increases by tens of times or even hundreds of times. The use of polypeptide polymers or peptidomimetics has proven to be less likely to induce microbial resistance.

Example 14: degradation testing of alpha-amino acid polymers

Degradation of the polypeptide polymer or peptidomimetic in the presence of enzymes will reduce its retention in the body and natural environment. The alpha-amino acid polymer of example 2 was tested for degradation by protease XXIII produced by Aspergillus oryzae present in large amounts in the soil (a in FIG. 2). The following test methods were mainly used for polymer degradation. The polymer was dissolved in Phosphate Buffered Saline (PBS) to give a final concentration of 4 mg/mL. Protease was added to the polymer in PBS and mixed to give a final protease concentration of 160. mu.g/mL. The mixture was then transferred to a water bath and heated (temperature is the optimum temperature for the enzyme). The reaction solution was removed at various time points and heated in a water bath at 100 ℃ for 10 minutes to inactivate the protease. And finally, freeze-drying the reaction solution on a freeze dryer and then characterizing by nuclear magnetism. The Minimum Inhibitory Concentration (MIC) after degradation was tested as in example 8. The protease XXIII was found to completely degrade the alpha-amino acid polymer in example 2 within 6 hours, and comparing the MICs (b in FIG. 2) of the amino acid polymer before and after degradation for Staphylococcus aureus and Escherichia coli, it was found that the alpha-amino acid polymer had a high antibacterial activity before degradation, and the antibacterial activity of the polymer disappeared after degradation. The results show that the alpha-amino acid polymer can be degraded in the presence of enzymes, and the residual quantity is reduced, so that persistent irritation to bacteria is avoided, and the possibility of drug resistance generation is further reduced.

Example 15: hemolytic Activity test of a polypeptide Polymer or a polypeptide mimetic on Red blood cells

Since the polypeptide polymer or the peptidomimetic enters the blood circulation of animals when used as an injection, it is necessary to verify the hemolytic activity against red blood cells. Examples 1-5 were selected and tested for hemolytic activity on red blood cells. Hemolytic activity is expressed as fifty percent hemolytic rate (HC)₅₀) The following test methods were mainly used for the demonstration. Fresh human blood from volunteers was stored at 4 ℃ until use. Testing ofTaking enough human blood, adding a proper amount of triethanolamine buffered saline solution (TBS) for dilution, centrifuging for 3 minutes at the rotating speed of 4000rpm on a centrifuge, pouring out supernatant, adding TBS, shaking up red blood cells at the bottom, continuing centrifugation, repeating the process for 3 times, and adding TBS to dilute the red blood cells to 5% for later use. And adding 10 mu L of alpha-amino acid polymer solution to be detected into the first row of a 96-well plate, adding 90 mu L of TBS, uniformly mixing, diluting 50 mu L of the mixture from the row B to the row H step by step, and taking 0.1% of polyethylene glycol octyl phenyl ether as a positive control and the TBS as a negative control. After that, 50. mu.L of red blood cells diluted with TBS were added to each well and incubated at 37 ℃ for 1 hour. The 96-well plate was transferred to a centrifuge for 5 minutes at 3700rpm, and 80. mu.L of each well was pipetted into another fresh 96-well plate and read on a microplate reader at 405 nm. Finally, the hemolysis rate is equal to (OD)^{Polymer and method of making same}-OD^{Blank space})/(OD^Control-OD^{Blank space}) X 100% calculation, two replicates per sample in the hemolytic activity assay and three replicates were performed. Experimental results As shown in Table 5, the HC of examples 1 to 5 tested₅₀The values are all more than 2000 mug/mL, which proves that the compound has no obvious hemolytic activity to red blood cells, and does not cause hemolysis and the like of the red blood cells even if the compound enters the in vivo circulation of the breeding animals.

TABLE 5 hemolytic Activity of amino acid polymers (HC)₅₀,μg/mL)

Sample(s)	Example 1	Example 2	Example 3	Example 4	Example 5
						HC₅₀	>2000	>2000	>2000	>2000	>2000

Example 16: cytotoxicity testing of polypeptide polymers or polypeptide mimetics against mammalian cells

The polypeptide polymer or the polypeptide mimic can be in direct contact with the breeding animals in the using process, and the cytotoxicity is an important basis for verifying the safety of the polypeptide polymer or the polypeptide mimic. Examples 2, 4 and 5 were selected and tested for cytotoxicity against mouse fibroblasts. Cytotoxicity in terms of fifty percent cell survival (IC)₅₀) The following test methods were mainly used for the demonstration. The monolayer of cells was first trypsinized, collected after shedding, centrifuged at 1200rpm for 4 minutes in a centrifuge to allow the cells to settle, the supernatant was decanted and the cells were counted in culture medium. Cells were diluted to 10⁵cells/mL, 100. mu.L per well were transferred to a 96-well plate, after which the 96-well plate was placed at 37 ℃ in 5% CO₂Incubate overnight in a concentration incubator. Adding the alpha-amino acid polymer to be tested into the first row of a new 96-well plate on the next day, diluting step by step for later use, sucking away the culture medium in the original pre-cultured 96-well plate, transferring the diluted polymer solution into the original 96-well plate, and performing 5% CO treatment at 37 deg.C₂Culturing in an incubator with concentration. After 24 hours, the medium in the well plate was aspirated, 100. mu.L of thiazole blue (MTT) dye (0.5mg/mL) was added and incubated in an incubator for 4 hours to stain, after which the MTT dye was aspirated, 150. mu.L of dimethyl sulfoxide was added, the 96 well plate was placed on a shaker for 15 minutes to mix well and then placed in a microplate reader for reading at 570 nm. Most preferablyCell viability (% OD) after^{Polymer and method of making same}-OD^{Blank space})/(OD^Control-OD^{Blank space}) X 100% calculation, three replicates per sample in the cytotoxicity test and three replicates were run. Finally obtained IC₅₀The values are shown in table 6, indicating that the examples tested are not significantly cytotoxic to mammalian cells.

TABLE 6 cytotoxicity (IC) of amino acid polymers₅₀,μg/mL)

Sample (I)	Example 2	Example 4	Example 6
				IC₅₀	175	50	200

Example 17: toxicity testing of polypeptide polymers or polypeptide mimetics against adult zebrafish

The polypeptide polymer or the polypeptide mimic inevitably dissolves in the culture water body during use. In order to verify that the polypeptide polymer or the polypeptide mimic in the aquaculture water body does not influence the life activities of fishes. Example 2 was selected for testing for acute toxicity in adult zebrafish. The following test methods were mainly used. Acute toxicity testing of the polymers on adult zebrafish was performed according to OECD standard protocol. We selected healthy adult zebrafish (2.5 + -0.5 cm long, body weight)0.2 ± 0.1 g). Zebrafish were acclimated in the laboratory for 1 week prior to the experiment. And then randomly divided into an experimental group and a control group. The polymer solutions in the experimental groups were diluted to different concentration gradients. Each group contained 2L of the soaking solution and 10 fish. And the soaking solution was renewed every 24 hours. The whole soaking process was maintained at 28 ℃ in a culture environment with 14 h 10 h light/dark cycle. The number of zebrafish survived was recorded at 24, 48, 72 and 96 hours. The criteria for determining death were no visible breath or no bullet motion when touching the tail. Example 2 results of acute toxicity to adult zebrafish as shown in fig. 3, it was found that adult zebrafish can survive totally at a polymer concentration of 50mg/L or less, and the resulting median Lethal Concentration (LC) was calculated₅₀) It was 98.14 mg/mL. The polypeptide polymer or the polypeptide mimic has no significant influence on the life activities of animals in the using process when the concentration of the polypeptide polymer or the polypeptide mimic in the aquaculture water body is lower than 50 mg/L.

Example 18: therapeutic effect of polypeptide polymers or peptidomimetics on bacterially infected zebrafish

Example 2 was selected to test its therapeutic effect on zebrafish after bacterial infection. The following test methods were mainly used. Firstly, culturing Vibrio anguillarum with 2216E culture medium in a shaker at 30 deg.C until the growth period is 4 × 10⁸CFU/mL is ready for use. The zebra fish were randomly divided into four groups of a bacterial infection and injection phosphate buffer group, a bacterial infection and injection 10mg/kg polymer group, a bacterial infection and injection 20mg/kg polymer group, and an injection phosphate buffer only group, wherein each group had three replicate groups, and 10 zebra fish were in each replicate group. In the infection process, 10 mu L of bacterial liquid is injected into the zebra fish body through the abdominal cavity, and then the polymer solution is injected. Zebrafish survival was recorded every 12 hours for the following week. As a result, as shown in FIG. 4, the zebrafish injected with only phosphate buffer after bacterial infection showed massive death, and hemorrhagic septicemia after Vibrio anguillarum infection was observed. The survival rates of two groups treated by the alpha-amino acid polymer after bacterial infection are obviously improved, the survival rate in the 20mg/kg group is improved to 90 percent, and relevant symptoms of vibrio infection do not appear. This demonstrates the inventionThe polypeptide polymer or the polypeptide mimic has good treatment effect on related diseases of fishes after being infected by bacteria.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of a polypeptide polymer or peptidomimetic or its salt as an aquaculture antimicrobial agent for killing or inhibiting the growth of pathogenic bacteria; or for the preparation of a medicament for the treatment of bacterial infections in animals in the breeding industry.

2. Use according to claim 1, wherein the aquaculture is aquaculture, poultry farming, livestock farming or domesticated animal farming.

3. Use according to claim 2, wherein the aquaculture is fish farming.

4. The use according to claim 3, wherein the fish is selected from the group consisting of: zebrafish, tilapia, turbot, herring, grass carp, silver carp, bighead carp, rainbow trout, catfish, carp, crucian carp, weever, reeves shad, yellow croaker, bream, mandarin fish, salmon, eel and grouper.

5. The use according to claim 1, wherein the pathogenic bacteria is one or a combination of two or more of staphylococcus aureus, streptococcus suis, nocardia asteroides, streptococcus agalactiae, escherichia coli, vibrio anguillarum, vibrio alginolyticus, vibrio parahaemolyticus, vibrio harveyi, vibrio cholerae, mannheimia haemolyticus, salmonella pullorum, salmonella typhimurium, salmonella choleraesuis, vibrio fluvialis, aeromonas hydrophila, edwardsiella tarda, pseudomonas fluorescens, and salmonella ducks.

6. The use of claim 1, wherein the polypeptide polymer or peptidomimetic is one or more homopolymers, copolymers or multipolymers selected from the group consisting of: alpha-amino acid, beta-amino acid, gamma-amino acid and oxazoline.

7. The use of claim 6, wherein the polypeptide polymer or peptidomimetic is an oxazoline polymer, an α -amino acid polymer, an α/β -amino acid polymer, a γ -amino acid polymer, or a β -amino acid polymer.

8. The use of claim 6, wherein the polypeptide polymer or peptidomimetic is a homopolymer, copolymer or multipolymer of the following A, A ', B, B', C, C ', C ", D, D', E, E ', F, F' and G structures, the total number of repeat units being a positive integer from 5 to 5000:

wherein r is each independently at occurrence 0, 1, 2, 3, 4 or 5; r' is independently at each occurrence 1, 2 or 3; r "is independently at each occurrence 0, 1, 2 or 3;

in the formula₁、R₂、R₃、R₄、R₅And R₆Each independently at each occurrence is selected from the group consisting of: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylhydroxy, C1-C6 alkoxy, C1-C6 alkylsulfonyl, C1-C6 alkylguanidino, C1-C6 alkyl ester, thio C1-C6 alkyl ester, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, C6-C12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, C1-C6 alkyl-C6-C12 aryl, amino and

P₁are protecting groups, each independently at the occurrence, selected from the group consisting of: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), phthaloyl (Pht), acetyl (Ac), trifluoroacetyl (Tfa), benzyl (Bn), triphenylmethyl (Tr);

P₂each independently at occurrence is selected from the group consisting of: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;

each L, at occurrence, is independently selected from: -CHR'₁-、-CO-、-COO-、-S(＝O)₂-; q is an integer of 0 to 6;

R'₁each occurrence is independently selected from the group consisting of substituted or unsubstituted: hydrogen, amino, C1-C15 alkyl, C1-C15 alkylamino, C1-C15 alkylhydroxy, C1-C15 alkylaldehyde group, C1-C15 alkyl ester group, thio C1-C15 alkyl ester group, C6-C15 aryl, C2-C15 alkenyl, C2-C15 alkynyl, -Rc-COO-Rc ", -Rc-CO-Rc", -Rc-O-Rc-, -Rc-S-Rc ", 5-15 membered heteroaryl, 5-12 membered heterocyclic group;

9. The use of claim 1, wherein the polypeptide polymer or peptidomimetic is selected from the group consisting of:

x is more than 0% and less than or equal to 100%, y is more than or equal to 0% and less than or equal to 100%, and x + y is 100%;

R_zeach independently at each occurrence is selected from the group consisting of: halogen, carboxyl, active ester groups, acid chlorides, alkylene oxides, sulfydryl, C2-C15 alkylene groups, C2-C15 alkynyl, azide, maleimide, ortho-dithiopyridyl (OPSS), cyclodextrin, adamantane;

R_seach occurrence independently of the others is hydrogen or

R_weach independently at the occurrence is

Wherein

Is a joint; r' "are each independently at the occurrence 1, 2 or 3; r₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃And R₁₄Each independently at each occurrence is selected from the group consisting of: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylhydroxy, C1-C6 alkoxy, C1-C6 alkylsulfonyl, C1-C6 alkylguanidino, C1-C6 alkylester, thio C1-C6 alkylester, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, C6-C12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, C1-C6 alkyl-C6-C12 aryl, amino and

10. The use of claim 1, wherein the polypeptide polymer or peptidomimetic is selected from the group consisting of: