阅读说明：本技术 支化抗菌聚氨基酸及其制备方法与应用 (Branched antibacterial polyamino acid and preparation method and application thereof ) 是由 陆超 吴传斌 于 2019-04-11 设计创作，主要内容包括：本发明提供一种支化抗菌聚氨基酸,所述支化抗菌聚氨基酸为：星型支化聚合物、刷状支化聚合物,由内核与支链组成；所述内核为含有4～15个氨基的支化聚合物,所述支链通过肽键与内核偶联；所述支链为亲水型聚合物嵌段,所述亲水型聚合物嵌段的聚合度为5～20,且所述纯亲水型聚合物嵌段的单体不包括疏水型单体。该支化抗菌聚氨基酸具有很好的抗菌活性的同时,不易发生溶血,具有很好的生物安全性。(The invention provides a branched antibacterial polyamino acid, which is prepared from the following components in parts by weight: the star-shaped branched polymer and the brush-shaped branched polymer consist of an inner core and a branched chain; the inner core is a branched polymer containing 4-15 amino groups, and the branched chain is coupled with the inner core through a peptide bond; the branched chain is a hydrophilic polymer block, the degree of polymerization of the hydrophilic polymer block is 5-20, and the monomers of the pure hydrophilic polymer block do not include hydrophobic monomers. The branched antibacterial polyamino acid has good antibacterial activity, is not easy to cause hemolysis, and has good biological safety.)

1. A branched antimicrobial polyamino acid, wherein the branched antimicrobial polyamino acid is: the star-shaped branched polymer or the brush-shaped branched polymer consists of an inner core and a branched chain;

the inner core is a branched polymer containing 4-15 amino groups, and the branched chain is coupled with the inner core through a peptide bond; the branched chain is a pure hydrophilic polymer block, the polymerization degree of the pure hydrophilic polymer block is 5-20, and the monomer of the pure hydrophilic polymer block does not include a hydrophobic monomer.

2. The branched antimicrobial polyamino acid of claim 1, wherein said inner core is selected from the group consisting of: hyperbranched polyethyleneimine, dendritic polylysine; and/or

The purely hydrophilic polymer block is selected from: polylysine.

3. The branched antibacterial polyamino acid according to claim 2, wherein the inner core is hyperbranched polyethyleneimine containing 8-15 amino groups, and the pure hydrophilic polymer block is polylysine with a polymerization degree of 5-20; or

The inner core is dendritic polylysine containing 4-8 amino groups, and the pure hydrophilic polymer block is polylysine with the polymerization degree of 5-10.

4. A branched antimicrobial polyamino acid according to any one of claims 1 to 3, wherein the surface electrostatic potential of the branched antimicrobial polyamino acid is positive and the Zeta potential is > 5 mV.

5. A preparation method of branched antibacterial polyamino acid is characterized by comprising the following steps:

taking a branched polymer containing 8-15 amino groups, and initiating a monomer of a pure hydrophilic polymer with a protected terminal amino group to perform a ring-opening polymerization reaction through the amino groups on the branched polymer;

adjusting the feed ratio of the monomers of the branched polymer and the pure hydrophilic polymer with the protected terminal amino group to ensure that the polymerization degree of the hydrophilic polymer is 5-20, and finally removing the protecting group of the terminal amino group; the branched antibacterial polyamino acid with the branched polymer block as the inner core and the hydrophilic polymer as the branched chain is obtained.

6. The method of preparing a branched antibacterial polyamino acid according to claim 5, wherein the monomer of the pure hydrophilic polymer whose terminal amino group is protected is-benzyloxycarbonyl-lysine-N-carboxy-cyclic anhydride; the branched polymer core containing 8-15 amino groups is hyperbranched polyethyleneimine.

7. The method of claim 6, wherein the branched antimicrobial polyamino acid is prepared by the steps of:

(1) dissolving hyperbranched polyethyleneimine to obtain an initiator solution, and initiating ring-opening polymerization of lysine-N-carboxyl-cyclic internal anhydride, wherein the lysine-N-carboxyl-cyclic internal anhydride contains a benzyloxycarbonyl side chain protecting group;

(2) and (2) removing the carbobenzoxy side chain protecting group in the polyethyleneimine grafted polylysine star block copolymer prepared in the step (1) by using a deprotection agent, and purifying to obtain a target product polyethyleneimine grafted polylysine star block copolymer, namely the branched antibacterial polyamino acid.

8. A preparation method of branched antibacterial polyamino acid is characterized by comprising the following steps:

(1) modifying a branched polymer on resin, wherein the branched polymer contains 4-8 amino groups;

(2) taking the branched polymer as an inner core, and adopting N_αfluorenylmethoxycarbonyl-NCoupling reaction is carried out by taking tert-butyloxycarbonyl-lysine as a reactant, DMF as a solvent and 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide as a condensing agent;

said N is_αfluorenylmethoxycarbonyl-N-tert-butoxycarbonyl-lysine alpha-amino protected with Fmoc and-amino protected with Boc;

(3) removing the Fmoc protecting group by using a piperidine-containing DMF solution;

(4) repeating the step (2) and the step (3) to form a pure hydrophilic polymer block branched chain of the branched antibacterial polyamino acid, wherein the polymerization degree of the branched chain is controlled to be 5-10;

(5) and (3) cutting the polypeptide from the resin by using TFA/DCM mixed solution, removing Boc protecting group, and purifying to obtain the branched antibacterial polyamino acid.

9. The method of producing a branched antibacterial polyamino acid according to claim 8, wherein the branched polymer is a dendritic polylysine; the method for modifying the branched polymer on the resin in the step (1) comprises the following steps:

removing Fmoc protective groups of Rink Amide-MBHA Resin by using a DMF solution containing piperidine, adding N, N '-bifluorene methoxycarbonyl-lysine, performing coupling reaction by using DMF as a solvent and 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide as a condensing agent, and removing Fmoc protective groups by using the DMF solution; and repeating the coupling reaction and removing the Fmoc protecting group for 2-3 times to obtain the dendritic poly-lysine containing 4-8 amino groups.

10. Use of a branched antimicrobial polyamino acid according to any of claims 1 to 4 for the preparation of an antimicrobial polymer, an antimicrobial medicament, an antimicrobial detergent, an antimicrobial coating.

Technical Field

The invention belongs to the field of medicines, and particularly relates to a branched antibacterial polyamino acid, and a preparation method and application thereof.

Background

The emergence of antibiotics is one of the most prominent findings in human medicine history. Since the discovery, countless lives have been saved. However, in recent years, the abuse of antibiotics has led to the development of a number of resistant and even lethal pathogens, such as methicillin-resistant staphylococcus aureus and antibiotic-resistant pseudomonas aeruginosa. These "superbacteria" with superior resistance to antibiotics are now widespread worldwide. In the face of this enormous health risk, antimicrobial peptides (e.g., defensins, humanized antimicrobial peptide LL-37, bombesin, etc.) hold promise for killing these drug-resistant pathogens. The antibacterial peptide is also called antimicrobial peptide or host defense peptide, and is a polypeptide substance which exists in organisms and has antibacterial activity. Compared with the traditional antibiotics, the antibacterial peptide has wide antibacterial spectrum, can effectively kill various drug-resistant bacteria, has high sterilization rate, is not easy to generate drug resistance, has no teratogenesis effect and is not easy to generate accumulation poisoning. Since they are bactericidal by disrupting the bacterial cell membrane, a mechanism completely different from that of conventional antibiotics, the resistance mechanism of bacteria to existing antibiotics does not have a blocking effect on them.

The way in which an antimicrobial peptide kills bacteria generally depends on its interaction with the bacterial cell membrane, i.e., the positively charged residues of the antimicrobial peptide interact electrostatically with the negatively charged bacterial cell membrane. After the antibacterial peptide with positive charges is combined with bacterial cell membranes through electrostatic interaction, the antibacterial peptide can induce the bacterial cell membranes to generate holes, so that bacterial contents flow out, and bacteria are killed finally, as shown in figure 1. The composition of mammalian cell membranes and bacterial cell membranes are different. The lipid on the outer layer of the mammalian cell membrane only consists of neutral amphoteric phospholipid, so the lipid is not charged; the phospholipid double-layer membranes of the bacteria contain a large amount of negatively charged phospholipids, and the outer walls of the cells contain a large amount of negatively charged lipopolysaccharides or teichoic acid, so that the surfaces of the bacteria are electronegative. Therefore, in the in vivo antibacterial process, the antibacterial peptide can selectively combine and kill negatively charged bacteria on the premise of basically not influencing the normal survival state of mammalian cells, thereby ensuring the biological safety of the antibacterial peptide and having great application value.

The common antibacterial peptide in the current market has the problems of low antibacterial activity, high toxicity, serious hemolysis, short half-life in vivo, poor enzymolysis stability, high cost, low yield and the like, so the application of the common antibacterial peptide is greatly limited. Because the mammalian cells are neutral, increasing the proportion of the segment hydrophobic block can promote the antibacterial peptide or polymer to pass through the bacterial cell membrane, and simultaneously can also improve the possibility that the antibacterial peptide or polymer is combined with the neutral mammalian cells, thereby reducing the selectivity and increasing the toxic and side effects, which are mainly reflected by hemolysis. Therefore, antimicrobial peptides or polymers with too large a proportion of hydrophobic blocks tend to have too large a hemolytic side effect, limiting their clinical use. In contrast, controlling the hydrophobic block ratio of the antimicrobial peptide or polymer within a certain range, and appropriately increasing the number and density of positive charges thereof, is an effective way to simultaneously improve the activity, selectivity, and reduce hemolytic side effects of the antimicrobial peptide or polymer. In addition, under physiological conditions, the presence of salt ions in the blood and tissues of organisms can cause charge shielding effect, weaken mutual attraction between the antibacterial peptide or polymer and bacterial cell membranes, and cause reduction and even loss of antibacterial activity. Therefore, the antibacterial peptide or polymer with high charge density has greater advantages when competing with salt ions to adsorb bacterial cell membranes, and has better salt tolerance. However, the conventional linear antimicrobial peptides are limited in their structures, and the charge density thereof is difficult to significantly increase as the number of charges increases, which has been a problem hindering the development of antimicrobial peptides or polymers.

Disclosure of Invention

Based on this, the present invention aims to provide a branched antibacterial polyamino acid which has the characteristics of high charge density, good antibacterial activity, low toxicity, good stability, no hemolysis in vivo and high safety.

In order to achieve the purpose, the invention provides the following technical scheme:

a branched antimicrobial polyamino acid, said branched antimicrobial polyamino acid being: the star-shaped branched polymer or the brush-shaped branched polymer consists of an inner core and a branched chain;

the inner core is a branched polymer containing 4-15 amino groups, and the branched chain is coupled with the inner core through a peptide bond; the branched chain is a pure hydrophilic polymer block, the polymerization degree of the pure hydrophilic polymer block is 5-20, and the monomer of the pure hydrophilic polymer block does not include a hydrophobic monomer.

The invention also provides a preparation method of the branched antibacterial polyamino acid, which comprises the following steps:

a preparation method of a branched antibacterial polyamino acid comprises the following steps:

taking a branched polymer containing 8-15 amino groups, and initiating a monomer of a pure hydrophilic polymer with a protected terminal amino group to perform a ring-opening polymerization reaction through the amino groups on the branched polymer;

adjusting the feeding ratio of the branched polymer to the monomer of the pure hydrophilic polymer with the protected terminal amino group to ensure that the polymerization degree of the hydrophilic polymer is 5-20, and finally removing the protecting group of the terminal amino group; the branched antibacterial polyamino acid with the branched polymer block as the inner core and the hydrophilic polymer as the branched chain is obtained.

A preparation method of a branched antibacterial polyamino acid comprises the following steps:

(1) modifying a branched polymer on resin, wherein the branched polymer contains 4-8 amino groups;

(2) taking the branched polymer as an inner core, and adopting N_αfluorenylmethoxycarbonyl-NCoupling reaction is carried out by taking tert-butyloxycarbonyl-lysine as a reactant, DMF as a solvent and 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide as a condensing agent;

said N is_αfluorenylmethoxycarbonyl-N-tert-butoxycarbonyl-lysine alpha-amino protected with Fmoc and-amino protected with Boc;

(3) removing the Fmoc protecting group by using a piperidine-containing DMF solution;

(4) repeating the step (2) and the step (3) to form a pure hydrophilic polymer block branched chain of the branched antibacterial polyamino acid, wherein the polymerization degree of the branched chain is controlled to be 5-10;

(5) and (3) cutting the polypeptide from the resin by using TFA/DCM mixed solution, removing Boc protecting group, and purifying to obtain the branched antibacterial polyamino acid.

The invention also provides the application of the branched antibacterial polyamino acid in preparing antibacterial polymers, antibacterial drugs, antibacterial detergents and antibacterial coatings.

Based on the technical scheme, the invention has the following beneficial effects:

according to the invention, a series of branched antibacterial polyamino acids with high charge density are obtained by designing a branched antibacterial polyamino acid through a great amount of creative work of an inventor, taking a branched polymer as an inner core and a pure hydrophilic polymer block as a branched chain, and controlling the number and the polymerization degree of the branched chain, wherein the charge properties such as surface electrostatic potential, zeta potential and the like of the branched antibacterial polyamino acid are obviously greater than those of linear contrast, so that the combination of the antibacterial polyamino acid and a negatively charged bacterial cytoplasmic membrane is facilitated, and the antibacterial activity of the branched antibacterial polyamino acid is improved. The branched chain of the branched antibacterial polyamino acid is a pure hydrophilic polymer block, wherein an amino acid monomer for synthesizing the block does not contain a hydrophobic monomer, and the branched antibacterial polyamino acid disclosed by the invention avoids the toxic effect of the branched antibacterial polyamino acid while obtaining good antibacterial activity by controlling the pure hydrophilic characteristic of the branched chain monomer and combining the reasonable range of the number and the polymerization degree of the branched chains of the pure hydrophilic polymer block, so that hemolysis is not easy to occur, and the branched antibacterial polyamino acid has high biological safety.

Drawings

FIG. 1 is a schematic diagram of the mechanism of action of an antimicrobial polyamino acid;

FIG. 2 is a diagram of the star PLL synthesized in example 1¹H NMR characterization and GPC characterization;

FIG. 3 shows the molecular dynamics simulations of P1(A), P2(B), P5(C) and linear PLL in example 3

(D) Schematic of surface electrostatic potential of (a);

FIG. 4 shows the zeta potential test results of the star PLL in example 3;

FIG. 5 is a graph of the sterilization kinetics of a star PLL;

fig. 6 shows the results of the star PLL treatment and the establishment of a skin infection model.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It will be appreciated that the experimental procedures for the following examples, where specific conditions are not indicated, are generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various reagents used in the examples are commercially available.

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 to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention provides a branched antibacterial polyamino acid, which consists of an inner core and branched chains; the branched antibacterial polyamino acid is star-branched polymer or dendritic branched polymer. Compared with the conventional linear antibacterial polyamino acid, the branched antibacterial polyamino acid has the characteristics of high charge density, good antibacterial activity and difficult hemolysis. The inner core is a branched polymer containing 4-15 amino groups, and the branched chain is coupled with the inner core through a peptide bond; the branched chain is a pure hydrophilic polymer block, the polymerization degree of the pure hydrophilic polymer block is 5-20, and the monomer of the pure hydrophilic polymer block does not include a hydrophobic monomer.

Optionally, the core of the branched antimicrobial polyamino acid is selected from: hyperbranched Polyethyleneimine (PEI), dendritic polylysine; and/or the branched hydrophilic polymer block of the branched antimicrobial polyamino acid is selected from: polylysine.

Specifically, in some embodiments, the inner core is hyperbranched polyethyleneimine containing 8-15 amino groups, and the hydrophilic polymer block is polylysine with the polymerization degree of 5-20. For example, the core may be hyperbranched polyethyleneimine having 8 amino groups or hyperbranched polyethyleneimine having 15 amino groups, and the degree of polymerization of the hydrophilic polymer block may be 5, 10, 15, or 20.

In other embodiments, the inner core of the antibacterial polyamino acid can also be a dendritic polylysine containing 4-8 amino groups, and a polylysine hydrophilic polymer block with the polymerization degree of 5-10 is taken as a branched chain. For example, the inner core of the antimicrobial amino acid can be a dendritic polylysine containing 4 amino groups or can be a dendritic polylysine containing 8 amino groups.

Preferably, the surface electrostatic potential of the branched antimicrobial polyamino acid is positive and the Zeta potential is greater than 5 mV.

The invention also provides a preparation method of the branched antibacterial polyamino acid, which comprises a liquid phase synthesis method (an amino acid N-carboxyl-cyclic internal anhydride (NCA) ring-opening polymerization method) and a solid phase synthesis method. The branched antibacterial polyamino acid synthesized by a liquid phase synthesis method (an amino acid N-carboxyl-cyclic internal anhydride ring-opening polymerization method) has the advantages of simple operation, high yield, low production cost, no racemization phenomenon, easiness in obtaining high-molecular polyamino acid and the like, but the molecular weight distribution of the synthesized polymer is relatively wide. The sequence and molecular weight of the polypeptide can be accurately controlled by a solid phase synthesis method, but the synthesis time is long, the cost is high, and the directly synthesized sequence is short. Therefore, although the antibacterial polyamino acid synthesized by the two methods has the characteristics of avoiding toxic effect, being not easy to cause hemolysis and having high biological safety, the specific structural characteristics and the production cost of the antibacterial polyamino acid are different, and a proper synthesis method can be selected to synthesize the branched antibacterial polyamino acid according to the application requirements and specific conditions.

In some of these embodiments, the liquid phase synthesis method comprises the steps of: taking a branched polymer containing 8-15 amino groups, and initiating a monomer of a pure hydrophilic polymer with a protected terminal amino group to perform a ring-opening polymerization reaction through the amino groups on the branched polymer;

adjusting the feeding ratio of the branched polymer to the monomer of the pure hydrophilic polymer with the protected terminal amino group to ensure that the polymerization degree of the hydrophilic polymer is 5-20, and finally removing the protecting group of the terminal amino group; the branched antibacterial polyamino acid with the branched polymer block as the inner core and the hydrophilic polymer as the branched chain is obtained. In some of these embodiments, the degree of polymerization of the hydrophilic polymer may be 5, 10, 15, or 20.

Preferably, the monomer of the pure hydrophilic polymer with protected terminal amino group is-benzyloxycarbonyl-lysine-N-carboxy-cyclic anhydride; the branched polymer core containing 8-15 amino groups is hyperbranched polyethyleneimine.

Specifically, the preparation steps of the branched antibacterial polyamino acid are as follows:

(1) dissolving hyperbranched polyethyleneimine to obtain an initiator solution, and initiating ring-opening polymerization of lysine-N-carboxyl-cyclic internal anhydride, wherein the lysine-N-carboxyl-cyclic internal anhydride contains a benzyloxycarbonyl side chain protecting group;

(2) and (2) removing a carbobenzoxy side chain protecting group in the polyethyleneimine grafted polylysine star block copolymer prepared in the step (1) by using a deprotection agent, and purifying to obtain a target product polyethyleneimine grafted polylysine star block copolymer, namely the branched antibacterial polyamino acid.

In some of these embodiments, the solid phase synthesis method comprises the steps of:

(1) modifying a branched polymer on resin, wherein the branched polymer contains 4-8 amino groups;

(2) taking the branched polymer as an inner core, and adopting N_αfluorenylmethoxycarbonyl-N-tert-butyloxycarbonyl-lysine as a reactant, Dimethylformamide (DMF) as a solvent, and 1-hydroxybenzotriazole (HOBt) and N, N' -Diisopropylcarbodiimide (DIC) as a condensing agent to perform coupling reaction;

said N is_αfluorenylmethoxycarbonyl-N-tert-butoxycarbonyl-lysine alpha-amino protected with fluorenylmethyloxycarbonyl (Fmoc) and-amino protected with tert-butoxycarbonyl (Boc);

(3) removing the Fmoc protecting group by using a piperidine-containing DMF solution;

(4) repeating the step (2) and the step (3) to form a pure hydrophilic polymer block branched chain of the branched antibacterial polyamino acid, wherein the polymerization degree of the branched chain is controlled to be 5-10;

(5) and (3) cutting the polypeptide from the resin by using TFA/DCM mixed solution, removing Boc protecting group, and purifying to obtain the branched antibacterial polyamino acid.

Preferably, the branched polymer is a dendritic polylysine.

Preferably, the modifying the branched polymer on the resin comprises the following steps:

removing an Fmoc protective group of Rink Amide-MBHA Resin by using a DMF solution containing piperidine, adding N, N '-bifluorenylmethoxycarbonyl-lysine, performing coupling reaction by using DMF as a solvent and 1-hydroxybenzotriazole (and N, N' -diisopropylcarbodiimide as a condensing agent, removing the Fmoc protective group by using the DMF solution, and repeating the coupling reaction and removing the Fmoc protective group for 2-3 times to obtain the dendritic polylysine containing 4-8 amino groups.

In some embodiments, the coupling reaction and removal of the Fmoc protecting group as described above is repeated 2 times to provide a dendritic polylysine containing 4 amino groups. Or repeating the coupling reaction and removing the Fmoc protecting group for 3 times to obtain the dendritic polylysine containing 8 amino groups.

The branched antibacterial polyamino acid has the characteristics of high charge density, good antibacterial activity and difficult hemolysis, and solves the problems of low antibacterial activity, high toxicity, serious blood dissolution, high cost, low yield and the like of the existing linear antibacterial polyamino acid.

The polylysine of the present invention may be poly-L-lysine or poly-D-lysine when used as the core or branch of the branched polymer. In the following specific examples, poly-L-lysine (PLL) is used as an example to more fully illustrate the invention.