阅读说明：本技术 基于多联苯芳烃的多肽缀合物、其制备方法和应用 (Polypeptides conjugate based on polybiphenyl arene, preparation method and application thereof ) 是由 袁行义 李春举 崔雷 于 2020-05-29 设计创作，主要内容包括：本发明公开了一种基于多联苯芳烃的多肽缀合物、其制备方法和应用,以及该化合物在抗菌方面的应用。该缀合物的结构通式如下：<Image he="657" wi="700" file="DDA0002514546870000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明多联苯芳烃-多肽缀合物,其中多肽部分赋予其阳离子性,亲水性,提供了缀合物和细菌细胞膜的识别位点,促进其与细菌细胞膜结合；多联苯芳烃片段赋予其疏水性,降低缀合物溶血毒性,赋予缀合物良好的生物相容性,缀合物通过破坏细菌细胞膜的完整性,导致细胞内容物外泄,最终诱导细菌死亡。(The invention discloses a polypeptide conjugate based on polybiphenyl arene, a preparation method and application thereof, and application of the compound in the aspect of antibiosis. The structural general formula of the conjugate is as follows: the polybiphenyl arene-polypeptide conjugate has cationic and hydrophilic properties endowed by the polypeptide part, provides a recognition site for the conjugate and bacterial cell membranes, and promotes the combination of the conjugate and the bacterial cell membranes; the polybiphenyl arene segment endows the conjugate with hydrophobicity, reduces hemolytic toxicity of the conjugate, and endows the conjugate with good biocompatibility, and the conjugate causes the leakage of cell contents by destroying the integrity of bacterial cell membranes, and finally induces bacterial death.)

1. A polybiphenyl arene-based polypeptide conjugate comprises two parts of polybiphenyl arene and polypeptide which are connected through covalent bonds, and is characterized in that the structural general formula of the polybiphenyl arene-based polypeptide conjugate is shown as the following formula I or formula II:

the formula I or the formula II is shown in the specification, wherein n is 0, 1 or 2.

2. A method for preparing the polybiphenyl arene-based polypeptide conjugate according to claim 1, wherein: alkynyl-modified polybiphenyl arenes and polypeptide RRR (N)₃) Forming a polybiphenyl arene-based polypeptide conjugate by click chemistry comprising the steps of:

(1) under the protection of nitrogen, adding alkynyl-modified polybiphenyl aromatic hydrocarbon and azide-modified polypeptide compounds into DMF (dimethyl formamide), adding copper tetra-acetonitrile hexafluorophosphate into the solution, stirring at normal temperature for 2 hours, uniformly mixing and reacting; the molar ratio of the polybiphenyl to the polypeptide to the copper tetra-acetonitrile hexafluorophosphate is 1 (0.9-2.5) to 0.05;

(2) after the reaction is finished, adding anhydrous ether into the reaction solution, and stirring vigorously to fully separate out a product; and centrifuging, removing supernatant, adding not less than 5mL of water to redissolve the crude product, and purifying by preparative high performance liquid chromatography to obtain the polybiphenyl arene-based polypeptide conjugate.

3. The method for preparing the polybiphenyl arene-based polypeptide conjugate according to claim 2, wherein: in the step (1), when the molar ratio of the polybiphenyl, the polypeptide and the copper tetraacetonitrile hexafluorophosphate is 1:0.9:0.05, the main product is the conjugate shown in the formula I; when the molar ratio of the polybiphenyl to the polypeptide to the copper tetraacetonitrile hexafluorophosphate is 1:2.5:0.05, the main product is the conjugate of formula II.

4. The method for preparing the polybiphenyl arene-based polypeptide conjugate according to claim 2, wherein: the alkynyl-modified polybiphenyl arene in the step (1) is prepared by the following method, and the method comprises the following steps:

a. under the protection of nitrogen, adding a boron tribromide solution into a dichloromethane solution of alkoxy polybiphenyl aromatic hydrocarbon, stirring at normal temperature for at least 12 hours, slowly dropwise adding a reaction solution into ice water to separate out white solid matters, and drying in a vacuum drying oven at the temperature of not lower than 50 ℃ to obtain the polybiphenyl;

b. adding K into the acetone solution of the poly-diphenol prepared in the step a under the protection of nitrogen₂CO₃Stirring and refluxing at 60 ℃ for at least 1h, adding 3-bromopropyne solution, continuously stirring and refluxing for at least 24h, carrying out thin-layer chromatography tracking reaction, cooling the reaction solution to room temperature after the reaction is finished, and carrying out suction filtration to remove K₂CO₃And repeatedly washing the filter cake with acetone, combining organic phases, and distilling the dry solvent under reduced pressure to obtain the target alkynyl-modified polybiphenyl aromatic hydrocarbon.

5. The method for preparing the polybiphenyl arene-based polypeptide conjugate according to claim 4, wherein: in the step b, the molar ratio of the alkoxy polybiphenyl aromatic hydrocarbon to the boron tribromide is 1: 3.

6. The method for preparing the polybiphenyl arene-based polypeptide conjugate according to claim 4, wherein: in the step b, the poly-diphenol, 3-bromopropyne and K₂CO₃In a molar ratio of 1:3: 3.

7. The method for preparing the polybiphenyl arene-based polypeptide conjugate according to claim 2, wherein: the azide-modified polypeptide compound in the step (1) is prepared by the following method, comprising the following steps:

① according to the polypeptide sequence RRR (N)₃) Synthesis of Arg-Arg-Arg-N₃The resin compound is gradually synthesized from the C end to the N end by adopting a standard Fmoc protection strategy solid-phase synthesis method, Rink amide resin with the loading capacity of 0.53mmol/g is used as a carrier, HBTU/HOBt/DIEA is used as a condensation reagent;

after the synthesis is finished, cracking the polypeptide on the resin compound obtained in the step I by using at least 10mL of cracking solution, reacting for at least 30min under an ice bath condition, and then continuing to react for at least 150min at normal temperature; adding at least 300mL of anhydrous ether, and fully stirring to separate out the polypeptide; and then carrying out suction filtration by using a G4 floxacin funnel, washing out the polypeptide by using water, and purifying the solution by using preparative high performance liquid chromatography to obtain the target azide-modified polypeptide compound.

8. The method for preparing the polybiphenyl arene-based polypeptide conjugate according to claim 2, wherein: in the step (1), the cracking solution is prepared from trifluoroacetic acid, m-cresol, anisole and water according to the mass ratio of 17:1:1: 1.

9. Use of the polybiphenyl arene-based polypeptide conjugate according to claim 1, wherein: the polypeptide conjugate of the polybiphenyl arene is used for preparing a medicament for resisting bacterial infection by destroying bacterial cell membranes to cause leakage of cell contents and further cause bacterial death.

10. Use according to claim 9, characterized in that: the bacteria are gram-positive bacteria or gram-negative bacteria; the gram-positive bacteria is at least one of staphylococcus, streptococcus, diplococcus pneumoniae, bacillus anthracis, corynebacterium diphtheriae and tetanus bacillus; the gram-negative bacteria is at least one of escherichia coli, proteus, shigella dysenteriae, pneumobacillus, brucella, haemophilus influenzae, haemophilus parainfluenza, moraxella catarrhalis, acinetobacter, yersinia, legionella pneumophila, bordetella pertussis, bordetella, shigella, pasteurella, vibrio cholerae, haemophilus parahaemolyticus, and shigella.

Technical Field

The invention relates to an antibacterial peptide substance, a preparation method and application thereof, in particular to an antibacterial peptide analogue, a preparation method and application thereof, which are applied to the technical field of antibacterial conjugates and biomedicines.

Background

Infectious diseases caused by pathogenic microorganisms such as bacteria, viruses and parasites seriously threaten the life and health of human beings. Bacterial infections are most common among these infectious diseases, with millions of patients suffering from these life-threatening bacterial diseases each year. Although our existing antibiotics and antibacterial drugs can effectively control and rapidly treat bacterial infections, the bacteria have drug resistance to the existing antibiotics due to the excessive use and dependence of our antibiotics. In particular, the emergence of multidrug-resistant bacteria makes the problem of bacterial drug resistance more difficult to solve. Therefore, there is an urgent need to design and synthesize antibacterial agents having a novel antibacterial mechanism and not easily causing the generation of drug resistance.

In recent years, antibacterial peptides have attracted extensive attention of researchers at home and abroad due to their broad-spectrum antibacterial activity, rapid bactericidal effect and specific antibacterial mechanism. Unlike conventional antibiotics, the cationic and amphiphilic structural characteristics of antibacterial peptides are key factors in their antibacterial activity. The antibacterial activity of the antibacterial peptide mainly depends on the interaction with bacterial cell membranes, and the special antibacterial mechanism ensures that the antibacterial peptide has the advantages of difficult generation of drug resistance and the like. However, the antibacterial peptide has many limitations and disadvantages: potential cytotoxicity, extreme instability under physiological conditions, extreme sensitivity to proteases, plasma, etc., and high synthesis cost.

An antibacterial peptide analogue is widely researched as a compound which inherits the advantages of an antibacterial peptide and improves the defects of the antibacterial peptide, a saturated alkyl chain is generally used as a hydrophobic segment, a polypeptide rich in cationic amino is used as a hydrophilic segment, and a conjugate obtained by coupling the saturated alkyl chain and the polypeptide through a chemical reaction often has better plasma or protease stability and broad-spectrum antibacterial activity. The literature reports that, compared with saturated alkane, a conjugate formed by unsaturated alkane as a hydrophobic segment has lower hemolytic toxicity, but the existing conjugate has unsatisfactory bacterial cell membrane binding capacity and stability, complex preparation method and high cost, which is a technical problem to be solved.

Disclosure of Invention

In order to solve the problems of the prior art, the invention aims to overcome the defects in the prior art and provide a polypeptide conjugate based on polybiphenyl arene, a preparation method and application thereof.

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

a polybiphenyl arene-based polypeptide conjugate comprises two parts of polybiphenyl arene and polypeptide which are connected through covalent bonds, wherein the structural general formula of the polybiphenyl arene-based polypeptide conjugate is shown as the following formula I or formula II:

the formula I or the formula II is shown in the specification, wherein n is 0, 1 or 2.

The invention relates to a preparation method of a polybiphenyl arene-based polypeptide conjugate, which is prepared from alkynyl-modified polybiphenyl arene and polypeptide RRR (N)₃) Forming a polybiphenyl arene-based polypeptide conjugate by click chemistry comprising the steps of:

(1) under the protection of nitrogen, adding alkynyl-modified polybiphenyl aromatic hydrocarbon and azide-modified polypeptide compounds into DMF (dimethyl formamide), adding copper tetra-acetonitrile hexafluorophosphate into the solution, stirring at normal temperature for 2 hours, uniformly mixing and reacting; the molar ratio of the polybiphenyl to the polypeptide to the copper tetra-acetonitrile hexafluorophosphate is 1 (0.9-2.5) to 0.05;

(2) after the reaction is finished, adding anhydrous ether into the reaction solution, and stirring vigorously to fully separate out a product; and centrifuging, removing supernatant, adding not less than 5mL of water to redissolve the crude product, and purifying by preparative high performance liquid chromatography to obtain the polybiphenyl arene-based polypeptide conjugate.

As a preferred technical solution of the present invention, in the step (1), when the molar ratio of the polybiphenyl, the polypeptide and the copper tetraacetonitrile hexafluorophosphate is 1:0.9:0.05, the main product is the conjugate of formula I; when the molar ratio of the polybiphenyl to the polypeptide to the copper tetraacetonitrile hexafluorophosphate is 1:2.5:0.05, the main product is the conjugate of formula II.

As a preferred technical solution of the present invention, the alkynyl-modified polybiphenyl arene in the step (1) is prepared by the following method, which comprises the following steps:

a. under the protection of nitrogen, adding a boron tribromide solution into a dichloromethane solution of alkoxy polybiphenyl aromatic hydrocarbon, stirring at normal temperature for at least 12 hours, slowly dropwise adding a reaction solution into ice water to separate out white solid matters, and drying in a vacuum drying oven at the temperature of not lower than 50 ℃ to obtain the polybiphenyl;

b. adding K into the acetone solution of the poly-diphenol prepared in the step a under the protection of nitrogen₂CO₃Stirring and refluxing at 60 ℃ for at least 1h, adding 3-bromopropyne solution, continuously stirring and refluxing for at least 24h, carrying out thin-layer chromatography tracking reaction, cooling the reaction solution to room temperature after the reaction is finished, and carrying out suction filtration to remove K₂CO₃And repeatedly washing the filter cake with acetone, combining organic phases, and distilling the dry solvent under reduced pressure to obtain the target alkynyl-modified polybiphenyl aromatic hydrocarbon.

In the step b, the molar ratio of the alkoxy polybiphenyl arene to the boron tribromide is 1: 3.

In a preferred embodiment of the present invention, in the step b, the poly-diphenol, 3-bromopropyne and K are₂CO₃In a molar ratio of 1:3: 3.

As a preferable technical scheme, the polybiphenyl aromatic hydrocarbon is 2,2 ' -biphenyl, 2 ' -terphenyl or 2,2 ' -quaterphenyl, and the structural formula of the polybiphenyl aromatic hydrocarbon is shown as the following formula III:

wherein n is 0, 1 or 2, wherein when n is 0, 2 ' -biphenyl represents, 2 ' -terphenyl when n is 1, and 2,2 ' -quaterphenyl when n is 2.

As a preferred embodiment of the present invention, the azide-modified polypeptide compound in the step (1) is prepared by the following method, which comprises the following steps:

① according to the polypeptide sequence RRR (N)₃) Synthesis of Arg-Arg-Arg-N₃The resin compound is gradually synthesized from the C end to the N end by adopting a standard Fmoc protection strategy solid-phase synthesis method, Rink amide resin with the loading capacity of 0.53mmol/g is used as a carrier, HBTU/HOBt/DIEA is used as a condensation reagent;

after the synthesis is finished, cracking the polypeptide on the resin compound obtained in the step I by using at least 10mL of cracking solution, reacting for at least 30min under an ice bath condition, and then continuing to react for at least 150min at normal temperature; adding at least 300mL of anhydrous ether, and fully stirring to separate out the polypeptide; and then carrying out suction filtration by using a G4 floxacin funnel, washing out the polypeptide by using water, and purifying the solution by using preparative high performance liquid chromatography to obtain the target azide-modified polypeptide compound.

In a preferred embodiment of the present invention, in the step (1), the lysis solution is prepared from trifluoroacetic acid, m-cresol, anisole and water at a mass ratio of 17:1:1: 1.

In a preferred embodiment of the present invention, the polypeptide sequence is RRR (N)₃) R (arginine) as a cationic amino acid, having a pKa value in water of 12 to 13.7, plays an irretrievable or absent role in the antimicrobial peptide sequence, is capable of sensing the potential on the cell membrane and interacting with the cell membrane, and polyarginine has a more effective bacterial cell membrane binding ability than the other two cationic amino acids (lysine and histidine); thus, polycationic arginine fragments can confer high biocompatibility to the conjugate, provide the necessary cationicity, and provide both the conjugate and bacterial cell membranesThe recognition site of (3); the structure is as the following IV formula:

the structure of the polybiphenyl-polypeptide conjugate is polybiphenyl arene modified by alkynyl and polypeptide RRR (N)₃) Forming a stable compound through click chemistry reaction, wherein the structure of the stable compound is shown in the following formulas V and VI:

wherein n is 0, 1 or 2.

The invention relates to application of a polybiphenyl arene-based polypeptide conjugate, which is used for preparing a medicament for resisting bacterial infection by destroying bacterial cell membranes, causing the leakage of cell contents and further causing bacterial death.

As a preferred technical scheme of the invention, the bacteria are gram-positive bacteria or gram-negative bacteria; the gram-positive bacteria is at least one of staphylococcus, streptococcus, diplococcus pneumoniae, bacillus anthracis, corynebacterium diphtheriae and tetanus bacillus; the gram-negative bacteria is at least one of escherichia coli, proteus, shigella dysenteriae, pneumobacillus, brucella, haemophilus influenzae, haemophilus parainfluenza, moraxella catarrhalis, acinetobacter, yersinia, legionella pneumophila, bordetella pertussis, bordetella, shigella, pasteurella, vibrio cholerae, haemophilus parahaemolyticus, and shigella.

Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:

1. the polybiphenyl arene-polypeptide conjugate has cationic and hydrophilic properties endowed by the polypeptide part, provides a recognition site for the conjugate and bacterial cell membranes, and promotes the combination of the conjugate and the bacterial cell membranes;

2. the polybiphenyl arene segment endows the conjugate with hydrophobicity, reduces hemolytic toxicity of the conjugate, endows the conjugate with good biocompatibility, leads to leakage of cell contents by destroying the integrity of a bacterial cell membrane of the conjugate, and finally induces bacterial death;

3. the method is simple and easy to implement, low in cost and suitable for popularization and application.

Drawings

FIG. 1 shows the preparation of 2, 2' -BP-OH in the first embodiment of the present invention¹H-NMR spectrum.

FIG. 2 shows the method of preparing 2, 2' -BP-yn according to one embodiment of the present invention¹H-NMR spectrum.

FIG. 3 shows 2, 2' -TP-OH in the first embodiment of the present invention¹H-NMR spectrum.

FIG. 4 shows 2,2 "-TP-yn in one embodiment of the present invention¹H-NMR spectrum.

FIG. 5 shows a graph of 2, 2' -QP-OH in one embodiment of the present invention¹H-NMR spectrum.

FIG. 6 shows a graph of 2, 2' -QP-yn in accordance with one embodiment of the present invention¹H-NMR spectrum.

FIG. 7 shows a polypeptide compound RRR (N) in accordance with one embodiment of the present invention₃) Mass spectrum of (2).

FIG. 8 is a mass spectrum of the conjugate 2, 2' -BP-1 in the first embodiment of the present invention.

FIG. 9 is a mass spectrum of the conjugate 2, 2' -BP-2 in the first embodiment of the present invention.

FIG. 10 is a mass spectrum of the conjugate 2, 2' -TP-1 in the first embodiment of the present invention.

FIG. 11 is a mass spectrum of the conjugate 2,2 "-TP-2 in the first embodiment of the present invention.

FIG. 12 is a mass spectrum of conjugate 2, 2' -QP-1 in accordance with one embodiment of the present invention.

FIG. 13 is a mass spectrum of conjugate 2, 2' -QP-2 in accordance with one embodiment of the present invention.

FIG. 14 shows the results of cytotoxicity test in the third example of the present invention.

FIG. 15 shows the results of hemolytic toxicity test in example three of the present invention.

FIG. 16 is a standard curve of 2, 2' -QP-1 concentration-absorption peak area in the fourth example of the present invention.

FIG. 17 is a positive control peixiaganan concentration-absorption peak area standard curve in the fourth example of the present invention.

FIG. 18 shows the results of plasma stability tests on 2, 2' -QP-1 and peixiananan in the fourth embodiment of the present invention.

Detailed Description

The following detailed description of embodiments of the invention is intended to be illustrative, but not limiting, of the invention. The examples, where no specific techniques or conditions are indicated, follow conventional techniques or conditions described in the literature in the art and suggested by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The source, trade name and composition of the reagents used are indicated at the first appearance and the same reagents used thereafter are the same as indicated for the first time unless otherwise specified.

The above-described scheme is further illustrated below with reference to specific embodiments, which are detailed below: