CN111848731B - In-situ self-assembled antibacterial molecule and preparation method and application thereof - Google Patents
In-situ self-assembled antibacterial molecule and preparation method and application thereof Download PDFInfo
- Publication number
- CN111848731B CN111848731B CN201910320336.9A CN201910320336A CN111848731B CN 111848731 B CN111848731 B CN 111848731B CN 201910320336 A CN201910320336 A CN 201910320336A CN 111848731 B CN111848731 B CN 111848731B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- dimethylformamide
- amino acid
- carrier resin
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Oncology (AREA)
- Animal Behavior & Ethology (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to an in-situ self-assembled antibacterial molecule, a preparation method and application thereof, wherein the antibacterial molecule comprises a polypeptide and an active oxygen response molecule positioned at the nitrogen tail end of the polypeptide; the amino acids that make up the polypeptide include glycine and at least two linked phenylalanines. The preparation method adopts a classical solid-phase synthesis method, has simple operation and high total yield, and the prepared antibacterial molecules can be self-assembled in situ at the infected part to form a nano-assembly body with a reticular nano-wire structure, and the structure can capture microbes such as bacteria and the like in vivo to achieve the bacteriostatic effect, is not easy to generate drug resistance and can realize broad-spectrum antibacterial. Meanwhile, the polypeptide in the molecular structure can act on the cell membrane of bacteria, and the cell is broken and killed through the physical membrane breaking effect, and in addition, the biocompatibility and the water solubility of the antibacterial molecule are good.
Description
Technical Field
The invention belongs to the field of chemical medicine, particularly relates to an antibacterial molecule and a preparation method and application thereof, and particularly relates to an antibacterial molecule capable of being self-assembled in situ and a preparation method and application thereof.
Background
The discovery of antibiotics is considered one of the most significant medical achievements of the 20 th century, and has saved the lives of millions, greatly improving the efficacy of surgery and cancer chemotherapy. Due to the widespread use of antibiotics, the generation and development of drug-resistant bacteria have shown a global trend, and the generation of drug-resistant bacteria in hospitals and emergency centers has increased the threat to treatment and emergency treatment. At present, the focus of research in the antibacterial field is mainly on the nano-antibacterial aspect. Some nanoparticles, such as gold, silver, copper oxide, zinc oxide or nanoparticles carrying cell-penetrating peptides, have high antibacterial activity due to their small size and large surface area, so that the nanoparticles can contact bacteria in a large area to destroy the permeability and respiratory function of the bacterial membrane, thereby further affecting the activity of the bacteria and finally achieving the purpose of treatment. However, nanoparticles of metals and metal oxides have limited their further widespread use due to their high biotoxicity.
In addition to antibiotics, organisms themselves rely on the autoimmune system for their own immune mechanisms in defending against bacterial infections. For example, neutrophils inhibit bacterial and fungal infections by extracellular traps (NETs). NETs are reported to have high viscosity and to be capable of capturing extracellular microorganisms in vivo. In recent years, research on artificial design and synthesis of self-assembled antibacterial peptide becomes a research hotspot in the fields of materials science, biomedicine and the like, and the self-assembled antibacterial peptide consisting of L-type natural amino acid has the advantages of high biocompatibility, low cytotoxicity, controllable degradability, high intracellular delivery efficiency and the like, and can reduce the toxic and side effects of medicaments, so the self-assembled antibacterial peptide has a huge development prospect in the aspect of development of antibacterial medicaments. Researches show that the antibacterial capacity of the self-assembled antibacterial peptide is closely related to the unique self-assembled structure of the self-assembled antibacterial peptide, and the self-assembled antibacterial peptide mainly acts on the cell membrane of bacteria, so that the cells are broken and killed through the physical membrane breaking effect, and the drug resistance is not easy to generate.
CN103467579A discloses a cationic amphiphilic self-assembled nano antibacterial peptide and its application, the general formula of its sequence is as follows: cn-Trp-Ile-Leu- (Ala) a- (Gly) b-X-Y, wherein all the amino acids are L-type amino acids; x is a cationic oligopeptide moiety; y is a protein transduction domain; cn is a fatty acid chain with 10-20 carbon atoms; a and b represent the number of amino acids. The long fatty acid chain modified at the N end of the polypeptide sequence can enhance the hydrophobic interaction in the self-assembly process and promote the self-assembly. The cationic amphiphilic self-assembled nano antibacterial peptide has a good antibacterial effect on staphylococcus aureus and can be used for developing novel nano antibacterial drugs. The preparation process is simple and feasible, the cost is relatively low, and the application of the antibacterial agent can enrich the types of the existing antibacterial agents.
CN106540238A discloses a self-assembly antibacterial lipopeptide nano-silver particle and a preparation method thereof, wherein an active peptide fragment is synthesized by a solid phase synthesis method, and the active peptide fragment and a hydrophobic fragment are coupled by a carbodiimide method to obtain the antibacterial lipopeptide. Alternatively, a spacer is added between the active peptide fragment and the hydrophobic fragment, the spacer being a short peptide containing two or more glycine residues. The nano-silver particles are prepared by an alcohol-thermal method, hydrophobic alkyl chains are fixed on the surfaces of the nano-silver particles through sulfydryl, and the nano-silver particles and the antibacterial lipopeptide are self-assembled through hydrophobic interaction. The antibacterial lipopeptide has the characteristics of wide antibacterial spectrum, high activity, difficulty in generating drug resistance and the like, and has remarkable effects on resisting bacteria and preventing infection by utilizing the synergistic effect of the silver nanoparticles and the antibacterial lipopeptide.
CN105749326A discloses a polypeptide antibacterial self-assembly composite material and a preparation method thereof, wherein the polypeptide antibacterial self-assembly composite material is CIP @ Fmoc-7AAP/PTA. The preparation method comprises the following steps: adding CIP into PTA aqueous solution to obtain mixed solution; and then adding the mixed solution into 1, 3-hexafluoro-2-propanol solution of Fmoc-7AAP, standing, centrifuging, and freeze-drying to obtain the Fmoc-7 AAP. The anti-infection self-assembly composite material has good drug loading and drug slow release effects, can be used for drug controlled release research, forms and creates a microenvironment for promoting wound healing, and has good practical value.
In summary, the existing antibacterial peptide or antibacterial nano material is mainly combined with glycoprotein on the cell membrane of bacteria through antibacterial polypeptide molecules, so that the bacteria die, or the bacteria die from natural antibacterial materials such as silver nanoparticles, and the materials can only be used for certain bacteria, but cannot be widely applied to various bacteria, such as positive bacteria, negative bacteria and drug-resistant bacteria, because the difference of the surface glycoprotein of different bacteria is large, the broad spectrum property is difficult to achieve by the identification mode. Moreover, the self-assembly molecules with antibacterial function in the prior art are difficult to realize in-situ self-assembly at the infected part, namely, the self-assembly molecules generate obvious antibacterial effect in situ. Therefore, it is very interesting to develop a novel antimicrobial molecule that is not susceptible to drug resistance and has a broad spectrum, and that can achieve in situ self-assembly.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an antibacterial molecule and a preparation method and application thereof, and particularly provides an antibacterial molecule capable of in-situ self-assembly and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides an in situ self-assembled antimicrobial molecule comprising a polypeptide and a reactive oxygen species-responsive molecule at the nitrogen terminus of the polypeptide; the amino acids that make up the polypeptide include glycine and at least two linked phenylalanines.
The in-situ self-assembled antibacterial molecule related by the invention has definite functional units, wherein active oxygen response molecules are used as the active oxygen response units, at least two connected phenylalanines are used as pi-pi hydrophobic interaction units, namely auxiliary self-assembly units, and glycine can help self-assembly precursor molecules to be better dissolved in a water environment. After the antibacterial molecules enter the infected part, active oxygen molecules over-expressed by the infected part can react with active oxygen response molecules in a molecular structure, and the generated new molecules can form intramolecular hydrogen bonds, so that the whole body is more planar, the pi-pi accumulation effect is facilitated, and in addition, the auxiliary effect of polypeptides in the molecular structure can form a nano assembly body with a net-shaped nano wire structure in situ self-assembly at the infected part, the structure is similar to an extracellular trapping net of neutrophils, the microbes such as bacteria and the like can be trapped in vivo, and the bacteriostatic effect can be achieved through the mechanical effect and the adhesion effect of the nano assembly body on the bacteria. Meanwhile, the polypeptide in the molecular structure can act on the cell membrane of bacteria, and cell rupture and cell death are caused by physical membrane rupture. And the antibacterial mechanism is not easy to generate drug resistance, and broad-spectrum antibacterial is realized. In addition, the antibacterial molecule is only a water-soluble molecule before assembly and has no antibacterial capability, so that the biocompatibility and biosafety of the antibacterial molecule are high.
The glycine and the phenylalanine in the polypeptide can be randomly arranged according to the actual requirement, but at least two phenylalanines are continuously connected, namely two, three, four, five or six phenylalanines are continuously connected, so as to ensure that the polypeptide can be self-assembled to form a nano assembly with a reticular nanowire structure through hydrogen bonds and pi-pi stacking action.
Preferably, the polypeptide further comprises any one of lysine, arginine, tyrosine, serine or tryptophan, or a combination of at least two such as arginine for lysine, tyrosine and serine and tryptophan, and the like.
The attachment position of the lysine, arginine, tyrosine, serine or tryptophan in the polypeptide can be arbitrarily selected according to actual needs, and the number of the attachment positions can be arbitrarily increased or decreased according to actual needs.
Preferably, the number of amino acid residues of the polypeptide is 4-7, such as 4, 5, 6 or 7.
The number of amino acid residues of the polypeptide is 4-7, so that the polypeptide has a better effect, and the antibacterial molecule can be self-assembled due to overlong polypeptide sequence, so that the self-assembly capability of active oxygen response is lost; the short peptide sequence may result in incomplete dissolution of the antibacterial molecule in neutral aqueous solution, and this may affect practical application.
Preferably, the active oxygen response molecule is a modified quinazolinone molecule, namely a quinazolinone derivative, and the specific modification mode is shown in the following molecular structure:
the active oxygen response molecule takes a quinazolinone molecule (Elf-97) as a main body, and a quinazolinone derivative is synthesized through proper chemical design, so that the active oxygen response molecule is obtained. This is because Elf-97 is a class of molecules with Aggregation Induced Emission (AIE) effect, which emits fluorescence because of the formation of intramolecular hydrogen bonds, which make the molecule more planar overall. The molecules of the above structure are distorted as a whole due to steric hindrance and the like before being oxidized by active oxygen, are not coplanar, and after being oxidized, the planarization thereof facilitates pi-pi stacking action, which provides a motive force for promoting self-assembly of antibacterial molecules.
When the active oxygen response molecule with the structure is stimulated by active oxygen, the active oxygen and phenylboronic acid undergo oxidation reaction and self-elimination to generate an oxidation product with green fluorescence and a benzoquinone molecule, and the reaction equation is shown as follows (wherein peptide represents a polypeptide chain in the antibacterial molecule):
preferably, the reactive oxygen species-responsive molecule is attached to the nitrogen terminus of the polypeptide by an amide bond.
In another aspect, the present invention provides a method for preparing the antibacterial molecule, the method comprising: the 2-chloro-trityl chloride resin is adopted as carrier resin, amino acid with protected terminal amino group and side chain amino group is adopted as raw material, polypeptide is synthesized through a solid phase synthesis method, active oxygen response molecules are connected with the prepared polypeptide, the active oxygen response molecules are removed from the carrier resin, side chain amino group protecting groups are removed, and the antibacterial molecules are obtained.
The preparation method adopts a classical solid-phase synthesis method, is simple to operate, and the obtained antibacterial molecules have the advantages of high chemical purity, high total yield and the like.
In the invention, the preparation method specifically comprises the following steps:
(1) Swelling the carrier resin in dichloromethane;
(2) Adopting amino acid with Fmoc protection obtained from terminal amino group and protected amino acid with side chain amino group as raw material, adding the first amino acid into carrier resin according to the amino acid sequence of the polypeptide to be synthesized to react with the carrier resin and connect;
(3) Removing the Fmoc protecting group on the first amino acid;
(4) Adding a second amino acid to the support resin for reaction and ligation;
(5) Removing the Fmoc protecting group from the second amino acid;
(6) Adding the next amino acid into the carrier resin for reaction and connection, and repeating the operations (4) and (5) until the condensation of all the amino acids is completed to obtain the polypeptide;
(7) Removing the polypeptide obtained in the step (6) from the carrier resin, and removing side chain amino protecting groups;
(8) And (4) reacting and connecting the active oxygen response molecule with the polypeptide prepared in the step (7).
Preferably, the swelling time in step (1) is 20-40min, such as 20min, 25min, 28min, 30min, 33min, 35min or 40min.
The amino acid with the protected side chain amino group in the step (2) refers to the amino acid with the protected side chain amino group obtained by Boc reagent.
Preferably, before the first amino acid is added to the carrier resin in step (2), the first amino acid and N, N-diisopropylethylamine are dissolved in N, N-dimethylformamide and then added to the carrier resin.
Preferably, the reaction of step (2) is carried out under a protective atmosphere.
Preferably, the protective atmosphere is nitrogen.
Preferably, the reaction time in step (2) is 1.5-3h, such as 1.5h, 1.8h, 2h, 2.2h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h or 3h, etc.
Preferably, the temperature of the reaction in step (2) is 20-30 ℃, such as 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃, etc.
Preferably, after the reaction in step (2) is finished, filtering is carried out, and the reaction product is washed by N, N-dimethylformamide, and then a mixed solution of methanol and dichloromethane is added into the resin for reaction, filtering is carried out, and the reaction product is washed by N, N-dimethylformamide.
Preferably, the volume ratio of methanol to dichloromethane is 1.
Preferably, the Fmoc protecting group on the first amino acid is removed in step (3) by the following method: and adding the mixed solution of piperidine and N, N-dimethylformamide into the carrier resin for reaction.
Preferably, the volume ratio of piperidine to N, N-dimethylformamide is 1.
Preferably, the reaction time is 20-40min, such as 20min, 22min, 25min, 30min, 32min, 35min, 36min, 38min or 40min and the like.
Preferably, the temperature of the reaction is 20-30 ℃, such as 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃.
Preferably, the reaction is filtered after completion and washed with N, N-dimethylformamide.
Preferably, before the second amino acid is added to the carrier resin in step (4), the second amino acid, benzotriazole-N, N' -tetramethyluronium hexafluorophosphate and N, N-diisopropylethylamine are dissolved in N, N-dimethylformamide and then added to the carrier resin.
Preferably, the reaction of step (4) is carried out under a protective atmosphere.
Preferably, the protective atmosphere is nitrogen.
Preferably, the reaction time in step (4) is 0.5-2h, such as 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.5h, 1.8h, 2h, etc.
Preferably, the temperature of the reaction in step (4) is 20-30 ℃, such as 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃, etc.
Preferably, the reaction in step (4) is filtered after completion and washed with N, N-dimethylformamide.
Preferably, the Fmoc protecting group of the second amino acid is removed in step (5) in the same manner as in step (3).
Preferably, the specific operation of step (7) is: adding trifluoroacetic acid into carrier resin, reacting, filtering, adding ethyl acetate into filtrate, and filtering to obtain white solid, namely polypeptide.
Preferably, the reaction is carried out under a protective atmosphere.
Preferably, the protective atmosphere is nitrogen.
Preferably, the reaction time is 1.5 to 3h, such as 1.5h, 1.8h, 2h, 2.2h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, or 3h, etc.
Preferably, the temperature of the reaction is 20-30 ℃, such as 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃.
Preferably, the adding manner of the glacial ethyl ether is dropwise adding.
Preferably, the white solid is obtained and then purified by a semi-preparative high performance liquid chromatograph.
Preferably, before the reactive oxygen species response molecule is reacted with the polypeptide prepared in step (7) in step (8), the reactive oxygen species response molecule and N, N-diisopropylethylamine are dissolved in N, N-dimethylformamide and then reacted with the polypeptide.
Preferably, the reaction time in step (8) is 18-30h, such as 18h, 20h, 21h, 22h, 24h, 25h, 26h, 28h or 30h, etc.
Preferably, the temperature of the reaction in step (8) is 40-60 deg.C, such as 40 deg.C, 42 deg.C, 45 deg.C, 48 deg.C, 50 deg.C, 52 deg.C, 55 deg.C, 58 deg.C or 60 deg.C.
Preferably, after the reaction in the step (8) is finished, the product is purified by semi-preparative high performance liquid chromatography and is frozen and dried.
As a preferred technical scheme of the invention, the preparation method specifically comprises the following steps:
(1) Swelling the carrier resin in dichloromethane for 20-40min;
(2) Adopting amino acid with Fmoc protection obtained from terminal amino group and protected amino acid with side chain as raw materials, dissolving the first amino acid and N, N-diisopropylethylamine in N, N-dimethylformamide according to the amino acid sequence of the polypeptide to be synthesized, adding into carrier resin, introducing nitrogen, reacting with the carrier resin at 20-30 ℃ for 1.5-3h, and connecting; filtering, washing with N, N-dimethylformamide, mixing methanol and dichloromethane in a volume ratio of 1;
(3) Mixing piperidine and N, N-dimethylformamide in a volume ratio of 1;
(4) Dissolving the second amino acid, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine in N, N-dimethylformamide, adding carrier resin, introducing nitrogen, reacting with the carrier resin at 20-30 deg.C for 0.5-2h, and connecting; filtering and washing by using N, N-dimethylformamide;
(5) Mixing piperidine and N, N-dimethylformamide in a volume ratio of 1;
(6) Adding the next amino acid into the carrier resin for reaction and connection, and repeating the operations (4) and (5) until the condensation of all the amino acids is completed to obtain the polypeptide;
(7) Adding trifluoroacetic acid into carrier resin, introducing nitrogen, reacting at 20-30 deg.C for 1.5-3h, filtering, adding dropwise glacial ethyl ether into the filtrate, filtering to obtain white solid, and purifying with semi-preparative high performance liquid chromatograph;
(8) Dissolving active oxygen response molecules and N, N-diisopropylethylamine in N, N-dimethylformamide, and reacting with the purified polypeptide at 40-60 ℃ for 18-30 h; purifying by semi-preparative high performance liquid chromatography, and freeze-drying to obtain the antibacterial molecule.
In a further aspect, the present invention provides the use of an antibacterial molecule as described above in the preparation of an antibacterial medicament.
Compared with the prior art, the invention has the following beneficial effects:
(1) The antibacterial molecules can form a nano assembly body with a net-shaped nano wire structure in an infected part through the triggering in-situ self-assembly of active oxygen, the structure can capture microorganisms such as bacteria and the like in a living body, and the antibacterial effect is achieved through the mechanical effect and the adhesion effect of the antibacterial molecules on the bacteria; meanwhile, the polypeptide in the antibacterial molecular structure can act on the cell membrane of bacteria, so that the cells are cracked and killed through the physical membrane breaking effect, and the antibacterial mechanism is not easy to generate drug resistance and can realize broad-spectrum antibacterial. In addition, the antibacterial molecule is only a water-soluble molecule before assembly and has no antibacterial capacity, so that the biocompatibility and the biosafety of the antibacterial molecule are high.
(2) The preparation method of the antibacterial molecule adopts a classical solid-phase synthesis method, is simple to operate, and the obtained antibacterial molecule has the advantages of high chemical purity, high total yield and the like and is good in water solubility.
Drawings
FIG. 1 is a graph showing the effect of dissolving the antibacterial molecule prepared in example 1 in PBS;
FIG. 2 is a graph showing the effect of dissolving the antibacterial molecule prepared in example 2 in PBS;
FIG. 3 is a graph showing the effect of dissolving the antibacterial molecule prepared in example 3 in PBS;
fig. 4 is a graph showing the results of an in vitro hydrogen peroxide triggering experiment (where a is a sample graph of a PBS group under natural light, b is a sample graph of a hydrogen peroxide group under natural light, c is a sample graph of a PBS group under an ultraviolet lamp, and d is a sample graph of a hydrogen peroxide group under an ultraviolet lamp);
FIG. 5 is an electron micrograph of the antimicrobial molecule prepared in example 1 after triggering with hydrogen peroxide;
FIG. 6 is an electron micrograph of the antimicrobial molecule prepared in example 2 after hydrogen peroxide triggering;
FIG. 7 is an electron micrograph of the antimicrobial molecule prepared in example 3 after hydrogen peroxide triggering;
FIG. 8 is an electron micrograph of the antimicrobial molecule prepared in example 4 after hydrogen peroxide triggering;
FIG. 9 is a graph of the in vitro bacterial survival statistics for the antimicrobial molecules prepared in example 2;
FIG. 10 is a distribution of the antimicrobial molecules prepared in example 1 at the site of infection in mice;
FIG. 11 is an electron micrograph of a hydrogel taken from a mouse;
fig. 12 is a partially enlarged electron microscopic view of fig. 11.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
This example provides an in situ self-assembled antimicrobial molecule comprising a polypeptide and a reactive oxygen species-responsive molecule at the nitrogen terminus of the polypeptide; the amino acids composing the polypeptide comprise glycine, phenylalanine and tyrosine, and the molecular structure of the polypeptide is as follows:
the preparation method comprises the following steps:
(1) In a solid phase synthesis tube, 1.0g of 2-chloro-trityl chloride resin was swollen in 10mL of dichloromethane for 30min;
(2) Adopting glycine, phenylalanine and tyrosine with Fmoc protection obtained from terminal amino group as raw materials, dissolving 1.45g of Fmoc protected tyrosine and 992 muL of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide according to the amino acid sequence of the polypeptide, adding into carrier resin, introducing nitrogen, reacting with the carrier resin at 25 ℃ for 2h, and connecting; filtering and washing with N, N-dimethylformamide, mixing methanol and dichloromethane in a volume ratio of 1;
(3) Mixing piperidine and N, N-dimethylformamide at a volume ratio of 1;
(4) Dissolving the next amino acid, namely 1.16g of Fmoc-protected phenylalanine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992 mu L of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, adding a carrier resin, introducing nitrogen, reacting with the carrier resin at 25 ℃ for 1h, and connecting; filtering and washing with N, N-dimethylformamide;
(5) Mixing piperidine and N, N-dimethylformamide at a volume ratio of 1;
(6) Dissolving the next amino acid, namely 1.16g of Fmoc-protected phenylalanine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992 microliter of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, adding carrier resin, introducing nitrogen, and reacting with the carrier resin at 25 ℃ for 1 hour for connection; filtering and washing with N, N-dimethylformamide;
(7) Then 892mg of Fmoc-protected glycine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992. Mu.L of N, N-diisopropylethylamine were dissolved in 5mL of N, N-dimethylformamide, and then the carrier resin was added, nitrogen was introduced, and the mixture was reacted with the carrier resin at 25 ℃ for 1 hour and connected; filtering and washing by using N, N-dimethylformamide;
(8) Then 892mg of Fmoc-protected glycine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992. Mu.L of N, N-diisopropylethylamine were dissolved in 5mL of N, N-dimethylformamide, and then the carrier resin was added, nitrogen was introduced, and the mixture was reacted with the carrier resin at 25 ℃ for 1 hour and connected; filtering and washing by using N, N-dimethylformamide;
(9) Adding 5mL of trifluoroacetic acid into carrier resin, introducing nitrogen, reacting at 25 ℃ for 2h, filtering, dropwise adding 30mL of ethyl glacial ether into the filtrate, filtering to obtain a white solid, and purifying the white solid by using a semi-preparative high performance liquid chromatograph, wherein the yield is 82.3%;
(10) Dissolving 51.2mg of activated oxygen response molecule after carboxyl activation and 48 mu L of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, and reacting with 200mg of the purified polypeptide at 50 ℃ for 24 h; by semi-preparation of highly effective liquidsPhase chromatography (C) 18 Column), and freeze-drying for three days to obtain the antibacterial molecule with a yield of 65.2%.
Subjecting the obtained antibacterial molecule to 1 H-NMR、 13 C-NMR and electrospray-mass spectrometry (ESI-MS) analysis gave the following results:
(1) 1 H NMR(DMSO-d6,400MHz)δ(ppm):8.84-8.87(m,1H),8.25-8.27(m,2H),8.06-8.18(m,5H),7.98-8.00(d,1H),7.83-7.87(t,1H),7.70-7.78(m,3H),7.53-7.57(t,1H),7.44-7.46(d,2H),7.37-7.40(m,1H),7.02-7.25(m,15H),5.29(s,2H),4.41-4.58(m,3H),3.57-3.75(m,4H),2.67-3.06(m,6H)；
(2) 13 C NMR(DMSO-d6,400MHz)δ(ppm):174.7,173.14,171.47,169.81,168.82,166.02,161.64,159.03,147.4,138.57,138.19,137.87,135.02,134.65,134.09,132.03,130.58,130.2,129.64,129.58,129.2,128.66,128.47,127.64,127.30,126.90,126.73,126.68,126.34,123.16,121.44,118.2,113.24,70.59,59.2,54.03,43.24,42.28,38.01,37.12,36.5；
(3)ESI MS(m/z):calcd.for C 53 H 51 BN 7 O 15 P,1067.33；found[M-H] - ,1066.33。
the above characterization results demonstrate that the antibacterial molecule with the structure is successfully synthesized.
Example 2
This example provides an in situ self-assembled antimicrobial molecule comprising a polypeptide and a reactive oxygen species-responsive molecule at the nitrogen terminus of the polypeptide; the amino acids composing the polypeptide comprise glycine, lysine and phenylalanine, and the molecular structure is as follows:
the preparation method comprises the following steps:
(1) In a solid phase synthesis tube, 1.0g of 2-chloro-trityl chloride resin was swollen in 10mL of dichloromethane for 30min;
(2) Adopting glycine and phenylalanine with Fmoc protection obtained from terminal amino group and lysine with side chain protected by Boc as raw materials, dissolving 1.16g of Fmoc protected phenylalanine and 992 muL of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide according to the amino acid sequence of the polypeptide, adding into carrier resin, introducing nitrogen, reacting with the carrier resin at 25 ℃ for 2h, and connecting; filtering and washing with N, N-dimethylformamide, mixing methanol and dichloromethane in a volume ratio of 1;
(3) Mixing piperidine and N, N-dimethylformamide in a volume ratio of 1:4 (total volume 10 mL) into a carrier resin, introducing nitrogen, reacting at 25 ℃ for 30min, filtering, and washing with N, N-dimethylformamide;
(4) Dissolving the next amino acid, namely 1.16g of Fmoc-protected phenylalanine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992 mu L of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, adding a carrier resin, introducing nitrogen, reacting with the carrier resin at 25 ℃ for 1h, and connecting; filtering and washing with N, N-dimethylformamide;
(5) Mixing piperidine and N, N-dimethylformamide in a volume ratio of 1:4 (total volume 10 mL) into a carrier resin, introducing nitrogen, reacting at 25 ℃ for 30min, filtering, and washing with N, N-dimethylformamide;
(6) Dissolving the next amino acid, namely 1.41g of lysine with the Fmoc-protected side chain protected by Boc, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992 muL of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, adding carrier resin, introducing nitrogen, reacting with the carrier resin at 25 ℃ for 1h, and connecting; filtering and washing with N, N-dimethylformamide;
(7) Then 892mg of Fmoc-protected glycine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992. Mu.L of N, N-diisopropylethylamine were dissolved in 5mL of N, N-dimethylformamide, and then the carrier resin was added, nitrogen was introduced, and the mixture was reacted with the carrier resin at 25 ℃ for 1 hour and connected; filtering and washing by using N, N-dimethylformamide;
(8) Adding 5mL of the mixed solution (trifluoroethanol: dichloromethane =1: 4) to a carrier resin, introducing nitrogen, carrying out a reaction at 25 ℃ for 2h, filtering, dropwise adding 30mL of diethyl ether to the filtrate, and filtering to obtain a white solid, and purifying the white solid by using a semi-preparative high performance liquid chromatograph, wherein the yield is 78.3%;
(9) Dissolving 51.2mg of activated oxygen response molecule after carboxyl activation and 48 mu L of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, and reacting with 200mg of the purified polypeptide at 50 ℃ for 24 h; by semi-preparative high performance liquid chromatography (C) 18 Column), and freeze-dried for three days, and the resulting compound was added with 3mL of trifluoroacetic acid to remove the Boc group, to obtain the antibacterial molecule with a yield of 60.2%.
Subjecting the obtained antibacterial molecule to 1 H-NMR、 13 C-NMR and electrospray-mass spectrometry (ESI-MS) analysis gave the following results:
(1) 1 H NMR(DMSO-d6,400MHz)δ(ppm):8.83-8.85(t,1H),8.22-8.27(m,2H),8.16-8.18(d,1H),7.98-8.13(m,4H),7.84-7.88(t,1H),7.73-7.80(m,3H),7.62-7.62(m,3H),7.54-7.58(t,1H),7.45-7.47(d,2H),7.37-7.39(d,1H),7.11-7.27(m,10H),5.30(s,2H),4.50-4.57(m,1H),4.38-4.43(m,1H),4.21-4.27(m.1H),3.88-3.92(m,2H),3.01-3.07(m,2H),2.68-2.93(m,6H),1.40-1.59(m,4H),1.17-1.29(m,4H)；
(2) 13 C NMR(DMSO-d6,400MHz)δ(ppm):173.24,170.48,168.71,168.22,164.12,160.64,157.08,136.58,134.29,137.87,135.02,134.65,134.09,132.03,130.58,129.64,129.58,128.66,128.47,127.64,127.30,126.90,126.73,126.68,126.34,123.16,121.44,113.24,70.59,59.22,58.73,57.11,43.56,42.04,37.12,36.56,31.52,28.61,22.35；
(3)ESI MS(m/z):calcd.for C 53 H 51 BN 7 O 15 P,895.37；found[M-H] - ,864.37。
the above characterization results prove that the antibacterial molecules with the structure are successfully synthesized.
Example 3
This example provides an in situ self-assembled antimicrobial molecule comprising a polypeptide and a reactive oxygen species-responsive molecule at the nitrogen terminus of the polypeptide; the amino acids composing the polypeptide comprise glycine and phenylalanine, and the molecular structure of the amino acids is shown as follows:
the preparation method comprises the following steps:
(1) In a solid phase synthesis tube, 1.0g of 2-chloro-trityl chloride resin was swollen in 10mL of dichloromethane for 30min;
(2) Adopting glycine and phenylalanine with Fmoc protection obtained from terminal amino group as raw materials, dissolving 1.16g of Fmoc protected phenylalanine and 992 muL of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide according to the amino acid sequence of the polypeptide, adding into carrier resin, introducing nitrogen, reacting with the carrier resin at 25 ℃ for 2h, and connecting; filtering and washing with N, N-dimethylformamide, mixing methanol and dichloromethane in a volume ratio of 1;
(3) Mixing piperidine and N, N-dimethylformamide in a volume ratio of 1:4 (total volume 10 mL) into a carrier resin, introducing nitrogen, reacting at 25 ℃ for 30min, filtering, and washing with N, N-dimethylformamide;
(4) Dissolving the next amino acid, namely 1.16g of Fmoc-protected phenylalanine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992 microliter of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, adding carrier resin, introducing nitrogen, and reacting with the carrier resin at 25 ℃ for 1 hour for connection; filtering and washing with N, N-dimethylformamide;
(5) Mixing piperidine and N, N-dimethylformamide at a volume ratio of 1;
(6) Then 892mg of Fmoc-protected glycine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992. Mu.L of N, N-diisopropylethylamine were dissolved in 5mL of N, N-dimethylformamide, and then the carrier resin was added, nitrogen was introduced, and the mixture was reacted with the carrier resin at 25 ℃ for 1 hour and connected; filtering and washing with N, N-dimethylformamide;
(7) Then 892mg of Fmoc-protected glycine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992. Mu.L of N, N-diisopropylethylamine were dissolved in 5mL of N, N-dimethylformamide, and then the carrier resin was added, nitrogen was introduced, and the mixture was reacted with the carrier resin at 25 ℃ for 1 hour and connected; filtering and washing by using N, N-dimethylformamide;
(8) Adding 5mL of trifluoroacetic acid into carrier resin, introducing nitrogen, reacting at 25 ℃ for 2h, filtering, dropwise adding 30mL of glacial ethyl ether into the filtrate, filtering to obtain a white solid, and purifying the white solid by using a semi-preparative high performance liquid chromatograph, wherein the yield is 95.3%;
(9) Dissolving 51.2mg of activated oxygen response molecule after carboxyl activation and 48 mu L of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, and reacting with 200mg of the purified polypeptide at 50 ℃ for 24 h; by semi-preparative high performance liquid chromatography (C) 18 Column), and freeze-dried for three days, and the resulting compound was added with 3mL of trifluoroacetic acid to remove the Boc group, to obtain the antibacterial molecule with a yield of 68.4%.
Subjecting the obtained antibacterial molecule to 1 H-NMR、 13 C-NMR and electrospray-mass spectrometry (ESI-MS) analysis gave the following results:
(1) 1 H NMR(DMSO-d6,400MHz)δ(ppm):8.87-8.84(t,1H),8.31-8.27(m,2H),8.17-8.06(m,3H),8.02-8.00(d,1H),7.87-7.83(t,1H),7.77-7.73(t,3H),7.57-7.53(t,1H),7.46-7.44(d,2H),7.39-7.37(d,1H),7.28-7.13(m,9H),5.28(s,2H),4.57-4.541(m,1H),4.44-4.39(m,1H),3.89-3.88(d,2H),3.75-3.69(m,1H),3.62-3.57(m,1H),3.08-2.98(m,2H),2.98-2.88(m,1H)；
(2) 13 C NMR(DMSO-d6,400MHz)δ(ppm):173.14,171.47,169.81,168.82,166.02,161.64,159.03,138.57,138.19,137.87,135.02,134.65,134.09,132.03,130.58,129.64,129.58,128.66,128.47,127.64,127.30,126.90,126.73,126.68,126.34,123.16,121.44,113.24,70.59,54.03,43.24,42.28,38.01,37.12；
(3)ESI MS(m/z):calcd.for C 44 H 41 BN 6 O 10 ,824.3；found[M-H] - ,823.3。
the above characterization results prove that the antibacterial molecules with the structure are successfully synthesized.
Example 4
This example provides an in situ self-assembled antimicrobial molecule comprising a polypeptide and a reactive oxygen species-responsive molecule at the nitrogen terminus of the polypeptide; the amino acids constituting the polypeptide include glycine and phenylalanine. The molecular structure is shown as follows:
the preparation method comprises the following steps:
(1) In a solid phase synthesis tube, 1.0g of 2-chloro-trityl chloride resin was swollen in 10mL of dichloromethane for 30min;
(2) Adopting glycine and phenylalanine with Fmoc protection obtained from terminal amino group as raw materials, dissolving 892mg of Fmoc protected glycine and 992 μ L of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide according to the amino acid sequence of the polypeptide, adding into a carrier resin, introducing nitrogen, reacting with the carrier resin at 25 ℃ for 2h, and connecting; filtering and washing with N, N-dimethylformamide, mixing methanol and dichloromethane in a volume ratio of 1;
(3) Mixing piperidine and N, N-dimethylformamide at a volume ratio of 1;
(4) Dissolving next amino acid namely 892Fmoc protected glycine and 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992 mu L of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, adding carrier resin, introducing nitrogen, reacting with the carrier resin for 1h at 25 ℃, and connecting; filtering and washing with N, N-dimethylformamide;
(5) Mixing piperidine and N, N-dimethylformamide at a volume ratio of 1;
(6) Dissolving the next amino acid, namely 1.16g of Fmoc-protected phenylalanine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992 mu L of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, adding a carrier resin, introducing nitrogen, reacting with the carrier resin at 25 ℃ for 1h, and connecting; filtering and washing by using N, N-dimethylformamide;
(7) Mixing piperidine and N, N-dimethylformamide in a volume ratio of 1:4 (total volume 10 mL) into a carrier resin, introducing nitrogen, reacting at 25 ℃ for 30min, filtering, and washing with N, N-dimethylformamide;
(8) Dissolving the next amino acid, namely 1.16g of Fmoc-protected phenylalanine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992 microliter of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, adding carrier resin, introducing nitrogen, and reacting with the carrier resin at 25 ℃ for 1 hour for connection; filtering and washing with N, N-dimethylformamide;
(9) Mixing piperidine and N, N-dimethylformamide in a volume ratio of 1:4 (total volume 10 mL) into a carrier resin, introducing nitrogen, reacting at 25 ℃ for 30min, filtering, and washing with N, N-dimethylformamide;
(10) Then 892mg of Fmoc-protected glycine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992. Mu.L of N, N-diisopropylethylamine were dissolved in 5mL of N, N-dimethylformamide, and then carrier resin was added, nitrogen was introduced, and 1 hour of reaction was carried out with the carrier resin at 25 ℃ and connection was carried out; filtering and washing by using N, N-dimethylformamide;
(11) Then 892mg of Fmoc-protected glycine, 1.14g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 992. Mu.L of N, N-diisopropylethylamine were dissolved in 5mL of N, N-dimethylformamide, and then the carrier resin was added, nitrogen was introduced, and the mixture was reacted with the carrier resin at 25 ℃ for 1 hour and connected; filtering and washing with N, N-dimethylformamide;
(12) Adding 5mL of trifluoroacetic acid into carrier resin, introducing nitrogen, reacting at 25 ℃ for 2h, filtering, dropwise adding 30mL of diethyl ether into the filtrate, filtering to obtain a white solid, and purifying the white solid by using a semi-preparative high performance liquid chromatograph, wherein the yield is 95.3%;
(13) Dissolving 51.2mg of activated oxygen-responsive molecule after carboxyl activation and 48 mu L of N, N-diisopropylethylamine in 5mL of N, N-dimethylformamide, and reacting with 230mg of the purified polypeptide at 50 ℃ for 24 h; by semi-preparative high performance liquid chromatography (C) 18 Column), and freeze-dried for three days, and the resulting compound was added with 3mL of trifluoroacetic acid to remove the Boc group, to obtain the antibacterial molecule with a yield of 59.1%.
Subjecting the obtained antibacterial molecule to 1 H-NMR and electrospray-mass spectrometry (ESI-MS) analysis, the results were as follows:
(1) 1 H NMR(DMSO-d6,400MHz)δ(ppm):8.89-8.85(t,1H),8.28-8.13(m,5H),8.10-8.02(m,3H),7.88-7.84(t,1H),7.78-7.73(m,3H),7.58-7.54(t,1H),7.47-7.45(d,2H),7.40-7.37(d,1H),7.27-7.13(m,10H),5.29(s,2H),4.55-4.47(m,2H),3.91-3.90(d,2H),3.82-3.68(m,5H),3.64-3.59(m,1H),3.08-3.04(m,1H),2.99-2.95(m,1H),2.86-2.80(m,1H),2.75-2.72(m,1H)
(2)ESI MS(m/z):calcd.for C 48 H 47 BN 8 O 12 ,838.3；found[M-H] - ,837.3。
the above characterization results demonstrate that the antibacterial molecule with the structure is successfully synthesized.
Example 5
Solubility test
The antibacterial molecules prepared in examples 1 to 3 were dissolved in PBS (concentration: 10mM each) and whether or not they were completely dissolved was observed, as shown in FIGS. 1 to 3: the antimicrobial molecules prepared in examples 1-3 were completely soluble in PBS, demonstrating the good water solubility of the antimicrobial molecules of the present invention.
Example 6
In vitro hydrogen peroxide triggering hydrogel formation experiment:
in this embodiment, the change of the antibacterial molecules prepared in embodiments 1 to 4 triggered by hydrogen peroxide in vitro is observed, and the microstructure of the antibacterial molecules is observed under a transmission electron microscope, and the specific operation method is as follows:
the antibacterial molecules prepared in examples 1 to 4 were dissolved in PBS to prepare 10mM solutions, 400 μ L of the solutions were placed in 4mL different sample bottles, 1 μ L of hydrogen peroxide (4 mol/L) was added thereto, or PBS with the same volume was added thereto, and after 120min, the samples were photographed by irradiating with a portable ultraviolet lamp at 365nm or by natural light, and the test results of example 1 are shown in fig. 4 (where a is a sample diagram of a PBS group under natural light, b is a sample diagram of a hydrogen peroxide group under natural light, c is a sample diagram of a PBS group under an ultraviolet lamp, and d is a sample diagram of a hydrogen peroxide group under an ultraviolet lamp): the antibacterial molecules macroscopically form green fluorescent hydrogel under the triggering of hydrogen peroxide.
Samples of the hydrogels formed in examples 1-4 were each sampled at 5 μ L on a carbon-supported copper mesh, stained with 3% uranyl acetate, and placed under a transmission electron microscope for microscopic morphology, as shown in fig. 5-8: the antimicrobial molecules of examples 1-4 microscopically formed a network nanowire structure under the trigger of hydrogen peroxide.
Example 7
In vitro antibacterial experiments:
in this example, the in vitro antibacterial experiment of the antibacterial molecule prepared in example 2 was performed by the following specific operation method: after reacting the antimicrobial molecules prepared in example 2 at different concentrations (2 mM and 10 mM) with hydrogen peroxide at a molar ratio of 1 5 CFU/mL) in a bacterial incubator for 24h, and then measuring the absorbance of each medium at 600nm by using a microplate reader to calculate the survival rate of each bacterium.
The experimental results are shown in fig. 9 (in the figure, e.coli, M-e.coli, PA, M-PA, SA, M-SA, SE, and M-SE represent e.coli, multi-drug resistant e.coli, pseudomonas aeruginosa, multi-drug resistant pseudomonas aeruginosa, staphylococcus aureus, multi-drug resistant staphylococcus aureus, staphylococcus epidermidis, and multi-drug resistant staphylococcus epidermidis, respectively), and it can be seen from the figure that: at low concentration (2 mM), the hydrogel had a limited inhibitory effect on bacteria due to the small number of nanowires formed, whereas at high concentration (10 mM), the hydrogel was stable and contained abundant nanowires, and thus its inhibitory effect was very significant.
Example 8
In vivo antibacterial experiments:
in this example, the following specific procedures were followed to perform in vivo antibacterial experiments on the antibacterial molecules prepared in example 1: mixing 100 μ L (2X 10) 8 CFU/mL) escherichia coli was injected into mice (type: balb/c female mouse, 6-8 weeks old) was subcutaneously injected on the right back side for 30min, and then 100. Mu.L (10 mg/mL) of the antibacterial molecule prepared in example 1 was injected into the same site of the mouse. After 30min, the mice were sacrificed and photographs taken under a hand-held uv lamp, as shown in fig. 10: the green fluorescent hydrogel can be clearly seen at the mouse injection site, indicating that the site of bacterial infection is indeed able to initiate self-assembly of the antimicrobial molecule.
The green hydrogel was taken out, dissolved in 200. Mu.L of PBS and stirred uniformly, 5. Mu.L of the supernatant was sampled on a carbon-supported copper mesh, stained with 3% uranyl acetate, and observed for microscopic morphology using a transmission electron microscope, as shown in FIGS. 11 and 12 (FIG. 12 is a partial enlarged view of the region indicated by the frame line in FIG. 11): a large number of nanowires exist around bacteria, and the nanowires have adhesion to the bacteria, can capture the bacteria in vivo and assist immune cells to kill the bacteria, thereby achieving the purpose of treatment.
The applicant states that the present invention is illustrated by the above examples to the in situ self-assembled antibacterial molecule of the present invention and the preparation method and application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.
Claims (37)
1. An in situ self-assembled antimicrobial molecule, comprising a polypeptide and a reactive oxygen species-responsive molecule at the nitrogen terminus of the polypeptide; the amino acids that make up the polypeptide include glycine and at least two linked phenylalanines;
the structure of the active oxygen response molecule is shown as follows, and the active oxygen response molecule is connected with the nitrogen tail end of the polypeptide through an amido bond;
the number of amino acid residues of the polypeptide is 4-6.
2. The antimicrobial molecule of claim 1, wherein said polypeptide further comprises any one of lysine, tyrosine, or a combination of at least two thereof.
3. A method of preparing an antibacterial molecule according to claim 1 or 2, wherein the method comprises: the 2-chloro-trityl chloride resin is adopted as carrier resin, amino acid with protected terminal amino group and side chain amino group is adopted as raw material, polypeptide is synthesized through a solid phase synthesis method, active oxygen response molecules are connected with the prepared polypeptide, the active oxygen response molecules are removed from the carrier resin, side chain amino group protecting groups are removed, and the antibacterial molecules are obtained.
4. The preparation method according to claim 3, comprising in particular the steps of:
(1) Swelling the carrier resin in dichloromethane;
(2) Adopting amino acid with Fmoc protection obtained from terminal amino group and protected amino acid with side chain amino group as raw material, adding the first amino acid into carrier resin according to the amino acid sequence of the polypeptide to be synthesized to react with the carrier resin and connect;
(3) Removing the Fmoc protecting group on the first amino acid;
(4) Adding a second amino acid to the support resin for reaction and ligation;
(5) Removing the Fmoc protecting group from the second amino acid;
(6) Adding the next amino acid into the carrier resin for reaction and connection, and repeating the operations (4) and (5) until the condensation of all the amino acids is completed to obtain the polypeptide;
(7) Removing the polypeptide obtained in the step (6) from the carrier resin, and removing side chain amino protecting groups;
(8) And (3) reacting and connecting the active oxygen response molecule with the polypeptide prepared in the step (7).
5. The method according to claim 4, wherein the swelling time in the step (1) is 20 to 40min.
6. The method of claim 4, wherein the first amino acid is dissolved in N, N-dimethylformamide with N, N-diisopropylethylamine and added to the carrier resin before being added to the carrier resin in step (2).
7. The method of claim 4, wherein the reaction of step (2) is carried out under a protective atmosphere.
8. The method of claim 7, wherein the protective atmosphere is nitrogen.
9. The method according to claim 4, wherein the reaction time in the step (2) is 1.5 to 3 hours.
10. The method according to claim 4, wherein the temperature of the reaction in the step (2) is 20 to 30 ℃.
11. The method according to claim 4, wherein the reaction in the step (2) is completed, followed by filtration and washing with N, N-dimethylformamide, and the mixture of methanol and methylene chloride is added to the resin to be reacted, followed by filtration and washing with N, N-dimethylformamide.
12. The method according to claim 11, wherein the volume ratio of methanol to dichloromethane is 1.
13. The method of claim 4, wherein the Fmoc protecting group of the first amino acid is removed in step (3) by: and adding the mixed solution of piperidine and N, N-dimethylformamide into the carrier resin for reaction.
14. The method of claim 13, wherein the volume ratio of piperidine to N, N-dimethylformamide is 1.
15. The method of claim 13, wherein the reaction time is 20-40min.
16. The method of claim 13, wherein the reaction temperature is 20-30 ℃.
17. The method according to claim 13, wherein the reaction is terminated and then filtered and washed with N, N-dimethylformamide.
18. The method according to claim 4, wherein the second amino acid is dissolved in N, N-dimethylformamide together with benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate and N, N-diisopropylethylamine before being added to the carrier resin in the step (4).
19. The method of claim 4, wherein the reaction of step (4) is carried out under a protective atmosphere.
20. The method of claim 19, wherein the protective atmosphere is nitrogen.
21. The method according to claim 4, wherein the reaction time in the step (4) is 0.5 to 2 hours.
22. The method according to claim 4, wherein the temperature of the reaction in the step (4) is 20 to 30 ℃.
23. The method according to claim 4, wherein the reaction in the step (4) is completed, followed by filtration and washing with N, N-dimethylformamide.
24. The method of claim 4, wherein the Fmoc protecting group of the second amino acid is removed in step (5) by the same method as in step (3).
25. The preparation method according to claim 4, wherein the specific operation of step (7) is: adding trifluoroacetic acid into carrier resin, reacting, filtering, adding ethyl acetate into filtrate, and filtering to obtain white solid, namely polypeptide.
26. The method of claim 25, wherein the reaction is conducted under a protective atmosphere.
27. The method of claim 26, wherein the protective atmosphere is nitrogen.
28. The method of claim 25, wherein the reaction time is 1.5 to 3 hours.
29. The method of claim 25, wherein the reaction temperature is 20-30 ℃.
30. The method of claim 25, wherein the addition of the glacial ethyl ether is performed in a dropwise manner.
31. The method of claim 25, wherein the white solid is obtained and purified by semi-preparative hplc.
32. The method of claim 4, wherein the reactive oxygen species of step (8) is reacted with the polypeptide of step (7) by dissolving it in N, N-diisopropylethylamine in N, N-dimethylformamide prior to reacting with the polypeptide.
33. The method according to claim 4, wherein the reaction time in the step (8) is 18 to 30 hours.
34. The method according to claim 4, wherein the temperature of the reaction in the step (8) is 40 to 60 ℃.
35. The method according to claim 4, wherein the purification by semi-preparative high performance liquid chromatography and freeze-drying are carried out after the completion of the reaction in the step (8).
36. The preparation method according to claim 4, comprising the following steps:
(1) Swelling the carrier resin in dichloromethane for 20-40min;
(2) Adopting amino acid with Fmoc protection obtained from terminal amino group and protected amino group of side chain as raw materials, dissolving the first amino acid and N, N-diisopropylethylamine in N, N-dimethylformamide according to the amino acid sequence of the polypeptide to be synthesized, adding into carrier resin, introducing nitrogen, reacting with the carrier resin at 20-30 ℃ for 1.5-3h, and connecting; filtering, washing with N, N-dimethylformamide, mixing methanol and dichloromethane in a volume ratio of 1;
(3) Mixing piperidine and N, N-dimethylformamide according to a volume ratio of 1;
(4) Dissolving the second amino acid, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine in N, N-dimethylformamide, adding carrier resin, introducing nitrogen, reacting with the carrier resin at 20-30 ℃ for 0.5-2h, and connecting; filtering and washing with N, N-dimethylformamide;
(5) Mixing piperidine and N, N-dimethylformamide according to a volume ratio of 1;
(6) Adding the next amino acid into the carrier resin for reaction and connection, and repeating the operations (4) and (5) until the condensation of all the amino acids is completed to obtain the polypeptide;
(7) Adding trifluoroacetic acid into carrier resin, introducing nitrogen, reacting at 20-30 deg.C for 1.5-3h, filtering, adding dropwise ethyl acetate into the filtrate, filtering to obtain white solid, and purifying with semi-preparative high performance liquid chromatograph;
(8) Dissolving active oxygen response molecules and N, N-diisopropylethylamine in N, N-dimethylformamide, and reacting with the purified polypeptide at 40-60 ℃ for 18-30 h; purifying by semi-preparative high performance liquid chromatography, and freeze-drying to obtain the antibacterial molecule.
37. Use of an antibacterial molecule according to claim 1 or 2 in the manufacture of an antibacterial medicament.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910320336.9A CN111848731B (en) | 2019-04-19 | 2019-04-19 | In-situ self-assembled antibacterial molecule and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910320336.9A CN111848731B (en) | 2019-04-19 | 2019-04-19 | In-situ self-assembled antibacterial molecule and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111848731A CN111848731A (en) | 2020-10-30 |
| CN111848731B true CN111848731B (en) | 2022-11-29 |
Family
ID=72951942
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910320336.9A Active CN111848731B (en) | 2019-04-19 | 2019-04-19 | In-situ self-assembled antibacterial molecule and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111848731B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114907546B (en) * | 2022-05-12 | 2023-06-02 | 四川大学 | Multifunctional polyurea acid ester derivative and preparation method and application thereof |
| CN114767884B (en) * | 2022-05-17 | 2024-01-26 | 国家纳米科学中心 | Visualized prodrug activating compound, prodrug system, preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105753941A (en) * | 2016-04-26 | 2016-07-13 | 中国石油大学(华东) | Self-assembly antibacterial peptide |
-
2019
- 2019-04-19 CN CN201910320336.9A patent/CN111848731B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105753941A (en) * | 2016-04-26 | 2016-07-13 | 中国石油大学(华东) | Self-assembly antibacterial peptide |
Non-Patent Citations (3)
| Title |
|---|
| Two Functional Motifs Define the Interaction，Internalization and Toxicity of the Cell-Penetrating Antifungal Peptide PAF26 on Fungal Cells;Alberto等;《PLOS ONE》;20130131;第8卷(第1期);全文 * |
| 喹唑啉酮及其衍生物的生物学活性研究进展;方莎莎等;《天然产物研究与开发》;20151231;第27卷;第1973页左栏第1段 * |
| 新型自组装纳米抗菌肽及抗菌肽药物研究;马诗怡等;《中国新药杂志》;20141231;第23卷(第2期);第1.2节，表1-表2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111848731A (en) | 2020-10-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3266311B2 (en) | Novel polypeptide and anti-HIV agent using the same | |
| CN111848731B (en) | In-situ self-assembled antibacterial molecule and preparation method and application thereof | |
| CN111718395B (en) | Prodrug activating compound, prodrug system, preparation method and application thereof | |
| US8217137B2 (en) | Fullerene-based amino acids | |
| WO2015161820A1 (en) | Amphiphilic synthetic antimicrobial peptide, and pharmaceutical composition and use thereof | |
| CN116655766B (en) | Staple peptide and preparation method and application thereof | |
| CN113797335B (en) | Polypeptide-based self-assembled photosensitive nanofiber material, preparation method thereof and application thereof in preparation of antitumor drugs | |
| CN108478802B (en) | High-stability combined drug delivery system for cancer treatment and construction method | |
| Wu et al. | Advances in the study of structural modification and biological activities of anoplin | |
| CN110330553B (en) | Mutant of antibacterial peptide VL25-1 and preparation method and application thereof | |
| CN115192730A (en) | Bispecific polypeptide nano-drug targeting PD-L1 and preparation method and application thereof | |
| CN114699387A (en) | Drug-loaded nanoparticle with core-shell structure and preparation method and application thereof | |
| WO2012153576A1 (en) | Organic nanotube having hydrophobized inner surface, and encapsulated medicinal agent prepared using the nanotube | |
| CN112316203B (en) | Cationic antibacterial peptide and hyaluronic acid composite hydrogel and preparation method thereof | |
| KR101670842B1 (en) | Nano sensor comprising Nanofibril conjugate for cell imaging and antimicrobial activity | |
| CN110642968B (en) | Double-enzyme responsive dumbbell-shaped super-amphiphilic molecule and preparation method and application thereof | |
| CN110028557B (en) | Ce 6-labeled double-chain antibacterial peptide and synthesis method and application thereof | |
| WO2020119773A1 (en) | Amphotericin b peptide derivative | |
| KR101657040B1 (en) | Nanofibril conjugate for detection of metal ion and preparation method thereof | |
| WO2010051667A1 (en) | Pharmaceutical compositions comprising cyclic peptide nanotubes and the uses thereof | |
| CN115317622A (en) | Preparation and application of anti-tumor polypeptide coupled drug | |
| CN112480213B (en) | Amphiphilic anti-tumor polypeptide and application thereof | |
| CN114767884B (en) | Visualized prodrug activating compound, prodrug system, preparation method and application thereof | |
| CN106749522B (en) | Anti-cancer polypeptide containing polyhistidine and preparation and application thereof | |
| CN117720661A (en) | Self-assembled thio cyclic peptide with neuroprotection effect, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |