CN116789771B - Antibacterial polypeptide modified protein derivative and preparation method and application thereof - Google Patents
Antibacterial polypeptide modified protein derivative and preparation method and application thereof Download PDFInfo
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- CN116789771B CN116789771B CN202311084038.7A CN202311084038A CN116789771B CN 116789771 B CN116789771 B CN 116789771B CN 202311084038 A CN202311084038 A CN 202311084038A CN 116789771 B CN116789771 B CN 116789771B
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- coupling reagent
- aryl aldehyde
- antibacterial peptide
- carrier protein
- pyridine disulfide
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- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 15
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 13
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 8
- 102000035118 modified proteins Human genes 0.000 title claims abstract description 6
- 108091005573 modified proteins Proteins 0.000 title claims abstract description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 155
- 238000010168 coupling process Methods 0.000 claims abstract description 120
- 230000008878 coupling Effects 0.000 claims abstract description 119
- 238000005859 coupling reaction Methods 0.000 claims abstract description 119
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 86
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims abstract description 82
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims abstract description 73
- 239000003910 polypeptide antibiotic agent Substances 0.000 claims abstract description 67
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 55
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 55
- 125000003118 aryl group Chemical group 0.000 claims abstract description 52
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 125000003396 thiol group Chemical group [H]S* 0.000 claims abstract description 21
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 20
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 20
- 125000000524 functional group Chemical group 0.000 claims abstract description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 8
- 239000002262 Schiff base Substances 0.000 claims abstract description 4
- 150000004753 Schiff bases Chemical class 0.000 claims abstract description 4
- 150000001299 aldehydes Chemical class 0.000 claims description 67
- 239000000178 monomer Substances 0.000 claims description 45
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- 150000003934 aromatic aldehydes Chemical class 0.000 claims description 20
- -1 p-methoxydimethylbenzene silane Chemical compound 0.000 claims description 19
- 239000008055 phosphate buffer solution Substances 0.000 claims description 19
- 239000000126 substance Substances 0.000 claims description 18
- XJUVFUPQCATDHB-UHFFFAOYSA-N 2,3,4-trimethoxypyridine Chemical compound COC1=CC=NC(OC)=C1OC XJUVFUPQCATDHB-UHFFFAOYSA-N 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 17
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 15
- 229910000077 silane Inorganic materials 0.000 claims description 15
- 230000000845 anti-microbial effect Effects 0.000 claims description 14
- 125000004119 disulfanediyl group Chemical group *SS* 0.000 claims description 14
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- VJOOEHFQQLYDJI-UHFFFAOYSA-N methoxy(dimethyl)silane Chemical compound CO[SiH](C)C VJOOEHFQQLYDJI-UHFFFAOYSA-N 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 10
- 239000003054 catalyst Substances 0.000 claims description 9
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 8
- 230000009471 action Effects 0.000 claims description 8
- HAXFWIACAGNFHA-UHFFFAOYSA-N aldrithiol Chemical compound C=1C=CC=NC=1SSC1=CC=CC=N1 HAXFWIACAGNFHA-UHFFFAOYSA-N 0.000 claims description 8
- RLOWWWKZYUNIDI-UHFFFAOYSA-N phosphinic chloride Chemical compound ClP=O RLOWWWKZYUNIDI-UHFFFAOYSA-N 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000004599 antimicrobial Substances 0.000 claims description 6
- 230000003301 hydrolyzing effect Effects 0.000 claims description 6
- 239000003112 inhibitor Substances 0.000 claims description 6
- 238000006116 polymerization reaction Methods 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 238000005874 Vilsmeier-Haack formylation reaction Methods 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- TXUICONDJPYNPY-UHFFFAOYSA-N (1,10,13-trimethyl-3-oxo-4,5,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-17-yl) heptanoate Chemical compound C1CC2CC(=O)C=C(C)C2(C)C2C1C1CCC(OC(=O)CCCCCC)C1(C)CC2 TXUICONDJPYNPY-UHFFFAOYSA-N 0.000 claims description 4
- UUEWCQRISZBELL-UHFFFAOYSA-N 3-trimethoxysilylpropane-1-thiol Chemical compound CO[Si](OC)(OC)CCCS UUEWCQRISZBELL-UHFFFAOYSA-N 0.000 claims description 4
- 229910021626 Tin(II) chloride Inorganic materials 0.000 claims description 4
- 239000001119 stannous chloride Substances 0.000 claims description 4
- 235000011150 stannous chloride Nutrition 0.000 claims description 4
- 229940073455 tetraethylammonium hydroxide Drugs 0.000 claims description 4
- LRGJRHZIDJQFCL-UHFFFAOYSA-M tetraethylazanium;hydroxide Chemical compound [OH-].CC[N+](CC)(CC)CC LRGJRHZIDJQFCL-UHFFFAOYSA-M 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 238000009833 condensation Methods 0.000 claims description 3
- 230000005494 condensation Effects 0.000 claims description 3
- 238000006482 condensation reaction Methods 0.000 claims description 3
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000006170 formylation reaction Methods 0.000 claims description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- 239000012460 protein solution Substances 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 241000191967 Staphylococcus aureus Species 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 150000003573 thiols Chemical class 0.000 claims 2
- RCJVRSBWZCNNQT-UHFFFAOYSA-N dichloridooxygen Chemical compound ClOCl RCJVRSBWZCNNQT-UHFFFAOYSA-N 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 abstract 5
- 239000000047 product Substances 0.000 description 39
- 235000018102 proteins Nutrition 0.000 description 38
- 229910052739 hydrogen Inorganic materials 0.000 description 15
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 11
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 11
- 238000005481 NMR spectroscopy Methods 0.000 description 10
- 229910052799 carbon Inorganic materials 0.000 description 10
- 229910052760 oxygen Inorganic materials 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 238000003756 stirring Methods 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- 229910052717 sulfur Inorganic materials 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- 238000010907 mechanical stirring Methods 0.000 description 4
- 238000001291 vacuum drying Methods 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000000921 elemental analysis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000003208 petroleum Substances 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229940124350 antibacterial drug Drugs 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000011056 performance test Methods 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 125000003345 AMP group Chemical group 0.000 description 1
- 238000007164 Haack reaction Methods 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 229910008051 Si-OH Inorganic materials 0.000 description 1
- 229910006358 Si—OH Inorganic materials 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000002802 antimicrobial activity assay Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000012456 homogeneous solution Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229920002050 silicone resin Polymers 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- 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
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of synthesis of novel antibacterial materials, in particular to an antibacterial polypeptide modified protein derivative, a preparation method and application thereof, wherein the antibacterial polypeptide modified protein derivative is as follows: antibacterial peptide-aryl aldehyde pyridine disulfide coupling reagent-carrier protein coupling product; the antibacterial peptide contains a primary amino functional group; the carrier protein comprises free sulfhydryl functional groups; the activated sulfhydryl functional group of the aryl aldehyde pyridine disulfide coupling reagent and the free sulfhydryl functional group of the carrier protein are utilized to generate sulfhydryl exchange reaction to form asymmetric disulfide bonds, then Schiff base reaction is carried out between the aryl aldehyde functional group of the aryl aldehyde pyridine disulfide coupling reagent and the primary amino functional group of the antibacterial peptide to form dynamic imine bonds, and the antibacterial peptide-aryl aldehyde pyridine disulfide coupling reagent-carrier protein coupling product is prepared, which enhances the selectivity and the binding force between multiple antibacterial peptide molecules and bacterial cells and improves the antibacterial activity of the antibacterial peptide by modifying the multiple antibacterial peptide.
Description
Technical Field
The invention relates to the technical field of synthesis of novel antibacterial materials, in particular to an antibacterial polypeptide modified protein derivative, a preparation method and application thereof.
Background
Antibacterial peptides (AMPs) with broad-spectrum antibacterial properties and resistance to drugs have evolved as one of the most potential antibiotic alternatives. The main antibacterial mechanism of the antibacterial peptide is that polypeptide molecules interact with bacterial membranes electrostatically, so that bacterial membrane breakage and cell content leakage are induced, and bacteria are killed.
The delivery of antimicrobial peptides is extremely challenging due to their chain length, net positive charge, and proteolytic sensitivity. Therefore, it is important to design a good delivery system that can effectively improve the antimicrobial activity, stability and biotoxicity of the antimicrobial peptide by covalently linking or non-covalently encapsulating the antimicrobial peptide in the delivery system.
Many materials are now found to be useful as delivery systems for antimicrobial peptides, with common materials for drug delivery being proteins, polymers, lipids, micelles, etc., bovine serum albumin BSA-based delivery systems having increased interest due to their biodegradability, non-immunogenicity, biocompatibility and longer half-life. Bovine serum albumin BSA consists of 581 amino acid residues, 35 cysteines of which constitute 17 disulfide bonds, and a free thiol group at position 34 of the peptide chain.
The present invention refers to the following references:
the research on the heat resistance of hydroxyl-containing POSS and modified silicone resin of the same is published in the university of Harbin industry in 2019 by the university of Shuoshi institute of Hao Yuxiu, which discloses the chemical structure and the synthesis method of trisilanolphenyl POSS;
the chemical structure, synthesis method and peptide sequence of SAMP-A4 are disclosed in the "antibacterial peptide SAMP-A4 and derivative design synthesis and pharmaceutical preliminary study" published by Henan university of industry in 2021.
Disclosure of Invention
The invention designs and synthesizes a novel aryl aldehyde pyridine disulfide coupling reagent, which not only can modify multiple antibacterial peptides, but also can be used as a bridge to connect the antibacterial peptides to carrier proteins, so as to improve the antibacterial activity of the antibacterial peptides, and can be applied to the research and development of antibacterial drugs, and the technical scheme is as follows:
in a first aspect, the present invention provides an antimicrobial polypeptide-modified protein derivative, which is: antibacterial peptide-aryl aldehyde pyridine disulfide coupling reagent-carrier protein coupling product;
wherein the antimicrobial peptide comprises a primary amino functional group;
the carrier protein comprises free sulfhydryl functional groups;
the active mercapto functional group of the aryl aldehyde pyridine disulfide coupling reagent and the free mercapto functional group of the carrier protein are utilized to generate mercapto exchange reaction to form asymmetric disulfide bond, and then the aryl aldehyde functional group of the aryl aldehyde pyridine disulfide coupling reagent and the primary amino functional group of the antibacterial peptide are utilized to generate Schiff base reaction to form dynamic imine bond, so that the antibacterial peptide-aryl aldehyde pyridine disulfide coupling reagent-carrier protein coupling product is prepared.
Preferably, the aryl aldehyde pyridine disulfide coupling reagent comprises an aryl aldehyde pyridine disulfide coupling reagent I or/and an aryl aldehyde pyridine disulfide coupling reagent II.
Preferably, the preparation method of the aryl aldehyde pyridine disulfide coupling reagent I comprises the following steps:
step S1, preparing trimethoxy pyridine dithio silane monomer through thiol activation exchange reaction of 2,2' -dithio dipyridine and thiol functional groups of mercaptopropyl trimethoxy silane;
step S2, using Vilsmeier-Haack reaction with N, N-dimethylformamide DMF as formylating reagent, acetonitrile CH 3 CN is solvent, in phosphorus oxychloride POCl 3 Under the action of a catalyst, formylating the p-methoxydimethylbenzene silane to prepare an aromatic aldehyde methoxy dimethylbenzene silane monomer;
and S3, using hydroquinone as a polymerization inhibitor and stannous chloride as an auxiliary polymerization inhibitor, and directly performing hydrolytic condensation reaction on trimethoxy pyridine dithio silane monomer and aromatic aldehyde methoxy dimethyl silane monomer by using a hydrolytic condensation method to prepare the aromatic aldehyde pyridine dithio coupling reagent I.
Preferably, the preparation method of the aryl aldehyde pyridine disulfide coupling reagent II comprises the following steps:
step S1, vilsmeier-Haack reaction is used, N-dimethylformamide DMF is used as formylating reagent, acetonitrile CH 3 CN is solvent, in phosphorus oxychloride POCl 3 Under the action of a catalyst, carrying out formylation reaction on trisilyl phenyl POSS to prepare an aryl aldehyde trisilyl POSS monomer;
and S2, using a vertex angle capping method, using an aromatic aldehyde trisilanol POSS monomer as a raw material, toluene as a solvent and tetraethylammonium hydroxide as a catalyst, and using a trimethoxy pyridine dithio silane monomer as a capping reagent to prepare the aromatic aldehyde pyridine dithio coupling reagent II.
In another aspect, the present invention provides a method for preparing an antimicrobial polypeptide-modified protein derivative, comprising the steps of:
step S1, converting a buffer solution for dissolving carrier protein into a phosphate buffer solution by using a filter, and regulating the protein concentration;
step S2, dissolving an aryl aldehyde pyridine disulfide coupling reagent by using a phosphate buffer solution, and adding the dissolved aryl aldehyde pyridine disulfide coupling reagent into the carrier protein solution in the step S1;
step S3, converting the buffer solution into a phosphate buffer solution after the slow-shaking reaction, and adjusting the concentration of the aryl aldehyde pyridine disulfide coupling reagent-carrier protein;
s4, dissolving the antibacterial peptide by using a phosphate buffer solution, and preparing an antibacterial peptide solution;
and S5, adding an excessive antibacterial peptide solution into the aryl aldehyde pyridine disulfide coupling reagent-carrier protein, covering nitrogen on the solution, carrying out slow shaking reaction, and then fully dialyzing the solution to remove unreacted antibacterial peptide to obtain an antibacterial peptide-aryl aldehyde pyridine disulfide coupling reagent-carrier protein coupling product.
Preferably, the step S1: the pH value of the phosphate buffer solution is 4.0-5.0;
preferably, the step S2: the pH value of the phosphate buffer solution is 4.0-5.0;
preferably, the step S3: the pH value of the phosphate buffer solution is 8.0-10.0, and the concentration is 0.5M;
preferably, the step S4: the pH of the phosphate buffer solution is 8.0-10.0, and the concentration is 0.5M.
Preferably, the carrier protein is selected from one or two of bovine serum albumin BSA and recombinant human serum albumin HSA.
Preferably, the antimicrobial peptide is selected from the group consisting of antimicrobial peptide (SAMP-A4).
In a third aspect, the present invention provides an antimicrobial polypeptide-modified protein derivative which can be used in the development of antimicrobial drugs.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention comprises the following steps: first, synthesizing an aryl aldehyde pyridine disulfide coupling reagent: aryl aldehyde pyridine disulfide coupling reagent I and aryl aldehyde pyridine disulfide coupling reagent II;
secondly, using an aryl aldehyde pyridine disulfide coupling reagent to connect carrier protein containing free sulfhydryl functional groups with antibacterial peptide containing primary amino functional groups to prepare an antibacterial peptide-aryl aldehyde pyridine disulfide coupling reagent-carrier protein coupling product, wherein multiple antibacterial peptides can be modified on the product, the selectivity and the binding force between multiple antibacterial peptide molecules and bacterial cells are enhanced, and the antibacterial efficiency of the antibacterial peptide is improved;
the invention provides a new method for developing antibacterial drugs.
Drawings
FIG. 1 is a chemical structural formula of trimethoxy pyridine dithio silane monomer;
FIG. 2 is a chemical structural formula of an araldehyde methoxydimethylsilane monomer;
FIG. 3 is a chemical structural formula of an araldehyde pyridyldithio coupling reagent I;
FIG. 4 is a chemical structural formula of an araldehyde trisilanol POSS monomer;
FIG. 5 is a chemical structural formula of an araldehyde-pyridyldithio coupling reagent II;
FIG. 6 is a chemical structural formula of an antibacterial peptide (SAMP-A4) -araldehyde-pyridyldithio coupling reagent I-carrier protein (BSA) coupled product I;
FIG. 7 is a chemical structural formula of an antibacterial peptide (SAMP-A4) -araldehyde-pyridyldithio coupling reagent II-carrier protein (BSA) coupled product II;
FIG. 8 shows the results of an antimicrobial assay for the product of the antimicrobial peptide (SAMP-A4) -aralkylthiopyridyldisulfide coupling reagent-carrier protein (BSA) coupling.
Detailed Description
Experimental example I:
the preparation method of the aryl aldehyde pyridine disulfide coupling reagent I comprises the following specific preparation processes:
experimental example I-1:
the trimethoxy pyridine dithio silane monomer is prepared by the following preparation mechanism: the trimethoxy pyridine dithio silane monomer is prepared by the mercapto-activated exchange reaction of 2,2' -dithio dipyridine and mercapto functional groups of mercaptopropyl trimethoxy silane;
the specific preparation steps of the trimethoxy pyridine dithio silane monomer are as follows: adding 2.2g of 2,2' -dithiodipyridine and 100mL of dichloromethane into a reactor under the protection of nitrogen, stirring and mixing for 0.5h, dropwise adding 100mL of dichloromethane dissolved with 2.94g of mercaptopropyl trimethoxysilane into the reaction system at the speed of 2mL/min under the speed of 180rpm of mechanical stirring, mechanically stirring at room temperature for reaction for 24h after the dropwise adding, removing the solvent by a rotary evaporator, separating the product by a silica gel column chromatography (mobile phase: 1 part by volume of ethyl acetate and 9 parts by volume of petroleum ether), and vacuum drying at 60 ℃ to constant weight to prepare trimethoxy pyridine dithiosilane monomer, wherein the chemical structural formula of the trimethoxy pyridine dithiosilane monomer is shown in figure 1;
the nuclear magnetic resonance hydrogen spectrum of trimethoxy pyridine dithio silane monomer is characterized as follows:
1 H NMR(400MHz，CDCl 3 ，δ)：0.56(t，2H，-Si-CH 2 -)，1.55(m，2H，-CH 2 -)，2.54(t，2H，-CH 2 -S-)，3.55(s，9H，-O-CH 3 )，7.21-8.26(m，4H，Ar-H)；
trimethoxy groupPyridinedichiosilane monomer (C) 11 H 19 NO 3 S 2 Si) is as follows:
detection value: 43.28% C,6.23% H,4.59% N,15.74% O,20.98% S,9.18% Si;
theoretical value: 43.25% C,6.27% H,4.59% N,15.71% O,20.99% S,9.19% Si.
Experimental example I-2:
the preparation mechanism of the aromatic aldehyde methoxy dimethyl silane monomer is as follows: the Vilsmeier-Haack reaction is used, N-dimethylformamide DMF is used as formylating reagent, acetonitrile CH 3 CN is solvent, in phosphorus oxychloride POCl 3 Under the action of a catalyst, formylating the p-methoxydimethylbenzene silane to prepare an aromatic aldehyde methoxy dimethylbenzene silane monomer;
the specific preparation steps of the aromatic aldehyde methoxy dimethyl silane monomer are as follows: under the protection of nitrogen, 1.5mLN, N-dimethylformamide DMF and 2mL phosphorus oxychloride POCl at-5 DEG C 3 100mL acetonitrile CH 3 Adding CN into a reactor, stirring for 20min, adding 3.33g of methoxydimethylbenzene silane into the reaction system under the action of mechanical stirring, reacting for 4h under mechanical stirring, filtering and separating the product, washing with cold acetonitrile, separating the product by silica gel column chromatography (mobile phase: 1 part by volume of acetonitrile and 10 parts by volume of petroleum ether), and vacuum drying at 40 ℃ to constant weight to obtain an aromatic aldehyde methoxy dimethylsilane monomer, wherein the chemical structural formula is shown in figure 2;
the nuclear magnetic resonance hydrogen spectrum of the araldehyde methoxydimethylsilane monomer is characterized as follows:
1 H NMR(400MHz，CDCl 3 ，δ)：0.64(s，6H，Si-CH 3 )，3.56(s，3H，Si-O-CH 3 )，7.35-7.84(m，4H，Ar-H)，9.98(s，1H，Ar-CHO)；
aromatic aldehyde methoxy dimethyl silane monomer (C) 10 H 14 O 2 Si) is as follows:
detection value: 61.86% C,7.22% H,16.49% O,14.43% Si;
theoretical value: 61.82% C,7.26% H,16.47% O,14.45% Si.
Experimental example I-3:
the preparation method of the aryl aldehyde pyridine disulfide coupling reagent I comprises the following preparation mechanisms: hydroquinone is used as a polymerization inhibitor, stannous chloride is used as an auxiliary polymerization inhibitor, and an aryl aldehyde pyridine dithio coupling reagent I is prepared by directly carrying out hydrolytic condensation reaction on trimethoxy pyridine dithio silane monomer and aryl aldehyde methoxy dimethyl silane monomer by utilizing a hydrolytic condensation method;
the preparation method of the aryl aldehyde pyridine disulfide coupling reagent I comprises the following steps: adding 3.05g of trimethoxy pyridine dithio silane monomer and 5.94g of aromatic aldehyde methoxy dimethyl silane monomer into a reactor, stirring to form a homogeneous solution, adding 135mg of hydroquinone and 180mg of stannous chloride at the temperature of 40 ℃ in an oil bath, heating to the temperature of 150 ℃ in the oil bath, stirring and reacting for 6 hours, heating to the temperature of 200 ℃ and vacuumizing for 1 hour to prepare an aromatic aldehyde pyridine dithio coupling reagent I, wherein the chemical structural formula of the aromatic aldehyde pyridine dithio coupling reagent I is shown in figure 3;
the nuclear magnetic resonance hydrogen spectrum of the aryl aldehyde pyridine disulfide coupling reagent I is characterized as follows:
1 H NMR(400MHz，CDCl 3 ，δ)：0.55(t，2H，-Si-CH 2 -)，0.62(s，18H，Si-CH 3 )，1.54(m，2H，-CH 2 -)，2.52(t，2H，-CH 2 -S-)，7.20-8.28(m，16H，Ar-H)，10.01(s，3H，Ar-CHO)；
aryl aldehyde pyridine disulfide coupling reagent I (C) 35 H 43 NO 6 S 2 Si 4 ) The elemental analysis results of (a) are as follows:
detection value: 56.07% C,5.74% H,1.87% N,12.82% O,8.54% S,14.95% Si;
theoretical value: 56.04% C,5.78% H,1.87% N,12.80% O,8.55% S,14.97% Si.
Experimental example II:
the preparation method of the aryl aldehyde pyridine disulfide coupling reagent II comprises the following specific preparation processes:
example II-1:
the preparation mechanism of the aromatic aldehyde trisilanol POSS monomer is as follows: usingVilsmeier-Haack reaction with N, N-dimethylformamide DMF as formylating agent and acetonitrile CH 3 CN is solvent, in phosphorus oxychloride POCl 3 Under the action of a catalyst, carrying out formylation reaction on trisilyl phenyl POSS to prepare an aryl aldehyde trisilyl POSS monomer;
the specific preparation steps of the aryl aldehyde trisilanol POSS monomer are as follows: under the protection of nitrogen, 0.375mL of LN, N-dimethylformamide DMF and 0.5mL of phosphorus oxychloride POCl are added at the temperature of minus 5 DEG C 3 100mL acetonitrile CH 3 Adding CN into a reactor, stirring for 20min, adding 4.65g of methoxydimethylbenzene silane into the reaction system under the action of mechanical stirring, mechanically stirring for reacting for 4h, filtering and separating a product, washing the product with cold acetonitrile, adding the precipitated product into 30mL of deionized water, heating to 55 ℃ for hydrolysis for 3h, separating the product by using a silica gel column chromatography (mobile phase: 1 volume part of acetonitrile and 10 volume parts of petroleum ether), and vacuum drying at 40 ℃ to constant weight to obtain an aromatic aldehyde trisilanol POSS monomer, wherein the chemical structural formula of the aromatic aldehyde trisilanol POSS monomer is shown in figure 4;
the nuclear magnetic resonance hydrogen spectrum of the aromatic aldehyde trisilanol POSS monomer is characterized as follows:
1 H NMR(400MHz，CDCl 3 ，δ)：1.18(s，3H，Si-OH)，7.36-7.87(m，28H，Ar-H)，10.02(s，7H，Ar-CHO)；
aromatic aldehyde trisilanol POSS monomer (C 49 H 38 O 19 Si 7 ) The elemental analysis results of (a) are as follows:
detection value: 52.22% C,3.37% H,27.00% O,17.41% Si;
theoretical value: 52.20% C,3.40% H,26.96% O,17.44% Si.
Example II-2:
the preparation method of the aryl aldehyde pyridine disulfide coupling reagent II comprises the following preparation mechanisms: using a top angle cap method, taking an aromatic aldehyde trisilanol POSS monomer as a raw material, toluene as a solvent and tetraethylammonium hydroxide as a catalyst, and taking a trimethoxy pyridine disulfide silane monomer as a cap reagent to prepare an aromatic aldehyde pyridine disulfide coupling reagent II;
the preparation method of the aryl aldehyde pyridine disulfide coupling reagent II comprises the following steps: 1.889g of aromatic aldehyde trisilanol POSS monomer is dissolved in 6mL of toluene, the temperature of a reaction system is controlled to be minus 10 ℃, 0.549g of trimethoxy pyridine dithio silane monomer and 50 mu L of 35% tetraethyl ammonium hydroxide aqueous solution are added, stirring reaction is carried out for 12 hours at room temperature, methanol precipitation, dichloromethane dissolution and vacuum drying are carried out on the product at 60 ℃ until the weight is constant, thus obtaining an aromatic aldehyde pyridine dithio coupling reagent II, the chemical structural formula of which is shown in figure 5;
the nuclear magnetic resonance hydrogen spectrum of the aryl aldehyde pyridine disulfide coupling reagent II is characterized as follows:
1 H NMR(400MHz，CDCl 3 ，δ)：0.54(t，2H，-Si-CH 2 -)，1.54(m，2H，-CH 2 -)，2.55(t，2H，-CH 2 -S-)，7.19-8.27(m，32H，Ar-H)，10.03(s，7H，Ar-CHO)；
aryl aldehyde pyridine disulfide coupling reagent II (C) 57 H 45 NO 19 S 2 Si 8 ) The elemental analysis results of (a) are as follows:
detection value: 51.24% C,3.37% H,1.05% N,22.77% O,4.79% S,16.78% Si;
theoretical value: 51.21% C,3.39% H,1.05% N,22.74% O,4.80% S,16.81% Si.
Experimental example III:
preparation of antibacterial peptide (SAMP-A4) -araldehyde pyridine disulfide-based coupling reagent-carrier protein (BSA) coupled product, the preparation mechanism is as follows:
the activated sulfhydryl functional group of the aryl aldehyde pyridine disulfide coupling reagent is utilized to generate sulfhydryl exchange reaction with the free sulfhydryl functional group of carrier protein (BSA) to form asymmetric disulfide bond, and then the aryl aldehyde functional group of the aryl aldehyde pyridine disulfide coupling reagent is utilized to generate Schiff base reaction with the primary amino functional group of the antibacterial peptide (SAMP-A4) to form dynamic imine bond, thus obtaining the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent-carrier protein (BSA) coupling product.
Example III-1:
preparation of antibacterial peptide (SAMP-A4) -araldehyde pyridine dithio coupling reagent I-Carrier protein (BSA) coupled product I, comprising the following specific steps:
step S1, using a pore size M r A 5000 filter converts the buffer solution dissolving carrier protein (bovine serum albumin BSA) into phosphate buffer solution (pH 5.0) and adjusts the protein concentration to 5mg/mL;
s2, dissolving the aryl aldehyde pyridine disulfide coupling reagent I by using a phosphate buffer solution (pH 5.0), and adding the dissolved aryl aldehyde pyridine disulfide coupling reagent I into the carrier protein solution in the step S1 to ensure that the final concentration of the aryl aldehyde pyridine disulfide coupling reagent I is 2mmol/L;
step S3, slowly shaking at 25 ℃ for 4 hours, then converting the buffer solution into a phosphate buffer solution (0.5M, pH 8.5), and regulating the concentration of the aryl aldehyde pyridine disulfide coupling reagent I-carrier protein (BSA) to 5mg/mL;
step S4, dissolving the antibacterial peptide (SAMP-A4) by using phosphate buffer (0.5M, pH 8.5) and preparing an antibacterial peptide (SAMP-A4) solution with the concentration of 1.5 mg/mL;
step S5, adding an excessive antibacterial peptide (SAMP-A4) solution into an araldehyde pyridine disulfide-based coupling reagent I-carrier protein (BSA), covering nitrogen on the solution, slowly shaking at 20 ℃ for 2 hours, and then fully dialyzing the solution to remove unreacted antibacterial peptide (SAMP-A4) to obtain an antibacterial peptide (SAMP-A4) -araldehyde pyridine disulfide-based coupling reagent I-carrier protein (BSA) coupling product I, wherein the chemical structural formula of the antibacterial peptide (SAMP-A4) -araldehyde pyridine disulfide-based coupling reagent I-carrier protein (BSA) coupling product I is shown in figure 6;
the mass spectrometry analysis result of the antibacterial peptide (SAMP-A4) -aralkyldithiopyridine coupling reagent I-carrier protein (BSA) coupled product I is as follows:
the relative molecular mass of the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent I-carrier protein (BSA) coupling product I is 68525, which is close to the sum 68654 of the relative molecular masses of three single components contained in the product I, and the difference between the molecular masses is about the relative molecular mass of byproducts generated in the coupling process;
wherein the relative molecular mass 1080 of the antimicrobial peptide (SAMP-A4);
relative molecular mass 749 of aryl aldehyde pyridine disulfide coupling reagent I;
relative molecular mass of carrier protein (BSA) 66825;
the following is given: the aralkylated pyridine disulfide coupling reagent I has successfully coupled an antibacterial peptide (SAMP-A4) to a carrier protein (BSA) to form an antibacterial peptide (SAMP-A4) -aralkylated pyridine disulfide coupling reagent I-carrier protein (BSA) coupled product I.
Example III-2:
preparation of antibacterial peptide (SAMP-A4) -araldehyde pyridine dithio coupling reagent II-carrier protein (BSA) coupled product II, the specific procedure of which is shown in example III-1, the chemical structural formula of which is shown in FIG. 7;
the mass spectrum analysis result of the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent II-carrier protein (BSA) coupling product II is as follows:
the relative molecular mass of the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent II-carrier protein (BSA) coupling product II is 72297, which is obviously larger than the sum 69240 of the relative molecular masses of three single components contained in the antibacterial peptide;
wherein the relative molecular mass 1080 of the antimicrobial peptide (SAMP-A4);
the relative molecular mass 1335 of the aryl aldehyde pyridine disulfide coupling reagent II;
relative molecular mass of carrier protein (BSA) 66825;
the following is given: the aralkylated pyridine disulfide coupling reagent II has successfully coupled an antibacterial peptide (SAMP-A4) to a carrier protein (BSA) to form an antibacterial peptide (SAMP-A4) -aralkylated pyridine disulfide coupling reagent II-carrier protein (BSA) coupled product II.
Performance test experiment:
the in vitro antibacterial performance test of the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent-carrier protein (BSA) coupling product comprises the following specific experimental procedures:
step S1, preparing bacterial suspension, wherein the specific experimental steps are as follows: before the bacteria experiment, the used experimental consumables were sterilized in a high-pressure steam sterilizing pot (120 ℃ C., 120 kPa) for 0.5h; gram positive bacteria (staphylococcus aureus S.aureus) and gram negative bacteria (escherichia coli E.coli) are selected as test strains, and the bacterial strains are mixed with LB and 50% glycerol and frozen in an ultralow temperature refrigerator at the temperature of-81 ℃; when in use, the solution is carried outInoculating frozen strain into LB solid culture medium, incubating in 37 deg.C incubator for 48 hr to grow colony, selecting single strain, placing in 3mL LB liquid culture medium, placing in shaking table (37 deg.C, 160 rpm) for culturing for 12 hr, centrifuging (4000 rpm,1 min) bacterial liquid, re-suspending bacteria with PBS buffer (pH 7.2), measuring bacterial liquid concentration with ultraviolet spectrophotometer, diluting to make OD 600 =0.1, yielding a corresponding bacterial concentration of 1×10 8 CFU/mL of bacterial suspension;
step S2, the antibacterial capacity of the antibacterial product is measured by adopting a colony counting method, and the specific experimental steps are as follows:
preparing an antibacterial solution:
an antibacterial peptide (SAMP-A4) -PBS buffer (pH 7.2) solution containing antibacterial peptide (SAMP-A4) at a concentration of 600. Mu.M, 300. Mu.M, 100. Mu.M, 50. Mu.M, 10. Mu.M, 5. Mu.M;
an I-carrier protein (BSA) conjugate product in PBS buffer (pH 7.2) containing the antimicrobial peptide (SAMP-A4) at a concentration of 600. Mu.M, 300. Mu.M, 100. Mu.M, 50. Mu.M, 10. Mu.M, 5. Mu.M;
an antibacterial peptide (SAMP-A4) -araldehyde-pyridine dithio coupling reagent II-carrier protein (BSA) coupling product II-PBS buffer (pH 7.2) solution containing antibacterial peptide (SAMP-A4) at a concentration of 600. Mu.M, 300. Mu.M, 100. Mu.M, 50. Mu.M, 10. Mu.M, 5. Mu.M;
taking 1mL of the above antibacterial solution and 1mL of 1×10 concentration 8 The CFU/mL bacterial suspension is mixed, PBS buffer (pH 7.2) is used as a blank control group, and the mixed solution is subjected to shaking culture at a constant temperature of 37 ℃ and 160rpm for 12 hours;
diluting 0.1mL of the cultured mixed bacterial liquid into 5mL of LPBS buffer solution (pH 7.2), fully mixing, uniformly coating 0.1mL of the diluted liquid on an agar plate, culturing the coated agar plate in a biological incubator at 37 ℃ for 24 hours, counting, and calculating the survival rate of bacteria, wherein the result is shown in figure 8;
by analyzing fig. 8, the following can be concluded:
first: under the condition that the concentration of the antibacterial peptide (SAMP-A4) is the same, the antibacterial performance of the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent I-carrier protein (BSA) coupling product I and the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent II-carrier protein (BSA) coupling product II is obviously superior to that of the free antibacterial peptide (SAMP-A4);
second,: under the same condition that the concentration of the antibacterial peptide (SAMP-A4) is contained, particularly at the concentration higher than 50 mu M, the antibacterial effect of the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent I-carrier protein (BSA) coupling product I and the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent II-carrier protein (BSA) coupling product II on gram-negative bacteria (E.coli) is obviously better than that on gram-positive bacteria (S.aureus);
third,: the antibacterial performance of the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent II-carrier protein (BSA) coupling product II is superior to that of the antibacterial peptide (SAMP-A4) -aryl aldehyde pyridine disulfide coupling reagent I-carrier protein (BSA) coupling product I as a whole.
Claims (7)
1. An antimicrobial polypeptide-modified protein derivative, wherein the antimicrobial polypeptide-modified protein derivative is: antibacterial peptide-aryl aldehyde pyridine disulfide coupling reagent-carrier protein coupling product;
wherein the antibacterial peptide contains a primary amino functional group, and the antibacterial peptide SAMP-A4 is selected to be used;
the carrier protein contains free sulfhydryl functional groups, and bovine serum albumin BSA is selected to be used;
the aryl aldehyde pyridine disulfide coupling reagent is an aryl aldehyde pyridine disulfide coupling reagent I or an aryl aldehyde pyridine disulfide coupling reagent II;
wherein, the chemical structural formula of the aryl aldehyde pyridine disulfide coupling reagent I is as follows:
；
the chemical structural formula of the aryl aldehyde pyridine disulfide coupling reagent II is as follows:
；
the preparation method of the antibacterial polypeptide modified protein derivative comprises the following steps: the active mercapto functional group of the aryl aldehyde pyridine disulfide coupling reagent and the free mercapto functional group of the carrier protein are utilized to generate mercapto exchange reaction to form asymmetric disulfide bond, and then the aryl aldehyde functional group of the aryl aldehyde pyridine disulfide coupling reagent and the primary amino functional group of the antibacterial peptide are utilized to generate Schiff base reaction to form dynamic imine bond, so that the antibacterial peptide-aryl aldehyde pyridine disulfide coupling reagent-carrier protein coupling product is prepared.
2. The antimicrobial polypeptide-modified protein derivative of claim 1, wherein the preparation method of the araldehyde-pyridyldithio coupling reagent i comprises the following steps:
step S1, preparing trimethoxy pyridine dithio silane monomer through thiol activation exchange reaction of 2,2' -dithio dipyridine and thiol functional groups of mercaptopropyl trimethoxy silane;
step S2, using Vilsmeier-Haack reaction with N, N-dimethylformamide DMF as formylating reagent, acetonitrile CH 3 CN is solvent, in phosphorus oxychloride POCl 3 Under the action of a catalyst, formylating the p-methoxydimethylbenzene silane to prepare an aromatic aldehyde methoxy dimethylbenzene silane monomer;
and S3, using hydroquinone as a polymerization inhibitor and stannous chloride as an auxiliary polymerization inhibitor, and directly performing hydrolytic condensation reaction on trimethoxy pyridine dithio silane monomer and aromatic aldehyde methoxy dimethyl silane monomer by using a hydrolytic condensation method to prepare the aromatic aldehyde pyridine dithio coupling reagent I.
3. The antimicrobial polypeptide-modified protein derivative of claim 1, wherein the preparation method of the araldehyde-pyridyldithio coupling reagent ii comprises the following steps:
step S1, vilsmeier-Haack reaction is used, N-dimethylformamide DMF is used as formylating reagent, acetonitrile CH 3 CN as solvent, in threePhosphorus oxychloride POCl 3 Under the action of a catalyst, carrying out formylation reaction on trisilyl phenyl POSS to prepare an aryl aldehyde trisilyl POSS monomer;
and S2, using a vertex angle capping method, using an aromatic aldehyde trisilanol POSS monomer as a raw material, toluene as a solvent and tetraethylammonium hydroxide as a catalyst, and using a trimethoxy pyridine dithio silane monomer as a capping reagent to prepare the aromatic aldehyde pyridine dithio coupling reagent II.
4. The antimicrobial polypeptide-modified protein derivative of claim 1, wherein the antimicrobial polypeptide-modified protein derivative is prepared by the steps of:
step S1, converting a buffer solution for dissolving carrier protein into a phosphate buffer solution by using a filter, and regulating the protein concentration;
step S2, dissolving an aryl aldehyde pyridine disulfide coupling reagent by using a phosphate buffer solution, and adding the dissolved aryl aldehyde pyridine disulfide coupling reagent into the carrier protein solution in the step S1;
step S3, converting the buffer solution into a phosphate buffer solution after the slow-shaking reaction, and adjusting the concentration of the aryl aldehyde pyridine disulfide coupling reagent-carrier protein;
s4, dissolving the antibacterial peptide by using a phosphate buffer solution, and preparing an antibacterial peptide solution;
and S5, adding an excessive antibacterial peptide solution into the aryl aldehyde pyridine disulfide coupling reagent-carrier protein, covering nitrogen on the solution, carrying out slow shaking reaction, and then fully dialyzing the solution to remove unreacted antibacterial peptide to obtain an antibacterial peptide-aryl aldehyde pyridine disulfide coupling reagent-carrier protein coupling product.
5. The antimicrobial polypeptide-modified protein derivative according to claim 4, wherein step S1: the pH value of the phosphate buffer solution is 4.0-5.0;
the step S2: the pH of the phosphate buffer solution is 4.0-5.0.
6. The antimicrobial polypeptide-modified protein derivative according to claim 4, wherein the step S3: the pH value of the phosphate buffer solution is 8.0-10.0, and the concentration is 0.5M;
the step S4: the pH of the phosphate buffer solution is 8.0-10.0, and the concentration is 0.5M.
7. The use of the protein derivative modified by an antimicrobial polypeptide according to claim 1 for the preparation of an antimicrobial drug, wherein the bacterium is escherichia coli or staphylococcus aureus.
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