CN108017690B - Column [5] aromatic artificial transmembrane channel with antibacterial activity and preparation method and application thereof - Google Patents
Column [5] aromatic artificial transmembrane channel with antibacterial activity and preparation method and application thereof Download PDFInfo
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- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 125000003118 aryl group Chemical group 0.000 title claims description 11
- 150000004945 aromatic hydrocarbons Chemical class 0.000 claims abstract description 27
- 241000894006 Bacteria Species 0.000 claims abstract description 15
- 150000001875 compounds Chemical class 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 8
- 201000010099 disease Diseases 0.000 claims abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 33
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 17
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 15
- 229920001184 polypeptide Polymers 0.000 claims description 13
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 10
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 9
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 9
- 229960005055 sodium ascorbate Drugs 0.000 claims description 9
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 244000063299 Bacillus subtilis Species 0.000 claims description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 4
- 241000191967 Staphylococcus aureus Species 0.000 claims description 4
- 241000191963 Staphylococcus epidermidis Species 0.000 claims description 4
- -1 arene compound Chemical class 0.000 claims description 4
- 239000003054 catalyst Substances 0.000 claims description 4
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 abstract description 7
- 229940088710 antibiotic agent Drugs 0.000 abstract description 6
- 230000002949 hemolytic effect Effects 0.000 abstract description 6
- 230000001988 toxicity Effects 0.000 abstract description 5
- 231100000419 toxicity Toxicity 0.000 abstract description 5
- 241001465754 Metazoa Species 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 4
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 102000004310 Ion Channels Human genes 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000005160 1H NMR spectroscopy Methods 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 7
- 210000003743 erythrocyte Anatomy 0.000 description 6
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 229910052927 chalcanthite Inorganic materials 0.000 description 5
- 239000012043 crude product Substances 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- CELJCNRXKZPTCX-XPUUQOCRSA-N Val-Gly-Ala Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O CELJCNRXKZPTCX-XPUUQOCRSA-N 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 108010038745 tryptophylglycine Proteins 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 3
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- FPFOYSCDUWTZBF-IHPCNDPISA-N Leu-Trp-Leu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H]([NH3+])CC(C)C)C(=O)N[C@@H](CC(C)C)C([O-])=O)=CNC2=C1 FPFOYSCDUWTZBF-IHPCNDPISA-N 0.000 description 3
- 239000000232 Lipid Bilayer Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000003385 bacteriostatic effect Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 239000007832 Na2SO4 Substances 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- YTZYHKOSHOXTHA-TUSQITKMSA-N Trp-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=3C4=CC=CC=C4NC=3)CC(C)C)C(O)=O)=CNC2=C1 YTZYHKOSHOXTHA-TUSQITKMSA-N 0.000 description 2
- WHNSHJJNWNSTSU-BZSNNMDCSA-N Val-Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 WHNSHJJNWNSTSU-BZSNNMDCSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- XMPZTFVPEKAKFH-UHFFFAOYSA-P ceric ammonium nitrate Chemical compound [NH4+].[NH4+].[Ce+4].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O XMPZTFVPEKAKFH-UHFFFAOYSA-P 0.000 description 2
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 2
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- XXFUZSHTIOFGNV-UHFFFAOYSA-N 1-bromoprop-1-yne Chemical compound CC#CBr XXFUZSHTIOFGNV-UHFFFAOYSA-N 0.000 description 1
- 241001120493 Arene Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- ZLMFVXMJFIWIRE-FHWLQOOXSA-N Val-Trp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](C(C)C)N ZLMFVXMJFIWIRE-FHWLQOOXSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 206010023332 keratitis Diseases 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 230000032895 transmembrane transport Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses a column [5] with antibacterial activity]An aromatic hydrocarbon artificial transmembrane channel, a preparation method and application thereof, belonging to the technical field of synthesis of compounds with antibacterial activity. The technical scheme provided by the invention has the key points that: column [5] with antibacterial activity]The aromatic hydrocarbon artificial transmembrane channel has a structural general formula as follows:
Description
Technical Field
The invention belongs to the technical field of synthesis of compounds with antibacterial activity, and particularly relates to a column [5] aromatic artificial transmembrane channel with antibacterial activity, and a preparation method and application thereof.
Background
Pathogenic infectious diseases caused by bacteria, viruses, fungi, etc. pose serious threats to the health of human beings and animals. To date, conventional antibiotics are the first choice for the treatment of such infectious diseases. However, with the use of antibiotics, it has been found that the antibiotics are liable to remain, cause allergic reactions, and pollute the environment. In particular, in recent years, the rapid increase of multi-drug resistant pathogenic bacteria has become a serious threat to human society, and the development of novel antibacterial agents has become an urgent task.
Antimicrobial peptides (AMPs) are a class of bioactive peptides widely existing in the biological world, are polypeptide active substances generated by an animal immune defense system to resist exogenous pathogens, and play an important role in regulating the immune function of an organism. The antibacterial peptide is a polypeptide substance generally composed of 12-80 amino acids, has broad-spectrum antibacterial activity on bacteria, fungi and the like, and has the characteristics of strong stability, no immunogenicity and the like. Unlike traditional antibiotics which usually act on a specific step of biosynthesis, the mainstream research at present considers that the sterilization mechanism of the antibacterial peptide is mainly to act on the cell membrane of the bacteria, destroy the integrity of the cell membrane and generate a perforation phenomenon, so that the cell content flows out of the cell and dies, and the unique action mechanism makes the bacteria difficult to generate the drug resistance. Although antibacterial peptide has the advantages of strong antibacterial activity, wide antibacterial spectrum and the like, the antibacterial peptide can affect mammalian cells (such as red blood cells and the like) so as to cause hemolytic toxicity. Therefore, the antibacterial peptide (such as the graminidin A and the like) entering clinical application is mainly used as an external medicine for treating diseases such as skin surface infection, keratitis and the like. Therefore, how to develop antibacterial peptides capable of being applied to the whole body is a problem to be solved urgently by vast pharmaceutical chemists.
The artificial transmembrane channel is an organic molecule obtained by artificial synthesis, and through reasonable design, the molecule can form a nanopore penetrating through a lipid bilayer on a phospholipid bilayer and can realize transmembrane delivery of anions and cations and polar substances. Since the artificial transmembrane channel and the antibacterial peptide have similar mechanisms of action, the transmembrane channel can be formed on the lipid bilayer. However, the artificial transmembrane channel can realize transmembrane transport function by forming a stable nanoscale pore channel in a lipid bilayer, so the structure of the artificial transmembrane channel is often very complex. How to efficiently construct an artificial transmembrane channel is a current research difficulty. The artificial transmembrane channel has the characteristics of high stability, high conveying efficiency, structural diversity and the like. Therefore, in recent years, research on biological activities of the artificial transmembrane channel such as antibacterial activity and anticancer activity has attracted attention. To date, although individual artificial transmembrane channels exhibit antimicrobial activity similar to that of antimicrobial peptides, they all face the same problems: (1) the antibacterial activity is not high; (2) hemolytic toxicity is difficult to eliminate.
In summary, the existing preparation method for the artificial transmembrane channel is long in steps, and a method for efficiently preparing the artificial transmembrane channel is rarely reported. In addition, artificial transmembrane channels having both high antibacterial activity and low hemolytic toxicity have not been reported.
Disclosure of Invention
The invention solves the technical problem of providing a column [5] arene artificial transmembrane channel with antibacterial activity and a preparation method thereof, and the column [5] arene artificial transmembrane channel prepared by the method can be used for preparing medicines for treating or preventing diseases caused by bacteria (such as bacillus subtilis, staphylococcus aureus and staphylococcus epidermidis).
The invention adopts the following technical scheme for solving the technical problems, and the column [5] aromatic artificial transmembrane channel with antibacterial activity is characterized in that the general structural formula is as follows:
wherein R is a polypeptide side chain, and the polypeptide sequence of the polypeptide side chain is as follows:
Val-Gly-Ala-D-Leu-Trp-D-Leu-Trp-Gly-COOH；
Val-Gly-Ala-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH；
Val-Gly-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH；
Val-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH；
Val-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH。
the invention relates to a preparation method of a column [5] aromatic artificial transmembrane channel with antibacterial activity, which is characterized by comprising the following specific steps: sequentially adding a diacetylene-modified column [5] arene compound, an azide-modified polypeptide compound, a catalyst copper sulfate pentahydrate and sodium ascorbate into a reaction vessel, adding DMSO for dissolving, reacting at room temperature for 12 hours, monitoring by TLC that raw materials react completely, filtering, drying and purifying by column chromatography to obtain a column [5] arene artificial transmembrane channel with antibacterial activity, wherein the reaction equation in the preparation process is as follows:
further preferably, the feeding molar ratio of the dialkynyl-modified column [5] arene compound, the azide-modified polypeptide compound and the catalyst copper sulfate pentahydrate to sodium ascorbate is 8:25:0.8: 4.
The invention relates to application of a column [5] aromatic artificial transmembrane channel with antibacterial activity in preparing a medicament for treating or preventing diseases caused by bacteria, wherein the bacteria are bacillus subtilis, staphylococcus aureus or staphylococcus epidermidis.
According to the invention, column [5] arene is chemically modified through Click Chemistry, a polypeptide side chain with beta helical conformation is introduced into a column [5] arene skeleton, a monomolecular artificial transmembrane channel with a tubular structure is efficiently constructed, and the compound can be efficiently embedded into a bacterial cell membrane to change the permeability of the cell membrane, so that the bacterial growth is inhibited; the compound has excellent antibacterial performance, can be better applied to the prevention and the treatment of animal and plant diseases caused by bacteria (such as bacillus subtilis, staphylococcus aureus, staphylococcus epidermidis and the like), has low hemolytic toxicity, and can be used as a candidate drug of ion channel antibiotics applied to the whole body.
Detailed Description
The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.
Example 1
(1) Preparation of pillar [4] arene [1] quinone
Will column [5]Dissolving aromatic hydrocarbon (3g,4mmol) in dichloromethane (50mL), adding ceric ammonium nitrate (2.19g, 4mmol), stirring at room temperature for 30min, adding water for extraction, and extracting with anhydrous Na2SO4Drying, filtering and spin-drying the filtrate. Column chromatography (ethyl acetate: petroleum ether) gave 1.44g of a solid in 50% yield.
1H NMR(DMSO-d6,600MHz):8.22(s,2H),6.84(s,2H),6.83(s,2H),6.79(s,2H),6.78(s,2H),6.54(s,2H),3.71(s,6H),3.68(s,6H),3.65(s,12H),3.63(s,6H),3.55(s,4H).HRMS:Calcd for C43H44NaO10[M+Na]+:743.2832.Found:743.2867。
(2) Preparation of dihydroxy column [5] arenes
Will column [4]Aromatic hydrocarbons [1]]Quinone (0.5g,0.693mmol), sodium borohydride (0.13g,3.448mmol) were dissolved in tetrahydrofuran and stirred at room temperature. Extracting with ethyl acetate, washing the organic phase with saturated sodium chloride solution, and adding anhydrous Na2SO4Drying, filtering and spin-drying the filtrate. Column chromatography gave 0.27g of a pale yellow solid in 54% yield.
1H NMR(DMSO-d6,600MHz):8.23(s,2H),6.85(s,2H),6.84(s,2H),6.80(s,4H),3.72(s,6H),3.69(s,6H),3.66(s,12H),3.64(s,2H),3.56(s,2H).ESI-HRMS for C43H46NaO10[M+Na]+calcd:745.2989,found:745.2997。
(3) Preparation of diyne column [5] arene
Compound 2-19(0.1g,0.139mmol) and potassium carbonate (0.3g,0.694mmol) were weighed into a flask, acetonitrile (2.5mL), bromopropyne (0.12mL,1.108mmol) were added, and reacted for 2 h. And after the reaction is complete, carrying out suction filtration, and spin-drying the filtrate. Column chromatography (ethyl acetate: petroleum ether) gave 0.06g of red solid compound 2-20 in 52% yield.
1H NMR(DMSO-d6,600MHz):6.87(s,2H),6.82(s,2H),6.79(s,4H),6.78(s,2H),4.66(s,2H),3.70-3.66(m,34H),3.31(s,2H).ESI-HRMS for C49H50O10[M+Na]+calcd:821.3302,found:821.3261。
Example 2
Preparation of double octapeptide side chain modified column [5] arene
Azide-modified octapeptide (48mg,0.05mmol,3equiv) was dissolved in DMSO (3mL) and applied to a diyne column [5]]Arene (13mg,0.016mmol), sodium ascorbate (1.5mg,0.008mmol,0.5equiv) and CuSO4·5H2O (0.4mg, 1.6. mu. mol,0.1 equiv). After stirring for 12h, spin-dry. The crude product was purified by HPLC to give 30mg of a white solid in 67% yield.
1H NMR(DMSO-d6,600MHz):12.55(s,2H),10.76(s,4H),8.87-7.96(m,20H),7.61(t,J＝6.0Hz,4H),7.30(t,J＝6.0Hz,4H),7.13-6.92(m,14H),6.78(d,J＝6.0Hz,4H),6.75(d,J＝6.0Hz,4H),5.29-4.97(m,9H),4.55-3.79(m,22H),3.51(d,J＝6.0Hz,8H),3.20-2.87(m,11H),2.02-1.97(m,3H),1.47-1.44(m,5H),1.24-1.06(m,27H),0.88(m,12H),0.69-0.59(m,25H).ESI-HRMS for C145H180N26O30[M+2H]2+calcd:1384.1769,found:1384.1838。
Example 3
Preparation of pillar [5] arene modified by side chain of didecapeptide
Azide-modified decapeptide (64mg,0.05mmol,3equiv) was dissolved in DMSO (3mL) and applied to a diyne column [5]]Arene (13mg,0.016mmol), sodium ascorbate (1.5mg,0.008mmol,0.5equiv) and CuSO4·5H2O (0.4mg, 1.6. mu. mol,0.1 equiv). After stirring for 12h, spin-dry. The crude product was purified by HPLC to yield 32mg of a white solid in 59% yield.
1H NMR(DMSO-d6,600MHz):10.76(s,2H),10.72(s,2H),10.71(s,2H),8.48(d,2H),8.38(t,2H),8.34-8.31(m,4H),8.18(s,2H),8.15(d,4H),8.04(d,2H),7.99(d,2H),7.92(d,2H),7.83(d,2H),7.59(q,6H),7.30-7.27(m,6H),7.12(s,2H),7.08(s,4H),7.02-6.99(m,8H),6.96-6.91(m,6H),6.78(d,4H),6.75(d,4H),5.32(t,2H),5.28-5.19(m,4H),5.02-4.96(m,4H),4.60-4.52(m,6H),4.40-4.35(m,2H),4.25-4.15(m,8H),3.80(d,4H),3.66-3.65(m,24H),3.51(d,6H),3.21-3.18(m,4H),3.09-3.07(m,4H),2.93-2.86(m,6H),2.02-1.96(m,6H),1.82-1.77(m,2H),1.48-1.43(m,2H),1.30-1.28(m,4H),1.16(d,6H),1.08-1.04(m,6H),1.01-0.97(m,2H),0.93-0.90(m,2H),0.87(d,12H),0.63(d,6H),0.58-0.56(m,16H),0.53(d,10H).HRMS:calcd for C177H220N32O34[M+2H]2+1669.3252,found 1669.3451。
Example 4
Preparation of pillar [5] arene modified by side chain of didodecyl peptide
Azide-modified dodecapeptide (74mg,0.05mmol,3equiv) was dissolved in DMSO (3mL) and applied to a diyne column [5]]Arene (13mg,0.016mmol), sodium ascorbate (1.5mg,0.008mmol,0.5equiv) and CuSO4·5H2O (0.4mg, 1.6. mu. mol,0.1 equiv). After stirring for 12h, spin-dry. The crude product was purified by HPLC to give 41mg of a white solid in 68% yield.
1H NMR(DMSO-d6,600MHz):10.79(s,2H),10.76(s,2H),10.71(s,2H),8.51(d,2H),8.42(t,2H),8.36-8.33(m,4H),8.20(br,6H),8.07-8.06(m,2H),8.00-7.89(m,8H),7.81(d,2H),7.59(d,4H),7.55(d,2H),7.30-7.26(m,6H),7.12(s,2H),7.08(s,4H),7.04-6.99(m,8H),6.96-6.90(m,6H),6.78(d,4H),6.75(d,4H),5.33-5.21(m,6H),5.02-4.97(m,4H),4.59-4.52(m,6H),4.41-4.39(m,2H),4.32-4.30(m,4H),4.23(br,4H),4.18-4.15(m,4H),3.80(d,4H),3.67-3.66(m,24H),3.52(d,6H),3.19(d,2H),3.10-3.08(m,4H),2.92-2.86(m,6H),2.03-1.98(m,8H),1.80-1.75(m,2H),1.29-1.27(m,2H),1.17(d,6H),1.10-1.04(m,10H),0.98-0.94(m,2H),0.88(br,12H),0.85-0.83(m,4H),0.80-0.78(m,24H),0.62(d,6H),0.57-0.53(m,22H),0.50(d,6H).HRMS:calcd for C197H256N36O38[M+2H]2+1867.9637,found 1867.9733。
Example 5
Preparation of pillar [5] arene modified by side chain of didetetradecapeptide
Azide-modified tetradecapeptide (83mg,0.05mmol,3equiv) was dissolved in DMSO (3mL) and applied to a diyne column [5]]Arene (13mg,0.016mmol), sodium ascorbate (1.5mg,0.008mmol,0.5equiv) and CuSO4·5H2O (0.4mg, 1.6. mu. mol,0.1 equiv). After stirring for 12h, spin-dry. The crude product was purified by HPLC to yield finally 38mg of a white solid in 57% yield.
1H NMR(DMSO-d6,600MHz):12.56(br,2H),10.76(br,4H),10.68(s,2H),8.51(d,2H),8.37(br,4H),8.32(br,2H),8.19(s,2H),8.18-8.15(m,4H),8.04(br,2H),7.95-7.91(m,8H),7.81(br,2H),7.73(br,2H),7.58(d,4H),7.54(d,2H),7.30-7.26(m,6H),7.11(s,2H),7.08(s,4H),7.04-6.99(m,8H),6.96-6.89(m,6H),6.78(d,4H),6.75(d,4H),5.33-5.21(m,4H),5.02-4.97(m,4H),4.55-4.53(m,6H),4.31-4.17(m,18H),3.79-3.75(m,6H),3.70-3.64(m,30H),3.52(d,6H),3.21-3.16(m,2H),3.09(br,4H),2.90-2.88(m,6H),2.03-1.94(m,8H),1.81(br,2H),1.57-1.52(m,2H),1.46-1.44(m,4H),1.22(s,4H),1.20(d,6H),1.16(d,6H),1.09(br,8H),0.96(br,2H),0.89(d,12H),0.84(d,8H),0.81-0.77(m,28H),0.63(d,6H),0.57-0.52(m,28H).HRMS:calcd for C215H288N40O42[M+2H]2+2052.0849,found 2052.1042。
Example 6
Preparation of dicetyl peptide side chain modified column [5] arene
Azide-modified hexadecapeptide (97mg,0.05mmol,3equiv) was dissolved in DMSO (3mL) and applied to a diyne column [5]]Arene (13mg,0.016mmol), sodium ascorbate (1.5mg,0.008mmol,0.5equiv) and CuSO4·5H2O (0.4mg, 1.6. mu. mol,0.1 equiv). After stirring for 12h, spin-dry. The crude product was purified by HPLC to yield 46mg of a white solid in 61% yield.
1H NMR(DMSO-d6,600MHz):12.55(br,2H),10.76(br,6H),10.69(s,2H),8.51(d,2H),8.37(br,6H),8.19-8.14(m,10H),7.99-7.93(m,12H),7.77-7.74(m,4H),7.58-7.52(m,8H),7.29-7.27(m,8H),7.11(s,2H),7.08(s,6H),7.03-7.00(m,10H),6.95-6.89(m,8H),6.78(br,4H),6.75(d,4H),5.33-5.21(m,4H),5.03-4.97(m,4H),4.2(br,8H),4.30-4.15(m,20H),3.79-3.75(m,6H),3.70-3.64(m,30H),3.52(d,6H),3.25-3.21(m,2H),3.17-3.12(m,6H),2.90(br,8H),2.03-1.97(m,6H),1.81-1.77(m,2H),1.56-1.52(m,2H),1.46(br,4H),1.22-1.12(m,28H),0.89-0.77(m,52H),0.63-0.51(m,46H).HRMS:calcd for C249H329KN46O46[M+H+K]2+2370.2262,found 2370.2216。
Example 7
Bacteriostatic activity test for bacteria
(1) Preparation of liquid culture Medium
0.2515g of yeast extract, 0.5020g of tryptone and 0.5030g of NaCl are weighed into a glass bottle by an electronic balance, respectively, and 50mL of secondary distilled water is added until complete dissolution, and the pH is adjusted to 7.51 by NaOH. 10mL of the culture medium was pipetted into the test tubes with pipettes equipped with 500. mu.L pipette tips.
(2) Activation of bacterial species and preparation of bacterial solution
On two days before the test, four test bacteria are respectively inoculated on test inclined planes, the test inclined planes are placed at the constant temperature of 28 ℃ for culturing for 48 hours, 2mL of sterilized normal saline with the weight percent of 0.85 percent is used for washing the bacterial lawn on each inclined plane, and the bacterial lawn is evenly shaken for standby.
(3) Floor board
A96-well plate is taken, a proper amount of liquid culture medium is added into the plate by using a pipette gun, the plate is gently shaken to be uniformly mixed, 200 mu L of bacterial liquid is added, and 2.5 mu L of pure DMSO and sample liquid with different concentrations are respectively added.
(4) Measuring method
The prepared 96-well plate was shaken in a constant temperature shaker (37 ℃) for 12 hours, and then absorbance was measured with a microplate reader. Three replicates were set up each time, and the same conditions were done three times.
Bacterial survival (%) ═ (OD)sample+bac-ODbroth only)/(ODDMSO+bac-ODbroth only)×100％。
Example 8
Hemolytic toxicity test for red blood cells
(1) Preparation of red blood cell suspension
Fresh SD rat blood was taken and rat red blood cells were isolated by centrifugation at 3500 rpm for 5 min. The separated red blood cells were washed with PBS buffer to clarify the supernatant, and then redispersed in PBS buffer (1%, v/v) for further use.
(2) Floor board
A 96-well plate was taken and 200 μ L of red blood cell suspension was added to each well followed by 2.5 μ L of sample molecules in DMSO solution or neat DMSO in triplicate. The 96-well plate was gently shaken and incubated at 37 ℃ for 30 min.
(3) Measuring method
The above 96-well plate was centrifuged at 3500 rpm for 10min, and an equal amount (50. mu.L) of the supernatant in each well was added to a new 96-well plate, followed by dilution to 100. mu.L with PBS buffer. The 96-well plate was then measured using a microplate reader at 562 nm.
Hemolysis rate (%) - (A)sample-ADMSO)/(Atriton X-100-ADMSO)×100％。
TABLE 1 numbering, chemical Structure, yield of typical Compounds
Table 2 shows the results of the bacteriostatic activity test of typical compounds against representative bacteria, and compound numbers 1 to 5 in table 2 correspond to those in table 1.
TABLE 2 bacteriostatic Activity of typical Compounds on representative bacteria
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.
SEQUENCE LISTING
<110> university of south Henan university
<120> column [5] aromatic artificial transmembrane channel with antibacterial activity, and preparation method and application thereof
<130> 2017
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> RNA
<213> Artificial sequence (Artificial sequence)
<400> 1
Val Gly Ala Leu Trp Leu Trp Gly
<210> 2
<211> 10
<212> RNA
<213> Artificial sequence (Artificial sequence)
<400> 1
Val Gly Ala Val Trp Leu Trp Leu Trp Gly
<210> 3
<211> 12
<212> RNA
<213> Artificial sequence (Artificial sequence)
<400> 1
Val Gly Ala Val Val Val Trp Leu Trp Leu Trp Gly
<210> 4
<211> 14
<212> RNA
<213> Artificial sequence (Artificial sequence)
<400> 1
Val Gly Ala Leu Ala Val Val Val Trp Leu Trp Leu Trp Gly
<210> 5
<211> 16
<212> RNA
<213> Artificial sequence (Artificial sequence)
<400> 1
Val Gly Ala Leu Ala Val Val Val Trp Leu Trp Leu Trp Leu Trp Gly
Claims (4)
1. The column [5] aromatic artificial transmembrane channel with antibacterial activity is characterized by having a structural general formula as follows:
wherein R is a polypeptide side chain, and the polypeptide sequence of the polypeptide side chain is as follows:
Val-Gly-Ala-D-Leu-Trp-D-Leu-Trp-Gly-COOH；
Val-Gly-Ala-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH；
Val-Gly-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH；
Val-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH；
Val-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH。
2. the preparation method of the column [5] aromatic artificial transmembrane channel with antibacterial activity, which is characterized by comprising the following steps: sequentially adding a diacetylene-modified column [5] arene compound, an azide-modified polypeptide compound, a catalyst copper sulfate pentahydrate and sodium ascorbate into a reaction vessel, adding DMSO for dissolving, reacting at room temperature until TLC monitors that raw materials are completely reacted, filtering, drying, and purifying by column chromatography to obtain a column [5] arene artificial transmembrane channel with antibacterial activity as a target product, wherein the reaction equation in the preparation process is as follows:
3. the method for preparing column [5] aromatic artificial transmembrane channel with antibacterial activity according to claim 2, which is characterized in that: the feeding molar ratio of the dialkynyl-modified column [5] arene compound, the azide-modified polypeptide compound and the catalyst copper sulfate pentahydrate to sodium ascorbate is 8:25:0.8: 4.
4. The use of the column [5] aromatic artificial transmembrane channel having antibacterial activity according to claim 1 for the preparation of a medicament for treating or preventing diseases caused by bacteria, such as bacillus subtilis, staphylococcus aureus or staphylococcus epidermidis.
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| CN102911071A (en) * | 2012-09-29 | 2013-02-06 | 上海大学 | Super-molecule complex containing amino acid and water-solubility pillar [5] arene and preparation method for complex |
| WO2017025951A1 (en) * | 2015-08-10 | 2017-02-16 | Ramot At Tel-Aviv University Ltd. | Pillararenes and uses thereof |
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| CN102911071A (en) * | 2012-09-29 | 2013-02-06 | 上海大学 | Super-molecule complex containing amino acid and water-solubility pillar [5] arene and preparation method for complex |
| WO2017025951A1 (en) * | 2015-08-10 | 2017-02-16 | Ramot At Tel-Aviv University Ltd. | Pillararenes and uses thereof |
| CN106974897A (en) * | 2017-02-20 | 2017-07-25 | 西北农林科技大学 | One kind targeting multi-functional cerium dioxide nano medicine-carried system of stimulating responsive |
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