CN102329375A - Group of terminal-amidated antibacterial peptides - Google Patents
Group of terminal-amidated antibacterial peptides Download PDFInfo
- Publication number
- CN102329375A CN102329375A CN201110291548A CN201110291548A CN102329375A CN 102329375 A CN102329375 A CN 102329375A CN 201110291548 A CN201110291548 A CN 201110291548A CN 201110291548 A CN201110291548 A CN 201110291548A CN 102329375 A CN102329375 A CN 102329375A
- Authority
- CN
- China
- Prior art keywords
- lys
- arg
- val
- phe
- leu
- 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.)
- Pending
Links
- 230000000844 anti-bacterial effect Effects 0.000 title abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 title description 23
- 102000004196 processed proteins & peptides Human genes 0.000 title description 17
- 239000003910 polypeptide antibiotic agent Substances 0.000 claims abstract description 64
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 239000003814 drug Substances 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims abstract description 5
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims abstract 15
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims abstract 15
- 206010017533 Fungal infection Diseases 0.000 claims abstract 2
- 208000031888 Mycoses Diseases 0.000 claims abstract 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 11
- QYLJIYOGHRGUIH-CIUDSAMLSA-N Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCNC(N)=N QYLJIYOGHRGUIH-CIUDSAMLSA-N 0.000 claims description 8
- WYBVBIHNJWOLCJ-IUCAKERBSA-N Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCNC(N)=N WYBVBIHNJWOLCJ-IUCAKERBSA-N 0.000 claims description 8
- PQBHGSGQZSOLIR-RYUDHWBXSA-N Arg-Phe Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PQBHGSGQZSOLIR-RYUDHWBXSA-N 0.000 claims description 8
- DAQIJMOLTMGJLO-YUMQZZPRSA-N Arg-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCNC(N)=N DAQIJMOLTMGJLO-YUMQZZPRSA-N 0.000 claims description 8
- FMIIKPHLJKUXGE-GUBZILKMSA-N Lys-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN FMIIKPHLJKUXGE-GUBZILKMSA-N 0.000 claims description 8
- ATIPDCIQTUXABX-UWVGGRQHSA-N Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN ATIPDCIQTUXABX-UWVGGRQHSA-N 0.000 claims description 8
- QCZYYEFXOBKCNQ-STQMWFEESA-N Lys-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QCZYYEFXOBKCNQ-STQMWFEESA-N 0.000 claims description 8
- YQAIUOWPSUOINN-IUCAKERBSA-N Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN YQAIUOWPSUOINN-IUCAKERBSA-N 0.000 claims description 8
- WYBVBIHNJWOLCJ-UHFFFAOYSA-N N-L-arginyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCCN=C(N)N WYBVBIHNJWOLCJ-UHFFFAOYSA-N 0.000 claims description 8
- IBIDRSSEHFLGSD-YUMQZZPRSA-N Val-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-YUMQZZPRSA-N 0.000 claims description 8
- WGHVMKFREWGCGR-SRVKXCTJSA-N Val-Arg-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N WGHVMKFREWGCGR-SRVKXCTJSA-N 0.000 claims description 8
- PAPWZOJOLKZEFR-AVGNSLFASA-N Val-Arg-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N PAPWZOJOLKZEFR-AVGNSLFASA-N 0.000 claims description 8
- JKHXYJKMNSSFFL-IUCAKERBSA-N Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN JKHXYJKMNSSFFL-IUCAKERBSA-N 0.000 claims description 8
- GVJUTBOZZBTBIG-AVGNSLFASA-N Val-Lys-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N GVJUTBOZZBTBIG-AVGNSLFASA-N 0.000 claims description 8
- YMTOEGGOCHVGEH-IHRRRGAJSA-N Val-Lys-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O YMTOEGGOCHVGEH-IHRRRGAJSA-N 0.000 claims description 8
- 108010036533 arginylvaline Proteins 0.000 claims description 8
- 108010038320 lysylphenylalanine Proteins 0.000 claims description 8
- 108010017391 lysylvaline Proteins 0.000 claims description 8
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 claims description 8
- 108010073969 valyllysine Proteins 0.000 claims description 8
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 6
- 125000003290 L-leucino group Chemical group [H]OC(=O)[C@@]([H])(N([H])[*])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] 0.000 claims description 4
- -1 Val Chemical compound 0.000 claims description 4
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 208000035143 Bacterial infection Diseases 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 208000022362 bacterial infectious disease Diseases 0.000 claims 1
- 239000003085 diluting agent Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 11
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000007790 solid phase Substances 0.000 abstract description 2
- 241000192125 Firmicutes Species 0.000 abstract 1
- 230000009435 amidation Effects 0.000 abstract 1
- 238000007112 amidation reaction Methods 0.000 abstract 1
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 208000027096 gram-negative bacterial infections Diseases 0.000 abstract 1
- 208000027136 gram-positive bacterial infections Diseases 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 description 44
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 42
- 229920005989 resin Polymers 0.000 description 16
- 239000011347 resin Substances 0.000 description 16
- 241000894006 Bacteria Species 0.000 description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 14
- 229920001184 polypeptide Polymers 0.000 description 11
- 230000000855 fungicidal effect Effects 0.000 description 10
- 230000002949 hemolytic effect Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 238000005859 coupling reaction Methods 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 230000008878 coupling Effects 0.000 description 7
- 238000010168 coupling process Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 4
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 229960003311 ampicillin trihydrate Drugs 0.000 description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 4
- KGZGFSNZWHMDGZ-KAYYGGFYSA-N pexiganan Chemical compound C([C@H](NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=CC=C1 KGZGFSNZWHMDGZ-KAYYGGFYSA-N 0.000 description 4
- 229950001731 pexiganan Drugs 0.000 description 4
- 108010062940 pexiganan Proteins 0.000 description 4
- 230000008961 swelling Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 230000000845 anti-microbial effect Effects 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 206010061217 Infestation Diseases 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 150000003053 piperidines Chemical class 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 1
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000006035 Tryptophane Substances 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 230000002070 germicidal effect Effects 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- MRJJQAVHEGLQJJ-UHFFFAOYSA-N n-formylacetamide Chemical class CC(=O)NC=O MRJJQAVHEGLQJJ-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004262 preparative liquid chromatography Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明提供一组末端酰胺化的新抗菌肽，这些抗菌肽对耐药细菌菌株具有强的杀菌活性。本发明合成抗菌肽的制备方法可以是固相化学法或是通过基因工程表达获得。本发明合成抗菌肽可以用于制备治疗革兰氏阳性菌、革兰氏阴性菌和真菌感染引起的疾病的药物。The invention provides a group of novel antimicrobial peptides with terminal amidation, and these antimicrobial peptides have strong bactericidal activity on drug-resistant bacterial strains. The preparation method of the synthetic antimicrobial peptide of the present invention can be a solid-phase chemical method or obtained through genetic engineering expression. The synthetic antibacterial peptide of the invention can be used to prepare medicines for treating diseases caused by Gram-positive bacteria, Gram-negative bacteria and fungal infections.
Description
Technical field
The present invention relates to the amidated antibacterial peptide of a group end, its preparation method and the application in the medicine of preparation treatment bacterium, fungi infestation, this antibacterial peptide has significant fungicidal activity to bacterium and fungi and Resistant strain thereof.
Background technology
Antibacterial peptide is an organism through the micromolecule polypeptide of a kind of biologically active of inducing generation, generally is made up of 20-60 amino acid, and molecular weight is about 2000-7000D.Along with Medical Immunology and molecular biological developing rapidly, the research of antibacterial peptide more and more becomes the heat subject in biotechnology and the biomedicine field.Up to now, oneself has found to surpass more than 200 kind of antibacterial peptide on many animals (especially insect), plant, mikrobe and human body, and this type small peptide not only has the germicidal action of wide spectrum to bacterium, fungi, and virus, protozoon and cancer cells are also had attack function.Clinical trial also shows, maybe possibly cause under the situation of courses of infection the organism infection germ, and antibiotic Toplink is killed the germ of having invaded fast, and can stop germ continue infect.
Along with to the deepening continuously of antibacterial peptide primary structure and higher structure research, oneself has many investigators that the 26S Proteasome Structure and Function of these antibacterial peptides is studied, and finds that alpha-helix degree and its fungicidal activity in the molecule are closely related under the hydrophobic environment of analogue membrane.Result of study shows that antibacterial peptide is to make bacterial cell membrane seepage and killing bacteria (Nakajima Y.et al., J.Biol.Chem, 262:1665-1669 through the integrity of destroying film in addition; ZasloffM.Nature, 2002,415:389-395).Therefore there is the people to attempt through α-Luo Xuanjiegou in the increase molecule or improve to contain polypeptide (the Broth W.B.et al. that the amino acid whose ratio of positive charge is sought stronger anti-microbial activity in the polypeptide; Antimicrobial Agents Chemotherapy; 2001,45:1894-1895; Hong S.Y. etal., Peptides, 2001,22:1669-1674).
Summary of the invention
The present invention relates to a kind of antibacterial peptide of forming by the aminoacid sequence of SEQ.ID NO:1 formula shown I:
R1-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-R2-NH
2(SEQ.ID?NO：1)
Formula I
Wherein:
R1: be Leu, Ile, Val, Phe, Lys-Leu, Lys-Ile, Lys-Val, Lys-Phe, Arg-Leu, Arg-Ile, Arg-Val, Arg-Phe or do not exist;
R2:Val, Val-Lys, Val-Arg, Val-Lys-Lys, Val-Lys-Arg; Val-Lys-Lys-Leu, Val-Lys-Lys-Ile, Val-Lys-Lys-Val, Val-Lys-Lys-Phe, Val-Lys-Arg-Leu; Val-Lys-Arg-Ile, Val-Lys-Arg-Val, Val-Lys-Arg-Phe, Val-Arg-Lys, Val-Arg-Arg; Val-Arg-Lys-Leu, Val-Arg-Lys-Ile, Val-Arg-Lys-Val, Val-Arg-Lys-Phe; Val-Arg-Arg-Leu, Val-Arg-Arg-Ile, Val-Arg-Arg-Val, Val-Arg-Arg-Phe or do not exist.
Said antibacterial peptide is further characterized in that the structure of this antibacterial peptide is following 4 kinds:
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：2)，
R1-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：3)，
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-R2-NH
2(SEQ.ID?NO：4)，
R1-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-S er-R2-NH
2One of (SEQ.ID NO:5),
R1: be Leu, Ile, Val, Phe, Lys-Leu, Lys-Ile, Lys-Val, Lys-Phe, Arg-Leu, Arg-Ile, Arg-Val or Arg-Phe;
R2: be Val, Val-Lys, Val-Arg, Val-Lys-Lys, Val-Lys-Arg; Val-Lys-Lys-Leu, Val-Lys-Lys-Ile, Val-Lys-Lys-Val, Val-Lys-Lys-Phe; Val-Lys-Arg-Leu, Val-Lys-Arg-Ile, Val-Lys-Arg-Val, Val-Lys-Arg-Phe; Val-Arg-Lys, Val-Arg-Arg, Val-Arg-Lys-Leu, Val-Arg-Lys-Ile, Val-Arg-Lys-Val, Val-Arg-Lys-Phe, Val-Arg-Arg-Leu, Val-Arg-Arg-Ile, Val-Arg-Arg-Val or Val-Arg-Arg-Phe.
Said antibacterial peptide is further characterized in that the structure of this antibacterial peptide does
R1-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-S er-NH
2, wherein R1 is Leu, Ile, Val, Phe, Lys-Leu, Lys-Ile, Lys-Val, Lys-Phe, Arg-Leu, Arg-Ile, Arg-Val or Arg-Phe.
Said antibacterial peptide is further characterized in that the structure of this antibacterial peptide does
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-R2-NH
2, wherein R2 is Val, Val-Lys, Val-Arg; Val-Lys-Lys, Val-Lys-Arg, Val-Lys-Lys-Leu, Val-Lys-Lys-Ile; Val-Lys-Lys-Val, Val-Lys-Lys-Phe, Val-Lys-Arg-Leu; Val-Lys-Arg-Ile, Val-Lys-Arg-Val, Val-Lys-Arg-Phe; Val-Arg-Lys, Val-Arg-Arg, Val-Arg-Lys-Leu, Val-Arg-Lys-Ile, Val-Arg-Lys-Val, Val-Arg-Lys-Phe, Val-Arg-Arg-Leu, Val-Arg-Arg-Ile, Val-Arg-Arg-Val or Val-Arg-Arg-Phe.
Said antibacterial peptide is further characterized in that the structure of this antibacterial peptide does
R1-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-S er-R2-NH
2, wherein R1 is Leu, Ile, Val, Phe, Lys-Leu, Lys-Ile, Lys-Val, Lys-Phe, Arg-Leu, Arg-Ile, Arg-Val or Arg-Phe; R2 is Val, Val-Lys, Val-Arg, Val-Lys-Lys, Val-Lys-Arg; Val-Lys-Lys-Leu, Val-Lys-Lys-Ile, Val-Lys-Lys-Val, Val-Lys-Lys-Phe; Val-Lys-Arg-Leu, Val-Lys-Arg-Ile, Val-Lys-Arg-Val, Val-Lys-Arg-Phe; Val-Arg-Lys, Val-Arg-Arg, Val-Arg-Lys-Leu, Val-Arg-Lys-I le, Val-Arg-Lys-Val, Val-Arg-Lys-Phe, Val-Arg-Arg-Leu, Val-Arg-Arg-Ile, Val-Arg-Arg-Val or Val-Arg-Arg-Phe.
Said antibacterial peptide is further characterized in that to have following sequence:
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-NH
2(SEQ.ID?NO：6)
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-NH
2(SEQ.ID?NO：7)
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.ID?NO：8)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-NH
2(SEQ.ID?NO：9)
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：10)
Lys-Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：11)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.ID?NO：12)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-NH
2(SEQ.ID?NO：13)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Lys-NH
2(SEQ.ID?NO：14)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.ID?NO：15)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-Ile-NH
2(SEQ.ID?NO：16)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-Val-NH
2(SEQ.ID?NO：17)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-Phe-NH
2(SEQ.ID?NO：18)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-Ile-NH
2(SEQ.ID?NO：19)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-Leu-NH
2(SEQ.ID?NO：20)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-Val-NH
2(SEQ.ID?NO：21)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-Phe-NH
2(SEQ.ID?NO：22)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-Ile-NH
2(SEQ.ID?NO：23)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-Leu-NH
2(SEQ.ID?NO：24)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-Val-NH
2(SEQ.ID?NO：25)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-Phe-NH
2(SEQ.ID?NO：26)
Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.ID?NO：27)
Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.ID?NO：28)
Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.ID?NO：29)
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.ID?NO：30)
Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.ID?NO：31)
Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.ID?NO：32)
Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.ID?NO：33)
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.ID?NO：34)
Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.ID?NO：35)
Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.ID?NO：36)
Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.ID?NO：37)
Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：38)
Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：39)
Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：40)
Arg-Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：41)
Arg-Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：42)
Arg-Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：43)
Arg-Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID?NO：44)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-NH
2(SEQ.ID?NO：45)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.ID?NO：46)
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-Leu-NH
2(SEQ.ID?NO：47)。
Said antibacterial peptide is further characterized in that, a kind of pharmaceutical composition comprises each described antibacterial peptide or its pharmacy acceptable salt and pharmaceutically acceptable carrier or the thinner of the claim 1-6 that treats significant quantity.
Said antibacterial peptide is further characterized in that preparation is used for treating the purposes of bacterium or fungi infestation medicine.
The invention provides the preparation method of this type of antibacterial peptide, the present invention adopts microwave to promote the efficient synthetic apace peptide chain that obtains this type of antibacterial peptide of Fmoc/tBu orthogonally protect solid phase synthesis strategy.
Adopt microwave to promote Fmoc/tBu orthogonally protect solid phase synthesis strategy; Earlier syntheticly on solid phase carrier obtain being loaded with first Fmoc and protect amino acid whose resin, ninhydrin method detects sloughs the resin that Fmoc protection base obtains being loaded with first amino-acid residue after negative; Get into next coupling circulation again; Repeat the step of coupling and deprotection with different protection amino acid according to corresponding peptide preface; Prolong required aminoacid sequence successively, the synthetic resin that obtains being loaded with corresponding polypeptide cuts down polypeptide with cutting agent at last and obtains the polypeptide bullion from resin.Bullion is purified, and freeze-drying gets the pure article of polypeptide.
One group of antibacterial peptide provided by the invention, its preparation method can be a mechanochemical method, also can the encoding sox of antibacterial peptide be cloned on the carrier, in host cell, expresses the back then and obtains.Wherein expression vector can be a kind of in plasmid or the virus; Host cell can be a prokaryotic cell prokaryocyte; Comprise intestinal bacteria, subtilis etc., host cell can be eukaryotic cell also, comprise yeast cell, vegetable cell, insect cell and mammalian cell etc.The antibacterial peptide of preparation can be identified through mass spectrum.
Utilize 96 well plate method to detect fungicidal activity (the In Yup Park etc of polypeptide; FEBS Letters; 437 (1998) 258-262) be contrast with synthetic natural antibacterial peptide Pexiganan and microbiotic Ampicillin Trihydrate and qingfengmeisu qiong in advance, carry out fungicidal activity and detect.The result shows that the fungicidal activity of synthetic antibacterial peptide of the present invention is better than the fungicidal activity of said two kinds of natural antibacterial peptides.
Antibacterial peptide also might act on high organism and comprise human body cell in efficient sterilizing, because the mode of action of antibacterial peptide all is that perforation makes cell generation seepage dead on cytolemma.So can make red corpuscle generation seepage as its virose standard whether antibacterial peptide, if antibiotic Toplink makes the oxyphorase generation seepage in the red corpuscle, just can be through detecting OD490.Value is confirmed toxic size.Therefore the present invention has also detected the hemolytic activity of synthetic antibacterial peptide to human erythrocyte, and experiment shows that antibacterial peptide hemolysis rate value is very low, confirms that the hemolytic toxicity of synthetic antibacterial peptide of the present invention is minimum.
Embodiment
Abbreviation below in this specification sheets full text, adopting: DIEA:N, N '-diisopropylethylamine; DMF: N; DCM: methylene dichloride; The Fmoc:N-9-fluorenylmethyloxycarbonyl; DIC:N, N '-DIC; The DMAP:4-Dimethylamino pyridine; HB TU: benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester; HOBT:1-hydroxyl-benzotriazole; HPLC: performance liquid chromatography; ESI-MS: electrospray ionization mass spectrum; Gly: glycocoll; Ser: Serine; Ala: L-Ala; Thr: Threonine; Val: Xie Ansuan; Ile: Isoleucine; Leu: leucine; Tyr: tyrosine; Phe: phenylalanine(Phe); His: Histidine; Pro: proline(Pro); Asp: aspartic acid; Met: methionine(Met); Glu: L-glutamic acid; Trp: tryptophane; Lys: Methionin; Arg: l-arginine.Asn: l-asparagine; Gln: Stimulina.
The present invention describes through the following example, but these embodiment do not do any restriction explanation of the present invention.
Embodiment 1
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2The microwave of (SEQ.ID NO:2) promotes solid phase synthesis
(1) swelling of resin
Take by weighing Fmoc-Rink amide-MBHA Resin 50mg (replacement amount 0.4mmol/g), through 7mL DCM swelling 30min, suction filtration removes DCM, uses 10mLNMP swelling 30min again, uses NMP at last respectively, DCM, and NMP 7mL rinses well.
(2) microwave promotes removing of Fmoc protection base
The resin that swelling is good is put into reactor drum, adds 25% piperidines/NMP (V/V) solution that 7mL contains 0.1M HOBT, in microwave reactor, reacts 1min; Microwave power is 15W; Temperature of reaction is controlled in 50 ℃, uses the cooling of air pressurized air, and reaction finishes back elimination solution; Add 25% piperidines/NMP (V/V) solution that 7mL contains 0.1M HOBT again and in microwave reactor, react 4min again, microwave power is 25W, and temperature of reaction is controlled at 50 ℃, uses the cooling of air pressurized air.Reaction finishes back elimination solution, uses the NMP washes clean.Obtain sloughing the basic resin of Fmoc protection of initial connection.
(3) microwave promotes synthesizing of Fmoc-Val-Rink amide-MBHA Resin
With Fmoc-Ser (tBu)-OH (0.04mmol); HBTU (0.04mmol), HOBT (0.04mmol) and DIPEA (0.08mmol) are dissolved among the 10mL NMP, in the resin above again this solution being added; In microwave reactor, react 7min; Microwave power is 25W, and temperature of reaction is controlled at 50 ℃, uses the cooling of air pressurized air.Reaction finishes back filtering reaction solution, with DCM and each 7mL washing resin of NMP 3 times.
(4) detection of coupling efficiency
With the coupling efficiency of ninhydrin method or bromjophenol blue method qualitative detection resin, coupling reaction is negative can to get into next coupling circulation.
Ninhydrin method: the resin particle that takes a morsel is used washing with alcohol; Put into transparent bottle and add respectively 2 of 5% triketohydrindene hydrate ethanol, KCN pyridine solution (2ml 0.001M KCN is diluted in the 98ml pyridine), 80% phenol ethanolic solns; In 100 ℃ of heating 5 minutes, promptly positive if resin shows blueness.
The bromjophenol blue method: the resin particle that takes a morsel washs with two formyl ethanamides, puts into the tetrabromophenol sulfonphthalein dimethylacetamide solution that transparent bottle adds 3 1%, and jolting is 3 minutes under the normal temperature, and is promptly positive if resin shows blueness.
(5) prolongation of peptide chain
According to the sequence of SEQ.ID NO:2, the steps in sequence that repeats above-mentioned deprotection and coupling is connected corresponding amino acid, and the coupling microwave promotes that reaction times 5～20min does not wait.Obtain being connected with the resin of peptide chain.
(6) cracking of polypeptide on the resin
The above-mentioned resin that is connected with peptide chain that obtains is put into reaction flask, and each adds cracking agent Reagent K, and (TFA/ thioanisole/water/phenol/EDT, 82.5: 5: 5: 5: 2.5, V/V) 10mL earlier at 0 ℃ of following jolting 30min, reacted 3h more at normal temperatures.Reaction finishes the back suction filtration, adds a small amount of TFA and DCM washing three times, merging filtrate.Separate out white flocks in the ice ether that the filtrating adding is a large amount of, frozen centrifugation obtains the bullion of target polypeptides.Finally obtain SEQ.ID NO:2 bullion 72.5mg, yield is 93.6%.
Use HPLC to measure purity.Chromatographic condition is: C18 reversed-phase column (150mm * 4.6mm, 5 μ m); Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V); Eluent gradient: Mobile phase B 10%～80%, 20min; Flow velocity is 1mL/min; Column temperature is 40 ℃; The detection wavelength is 214nm.Use the preparative liquid chromatography purifying, chromatographic condition is: C18 reversed-phase column (320mm * 28mm, 5 μ m); Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V); Eluent gradient: Mobile phase B 40%～90%, 20min; Flow velocity is that 6mL/min detection wavelength is 214nm.The solution freeze-drying of collecting gets pure article.Theoretical relative molecular mass is 1937.26.ESI-MS m/z: calculated value [M+H]
+: 1839.19, measured value of experiment [M+H]
+: 1839.2
Embodiment 2
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-NH
2(SEQ.ID NO:6); ESI-MS m/z: calculated value [M+H]
+: 2051.34, [M+3H]
3+: 684.45, [M+4H]
4+: 513.585, measured value of experiment [M+H]
+: 2051.3, [M+3H]
3+: 684.4, [M+4H]
4+: 513.6
Embodiment 3
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-NH
2(SEQ.ID NO:7); ESI-MS m/z: calculated value [M+H]
+: 2179.81, [M+2H]
2+: 1090.4, [M+3H]
3+: 727.27, measured value of experiment [M+H]
+: 2179.9, [M+2H]
2+: 1090.5, [M+3H]
3+: 727.3
Embodiment 4
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.IDNO:8); ESI-MS m/z: calculated value [M+H]
+: 2307.98, [M+2H]
2+: 1154.49, [M+3H]
3+: 769.99, measured value of experiment [M+H]
+: 2308.1, [M+2H]
2+: 1154.6, [M+3H]
3+: 770.0
Embodiment 5
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-NH
2(SEQ.ID NO:9); ESI-MSm/z: calculated value [M+H]
+: 1938.47, [M+2H]
2+: 969.74, [M+3H]
3+: 646.82, measured value of experiment [M+H]
+: 1938.6, [M+2H]
2+: 969.8, [M+3H]
3+: 646.9
Embodiment 6
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID NO:10); ESI-MS m/z: calculated value [M+H]
+: 1952.5, [M+2H]
2+: 976.75, [M+3H]
3+: 651.5, measured value of experiment [M+H]
+: 1952.6, [M+2H]
2+: 976.8, [M+3H]
3+: 651.5
Embodiment 7
Lys-Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID NO:11); ESI-MS m/z: calculated value [M+H]
+: 2080.67, [M+2H]
2+: 1040.84, [M+3H]
3+: 694.22, measured value of experiment [M+H]
+: 2080.8, [M+2H]
2+: 1040.9, [M+3H]
3+: 694.3
Embodiment 8
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.ID NO:12); ESI-MS m/z: calculated value [M+H]
+: 2222.83, [M+2H]
2+: 1111.92, [M+3H]
3+: 741.61, measured value of experiment [M+H]
+: 2222.9, [M+2H]
2+: 1112.0, [M+3H]
3+: 741.7
Embodiment 9
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-NH
2(SEQ.ID NO:13); ESI-MS m/z: calculated value [M+H]
+: 2094.66, [M+2H]
2+: 1047.83, [M+3H]
3+: 698.89, measured value of experiment [M+H]
+: 2094.8, [M+2H]
2+: 1047.9, [M+3H]
3+: 698.9
Embodiment 10
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Lys-NH
2(SEQ.ID NO:14); ESI-MS m/z: calculated value [M+H]
+: 2222.83, [M+2H]
2+: 1111.92, [M+3H]
3+: 741.61, measured value of experiment [M+H]
+: 2222.9, [M+2H]
2+: 1112.0, [M+3H]
3+: 741.7
Embodiment 11
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.ID NO:15); ESI-MS m/z: calculated value [M+H]
+: 2250.85, [M+2H]
2+: 1125.92, [M+3H]
3+: 750.95, measured value of experiment [M+Hr
+: 2251.0, [M+2H]
2+: 1126.0, [M+3H]
3+: 751.0
Embodiment 12
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-Ile-NH
2(SEQ.ID NO:16); ESI-MS m/z: calculated value [M+H]
+: 2307.98, [M+2H]
2+: 1154.49, [M+3H]
3+: 769.99, measured value of experiment [M+H]
+: 2308.1, [M+2H]
2+: 1154.6, [M+3H]
3+: 770.0
Embodiment 13
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-Val-NH
2(SEQ.IDNO:17); ESI-MS m/z: calculated value [M+H]
+: 2293.95, [M+2H]
2+: 1147.48, [M+3H]
3+: 765.32, measured value of experiment [M+H]
+: 2294.1, [M+2H]
2+: 1147.6, [M+3H]
3+: 765.4
Embodiment 14
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-Phe-NH
2(SEQ.IDNO:18); ESI-MS m/z: calculated value [M+H]
+: 2342, [M+2H]
2+: 1171.5, [M+3H]
3+: 781.33, measured value of experiment [M+H]
+: 2342.1, [M+2H]
2+: 1171.6, [M+3H]
3+: 781.4
Embodiment 15
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-Ile-NH
2(SEQ.ID NO:19); ESI-MS m/z: calculated value [M+H]
+: 2336, [M+2H]
2+: 1168.5, [M+3H]
3+: 779.33, measured value of experiment [M+H]
+: 2336.1, [M+2H]
2+: 1168.6, [M+3H]
3+: 779.4
Embodiment 16
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-Leu-NH
2(SEQ.IDNO:20); ESI-MS m/z: calculated value [M+H]
+: 2336, [M+2H]
2+: 1168.5, [M+3H]
3+: 779.33, measured value of experiment [M+H]
+: 2336.1, [M+2H]
2+: 1168.6, [M+3H]
3+: 779.4
Embodiment 17
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-Val-NH
2(SEQ.IDNO:21); ESI-MS m/z: calculated value [M+H]
+: 2321.97, [M+2H]
2+: 1161.48, [M+3H]
3+: 774.66, measured value of experiment [M+H]
+: 2322.1, [M+2H]
2+: 1161.6, [M+3H]
3+: 774.7
Embodiment 18
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-Phe-NH
2(SEQ.IDNO:22); ESI-MS m/z: calculated value [M+H]
+: 2370.01, [M+2H]
2+: 1185.51, [M+3H]
3+: 790.67, measured value of experiment [M+H]
+: 2370.1, [M+2H]
2+: 1185.6, [M+3H]
3+: 790.7
Embodiment 19
23); ESI-MS m/z: calculated value [M+H]
+: 2364.01, [M+2H]
2+: 1182.5, [M+3H]
3+: 788.67, measured value of experiment [M+H]
+: 2364.1, [M+2H]
2+: 1182.6, [M+3H]
3+: 788.7
Embodiment 20
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-Leu-NH
2(SEQ.IDNO:24); ESI-MS m/z: calculated value [M+H]
+: 2364.01, [M+2H]
2+: 1182.5, [M+3H]
3+: 788.67, measured value of experiment [M+H]
+: 2364.1, [M+2H]
2+: 1182.6, [M+3H]
3+: 788.7
Embodiment 21
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-Val-NH
2(SEQ.IDNO:25); ESI-MS m/z: calculated value [M+H]
+: 2349.98, [M+2H]
2+: 1175.49, [M+3H]
3+: 783.99, measured value of experiment [M+H]
+: 2350.1, [M+2H]
2+: 1175.6, [M+3H]
3+: 784.0
Embodiment 22
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-Phe-NH
2(SEQ.IDNO:26); ESI-MS m/z: calculated value [M+H]
+: 2398.03, [M+2H]
2+: 1199.51, [M+3H]
3+: 800.01, measured value of experiment [M+H]
+: 2398.1, [M+2H]
2+: 1199.6, [M+3H]
3+: 800.1
Embodiment 23
Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.ID NO:27); ESI-MS m/z: calculated value [M+H]
+: 2307.98, [M+2H]
2+: 1154.49, [M+3H]
3+: 769.99, measured value of experiment [M+H]
+: 2308.1, [M+2H]
2+: 1154.6, [M+3H]
3+: 770.0
Embodiment 24
Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.IDNO:28); ESI-MS m/z: calculated value [M+H]
+: 2293.95, [M+2H]
2+: 1147.48, [M+3H]
3+: 765.32, measured value of experiment [M+H]
+: 2294.1, [M+2H]
2+: 1147.62, [M+3H]
3+: 765.4
Embodiment 25
Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.IDNO:29); ESI-MS m/z: calculated value [M+H]
+: 2342, [M+2H]
2+: 1171.5, [M+3H]
3+: 781.33, measured value of experiment [M+H]
+: 2342.1, [M+2H]
2+: 1171.6, [M+3H]
3+: 781.4
Embodiment 26
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.IDNO:30); ESI-MS m/z: calculated value [M+H]
+: 2336, [M+2H]
2+: 1168.5, [M+3H]
3+: 779.33, measured value of experiment [M+H]
+: 2336.1, [M+2H]
2+: 1168.6, [M+3H]
3+: 779.4
Embodiment 27
Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.ID NO:31); ESI-MS m/z: calculated value [M+H]
+: 2336, [M+2H]
2+: 1168.5, [M+3H]
3+: 779.33, measured value of experiment [M+H]
+: 2336.1, [M+2H]
2+: 1168.6, [M+3H]
3+: 779.42
Embodiment 28
Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.IDNO:32); ESI-MS m/z: calculated value [M+H]
+: 2321.97, [M+2H]
2+: 1161.48, [M+3H]
3+: 774.66, measured value of experiment [M+H]
+: 2322.1, [M+2H]
2+: 1161.6, [M+3H]
3+: 774.7
Embodiment 29
Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Arg-NH
2(SEQ.IDNO:33); ESI-MS m/z: calculated value [M+H]
+: 2370.01, [M+2H]
2+: 1185.51, [M+3H]
3+: 790.67, measured value of experiment [M+H]
+: 2370.1, [M+2H]
2+: 1185.6, [M+3H]
3+: 790.7
Embodiment 30
Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.IDNO:34); ESI-MS m/z: calculated value [M+H]
+: 2364.01, [M+2H]
2+: 1182.5, [M+3H]
3+: 788.67, measured value of experiment [M+H]
+: 2364.1, [M+2H]
2+: 1182.6, [M+3H]
3+: 788.7
Embodiment 31
Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.ID NO:35); ESI-MS m/z: calculated value [M+H]
+: 2364.01, [M+2H]
2+: 1182.5, [M+3H]
3+: 788.67, measured value of experiment [M+H]
+: 2364.1, [M+2H]
2+: 1182.6, [M+3H]
3+: 788.7
Embodiment 32
Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.IDNO:36); ESI-MS m/z: calculated value [M+H]
+: 2349.98, [M+2H]
2+: 1175.49, [M+3H]
3+: 783.99, measured value of experiment [M+H]
+: 2350.1, [M+2H]
2+: 1175.6, [M+3H]
3+: 784.0
Embodiment 33
Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Arg-Arg-NH
2(SEQ.IDNO:37); ESI-MS m/z: calculated value [M+H]
+: 2398.03, [M+2H]
2+: 1199.51, [M+3H]
3+: 800.01, measured value of experiment [M+H]
+: 2398.1, [M+2H]
2+: 1199.6, [M+3H]
3+: 800.1
Embodiment 34
Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID NO:38); ESI-MSm/z: calculated value [M+H]
+: 1952.5, [M+2H]
2+: 976.75, [M+3H]
3+: 651.5, measured value of experiment [M+H]
+: 1952.6, [M+2H]
2+: 976.8, [M+3H]
3+: 651.5
Embodiment 35
Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID NO:39); ESI-MS m/z: calculated value [M+H]
+: 1938.47, [M+2H]
2+: 969.74, [M+3H]
3+: 646.82, measured value of experiment [M+H]
+: 1938.6, [M+2H]
2+: 969.8, [M+3H]
3+: 646.9
Embodiment 36
Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID NO:40); ESI-MS m/z: calculated value [M+H]
+: 1986.51, [M+2H]
2+: 993.76, [M+3H]
3+: 662.84, measured value of experiment [M+H]
+: 1986.6, [M+2H]
2+: 993.9, [M+3H]
3+: 662.9
Embodiment 37
Arg-Ile-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID NO:41); ESI-MS m/z: calculated value [M+H]
+: 2108.69, [M+2H]
2+: 1054.84, [M+3H]
3+: 703.56, measured value of experiment [M+H]
+: 2108.8, [M+2H]
2+: 1054.9, [M+3H]
3+: 703.6
Embodiment 38
Arg-Val-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID NO:42); ESI-MS m/z: calculated value [M+H]
+: 2094.66, [M+2H]
2+: 1047.83, [M+3H]
3+: 698.89, measured value of experiment [M+H]
+: 2094.8, [M+2H]
2+: 1047.9, [M+3H]
3+: 698.9
Embodiment 39
Arg-Phe-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID NO:43); ESI-MS m/z: calculated value [M+H]
+: 2142.7, [M+2H]
2+: 1071.85, [M+3H]
3+: 714.9, measured value of experiment [M+H]
+: 2142.8, [M+2H]
2+: 1071.9, [M+3H]
3+: 714.9
Embodiment 40
Arg-Leu-Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-NH
2(SEQ.ID NO:44); ESI-MS m/z: calculated value [M+H]
+: 2108.69, [M+2H]
2+: 1054.84, [M+3H]
3+: 703.56, measured value of experiment [M+H]
+: 2108.8, [M+2H]
2+: 1054.9, [M+3H]
3+: 703.6
Embodiment 41
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-NH
2(SEQ.ID NO:45); ESI-MS m/z: calculated value [M+H]
+: 2066.65, [M+2H]
2+: 1033.82, [M+3H]
3+: 689.55, measured value of experiment [M+H]
+: 2066.8, [M+2H]
2+: 1033.9, [M+3H]
3+: 689.6
Embodiment 42
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-NH
2(SEQ.ID NO:46); ESI-MS m/z: calculated value [M+H]
+: 2194.82, [M+2H]
2+: 1097.91, [M+3H]
3+: 732.27, measured value of experiment [M+H]
+: 2194.9, [M+2H]
2+: 1098.0, [M+3H]
3+: 732.3
Embodiment 43
Val-Lys-Arg-Phe-Lys-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-Lys-Lys-Leu-NH
2(SEQ.IDNO:47); ESI-MS m/z: calculated value [M+H]
+: 2307.98, [M+2H]
2+: 1154.49, [M+3H]
3+: 769.99, measured value of experiment [M+H]
+: 2308.1, [M+2H]
2+: 1154.6, [M+3H]
3+: 770.0
The fungicidal activity of embodiment 44 synthetic antibacterial peptides detects
Employed various bacterial strains are purchased in Chinese biological goods calibrating institute in following examples.
Adopt 96 well plate method that the fungicidal activity of synthetic antibacterial peptide is detected, and with Ampicillin Trihydrate, qingfengmeisu qiong and mechanochemical method synthetic antibacterial peptide pexiganan as contrast, to estimate among the present invention fungicidal activity as ten antibacterial peptides of giving an example.
Measure the fungicidal activity of antibacterial peptide according to the following steps:
The bacterial classification recovery, the 37 ℃ of overnight cultures in inoculation inclined-plane connect bacterium in common LB substratum, 37 ℃ of overnight cultures, it is 10 that dilution bacterium liquid makes bacteria concentration
4-10
5CFU/ml is inoculated in 96 orifice plates by every hole 100ul bacterium liquid, after polypeptide is diluted with certain proportion, adds 10ul in every hole, and 96 orifice plates are placed 37 ℃ of overnight cultures, and ELIASA detects OD
620Value (In Yup Park etc; FEBS Letters; 437 (1998) 258-262).Detected result is seen table 1.
Growth concentration (the OD that contains the bacterium of antibacterial peptide
620) be minimal inhibitory concentration (minimal inhibitory concentration (MIC) is defined as the minimum concentration of remarkable bacteria growing inhibiting) with the ratio of the growth concentration of the bacterium that does not add antibacterial peptide greater than 90% o'clock antibacterial peptide concentration.
Antibacterial peptide is to the comparison of the anti-microbial activity minimal inhibitory concentration (MIC) of different bacterium among table 1 embodiment 1～43
IS: clinical isolates strain, DR: anti-Ampicillin Trihydrate and penicillium candidum strain
In the last table the minimal inhibitory concentration value more little, then represent antibacterial ability strong more.Can find out that from last table antibacterial peptide of the present invention obviously is better than positive drug Ampicillin Trihydrate and qingfengmeisu qiong to the anti-microbial activity of resistant organism.
Embodiment 45 external hemolytic activities detect
Present embodiment is used to detect antibacterial peptide whether human erythrocyte is had hemolytic activity, and with mechanochemical method synthetic antibacterial peptide pexiganan as contrast.Use blood sample be taken at normal human blood.
The detection step of antibacterial peptide hemolytic activity is:
The release of fresh red blood cell suspension oxyphorase under 414nm of 4% detects.The HRBC is through PBS (PBS:35mM phosphoric acid buffer/0.15M NaCl; PH7.0) washing, 8% HRBC's suspension of getting 100ul add 100ul antibacterial peptide solution in every hole in 96 orifice plates; 37 ℃ after one hour; Centrifugal 5 minutes of 1500rpm shifts the 100ul supernatant in 96 new orifice plates, detects the absorption under the 414nm through ELIASA.Negative control is used PBS, and positive control is used 0.1%Triton X-100.Hemolytic action is used the concentration (LC that causes half red corpuscle generation hemolytic action
50) expression, detected result is seen table 2
The hemolytic action of antibacterial peptide among table 2 embodiment 1～10 (μ g/mL)
The LC of antibacterial peptide in the last table
50Be worth greatly more, then represent the hemolytic toxicity of antibacterial peptide more little.Can find out that from last table the hemolytic action of antibacterial peptide of the present invention is starkly lower than Pexiganan.
Claims (8)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110291548A CN102329375A (en) | 2011-09-30 | 2011-09-30 | Group of terminal-amidated antibacterial peptides |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110291548A CN102329375A (en) | 2011-09-30 | 2011-09-30 | Group of terminal-amidated antibacterial peptides |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102329375A true CN102329375A (en) | 2012-01-25 |
Family
ID=45481360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110291548A Pending CN102329375A (en) | 2011-09-30 | 2011-09-30 | Group of terminal-amidated antibacterial peptides |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102329375A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107080831A (en) * | 2012-03-20 | 2017-08-22 | 赫里克斯生物医疗公司 | Short antimicrobial lipopeptid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101386640A (en) * | 2008-09-27 | 2009-03-18 | 中国科学院昆明动物研究所 | Cathelicidin-BF variant of antibacterial peptide cathelicidin-BF15 and its application |
-
2011
- 2011-09-30 CN CN201110291548A patent/CN102329375A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101386640A (en) * | 2008-09-27 | 2009-03-18 | 中国科学院昆明动物研究所 | Cathelicidin-BF variant of antibacterial peptide cathelicidin-BF15 and its application |
Non-Patent Citations (3)
| Title |
|---|
| 20060916 Yipeng Wang等 Snake Cathelicidin from Bungarus fasciatus Is a Potent , * |
| YIPENG WANG等: "Snake Cathelicidin from Bungarus fasciatus Is a Potent", <PLOS ONE>, 16 September 2006 (2006-09-16) * |
| 牛明福等: "抗菌肽的分子设计研究进展", 《上海畜牧兽医通讯》, 30 April 2008 (2008-04-30) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107080831A (en) * | 2012-03-20 | 2017-08-22 | 赫里克斯生物医疗公司 | Short antimicrobial lipopeptid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101570569B (en) | Synthetic antibacterial peptide and preparation method and application thereof | |
| ES2429108T3 (en) | Antibiotic peptides | |
| CN106232616B (en) | Amphiphilic synthetic antimicrobial peptide, its pharmaceutical composition and its use | |
| CN101717450B (en) | Antimicrobial peptide LFB-ME and its preparation method | |
| CN104292301A (en) | Micromolecule synthesized anti-microbial peptide, as well as preparation method and application thereof | |
| WO2004020461A1 (en) | A group of novel synthetic antibiotic peptides | |
| CN101215325B (en) | Antibiotic peptides, preparation method and application thereof | |
| Zhang et al. | Rational design and synthesis of Oreoch-2 analogues as efficient broad-spectrum antimicrobial peptides | |
| CN102391362A (en) | Group of animal-derived cationic antibacterial peptides and its application | |
| CN100365018C (en) | Antibiotic peptides and their prepn process and application | |
| TWI577697B (en) | Salt and protease-resistance of antimicrobial peptide and the manufacture thereof | |
| CN101182351A (en) | Antibiotic peptide as well as preparation method and application thereof | |
| CN102329375A (en) | Group of terminal-amidated antibacterial peptides | |
| US20200071357A1 (en) | Antimicrobial peptides | |
| KR20030077950A (en) | Physiologically active complex | |
| CN111116714B (en) | Wasp venom peptide reverse sequence analogue WVC-II and preparation method and application thereof | |
| CN101781359B (en) | Novel antibacterial peptide and preparation method and application thereof | |
| CN111153966B (en) | A kind of vespidin reverse sequence analogue WVF-II and its preparation method and application | |
| CN102391364B (en) | New antibacterial peptides as well as preparation method and application of the same | |
| CN101781358B (en) | Novel antibacterial peptide and preparation method and application thereof | |
| CN101775067A (en) | New synthesized antibacterial peptide as well as preparation method and application thereof | |
| CN101781366B (en) | Novel antibacterial peptide and preparation method and application thereof | |
| CN104356202A (en) | Cationic antibacterial peptide as well as preparation method and application thereof | |
| KR100980300B1 (en) | Fishid derivatives having novel bacterial selectivity comprising lysine peptoid residues and their use | |
| CN102391370B (en) | Antimicrobial peptides, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120125 |