CN117143196A - Alpha-helical polypeptide antibiotic bound by full-hydrocarbon side chain, and preparation method and application thereof - Google Patents
Alpha-helical polypeptide antibiotic bound by full-hydrocarbon side chain, and preparation method and application thereof Download PDFInfo
- Publication number
- CN117143196A CN117143196A CN202211414907.3A CN202211414907A CN117143196A CN 117143196 A CN117143196 A CN 117143196A CN 202211414907 A CN202211414907 A CN 202211414907A CN 117143196 A CN117143196 A CN 117143196A
- Authority
- CN
- China
- Prior art keywords
- reagent
- alpha
- resin
- polypeptide antibiotic
- amino acid
- 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
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 150000001413 amino acids Chemical class 0.000 claims abstract description 24
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 21
- 241000894006 Bacteria Species 0.000 claims abstract description 15
- 238000007363 ring formation reaction Methods 0.000 claims abstract description 11
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 8
- 230000003110 anti-inflammatory effect Effects 0.000 claims abstract description 8
- 238000005865 alkene metathesis reaction Methods 0.000 claims abstract description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 38
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 21
- 229920005989 resin Polymers 0.000 claims description 20
- 239000011347 resin Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 238000010511 deprotection reaction Methods 0.000 claims description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 11
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 9
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 9
- 239000003814 drug Substances 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 8
- 229920003180 amino resin Polymers 0.000 claims description 7
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000003776 cleavage reaction Methods 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 230000007017 scission Effects 0.000 claims description 5
- 239000012345 acetylating agent Substances 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 125000006239 protecting group Chemical group 0.000 claims description 4
- 238000011282 treatment Methods 0.000 claims description 4
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000029663 wound healing Effects 0.000 claims description 3
- 208000035473 Communicable disease Diseases 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000006798 ring closing metathesis reaction Methods 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 235000001014 amino acid Nutrition 0.000 abstract description 18
- 241000699670 Mus sp. Species 0.000 abstract description 10
- 239000003242 anti bacterial agent Substances 0.000 abstract description 8
- 229940088710 antibiotic agent Drugs 0.000 abstract description 8
- 231100000419 toxicity Toxicity 0.000 abstract description 8
- 230000001988 toxicity Effects 0.000 abstract description 8
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 7
- 230000003013 cytotoxicity Effects 0.000 abstract description 7
- 230000002949 hemolytic effect Effects 0.000 abstract description 7
- 210000002966 serum Anatomy 0.000 abstract description 7
- 208000035143 Bacterial infection Diseases 0.000 abstract description 5
- 208000022362 bacterial infectious disease Diseases 0.000 abstract description 5
- 230000035876 healing Effects 0.000 abstract description 5
- 210000003743 erythrocyte Anatomy 0.000 abstract description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 abstract description 4
- 241000192125 Firmicutes Species 0.000 abstract description 3
- 210000002950 fibroblast Anatomy 0.000 abstract description 3
- AERCCJGORROTKW-QMMMGPOBSA-N (2s)-2-amino-2-methylhept-6-enoic acid Chemical compound OC(=O)[C@](N)(C)CCCC=C AERCCJGORROTKW-QMMMGPOBSA-N 0.000 abstract description 2
- 125000000539 amino acid group Chemical group 0.000 abstract description 2
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 2
- 238000006555 catalytic reaction Methods 0.000 abstract 1
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 30
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 239000000872 buffer Substances 0.000 description 9
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical group CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 8
- 238000004007 reversed phase HPLC Methods 0.000 description 8
- 230000000844 anti-bacterial effect Effects 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 108700022109 ropocamptide Proteins 0.000 description 5
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 206010052428 Wound Diseases 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 206010072170 Skin wound Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 150000002430 hydrocarbons Chemical group 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 150000003904 phospholipids Chemical class 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- -1 cationic amino acids Chemical class 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000017306 interleukin-6 production Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000006433 tumor necrosis factor production Effects 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- UIRFWOOIGULZQN-UHFFFAOYSA-L [Ru](Cl)Cl.C1(=CC=CC=C1)C([P](C1CCCCC1)(C1CCCCC1)C1CCCCC1)[P](C1CCCCC1)(C1CCCCC1)C1CCCCC1 Chemical compound [Ru](Cl)Cl.C1(=CC=CC=C1)C([P](C1CCCCC1)(C1CCCCC1)C1CCCCC1)[P](C1CCCCC1)(C1CCCCC1)C1CCCCC1 UIRFWOOIGULZQN-UHFFFAOYSA-L 0.000 description 1
- 230000000397 acetylating effect Effects 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000012042 active reagent Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- LCFXLZAXGXOXAP-QPJJXVBHSA-N ethyl (2e)-2-cyano-2-hydroxyiminoacetate Chemical compound CCOC(=O)C(=N\O)\C#N LCFXLZAXGXOXAP-QPJJXVBHSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000012988 high-throughput synthesis Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- NIHNNTQXNPWCJQ-UHFFFAOYSA-N o-biphenylenemethane Natural products C1=CC=C2CC3=CC=CC=C3C2=C1 NIHNNTQXNPWCJQ-UHFFFAOYSA-N 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000004260 plant-type cell wall biogenesis Effects 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 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
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses an alpha-helical polypeptide antibiotic bound by a full-hydrocarbon side chain, and a preparation method and application thereof. The polypeptide antibiotics are represented by SEQ ID NO:1, firstly obtaining a linear polypeptide antibiotic by Fmoc solid-phase synthesis; and then, on the basis of keeping key amino acid residues, replacing the original amino acid with (S) -2- (4-pentenyl) alanine at a specific position, carrying out olefin metathesis reaction under the catalysis of Grubbs I reagent, and carrying out binding cyclization on the ith position and the (i+4) th position of the linear polypeptide antibiotics to obtain the target polypeptide antibiotics. The invention can inhibit the growth and reproduction of gram-negative bacteria and gram-positive bacteria, has low hemolytic toxicity to red blood cells and cytotoxicity to embryo fibroblasts, has high serum stability, has anti-inflammatory activity, promotes the healing of the wound surface of the skin of mice infected by bacteria, and is expected to replace the traditional antibiotics for treating diseases related to bacterial infection.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an alpha-helical polypeptide antibiotic with all-hydrocarbon side chains bound, and a preparation method and application thereof.
Background
Bacterial infection refers to the process by which pathogenic bacteria invade host tissues or organs, and cause pathological reactions to tissues and organs to varying degrees through massive growth and reproduction and release of toxic substances (toxins). Bacteria can infect any tissue and organ of the human body and can even invade the blood circulation system, causing acute systemic infections and developing sepsis or sepsis. Currently, antibiotics remain the primary drug for the treatment of bacterial infections. It generally exerts an antibacterial effect by affecting bacterial metabolic processes, such as inhibiting bacterial cell wall, protein, and deoxyribonucleic acid synthesis. Bacteria resist their antimicrobial action by corresponding means, such as altering cell wall composition to reduce the binding site of antibiotics, using efflux pumps to exclude antibiotics from the cell, and producing specific active enzymes to degrade antibiotics, resulting in resistance. From the evolutionary point of view, the generation of bacterial drug resistance is an unavoidable natural selection process, and the existing antibiotics which can effectively act on drug resistant bacteria are inevitably reduced continuously. While the development of new antibiotics by high throughput screening and synthesis using natural products and chemical libraries is extremely slow, it will not be sufficient to address the challenges presented by the dramatically increasing number of resistant bacteria worldwide. Thus, there is increasing interest in natural "antibacterial" weapon antibacterial peptides that are found in large numbers in nature.
The antibacterial peptide is generally formed by combining 20-60 cationic amino acids and hydrophobic amino acids in a certain sequence, mainly plays an antibacterial role by destroying the structural integrity of bacterial cell membranes, and has broad-spectrum antibacterial activity. Among them, LL-37 is the only antibacterial peptide found in the human body at present, and an amphipathic alpha-helix structure is formed by 37 amino acids. LL-37 with electropositivity is firstly adsorbed on the surface of a bacterial membrane with electronegativity through electrostatic action, then a hydrophobic structure of the LL-37 is inserted into a bacterial phospholipid membrane layer to damage the fluidity of the phospholipid membrane layer, and meanwhile, ion channels are continuously enriched in the phospholipid membrane layer to leak bacterial contents, so that bacterial death is caused. Therefore, drug resistance is less likely to occur than conventional antibiotics. However, in clinical transformation applications, LL-37 antibacterial peptides still face the following problems: (1) The number of peptide chain amino acids is large, and the solid phase synthesis cost is high; (2) is easily degraded by protease, and has poor in vivo stability; (3) The selectivity is low, and the hemolytic toxicity and the normal cytotoxicity are high.
The related research shows that the formation of side chain cyclization structure by using all-carbon skeleton can stabilize the active conformation of alpha-helical polypeptide and improve the cell membrane penetrability and enzyme stability. And meanwhile, the selective toxicity of the polymer can be improved due to the change of the charge quantity caused by side chain cyclization. Therefore, the full-hydrocarbon side chain binding strategy is hopeful to be the most direct and effective method for overcoming the clinical application limitation of LL-37 antibacterial peptide, and related research reports are not yet seen at present.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: provides an alpha-helical polypeptide antibiotic with bound full-hydrocarbon side chain, a preparation method and application thereof, which solves the problems of LL-37 antibacterial peptide in clinical transformation application.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, the invention provides a fully hydrocarbon side-chain bound alpha-helical polypeptide antibiotic, wherein the polypeptide sequence is the shortest sequence with antibacterial activity in LL-37 antibacterial peptide, and comprises amino acid residues 12 between 18 th and 29 th positions. Using the polypeptide sequence as a peptide chain template, replacing the ith and (i+4) th amino acids with (S) 2 (4 pentenyl) alanine (S5), and binding a full hydrocarbon side chain;
preferably, the all-hydrocarbon side chain bound α -helical polypeptide antibiotic is selected from one of the following:
a) Ac-KRIVQRIKDFLR-NH 2 Is a peptide chain template, 3 of which I And 7 I Quilt S 5 Replacing and cyclization;
b) Ac-KRIVQRIKDFLR-NH 2 Is a peptide chain template, 5 of which Q And 9 D Quilt S 5 Replacing and cyclization;
c) Ac-KRIVQRIKDFLR-NH 2 Is a peptide chain template, 7 of which I And 11 (V) L Quilt S 5 Replacing and cyclization;
the invention provides an alpha-helical polypeptide antibiotic with bound full-hydrocarbon side chains, which has the following structural formula:
KR(I 3 ，I 7 )
KR(Q 5 ，D 9 )
KR(I 7 ,L 11 )
in a second aspect, the invention provides a method for preparing an alpha-helical polypeptide antibiotic with bound all-hydrocarbon side chains, comprising the steps of:
step 1): removing Fmoc protecting groups on the amino resin using a deprotection reagent;
step 2): coupling the carboxyl group of the Fmoc-protected amino acid with the exposed amino group on the Rink Amide MBHA resin using a condensing reagent;
step 3): removing Fmoc protecting groups on the amino acid using a deprotection reagent;
step 4): repeating the coupling-deprotection operation in the step 2) and the step 3), and synthesizing a peptide chain according to an amino acid sequence, wherein the amino acids at the i and i+4 ring closure sites are respectively replaced by S5;
step 5): modifying the deprotected amino group of the last amino acid on the peptide chain by using an acetylating agent;
step 6): catalyzing the side chains at the S5 positions i and i+4 to generate olefin metathesis reaction by using a cyclization reagent;
step 7): cleaving the peptide chain from the resin using a cleavage reagent and precipitating with glacial ethyl ether to obtain a crude polypeptide antibiotic;
step 8): and (3) separating and purifying the crude polypeptide antibiotics by using high performance liquid chromatography.
As a preferred example of the present invention, the loading amount of the resin in the solid phase synthesis in step 1) is 0.3mmol/g.
As another preferred example of the present invention, the deprotection reagent in step 1) is a mixed solution of piperidine and DMF in a ratio of 1:4 (v/v).
As another preferred example of the present invention, the deprotection in step 1) is performed for 5 minutes by using a deprotection reagent and then performed for 5 minutes again; the reaction temperature for Fmoc group removal is 20 to 30℃and more preferably 25 ℃.
As another preferred example of the present invention, the condensing agent used in the step 2) is DIC-Oxyme condensation system, the activator is DIC, and DMF is solvent.
More preferably, the ratio of amino acids, oxime, DIC and DMF in step 2) is 1:1:1:6 (mol/mol/mL).
As another preferred example of the present invention, the temperature of the coupling reaction in step 2) is 50 to 60 ℃, more preferably 55 ℃; the coupling reaction time is 20 to 30min, more preferably 20min.
As another preferable example of the present invention, S 5 The reaction time of the first amino acid is 1h, and the reaction is repeated once under the same condition to carry out the next operation.
As another preferred example of the invention, in the step 5), the acetylating agent is mixed solution of acetic anhydride, DIEA and DMF, and the feeding ratio is 1:1:8 (v/v/v).
As another preferred example of the present invention, the acetylation in step 5) is performed by reacting the resin in an acetylating reagent for 20min; the reaction temperature is 20 to 30 ℃, more preferably 25 ℃.
As another preferable example of the present invention, the cyclic agent in the step 6) is a solution of DCE of Grubbs I reagent, and the feeding ratio is resin loading amount of Grubbs I reagent, DCE=0.3:58:6 (mmol/mg/mL).
As another preferred example of the present invention, the cyclization in step 6) is that the resin is oscillated twice in the cyclization agent for 2 hours each time; the reaction temperature is 20 to 30 ℃, more preferably 25 ℃.
As another preferable example of the present invention, in the step 7), the cleavage reagent is TIPS, H 2 A mixed solution of O and TFA in a volume ratio of 2.5:2.5:95; the volume to mass ratio of the cleavage reagent to the resin was 1:50 (mL/mg).
As another preferred example of the present invention, in step 7), the cutting temperature is 20 to 30 ℃, more preferably 25 ℃; the time for cutting was 4h.
As another preferred example of the present invention, the purification method used in the present invention is reverse phase high performance liquid chromatography (SHIMADZU (LC-6A) reverse phase high performance liquid chromatography (RP-HPLC), C18 column (Daisogel, 20X 250 mm) with a flow rate of 10mL/min. Mobile phase buffer a was acetonitrile plus 0.1% TFA, and mobile phase buffer B was water plus 0.1% TFA. Starting from 10% buffer A, a linear gradient elution was performed over 50 minutes to 75% buffer A to obtain the target polypeptide antibiotic.
In a third aspect, the invention provides the use of an alpha-helical polypeptide antibiotic bound in the side chain of all hydrocarbons.
Preferably, the present invention provides the use of said polypeptide antibiotics for the manufacture of a medicament for inhibiting the growth and reproduction of gram positive and negative bacteria.
Preferably, the present invention provides the use of said polypeptide antibiotics for the manufacture of a medicament for the treatment of infectious diseases caused by gram positive or/and negative bacteria.
Preferably, the invention provides the use of said polypeptide antibiotics for the preparation of a medicament for anti-inflammatory and wound healing.
Compared with the existing LL-37 antibacterial peptide, the polypeptide antibiotic provided by the invention has the following beneficial effects:
(1) The polypeptide antibiotics comprise fewer amino acid sequences, the preparation method is simple and feasible, and the obtained product has high yield and high purity;
(2) The polypeptide antibiotics can remarkably inhibit the growth and reproduction of gram-negative bacteria and gram-positive bacteria;
(3) The polypeptide antibiotics have lower hemolytic toxicity to erythrocytes and cytotoxicity to embryonic fibroblasts;
(4) The serum stability of the polypeptide antibiotics is higher;
(5) The polypeptide antibiotic has more remarkable anti-inflammatory activity, and can more remarkably promote the healing of the skin wound surface of the mice infected by bacteria.
Drawings
FIG. 1 is a schematic sequence diagram of a polypeptide antibiotic prepared in example 1;
FIG. 2 is a synthetic route diagram of example 1;
FIGS. 3-7 are high performance liquid chromatograms and mass chromatograms of the polypeptide antibiotics prepared in example 1;
FIG. 8 is a summary table of minimum inhibitory concentrations of the polypeptide antibiotics prepared in example 1;
FIG. 9 is a graph showing the hemolytic toxicity of the polypeptide antibiotic prepared in example 1;
FIG. 10 is a cytotoxicity map of the polypeptide antibiotic prepared in example 1;
FIG. 11 is a graph showing the serum stability of the polypeptide antibiotics prepared in example 1;
FIG. 12 is a graph showing anti-inflammatory activity of the polypeptide antibiotics prepared in example 1;
FIG. 13 is a graph showing the experimental result of promoting wound healing of bacterial infection of mice by using the polypeptide antibiotics prepared in example 1.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
The following examples are presented with the abbreviations that are explained as follows:
fmoc: fluorene methoxycarbonyl group
DCM: dichloromethane (dichloromethane)
DCE:1, 2-dichloroethane
DMF: n, N-dimethylformamide
Oxyme：Ethyl Cyanoglyoxylate-2-Oxime
DIEA: n, N-diisopropylethylamine
DIC: n, N-diisopropylcarbodiimide
S5: (S) -2-amino-2-methyl-4-pentanoic acid
TFA: trifluoroacetic acid
TIPS: triisopropylsilane
Grubbs i: phenyl methylene bis (tricyclohexylphosphorus) ruthenium dichloride
Percent: unless otherwise specified, the mass percentages are all indicated in the invention.
The experimental materials involved were derived as follows:
amino acid, rink Amide MBHA resin was purchased from shanghai jier biochemical limited; n, N-Diisopropylcarbodiimide (DIC), ethyl Cyanoglyoxylate-2-oxo, trifluoroacetic acid (TFA), acetonitrile (chromatographic purity) were purchased from Beijing carbofuran technologies Co., ltd; n, N-Diisopropylethylamine (DIEA), N-Dimethylformamide (DMF), dehydrated ether, dichloromethane (DCM), 1, 2-Dichloroethane (DCE), piperidine, and phenol were all analytically pure and purchased from Beijing, inc. as a national pharmaceutical chemicals group.
Example 1
The invention provides an alpha-helical polypeptide antibiotic bound by a full-hydrocarbon side chain, which is prepared by the following steps:
1. synthesis of polypeptide antibiotics (shown in FIG. 2)
(1) Preparation of Compound 1
500mg (sample loading amount of 0.30 mmol/g) of amino resin is taken and added into a solid phase synthesis reaction tube, and the resin is fully swelled by soaking in DCM for 20min and pumped for later use.
A20% piperidine-DMF solution was added until the resin was completely submerged, shaking was performed at 25℃for 5min X2 to remove Fmoc from the resin, and the resin was washed 3 times with DCM and DMF in sequence.
(2) Preparation of Compound 2
The first amino acid in the sequence (1 mmol), oxyme (142 mg,1 mmol) and DIC (155. Mu.L, 1 mmol) were dissolved in 6mL DMF and added to the resin and shaken for 20min at 60℃ (S) 5 The latter amino acid was reacted for 1h and the reaction was repeated 1 time, washing the resin 3 times each with DCM and DMF in sequence.
(3) Preparation of Compound 3
Repeating steps (1) and (2), sequentially mixing Fmoc amino acid (1 mmol), oxyme (142 mg) and DIC (155 μl) according to polypeptide sequence in 6mL DMF, adding into resin, oscillating at 60deg.C for 20min, and repeating deprotection, condensation and deprotection until all amino acids are connected. After deprotection of the last amino acid, 6mL of a mixture of acetic anhydride: DIEA: DMF (1:1:8) was added and the resin was washed 3 times with DCM and DMF sequentially with shaking at 25℃for 20min.
(4) Preparation of Compound 4
A solution of Grubbs I (58 mg) in DCE (6 mL) was added and the reaction was run twice at 25℃for 2h each, after which the resin was washed 3 times with DCM, DMF and anhydrous diethyl ether, and the resin was dried in vacuo.
(5) Preparation of target Compounds
Cleaning resin, draining, adding TIPS H 2 O tfa=2.5:2.5:95 (V/V) 10mL, shaking for 4h at normal temperature, filtering, washing the resin with a little TFA, and collecting the filtrate. And (3) blowing off excessive TFA by argon bubbling, pouring into glacial ethyl ether for precipitation and centrifugation, discarding supernatant, repeatedly washing and centrifuging for 3 times by using the glacial ethyl ether, and drying by argon to obtain a crude product of the staple peptide.
2. Purification of target polypeptide antibiotics
The crude peptide was dissolved with acetonitrile and water and purified by preparative RP-HPLC. The separation conditions were as follows:
instrument: SHIMADZU (LC-6A) reverse phase high performance liquid chromatography (RP-HPLC)
Chromatographic column: c18 column (Daisogel, 20X 250 mm)
Mobile phase: mobile phase a is an aqueous solution with a volume fraction of 0.1% TFA, mobile phase B is an acetonitrile solution with a volume fraction of 0.1% TFA;
the steps and parameters are as follows: starting from 10% buffer A, a linear gradient elution was performed over 50 minutes to 75% buffer A at a flow rate of 10mL/min and a sample loading of 5mL at a detection wavelength of 214nm.
Example 2: identification and purity analysis of the product
The product from step 2 of example 1 was analyzed for purity by HPLC and the molecular weight of the product was identified by ESI-MS, which was measured on a shimadzu LCMS-8040 mass spectrometer. Scanning mode: a positive ion; measurement time: 0-2min; measurement range: 400-2000; data acquisition time: 2min; total flow rate: 0.2mL/min; buffer a was water plus 0.1% formic acid, buffer B was acetonitrile plus 0.1% formic acid, and 80% buffer B was maintained for 2min for compound analysis. The time of the staple peptide is consistent with the time of the main peak of the crude product, and the purity of the staple peptide prepared by the method is more than 98 percent. The HPLC and ESI-MS mass spectrometer analysis results are shown in FIGS. 3-5.
Example 3: determination of the antibacterial Activity of the polypeptide antibiotics of the present invention
The Minimum Inhibitory Concentration (MIC) of the stapled peptides was determined using a microbial dilution method according to the method of the Clinical and Laboratory Standards Institute (CLSI). Preparation of 10 in MH-II broth 6 CFU/mL bacterial suspension, peptide solutions of different concentrations were prepared in Phosphate Buffered Saline (PBS), then 50 μl of peptide solution was mixed with 50 μl of bacterial suspension in 96-well plates, and the plates were incubated at room temperature of 37 ℃ for 24 hours. To each well 10. Mu.L of AlamarBlue cell active reagent (Siamer Feishmanic science Co., UK) was added and the plate was incubated at 37℃for an additional 2 hours. MIC was recorded as the lowest peptide concentration required to completely inhibit bacterial growth. The assays were performed in three independent experiments. From the results, the polypeptide antibiotic KR (I 3 ,I 7 )、KR(Q 5 ,D 9 )、KR(I 7 ,L 11 ) Can significantly inhibit the growth and reproduction of gram positive and gram negative bacteria, wherein KR (Q 5 ,D 9 ) The effect was optimal and the results are shown in Table 6.
Example 4: determination of the haemolytic toxicity of the polypeptide antibiotics prepared in example 1
Peptide solutions at a concentration of 256 μg/mL were serially diluted in 96-well plates and then mixed with an equal volume of 4% (v/v) rabbit red blood cell suspension. Plates were incubated at 37℃for 1 hour and then centrifuged at 1000rpm for 10 minutes to separate the supernatant. The amount of released hemoglobin was determined using a microplate reader (BioTek, USA). Absorbance at 570nm wavelength was measured and percent hemolysis was calculated as: hemolysis (%) = (Abspeptide-absblanc)/(abscon-absblanc), where absblanc and abscon are absorbance of samples treated with PBS and 0.1% triton X-100, respectively. From the results, the polypeptide antibiotic KR (I 3 ,I 7 )、KR(Q 5 ,D 9 )、KR(I 7 ,L 11 ) Has lower hemolytic toxicity than LL-37, and the results are shown in FIG. 7.
Example 5: determination of cytotoxicity of polypeptide antibiotics prepared in example 1
NIH 3T3 cells were grown at 1.5X10 4 The density of individual cells/wells was seeded in 96-well plates and incubated at 37 ℃ for 24 hours. Cells were then treated with peptide solutions of varying concentrations in fresh DMEM and incubated for 24 hours, followed by the addition of 10 μl CCK-8 and further incubation for 1 hour. The absorbance was measured at a wavelength of 450nm using a station 5 microplate reader (BioTek, USA), and the percent cell viability was calculated as follows: cell viability (%) = (Abspeptide-absblanck)/(abscoprol-absblanck) ×100%, where abscoprol is the absorbance of cells treated with PBS and absblanck is the absorbance of cell-free medium. Fitting data using nonlinear regression in Graphpad Prism (curve fitting) to obtain IC 50 Values. The measurements were performed in three independent experiments, and standard error (SD) is shown in the histogram. From the results, the polypeptide antibiotic KR (I 3 ,I 7 )、KR(Q 5 ,D 9 )、KR(I 7 ,L 11 ) Has lower cytotoxicity than LL-37, and the results are shown in FIG. 8.
Example 6: determination of the serological stability of the polypeptide antibiotics prepared in example 1
The serum stability of the stapled peptides was tested by reverse phase high performance liquid chromatography (RP-HPLC). A2 mg/mL peptide solution was prepared in PBS at pH 7.4 and incubated with human serum at 37℃at a final volume ratio of 1:4. Samples were taken at various time intervals and 50. Mu.L of the mixture was added to 50. Mu.L of ethanol to terminate the enzymatic hydrolysis. After centrifugation at 10000rpm for 10min at 4℃the supernatant was collected and analyzed by HPLC at a wavelength of 220nm. The integrated peak area of the intact peptide was recorded and the percentage of remaining peptide was calculated from the integrated area as follows: polypeptide remaining (%) = peak area of remaining polypeptide/peak area of intact polypeptide x 100% was measured in three independent experiments and standard error (SD) is shown in the histogram. From the results, the polypeptide antibiotic KR (I 3 ,I 7 )、KR(Q 5 ,D 9 )、KR(I 7 ,L 11 ) Has higher serum stability than LL-37, and the results are shown in FIG. 9.
Example 7: determination of anti-inflammatory Activity of polypeptide antibiotics prepared in example 1
RAW264.7 cells were cultured at 4X 10 4 The density of individual cells/wells was seeded in 48-well plates and incubated at 37 ℃ for 24 hours. Cells were then treated with different concentrations of peptide in fresh DMEM and incubated for 2 hours. mu.L of LPS (10. Mu.g/mL) was added and incubated for an additional 6 hours. After centrifugation at 1809rpm for 10 minutes, cell supernatants were collected and levels of TNF- α and IL-6 production were measured by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Lianke, china) according to the manufacturer's instructions. Measurements were performed in three independent experiments, standard error (SD) is shown in the histogram. From the results, the polypeptide antibiotic KR (Q 5 ,D 9 ) Has higher anti-inflammatory activity than LL-37, and can significantly reduce TNF-alpha and IL-6 production, as shown in FIG. 10.
Example 8: experiment of polypeptide antibiotics prepared in example 1 for promoting healing of bacterial infection wound of mice
Animal experiments were approved by the university of Shanghai Ethical Committee (ECSHU). Adult female BALB/c mice (6-8 weeks, 15-20 g) were purchased from Jiangsu Hua Xinnuo medical science and technology Co., ltd (Jiangsu, china) and housed in standard plastic rodent cages (25 ℃, 50% -70% humidity, 12 hour light/dark cycle) under manual control. All mice were anesthetized with a 10mL/kg dose of 4% chloral hydrate for intraperitoneal injection, then shaved with a razor and rinsed with 75% ethanol. Full-thickness wounds of 8mm diameter were then prepared on the back of mice using a biopsy punch and were dense at 2.5X10 in PBS pH 7.4 8 CFU/mL of 20. Mu.L of E.coli suspension. The infected wound was covered with sterile gauze and secured with an elastic adhesive bandage. After 2 days of continuous infection, mice were divided into 3 groups of 10 mice each, each group was treated with 20. Mu.L of LPBS, 20. Mu.L of LL-37 solution (12.8 mg/mL,100 XMIC) and 20. Mu.L of KR (Q) 5 ,D 9 ) Solution (0.8 mg/mL,100 XMIC). Treatments were repeated on days 3,7 and 10 and wound areas of mice were recorded on days-2, 0, 3, 5, 7, 10, 12 and 14. From the results, the polypeptide antibiotic KR (Q) 5 ,D 9 ) The healing of bacterial infected skin wounds was promoted more significantly than LL-37, and the results are shown in figure 11.
The above examples show that the invention successfully prepares the peptide Ac-KRIVQRIKDFLR-NH based on the linear template 2 The polypeptide antibiotic can obviously inhibit the growth and reproduction of gram-negative bacteria and gram-positive bacteria, has low hemolytic toxicity to red blood cells and cytotoxicity to embryo fibroblasts, high serum stability and certain anti-inflammatory activity, can obviously promote the healing of skin wound surfaces of mice infected by bacteria, and has good application prospect.
Claims (10)
1. An all-hydrocarbon side-chain bound alpha-helical polypeptide antibiotic, wherein said polypeptide antibiotic is selected from one of the following:
a) Ac-KRIVQRIKDFLR-NH 2 Is a peptide chain template in which 3I and 7I are replaced by S5 and are cyclized;
b) Ac-KRIVQRIKDFLR-NH 2 Is used as a template of the peptide chain,wherein 5Q and 9D are replaced with S5 and are ring-closed;
c) Ac-KRIVQRIKDFLR-NH 2 Is a peptide chain template in which 7I and 11L are replaced by S5 and are cyclized.
2. The method for preparing the alpha-helical polypeptide antibiotic according to claim 1, comprising the steps of:
step 1): removing Fmoc protecting groups on the Rink Amide MBHA resin by using a deprotection reagent;
step 2): coupling the carboxyl group of the Fmoc-protected amino acid with the exposed amino group on the resin using a condensing reagent;
step 3): removing Fmoc protecting groups on the amino acid using a deprotection reagent;
step 4): repeating the coupling-deprotection operation in the step 2) and the step 3), and synthesizing a peptide chain according to an amino acid sequence, wherein the amino acids at the i and i+4 ring closure sites are respectively replaced by S5;
step 5): modifying the deprotected amino group of the last amino acid on the peptide chain by using an acetylating agent;
step 6): catalyzing the side chains at the S5 positions i and i+4 to generate olefin metathesis reaction by using a cyclization reagent;
step 7): cleaving the peptide chain from the resin using a cleavage reagent and precipitating with glacial ethyl ether to obtain a crude polypeptide antibiotic;
step 8): and (3) separating and purifying the crude polypeptide antibiotics by using high performance liquid chromatography.
3. The method according to claim 2, wherein the deprotecting reagent in step 1) is piperidine and DMF in a volume ratio of 1:4, the feed ratio of the mixed solution relative to the amino resin is 20:1mL/mmol.
4. The method according to claim 2, wherein the condensing agent in the step 2) is a mixed solution of amino acid, oxyme, DIC and DMF, and the feeding ratio of the mixed solution to the amino resin is 24:1mL/mmol; wherein the ratio of amino acids to Oxyme, DIC, DMF is 1mol:1mol:6mL.
5. The method according to claim 2, wherein the acetylating agent in step 5) is acetic anhydride, DIEA and DMF in a volume ratio of 1:1:8, the feed ratio of the mixed solution relative to the amino resin is 20:1mL/mmol.
6. The method of claim 2, wherein the cyclization reagent in step 6) is a DCE solution of Grubbs I reagent having a concentration of 29mg/3mL, and the feed ratio of the DCE solution to the amino resin is 20:1mL/mmol.
7. The method of claim 2, wherein the cleavage agent in step 7) is 2.5:2.5: TIPS, H in 95 volume ratio 2 The feed ratio of the mixed solution of O and TFA to the amino resin was 20:1mL/mmol.
8. Use of an alpha-helical polypeptide antibiotic according to claim 1 for the preparation of a medicament for inhibiting the growth and reproduction of gram-positive and negative bacteria.
9. Use of an alpha-helical polypeptide antibiotic according to claim 1 for the manufacture of a medicament for the treatment of an infectious disease caused by gram-positive or/and negative bacteria.
10. Use of an alpha-helical polypeptide antibiotic according to claim 1 for the preparation of a medicament for anti-inflammatory and pro-wound healing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211414907.3A CN117143196A (en) | 2022-11-11 | 2022-11-11 | Alpha-helical polypeptide antibiotic bound by full-hydrocarbon side chain, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211414907.3A CN117143196A (en) | 2022-11-11 | 2022-11-11 | Alpha-helical polypeptide antibiotic bound by full-hydrocarbon side chain, and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117143196A true CN117143196A (en) | 2023-12-01 |
Family
ID=88910690
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211414907.3A Pending CN117143196A (en) | 2022-11-11 | 2022-11-11 | Alpha-helical polypeptide antibiotic bound by full-hydrocarbon side chain, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117143196A (en) |
-
2022
- 2022-11-11 CN CN202211414907.3A patent/CN117143196A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111748018B (en) | Biocompatible antibacterial peptide with self-assembly potential, and preparation method and application thereof | |
| CN110627871A (en) | Cationic bridged staple peptides and uses thereof | |
| CN115028685B (en) | Cationic bicyclic antibacterial peptide and application thereof | |
| CN115960261A (en) | Tryptophan and phenylalanine cross-chain interaction beta-hairpin antibacterial peptide WFL and preparation method and application thereof | |
| CN112694525B (en) | Small molecular polypeptide and antibacterial and antiviral application thereof | |
| CN116804046B (en) | Cyclic cation antibacterial peptide and application thereof | |
| CN110283245B (en) | Pig marrow derived PMAP-23 derived antibacterial peptide, preparation method and application | |
| CN111253474B (en) | Antibacterial peptide RG-27 and application thereof | |
| CN112625106A (en) | Antibacterial polypeptide compound, synthesis method and application thereof | |
| CN116217669A (en) | Stapler peptide capable of improving broad-spectrum antibacterial activity, and preparation method and application thereof | |
| CN110066320B (en) | Cyclic peptide resisting multiple drug-resistant bacteria and preparation method and application thereof | |
| CN117024552A (en) | Small molecular antibacterial polypeptide, preparation method and application thereof | |
| CN110054664B (en) | Side chain fatty acid modified antibacterial peptide analogue containing D-type amino acid and synthesis and application thereof | |
| CN116987150A (en) | Antibacterial polypeptide, polypeptide derivative, and preparation method and application thereof | |
| CN117143196A (en) | Alpha-helical polypeptide antibiotic bound by full-hydrocarbon side chain, and preparation method and application thereof | |
| CN114805495B (en) | Enzymolysis-resistant branched antibacterial peptide Pal-CRKP, and preparation method and application thereof | |
| US20040072990A1 (en) | Antimicrobial peptides with reduced hemolysis and methods of their use | |
| CN116178506A (en) | Stapler peptide and application thereof | |
| CN111410681B (en) | Antibacterial peptide analogue with small repeat unit sequence and application thereof | |
| CN113185577A (en) | Low-toxicity broad-spectrum antibacterial peptide with different charge arrangement modes and different charge types and application thereof | |
| CN106699862B (en) | Three cyclic polypeptides from palaemon carinicauda ALF and application and antibacterial agent thereof | |
| CN113248572B (en) | Anti-multidrug-resistant bacteria cyclopeptide and application thereof | |
| CN111116714B (en) | Wasp venom peptide reverse sequence analogue WVC-II and preparation method and application thereof | |
| CN111153966B (en) | Wasp venom peptide reverse sequence analogue WVF-II and preparation method and application thereof | |
| CN115746094B (en) | Antibacterial peptide and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |