CN116444603A - Tryptophan-containing antibacterial peptide and synthesis method and application thereof - Google Patents
Tryptophan-containing antibacterial peptide and synthesis method and application thereof Download PDFInfo
- Publication number
- CN116444603A CN116444603A CN202310434934.5A CN202310434934A CN116444603A CN 116444603 A CN116444603 A CN 116444603A CN 202310434934 A CN202310434934 A CN 202310434934A CN 116444603 A CN116444603 A CN 116444603A
- Authority
- CN
- China
- Prior art keywords
- fmoc
- resin
- tryptophan
- kwk
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 title claims abstract description 38
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 34
- 238000001308 synthesis method Methods 0.000 title claims abstract description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 103
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 92
- 229920001184 polypeptide Polymers 0.000 claims abstract description 85
- 241000894006 Bacteria Species 0.000 claims abstract description 18
- 238000001338 self-assembly Methods 0.000 claims abstract description 11
- 229920005989 resin Polymers 0.000 claims description 94
- 239000011347 resin Substances 0.000 claims description 94
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 43
- 238000006243 chemical reaction Methods 0.000 claims description 42
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 29
- 150000001413 amino acids Chemical class 0.000 claims description 29
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 26
- 239000003153 chemical reaction reagent Substances 0.000 claims description 23
- 238000005406 washing Methods 0.000 claims description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- 238000006482 condensation reaction Methods 0.000 claims description 18
- 238000010511 deprotection reaction Methods 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 17
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 15
- 125000006239 protecting group Chemical group 0.000 claims description 15
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 14
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 14
- 238000005086 pumping Methods 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 11
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 8
- 230000005587 bubbling Effects 0.000 claims description 7
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- MGHMWKZOLAAOTD-DEOSSOPVSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1h-indol-3-yl)propanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(=O)O)CC1=CNC2=CC=CC=C12 MGHMWKZOLAAOTD-DEOSSOPVSA-N 0.000 claims description 5
- 238000005336 cracking Methods 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 239000002121 nanofiber Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 230000002194 synthesizing effect Effects 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 4
- 238000000197 pyrolysis Methods 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 230000007017 scission Effects 0.000 claims description 3
- QXTIBZLKQPJVII-UHFFFAOYSA-N triethylsilicon Chemical compound CC[Si](CC)CC QXTIBZLKQPJVII-UHFFFAOYSA-N 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims 1
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 26
- 230000001580 bacterial effect Effects 0.000 abstract description 20
- 210000000170 cell membrane Anatomy 0.000 abstract description 18
- 239000004472 Lysine Substances 0.000 abstract description 10
- 239000003242 anti bacterial agent Substances 0.000 abstract description 9
- 239000000463 material Substances 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 5
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 abstract description 5
- 230000003044 adaptive effect Effects 0.000 abstract description 4
- 238000010276 construction Methods 0.000 abstract description 4
- 230000008105 immune reaction Effects 0.000 abstract description 4
- 239000002086 nanomaterial Substances 0.000 abstract description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 67
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 51
- 239000000243 solution Substances 0.000 description 39
- 238000000429 assembly Methods 0.000 description 21
- 230000000712 assembly Effects 0.000 description 21
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 10
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 9
- 206010018910 Haemolysis Diseases 0.000 description 9
- 230000008588 hemolysis Effects 0.000 description 9
- 125000003345 AMP group Chemical group 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 8
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 238000003917 TEM image Methods 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 210000003743 erythrocyte Anatomy 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 239000000178 monomer Substances 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000845 anti-microbial effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000009833 condensation Methods 0.000 description 3
- 230000005494 condensation Effects 0.000 description 3
- 230000009881 electrostatic interaction Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 241000194032 Enterococcus faecalis Species 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 238000001994 activation Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 125000000129 anionic group Chemical group 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- -1 lipid phosphatidylethanolamine Chemical class 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- RAVVEEJGALCVIN-AGVBWZICSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-5-amino-2-[[(2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2-[[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]hexanoyl]amino]hexanoyl]amino]-5-(diamino Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RAVVEEJGALCVIN-AGVBWZICSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000943303 Enterococcus faecalis ATCC 29212 Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 108700000788 Human immunodeficiency virus 1 tat peptide (47-57) Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229920002988 biodegradable polymer Polymers 0.000 description 1
- 239000004621 biodegradable polymer Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000002815 broth microdilution Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- ZGSPNIOCEDOHGS-UHFFFAOYSA-L disodium [3-[2,3-di(octadeca-9,12-dienoyloxy)propoxy-oxidophosphoryl]oxy-2-hydroxypropyl] 2,3-di(octadeca-9,12-dienoyloxy)propyl phosphate Chemical compound [Na+].[Na+].CCCCCC=CCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COP([O-])(=O)OCC(O)COP([O-])(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COC(=O)CCCCCCCC=CCC=CCCCCC ZGSPNIOCEDOHGS-UHFFFAOYSA-L 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012567 medical material Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000017066 negative regulation of growth Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 150000003408 sphingolipids Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- AQRLNPVMDITEJU-UHFFFAOYSA-N triethylsilane Substances CC[SiH](CC)CC AQRLNPVMDITEJU-UHFFFAOYSA-N 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0815—Tripeptides with the first amino acid being basic
-
- 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)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Communicable Diseases (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a self-assembly induced tryptophan-containing antibacterial peptide, a synthesis method and application thereof, belonging to the technical field of antibacterial agent preparation; according to the invention, an antibacterial peptide containing tryptophan with antibacterial performance is synthesized firstly, namely, fmoc-protected lysine-tryptophan-lysine tripeptide (Fmoc-KWK) is used, and then Fmoc-KWK is self-assembled to obtain an Fmoc-KWK assembly with better antibacterial performance; the Fmoc-KWK assembly is nanofiber-shaped, and promotes the polypeptide nanostructure to be combined with bacterial cell membranes through chiral arrangement of tryptophan residues induced by self-assembly, so that the aim of killing bacteria is fulfilled; the Fmoc-KWK and Fmoc-KWK assembly body disclosed by the invention have the advantages that the structure is clear, the sequence is controllable, the Fmoc-KWK assembly body does not contain an epitope which can possibly trigger specific recognition, the adaptive immune reaction can be avoided, and the Fmoc-KWK assembly body has more advantages than long-chain polypeptides when being used as an antibacterial material construction element.
Description
Technical Field
The invention belongs to the technical field of preparation of antibacterial agents, and particularly relates to an antibacterial peptide containing tryptophan as well as a synthesis method and application thereof.
Background
Currently, the widespread resistance of pathogenic bacteria to antibiotics has become a global threat. Many antibiotics under clinical development still belong to the existing family of compounds, and their availability may be reduced by the rapid development of clinical strain resistance. Therefore, there is an urgent need to develop antibacterial agents having new mechanisms of action.
Unlike traditional antibiotics, which act primarily through specific receptor-protein interactions, antibacterial peptides (Antimicrobial peptides, AMPs) generally exert antibacterial activity through non-receptor mediated membrane cleavage of pathogens, so they do not render bacteria resistant. Most of the antibacterial peptides have both cationic and hydrophobic amino acid residues, so that the antibacterial peptides have amphipathy in polar organism environments, and further can destroy bacterial membranes. The antibacterial principle of AMPs is: the bacterial membrane and AMPs have electrostatic interactions because the bacterial membrane is composed of anionic lipids such as phosphatidylglycerol and cardiolipin and amphiphilic ionic lipid phosphatidylethanolamine, once the polar side of AMPs rich in charged residues is combined with the anionic bacterial membrane, the hydrophobic region of the peptide chain is further inserted into the hydrophobic region in the phospholipid bilayer, thereby inducing membrane permeability and killing bacteria. Also, unlike the negatively charged cell membrane of bacteria, the normal cell surface of mammals is mainly composed of neutral charged phospholipids, such as sphingolipids or phosphatidylcholine, so AMPs only selectively kill bacteria without damaging normal cells. AMPs have broad-spectrum antibacterial activity due to their high specificity, lack of drug resistance propensity, good biocompatibility, etc., and have received great attention as potential alternatives to traditional antibiotics.
In recent years, the designs of antibacterial peptides by scholars at home and abroad are mostly concentrated on natural and long-chain polypeptides or coupled with other polymers and metal nano examples with antibacterial capability. For example, the HnMC peptide composite micelle is formed by self-assembly of chimeric antibacterial lipid polypeptide (DSPE-PEG-HnMC) and amphiphilic biodegradable polymer, has very high specificity, can easily detect wide bacterial infection, and is a good targeted antibacterial agent for drug-resistant bacterial infection; the composite material AgPW@PDA@Nisin with a shell-core structure can destroy cell membranes of staphylococcus aureus, so that nucleotide leakage is caused, the permeability is changed, the cell integrity is destroyed, and bacteria are killed; TAT (YGRKKRRQRRR) is a peptide derived from HIV glycoprotein, TAT is used as an antibacterial peptide against drug resistant bacteria, and as a transporter for other polypeptides, proteins, nanoparticles or anticancer drugs, which are very effective against staphylococcus aureus in vivo. However, applications involving AMPs as described above have focused mainly on naturally occurring complex long-chain polypeptides and their derivatives, mostly comprising 20-50 amino acid residues, and are difficult to synthesize and costly to produce. Therefore, it is desirable to provide a method for synthesizing short peptide AMPs with definite structure and controllable sequence.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a self-assembly induced tryptophan-containing antibacterial peptide, and a synthesis method and application thereof; according to the invention, an antibacterial peptide containing tryptophan with antibacterial performance is synthesized firstly, namely, fmoc-protected lysine-tryptophan-lysine tripeptide (Fmoc-KWK) is used, and then Fmoc-KWK is self-assembled to obtain an Fmoc-KWK assembly with better antibacterial performance; the Fmoc-KWK assembly is nanofiber-shaped, and promotes the polypeptide nanostructure to be combined with bacterial cell membranes through chiral arrangement of tryptophan residues induced by self-assembly, so that the aim of killing bacteria is fulfilled; the Fmoc-KWK and Fmoc-KWK assembly body disclosed by the invention have the advantages that the structure is clear, the sequence is controllable, the Fmoc-KWK assembly body does not contain an epitope which can possibly trigger specific recognition, the adaptive immune reaction can be avoided, and the Fmoc-KWK assembly body has more advantages than long-chain polypeptides when being used as an antibacterial material construction element.
In order to achieve the above technical object, the present invention adopts the following technical means.
The invention firstly provides a tryptophan-containing antibacterial peptide, which is Fmoc-KWK, and has the structural formula:
the invention also provides a synthesis method of the tryptophan-containing antibacterial peptide, which comprises the following steps:
(1) Adding activated resin into a polypeptide solid-phase synthesis column, then adding an Fmoc deprotection reagent into the activated resin to perform Fmoc protection group removal reaction on the activated resin, and repeating the Fmoc protection group removal reaction for a plurality of times to obtain resin A;
(2) Fmoc-protected lysine (Fmoc-Lys (Boc) -OH), condensing agent benzotriazole-N, N, N ', N' -tetramethylurea Hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIPEA) were dissolved in Dimethylformamide (DMF) to give amino acid solution A;
adding an amino acid solution A into a polypeptide solid-phase synthesis column containing resin A, then placing the polypeptide solid-phase synthesis column containing resin A into a polypeptide synthesizer for condensation reaction, washing and pumping after the reaction is finished to obtain resin B;
(3) Fmoc-protected tryptophan (Fmoc-Trp-OH (Boc) -OH), condensing agent HBTU, HOBt and DIPEA were dissolved in DMF to obtain amino acid solution B;
repeating the step (1) to remove Fmoc protecting groups in the resin B, adding an amino acid solution B into a polypeptide solid-phase synthesis column containing the resin B, then placing the polypeptide solid-phase synthesis column containing the resin B into a polypeptide synthesizer for condensation reaction, and washing and pumping after the reaction is finished to obtain resin C;
(4) Fmoc-protected lysine (Fmoc-Lys (Boc) -OH), condensing agent HBTU, HOBt and DIPEA were dissolved in DMF to give amino acid solution C;
repeating the step (1) to remove Fmoc protecting groups in the resin C, adding an amino acid solution C into a polypeptide solid-phase synthesis column containing the resin C, then placing the polypeptide solid-phase synthesis column containing the resin C into a polypeptide synthesizer for condensation reaction, and washing and pumping out after the reaction is finished to obtain resin D;
(5) Adding a deprotection reagent into a polypeptide solid-phase synthesis column containing resin D, then placing the polypeptide solid-phase synthesis column into a polypeptide synthesizer for deprotection reaction, and flushing and pumping out after the reaction is finished; repeating the steps for a plurality of times to obtain resin E;
(6) Adding a pyrolysis liquid into a polypeptide solid-phase synthesis column containing resin E, then placing the polypeptide solid-phase synthesis column into a polypeptide synthesizer for pyrolysis reaction, and filtering to obtain a filtrate after the reaction is finished;
then nitrogen bubbling is carried out on the filtrate, precooled diethyl ether is added into the filtrate after the bubbling is finished, and the precipitate is centrifugally taken out, washed and dried to obtain a crude polypeptide product;
and (3) separating and purifying the crude polypeptide product by high performance liquid chromatography, and freeze-drying after purification to obtain the tryptophan-containing antibacterial peptide, which is marked as Fmoc-KWK.
Preferably, in step (1), the resin comprises Rink-Amide-AM resin;
the Fmoc deprotection reagent is prepared from piperidine and DMF in a volume ratio of 1:4;
the dosage ratio of the activated resin to Fmoc deprotection reagent is 5mL:100mg;
the Fmoc protecting group removal reaction is carried out for 15-20 min at room temperature, and the Fmoc protecting group removal reaction is repeated for 2-3 times.
Preferably, in step (2), the ratio of Fmoc-Lys (Boc) -OHHBTU, HOBt, DIPEA to DMF is 92mg:74mg:30mg:50mg:3.5mL;
the dosage ratio of the amino acid solution A to the activated resin in the step (1) is 5mL:100mg;
the condensation reaction is carried out for 4-8 hours at room temperature, and DMF and Dichloromethane (DCM) are alternately used for washing after the reaction is finished.
Preferably, in step (3), the ratio of Fmoc-Trp-OH (Boc) -OH, HOBt, DIPEA to DMF is 92mg:74mg:30mg:50mg:3.5mL;
the dosage ratio of the amino acid solution B to the activated resin in the step (1) is 5mL:100mg;
the condensation reaction is carried out for 4-8 hours at room temperature, and DMF and DCM are alternately used for washing after the reaction is finished.
Preferably, in step (4), the ratio of Fmoc-Lys (Boc) -OH, HOBt, DIPEA to DMF is 92mg:74mg:30mg:50mg:3.5mL;
the dosage ratio of the amino acid solution B to the activated resin in the step (1) is 5mL:100mg;
the condensation reaction is carried out for 4-8 hours at room temperature, and DMF and DCM are alternately used for washing after the reaction is finished.
Preferably, in step (5), the deprotecting reagent consists of a volume ratio of 10:90 trifluoroacetic acid (TFA) and DCM,
the ratio of the amount of the deprotection reagent to the activated resin in step (1) was 5mL:100mg;
repeating the steps for a plurality of times for 2-3 times.
Preferably, in step (6), the lysate consists of TFA, triethylsilane (TES) and H in a volume ratio of 94:5:1 2 O is prepared;
the condition of the cracking reaction is that the reaction is carried out for 1.5 to 2 hours at room temperature;
the nitrogen bubbling time is 10-20 min;
the dosage ratio of the precooled diethyl ether to the activated resin in the step (1) is 100mL:100mg.
The invention also provides a tryptophan-containing antibacterial peptide assembly, which is formed by self-assembling the tryptophan-containing antibacterial peptide; the tryptophan-containing antibacterial peptide assembly has a diameter of 7-10nm and is in a nanofiber shape and is marked as an Fmoc-KWK assembly.
The invention also provides a synthesis method of the Fmoc-KWK assembly, which comprises the steps of adjusting the pH value of the Fmoc-KWK prepared by the method to 7-7.5, and incubating for 24 hours at room temperature to obtain the Fmoc-KWK assembly.
The invention also provides application of the tryptophan-containing antibacterial peptides Fmoc-KWK and Fmoc-KWK assembly in bacterial killing.
Compared with the prior art, the invention has the beneficial effects that:
in Fmoc-KWK, the surface of lysine is positively charged, so that bacterial cell membranes can be targeted, tryptophan is nonpolar amino acid and can be inserted into the bacterial cell membranes, and cell lysis and death are caused. And, the Fmoc-protected lysine-tryptophan-lysine tripeptide has a pH-induced self-assembly phenomenon, and Fmoc-KWK assembly can be obtained through self-assembly. Fmoc-KWK assemblies have a greater antimicrobial capacity than Fmoc-KWK. Fmoc-KWK and Fmoc-KWK assemblies both have potential as antibiotic substitutes.
The antibacterial mechanism of the Fmoc-KWK and Fmoc-KWK assembly provided by the invention is as follows: the charged lysine residue (K) targets the bacterial cell membrane through electrostatic interaction, is enriched on the surface of the bacterial cell membrane, and after a certain threshold value is reached, the polypeptide starts to be inserted into the cell membrane, and a stable barrel-shaped hole is formed on the cell membrane, so that the cell death is caused. The ordered arrangement of tryptophan residues in Fmoc-KWK assemblies is more favorable for insertion into cell membranes than Fmoc-KWK, and thus has a greater antibacterial capacity and can kill bacteria at a lower dose and faster rate. Since the process of killing bacteria by Fmoc-KWK and Fmoc-KWK assemblies is the targeted bacterial cell membrane, bacteria cannot evolve resistance to Fmoc-KWK and Fmoc-KWK assemblies.
In addition, the Fmoc-KWK and Fmoc-KWK assembly provided by the invention are short peptides composed of three amino acids, are low in cost, convenient to prepare and have the effect equivalent to that of long-chain antibacterial peptides. The Fmoc-KWK and Fmoc-KWK assemblies have definite structure and controllable sequence, do not contain epitope which possibly causes specific recognition, can avoid causing adaptive immune reaction, and have more advantages than long-chain polypeptides when being used as antibacterial material construction motifs.
Drawings
FIG. 1 is a schematic diagram of the synthetic pathway of Fmoc-KWK.
FIG. 2 is an electrospray ionization mass spectrometry (ESI-MS) diagram of Fmoc-KWK.
FIG. 3 is a high performance liquid chromatogram of Fmoc-KWK.
FIG. 4 is a TEM image of Fmoc-KWK at pH 2.7.
FIG. 5 is a TEM image of Fmoc-KWK assemblies at 100nm scale.
FIG. 6 is a TEM image of Fmoc-KWK assembly at a scale of 1 μm.
FIG. 7 is a TEM image of Fmoc-KWK assemblies at 200nm scale.
FIG. 8 is a MIC pattern of Fmoc-KWK assemblies (a) and Fmoc-KWK (b) for enterococcus faecalis ATCC 29212, with the right-most control group of normally growing bacteria.
FIG. 9 is a MIC diagram of Fmoc-KWK assemblies (a) and Fmoc-KWK (b) for Bacillus subtilis ATCC 6633, with the rightmost control group of normally growing bacteria.
FIG. 10 is a MIC diagram of Fmoc-KWK assemblies (a) and Fmoc-KWK (b) for E.coli ATCC 25922, with the rightmost control group of normally growing bacteria.
FIG. 11 is a MIC pattern of Fmoc-KWK assemblies (a) and Fmoc-KWK (b) against E.coli ATCC 8739, the rightmost side of which is a control group of normally growing bacteria.
FIG. 12 is an ultraviolet absorbance graph of TX-100 positive control group.
FIG. 13 is a drawing of Fmoc-KWK assembly and a hemolysis experiment of Fmoc-KWK, wherein the inset is a blood sample after centrifugation.
FIG. 14 shows Fmoc-KWK assemblies and the antibacterial principle of Fmoc-KWK.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto. In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The sources of the reagents used, the trade names and the components of the reagents are shown when the reagents appear for the first time, and the reagents which are the same as the sources shown for the first time are not specially indicated; the reagents, materials, etc. involved are commercially available without any particular explanation.
Example 1:
the synthetic route of Fmoc-KWK tripeptide sequence in this example is schematically shown in FIG. 1, and the following specifically describes the synthetic process of Fmoc-KWK tripeptide sequence.
(1) Resin activation:
washing a polypeptide solid-phase synthesis column, weighing 100mg Rink-Amide-AM resin (the loading amount is 0.35mg/mL, hereinafter referred to as resin) into the polypeptide solid-phase synthesis column, adding DMF (dimethyl formamide) washing resin into the polypeptide solid-phase synthesis column for three times, washing impurities on the resin surface, and pumping liquid in the polypeptide solid-phase synthesis column by using a vacuum pump.
5mL DCM is added into the polypeptide solid-phase synthesis column, resin is soaked for 1-2h at 25 ℃, then DCM is added into the polypeptide solid-phase synthesis column for three times, and liquid in the polypeptide solid-phase synthesis column is used for obtaining activated resin. The activation process mainly causes the resin pellets to swell, so that the groups carried by the resin pellets are fully exposed, and the resin pellets are easier to carry out condensation reaction with amino acid.
(2) Removal of Fmoc protecting groups on resin:
5mL of Fmoc deprotection reagent prepared by piperidine and DMF according to a volume ratio of 1:4 is added into a polypeptide solid-phase synthesis column, then the polypeptide solid-phase synthesis column is placed into a polypeptide synthesizer (Intelli-MixerTMRM-2, shanghai micro hundred technology) for deprotection reaction for 20min at 25 ℃, and after the reaction is finished, the resin is alternately washed three times by DMF and DCM, and the liquid in the polypeptide solid-phase synthesis column is pumped out. To ensure complete removal of the Fmoc group, the deprotection reaction was repeated three times to give resin a.
In this step, the resin was alternately washed three times with DMF and DCM, the first DMF and DCM alternately washing to remove the reaction solvent and allow the resin to swell again; the second alternate washing is to remove residual DMF which is coated in the resin; the third alternate flushing was to completely remove the residual reaction solution. The DCM is used for final flushing to ensure that the resin is in a swelling state, and the next reaction is facilitated. And simultaneously, DCM has strong volatility and is easy to pump out, so that the resin is kept in a dry state, and the concentration of a subsequent reaction solution is not reduced.
(3) Amino acid condensation of polypeptide sequences:
fmoc-protected lysine Fmoc-Lys (Boc) -OH (92 mg,3 eq) and condensing agent HBTU (74 mg,3 eq), HOBt (30 mg,3 eq), DIPEA (50 mg,6 eq) were weighed and added to a 5mL glass bottle, then 3.5mL pure DMF was added and dissolved under ultrasound to give amino acid solution A.
Then adding amino acid solution A into the polypeptide solid-phase synthesis column containing resin A, then carrying out condensation reaction in a polypeptide solid-phase synthesis column polypeptide synthesizer containing resin A at 25 ℃ for 4 hours, and after the reaction is finished, alternately washing the resin with DMF and DCM for three times and pumping out to obtain resin B.
To ensure that the condensation reaction is complete, the extent of reaction can be determined by qualitatively detecting the presence of free amino groups on the resin by ninhydrin detection (Kaiser). The Kaiser detection steps are as follows: taking a small amount of cleaned resin by using a capillary tube, sequentially adding 2 drops of reagent 1 and 2 drops of reagent 2, uniformly mixing, heating in an oil bath pot at 100 ℃, taking out the small glass tube after heating for 3min to observe the color of the resin, and colorless to indicate that the condensation reaction is complete.
Wherein reagent 1:5g of ninhydrin was dissolved in 100mL of ethanol; reagent 2:80g of phenol was dissolved in 20mL of ethanol.
(4) Condensation of Fmoc-protected tryptophan:
the Fmoc protecting group removal step on resin described in step (2) was repeated to remove Fmoc protecting group in resin C in step (3), then Fmoc-Trp-OH (92 mg,3 eq) of Fmoc-protected tryptophan and condensing agent HBTU (74 mg,3 eq), HOBt (30 mg,3 eq), DIPEA (50 mg,6 eq) were weighed and added to a 5mL glass bottle, then 3.5mL pure DMF was added and dissolved under ultrasonic conditions to give amino acid solution B.
Then adding amino acid solution B into the polypeptide solid-phase synthesis column containing the resin B, then carrying out condensation reaction in a polypeptide solid-phase synthesis column polypeptide synthesizer containing the resin B at 25 ℃ for 4 hours, and after the reaction is finished, alternately washing the resin with DMF and DCM for three times and pumping out to obtain resin C.
(5) Condensation of Fmoc protected lysine:
the Fmoc protecting group removal step on resin described in step (2) was repeated to remove Fmoc protecting group in resin C in step (3), then Fmoc-protected lysine Fmoc-Lys (Boc) -OH (92 mg,3 eq) and condensing agent HBTU (74 mg,3 eq), HOBt (30 mg,3 eq), DIPEA (50 mg,6 eq) were weighed and added to a 5mL glass bottle, then 3.5mL pure DMF was added and dissolved under ultrasound to give amino acid solution C.
Then adding amino acid solution C into the polypeptide solid-phase synthesis column containing the resin C, then carrying out condensation reaction in a polypeptide solid-phase synthesis column polypeptide synthesizer containing the resin C at 25 ℃ for 4 hours, and after the reaction is finished, alternately washing the resin with DMF and DCM for three times and pumping out to obtain the resin D.
(6) Removal of lysine side chain Boc protecting group:
adding 5mL of deprotection reagent prepared from TFA and DCM according to a volume ratio of 10:90 into a polypeptide solid-phase synthesis column containing resin D, carrying out deprotection reaction at 25 ℃ for 20min in a polypeptide solid-phase synthesis column polypeptide synthesizer, flushing the resin three times with DCM after the reaction is finished, and pumping out liquid in the polypeptide solid-phase synthesis column. To ensure complete removal of the lysine side chain Boc protecting group, the deprotection reaction was repeated three times to give resin E.
(7) Cleavage of peptide chain on resin:
5mL of a solid phase synthesis column containing resin E from TFA, TES and H was added to the column 2 And (3) carrying out a cracking reaction for 1.5-2 hours at 25 ℃ in a polypeptide solid-phase synthesis column polypeptide synthesizer on the cracking solution prepared by O according to the volume ratio of 94:5:1, wherein the cracking reaction is not suitable for too long, so that the degradation of peptide chains by strong acid is avoided. After the reaction, the peptide chain will be detached from the resin and stored in the lysate. The resin was then removed by filtration, the lysate was collected in an Erlenmeyer flask and excess TFA was removed by bubbling pure nitrogen for 10 min.
(8) Polypeptide precipitation:
100mL of precooled diethyl ether was added to the conical flask containing the lysate, and the peptide chain was insoluble in diethyl ether, which promoted its precipitation. Then transferring the solution into a 50mL centrifuge tube, putting the centrifuge into a centrifuge for centrifugation at 9000rpm/min for 5min, removing the supernatant after centrifugation to obtain polypeptide precipitate, and completing one-time washing. Repeating the washing for 3 times to remove most of impurities in the polypeptide precipitate, and drying the polypeptide precipitate in a vacuum drying dish to obtain a powdery crude polypeptide product.
(9) Purification of crude peptide:
separating the powdered crude polypeptide product by High Performance Liquid Chromatography (HPLC) (1 mL/min) at ambient temperature (25deg.C)Purification by separation using CH containing 0.1% TFA 3 CN/H 2 O is subjected to linear gradient elution, the time line is set to be 0-20min, the powder polypeptide product is obtained through freeze drying after the elution is finished, and the powder polypeptide product is stored in a refrigerator at the temperature of minus 8 ℃ for standby, so that the tryptophan-containing antibacterial peptide in a monomer form is obtained and is marked as Fmoc-KWK.
FIG. 2 is an electrospray ionization mass spectrometry (ESI-MS) image of Fmoc-KWK, from which it can be seen that Fmoc-KWK has a calculated molecular weight [ M+H ]] + As 683.82, it can be seen from the figure that the actual measurement [ M+H ]] + This indicated that Fmoc-KWK was successfully synthesized as 683.54.
FIG. 3 shows a high performance liquid chromatogram of Fmoc-KWK, from which it can be seen that the synthesized Fmoc-KWK has a purity of 99.33%.
FIG. 4 is a TEM image of Fmoc-KWK at pH 2.7, from which it can be seen that Fmoc-KWK does not self-assemble under this condition, since TEM can only observe some random agglomerates and does not show an organized structure.
Example 2:
the ability of the non-self-assembled Fmoc-KWK prepared in example 1 and Fmoc-KWK assemblies obtained by self-assembling Fmoc-KWK to inhibit the growth of Escherichia coli (E.coli, ATCC 8739), escherichia coli (E.coli, ATCC 25922), bacillus subtilis (B.subtilis, ATCC 6633) and enterococcus faecalis (E.faecalis, ATCC 29212) were examined by a broth microdilution method, respectively, in this example.
(1) Preparation of the samples:
since tryptophan-containing antimicrobial peptides in a monomer form may be aggregated into oligomers such as dimers and trimers during storage, the oligomers are no longer in a monomer form, and therefore, before use, TFE treatment is required to ensure that all Fmoc-KWK is in a monomer form, and therefore, TFE treatment is required to treat Fmoc-KWK obtained in example 1, and the treatment steps are as follows: 100mg of Fmoc-KWK prepared in example 1 was dissolved in 10mL of TFE, the solvent was removed by spin evaporation, and then freeze-dried to give Fmoc-KWK in the completely monomeric form.
The solution of Fmoc-KWK and the solution of Fmoc-KWK assembly which are not self-assembled are prepared respectively for standby.
Fmoc-KWK solution: fmoc-KWK 6.8mg is weighed into a 3mL vial at room temperature of 25 ℃, 2mL of HPLC grade water is added, ultrasonic mixing is carried out uniformly, the pH of the solution is 2.7, natural incubation is carried out for 24h at room temperature, and 2mL of Fmoc-KWK solution with the concentration of 5mM is obtained.
Fmoc-KWK assembly solution: fmoc-KWK pure monomer 6.8mg is weighed into a 3mL vial at room temperature of 25 ℃, 2mL of HPLC grade water is added, and the mixture is uniformly mixed by ultrasonic waves to obtain 2mL of Fmoc-KWK aqueous solution with the concentration of 5 mM. The pH of the Fmoc-KWK aqueous solution is adjusted to 7.4 by using 1M NaOH/HCl solution, and the Fmoc-KWK aqueous solution is naturally incubated for 24 hours at room temperature, so that a hydrogel Fmoc-KWK assembly is obtained.
FIGS. 5-7 are TEM images of Fmoc-KWK assemblies at pH7.4 after self-assembly at different scales. From the figure it can be seen that Fmoc-KWK in monomeric form self-assembles into continuous nanofibers of diameter 7-10nm, several tens of microns in length. And Fmoc-KWK nanofibers are present as well-structured monofilaments, probably because the nanofibers have a charged surface and thus can maintain a uniformly dispersed morphology.
(2) Investigation of bacteriostatic ability:
2mL of Fmoc-KWK assembly was added to 18mL of LB broth, and then the Fmoc-KWK assembly was diluted to a concentration of 0.5mM; 2mL of Fmoc-KWK solution was added to 9mL of LB broth, and the concentration was diluted to 0.1mg/mL, about 1.464mM, for further use.
100. Mu.L of LB liquid medium (bacterial content 10) was added to the second to twelfth wells of the sterile 96-well plate 6 CFU/mL), take respectively
200 mu L of diluted Fmoc-KWK solution and Fmoc-KWK assembly are added into a first hole, then 100 mu L of solution is taken from the first hole and added into a second hole, uniformly mixed, 100 mu L of uniformly mixed sample solution is added into a third hole, uniformly mixed, and the like to an eleventh hole, the sample solution is diluted twice, and finally 100 mu L of uniformly mixed sample solution is taken from the eleventh hole and discarded. The twelfth well was 100. Mu.L of LB liquid medium (bacterial content 10) 6 CFU/mL) as a control for normally growing bacteria.
MIC values (minimum inhibitory concentration), which are the minimum polypeptide concentration that prevents turbidity of the bacterial culture after incubation, were measured after incubation of 96-well plates in a thermostated incubator at 37 ℃ for 24h, all MIC measurements being the average of 3 independent experiments. The examination results are shown in table 1 and fig. 8 to 11:
TABLE 1 inhibition of Fmoc-KWK Assembly against different bacterial growth
As can be seen in combination with table 1 and fig. 8-11, fmoc-KWK assemblies show significant inhibition of growth of both common pathogenic gram-negative and gram-positive bacteria, and exhibit a broad spectrum of antimicrobial activity and greater antimicrobial capacity than the use limitations of existing antibiotics. Compared to a polypeptide analogue (KW) also comprising lysine and tryptophan n Fmoc-KWK assemblies can be seen with longer peptide polypeptides such as octapeptides: (KW) 4 Decapeptides: (KW) 5 The antibacterial ability is equivalent and even better. However, the relationship between the chain length of the antimicrobial peptide and the rate of hemolysis is also considered, and in general, the longer the chain length is, the higher the rate of hemolysis is. Wherein the method comprises the steps of (KW) 5 Obvious hemolysis phenomenon can be caused, and the medical requirements are not met.
The MIC of Fmoc-KWK is 5-10 times greater than that of Fmoc-KWK assembly, indicating that the spatial configuration of Fmoc-KWK assembly evolving with self-assembly, i.e. ordered arrangement of tryptophan residues, greatly contributes to the antibacterial ability thereof, so that the Fmoc-KWK assembly is easier to insert into bacterial cell membranes, resulting in bacterial lysis and death.
The antibacterial mechanism of Fmoc-KWK and Fmoc-KWK assemblies is shown in FIG. 14, and it can be seen from the graph that charged lysine residues (K) target bacterial cell membranes through electrostatic interaction, are enriched on the surfaces of the bacterial cell membranes, and after a certain threshold value is reached, polypeptides begin to be inserted into the cell membranes to form a stable barrel-shaped hole on the cell membranes, so that the cells die. The ordered arrangement of tryptophan residues in Fmoc-KWK assemblies is more favorable for insertion into cell membranes than Fmoc-KWK, and thus has a greater antibacterial capacity and can kill bacteria at a lower dose and faster rate.
Implementation of the embodiments example 3:
in this example, human red blood cell (hRBC) hemolysis experiments were performed to initially evaluate toxicity of Fmoc-KWK assemblies and unassembled Fmoc-KWK solutions, using PBS solution as a negative control group (zero hemolysis) and 0.3wt% aqueous solution of triton-100 (TX-100) as a positive control (100% hemolysis), as follows:
(1) Preparation of erythrocyte suspensions:
2mL of anticoagulated whole blood containing ethylenediamine tetraacetic acid is centrifuged in a centrifuge tube at 1000rpm for 10min, then 0.2mL of lower layer erythrocyte sediment is added into a 1.5mL centrifuge tube, then 1mL of physiological saline is added, the mixture is gently reversed, centrifugation is carried out at 1000rpm for 10min again to remove supernatant, and finally 0.25mL of physiological saline is added into the centrifuge tube to dilute erythrocytes, thus obtaining erythrocyte suspension for standby.
(2) Determination of wavelength of maximum absorbance value:
the absorption spectrum of the positive control supernatant was measured by an ultraviolet spectrometer in the wavelength range of 400 to 600nm, and the absorption spectrum is shown in FIG. 12.
As can be seen from fig. 12, the absorbance was proportional to the amount of hemoglobin released from the hrscs, and the wavelength of the positive control supernatant at the maximum absorbance value was determined to be 414nm. Therefore, the measurement wavelength was 414nm.
(3) Sample measurement:
deionized water was added to the Fmoc-KWK assembly solution and the unassembled Fmoc-KWK solution obtained in example 2, respectively, to dilute to 100 μm for use. 1mL of Fmoc-KWK assembly solution and Fmoc-KWK solution were taken and added to 20. Mu.L of red blood cell suspension, followed by incubation at 37℃for 60min with gentle stirring, centrifugation at 3500rpm (centrifugal force of about 1000 g) for 5min after completion of incubation, and the supernatant was aspirated and transferred into a 96-well plate to obtain a test sample 1 containing Fmoc-KWK assembly solution and a test solution 2 containing unassembled Fmoc-KWK monomer solution.
Then, absorbance values of the supernatants of the test sample 1, test sample 2, negative control and positive control at a wavelength of 414nm were measured, and three replicates were set for each sample and control group. The hemolysis ratio was calculated according to the following formula.
In which A s For the absorbance value of the experimental sample to be tested at the wavelength of 414nm, A nc Absorbance at 414nm for negative control group, A pc Is the absorbance value of the positive control group at a wavelength of 414nm.
The test results are shown in FIG. 13 (the inset shows a blood sample after centrifugation), and from the figure, it can be seen that after treatment with 100. Mu.M (about 5 times the MIC of Fmoc-KWK) Fmoc-KWK assembly solution and Fmoc-KWK solution, the blood cell integrity rates are respectively >95% and >90%, which shows that Fmoc-KWK has significant biocompatibility, and especially the hemolysis rate of Fmoc-KWK assembly after self-assembly is <5%, which meets the requirements of medical materials.
In conclusion, the Fmoc-KWK and Fmoc-KWK assembly provided by the invention is a short peptide consisting of three amino acids, and has the advantages of low cost, convenience in preparation and equivalent effect to long-chain antibacterial peptide. The Fmoc-KWK and Fmoc-KWK assemblies have definite structure and controllable sequence, do not contain epitope which possibly causes specific recognition, can avoid causing adaptive immune reaction, and have more advantages than long-chain polypeptides when being used as antibacterial material construction motifs.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (10)
1. The tryptophan-containing antibacterial peptide is characterized by having the structural formula:
2. the method for synthesizing tryptophan-containing antimicrobial peptide according to claim 1, comprising:
(1) Adding activated resin into a polypeptide solid-phase synthesis column, then adding an Fmoc deprotection reagent into the activated resin to perform Fmoc protection group removal reaction on the activated resin, and repeating the Fmoc protection group removal reaction for a plurality of times to obtain resin A;
(2) Fmoc-Lys (Boc) -OH, HBTU, HOBt and DIPEA were dissolved in DMF to give amino acid solution A;
adding an amino acid solution A into a polypeptide solid-phase synthesis column containing resin A, then placing the polypeptide solid-phase synthesis column containing resin A into a polypeptide synthesizer for condensation reaction, washing and pumping after the reaction is finished to obtain resin B;
(3) Fmoc-Trp-OH (Boc) -OH, HBTU, HOBt and DIPEA were dissolved in DMF to give amino acid solution B;
repeating the operation of the step (1) to remove Fmoc protecting groups in the resin B, adding an amino acid solution B into a polypeptide solid-phase synthesis column containing the resin B, then placing the polypeptide solid-phase synthesis column containing the resin B into a polypeptide synthesizer for condensation reaction, and washing and pumping after the reaction is finished to obtain resin C;
(4) Fmoc-Lys (Boc) -OH, HBTU, HOBt and DIPEA were dissolved in DMF to give amino acid solution C;
repeating the operation of the step (1) to remove Fmoc protecting groups in the resin C, adding an amino acid solution C into a polypeptide solid-phase synthesis column containing the resin C, then placing the polypeptide solid-phase synthesis column containing the resin C into a polypeptide synthesizer for condensation reaction, and washing and pumping out after the reaction is finished to obtain resin D;
(5) Adding a deprotection reagent into a polypeptide solid-phase synthesis column containing resin D, then placing the polypeptide solid-phase synthesis column into a polypeptide synthesizer for deprotection reaction, and flushing and pumping out after the reaction is finished; repeating the steps for a plurality of times to obtain resin E;
(6) Adding a pyrolysis liquid into a polypeptide solid-phase synthesis column containing resin E, then placing the polypeptide solid-phase synthesis column into a polypeptide synthesizer for pyrolysis reaction, and filtering to obtain a filtrate after the reaction is finished;
then nitrogen bubbling is carried out on the filtrate, precooled diethyl ether is added into the filtrate after the bubbling is finished, and the precipitate is centrifugally taken out, washed and dried to obtain a crude polypeptide product;
and (3) separating and purifying the crude polypeptide product by high performance liquid chromatography, and freeze-drying after purification to obtain the tryptophan-containing antibacterial peptide, which is marked as Fmoc-KWK.
3. The method of claim 2, wherein in step (1), the Fmoc deprotection reagent is prepared from piperidine and DMF in a volume ratio of 1:4;
the dosage ratio of the activated resin to Fmoc deprotection reagent is 5mL:100mg;
the Fmoc protecting group removal reaction is carried out for 15-20 min at room temperature, and the Fmoc protecting group removal reaction is repeated for 2-3 times.
4. The method for synthesizing tryptophan-containing antimicrobial peptide according to claim 2, wherein in the step (2), the ratio of Fmoc-Lys (Boc) -OHHBTU, HOBt, DIPEA to DMF is 92mg:74mg:30mg:50mg:3.5mL;
the dosage ratio of the amino acid solution A to the activated resin in the step (1) is 5mL:100mg;
the condensation reaction condition is that the reaction is carried out for 4 to 8 hours at room temperature.
5. The method for synthesizing tryptophan-containing antimicrobial peptide according to claim 2, wherein in the step (3), the ratio of Fmoc-Trp-OH (Boc) -OH, HOBt, DIPEA to DMF is 92mg:74mg:30mg:50mg:3.5mL;
the dosage ratio of the amino acid solution B to the activated resin in the step (1) is 5mL:100mg;
the condensation reaction is carried out for 4-8 hours at room temperature, and DMF and DCM are alternately used for washing after the reaction is finished.
6. The method for synthesizing tryptophan-containing antimicrobial peptide according to claim 2, wherein in the step (4), the ratio of Fmoc-Lys (Boc) -OH, HOBt, DIPEA to DMF is 92mg:74mg:30mg:50mg:3.5mL;
the dosage ratio of the amino acid solution B to the activated resin in the step (1) is 5mL:100mg;
the condensation reaction is carried out for 4-8 hours at room temperature, and DMF and DCM are alternately used for washing after the reaction is finished.
7. The method of claim 2, wherein in step (5), the deprotecting reagent comprises the following components in a volume ratio of 10: the TFA and DCM of 90 were formulated to give,
the ratio of the amount of the deprotection reagent to the activated resin in step (1) was 5mL:100mg;
repeating the steps for a plurality of times for 2-3 times.
8. The method of claim 2, wherein in step (6), the cleavage liquid comprises TFA, triethylsilane (TES) and H in a volume ratio of 94:5:1 2 O is prepared;
the condition of the cracking reaction is that the reaction is carried out for 1.5 to 2 hours at room temperature;
the nitrogen bubbling time is 10-20 min;
the dosage ratio of the precooled diethyl ether to the activated resin in the step (1) is 100mL:100mg.
9. A tryptophan-containing antimicrobial peptide assembly, characterized in that the tryptophan-containing antimicrobial peptide assembly is formed by self-assembly from the tryptophan-containing antimicrobial peptide of claim 1 or the tryptophan-containing antimicrobial peptide prepared by the method of any one of claims 2 to 8; the tryptophan-containing antibacterial peptide assembly has a diameter of 7-10nm and is in a nanofiber shape;
the synthesis method of the tryptophan-containing antibacterial peptide assembly comprises the following steps: and regulating the pH value of the tryptophan-containing antibacterial peptide to 7-7.5, and incubating for 20-24 hours at room temperature to obtain the tryptophan-containing antibacterial peptide assembly.
10. Use of the tryptophan-containing antimicrobial peptide of claim 1, the tryptophan-containing antimicrobial peptide prepared by the method of any one of claims 2 to 8, or the tryptophan-containing antimicrobial peptide assembly of claim 9 to kill bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310434934.5A CN116444603A (en) | 2023-04-21 | 2023-04-21 | Tryptophan-containing antibacterial peptide and synthesis method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310434934.5A CN116444603A (en) | 2023-04-21 | 2023-04-21 | Tryptophan-containing antibacterial peptide and synthesis method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116444603A true CN116444603A (en) | 2023-07-18 |
Family
ID=87125355
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310434934.5A Pending CN116444603A (en) | 2023-04-21 | 2023-04-21 | Tryptophan-containing antibacterial peptide and synthesis method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116444603A (en) |
-
2023
- 2023-04-21 CN CN202310434934.5A patent/CN116444603A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2468033C2 (en) | Antibacterial peptides | |
| EP0955312B1 (en) | Antimicrobial peptide | |
| CN109232719B (en) | PH-responsive antibacterial peptide and preparation method and application thereof | |
| WO2018130000A1 (en) | Self-assembled serially-connected cell-penetrating peptide nano-particulate antibacterial agent capable of penetrating blood brain barrier, preparation method therefor, and use thereof | |
| CN111748018B (en) | Biocompatible antibacterial peptide with self-assembly potential, and preparation method and application thereof | |
| JPH09503489A (en) | Peptide for neutralizing lipid A toxicity | |
| CN110066320B (en) | Cyclic peptide resisting multiple drug-resistant bacteria and preparation method and application thereof | |
| CN110330553B (en) | Mutant of antibacterial peptide VL25-1 and preparation method and application thereof | |
| CN112625106B (en) | Antibacterial polypeptide compound, synthesis method and application thereof | |
| CN114106106A (en) | Self-assembled dendritic antibacterial peptide Pal3RP, preparation method thereof, self-assembled nanoparticles thereof and application | |
| CN112625092B (en) | Antibacterial polypeptide compound based on polybia-MPI and synthesis and application thereof | |
| CN112430262B (en) | Antifungal peptides and application thereof | |
| Xie et al. | Recent progress in ionic coassembly of cationic peptides and anionic species | |
| CN116444603A (en) | Tryptophan-containing antibacterial peptide and synthesis method and application thereof | |
| CN111777670B (en) | PH-adjusted self-assembled antibacterial peptide and preparation method and application thereof | |
| CN106957370B (en) | Polypeptide capable of inducing toxicity in tumor cells | |
| CN112409457B (en) | Antibacterial polypeptide and application thereof | |
| CN113896767B (en) | Self-assembled antibacterial peptide nanoparticle and application thereof | |
| CN108610428B (en) | Antibacterial fusion peptide and preparation method and application thereof | |
| CN114195863B (en) | Side chain esterified antibacterial peptide WLC6 and preparation method and application thereof | |
| CN115925802B (en) | Antibacterial peptide with enhanced antibacterial activity and serum stability and application thereof | |
| CN113248572B (en) | Anti-multidrug-resistant bacteria cyclopeptide and application thereof | |
| CN117986342A (en) | Black orbital toad source derived antibacterial peptide, preparation method and application | |
| CN112480213B (en) | Amphiphilic anti-tumor polypeptide and application thereof | |
| Yamaguchi et al. | The position of Aib residues defines the antimicrobial activity of Aib-containing peptides |
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 |