CN116003453A - Reversible cyclized peptide based on double 1, 4-elimination reaction and application thereof - Google Patents
Reversible cyclized peptide based on double 1, 4-elimination reaction and application thereof Download PDFInfo
- Publication number
- CN116003453A CN116003453A CN202211619035.4A CN202211619035A CN116003453A CN 116003453 A CN116003453 A CN 116003453A CN 202211619035 A CN202211619035 A CN 202211619035A CN 116003453 A CN116003453 A CN 116003453A
- Authority
- CN
- China
- Prior art keywords
- peptide
- polypeptide
- resin
- reversible
- linker
- 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.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 177
- 230000002441 reversible effect Effects 0.000 title claims abstract description 36
- 238000003379 elimination reaction Methods 0.000 title claims abstract description 15
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 29
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 29
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 15
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 115
- 229920001184 polypeptide Polymers 0.000 claims description 100
- 150000001413 amino acids Chemical class 0.000 claims description 73
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 72
- 229920005989 resin Polymers 0.000 claims description 67
- 239000011347 resin Substances 0.000 claims description 67
- 239000007788 liquid Substances 0.000 claims description 48
- 238000005406 washing Methods 0.000 claims description 39
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 35
- 239000003814 drug Substances 0.000 claims description 32
- 229940079593 drug Drugs 0.000 claims description 25
- 238000005520 cutting process Methods 0.000 claims description 24
- 238000005086 pumping Methods 0.000 claims description 24
- 239000007791 liquid phase Substances 0.000 claims description 22
- 125000006239 protecting group Chemical group 0.000 claims description 22
- 238000009833 condensation Methods 0.000 claims description 21
- 230000005494 condensation Effects 0.000 claims description 21
- 102000001189 Cyclic Peptides Human genes 0.000 claims description 20
- 108010069514 Cyclic Peptides Proteins 0.000 claims description 20
- 238000007363 ring formation reaction Methods 0.000 claims description 20
- 238000010511 deprotection reaction Methods 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 17
- 239000000843 powder Substances 0.000 claims description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 238000003776 cleavage reaction Methods 0.000 claims description 12
- 230000001376 precipitating effect Effects 0.000 claims description 12
- 230000007017 scission Effects 0.000 claims description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 11
- 238000004108 freeze drying Methods 0.000 claims description 10
- 238000001819 mass spectrum Methods 0.000 claims description 10
- 238000007664 blowing Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 8
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 229920001971 elastomer Polymers 0.000 claims description 7
- 239000003960 organic solvent Substances 0.000 claims description 7
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 6
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 6
- 229930182555 Penicillin Natural products 0.000 claims description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 6
- 150000001408 amides Chemical class 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- 238000010009 beating Methods 0.000 claims description 6
- 230000008878 coupling Effects 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 229940049954 penicillin Drugs 0.000 claims description 6
- IVDFJHOHABJVEH-UHFFFAOYSA-N pinacol Chemical compound CC(C)(O)C(C)(C)O IVDFJHOHABJVEH-UHFFFAOYSA-N 0.000 claims description 6
- 229920003023 plastic Polymers 0.000 claims description 6
- 239000004033 plastic Substances 0.000 claims description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 6
- 230000008961 swelling Effects 0.000 claims description 6
- UYVXZUTYZGILQG-UHFFFAOYSA-N methoxyboronic acid Chemical compound COB(O)O UYVXZUTYZGILQG-UHFFFAOYSA-N 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 abstract description 43
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 9
- 230000003013 cytotoxicity Effects 0.000 abstract description 9
- 230000002949 hemolytic effect Effects 0.000 abstract description 8
- 102000004190 Enzymes Human genes 0.000 abstract description 6
- 108090000790 Enzymes Proteins 0.000 abstract description 6
- 230000001988 toxicity Effects 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 238000005336 cracking Methods 0.000 abstract description 2
- 230000004044 response Effects 0.000 abstract description 2
- 229940024606 amino acid Drugs 0.000 description 62
- 239000000243 solution Substances 0.000 description 62
- 230000001580 bacterial effect Effects 0.000 description 26
- 241000894006 Bacteria Species 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 18
- 239000000203 mixture Substances 0.000 description 18
- 230000000694 effects Effects 0.000 description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 239000002502 liposome Substances 0.000 description 11
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 10
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 9
- 229960002378 oftasceine Drugs 0.000 description 9
- 108090000631 Trypsin Proteins 0.000 description 8
- 102000004142 Trypsin Human genes 0.000 description 8
- 239000012588 trypsin Substances 0.000 description 8
- 206010018910 Haemolysis Diseases 0.000 description 7
- 230000003115 biocidal effect Effects 0.000 description 7
- 239000011521 glass Substances 0.000 description 7
- 230000008588 hemolysis Effects 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 230000007246 mechanism Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 5
- 125000003345 AMP group Chemical group 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 230000000845 anti-microbial effect Effects 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 5
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 238000002604 ultrasonography Methods 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000010413 mother solution Substances 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- KUMMBDBTERQYCG-UHFFFAOYSA-N 2,6-bis(hydroxymethyl)-4-methylphenol Chemical compound CC1=CC(CO)=C(O)C(CO)=C1 KUMMBDBTERQYCG-UHFFFAOYSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 3
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 3
- 108010067770 Endopeptidase K Proteins 0.000 description 3
- 102000057297 Pepsin A Human genes 0.000 description 3
- 108090000284 Pepsin A Proteins 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 206010041925 Staphylococcal infections Diseases 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000000601 blood cell Anatomy 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 229940111202 pepsin Drugs 0.000 description 3
- 230000009870 specific binding Effects 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 239000012230 colorless oil Substances 0.000 description 2
- 229940125904 compound 1 Drugs 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 231100001083 no cytotoxicity Toxicity 0.000 description 2
- KJIFKLIQANRMOU-UHFFFAOYSA-N oxidanium;4-methylbenzenesulfonate Chemical compound O.CC1=CC=C(S(O)(=O)=O)C=C1 KJIFKLIQANRMOU-UHFFFAOYSA-N 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 description 1
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- OTLNPYWUJOZPPA-UHFFFAOYSA-M 4-nitrobenzoate Chemical compound [O-]C(=O)C1=CC=C([N+]([O-])=O)C=C1 OTLNPYWUJOZPPA-UHFFFAOYSA-M 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-N Carbamic acid Chemical group NC(O)=O KXDHJXZQYSOELW-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101710151387 Serine protease 1 Proteins 0.000 description 1
- 102100032491 Serine protease 1 Human genes 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 101710119665 Trypsin-1 Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- UPQMBUAVJFJUDE-UHFFFAOYSA-N [4-(bromomethyl)phenoxy]boronic acid Chemical compound OB(O)OC1=CC=C(CBr)C=C1 UPQMBUAVJFJUDE-UHFFFAOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 230000008262 antibiotic resistance mechanism Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000037358 bacterial metabolism Effects 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000000887 hydrating effect Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 231100000065 noncytotoxic Toxicity 0.000 description 1
- 230000002020 noncytotoxic effect Effects 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 235000015320 potassium carbonate Nutrition 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000003521 protein stability assay Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000006462 rearrangement reaction Methods 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 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
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Images
Classifications
-
- 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
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a reversible cyclized peptide based on double 1, 4-elimination reaction and application thereof, wherein the reversible cyclized peptide passes through a linker 1 pairThe synthesis of the chain peptide and the synthesis process of the linker 1 and the reversible cyclized peptide are also disclosed. The synthesized reversible cyclized antibacterial peptide greatly reduces the hemolytic toxicity and the cytotoxicity of the antibacterial peptide and greatly enhances the stability of enzyme protein; at the same time, the reversible antibacterial peptide has no antibacterial activity per se, but is in H 2 O 2 The response factor can be used for cracking and releasing the antibacterial peptide and recovering the antibacterial activity.
Description
Technical Field
The invention belongs to the field of biochemistry, and in particular relates to a reversible cyclized peptide based on double 1, 4-elimination reaction and application thereof.
Background
Antibiotic resistance has become one of the major problems threatening public health. According to the report of the us center 2019 for disease control and prevention, more than 280 tens of thousands of people in the united states are infected with antibiotic-resistant bacteria each year, and more than 3.5 tens of thousands of people die directly from these infections. World health organization warns that by 2050, the number of deaths each year worldwide due to antibiotic-resistant pathogens is expected to reach 1000 tens of thousands. There have been reports of various antibiotic resistance mechanisms. Most conventional antibiotics bind to targets through site-specific binding mechanisms, thereby exerting pressure on the metabolism and proliferation of bacteria to inhibit or kill the bacteria. However, this site-specific binding mechanism is also affected by the rapid development of antibiotic resistance, as simple mutations in the binding site or modifications of the antibiotic structure inactivate the antibiotic. Furthermore, since many antibiotics act on intracellular targets, reduced membrane permeability and increased efflux pump activity are also important mechanisms of drug resistance. There is an urgent need for new potent antimicrobial agents that act through different mechanisms to cope with a wide range of antibiotic resistances.
In the search for new generation antibiotics, antibacterial peptides (AMPs) have received great attention over the last 30 years. AMPs coexist with microorganisms for millions of years, but have not been widely reported for resistance, which strongly suggests that they have unique antimicrobial mechanisms that may be able to evade the development of resistance. Unlike most traditional antibiotics, AMPs kill or inhibit bacteria primarily through a mechanism of membrane activity, neither involving site-specific binding nor interfering with bacterial metabolism. Although various drug resistance mechanisms have been reported, it is still very difficult for such membrane-active antimicrobial mechanisms to develop resistance to bacteria. Some AMPs have a broad range of biological activities against bacteria, fungi, parasites, insects, viruses and even cancer cells. Thus, antibacterial peptides and synthetic mimics of antibacterial peptides (SMAMPs) have been widely studied over the past 30 years as new generation antibiotics. However, current clinical transformation progress is limited, in part, due to the large toxicity, low in vivo efficiency and poor enzyme stability of the antibacterial peptides designed by the existing strategies.
Cyclization is an effective method for improving stability of antibacterial peptide proteases. The arrangement of the side chains of amino acids can be more compact through cyclization, and the steric hindrance is formed to stabilize the steric structure of the antibacterial peptide and slow down the cleavage effect of protease.
Inspired by the fact that cyclization strategies can disrupt the secondary structure of the antibacterial peptide, and in combination with the relevant reports, the present invention envisages whether or not the scientific assumption of the spiral conformation variable antibacterial peptide can be designed by reversible cyclization strategies. Briefly, the present study hopes to disrupt the secondary structure of the antimicrobial peptide by a reversible cyclization strategy, thereby obtaining a metabolically stable but biologically inactive reversible cyclized antimicrobial peptide (Reversibly Stapled AMPs, RStAMPs). At the bacterial infection site, RStAMPs can release active antibacterial peptide again under the stimulation of a stimulating factor, so that the aim of targeted administration is fulfilled. Designing a drug cascade release system or diagnostic reagent based on 1, 4-or 1, 6-benzyl elimination reaction (1, 4-or 1, 6-benzyl elimination) is a common synthetic means. Under the stimulation of the stimulation factors of the target positions, the system can spontaneously perform elimination/rearrangement reactions for multiple times, so as to achieve the purpose of releasing multiple compounds. Based on the above, the present invention is intended to construct a reversible cyclized antibacterial peptide system by a double 1, 4-benzyl elimination reaction.
Disclosure of Invention
The invention realizes the above purpose through the following technical scheme: the invention aims to construct a reversible cyclization antibacterial peptide system realized by double 1, 4-benzyl elimination reaction, which takes cheap and easily available 2, 6-bis (hydroxymethyl) p-cresol as a core, and constructs a polypeptide macrocyclic structure through forming a carbamate structure with the terminal of the antibacterial peptide or lysine side chain amino group.
The reversible cyclized antibacterial peptide (hereinafter referred to as cyclic peptide) is synthesized by coupling an antibacterial chain peptide with a linker 1, wherein the structural formula of the linker 1 is as follows:
the antibacterial chain peptide and the reversible cyclized antibacterial peptide have the following sequences:
chain peptide TL: FVQWFSKFLGRIL-NH 2 ；
Chain peptide TL-2: FVQWFSRFLGKIL-NH 2 ；
Cyclic peptide RCTL: f- (linker 2) VQWFSKFLGRIL-NH 2 ；
Cyclic peptide RCTL-2: f- (linker 2) VQWFSRFLGKIL-NH 2 。
Wherein F or F is Phe (phenylalanine), V is Val (valine), Q is Gln (glutamine), W is Trp (tryptophan), S is Ser (serine), K or K is Lys (lysine), L is Leu (leucine), G is Glu (glycine), R is Arg (arginine), and I is Ile (isoleucine). F and K in the cyclic peptides RCTL and RCTL-2 refer to the site of coupling of linker 1, and linker 1 cyclizes the polypeptide by coupling the two amino acids F and K.
Wherein linker 2 is a molecule obtained by cyclizing chain peptide TL and chain peptide TL-2 with linker 1 and then removing pinacol.
Linker 1 after coupling the chain peptide, at H 2 O 2 Under the action of the double 1, 4-elimination reaction, the chain peptide is released, and the active chain antibacterial peptide is obtained.
The invention also provides application of the reversible cyclized peptide in preparation of antibacterial targeted drugs.
The synthetic route of the linker 1 of the present invention is as follows:
the synthesis method of the reversible cyclization antibacterial peptide comprises the following steps:
1. swelling of the resin: 110mg of amide resin with 0.1mmol substitution value of 0.91mmol/g is precisely weighed and put into a T-type polypeptide solid phase synthesis tube, 4-5ml of dry DMF is added, the mixture is sleeved on a cover by a self-sealing bag to be screwed up, and the mixture is put into a shaking table (220 rpm/min) to shake for 15 minutes.
2. Removal of Fmoc protecting groups of the resin: pumping the fully swelled resin, adding 4-5ml of prepared deprotection liquid (50% morpholine/DMF), placing into a shaking table, shaking for 10 minutes, pumping the deprotection liquid to dryness after the time is up, and adding 4-5ml of deprotection liquid, and shaking for 10 minutes.
3. Resin washing: taking the resin without Fmoc protecting group out of the shaking table, pumping the deprotected solution, washing with DMF once, methanol twice and DCM twice respectively, washing with DMF twice finally, pumping after the resin is washed completely, and carrying out the next step.
4. Condensation of amino acids: according to the antibacterial peptide sequence, 0.5mmol of the first required Fmoc protected amino acid is weighed and added into a 10 ml penicillin bottle, then 0.5mmol of HBTU, HOBT and 1 mmole of DiEA are weighed and added, then 4-5ml of dry DMF is added for dissolving, ultrasound is carried out for 2 minutes, so that the first required Fmoc protected amino acid is fully dissolved, and the solution is sealed for standby. And (3) washing the resin with the protective groups removed in the same way, adding the prepared amino acid into a polypeptide synthesis tube, and oscillating for 1 hour. When the steric hindrance of the amino acid is larger (the side chain of the amino acid has a larger protecting group such as Fmoc-Arg (Pbf) -OH), the amino acid should be condensed for 2 times, namely, the amino acid is pumped out after the first condensation is finished, and the condensation is repeated once again, so that the amino acid is completely coupled.
5. Synthesis of amino acid sequence: washing the resin with the first amino acid after condensation, adding deprotected solution to remove Fmoc on amino, washing the resin, adding the second amino acid, and repeating the synthesis according to the polypeptide sequence.
6. Polypeptide cleavage: the method comprises the steps of preparing a cutting reagent of 95% TFA, 2.5% Tis and 2.5% water in advance, removing Fmoc groups of the last amino acid synthesized, cleaning resin, adding 4-5 ml of the cutting reagent, placing into a shaking table to shake for 30 minutes, cutting twice, collecting the cutting liquid obtained by the two times into a 50 ml centrifuge tube, and drying with nitrogen.
7. Purifying polypeptide: adding 40 ml of methyl tertiary butyl ether into a centrifuge tube which is blown to a centrifuge tube with 1 ml of solvent left, blowing and beating uniformly by a plastic rubber head dropper, freezing and precipitating for 15 minutes in a refrigerator with the temperature of minus 20 ℃, centrifuging by a centrifuge, pouring out supernatant, dissolving and precipitating by an organic solvent, taking a trace sample, determining the molecular weight of the polypeptide by using mass spectrum, analyzing the purity of the polypeptide by using reverse high-efficiency analysis liquid phase full gradient, and purifying the polypeptide by using a preparation liquid phase.
8. Polypeptide curing: and freeze-drying the purified polypeptide by using a low-temperature freeze dryer to obtain polypeptide solid powder, namely chain peptide. And analyzing the purity of the polypeptide by analyzing the liquid phase.
9. Cyclization of polypeptide: precisely weighing chain peptide freeze-dried powder and a linker 1 (chain peptide: linker 1=1:1.2 molar ratio) to dissolve in DMSO, adding DiEA, reacting in a water bath kettle at 40 ℃ for 2 hours, freeze-drying the DMSO after the reaction is completed, adding a proper amount of methyl boric acid, dissolving with 5% TFA/DCM, reacting for 2 hours at normal temperature, removing a solvent by using a rotary evaporator, adding a proper amount of methanol for dissolving, purifying by using a preparation liquid phase, and solidifying the obtained cyclic peptide preparation liquid by using a low-temperature freeze-dryer to finally obtain white cyclic peptide solid powder.
The invention has the beneficial effects that:
1. the invention provides a connector 1 molecule based on double 1, 4-elimination reaction and a synthesis method thereof, and the method has the advantages of few synthesis steps, simple synthesis method, low manufacturing cost and the like.
2. The synthesized reversible cyclized antibacterial peptide greatly reduces the hemolytic toxicity of the antibacterial peptide (the test does not generate hemolysis at 100 mu M) and the cytotoxicity of the antibacterial peptide (the test does not generate cytotoxicity at 64 mu M), and greatly enhances the stability of enzyme protein; at the same time, the reversible antibacterial peptide has no antibacterial activity and is in H 2 O 2 The response factor can be used for cracking and releasing the antibacterial peptide and recovering the antibacterial activity.
Drawings
FIG. 1 is a mass spectrum of a chain peptide TL provided by the invention;
FIG. 2 is a mass spectrum of the chain peptide TL-2 provided by the invention;
FIG. 3 is a mass spectrum of the cyclic peptide RCTL provided by the invention;
FIG. 4 is a mass spectrum of the cyclic peptide RCTL-2 provided by the invention;
FIG. 5 is a graph showing the results of the antibacterial peptide liposome cleavage experiment provided by the invention;
FIG. 6 is a graph showing the results of an antibacterial peptide hemolysis experiment provided by the invention;
FIG. 7 is a graph showing the cytotoxicity test results of the antibacterial peptide provided by the invention;
FIG. 8 is a graph showing the experimental results of the stability of the antibacterial peptidase protein;
FIG. 9 is a graph of the experimental result of the antibacterial peptide scanning electron microscope provided by the invention;
fig. 10 is a graph showing the experimental result of the bacterial survival rate of the antibacterial peptide provided by the invention.
Detailed Description
The invention will be further described with reference to examples and with reference to the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
1. Synthesis of linker 1 molecule
The linker 1 molecule based on double 1, 4-elimination reaction is obtained by taking cheap and easily available 2, 6-bis (hydroxymethyl) p-cresol as a core and performing short synthesis in several steps, and the specific reaction flow is as follows:
Synthesis of Compound 1: 2, 6-bis- (hydroxymethyl) -p-cresol (5.00 g, 29.7 mmol) and imidazole (4.65 g, 68.3 mmol) were dissolved in anhydrous DMF (30 mL) at 0deg.C. Addition of tert-butyl Di (butyl) dissolved in anhydrous DMF (15 mL)Methylchlorosilanes (9.85 g, 65.34 mmol). The solution was stirred at room temperature for 2 h, then diluted with diethyl ether and washed 3 times with water. After drying the organic phase over anhydrous MgSO4, filtering and concentrating. The crude product was purified by silica gel column (petroleum ether: ethyl acetate=90:10) to give compound S1 (11.01 g, yield 94%) as a colorless oil. 1 H NMR (600 MHz, CDCl 3 ): δ 8.09 (s, 1H), 6.98 (s, 2H), 4.90 (s, 4H), 2.33 (s, 3H), 1.03 (s, 18H), 0.21 (s, 12H). 13 C (150 MHz, CDCl 3 ): δ 151.0, 128.3, 126.3, 125.8, 63.1, 26.0, 20.7, 18.4, -5.4. ESI-MS: calcd. for C 21 H 40 NaO 3 Si 2 [M + Na] + 419.24; found 419.24。
Synthesis of Compound 2: compound 1 (2.00 g, 5.0 mmol) was dissolved in dry DMF (15 mL) and K2CO3 (0.84 g, 6.0 mmol) was added. The mixed solution was stirred at 0℃for 15 min, followed by the addition of pinacol 4-bromomethylphenyl borate (1.49, g, 5.0 mmol). The resulting mixture was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was diluted with diethyl ether and washed with saturated NH4Cl and saturated brine, respectively. The organic phase was collected, dried over anhydrous MgSO4, filtered and concentrated. The residue was purified by chromatography on a silica gel column (petroleum ether: ethyl acetate=90:10) to give pure compound S2 (2.51 g, yield 82%) as a colorless oil. 1 H NMR (600 MHz, CDCl 3 ): δ7.92 (d, J = 7.6 Hz, 2H), 7.50 (d, J = 7.6 Hz, 2H), 7.25 (s, 2H), 4.97 (s, 2H), 4.78 (s, 4H), 2.41 (s, 3H), 1.42 (s, 12H), 0.99 (s, 18H), 0.14 (s, 12H). 13 C (150 MHz, CDCl 3 ): δ151.2, 140.8, 135.1, 133.8, 133.7, 128.0, 127.0, 83.8, 76.2, 60.4, 26.1, 24.9, 21.3, 18.5, -5.2. ESI-MS: calcd. for C 34 H 57 BNaO 5 Si 2 [M + Na] + 635.37; found 635.71。
Synthesis of Compound 3: to a solution of compound 2 (1.20 g, 3.0 mmol) in methanol (10 mL) was added a catalytic amount of p-toluenesulfonic acid monohydrate (0.10 g, 0.6 mmol). The resulting mixture was stirred at room temperature for 1 h. After completion, the mixture was removed under reduced pressureAn organic solvent. The residue was purified by chromatography on a silica gel column (petroleum ether: ethyl acetate=50:50) to give pure compound S3 (1.02 g, yield 89%) as a white solid. 1 H NMR (600 MHz, CDCl 3 ): δ7.82 (d, J = 7.9 Hz, 2H), 7.37 (d, J = 7.9 Hz, 2H), 7.11 (s, 2H), 4.81 (s, 2H), 4.58 (s, 4H), 3.10 (s, 2H), 2.26 (s, 3H), 1.36 (s, 12H). 13 C (150 MHz, CDCl 3 ): δ152.2, 140.1, 135.1, 134.2, 133.8, 129.3, 127.2, 84.0, 76.6, 60.4, 24.9, 20.9. ESI-MS: calcd. for C 22 H 29 BNaO 5 [M + Na] + 407.20; found 407.73。
Synthesis of linker 1: compound 3 (0.72 g, 1.9 mmol), DMAP (22.9 mg, 0.19 mmol) and DiEA (1.21 g, 9.4 mmol) were dissolved in anhydrous DCM and cooled to 0 ℃. Then, p-nitrobenzoate (0.83 g, 4.1 mmol) was added. The resulting solution was stirred at 0 ℃ for 10 min and then at room temperature for 4 h. After completion of the reaction, the reaction mixture was diluted with DCM and washed with 1N HCl and brine. The organic phase was separated, dried over MgSO4, filtered and concentrated. The residue was purified by chromatography on a silica gel column (petroleum ether: ethyl acetate=80:20) to give protected linker (linker 1) as a white solid (0.82 g, 61% yield). 1 H NMR (600 MHz, CDCl 3 ): δ8.27 (d, J = 9.0 Hz, 4H), 7.87 (d, J = 7.6 Hz, 2H), 7.50 (d, J = 7.6 Hz, 2H), 7.37 (s, 2H), 7.32 (d, J = 9.0 Hz, 4H), 5.36 (s, 4H), 5.09 (s, 2H), 2.41 (s, 3H), 1.39 (s, 12H). 13 C (150 MHz, CDCl 3 ): δ155.5, 154.6, 152.4, 145.4, 139.7, 135.2, 134.8, 132.8, 128.2, 126.7, 125.3, 121.8, 84.0, 77.7, 66.3, 24.9, 20.8.ESI-MS: calcd. for C 36 H 35 BN 2 NaO 13 [M + Na] + 737.21; found 737.48。
2. Synthesis of chain antibacterial peptide and cyclized antibacterial peptide
The chain antibacterial peptide and the cyclized antibacterial peptide synthesized by the invention have the following structures:
Basic parameters of the polypeptides of Table 1
The mass spectrograms of the four polypeptides are shown in figures 1, 2, 3 and 4.
The polypeptide synthesized by the invention adopts a solid-phase and liquid-phase combined mode, wherein the solid-phase synthesis part extends a chain in a polypeptide solid-phase synthesis tube according to a sequence by manually shaking a table from resin. Linker 1 after coupling the chain peptide, at H 2 O 2 Under the action of the above-mentioned linker 1 releases the chain peptide by means of double 1, 4-elimination reaction so as to obtain the active chain peptide.
Synthesis of (a) chain peptide TL:
1. swelling of the resin: 110mg of amide resin with 0.1mmol substitution value of 0.91mmol/g is precisely weighed and put into a T-type polypeptide solid phase synthesis tube, 4-5ml of dry DMF is added, the mixture is sleeved on a cover by a self-sealing bag to be screwed up, and the mixture is put into a shaking table (220 rpm/min) to shake for 15 minutes.
2. Removal of Fmoc protecting groups of the resin: pumping the fully swelled resin, adding 4-5ml of prepared deprotection liquid (50% morpholine/DMF), placing into a shaking table, shaking for 10 minutes, pumping the deprotection liquid to dryness after the time is up, and adding 4-5ml of deprotection liquid, and shaking for 10 minutes.
3. Resin washing: taking the resin without Fmoc protecting group out of the shaking table, pumping the deprotected solution, washing with DMF once, methanol twice and DCM twice respectively, washing with DMF twice finally, pumping after the resin is washed completely, and carrying out the next step.
4. Condensation of amino acids: according to the antibacterial peptide sequence (FVQWFSKFLGRIL-NH 2 ) 0.5mmol of the first required Fmoc protected amino acid is weighed into a 10 ml penicillin bottle, then 0.5mmol of HBTU, HOBT and 1 mmole of DiEA are weighed and added, then 4-5ml of dry DMF is added for dissolution, ultrasound is carried out for 2 minutes, so that the first required Fmoc protected amino acid is fully dissolved, and the solution is sealed for standby. The protective group-removed resin is prepared by the same methodThe method is used for washing cleanly, and the prepared amino acid is added into a polypeptide synthesis tube and oscillated for 1 hour. When the steric hindrance of the amino acid is larger (the side chain of the amino acid has a larger protecting group such as Fmoc-Arg (Pbf) -OH), the amino acid should be condensed for 2 times, namely, the amino acid is pumped out after the first condensation is finished, and the condensation is repeated once again, so that the amino acid is completely coupled.
5. Synthesis of amino acid sequence: washing the resin with the first amino acid after condensation, adding deprotected solution to remove Fmoc on amino, washing the resin, adding the second amino acid, and repeating the synthesis according to the polypeptide sequence.
6. Polypeptide cleavage: the method comprises the steps of preparing a cutting reagent of 95% TFA, 2.5% Tis and 2.5% water in advance, removing Fmoc groups of the last amino acid synthesized, cleaning resin, adding 4-5ml of the cutting reagent, placing into a shaking table to shake for 30 minutes, cutting twice, collecting the cutting liquid obtained by the two times into a 50 ml centrifuge tube, and drying with nitrogen.
7. Purifying polypeptide: adding 40 ml of methyl tertiary butyl ether into a centrifuge tube which is blown to a centrifuge tube with 1 ml of solvent left, blowing and beating uniformly by a plastic rubber head dropper, freezing and precipitating for 15 minutes in a refrigerator with the temperature of minus 20 ℃, centrifuging by a centrifuge, pouring out supernatant, dissolving and precipitating by an organic solvent, taking a trace sample, determining the molecular weight of the polypeptide by using mass spectrum, analyzing the purity of the polypeptide by using reverse high-efficiency analysis liquid phase full gradient, and purifying the polypeptide by using a preparation liquid phase.
8. Polypeptide curing: and freeze-drying the purified polypeptide by using a low-temperature freeze dryer to obtain polypeptide solid powder. And analyzing the purity of the polypeptide by analyzing the liquid phase.
Synthesis of (II) chain peptide TL-2:
1. swelling of the resin: 110mg of amide resin with 0.1mmol substitution value of 0.91mmol/g is precisely weighed and put into a T-type polypeptide solid phase synthesis tube, 4-5ml of dry DMF is added, the mixture is sleeved on a cover by a self-sealing bag to be screwed up, and the mixture is put into a shaking table (220 rpm/min) to shake for 15 minutes.
2. Removal of Fmoc protecting groups of the resin: pumping the fully swelled resin, adding 4-5ml of prepared deprotection liquid (50% morpholine/DMF), placing into a shaking table, shaking for 10 minutes, pumping the deprotection liquid to dryness after the time is up, and adding 4-5ml of deprotection liquid, and shaking for 10 minutes.
3. Resin washing: taking the resin without Fmoc protecting group out of the shaking table, pumping the deprotected solution, washing with DMF once, methanol twice and DCM twice respectively, washing with DMF twice finally, pumping after the resin is washed completely, and carrying out the next step.
4. Condensation of amino acids: according to the antibacterial peptide sequence (FVQWFSRFLGKIL-NH 2 ) 0.5mmol of the first required Fmoc protected amino acid is weighed into a 10 ml penicillin bottle, then 0.5mmol of HBTU, HOBT and 1 mmole of DiEA are weighed and added, then 4-5ml of dry DMF is added for dissolution, ultrasound is carried out for 2 minutes, so that the first required Fmoc protected amino acid is fully dissolved, and the solution is sealed for standby. And (3) washing the resin with the protective groups removed in the same way, adding the prepared amino acid into a polypeptide synthesis tube, and oscillating for 1 hour. When the steric hindrance of the amino acid is larger (the side chain of the amino acid has a larger protecting group such as Fmoc-Arg (Pbf) -OH), the amino acid should be condensed for 2 times, namely, the amino acid is pumped out after the first condensation is finished, and the condensation is repeated once again, so that the amino acid is completely coupled.
5. Synthesis of amino acid sequence: washing the resin with the first amino acid after condensation, adding deprotected solution to remove Fmoc on amino, washing the resin, adding the second amino acid, and repeating the synthesis according to the polypeptide sequence.
6. Polypeptide cleavage: the method comprises the steps of preparing a cutting reagent of 95% TFA, 2.5% Tis and 2.5% water in advance, removing Fmoc groups of the last amino acid synthesized, cleaning resin, adding 4-5ml of the cutting reagent, placing into a shaking table to shake for 30 minutes, cutting twice, collecting the cutting liquid obtained by the two times into a 50 ml centrifuge tube, and drying with nitrogen.
7. Purifying polypeptide: adding 40 ml of methyl tertiary butyl ether into a centrifuge tube which is blown to a centrifuge tube with 1 ml of solvent left, blowing and beating uniformly by a plastic rubber head dropper, freezing and precipitating for 15 minutes in a refrigerator with the temperature of minus 20 ℃, centrifuging by a centrifuge, pouring out supernatant, dissolving and precipitating by an organic solvent, taking a trace sample, determining the molecular weight of the polypeptide by using mass spectrum, analyzing the purity of the polypeptide by using reverse high-efficiency analysis liquid phase full gradient, and purifying the polypeptide by using a preparation liquid phase.
8. Polypeptide curing: and freeze-drying the purified polypeptide by using a low-temperature freeze dryer to obtain polypeptide solid powder. And analyzing the purity of the polypeptide by analyzing the liquid phase.
(III) Synthesis of the cyclic peptide RCTL:
1. swelling of the resin: 110mg of amide resin with 0.1mmol substitution value of 0.91mmol/g is precisely weighed and put into a T-type polypeptide solid phase synthesis tube, 4-5ml of dry DMF is added, the mixture is sleeved on a cover by a self-sealing bag to be screwed up, and the mixture is put into a shaking table (220 rpm/min) to shake for 15 minutes.
2. Removal of Fmoc protecting groups of the resin: pumping the fully swelled resin, adding 4-5ml of prepared deprotection liquid (50% morpholine/DMF), placing into a shaking table, shaking for 10 minutes, pumping the deprotection liquid to dryness after the time is up, and adding 4-5ml of deprotection liquid, and shaking for 10 minutes.
3. Resin washing: taking the resin without Fmoc protecting group out of the shaking table, pumping the deprotected solution, washing with DMF once, methanol twice and DCM twice respectively, washing with DMF twice finally, pumping after the resin is washed completely, and carrying out the next step.
4. Condensation of amino acids: according to the antibacterial peptide sequence (F- (linker 2) VQWFSKFLGRIL-NH) 2 ) 0.5mmol of the first required Fmoc protected amino acid is weighed into a 10 ml penicillin bottle, then 0.5mmol of HBTU, HOBT and 1 mmole of DiEA are weighed and added, then 4-5ml of dry DMF is added for dissolution, ultrasound is carried out for 2 minutes, so that the first required Fmoc protected amino acid is fully dissolved, and the solution is sealed for standby. And (3) washing the resin with the protective groups removed in the same way, adding the prepared amino acid into a polypeptide synthesis tube, and oscillating for 1 hour. When the steric hindrance of the amino acid is larger (the side chain of the amino acid has a larger protecting group such as Fmoc-Arg (Pbf) -OH), the amino acid should be condensed for 2 times, namely, the amino acid is pumped out after the first condensation is finished, and the condensation is repeated once again, so that the amino acid is completely coupled.
5. Synthesis of amino acid sequence: washing the resin with the first amino acid after condensation, adding deprotected solution to remove Fmoc on amino, washing the resin, adding the second amino acid, and repeating the synthesis according to the polypeptide sequence.
6. Polypeptide cleavage: the method comprises the steps of preparing a cutting reagent of 95% TFA, 2.5% Tis and 2.5% water in advance, removing Fmoc groups of the last amino acid synthesized, cleaning resin, adding 4-5 ml of the cutting reagent, placing into a shaking table to shake for 30 minutes, cutting twice, collecting the cutting liquid obtained by the two times into a 50 ml centrifuge tube, and drying with nitrogen.
7. Purifying polypeptide: adding 40 ml of methyl tertiary butyl ether into a centrifuge tube which is blown to a centrifuge tube with 1 ml of solvent left, blowing and beating uniformly by a plastic rubber head dropper, freezing and precipitating for 15 minutes in a refrigerator with the temperature of minus 20 ℃, centrifuging by a centrifuge, pouring out supernatant, dissolving and precipitating by an organic solvent, taking a trace sample, determining the molecular weight of the polypeptide by using mass spectrum, analyzing the purity of the polypeptide by using reverse high-efficiency analysis liquid phase full gradient, and purifying the polypeptide by using a preparation liquid phase.
8. Polypeptide curing: and freeze-drying the purified polypeptide by using a low-temperature freeze dryer to obtain polypeptide solid powder. And analyzing the purity of the polypeptide by analyzing the liquid phase.
9. Cyclization of polypeptide: precisely weighing chain peptide freeze-dried powder and a linker 1 (chain peptide: linker 1=1:1.2 molar ratio) to dissolve in DMSO, adding DiEA, reacting in a water bath kettle at 40 ℃ for 2 hours, freeze-drying the DMSO after the reaction is completed, adding a proper amount of methyl boric acid, dissolving with 5% TFA/DCM, reacting for 2 hours at normal temperature, removing a solvent by using a rotary evaporator, adding a proper amount of methanol for dissolving, purifying by using a preparation liquid phase, and solidifying the obtained cyclic peptide preparation liquid by using a low-temperature freeze-dryer to finally obtain white cyclic peptide solid powder.
(IV) Synthesis of the cyclic peptide RCTL-2:
1. swelling of the resin: 110mg of amide resin with 0.1mmol substitution value of 0.91mmol/g is precisely weighed and put into a T-type polypeptide solid phase synthesis tube, 4-5ml of dry DMF is added, the mixture is sleeved on a cover by a self-sealing bag to be screwed up, and the mixture is put into a shaking table (220 rpm/min) to shake for 15 minutes.
2. Removal of Fmoc protecting groups of the resin: pumping the fully swelled resin, adding 4-5ml of prepared deprotection liquid (50% morpholine/DMF), placing into a shaking table, shaking for 10 minutes, pumping the deprotection liquid to dryness after the time is up, and adding 4-5ml of deprotection liquid, and shaking for 10 minutes.
3. Resin washing: taking the resin without Fmoc protecting group out of the shaking table, pumping the deprotected solution, washing with DMF once, methanol twice and DCM twice respectively, washing with DMF twice finally, pumping after the resin is washed completely, and carrying out the next step.
4. Condensation of amino acids: according to the antibacterial peptide sequence (F- (linker 2) VQWFSRFLGKIL-NH) 2 ) 0.5mmol of the first required Fmoc protected amino acid is weighed into a 10 ml penicillin bottle, then 0.5mmol of HBTU, HOBT and 1 mmole of DiEA are weighed and added, then 4-5ml of dry DMF is added for dissolution, ultrasound is carried out for 2 minutes, so that the first required Fmoc protected amino acid is fully dissolved, and the solution is sealed for standby. And (3) washing the resin with the protective groups removed in the same way, adding the prepared amino acid into a polypeptide synthesis tube, and oscillating for 1 hour. When the steric hindrance of the amino acid is larger (the side chain of the amino acid has a larger protecting group such as Fmoc-Arg (Pbf) -OH), the amino acid should be condensed for 2 times, namely, the amino acid is pumped out after the first condensation is finished, and the condensation is repeated once again, so that the amino acid is completely coupled.
5. Synthesis of amino acid sequence: washing the resin with the first amino acid after condensation, adding deprotected solution to remove Fmoc on amino, washing the resin, adding the second amino acid, and repeating the synthesis according to the polypeptide sequence.
6. Polypeptide cleavage: the method comprises the steps of preparing a cutting reagent of 95% TFA, 2.5% Tis and 2.5% water in advance, removing Fmoc groups of the last amino acid synthesized, cleaning resin, adding 4-5ml of the cutting reagent, placing into a shaking table to shake for 30 minutes, cutting twice, collecting the cutting liquid obtained by the two times into a 50 ml centrifuge tube, and drying with nitrogen.
7. Purifying polypeptide: adding 40 ml of methyl tertiary butyl ether into a centrifuge tube which is blown to a centrifuge tube with 1 ml of solvent left, blowing and beating uniformly by a plastic rubber head dropper, freezing and precipitating for 15 minutes in a refrigerator with the temperature of minus 20 ℃, centrifuging by a centrifuge, pouring out supernatant, dissolving and precipitating by an organic solvent, taking a trace sample, determining the molecular weight of the polypeptide by using mass spectrum, analyzing the purity of the polypeptide by using reverse high-efficiency analysis liquid phase full gradient, and purifying the polypeptide by using a preparation liquid phase.
8. Polypeptide curing: and freeze-drying the purified polypeptide by using a low-temperature freeze dryer to obtain polypeptide solid powder. And analyzing the purity of the polypeptide by analyzing the liquid phase.
9. Cyclization of polypeptide: precisely weighing chain peptide freeze-dried powder and a linker 1 (chain peptide: linker 1=1:1.2 molar ratio) to dissolve in DMSO, adding DiEA, reacting in a water bath kettle at 40 ℃ for 2 hours, freeze-drying the DMSO after the reaction is completed, adding a proper amount of methyl boric acid, dissolving with 5% TFA/DCM, reacting for 2 hours at normal temperature, removing a solvent by using a rotary evaporator, adding a proper amount of methanol for dissolving, purifying by using a preparation liquid phase, and solidifying the obtained cyclic peptide preparation liquid by using a low-temperature freeze-dryer to finally obtain white cyclic peptide solid powder.
3. In vitro application of reversible cyclized antibacterial peptide in preparation of clinical antibacterial drugs
1. Liposome lysis assay
The calcein powder was weighed into a volumetric flask, added with 3/4 volume of Tris-HCl buffer (100 mM Tris-HCl 150 mM NaCl pH 7.4) and placed in an ultrasonic water bath for 10 minutes at 37 ℃. After the calcium carbonate is completely dissolved, the volume is fixed to obtain 80 mM calcein buffer solution for use in light shielding.
1, 2-dioleoyl-SN-propan-3-phosphatidylethanolamine (DOPE) and 4 mg of 1, 2-dioleoyl-SN-glyceryl-3-phosphate-RAC-glycerinum sodium salt (75/25, w/w) were weighed in amounts of 12 mg, dissolved in 3 mL chloroform/methanol (9:1, V/V) to give a colorless transparent solution, and the solvent was removed by rotary evaporation of 1 h at 37℃and the liposomes were dried, while forming a translucent film in a low pressure state. Adding calcein buffer solution 4 mL, water bathing at 37deg.C, and ultrasonic treating for 10 min. The cycle was then repeated for about 1 h by hydrating for 5 min in a 37℃water bath and vortexing for 30 min s. And (5) performing ultrasonic treatment for 30 min by using an ultrasonic cell disruption instrument to obtain the liposome with uniform diameter. Centrifuging for 15 min by using an ultrafiltration centrifuge tube (centrifugal force of 5000 xg), removing centrifugate, diluting with Tris-HCl buffer solution, cleaning liposome, centrifuging for 15 min, repeating for 3-4 times until the centrifugate has no obvious calcein, and collecting liposome in the centrifuge tube for use in dark place. Lipid concentrations were determined by the phosphorus assay.
Liposomes were diluted with Tris buffer (final lipid concentration: 0.5 mM) and the liposome solution (95. Mu.L) was then mixed with drug solution (5. Mu.L) on 96-well black microwell plates (grey, flat bottom); the final drug concentrations were made 2. Mu.M, 1. Mu.M, 0.5. Mu.M, 0.25. Mu.M, 0.125. Mu.M, 0.0625. Mu.M and 0.03125. Mu.M with 5% DMSO/Tris solution and Triton-X/Tris solution (0.1% v/v) as negative and positive controls, respectively. After incubation for 1 hour at 37 ℃ in the dark, the fluorescence intensity in each well was recorded using the instrument. The release rate of calcein was calculated based on the measured fluorescence intensity, thereby illustrating the destructive effect of the drug on the liposomes. The excitation and emission wavelengths of calcein were 490nm and 515 nm, respectively, and the calcein leakage rates were as follows: leakage (%) = [ (F-F0)/(FTX-F0) ] ×100%, F being fluorescence intensity; f0 Fluorescence intensity as negative control; FTX is the fluorescence intensity of the positive control. The detailed results are shown in FIG. 5.
As can be seen from FIG. 5, either the cyclic peptide RCTL or the cyclic peptide RCTL-2 requires H 2 O 2 Cleavage releases biological activity when no H is added 2 O 2 At this time, none of the calcein leaked in the concentrations tested, indicating no H 2 O 2 In this case, the cyclic peptides RCTL and RCTL-2 are not destroyed and have no biological activity. Both chain peptide TL and TL-2 show strong cleavage activity, the chain peptide TL and chain peptide RCTL-2 show cleavage activity at 0.125 mu M, calcein is completely released at 0.25 mu M, and the liposome is almost completely destroyed; and cyclized peptides RCTL and RCTL-2, upon addition of H 2 O 2 After incubation, the cleavage activity was shown to be almost completely equivalent to that of its parent peptide. It was shown that in liposomes, this concentration of H 2 O 2 Can completely hydrolyze cyclized peptide and release corresponding polypeptide, and cyclized peptide is cleaved by H 2 O 2 。
2. Hemolysis experiment
The blood used in this experiment was fresh human blood, 2 mL blood was drawn into a 15 mL centrifuge tube, 3 mL PBS (pH 7.4) was added, gently blown (to avoid vigorous blowing, causing red blood cell disruption) with a pipette, the supernatant was centrifuged with a centrifuge for 15 min (1000 rpm), and then the supernatant was washed with PBS again and clarified. The blood cells were dispersed in PBS solution to prepare a 4% (V/V) blood cell solution. The drug was precisely weighed and dissolved in DMSO to a mother liquor of 2 mM, half-diluted to the desired concentration (2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.125 mM and 0.0625 mM, respectively). With 5% DMSO as a blank negative control, 2% sds solution as a positive control, 95 uL blood cell solution, 5 uL drug solution were pipetted into 96 well plates at final drug concentrations of 100 μm, 50 μm, 25 μm, 12.5 μm, 6.25 μm and 3.125 μm, three wells per level. Placing the sample in a constant temperature incubator at 37 ℃ for incubation for 1 hour, transferring the incubated sample into an EP tube (the 96-well plate is inclined and the bottom of the sample cannot be touched by a gun head to avoid red blood cell rupture), centrifuging for 15 min (1000 rpm), sucking 50 uL supernatant into the corresponding 96-well plate, and measuring the absorbance (450 nm) by using an enzyme-labeled instrument. Hemolysis (%) = [ (base:Sub>A-base:Sub>A yin)/(base:Sub>A-base:Sub>A yang-base:Sub>A yin) ]×100%base:Sub>A is absorbance intensity,base:Sub>A yin is absorbance of negative control, andbase:Sub>A yang is absorbance of positive control.
The results of the hemolysis experiments are shown in FIG. 6, in which the linear native peptides TL and TL-2 exhibit strong hemolytic activity, with TL exhibiting hemolytic activity at 6.25. Mu.M and hemolyzing up to 85% at 12.5. Mu.M; the TL-2 hemolysis activity was stronger, and started to be present at 3.125. Mu.M, and was as high as 80% at 12.5. Mu.M. Whereas, after cyclizing the polypeptides, the cyclized polypeptides RCTL and RCTL-2 showed a very large decrease in hemolytic activity, even at 100 μm. The method shows that the hemolytic activity of the polypeptide can be greatly reduced after cyclization, and the method verifies the concept of the invention, thereby achieving the purpose of reducing the hemolytic toxic and side effects of the expected polypeptide after cyclization.
3. Cytotoxicity test
The cells selected in the experiment are LO2 cells and HUVEC cells with good cell growth state, and the specific experimental steps are as follows: LO2 cells and HUVEC cells were resuscitated and passaged to normal cell morphology, after vigorous growth (at least 2-3 passages), 10% FBS in 1640 medium cell suspension and transferred to 96 well plates for growth, 100 μl of each microwell cell suspension (cell numberFor purposes of 5X 10 3 ) 24 h was cultured in a carbon dioxide incubator (37 ℃). Dissolving polypeptide medicine with DMSO to prepare mother solution with concentration of 10 mM; the 1640 medium was warmed to 37℃in a water bath in advance, and the polypeptides (TL, TL-2, RCTL and RCTL-2) were formulated with 1640 medium in half-fold dilution to drug solutions of different concentrations (64. Mu.M, 32. Mu.M, 16. Mu.M, 8. Mu.M, 4. Mu.M and 2. Mu.M) (highest concentration DMOS content 0.64%). The original culture medium in the 96-well plate is carefully sucked out (avoiding sucking into cells) by a pipette, the prepared drug solution (100 mu L of each microwell) is added, meanwhile, 0.64% DMSO/1640 solution and 20% DMSO/1640 solution are respectively used as a negative control and a positive control, three compound wells are arranged at each level, and the culture medium is put into a carbon dioxide incubator for incubation (37 ℃) for 24 hours after the drug is added. The drug solution was aspirated, 100. Mu.L of CCK-8/1640 medium solution (10%) was added to each microwell, incubated in a carbon dioxide incubator for 4 hours, and absorbance at 450nm was measured by a microplate reader, reflecting the number of living cells.
As shown in FIG. 7, the cytotoxicity results are strong for the linear peptides TL and TL-2, wherein TL and TL-2 have almost 0% cell viability at 16 μM, 32 μM, 64 μM for LO2 cells and HUVEC cells, i.e., strong cytotoxicity, whereas TL has 63% HUVEC viability, 70% LO2 cell viability, 45% HUVEC viability and 62% LO2 cell viability at 8 μM; TL and TL-2 had almost 100% cell viability at LO2 cells and HUVEC cells at concentrations of 2. Mu.M, 4. Mu.M, with no cytotoxicity. In contrast, after cyclization of the polypeptide, the cell viability of the cyclized polypeptides RCTL and RCTL-2, both LO2 cells and HUVEC cells, was nearly 100% at the tested concentration conditions, without cytotoxicity. In summary, the linear peptides TL, TL-2 were non-cytotoxic at concentrations below 4. Mu.M, starting to exhibit cytotoxicity above 4. Mu.M, and exhibited strong cytotoxicity above 16. Mu.M; and after cyclizing the peptide, no cytotoxicity was exhibited at the concentrations tested; the polypeptide can reduce the toxic and side effects of hemolysis and reduce the cytotoxicity at the same time after cyclization.
4. Enzyme protein stability assay
Trypsin (Trypsin), pepsin (Pepsin), chymotrypsin (Chymotorypsin), proteinase-K (proteinase K) and FBS (fetal bovine serum) were precisely weighed and dissolved in PBS to prepare a Trypsin solution with a concentration of 0.25. Mu.g/ml, a Chymotrypsin solution with a concentration of 0.1. Mu.g/ml, a pH=2 Pepsin solution with a concentration of 10. Mu.g/ml, a proteinase-K solution with a concentration of 0.05. Mu.g/ml and a 30% FBS solution. The polypeptides (TL-2 and RCTL-2) were precisely weighed and dissolved in DMSO to prepare a stock solution at a concentration of 2 mM. To an EP tube of 1.5. mL, 940. Mu.L of PBS solution, 50. Mu.L of drug mother liquor, 10. Mu.L/mL of enzyme solution and FBS solution were added so that the final concentration of the drug was 100. Mu.M and the DMSO content was 5%, and the mixture was homogenized by pipetting and incubated in a water bath at 37 ℃. Samples were taken after incubation for 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, respectively. Mixing well before sampling, sampling 100 uL each time, immediately adding equal volume of glacial acetonitrile to terminate the reaction and mixing well, placing the mixture in an environment of-20 ℃ for 15 min, centrifuging the mixture for 15 min by using a 10000 rpm centrifuge, absorbing supernatant, and observing the change of TL peak areas before and after hydrolysis by RP-HPLC to judge the stability of TL in trypsin. The detailed results are shown in FIG. 8.
As can be seen from FIG. 8, the natural antibacterial peptide has stronger stability against enzymolysis after cyclization. In summary, the natural linear antibacterial peptide can greatly improve the enzymolysis resistance stability after being cyclized by the reversible cyclization strategy.
5. Minimum inhibitory concentration assay
10 mL glass test tubes were taken, 6 mL LB liquid medium solution (pH 7.4) was added, normal bacteria (ATCC 25923, ATCC 29213 and MRSA) were inoculated into the glass test tubes with an inoculating loop, rubber stoppers were added and sealed with a sealing film. Shaking at constant temperature (220 rpm,37 ℃) for 2-3 hr, culturing to logarithmic phase, collecting bacteria by centrifuge (4000 rpm,15 min), and diluting bacterial solution to bacterial concentration of 2×10 by adding LB liquid culture medium solution 5 CFU/mL, blow-down to suspend the bacterial suspension in a refrigerator at 4deg.C (maximum cannot exceed 1 h). Polypeptide medicine (TL, TL-2, RCTL-2) is dissolved in DMSO to prepare 10 mM mother solution, H 2 O 2 The solution was diluted with LB liquid medium to a mother liquor of 2 mM. By half-fold dilution, addingThe polypeptide drugs are diluted into polypeptide liquid medicine of 640 mu M, 320 mu M, 160 mu M, 80 mu M, 40 mu M and 20 mu M in DMSO solution. In the experiments for investigating the antibacterial activities of TL, TL-2, RCTL and RCTL-2, 95 mu L of bacterial liquid and 5 uL drugs are added into each microwell in a sterile 96-well plate, so that the final concentration of polypeptide drugs is respectively 32 mu M, 16 mu M, 8 mu M, 4 mu M, 2 mu M and 1 mu M, and the content of DMSO is 5%. In the investigation of 100 mu M H 2 O 2 +polypeptide drugs (TL, TL-2, RCTL-2) antibacterial Activity experiments, 90. Mu.L of bacterial liquid, 5 uL drug, 5 uL H were added to each microwell 2 O 2 Solution, ensuring the final concentration of polypeptide to be the required concentration, H 2 O 2 The concentration of the solution was 100. Mu.M and the DMSO content was 5%. Each 96-well plate was incubated in a water bath thermostatted shaker (37 ℃) for 24 h and its absorbance at 620nm was measured using a microplate reader. The detailed results are shown in Table 2 below.
Table 2 minimum inhibitory concentration results
As can be seen from Table 2, the antimicrobial activity of the cyclized peptides RCTL and RCTL-2 was inactive at the highest concentration of 32. Mu.M for the three bacteria tested in this experiment, indicating that the cyclized strategy can greatly reduce (at least 8-fold) the antimicrobial activity of the linear antimicrobial peptide drug, even without activity, as compared to the antimicrobial activity of the linear peptides TL and TL-2. When the polypeptides TL, TL-2, RCTL-2 are 100 mu M H 2 O 2 Under action, the linear peptides TL and TL-2 showed no change in activity in three bacteria, indicating H 2 O 2 Has no influence on the activity of the linear peptide, and the cyclized peptide RCTL, RCTL-2 is shown in H 2 O 2 The activity of the peptide is reduced by one time compared with that of the linear peptide. The experiment shows that the reversible cyclization strategy can greatly reduce the antibacterial activity of the antibacterial peptide, and meanwhile, the antibacterial peptide prodrug can be replaced by H 2 O 2 "release" biological activity, but its activity is doubled compared to a linear peptide.
6. Scanning electron microscope experiment
MRSA bacteria were inoculated from a bacterial culture dish with an inoculating loop into LB liquid medium (PH=7.4)Is incubated in a logarithmic growth phase (OD 620 = 0.4-0.6) by shaking at 220ppm in a constant temperature air shaker at 37℃for 3 h. Transferring the bacterial liquid into a centrifuge tube, centrifuging the bacterial liquid by using a low-temperature centrifuge (4000 ppm,10 min), removing the supernatant, adding an equal volume of PBS buffer (PH=7.4), blowing uniformly, standing for 15s, centrifuging again, and repeating washing three times. Concentrating bacteria into 1 x 10 x 9CFU/mL, and suspending with PBS to obtain bacterial liquid for later use. The bacterial liquid without drug treatment was set as a negative control group. 950uL of bacterial liquid and 50uL of the required drug (containing 5% DMSO) were added to a 2xMIC final concentration in a 1.5ml centrifuge tube, and then H was added separately 2 O 2 (100. Mu.M) or no addition of H 2 O 2 Then incubated at 37℃for 1h. After the incubation, the bacterial solution (4000 ppm,10 min) was centrifuged with a centrifuge, the supernatant was discarded, an equal volume of PBS buffer (ph=7.4) was added and blown up evenly, and after 15s standing, it was centrifuged again and washing was repeated three times. After the last washing, the supernatant was removed, and 1mL of an electron microscope fixing solution (2.5% glutaraldehyde) (the bacterial mass was not dispersed) was directly added, and the mixture was fixed in a refrigerator at 4℃overnight. The fixed sample was washed three times with PBS buffer for 10min each time and the fixative was removed thoroughly. Then dehydrated with ethanol gradient (30%, 50%, 60%, 70%, 80%, 90% and 100%), incubated at 4℃for 15min, and centrifuged for 15min (4000 ppm,4 ℃). And uniformly dispersing the dehydrated sample with absolute ethyl alcohol, sucking 10uL of bacterial liquid onto a slide, and baking the slide on an alcohol lamp. The surface of the sample is plated with gold for 30s, and finally observed under a scanning electron microscope.
The experimental results are shown in fig. 9, and the bacteria of the negative control group (control group in fig. 9) are normal in morphology, the bacterial cell membranes are kept intact, and the cells are full spheres without leakage of contents. At this concentration, however, the linear peptides TL, TL-2 of the dosing group were severely deformed in part of the bacterial morphology (at the white arrow in fig. 9), their cell membranes were broken, the contents were released, and no globular structure was observed, indicating that they were bacterial cell membrane destruction to kill bacteria. At this concentration, the cyclized peptides RCTL, RCTL-2 were normal in bacterial morphology, indicating that their direct interaction with the bacteria did not affect the bacteria. When cyclized peptide RCTL, RCTL-2 is added to H 2 O 2 After that, the bacterial morphology was severely deformed (at the white arrow in FIG. 9), and the results were consistent with TL, TL-2. In summary, the antibacterial peptides TL and TL-2 kill bacteria by destroying bacterial membranes, and the cyclized peptides RCTL and RCTL-2 directly act without affecting bacteria; and only add H 2 O 2 After that, the biological activity, namely the activity dependence H of the cyclized peptide RCTL and RCTL-2, can be displayed 2 O 2 。
7. Bacterial viability experiment
An LB nutrient agar ultrapure water solution was prepared according to the formulation, and the solution and a clean glass petri dish were sterilized in an autoclave (121 ℃) for 30 minutes. The glass culture dish is put into a baking oven to dry water, LB agar is carefully poured into the glass culture dish (20 mL each) after the temperature is reduced to about 60 ℃, the glass culture dish is irradiated under an ultraviolet lamp and naturally cooled, and the glass culture dish is cooled and solidified after 2-3 h for later use. Precisely weighing trypsin 1 mg, preparing into trypsin solution with concentration of 0.025 mg/mL by PBS solution, and preserving at low temperature (4 ℃) for standby. Taking a mother solution of TL-2 or RCTL-2 of 2 mM (DMSO dissolution), adding 940 mu L of PBS solution, 50 mu L of TL-2 or RCTL-2 mother solution of uL and 10 mu L/mL of trypsin solution (0.025 mg/mL) into an EP tube of 1.5 mL, so that the final concentration of TL-2 or RCTL-2 medicine is 100 mu M, the DMSO content is 5 percent, taking 5 percent DMSO PBS solution as a blank, blowing uniformly, and then incubating the mixed solution for 1 hour under the water bath condition of 37 ℃. Immediately placing the EP tube into a water bath kettle at 60 ℃ for 15 min to deactivate trypsin and terminate the reaction, obtaining enzymolysis liquid medicine or blank solution, and preserving the enzymolysis liquid medicine or blank solution in a refrigerator at 4 ℃ for later use. Selecting MRSA bacteria, culturing with LB culture medium to logarithmic phase, centrifuging with centrifuge to collect bacterial liquid (4000 rpm,15 min), diluting bacterial liquid with LB culture medium to 2×10 6 CFU/mL; diluting enzymolysis agent and H with LB culture medium 2 O 2 Solution, 490. Mu.L of bacterial liquid and 500. Mu.L of enzymatic drug solution 10. Mu.L of H were added to a 1.5 mL EP tube 2 O 2 The solution is such that the final concentration of the bacterial liquid is 1 multiplied by 10 6 CFU/mL, the final concentration of the enzymolysis liquid medicine is 25 mu M, 10 mu M and 5 mu M respectively, the final concentration of the H2O2 solution is 100 mu M, and the three are evenly mixed and incubated in a constant temperature incubator at 37 ℃ for 1H. At the same time dilute the bacteria 10 2 、10 3 、10 4 Fold, 100. Mu.L of incubation liquid was aspirated and carefully spread on LB agar plates at room temperature. Culturing in a constant temperature incubator for 24 hours, counting the colonies on the plate, and reflecting the inhibition condition of the drug on bacteria by the colony count.
The results of the experiment are shown in FIG. 10, in which TL-2 does not exhibit biological activity at each concentration, indicating that TL-2 is completely hydrolyzed by trypsin, whereas RCTL-2 completely kills bacteria at 25. Mu.M and exhibits killing of bacteria at both 10. Mu.M and 5. Mu.M.
Claims (7)
1. A double 1, 4-elimination reaction-based linker 1, characterized in that: the connector 1 has the following structure:
2. a reversible cyclized peptide synthesized by coupling of linker 1 according to claim 1, wherein: the linker 1 is coupled with a chain peptide to synthesize a reversible cyclized peptide, and the sequences of the chain peptide and the reversible cyclized peptide are as follows:
Chain peptide TL: FVQWFSKFLGRIL-NH 2 ；
Chain peptide TL-2: FVQWFSRFLGKIL-NH 2 ；
Reversible cyclization peptide RCTL: f- (linker 2) VQWFSKFLGRIL-NH 2 ；
Reversible cyclization peptide RCTL-2: f- (linker 2) VQWFSRFLGKIL-NH 2 ；
Wherein, the linker 2 is a molecule obtained by removing pinacol after cyclizing the chain peptide TL and the chain peptide TL-2 by the linker 1.
3. Connector 1 according to claim 1 or 2, characterized in that: the linker 1 is attached to the chain peptide at H 2 O 2 Under the influence of (a) the chain peptide can be released by a double 1, 4-elimination reaction.
4. A method of synthesizing the linker 1 according to claim 1, characterized in that: the synthetic route is as follows:
5. a method of synthesizing a reversible cyclized peptide according to claim 2, characterized in that: the method comprises the following steps:
(1) Swelling of the resin: precisely weighing 110mg of amide resin with 0.1mmol substitution value of 0.91mmol/g, placing into a T-type polypeptide solid-phase synthesis tube, adding 4-5ml dry DMF, sleeving a self-sealing bag on a cover, tightening, and placing into a shaking table to shake for 15 minutes;
(2) Removal of Fmoc protecting groups of the resin: pumping the fully swelled resin, adding 4-5ml of the prepared deprotection liquid, putting into a shaking table, shaking for 10 minutes, pumping the deprotection liquid to dryness after the time is up, and adding 4-5ml of the deprotection liquid, and shaking for 10 minutes;
(3) Resin washing: taking the resin without Fmoc protecting groups out of a shaking table, pumping the deprotected solution, washing with DMF once, methanol twice and DCM twice respectively, washing with DMF twice finally, pumping after the resin is washed completely, and then carrying out the next step;
(4) Condensation of amino acids: weighing 0.5mmol of first required Fmoc protected amino acid according to a target polypeptide sequence, adding into a 10 ml penicillin bottle, weighing and adding 0.5mmol of HBTU, HOBT and 1 mmole of DiEA, adding 4-5ml of dry DMF for dissolving, performing ultrasonic treatment for 2 minutes to fully dissolve, sealing for standby, washing the resin with the protective groups removed in the same manner, adding the prepared amino acid into a polypeptide synthesis tube, and vibrating for 1 hour;
(5) Synthesis of amino acid sequence: washing the resin with condensed first amino acid according to the steps, adding deprotection solution to remove Fmoc on amino, washing the resin, adding second amino acid, and synthesizing repeatedly according to polypeptide sequence;
(6) Polypeptide cleavage: pre-preparing a cutting reagent of 95% TFA, 2.5% Tis and 2.5% water, removing Fmoc groups of the last amino acid synthesized, cleaning resin, adding 4-5ml cutting reagent, putting into a shaking table for shaking for 30 minutes, cutting twice, collecting the cutting liquid of the two times in a 50 ml centrifuge tube, and drying with nitrogen;
(7) Purifying polypeptide: adding 40 ml of methyl tertiary butyl ether into a centrifuge tube which is blown to a centrifuge tube with 1 ml of solvent left, blowing and beating uniformly by a plastic rubber head dropper, putting into a refrigerator with the temperature of minus 20 ℃ for freezing and precipitating for 15 minutes, centrifuging by using a centrifuge, pouring out supernatant, dissolving and precipitating by using an organic solvent, taking a trace sample, determining the molecular weight of the polypeptide by using mass spectrum, analyzing the purity of the polypeptide by using a reverse high-efficiency analysis liquid phase full gradient, and purifying the polypeptide by using a preparation liquid phase;
(8) Polypeptide curing: freeze-drying the purified polypeptide by using a low-temperature freeze dryer to obtain polypeptide freeze-dried powder, namely chain peptide;
(9) Cyclization of polypeptide: precisely weighing chain peptide freeze-dried powder and a connector 1, dissolving in DMSO, adding DiEA, reacting in a water bath kettle at 40 ℃ for 2 hours, freeze-drying the DMSO after the reaction is completed, adding a proper amount of methyl boric acid, dissolving with 5% TFA/DCM, reacting for 2 hours at normal temperature, removing a solvent by using a rotary evaporator, adding a proper amount of methanol for dissolving, purifying by using a preparation liquid phase, and solidifying the obtained cyclic peptide preparation liquid by using a low-temperature freeze-dryer to finally obtain white cyclic peptide solid powder.
6. The method for synthesizing a reversible cyclized peptide according to claim 5, wherein: the molar ratio of the chain peptide to the linker 1 is 1:1.2.
7. Use of a reversible cyclic peptide according to claim 2 for the preparation of an antibacterial targeted drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211619035.4A CN116003453B (en) | 2022-12-16 | 2022-12-16 | Reversible cyclized peptide based on double 1, 4-elimination reaction and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211619035.4A CN116003453B (en) | 2022-12-16 | 2022-12-16 | Reversible cyclized peptide based on double 1, 4-elimination reaction and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116003453A true CN116003453A (en) | 2023-04-25 |
| CN116003453B CN116003453B (en) | 2024-07-30 |
Family
ID=86034603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211619035.4A Active CN116003453B (en) | 2022-12-16 | 2022-12-16 | Reversible cyclized peptide based on double 1, 4-elimination reaction and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116003453B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5811392A (en) * | 1994-06-08 | 1998-09-22 | Yissum Research Development Co. Of The Hebrew University | Conformationally constrained backbone cyclized peptide analogs |
| CN107400161A (en) * | 2017-09-19 | 2017-11-28 | 中国工程物理研究院核物理与化学研究所 | Equal cyclic peptide Cyclo [(Asp)5Gly] preparation method |
| CN108315308A (en) * | 2018-02-26 | 2018-07-24 | 中南民族大学 | A kind of biologically active COX18 polypeptides and its synthetic method and purposes |
| CN112625092A (en) * | 2021-01-13 | 2021-04-09 | 兰州大学 | Antibacterial polypeptide compound based on polybia-MPI and synthesis and application thereof |
| CN112625106A (en) * | 2021-01-13 | 2021-04-09 | 兰州大学 | Antibacterial polypeptide compound, synthesis method and application thereof |
-
2022
- 2022-12-16 CN CN202211619035.4A patent/CN116003453B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5811392A (en) * | 1994-06-08 | 1998-09-22 | Yissum Research Development Co. Of The Hebrew University | Conformationally constrained backbone cyclized peptide analogs |
| CN107400161A (en) * | 2017-09-19 | 2017-11-28 | 中国工程物理研究院核物理与化学研究所 | Equal cyclic peptide Cyclo [(Asp)5Gly] preparation method |
| CN108315308A (en) * | 2018-02-26 | 2018-07-24 | 中南民族大学 | A kind of biologically active COX18 polypeptides and its synthetic method and purposes |
| CN112625092A (en) * | 2021-01-13 | 2021-04-09 | 兰州大学 | Antibacterial polypeptide compound based on polybia-MPI and synthesis and application thereof |
| CN112625106A (en) * | 2021-01-13 | 2021-04-09 | 兰州大学 | Antibacterial polypeptide compound, synthesis method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| OZLEM SEVEN ET.AL: ""Self Immolative Dioxetane Based Chemiluminescent Probe for H2O2Detection"", 《SENSORS AND ACTUATORS B》, 20 September 2016 (2016-09-20), pages 16 * |
| TATSUYUKI YOSHII ET.AL: ""Chemically Reactive Supramolecular Hydrogel Coupled with a Signal Amplification System for Enhanced Analyte Sensitivity"", 《JOURAL OF AMERICAN CHEMICAL SOCIETY》, 13 February 2015 (2015-02-13), pages 3363 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116003453B (en) | 2024-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109232719B (en) | PH-responsive antibacterial peptide and preparation method and application thereof | |
| Jiang et al. | Self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity | |
| WO2006058436A1 (en) | Antimicrobial peptides with reduced hemolysis and methods of their use | |
| CN105566452A (en) | Antibacterial peptide with annular structure and preparation method and application thereof | |
| CN103421090B (en) | A kind of novel antimicrobial peptide | |
| CN116874613B (en) | Broad-spectrum efficient antibacterial polypeptide APH143 and preparation method and application thereof | |
| CN111363010A (en) | Symmetric short-sequence antibacterial peptide analogue and application thereof | |
| RU2415868C1 (en) | Hemin derivatives, having antimicrobial activity or pharmaceutically acceptable salts thereof, synthesis method, pharmaceutical composition and use | |
| JP6124139B2 (en) | Low erythrocyte lytic antimicrobial peptide, pharmaceutical composition and use thereof | |
| US9090655B2 (en) | Low hemolytic antimicrobial peptide, pharmaceutical composition and use thereof | |
| CN116003453B (en) | Reversible cyclized peptide based on double 1, 4-elimination reaction and application thereof | |
| CN112625092B (en) | Antibacterial polypeptide compound based on polybia-MPI and synthesis and application thereof | |
| CN110054664B (en) | Side chain fatty acid modified antibacterial peptide analogue containing D-type amino acid and synthesis and application thereof | |
| CN110066320B (en) | Cyclic peptide resisting multiple drug-resistant bacteria and preparation method and application thereof | |
| CN112625106A (en) | Antibacterial polypeptide compound, synthesis method and application thereof | |
| CN116947999A (en) | Small molecular antibacterial polypeptide, preparation method and application thereof | |
| CN104672307B (en) | A kind of method for improving cation small peptide antibiotic property and stability | |
| JP2005247721A (en) | Antimicrobial peptide having reduced hemolysis, and method for using the same | |
| WO2020119773A1 (en) | Amphotericin b peptide derivative | |
| CN107033219A (en) | A kind of self-assembling peptides and its application as DNA agglomeration reagents | |
| RU2758060C1 (en) | Bacillus subtilis strain - producer of a low-molecular antimicrobial peptide and method for producing a low-molecular antimicrobial peptide | |
| CN110437308A (en) | One kind has antibacterial peptide analogues containing beta amino acids and its application of specific activity to pseudomonas aeruginosa | |
| CN112341524B (en) | Cyclic antibacterial peptide analogue rich in positive charges and application thereof | |
| CN118480089A (en) | Gemini type surfactant-like antibacterial peptide resistant to enzymolysis, and preparation method and application thereof | |
| CN118307634A (en) | Antibacterial peptide derivative, synthesis method thereof and application thereof in resisting drug-resistant bacteria |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |