CN117486993B - Staple peptide and preparation method and application thereof - Google Patents
Staple peptide and preparation method and application thereof Download PDFInfo
- Publication number
- CN117486993B CN117486993B CN202311448578.9A CN202311448578A CN117486993B CN 117486993 B CN117486993 B CN 117486993B CN 202311448578 A CN202311448578 A CN 202311448578A CN 117486993 B CN117486993 B CN 117486993B
- Authority
- CN
- China
- Prior art keywords
- amino acid
- slp
- tfa
- peptide
- reagent
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 88
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 34
- 239000003814 drug Substances 0.000 claims abstract description 24
- 238000007363 ring formation reaction Methods 0.000 claims abstract description 18
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 16
- 239000007790 solid phase Substances 0.000 claims abstract description 8
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 7
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 7
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 229920003180 amino resin Polymers 0.000 claims abstract description 6
- 241000588626 Acinetobacter baumannii Species 0.000 claims abstract description 5
- 150000001408 amides Chemical class 0.000 claims abstract description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 43
- 150000001413 amino acids Chemical class 0.000 claims description 21
- 239000012071 phase Substances 0.000 claims description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000000746 purification Methods 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 13
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 8
- 125000006239 protecting group Chemical group 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 7
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000005520 cutting process Methods 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 5
- 125000004122 cyclic group Chemical group 0.000 claims description 5
- 238000011068 loading method Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000000397 acetylating effect Effects 0.000 claims description 4
- 230000009471 action Effects 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 108700042658 GAP-43 Proteins 0.000 claims description 3
- 230000021736 acetylation Effects 0.000 claims description 3
- 238000006640 acetylation reaction Methods 0.000 claims description 3
- 238000010511 deprotection reaction Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 238000004007 reversed phase HPLC Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 239000012190 activator Substances 0.000 claims description 2
- 150000001336 alkenes Chemical class 0.000 claims description 2
- 210000004899 c-terminal region Anatomy 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims description 2
- 238000000354 decomposition reaction Methods 0.000 claims description 2
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 claims description 2
- 238000006798 ring closing metathesis reaction Methods 0.000 claims description 2
- 230000007017 scission Effects 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 37
- 241000894006 Bacteria Species 0.000 abstract description 23
- 229920001184 polypeptide Polymers 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 8
- 230000005764 inhibitory process Effects 0.000 abstract description 4
- 208000015181 infectious disease Diseases 0.000 abstract description 3
- 230000004048 modification Effects 0.000 abstract description 3
- 238000012986 modification Methods 0.000 abstract description 3
- 229940121657 clinical drug Drugs 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 67
- 238000004128 high performance liquid chromatography Methods 0.000 description 29
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 20
- 229940079593 drug Drugs 0.000 description 20
- 229920005989 resin Polymers 0.000 description 19
- 239000011347 resin Substances 0.000 description 19
- 238000012512 characterization method Methods 0.000 description 15
- 238000001819 mass spectrum Methods 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 102100024173 Stomatin-like protein 1 Human genes 0.000 description 6
- 101710082021 Stomatin-like protein 1 Proteins 0.000 description 6
- 102100024172 Stomatin-like protein 2, mitochondrial Human genes 0.000 description 6
- 101710196524 Stomatin-like protein 2, mitochondrial Proteins 0.000 description 6
- 102100024234 Stomatin-like protein 3 Human genes 0.000 description 6
- 108050003907 Stomatin-like protein 3 Proteins 0.000 description 6
- 125000003345 AMP group Chemical group 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 208000022362 bacterial infectious disease Diseases 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 208000035143 Bacterial infection Diseases 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 239000012345 acetylating agent Substances 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940124350 antibacterial drug Drugs 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical group CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- AERCCJGORROTKW-UHFFFAOYSA-N 2-amino-2-methylhept-6-enoic acid Chemical compound OC(=O)C(N)(C)CCCC=C AERCCJGORROTKW-UHFFFAOYSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- -1 Fmoc amino acid Chemical class 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- UIRFWOOIGULZQN-UHFFFAOYSA-L [Ru](Cl)Cl.C1(=CC=CC=C1)C([P](C1CCCCC1)(C1CCCCC1)C1CCCCC1)[P](C1CCCCC1)(C1CCCCC1)C1CCCCC1 Chemical compound [Ru](Cl)Cl.C1(=CC=CC=C1)C([P](C1CCCCC1)(C1CCCCC1)C1CCCCC1)[P](C1CCCCC1)(C1CCCCC1)C1CCCCC1 UIRFWOOIGULZQN-UHFFFAOYSA-L 0.000 description 1
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 1
- 238000005865 alkene metathesis reaction Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- PNPBGYBHLCEVMK-UHFFFAOYSA-N benzylidene(dichloro)ruthenium;tricyclohexylphosphanium Chemical compound Cl[Ru](Cl)=CC1=CC=CC=C1.C1CCCCC1[PH+](C1CCCCC1)C1CCCCC1.C1CCCCC1[PH+](C1CCCCC1)C1CCCCC1 PNPBGYBHLCEVMK-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012733 comparative method Methods 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- LCFXLZAXGXOXAP-QPJJXVBHSA-N ethyl (2e)-2-cyano-2-hydroxyiminoacetate Chemical compound CCOC(=O)C(=N\O)\C#N LCFXLZAXGXOXAP-QPJJXVBHSA-N 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention belongs to the field of polypeptide medicaments, and particularly relates to a staple peptide and a preparation method and application thereof. The invention takes Rink amide MBHA amino resin as a solid phase carrier, and according to template polypeptide SLP-0:Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Modification of the amino acid sequence in which amino acid residues 16W and 20K are S 5 And (5) replacing and cyclization to obtain the target stapling peptide. The staple peptide can obviously improve the inhibition activity to staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii, and has potential application value in the treatment of related diseases such as clinical drug-resistant bacteria infection and the like.
Description
Technical Field
The invention belongs to the field of polypeptide medicaments, and in particular relates to a stapler peptide capable of improving gram-positive and gram-negative bacteria antibacterial activity on the basis of a template polypeptide, and a preparation method and application thereof.
Background
The abuse of antibiotics has led to the emergence of drug-resistant strains, and infectious diseases caused by drug-resistant strains have become a serious threat to public health worldwide. Clinically resistant strains such as staphylococcus aureus, pseudomonas aeruginosa, acinetobacter baumannii and the like can form a layer of biological film on the surface of the strain, and the tolerance of bacteria wrapped by the biological film to conventional antibiotics is 10-1000 times that of other bacteria, so that clinically available antibiotics have certain limitation in resisting the resistant bacteria. In recent years, development of antibacterial drugs has been slow, and part of the antibiotic drugs on the market have developed drug-resistant bacteria in a short time. It is estimated that the worldwide number of deaths caused by drug-resistant bacteria in 2050 can reach 1000 ten thousand, and the economic loss caused by them can reach 1005 trillion dollars, and bacterial infection has become the second leading cause of death in humans following ischemic heart disease. For this reason, there is an urgent need to find new therapeutic agents to cope with infectious diseases caused by drug-resistant bacteria.
Antibacterial peptides (AMPs) are a class of host defensive peptides that kill bacteria through a variety of pathways, such as bacterial membrane lysis, oxidative damage, inhibition of biofilm formation, etc., do not involve the binding of specific proteins, are not prone to drug resistance, and can combat bacterial infections that are not effective with conventional antibiotics, thus AMPs have the potential to be a new generation of antibiotics for the treatment of drug-resistant bacterial infectious diseases. Most AMPs are linear polypeptides, however, are easily degraded by proteases in vivo, are structurally unstable, and greatly limit their clinical applications. The structural stability of polypeptides has been improved and certain progress has been made by previous chemical modifications to AMPs, such as backbone changes, D-type and unnatural amino acid substitutions. However, in the last decades, the structural stability of AMP has remained an important factor that prevents this class of drugs from entering clinical applications, so there is a need to find more efficient strategies to modify AMP to better address the problem of drug-resistant bacterial infection.
LL-37 is one of the major human AMPs and plays an important role in the defense against local and systemic infections. In 2018, nibbering group was based on the human LL-37 engineered antimicrobial peptide SAAP-148 (SLP-0), containing 24 residues, with an alpha helix structure, which exhibited a higher resistance to drug-resistant bacteria in vivo than model peptide LL-37. But the linear polypeptide is easy to be hydrolyzed, has unstable structure and limited drug-resistant bacteria activity, and can be realized only by high concentration for inhibiting staphylococcus aureus, pseudomonas aeruginosa and escherichia coli, thus preventing the drug development.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art and provides a staple peptide for improving the activity and stability of drug-resistant bacteria.
It is another object of the present invention to provide a method for preparing the stapled peptide.
It is a further object of the present invention to provide the use of the stapling peptides.
In order to achieve the first object, the invention adopts the following technical scheme:
a staple peptide selected from one of the following:
a) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 1L and 5W are S 5 Replacing and cyclization;
b) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 4V and 8V are S 5 Replacing and cyclization;
c) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 5W and 9F are S 5 Replacing and cyclization;
d) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 7R and 11L are S 5 Replacing and cyclization;
e) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 8V and 12L are S 5 Replacing and cyclization;
f) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 9F and 13K are S 5 Replacing and cyclization;
g) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 11L and 15Y are S 5 Replacing and cyclization;
h) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 12L and 16W are S 5 Replacing and cyclization;
i) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 14R and 18Q are S 5 Replacing and cyclization;
j) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 15Y and 19L are S 5 Replacing and cyclization;
k) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 16W and 20K are S 5 Replacing and cyclization;
l) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 18Q and 22P are S 5 Replacing and cyclization;
m) Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 19L and 23V are S 5 And (5) replacing and cyclization.
The sequences of the template polypeptides and engineered staple peptides of the invention are shown in the following table:
TABLE 1 sequences of template polypeptides and engineered staple peptides of the invention
In order to achieve the second purpose, the invention adopts the following technical scheme:
the preparation method of the staple peptide comprises the following steps:
(1) The Rink amide MBHA amino resin is taken as a carrier, the sample loading amount is 0.30mmol/g, and the C terminal first amino acid is respectively coupled with a solid phase carrier under the action of a DIC/Oxyme condensing agent activated by an activating agent;
(2) Removing Fmoc protecting groups on the amino acids using a deprotection reagent;
(3) Condensing amino acids using a condensing agent;
(4) Repeating the operation of removing the protecting group and condensing the amino acid, and synthesizing a peptide chain according to the amino acid sequence; wherein the ring closure site is S 5 To replace amino acids i and i+4;
(5) After removing Fmoc protecting groups from the last amino acid, acetylation is carried out;
(6) Under the action of cyclic mixture, S at positions i and i+4 5 The amino acid undergoes olefin double decomposition reaction to synthesize the staple peptide;
(7) The peptide chain is cut off from the carrier by using a cutting reagent, and the corresponding staple peptide is obtained by reversed-phase high-efficiency preparation and liquid-phase purification.
Wherein, as another preferable example of the present invention, the chromatographic conditions of the reversed phase high performance liquid preparation in the step (7) are as follows: chromatographic column: a UltimateXB-C18 column; mobile phase: mobile phase a was 0.1% tfa/acetonitrile, mobile phase B was 0.1% tfa/water; gradient elution procedure: eluting for 0-3 min with 90% B and 40min with 90% B-50% B; the flow rate was 8ml/min, the sample volume was 3ml, and the detection wavelengths were 214nm and 254nm.
As another preferable example of the present invention, the condensing agent in the step (1) is DIC-Oxyme condensing system, the activator of the condensing system is DIC, and DMF is solvent.
As another preferred example of the present invention, the deprotection reagent in the step (2) is a mixed solution of piperidine and DMF, and the volume ratio of the piperidine to the DMF is 1:4.
As another preferable example of the present invention, the acetylating agent of the acetylating step in the step (5) is Ac 2 O, DIEA, DMF mixture, the Ac 2 O, DIEA, DMF in a volume ratio of 1:1:8.
As another preferred example of the present invention, the cyclic mixture in the step (6) is a solution of 1, 2-dichloroethane of Grubbs I reagent, and the ratio of the amount of Grubbs I reagent to the amount of 1, 2-dichloroethane is 60:7 in mg/ml.
As another preferable example of the present invention, in the step (7), the cleavage reagent is TFA, H 2 Mixed solution of O, phenol and TIPS, TFA, H 2 The volume ratio of O, phenol and TIPS is 88.75:0.5:0.5:0.25; the dosage ratio of the cutting reagent to the carrier is 1:20, and the unit is mL/mg.
As another preferable example of the present invention, in the solid phase synthesis in the step (1), the loading amount of the solid phase resin was 0.30mmol/g.
As another preferable example of the present invention, the temperature of the coupling reaction in the step (1) is 50 to 60 ℃, preferably the reaction temperature is 60 ℃; the coupling reaction time is 20-30min, preferably 30min.
As another preferable example of the present invention, in the step (2), fmoc protection is removed by reacting for 5min with a protecting reagent, and then reacting for 5min again; the reaction temperature for Fmoc group removal is 20 to 40℃and more preferably 37 ℃.
As another preferable example of the present invention, S 5 The reaction time of the first amino acid is 1h, and the reaction is repeated once under the same condition to carry out the next operation.
As another preferable example of the present invention, the acetylation in the step (5) is that the resin is reacted in an acetylating agent for 15min and then reacted again for 15min; the reaction temperature is 20 to 40℃and preferably 37 ℃.
As another preferred example of the present invention, the cyclic mixture in the step (6) is a solution of Grubbs I reagent in 1, 2-dichloroethane, and the feeding ratio is the solid phase resin loading amount of Grubbs I: 1, 2-dichloroethane=0.35:60:7 (mmol/mg/ml).
As another preferable example of the present invention, the cyclization in step (6) is that the resin is reacted in a cyclization reagent in a constant temperature shaker twice for 2 hours each time; the reaction temperature is 20 to 40℃and preferably 37 ℃.
As another preferable example of the present invention, in the step (7), the cutting temperature is 20 to 40 ℃, more preferably 37 ℃; the time for cutting was 3h.
The application of the staple peptide in preparing medicines for inhibiting staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii.
In the present invention, the abbreviations involved are explained as follows:
fmoc: fluorenylmethoxycarbonyl; DCM: dichloromethane (dichloromethane)
DCE:1, 2-dichloroethane
DMF: n, N-dimethylformamide
Oxyme：Ethyl Cyanoglyoxylate-2-Oxime
DIC: n, N-diisopropylcarbodiimide
S 5 ：2-amino-2-methylhept-6-enoic acid
TFA: trifluoroacetic acid
TIPS: triisopropylsilane
Grubbs i: phenyl methylene bis (tricyclohexylphosphorus) ruthenium dichloride
MS: mass spectrometry
HR-Q-TOF-MS: high resolution matrix assisted laser desorption ionization time-of-flight mass spectrometry.
The invention has the advantages that:
1. the invention provides a series of novel staple peptides, and SAAP-148 (SLP-0) is structurally modified to obtain the novel staple peptides, so that the novel staple peptides have higher drug-resistant bacteria activity, and pharmacological experiments show that the novel staple peptides can remarkably improve the inhibitory activity on staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii and have potential application value in the treatment of related diseases such as clinical drug-resistant bacteria infection and the like.
2. The invention takes Rink amide MBHA amino resin as a solid phase carrier, and according to template polypeptide SLP-0:Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Modifying amino acid sequence, synthesizing to obtain peptide chain by Fmoc solid phase synthesis in DIC-Oxyme condensation system, and using S at i, i+4 amino acid position based on the remaining key amino acid residue 5 Instead of the original amino acid, after the synthesis of the linear peptide on the solid phase is completed, grubbs I mixed solution is added to the solid phase carrier to carry out olefin metathesis reaction cyclization, target stapling peptides are cut off through TFA mixed solution, and the obtained compound is purified through reversed-phase high-performance liquid phase and is subjected to characterization analysis by adopting HPLC, MS and other spectrums. The method comprisesThe method is simple and easy to implement, the purity of the obtained staple peptide is more than 95%, and the yield is high.
3. In order to improve the stability and the drug-resistant bacteria activity of the polypeptide, the SLP-0 is stapled and modified, the design is carried out in an i, i+4 mode on the basis of keeping key residues, and S is used at the non-key residue position of a peptide chain 5 And respectively replacing amino acids at positions i and i+4, and cyclizing to obtain the stapler peptide with stable structure. 13 staples (SLP-1-SLP-13) are synthesized together, and the screened polypeptide improves the antibacterial activity and can produce the forward regulation of the inhibition of harmful drug-resistant bacteria. The antibacterial effect of SLP-9 on staphylococcus aureus is obviously improved, and the inhibition effects of SLP-12 and SLP-13 on pseudomonas aeruginosa and escherichia coli are obviously enhanced.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following description of the drawings required in the examples and comparative examples will be briefly presented, with the understanding that the following shows comparative results and comparative method verification presentations of the examples and comparative examples of the present invention. FIG. 1 is a synthetic route for the peptides of the invention.
FIG. 2 is a schematic amino acid sequence of SLP-0 and its characterization, SLP-0 being purified by HPLC (purification conditions: 10% -55% CH) 3 CN (0.1% TFA) in H 2 O (0.1% TFA) over 55 minutes on a Welch C18 column. Wherein A is the amino acid sequence of SLP-0, B is the HPLC chromatogram of SLP-0, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is SLP-0 mass spectrum.
FIG. 3 is a schematic amino acid sequence of SLP-1 and its characterization, SLP-1 is purified by HPLC (purification conditions: 10% -60% CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) was applied to a Welch C18 column for 55 minutes to give SLP-1 with a separation of 24.4%. Wherein A is the amino acid sequence of SLP-1, B is the HPLC chromatogram of SLP-1, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-1.
FIG. 4 is a schematic amino acid sequence and characterization map of SLP-2, SLP-2 purified by HPLC (purification conditions: 10% -55% CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) was applied to a Welch C18 column for 55 minutes to give SLP-2 with a 14.7% separation. Wherein A is the amino acid sequence of SLP-2, B is the HPLC chromatogram of SLP-2, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-2.
FIG. 5 is a schematic representation of the amino acid sequence and characterization of SLP-3, SLP-3 purified by HPLC (purification conditions: 10% -60% CH) 3 CN (0.1% TFA) in H 2 O (0.1% TFA) was applied to a Welch C18 column for 55 minutes to give SLP-3 with a 14.6% separation. Wherein A is the amino acid sequence of SLP-3, B is the HPLC chromatogram of SLP-3, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-3.
FIG. 6 is a schematic representation of the amino acid sequence and characterization of SLP-4, SLP-4 purified by HPLC (purification conditions: 10% -60% CH) 3 CN (0.1% TFA) in H 2 O (0.1% TFA) was applied to a Welch C18 column for 55 minutes to give SLP-4 with a resolution of 19.6%. Wherein A is the amino acid sequence of SLP-4, B is the HPLC chromatogram of SLP-4, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-4.
FIG. 7 is a schematic representation of the amino acid sequence and characterization of SLP-5, SLP-5 purified by HPLC (purification conditions: 10% -55% CH) 3 CN (0.1% TFA) in H 2 O (0.1% TFA) over 55 minutes on a Welch C18 column. SLP-5 was obtained. Wherein A is the amino acid sequence of SLP-5, B is the HPLC chromatogram of SLP-5, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-5.
FIG. 8 is a schematic representation of the amino acid sequence and characterization of SLP-6, SLP-6 purified by HPLC (pureThe chemical conditions are as follows: 10% -60% CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) was applied to a Welch C18 column for 55 minutes to give SLP-6 with a resolution of 15.6%. Wherein A is the amino acid sequence of SLP-6, B is the HPLC chromatogram of SLP-6, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-6.
FIG. 9 is a schematic amino acid sequence diagram of SLP-7 and its characterization, SLP-7 is purified by HPLC (purification conditions: 10% -60% CH) 3 CN (0.1% TFA) at H 2 O (0.1% TFA) was applied to a Welch C18 column for 55 minutes to give SLP-7 with a separation of 6.7%. Wherein A is the amino acid sequence of SLP-7, B is the HPLC chromatogram of SLP-7, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-7.
FIG. 10 is a schematic amino acid sequence diagram of SLP-8 and its characterization map, SLP-8 is purified by HPLC (purification conditions: 10% -60% CH) 3 CN (0.1% TFA) in H 2 O (0.1% TFA) was applied to a Welch C18 column for 55 minutes to give SLP-8 with a resolution of 19.8%. Wherein A is the amino acid sequence of SLP-8, B is the HPLC chromatogram of SLP-8, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-8.
FIG. 11 is a schematic amino acid sequence of SLP-9 and its characterization, SLP-9 being purified by HPLC (purification conditions: 10% -60% CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) was applied to a Welch C18 column for 55 minutes to give SLP-9 with a resolution of 15.1%. Wherein A is the amino acid sequence of SLP-9, B is the HPLC chromatogram of SLP-9, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-9.
FIG. 12 is a schematic amino acid sequence diagram of SLP-10 and its characterization map, SLP-10 is purified by HPLC (purification conditions: 10% -60% CH) 3 CN (0.1% TFA) in H 2 O (0.1% TFA) was applied to a Welch C18 column for 55 minutes to give SLP-10 with a 9.3% separation. Wherein A is the amino acid sequence of SLP-10, B is the HPLC chromatogram of SLP-10, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-10.
FIG. 13 is a schematic amino acid sequence of SLP-11 and its characterization, SLP-11 is purified by HPLC (purification conditions: 10% -65% CH) 3 CN (0.1% TFA) in H 2 O (0.1% TFA) over 55 minutes on a Welch C18 column. Wherein A is the amino acid sequence of SLP-11, B is the HPLC chromatogram of SLP-11, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-11.
FIG. 14 is a schematic amino acid sequence of SLP-12 and its characterization, SLP-12 purified by HPLC (purification conditions: 10% -65% CH) 3 CN (0.1% TFA) in H 2 O (0.1% TFA) over 55 minutes on a Welch C18 column. Wherein A is the amino acid sequence of SLP-12, B is the HPLC chromatogram of SLP-12, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-12.
FIG. 15 is a schematic amino acid sequence of SLP-13 and its characterization, SLP-13 purified by HPLC (purification conditions: 10% -60% CH) 3 CN (0.1% TFA) in H 2 O (0.1% TFA) over 55 minutes on a Welch C18 column gave peptide 13 with a 2.4% separation. Wherein A is the amino acid sequence of SLP-13, B is the HPLC chromatogram of SLP-13, analytical column: welch C18, gradient: 10% -90% CH 3 CN (0.1% TFA) at H 2 O (0.1% TFA), 25min (λ=214 nm). C is the mass spectrum of SLP-13.
Detailed Description
The present application will be further described with reference to the drawings and detailed description so as to be more readily apparent to those skilled in the art, but such examples are intended to illustrate the invention and not to limit the scope of the invention, i.e. the examples described are only some, but not all, of the examples of the invention.
Thus, the following detailed description of certain embodiments of the present invention is not intended to limit the scope of the invention, as claimed, but is merely a selection of embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.
The invention relates to a template polypeptide SLP-0:Ac-LKRVWKRVFKLLKRYWRQL KKPVR-NH 2 Amino acid sequence design and synthesis of 13 staples. The structure and molecular weight of the stapled peptides are shown in FIG. 1.
The experimental materials involved in the embodiment of the invention are derived from the following sources:
fmoc-amino acids, rink amide MBHA amino resins were purchased from Nanking Synthesis Inc.; NMP, DIC, oxyme, TFA acetonitrile (chromatographic purity) from an exploration platform; DMF, anhydrous diethyl ether, DCM, DCE, piperidine and phenol are all analytically pure and purchased from Shanghai Co., ltd
EXAMPLE 1 SAAP-148 (SLP-0) based preparation of staple peptides
1. Synthesis of staple peptides
As shown in fig. 2:
(1) Preparation of Compound 1
400mg of amino resin (sample loading of 0.30 mmol.g) -1 ) Adding the mixture into a solid phase synthesis reaction tube, soaking the mixture in DCM for 30min to fully swell the resin, and pumping the resin for later use.
7ml of 20% piperidine-DMF solution was added to the resin and the Fmoc protecting groups on the resin were removed by shaking 5min X2 at 35℃and the resin was washed 3 times each with DMF, DCM, DMF.
(2) Preparation of Compound 2
The first amino acid (1 mmol), oxyme (142 mg,1 mmol) and DIC (200. Mu.L) of the sequence were dissolved in 7ml DMF and added to the resin and shaken at 60℃for 20min (S) 5 The latter amino acid was reacted for 1 hour and the reaction was repeated 1 time, and the resin was washed 3 times with DMF, DCM, DMF each in turn.
(3) Preparation of Compound 3
Repeating the steps (1) and (2), sequentially dissolving Fmoc amino acid (1 mmol), oxyme (142 mg) and DIC (200 μl) in 7ml DMF according to the polypeptide sequence, adding to the resin, and performing oscillation reaction at 60deg.C for 30min, and repeating the processes of Fmoc protection removal, condensation and Fmoc protection removal until all amino acids are condensed. After the Fmoc protecting group of the last amino acid is removed, 7ml of acetic anhydride/DIEA/DMF (1:1:8) mixed solution is added, and the mixture is vibrated at 37 ℃ for 15min, pumped down, added with an acetylating reagent again, reacted for 15min, washed with DMF, DCM, DMF for 3 times each resin in turn, and pumped down by an oil pump to dry the resin.
(4) Preparation of Compound 4
After the resin was completely dried, 1, 2-dichloroethane solution (7 ml) of Grubbs I (58 mg) reagent was added, and the reaction was carried out by shaking twice at 37℃for 2 hours each, and after the completion of the reaction, the resin was washed 3 times each by DMF, DCM, DMF in sequence, and the resin was dried by pumping vacuum.
(5) Preparation of target Compounds
The resin was first washed and drained and TFA, phenol, H were added 2 Tips=88.75:0.5:0.5:0.25 (V/V) 20ml, shaking 3h at 37 ℃, filtering, washing the resin with a little TFA, and collecting the filtrate. And (3) blowing off excessive TFA by bubbling argon, pouring into a glacial ethyl ether to precipitate and centrifuge, discarding supernatant, repeating the steps, washing and centrifuging the glacial ethyl ether to precipitate for three times, and naturally volatilizing under a fume hood to obtain a crude polypeptide sample.
2. Purification of stapled peptide samples
Dissolving the crude polypeptide with a mixed solvent of acetonitrile and water, and purifying by reverse phase preparative RP-HPLC to obtain a purified pure product of the staple peptide. The separation conditions were as follows:
instrument: the Shimadzu LC-20A reversed phase high performance liquid chromatograph;
chromatographic column: ultimateXB-C18, 21.2X250 mm,5 μm;
mobile phase: mobile phase a was acetonitrile solution with a volume fraction of 0.1% tfa, and mobile phase B was aqueous solution with a volume fraction of 0.1% tfa;
the steps and parameters are as follows: eluting with 90% B for 3min, and eluting with 90% B-50% B for 40min; the flow rate was 8ml/min, the sample injection amount was 3ml, and the detection wavelengths were 214nm and 254nm.
Example 2 identification and Structure analysis of the product
The product from step 2 of example 1 was identified by reverse phase HPLC and analyzed for structure by HR-Q-TOF-MS, the chromatographic mobile phase being acetonitrile and water. Mobile phase A is acetonitrile solution with volume fraction of 0.1% TFA, mobile phase B is water solution with volume fraction of 0.1% TFA, gradient elution is carried out (0-2 min, mobile phase B:90%, 3-25min, mobile phase B:90% -10%); the flow rate is 1.0 mL-min -1 The method comprises the steps of carrying out a first treatment on the surface of the The detection wavelengths were 214nm and 254nm, and the sample volume was 24. Mu.l. The time of the main peak of the obtained product is consistent with that of the crude product, and the purity of the staple peptide prepared by the method>95% and the mass spectrum analysis results are shown in FIGS. 3-15. The structure of the resulting stapled peptides is shown in Table 1.
TABLE 1 sequences of template polypeptides and engineered staple peptides of the invention
EXAMPLE 3 experiment of the inventive staple peptide against gram-Positive and gram-negative bacteria
In vitro anti-drug-resistant bacteria test: preparing a solid LB culture medium, plating the solid LB culture medium after autoclaving, and preparing an LB liquid culture medium for later use in a refrigerator at 4 ℃. Coating the bacterial liquid on a solid LB culture medium, and culturing the bacterial liquid in an incubator at 37 ℃ in an inverted way overnight; taking a monoclonal, adding the monoclonal into 3mL of liquid LB culture medium, culturing for 6 hours at 37 ℃ and 220rpm in a constant-temperature shaking table, and enabling bacteria to grow to a logarithmic phase; 1mL of the bacterial liquid is taken, centrifuged at 4000rpm for 5min, the supernatant is discarded, PBS is added, and the bacterial liquid concentration is adjusted to 2X 106CFU/mL by OD value. Antibacterial peptides with different concentrations are added into a 96-well plate, bacterial liquid is added into the 96-well plate at the same time, the bacterial liquid is cultured for 8 hours at 37 ℃, the enzyme label instrument adopts 595nm for detection, the detection is repeated three times, and MIC values are statistically analyzed.
The results are shown in Table 2.
TABLE 2 results of experiment of the inventive staple peptides against gram-positive and gram-negative bacteria
The results in Table 2 show that the stapled peptides of the invention can improve the drug-resistant bacteria activity of the template polypeptide, wherein the SLP-12 and SLP-13 have the most prominent drug-resistant bacteria resistant effects.
The embodiment shows that the SAAP-148 (SLP-0) -based modified staple peptide is successfully prepared, and the in-vitro bacteriostasis experiment proves that the synthesized staple peptide can obviously inhibit the growth and propagation of pathogenic drug-resistant bacteria, and has an application prospect of being developed into a novel antibacterial drug.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.
Claims (9)
1. A staple peptide, wherein the staple peptide is:
Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH 2 Is a peptide chain template in which amino acid residues 16W and 20K are S 5 And (5) replacing and cyclization.
2. The method for preparing the stapled peptide as claimed in claim 1, comprising the steps of:
(1) The Rink amide MBHA amino resin is taken as a carrier, the sample loading amount is 0.30mmol/g, and the C terminal first amino acid is respectively coupled with a solid phase carrier under the action of a DIC/Oxyme condensing agent activated by an activating agent;
(2) Removing Fmoc protecting groups on the amino acids using a deprotection reagent;
(3) Condensing amino acids using a condensing agent;
(4) Repeating the operation of removing the protecting group and condensing the amino acid, and synthesizing a peptide chain according to the amino acid sequence; wherein the ring closure site is S 5 To replace amino acids i and i+4;
(5) After removing Fmoc protecting groups from the last amino acid, acetylation is carried out;
(6) Under the action of cyclic mixture, S at positions i and i+4 5 The amino acid undergoes olefin double decomposition reaction to synthesize the staple peptide;
(7) The peptide chain is cut off from the carrier by using a cutting reagent, and the corresponding staple peptide is obtained by reversed-phase high-efficiency preparation and liquid-phase purification.
3. The method of claim 2, wherein the reversed phase high performance liquid chromatography in step (7) is performed under the following conditions: chromatographic column: a UltimateXB-C18 column; mobile phase: mobile phase a was 0.1% tfa/acetonitrile, mobile phase B was 0.1% tfa/water; gradient elution procedure: eluting for 0-3 min with 90% B and 40min with 90% B-50% B; the flow rate was 8ml/min, the sample volume was 3ml, and the detection wavelengths were 214nm and 254nm.
4. The method according to claim 2, wherein the condensing agent in the step (1) is DIC-Oxyme condensing system, the activator of the condensing system is DIC, and DMF is solvent.
5. The method of claim 2, wherein the deprotecting reagent in step (2) is a mixed solution of piperidine and DMF in a volume ratio of 1:4.
6. The method of claim 2, wherein the acetylating reagent of the acetylating step in step (5) is Ac 2 O, DIEA, DMF mixture, the Ac 2 O, DIEA, DMF in a volume ratio of 1:1:8.
7. The method of claim 2, wherein the cyclic agent in step (6) is a solution of 1, 2-dichloroethane of Grubbs i reagent in a ratio of 60:7 of the amount of Grubbs i reagent to 1, 2-dichloroethane in mg/ml.
8. The method for producing a staple peptide according to claim 2,wherein in the step (7), the cleavage reagent is TFA or H 2 Mixed solution of O, phenol and TIPS, TFA, H 2 The volume ratio of O, phenol and TIPS is 88.75:0.5:0.5:0.25; the dosage ratio of the cutting reagent to the carrier is 1:20, and the unit is mL/mg.
9. The use of the staple peptide of claim 1 in the preparation of a medicament for inhibiting staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311448578.9A CN117486993B (en) | 2023-04-23 | 2023-04-23 | Staple peptide and preparation method and application thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310438710.1A CN116655766B (en) | 2023-04-23 | 2023-04-23 | Staple peptide and preparation method and application thereof |
| CN202311448578.9A CN117486993B (en) | 2023-04-23 | 2023-04-23 | Staple peptide and preparation method and application thereof |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310438710.1A Division CN116655766B (en) | 2023-04-23 | 2023-04-23 | Staple peptide and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117486993A CN117486993A (en) | 2024-02-02 |
| CN117486993B true CN117486993B (en) | 2024-03-26 |
Family
ID=87717957
Family Applications (6)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311448578.9A Active CN117486993B (en) | 2023-04-23 | 2023-04-23 | Staple peptide and preparation method and application thereof |
| CN202311452121.5A Active CN117486994B (en) | 2023-04-23 | 2023-04-23 | Antibacterial stapling peptide and preparation method and application thereof |
| CN202311453511.4A Active CN117603330B (en) | 2023-04-23 | 2023-04-23 | Staple peptides, methods of making and uses thereof |
| CN202310438710.1A Active CN116655766B (en) | 2023-04-23 | 2023-04-23 | Staple peptide and preparation method and application thereof |
| CN202311547292.6A Active CN117567590B (en) | 2023-04-23 | 2023-04-23 | Stapler peptide capable of improving activity of resisting drug-resistant bacteria, and preparation method and application thereof |
| CN202311547187.2A Active CN117567589B (en) | 2023-04-23 | 2023-04-23 | Staple peptide with antibacterial activity and preparation method and application thereof |
Family Applications After (5)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311452121.5A Active CN117486994B (en) | 2023-04-23 | 2023-04-23 | Antibacterial stapling peptide and preparation method and application thereof |
| CN202311453511.4A Active CN117603330B (en) | 2023-04-23 | 2023-04-23 | Staple peptides, methods of making and uses thereof |
| CN202310438710.1A Active CN116655766B (en) | 2023-04-23 | 2023-04-23 | Staple peptide and preparation method and application thereof |
| CN202311547292.6A Active CN117567590B (en) | 2023-04-23 | 2023-04-23 | Stapler peptide capable of improving activity of resisting drug-resistant bacteria, and preparation method and application thereof |
| CN202311547187.2A Active CN117567589B (en) | 2023-04-23 | 2023-04-23 | Staple peptide with antibacterial activity and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (6) | CN117486993B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117486993B (en) * | 2023-04-23 | 2024-03-26 | 山东第一医科大学(山东省医学科学院) | Staple peptide and preparation method and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105820215A (en) * | 2016-05-09 | 2016-08-03 | 中国人民解放军第二军医大学 | Method for efficient synthesis of Stylissatin A natural cyclo-heptapeptide |
| CN106164087A (en) * | 2013-12-12 | 2016-11-23 | 莱顿大学学术医院以Lumc 的名义运作 | Antimicrobial polypeptide and application thereof |
| CN110627871A (en) * | 2019-09-29 | 2019-12-31 | 重庆大学 | Cationic bridged staple peptides and uses thereof |
| CN111233977A (en) * | 2020-02-24 | 2020-06-05 | 中国人民解放军第二军医大学 | Stapler peptide for inhibiting osteoclast differentiation and preparation method and application thereof |
| CN113651871A (en) * | 2021-08-03 | 2021-11-16 | 东北农业大学 | Anti-enzymolysis alpha-helical antibacterial peptide bound by all-carbon hydrogen side chain, preparation method and application |
| CN113651874A (en) * | 2021-08-02 | 2021-11-16 | 上海大学 | Stapler peptide with candida growth and reproduction inhibiting effect and preparation method and application thereof |
| WO2022226297A1 (en) * | 2021-04-23 | 2022-10-27 | Board Of Regents Of The University Of Nebraska | Antimicrobial peptides |
| WO2022237853A1 (en) * | 2021-05-12 | 2022-11-17 | 香港理工大学深圳研究院 | Beclin1-targeted stapled peptide and pharmaceutical composition |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012532929A (en) * | 2009-07-13 | 2012-12-20 | プレジデント アンド フェロウズ オブ ハーバード カレッジ | Bifunctional stapled polypeptides and their use |
| CN106749559B (en) * | 2016-11-23 | 2020-09-04 | 郑州大学 | Antibacterial peptide based on cell-penetrating peptide Tat (49-57) and synthesis method thereof |
| CN110240633A (en) * | 2018-03-09 | 2019-09-17 | 韩苏 | Polypeptide compound and preparation method thereof |
| CN111892646B (en) * | 2020-08-13 | 2023-05-02 | 中国人民解放军军事科学院军事医学研究院 | Antibacterial peptide derivative and application thereof in preparation of antibacterial infection medicines |
| WO2022236003A1 (en) * | 2021-05-07 | 2022-11-10 | Lytica Therapeutics, Inc. | Stapled antimicrobial peptides (stamps) and uses thereof |
| CN117279933A (en) * | 2021-05-07 | 2023-12-22 | 中外制药株式会社 | Cyclic compounds having selective KRAS inhibition relative to HRAS and NRAS |
| CN114014914B (en) * | 2021-09-16 | 2023-05-09 | 上海大学 | Tapelin targeting spike protein HR1, preparation method and application of pelin targeting spike protein HR1 in resisting coronavirus |
| CN113817026B (en) * | 2021-09-16 | 2023-06-30 | 中国人民解放军海军军医大学 | Tapelin of targeting spike protein HR1, preparation method and application of pelin in resisting novel coronavirus |
| CN114751962B (en) * | 2022-03-17 | 2023-11-07 | 北京大学 | Staple peptide, preparation method and pharmaceutical application thereof |
| CN115340594A (en) * | 2022-07-27 | 2022-11-15 | 上海大学绍兴研究院 | Stapler peptide for inhibiting osteoclast differentiation and preparation method and application thereof |
| CN115894715A (en) * | 2022-11-04 | 2023-04-04 | 石河子大学 | Preparation of fusion antibacterial peptide for resisting main pathogenic bacteria of cow mastitis |
| CN117486993B (en) * | 2023-04-23 | 2024-03-26 | 山东第一医科大学(山东省医学科学院) | Staple peptide and preparation method and application thereof |
-
2023
- 2023-04-23 CN CN202311448578.9A patent/CN117486993B/en active Active
- 2023-04-23 CN CN202311452121.5A patent/CN117486994B/en active Active
- 2023-04-23 CN CN202311453511.4A patent/CN117603330B/en active Active
- 2023-04-23 CN CN202310438710.1A patent/CN116655766B/en active Active
- 2023-04-23 CN CN202311547292.6A patent/CN117567590B/en active Active
- 2023-04-23 CN CN202311547187.2A patent/CN117567589B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106164087A (en) * | 2013-12-12 | 2016-11-23 | 莱顿大学学术医院以Lumc 的名义运作 | Antimicrobial polypeptide and application thereof |
| CN105820215A (en) * | 2016-05-09 | 2016-08-03 | 中国人民解放军第二军医大学 | Method for efficient synthesis of Stylissatin A natural cyclo-heptapeptide |
| CN110627871A (en) * | 2019-09-29 | 2019-12-31 | 重庆大学 | Cationic bridged staple peptides and uses thereof |
| CN111233977A (en) * | 2020-02-24 | 2020-06-05 | 中国人民解放军第二军医大学 | Stapler peptide for inhibiting osteoclast differentiation and preparation method and application thereof |
| WO2022226297A1 (en) * | 2021-04-23 | 2022-10-27 | Board Of Regents Of The University Of Nebraska | Antimicrobial peptides |
| WO2022237853A1 (en) * | 2021-05-12 | 2022-11-17 | 香港理工大学深圳研究院 | Beclin1-targeted stapled peptide and pharmaceutical composition |
| CN113651874A (en) * | 2021-08-02 | 2021-11-16 | 上海大学 | Stapler peptide with candida growth and reproduction inhibiting effect and preparation method and application thereof |
| CN113651871A (en) * | 2021-08-03 | 2021-11-16 | 东北农业大学 | Anti-enzymolysis alpha-helical antibacterial peptide bound by all-carbon hydrogen side chain, preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| 订书肽的合成与活性研究进展;李博等;药学学报;20170512;第52卷(第05期);第685-698页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117567590A (en) | 2024-02-20 |
| CN117567589B (en) | 2024-03-26 |
| CN117567589A (en) | 2024-02-20 |
| CN117603330B (en) | 2024-04-16 |
| CN116655766A (en) | 2023-08-29 |
| CN117486994B (en) | 2024-03-12 |
| CN116655766B (en) | 2023-12-12 |
| CN117567590B (en) | 2024-04-12 |
| CN117486994A (en) | 2024-02-02 |
| CN117486993A (en) | 2024-02-02 |
| CN117603330A (en) | 2024-02-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Andreu et al. | Shortened cecropin A‐melittin hybrids Significant size reduction retains potent antibiotic activity | |
| JP3266311B2 (en) | Novel polypeptide and anti-HIV agent using the same | |
| CN117486993B (en) | Staple peptide and preparation method and application thereof | |
| Fairweather et al. | Synthesis of the cyclic heptapeptide axinellin A | |
| CN111233977B (en) | Stapler peptide for inhibiting osteoclast differentiation and preparation method and application thereof | |
| AU2017204122B2 (en) | Template -fixed peptidomimetics as inhibitors of FPR1 | |
| CN104703997A (en) | Solution phase processes for the manufacture of macrocyclic depsipeptides and new intermediates | |
| JP3178835B2 (en) | Novel polypeptide and anti-HIV agent using the same | |
| EP2427476B1 (en) | Beta-hairpin peptidomimetics having cxcr4 antagonizing activity | |
| JP7292751B2 (en) | Method for preparing drug linker MC-MMAF for antibody-drug conjugate and its intermediate | |
| CN114096554A (en) | Peptide and method for producing same | |
| CN113651874A (en) | Stapler peptide with candida growth and reproduction inhibiting effect and preparation method and application thereof | |
| KR100219256B1 (en) | Novel polypeptides with affinity to lipopolysaccharides and their uses | |
| WO2004018644A2 (en) | HETEROGENEOUS FOLDAMERS CONTAINING α, β, AND/OR Ϝ-AMINO ACIDS | |
| CN102127155B (en) | Preparation of somatostatin peptides | |
| CN105777864B (en) | Pentacyclic triterpene-peptide conjugate, preparation method, pharmaceutical composition and application thereof | |
| EP4132481A1 (en) | Bicyclic peptide ligands specific for tslp | |
| ANWER et al. | Backbone modifications in cyclic pep tides Conformational analysis of a cyclic pseudopentapeptide containing a thiomethylene ether amide bond replacement | |
| Caddy et al. | Introduction of phosphine-gold (I) precursors into a Cys-modified enkephalin neuropeptide as part of solid phase peptide synthesis | |
| JP2005247721A (en) | Antimicrobial peptide having reduced hemolysis, and method for using the same | |
| Gardiner et al. | β‐Peptide Conjugates: Syntheses and CD and NMR Investigations of β/α‐Chimeric Peptides, of a DPA‐β‐Decapeptide, and of a PEGylated β‐Heptapeptide | |
| EP2958934A1 (en) | Peptidomimetics possessing photo-controlled biological activity | |
| CN117756905A (en) | Staple peptide and pharmaceutical application thereof | |
| WO1997047313A1 (en) | Total synthesis of bacitracin polypetides | |
| CN110305197B (en) | Polypeptide for inhibiting lung metastasis in tumor cells, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |