CN102766197A - Novel HRP5 analogues and its preparation method - Google Patents
Novel HRP5 analogues and its preparation method Download PDFInfo
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- CN102766197A CN102766197A CN2011101166439A CN201110116643A CN102766197A CN 102766197 A CN102766197 A CN 102766197A CN 2011101166439 A CN2011101166439 A CN 2011101166439A CN 201110116643 A CN201110116643 A CN 201110116643A CN 102766197 A CN102766197 A CN 102766197A
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- polypeptide
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- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- UMHAZRHZAQYINK-UCLGWUJRSA-N hrp-5 Chemical class C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1N=CN=C1)C(=O)N[C@@H](CC1N=CN=C1)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)OC(=O)CC[C@H](NC(=O)[C@H](CC1N=CN=C1)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1N=CN=C1)C(=O)N[C@@H](CC1N=CN=C1)C(=O)OC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC1N=CN=C1)NC(=O)[C@@H](N)CO)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(O)=O)C1C=NC=N1 UMHAZRHZAQYINK-UCLGWUJRSA-N 0.000 title abstract description 16
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- ODKSFYDXXFIFQN-SCSAIBSYSA-N D-arginine Chemical compound OC(=O)[C@H](N)CCCNC(N)=N ODKSFYDXXFIFQN-SCSAIBSYSA-N 0.000 claims description 14
- COLNVLDHVKWLRT-MRVPVSSYSA-N D-phenylalanine Chemical compound OC(=O)[C@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-MRVPVSSYSA-N 0.000 claims description 14
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- 241000228143 Penicillium Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 235000004789 Rosa xanthina Nutrition 0.000 description 1
- 241000109329 Rosa xanthina Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000194019 Streptococcus mutans Species 0.000 description 1
- 101000802471 Sylvirana guentheri Brevinin-2GHb Proteins 0.000 description 1
- 101000802470 Sylvirana guentheri Brevinin-2GHc Proteins 0.000 description 1
- 101000906575 Sylvirana guentheri Guentherin Proteins 0.000 description 1
- 101000851879 Sylvirana guentheri Temporin-GH Proteins 0.000 description 1
- 239000003875 Wang resin Substances 0.000 description 1
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000000903 blocking effect Effects 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
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 229940124280 l-arginine Drugs 0.000 description 1
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 229940100630 metacresol Drugs 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000003681 parotid gland Anatomy 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000001913 submandibular gland Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- HODRFAVLXIFVTR-RKDXNWHRSA-N tevenel Chemical compound NS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CO)NC(=O)C(Cl)Cl)C=C1 HODRFAVLXIFVTR-RKDXNWHRSA-N 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
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
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
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明属于医药领域，具体而言涉及一组新型的具有广谱抗菌活性的HRP5类似物及其制备方法。本发明所涉及的HRP5类似物的氨基酸序列通式如SEQ ID NO：2所示：Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Leu-Xaa11-Lys-Tyr-Leu-Arg-Xaa16-Xaa17-Xaa18。The invention belongs to the field of medicine, and in particular relates to a group of novel HRP5 analogues with broad-spectrum antibacterial activity and a preparation method thereof. The general formula of the amino acid sequence of the HRP5 analog involved in the present invention is shown in SEQ ID NO: 2: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Leu-Xaa11-Lys-Tyr-Leu- Arg-Xaa16-Xaa17-Xaa18.
Description
Technical field
The invention belongs to field of medicaments, novel have HRP5 analogue of broad spectrum antibiotic activity and preparation method thereof in particular to one group.
Background technology
Since penicillium mould is found; Microbiotic is the powerful mean of human treatment's cause pathogeny imcrobe infection always; But along with the antibiotic abuse of tradition; Increasing pathogenic bacteria begins traditional microbiotic is produced resistance, uses so the brand-new antibacterials of urgent need searching-class substitute microbiotic.
Cationic antibacterial peptide (cationic antimicrobial peptides) is cationic (being rich in l-arginine and Methionin) polypeptide of generally being made up of 12-50 amino-acid residue that plant and animal produces, and can protect the host not receive extraneous cause pathogeny imcrobe infection.The antimicrobial spectrum of cationic antibacterial peptide is wideer than traditional microbiotic, not only gram-positive microorganism and Gram-negative bacteria is had anti-microbial effect from insect, pig, the frog and people's cationic antibacterial peptide, but also has antimycotic and antiviral activity.The tradition microbiotic passes through to eliminate microorganism growth or existence essential condition, as makes enzyme denaturation, reach germ-resistant purpose, but bacterium just can be resisted this type of antibiotic attack after needing only a kind of transgenation.With method and the bacterial cell membrane interaction of electric charge, penetrate killing bacteria during cationic antibacterial peptide then passes through, greatly reduced bacterium and produced chemical sproof possible with this.But natural cationic antibacterial peptide is intact no all roses, and the part antibacterial peptide has certain toxicity to eukaryote, the cause of disease object height is killed the active while often be accompanied by Eukaryotic hemolytic action.Therefore how improving its activity and at utmost reducing its toxicity is that the difficult point that present antimicrobial peptide medicaments is developed belongs to hoping.Present research mainly with the both sexes antibacterial peptide of alpha-helix as research object; Around cationic amino acid and hydrophobic amino acid quantity and position; Antibacterial peptide is carried out structure of modification; Like the partial sequence of residue replacement, intercepting natural antibacterial peptide and the positive charge content of increase peptide chain etc., obtained bigger progress.
Histatins (histidine rich protein, HRPs or histatins) is-class is present in the cationic polypeptide that is rich in Histidine in the primate parotid gland and the submaxillary gland secretory product.Isolated at least 12 kinds of people HRPs (HRP1-12), wherein HRP1, HRP3 and HRP5 are main histatinses at present, account for the 85%-90% of total HRPs, and respectively by 38,32,24 amino acid are formed.HRP1 and HRP3 are encoded by different gene, and HRP5 is the product of HRP3 through protease hydrolysis, are included in 24 residues of N-terminal of HRP3.Coding HRP1,3 gene are checked order and are positioned on the human No. 4 karyomit(e) q13.Existing research shows: HRPs has multiple active biological function, and its anti-microbial effect is particularly important, as suppressing, kill streptococcus mutans and Candida albicans etc.
HRP5 contains 24 amino-acid residues, and molecular weight is 3037D.Its aminoacid sequence is Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr (SEQ ID NO:1).Scholars such as Raj think that the characteristic of HRP5 anti-candida albicans is relevant with its aminoacid sequence; They respectively with HRP5 and residue segment N16 (1-16 amino acids), C16 (9-24 amino acids), C14 (11-24 amino acids), C12 (13-24 amino acids), C10 (15-24 amino acids) and M10 (7-16 amino acids) respectively with the Candida albicans effect of different concns, detect the ability of its anti-Candida albicans.Test-results shows that C16 and C14 have the ability of stronger inhibition albicans growth, and the active basically identical of the anti-Candida albicans of HRP5 and C16, and the activity that contains 16 amino acid whose N16 anti-candida albicanses equally is well below C16.The functional zone of the anti-candida albicans of this results suggest HRP5 are positioned at carboxyl terminal.For the further relation of the 26S Proteasome Structure and Function of research HRP5, many scholars have obtained a series of varient through the residue replacement, wherein more importantly dhvar1, dhvar2, dhvar4, dhvar5.They are HRP5 active zone (11-24 amino acids) varients.
Summary of the invention
Polypeptide or its pharmaceutical salts, said polypeptide has following aminoacid sequence:
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Leu-Xaa11-Lys-Tyr-Leu-Arg-Xaa16-Xaa17-Xaa18(SEQ?ID?NO：2)
Wherein Xaa1 is Cys or disappearance;
Xaa2 is Lys, D-Lys, Arg, D-Arg, HoR or disappearance;
Xaa3 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe, Thi or disappearance;
Xaa4 is Lys, D-Lys, Arg, D-Arg, Aib or HoR;
Xaa5 is Lys, D-Lys, Arg, D-Arg or HoR;
Xaa6 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi;
Xaa7 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi;
Xaa8 is Lys, D-Lys, Arg, D-Arg or HoR;
Xaa9 is Lys, D-Lys, Arg, D-Arg or HoR;
Xaa11 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi;
Xaa16 is Lys, D-Lys, Arg, D-Arg or HoR;
Xaa17 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi;
Xaa18 is Cys or disappearance, and when Xaa1 and Xaa18 were all Cys, this polypeptide can form cyclic peptide.
Be all Cys or disappearance simultaneously as a kind of preferred version Xaa1 of the present invention and Xaa18; The preferred Arg of Xaa2, D-Arg or disappearance; The preferred Phe of Xaa3, D-Phe or disappearance; The preferred Lys of Xaa4, D-Lys, Arg, Aib or HoR; The preferred Lys of Xaa5, Arg or D-Arg; The preferred Leu of Xaa6, Ile or Nle; The preferred Trp of Xaa7, Phe, D-Phe or Thi; The preferred Lys of Xaa8, D-Lys or Arg; The preferred Lys of Xaa9, D-Lys or Arg; The preferred Leu of Xaa11, D-Phe or Phe; The preferred Lys of Xaa16, D-Lys or Arg; The preferred Trp of Xaa17, Phe, D-Phe or Thi.
Amino-acid residue involved in the present invention had both comprised natural amino acid, also comprised alpha-non-natural amino acid.The pairing trigram code table of natural amino acid involved in the present invention is as shown in table 1, and pairing trigram code table of alpha-non-natural amino acid involved in the present invention and structure are as shown in table 2.
All amino-acid residues involved in the present invention are represented L type amino acid if do not limit its configuration especially.
Polypeptide involved in the present invention both can be a linear peptides, also can be cyclic peptide.When Xaa1 and Xaa18 are all Cys, can make and form disulfide linkage between Xaa1 and the Xaa18 that this moment, this peptide became cyclic peptide by linear peptides through adding oxygenant.
When polypeptide involved in the present invention was linear peptides, its C-terminal both can be the form of carboxylic acid, also can be the form of acid amides, and preferably the form with acid amides exists.
Table 1: natural amino acid trigram code table
Table 2: alpha-non-natural amino acid trigram code table and structure
Disclose a kind of polypeptide (AMP-1) of linearity in the preferred embodiment of the present invention, its aminoacid sequence is: Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH
2(SEQ ID NO:3)
Another discloses one group of linear polypeptide (AMP-2, AMP-3, AMP-4 the present invention preferred embodiment; AMP-5, AMP-6, AMP-7; AMP-8, AMP-9, AMP-10; AMP-11, AMP-12), its aminoacid sequence is respectively: Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe-NH
2(SEQ ID NO:4) D-Lys-D-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Ly s-D-Phe-NH
2(SEQ ID NO:5) D-Lys-D-Arg-Leu-Phe-D-Lys-D-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe-NH
2(SEQ IDNO:6)
Phe-Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH
2(SEQ?ID?NO：7)
Lys-Arg-Leu-Phe-Lys-Arg-Leu-Phe-Lys-Tyr-Leu-Arg-Arg-Phe-NH
2(SEQ?ID?NO：8)
Arg-Arg-Ile-Phe-Lys-Arg-Leu-Phe-Lys-Tyr-Leu-Arg-Arg-Phe-NH
2(SEQ?ID?NO：9)
Arg-Leu-Arg-Arg-Ile-Phe-Lys-Arg-Leu-Phe-Lys-Tyr-Leu-Arg-Arg-Phe-NH
2(SEQID?NO：10)
Lys-Arg-Leu-Thi-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH
2(SEQ?ID?NO：11)
Lys-Arg-Nle-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH
2(SEQ?ID?NO：12)
Aib-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH
2(SEQ?ID?NO：13)
HoR-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH
2(SEQ?ID?NO：14)
Of the present invention another preferred embodiment discloses a kind of annular polypeptide (AMP-13), and its aminoacid sequence is:
The present invention also has a purpose just to provide the preparation method of above-mentioned linearity and ring type polypeptide, and the solid state chemistry synthetic technology that can adopt those skilled in the art to be familiar with prepares linear peptides and on the basis of linear peptides, prepares cyclic peptide through hydrogen peroxide oxidation method.
The preparation process of solid state chemistry synthetic technology is following:
(1) solid-phase synthetic peptide on resin;
(2) product with step (1) carries out cracking in trifluoroacetic acid or hydrofluoric acid, preferred trifluoroacetic acid, and add the Side chain protective group scavenging agent; With the ice ether sedimentation polypeptide of 5-20 times of volume, centrifugal then, abandon supernatant; Precipitate 4-5 time with ice ether repetitive scrubbing, vacuum-drying obtains thick peptide again;
If peptide C involved in the present invention terminal for carboxylic acid form then step (1) adopt the Wang resin to synthesize; If peptide C involved in the present invention terminal for amide form then step (1) adopt Rink Amide mbha resin to synthesize.
Step (1) is in liquid phase environment, to carry out, and specifically comprises: soak resin-remove amino protecting group-washing-coupling amino acid-monitor-wash-remove amino protecting group (coupling remaining amino acid in proper order)-dry resin.
Wherein amino protecting group is meant the chemical group of introducing for the amino of protection participation condensation reaction.Described amino protecting group is selected from: tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (Z) and 9-fluorenyl-methyl carbonyl (Fmoc), preferred 9-fluorenyl-methyl carbonyl (Fmoc).
As an advantage of solid-phase polypeptide synthetic technology, can protect through introducing chemical group the side chain of partial amino-acid, for example Arg and HoR can adopt pentamethyl-benzo furans-5-alkylsulfonyl (Pbf); Cys can adopt trityl (Trt); Lys and Trp can adopt tertbutyloxycarbonyl (Boc); Tyr can adopt the tertiary butyl (tBu).Described blocking group is not limited thereto, and can carry out choose reasonable according to this area conventional scheme.
The liquid phase environment solvent for use of step (1) is selected from: N (DMF) or methylene dichloride (DCM), preferred DMF.
Remove the agent that removes that amino protecting group need add amino protecting group in the step (1), piperidines (PIP) solution is selected in the agent that removes of amino protecting group for use, concentration 10-40% (PIP/DMF), and the time of removing is 20-50min.Preferred concentration is 20-25% (PIP/DMF), removes time 25-35min.
Amino acid whose coupling need add coupling reagent in the step (1), coupling reagent by: carbodiimide type reagent or benzotriazole salt type reagent and I-hydroxybenzotriazole (HOBt) are formed.
Carbodiimide type reagent comprises NSC 57182 (DCC), DIC (DIC) and N-diamino-propyl group-N-ethyl carbodiimide (EDC).
Benzotriazole salt type reagent comprises 2-(1H-benzo trisazo-L-1-yl)-1; 1; 3,3-tetramethyl-urea Tetrafluoroboric acid ester (TBTU), O-benzotriazole-N, N; N ', N '-tetramethyl-urea hexafluorophosphate (HBTU), phosphofluoric acid benzotriazole-1-oxygen base three (dimethylamino) phosphorus (BOP), phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus (PyBOP) etc.
The preferred DIC of coupling reagent (DIC) and I-hydroxybenzotriazole (HOBt), 2-(1H-benzo trisazo-L-1-yl)-1; 1; 3; 3-tetramethyl-urea Tetrafluoroboric acid ester (TBTU) and I-hydroxybenzotriazole (HOBt), most preferably DIC (DIC) and I-hydroxybenzotriazole (HOBt).
The condensation reaction of triketohydrindene hydrate detection method monitoring polypeptide is adopted in " monitoring " in the step (1).
The amino acid that is linked in sequence in the step (1) is meant according to polypeptid acid sequence and connects amino acid from the C end one by one to the N end.
The described Side chain protective group scavenging agent of step (2) is to be selected from that two or more combination and prepare by 5-20% (V/V) with trifluoroacetic acid or hydrofluoric acid obtains like thioanisole, tri isopropyl silane, phenol, water, 1, meta-cresol etc.Preferred trifluoroacetic acid (TFA): thioanisole: 1: 75% phenol: water=85: 5: 5: 4: 1.
Useful especially is for satisfying the specification of quality of medicinal use, and polypeptide preparation method provided by the present invention also comprises and adopts conventional means to carry out purifying.The purification process that is adopted can be reverse-phase chromatography or ion exchange chromatography, preferred reverse-phase chromatography.
The antibacterial activity in vitro of polypeptide involved in the present invention can be identified through measuring its minimum inhibitory concentration (MIC).The stdn council of U.S. clinical labororatory (NCCLS) recommends to adopt the constant broth dilution method to measure the minimum inhibitory concentration (MIC) of each antibacterial peptide, and substratum adopts Mueller-Hinton (MH) broth culture.With amphotericin B, Polymyxin E and vancomyein as positive control.Active determination in vitro shows; Polypeptide provided by the invention all has significant sterilization effect to gram-positive microorganism, Gram-negative bacteria and fungi; Isolating resistance gram-positive microorganism, resistance Gram-negative bacteria and resistance fungi are clinically had significant sterilization effect equally, have wide spectrum, characteristics of high efficiency.Therefore polypeptide involved in the present invention can substitute traditional microbiotic as activeconstituents clinically and treat.
The aspect that on behalf of the present invention, following examples only set forth is not the limitation of theme of the present invention.
Embodiment
The preparation of embodiment 1:AMP-1 and purifying
Sequence: Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH
2(SEQ ID NO:3)
(1) material and reagent
Rink Amide mbha resin, substitution value 0.41mmol/g.
The amino acid of required band protection is Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Phe-OH and Fmoc-L-Tyr (tBu)-OH.
Reagent: HOBt, DIC, DMF, piperidines.
(2) instrument
PSI300 type Peptide synthesizer, Waters600 half preparative high performance liquid chromatography appearance, magnetic stirring apparatus.
(3) operation steps (is example with 0.25mmol)
A. solid state chemistry synthesizes polypeptide
Take by weighing Rink Amide mbha resin 0.61g, place the reactor drum of Peptide synthesizer, add 10mLDMF; Soak 2h, add 20%PIP/DMF solution 15mL then, mix 30min and remove amino protecting agent; With DMF washing resin 7 times; In reactor drum, add 387.4mg Fmoc-L-Phe-OH, equimolar coupling reagent DIC (0.33mol/L) and HOBt (0.33mol/L) then and react, temperature of reaction is a room temperature, with ninhydrin reaction monitoring reaction process; Guarantee that amino acid is coupled on the resin, with DMF washing resin 7 times.After first amino acid is coupled on the resin, can proceed next amino acid whose linked reaction according to the method described above, so circulation is accomplished until whole amino acid couplings.
B. cracking and deposition
Behind the peptide end of synthesis, the vacuum-drying resin is weighed.Ratio according to 1g resin 10mL lytic reagent adds lytic reagent, and the reagent proportioning is TFA: thioanisole: 1: 75% phenol: water=85: 5: 5: 4: 1, and stirring at room reaction 3 hours, suction filtration.Then in cracking suction filtration liquid, add the ice ether sedimentation polypeptide of 10 times of volumes, centrifugal, abandon supernatant, precipitate vacuum-drying, the thick peptide of weighing 4～5 times with ice ether repetitive scrubbing again.
C. reverse-phase chromatography purifying
Use preparation HPLC, adopt reverse-phase chromatography, the above-mentioned thick peptide of purifying.
The HPLC condition is following:
Chromatographic column: XBridgeTM Prep C18 5 μ m OBDTM 19 * 150mm
Flow velocity: 10mL/min
Detect wavelength: 210nm
Moving phase: A: contain the 0.1%TFA aqueous solution
B: the acetonitrile that contains 0.1%TFA
Gradient elution program such as table 3:
Table 3 gradient elution table
Collect target peptide purity greater than 99% part, 50 ℃ are evaporated to dried then.Measure through ESI-MS, the molecular weight of this peptide is 1880.28, and theoretical value is 1880.43.
Embodiment 2: the preparation of HRP5 analogue and purifying in the table 4
(1) material and reagent
Rink Amide mbha resin, substitution value 0.41mmol/g.
Amino acid Fmoc-L-Arg (Pbf)-OH, Fmoc-D-Arg (Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-HoR (Pbf)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-D-Lys (Boc)-OH, Fmoc-L-Phe-OH, Fmoc-D-Phe-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ile-OH, Fmoc-L-Nle-OH, Fmoc-L-Thi-OH and the Fmoc-L-Aib-OH of required band protection.
Reagent: HOBt, DIC, DMF, piperidines.
(2) instrument
PSI300 type Peptide synthesizer, Waters600 half preparative high performance liquid chromatography appearance, magnetic stirring apparatus.
(3) operation steps (is example with 0.25mmol)
With the polypeptide in preparation of the method for operation steps a-c in the similar embodiment 1 and the purifying table 4, collect purity greater than 99% part, 50 ℃ are evaporated to dried then.The ESI-MS measured value is as shown in table 4.
Table 4HRP5 analogue
Embodiment 3: the preparation of cyclic peptide AMP-13 and purifying
(1) material and reagent
Rink Amide mbha resin, substitution value 0.41mmol/g.
The amino acid of required band protection is Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Phe-OH, Fmoc-L-Tyr (tBu)-OH and Fmoc-L-Cys (Trt)-OH.
Reagent: HOBt, DIC, DMF, piperidines, 30%H
2O
2
(2) instrument
PSI300 type Peptide synthesizer, Waters600 half preparative high performance liquid chromatography appearance, magnetic stirring apparatus.
(3) operation steps
A. method preparation and the purifying with operation steps a-c in the similar embodiment 1 has following sequence (Cys-Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-Cys-NH
2) polypeptide, collect purity greater than 99% part, 50 ℃ are evaporated to dried then.Measure through ESI-MS, the molecular weight of this peptide is 2086.24, and theoretical value is 2086.72.
B. the formation of intramolecular disulfide bond
Take by weighing polypeptide 100.0mg among the present embodiment operation steps a, be dissolved in 200ml water, be mixed with the solution that concentration is 0.5mg/ml; Adding 3.33ml concentration is 30% hydrogen peroxide; Stirring reaction under the room temperature is with the carrying out of HPLC monitoring reaction, until being converted into oxidized form fully.
C. reverse chromatograms purifying
With the method purifying of operation steps c in the similar embodiment 1, collect cyclic peptide AMP-13 purity greater than 99% part, 50 ℃ are evaporated to dried then.Measure through ESI-MS, the molecular weight of this peptide is 2084.25, and theoretical value is 2084.72.
Embodiment 4: antibacterial activity in vitro is measured
The minimum inhibitory concentration (MIC) of each antibacterial peptide of constant broth dilution method determination of recommending according to the stdn council of U.S. clinical labororatory (NCCLS), substratum adopts Mueller-Hinton (MH) broth culture.
Concrete steps are:
(1) antibacterials stock solution preparation:
Accurately compound concentration is the above-mentioned HRP5 analogue AMP 1～13 and positive reference substance amphotericin B, Polymyxin E and vancomyein of 1280 μ g/mL.It is subsequent use that each stock solution for preparing places-20 ℃ of environment to preserve.
(2) culture medium preparation:
Take by weighing MH broth culture 21g, be dissolved in the zero(ppm) water and be settled to 1L, 121 ℃ of high-temperature sterilization 30min.
(3) preparation of inoculum:
With 3～5 of transfering loop picking plesiomorphism bacterium colonies to be checked, be inoculated in the MH meat soup of 4～5mL, hatch 2～6h for 35 ℃.Increase logarithmic phase bacterium liquid behind the bacterium with saline water or MH meat soup corrected concentrations to 0.5 Maxwell than turbid standard, contain 1～2 * 10 approximately
8CFU/mL.It is back subsequent use with MH meat soup above-mentioned bacteria suspension to be carried out dilution in 1: 100.
(4) preparation of dilution antibacterials and the inoculation of bacterium liquid:
Get sterile test tube (13 * 100mm) 13 are arranged in a row adding 1.6mL MH meat soup in the 1st pipe; Respectively add 1mL MH meat soup in the 2-13 pipe, add antibacterials stostes (1280 μ g/mL) 0.4mL, mixing to the 1st pipe then; Then draw 1mL to the 2 pipes, from the 2nd pipe, draw 1mL to the 3 pipes, doubling dilution to the 11 pipes so continuously behind the mixing again; And absorption 1mL discards from the 11st pipe;, in 1-11 pipe and the 13rd pipe, add above-mentioned each 1mL of the inoculum for preparing then, make the final bacterial concentration of every pipe be about 5 * 10
5CFU/mL.1-11 pipe drug level is respectively 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL; The 12nd pipe is not for containing the blank of antibacterials and inoculum, and the 13rd pipe is not for containing the negative control of antibacterials.
(5) hatch: will inoculate the good good stopper of dilution pipe close, and put in 35 ℃ of normal air incubators and hatch 16～20h.
(6) result: with visual inspection, the lowest drug concentration of no bacterial growth is the minimum inhibitory concentration of this sample (MIC).It is as shown in table 5 that the MIC of each antibacterial peptide measures the result.
The MIC of table 5HRP5 analogue measures the result
Claims (10)
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