CN109485713A - Dimerization modified antimicrobial peptide analogues and its synthesis and application with strong antibacterial activity and hypotoxicity - Google Patents
Dimerization modified antimicrobial peptide analogues and its synthesis and application with strong antibacterial activity and hypotoxicity Download PDFInfo
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Abstract
Present invention design has synthesized the dimerization modified antimicrobial peptide analogues with strong antibacterial activity and hypotoxicity, it is to replace analog Anoplin-D4 to the part D type of natural antibacterial peptide Anoplin, 7, Anoplin-D5,7 and Anoplin-D9,10,3 carry out " end C-C " connects with " end C-N " intermolecular side chain, a series of obtained dimerization antibacterial peptide analogues.Antibacterial experiment in vitro, PI decoration method flow cytometry tests, hemolytic experiment show, the dimerization modified antimicrobial peptide analogues that the present invention designs synthesis have strong antibacterial activity and hypotoxicity, therefore, the antibacterial peptide analogues that the present invention obtains have a good application prospect in terms of the exploitation of clinical antibacterials.
Description
Technical field
The present invention relates to technological field of biochemistry, be related to a kind of new structural dimerization modified antimicrobial peptide analogues and
It is synthesized and application, in particular to a kind of dimerization modified antimicrobial peptide analogues and its conjunction with strong antibacterial activity and hypotoxicity
At and application.
Background technique
It is largely infected using antibiotic treatment, bacterium is caused to rapidly develop (Lancet to the drug resistance of Conventional antibiotic
Infectious Diseases, 2013,13:1057-1098).Due to that cannot use Conventional antibiotic, new antibody-resistant bacterium is not
It is disconnected to occur, it has also become the problem of whole world is paid close attention to.According to the monitoring of the data of infectious disease association, the U.S. and hospital
Report, by these be easy drug resistant pathogen be known as " ESKAPE " (Clinical Infectious Diseases, 2009,48:
1-12)." ESKAPE " pathogen includes: enterococcus faecium (Enterococcus faecium), staphylococcus aureus
(Staphylococcus aureus), Klebsiella Pneumoniae (Klebsiella pneumoniae), acinetobacter calcoaceticus
(Acinetobacter), pseudomonas aeruginosa (Pseudomonas aeruginosa) and enterobacteria (Enterobacter).This
A little bacterial communities can resist the antibacterial ability of antibiotic, or even generate bacterial resistance, be the main original of whole world nosocomial infection
Cause, for this purpose, the exploitation of novel antibacterial drug is extremely urgent.Antibacterial peptide (AMPs) has positive charge and amphipathic structure, in vitro
Antibacterial activity with higher, and due to unique mechanism of action, it is not easy to induce bacterial resistance, it is considered to be ideal candidate
Drug (Biological Chemistry, 2001,382:597-619).AMPs be include animal, plant, insect and micro- life
A part in biologic artifact innate defence system including object has extensive antibacterial activity to bacterium, fungi and virus,
In addition, part antibacterial peptide is even also proved to anti-tumor activity (PLoS Pathog.2010.6:e1001067；Amino
Acids, 2011,40:51-59).Under normal circumstances, AMPs is made of 10 to 50 amino acid, these amino acid include hydrophobicity
And hydrophilic amino-acid residue, amphiphilic α-helixstructure can be formed in bacterial cell membrane environment, with bacterial cell membrane
Interaction makes bacterial cell membrane by rapid damage and bacterium content leaks, leads to bacterial death, this mechanism of action with
It is significantly different for the antibiotic mechanism of action of pathogen specific molecular receptor, thus be not easy to induce bacterial resistance
(Antimicrob Agents Chemother, 2012,56 (6): 3004-3010).
Although the appearance of antibacterial peptide is overcomes bacterial drug resistance to provide opportunity, since natural antibacterial peptide is easily by internal egg
The disadvantages of white enzyme identification, half-life short, antibacterial activity be strong, poor selectivity, makes it receive limit in clinical application and development
System.Studies have shown that antibacterial peptide is carried out intermolecular dimerization modification, antibacterial activity, the stability of antibacterial peptide can be significantly improved
(Journal of Peptide Science, 2002,8:570-577)；At the same time, amino acid side chain " azido " official is utilized
It can roll into a ball and " propargyl " functional group, 1, the 3- Dipolar Cycloaddition through click chemistry form " triazole ring " structure, help to increase
Strong antibacterial peptide improves antibacterial activity (Peptides, 2017,88:115-125) to the penetration capacity of bacterial cell membrane；It removes
Except this, the D type enantiomer of l-amino acid residue is introduced into antibacterial peptide, can be avoided the identification of protease, hence it is evident that increases antibacterial
The stability (Frontiers in Chemistry, 2017,5:40) of peptide；Also, the polypeptide containing D type amino acid residue is not allowed
(Antimicrob Agents Chemother, 2004,48 (8): 3127-3129) are easily identified by internal antigen presenting cell.
Summary of the invention
An object of the present invention: it provides a kind of similar with the dimerization modified antimicrobial peptide of hypotoxicity with strong antibacterial activity
Object.
The second object of the present invention: it provides above-mentioned similar with the dimerization modified antimicrobial peptide of hypotoxicity with strong antibacterial activity
Application of the object in the exploitation of clinical antibacterials.
The third object of the present invention: it provides above-mentioned similar with the dimerization modified antimicrobial peptide of hypotoxicity with strong antibacterial activity
The synthetic method of object.
(1) with the dimerization modified antimicrobial peptide analogues of strong antibacterial activity and hypotoxicity
The present invention has the dimerization modified antimicrobial peptide analogues of strong antibacterial activity and hypotoxicity, including " end C-C " two
Dimerization modified antimicrobial peptide analogues and " end C-N " dimerization modified antimicrobial peptide analogues." end C-C " dimerization modified antimicrobial
Peptide analogues are part D type amino acid substitution analog Anoplin-D4,7, Anoplin-D9,10,3 in female peptide Anoplin
Non-natural special acid Fmoc-L-Propargylgly-OH or Fmoc- is introduced respectively with the end C- of Anoplin-D5,7
Then L-Lys (Mtt)-OH carries out acetylation modification to its end N- respectively, obtains precursor peptide Ac-D4,7-Pra, Ac-D9,
10,3-Pra、Ac-D4,7-Lys(N3) and Ac-D5,7-Lys (N3), then intermolecular side is carried out to precursor peptide by " click chemistry "
Chain link, obtain " end C-C " dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D4,7), JC-AA- (D4,7+D5,
7),JC-AA-(D9,10,3+D5,7)；" end C-N " dimerization modified antimicrobial peptide analogues are the part D in female peptide Anoplin
The end C- of type amino acid substitution analog Anoplin-D4,7, Anoplin-D9,10,3 introduce non-natural special ammonia respectively
Then base acid Fmoc-L-Propargylgly-OH carries out acetylation modification to its end N- respectively, obtains precursor peptide Ac-D4,
7-Pra, Ac-D9,10,3-Pra, in the part D type amino acid substitution analog Anoplin-D4 of female peptide Anoplin, 7,
Anoplin-D5,7 end N- introduces non-natural special acid Fmoc-L-Lys (Mtt)-OH respectively, then respectively to it
The end N- carries out acetylation modification, obtains precursor peptide Ac-Lys (N3)-D4,7 and Ac-Lys (N3)-D5,7, then pass through " clickization
Learn " carry out intermolecular side chain connection respectively to precursor peptide, obtain " end C-N " dimerization modified antimicrobial peptide analogues JCN-AA-
(D4,7+D4,7)、JCN-AA-(D4,7+D5,7)、JCN-AA-(D9,10,3+D5,7)。
The structural formula difference of above-mentioned dimerization modified antimicrobial peptide analogues is as follows:
" end C-C " dimerization modified antimicrobial peptide analogues:
(1)JC-AA-(D4,7+D4,7)
(2)JC-AA-(D4,7+D5,7)
(3)JC-AA-(D9,10,3+D5,7)；
" end C-N " dimerization modified antimicrobial peptide analogues:
(4)JCN-AA-(D4,7+D4,7)
(5)JCN-AA-(D4,7+D5,7)
(6)JCN-AA-(D9,10,3+D5,7)。
The synthetic method of dimerization modified antimicrobial peptide analogues of the present invention, comprises the following steps that:
1, the synthesis of " end C-C " dimerization modified antimicrobial peptide analogues
A, unnatural amino acid is introduced in the end C- of Anoplin-D4,7 and Anoplin-D9,10,3 sequence respectively
Fmoc-L-Propargylgly-OH, obtains Anoplin-D4,7-Pra and Anoplin-D9, and 10,3-Pra；Respectively with
Anoplin-D4,7-Pra and Anoplin-D9,10,3-Pra be matrix, synthesizes Anoplin- using classical solid phase synthesis process
D4,7-Pra-resin and Anoplin-D9,10,3-Pra-resin, the terminated acetylated modification of N- is then carried out respectively, is obtained
Ac-D4,7-Pra-resin and Ac-D9,10,3-Pra-resin, it cut respectively, obtain precursor peptide Ac-D4,7- after purification
Pra and Ac-D9,10,3-Pra；
B, respectively in Anoplin-D4, the end C- of 7, Anoplin-D5,7 sequences introduces unnatural amino acid Fmoc-Lys
(Mtt)-OH obtains Anoplin-D4,7-Lys (Mtt) and Anoplin-D5,7-Lys (Mtt)；With Anoplin-D4,7-Lys
(Mtt) and Anoplin-D5,7-Lys (Mtt) are matrix, synthesize Anoplin-D4,7-Lys using classical solid phase synthesis process
(Mtt) then-resin and Anoplin-D5,7-Lys (Mtt)-resin carries out the terminated acetylated modification of N- respectively, obtains Ac-
D4,7-Lys (Mtt)-resin and Ac-D5,7-Lys (Mtt)-resin；Respectively to Ac-D4,7-Lys (Mtt)-resin and Ac-
D5,7-Lys (Mtt)-resin carry out the modification of side chain Azide, and cutting obtains precursor peptide Ac-D4,7-Lys (N after purification3) with
Ac-D5,7-Lys(N3)；
C, using 1, the 3- Dipolar Cycloaddition of click chemistry, make precursor peptide Ac-D4, " alkynyl " official of 7-Pra side chain
Can group respectively with precursor peptide Ac-D4,7-Lys (N3) or Ac-D5,7-Lys (N3) side chain " azido " functional group reactions, by two
Precursor peptide carries out intermolecular side chain connection, obtain " end C-C " dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D4,
And JC-AA- (D4,7+D5,7) 7)；Using 1, the 3- Dipolar Cycloaddition of click chemistry, make precursor peptide Ac-D9,10,3-Pra
" alkynyl " functional group of side chain and precursor peptide Ac-D5,7-Lys (N3) side chain " azido " functional group reactions, by two precursors
Peptide carries out intermolecular side chain connection, obtains " end C-C " dimerization modified antimicrobial peptide analogues JC-AA- (D9,10,3+D5,7)；
2, the synthesis of " end C-N " dimerization modification dimer antibacterial peptide analogues
A, precursor peptide Ac- is obtained using method in such as synthesis step a of " end C-C " dimerization modified antimicrobial peptide analogues
D4,7-Pra and Ac-D9,10,3-Pra；
B, respectively in Anoplin-D4, the end N- of 7, Anoplin-D5,7 sequences introduces unnatural amino acid Fmoc-Lys
(Mtt)-OH obtains Anoplin-Lys (Mtt)-D4,7 and Anoplin-Lys (Mtt)-D5,7；With Anoplin-Lys (Mtt)-
D4,7 and Anoplin-Lys (Mtt)-D5,7 be matrix, synthesizes Anoplin-Lys (Mtt)-using classical solid phase synthesis process
Then D4,7-resin and Anoplin-Lys (Mtt)-D5,7-resin carries out the terminated acetylated modification of N- respectively, obtains Ac-
Lys (Mtt)-D4,7-resin and Ac-Lys (Mtt)-D5,7-resin；Respectively to Ac-Lys (Mtt)-D4,7-resin and Ac-
Lys (Mtt)-D5,7-resin carry out the modification of side chain Azide, and cutting, purifying obtain precursor peptide Ac-Lys (N3)-D4,7 and Ac-
Lys(N3)-D5,7；
C, using 1, the 3- Dipolar Cycloaddition of click chemistry, make precursor peptide Ac-D4, " alkynyl " official of 7-Pra side chain
Can group respectively with precursor peptide Ac-Lys (N3)-D4,7 or Ac-Lys (N3)-D5, " azido " functional group reactions of 7 side chains, by two
Precursor peptide carries out intermolecular side chain connection, obtains " end C-N " dimerization modified antimicrobial peptide analogues JCN-AA- (D4,7+
D4,7) with JCN-AA- (D4,7+D5,7)；Using 1, the 3- Dipolar Cycloaddition of click chemistry, make precursor peptide Ac-D9,10,
" alkynyl " functional group of 3-Pra side chain and precursor peptide Ac-Lys (N3)-D5, " azido " functional group reactions of 7 side chains, by two
Precursor peptide carries out intermolecular side chain connection, obtain " end C-N " dimerization modified antimicrobial peptide analogues JCN-AA- (D9,10,3+
D5,7)。
More specifically, the synthetic method of dimerization modified antimicrobial peptide analogues of the present invention, comprises the following steps that:
1, the synthesis of " end C-C " dimerization modified antimicrobial peptide analogues
a、Ac-D4,7-Lys(N3) synthesis
Fmoc-Lys (Mtt)-OH, HOBT, HBTU, DIEA are dissolved into mixing in DMF, and with slough Fmoc protecting group
MBHA resin carries out condensation reaction, obtains Fmoc-Lys (Mtt)-resin；With method successively condensation reaction amino acid Fmoc-Leu-
OH、Fmoc-Leu-OH、Fmoc-Thr(tBu)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Ile-OH、Fmoc-Arg(pbf)-
OH, Fmoc-D-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, obtain Fmoc-Gly-Leu-
Leu-D-Lys-Arg-Ile-D-Lys-Thr-Leu-Leu-Lys (Mtt)-resin is Fmoc-D4,7-Lys (Mtt)-
resin；Then end Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction, obtains Anoplin-D4,7-Lys
(Mtt)-resin；Acetic anhydride, DIEA are dissolved to mixing in DMF, and carried out with Anoplin-D4,7-Lys (Mtt)-resin
Condensation reaction obtains Ac-D4,7-Lys (Mtt)-resin；By Ac-D4,7-Lys (Mtt)-resin volume fraction 1%TFA
DCM solution slough side chain Mtt protecting group, and carry out side chain Azide modification, obtain side chain azido compound Ac-D4,7-Lys
(N3)-resin, cutting, purifying obtain precursor peptide Ac-D4,7-Lys (N3)；
b、Ac-D5,7-Lys(N3) synthesis
Fmoc-Lys (Mtt)-OH, HOBT, HBTU, DIEA are dissolved into mixing in DMF, and with slough Fmoc protecting group
MBHA resin carries out condensation reaction, obtains Fmoc-Lys (Mtt)-resin；With method successively condensation reaction amino acid Fmoc-Leu-
OH、Fmoc-Leu-OH、Fmoc-Thr(tBu)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Ile-OH、Fmoc-D-Arg
(pbf)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, obtain Fmoc-Gly-
Leu-Leu-Lys-D-Arg-Ile-D-Lys-Thr-Leu-Leu-Lys (Mtt)-resin is Fmoc-D5,7-Lys (Mtt)-
resin；Then end Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction, obtains Anoplin-D5,7-Lys
(Mtt)-resin；Acetic anhydride, DIEA are dissolved to mixing in DMF, and carried out with Anoplin-D5,7-Lys (Mtt)-resin
Condensation reaction obtains Ac-D5,7-Lys (Mtt)-resin；By DCM of Ac-D5,7-Lys (the Mtt)-resin containing 1%TFA
Solution sloughs side chain Mtt protecting group, and carries out side chain Azide modification, obtains side chain azido compound Ac-D5,7-Lys (N3)-
Resin, cutting, purifying obtain precursor peptide Ac-D5,7-Lys (N3)；
C, dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D4,7), JC-AA- (D4,7+D5,7), JC-AA-
(D9,10,3+D5,7) synthesis
By precursor peptide Ac-D4,7-Pra respectively with Ac-D4,7-Lys (N3) or Ac-D5,7-Lys (N3) it is dissolved in H2In O,
The CuSO containing 5%DMF is added4Solution makees antioxidant, argon gas protection with sodium ascorbate, and room temperature is protected from light 24-28h；Through
RP-HPLC purifying, obtains dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D4,7), JC-AA- (D4,7+D5,7)；
By precursor peptide Ac-D9,10,3-Pra and Ac-D5,7-Lys (N3) it is dissolved in H2In O, the CuSO containing 5%DMF is added4
Solution makees antioxidant, argon gas protection with sodium ascorbate, and room temperature is protected from light 24-28h；It is purified through RP-HPLC, obtains two
Dimerization modified antimicrobial peptide analogues JC-AA- (D9,10,3+D5,7).
2, the synthesis of " end C-N " dimerization modified antimicrobial peptide analogues
a、Ac-Lys(N3)-D4,7 synthesis
Fmoc-Leu-OH, HOBT, HBTU, DIEA dissolve to mixing in DMF, and with the MBHA that sloughs Fmoc protecting group
Resin carries out condensation reaction, obtains Fmoc-Leu-resin；With method successively condensation reaction amino acid Fmoc-Leu-OH, Fmoc-
Thr(tBu)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Ile-OH、Fmoc-Arg(pbf)-OH、Fmoc-D-Lys(Boc)-
OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys (Mtt)-OH, obtains Fmoc-Lys (Mtt)-
Gly-Leu-Leu-D-Lys-Arg-Ile-D-Lys-Thr-Leu-Leu-resin, i.e. Fmoc-Lys (Mtt)-D4,7-resin；
Then end Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction, obtains Anoplin-Lys (Mtt)-D4,7-
resin；Acetic anhydride, DIEA are dissolved to mixing in DMF, and be condensed instead with Anoplin-Lys (Mtt)-D4,7-resin
It answers, obtains Ac-Lys (Mtt)-D4,7-resin；Ac-Lys (Mtt)-D4,7-resin is molten with the DCM of volume fraction 1%TFA
Liquid sloughs side chain Mtt protecting group, and carries out side chain Azide modification, obtains side chain azido compound Ac-Lys (N3)-D4,7-
Resin, cutting, purifying obtain precursor peptide Ac-Lys (N3)-D4,7；
b、Ac-Lys(N3)-D5,7 synthesis
Fmoc-Leu-OH, HOBT, HBTU, DIEA dissolve to mixing in DMF, and with the MBHA that sloughs Fmoc protecting group
Resin carries out condensation reaction, obtains Fmoc-Leu-resin；With method successively condensation reaction amino acid Fmoc-Leu-OH, Fmoc-
Thr(tBu)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Ile-OH、Fmoc-D-Arg(pbf)-OH、Fmoc-Lys(Boc)-
OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys (Mtt)-OH, obtains Fmoc-Lys (Mtt)-
Gly-Leu-Leu-Lys-D-Arg-Ile-D-Lys-Thr-Leu-Leu-resin is Fmoc-Lys (Mtt)-D5,7-
resin；Then end Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction, obtains Anoplin-Lys (Mtt)-
D5,7-resin；Acetic anhydride, DIEA are dissolved to mixing in DMF, and contracted with Anoplin-Lys (Mtt)-D5,7-resin
Reaction is closed, Ac-Lys (Mtt)-D5,7-resin is obtained；By Ac-Lys (Mtt)-D5,7-resin with volume fraction 1%TFA's
DCM solution sloughs side chain Mtt protecting group, and carries out side chain Azide modification, obtains side chain azido compound Ac-Lys (N3)-
D5,7-resin, cutting, purifying obtain precursor peptide Ac-Lys (N3)-D5,7；
C, dimerization modified antimicrobial peptide analogues JCN-AA- (D4,7+D4,7), JCN-AA- (D4,7+D5,7), JCN-AA-
(D9,10,3+D5,7) synthesis
By precursor peptide Ac-D4,7-Pra respectively with Ac-Lys (N3)-D4,7 or Ac-Lys (N3)-D5,7 is dissolved in H2In O,
The CuSO containing 5%DMF is added4Solution makees antioxidant, argon gas protection with sodium ascorbate, and room temperature is protected from light 24-28h；Through
RP-HPLC purifying, obtains dimerization modified antimicrobial peptide analogues JCN-AA- (D4,7+D4,7), JCN-AA- (D4,7+D5,7)；
By precursor peptide Ac-D9,10,3-Pra and Ac-Lys (N3)-D5,7 is dissolved in H2In O, the CuSO containing 5%DMF is added4
Solution makees antioxidant, argon gas protection with sodium ascorbate, and room temperature is protected from light 24-28h；It is purified through RP-HPLC, obtains two
Dimerization modified antimicrobial peptide analogues JCN-AA- (D9,10,3+D5,7).
As the preferred of technical solution of the present invention, precursor peptide Ac-D4,7-Pra and Ac-D9 described above, 10,3-Pra
Synthetic method specifically:
(1) synthesis of Ac-D4,7-Pra
Fmoc-L-Propargylgly-OH, HOBT, HBTU, DIEA are dissolved to mixing in DMF, and protected with Fmoc is sloughed
The MBHA resin for protecting base carries out condensation reaction, obtains Fmoc-L-Propargylgly-resin, i.e. Fmoc-Pra-resin；Together
Method successively condensation reaction subsequent amino-acid Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Thr (tBu)-OH, Fmoc-D-Lys
(Boc)-OH、Fmoc-Ile-OH、Fmoc-Arg(pbf)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-
Leu-OH, Fmoc-Gly-OH obtain Fmoc-Gly-Leu-Leu-D-Lys-Arg-Ile-D-Lys-Thr-Leu-Leu-Pra-
Resin, i.e. Fmoc-Anoplin-D4,7-Pra-resin；Then end is sloughed with the DMF solution of 20% piperidines of volume fraction
Fmoc protecting group obtains Anoplin-D4,7-Pra-resin；Acetic anhydride, DIEA dissolve to mixing in DMF, and with
Anoplin-D4,7-Pra-resin carry out condensation reaction, obtain Ac-D4,7-Pra-resin, and cutting, purifying obtain precursor peptide
Ac-D4,7-Pra；
(2) synthesis of Ac-D9,10,3-Pra
Fmoc-L-Propargylgly-OH, HOBT, HBTU, DIEA are dissolved to mixing in DMF, and protected with Fmoc is sloughed
The MBHA resin for protecting base carries out condensation reaction, obtains Fmoc-L-Propargylgly-resin, i.e. Fmoc-Pra-resin；Together
Method successively condensation reaction subsequent amino-acid Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Thr (tBu)-OH, Fmoc-Lys
(Boc)-OH、Fmoc-Ile-OH、Fmoc-Arg(pbf)-OH、Fmoc-Lys(Boc)-OH、Fmoc-D-Leu-OH、Fmoc-
Leu-OH, Fmoc-Gly-OH obtain Fmoc-Gly-Leu-D-Leu-Lys-Arg-Ile-Lys-Thr-D-Leu-D-Leu-
Pra-resin, i.e. Fmoc-Anoplin-D9,10,3-Pra-resin, it is then de- with the DMF solution of 20% piperidines of volume fraction
End Fmoc protecting group is gone, obtains Anoplin-D9,10,3-Pra-resin；Acetic anhydride, DIEA are dissolved to mixing in DMF,
And and Anoplin-D9,10,3-Pra-resin carry out condensation reactions, obtain Ac-D9,10,3-Pra-resin, and cutting, purifying
Obtain precursor peptide Ac-D9,10,3-Pra.
Each amino acid, HOBT, HBTU and to slough the molal weight ratio of the MBHA resin of Fmoc protecting group be 3:1-
The molal weight ratio of 4:1, acetic anhydride and the MBHA resin for sloughing Fmoc protecting group is 12:1-26:1, and DIEA is protected with Fmoc is sloughed
The molal weight ratio for protecting the MBHA resin of base is 6:1.
The side chain azide method are as follows: first by NaN3With Tf2O is in H22-2.5h is reacted at room temperature in O/DCM mixed liquor, instead
Answer liquid through DCM, Na2CO3And Na2SO4Moisture is removed, Tf is obtained2N3Solution；Mtt protection is sloughed with the DCM solution containing 1%TFA
The resin of base is expanded through DCM and CH3OH compression, after vacuum is drained, with CuSO4、K2CO3And CH3OH mixing, then with it is obtained above
Tf2N3Solution reacts at room temperature 46-48h, obtains side chain azido compound, and cutting, purifying obtain side chain Azide precursor peptide；Wherein
H2The volume ratio of O and DCM is 1:2, NaN3、Tf2The molal weight ratio of O and the MBHA resin for sloughing Mtt protecting group are respectively 100:
1-200:1,30:1-50:1, CuSO4、K2CO3It is respectively 0.1:1- with the MBHA resin molal weight ratio for sloughing Mtt protecting group
0.3:1、0.2:1-0.5:1。
The precursor peptide Ac-D4,7-Pra or Ac-D9,10,3-Pra and side chain Azide precursor peptide Ac-D4,7-Lys
(N3) or Ac-D5,7-Lys (N3) or Ac-Lys (N3)-D4,7 or Ac-Lys (N3) the molal weight ratio of-D5,7 is 1:1-
1.3:1, the concentration of precursor peptide in water are 8-10mg/mL, CuSO4, sodium ascorbate and side chain Azide precursor peptide rub
Your mass ratio is respectively 3:1-8:1,15:1-20:1.
The cutting reagent is TFA, TIS and H that volume ratio is 95:2.5:2.52The mixed solution of O.
The purification process is first to carry out RP-HPLC separation, be then freeze-dried；RP-HPLC purification condition is to flow
The aqueous solution of phase A:0.05%TFA, Mobile phase B: the acetonitrile solution of 0.05%TFA；Linear gradient elution 30min is collected main
The efflux of absorption peak.
It is described to slough the DMF solution that reagent used by MBHA resin Fmoc protecting group is 20% piperidines of volume fraction.
Through Mass Spectrometric Identification, the method for the present invention successfully synthesizes " end C-C " dimerization modified antimicrobial peptide analogues JC-AA-
(D4,7+D4,7), JC-AA- (D4,7+D5,7) and JC-AA- (D9,10,3+D5,7), and the modification of " end C-N " dimerization are anti-
Bacterium peptide analogues JCN-AA- (D4,7+D4,7), JCN-AA- (D4,7+D5,7) and JCN-AA- (D9,10,3+D5,7).
(2) the outer activity research of dimerization modified antimicrobial peptide similar object
1, bacteriostatic experiment
The minimal inhibitory concentration of above-mentioned antibacterial peptide analogues, i.e. MIC value are measured using two times of micro-dilution methods of classics.Specifically
Method is: experiment bacterium bacterial strain is inoculated in respectively in MH culture medium and is incubated overnight to logarithmic phase, and is diluted to 1 × 106CFU/
The bacterial suspension of mL concentration；Above-mentioned antibacterial peptide analogues are dissolved in aseptic deionized water, are matched with doubling dilution
It at the peptide solution of 128-1 μm of ol/L various concentration, mixes with above-mentioned bacterial suspension, is incubated for 37 DEG C in 96 well culture plates in equal volume
18h is educated, is observed；Visually the Cmin of visible no bacterial growth is minimal inhibitory concentration MIC；Antibiotic Rifampicin makees
Positive control drug；Above-mentioned experiment is repeated in parallel three times, as a result such as table 1.
Table 1 fights the minimal inhibitory concentration of common bacterial strain
Table 1 the result shows that, the dimerization modified antimicrobial peptide analogues that the present invention designs synthesis have common bacteria good
Antibacterial activity, antibacterial activity is significantly improved compared to female peptide Anoplin, and is better than to the antibacterial activity of part bacterium
Conventional antibiotic.
2, flow cytometry tests
Using standard E. coli (ATCC25922) bacterial strain, it is inoculated in MH culture medium and is incubated overnight to logarithmic phase,
It is diluted to 10 × 108CFU/mL, after PBS (10mM, pH7.4) washing, half volume is resuspended；Above-mentioned antibacterial peptide analogues are dissolved
In PBS, make peptide solution 4 × MIC of concentration, then mixed in equal volume with above-mentioned bacterial suspension, 37 DEG C are incubated for 1h altogether, through iodate
After pyridine (PI) is protected from light dyeing 15min, PBS washing removes residue PI dyestuff, most contaminates afterwards through flow cytomery bacterium PI
Expect the intake ability of fluorescence, and then quantitative analysis antibacterial peptide analogues, to the damage capability of bacterial cell membrane, Rifampicin makees
Positive control, as a result such as Figure 13.
Figure 13 the result shows that, the present invention design synthesis dimerization modified antimicrobial peptide analogues have preferable bacterial cell
Film damage capability, damage capability is suitable with mother peptide Anoplin, and antibiotic Rifampicin is broken without apparent bacterial cell membrane
Bad ability.
3, hemolytic experiment
Fresh healthy mice blood is taken, 1000g is centrifuged 10min (4 DEG C), discards upper serum, and it is blood red thin to retain lower layer
Born of the same parents are configured to the suspension containing 8% haemocyte, are added on 96 orifice plates after cleaning haemocyte with PBS (10mM, pH7.4)
In；Above-mentioned antibacterial peptide analogues are dissolved in PBS, and are configured to the peptide of 512-4 μm of ol/L various concentration with doubling dilution
It is added in 96 orifice plates that haemocyte suspension has been added by solution in equal volume, 37 DEG C of incubation 1h；Object 1200g centrifugation is incubated altogether
After 15min, supernatant is transferred to 96 new orifice plates, microplate reader detects the light absorption value at its 490nm；PBS blank solution makees yin
Property control, 1%TritonX-100 makees positive control, according to formula: Hemolysis rate (%)=[(OD490nm peptides-
OD490nm negative control)/(OD490nm positive control-OD490nm negative control)] × 100%, calculate hemolysis rate, knot
Fruit such as Figure 14.
Figure 14 the result shows that, it is living that the dimerization modified antimicrobial peptide analogues that the present invention designs synthesis all have extremely low haemolysis
Property, hemolysis rate is below 1%, or even in 256 μm of ol/L of highest experimental concentration, still without apparent hemolytic activity, that is, shows
Hypotoxicity out has certain safety.
To sum up, the present invention is to the part natural antibacterial peptide Anoplin D type amino acid substitution analog Anoplin-D4 7,
The end C- of Anoplin-D5,7 and Anoplin-D9,10,3 or the end N- introduce unnatural amino acid Fmoc-L-
Propargylgly-OH or Fmoc-Lys (Mtt)-OH, and acetylation modification is carried out to its end N- respectively, obtain precursor peptide
Ac-D4,7-Pra、Ac-D9,10,3-Pra、Ac-D4,7-Lys(N3)、Ac-D5,7-Lys(N3)、Ac-Lys(N3)-D4,7 and
Ac-Lys(N3)-D5,7；Then pass through 1, the 3- Dipolar Cycloaddition of click chemistry, with " end C-C " and " end C-N " shape
Formula carries out intermolecular side chain connection, obtains a kind of dimerization modified antimicrobial peptide analogues.External activity result of study shows this hair
The dimerization modified antimicrobial peptide analogues of bright design synthesis have strong antibacterial activity and hypotoxicity, in the exploitation of clinical antibacterials
It has a good application prospect.
Detailed description of the invention
Fig. 1 is Ac-D4,7-Pra mass spectrogram；
Fig. 2 is Ac-D4,7-Lys (N3) mass spectrogram；
Fig. 3 is JC-AA- (D4,7+D4,7) mass spectrogram；
Fig. 4 is Ac-D5,7-Lys (N3) mass spectrogram；
Fig. 5 is JC-AA- (D4,7+D5,7) mass spectrogram；
Fig. 6 is Ac-D9,10,3-Pra mass spectrogram；
The position Fig. 7 JC-AA- (D9,10,3+D5,7) mass spectrogram；
Fig. 8 is Ac-Lys (N3)-D4,7 mass spectrogram；
Fig. 9 is JCN-AA- (D4,7+D4,7) mass spectrogram；
Figure 10 is Ac-Lys (N3)-D5,7 mass spectrogram；
Figure 11 is JCN-AA- (D4,7+D5,7) mass spectrogram
Figure 12 is JCN-AA- (D9,10,3+D5,7) mass spectrogram
Figure 13 is antibacterial peptide analogues PI flow cytometry tests result figure；With left-to-right in figure, the sequence of top to bottm according to
Secondary is control group, Anoplin group, JC-AA- (D4,7+D4,7) group, JC-AA- (D4,7+D5,7) group, JC-AA- (D9,10,3+
D5,7) group, JCN-AA- (D4,7+D4,7) group, JCN-AA- (D4,7+D5,7), JCN-AA- (D9,10,3+D5,7) group and
Rifampicin group.
Figure 14 is antibacterial peptide analogues hemolytic experiment result figure.
Specific embodiment
There are below by specific embodiment to the present invention dimerization modified antimicrobial peptides of strong antibacterial activity and hypotoxicity
It is described further like the synthetic method of object.
Embodiment 1: the synthesis of dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D4,7)
(1) synthesis of Ac-D4,7-Pra
A) activation and pretreatment of resin
MBHA resin (0.43mmol/g) 1.16g is accurately weighed in Solid-phase synthesis peptides instrument, is sufficiently swollen through DCM solution
After 30min, ninhydrin is examined, and resin is colorless and transparent, shows that resin is normal, can carry out subsequent experimental.
B) synthesis of Fmoc-Anoplin-D4,7-Pra-resin
Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction to the normal MBHA resin of above-mentioned inspection；It will
Fmoc-L-Propargylgly-OH (503mg), HOBT (204mg), HBTU (569mg), DIEA (0.5mL) dissolve in DMF
It mixes, and the MBHA resin for sloughing Fmoc protecting group with the DMF solution by 20% piperidines of volume fraction carries out condensation reaction
1.5h, ninhydrin are examined, and resin is colorless and transparent, then show condensation reaction success, obtain Fmoc-L-
Propargylgly-resin, i.e. Fmoc-Pra-resin；With method successively condensation reaction subsequent amino-acid Fmoc-Leu-OH
(530mg)、Fmoc-Leu-OH(530mg)、Fmoc-Thr(tBu)-OH(597mg)、Fmoc-D-Lys(Boc)-OH(703mg)、
Fmoc-Ile-OH(530mg)、Fmoc-Arg(pbf)-OH(974mg)、Fmoc-D-Lys(Boc)-OH(703mg)、Fmoc-
Leu-OH (530mg), Fmoc-Leu-OH (530mg), Fmoc-Gly-OH (447mg), reaction time are 1h, HOBT,
HBTU and DIEA dosage is same as above, and obtains Fmoc-Gly-Leu-Leu-D-Lys-Arg-Ile-D-Lys-Thr-Leu-Leu-Pra-
Resin, i.e. Fmoc-Anoplin-D4,7-Pra-resin；
C) synthesis of Ac-D4,7-Pra-resin
Fmoc-Anoplin-D4,7-Pra-resin obtained above is taken off with the DMF solution of 20% piperidines of volume fraction
Fmoc protecting group is gone, acetic anhydride (1.3mL) and DIEA (0.5mL), condensation reaction 1.5h is added, ninhydrin is examined, tree
Rouge is colorless and transparent, then shows condensation reaction success, obtain Ac-D4,7-Pra-resin；
D) polypeptide is cut
Ac-D4,7-Pra-resin are cut, (three is different by TFA, TIS that cutting reagent is volume ratio 95:2.5:2.5
Propyl silane) and H2O mixed solution 15mL, after ether and water extraction, freeze-drying；
E) peptide purification
RP-HPLC separation is first carried out, is then freeze-dried, obtains precursor peptide Ac-D4,7-Pra through Mass Spectrometric Identification, molecular weight is
1290Da, mass spectrogram are shown in Fig. 1；Wherein, RP-HPLC purification condition: mobile phase A: 0.05%TFA/ water；Mobile phase B: 0.05%
TFA/ acetonitrile；Linear gradient elution 30min collects the efflux of major absorbance peak.
(2)Ac-D4,7-Lys(N3) synthesis
A) activation and pretreatment of resin
MBHA resin (0.43mmol/g) 1.16g is accurately weighed in Solid-phase synthesis peptides instrument, is sufficiently swollen through DCM solution
After 30min, ninhydrin is examined, and resin is colorless and transparent, shows that resin is normal, can carry out subsequent experimental；
B) synthesis of Fmoc-Anoplin-D4,7-Lys (Mtt)-resin
Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction to the normal MBHA resin of above-mentioned inspection；It will
Fmoc-Lys (Mtt)-OH (938mg), HOBT (204mg), HBTU (569mg), DIEA (0.5mL) dissolve mixing in DMF, and
The MBHA resin for sloughing Fmoc protecting group with the DMF solution by 20% piperidines of volume fraction carries out condensation reaction 1.5h, indenes three
Ketone development process is examined, and resin is colorless and transparent, then shows condensation reaction success, obtain Fmoc-Lys (Mtt)-resin；Same method
Successively condensation reaction subsequent amino-acid Fmoc-Leu-OH (530mg), Fmoc-Leu-OH (530mg), Fmoc-Thr (tBu)-OH
(597mg)、Fmoc-D-Lys(Boc)-OH(703mg)、Fmoc-Ile-OH(530mg)、Fmoc-Arg(pbf)-OH(974mg)、
Fmoc-D-Lys(Boc)-OH(703mg)、Fmoc-Leu-OH(530mg)、Fmoc-Leu-OH(530mg)、Fmoc-Gly-OH
(447mg), reaction time are 1h, and HOBT, HBTU and DIEA dosage are same as above, and obtain Fmoc-Gly-Leu-Leu-D-Lys-
Arg-Ile-D-Lys-Thr-Leu-Leu-Lys (Mtt)-resin, i.e. Fmoc-Anoplin-D4,7-Lys (Mtt)-resin；
C) synthesis of Ac-D4,7-Lys (Mtt)-resin
Fmoc-Anoplin-D4,7-Lys obtained above (Mtt)-resin is molten with the DMF of 20% piperidines of volume fraction
Liquid sloughs Fmoc protecting group, and acetic anhydride (1.3mL) and DIEA (0.5mL), condensation reaction 1.5h, ninhydrin inspection is added
It tests, resin is colorless and transparent, then shows to be condensed successfully, obtains Ac-D4,7-Lys (Mtt)-resin；
d)Ac-D4,7-Lys(N3)-resin synthesis
By Ac-D4 obtained above, 7-Lys (Mtt)-resin sloughs side chain Mtt protecting group, and carries out side chain Azide
Modification.The wherein method of side chain Azide are as follows: by 6.5g NaN3(sodium azide) and 2.86mL Tf2O (trifluoromethanesulfanhydride anhydride) exists
17mL H22h is reacted at room temperature in the mixed liquor of O and 33mL DCM, then through DCM, NaCO3And Na2SO4Moisture is removed, Tf is obtained2N3
(trifluoro nitrine) solution；Ac-D4,7-Lys (Mtt)-resin slough Mtt protecting group, resin with the DCM solution containing 1%TFA
Through DCM expansion and CH3OH compression, after vacuum is drained, with 25mg CuSO4、25mg K2CO3、7mL CH3OH mixing, then with it is above-mentioned
Obtained Tf2N3Solution reacts at room temperature 48h, obtains side chain azido compound Ac-D4,7-Lys (N3)-resin。
E) polypeptide is cut
By Ac-D4,7-Lys (N3)-resin cut, TFA, TIS that cutting reagent is volume ratio 95:2.5:2.5 with
H2O mixed solution 15mL, after ether and water extraction, freeze-drying；
F) peptide purification
RP-HPLC separation is first carried out, is then freeze-dried, obtains precursor peptide Ac-D4,7-Lys (N through Mass Spectrometric Identification3)-
Resin, molecular weight 1349Da, mass spectrogram are shown in Fig. 2；Wherein, RP-HPLC purification condition: mobile phase A: 0.05%TFA/ water；Stream
Dynamic phase B:0.05%TFA/ acetonitrile；Linear gradient elution 30min collects the efflux of major absorbance peak.
(3) synthesis of JC-AA- (D4,7+D4,7)
With precursor peptide Ac-D4,7-Lys (N3) on the basis of, by 13.51mgAc-D4,7-Pra and 12.65mgAc-D4,7-
Lys(N3) be dissolved in 2616 μ L aqueous solutions, make peptide concentration 10mg/mL, it is water-soluble that 1171 copper sulphate of the μ L containing 5%DMF are added
Liquid, 37.15mg sodium ascorbate make antioxidant, and argon gas protection, room temperature is protected from light 26h, purifies through RP-HPLC, final to pass through
Mass Spectrometric Identification obtains dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D4,7), and molecular weight is respectively 2639Da, mass spectrogram
See Fig. 3.
Embodiment 2: the synthesis of dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D5,7)
Precursor peptide Ac-D5,7-Lys (N3) synthesis: Fmoc-Lys (Mtt)-OH (938mg), HOBT (204mg), HBTU
(569mg), DIEA (0.5mL) dissolve mixing in DMF, and slough Fmoc with by the DMF solution of 20% piperidines of volume fraction
The MBHA resin of protecting group carries out condensation reaction, obtains Fmoc-Lys (Mtt)-resin, then condensation reaction subsequent amino-acid:
Fmoc-Leu-OH(530mg)、Fmoc-Leu-OH(530mg)、Fmoc-Thr(tBu)-OH(597mg)、Fmoc-D-Lys
(Boc)-OH(703mg)、Fmoc-Ile-OH(530mg)、Fmoc-D-Arg(pbf)-OH(974mg)、Fmoc-Lys(Boc)-OH
(703mg),Fmoc-Leu-OH(530mg),Fmoc-Leu-OH(530mg),Fmoc-Gly-OH(447mg)；
Other processes are the same as embodiment 1.Through Mass Spectrometric Identification, precursor peptide Ac-D4,7-Pra are respectively obtained, molecular weight is
1290Da, mass spectrogram are shown in Fig. 1；Precursor peptide Ac-D5,7-Lys (N3), molecular weight 1349Da, mass spectrogram is shown in Fig. 4；Dimerization is repaired
It adorns antibacterial peptide analogues JC-AA- (D4,7+D5,7), molecular weight is respectively 2639Da, and mass spectrogram is shown in Fig. 5.
Embodiment 3: the synthesis of dimerization modified antimicrobial peptide analogues JC-AA- (D9,10,3+D5,7)
The synthesis of precursor peptide Ac-D9,10,3-Pra: Fmoc-L-Propargylgly-OH (503mg), HOBT (204mg),
HBTU (569mg), DIEA (0.5mL) dissolve mixing in DMF, and slough with by the DMF solution of 20% piperidines of volume fraction
The MBHA resin of Fmoc protecting group carries out condensation reaction, obtains Fmoc-Pra-resin, then condensation reaction subsequent amino-acid:
Fmoc-D-Leu-OH(530mg)、Fmoc-D-Leu-OH(530mg)、Fmoc-Thr(tBu)-OH(597mg)、Fmoc-Lys
(Boc)-OH(703mg)、Fmoc-Ile-OH(530mg)、Fmoc-Arg(pbf)-OH(974mg)、Fmoc-Lys(Boc)-OH
(703mg),Fmoc-D-Leu-OH(530mg),Fmoc-Leu-OH(530mg),Fmoc-Gly-OH(447mg)；
Precursor peptide Ac-D5,7-Lys (N3) synthesis with embodiment 2；
Other processes are the same as embodiment 1.Through Mass Spectrometric Identification, precursor peptide Ac-D9 is respectively obtained, 10,3-Pra, molecular weight is
1290Da, mass spectrogram are shown in Fig. 6；Precursor peptide Ac-D5,7-Lys (N3), molecular weight 1349Da, mass spectrogram is shown in Fig. 4；Dimerization is repaired
It adorns antibacterial peptide analogues JC-AA- (D9,10,3+D5,7), molecular weight is respectively 2639Da, and mass spectrogram is shown in Fig. 7.
Embodiment 4: the synthesis of dimerization modified antimicrobial peptide analogues JCN-AA- (D4,7+D4,7)
Precursor peptide Ac-Lys (N3)-D4,7 synthesis: sloughing the MBHA resin of Fmoc protecting group, and first condensation reaction obtains
Fmoc-Leu-resin；With method successively condensation reaction subsequent amino-acid Fmoc-Leu-OH (530mg), Fmoc-Thr (tBu)-OH
(597mg)、Fmoc-D-Lys(Boc)-OH(703mg)、Fmoc-Ile-OH(530mg)、Fmoc-Arg(pbf)-OH(974mg)、
Fmoc-D-Lys(Boc)-OH(703mg)、Fmoc-Leu-OH(530mg)、Fmoc-Leu-OH(530mg)、Fmoc-Gly-OH
(447mg),Fmoc-Lys(Mtt)-OH(938mg)；
Other processes are the same as embodiment 1.Through Mass Spectrometric Identification, precursor peptide Ac-D4,7-Pra are respectively obtained, molecular weight is
1290Da, mass spectrogram are shown in Fig. 1；Precursor peptide Ac-Lys (N3)-D4,7, molecular weight 1349Da, mass spectrogram is shown in Fig. 8；Dimerization is repaired
It adorns antibacterial peptide analogues JCN-AA- (D4,7+D4,7), molecular weight is respectively 2639Da, and mass spectrogram is shown in Fig. 9.
Embodiment 5: the synthesis of dimerization modified antimicrobial peptide analogues JCN-AA- (D4,7+D5,7)
Precursor peptide Ac-Lys (N3)-D5,7 synthesis: sloughing fmoc-protected MBHA resin, and first condensation reaction obtains
Fmoc-Leu-resin；With method successively condensation reaction subsequent amino-acid: Fmoc-Leu-OH (530mg), Fmoc-Thr (tBu)-OH
(597mg)、Fmoc-D-Lys(Boc)-OH(703mg)、Fmoc-Ile-OH(530mg)、Fmoc-D-Arg(pbf)-OH
(974mg)、Fmoc-Lys(Boc)-OH(703mg)、Fmoc-Leu-OH(530mg)、Fmoc-Leu-OH(530mg)、Fmoc-
Gly-OH(447mg),Fmoc-Lys(Mtt)-OH(938mg)；
Other processes are the same as embodiment 1.Through Mass Spectrometric Identification, precursor peptide Ac-D4,7-Pra are respectively obtained, molecular weight is
1290Da, mass spectrogram are shown in Fig. 1；Precursor peptide Ac-Lys (N3)-D5,7, molecular weight 1349Da, mass spectrogram is shown in Figure 10；Dimerization is repaired
It adorns antibacterial peptide analogues JCN-AA- (D4,7+D5,7), molecular weight is respectively 2639Da, and mass spectrogram is shown in Figure 11.
Embodiment 6: the synthesis of dimerization modified antimicrobial peptide analogues JCN-AA- (D9,10,3+D5,7)
The synthesis of precursor peptide Ac-D9,10,3-Pra is the same as embodiment 3；Precursor peptide Ac-Lys (N3)-D5,7 synthesis it is same
Embodiment 5；
Other processes are the same as embodiment 1.Through Mass Spectrometric Identification, precursor peptide Ac-D9 is respectively obtained, 10,3-Pra, molecular weight is
1290Da, mass spectrogram are shown in Fig. 6；Precursor peptide Ac-Lys (N3)-D5,7, molecular weight 1349Da, mass spectrogram is shown in Figure 10；Dimerization is repaired
It adorns antibacterial peptide analogues JCN-AA- (D9,10,3+D5,7), molecular weight is respectively 2639Da, and mass spectrogram is shown in Figure 13.
Claims (10)
1. the dimerization modified antimicrobial peptide analogues with strong antibacterial activity and hypotoxicity, characterized in that the antibacterial peptide analogues
Including " end C-C " dimerization modified antimicrobial peptide analogues and " end C-N " dimerization modified antimicrobial peptide analogues, wherein " C-
C-terminal " dimerization modified antimicrobial peptide analogues include with flowering structure:
(1)JC-AA-(D4,7+D4,7)
(2)JC-AA-(D4,7+D5,7)
(3)JC-AA-(D9,10,3+D5,7)；
" end C-N " dimerization modified antimicrobial peptide analogues include with flowering structure:
(4)JCN-AA-(D4,7+D4,7)
(5)JCN-AA-(D4,7+D5,7)
(6)JCN-AA-(D9,10,3+D5,7)。
2. the dimerization modified antimicrobial peptide analogues as described in claim 1 with strong antibacterial activity and hypotoxicity are anti-in clinic
Application in bacterium drug development.
3. the synthesis side of the dimerization modified antimicrobial peptide analogues as described in claim 1 with strong antibacterial activity and hypotoxicity
Method, it is characterized in that:
(1) synthesis of " end C-C " dimerization modified antimicrobial peptide analogues
A, unnatural amino acid Fmoc-L- is introduced in the end C- of Anoplin-D4,7 and Anoplin-D9,10,3 sequence respectively
Propargylgly-OH, obtains Anoplin-D4,7-Pra and Anoplin-D9, and 10,3-Pra；Respectively with Anoplin-D4,7-
Pra and Anoplin-D9,10,3-Pra be matrix, synthesizes Anoplin-D4,7-Pra-resin using classical solid phase synthesis process
With Anoplin-D9,10,3-Pra-resin, then respectively carry out the terminated acetylated modification of N-, obtain Ac-D4,7-Pra-
Resin and Ac-D9,10,3-Pra-resin, respectively through cutting, obtain precursor peptide Ac-D4,7-Pra and Ac-D9 after purification, 10,
3-Pra；
B, respectively in Anoplin-D4, the end C- of 7, Anoplin-D5,7 sequences introduces unnatural amino acid Fmoc-Lys
(Mtt)-OH obtains Anoplin-D4,7-Lys (Mtt) and Anoplin-D5,7-Lys (Mtt)；With Anoplin-D4,7-Lys
(Mtt) and Anoplin-D5,7-Lys (Mtt) are matrix, synthesize Anoplin-D4,7-Lys using classical solid phase synthesis process
(Mtt) then-resin and Anoplin-D5,7-Lys (Mtt)-resin carries out the terminated acetylated modification of N- respectively, obtains Ac-
D4,7-Lys (Mtt)-resin and Ac-D5,7-Lys (Mtt)-resin；Respectively to Ac-D4,7-Lys (Mtt)-resin and Ac-
D5,7-Lys (Mtt)-resin carry out the modification of side chain Azide, and cutting obtains precursor peptide Ac-D4,7-Lys (N after purification3) with
Ac-D5,7-Lys(N3)；
C, using 1, the 3- Dipolar Cycloaddition of click chemistry, make precursor peptide Ac-D4, " alkynyl " functional group of 7-Pra side chain
Respectively with precursor peptide Ac-D4,7-Lys (N3) or Ac-D5,7-Lys (N3) side chain " azido " functional group reactions, before two
Body peptide carries out intermolecular side chain connection, obtain " end C-C " dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D4,7) and
JC-AA-(D4,7+D5,7)；Using 1, the 3- Dipolar Cycloaddition of click chemistry, make precursor peptide Ac-D9,10,3-Pra side chains
" alkynyl " functional group and precursor peptide Ac-D5,7-Lys (N3) side chain " azido " functional group reactions, by two precursor peptides into
The intermolecular side chain connection of row, obtains " end C-C " dimerization modified antimicrobial peptide analogues JC-AA- (D9,10,3+D5,7)；
(2) synthesis of " end C-N " dimerization modification dimer antibacterial peptide analogues
A, precursor peptide Ac- is obtained using method in the synthesis step a of such as (1) " end C-C " dimerization modified antimicrobial peptide analogues
D4,7-Pra and Ac-D9,10,3-Pra；
B, respectively in Anoplin-D4, the end N- of 7, Anoplin-D5,7 sequences introduces unnatural amino acid Fmoc-Lys
(Mtt)-OH obtains Anoplin-Lys (Mtt)-D4,7 and Anoplin-Lys (Mtt)-D5,7；With Anoplin-Lys (Mtt)-
D4,7 and Anoplin-Lys (Mtt)-D5,7 be matrix, synthesizes Anoplin-Lys (Mtt)-using classical solid phase synthesis process
Then D4,7-resin and Anoplin-Lys (Mtt)-D5,7-resin carries out the terminated acetylated modification of N- respectively, obtains Ac-
Lys (Mtt)-D4,7-resin and Ac-Lys (Mtt)-D5,7-resin；Respectively to Ac-Lys (Mtt)-D4,7-resin and Ac-
Lys (Mtt)-D5,7-resin carry out the modification of side chain Azide, and cutting, purifying obtain precursor peptide Ac-Lys (N3)-D4,7 and Ac-
Lys(N3)-D5,7；
C, using 1, the 3- Dipolar Cycloaddition of click chemistry, make precursor peptide Ac-D4, " alkynyl " functional group of 7-Pra side chain
Respectively with precursor peptide Ac-Lys (N3)-D4,7 or Ac-Lys (N3)-D5, " azido " functional group reactions of 7 side chains, before two
Body peptide carries out intermolecular side chain connection, obtains " end C-N " dimerization modified antimicrobial peptide analogues JCN-AA- (D4,7+D4,7)
With JCN-AA- (D4,7+D5,7)；Using 1, the 3- Dipolar Cycloaddition of click chemistry, make precursor peptide Ac-D9,10,3-Pra
" alkynyl " functional group of side chain and precursor peptide Ac-Lys (N3)-D5, " azido " functional group reactions of 7 side chains, by two precursors
Peptide carries out intermolecular side chain connection, obtain " end C-N " dimerization modified antimicrobial peptide analogues JCN-AA- (D9,10,3+D5,
7)。
4. the synthesis side of the dimerization modified antimicrobial peptide analogues as claimed in claim 3 with strong antibacterial activity and hypotoxicity
Method, characterized in that precursor peptide Ac-D4,7-Pra and the Ac-D9,10,3-Pra synthetic method specifically:
(1) synthesis of Ac-D4,7-Pra
Fmoc-L-Propargylgly-OH, HOBT, HBTU, DIEA dissolve to mixing in DMF, and with slough Fmoc protecting group
MBHA resin carry out condensation reaction, obtain Fmoc-L-Propargylgly-resin, i.e. Fmoc-Pra-resin；With method according to
Secondary condensation reaction subsequent amino-acid Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Thr (tBu)-OH, Fmoc-D-Lys (Boc)-
OH、Fmoc-Ile-OH、Fmoc-Arg(pbf)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Leu-OH、
Fmoc-Gly-OH obtains Fmoc-Gly-Leu-Leu-D-Lys-Arg-Ile-D-Lys-Thr-Leu-Leu-Pra-res in, i.e.,
Fmoc-Anoplin-D4,7-Pra-resin；Then end Fmoc protection is sloughed with the DMF solution of 20% piperidines of volume fraction
Base obtains Anoplin-D4,7-Pra-resin；Acetic anhydride, DIEA are dissolved to mixing, and and Anoplin-D4,7- in DMF
Pra-resin carries out condensation reaction, obtains Ac-D4,7-Pra-resin, and cutting, purifying obtain precursor peptide Ac-D4,7-Pra；
(2) synthesis of Ac-D9,10,3-Pra
Fmoc-L-Propargylgly-OH, HOBT, HBTU, DIEA dissolve to mixing in DMF, and with slough Fmoc protecting group
MBHA resin carry out condensation reaction, obtain Fmoc-L-Propargylgly-resin, i.e. Fmoc-Pra-resin；With method according to
Secondary condensation reaction subsequent amino-acid Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Thr (tBu)-OH, Fmoc-Lys
(Boc)-OH、Fmoc-Ile-OH、Fmoc-Arg(pbf)-OH、Fmoc-Lys(Boc)-OH、Fmoc-D-Leu-OH、Fmoc-
Leu-OH, Fmoc-Gly-OH obtain Fmoc-Gly-Leu-D-Leu-Lys-Arg-Ile-Lys-Thr-D-Leu-D-Leu-
Pra-resin, i.e. Fmoc-Anoplin-D9,10,3-Pra-resin, it is then de- with the DMF solution of 20% piperidines of volume fraction
End Fmoc protecting group is gone, obtains Anoplin-D9,10,3-Pra-resin；Acetic anhydride, DIEA are dissolved to mixing in DMF,
And and Anoplin-D9,10,3-Pra-resin carry out condensation reactions, obtain Ac-D9,10,3-Pra-resin, and cutting, purifying
Obtain precursor peptide Ac-D9,10,3-Pra.
5. the synthetic method of the dimerization modified antimicrobial peptide analogues with strong antibacterial activity and hypotoxicity, characterized in that utilize
Method as claimed in claim 4 synthesize precursor peptide Ac-D4,7-Pra and Ac-D9,10,3-Pra, the dimerization modified antimicrobial
The synthetic method of peptide analogues specifically:
(1) synthesis of " end C-C " dimerization modified antimicrobial peptide analogues
a、Ac-D4,7-Lys(N3) synthesis
Fmoc-Lys (Mtt)-OH, HOBT, HBTU, DIEA are dissolved into mixing in DMF, and with the MBHA that sloughs Fmoc protecting group
Resin carries out condensation reaction, obtains Fmoc-Lys (Mtt)-resin；With method successively condensation reaction amino acid Fmoc-Leu-OH,
Fmoc-Leu-OH、Fmoc-Thr(tBu)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Ile-OH、Fmoc-Arg(pbf)-OH、
Fmoc-D-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, obtain Fmoc-Gly-Leu-Leu-
D-Lys-Arg-Ile-D-Lys-Thr-Leu-Leu-Lys (Mtt)-resin is Fmoc-D4,7-Lys (Mtt)-resin；So
End Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction afterwards, obtains Anoplin-D4,7-Lys (Mtt)-
resin；Acetic anhydride, DIEA dissolve to mixing in DMF, and and Anoplin-D4,7-Lys (Mtt)-resin be condensed it is anti-
It answers, obtains Ac-D4,7-Lys (Mtt)-resin；Ac-D4,7-Lys (Mtt)-resin is molten with the DCM of volume fraction 1%TFA
Liquid sloughs side chain Mtt protecting group, and carries out side chain Azide modification, obtains side chain azido compound Ac-D4,7-Lys (N3)-
Resin, cutting, purifying obtain precursor peptide Ac-D4,7-Lys (N3)；
b、Ac-D5,7-Lys(N3) synthesis
Fmoc-Lys (Mtt)-OH, HOBT, HBTU, DIEA are dissolved into mixing in DMF, and with the MBHA that sloughs Fmoc protecting group
Resin carries out condensation reaction, obtains Fmoc-Lys (Mtt)-resin；With method successively condensation reaction amino acid Fmoc-Leu-OH,
Fmoc-Leu-OH、Fmoc-Thr(tBu)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Ile-OH、Fmoc-D-Arg(pbf)-
OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, obtain Fmoc-Gly-Leu-Leu-
Lys-D-Arg-Ile-D-Lys-Thr-Leu-Leu-Lys (Mtt)-resin is Fmoc-D5,7-Lys (Mtt)-resin；So
End Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction afterwards, obtains Anoplin-D5,7-Lys (Mtt)-
resin；Acetic anhydride, DIEA dissolve to mixing in DMF, and and Anoplin-D5,7-Lys (Mtt)-resin be condensed it is anti-
It answers, obtains Ac-D5,7-Lys (Mtt)-resin；Ac-D5,7-Lys (Mtt)-resin is de- with the DCM solution containing 1%TFA
Side chain Mtt protecting group is gone, and carries out side chain Azide modification, obtains side chain azido compound Ac-D5,7-Lys (N3)-resin,
Cutting, purifying obtain precursor peptide Ac-D5,7-Lys (N3)；
C, dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D4,7), JC-AA- (D4,7+D5,7), JC-AA- (D9,10,
Synthesis 3+D5,7)
By precursor peptide Ac-D4,7-Pra respectively with Ac-D4,7-Lys (N3) or Ac-D5,7-Lys (N3) it is dissolved in H2In O, it is added
CuSO containing 5%DMF4Solution makees antioxidant, argon gas protection with sodium ascorbate, and room temperature is protected from light 24-28h；Through RP-
HPLC purifying, obtains dimerization modified antimicrobial peptide analogues JC-AA- (D4,7+D4,7), JC-AA- (D4,7+D5,7)；
By precursor peptide Ac-D9,10,3-Pra and Ac-D5,7-Lys (N3) it is dissolved in H2In O, the CuSO containing 5%DMF is added4It is molten
Liquid makees antioxidant, argon gas protection with sodium ascorbate, and room temperature is protected from light 24-28h；It is purified through RP-HPLC, obtains dimerization
Change modified antimicrobial peptide analogues JC-AA- (D9,10,3+D5,7).
(2) synthesis of " end C-N " dimerization modified antimicrobial peptide analogues
a、Ac-Lys(N3)-D4,7 synthesis
Fmoc-Leu-OH, HOBT, HBTU, DIEA dissolve to mixing in DMF, and with the MBHA resin of sloughing Fmoc protecting group
Condensation reaction is carried out, Fmoc-Leu-resin is obtained；With method successively condensation reaction amino acid Fmoc-Leu-OH, Fmoc-Thr
(tBu)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Ile-OH、Fmoc-Arg(pbf)-OH、Fmoc-D-Lys(Boc)-OH、
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys (Mtt)-OH obtains Fmoc-Lys (Mtt)-Gly-
Leu-Leu-D-Lys-Arg-Ile-D-Lys-Thr-Leu-Leu-resin, i.e. Fmoc-Lys (Mtt)-D4,7-resin；Then
End Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction, obtains Anoplin-Lys (Mtt)-D4,7-
resin；Acetic anhydride, DIEA are dissolved to mixing in DMF, and be condensed instead with Anoplin-Lys (Mtt)-D4,7-resin
It answers, obtains Ac-Lys (Mtt)-D4,7-resin；Ac-Lys (Mtt)-D4,7-resin is molten with the DCM of volume fraction 1%TFA
Liquid sloughs side chain Mtt protecting group, and carries out side chain Azide modification, obtains side chain azido compound Ac-Lys (N3)-D4,7-
Resin, cutting, purifying obtain precursor peptide Ac-Lys (N3)-D4,7；
b、Ac-Lys(N3)-D5,7 synthesis
Fmoc-Leu-OH, HOBT, HBTU, DIEA dissolve to mixing in DMF, and with the MBHA resin of sloughing Fmoc protecting group
Condensation reaction is carried out, Fmoc-Leu-resin is obtained；With method successively condensation reaction amino acid Fmoc-Leu-OH, Fmoc-Thr
(tBu)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Ile-OH、Fmoc-D-Arg(pbf)-OH、Fmoc-Lys(Boc)-OH、
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys (Mtt)-OH obtains Fmoc-Lys (Mtt)-Gly-
Leu-Leu-Lys-D-Arg-Ile-D-Lys-Thr-Leu-Leu-resin is Fmoc-Lys (Mtt)-D5,7-resin；So
End Fmoc protecting group is sloughed with the DMF solution of 20% piperidines of volume fraction afterwards, obtains Anoplin-Lys (Mtt)-D5,7-
resin；Acetic anhydride, DIEA are dissolved to mixing in DMF, and be condensed instead with Anoplin-Lys (Mtt)-D5,7-resin
It answers, obtains Ac-Lys (Mtt)-D5,7-resin；Ac-Lys (Mtt)-D5,7-resin is molten with the DCM of volume fraction 1%TFA
Liquid sloughs side chain Mtt protecting group, and carries out side chain Azide modification, obtains side chain azido compound Ac-Lys (N3)-D5,7-
Resin, cutting, purifying obtain precursor peptide Ac-Lys (N3)-D5,7；
C, dimerization modified antimicrobial peptide analogues JCN-AA- (D4,7+D4,7), JCN-AA- (D4,7+D5,7), JCN-AA- (D9,
Synthesis 10,3+D5,7)
By precursor peptide Ac-D4,7-Pra respectively with Ac-Lys (N3)-D4,7 or Ac-Lys (N3)-D5,7 is dissolved in H2In O, it is added
CuSO containing 5%DMF4Solution makees antioxidant, argon gas protection with sodium ascorbate, and room temperature is protected from light 24-28h；Through RP-
HPLC purifying, obtains dimerization modified antimicrobial peptide analogues JCN-AA- (D4,7+D4,7), JCN-AA- (D4,7+D5,7)；
By precursor peptide Ac-D9,10,3-Pra and Ac-Lys (N3)-D5,7 is dissolved in H2In O, the CuSO containing 5%DMF is added4It is molten
Liquid makees antioxidant, argon gas protection with sodium ascorbate, and room temperature is protected from light 24-28h；It is purified through RP-HPLC, obtains dimerization
Change modified antimicrobial peptide analogues JCN-AA- (D9,10,3+D5,7).
6. the conjunction of the dimerization modified antimicrobial peptide analogues as described in claim 4 or 5 with strong antibacterial activity and hypotoxicity
At method, it is characterized in that:
(1) each amino acid, HOBT, HBTU and to slough the molal weight ratio of the MBHA resin of Fmoc protecting group be 3:1-4:
1, acetic anhydride and the molal weight ratio for sloughing the MBHA resin of Fmoc protecting group are 12:1-26:1, and DIEA is protected with Fmoc is sloughed
The molal weight ratio of the MBHA resin of base is 6:1；
(2) the side chain azide method are as follows: first by NaN3With Tf2O is in H22-2.5h is reacted at room temperature in O/DCM mixed liquor, is reacted
Liquid is through DCM, Na2CO3And Na2SO4Moisture is removed, Tf is obtained2N3Solution；Mtt protecting group is sloughed with the DCM solution containing 1%TFA
Resin through DCM expand and CH3OH compression, after vacuum is drained, with CuSO4、K2CO3And CH3OH mixing, then with it is obtained above
Tf2N3Solution reacts at room temperature 46-48h, obtains side chain azido compound, and cutting, purifying obtain side chain Azide precursor peptide；Wherein
H2The volume ratio of O and DCM is 1:2, NaN3、Tf2The molal weight ratio of O and the MBHA resin for sloughing Mtt protecting group are respectively 100:
1-200:1,30:1-50:1, CuSO4、K2CO3It is respectively 0.1:1- with the MBHA resin molal weight ratio for sloughing Mtt protecting group
0.3:1、0.2:1-0.5:1。
7. the conjunction of the dimerization modified antimicrobial peptide analogues as described in claim 4 or 5 with strong antibacterial activity and hypotoxicity
At method, characterized in that precursor peptide Ac-D4,7-Pra or the Ac-D9,10,3-Pra and side chain Azide precursor peptide Ac-D4,
7-Lys(N3) or Ac-D5,7-Lys (N3) or Ac-Lys (N3)-D4,7 or Ac-Lys (N3) the molal weight ratio of-D5,7 is 1:
1-1.3:1, the concentration of precursor peptide in water are 8-10mg/mL, CuSO4, sodium ascorbate and side chain Azide precursor peptide
Molal weight is than being respectively 3:1-8:1,15:1-20:1.
8. such as the described in any item dimerization modified antimicrobial peptide analogues with strong antibacterial activity and hypotoxicity of claim 3-5
Synthetic method, characterized in that the cutting reagent is TFA, TIS and H that volume ratio is 95:2.5:2.52The mixed solution of O.
9. such as the described in any item dimerization modified antimicrobial peptide analogues with strong antibacterial activity and hypotoxicity of claim 3-5
Synthetic method, characterized in that the purification process is, first carries out RP-HPLC separation, is then freeze-dried；RP-HPLC purifying
Condition is mobile phase A: the aqueous solution of 0.05%TFA, Mobile phase B: the acetonitrile solution of 0.05%TFA；Linear gradient elution
30min collects the efflux of major absorbance peak.
10. having strong antibacterial activity similar with the dimerization modified antimicrobial peptide of hypotoxicity as claim 3-5 is described in any item
The synthetic method of object, characterized in that described to slough reagent used by MBHA resin Fmoc protecting group as 20% piperazine of volume fraction
The DMF solution of pyridine.
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