Antibacterial peptide dimer analogues containing D type amino acids as well as synthesis and application of dimer analogues

A peptide dimer and amino acid technology, applied in the field of biochemistry, can solve the problems of reduced antibacterial activity and no improvement of antibacterial activity

Active Publication Date: 2017-09-05
倪京满
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the synthesized hydrophilic D-peptide analogs showed strong ability to inhibit bacterial biofilm formation and trypsin resistance, their antibacterial activity was not improved, and the antibacterial activity of the original parent peptide was retained. ; The stability and resistance to trypsin of the synthesized D-type replacement analogue of the hydrophobic surface were significantly improved, but its antibacterial activity was lower than that of the parent peptide

Method used

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  • Antibacterial peptide dimer analogues containing D type amino acids as well as synthesis and application of dimer analogues
  • Antibacterial peptide dimer analogues containing D type amino acids as well as synthesis and application of dimer analogues
  • Antibacterial peptide dimer analogues containing D type amino acids as well as synthesis and application of dimer analogues

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the synthesis of analogue J-AA-(D4,7) 2

[0044] Weigh Rink-MBHA resin, the substitution value is 0.43mmol / g, and the amount of peptide is 0.15mmol, add redistilled dichloromethane solution, fully stir and swell the resin for 30min, then add 10% hexahydropyridine to remove the resin Fmoc protection, wash 2 minutes each time, wash 4 times in total, then wash the resin with redistilled DMF solution, the same 2 minutes each time, wash 4 times in total, and finally use indene detection indicator (phenol: potassium cyanide pyridine: ninhydrin = 1:2:1) To check whether the resin has removed the Fmoc protecting group, boil the resin for 1 minute, observe immediately, and the resin turns blue, which proves that the Fmoc group has been removed. According to the sequence of the peptide, connect the amino acid from the C-terminus, Prepared by RP-HPLC and purified by C18 column, the elution condition is 20%~60% acetonitrile, 8ml / min, and the precursor peptides Ac-Pra-...

Embodiment 2

[0045] Embodiment 2, the synthesis of analogue J-AA-(D4,7+D5,7)

[0046] Weigh Rink-MBHA resin, the substitution value is 0.43mmol / g, and the amount of peptide is 0.25mmol, add redistilled dichloromethane solution, fully stir and swell the resin for 30min, then add 15% hexahydropyridine to remove the resin Fmoc protection, wash for 2 minutes each time, wash 4 times in total, then wash the resin with redistilled DMF solution, the same 2 minutes each time, wash 4 times in total, and then detect the indicator (phenol: potassium cyanide pyridine: ninhydrin = 1 :2:1) test, the resin is blue, which proves that the Fmoc group on the resin has been removed. According to the sequence of the peptide, amino acids are connected from the C-terminus, prepared by RP-HPLC, and purified using a C18 column. The elution condition is: 20%-60% acetonitrile, 8ml / min purification, to get the precursor peptide Ac-Pra-D4,7 and Ac-Lys(N3)-D5,7. The obtained precursor peptides were subjected to click c...

Embodiment 3

[0047] Embodiment 3, the synthesis of analogue J-AA-(D9,10,3+D5,7)

[0048] Weigh the Rink-MBHA resin, the substitution value is 0.43mmol / g, the amount of peptide synthesis is 0.2mmol, add redistilled dichloromethane solution, fully stir and swell the resin for 30min, then add 15% hexahydropyridine to remove the resin Fmoc protection, wash 2 minutes each time, wash 4 times in total, then wash the resin with redistilled DMF solution, the same 2 minutes each time, wash 4 times in total, and then detect the indicator (phenol: potassium cyanide pyridine: ninhydrin = 1 :2:1) test, the resin is blue, which proves that the Fmoc group has been removed. According to the sequence of the peptide, amino acids are connected from the C-terminus, prepared by RP-HPLC, and purified using a C18 column, and the elution condition is 20%- 60% acetonitrile, 8ml / min purification, to obtain the precursor peptide Ac-Pra-D9,10,3 and Ac-Lys(N3)-D5,7. The obtained precursor peptides were subjected to cl...

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Abstract

The invention discloses antibacterial peptide dimer analogues containing D type amino acids. The dimer analogues are prepared by respectively performing N-terminal side chain polymerization on D type amino acid-containing precursor peptides of a naturally antibacterial peptide Anoplin. According to determination of the lowest minimum inhibitory concentration of common standard microorganisms, a biofilm formation test is inhibited. An enzymatic stability test shows that the antibacterial activities of the antibacterial peptide dimer analogues containing D type amino acids synthesized according to the invention are enhanced by 4-16 times compared with those of parent peptides, and the dimer analogues have strong abilities of inhibiting bacterial biofilm formation. In addition, the stabilities of the antibacterial peptide dimer analogues containing D type amino acids synthesized according to the invention are obviously improved. Compared with the parent peptide Anoplin, the tolerance to trypsin enzymolysis is improved by 104-105 times, and the tolerance to chymotrypsin is improved by 102 times. Therefore, the antibacterial peptide dimer analogues containing D type amino acids have excellent application prospects in preparation of long-lasting clinical antibacterial drugs.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and relates to a synthesis method of a kind of antibacterial peptide dimer analog containing D-type amino acid with strong antibacterial activity and resistance to enzymatic degradation, and is mainly used for preparing long-acting clinical antibacterial drugs. Background technique [0002] In recent years, the problem of drug resistance caused by the abuse of antibiotics has become increasingly serious, posing a huge threat to human diseases. It is imminent to find new drugs that can replace antibiotics. Antimicrobial peptides are a class of small molecular peptides with antibacterial activity induced by organisms. They come from a wide range of sources, and their molecular weight is small, about 3-6 kD. They are heat-resistant, acid-resistant, and water-soluble. Well, rapid bactericidal ability and other characteristics (Hancock REW, Scott MG. ProcNatlAcadSci USA, 2000, 97 (16): 8856~8861...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/107A61K38/08A61P31/04
CPCA61K38/00C07K7/06
Inventor 倪京满刘倍均王一杰王锐
Owner 倪京满
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