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Preparation method of beta-defensin 2 antibacterial peptide

A technology of antimicrobial peptides and defensins, which is applied in the field of preparation of β-defensin 2 antimicrobial peptides, can solve the problems of difficult production, many side reactions, and increase the difficulty of production, so as to reduce the generation of impurities and reduce the discharge of "three wastes" , highly operable effect

Pending Publication Date: 2022-02-15
HANGZHOU GOTOP BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] But at the same time, because there are three pairs of disulfide bonds in the structural formula, it inevitably increases the difficulty of production, especially in the operation of disulfide bond ring formation, the impurities produced by oxidation or synthesis will have a considerable impact, so The present invention is a new process designed based on overcoming the difficulty in producing the above-mentioned β-defensin 2 antimicrobial peptides and many side reactions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Method for preparing β-defensin 2 antibacterial peptide

[0033] Specific steps are as follows:

[0034] S1, respectively, for the replacement of 0.25 mmol / g, 0.35 mmol / g, 0.45 mmol / g and 0.55 mmol / g of FMoc-Pro-2-Chlorotrityl Chloride Resin resin each each was 100g, 71.43 g, 55.56g, 45.45g, No. 1 peptide, 2 peptide, 3 peptide peptide and peptide. After the numbered completion, the resin was added to the reaction column, and the appropriate amount of DCM was added for 15 min, and after evolving with an appropriate amount of DMF washed 3 times, it was dried, and the appropriate amount of desorbing liquid (DBLK / DMF volume ratio was 1: 4) 5 min, 10 min, and wash it once with DMF.

[0035] S2, weigh 4 parts of FMOC-Lys (Boc) -OH (29.28g) and TBTU (20.07 g), add an appropriate amount of DMF, fully dissolved in 4 beaker, slowly add DIEA under ice bath conditions (20.66ml ) The dried dry was washed 3 times, and the sequence of the β-defensin 2 antibacterial peptide was s...

Embodiment 2

[0043] S1, which was weighed with a total of 3 parts of FMoc-Pro-2-Chlorotrityl Chloride Resin resin having a replacement of 0.35 mmol / g, named 5 peptide, 6 peptide, and 7 peptide. After the numbered completion, the resin was added to the reaction column, and the appropriate amount of DCM was added for 15 min, and after evolving with an appropriate amount of DMF washed 3 times, it was dried, and the appropriate amount of desorbing liquid (DBLK / DMF volume ratio was 1: 4) 5 min, 10 min, and wash it once with DMF.

[0044] S2, 3 copies of FMOC-Lys (Boc) -OH (29.28g) and TBTU (20.07 g), add appropriate amount of DMF, fully dissolved in three beaker, slowly add Diea under ice bath conditions (20.66ml ) Wash 3 times with DMF. The sequence according to the antibacterial peptide of the beta-defensin 2 is sequentially coupled until the last amino acid Fmoc-Gly-oh is coupled, and the amount of desorbing liquid (DBLK / DMF volume ratio is 1: 4) is in turn, after 10min. Use DMF, DCM, meth...

Embodiment 3

[0055] S1, which is called 71.43 g of FMOC-PRO-2-Chlorotrityl Chloride Resin resin in replacement of 0.35 mmol / g, and is numbered No. 8 peptide, 9 peptide, 10 peptide, 11 peptide. After the numbered completion, the resin was added to the reaction column, and the appropriate amount of DCM was added for 15 min, and after evolving with an appropriate amount of DMF washed 3 times, it was dried, and the appropriate amount of desorbing liquid (DBLK / DMF volume ratio was 1: 4) 5 min, 10 min, and wash it once with DMF.

[0056] S2, weigh 4 parts of FMOC-Lys (Boc) -OH (29.28g) and TBTU (20.07 g), add an appropriate amount of DMF, fully dissolved in 4 beaker, slowly add DIEA under ice bath conditions (20.66ml ) Wash 3 times with DMF. The sequence according to the antibacterial peptide of the beta-defensin 2 is sequentially coupled until the last amino acid Fmoc-Gly-oh is coupled, and the amount of desorbing liquid (DBLK / DMF volume ratio is 1: 4) is in turn, after 10min. Use DMF, DCM, m...

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Abstract

The invention discloses a preparation method of a beta-defensin 2 antibacterial peptide. The preparation method comprises the following steps: S1, selecting Fmoc-Pro-2-Chloro-Resin resin, carrying out swelling, and then removing an Fmoc protecting group; and S2, according to the sequence of the beta-defensin 2 antibacterial peptide, sequentially carrying out coupling from the C terminal to the N terminal until the last amino acid Fmoc-Gly-OH, and removing the Fmoc protecting group to obtain the beta-defensin 2 antibacterial peptide resin peptide. According to the preparation method, an Fmoc solid phase method is used, short dipeptide and short tripeptide are selected as reaction raw materials for coupling, generation of impurities is reduced, the idea that a pair of disulfide bonds is formed through oxidation to form a relatively stable structure, and then the remaining two pairs of disulfide bonds are formed through natural oxidation is adopted, so that the purposes of high purity and high yield of a crude product are achieved; the method has the advantages of high operability and reduction of emission of three wastes, can be used for large-scale production, has high theoretical guidance value and considerable economic and practical value, and is wide in application prospect.

Description

Technical field [0001] The present invention relates to the field of polypeptide synthesis, and more particularly to a preparation method of beta-defensin 2 antimicrobial peptide. Background technique [0002] Humans have found antibacterial peptides in 1980, such antimicrobial active polypeptide substances are always prepared, due to antimicrobial peptides have a strong killing effect on bacteria, and it is not easy to generate resistance, and the potential is huge. So people have found and separate all kinds of antimicrobial peptides from bacteria, fungi, amphibia, insects, higher plants, mammals, humans, where defensin is widely distributed in fungi, plants and animals, β-defensin 2 antibacterial The peptide is one of them. [0003] The β-defensin 2 antimicrobial peptide is found in the tracheal mucosal epithelial cells of the cow in 1991, which is a polypeptide consisting of 41 amino acids, and includes three pairs of disulfide bonds, and the sequence is: [0004] H-GLY-ILE-G...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/36C07K1/34C07K1/16C07K1/06C07K1/04
CPCC07K14/4723Y02P20/55
Inventor 沈永刚肖攀谢振亮付坤沈永良程益明胡桂香
Owner HANGZHOU GOTOP BIOTECH
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