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Method for synthesizing cyclic peptides through enzyme method

A cyclic peptide and enzymatic activity technology, applied in the field of enzymatic synthesis of cyclic peptides, can solve the problems of complex steps, toxic and side effects, unfavorable to environmental protection, etc., and achieve the effects of high yield and simple steps

Active Publication Date: 2017-01-25
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When enzymatically synthesizing cyclic peptides, when the linear peptide is cleaved from the solid phase carrier with trifluoroacetic acid, the side chain protecting group has been removed at the same time. After HPLC purification, the obtained linear peptide is directly cyclized with an enzyme, and then cyclized by an enzyme. The cyclic peptide product can be obtained by HPLC purification. Compared with the enzymatic synthesis, the traditional cyclic peptide synthesis method has one more step of HPLC purification, the steps are relatively complicated, and the yield of the final product is low. In addition, the condensation agent used in the traditional method of cyclization, For example: HBTU, DIC, etc., have toxic and side effects and are not conducive to environmental protection

Method used

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  • Method for synthesizing cyclic peptides through enzyme method
  • Method for synthesizing cyclic peptides through enzyme method
  • Method for synthesizing cyclic peptides through enzyme method

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Experimental program
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Effect test

Embodiment 1

[0015] The design of embodiment 1 linear peptide

[0016] (1) For the linear peptide containing the RGD sequence, design a polypeptide sequence containing 17 amino acids, including the sortaseA recognition sequence LPETGGS at the C segment and the polyglycine sequence at the N-terminal, which is convenient for sortaseA to recognize and cyclize, and the sequence contains 1-3 RGD sequence, multivalent RGD in order to improve the biological activity of the peptide; the prepared linear peptide sequence is Gn(RGD)mLPETGGS; where n=7, m=1; or n=4, m=2; or n=1, m = 3; the synthetic linear peptide was able to inhibit the interaction of integrin αvβ3 with the extracellular matrix.

[0017] (2) For the linear peptide containing the antimicrobial peptide AKRHHGYKRKFH, the sortase A recognition sequence LPETGGS is carried on the C segment of the antimicrobial peptide, and the N segment is a polyglycine sequence, and the last glycine in the polyglycine sequence is structurally modified. G...

Embodiment 2

[0018] Example 2 The linear peptide G7RGDLPETGGS synthesizes RGD cyclic peptide under the catalysis of sortaseA enzyme

[0019] Linear peptide synthesis: Weigh Fmoc-Ser(tBu)-Wang resin and swell overnight in dimethylformamide (DMF) solution, remove Fmoc with 20% (V / V) piperidine, and dichloromethane (DCM) , MeOH and DMF to clean the resin, take a small amount of resin and detect it with ninhydrin chromogenic method, and heat it to 100°C to detect that the resin turns blue, indicating that Fmoc has been removed completely. 5eq (resin is 1eq) of amino acid, 5eq condensing agent O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) and 10eq diisopropyl ethyl Amine (DIPEA) was dissolved in DMF, added to the solid-phase synthesis tube and reacted for 2 hours, followed by washing the resin with DCM, MeOH, and DMF, taking a small amount of resin and using the ninhydrin color method to detect the yellow color of the resin, indicating that the free amino group and amino...

Embodiment 3

[0021] Example 3 Linear peptide G4 (RGD) 2LPETGGS synthesizes RGD cyclic peptide under the catalysis of sortaseA enzyme

[0022] Linear peptide synthesis: the method is the same as Example 1

[0023] Enzyme-catalyzed synthesis of cyclic peptides: in 100 μL enzyme reaction system, take linear peptide G4(RGD)2LPETGGS 0.25 mM; sortase A 10 μM; reaction buffer is 0.3M Tris-HCl (PH=7.5), 0.15M NaCl, 5mM CaCl 2 , 2mM mercaptoethanol, reacted at 37°C for 20h. After the reaction, the enzyme was filtered by ultrafiltration, and the resulting filtrate was purified by HPLC and identified by mass spectrometry, and HPLC gradient elution: mobile phase A was eluted from 5% to 50% for 20 minutes; the molecular weight of the cyclic peptide obtained by the G4(RGD)2LPETGGS reaction was respectively 1325.4, 2650.7, which are characterized by inhibiting the interaction between integrin αvβ3 and extracellular matrix. The yields are shown in Table 1.

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Abstract

The invention disclsoes a method for synthesizing cyclic peptides through an enzyme method and belongs to the technical field of cyclic peptide synthesis. The method comprises: mixing linear peptides and sortase A at the mol ratio of 25 to 1, and reacting at 37 DEG C to prepare the cyclic peptides. The yield of the cyclic peptides is 70 percent to 93 percent; the method has the advantages of high yield, simple steps and the like and is green and environmentally friendly. The prepared RGD (arginine-glycine-aspartic acid) linear peptides and the cyclic peptides have a property of inhibiting the mutual effect of integrin alphavbeta3 and extracellular matrixes; the prepared AKRHHGYKRKFH linear peptides and cyclic peptides have a property of inhibiting candida albicans, pseudomonas aeruginosa and staphylococcus aureus and have wide application value.

Description

technical field [0001] The invention relates to a method for enzymatically synthesizing cyclic peptides, belonging to the technical field of cyclic peptide synthesis. Background technique [0002] Cyclic peptide compounds have various biological activities, including anti-tumor, anti-immune and other biological activities. Compared with the corresponding linear peptide, it has better resistance to enzymatic and chemical degradation in vivo, and can prolong the half-life in vivo to exert long-acting effects. The commonly used cyclosynthesis method is to first synthesize the linear peptide by solid-phase method, and then cut the linear peptide from the resin with a cleavage solution containing trifluoroacetic acid after removing Fmoc, and perform HPLC purification to obtain a relatively pure peptide with side chain protecting groups. The linear peptide is then condensed with a condensing agent in the liquid phase to form a cyclic peptide. The cyclic peptide containing a prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K7/64C12P21/04
Inventor 吴志猛程孝中刘少中
Owner JIANGNAN UNIV
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