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Penicillamine-based polypeptide disulfide bond synthesis method and application thereof

A synthesis method and penicillamine technology, applied in the field of chemical biology research, can solve problems such as difficulty in correct recombination of disulfide bonds

Active Publication Date: 2021-04-20
PEKING UNIV SHENZHEN GRADUATE SCHOOL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the stability of the disulfide bond itself and the difficulty of correct recombination of disulfide bonds in polypeptides or proteins that are naturally rich in disulfide bonds, the application and development of disulfide bonds have been limited.

Method used

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  • Penicillamine-based polypeptide disulfide bond synthesis method and application thereof
  • Penicillamine-based polypeptide disulfide bond synthesis method and application thereof
  • Penicillamine-based polypeptide disulfide bond synthesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 The synthesis of the polypeptide disulfide bond based on penicillamine

[0049] 1. Use WANG resin to synthesize resin A

[0050] Peptide solid-phase synthesis (taking WANG resin as an example): Weigh WANG resin into a peptide tube, add DCM, and blow nitrogen for 10 minutes to make it swell. Add 50% (v / v) morpholine in DMF and blow nitrogen gas for 30 minutes (twice) to remove the Fmoc protecting group. The resin was alternately washed 8 times with DMF and DCM, and the corresponding Fmoc-AA-OH (5eq), HCTU (5eq), and DIPEA (10eq) were dissolved in DMF and added to the resin for 1 hour under nitrogen blowing. Then alternately wash with DMF and DCM for 8 times, continue deprotection according to the above method, and connect another amino acid until the synthesis of the polypeptide sequence is completed.

[0051] 2. Synthesis and separation of polypeptides containing disulfide bonds (taking two main routes as examples):

[0052] Route 2: Take the resin (100m...

Embodiment 2

[0064] Example 2 Stability verification of polypeptides containing disulfide bonds in the presence of reducing agents:

[0065] Dissolve the polypeptide (1mM) synthesized according to the method of Example 1 in 100mM PBS (phosphate buffer saline) with a pH value of 7.4, then add 10mM GSH or Pys, and incubate at 37°C in a water bath. The reaction solution after 24 hours and 48 hours was analyzed by liquid chromatography-mass spectrometry to observe the reduction of disulfide bonds.

[0066] The LCMS analysis pictures of the polypeptide Ac-Cyclo (PenSPC) NIYYKV-COOH (MW: 1257) at different times in the presence of GSH are as follows figure 1 , which are respectively identified by LCMS after co-incubating the polypeptide with GSH for 24 hours and 48 hours. It can be seen from the figure that the polypeptide has not been substantially reduced.

[0067] The LCMS analysis pictures of the polypeptide Ac-Cyclo (PenSPC) NIYYKV-COOH (MW: 1257) at different times in the presence of Pys...

Embodiment 3

[0069] Example 3 Stability verification of polypeptides containing disulfide bonds in the presence of alkylating reagents (taking 1,3-2 bromomethylbenzene as an example):

[0070] The polypeptide synthesized according to the method of Example 1 was dissolved in 100 mM PBS with a pH value of 7.4, and the alkylating agent (1.5 mM) dissolved in DMSO (2-methyl sulfoxide) was added, and reacted in the presence of 1% formic acid for 24 hours, followed by analysis by liquid chromatography-mass spectrometry to observe the changes in the reaction system.

[0071] The LCMS analysis diagram of the polypeptide Ac-cycle (CSPAPen) IYYKV-COOH (MW: 1215) reacted for 24 hours in the presence of an alkylating reagent is shown in Figure 4 , that is, the peptide and the alkylating agent 1,3-dibromomethylbenzene were reacted for 24 hours in the presence of 1% formic acid, and then analyzed by LCMS. It can be seen from the figure that the polypeptide is basically unchanged.

[0072] The LCMS ana...

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Abstract

The invention provides a penicillamine-based polypeptide disulfide bond synthesis method. The method adopts a route 1 or 2, wherein the route 1 comprises the following steps: dissolving iodine in N,N-dimethylformamide, adding the mixture into resin A with synthesized related sequences, performing a nitrogen blowing reaction, cutting off polypeptide by using shearing liquid, and separating and purifying the mixture with high performance liquid chromatography to obtain a target product; and the route 2 comprises the following steps: cutting off the polypeptide with the synthesized related sequence from the resin A by utilizing the shearing liquid, then separating out non-oxidized and ring-closed polypeptide by using the high performance liquid chromatography, dissolving the polypeptide in a phosphate buffer solution with the pH value of 7.4-8.0, adding an oxidant into the mixture for oxidation, and carrying out separation and purification by using the high performance liquid chromatography to obtain the target product. The invention provides the novel stable disulfide bond designing and synthesizing strategy, and the disulfide bond serving as the bullet of an effective covalent reaction is applied to the field of chemical biology.

Description

technical field [0001] The invention belongs to the field of stable polypeptide methodology and design and development of chemical biology polypeptide inhibitors, specifically relates to a penicillamine-based polypeptide disulfide bond synthesis method and its application, especially relates to the design of a novel stable disulfide bond , synthesis, and the development of covalent inhibitors of peptides as an example to explore their application in the field of chemical biology research. Background technique [0002] Peptide drugs are between traditional small molecule drugs (<500Da) and biomacromolecular drugs (>5000Da), combining many advantages of both, such as better affinity and specificity and better biological Compatibility and safety, etc., and can effectively target proteins and protein interaction (Protein-Protein Interaction, PPI) interfaces that are difficult to be targeted by other drugs, filling the corresponding vacancies. In recent years, peptide drug...

Claims

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Application Information

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IPC IPC(8): C07K1/36C07K1/30C07K1/16C07K1/113C07K1/06C07K1/04
CPCY02P20/55
Inventor 李子刚尹丰詹美苗
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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