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A kind of polypeptide synthesis method and application thereof containing tyrosine sulfate modification

A technology for peptide synthesis and tyrosine, which is applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problems of complex process, low yield and high cost, and achieve the effect of broadening the scope.

Active Publication Date: 2021-10-22
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, no conotoxin containing tyrosine sulfate modification has been obtained by chemical synthesis. The method of obtaining it is generally the venom separation method. This method is complicated in process, high in cost, and low in yield, so it is not suitable for Large-scale preparation and application

Method used

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  • A kind of polypeptide synthesis method and application thereof containing tyrosine sulfate modification
  • A kind of polypeptide synthesis method and application thereof containing tyrosine sulfate modification
  • A kind of polypeptide synthesis method and application thereof containing tyrosine sulfate modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1: Exploration of the conditions for removing the protective group-neopentyl group on the tyrosine sulfate radical:

[0100] Fmoc-Tyr(OSO 3 nP)-OH were dissolved in 100% H 2 O (incomplete dissolution), 90% H 2 O / ACN, 80%H 2 O / ACN, 70%H 2 O / ACN, 60%H 2 O / ACN, 50%H 2 O / ACN, 40%H 2 O / ACN, 30%H 2 O / ACN, 20%H 2 O / ACN, 10%H 2 O / ACN, 100% ACN solution to a concentration of 1mg / ml, room temperature shaking reaction for 2 days, estimated its neopentyl removal rate by the peak area of ​​liquid chromatography mass spectrometry (LC-MS), its removal The rates were 100%, 83%, 75%, 59%, 22%, 18%, 11%, 2%, 4%, 1%, and 0%, respectively. It can be seen that as the water component in the solution increases, the neopentyl removal rate is higher in the same time period.

[0101] Fmoc-Tyr(OSO 3 nP)-OH was dissolved in a small amount of ACN (poor water solubility), and buffered saline solution was added until the acetonitrile ratio was 20%, the concentration was 0.1mg / ml, ...

Embodiment 2

[0103] Embodiment 2: the preparation method of α-conotoxin EpI[Y15sY]:

[0104] (1) Preparation of Fmoc-Cys(Acm)-MBHA resin: Take 0.5g Rink Amide MBHA resin in a peptide synthesis tube with a loading capacity of 0.2-0.8mmol / g, add 10mL DMF to swell twice at room temperature, 15min each time , pumped dry, add 5mL 20% piperidine / DMF to the resin, shake reaction at room temperature for 5min, wash twice with DMF, add 5mL 20%piperidine / DMF again, shake reaction at room temperature for 5min, wash with DMF, DCM in turn , DMF were washed twice each, and the solvent was drained to obtain the resin obtained by removing the Fmoc protection from the amino group. Fmoc-Cys (Acm)-OH (0.3mmol), HOBT (0.6mmol) and DIC (0.3mmol) were weighed, Fmoc- Dissolve Cys(Acm)-OH and HOBT with a small amount of DMF, add DIC, and activate the carboxyl group by shaking at room temperature for 20 minutes, then add the activated amino acid to the resin, shake and react at room temperature for 2 hours, wash wi...

Embodiment 3

[0108] Embodiment 3: the preparation method of α-conotoxin AnIA[Y14sY]:

[0109] (1) Preparation of Fmoc-Cys(Acm)-MBHA resin: According to the method of Example 2, a dry resin with a loading capacity of 0.6g0.5mmol / g was obtained.

[0110](2) Preparation of AnIA[Y16sY]-MBHA resin: Add 0.6g of the obtained Fmoc-Cys(Acm)-MBHA resin to 10mL DMF and swell twice at room temperature, 15min each time, drain, and then add 5mL 20 % acetic anhydride / DMF, shake at room temperature for 20 minutes to block the amino group of the resin that is not coupled with amino acid, prevent its next reaction, wash twice with DMF, DCM, DMF in turn, add 5mL 20% piperidine / DMF to the resin , shaken at room temperature for 5 minutes, washed twice with DMF, added 5 mL of 20% piperidine / DMF again, shaken for 5 minutes at room temperature, washed twice with DMF, DCM, DMF in turn, drained the solvent, and obtained Fmoc-protected resin, weigh Fmoc-L-Tyr (OSO 3 nP)-OH (0.6mmol), HOBT (1.2mmol) and DIC (0.6mmo...

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Abstract

The invention provides a method for synthesizing a polypeptide containing tyrosine sulfate modification and its application. The method of the present invention adopts the Fmoc solid-phase polypeptide synthesis method, uses the Fmoc amino resin as a carrier, and sequentially condenses the Fmoc protected amino acids from the C-terminal to the N-terminal according to the polypeptide sequence containing the tyrosine sulfate modification site, to obtain a linear peptide amino resin; Wherein, the tyrosine side chain sulfate uses neopentyl as a protecting group, and the cysteine ​​side chain uses trityl and / or acetamidomethyl as a protecting group; then removes the side chain protecting group and The target polypeptide is cleaved from the resin and purified to obtain a tyrosine sulfate-modified polypeptide. The invention helps us to study the influence of the tyrosine sulfate modification on the biological activity of the polypeptide and related mechanisms, and lays a good foundation for the diagnosis and treatment of related diseases.

Description

technical field [0001] The invention relates to the technical field of polypeptide synthesis and preparation, in particular to a method for synthesizing a polypeptide containing tyrosine sulfate modification and its application. Background technique [0002] Protein tyrosine sulfation (PTS) is a common post-translational modification, and the combination of inorganic sulfate and biomolecules plays an important role in biological systems. Some researchers modified the Anophelin protein with double sulfation, and the results showed that this modification led to a 100-fold increase in its thrombin inhibitory activity (Watson, E.E.; Liu, X.; Thompson, R.E.ACS.Cent.Sci.2018, 4, 468-476). Therefore, the effect of protein tyrosine sulfate modification on its biological activity is particularly important. [0003] Conotoxin is known as the "treasure house of marine drugs". It is a biologically active cyclic polypeptide compound derived from marine cone snails. Up to now, thousand...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/04C07K1/06
CPCC07K7/08Y02P20/55
Inventor 何春茂李长鹏
Owner SOUTH CHINA UNIV OF TECH
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