A kind of purification method of phosphorylated muscarin

A technology of muscarin and purification method, which is applied in the field of chemical purification, can solve the problems of insufficient purification of a large amount of phosphorylated polypeptides, and achieve the effect of good purification, economical and efficient effect, and shortened time

Active Publication Date: 2016-03-02
中国人民解放军防化学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the technologies reported so far are mainly in the field of proteomics to carry out trace relative enrichment of natural phosphorylated polypeptides in biological samples (application numbers 201310253448.X, 200810202189.7, 200810211727.9), in order to improve the detection of phosphorylated polypeptides in mass spectrometry The purification rate still stays at the analytical level, which cannot meet the requirements of a large amount of purification of phosphorylated polypeptides synthesized by chemical methods, especially the purification method of phosphorylated muscotin has not been reported yet.

Method used

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  • A kind of purification method of phosphorylated muscarin
  • A kind of purification method of phosphorylated muscarin
  • A kind of purification method of phosphorylated muscarin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Purification of a simple sample of phosphorylated muscotin mixed with two other non-phosphopeptides:

[0027] Weigh 100 mg of titanium dioxide into a purified glass tube with a sieve plate, add 10 mL of 70% acetonitrile / 3% TFA solution, shake up and down for 1 minute, filter the solution under reduced pressure at 0.05-0.09 mbar, and wait for the sample; weigh the pure product of phosphorylated polypeptide 1 Pyr-Pro-Ser-Lys-Asp-Ala-Phe-Tyr (PO 3 H)-Gly-Leu-Met-NH 2 and two pure non-phosphorylated polypeptides [Pyr-Pro-Ser-Lys-Asp-Ala-Phe-Phe(Cl)-Gly-Leu-Met-NH 2 and Pyr-Pro-Ser-Lys-Asp-Ala-Phe-Tyr-Gly-Leu-Met-NH 2] each 10 mg, after mixing, dissolve in 20 mL of 70% acetonitrile / 3% TFA solution, shake up and down for 5 minutes, filter the solution under reduced pressure at 0.05-0.09 mbar; add 60% acetonitrile / 0.1% TFA 20mL of the solution, shake up and down for 1 minute, filter the solution under reduced pressure; add 20mL of the solution of 50% acetonitrile / 0.1% TFA, ...

Embodiment 2

[0029] Purification and preparation of phosphorylated muscotin chemically synthesized by monomer method:

[0030] Weigh 1 g of titanium dioxide into a purified glass tube with a sieve plate, add 10 mL of a solution of 70% acetonitrile / 3% TFA, shake up and down for 1 minute, filter the solution under reduced pressure at 0.05-0.09 mbar, and wait for Loading the sample; Weigh the phosphorylated muscarin [Pyr-Pro-Ser(PO 3 H)-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH 2 ] Crude product, dissolve it in 20mL of a solution of 70% acetonitrile / 3% TFA, shake it up and down for 5 minutes, filter the solution under reduced pressure at 0.05-0.09mbar; add a solution of 60% acetonitrile / 0.1% TFA 20mL, oscillate up and down for 1 minute, filter the solution under reduced pressure at 0.05-0.09mbar; add 20mL of 50% acetonitrile / 0.1% TFA solution, oscillate up and down for 1 minute, filter the solution under reduced pressure; add 20mL of 50% acetonitrile solution, shake up and down for 1 minute, filte...

Embodiment 3

[0032] Purification and preparation of phosphorylated muscotin chemically synthesized by overall method:

[0033] Weigh 1 g of titanium dioxide into a purified glass tube with a sieve plate, add 10 mL of a solution of 70% acetonitrile / 3% TFA, shake up and down for 1 minute, filter the solution under reduced pressure at 0.05-0.09 mbar, and wait for Loading the sample; Weigh the phosphorylated muscarin [Pyr-Pro-Ser(PO 3 H)-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH 2 ] Crude product, dissolve it in 20mL of a solution of 70% acetonitrile / 3% TFA, shake it up and down for 5 minutes, filter the solution under reduced pressure at 0.05-0.09mbar; add a solution of 60% acetonitrile / 0.1% TFA 20mL, shake up and down for 1 minute, filter the solution under reduced pressure at 0.05-0.09mbar; add 20mL of 50% acetonitrile / 0.1%TFA solution, shake up and down for 1 minute, and filter the solution at Filter under reduced pressure; add 20mL of 50% acetonitrile solution, shake up and down for 1 minute, ...

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Abstract

The invention relates to a method for purifying phosphorylated eledoisin, and belongs to the technical field of chemical purification. The phosphorylated eledoisin has very strong antihypertensive activity, and a chemosynthetic crude product of the phosphorylated eledoisin is difficult to purify in quantity, so that the application of the phosphorylated eledoisin is limited. The purification and preparation of the chemosynthetic phosphorylated eledoisin are realized by dint of technologies, namely vacuum filtration and the like, through three key steps of sample loading, cleaning and elution by utilizing a principle that titanium dioxide and the phosphorylated eledoisin generate higher Lewis acid-base effect in an acid environment and the Lewis acid-base effect disappears in an alkaline environment and adopting an independently researched phosphorylated polypeptide purification device (an application number: 201420158316.9). The method disclosed by the invention is easy and convenient to operate, economy and high-efficiency, and provides an effective method for the purification of the phosphorylated eledoisin, thereby promoting the application of the phosphorylated eledoisin in the fields of medicines and the like.

Description

technical field [0001] The invention relates to a method for purifying phosphorylated muscothin, which belongs to the technical field of chemical purification and mainly provides conditions for the effective application of phosphorylated polypeptides in the fields of medicine and the like. Background technique [0002] Eledoisin is a polypeptide containing 11 amino acids extracted from Eledone, and its sequence is Pyr-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH 2 , has strong peripheral vasodilation and increased coronary blood flow, is the most effective antihypertensive substance among known peptides, and its effect is thousands of times stronger than nitroglycerin. Phosphorylated muscotin is obtained by phosphorylating Ser at the 3rd position of its N-terminus, and the sequence is Pyr-Pro-Ser(PO 3 H)-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH 2 , its activity is more than two times higher than that of muscine (a powerful antihypertensive phosphopeptide and its preparation method...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K1/22A61P9/12
Inventor 隋少卉董俊军游炎富李建吴为辉李善茂刘基伟张鸣蒋辉刘尚义
Owner 中国人民解放军防化学院
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