Preparation of C-terminal ethylamine polypeptides and derivates thereof

A derivative, the technology of terminal ethylamine, applied in the field of polypeptide chemistry, can solve the problems of cumbersome process, difficult to scale up, pollution costs and other problems, and achieve the effect of simple process, reduced preparation cost and low price

Active Publication Date: 2008-10-08
GL BIOCHEM SHANGHAI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a simple, efficient and low-cost preparation method of C-terminal ethylaminated polypeptides, which solves the problem of tedious process, long cycle, serious pollution or The problem of high cost and not easy to enlarge

Method used

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  • Preparation of C-terminal ethylamine polypeptides and derivates thereof
  • Preparation of C-terminal ethylamine polypeptides and derivates thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: Preparation of leuprolide (pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt) (1) Preparation of ethylamine resin: weighing 2-chlorotrityl Put 1 g of chlorine resin (1 mmol / g) in a glass sand core reactor, add 15 mL of DCM, and let stand to swell the resin for 0.5 h. Introduce 0.326mL ethylamine gas. React at 10°C for 1h. After the reaction, filter. Then 15 mL of DMF was added to the resin to wash the resin and filtered. Then add 15mL THF to wash the resin, and filter.

[0023] (2) Connect the C-terminal amino acid Pro

[0024] Weigh 2 mmol each of Fmoc-Pro-OH, HBTU and HOBt, and add 10 mL of DMF to dissolve. Then the mixed solution was added to the resin in (1), and 4 mmol of diisopropylethylamine (DIEA) was added at the same time, and reacted at 20° C. for 1 h. After the reaction, filter. Then 15 mL of DMF was added to the resin to wash the resin and filtered. Add 15 mL of NMP to wash the resin and filter.

[0025] (3) Remove the Pro amino protecting ...

Embodiment 2

[0033]Example 2: Preparation of histrelin (Pyr-His-Trp-Ser-Tyr-D-His(Bzl)-Leu-Arg-Pro-NHEt)

[0034] (1) Preparation of ethylamine resin: Weigh 2 g of 4-methyl-2-chlorotrityl chloride resin (1 mmol / g), place it in a glass sand core reactor, add 20 mL of DMF, and let stand to swell the resin for 1 h . Introduce 0.653mL ethylamine gas. Reaction at 30°C for 2h. After the reaction, filter. 20 mL of DCM was then added to the resin to wash the resin and filtered. Another 20 mL of DCM was added to wash the resin and filtered.

[0035] (2) Connect the C-terminal amino acid Pro

[0036] Weigh Fmoc-Pro-OH, HATU and HOAt each 8mmol, 10mmol, 10mmol, add 20mL DMF to dissolve. Then the mixed solution was added to the resin in (1), and 16 mmol DIEA was added at the same time, and reacted at 40° C. for 5 h. After the reaction, filter. 20 mL of NMP was then added to the resin to wash the resin and filtered. Add 20 mL of NMP to wash the resin, and filter.

[0037] (3) Remove the Pro a...

Embodiment 3

[0045] Example 3: Preparation of Alarelin (pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt)

[0046] (1) Preparation of ethylamine resin: Weigh 3 grams of 2-chlorotrityl chloride resin (1 mmol / g), place it in a glass sand core reactor, add THF 20 mL, and let stand to swell the resin for 2 hours. Introduce 1.306mL ethylamine gas. Reaction at 50°C for 3h. After the reaction, filter. 20 mL of DCM was then added to the resin to wash the resin and filtered. Another 20 mL of DCM was added to wash the resin and filtered.

[0047] (2) Connect the C-terminal amino acid Pro

[0048] Weigh 15mmol, 22.5mmol and 22.5mmol of Fmoc-Pro-OH, PyBOP and HOBt respectively, and add 25mL NMP to dissolve. Then the mixture was added to the resin in (1), and 30 mmol DIEA was added at the same time, and reacted at 60° C. for 10 h. After the reaction, filter. 20 mL of NMP was then added to the resin to wash the resin and filtered. Add 20 mL of DMF to wash the resin, and filter.

[0049] (3) Remove t...

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Abstract

The invention relates to a method to prepare a polypeptide and in particular relates to a method to prepare C terminal ethylaminated polypeptide, solving the problems of long period in liquid phase synthesis, the use of poisonous reagent in Boc solid-liquid method and high cost in Fmoc method. The preparation method includes the following steps(1)preparing solid pahse carrier; (2)connecting terminal amino acids at the C terminal with the solid pahse carrier; (3) desorbing the amido protecting groups of the amino acids at the C terminal; (4)condensating the second amino acid and the first amino acid at the C terminal; (5)repeating the processes in step(3)and step(4) according to the sequence from C terminal to N terminal of the polypeptide of the derivative thereof to synthesize the polypeptide or the derivative peptide chain e thereof; (6)adding reagent to cleave the peptide resin and purifying through high efficiency liquid chromatography; in this way the target product f is obtained. The method is suitable for producing C terminal ethylaminated polypeptide with low cost and high efficiency.

Description

Technical field: [0001] The invention belongs to the field of polypeptide chemistry, and in particular relates to a solid-phase preparation method of C-terminal ethylaminated polypeptide and derivatives thereof. Background technique: [0002] A polypeptide is a peptide chain formed by condensation of amino acids in a certain order, and the peptide chain is modified to form a polypeptide derivative. After the C-terminus of the polypeptide or its derivative is modified by ethylamination, its biological activity and proteolytic stability can be enhanced, thereby improving its utilization rate in vivo. C-terminally ethylated peptides are mainly analogs of luteinizing hormone releasing hormone, including leuprolide, histrelin, alarelin, deserelin, etc., the main function is to effectively inhibit the function of the pituitary-gonadal system , clinically mainly used for the treatment of prostate cancer and endometriosis, with an annual sales of more than 1 billion US dollars. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/04C07K1/16
CPCY02P20/55
Inventor 徐红岩王卫国
Owner GL BIOCHEM SHANGHAI
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