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A kind of solid phase synthesis method of bremelanotide

A technology of bumenotide and solid-phase synthesis, which is applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., and can solve the problems of large steric hindrance and difficulty in cyclization.

Inactive Publication Date: 2019-02-05
JINAN KANGHE MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ring formation in the liquid phase is easy to produce a large number of intermolecular coupling products, and the solid phase ring formation method is difficult due to too much steric hindrance

Method used

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  • A kind of solid phase synthesis method of bremelanotide
  • A kind of solid phase synthesis method of bremelanotide
  • A kind of solid phase synthesis method of bremelanotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: HO-(CH 2 ) 7 - Preparation of CONH-Rink Amide Resin

[0071] Accurately weigh 100g of Rink Amide Resin with a substitution degree of 0.52mmol / g and place it in a solid-phase synthesis kettle, wash twice with DMF, 500ml / time, 3min / time, after washing, add 500ml DCM to swell the resin for 30min.

[0072] Rink Amide Resin was deprotected with 20% piperidine / DMF, 400ml / time, the first reaction was 5 minutes, and the second reaction was 15 minutes. After deprotection twice, the resin was washed six times with DMF, 500ml / time, 3min / time. Take a small sample of the resin and detect it with ninhydrin reagent, the resin is dark blue.

[0073] Weigh HO-(CH 2 ) 7 -COOH 24.9g (156mmol, 3eq), HOBt 23.2g (171.6mmol, 3.3eq) was placed in a dissolving bottle, dissolved in 200ml DMF, pre-cooled at 0-5°C for 10min, and DIC 27.5ml (171.6mmol, 3.3eq) was added Activation for 5 minutes, after the activation is completed, put into the resin, stir and react at 25°C for 2 hour...

Embodiment 2

[0074] Example 2: HO-(CH 2 ) 15 - Preparation of CONH-RinkAmide Resin

[0075] Accurately weigh 100g of Rink Amide Resin with a substitution degree of 0.52mmol / g and place it in a solid-phase synthesis kettle, wash twice with DMF, 500ml / time, 3min / time, after washing, add 500ml DCM to swell the resin for 30min.

[0076] RinkAmide Resin was deprotected with 20% piperidine / DMF, 400ml / time, the first reaction was 5min, and the second reaction was 15min. After deprotection twice, the resin was washed six times with DMF, 500ml / time, 3min / time. Take a small sample of the resin and detect it with ninhydrin reagent, the resin is dark blue.

[0077] Weigh HO-(CH 2 ) 15-COOH 42.4g (156mmol, 3eq), HOBt 23.2g (171.6mmol, 3.3eq) was placed in a dissolving bottle, dissolved in 200ml DMF, pre-cooled at 0-5°C for 10min, and DIC 27.5ml (171.6mmol, 3.3eq) was added Activation for 5 minutes, after the activation is completed, put into the resin, stir and react at 25°C for 2 hours. After 2...

Embodiment 3

[0078] Example 3: Fmoc-Lys(Mtt)-O-(CH 2 ) 7 - Preparation of CONH-RinkAmide Resin

[0079] Accurately weigh 97.344g (156mmol, 3eq) of Fmoc-Lys(Mtt)-OH and 23.2g (171.6mmol, 3.3eq) of HOBt into a dissolving bottle, dissolve in 300ml DMF, pre-cool at 0-5°C for 10min, add DIC 27.5ml (171.6mmol, 3.3eq) activation 5min, drop into the synthetic resin HO-(CH 2 ) 7 -CONH-RinkAmide Resin, after stirring for 10 minutes, add DMAP 1.9g (15.6mmol, 0.3eq), and stir for 5 hours at 25°C. After 5h, the resin was washed and dried in a vacuum drying phase (<-0.1Mpa, 8h). A sample was taken to measure the degree of substitution, and the measured value was 0.33mmol / g.

[0080] Weigh 90g of the above dry resin (substitution degree is 0.33mmol / g, 30mmol) and place it in a solid-phase synthesis kettle, wash twice with DMF, 500ml / time, 3min / time, after washing, add 500ml DCM to swell the resin for 30min.

[0081] Measure 45ml (450mmol, 15eq) of acetic anhydride and 38ml (450mmol, 15eq) of pyridi...

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Abstract

The invention belongs to the field of polypeptide synthesis, and specifically relates to using Rink Amide Resin as a solid-phase carrier, and using HO-(CH2)n-COOH as a Linker connecting the solid-phase carrier and a target peptide, wherein the carboxyl terminal is connected to the solid-phase carrier through an amide bond , the hydroxyl end is linked to the target peptide through an ester bond. Compared with the existing technology, this technical solution greatly optimizes the space environment for solid-phase cyclization of the target peptide by introducing long-chain fatty acids between the solid-phase carrier and bremelanotide, and avoids the occurrence of cyclic formation in the liquid phase. Intermolecular coupling, the purity of the final target peptide-Linker can reach more than 85%, the total purification yield can reach more than 50%, and the purity of the refined peptide can reach more than 99%.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a method for solid-phase synthesis of bremelanotide by using long-chain fatty acids as Linker. Background technique [0002] Bremelanotide, the English name is Bremelanotide, also known as Bremelanotide, PT141, which consists of 7 amino acids and has the same core sequence as α-melanocyte-stimulating hormone, whose receptor is in appetite and sexual desire arousal makes an important impact. Its peptide sequence is AC-Nle-[Asp-His-D-Phe-Arg-Trp-Lys] cycle -OH, the structural formula is as follows: [0003] [0004] Sexual dysfunction is divided into male sexual dysfunction and female sexual dysfunction. Most of the drugs currently used to treat male sexual dysfunction are phosphodiesterase 5 (PDE-5) inhibitors, such as cetenafil and tadalafil , vardenafil. The mechanism of action of these drugs is to maintain or increase the concentration of cyclic adenosine monophospha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/56C07K1/10C07K1/06C07K1/04
CPCY02P20/55
Inventor 张颖陈雷王仁友李同金石鑫磊
Owner JINAN KANGHE MEDICAL TECH