Solid-phase synthesis of ATL peptides

A technology of solid-phase synthesis and solid-phase carrier, which is applied in peptide preparation methods, chemical instruments and methods, peptides, etc., and can solve problems that are not suitable for large-scale preparation and industrial production, production scale limitations, and sources of amino acid side chain protecting groups. and difficulty in preparation

Inactive Publication Date: 2008-09-03
济南环肽医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the liquid-phase synthesis method has (1) many reaction steps, and the route is tedious
(2) The intermediate is not easy to purify and separate, and it is difficult to evaporate the solvent in actual operation
(3) The source and preparation of the reagents and the amino acid side chain protecting groups used are relatively difficult, etc.
Merrifield type resin is used as the carrier. After synthesizing the protective ATL resin, the conditions required to cut the peptide from the resin are relatively harsh. This method uses HF cracking, and a HF gas generating device is required to achieve cracking. For a large number of peptide resins It can only be cracked twice, and the production scale is limited
In addition, HF is a highly corrosive gas, once it leaks, it will cause great harm to people
Furthermore, this synthesis method cracks the peptide from the resin, extracts the HF and extracts it with dilute acetic acid, and after the extract is freeze-dried, the production cycle is long and the cost is high
TentaGel Resin (PEG-PS) type resin is currently expensive and not suitable for mass production and industrial production

Method used

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  • Solid-phase synthesis of ATL peptides
  • Solid-phase synthesis of ATL peptides
  • Solid-phase synthesis of ATL peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] h 2 Preparation of N-Lys(2-Cl-Z)-MBHA

[0043] (1) Put 50g (40mmol) of MBHA resin in a polypeptide synthesis reactor, wash it twice with 5% triethylamine DCM solution 5-7ml / g, each time for 8 minutes; alternately wash with 300ml each of dichloromethane and ethanol 2 times, 2 minutes / time, drain.

[0044] (2) Mix Boc-Lys(2-Cl-Z)-OH 82.8g and HOBt 27.0g, N,N-cyclohexylcarbodiimide 41.2g with 500ml dichloromethane / DMF solution (dichloromethane:N, N-dimethylformamide (volume ratio: 1:0.8) was dissolved, added to the resin, stirred at room temperature for 5 hours, and drained.

[0045] (3) The product of (2) was alternately washed 3 times with 300 ml of dichloromethane and 300 ml of ethanol, each time for 2 minutes, and drained.

[0046] (4) Wash once with 50% trifluoroacetic acid / dichloromethane (DCM) 10ml / g for 20 minutes; wash twice with dichloromethane 5-7ml / g for 2 minutes each time; get: H 2 N-Lys(2-Cl-Z)-MBHA.

Embodiment 2

[0048] h 2 Preparation of N-Lys(2-Cl-Z)-Lys(2-Cl-Z)-MBHA

[0049] (1) to resin H 2 Add 5% triethylamine DCM solution 5-7ml / g to N-Lys(2-Cl-Z)-MBHA and wash twice, 8 minutes each time; alternately wash twice with 300ml dichloromethane and ethanol, 2 minutes / times, drained.

[0050] (2) Mix Boc-Lys(2-Cl-Z)-OH 82.8g and HOBt 27.0g, N,N-cyclohexylcarbodiimide 41.2g with 500ml dichloromethane / DMF solution (dichloromethane:N, N-dimethylformamide (volume ratio: 1:0.9) was dissolved, added to the resin, stirred at room temperature for 5 hours, and drained.

[0051] (3) The product of (2) was alternately washed 3 times with 300 ml of dichloromethane and 300 ml of ethanol, each time for 2 minutes, and drained.

[0052] (4) Wash once with 50% trifluoroacetic acid / dichloromethane (DCM) 10ml / g for 20 minutes;

[0053] Wash twice with dichloromethane 5-7ml / g, 2 minutes each time;

[0054] Got: H 2 N-Lys(2-Cl-Z)-Lys(2-Cl-Z)-MBHA.

Embodiment 3

[0056] h 2 The preparation operation of N-Lys(2-Cl-Z)-Lys(2-Cl-Z)-Lys(2-Cl-Z)-MBHA: (1) to resin H 2 Add 5% triethylamine DCM solution 5-7ml / g to N-Lys(2-Cl-Z)-Lys(2-Cl-Z)-MBHA and wash twice, each time for 8 minutes; Each 300ml was alternately washed twice, 2 minutes each time, and drained.

[0057] (2) Mix Boc-Lys(2-Cl-Z)-OH 82.8g and HOBt 27.0g, N,N-cyclohexylcarbodiimide 41.2g with 500ml dichloromethane / DMF (dichloromethane:N,N - dimethylformamide (volume ratio: 1:0.9) solution was dissolved, added to the resin, stirred at room temperature for 5 hours, and drained.

[0058] (3) The product of (2) was alternately washed 3 times with 300 ml of dichloromethane and 300 ml of ethanol, each time for 2 minutes, and drained.

[0059] (4) Wash once with 50% trifluoroacetic acid / dichloromethane (DCM) 10ml / g for 20 minutes;

[0060] Wash twice with dichloromethane 5-7ml / g, 2 minutes each time;

[0061] Got: H 2 N-Lys(2-Cl-Z)-Lys(2-Cl-Z)-Lys(2-Cl-Z)-MBHA.

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Abstract

The invention disclose a solid phase synthesis process for a ATL peptide. The process comprises using MBHA resin as a solid phase carrier, combines protective lysyl Boc-Lys(2-Cl-Z) on the resin, completing the synthesis of ATL by stepwise peptide combining method for protecting aminophenol with Boc, modifying N end with acetic anhydride by acetylizing to obtain all-protected peptide resin; depriving the side chain protecting group of the formacyl of Trp with piperidine, and at last, depriving side chain protection group with fluoroform sulfonic acids and incising the resin to obtain ATL coarse peptide; and then purifying the crude product with the highly effective liquid phase of reversed phase C18 filler, separating and purifying with methanol-water gradient eluting process to obtain ATL peptide refining product. The process of the invention with single-step condensation reaction, has high yield and low by-product, and the final crude product has high purity and small impurity content, the operation is simple, and the total yield is high.

Description

technical field [0001] The present invention relates to the preparation method of polypeptide medicine, especially relates to a kind of ATL peptide (C 85 h 141 N 27 o 15 ) of the solid-phase synthesis method. It belongs to the field of biotechnology. Background technique [0002] ATL peptide chemical name: acetyl-arginyl-tyrosyl-tyrosyl-arginyl-tryptophanyl-lysyl-lysyl-lysyl-lysyl-lysyl-lysyl -Lysinamide (Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH 2 ), [0003] The chemical structural formula is: [0004] [0005] Molecular formula: C 85 h 141 N 27 o 15 [0006] Molecular weight: 1781.24. [0007] ATL peptide is in Ac-RYYRWK-NH 2 Hexapeptide is modified on the basis of hexapeptide, which is a peptide selectively screened by NOP (Nociceptin / orphanin FQ peptide receptor) from a synthetic combinatorial chemical peptide library. Orphanin (nocieeptin or orphanin FQ) is an endogenous ligand of opioid receptor-like receptors (ORL1 or LC 132), which is i...

Claims

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Application Information

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IPC IPC(8): C07K1/04C07K1/06
CPCY02P20/55
Inventor 厉保秋
Owner 济南环肽医药科技有限公司
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