Solid phase synthesis method of ZP120

A technology of ZP120 and solid-phase synthesis, which is applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems of long production cycle, lengthy route, high cost, etc., and achieve industrial production, good product quality, and low spin effect

Inactive Publication Date: 2009-04-15
ADLAI NORTYE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The liquid phase synthesis method has (1) many reaction steps and lengthy routes
(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 reagents and amino acid side chain protecting groups used are relatively difficult
[0010] Merrifield type resin is used as the carrier. After synthesizing the protective ZP120 resin, the conditions required to cut the peptide from the resin are relatively harsh. This method uses HF cracking method, and a HF gas generating device is required to achieve cracking. For a large number of peptide resins Can only be cracked twice, 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
[0011] 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 method of ZP120
  • Solid phase synthesis method of ZP120
  • Solid phase synthesis method of ZP120

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Preparation of Fmoc-Lys(Boc)-Rink Amide MBHA

[0053] operate:

[0054] (1) Put 50g (30mmol) of Fmoc-Rink Amide-MBHA resin in the polypeptide synthesis reactor, alternately wash twice with 300ml of dichloromethane and 300ml of ethanol, 2 minutes each time, and drain. Add 500 ml of 20% (v / v) piperidine / DMF solution, stir and react at room temperature for 20 minutes, and drain. The resin was alternately washed 3 times with dichloromethane and 300ml ethanol, 2 minutes each time, and drained. Kaiser reagent test, the result was positive, Fmoc- has been removed.

[0055] (2) After dissolving 70.28g of Fmoc-Lys(Boc)-OH and 20.25g of HOBt with 500ml of dichloromethane / DMF solution (the volume ratio of dichloromethane and DMF is 1:1, the same below), add them to the resin, and then Add 23 ml of N,N-diisopropylcarbodiimide dropwise, and stir at room temperature for 5 hours after the drop is complete.

[0056] (3) Drain. The resin was alternately washed 3 times...

Embodiment 2

[0059] Embodiment 2: Preparation of Fmoc-Lys(Boc)-Lys(Boc)-Rink Amide MBHA

[0060] operate:

[0061] (1) Add 500 ml of 20% piperidine / DMF solution to the resin, stir and react at room temperature for 20 minutes, and drain. The resin was alternately washed 3 times with dichloromethane and 300ml ethanol, 2 minutes each time, and drained. Kaiser reagent test, the result was positive, Fmoc- has been removed.

[0062] (2) After dissolving 70.28g of Fmoc-Lys(Boc)-OH and 20.25g of HOBt with 500ml of dichloromethane / DMF solution, add it to the resin, then add 23ml of N,N-diisopropylcarbodiimide dropwise, After the drop was completed, it was stirred at room temperature for 5 hours.

[0063] (3) Drain. The resin was alternately washed 3 times with 300ml of dichloromethane and 300ml of ethanol, each time for 2 minutes, and drained.

[0064] (4) Kaiser reagent was used for color development, and the test was negative. The reaction is over.

[0065] Get: Fmoc-Lys(Boc)-Lys(Boc)-Rink...

Embodiment 3

[0066] Embodiment 3: Preparation of Fmoc-Lys(Boc)-Lys(Boc)-Lys(Boc)-Rink Amide MBHA

[0067] operate:

[0068] (1) Add 550 ml of 20% piperidine / DMF solution to the resin, stir and react at room temperature for 20 minutes, and drain. The resin was alternately washed 3 times with dichloromethane and 350 ml of ethanol, 2 minutes each time, and drained. Kaiser reagent test, the result was positive, Fmoc- has been removed.

[0069] (2) After dissolving 70.28g of Fmoc-Lys(Boc)-OH and 20.25g of HOBt with 500ml of dichloromethane / DMF solution, add it to the resin, then add 23ml of N,N-diisopropylcarbodiimide dropwise, After the drop was completed, it was stirred at room temperature for 5 hours.

[0070] (3) Drain. The resin was alternately washed 3 times with 350ml of dichloromethane and 350ml of ethanol, each time for 2 minutes, and drained.

[0071] (4) Kaiser reagent was used for color development, and the test was negative. The reaction is over.

[0072] Get: Fmoc-Lys(Boc)-...

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Abstract

The invention provides a solid phase synthetic method of ZP120: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2, which uses Fmoc-Rink Amide-MBHA resin as a material to carry out procedure reaction, and the salt of the ZP120 can be obtained by sequentially connecting amino protecting groups and then removing the groups, acidifying, and removing and cutting side chain protecting groups and resin, and then the ZP120 can be obtained further. The beneficial effect of the invention mainly embodies on: (1) the invention that uses the Fmoc Rink Amide-MBHA resin as a solid phase synthetic carrier is low in cost; (2) N,N-diisopropyl carbodiimide is used as a peptide synthesis condensing agent, and the product process is low in racemization, and the product is good in quality and high in yield; (3) trifluoroacetate is used for cutting the ZP120 peptide resin, and the form of peptide amide can be obtained directly after the cutting, and the subsequent treatment is simple; and (4) the technique is simple and systematic, and is beneficial to industrialization production.

Description

technical field [0001] The present invention relates to a preparation method of polypeptide drug ZP120. It belongs to the field of biotechnology. Background technique [0002] ZP120 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-NH2) [0003] The chemical structural formula is: [0004] [0005] Molecular formula: C 85 h 141 N 27 o 15 [0006] Molecular weight: 1781.24 [0007] Orphanin (nocieeptin or orphanin FQ) was discovered at the end of 1995. It is an endogenous ligand of opioid receptor-like receptors (ORL1 or LC 132), and plays a role in pain regulation, cardiovascular system, ion channels, dependence and tolerance , learning and memory have a wide range of biological activities. ZP120 is a novel ligand polypeptide for NOP (Nociceptin / orphanin FQ peptidereceptor) receptor designed by SIP (Structure inducing probes) technology. Ac-RYYRW...

Claims

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Application Information

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IPC IPC(8): C07K1/06C07K1/04C07K7/08
CPCY02P20/55
Inventor 李新宇杨东晖
Owner ADLAI NORTYE BIOPHARMA CO LTD
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