Method for preparing ziconotide by solid-liquid combination

A ziconotide, solid-liquid phase technology, applied in the field of peptide compound synthesis, can solve the problems of long synthesis cycle, low product yield, unfavorable industrialization amplification, and high production cost, and achieve easy operation, shorten synthesis cycle, improve purity and Yield effect

Inactive Publication Date: 2018-12-18
滨海吉尔多肽有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to provide a method for preparing ziconotide by combining solid-liquid phases, which mainly solves the technica

Method used

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  • Method for preparing ziconotide by solid-liquid combination
  • Method for preparing ziconotide by solid-liquid combination
  • Method for preparing ziconotide by solid-liquid combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Boc-Cys(trt)-Lys(boc)-Gly-Lys(boc)-Gly-Ala-Lys(boc)-Cys(trt)-Ser(tbu)-Arg(pbf)-Leu-Met - Preparation of Tyr(tbu)-Asp(otbu)-COOH;

[0054] Weigh 20g (0.3mmol / g) of Fmoc-Asp(otbu)-2Cl-Trt-Cl-resin and place it in the reaction column, add 150ml of 20% piperidine / DMF solution by volume percentage, react for 30 minutes, and drain. Wash with DMF for 5 times, then weigh the protected amino acid and condensing agent, add to the reaction column, then add 100ml of DMF, react for 30-60 minutes, detect the end of the reaction with ninhydrin, drain the reaction solution after the reaction, wash with DMF for 3 times, Repeat the cycle until the last amino acid is connected to get Boc-Cys(trt)-Lys(boc)-Gly-Lys(boc)-Gly-Ala-Lys(boc)-Cys(trt)-Ser(tbu)-Arg (pbf)-Leu-Met-Tyr(tbu)-Asp(otbu)-2Cl-Trt-Cl-resin 36g.

[0055] The amount of amino acid added in each step of condensation reaction is respectively:

[0056] Fmoc-Tyr(tbu)-OH 5.52g,

[0057] Fmoc-Met-OH 4.46g,

[0058] ...

Embodiment 2

[0071] Embodiment 2: Preparation of Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Sbzl;

[0072] Get 4.2g of fully protected polypeptide fragment A in Example 1 and place it in a flask, add 100ml DMF to dissolve, then add 0.37g benzyl mercaptan, 1.24g HCTU, 5ml DIEA, react for 1 hour, add water to separate out a white solid, filter to remove the filtrate , after the filter cake is dried, add 200ml of secondary lysate, the ratio (V%) consists of: 87.5% TFA + 5% sulfide anisole + 2.5% ethanedithiol + 2.5% phenol + 2.5% water, the reaction After 2 hours, 800ml of ether was added to crystallize, and a white solid was precipitated. After drying, the polypeptide fragment A was obtained: Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Sbzl 2.25 g, MW: 1666, see product mass spectrum figure 1 .

Embodiment 3

[0073] Embodiment 3: Preparation of Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Sbzl;

[0074] Get 4.2g of fully protected polypeptide fragment A in Example 1 and place it in a flask, add 100ml NMP to dissolve, then add 0.37g benzyl mercaptan, 1.24g HCTU, 5ml DIEA, react for 2 hours, add water to separate out a white solid, filter to remove the filtrate , after the filter cake is dried, add 200ml of secondary lysate, the ratio (V%) consists of: 87.5% TFA + 5% sulfide anisole + 2.5% ethanedithiol + 2.5% phenol + 2.5% water, the reaction After 2 hours, 800ml of ether was added to crystallize, and a white solid was precipitated. After drying, the polypeptide fragment A was obtained: Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Sbzl 2.35 g.

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Abstract

The invention discloses a method for preparing ziconotide by solid-liquid combination and mainly solves the technical problems that an existing synthesizing method has a long synthesizing period, highproduction cost, a low product yield and no favorability for later industrialized enlargement. The method disclosed by the invention comprises the following steps: (1) preparing modified polypeptidefragment A by solid-liquid combination; (2) preparing polypeptide fragment B by a solid phase method; (3) linking the modified polypeptide fragment A to the polypeptide fragment B by a natural linkingmethod under the liquid phase condition to prepare and obtain a ziconotide linear peptide crude product solution; (4) adding an oxidizing agent under the same system oxidizing to obtain a ziconotidecrude product water solution and purifying and dry freezing through a high performance liquid chromatography to obtain a ziconotide fine product.

Description

technical field [0001] The invention relates to the technical field of polypeptide compound synthesis, in particular to a method for preparing ziconotide through solid-liquid phase combination. Background technique [0002] Ziconotide, the English name of Ziconotide, is a polypeptide containing 3 disulfide bonds and 25 amino acids. Molecular formula: C 102 h 172 N 36 o 32 S 7 , molecular weight: 2639.13, sequence and structure as shown in formula I [0003] [0004] Formula 1 [0005] Ziconotide is a nonopioid analgesic indicated for use in patients with severe chronic pain who are administered intrathecally and who are intolerant or refractory to other treatments such as systemic analgesics, adjuvant therapy, or intrathecal morphine. Ziconotide is a polypeptide containing 3 disulfide bonds and 25 amino acids. This product binds to the N-type calcium channel on the primary nociceptive afferent nerve located in the superficial layer of the dorsal horn of the spinal...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/20C07K1/06C07K1/04C07K1/02
CPCC07K14/43504Y02P20/55
Inventor 徐红岩竺剑峰
Owner 滨海吉尔多肽有限公司
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