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Solid-phase synthesis method of Ziconotide

A solid-phase synthesis, ziconotide technology, applied in the field of biochemistry, can solve the problems of low oxidation intensity of dimethyl sulfoxide and anisole, difficult to repeat tricyclic peptides, long reaction time, etc. Economical and practical value, easy preparation and purification, single product effect

Inactive Publication Date: 2013-08-14
Jiangsu Shimeikang Pharmaceutical Co Ltd
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the first two disulfide bonds are formed at the same time, three isomers with different disulfide bond connection methods will be formed, and these three isomers have little difference in polarity, which makes it difficult to separate them in HPLC preparation and purification, resulting in The final product has low purity and many impurities
Moreover, the oxidation intensity of dimethyl sulfoxide and anisole is not high, the process of forming tricyclic peptide is difficult to repeat, and the reaction time is long, and it is easy to form dimers

Method used

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  • Solid-phase synthesis method of Ziconotide
  • Solid-phase synthesis method of Ziconotide
  • Solid-phase synthesis method of Ziconotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Preparation of Ziconotide Linear Fully Protected Peptide Resin.

[0061] (1) Resin swelling: Weigh 0.5g of Fmoc-Rink Amide-MBHA resin (SD=0.5mmol / g, 0.25mmol), add it to a 20ml BD syringe with a sieve plate, and use dimethylformamide and dimethicone Chloromethane was alternately washed 2 times each, 5ml / time, 3 minutes / time, and drained. Add dichloromethane twice the volume of the resin, shake at 640r / min for about 2 hours, and remove the dichloromethane by suction filtration. Then add dimethylformamide to wash the resin 3 times, 5ml / time, 3 minutes / time, and drain.

[0062] (2) Removal of the Fmoc protecting group: 5 ml of 20% piperidine / DMF (v / v) solution was added to the reactor, stirred at room temperature for 5 minutes, and drained. Repeat twice for 5 minutes and 15 minutes respectively. The ninhydrin method was used to detect the removal of Fmoc. Then alternately wash with dimethylformamide and dichloromethane 3 times each, 5 ml / time, 3 minutes / time...

Embodiment 2

[0070] Example 2: Cleavage of Ziconotide Linear Fully Protected Peptide Resin.

[0071] (1) Add the linear fully protected peptide resin obtained in Example 1 into a 50ml round bottom flask, and configure cutting agent 1 (mix trifluoroacetic acid, phenol, water, and TIS according to the volume ratio of 88:5:5:2) or Cutting agent 2 (mix trifluoroacetic acid, thioanisole, ethanedithiol, and anisole in a volume ratio of 90:5:3:2), add 12ml (10ml / g) cutting agent to the round bottom flask, 720r / min shaking reaction for 2 hours.

[0072] (2) After the cutting reaction is completed, drop the cutting solution into 10 times the volume of glacial ether (-20°C), then add 2 to 3ml of cutting agent 1 or 2 to the reactor to clean the resin, drop it into glacial ether, and A large number of peptides were precipitated and left to settle for half an hour. Centrifuge at 8000r / min at 4°C for 15min. Discard the supernatant, re-add glacial ether, oscillate and ultrasonically dissolve, balance,...

Embodiment 3

[0073] Example 3: Preparation of Ziconotide Single Disulfide Ring Crude Peptide.

[0074] (1) Add 30 mg of the linear peptide obtained in Example 2 into a 100 ml round-bottomed flask, add 60 ml of 5% acetic acid aqueous solution to dissolve to a concentration of 0.5 mg / ml, adjust the pH to 6 with a saturated ammonium carbonate aqueous solution, add 15 ml of DMSO, 640 r / ml Min room temperature shaking reaction for 2 hours.

[0075] (2) After the reaction is over, add 150ml of 0.05% trifluoroacetic acid-5% acetonitrile solution to terminate the reaction. The solution was lyophilized to obtain 28.5 mg of crude ziconotide monodisulfide ring peptide, with a crude yield of 95%.

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Abstract

The invention discloses a solid-phase synthesis method of Ziconotide. The method comprises the following steps of: performing condensation reaction by taking Fmoc-amino resin as a solid-phase carrier and connecting 25 side chain protected amino acids to obtain linear fully protected resin, wherein three groups of Cys which form disulfide bonds are connected with Trt, Acm or tBu protecting groups respectively; linearly cutting the resin and removing the side chain protected groups of other amino acids except for Cys(Acm) and Cys(tBu); oxidizing linear peptide to form the first pair of disulfide bonds to obtain single disulfide cyclopeptide; removing the Acm protected group of the Cys(Acm) from the single disulfide cyclopeptide and cyclizing to form the second pair of disulfide bonds to obtain double disulfide cyclopeptide; removing the tBu protected group of the Cys(tBu) from the double disulfide cyclopeptide and cyclizing to form the third pair of disulfide bonds to obtain triple disulfide cyclopeptide; and purifying and freeze-drying the triple disulfide cyclopeptide to obtain the Ziconotide.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a solid phase synthesis method of ziconotide. Background technique [0002] Ziconotide (ziconotide) is a synthetic form of ω-conotoxin MVIIA, which naturally exists in the South Pacific marine snail (Conusmagus). It is a cationic polypeptide composed of 25 amino acids, including 3 pairs of disulfide bonded 6 cysteine ​​residues with a molecular weight of 2639.12. [0003] It is a potent, specific, selective, and reversible N-type voltage-sensitive calcium channel (N-type voltage-sensitive calcium channels, N-type VSCCs) blocker, which can block primary pain afferent neurons in the spinal cord The influx of calcium ions inhibits the release of neurotransmitters such as substance P, calcitonin gene-related peptide, and glutamic acid, thereby preventing or reducing the transmission of pain signals, and has no obvious affinity for other ion channels. Intrathecal in...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/06C07K1/04C07K1/02
CPCY02P20/55
Inventor 董守良曹硕陈强常民彭雅丽
Owner Jiangsu Shimeikang Pharmaceutical Co Ltd
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