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Efficient ziconotide synthesis and preparation method

A high-efficiency technology of ziconotide, applied in the field of polypeptide drug preparation, can solve the problems of unfavorable large-scale production of ziconotide, long synthesis cycle, and many synthesis steps, and achieve industrial production, short synthesis cycle and simple operation Effect

Inactive Publication Date: 2017-03-15
合肥国肽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The existing ziconotide preparation method has a long synthesis period, high production cost and low product yield, which is not conducive to the large-scale production of ziconotide
Application publication number CN 104974237 A and a method for solid-phase synthesis of ziconotide by fragment method. First, four fragment peptides [19-25]A, [12-18]B, [12-18]B, and [6-11]C, [1-5]D; then connect the fully protected peptide fragments B, C, and D to the peptide resin A in sequence, and crack to obtain the linear peptide of ziconotide; finally, through mild conditions, the operation Streamlined, easy post-processing, and one-step oxidation in an environmentally friendly liquid phase to obtain ziconotide; the disadvantages of this scheme are: the four fragment peptides of ziconotide are synthesized in a distributed manner, with many synthesis steps and cumbersome operation

Method used

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  • Efficient ziconotide synthesis and preparation method
  • Efficient ziconotide synthesis and preparation method
  • Efficient ziconotide synthesis and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Synthesis of Ziconotide Precursor Peptide-Amino Resin

[0038] 1. Swelling of amino resin

[0039] Weigh 0.1g of Rink-MBHA-Resin with a degree of substitution of 0.38mmol / g, add it to the polypeptide synthesis reactor from the open end, take the DCM reagent and add it to the reactor, so that the resin is completely immersed in the DCM solvent, fully mixed with the solvent Contact, swelling for 2h.

[0040] 2. Synthesis of ziconotide precursor peptide-amino resin

[0041] Ziconotide precursor peptide-amino resin is:

[0042] Fmoc-CKGKGAKCSRLMYDCCTGSCRSGKC-Rink Resin

[0043] The protected amino acids used in this example are listed in Table 1 below for the protected amino acids and molecular weights corresponding to the 1-25th amino acid from the resin:

[0044] Table 1

[0045]

[0046]

[0047]Some commonly used abbreviations in the present invention have the following meanings:

[0048]

[0049] Activation methods for protected amino acids

[0050] Tak...

Embodiment 2

[0055] Synthesis of Ziconotide Precursor Peptide Ⅰ-Amino Resin

[0056] The ziconotide precursor peptide-amino resin was eluted to remove the side chain protecting groups of Cys8 and Cys20, and the elution reaction reagent was a mixed solution of 0.5% TFA+10% dichloroacetic acid+DCM, and the elution reaction was 20min Drain the eluate, then form the first pair of disulfide bonds of ziconotide with the solution cyclization reaction of DCM:DMF=1:1, add triethylamine to adjust the pH to 9, and obtain the ziconotide precursor Peptide I - amino resin.

Embodiment 3

[0058] Synthesis of Ziconotide Precursor Peptide Ⅱ

[0059] The ziconotide precursor peptide I-amino resin was eluted to remove the side chain protecting groups of Cys15 and Cys25, and the elution reagent was 6% TFA + 2.5% H 2 O + 2.5% Tis + 1% EDT + 88% DCM mixed solution, elution reaction for 40 minutes; the reaction solution was centrifugally settled and freeze-dried with anhydrous ether pre-cooled at -20°C, and the obtained sample was reversed-phase high-efficiency Purified by liquid chromatography, freeze-dried to obtain a pure product sample; the sample was dissolved and then dissolved in (NH 4 ) 2 CO 3 Adjust the pH to 8.6 to carry out the second disulfide bond cyclization reaction of ziconotide, and adjust the pH to 5 with 5% acetic acid aqueous solution to obtain the ziconotide precursor peptide II.

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Abstract

The invention discloses an efficient ziconotide synthesis and preparation method. The efficient ziconotide synthesis and preparation method comprises the following synthesis and preparation steps: enabling reaction between amino resin subjected to swelling, de-protection and washing and an activated protected amino acid solution in a constant-temperature oscillator, bonding protected amino acids corresponding to the first to the twenty-fifth amino acids from the resin in sequence to obtain ziconotide precursor peptide-amino resin, removing side-chain protecting groups of cysteine separately, performing disulfide bond cyclization reaction, purifying and freeze-drying to obtain ziconotide pure product polypeptide. The method adopts common, easily available and low-cost raw materials and reagents to perform synthesis and preparation, the yield of the ziconotide pure product reaches 30 to 40 percent through process optimization, and the yield of the ziconotide is greatly increased; and the preparation method is simple to operate, convenient to sample in the reaction process, easy in central control, short in synthesis cycle, low in production cost, high in product yield and favorable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of preparation methods of polypeptide medicines, and in particular relates to a high-efficiency synthesis and preparation method of ziconotide. Background technique [0002] Chinese name: Ziconotide [0003] English name: Ziconotide [0004] Molecular formula C 102 h 172 N 36 o 32 S 7 [0005] Molecular weight: 2639.13 [0006] Structural formula: [0007] H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys- Cys-NH2 [0008] [0009] Ziconotide (Ziconotide) is a synthetic non-atropine analgesic polypeptide drug, and it is the first new type of neuron-specific non-morphine analgesic used in clinical practice. It achieves the purpose of analgesia by blocking N-type calcium ion channels, and can relieve pain that other treatments including intrathecal injection of morphine are ineffective, and long-term use of the drug will not produce tolerance and ad...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/06C07K1/04
CPCY02P20/55C07K14/43504
Inventor 郑范娜陈为光
Owner 合肥国肽生物科技有限公司
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