Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of solid-phase synthesis method of ziconotide

A technology of solid-phase synthesis and ziconotide, which is applied in the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems of low yield, low accuracy of disulfide bond connection, and great difficulty in separation and purification, etc. problems, to achieve the effect of streamlining steps, easy preparation and purification, and the same number of steps

Active Publication Date: 2011-12-07
NANTONG SHIMEIKANG PHARMA CHEM
View PDF2 Cites 20 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The synthetic route II of ziconotide has the disadvantages of low accuracy and low yield of disulfide bonds, and the total yield is only 30%.
The final separation and purification is also very difficult

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of solid-phase synthesis method of ziconotide
  • A kind of solid-phase synthesis method of ziconotide
  • A kind of solid-phase synthesis method of ziconotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of Ziconotide Linear Peptide Resin.

[0041] Resin swelling: Weigh 1g of Fmoc-Rink-Amide-MBHA resin and place it in a 20ml BD syringe with a sieve plate (ordinary glass peptide reactor is also acceptable), and use 3 times the volume of the resin to swell with DCM for 1-4 hours until the resin is completely swollen After that, 20% (v / v) piperidine / DMF solution was used to remove the Fmoc protecting group twice for 5 min and 20 min respectively, and washed 6 times alternately with DCM and DMF.

[0042] Amino acid connection: Dissolve 2mmol of Fmoc-protected amino acid, 2mmol of HoBt, 2mmol of HBTU, and 4mmol of DIPEA in DMF, preactivate for 5 minutes, then add to the swollen resin (resin mass*substitution value=0.5mmol), shake at room temperature React for 1.5-2.5 hours, see Table 1 for the dosage and specific reaction time of each amino acid and condensing agent. Remove the Fmoc protection group before connecting the next amino acid with piperidin...

Embodiment 2

[0049] Example 2: Cleavage of resin and removal of some side chain protecting groups.

[0050] Mix trifluoroacetic acid, thioanisole, dimercaptoethane and anisole in a volume ratio of 90:5:2.5:2.5, or mix trifluoroacetic acid, methanol, water and triisopropylsilane in a volume ratio Mix 88:5:5:2, add the linear fully protected peptide resin prepared in Example 1 to any one of the above two mixed solutions, use a volume of 10 mL of the mixed solution per 1 g of linear fully protected peptide resin, shake After 2 hours of reaction, the reaction solution was poured into -20°C cold diethyl ether for precipitation. After centrifugation, the white precipitate was collected and air-dried to obtain a linear crude peptide containing Acm and tBu in the Cys side chain, weighing 1.4 g, and the crude yield was 94%.

Embodiment 3

[0051] Example 3: Formation of Ziconotide Bicyclic Peptide.

[0052] 1.0 g of the linear peptide prepared in Example 2 was dissolved in acetic acid so that the concentration of the linear peptide was 0.5 mg / mL, and then 10 times the molar amount of I 2 (dissolved in methanol), stirred and reacted at room temperature for 1 hour, then added 20% water of the reaction solution volume, and whether the reaction was complete was detected by HPLC. After the reaction was complete, 0.1M ascorbic acid was added until the solution became colorless, and the reaction mixture was frozen. After drying, the precipitate was washed with ether to obtain a crude bicyclic peptide containing tBu in the Cys side chain, weighing 0.91 g, and the crude yield of this step was 91%.

[0053] Analytical HPLC detection method is:

[0054] Equipment: C18 analytical column: 4.6×150mm;

[0055] Eluent A: 0.1% (v / v) TFA / H 2 O;

[0056] Eluent B: 0.08% (v / v) TFA / acetonitrile;

[0057] Flow rate: 1ml / min;

...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Wavelengthaaaaaaaaaa
Login to View More

Abstract

The invention discloses a solid-phase synthesis method of ziconotide, and the method comprises the following steps: with Fmoc(9-fluorenylmethyloxycarbonyl)-amino resin as a solid-phase carrier, successively carrying out condensation reaction for connecting 25 protected amino acids to obtain linear fully-protected peptide resin, wherein three groups of Cys (cysteine) with disulfide bond formed areseparately connected with Trt (triphenylmethyl), Acm (acetamidomethyl), or tBu (t-butyl) protecting group; cutting resin, and simultaneously removing all amino acid protecting groups except for Acm and tBu to obtain a linear peptide containing Acm and tBu; oxidizing the linear peptide to form a first pair of disulfide bonds, and simultaneously removing Acm and forming a second pair of disulfide bonds to obtain bicyclic peptide resin; and removing tBu of the bicyclic peptide resin, and simultaneously carrying out cyclization to form a third pair of disulfide bonds and to obtain ziconotide. In the method disclosed by the invention, Trt, Acm and tBu are selected to protect the three groups of Cys, thereby improving the formation accuracy of the disulfide bonds; and after resin is cut off, three pairs of disulfide bonds are sequentially formed through two-step reaction, thereby simplifying steps and improving productivity.

Description

technical field [0001] The invention relates to a solid phase synthesis method of ziconotide. Background technique [0002] Ziconotide (ziconotide), whose trade name is Prialt, is developed and marketed by Elan Corporation of the United States. It was first launched in the United States in January 2005, and was licensed by the European Union in February 2005. The main indications are: it has a strong analgesic effect on acute, chronic and neuropathic pain models in rodents, and can safely and effectively relieve the pain of malignant and non-malignant pain patients, including patients who are ineffective in opioid therapy and tolerated It has good properties and is also used for some patients with severe chronic pain who are poorly cured by other analgesic treatments. The drug effect is superior to other analgesic products, and its analgesic effect is 1000 times stronger than morphine when injected intrathecally, and there will be no side effects when using morphine, such a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/435C07K1/06C07K1/04C07K1/02
CPCY02P20/55
Inventor 董守良李梅兴
Owner NANTONG SHIMEIKANG PHARMA CHEM
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com