Method for synthesizing and purifying linaclotide

A technology of linaclotide and purification method, which is applied in the field of synthesizing linaclotide, can solve the problems of unsuitability for scale-up production, complicated process, low total yield, etc., and achieves practical value and application prospect, good purity, improved The effect of total yield

Inactive Publication Date: 2016-11-30
ZHEJIANG PEPTITES BIOTECH CO LTD
View PDF7 Cites 16 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Compared with patents CN105017387A and CN104974229A, the total yield of patent CN104231051A is lower, but considering the complexity of the process, the other t

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the synthesis of Fmoc-Tyr (tBu)-Wang resin

[0041] Take 600 g of Wang resin with a substitution degree of 0.6 mmol / g, and add DMF to swell the resin. Take 0.72mol Fmoc-Tyr(tBu)-OH, dissolve it with an appropriate amount of DMF, add it to the above resin, stir evenly, then add 0.72mol DIC, 0.72molHOBt, 0.072mol DMAP, stir for 5 hours, and filter off the reaction solution. After washing 3 times with DMF, and 3 times with DCM, Fmoc-Tyr(tBu)-Wang resin was obtained. Its substitution value is 0.41 mmol / g.

Embodiment 2

[0042] Embodiment 2: Preparation of linaclotide resin

[0043] Take 0.2 mol of the Fmoc-Tyr(tBu)-Wang resin of Example 1, deprotect it with 20% PIP / DMF solution for 20 minutes, wash and filter to obtain the H-Tyr(tBu)-Wang resin from which Fmoc has been removed.

[0044] Take 0.25mol Fmoc-Cys(tBu)-OH, dissolve it with an appropriate amount of DMF, and add it to the reactor equipped with the above-mentioned H-Tyr(tBu)-Wang resin, and take another 0.24mol HBTU and 0.25mol DIPEA, and add them slowly Into the above-mentioned reactor which is blown with nitrogen. Coupling reaction for 30-120 minutes, the end point of the reaction is determined by the ninhydrin method, washed and filtered, then deprotected with 20% PIP / DMF solution for 20 minutes, washed and filtered to obtain H-Cys(Trt)-Tyr(tBu) -Wang Resin.

[0045] In the same way as above, the remaining protected amino acids in Table 3 were sequentially inserted to obtain the linaclotide peptide resin:

[0046] H-Cys(Trt)-Cys...

Embodiment 3

[0049] Example 3: Preparation of crude linaclotide linear peptide

[0050] Take the linaclotide resin obtained in Example 2, add the mixed acid solution with a volume ratio of TFA:EDT:Tis:H2O=89:5:1:5 (the amount is 8ml of acid solution per gram of linaclotide resin) , reacted on a shaking table at room temperature for 3 hours, filtered the reaction mixture with a sand core funnel, collected the filtrate, washed the resin 3 times with a small amount of TFA, added anhydrous ether to precipitate after combining the filtrate, washed the precipitate 3 times with anhydrous ether, and drained to obtain Off-white powder, vacuum-dried to constant weight, yielded 325.1 g of crude linaclotide linear peptide with a purity of 78.2%.

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

No PUM Login to view more

Abstract

The invention relates to the field of medicine synthesis, in particular to a method for synthesizing linaclotide. An amino acid sequence as shown in SEQ ID NO:1 by solid-phase synthesis is adopted, protecting groups are coupled to side chains of Thr, Cys, Asn, Tyr and Glu, and linaclotide resin of a resin carrier is coupled to a C-terminal; linaclotide resin pyrolysis is performed to remove all the protecting groups and the resin carrier, uncyclized linaclotide linear crude peptides are obtained, a guanidine hydrochloride oxidation system of Cystine/Cysteine performs oxidation reaction on the linaclotide linear crude peptides, three disulfide bonds are formed from an N-terminal to the C-terminal, crude linaclotide is obtained, and linaclotide is obtained after purification. The method starts from the aspect of the oxidation system, the method for producing linaclotide is improved, the total yield of linaclotide is improved through simple, convenient and rapid process steps, the total yield is 38% or above, the product purity is stabilized to be 99% or above, and single purities are controlled below 0.1%.

Description

technical field [0001] The invention relates to the field of pharmaceutical synthesis, in particular to a method for synthesizing linaclotide. Background technique [0002] Linaclotide (linaclotide), a new product developed for ironwood, is a new type of GC-C (intestinal cell uridylate cyclase C) receptor agonist. The compound is a polypeptide composed of 14 amino acids. obtained by phase synthesis technique. The FDA approved linaclotide for the treatment of adults with chronic idiopathic constipation and irritable bowel syndrome with constipation (IBS-C). The concentration of linaclotide in plasma is basically undetectable, but after binding to intestinal GC-C, it can lead to increased intracellular and extracellular cyclic guanosine monophosphate (cGMP) concentrations. Elevated intracellular cGMP can stimulate intestinal fluid secretion, accelerate gastrointestinal transit, and thus increase defecation frequency. The structural sequence of linaclotide is as follows: ...

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): C07K7/08C07K1/20C07K1/06C07K1/04
CPCY02P20/55C07K7/08
Inventor 刘志国李雪豪纪东亮秦德志
Owner ZHEJIANG PEPTITES BIOTECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap