CN105884864A - Method for synthesizing linaclotide - Google Patents
Method for synthesizing linaclotide Download PDFInfo
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- CN105884864A CN105884864A CN201610328263.4A CN201610328263A CN105884864A CN 105884864 A CN105884864 A CN 105884864A CN 201610328263 A CN201610328263 A CN 201610328263A CN 105884864 A CN105884864 A CN 105884864A
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- linaclotide
- aqueous solution
- dmso
- crude product
- linear
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for synthesizing linaclotide, and relates to the field of medicine synthesis. The method mainly comprises the following steps: improving a linear cyclizing system of linaclotide, cyclizing crude linear peptide by using a ammonium carbonate/DMSO/aqueous solution together with hydroquinone or TCEP so as to obtain a crude linaclotide product, and purifying the crude linaclotide product, thereby obtaining a finished product. By improving the cyclizing system, the yield of linaclotide is increased, the cyclizing time is shortened, the cost of raw materials is lowered, and the method is applicable to industrial production.
Description
Technical field
The present invention relates to the preparation method of a peptide species, be specifically related to a kind of method synthesizing Linaclotide, belong to medicine
Synthesis field.
Background technology
Linaclotide (linaclotide), trade name Linzess, is first guanylate cyclase agonist (GCCA)
Class medicine, is researched and developed by American I ronwood company, obtains the approval listing of U.S. FDA in August, 2012.Linaclotide is one
14-amino acid peptide, after it is combined with the guanosine cyclic mono-phosphate being positioned in intestinal, makes intracellular and extracellular loop guanosine 5-monophosphate
(cGMP) concentration increases.It is generally acknowledged that the increase of intracellular cGMP concentration can stimulate the secretion of intestinal juice and promote that gastrointestinal is movable,
Thus cause defecation frequency to increase；Extracellular cGMP concentration rising can reduce the sensitivity of pain nerve, reduce intestinal pain.Profit
The effect of that Lip river peptide be by reduce visceral hypersensitivity and increase intestinal secretion, accelerate intestinal transport improve constipation symptom and
Alleviating stomachache, Linaclotide is also first medicine with this double action mechanism found at present.
Containing three disulfide bond in Linaclotide, its structure sequence is as follows:
In Linaclotide, the annulation of three disulfide bond is the difficult point place closing cost product, and each inventor has been also carried out many
Explore.
Chinese patent CN104844693A discloses a kind of method synthesizing Linaclotide, and it passes through solid phase synthesis at resin
Upper synthesizing linear peptide, acidolysis deprotection base and resin carrier obtain Linaclotide linearly thick peptide, use cysteine salt/DMSO
Linear thick peptide is cyclized by buffer solution, obtains Linaclotide crude product, and purifying crude gets product after turning acetate.Want
Reaching the productivity described in this invention and shorter cyclisation time, it is higher to the purity requirement of linear thick peptide, its linear thick peptide
Purity is more than 80%, and the technological deficiency such as its expensive starting materials.
For the deficiencies in the prior art, the invention provides a kind of method synthesizing Linaclotide, the method for the invention
Mainly linear by improving Linaclotide cyclisation system, uses amine carbonate/additional hydroquinone of DMSO/ aqueous solution or TCEP
Linear thick peptide is cyclized by (Chinese is entitled: three (2-carboxyethyl) phosphine), obtains Linaclotide crude product, i.e. obtains into after purifying crude
Product.The present invention, by the improvement to cyclisation system, reduces the reaction condition i.e. present invention purity to Linaclotide linearly thick peptide
Less demanding, improve the yield of Linaclotide, shorten the cyclisation time, be suitable for industrialized production.
Summary of the invention
The invention provides a kind of method synthesizing Linaclotide.
The present invention adopts the following technical scheme that
By the linearly thick peptide of the Linaclotide without protection group, join in amine carbonate/DMSO/ aqueous solution, be subsequently adding benzene two
Phenol or TCEP are cyclized, and are cyclized 1-2 hour, obtain Linaclotide crude product.Wherein the consumption of amine carbonate/DMSO/ aqueous solution is
Every 1 gram of linear crude product of Linaclotide uses 50-70mL amine carbonate/DMSO/ aqueous solution；Amine carbonate/each composition of DMSO/ aqueous solution
Ratio is 100mg ammonium carbonate/1mlDMSO/10ml water；Hydroquinone is 0.8-with the mass ratio of the linear crude product of Linaclotide
1.5:1；TCEP is 0.5-1:1 with the mass ratio of the linear crude product of Linaclotide.By Linaclotide crude product through preparative high-efficient liquid
After phase chromatographic separation and purification, lyophilization obtains Linaclotide sterling.Sterling being detected, purity is more than 98%, the response rate
70%-90%。
Cyclisation system used by the present invention is simple, and reaction condition is easy, it is adaptable to Li Luona peptide linearly thick peptide purity is at 30-
Raw material between 85%, the yield of Linaclotide is high, and the cyclisation time is short, is suitable for industrialized production.
Embodiment one:
Weighing the 1g Linaclotide without protection group linearly thick peptide, carry out purity detecting, obtaining linear thick peptide purity is 45%, adds
Enter to 55mL in the amine carbonate/DMSO/ aqueous solution being equipped with, amine carbonate/DMSO/ aqueous solution be according to 100mg ammonium carbonate/
The ratio of 1mlDMSO/10ml water prepares in advance.In solution, add 0.8g hydroquinone the most again start cyclisation, be cyclized 2
Hour, the oxidation of three pairs of disulfide bond can be completed, filter.Linaclotide crude product solution after filtering, injects preparative efficient
Chromatograph of liquid.Preparation condition be A phase be the TFA aqueous solution of 0.1%, B phase is the TFA acetonitrile solution of 0.1%, select C18 diameter
50MMDAC prepares post, flow velocity be 40ml/min column temperature be 25 degree.Collecting flowing phase, lyophilization obtains Linaclotide sterling, weighs
For 317mg.Sterling being detected, purity is 99.3%, and the response rate is 70.4 %.
Embodiment two:
Weighing the 1g Linaclotide without protection group linearly thick peptide, carry out purity detecting, obtaining linear thick peptide purity is 65%, adds
Enter to 70mL in the amine carbonate/DMSO/ aqueous solution being equipped with, amine carbonate/DMSO/ aqueous solution be according to 100mg ammonium carbonate/
The ratio of 1mlDMSO/10ml water prepares in advance.In solution, add 0.5gTCEP the most again start cyclisation, be cyclized 1 hour,
The oxidation of three pairs of disulfide bond can be completed, filter.Linaclotide crude product solution after filtering, injects preparative high-efficient liquid phase color
Spectrometer.Preparation condition be A phase be the TFA aqueous solution of 0.1%, B phase is the TFA acetonitrile solution of 0.1%, select C18 diameter 50MMDAC
Prepare post, flow velocity be 40ml/min column temperature be 25 degree.Collecting flowing phase, lyophilization obtains Linaclotide sterling, weighs and is
579mg.Sterling being detected, purity is more than 99.1%, and the response rate is 89%.
Embodiment three:
Weighing the 1g Linaclotide without protection group linearly thick peptide, carry out purity detecting, obtaining linear thick peptide purity is 45%, adds
Enter to 55mL in the amine carbonate/DMSO/ aqueous solution being equipped with, amine carbonate/DMSO/ aqueous solution be according to 100mg ammonium carbonate/
The ratio of 1mlDMSO/10ml water prepares in advance.In solution, add 0.8g hydroquinone the most again start cyclisation, be cyclized 6
Hour, filter.Linaclotide crude product solution after filtering, injects preparative high performance liquid chromatography instrument.Preparation condition is that A phase is
The TFA aqueous solution of 0.1%, B phase is the TFA acetonitrile solution of 0.1%, selects C18 diameter 50MMDAC to prepare post, and flow velocity is 40ml/
Min column temperature is 25 degree.Collecting flowing phase, lyophilization obtains Linaclotide sterling, weighs as 321mg.Sterling is detected, pure
Degree is 99 %, and the response rate is 71.3 %.Compared with embodiment one, this experiment is by extending the cyclisation time, and Li Luona peptide is final
Purity and the response rate be basically unchanged, illustrate that, in the response time of first 2 hours, the cyclization of Li Luona peptide is the completeest
Entirely.
Claims (4)
1. the method synthesizing Linaclotide, it is characterised in that: by the linearly thick peptide of the Linaclotide without protection group, join
In amine carbonate/DMSO/ aqueous solution, add hydroquinone or TCEP carries out cyclisation and obtains Linaclotide crude product.
Method the most according to claim 1, it is characterised in that the consumption of described amine carbonate/DMSO/ aqueous solution is every 1 gram
The linear crude product of Linaclotide uses 50-70mL amine carbonate/DMSO/ aqueous solution；Described hydroquinone and the linear crude product of Linaclotide
Mass ratio be 0.8-1.5:1；Described TCEP is 0.5-1:1 with the mass ratio of the linear crude product of Linaclotide.
Method the most according to claim 1 and 2, it is characterised in that the proportioning of described amine carbonate/DMSO/ aqueous solution is:
100mg ammonium carbonate/1mlDMSO/10ml water.
Method the most according to claim 1, it is characterised in that the cyclisation time is 1-2 hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610328263.4A CN105884864A (en) | 2016-05-18 | 2016-05-18 | Method for synthesizing linaclotide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610328263.4A CN105884864A (en) | 2016-05-18 | 2016-05-18 | Method for synthesizing linaclotide |
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| CN105884864A true CN105884864A (en) | 2016-08-24 |
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| CN201610328263.4A Withdrawn CN105884864A (en) | 2016-05-18 | 2016-05-18 | Method for synthesizing linaclotide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107936094A (en) * | 2017-12-30 | 2018-04-20 | 江苏诺泰澳赛诺生物制药股份有限公司 | The synthetic method that a kind of solid liquid phase of Li Laluo peptides is combined |
| CN111732632A (en) * | 2020-07-16 | 2020-10-02 | 台州吉诺生物科技有限公司 | Synthesis method of linaclotide |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709082A (en) * | 2009-12-08 | 2010-05-19 | 深圳市翰宇药业有限公司 | Method for preparing ziconotide |
| CN102875655A (en) * | 2012-09-29 | 2013-01-16 | 深圳翰宇药业股份有限公司 | Linaclotide synthesis method |
| CN103304655A (en) * | 2013-05-27 | 2013-09-18 | 成都圣诺生物制药有限公司 | Method for preparing ziconotide |
| CN104231051A (en) * | 2013-06-06 | 2014-12-24 | 深圳翰宇药业股份有限公司 | Preparation method for linaclotide |
| CN104341496A (en) * | 2013-08-09 | 2015-02-11 | 深圳翰宇药业股份有限公司 | Method for synthesizing leconotide |
| CN104844693A (en) * | 2015-06-10 | 2015-08-19 | 成都圣诺生物科技股份有限公司 | Method for synthesizing linaclotide |
| CN104974229A (en) * | 2015-07-06 | 2015-10-14 | 泰州施美康多肽药物技术有限公司 | Linaclotide solid-phase synthesis method |
| CN105017387A (en) * | 2015-07-31 | 2015-11-04 | 济南康和医药科技有限公司 | Method for preparing linaclotide |
-
2016
- 2016-05-18 CN CN201610328263.4A patent/CN105884864A/en not_active Withdrawn
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709082A (en) * | 2009-12-08 | 2010-05-19 | 深圳市翰宇药业有限公司 | Method for preparing ziconotide |
| CN102875655A (en) * | 2012-09-29 | 2013-01-16 | 深圳翰宇药业股份有限公司 | Linaclotide synthesis method |
| CN103304655A (en) * | 2013-05-27 | 2013-09-18 | 成都圣诺生物制药有限公司 | Method for preparing ziconotide |
| CN104231051A (en) * | 2013-06-06 | 2014-12-24 | 深圳翰宇药业股份有限公司 | Preparation method for linaclotide |
| CN104341496A (en) * | 2013-08-09 | 2015-02-11 | 深圳翰宇药业股份有限公司 | Method for synthesizing leconotide |
| CN104844693A (en) * | 2015-06-10 | 2015-08-19 | 成都圣诺生物科技股份有限公司 | Method for synthesizing linaclotide |
| CN104974229A (en) * | 2015-07-06 | 2015-10-14 | 泰州施美康多肽药物技术有限公司 | Linaclotide solid-phase synthesis method |
| CN105017387A (en) * | 2015-07-31 | 2015-11-04 | 济南康和医药科技有限公司 | Method for preparing linaclotide |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107936094A (en) * | 2017-12-30 | 2018-04-20 | 江苏诺泰澳赛诺生物制药股份有限公司 | The synthetic method that a kind of solid liquid phase of Li Laluo peptides is combined |
| CN111732632A (en) * | 2020-07-16 | 2020-10-02 | 台州吉诺生物科技有限公司 | Synthesis method of linaclotide |
| CN111732632B (en) * | 2020-07-16 | 2021-12-21 | 台州吉诺生物科技有限公司 | Synthesis method of linaclotide |
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