CN106167514A - The synthesis of a kind of Linaclotide and purification process - Google Patents
The synthesis of a kind of Linaclotide and purification process Download PDFInfo
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- CN106167514A CN106167514A CN201610739783.4A CN201610739783A CN106167514A CN 106167514 A CN106167514 A CN 106167514A CN 201610739783 A CN201610739783 A CN 201610739783A CN 106167514 A CN106167514 A CN 106167514A
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- linaclotide
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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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The present invention relates to medicine synthesis field; it is specifically related to a kind of method synthesizing Linaclotide; use solid phase synthesis at aminoacid sequence shown in SEQ ID NO:1, the protected base of coupling on Thr, Cys, Asn, Tyr, Glu side chain, have the Linaclotide resin of resin carrier in C end coupling；Linaclotide pitch shake solution removes all protection groups and resin carrier; obtain not cyclized Linaclotide linearly thick peptide; then the guanidine hydrochloride oxidation system using Cystine/Cysteine carries out oxidation reaction to Linaclotide linearly thick peptide; three disulfide bond are formed from N end to C end; obtain Linaclotide crude product, the most i.e. obtain Linaclotide.The method is set about in terms of oxidation system, improve the production method of Linaclotide, with easier, more efficiently processing step improve the total recovery of Linaclotide, total recovery is more than 38%, and product purity is stable more than 99%, single contaminant controls below 0.1%.
Description
Technical field
The present invention relates to medicine synthesis field, be specifically related to a kind of method synthesizing Linaclotide.
Background technology
Linaclotide (linaclotide) is the new product of ironwood exploitation, is a kind of novel GC-C(enterocyte
Uridylic acid cyclase C) receptor stimulating agent, this compound is the polypeptide of 14 aminoacid compositions, is obtained by solid phase synthesis technique.
U.S. FDA have approved Linaclotide for treating Adult chronic idiopathic constipation and constipation type irritable bowel syndrome (IBS-C).
Linaclotide concentration in blood plasma cannot be measured substantially, but after being combined with intestinal GC-C, can cause intracellular and extracellular loop
Guanyl (cGMP) concentration raises.Intracellular cGMP raises can stimulate intestinal fluid secretion, accelerates gastrointestinal tract and divides a word with a hyphen at the end of a line, thus increases
Stool.Linaclotide structure sequence is as follows:
H-Cys1-Cys2-Glu3-Tyr4-Cys5-Cys6-Asn7-Pro8-Ala9-Cys10-Thr11-Gly12-Cys13-Tyr14-OH
(3 pairs of disulfide bond are: 1-6,2-10,5-13).
Chinese patent CN104974229A discloses a kind of method synthesizing Linaclotide, and it is with ACM, Trt, tBu respectively
Cys 3 is synthesized by protection, and the later stage uses three-step approach oxidation to obtain Linaclotide finished product, its total recovery according to record the highest can
Reach 71.2%.
Chinese patent CN105017387A discloses a kind of method synthesizing Linaclotide, and first it synthesize 6 fragments of peptides, ring
Change the fragment obtained containing a pair disulfide bond；The Linaclotide peptide tree containing a pair disulfide bond it is condensed to yield again with another fragment peptide resin
Fat, obtains the thick peptide of Linaclotide containing a pair disulfide bond, the Linaclotide crude product after step-by-step oxidation is cyclized through cracking.
Purified acquisition Linaclotide.Its total recovery reaches as high as 59% according to record.But the method uses fragment condensation, step by step oxygen
Changing, it operates complexity, and cost is high, and the side of being not suitable for produces greatly.
Chinese patent CN104231051A discloses the preparation method of a kind of Linaclotide, enters with special Cys protection group
Row synthesis, uses EDTA and DMSO to carry out during oxidation, oxidization time is 8-15 hour, and its total recovery reaches as high as according to record
32.15%。
Comparing CN105017387A, CN104974229A patent, patent CN104231051A is relatively low in terms of total recovery, but
Being to consider complex process degree, other two techniques, due to complex process, are not suitable for amplifying production.Patent
CN104231051A uses special acid synthesis, relatively costly.How Simplified flowsheet on the basis of prior art, improves total receipts
Rate, avoids using special material simultaneously, reduces production cost, develop and be easy to amplify the technique produced, be our mainly grind
Study carefully direction.
Summary of the invention
In view of this, it is an object of the invention to provide a kind of method synthesizing Linaclotide so that side of the present invention
Method can significantly simplify production technology, improves the total recovery of Linaclotide, reduces the volume of oxidation system simultaneously, is suitable for amplifying
Produce.
For achieving the above object, the present invention provides following technical scheme:
A kind of method synthesizing Linaclotide, comprises the following steps:
Step 1, solid phase synthesis are at aminoacid sequence shown in SEQ ID NO:1, and on Thr, Cys, Asn, Tyr, Glu side chain, coupling has
Protection group, has the Linaclotide resin of resin carrier in C end coupling.
Step 2, Linaclotide pitch shake solution remove all protection groups and resin carrier, obtain not cyclized Linaclotide line
Property thick peptide, then use the guanidine hydrochloride oxidation system of Cystine/Cysteine that Linaclotide linearly thick peptide is carried out oxidation anti-
Should, according to the amino acid sequence of its N end to C end, form Cys1 and Cys6, Cys2 and Cys10, the disulfide bond of Cys5 and Cys13,
Obtain Linaclotide crude product, the most i.e. obtain Linaclotide.
In the method for the invention, according to amino acid sequence (the i.e. SEQ of Linaclotide linear peptides main chain N end to C end
Aminoacid sequence shown in ID NO:1), such as following formula:
H-Cys1-Cys2-Glu3-Tyr4-Cys5-Cys6-Asn7-Pro8-Ala9-Cys10-Thr11-Gly12-Cys13-Tyr14-OH
As preferably, the guanidine hydrochloride solution pH value Tris of described Cystine/Cysteine or aqueous hydrochloric acid solution are adjusted to 7.0-
The concentration of 9.0, Cystine be the mol ratio of 0.5-5mM, Cystine and Cysteine be 1:1 ~ 1:5, linear Linaclotide is dense
Degree is 0.1-5mg/ml.It is furthermore preferred that the guanidine hydrochloride solution pH value Tris of described Cystine/Cysteine or hydrochloric acid are water-soluble
It is 1:1 ~ 1:5 that liquid is adjusted to the mol ratio that concentration is 1-3mM, Cystine and Cysteine of 7.0-9.0, Cystine, linearly profit
That Lip river peptide concentration is 0.1-5mg/ml.Further preferred, the guanidine hydrochloride solution pH value of described Cystine/Cysteine is used
Tris or aqueous hydrochloric acid solution are adjusted to 8.5, the concentration of Cystine be the mol ratio of 1-3mM, Cystine and Cysteine be 1:1 ~
1:5, linear Linaclotide concentration is 0.1-5mg/ml.
As preferably, step 1 is:
In the presence of activation system, by Fmoc-Tyr(tBu) de-Fmoc protection group obtains H-Tyr after-OH and resin carrier coupling
(tBu)-resin carrier, then according to Linaclotide linear amino acid sequence, according to aminoacid sequence shown in SEQ ID NO:1,
From C end to the order of N end, the aminoacid that N end coupling has Fmoc protection group and side chain are connected with protection group the most one by one connects
On peptide resin, obtain Linaclotide resin: H-Cys (Trt)-Cys (Trt)-Glu (OtBu)-Tyr (tBu)-Cys (Trt)-
Cys (Trt)-Asn (Trt)-Pro-Ala-Cys (Trt)-Thr (tBu)-Gly-Cys (Trt)-Tyr (tBu)-resin solid phase carries
Body.
Described residue protected amino acid N end protection group is Fmoc protection group.
Protection group of the present invention is ammonia on the conventional protected amino acid backbone amino of Peptides Synthesis and side chain
The blocking group of the group of the interference synthesis such as base, carboxyl, hydroxyl, sulfydryl, prevents above-mentioned active group from preparing target product mistake
Journey reacts, generates impurity.N end protection group in the present invention is Fmoc, for needing to protect the ammonia of side chain in the present invention
For base acid, its side-chain structure as well known to those skilled in the art and know the conventional protection group of employing and come on protected amino acid side chain
Amino, carboxyl, hydroxyl, the group such as sulfydryl, as preferably, currently preferred side chain protected aminoacid is shown in Table 1.
Table 1
| Fmoc-Tyr(tBu) | Fmoc-Cys(Trt) | Fmoc-Thr(tBu) | Fmoc-Asn(Trt) | Fmoc-Glu(OtBu) |
In synthesis Fmoc-Tyr (tBu)-resin carrier, its substitution value is the resin of 0.2-1.2mmol/g, preferred replacement
Value is the resin of 0.3-0.7mmol/g.
When coupling, each protected amino acid consumption is preferably resin or 1.5-6 times of peptide resin molal quantity, more preferably
2.5-3.5 again；The described coupling reaction time is preferably 60-240 minute, more preferably 120-180 minute.
After coupling, use commonsense method to carry out removing N end protection group and carry out next step coupling again.
As preferably, described resin is CTC resin or hydroxy kind resin.It is highly preferred that use hydroxy kind resin, more enter one
Step preferably, uses hydroxy kind Wang resin.
As preferably, described condensation reagent is preferably the one in DIC, DCC, HBTU/ organic base, HATU/ organic base.Institute
State the mole of condensation reagent and be preferably in peptide resin 1.5-6 times of total mole number, more preferably 2.5-3.5 times.
As preferably, described organic base is: DIPEA, NMM, TEA, more preferably DIPEA.
As preferably, in building-up process of the present invention, use DMF as solvent.
As preferably, it is the TFA of 80-90% that described cracking uses by percent by volume, and percent by volume is the EDT of 1-8%,
Percent by volume is the TIS of 1%, and surplus is the mixing acid hydrolysis solution acidolysis of water composition.
As preferably, Linaclotide crude product, use the 0.45 organic membrane filtration of μm micropore after oxidation, purification is standby；
Employing high performance liquid chromatography is purified, and purification chromatograph packing material is anti-phase Luna C18(10 μm), buffer system is
0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, 100*250mm column flow rate is 200ml/min, uses linear gradient to wash
De-.Take crude product liquid to be splined in chromatographic column, start flowing phase eluting, collect main peak and with analyzing Liquid Detection purity, merge pure
Degree reaches the collection liquid of requirement and obtains Linaclotide sample liquid；
Using high performance liquid chromatography to carry out changing salt, buffer system is 0.5% acetic acid/water solution-acetonitrile, purification chromatograph packing material
For anti-phase Luna C18(10 μm), 100*250mm column flow rate is 200ml/min, uses linear gradient elution.Take sample liquid
Being splined in chromatographic column, start flowing phase eluting, collect main peak and with analyzing Liquid Detection purity, merging is changed salt main peak solution and is obtained
Linaclotide refined liquid, concentrating under reduced pressure, obtain Linaclotide aqueous acetic acid, lyophilization, obtain Linaclotide finished product.
From above technical scheme, the invention has the beneficial effects as follows: the present invention, when linear peptide symthesis, uses price phase
To cheap Fmoc-Cys (Trt)-OH as reaction raw materials, greatly reduce synthesis cost；Oxidation step side is used during cyclisation
Method prepares Linaclotide, it is to avoid the loaded down with trivial details oxidizing process of step-by-step oxidation；Meanwhile, the present invention uses Cystine/Cysteine's
Guanidine hydrochloride oxidation system, peptide concentration, up to 5mg/ml, can effectively reduce the volume of oxidation system, is more suitable for amplifying production；
And oxidation after thick peptide purity good, be conducive to next step purge process.
Described method is set about in terms of oxidation system, improves the production method of Linaclotide, with easier, more efficiently
Processing step improves the total recovery of Linaclotide, and total recovery is more than 38%, and product purity is stable more than 99%, single miscellaneous
Quality Control system is below 0.1%.Simultaneously when oxidation, owing to peptide concentration can improve to 5mg/ml, considerably reduce oxidation system
Volume, be more beneficial for amplify produce, the most more there is practical value and application prospect.
Detailed description of the invention
The invention discloses a kind of method synthesizing Linaclotide, those skilled in the art can use for reference present disclosure, suitable
When realizing diverted via technological parameter.Special needs to be pointed out is, all similar replacements and change are for a person skilled in the art
Being apparent from, they are considered as being included in the present invention.The method of the present invention is retouched by preferred embodiment
Stating, related personnel substantially can be to compound as herein described and preparation method in without departing from present invention, spirit and scope
It is modified and suitably changes and combine, realize and apply the technology of the present invention.
In the specific embodiment of the invention, all protected amino acids all can be by commercially available acquisition, the protection in the present invention
Aminoacid is purchased from Suzhou Mantidis fine chemicals company limited, and resin used has purchased from Xi'an indigo plant science and technology dawn new material share
Limit company, the Chinese implication that in application documents, english abbreviation used is corresponding is shown in Table 2.
Table 2 english abbreviation lexical or textual analysis
| English abbreviation | Chinese | English abbreviation | Chinese |
| Fmoc | 9-fluorenylmethyloxycarbonyl | NMM | N-methylmorpholine |
| tBu | The tert-butyl group | TEA | Triethylamine |
| OtBu | Tert-butoxy | PIP | Piperidines |
| Trt | Trityl | DMF | N,N-dimethylformamide |
| DIC | N, N-DIC | DMAP | 4-N, N-lutidines |
| DCC | N, N-dicyclohexylcarbodiimide | Cys | Cysteine |
| HBTU | BTA-N, N, N ', N ' ,-tetramethylurea hexafluorophosphate | Glu | Glutamic acid |
| HATU | 2-(7-azo BTA)-N, N, N ', N ' ,-tetramethylurea hexafluorophosphoric acid ester | Tyr | Tyrosine |
| CTC | Trityl chloride | Pro | Proline |
| DIPEA | N, N-diisopropylethylamine | Ala | Alanine |
| Cystine | Cystine | Thr | Threonine |
| Cysteine | Cysteine | Gly | Glycine |
Below in conjunction with embodiment, the present invention is expanded on further.
The synthesis of embodiment 1:Fmoc-Tyr (tBu)-Wang resin
Take the Wang resin that 600g substitution value is 0.6mmol/g, add DMF swellable resins.Take 0.72mol Fmoc-Tyr
(tBu)-OH, dissolves with appropriate DMF, joins in above-mentioned resin, add 0.72mol DIC, 0.72mol after stirring
HOBt, 0.072mol DMAP, stirring reaction 5 hours, leach out reactant liquor, then after washing 3 times with DMF, then wash 3 with DCM
Secondary, obtain Fmoc-Tyr (tBu)-Wang resin.Its substitution value is 0.41mmol/g.
Embodiment 2: the preparation of Linaclotide resin
Take Fmoc-Tyr (the tBu)-Wang resin of 0.2mol embodiment 1, deprotect 20 minutes with 20%PIP/DMF solution, washing
Filter, obtain removing H-Tyr (the tBu)-Wang resin of Fmoc.
Take 0.25mol Fmoc-Cys (tBu)-OH, with appropriate DMF dissolve after join equipped with above-mentioned H-Tyr (tBu)-
In the reactor of Wang resin, separately take 0.24mol HBTU and 0.25mol DIPEA, be slowly added to the above-mentioned of drum nitrogen respectively
In reactor.Coupling reaction 30 ~ 120 minutes, reaction end is as the criterion with ninhydrin method detection, washing and filtering, then uses 20% PIP/
DMF solution removes protection 20 minutes, washing and filtering, obtains H-Cys (Trt)-Tyr (tBu)-Wang resin.
Remain protected amino acid during ibid method is sequentially ingressed into table 3, obtain Linaclotide peptide resin:
H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-
Ala-Cys (Trt)-Thr (tBu)-Gly-Cys (Trt)-Tyr (tBu)-Wang resin.
Table 3
| Numbering | Aminoacid | Numbering | Aminoacid |
| 3 | Fmoc-Gly | 9 | Fmoc-Cys(Trt) |
| 4 | Fmoc-Thr(tBu) | 10 | Fmoc-Cys(Trt) |
| 5 | Fmoc-Cys(Trt) | 11 | Fmoc-Tyr(tBu) |
| 6 | Fmoc-Ala | 12 | Fmoc-Glu(OtBu) |
| 7 | Fmoc-Pro | 13 | Fmoc-Cys(Trt) |
| 8 | Fmoc-Asn(Trt) | 14 | Fmoc-Cys(Trt) |
Embodiment 3: the preparation of Linaclotide linear peptides crude product
Example 2 gained Linaclotide resin, adds the mixing acidolysis that volume ratio is TFA:EDT:Tis:H2O=89:5:1:5
Liquid (consumption is that every gram of Linaclotide resin adds acid hydrolysis solution 8ml), room temperature shaker reacts 3 hours, and reactant mixture uses core
Funnel filters, and collects filtrate, and resin washs 3 times with a small amount of TFA again, adds absolute ether precipitation after merging filtrate, then with anhydrous
Ether washing precipitation 3 times, drains and obtains off-white powder, and vacuum decompression is dried to constant weight, obtains Linaclotide linear peptides crude product
325.1g, purity is 78.2%.
Embodiment 4: the preparation of Linaclotide crude product
Example 3 gained Linaclotide linear peptides crude product 100g, the guanidine hydrochloride with 1mM Cystine/5mM Cysteine is molten
Liquid dissolves Linaclotide linear peptides crude product, and is diluted to 3mg/ml, then adjusts pH to 8.5 with Tris or aqueous hydrochloric acid solution, stirs at a slow speed
Mix 12 hours, obtain Linaclotide crude product liquid.
Embodiment 5: the preparation of Linaclotide crude product
Example 3 gained Linaclotide linear peptides crude product 100g, the guanidine hydrochloride with 1mM Cystine/5mM Cysteine is molten
Liquid dissolves Linaclotide linear peptides crude product, and is diluted to 5mg/ml, then adjusts pH to 9.0 with Tris or aqueous hydrochloric acid solution, stirs at a slow speed
Mix 12 hours, obtain Linaclotide crude product liquid.
Embodiment 6: the preparation of Linaclotide crude product
Example 3 gained Linaclotide linear peptides crude product 100g, the guanidine hydrochloride with 1mM Cystine/5mM Cysteine is molten
Liquid dissolves Linaclotide linear peptides crude product, and is diluted to 5mg/ml, then adjusts pH to 7.0 with Tris or aqueous hydrochloric acid solution, stirs at a slow speed
Mix 12 hours, obtain Linaclotide crude product liquid.
Embodiment 7: the purification of Linaclotide crude product
Example 4 gained Linaclotide crude product is with 0.45 μm filtering with microporous membrane, and purification is standby.
Employing high performance liquid chromatography is purified, and purification chromatograph packing material is anti-phase Luna C18(10 μm), buffer body
System is 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, and 100*250mm column flow rate is 200ml/min, uses linear ladder
Degree eluting.Take crude product liquid to be splined in chromatographic column, start flowing phase eluting, collect main peak and with analyzing Liquid Detection purity, close
And purity reaches the collection liquid of requirement and obtains Linaclotide sample liquid；
Using high performance liquid chromatography to carry out changing salt, buffer system is 0.5% acetic acid/water solution-acetonitrile, purification chromatograph packing material
For anti-phase Luna C18(10 μm), 100*250mm column flow rate is 200ml/min, uses linear gradient elution.Take sample liquid
Being splined in chromatographic column, start flowing phase eluting, collect main peak and with analyzing Liquid Detection purity, merging is changed salt main peak solution and is obtained
Linaclotide refined liquid, concentrating under reduced pressure, obtain Linaclotide aqueous acetic acid, lyophilization, obtain Linaclotide finished product 39.1g,
Total recovery is 41.4%, molecular weight: 1527.8(100%M+H), purity: 99.6%, maximum single contaminant is 0.07%.
Embodiment 8: the purification of Linaclotide crude product
Example 5 gained Linaclotide crude product is with 0.45 μm filtering with microporous membrane, and purification is standby.
Employing high performance liquid chromatography is purified, and purification chromatograph packing material is anti-phase Luna C18(10 μm), buffer body
System is 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, and 100*250mm column flow rate is 200ml/min, uses linear ladder
Degree eluting.Take crude product liquid to be splined in chromatographic column, start flowing phase eluting, collect main peak and with analyzing Liquid Detection purity, close
And purity reaches the collection liquid of requirement and obtains Linaclotide sample liquid；
Using high performance liquid chromatography to carry out changing salt, buffer system is 0.5% acetic acid/water solution-acetonitrile, purification chromatograph packing material
For anti-phase Luna C18(10 μm), 100*250mm column flow rate is 200ml/min, uses linear gradient elution.Take sample liquid
Being splined in chromatographic column, start flowing phase eluting, collect main peak and with analyzing Liquid Detection purity, merging is changed salt main peak solution and is obtained
Linaclotide refined liquid, concentrating under reduced pressure, obtain Linaclotide aqueous acetic acid, lyophilization, obtain Linaclotide finished product 38.6g,
Total recovery is 40.9%, molecular weight: 1527.8(100%M+H), purity: 99.5%, maximum single contaminant is 0.08%.
Embodiment 9: the purification of Linaclotide crude product
Example 6 gained Linaclotide crude product is with 0.45 μm filtering with microporous membrane, and purification is standby.
Employing high performance liquid chromatography is purified, and purification chromatograph packing material is anti-phase Luna C18(10 μm), buffer body
System is 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, and 100*250mm column flow rate is 200ml/min, uses linear ladder
Degree eluting.Take crude product liquid to be splined in chromatographic column, start flowing phase eluting, collect main peak and with analyzing Liquid Detection purity, close
And purity reaches the collection liquid of requirement and obtains Linaclotide sample liquid；
Using high performance liquid chromatography to carry out changing salt, buffer system is 0.5% acetic acid/water solution-acetonitrile, purification chromatograph packing material
For anti-phase Luna C18(10 μm), 100*250mm column flow rate is 200ml/min, uses linear gradient elution.Take sample liquid
Being splined in chromatographic column, start flowing phase eluting, collect main peak and with analyzing Liquid Detection purity, merging is changed salt main peak solution and is obtained
Linaclotide refined liquid, concentrating under reduced pressure, obtain Linaclotide aqueous acetic acid, lyophilization, obtain Linaclotide finished product 36.5g,
Total recovery is 38.6%, molecular weight: 1527.8(100%M+H), purity: 99.5%, maximum single contaminant is 0.08%.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. the synthesis of a Linaclotide and purification process, it is characterised in that comprise the steps:
1) in the presence of activation system, by Fmoc-Tyr(tBu) de-Fmoc protection group obtains H-after-OH and resin carrier coupling
Tyr (tBu)-resin carrier, then according to Linaclotide linear amino acid sequence, according to aminoacid sequence shown in SEQ ID NO:1
Row, from C end to the order of N end, have Fmoc protection group and side chain to be connected with the aminoacid of protection group even the most one by one by N end coupling
Receive on peptide resin, obtain Linaclotide resin: H-Cys (Trt)-Cys (Trt)-Glu (OtBu)-Tyr (tBu)-Cys
(Trt)-Cys (Trt)-Asn (Trt)-Pro-Ala-Cys (Trt)-Thr (tBu)-Gly-Cys (Trt)-Tyr (tBu)-resin
Solid phase carrier；
2) the Linaclotide pitch shake solution that obtains in step 1) removes all protection groups and resin carrier, obtain not cyclized profit that
Lip river peptide linearly thick peptide, then uses the guanidine hydrochloride oxidation system of Cystine/Cysteine that Linaclotide linearly thick peptide is carried out oxygen
Change reaction, according to the amino acid sequence of its N end to C end, form Cys1And Cys6、Cys2And Cys10、Cys5And Cys13Two sulfur
Key, obtains Linaclotide crude product；
3) take step 2) in the Linaclotide crude product that obtains, be prepared as Linaclotide crude product liquid, standby through membrane filtration purification, adopt
Carrying out isolated and purified by high performance liquid chromatography to the crude product after filtering, the collection liquid that purity reaches requirement uses efficient liquid phase
Chromatography carries out changing salt；Collecting main peak and with analyzing Liquid Detection purity, merging is changed salt main peak solution and is obtained Linaclotide refined liquid,
Concentrating under reduced pressure, obtains Linaclotide aqueous acetic acid, lyophilization, obtains Linaclotide finished product.
The synthesis of a kind of Linaclotide the most according to claim 1 and purification process, it is characterised in that: described resin is
CTC resin or hydroxy kind resin, its substitution value be 0.2-1.2mmol/g, preferably substitution value be 0.3-0.7mmol/g.
Method the most according to claim 1, it is characterised in that described condensation reagent be DIC, DCC, HBTU/ organic base,
One in HATU/ organic base, organic base is the one in DIPEA, NMM, TEA.
Method the most according to claim 1, it is characterised in that the mole of described condensation reagent is total mole number in peptide resin
1.5-6 times, preferably 2.5-3.5 times.
Method the most according to claim 1, it is characterised in that described activation system uses in NMP, DCM, DMF and DMSO appoints
Anticipating, one or more dissolve.
Method the most according to claim 1, it is characterised in that in Cystine/Cysteine oxidation system described in step 2
The mol ratio of Cystine and Cysteine is 1:1 ~ 1:5.
Method the most according to claim 1, it is characterised in that step 2) in the concentration of guanidine hydrochloride of oxidation system be 0.5M-4M,
Its pH value is 7.0-9.0；Peptide concentration is 0.1mg/ml-5mg/ml；The temperature of oxidation reaction is 0-35 DEG C；Oxidization time is
12-24 hour.
Method the most according to claim 1, it is characterised in that step 2) cracking uses is 80-90%'s by percent by volume
TFA, percent by volume is the EDT of 1-8%, and percent by volume is the TIS of 1%, and surplus is the mixing acid hydrolysis solution acidolysis of water composition.
Method the most according to claim 1, it is characterised in that in step 3), Linaclotide crude product liquid uses 0.45 μm micropore
Organic membrane filtration.
Method the most according to claim 1, it is characterised in that high performance liquid chromatography buffer in purification step in step 3)
System is 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution；Changing high performance liquid chromatography buffer system in salt step is 0.5%
Acetic acid/water solution-acetonitrile.
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| CN107266535A (en) * | 2017-08-08 | 2017-10-20 | 南京工业大学 | A kind of purification process of Linaclotide |
| CN108107141A (en) * | 2017-12-19 | 2018-06-01 | 浙江丰安生物制药有限公司 | The extracting method of polypeptide in a kind of spleen aminopeptide |
| CN108250265A (en) * | 2016-12-29 | 2018-07-06 | 北京中科亚光生物科技有限公司 | A kind of new process for synthesizing the polypeptide containing intramolecular disulfide bond |
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| CN111732632A (en) * | 2020-07-16 | 2020-10-02 | 台州吉诺生物科技有限公司 | Synthesis method of linaclotide |
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| CN113956333A (en) * | 2021-12-22 | 2022-01-21 | 浙江湃肽生物有限公司南京分公司 | Synthesis and purification method of linaclotide |
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| CN108250265A (en) * | 2016-12-29 | 2018-07-06 | 北京中科亚光生物科技有限公司 | A kind of new process for synthesizing the polypeptide containing intramolecular disulfide bond |
| CN107266535A (en) * | 2017-08-08 | 2017-10-20 | 南京工业大学 | A kind of purification process of Linaclotide |
| WO2019113872A1 (en) * | 2017-12-14 | 2019-06-20 | 深圳市健元医药科技有限公司 | Method for synthesizing linaclotide |
| CN108107141A (en) * | 2017-12-19 | 2018-06-01 | 浙江丰安生物制药有限公司 | The extracting method of polypeptide in a kind of spleen aminopeptide |
| CN109053863A (en) * | 2018-08-27 | 2018-12-21 | 杭州肽佳生物科技有限公司 | A kind of method of low cost preparation high-purity Linaclotide |
| CN113039193A (en) * | 2018-11-16 | 2021-06-25 | 味之素株式会社 | Method for producing cyclized peptide having intramolecular S-S bond |
| US11939404B2 (en) | 2018-11-16 | 2024-03-26 | Ajinomoto Co., Inc. | Method for producing cyclized peptide having intramolecular S-S bond |
| 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 |
| CN113956333A (en) * | 2021-12-22 | 2022-01-21 | 浙江湃肽生物有限公司南京分公司 | Synthesis and purification method of linaclotide |
| CN114350587A (en) * | 2022-01-24 | 2022-04-15 | 修实生物医药(南通)有限公司 | Engineering bacterium for gene recombination tandem expression of linaclotide |
| CN114350587B (en) * | 2022-01-24 | 2023-10-31 | 修实生物医药(南通)有限公司 | Engineering bacterium for expressing linaclotide by gene recombination in series |
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