CN106831950A - A kind of method for preparing Linaclotide - Google Patents
A kind of method for preparing Linaclotide Download PDFInfo
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- CN106831950A CN106831950A CN201710162507.0A CN201710162507A CN106831950A CN 106831950 A CN106831950 A CN 106831950A CN 201710162507 A CN201710162507 A CN 201710162507A CN 106831950 A CN106831950 A CN 106831950A
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- Prior art keywords
- linaclotide
- cys
- disulfide bond
- tbu
- dmf
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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
-
- 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 invention discloses a kind of method for preparing Linaclotide, including, oxidation reaction is carried out to the Linaclotide intermediate containing two pairs of disulfide bond, obtain Linaclotide；Wherein, described containing two pairs of Linaclotide intermediates of disulfide bond, it is in the presence of the 2nd, 10 disulfide bond and the 5th, 13 disulfide bond of Cys of Cys.Reaction scheme of the present invention is short, and raw material is cheap and easy to get, and reaction condition is gentle, reduces energy consumption, reduces production cost.Compared to the route that forefathers deliver, the reaction selectivity of this route is higher with conversion ratio, reduces the waste of raw material, there is stronger economy.The preparation method that the present invention is provided, optimum reacting time is 30min, and products therefrom purity is up to 85%.Reduce the discharge of the three wastes, green safety.
Description
Technical field
The invention belongs to medical synthesis technical field, and in particular to a kind of method for preparing Linaclotide.
Background technology
Linaclotide (Linaclotide) is a kind of oral selective guanosine cyclic mono-phosphate receptor stimulating agent, main to make
For enteron aisle, intestinal fluid secretion is can induce, for treating irritable bowels syndrome and prolonged constipation based on constipation.Its trade name
Linzess, is researched and developed by Ironwood Pharma companies of the U.S., and in August, 2012 obtains U.S. FDA approval listing.Linaclotide is bird
The capsule preparations of thuja acid cyclase C (GC-C) activator, daily orally once.Linaclotide and intestines that PC can't detect
After road GC-C is combined, intracellular and extracellular loop guanylic acid (cGMP) concentration is caused to raise.Intracellular cGMP is raised can stimulate
Intestinal fluid secretion, accelerates intestines and stomach and divides a word with a hyphen at the end of a line, so as to increase stool interval.Extracellular cGMP concentration is raised and can reduce pain nerve
Sensitivity, and shown according to Research of Animal Model for Study, this can lower enteron aisle pain.Linaclotide is first with this kind of mechanism of action
Constipation therapy medicine.
Linaclotide is a 14- amino acid polypeptide, and containing three disulfide bond, its sequential structure is as follows：
Structural formula (amino acid sequence)
The annulation of three disulfide bond is the difficult point of this product synthesis in Linaclotide.
The synthetic route of existing document report mainly has three kinds：
Method one：Random oxidation (random oxidation) method
By conventional solid polypeptide synthesis method on resin synthesizing linear peptide, acidolysis deprotection base and resin carrier are obtained
To the linear thick peptide of Linaclotide, then in liquid phase buffer solution, using the method for random oxidation (random oxidation)
One step forms three pairs of disulfide bond (see reaction schematic diagram).As patent CN201310556661.8, CN201510314459.3,
CN201610739783.4、CN201610644791.0、CN201610328263.4、CN201210375246.8、
CN201310223243.7, WO2014188011A2, WO2016038497A1 etc..
Solid phase coupling-random oxidation reaction schematic diagram is as shown in Figure 5.
In method one, synthesis has only used a kind of blocking group of Cys, finally only needs a step to aoxidize cyclization at random and obtains mesh
Mark product, it is simple to operate.But, for needing positioning to form three for the peptide of disulfide bond, random oxidation can obtain many two
The isomers of sulfide linkage mispairing, although as big as possible during oxidizing process can be caused by some buffer solution systems be changed into
Target molecule, but the generation of other mispairing isomers cannot be avoided all the time.This operation easily causes the thick peptide purification of target peptide
Difficulty, is unfavorable for the control of product quality.Meanwhile, aoxidized using the method, it is necessary in the solution of high dilution, externally
The dependence of boundary's condition such as temperature etc. is also very high, in different environments, the product yield difference obtained by autoxidation
Also it is very big, it is difficult to be mass produced.
Method two：Step-by-step oxidation method (Step Oxidation)
Using three couples of Cys with different Side chain protective groups, by synthesis in solid state on resin synthesizing linear peptide, then adopt
Three pairs of disulfide bond are formed with the method for substep removing oxidation.Such as patent CN201510391880.4, Trt, Acm, tBu tri- is employed
To different protection groups.
Solid phase coupling-step-by-step oxidation reaction schematic diagram is as shown in Figure 6.
Patent CN201310617050.X employs tri- pairs of different protection groups of Trt, Acm, Hmq.
Step-by-step oxidation method, cysteine uses three pairs of different protection groups, synthesizes high cost；Iodine is strong oxidizer, can be broken
Bad two pairs of disulfide bond being previously formed, form mispairing product；Using the cysteine of different Side chain protective groups, production cost is high；
Step-by-step oxidation operation is complicated, and yield is extremely low.
Method three：By solid-liquid combination, fragment condensation, then synthesizing linear peptide is closed by random oxidation or step-by-step oxidation
Into Linaclotide.As patent CN201510467488.3, CN201410357720.3, WO2015022575A2,
WO2012118972A2。
This method operation is complicated, and efficiency is low, high cost.
To sum up, random oxidizing process mispairing probability is high, and impurity is more and whard to control, and oxide whiskers are used as far as possible in weak solution
Gentle oxidation system, the cyclisation time is long, low production efficiency；Step-by-step oxidation method, cysteine needs three pairs of different protection groups,
Synthetic operation step is complicated, low yield, high cost.The oxidation product crude product purity of random oxidation is general 30~40% or so.
Prepared by step-by-step oxidation method, if each step is all made of sterling, last products therefrom purity also only can reach 50~60%, while it is walked
It is rapid cumbersome.
So that finding a more simple, efficient synthetic route, the inventive method preferably solves this and asks
Topic, is adapted to industrialized production.
The content of the invention
The purpose of this part is to summarize some aspects of embodiments of the invention and briefly introduce some preferable implementations
Example.May be done in this part and the description of the present application summary and denomination of invention a little simplified or omitted to avoid making our department
Point, the purpose of specification digest and denomination of invention obscure, and this simplification or omission cannot be used for limiting the scope of the present invention.
In view of above-mentioned and/or existing technological gap, it is proposed that the present invention.
In order to solve the above technical problems, the invention provides following technical scheme：Including to containing two pairs of profits of disulfide bond
That Lip river peptide intermediate carries out oxidation reaction, obtains Linaclotide；Wherein, it is described containing two pairs of Linaclotides of disulfide bond in the middle of
Body, it is in the presence of the 2nd, 10 disulfide bond and the 5th, 13 disulfide bond of Cys of Cys.
As a kind of preferred scheme of preparation method of the present invention, wherein：It is described to contain two couples of Li Naluo of disulfide bond
Peptide intermediate, it is have blocking group on the 1st, 6 Cys.
As a kind of preferred scheme of preparation method of the present invention, wherein：It is described to contain two couples of Li Naluo of disulfide bond
Peptide intermediate, it is to carry out the 2nd, 10 Cys and the 5th, 13 Cys to the linear thick peptide of Linaclotide to carry out oxidation cyclization and be obtained.
As a kind of preferred scheme of preparation method of the present invention, wherein：The linear thick peptide of the Linaclotide, its be
There is blocking group on 1st, 6 Cys.
As a kind of preferred scheme of preparation method of the present invention, wherein：The linear thick peptide of the Linaclotide, it is profit
That Lip river peptide resin cracking is obtained；Wherein, the Linaclotide resin, it is that 1,6 Cys have common blocking group, 2,10
There is Trt with 5,13 Cys.
As a kind of preferred scheme of preparation method of the present invention, wherein：The oxidation cyclization, pH is 6.5~7.5,
Time is 10~14h.
As a kind of preferred scheme of preparation method of the present invention, wherein：The blocking group, it is tBu, Meb,
One kind in Mob.
As a kind of preferred scheme of preparation method of the present invention, wherein：The blocking group, it is tBu.
As a kind of preferred scheme of preparation method of the present invention, wherein：The oxidation reaction, including, it is directed to
TBu, specially with one or more in TFA or 5~20%DMSO as solvent, temperature be 15~30 DEG C, the time be 25~
40min；It is directed to Meb, and specially with one or more in TFA or 5~20%DMSO as solvent, temperature is 40~60 DEG C, when
Between be 3~4h；It is directed to Mob, is 94 specially with volume ratio:5:1 TFA, DMSO, methyl phenyl ethers anisole intermixture are solvent, temperature
It it is 55~65 DEG C, the time is 30min~90min.
As a kind of preferred scheme of preparation method of the present invention, wherein：The cracking, it is with TFA, EDT, benzene first
Ether, thioanisole and water are lysate, and its volume ratio is 90:2.5:2.5:2.5:2.5；Its Linaclotide resin is with lysate
Volume ratio is 1:8.
Beneficial effects of the present invention：
(1) the technological reaction route is short, and raw material is cheap and easy to get, and reaction condition is gentle, reduces energy consumption, reduces and is produced into
This.
(2) route delivered compared to forefathers, the reaction selectivity of this route is higher with conversion ratio, reduces raw material
Waste, there is stronger economy.The preparation method that the present invention is provided, optimum reacting time is 30min, and products therefrom purity is reachable
85%.
(3) discharge of the three wastes, green safety are reduced.
Brief description of the drawings
Technical scheme in order to illustrate more clearly the embodiments of the present invention, below will be to that will use needed for embodiment description
Accompanying drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for this
For the those of ordinary skill of field, without having to pay creative labor, other can also be obtained according to these accompanying drawings
Accompanying drawing.Wherein：
Fig. 1 is the prepared Linaclotide MS figures for obtaining.
Fig. 2 is the prepared Linaclotide HPLC figures for obtaining.
Fig. 3 is that the prepared two couples of MS of the Linaclotide intermediate of disulfide bond that contain for obtaining scheme.
Fig. 4 is that the prepared two couples of HPLC of the Linaclotide intermediate of disulfide bond that contain for obtaining scheme.
Fig. 5 is solid phase coupling-random oxidation reaction schematic diagram.
Fig. 6 is that solid phase coupling-step-by-step oxidation reacts schematic diagram.
Specific embodiment
To enable the above objects, features and advantages of the present invention more obvious understandable, with reference to specific embodiment pair
Specific embodiment of the invention is described in detail.
Many details are elaborated in the following description in order to fully understand the present invention, but the present invention can be with
Other manner described here is different from using other to implement, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by following public specific embodiment.
Secondly, " one embodiment " or " embodiment " referred to herein refers to that may be included at least one realization side of the invention
Special characteristic, structure or characteristic in formula." in one embodiment " that different places occur in this manual not refers both to
Same embodiment, nor the single or selective embodiment mutually exclusive with other embodiment.
Embodiment 1：
The preparation of Linaclotide resin
H-Cys(tBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(tBu)-Asn(Trt)-
The preparation of Pro-Ala-Cys (Trt)-Thr (tBu)-Gly-Cys (Trt)-Tyr (tBu)-Resin
Weigh 10g substitution degrees be 1.14mmol/g Wang resins in solid phase reactor, with the swelling Wang resins 20 of DCM
Minute, drain.
To in solid phase reactor add Fmoc-Tyr (tBu)-OH (15.7g, 13.68mmol), HOBt (5.55g,
4.1mmol), DIC (6.35mL, 4.1mmol), DMAP (0.5g, 4.1mmol) are dissolved in DMF (70ml), 15 points of room temperature reaction
Clock.
Resin washing is drained, Fmoc-Tyr (tBu)-Wang resins are obtained final product, resin substitution degree is measured for 0.426mmol/
g.To addition closed reagent 70mL (acetic anhydride (mmol) in resin:DIPEA (mmol)=1:1) 10h, is reacted, is closed remaining
Amino, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
Add 20%PIP/DMF solution reactions 20min removing Fmoc protection groups, respectively with DCM (1 time), MeOH (1 time) with
(3 times) washings of DMF, obtain H-Tyr (tBu)-Wang resins；Continuously add Fmoc-Cys (Trt)-OH (7.48g, 12.78mmol),
HOBt (2.07g, 15.34mmol), DIC (2.37mL, 15.34mmol), DMF (70mL), room temperature reaction 2h.
Coupling completeness can test detection using Kaiser；After detection passes through, with 20%PIP/DMF solution removals Fmoc
Protection group 5+15min, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
In this approach, according to target product correspondence institute palpus amino acid sequence, remaining amino acid is coupled successively, obtain side chain
Full guard polypeptide resin.
This side chain full guard polypeptide resin is reacted into 5min with 20%PIP/DMF solution 5mL, DMF washed once, add
20%PIP/DMF solution 5mL reacts 15min, drains, and is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively, drains,
Weigh to obtain 22.95g peptide resins.
The preparation of the linear thick peptide of Linaclotide
H-Cys(tBu)-Cys-Glu-Tyr-Cys-Cys(tBu)-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-
It is prepared by OH
Configuration lysate, its each component volume ratio is TFA:EDT:Methyl phenyl ethers anisole:Thioanisole:Water=90:2.5:2.5:
2.5:2.5；By lysate add solid phase reactor in, with above-mentioned 5g peptide resins (V peptide resins:V cutting liquid=1:8) at room temperature
After concussion reaction 2h, reaction solution is injected into ether (V reaction solutions:V ether=1:6) in, precipitation, collected after centrifugation white solid sinks
Form sediment, vacuum drying obtains final product the linear thick peptide 0.82g of Cys (tBu).
Two pairs of preparations of disulfide bond intermediate
The thick peptides of 50mg are taken, 2M guanidine hydrochlorides (50ml), 0.1M tertiary sodium phosphates regulation PH to 7, room temperature reaction 12h is added, obtained
Containing two pairs of disulfide bond intermediates (IV).
Above-mentioned reaction solution is purified using the semi-preparative HPLC of Yi Lite.Post is prepared using C18 (20mm*250mm)；
Flow velocity:9ml/min；Mobile phase：A phases are water (1 ‰ TFA), and B phases are acetonitrile；Analysis condition：Acetonitrile：5-20-30-95%, when
Between：0-5-45-50min.Preparation solution is collected, freeze-drying obtains 15mg two to disulfide bond intermediate (IV).
The preparation of Linaclotide (I)
Above-mentioned two pairs of disulfide bond intermediates (IV) sterlings of 10mg are taken, TFA (3ml), 5%DMSO is added, reacted at 15 DEG C
30 minutes, Linaclotide (I) is obtained, carry out mass spectral analysis, while compared with standard items, as a result correctly.Its HPLC purity is
85%.
Embodiment 2：
The preparation of Linaclotide resin
H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(tBu)-Cys(Trt)-Asn(Trt)-
The preparation of Pro-Ala-Cys (Trt)-Thr (tBu)-Gly-Cys (tBu)-Tyr (tBu)-Resin
Weigh 10g substitution degrees be 1.14mmol/g Wang resins in solid phase reactor, with the swelling Wang resins 20 of DCM
Minute, drain.
To in solid phase reactor add Fmoc-Tyr (tBu)-OH (15.7g, 13.68mmol), HOBt (5.55g,
4.1mmol), DIC (6.35mL, 4.1mmol), DMAP (0.5g, 4.1mmol) are dissolved in DMF (70ml), 15 points of room temperature reaction
Clock.
Resin washing is drained, Fmoc-Tyr (tBu)-Wang resins are obtained final product, resin substitution degree is measured for 0.426mmol/
g.To addition closed reagent 70mL (acetic anhydride (mmol) in resin:DIPEA (mmol)=1:1) 10h, is reacted, is closed remaining
Amino, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
Add 20%PIP/DMF solution reactions 20min removing Fmoc protection groups, respectively with DCM (1 time), MeOH (1 time) with
(3 times) washings of DMF, obtain H-Tyr (tBu)-Wang resins；Continuously add Fmoc-Cys (tBu)-OH (5.10g,
12.78mmol), HOBt (2.07g, 15.34mmol), DIC (2.37mL, 15.34mmol), DMF (70mL), room temperature reaction 2h.
Coupling completeness can test detection using Kaiser；After detection passes through, with 20%PIP/DMF solution removals Fmoc
Protection group 5+15min, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
In this approach, according to target product correspondence institute palpus amino acid sequence, remaining amino acid is coupled successively, obtain side chain
Full guard polypeptide resin.
This side chain full guard polypeptide resin is reacted into 5min with 20%PIP/DMF solution 5mL, DMF washed once, add
20%PIP/DMF solution 5mL reacts 15min, drains, and is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively, drains,
Weigh to obtain 21.12g peptide resins.
The preparation of the linear thick peptide of Linaclotide
H-Cys-Cys-Glu-Tyr-Cys(tBu)-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys(tBu)-Tyr-
It is prepared by OH
Configuration lysate, its each component volume ratio is TFA:EDT:Methyl phenyl ethers anisole:Thioanisole:Water=90:2.5:2.5:
2.5:2.5；By lysate add solid phase reactor in, with above-mentioned 5g peptide resins (V peptide resins:V cutting liquid=1:8) at room temperature
After concussion reaction 2h, reaction solution is injected into ether (V reaction solutions:V ether=1:6) in, precipitation, collected after centrifugation white solid sinks
Form sediment, vacuum drying obtains final product the linear thick peptide 0.78g of Cys (tBu).
Two pairs of preparations of disulfide bond intermediate
The thick peptides of 50mg are taken, 2M guanidine hydrochlorides (50ml), 0.1M tertiary sodium phosphates regulation PH to 7, room temperature reaction 12h is added, obtained
Containing two pairs of disulfide bond intermediates (IV).
Above-mentioned reaction solution is purified using the semi-preparative HPLC of Yi Lite.Post is prepared using C18 (20mm*250mm)；
Flow velocity：9ml/min；Mobile phase：A phases are water (1 ‰ TFA), and B phases are acetonitrile；Analysis condition：Acetonitrile：5-20-30-95%, when
Between：0-5-45-50min.Preparation solution is collected, freeze-drying obtains 14mg two to disulfide bond intermediate (IV).
The preparation of Linaclotide (I)
Above-mentioned two pairs of disulfide bond intermediates (IV) sterlings of 10mg are taken, TFA (3ml), 15%DMSO is added, reacted at 30 DEG C
25 minutes, Linaclotide (I) is obtained, carry out mass spectral analysis, while compared with standard items, as a result correctly.Its HPLC purity is
48%.
Embodiment 3：
The preparation of Linaclotide resin
H-Cys(Trt)-Cys(tBu)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-
The preparation of Pro-Ala-Cys (tBu)-Thr (tBu)-Gly-Cys (Trt)-Tyr (tBu)-Resin
Weigh 10g substitution degrees be 1.14mmol/g Wang resins in solid phase reactor, with the swelling Wang resins 20 of DCM
Minute, drain.
To in solid phase reactor add Fmoc-Tyr (tBu)-OH (15.7g, 13.68mmol), HOBt (5.55g,
4.1mmol), DIC (6.35mL, 4.1mmol), DMAP (0.5g, 4.1mmol) are dissolved in DMF (70ml), 15 points of room temperature reaction
Clock.
Resin washing is drained, Fmoc-Tyr (tBu)-Wang resins are obtained final product, resin substitution degree is measured for 0.426mmol/
g.To addition closed reagent 70mL (acetic anhydride (mmol) in resin:DIPEA (mmol)=1:1) 10h, is reacted, is closed remaining
Amino, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
Add 20%PIP/DMF solution reactions 20min removing Fmoc protection groups, respectively with DCM (1 time), MeOH (1 time) with
(3 times) washings of DMF, obtain H-Tyr (tBu)-Wang resins；Continuously add Fmoc-Cys (Trt)-OH (7.48g, 12.78mmol),
HOBt (2.07g, 15.34mmol), DIC (2.37mL, 15.34mmol), DMF (70mL), room temperature reaction 2h.
Coupling completeness can test detection using Kaiser；After detection passes through, with 20%PIP/DMF solution removals Fmoc
Protection group 5+15min, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
In this approach, according to target product correspondence institute palpus amino acid sequence, remaining amino acid is coupled successively, obtain side chain
Full guard polypeptide resin.
This side chain full guard polypeptide resin is reacted into 5min with 20%PIP/DMF solution 5mL, DMF washed once, add
20%PIP/DMF solution 5mL reacts 15min, drains, and is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively, drains,
Weigh to obtain 23.12g peptide resins.
The preparation of the linear thick peptide of Linaclotide
H-Cys-Cys(tBu)-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys(tBu)-Thr-Gly-Cys-Tyr-
It is prepared by OH
Configuration lysate, its each component volume ratio is TFA:EDT:Methyl phenyl ethers anisole:Thioanisole:Water=90:2.5:2.5:
2.5:2.5；By lysate add solid phase reactor in, with above-mentioned 5g peptide resins (V peptide resins:V cutting liquid=1:8) in room temperature
After lower concussion reaction 2h, reaction solution is injected into ether (V reaction solutions:V ether=1:6) in, precipitation, collected after centrifugation white solid
Precipitation, vacuum drying, obtains final product the linear thick peptide 0.84g of Cys (tBu).
Two pairs of preparations of disulfide bond intermediate
The thick peptides of 50mg are taken, 2M guanidine hydrochlorides (50ml), 0.1M tertiary sodium phosphates regulation PH to 7, room temperature reaction 12h is added, obtained
Containing two pairs of disulfide bond intermediates (IV).
Above-mentioned reaction solution is purified using the semi-preparative HPLC of Yi Lite.Post is prepared using C18 (20mm*250mm)；
Flow velocity：9ml/min；Mobile phase：A phases are water (1 ‰ TFA), and B phases are acetonitrile；Analysis condition：Acetonitrile：5-20-30-95%, when
Between：0-5-45-50min.Preparation solution is collected, freeze-drying obtains 15mg two to disulfide bond intermediate (IV).
The preparation of Linaclotide (I)
Above-mentioned two pairs of disulfide bond intermediates (IV) sterlings of 10mg are taken, TFA (3ml), 20%DMSO is added, reacted at 25 DEG C
40 minutes, Linaclotide (I) is obtained, carry out mass spectral analysis, while compared with standard items, as a result correctly.Its HPLC purity is
34%.
Embodiment 4：
The preparation of Linaclotide resin
H-Cys(Meb)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Meb)-Asn(Trt)-
The preparation of Pro-Ala-Cys (Trt)-Thr (tBu)-Gly-Cys (Trt)-Tyr (tBu)-Resin
Weigh 10g substitution degrees be 1.14mmol/g Wang resins in solid phase reactor, with the swelling Wang resins 20 of DCM
Minute, drain.
To in solid phase reactor add Fmoc-Tyr (tBu)-OH (15.7g, 13.68mmol), HOBt (5.55g,
4.1mmol), DIC (6.35mL, 4.1mmol), DMAP (0.5g, 4.1mmol) are dissolved in DMF (70ml), 15 points of room temperature reaction
Clock.
Resin washing is drained, Fmoc-Tyr (tBu)-Wang resins are obtained final product, resin substitution degree is measured for 0.426mmol/
g.To addition closed reagent 70mL (acetic anhydride (mmol) in resin:DIPEA (mmol)=1:1) 10h, is reacted, is closed remaining
Amino, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
Add 20%PIP/DMF solution reactions 20min removing Fmoc protection groups, respectively with DCM (1 time), MeOH (1 time) with
(3 times) washings of DMF, obtain H-Tyr (tBu)-Wang resins；Continuously add Fmoc-Cys (Trt)-OH (7.48g, 12.78mmol),
HOBt (2.07g, 15.34mmol), DIC (2.37mL, 15.34mmol), DMF (70mL), room temperature reaction 2h.
Coupling completeness can test detection using Kaiser；After detection passes through, with 20%PIP/DMF solution removals Fmoc
Protection group 5+15min, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
In this approach, according to target product correspondence institute palpus amino acid sequence, remaining amino acid is coupled successively, obtain side chain
Full guard polypeptide resin.
This side chain full guard polypeptide resin is reacted into 5min with 20%PIP/DMF solution 5mL, DMF washed once, add
20%PIP/DMF solution 5mL reacts 15min, drains, and is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively, drains,
Weigh to obtain 24.65g peptide resins.
The preparation of the linear thick peptide of Linaclotide
H-Cys(Meb)-Cys-Glu-Tyr-Cys-Cys(Meb)-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-
It is prepared by OH
Configuration lysate, its each component volume ratio is TFA:EDT:Methyl phenyl ethers anisole:Thioanisole:Water=90:2.5:2.5:
2.5:2.5；By lysate add solid phase reactor in, with above-mentioned 5g peptide resins (V peptide resins:V cutting liquid=1:8) at room temperature
After concussion reaction 2h, reaction solution is injected into ether (V reaction solutions:V ether=1:6) in, precipitation, collected after centrifugation white solid sinks
Form sediment, vacuum drying obtains final product the linear thick peptide 0.96g of Cys (Meb).
Two pairs of preparations of disulfide bond intermediate
The thick peptides of 50mg are taken, 2M guanidine hydrochlorides (50ml), 0.1M tertiary sodium phosphates regulation PH to 7, room temperature reaction 12h is added, obtained
Containing two pairs of disulfide bond intermediates (IV).
Above-mentioned reaction solution is purified using the semi-preparative HPLC of Yi Lite.Post is prepared using C18 (20mm*250mm)；
Flow velocity：9ml/min；Mobile phase：A phases are water (1 ‰ TFA), and B phases are acetonitrile；Analysis condition：Acetonitrile：5-20-30-95%, when
Between：0-5-45-50min.Preparation solution is collected, freeze-drying obtains 16mg two to disulfide bond intermediate (IV).
The preparation of Linaclotide (I)
Above-mentioned two pairs of disulfide bond intermediates (IV) sterlings of 10mg are taken, TFA (3ml), 5%DMSO is added, at 40 DEG C, reaction
4h, obtains Linaclotide (I), carries out mass spectral analysis, while compared with standard items, as a result correctly.Its HPLC purity is 68%.
Embodiment 5：
The preparation of Linaclotide resin
H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Meb)-Cys(Trt)-Asn(Trt)-
The preparation of Pro-Ala-Cys (Trt) Thr (tBu)-Gly-Cys (Meb)-Tyr (tBu)-Resin
Weigh 10g substitution degrees be 1.14mmol/g Wang resins in solid phase reactor, with the swelling Wang resins 20 of DCM
Minute, drain.
To in solid phase reactor add Fmoc-Tyr (tBu)-OH (15.7g, 13.68mmol), HOBt (5.55g,
4.1mmol), DIC (6.35mL, 4.1mmol), DMAP (0.5g, 4.1mmol) are dissolved in DMF (70ml), 15 points of room temperature reaction
Clock.
Resin washing is drained, Fmoc-Tyr (tBu)-Wang resins are obtained final product, resin substitution degree is measured for 0.426mmol/
g.To addition closed reagent 70mL (acetic anhydride (mmol) in resin:DIPEA (mmol)=1:1) 10h, is reacted, is closed remaining
Amino, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
Add 20%PIP/DMF solution reactions 20min removing Fmoc protection groups, respectively with DCM (1 time), MeOH (1 time) with
(3 times) washings of DMF, obtain H-Tyr (tBu)-Wang resins；Continuously add Fmoc-Cys (Meb) OH (5.71g, 12.78mmol),
HOBt (2.07g, 15.34mmol), DIC (2.37mL, 15.34mmol), DMF (70mL), room temperature reaction 2h.
Coupling completeness can test detection using Kaiser；After detection passes through, with 20%PIP/DMF solution removals Fmoc
Protection group 5+15min, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
In this approach, according to target product correspondence institute palpus amino acid sequence, remaining amino acid is coupled successively, obtain side chain
Full guard polypeptide resin.
This side chain full guard polypeptide resin is reacted into 5min with 20%PIP/DMF solution 5mL, DMF washed once, add
20%PIP/DMF solution 5mL reacts 15min, drains, and is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively, drains,
Weigh to obtain 24.86g peptide resins.
The preparation of the linear thick peptide of Linaclotide
H-Cys-Cys-Glu-Tyr-Cys(Meb)-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys(Meb)-Tyr-
It is prepared by OH
Configuration lysate, its each component volume ratio is TFA:EDT:Methyl phenyl ethers anisole:Thioanisole:Water=90:2.5:2.5:
2.5:2.5；By lysate add solid phase reactor in, with above-mentioned 5g peptide resins (V peptide resins:V cutting liquid=1:8) at room temperature
After concussion reaction 2h, reaction solution is injected into ether (V reaction solutions:V ether=1:6) in, precipitation, collected after centrifugation white solid sinks
Form sediment, vacuum drying obtains final product the linear thick peptide 0.93g of Cys (Meb).
Two pairs of preparations of disulfide bond intermediate
The thick peptides of 50mg are taken, 2M guanidine hydrochlorides (50ml), 0.1M tertiary sodium phosphates regulation PH to 7, room temperature reaction 12h is added, obtained
Containing two pairs of disulfide bond intermediates (IV).
Above-mentioned reaction solution is purified using the semi-preparative HPLC of Yi Lite.Post is prepared using C18 (20mm*250mm)；
Flow velocity：9ml/min；Mobile phase：A phases are water (1 ‰ TFA), and B phases are acetonitrile；Analysis condition：Acetonitrile：5-20-30-95%, when
Between：0-5-45-50min.Preparation solution is collected, freeze-drying obtains 15mg two to disulfide bond intermediate (IV).
The preparation of Linaclotide (I)
Above-mentioned two pairs of disulfide bond intermediates (IV) sterlings of 10mg are taken, TFA (3ml), 15%DMSO is added, at 60 DEG C, instead
3h is answered, Linaclotide (I) is obtained, mass spectral analysis is carried out, while compared with standard items, as a result correctly.Its HPLC purity is 42%.
Embodiment 6：
The preparation of Linaclotide resin
H-Cys(Trt)-Cys(Meb)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-
The preparation of Pro-Ala-Cys (Meb)-Thr (tBu)-Gly-Cys (Trt)-Tyr (tBu)-Resin
Weigh 10g substitution degrees be 1.14mmol/g Wang resins in solid phase reactor, with the swelling Wang resins 20 of DCM
Minute, drain.
To in solid phase reactor add Fmoc-Tyr (tBu)-OH (15.7g, 13.68mmol), HOBt (5.55g,
4.1mmol), DIC (6.35mL, 4.1mmol), DMAP (0.5g, 4.1mmol) are dissolved in DMF (70ml), 15 points of room temperature reaction
Clock.
Resin washing is drained, Fmoc-Tyr (tBu)-Wang resins are obtained final product, resin substitution degree is measured for 0.426mmol/
g.To addition closed reagent 70mL (acetic anhydride (mmol) in resin:DIPEA (mmol)=1:1) 10h, is reacted, is closed remaining
Amino, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
Add 20%PIP/DMF solution reactions 20min removing Fmoc protection groups, respectively with DCM (1 time), MeOH (1 time) with
(3 times) washings of DMF, obtain H-Tyr (tBu)-Wang resins；Continuously add Fmoc-Cys (Trt)-OH (7.48g, 12.78mmol),
HOBt (2.07g, 15.34mmol), DIC (2.37mL, 15.34mmol), DMF (70mL), room temperature reaction 2h.
Coupling completeness can test detection using Kaiser；After detection passes through, with 20%PIP/DMF solution removals Fmoc
Protection group 5+15min, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
In this approach, according to target product correspondence institute palpus amino acid sequence, remaining amino acid is coupled successively, obtain side chain
Full guard polypeptide resin.
This side chain full guard polypeptide resin is reacted into 5min with 20%PIP/DMF solution 5mL, DMF washed once, add
20%PIP/DMF solution 5mL reacts 15min, drains, and is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively, drains,
Weigh to obtain 25.24g peptide resins.
The preparation of the linear thick peptide of Linaclotide
H-Cys-Cys(Meb)-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys(Meb)-Thr-Gly-Cys-Tyr-
It is prepared by OH
Configuration lysate, its each component volume ratio is TFA:EDT:Methyl phenyl ethers anisole:Thioanisole:Water=90:2.5:2.5:
2.5:2.5；By lysate add solid phase reactor in, with above-mentioned 5g peptide resins (V peptide resins:V cutting liquid=1:8) at room temperature
After concussion reaction 2h, reaction solution is injected into ether (V reaction solutions:V ether=1:6) in, precipitation, collected after centrifugation white solid sinks
Form sediment, vacuum drying obtains final product the linear thick peptide 0.98g of Cys (Meb).
Two pairs of preparations of disulfide bond intermediate
The thick peptides of 50mg are taken, 2M guanidine hydrochlorides (50ml), 0.1M tertiary sodium phosphates regulation PH to 7, room temperature reaction 12h is added, obtained
Containing two pairs of disulfide bond intermediates (IV).
Above-mentioned reaction solution is purified using the semi-preparative HPLC of Yi Lite.Post is prepared using C18 (20mm*250mm)；
Flow velocity：9ml/min；Mobile phase：A phases are water (1 ‰ TFA), and B phases are acetonitrile；Analysis condition：Acetonitrile：5-20-30-95%, when
Between：0-5-45-50min.Preparation solution is collected, freeze-drying obtains 15mg two to disulfide bond intermediate (IV).
The preparation of Linaclotide (I)
Above-mentioned two pairs of disulfide bond intermediates (IV) sterlings of 10mg are taken, TFA (3ml), 20%DMSO is added, at 50 DEG C, instead
3.5h is answered, Linaclotide (I) is obtained, mass spectral analysis is carried out, while compared with standard items, as a result correctly.Its HPLC purity is
19%.
Embodiment 7：
The preparation of Linaclotide resin
H-Cys(Mob)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Mob)-Asn(Trt)-
The preparation of Pro-Ala-Cys (Trt)-Thr (tBu)-Gly-Cys (Trt)-Tyr (tBu)-Resin
Weigh 10g substitution degrees be 1.14mmol/g Wang resins in solid phase reactor, with the swelling Wang resins 20 of DCM
Minute, drain.
To in solid phase reactor add Fmoc-Tyr (tBu)-OH (15.7g, 13.68mmol), HOBt (5.55g,
4.1mmol), DIC (6.35mL, 4.1mmol), DMAP (0.5g, 4.1mmol) are dissolved in DMF (70ml), 15 points of room temperature reaction
Clock.
Resin washing is drained, Fmoc-Tyr (tBu)-Wang resins are obtained final product, resin substitution degree is measured for 0.426mmol/
g.To addition closed reagent 70mL (acetic anhydride (mmol) in resin:DIPEA (mmol)=1:1) 10h, is reacted, is closed remaining
Amino, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
Add 20%PIP/DMF solution reactions 20min removing Fmoc protection groups, respectively with DCM (1 time), MeOH (1 time) with
(3 times) washings of DMF, obtain H-Tyr (tBu)-Wang resins；Continuously add Fmoc-Cys (Trt)-OH (7.48g, 12.78mmol),
HOBt (2.07g, 15.34mmol), DIC (2.37mL, 15.34mmol), DMF (70mL), room temperature reaction 2h.
Coupling completeness can test detection using Kaiser；After detection passes through, with 20%PIP/DMF solution removals Fmoc
Protection group 5+15min, is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively.
In this approach, according to target product correspondence institute palpus amino acid sequence, remaining amino acid is coupled successively, obtain side chain
Full guard polypeptide resin.
This side chain full guard polypeptide resin is reacted into 5min with 20%PIP/DMF solution 5mL, DMF washed once, add
20%PIP/DMF solution 5mL reacts 15min, drains, and is washed with DCM (1 time), MeOH (1 time) and DMF (3 times) respectively, drains,
Weigh to obtain 25.34g peptide resins.
The preparation of the linear thick peptide of Linaclotide
H-Cys(Mob)-Cys-Glu-Tyr-Cys-Cys(Mob)-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-
It is prepared by OH
Configuration lysate, its each component volume ratio is TFA:EDT:Methyl phenyl ethers anisole:Thioanisole:Water=90:2.5:2.5:
2.5:2.5；By lysate add solid phase reactor in, with above-mentioned 5g peptide resins (V peptide resins:V cutting liquid=1:8) at room temperature
After concussion reaction 2h, reaction solution is injected into ether (V reaction solutions:V ether=1:6) in, precipitation, collected after centrifugation white solid sinks
Form sediment, vacuum drying obtains final product the linear thick peptide 1.02g of Cys (Mob).
Two pairs of preparations of disulfide bond intermediate
The thick peptides of 50mg are taken, 2M guanidine hydrochlorides (50ml), 0.1M tertiary sodium phosphates regulation PH to 7, room temperature reaction 12h is added, obtained
Containing two pairs of disulfide bond intermediates (IV).
Above-mentioned reaction solution is purified using the semi-preparative HPLC of Yi Lite.Post is prepared using C18 (20mm*250mm)；
Flow velocity:9ml/min；Mobile phase：A phases are water (1 ‰ TFA), and B phases are acetonitrile；Analysis condition：Acetonitrile：5-20-30-95%, when
Between：0-5-45-50min.Preparation solution is collected, freeze-drying obtains 15mg two to disulfide bond intermediate (IV).
The preparation of Linaclotide (I)
Above-mentioned two pairs of disulfide bond intermediates (IV) sterlings of 10mg are taken, is 94 with volume ratio:5:1 TFA, DMSO, methyl phenyl ethers anisole
Intermixture is solvent, and temperature is 60 DEG C, and the time is 600min, obtains Linaclotide (I), carries out mass spectral analysis, while and standard
Product are compared, as a result correctly.Its HPLC purity is 23%.
As seen from the above-described embodiment, with tBu as blocking group, the Cys for first completing the 2nd, 10 and the 5th, 13 is oxidized to
Ring, is obtained containing two pairs of Linaclotide intermediates of disulfide bond, is further oxidized to target product Linaclotide, the synthesis road
Line, its preparation time, and preparation product purity is optimal.
Embodiment 1~3 is one group, and 4~6 make comparisons for one group, it is seen then that for containing in two pairs of Linaclotides of disulfide bond
Mesosome, when its disulfide bond is present in 2,10 and 5,13, effect is the most excellent.Because, the selection of different cyclization strategies, by
In its steric hindrance, many influences such as group conjugated system, there can be complexity to reacting the final Linaclotide that is oxidized to
Influence.On the one hand, the group of preferential bonding has space steric effect in itself, has inhibition, the opposing party to further cyclization
Face, the group of preferential bonding has been provided with more stable conjugated system, serves what is promoted and consolidate to desirable oxidation reaction
Effect.Complexing action of both comprehensive, finally, in the disulfide bond first prepared in the presence of 2,10 and 5,13 of present invention offer
Mesosome, further prepares target product, highly efficient, economy.
Embodiment 1,4,7 is made comparisons for one group, it is seen then that used as blocking group, and tBu is blocking group synthetic method, effect
It is more excellent.Because, used as blocking group, it can provide appropriate steric hindrance to tBu.On the one hand, blocking group pair
Cys plays enough protective effects, but still further aspect, and blocking group makees due to steric hindrance and conjugated system electrophilic etc.
With key to other key mappings and also negative impact, it is most important that, blocking group turns in final intermediate to target product
During change, the removing to stablize.The consideration of comprehensive several respects, finally, tBu preferred for this invention can be presented optimal effect
Really.
Mentioned reagent title abbreviation full name control such as table 1 below：
Table 1- reagent name lists
| Referred to as | Full name |
| DMF | N,N-dimethylformamide |
| DCM | Dichloromethane |
| DMAP | DMAP |
| DMSO | Dimethyl sulfoxide (DMSO) |
| DIC | N, N '-DIC |
| TFA | Trifluoroacetic acid |
| NMM | N-methylmorpholine |
| HOBt | I-hydroxybenzotriazole |
| HOAt | N- hydroxyl -7- azepine BTAs |
| HBTU | BTA-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester |
| HATU | 2-7 (azo BTA)-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester |
| PyBOP | Hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus |
| EDT | 1,2- dithioglycols |
| PIP | Piperidines |
| GuHCl | Guanidine hydrochloride |
| TSP | Tri-sodium phosphate |
It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to preferably
Embodiment has been described in detail to the present invention, it will be understood by those within the art that, can be to technology of the invention
Scheme is modified or equivalent, and without deviating from the spirit and scope of technical solution of the present invention, it all should cover in this hair
In the middle of bright right.
Claims (10)
1. a kind of method for preparing Linaclotide, it is characterised in that：Including,
Oxidation reaction is carried out to the Linaclotide intermediate containing two pairs of disulfide bond, Linaclotide is obtained；
Wherein, it is described containing two pairs of Linaclotide intermediates of disulfide bond, its be in the presence of the disulfide bond of the 2nd, 10 Cys and the 5th,
13 disulfide bond of Cys.
2. the method for preparing Linaclotide as claimed in claim 1, it is characterised in that：It is described to contain two couples of Li Naluo of disulfide bond
Peptide intermediate, it is have blocking group on the 1st, 6 Cys.
3. the method for preparing Linaclotide as claimed in claim 1 or 2, it is characterised in that：It is described to contain two pairs of profits of disulfide bond
That Lip river peptide intermediate, it is to carry out the 2nd, 10 Cys and the 5th, 13 Cys to the linear thick peptide of Linaclotide to carry out aoxidizing cyclic system
.
4. the method for preparing Linaclotide as claimed in claim 3, it is characterised in that：The linear thick peptide of the Linaclotide, it is
There is blocking group on the 1st, 6 Cys.
5. the method for preparing Linaclotide as claimed in claim 4, it is characterised in that：The linear thick peptide of the Linaclotide, it is
The cracking of Linaclotide resin is obtained；Wherein, the Linaclotide resin, it is that 1,6 Cys have common blocking group, 2,10
There is Trt on position and 5,13 Cys.
6. the method for preparing Linaclotide as claimed in claim 5, it is characterised in that：The oxidation cyclization, pH is 6.5~9.5,
Time is 10~14h.
7. the method that Linaclotide is prepared as any one of claim 2,4,5 or 6, it is characterised in that：The protection group
Group, it is tBu, Meb, the one kind in Mob.
8. the method for preparing Linaclotide as claimed in claim 7, it is characterised in that：The blocking group, it is tBu.
9. the method for preparing Linaclotide as claimed in claim 8, it is characterised in that：The oxidation reaction, including, it is directed to
TBu, specially with one or more in TFA or 5%-20%DMSO as solvent, temperature be 15~30 DEG C, the time be 25~
40min；It is directed to Meb, and specially with one or more in TFA or 5%-20%DMSO as solvent, temperature is 40~60 DEG C, when
Between be 3~4h；It is directed to Mob, is 94 specially with volume ratio:5:1 TFA, DMSO, methyl phenyl ethers anisole intermixture are solvent, temperature
It it is 55~65 DEG C, the time is 30min~90min.
10. the method that Linaclotide is prepared as any one of claim 5,6,8 or 9, it is characterised in that：The cracking,
It is that, with TFA, EDT, methyl phenyl ethers anisole, thioanisole and water as lysate, its volume ratio is 90:2.5:2.5:2.5:2.5；Its profit that
Lip river peptide resin is 1 with the volume ratio of lysate:8.
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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 |
| 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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|---|---|---|---|---|
| WO2011103311A2 (en) * | 2010-02-17 | 2011-08-25 | Ironwood Pharmaceuticals, Inc | Treatments for gastrointestinal disorders |
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2017
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| WO2011103311A2 (en) * | 2010-02-17 | 2011-08-25 | Ironwood Pharmaceuticals, Inc | Treatments for gastrointestinal disorders |
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| MIRIAM等: "Optimized Fmoc Solid-Phase Synthesis of the Cysteine-Rich", 《BIOPOLYMERS》 * |
| 王德心等: "环肽的合成研究进展", 《有机化学》 * |
| 黄蓓: "半胱氨酸的保护及研究进展", 《河南化工》 * |
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 |
| 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 |
| CN113956333B (en) * | 2021-12-22 | 2022-03-29 | 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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