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CN108003222A - A kind of solid phase synthesis process of plecanatide - Google Patents

A kind of solid phase synthesis process of plecanatide Download PDF

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Publication number
CN108003222A
CN108003222A CN201711495911.6A CN201711495911A CN108003222A CN 108003222 A CN108003222 A CN 108003222A CN 201711495911 A CN201711495911 A CN 201711495911A CN 108003222 A CN108003222 A CN 108003222A
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Prior art keywords
cys
leu
glu
asn
synthesis
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徐峰
苏军
谷海涛
周金玉
刘标
孙美禄
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Jiangsu Sinopep Macao Zaino Biological Pharmaceutical Ltd By Share Ltd
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Jiangsu Sinopep Macao Zaino Biological Pharmaceutical Ltd By Share Ltd
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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

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention relates to the synthetic method of field of medicaments product plecanatide.This method is combined to method synthesis plecanatide using solid liquid phase, directly obtains plecanatide crude product by cracking, product is obtained by purifying.The present invention is first using liquid phase synthesizing method synthesis hexapeptide fragment; hexapeptide fragment is coupled on solid-phase resin using enzymatic method; pass through the protection group to Cys diverse locations on solid-phase resin using the false dilution effect of solid-phase resin; it is done directly the selectivity synthesis of two two sulphur rings; it is cut down from resin afterwards, is directly purified.Since liquid phase, solid phase carry out synthesis at the same time, enzyme' s catalysis substantially increases combined coefficient and synthesis yield, not only greatly reduces the generation of waste water, spent organic solvent, exhaust gas, while solid-liquid is combined to and greatly reduces production cost, improves product quality.

Description

A kind of solid phase synthesis process of plecanatide
Technical field
The present invention is the synthesis preparation method of Peptide systhesis preparation field, more particularly to plecanatide.
Technical background
Plecanatide structural formula is as follows：
H-Asn-Asp-Glu-Cys(1)-Glu-Leu-Cys(2)-Val-Asn-Val-Ala-Cys(1)-Thr-Gly-Cys (2)-Leu-OH-cyclic (4→12),(7→15)-bis(disulfide)
Plecanatide is researched and developed successfully by Synergy companies of the U.S., and the approval of FDA is obtained on January 19th, 2017, which is A kind of oral guanosine cyclic mono-phosphate (GC-C) receptor stimulating agent, can play a role in upper digestive tract to stimulate secretion intestinal juice, So as to maintain function of intestinal canal normal.Its amino acid peptide sequence is H-Asn-Asp-Glu-Cys (1)-Glu-Leu-Cys (2)-Val- Asn-Val-Ala-Cys (1)-Thr-Gly-Cys (2)-Leu-OH, wherein sequence No. 4 position and No. 10 positions, No. 7 positions with No. 15 Position forms two pair of two sulphur ring respectively.It is mainly used for treating constipation intestinal irritable syndrome (IBS-C) and chronic idiopathic constipation (CIC), it be so far second be used to treat the oral polypeptide drugs of constipation.Synthesis patent currently on the medicine is few, Mainly there are two patents of CN201310209459.8 and CN201310680490.X of Shenzhen writing brush space medicine company, and Nanjing industry is greatly One learned prepares patent CN201510104385.0.Two patents of Shenzhen writing brush space medicine company are respectively adopted different solid-liquids and are harmonious Into strategy, although the different side chain protected agent all used, two pairs of disulfide bond of controlled syntheses, as a result of last liquid Mutually the method for cyclisation, the concentration of synthesis reaction solution are extremely restricted, and can produce substantial amounts of waste water, and complicated trouble is uncomfortable Close industrialization production.The method that Nanjing University of Technology employs solid phase cyclization, improves to some extent, but pure synthesis in solid state is, it is necessary to height Multiple material is put into, and produces the waste of a large amount of raw material, atom utilization is very poor, and the reaction time is relatively long.Yield of the present invention Substantially maintain an equal level with existing literature report or slightly above report, but overall time cost, Material Cost etc. substantially reduce, and fit For industrialized production.
The content of the invention
The technical problems to be solved by the invention are in view of the deficiencies of the prior art, there is provided a kind of new plecanatide Solid phase synthesis process, this method combined coefficient is high, and Material Cost substantially reduces, and is suitable for industrialized production.
The technical problems to be solved by the invention are realized by following technical solution.The present invention is a kind of Puli Block the solid phase synthesis process of that peptide, its main feature is that：First using liquid phase synthesizing method synthesis hexapeptide fragment, using enzymatic method by hexapeptide piece Section is coupled on solid-phase resin, passes through the guarantor to Cys diverse locations on solid-phase resin using the false dilution effect of solid-phase resin Base is protected, the selectivity synthesis of two two sulphur rings is done directly, it is cut down from resin afterwards, is directly purified.
A kind of solid phase synthesis process of plecanatide of the present invention, its further preferred technical scheme steps It is：
A, fragment condensation is into all kinds of plecanatide intermediates：
(1)R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)-Val- Ala-
The synthesis of Cys (R1)-Thr (R4)-Gly-Cys (R1)-Leu-R3；
（2）R-Asn (R1)-Asp (R2)-Glu (R2)-Cys (R1)-Glu (R2)-Leu- is completed using the false dilution effect of resin
Cys(R1)-Val-Asn(R1)-Val-Ala-Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4 → 12) two cysteines of the synthesis of-disulfide, No. 4 positions and No. 12 positions are into thioether bond ring；
(3)R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)-Val- Ala-
Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4→12), (7→15)-bis(disulfide) Synthesis；Two cysteines of No. 4 positions and No. 12 positions are on the basis of thioether ring, two cysteines of No. 7 positions and No. 15 positions Again into thioether ring；
In all kinds of amino acid protecting groups：
R for Z, Boc, Cl-Z, Fmoc, oNBS, dNBS, Troc, Dts, pNZ, oNZ, NVOC, NPPOC, HFA, Ddz, Bpoc, Nps, Nsc, Bsmoc, α-Nsmoc, ivDde, Fmoc*, MTT, Alloc wherein any one；
R1 is Meb, Mob, TRT, Tmob, Mmt, Xan, Pmbf, Bn, tBu, Fm, Dnpe, Fmoc, Acm, Phacm, StBu、 Npys, Alloc wherein any one；
R2 for H, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb wherein any one；
R3 is H, OMe, Oet, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb, wang tree Fat, CTC resins wherein any one；
R4 for Bn, cHx, tBu, Trt, TBDMS, Dmnb, Poc wherein any one；
B, the synthesis of the thick peptide of plecanatide；
H-Asn-Asp-Glu-Cys(1)-Glu-Leu-Cys(2)-Val-Asn-Val-Ala-Cys(1)-Thr-Gly-Cys (2)-Leu-OH-cyclic (4→12),(7→15)-bis(disulfide)
C, the purifying of plecanatide and lyophilized.
Related terms in the present invention are explained as follows：
The solid phase synthesis process of plecanatide of the present invention, its further preferred technical solution are：Synthesis in solid state mistake The coupling system used in journey is：
（1）, single condensing agent：DIC, EDC, chloracetyl chloride, ethyl chloroformate, isobutylchloroformate, isobutyl chlorocarbonate, TBTU, PyBOP, HBTU, papain, trypsase；
（2）Two system condensing agents：DIC/HOBT、EDC/HOBT、DIC/HOSu、EDC/ HOSu；
（3）Cyclization reagent：Piperidines/DMF, H2O2/DMF、I2/DMF、DMSO/DMF、H2O2/H2O、I2/ H2O、DMSO/ H2O；
The solid phase synthesis process of plecanatide of the present invention, its further preferred technical solution are：R-Asn(R1)- Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-
Val-Asn(R1)-Val-Ala-Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4→12), (7 → 15) synthesis of-bis (disulfide) comprises the following steps that：
（1）Using Fmoc-Leu-OH as first amino acid, with wang resins or CTC resins, the resin substitution degree being related to preferably from The mmol/g of 0.2mmol/g ~ 1.8 carries out condensation and forms Fmoc-Leu- resins, then passes through the method for coupling amino acid one by one, even Connection completes 10 amino-acid resins；R-Asn (R1)-Asp (R2)-Glu (R2)-Cys (R1) that liquid phase process is synthesized afterwards- Glu (R2)-Leu-R3 is directly coupled on peptide resin using papain, completes the synthesis of full guard peptide resin；
（2）、R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)-Val- Ala-
Cys (1)-Thr (R4)-Gly-Cys (R1)-Leu-R3-cyclic (4 → 12), the synthesis of disulfide
The cysteine protected using No. 4 positions, No. 12 positions Mmt or ACM, is cracked with the TFA of various concentrations, removes Mmt Oxidation cyclization either is carried out with one kind in the oxidants such as DMSO, air, hydrogen peroxide or composition after ACM, obtains target production Product；
（3）、R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)- Val-Ala-
Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4→12), (7→15)-bis(disulfide) Synthesis
The cysteine protected using No. 7 positions, No. 15 positions TRT, Tmob, Pmbf or ACM, uses I2/ DMF be deprotected simultaneously same Shi Jinhang is oxidized to two sulphur rings, obtains target product；
（4）H-Asn-Asp-Glu-Cys(1)-Glu-Leu-Cys(2)-Val-Asn-Val-Ala-Cys(1)-Thr-Gly-Cys (2)-
Leu-OH-cyclic (4 → 12), the thick peptide symthesis of (7 → 15)-bis (disulfide)
Cracked using the TFA of different proportion, proton capture agent or protection group capturing agent are selected from：TIS, EDT, water, dimethyl sulfide Ether, thioanisole etc., the ratio of TFA are more than 90%.
In the present invention, papain can not only be catalyzed the reaction, effective to remaining all kinds of amide linkage Chengdu, ammonia Base acid range includes all 20 kinds of conventional amino acids.In coupling process, used DMF:Boric acid sodium hydroxide（Or hydroxide Potassium）Scope is 90:10~99:1；DMF:Sodium dihydrogen phosphate scope is 92:8~99:1, wherein, boric acid sodium hydroxide（Or hydroxide Potassium）Solution range is 0.5% ~ 20%, and sodium dihydrogen phosphate scope is 1% ~ 13%.
Compared with prior art, the method for the present invention carries out synthesis due to liquid phase, solid phase at the same time, and enzyme' s catalysis substantially increases Combined coefficient and synthesis yield, not only greatly reduce the generation of waste water, spent organic solvent, exhaust gas, while solid-liquid is combined to Production cost is greatly reduced, improves product quality.
Brief description of the drawings
Fig. 1 is that plecanatide crude product HPLC obtained by embodiment schemes；
Fig. 2 is the plecanatide high purity product HPLC figures obtained by embodiment；
Fig. 3 is the plecanatide high purity product mass spectrogram obtained by embodiment.
Embodiment
Referring to the drawings, concrete technical scheme of the invention described further below, in order to those skilled in the art Member is further understood that the present invention, without forming the limitation to its right.
Embodiment 1, a kind of solid phase synthesis process of plecanatide, first synthesizes hexapeptide fragment, profit using liquid phase synthesizing method Hexapeptide fragment is coupled on solid-phase resin with enzymatic method, using the false dilution effect of solid-phase resin on solid-phase resin by right The protection group of Cys diverse locations, is done directly the selectivity synthesis of two two sulphur rings, afterwards cuts down it from resin, Directly purified.
Embodiment 2, a kind of solid phase synthesis process of plecanatide described in embodiment 1, it is comprised the concrete steps that：
A, fragment condensation is into all kinds of plecanatide intermediates：
(1)R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)-Val- Ala-
The synthesis of Cys (R1)-Thr (R4)-Gly-Cys (R1)-Leu-R3；
（2）R-Asn (R1)-Asp (R2)-Glu (R2)-Cys (R1)-Glu (R2)-Leu- is completed using the false dilution effect of resin
Cys(R1)-Val-Asn(R1)-Val-Ala-Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4 → 12) two cysteines of the synthesis of-disulfide, No. 4 positions and No. 12 positions are into thioether bond ring；
(3)R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)-Val- Ala-
Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4→12), (7→15)-bis(disulfide) Synthesis；Two cysteines of No. 4 positions and No. 12 positions are on the basis of thioether ring, two cysteines of No. 7 positions and No. 15 positions Again into thioether ring；
In all kinds of amino acid protecting groups：
R for Z, Boc, Cl-Z, Fmoc, oNBS, dNBS, Troc, Dts, pNZ, oNZ, NVOC, NPPOC, HFA, Ddz, Bpoc, Nps, Nsc, Bsmoc, α-Nsmoc, ivDde, Fmoc*, MTT, Alloc wherein any one；
R1 is Meb, Mob, TRT, Tmob, Mmt, Xan, Pmbf, Bn, tBu, Fm, Dnpe, Fmoc, Acm, Phacm, StBu、 Npys, Alloc wherein any one；
R2 for H, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb wherein any one；
R3 is H, OMe, Oet, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb, wang tree Fat, CTC resins wherein any one；
R4 for Bn, cHx, tBu, Trt, TBDMS, Dmnb, Poc wherein any one；
B, the synthesis of the thick peptide of plecanatide；
C, the purifying of plecanatide and lyophilized.
The coupling system used during synthesis in solid state is：
（1）, single condensing agent：DIC, EDC, chloracetyl chloride, ethyl chloroformate, isobutylchloroformate, isobutyl chlorocarbonate, TBTU, PyBOP, HBTU, papain, trypsase；
（2）Two system condensing agents：DIC/HOBT、EDC/HOBT、DIC/HOSu、EDC/ HOSu；
（3）Cyclization reagent：Piperidines/DMF, H2O2/DMF、I2/DMF、DMSO/DMF、H2O2/H2O、I2/ H2O、DMSO/ H2O；
Embodiment 3, in a kind of solid phase synthesis process of plecanatide described in embodiment 1 or 2：
R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-
Val-Asn(R1)-Val-Ala-Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4→12), (7 → 15) synthesis of-bis (disulfide) comprises the following steps that：
（1）Using Fmoc-Leu-OH as first amino acid, with wang resins or CTC resins, the resin substitution degree being related to preferably from The mmol/g of 0.2mmol/g ~ 1.8 carries out condensation and forms Fmoc-Leu- resins, then passes through the method for coupling amino acid one by one, even Connection completes 10 amino-acid resins；R-Asn (R1)-Asp (R2)-Glu (R2)-Cys (R1) that liquid phase process is synthesized afterwards- Glu (R2)-Leu-R3 is directly coupled on peptide resin using papain, completes the synthesis of full guard peptide resin；
（2）、R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)-Val- Ala-
Cys (1)-Thr (R4)-Gly-Cys (R1)-Leu-R3-cyclic (4 → 12), the synthesis of disulfide
The cysteine protected using No. 4 positions, No. 12 positions Mmt or ACM, is cracked with the TFA of various concentrations, removes Mmt Oxidation cyclization either is carried out with one kind in the oxidants such as DMSO, air, hydrogen peroxide or composition after ACM, obtains target production Product；
（3）、R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)- Val-Ala-
Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4→12), (7→15)-bis(disulfide) Synthesis
The cysteine protected using No. 7 positions, No. 15 positions TRT, Tmob, Pmbf or ACM, uses I2/ DMF be deprotected simultaneously same Shi Jinhang is oxidized to two sulphur rings, obtains target product；
（4）H-Asn-Asp-Glu-Cys(1)-Glu-Leu-Cys(2)-Val-Asn-Val-Ala-Cys(1)-Thr-Gly-Cys (2)-
Leu-OH-cyclic (4 → 12), the thick peptide symthesis of (7 → 15)-bis (disulfide)
Cracked using the TFA of different proportion, proton capture agent or protection group capturing agent are selected from：TIS, EDT, water, dimethyl sulfide Ether, thioanisole etc., the ratio of TFA are more than 90%.
Embodiment 4, a kind of solid phase synthesis process methods experiment of plecanatide：
First, the synthesis of Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-Glu (OtBu)-Leu-OMe
(1), the synthesis of Boc-Asn (Trt)-Asp (OtBu)-OH
Solution 1：Boc-Asn (Trt)-OH, the HOSu of 110 mmoles of 100 mmoles are weighed, is placed in the round-bottomed flask of 500ml, adds Enter 200mlTHF dissolvings, be cooled under magnetic agitation 0-5 DEG C it is spare.
Solution 2：Weigh the DCC of 110 mmoles, be placed in small beaker, dissolved with the THF of 100ml, be cooled to 0-5 DEG C it is spare
Solution 3：Weigh the NH of 120 mmoles2- Asp (OtBu)-OH, is dissolved in 10% sodium carbonate liquor of 100ml, standby at room temperature With
At 0-5 DEG C, by DCC solution（Solution 2）Slowly it is added drop-wise in the solution 1 of stirring, reaction reacts 2 at 15 minutes, 25 DEG C Hour, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.White precipitate is filtered to remove, uses 30mlTHF Washing is precipitated and filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, is beaten with saturated sodium bicarbonate 500ml Washing, filters, repeated washing 3 times, obtains white solid.White solid is dissolved in 200mlTHF, constant pressure funnel is used at 25 DEG C Being added drop-wise in solution 3 slowly, after being added dropwise reaction the reaction was continued 1 it is small when, reaction terminates（It is subject to contact plate）.With dilute lemon Acid adjusts pH value, and revolving removes THF to 7, is filtered again with dilute citric acid tune pH value to a large amount of white precipitates between 3-4, are obtained, It is beaten and is washed with dilute citric acid 500ml, filtering, obtains white solid after repeated washing 3 times.Product is white solid, purity： 99.2%
(2), the synthesis of Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-OH
Solution 1：Boc-Asn (Trt)-Asp (OtBu)-OH, the HOSu of 110 mmoles of 100 mmoles are weighed, is placed in the circle of 500ml In the flask of bottom, add 200mlTHF dissolvings, be cooled under magnetic agitation 0-5 DEG C it is spare.
Solution 2：Weigh the DCC of 110 mmoles, be placed in small beaker, dissolved with the THF of 100ml, be cooled to 0-5 DEG C it is spare
Solution 3：Weigh the NH of 120 mmoles2- Glu (OtBu)-OH, is dissolved in 10% sodium carbonate liquor of 100ml, standby at room temperature With
At 0-5 DEG C, by DCC solution（Solution 2）Slowly it is added drop-wise in the solution 1 of stirring, reaction reacts 2 at 15 minutes, 25 DEG C Hour, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.White precipitate is filtered to remove, uses 30mlTHF Washing is precipitated and filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, is beaten with saturated sodium bicarbonate 500ml Washing, filters, repeated washing 3 times, obtains white solid.White solid is dissolved in 200mlTHF, constant pressure funnel is used at 25 DEG C Being added drop-wise in solution 3 slowly, after being added dropwise reaction the reaction was continued 1 it is small when, reaction terminates（It is subject to contact plate）.With dilute lemon Acid adjusts pH value, and revolving removes THF to 7, is filtered again with dilute citric acid tune pH value to a large amount of white precipitates between 3-4, are obtained, It is beaten and is washed with dilute citric acid 500ml, filtering, obtains white solid after repeated washing 3 times.Product is white solid, purity： 99.1%。
(3), the synthesis of Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-OH
Solution 1：Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-OH, the HOSu of 110 mmoles of 100 mmoles are weighed, is placed in In the round-bottomed flask of 500ml, add 200mlTHF dissolvings, be cooled under magnetic agitation 0-5 DEG C it is spare.
Solution 2：Weigh the DCC of 110 mmoles, be placed in small beaker, dissolved with the THF of 100ml, be cooled to 0-5 DEG C it is spare
Solution 3：Weigh the NH of 120 mmoles2- Cys (Mmt)-OH, is dissolved in 10% sodium carbonate liquor of 100ml, standby at room temperature With
At 0-5 DEG C, by DCC solution（Solution 2）Slowly it is added drop-wise in the solution 1 of stirring, reaction reacts 2 at 15 minutes, 25 DEG C Hour, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.White precipitate is filtered to remove, uses 30mlTHF Washing is precipitated and filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, is beaten with saturated sodium bicarbonate 500ml Washing, filters, repeated washing 3 times, obtains white solid.White solid is dissolved in 200mlTHF, constant pressure funnel is used at 25 DEG C Being added drop-wise in solution 3 slowly, after being added dropwise reaction the reaction was continued 1 it is small when, reaction terminates（It is subject to contact plate）.With dilute lemon Acid adjusts pH value, and revolving removes THF to 7, is filtered again with dilute citric acid tune pH value to a large amount of white precipitates between 3-4, are obtained, It is beaten and is washed with dilute citric acid 500ml, filtering, obtains white solid after repeated washing 3 times.Product is white solid, purity： 99.3%。
(4), the synthesis of Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-Glu (OtBu)-OH
Solution 1：Weigh Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-OH of 100 mmoles, 110 mmoles HOSu, is placed in the round-bottomed flask of 500ml, adds 200mlTHF dissolvings, be cooled under magnetic agitation 0-5 DEG C it is spare.
Solution 2：Weigh the DCC of 110 mmoles, be placed in small beaker, dissolved with the THF of 100ml, be cooled to 0-5 DEG C it is spare
Solution 3：Weigh the NH of 120 mmoles2- Glu (OtBu)-OH, is dissolved in 10% sodium carbonate liquor of 100ml, standby at room temperature With
At 0-5 DEG C, by DCC solution（Solution 2）Slowly it is added drop-wise in the solution 1 of stirring, reaction reacts 2 at 15 minutes, 25 DEG C Hour, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.White precipitate is filtered to remove, uses 30mlTHF Washing is precipitated and filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, is beaten with saturated sodium bicarbonate 500ml Washing, filters, repeated washing 3 times, obtains white solid.White solid is dissolved in 200mlTHF, constant pressure funnel is used at 25 DEG C Being added drop-wise in solution 3 slowly, after being added dropwise reaction the reaction was continued 1 it is small when, reaction terminates（It is subject to contact plate）.With dilute lemon Acid adjusts pH value, and revolving removes THF to 7, is filtered again with dilute citric acid tune pH value to a large amount of white precipitates between 3-4, are obtained, It is beaten and is washed with dilute citric acid 500ml, filtering, obtains white solid after repeated washing 3 times.Product is white solid, purity： 98.7%。
(5), the conjunction of Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-Glu (OtBu)-Leu-OMe Into
Solution 1：Weigh Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-Glu (OtBu) of 100 mmoles- The HOSu of OH, 110 mmoles, are placed in the round-bottomed flask of 500ml, add 200mlTHF dissolvings, 0-5 DEG C is cooled under magnetic agitation It is spare.
Solution 2：Weigh the DCC of 110 mmoles, be placed in small beaker, dissolved with the THF of 100ml, be cooled to 0-5 DEG C it is spare
Solution 3：Weigh the NH of 110 mmoles2- Leu-OMe, is dissolved in the THF of 100ml, spare at room temperature
At 0-5 DEG C, by DCC solution（Solution 2）Slowly it is added drop-wise in the solution 1 of stirring, reaction reacts 2 at 15 minutes, 25 DEG C Hour, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.White precipitate is filtered to remove, uses 30mlTHF Washing is precipitated and filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, is beaten with saturated sodium bicarbonate 500ml Washing, filters, repeated washing 3 times, obtains white solid.White solid is dissolved in 200mlTHF, constant pressure funnel is used at 25 DEG C Being added drop-wise in solution 3 slowly, after being added dropwise reaction the reaction was continued 1 it is small when, reaction terminates（It is subject to contact plate）.Revolving removes THF solvents, obtain a large amount of white precipitates, are dissolved, are washed with saturated sodium bicarbonate, repeated washing with 1.5 liters of ethyl acetate 500ml*3, with dilute lemon acid elution, repeated washing 500ml*3, with saturated common salt water washing 500ml*3, rotates after dry and removes Ethyl acetate, obtains a large amount of white solids, purity：98.8 %.
2nd, Fmoc-Cys (Trt)-Val-Asn (Trt)-Val-Ala-Cys (Mmt)-Thr (R4)-Gly-Cys (Trt)-Leu- The synthesis of wang resins
2.1st, first amino acid couplings
2.1.1 into reaction kettle, the wang resins and 6L DMF of 200mmol are added, is stirred 10-15 minutes, filters and removes liquid； 6L DCM are added, stirred under nitrogen atmosphere is swollen 25 ~ 35 minutes, is filtered and is removed liquid.
2.1.2 400mmol Fmoc-Leu-OH, 400mmol HOBT are weighed, are dissolved with the DMF of 5L, are turned after dissolving completely Move in activation filling.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL, control temperature is 0 ~ 10 at 0 ~ 10 DEG C DEG C, priming reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Open reaction kettle stirring and upper drum Nitrogen, 20-35 DEG C of coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.Methanol is added to be blocked, After end-blocking, filter and remove reaction solution, wash resin, detect the substitution degree of resin, according to substitution degree feed intake in next step.
2.2nd, second amino acid couplings
2.2.1 deprotection
20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, is added in main reaction kettle and is led to nitrogen and stir Controlling reaction temperature is mixed at 15 ~ 30 DEG C, 5 ~ 7 min is reacted, extracts reaction solution, drain 3 ~ 5 min.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.
Washed 6 times after deprotection
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
2.2.2 coupling
400mmol Fmoc-Cys (Trt)-OH, 400mmol HOBT is weighed, is dissolved with the DMF of 5L, is transferred to after dissolving completely During activation fills.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL at 0 ~ 10 DEG C, and control temperature is living at 0 ~ 10 DEG C Change reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Reaction kettle stirring and upper bulging nitrogen are opened, 20-35 DEG C of coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.
2.3rd, the 3rd amino acid couplings
2.3.1 deprotection
20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, is added in main reaction kettle and is led to nitrogen and stir Controlling reaction temperature is mixed at 15 ~ 30 DEG C, 5 ~ 7 min is reacted, extracts reaction solution, drain 3 ~ 5 min.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.
Washed 6 times after deprotection
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
2.3.2 coupling
400mmol Fmoc-Gly-OH, 400mmol HOBT are weighed, is dissolved with the DMF of 5L, activation is transferred to after dissolving completely and is filled In.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL at 0 ~ 10 DEG C, and control temperature is at 0 ~ 10 DEG C, priming reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Open reaction kettle stirring and upper bulging nitrogen, 20-35 DEG C coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.
2.4th, the 4th amino acid couplings
2.4.1 deprotection
20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, is added in main reaction kettle and is led to nitrogen and stir Controlling reaction temperature is mixed at 15 ~ 30 DEG C, 5 ~ 7 min is reacted, extracts reaction solution, drain 3 ~ 5 min.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.
Washed 6 times after deprotection
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
2.4.2 coupling
400mmol Fmoc-Thr (tbu)-OH, 400mmol HOBT is weighed, is dissolved with the DMF of 5L, is transferred to after dissolving completely During activation fills.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL at 0 ~ 10 DEG C, and control temperature is living at 0 ~ 10 DEG C Change reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Reaction kettle stirring and upper bulging nitrogen are opened, 20-35 DEG C of coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.
2.5th, five amino acid is coupled
2.5.1 deprotection
20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, is added in main reaction kettle and is led to nitrogen and stir Controlling reaction temperature is mixed at 15 ~ 30 DEG C, 5 ~ 7 min is reacted, extracts reaction solution, drain 3 ~ 5 min.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.
Washed 6 times after deprotection
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
2.5.2 coupling
400mmol Fmoc-Cys (Mmt)-OH, 400mmol HOBT is weighed, is dissolved with the DMF of 5L, is transferred to after dissolving completely During activation fills.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL at 0 ~ 10 DEG C, and control temperature is living at 0 ~ 10 DEG C Change reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Reaction kettle stirring and upper bulging nitrogen are opened, 20-35 DEG C of coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.
2.6th, the 6th amino acid couplings
2.6.1 deprotection
20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, is added in main reaction kettle and is led to nitrogen and stir Controlling reaction temperature is mixed at 15 ~ 30 DEG C, 5 ~ 7 min is reacted, extracts reaction solution, drain 3 ~ 5 min.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.
Washed 6 times after deprotection
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
2.6.2 coupling
400mmol Fmoc-Ala-OH, 400mmol HOBT are weighed, is dissolved with the DMF of 5L, activation is transferred to after dissolving completely and is filled In.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL at 0 ~ 10 DEG C, and control temperature is at 0 ~ 10 DEG C, priming reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Open reaction kettle stirring and upper bulging nitrogen, 20-35 DEG C coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.
2.7th, seven amino acid is coupled
2.7.1 deprotection
20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, is added in main reaction kettle and is led to nitrogen and stir Controlling reaction temperature is mixed at 15 ~ 30 DEG C, 5 ~ 7 min is reacted, extracts reaction solution, drain 3 ~ 5 min.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.
Washed 6 times after deprotection
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
2.7.2 coupling
400mmol Fmoc-Val-OH, 400mmol HOBT are weighed, is dissolved with the DMF of 5L, activation is transferred to after dissolving completely and is filled In.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL at 0 ~ 10 DEG C, and control temperature is at 0 ~ 10 DEG C, priming reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Open reaction kettle stirring and upper bulging nitrogen, 20-35 DEG C coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.
2.8th, the 8th amino acid couplings
2.8.1 deprotection
20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, is added in main reaction kettle and is led to nitrogen and stir Controlling reaction temperature is mixed at 15 ~ 30 DEG C, 5 ~ 7 min is reacted, extracts reaction solution, drain 3 ~ 5 min.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.
Washed 6 times after deprotection
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
2.8.2 coupling
400mmol Fmoc-Asn-OH, 400mmol HOBT are weighed, is dissolved with the DMF of 5L, activation is transferred to after dissolving completely and is filled In.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL at 0 ~ 10 DEG C, and control temperature is at 0 ~ 10 DEG C, priming reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Open reaction kettle stirring and upper bulging nitrogen, 20-35 DEG C coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.
2.9th, the 8th amino acid couplings
2.9.1 deprotection
20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, is added in main reaction kettle and is led to nitrogen and stir Controlling reaction temperature is mixed at 15 ~ 30 DEG C, 5 ~ 7 min is reacted, extracts reaction solution, drain 3 ~ 5 min.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.
Washed 6 times after deprotection
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
2.9.2 coupling
400mmol Fmoc-Val-OH, 400mmol HOBT are weighed, is dissolved with the DMF of 5L, activation is transferred to after dissolving completely and is filled In.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL at 0 ~ 10 DEG C, and control temperature is at 0 ~ 10 DEG C, priming reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Open reaction kettle stirring and upper bulging nitrogen, 20-35 DEG C coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.
2.10th, the 8th amino acid couplings
2.10.1 deprotection
20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, is added in main reaction kettle and is led to nitrogen and stir Controlling reaction temperature is mixed at 15 ~ 30 DEG C, 5 ~ 7 min is reacted, extracts reaction solution, drain 3 ~ 5 min.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.
Washed 6 times after deprotection
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
2.10.2 coupling
400mmol Fmoc-Cys (Trt)-OH, 400mmol HOBT is weighed, is dissolved with the DMF of 5L, is transferred to after dissolving completely During activation fills.Stirring, refrigerant are opened, control temperature is slowly added to the DIC of 66mL at 0 ~ 10 DEG C, and control temperature is living at 0 ~ 10 DEG C Change reaction 10 ~ 15 minutes.After the completion of priming reaction, activating solution is transferred in reaction kettle.Reaction kettle stirring and upper bulging nitrogen are opened, 20-35 DEG C of coupling reaction 2h.Filter after reaction and remove reaction liquid, wash resin.
3rd, Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-Glu (OtBu)-Leu-Cys (Trt)- Val-
The synthesis of Asn (Trt)-Val-Ala-Cys (Mmt)-Thr (R4)-Gly-Cys (Trt)-Leu-wang resins
3.1 deprotection
Weigh Fmoc-Cys (Trt)-Val-Asn (Trt)-Val-Ala-Cys (Mmt)-Thr (R4)-Gly-Cys (Trt)-Leu-
Wang resin 100mmol, 20% hexahydropyridine/DMF solution 5L is added into measuring tank as deprotection liquid, main reaction kettle Interior logical nitrogen simultaneously at 15 ~ 30 DEG C, react 5 ~ 7 min, extract reaction solution, drain 3 ~ 5 min by mixing control reaction temperature.
20% hexahydropyridine/DMF solution 5L is added into measuring tank again as deprotection liquid, adds in main reaction kettle and leads to Nitrogen simultaneously at 15 ~ 30 DEG C, react 8 ~ 12 min, extract reaction solution, drain 3 ~ 5min by mixing control reaction temperature.Washed after deprotection Wash 6 times
DMF 5L are measured every time and add main reaction kettle, are led to nitrogen and 3 ~ 5min of agitator treating, are drained 3 ~ 5min.Ninhydrin detection sun Property terminates to wash.
3.2 coupling
Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-Glu (OtBu)-Leu-OMe of 100mmol is weighed, The papain of 1mmol, NH2-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Mmt)-Thr(R4)-Gly-Cys (Trt)-Leu-wang resins 100mmol, is dissolved in DMF:Boric acid sodium hydroxide solution=98.5:In 1.5, the reaction time is small for 3 When.Reaction was completed after the reaction was complete for monitoring, filters remove reaction liquid after reaction, and resin, acquisition light brown washs with DMF Polypeptide resin.
4th, Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-Glu (OtBu)-Leu-Cys (Trt)- Val-
Asn(Trt)-Val-Ala-Cys(Mmt)-Thr(R4)-Gly-Cys(Trt)-Leu-wang-cyclic (4→12) The synthesis of disulfide
Weigh Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (Mmt)-Glu (OtBu)-Leu-Cys (Trt)-Val- 100 mmoles of Asn (Trt)-Val-Ala-Cys (Mmt)-Thr (R4)-Gly-Cys (Trt)-Leu-wang resins, into measuring tank The TFA/DCM solution 5L for adding 1-5% is used as reaction solution, lead in main reaction kettle nitrogen simultaneously mixing control reaction temperature 15 ~ 30 DEG C, react 40 minutes, extract reaction solution, drain reaction solution, washed 1 time with DMF, add the TFA/ of 1-5% into measuring tank again DCM solution 5L leads to nitrogen in main reaction kettle and mixing control reaction temperature is at 15 ~ 30 DEG C as reaction solution, reacts 60 minutes, takes out Except reaction solution, washed 3 times with DCM.DMF and H is added into measuring tank2O2Solution 5L leads to nitrogen as reaction solution in main reaction kettle And mixing control reaction temperature when reaction 3 is small, extracts reaction solution, is washed 3 times with DCM, reaction terminates, and obtains at 15 ~ 30 DEG C Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(1)-Glu(OtBu)-Leu-Cys(Trt)-Val-Asn(Trt)- Val-Ala-Cys (1)-Thr (R4)-Gly-Cys (Trt)-Leu-wang-cyclic (4 → 12) disulfide products.
5th, Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (1)-Glu (OtBu)-Leu-Cys (Trt)-Val-Asn (Trt)-
Val-Ala-Cys(1)-Thr(R4)-Gly-Cys(Trt)-Leu-wang-cyclic (4→12) (7-15)bis (disulfide) synthesis
Weigh Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (1)-Glu (OtBu)-Leu-Cys (Trt)-Val- Asn(Trt)-Val-Ala-Cys(1)-Thr(R4)-Gly-Cys(Trt)-Leu-wang-cyclic (4→12)disulfide 100 mmoles of compound, I is added into measuring tank2/ DMF solution 5L leads to nitrogen and mixing control as reaction solution in main reaction kettle Reaction temperature reacts 30 minutes at 20 ~ 30 DEG C, extracts reaction solution, drain reaction solution, washed 1 time with DMF, again to measuring tank Middle addition I2/ DMF solution 5L leads to nitrogen in main reaction kettle and mixing control reaction temperature is at 20 ~ 30 DEG C as reaction solution, reaction 2 it is small when, extract reaction solution, drain reaction solution, washed 3 times with DMF, reaction terminates, and obtains target product.
6th, NH2-Asn-Asp-Glu-Cys(1)-Glu-Leu-Cys(2)-Val-Asn-Val-Ala-Cys(1)-Thr-Gly- Cys(2)-
The preparation of Leu-OH-cyclic (4 → 12) (7-15)-bis (disulfide)
By Boc-Asn (Trt)-Asp (OtBu)-Glu (OtBu)-Cys (1)-Glu (OtBu)-Leu-Cys of 100 mmoles (Trt)-
Val-Asn(Trt)-Val-Ala-Cys(1)-Thr(R4)-Gly-Cys(Trt)-Leu-wang-cyclic (4→12) (7-15) bis (disulfide) is placed in 1000 milliliters of reaction bulb, uses EDT：TIS:H2O：TFA=3:1:1:95 ratios are split 500 milliliters of liquid of solution is cracked, and when cracking 2-4 is small at room temperature, is filtered to remove resin and is obtained peptide solution, be poured into 5 liters In ice anhydrous ether, a large amount of white precipitates are produced, are centrifuged, ice anhydrous ether washs 3-6 times, obtains solid.Crude product chromatography Figure is with reference to Fig. 1；Afterwards, purification system is done with 0.1% TFA aqueous solutions and acetonitrile, purifier apparatus is done with C18 columns, after revolving is lyophilized The high purity product 71.22g that purity is 99.79% is obtained, total recovery reaches 42.34%.Sterling chromatogram is with reference to Fig. 2；Mass spectrum confirms Molecular weight is 1681.88, and desalination is done with acetonitrile and water before doing mass spectrum, and methanol makees solvent and carries out mass spectrum confirmation.The general figure reference of matter Fig. 3.

Claims (4)

  1. A kind of 1. solid phase synthesis process of plecanatide, it is characterised in that：First using liquid phase synthesizing method synthesis hexapeptide fragment, profit Hexapeptide fragment is coupled on solid-phase resin with enzymatic method, using the false dilution effect of solid-phase resin on solid-phase resin by right The protection group of Cys diverse locations, is done directly the selectivity synthesis of two two sulphur rings, afterwards cuts down it from resin, Directly purified.
  2. 2. the solid phase synthesis process of a kind of plecanatide according to claim 1, it is characterised in that its step is：
    A, fragment condensation is into all kinds of plecanatide intermediates：
    (1)R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)-Val- Ala-
    The synthesis of Cys (R1)-Thr (R4)-Gly-Cys (R1)-Leu-R3；
    （2）R-Asn (R1)-Asp (R2)-Glu (R2)-Cys (R1)-Glu (R2)-Leu- is completed using the false dilution effect of resin
    Cys(R1)-Val-Asn(R1)-Val-Ala-Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4 → 12) two cysteines of the synthesis of-disulfide, No. 4 positions and No. 12 positions are into thioether bond ring；
    (3)R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)-Val- Ala-
    Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4→12), (7→15)-bis(disulfide) Synthesis；Two cysteines of No. 4 positions and No. 12 positions are on the basis of thioether ring, two cysteines of No. 7 positions and No. 15 positions Again into thioether ring；
    In all kinds of amino acid protecting groups：
    R for Z, Boc, Cl-Z, Fmoc, oNBS, dNBS, Troc, Dts, pNZ, oNZ, NVOC, NPPOC, HFA, Ddz, Bpoc, Nps, Nsc, Bsmoc, α-Nsmoc, ivDde, Fmoc*, MTT, Alloc wherein any one；
    R1 is Meb, Mob, TRT, Tmob, Mmt, Xan, Pmbf, Bn, tBu, Fm, Dnpe, Fmoc, Acm, Phacm, StBu、Npys、 Alloc wherein any one；
    R2 for H, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb wherein any one；
    R3 is H, OMe, Oet, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb, wang tree Fat, CTC resins wherein any one；
    R4 for Bn, cHx, tBu, Trt, TBDMS, Dmnb, Poc wherein any one；
    B, the synthesis of the thick peptide of plecanatide；
    C, the purifying of plecanatide and lyophilized.
  3. 3. the solid phase synthesis process of plecanatide according to claim 1 or 2, its feature is as follows：Synthesis in solid state process In the coupling system used be：
    （1）, single condensing agent：DIC, EDC, chloracetyl chloride, ethyl chloroformate, isobutylchloroformate, isobutyl chlorocarbonate, TBTU, PyBOP, HBTU, papain, trypsase；
    （2）Two system condensing agents：DIC/HOBT、EDC/HOBT、DIC/HOSu、EDC/ HOSu；
    （3）Cyclization reagent：Piperidines/DMF, H2O2/DMF、I2/DMF、DMSO/DMF、H2O2/H2O、I2/ H2O、DMSO/ H2O。
  4. 4. the solid phase synthesis process of plecanatide according to claim 1 or 2, it is characterised in that：R-Asn(R1)-Asp (R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-
    Val-Asn(R1)-Val-Ala-Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4→12), (7 → 15) synthesis of-bis (disulfide) comprises the following steps that：
    （1）Using Fmoc-Leu-OH as first amino acid, with wang resins or CTC resins, the resin substitution degree being related to preferably from The mmol/g of 0.2mmol/g ~ 1.8 carries out condensation and forms Fmoc-Leu- resins, then passes through the method for coupling amino acid one by one, even Connection completes 10 amino-acid resins；R-Asn (R1)-Asp (R2)-Glu (R2)-Cys (R1) that liquid phase process is synthesized afterwards- Glu (R2)-Leu-R3 is directly coupled on peptide resin using papain, completes the synthesis of full guard peptide resin；
    （2）、R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)-Val- Ala-
    Cys (1)-Thr (R4)-Gly-Cys (R1)-Leu-R3-cyclic (4 → 12), the synthesis of disulfide
    The cysteine protected using No. 4 positions, No. 12 positions Mmt or ACM, is cracked with the TFA of various concentrations, removes Mmt Oxidation cyclization either is carried out with one kind in the oxidants such as DMSO, air, hydrogen peroxide or composition after ACM, obtains target production Product；
    （3）、R-Asn(R1)-Asp(R2)-Glu(R2)-Cys(R1)-Glu(R2)-Leu-Cys(R1)-Val-Asn(R1)- Val-Ala-
    Cys(R1)-Thr(R4)-Gly-Cys(R1)-Leu-R3-cyclic (4→12), (7→15)-bis(disulfide) Synthesis
    The cysteine protected using No. 7 positions, No. 15 positions TRT, Tmob, Pmbf or ACM, uses I2/ DMF be deprotected simultaneously same Shi Jinhang is oxidized to two sulphur rings, obtains target product；
    （4）H-Asn-Asp-Glu-Cys(1)-Glu-Leu-Cys(2)-Val-Asn-Val-Ala-Cys(1)-Thr-Gly-Cys (2)-
    Leu-OH-cyclic (4 → 12), the thick peptide symthesis of (7 → 15)-bis (disulfide)
    Cracked using the TFA of different proportion, proton capture agent or protection group capturing agent are selected from：TIS, EDT, water, dimethyl sulfide Ether, thioanisole, the ratio of TFA are more than 90%.
CN201711495911.6A 2017-12-31 2017-12-31 A kind of solid phase synthesis process of plecanatide Pending CN108003222A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020034286A1 (en) * 2018-08-15 2020-02-20 深圳翰宇药业股份有限公司 Method for preparing plecanatide
CN110981939A (en) * 2018-11-13 2020-04-10 杭州肽佳生物科技有限公司 Preparation method of polycaprolactam
WO2020250102A1 (en) * 2019-06-10 2020-12-17 Auro Peptides Ltd An improved process for the preparation of plecanatide

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Application publication date: 20180508