CN102199188A - Liquid-phase synthesis method for polypeptide - Google Patents
Liquid-phase synthesis method for polypeptide Download PDFInfo
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- CN102199188A CN102199188A CN201110051933XA CN201110051933A CN102199188A CN 102199188 A CN102199188 A CN 102199188A CN 201110051933X A CN201110051933X A CN 201110051933XA CN 201110051933 A CN201110051933 A CN 201110051933A CN 102199188 A CN102199188 A CN 102199188A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a liquid-phase synthesis method for a polypeptide, and relates to the synthesis of a polypeptide. The method comprises the following steps: carrying out condensation reaction on an amino acid derivative in N,N-dimethyl formylamine to obtain a reaction solution; adding the reaction solution into water to precipitate solid; filtering, washing, and drying to obtain an intermediate product; removing the amino protecting group of the intermediate product, adding into water, regulating the pH value of the solution to precipitate the intermediate product without the amino protecting group, and drying; and carrying out condensation reaction on the intermediate product without the amino protecting group and the amino acid derivative to be condensed in N,N-dimethyl formylamine, repeating the above processes until a fully-protected polypeptide is obtained, and removing the protecting groups of the fully-protected polypeptide to obtain a target polypeptide. The liquid-phase synthesis method disclosed by the invention avoids the use of numerous organic solvents for after-reaction treatment and uses water as the solvent to precipitate the protected polypeptide intermediate product, thus lowering the production cost of the polypeptide and reducing the environmental pollution caused by the use of numerous organic solvents.
Description
Technical field
The present invention relates to the synthetic of polypeptide, particularly relate to a kind of polypeptide liquid-phase synthesis process.
Background technology
Along with the raising of scientific and technological level and human attention and research and development to polypeptide, increasing polypeptide drugs are developed and are applied to clinically, for improving level of human health and treatment, preventing disease, provide the newtype drug and the vaccine of highly effective and safe.Along with people increase gradually to the demand of polypeptide drugs, to the chemist, particularly how Pharmaceutical Chemist efficiently synthesizes cheap polypeptide drugs, is a technical problem that needs to be resolved hurrily.The chemosynthesis of polypeptide has become the most important approach that obtains polypeptide drugs.At present, the synthetic polypeptide of liquid phase is the very efficient cheap preparation means of synthetic small peptide and fragment peptide, and in conjunction with the fragment condensation technology of solid phase synthesis and the synthetic advantage of liquid phase, compares with the method for the synthetic polypeptide of liquid phase, and is then cheap especially and efficient.But; when adopting traditional liquid-phase synthesis process to synthesize polypeptide; except must using protection amino acid, condensing agent and reaction solvent (being generally organic solvent); also need to use a large amount of organic solvents to be used for reacted processing; organic solvent commonly used comprises sherwood oil, ether, methyl tertiary butyl ether, ethyl acetate, chloroform, methylene dichloride etc., easily contaminate environment.Owing to using a large amount of organic solvents, not only improve the production cost of polypeptide, and caused very serious environmental pollution, therefore, how to reduce the cost and the pollution that reduces environment of the synthetic polypeptide of liquid phase, be the technical problem that this area needs to be resolved hurrily.
Summary of the invention
The objective of the invention is provides a kind of polypeptide liquid-phase synthesis process in order to overcome the deficiency of above-mentioned background technology, can reduce the production cost of polypeptide significantly, and reduces because of pollution on the environment with an organic solvent in a large number.
Polypeptide liquid-phase synthesis process provided by the invention, may further comprise the steps: amino acid derivative is placed N, condensation in the dinethylformamide (DMF) adds reaction soln in the entry, separate out solid after, washing after filtration,, drying, obtain intermediate, add in the entry after removing the amino protecting group of described intermediate, and the regulator solution acid-basicity, the intermediate that removes amino protecting group is separated out drying; Remove the intermediate of amino protecting group and treat that the amino acid derivative of condensation places N described; condensation in the dinethylformamide (DMF) repeats said process, then until obtaining the full guard polypeptide; remove the protecting group of described full guard polypeptide again, obtain target polypeptides.
In technique scheme; the amino protecting group of described intermediate comprises tertbutyloxycarbonyl (Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc); adopt trifluoroacetic acid (TFA) to remove tertbutyloxycarbonyl (Boc), adopt the mixing solutions of piperidines and DMF to remove 9-fluorenylmethyloxycarbonyl (Fmoc).
In technique scheme, the reaction soln that will remove behind the tertbutyloxycarbonyl (Boc) adds in the entry, comes the regulator solution acid-basicity by weak base again, and the intermediate that removes tertbutyloxycarbonyl (Boc) is separated out, and filters then, washs, drying.
In technique scheme, the reaction soln that will remove behind the 9-fluorenylmethyloxycarbonyl (Fmoc) adds in the entry, comes the regulator solution acid-basicity by weak acid again, and the intermediate that removes 9-fluorenylmethyloxycarbonyl (Fmoc) is separated out, and filters then, washs, drying.
Compared with prior art, advantage of the present invention is as follows:
The present invention avoids with an organic solvent carrying out in a large number reacted processing, uses water instead as the solvent of separating out protection polypeptide intermediate.The present invention is applicable to the large-scale production micromolecule polypeptide; do not need to change on a large scale original technology; only need in synthetic link, to adopt N soluble in water; dinethylformamide (DMF) is as reaction solvent; in removing the process of amino protecting group; select for use trifluoroacetic acid soluble in water (TFA) to remove tertbutyloxycarbonyl (Boc), select for use the mixing solutions of piperidines and DMF to remove 9-fluorenylmethyloxycarbonyl (Fmoc).Therefore, the present invention not only can reduce the production cost of polypeptide significantly, and can reduce because of pollution on the environment with an organic solvent in a large number.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
The polypeptide liquid-phase synthesis process that the embodiment of the invention provides, may further comprise the steps: amino acid derivative is placed N, condensation in the dinethylformamide (DMF) adds reaction soln in the entry, separate out solid after, washing after filtration,, drying, obtain intermediate, add in the entry after removing the amino protecting group of described intermediate, and the regulator solution acid-basicity, the intermediate that removes amino protecting group is separated out drying; Remove the intermediate of amino protecting group and treat that the amino acid derivative of condensation places N described; condensation in the dinethylformamide (DMF) repeats said process, then until obtaining the full guard polypeptide; remove the protecting group of described full guard polypeptide again, obtain target polypeptides.
Wherein, the amino protecting group of described intermediate comprises tertbutyloxycarbonyl (Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc), adopts trifluoroacetic acid (TFA) to remove tertbutyloxycarbonyl (Boc), adopts the mixing solutions of piperidines and DMF to remove 9-fluorenylmethyloxycarbonyl (Fmoc).The reaction soln that will remove behind the tertbutyloxycarbonyl (Boc) adds in the entry, comes the regulator solution acid-basicity by weak base again, and the intermediate that removes tertbutyloxycarbonyl (Boc) is separated out, and filters then, washs, drying.The reaction soln that will remove behind the 9-fluorenylmethyloxycarbonyl (Fmoc) adds in the entry, comes the regulator solution acid-basicity by weak acid again, and the intermediate that removes 9-fluorenylmethyloxycarbonyl (Fmoc) is separated out, and filters then, washs, drying.
The concrete enforcement general formula of reaction process is as follows:
Condensation → all risk insurance of No. 1 protected amino acid+No. 2 protected amino acid → in dimethyl formamide (DMF) protects separates out all risk insurance and protects dipeptides (solid) → filtration washing and obtain all risk insurance and protect dipeptides (the protection dipeptides of purifying) → drying in two peptide solutions → adding pure water; Remove in side chain protected two peptide solutions of the amino protecting group that all risk insurance protects dipeptides → remove amino protecting group → adding pure water and with weak acid (or weak base) regulator solution Acidity of Aikalinity make the side chain protected dipeptides separate out fully → filtration washing gets side chain protected dipeptides → drying, protects polypeptide → deprotection base → polypeptide with No. 3 protected amino acid condensation → processes all risk insurance that circulates → get
Elaborate concrete application of the present invention below by 4 embodiment.
Embodiment 1, the application of the present invention in the synthetic thymopeptide-5 of mixed anhydride method.
Step 101: synthetic dipeptides.
21.7g (100mmol) tertbutyloxycarbonyl Xie Ansuan (Boc-Val-OH) is dissolved among the 100ml DMF, behind-15 ℃ of following adding 30g (300mmol) N-methylmorpholines (NMM), drips 20.4g (150mmol) isobutyl chlorocarbonate (ClCO2
iBu), stirring reaction is after 30 minutes, and (H2N-Tyr (OBzl)-OBzl), stirring reaction also removes cryostat, with liquid chromatography HPLC detection reaction complete (Boc-Val-OH consumes fully) to add 39.7g (110mmol) tyrosine (Bian ether) Bian ester.Reaction solution under agitation added in the 2000ml cold water slowly stirred 20 minutes; treat that product separates out fully; suction filtration; twice of 100ml water washing of filter cake; change in the 500ml round-bottomed flask,, get 51g powdery solid full guard dipeptides (Boc-Val-Try (OBzl)-OBzl) 50 ℃ of following vacuum-dryings 4 hours; yield 91%, purity 97% (area normalization).
Step 102: synthetic tripeptides.
(Boc-Val-Try (OBzl)-OBzl) is dissolved in the 50ml trifluoroacetic acid with 50g full guard dipeptides; stir under the room temperature; reaction under agitation adds reaction solution in the 1000ml water slowly to fully, is continuing slowly to add 36g yellow soda ash to pH～7 under the stirring then.Suction filtration, after filter cake was used 50ml 5% sodium bicarbonate aqueous solution washed twice, 40 ℃ of following vacuum-dryings got dipeptides (H2N-Val-Try (OBzl)-OBzl), the yield 97% that the 40g powdery solid removes amino protecting group.
40g (124mmol) N-tertbutyloxycarbonyl (Bian ester) aspartic acid is dissolved among the 100mlDMF, adds 37g (372mol) NMM, be cooled to-15 ℃, stir and slowly splash into 33.8g (248mmol) ClCO2 down
iBu, stirring reaction be after 30 minutes, and 40g (87mmol) is removed the dipeptides of amino protecting group, and (H2N-Val-Try (OBzl)-OBzl) is dissolved among the 50ml DMF, stirs also to be warming up to room temperature (21 ℃) slowly.Behind the monitoring reaction complete (H2N-Val-Try (OBzl)-OBzl consumes fully), add in the 2000ml water that continues stirring, stirred 20 minutes, suction filtration, filter cake is used twice, 50 ℃ of following vacuum-drying of 100ml water washing 4 hours.Get 61.2g white powdery solid full guard tripeptides (Boc-Asp (OBzl)-Val-Try (OBzl)-OBzl), yield 91.8%, purity 95%.
Step 103: synthetic tetrapeptide.
60g (78mmol) full guard tripeptides is dissolved in the 60ml trifluoroacetic acid, and stirring reaction under agitation adds reaction solution in the 1000ml water slowly to fully under the room temperature, is continuing slowly to add 43g yellow soda ash to pH～7 under the stirring then.Suction filtration, filter cake is with after the sodium bicarbonate aqueous solution washed twice of 80ml5%, and 40 ℃ of following vacuum-dryings are spent the night to such an extent that the 49.8g powdery solid removes amido protecting tripeptides (H2N-Asp (OBzl)-Val-Try (OBzl)-OBzl), yield 95.4%.
(Boc-Lys (2-Cl-Z)-OH) is dissolved among the 100ml DMF with 35.7g (86mmol) N-tertbutyloxycarbonyl (2-benzyloxycarbonylchloride base) Methionin, add 26g (260mmol) N-methylmorpholine (NMM), be cooled to-15 ℃, stir and slowly splash into 23.5g (172mmol) isobutyl chlorocarbonate (ClCO2 down
iBu), stirring reaction after 30 minutes removes 48g (72mmol) the amido protecting tripeptides (solution that H2N-Asp (OBzl)-Val-Try (OBzl)-OBzl) is dissolved in the 60mlDMF gained adds, and stirs also to be warming up to room temperature (20 ℃) slowly.Behind liquid chromatography monitoring reaction complete (H2N-Asp (OBzl)-Val-Try (OBzl)-OBzl consumes fully); add in the 2500ml water that continues stirring; stirred 20 minutes; suction filtration; filter cake gets 70.3g white powdery solid full guard tetrapeptide (Boc-Lys (2-Cl-Z)-Asp (OBzl)-Val-Try (OBzl)-OBzl) with twice, 50 ℃ of following vacuum-drying of 100ml water washing 4 hours; yield 94.9%, purity 96%.
Step 104: synthetic pentapeptide.
70g (66mmol) full guard tetrapeptide is dissolved in the 90ml trifluoroacetic acid, and stirring reaction under agitation adds reaction solution in the 1000ml water slowly to fully under the room temperature, is 7 continuing slowly to add yellow soda ash 65g to pH value under the stirring then.Suction filtration; after filter cake was used 80ml 5% sodium bicarbonate aqueous solution washed twice, 40 ℃ of following vacuum-dryings were spent the night, and got the 56.2g powdery solid and removed amido protecting tetrapeptide (H2N-Lys (2-Cl-Z)-Asp (OBzl)-Val-Try (OBzl)-OBzl); yield 88.6%, purity 94.6%.
(Z-Arg (NO2)-OH) is dissolved among the 60ml DMF, adds 20.5g (200mmol) N-methylmorpholine (NMM), is cooled to-15 ℃, stirs slowly to splash into 13.9g (102mmol) ClCO2 down with 24.2g (68mmol) N-carbobenzoxy-(Cbz) (nitro) arginine
iBu, stirring reaction removed the amido protecting tetrapeptide with 55g (57mmol) after 30 minutes, and (solution that H2N-Lys (2-Cl-Z)-Asp (OBzl)-Val-Try (OBzl)-OBzl) is dissolved in 80ml DMF gained adds, and stirs and be warming up to slowly room temperature (22 ℃).Behind the monitoring reaction complete (H2N-Lys (2-Cl-Z)-Asp (OBzl)-Val-Try (OBzl)-OBzl consumes fully); add in the 2000ml water that continues stirring; stirred 20 minutes; suction filtration; filter cake gets 67.1g white powdery solid full guard pentapeptide (Z-Arg (NO2)-Lys (2-Cl-Z)-Asp (OBzl)-Val-Try (OBzl)-OBzl) with twice, 50 ℃ of following vacuum-drying of 100ml water washing 4 hours; yield 90.6%, purity 97%.
67g (51mmol) full guard pentapeptide is suspended in 1000ml methyl alcohol and the 200ml acetic acid mixed solvent, adds 7g 10% palladium carbon, insert in the 2L autoclave, logical hydrogen (10kg/cm2), hydrogenation is complete to raw material consumption.Pressure release, filtration catalizer, mother liquor is evaporated to 260ml, is cooled to room temperature, under vigorous stirring, slowly add in the 2000ml ether that is cooled to 0 ℃ in advance, separate out thick peptide, suction filtration, vacuum-drying (25 ℃) is spent the night, get the 33.4g blocks of solid, productive rate 95.0%, purity 89%.
(4) purifying: the thick peptide of 30g is dissolved in the 60ml acetic acid, with preparative high-performance liquid chromatographic purifying (parameter: 100 * 250mm C18,10 μ, monitoring wavelength: 254nm, gradient elution: 2% acetonitrile～40% acetonitrile, 30 minutes).Collect pure product cut, be evaporated to 20～25mg/ml, freeze-drying gets the pure product of 22g white snow shape solid thymopeptide-5, total recovery 32.3%, purity 98.6%.
Embodiment 2, the application of the present invention in the synthetic thymopeptide-5 (Fmoc method) of DIC (N, N '-DIC) method.
Step 201: synthetic dipeptides.
With 30g (88mmol) N-fluorenylmethyloxycarbonyl Xie Ansuan (Fmoc-Val-OH) in 200mlDMF with 11.9g (88mmol) 2-hydroxy benzo ribavirin (HOBt) and 13.3g (106mmol) N; N '-DIC (DIC) reacted 30 minutes down at-5 ℃; add 31.0g (106mmol) tyrosine (tert.-butoxy) tert-butyl ester (H2N-Try (OtBu)-OtBu) then; stir and be warming up to room temperature down slowly; react monitoring reaction complete (Fmoc-Val-OH disappearance) after 2 hours; reaction solution is added in the 1500ml water; separate out full guard dipeptides (solid; Fmoc-Val-Try (OtBu)-OtBu); filtration washing; drying gets anhydrous full guard dipeptides.
Step 202: synthetic tripeptides.
The full guard dipeptides powder dissolution that obtains in the step 201 was reacted 10 minutes in 100ml 20% piperidines/DMF solution; remove amino protecting group, monitoring reaction is complete, and reaction solution adds in the 1000ml water; separate out solids removal amido protecting dipeptides (H2N-Val-Try (OtBu)-OtBu); filter, filter cake washs with 5% weak ammonia, and vacuum-drying is filtered; get 30g white powdery solid and remove amido protecting dipeptides (H2N-Val-Try (OtBu)-OtBu); 76mmol, yield 86.9%, purity 95%.(Fmoc-Asp (OtBu)-OH) and 12.2g (91mmol) HOBt and 17.2g (136mmol) DIC reacted 50 minutes down at-10 ℃ to add 37.5g (91mmol) N-fluorenylmethyloxycarbonyl (tert.-butoxy) aspartic acid in 100ml DMF; add 30g (76mmol) then and remove amido protecting dipeptides (H2N-Val-Try (OtBu)-OtBu); stirring reaction also slowly is warming up to room temperature; make react completely (removing the amido protecting dipeptides disappears); reaction solution is added in the 1000ml water; separate out full guard tripeptides (Fmoc-Asp (OtBu)-Val-Try (OtBu)-OtBu); filter; twice of 50ml water washing of filter cake; vacuum-drying 4 hours; the gained white powder is dissolved in 100ml 20% piperidines/DMF solution reaction removes amino protecting group; reaction solution adds in the 1000ml water; separate out and remove amido protecting tripeptides solid (H2N-Asp (OtBu)-Val-Try (OtBu)-OtBu); filter; use 5% ammonia scrubbing; dry; obtain anhydrous 39.2g white powder and remove amido protecting tripeptides (H2N-Asp (OtBu)-Val-Try (OtBu)-OtBu); yield 91.5%, purity 96%.
Step 203: synthetic tetrapeptide.
38.8g (83mmol) (Fmoc-Lys (Boc)-OH) is dissolved among the 120ml DMF N-fluorenylmethyloxycarbonyl (tertbutyloxycarbonyl) Methionin;-15 ℃ added 11.2g (83mmol) HOBt and 15.7g (124mmol) DIC and stirring reaction 3 hours down; add 39g (69mmol) then and remove amido protecting tripeptides (H2N-Asp (OtBu)-Val-Try (OtBu)-OtBu); stirring reaction also slowly is warming up to room temperature; make and react completely; reaction solution is added in the 1500ml water; separate out white solid; suction filtration; filter cake washes with water; vacuum-drying 6 hours; get 62.3g (61mmol) white powder full guard tetrapeptide (Fmoc-Lys (Boc)-Asp (OtBu)-Val-Try (OtBu)-OtBu); yield 88.4%, purity 95.2%.This white powder was dissolved in 100ml 20% piperidines/DMF solution reaction 20 minutes; reaction solution adds in the 1500ml water; the white solid of separating out is through suction filtration; washing (5% ammoniacal liquor); 40 ℃ of following vacuum-dryings are filtered; get the 47.2g white powder and remove amido protecting tetrapeptide (H2N-Lys (Boc)-Asp (OtBu)-Val-Try (OtBu)-OtBu), yield 97.7%, purity 95.1%.
Step 204: synthetic pentapeptide.
(Fmoc-Arg (Pbf)-OH) is dissolved among the 100ml DMF 37g (70mmol) N-fluorenylmethyloxycarbonyl arginine;-10 ℃ add 11.4g (74mmol) HOBt and 13.2g (106mmol) DIC reaction 3 hours down; add 47.0g (59.3mmol) then and remove amido protecting tetrapeptide (H2N-Lys (Boc)-Asp (OtBu)-Val-Try (OtBu)-OtBu); slowly be warming up to room temperature; make and react completely; reaction solution is added in the 1200ml water; separate out white solid; suction filtration; filter cake 100ml water washing; 35 ℃ of following vacuum-dryings are filtered; get 70.3g (54.0mmol) white powder full guard pentapeptide (Fmoc-Arg (Pbf)-Lys (Boc)-Asp (OtBu)-Val-Try (OtBu)-OtBu), yield 91.2%.
With 70g (53.8mmol) full guard pentapeptide and piperidines/DMF solution reaction; remove amino protecting group; reaction solution is added in the entry; separate out and remove amido protecting pentapeptide (H2N-Arg (Pbf)-Lys (Boc)-Asp (OtBu)-Val-Try (OtBu)-OtBu); filtration washing; dry; to remove the reaction of amido protecting pentapeptide and 150ml trifluoroacetic acid again; stirring reaction is 2 hours under the room temperature; remove all protecting groups, reaction solution adds in the 1500ml ether, separates out white colloidal solid; with ether washing 3 times; centrifugation, gained solid at room temperature vacuum-drying spends the night, and gets 30.1g (44.2mmol) the thick peptide of white blocky thymopeptide-5 (H2N-Arg-Lys-Asp-Val-Try-OH); yield 82.1%, purity 85%.The thick peptide of this thymopeptide-5 is dissolved in the 140ml acetic acid, uses the preparative high-performance liquid chromatographic purifying, freeze-drying gets 21.3g (31.3mmol) the snow shape pure product of white solid thymopeptide-5 (H2N-Arg-Lys-Asp-Val-Try-OH), total recovery 29.6%, purity 98.3%.
Embodiment 3, the application of the present invention in the synthetic Thymosin alpha 1 (28 peptide) of fragment method, 1～5 fragment (Fmoc-Ser (OBzl)-Asp (OBzl)-Ala-Ala-Val-OH) of synthetic Thymosin alpha 1.
Step 301: the preparation dipeptides tert-butyl ester (H2N-Ala-Val-OtBu).
31.1g (100mmol) N-fluorenylmethyloxycarbonyl L-Ala (Fmoc-Ala-OH) is dissolved among the 150ml DMF;-5 ℃ add 20.2g (150mmol) HOBt and 18.9g (150mmol) DIC reaction down; add 19.1g (110mmol) the Xie Ansuan tert-butyl ester (H2N-Val-OtBu) then; stirring reaction 3 hours; (Fmoc-Ala-OH disappearance) reacts completely; reaction solution is added in the 1500ml water; separate out full guard dipeptides (Fmoc-Ala-Val-OtBu); filtration washing; dry; get white powdery solid 42.1g (90mmol), yield 90%, purity 95%.Should the white powdery solid being dissolved in 100ml 20% piperidines/DMF reacts completely, reaction solution adds in the 1000ml water, separate out the solid dipeptides tert-butyl ester (H2N-Ala-Val-OtBu), suction filtration, filter cake is with 5% weak ammonia 50ml washed twice, and vacuum-drying is spent the night, 21.2g (86mmol) the white powdery solid dipeptides tert-butyl ester (H2N-Ala-Val-OtBu), yield 95%, purity 95%.
Step 302: the preparation tripeptides tert-butyl ester (H2N-Ala-Ala-Val-OtBu).
In 100ml DMF, dissolve 32.1g (103mmol) N-fluorenylmethyloxycarbonyl L-Ala (Fmoc-Ala-OH), be cooled to-10 ℃ then, add 20.8g (154mmol) HOBt and 19.4g (154mmol) DIC reaction after 30 minutes, add 21.0g (85mmol) the dipeptides tert-butyl ester (H2N-Ala-Val-OtBu), stir down and be warming up to room temperature slowly, afterreaction was complete in 3 hours.Reaction solution added separate out full guard tripeptides (Fmoc-Ala-Ala-Val-OtBu) in the 1500ml water, suction filtration, filter cake washes with water, and vacuum-drying obtains the white powdery solid of 43.1g (80mmol), yield 94%.Should be dissolved in 100ml 20% piperidines/DMF solution reaction by the white powdery solid, add in the 2000ml water after reacting completely and separate out, suction filtration, filter cake washs with 5% weak ammonia, vacuum-drying is spent the night, obtain 24.9g (78.9mmol) the tripeptides tert-butyl ester (H2N-Ala-Ala-Val-OtBu), yield 98%.
Step 303: the preparation tetrapeptide tert-butyl ester (H2N-Asp (OtBu)-Ala-Ala-Val-OtBu).
(Fmoc-Asp (OBzl)-OH) is dissolved among the 130ml DMF with 42.3g (96mmol) fluorenylmethyloxycarbonyl (benzyl) aspartic acid, added 16g (118mmol) HOBt and 14.9g (118mmol) DIC stirring reaction after being cooled to-10 ℃ 40 minutes, add the tripeptides tert-butyl ester (H2N-Ala-Ala-Val-OtBu) powder then, stirring reaction also slowly is warming up to room temperature, and monitoring reaction is complete after 4 hours.Reaction solution adds in the 2000ml water separates out white solid, suction filtration, and filter cake washes with water, and dry filter obtains 52g (70mmol) powdery white solid full guard tetrapeptide (Fmoc-Asp (OBzl)-Ala-Ala-Val-OtBu), yield 88.6%, purity 93%.This powdery white solid is dissolved in 100ml 20% piperidines/DMF to react 40 minutes, reaction solution adds separates out solid in the entry, suction filtration, filter cake washes the final vacuum dried overnight with water, obtain 35.4g (68mmol) the tetrapeptide tert-butyl ester (H2N-Asp (OtBu)-Ala-Ala-Val-OtBu), yield 97.1%, purity 92%.
Step 304: preparation pentapeptide fragment (Fmoc-Ser (OBzl)-Asp (OBzl)-Ala-Ala-Val-OH).
(Fmoc-Ser (OBzl)-OH) is dissolved in 100ml DMF to 34g (82mmol) fluorenylmethyloxycarbonyl (benzyl) Serine, add 16.6g (123mmol) HOBt, after being cooled to-10 ℃, solution adds 15.5g (123mmol) DIC reaction, stirring reaction 1 hour, add the tetrapeptide tert-butyl ester (H2N-Asp (OtBu)-Ala-Ala-Val-OtBu) solid then, stirring reaction also slowly is warming up to room temperature, continue reaction after 3 hours monitoring reaction complete.Reaction solution added in the 2000ml water separate out white solid; suction filtration, filter cake washes with water, and vacuum-drying obtains 57g (62mmol) drying solid full guard pentapeptide (Fmoc-Ser (OBzl)-Asp (OBzl)-Ala-Ala-Val-OtBu) after 12 hours; yield 91.1%, purity 91%.This drying solid is dissolved in the 100ml trifluoroacetic acid reaction 1 hour, reaction solution adds separates out solid in the entry, suction filtration, filter cake washes with water three times, vacuum-drying 48 hours, obtain the white powdery solid pentapeptide of 51.8g (59.9mmol) fragment (Fmoc-Ser (OBzl)-Asp (OBzl)-Ala-Ala-Val-OH), yield 96.6%, purity 90%.This white powdery solid pentapeptide fragment is used for synthetic Thymosin alpha 1.
Embodiment 4, the present invention be the application in (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2) in synthetic beauty treatment polypeptide A Ji Rayleigh (six peptides).
Step 401: synthetic dipeptides.
(Boc-Arg (NO2)-OH) is dissolved among the 50ml DMF 16g (50mmol) tertbutyloxycarbonyl (nitro) arginine, adds 10g (100mmol) NMM, is cooled to-15 ℃, slowly drips 14g (100mmol) ClCO2
iBu; behind the stirring reaction 1 hour; add 25g (115mmol) nitro arginine amide (H2N-Arg (NO2)-NH2); slowly be warming up to room temperature under stirring and reacted 4 hours again, react completely, reaction solution adds in the 1000ml water separates out white solid; suction filtration; filter cake washes the final vacuum drying with water, obtains 22g (42.4mmol) white powder full guard dipeptides (Boc-Arg (NO2)-Arg (NO2)-NH2), yield 84.8%.22g full guard dipeptides dipeptides is dissolved in the 50ml trifluoroacetic acid reaction 1 hour; reaction solution adds in the entry; with strong aqua regulator solution pH to 8～9; separate out white solid; suction filtration; filter cake washs the final vacuum dried overnight with 5% weak ammonia, and the white solid 16g (38.2mmol) that obtains very easily pulverizing removes amido protecting dipeptides (H2N-Arg (NO2)-Arg (NO2)-NH2), yield 90%.
Step 402: synthetic tripeptides.
12g (48.7mmol) tertbutyloxycarbonyl (nitro) arginine (Boc-Gln-OH) is dissolved among the 50ml DMF, adds 10g (100mmol) NMM, is cooled to-15 ℃, slowly drips 14g (100mmol) ClCO2
iBu; behind the stirring reaction 1 hour; adding 16g removes the amido protecting dipeptides, and (H2N-Arg (NO2)-Arg (NO2)-NH2) is warming up to room temperature slowly under stirring and reacted 5 hours again, reacts completely; reaction solution adds in the 1000ml water separates out pale solid full guard tripeptides (Boc-Gln-Arg (NO2)-Arg (NO2)-NH2); suction filtration, filter cake wash the final vacuum drying with water, obtain 21g pale powder full guard tripeptides; 33.3mmol, yield 86.4%.21g full guard tripeptides is dissolved in the 50ml trifluoroacetic acid reaction 1 hour; reaction solution adds in the entry; with strong aqua regulator solution pH to 8～9; separate out white solid and remove amido protecting tripeptides (H2N-Gln-Arg (NO2)-Arg (NO2)-NH2); suction filtration; filter cake obtains 16.4g (30.2mmol) and removes amido protecting tripeptides (H2N-Gln-Arg (NO2)-Arg (NO2)-NH2), yield 90.7% with 5% weak ammonia washing final vacuum dried overnight.
Step 403: synthetic six peptides.
(Ac-Glu (OBzl)-Glu (OBzl)-Met-OH) (synthetic in advance) is dissolved among the 50ml DMF (10ml DMSO), adds 10g (100mmol) NMM, is cooled to-15 ℃, slowly drips 14g (100mmol) ClCO2 with 25g (39mmol) tripeptide fragment
iBu; behind the stirring reaction 1.5 hours; add 16g and remove amido protecting tripeptides (H2N-Gln-Arg (NO2)-Arg (NO2)-NH2); slowly being warming up to room temperature under stirring reacted 8 hours again; react completely; reaction solution is added in the 1500ml water; separate out full guard six peptides (Ac-Glu (OBzl)-Glu (OBzl)-Met-Gln-Arg (NO2)-Arg (NO2)-NH2); suction filtration; after filter cake washed with water, vacuum-drying obtained 28g (25.2mmol) pale solid full guard six peptides; yield 80%, purity 82%.(Ac-Glu (OBzl)-Glu (OBzl)-Met-Gln-Arg (NO2)-Arg (NO2)-NH2) is dissolved in methyl alcohol acetic acid mixed solvent (600ml methyl alcohol with 25g (21.6mmol) full guard six peptides; 100ml acetic acid) in; with palladium carbon shortening; remove protecting group; obtain target product, separate out thick peptide with ether.Use macroporous resin purification, obtain the purified peptide (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2) that 13g (14.6mmol) satisfies the demands, total recovery 29.2%, purity 91%.
Obviously, those skilled in the art can carry out various changes and modification to the present invention and not break away from the spirit and scope of the present invention.Like this, if of the present invention these are revised and modification belongs within the scope of claim of the present invention and equivalent technologies thereof, then the present invention also is intended to comprise these changes and modification interior.The content that is not described in detail in this specification sheets belongs to this area professional and technical personnel's known prior art.
Claims (4)
1. polypeptide liquid-phase synthesis process, it is characterized in that may further comprise the steps: amino acid derivative is placed N, condensation in the dinethylformamide (DMF) adds reaction soln in the entry, separate out solid after, washing after filtration,, drying, obtain intermediate, add in the entry after removing the amino protecting group of described intermediate, and the regulator solution acid-basicity, the intermediate that removes amino protecting group is separated out drying; Remove the intermediate of amino protecting group and treat that the amino acid derivative of condensation places N described; condensation in the dinethylformamide (DMF) repeats said process, then until obtaining the full guard polypeptide; remove the protecting group of described full guard polypeptide again, obtain target polypeptides.
2. polypeptide liquid-phase synthesis process as claimed in claim 1; it is characterized in that: the amino protecting group of described intermediate comprises tertbutyloxycarbonyl (Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc); adopt trifluoroacetic acid (TFA) to remove tertbutyloxycarbonyl (Boc), adopt the mixing solutions of piperidines and DMF to remove 9-fluorenylmethyloxycarbonyl (Fmoc).
3. polypeptide liquid-phase synthesis process as claimed in claim 2, it is characterized in that: the reaction soln that will remove behind the tertbutyloxycarbonyl (Boc) adds in the entry, come the regulator solution acid-basicity by weak base again, the intermediate that removes tertbutyloxycarbonyl (Boc) is separated out, filter then, wash, drying.
4. polypeptide liquid-phase synthesis process as claimed in claim 2, it is characterized in that: the reaction soln that will remove behind the 9-fluorenylmethyloxycarbonyl (Fmoc) adds in the entry, come the regulator solution acid-basicity by weak acid again, the intermediate that removes 9-fluorenylmethyloxycarbonyl (Fmoc) is separated out, filter then, wash, drying.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103421087A (en) * | 2013-04-12 | 2013-12-04 | 上海捌加壹医药科技有限公司 | Liquid-phase synthesizing method for polypeptide |
| CN111675805A (en) * | 2020-07-22 | 2020-09-18 | 万华化学集团股份有限公司 | Toughened hard thermoplastic polyurethane elastomer and preparation method thereof |
| CN113549130A (en) * | 2021-07-28 | 2021-10-26 | 武汉桀升生物科技有限公司 | Liquid phase synthesis method of breast enlarging peptide |
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| CN101747412A (en) * | 2009-12-30 | 2010-06-23 | 江苏诺泰制药技术有限公司 | Synthesis and preparation process of eptifibatide |
| WO2010113939A1 (en) * | 2009-03-30 | 2010-10-07 | 味の素株式会社 | Diphenylmethane compound |
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| WO2010113939A1 (en) * | 2009-03-30 | 2010-10-07 | 味の素株式会社 | Diphenylmethane compound |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421087A (en) * | 2013-04-12 | 2013-12-04 | 上海捌加壹医药科技有限公司 | Liquid-phase synthesizing method for polypeptide |
| CN111675805A (en) * | 2020-07-22 | 2020-09-18 | 万华化学集团股份有限公司 | Toughened hard thermoplastic polyurethane elastomer and preparation method thereof |
| CN111675805B (en) * | 2020-07-22 | 2022-07-12 | 万华化学集团股份有限公司 | Toughened hard thermoplastic polyurethane elastomer and preparation method thereof |
| CN113549130A (en) * | 2021-07-28 | 2021-10-26 | 武汉桀升生物科技有限公司 | Liquid phase synthesis method of breast enlarging peptide |
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