CN104098688A - Method for synthesizing thymalfasin - Google Patents

Method for synthesizing thymalfasin Download PDF

Info

Publication number
CN104098688A
CN104098688A CN201410333844.8A CN201410333844A CN104098688A CN 104098688 A CN104098688 A CN 104098688A CN 201410333844 A CN201410333844 A CN 201410333844A CN 104098688 A CN104098688 A CN 104098688A
Authority
CN
China
Prior art keywords
otbu
fmoc
glu
tbu
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201410333844.8A
Other languages
Chinese (zh)
Inventor
文永均
郭德文
董华建
曾德志
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chengdu Shengnuo Biopharm Co., Ltd.
Original Assignee
CHENGDU SHENGNUO BIOTEC Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CHENGDU SHENGNUO BIOTEC Co Ltd filed Critical CHENGDU SHENGNUO BIOTEC Co Ltd
Priority to CN201410333844.8A priority Critical patent/CN104098688A/en
Publication of CN104098688A publication Critical patent/CN104098688A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57581—Thymosin; Related peptides
    • 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

Landscapes

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

Abstract

The invention relates to the field of pharmaceutical synthesis, in particular to a method for synthesizing thymalfasin, and aims to solve the technical problems of difficulty in separation and purification, low total yield and high production cost of a conventional method. According to the scheme, the method for synthesizing thymalfasin comprises the steps as follows: a, a polypeptide fragment 1 and a polypeptide fragment 2 provided with protecting groups on side chains are synthesized; b, a C terminal of the polypeptide fragment 1 and an N terminal of the polypeptide fragment 2 are coupled, and the protecting group at the N terminal is removed to obtain polypeptide resin I; c, according to an amino acid sequence of thymalfasin, amino acids from the eleventh to the first are sequentially coupled one by one according to the order from the C terminal to the N terminal, then the protecting group at the N terminal is removed, and acetylation is performed to obtain thymalfasin resin; and d, the thymalfasin resin is subjected to acidolysis to remove the C terminal resin and all protecting groups to obtain a coarse thymalfasin product, and thymalfasin is obtained after purification. With the adoption of the method, the product yield can be greatly improved, and the synthesis cycle is shortened.

Description

The method of synthetic Thymosin-Alpha1
Technical field
The present invention relates to the synthetic field of medicine, be specifically related to the method for synthetic Thymosin-Alpha1.
Background technology
Thymosin-Alpha1 is a kind of immune-regulating factor, formed by 28 amino acid, it is inducing T cell Lyt1+ specifically, 2+, the generation of 3+ surface marker, impel the differentiation of T cell and natural killer cell (NK) with ripe, impel the T cell of sensitization to activate the rear various lymphokines that produce at various antigen or mitogen, as interleukin-22 (IL2), interferon-alpha (IFN α), the lymphokines such as IFN-γ (IFN γ), impel the expression of IL2 high-affinity receptor is increased, strengthen the function of Th cell, the lymphocyte reaction that it also mixes by the activation of T4 cell being strengthened to allosome and the autologous mankind simultaneously.Thymosin-Alpha1 may affect raising of NK precursor cell, and this precursor cell is exposed to after Interferon, rabbit and becomes and more have cytotoxicity, makes body can effectively bring into play immune protection function.These product have the T of promotion cytodifferentiation, maturation, improve NK cytoactive, promote all kinds of lymphokines of T emiocytosis, improve the effects such as the avidity of IL-2 acceptor to IL-2.Early clinic application mainly concentrates on antitumor, immunologic hypofunction aspect; pharmacodynamic study result shows simultaneously in recent years, and this product has more the effect of protection liver, the growth that suppresses specifically the HepG2-Nu2 cell that is subject to HBV infection and inhibition marmot WHV virus replication.Show to use separately the curative effect of these product identical with Interferon, rabbit in the multicenter trial showed that is used for the treatment of chronic hepatitis, but side reaction is than Interferon, rabbit much less.Clinically, be successfully applied to the disease for the treatment of second, hepatitis C and other immune deficiency.
Pharmacokinetic document shows, this product Healthy People single subcutaneous injection Thymosin-Alpha1 1.6mg, and blood peak concentration of drug is about 37.51ng/ml, and peak time is about 1.67 hours, and AUC0-15 is about 152.15ng/mlh, and the transformation period is about 1.65 hours.In the experiment being compared by Zadaxin, Timosina and 1 three kinds of samples of T α of one group of relevant T α 1 pharmacokinetics aspect, show, T α 1 discharges (Tmax is 1-2h) fast, metabolism is (16-18h) slowly, although 80% is excreted by urinary system, but Cmax is 30-80ug/L, still exceed tens times than normal T α 1 blood concentration 0.5～0.75ug/L.
The preparation method of current Thymosin-Alpha1 is mainly solid-phase synthesis, but owing to there being a large amount of β-pleated sheet structure structures in Thymosin-Alpha1 molecular structure, form difficult sequences, as amino acid one by one holds N end to connect peptide from C, while being coupled to the 21st amino acid from the 28th amino acid, just there is more than 20% contraction in peptide resin volume, while continuing to receive the 11st amino acid, just recover normal simultaneously, this contraction has seriously reduced follow-up amino acid whose coupling yield, increase the difficulty of separation and purification, seriously reduce the yield of product simultaneously, from the preparation method who has reported, for the Thymosin-Alpha1 of 98%HPLC purity, current preparation total recovery is generally all in 20% left and right.
The amino-acid sequence of Thymosin-Alpha1 is as follows:
Ac-Ser 1-Asp 2-Ala 3-Ala 4-Val 5-Asp 6-Thr 7-Ser 8-Ser 9-Glu 10-Ile 11-Thr 12-Thr 13-Lys 14-Asp 15-Leu 16-Lys 17-Glu 18-Lys 19-Lys 20-Glu 21-Val 22-Val 23-Glu 24-Glu 25-Ala 26-Glu 27-Asn 28-OH
Summary of the invention
The technical problem to be solved in the present invention is existing method separation and purification difficulty, total recovery is low, production cost is high.
The scheme that the present invention solves the problems of the technologies described above is to provide a kind of method of synthetic Thymosin-Alpha1, and the method for the invention is being ensured under the prerequisite of quality product, reduces the complexity of synthetic method and the total recovery of raising product.
The method of above-mentioned synthetic Thymosin-Alpha1, comprises the following steps:
A, synthesize and on side chain, have the polypeptide fragment of protecting group 1:
Fmoc-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-OH；
Synthesize and on side chain, have the polypeptide fragment of protecting group 2:
NH 2-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-aminoresin;
B, by the N end coupling of the C end of polypeptide fragment 1 and polypeptide fragment 2, then remove the N end Fmoc protecting group of polypeptide fragment, obtain polypeptide resin I:
NH 2-Thr (tBu)-Thr (tBu)-Lys (Boc)-Asp (OtBu)-Leu-Lys (Boc)-Glu (OtBu)-Lys (Boc)-Lys (Boc)-Glu (OtBu)-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-aminoresin;
C, according to the aminoacid sequence of Thymosin-Alpha1, according to the order of holding N end from C successively the 11st to the 1st amino acid of coupling one by one, then remove N end protecting group, then carry out acetylization reaction, obtain Thymosin-Alpha1 resin:
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (α-OtBu)-aminoresin,
D, Thymosin-Alpha1 resinous acid solution remove C end resin and all protecting groups obtain Thymosin-Alpha1 crude product, after Thymosin-Alpha1 purifying crude, obtain Thymosin-Alpha1.
In the method for the invention, be first divided into 3 parts according to Thymosin-Alpha1 peptide sequence, be divided into synthetic and other amino acid whose couplings one by one of 2 fragments, the amino-acid sequence numbering of holding C end with Thymosin-Alpha1 main chain N, is shown below:
Ac-Ser 1-Asp 2-Ala 3-Ala 4-Val 5-Asp 6-Thr 7-Ser 8-Ser 9-Glu 10-Ile 11-Thr 12-Thr 13-Lys 14-Asp 15-Leu 16-Lys 17-Glu 18-Lys 19-Lys 20-Glu 21-Val 22-Val 23-Glu 24-Glu 25-Ala 26-Glu 27-Asn 28-OH
The aminoacid sequence of polypeptide fragment 1 is the peptide sequence of numbering 12-21 in above formula:
Fmoc-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-OH。
The aminoacid sequence of polypeptide fragment 2 is the peptide sequence of numbering 22-28 in above formula:
NH 2-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-aminoresin.
As preferred version of the present invention, described in step a, synthetic polypeptide fragment 1 is specially: under coupling reagent exists, hold coupling to have Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) and vector resin coupling of tBu protecting group N, de-Fmoc protecting group obtains H-Glu (OtBu)-vector resin again, then adopt activating reagent and condensation reagent, according to Thymosin-Alpha1 aminoacid sequence, one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the Methionin (Fmoc-Lys (Boc)-OH) of Boc protecting group according to the order of holding N end from C, the coupling of N end has Fmoc protecting group and side chain coupling to have the Methionin (Fmoc-Lys (Boc)-OH) of Boc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Methionin (Fmoc-Lys (Boc)-OH) of Boc protecting group, the coupling of N end has the leucine (Fmoc-Leu-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Methionin (Fmoc-Lys (Boc)-OH) of Boc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have Soviet Union's winter propylhomoserin (Fmoc-Thr (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group to extend coupling, lysate cracking after coupling, remove vector resin and obtain polypeptide fragment 1:
Fmoc-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-OH。
As preferred version of the present invention, described in step a, synthetic polypeptide fragment 2 is specially: under coupling reagent exists, N is held to the aminoresin coupling after aspartic acid (Fmoc-Asp (α-tBu)-OH) that coupling has a Fmoc protecting group and deprotection, de-Fmoc protecting group obtains H-Asp (α-tBu)-aminoresin, then adopt activating reagent and condensation reagent, according to Thymosin-Alpha1 aminoacid sequence, one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of tBu protecting group according to the order of holding N end from C, the coupling of N end has the L-Ala (Fmoc-Ala-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of tBu protecting group, the coupling of N end has the α-amino-isovaleric acid (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has the α-amino-isovaleric acid (Fmoc-Val-OH) of Fmoc protecting group to extend coupling, remove N section with Fmoc protecting group obtain polypeptide fragment 2:
NH 2-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-aminoresin.
As preferred version, step c is specially:
Adopt activating reagent and condensation reagent, according to Thymosin-Alpha1 aminoacid sequence, first N being held coupling to have the C end of the Isoleucine (Fmoc-Ile-OH) of Fmoc protecting group to hold coupling with the N of polypeptide resin I according to the order of holding N end from C, de-Fmoc protecting group obtains H-Ile-polypeptide resin I, then one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has the α-amino-isovaleric acid (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has the L-Ala (Fmoc-Aal-OH) of Fmoc protecting group, the coupling of N end has the L-Ala (Fmoc-Aal-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group to extend coupling, after coupling, remove N end with Fmoc protecting group, carry out obtaining after acetylization reaction Thymosin-Alpha1 resin:
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (α-OtBu)-aminoresin.
In the synthetic polypeptide fragment 1 of aforesaid method step a, the polypeptide resin substitution value that first protected amino acid and vector resin coupling form is 0.3～1.2mmol/g polypeptide resin; Preferred substitution value is 0.6～1.0mmol/g polypeptide resin.
In the synthetic polypeptide fragment 2 of aforesaid method step a, the polypeptide resin substitution value that first protected amino acid and aminoresin coupling form is 0.2～0.8mmol/g polypeptide resin; Preferred substitution value is 0.4～0.6mmol/g polypeptide resin.
Extension coupling of the present invention refers to after first amino acid and amino carrier coupling, and remaining amino acid carries out coupling with the amino acid generation condensation reaction of previous coupling (condensation reaction of the amino and carboxyl of main chain) one by one according to the order of sequence separately.When coupling of the present invention, each protected amino acid or polypeptide fragment consumption are preferably 1～6 times of polypeptide resin mole number; More preferably 2.5～3.5 times.The reaction times of described coupling is 60～300 minutes; Be preferably 100～140 minutes.
Extending in coupling, because each amino acid whose N end has protecting group, therefore need to spend N end protecting group reagent and first remove the coupling again of N end protecting group.PIP/DMF for the present invention (piperidines/DMF) mixing solutions is as going N end protecting group reagent to remove N end protecting group; In described mixing solutions, be 10～30% (V) containing piperidines, all the other are DMF.The consumption of the described N of going end protecting group reagent is every gram of polypeptide resin 5～15mL, preferably every gram of polypeptide resin 8～12mL.Going the time of N end protecting group is 10～60 minutes, preferably 15～25 minutes.
It should be noted that, polypeptide resin of the present invention refer to any number protected amino acid be connected with amino carrier according to Thymosin-Alpha1 amino-acid sequence form polypeptide resin, this wherein also comprises polypeptide resin I, Thymosin-Alpha1 resin.
In the synthetic polypeptide fragment 1 of aforesaid method step a, described vector resin is Trityl-Cl (trityl chloride) resinoid.Preferably, described Trityl-Cl resinoid is any one in Trityl-Cl resin, 4-Methyltrityl-Cl (4-methyl trityl chloride) resin, 4-Methoxytrityl-Cl (4-methoxyl group trityl chloride) resin or 2-Cl Trity-Cl (2-chlorine trityl chloride) resin.
Further preferred, in the time that vector resin is Trityl-Cl resinoid, the coupling method of protected amino acid and vector resin is: the Cl substituted alkyl in carboxyl and the resin of protected amino acid under the effect of alkali, esterification occurs and accesses protected amino acid.
In the synthetic polypeptide fragment 2 of aforesaid method step a, described aminoresin is selected from any one in Rink Amide AM resin, Rink Amide resin and Rink mbha resin, is preferably Rink Amide mbha resin.
In the synthetic polypeptide fragment 1 of aforesaid method step a, described lysate is the dichloromethane solution of the trifluoroethanol of volume percent 25%.
As preferred scheme, described condensation reagent is N, N-DIC (DIC), N, N-dicyclohexylcarbodiimide (DCC), phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus/organic bases (PyBOP/ organic bases), 2-(7-azepine-1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyl-urea phosphofluoric acid ester/organic bases (HATU/ organic bases), benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate/organic bases (HBTU/ organic bases) or O-benzotriazole-N, N, N', any one in N'-tetramethyl-urea Tetrafluoroboric acid ester/organic bases (TBTU/ organic bases).The mole dosage of described condensation reagent is 1～6 times of amino total mole number in polypeptide resin; Be preferably 2.5～3.5 times.
Organic bases described in above-mentioned condensation reagent is any one in DIPEA (DIPEA), triethylamine (TEA) or N-methylmorpholine (NMM); Be preferably DIPEA.
It should be noted that, described PyBOP/ organic bases, HATU/ organic bases, HBTU/ organic bases, TBTU/ organic bases, belong in the present invention the condensation reagent of four kinds of binary systems, PyBOP, HATU, HBTU need to combine and become a kind of condensation reagent use with organic bases respectively in use.The mol ratio of wherein said organic bases and PyBOP, HATU, HBTU, TBTU is 1.3～3.0:1; More preferably 1.3～1.5:1.
As preferred scheme, described activating reagent is I-hydroxybenzotriazole (HOBt) or N-hydroxyl-7-azepine benzotriazole (HOAt).The consumption of described activating reagent is 1.2～6 times of amino total mole number in polypeptide resin; Be preferably 2.5～3.5 times.
As preferred scheme, described coupling reagent is DIPEA (DIPEA), N, one of two kinds of N-DIC/DMAPs (DIC/DMAP).
In building-up process of the present invention, preferably adopt DMF solvent to dissolve.
Except the above-mentioned synthetic method of enumerating, the present invention also can adopt liquid phase synthesizing method to synthesize according to segment condense strategy of the present invention.
As preferred version, the acidolysis described in steps d, is that to adopt the TFA that is 80～95% by volume percent, EDT, the surplus that volume percent is 1～10% be that the mixing acid hydrolysis solution that water forms carries out acidolysis.Preferably, use by volume percent the TFA that is 90%, EDT, the surplus that volume percent is 5% be that the mixing acid hydrolysis solution that water forms carries out acidolysis.Described mixing acid hydrolysis solution consumption is that every gram of Thymosin-Alpha1 resin needs 4～15mL; Be preferably 9～11mL.The time of described acidolysis is under room temperature condition 1～6 hour; Be preferably under room temperature condition 3～4 hours.
As preferred scheme, the purifying described in steps d is specially:
Thymosin-Alpha1 crude product, the stirring that adds water, with ammoniacal liquor tune, pH7.0 extremely dissolves completely, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Thymosin-Alpha1 purify intermediates concentrated solution;
Get Thymosin-Alpha1 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, is splined in chromatographic column, starts moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collects and changes salt main peak and with analyzing Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Thymosin-Alpha1 aqueous acetic acid, lyophilize, obtains Thymosin-Alpha1 sterling.
The Thymosin-Alpha1 being synthesized by method provided by the invention, it is more than 99% detecting purity through HPLC, total recovery is more than 30%, and in the method for the invention, 2 fragments can be synthesized simultaneously, compared with the technical scheme of existing synthesizing amino acid one by one, shorten synthesis cycle, improved total recovery.
Brief description of the drawings
The structure of Fig. 1 Rink Amide AM resin.
The structure of Fig. 2 Rink Amide resin.
The structure of Fig. 3 Rink mbha resin.
Embodiment
The method of synthetic Thymosin-Alpha1, comprises the following steps:
A, under coupling reagent exists, by Fmoc-Glu (OtBu)-OH and vector resin coupling, de-Fmoc protecting group obtains H-Glu (OtBu)-vector resin again, then adopt activating reagent and condensation reagent, according to Thymosin-Alpha1 aminoacid sequence, according to hold N to hold successively one by one by Fmoc-Lys (Boc)-OH from C, Fmoc-Lys (Boc)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Thr (tBu)-OH extends coupling, lysate cracking after coupling, remove vector resin and obtain polypeptide fragment 1:
Fmoc-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-OH；
Under coupling reagent exists, by the aminoresin coupling after Fmoc-Asp (α-tBu)-OH and deprotection, de-Fmoc protecting group obtains H-Asp (α-tBu)-aminoresin, then adopt activating reagent and condensation reagent, according to according to Thymosin-Alpha1 aminoacid sequence, according to hold N to hold successively one by one by Fmoc-Glu (OtBu)-OH from C, Fmoc-Ala-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Val-OH, Fmoc-Val-OH extends coupling, remove N end with Fmoc protecting group obtain polypeptide fragment 2:
NH 2-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-aminoresin;
B, the N end of the C end of polypeptide fragment 1 and polypeptide fragment 2 is connected, then removes N and hold Fmoc protecting group, obtain polypeptide resin I:
NH 2-Thr (tBu)-Thr (tBu)-Lys (Boc)-Asp (OtBu)-Leu-Lys (Boc)-Glu (OtBu)-Lys (Boc)-Lys (Boc)-Glu (OtBu)-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-aminoresin;
C, according to the aminoacid sequence of Thymosin-Alpha1, connect one by one successively the 11st to the 1st amino acid according to the order of holding N end from C, after first the C end of Fmoc-Ile-OH being held to coupling with the N of polypeptide resin I, de-Fmoc protecting group obtains H-Ile-polypeptide resin I, then successively one by one by Fmoc-Glu (OtBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Val-OH, Fmoc-Aal-OH, Fmoc-Aal-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Ser (tBu)-OH extends coupling, after coupling, remove N end with Fmoc protecting group, carry out obtaining after acetylization reaction Thymosin-Alpha1 resin:
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (α-OtBu)-aminoresin,
D, Thymosin-Alpha1 resinous acid solution remove C end resin and all protecting groups obtain Thymosin-Alpha1 crude product, after Thymosin-Alpha1 purifying crude, obtain Thymosin-Alpha1.
Thymosin-Alpha1 structure has 28 amino-acid residues, obtain total recovery and significantly improve, and must overcome the contraction problem of peptide resin in building-up process.For this reason, applicant, according to long-term experimental study, has proposed the method for the invention and has prepared Thymosin-Alpha1, in improving the quality of products, improves preparation total recovery.
The present invention is aminoacid sequence shown in synthetic polypeptide fragment 1 in step a, coupling protecting group respectively on its N end, Thr side chain, on Lys side chain, on Asp side chain and on Glu side chain; Aminoacid sequence shown in synthetic polypeptide fragment 2, on its N end, Asp side chain and, coupling protecting group respectively on Glu side chain; And remaining other amino acid; to form after polypeptide resin I in polypeptide fragment 1 and polypeptide fragment 2 couplings; hold the order of N end according to aminoacid sequence C shown in Thymosin-Alpha1 1～11 peptide sequence; first N is held coupling to have the leucic C section of protecting group and the coupling of the N of polypeptide resin I end; then one by one other corresponding protected amino acids are extended to coupling successively; after coupling, remove N end protecting group, after acetylization reaction, obtain whole Thymosin-Alpha1 resin.
In the synthetic polypeptide fragment 1 of aforesaid method step a, the polypeptide resin substitution value that first protected amino acid and vector resin coupling form is preferably 0.3～1.2mmol/g polypeptide resin; Preferred substitution value is 0.6～1.0mmol/g polypeptide resin.
In the synthetic polypeptide fragment 2 of aforesaid method step a, the polypeptide resin substitution value that first protected amino acid and aminoresin coupling form is preferably 0.2～0.8mmol/g polypeptide resin; Preferred substitution value is 0.4～0.6mmol/g polypeptide resin.
Extension coupling of the present invention refers to after first amino acid and amino carrier coupling, and remaining amino acid carries out coupling with the amino acid generation condensation reaction of previous coupling (condensation reaction of the amino and carboxyl of main chain) one by one according to the order of sequence separately.When coupling of the present invention, each protected amino acid or polypeptide fragment consumption are preferably 1～6 times of polypeptide resin mole number; More preferably 2.5～3.5 times.The reaction times of described coupling is preferably 60～300 minutes; More preferably 100～140 minutes.
Extending in coupling, because each amino acid N end has protecting group, therefore need to spend N end protecting group reagent and first remove the coupling again of N end protecting group.The present invention preferably uses PIP/DMF (piperidines/DMF) mixing solutions as going N end protecting group reagent to remove N end protecting group; In described mixing solutions, be 10～30% (V) containing piperidines, all the other are DMF.The consumption of the described N of going end protecting group reagent is every gram of polypeptide resin 5～15mL, preferably every gram of polypeptide resin 8～12mL.Going the time of N end protecting group is 10～60 minutes, preferably 15～25 minutes.
It should be noted that, polypeptide resin of the present invention refer to any number protected amino acid be connected with amino carrier according to Thymosin-Alpha1 amino-acid sequence form polypeptide resin, this wherein also comprises polypeptide resin I, Thymosin-Alpha1 resin.
In the synthetic polypeptide fragment 1 of aforesaid method step a, described vector resin is Trityl-Cl (trityl chloride) resinoid.Preferably, described Trityl-Cl resinoid is any one in Trityl-Cl resin, 4-Methyltrityl-Cl (4-methyl trityl chloride) resin, 4-Methoxytrityl-Cl (4-methoxyl group trityl chloride) resin or 2-Cl Trity-Cl (2-chlorine trityl chloride) resin.
Wherein, further preferably, in the time that vector resin is Trityl-Cl resinoid, the coupling method of protected amino acid and vector resin is: the Cl substituted alkyl in carboxyl and the resin of protected amino acid under the effect of alkali, esterification occurs and accesses protected amino acid.
In the synthetic polypeptide fragment 2 of aforesaid method step a, described aminoresin is selected from any one in Rink Amide AM resin, Rink Amide resin and Rink mbha resin, is preferably Rink Amide mbha resin.
In the synthetic polypeptide fragment 1 of aforesaid method step a, described lysate is the dichloromethane solution of the trifluoroethanol of volume percent 25%.
As preferred embodiments of the present invention, described condensation reagent is N, N-DIC (DIC), N, N-dicyclohexylcarbodiimide (DCC), phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus/organic bases (PyBOP/ organic bases), 2-(7-azepine-1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyl-urea phosphofluoric acid ester/organic bases (HATU/ organic bases), benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate/organic bases (HBTU/ organic bases) or O-benzotriazole-N, N, N', any one in N'-tetramethyl-urea Tetrafluoroboric acid ester/organic bases (TBTU/ organic bases).The mole dosage of described condensation reagent is 1～6 times of amino total mole number in polypeptide resin; Be preferably 2.5～3.5 times.
Organic bases described in above-mentioned condensation reagent is any one in DIPEA (DIPEA), triethylamine (TEA) or N-methylmorpholine (NMM); Be preferably DIPEA.
It should be noted that, described PyBOP/ organic bases, HATU/ organic bases, HBTU/ organic bases, TBTU/ organic bases, belong in the present invention the condensation reagent of four kinds of binary systems, PyBOP, HATU, HBTU need to combine and become a kind of condensation reagent use with organic bases respectively in use.The mol ratio of wherein said organic bases and PyBOP, HATU, HBTU, TBTU is 1.3～3.0:1; More preferably 1.3～1.5:1.
As preferably, described activating reagent is I-hydroxybenzotriazole (HOBt) or N-hydroxyl-7-azepine benzotriazole (HOAt).The consumption of described activating reagent is 1.2～6 times of amino total mole number in polypeptide resin; Be preferably 2.5～3.5 times.
As preferably, coupling reagent of the present invention is DIPEA (DIPEA), N, one of two kinds of N-DIC/DMAPs (DIC/DMAP).
In building-up process of the present invention, preferably adopt DMF solvent to dissolve.
Except the above-mentioned synthetic method of enumerating, the present invention also can adopt liquid phase synthesizing method to synthesize according to segment condense strategy of the present invention.
As preferred version, the acidolysis described in steps d, is that to adopt the TFA that is 80～95% by volume percent, EDT, the surplus that volume percent is 1～10% be that the mixing acid hydrolysis solution that water forms carries out acidolysis.Preferably, use by volume percent the TFA that is 90%, EDT, the surplus that volume percent is 5% be that the mixing acid hydrolysis solution that water forms carries out acidolysis.Described mixing acid hydrolysis solution consumption is that every gram of Thymosin-Alpha1 resin needs 4～15mL; Be preferably 9～11mL.The time of described acidolysis is under room temperature condition 1～6 hour; Be preferably under room temperature condition 3～4 hours.
As preferably, purifying is specially described in steps d:
Thymosin-Alpha1 crude product, the stirring that adds water, with ammoniacal liquor tune, pH7.0 extremely dissolves completely, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Thymosin-Alpha1 purify intermediates concentrated solution;
Get Thymosin-Alpha1 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, is splined in chromatographic column, starts moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collects and changes salt main peak and with analyzing Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Thymosin-Alpha1 aqueous acetic acid, lyophilize, obtains Thymosin-Alpha1 sterling.
Protecting group of the present invention is the blocking group of the synthetic group of the interference such as amino, carboxyl on the protected amino acid main chain conventional in the synthetic field of amino acid and side chain, prevent that amino, carboxyl etc. from reacting preparing in target product process, generate impurity, for the amino acid of the side chain that needs protection in the present invention, its side-chain structure as well known to those skilled in the art and know and adopt conventional protecting group to carry out the group such as amino, carboxyl on protected amino acid side chain, as preferably, the present invention is by the side chain of OtBu protecting group protection L-glutamic acid, aspartic acid; By the side chain of tBu protecting group protection Threonine, Serine, tyrosine; By the side chain of Boc protecting group protection Methionin.In addition,, in the amino acid relating in the method for the invention, amino acid N end is all preferably protected by Fmoc protecting group.And the amino acid of protected base protection is called protected amino acid.
The invention discloses a kind of method of synthetic Thymosin-Alpha1, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can, not departing from content of the present invention, spirit and scope compound as herein described and preparation method changes or suitably change and combination, realize and apply the technology of the present invention.
In the specific embodiment of the invention; all couplings all can be by commercially available acquisition by the amino acid of protecting group; protected amino acid in the present invention is purchased from Hui Rong bio tech ltd, Chengdu; resin used is purchased from Shangyu pul resin company limited; wherein polypeptide fragment 1 of the present invention also can be by commercially available acquisition, and the Chinese implication that in application documents, english abbreviation used is corresponding is in table 1.
The lexical or textual analysis of table 1 english abbreviation
English abbreviation Chinese English abbreviation Chinese
Ala L-Ala Lys Methionin
Asp Aspartic acid Ser Serine
Glu L-glutamic acid Thr Threonine
Ile Isoleucine Val α-amino-isovaleric acid
Leu Leucine OtBu Tert.-butoxy
Fmoc 9-fluorenylmethyloxycarbonyl Boc Tertiary butyloxycarbonyl acyl group
tBu The tertiary butyl Ac 2O Acetic anhydride
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: polypeptide fragment 1 synthetic
Taking substitution degree is the 2-CTC resin 0.5Kg of 0.6mol/g, join in solid phase synthetic instrument reactor, with DMF washing 2 times, after 30 minutes, drain with the swelling resin of DMF, getting 0.6mol Fmoc-Glu (OtBu)-OH DMF dissolves, add in the above-mentioned reaction column that resin is housed, add again 1.2mol DIPEA, react and drain after 2 hours, add the DMF solution that contains 1.2mol anhydrous methanol, stirring reaction 1 hour, with DMF washing 6 times, obtains Fmoc-Glu (OtBu)-2-CTC resin.
Remove Fmoc protecting group in Fmoc-Glu (OtBu)-2-CTC resin 20 minutes with every gram of resin 10mL20% piperidines/DMF solution, then, with DMF washing 6 times, obtain H-Glu (OtBu)-2-CTC resin.
Get 0.9mol Fmoc-Lys (Boc)-OH and 0.9mol HOBt, with DMF dissolving, under stirring, add 0.9mol DIC, continue stirring reaction 1 hour, add in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency reacts completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Lys (Boc)-Glu (OtBu)-2-CTC resin.
According to Thymosin-Alpha1 aminoacid sequence the 20th to the 12nd amino-acid sequence; repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, complete successively the extension coupling of the extension coupling of Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Thr (tBu)-OH.
Reaction finishes that rear resin vacuum-drying is spent the night by methanol wash 6 times, weighs and obtains 1.21Kg and do not remove the polypeptide fragment 1 of 2-CTC resin, is added in 25L glass reactor.
Configuration lysate 25%TFE/ dichloromethane solution 12L, pours lytic reagent in flask into room temperature reaction 2h.Reaction finishes, and filters resin, collects filtrate.Filtrate volume is revolved after evaporate to dryness, add 3L DCM, mixed solution is dropped in 15L ether, separate out white solid, filter collecting precipitation, anhydrous diethyl ether washing, and vacuum-drying, obtain 0.58Kg polypeptide fragment 1, purity 95.1%, yield 90.7%.
Fmoc-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-OH。
Embodiment 2: polypeptide fragment 1 synthetic
Taking substitution degree is the 2-CTC resin 0.5Kg of 0.6mol/g, join in solid phase synthetic instrument reactor, with DMF washing 2 times, after 30 minutes, drain with the swelling resin of DMF, getting 0.6mol Fmoc-Glu (OtBu)-OH DMF dissolves, add in the above-mentioned reaction column that resin is housed, add again 1.2mol DIPEA, react and drain after 2 hours, add the DMF solution that contains 1.2mol anhydrous methanol, stirring reaction 1 hour, with DMF washing 6 times, obtains Fmoc-Glu (OtBu)-2-CTC resin.
Remove Fmoc protecting group in Fmoc-Glu (OtBu)-2-CTC resin 20 minutes with every gram of resin 10mL20% piperidines/DMF solution, then, with DMF washing 6 times, obtain H-Glu (OtBu)-2-CTC resin.
Get 0.9mol Fmoc-Lys (Boc)-OH and 0.9mol HOBt, dissolve with DMF, under stirring, add 0.86mol HBTU, continue stirring reaction and after 1 hour, add again 1.3molDIPEA, after mixing, join in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Lys (Boc)-Glu (OtBu)-2-CTC resin.
According to Thymosin-Alpha1 aminoacid sequence the 20th to the 12nd amino-acid sequence; repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, complete successively the extension coupling of the extension coupling of Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Thr (tBu)-OH.
Reaction finishes that rear resin vacuum-drying is spent the night by methanol wash 6 times, weighs and obtains 1.17Kg and do not remove the polypeptide fragment 1 of 2-CTC resin, is added in 25L glass reactor.
Configuration lysate 25%TFE/ dichloromethane solution 12L, pours lytic reagent in flask into room temperature reaction 2h.Reaction finishes, and filters resin, collects filtrate.Filtrate volume is revolved after evaporate to dryness, add 3L DCM, mixed solution is dropped in 15L ether, separate out white solid, filter collecting precipitation, anhydrous diethyl ether washing, and vacuum-drying, obtain 0.55Kg polypeptide fragment 1, purity 91.7%, yield 86.4%.
Fmoc-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-OH。
Embodiment 3: polypeptide fragment 2 synthetic
Taking substitution degree is the Rink mbha resin 0.2Kg of 0.5mol/g; join in solid phase synthetic instrument reactor; with DMF washing 2 times; after 30 minutes, drain with the swelling resin of DMF; by the Fmoc protecting group in every gram of resin 10mL20% piperidines/DMF solution deresinate 20 minutes; then with DMF washing 6 times, obtain de-protected Rink mbha resin.
Get 0.3mol Asp (α-OtBu)-OH and 0.3mol HOBt, with DMF dissolving, under stirring, add 0.3mol DIC, continue stirring reaction 1 hour, add in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency reacts completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Asp (α-OtBu)-Rink mbha resin.
According to Thymosin-Alpha1 aminoacid sequence the 27th to the 22nd amino-acid sequence; repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, complete successively the extension coupling of the extension coupling of Fmoc-Glu (OtBu)-OH, Fmoc-Ala, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Val, Fmoc-Val-OH.Finally with every gram of aminoresin 15mL PIP (20%)/DMF (80%) mixing solutions remove N section with Fmoc protecting group 20min, then, with DMF washing 6 times, obtain polypeptide fragment 2:
NH 2-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-Rink mbha resin.
Embodiment 4: polypeptide fragment 2 synthetic
Taking substitution degree is the Rink mbha resin 0.2Kg of 0.5mol/g; join in solid phase synthetic instrument reactor; with DMF washing 2 times; after 30 minutes, drain with the swelling resin of DMF; by the Fmoc protecting group in every gram of resin 10mL20% piperidines/DMF solution deresinate 20 minutes; then with DMF washing 6 times, obtain de-protected Rink mbha resin.
Get 0.3mol Fmoc-Lys (Boc)-OH and 0.3mol HOBt, dissolve with DMF, under stirring, add 0.28mol HBTU, continue stirring reaction and after 1 hour, add again 0.45molDIPEA, after mixing, join in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Asp (α-OtBu)-Rink mbha resin.
According to Thymosin-Alpha1 aminoacid sequence the 27th to the 22nd amino-acid sequence; repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, complete successively the extension coupling of the extension coupling of Fmoc-Glu (OtBu)-OH, Fmoc-Ala, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Val, Fmoc-Val-OH.Finally with every gram of aminoresin 15mL PIP (20%)/DMF (80%) mixing solutions remove N section with Fmoc protecting group 20min, then, with DMF washing 6 times, obtain polypeptide fragment 2:
NH 2-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-Rink mbha resin.
Embodiment 5: polypeptide resin I synthetic
Get the polypeptide fragment 1 of 0.25mol embodiment 1, polypeptide fragment 1 and the 0.25mol HOAt of 0.1mol embodiment 3; dissolve with DMF; under stirring, add 0.25mol DIC; (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 6h; if resin water white transparency; react completely; resin colour developing; represent that reaction not exclusively; need extend again the linked reaction time), then use every gram of resin 10mL PIP (20%)/DMF (80%) mixing solutions to take off Fmoc protecting group, the time is 20min; with DMF washing 6 times, obtain polypeptide resin I:
NH 2-Thr (tBu)-Thr (tBu)-Lys (Boc)-Asp (OtBu)-Leu-Lys (Boc)-Glu (OtBu)-Lys (Boc)-Lys (Boc)-Glu (OtBu)-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-Rink mbha resin.
Embodiment 6: polypeptide resin I synthetic
Get polypeptide fragment 1 and the 0.25mol HOAt of 0.25mol embodiment 2, dissolve with DMF, under stirring, add 0.24mol HBTU, continue stirring reaction and after 1 hour, add again 0.4molDIPEA, after stirring, add the polypeptide fragment 2 of 0.1mol embodiment 4, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 6h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, need extend again the linked reaction time), use again every gram of resin 10mL PIP (20%)/DMF (80%) mixing solutions to take off Fmoc protecting group, time is 20min, with DMF washing 6 times, obtain polypeptide resin I:
NH 2-Thr (tBu)-Thr (tBu)-Lys (Boc)-Asp (OtBu)-Leu-Lys (Boc)-Glu (OtBu)-Lys (Boc)-Lys (Boc)-Glu (OtBu)-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-Rink mbha resin.
Embodiment 7: Thymosin-Alpha1 peptide resin synthetic
Get polypeptide resin I and the 0.3mol HOBt of 0.3mol Fmoc-Ile-OH, 0.1mol embodiment 5, dissolve with DMF, under stirring, add 0.3mol DIC, continue stirring reaction 1 hour, add in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 3h, if resin water white transparency, reacts completely, resin colour developing, represent that reaction not exclusively, extends the linked reaction time).
Protected amino acid according to Thymosin-Alpha1 aminoacid sequence the 1st to ten amino acid sequence, hold the order of N end to extend one by one successively coupling from C, one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has the α-amino-isovaleric acid (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has the L-Ala (Fmoc-Aal-OH) of Fmoc protecting group, the coupling of N end has the L-Ala (Fmoc-Aal-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group to extend coupling, after coupling, remove N end with Fmoc protecting group, use 25%Ac 2o/DMF solution carries out acetylization reaction 2 hours, uses respectively DMF and methanol wash 6 times, obtains Thymosin-Alpha1 resin 578g after drying under reduced pressure, and resin total recovery is 89.6%.
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (α-OtBu)-Rink mbha resin.
Embodiment 8: Thymosin-Alpha1 peptide resin synthetic
Get 0.3mol Fmoc-Ile-OH and 0.3mol HOBt, with DMF dissolving, under stirring, add 0.28mol HBTU, continue stirring reaction and after 1 hour, add again 0.45molDIPEA, be transferred in solid phase synthetic instrument reactor, after stirring, add the polypeptide resin I of 0.1mol embodiment 6, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 3h, if resin water white transparency, react completely, resin colour developing, represents that reaction not exclusively, extends the linked reaction time).
Protected amino acid according to Thymosin-Alpha1 aminoacid sequence the 1st to ten amino acid sequence, hold the order of N end to extend one by one successively coupling from C, one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has the α-amino-isovaleric acid (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has the L-Ala (Fmoc-Aal-OH) of Fmoc protecting group, the coupling of N end has the L-Ala (Fmoc-Aal-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group to extend coupling, after coupling, remove N end with Fmoc protecting group, use 25%Ac 2o/DMF solution carries out acetylization reaction 2 hours, uses respectively DMF and methanol wash 6 times, obtains Thymosin-Alpha1 resin 561g after drying under reduced pressure, and resin total recovery is 85.5%.
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (α-OtBu)-Rink mbha resin.
Embodiment 9: the preparation of Thymosin-Alpha1 crude product
Get embodiment 7 gained Thymosin-Alpha1 resins, adding volume ratio is the mixing acid hydrolysis solution (consumption 10mL/ gram of Thymosin-Alpha1 resin) of TFA ︰ water ︰ EDT=90 ︰ 5 ︰ 5, stir, stirring at room temperature reaction 3 hours, reaction mixture uses sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, adds anhydrous diethyl ether precipitation, then washes and precipitate 3 times with anhydrous diethyl ether, drain to obtain off-white powder, vacuum decompression is dried to constant weight, obtains Thymosin-Alpha1 crude product 341g, and crude product purity is 66.9%.
Embodiment 10: the preparation of Thymosin-Alpha1 crude product
Get embodiment 8 gained Thymosin-Alpha1 resins, adding volume ratio is the mixing acid hydrolysis solution (consumption 10mL/ gram of Thymosin-Alpha1 resin) of TFA ︰ water ︰ EDT=90 ︰ 5 ︰ 5, stir, stirring at room temperature reaction 3 hours, reaction mixture uses sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, adds anhydrous diethyl ether precipitation, then washes and precipitate 3 times with anhydrous diethyl ether, drain to obtain off-white powder, vacuum decompression is dried to constant weight, obtains Thymosin-Alpha1 crude product 328g, and crude product purity is 62.6%.
Embodiment 11: Thymosin-Alpha1 purifying crude
Get embodiment 9 gained Thymosin-Alpha1 crude products, the stirring that adds water, with ammoniacal liquor tune, pH7.0 extremely dissolves completely, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 100mm*250mm is 120mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Thymosin-Alpha1 purify intermediates concentrated solution;
Get Thymosin-Alpha1 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 100mm*250mm is 120mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Thymosin-Alpha1 aqueous acetic acid, lyophilize, obtain Thymosin-Alpha1 sterling 106.1g, preparation total recovery is 34.1%, molecular weight: 3108.8 (100%M+H), purity: 99.4%, maximum single contaminant 0.13%.
Embodiment 12: Thymosin-Alpha1 purifying crude
Get embodiment 10 gained Thymosin-Alpha1 crude products, the stirring that adds water, with ammoniacal liquor tune, pH7.0 extremely dissolves completely, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 100mm*250mm is 120mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Thymosin-Alpha1 purify intermediates concentrated solution;
Get Thymosin-Alpha1 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 100mm*250mm is 120mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Thymosin-Alpha1 aqueous acetic acid, lyophilize, obtain Thymosin-Alpha1 sterling 96.9g, preparation total recovery is 31.2%, molecular weight: 3108.6 (100%M+H), purity: 99.5%, maximum single contaminant 0.19%.
Embodiment 13 adopts 2nd～11 protected amino acid fragments of Thymosin-Alpha1 to prepare Thymosin-Alpha1
Ac-Ser 1-Asp 2-Ala 3-Ala 4-Val 5-Asp 6-Thr 7-Ser 8-Ser 9-Glu 10-Ile 11-Thr 12-Thr 13-Lys 14-Asp 15-Leu 16-Lys 17-Glu 18-Lys 19-Lys 20-Glu 21-Val 22-Val 23-Glu 24-Glu 25-Ala 26-Glu 27-Asn 28-OH
Taking substitution degree is the Rink mbha resin 0.2Kg of 0.5mol/g; join in solid phase synthetic instrument reactor; with DMF washing 2 times; after 30 minutes, drain with the swelling resin of DMF; by the Fmoc protecting group in every gram of resin 10mL20% piperidines/DMF solution deresinate 20 minutes; then with DMF washing 6 times, obtain de-protected Rink mbha resin.
Get 0.3mol Asp (α-OtBu)-OH and 0.3mol HOBt, with DMF dissolving, under stirring, add 0.3mol DIC, continue stirring reaction 1 hour, add in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency reacts completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Asp (α-OtBu)-Rink mbha resin.
According to Thymosin-Alpha1 aminoacid sequence, from C end to N end, repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, once complete the extension coupling of corresponding amino acid or fragment by the order of table 2.
Table 2
Finally with every gram of aminoresin 15mL PIP (20%)/DMF (80%) mixing solutions remove N section with Fmoc protecting group 20min, then, with DMF washing 6 times, use 25%Ac 2o/DMF solution carries out acetylization reaction 2 hours, uses respectively DMF and methanol wash 6 times, obtains Thymosin-Alpha1 resin 521g after drying under reduced pressure, and resin total recovery is 76.1%.
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (α-OtBu)-Rink mbha resin.
Get above-mentioned Thymosin-Alpha1 resin, adding volume ratio is the mixing acid hydrolysis solution (consumption 10mL/ gram of Thymosin-Alpha1 resin) of TFA ︰ water ︰ EDT=90 ︰ 5 ︰ 5, stir, stirring at room temperature reaction 3 hours, reaction mixture uses sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, adds anhydrous diethyl ether precipitation, then washes and precipitate 3 times with anhydrous diethyl ether, drain to obtain off-white powder, vacuum decompression is dried to constant weight, obtains Thymosin-Alpha1 crude product 303g, and crude product purity is 43.8%.
Get above-mentioned Thymosin-Alpha1 crude product, the stirring that adds water, with ammoniacal liquor tune, pH7.0 extremely dissolves completely, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 100mm*250mm is 120mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Thymosin-Alpha1 purify intermediates concentrated solution;
Get Thymosin-Alpha1 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 100mm*250mm is 120mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Thymosin-Alpha1 aqueous acetic acid, lyophilize, obtain Thymosin-Alpha1 sterling 48.9g, preparation total recovery is 15.7%, molecular weight: 3108.4 (100%M+H), purity: 97.9%, maximum single contaminant 0.9%.
Embodiment 14 adopts 7th～16 protected amino acid fragments of Thymosin-Alpha1 to prepare Thymosin-Alpha1
Ac-Ser 1-Asp 2-Ala 3-Ala 4-Val 5-Asp 6-Thr 7-Ser 8-Ser 9-Glu 10-Ile 11-Thr 12-Thr 13-Lys 14-Asp 15-Leu 16-Lys 17-Glu 18-Lys 19-Lys 20-Glu 21-Val 22-Val 23-Glu 24-Glu 25-Ala 26-Glu 27-Asn 28-OH
Taking substitution degree is the Rink mbha resin 0.2Kg of 0.5mol/g; join in solid phase synthetic instrument reactor; with DMF washing 2 times; after 30 minutes, drain with the swelling resin of DMF; by the Fmoc protecting group in every gram of resin 10mL20% piperidines/DMF solution deresinate 20 minutes; then with DMF washing 6 times, obtain de-protected Rink mbha resin.
Get 0.3mol Asp (α-OtBu)-OH and 0.3mol HOBt, with DMF dissolving, under stirring, add 0.3mol DIC, continue stirring reaction 1 hour, add in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency reacts completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Asp (α-OtBu)-Rink mbha resin.
According to Thymosin-Alpha1 aminoacid sequence, from C end to N end, repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, once complete the extension coupling of corresponding amino acid or fragment by the order of table 3.
Table 3
Finally with every gram of aminoresin 15mL PIP (20%)/DMF (80%) mixing solutions remove N section with Fmoc protecting group 20min, then, with DMF washing 6 times, use 25%Ac 2o/DMF solution carries out acetylization reaction 2 hours, uses respectively DMF and methanol wash 6 times, obtains Thymosin-Alpha1 resin 535g after drying under reduced pressure, and resin total recovery is 79.4%.
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (α-OtBu)-Rink mbha resin.
Get above-mentioned Thymosin-Alpha1 resin, adding volume ratio is the mixing acid hydrolysis solution (consumption 10mL/ gram of Thymosin-Alpha1 resin) of TFA ︰ water ︰ EDT=90 ︰ 5 ︰ 5, stir, stirring at room temperature reaction 3 hours, reaction mixture uses sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, adds anhydrous diethyl ether precipitation, then washes and precipitate 3 times with anhydrous diethyl ether, drain to obtain off-white powder, vacuum decompression is dried to constant weight, obtains Thymosin-Alpha1 crude product 317g, and crude product purity is 47.2%.
Get above-mentioned Thymosin-Alpha1 crude product, the stirring that adds water, with ammoniacal liquor tune, pH7.0 extremely dissolves completely, 0.45 μ m filtering with microporous membrane for solution, purifying is for subsequent use;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 100mm*250mm is 120mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, obtain Thymosin-Alpha1 purify intermediates concentrated solution;
Get Thymosin-Alpha1 purify intermediates concentrated solution, for subsequent use with 0.45 μ m filter membrane filtration;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 100mm*250mm is 120mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Thymosin-Alpha1 aqueous acetic acid, lyophilize, obtain Thymosin-Alpha1 sterling 57.8g, preparation total recovery is 18.6%, molecular weight: 3108.6 (100%M+H), purity: 98.2%, maximum single contaminant 0.6%.
Above-described embodiment shows, the polypeptide fragment 1 and 2 that the present invention adopts synthesize Thymosin-Alpha1, and through HPLC detection purity 99.5%, total recovery can reach more than 34%, can not reach similar effect and adopt other fragments to synthesize Thymosin-Alpha1.Method provided by the invention is synthesized Thymosin-Alpha1, has shortened synthesis cycle, has improved purity and total recovery.

Claims (10)

1. the method for synthetic Thymosin-Alpha1, is characterized in that, comprises the following steps:
A, synthesize and on side chain, have the polypeptide fragment of protecting group 1:
Fmoc-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-OH；
Synthesize and on side chain, have the polypeptide fragment of protecting group 2:
NH 2-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-aminoresin;
B, by the N end coupling of the C end of polypeptide fragment 1 and polypeptide fragment 2, then remove the N end Fmoc protecting group of polypeptide fragment, obtain polypeptide resin I:
NH 2-Thr (tBu)-Thr (tBu)-Lys (Boc)-Asp (OtBu)-Leu-Lys (Boc)-Glu (OtBu)-Lys (Boc)-Lys (Boc)-Glu (OtBu)-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-aminoresin;
C, according to the aminoacid sequence of Thymosin-Alpha1, according to the order of holding N end from C successively the 11st to the 1st amino acid of coupling one by one, then remove N end protecting group, then carry out acetylization reaction, obtain Thymosin-Alpha1 resin:
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (α-OtBu)-aminoresin,
D, Thymosin-Alpha1 resinous acid solution remove C end resin and all protecting groups obtain Thymosin-Alpha1 crude product, after Thymosin-Alpha1 purifying crude, obtain Thymosin-Alpha1.
2. method according to claim 1, is characterized in that: described in step a, the step of synthetic polypeptide fragment 1 is:
Under coupling reagent exists, by Fmoc-Glu (OtBu)-OH and vector resin coupling, de-Fmoc protecting group obtains H-Glu (OtBu)-vector resin again, then adopt activating reagent and condensation reagent, according to the order of holding N end from C successively coupling Fmoc-Lys (Boc)-OH one by one, Fmoc-Lys (Boc)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Thr (tBu)-OH, after coupling, use lysate cracking, remove vector resin and obtain polypeptide fragment 1:
Fmoc-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-OH。
3. method according to claim 1, is characterized in that: described in step a, the step of synthetic polypeptide fragment 2 is:
Under coupling reagent exists; by the aminoresin coupling after Fmoc-Asp (α-tBu)-OH and deprotection; de-Fmoc protecting group obtains H-Asp (α-tBu)-aminoresin; then adopt activating reagent and condensation reagent; according to the order of holding N end from C successively coupling Fmoc-Glu (OtBu)-OH, Fmoc-Ala-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Val-OH, Fmoc-Val-OH one by one, the Fmoc protecting group that removes N end obtains polypeptide fragment 2:
NH 2-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asp (α-OtBu)-aminoresin.
4. method according to claim 1, is characterized in that: the step of step c is:
Adopt activating reagent and condensation reagent, first the C end of Fmoc-Ile-OH is held after coupling with the N of polypeptide resin I, de-Fmoc protecting group obtains H-Ile-polypeptide resin I, then according to the order of holding N end from C successively coupling Fmoc-Glu (OtBu)-OH one by one, Fmoc-Ser (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Val-OH, Fmoc-Aal-OH, Fmoc-Aal-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Ser (tBu)-OH, after coupling, remove the Fmoc protecting group of N end, carry out obtaining after acetylization reaction Thymosin-Alpha1 resin:
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (α-OtBu)-aminoresin.
5. according to the method described in claim 1,3 or 4 any one, it is characterized in that: described aminoresin is selected from any one in Rink mbha resin, Rink Amide AM resin and Rink Amide resin; Be preferably Rink Amide mbha resin.
6. method according to claim 2, is characterized in that: described vector resin is selected from Trityl-Cl resinoid; Be preferably 2-Cl Trity-Cl resin.
7. method according to claim 2, is characterized in that: described lysate is the dichloromethane solution of the trifluoroethanol of volume percent 25%.
8. according to the method described in claim 1～4 any one, it is characterized in that: described condensation reagent is N, N-DIC, N, N-dicyclohexylcarbodiimide, phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus/N, N-diisopropylethylamine, 2-(7-azepine-1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyl-urea phosphofluoric acid ester/N, N-diisopropylethylamine, benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate/N, N-diisopropylethylamine or O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid ester/N, any one in N-diisopropylethylamine.
9. according to the method described in claim 1～4 any one, it is characterized in that: described activating reagent is I-hydroxybenzotriazole or N-hydroxyl-7-azepine benzotriazole.
10. method according to claim 1, is characterized in that: the acidolysis described in steps d is that to adopt the TFA that is 80～95% by volume percent, EDT, the surplus that volume percent is 1-10% be that the mixing acid hydrolysis solution that water forms carries out acidolysis.
CN201410333844.8A 2014-07-14 2014-07-14 Method for synthesizing thymalfasin Pending CN104098688A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410333844.8A CN104098688A (en) 2014-07-14 2014-07-14 Method for synthesizing thymalfasin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410333844.8A CN104098688A (en) 2014-07-14 2014-07-14 Method for synthesizing thymalfasin

Publications (1)

Publication Number Publication Date
CN104098688A true CN104098688A (en) 2014-10-15

Family

ID=51667264

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410333844.8A Pending CN104098688A (en) 2014-07-14 2014-07-14 Method for synthesizing thymalfasin

Country Status (1)

Country Link
CN (1) CN104098688A (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107417786A (en) * 2017-05-04 2017-12-01 苏州强耀生物科技有限公司 A kind of preparation method of Thymosin alpha 1
CN108218980A (en) * 2016-12-21 2018-06-29 鲁南制药集团股份有限公司 A kind of synthetic method of thymalfasin
CN108314725A (en) * 2018-04-13 2018-07-24 成都圣诺生物制药有限公司 A kind of synthetic method of thymalfasin
WO2020124975A1 (en) * 2018-12-20 2020-06-25 深圳翰宇药业股份有限公司 Method for preparing thymalfasin
CN113461775A (en) * 2021-08-23 2021-10-01 成都诺和晟泰生物科技有限公司 Preparation method of polypeptide compound
CN116199765A (en) * 2022-12-29 2023-06-02 江苏诺泰澳赛诺生物制药股份有限公司 Method for synthesizing thymus by using modified dipeptide

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101104638A (en) * 2007-06-18 2008-01-16 苏州中科天马肽工程中心有限公司 Solid phase synthetic technique for thymosin alpha1
CN102199205A (en) * 2011-03-22 2011-09-28 海南双成药业股份有限公司 Synthetic method of polypeptide thymosin alpha1
CN103497245A (en) * 2013-06-25 2014-01-08 深圳翰宇药业股份有限公司 Method for synthesizing thymalfasin
CN103880945A (en) * 2013-12-28 2014-06-25 郑州大明药物科技有限公司 Method for preparing high-purity thymalfasin

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101104638A (en) * 2007-06-18 2008-01-16 苏州中科天马肽工程中心有限公司 Solid phase synthetic technique for thymosin alpha1
CN102199205A (en) * 2011-03-22 2011-09-28 海南双成药业股份有限公司 Synthetic method of polypeptide thymosin alpha1
CN103497245A (en) * 2013-06-25 2014-01-08 深圳翰宇药业股份有限公司 Method for synthesizing thymalfasin
CN103880945A (en) * 2013-12-28 2014-06-25 郑州大明药物科技有限公司 Method for preparing high-purity thymalfasin

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
刘玉英等: "胸腺肽α1的研究进展", 《上海医药》 *
张鸿鹏: "胸腺素α1的固相片段合成及鉴定分析", 《中国优秀硕士学位论文全文数据库》 *
韩香等: "多肽的固相合成", 《天津药学》 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108218980A (en) * 2016-12-21 2018-06-29 鲁南制药集团股份有限公司 A kind of synthetic method of thymalfasin
CN108218980B (en) * 2016-12-21 2020-12-08 鲁南制药集团股份有限公司 Synthesis method of thymalfasin
CN107417786A (en) * 2017-05-04 2017-12-01 苏州强耀生物科技有限公司 A kind of preparation method of Thymosin alpha 1
CN107417786B (en) * 2017-05-04 2021-03-09 苏州强耀生物科技有限公司 Preparation method of thymosin alpha 1
CN108314725A (en) * 2018-04-13 2018-07-24 成都圣诺生物制药有限公司 A kind of synthetic method of thymalfasin
WO2020124975A1 (en) * 2018-12-20 2020-06-25 深圳翰宇药业股份有限公司 Method for preparing thymalfasin
CN113461775A (en) * 2021-08-23 2021-10-01 成都诺和晟泰生物科技有限公司 Preparation method of polypeptide compound
CN116199765A (en) * 2022-12-29 2023-06-02 江苏诺泰澳赛诺生物制药股份有限公司 Method for synthesizing thymus by using modified dipeptide

Similar Documents

Publication Publication Date Title
CN103497245B (en) Method for synthesizing thymalfasin
CN104098688A (en) Method for synthesizing thymalfasin
WO2018032521A1 (en) Method for synthesizing liraglutide
CN104004083B (en) A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
CN106146648B (en) Synthetic method of parathyroid hormone analogue
CN103224558B (en) A kind of preparation method of Exenatide
CN104987382A (en) Method for preparing thymalfasin through dipeptide fragment liquid-solid bonding
CN101104638B (en) Solid phase synthetic technique for thymosin alpha1
CN112679602A (en) Solid-phase synthesis method of Somaloutide
CN108047329A (en) A kind of preparation method of A Bapa peptides
CN104788546A (en) Preparation method of linear peptides containing 24 amino acid residues
CN104844706B (en) A method of synthesis lixisenatide
CN112125970A (en) Synthesis method of semaglutide
CN109180803B (en) Somatostatin and preparation method and pharmaceutical composition thereof
CN103265620B (en) Somatostatin and preparation method thereof
CN109306366B (en) Method for synthesizing PT141
CN113412272A (en) Improved process for preparing procatide
CN115746102A (en) Method for preparing Bulevirtide by combining solid phase with liquid phase
CN113754753A (en) Synthetic method of somaglutide
CN103694336B (en) A kind of solid liquid phase fragment condensation prepares the method for Thymosin alpha 1
Dalcol et al. Convergent solid phase peptide synthesis: an efficient approach to the synthesis of highly repetitive protein domains
CN108314725A (en) A kind of synthetic method of thymalfasin
CN112125971B (en) Method for rapidly synthesizing semaglutide by ultrasonic wave
CN109912709B (en) Preparation method of acid-sensitive ion channel inhibitor
CN114249810A (en) Synthetic method of somaglutide

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C41 Transfer of patent application or patent right or utility model
TA01 Transfer of patent application right

Effective date of registration: 20160307

Address after: 611330 section, Industrial Avenue, Dayi County, Sichuan, Chengdu

Applicant after: Chengdu Shengnuo Biopharm Co., Ltd.

Address before: 611330, No. 258, snow capped Avenue, Dayi County, Chengdu, Sichuan

Applicant before: Chengdu Shengnuo Biotec Co., Ltd.

RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20141015