CN103923210A - Solid-phase synthesis method of thymalfasin - Google Patents

Solid-phase synthesis method of thymalfasin Download PDF

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Publication number
CN103923210A
CN103923210A CN201310013306.6A CN201310013306A CN103923210A CN 103923210 A CN103923210 A CN 103923210A CN 201310013306 A CN201310013306 A CN 201310013306A CN 103923210 A CN103923210 A CN 103923210A
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China
Prior art keywords
resin
thymosin
alpha1
fmoc
glu
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Chinese (zh)
Inventor
赵志全
李铁健
颜凯
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Shandong New Time Pharmaceutical Co Ltd
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Shandong New Time Pharmaceutical Co Ltd
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Priority to CN201310013306.6A priority Critical patent/CN103923210A/en
Publication of CN103923210A publication Critical patent/CN103923210A/en
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    • 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

Abstract

The invention relates to a solid-phase synthesis method of thymalfasin. The solid-phase synthesis method comprises the process steps: solid-phase linear synthesis of linear thymalfasin is carried out, a DMF solution of a salt composed of bulky anions and monovalence cations is used as a reaction solvent in a difficult sequence, hydrogen bond formation is interfered, and a beta structure of a peptide chain is destroyed. The process can overcome the polypeptide synthesized difficult sequence, improves the coupling rate of the difficult sequence so as to improve the yield of thymalfasin, reduces the production cost, and is beneficial for industrialized production.

Description

A kind of process for solid phase synthesis of Thymosin-Alpha1
Technical field
The present invention relates to Peptides Synthesis, refer more particularly to the solid phase synthesis process of Thymosin-Alpha1.
Background technology
Thymosin-Alpha1 is name thymosin α1 Thymosin alpha 1 once, first from Zadaxin component 5, is isolated and is determined that its primary structure is made up of 28 amino acid by Goldstein in 1977, and N holds acetylize, and molecular mass is 3108Da, iso-electric point 4.2.Thymosin-Alpha1 is a kind of Novel immune regulatory factor, it is converted into mature T lymphocyte by induced dry-cell, stimulates lymphsystem, improves body's immunity, apply clinically the immunologic function that Thymosin-Alpha1 improves T cell, treatment viral hepatitis and the generation and the development that delay some senile disease.Thymosin-Alpha1 is anti-infective, antiviral and antineoplastic effect in addition, especially the immunity of cancer is treated to also tool and has certain effect.
Thymosin-Alpha1 1997 is by the exploitation listing of raw (Sciclone) company of Italy's match, and trade name " Zadaxin " (Zadaxin).As a kind of cellular immunization toughener, be mainly used in clinically: 1. the treatment 3. that the treatment 2. that is applied to chronic viral hepatitis is applied to tumour is applied to immunologic hypofunction crowd as vaccine toughener
Thymosin-Alpha1 solid phase synthesis has mainly contained two kinds, and one is Boc strategy, and one is Fmoc strategy.Boc strategy is owing to using hypertoxic HF, less use now.There is more report about Fmoc strategy, as the 13rd the 3rd phase of volume of " Tianjin pharmacy " calendar year 2001 " the novel solid phase method synthesizing thymosins of Fmoc α 1 and reaction path thereof " has described taking HMP resin as carrier, the method for synthetic Thymosin-Alpha1 taking DCC-HOBT as couplant; " Journal of Chemical Industry and Engineering " the 55th the 2nd phase of volume in 2004 " the DIC solid state chemistry of thymosin α1 synthesizes and qualification " has described taking king's resin as carrier, the method for synthetic Thymosin-Alpha1 taking DIC-HOBT as couplant; Patent CN101104638A has described the method with Fmoc-RinkAmideMBHA resin and the synthetic Thymosin-Alpha1 of Fmoc-Asp-X; Patent CN102199205A has described the preparation taking hydroxy functional group resin as carrier, the method for synthetic Thymosin-Alpha1 taking DIC-HOBT as couplant.But due to the existence of difficult sequences (from C end in peptide chain length greatly about 6-16 amino acid whose sequence), make that these methods exist that yield is low, cost is high, impurity and product polarity too near and cannot obtain the problems such as high purity product.And these methods are mainly round the selection of resin, coupled modes, purification strategy.
The implication of abbreviation used in the present invention or English full name sees following table:
English is write a Chinese character in simplified form Chinese
Fmoc 9-fluorenylmethyloxycarbonyl
DMAP To Dimethylamino pyridine
DIC N, N-DIC
HOBT 1-hydroxy benzo triazole
DIEA DIPEA
NMM N-methylmorpholine
Ac 2O Diacetyl oxide
TFA Trifluoroacetic acid
Tis Tri isopropyl silane
Boc Tertbutyloxycarbonyl
tBu The tertiary butyl
OtBu The oxygen tertiary butyl
Trt Trityl
H 2O Water
Summary of the invention
The object of the invention is to solve the problems referred to above that prior art exists, provide that a kind of yield is higher, cost is lower, be more conducive to the Thymosin-Alpha1 solid phase synthesis process of suitability for industrialized production.
The invention provides a kind ofly suc as formula the Thymosin-Alpha1 process for solid phase synthesis shown in I, comprise the following steps:
1) synthetic Fmoc-Asn (Trt)-king's resin
Fmoc-Asn (Trt)-OH is dissolved in to DMF and substitution value is 0.3mmol/g-1.0mmol/g king's mixed with resin, obtains Fmoc-Asn (Trt)-king's resin through esterification;
2) all the other 27 amino acid whose couplings
Taking gained Fmoc-Asn (Trt)-king's resin in step 1) as substrate, taking DIC as coupling reagent, DMF are as reaction solvent, adopt Fmoc strategy, all the other 27 amino acid suc as formula I, with ordinary method successively solid phase synthesis, are obtained to the Thymosin-Alpha1 resin of the Fmoc protection shown in formula II;
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
I
FmocSer 1(tBu)Asp 2(OtBu)Ala 3Ala 4Val 5Asp 6(OtBu)Thr 7(tBu)Ser 8(tBu)Ser 9(tBu)Glu 10(OtBu)Ile 11Thr 12(tBu)Thr 13(tBu)Lys 14(Boc)Asp 15(OtBu)Leu 16Lys 17(Boc)Glu 18(OtBu)Lys 19(Boc)Lys 20(Boc)Glu 21(OtBu)Val 22Val 23Glu 24(OtBu)Glu 25(OtBu)Ala 26Glu 27(OtBu)Asn 28-Wang?Resin
II
Wherein, from C end, when 6-16 amino acid whose coupling, in solution, add KSCN, NaClO 4or LiCl;
3) by 2) in after the de-Fmoc protection of N terminal amino acid of Thymosin-Alpha1 resin of gained Fmoc protection, acetylize N hold amino, obtains Thymosin-Alpha1 resin;
4) by 3) in gained Thymosin-Alpha1 resin cut with cutting reagent, filtering resin, filtrate is through precipitating and obtaining the thick peptide of Thymosin-Alpha1;
5) by 4) in the thick peptide of gained carry out purifying, freeze-drying, obtain Thymosin-Alpha1.
Wherein, step 1) in esterification be that Fmoc-Asn (Trt)-OH, king's resin, DMAP, DIC are mixed and carried out in DMF.
Contriver finds by lot of experiments, in step 2) in, the amino acid in difficult sequences fragment adds the salt of bulky negatively charged ion and 1 valency cation composition can greatly improve productive rate while coupling; The preferred KSCN of described salt, NaClO 4or LiCl; The mode that adds of preferably salt is the DMF solution of salt; The DMF strength of solution of salt is 0.1mol/L-1mol/L; Preferably 0.4-0.6mol/L, further preferred NaClO 4dMF solution, strength of solution is preferably 0.4mol/L.
In step 3) when acetylize by diacetyl oxide and organic bases to amino acidylate.Wherein at least one in the preferred NMM of organic bases, DIEA; The amount of substance consumption of diacetyl oxide is 5-10 times of king's resin replacement amount, and the amount of substance consumption of organic bases is 1-5 times of king's resin replacement amount.Replacement amount equals the charging capacity of king's resin and king's resin substitution value is long-pending.
Step 4) cutting reagent is by TFA, Tis and H 2o is that 95:2.5:2.5 is formulated according to volume ratio; On cutting reagent volumetric usage numerical value, equal the 2-12 of king's resin quality consumption doubly; Cutting is to react 2-3 hour under ice-water bath; Filtering resin, adds in ice ether and precipitates after filtrate is concentrated.
Described difficult sequences refers to that at peptide chain length be approximately that the 23rd formula I is to the 13rd at 6-16(from C end) aminoacid sequence.Due to this section of sequence when coupling very easily occur in molecule or intermolecular hydrogen bond to form and form beta sheet be main secondary structure, in addition, in solid phase synthesis, also there is the molecular aggregates phenomenon between peptide chain and resin.β-corner and beta sheet make the N end of peptide molecule be buried the depths in secondary structure, carry out coupling reaction with regard to being difficult to carboxyl like this, still extend the reaction times and even repeat coupling and all cannot obtain satisfied result by increasing the protected amino acid multiple that feeds intake, coupling efficiency is obviously reduced, and the chromatographic purity of thick peptide is poor, the highlyest can only reach 60%, yield is the highest can only reach 35%, and the impurity of main peak front and back is more, is difficult for purifying, purity is difficult to reach 98%, and yield is not high; And the present invention uses the DMF solution of bulky negatively charged ion and 1 valency cation composition salt to interrupt β-pleated sheet structure by destroying hydrogen bond, make resin and peptide chain become unordered curling and sufficient solvation from assembling to be retracted to, make to need the N end of coupling fully to expose, the efficiency of coupling improves greatly, and crude product yield can reach more than 45%; More excellent part is, the impurity before and after main peak greatly reduces, and is conducive to be further purified, and makes the purity of finished product more than 99%, and total recovery is corresponding improve also.
Testing conditions for the product after Thymosin-Alpha1 crude product and purifying is: with octadecylsilane chemically bonded silica be weighting agent; Taking acetonitrile-water-phosphoric acid (140:860:1) as mobile phase A, taking acetonitrile-water-phosphoric acid (250:750:1) as Mobile phase B; Detection wavelength is 210nm.According to the form below carries out gradient elution:
Time (minute) Moving phase (A%) Moving phase (B%)
0 100 0
30 50 50
31 0 100
35 0 100
35.1 100 0
45 100 0
Wherein, the HPLC purity of crude product is that area normalization method is calculated and obtained; Content adopts external standard method.
Compared with traditional technology, the present invention has following excellent effect:
1. the present invention adopts Fmoc solid phase synthesis strategy to synthesize Thymosin-Alpha1, when amino acid in difficult sequences is coupled, the DMF solution that uses the salt of bulky negatively charged ion and 1 valency cation composition is reaction solvent, overcome difficult sequences, reaction yield is improved, and finished product yield can reach more than 25%.
2. technical solution of the present invention is easy to chromatographic separation, has significantly improved the purity of product.The chromatographic purity of thick peptide can reach more than 45%; After purifying, can reach more than 99%.
The assorted peak of chromatogram of the thick peptide of gained of the present invention reduces, and the assorted peak, main peak front and back that particularly affects separation and purification obviously reduces, thereby is easy to chromatographic separation, and the purity after separation and purification is reached more than 99%.
3. in technical solution of the present invention, acetylize adopts DIEA or NMM and diacetyl oxide, has avoided that toxicity is large, stench, carcinogenic pyridine, is more conducive to producers' labour protection, and has greatly alleviated environmental protection pressure.
4. reduced cost, step is simple and easy, is easy to industrialization.
Brief description of the drawings
The schema of Fig. 1 solid phase synthesis Thymosin-Alpha1.
Embodiment
Following content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention; without departing from the inventive concept of the premise; can also make some simple deduction or replace; all should be considered as belonging to protection scope of the present invention; the present invention uses but the technology and the indexing section that do not describe, is prior art.
Below technical solution of the present invention is described in further detail.
(1) synthetic Fmoc-Asn (Trt)-king's resin
The resin that is 0.3mmol/g-1.0mmol/g by substitution value adds Solid-phase Polypeptide composite tube, with the swelling 1-2 hour of DMF, add Fmoc-Asn (Trt)-OH, DIC, HOBt, DMAP, make solvent with DMF, room temperature reaction 3-6 hour, drain reaction solution, use respectively DMF washing resin three times, obtain Fmoc-Asn (Trt)-WangResin;
(2) all the other 27 amino acid whose couplings
By gained Fmoc-Asn (Trt)-WangResin in step 1), with the de-Fmoc protection of 20% piperidines/DMF solution, with DMF washing 6 times, add Fmoc-Glu (OtBu)-OH, HOBT, DIC DMF to make solvent, room temperature reaction 2-4 hour; Take out after reaction solution with after DMF washing 6 times and remove Fmoc, repeat circulation above, complete successively all the other 26 amino acid whose solid phase synthesis, obtain the Thymosin-Alpha1 resin of Fmoc protection.When 6-16 amino acid is coupled from C end, use the DMF solution of bulky negatively charged ion and 1 valency cation composition salt as solvent.
(3) acetylize of N-terminal Ser
By after de-the N terminal amino acid of the Thymosin-Alpha1 resin of gained Fmoc protection in (2) Fmoc protection; add diacetyl oxide and organic bases room temperature reaction 2-3 hour; acetylize N holds amino; take out reaction reagent; with DMF washing 3 times; with methyl alcohol shrinkage resin 3 times, vacuum decompression is dry, obtains Thymosin-Alpha1 resin.
(4) cutting resin
Thymosin-Alpha1 resin is joined in round-bottomed flask, add cutting reagent (by TFA, Tis and H 2o is that 95:2.5:2.5 is formulated according to volume ratio), under ice-water bath, react 2-3 hour.Filter resin filtrate is separated, after filtrate is concentrated, adds in ice ether and precipitate, filter, after ice ether washing leaching cake 3 times, after drying under reduced pressure, obtain the thick peptide of Thymosin-Alpha1.
(5) purifying
Concrete purge process can be of the prior art any one.Can adopt preparative chromatography to carry out purifying, as adopted the RP-HPLC purification process in CN101104638B.
Flow process is referring to accompanying drawing 1.
embodiment 1
1) Fmoc-Asn (Trt)-king's resin is synthetic
Taking substitution value is that 0.4mmol/g king's resin 10g 100mlDMF drains after swelling 2 hours, add the 100mlDMF solution containing Fmoc-Asn (Trt)-OH7.2g, DIC1.9ml, HOBT1.7g, DMAP0.3g, room temperature reaction 3 hours, drain reaction solution, use respectively 100mlDMF washing resin three times, obtain Fmoc-Asn (Trt)-king's resin.
2) coupling
By gained Fmoc-Asn (Trt)-WangResin in step 1); use respectively twice of the de-Fmoc protection of 100ml20% piperidines/DMF solution; then use respectively 100mlDMF washing resin 6 times; then the DMF reaction soln 100ml that adds Fmoc-Glu (OtBu)-OH5.1g and contain DIC1.9ml, HOBT1.7g; reaction at 28 DEG C, the basis for estimation using triketohydrindene hydrate color reaction as reaction end.After reaction finishes, drain reaction solution, use DMF washing resin 3 times.Repeat above-mentioned steps, by all the other 26 amino acid solid phase synthesis successively suc as formula I, obtain the Thymosin-Alpha1 resin of the Fmoc protection shown in formula II; Wherein, coupling is from C end when 6-16 amino acid, and DMF reaction soln dissolves in 0.4mmol KSCN.
3) by step 2) in after the de-Fmoc protection of N terminal amino acid of Thymosin-Alpha1 resin of gained Fmoc protection; add 12ml diacetyl oxide, 8mlDIEA, 100mlDMF acetylize 2 hours, DMF washing resin 3 times, methyl alcohol shrinkage resin 3 times; be dried to constant weight, obtain Thymosin-Alpha1 resin.
4) cutting
Gained Thymosin-Alpha1 resin in step 3) is added to round-bottomed flask, add by TFA, Tis and H 2o is the formulated cutting reagent 260ml of 95:2.5:2.5 according to volume ratio, stirring reaction 3 hours under ice bath, filtering resin, filtrate is concentrated in the ice ether that adds 300ml after 50ml to be precipitated, filter, ice ether washing leaching cake three times, drying under reduced pressure obtains the thick peptide of Thymosin-Alpha1.Thick peptide HPLC(area normalization method) purity is 63.4%, content (external standard method) 15.3%, yield is 46.7%.
5) purifying
By 4) in the thick peptide of gained carry out purifying by preparative chromatography, obtain Thymosin-Alpha1 product, its purity is 99.1%, total recovery 28.02%.
embodiment 2
1) Fmoc-Asn (Trt)-king's resin is synthetic
Taking substitution value is that 0.4mmol/g king's resin 10g 100mlDMF drains after swelling 2 hours, add the 100mlDMF solution containing Fmoc-Asn (Trt)-OH7.2g, DIC1.9ml, HOBT1.7g, DMAP0.3g, room temperature reaction 3 hours, drain reaction solution, use respectively 100mlDMF washing resin three times, obtain Fmoc-Asn (Trt)-king's resin.
2) coupling
By gained Fmoc-Asn (Trt)-WangResin in step 1); use respectively twice of the de-Fmoc protection of 100ml20% piperidines/DMF solution; then use respectively 100mlDMF washing resin 6 times; then the DMF reaction soln 100ml that adds Fmoc-Glu (OtBu)-OH5.1g and contain DIC1.9ml, HOBT1.7g; reaction at 28 DEG C, the basis for estimation using triketohydrindene hydrate color reaction as reaction end.After reaction finishes, drain reaction solution, use DMF washing resin 3 times.Repeat above-mentioned steps, by all the other 26 amino acid solid phase synthesis successively suc as formula I, obtain the Thymosin-Alpha1 resin of the Fmoc protection shown in formula II; Wherein, coupling is from C end when 6-16 amino acid, and DMF reaction soln dissolves in 0.04mmol KSCN.
3) by step 2) in after the de-Fmoc protection of N terminal amino acid of Thymosin-Alpha1 resin of gained Fmoc protection; add 12ml diacetyl oxide, 8mlDIEA, 100mlDMF acetylize 2 hours, DMF washing resin 3 times, methyl alcohol shrinkage resin 3 times; be dried to constant weight, obtain Thymosin-Alpha1 resin.
4) cutting
Gained Thymosin-Alpha1 resin in step 3) is added to round-bottomed flask, add by TFA, Tis and H 2o is the formulated cutting reagent 260ml of 95:2.5:2.5 according to volume ratio, stirring reaction 3 hours under ice bath, filtering resin, filtrate is concentrated in the ice ether that adds 600ml after 50ml to be precipitated, filter, ice ether washing leaching cake three times, drying under reduced pressure obtains the thick peptide of Thymosin-Alpha1.Thick peptide HPLC(area normalization method) purity is 71.4%, and content (external standard method) is 21.7%, and yield is 48.6%.
5) purifying
By 4) in the thick peptide of gained carry out purifying by preparative chromatography, obtain Thymosin-Alpha1 product, its purity is 99.2%, total recovery 29.36%.
embodiment 3
1) Fmoc-Asn (Trt)-king's resin is synthetic
Taking substitution value is that 0.5mmol/g king's resin 10g 100mlDMF drains after swelling 2 hours, add the 100mlDMF solution containing Fmoc-Asn (Trt)-OH9.0g, DIC2.3ml, HOBT2.0g, DMAP0.4g, room temperature reaction 3 hours, drain reaction solution, use respectively 100mlDMF washing resin three times, obtain Fmoc-Asn (Trt)-king's resin.
2) coupling
By gained Fmoc-Asn (Trt)-WangResin in step 1); use respectively twice of the de-Fmoc protection of 100ml20% piperidines/DMF solution; then use respectively 100mlDMF washing resin 6 times; then the DMF reaction soln 100ml that adds Fmoc-Glu (OtBu)-OH6.4g and contain DIC2.3ml, HOBT2.0g; reaction at 28 DEG C, the basis for estimation using triketohydrindene hydrate color reaction as reaction end.After reaction finishes, drain reaction solution, use DMF washing resin 3 times.Repeat above-mentioned steps, by all the other 26 amino acid solid phase synthesis successively suc as formula I, obtain the Thymosin-Alpha1 resin of the Fmoc protection shown in formula II; Wherein, coupling is from C end when 6-16 amino acid, and DMF reaction soln dissolves in 0.05mmolNaClO 4.
3) by step 2) in after the de-Fmoc protection of N terminal amino acid of Thymosin-Alpha1 resin of gained Fmoc protection; add 25ml diacetyl oxide, 15mlNMM, 100mlDMF acetylize 2 hours, DMF washing resin 3 times, methyl alcohol shrinkage resin 3 times; be dried to constant weight, obtain Thymosin-Alpha1 resin.
4) cutting
Gained Thymosin-Alpha1 resin in step 3) is added to round-bottomed flask, add by TFA, Tis and H 2o is the formulated cutting reagent 300ml of 95:2.5:2.5 according to volume ratio, stirring reaction 3 hours under ice bath, filtering resin, filtrate is concentrated in the ice ether that adds 800ml after 60ml to be precipitated, filter, ice ether washing leaching cake three times, drying under reduced pressure obtains the thick peptide of Thymosin-Alpha1.Thick peptide HPLC(area normalization method) purity is 78.6%, and content (external standard method) is 29.2%, and yield is 52.6%.
5) purifying
By 4) in the thick peptide of gained carry out purifying by preparative chromatography, obtain Thymosin-Alpha1 product, its purity is 99.8%, total recovery 33.91%.
embodiment 4
1) Fmoc-Asn (Trt)-king's resin is synthetic
Taking substitution value is that 0.5mmol/g king's resin 10g 100mlDMF drains after swelling 2 hours, add the 100mlDMF solution containing Fmoc-Asn (Trt)-OH9.0g, DIC2.3ml, HOBT2.0g, DMAP0.4g, room temperature reaction 3 hours, drain reaction solution, use respectively 100mlDMF washing resin three times, obtain Fmoc-Asn (Trt)-king's resin.
2) coupling
By gained Fmoc-Asn (Trt)-WangResin in step 1); use respectively twice of the de-Fmoc protection of 100ml20% piperidines/DMF solution; then use respectively 100mlDMF washing resin 6 times; then the DMF reaction soln 100ml that adds Fmoc-Glu (OtBu)-OH7.0g and contain DIC1.9ml, HOBT2.0g; reaction at 28 DEG C, the basis for estimation using triketohydrindene hydrate color reaction as reaction end.After reaction finishes, drain reaction solution, use DMF washing resin 3 times.Repeat above-mentioned steps, by all the other 26 amino acid solid phase synthesis successively suc as formula I, obtain the Thymosin-Alpha1 resin of the Fmoc protection shown in formula II; Wherein, coupling is from C end when 6-16 amino acid, and DMF reaction soln dissolves in 0.05mmolNaClO 4.
3) by step 2) in after the de-Fmoc protection of N terminal amino acid of Thymosin-Alpha1 resin of gained Fmoc protection; add 20ml diacetyl oxide, 15mlNMM, 100mlDMF acetylize 2 hours, DMF washing resin 3 times, methyl alcohol shrinkage resin 3 times; be dried to constant weight, obtain Thymosin-Alpha1 resin.
4) cutting
Gained Thymosin-Alpha1 resin in step 3) is added to round-bottomed flask, add by TFA, Tis and H 2o is the formulated cutting reagent 280ml of 95:2.5:2.5 according to volume ratio, stirring reaction 3 hours under ice bath, filtering resin, filtrate is concentrated in the ice ether that adds 600ml after 50ml to be precipitated, filter, ice ether washing leaching cake three times, drying under reduced pressure obtains the thick peptide of Thymosin-Alpha1.Thick peptide HPLC(area normalization method) purity is 73.4%, and content (external standard method) is 26.7%, and yield is 48.2%.
5) purifying
By 4) in the thick peptide of gained carry out purifying by preparative chromatography, obtain Thymosin-Alpha1 product, its purity is 99.6%, total recovery 29.3%.
embodiment 5
1) Fmoc-Asn (Trt)-king's resin is synthetic
Taking substitution value is that 0.6mmol/g king's resin 10g 100mlDMF drains after swelling 2 hours, add the 100mlDMF solution containing Fmoc-Asn (Trt)-OH10.7g, DIC2.8ml, HOBT2.5g, DMAP0.5g, room temperature reaction 3 hours, drain reaction solution, use respectively 100mlDMF washing resin three times, obtain Fmoc-Asn (Trt)-king's resin.
2) coupling
By gained Fmoc-Asn (Trt)-WangResin in step 1); use respectively twice of the de-Fmoc protection of 100ml20% piperidines/DMF solution; then use respectively 100mlDMF washing resin 6 times; then the DMF reaction soln 100ml that adds Fmoc-Glu (OtBu)-OH7.7g and contain DIC2.8ml, HOBT2.5g; reaction at 28 DEG C, the basis for estimation using triketohydrindene hydrate color reaction as reaction end.After reaction finishes, drain reaction solution, use DMF washing resin 3 times.Repeat above-mentioned steps, by all the other 26 amino acid solid phase synthesis successively suc as formula I, obtain the Thymosin-Alpha1 resin of the Fmoc protection shown in formula II; Wherein, coupling is from C end when 6-16 amino acid, and DMF reaction soln dissolves in 0.06mmolNaClO 4.
3) by step 2) in after the de-Fmoc protection of N terminal amino acid of Thymosin-Alpha1 resin of gained Fmoc protection; add 36ml diacetyl oxide, 24mlNMM, 100mlDMF acetylize 2 hours, DMF washing resin 3 times, methyl alcohol shrinkage resin 3 times; be dried to constant weight, obtain Thymosin-Alpha1 resin.
4) cutting
Gained Thymosin-Alpha1 resin in step 3) is added to round-bottomed flask, add by TFA, Tis and H 2o is the formulated cutting reagent 300ml of 95:2.5:2.5 according to volume ratio, stirring reaction 3 hours under ice bath, filtering resin, filtrate is concentrated in the ice ether that adds 500ml after 50ml to be precipitated, filter, ice ether washing leaching cake three times, drying under reduced pressure obtains the thick peptide of Thymosin-Alpha1.Thick peptide HPLC(area normalization method) purity is 69.6%, and content (external standard method) is 22.8%, and yield is 47.2%.
5) purifying
By 4) in the thick peptide of gained carry out purifying by preparative chromatography, obtain Thymosin-Alpha1 product, its purity is 99.2%, total recovery 28.52%.
embodiment 6
1) Fmoc-Asn (Trt)-king's resin is synthetic
Taking substitution value is that 1.0mmol/g king's resin 10g 100mlDMF drains after swelling 2 hours, add the 100mlDMF solution containing Fmoc-Asn (Trt)-OH17.9g, DIC4.6ml, HOBT4.1g, DMAP0.7g, room temperature reaction 3 hours, drain reaction solution, use respectively 100mlDMF washing resin three times, obtain Fmoc-Asn (Trt)-king's resin.
2) coupling
By gained Fmoc-Asn (Trt)-WangResin in step 1); use respectively twice of the de-Fmoc protection of 100ml20% piperidines/DMF solution; then use respectively 100mlDMF washing resin 6 times; then the DMF reaction soln 100ml that adds Fmoc-Glu (OtBu)-OH12.8g and contain DIC4.6ml, HOBT4.1g; reaction at 28 DEG C, the basis for estimation using triketohydrindene hydrate color reaction as reaction end.After reaction finishes, drain reaction solution, use DMF washing resin 3 times.Repeat above-mentioned steps, by all the other 26 amino acid solid phase synthesis successively suc as formula I, obtain the Thymosin-Alpha1 resin of the Fmoc protection shown in formula II; Wherein, coupling is from C end when 6-16 amino acid, and DMF reaction soln dissolves in 0.04mmol LiCl.
3) by step 2) in after the de-Fmoc protection of N terminal amino acid of Thymosin-Alpha1 resin of gained Fmoc protection; add 60ml diacetyl oxide, 50ml pyridine, 100mlDMF acetylize 2 hours, DMF washing resin 3 times, methyl alcohol shrinkage resin 3 times; be dried to constant weight, obtain Thymosin-Alpha1 resin.
4) cutting
Gained Thymosin-Alpha1 resin in step 3) is added to round-bottomed flask, add by TFA, Tis and H 2o is the formulated cutting reagent 300ml of 95:2.5:2.5 according to volume ratio, stirring reaction 3 hours under ice bath, filtering resin, filtrate is concentrated in the ice ether that adds 600ml after 50ml to be precipitated, filter, ice ether washing leaching cake three times, drying under reduced pressure obtains the thick peptide of Thymosin-Alpha1.Thick peptide HPLC(area normalization method) purity is 68.3%, and content (external standard method) is 21.6%, and yield is 46.1%.
5) purifying
By 4) in the thick peptide of gained carry out purifying by preparative chromatography, obtain Thymosin-Alpha1 product, its purity is 99.3%, total recovery 27.51%.
comparative example
1) Fmoc-Asn (Trt)-king's resin is synthetic
Taking substitution value is that 0.5mmol/g king's resin 10g 100mlDMF drains after swelling 2 hours, add the 100mlDMF solution containing Fmoc-Asn (Trt)-OH9.0g, DIC2.3ml, HOBT2.0g, DMAP0.4g, room temperature reaction 3 hours, drain reaction solution, use respectively 100mlDMF washing resin three times, obtain Fmoc-Asn (Trt)-king's resin.
2) coupling
By gained Fmoc-Asn (Trt)-WangResin in step 1); use respectively twice of the de-Fmoc protection of 100ml20% piperidines/DMF solution; then use respectively 100mlDMF washing resin 6 times; then the DMF reaction soln 100ml that adds Fmoc-Glu (OtBu)-OH7.0g and contain DIC1.9ml, HOBT2.0g; reaction at 28 DEG C, the basis for estimation using triketohydrindene hydrate color reaction as reaction end.After reaction finishes, drain reaction solution, use DMF washing resin 3 times.Repeat above-mentioned steps, by all the other 26 amino acid solid phase synthesis successively suc as formula I, obtain the Thymosin-Alpha1 resin of the Fmoc protection shown in formula II.
3) by step 2) in after the de-Fmoc protection of N terminal amino acid of Thymosin-Alpha1 resin of gained Fmoc protection; add 25ml diacetyl oxide, 15ml pyridine, 100mlDMF acetylize 2 hours, DMF washing resin 3 times, methyl alcohol shrinkage resin 3 times; be dried to constant weight, obtain Thymosin-Alpha1 resin.
4) cutting
Gained Thymosin-Alpha1 resin in step 3) is added to round-bottomed flask, add by TFA, Tis and H 2o is the formulated cutting reagent 260ml of 95:2.5:2.5 according to volume ratio, stirring reaction 3 hours under ice bath, filtering resin, filtrate is concentrated in the ice ether that adds 500ml after 50ml to be precipitated, filter, ice ether washing leaching cake three times, drying under reduced pressure obtains the thick peptide of Thymosin-Alpha1.Thick peptide HPLC(area normalization method) purity is 59.7%, and content (external standard method) is 15.1%, and yield is 37.7%.
5) purifying
By 4) in the thick peptide of gained carry out purifying by preparative chromatography, obtain Thymosin-Alpha1 product, its purity is 97.8%, total recovery 19.91%.

Claims (10)

1. suc as formula a synthesis technique for the Thymosin-Alpha1 shown in I, this technique comprises the following steps:
1) synthetic Fmoc-Asn (Trt)-king's resin
Fmoc-Asn (Trt)-OH is dissolved in to DMF, and substitution value is 0.3mmol/g-1.0mmol/g king's mixed with resin, obtains Fmoc-Asn (Trt)-king's resin through esterification;
2) all the other 27 amino acid whose couplings
Taking gained Fmoc-Asn (Trt)-king's resin in step 1) as substrate, taking DIC as coupling reagent, DMF are as reaction solvent, adopt Fmoc strategy, all the other 27 amino acid, with ordinary method successively solid phase synthesis, are obtained to the Thymosin-Alpha1 resin of the Fmoc protection shown in formula II;
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-L
ys 17-Glu 18-Lys 19-Lys 20-Glu 21-Val 22-Val 23-Glu 24-Glu 25-Ala 26-Glu 27-Asn 28-OH
I
FmocSer 1(tBu)Asp 2(OtBu)Ala 3Ala 4Val 5Asp 6(OtBu)Thr 7(tBu)Ser 8(tBu)Ser 9(tBu)Glu 10(OtBu)
Ile 11Thr 12(tBu)Thr 13(tBu)Lys 14(Boc)Asp 15(OtBu)Leu 16Lys 17(Boc)Glu 18(OtBu)Lys 19(Boc)Lys 2
0(Boc)Glu 21(OtBu)Val 22Val 23Glu 24(OtBu)Glu 25(OtBu)Ala 26Glu 27(OtBu)Asn 28-Wang?Resin
II
Wherein, coupling when 6-16 amino acid, adds KSCN, NaClO from C end in reaction soln 4or LiCl;
3) by 2) in after the de-Fmoc protection of N terminal amino acid of Thymosin-Alpha1 resin of gained Fmoc protection, acetylize N hold amino, obtains Thymosin-Alpha1 resin;
4) by 3) in gained Thymosin-Alpha1 resin cut with cutting reagent, filtering resin, filtrate is through precipitating and obtaining the thick peptide of Thymosin-Alpha1;
5) by 4) in the thick peptide of gained carry out purifying, freeze-drying, obtain Thymosin-Alpha1.
2. the process for solid phase synthesis of Thymosin-Alpha1 according to claim 1, is characterized in that: step 2) middle KSCN, NaClO 4or LiCl adds with the form of DMF solution.
3. the process for solid phase synthesis of Thymosin-Alpha1 according to claim 2, is characterized in that: step 2) middle KSCN, NaClO 4or the DMF strength of solution of LiCl is 0.1mol/L-1mol/L.
4. the process for solid phase synthesis of Thymosin-Alpha1 according to claim 3, is characterized in that: step 2) middle KSCN, NaClO 4or the DMF strength of solution of LiCl is 0.4-0.6mol/L.
5. the process for solid phase synthesis of Thymosin-Alpha1 according to claim 4, is characterized in that: step 2) in add NaClO 4dMF strength of solution be 0.4mol/L.
6. the process for solid phase synthesis of Thymosin-Alpha1 according to claim 1, is characterized in that: when acetylize N end is amino in step 3), select diacetyl oxide and organic bases.
7. the process for solid phase synthesis of Thymosin-Alpha1 according to claim 6, is characterized in that: in step 3), organic bases is at least one in NMM, DIEA.
8. the process for solid phase synthesis of Thymosin-Alpha1 according to claim 6, is characterized in that: in step 3), the amount of substance consumption of diacetyl oxide is 5-10 times for king's resin replacement amount.
9. the process for solid phase synthesis of Thymosin-Alpha1 according to claim 6, is characterized in that: in step 3) the amount of substance consumption of organic bases be king's resin replacement amount 1-5 doubly.
10. according to the process for solid phase synthesis of the Thymosin-Alpha1 described in the arbitrary claim of claim 1-9, it is characterized in that: step 4) cutting reagent is TFA, Tis and H 2o is that 90:2.5:2.5 is formulated according to volume ratio; On cutting reagent volumetric usage numerical value, equal the 2-12 of king's resin quality consumption doubly; Cutting is to react 2-3 hour under ice-water bath; Filtrate ether sedimentation.
CN201310013306.6A 2013-01-14 2013-01-14 Solid-phase synthesis method of thymalfasin Pending CN103923210A (en)

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CN104327181A (en) * 2014-09-28 2015-02-04 上海昂博生物技术有限公司 Solid-phase synthesis of thymosin [alpha]1
CN104558149A (en) * 2015-01-22 2015-04-29 苏州天马医药集团天吉生物制药有限公司 Synthesis method of solid-phase segment of thymosin alpha1
WO2020124975A1 (en) * 2018-12-20 2020-06-25 深圳翰宇药业股份有限公司 Method for preparing thymalfasin

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104327181A (en) * 2014-09-28 2015-02-04 上海昂博生物技术有限公司 Solid-phase synthesis of thymosin [alpha]1
CN104558149A (en) * 2015-01-22 2015-04-29 苏州天马医药集团天吉生物制药有限公司 Synthesis method of solid-phase segment of thymosin alpha1
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Application publication date: 20140716