CN103880945B - The method of preparation high-purity thymalfasin - Google Patents
The method of preparation high-purity thymalfasin Download PDFInfo
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- CN103880945B CN103880945B CN201310735479.9A CN201310735479A CN103880945B CN 103880945 B CN103880945 B CN 103880945B CN 201310735479 A CN201310735479 A CN 201310735479A CN 103880945 B CN103880945 B CN 103880945B
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- NZVYCXVTEHPMHE-ZSUJOUNUSA-N thymalfasin Chemical compound CC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NZVYCXVTEHPMHE-ZSUJOUNUSA-N 0.000 title claims abstract description 78
- 108010078233 Thymalfasin Proteins 0.000 title claims abstract description 77
- 229960004231 thymalfasin Drugs 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 58
- 238000002360 preparation method Methods 0.000 title claims abstract description 41
- 239000011347 resin Substances 0.000 claims abstract description 225
- 229920005989 resin Polymers 0.000 claims abstract description 225
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 159
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 154
- 229920001184 polypeptide Polymers 0.000 claims abstract description 153
- 239000012634 fragment Substances 0.000 claims abstract description 150
- 238000006243 chemical reaction Methods 0.000 claims abstract description 54
- 239000003875 Wang resin Substances 0.000 claims abstract description 43
- 239000000047 product Substances 0.000 claims abstract description 33
- 239000012043 crude product Substances 0.000 claims abstract description 11
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000000746 purification Methods 0.000 claims abstract description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 364
- -1 Fmoc-Glu(OtBu)-OH amino acid Chemical class 0.000 claims description 84
- 238000010511 deprotection reaction Methods 0.000 claims description 66
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 60
- 238000005406 washing Methods 0.000 claims description 60
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 54
- 239000003054 catalyst Substances 0.000 claims description 48
- 230000008961 swelling Effects 0.000 claims description 45
- 239000003795 chemical substances by application Substances 0.000 claims description 39
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 38
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 36
- 150000003053 piperidines Chemical class 0.000 claims description 36
- 238000009833 condensation Methods 0.000 claims description 30
- 230000005494 condensation Effects 0.000 claims description 30
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 25
- 239000007821 HATU Substances 0.000 claims description 24
- 239000006166 lysate Substances 0.000 claims description 24
- 238000005336 cracking Methods 0.000 claims description 21
- 239000012071 phase Substances 0.000 claims description 21
- 239000000706 filtrate Substances 0.000 claims description 20
- 238000006482 condensation reaction Methods 0.000 claims description 18
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 18
- 239000002994 raw material Substances 0.000 claims description 15
- 239000000376 reactant Substances 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 12
- LJGFXAKKZLFEDR-LBPRGKRZSA-N 9h-fluoren-9-ylmethyl n-[(2s)-1-oxopropan-2-yl]carbamate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C=O)C3=CC=CC=C3C2=C1 LJGFXAKKZLFEDR-LBPRGKRZSA-N 0.000 claims description 11
- 230000006837 decompression Effects 0.000 claims description 11
- 238000004821 distillation Methods 0.000 claims description 11
- BCIPGSZQUDLGSY-INIZCTEOSA-N tert-butyl (4s)-4-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxopentanoate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C=O)C3=CC=CC=C3C2=C1 BCIPGSZQUDLGSY-INIZCTEOSA-N 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 8
- VNCNWQPIQYAMAK-ACZMJKKPSA-N Glu-Ser-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O VNCNWQPIQYAMAK-ACZMJKKPSA-N 0.000 claims description 6
- ZUUDNCOCILSYAM-KKHAAJSZSA-N Thr-Asp-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZUUDNCOCILSYAM-KKHAAJSZSA-N 0.000 claims description 6
- 239000007791 liquid phase Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 230000002441 reversible effect Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 230000021736 acetylation Effects 0.000 claims description 3
- 238000006640 acetylation reaction Methods 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 abstract description 4
- 238000010926 purge Methods 0.000 abstract 1
- 238000001291 vacuum drying Methods 0.000 abstract 1
- 235000001014 amino acid Nutrition 0.000 description 61
- 150000001413 amino acids Chemical class 0.000 description 5
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 4
- 208000002672 hepatitis B Diseases 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000003277 amino acid sequence analysis Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 230000002992 thymic effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 2
- 230000000405 serological effect Effects 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 101710142246 External core antigen Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000018528 secretion by tissue Effects 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
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
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- 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 discloses a kind of method preparing high-purity thymalfasin.Prepare polypeptide fragment one resin, polypeptide fragment two resin, polypeptide fragment three resin, polypeptide fragment four resin and polypeptide fragment five resin the most respectively；Polypeptide fragment two, three, four and five resin is cracked, obtains each thick polypeptide fragment；The thick polypeptide fragment two, three, four and five obtained is purified respectively；Each polypeptide fragment after purification is connected on polypeptide fragment one resin；Polypeptide fragment resin after connection is carried out acetylization reaction, obtains thymalfasin wang resin；Crack, obtain thymalfasin crude product；Gained thymalfasin crude product is carried out twice purifying；Collecting the flowing phase evaporated under reduced pressure containing thymalfasin in purge process, obtain thymalfasin after being centrifuged, vacuum drying obtains thymalfasin finished product.Utilize the present invention to prepare thymalfasin, be effectively shortened the reaction time, improve the quality of reaction yield and final products；The thymalfasin purity of preparation reaches more than 99%.
Description
Technical field
The present invention relates to Solid-phase synthesis peptides technical field, be specifically related to a kind of method preparing high-purity thymalfasin.
Background technology
Thymic peptide is one group of polypeptide with physiologically active of thymic tissue secretion, can promote lymphocyte transformation, strengthens macrophage phagocytic activity, can be used for treating panimmunity defect sick.
The chemical structural formula of thymalfasin is:
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH。
There are some researches show inside and outside in alone thymalfasin treatment chronic hepatitis B, alone thymalfasin treatment chronic hepatitis B, lasting response rate is about 37%, close with alone interferon, but does not interferes with the toxic and side effect of element.
Clinical research at TaiWan, China application thymalfasin treatment chronic hepatitis B shows: thymalfasin has the response to treatment of delay.After thymalfasin treatment terminates, ALT has transient rising, and simultaneously with the serological conversion of HBeAg, HBV DNA disappears.The result of study of HBV DNA negative conversion rate and serological conversion superiority shows: alone thymalfasin treatment chronic hepatitis B has long-term efficacy, and does not has obvious adverse reaction.For patients with liver fibrosis in late period, the thymalfasin result for the treatment of of high dose is preferable.
Chemical synthesis thymic peptide mainly has solid-phase synthesis at present, though the method comparative maturity, using this solid-phase synthesis is extremely difficult to synthesize polypeptide.
Summary of the invention
The technical problem to be solved in the present invention is: a kind of method providing efficient preparation high-purity thymalfasin.Utilize technical solution of the present invention to prepare thymalfasin, be effectively shortened the reaction time, improve the quality of reaction yield and final products.The thymalfasin purity utilizing technical solution of the present invention to prepare reaches more than 99%.
In order to solve the problems referred to above, the technical solution used in the present invention is:
The present invention provides a kind of method preparing high-purity thymalfasin, and described preparation method comprises the following steps:
(1), the preparation of polypeptide fragment one Asn-Glu-Ala-Glu-Glu-Val-Val-Glu-Lys-Lys-Glu resin:
A, first by Fmoc-Asn(Trt)-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, being added by step a resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Asn(Trt)-wang
Resin resin adds 8～12mL mixed liquors, uses dimethylformamide to wash after deprotection 5～8 times；
C, step b deprotection is washed after resin add in reactor, be subsequently adding Fmoc-Glu(OtBu)-OH amino acid, condensing agent HATU and condensation catalyst DIPEA, described Fmoc-Asn(Trt)-wang
Resin resin, Fmoc-Glu(OtBu)-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method successively by Fmoc-Ala-OH amino acid, Fmoc-Glu(OtBu)-OH amino acid, Fmoc-Glu(OtBu)-OH amino acid, Fmoc-Val-OH amino acid, Fmoc-Val-OH amino acid, Fmoc-Glu(OtBu)-OH amino acid, Fmoc-Lys(Boc)-OH amino acid, Fmoc-Lys(Boc)-OH amino acid and Fmoc-Glu(OtBu)-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment one Asn-Glu-Ala-Glu-Glu-Val-Val-Glu-Lys-Lys-Glu resin；
(2), the preparation of polypeptide fragment two Lys-Leu-Asp-Lys-Thr-Thr-Ile resin:
A, first by Fmoc-Lys(Boc)-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, being added by step a resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Lys(Boc)-wang
Resin resin adds 8～12mL mixed liquors, uses dimethylformamide to wash after deprotection 5～8 times；
C, the resin after step b deprotection, washing is added in reactor, is subsequently adding Fmoc-Leu-OH amino acid, condensing agent HATU and condensation catalyst DIPEA, described Fmoc-Lys(Boc)-wang
Resin resin, Fmoc-Leu-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Then products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method successively by Fmoc-Asp(OtBu)-OH amino acid, Fmoc-Lys(Boc)-OH amino acid, Fmoc-Thr(tBu)-OH amino acid, Fmoc-Thr(tBu)-OH amino acid and Fmoc-Ile-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment two Lys-Leu-Asp-Lys-Thr-Thr-Ile resin；
(3), the preparation of polypeptide fragment three Glu-Ser-Ser resin:
A, first by Fmoc-Glu-(OtBu)-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, being added by the resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Glu-(OtBu)-wang
Resin resin adds 8～12mL mixed liquors, uses dimethylformamide to wash after deprotection 5～8 times；
C, the resin after step b deprotection, washing is added in reactor, is subsequently adding Fmoc-Ser(tBu)-OH amino acid, condensing agent HATU and condensation catalyst DIPEA, described Fmoc-Glu-(OtBu)-wang
Resin resin, Fmoc-Ser(tBu)-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Products therefrom after washing being carried out deprotection, washing according to step b same method, according to step c same method by Fmoc-Ser(tBu)-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment three Glu-Ser-Ser resin；
(4), the preparation of polypeptide fragment four Thr-Asp-Val resin:
A, first by Fmoc-Thr(tBu)-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, being added by the resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Thr(tBu)-wang
Resin resin adds 8～12mL mixed liquors, uses dimethylformamide to wash after deprotection 5～8 times；
C, the resin after step b deprotection, washing is added in reactor, is subsequently adding Fmoc-Asp(OtBu)-OH amino acid, condensing agent HATU and condensation catalyst DIPEA, described Fmoc-Thr(tBu)-wang
Resin resin, Fmoc-Asp(OtBu)-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method, Fmoc-Val-OH amino acid is connected on polypeptide fragment, obtain polypeptide fragment four Thr-Asp-Val resin；
(5), the preparation of polypeptide fragment five Ala-Ala-Asp-Ser resin:
A, first by Fmoc-Ala-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, being added by the resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Ala-wang
Resin resin adds 8～12mL mixed liquors, uses dimethylformamide to wash after deprotection 5～8 times；
C, the resin after step b deprotection, washing is added in reaction vessel, be subsequently adding Fmoc-Ala-OH amino acid, condensing agent HATU and catalyst DIPEA, described Fmoc-Ala-wang
Resin resin, Fmoc-Ala-OH amino acid, between condensing agent and catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Then products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method successively by Fmoc-Asp(OtBu)-OH amino acid and Fmoc-Ser(tBu)-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment five Ala-Ala-Asp-Ser resin；
(6), the cracking of polypeptide fragment:
Polypeptide fragment two resin of above-mentioned preparation is added in reaction vessel, according to 1g polypeptide fragment two resin: the ratio of 8～12mL lysates adds lysate and cracks, lysate is mixed according to the ratio that mass ratio is 18:1:1 by trifluoroacetic acid, water and 1,2-dithioglycol；Carry out after adding lysate cracking 2h under the conditions of 25 DEG C, by cracking, polypeptide fragment two is cleaved from resin；Filtering after cracking, gained filtrate carries out decompression distillation, is subsequently adding ether, is centrifuged and obtains thick polypeptide fragment two；
Repeat step (6) and process polypeptide fragment three resin of above-mentioned preparation, polypeptide fragment four resin and polypeptide fragment five resin successively, obtain thick polypeptide fragment three, thick polypeptide fragment four and thick polypeptide fragment five；
(7) purifying of polypeptide fragment:
The thick polypeptide fragment two, three, four and five step (6) obtained dissolves respectively by mobile phase A, makes the solution of 10mg/ml, is purified respectively by preparation liquid phase；
(8) connection of polypeptide fragment:
Polypeptide fragment one Asn-Glu-Ala-Glu-Glu-Val-Val-Glu-Lys-Lys-Glu resin step (1) obtained adds piperidines and carries out deprotection 5～8min in the mixed solution of dimethylformamide; in mixed liquor, piperidines and dimethylformamide mass ratio therebetween are 1:1, and the addition ratio between described polypeptide fragment one resin and mixed liquor is 1g polypeptide fragment one resin: 8～12mL mixed liquors；Dimethylformamide is used to carry out washing 5～8 times after deprotection, resin after washing is added in reaction vessel, and add step (7) polypeptide fragment two after purification, condensing agent HATU and condensation catalyst DIPEA carries out condensation reaction, resin after washing, polypeptide fragment two, between condensing agent HATU and condensation catalyst DIPEA, the molar ratio of addition is 1:2:2:4, carry out after addition contracting and reacting 3h at 25 DEG C, reaction is filtered after terminating, filter gained resin dimethylformamide to wash 5～8 times, the product that polypeptide fragment two is connected on polypeptide fragment one resin is obtained after washing；
Repeat step (8), successively step (7) polypeptide fragment three, four and five after purification is connected to polypeptide fragment one resin, the polypeptide fragment resin after being connected；
(9) acetylation:
During after the connection obtain step (8), polypeptide fragment resin adds reaction vessel, and adding acetic anhydride, catalyst DIPEA and dimethylformamide, after described connection, polypeptide fragment resin, acetic anhydride, catalyst and dimethylformamide additional proportion are polypeptide fragment resin after 1g connects: 10mL acetic anhydride: 20mL catalyst: 20mL dimethylformamide；Being stirred at room temperature 30～40min after addition and carry out acetylization reaction, detect reaction process by ninhydrin, testing result is that negative reaction terminates；Filtering after reaction, products therefrom first washs 5～8 times with dimethylformamide, then with absolute ethanol washing 3～5 times, obtains thymalfasin wang resin after washing；
(10) cracking of thick peptide:
Thymalfasin wang resin step (9) obtained adds in reaction vessel, according to 1g thymalfasin wang resin: the ratio of 8～12mL lysates adds lysate, described lysate is mixed according to the ratio that mass ratio is 18:1:1 by trifluoroacetic acid, water and 1,2-dithioglycol；Under the conditions of 25 DEG C, crack 2h after adding lysate, by cracking, polypeptide fragment is cleaved from resin；Filtering after cracking, gained filtrate, in vacuum 0.095MPa, evaporated under reduced pressure under the conditions of 40 DEG C, is evaporated in rear products therefrom addition ether and is centrifuged, centrifugal after obtain thymalfasin crude product；
(11) purifying of thymalfasin crude product:
Thymalfasin crude product step (10) obtained uses mobile phase A to dissolve, and makes the solution of 10mg/ml, carries out twice purifying by preparation liquid phase；
(12) it is dried:
Collect the step (11) the flowing phase containing thymalfasin, in vacuum 0.095MPa, evaporated under reduced pressure at 30 DEG C, be subsequently adding ether and be centrifuged, centrifugal after obtain thymalfasin, then in vacuum 0.095MPa, it is vacuum dried 24h at 30 DEG C, obtains thymalfasin finished product.
According to the method for above-mentioned preparation high-purity thymalfasin, described Fmoc-Asn(Trt)-wang
Resin resin, Fmoc-Lys(Boc)-wang
Resin resin, Fmoc-Glu-(OtBu)-wang
Resin resin, Fmoc-Thr(tBu)-wang
Resin resin and Fmoc-Ala-wang
Resin resin is provided by gill biochemistry (Shanghai) Co., Ltd..
According to the method for above-mentioned preparation high-purity thymalfasin, described condensing agent HATU is 2-(7-azo BTA)-N, and N, N', N'-tetramethylurea hexafluorophosphoric acid ester, described catalyst DIPEA is DIPEA.
According to the method for above-mentioned preparation high-purity thymalfasin, gained filtrate described in step (6) carry out decompression distillation, i.e. vacuum be 0.095MPa, temperature be 40 DEG C under the conditions of be evaporated；Described addition ether, the addition of its ether is the 1/2 of gained filtrate quality.
According to chromatographic condition when purifying in the method for above-mentioned preparation high-purity thymalfasin, step (7) it is:
Mobile phase A is the trifluoroacetic acid aqueous solution of mass percentage concentration 0.1%, and Mobile phase B is acetonitrile solution；Chromatographic column is the reverse post of C18 (20 × 250mm), and linear gradient is 0~60min, 10~35%B, flow velocity is 14ml/min；Described acetonitrile solution be mass percentage concentration be the mixed liquor of 0.1% trifluoroacetic acid aqueous solution and acetonitrile, its mixing quality is than for 10:90.
Method according to above-mentioned preparation high-purity thymalfasin, filter after described in step (8), reaction terminates, containing excessive each polypeptide fragment in gained filtrate, by decompression Distillation recovery filtrate in each polypeptide fragment, decompression distillation i.e. vacuum be 0.095MPa, temperature be 40 DEG C under conditions of be evaporated.
According to the method for above-mentioned preparation high-purity thymalfasin, being evaporated in rear products therefrom addition ether and be centrifuged described in step (10), the addition of its ether is the 1/2 of gained filtrate quality.
According to chromatographic condition when purifying in the method for above-mentioned preparation high-purity thymalfasin, step (11) it is:
Mobile phase A is the trifluoroacetic acid aqueous solution of mass percentage concentration 0.1%, and Mobile phase B is acetonitrile solution；Chromatographic column is the reverse post of C18 (20 × 250mm), and linear gradient is 0~60min, 10~35%B, flow velocity 14ml/min；Described acetonitrile solution is the mixed liquor of trifluoroacetic acid aqueous solution and the acetonitrile of mass percentage concentration 0.1%, and its mixing quality ratio is for 10:90.
The amino acid that the present invention relates to and reagent abbreviation thereof refer to table 1.
The positive beneficial effect of the present invention:
1, in existing thymalfasin traditional handicraft, being connected one by one by amino acid successively according to polypeptide amino acid composition, in connection procedure, the Connection Time of different aminoacids is inconsistent so that the reaction time extends guarantee reaction and can complete.The present invention combines practical experience, by suitable segmentation, thymalfasin is divided into five sections, synthesize polypeptide fragment one by one, again polypeptide fragment is connected together so that react amino acid faster and be placed in a polypeptide fragment and synthesize, be effectively shortened the reaction time.
2, technical solution of the present invention uses subsection synthesis thymalfasin, and polypeptide fragment can synthesize simultaneously, substantially reduces the time producing thymalfasin.
3, technical solution of the present invention uses subsection synthesis thymalfasin, can be effectively improved the yield of thymalfasin.Related data refers to table 2.
4, technical solution of the present invention uses subsection synthesis thymalfasin, can be effectively improved the quality level of thymalfasin finished product.The thymalfasin purity using technical solution of the present invention to prepare reaches more than 99%.
Four, accompanying drawing explanation:
Fig. 1 uses the chromatograms of thymalfasin prepared by the inventive method.
Fig. 2 uses amino acid sequence analysis Fig. 1 of thymalfasin prepared by the inventive method.
Fig. 3 uses amino acid sequence analysis Fig. 2 of thymalfasin prepared by the inventive method.
Five, detailed description of the invention:
The present invention is expanded on further below in conjunction with embodiment, but is not limiting as present disclosure.
Embodiment:
The present invention prepares the method for high-purity thymalfasin, and the detailed step of this preparation method is as follows:
(1), the preparation of polypeptide fragment one Asn-Glu-Ala-Glu-Glu-Val-Val-Glu-Lys-Lys-Glu resin:
A, first by Fmoc-Asn(Trt)-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 10mL dimethylformamide；
B, being added by step a resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Asn(Trt)-wang
Resin resin adds 10mL mixed liquor, uses dimethylformamide to wash after deprotection 5 times；
C, step b deprotection is washed after resin add in reactor, be subsequently adding Fmoc-Glu(OtBu)-OH amino acid, condensing agent HATU and condensation catalyst DIPEA, described Fmoc-Asn(Trt)-wang
Resin resin, Fmoc-Glu(OtBu)-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5 times；
Products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method successively by Fmoc-Ala-OH amino acid, Fmoc-Glu(OtBu)-OH amino acid, Fmoc-Glu(OtBu)-OH amino acid, Fmoc-Val-OH amino acid, Fmoc-Val-OH amino acid, Fmoc-Glu(OtBu)-OH amino acid, Fmoc-Lys(Boc)-OH amino acid, Fmoc-Lys(Boc)-OH amino acid and Fmoc-Glu(OtBu)-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment one Asn-Glu-Ala-Glu-Glu-Val-Val-Glu-Lys-Lys-Glu resin；
(2), the preparation of polypeptide fragment two Lys-Leu-Asp-Lys-Thr-Thr-Ile resin:
A, first by Fmoc-Lys(Boc)-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 10mL dimethylformamide；
B, being added by step a resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Lys(Boc)-wang
Resin resin adds 10mL mixed liquor, uses dimethylformamide to wash after deprotection 5 times；
C, the resin after step b deprotection, washing is added in reactor, is subsequently adding Fmoc-Leu-OH amino acid, condensing agent HATU and condensation catalyst DIPEA, described Fmoc-Lys(Boc)-wang
Resin resin, Fmoc-Leu-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5 times；
Then products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method successively by Fmoc-Asp(OtBu)-OH amino acid, Fmoc-Lys(Boc)-OH amino acid, Fmoc-Thr(tBu)-OH amino acid, Fmoc-Thr(tBu)-OH amino acid and Fmoc-Ile-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment two Lys-Leu-Asp-Lys-Thr-Thr-Ile resin；
(3), the preparation of polypeptide fragment three Glu-Ser-Ser resin:
A, first by Fmoc-Glu-(OtBu)-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 10mL dimethylformamide；
B, being added by the resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Glu-(OtBu)-wang
Resin resin adds 10mL mixed liquor, uses dimethylformamide to wash after deprotection 5 times；
C, the resin after step b deprotection, washing is added in reactor, is subsequently adding Fmoc-Ser(tBu)-OH amino acid, condensing agent HATU and condensation catalyst DIPEA, described Fmoc-Glu-(OtBu)-wang
Resin resin, Fmoc-Ser(tBu)-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5 times；
Products therefrom after washing being carried out deprotection, washing according to step b same method, according to step c same method by Fmoc-Ser(tBu)-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment three Glu-Ser-Ser resin；
(4), the preparation of polypeptide fragment four Thr-Asp-Val resin:
A, first by Fmoc-Thr(tBu)-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 10mL dimethylformamide；
B, being added by the resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Thr(tBu)-wang
Resin resin adds 10mL mixed liquor, uses dimethylformamide to wash after deprotection 5 times；
C, the resin after step b deprotection, washing is added in reactor, is subsequently adding Fmoc-Asp(OtBu)-OH amino acid, condensing agent HATU and condensation catalyst DIPEA, described Fmoc-Thr(tBu)-wang
Resin resin, Fmoc-Asp(OtBu)-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5 times；
Products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method, Fmoc-Val-OH amino acid is connected on polypeptide fragment, obtain polypeptide fragment four Thr-Asp-Val resin；
(5), the preparation of polypeptide fragment five Ala-Ala-Asp-Ser resin:
A, first by Fmoc-Ala-wang
Resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, and swelling time is 30min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 10mL dimethylformamide；
B, being added by the resin after swelling in the piperidines mixed liquor with dimethylformamide and carry out deprotection, in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1, every gram of Fmoc-Ala-wang
Resin resin adds 8～12mL mixed liquors, uses dimethylformamide to wash after deprotection 5 times；
C, the resin after step b deprotection, washing is added in reaction vessel, be subsequently adding Fmoc-Ala-OH amino acid, condensing agent HATU and catalyst DIPEA, described Fmoc-Ala-wang
Resin resin, Fmoc-Ala-OH amino acid, between condensing agent and catalyst add mol ratio be 1:2:2:4, various raw materials add after under the conditions of 25 DEG C, carry out condensation reaction, be detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5 times；
Then products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method successively by Fmoc-Asp(OtBu)-OH amino acid and Fmoc-Ser(tBu)-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment five Ala-Ala-Asp-Ser resin；
(6), the cracking of polypeptide fragment:
Polypeptide fragment two resin of above-mentioned preparation is added in reaction vessel, according to 1g polypeptide fragment two resin: the ratio of 8～12mL lysates adds lysate and cracks, lysate is mixed according to the ratio that mass ratio is 18:1:1 by trifluoroacetic acid, water and 1,2-dithioglycol；Carry out after adding lysate cracking 2h under the conditions of 25 DEG C, by cracking, polypeptide fragment two is cleaved from resin；Filter after cracking, gained filtrate carry out decompression distillation (vacuum be 0.095MPa, temperature be 40 DEG C under the conditions of be evaporated), be subsequently adding ether (addition of ether is the 1/2 of gained filtrate quality), centrifugal obtain thick polypeptide fragment two；
Repeat step (6) and process polypeptide fragment three resin of above-mentioned preparation, polypeptide fragment four resin and polypeptide fragment five resin successively, obtain thick polypeptide fragment three, thick polypeptide fragment four and thick polypeptide fragment five；
(7) purifying of polypeptide fragment:
The thick polypeptide fragment two, three, four and five step (6) obtained dissolves respectively by mobile phase A, makes the solution of 10mg/ml, is purified respectively by preparation liquid phase；
Chromatographic condition during purifying is:
Mobile phase A is the trifluoroacetic acid aqueous solution of mass percentage concentration 0.1%, and Mobile phase B is acetonitrile solution；Chromatographic column is the reverse post of C18 (20 × 250mm), and linear gradient is 0~60min, 10~35%B, flow velocity is 14ml/min；Described acetonitrile solution be mass percentage concentration be the mixed liquor of 0.1% trifluoroacetic acid aqueous solution and acetonitrile, its mixing quality is than for 10:90；
(8) connection of polypeptide fragment:
Polypeptide fragment one Asn-Glu-Ala-Glu-Glu-Val-Val-Glu-Lys-Lys-Glu resin step (1) obtained adds piperidines and carries out deprotection 5min in the mixed solution of dimethylformamide; in mixed liquor, piperidines and dimethylformamide mass ratio therebetween are 1:1, and the addition ratio between described polypeptide fragment one resin and mixed liquor is 1g polypeptide fragment one resin: 10mL mixed liquor；nullDimethylformamide is used to carry out washing 5 times after deprotection，Resin after washing is added in reaction vessel，And add step (7) polypeptide fragment two after purification、Condensing agent HATU and condensation catalyst DIPEA carries out condensation reaction，Resin after washing、Polypeptide fragment two、Between condensing agent HATU and condensation catalyst DIPEA, the molar ratio of addition is 1:2:2:4，Carry out after addition contracting and reacting 3h at 25 DEG C，Reaction is filtered (containing excessive each polypeptide fragment in gained filtrate after terminating，By each polypeptide fragment in decompression Distillation recovery filtrate，Decompression distillation is i.e. 0.095MPa in vacuum、Temperature is evaporated under conditions of being 40 DEG C)，Filter gained resin dimethylformamide to wash 5 times，The product that polypeptide fragment two is connected on polypeptide fragment one resin is obtained after washing；
Repeat step (8), successively step (7) polypeptide fragment three, four and five after purification is connected to polypeptide fragment one resin, the polypeptide fragment resin after being connected；
(9) acetylation:
During after the connection obtain step (8), polypeptide fragment resin adds reaction vessel, and adding acetic anhydride, catalyst DIPEA and dimethylformamide, after described connection, polypeptide fragment resin, acetic anhydride, catalyst and dimethylformamide additional proportion are polypeptide fragment resin after 1g connects: 10mL acetic anhydride: 20mL catalyst: 20mL dimethylformamide；Being stirred at room temperature 30min after addition and carry out acetylization reaction, detect reaction process by ninhydrin, testing result is that negative reaction terminates；Filtering after reaction, products therefrom first washs 5 times with dimethylformamide, then with absolute ethanol washing 3 times, obtains thymalfasin wang resin after washing；
(10) cracking of thick peptide:
Thymalfasin wang resin step (9) obtained adds in reaction vessel, according to 1g thymalfasin wang resin: the ratio of 10mL lysate adds lysate, described lysate is mixed according to the ratio that mass ratio is 18:1:1 by trifluoroacetic acid, water and 1,2-dithioglycol；Under the conditions of 25 DEG C, crack 2h after adding lysate, by cracking, polypeptide fragment is cleaved from resin；Filtering after cracking, gained filtrate, in vacuum 0.095MPa, evaporated under reduced pressure under the conditions of 40 DEG C, is evaporated in rear products therefrom addition ether and is centrifuged (addition of ether is the 1/2 of gained filtrate quality), centrifugal after obtain thymalfasin crude product；
(11) purifying of thymalfasin crude product:
Thymalfasin crude product step (10) obtained uses mobile phase A to dissolve, and makes the solution of 10mg/ml, carries out twice purifying by preparation liquid phase；
Chromatographic condition during purifying is:
Mobile phase A is the trifluoroacetic acid aqueous solution of mass percentage concentration 0.1%, and Mobile phase B is acetonitrile solution；Chromatographic column is the reverse post of C18 (20 × 250mm), and linear gradient is 0~60min, 10~35%B, flow velocity 14ml/min；Described acetonitrile solution is the mixed liquor of trifluoroacetic acid aqueous solution and the acetonitrile of mass percentage concentration 0.1%, and its mixing quality ratio is for 10:90；
(12) it is dried:
Collect the step (11) the flowing phase containing thymalfasin, in vacuum 0.095MPa, evaporated under reduced pressure at 30 DEG C, be subsequently adding ether and be centrifuged, centrifugal after obtain thymalfasin, then in vacuum 0.095MPa, it is vacuum dried 24h at 30 DEG C, obtains thymalfasin finished product.
The Fmoc-Asn(Trt that the present embodiment uses)-wang
Resin resin, Fmoc-Lys(Boc)-wang
Resin resin, Fmoc-Glu-(OtBu)-wang
Resin resin, Fmoc-Thr(tBu)-wang
Resin resin and Fmoc-Ala-wang
Resin resin is provided by gill biochemistry (Shanghai) Co., Ltd.；
The condensing agent HATU used is 2-(7-azo BTA)-N, and N, N', N'-tetramethylurea hexafluorophosphoric acid ester, catalyst DIPEA is DIPEA.
The liquid chromatogram of the thymalfasin prepared by the present embodiment refers to accompanying drawing 1, and amino acid sequence analysis refers to accompanying drawing 2,3.
Claims (5)
1. the method preparing thymalfasin, it is characterised in that described preparation method comprises the following steps:
(1), the preparation of polypeptide fragment one Asn-Glu-Ala-Glu-Glu-Val-Val-Glu-Lys-Lys-Glu resin:
A, first by Fmoc-Asn(Trt)-wang resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, step a resin after swelling is added in the piperidines mixed liquor with dimethylformamide and carry out deprotection; in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1; every gram of Fmoc-Asn(Trt)-wang resin resin addition 8～12mL mixed liquors, use dimethylformamide to wash after deprotection 5～8 times；
C, step b deprotection is washed after resin add in reactor; be subsequently adding Fmoc-Glu(OtBu)-OH amino acid, condensing agent HATU and condensation catalyst DIPEA; described Fmoc-Asn(Trt)-wang resin resin, Fmoc-Glu(OtBu)-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4; various raw materials carry out condensation reaction after adding under the conditions of 25 DEG C; being detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method successively by Fmoc-Ala-OH amino acid, Fmoc-Glu(OtBu)-OH amino acid, Fmoc-Glu(OtBu)-OH amino acid, Fmoc-Val-OH amino acid, Fmoc-Val-OH amino acid, Fmoc-Glu(OtBu)-OH amino acid, Fmoc-Lys(Boc)-OH amino acid, Fmoc-Lys(Boc)-OH amino acid and Fmoc-Glu(OtBu)-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment one Asn-Glu-Ala-Glu-Glu-Val-Val-Glu-Lys-Lys-Glu resin；
(2), the preparation of polypeptide fragment two Lys-Leu-Asp-Lys-Thr-Thr-Ile resin:
A, first by Fmoc-Lys(Boc)-wang resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, step a resin after swelling is added in the piperidines mixed liquor with dimethylformamide and carry out deprotection; in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1; every gram of Fmoc-Lys(Boc)-wang resin resin addition 8～12mL mixed liquors, use dimethylformamide to wash after deprotection 5～8 times；
C, the resin after step b deprotection, washing is added in reactor; it is subsequently adding Fmoc-Leu-OH amino acid, condensing agent HATU and condensation catalyst DIPEA; described Fmoc-Lys(Boc)-wang resin resin, Fmoc-Leu-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4; various raw materials carry out condensation reaction after adding under the conditions of 25 DEG C; being detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Then products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method successively by Fmoc-Asp(OtBu)-OH amino acid, Fmoc-Lys(Boc)-OH amino acid, Fmoc-Thr(tBu)-OH amino acid, Fmoc-Thr(tBu)-OH amino acid and Fmoc-Ile-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment two Lys-Leu-Asp-Lys-Thr-Thr-Ile resin；
(3), the preparation of polypeptide fragment three Glu-Ser-Ser resin:
A, first by Fmoc-Glu-(OtBu)-wang resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, the resin after swelling is added in the piperidines mixed liquor with dimethylformamide and carry out deprotection; in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1; every gram of Fmoc-Glu-(OtBu)-wang resin resin addition 8～12mL mixed liquors, use dimethylformamide to wash after deprotection 5～8 times；
C, the resin after step b deprotection, washing is added in reactor; be subsequently adding Fmoc-Ser(tBu)-OH amino acid, condensing agent HATU and condensation catalyst DIPEA; described Fmoc-Glu-(OtBu)-wang resin resin, Fmoc-Ser(tBu)-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4; various raw materials carry out condensation reaction after adding under the conditions of 25 DEG C; being detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Products therefrom after washing being carried out deprotection, washing according to step b same method, according to step c same method by Fmoc-Ser(tBu)-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment three Glu-Ser-Ser resin；
(4), the preparation of polypeptide fragment four Thr-Asp-Val resin:
A, first by Fmoc-Thr(tBu)-wang resin resin adds in container, is subsequently adding dimethylformamide and carries out swelling, swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, the resin after swelling is added in the piperidines mixed liquor with dimethylformamide and carry out deprotection; in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1; every gram of Fmoc-Thr(tBu)-wang resin resin addition 8～12mL mixed liquors, use dimethylformamide to wash after deprotection 5～8 times；
C, the resin after step b deprotection, washing is added in reactor; be subsequently adding Fmoc-Asp(OtBu)-OH amino acid, condensing agent HATU and condensation catalyst DIPEA; described Fmoc-Thr(tBu)-wang resin resin, Fmoc-Asp(OtBu)-OH amino acid, between condensing agent and condensation catalyst add mol ratio be 1:2:2:4; various raw materials carry out condensation reaction after adding under the conditions of 25 DEG C; being detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method, Fmoc-Val-OH amino acid is connected on polypeptide fragment, obtain polypeptide fragment four Thr-Asp-Val resin；
(5), the preparation of polypeptide fragment five Ala-Ala-Asp-Ser resin:
A, first being added in container by Fmoc-Ala-wang resin resin, be subsequently adding dimethylformamide and carry out swelling, swelling time is 30～60min；Described resin is 1g resin with the ratio of dimethylformamide addition therebetween: 8～12mL dimethylformamides；
B, the resin after swelling is added in the piperidines mixed liquor with dimethylformamide and carry out deprotection; in mixed liquor, the mass ratio between piperidines and dimethylformamide is 1:1; every gram of Fmoc-Ala-wang resin resin adds 8～12mL mixed liquors, uses dimethylformamide to wash after deprotection 5～8 times；
C, the resin after step b deprotection, washing is added in reaction vessel; it is subsequently adding Fmoc-Ala-OH amino acid, condensing agent HATU and catalyst DIPEA; described Fmoc-Ala-wang resin resin, Fmoc-Ala-OH amino acid, between condensing agent and catalyst add mol ratio be 1:2:2:4; various raw materials carry out condensation reaction after adding under the conditions of 25 DEG C; being detected as feminine gender by ninhydrin, reaction terminates；After end, gained reactant is filtered, filter gained resin dimethylformamide and wash 5～8 times；
Then products therefrom after washing is carried out deprotection, washing according to step b same method, according to step c same method successively by Fmoc-Asp(OtBu)-OH amino acid and Fmoc-Ser(tBu)-OH amino acid is connected on polypeptide fragment；Obtain polypeptide fragment five Ala-Ala-Asp-Ser resin；
(6), the cracking of polypeptide fragment:
Polypeptide fragment two resin of above-mentioned preparation is added in reaction vessel, according to 1g polypeptide fragment two resin: the ratio of 8～12mL lysates adds lysate and cracks, lysate is mixed according to the ratio that mass ratio is 18:1:1 by trifluoroacetic acid, water and 1,2-dithioglycol；Carry out after adding lysate cracking 2h under the conditions of 25 DEG C, by cracking, polypeptide fragment two is cleaved from resin；Filtering after cracking, gained filtrate carries out decompression distillation, is subsequently adding ether, is centrifuged and obtains thick polypeptide fragment two；
Repeat step (6) and process polypeptide fragment three resin of above-mentioned preparation, polypeptide fragment four resin and polypeptide fragment five resin successively, obtain thick polypeptide fragment three, thick polypeptide fragment four and thick polypeptide fragment five；
(7) purifying of polypeptide fragment:
The thick polypeptide fragment two, three, four and five step (6) obtained dissolves respectively by mobile phase A, makes the solution of 10mg/ml, is purified respectively by preparation liquid phase；
Chromatographic condition during purifying is: mobile phase A is the trifluoroacetic acid aqueous solution of mass percentage concentration 0.1%, and Mobile phase B is acetonitrile solution；Chromatographic column is the reverse post of C18,20 × 250mm, and linear gradient is 0~60min, 10~35%B, flow velocity is 14ml/min；Described acetonitrile solution be mass percentage concentration be the mixed liquor of 0.1% trifluoroacetic acid aqueous solution and acetonitrile, its mixing quality is than for 10:90；
(8) connection of polypeptide fragment:
Polypeptide fragment one Asn-Glu-Ala-Glu-Glu-Val-Val-Glu-Lys-Lys-Glu resin step (1) obtained adds piperidines and carries out deprotection 5～8min in the mixed solution of dimethylformamide; in mixed liquor, piperidines and dimethylformamide mass ratio therebetween are 1:1, and the addition ratio between described polypeptide fragment one resin and mixed liquor is 1g polypeptide fragment one resin: 8～12mL mixed liquors；Dimethylformamide is used to carry out washing 5～8 times after deprotection, resin after washing is added in reaction vessel, and add step (7) polypeptide fragment two after purification, condensing agent HATU and condensation catalyst DIPEA carries out condensation reaction, resin after washing, polypeptide fragment two, between condensing agent HATU and condensation catalyst DIPEA, the molar ratio of addition is 1:2:2:4, carry out after addition contracting and reacting 3h at 25 DEG C, reaction is filtered after terminating, filter gained resin dimethylformamide to wash 5～8 times, the product that polypeptide fragment two is connected on polypeptide fragment one resin is obtained after washing；
Repeat step (8), successively step (7) polypeptide fragment three, four and five after purification is connected to polypeptide fragment one resin, the polypeptide fragment resin after being connected；
(9) acetylation:
During after the connection obtain step (8), polypeptide fragment resin adds reaction vessel, and adding acetic anhydride, catalyst DIPEA and dimethylformamide, after described connection, polypeptide fragment resin, acetic anhydride, catalyst and dimethylformamide additional proportion are polypeptide fragment resin after 1g connects: 10mL acetic anhydride: 20mL catalyst: 20mL dimethylformamide；Being stirred at room temperature 30～40min after addition and carry out acetylization reaction, detect reaction process by ninhydrin, testing result is that negative reaction terminates；Filtering after reaction, products therefrom first washs 5～8 times with dimethylformamide, then with absolute ethanol washing 3～5 times, obtains thymalfasin wang resin after washing；
(10) cracking of thick peptide:
Thymalfasin wang resin step (9) obtained adds in reaction vessel, according to 1g thymalfasin wang resin: the ratio of 8～12mL lysates adds lysate, described lysate is mixed according to the ratio that mass ratio is 18:1:1 by trifluoroacetic acid, water and 1,2-dithioglycol；Under the conditions of 25 DEG C, crack 2h after adding lysate, by cracking, polypeptide fragment is cleaved from resin；Filtering after cracking, gained filtrate, in vacuum 0.095MPa, evaporated under reduced pressure under the conditions of 40 DEG C, is evaporated in rear products therefrom addition ether and is centrifuged, centrifugal after obtain thymalfasin crude product；
(11) purifying of thymalfasin crude product:
Thymalfasin crude product step (10) obtained uses mobile phase A to dissolve, and makes the solution of 10mg/ml, carries out twice purifying by preparation liquid phase；
Chromatographic condition during purifying is: mobile phase A is the trifluoroacetic acid aqueous solution of mass percentage concentration 0.1%, and Mobile phase B is acetonitrile solution；Chromatographic column is the reverse post of C18,20 × 250mm, and linear gradient is 0~60min, 10~35%B, flow velocity 14ml/min；Described acetonitrile solution is the mixed liquor of trifluoroacetic acid aqueous solution and the acetonitrile of mass percentage concentration 0.1%, and its mixing quality ratio is for 10:90；
(12) it is dried:
Collect the step (11) the flowing phase containing thymalfasin, in vacuum 0.095MPa, evaporated under reduced pressure at 30 DEG C, be subsequently adding ether and be centrifuged, centrifugal after obtain thymalfasin, then in vacuum 0.095MPa, it is vacuum dried 24h at 30 DEG C, obtains thymalfasin finished product.
The method preparing thymalfasin the most according to claim 1, it is characterised in that: described condensing agent HATU is 2-(7-azo BTA)-N, N, N', N'-tetramethylurea hexafluorophosphoric acid ester, described catalyst DIPEA is DIPEA.
The method preparing thymalfasin the most according to claim 1, it is characterised in that gained filtrate described in step (6) carry out decompression distillation, i.e. vacuum be 0.095MPa, temperature be 40 DEG C under the conditions of be evaporated；Described addition ether, the addition of its ether is the 1/2 of gained filtrate quality.
The method preparing thymalfasin the most according to claim 1, it is characterized in that, filter after described in step (8), reaction terminates, containing excessive each polypeptide fragment in gained filtrate, by decompression Distillation recovery filtrate in each polypeptide fragment, decompression distillation i.e. vacuum be 0.095MPa, temperature be 40 DEG C under conditions of be evaporated.
The method preparing thymalfasin the most according to claim 1, it is characterised in that being evaporated in rear products therefrom addition ether described in step (10) and be centrifuged, the addition of its ether is the 1/2 of gained filtrate quality.
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| CN104558149B (en) * | 2015-01-22 | 2018-10-26 | 苏州天马医药集团天吉生物制药有限公司 | The solid phase segment synthetic method of thymosin α1 |
| CN106543279A (en) * | 2016-11-01 | 2017-03-29 | 岳阳新华达制药有限公司 | A kind of thymalfasin synthesis technique |
| CN108218980B (en) * | 2016-12-21 | 2020-12-08 | 鲁南制药集团股份有限公司 | Synthesis method of thymalfasin |
| 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 |
| CN108640975A (en) * | 2018-03-17 | 2018-10-12 | 中国海洋大学 | A kind of synthetic method of N-terminal acetylation antibacterial peptide |
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| CN101412755A (en) * | 2007-12-26 | 2009-04-22 | 杭州诺泰制药技术有限公司 | Solid phase synthesis method for thymosin beta 4 |
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