CN104045694B - Method for preparing icaritin - Google Patents
Method for preparing icaritin Download PDFInfo
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- CN104045694B CN104045694B CN201310082987.1A CN201310082987A CN104045694B CN 104045694 B CN104045694 B CN 104045694B CN 201310082987 A CN201310082987 A CN 201310082987A CN 104045694 B CN104045694 B CN 104045694B
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- phe
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- 238000000034 method Methods 0.000 title claims abstract description 49
- TUUXBSASAQJECY-UHFFFAOYSA-N 3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)chromen-4-one Chemical compound C1=CC(OC)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C(CC=C(C)C)=C2O1 TUUXBSASAQJECY-UHFFFAOYSA-N 0.000 title abstract 6
- CTGVBHDTGZUEJZ-UHFFFAOYSA-N Noricaritin Natural products CC(C)(O)CCC1=C(O)C=C(O)C(C(C=2O)=O)=C1OC=2C1=CC=C(O)C=C1 CTGVBHDTGZUEJZ-UHFFFAOYSA-N 0.000 title abstract 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 221
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 120
- 239000011347 resin Substances 0.000 claims description 114
- 229920005989 resin Polymers 0.000 claims description 114
- 229920001184 polypeptide Polymers 0.000 claims description 50
- 239000012634 fragment Substances 0.000 claims description 43
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 40
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 37
- 238000006243 chemical reaction Methods 0.000 claims description 32
- 239000000203 mixture Substances 0.000 claims description 30
- 230000004224 protection Effects 0.000 claims description 27
- 150000001413 amino acids Chemical class 0.000 claims description 26
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 25
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 claims description 21
- 238000006467 substitution reaction Methods 0.000 claims description 21
- 239000006166 lysate Substances 0.000 claims description 19
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 18
- 229960003180 glutathione Drugs 0.000 claims description 18
- CSMYOORPUGPKAP-IBGZPJMESA-N (2r)-3-(acetamidomethylsulfanyl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CSCNC(=O)C)C(O)=O)C3=CC=CC=C3C2=C1 CSMYOORPUGPKAP-IBGZPJMESA-N 0.000 claims description 16
- 210000004899 c-terminal region Anatomy 0.000 claims description 15
- 239000007790 solid phase Substances 0.000 claims description 15
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 13
- 239000007821 HATU Substances 0.000 claims description 13
- 238000010647 peptide synthesis reaction Methods 0.000 claims description 13
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 claims description 12
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 claims description 12
- 108010024636 Glutathione Proteins 0.000 claims description 12
- 230000015572 biosynthetic process Effects 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 12
- 239000007822 coupling agent Substances 0.000 claims description 12
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 12
- 238000003786 synthesis reaction Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 claims description 11
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 238000005336 cracking Methods 0.000 claims description 11
- 230000003647 oxidation Effects 0.000 claims description 11
- 238000007254 oxidation reaction Methods 0.000 claims description 11
- 239000003875 Wang resin Substances 0.000 claims description 10
- 239000012071 phase Substances 0.000 claims description 10
- 238000004007 reversed phase HPLC Methods 0.000 claims description 10
- KJYAFJQCGPUXJY-UMSFTDKQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-(tritylamino)butanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KJYAFJQCGPUXJY-UMSFTDKQSA-N 0.000 claims description 9
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 claims description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- DVBUCBXGDWWXNY-SFHVURJKSA-N (2s)-5-(diaminomethylideneamino)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C3=CC=CC=C3C2=C1 DVBUCBXGDWWXNY-SFHVURJKSA-N 0.000 claims description 7
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 claims description 7
- 235000010894 Artemisia argyi Nutrition 0.000 claims description 7
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 7
- 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 description 7
- 244000030166 artemisia Species 0.000 claims description 7
- WDGICUODAOGOMO-DHUJRADRSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(tritylamino)pentanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)CC(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 WDGICUODAOGOMO-DHUJRADRSA-N 0.000 claims description 6
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 6
- 230000000903 blocking effect Effects 0.000 claims description 6
- TYPIQBXHJBDEBC-HNNXBMFYSA-N tert-butyl (3s)-3-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxobutanoate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C=O)C3=CC=CC=C3C2=C1 TYPIQBXHJBDEBC-HNNXBMFYSA-N 0.000 claims description 6
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 claims description 6
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 claims description 5
- BUBGAUHBELNDEW-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylsulfanylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCSC)C(O)=O)C3=CC=CC=C3C2=C1 BUBGAUHBELNDEW-SFHVURJKSA-N 0.000 claims description 5
- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 claims description 4
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 claims description 4
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 claims description 3
- 239000003513 alkali Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 230000005526 G1 to G0 transition Effects 0.000 claims description 2
- 239000012317 TBTU Substances 0.000 claims description 2
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 claims description 2
- QRUBYZBWAOOHSV-UHFFFAOYSA-M silver trifluoromethanesulfonate Chemical group [Ag+].[O-]S(=O)(=O)C(F)(F)F QRUBYZBWAOOHSV-UHFFFAOYSA-M 0.000 claims description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims 3
- RZJRJXONCZWCBN-UHFFFAOYSA-N octadecane Chemical compound CCCCCCCCCCCCCCCCCC RZJRJXONCZWCBN-UHFFFAOYSA-N 0.000 claims 2
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims 1
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical group OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 claims 1
- 229940038384 octadecane Drugs 0.000 claims 1
- 238000000197 pyrolysis Methods 0.000 claims 1
- 229910000077 silane Inorganic materials 0.000 claims 1
- 238000006257 total synthesis reaction Methods 0.000 abstract description 5
- 239000007791 liquid phase Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical class OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 11
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- 238000001514 detection method Methods 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
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- 206010019860 Hereditary angioedema Diseases 0.000 description 6
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 6
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- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
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- ITFICYZHWXDVMU-IPTZIORSSA-N (2S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-6-amino-2-[[(2S,3S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-1-[(2S)-2-[[(2S,3R)-2-[[(2S,3S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-amino-4-carboxybutanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxybutanoyl]amino]-3-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-3-sulfanylpropanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-3-methylpentanoyl]amino]hexanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-3-methylpentanoyl]amino]-4-oxobutanoyl]amino]-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-oxopentanoyl]amino]pentanedioic acid Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CS)NC(=O)[C@@H]4CCCN4C(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(=O)O)N ITFICYZHWXDVMU-IPTZIORSSA-N 0.000 description 3
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- XXMYDXUIZKNHDT-QNGWXLTQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1-tritylimidazol-4-yl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(N=C1)=CN1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XXMYDXUIZKNHDT-QNGWXLTQSA-N 0.000 description 2
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical class N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 229960001174 ecallantide Drugs 0.000 description 2
- 230000002101 lytic effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011435 rock Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- SWZCTMTWRHEBIN-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=C(O)C=C1 SWZCTMTWRHEBIN-QFIPXVFZSA-N 0.000 description 1
- ADOHASQZJSJZBT-SANMLTNESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[1-[(2-methylpropan-2-yl)oxycarbonyl]indol-3-yl]propanoic acid Chemical compound C12=CC=CC=C2N(C(=O)OC(C)(C)C)C=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ADOHASQZJSJZBT-SANMLTNESA-N 0.000 description 1
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 208000028185 Angioedema Diseases 0.000 description 1
- 102100028501 Galanin peptides Human genes 0.000 description 1
- 101710169265 Galanin peptides Proteins 0.000 description 1
- 241000521257 Hydrops Species 0.000 description 1
- 208000025047 Non-histaminic angioedema Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 150000004075 acetic anhydrides Chemical class 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- YWKKNVVRDIKEHZ-UHFFFAOYSA-N benzene;methylsulfanylmethane Chemical compound CSC.C1=CC=CC=C1 YWKKNVVRDIKEHZ-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001808 coupling effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229940018902 kalbitor Drugs 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- BRMYZIKAHFEUFJ-UHFFFAOYSA-L mercury diacetate Chemical compound CC(=O)O[Hg]OC(C)=O BRMYZIKAHFEUFJ-UHFFFAOYSA-L 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- ZTRPYTHOEREHEN-UHFFFAOYSA-N piperazine pyridine Chemical compound N1CCNCC1.N1=CC=CC=C1.N1=CC=CC=C1 ZTRPYTHOEREHEN-UHFFFAOYSA-N 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Peptides Or Proteins (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Cosmetics (AREA)
Abstract
The invention relates to a solid-liquid phase total synthesis method for preparing icaritin, which has high yield and convenient post-treatment and provides a new idea for industrial scale production of the icaritin.
Description
Technical field
The present invention relates to a kind of method for preparing polypeptide, and in particular to one kind prepares Ecallantide（Ai Kala peptides）'s
Method.
Background technology
Ecallantide（Hereinafter referred to as " Ai Kala peptides "）For a kind of long-chain polypeptide, it is made up of 60 amino acid sequences,
And contain three pairs of disulfide bond.The sequence of Ai Kala peptides such as SEQ ID NO.：Shown in 1:
Food and medicine Surveillance Authority of the U.S. is in approval Oyax companies new drug Chinese mugwort OK a karaoke club peptide on December 1st, 2009（Trade name：
Kalbitor）For hereditary angioedema（HAE）Burst or the treatment of mortality hydrops.Ai Kala peptides are in U.S.'s listing
The medicine of second for the treatment of HAE breaking-out.The medicine is subcutaneous injection agent, it is adaptable to the trouble for having HAE histories of attack in 16 years old and more than 16 years old
Person
But, on June 14th, 2012, Dyax companies, which have issued Ai Kala peptides, to be used to treat angiotensin converting enzyme
（ACE）The interim analysis results of angioneurotic edema II clinical trial phases caused by mortifier.Based on its term results not right and wrong
Convention thinks that Dyax companies determine to interrupt the clinical research of this medicine, turn to based on the further Depth Study of Ai Kala peptides and exploitation.
Although, Ai Kala peptides showed in random, double blinding, the II clinical trial phases of placebo it is not anticipated that it is good
It is good, but it provides a promising direction for treatment hereditary angioedema, it is contemplated that later in Ai Kala peptides
On the basis of will derive many new medicines be used for treat hereditary angioedema, accordingly, it would be desirable to set up one kind prepare Chinese mugwort card
The method of peptide is drawn to meet ever-increasing scientific research needs.
Because the peptide sequence of Ai Kala peptides is longer and wherein containing three pairs of disulfide bond, peptide chain easily occurs in building-up process
Curling, reaction site be difficult it is exposed, therefore rarely have chemical synthesis method report.At present, the related patent of Ai Kala peptides mainly collects
In in terms of pharmacological activity and treatment, such as in WO2007/106746A2, US2007/0213275A1 and EP20070758271
Record.So far, the fully synthetic report of chemistry is not carried out to Ai Kala peptides still.
The content of the invention
In the Solid phase peptide synthssis of prior art, conventional coupling method one by one is tended to vary with when carrying out the coupling of long peptide
The number for coupling amino acid increases and easily makes peptide chain occur to fold curling, and the exposed deficiency of reaction site causes coupling effect to be got over
Come poorer.Further, since comprising three pairs of disulfide bond in the peptide chain of Ai Kala peptides, conventional method as a result of multipair disulfide bond by
To be oriented method for oxidation, used a variety of side chains to remove and oxidising agent, each step necessarily brings the increasing of impurity
It is many, therefore be unfavorable for obtaining high-purity, product in high yield is also unfavorable for the amplification of technique.
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of total synthesis method of Ai Kala peptides, specifically carry
For a kind of solid-liquid total synthesis method of Ai Kala peptides, it comprises the steps：
（1）Following peptide resin is respectively synthesized by carrier of 2-CTC resins：1st, Fmoc-Gly-CTC resins；2、Fmoc-Arg
(Pbf)-CTC resins；3rd, Fmoc-Pro-CTC resins；4th, Fmoc-Ala-CTC resins；Then solid-phase peptide synthesis are used
Peptide resin 1,2,3,1 and 4 is coupled the amino acid with Fmoc protection groups one by one from C-terminal to N-terminal respectively, then split through lysate
Solution obtains corresponding polypeptide fragments A, B, C, D and E, wherein
Polypeptide fragment A is
Fmoc-Gln(Trt)-Cys(Acm)-Glu(OtBu)-Glu(OtBu)-Phe-Ile-Tyr(tBu)-Gly-OH；
Polypeptide fragment B is
Fmoc-Arg(Pbf)-Trp(Boc)-Phe-Phe-Asn(Trt)-Ile-Phe-Thr(tBu)-Arg(Pbf)-OH；
Polypeptide fragment C is
Fmoc-Pro-Cys(Acm)-Arg(Pbf)-Ala-Ala-His(Trt)-Pro-OH；
Polypeptide fragment D is
Fmoc-Phe-Lys(Boc)-Ala-Asp(OtBu)-Asp(OtBu)-Gly-OH；
Polypeptide fragment E is
Fmoc-Glu(OtBu)-Ala-Met-His(Trt)-Ser(tBu)-Phe-Cys(Acm)-Ala-OH；
（2）Fmoc-Asp (OtBu)-Wang resin is synthesized by carrier of Wang resin, then will using solid-phase peptide synthesis
It is coupled the amino acid with Fmoc protection groups one by one from C-terminal to N-terminal, peptide resin F is obtained, wherein the ammonia with Fmoc protection groups
Base acid be followed successively by Fmoc-Arg (Pbf)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Cys (Acm)-OH, Fmoc-Met-OH,
Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu
(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Ser(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Phe-OH、Fmoc-Arg
(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Fmoc-
Glu (OtBu)-OH, Fmoc-Cys (Acm)-OH and Fmoc-Gly-OH；
（3）By step（1）The middle polypeptide fragments with protection group are according to A, B, C, D and E order successively with peptide resin F with solid
The coupling of phase polypeptide synthesis method obtains the linear peptide resin of Ai Kala peptides；
（4）To step（3）In the linear peptide resin of Ai Kala peptides cracked, obtain comprising six-Cys (Acm)-fragments
Ai Kala peptide linear peptides；
（5）Remove step（4）Product in Acm protection groups in-Cys (Acm)-fragment, obtain Ai Kala peptide linear peptides；
（6）Using suitable oxidation system by step（5）Ai Kala peptide linear peptides three pairs of disulfide bond cyclisation, ended
OK a karaoke club peptide.
The solid-liquid total synthesis method that the present invention prepares Ai Kala peptides is the full conjunction of unique chemistry of Ai Kala peptides so far
Into method, its simple to operate, reaction condition is gentle, post processing is easy, raw material is easy to get, and the large-scale production for Ai Kala peptides is provided
A kind of brand-new thinking.
Brief description of the drawings
Fig. 1 is the synthetic route chart of Ai Kala peptides preparation method of the present invention.
Fig. 2 is that the thick peptide of Ai Kala peptides passes through the chromatogram that RP-HPLC is purified, in the spectrogram, retention time t=14.108
It is the signal peak of Ai Kala peptides at minute, peak area is 99.13%.
Embodiment
Term is explained
Term " solid-phase peptide synthesis " herein in the whole text refers to Fmoc synthesis in solid state sides known to Peptides Synthesis
Method, for example, be recorded in Fmoc Solid Phase Peptide Synthesis:A Practical Approach,
W.C.Chan, Peter D.White write, March2,2000（ISBN-10:0199637245）, Britain Oxford
University Press。
Term " peptide resin " herein in the whole text refers to that the C-terminal of polypeptide is connected with resin, the amino of N-terminal is connected with Fmoc protection groups
Polypeptide resin.
Term " polypeptide fragment " herein in the whole text refers to that " peptide resin " mentioned above removing C-terminal resin exposes free carboxylic
Base, the amino of N-terminal are connected with the polypeptide of Fmoc protection groups.
Term " Ai Kala peptides linear peptides " herein in the whole text refers to that " peptide resin " mentioned above removing C-terminal resin exposes trip
There is no the Ai Kala galanin peptides of cyclization from disulfide bond in carboxyl and its molecular structure.
The implication of english abbreviation is listed in the following table used in specification and claims：
The invention provides a kind of solid-liquid total synthesis method of Ai Kala peptides, comprise the steps：
（1）Peptide resin 1, Fmoc-Gly-CTC resins are respectively synthesized by carrier of 2-CTC resins；2、Fmoc-Arg(Pbf)-
CTC resins；3rd, Fmoc-Pro-CTC resins；4th, Fmoc-Ala-CTC resins, then use solid-phase peptide synthesis respectively will
1st, 2,3,1 and 4 peptide resins are coupled the amino acid with Fmoc protection groups one by one from C-terminal to N-terminal, then are obtained through lysate cracking
Corresponding polypeptide fragments A, B, C, D and E, wherein
Polypeptide fragment A is
Fmoc-Gln(Trt)-Cys(Acm)-Glu(OtBu)-Glu(OtBu)-Phe-Ile-Tyr(tBu)-Gly-OH；
Polypeptide fragment B is
Fmoc-Arg(Pbf)-Trp(Boc)-Phe-Phe-Asn(Trt)-Ile-Phe-Thr(tBu)-Arg(Pbf)-OH；
Polypeptide fragment C is
Fmoc-Pro-Cys(Acm)-Arg(Pbf)-Ala-Ala-His(Trt)-Pro-OH；
Polypeptide fragment D is
Fmoc-Phe-Lys(Boc)-Ala-Asp(OtBu)-Asp(OtBu)-Gly-OH；
Polypeptide fragment E is
Fmoc-Glu(OtBu)-Ala-Met-His(Trt)-Ser(tBu)-Phe-Cys(Acm)-Ala-OH；
（2）Fmoc-Asp (OtBu)-Wang resin is synthesized by carrier of Wang resin, then will using solid-phase peptide synthesis
It is coupled the amino acid with Fmoc protection groups one by one from C-terminal to N-terminal, peptide resin F is obtained, wherein the ammonia with Fmoc protection groups
Base acid be followed successively by Fmoc-Arg (Pbf)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Cys (Acm)-OH, Fmoc-Met-OH,
Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu
(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Ser(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Phe-OH、Fmoc-Arg
(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Fmoc-
Glu (OtBu)-OH, Fmoc-Cys (Acm)-OH and Fmoc-Gly-OH；
（3）By step（1）The middle polypeptide fragments with protection group are according to A, B, C, D and E order successively with peptide resin F with solid
The coupling of phase polypeptide synthesis method obtains the linear peptide resin of Ai Kala peptides；
（4）To step（3）In the linear peptide resin of Ai Kala peptides cracked, obtain comprising six-Cys (Acm)-fragments
Ai Kala peptide linear peptides；
（5）Remove step（4）Product in Acm protection groups in-Cys (Acm)-fragment, obtain Ai Kala peptide linear peptides；
（6）Using suitable oxidation system by step（5）Ai Kala peptide linear peptides three pairs of disulfide bond cyclisation, ended
OK a karaoke club peptide.
Wherein, in step（1）The synthesis of middle peptide resin 1,2,3 and 4 is carried out in the basic conditions, the alkali be DIPEA or
TMP, preferably DIPEA；The substitution degree of CTC resins is 0.5-1.0mmol/g, preferably 0.7mmol/g；In step（2）In Wang Shu
The substitution degree of fat is 0.1-0.2mmol/g, preferably 0.15mmol/g.
In step（1）It is middle synthesis peptide resin 1,2,3 and 4 amino acid be respectively Fmoc-Gly-OH, Fmoc-Arg (Pbf)-
OH, Fmoc-Pro-OH and Fmoc-Ala-OH；Synthesis polypeptide segment A amino acid is successively（According to reaction sequence）For：Fmoc-
Tyr(tBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-
Cys (Acm)-OH and Fmoc-Gln (Trt)-OH；Synthesis polypeptide segment B amino acid is successively（According to reaction sequence）For：Fmoc-
Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Ile-OH、Fmoc-Asn(Trt)-OH、Fmoc-Phe-OH、Fmoc-Phe-OH、
Fmoc-Trp (Boc)-OH and Fmoc-Arg (Pbf)-OH；Synthesis polypeptide segment C amino acid is successively（According to reaction sequence）For：
Fmoc-His (Trt)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Cys (Acm)-OH and
Fmoc-Pro-OH；Synthesis polypeptide segment D amino acid is successively（According to reaction sequence）For：Fmoc-Asp(OtBu)-OH、Fmoc-
Asp (OtBu)-OH, Fmoc-Ala-OH, Fmoc-Lys (Boc)-OH and Fmoc-Phe-OH；Synthesis polypeptide segment E amino acid
Successively（According to reaction sequence）For：Fmoc-Cys(Acm)-OH、Fmoc-Phe-OH、Fmoc-Ser(tBu)-OH、Fmoc-His
(Trt)-OH, Fmoc-Met-OH, Fmoc-Ala-OH and Fmoc-Glu (OtBu)-OH；Above-mentioned amino acid is commercially available available, example
Such as purchased from the biochemical Shanghai Co., Ltd of gill;
In step（2）In peptide resin F be：
Fmoc-Gly-Cys(Acm)-Glu(OtBu)-Gly-Asn(Trt)-Gln(Trt)-Asn(Trt)-Arg(Pbf)-
Phe-Glu(OtBu)-Ser(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Cys(Ac m)-Lys(Boc)-Lys(Boc)-
Met-Cys (Acm)-Thr (tBu)-Arg (Pbf)-Asp (OtBu)-Wang resin, used amino acid is commercially available available, example
Such as purchased from the biochemical Shanghai Co., Ltd of gill.
In step（1）In and the step of hereafter use solid-phase peptide synthesis described in solid-phase peptide synthesis
Including：1）Fmoc is removed, then resin is washed with solvent, untill removing Fmoc completely is detected with detection method；2）Will
It is proper amount of to be added to together in solid reaction post after coupling amino acid and coupling agent dissolve and activated in a solvent, until
Untill reaction terminating being detected with detection method；3）Repeat 1）With 2）.
Wherein removing Fmoc reagent can be known in the art any reagent of the achievable purpose, preferably 20% piperazine
Pyridine/DMF solution（DBLK）, i.e. piperidines：DMF（Volume ratio）For 1:4 mixed solution.
In step（1）In coupling agent be DIPCDI and HOBT composition, wherein the ratio of each composition is with molar ratio computing
For DIPCDI:HOBT=1:1；Step（2）In coupling agent for DIPCDI and compound a composition or DIPEA and compound a
With compound b composition, wherein compound a be HOBt or HOAt, compound b be PyBOP, PyAOP, HATU, HBTU or
The ratio of each composition is using molar ratio computing as DIPCDI in TBTU, preferably HATU, wherein coupling agent:a=1:1 and DIPEA:a:b=2:
1:1；In step（3）In coupling agent for DIPCDI and HOBT composition or HATU, HOAt and DIPEA composition, preferably
The ratio of each composition is using molar ratio computing as HATU in HATU, HOAt and DIPEA composition, wherein coupling agent：HOAt：DIPEA=
1.0:1.2:2.0。
Solid-phase peptide synthesis mentioned above are carried out in solid phase reaction post.Solid phase reaction post is not particularly limited,
It can be any solid phase reaction post that this purpose can be achieved.In addition, the time that every kind of amino acid carries out coupling reaction is usually 1.5-4
Hour, preferably 2-3 hours；Pressure is preferably normal pressure, can also be carried out under the pressure for properly increasing or reducing；Temperature is preferably room
Temperature（I.e. 20 ± 5 DEG C）, can also be carried out at a temperature of properly increasing or reducing.
Resin is preferably swelled by solid-phase peptide synthesis mentioned above before coupling, described to wash and be swelled
Step this area can using realize the purpose any reagent carry out, preferably DMF.The detection method applied in the reaction is
Any means known in the art that this purpose can be achieved, such as chromatography or Chemical Calibration preferably use and can determine that reaction
The reagent of terminal, preferably ninhydrin, when using ninhydrin, illustrate there is free acid amides, i.e. acyl in polypeptide if resin develops the color
Unprotect base on amine nitrogen.
In step（1）In cracking process carried out in the presence of lysate.The lysate is TFE：DCM=20：
80（V/V）Mixed liquor, consumption is 1g peptide resins correspondence 10-20ml lysates, preferably 1g peptide resins correspondence 15ml lysates, is split
The solution time is 2-5 hours.
In step（4）In cracking process carried out in the presence of lysate.The lysate is trifluoroacetic acid, benzene
Methyl sulfide, phenol, 1,2- dithioglycols（EDT）, water and tri isopropyl silane mixture, wherein, each composition in mixture
Match and be：TFA71-79%（V/V）, thioanisole 7-10%（V/V）, phenol 7-10%（V/V）、TIS1-3%（V/V）、EDT4-8%
（V/V）, remaining be water, each component summation be 100%.The proportioning is preferably TFA：Thioanisole：Phenol：Water：EDT：TIS=
72：8：8：6：4：2（V/V）.
The step of the present invention（5）In, removing removing Acm blocking groups are carried out under conditions of acidity, and the reagent of use can
Think AgOTf, Hg (OAc)2Or Ti (TFA)3, preferred 0.1mmol/L mercuric acetate, the pH value of reaction is 3-4.
Step of the present invention（6）Middle oxidation system is O2/ DTT/ ammonium acetates, DMSO/ ammonium sulfate or glutathione/oxidation
Type glutathione（GSH/GSSG）Each composition adds in buffer solution, preferably GSH/GSSG/ buffer solutions, wherein linear peptides and oxidation system
The concentration entered（Mass concentration or molar concentration, i.e., add the quality of each composition per mL or per L buffer solutions（mg）Or mole
（mmol））Ratio be linear peptides:GSH:GSSG=1:100~150:10, preferably linear peptides：GSH:GSSG=1:100:10,
The wherein concentration of linear peptides is in terms of mg/mL, and GSH concentration is in terms of mmol/L, and GSSG concentration is in terms of mmol/L, this buffer system
Gently, the Met in peptide chain can be prevented to be oxidized, be conducive to improving yield.；
The preparation method of the present invention first uses linear Ai Kala peptide of the synthesis in solid state with Acm blocking groups, directly " one pot
Method " disposably formed three pairs of disulfide bond, it is to avoid use bulky Trt blocking groups, be easy to the space of long peptide chain unfold and
Be conducive to the progress of coupling reaction
The present invention preferably also includes the purification step of Ai Kala peptides.The purification step can be using known in the art any
Polypeptide purification techniques are carried out, it is preferred to use reversed-phase high pressure liquid chromatography.Further, the reversed-phase high pressure liquid chromatography bag
Include：Using anti-phase octadecylsilane as stationary phase, with 0.1% trifluoroacetic acid/water, acetonitrile is respectively mobile phase, collects purpose peak and evaporates
Point, it is concentrated freeze-dried.
Embodiment
For a further understanding of the present invention, with reference to specific embodiment, the present invention is described in detail, it should be appreciated that under
State embodiment and be intended to explanation, do not limit the invention.
The Wang resin and 2-CTC resins used in this manual is purchased from Tianjin Nankai and into Co., Ltd, and various bands are protected
The amino acid of base is protected purchased from the biochemical Shanghai Co., Ltd of gill, coupling reagent is purchased from Suzhou heavenly steed Co., Ltd, other solvents and
Reagent is common commercially available product.
Embodiment 1：The preparation of substitution degree 0.5mmol/g-1.0mmol/g Fmoc-Gly-CTC resins
Substitution degree is the preparation of 0.7mmol/g Fmoc-Gly-CTC resins
Weigh the 2-CTC resins 300g that substitution degree is 1.0mmol/g（300mmol）, it is added in solid phase reaction post, uses
DMF is washed 2 times, after DMF swellable resins 30 minutes, takes 160.4g（540mmol）Fmoc-Gly-OH DMF dissolve, ice-water bath
Lower addition 116.6g（900mmol）After DIPEA activation, add in the above-mentioned reaction column equipped with resin, after reacting 2 hours, add
360ml absolute methanols close 30min.Washed with DMF 3 times, DCM is washed 3 times, methanol, which shrinks, to be drained, and obtains Fmo-Gly-CTC trees
Fat, detection substitution degree is 0.705mmol/g.
Substitution degree is the preparation of 0.5mmol/g Fmoc-Gly-CTC resins
Weigh the 2-CTC resins 300g that substitution degree is 1.0mmol/g（300mmol）, it is added in solid phase reaction post, uses
DMF is washed 2 times, after DMF swellable resins 30 minutes, takes 133.8g（450mmol）Fmoc-Gly-OH DMF dissolve, ice-water bath
Lower addition 116.6g（900mmol）After DIPEA activation, add in the above-mentioned reaction column equipped with resin, after reacting 2 hours, add
360ml absolute methanols close 30min.Washed with DMF 3 times, DCM is washed 3 times, methanol, which shrinks, to be drained, and obtains Fmo-Gly-CTC trees
Fat, detection substitution degree is 0.505mmol/g.
Substitution degree is the preparation of 1.0mmol/g Fmoc-Gly-CTC resins
Weigh the 2-CTC resins 250g that substitution degree is 1.2mmol/g（300mmol）, it is added in solid phase reaction post, uses
DMF is washed 2 times, after DMF swellable resins 30 minutes, takes 267.6g（900mmol）Fmoc-Gly-OH DMF dissolve, ice-water bath
Lower addition 116.6g（900mmol）After DIPEA activation, add in the above-mentioned reaction column equipped with resin, after reacting 2 hours, add
360ml absolute methanols close 30min.Washed with DMF 3 times, DCM is washed 3 times, methanol, which shrinks, to be drained, and obtains Fmo-Gly-CTC trees
Fat, detection substitution degree is 0.989mmol/g.
The peptide resin 2,3 and 4 used in the method for the present invention is carried out according to the method for embodiment 1.
Embodiment 2：The preparation of polypeptide fragment A peptide resins
Weigh the Fmoc-Gly-CTC resins 156g that substitution degree is 0.705mmol/g（110mmol）, it is added to solid phase reaction
In post, washed with DMF 2 times, after being swelled Fmoc-Gly-CTC resins 30 minutes with DMF, with DBLK removing Fmoc protections, Ran Houyong
DMF is washed 6 times.By 151.6g Fmoc-Tyr (tBu)-OH（330mmol）, 53.6gHOBt（396mmol）, 50.1g DIC
（396mmol）DMF solution is dissolved in, is added in solid phase reaction post, 2h is reacted at room temperature（Reaction end is defined by ninhydrin method detection,
If resin water white transparency, reaction is complete, resin colour developing, represents that reaction is incomplete, needs coupling reaction 1hrs again.Obtain
Fmoc-Tyr(tBu)-Gly-CTC Resin.The step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, presses
The order of photo section, sequentially completes Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu
(OtBu)-OH, Fmoc-Cys (Acm)-OH and Fmoc-Gln (Trt)-OH coupling, obtain 343g polypeptide fragment A peptide resins：
Fmoc-Gln (Trt)-Cys (Acm)-Glu (OtBu)-Glu (OtBu)-Phe-Ile-Tyr (tBu)-Gly-CTC is set
Fat.
The method of the preparation be the same as Example 2 of polypeptide fragment B, C, D and E peptide resin.
Embodiment 3：Polypeptide fragment A：
Fmoc-Gln (Trt)-Cys (Acm)-Glu (OtBu)-Glu (OtBu)-Phe-Ile-Tyr (tBu)-Gly-OH's
Prepare
343g polypeptide fragment A peptide resins are placed in cracking reaction bottle, lytic reagent is added with the ratio of 15ml/g resins
（TFE：DCM=20：80（V/V））, 2.5h is stirred at room temperature.Reactant is filtered with sand core funnel, collects filtrate, resin is again with a small amount of
DCM is washed 3 times, is concentrated under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, is washed with absolute ether 3 times, vacuum is done
It is dry to obtain the thick peptide 188.7g of white powder solid, i.e. polypeptide fragment A.Weight yield is that 99.5%, RP-HPLC purity is 96.4%,
Mass signal is 1723.912.
Next, using feed molar number and reactant equivalent proportion same as Example 3, being prepared with identical method many
Fragments of peptides B, C, D and E obtain the thick peptide 269.7g of polypeptide fragment B.Weight yield is that 101.7%, RP-HPLC purity is 97.4%, matter
Spectrum signal is 2413.125；The thick peptides 178.4 of polypeptide fragment C.Weight yield is that 105.3%, RP-HPLC purity is 96.5%, mass spectrum
Signal is 1540.935；The thick peptide 124.21g of polypeptide fragment D.Weight yield is that 100.7%, RP-HPLC purity is 97.4%, mass spectrum
Signal is 1122.512；The thick peptide 289.4g of polypeptide fragment E.Weight yield is that 102.9%, RP-HPLC purity is 95.4%, mass spectrum letter
Number be 25567.325.
Embodiment 4：Substitution degree is the preparation of 0.15mmol/g Fmoc-Asp (OtBu)-Wang resin
Weigh the Wang resin 333.3g that substitution degree is 0.6mmol/g（200mmol）, add in solid phase reaction post, washed with DMF
Wash 2 times, after DMF swellable resins 30 minutes, take 246.9g（600mmol）Fmoc-Asp(OtBu)-OH、97.3g（720mmol）
HOBt、90.86g（720mmol）DIC、7.32g（60mmol）It is 1 that DMAP, which is dissolved in volume ratio,:1 DCM and DMF mixed solutions, plus
Enter in solid phase reaction post, react at room temperature 2h.Reaction is washed 4 times after terminating with DMF, and DCM is washed 2 times.Then 316.4g pyridines are added
With 408.4g acetic anhydrides mixed liquor closing resin 6h.Washed with DMF 4 times, after DCM is washed 2 times, methanol, which shrinks, to be drained, and is obtained
Fmoc-Asp (OtBu)-Wang resin 367.5g, detection substitution degree is 0.153mmol/g.
Embodiment 5：The preparation of polypeptide fragment F peptide resins
Weigh Fmoc-Asp (OtBu)-Wang resin 392.2g that substitution degree is 0.153mmol/g（60mmol）, add solid phase
In reaction column, washed with DMF 2 times, after DMF swellable resins 30 minutes, with DBLK removing Fmoc protections, then wash 4 with DMF
Secondary, DCM is washed 2 times.By 116.8g Fmoc-Arg (Pbf)-OH（180mmol）, 29.1g HOBt（216mmol）, 27.3g
DIPCDI（216mmol）It is 1 to be dissolved in volume ratio:1 DCM and DMF mixed solutions, are added in solid phase reaction post, react at room temperature 2h
（Reaction end is defined by ninhydrin method detection, if resin water white transparency, and reaction is complete, resin colour developing, represents that reaction is endless
Entirely, coupling reaction 1h again is needed.The step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, according to the suitable of fragment
Sequence, uses coupling agent for DIPCDI and compound HOBt composition or DIPEA and HOAt and HATU composition, wherein being coupled
The ratio of each composition is using molar ratio computing as DIPCDI in agent:HOBt=1:1 and DIPEA:HOAt:HATU=2:1:1, it is sequentially completed
Fmoc-Thr(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Met-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-
OH、Fmoc-Cys(Acm)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Ser
(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Phe-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、
Fmoc-Gln(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys
(Acm)-OH and Fmoc-Gly-OH coupling.Reaction is shunk after terminating with methanol, and resin vacuum is dried overnight, polypeptide piece of weighing
Section F peptide resins：Fmoc-Gly-Cys(Acm)-Glu(OtBu)-Gly-Asn(Trt)-Gln(Trt)-Asn(Trt)-Arg
(Pbf)-Phe-Glu(OtBu)-Ser(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Cys(Acm)-L ys(Boc)-Lys
(Boc)-Met-Cys (Acm)-Thr (tBu)-Arg (Pbf)-Asp (OtBu)-Wang resin 685.2g.
Embodiment 6：The preparation of Ai Kala peptide peptide resins
685.2g polypeptide fragment F peptide resins are weighed, are added in solid phase reaction post, is washed 2 times, is swelled with DMF with DMF
After resin 30 minutes, with DBLK removing Fmoc protections, then washed with DMF 6 times.By 310.2g polypeptide fragments A（180mmol）,
29.4g HOAt（216mmol）, 68.4g HATU（180mmol）With 59.5ml DIPEA（360mmol）It is dissolved in DMF, adds
In solid phase reaction post, 2h is reacted at room temperature（Reaction end is defined by ninhydrin method detection, if resin water white transparency, has reacted
Entirely, resin develops the color, and represents that reaction is incomplete, needs coupling reaction 1h again）.Reaction is shunk after terminating with methanol, and peptide resin vacuum is done
It is dry to stay overnight, obtain the following sequence of polypeptide peptide resins of 788.1g：Fmoc-Gln(Trt)-Cys(Acm)-Glu(OtBu)-Glu
(OtBu)-Phe-Ile-Tyr(tBu)-Gl y-Gly-Cys(Acm)-Glu(OtBu)-Gly-Asn(Trt)-Gln(Trt)-Asn
(Trt)-Arg(Pbf)-Phe-Glu(OtBu)-Ser(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Cys(Acm)-Lys(B
Oc)-Lys (Boc)-Met-Cys (Acm)-Thr (tBu)-Arg (Pbf)-Asp (OtBu)-Wang resin.
Next, according to the method described above, polypeptide fragment B, C, D and the E prepared in embodiment 3 being coupled successively, is finally given
1241.1g end OK a karaoke club peptide peptide resin：
Fmoc-Phe-Lys(Boc)-Ala-Asp(OtBu)-Asp(OtBu)-Gly-Phe-Lys(Boc)-Ala-Asp
(OtBu)-Asp(OtBu)-Gly-Pro-Cys(Acm)-Arg(Pbf)-Ala-Ala-His(Trt)-Pro-Arg(Pbf)-Trp
(Boc)-Phe-Phe-Asn(Trt)-Ile-Phe-Thr(tBu)-Arg(Pbf)-Gln(Trt)-Cys(Acm)-Glu(OtBu)-
Glu(OtBu)-Phe-Ile-Tyr(tBu)-Gly-Gly-Cys(Acm)-Glu(OtBu)-Gly-Asn(Trt)-Gln(Trt)-
Asn(Trt)-Arg(Pbf)-Phe-Glu(OtBu)-Ser(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Cys(Acm)-Lys
(B oc)-Lys (Boc)-Met-Cys (Acm)-Thr (tBu)-Arg (Pbf)-Asp (OtBu)-king's peptide resin.
Embodiment 7：The preparation of the linear thick peptide of Ai Kala peptides with Acm blocking groups
1241.1g Chinese mugwort OK a karaoke club peptide peptide resin peptide resins are placed in cracking reaction wherein, added with the ratio of 15ml/g resins
Lytic reagent（TFA：Thioanisole：Phenol：Water：EDT：TIS=72：8：8：6：4：2（V/V））, 3.0h is stirred at room temperature.Reactant
Filtered with sand core funnel, collect filtrate, resin is washed 3 times with a small amount of TFA, is concentrated under reduced pressure after merging filtrate again.Add frost
Absolute ether is precipitated, and is washed with absolute ether 3 times, and vacuum drying obtains white powder solid, the i.e. Ai Kala with Acm protection groups
The linear thick peptide 532.2g of peptide.Weight yield is that 100.5%, HPLC purity is 82.1%, and mass signal is 8820.726.
Embodiment 8：Remove the Acm blocking groups of the linear thick peptide of Ai Kala peptides
It is 7-12mg/ml that the 200.0g linear thick peptides of Ai Kala peptides for containing Acm groups are configured into concentration with 10% acetic acid
Solution.The pH of solution is accurately adjusted to 3.5 with glacial acetic acid.Add 0.1mmol/L mercuric acetates（12eq./Acm）, use acetic acid
Or pH is adjusted to 3.5 by ammoniacal liquor again.Inflated with nitrogen, at room temperature gentle agitation reaction.Beta -mercaptoethanol (25eq/Acm) is added, is put
Put 3h.Centrifugation, removes precipitation, by supernatant HPLC desalinations.Vacuum drying, finally gives the linear thick peptide of Ai Kala peptides
174.9g, mass signal is 8387.625.
Embodiment 9：The preparation of the thick peptide of Ai Kala peptides
The linear thick peptide of 150g Chinese mugwort OK a karaoke club peptides is dissolved in 150L buffer system, the buffer system be 0.1mM ammonium acetates,
3 times are measured in the DTT of linear thick peptide (amount of the material) aqueous solution, and pH is 7.8, and opening is placed in room temperature, is stirred 36 hours, on the rocks
Second acid for adjusting pH=3~5 are quenched, that is, obtain the thick peptide solution of Ai Kala peptides, and carry out purifying preparation simultaneously with reference to the method for embodiment 12
It is lyophilized, white fine peptide solid 35.7g is obtained, cyclisation yield is 23.8%, and mass signal is 8387.654.
Embodiment 10：The preparation of the thick peptide of Ai Kala peptides
The linear thick peptide of 150g Chinese mugwort OK a karaoke club peptides is dissolved in 150L buffer system, the buffer system be 0.2mM ammonium sulfate/
The 5%DMSO aqueous solution, pH is 8.2, and opening is placed in room temperature, is stirred 24 hours, and ice acetic acid regulation pH=3~5 are quenched, and reference
The method of embodiment 12 carries out purifying preparation and freezed, and obtains white fine peptide solid 37.1g, and cyclisation yield is 24.7%, mass spectrum letter
Number be 8387.627..
Embodiment 11：The preparation of the thick peptide of Ai Kala peptides
The linear thick peptide of 150g Chinese mugwort OK a karaoke club peptides is dissolved in 150L buffer system, the buffer system is 0.1mM ammonium phosphate
With GSH/GSSG（100:10, in terms of mmol/L）The aqueous solution, pH is 8.2, and opening is placed in room temperature, is stirred 24 hours, second on the rocks
Acid for adjusting pH=3~5 are quenched, and purify and prepare according to embodiment 12, white fine peptide solid 40.78g, and yield is 27.19%, mass spectrum letter
Number be 8387.637.
Embodiment 12：The purifying of the thick peptide of Ai Kala peptides
The thick peptide of Ai Kala peptides that Example 11 is prepared, using NOVASEP RP-HPLC systems, wavelength 220nm, color
Spectrum post is anti-phase C18 posts, and conventional 0.1%TFA/ water, acetonitrile are respectively mobile phase purifying, and desalination collects purpose peak cut, rotation
It is concentrated by evaporation, lyophilized to obtain white fine peptide solid 40.78g, yield is 27.19%, and mass signal is 8387.637.HPLC purity
99.54%.The spectrogram and data of specific fine peptide are referring to Fig. 2
Although with reference to particular, the present invention is described, those skilled in the art will recognize that
It is that in the case of without departing from spirit and scope of the present invention, the embodiment can be changed or be improved, the scope of the invention
Limited by appended claims.
Claims (19)
1. a kind of method for preparing Ai Kala peptides, comprises the steps：
(1) following peptide resin is respectively synthesized by carrier of 2-CTC resins：Peptide resin 1, i.e. Fmoc-Gly-CTC resins；Peptide resin
2, i.e. Fmoc-Arg (Pbf)-CTC resins；Peptide resin 3, i.e. Fmoc-Pro-CTC resins；Peptide resin 4, i.e. Fmoc-Ala-CTC trees
Fat；Then solid-phase peptide synthesis are used, peptide resin 1 is coupled the amino with Fmoc protection groups one by one from C-terminal to N-terminal
Acid, obtains Fmoc-Gln (Trt)-Cys (Acm)-Glu (OtBu)-Glu (OtBu)-Phe-Ile-Tyr (tBu)-Gly-CTC trees
Fat, then obtain polypeptide fragment A through lysate cracking：Fmoc-Gln(Trt)-Cys(Acm)-Glu(OtBu)-Glu(OtBu)-
Phe-Ile-Tyr(tBu)-Gly-OH；Peptide resin 2 is coupled the amino acid with Fmoc protection groups one by one from C-terminal to N-terminal,
Obtain Fmoc-Arg (Pbf)-Trp (Boc)-Phe-Phe-Asn (Trt)-Ile-Phe-Thr (tBu)-Arg (Pbf)-CTC trees
Fat, then obtain polypeptide fragment B through lysate cracking：Fmoc-Arg(Pbf)-Trp(Boc)-Phe-Phe-Asn(Trt)-Ile-
Phe-Thr(tBu)-Arg(Pbf)-OH；Peptide resin 3 is coupled the amino acid with Fmoc protection groups one by one from C-terminal to N-terminal,
Fmoc-Pro-Cys (Acm)-Arg (Pbf)-Ala-Ala-His (Trt)-Pro-CTC resins are obtained, then are cracked through lysate
To polypeptide fragment C：Fmoc-Pro-Cys(Acm)-Arg(Pbf)-Ala-Ala-His(Trt)-Pro-OH；By peptide resin 1 from C-terminal
It is coupled the amino acid with Fmoc protection groups one by one to N-terminal, obtains Fmoc-Phe-Lys (Boc)-Ala-Asp (OtBu)-Asp
(OtBu)-Gly-CTC resins, then obtain polypeptide fragment D through lysate cracking：Fmoc-Phe-Lys(Boc)-Ala-Asp
(OtBu)-Asp(OtBu)-Gly-OH；Peptide resin 4 is coupled the amino acid with Fmoc protection groups one by one from C-terminal to N-terminal,
Fmoc-Glu (OtBu)-Ala-Met-His (Trt)-Ser (tBu)-Phe-Cys (Acm)-Ala-CTC resins are obtained, then through splitting
Solution liquid cracking obtains polypeptide fragment E：Fmoc-Glu(OtBu)-Ala-Met-His(Trt)-Ser(tBu)-Phe-Cys(Acm)-
Ala-OH；
(2) Fmoc-Asp (OtBu)-Wang resin is synthesized by carrier of Wang resin, then using solid-phase peptide synthesis by its from
C-terminal is coupled the amino acid with Fmoc protection groups to N-terminal one by one, peptide resin F is obtained, wherein the amino acid with Fmoc protection groups
It is followed successively by Fmoc-Arg (Pbf)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Cys (Acm)-OH, Fmoc-Met-OH, Fmoc-Lys
(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-
OH、Fmoc-Leu-OH、Fmoc-Ser(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Phe-OH、Fmoc-Arg(Pbf)-
OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu
(OtBu)-OH, Fmoc-Cys (Acm)-OH and Fmoc-Gly-OH；
(3) solid-phase peptide synthesis are used, make from C-terminal to N-terminal peptide resin F successively with the polypeptide with protection group in step (1)
Fragment A, B, C, D, E are coupled, i.e., from C-terminal to N-terminal F connections A, and A reconnects B, and B reconnects C, and C reconnects D, and D reconnects E, obtained
The linear peptide resin of Ai Kala peptides；
(4) the linear peptide resin of Ai Kala peptides in step (3) is cracked, obtains including the Chinese mugwort of six-Cys (Acm)-fragments
OK a karaoke club peptide linear peptides；
(5) the Acm protection groups in the product of removing step (4) in-Cys (Acm)-fragment, obtain Ai Kala peptide linear peptides；
(6) make Ai Kala peptides linear peptides three pairs of disulfide bond of formation of step (5) using suitable oxidation system, obtain Ai Kala
Peptide, the oxidation system is the buffer solution being made up of glutathione and oxidized form of glutathione.
2. the method for claim 1 wherein the synthesis of peptide resin 1, peptide resin 2, peptide resin 3 and peptide resin 4 in step (1)
Carry out in the basic conditions, the alkali is DIPEA or TMP；The substitution degree of CTC resins is 0.5-1.0mmol/g；In step (2)
In Wang resin substitution degree be 0.1-0.2mmol/g.
3. the method for claim 2, wherein the alkali is DIPEA.
4. the method for claim 2, wherein the substitution degree of CTC resins is 0.7mmol/g in step (1).
5. the method for claim 2, wherein the substitution degree of the Wang resin in step (2) is 0.15mmol/g.
6. the method for claim 1 wherein the amino acid of synthesis peptide resin 1,2,3 and 4 is respectively Fmoc-Gly- in step (1)
OH, Fmoc-Arg (Pbf)-OH, Fmoc-Pro-OH and Fmoc-Ala-OH.
7. the method for claim 1 wherein, coupling agent in step (1) is DIPCDI and HOBt composition, wherein it is each into
The ratio divided is using molar ratio computing as DIPCDI:HOBt=1:1；Coupling agent in step (2) is the combination of DIPCDI and compound a
The composition of thing or DIPEA and compound a and compound b, wherein compound a be HOBt or HOAt, compound b be PyBOP,
PyAOP, HATU, HBTU or TBTU, wherein, the ratio of each composition is using molar ratio computing as DIPCDI in coupling agent:A=1:1 He
DIPEA:a:B=2:1:1；Composition or HATU, HOAt and DIPEA that coupling agent in step (3) is DIPCDI and HOBT
Composition.
8. the method for claim 7, the compound b is HATU.
9. the coupling agent in the method for claim 7, the step (3) is HATU, HOAt and DIPEA composition, wherein occasionally
Join the ratio of each composition in agent using molar ratio computing as HATU：HOAt：DIPEA=1.0:1.2:2.0.
10. the method for claim 1 wherein the cracking process in step (1) is carried out in the presence of lysate, institute
It is trifluoroethanol to state lysate：The volume ratio of dichloromethane is 20：80 mixture, consumption is 1g peptide resins correspondence 10-20ml
Lysate, pyrolysis time is 2-5 hours.
11. the method for claim 10, wherein lysate consumption are 1g peptide resins correspondence 15ml lysates.
12. the method for claim 1 wherein the cracking process of step (4) is carried out in the presence of lysate, described to split
Solution liquid is the mixture of trifluoroacetic acid, thioanisole, phenol, 1,2- dithioglycols, water and tri isopropyl silane, wherein, respectively into
Point proportioning using volume basis as：Trifluoroacetic acid 71-79%, thioanisole 7-10%, phenol 7-10%, tri isopropyl silane 1-
3%th, 1,2- dithioglycols 4-8%, remaining be water, each component summation be 100%.
13. the method for claim 12, the proportioning of each composition of lysate is using volume basis as trifluoroacetic acid：Thioanisole：
Phenol：Water：1,2- dithioglycols：Tri isopropyl silane=72：8：8：6：4：2.
14. the method for claim 1 wherein in step (5), removing Acm blocking groups are carried out under conditions of acidity, are adopted
Reagent is AgOTf, Hg (OAc)2Or Ti (TFA)3, the pH value of reaction is 3-4.
15. the method for claim 14, wherein the reagent is 0.1mmol/L Hg (OAc)2。
16. the method for claim 1 wherein the oxidation system described in step (6) is by glutathione and oxidized form gluathione
The buffer solution of peptide composition, the ratio of the addition concentration of each composition is linear peptides wherein in linear peptides and oxidation system:Glutathione:
Oxidized form of glutathione=1:100~150:10, the wherein concentration of linear peptides is in terms of mg/mL, and the concentration of glutathione is with mmol/
L is counted, and the concentration of oxidized form of glutathione is in terms of mmol/L.
17. the ratio of the addition concentration of each composition is linear peptides in the method for claim 16, wherein linear peptides and oxidation system：
Glutathione:Oxidized form of glutathione=1:100:10, the wherein concentration of linear peptides in terms of mg/mL, the concentration of glutathione with
Mmol/L is counted, and the concentration of oxidized form of glutathione is in terms of mmol/L.
18. any one of claim 1-17 method, includes the purification step of Ai Kala peptides.
19. the method for claim 18, wherein purification step use reversed-phase high pressure liquid chromatography, including：With anti-phase octadecane
Base silane is stationary phase, is respectively mobile phase with 0.1% trifluoroacetic acid aqueous solution, acetonitrile, collects purpose peak cut, and concentration is frozen
It is dry.
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