CN109096388A - A kind of preparation method of Teriparatide - Google Patents
A kind of preparation method of Teriparatide Download PDFInfo
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- CN109096388A CN109096388A CN201710607171.4A CN201710607171A CN109096388A CN 109096388 A CN109096388 A CN 109096388A CN 201710607171 A CN201710607171 A CN 201710607171A CN 109096388 A CN109096388 A CN 109096388A
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- Prior art keywords
- teriparatide
- resin
- fmoc
- reaction
- reagent
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- 108010049264 Teriparatide Proteins 0.000 title claims abstract description 75
- 229960005460 teriparatide Drugs 0.000 title claims abstract description 75
- OGBMKVWORPGQRR-UMXFMPSGSA-N teriparatide Chemical compound C([C@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)[C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 OGBMKVWORPGQRR-UMXFMPSGSA-N 0.000 title claims abstract description 74
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 239000011347 resin Substances 0.000 claims abstract description 91
- 229920005989 resin Polymers 0.000 claims abstract description 91
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 49
- 239000012043 crude product Substances 0.000 claims abstract description 19
- 238000005520 cutting process Methods 0.000 claims abstract description 17
- 125000006239 protecting group Chemical group 0.000 claims abstract description 17
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 14
- 230000032050 esterification Effects 0.000 claims abstract description 5
- 238000005886 esterification reaction Methods 0.000 claims abstract description 5
- 230000001376 precipitating effect Effects 0.000 claims abstract description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 92
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 238000009833 condensation Methods 0.000 claims description 36
- 230000005494 condensation Effects 0.000 claims description 36
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 27
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 27
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 229940024606 amino acid Drugs 0.000 claims description 19
- 235000001014 amino acid Nutrition 0.000 claims description 19
- 150000001413 amino acids Chemical class 0.000 claims description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 16
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical class COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 claims description 14
- -1 benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester Chemical class 0.000 claims description 13
- 210000004899 c-terminal region Anatomy 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 238000006482 condensation reaction Methods 0.000 claims description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 11
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 10
- 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 9
- 238000010511 deprotection reaction Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000012964 benzotriazole Substances 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 230000035484 reaction time Effects 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- AQRLNPVMDITEJU-UHFFFAOYSA-N triethylsilane Chemical compound CC[SiH](CC)CC AQRLNPVMDITEJU-UHFFFAOYSA-N 0.000 claims description 8
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 7
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 6
- XZXYQEHISUMZAT-UHFFFAOYSA-N 2-[(2-hydroxy-5-methylphenyl)methyl]-4-methylphenol Chemical compound CC1=CC=C(O)C(CC=2C(=CC=C(C)C=2)O)=C1 XZXYQEHISUMZAT-UHFFFAOYSA-N 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 5
- 229940107816 ammonium iodide Drugs 0.000 claims description 5
- 239000000460 chlorine Substances 0.000 claims description 5
- 229910052801 chlorine Inorganic materials 0.000 claims description 5
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 claims description 5
- 229910052698 phosphorus Inorganic materials 0.000 claims description 5
- 239000011574 phosphorus Substances 0.000 claims description 5
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 claims description 4
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 4
- 150000002148 esters Chemical class 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 4
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 claims description 4
- 150000003053 piperidines Chemical class 0.000 claims description 3
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 3
- 238000011282 treatment Methods 0.000 claims description 3
- 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 3
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 3
- 125000003287 1H-imidazol-4-ylmethyl group Chemical group [H]N1C([H])=NC(C([H])([H])[*])=C1[H] 0.000 claims description 2
- SLROPVABSCVNOL-UHFFFAOYSA-N 2,2,4,6,7-pentamethyl-3a,4-dihydro-3h-1-benzofuran Chemical class CC1C=C(C)C(C)=C2OC(C)(C)CC12 SLROPVABSCVNOL-UHFFFAOYSA-N 0.000 claims description 2
- 125000000979 2-amino-2-oxoethyl group Chemical group [H]C([*])([H])C(=O)N([H])[H] 0.000 claims description 2
- 125000003974 3-carbamimidamidopropyl group Chemical group C(N)(=N)NCCC* 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 235000003704 aspartic acid Nutrition 0.000 claims description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-L glutamate group Chemical group N[C@@H](CCC(=O)[O-])C(=O)[O-] WHUUTDBJXJRKMK-VKHMYHEASA-L 0.000 claims description 2
- 238000007654 immersion Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims description 2
- 238000004007 reversed phase HPLC Methods 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 claims 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical group OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical group NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims 1
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 claims 1
- 239000004472 Lysine Chemical group 0.000 claims 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Chemical group NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims 1
- 125000004744 butyloxycarbonyl group Chemical group 0.000 claims 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Chemical group OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 15
- 238000007086 side reaction Methods 0.000 abstract description 6
- 239000007791 liquid phase Substances 0.000 abstract description 5
- 239000007790 solid phase Substances 0.000 abstract description 3
- 238000010647 peptide synthesis reaction Methods 0.000 abstract 1
- 238000001914 filtration Methods 0.000 description 20
- 239000000243 solution Substances 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 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 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 6
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 6
- ICIWUVCWSCSTAQ-UHFFFAOYSA-M iodate Chemical compound [O-]I(=O)=O ICIWUVCWSCSTAQ-UHFFFAOYSA-M 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 208000001132 Osteoporosis Diseases 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 208000010392 Bone Fractures Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 210000000963 osteoblast Anatomy 0.000 description 3
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-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
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- 108010016626 Dipeptides Proteins 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- QRUDEWIWKLJBPS-UHFFFAOYSA-N benzotriazole Chemical compound C1=CC=C2N[N][N]C2=C1 QRUDEWIWKLJBPS-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000006340 racemization Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- MGHMWKZOLAAOTD-DEOSSOPVSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1h-indol-3-yl)propanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(=O)O)CC1=CNC2=CC=CC=C12 MGHMWKZOLAAOTD-DEOSSOPVSA-N 0.000 description 1
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 description 1
- COHQCOIIBYMTJT-UHFFFAOYSA-N 2h-benzotriazole;1,1,3,3-tetramethylurea Chemical compound CN(C)C(=O)N(C)C.C1=CC=C2NN=NC2=C1 COHQCOIIBYMTJT-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 206010065687 Bone loss Diseases 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- 101001135770 Homo sapiens Parathyroid hormone Proteins 0.000 description 1
- 101001135995 Homo sapiens Probable peptidyl-tRNA hydrolase Proteins 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 208000007101 Muscle Cramp Diseases 0.000 description 1
- DXRFZHILMCWCNG-UHFFFAOYSA-N N,N-dimethyl-1,8-naphthyridin-2-amine Chemical compound C1=CC=NC2=NC(N(C)C)=CC=C21 DXRFZHILMCWCNG-UHFFFAOYSA-N 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- UKFWSNCTAHXBQN-UHFFFAOYSA-N ammonium iodide Chemical compound [NH4+].[I-] UKFWSNCTAHXBQN-UHFFFAOYSA-N 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 235000015177 dried meat Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 102000058004 human PTH Human genes 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- UIDUKLCLJMXFEO-UHFFFAOYSA-N propylsilane Chemical compound CCC[SiH3] UIDUKLCLJMXFEO-UHFFFAOYSA-N 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000011519 second-line treatment Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003568 thioethers Chemical class 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
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
Abstract
The invention discloses a kind of preparation methods of Teriparatide, comprising the following steps: (1) uses resin for solid phase carrier, with Teriparatide C-terminal amino acid esterification, prepare amino acid-resin complexes；(2) Fmoc strategy solid-phase peptide synthesis is used, Teriparatide-resin complexes of protection are prepared；(3) Side chain protective group is removed using cutting reagent and is cleaved Teriparatide from resin, obtain Teriparatide crude product after ether precipitating；(4) crude product is purified using high-efficient liquid phase technique, obtains Teriparatide fine work.The present invention is suitble to simplicity, efficiently prepares Teriparatide.The present invention solves the problems, such as that existing method is complicated, side reaction is more.
Description
Technical field
The invention belongs to chemiluminescent polypeptide field, in particular to a kind of preparation method of Teriparatide.
Background technique
Teriparatide sequence is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF, is one containing 34 amino acid
Polypeptide.Teriparatide is developed by Lilly Co., Eli., and U.S. Food and Drug Administration had approved on November 26th, 2002
Teriparatide is used to treat the osteoporosis with height risk of bone fracture of man and menopausal women, is also used for treatment man
It is primary and/or as caused by sexual hypofunction with high risk of bone fracture osteoporosis.Current conventional medicine for treating osteoporosis
It typically just acts on osteoclast and slows down or block bone-loss, and the derivative of this parathyroid hormone of Teriparatide
Play the role of increasing osteoblast number, enhance its activity and preventing TNF-a Induced Apoptosis in Osteoblasts.Because it can increase the work of osteoblast
Property and quantity, the second line treatment so as to promote bone uptake, for severe osteoporosis.Furthermore Teriparatide side effect is very
It is small, it is generally only nausea, dizziness and leg cramps.Therefore before Teriparatide has very high medical value and wide market
Scape.
The preparation of Teriparatide mainly uses chemical method to synthesize.According to " synthesis chemistry " magazine 2007,15 (3): 388-390's
Report, using Wang Shuzhi, using DCC/HOBT as condensing agent, tryptophan Fmoc-Trp-OH has synthesized Teriparatide.The method
DCU precipitating, post-processing trouble are easy to produce in middle DCC condensation course；Racemization is easy to happen when amino acid is connect with Wang Shuzhi；Color
The side chain no protective of propylhomoserin, can all occur side reaction in condensation and cutting process, and final yield only has 23.3%.Patent
CN201210213044.3 synthesizes Teriparatides using 5 polypeptide fragment condensation methods, complex process, and condensation efficiency is low.Patent
CN201310403743.9 synthesizes Teriparatide using the method for ester bond substitution amido bond, and the reaction effect of ester bond is formed in this method
Rate is lower, and ester bond is unstable, the easy fracture in duplicate Fmoc deprotection reaction.Patent CN201410262511.0 is using pseudo-
Dried meat dipeptides considerably increases synthesis cost, and improves process complexity.Patent CN201511024053.8 is multiple using being condensed
The mode of dipeptides or tripeptide fragment synthesizes Teriparatide, increases process complexity.Patent CN201510005427.5 is with Wang Shu
Rouge or chlorine resin are carrier, and DIC/HOBT is that condensing agent synthesizes Teriparatide；Wherein His, Arg, Asn and Trp pendant reactive base
Group is not protected, and side reaction is easy to happen in synthesis and cleavage reaction, and cutting reagent used not can avoid Met (O)
The generation of impurity.
Summary of the invention
The object of the present invention is to provide a kind of easy, efficient Teriparatide preparation methods, solve current Teriparatide system
The problem of side reaction occurs when standby cumbersome, synthesis and cutting.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of preparation method of Teriparatide, comprising the following steps:
(1) Fmoc-Phe- resin complexes are prepared: dissolving first ammonia of Teriparatide C-terminal with N,N-dimethylformamide
After base acid Fmoc-Phe-OH and esterifying reagent, it is added to carrying out ester in the resin after n,N-Dimethylformamide immersion treatment
Change reaction, after reaction, be added 10 times of resin volume methanol/n,N-diisopropylethylamine mixed solution (volume ratio 1:
1) it is filtered after, reacting at room temperature 1h.Then resin is washed with n,N-Dimethylformamide, obtains Fmoc-Phe- resin complexes；
(2) Teriparatide-resin complexes of preparation protection: in the Fmoc-Phe- resin complexes obtained to step (1)
Fmoc deprotecting regent is added and removes Fmoc protecting group, filters after reaction and washs resin with dimethylformamide, then
Second amino acid of C-terminal and condensation reagent that Teriparatide is added into Phe- resin complexes carry out condensation reaction, and condensation is anti-
Resin is washed with dimethylformamide after answering；According to sequence of the Teriparatide from C-terminal to N-terminal, amino acid is replaced, is repeated
Fmoc deprotection reaction and condensation reaction, the Teriparatide-resin complexes protected；
(3) it prepares Teriparatide crude product: cutting reagent being added into Teriparatide-resin complexes of protection, remove more
Teriparatide is simultaneously cleaved by peptide Side chain protective group from resin, obtains Teriparatide crude product after ether precipitating；
(4) it prepares Teriparatide fine work: Teriparatide crude product is isolated and purified using reversed-phase high performance liquid chromatography, obtain special vertical
Pa peptide fine work.
The preferred Wang Shuzhi of resin described in step (1) or 2- chlorine resin；Further preferred 2- chlorine trityl chloride resin.
The preferred N of esterifying reagent, N- diisopropylethylamine described in step (1)；
The preferred n of ratio (Fmoc-Phe-OH): n (N, N- diisopropyl second of the amount of the substance in step (1) between each substance
Amine): n (2- chlorine trityl chloride resin)=3~0.1:60~2:1；
Preferably 20 DEG C~60 DEG C of the temperature of esterification described in step (1), the reaction time preferably 1 hour~10 hours.
Fmoc deprotecting regent preferred volume ratio described in step (2) is the piperidines and dimethylformamide of 1:1~4
Mixture, preferably 20 DEG C~60 DEG C of the temperature of the Fmoc deprotection reaction, reaction time preferred 10min~60min.
It is Fmoc protection that the protecting group of amino acid described in step (2), which is respectively as follows: all amino acid α amino protecting groups,
Base, lysine side chains hydroxyl protection base are tert-butyl, and aspartic acid and glutamate side chain carboxyl-protecting group are tert-butyl, glutamy
Amine and asparagine side chain protecting group are trityl, and histidine side chains protecting group is trityl or tertbutyloxycarbonyl, rely ammonia
Acid and Trp side chain protecting group are tertbutyloxycarbonyl, and arginine side chain protecting group is 2,2,4,6,7- pentamethyl dihydrobenzo furans
It mutters -5- sulfonyl.
The combination of condensation reagent described in step (2) preferred condensation reagent 1, condensation reagent 2 and condensation reagent 3, condensation
Reagent 1 is selected from N, N- dicyclohexylcarbodiimide, N, N- diisopropylcarbodiimide, benzotriazole-N, N, N', N'- tetramethyl
Base urea hexafluorophosphoric acid ester, 2- (7- azo benzotriazole)-N, N, N', the special condensing agent of N'- tetramethylurea hexafluorophosphoric acid ester, card,
One of hexafluorophosphoric acid benzotriazole -1- base-oxygroup tripyrrole alkyl phosphorus, condensation reagent 2 be selected from 1- hydroxy benzo triazole,
One of N- hydroxyl -7- azo benzotriazole；Condensation reagent 3 is selected from N, N- diisopropylethylamine；And N, N- dicyclohexyl
Carbodiimide or N, N- diisopropylcarbodiimide not with diisopropylethylamine combined application.
The ratio of the amount of the substance of each reagent described in step (2) is preferred: amino acid: condensation reagent 1: condensation reagent 2:
Condensation reagent 3: resin=6~3:6~3:6~3:12~6:1；Preferably 20 DEG C~60 DEG C of the temperature of the condensation reaction, reaction
Time preferably 1 hour~4 hours.
The preferred redistilled water of cutting reagent, triethylsilane, 1,2 dithiothreitol (DTT)s, phenol, benzene first described in step (3)
The mixed liquor of at least one of thioether, ammonium iodide, tri isopropyl silane, methyl phenyl ethers anisole and dithioglycol and trifluoroacetic acid, it is described
Mixed liquor in trifluoroacetic acid volume ratio be not less than 80%；Cutting examination is added into Teriparatide-resin complexes of protection
Preferably 0 DEG C~60 DEG C of the temperature that agent is reacted, the reaction time preferably 1 hour~4 hours.
Purification condition described in step (4) is preferred: preparing column packing is that carbon carries silica gel type, and mobile phase is that mass ratio is equal
For the water, acetonitrile or methanol solution of 0.1%TFA.
Preparation method of the present invention further preferably includes the following steps:
(1) Fmoc-Phe- resin complexes are prepared
Resin is added into the reaction vessel with filter device.Solvent DMF is added into the container, submerges resin,
1 hour is stood, filtering.
After dissolving Fmoc-Phe-OH and esterifying reagent with DMF, be added in resin, reacted at 20 DEG C~60 DEG C 1h~
10h.After reaction, 10 times of resin volume of methanol/n,N-diisopropylethylamine mixed solution (volume ratio 1:1) is added,
It is filtered after room temperature reaction 1h.Then solvent DMF is added, washs resin six times, filtering.Wherein the resin is 2- chlorine triphen first
Base chlorine resin, the esterifying reagent are DIEA, and the ratio between each substance is n (Fmoc-Phe-OH): n (DIEA): n (resin)
=3~0.1:60~2:1.
(2) Teriparatide-resin complexes of preparation protection
The PIP/DMF solution that volume ratio is 1:1~4 is added in above-mentioned Fmoc-Phe- resin complexes, carries out remove-insurance
Shield is reacted, and 10min~60min is reacted at 20 DEG C~60 DEG C.After completion of the reaction, it filters.Then DMF is added and washs resin, filtering.
Second amino acid of C-terminal [Fmoc-Asn (Trt)-OH] and condensation reagent of Teriparatide are added into reactor.
1h~4h is reacted at 20 DEG C~60 DEG C.DMF is added after reaction and washs resin, filtering.The condensation reagent is condensation examination
Agent 1 (N, N- dicyclohexylcarbodiimide, N, N- diisopropylcarbodiimide, benzotriazole-N, N, N', N'- tetramethylurea six
Fluorophosphoric acid ester, 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester block special condensing agent, hexafluoro phosphorus
One of sour benzotriazole -1- base-oxygroup tripyrrole alkyl phosphorus), condensation reagent 2 (1- hydroxy benzo triazole, N- hydroxyl -
One of 7- azo benzotriazole) and condensation reagent 3 (diisopropylethylamine) combination, and DIC or DCC not with DIEA group
Close application.Wherein the ratio between the amount of reactant species are as follows: n [Fmoc-Asn (Trt)-OH]: n (condensation reagent 1): n (condensation
Reagent 2): n (condensation reagent 3): n (resin)=6~3:6~3:6~3:12~6:1.
According to the sequence of Teriparatide C-terminal to N-terminal, amino acid is replaced, recycles the operation of deprotection reaction and condensation reaction,
Synthesize Teriparatide-resin complexes of protection.Wherein the ratio between described amount of reactant species and reaction condition are same as
Fmoc-Asn(Trt)-OH。
(3) Teriparatide crude product is prepared
Cutting reagent is added in above-mentioned resin, reaction condition is to react 1 hour~4 hours at 0 DEG C~60 DEG C.Described
Cutting reagent is trifluoroacetic acid, redistilled water, triethylsilane, 1,2- dithiothreitol (DTT), phenol, thioanisole, ammonium iodide, three different
The mixed liquor of at least one of propyl silane, methyl phenyl ethers anisole and dithioglycol and trifluoroacetic acid, the body of trifluoroacetic acid in mixed liquor
Product is than being not less than 80%.Filtering, filtrate is added in ether and is precipitated, Teriparatide crude product is obtained after centrifugation after having reacted.
(4) Teriparatide fine work is prepared
Teriparatide crude product volume ratio is dissolved for 5% methanol or acetonitrile solution, using preparative efficient liquid phase
Chromatogram purification obtains Teriparatide fine work.Preparing column is C4/C8/C18Type reverse-phase chromatographic column, mobile phase are containing 0.1%TFA's
One or more of water, the acetonitrile containing 0.1%TFA and methanol containing 0.1%TFA.
The beneficial effects of the present invention are: use 2- chlorine trityl chloride resin for solid phase carrier, avoid C-terminal amino acid with
The risk of racemization occurs for resin esterification；Using the amino acid of pendant reactive group full guard as raw material, reduces in synthesis process and send out
The probability of raw side reaction；Ammonium iodide is added in cutting reagent, avoids the generation of oxidation impurities Met (O)；In conclusion this
Invention provides a simplicity, the few Teriparatide synthesis technology of side reaction.
Some common abbreviations have following meaning in the present invention:
Fmoc: fluorenes methoxy carbonyl acyl group
TFA: trifluoracetic acid
EDT: dithioglycol
Phenol: phenol
Thioanisole: thioanisole
TES: triethylsilane
TIS: tri isopropyl silane
DTT:1,2 dithiothreitol (DTT)
Anisole: methyl phenyl ethers anisole
NH4I: ammonium iodide
MeOH: methanol
DCM: methylene chloride
DMF:N, dinethylformamide
PIP: piperidines
CTC resin:2- chlorine trityl chloride resin
DMAP: dimethylamino naphthyridine
DIEA:N, N- diisopropylethylamine
BOP: block special condensing agent
HOBt:1- hydroxy benzo triazole
DIC:N, N- Diisopropylcarbodiimide
DCC:N, N- dicyclohexylcarbodiimide
HOAt:N- hydroxyl -7- azo benzotriazole
HBTU: benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
HATU:2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
PyBOP: hexafluorophosphoric acid benzotriazole -1- base-oxygroup tripyrrole alkyl phosphorus
Fmoc-Asn (Trt)-OH:N- (9-fluorenylmethyloxycarbonyl)-N γ-trityl-asparagine
Specific embodiment
The present invention is further illustrated by following instance, these examples should not be construed as limitation of the present invention.
Embodiment 1: Teriparatide is prepared
(1) Fmoc-Phe- resin complexes are prepared
CTC resin (1.0mmol/g) 1g is added into the reactor with filter device, injects 10ml DMF, stands
1h, filtering.
3mmol Fmoc-Phe-OH and 60mmol DIEA is taken, is dissolved in 10ml DMF, is added in resin, it is anti-at 20 DEG C
Answer 1h.After reaction, it filters.10ml methanol/n,N-diisopropylethylamine mixed solution (volume ratio 1:1), room temperature is added
It is filtered after reaction 1h.Then 10ml DMF is added, washs resin six times, filtering.
(2) Teriparatide-resin complexes of preparation protection
The PIP/DMF solution that 10ml volume ratio is 1:1 is added into reactor, reacts 10min at 20 DEG C.End of reaction
Afterwards, it filters.Then 10ml DMF is added to wash resin six times, filtering.
3mmol Fmoc-Asn (Trt)-OH, 3mmol HBTU, 3mmol HOBt and 6mmol DIEA are taken, 10ml is dissolved in
In DMF, acquired solution is added in reactor, reacts 1h at 20 DEG C.After reaction, it filters.Then 10ml DMF is added,
Washing resin six times, filtering.
According to the sequence of Teriparatide C-terminal to N-terminal, amino acid is replaced, recycles the operation of deprotection reaction and condensation reaction,
Synthesize Teriparatide-resin complexes of protection.
(3) Teriparatide crude product is prepared
Into Teriparatide-resin complexes of above-mentioned protection be added 10ml cutting reagent (based on volumn concentration,
TFA:phenol:thioansole: iodate aqueous ammonium=87.5%:5%:2.5%:5%, the concentration of iodate aqueous ammonium are
100mg/ml), 2h is reacted at 0 DEG C.Filtering.Filtrate is added in the ether of 10 times of volumes and is precipitated, Te Lipa is obtained after centrifugation
Peptide crude product.
(4) Teriparatide fine work is prepared
Teriparatide crude product is dissolved in the methanol aqueous solution that volume ratio is 5%, obtains spy through high-efficient liquid phase chromatogram purification
Vertical pa peptide fine work, obtains 1.33g, purity 99.1%, yield 32.1% after freeze-drying.Preparation condition are as follows: C18Prepare column, two kinds
Mobile phase is respectively that mass ratio is the water of 0.1%TFA, methanol solution.
Embodiment 2: Teriparatide is prepared
(1) Fmoc-Phe- resin complexes are prepared
CTC resin (1.0mmol/g) 1g is added into the reactor with filter device, injects 10ml DMF, stands
1h, filtering.
0.4mmol Fmoc-Phe-OH and 8mmol DIEA is taken, is dissolved in 10ml DMF, is added in resin, at 30 DEG C
React 2h.After reaction, it filters.10ml methanol/n,N-diisopropylethylamine mixed solution (volume ratio 1:1), room is added
It is filtered after temperature reaction 1h.Then 10ml DMF is added, washs resin six times, filtering.
(2) Teriparatide-resin complexes of preparation protection
The PIP/DMF solution that 10ml volume ratio is 1:4 is added into reactor, reacts 10min at 30 DEG C.End of reaction
Afterwards, it filters.Then 10ml DMF is added to wash resin six times, filtering.
6mmol Fmoc-Asn (Trt)-OH, 6mmol DIC, 6mmol HOBt is taken, is dissolved in 10ml DMF, gained is molten
Liquid is added in reactor, reacts 2h at 30 DEG C.After reaction, it filters.Then 10ml DMF is added, washs resin six times,
Filtering.
According to the sequence of Teriparatide C-terminal to N-terminal, amino acid is replaced, recycles the operation of deprotection reaction and condensation reaction,
Synthesize Teriparatide-resin complexes of protection.
(3) Teriparatide crude product is prepared
Into Teriparatide-resin complexes of above-mentioned protection be added 10ml cutting reagent (based on volumn concentration,
TFA:phenol:thioansole:EDT: iodate aqueous ammonium=82.5%:5%:5%:2.5%:5%, iodate aqueous ammonium
Concentration be 100mg/ml), react 3h at 20 DEG C.Filtering.Filtrate is added in the ether of 10 times of volumes and is precipitated, after centrifugation
To Teriparatide crude product.
(4) Teriparatide fine work is prepared
Teriparatide crude product is dissolved in the acetonitrile solution that volume ratio is 5%, obtains spy through high-efficient liquid phase chromatogram purification
Vertical pa peptide fine work, obtains 0.41g, purity 99.2%, yield 33.2% after freeze-drying.Preparation condition are as follows: C18Prepare column, two kinds
Mobile phase is respectively that mass ratio is the water of 0.1%TFA, acetonitrile solution.
Embodiment 3: Teriparatide is prepared
(1) Fmoc-Phe- resin complexes are prepared
CTC resin (1.0mmol/g) 1g is added into the reactor with filter device, injects 10ml DMF, stands
1h, filtering.
0.1mmol Fmoc-Phe-OH and 2mmol DIEA is taken, is dissolved in 10ml DMF, is added in resin, at 60 DEG C
React 10h.After reaction, it filters.10ml methanol/n,N-diisopropylethylamine mixed solution (volume ratio 1:1), room is added
It is filtered after temperature reaction 1h.Then 10ml DMF is added, washs resin six times, filtering.
(2) Teriparatide-resin complexes of preparation protection
The PIP/DMF solution that 10ml volume ratio is 1:2 is added into reactor, reacts 60min at 60 DEG C.End of reaction
Afterwards, it filters.Then 10ml DMF is added to wash resin six times, filtering.
4mmol Fmoc-Asn (Trt)-OH, 4mmol PyBOP, 4mmol HOBt and 8mmol DIEA are taken, 10ml is dissolved in
In DMF, acquired solution is added in reactor, reacts 4h at 60 DEG C.After reaction, it filters.Then 10ml DMF is added,
Washing resin six times, filtering.
According to the sequence of Teriparatide C-terminal to N-terminal, amino acid is replaced, recycles the operation of deprotection reaction and condensation reaction,
Synthesize Teriparatide-resin complexes of protection.
(3) Teriparatide crude product is prepared
Into Teriparatide-resin complexes of above-mentioned protection be added 10ml cutting reagent (based on volumn concentration,
TFA:phenol:EDT: iodate aqueous ammonium=87.5%:5%:2.5%:5%, the concentration of iodate aqueous ammonium are 100mg/
Ml), 4h is reacted at 60 DEG C.Filtering.Filtrate is added in the ether of 10 times of volumes and is precipitated, it is thick that Teriparatide is obtained after centrifugation
Product.
(4) Teriparatide fine work is prepared
Teriparatide crude product is dissolved in the methanol aqueous solution that volume ratio is 5%, obtains spy through high-efficient liquid phase chromatogram purification
Vertical pa peptide fine work, obtains 0.14g, purity 99.3%, yield 34.0% after freeze-drying.Preparation condition are as follows: C18Prepare column, two kinds
Mobile phase is respectively that mass ratio is the water of 0.1%TFA, methanol solution.
Embodiment 4: check experiment
According to document " synthesis in solid state of N-Terminal 1-34 Peptide of Human Parathyroid Hormone, " synthesis chemistry " magazine 2007,15 (3): 388-390 "
Disclosed in method synthesize Teriparatide, in addition to resin used, Trp raw material, condensation reagent and cutting scheme, remaining condition and this
Invention the method is essentially identical, and final product purity is greater than 99%, yield 23.3%.And method of the invention is used,
While guaranteeing that purity is greater than 99%, yield can be made to improve about 10 percentage points.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of preparation method of Teriparatide, which comprises the following steps:
(1) Fmoc-Phe- resin complexes are prepared: dissolving first amino acid of Teriparatide C-terminal with N,N-dimethylformamide
After Fmoc-Phe-OH and esterifying reagent, it is added to anti-with carrying out being esterified in the resin after n,N-Dimethylformamide immersion treatment
It answers, after reaction, filters after methanol/n,N-diisopropylethylamine mixed liquor (volume ratio 1:1) reaction 1h is added, then use
N,N-Dimethylformamide washs resin, obtains Fmoc-Phe- resin complexes；
(2) it Teriparatide-resin complexes of preparation protection: is added in the Fmoc-Phe- resin complexes obtained to step (1)
Fmoc deprotecting regent removes Fmoc protecting group, filters after reaction and washs resin with n,N-Dimethylformamide and obtain
Then second amino acid of C-terminal and condensation reagent of Teriparatide is added in Phe- resin complexes into Phe- resin complexes
Condensation reaction is carried out, washs resin with dimethylformamide after condensation reaction；It is suitable from C-terminal to N-terminal according to Teriparatide
Sequence replaces amino acid, repeats Fmoc deprotection reaction and condensation reaction, the Teriparatide-resin complexes protected；
(3) it prepares Teriparatide crude product: cutting reagent being added into Teriparatide-resin complexes of protection, remove polypeptide side
Teriparatide is simultaneously cleaved by chain protecting group from resin, obtains Teriparatide crude product after ether precipitating；
(4) it prepares Teriparatide fine work: Teriparatide crude product being isolated and purified using reversed-phase high performance liquid chromatography, obtains Teriparatide
Fine work.
2. the preparation method of Teriparatide according to claim 1, which is characterized in that resin described in step (1) is
Wang Shuzhi or 2- chlorine resin；It is preferred that 2- chlorine trityl chloride resin.
3. the preparation method of Teriparatide according to claim 1, which is characterized in that the examination of esterification described in step (1)
Agent is n,N-diisopropylethylamine, and the ratio of the amount of the substance between each substance is n (Fmoc-Phe-OH): n (N, N- bis-
Wopropyl ethyl amine): n (2- chlorine trityl chloride resin)=3~0.1:60~2:1；The temperature of the esterification be 20 DEG C~
60 DEG C, the reaction time is 1 hour~10 hours.
4. the preparation method of Teriparatide according to claim 1, which is characterized in that Fmoc described in step (2) is de-
Protection reagent is the mixture of the piperidines that volume ratio is 1:1~4 and dimethylformamide, the temperature of the Fmoc deprotection reaction
It is 20 DEG C~60 DEG C, the reaction time is 10min~60min.
5. the preparation method of Teriparatide according to claim 1, which is characterized in that amino acid described in step (2)
Protecting group to be respectively as follows: the α amino of all amino acid be Fmoc protecting group, lysine side chains hydroxyl protection base is tert-butyl,
Aspartic acid and glutamate side chain carboxyl-protecting group are tert-butyl, and glutamine and asparagine side chain protecting group are triphen first
Base, histidine side chains protecting group are trityl or tertbutyloxycarbonyl, and lysine and Trp side chain protecting group are tertiary butyloxycarbonyl
Base, arginine side chain protecting group are 2,2,4,6,7- pentamethyl Dihydrobenzofuranes -5- sulfonyls.
6. the preparation method of Teriparatide according to claim 1, which is characterized in that the examination of condensation described in step (2)
Agent be condensation reagent 1, condensation reagent 2 and condensation reagent 3 combination, condensation reagent 1 be selected from N, N- dicyclohexylcarbodiimide, N,
N- diisopropylcarbodiimide, benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester, 2- (three nitrogen of 7- azo benzo
Azoles)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester, card special condensing agent, hexafluorophosphoric acid benzotriazole -1- base-oxygroup tripyrrole
One of alkyl phosphorus, condensation reagent 2 are selected from one of 1- hydroxy benzo triazole, N- hydroxyl -7- azo benzotriazole；
Condensation reagent 3 is selected from N, N- diisopropylethylamine；And N, N- dicyclohexylcarbodiimide or N, N- diisopropylcarbodiimide are not
With diisopropylethylamine combined application.
7. the preparation method of Teriparatide according to claim 1, which is characterized in that each reagent described in step (2)
Substance amount ratio are as follows: amino acid: condensation reagent 1: condensation reagent 2: condensation reagent 3: resin=6~3:6~3:6~3:
12~6:1；The temperature of the condensation reaction is 20 DEG C~60 DEG C, and the reaction time is 1 hour~4 hours.
8. the preparation method of Teriparatide according to claim 1, which is characterized in that the examination of cutting described in step (3)
Agent is redistilled water, triethylsilane, 1,2 dithiothreitol (DTT)s, phenol, thioanisole, ammonium iodide, tri isopropyl silane, methyl phenyl ethers anisole
Mixed liquor at least one of dithioglycol with trifluoroacetic acid, the volume ratio of trifluoroacetic acid is not less than in the mixed liquor
80%；It is 0 DEG C~60 DEG C that the temperature that cutting reagent is reacted is added into Teriparatide-resin complexes of protection, reaction
Time is 1 hour~4 hours.
9. the preparation method of Teriparatide according to claim 1, which is characterized in that purifying item described in step (4)
Part are as follows: preparing column packing is that carbon carries silica gel type, and mobile phase is water, acetonitrile or the methanol solution that mass ratio is 0.1%TFA.
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| CN109879954A (en) * | 2019-03-27 | 2019-06-14 | 哈尔滨吉象隆生物技术有限公司 | A kind of synthetic method of teriparatide |
| CN109897099A (en) * | 2019-03-27 | 2019-06-18 | 哈尔滨吉象隆生物技术有限公司 | A kind of preparation method of Teriparatide |
| WO2021114788A1 (en) * | 2019-12-10 | 2021-06-17 | 深圳翰宇药业股份有限公司 | Teriparatide impurity f |
| CN114456254A (en) * | 2021-12-29 | 2022-05-10 | 江苏诺泰澳赛诺生物制药股份有限公司 | Synthesis method of C-type natriuretic peptide analogue Vosolitide |
| CN116640100A (en) * | 2023-05-18 | 2023-08-25 | 河北圣雪大成制药有限责任公司 | A kind of solid-phase synthesis method of Freilaner |
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| CN109879954A (en) * | 2019-03-27 | 2019-06-14 | 哈尔滨吉象隆生物技术有限公司 | A kind of synthetic method of teriparatide |
| CN109897099A (en) * | 2019-03-27 | 2019-06-18 | 哈尔滨吉象隆生物技术有限公司 | A kind of preparation method of Teriparatide |
| CN109879954B (en) * | 2019-03-27 | 2023-03-31 | 哈尔滨吉象隆生物技术有限公司 | Synthetic method of teriparatide |
| WO2021114788A1 (en) * | 2019-12-10 | 2021-06-17 | 深圳翰宇药业股份有限公司 | Teriparatide impurity f |
| CN114456254A (en) * | 2021-12-29 | 2022-05-10 | 江苏诺泰澳赛诺生物制药股份有限公司 | Synthesis method of C-type natriuretic peptide analogue Vosolitide |
| CN114456254B (en) * | 2021-12-29 | 2023-06-16 | 江苏诺泰澳赛诺生物制药股份有限公司 | Synthesis method of C-type natriuretic peptide analogue Vosoritide |
| CN116640100A (en) * | 2023-05-18 | 2023-08-25 | 河北圣雪大成制药有限责任公司 | A kind of solid-phase synthesis method of Freilaner |
| CN119039375A (en) * | 2024-10-28 | 2024-11-29 | 成都圣诺生物多肽科技有限公司 | Solid phase synthesis method of peptides |
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