CN116813693B - Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis - Google Patents
Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis Download PDFInfo
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- CN116813693B CN116813693B CN202311093705.8A CN202311093705A CN116813693B CN 116813693 B CN116813693 B CN 116813693B CN 202311093705 A CN202311093705 A CN 202311093705A CN 116813693 B CN116813693 B CN 116813693B
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- Prior art keywords
- polypeptide
- arg
- linker
- pbf
- boc
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 83
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 82
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 81
- 150000001875 compounds Chemical class 0.000 title claims abstract description 43
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 43
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 41
- 150000001721 carbon Chemical class 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 23
- 150000001413 amino acids Chemical class 0.000 claims abstract description 9
- 238000010511 deprotection reaction Methods 0.000 claims abstract description 7
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 11
- 238000005859 coupling reaction Methods 0.000 claims description 11
- UPMGJEMWPQOACJ-UHFFFAOYSA-N 2-[4-[(2,4-dimethoxyphenyl)-(9h-fluoren-9-ylmethoxycarbonylamino)methyl]phenoxy]acetic acid Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(O)=O)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 UPMGJEMWPQOACJ-UHFFFAOYSA-N 0.000 claims description 10
- 230000008878 coupling Effects 0.000 claims description 10
- 238000010168 coupling process Methods 0.000 claims description 10
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 10
- -1 hexafluorophosphate Chemical compound 0.000 claims description 8
- VVQIIIAZJXTLRE-QMMMGPOBSA-N (2s)-2-amino-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound CC(C)(C)OC(=O)NCCCC[C@H](N)C(O)=O VVQIIIAZJXTLRE-QMMMGPOBSA-N 0.000 claims description 7
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 6
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000009833 condensation Methods 0.000 claims description 4
- 230000005494 condensation Effects 0.000 claims description 4
- 239000012043 crude product Substances 0.000 claims description 4
- 125000006239 protecting group Chemical group 0.000 claims description 4
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 3
- TZCYLJGNWDVJRA-UHFFFAOYSA-N 6-chloro-1-hydroxybenzotriazole Chemical compound C1=C(Cl)C=C2N(O)N=NC2=C1 TZCYLJGNWDVJRA-UHFFFAOYSA-N 0.000 claims description 3
- 239000007821 HATU Substances 0.000 claims description 3
- 239000012317 TBTU Substances 0.000 claims description 3
- 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 3
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims description 3
- AJDPNPAGZMZOMN-UHFFFAOYSA-N diethyl (4-oxo-1,2,3-benzotriazin-3-yl) phosphate Chemical compound C1=CC=C2C(=O)N(OP(=O)(OCC)OCC)N=NC2=C1 AJDPNPAGZMZOMN-UHFFFAOYSA-N 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 210000004899 c-terminal region Anatomy 0.000 abstract description 8
- 150000001408 amides Chemical group 0.000 abstract description 4
- 238000007098 aminolysis reaction Methods 0.000 abstract description 2
- 150000002148 esters Chemical class 0.000 abstract 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 61
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 57
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 24
- 239000007787 solid Substances 0.000 description 24
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 12
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 9
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 7
- 125000004185 ester group Chemical group 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 230000007017 scission Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 125000001033 ether group Chemical group 0.000 description 6
- 238000001819 mass spectrum Methods 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 6
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 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 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000000543 intermediate Substances 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 5
- 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 description 4
- 108010016626 Dipeptides Proteins 0.000 description 4
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000012295 chemical reaction liquid Substances 0.000 description 4
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- JWUJQDFVADABEY-UHFFFAOYSA-N 2-methyltetrahydrofuran Chemical compound CC1CCCO1 JWUJQDFVADABEY-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- GPDHNZNLPKYHCN-DZOOLQPHSA-N [[(z)-(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy-morpholin-4-ylmethylidene]-dimethylazanium;hexafluorophosphate Chemical compound F[P-](F)(F)(F)(F)F.CCOC(=O)C(\C#N)=N/OC(=[N+](C)C)N1CCOCC1 GPDHNZNLPKYHCN-DZOOLQPHSA-N 0.000 description 3
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000005086 pumping Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- DYWUPCCKOVTCFZ-LBPRGKRZSA-N (2s)-2-amino-3-[1-[(2-methylpropan-2-yl)oxycarbonyl]indol-3-yl]propanoic acid Chemical compound C1=CC=C2N(C(=O)OC(C)(C)C)C=C(C[C@H](N)C(O)=O)C2=C1 DYWUPCCKOVTCFZ-LBPRGKRZSA-N 0.000 description 2
- GVIXTVCDNCXXSH-AWEZNQCLSA-N (2s)-2-amino-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]pentanoic acid Chemical compound OC(=O)[C@@H](N)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C GVIXTVCDNCXXSH-AWEZNQCLSA-N 0.000 description 2
- OHVLMTFVQDZYHP-UHFFFAOYSA-N 1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-2-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound N1N=NC=2CN(CCC=21)C(CN1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)=O OHVLMTFVQDZYHP-UHFFFAOYSA-N 0.000 description 2
- PMKKIDFHWBBGDA-UHFFFAOYSA-N 2-(2,5-dioxopyrrol-1-yl)ethyl methanesulfonate Chemical compound CS(=O)(=O)OCCN1C(=O)C=CC1=O PMKKIDFHWBBGDA-UHFFFAOYSA-N 0.000 description 2
- WZFUQSJFWNHZHM-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)N1CC2=C(CC1)NN=N2 WZFUQSJFWNHZHM-UHFFFAOYSA-N 0.000 description 2
- JQMFQLVAJGZSQS-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-N-(2-oxo-3H-1,3-benzoxazol-6-yl)acetamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)NC1=CC2=C(NC(O2)=O)C=C1 JQMFQLVAJGZSQS-UHFFFAOYSA-N 0.000 description 2
- YJLUBHOZZTYQIP-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=N2 YJLUBHOZZTYQIP-UHFFFAOYSA-N 0.000 description 2
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 2
- ZTQNECFPCYNOQY-UHFFFAOYSA-N COC12C(C=CC=C1)S2 Chemical compound COC12C(C=CC=C1)S2 ZTQNECFPCYNOQY-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- KPFBUSLHFFWMAI-HYRPPVSQSA-N [(8r,9s,10r,13s,14s,17r)-17-acetyl-6-formyl-3-methoxy-10,13-dimethyl-1,2,7,8,9,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-17-yl] acetate Chemical compound C1C[C@@H]2[C@](CCC(OC)=C3)(C)C3=C(C=O)C[C@H]2[C@@H]2CC[C@](OC(C)=O)(C(C)=O)[C@]21C KPFBUSLHFFWMAI-HYRPPVSQSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 125000003545 alkoxy group Chemical group 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 125000005605 benzo group Chemical group 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000011056 performance test Methods 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 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 description 1
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 description 1
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical class C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- VSTXCZGEEVFJES-UHFFFAOYSA-N 1-cycloundecyl-1,5-diazacycloundec-5-ene Chemical compound C1CCCCCC(CCCC1)N1CCCCCC=NCCC1 VSTXCZGEEVFJES-UHFFFAOYSA-N 0.000 description 1
- VUCNQOPCYRJCGQ-UHFFFAOYSA-N 2-[4-(hydroxymethyl)phenoxy]acetic acid Chemical compound OCC1=CC=C(OCC(O)=O)C=C1 VUCNQOPCYRJCGQ-UHFFFAOYSA-N 0.000 description 1
- ZKDXHLFLKKWCBY-UHFFFAOYSA-N 4-Hydroxymethyl-3-methoxyphenoxyacetic acid Chemical compound COC1=CC(OCC(O)=O)=CC=C1CO ZKDXHLFLKKWCBY-UHFFFAOYSA-N 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- LCFXLZAXGXOXAP-UHFFFAOYSA-N ethyl 2-cyano-2-hydroxyiminoacetate Chemical compound CCOC(=O)C(=NO)C#N LCFXLZAXGXOXAP-UHFFFAOYSA-N 0.000 description 1
- ZIUSEGSNTOUIPT-UHFFFAOYSA-N ethyl 2-cyanoacetate Chemical compound CCOC(=O)CC#N ZIUSEGSNTOUIPT-UHFFFAOYSA-N 0.000 description 1
- FAMRKDQNMBBFBR-UHFFFAOYSA-N ethyl n-ethoxycarbonyliminocarbamate Chemical compound CCOC(=O)N=NC(=O)OCC FAMRKDQNMBBFBR-UHFFFAOYSA-N 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- GNOIPBMMFNIUFM-UHFFFAOYSA-N hexamethylphosphoric triamide Chemical compound CN(C)P(=O)(N(C)C)N(C)C GNOIPBMMFNIUFM-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- WOOWBQQQJXZGIE-UHFFFAOYSA-N n-ethyl-n-propan-2-ylpropan-2-amine Chemical compound CCN(C(C)C)C(C)C.CCN(C(C)C)C(C)C WOOWBQQQJXZGIE-UHFFFAOYSA-N 0.000 description 1
- VWBWQOUWDOULQN-UHFFFAOYSA-N nmp n-methylpyrrolidone Chemical compound CN1CCCC1=O.CN1CCCC1=O VWBWQOUWDOULQN-UHFFFAOYSA-N 0.000 description 1
- NLCYPSDJSMBAMU-UHFFFAOYSA-N octacosan-4-ol Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCC(O)CCC NLCYPSDJSMBAMU-UHFFFAOYSA-N 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 108010004034 stable plasma protein solution Proteins 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- OHSJPLSEQNCRLW-UHFFFAOYSA-N triphenylmethyl radical Chemical compound C1=CC=CC=C1[C](C=1C=CC=CC=1)C1=CC=CC=C1 OHSJPLSEQNCRLW-UHFFFAOYSA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a tag compound for polypeptide synthesis and application thereof in polypeptide synthesis, and relates to the technical field of organic synthesis. The tag compound introduces amino acid containing longer side chains on the 2, 4-disubstituted carbon-based tags, reduces the degree of ester-based aminolysis in the deprotection process, and increases the synthesis yield of the polypeptide. In addition, the invention introduces the C-terminal amide linker to prepare the polypeptide with the C-terminal amide, thereby expanding the application range of the 2, 4-disubstituted carbon-based label in the prior art.
Description
Technical Field
The invention relates to the technical field of organic synthesis, in particular to a tag compound for polypeptide synthesis and application thereof in polypeptide synthesis.
Background
Polypeptide compounds are mostly prepared by conventional solid-phase polypeptide synthesis (solid-phase-phase peptide synthesis, SPPS). However, solid-phase polypeptide synthesis methods suffer from several drawbacks, including: the purity of the final product is lower, the consumption of raw materials is overlarge, and more three wastes are generated. The Liquid phase polypeptide synthesis method (LPPS) meets the requirements of green chemistry and large-scale production, and the current Liquid phase synthesis method adds a soluble C-terminal Tag (Tag) to solidify the growing polypeptide chain. However, because of the great lipophilicity of the labels, the polypeptide-label adducts formed are soluble in organic solvents and are significantly different from reagents and byproducts, and the polypeptide-label adduct intermediates can be obtained by simple precipitation, filtration or extraction, thereby achieving the purpose of removing excess reagents and byproducts by elution similar to solid phase synthesis. Representative C-terminal tags are shown below:
LPPS can be produced on a kilogram or ton scale, can be automated, and has low energy input in the production process; the labor force is less; low energy and material consumption cost. The presence of long fatty chains can significantly increase the hydrophobicity of the polypeptide-tag, which is not greatly affected by the growth of the polypeptide chain, and therefore, long fatty chain based tags have also been used in LPPS. These carbon-based tags can be precipitated in a polar solvent such as acetonitrile or methanol, providing a basis for the separation and extraction of intermediates in amino acid condensation and Fmoc deprotection. Methods for producing polypeptides using C-terminal tags are disclosed in patent US20180215782, CN107406480B, US20140107319A 1.
The representative C-terminal tags described above have unique properties. More than 80% trifluoroacetic acid is required for releasing the polypeptide from the carrier, and in the preparation of the polypeptide, the 3, 5-disubstituted carbon-based label is not subjected to color reaction by acid treatment. The 3, 5-disubstituted carbon-based label is suitable for an acid-resistant Boc method polypeptide synthesis strategy. The polypeptide-carrier formed by the 2, 4-disubstituted carbon-based label is sensitive to acidic conditions, and only 2% TFA can release the polypeptide from the carrier, so that the polypeptide-carrier is suitable for preparing polypeptide fragments containing protecting groups. And the 2, 4-disubstituted carbon-based label shows deeper red when meeting acid, and the reaction is conveniently tracked in the process of preparing the polypeptide. The carbon-based tag is only suitable for the preparation of middle-short peptides because of the reduced properties of long-chain alkanes, the enhanced properties of polypeptides, and the difficulty of subsequent operations with the growth of peptide chains attached to the carbon-based tag.
However, in the process of preparing the protected polypeptide fragment by actually applying the 2, 4-disubstituted carbon-based label, the condition that the carrier is removed during the coupling and Fmoc removal of the polypeptide is found, so that the final weight gain of the polypeptide-carrier is small, and particularly, in the Fmoc removal step of the dipeptide, some dipeptides are separated more from the synthetic carrier. In order to overcome the above-mentioned defects of 2, 4-disubstituted carbon-based labels and utilize the characteristic of convenient central control, the method is effectively applied to the preparation of polypeptides, and the improvement of the polypeptide synthesis vector is very necessary.
Disclosure of Invention
The invention aims to provide a tag compound for polypeptide synthesis and application thereof in polypeptide synthesis, and the tag compound increases the yield of polypeptide synthesis, can regulate the solubility of a subsequent polypeptide-carrier, and is convenient for polypeptide coupling or purification.
Abbreviations used in the present invention and meanings corresponding to English are shown in the following table:
DIEA | n, N-diisopropylethylamine |
DMF | N, N-dimethylformamide |
NMP | N-methylpyrrolidone |
Fmoc | 9-fluorenylmethoxycarbonyl |
DBU | 1, 8-diazabicyclo undec-7-ene |
DIC | N, N' -diisopropylcarbodiimide |
HOBt | 1-hydroxybenzotriazoles |
HATU | 2- (7-Oxybenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate |
HBTU | Benzo (E) benzo (Etriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate |
TBTU | O- (IH-benzotriazol-1-yl) -N, N, N ', N' -tetramethylisourea boron tetrafluoride |
COMU | (2-oximino-cyanoacetic acid ethyl ester) -N, N-dimethyl-morpholinyl urea hexafluorophosphate |
Trt | Triphenylmethyl radical |
Aloc | (2-propenoxy) carbonyl group |
OtBu | Tert-butyl ester group |
tBu | Tert-butyl group |
Boc | Boc-group |
Cbz | Benzyloxycarbonyl group |
Z | Benzyloxycarbonyl group |
Oxyma pure | 2-Oxime cyanoacetic acid ethyl ester |
Pbf | Azodicarboxylic acid diethyl ester |
HMPA Linker | 4-hydroxymethyl-3-methoxyphenoxyacetic acid |
Rink amide | 4- [ (2, 4-Dimethoxyphenyl) (Fmoc-amino) methyl]Phenoxyacetamide |
Fmoc-Rink amide Linker | 4- [ (2, 4-Dimethoxyphenyl) (FMOC-amino) methyl]Phenoxyacetic acid |
In order to achieve the above object, the present invention has the following technical scheme:
in one aspect, the present invention provides a tag compound for polypeptide synthesis, wherein the chemical structural formula of the tag compound is as follows:
wherein R is a linear alkane of 10 to 30C atoms or an alkane containing a branched chain with a main chain length of 14 to 40C atoms;
-O-A-L is-O-AA 1 -AA 2 -L or-O-AA 1 -AA 2 -AA 3 -L;
AA 1 Selected from Lys (Boc), lys (Z), lys (Aloc) or Nle;
AA 2 selected from Arg (pbf), lys (Boc), lys (Z), glu (OtBu), asp (OtBu), trp (Boc), gln (Trt), ser (tBu), thr (tBu), leu, val, ala, met, ile, pro, gly, tyr (tBu), asn (Trt) or Cys (Trt);
AA 3 selected from Arg (pbf), lys (Boc), lys (Z), glu (OtBu), asp (OtBu), trp (Boc), gln (Trt), ser (tBu), thr (tBu), leu, val, ala, met, ile, pro, gly, tyr (tBu), asn (Trt) or Cys (Trt).
In one aspect of the inventionIn some embodiments, the AA 1 And AA (alpha) 2 The materials in (a) can be repeatedly selected.
Preferably, L in the general formula is a Linker synthesized by polypeptide, and the Linker is selected from one or more of HMPA Linker and Rink amide Linker;
the HMPA Linker structure is as follows:
the structure of Rink amide Linker is as follows:
。
preferably, in the general formula of the tag compound described above, R is a linear alkane having 14 to 30C atoms or an alkane having a branched chain and a main chain length of 14 to 30C atoms.
Preferably, the present invention provides a method for preparing the above-mentioned tag compound, the method comprising the steps of:
(1) Introducing Fmoc-amino acid 1 into a tag compound to obtain Fmoc-amino acid-2, 4 disubstituted tag compound;
(2) Fmoc is removed by using organic alkali to obtain an H-amino acid 1-2,4 disubstituted label compound;
(3) Condensing Fmoc-amino acid 2 with an H-amino acid 1-tag compound to obtain Fmoc-amino acid 2-amino acid 1-2,4 disubstituted tag compounds;
(4) Fmoc is removed by using organic alkali to obtain an H-amino acid 2-amino acid 1-2,4 disubstituted label compound;
(5) Introducing a polypeptide synthesis linker to obtain a synthon-amino acid 2-amino acid 1-2,4 disubstituted label compound;
(6) Repeating (4) and (5) to obtain the synthon-amino acid 3-amino acid 2-amino acid 1-2,4 disubstituted label compound.
Further preferably, taking a as an example of dipeptide a, when the Linker is HMPA Linker, the reaction scheme of the tag compound is as follows:
。
further preferably, the preparation method comprises the following steps:
(1) 2,4 disubstituted carbon based tags and Fmoc-AA 1 Obtaining 2, 4-disubstituted carbon-based label-Fmoc-AA through PCB condensation 1 A lipid;
(2) 2,4 disubstituted carbon based label-Fmoc-AA 1 Fmo group removal of fat by piperidine to obtain 2, 4-disubstituted carbon-based label-AA 1 -NH 2 ;
(3) Fmoc-AA using polypeptide condensing reagent 2 With 2, 4-disubstituted carbon label-AA 1 -NH 2 Coupling to obtain 2, 4-disubstituted carbon label-AA 1 -AA 2 -Fmoc;
(4) 2,4 disubstituted carbon based label-AA 1 -AA 2 Fmoc protecting group is removed by piperidine to obtain 2, 4-disubstituted carbon-based label-AA 1 -AA 2 -NH 2 ;
(5) Coupling of Linker HMPA Linker to 2,4 disubstituted carbon based label-AA with polypeptide condensing agent 1 -AA 2 -NH 2 Obtaining 2, 4-disubstituted carbon-based label-AA 1 -AA 2 -NH-HMPA Linker 8。
Still further preferably, the 2, 4-disubstituted carbon based label of step (1) is attached to Fmoc-AA 1 The molar ratio of (2) is 1:2.
still further preferably, the 2, 4-disubstituted carbon label of step (2) -Fmoc-AA 1 The molar ratio of lipid to piperidine was 1:15.
still more preferably, the concentration of piperidine is 10%.
Still further preferably, fmoc-AA in step (3) 2 With 2, 4-disubstituted carbon label-AA 1 -NH 2 The molar ratio of (2) is 1.5:1.
still further preferably, the 2, 4-disubstituted carbon-based label-AA in step (3) 1 -NH 2 The mass volume ratio of the polypeptide condensation reagent is 1:1.5.
still further preferably, the 2, 4-disubstituted carbon-based label-AA in step (4) 1 -AA 2 -Fmoc to piperidine molar ratio of 1:15.
still more preferably, the piperidine concentration is 10%.
Still further preferably, the Linker HMPA Linker in step (5) is used with the 2, 4-disubstituted carbon based tag-AA 1 -AA 2 -NH 2 The molar ratio of (2) is 1.5:1.
still further preferably, the 2, 4-disubstituted carbon-based label-AA in step (5) 1 -AA 2 -NH 2 The molar ratio of the polypeptide condensing agent to the polypeptide condensing agent is 1:1.5.
still further preferably, the polypeptide condensing agent in step (3) and step (5) is selected from one or more of DIC, HOBt, 6-chloro-1-hydroxy-benzotriazol (6-Cl-HOBt), 1-hydroxy-7-azabenzotriazol (HOAt), N' -Dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), carbonyldiimidazole (CDI), benzotriazol-1-yl-oxy-tripyrrolidinyl (PyBOP), tripyrrolidinylphosphonium bromide hexafluorophosphate (PyBroP), 3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4-one (debt), TBTU, HATU and HBTU.
Preferably, taking a as an example of dipeptide a, when the linker is Rink amide Linker, the reaction scheme of the tag compound is as follows:
。
further preferably, the method for preparing the tag compound is as follows: after introduction of linker Rink amide Linker 9 according to step (5) above, intermediate 10 was freed of Fmoc protecting group with piperidine solution to give 2, 4-disubstituted carbon label-AA 1 -AA 2 -NH-Rink amide Linker 11。
In the structural formulae of Compound 8 and Compound 11, the group-OA 1 A-AA 2 the-NH-of the-NH-is intended to represent AA 2 Specific connection form of amino group and linker HMPA, compoundThe label compounds represented by 8 and 11 correspond to the general formula of the label compounds described above.
Still more preferably, the piperidine concentration is 5-15%.
Still more preferably, the piperidine concentration is 10%.
Still further preferably, the molar ratio of intermediate 10 to piperidine is 1: (10-20).
Still more preferably, the molar ratio of intermediate 10 to piperidine is 1:15.
in yet another aspect, the present invention provides the use of the above-described tag compounds in polypeptide synthesis.
Preferably, the use of the compounds of the invention for polypeptide synthesis comprises the steps of:
(1) Introducing a first protected amino acid onto the tag compound;
(2) And obtaining a polypeptide crude product through coupling, deprotection and acidolysis.
It is further preferred that the tag compounds described in step (1) include, but are not limited to, 2,4 disubstituted carbon based tags-AA 1-AA2-NH-HMPA Linker 8, 2,4 disubstituted carbon based tags-AA 1-AA2-NH-Rink amide Linker 11.
Further preferably, the coupling, deprotection and acidolysis described in step (2) are conventional methods in the art.
The beneficial effects of the invention are as follows:
(1) According to the invention, amino acids (Lys and Nle) containing longer side chains are introduced on the 2, 4-disubstituted carbon-based label, so that the ester group at the root of the 2, 4-disubstituted carbon-based label is shielded, the degree of ester group aminolysis in the deprotection process is greatly reduced, the polypeptide is not easy to fall off from the carrier, and the polypeptide synthesis yield is increased.
(2) The number and combination of amino acids between the 2, 4-disubstituted carbon-based tag and the linker can be selected to adjust the solubility of the subsequent polypeptide-carrier, thereby facilitating the coupling or purification of the polypeptide.
(3) The C-terminal amide linker is introduced to prepare the polypeptide with the C-terminal amide, so that the application range of the 2, 4-disubstituted carbon-based label in the prior art is enlarged.
(4) The 2, 4-disubstituted carbon-based tag-A-L polypeptide synthesis carrier improves the polypeptide synthesis performance of the 2, 4-disubstituted carbon-based tag.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -HMPA in example 1.
FIG. 2 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide Linker in example 2.
FIG. 3 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Nle-Arg (pbf) -HMPA of example 3.
FIG. 4 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Nle-Leu-HMPA in example 3.
FIG. 5 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -HMPA in example 3.
FIG. 6 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -Rink Amide Linker in example 3.
FIG. 7 is a high performance liquid chromatogram of crude Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH in example 4.
FIG. 8 is a mass spectrum of crude Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH in example 4.
FIG. 9 shows the reaction sequence of Ac-Ser-Val-Val-Val-Arg-Thr-NH according to example 5 2 High performance liquid chromatography.
FIG. 10 shows the structure of Ac-Ser-Val-Val-Val-Arg-Thr-NH according to example 5 2 Mass spectrum of crude product.
FIG. 11 is a high performance liquid chromatogram of the crude H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-OH in example 6.
FIG. 12 is a mass spectrum of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-OH crude product in example 6.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purpose and the effect of the present invention easy to understand, the present invention will be further elucidated with reference to the specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all of them. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. In the following examples, unless otherwise specified, the methods of operation used were conventional, the equipment used was conventional, and the materials used in the examples were the same.
Example 1 1-methoxy-2, 4-twenty-eight alkoxy benzene-Lys (Boc) -Arg (pbf) -HMPALink
1.1Fmoc-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene Synthesis
Fmoc-Lys (Boc) -OH 93.7g (0.2 mol), DIC 25.2g (0.2 mol), HOBt 27g (0.2 mol) and 2, 4-octacosanol 64.5g (0.1 mol) were mixed in 700mL of methylene chloride and reacted at room temperature for 16 hours. The solvent was dried under reduced pressure and the resulting paste was stirred with 1000mL of acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, then filtered, and the obtained solid was slurried once again with acetonitrile, filtered and dried to obtain 101g of purified Fmoc-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene in 88.6% yield.
Synthesis of 1.2H-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene
Fmoc-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 100g (0.9 mol) was dissolved in 1000mL of dichloromethane, DBU 136.6g (0.9 mol) and 80mL of piperidine were added sequentially, the reaction was stirred at room temperature for 20 min and Fmoc was removed completely. Cooling the reaction liquid to 5-10 ℃, regulating the pH of the reaction liquid to 7.0 by phosphoric acid, drying the solvent by spin-drying under reduced pressure, and stirring the obtained paste by acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, and then the solid obtained by filtration was slurried with water 2 times and then with acetonitrile 2 times, and H-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 754g was obtained after filtration and air drying in 94% yield.
1.3 Fmoc-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene Synthesis
Fmoc-Arg (pbf) -OH 818.6g (1.26 mol), DIC 162.6g (1.26 mol), oxyma pure 179g (1.26 mol) and H-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 750g (0.84 mol) were mixed in 8000mL of dichloromethane and stirred at room temperature for 40 minutes. The solvent was dried under reduced pressure and the resulting paste was stirred with acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, then filtered, and the obtained solid was slurried twice with acetonitrile to obtain 1151g of Fmoc-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene in a yield of 90%.
1.4 Synthesis of H-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene
1151g of Fmoc-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 914g was synthesized according to the feed ratio and the procedure of step 1.2 of example 1 to yield 914g of H-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene in 94% yield.
1.5 Synthesis of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -HMPA Linker
140.3g (0.77 mol) of p-hydroxymethylphenoxyacetic acid (MW: 182.17), 99.3g (0.77 mol) of DIC, 104g (0.77 mol) of HOBt and 910g (0.7 mol) of H-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene were mixed in 8000mL of methylene chloride and stirred at room temperature for 70 minutes. The solvent was dried under reduced pressure and the resulting paste was stirred with 5000mL of acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, then filtered, and the obtained solid was slurried twice with acetonitrile to obtain 974g of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -HMPA, the nuclear magnetic pattern of which is shown in FIG. 1, and the yield was 95%. The structural formula is as follows:
。
example 2 1-methoxy-2, 4-twenty-eight-alkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide Linker
2.1 Fmoc-Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene synthesis
Fmoc-Rink Amide Linker 701.5.5 g (0.13 mol), H-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxy obtained in example 1.4 (130 g (0.1 mol), HBTU 49.3g (0.13 mol), HOBt 17.6g (0.13 mol) and DIEA 53mL (0.3 mol) were mixed in 7500mL of dichloromethane and stirred at room temperature for 40 minutes. The solvent was dried under reduced pressure and the resulting paste was stirred with 5000mL of acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, then filtered, and the obtained solid was slurried twice with acetonitrile to obtain 229g of Fmoc-Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene in a yield of 95%.
2.2 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide Linker
220g (0.12 mol) of Fmoc-Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 1100mL of tetrahydrofuran, 21g of DBU and 100mL of piperidine were added sequentially, and the reaction was stirred at room temperature for 20 minutes, and Fmoc was removed completely. Cooling the reaction liquid to 5-10 ℃, regulating the pH value of the reaction liquid to 7.0 by phosphoric acid, decompressing and spin-drying the solvent, and stirring the obtained paste by acetonitrile to form a solid. The solid is stirred with 1200mL of acetonitrile for 10 minutes, the solid obtained by filtration is pulped once again by acetonitrile, and 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide Linker g is obtained after filtration and air drying, the nuclear magnetic spectrum is shown in figure 2, and the yield is 87%. The structural formula is as follows:
example 3
Following the dosing ratios and operating methods of example 1 and example 2, the following compounds were prepared:
1-methoxy-2, 4-twenty-eight alkoxy benzene-Nle-Arg (pbf) -HMPA, its nuclear magnetic spectrum is shown in figure 3.
The nuclear magnetic spectrum of the 1-methoxy-2, 4-octacosalkoxybenzene-Nle-Leu-HMPA is shown in figure 4.
The nuclear magnetic spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -HMPA is shown in figure 5.
The nuclear magnetic spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -Rink Amide Linker is shown in FIG. 6.
EXAMPLE 4 Synthesis of Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH
4.1 Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -HMPa-Arg (pbf) -Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-twenty-eight-alkoxybenzene;
starting material 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -HMPA 186g (0.1 mol) was synthesized using the procedure of steps 1.1-1.4 of example 1 to yield 194.1g with a total yield of 78.03%.
4.2 Synthesis of Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH
150g of Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -HMPa-Arg (pbf) -Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 1800mL of a polypeptide cleavage solution (80% TFA:5% anisole: 5% phenol: 5% water: 3% EDT:2% TIS), and the solution was stirred at room temperature for 3 hours, and 3000mL of cold diethyl ether was added to the reaction solution to precipitate a polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 41.1g of crude polypeptide is obtained after vacuum drying at room temperature, and the purity is 92.7%. The high performance liquid chromatogram is shown in FIG. 7, and the mass spectrum is shown in FIG. 8.
Comparative example 4-1
1) Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -1-methoxy-2, 4-twenty-eight-alkoxy benzene
54.5g (0.1 mol) of starting material 2, 4-octaalkoxybenzyl alcohol were synthesized using the procedure of steps 1.1 to 1.4 of example 1 to give 131.1g, with a total yield of 41%.
2) 210g of Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -1-methoxy-2, 4-twenty-eight-alkoxy benzene was dissolved in 2600mL of polypeptide cleavage liquid (80% TFA:5% anisole: 5% phenol: 5% water: 3% edt:2% tis), stirring at room temperature for 3 hours, and adding 5000mL of cold diethyl ether into the reaction solution to precipitate polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 79.5g of crude polypeptide is obtained after vacuum drying at room temperature, and the purity is 92.3%.
Comparative example 4-2
1) Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -HMPA-Leu-Nle-1-methoxy-2, 4-octacosalkoxybenzene
104g (0.1 mol) of 1-methoxy-2, 4-octacosalkoxybenzene-Nle-Leu-HMPA was synthesized using the procedure of steps 1.1-1.4 of example 1 to give 247.9. 247.9 g in a total yield of 69.14%.
2) Synthesis of Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH
170g of Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -HMPA-Leu-Nle-1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 2000ml of a polypeptide cleavage solution (80% TFA:5% anisole: 5% phenol: 5% water: 3% EDT:2% TIS), stirred at room temperature for 3 hours, 4000ml of cold diethyl ether was added to the reaction solution, and a polypeptide solid was precipitated. After repeated centrifugation and methyl tertiary ether washing, 57.1g of crude polypeptide with the purity of 92.1% is obtained after vacuum pumping at room temperature.
The above comparative examples 4-1 and 4-2 were each compared with example 4.
The data for example 4 above and comparative examples 4-1 and 4-2 are summarized in Table 1.
Table 1.
As can be seen from the above table, both comparative example 4-1 and example 4 differ little in product purity, but the unmodified 1-methoxy-2, 4-octacosalkoxybenzene polypeptide synthesis vector is significantly lower in yield than the Lys (Boc) -Arg (pbf) -Arg (pbf) modified vector. The comparison test shows that Lys (Boc) with longer side chain can shade ester group at root of 2, 4-disubstituted carbon-based label, greatly reduce the degree of degradation of ester group by amine during deprotection process and improve product yield.
The yield in comparative example 4-2 was 69.14%, which indicates that the cleavage of the ester group was largely suppressed by blocking the ester group at the root of the label with Nle.
Example 5 Ac-Ser-Val-Val-Val-Arg-Thr-NH 2 Synthesis
5.1 Ac-Ser (tBu) -Val-Val-Val-Arg (pbf) -Thr (tBu) -Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene Synthesis
Starting material 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide 133g (0.1 mol) was synthesized by the method of example 1, steps 1.2-1.4 to give Ac-Ser (tBu) -Val-Val-Val-Arg (pbf) -Thr (tBu) -Rink Amide-Arg (pbf) -Nle-1-methoxy-2, 4-octacosalkoxybenzene 192.7. 192.7 g in 81.2% yield.
5.2 Ac-Ser-Val-Val-Val-Arg-Thr-NH 2 Synthesis
197g Ac-Ser (tBu) -Val-Val-Val-Arg (pbf) -Thr (tBu) -Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 2100mL polypeptide cleavage solution (80% TFA:5% anisole: 5% phenol: 5% water: 5% TIS), stirred at room temperature for 2.5 hours, and 4000mL cold diethyl ether was added to the reaction solution to precipitate a polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 46.5g of crude polypeptide is obtained after vacuum drying at room temperature, and the purity is 95.1%. The high performance liquid chromatogram is shown in FIG. 9, and the mass spectrum is shown in FIG. 10.
EXAMPLE 6 Synthesis of H-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
6.1 H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene
144.6g (0.1 mol) of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -HMPA (prepared by the method of example 1, steps 1.1-1.4) was synthesized to yield 278.6g, 82.1% yield.
6.2 Synthesis of H-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 490g was dissolved in 6000mL of the polypeptide cleavage solution (80% TFA:5% anisole sulfide: 5% phenol: 5% water: 3% EDT:2% TIS), stirred for 2 hours, and 10 liters of cold diethyl ether was added to the reaction solution to precipitate a polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 143.8g of crude polypeptide is obtained after vacuum pumping at room temperature, and the purity is 91.2%. The high performance liquid chromatogram is shown in FIG. 11, and the mass spectrum is shown in FIG. 12.
6.3 Performance test of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene
100mg of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene were dissolved in 0.5mL of each of dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydrofuran: DMF was 1:3 to obtain a clear and transparent solution. The polypeptide carrier can be continuously coupled with Fmoc-Lys (Boc) -OH, fmoc-Ala-OH and other protective amino acids by adopting DIC/HOBt, HBTU/HOBT and COMU condensing agents to form longer polypeptides.
Comparative example 6-1
1) H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-1-methoxy-2, 4-octacosalkoxybenzene
Starting material 2, 4-octacosalkoxybenzyl alcohol 64.5g (0.1 mol) the method of step 1.1-1.4 of example 1 was used, and the product was found to be poorly soluble in dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, DMF in the penultimate step, i.e. Fmoc-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-1-methoxy-2, 4-octacosalkoxybenzene. After the completion of the treatment, 99.45g of a product was obtained, and the yield was 51%.
2) Synthesis of H-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
80g H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 1000mL of a solution of the polypeptide in cleavage liquid (80% TFA:5% anisole sulfide: 5% phenol: 5% water: 3% EDT:2% TIS), stirred for 2 hours, and 1.2L of cold diethyl ether was added to the reaction solution to precipitate a polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 31.7g of crude polypeptide is obtained after vacuum pumping at room temperature, and the purity is 90.9%.
3) Performance test of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-1-methoxy-2, 4-twenty-eight-alkoxybenzene
100mg of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene was dissolved in each of 2mL of dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydrofuran/DMF (1:3) and not completely dissolved. The polypeptide carrier and Fmoc-Lys (Boc) -OH, fmoc-Ala-OH and other protective amino acids cannot react by adopting DIC/HOBt, HBTU/HOBT and COMU condensing agents.
The data for example 6 and comparative example 6-1 are summarized in Table 2.
Table 2.
As can be seen from the above table, the unmodified 1-methoxy-2, 4-octacosalkoxybenzene polypeptide synthesized vector was not only significantly lower in yield than the modified vector, but the coupling reaction was terminated when the performance of the whole polypeptide-vector reached a certain stage. From this, it can be seen that the solubility of the subsequent polypeptide-carrier can be adjusted by selecting the number and combination of amino acids between the 2, 4-disubstituted carbon based tag and the linker, facilitating the coupling or purification of the polypeptide.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. A tag compound for polypeptide synthesis, said tag compound having the chemical structural formula:
wherein R is a linear alkane of 18 to 22C atoms;
-O-A-L is-O-AA 1 -AA 2 -L or-O-AA 1 -AA 2 -AA 3 -L;
AA 1 -AA 2 Is Lys (Boc) -Arg (pbf) or Nle-Leu;
AA 1 -AA 2 -AA 3 lys (Boc) -Arg (pbf);
l in the general formula is a Linker synthesized by polypeptide, wherein the Linker is selected from one or more of HMPA Linker and Rink amide Linker;
the HMPA Linker structure is as follows:
the structure of Rink amide Linker is as follows:
。
2. a process for producing A labeled compound according to claim 1, wherein when-O-A-L is-O-AA 1 -AA 2 When the Linker is HMPA Linker, the reaction scheme of the tag compound is as follows:
。
3. the method according to claim 2, wherein Fmoc-AA is condensed with a polypeptide condensing reagent in step (3) 2 With 2, 4-disubstituted carbon label-AA 1 -NH 2 Coupling to obtain 2, 4-disubstituted carbon label-AA 1 -AA 2 -Fmoc; the polypeptide condensation reagent is selected from one or more of DIC, HOBt, 6-chloro-1-hydroxy-benzotriazole, 1-hydroxy-7-azabenzotriazole, N' -dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, carbonyldiimidazole, benzotriazol-1-yl-oxy-tripyrrolidinyl hexafluorophosphate, tripyrrolidinylphosphonium bromide hexafluorophosphate, 3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4-one, TBTU, HATU and HBTU.
4. The method for producing a labeled compound according to claim 1, wherein when the linker is Rink amide Linker, the reaction scheme of the labeled compound is as follows:
。
5. the process of claim 4, wherein intermediate 10 is prepared by removing Fmoc protecting groups from a piperidine solution to provide 2, 4-disubstituted carbon label-AA 1 -AA 2 -NH-Rink amide Linker 11; the mass concentration of the piperidine is 5-15%.
6. The process of claim 5, wherein the molar ratio of intermediate 10 to piperidine is 1: (10-20).
7. Use of a tag compound according to claim 1 or a tag compound according to any one of claims 2 to 6 in polypeptide synthesis.
8. The use according to claim 7, wherein the use of a tag compound for polypeptide synthesis comprises the steps of:
(1) Introducing a first protected amino acid onto the tag compound;
(2) And obtaining a polypeptide crude product through coupling, deprotection and acidolysis.
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A Pot-economical Liquid-phase Peptide Nucleic Acid Synthesis Enabled by a Soluble Tag-assisted Method;Keisuke Ogami 等;Chem. Lett.;第138-140页 * |
Tag-Assisted Liquid-Phase Peptide Synthesis Using Hydrophobic Benzyl Alcohols as Supports;Yohei Okada 等;The Journal of Organic Chemistry;第320-327页 * |
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