KR20240086726A - A Convenient Synthetic Method of Polymyxin E and B - Google Patents
A Convenient Synthetic Method of Polymyxin E and B Download PDFInfo
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- KR20240086726A KR20240086726A KR1020220158644A KR20220158644A KR20240086726A KR 20240086726 A KR20240086726 A KR 20240086726A KR 1020220158644 A KR1020220158644 A KR 1020220158644A KR 20220158644 A KR20220158644 A KR 20220158644A KR 20240086726 A KR20240086726 A KR 20240086726A
- Authority
- KR
- South Korea
- Prior art keywords
- formula
- polymyxin
- peptide
- dab
- group
- Prior art date
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- 108010078777 Colistin Proteins 0.000 title claims abstract description 33
- 108010093965 Polymyxin B Proteins 0.000 title claims abstract description 27
- JORAUNFTUVJTNG-BSTBCYLQSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Polymers CC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O.CCC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O JORAUNFTUVJTNG-BSTBCYLQSA-N 0.000 title claims abstract description 27
- XDJYMJULXQKGMM-UHFFFAOYSA-N polymyxin E1 Natural products CCC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O XDJYMJULXQKGMM-UHFFFAOYSA-N 0.000 title claims abstract description 27
- YKQOSKADJPQZHB-YNWHQGOSSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1s)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Polymers CCC(C)CCCC(=O)N[C@@H](CCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O YKQOSKADJPQZHB-YNWHQGOSSA-N 0.000 title claims abstract description 24
- 238000010189 synthetic method Methods 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract description 27
- 125000006239 protecting group Chemical group 0.000 claims abstract description 25
- 229920000024 polymyxin B Polymers 0.000 claims abstract description 21
- 229960005266 polymyxin b Drugs 0.000 claims abstract description 21
- 238000004519 manufacturing process Methods 0.000 claims abstract description 20
- 238000007363 ring formation reaction Methods 0.000 claims abstract description 14
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 14
- 238000001308 synthesis method Methods 0.000 claims abstract description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 131
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 71
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 62
- 239000011347 resin Substances 0.000 claims description 54
- 229920005989 resin Polymers 0.000 claims description 54
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 125000006242 amine protecting group Chemical group 0.000 claims description 14
- 238000010511 deprotection reaction Methods 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 12
- 239000003054 catalyst Substances 0.000 claims description 9
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 239000012071 phase Substances 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical group ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 230000002378 acidificating effect Effects 0.000 claims description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 6
- 229940098779 methanesulfonic acid Drugs 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- 239000001257 hydrogen Substances 0.000 claims description 5
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- 239000003929 acidic solution Substances 0.000 claims description 4
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 claims description 4
- 229940092714 benzenesulfonic acid Drugs 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 4
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 3
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 3
- 229940013688 formic acid Drugs 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- ZKGNPQKYVKXMGJ-UHFFFAOYSA-N N,N-dimethylacetamide Chemical compound CN(C)C(C)=O.CN(C)C(C)=O ZKGNPQKYVKXMGJ-UHFFFAOYSA-N 0.000 claims 2
- 238000006243 chemical reaction Methods 0.000 abstract description 47
- 108010040201 Polymyxins Proteins 0.000 abstract description 7
- 229940041153 polymyxins Drugs 0.000 abstract description 5
- 238000000746 purification Methods 0.000 abstract description 5
- 230000002194 synthesizing effect Effects 0.000 abstract description 5
- 239000007791 liquid phase Substances 0.000 abstract description 3
- 238000009825 accumulation Methods 0.000 abstract description 2
- 239000012535 impurity Substances 0.000 abstract description 2
- -1 tert-butyl ( tert -Butyl) Chemical group 0.000 description 69
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 33
- 238000001914 filtration Methods 0.000 description 25
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 23
- 238000003786 synthesis reaction Methods 0.000 description 22
- GPOPHQSTNHUENT-UHFFFAOYSA-N 6-Methyl caprylic acid Chemical compound CCC(C)CCCCC(O)=O GPOPHQSTNHUENT-UHFFFAOYSA-N 0.000 description 20
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 20
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 20
- 230000015572 biosynthetic process Effects 0.000 description 20
- 150000001413 amino acids Chemical class 0.000 description 15
- 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 11
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 10
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 8
- 239000003242 anti bacterial agent Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 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 description 6
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 125000000524 functional group Chemical group 0.000 description 5
- LIWKOFAHRLBNMG-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxycarbonylamino]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 LIWKOFAHRLBNMG-FQEVSTJZSA-N 0.000 description 4
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 description 4
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 description 4
- LJCZNYWLQZZIOS-UHFFFAOYSA-N 2,2,2-trichlorethoxycarbonyl chloride Chemical compound ClC(=O)OCC(Cl)(Cl)Cl LJCZNYWLQZZIOS-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- BSXPDVKSFWQFRT-UHFFFAOYSA-N 1-hydroxytriazolo[4,5-b]pyridine Chemical compound C1=CC=C2N(O)N=NC2=N1 BSXPDVKSFWQFRT-UHFFFAOYSA-N 0.000 description 3
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 3
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229960003346 colistin Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- KNIWPHSUTGNZST-UHFFFAOYSA-N polymyxin E2 Natural products CC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O KNIWPHSUTGNZST-UHFFFAOYSA-N 0.000 description 3
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 3
- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 description 2
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 2
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 2
- PECYZEOJVXMISF-UHFFFAOYSA-N 3-aminoalanine Chemical compound [NH3+]CC(N)C([O-])=O PECYZEOJVXMISF-UHFFFAOYSA-N 0.000 description 2
- TZCYLJGNWDVJRA-UHFFFAOYSA-N 6-chloro-1-hydroxybenzotriazole Chemical compound C1=C(Cl)C=C2N(O)N=NC2=C1 TZCYLJGNWDVJRA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- CBPJQFCAFFNICX-LJQANCHMSA-N Fmoc-D-Leu-OH Chemical compound C1=CC=C2C(COC(=O)N[C@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-LJQANCHMSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- SIEILFNCEFEENQ-UHFFFAOYSA-N dibromoacetic acid Chemical compound OC(=O)C(Br)Br SIEILFNCEFEENQ-UHFFFAOYSA-N 0.000 description 2
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- 125000005928 isopropyloxycarbonyl group Chemical group [H]C([H])([H])C([H])(OC(*)=O)C([H])([H])[H] 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 2
- AQRLNPVMDITEJU-UHFFFAOYSA-N triethylsilane Chemical compound CC[SiH](CC)CC AQRLNPVMDITEJU-UHFFFAOYSA-N 0.000 description 2
- JBWKIWSBJXDJDT-UHFFFAOYSA-N triphenylmethyl chloride Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(Cl)C1=CC=CC=C1 JBWKIWSBJXDJDT-UHFFFAOYSA-N 0.000 description 2
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 2
- SJVFAHZPLIXNDH-JOCHJYFZSA-N (2r)-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-JOCHJYFZSA-N 0.000 description 1
- RXKBBKLPMHPIKD-BHVANESWSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[[(4-methylphenyl)-diphenylmethyl]amino]butanoic acid Chemical compound C1=CC(C)=CC=C1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)NCC[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 RXKBBKLPMHPIKD-BHVANESWSA-N 0.000 description 1
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 1
- ZZDRDGKSMGGBDI-KRWDZBQOSA-N (2s)-4-azaniumyl-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanoate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCN)C(O)=O)C3=CC=CC=C3C2=C1 ZZDRDGKSMGGBDI-KRWDZBQOSA-N 0.000 description 1
- LNOLJFCCYQZFBQ-BUHFOSPRSA-N (ne)-n-[(4-nitrophenyl)-phenylmethylidene]hydroxylamine Chemical compound C=1C=C([N+]([O-])=O)C=CC=1C(=N/O)/C1=CC=CC=C1 LNOLJFCCYQZFBQ-BUHFOSPRSA-N 0.000 description 1
- OBOHMJWDFPBPKD-UHFFFAOYSA-N 1-[chloro(diphenyl)methyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 OBOHMJWDFPBPKD-UHFFFAOYSA-N 0.000 description 1
- VUTZFAOGDXUYEJ-UHFFFAOYSA-N 1-[chloro(diphenyl)methyl]-4-methylbenzene Chemical compound C1=CC(C)=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 VUTZFAOGDXUYEJ-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- HLHNOIAOWQFNGW-UHFFFAOYSA-N 3-bromo-4-hydroxybenzonitrile Chemical compound OC1=CC=C(C#N)C=C1Br HLHNOIAOWQFNGW-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000588626 Acinetobacter baumannii Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 1
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- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
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- 208000015181 infectious disease Diseases 0.000 description 1
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- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
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- 125000006503 p-nitrobenzyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1[N+]([O-])=O)C([H])([H])* 0.000 description 1
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 1
- 229940044654 phenolsulfonic acid Drugs 0.000 description 1
- PARWUHTVGZSQPD-UHFFFAOYSA-N phenylsilane Chemical compound [SiH3]C1=CC=CC=C1 PARWUHTVGZSQPD-UHFFFAOYSA-N 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 125000000025 triisopropylsilyl group Chemical group C(C)(C)[Si](C(C)C)(C(C)C)* 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
- C07K7/62—Polymyxins; Related peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
- C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
본 발명은 폴리믹신 E 및 폴리믹신 B의 제조방법에 관한 것으로서, 더욱 상세하게는 고체상 (solid-phase) 합성법과 액체상 (solution-phase) 합성법으로 폴리믹신 E 및 폴리믹신 B를 용이하게 합성하는 방법에 관한 것이다.
본 발명에 따른 폴리믹신의 제조방법을 이용하면 R₃아민 보호기를 선택적으로 제거하고, 저농도에서 고리화 반응을 최적화하여 불순물의 발생 및 누적을 최소화할 수 있고, 반응종료 후 폴리믹신 E 및 B의 분리 및 정제가 용이하므로, 고순도의 폴리믹신 폴리믹신 E 및 B를 상업적으로 대량 생산할 수 있다.The present invention relates to a method for producing polymyxin E and polymyxin B, and more specifically, to a method for easily synthesizing polymyxin E and polymyxin B using a solid-phase synthesis method and a liquid-phase synthesis method. It's about.
Using the method for producing polymyxin according to the present invention, it is possible to selectively remove the R₃amine protecting group, optimize the cyclization reaction at low concentration, minimize the generation and accumulation of impurities, and separate polymyxins E and B after completion of the reaction. And because purification is easy, high-purity polymyxin polymyxin E and B can be mass-produced commercially.
Description
본 발명은 폴리믹신 E 및 폴리믹신 B의 제조방법에 관한 것으로서, 더욱 상세하게는 고체상 (solid-phase) 합성법과 액체상 (solution-phase) 합성법으로 폴리믹신 E 및 폴리믹신 B를 용이하게 합성하는 방법에 관한 것이다.The present invention relates to a method for producing polymyxin E and polymyxin B, and more specifically, to a method for easily synthesizing polymyxin E and polymyxin B using a solid-phase synthesis method and a liquid-phase synthesis method. It's about.
Colistin (Polymyxin E)는 Polypeptide계 항균제로 1950년대에 개발되어 1970년대 초까지 사용되었으며 그람음성균에 특히 효과적이나 전신투여시 신독성, 신경독성 등의 부작용으로 인해 외용약제 외에는 거의 처방되지 않았던 약제이다.Colistin (Polymyxin E) is a polypeptide-based antibacterial agent that was developed in the 1950s and used until the early 1970s. It is particularly effective against Gram-negative bacteria, but due to side effects such as nephrotoxicity and neurotoxicity when administered systemically, it is rarely prescribed except as a topical medication.
그러나, 2000년대 이후 Carbapenem계 약물에 내성을 보이는 그람음성균주가 점차 확산됨에 따라 이에 대해 사용할 수 있는 항균제가 많지 않아 Colistin의 사용량이 증가하고 있다. 기존의 모든 항생제에 내성을 보이는 Pseudomonas aeruginosa, Acinetobacter baumannii에 의한 감염에 대하여 사용되는 약물로 다른 내성 균주 발현 예방을 위해 제한적 사용되고 있다.However, as Gram-negative bacteria resistant to carbapenems have gradually spread since the 2000s, there are not many antibacterial agents that can be used against them, so the use of Colistin is increasing. It is a drug used for infections caused by Pseudomonas aeruginosa and Acinetobacter baumannii, which are resistant to all existing antibiotics, and is of limited use to prevent the development of other resistant strains.
Polymyxin B는 Colistin과 거의 비슷한 구조를 가지며 최근에 개발되어 동일한 목적으로 사용되는 치료제이다.Polymyxin B has a structure almost similar to Colistin and is a recently developed treatment used for the same purpose.
두 가지 항생제는 최근 최후의 항생제로 사용중인 카바페넴계 항생제 마저 다양한 내성균주들이 발현됨에 따라 독성이 있음에도 불구하고 제한적으로 최후의 항생제로 사용되고 있어 그 제조방법 또한 개발이 매우 중요한 실정이다.These two antibiotics are being used in a limited manner as last-line antibiotics despite their toxicity as various resistant strains have emerged, including carbapenem antibiotics, which are currently being used as last-line antibiotics. Therefore, the development of methods for producing them is also very important.
Pamela Brown 등은 Polymyxin B 유도체를 합성하는 방법을 개시하였는데, Polymyxin B의 효소 분해에 의해 주요 구조인 cyclic heptapeptide를 얻은 다음 화학 제조방법으로 유도체를 붙여 합성하는 방법으로서, 합성 공정에 위험하며, 고가인 10% Pd/C 촉매를 사용하는 등 상업적으로 적용하는데 문제가 있었다 (Pamela Brown et al., American Chemical Society Infectious Diseases, 2019, 5, 1645-1656).Pamela Brown et al. disclosed a method of synthesizing Polymyxin B derivatives. This is a method of obtaining the main structure, cyclic heptapeptide, by enzymatic decomposition of Polymyxin B, and then attaching the derivative through a chemical manufacturing method to synthesize it, which is dangerous and expensive in the synthesis process. There were problems with commercial application, such as using 10% Pd/C catalyst (Pamela Brown et al., American Chemical Society Infectious Diseases, 2019, 5, 1645-1656).
또한, Alejandra Gallardo-Godoy 등은 전체 합성을 고체상으로 합성하는 방법을 개시하였으나, 고리화의 핵심 아미노산 7번의 보호기를 Alloc을 사용하였으며, 이의 탈보호화시 상업적 활용이 제한되는 Pd(PPh3), PhSiH3을 사용하여 공정이 번거로울 뿐만 아니라 상업적으로 적용하는데 문제가 있었다(Alejandra Gallardo-Godoy et al., Journal of Medicinal Chemistry, 2016, 59, 1068~1077). In addition, Alejandra Gallardo-Godoy et al. disclosed a method of synthesizing the entire synthesis in solid phase, but used Alloc as the protecting group at position 7 of the key amino acid for cyclization, and when deprotected, Pd (PPh 3 ) and PhSiH, which have limited commercial use, were used. 3 Not only was the process cumbersome, but there were problems with commercial application (Alejandra Gallardo-Godoy et al., Journal of Medicinal Chemistry, 2016, 59, 1068~1077).
또한, Suhas Ramesh 등은 고체상 반응으로 제조하는 기술로써 정제수율은 기재하지 않았으나 산업적으로 이용이 제한된 phenylsilane이나 tetrakis(triphenylphosphine) Pd(0) 등 상업적으로 이용 가능성이 어려운 고가의 시약을 사용하여 상업적으로 적용하기 어려운 문제점이 있었다(Suhas Ramesh et al., Tetrahedron Letters, Volume 57, Issue 17, 27 April 2016, Pages 1885-1888).In addition, Suhas Ramesh et al. did not describe the purification yield as a solid-phase reaction manufacturing technology, but it is commercially applied using expensive reagents that are difficult to commercially exploit, such as phenylsilane and tetrakis(triphenylphosphine) Pd(0), which have limited industrial use. There was a problem that made it difficult to do (Suhas Ramesh et al., Tetrahedron Letters, Volume 57, Issue 17, 27 April 2016, Pages 1885-1888).
이에, 본 발명자들은 상기 문제점을 해결하기 위하여 노력한 결과, 아미노산 을 사용하면서, 고체상 (solid-phase) 합성법으로 레진이 부착된 펩타이드를 수득한 다음, 레진으로부터 떼어내어 보호화된 펩타이드를 수득하고, 액상 (solution-phase) 합성법으로 고리화한 다음, 최종 탈보호시킬 경우, 고수율로 고순도의 폴리믹신 E 및 폴리믹신 B를 제조할 수 있다는 것을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors tried to solve the above problem, and as a result, obtained a peptide with a resin attached using a solid-phase synthesis method using amino acids, then separated it from the resin to obtain a protected peptide, and obtained a protected peptide using a solid-phase synthesis method. It was confirmed that high purity polymyxin E and polymyxin B could be produced in high yield when cyclized using a (solution-phase) synthesis method and then subjected to final deprotection, thereby completing the present invention.
본 발명의 목적은 고순도의 폴리믹신 E 및 폴리믹신 B를 간단하게 제조하는 방법을 제공하는데 있다.The purpose of the present invention is to provide a simple method for producing high purity polymyxin E and polymyxin B.
상기 목적을 달성하기 위하여, 본 발명은 (a) 고체상 (solid-phase) 합성법으로 화학식 I-a로 표시되는 레진이 부착된 펩타이드를 수득하는 단계; (b) 상기 (a) 단계에서 수득된 펩타이드에서 레진을 제거하여 화학식 I-b로 표시되는 보호화된 펩타이드를 수득하는 단계; (c) 상기 (b) 단계에서 수득된 펩타이드를 액상 (solution-phase) 합성법으로 촉매를 사용하여 고리화 반응시켜, 화학식 I-c로 표시되는 보호화된 고리화 펩타이드를 수득하는 단계; 및 (d) 상기 (c) 단계에서 수득한 펩타이드에서 탈 보호화 반응을 수행하여 화학식 Ⅰ로 표시되는 폴리믹신 E를 수득하는 단계를 포함하는 폴리믹신 E의 제조방법을 제공한다.In order to achieve the above object, the present invention includes the steps of (a) obtaining a resin-attached peptide represented by Formula I-a by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in step (a) to obtain a protected peptide represented by Formula I-b; (c) cyclizing the peptide obtained in step (b) using a catalyst using a solution-phase synthesis method to obtain a protected cyclized peptide represented by Formula I-c; and (d) performing a deprotection reaction on the peptide obtained in step (c) to obtain polymyxin E represented by Formula I.
[화학식 I][Formula I]
[화학식 I-a][Formula I-a]
[화학식 I-b][Formula I-b]
[화학식 I-c][Formula I-c]
상기 화학식에서, R₁는 아민 보호기, R₂는 수소 또는 수산기 보호기, R₃는 아민 보호기이다.In the above formula, R₁ is an amine protecting group, R₂ is a hydrogen or hydroxyl group protecting group, and R₃ is an amine protecting group.
본 발명은 또한, (a) 고체상 (solid-phase) 합성법으로 화학식 Ⅱ-a로 표시되는 레진이 부착된 펩타이드를 수득하는 단계; (b) 상기 (a) 단계에서 수득된 펩타이드에서 레진을 제거하여 화학식 Ⅱ-b로 표시되는 보호화된 펩타이드를 수득하는 단계; (c) 상기 (b) 단계에서 수득된 펩타이드를 액상 (solution-phase) 합성법으로 촉매를 사용하여 고리화 반응시켜, 화학식 Ⅱ-c로 표시되는 보호화된 고리화 펩타이드를 수득하는 단계; 및 (d) 상기 (c) 단계에서 수득한 펩타이드에서 탈 보호화 반응을 수행하여 화학식 Ⅱ로 표시되는 폴리믹신 B를 수득하는 단계를 포함하는 폴리믹신 B의 제조방법을 제공한다.The present invention also includes the steps of (a) obtaining a resin-attached peptide represented by Formula II-a by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in step (a) to obtain a protected peptide represented by Formula II-b; (c) cyclizing the peptide obtained in step (b) using a catalyst using a solution-phase synthesis method to obtain a protected cyclized peptide represented by Formula II-c; and (d) performing a deprotection reaction on the peptide obtained in step (c) to obtain polymyxin B represented by Formula II.
[화학식 Ⅱ][Formula Ⅱ]
[화학식 Ⅱ-a][Formula Ⅱ-a]
[화학식 Ⅱ-b][Formula Ⅱ-b]
[화학식 Ⅱ-c][Formula Ⅱ-c]
상기 화학식에서, R₁는 아민 보호기, R₂는 수소 또는 수산기 보호기, R₃는 아민 보호기이다.In the above formula, R₁ is an amine protecting group, R₂ is a hydrogen or hydroxyl group protecting group, and R₃ is an amine protecting group.
본 발명에 따른 폴리믹신의 제조방법을 이용하면 R₃아민 보호기를 선택적으로 제거하고, 저농도에서 고리화 반응을 최적화하여 불순물의 발생 및 누적을 최소화할 수 있고, 반응종료 후 폴리믹신 E 및 B의 분리 및 정제가 용이하므로, 고순도의 폴리믹신 폴리믹신 E 및 B를 상업적으로 대량 생산할 수 있다.Using the method for producing polymyxin according to the present invention, it is possible to selectively remove the R₃amine protecting group, optimize the cyclization reaction at low concentration, minimize the generation and accumulation of impurities, and separate polymyxins E and B after completion of the reaction. And because purification is easy, high-purity polymyxin polymyxin E and B can be mass-produced commercially.
도 1은 폴리믹신 E 및 B를 제조하는 전체 공정을 나타낸 모식도이다.Figure 1 is a schematic diagram showing the overall process for producing polymyxins E and B.
본 명세서에서 특별한 표시가 없는 한, 아미노산 및 보호기의 지정에 사용되는 약어는 IUPAC-IUB의 생화학 용어 위원회 (Commission of Biochemical Nomenclature)에서 권장하는 용어에 기초한다 (Biochemistry, 11:1726-1732(1972); Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984).Unless specifically indicated herein, the abbreviations used to designate amino acids and protecting groups are based on the terminology recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB ( Biochemistry, 11:1726-1732 (1972) Pure & Appl., Vol. 56, pp. 595-624.
본 명세서에서 사용한 보호기 및 아미노산의 약어는 다음과 같다:The abbreviations for protecting groups and amino acids used herein are as follows:
t-Bu: 터트-부틸 (tert-Butyl)t-Bu: tert-butyl ( tert -Butyl)
Boc: 복 (tert-Butyloxycarbonyl)Boc: Boc ( tert -Butyloxycarbonyl)
Dab: 2,3-디아미노프로피온산 (2,3-Diaminopropionic acid)Dab: 2,3-Diaminopropionic acid
Ser: 세린 (Serine)Ser: Serine
Thr: 쓰레오닌 (Threonine)Thr: Threonine
Fmoc: 9-플루오레닐옥시카보닐 (9-Fluorenyloxycarbonyl)Fmoc: 9-Fluorenyloxycarbonyl
Mtt: 4-메틸트리틸 (4-Methyltrityl)Mtt: 4-Methyltrityl
본 발명에서는 아미노산을 사용하여 고체상 (solid-phase) 합성법으로 레진이 부착된 펩타이드를 수득한 다음, 레진으로부터 보호화된 펩타이드를 얻고, 액상 (solution-phase) 합성법으로 고리화한 다음, 최종 탈보호시킬 경우, 고수율로 고순도의 폴리믹신 E 및 B를 제조할 수 있다는 것을 확인하고자 하였다.In the present invention, a peptide to which a resin is attached is obtained by solid-phase synthesis using amino acids, then a peptide protected from the resin is obtained, cyclized by a liquid-phase synthesis method, and then finally deprotected. The purpose of this study was to confirm that high purity polymyxins E and B can be produced in high yield.
본 발명에서는 아미노산을 사용하여, 고체상 (solid-phase) 합성법으로 레진이 부착된 펩타이드 (화학식 I-a 및 화학식 Ⅱ-a)를 수득한 다음, 레진으로부터 보호화된 펩타이드 (화학식 I-b 및 화학식 Ⅱ-b)를 수득하고, 액상 (solution-phase) 합성법으로 촉매를 사용하여 고리화 반응시켜 고리화된 펩타이드 (화학식 I-c 및 화학식 Ⅱ-c)를 수득 후, 탈 보호화하여 최종적으로 폴리믹신 E(화학식 Ⅰ) 및 폴리믹신 B(화학식 Ⅱ)를 합성하였다. 그 결과, 폴리믹신 E 및 폴리믹신 B를 상업적 대량 생산이 가능할 정도의 고수율로 합성할 수 있음을 확인하였다. In the present invention, resin-attached peptides (Formula I-a and Formula II-a) are obtained by solid-phase synthesis using amino acids, and then peptides protected from resin (Formula I-b and Formula II-b) are obtained. was obtained, and a cyclization reaction was performed using a catalyst using a solution-phase synthesis method to obtain cyclized peptides (Formula I-c and Formula II-c), which were then deprotected to finally produce polymyxin E (Formula I). and polymyxin B (Formula II) was synthesized. As a result, it was confirmed that polymyxin E and polymyxin B could be synthesized in high yield enough to enable commercial mass production.
따라서, 본 발명은 일 관점에서, (a) 고체상 (solid-phase) 합성법으로 화학식 I-a로 표시되는 레진이 부착된 펩타이드를 수득하는 단계; (b) 상기 (a) 단계에서 수득된 펩타이드에서 레진을 제거하여 화학식 I-b로 표시되는 보호화된 펩타이드를 수득하는 단계; (c) 상기 (b) 단계에서 수득된 펩타이드를 액상 (solution-phase) 합성법으로 촉매를 사용하여 고리화 반응시켜, 화학식 I-c로 표시되는 보호화된 고리화 펩타이드를 수득하는 단계; 및 (d) 상기 (c) 단계에서 수득한 펩타이드에서 탈 보호화 반응을 수행하여 화학식 Ⅰ로 표시되는 폴리믹신 E를 수득하는 단계를 포함하는 폴리믹신 E의 제조방법에 관한 것이다.Therefore, in one aspect, the present invention includes the steps of (a) obtaining a resin-attached peptide represented by Formula I-a by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in step (a) to obtain a protected peptide represented by Formula I-b; (c) cyclizing the peptide obtained in step (b) using a catalyst using a solution-phase synthesis method to obtain a protected cyclized peptide represented by Formula I-c; and (d) performing a deprotection reaction on the peptide obtained in step (c) to obtain polymyxin E represented by Formula I.
[화학식 I][Formula I]
[화학식 I-a][Formula I-a]
[화학식 I-b][Formula I-b]
[화학식 I-c][Formula I-c]
본 발명은 또한, (a) 고체상 (solid-phase) 합성법으로 화학식 Ⅱ-a로 표시되는 레진이 부착된 펩타이드를 수득하는 단계; (b) 상기 (a) 단계에서 수득된 펩타이드에서 레진을 제거하여 화학식 Ⅱ-b로 표시되는 보호화된 펩타이드를 수득하는 단계; (c) 상기 (b) 단계에서 수득된 펩타이드를 액상 (solution-phase) 합성법으로 촉매를 사용하여 고리화 반응시켜, 화학식 Ⅱ-c로 표시되는 보호화된 고리화 펩타이드를 수득하는 단계; 및 (d) 상기 (c) 단계에서 수득한 펩타이드에서 탈 보호화 반응을 수행하여 화학식 Ⅱ로 표시되는 폴리믹신 B를 수득하는 단계를 포함하는 폴리믹신 B의 제조방법에 관한 것이다.The present invention also includes the steps of (a) obtaining a resin-attached peptide represented by Formula II-a by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in step (a) to obtain a protected peptide represented by Formula II-b; (c) cyclizing the peptide obtained in step (b) using a catalyst using a solution-phase synthesis method to obtain a protected cyclized peptide represented by Formula II-c; and (d) performing a deprotection reaction on the peptide obtained in step (c) to obtain polymyxin B represented by Formula II.
[화학식 Ⅱ][Formula Ⅱ]
[화학식 Ⅱ-a][Formula Ⅱ-a]
[화학식 Ⅱ-b][Formula Ⅱ-b]
[화학식 Ⅱ-c][Formula Ⅱ-c]
상기 화학식에서 R₁은 당 업계에서 통상적으로 이용하는 아민 보호기를 이용할 수 있다. 상기 아민 보호기는 아세틸 (Acetyl)기, 벤조일(Benzoyl), 벤질옥시메틸 (Benzyloxymethyl)기, 트리틸 (Trityl)기, Ddz (α,α-Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz))기, Bpoc (2-(4-Biphenyl)isopropoxycarbonyl (Bpoc))기, Nps (2-Nitrophenylsulfenyl (Nps))기, Fmoc (9-Fluorenylmethoxycarbonyl (Fmoc))기, Nsc (2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc))기, Bsmoc (1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc))기, α-Nsmoc ( (1,1-Dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α-Nsmoc))기, Dde & ivDde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) and 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-mehtylbutyl (ivDde))기, Fmoc* (2,7-Di-tert-butyl-Fmoc (Fmoc*))기, Fmoc(2F) (2-Fluoro-Fmoc (Fmoc(2F)))기, 알릴 (allyl)기, mio-Fmoc & dio-Fmoc (2-Monoisooctyl-Fmoc (mio-Fmoc) and 2,7-Diisooctyl-Fmoc (dio-Fmoc))기, TCP (Tetrachlorophthaloyl (TCP))기, Pms (2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms))기, Esc (Ethanesulfonylethoxycarbonyl (Esc))기, Sps (2-(4-Sulfophenylsulfonyl)ethoxycarbonyl (Sps))기, Z (Benzyloxycarbonyl (Z))기, Alloc (Allyloxycarbonyl (Alloc))기, oNBS & pNBS (o-Nitrobenzenesulfonyl (oNBS) and p-nitrobenzenesulfonyl (pNBS)기, dNBS (2,4-Dinitrobenzenesulfonyl (dNBS))기, Bts (Benzothiazole-2-sulfonyl (Bts))기, Troc (2,2,2-Trichloroethyloxycarbonyl (Troc))기, Dts (Dithiasuccinoyl (Dts))기, pNZ (p-Nitrobenzyloxycarbonyl (pNZ))기, Poc (Propargyloxycarbonyl (Poc))기, oNZ & NVOC (o-Nitrobenzyloxycarbonyl (oNZ) and 6-Nitroveratryloxycarbonyl (NVOC))기, NPPOC (2-(2-Nitrophenyl)propyloxycarbonyl (NPPOC))기, MNPPOC ( 2-(3,4-Methylenedioxy-6-nitrophenyl)propyloxycarbonyl (MNPPOC))기, BrPhF (9-(4-Bromophenyl)-9-fluorenyl (BrPhF))기, Azoc (Azidomethyloxycarbonyl (Azoc))기, Cl-Z (2-Chlorobenzyloxycarbonyl (Cl-Z))기, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기 등을 예시할 수 있으며, Azoc (Azidomethyloxycarbonyl (Azoc))기, Cl-Z (2-Chlorobenzyloxycarbonyl (Cl-Z))기, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기를 이용하는 것이 바람직하며, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기를 이용하는 것이 보다 바람직하며, 아민 보호기가 Boc (tert-Butyloxycarbonyl (Boc))기를 이용하는 것이 가장 바람직하다.In the above chemical formula, R₁ can use an amine protecting group commonly used in the industry. The amine protecting group is acetyl group, benzoyl group, benzyloxymethyl group, trityl group, Ddz (α,α-Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz)) group, Bpoc (2-(4-Biphenyl)isopropoxycarbonyl (Bpoc)) group, Nps (2-Nitrophenylsulfenyl (Nps)) group, Fmoc (9-Fluorenylmethoxycarbonyl (Fmoc)) group, Nsc (2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc) ) group, Bsmoc (1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc)) group, α-Nsmoc ( (1,1-Dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α -Nsmoc)) group, Dde & ivDde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) and 1-(4,4-Dimethyl-2, 6-dioxocyclohex-1-ylidene)-3-mehtylbutyl (ivDde)) group, Fmoc* (2,7-Di-tert-butyl-Fmoc (Fmoc*)) group, Fmoc(2F) (2-Fluoro-Fmoc ( Fmoc(2F))) group, allyl group, mio-Fmoc & dio-Fmoc (2-Monoisooctyl-Fmoc (mio-Fmoc) and 2,7-Diisooctyl-Fmoc (dio-Fmoc)) group, TCP ( Tetrachlorophthaloyl (TCP)) group, Pms (2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms)) group, Esc (Ethanesulfonylethoxycarbonyl (Esc)) group, Sps (2-(4-Sulfophenylsulfonyl)ethoxycarbonyl (Sps)) group , Z (Benzyloxycarbonyl (Z)) group, Alloc (Allyloxycarbonyl (Alloc)) group, oNBS & pNBS (o-Nitrobenzenesulfonyl (oNBS) and p-nitrobenzenesulfonyl (pNBS) group, dNBS (2,4-Dinitrobenzenesulfonyl (dNBS)) group , Bts (Benzothiazole-2-sulfonyl (Bts)) group, Troc (2,2,2-Trichloroethyloxycarbonyl (Troc)) group, Dts (Dithiasuccinoyl (Dts)) group, pNZ (p-Nitrobenzyloxycarbonyl (pNZ)) group, Poc (Propargyloxycarbonyl (Poc)) group, oNZ & NVOC (o-Nitrobenzyloxycarbonyl (oNZ) and 6-Nitroberatryloxycarbonyl (NVOC)) group, NPPOC (2-(2-Nitrophenyl)propyloxycarbonyl (NPPOC)) group, MNPPOC (2-(3) ,4-Methylenedioxy-6-nitrophenyl)propyloxycarbonyl (MNPPOC)) group, BrPhF (9-(4-Bromophenyl)-9-fluorenyl (BrPhF)) group, Azoc (Azidomethyloxycarbonyl (Azoc)) group, Cl-Z (2- Examples include Chlorobenzyloxycarbonyl (Cl-Z)) group, Boc (tert-Butyloxycarbonyl (Boc)) group, Mtt (4-Methyltrityl (Mtt)) group, Azoc (Azidomethyloxycarbonyl (Azoc)) group, Cl-Z ( It is preferable to use 2-Chlorobenzyloxycarbonyl (Cl-Z)) group, Boc (tert-Butyloxycarbonyl (Boc)) group, and Mtt (4-Methyltrityl (Mtt)) group, and Boc (tert-Butyloxycarbonyl (Boc)) group, Mtt ( It is more preferable to use the 4-Methyltrityl (Mtt)) group, and it is most preferable to use the Boc (tert-Butyloxycarbonyl (Boc)) group as the amine protecting group.
상기 화학식에서 R₂는 당 업계에서 통상적으로 이용하는 수소 또는 수산기 보호기를 이용할 수 있다. 상기 수산기 보호기는 터트-부틸 (tert-butyl)기, 트리페닐메틸 (triphenylmethyl 또는 trityl)기, 아세트아미노메틸 (acetaminomethyl)기, 벤조일 (benzoyl)기, 디페닐메틸 (diphenylmethyl)기, 벤질 (benzyl)기, 파라-메톡시벤질 (para-methoxybenzyl)기, 벤질옥시카보닐 (benzyloxycarbonyl)기, 파라-니트로벤질 (para-nitrobenzyl)기, 알릴 (allyl)기, 디메틸실릴 (dimethylsilyl)기, 터트-부틸디메틸실릴 (tert-butyldimethylsilyl)기, 트리이소프로필실릴 (triisopropylsilyl)기, 알킬 (alkyl, 탄소수 10개 이하)기 등을 예시할 수 있으며, 터트-부틸 (tert-butyl)기, 트리페닐메틸 (triphenylmethyl)기 또는 아세트아미노메틸 (acetaminomethyl)기가 바람직하며, 터트-부틸 (tert-butyl)기를 이용하는 것이 보다 바람직하다.In the above chemical formula, R₂ can use a hydrogen or hydroxyl protecting group commonly used in the industry. The hydroxyl protecting group is tert -butyl group, triphenylmethyl or trityl group, acetaminomethyl group, benzoyl group, diphenylmethyl group, and benzyl group. group, para -methoxybenzyl group, benzyloxycarbonyl group, para -nitrobenzyl group, allyl group, dimethylsilyl group, tert-butyl group Examples include dimethylsilyl ( tert -butyldimethylsilyl) group, triisopropylsilyl group, alkyl (10 or less carbon atoms) group, tert-butyl ( tert -butyl) group, triphenylmethyl ) group or acetaminomethyl group is preferable, and it is more preferable to use a tert -butyl group.
상기 화학식에서 R₃은 당 업계에서 통상적으로 이용하는 아민 보호기를 이용할 수 있다. 아세틸 (Acetyl)기, 벤조일(Benzoyl), 벤질옥시메틸 (Benzyloxymethyl)기, 트리틸 (Trityl)기, Ddz (α,α-Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz))기, Bpoc (2-(4-Biphenyl)isopropoxycarbonyl (Bpoc))기, Nps (2-Nitrophenylsulfenyl (Nps))기, Fmoc (9-Fluorenylmethoxycarbonyl (Fmoc))기, Nsc (2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc))기, Bsmoc (1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc))기, α-Nsmoc ( (1,1-Dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α-Nsmoc))기, Dde & ivDde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) and 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-mehtylbutyl (ivDde))기, Fmoc* (2,7-Di-tert-butyl-Fmoc (Fmoc*))기, Fmoc(2F) (2-Fluoro-Fmoc (Fmoc(2F)))기, 알릴 (allyl)기, mio-Fmoc & dio-Fmoc (2-Monoisooctyl-Fmoc (mio-Fmoc) and 2,7-Diisooctyl-Fmoc (dio-Fmoc))기, TCP (Tetrachlorophthaloyl (TCP))기, Pms (2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms))기, Esc (Ethanesulfonylethoxycarbonyl (Esc))기, Sps (2-(4-Sulfophenylsulfonyl)ethoxycarbonyl (Sps))기, Z (Benzyloxycarbonyl (Z))기, Alloc (Allyloxycarbonyl (Alloc))기, oNBS & pNBS (o-Nitrobenzenesulfonyl (oNBS) and p-nitrobenzenesulfonyl (pNBS)기, dNBS (2,4-Dinitrobenzenesulfonyl (dNBS))기, Bts (Benzothiazole-2-sulfonyl (Bts))기, Troc (2,2,2-Trichloroethyloxycarbonyl (Troc))기, Dts (Dithiasuccinoyl (Dts))기, pNZ (p-Nitrobenzyloxycarbonyl (pNZ))기, Poc (Propargyloxycarbonyl (Poc))기, oNZ & NVOC (o-Nitrobenzyloxycarbonyl (oNZ) and 6-Nitroveratryloxycarbonyl (NVOC))기, NPPOC (2-(2-Nitrophenyl)propyloxycarbonyl (NPPOC))기, MNPPOC ( 2-(3,4-Methylenedioxy-6-nitrophenyl)propyloxycarbonyl (MNPPOC))기, BrPhF (9-(4-Bromophenyl)-9-fluorenyl (BrPhF))기, Azoc (Azidomethyloxycarbonyl (Azoc))기, Cl-Z (2-Chlorobenzyloxycarbonyl (Cl-Z))기, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기 등을 예시할 수 있으며, Azoc (Azidomethyloxycarbonyl (Azoc))기, Cl-Z (2-Chlorobenzyloxycarbonyl (Cl-Z))기, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기를 이용하는 것이 바람직하며, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기를 이용하는 것이 보다 바람직하며, 아민 보호기가 Mtt (4-Methyltrityl (Mtt))기를 이용하는 것이 가장 바람직하다.In the above chemical formula, R₃ can use an amine protecting group commonly used in the industry. Acetyl group, Benzoyl, Benzyloxymethyl group, Trityl group, Ddz (α,α-Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz)) group, Bpoc (2-( 4-Biphenyl)isopropoxycarbonyl (Bpoc)) group, Nps (2-Nitrophenylsulfenyl (Nps)) group, Fmoc (9-Fluorenylmethoxycarbonyl (Fmoc)) group, Nsc (2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc)) group, Bsmoc (1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc)) group, α-Nsmoc ( (1,1-Dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α-Nsmoc)) Group, Dde & ivDde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) and 1-(4,4-Dimethyl-2,6-dioxocyclohex- 1-ylidene)-3-mehtylbutyl (ivDde)) group, Fmoc* (2,7-Di-tert-butyl-Fmoc (Fmoc*)) group, Fmoc(2F) (2-Fluoro-Fmoc (Fmoc(2F) )) group, allyl group, mio-Fmoc & dio-Fmoc (2-Monoisooctyl-Fmoc (mio-Fmoc) and 2,7-Diisooctyl-Fmoc (dio-Fmoc)) group, TCP (Tetrachlorophthaloyl (TCP) ) group, Pms (2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms)) group, Esc (Ethanesulfonylethoxycarbonyl (Esc)) group, Sps (2-(4-Sulfophenylsulfonyl)ethoxycarbonyl (Sps)) group, Z (Benzyloxycarbonyl (Z)) group, Alloc (Allyloxycarbonyl (Alloc)) group, oNBS & pNBS (o-Nitrobenzenesulfonyl (oNBS) and p-nitrobenzenesulfonyl (pNBS) group, dNBS (2,4-Dinitrobenzenesulfonyl (dNBS)) group, Bts (Benzothiazole -2-sulfonyl (Bts)) group, Troc (2,2,2-Trichloroethyloxycarbonyl (Troc)) group, Dts (Dithiasuccinoyl (Dts)) group, pNZ (p-Nitrobenzyloxycarbonyl (pNZ)) group, Poc (Propargyloxycarbonyl (Poc) )) group, oNZ & NVOC (o-Nitrobenzyloxycarbonyl (oNZ) and 6-Nitroveratryloxycarbonyl (NVOC)) group, NPPOC (2-(2-Nitrophenyl)propyloxycarbonyl (NPPOC)) group, MNPPOC ( 2-(3,4-Methylenedioxy -6-nitrophenyl)propyloxycarbonyl (MNPPOC)) group, BrPhF (9-(4-Bromophenyl)-9-fluorenyl (BrPhF)) group, Azoc (Azidomethyloxycarbonyl (Azoc)) group, Cl-Z (2-Chlorobenzyloxycarbonyl (Cl-) Z)) group, Boc (tert-Butyloxycarbonyl (Boc)) group, Mtt (4-Methyltrityl (Mtt)) group, etc., Azoc (Azidomethyloxycarbonyl (Azoc)) group, Cl-Z (2-Chlorobenzyloxycarbonyl ( Cl-Z)) group, Boc (tert-Butyloxycarbonyl (Boc)) group, and Mtt (4-Methyltrityl (Mtt)) group are preferably used. Boc (tert-Butyloxycarbonyl (Boc)) group, Mtt (4-Methyltrityl ( It is more preferable to use the Mtt)) group, and it is most preferable to use the Mtt (4-Methyltrityl (Mtt)) group as the amine protecting group.
상기 작용기에 대한 보호기는 "Protecting Groups in Organic Synthesis (Greene and Wuts, John Wiley & Sons, 1991)"와 "Amino acid-Protecting Groups (A Isidro Llobet 저술, 2009)에 상세히 기재되어 있다.Protecting groups for the above functional groups are described in detail in “Protecting Groups in Organic Synthesis (Greene and Wuts, John Wiley & Sons, 1991)” and “Amino acid-Protecting Groups” (written by A Isidro Llobet, 2009).
본 명세서에서 용어 "펩타이드"는 펩타이드 결합에 의해 아미노산 잔기들이 서로 결합되어 형성된 선형의 분자를 의미한다.As used herein, the term “peptide” refers to a linear molecule formed by linking amino acid residues together through peptide bonds.
도 1을 참조로 하여 본 발명의 제조방법을 각각의 단계별로 상세하게 설명하면 다음과 같다.With reference to Figure 1, the manufacturing method of the present invention will be described in detail in each step as follows.
(a) (a) 화학식 Ⅰ-a 및 화학식 Ⅱ-a로 표시되는 레진이 부착된 펩타이드 수득Obtaining resin-attached peptides represented by Formula I-a and Formula II-a
화학식 Ⅰ-a 및 화학식 Ⅱ-a로 표시되는 레진이 부착된 펩타이드는 당 업계에서 통상적으로 사용하는 고체상 (solid- phase) 합성법에 의해 제조될 수 있다 (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154(1963), Kaiser, E., Colescot, R. L., Bossinger, C. D., Cook, P.I., Anal. Biochem., 34:595-598(1970)). 즉, 알파-아미노 및 측쇄 작용기가 보호화된 아미노산을 레진에 결합시킨 후, 알파-아미노 보호기를 제거하고 남은 알파-아미노 및 측쇄 작용기가 보호화된 아미노산을 원하는 순서로 단계적으로 결합하여 중간체를 얻는다. Resin-attached peptides represented by Formula I-a and Formula II-a can be prepared by solid-phase synthesis methods commonly used in the art (Merrifield, RB, J. Am. Chem. Soc ., 85:2149-2154 (1963), Kaiser, E., Colescot, RL, Bossinger, CD, Cook, PI, Anal. Biochem., 34:595-598 (1970). That is, after binding the amino acid with protected alpha-amino and side chain functional groups to the resin, the alpha-amino protecting group is removed and the remaining amino acids with protected alpha-amino and side chain functional groups are combined step by step in the desired order to obtain an intermediate. .
본 발명에서는 펩타이드 서열 중 7번째 아미노산은 아민 보호기가 Mtt인 것을 사용하는 것을 특징으로 한다. 상기 아미노산은 Fmoc-Dab(Mtt)-OH일 수 있다. Mtt를 보호기로 사용할 경우, 레진으로부터 보호화된 펩타이드를 떼어내면서 Mtt 보호기도 동시에 선택적으로 떼어낼수 있다.In the present invention, the 7th amino acid in the peptide sequence is characterized in that the amine protecting group is Mtt. The amino acid may be Fmoc-Dab(Mtt)-OH. When Mtt is used as a protecting group, the Mtt protecting group can be selectively removed at the same time as the protected peptide is removed from the resin.
적절한 보호기의 선택은 보호되는 작용기, 보호기가 노출되는 조건 및 그 분자 내에 존재할 수 있는 다른 작용기에 따라 달라진다. 보호기는 합성 각 단계에서 ㈀ 알파-아미노 보호기를 제거하기 위해 선택한 반응조건 및 시약에 대해 안정해야 하고, ㈁ 결합반응에서 탈 보호화 반응이 일어나지 않아야 하며, ㈂ 원하는 아미노산 사슬을 포함하는 합성이 완결되었을 때 레진과의 분해 조건에서 안정하여야 한다.The selection of an appropriate protecting group depends on the functional group being protected, the conditions under which the protecting group is exposed, and other functional groups that may be present in the molecule. The protecting group must be stable against the reaction conditions and reagents selected to remove the alpha-amino protecting group at each stage of synthesis, ㈁ no deprotection reaction occurs in the coupling reaction, and ㈂ the synthesis containing the desired amino acid chain must be complete. It must be stable under conditions of decomposition with resin.
본 발명의 바람직한 구현 예에 따르면, 화학식 Ⅰ-a 및 화학식 Ⅱ-a로 표시되는 펩타이드를 합성하는 과정에서 사용될 수 있는 레진은 제조된 펩타이드의 측쇄 보호기를 완전히 보존시킬 수 있는 온화한 산성 조건하에서 쉽게 분해될 수 있는 통상적인 레진을 사용할 수 있다. 바람직하게는, 상기 레진은 트리틸클로라이드 레진 (tritylchloride resin), 2-클로로트리틸클로라이드 레진 (2-chlorotritylchloride resin), 4-메틸트리틸클로라이드 레진 (4-methyltritylchloride resin) 또는 4-메톡시트리틸클로라이드 레진 (4-methoxytritylchloride resin), PAM 레진 (PAM Resin), 왕 레진 (Wang Resin), 옥심 레진 (Oxime Resin), HMBA 레진 (HMBA Resin), 이고, 보다 바람직하게는 왕 레진 (Wang Resin), 트리틸클로라이드 레진 (tritylchloride resin) 또는 2-클로로트리틸클로라이드 레진 (2-chlorotritylchloride resin)이며, 가장 바람직하게는 2-클로로트리틸클로라이드 레진 (2-chlorotritylchloride resin)이다.According to a preferred embodiment of the present invention, the resin that can be used in the process of synthesizing peptides represented by Formula I-a and Formula II-a is easily decomposed under mild acidic conditions that can completely preserve the side chain protecting group of the prepared peptide. Any conventional resin can be used. Preferably, the resin is tritylchloride resin, 2-chlorotritylchloride resin, 4-methyltritylchloride resin, or 4-methoxytrityl. 4-methoxytritylchloride resin, PAM Resin, Wang Resin, Oxime Resin, HMBA Resin, more preferably Wang Resin, It is tritylchloride resin or 2-chlorotritylchloride resin, most preferably 2-chlorotritylchloride resin.
(b) (b) 화학식 I-b 및 화학식 Ⅱ-b로 표시되는 펩타이드의 수득Obtaining peptides represented by Formula I-b and Formula II-b
상기 화학식 I-b 및 화학식 Ⅱ-b로 표시되는 화합물은 상기 단계 (a)에서 얻은 펩타이드로부터 온화한 산성 조건 하에서 레진을 제거하여 얻을 수 있다. 이 때, 사용할 수 있는 산성 조건은 아미노산 사슬의 측쇄 보호기가 유지될 수 있는 온화한 조건이어야 한다.Compounds represented by Formula I-b and Formula II-b can be obtained by removing the resin from the peptide obtained in step (a) under mild acidic conditions. At this time, the acidic conditions that can be used must be mild conditions in which the side chain protecting group of the amino acid chain can be maintained.
본 발명의 바람직한 구현 예에 따르면, 레진을 제거하는 과정은 산성 용액의 존재 하에서 수행할 수 있다. 상기 산성 용액은 아세토니트릴 (Acetonitrile), 디클로로메탄 (dichloromethane), 삼불화 초산 (Trifluoroacetic acid), 삼염화 초산 (Trichloroacetic acid), 염화초산 (Chloroacetic acid), 이염화 초산 (Dichloroacetic acid), 브롬화 초산 (Bromoacetic acid), 이브롬화 초산 (Dibromoacetic acid), 삼브롬화 초산 (Tribromoacetic acid), 아세트산 (acetic acid), 포름산 (Formic acid), 메탄술폰산 (Methanesulfonic acid), 벤젠술폰산 (Benzenesulfonic acid), 파라-톨루엔술폰산 (para-Toluenesulfonic acid), 메탄올 (Methanol), 에탄올 (Ethanol), 이소프로판올 (Isopropanol), 트리플루오로에탄올 (trifluoroethanol) 등의 단독 용액 또는 이들의 혼합용액을 이용할 수 있다.According to a preferred embodiment of the present invention, the process of removing the resin can be performed in the presence of an acidic solution. The acidic solution includes Acetonitrile, dichloromethane, Trifluoroacetic acid, Trichloroacetic acid, Chloroacetic acid, Dichloroacetic acid, and Bromoacetic acid. acid), Dibromoacetic acid, Tribromoacetic acid, acetic acid, Formic acid, Methanesulfonic acid, Benzenesulfonic acid, para-toluenesulfonic acid ( Para-Toluenesulfonic acid, methanol, ethanol, isopropanol, trifluoroethanol, etc. can be used alone or mixed solutions thereof.
바람직하게는, 산성 조건은 2 내지 5부피% 삼염화 초산 (Trichloroacetic acid) 용액, 2 내지 5부피% 트리플루오로아세트산 (trifluoroacetic acid) 용액, 0.1%의 포름산 (Formic acid), 메탄술폰산 (Methanesulfonic acid), 벤젠술폰산 (Benzenesulfonic acid) 또는 파라-톨루엔술폰산 (para-Toluenesulfonic acid)이 포함된 디클로로메탄 (dichloromethane) 용액을 단독 또는 혼합 사용하는 것을 예시할 수 있다.Preferably, the acidic conditions include a 2 to 5% by volume solution of Trichloroacetic acid, a 2 to 5% by volume solution of trifluoroacetic acid, 0.1% of Formic acid, and Methanesulfonic acid. , a dichloromethane solution containing benzenesulfonic acid or para-Toluenesulfonic acid may be used alone or in combination.
(c) (c) 화학식 I-c 및 화학식 Ⅱ-c로 표시되는 고리화 펩타이드의 수득Obtaining cyclized peptides represented by formula I-c and formula II-c
화학식 I-c 및 화학식 Ⅱ-c로 표시되는 화합물은 상기 단계 (b)에서 얻은 펩타이드를 촉매 존재하에 고리화 반응을 수행하여 얻는다.Compounds represented by Formula I-c and Formula II-c are obtained by performing a cyclization reaction of the peptide obtained in step (b) in the presence of a catalyst.
본 발명의 바람직한 구현 예에 따르면, 단계 (c)에서 이용 가능한 촉매 시약은 DCC (N,N-dicyclohexylcarbodiimide), DIC (N,N-diisopropylcarbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphatemethanaminium), TATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroboratemethanaminium), CDI (carbonyldiimidazole), EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), Oxyma (Ethyl cyanohydroxyiminoacetate), HOBt (1-Hydroxybenzotriazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole) 등을 단독 또는 혼합하여 사용할 수 있고, PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), HOBt (1-Hydroxybenzotriazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole)를 사용하는 것이 바람직하고, EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), HOAt (1-Hydroxyazabenzotriazole)를 사용하는 것이 가장 바람직하다.According to a preferred embodiment of the present invention, the catalytic reagents available in step (c) include DCC (N,N-dicyclohexylcarbodiimide), DIC (N,N-diisopropylcarbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-( dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), TBTU (O-(Benzotriazol-1-yl)-N ,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphatemethanaminium), TATU (2-(1H-7-Azabenzotriazol- 1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroboratemethanaminium), CDI (carbonyldiimidazole), EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), DEPBT (3-(diethoxyphosphoryloxy)-1, 2,3-benzotriazin-4(3H)-one), Oxyma (Ethyl cyanohydroxyiminoacetate), HOBt (1-Hydroxybenzotriazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole), etc. Can be used alone or in combination, PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), HOBt (1-Hydroxybenzotriazole), 6-ClHOBt (1 -Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole) is preferably used, and EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), HOAt (1-Hydroxyazabenzotriazole) is used. It is most desirable to do so.
본 발명의 바람직한 구현 예에 따르면, 고리화 반응에서 사용되는 용매는 1,2-디클로로에탄 (1,2-dichloroethane), 클로로포름 (chloroform), 디클로로메탄 (Dichloromethane), 1,2-디클로로메탄 (1,2-Dichloromethane), 테트하이드로퓨란 (Tetrahydrofurane), 1,4-디옥산 (1,4-Dioxane), 아세토니트릴 (Acetonitrile), 디메틸설폭시드 (Dimethylsulfoxide), N,N-디메틸포름아미드 (N,N-Dimethylformamide), N,N-디메틸아세트아미드 (N,N-Dimethylacetamide)이며, 바람직하게는 디클로로메탄 (dichloromethane)과 N,N-디메틸포름아미드 (N,N-dimethylformamide)이며, 가장 바람직하게는 디클로로메탄 (dichloromethane)과 N,N-디메틸포름아미드 (N,N-dimethylformamide)의 혼합용액이다.According to a preferred embodiment of the present invention, the solvent used in the cyclization reaction is 1,2-dichloroethane, chloroform, dichloromethane, 1,2-dichloromethane (1 ,2-Dichloromethane, Tetrahydrofurane, 1,4-Dioxane, Acetonitrile, Dimethylsulfoxide, N,N-dimethylformamide (N, N-Dimethylformamide), N,N-dimethylacetamide, preferably dichloromethane and N,N-dimethylformamide, most preferably It is a mixed solution of dichloromethane and N,N-dimethylformamide.
상기 고리화 반응은 -20~50℃에서 수행할 수 있으며, 0~30℃에서 수행하는 것이 바람직하다. 또한 고리화 반응의 농도는 펩타이드로 환산하여 10-2몰에서 10-9몰 농도 범위내에서 수행 가능하며, 바람직하게는 10-5몰에서 10-7몰 농도에서 수행하는 것이 바람직하지만 이에 한정되는 것은 아니다.The cyclization reaction can be performed at -20 to 50°C, and is preferably performed at 0 to 30°C. In addition, the concentration of the cyclization reaction can be performed within the range of 10 -2 mole to 10 -9 mole in terms of peptide, and is preferably carried out at a concentration of 10 -5 mole to 10 -7 mole, but is limited thereto. That is not the case.
(d) (d) 화학식 I로 표시되는 폴리믹신 E 및 화학식 Ⅱ으로 표시되는 폴리믹신 B펩타이드의 수득Obtaining polymyxin E represented by formula I and polymyxin B peptide represented by formula II
화학식 I로 표시되는 폴리믹신 E 및 화학식 Ⅱ으로 표시되는 폴리믹신 B펩타이드는 상기 단계 (c)에서 얻은 펩타이드로부터 당 업계에서 통상적으로 이용하는 반응 조건하에서 탈 보호화 반응을 수행하여 얻을 수 있다.Polymyxin E represented by Formula I and polymyxin B peptide represented by Formula II can be obtained from the peptide obtained in step (c) by performing a deprotection reaction under reaction conditions commonly used in the art.
탈 보호화 반응은 (1) 트리플루오로아세트산 (trifuoroacetic acid), 물 (H₂O), 페놀 (phenol), 티오아니솔 (thioanisole) 및 1,2-에탄디티올 (1,2-ethanedithiol)의 혼합물; (2) 트리플루오로아세트산 (trifuoroacetic acid), 물 (H₂O), 페놀 (phenol), 트리이소프로필실란 (Triisopropylsilane)의 혼합물; (3) 트리플루오로아세트산 (trifuoroacetic acid), 물 (H₂O), 페놀 (phenol), 티오아니솔 (thioanisole) 및 1-도데칸티올 (1-dodecanethiol)의 혼합물; (4) 트리플루오로아세트산 (trifuoroacetic acid), DTT (Dithiothreonitol), 물 (H₂O), 트리이소프로필실란 (Triisopropylsilane)의 혼합물; (5) 트리플루오로아세트산 (trifuoroacetic acid), 페놀 (phenol)의 혼합물; (6) 트리플루오로아세트산 (trifuoroacetic acid), 페놀 (phenol), 메탄술폰산 (Methanesulfonic acid)의 혼합물; (7) 트리플루오로아세트산 (trifuoroacetic acid), 티오아니솔 (thioanisole) 및 1,2-에탄디티올 (1,2-ethanedithiol), 아니솔 (anisole)의 혼합물; (8) 트리플루오로아세트산 (trifluoroacetic acid), 트리에틸실란 (triethylsilane)의 혼합물; (9) 트리플루오로아세트산 (trifluoroacetic acid), 물 (H₂O)의 혼합물; (10) 트리플루오로아세트산 (trifluoroacetic acid), 디클로로메탄 (Dichloromerhane), 인돌 (indole)의 혼합물 하에서 수행될 수 있으며, 트리플루오로아세트산 (trifluoroacetic acid), 물 (H₂O)의 혼합물 하에서 수행되는 것이 바람직하다.The deprotection reaction is (1) a mixture of trifluoroacetic acid, water (H₂O), phenol, thioanisole, and 1,2-ethanedithiol. ; (2) a mixture of trifluoroacetic acid, water (H₂O), phenol, and triisopropylsilane; (3) a mixture of trifluoroacetic acid, water (H₂O), phenol, thioanisole and 1-dodecanethiol; (4) a mixture of trifuoroacetic acid, DTT (Dithiothreonitol), water (H₂O), and triisopropylsilane; (5) mixture of trifluoroacetic acid and phenol; (6) mixtures of trifluoroacetic acid, phenol, and methanesulfonic acid; (7) mixtures of trifluoroacetic acid, thioanisole and 1,2-ethanedithiol, anisole; (8) mixture of trifluoroacetic acid and triethylsilane; (9) mixture of trifluoroacetic acid and water (H₂O); (10) It can be performed in a mixture of trifluoroacetic acid, dichloromethane, and indole, and is preferably performed in a mixture of trifluoroacetic acid and water (H₂O). do.
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.
본 명세서 전체에 거쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 한 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and liquid (weight/volume) %, unless otherwise specified. /liquid is (volume/volume) %.
실시 예 1 : 폴리믹신 E 및 B의 합성Example 1: Synthesis of polymyxins E and B
1-1: 화학식 I로 표시되는 폴리믹신 E 펩타이드의 합성1-1: Synthesis of polymyxin E peptide represented by formula I
[화학식 I][Formula I]
1-1-1: H-Thr(tBu)-2-클로로트리틸 레진의 합성1-1-1: Synthesis of H-Thr(tBu)-2-chlorotrityl resin
여과막이 장착된 고체상 (solid-phase)합성 반응기에 2-클로로트리틸 클로라이드 레진 (치환율 = 1.08 mmol/g의 레진, 100 mmol) 및 디클로로메탄 (1000 ml)를 넣고, 15분간 레진을 팽창시킨 후, 감압 하에서 여과막을 통하여 용매를 제거하였다. Fmoc-Thr(tBu)-OH (분자량 = 297.5 g/mol) (59.5 g, 200 mmol, 2.0 당량)이 포함된 디클로로메탄 (1000 ml)을 넣고, 이어서 N,N-디이소프로필에틸아민 (분자량 = 129.25 g/mol) (32.31 g, 250 mmol, 2.5 당량)를 첨가한 후, 실온에서 12시간 동안 반응시켰다. 감압 여과하여 반응액을 제거하고, 레진을 디클로로메탄으로 1회 세척한 후, 레진에 디클로로메탄 : 메탄올 : N,N-디이소프로필에틸아민 = 17 : 2 : 1 (1000 ml)을 넣고 20분간 교반시켰다. 감압 여과하여 반응액을 제거하고, 레진을 디클로로메탄으로 3회 세척한 후, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)을 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Add 2-chlorotrityl chloride resin (ratio of substitution = 1.08 mmol/g of resin, 100 mmol) and dichloromethane (1000 ml) to a solid-phase synthesis reactor equipped with a filtration membrane, and allow the resin to swell for 15 minutes. , the solvent was removed through a filtration membrane under reduced pressure. Dichloromethane (1000 ml) containing Fmoc-Thr(tBu)-OH (molecular weight = 297.5 g/mol) (59.5 g, 200 mmol, 2.0 equiv) was added, followed by N,N-diisopropylethylamine (molecular weight = 129.25 g/mol) (32.31 g, 250 mmol, 2.5 equivalents) was added and reacted at room temperature for 12 hours. The reaction solution was removed by filtration under reduced pressure, the resin was washed once with dichloromethane, and then dichloromethane : methanol : N, N-diisopropylethylamine = 17 : 2 : 1 (1000 ml) was added to the resin and incubated for 20 minutes. It was stirred. The reaction solution was removed by filtration under reduced pressure, and the resin was washed three times with dichloromethane. Then, N,N-dimethylformamide (1000 ml) containing 20% (v/v) piperidine was added and the Fmoc concentration was maintained for 15 minutes. After performing the removal reaction, the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to obtain H-Thr(tBu)-2-chlorotrityl resin (yield > 98%).
1-1-2: H-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진의 합성1-1-2: Synthesis of H-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin
상기 반응에서 얻은 H-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 Fmoc-Dab(Boc)-OH (분자량 = 440.49 g/mol) (132.15 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.Fmoc-Dab(Boc)-OH (molecular weight = 440.49 g/mol) (132.15 g, 300 mmol, 3 equivalents) and Oxyma were added to H-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction. (Molecular weight = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 equivalent) of N,N-dimethylformamide (1000 ml) was added, followed by N,N-diisopropylcarbodiimide (molecular weight = 126.20 g/ mol, 41.65 g, 3.3 equivalent) was added and reacted at room temperature for 4 hours.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)를 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, the resin was washed twice with N,N-dimethylformamide (1000 ml), and then washed with N,N-dimethylformamide containing 20% (v/v) piperidine. Formamide (1000 ml) was added to remove Fmoc for 15 minutes, and then the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to produce H-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin. Obtained (yield >98%).
1-1-3: H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진의 합성1-1-3: Synthesis of H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin
상기 반응에서 얻은 H-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 Fmoc-Dab(Boc)-OH (분자량 = 440.49 g/mol) (132.15 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.Fmoc-Dab(Boc)-OH (molecular weight = 440.49 g/mol) (132.15 g, 300 mmol, 3 equivalents) and N,N-dimethylformamide (1000 ml) of Oxyma (molecular weight = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 equivalents) were added, followed by N,N-diisopropylcarbodiimide ( Molecular weight = 126.20 g/mol, 41.65 g, 3.3 equivalent) was added and reacted at room temperature for 4 hours.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)를 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, the resin was washed twice with N,N-dimethylformamide (1000 ml), and then washed with N,N-dimethylformamide containing 20% (v/v) piperidine. Formamide (1000 ml) was added to remove Fmoc for 15 minutes, and then the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to remove H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2- Chlorotrityl resin was obtained (yield >98%).
1-1-4: H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진의 합성1-1-4: Synthesis of H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin
상기 반응에서 얻은 H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 Fmoc-Leu-OH (분자량 = 353.4 g/mol) (106.02 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.Fmoc-Leu-OH (molecular weight = 353.4 g/mol) (106.02 g, 300 mmol) was added to H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction. mmol, 3 equivalents) and N,N-dimethylformamide (1000 ml) of Oxyma (molecular weight = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 equivalents) were added, followed by N,N-diisopropylcarbodi Mead (molecular weight = 126.20 g/mol, 41.65 g, 3.3 equivalent) was added, and then reacted at room temperature for 4 hours.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)를 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, the resin was washed twice with N,N-dimethylformamide (1000 ml), and then washed with N,N-dimethylformamide containing 20% (v/v) piperidine. Formamide (1000 ml) was added to remove Fmoc for 15 minutes, and then the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to remove H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)- 2-Chlorotrityl resin was obtained (yield >98%).
1-1-5: H-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진의 합성1-1-5: Synthesis of H-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin
상기 반응에서 얻은 H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 Fmoc-D-Leu-OH (분자량 = 353.4 g/mol) (106.02 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.Fmoc-D-Leu-OH (molecular weight = 353.4 g/mol) was added to H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction ( 106.02 g, 300 mmol, 3 equivalents) and N,N-dimethylformamide (1000 ml) of Oxyma (molecular weight = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 equivalents) were added, followed by N,N-dimethylformamide Isopropylcarbodiimide (molecular weight = 126.20 g/mol, 41.65 g, 3.3 equivalents) was added, and then reacted at room temperature for 4 hours.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)를 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, the resin was washed twice with N,N-dimethylformamide (1000 ml), and then washed with N,N-dimethylformamide containing 20% (v/v) piperidine. Formamide (1000 ml) was added to remove Fmoc for 15 minutes, and then the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to remove H-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu). )-2-Chlorotrityl resin was obtained (yield >98%).
1-1-6: H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진의 합성1-1-6: Synthesis of H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin
상기 반응에서 얻은 H-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 Fmoc-Dab(Boc)-OH (분자량 = 440.49 g/mol) (132.15 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.Fmoc-Dab(Boc)-OH (molecular weight = 440.49 g/ mol) (132.15 g, 300 mmol, 3 equivalents) and Oxyma (molecular weight = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 equivalents) of N,N-dimethylformamide (1000 ml) were added, followed by N, N-diisopropylcarbodiimide (molecular weight = 126.20 g/mol, 41.65 g, 3.3 equivalents) was added, and then reacted at room temperature for 4 hours.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)를 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, the resin was washed twice with N,N-dimethylformamide (1000 ml), and then washed with N,N-dimethylformamide containing 20% (v/v) piperidine. Formamide (1000 ml) was added to remove Fmoc for 15 minutes, and then the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab. (Boc)-Thr(tBu)-2-chlorotrityl resin was obtained (yield >98%).
1-1-7: H-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr (tBu)-2-클로로트리틸 레진의 합성1-1-7: Synthesis of H-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr (tBu)-2-chlorotrityl resin
상기 반응에서 얻은 H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 Fmoc-Dab(Mtt)-OH (분자량 = 582.7 g/mol) (174.81 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.Fmoc-Dab(Mtt)- N,N-dimethylformamide (1000 ml) of OH (molecular weight = 582.7 g/mol) (174.81 g, 300 mmol, 3 eq) and Oxyma (molecular weight = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 eq) ) was added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g/mol, 41.65g, 3.3 equivalent) was added, and then reacted at room temperature for 4 hours.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)를 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, the resin was washed twice with N,N-dimethylformamide (1000 ml), and then washed with N,N-dimethylformamide containing 20% (v/v) piperidine. Formamide (1000 ml) was added to remove Fmoc for 15 minutes, and then the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab. (Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin was obtained (yield >98%).
1-1-8: H-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab (Boc)-Thr(tBu)-2-클로로트리틸 레진의 합성1-1-8: H-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab (Boc)-Thr(tBu)-2-chlorotrityl resin synthesis of
상기 반응에서 얻은 H-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 Fmoc-Dab(Boc)-OH (분자량 = 440.49 g/mol) (132.15 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.Fmoc- N,N-dimethyl of Dab(Boc)-OH (molecular weight = 440.49 g/mol) (132.15 g, 300 mmol, 3 eq) and Oxyma (molecular weight = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 eq) Formamide (1000 ml) was added, followed by N,N-diisopropylcarbodiimide (molecular weight = 126.20 g/mol, 41.65g, 3.3 equivalent), and then reacted at room temperature for 4 hours.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)를 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, the resin was washed twice with N,N-dimethylformamide (1000 ml), and then washed with N,N-dimethylformamide containing 20% (v/v) piperidine. Formamide (1000 ml) was added to remove Fmoc for 15 minutes, and then the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to remove H-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D- Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin was obtained (yield >98%).
1-1-9: H-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab (Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진의 합성1-1-9: H-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab (Boc)-Dab(Boc)-Thr(tBu)-2 -Synthesis of chlorotrityl resin
상기 반응에서 얻은 H-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 Fmoc-Thr(tBu)-OH (분자량 = 297.5 g/mol) (89.25 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.H-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin obtained in the above reaction (100 mmol) of Fmoc-Thr(tBu)-OH (molecular weight = 297.5 g/mol) (89.25 g, 300 mmol, 3 equiv) and Oxyma (molecular weight = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 equiv) Add N,N-dimethylformamide (1000 ml), then add N,N-diisopropylcarbodiimide (molecular weight = 126.20 g/mol, 41.65g, 3.3 equivalent), and react at room temperature for 4 hours. I ordered it.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)를 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, the resin was washed twice with N,N-dimethylformamide (1000 ml), and then washed with N,N-dimethylformamide containing 20% (v/v) piperidine. Formamide (1000 ml) was added to remove Fmoc for 15 minutes, and then the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to remove H-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab( Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin was obtained (yield >98%).
1-1-10: H-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu- Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진의 합성1-1-10: H-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu- Leu-Dab(Boc)-Dab(Boc)-Thr Synthesis of (tBu)-2-chlorotrityl resin
상기 반응에서 얻은 H-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 Fmoc-Dab(Boc)-OH (분자량 = 440.49 g/mol) (132.15 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.H-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chloro obtained from the above reaction Fmoc-Dab(Boc)-OH (molecular weight = 440.49 g/mol) (132.15 g, 300 mmol, 3 equiv) and Oxyma (molecular weight = 142.11 g/mol) (46.9 g, 330 mmol) in trityl resin (100 mmol) , 3.3 equivalents) of N,N-dimethylformamide (1000 ml) was added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g/mol, 41.65g, 3.3 equivalents) was added, and then incubated at room temperature. It was reacted for 4 hours.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 20% (v/v) 피페리딘이 포함된 N,N-디메틸포름아미드 (1000 ml)를 넣어 15분간 Fmoc의 제거 반응을 수행한 후, 감압 여과하여 반응액을 제거하였다. 상기 Fmoc의 제거 반응을 1회 반복하고, 이어서 레진을 차례로 N,N-디메틸포름아미드 (1000 ml)로 6회 세척하여 H-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, the resin was washed twice with N,N-dimethylformamide (1000 ml), and then washed with N,N-dimethylformamide containing 20% (v/v) piperidine. Formamide (1000 ml) was added to remove Fmoc for 15 minutes, and then the reaction solution was removed by filtration under reduced pressure. The Fmoc removal reaction was repeated once, and the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to remove H-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab ( Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin was obtained (yield >98%).
1-1-11: (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab (Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진의 제조1-1-11: (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab (Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab Preparation of (Boc)-Thr(tBu)-2-chlorotrityl resin
상기 반응에서 얻은 H-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진 (100 mmol)에 6-methyl-octanoic acid (분자량 = 158.24 g/mol) (47.47 g, 300 mmol, 3 당량) 및 Oxyma (분자량 = 142.11 g/mol) (46.9 g, 330 mmol, 3.3 당량)의 N,N-디메틸포름아미드 (1000 ml)를 넣고, 이어서 N,N-디이소프로필카르보디이미드 (분자량 = 126.20 g/mol, 41.65g, 3.3당량)을 첨가한 후, 실온에서 4시간 동안 반응시켰다.H-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu) obtained from the above reaction. )-2-chlorotrityl resin (100 mmol) with 6-methyl-octanoic acid (molecular weight = 158.24 g/mol) (47.47 g, 300 mmol, 3 equivalents) and Oxyma (molecular weight = 142.11 g/mol) (46.9 g) , 330 mmol, 3.3 equivalent) of N,N-dimethylformamide (1000 ml) was added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g/mol, 41.65g, 3.3 equivalent) was added. Afterwards, it was reacted at room temperature for 4 hours.
다음으로, 감압 여과하여 반응액을 제거하고, 레진을 N,N-디메틸포름아미드 (1000 ml)로 2회 세척한 다음, 디클로로메탄 (1000 ml)으로 3회 세척한 다음 건조하여 (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진을 수득하였다 (수율 >98%).Next, the reaction solution was removed by filtration under reduced pressure, and the resin was washed twice with N,N-dimethylformamide (1000 ml), then three times with dichloromethane (1000 ml), and dried (6-methyloctanoic acid). acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2 -Chlorotrityl resin was obtained (yield >98%).
1-1-12: (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab -Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH의 제조1-1-12: (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab -Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc) Preparation of -Thr(tBu)-OH
상기 반응에서 얻은 (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-클로로트리틸 레진에 디클로로메탄 (1000 ml), 5% 삼염화초산 (50 ml) 및 0.1% 메탄술폰산 (1 ml)를 넣고 30분 동안 교반하였다. 감압 여과하여 레진을 제거하고, 여과액을 감압 농축하여 보호화된 펩타이드 (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH (분자량: 1800.27 g/mol) 268.24 g (수율: 104.9%, 탈보호화 후 순도: 76.7%)을 수득하였다.(6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc) obtained from the above reaction. )-Thr(tBu)-2-chlorotrityl resin was added with dichloromethane (1000 ml), 5% trichloroacetic acid (50 ml), and 0.1% methanesulfonic acid (1 ml) and stirred for 30 minutes. The resin was removed by filtration under reduced pressure, and the filtrate was concentrated under reduced pressure to protect the protected peptide (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Leu. -Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH (molecular weight: 1800.27 g/mol) 268.24 g (yield: 104.9%, purity after deprotection: 76.7%) was obtained.
1-1-13: (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab- Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu) (1-7 cyclized) : 화학식 I의 보호기를 가진 폴리믹신 E의 합성1-1-13: (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab- Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc) -Thr(tBu) (1-7 cyclized): Synthesis of polymyxin E with protecting group of formula I
상기 반응에서 얻은 (6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH (268 g, 0.15mole)을 디클로로메탄/N,N-디메틸포름아미드 (9:1, 2000 ml)를 용해한 다음 EDC.HCl (분자량 = 191.7 g/mol, 86.26 g, 3당량), HOAt (분자량 = 136.11 g/mol, 61.25 g, 3당량) 넣고 12시간 동안 실온에서 교반하였다. 감압 농축하여 부피를 3/1 이하로 줄인 다음 에틸에테르 2500 ml에 결정화한 다음 석출된 고체를 여과하고 진공 건조하여 보호기를 가진 고리화된 폴리믹신 E 펩타이드 (분자량: 1782.25 g/mol) 243 g (수율: 90.7%, 탈보호화 후 순도: 70.4%)을 수득하였다.(6-methyloctanoic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr obtained from the above reaction. (tBu)-OH (268 g, 0.15 mole) was dissolved in dichloromethane/N,N-dimethylformamide (9:1, 2000 ml) and then dissolved in EDC.HCl (molecular weight = 191.7 g/mol, 86.26 g, 3 equivalents) ), HOAt (molecular weight = 136.11 g/mol, 61.25 g, 3 equivalents) were added and stirred at room temperature for 12 hours. Concentrate under reduced pressure to reduce the volume to less than 3/1, crystallize in 2500 ml of ethyl ether, filter the precipitated solid, and dry in vacuum to obtain 243 g of cyclized polymyxin E peptide with a protecting group (molecular weight: 1782.25 g/mol) ( Yield: 90.7%, purity after deprotection: 70.4%) was obtained.
1-1-14: 화학식 I의 폴리믹신 E의 합성1-1-14: Synthesis of polymyxin E of formula I
상기 반응에서 얻은 보호기를 가진 고리화된 폴리믹신 E 펩타이드 (243 g)을 트리플루오로아세트산 : 정제수 = 95 : 5의 혼합액 (3000 ml)를 넣고 3시간 상온에서 동안 교반하였다. 반응액을 에틸에테르 9000 ml에 서서히 부어넣어 펩타이드를 석출시키고 30분 동안 교반하였다. 생성된 펩타이드를 감압 여과하여 진공 건조 후 화학식 I의 폴리믹신 E 펩타이드 (분자량: 1389.51 g/mol) 178 g (이론양 138.95g, 수율: 128.10%, 순도: 67.3%, 정제 후 순도 98.8%)을 수득하였다.The cyclized polymyxin E peptide (243 g) with a protective group obtained in the above reaction was added to a mixture of trifluoroacetic acid and purified water = 95:5 (3000 ml) and stirred at room temperature for 3 hours. The reaction solution was slowly poured into 9000 ml of ethyl ether to precipitate the peptide and stirred for 30 minutes. The resulting peptide was filtered under reduced pressure, dried under vacuum, and then 178 g of the polymyxin E peptide of Formula I (molecular weight: 1389.51 g/mol) (theoretical weight 138.95 g, yield: 128.10%, purity: 67.3%, purity after purification 98.8%) was obtained. Obtained.
참고로, 상기 화학식 I로 표시되는 펩타이드는 6-methyloctanoic acid가 서열번호 1로 표시되는 아미노산 서열 (Dab-Thr-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr(1-7 cyclized))에 결합된 것이다.For reference, the peptide represented by Formula I has 6-methyloctanoic acid having the amino acid sequence represented by SEQ ID NO: 1 (Dab-Thr-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr (1-7 cyclized)).
1-2: 화학식 Ⅱ으로 표시되는 폴리믹신 B 펩타이드의 합성1-2: Synthesis of polymyxin B peptide represented by formula II
[화학식 Ⅱ][Formula Ⅱ]
상기 실시예 1-1의 화학식 I로 표시되는 폴리믹신 E의 합성 방법과 동일한 방법으로 합성하였으며, 단지 5번째 아미노산은 Fmoc-D-Leu-OH 대신 Fmoc-D-Phe-OH를 사용하여 합성하였다. 그 외 모든 제조방법을 동일하게 진행하였다. 합성결과 동일한 합성 Scale로 합성을 시작하여 화학식 Ⅱ로 표시되는 폴리믹신 B (분자량: 1423.52 g/mol) 168 g (이론양 142.35g, 수율: 118.02%, 순도: 70.6%, 정제 후 순도 98.2%)을 수득하였다.It was synthesized in the same manner as the synthesis method of polymyxin E represented by Formula I in Example 1-1, except that the 5th amino acid was synthesized using Fmoc-D-Phe-OH instead of Fmoc-D-Leu-OH. . All other manufacturing methods were performed in the same manner. Synthesis results: Polymyxin B (molecular weight: 1423.52 g/mol), 168 g (theoretical amount: 142.35g, yield: 118.02%, purity: 70.6%, purity after purification: 98.2%), represented by Chemical Formula II, starting with the same synthesis scale. was obtained.
참고로, 상기 화학식 Ⅱ로 표시되는 펩타이드는 6-methyloctanoic acid가 서열번호 2로 표시되는 아미노산 서열(Dab-Thr-Dab-Dab-Dab-Phe-Leu-Dab-Dab-Thr(1-7 cyclized))에 결합된 것이다.For reference, the peptide represented by Formula II has the amino acid sequence of 6-methyloctanoic acid shown in SEQ ID NO: 2 (Dab-Thr-Dab-Dab-Dab-Phe-Leu-Dab-Dab-Thr (1-7 cyclized) ) is combined with.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당 업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
서열목록 전자파일 첨부Sequence list electronic file attached
Claims (12)
(a) 고체상 (solid-phase) 합성법으로 화학식 I-a로 표시되는 레진이 부착된 펩타이드를 수득하는 단계;
(b) 상기 (a) 단계에서 수득된 펩타이드에서 레진을 제거하여 화학식 I-b로 표시되는 보호화된 펩타이드를 수득하는 단계;
(c) 상기 (b) 단계에서 수득된 펩타이드를 액상 (solution-phase) 합성법으로 촉매를 사용하여 고리화 반응시켜, 화학식 I-c로 표시되는 보호화된 고리화 펩타이드를 수득하는 단계; 및
(d) 상기 (c) 단계에서 수득한 펩타이드에서 탈 보호화 반응을 수행하여 화학식 Ⅰ로 표시되는 폴리믹신 E를 수득하는 단계:
[화학식 I]
[화학식 I-a]
[화학식 I-b]
[화학식 I-c]
상기 화학식에서, R₁는 아민 보호기, R₂는 수소 또는 수산기 보호기, R₃는 아민 보호기이다.
Method for preparing polymyxin E represented by formula I comprising the following steps:
(a) obtaining a resin-attached peptide represented by Formula Ia by solid-phase synthesis;
(b) removing the resin from the peptide obtained in step (a) to obtain a protected peptide represented by Formula Ib;
(c) cyclizing the peptide obtained in step (b) using a catalyst using a solution-phase synthesis method to obtain a protected cyclized peptide represented by Formula Ic; and
(d) performing a deprotection reaction on the peptide obtained in step (c) to obtain polymyxin E represented by Formula I:
[Formula I]
[Formula Ia]
[Formula Ib]
[Formula Ic]
In the above formula, R₁ is an amine protecting group, R₂ is a hydrogen or hydroxyl group protecting group, and R₃ is an amine protecting group.
The method of claim 1, wherein the acidic conditions for removing the protected peptide and optionally the R₃amine protecting group from the resin include (i) 2 to 5% by volume Trichloroacetic acid or trifluoroacetic acid solution; and (ii) selected from the group consisting of a dichloromethane solution containing 0.1% formic acid, methanesulfonic acid, benzenesulfonic acid, or para-Toluenesulfonic acid. A method for producing polymyxin E, characterized in that it is used alone or in combination.
The method for producing polymyxin E according to claim 1, wherein the cyclization reaction is performed at a temperature of -20 to 50°C.
The method for producing polymyxin E according to claim 1, wherein the concentration of the protected peptide in the cyclization reaction is within the range of 10 -2 mol to 10 -9 mol in terms of peptide.
The method of claim 1, wherein the solvent for the cyclization reaction of the peptide is 1,2-dichloroethane, chloroform, dichloromethane, 1,2-dichloromethane (1,2- Dichloromethane, Tetrahydrofurane, 1,4-Dioxane, Acetonitrile, Dimethylsulfoxide, N,N-Dimethylformamide ) and N,N-dimethylacetamide (N,N-Dimethylacetamide). A method for producing polymyxin E, characterized in that the solvent is used alone or in combination.
The method for producing polymyxin E according to claim 1, wherein the acidic solution for the deprotection reaction is a mixture containing trifluoroacetic acid and purified water (95:5).
(a) 고체상 (solid-phase) 합성법으로 화학식 Ⅱ-a로 표시되는 레진이 부착된 펩타이드를 수득하는 단계;
(b) 상기 (a) 단계에서 수득된 펩타이드에서 레진을 제거하여 화학식 Ⅱ-b로 표시되는 보호화된 펩타이드를 수득하는 단계;
(c) 상기 (b) 단계에서 수득된 펩타이드를 액상 (solution-phase) 합성법으로 촉매를 사용하여 고리화 반응시켜, 화학식 Ⅱ-c로 표시되는 보호화된 고리화 펩타이드를 수득하는 단계; 및
(d) 상기 (c) 단계에서 수득한 펩타이드에서 탈 보호화 반응을 수행하여 화학식 Ⅱ로 표시되는 폴리믹신 B를 수득하는 단계:
[화학식 Ⅱ]
[화학식 Ⅱ-a]
[화학식 Ⅱ-b]
[화학식 Ⅱ-c]
상기 화학식에서, R₁는 아민 보호기, R₂는 수소 또는 수산기 보호기, R₃는 아민 보호기이다.
A method for preparing polymyxin B represented by formula II comprising the following steps:
(a) obtaining a resin-attached peptide represented by Formula II-a by solid-phase synthesis;
(b) removing the resin from the peptide obtained in step (a) to obtain a protected peptide represented by Formula II-b;
(c) cyclizing the peptide obtained in step (b) using a catalyst using a solution-phase synthesis method to obtain a protected cyclized peptide represented by Formula II-c; and
(d) performing a deprotection reaction on the peptide obtained in step (c) to obtain polymyxin B represented by Formula II:
[Formula Ⅱ]
[Formula Ⅱ-a]
[Formula Ⅱ-b]
[Formula Ⅱ-c]
In the above formula, R₁ is an amine protecting group, R₂ is a hydrogen or hydroxyl group protecting group, and R₃ is an amine protecting group.
The method of claim 7, wherein the acidic conditions for removing the protected peptide and optionally the R₃amine protecting group from the resin include (i) 2 to 5% by volume Trichloroacetic acid or trifluoroacetic acid solution; and (ii) in the group consisting of a dichloromethane solution containing 0.1% of formic acid, methanesulfonic acid, benzenesulfonic acid, or para-Toluenesulfonic acid. A method for producing polymyxin B, characterized in that the selected substances are used alone or in combination.
The method for producing polymyxin B according to claim 7, wherein the cyclization reaction is performed at a temperature of -20 to 50°C.
The method for producing polymyxin B according to claim 7, wherein the concentration of the protected peptide in the cyclization reaction is within the range of 10 -2 mol to 10 -9 mol in terms of peptide.
The method of claim 7, wherein the solvent for the cyclization reaction of the peptide is 1,2-dichloroethane, chloroform, dichloromethane, 1,2-dichloromethane (1,2- Dichloromethane, Tetrahydrofurane, 1,4-Dioxane, Acetonitrile, Dimethylsulfoxide, N,N-Dimethylformamide ) and N,N-dimethylacetamide (N,N-Dimethylacetamide). A method for producing polymyxin B, characterized in that the solvent is used alone or in combination.
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