JPS6058232B2 - Novel disulfide derivative - Google Patents
Novel disulfide derivativeInfo
- Publication number
- JPS6058232B2 JPS6058232B2 JP53085900A JP8590078A JPS6058232B2 JP S6058232 B2 JPS6058232 B2 JP S6058232B2 JP 53085900 A JP53085900 A JP 53085900A JP 8590078 A JP8590078 A JP 8590078A JP S6058232 B2 JPS6058232 B2 JP S6058232B2
- Authority
- JP
- Japan
- Prior art keywords
- group
- groups
- acid
- structural formula
- value
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 150000002019 disulfides Chemical class 0.000 title claims description 37
- -1 2-benzothiazolyl group Chemical group 0.000 claims description 58
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 21
- 150000001413 amino acids Chemical group 0.000 claims description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 10
- CEIPQQODRKXDSB-UHFFFAOYSA-N ethyl 3-(6-hydroxynaphthalen-2-yl)-1H-indazole-5-carboximidate dihydrochloride Chemical group Cl.Cl.C1=C(O)C=CC2=CC(C3=NNC4=CC=C(C=C43)C(=N)OCC)=CC=C21 CEIPQQODRKXDSB-UHFFFAOYSA-N 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
- 125000002947 alkylene group Chemical group 0.000 claims description 5
- 125000000524 functional group Chemical group 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 92
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 48
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 48
- 150000001875 compounds Chemical class 0.000 description 40
- 238000006243 chemical reaction Methods 0.000 description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 26
- 125000003277 amino group Chemical group 0.000 description 17
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 16
- 229920002239 polyacrylonitrile Polymers 0.000 description 15
- OXNBMYPEYRSPMC-UHFFFAOYSA-N acetic acid;butan-1-ol Chemical compound CC(O)=O.CC(O)=O.CCCCO OXNBMYPEYRSPMC-UHFFFAOYSA-N 0.000 description 14
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 11
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 11
- 239000008363 phosphate buffer Substances 0.000 description 11
- 239000000741 silica gel Substances 0.000 description 11
- 229910002027 silica gel Inorganic materials 0.000 description 11
- 238000004809 thin layer chromatography Methods 0.000 description 11
- 125000002560 nitrile group Chemical group 0.000 description 10
- 125000003396 thiol group Chemical group [H]S* 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 9
- 102000005936 beta-Galactosidase Human genes 0.000 description 9
- 108010005774 beta-Galactosidase Proteins 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 150000002148 esters Chemical class 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 239000013078 crystal Substances 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 235000019260 propionic acid Nutrition 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 239000004677 Nylon Substances 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 229920001778 nylon Polymers 0.000 description 7
- PMNLUUOXGOOLSP-UHFFFAOYSA-N 2-mercaptopropanoic acid Chemical compound CC(S)C(O)=O PMNLUUOXGOOLSP-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 102000004877 Insulin Human genes 0.000 description 6
- 108090001061 Insulin Proteins 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 6
- 229940125396 insulin Drugs 0.000 description 6
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 6
- 229960002317 succinimide Drugs 0.000 description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 6
- 101001011741 Bos taurus Insulin Proteins 0.000 description 5
- IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- NVIAYEIXYQCDAN-CLZZGJSISA-N 7beta-aminodeacetoxycephalosporanic acid Chemical compound S1CC(C)=C(C(O)=O)N2C(=O)[C@@H](N)[C@@H]12 NVIAYEIXYQCDAN-CLZZGJSISA-N 0.000 description 4
- 241000700199 Cavia porcellus Species 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000004952 Polyamide Substances 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
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- 229920002647 polyamide Polymers 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 125000006239 protecting group Chemical group 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 4
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 4
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 3
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 3
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 3
- 102000002464 Galactosidases Human genes 0.000 description 3
- 108010093031 Galactosidases Proteins 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- RGKRCYWSKVINLL-UHFFFAOYSA-N acetic acid benzene Chemical compound CC(O)=O.CC(O)=O.C1=CC=CC=C1 RGKRCYWSKVINLL-UHFFFAOYSA-N 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
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- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
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- 150000003839 salts Chemical class 0.000 description 3
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 3
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 3
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- LJRDOKAZOAKLDU-UDXJMMFXSA-N (2s,3s,4r,5r,6r)-5-amino-2-(aminomethyl)-6-[(2r,3s,4r,5s)-5-[(1r,2r,3s,5r,6s)-3,5-diamino-2-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-hydroxycyclohexyl]oxy-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl]oxyoxane-3,4-diol;sulfuric ac Chemical compound OS(O)(=O)=O.N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)O[C@@H]1CO LJRDOKAZOAKLDU-UDXJMMFXSA-N 0.000 description 2
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- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
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- 102000016938 Catalase Human genes 0.000 description 2
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- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
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- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 2
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- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
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- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
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- 125000002774 3,4-dimethoxybenzyl group Chemical group [H]C1=C([H])C(=C([H])C(OC([H])([H])[H])=C1OC([H])([H])[H])C([H])([H])* 0.000 description 1
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- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
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- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
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- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
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- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
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- 238000000502 dialysis Methods 0.000 description 1
- AFZSMODLJJCVPP-UHFFFAOYSA-N dibenzothiazol-2-yl disulfide Chemical compound C1=CC=C2SC(SSC=3SC4=CC=CC=C4N=3)=NC2=C1 AFZSMODLJJCVPP-UHFFFAOYSA-N 0.000 description 1
- BADXJIPKFRBFOT-UHFFFAOYSA-N dimedone Chemical compound CC1(C)CC(=O)CC(=O)C1 BADXJIPKFRBFOT-UHFFFAOYSA-N 0.000 description 1
- QILSFLSDHQAZET-UHFFFAOYSA-N diphenylmethanol Chemical compound C=1C=CC=CC=1C(O)C1=CC=CC=C1 QILSFLSDHQAZET-UHFFFAOYSA-N 0.000 description 1
- 125000005982 diphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- ZHDBTKPXEJDTTQ-UHFFFAOYSA-N dipyrithione Chemical compound [O-][N+]1=CC=CC=C1SSC1=CC=CC=[N+]1[O-] ZHDBTKPXEJDTTQ-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 150000002081 enamines Chemical class 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
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- 150000008195 galaktosides Chemical class 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 238000006698 hydrazinolysis reaction Methods 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000003326 hypnotic agent Substances 0.000 description 1
- 230000000147 hypnotic effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
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- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
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- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000010446 mirabilite Substances 0.000 description 1
- KINULKKPVJYRON-PVNXHVEDSA-N n-[(e)-[10-[(e)-(4,5-dihydro-1h-imidazol-2-ylhydrazinylidene)methyl]anthracen-9-yl]methylideneamino]-4,5-dihydro-1h-imidazol-2-amine;hydron;dichloride Chemical compound Cl.Cl.N1CCN=C1N\N=C\C(C1=CC=CC=C11)=C(C=CC=C2)C2=C1\C=N\NC1=NCCN1 KINULKKPVJYRON-PVNXHVEDSA-N 0.000 description 1
- LKPFBGKZCCBZDK-UHFFFAOYSA-N n-hydroxypiperidine Chemical compound ON1CCCCC1 LKPFBGKZCCBZDK-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 125000005740 oxycarbonyl group Chemical group [*:1]OC([*:2])=O 0.000 description 1
- VEDDBHYQWFOITD-UHFFFAOYSA-N para-bromobenzyl alcohol Chemical compound OCC1=CC=C(Br)C=C1 VEDDBHYQWFOITD-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 150000002989 phenols Chemical group 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 125000003170 phenylsulfonyl group Chemical group C1(=CC=CC=C1)S(=O)(=O)* 0.000 description 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 125000000612 phthaloyl group Chemical group C(C=1C(C(=O)*)=CC=CC1)(=O)* 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229940097325 prolactin Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- AHTFMWCHTGEJHA-UHFFFAOYSA-N s-(2,5-dioxooxolan-3-yl) ethanethioate Chemical compound CC(=O)SC1CC(=O)OC1=O AHTFMWCHTGEJHA-UHFFFAOYSA-N 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 229960002101 secretin Drugs 0.000 description 1
- OWMZNFCDEHGFEP-NFBCVYDUSA-N secretin human Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(N)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)C1=CC=CC=C1 OWMZNFCDEHGFEP-NFBCVYDUSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000004756 silanes Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 description 1
- RSIJVJUOQBWMIM-UHFFFAOYSA-L sodium sulfate decahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].[O-]S([O-])(=O)=O RSIJVJUOQBWMIM-UHFFFAOYSA-L 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 150000003573 thiols Chemical group 0.000 description 1
- QERYCTSHXKAMIS-UHFFFAOYSA-N thiophene-2-carboxylic acid Chemical compound OC(=O)C1=CC=CS1 QERYCTSHXKAMIS-UHFFFAOYSA-N 0.000 description 1
- 229940035722 triiodothyronine Drugs 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/89—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members with hetero atoms directly attached to the ring nitrogen atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
- C07D277/70—Sulfur atoms
- C07D277/76—Sulfur atoms attached to a second hetero atom
- C07D277/78—Sulfur atoms attached to a second hetero atom to a second sulphur atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Plural Heterocyclic Compounds (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pyridine Compounds (AREA)
- Polyamides (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Cephalosporin Compounds (AREA)
Description
【発明の詳細な説明】
本発明は下記一般式〔I〕
R、−S−S−R2→Co−R3分nR、〔I〕(た
だし、式中、R、は2−ベンゾチアゾリル基または2−
ピリジールーN−オキサイド基、R2は遊離または保護
された官能基を有してもよいアルキレン基、R3はアミ
ノ酸または低及ポリペプタイドのカルボキシル残基、R
4はカルボキシル基、その反応性誘導体または保護され
たカルボキシル基もしくはイミデート基、nはoまたは
1を示す)で表わされる新規なジスルフィド誘導体に関
するものである。Detailed Description of the Invention The present invention relates to the following general formula [I] R, -S-S-R2→Co-R3minR, [I] (wherein, R is a 2-benzothiazolyl group or a 2-
pyridyl-N-oxide group, R2 is an alkylene group that may have a free or protected functional group, R3 is a carboxyl residue of an amino acid or polypeptide, R
4 relates to a novel disulfide derivative represented by a carboxyl group, a reactive derivative thereof, or a protected carboxyl group or imidate group, where n represents o or 1.
この新規なジスルフィド誘導体はその5−5交換反応性
に有用な試薬であり、さらにこの新規なジスルフィド誘
導体のカルボキ゛シル基の反応性誘導体を用いることに
よりチオール基導入試薬として利用し得るものてある。
従来、チオール基を有する化合物に対してS一S交換
反応性を示すジスルフィド誘導体としては種々知られて
おり、近年コバレント・クロマトグ門ラフイーに利用さ
れている。〔TheBiochemicalJourn
al(1973)L圓、573〜584、実用と応用
アフイニテイークロマトグラフイー第64〜65頁(1
97岬9月10日株式会社講談社発行)、フアルマシア
(1978)J4(1)第47〜52頁〕もので、さら
に」部のジスルフィド誘導体はこのS−S交換反応性に
基いてチオール基を有する蛋白質とアミノ基を有する蛋
白質との架橋試薬として利用されている〔B】0che
m1stry.U7(8)1499〜1506(197
8)〕。しかしながら、従来のジスルフィド誘導体のS
−S交換反応速度は著しく悪く、その反応において長時
間を要すものてあつて、満足のいく化合物ではなかつた
。本発明者らは、種々のジスルフィド誘導体について種
々研究した結果、下記一般式〔1〕R1−S−S−R2
+CO−R3+−0R4〔1〕(たた七、式中、R,、
R2、R3、R4、nは前記と同じ意味を示す)で表わ
される新規なジスルフィド誘導体が、その式中R1で示
される基において2−ベンゾチアゾリル基または2−ピ
リジールーN−オキサイド基、かつ式中R2で示される
基においてアルキレン基である場合に、そのS−S交換
反応に反応速度は従来の化合物のそれに比べ2〜1皓程
度早い反応速度てある有用な化合物てあることを見い出
した。This novel disulfide derivative is a useful reagent due to its 5-5 exchange reactivity, and furthermore, by using a reactive derivative of the carboxyl group of this novel disulfide derivative, it can be used as a reagent for introducing a thiol group.
Conventionally, various disulfide derivatives have been known that exhibit S1S exchange reactivity with compounds having a thiol group, and have recently been utilized in covalent chromatographies. [The Biochemical Journal
al (1973) L-en, 573-584, practical and application
Affinity Chromatography, pages 64-65 (1
97 Misaki, September 10, published by Kodansha Co., Ltd.), Pharmacia (1978) J4 (1), pp. 47-52], and furthermore, the disulfide derivative of the "" part has a thiol group based on this S-S exchange reactivity. [B] 0che is used as a crosslinking reagent between proteins and proteins with amino groups.
m1stry. U7 (8) 1499-1506 (197
8)]. However, the S of conventional disulfide derivatives
The -S exchange reaction rate was extremely poor, and the reaction required a long time, so the compound was not satisfactory. As a result of various studies on various disulfide derivatives, the present inventors found that the following general formula [1] R1-S-S-R2
+CO-R3+-0R4 [1] (Tata7, in the formula, R,,
R2, R3, R4, n have the same meanings as above), wherein the group represented by R1 in the formula is a 2-benzothiazolyl group or a 2-pyridyl-N-oxide group, and R2 in the formula It has been found that when the group represented by is an alkylene group, the reaction rate for the S--S exchange reaction is about 2 to 1 times faster than that of conventional compounds, making it a useful compound.
本発明は上記の知見に基いて完成されたもので、下記一
般式〔1〕R1−S−S−R2+CO−R3+NR,〔
1〕(ただし、式中、R1、R2、R3、R4、nは前
記を同じ意味を示す)て表わされる新規なジスルフィド
誘導体である。The present invention was completed based on the above findings, and has the following general formula [1] R1-S-S-R2+CO-R3+NR, [
1] (wherein R1, R2, R3, R4, and n have the same meanings as above).
ます、本発明における一般式〔1〕で表わされる新規な
ジスルフィド誘導体(以下、ジスルフィド誘導体〔1〕
と称す)における基R1としては2−ベンゾチアゾリル
基、2−ピリジールーN−オキサイド基を示し、基R2
としては遊離したまたは保護された官能基を有するか、
または有しない直鎖状または分枝鎖状のアルキレン基を
示すもので、さらにその官能性としては例えばアミノ基
、カルボキシル基が挙げられ、さらに基R3としてはS
−S交換反応に影響を与えないスペーサーであつて、通
常アミノ酸または低級ポリペプタイドのカルボキシル残
基を示すもので、このアミノ酸としては公知の種々のα
−アミノ酸やω−アミノ酸などが挙げられ、また低級ポ
リペプタイドとしては、2〜5ケのアミノ酸からなるペ
プタイドが挙げられ、基R4としてはカルボキシル基、
またはその反応性誘導体、例えば活性エステル、酸ハロ
ゲンなどの誘導体、さらに保護されたカルボキシル基、
もしくはイミデート基を示すものであり、nは0または
1を示すものである。First, a novel disulfide derivative represented by the general formula [1] in the present invention (hereinafter referred to as disulfide derivative [1])
The group R1 in (referred to as
as having free or protected functional groups;
or a straight-chain or branched alkylene group, and its functionality includes, for example, an amino group and a carboxyl group.
A spacer that does not affect the -S exchange reaction, and usually represents an amino acid or a carboxyl residue of a lower polypeptide.
-amino acids and ω-amino acids, and lower polypeptides include peptides consisting of 2 to 5 amino acids, and the group R4 is a carboxyl group,
or reactive derivatives thereof, such as active esters, acid halogens, and further protected carboxyl groups,
or represents an imidate group, and n represents 0 or 1.
また本発明のジスルフィド誘導体について、それらの基
を具体的に例示すれば第1表に示す如くである。さらに
、本発明のジスルフィド誘導体〔1〕は、そのベンゾチ
アゾリル基、ピリジールーN−オキサイド基の式R1の
基とアルキレン基の式R2の基の各基を有しているため
、そのS−S交換反応性は著しく良好なものである。次
いで、本発明のジスルフィド誘導体〔1〕を得るに当つ
て、例示すれば、次式の反応によつて得られるものてあ
る。Regarding the disulfide derivatives of the present invention, specific examples of these groups are shown in Table 1. Further, since the disulfide derivative [1] of the present invention has a benzothiazolyl group, a pyridyl-N-oxide group of formula R1, and an alkylene group of formula R2, the S-S exchange reaction The properties are extremely good. Next, to obtain the disulfide derivative [1] of the present invention, for example, it can be obtained by the reaction of the following formula.
(ただし、式中、R4はR3と同一の基またはイミデー
ト基に変換しうる基、R1、R2、R3、R4、nは前
記と同じ意味を示す)上記反応における、一般式〔■〕
で表わされる化合物、R1−S−S−R1としては、例
えば2・2゛ージチオビス(ベンゾチアゾール)、2・
7ージチオビス(ピリジンーN−オキサイド)が挙げら
れる。(However, in the formula, R4 is the same group as R3 or a group that can be converted into an imidate group, and R1, R2, R3, R4, and n have the same meanings as above) General formula [■] in the above reaction
Examples of the compound R1-S-S-R1 include 2,2-dithiobis(benzothiazole), 2-S-S-R1,
7-dithiobis(pyridine-N-oxide) is mentioned.
また一般式〔■〕で表わされる化合物、HS−R2−+
CO−R3′+−NR5としては、上記一般式〔■〕で
表わされる化合物と反応するチオールカルボン酸化合物
またはそのカルボ7酸誘導体、例えば活性エステル、酸
ハロゲンなどの反応性誘導体や保護誘導体や、チオール
ニトリル化合物などのイミデート基に変換しうるニトリ
ル基を有する化合物であればよく、例えばチオグリコー
ル酸、β−メルカプトプロピオン酸、チオ乳酸(α−メ
ルカプトプロピオン酸)、チオリンゴ酸、システイン、
ペニシラミン、グルタチオン、β−メルカプトプロピオ
ン酸−ε−アミノカプロン酸縮合生成物、β−メルカプ
トプロビオニトリルなどやそのカルボン酸誘導体などが
挙げられる。Also, a compound represented by the general formula [■], HS-R2-+
CO-R3'+-NR5 is a thiol carboxylic acid compound or its carboxylic acid derivative which reacts with the compound represented by the above general formula [■], such as a reactive derivative or a protected derivative such as an active ester or an acid halogen; Any compound having a nitrile group that can be converted into an imidate group such as a thiolnitrile compound may be used, such as thioglycolic acid, β-mercaptopropionic acid, thiolactic acid (α-mercaptopropionic acid), thiomalic acid, cysteine,
Examples include penicillamine, glutathione, β-mercaptopropionic acid-ε-aminocaproic acid condensation product, β-mercaptoprobionitrile, and carboxylic acid derivatives thereof.
また上記の化合物において、その分子内に有するアミノ
基やカルボキシル基などの官能基は、必要に応じて、有
機または無機の酸または塩基を用いて塩形成せしめて保
護するか、または公知の保護基を形成せしめ.てもよい
。またこれらの各種保護基はペプチド合成で既知なもの
、したがつて加水分解、酸分解、還元、アミノリシスま
たはヒドラジノリシスのような既知手段によつて容易に
脱離することのできる保護基が用いられる。例えば、ア
ミノ基に使用くする保護基としては、ホルミル基、トリ
フルオロアセチル基、フタロイル基、ベンゼンスルホニ
ル基、p−トルエンスルホニル基、o−ニトロフエニル
スルフエニル基、2●4−ジニトロフエニルスルフエニ
ル基などのアシル基、ベンジル基、ジフェニルメチル基
、トリフェニルメチル基(これらの基は場合によつては
o−メトキシ基、p−メトキシ基などの低級アルコキシ
基によつて置換さ7れている)などのアラルキシ基、ベ
ンジルオキシカルボニル基、o−ブロモベンジルオキシ
カルボニル基、p−ブロモベンジルオキシカルボニル基
、o−クロロベンジルオキシカルボニル基、p−クロロ
ベンジルオキシカルボニル基、p−ニトO口ベンジルオ
キシカルボニル基、p−メトキシベンジルオキシカルボ
ニル基、p−フェニルアゾーベンジルオキシカルボニル
基、p−(p′−メトキシフェニルアゾ)−ベンジルオ
キシカルボニル基などのベンジルオキシカルボニル基、
シクロペン5チルオキシカルボニル基、トリクロロエチ
ルオキシカルボニル基、t−アミルオキシカルボニル基
、t−ブトキシカルボニル基、ジイソプロピルメトキシ
カルボニル基などの脂肪族オキシカルボニル基、2−フ
エニルーイソプロポキシカルボニ)ル基、2−トリルー
イソプロポキシカルボニル基、2−p−ジフエニルーイ
ソプロポキシカルボニル基などのアラルキルオキシカル
ボニル基などがある。またこれらアミノ基をベンゾイル
アセトン、アセチルアセトン、ジメドンなどの1・3ー
ジケトンと反応させることによつて得られるエナミンの
形成により保護することができる。カルボキシル基は、
アミド形成、ヒドラチド形成またはエステル化によつて
保護される。すなわちアミド基は3・4ージメトキシベ
ンジル基、ビスー(p−メトキシフェニル)メチル基な
どによつて置換される。ヒドラチド基はベンジルオキシ
カルボニル基、トリクロロエチルオキシカルボニル基、
トリフルオロアセチル基、t−ブトキシカルボニル基、
トリチル基、2−p−ジフエニルーイソプロポキシカル
ボニル基などによつて置換される。エステル基はメタノ
ール、エタノール、t−ブタノール、シアノメチルアル
コールなどのアルカノール、ベンジルアルコール、p−
ブロモベンジルアルコール、p−クロロベンジルアルコ
ール、p−メトキシベンジルアルコール、p−ニトロベ
ンジルアルコール、2◆4●6−トリメチルベンジルア
ルコール、ベンズヒドリルアルコール、ベンゾイルメチ
ルアルコール、p−ブロモベンゾイルメチルアルコール
、p−クロロベンゾイルメチルアルコールなどのアラル
カノール、2●4●6−トリクロロフェノール、2・4
・5−トリクロロフェノール、ペンタクロロフェノール
、p−トロフエノール、2●4ージニトロフェノール、
p−シアノフェノール、p−メタンスルホニルフェノー
ルなどのフェノール、チオフェノール、チオクレゾール
、p−ニトロチオフェノールなどのチオフェノールなど
によつて置換される。その他、水酸基は、例えばエステ
ル化またはエーテル化によつて保護することとができる
。In addition, in the above compounds, functional groups such as amino groups and carboxyl groups in the molecule may be protected by forming salts using organic or inorganic acids or bases, or by using known protective groups. Let it form. It's okay. In addition, these various protecting groups are those known in peptide synthesis, and therefore protecting groups that can be easily removed by known means such as hydrolysis, acidolysis, reduction, aminolysis or hydrazinolysis are used. It will be done. For example, protective groups used for amino groups include formyl group, trifluoroacetyl group, phthaloyl group, benzenesulfonyl group, p-toluenesulfonyl group, o-nitrophenylsulfenyl group, 2●4-dinitrophenyl group. Acyl groups such as phenyl groups, benzyl groups, diphenylmethyl groups, triphenylmethyl groups (these groups may be substituted with lower alkoxy groups such as o-methoxy groups and p-methoxy groups). ), benzyloxycarbonyl group, o-bromobenzyloxycarbonyl group, p-bromobenzyloxycarbonyl group, o-chlorobenzyloxycarbonyl group, p-chlorobenzyloxycarbonyl group, p-nito Benzyloxycarbonyl groups such as benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, p-phenylazobenzyloxycarbonyl group, p-(p'-methoxyphenylazo)-benzyloxycarbonyl group,
Aliphatic oxycarbonyl groups such as cyclopentyloxycarbonyl group, trichloroethyloxycarbonyl group, t-amyloxycarbonyl group, t-butoxycarbonyl group, diisopropylmethoxycarbonyl group, 2-phenylisopropoxycarbonyl group, Examples include aralkyloxycarbonyl groups such as 2-tolyluisopropoxycarbonyl group and 2-p-diphenylisopropoxycarbonyl group. These amino groups can also be protected by the formation of enamines obtained by reacting with 1,3-diketones such as benzoylacetone, acetylacetone, dimedone and the like. The carboxyl group is
Protected by amide formation, hydratide formation or esterification. That is, the amide group is substituted with a 3,4-dimethoxybenzyl group, a bis(p-methoxyphenyl)methyl group, or the like. Hydratide group is benzyloxycarbonyl group, trichloroethyloxycarbonyl group,
trifluoroacetyl group, t-butoxycarbonyl group,
Substituted with trityl group, 2-p-diphenyl-isopropoxycarbonyl group, etc. Ester groups include alkanols such as methanol, ethanol, t-butanol, and cyanomethyl alcohol, benzyl alcohol, p-
Bromobenzyl alcohol, p-chlorobenzyl alcohol, p-methoxybenzyl alcohol, p-nitrobenzyl alcohol, 2◆4●6-trimethylbenzyl alcohol, benzhydryl alcohol, benzoylmethyl alcohol, p-bromobenzoylmethyl alcohol, p- Aralkanols such as chlorobenzoyl methyl alcohol, 2●4●6-trichlorophenol, 2.4
・5-trichlorophenol, pentachlorophenol, p-trophenol, 2●4-dinitrophenol,
It is substituted with phenols such as p-cyanophenol and p-methanesulfonylphenol, and thiophenols such as thiophenol, thiocresol and p-nitrothiophenol. In addition, hydroxyl groups can be protected, for example by esterification or etherification.
このエステル化に適する基としては、例えばアセチル基
などの低級アルカノイル基、ベンゾイル基などのアロイ
ル基、ベンジルオキシカルボニル基、エチルオキシカル
ボニル基などの炭酸から誘導される基である。またエー
テル化に適する基としては、例えばベンジル基、テトラ
ヒドロピラニル基、t−ブチル基などである。これら水
酸基の保護には2・2・2−トリフルオロー1−ブチル
オキシカルボニルアミノエチル基、2●2・2−トリフ
ルオロー1−ベンジルオキシカルボニルアミノエチル基
も適する。しかしながら、これら水酸基を必ずしも保護
する必要はない。さらにイミノ基を保護するのに使用す
る基としては、例えばベンジル基、トリチル基、ベンジ
ルオキシカルボニル基、トノル基、アダマンチルオキシ
カルボニル基、2・2・2−トリフルオロー1−t−ブ
チルオキシカルボニルアミノエチル基、2●2●2−ト
リフルオロー1−ベンジルオキシカルボニルアミノエチ
ル基などであるが、このイミノ基を必ずしも保護する必
要はない。さらにまた、本発明において、個々のアミノ
酸もしくは2〜4個のアミノ酸からなる低級ペプチドの
縮合体からなるチオールカルボン酸を用いる場合は、例
えば、保護されたα−アミノ基および活性化末端カルボ
キシル基をもつアミノ酸またはまたはペプチドと遊離α
−アミノ基および保護された末端カルボキシル基をもつ
アミノ酸またはペプチドとを反応させるか、あるいは活
性化α−アミノ基および保護された末端カルボキシル基
をもつアミノ酸またはペプチドと遊離の末端カルボキシ
ル基および保護されたα−アミノ基をもつアミノ酸また
はペプチドを反応させることにより得られたものであつ
てもよい。次いで、この一般式〔■〕で表わされる化合
物、R1−S−S−R1と、一般式〔■〕て表わされる
化合物、HS−R2(.CO−R3),、R5とを反応
せしめるのであるが、反応に当つて、通常メタノール、
エタノール、アセトン、ベンゼン、クロロホルム、四塩
化炭素などの溶媒が用いられ、この溶媒に各々の化合物
を等モル程度添加し、10〜70℃程度、好ましくは7
0℃程度、1紛間〜5時間程度、好ましくは2〜3時間
程度の条件下反応せしめ、反応終了後冷却または抽出な
どの通常の採取手段により反応生成物たる一般式〔■〕
で表わされる化合物、R1−S−S−R2−(CO−R
3.+−NR5を得ればよい。Groups suitable for this esterification include, for example, lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, and groups derived from carbonic acid such as benzyloxycarbonyl groups and ethyloxycarbonyl groups. Examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, and t-butyl group. A 2.2.2-trifluoro-1-butyloxycarbonylaminoethyl group and a 2.multidot.2.2-trifluoro-1-benzyloxycarbonylaminoethyl group are also suitable for protecting these hydroxyl groups. However, it is not necessary to protect these hydroxyl groups. Further, examples of groups used to protect the imino group include benzyl group, trityl group, benzyloxycarbonyl group, tonol group, adamantyloxycarbonyl group, 2,2,2-trifluoro-1-t-butyloxycarbonylamino These include ethyl group, 2●2●2-trifluoro-1-benzyloxycarbonylaminoethyl group, but it is not necessary to protect this imino group. Furthermore, in the present invention, when using a thiol carboxylic acid consisting of an individual amino acid or a condensate of a lower peptide consisting of 2 to 4 amino acids, for example, a protected α-amino group and an activated terminal carboxyl group are used. amino acids or peptides with free α
- reacting an amino acid or peptide with an amino group and a protected terminal carboxyl group, or reacting an amino acid or peptide with an activated α-amino group and a protected terminal carboxyl group with a free terminal carboxyl group and a protected terminal carboxyl group; It may be obtained by reacting an amino acid or peptide having an α-amino group. Next, the compound represented by the general formula [■], R1-S-S-R1, is reacted with the compound represented by the general formula [■], HS-R2(.CO-R3), R5. However, in the reaction, methanol,
A solvent such as ethanol, acetone, benzene, chloroform, or carbon tetrachloride is used, and equimolar amounts of each compound are added to this solvent, and the mixture is heated to about 10 to 70°C, preferably 70°C.
The general formula [■] which is the reaction product is reacted at about 0°C for 1 to 5 hours, preferably about 2 to 3 hours, and after the reaction is completed, the reaction product is collected by a usual method such as cooling or extraction.
The compound represented by R1-S-S-R2-(CO-R
3. It is sufficient to obtain +-NR5.
また、この反応生成物は、必要に応じて、そのカルボキ
シル基やニトリル基は通常の手段を用いてカルボキシル
基の反応性誘導体となすか、保護せしめるか、さらにニ
トリル基をイミデート基に変換せしめればよい。特に、
カルボキシル基の反応性誘導体としては、一般に使用さ
れる誘導体、例えば酸アジド、酸無水物、酸イミグゾリ
ド、活性エステル、酸ハロゲナイド、例えばシアノメチ
ルエステル、チオフェニルエステル、p−ニトロチオフ
ェニルエステル、p−メタンスルホニルフェニルエステ
ル、チオジルエステル、p−ニトロフェニルエステル、
2●4ージニトロフェニルエステル、2◆4●5−トリ
クロロフェニルエステル、2●4◆6−トリクロロフェ
ニルエステル、ペンタクロロフェニルエステル、N−ヒ
ドロキシコハク酸イミドエステル、N−ヒドロキシフタ
ル酸イミドエステル、8−ヒドロキシキノリンエステル
またはN−ヒドロキシピペリジン“エステルなどに変換
することによつて、あるいはカルボジイミド、N−N″
一カルボニルージイミダゾールまたはイソオキゾリウム
塩、例えばウッドワード反応剤などを使用して反応させ
ることによつてその反応性誘導体となすことができる。
このようにして得られた、本発明のジスルフィド誘導体
〔1〕は極めて良好なS−S交換反応性を示す優れたも
のである。次に、本発明のジスルフィド誘導体〔1〕を
例示し、またそのS−S交換反応速度を、対照としノて
の3−ピリジンー2″−イルジチオ)プロピオン酸と比
較して述べる。In addition, in this reaction product, the carboxyl group or nitrile group can be converted into a reactive derivative of the carboxyl group using conventional means, or the nitrile group can be protected, or the nitrile group can be converted into an imidate group, if necessary. Bye. especially,
As reactive derivatives of carboxyl groups, commonly used derivatives such as acid azides, acid anhydrides, acid imigzolides, active esters, acid halogenides such as cyanomethyl ester, thiophenyl ester, p-nitrothiophenyl ester, p- Methanesulfonylphenyl ester, thiodyl ester, p-nitrophenyl ester,
2●4-dinitrophenyl ester, 2◆4●5-trichlorophenyl ester, 2●4◆6-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, N-hydroxyphthalic acid imide ester, 8- or by converting into hydroxyquinoline ester or N-hydroxypiperidine “ester” or carbodiimide, N-N”
Reactive derivatives thereof can be obtained by reaction with monocarbonyl diimidazole or isoxolium salts, such as Woodward reagents.
The disulfide derivative [1] of the present invention thus obtained is an excellent product exhibiting extremely good SS exchange reactivity. Next, the disulfide derivative [1] of the present invention will be illustrated, and its SS exchange reaction rate will be described in comparison with 3-pyridin-2''-yldithio)propionic acid as a control.
なお、S−S交換反応速度の測定は、S−S交換反応を
行なわしめるチオール基をを有する化合物としてジチオ
スライトールの17TLMEDTA含有0.2Mトリス
塩酸緩衝液(PH7.5)を用いて反応せしめ、その結
果増加するその吸収をその極大吸収波長に測定し、1分
間当りのS−S交換反応のモル数を求めたものであその
結果、本発明のジスルフィド誘導体は、対照の化合物に
比べ、極めて良好なS−S反応速度を示すものであつた
。さらに、本発明のジスルフィド誘導体〔1〕は、チオ
ール基導入試薬や架橋試薬として有用な化合物である。In addition, the S-S exchange reaction rate was measured using a 0.2 M Tris-HCl buffer (PH 7.5) containing 17TLMEDTA of dithiothreitol as a compound having a thiol group that performs the S-S exchange reaction. The resulting increase in absorption was measured at its maximum absorption wavelength to determine the number of moles of S-S exchange reaction per minute.As a result, the disulfide derivative of the present invention had a It showed an extremely good SS reaction rate. Furthermore, the disulfide derivative [1] of the present invention is a compound useful as a thiol group-introducing reagent or a crosslinking reagent.
例えば、ジスルフィド誘導体〔1〕と、これと反応し得
る水酸基を有する化合物やアミン化合物とを、溶媒例え
ばベンゼン、トルエン、クロロホルム、アセトン、テト
ラヒドロフラン、ジメチルホルムアミド中にて、必要に
応じて縮合剤の存在下反応せしめて本発明のジスルフィ
ド誘導体〔1〕の反応基たるR4の基と水酸基を有する
化合物やアミン化合物の水酸基、アミノ基などの基とに
基づいて、エステル結合、アミジノ結合またはアミド結
合などにて両者を結合し、次いでこれをアルカリ性条件
下室温ないし加温下にてそのジスルフィド結合を加水分
解して、その水素基を有する化合物やアミン化合物にチ
オール基を導入せしめる、またはこの上記のエステル結
合またはアミド結合などにて両者が結合した化合物に、
チオール基を有する化合物を、水性媒体下PH7〜8付
近のPH条件にてS−S交換反応を行なわせしめてチオ
ール基を有する化合物と水酸基を有する化合物またはア
ミン化合物とを架橋結合せしめるものである。またこの
用途における、水酸基を有する化合物やアミン化合物と
しては種々の化合物が挙られ、インスリン、アルブミン
、成長ホルモン、カルチトニン、プロラクチン、ACT
H,.PTHlグルカゴン、ガストリシン、セクレチン
、γ−グロブリン、またはIgGlIgM.IgA、第
二抗体、エストロゲン、ATP、カテコールアミン、ト
リヨードサイロニン、その他抗生物質、催眠剤などの抗
原、抗体、第二抗体、ハプテンなどの免疫成分やベルオ
キシダーゼ、カタラーゼ、コレステロールオキシダーゼ
、グリセロールデヒドロゲナーゼ、コリンオキシグーゼ
などの酸化還元酵素、アルカリフォスファターゼ、グル
コアミラーゼ、ホスフオリパーゼD1β−ガ,ラクトシ
ダーゼ、リゾチームなどの加水分解酵素やその他転位酵
素、リアーゼ、イソメラーゼなどの酵素、セルロース、
アミノ化セルロース、アガロース、デキストリン、、デ
キストランなどのポリサッカライドまたはそれらの水不
溶性担体、そ,の他アミノ化せしめたポリアミド、ポリ
アクリロニトリルまたはシラン化合物などの水酸基また
はアミノ基を有してなる水不溶性担体が挙げられるもの
で、公知化合物のみならず新規な化合物であつても水酸
基またはアミノ基などのジスルフィド化合物〔1〕と反
応し得るものであれぱすべてよく、また新規なアミノ基
を有する化合物としては、例えば6・6−ナイロン、6
−ナイロン、などのポリアミドをγ−アミノプロピルー
トリエトキシシランにて100℃、3時間加熱反応後泊
取、水洗、乾燥することにより6・6−ナイロン、6−
ナイロンなどのポリアミドの一部にγ−アミノプロピル
基が導入されたγ−アミノプロピル化ポリアミド、ポリ
アクリロニトリルまたはポリアクリロニトリル系ポリマ
ーをジエチルエーテル、ジオキサン、テトラヒドロフラ
ンなどの媒体中水素化リチウムアルミニウムにて1〜化
時間加熱還流してそのニトリル基の一部を還元せしめて
アミノ基となしたアミノ化ポリアクリロニトリルまたは
ポリアクリロニトリル系ポリマーなどが挙られる。また
チオール基を有する化合物としては、、例えばベルオキ
シダーゼ、カタラーゼ、β−ガラクトシダーゼ、アルカ
リフォスファターゼなどの酵素や、s−アセチルメルカ
プトサクシニツクアンハイドライドによるチオール基を
導入〔Ar′Ch.BlOchem.BlOphy.?
605〜612(1962)〕せしめた種々の化合物、
や上記の加水分解により得られるジスルフィド化合物〔
1〕からの水酸基を有する化合物やアミン化合物へのチ
オール基導入化合物が挙げられる。このような種々の化
合物を適宜組合せて、S−S交換反応、通常水性媒体、
例えばPH7〜8程度の緩衝液中にて室温下行なわせし
め、その後反応生成物は公知の分離、精製手段、例えは
塩析、相分離、抽出、透析、吸着クロマトグラフィーな
どの手段を用いて回収すればよい。このようにして得ら
れた生成物は、酵素と水不溶性担体との架橋結合体てあ
る固定化酵素、抗原または抗体などと水溶性担体との架
橋結合体である固定化抗原、抗体、蛋白質とハプテンと
の架橋結合体である抗原性ハプテン、酵素と免疫成分と
の架橋結合体である酵素免疫測定用成分などの有用な化
合物である。次に本発明の実施例および参考例を挙げて
具体的に述べるが、本発明はこれらによつて何んら限定
されるものではない。For example, the disulfide derivative [1] and a compound having a hydroxyl group or an amine compound that can react with the disulfide derivative [1] are mixed in a solvent such as benzene, toluene, chloroform, acetone, tetrahydrofuran, or dimethylformamide, if necessary in the presence of a condensing agent. The following reaction is performed to form an ester bond, amidino bond, or amide bond based on the group R4, which is the reactive group of the disulfide derivative [1] of the present invention, and the hydroxyl group, amino group, etc. of a compound having a hydroxyl group or an amine compound. Then, the disulfide bond is hydrolyzed under alkaline conditions at room temperature or under heating to introduce a thiol group into the compound or amine compound having a hydrogen group, or the above-mentioned ester bond is Or to a compound in which the two are bonded together through an amide bond, etc.
A compound having a thiol group is subjected to an S--S exchange reaction in an aqueous medium at a pH of around 7 to 8 to form a crosslinking bond between the compound having a thiol group and a compound having a hydroxyl group or an amine compound. In addition, various compounds can be mentioned as compounds having a hydroxyl group and amine compounds for this purpose, such as insulin, albumin, growth hormone, calcitonin, prolactin, ACT, etc.
H,. PTHl glucagon, gastricin, secretin, γ-globulin, or IgGlIgM. IgA, secondary antibodies, estrogen, ATP, catecholamines, triiodothyronine, other antibiotics, antigens such as hypnotics, antibodies, secondary antibodies, immune components such as haptens, peroxidase, catalase, cholesterol oxidase, glycerol dehydrogenase, Oxidoreductases such as choline oxyguse, alkaline phosphatase, glucoamylase, phospholipase D1β-ga, lactosidase, hydrolytic enzymes such as lysozyme, other enzymes such as transposase, lyase, isomerase, cellulose,
Polysaccharides such as aminated cellulose, agarose, dextrin, and dextran or their water-insoluble carriers, and other water-insoluble carriers having hydroxyl or amino groups such as aminated polyamides, polyacrylonitrile, or silane compounds Not only known compounds but also new compounds may be used as long as they can react with disulfide compounds [1] such as hydroxyl groups or amino groups. , e.g. 6.6-nylon, 6
- By heating polyamide such as nylon with γ-aminopropyltriethoxysilane at 100°C for 3 hours, overnight, washing with water, and drying, 6,6-nylon, 6-
A γ-aminopropylated polyamide, polyacrylonitrile, or a polyacrylonitrile-based polymer in which a γ-aminopropyl group has been introduced into a part of a polyamide such as nylon is mixed with lithium aluminum hydride in a medium such as diethyl ether, dioxane, or tetrahydrofuran. Examples include aminated polyacrylonitrile or polyacrylonitrile-based polymers in which a portion of the nitrile groups are reduced to amino groups by heating under reflux for a certain period of time. Examples of compounds having a thiol group include enzymes such as peroxidase, catalase, β-galactosidase, and alkaline phosphatase, and introduction of a thiol group using s-acetylmercaptosuccinic anhydride [Ar'Ch. BlOchem. BlOphy. ?
605-612 (1962)] various compounds,
and disulfide compounds obtained by the above hydrolysis [
1] and a compound having a hydroxyl group and a compound introducing a thiol group into an amine compound. By appropriately combining such various compounds, S-S exchange reaction, usually in an aqueous medium,
For example, the reaction is carried out at room temperature in a buffer solution with a pH of about 7 to 8, and the reaction product is then recovered using known separation and purification methods, such as salting out, phase separation, extraction, dialysis, and adsorption chromatography. do it. The products thus obtained include immobilized enzymes, antigens or antibodies, which are cross-linked bodies of enzymes and water-insoluble carriers, and immobilized antigens, antibodies, proteins, which are cross-linked bodies of water-soluble carriers, etc. They are useful compounds such as antigenic haptens, which are cross-linked compounds with haptens, and components for enzyme immunoassays, which are cross-linked compounds with enzymes and immune components. Next, the present invention will be specifically described with reference to Examples and Reference Examples, but the present invention is not limited thereto.
実施例1
2・2″ージチオビス(ベンゾチアゾール)13.2y
をベンゼン400m1に加え、さらにβ−メルカプトプ
ロピオン酸6yを加えて、70℃、3時間加熱攪拌し、
その後反応液を氷水浴にて冷却して析出せしめて13.
8yの粗結晶を得、さらにこれをベンゼンにて再結晶し
て12′の3−(ベンゾチアゾールー2″−イルジチオ
)プロピオン酸の結晶を得た。Example 1 2.2″-dithiobis(benzothiazole) 13.2y
was added to 400ml of benzene, further added 6y of β-mercaptopropionic acid, heated and stirred at 70°C for 3 hours,
After that, the reaction solution was cooled in an ice water bath to precipitate 13.
Crude crystals of 8y were obtained, which were recrystallized from benzene to obtain crystals of 12'3-(benzothiazol-2''-yldithio)propionic acid.
構造式:
融点:162〜164)C
λMaX:272r1TrL.(メタノール)Rf値:
0.33(ベンゼンニ酢酸エチルニ1:2によるシリカ
ゲル薄層クロマトグラフィー)実施例2
2・2″ージチオビス(ピリジンーN−オキサイド)4
.3yおよびβ−メルカプトプロピオン酸3yを用い、
以下実施例1と同様にして3−(ピリジンーN−オキサ
イドー2−イルジチオ)プロピオン酸4.1yの結晶を
得た。Structural formula: Melting point: 162-164) C λMaX: 272r1TrL. (Methanol) Rf value:
0.33 (Silica gel thin layer chromatography with benzene ethyl diacetate 1:2) Example 2 2.2″-dithiobis(pyridine-N-oxide) 4
.. 3y and β-mercaptopropionic acid 3y,
Thereafter, in the same manner as in Example 1, crystals of 3-(pyridine-N-oxide-2-yldithio)propionic acid 4.1y were obtained.
構造式: ※融点:
114〜115 C(補正値121〜123℃)λMa
x:270nm(PH7.5、10%ジメチルホルムア
ミド水溶液)Rf値:0.53(ベンゼンニ酢酸エチル
ニ3:1によるシリカゲル薄層クロマトグラフィー)実
施例4
実施例1と同様にして得られた3−(ベンゾチアゾール
ー2″−イルジチオ)プロピオン酸3y1,融点:12
6〜128℃λRna)c:270nTrL(メタノー
ル)Rf値:0.66(ブタノールニ酢酸:水=4:1
:1によるシリカゲル薄層クロマトグラフィー)実施例
3実施例1と同様にして得られた3−(ベンゾチアゾー
ルー2″−イルジチオ)プロピオン酸3fIを酢酸エチ
ル20m1に溶解し、これにN−ヒドロキシスクシンイ
ミド1qおよびジシクロヘキシルカルボジイミド1.7
ダを加えて3時間、室温中で攪拌し、生成するジシクロ
ヘキシル尿素を戸別した後その酢酸エチル層を回収し、
さらにこれをPH7.5のリン酸緩衝液で洗浄して未反
応の遊離酸を除去し、さらにこの酢酸エチル層に芒硝を
加えて脱水した後乾固し、さらにこれを熱石油エールに
溶解した後冷却して3−(ベンゾチアゾールー2″−イ
ルジチオ)プロピオン酸−スクシンイミドエステル2.
4yの結晶を得た。Structural formula: *Melting point:
114 to 115 C (corrected value 121 to 123 C) λMa
x: 270 nm (PH 7.5, 10% dimethylformamide aqueous solution) Rf value: 0.53 (silica gel thin layer chromatography with benzene ethyl diacetate 3:1) Example 4 3-( obtained in the same manner as in Example 1) Benzothiazole-2″-yldithio)propionic acid 3y1, melting point: 12
6-128°C λRna) c: 270nTrL (methanol) Rf value: 0.66 (butanol diacetic acid: water = 4:1
Example 3 3fI of 3-(benzothiazol-2''-yldithio)propionic acid obtained in the same manner as in Example 1 was dissolved in 20 ml of ethyl acetate, and N-hydroxysuccinimide was added to the solution. 1q and dicyclohexylcarbodiimide 1.7
After adding dicyclohexyl urea and stirring at room temperature for 3 hours, the generated dicyclohexyl urea was collected from each house and the ethyl acetate layer was collected.
This was further washed with a phosphate buffer solution of pH 7.5 to remove unreacted free acid, and the ethyl acetate layer was further dehydrated by adding Glauber's salt to dryness, and this was further dissolved in hot petroleum ale. After cooling, 3-(benzothiazol-2''-yldithio)propionic acid-succinimide ester2.
Crystals of 4y were obtained.
構造式:
*p−ニトロフェノール1.2fおよびジシクロヘキシ
ルカルボジイミド2.1qを酢酸エチル20m1に溶解
し、室温下3時間攪拌し、以下実施例3と同様に行なつ
て、3−(ベンゾチアゾールー2″−イル7ジチオ)プ
ロピオン酸−p−ニトロフェニルエステル1.85yの
結晶を得た。Structural formula: *1.2f of p-nitrophenol and 2.1q of dicyclohexylcarbodiimide were dissolved in 20ml of ethyl acetate, stirred at room temperature for 3 hours, and the same procedure as in Example 3 was carried out to prepare 3-(benzothiazole-2 Crystals of 1.85y of ``-yl7dithio)propionic acid-p-nitrophenyl ester were obtained.
構造式:
融点:113〜114をC
λ.,AX:279r17TL,(PH7.5、10%
ジメチルホルムアミド水溶液)Rf値:0.84(ベン
ゼンニ酢酸エチルニ5:1によるシリカゲル薄層クロマ
トグラフィー)実施例5
3−(ベンゾチアゾールー7−イルジチオ)プロピオン
酸3yを塩化チオニル10m1に溶解し、25℃、2時
間加熱した後減圧下にて塩化チオニルを留去して、3−
(ベンゾチアゾールー7−イルジチオ)プロピオン酸の
酸クロライド物を油状にて得た。Structural formula: Melting point: 113-114 C λ. , AX:279r17TL, (PH7.5, 10%
Dimethylformamide aqueous solution) Rf value: 0.84 (Silica gel thin layer chromatography with benzene ethyl diacetate 5:1) Example 5 3-(benzothiazol-7-yldithio)propionic acid 3y was dissolved in 10 ml of thionyl chloride, and the mixture was heated at 25°C. After heating for 2 hours, thionyl chloride was distilled off under reduced pressure to obtain 3-
An acid chloride of (benzothiazol-7-yldithio)propionic acid was obtained in the form of an oil.
構造式:
λ.へニ272r1m(メタノール)
Rf値:0.25(ベンゼンによるシリカゲル薄層クロ
マトグラフィー)実施例6、7
実施例3と同様にして得られた3−(ベンゾチアゾール
ー7−イルジチオ)プロピオン酸−スクシンイミドエス
テル1.3fおよびε−アミノカプロン酸0.6yをテ
トラヒドロフラン50m1に加えて室温下一夜反応せし
め、次いで減圧下テトラヒドロフランを留去した後熱イ
ソプo/ぐノールに溶解し、これを冷却して6−N〔3
−(ベンゾチアゾールー2″−イルジチオ)プロピオニ
ル〕力フロン酸の結晶0.8yを得た。Structural formula: λ. Heni 272r1m (methanol) Rf value: 0.25 (silica gel thin layer chromatography with benzene) Examples 6 and 7 3-(benzothiazol-7-yldithio)propionic acid-succinimide obtained in the same manner as in Example 3 Ester 1.3f and ε-aminocaproic acid 0.6y were added to 50 ml of tetrahydrofuran and reacted overnight at room temperature.Then, after distilling off the tetrahydrofuran under reduced pressure, it was dissolved in hot isopropyl alcohol, and this was cooled to give 6- N [3
-(Benzothiazole-2''-yldithio)propionyl]-(benzothiazole-2''-yldithio)propionyl) crystals of 0.8y were obtained.
構造式:
λRr.ax:272r1TrL(メタノール)Rf値
:0.07(ベンゼンニ酢酸エチルニ1:2によるシリ
カゲル薄層クロマトグラフィー)さらに本品500m9
を、N−ヒドロキシスクシンイミド200m9、ジシク
ロヘキシルカルボジイミド340m9とともにテトラヒ
ドロフラン10mLに溶解し、室温下3時間反応せしめ
、生成するジシクロヘシル尿素をp別した後テトラヒド
ロフランを留去し、残渣を熱石油エーテルに溶解し、冷
却して6−N〔3−(ベンゾチアゾールー7−イルジチ
オ)プロピオニル〕力フロン酸−スクシンイミドエステ
ル430m9の結晶を得た。Structural formula: λRr. ax: 272r1TrL (methanol) Rf value: 0.07 (silica gel thin layer chromatography with benzene diacetate 1:2) Furthermore, this product 500m9
was dissolved in 10 mL of tetrahydrofuran with 200 m9 of N-hydroxysuccinimide and 340 m9 dicyclohexylcarbodiimide, and reacted at room temperature for 3 hours. After separating the dicyclohexyl urea formed, tetrahydrofuran was distilled off, and the residue was dissolved in hot petroleum ether and cooled. Thus, 430 m9 of crystals of 6-N[3-(benzothiazol-7-yldithio)propionyl]furoic acid-succinimide ester were obtained.
構造式:
λMa).:271nTrL
Rf値:0.42(ベンゼンニ酢酸エチルニ3:1によ
るシリカゲル薄層クロマトグラフィー)実施例8、9、
10
実施例2と同様にして得られた3−ピリジンーN−オキ
サイドー2″−イルジチオ)プロピオン酸を用いて、上
記実施例3、実施例4、実施例5の如くして、各々、そ
のスクシンイミドエステル、p−ニトロフェニルエステ
ル、酸クロライドを得た。Structural formula: λMa). :271nTrL Rf value: 0.42 (silica gel thin layer chromatography with benzene ethyl diacetate 3:1) Examples 8, 9,
10 Using 3-pyridine-N-oxide-2''-yldithio)propionic acid obtained in the same manner as in Example 2, the succinimide ester thereof was prepared as in Example 3, Example 4, and Example 5, respectively. , p-nitrophenyl ester, and acid chloride were obtained.
スクシンイミドエステル体
構造式:
λMax:260n7n(メタノール)
Rf値:0.25(ベンゼンニ酢酸エチルニ3:1によ
るシリカゲル薄層クロマトグラフィー)p−ニトロフェ
ニルエステル体
構造式:
λTr.ax:391nn1.(メタノール)Rf値:
0.82(ベンゼンニ酢酸エチルニ3:1によりシリカ
ゲル薄層クロマトグラフィー)酸クロライド体
構造式:
λNlax:270n7T1.(メタノール)Rf値:
0.15(ベンゼンニ酢酸エチルニ3:1によるシリカ
ゲル薄層クロマトグラフィー)実施例11
2−2″ージチオビス(ベンゾチアゾール)1.1yお
よびβ−メルカプトプロビオニトリルをベンゼン50m
1に溶解し、70℃、3時間攪拌反応せしめ、次いでこ
れを氷水浴にて冷却して粗結晶を得、さらにベンゼンに
て再結晶して3−(ベンゾチアゾールー2″−イルジチ
オ)プロビオニトリル750m9を得、次いでこの70
0m9を塩酸19y含有メタノール50mLに加えて一
夜5℃にて反応せしめ、その後溶媒を減圧留去して粗粉
末を得、これをベンゼンにて洗浄して、メチル・3−(
ベンゾチアゾールー2″−イルジチオ)プロピオニルイ
ミテート・塩酸塩7207F!9を得た。Succinimide ester structural formula: λMax: 260n7n (methanol) Rf value: 0.25 (silica gel thin layer chromatography with benzene ethyl diacetate 3:1) p-nitrophenyl ester structural formula: λTr. ax:391nn1. (Methanol) Rf value:
0.82 (Silica gel thin layer chromatography using benzene ethyl diacetate 3:1) Acid chloride Structural formula: λNlax: 270n7T1. (Methanol) Rf value:
0.15 (Silica gel thin layer chromatography with benzene ethyl diacetate 3:1) Example 11 2-2''-dithiobis(benzothiazole) 1.1y and β-mercaptoprobionitrile in benzene 50m
1 and reacted with stirring at 70°C for 3 hours, then cooled in an ice water bath to obtain crude crystals, which were further recrystallized from benzene to obtain 3-(benzothiazol-2″-yldithio)probionitrile. 750 m9 and then this 70
0m9 was added to 50 mL of methanol containing 19y hydrochloric acid and reacted overnight at 5°C, and then the solvent was distilled off under reduced pressure to obtain a crude powder, which was washed with benzene to obtain methyl 3-(
Benzothiazole-2''-yldithio)propionyl imitate hydrochloride 7207F!9 was obtained.
構造式:
λMaX:272r1TrL(メタノール)Rf値:0
.05(ベンゼンニ酢酸エチルニ1:2によりシリカゲ
ル薄層クロマトグラフィー)実施例12〜35
上記の2・2″ージチオビス(ベンゾチアゾール)また
は2・2ージチオビス(ピリジンーN−オキサイド)を
用い、かつチオグリコール酸、チオ乳酸、システイル、
チオリンゴ酸、ペニシラミン、N−(2−メルカプトプ
ロピオニル)グリシン、グルタチオンなどの種々の化合
物を用いて、上記実施例と同様にして、以下の通りの目
的物を得た。Structural formula: λMaX: 272r1TrL (methanol) Rf value: 0
.. 05 (Silica gel thin layer chromatography with benzene ethyl diacetate 1:2) Examples 12 to 35 Using the above 2,2''-dithiobis(benzothiazole) or 2,2-dithiobis(pyridine-N-oxide), and thioglycolic acid, Thiolactic acid, cysteine,
Using various compounds such as thiomalic acid, penicillamine, N-(2-mercaptopropionyl)glycine, and glutathione, the following target products were obtained in the same manner as in the above examples.
なお、Rf値はシリカゲル薄層クロマトグノラフイーに
よるものである。@構造体:
λT!1aX:272r1m(メタノール)Rf値:0
.36(ベンゼンニメタノールニ1:2)S−S交換反
応速度35.6μMOle/分◎構造式:λ.Nax:
272r1m,(メタノール)Rf値:0.38(ベン
ゼンニメタノールニ1:2)9構造式:λRTlax:
271n7T1.(メタノール)Rf値:0.08(ベ
ンゼンニメタノールニ1:2)、6.60(ブタノール
ニ酢酸:水=4:1:5の上層)[相]構造式:
λTna)C:271nW1,(メタノール)Rf値:
0.15(ベンゼンニ酢酸エチルニ1:2)[相]構造
式:Xmax:271nwL(PH7.5.リン酸バッ
ファー)Rf値:0.60(ブタノールニ酢酸:水=4
:1:の上層)8構造式:
λMax:271nwL(メタノール)
Rf値:0.30(ブタノールニ酢酸:水=4:1:の
上層)S−S交換反応速度35.5μMOle/分[相
]構造式: 1112+S−S−CH−CO−CH
λTnax:271nm,、418r17TL(メタノ
ール)Rf値:0.23(ブタノールニ酢酸:水=4:
1:5θ の上層)[相]構造式:
λTrlax:271n7T1,(メタノール)Rf値
:0.48(ベンゼンニ酢酸エチルニ3:1)[相]構
造式:1。Note that the Rf value is based on silica gel thin layer chromatography. @Structure: λT! 1aX: 272r1m (methanol) Rf value: 0
.. 36 (benzenimethanol 1:2) S-S exchange reaction rate 35.6 μMole/min ◎ Structural formula: λ. Nax:
272r1m, (methanol) Rf value: 0.38 (benzenimethanol 1:2) 9 Structural formula: λRTlax:
271n7T1. (Methanol) Rf value: 0.08 (benzenimethanol di 1:2), 6.60 (upper layer of butanol diacetic acid: water = 4:1:5) [Phase] Structural formula: λTna)C: 271nW1, ( methanol) Rf value:
0.15 (Benzene diacetate: ethyl diacetate 1:2) [Phase] Structural formula: Xmax: 271nwL (PH 7.5. Phosphate buffer) Rf value: 0.60 (Butanol diacetate: Water = 4
: 1: upper layer) 8 Structural formula: λMax: 271 nwL (methanol) Rf value: 0.30 (butanol diacetic acid: water = 4:1: upper layer) S-S exchange reaction rate 35.5 μMole/min [phase] Structural formula: 1112+S-S-CH-CO-CH
λTnax: 271 nm, 418r17TL (methanol) Rf value: 0.23 (butanol diacetic acid: water = 4:
1:5θ upper layer) [Phase] Structural formula: λTrlax: 271n7T1, (methanol) Rf value: 0.48 (Benzene ethyl diacetate 3:1) [Phase] Structural formula: 1.
)3CH2C00HλMax:271nTrL(メタノ
ール)Rf値:0.12(ベンゼンニ酢酸エチルニ3:
1)S−S交換反応速度35.0μMOle/分3構造
式:λMax:278r1TL
Rf値:0.80(ベンゼンニ酢酸エチルニ3:1)
/\/5、O構造式:
局。)3CH2C00HλMax: 271nTrL (methanol) Rf value: 0.12 (ethyl benzene diacetate 3:
1) S-S exchange reaction rate 35.0μMole/min 3 Structural formula: λMax: 278r1TL Rf value: 0.80 (Benzene ethyl diacetate 3:1)
/\/5, O structural formula: Bureau.
:271mm(メタノール)7Rf値:0.15(ベン
ゼンニ酢酸エチルニ3:1)S−S交換反応速度36.
6μMOle/分◎構造式:λm&x:271nwL(
メタノール)Rf値:0.80(ベンゼンニ酢酸エチル
ニ3:1)[相]構造式:゛0
XmaX:271n7TI.(メタノール)Rf値:0
.10(ベンゼンニ酢酸エチルニ3:1)S−S交換反
応速度35.7μMOle/分(ハ)構造式:λ111
aX:271nTrL(メタノール)Rf値:0.05
(ベンゼンニ酢酸エチルニ3:1)@構造式:局Ax:
272r1m,(メタノール)Rf値:0.31(ブタ
ノールニ酢酸:水=4:1:の上層)s−S交換反応速
度35.2μMOle/分@構造式:λRr.ax:2
70r1m(メタノール)Rf値:0.7(ブタノール
ニ酢酸:水=4:1:の上層)S−S交換反応速度92
.8μMOIe/分@構造式:λMax:270r1T
rL(メタノール)Rf値:0.66(ブタノールニ酢
酸:水=4:11)S−S交換反応速度93.2μMO
le/分@構造式:λMax:310r1j1.(メタ
ノール)Rf値:0.82(ベンゼンニ酢酸エチルニ3
:1)[相]構造式:0XT11aX:271nTr!
.(メタノール)Rf値:0.41(ブタノールニ酢酸
:水=4:ニ1) S−S交換反応速度92.6μMO
le/分9構造式:λMa)c:271nrrL,(メ
タノール)0Rf値:0.25(ブタノールニ酢酸:水
=4:11)O構造式:
λRnax:270r)7n,(メタノール)Rf値:
0.32(ブタノールニ酢酸:水=4:1:O11)S
−S交換反応速度90.1μMOle/分O構造式:λ
NlaX:270r1TrLRf値:0.20(ブタノ
ールニ酢酸:水=4:1:1)S−S交換反応速度91
.5μMOle/分[有]構造式:λMax:270r
17n
Rf値:0.41(ブタノールニ酢酸:水=4:1:1
)[相]構造式:
λMa:X:270r1TrL
Rf値:0.25(ブタノールニ酢酸:水=4:1:1
)参考例1
500m1容三つロフラスコを約35℃の恒温水浴にひ
たし、約1紛間窒素で置換せしめ、次いで、フラスコ内
に120m1の蒸留水を加え、さらにアルキルスルホン
酸ナトリウム2y1アクリロニトリル80ダ、過硫酸ナ
トリウム0.1y1亜硫酸水素ナトリウム0.033y
を加え、約3時間攪拌せしめて乳濁液を得、次いでこれ
を約500m1の水に注ぎ、攪拌下塩を加えて凝固せし
めて生成物を析出し、これを枦別、水洗し、通風乾燥し
たポリアクリロニトリル(イ).5%、30℃における
ジメチルホルムアミドでの対数粘度は約10.5である
)を得た。:271mm (methanol) 7Rf value: 0.15 (benzene diacetate 3:1) S-S exchange reaction rate 36.
6μMOle/min◎Structural formula: λm&x:271nwL(
Methanol) Rf value: 0.80 (Benzene ethyl diacetate 3:1) [Phase] Structural formula: ゛0 XmaX: 271n7TI. (Methanol) Rf value: 0
.. 10 (Benzene ethyl diacetate 3:1) S-S exchange reaction rate 35.7 μMole/min (c) Structural formula: λ111
aX: 271nTrL (methanol) Rf value: 0.05
(Benzene ethyl acetate 3:1) @ Structural formula: Ax:
272r1m, (methanol) Rf value: 0.31 (upper layer of butanol diacetic acid: water = 4:1) s-S exchange reaction rate 35.2 μMole/min @ structural formula: λRr. ax: 2
70r1m (methanol) Rf value: 0.7 (upper layer of butanol diacetic acid: water = 4:1) S-S exchange reaction rate 92
.. 8 μMOIe/min @ Structural formula: λMax: 270r1T
rL (methanol) Rf value: 0.66 (butanol diacetic acid: water = 4:11) S-S exchange reaction rate 93.2 μMO
le/min @ Structural formula: λMax: 310r1j1. (methanol) Rf value: 0.82 (ethyl benzene diacetate
:1) [Phase] Structural formula: 0XT11aX:271nTr!
.. (Methanol) Rf value: 0.41 (butanol diacetic acid: water = 4: 1) S-S exchange reaction rate 92.6 μMO
le/min9 Structural formula: λMa) c: 271nrrL, (methanol) 0 Rf value: 0.25 (butanol diacetic acid: water = 4:11) O structural formula: λRnax: 270r) 7n, (methanol) Rf value:
0.32 (butanol diacetic acid:water = 4:1:O11)S
-S exchange reaction rate 90.1 μM Ole/min O structural formula: λ
NlaX: 270r1TrLRf value: 0.20 (butanol diacetic acid: water = 4:1:1) S-S exchange reaction rate 91
.. 5μMOle/min [Yes] Structural formula: λMax: 270r
17n Rf value: 0.41 (butanol diacetic acid: water = 4:1:1
) [Phase] Structural formula: λMa:X:270r1TrL Rf value: 0.25 (butanol diacetic acid: water = 4:1:1
) Reference Example 1 A 500 ml three-necked flask was immersed in a constant temperature water bath at about 35°C, and the air was replaced with about 1 ml of nitrogen powder.Next, 120 ml of distilled water was added to the flask, and sodium alkylsulfonate 2y1 acrylonitrile 80 da, Sodium persulfate 0.1y1 Sodium bisulfite 0.033y
was added and stirred for about 3 hours to obtain an emulsion, which was then poured into about 500 ml of water, and while stirring, salt was added to coagulate to precipitate the product, which was separated, washed with water, and dried with ventilation. polyacrylonitrile (a). 5%, the logarithmic viscosity in dimethylformamide at 30° C. is about 10.5) was obtained.
次いでこのポリアクリロニトリル10yをジメチルホル
ムアミド150m1に溶解し、これを、20%ジメチル
ホルムアミド含有水浴中に、糸状に成形して、多孔質構
造を有するフィラメント状のポリアクリロニトリルを得
た。同様に、ポリアクリロニトリル10yを20%ジメ
チルホルムアミド含有水浴中に、アトマイザ−カップを
用いて滴下して、多孔質構”造を有する粒状のポリアク
リロニトリルを得た。次いで、水素化リチウムアルミニ
ウム2.5gを三つロフラスコに加え、乾燥エーテル1
00m1を添加・攪拌し、これに上記の多孔質構造を有
する粒状のポリアクリロニトリル2qを加えて50℃に
て托時間加熱還流し、反応後氷冷下、水を滴下して未反
応の水素化リチウムアルミニウムを分解せしめ、さらに
1NHC1を滴下して、その分解物を溶解せしめ、次い
でこれを沖別して、アミノ化された該ポリアクリロニト
リルを回収し、次いで1NHCI1水、1NNa0H1
水、0.IMリン酸緩衝液(PH7.5)の順で洗浄し
て遊離アミノ基およびニトリル基を有する多孔質構造の
粒状物を得た。同様に、上記の多孔質構造を有する粒状
のポリアクリロニトリルの代りに、多孔質構造を有する
フィラメント状のポリアクリロニトリルを用いて行なつ
た結果、遊離アミノ基およびニトリル基を有する多孔質
構造のフィラメント状物を得た。このようにして得られ
た遊離アミノ基およびニトリル基を有する多孔質構造物
を、12.5%グルタルアルデヒド/ホウ酸緩衝液(P
H8.5)に加えて0℃、2紛間反応応せしめ、次いで
これをp取し、ホウ酸緩衝液にて洗浄後、さらにこれを
7−ADCA(7−アミノデスアセトキシセフアロスポ
ラン酸)/0.1Mリン酸緩衝液(PH7.5)に加え
て30℃、6吟間振盪して反応せしめ、その後その上清
中に残存する7−ADCAの量を液体クロマトグラフィ
ーにより求めて、該遊離アミノ基およびニトリル基を有
する多孔質構造物1y当り7一,ADCA33〜35μ
Mを結合し得るアミノ基を有している構造物であつた。
また、上記のグルタルアルデヒド処理後、0.2Mヘキ
サメチレンジアミン(PH9.5)を室温下2時間処理
しさらにグルタルアルデヒドを反応せしてた後、7−A
DCAを同様反応せしめて求めた結果7−ADCAの結
合量は42〜46μMlyであつた。Next, this polyacrylonitrile 10y was dissolved in 150 ml of dimethylformamide, and this was formed into a thread in a water bath containing 20% dimethylformamide to obtain a filament-like polyacrylonitrile having a porous structure. Similarly, polyacrylonitrile 10y was dropped into a water bath containing 20% dimethylformamide using an atomizer cup to obtain granular polyacrylonitrile having a porous structure. Then, 2.5 g of lithium aluminum hydride was obtained. into the flask, add 1 part of dry ether
00ml was added and stirred, and 2q of granular polyacrylonitrile having the above-mentioned porous structure was added thereto and heated under reflux at 50°C for an hour. After the reaction, water was added dropwise under ice cooling to hydrogenate unreacted Lithium aluminum was decomposed, 1N HCl was added dropwise to dissolve the decomposed product, and then the aminated polyacrylonitrile was recovered, and then 1N HCl1 water, 1N Na0H1
Water, 0. The particles were sequentially washed with IM phosphate buffer (PH 7.5) to obtain granules with a porous structure having free amino groups and nitrile groups. Similarly, as a result of using filamentary polyacrylonitrile with a porous structure instead of the granular polyacrylonitrile with a porous structure described above, a filamentary polyacrylonitrile with a porous structure having free amino groups and nitrile groups was used. I got something. The thus obtained porous structure with free amino groups and nitrile groups was dissolved in a 12.5% glutaraldehyde/borate buffer (P
H8.5) was added to 0°C for a two-powder reaction, then this was taken out, washed with a boric acid buffer, and further added to 7-ADCA (7-aminodesacetoxycephalosporanic acid). /0.1M phosphate buffer (PH7.5) and shaken at 30°C for 6 minutes to react, and then the amount of 7-ADCA remaining in the supernatant was determined by liquid chromatography. 71, ADCA 33-35μ per y of porous structure with free amino groups and nitrile groups
The structure had an amino group capable of binding M.
In addition, after the above glutaraldehyde treatment, 0.2M hexamethylene diamine (PH9.5) was treated at room temperature for 2 hours, and glutaraldehyde was further reacted.
The amount of 7-ADCA bound was determined to be 42 to 46 μMly by performing the same reaction with DCA.
本発明においては、この新規な遊離アミノ基およびニト
リル基を有する多孔性構造物を、アミノ基を有する化合
物として使用しうるものてある。In the present invention, this novel porous structure having free amino groups and nitrile groups can be used as a compound having an amino group.
参考例96・6−ナイロンピース(径4.8Twt)1
00yをγーアミノプロピルトリエトキシシラン100
mL中に分散して、100℃、3時間加熱処理した後該
ピースを沖取し、水洗して乾燥してアミノプロピル化し
た該ビーズを得た。Reference example 96.6-nylon piece (diameter 4.8Twt) 1
00y is γ-aminopropyltriethoxysilane 100
After dispersing in mL and heating at 100° C. for 3 hours, the pieces were taken off, washed with water, and dried to obtain the aminopropylated beads.
本化合物を、無水コハク酸20q/150m1ジメチル
ホルムアミド中に加え、一夜放置反応せしめ、戸取し、
ジメチルホルムアミドにて洗浄し、これをジシクロヘキ
シルカルボジイミド20.6yおよびN−ヒドロキシス
クシンイミド11.5y/ジメチルホルムアミド150
m1にて一夜反応せしめてジメチルホルムアミドにて洗
浄し、さらにこのピースを0.05Mリン酸緩衝液(P
H7.4)(イ).1%NaN3、0.25%牛血清
アルブミン、37TLMMgC1。、0.15r!4N
acI含有:以下抗原抗体用緩衝液と略す)で3回洗浄
し、これにウサギのモルモツトIgG抗体(以下Ab2
と略す)を加えて5℃、17時間放置してp取し、抗原
抗体用緩衝液で3回洗浄してAb2B結合該ピースを得
、さらにこの2ビーズを用いて、これに、モルモツトの
牛インスリン抗体(以下Ablと略す)2γを用いるβ
−ガラクトシダーゼ架橋結合体(参考例4参照)100
mLを加えて20!11寺間、5℃下放置し、抗原抗体
用緩衝液で洗浄し、5m9ノmlのオルトニトロフェニ
ルガラクトサイド(イ).1%牛血清アルブミン、10
TrL,Mメルカプトエタノール含有PH6.7、0.
1Mリン酸緩衝液)200μeからなるβ−ガラクトシ
ダーゼ呈色反応を用いて44eC2吟間反応せしめ、直
ちにグリシン緩衝液(PHlO.5、0.1M)25m
1を加えて反応を停止せしめ、これを420r17Tt
.に比色分析して、少なくとも1ピース当り54γのA
b2を結合せしめ得るアミノプロピル基を導入された化
合物であつた。本発明において、この新規なアミノプロ
ピル化せしめたナイロンビーズも利用し得るものである
。This compound was added to 20q of succinic anhydride/150ml of dimethylformamide, left to react overnight, and taken out.
Washed with dimethylformamide and mixed with 20.6y of dicyclohexylcarbodiimide and 11.5y of N-hydroxysuccinimide/150y of dimethylformamide.
The piece was reacted overnight in M1, washed with dimethylformamide, and then added to 0.05M phosphate buffer (P
H7.4) (a). 1% NaN3, 0.25% bovine serum
Albumin, 37TLMMgC1. , 0.15r! 4N
Wash three times with acI-containing buffer (hereinafter referred to as antigen-antibody buffer), and add rabbit guinea pig IgG antibody (hereinafter referred to as Ab2) to this.
) was added and left at 5°C for 17 hours to remove the beads, and washed three times with antigen-antibody buffer to obtain the Ab2B-bound piece.Furthermore, using these two beads, β using insulin antibody (hereinafter abbreviated as Abl) 2γ
- Galactosidase cross-linked conjugate (see Reference Example 4) 100
mL of orthonitrophenyl galactoside (a). 1% bovine serum albumin, 10
TrL,M mercaptoethanol containing PH6.7, 0.
A β-galactosidase color reaction consisting of 200 μe of 1M phosphate buffer was used to react for 44 eC2, and immediately 25 μe of glycine buffer (PHlO.5, 0.1 M) was added.
1 was added to stop the reaction, and this was added to 420r17Tt.
.. Colorimetrically analyzed, A of at least 54γ per piece
It was a compound into which an aminopropyl group capable of binding b2 was introduced. The novel aminopropylated nylon beads can also be used in the present invention.
参考例3
牛インスリン60mgを0.1Mベロナール緩衝液(P
H7.5)10mtに溶解し、これに、3(−ベンゾチ
アゾールー2−イルジチオ)プロピオン酸スクシンイミ
ドエステル3.1m9溶解1m1ジメチルホルムアミド
溶液を加えて室温下4時間攪拌せしめ、その後PHを5
.0に調整して生成物を析出せしめ、これを3000r
′Pmll紛間遠心分離して3−(ベンゾチアゾールー
2″−イルジチオ)プロピオニル基を導入せしめたイン
スリン誘導体を得、さらにクエン酸緩衝液(PH5)で
洗浄後、これを10mLの0.1Mリン酸緩衝液(PH
7.5)に溶解し、その0.1mL・を分取し、これに
、あらかじめリン酸緩衝液(PH7.5)5m1に5T
fLgのβ−ガラクトシダーゼを溶解した溶液を加えて
室温下、1時間反応せしめて、次いでこの反応液をセフ
アデツクスG−100(商品名)(1×80C77りに
チャージし、0.15MNac1含有0.01Mリン酸
緩衝液(PH7.5)にて、1フラクシヨン4m1づつ
分画して、第7〜8フラクシヨンの活性画分を回収して
インスリンとβ−ガラクトシダーゼとを架橋結合せしめ
た化合物を得た。Reference Example 3 60 mg of bovine insulin was added to 0.1 M veronal buffer (P
H7.5) was dissolved in 10 mt of succinimide 3(-benzothiazol-2-yldithio)propionic acid, and to this was added 1 ml of dimethylformamide solution dissolved in 3.1 m of 3(-benzothiazol-2-yldithio)propionic acid succinimide ester, and stirred at room temperature for 4 hours.
.. 0 to precipitate the product, and heated it at 3000 r.
'Pml powder centrifugation to obtain an insulin derivative into which a 3-(benzothiazol-2''-yldithio)propionyl group was introduced, and after further washing with citric acid buffer (PH5), this was added to 10 mL of 0.1 M phosphorus. Acid buffer (PH
7.5), aliquot 0.1 mL of it, and add 5T to 5 ml of phosphate buffer (PH 7.5) in advance.
A solution of fLg β-galactosidase was added and allowed to react at room temperature for 1 hour, and then this reaction solution was charged to Cephadex G-100 (trade name) (1 x 80C77, 0.01M containing 0.15M Nacl). The mixture was fractionated into 4 ml fractions using a phosphate buffer (PH 7.5), and the active fractions of the 7th and 8th fractions were collected to obtain a compound in which insulin and β-galactosidase were cross-linked.
さらに0.02ny〜10r1Vの牛インスリンを試験
管に取り、これらに上記のインスリンーβ−ガラクトシ
ダーゼ架橋結合物とモルモツトの抗牛インスリン血清と
を加えて5℃、一夜反応せしめ、さらに第2抗体として
ウサギの抗モルモツトIgG血清を加えて5℃、一夜反
応せしめ、これを3000rpm110分間遠心分離し
、得られた沈澱物を、上記参考例2と同一のβ−ガラク
トシダーゼの活性測定手段を用いて、上記のインスリン
ーβ−ガラクトシダーゼ架橋結合体とインスリンとを用
いてなるインスリン抗血清の第2抗体法による酵素免疫
を行なつた。その結果は第1図に示す通りであり、良好
な測定結果を示した。参考例4
参考例3の牛インスリンの代りに、モルモツトの牛イン
スリン抗体を用いて、3−(ベンゾチアゾールー2′−
イルジチオ)プロピオン酸スクシンイミドエステルと反
応せしめ、以下参考例3と同様に行なつてモルモツトの
牛インスリン抗体−βーガラクトシダーゼ架橋結合体を
得た。Further, 0.02 ny to 10 r1 V of bovine insulin was placed in a test tube, and the above insulin-β-galactosidase cross-linked product and guinea pig anti-bovine insulin serum were added thereto and reacted overnight at 5°C. anti-guinea pig IgG serum was added to react overnight at 5°C, and the mixture was centrifuged at 3000 rpm for 110 minutes. Enzyme immunization was performed using a second antibody method using an insulin antiserum using an insulin-β-galactosidase cross-linked conjugate and insulin. The results are as shown in FIG. 1, showing good measurement results. Reference Example 4 Instead of the bovine insulin of Reference Example 3, guinea pig bovine insulin antibody was used to produce 3-(benzothiazole-2'-
The reaction was carried out in the same manner as in Reference Example 3 to obtain a guinea pig bovine insulin antibody-β-galactosidase crosslinked conjugate.
本品は上記参考例2に記載の通り利用されたものである
。参考例5参考例2で得られたアミノプロピル化した6
.6ーナイロンピース2唯を5瓦1ジメチルホルムアミ
ドに加え、さらにこれに3−(ベンゾチアゾールー2″
−イルジチオ)プロピオン酸シクシンイミドエステル2
.0m9含有ジメチルホルムアミド1mιを加えて室温
下4時間攪拌し、その後これを戸取し、ジメチルホルム
アミドー0.IMリン酸緩衝液(PH7.5)にて洗浄
し、さらにこれを0.IMリン酸緩衝液(PH7.5)
5mLに加え、次いでこれに3m9をβ−ガラクトシダ
ーゼ含有0.IMリン酸緩衝液(PH7.5)を加えて
室温下2時間反応せしめ、これを戸取し、0.IMリン
酸緩衝液(PH7.5)にて洗浄して、β−ガラクトシ
ダーゼの不溶化ビーズを得た(1−ビーズ当りβ−ガラ
クトシダーゼ6U.の活性を有していた)。This product was used as described in Reference Example 2 above. Reference Example 5 Aminopropylated 6 obtained in Reference Example 2
.. Add 2 pieces of 6-nylon to 1 piece of dimethylformamide, and add 2 pieces of 3-(benzothiazole) to this.
-yldithio)propionic acid succinimide ester 2
.. Add 1 mι of dimethylformamide containing 0.0 m9 and stir at room temperature for 4 hours, then take it out and add 0.0 m9 of dimethylformamide. Washed with IM phosphate buffer (PH7.5) and further diluted with 0. IM phosphate buffer (PH7.5)
5 mL and then add 3m9 to this 0.5 mL containing β-galactosidase. IM phosphate buffer (PH7.5) was added and reacted at room temperature for 2 hours, taken out and 0. By washing with IM phosphate buffer (PH 7.5), β-galactosidase insolubilized beads were obtained (having an activity of 6 U. β-galactosidase per bead).
参考例6
5m9のβ−ガラクトシダーゼ含有0.IMベロナール
緩衝液(PH7.5)57711に、3.1m9の3−
(ベンゾチアゾールー7−イルジチオ)プロピオン酸ス
クシンイミドエステル含有1m1ジメチルホルムアミド
を加えて室温下、4時間攪拌反応せしめ、3−(ベンゾ
チアゾールー2’−イルジチオ)プロピオニル化したβ
−ガラクトシダーゼを得その後これを0.IN炭酸ナト
リウム水溶液を用いて、PH9.Oに調整した水溶液に
加え2時間放置して、そのS−S結合を加水分解してβ
−チオプロピオニル化されたβ−ガラクトシダーゼ(β
−ガラクトシダーゼ1分子中β−チオプロピオニル基2
紛子導入)を得た。Reference Example 6 5m9 β-galactosidase containing 0. 3.1 m9 of 3-
1ml of dimethylformamide containing (benzothiazol-7-yldithio)propionic acid succinimide ester was added and reacted with stirring at room temperature for 4 hours to form 3-(benzothiazol-2'-yldithio)propionylated β.
- Obtain galactosidase and then add it to 0. pH 9. using IN sodium carbonate aqueous solution. Add it to an aqueous solution adjusted to O and leave it for 2 hours to hydrolyze the S-S bond and form β
-thiopropionylated β-galactosidase (β
-2 β-thiopropionyl groups in one molecule of galactosidase
(introduced a miscellaneous child).
第1図は酵素免疫法におけるインスリンの測定曲線を示
す。FIG. 1 shows a measurement curve for insulin in the enzyme immunoassay.
Claims (1)
式中、R_1は2−ベンゾチアゾリル基または2−ピリ
ジール−N−オキサイド基、R_2は遊離または保護さ
れた官能基を有してもよいアルキレン基、R_3はアミ
ノ酸または低級ポリペプタイドのカルボキシル残基、R
_4はカルボキシル基、その反応性誘導体または保護さ
れたカルボキシル基もしくはイミデート基、nは0また
は1を示す)で表わされる新規なジスルフィド誘導体。[Claims] 1 The following general formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] (However,
In the formula, R_1 is a 2-benzothiazolyl group or a 2-pyridyl-N-oxide group, R_2 is an alkylene group that may have a free or protected functional group, R_3 is an amino acid or a carboxyl residue of a lower polypeptide, R
_4 is a carboxyl group, a reactive derivative thereof, or a protected carboxyl group or imidate group; n is 0 or 1); a novel disulfide derivative;
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP53085900A JPS6058232B2 (en) | 1978-07-13 | 1978-07-13 | Novel disulfide derivative |
FR7918007A FR2430943A1 (en) | 1978-07-13 | 1979-07-11 | NEW DISULFIDE DERIVATIVES |
GB7924336A GB2029825B (en) | 1978-07-13 | 1979-07-12 | Benzothiazolyl and pyndyl n oxide disulphide derivatives |
DE19792928384 DE2928384A1 (en) | 1978-07-13 | 1979-07-13 | DISULFID DERIVATIVES |
US06/057,502 US4258193A (en) | 1978-07-13 | 1979-07-13 | Disulfide derivatives having S--S exchange reactivity |
SE7906233A SE448095B (en) | 1978-07-13 | 1979-07-19 | disulfide |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP53085900A JPS6058232B2 (en) | 1978-07-13 | 1978-07-13 | Novel disulfide derivative |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP16490978A Division JPS5512178A (en) | 1978-04-28 | 1978-12-28 | Carrier having s-s exchange reactivity |
JP16491178A Division JPS5522657A (en) | 1978-07-13 | 1978-12-28 | Reactive compound |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS5517302A JPS5517302A (en) | 1980-02-06 |
JPS6058232B2 true JPS6058232B2 (en) | 1985-12-19 |
Family
ID=13871729
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP53085900A Expired JPS6058232B2 (en) | 1978-07-13 | 1978-07-13 | Novel disulfide derivative |
Country Status (2)
Country | Link |
---|---|
JP (1) | JPS6058232B2 (en) |
SE (1) | SE448095B (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SE8102193L (en) * | 1981-04-06 | 1982-10-07 | Pharmacia Ab | THERAPEUTIC ACTIVE ORGANIC ASSOCIATION AND ITS USE |
SE8102194L (en) * | 1981-04-06 | 1982-10-07 | Pharmacia Ab | THERAPEUTIC ACTIVE ORGANIC ASSOCIATION AND PHARMACEUTICAL PREPARATION INCLUDING THIS |
JPS5880558A (en) * | 1981-11-09 | 1983-05-14 | Sekisui Chem Co Ltd | Immunochemical measuring reagent |
JPS6391563A (en) * | 1986-10-07 | 1988-04-22 | Nitsusui Seiyaku Kk | Examination of protein specific antigen derived from bacteria |
GB0420062D0 (en) * | 2004-09-09 | 2004-10-13 | Akubio Ltd | Assay methods,materials and preparations |
WO2009134977A1 (en) * | 2008-04-30 | 2009-11-05 | Immunogen, Inc. | Cross-linkers and their uses |
-
1978
- 1978-07-13 JP JP53085900A patent/JPS6058232B2/en not_active Expired
-
1979
- 1979-07-19 SE SE7906233A patent/SE448095B/en not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
SE7906233L (en) | 1981-01-20 |
SE448095B (en) | 1987-01-19 |
JPS5517302A (en) | 1980-02-06 |
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