JPH0321038B2 - - Google Patents
Info
- Publication number
- JPH0321038B2 JPH0321038B2 JP57121988A JP12198882A JPH0321038B2 JP H0321038 B2 JPH0321038 B2 JP H0321038B2 JP 57121988 A JP57121988 A JP 57121988A JP 12198882 A JP12198882 A JP 12198882A JP H0321038 B2 JPH0321038 B2 JP H0321038B2
- Authority
- JP
- Japan
- Prior art keywords
- group
- glutathione
- formula
- trigsh
- glu
- 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 - Lifetime
Links
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 105
- 108010024636 Glutathione Proteins 0.000 claims description 45
- 229960003180 glutathione Drugs 0.000 claims description 43
- 150000001875 compounds Chemical class 0.000 claims description 33
- 239000013638 trimer Substances 0.000 claims description 30
- 229910052751 metal Inorganic materials 0.000 claims description 23
- 239000002184 metal Substances 0.000 claims description 23
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 13
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 8
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 5
- 235000001014 amino acid Nutrition 0.000 claims description 5
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 5
- 229910052793 cadmium Inorganic materials 0.000 claims description 4
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 4
- 229910052742 iron Inorganic materials 0.000 claims description 3
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 3
- 229910052725 zinc Inorganic materials 0.000 claims description 3
- 125000006307 alkoxy benzyl group Chemical group 0.000 claims description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 2
- 229910052759 nickel Inorganic materials 0.000 claims description 2
- 125000003710 aryl alkyl group Chemical group 0.000 claims 2
- 230000009920 chelation Effects 0.000 claims 1
- 229910052748 manganese Inorganic materials 0.000 claims 1
- 229910052763 palladium Inorganic materials 0.000 claims 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 78
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 60
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 43
- 238000006243 chemical reaction Methods 0.000 description 30
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 30
- 239000000243 solution Substances 0.000 description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 23
- 238000002360 preparation method Methods 0.000 description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 20
- 239000002904 solvent Substances 0.000 description 20
- 238000002844 melting Methods 0.000 description 18
- 230000008018 melting Effects 0.000 description 18
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 16
- 238000000034 method Methods 0.000 description 15
- 239000013078 crystal Substances 0.000 description 14
- 239000003921 oil Substances 0.000 description 14
- 125000006239 protecting group Chemical group 0.000 description 14
- 238000000921 elemental analysis Methods 0.000 description 13
- 239000000047 product Substances 0.000 description 12
- 238000001816 cooling Methods 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 150000002148 esters Chemical class 0.000 description 9
- 125000003277 amino group Chemical group 0.000 description 8
- 238000001035 drying Methods 0.000 description 8
- 229910001385 heavy metal Inorganic materials 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- 150000002739 metals Chemical class 0.000 description 7
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- -1 and Co Substances 0.000 description 6
- 239000013522 chelant Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 230000000704 physical effect Effects 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- 238000004809 thin layer chromatography Methods 0.000 description 6
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 5
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical group OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- XYJWEQWNNKNSFU-STQMWFEESA-N (2s)-2-amino-5-[[(2r)-3-benzylsulfanyl-1-(carboxymethylamino)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSCC1=CC=CC=C1 XYJWEQWNNKNSFU-STQMWFEESA-N 0.000 description 4
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 108700021109 S-benzylglutathione Proteins 0.000 description 4
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000006227 byproduct Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- YWEUIGNSBFLMFL-UHFFFAOYSA-N diphosphonate Chemical compound O=P(=O)OP(=O)=O YWEUIGNSBFLMFL-UHFFFAOYSA-N 0.000 description 4
- 238000010494 dissociation reaction Methods 0.000 description 4
- 230000005593 dissociations Effects 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- DLYUQMMRRRQYAE-UHFFFAOYSA-N phosphorus pentoxide Inorganic materials O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 239000006188 syrup Substances 0.000 description 4
- 235000020357 syrup Nutrition 0.000 description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- 231100000419 toxicity Toxicity 0.000 description 4
- 230000001988 toxicity Effects 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- IFVORPLRHYROAA-LBPRGKRZSA-N (2r)-3-benzylsulfanyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CSCC1=CC=CC=C1 IFVORPLRHYROAA-LBPRGKRZSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical class C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- VQTUBCCKSQIDNK-UHFFFAOYSA-N Isobutene Chemical group CC(C)=C VQTUBCCKSQIDNK-UHFFFAOYSA-N 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- HSDAJNMJOMSNEV-UHFFFAOYSA-N benzyl chloroformate Chemical compound ClC(=O)OCC1=CC=CC=C1 HSDAJNMJOMSNEV-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 235000008504 concentrate Nutrition 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- 238000004821 distillation Methods 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- QQWYQAQQADNEIC-RVDMUPIBSA-N tert-butyl [(z)-[cyano(phenyl)methylidene]amino] carbonate Chemical compound CC(C)(C)OC(=O)O\N=C(/C#N)C1=CC=CC=C1 QQWYQAQQADNEIC-RVDMUPIBSA-N 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- QKAFVKIVSGVOPT-KBPBESRZSA-N (2s)-2-amino-5-[[(2r)-1-(carboxymethylamino)-3-[(4-methoxyphenyl)methylsulfanyl]-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound COC1=CC=C(CSC[C@H](NC(=O)CC[C@H](N)C(O)=O)C(=O)NCC(O)=O)C=C1 QKAFVKIVSGVOPT-KBPBESRZSA-N 0.000 description 2
- RNHDAKUGFHSZEV-UHFFFAOYSA-N 1,4-dioxane;hydrate Chemical compound O.C1COCCO1 RNHDAKUGFHSZEV-UHFFFAOYSA-N 0.000 description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- MSHFRERJPWKJFX-UHFFFAOYSA-N 4-Methoxybenzyl alcohol Chemical compound COC1=CC=C(CO)C=C1 MSHFRERJPWKJFX-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 235000019445 benzyl alcohol Nutrition 0.000 description 2
- AGEZXYOZHKGVCM-UHFFFAOYSA-N benzyl bromide Chemical compound BrCC1=CC=CC=C1 AGEZXYOZHKGVCM-UHFFFAOYSA-N 0.000 description 2
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 2
- YKYOUMDCQGMQQO-UHFFFAOYSA-L cadmium dichloride Chemical compound Cl[Cd]Cl YKYOUMDCQGMQQO-UHFFFAOYSA-L 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- PSLIMVZEAPALCD-UHFFFAOYSA-N ethanol;ethoxyethane Chemical compound CCO.CCOCC PSLIMVZEAPALCD-UHFFFAOYSA-N 0.000 description 2
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000006722 reduction reaction Methods 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- DCKVNWZUADLDEH-UHFFFAOYSA-N sec-butyl acetate Chemical group CCC(C)OC(C)=O DCKVNWZUADLDEH-UHFFFAOYSA-N 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- QCQCHGYLTSGIGX-GHXANHINSA-N 4-[[(3ar,5ar,5br,7ar,9s,11ar,11br,13as)-5a,5b,8,8,11a-pentamethyl-3a-[(5-methylpyridine-3-carbonyl)amino]-2-oxo-1-propan-2-yl-4,5,6,7,7a,9,10,11,11b,12,13,13a-dodecahydro-3h-cyclopenta[a]chrysen-9-yl]oxy]-2,2-dimethyl-4-oxobutanoic acid Chemical compound N([C@@]12CC[C@@]3(C)[C@]4(C)CC[C@H]5C(C)(C)[C@@H](OC(=O)CC(C)(C)C(O)=O)CC[C@]5(C)[C@H]4CC[C@@H]3C1=C(C(C2)=O)C(C)C)C(=O)C1=CN=CC(C)=C1 QCQCHGYLTSGIGX-GHXANHINSA-N 0.000 description 1
- JXYACYYPACQCDM-UHFFFAOYSA-N Benzyl glycinate Chemical compound NCC(=O)OCC1=CC=CC=C1 JXYACYYPACQCDM-UHFFFAOYSA-N 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 125000000415 L-cysteinyl group Chemical group O=C([*])[C@@](N([H])[H])([H])C([H])([H])S[H] 0.000 description 1
- 125000003338 L-glutaminyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C(=O)N([H])[H] 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- GHBAYRBVXCRIHT-VIFPVBQESA-N S-benzyl-L-cysteine zwitterion Chemical compound OC(=O)[C@@H](N)CSCC1=CC=CC=C1 GHBAYRBVXCRIHT-VIFPVBQESA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 241001061127 Thione Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- YEESUBCSWGVPCE-UHFFFAOYSA-N azanylidyneoxidanium iron(2+) pentacyanide Chemical compound [Fe++].[C-]#N.[C-]#N.[C-]#N.[C-]#N.[C-]#N.N#[O+] YEESUBCSWGVPCE-UHFFFAOYSA-N 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000010531 catalytic reduction reaction Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- MHDVGSVTJDSBDK-UHFFFAOYSA-N dibenzyl ether Chemical compound C=1C=CC=CC=1COCC1=CC=CC=C1 MHDVGSVTJDSBDK-UHFFFAOYSA-N 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- MDKXBBPLEGPIRI-UHFFFAOYSA-N ethoxyethane;methanol Chemical compound OC.CCOCC MDKXBBPLEGPIRI-UHFFFAOYSA-N 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000010446 mirabilite Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229960002460 nitroprusside Drugs 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- KJIFKLIQANRMOU-UHFFFAOYSA-N oxidanium;4-methylbenzenesulfonate Chemical compound O.CC1=CC=C(S(O)(=O)=O)C=C1 KJIFKLIQANRMOU-UHFFFAOYSA-N 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 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
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 238000011110 re-filtration Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012056 semi-solid material Substances 0.000 description 1
- UQDJGEHQDNVPGU-UHFFFAOYSA-N serine phosphoethanolamine Chemical compound [NH3+]CCOP([O-])(=O)OCC([NH3+])C([O-])=O UQDJGEHQDNVPGU-UHFFFAOYSA-N 0.000 description 1
- 239000010703 silicon Substances 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
- 239000012265 solid product Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- QQWYQAQQADNEIC-UHFFFAOYSA-N tert-butyl [[cyano(phenyl)methylidene]amino] carbonate Chemical compound CC(C)(C)OC(=O)ON=C(C#N)C1=CC=CC=C1 QQWYQAQQADNEIC-UHFFFAOYSA-N 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000000954 titration curve Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- LZTRCELOJRDYMQ-UHFFFAOYSA-N triphenylmethanol Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(O)C1=CC=CC=C1 LZTRCELOJRDYMQ-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009834 vaporization Methods 0.000 description 1
- 230000008016 vaporization Effects 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
本発明は、γ−Ｌ−グルタミルＬ−システイニ
ルグリシン（別称：グルタチオン；以下、グルタ
チオンと呼ぶ）の従来公知文献未記載の三量体に
関し、公知グルタチオン単量体に比べて極めて優
れた金属結合能を有し、重金属類の生体内、毒性
発現の防除、生体内金属の移行制御関与物質とし
て医学及び薬学の広い分野における利用などに有
用且つ注目されるグルタチオン三量体に関する。
更に詳しくは、本発明は従来公知文献未記載の
グルタチオン三量体（グルタチオン単位における
システイン残基の−SH基が遊離状態であるグル
タチオン三量体）及び少なくともその−SH基の
保護された誘導体に関する。更に又、本発明はそ
れ自体公知の手法を利用して該グルタチオン三量
体及び該誘導体を製造する方法にも関する。
グルタチオンは公知物質であつて、下記式で示
すようにＬ−グルタミン酸単位部分、Ｌ−システ
イン単位部分及びグリシン単位部分から成り（以
下、グリシン以外のアミノ酸はＬ−体を示す）、
グルタチオン−単位についてペプチド結合により
重合可能な位置が下記式にアンダーラインを施し
た３ケ所存在する。従つて、その三量体の位置異
性体の存在様式は５種類であり、本発明はこれら
５種類の位置異性体の中後記のTri−GSH−Ｉで
表わされるグルタチオン三量体に係るが、以下に
参考として残りの４種類の位置異性体についても
説明する。
グルタチオン：−
グルタチオンは、例えば酵母や動物の肝臓、筋
肉などに広く分布する公知物質であつて、生体内
酸化還元に重要な役割を果していることが知られ
ている。又、金属に対して結合能を有する物質で
あることも知られている。従来、グルタチオンの
酸化体である−Ｓ−Ｓ−結合により結合された二
量体は知られている。しかしながら、ペプチド結
合により結合されたグルタチオンオリゴマーにつ
いては従来全く知られていない。
本発明者等はグルタチオン及びその誘導体に関
して研究を行つてきた。その結果、ペプチド結合
により結合されたグルタチオンの三量体及び少な
くともその−SH基の保護された誘導体が存在し
得て且つそれ自体公知の反応手法を組み合わせる
ことにより合成できることを発見した。
更に、得られるグルタチオン三量体は従来公知
文献未記載の化合物であつて、グルタチオン単量
体に比して極めて優れた金属結合能を有し、細胞
に対する重金属類の毒性に対する毒性低減能も強
いことを発見した。
又更に、上記特性に由来して、重金属類による
生体内毒性発現を防除する作用、生体細胞の機能
維持に重要な役割を演ずるホルモン、酵素類など
の生理活性物質に密接に関与する銅、亜鉛、鉄、
コバルト、ニツケル、硅素などの金属の移行制御
作用、などに関係を有する物質として医学及び薬
学の広い応用分野に於て有用であり、更に、本発
明新規化合物は生体内における金属の移行制御に
関与する物質として公知で且つ生物界に広く存在
する金属結合蛋白“メタロチオネイン”の金属結
合部位と構造的な類似性を有する点でも興味ある
注目すべき化合物であることを知つた。
従つて本発明の目的は新規グルタチオン三量体
及びその誘導体を提供するにある。
本発明の他の目的は上記化合物の製法を提供す
るにある。
本発明の上記目的及び更に多くの他の目的なら
びに利点は以下の記載から一層明らかとなるであ
ろう。
グルタチオン三量体は、グルタチオン単位中の
グルタミン酸残基のカルボキシル基及び／又はグ
リシン残基のカルボキシル基と、他のグルタチオ
ン単位のグルタミン酸残基のアミノ基との間のペ
プチド結合を形成する組合わせに応じて５種のグ
ルタチオン三量体〔以下TriGSHと略記すること
がある〕を包含する。そしてこれら５種のグルタ
チオン三量体の分子構造を表示するため、グルタ
チオン単位の結合様式を次のごとく定義する。ア
ミノ基が遊離状態であるグルタチオン単位を母体
として、第２位のグルタチオン単位のアミノ基が
グルタミン酸残基のα−カルボキシル基とペプチ
ド結合により連結している状態をα−、グリシン
残基のカルボキシル基とペプチド結合している状
態をω−、第３位のグルタチオン単位が、第２位
のグルタチオン単位のグルタミン酸残基のα−カ
ルボキシル基、グリシン残基のカルボキシル基と
結合している状態をそれぞれα′−、ω′−と表示
し、下記のとおり５種のTriGSHの構造と名称を
提示する。
(1) TriGSH−（α，ω−グルタチオニルジグ
ルタチオン）〔本発明〕：−
但し式中−Glu−または−Glu−はグルタミン
酸残基のうち−NH2のＨ及び−COOHのOHを
除いた残基
を示し、−Cys−は、システイン酸基のうち
を示し、そして−Gly−は、グリシン残基のう
ち−HN−CH2−CO−
を示し、本化合物に於けるアミノ酸略号間の実
線に各々アミノ酸がペプチド結合で連絡されて
いることを示す。GluまたはGluの上、下に出
た実線はγ−カルボキシル基からOHを除いた
カルボニル基、Glu−の右横実線はα−カルボ
キシル基からOHを除いたカルボニル基を示
す。
またシステイン残基中−SH保護基にて保護
されている場合（ ）内にその保護基の略号を
示し−SHが遊離の場合は、（Ｈ）を付して表示
した。
(2) TriGSH−（ω−グルタチオニル−ω′−グ
ルタチオニルグルタチオン）〔参考〕：−
但し式中、−Glu−または−Glu−、−Cys（Ｈ）−
及び−Gly−は上記したと同義。
(3) TriGSH−（ω−グルタチオニルα′−グル
タチオニルグルタチオン）〔参考〕：−
但し式中、−Glu−または−Glu−、−Cys（Ｈ）−
及び−Gly−は上記したと同義。
(4) TriGSH−（α−グルタチオニル−ω′−グ
ルタチオニルグルタチオン）〔参考〕：−
但し式中、−Glu−または−Glu−、−Cys（Ｈ）−
及び−Gly−は上記したと同義。
(5) TriGSH−（α−グルタチオニル−α′−グ
ルタチオニルグルタチオン）〔参考〕：−
但し式中、−Glu−または−Glu−、−Cys（Ｈ）−
及び−Gly−は上記したと同義。
本発明はまた、上記グルタチオン三量体の少な
くとも−SH基の保護された誘導体を包含し、例
えば、該三量体の金属キレート化合物もしくは−
SH保護基で保護された化合物を挙げることがで
きる。−SH保護基で保護された化合物は、前記式
(1)〜(5)中、−Gly−OHで示されるグリシン単位カ
ルボキシル基（−COOH）、Glu−OHで示される
グルタミン酸単位α−カルボキシル基（−
COOH）、及びＨ−Glu−で示されるグルタミン
酸単位アミノ基（NH2−）も保護されていてよ
い。更に又、金属キレート化合物の形で少なくと
も−SH基が保護された誘導体は、−SH基と結合
能を有する少なくとも二価の任意の金属との金属
キレート化合物であることができる。このような
金属としては、Cd、Hg、Zn、Cu、Fe、Coなど
の如き重金属類及びCa、Mg、Siなどの如き金属
類を例示できる。二価金属が好ましい。
上記アミノ基（NH2 -）の保護基の例として
は、たとえばベンジルオキシカルボニル基〔以
下、Ｚと略示することがある〕、第三ブトキシカ
ルボニル基〔以下、Bocと略示することがある〕
などの如きウレタン型保護基やトリチル基〔以
下、Trtと略示することがある〕の如きアラルキ
ル型保護基を例示することができる。又、上記α
−カルボキシル基の（−COOH）の保護の例と
しては、たとえばベンジルエステル〔以下、
OBzlと略示することがある〕、第三ブチルエステ
ル〔以下、OButと略示することがある〕などを
例示することができる。更に、上記システイン単
位の側鎖−SH基の保護基としては、たとえばベ
ンジル基〔Bzl〕、低級アルコキシベンジル基例
えばｐ−メトキシベンジル基〔以下、MBzlと略
示することがある〕、トリチル基〔Trt〕などを
例示することができる。
本発明及び参考として示すグルタチオン三量体
及び少なくともその−SH基の保護された誘導体
は、それ自体公知のペプチド合成単位反応手段を
適宜に組み合わせて、たとえば、後記図式１〜４
に例示する如き合成経路により合成することがで
きる。
このような公知の合成単位反応手段としては、
例えば、ラセミ化を抑制するために、Ｎ−ヒドロ
キシサクシンイミド〔以下、HOSuと略記するこ
とがある〕や１−ヒドロキシベンゾトリアゾール
〔以下、HOBtと略記することがある〕の如き試
薬を添加するジシクロヘキシルカルボイミド法
（DCC法）や水溶性カルボジイミド法（EDC法）
またＮ−ヒドロキシサクシンイミドエステル〔以
下、−OSuと略記することがある〕などの活性エ
ステルの如き公知方法を利用したカツプリング反
応；例えば、ベンジルアルコールやイソブチレン
の如き化合物を用いて前記例示の如きカルボキシ
基の（−COOH）を保護してベンジルエステル
や第三ブチルエステルに転化するカルボキシル保
護基導入反応、上記アミノ基の（NH2−）の保
護のため、例えば、塩化ベンジルオキシカルボニ
ル〔以下、Ｚ−Clと略記することがある〕や２−
第三ブトキシカルボニルオキシイミノ−２−フエ
ニルアセトニトリル〔以下、Boc−ONと略記す
ることがある〕の如き化合物を用いて前記例示の
如きアミノ基の（NH2−）を保護してＺあるい
はBoc基を導入するアミノ保護基導入反応、上記
側鎖−SH基の保護のため、例えば臭化ベンジル、
ｐ−メトキシベンジルアルコールやトリフエニル
カルビノールの如き化合物を用いてチオベンジル
基、チオｐ−メトキシベンジル基、チオトリチル
基に転化するメルカプト保護基導入反応などの如
き反応目的の官能基以外の官能基部位を保護する
保護基導入反応；上記保護基導入反応により反応
目的の官能基以外の官能基部位を保護したのち、
前記例示の如きカツプリング反応を行い、次いで
保護基を除去する保護基除去反応、例えば、
1NHCl／酢酸（AcOH）、トリフルオロ酢酸
（TFA）、HCl／有機溶媒、HFなどの各種の酸や
液体アンモニア中金属Naによる還元又はH2／Pd
の如き接触還元手段による保護基除去反応などの
保基除去反応；及び金属キレート形成反応；など
の反応手段を例示できる。
上記例示の如き合成単位反応手段及び各単位手
段における反応条件は当業者によく知られてお
り、本発明新規化合物の合成について後記図式１
〜４に例示する合成経路の単位反応において利用
できる。
以下、本発明及び参考として示す新規化合物の
合成方法の数例について図式を用いて更に詳しく
説明する。
以下の図式に於て、各略号の意味は以下のとお
りである（既にのべた略号も一緒に示してある。
The present invention relates to a trimer of γ-L-glutamyl L-cysteinylglycine (also known as glutathione; hereinafter referred to as glutathione), which has not been described in any known literature. The present invention relates to a glutathione trimer that has a binding ability and is useful and attracts attention for its use in a wide range of medical and pharmaceutical fields as a substance involved in controlling the toxicity of heavy metals in vivo and controlling the transfer of metals in vivo. More specifically, the present invention relates to a glutathione trimer (a glutathione trimer in which the -SH group of the cysteine residue in the glutathione unit is in a free state) which has not been previously described in any known literature, and at least derivatives with protected -SH groups thereof. . Furthermore, the present invention also relates to a method for producing the glutathione trimer and the derivatives using techniques known per se. Glutathione is a known substance and consists of an L-glutamic acid unit, an L-cysteine unit, and a glycine unit as shown in the following formula (hereinafter, amino acids other than glycine are in the L-form):
There are three positions where the glutathione unit can be polymerized by a peptide bond, as indicated by underlining in the formula below. Therefore, there are five types of positional isomers of the trimer, and the present invention relates to the glutathione trimer represented by Tri-GSH-I described below among these five positional isomers. The remaining four positional isomers will also be explained below for reference. Glutathione:- Glutathione is a known substance that is widely distributed, for example, in yeast, animal liver, muscle, etc., and is known to play an important role in in vivo redox. It is also known that it is a substance that has the ability to bind to metals. A dimer bonded by an -S-S- bond, which is an oxidized form of glutathione, has been known. However, glutathione oligomers bound by peptide bonds have not been known at all. The present inventors have conducted research on glutathione and its derivatives. As a result, it was discovered that a trimer of glutathione bound by a peptide bond and a derivative thereof with at least the -SH group protected can exist and can be synthesized by combining reaction techniques known per se. Furthermore, the obtained glutathione trimer is a compound that has not been previously described in any known literature, and has extremely superior metal binding ability compared to glutathione monomer, and also has a strong ability to reduce the toxicity of heavy metals to cells. I discovered that. Furthermore, due to the above-mentioned properties, copper and zinc are closely involved in physiologically active substances such as hormones and enzymes that play an important role in preventing in vivo toxicity caused by heavy metals and maintaining the functions of living cells. ,iron,
The novel compound of the present invention is useful in a wide range of medical and pharmaceutical fields as a substance that is involved in controlling the transfer of metals such as cobalt, nickel, and silicon. We found that this compound is interesting and noteworthy because it has structural similarity to the metal-binding site of the metal-binding protein "metallothionein," which is known as a substance that binds metals and is widely present in the biological world. Therefore, an object of the present invention is to provide a novel glutathione trimer and its derivatives. Another object of the present invention is to provide a method for preparing the above-mentioned compounds. The above objects and many other objects and advantages of the present invention will become more apparent from the following description. Glutathione trimers consist of a combination that forms a peptide bond between the carboxyl group of a glutamic acid residue in a glutathione unit and/or the carboxyl group of a glycine residue and the amino group of a glutamic acid residue in another glutathione unit. Accordingly, it includes five types of glutathione trimers (hereinafter sometimes abbreviated as TriGSH). In order to display the molecular structures of these five types of glutathione trimers, the binding mode of glutathione units is defined as follows. The state in which the amino group of the glutathione unit at the second position is connected to the α-carboxyl group of the glutamic acid residue through a peptide bond, with the glutathione unit in which the amino group is in a free state as the base, is the α-carboxylic group of the glycine residue. The state in which the third-position glutathione unit is bonded to the α-carboxyl group of the glutamic acid residue and the carboxyl group of the glycine residue in the second-position glutathione unit is α-, respectively. '-, ω'-, and the structures and names of five types of TriGSH are presented below. (1) TriGSH-(α,ω-glutathionyldiglutathione) [this invention]:- However, in the formula, -Glu- or -Glu- is a glutamic acid residue excluding the H of -NH 2 and the OH of -COOH. -Cys- represents the cysteic acid group. and -Gly- indicates -HN- CH2 -CO- among glycine residues, and the solid line between the amino acid abbreviations in this compound indicates that each amino acid is connected by a peptide bond. The solid lines above and below Glu or Glu indicate a carbonyl group obtained by removing OH from a γ-carboxyl group, and the horizontal solid line to the right of Glu- indicates a carbonyl group obtained by removing OH from an α-carboxyl group. In addition, when a cysteine residue is protected with an -SH protecting group, the abbreviation of the protecting group is shown in parentheses, and when -SH is free, it is indicated with (H) attached. (2) TriGSH− (ω-glutathionyl-ω′-glutathionylglutathione) [Reference]: − However, in the formula, -Glu- or -Glu-, -Cys(H)-
and -Gly- have the same meaning as above. (3) TriGSH− (ω-glutathionyl α′-glutathionylglutathione) [Reference]: − However, in the formula, -Glu- or -Glu-, -Cys(H)-
and -Gly- have the same meaning as above. (4) TriGSH− (α-glutathionyl-ω′-glutathionylglutathione) [Reference]: − However, in the formula, -Glu- or -Glu-, -Cys(H)-
and -Gly- have the same meaning as above. (5) TriGSH− (α-glutathionyl-α′-glutathionylglutathione) [Reference]: − However, in the formula, -Glu- or -Glu-, -Cys(H)-
and -Gly- have the same meaning as above. The present invention also includes protected derivatives of at least the -SH group of the glutathione trimer, such as metal chelate compounds or -SH groups of the trimer.
Mention may be made of compounds protected with SH protecting groups. The compound protected with the -SH protecting group has the formula
In (1) to (5), -Gly-OH represents the glycine unit carboxyl group (-COOH), Glu-OH represents the glutamic acid unit α-carboxyl group (-
COOH) and the glutamic acid unit amino group ( NH2- ) represented by H-Glu- may also be protected. Furthermore, the derivative in which at least the -SH group is protected in the form of a metal chelate compound can be a metal chelate compound with any divalent metal capable of binding to the -SH group. Examples of such metals include heavy metals such as Cd, Hg, Zn, Cu, Fe, and Co, and metals such as Ca, Mg, and Si. Divalent metals are preferred. Examples of protecting groups for the above amino group (NH 2 - ) include benzyloxycarbonyl group [hereinafter sometimes abbreviated as Z], tert-butoxycarbonyl group [hereinafter sometimes abbreviated as Boc] ]
Examples thereof include urethane-type protecting groups such as and aralkyl-type protecting groups such as trityl group (hereinafter sometimes abbreviated as Trt). Also, the above α
- As an example of protection of (-COOH) of carboxyl group, for example, benzyl ester [hereinafter referred to as
OBzl], tertiary butyl ester (hereinafter sometimes abbreviated as OBu t ), and the like. Furthermore, as the protecting group for the side chain -SH group of the cysteine unit, for example, a benzyl group [Bzl], a lower alkoxybenzyl group such as a p-methoxybenzyl group [hereinafter sometimes abbreviated as MBzl], a trityl group [ Trt], etc. The glutathione trimer and at least its −SH group-protected derivative shown in the present invention and as a reference can be prepared by appropriately combining known peptide synthesis unit reaction means, for example, in the following schemes 1 to 4.
It can be synthesized by a synthetic route as exemplified in . Such known synthetic unit reaction means include:
For example, dicyclohexyl to which reagents such as N-hydroxysuccinimide (hereinafter sometimes abbreviated as HOSu) and 1-hydroxybenzotriazole (hereinafter sometimes abbreviated as HOBt) are added to suppress racemization. Carboimide method (DCC method) and water-soluble carbodiimide method (EDC method)
In addition, coupling reactions using known methods such as active esters such as N-hydroxysuccinimide ester (hereinafter sometimes abbreviated as -OSu); for example, coupling reactions using compounds such as benzyl alcohol and isobutylene to For example, benzyloxycarbonyl chloride [ hereinafter referred to as Z -Cl] or 2-
Z or Boc is obtained by protecting (NH 2 -) of the amino group as exemplified above using a compound such as tert-butoxycarbonyloxyimino-2-phenylacetonitrile (hereinafter sometimes abbreviated as Boc-ON). For example, benzyl bromide,
A functional group site other than the functional group targeted for reaction, such as a mercapto protecting group introduction reaction in which a compound such as p-methoxybenzyl alcohol or triphenyl carbinol is used to convert a thiobenzyl group, thio p-methoxybenzyl group, or thiotrityl group. Protecting group introduction reaction: After protecting the functional group site other than the functional group targeted for reaction by the above protecting group introduction reaction,
A protecting group removal reaction in which a coupling reaction as exemplified above is performed and then a protecting group is removed, for example,
Reduction with various acids such as 1NHCl/acetic acid (AcOH), trifluoroacetic acid (TFA), HCl/organic solvent, HF, metal Na in liquid ammonia, or H 2 /Pd
Examples of reaction means include a protective group removal reaction such as a protecting group removal reaction using a catalytic reduction method; and a metal chelate formation reaction. The synthetic unit reaction means and the reaction conditions in each unit means as illustrated above are well known to those skilled in the art, and the synthesis of the novel compound of the present invention is described in Scheme 1 below.
It can be used in the unit reactions of the synthetic routes exemplified in ~4. Hereinafter, the present invention and several examples of methods for synthesizing new compounds shown as reference will be explained in more detail using diagrams. In the diagram below, the meaning of each abbreviation is as follows (already mentioned abbreviations are also shown).
【表】【table】
【表】
上述のようにして得ることのできる従来公知文
献未記載の前記式(1)〜式(5)で表わされる本発明及
び参考として示すグルタチオン三量体は、極めて
優れた金属結合能を有する。更に又、重金属類の
毒性に対する優れた毒性低減能を示す。そのテス
トの一例を以下に示す。
〔Ａ〕 グルタチオン三量体の抗Cd毒性作用試
験：−重金属カドミウムに対する抗毒性作用試
験を以下の方法で行つた。
培養３日目のVero細胞（アフリカミドリザ
ルの腎臓由来の株化細胞）に種々の濃度のグル
タチオン三量体溶液を加える。続いて、塩化カ
ドミウム溶液を最終濃度5ppmになるように添
加してさらに培養を継続し、１日後に生残細胞
数を測定し、その結果に基づいて用量−反応曲
線を描き、グルタチオン三量体の抗Cd毒性作
用のED50値を求め、グルタチオンのED50値と
比較した。
その結果を下掲表〔Ａ〕に示した。
表〔Ａ〕（抗Cd毒性作用）
化合物 ED50（mμmole／ml）
式(1)TriGSH− 40.7
式(2)TriGSH− 20.3
式(3)TriGSH− 32.7
式(4)TriGSH− 28.2
式(5)TriGSH− 28.2
グルタチオン（比較） 164.6
上掲表〔Ａ〕の結果に明らかなように、本発
明及び参考として示す化合物の式(1)〜(5)グルタ
チオン三量体はいずれも、グルタチオンに比し
て顕著に高い抗Cd毒性作用を示し、最も高い
式(2)化合物に於てはグルタチオンの約８倍、最
も低い式(5)化合物に於てもグルタチオン約４倍
という、全く予想外の且つ驚くべき高活性の抗
Cd毒性作用を示すことがわかる。
〔Ｂ〕 グルタチオン三量体のCd捕捉作用試
験：−重金属カドミウムに対するグルタチオン
三量体のCd捕捉作用試験をA.Yoshidaらの方
法〔Proc.Natl.Acad.Sci.U.S.A.，76 486〜490
（1979）〕に準じて行つた。解離定数が小さいほ
ど捕捉作用が大であることを意味する。結果を
下掲表〔Ｂ〕に示した。
表〔Ｂ〕
化合物 Cdの解離定数（μM）
式(1)TriGSH− 11.3
式(2)TriGSH− 1.23
式(3)TriGSH− 2.21
式(4)TriGSH− 1.91
式(5)TriGSH− 1.85
グルタチオン（比較） 190.9
上掲表〔Ｂ〕の結果に明らかなとおり、本発
明及び参考として示す化合物の式(1)〜(5)グルタ
チオン三量体はCdの解離定数で示したCdの捕
捉作用がグルタチオンに比して、全く予想外且
つ驚くべき強さであることがわかるる。
これら表〔Ａ〕及び表〔Ｂ〕に示した結果か
ら、本発明及び参考として示す化合物は重金属類
の生体内毒素発現の防除に、極めて高い且つ有用
な性質を示す新規化合物であることが理解され
る。
以下、実施例により本発明及び参考として示す
グルタチオン三量体及び少なくともその−SH基
の保護された誘導体及びその製造について更に具
体的に例示する。尚、以下の例において、グルタ
チオン三量体（グルタチオン単位におけるシステ
イン残基の−SH基が遊離状態である化合物）は、
比較的不安定であつて、融点を測定することは物
理化学恒数の特定として無意味であるため、他の
物性でその確認データーを示した。又、本発明及
び参考として示すグルタチオン三量体の前記式(1)
〜(5)で示した構造は、その合成経路及び下記の手
段を利用して確認された。
実施例１ 式(1)TriGSH−及びその−SH基の
保護された誘導体の合成。〔図式１の合成経路〕
Ｓ−ｐ−メトキシベンジルグルタチオン〔GS
（MBzl）〕，１−(1)の調整。グルタチオン〔興人
（株）販売〕4.54ｇ（14.8ｍmol），1NHCl／
AcOH20ml、及びｐ−メトキシベンジルアルコー
ル〔井上香料（株）販売〕2.2ml（17.8ｍmol）の
混合懸濁液を、65／70℃で30分加熱すると澄明な
溶液に変る。室温に冷却し減圧下に蒸発分を溜去
して黄色残渣を得る。該残渣のエーテル洗滌を３
度繰返したのちKOH上で減圧乾燥して半固形物
を得る。この半固形物を温2NAcOH（200ml）に
とかし、Dowexl（AcO-型、30ml）を加え加温下
に10分撹拌し、Dowexlを別する。液を減圧
濃縮して結晶を得る。この結晶を200mlのH2Oに
懸濁溶解、濃縮し、氷室放置して再結晶せしめ
GS（MBzl）4.43ｇ（収率70％）を得る。その物
性値は以下のとおりであつた。
融点 ：192〜193℃（分解）
〔α〕20 D：−20.4℃（C0.5、1NNaOH）
Rf2 ：0.17
元素分析 ：C18H25O7N3Sとして
Ｃ Ｈ Ｎ
（計算値）50.58 5.90 9.83％
（実測値）50.63 5.91 9.59％
N〓−Ｚ−Ｓ−ｐ−メトキシベンジルグルタチ
オン〔Ｚ・GS（MBzl）〕、１−(2)の調製。
１−(1)で得たGS（MBzl）427mg（１ｍmol）に
０℃に於て1NNaOH、２ml及び0.2mlのエチルエ
ーテルを加え澄明溶液とし、はげしく撹拌しなが
ら1NNaOH1.2mlと塩化ベンジルオキシカルボニ
ル（Ｚ−Cl）0.17ml（1.2ｍmol）とを０℃に於て
徐々に加え（30分を要す）、更に室温下で２時間
撹拌を続ける。
水層をエチルエーテルで抽出し、抽出残分（水
層）を濃HClにて酸性化すると油状物が析出す
る。この油状物を酢酸エチル40mlで２回抽出し酢
酸エチル層を水洗し、芒硝にて乾燥する。乾燥酢
酸エチル層を減圧下に溜去し油状残留物を得る。
この油状物をエチルエーテルから結晶化して、Ｚ
−GS（MBzl）516mg（収率92％）を得る。その物
性値は以下のとおりであつた。
融点 ：117−118℃
〔α〕20 D：−36.4゜（C1、MeOH）
R2 f ：0.70、R1 f：0.16、R3 f：
0.45
元素分析 ：C26H31O9N3S・1/2
H2Oとして
Ｃ Ｈ Ｎ
（計算値）54.73 5.65 7.36％
（実測値）54.90 5.56 7.27％
Ｓ−ｐ−メトキシベンジルグルタチオン・ジベ
ンジルエステル〔GS（MBzl）−（OBzl）2〕１−(3)
の調製。
Dean−Stark装置を用い１−(1)で得たGS
（MBzl）855mg（２ｍmol）、ｐ−トルエンスルホ
ン酸、１水和物457mg（2.4ｍmol）、ベンジルア
ルコール２ml及びベンゼン４mlを加えて澄明粘稠
な溶液となし、油浴上110℃で６時間還流を行う。
冷却後、反応液を40mlのエチルエーテル中に注
ぎ、生じた油状物質をエチルエーテル洗滌する。
この油状物質を40mlの酢酸エチルに溶解し、
0.5M・NaHCO3及びH2Oにて洗滌し、酢酸エチ
ル層をNa2SO4上で乾燥し、酢酸エチルを減圧下
に溜去して油状のGS（MBzl）−（OBzl）21.06ｇ
（収率87％）を得た。この物質の結晶化は出来な
かつた。
物性値R1 f：0.70、R2 f：0.66、R3 f：0.83−SH基
の保護された式(1)TriGSH−、１−(4)の調製。
１−(2)で得たＺ−GS（MBzl）281mg（0.5ｍ
mol）、１−(3)で得たGS（MBzl）−（OBzl）2730mg
（1.2mmol）、１−ヒドロキシベンゾトリアゾール
（HOBt）〔（株）ペプチド研究所販売〕230mg
（1.7ｍmol）を０℃で５mlのDMFに加える。次い
で１−エチル−３−（３−ジメチルアミノプロピ
ル）−カルボジイミド塩酸（EDC・HCl）〔和光純
薬工業（株）販売〕326mg（1.7ｍmol）加え、０
℃で１時間、室温下２日間撹拌を続ける。反応物
を減圧乾固し、40mlの0.5MNaHCO3を加えると
半固形状物質を得る。この半固形状物質を水洗、
0.5Mクエン酸洗滌及び水洗を行つた後、２mlの
DMFに溶解し40mlの酢酸エチルを加えるとゲル
状沈殿を得る。このゲル状沈殿を別、冷酢酸エ
チル洗滌し乾燥し、704mg（収率81％）のSH基の
保護された式(1)TriGSH−（Protected
TriGSH−）、（１−４）を得る。その物性値は
以下のとおりであつた。
融点 ：183〜184℃
〔α〕20 D：−26.4゜（C1、DMF）
R1 f ：0.82
元素分析 ：C90H101O21NaS3
として
Ｃ Ｈ Ｎ
（計算値）62.09 5.85 7.42％
（実測値）62.00 5.86 7.33％
得られたProtected TriGSH−は下記式で示す
ことができる。
式(1)TriGSH−、１−(5)の調製。
１−(4)で得たProtected TriGSH−348mg
（0.2mmol）にアニソール0.35ml（3.2ｍmol）を
加える。次いで、HF5mlを加えて０℃で1.5時間
撹拌反応する。反応液を最初アスピレータにて０
℃減圧濃縮し、次いで、真空ポンプを用いて室温
下で18時間減圧乾固する。分液斗中窒素飽和脱
イオン水15ml、エチルエーテル10mlで得られた乾
固物からアニソールを抽出除去し、水層を
Dowex1×８（AcO-型、1.8×３cm）のカラムを
通過せしめ、次いで、カラムを20mlの2NAcOH
（N2飽和）、６mlの25％AcOH（N2飽和）、10mlの
50％AcOH（N2飽和）溶液で須次溶出、その全溶
出液を合せて直ちに凍結乾燥し136mg（収率77％）
の式(1)TriGSH−を得る。その物性値は以下の
とおりであつた。
R2 f：0.03、R3 f：0.03、R5 f：0.15
R2 f（ppc）：0.02R5 f（ppc）：0.70
ニンヒドリン反応：（−NH2）
ニトロプルシド反応：（−SH）
参考例１ 式(2)TriGSH−、式(3)TriGSH−
、式(4)TriGSH−、式(5)TriGSH−及び
それらの−SH基の保護された誘導体の合成。
〔図式４のステツプワイズ合成経路〕
グルタミン酸−γ−ベンジル−α−第三ブチル
エステル[Table] The glutathione trimers of the present invention and shown as reference, which have not been described in any prior known literature and which can be obtained as described above, have extremely excellent metal binding ability. have Furthermore, it shows an excellent ability to reduce the toxicity of heavy metals. An example of the test is shown below. [A] Anti-Cd toxicity test of glutathione trimer: - An anti-Cd toxicity test against the heavy metal cadmium was conducted in the following manner. Glutathione trimer solutions at various concentrations are added to Vero cells (an established cell line derived from African green monkey kidney) on the third day of culture. Next, a cadmium chloride solution was added to a final concentration of 5 ppm, and the culture was continued. After one day, the number of surviving cells was measured. Based on the results, a dose-response curve was drawn, and glutathione trimer The ED 50 value of anti-Cd toxic effect was determined and compared with the ED 50 value of glutathione. The results are shown in Table [A] below. Table [A] (Anti-Cd toxic effect) Compound ED 50 (mμmole/ml) Formula (1) TriGSH- 40.7 Formula (2) TriGSH- 20.3 Formula (3) TriGSH- 32.7 Formula (4) TriGSH- 28.2 Formula (5) TriGSH- 28.2 Glutathione (comparison) 164.6 As is clear from the results in Table [A] above, the glutathione trimers of formulas (1) to (5) of the compounds shown in the present invention and as a reference all have a higher concentration than glutathione. It shows a significantly high anti-Cd toxic effect, with the highest compound of formula (2) being about 8 times that of glutathione, and the lowest compound of formula (5) being about 4 times that of glutathione, which is completely unexpected and surprising. Highly active anti-
It can be seen that Cd exhibits a toxic effect. [B] Cd scavenging effect test of glutathione trimer: - Cd scavenging effect test of glutathione trimer against heavy metal cadmium was conducted using the method of A. Yoshida et al. [Proc. Natl. Acad. Sci. USA, 76 486-490
(1979)]. The smaller the dissociation constant, the greater the trapping effect. The results are shown in the table [B] below. Table [B] Dissociation constant of compound Cd (μM) Formula (1) TriGSH− 11.3 Formula (2) TriGSH− 1.23 Formula (3) TriGSH− 2.21 Formula (4) TriGSH− 1.91 Formula (5) TriGSH− 1.85 Glutathione (comparison) ) 190.9 As is clear from the results in Table [B] above, the glutathione trimers of the formulas (1) to (5) of the compounds shown in the present invention and as a reference have a Cd-trapping effect expressed by the Cd dissociation constant compared to that of glutathione. It turns out that it has a completely unexpected and surprising strength. From the results shown in Tables [A] and [B], it is understood that the compounds of the present invention and those shown as references are novel compounds that exhibit extremely high and useful properties for controlling the expression of toxins caused by heavy metals in living organisms. be done. EXAMPLES Hereinafter, the present invention and the glutathione trimer, at least its -SH group-protected derivative, and the production thereof will be illustrated in more detail with reference to Examples. In the following examples, the glutathione trimer (a compound in which the -SH group of the cysteine residue in the glutathione unit is in a free state) is
Because it is relatively unstable, measuring the melting point is meaningless in identifying physicochemical constants, so we showed confirmation data using other physical properties. In addition, the above formula (1) of the glutathione trimer shown in the present invention and as a reference
The structures shown in ~(5) were confirmed using the synthetic route and the following means. Example 1 Synthesis of formula (1) TriGSH- and its -SH group protected derivative. [Synthetic route of Scheme 1] S-p-methoxybenzylglutathione [GS
(MBzl)], 1-(1) adjustment. Glutathione [sold by Kojin Co., Ltd.] 4.54g (14.8mmol), 1NHCl/
When a mixed suspension of 20 ml of AcOH and 2.2 ml (17.8 mmol) of p-methoxybenzyl alcohol (sold by Inoue Perfume Co., Ltd.) is heated at 65/70°C for 30 minutes, it turns into a clear solution. The mixture was cooled to room temperature and the evaporated components were distilled off under reduced pressure to obtain a yellow residue. The residue was washed with ether for 3
After repeated drying over KOH under reduced pressure, a semi-solid product was obtained. Dissolve this semi-solid in warm 2NAcOH (200 ml), add Dowexl (AcO - form, 30 ml), stir for 10 minutes while warming, and separate Dowexl. Concentrate the liquid under reduced pressure to obtain crystals. The crystals were suspended and dissolved in 200 ml of H 2 O, concentrated, and left in an ice room to recrystallize.
4.43 g (yield 70%) of GS (MBzl) is obtained. Its physical property values were as follows. Melting point: 192-193℃ (decomposition) [α] 20 D : -20.4℃ (C0.5, 1NNaOH) Rf 2 : 0.17 Elemental analysis: C 18 H 25 O 7 N 3 S as C H N (calculated value) 50.58 5.90 9.83% (Actual value) 50.63 5.91 9.59% Preparation of N〓-Z-S-p-methoxybenzylglutathione [Z·GS(MBzl)], 1-(2). 1-(1) To 427 mg (1 mmol) of GS (MBzl) obtained in 1-(1) were added 1 N NaOH, 2 ml, and 0.2 ml of ethyl ether at 0°C to make a clear solution, and with vigorous stirring, 1.2 ml of 1 N NaOH and benzyloxycarbonyl chloride ( 0.17 ml (1.2 mmol) of Z-Cl) was gradually added at 0° C. (30 minutes were required), and stirring was continued for 2 hours at room temperature. The aqueous layer is extracted with ethyl ether, and the extracted residue (aqueous layer) is acidified with concentrated HCl to precipitate an oil. This oil was extracted twice with 40 ml of ethyl acetate, and the ethyl acetate layer was washed with water and dried over Glauber's salt. The dried ethyl acetate layer was distilled off under reduced pressure to obtain an oily residue.
This oil was crystallized from ethyl ether to give Z
- Obtain 516 mg (92% yield) of GS (MBzl). Its physical property values were as follows. Melting point: 117-118℃ [α] 20 D : -36.4゜ (C1, MeOH) R 2 f : 0.70, R 1 f : 0.16, R 3 f :
0.45 Elemental analysis: C 26 H 31 O 9 N 3 S・1/2
As H 2 O C H N (calculated value) 54.73 5.65 7.36% (actual value) 54.90 5.56 7.27% S-p-methoxybenzylglutathione dibenzyl ester [GS(MBzl)-(OBzl) 2 ] 1-(3)
Preparation of. GS obtained in 1-(1) using Dean-Stark apparatus
(MBzl) 855 mg (2 mmol), p-toluenesulfonic acid, monohydrate 457 mg (2.4 mmol), benzyl alcohol 2 ml and benzene 4 ml to make a clear viscous solution, which was refluxed on an oil bath at 110°C for 6 hours. I do.
After cooling, the reaction solution is poured into 40 ml of ethyl ether and the resulting oil is washed with ethyl ether.
Dissolve this oil in 40 ml of ethyl acetate and
Washing with 0.5M NaHCO 3 and H 2 O, drying the ethyl acetate layer over Na 2 SO 4 and distilling off the ethyl acetate under reduced pressure gave 1.06 g of oily GS(MBzl)-(OBzl) 2
(yield 87%). It was not possible to crystallize this substance. Physical properties: R 1 f : 0.70, R 2 f : 0.66, R 3 f : 0.83 - Preparation of formula (1) TriGSH-, 1-(4) with protected SH group. Z-GS (MBzl) obtained in 1-(2) 281mg (0.5m
mol), GS obtained in 1-(3) (MBzl) - (OBzl) 2 730mg
(1.2 mmol), 1-hydroxybenzotriazole (HOBt) [Sold by Peptide Institute Co., Ltd.] 230 mg
(1.7 mmol) in 5 ml DMF at 0°C. Next, 326 mg (1.7 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloric acid (EDC/HCl) [sold by Wako Pure Chemical Industries, Ltd.] was added, and 0
Stirring was continued for 1 hour at °C and for 2 days at room temperature. The reaction is dried under vacuum and 40 ml of 0.5M NaHCO 3 is added to obtain a semi-solid material. Wash this semi-solid substance with water,
After washing with 0.5M citric acid and water, 2ml of
Dissolve in DMF and add 40 ml of ethyl acetate to obtain a gel-like precipitate. This gel-like precipitate was separated, washed with cold ethyl acetate and dried, and 704 mg (yield 81%) of formula (1) TriGSH−
TriGSH-), (1-4) is obtained. Its physical property values were as follows. Melting point: 183-184℃ [α] 20 D : −26.4゜ (C1, DMF) R 1 f : 0.82 Elemental analysis: C 90 H 101 O 21 NaS 3
As C H N (calculated value) 62.09 5.85 7.42% (actual value) 62.00 5.86 7.33% The obtained Protected TriGSH- can be expressed by the following formula. Preparation of formula (1) TriGSH-, 1-(5). Protected TriGSH obtained in 1-(4) - 348mg
(0.2 mmol) and add 0.35 ml (3.2 mmol) of anisole. Next, 5 ml of HF was added and the reaction was stirred at 0°C for 1.5 hours. First, the reaction solution is aspirated to 0.
℃ and then concentrated to dryness under reduced pressure at room temperature using a vacuum pump for 18 hours. Anisole was extracted and removed from the dried product obtained with 15 ml of nitrogen-saturated deionized water and 10 ml of ethyl ether in a separating funnel, and the aqueous layer was removed.
Pass through a column of Dowex 1 x 8 (AcO - type, 1.8 x 3 cm), then replace the column with 20 ml of 2NAcOH
( N2 saturated), 6 ml 25% AcOH ( N2 saturated), 10 ml
Suji elution was carried out with 50% AcOH (saturated with N2 ) solution, and all the eluates were combined and immediately lyophilized to yield 136 mg (77% yield).
Equation (1) TriGSH− is obtained. Its physical property values were as follows. R 2 f : 0.03, R 3 f : 0.03, R 5 f : 0.15 R 2 f (ppc): 0.02R 5 f (ppc): 0.70 Ninhydrin reaction: (-NH 2 ) Nitroprusside reaction: (-SH) Reference example 1 Formula (2) TriGSH−, Formula (3) TriGSH−
, formula (4) TriGSH-, formula (5) TriGSH- and their -SH group protected derivatives synthesis.
[Stepwise synthesis route of Scheme 4] Glutamic acid-γ-benzyl-α-tert-butyl ester
【式】４−(1)の調製。
グルタミン酸γ−ベンジルエステル〔（株）ペ
プチド研究所販売〕25ｇをジオキサン100mlに懸
濁せしめ、濃硫酸10mlを加え−10℃〜−15℃の氷
塩浴上で冷却下にイソブチレンガスを吹き込み、
液量が100ml増量を確認した後、密閉容器中、室
温で18時間撹拌反応せしめる。未反応のイソブチ
レンを気化除去後、NaHCO3 60ｇを加えて中和
し溶媒等を減圧溜去して得られた残留状物を酢酸
エチルにて抽出、水洗乾燥後、酢酸エチルを留去
して20ｇの油状物としてPreparation of Formula 4-(1). Suspend 25 g of glutamic acid γ-benzyl ester [sold by Peptide Institute Co., Ltd.] in 100 ml of dioxane, add 10 ml of concentrated sulfuric acid, cool on an ice-salt bath at -10°C to -15°C, and blow isobutylene gas into it.
After confirming that the liquid volume has increased by 100 ml, stir the reaction in a closed container at room temperature for 18 hours. After removing unreacted isobutylene by vaporization, neutralize by adding 60 g of NaHCO 3 and distilling off the solvent etc. under reduced pressure. As 20g of oil
【式】を得
る。Rf1：0.83であつた。
グルタミン酸−α−第三ブチルエステル
Obtain [formula]. Rf 1 : 0.83. Glutamic acid-α-tert-butyl ester
【式】４−(2)の調製。 ５−(1)で得た油状のPreparation of Formula 4-(2). The oil obtained in 5-(1)
【式】15ｇ
（50ｍmol）を50％エタノール50mlに溶解し水素
気流中でPd黒を用いて24時間接触水素還元した。
Pdを去し、溶媒を留去後、酢酸エチルを加え
て結晶化し、融点143〜144℃の結晶
[Formula] 15g (50mmol) was dissolved in 50ml of 50% ethanol and subjected to catalytic hydrogen reduction using Pd black in a hydrogen stream for 24 hours.
After removing Pd and distilling off the solvent, ethyl acetate was added to crystallize the crystals with a melting point of 143-144℃.
【式】7.22ｇ（収率70％）を得た。
文献値融点143〜144℃ R.Roeske、J.Org.
Chem.，28 1251（1963）。
N〓−第三ブトキシカルボニルグルタミン酸−
α−第三ブチルエステル
[Formula] 7.22g (yield 70%) was obtained. Literature value Melting point 143-144℃ R.Roeske, J.Org.
Chem., 28 1251 (1963). N〓-tert-butoxycarbonylglutamic acid-
α-Tertiary butyl ester
【式】４−(3)の調製。M.Itoh
et al，Tetrahedron Lett.1975，4393（1975）
の方法に準じて合成する。
前記−(2)で得たPreparation of Formula 4-(3). M.Itoh et al, Tetrahedron Lett.1975, 4393 (1975)
Synthesize according to the method. Obtained in -(2) above
【式】7.11ｇ（35
ｍmol）をジオキサン−水（１：１、ｖ／ｖ）70
mlに懸濁しEt3N4.9ml及びBoc−ON8.75ｇを加え
て室温で１夜撹拌した。ジオキサンを留去しエチ
ルエーテルと振とう後、水層をクエン酸にて中
和。中和物を酢酸エチルにて抽出し、MgSO4で
乾燥後、溶媒を留去して7.51ｇ（71％）の油状物
として[Formula] 7.11g (35 mmol) of dioxane-water (1:1, v/v) 70
ml, 4.9 ml of Et 3 N and 8.75 g of Boc-ON were added, and the mixture was stirred at room temperature overnight. After distilling off dioxane and shaking with ethyl ether, the aqueous layer was neutralized with citric acid. The neutralized product was extracted with ethyl acetate, dried over MgSO4 , and the solvent was distilled off to obtain 7.51 g (71%) of an oil.
【式】を得た。R1 f：0.65で
あつた。この油状物の一部をDCHA塩とし結晶
化した。その融点151〜152℃で、〔α〕24 D：−13.8゜
（C1、MeOH）であつた。
元素分析 C26H48O6N2として
Ｃ Ｈ Ｎ
計算値 64.43 9.98 5.78％
実測値 64.03 9.84 5.74％
N〓−第三ブトキシカルボニル−Ｓ−ベンジル
システイン〔Boc−Cys（Bzl）−OH〕、４−(4)の
調製。
Ｓ−ベンジルシステイン12.7ｇ（60ｍmol）か
ら４−(3)の製法に準じて18ｇのBoc−Cys（Bzl）
−OHを得る。
R1 f：0.37であつた。
N〓−第三ブトキシカルボニル−Ｓ−ベンジル
システイニルグリシンベンジルエステル〔Boc−
Cys（Bzl）−Gly−OBzl〕、４−(5)の調製。
前記４−(4)で得たBoc−Cys（Bzl）−OH6.23ｇ
（20ｍmol）とＨ−Gly−OBzlのTosOH塩6.80ｇ
（20ｍmol）及びHOBt0.3ｇの40mlDMF溶液に、
冷却下2.8mlのEt3N、次いでDCC4.40ｇ（21ｍ
mol）を加え氷浴上５時間、室温で１夜反応せし
める。副生したDCUを去、DMFを留去後残留
物を酢酸エチルにとかし、10％クエン酸、４％
NaHCO3水で各３回振とう洗滌し、酢酸エチル
層を乾燥後、溶媒を減圧留去し、残渣に石油エー
テルを加えて結晶化し、8.30ｇ（収率90％）の
Boc−Cys（Bzl）−Gly−OBzlを得る。
融点 ：75〜76℃
R1 f ：0.85
〔α〕20 D：−23.2゜（C1、MeOH）
N〓−第三ブトキシカルボニル−Ｓ−ベンジル
システニイルグリシン〔Boc−Cys（Bzl）−Gly−
OH〕、４−(6)の調製。
前記４−(4)で得たBoc−Cys（Bzl）−OH6.23ｇ
（20mmol）とHOSu2.6ｇをジオキサン40mlに溶
かし、冷却下DCC4.50ｇを加え４℃で１夜反応す
る。DCUを去、ジオキサンを留去後残渣をエ
チルエーテル−石油エーテルで洗滌、減圧乾燥し
てBoc−Cys（Bzl）−OSu，４−（41）とする。こ
のBoc−Cys（Bzl）−OSuをDMFに溶解し、グリ
シン2.25ｇ（30ｍmol）、Et3N4.2mlを水20mlに溶
解した溶液を低温下（０〜４℃）で徐々に滴加し
一夜反応せしめる。反応物から溶媒を留去し酢酸
エチルで抽出し10％クエン酸、水で洗滌、乾燥後
溶媒を留去して油状のBoc−Cys（Bzl）−Gly−
OH 6.5ｇ（収率88％）を得る。
R1 f：0.66であつた。
N〓−第三ブトキシカルボニル−α−第三ブチ
ルエステル−γ−グルタミル−Ｓ−ベンジルシス
テイニルグリシンベンジルエステル
I got [formula]. R 1 f : 0.65. A portion of this oil was crystallized as a DCHA salt. Its melting point was 151-152°C, [α] 24 D : -13.8° (C1, MeOH). Elemental analysis C 26 H 48 O 6 N 2 as C H N Calculated value 64.43 9.98 5.78% Actual value 64.03 9.84 5.74% N〓-tert-butoxycarbonyl-S-benzylcysteine [Boc-Cys(Bzl)-OH], 4 - Preparation of (4). From 12.7 g (60 mmol) of S-benzylcysteine, 18 g of Boc-Cys (Bzl) was prepared according to the method of 4-(3).
−OH is obtained. R 1 f : 0.37. N〓-tert-butoxycarbonyl-S-benzylcysteinyl glycine benzyl ester [Boc-
Preparation of Cys(Bzl)-Gly-OBzl], 4-(5). 6.23 g of Boc-Cys(Bzl)-OH obtained in 4-(4) above
(20 mmol) and H-Gly-OBzl TosOH salt 6.80 g
(20 mmol) and 0.3 g of HOBt in 40 ml DMF solution,
2.8 ml of Et 3 N under cooling, then 4.40 g of DCC (21 m
mol) and allowed to react on an ice bath for 5 hours and at room temperature overnight. After removing the by-produced DCU and distilling off DMF, the residue was dissolved in ethyl acetate, 10% citric acid, 4%
After shaking and washing with NaHCO 3 water three times each, and drying the ethyl acetate layer, the solvent was distilled off under reduced pressure, and petroleum ether was added to the residue to crystallize.
Obtain Boc-Cys(Bzl)-Gly-OBzl. Melting point: 75-76℃ R 1 f : 0.85 [α] 20 D : -23.2゜ (C1, MeOH)
OH], preparation of 4-(6). 6.23 g of Boc-Cys(Bzl)-OH obtained in 4-(4) above
(20 mmol) and 2.6 g of HOSu were dissolved in 40 ml of dioxane, and while cooling, 4.50 g of DCC was added and reacted at 4°C overnight. After removing DCU and distilling off dioxane, the residue was washed with ethyl ether-petroleum ether and dried under reduced pressure to give Boc-Cys(Bzl)-OSu, 4-(41). This Boc-Cys(Bzl)-OSu was dissolved in DMF, and a solution of 2.25 g (30 mmol) of glycine and 4.2 ml of Et 3 N dissolved in 20 ml of water was gradually added dropwise at low temperature (0 to 4°C) overnight. Make it react. The solvent was distilled off from the reaction mixture, extracted with ethyl acetate, washed with 10% citric acid and water, and after drying, the solvent was distilled off to obtain an oily Boc-Cys(Bzl)-Gly-.
6.5 g of OH (88% yield) are obtained. R 1 f : 0.66. N〓-tert-butoxycarbonyl-α-tert-butyl ester-γ-glutamyl-S-benzylcysteinylglycine benzyl ester
【式】４−(7)の
調製。
１ ４−(5)で得たBoc−Cys（Bzl）−Gly−
OBzl6.88ｇ（15ｍmol）をTFA10mlに冷却溶
解30分後TFAを減圧留去し残留油状物に
HCl／ジオキサンを1.1当量加えHCl塩をエー
テルにて結晶化せしめる，４−（52）・HCl。
２ ４−(3)で得たBoc−Glu−OBut3.64ｇ（12ｍ
mol）及び１）の操作で得た結晶４−（52）
5.20ｇ（12ｍmol）をDMF20mlに溶解し、氷浴
上で冷却後Et3N1.7mlで中和し、HOBt0.5ｇ及
びDCC3.1ｇを加えて冷却下３時間室温で一夜
反応せしめる。生成したDCUを去、PMFを
減圧留去後残留分を酢酸エチルに溶解し、10％
クエン酸、４％NaHCO3、水で各３回振とう
洗滌し、酢酸エチル層を乾燥後、溶媒を留去し
残留油状物をエチルエーテル−石油エーテルで
結晶せしめ6.11ｇ（収率77％）の
Preparation of Formula 4-(7). 1 4-Boc-Cys(Bzl)-Gly- obtained in (5)
6.88g (15mmol) of OBzl was cooled and dissolved in 10ml of TFA for 30 minutes, and then the TFA was distilled off under reduced pressure to form a residual oil.
Add 1.1 equivalents of HCl/dioxane and crystallize the HCl salt from ether, 4-(52).HCl. 2 3.64g of Boc-Glu- OBut obtained in 4-(3) (12m
mol) and crystal 4-(52) obtained by operation 1)
5.20 g (12 mmol) was dissolved in 20 ml of DMF, cooled on an ice bath, neutralized with 1.7 ml of Et 3 N, added with 0.5 g of HOB t and 3.1 g of DCC, and reacted under cooling for 3 hours at room temperature overnight. After removing the generated DCU and distilling off the PMF under reduced pressure, the residue was dissolved in ethyl acetate and diluted with 10%
After shaking and washing three times each with citric acid, 4% NaHCO 3 and water, and drying the ethyl acetate layer, the solvent was distilled off and the remaining oil was crystallized from ethyl ether-petroleum ether, 6.11 g (yield 77%). of
【式】を得る。
融点 ：81〜82℃
R1 f ：0.48
〔α〕24 D：−39.2゜（C1、MeOH）
元素分析 C34H47O8N3S・1/2H2Oとして
Ｃ Ｈ Ｎ
計算値 61.24 7.26 6.30％
実測値 61.40 7.02 6.45％
N〓−第三ブトキシカルボニル−γ−グルタミ
ル−Ｓ−ベンジルシステイニルグリシンベンジル
エステルObtain [formula]. Melting point: 81-82℃ R 1 f : 0.48 [α] 24 D : -39.2゜ (C1, MeOH) Elemental analysis C 34 H 47 O 8 N 3 As S・1/2H 2 O C H N Calculated value 61.24 7.26 6.30% Actual value 61.40 7.02 6.45% N〓-tert-butoxycarbonyl-γ-glutamyl-S-benzylcysteinylglycine benzyl ester
【式】
４−(8)の調製。
５−(7)で得た結晶
Preparation of Formula 4-(8). 5-Crystal obtained in (7)
【式】5.92ｇ
（９ｍmol）を1NHCl／AcOH50mlに溶解し室温
に放置する。５時間後溶媒を留去し、エチルエー
テルを加え生じた沈殿を取乾燥する。乾燥物
4.4ｇ（８ｍmol）をジオキサン−水（１：１，
ｖ／ｖ）50mlに溶解しEt3N1.34ml及びBoc−ON
2.20ｇを加え室温で一夜撹拌反応する。副生物を
エーテル抽出により除去後、目的物を酢酸エチル
にて抽出し、水洗乾燥後酢酸エチルを留去して油
状の[Formula] 5.92g (9mmol) was dissolved in 1NHCl/AcOH50ml and left at room temperature. After 5 hours, the solvent was distilled off, ethyl ether was added, and the resulting precipitate was dried. dry matter
4.4g (8mmol) was mixed with dioxane-water (1:1,
v/v) 1.34 ml of Et 3 N and Boc-ON dissolved in 50 ml
Add 2.20g and react with stirring at room temperature overnight. After removing the by-products by ether extraction, the target product was extracted with ethyl acetate, washed with water, dried, and ethyl acetate was distilled off to obtain an oily product.
【式】3.6ｇ
（収率77％）を得た。
R1 f：0.72
上記油状物の一部をDCHA塩とし結晶化せし
める。
融点 ：72〜75℃
〔α〕20 D：−19.6゜（C1、MeOH）
元素分析 C29H37O8N3S・C12H23N・H2Oと
して
Ｃ Ｈ Ｎ
計算値 62.57 7.94 7.12％
実測値 62.32 8.05 7.18％
N〓−第三ブトキシカルボニル−α−第三ブチ
ルエステル−γ−グルタミル−Ｓ−ベンジルシス
テイニルグリシン
[Formula] 3.6g (yield 77%) was obtained. R 1 f : 0.72 Part of the above oil is converted into DCHA salt and crystallized. Melting point: 72-75℃ [α] 20 D : -19.6゜ (C1, MeOH) Elemental analysis C 29 H 37 O 8 N 3 S・C 12 H 23 N・H 2 O as C H N Calculated value 62.57 7.94 7.12 % Actual value 62.32 8.05 7.18% N〓-tert-butoxycarbonyl-α-tert-butyl ester-γ-glutamyl-S-benzylcysteinylglycine
【式】４−(9)の調
製。
１ ４−(6)で得たBoc−Cys（Bzl）−Gly・
OH6.00ｇ（15ｍmol）を氷冷下、TFA10mlに
とかし30分放置、TFAを留去しエチルエーテ
ルを加えて固定せしむ，４−（61）・TFA。
２ ４−(3)で得たPreparation of Formula 4-(9). 1 4-Boc−Cys(Bzl)−Gly・ obtained in (6)
Dissolve 6.00 g (15 mmol) of OH in 10 ml of TFA under ice cooling, leave for 30 minutes, distill off TFA, and fix by adding ethyl ether, 4-(61)・TFA. 2 Obtained from 4-(3)
【式】3.64ｇ
（12ｍmol）とHOSu1.45ｇ（12.6ｍmol）を
DMF20mlに溶解し、氷冷後DCC2.6ｇ（12）６
ｍmol）を加えて氷冷下６時間反応せしめる。
この反応物１）で得た固化物，４−（61）・
TFA 4.06ｇ（12ｍmol）とEt3N 3.5ml、
DMF20mlからなる溶液を徐々に添加し0゜〜４
℃で４時間、室温で１夜反応する。副生した
DCUを去し、DMFを留去し残渣を酢酸エチ
ルに溶解し、10％クエン酸、H2Oで各３回洗
滌し、酢酸エチル層を乾燥後濃縮した。生成油
状物をエチルエーテル−石油エーテルにて洗滌
乾燥し無定形の目的物
[Formula] 3.64g (12mmol) and HOSu1.45g (12.6mmol)
Dissolved in DMF20ml and cooled on ice, then DCC2.6g (12)6
mmol) and reacted for 6 hours under ice cooling.
The solidified product obtained from this reaction product 1), 4-(61)・
TFA 4.06g (12mmol) and Et 3 N 3.5ml,
Gradually add a solution consisting of 20 ml of DMF to
React for 4 hours at ℃ and overnight at room temperature. was a byproduct
DCU was removed, DMF was distilled off, and the residue was dissolved in ethyl acetate, washed three times each with 10% citric acid and H 2 O, and the ethyl acetate layer was dried and concentrated. The resulting oil is washed with ethyl ether-petroleum ether and dried to obtain an amorphous target product.
【式】4.71ｇ
（収率71％）を得る。
R1 f：0.61
上記無定形物120mgを酢酸エチルに溶解し
DCHA0.04mlを加え、減圧乾固し、エチルエー
テルにて結晶化する。
融点 ：148−149℃
〔α〕24 D：−37.6゜（C1、MeOH）
元素分析 C26H39O8N3S・C12H23N・1/2
H2Oとして
Ｃ Ｈ Ｎ
計算値 61.35 8.54 7.53％
実測値 61.46 8.41 7.54％
Ｓ−ベンジルグルタチオン
[Formula] 4.71g (yield 71%) is obtained. R 1 f : 0.61 Dissolve 120 mg of the above amorphous substance in ethyl acetate.
Add 0.04 ml of DCHA, dry under reduced pressure, and crystallize from ethyl ether. Melting point: 148−149℃ [α] 24 D : −37.6゜ (C1, MeOH) Elemental analysis C 26 H 39 O 8 N 3 S・C 12 H 23 N・1/2
As H 2 O C H N Calculated value 61.35 8.54 7.53% Actual value 61.46 8.41 7.54% S-Benzylglutathione
【式】２−(1)の調
製。
グルタチオン〔興人（株）販売〕1.84ｇ（６
ｍmol〕を氷冷下EtOH12mlと1NNaOH12ml
（12ｍmol）混合溶液に加え澄明溶液とする。
N2ガス飽和後、冷却撹拌しながら臭化ベンジ
ル0.79ml（6.6ｍmol）を加え１時間反応する。
濃・HClを用いて反応液のPHを３〜４に調整す
ると目的物の結晶が析出する。この結晶を
別、冷水、EtOH、エチルエーテル洗滌後、乾
燥し、1.87ｇ（収率78％）のＳ−ベンジルグル
タチオンを得る。
融点 ：200〜202℃（分
解）
〔α〕20 D：−6.4゜（C0.7，3NHCl）
R1 f ：0.02
R2 f ：0.21
R3 f ：0.21
Ｓ−保護−ω−グルタチオニルグルタチオン
誘導体
４−(10)の調整。
N〓−第三ブトキシカルボニル−α−第三ブ
チルエステル−γ−グルタミル−Ｓ−ベンジル
システイニルグリシン４−(9)2.21ｇ（４ｍ
mol）とHOSu506mg（4.4ｍmol）の10mlDMF
溶液に冷却下DCC865mg（4.2ｍmol）を加えて
０℃３時間、室温で２時間反応せしめ、活性エ
ステル，４−（91）とする。
先に用意したＳ−ベンジルグルタチオン1.75
ｇ（4.4ｍmol）とEt3N1.22ml（8.8ｍmol）の
６mlDMF溶液を冷却下添加、0゜〜４℃で一夜
反応せしめる。副生するDCUを去し、DMF
を留去後、残渣を酢酸エチルに溶解、10％クエ
ン酸及び冷水で各３回振とう洗滌する。酢酸エ
チル層を乾燥後減圧乾固すると目的のＳ−保護
−ω−グルタチオニルグルタチオン誘導体４−
(10)の結晶が析出する。
メタノールエチルエーテル再結後3.14ｇ（収
率83％）の目的物４−(10)を得る。
融点 ：128〜130℃
〔α〕24 D：−27.1゜（C0.7，MeOH）
R1 f ：0.62
元素分析 C43H60O13N6S2・3/2・H2Oとし
て
Ｃ Ｈ Ｎ
計算値 53.79 6.61 8.75％
実測値 53.95 6.51 9.05％
Ｓ−保護−α−グルタチオニルグルタチオン
誘導体
４−(11)の調整。
４−(8)のグルタチオン誘導体2.06ｇ（3.5ｍ
mol）と、HOSu460mg（４ｍmol）の10ml
DMF溶液にDCC824mg（４ｍmol）を加えて０
℃３時間、室温下２時間反応せしめる。副生す
るDCUを去し、DMFを減圧留去後残渣にエ
チルエーテルを加えて、生ずる沈殿を取し、
減圧乾燥して2.75ｇの活性エステル，４−（81）
を得る。
この活性エステル，４−（81）2.75ｇをＳ−
ベンジルグルタチオン1.59ｇ（４ｍmol）と
Et3N1.12ml（８ｍmol）の10mlDMF溶液に冷
却下加え、0゜〜４℃で一夜反応せしめる。
DMFを減圧留去後、残渣を酢酸エチルに抽瞬
し、10％クエン酸、H2Oで各３回洗滌、乾燥
後、溶媒を留去し、エチルエーテルを加えると
結晶が析出する。これら結晶を酢酸エチル−エ
チルエーテル系溶媒から再結し、3.05ｇ（収率
89％）の目的物、Ｓ−保護−α−グルタチオニ
ルグルタチオン誘導体、４−(11)を得る。
融点 ：137〜139℃
〔α〕24 D：−33.6゜（C1，MeOH）
R1 f ：0.66
元素分析 C46H58O13N6S2・1/2H2Oとして
Ｃ Ｈ Ｎ
計算値 56.60 6.09 8.61％
実測値 56.53 6.13 8.69％
保護トリグルタチオン−（Protected
TriGSH−）
４−(12)の調整。
１ 先に得た４−(10)3.00ｇ（3.16ｍmol）を
1NHCl／CH3COOH20mlに溶解し室温で３時
間放置してBoc−および−OBut基を除去し、
反応溶液を濃縮し、エチルエーテルを加えて固
化せしめた。固化物を取し、エチルエーテル
洗滌後減圧乾燥して2.56ｇ（収率100％）のペ
プチド塩酸塩４−（101）、HClを得た。
２ 先に得た４−(9)の保護グルタチオン誘導体
886ｇ（1.6ｍmol）を、４−(10)調製に準じて活
性エステル、４−（91）とする。
３ １）で得た４−（101）・HCl2.56ｇ（3.16ｍ
mol）とEt3N0.90ml（6.4ｍmol）の10mlDMF
溶液に２）の活性エステル４−（91）を加え、
0゜〜４℃で２日間反応せしめる。反応液から
DMFを留去し、残留分にEtOHを添加し、酢
酸エチルにて抽出し、すばやく10％クエン酸、
H2Oで振とう洗滌、酢酸エチル層を乾燥後、
溶媒を留去し、エチルエーテルにて目的物を粉
末化せしめる。2.1ｇの粉末を酢酸エチル−エ
チルエーテル系溶媒から再結（沈殿）せしめ、
不定形粉末の目的保護トリグルタチオン−、
４−(12)1.76ｇ（収率84％）を得る。
融点：117〜119℃（分解）
〔α〕24 D：−29.8（C1、DMF）
元素分析 C60H81O18N9S3・2H2Oとして
Ｃ Ｈ Ｎ
計算値 53.44 6.35 9.35％
実測値 53.26 6.17 10.01％
保護トリグルタチオン−
４−（13）の調整。
１ ４−(11)で得たＳ−保護−α−グルタチオニル
グルタチオン誘導体2.90ｇ（３ｍmol）を
1NHCl／AcOH 20mlに溶解し室温で２時間静
置する。減圧濃縮後、エチルエーテルを加え、
白沈を析出せしめ、この沈殿を取し、減圧下
乾燥して2.6ｇ（収率97％）の部分脱保護基ペ
プチド４−（111）の塩酸塩を得る。
２ ４−(9)のグルタチオン誘導体831mg（1.5ｍ
mol）を４−(10)調製に準じて活性エステル、４
−（91）とする。
この活性エステルを１）で準備した部分脱保
護基ペプチド４−（111）の塩酸塩1.35ｇ（1.5
ｍmol）を加え、10mlのDMFとEt3N（4.5ｍ
mol）溶液に加え0゜〜４℃で２日間反応を行
う。反応液を酢酸にて中和し、溶媒を留去し生
成する固形物をH2O 20mlに分散し、再過に
より取する。エタノール−エチルエーテル系
溶媒から再結晶化し1.64ｇ（収率78％）の保護
トリグルタチオン−、４−（13）を得る。
融点 ：174〜176℃（分解）
〔α〕24：−34.2°（C1、DMF）
R1 f：0.57、Rf6：0.59、R2 f：0.92、R3 f：0.77
R2 f：0.59
R3 f：0.92
R4 f：0.77
元素分析 C67H87O18N9S3・1/2H2Oとして
Ｃ Ｈ Ｎ
計算値 56.29 6.35 8.82％
実測値 56.12 6.24 9.08％
保護トリグルタチオン−、４−（14）の調製
１ ４−(10)で得たＳ−保護−ω−グルタチオニル
グルタチオン誘導体1.42ｇ（1.5ｍmol）を
1NHCl／AcOH10mlに溶解し、室温で３時間
放置した後、減圧濃縮し、エーテルを加え残留
分を固化せしめた。固形物を取し、エーテル
洗滌後、減圧乾燥して1.23ｇ（1.5ｍmol）の部
分脱保護基ペプチド４−（101）の塩酸塩を得
た。
２ ４−(8)のグルタチオン誘導体882mg（1.5ｍ
mol）から４−(11)の記載に準じて得られた活性
エステル、４−（81）1.18ｇ、４−（101）の部
分脱保護基ペプチド塩酸塩1.23ｇおよび0.84ml
（６ｍmol）のEt3Nを５mlのDMFに溶解して、
0゜〜４℃で２日間反応せしめる。DMFを減圧
留去後、少量のEtOHの存在下残留物を酢酸エ
チルで抽出しすばやく10％クエン酸、水で洗滌
し、後乾燥した。溶媒を減圧濃縮し、エチルエ
ーテルを加え結晶化させる。EtOH−酢酸エチ
ル−エチルエーテル系溶媒から再結晶して1.43
ｇ（収率71％）の目的物保護トリグルタチオン
−を得た。
融点：131〜133℃（分解）
〔α〕24 D：−30.4゜（C1、DMF）
R1 f：0.54、R6 f：0.44、R2 f：0.86
R3 f：0.72
元素分析 C63H79O18N9S3・2H2Oとして
Ｃ Ｈ Ｎ
計算値 54.73 6.05 9.12％
実測値 54.68 6.02 9.45％
保護トリグルタチオン−、４−（15）の調製。
１ ４−(11)で得たＳ−保護−α−グルタチオニル
グルタチオン誘導体1.35ｇ（1.4ｍmol）を
1NHCl／AcOH10mlに溶解し、室温で３時間
放置後減圧濃縮し、エチルエーテルを加え、残
留物を固化せしめた。固化物を取し、エチル
エーテル洗滌後減圧乾燥し、1.26ｇ（1.4ｍ
mol）の部分脱保護基ペプチド、４−（111）の
塩酸塩を得る。
２ ４−(8)のグルタチオン誘導体823mg（1.4ｍ
mol）より４−(11)の記載に準じて得られた活性
エステル、４−（81）1.1ｇと１）のペプチド塩
酸塩、４−（111）・HCl 1.26ｇおよび0.59ml
（4.2ｍmol）のEt3Nを５mlのDMFに溶解し、
0゜〜４℃で２目間反応させた。反応液に酢酸を
加え中和後、減圧濃縮し、固化物に水を加えて
取した。エタノール−エチルエーテル系溶媒
より再結晶して1.67ｇ（収率83％）の保護トリ
グルタチオン−、４−（15）を得た。
融点：173〜175℃
〔α〕24 D：−36.6゜（C1、DMF）
R1 f：0.71、R6 f：0.59、R2 f：0.97、R4 f：：
0.75
元素分析 C70H85O18N9S3・1/2H2Oとして
Ｃ Ｈ Ｎ
計算値 57.44 6.06 8.61％
実測値 57.46 6.00 8.78％
式(2)トリグルタチオン−（TriGSH−）４
−（16）の調製。
４−(12)で調製した保護トリグルタチオン−
800mg（0.61ｍmol）を1.2NHCl／AcOH5mlに溶
解し、１時間室温に放置する。溶媒を減圧留去
し、残渣にエチルエーテルを加えて、脱Boc−、
OBut、４−(12)塩酸塩700mg（収率96％）を無定形
粉末として得る。この脱Boc−、OBut、４−(12)
塩酸塩700mgを−50℃で液体アンモニア10mlに溶
解し、約120mgの金属Naを少量づつ加える。１分
間以上溶液が青色を持続することを確認した後、
−50℃でNH4Cl250mgを加え、15分間撹拌した
後、室温に戻しつつ窒素ガスを通じてNH3を留
去する。残渣を窒素ガス置換した2M酢酸５mlに
溶解し、SephadexG−25カラム（2.0×80cm）に
てゲル過法による脱塩を行いペプチド画分約50
ml（４mlフラクシヨン、フラクシヨン番号、45番
〜56番）を集めβ−メルカプトエタノール５滴加
えて減圧濃縮する。濃縮シロツプにエタノールを
加えて、析出する結晶を取し、五酸化リン上真
空乾燥して、目的のトリグルタチオン−
（TriGSH−）、４−（16）325mg（収率72％）を
得る。 TLCによるR2 f：0.02
紙電気泳動のRLys：0.27
式(3)トリグルタチオン−（TriGSH−）、
４−（17）の調製。 ４−（13）で調製した保護ト
リグルタチオン−800mg（0.57ｍmol）を４−
（16）に準じて処理し、脱Boc−、OBut４−（13）
誘導体塩酸塩720mg（収率99％）を得る。この化
合物を４−（16）に準じて金属Na／液体アンモニ
ア処理し、NH3留去後の残渣を窒素ガス置換し
た水10mlに溶解し、Dowex1×８（Aco-型）カラ
ム（200〜400メツシユ、2.0×10cm）にて吸着、
50％酢酸にて脱着し、溶出画分約40mlを集め、β
−メルカプトエタノール数滴加え減圧濃縮する。
濃縮シロツプにエタノールを加え析出する結晶を
取し、五酸化リン上真空乾燥して、目的のトリ
グルタチオン−、４−（17）410mg（収率82％）
を得る。
TLCによるR2 f：0.02
紙電気泳動のRLys：0.30
式(4)トリグルタチオン−（TriGSH−）、
４−（18）の調製。
４−（14）で得た保護トリグルタチオン−800
mg（0.59ｍmol）を４−（16）に準じて処理し脱
Boc4−（14）誘導体塩酸塩735mg（収率97％）を
得る。本化合物を４−（16）に準じて金属ナトリ
ウム／液体アンモニア処理し、アンモニア留去後
の残渣を窒素置換した水10mlに溶解しDowex1×
８カラム（Aco-型）（200〜400メツシユ、2.0×
10cm）に吸着し、50％酢酸溶液にて脱着し、溶出
画分約40mlをβ−メルカプトエタノール存在下に
濃縮し、生じたシロツプにエタノールを加えて析
出する結晶を取し、五酸化リン上真空乾燥し
て、目的のトリグルタチオン−、４−（18）450
mg（収率85％）を得る。
TLCによるR2 f：0.02
紙電気泳動のRLys：0.34であつた。
式(5)トリグルタチオン−（TriGSH−）、
４−（19）の調製。
４−（15）で調製した保護トリグルタチオン−
800mg（0.56ｍmol）を５−（16）に準じて処理
し脱Boc−４−（15）誘導体塩酸塩730mg（収率95
％）を得る。本化合物を４−（16）に準じて金属
ナトリウム／液体アンモニア処理し、NH3留去
後の残渣を窒素置換した2M酢酸溶液５mlに溶解
し、SephadexG−25カラム（2.0×80cm）にてゲ
ル過クロマトグラフイーを行い、ペプチド画分
約40ml（４mlフラクシヨン、フラクシヨンNo.41〜
50番）を集めβ−メルカプトエタノール５滴添加
して濃縮し、生じたシロツプにエタノールを加え
析出する結晶を取し、五酸化リン真空乾燥して
目的のトリグルタチオン−、４−（19）380mg
（収率77％）を得る。
TLCによるR2 f：0.02
紙電気泳動のRLys：0.37であつた。
薄層クロマトグラフイー（TLC）の移動度Rf
は下記の各溶媒系に於ける値を示す。紙での移
動度はRf（ppc）で示す。Preparation of Formula 2-(1). Glutathione [sold by Kojin Co., Ltd.] 1.84g (6
mmol] under ice-cooling with 12 ml of EtOH and 12 ml of 1NNaOH.
(12 mmol) Add to the mixed solution to make a clear solution.
After saturation with N 2 gas, 0.79 ml (6.6 mmol) of benzyl bromide was added while cooling and stirring, and the mixture was reacted for 1 hour.
When the pH of the reaction solution is adjusted to 3 to 4 using concentrated HCl, crystals of the target product will precipitate. The crystals were separated, washed with cold water, EtOH, and ethyl ether, and then dried to obtain 1.87 g (yield: 78%) of S-benzylglutathione. Melting point: 200-202℃ (decomposed) [α] 20 D : -6.4゜ (C0.7, 3NHCl) R 1 f : 0.02 R 2 f : 0.21 R 3 f : 0.21 S-protected-ω-glutathionylglutathione derivative 4-(10) Adjustment. 2.21 g (4 m
mol) and HOSu 506 mg (4.4 mmol) in 10 ml DMF
865 mg (4.2 mmol) of DCC was added to the solution under cooling and reacted for 3 hours at 0°C and 2 hours at room temperature to form active ester, 4-(91). S-benzylglutathione prepared earlier 1.75
A solution of 1.22 ml (8.8 mmol) of Et 3 N and 1.22 ml (8.8 mmol) of Et 3 N in 6 ml DMF was added under cooling, and the mixture was allowed to react at 0° to 4° C. overnight. Remove the by-product DCU and change to DMF
After distilling off, the residue was dissolved in ethyl acetate and washed with 10% citric acid and cold water three times each with shaking. After drying the ethyl acetate layer, the desired S-protected-ω-glutathionylglutathione derivative 4- is obtained by drying under reduced pressure.
Crystals of (10) precipitate. After reconsolidation with methanol ethyl ether, 3.14 g (yield 83%) of the desired product 4-(10) was obtained. Melting point: 128-130℃ [α] 24 D : -27.1゜ (C0.7, MeOH) R 1 f : 0.62 Elemental analysis C 43 H 60 O 13 N 6 S 2・3/2・H 2 O as C H N Calculated value 53.79 6.61 8.75% Actual value 53.95 6.51 9.05% S-protected-α-glutathionylglutathione derivative 4-(11) adjustment. 4-(8) Glutathione derivative 2.06g (3.5m
mol) and 10ml of 460mg (4mmol) of HOSu
Add 824 mg (4 mmol) of DCC to the DMF solution and
C. for 3 hours and at room temperature for 2 hours. After removing the by-product DCU and distilling off DMF under reduced pressure, ethyl ether was added to the residue and the resulting precipitate was collected.
Vacuum drying yields 2.75 g of active ester, 4-(81)
get. 2.75 g of this active ester, 4-(81), was added to S-
Benzylglutathione 1.59g (4mmol)
Add to a solution of 1.12 ml (8 mmol) of Et 3 N in 10 ml of DMF under cooling, and react overnight at 0° to 4°C.
After evaporating DMF under reduced pressure, the residue was extracted with ethyl acetate, washed three times each with 10% citric acid and H 2 O, and dried. The solvent was evaporated and ethyl ether was added to precipitate crystals. These crystals were recrystallized from ethyl acetate-ethyl ether solvent, and 3.05g (yield:
89%) of the target product, S-protected-α-glutathionylglutathione derivative, 4-(11) was obtained. Melting point: 137-139℃ [α] 24 D : -33.6゜(C1, MeOH) R 1 f : 0.66 Elemental analysis C 46 H 58 O 13 N 6 S 2・1/2H 2 O As C H N Calculated value 56.60 6.09 8.61% Actual value 56.53 6.13 8.69% Protected triglutathione
TriGSH−) 4-(12) adjustment. 1 3.00g (3.16mmol) of 4-(10) obtained earlier
Dissolve in 20 ml of 1NHCl/CH 3 COOH and leave at room temperature for 3 hours to remove Boc- and -OBu t groups.
The reaction solution was concentrated and solidified by adding ethyl ether. The solidified product was washed with ethyl ether and dried under reduced pressure to obtain 2.56 g (yield 100%) of peptide hydrochloride 4-(101), HCl. 2 Protected glutathione derivative of 4-(9) obtained earlier
886 g (1.6 mmol) was converted into active ester 4-(91) according to the preparation of 4-(10). 3 2.56g (3.16m) of 4-(101)・HCl obtained in 1)
mol) and Et3N0.90ml (6.4mmol) in 10mlDMF
Add active ester 4-(91) of 2) to the solution,
Allow to react at 0° to 4°C for 2 days. from reaction solution
DMF was distilled off, EtOH was added to the residue, extracted with ethyl acetate, and quickly extracted with 10% citric acid,
After shaking and washing with H 2 O and drying the ethyl acetate layer,
The solvent was distilled off, and the target product was powdered with ethyl ether. 2.1 g of powder was recrystallized (precipitated) from ethyl acetate-ethyl ether solvent,
Purpose-protected triglutathione in amorphous powder,
1.76 g (yield 84%) of 4-(12) is obtained. Melting point: 117-119℃ (decomposed) [α] 24 D : -29.8 (C1, DMF) Elemental analysis C 60 H 81 O 18 N 9 S 3・2H 2 O as C H N Calculated value 53.44 6.35 9.35% Actual value 53.26 6.17 10.01% Protected triglutathione 4-(13) adjustment. 1 2.90 g (3 mmol) of the S-protected-α-glutathionylglutathione derivative obtained in 4-(11)
Dissolve in 20 ml of 1NHCl/AcOH and let stand at room temperature for 2 hours. After concentrating under reduced pressure, add ethyl ether,
A white precipitate was precipitated, and this precipitate was collected and dried under reduced pressure to obtain 2.6 g (yield 97%) of the hydrochloride of partially deprotected peptide 4-(111). 2 4-(9) glutathione derivative 831mg (1.5m
mol) of the active ester according to the preparation of 4-(10), 4
−(91). This active ester was used as a hydrochloride salt of 1.35 g (1.5
mmol) and 10 ml DMF and Et 3 N (4.5 mmol).
mol) solution and react at 0° to 4°C for 2 days. The reaction solution was neutralized with acetic acid, the solvent was distilled off, and the resulting solid was dispersed in 20 ml of H 2 O and collected by refiltration. Recrystallization from an ethanol-ethyl ether solvent gives 1.64 g (yield 78%) of protected triglutathione-4-(13). Melting point: 174-176℃ (decomposition) [α] 24 : −34.2° (C1, DMF) R 1 f : 0.57, Rf 6 : 0.59, R 2 f : 0.92, R 3 f : 0.77 R 2 f : 0.59 R 3 f : 0.92 R 4 f : 0.77 Elemental analysis C 67 H 87 O 18 N 9 S 3・1/2H 2 O C H N Calculated value 56.29 6.35 8.82% Actual value 56.12 6.24 9.08% Protected triglutathione-, 4- Preparation of (14) 1 1.42 g (1.5 mmol) of the S-protected-ω-glutathionylglutathione derivative obtained in 4-(10)
The solution was dissolved in 10 ml of 1NHCl/AcOH, left at room temperature for 3 hours, concentrated under reduced pressure, and ether was added to solidify the residue. The solid was collected, washed with ether, and dried under reduced pressure to obtain 1.23 g (1.5 mmol) of the hydrochloride of partially deprotected peptide 4-(101). 2 4-(8) glutathione derivative 882mg (1.5m
1.18 g of 4-(81), 1.23 g and 0.84 ml of partially deprotected peptide hydrochloride of 4-(101)
(6 mmol) of Et 3 N was dissolved in 5 ml of DMF,
Allow to react at 0° to 4°C for 2 days. After evaporating DMF under reduced pressure, the residue was extracted with ethyl acetate in the presence of a small amount of EtOH, quickly washed with 10% citric acid and water, and then dried. The solvent is concentrated under reduced pressure, and ethyl ether is added for crystallization. Recrystallized from EtOH-ethyl acetate-ethyl ether solvent to 1.43
g (yield 71%) of the target protected triglutathione was obtained. Melting point: 131-133℃ (decomposition) [α] 24 D : -30.4゜ (C1, DMF) R 1 f : 0.54, R 6 f : 0.44, R 2 f : 0.86 R 3 f : 0.72 Elemental analysis C 63 H 79 O 18 N 9 S 3.2H 2 O as C H N Calculated value 54.73 6.05 9.12% Actual value 54.68 6.02 9.45% Preparation of protected triglutathione-,4-(15). 1 1.35 g (1.4 mmol) of the S-protected-α-glutathionylglutathione derivative obtained in 4-(11)
The solution was dissolved in 10 ml of 1NHCl/AcOH, left at room temperature for 3 hours, concentrated under reduced pressure, and ethyl ether was added to solidify the residue. The solidified material was removed, washed with ethyl ether, dried under reduced pressure, and weighed 1.26 g (1.4 m
mol) of partially deprotected peptide, 4-(111) hydrochloride. 2 4-(8) glutathione derivative 823mg (1.4m
1.1 g of active ester 4-(81) obtained according to the description in 4-(11) from 1) peptide hydrochloride of 1), 1.26 g and 0.59 ml of 4-(111).HCl
(4.2 mmol) of Et 3 N was dissolved in 5 ml of DMF,
The reaction was carried out at 0° to 4°C for 2 days. After neutralizing the reaction solution by adding acetic acid, it was concentrated under reduced pressure, and the solidified product was collected by adding water. Recrystallization from an ethanol-ethyl ether solvent gave 1.67 g (yield 83%) of protected triglutathione-4-(15). Melting point: 173-175℃ [α] 24 D : -36.6゜ (C1, DMF) R 1 f : 0.71, R 6 f : 0.59, R 2 f : 0.97, R 4 f ::
0.75 Elemental analysis C 70 H 85 O 18 N 9 S 3・1/2H 2 O C H N Calculated value 57.44 6.06 8.61% Actual value 57.46 6.00 8.78% Formula (2) Triglutathione-(TriGSH-) 4
- Preparation of (16). 4-Protected triglutathione prepared in (12)
Dissolve 800 mg (0.61 mmol) in 5 ml of 1.2NHCl/AcOH and leave at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and ethyl ether was added to the residue to remove Boc.
Obtain 700 mg (96% yield) of OBut , 4-(12) hydrochloride as an amorphous powder. This de-Boc-, OBu t , 4-(12)
Dissolve 700 mg of hydrochloride in 10 ml of liquid ammonia at -50°C, and add about 120 mg of metallic Na little by little. After confirming that the solution remains blue for more than 1 minute,
After adding 250 mg of NH 4 Cl at −50° C. and stirring for 15 minutes, NH 3 was distilled off by passing nitrogen gas while returning the mixture to room temperature. The residue was dissolved in 5 ml of 2M acetic acid purged with nitrogen gas, and desalted by gel filtration using a Sephadex G-25 column (2.0 x 80 cm) to obtain a peptide fraction of approximately 50.
ml (4 ml fraction, fraction numbers 45-56) are collected, 5 drops of β-mercaptoethanol are added, and the mixture is concentrated under reduced pressure. Add ethanol to the concentrated syrup, collect the precipitated crystals, vacuum dry over phosphorus pentoxide, and obtain the desired triglutathione.
(TriGSH-), 325 mg (yield 72%) of 4-(16) is obtained. R2f by TLC : 0.02 R Lys by paper electrophoresis: 0.27 Preparation of formula (3) triglutathione-(TriGSH-), 4-(17). 4-800 mg (0.57 mmol) of protected triglutathione prepared in (13) was added to 4-
(16), remove Boc-, OBu t 4-(13)
720 mg (99% yield) of derivative hydrochloride is obtained. This compound was treated with metallic Na/liquid ammonia according to 4-(16), and the residue after NH 3 distillation was dissolved in 10 ml of water purged with nitrogen gas. Adsorption with mesh, 2.0 x 10 cm),
Desorb with 50% acetic acid, collect approximately 40 ml of the eluate fraction, and
- Add a few drops of mercaptoethanol and concentrate under reduced pressure.
Add ethanol to the concentrated syrup, collect the precipitated crystals, and vacuum dry over phosphorus pentoxide to obtain 410 mg of the desired triglutathione-4-(17) (yield 82%).
get. R 2 f by TLC: 0.02 R Lys by paper electrophoresis: 0.30 Formula (4) Triglutathione- (TriGSH-),
4-Preparation of (18). 4-Protected triglutathione obtained in (14) -800
mg (0.59 mmol) was treated according to 4-(16) and desorbed.
735 mg (yield 97%) of Boc4-(14) derivative hydrochloride is obtained. This compound was treated with metallic sodium/liquid ammonia according to 4-(16), and the residue after ammonia distillation was dissolved in 10 ml of nitrogen-substituted water and Dowex 1×
8 columns (Aco - type) (200-400 meshes, 2.0×
10 cm), desorbed with 50% acetic acid solution, concentrated about 40 ml of the eluted fraction in the presence of β-mercaptoethanol, added ethanol to the resulting syrup to collect the precipitated crystals, and concentrated it on phosphorus pentoxide. Vacuum dry to obtain the desired triglutathione, 4-(18) 450
mg (yield 85%). R 2 f by TLC: 0.02 R Lys by paper electrophoresis: 0.34. Formula (5) triglutathione- (TriGSH-),
4-Preparation of (19). 4-Protected triglutathione prepared in (15)
800 mg (0.56 mmol) was treated according to 5-(16) to obtain 730 mg of de-Boc-4-(15) derivative hydrochloride (yield 95
%). This compound was treated with metallic sodium/liquid ammonia according to 4-(16), and the residue after NH 3 distillation was dissolved in 5 ml of a 2M acetic acid solution purged with nitrogen. Perform hyperchromatography and collect approximately 40 ml of peptide fraction (4 ml fraction, fraction No. 41~
Collect No. 50), add 5 drops of β-mercaptoethanol, concentrate, add ethanol to the resulting syrup, collect the precipitated crystals, vacuum dry phosphorus pentoxide, and obtain 380 mg of the desired triglutathione-4-(19).
(yield 77%). R 2 f by TLC: 0.02 R Lys by paper electrophoresis: 0.37. Mobility Rf of thin layer chromatography (TLC)
indicates the values for each solvent system below. Mobility on paper is expressed in Rf (ppc).
【表】
検出法：遊離アミノ基を有する化合物については
ニンヒドリン試薬の噴霧、加熱により生じた紫
色スポツトを確認し、アミノ基を保護した化合
物についてはI2蒸気又は10％H2SO4噴霧後乾熱
機中加熱し生じた灰暗色スポツトを確認した。
紙電気泳動は東洋紙No.52を用い溶媒（蟻
酸：酢酸：メタノール：水＝１：３：６：10v／
ｖPH1.8）を用い600volt、３時間に於ける各化合
物の移動距離をリシン（Lys）との比で測定し移
動度RLysとして表わした。
上記本発明及び参考として示す化合物の赤外線
吸収スペクトルの特異吸収を表１−(1)及び表１−
(2)に示す。[Table] Detection method: For compounds with free amino groups, spray with ninhydrin reagent and check the purple spots generated by heating; for compounds with protected amino groups, spray with I 2 steam or 10% H 2 SO 4 and dry. Dark gray spots that were produced by heating in a hot oven were observed. Paper electrophoresis uses Toyo Paper No. 52 with solvent (formic acid: acetic acid: methanol: water = 1:3:6:10v/
The migration distance of each compound in 3 hours at 600 volts was measured as a ratio to lysine (Lys) and expressed as mobility R Lys . Table 1-(1) and Table 1-
Shown in (2).
【表】【table】
【表】【table】
【表】
グルタチオン三量体GSH−、GSH−、
GSH−、GSH−、GSH−の−SH基が遊
離状態であることの確認は、Elleman法〔G.L.
Elleman：Arch.Biochem.Bisphys.，74，443
（1958）〕に従つて、１−(5)の操作により得た式(1)
TriGSH−、４−（16）の操作にて得た式(2)
TriGSH−、４−（17）の操作にて得た式(3)
TriGSH−、４−（18）の操作にて得た式(4)
TriGSH−及び４−（19）の操作にて得た式(5)
TriGSH−各2.77mgを6Mグアニジン塩酸／
0.01Mトリス塩酸PH8.0／0.05MEDTA水溶液3.1
mlに溶解して調製した１ｍＭ溶液２mlに５，5′−
ジチオービス（２−ニトロベンゾイツクアシド）
39.6mg／0.05Mリン酸緩衝液、PH7.0 10mlに溶解
して得た試薬溶液0.1mlを加え25℃で反応せしめ
生じたチオニトロフエノレイトアニオンの吸光を
412mmで経時的に測定して反応−SH基の量を測定
した。
測定の結果は表−に示す。[Table] Glutathione trimer GSH-, GSH-,
To confirm that the -SH group of GSH-, GSH-, and GSH- is in a free state, use the Elleman method [GL
Elleman: Arch.Biochem.Bisphys., 74, 443
(1958)], formula (1) obtained by operation 1-(5)
Formula (2) obtained by operating TriGSH-, 4-(16)
Formula (3) obtained by operating TriGSH-, 4-(17)
Formula (4) obtained by operating TriGSH-, 4-(18)
Formula (5) obtained by manipulating TriGSH- and 4-(19)
TriGSH - 2.77mg each in 6M guanidine hydrochloride/
0.01M Tris-HCl PH8.0/0.05MEDTA aqueous solution 3.1
5,5'-
Dithiobis (2-nitrobenzoitsuquaside)
Add 0.1 ml of the reagent solution obtained by dissolving 39.6 mg/0.05 M phosphate buffer in 10 ml of pH 7.0, react at 25°C, and measure the absorbance of the resulting thionitrophenolate anion.
The amount of reacted -SH groups was determined by measuring over time at 412 mm. The measurement results are shown in Table.
【表】
チオン
TriGSH各化合物のカルボキシメチル誘導体の
紙電気泳動。
TriGSH−、TriGSH−、TriGSH−、
TriGSH−、TriGSH−それぞれ各20mgをト
リス緩衝液（PH8.0）0.5mlに溶解し0.02Mモノヨ
ード酢酸水溶液0.7mlを加えてSH基のカルボキ
シ・メチル化を行う。余剰のモノヨート酢酸をヂ
チオエリスリトールで中和し、反応を止め、反応
液をセフアデツクスＧ−10のゲル過により脱塩
し、得られたカルボキシメチル化TriGSH各種
を、ビリジン／酢酸／水PH3.6で2000ボルト1.5時
間紙電気泳動を行つた。システイン酸を標準物
質として移動度〔Rf＝1.00とした時のカルボキシ
メチル化トリグルタチオンの移動度〕を表−に
示す。
表−
化合物 Rf
システイン酸 1.00
CM化TriGSH− 0.60
CM化TriGSH− 0.64
CM化TriGSH− 0.61
CM化TriGSH− 0.61
CM化TriGSH− 0.60
対象CM化システイン 0.51
更に、TriGSH−、同−、同−、同−
及びTriGSH−の各種金属キレート化合物を形
成し、その安定度定数を下掲表−に示した。[Table] Thione
Paper electrophoresis of carboxymethyl derivatives of each TriGSH compound. TriGSH−, TriGSH−, TriGSH−,
20 mg each of TriGSH- and TriGSH- are dissolved in 0.5 ml of Tris buffer (PH 8.0) and 0.7 ml of a 0.02 M monoiodoacetic acid aqueous solution is added to carry out carboxy methylation of the SH group. Excess monoiotic acetic acid was neutralized with dithioerythritol to stop the reaction, and the reaction solution was desalted by gel filtration through Sephadex G-10. Paper electrophoresis was performed at 2000 volts for 1.5 hours. Table 1 shows the mobility [mobility of carboxymethylated triglutathione when Rf = 1.00] using cysteic acid as a standard substance. Table - Compound Rf Cysteic acid 1.00 CM TriGSH- 0.60 CM TriGSH- 0.64 CM TriGSH- 0.61 CM TriGSH- 0.61 CM TriGSH- 0.60 Target CM cysteine 0.51 Furthermore, TriGSH-, same-, same-, same-
Various metal chelate compounds of TriGSH- and TriGSH- were formed, and their stability constants are shown in the table below.
【表】
上記安定度定数の測定は、Bjerrumの方法（金
属キレート〔〕239頁〜255頁；坂口武一、上野
景平編 南江堂）に従い４×10-3mole／ｌの試
料、１×10-3mole／ｌの各種金属イオンの混合
液30mlをイオン強度0.15、測定温度25℃にして
0.01NKOH標準液を用いて滴定したときのPH変
化を東洋科学産業販売ガラス電極PHメーター（型
式TD−15）にて測定し滴定曲線から生成関数
＝〔Ａ〕t−〔Ａ〕／〔Ｍ〕t、但し：〔Ａ〕t：配位子
の総濃度、
〔Ｍ〕t＝金属の総濃度とpA＝−40ｇ〔Ａ〕グラフ
による中点傾斜から総安定度定数β＝2
〔MZ2〕／〔Ｍ〕〔Ｚ〕2＝KMZ・KMZ2を算出し、平均安
定度
定数の対数値をもつて表した。計算に用いた
TriGSHの酸解離恒数pK1、pK2、pK3、pK4はそ
れぞれ次の通り
2.12、 2.35、 8.66、
（α−COOH） （ω−COOH） （N〓−NH2）
9.65（−SH）であつた。[Table] The above stability constant was measured according to Bjerrum's method (Metal Chelate [] pages 239-255; Takeichi Sakaguchi, Keihei Ueno, Nankodo) using a sample of 4 x 10 -3 mole/l, 1 x 10 -3 mole/l of a mixed solution of various metal ions (30 ml) at an ionic strength of 0.15 and a measurement temperature of 25°C.
The PH change during titration using 0.01NKOH standard solution was measured using a glass electrode PH meter (model TD-15) sold by Toyo Kagaku Sangyo, and the generation function = [A] t - [A] / [M] was determined from the titration curve. t , where: [A] t : total concentration of ligands, [M] t = total concentration of metal and pA = -40 g [A] Total stability constant β = 2 from the midpoint slope according to the graph
[MZ 2 ]/[M] [Z] 2 =K MZ ·K MZ2 was calculated and expressed as a logarithm value of the average stability constant. used for calculation
The acid dissociation constants pK 1 , pK 2 , pK 3 , and pK 4 of TriGSH are as follows, respectively: 2.12, 2.35, 8.66, (α−COOH) (ω−COOH) (N〓−NH 2 ) 9.65 (−SH) It was hot.
Claims (1)
ン酸残基のうち−NH2のＨ及び−COOHのOHを
除いた残基 を示し、−Cys−は、システイン残基のうち を示し、そして−Gly−は、グリシン残基のうち −HN−CH2−CO− を示し、本化合物に於けるアミノ酸略号間の実線
は各々アミノ酸がペプチド結合で連絡されている
ことを示す。GluまたはGluの上、下に出た実線
はγ−カルボキシル基からOHを除いたカルボニ
ル基、Glu−の右横実線はα−カルボキシル基か
らOHを除いたカルボニル基を示す。］ のγ−Ｌ−グルタミルＬ−システイニルグリシン
（別称：グルタチオン）三量体並びに少なくとも
その−SH基が(イ)随意置換されていてもよいアラ
ルキル基または(ロ)Hg、Cu、Pd、Cd、Ni、Zn、
Co、Fe及びMnからなる群から選ばれた金属によ
るキレート化によつて保護された該三量体の誘導
体。 ２ 該金属が二価金属である特許請求の範囲第１
項記載の誘導体。 ３ 随意置換されていてもよいアラルキル基が、
ベンジル基、低級アルコキシベンジル基またはト
リチル基である特許請求の範囲第１項記載の誘導
体。[Claims] 1 Formula (1) [However, in the formula, -Glu- or -Glu- is a glutamic acid residue excluding the H of -NH 2 and the OH of -COOH -Cys- indicates cysteine residues. and -Gly- indicates -HN- CH2 -CO- among glycine residues, and the solid line between the amino acid abbreviations in this compound indicates that each amino acid is connected by a peptide bond. The solid lines above and below Glu or Glu indicate a carbonyl group obtained by removing OH from a γ-carboxyl group, and the horizontal solid line to the right of Glu- indicates a carbonyl group obtained by removing OH from an α-carboxyl group. ] γ-L-glutamyl L-cysteinylglycine (also known as glutathione) trimer and at least its -SH group is (a) an optionally substituted aralkyl group or (b) Hg, Cu, Pd, Cd, Ni, Zn,
A derivative of said trimer protected by chelation with a metal selected from the group consisting of Co, Fe and Mn. 2 Claim 1 in which the metal is a divalent metal
Derivatives described in Section. 3 The optionally substituted aralkyl group is
The derivative according to claim 1, which is a benzyl group, a lower alkoxybenzyl group or a trityl group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57121988A JPS5913752A (en) | 1982-07-15 | 1982-07-15 | Glutathione trimer and its derivative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57121988A JPS5913752A (en) | 1982-07-15 | 1982-07-15 | Glutathione trimer and its derivative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5913752A JPS5913752A (en) | 1984-01-24 |
| JPH0321038B2 true JPH0321038B2 (en) | 1991-03-20 |
Family
ID=14824779
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57121988A Granted JPS5913752A (en) | 1982-07-15 | 1982-07-15 | Glutathione trimer and its derivative |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5913752A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4909944A (en) * | 1988-08-26 | 1990-03-20 | The United States Of America As Represented By The United States Department Of Energy | Removal of metal ions from aqueous solution |
| CN106030763B (en) | 2014-03-28 | 2019-06-28 | 琳得科株式会社 | The manufacturing method of protective film formation film and the semiconductor chip with protective film |
-
1982
- 1982-07-15 JP JP57121988A patent/JPS5913752A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5913752A (en) | 1984-01-24 |
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