金沢大学大学院自然科学研究科分子作用学金沢大学薬学

Nambara Toshio

Ohishi Kazuo

Shimada Kazutake

Chemical and Pharmaceutical Bulletin

Kanazawa University Repository for Academic Resources

Isolation and Characterization of NewBufotoxins from the Skin of Bufo melanostictusSCHNEIDER著者 Shimada Kazutake, Ohishi Kazuo, Nambara Toshiojournal orpublication titleChemical & pharmaceutical bulletinvolume 32number 11page range 4396-4401year 1984-11-25URL http://hdl.handle.net/2297/7071ｌｌ１ｌＦ－－－ ’，Ⅱ 4397 ■。・・■輝辮隷一瀞 Ｎｏ．11. 8－ skihsof80Formosantoads(Bz《/Ｍｌｅ伽osjicr"sScHNEIDER）lextractedwithEtOH 、む・Ｐ Ｆ１ residue EtOH-soIublepart l3謡:{鯛淫…ｎM……,aqlayer lpartitionedbetweenAcOEtandwater hexanelayer aq.］ayer lchromatographedonAmberliteXAD-2resin ＡｃＯＥｔｌａｙｅｒ Ⅱ 1100％ＭｅＯＨ Ｆｒ.５ 190％ＭｅＯＨ Ｆｒ４ 180％MeOH Pr3 ’７０％MeOH Fr､２ １Ａ Ｆｒｂ ｌＢ Ｆｎ７ １ｃ 160％MeOH Fr1 fl Ｆｒ､９ １， Fr､８ １， Fr，１３ Ｆｒ」２Ｆｒｌｌ ｌＥ Ｉｆｌｇ Ａ： Ｂ： Ｃ： Ｄ： Ｅ： Ｆｒ・１０ndlle chromatographyonsilicagel;eluent,AcOEt-MeOH chromatographyonsilicagel;eluent,CHC13-MeOH-H20 HPLCeluent,MeOH-03％(NH4)2CO3 HPLC;eluent,CH3CN-0.3％(NH4)2CO3 preparativeTLConsilicagel Chart１．seParationofBufbgeninConjugates I。authenticsample､8)Inordertoclarifythecompletestructure,bufbtalin3-suberoyl-L-and-D‐ histidineestersweresynthesizedfrombufbtalin3-hemisuberate（Ia）bytheactiveester method6)Theprotonnuclearmagneticresona､Ｃｅ('Ｈ－ＮＭＲ)spectraandchromatographic behavior(HPLC,TLC)ofthesesyntheticconjugateswereidenticalwiththoseofthenatural product，However，thediastereoisomerscouldnotbedifferentiatedfromeachotherby physicalmethods・Uponenzymichydrolysiswiththeaboveenzymepreparation,thesyntheticL-histidineester(Id)providedbufbtalin3-hemisuberate(Ia)whiletheD-isomerester(Ie)gave onlythegenin(bufbtalin)insteadoflaevenafterincubationfbraprolongedtime・Asimilarresultwasobtainedfbrenzymichydrolysisofepimericmarinobufagin3-suberoylglutamine esters､6）Theseresultssupportedtheassignmentofthestructurcbufbtalin3-suberoyl-L-histidineestertothenewbufbtoxin(Id)． Thesecondncwbufbtoxin(If),mpl70-174oC(dec.),[０.ザ＋5.6゜,gaveanegativeninhydrintest・Hydrolyticcleavagewith6Nhydrochloricacidfilrnishedsolely3-methylhis‐tidineasananunoacidcomponent、Enzymichydrolysisandsubsequentmethylationwithdiazomethaneyieldedbufbtalin3-hemisuberatemethylester(Ib)asdescribedabove.Inorder toestablishthecompletestructure，bufbtalin3-suberoyl-L-3-methylhistidineesterwas synthesizedasdescribedabove・ThesyntheticsampleprovedtobeidenticalwiththenaturalprOduct・TheabsoluteconfigUrationoftheaminoacidwasconsideredtobeL-asjudgedfrom！ ｌ 塵でNo.１１ 4399 (Kawasaki)．L-l-Methylhistidine，hogpancreaslipasepreparationandotherreagentswerepurchasedfrom Calbiochem-BehringCo.(LaJolla,ＣＡ),SigmaChemicalCo.(StLouis,ＭＯ)andNakaraiChemicalsLtd(Kyoto)， rcspectively､HPLCwascarriedoutonaWatersALC/GPC202chromatographequippedwitｈａＵＶｄｅｔｅｃｔｏｒ (280,ｍ)andａ似BondapakC18column(30cmx039cmi.｡.)(WatersAssoc.,Milfbrd,ＭＡ)ataflowrateoflml/minunlessotherwisestated・ExtractionofSteroidalComponents-Eightytoads(B2(/ｂ'MZz"Cs"α"sScHNEIDEIOobtainedfromVivariuｍ Ｃｏ.(Tokyo)weresacriiicedbyfreezingindry-iceTheskinswereimmediatelyHayedofTandextractedwithEtOH(16 1)fbraweekAfterremovalofinsolublematerialsbyfiltrationthroughalayerofCelite,thefiltratewasconcentrated j"wJcuJobelow50oC,ａｎｄｐartitionedwiththehexane-H20andthenAcOEt-HzOsystemsthreetimeseach､Sterols andftlttyacidswereextractedintothehexanelayerBufbgeninsandtheirconjugateswereextractedintotheAcOEt andaqueouslayers,respectively､2,3)19-Hydroxybufalin(10ｍｇ)，)wasobtainedfipomtheAcOEtlayerbycolumn chromatography・ColumnChromatographyonAmberliteXAD-2Resin-TheaqueouslayerwasconcentratedmvaczJobelow 50゜CtoremovetheorganicsolventanddilutedwithH20(101).TheresultingsolutionwaspercolatedthroughacolumnpackedwithAmberliteXAD-2resin(50ｃｍｘ３ｃｍｉ.。)toadsorbbufbgeninconjugatesThecolumnwaswashedwithH20(151),andthenelutedsuccessivelywith60％ＭｅＯＨ(500,,,70％ＭｅＯＨ(500ｍl),８０％ＭｅＯＨ (500ｍl),９０％ＭｅＯＨ(500ｍl),andanhydrousMeOH(11)2,3） IsoMonofBufbtaIin3-Suberoyl-L-histidineEster(I｡)－Fr､２(seeChartl)wasconcentratedmwzc"otoafIbrd abrownresidue(250ｍｇ),whichwaschromatographedonsilicagel(45ｃｍｘ２ｃｍＬｄ.)andelutedsuccessivelywith AcOEt（100ｍl),AcOEt-MeOH(2:1)(200ｍl),AcOEt-MeOH(1：1）(300ｍl),AcOEt-MeOH（1:2)(250ｍl),and anhydrousMeOH(100ｍl).Ｆｒ､６(120ｍｇ)wasrechromatographedonsilicagel(40ｃｍ×1.5ｃｍｉｄ)usingCHCl3-MeOH-HzO(80:20:２５)asasolventtogiveFr､７(70ｍｇ),whichwasseparatedbyHPLCusingMeOH-0.3％ (NH4)zCO3(5:4)asamobilephasetogiveFr､８andFr､9.Ｆr､８ｗａｓｈlrtherpurifiedbyHPLCusingCH3CN-0.3％ (NH4)zCO3(1:2)asamobilephasetogiveFrlOandFr､１１Ｆr､３(200ｍｇ)wasalsoprocessedinasimilarfaShion・TreatmentofdriedFr,１０ｃｏｍｂｉｎｅｄｗｉｔｈｔｈｅｃorrespondingfractionderivedfroｍＦｒ､３ｗｉｔｈＭｅＯＨ毛ｔｈｅｒｇａｖｅｌｄ(3ｍｇ)asacolorlessamorphoussubstancempl93-193.5°Ｃ(dec.).[００部-8.1゜(c＝0.09,MeOH).lH-NMR(CD30D/CDCl3)６:0.78(3Ｈ,s,18-CH3),0.98(3Ｈ,s,l9-CH3),1.85(3Ｈ,s,OCOCH3),5.06(1Ｈ,ｍ'3ａ－Ｈ),5.50(ｌＨ， ｍ,l6a-H),6.18(1Ｈ,ｄ,ノー10Ｈｚ,23-Ｈ),7.00(1Ｈ,brs,４'‐H),7.30(1Ｈ,。,Ｊ＝３Ｈｚ,21-Ｈ),８．１６(2Ｈ,ｍ,22-Hand2'‐Ｈ）Compoundldshowedthesamechromatogaphicbehaviorwithasyntheticsample.ＨPLC[CH3CN-0.3％ (NH4)zCO3(l:2);ＺＲ１２５ｍｉｎ],ＴＬＣ[CHCl3-MeOH-H20(80:20:2.5);Ｒ/014]・IsoMionofBufOtalin3-Suberoyl-し3-methylhistidine(If)and-1-methylhistidine(19)Esters--Fr､１１togetherwiththecorrespondingfractionfromFr､３wassubjectedtopreparativeTLCusingCHC13-MeOH-H20(80:20:2.5） asadevelopingsolventElutionoftheadsorbentcorrespondingtothespotofR/Ul8withCHCl3-MeOH-H20 (80:20:2.5)andrecrystallizationofthedriedeluatefromacetonegavelf(3ｍｇ)asacoIorlessamorphoussubstance・mpl70-174°Ｃ(dec.)[Ｃｌ：＋５６゜(c＝0.07,McOH)ｌＨ－ＮＭＲ(CD30D/CDCl3)６:0.78(3H,s,l8-CH3),0.98(3H，s,l9-CH3),１．８６(3Ｈ,s,OCOCH3),３．６５(3Ｈ,s,N-CH3),5.06(1Ｈ,ｍ,３０(-Ｈ),５．５０(1Ｈ,ｍ,１６α-Ｈ),６．１８(1Ｈ,。,ノー10Ｈｚ,23-H),7.00(1Ｈ,brs,４'一Ｈ),７．３２(1Ｈ,ｄ,Ｊ＝３Ｈｚ,21-Ｈ),800(1Ｈ,brs,２'一Ｈ),8.20(1Ｈ,．｡,Ｊ＝10,3Ｈｚ,２２‐Ｈ)ElutionoftheadsorbentcorrespondingtothespotofR/nl2withCHCl3-MeOH-H20(80:20:2.5)gavelg (2ｍｇ)asacolorlessoil1H-NMR(ＣＤ３０Ｄ/CDCl3）６：078(3Ｈ,ｓ,18-CH3),０．９８(3Ｈ,ｓ,19-CH3),Ｌ８６(3Ｈ,s， OCOCH3),３．８０(3Ｈ,s,N-CH3),5.06(1Ｈ,ｍ,３ａ－Ｈ),５．５０(1Ｈ,ｍ,l6a-H),６．１８(1Ｈ,。,Ｊ＝10Ｈｚ,23-Ｈ),７．００(1Ｈ，brs,４'一Ｈ),7.32(1Ｈ,。,Ｊ＝３Ｈｚ,21-Ｈ),8.08(1Ｈ,brs,２'‐Ｈ),8.20(1Ｈ,．｡,Ｊ＝10,3Ｈｚ,22-Ｈ).CompoundslfandIgshowedthesameHPLCchromatographicbehaviorasthecorrespondingsyntheticsamplesontwodifIbrentcolumns UBondapakC18,TSKgell20T(ToyoSodaCo.,Tokyo))with3solventsystems(MeOH-0.3％(NH4)2CO3(5:3)， CH3CN-0.3％(NH4)2CO3（1:2),tetrahydrofilran-03％(NH4)zCO3(2:5))． IsoMonofl9-Hydroxybufillin3-Suberoyl-IPhistidine（Ⅱ｡）ａｎｄ‐3-inethylhistidine（Ⅱe）Esters-Fr,gwas fnrtherpurifiedbyHPLCusingCH3CN-0.3％(NH4)2CO3(3:7)asamobilephasetogiveFr､１２andFr､１３The samefi｢actionswerealsoobtainedfiFomFr、３byalmostthesamepurificationprocedures・Ｆｒ、１２togetherwiththecorrespondingfractionfromFr3wascvaporatedmvac灘otogivelld(8ｍｇ)asacolorlessoilMざ＋3.0｡(c＝０１７，ＭｅＯＨ).ｌＨ－ＮＭＲ(pyridine-必/CDCl3)５：０．７９(3Ｈ,ｓ,18-CH3),３．５６ａｎｄ４．０２(ｅａｃｈｌＨ,ｅａｃｈｄ,Ｊ＝12Ｈｚ,１９‐CU20H),５．２１（1Ｈ,ｍＪ'i/2＝１１Ｈｚ,３ａ－Ｈ),６．２４(1Ｈ,ｄ,Ｊ＝１２Ｈｚ,23-Ｈ),７．０４(1Ｈ,brs,４'一Ｈ),７．２９(1Ｈ,s,21-Ｈ)，7.76(1Ｈ,brs,２'一Ｈ),８．１１（1Ｈ,。,Ｊ＝12Ｈｚ,22-Ｈ).Ｆｒ､１３togetherwiththecorrespondingfractionfiPomFr3wasevaporatedmvac"otogivelIe(10ｍｇ)asacolorlessoil[α]ＡＰ＋４８。(c＝026,MeOH).ｌＨ－ＮＭＲ(pyridineWCDC13)６:０．８４(3Ｈ,s,18-CH3),3.62and4.09(ｅａｃｈｌＨ,each。,Ｊ＝12Ｈｚ,l9-CLJ20H),5.28(1Ｈ,ｍ,WPi/2＝11Ｈｚ，3αＨ),６２５(1Ｈ,。,Ｊ＝12Ｈｚ,23-Ｈ),６．８３(1Ｈ,brs,４'一Ｈ),７．３５(1Ｈ,s,21-Ｈ),7.45(1Ｈ,brs,２'一Ｈ),８．１７(1Ｈ,。,Ｊ＝12Ｈｚ,22-H)． HydrolysisofBufDtoxinwith6NHCl-Bufbtoxin(1ｍｇ)washeatedwith6NHC1(0.5ｍl)inasealcdtubeat llOoCfbr8h.Ａｐortionoftheresultingsolutionwasappliedtotheaminoacidanalyzer、EnzymicHydrolysisofld,If,andIg-Id,IforIg(1ｍｇ)wasdissolvedmlO％ＭｅＯＨｉｎ1％ＮａCl(2.5ｍl)and incubatedwithhogpancreaslipaseprcparation(1ｍｇ)at37oCfbr2hTheincubationmixturewasconcentratedm wzcHoandextractedwithAcOEtTheextractwassubjectedtopreparativeTLCusingAcOEtasadevelopingsolvent． ｌ１ｉｉ 灘蕊liIlllilli 欝口lilI lIillllllliE 4401 Ｎｏ．１１ ReferencesandNotes PartCCIVof``StudiesonSteroids，，ｂｙＴ,Nambara;PartCCIII:Ｈ､Hosoda,Ｈ・Yokohama,ａｎｄＴ､Nambara，die､､ＰｈａｒｍＢ"".,32,4023（1984).Apartofthisworkhaspreviouslybeenpresentedasapreliminary communication:KShimada,Ｋ・Ohishi,ａｎｄＴ､Nambara,T1eZraAe小o〃Ｌｅ".,２５，５５１（1984)．Ｋ､Shimada,YFUjii,EYamashita,YNiizaki,YSato,andTNambara,Ｃ/1ｃｍ.Ｐ/zarm.Ｂ"ﾉﾉ.'25,714(1977)． Ｋ・ＳｈｉｍａｄａａｎｄＴ､Nambara,Ｃ/ｌｅｍＰ/ｌａｒｍＢ"ﾉﾉ.,27,1881（1979)．KShimada,Ｙ・FUjii,andTNambara,Ｚ１ｅ２ｒａｈｅ巾o"Ｌα/・'1974,2767;YFUjii,KShimada,andTNambara，die腕.ｈｃﾉ.(London),1976,614.Ｙ・FUjii,Ｋ・Shimada,YNiizaki,ａｎｄＴ・Nambara,Z1eZraAe巾o〃Ｌｅｎ.,1975,3017.Ｋ.ShimadaandTNambara,Ｃ/iem､ＰｈａｒｍＢＭ.,28,1559(1980)． Theenzymepreparationalsocontainsamylaseandpeptidase NH6riger,DZivanov,ＨＨＡＬｉｎｄｅ,andKMeyer,ＨＣ/v､ＣﾉZmAcra,53,1993(1970)． RVerpoorte,Phan-qu66-Kinh,ａｎｄＡ・BSvendsen,Ｊ､ﾉVZzZ"rαﾉProdms,４３，３４７(1980)．1） 誉 2） 3） 4） 5） 6） 7） 8） 9） ili 、

Isolation and Characterization of New Bufotoxins from the Skin of Bufo melanostictus SCHNEIDER

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Isolation and Characterization of New Bufotoxins from the Skin of Bufo melanostictus SCHNEIDER

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