thiol reactivity of curcumin and its oxidation products paula b. luis论文.pdf

该【thiol reactivity of curcumin and its oxidation products paula b. luis论文 】是由【小舍儿】上传分享，文档一共【38】页，该文档可以免费在线阅读，需要了解更多关于【thiol reactivity of curcumin and its oxidation products paula b. luis论文 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..essprovidedby-Accesspaidbythe|,,.,eptedManuscript?DOI:.7b00326?PublicationDate(Web):23Mar2018Downloadedfrom,2018epted“epted”manuscriptshavebeenpeer-,“epted”munitytoexpeditethedisseminationeptance.“epted”manuscriptsappearinpaniedbyanHTMLabstract.“epted”manuscriptshavebeenfullypeerreviewed,(DOI?).“epted”,the“epted”,itwillberemovedfromthe“epted”,“epted”.,Washington,?,,orworksmonwealthrealmCrowngovernmentinthecourseoftheirduties.:..,,andClausSchneider*17181920DepartmentofPharmacology,Division,ofClinicalPharmacology,andVanderbiltInstituteof2122ChemicalBiology,VanderbiltUniversityMedicalSchool,Nashville,Tennessee37232,-typeaddition,quinonemethide,turmeric,curcuminoids,glutathione,2627spiroepoxide282930313233343536373839404142434445464748495051525354555657585960ACSParagonPlusEnvironment1:..ChemicalResearchinToxicologyPage2of37123TOCgraphic456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960ACSParagonPlusEnvironment2:..--Michaeladductswiththreemodelthiols,glutathione,N-1415acetylcysteine,andβ-,however,--mercaptoethanolatthe21225’-positionoftheringgavea1,7-dihydroxycyclopentadione-5’thioether,additionatthe1’-2324positionresultedincleavageofthearomaticringfromthemolecule,formingmethoxyphenol--2829andbisdemethoxycurcumindidnotformallofthepossiblethioetheradducts,-likecellsactivated323334withphorbolesterformedcurcumin-glutathionylandthe1,7-dihydroxycyclopentadione-5’-,:..ChemicalResearchinToxicologyPage4of37123INTRODUCTION4567Researchoverthepasttwodecadeshasuncoveredawealthofbiologicalactivitiesof89curcumin,,anti-inflammatory,1011antimicrobial,andanti-,2121314Onehypothesistoexplainthepolypharmacologiceffectsofcurcuminiscovalentbindingto1516cellularproteininducingafunctionalchangetoenzymes,proteinkinases,transcriptionfactors,-likeadditionofaprotein19otheenoneelectrophileoftheheptadienedionechainofcurcumin(Scheme1).This2122ount,forexample,fortheinhibitionofNF-κBactivityby2425adductionofcurcumintotheupstreamactivatingkinase,,,,however,littlemechanisticinformationonhowcurcuminbindstoproteinor3637smallmoleculethiolsexceptforthestructuralanalysisofcurcuminandacloseanalogbindingto3839GSHorcysteamine,respectively,thatshowedtheexpectedadditionatC-,(Scheme1),:..Page5of37ChemicalResearchinToxicology123theenoneofcurcumin,thequinonemethideformeduponH-abstraction,andthespiroepoxide456intermediateresultingfromcyclizationandoxygenation(Scheme1).Inanattempttoestimate78thecontributionofoxidativetransformationofcurcumintoproteinbindingwehavesynthesized910alkynyl-,(s),,demethoxy-(DMC)andbisdemethoxycurcumin(BDMC),withthesmallmolecule363738thiolsGSH,NAc,,DMC,(hanol)werestoredat-20°(curcuminfromCurcumatlongapowder;C1386),HRP(type-II,5kU/mL,535455P8250),andphorbolester(12-O-tetradecanoylphorbol-13-acetate,PMA)wereobtainedfrom5657585960ACSParagonPlusEnvironment5:..,vinylether,andbicyclopentadionederivativesofcurcuminwereprepared45146byautoxidationofcurcuminfollowedbyRP-(50μM)wasaddedto50mMammoniumacetatebuffer1617(),NAc,orβME(1mM)were1819addedeitherbeforeor20minafterinitiationofautoxidation,-mgWatersHLBcartridgespre-2324conditionedwithMeOH,water,andcold50mMammoniumacetatebuffer()andeluted2526withMeOH(350μL).Theeluateswereevaporatedtodrynessunderastreamofnitrogenand272829reconstitutedinmethanolforanalysisbyRP-HPLCoranalyzeddirectly(withoutconcentration)3031byLC-MS,(50μM)in50mMammoniumacetatebuffer(pH38398)wereconductedat20°Cfor19hfollowedbyadditionofthiol(1mM)(10mlof50mMammoniumacetatebuffer,pH8)ofDMCandBDMC424344(50μM)usedHRP(200μlofa1:10,000,000dilution;)andH2O2(40μM).Reactions4546wereconductedfor30minbeforeadditionofthiols(1mM)-(50μM)wasdissolvedin50mlof20mMammonium5455acetatebuffer(pH8)-ME(10mM)wasaddedandallowedto5657585960ACSParagonPlusEnvironment6:..--HPLCusedaWatersSymmetryC18column78(;5μm)elutedwithagradientofsolventA(50mMammoniumacetatebuffer,pH9108)andB(acetonitrile)at1mL/%Ato70%111213Ain15minandthento20%%fetal2324bovineserumat37°Cin5%-(500nM)for5minfollowedbyincubationwithcurcumin(10μM),--(,)-95%%formicacidover2minfollowedby1min5354ofisocraticelutionwith95%%:..-20%over4min,20-45695%over2minand95%%,DMC,andBDMCwiththiolswereanalyzedintheMS1910mode,-()wereanalyzedintheSRMmodeusingm/-GSadduct;andm/-GS1617adduct(1,7-dihydroxy-cyclopentadione-5’-GS)./(9:1),CD3OD,--deuteratedacetonitrile(δ=),methanol(δ=),ordichloromethane(δ=)(50μM)-foldmolarexcessof4950GSH,NAc,-(m/z367)accountedforthemostabundantionchromatogramin5455allreactions().TheBCPoxidationproduct(m/z399)wasabouthalfasabundant,5657585960ACSParagonPlusEnvironment8:..-GSHadductandatm/z706indicatingthat78GSHhadalsoformedanadductwithadioxygenatedmetaboliteofcurcumin().9101112131415ThereactionofcurcuminwithNAcdidnotshowadistinctcurcumin-NAcadduct(m/z530)161417().Instead,twoNAcadductswithadioxygenatedcurcuminmetabolite(m/z562),βMEreactedmorereadilythanGSHorNActoformadducts202122withcurcumin(m/z