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首页 Development of a novel rhodamine type fluoresce…

Development of a novel rhodamine type fluorescent probe to determine peroxynitrite.pdf

Development of a novel rhodamin…

上传者: silly 2010-05-24 评分 0 0 0 0 0 0 暂无简介 简介 举报

简介:本文档为《Development of a novel rhodamine type fluorescent probe to determine peroxynitritepdf》,可适用于工程科技领域,主题内容包含Talanta()–Developmentofanovelrhodaminetypefluorescentprobetodetermineperox符等。

Talanta()–DevelopmentofanovelrhodaminetypefluorescentprobetodetermineperoxynitriteXiaoFengYang,XiangQunGuo*,YiBingZhaoDepartmentofChemistryandtheKeyLaboratoryofAnalyticalSciencesofMOE,XiamenUniersity,Xiamen,Fujian,PRChinaReceivedDecemberreceivedinrevisedformMarchacceptedMarchAbstractAnovelmethodforthedeterminationofperoxynitriteusingrhodamineBhydrazideasafluorogenicprobeisdescribedThemethodisbasedontheoxidationofrhodamineBhydrazide,acolorless,nonfluorescentsubstance,byperoxynitritetogiverhodamineBlikefluorescenceemissionThefluorescenceincreaseislinearlyrelatedtotheconcentrationofperoxynitriteintherangeof–mollwithadetectionlimitofmoll()TheoptimalconditionsforthedetectionofperoxynitritewereevaluatedandthepossibledetectionmechanismwasalsodiscussedinthispaperElsevierScienceBVAllrightsreservedKeywords:PeroxynitriteRhodamineBhydrazideFluorimetryProbewwwelseviercomlocatetalantaIntroductionPeroxynitrite,theproductofthecombinationreactionbetweennitricoxide(NO)andsuperoxideradical(O),isapotentbiologicaloxidantthatpromotesoxidativemolecularandtissuedamage,Peroxynitriteformationandreactionsareproposedtocontributetothepathogenesisofaseriesofdiseases,includinginflammatoryprocesses,ischemiareperfusion,septicshockandneurodegenerativedisorders–Studyoftheroleofperoxynitriteneedsrealtimeandsensitiveprobingofperoxynitriteinbothpathologicalandnormalconditionsinbiologicalsystems,whichisindeedextremelydifficultbecauseofthelowconcentration,highactivityandelusivenatureofperoxynitritePeroxynitritegenerationisusuallymeasuredbyUV–visiblespectrometry,chemiluminescence,,electrochemistry,andimmunohistochemistryTwofluorogenicprobes,dihydrodichlorofluorescein(DCFH)anddihydrorhodamine(DHR),havebeenwidelyemployedtomonitorperoxynitriteproductioninavarietyofsystems–Themethodsarebasedontheuseofchemicallyreduced,nonfluorescentformsofhighlyfluorescentdyessuchasfluoresceinandrhodaminethatareoxidizedbyperoxynitritetotheparentdyemolecule,resultinginadramaticincreaseinfluorescenceintensity*CorrespondingauthorTel:fax:Emailaddress:xqguojingxianxmueducn(XQGuo)$seefrontmatterElsevierScienceBVAllrightsreservedPII:S()XFYangetalTalanta()–Thus,these‘dihydro’derivatescanserveasfluorogenicprobesformonitoringperoxynitriteproductioninbiologicalsystemsRhodaminederivativesareprizedfortheirgreatphotostability,pHinsensitivityoverabroadrange(lowtoneutralpH),highquantumyieldinaqueoussolutionandtobeexcitableatlongwavelength,andhavebeenwidelyusedforstudyingbiologicalsystemsInthepresentstudy,thecharacteristicofanonfluorescentspiroformrhodamineBhydrazidewasstudiedandtheresultsshowedthatithasasensitiveresponsetoproxynitriteThereactionofperoxynitritewithrhodamineBhydrazide,acolorless,nonfluorescentcompoundresultsinadramaticincreaseinfluorescenceintensityofthereactionmixtureBasedonthisphenomenon,anovelfluorescentprobeforperoxynitritewasdevelopedTheobjectivesofthepresentstudyaretoevaluatetheabilityofrhodamineBhydrazideasaprobeforperoxynitriteanditspotentialuseinbiologicalsystemsExperimentalChemicalsStocksolutionofrhodamineBhydrazide(II,moll)waspreparedbydissolvingappropriateamountofIIinacetonitrile–watersolutionSuperoxidedismutase(SOD)(frombovineerythrocytes,Umg)wasobtainedfromSigmaAdimethylsulfoxide(DMSO,moll)solutionwaspreparedAmollofKHPO–NaHPObuffer(pH)wasalsopreparedAllthereagentswereofanalyticalreagentgrade,anddoublydistilledwaterwasusedthroughoutSynthesisofrhodamineBhydrazide(II)AmodificationoftheprocedureofDujolsetalforthesynthesisofIIwasemployedIIwassynthesizedbyaonestepreactionofrhodamineB(I)withhydrazinehydrateinmethanol(Scheme)ToagofrhodamineB(I)dissolvedinmlofmethanol,anexcessivehydrazinehydrate(ml)wasaddedandthenthereactionsolutionwasrefluxedtillthepinkcolordisappearedAfterthat,thecooledreactionsolutionwaspouredintodistilledwaterandextractedwithethylacetate(ml)Thecombinedextractsweredriedwithsodiumsulfateanhydrous,filtered,andthenevaporatedThesolventyieldedg()ofIIMS(CI)me,(MH)M,calculatedHNMR(CDCl):(m,H,ArH),(m,H,ArH),(m,H,ArH),(d,H,xantheneH),(d,H,xantheneH),(dd,H,xantheneH),(s,H,NH),(q,H,NCHCH),(t,H,NCHCH)SynthesisofperoxynitritePeroxynitritewassynthesizedbytheautoxidationofhydroxylamineinalkalinemediumTheprocedurewasasfollows:themixturesolutioncontainingmollofhydroxylamine,mollofsodiumhydroxideandmollofEDTA,wasstirredvigorouslyinaerobicconditionsforabout–h,thensomeMnOpowderSchemeXFYangetalTalanta()–SchemewasaddedtothemixturesolutiontoeliminateHOgeneratedinthereactionsolution,afterthatthemixturewasfilteredandstoredatCPeroxynitriteconcentrationwasdeterminedbyUVspectrometryatnm(=lmolcm)ApparatusThefluorescencespectraandrelativefluorescenceintensityweremeasuredwithaShimadzuRFspectrofluorimeter(Kyoto,Japan)withammquartzcuvetteTheexcitationandemissionwavelengthbandpasseswerebothsetatnmAbsorptionspectrawereobtainedonaBeckmanDUUltraviolet–VisibleSpectrophotometerAllpHvaluesweremeasuredwithapHSdigitalionmeterProcedureInasetofmlvolumetrictubescontainingpHofphosphatebuffersolution,mlofII(moll)anddifferentamountofperoxynitritewereaddedThereactionsolutionwaskeptatroomtemperatureformin,andthenthereactionsolutionwasdilutedtothemarkwithwaterThefluorescenceintensityofthesolutionwasrecordedatnmwiththeexcitationwavelengthsetatnmResultsanddiscussionPreparationofperoxynitriteThereareseveralmethodsreportedforthesynthesisofperoxynitriteThecommonlyusedmethodforthesynthesisofperoxynitriteisbasedonthefastnitrosationofHOinacidmediumtoyieldthesolutionoftheunstableperoxynitrousacid(ONOOH),towhichNaOHisaddedimmediatelytogeneratetheperoxynitriteanion(ONOO)However,theelusivenatureofONOOH(t,s)makesthe‘timing’ofbothprocesses(nitrosationandadditionofNaOH)thekeypointforthesuccessoftheprocedureTherefore,thismethodoftenrequirestheuseofaquenchedflowreactorAnothercommonlyusedmethodisbasedonthereactionofHOwithalkylnitritesinalkalinesolutionsThismethodisclean,simpleandfast,butalkylnitriteisnotwidelyavailableHughesetalhadreportedthattheautoxidationofhydroxylamineinalkalinesolutionsledtotheformationofperoxynitriteasthemajorproductHence,wesynthesizedperoxynitritebasedonthisreaction(Scheme)FigshowsthetypicalspectraofthesystematFigTypicalreactionspectrumshowingtheformationofperoxynitritefromtheautoxidationofhydroxylamineinalkalinesolutionHydroxylamine,mollNaOH,mollEDTA,mollThereactionwascarriedoutataerobicconditionatroomtemperature(C),withvigorousstirringThespectrumwasrecordedbytransferringmlofreactionmixturetoamlvolumetrictubeatdifferentreactiontimeanddilutingtothemarkwithwatert:(a)min(b)min(c)min(d)min(e)min(f)min(g)min(h)min(i)minXFYangetalTalanta()–FigFluorescenceexcitationandemissionspectraofthesystema,a,II(moll)b,b,II(moll)peroxynitrite(moll)c,c,rhodamineB(moll)ThereactionofperoxynitritewithIIwascarriedoutinphosphatebuffer(pH)atroomtemperature(C)forminAllthespectrawererecordedatpHofphosphatebufferexcitationnorfluorescenceemissionspectraHowever,amixtureofIIwithperoxynitriteshowsapparentspectralcharacteristicswithexcitationmaximumatnmandfluorescenceemissionmaximumatnmOptimizationofthegeneralprocedureEffectofreactiontimeThekineticcharacteristicsoftheproposeddetectionsystemwerestudiedUpontheadditionofperoxynitritetothesolutionofIIinphosphatebuffer,thefluorescenceintensityofthedetectionsystemwasrecordedasafunctionofreactiontime(asshowninFig)FromFig,onemayseethefluorescenceintensityofthedetectionsystemincreaseddramaticallyinafewsecondsandleveledoffasthereactionwenton,whilethefluorescencebackgroundofthedetectionsystemintheabsenceofperoxynitriteremainedunchangedatthesametimeThefluorescenceintensityofthedetectionsystemreacheditsmaximumvalueforaboutmin,afterthatthefluorescenceintensityofthedetectionsystemremainedalmostconstantToobtainahighsensitivityandreproducibleresults,aminreactiontimewasselectedinthefollowingexperimentdifferentstagesofreactionAsthereactionproceeds,anewbandcenteredon–nm,correspondingtoONOOisformedEDTAisaddedasthemetalionsequesteringtothereactionsolutiontoprecludethedecompositionofperoxynitriteTheperoxynitriteconcentrationisestimatedfromabsorbanceatnm,nitrateandperoxidescarcelyshowsabsorbanceinthisregion(=andlmolcm,respectively)Moreover,thecoexistingspecies,suchashydroxylamine,NO,NO,andHOshownoresponsetothefluorescenceincreaseofthesolutionofII(seeSection)Thepresentmethodforthepreparationofperoxynitriteiseasytooperate,needsnospecialequipmentandonlyinexpensiveandeasyavailablereagentsareusedSpectracharacteristicsIIisacolorless,nonfluorescentsubstanceWhenperoxynitritewasintroducedtothesolutionofII,afluorescenceemissionsimilartothatofrhodamineBwasobservedFigshowsthefluorescenceexcitationandemissionspectraofIIandthemixtureofIIwithperoxynitriteinphosphatesolution(pH)ItcanbeseenthatIIshowsnoobviousspectracharacteristics,neitherFigKineticbehaviorofthepresentsystem(a)II(b)IIperoxynitrite(moll)(c)IIperoxynitrite(moll)II,mollThefluorescencedevelopmentandmeasurementwerebothcarriedoutinpHofphosphatebuffersolutionXFYangetalTalanta()–FigEffectofpHonthefluorescencedevelopmentreactionofthesystemThereactionofII(moll)withperoxynitrite(moll)wascarriedoutinphosphatebufferwithdifferentpHvalueformin,thenpHofthesystemwasadjustedtoandthefluorescenceintensitywasrecordedconcentrationofIIwasuptomoll,therefore,mollofIIwasrecommendedforthesubsequentexperimentStabilityofIIThestabilityofIIwasstudiedatdifferentpHvaluesandtheexperimentsshowedthefluorescencebackgroundofthesystemvariedwithpHInstronglyacidmedia(mollsulfuricacid),thefluorescencebackgroundwasunstableandincreasedwithtime,presumablybecauseIIwaspartlydecomposedinstronglyacidmediaandhenceahighfluorescencebackgroundwasrecorded,whilethefluorescencebackgroundoftheproposedsystemwaslowandstablewhenthepHofthedetectionsystemwasTomaintainitsstability,IIshouldbekeptinneutralornearneutralpHsolution,andthewatersolutionofIIwasabletostandatroomtemperatureforweekswithoutapparentincreaseoffluorescencebackground(FurtherexperimentonthestabilityofIIwithtimewasnotcarriedout)PossibledetectionmechanismTheoxidationofthevarioussubstratesbyperoxynitrite(OONOONOOH)cantakeplaceviamultiplepathways:(i)peroxynitritemaydirectlyEffectofpHTheeffectofpHonthefluorogenicreactionwasstudiedintherangeof–inphosphatebuffersolution,andtheresultsareshowninFigItcanbeseenthatfluorescenceincrement(F)ofthedetectionsystemwasincreasedwithpHupto,remainedalmostthesameintherangeof–,anddecreasedwhenpHvaluewasaboveHence,pHofphosphatebufferwaschosenforfluorogenicreactioninthefollowingstudyItisreportedthatperoxynitritehasapKaof,soatpH,aboutofperoxynitriteispresentintheformofONOO,whileatpH,aboutofperoxynitriteispresentastheconjugateacid,ONOOHFromFig,itcanbeseenthatthefluorescenceincreaseofthesystembeobservedinasignificantdegreeatpHandat,respectively,indicatingthatbothONOOHandONOOcanoxidizeIItoyieldahighlyfluorescentproduct,whichisconsistentwiththeoxidizingnatureofbothspeciesEffectoftheconcentrationofIITheeffectoftheconcentrationofIIonFofthesystemwasstudiedandtheresultswereshowninFigFromFig,itcanbeseenthatFofthedetectionsystemwasincreasedwithincreasingtheconcentrationofIIfromtomollandremainedconstantwhentheFigEffectoftheconcentrationofIIonthefluorescenceincrement(F)ofthesystemII(differentconcentrations)peroxynitrite(moll)phosphatebuffer(pH)Thereactionmixturewascarriedoutatroomtemperature(C)formin,andthenthefluorescenceintensitywasrecordedXFYangetalTalanta()–Schemeoxidizethesubstrates(ii)Peroxynitritemaydecomposefirstlyintohighlyreactivespecies(OH,NO),whichsubsequentlyoxidizesthesubstrateorhydroxylatesandnitratesaromaticcompoundInthepresentstudy,thepossiblemechanismoftheoxidationofIIbyperoxynitriteisstudiedFirstly,DMSO(moll),aspecificscavengerforOH,wasintroducedtothereactionmixturebeforetheadditionofperoxynitrite,andwefoundtheFofthereactionmixturewasalmostunchanged,indicatingthatOHdoesnotcontributetothefluorescenceincreaseofthesystemSecond,SOD(Uml),aspecificscavengerforO,wasaddedtothereactionsystem,andthefluorescenceofthesystemshowednochangescomparedwiththatintheabsenceofSOD,provingthatOdoesnotmediatethefluorescenceincreaseofthesystemTheaboveexperimentssuggestthattheoxidationofIImightarisefromtheperoxynitriteitself,butnotitsdecomposedreactivespeciesThiscanbeexplainedthatthereactionrateofperoxynitratewithII(k)wasfasterthantheselfdecompositionrateofperoxynitriteviathepathwayofanddescribedinScheme(k,kareands,respectivelykisstillunknown,butaccordingtotheliterature,whichreportedthereactionrateofdirectoxidationofsomecompoundsbyperoxynitritewerelmols,weassumethatthevalueofkislmols)Third,comparisonoftheexcitationandemissionfluorescencespectraofthereactionsystemwiththatofauthenticrhodamineBshowsthattheyareidentical,bothhavingexcitationmaximumatnmandemissionmaximumatnm(showninFig),indicatingthatthefluorescentproductgeneratedinthereactionmixturemightberhodamineBBasedontheaboveexperimentalresults,apossiblemechanismoftheproposedmethodisgivenbySchemeLimitofdetection,linearconcentrationrangeandprecisionUndertheselectedconditionsgivenabove,thefluorescenceincrementshowsalinearrelationshipwiththeconcentrationofperoxynitriteintherangeof–moll(r=,n=)Thedetectionlimitismoll()Therelativestandarddeviation(n=)isformollofperoxynitriteInterferencestudyTheinterferencesoftheproposedprobeforperoxynitritewerestudiedAvarietyofinterferingagentswereaddedtothesolutionofIIinpHofphosphatebuffer,andtheresponseofIItothesecompoundsarelistedinTableInterestingly,itwasfoundthatashighasmollofCudidnotproducesignificantfluorescenceincrementofthedetectionsystemdescribedinthispaper,whichisnotcontradictedwiththefactdescribedbyDujolsetal,becausetheselectiveresponseofIItoCumustbecarriedoutinacetonitrile–watersolutionIfofacetonitrilewasaddedtothesolutionofIIcontainingCu,asimultaneousfluorescenceincrementwasobservedHence,theproposedprobeforperoxynitritewithoutacetonitrilecouldsuccessfullyavoidtheinterferencefromCuXFYangetalTalanta()–ConclusionInsummary,anovelfluorogenicprobeforperoxynitritewasproposedinthispaperThepresentmethodforthedeterminationofperoxynitriteissimple,fastandsensitiveTheresponsetimeoftheproposedprobeforperoxynitriteislessthans,andithasadetectionlimitofmollforperoxynitriteComparedwiththetwofluorescentprobes(DCFHandDHR)reportedintheliterature,theflorescentprobereportedherehavesomeadvantages:(i)longerexcitationwavelengthDCFHandDHRhavetheexcitationwavelengthofandnm,respectively,whiletheproposedprobehasthelongerwavelengthofnmThisisdesirablefortheprobingofperoxynitriteformationinbiologicalsamplesbecauseofthelowbackgroundfluorescencewiththelongerwavelengthexcitationandlesscytotoxicitycausedbylongerUVexcitation(ii)InsensitivetopHchangeinawiderrangeBecausetherhodaminefluorophoredoesnothavephenolichydroxylgroups,thefluorescenceofthereactionproductofIIwithperoxynitritewouldbeindependentofphysiologicalpHchangeandcanbeapplicableinawiderpHrange(iii)HigherstabilityIIdemonstratesgreaterphotostabilitythanthoseofDCFHandDHR,whichareextremelysensitivetolightinducedoxidationHence,webelievethattheproposedprobecanbeapplicabletostudytheperoxynitriteformationinbiologicalsystemsAcknowledgementsThisworkwassupportedbyNaturalScienceFoundationofFujianProvince(D)andNationalEducationCommitteeofChinaReferencesSGoldstein,GCzapski,FreeRadicalBiolMed()RRadi,JSBeckman,KMBush,BAFreeman,JBiolChem()RRadi,JSBeckman,KMBush,BAFreeman,ArchBiochemBiophys()JWReed,HHHo,WLJolly,JAmChemSoc()NFukuyama,YTakebayashi,MHida,HIshida,KIchimori,HNakazawa,FreeRadicalBiolMed()CSzabo,Shock(

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