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首页 Development of a novel rhodamine type fluorescen…

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

Development of a novel rhodamin…

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2010-05-24 0人阅读 0 0 0 暂无简介 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

Talanta()–DevelopmentofanovelrhodaminetypefluorescentprobetodetermineperoxynitriteXiaoFengYang,XiangQunGuo*,YiBingZhaoDepartmentofChemistryandtheKeyLaboratoryofAnalyticalSciencesofMOE,XiamenUni�ersity,Xiamen,Fujian,PRChinaReceivedDecemberreceivedinrevisedformMarchacceptedMarchAbstractAnovelmethodforthedeterminationofperoxynitriteusingrhodamineBhydrazideasafluorogenicprobeisdescribedThemethodisbasedontheoxidationofrhodamineBhydrazide,acolorless,nonfluorescentsubstance,byperoxynitritetogiverhodamineBlikefluorescenceemissionThefluorescenceincreaseislinearlyrelatedtotheconcentrationofperoxynitriteintherangeof×−–×−moll−withadetectionlimitof×−moll−(�)Theoptimalconditionsforthedetectionofperoxynitritewereevaluatedandthepossibledetectionmechanismwasalsodiscussedinthispaper©ElsevierScienceBVAllrightsreservedKeywords: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)$seefrontmatter©ElsevierScienceBVAllrightsreservedPII:S()XFYangetalTalanta()–Thus,these‘dihydro’derivatescanserveasfluorogenicprobesformonitoringperoxynitriteproductioninbiologicalsystemsRhodaminederivativesareprizedfortheirgreatphotostability,pHinsensitivityoverabroadrange(lowtoneutralpH),highquantumyieldinaqueoussolutionandtobeexcitableatlongwavelength,andhavebeenwidelyusedforstudyingbiologicalsystemsInthepresentstudy,thecharacteristicofanonfluorescentspiroformrhodamineBhydrazidewasstudiedandtheresultsshowedthatithasasensitiveresponsetoproxynitriteThereactionofperoxynitritewithrhodamineBhydrazide,acolorless,nonfluorescentcompoundresultsinadramaticincreaseinfluorescenceintensityofthereactionmixtureBasedonthisphenomenon,anovelfluorescentprobeforperoxynitritewasdevelopedTheobjectivesofthepresentstudyaretoevaluatetheabilityofrhodamineBhydrazideasaprobeforperoxynitriteanditspotentialuseinbiologicalsystemsExperimentalChemicalsStocksolutionofrhodamineBhydrazide(II,×−moll−)waspreparedbydissolvingappropriateamountofIIinacetonitrile–watersolutionSuperoxidedismutase(SOD)(frombovineerythrocytes,Umg−)wasobtainedfromSigmaAdimethylsulfoxide(DMSO,moll−)solutionwaspreparedAmoll−ofKHPO–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:themixturesolutioncontainingmoll−ofhydroxylamine,moll−ofsodiumhydroxideandmoll−ofEDTA,wasstirredvigorouslyinaerobicconditionsforabout–h,thensomeMnOpowderSchemeXFYangetalTalanta()–SchemewasaddedtothemixturesolutiontoeliminateHOgeneratedinthereactionsolution,afterthatthemixturewasfilteredandstoredat−°CPeroxynitriteconcentrationwasdeterminedbyUVspectrometryatnm(�=lmol−cm−)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,moll−NaOH,moll−EDTA,moll−Thereactionwascarriedoutataerobicconditionatroomtemperature(°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,correspondingtoONOO−isformedEDTAisaddedasthemetalionsequesteringtothereactionsolutiontoprecludethedecompositionofperoxynitriteTheperoxynitriteconcentrationisestimatedfromabsorbanceatnm,nitrateandperoxidescarcelyshowsabsorbanceinthisregion(�=andlmol−cm−,respectively)Moreover,thecoexistingspecies,suchashydroxylamine,NO−,NO−,andHOshownoresponsetothefluorescenceincreaseofthesolutionofII(seeSection)Thepresentmethodforthepreparationofperoxynitriteiseasytooperate,needsnospecialequipmentandonlyinexpensiveandeasyavailablereagentsareusedSpectracharacteristicsIIisacolorless,nonfluorescentsubstanceWhenperoxynitritewasintroducedtothesolutionofII,afluorescenceemissionsimilartothatofrhodamineBwasobservedFigshowsthefluorescenceexcitationandemissionspectraofIIandthemixtureofIIwithperoxynitriteinphosphatesolution(pH)ItcanbeseenthatIIshowsnoobviousspectracharacteristics,neitherFigKineticbehaviorofthepresentsystem(a)II(b)IIperoxynitrite(×−moll−)(c)IIperoxynitrite(×−moll−)II,×−moll−ThefluorescencedevelopmentandmeasurementwerebothcarriedoutinpHofphosphatebuffersolutionXFYangetalTalanta()–FigEffectofpHonthefluorescencedevelopmentreactionofthesystemThereactionofII(×−moll−)withperoxynitrite(×−moll−)wascarriedoutinphosphatebufferwithdifferentpHvalueformin,thenpHofthesystemwasadjustedtoandthefluorescenceintensitywasrecordedconcentrationofIIwasupto×−moll−,therefore,×−moll−ofIIwasrecommendedforthesubsequentexperimentStabilityofIIThestabilityofIIwasstudiedatdifferentpHvaluesandtheexperimentsshowedthefluorescencebackgroundofthesystemvariedwithpHInstronglyacidmedia(moll−sulfuricacid),thefluorescencebackgroundwasunstableandincreasedwithtime,presumablybecauseIIwaspartlydecomposedinstronglyacidmediaandhenceahighfluorescencebackgroundwasrecorded,whilethefluorescencebackgroundoftheproposedsystemwaslowandstablewhenthepHofthedetectionsystemwas�Tomaintainitsstability,IIshouldbekeptinneutralornearneutralpHsolution,andthewatersolutionofIIwasabletostandatroomtemperatureforweekswithoutapparentincreaseoffluorescencebackground(FurtherexperimentonthestabilityofIIwithtimewasnotcarriedout)PossibledetectionmechanismTheoxidationofthevarioussubstratesbyperoxynitrite(OONO−ONOOH)cantakeplaceviamultiplepathways:(i)peroxynitritemaydirectlyEffectofpHTheeffectofpHonthefluorogenicreactionwasstudiedintherangeof–inphosphatebuffersolution,andtheresultsareshowninFigItcanbeseenthatfluorescenceincrement(�F)ofthedetectionsystemwasincreasedwithpHupto,remainedalmostthesameintherangeof–,anddecreasedwhenpHvaluewasaboveHence,pHofphosphatebufferwaschosenforfluorogenicreactioninthefollowingstudyItisreportedthatperoxynitritehasapKaof,soatpH,aboutofperoxynitriteispresentintheformofONOO−,whileatpH,aboutofperoxynitriteispresentastheconjugateacid,ONOOHFromFig,itcanbeseenthatthefluorescenceincreaseofthesystembeobservedinasignificantdegreeatpHandat,respectively,indicatingthatbothONOOHandONOO−canoxidizeIItoyieldahighlyfluorescentproduct,whichisconsistentwiththeoxidizingnatureofbothspeciesEffectoftheconcentrationofIITheeffectoftheconcentrationofIIon�FofthesystemwasstudiedandtheresultswereshowninFigFromFig,itcanbeseenthat�FofthedetectionsystemwasincreasedwithincreasingtheconcentrationofIIfrom×−to×−moll−andremainedconstantwhentheFigEffectoftheconcentrationofIIonthefluorescenceincrement(�F)ofthesystemII(differentconcentrations)peroxynitrite(×−moll−)phosphatebuffer(pH)Thereactionmixturewascarriedoutatroomtemperature(°C)formin,andthenthefluorescenceintensitywasrecordedXFYangetalTalanta()–Schemeoxidizethesubstrates(ii)Peroxynitritemaydecomposefirstlyintohighlyreactivespecies(�OH,�NO),whichsubsequentlyoxidizesthesubstrateorhydroxylatesandnitratesaromaticcompoundInthepresentstudy,thepossiblemechanismoftheoxidationofIIbyperoxynitriteisstudiedFirstly,DMSO(moll−),aspecificscavengerfor�OH,wasintroducedtothereactionmixturebeforetheadditionofperoxynitrite,andwefoundthe�Fofthereactionmixturewasalmostunchanged,indicatingthat�OHdoesnotcontributetothefluorescenceincreaseofthesystemSecond,SOD(Uml−),aspecificscavengerforO−�,wasaddedtothereactionsystem,andthefluorescenceofthesystemshowednochangescomparedwiththatintheabsenceofSOD,provingthatO−�doesnotmediatethefluorescenceincreaseofthesystemTheaboveexperimentssuggestthattheoxidationofIImightarisefromtheperoxynitriteitself,butnotitsdecomposedreactivespeciesThiscanbeexplainedthatthereactionrateofperoxynitratewithII(k)wasfasterthantheselfdecompositionrateofperoxynitriteviathepathwayofanddescribedinScheme(k,kareands−,respectivelykisstillunknown,butaccordingtotheliterature,whichreportedthereactionrateofdirectoxidationofsomecompoundsbyperoxynitritewere�lmol−s−,weassumethatthevalueofkis�lmol−s−)Third,comparisonoftheexcitationandemissionfluorescencespectraofthereactionsystemwiththatofauthenticrhodamineBshowsthattheyareidentical,bothhavingexcitationmaximumatnmandemissionmaximumatnm(showninFig),indicatingthatthefluorescentproductgeneratedinthereactionmixturemightberhodamineBBasedontheaboveexperimentalresults,apossiblemechanismoftheproposedmethodisgivenbySchemeLimitofdetection,linearconcentrationrangeandprecisionUndertheselectedconditionsgivenabove,thefluorescenceincrementshowsalinearrelationshipwiththeconcentrationofperoxynitriteintherangeof×−–×−moll−(r=,n=)Thedetectionlimitis×−moll−(�)Therelativestandarddeviation(n=)isfor×−moll−ofperoxynitriteInterferencestudyTheinterferencesoftheproposedprobeforperoxynitritewerestudiedAvarietyofinterferingagentswereaddedtothesolutionofIIinpHofphosphatebuffer,andtheresponseofIItothesecompoundsarelistedinTableInterestingly,itwasfoundthatashighas−moll−ofCudidnotproducesignificantfluorescenceincrementofthedetectionsystemdescribedinthispaper,whichisnotcontradictedwiththefactdescribedbyDujolsetal,becausetheselectiveresponseofIItoCumustbecarriedoutinacetonitrile–watersolutionIfofacetonitrilewasaddedtothesolutionofIIcontainingCu,asimultaneousfluorescenceincrementwasobservedHence,theproposedprobeforperoxynitritewithoutacetonitrilecouldsuccessfullyavoidtheinterferencefromCuXFYangetalTalanta()–ConclusionInsummary,anovelfluorogenicprobeforperoxynitritewasproposedinthispaperThepresentmethodforthedeterminationofperoxynitriteissimple,fastandsensitiveTheresponsetimeoftheproposedprobeforperoxynitriteislessthans,andithasadetectionlimitof×−moll−forperoxynitriteComparedwiththetwofluorescentprobes(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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