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Reductive Metabolism of Nitroaromatic Compounds by Various Liver Microsomes

By Beatrice Anderson,2014-12-25 03:18
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Reductive Metabolism of Nitroaromatic Compounds by Various Liver Microsomes

    Reductive Metabolism of Nitroaromatic Compounds by Various Liver Microsomes CHEM.RES.CHINESEUNIVERSITIES2010,26(6),981985

    ReductiveMetabolismofNitroaromaticCompoundsby

    riousLiverMicrosomes

    WANGXing.yong,,CUIJing.nan,RENWeimin,ZHAOGuo.quan,LIFengandQIANXu-hong

    .StateKeyLaboratoryofFineChemicals,DalianUniversityofTechnology,Dalian116012,PR.China;

    2.SchoolofChemicalEngineeringandTechnology,ChinaUniversityofMining&Technology,

    Xuzhou221008,PR.China;3.ShanghaiKeyLaboratoryofChemicalBiology, EastChinaUniversityofScienceandTechnology,Shanghai200237,PR.China AbstraetNitroaromaticcompoundswerereductivelymetabolizedtothecorrespondingaminecompoundsviathe

    intermediatehydroxylaminesbylivermicrosomesfrompig,rat,chook,cattle,sheep,paralichthysolivaceusandcy.

    prinoidinvariedreactivity.Similarwithbaker'syeast,thepig,ratandsheeplivermicrosomesexhibitedhighreac

    tivitytoward4-nitro.12-dicyanbenzen(1a1,whilethecyprinoidlivermicrosomeswereinemcient.Contrastedto

    compoundla,monocyannitrobenzene(2a)wasdifficulttoreducebypiglivermicrosomes.Inoppositiontogrape

    cells.piglivermicrosomesexhibitedactivitiestowardsomearomatichydroxylaminecompounds.

    KeywordsLivermicrosome;Reduction;Nitroaromaticcompound;Aromatichydroxylamine

ArticleID1005.9040(20101.06-981.05

    1Introduction

    Nitroaromaticcompounds.assolventsandin

    termediates.areusedinthefieldsofpharmaceutica1. dye,plastics,agrochemicalsanddefenseindustries, andsomeofthemhavebeennoticedasimportanten. vironmentalpollutantsL'.Naturallyoccu~ingredox enzymessuchasdehydrogenasesandreductasesare knowntoreactwithanumberofnitroaromaticcom. poundst3,4J.Theseenzymesareubiquitousandcanbe obtainedfromyeast,microorganism,plantsandani

    mals.Becauseofthebiodiversity,theenzymesfrom dierentorganismsexhibitdifierentactivitiestoward thesalnenitroaromaticsubstrate.Andtowardthedif- ferentsubstrates,someoftheenzymesshouldhave direntsubstrateanities.rateofreactionandselec. tivity.Thereforc.itisimportanttounderstandtheme

    tabolismprocessOfnjtroaromaticcompoundsbythe enzymesalthoughthenitroaromaticcompoundsare nottherealsubstratesofthesenitroreductases.In general.aplausibleexplanationforthereductionOf nitroaromaticcompoundsbythenitroreductaseisas follows(Scheme11:thenitroaromaticcompoundais firstreducedviatwoelectronstoforinthearylnitrOSO bwhichacceptsanothertwoelectronstoformary1. hydroxylaminec:inthefinalreductionstep.the hydroxylamineisconvertedtoamined,byatwoelec

    tronreduction.

    ON.三旦?HO\N/H.

曼—}H\N/H

    lII

    ArArAr

    bcd

    SchemelMechanismforthereductionof

    nitroaromaticcompounds

    Itisknownthatnitroaromaticcompoundsandthe correspondinghydroxylamines,aminescouldbehave

    . verydifferentactivitiesagainsttheorganismst一?

    suchasplantcells.1ivercellsfrommammalianpisces andaves.Therefore.thestudyofreductivemetabo. 1ismofnitroaromaticcompoundsbyvariouslivermi. crosomesisveryusefulinthedesignoffunctional nitroaromaticchemicalsandthespeculationofthe metabolismprocess.Therehavebeenseveralreports onthemetabolismofsevera1nitroaromaticcom. poundsbvratlorrabbit[13livermicrosomes.

    whereastherehasbeenlittleornoknowledgeonthe reactionsOfnitrocompoundswithlivermicrosomes frompig,fish,cattle,sheepandchook.Wehave studiedthechemoselectivereductionofnitroaromatic compoundstothecorrespondinghydroxyamines usingbaker'syeast【】orplantcells[asreportedin

    previouspapers.Hereinliesreportaboutthescopeof Correspondingauthor.E-mail:jncui@dlut.edu.cn;xhqian@ecust.edu.ca

    ReceivedNovember16,2009;acceptedFebruary25,2010. SuppoSedbytheNationalBasicResearchProgramofChina(No.2009CB7247061andtheN

    ationalNaturalScienceFounda

    tionofChina(No.20876025).

    O

0

    982CHEM.

    RES.CHINESEUNIVERSITIESVb1.26

    thereductivemetabolismofnitroaromaticcompounds byvariouslivermicrosomes.2.4ReductionbyGrapeCells 2Experimental

    2.1GeneralProcedure

    MeltingpointsweredeterminedonaX..6mi.. cromeltingpointapparatusanduncorrected.Infra

    red(IR)spectraweremeasuredonaNicolet20DxB FR-IinfraredspectrometerviaKBrdisktechnique. High-resolutionmassspectra(HRMS)wereobtained onaHPLCQ-TofMS(Micro)spectrometer.NMR

    measurementwasperformedonaVianInovaspec

    trometeroperatedat400MHzorflAvancespectro. meteroperatedat500MHz.UVspectrawererecor- dedonanHP8453UV_visiblespectrophotometer. HPLCwasperformedwithAglentl100HPLC.DAD. ForthedataofHand"CNMR.IR.HRMS.EIand m.P.conferrefs.9,101.

    2.2ReductionbyLiverMicrosomes

    Thesubstrate(100mg)wasaddedtoasuspension offreshlycutliver(50in0.2mol/LTris.HClbufrer (pH6.5,100mE).Thereactionmixturewasvigo- rouslystirredat30.Candtheprocedureofthereac. tionwasmonitoredbyHPLC.Aftercompletionofthe reaction,thesuspensionwasfilteredoffandthe fiItratewasextractedwithethylacetate(200mLx3). Thenthecombinedorganicphasewasdriedoveran. hydrousMgSOd.Afterthefiltrationandtheremoval

    oforganicsolventunderreducedpressure,thecrude productswereanalyzedwithrespecttotheconver

    sion(viaHNM_RandHPLC).

    2.3ReductionbyBaker'sYeast

    Bakers'yeast(5.0g)wassuspendedin0.2mol/L phosphatebuffer(pH6.5,100mL)containingglucose (2.5g)andincubatedat30.Cfor0.5h,thenthesub

    strate(100mg)dissolvedinethanol(5mE)wasadded

     tothesuspension.Thereactionmixturewasvigo

    rouslystirredat30.Candtheprogressofthereaction wasmonitoredbyHPLC.Attheendofreaction,ethyl acetate(500mE)wasaddedtothereactionmixture. Theseparatedorganicphasewasfilteredthrougha celitepadanddriedoveranhydrousMgSO4.Afterthe filtrationandtheremovaloforganicsolventunder reducedpressure,thecrudeproductswereanalyzed withrespecttotheconversion(viaHNMRand HPLC).

    Thesubstrate(100mg)wasaddedtoasuspension offreshlycutplant(40g)in100mLofwateLandthe mixturewasstirredat25.C.Theprogressofthereac. tionwasmonitoredbvHPLC.Atierthecompletionof thereaction.thesuspensionwasfilteredoffandthe filtratewasextractedwithethylacetate(80mLx3). Thenthecombinedorganicphasewasdriedoveran. hydrousMgSO4.Afterthefiltrationandremovalof organicsolventunderreducedpressure,thecrude productswereanalyzedwithrespecttotheconver. sion(viaHNMIandHPLC).

    2.5HPLCData

Aglent1100HPLCDAD(Agilent,USA);Rxsi1,

    normalphasecolumn:5gin;4.6minx250mm

    (Agilent);reversedphasecolumn:HIQSILCl8,4.6 mm~250mm,5gm(Japan).

    Compounds1ald:normaleluent:V(nhexane):

    isopropano1)=80:20,UVdetectionat275mTl,flOW rate1.0mL/min,retentiontime:la.5.2min;lc.6.5 min;ld,12.3min;reversedeluent:citricacid(1.0g/L) inwater(solventA)+citricacid(1.0gm)inmethanol (solventB),alineargradientof30%(volumefraction) ofBto60%over26min.UVdetectionat286nn1. flowrate0.80mL/min,retentiontime:la.12.5min; lc,1O.2min;ld,l1.7min.

    Compounds2a-2d:normaleluent:V(n-hexane):

    isopropano1)80:20,UVdetectionat275nin,flow rate1.0mL/min.retentiontime:2a.4.1min;2c.6.4 min;2d.9.3min;reversedeluent:citricacid(1.0g/L) inwater(solventA)+citricacid(1.0g/L)inmethanol (solventB1.alineargradientof30%ofBt060%over 26min.UVdetectionat254nlT1.flOWrate0.80 mL/min,retentiontime:2a,l5.6min;2c,8.2min;2d 9.7min.

    Compounds3a_3d:normaleluent:V(n.hexane):

    V(isopropano1)80:20,uVdetectionat275nn'l,flow rate1.0mL/min,retentiontime:3a.3.7min;3c.5.9 min;3d,8.8min;reversedeluent:citricacid(1.0g/L) inwater(solventA)+citricacid(1.0gm)inmethanol (solventB1.alineargradientof30%0fBto60%over 26min.UVdetectionat254mT1.flOWrate0.80 mL/min,retentiontime:3a,19.9min;3c,l1.3min;3d.

l2.4min.

    Compounds4a4d:normaleluent:V(nhexane):

    isopropano1)80:20,UVdetectionat275ntn,flow No.6WANGXing-yongetal983

    rate1.0mL/rain,retentiontime:4a,3.0min;4c,4.7 min;4d.8.3rain;reversedeluent:citricacid(1.0g/L) inwater(solventA)+citricacid(1.0g/L)inmethanol (solventB1.alineargradientof3O%0fBto6O% over26min,UVdetectionat254nm,flowrate0.80 mL/minretentiontime:4a,20.0min;4c,l5.9min;4d. 19.0min.

    Compounds5a5d:normaleluent:V(n-hexane):

    V(isopropano1)=80:20,UVdetectionat275nln,flow rate1.0mL/min.retentiontime:5a,5.1min;5c.7.1 min;5d.9.7min;reversedeluent:citricacid(1.0g/L) inwater(solventA)+citricacid(1.0g/L)inmethanol (solventB),alineargradientof30%ofBto60%over 26min.UVdetectionat300nln.flowrate0.8O mL/min,retentiontime:5a,21.8min;5c,8.2min;5d 9.5min;1(4nitropheny1)ethanol,20.2min.

    Compounds6a6d:normaleluent:V(n.hexane):

    V(isopropano1)=80:20,uVdetectionat275nlTl,flOW rate1.0mL/min.retentiontime:6a,3.9min;6c.5.5 min;6d.8.0min;reversedeluent:citricacid(1.0g/L) inwater(solventA)+citricacid(1.0gm)inmethanol (solventB),alineargradientof30%ofBto51%over l8min.UVdetectionat254nin.flowrate0.80 mL/min,retentiontime:6a,14.3min;6c,6.5min;6d, 7.7min.

Compounds7a7d:normaleluent:V(nhexane):

    V(isopropano1)=80:20,UVdetectionat275nm,flow rate1.0mL/min.retentiontime:7a.4.6min;7c.8.1 min;7d.14.6min;reversedeluent:citricacid(1.0g/L) inwater(solventA)+citricacid(1.0g/L)inmethanol (solventB1,alineargradientof30%0fBto100% ofBover60min,UVdetectionat254nm.flowrate 0.80mL/min,retentiontime:7a,28.3min;7c,18.3 min;7d.20.0min.

    3ResultsandDiscussion

    Initially,4-nitro-l,2dicyanbenzene(1a)wascho-

    senasamodeltoexploretheprocessofthereduction. Inatypicalexperiment,compoundlawasaddedto piglivermicrosomesuspensionwhichwasprepared byfreshlycuttingliverin0.2mol/LTris.HClbuffer (pH=6.5),andthereactionmixturewasstirredwitha mechanicalstirrer.Thereactionprocesswasmoni

    toredbynormalorreversedphaseHPLCandthe NPHPLCprolifeisshowninFig.1.

    Asthepolaritysequenceofthecompoundsla,lc andldweredetectedbyHPLCatretentiontimeof5.2, 6.5and12.3min,respectively.Accordingtothere ductionconditions,compoundlawasreducedby 02468012l4l6

    t/min

    Fig.1Monitoringinthereductionof4-nitro-1,2-dicyan

    benzene(1a)bypiglivermicrosomesviaNP-HPLC Reactionconditions:fleshlycutpigliver50g,substrate100mg,0.2mol/L

    Tris-HClbuffer(pH=6.5)100mL,30.C,reactiontime4.0h. variouslivermicrosomesfromrat.chook,cattle,

    sheep,paralichthysolivaceusfishandcyprinoid fish.andthetime.courseofthereductionsarcshown inFig.2.Baker'syeastandgrapecellsmediatingre- ductionofcompoundlawasalsocarriedoutfora controlexperiment[9,10].Al1thelivermicrosomeswere abletometabolizecompoundslatoldalthoughtheir reactivityvariedsignificantlyfordifferentliverspe

    cies.Amongthetestedmammalianlivermicrosomes. thepig,ratandsheeplivers'microsomesexhibitedthe higherreactivity,andthemetabolizedintermediate hydroxylaminelcwasalsodetectedintheinitialstage Inthecaseofcattle1ivermicrosomesreductionsys. tem.onlycompoundldwasfoundinthefullprocess andtheconversionreached63%after48h.There. sultswerepossiblyduetotheimmediateconsumption ofcompoundlc,indicatingitshigherreactivityto? wardhydroxylaminethannitrocompound.Thereduc. tionofcompoundlabytheparalichthyolivaceus(a seafish)livermicrosomesexhibitedamoderatereac. tivity,andthecyprinoid(ariverfish)livermicrosome wasinemcientforthereductionalthoughthebOth organismsbelongtothesamespecies.Thetested chooklivermicrosomes.asaspeciesofaves.afrorded similarresultstothoseofpigmicrosomesbuttheac. tivitywaslowertowardcompoundla,andsimilar activitytowardcompoundlc.

    Sincethepiglivermicrosomesexhibitedhigh reactivitytowardcompoundla.itwaschosentoex. plorethereductivemetabolismofvariousnioaro.

maticcompoundsbearingelectronwithdrawing

    groups.andtheresultsandthosewithbaker'syeast9J

    andgrapecells.]asreducingagentsarelistedin Tlable1.Thereactiontimeinthegrapereduction systemneededabout45d.whichwasongerthan

    thatinthelivermicrosomesandbakers'yeastreduc. tionsystems.Toavoiddeactivationoftheenzymesin thegrapecells,thereactiontemperaturewassetat 25.C.Contrasttodicyannitrobenzenela.mono. cyanniti'obenzene(2wasdifficulttoreducebypig 984CHEM.RES.CHINESEUNIVERSITIESVb1.26 lOO

    80

    60

    40

    20

    100

    8O

    60

    40

    20

    100

    8O

    60

    40

    20

    Reactiontime/h

    Reactiontime/h

    Reactiontime/h

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