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Laccase-catalyzed dimerization of ferulic acid amplifies antioxidant activity

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Laccase-catalyzed dimerization of ferulic acid amplifies antioxidant activity
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   JournalofMolecularCatalysisB:Enzymatic 74 (2012) 29–35 ContentslistsavailableatSciVerseScienceDirect  Journal   of    Molecular   CatalysisB:   Enzymatic  journalhomepage:www.elsevier.com/locate/molcatb Laccase-catalyzed   dimerization   of    ferulic   acid   amplifies   antioxidant   activity Oluyemisi   E.   Adelakun a , Tukayi   Kudanga a , ∗ , Ayesha   Parker a , Ivan   R.   Green b ,   Marilize   le   Roes-Hill a ,Stephanie   G.   Burton a a BiocatalysisandTechnicalBiologyResearchGroup,CapePeninsulaUniversityofTechnology,BellvilleCampus,SymphonyWay,P.O.Box1906,Bellville7535,CapeTown,South Africa b ChemistryDepartment,UniversityoftheWesternCape,ModderdamRoad,PrivateBagX17,Bellville7530,SouthAfrica a   r   t   i   c   l   e   i   n   f   o  Articlehistory: Received29June2011Receivedinrevisedform2August2011Accepted26August2011 Available online 6 September 2011 Keywords: FerulicacidLaccaseAntioxidantactivity a   b   s   t   r   a   c   t Enzymatic   modification   can   be   used   to   enhance   the   bioactive   properties   of    phenolic   compounds.   Thepresent   study   employed   laccase   from   Trametes    pubescens   to   catalyze   the   modification   of    ferulic   acid   inamonophasic   or   biphasic   system,   as   away   of    enhancing   its   antioxidant   capacity.   Two   dimeric   products( m /  z    385.1)   were   purified   and   characterized   as   the   ˇ -5   and   ˇ – ˇ dimers.   Inthe   monophasic   system,   the ˇ -5dimer   was   preferentially   formed   in   dioxane   while   the   ˇ – ˇ   dimer   formation   was   enriched   in   ethanolasco-solvent.   Inthe   biphasic   system,   formation   of    the   dimers   increased   asthe   concentration   of    ethylacetate   was   increased   from   80%   to   95%.   The   ˇ -5   dimer   showed   higher   antioxidant   capacity   than   thesubstrate   as   demonstrated   by   standard   antioxidant   assays   (DPPH   and   TEAC).   These   results   demonstratethat   alteration   of    reaction   conditions   influences   the   laccase-mediated   oxidation   of    ferulic   acid   to   formdimers   with   higher   antioxidant   capacity   than   the   substrate. © 2011 Elsevier B.V. All rights reserved. 1.Introduction Plantphenolsandphenolicacidsareincreasinglybecomingasubjectofintensiveresearchduetotheirbioactivepropertieswhichincludeantioxidant,anti-mutagenic,anti-viralandanti-inflammatoryactivities[1–4].Amongthephenolicacids,ferulicacid,3-(4-hydroxy-3-methoxy-phenyl)-acrylicacid,isthemostabundanthydroxycin-namicacidintheplantworld[5],   constituting5gkg − 1 inwheatbran,9gkg − 1 insugarbeetpulp[6,7],15–28gkg − 1 ofricebranoil[8]and25gkg − 1 incornkernel[9].   Itisoneofthemajorphenolicligninmonomersfoundinwoodsandgrasses,andiswidelydistributedincereals,fruitsandvegetables[10,11].Some ofthesesourcesareusedtoprovideferulicacidasasubstrateforconversionintovalueaddedchemicalssuchasguaiacol,vanillin,vanillicacidandprotocatechuicacid[12].Ferulicacidisfound asthefreeacid,lowmolecularweightconjugates,esterswithcellwallheteroxylans,orcovalentlyboundtoligninandotherbiopolymers.FerulicacidisoneoftheactiveingredientsofmanyChinesetraditionalmedicinesusedinthepreventionandtreat-mentofvariousdiseases[13–15].   Itshowsmanyphysiologicalfunctions,includingantioxidant,antimicrobial,anti-inflammatory,anti-thrombosis,andanti-canceractivities[16,17]andcanbeeas- ilyabsorbedandmetabolizedinthehumanbody[18].Ithas ∗ Correspondingauthor.Tel.:+27219538497;fax:+27219538494. E-mailaddresses: tikudanga@yahoo.co.uk,kudangat@cput.ac.za(T.Kudanga). beenreportedtoalsoincreasespermviability,lowercholesterolinserumandliverandprotectagainstangina,hypertensiveandcoro-narydiseases[19].However,ithasbeenmostwidelyinvestigated foritsantioxidantpropertieswhichareimportantinthepreven-tionoflipidoxidationinfoodandalsointheputativepreventionof free-radical-induceddiseasessuchascancerandatherosclerosis,oragingcausedbyoxidativetissuedegeneration[20,21].Antioxi- dantpropertiesareascribedtoitsstructure(Fig.1)andspecifically toitsphenolicnucleuscoupledtoanextendedconjugatedsidechainwhichfacilitatetheformationofaresonance-stabilizedphe-noxyradical[22].Otherimportantstructuralpropertiesinclude electron-donatinggroupsonthebenzenering(3-methoxyland,moreimportantly,4-hydroxyl)whichgivetheadditionalprop-ertyofterminatingfreeradicalchainreactions;thecarboxylicacidgroupwithanadjacentunsaturatedC–Cdoublebondwhichcanprovideadditionalattacksitesforfreeradicalsthuspreventingthemfromattackingmembranes;andthecarboxylicacidgroupwhichactsasananchorgroupwhichbindsittothelipidbilayer,providingsomeprotectionagainstlipidperoxidation[20].Despite havingtheseattractiveproperties,theantioxidantcapacityoffer-ulicacidisgenerallylowcomparedtoconventionalantioxidantsandotherhydroxyl-cinnamicacids[23,24].Sánchez-Morenoetal. [25]alsoreportedthatferulicacidhasalowfreeradicalscavengingactivity,butshowedhigheractivityininhibitionoflipidoxidationandintheprotectionagainsthydroxylandperoxylradicaloxida-tioninsynaptosomalandneuronalcellculturesystems invitro [26].Recentlytherehavebeenanumberofattemptstoenzymaticallymodifyphenolicmoleculesasaway   ofimprovingantioxidantprop- 1381-1177/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2011.08.010  30  O.E.Adelakunetal./JournalofMolecularCatalysisB:Enzymatic  74 (2012) 29–35 Fig.1. Structureofferulicacidshowingtheimportantfeaturesforantioxidantactivity. erties.Laccasesareamongtheenzymesthatarecurrentlybeinginvestigated.Laccases(EC1.10.3.2)areenzymesthatarecapableofcatalyzingtheone-electronoxidationofphenolstoproducephenoxyrad-icalsandconcomitantlyreducemolecularoxygentowater[27].Oligomerizationorpolymerisationoftheradicalstoformoligomersorpolymerswithhigherantioxidantpropertiesthanthestart-ingmaterialshasbeenreported[28–31].   Consequently,researchinterestinenzymaticallytransformingferulicacidisnowincreas-ingwithsomeresearchershavingreportedthetransformationofferulicacidbylaccase[32–34].   Althoughpossiblestructuresoftheoxidationproductshavebeenproposed[33],noneofthe researchershasconclusivelyelucidatedthestructuresnorreportedtheantioxidantcapacityoftheproducts.Thispromptedustoinves-tigatethemodificationofferulicacidbylaccaseproducedfrom Trametespubescens (strainCBS696.94)asawayofenhancingtheantioxidantcapacityofferulicacid.Tothebestofourknowledge,ourworkforthefirsttimeconclusivelyelucidatesthestructureof twodimersformedfromlaccase-mediatedoxidationofferulicacidinorganicmedia,oneofwhichhashigherantioxidantcapacitythanthesubstrate. 2.Materialsandmethods  2.1.Chemicalsandenzyme FerulicacidandotherchemicalswerepurchasedfromSigma–Aldrich,SouthAfrica.Anairliftreactorwasusedtogrow T.pubescens (strainCBS696.94)fortheproductionoflaccase[29]whichwaspurifiedaccordingtothemethodofRyanetal.[35].  2.2.Enzymeactivity Laccaseactivitywasdeterminedspectrophometricallybymonitoringtheoxidationof2,2  -Azinobis(3-ethylbenzthiazoline-6-sulfonicacid)(ABTS, ε 420 =36,000M − 1 cm − 1 )asthesubstrate[36].Thereactionmixturecontained0.330mL    ABTS(5mM),   2.5mL 0.1Msodiumacetatebuffer(pH5.0)and0.17mL    laccase.OxidationoftheABTSwasmonitoredbymeasuringtheincreaseinabsorbanceat420nm.   Oneunitoflaccaseactivitywasdefinedastheamountofenzymerequiredtooxidise1  mol   ofABTSmin − 1 at25 ◦ C.  2.3.Oxidationofferulicacid Theoxidationreactionswerecarriedouteitherinabiphasicsystemcomprisingbufferwithethylacetateasco-solventorinamonophasicsystemwithmisciblesolvents(dioxane,methanol,ethanoloracetone)asco-solvents.Forthebiphasicsystemthereactionmixturecontainedferulicacid(10mM),laccase(10U)in100mM   sodiumacetatebuffer(pH5.0)andethylacetateatvariousconcentrations(80,85,90,95and96%,v/v).Forthemonopha-sicsystemthemisciblesolventswereusedat80%(previouslydeterminedastheoptimumforproductyieldandreducedsidereactions).Thereactionswerecarriedoutfor24hat28 ◦ Cwithshakingonanorbitalshakerat180rpm.Thereactionsweremoni-toredbyThinLayerChromatography(TLC)andHighPerformanceLiquidChromatography(HPLC)asdescribedbelow(Sections2.4and2.5).  2.4.Chromatographicseparationofreactionproducts TLCanalysiswas   performedonaluminium-backedsilicagel60F 254  (Merck)platesusingtoluene:dioxane:aceticacid(10:2.5:0.2,v/v/v)orethylacetate:dioxane:aceticacid(6:0.2:0.05,v/v/v)asthemobilephase.ThecompoundswerethenvisualizedbyexposuretoUVlightat254nm.  2.5.Highperformanceliquidchromatography(HPLC) PriortoHPLCanalysis,theenzymewasprecipitatedoutofthereactionsolutionbytheadditionofanequalvolumeofice-coldmethanol(onlywhenmisciblesolventswereused;inbiphasicsys-tem,theenzymewas   readilyseparatedfromtheproduct).Themixturewas   incubatedat0 ◦ Cfor20minandthencentrifugedat0 ◦ Cfor15min   at14,000 ×  g  .Thesupernatant(1.5mL    aliquots)wastransferredintocleanvialsandanalyzedbyHPLC.HPLCanal-ysiswascarriedoutusingaHitachiLaChromHPLCsystemfromMerk(Merck,Hitachi,Germany).Separationofthereactionprod-uctswascarriedoutonareversedphaseLUNA5  PFP(2)100A,250mm × 4.60mmcolumnunderisocraticconditionsusingace-tonitrile:water:aceticacid(25:75:0.1,v/v/v)asthemobilephaseataflowrateof1mL    min − 1 ,with1hrunningtime.Theproductsweredetectedat270nm.   Alternativelygradientelutionusing0.1%formicacid(solventA)andacetonitrile(solventB)was   usedinordertoreducetherunningtimeto23min.Thegradientsetupwasasfollows:98%Ato0%A(20min);0%Ato98%A(20–21min);98%A(21–23min).PeakswereanalyzedusingHPLCManager,MerckHitachimodelD700datasoftware.  2.6.Purificationofreactionproducts ThereactionproductswerepurifiedbyFlashChromatography.Themisciblesolventscontainingproductswereevaporatedusingarotaryevaporatorandtheproductextractedwithethylacetatefollowedbyseparationusingaseparationfunnel.Theaqueousphasewas   washedtwiceandmonitoredfortheabsenceofproduct.  O.E.Adelakunetal./JournalofMolecularCatalysisB:Enzymatic  74 (2012) 29–35 31 Theorganicphasewasdriedusingarotaryevaporator(Heidolph,Germany).Forthebiphasicsystem,theorganicphasewassep-aratedusingaseparationfunnelandtheaqueousphasewashedtwicewithethylacetate.TheorganicphasewasevaporatedunderreducedpressurewitharotaryevaporatorandthecruderesiduepurifiedbysilicaFlashChromatographyusingethylacetate:diox-ane:aceticacid(6:0.2:0.05,v/v/v)asmobilephaseforpurifyingproduct1(P1)ortoluene:dioxane:aceticacid(10:2.5:0.2,v/v/v)asmobilephaseforpurifyingproduct2(P2).Thepurefractionsweredriedusingarotaryevaporatorandtheproductssequentiallywashedwithacetone,methanolandthenacetoneagaintoremovetheaceticacid.  2.7.Characterizationofproducts Thepurifiedproducts(P1andP2)werecharacterizedbymassspectrometryandnuclearmagneticresonance(NMR)analysis.  2.7.1.Liquidchromatography–massspectrometry(LC–MS) LC–MSwasperformedonaDionexHPLCsystemequippedwithabinarysolventmanagerandautosamplercoupledtoaBruckerESIQ-TOFmassspectrometer.Theproductswereseparatedusingthesamelineargradientofacetonitrile(solventB)and0.1%formicacid(solventA)asdescribedinSection2.5above,ataflowrateof  1mL    min − 1 ,usinganinjectionvolumeof10  Landanoventem-peratureof30 ◦ C.MS   spectrawereacquiredinnegativemodeandelectrosprayvoltagewassetto+3500V.Drygasflowwassetto9Lmin − 1 withatemperatureof350 ◦ Candnebulizergaspressurewassetto35psi.  2.7.2.Nuclearmagneticresonance(NMR)analysis Nuclearmagneticresonance(NMR)spectrawererecordedusingaVARIAN200spectrometer( 1 H,200MHz;  13 C,50MHz).Thespectraweredeterminedatambienttemperatureindeuteratedchloroform(CDCl 3 )andmethanolsolutions,withCHCl 3  at ı 7.26for  1 HNMR    spectraandchloroform( ı 77.00)for  13 CNMR    spectraasinternalstandards.IntheNMR    spectra,assignmentsofsignalswiththesamesuperscriptsareinterchangeable.Splittingpatternsaredesignatedas“s”,“d”,“t”,“q”,“m”   and“bs”.Thesesymbolsindi-cate“singlet”,“doublet”,“triplet”,“quartet”,“multiplet”and“broadsinglet”.  2.8.Antioxidantactivitydetermination 2.8.1.DPPH(2,2  -diphenyl-1-picrylhydrazyl)scavengingeffect  AntioxidantcapacitywasdeterminedbymeasuringDPPHradical-scavengingactivity[37].Briefly,3.9mL    ofDPPHdissolvedinmethanol(0.025mg/mL)wasaddedto0.1mL    sample(dissolvedinmethanol)atvariousconcentrations.Themixturewasshakenvig-orouslyandincubatedatroomtemperatureinthedarkfor60min,andthedecreaseinabsorbanceat517nmdeterminedusingaspec-trometer.TheremainingconcentrationofDPPHinthereactionmediumwasthencalculatedfromacalibrationcurveobtainedwithDPPHat517nm.ThepercentageofremainingDPPH(DPPH R  )wascalculatedasfollows:%DPPH R   =   (DPPH) T  (DPPH) T  = 0  × 100where(DPPH) T   istheconcentrationofDPPHattime60min   and(DPPH) T  =0  istheconcentrationofDPPHattimezero(initialconcentration).ThepercentageofremainingDPPHagainstthesam-ple/standardconcentrationwasplottedtoobtaintheamountof antioxidant(mM)   necessarytodecreasetheinitialconcentrationofDPPHby50%(EC 50 ).  2.8.2.TEAC(Troloxequivalentantioxidantcapacity)assay TheABTSradicalscavengingactivityofferulicacidandthetwoproductsweredeterminedaccordingtothemethoddescribedbyReetal.[38].Thetroloxequivalentantioxidantcapacity(TEAC) methodisbasedontheabilityofantioxidantmoleculestoquenchABTS • + ,ablue–greenchromophorewithcharacteristicabsorptionat734nm,   comparedwiththatofTrolox,awatersolublevitaminEanalog.TheadditionofantioxidantstothepreformedradicalcationdecolourizestheABTS • + asitisreducedtoABTS.ABTS • + solutionwasprepared12–16hbeforeusebymixingABTSsalt(7mM)   withpotassiumpersulfate(2.45mM)   andthenstoredinthedarkuntiltheassaywasperformed.TheABTS • + solutionwasdilutedwithmethanoltogiveanabsorbanceof0.70 ± 0.002at734nm.   Eachsample(100  L)preparedatdifferentconcentrationwasmixedwith1100  LABTS • + solutionandtheabsorbancewas   readafter30min   incubationat25 ◦ C. 3.Resultsanddiscussion  3.1.Oxidationofferulicacidinorganicsolvents Biocatalysisisconductedinorganicmediaforanumberofrea-sons,mainlypoorsolubilityofsomecompoundsinaqueousmedia;abilitytocarryoutnewreactionswhichareimpossibleinwaterbecauseofkineticorthermodynamicrestrictions;relativeeaseof productrecoveryfromorganicsolventsascomparedtowater;andtheinsolubilityofenzymesinorganicmedia,whichpermitstheireasyrecoveryandreuse(thuseliminatingtheneedforimmobi-lization)[39,40].Ferulicacidisnotsolubleinaqueousmedia,thus organicsolventswereemployedforthisstudyineitheramonopha-sicorbiphasicsystem.Boththebiphasicsystemandmonophasicsystemsproducedthreemainoxidationproductswhichweredes-ignatedasP1( t  R  =10.535);P2( t  R  =12.202);P3( t  R  =13.446)(Fig.2).LC–MSanalysisoftheoxidationproductsinnegativemodeshoweddominantsignalsat m /  z  385.1143(P1)and m /  z  385.1077(P2)(Fig.3)whichsuggestedthattheseoxidationproductsweredimers offerulicacid(exactmass[M]   386.1).Whenthemassspectrom-eterwassettoruninpositivemode[M+H] + ionsignalswereobservedat m /  z    387.1,whilesignalsat m /  z    409.1indicatingNaadducts,confirmedthatthemoleculeswereindeeddimers.LC–MSresultsoftheproductP3( m /  z  579.2154)suggestoligomerizationtoformatrimer.Inaddition,atetramer( m /  z  769.1948; t  R  =13.7)wasobservedwiththemoresensitiveMS   detector(notobservedwithHPLC).Afterpurification,theyieldsofP1andP2were11.190%and38.189%,respectively.Workiscurrentlyunderwaytopurifyprod-uctP3.Significantamountsofinsolublepolymericproductswerealsoobserved.TheNMR    resultsindicatedthatP1isadimeroftwoferulicacidmonomerscovalentlyboundthrougha ˇ -5linkagewhiletheresultsforP2suggestdimerizationthrougha ˇ – ˇ linkage(Fig.4).ThesymmetryofthemoleculeP2madeassignmenteasyduetothesignalsbeingclearandseparatefromeachother.Inthe  1 HNMR    spectrum2-Happearedasasinglepeakat ı 3.56whilethemethoxygroupappearedasathreeprotonsingletat ı 3.91.ThephenolicOHappearedasasinglepeakat ı 5.86incloseproximityto3-Hwhichappearedasasinglepeakat ı 5.69.Couplingbetween2-Hand3-Hwasnotobservedduetothedihedralanglebeinginthevicinityof  ∼ 90 ◦ .Thearomaticregiondisplayedatypicalpatternofa1,3,4-trisubstitutedsystemviz.,a2-protonmultipletat ı 6.79assignedto2  -Hand6  -Hwhile5  -Happearedasan ortho coupleddoubletat ı 6.93with  J  =8.8Hz.Inthe 13 CNMR    spectrumallsignalscouldbeassignedaccordingtothestructurewithC2at ı 48.5,themethoxygroupat ı 56.3andC3at ı 82.0.Threestrongsignalsat ı 107.6,115.1and117.5wereassignedtoC2  ,C5  andC6  respectivelywhileC1  appearedat ı 130.0.Themoredeshieldedcarbonsignals  32  O.E.Adelakunetal./JournalofMolecularCatalysisB:Enzymatic  74 (2012) 29–35 Fig.2. HPLCchromatogramofproductsformedduringlaccase-catalyzedoxidationofferulicacid.P1,P2andP3–oxidationproducts. Fig.3. MassspectrumofProductP1(A)andP2(B)formedduringlaccase-mediatedoxidationofferulicacid. Fig.4. Dimers(P1andP2)formedduringlaccase-catalyzedoxidationofferulicacid.  O.E.Adelakunetal./JournalofMolecularCatalysisB:Enzymatic  74 (2012) 29–35 33 Fig.5. Laccase-catalyzedoxidationofferulicacidtoproduce ˇ – ˇ and ˇ -5dimers. ofC3  andC4  appearedat ı 146.5and147.2.Finallythelactonecarbonylappearedasexpectedat ı 175.0.AsexpectedthespectraweremorecomplicatedforP1duetoalackofsymmetryinthecompoundandthespectrumwasrunindeuteromethanol.Thetwomethoxygroupsappearedas3-protonsingletsat ı 3.83and3.91while3-Happearedasasharpdoubletat ı 4.01(  J  =2.6Hz).Acleardoubletat ı 5.61(  J  =2.6Hz)was   assignedto2-H.Aratherbroadmultipletcentredaround ı 6.70integratedfor5protonsandassignedto6-H,5  -H,1  -H,2  -Hand4  -OH.Adoubledoubletat ı 7.01(  J  =8.0and2.0Hz)wasassignedto6  -Hwhilethecorrespondingdoubletat ı 7.07(  J  =2.0Hz)wasassignedto2  -H.Adownfielddoubletat ı 7.57(  J  =1.8Hz)wasassignedto4-H.ThetwoCOOHhydrogenwerenotobservedduetoexchangewithprotonsinthedeuteromethanolsolvent.The  13 Cspectrumwasassignedasfollows:Abroadishsignalat ı 55.7isduetothetwomethoxycarbonsaswellasC3.Thesignalat ı 81.2isassignedtoC2.Strongsignalsinthearomaticregionhavebeenassignedasfollows: ı 108.3(C2  ),112.7(C8),115.1(C5  ),118.0(C4),118.7(C6  ).Thesp 2 carbonsoftheethylenesidechainwereassignedasfollows: ı 115.2(C2  )and146.3(C1  ).TheringjunctioncarbonsC3aandC7awereassignedasfollows: ı 125.5and148.9respectively.C5andC1  wereassignedtothesignalsat ı 125.9and131.2respectively.Thedeshieldedcarbonatomsattachedtotheoxygenswereassignedas:C7( ı 140.8),C4  ( ı 147.5)andC3  ( ı 148.9).Finallythetwo   carbonylsignalsat ı 169.2and172.6wereassignedtothetwocarboxylicacidgroups.BasedontheresultsfromLC–MSandNMR,theschemeshowninFig.5isproposedasthepossiblereactionpathwayforthedimerizationofferulicacidtoforma ˇ -5and ˇ – ˇ linkage[33].AsdepictedinFig.5,laccaseinitiatesthereactionbyoxidizingthe para-hydroxylgroupresultinginaradical.Throughresonancesta-bilization,theunpairedelectroncanoccupydifferentpositionsontheradicalizedmolecule[27].Theradicalsarestabilizedthrough couplingwithotherradicalstoformdimers.Thereforecouplingof    anunpairedelectronatthe ˇ positionwithanotheratpositionC5ofthenextradicalyieldsa ˇ -5linkeddimer.Similarlycovalentcouplingofradicalsbothwithunpairedelectronsatthe ˇ positionproducesthe ˇ – ˇ linkeddimer.Whiletherewereseveralpossibil-itiesforcouplingduetothelargenumberofmesomericforms,itappearsthatourreactionconditionsfavoredtheformationof  ˇ -5and ˇ – ˇ linkagesduetothestabilityofC–Cbonds[41]andthelowerheatofformationof  ˇ -5linkages[42].We   haveobservedsimilarfindingsinwhichC–Cbondspredominatedinthecouplingoffunctionalmoleculestoligninmodels,thoughmostly5–5link-ageswereformedduetotheabsenceofunsaturatedsidechains[43,44].Inaddition,recentreviewsagreethatindimerizationof asimilarmolecule,coniferylalcohol,theradicalsfavorcouplingattheir ˇ positions,resultingessentiallyinonlythe ˇ – ˇ , ˇ -O-4,and ˇ -5dimers[45,46]althoughthe ˇ -O-4arethepredominantlinkagesinpolymerizationreactionsduringligninsynthesis[47].  3.2.Effectoforganicsolvents Theeffectofthenatureoftheorganicsolventandtheconcentra-tionofthesolventwasstudiedinordertoobtainthebestreactionconditionsforproductformation.Inthebiphasicsystem,therewas   anincreaseintheformationof P2( ˇ – ˇ dimer)astheconcentrationofethylacetatewasincreasedfrom80%to90%afterwhichadeclineinproductformationwasobserved(Fig.6).Asimilarpatternwas   observedduringtheforma-tionofP1( ˇ -5dimer)exceptthatadeclinewasobservedafterthecompositionoforganicsolventwas   increasedbeyond95%(Fig.6).Inthemonophasicsystem,fourco-solventsnamelyacetone,dioxane,ethanolandmethanolwereinvestigated.AsshowninFig.7mostofthesubstratewasconvertedtoproductsinethylacetate:abiphasicsystem.Inthemonophasicsystem,theorderof substrateconversionwasethanol>acetone>dioxane>methanol.However,forproductformation, ˇ – ˇ dimerformationwas
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