Delft University of Technology
Towards preparative peroxygenase-catalyzed oxyfunctionalization reactions in organic
media
Fernández-Fueyo, Elena; Ni, Yan; Gomez Baraibar, Alvaro; Alcalde, Miguel; van Langen, Lukas M.;
Hollmann, Frank
DOI
10.1016/j.molcatb.2016.09.013
Publication date
2016
Document Version
Final published version
Published in
Journal of Molecular Catalysis B: Enzymatic
Citation (APA)
Fernández-Fueyo, E., Ni, Y., Gomez Baraibar, A., Alcalde, M., van Langen, L. M., & Hollmann, F. (2016).
Towards preparative peroxygenase-catalyzed oxyfunctionalization reactions in organic media. Journal of
Molecular Catalysis B: Enzymatic, 134, 347-352. https://doi.org/10.1016/j.molcatb.2016.09.013
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ContentslistsavailableatScienceDirect
Journal
of
Molecular
Catalysis
B:
Enzymatic
jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / m o l c a t b
Towards
preparative
peroxygenase-catalyzed
oxyfunctionalization
reactions
in
organic
media
Elena
Fernández-Fueyo
a,1,
Yan
Ni
a,1,
Alvaro
Gomez
Baraibar
a,
Miguel
Alcalde
b,
Lukas
M.
van
Langen
c,
Frank
Hollmann
a,∗aDepartmentofBiotechnology,DelftUniversityofTechnology,VanderMaasweg9,2629HZDelft,TheNetherlands bDepartmentofBiocatalysis,InstituteofCatalysis,CSIC,28049Madrid,Spain
cViaZymB.V.,Molengraaffsingel10,2629JDDelft,TheNetherlands
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received27July2016 Receivedinrevisedform 13September2016 Accepted13September2016 Availableonline15September2016 Keywords:
Biocatalysis Oxyfunctionalization Hydroxylation
Non-aqueousreactionmedia Peroxygenase
a
b
s
t
r
a
c
t
TheperoxygenasefromAgrocybeaegerita(AaeUPO)hasbeenevaluatedforstereoselective
oxyfunction-alizationchemistryundernon-aqueousreactionconditions.
Thestereoselectivehydroxylationofethylbenzeneto(R)-1-phenylethanolwasperformedinneat
substrateasreactionmediumtogetherwiththeimmobilizedbiocatalystandtertBuOOHasoxidant.
Stabilityandactivityissuesstillhavetobeaddressed.Nevertheless,gram-scaleproductionof
enan-tiopure(R)-1-phenylethanolwasachievedwithrespectable90,000turnoversofthebiocatalyst.
©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Peroxygenases catalyzea broad range ofsynthetically inter-estingoxyfunctionalizationreactions.[1,2]Amongstthem, stere-ospecifichydroxylationofalkylbenzenesisworthmentioningas chemical catalysts withcomparable selectivityand activity are stillmissing[3].Furthermore,peroxygenasesexceloverthe well-knownP450monooxygenasesbytheirsimplicityneedingsimple hydrogen peroxide or organic hydroperoxides as cosubstrates instead of the nicotinamide cofactor and complicated electron transportchains[4,5].
ThecholoroperoxidasefromCaldariomycesfumago(CfUPO) rep-resents the first example of an ‘unspecific’ peroxygenase (E.C. 1.11.2.1)exhibitingsignificantP450-likeactivity(e.g.C H-bond activation) [6,7]; and major researchefforts had been devoted totheexplorationofitsproperties,productspectrumand possi-bleapplications.Unfortunately,however,CfUPO’scatalyticactivity towardsnon-activatedorpoorlyactivatedC Hbondsis compa-rablylowimpairingitspreparativeusefulness.In2004thegroup
∗ Correspondingauthor.
E-mailaddress:f.hollmann@tudelft.nl(F.Hollmann).
1 Bothauthorscontributedequally.
aroundHofrichterreportedanotherperoxygenasefromthefungus Agrocybeaegerita (AaeUPO)exhibitingsignificantly higher activ-ity[8].Today,morethan300substrateshavebeenreportedfor AaeUPOthat oftenareconverted highlychemo-and enantiose-lectively[1,2].Furthermore,recombinantexpressionsystemsfor AaeUPOareavailable[9]enablingproteinengineering[10,11].Also acrystalstructureofAaeUPOisknownfacilitating(semi-)rational proteinengineering[12].
Overall,AaeUPOisanextremelypromisingcandidate biocata-lystforpreparative-scale,selectiveoxyfunctionalizationchemistry. OnemajorlimitationofAaeUPO(andofperoxygenase-catalysis ingeneral)howeverstillisitslimitationtoaqueousreaction con-ditions,whichposesamajorchallengetotheconversionofpoorly watersoluble,hydrophobicstartingmaterialssuchasalkyl ben-zenes.Both,fromaneconomicaland anenvironmentalpointof view,highersubstrateloadingsthantraditionallyusedarehighly desirable[13–15].Theuseofcosolventstoincreasethewater sol-ubilityofthehydrophobicstartingmaterialsortwo-liquid-phase approachesusingahydrophobiccosolventassubstratereservoir andproductsinkhavebeenproposed[16–19].Avoidingadditional solventsatallandperformingthetransformationsinneat condi-tions(i.e.withoutanycosolventwhatsoever)wouldbethemost elegantmethodology.
http://dx.doi.org/10.1016/j.molcatb.2016.09.013
348 E.Fernández-Fueyoetal./JournalofMolecularCatalysisB:Enzymatic134(2016)347–352 Therefore,inthepresentstudy,wesetouttoevaluatethe
fea-sibilityofperoxygenase-catalyzedoxyfunctionalizationreactions undernon-aqueousconditions.Asthemodel enzymewechose AaeUPO,recombinantlyexpressedinPichiapastoris(rAaeUPO)[9], themodelreactionwasthestereoselectivehydroxylationof ethyl-benzeneto(R)-1-phenylethanol.
2. Materials
2.1. Chemicalsandenzymes
AllchemicalswerepurchasedfromSigma-Aldrich(Zwijndrecht, TheNetherlands)inthehighestpurityavailableandusedwithout furtherpurificationexceptethylbenzenethatwasfreshlydistilled priorusetoremovethetracesofphenylethanolandacetophenone. TheenzymecarrierReliZymeTMHA403/M,amacroporousPMMA
resinwithamino-functionalizations,wasobtainedfromResindion S.r.l.,Italy.
2.2. Enzymeproduction
Therecombinantperoxygenasefromthebasidiomycetous fun-gusAgrocybe aegerita(rAaeUPO)wasproducedviaheterologous fermentationin Pichia pastorisfollowing a previously described procedure[9].
2.3. ConcentrationofrAaeUPO
TheconcentrationofrAaeUPOwasdeterminedusingthemolar extinction coefficient of 115mM−1cm−1 at 420nm.Absorption spectraintheUV/visrangewasrecordedinaBiomate5(Thermo) spectrophotometer(Fig.1).TheReinheitszahl(Rzvalue)istheratio ofabsorbancedue tohemin(A420, Soretregion)toabsorbance duetoprotein(A280) andtherewitha measurefor theprotein purity.TheRz-valueofthecurrentrAaeUPOpreparationwas1.6 correspondingwelltovaluesreportedintheliterature[9–11].
2.4. ImmobilizationofrAaeUPOonRelizymeTMHA403/Mresin
Toimmobilizetheperoxygenasethefollowingprocedurewas used:RelizymeTMHA 403/Mresin(1g)wastreatedwith50mL
of0.125%glutaraldehydesolutioninwaterfor2.5hinashaking deviceat16◦C.Theglutaraldehydesolutionwasthenremovedby centrifugation,andtheresinwaswashedthreetimeswith0.1M phosphatebufferatpH7.Thebufferwasthenremoved,and3mL ofpurerAaeUPO(1.35mg)and1mLof0.1Mphosphatebufferat pH7wereaddedtotheactivatedsupport.Themixturewas incu-batedinashakerfor24hat16◦C.Theresidualenzymaticactivityin thesolutionwasmonitoredbyusingtheABTSoxidationassay(vide infrafordetails).Theresinwasthenwashedwith50mMphosphate bufferatpH7,driedandstoredat4◦C.
2.5. rAaeUPOconcentrationinthebeads
TheamountoftherAaeUPOboundtotheresinwasdetermined bysubtractingtheamountofenzymepresentinthesupernatant afterimmobilizationfromthetotalamountofenzymepresent orig-inally(prioradditionof theresin).Themeasurement wasdone spectrophotometrically(Fig.1)usingthemolarextinction coeffi-cientof 115mM−1cm−1 at420nm.Quite reproducibly,1.35mg rAaeUPOpergramofresinwasbound(i.e.quantitative immobi-lization).
2.6. ActivitydeterminationofrAaeUPO
InordertoquantifythespecificactivityofrAaeUPO,weused ABTSasasubstrateinaqueousmedia.Absorbancechangesduring ABTSoxidationin0.1McitratebufferpH5wererecordedat25◦C ina Biomate5(Thermo)spectrophotometer. Thereactionswere initiatedbytheadditionof5mMH2O2.OxidationofABTSwas
fol-lowedbytheformationofthecationradical(40536.8mM−1cm−1).
DifferentconcentrationsofrAaeUPOwereusedtocreatea calibra-tionlinebasedontheactivitytowardsABTS(0.5mMABTSin0,1M citratebufferand5mMH2O2).
Theactivityoftheimmobilizedenzymewasestimatedina reac-tionmixtureof5mLcontaining7mgbeads,0.5mMABTSand5mM H2O2incitratebuffer(pH5.0)magneticallystirredatroom
tem-perature.Aliquotswerewithdrawnevery30sandmeasuredat 405nm.Reactionswereperformedinduplicates.
2.7. Hydroxylationofethylbenzene
Reactionswereperformedat 30◦C and ambientatmosphere in1mLethylbenzenecontainingdifferentamountofimmobilized rAaeUPO.Every30mintertBuOOHwasaddedintothereaction
mix-tureandsampleswerecollected.Samplesweremixedwithethyl acetate(containing5mM1-octanolasinternalstandard)and ana-lysedbyGC.Reactionswereperformedinduplicates.
2.8. StabilityofimmobilizedrAaeUPOinethylbenzene
Theimmobilized rAaeUPOwasincubatedin ethylbenzeneat 30◦C for24hand theresidualactivitywasmeasuredfollowing ethylbenzenehydroxylationaftertheadditionof10mMtertBuOOH
and1hofincubation.
2.9. StabilityofimmobilizedrAaeUPOagainstperoxide
TheimmobilizedrAaeUPO(7mg)wasincubatedinphosphate buffer(pH5.0)containing10mMtertBuOOHandtheresidual
activ-itytowardsABTSwasmeasuredasdescribedabove. 2.10. ImmobilizationofPpAOxonRelizymeTMHA403/Mresin
TheRelizymeTMHA403/Mresin(1g)wastreatedwith50mL
of0.125%glutaraldehydesolutioninwaterfor2.5hinashaking deviceat18◦C.Theglutaraldehydesolutionwasthenremovedby centrifugation,andtheresinwaswashedthreetimeswith0.1M phosphatebufferatpH7.Thebufferwasthenremoved,and9mL ofPpAOx(15mg)and1mLof0.1MphosphatebufferatpH7were addedtotheactivatedsupport.Themixturewasincubatedina shakerfor24hat18◦C:theresidualenzymaticactivityinthe solu-tionwasassayedbyABTSoxidationassay(0.1MphosphatepH7, 0.033%methanol,2mMABTSand2.5UHRP)describedbySigma Aldrich.Theresinwasthenwashedwith50mMphosphatebuffer atpH7,driedandstoredat4◦C.
2.11. ConcentrationofPpAOxonthebeads
Theamountoftheenzymeaddedfortheimmobilizationand theenzymeretainedinthesupernatantwasquantifywiththeBSA assay.Basedonthisassay,15mgofproteinwasboundpermgof beads(i.e.quantitativeimmobilization).
2.12. ActivityofPpAOx
In order to quantify the specific activity of the immobi-lized PpAOxwe used the assay recommended by the supplier (SigmaAldrich):thereactionconditionswere: 0.1mphosphate
Fig.1.UV/visspectrumofpurifiedrAaeUPO.
pH7,0.033%methanol,2mMABTSand 2.5UHRP.Oxidationof ABTSwasfollowed bytheformationof thecationradical(405
36.8mM−1cm−1).
2.13. Reactionsintwo-liquidphasesystem(2LPS)
The two-liquid-phase system consisted of ethylbenzene and phosphate buffer (pH 7.0) (phase ratio=1:1) containing aqueousconcentrationsof[rAaeUPO]=100nM,[PpAOX]=60nM, [FDM]=0.2gL−1and200mMmethanol.Aliquotswerewithdrawn fromclearlyseparatedorganicphaseatinterval,mixedwithequal volumeofethylacetate(containing5mMdodecane)andanalysed byGC.Reactionswereperformedinduplicates.
3. Methods 3.1. GCanalytics
Atintervals,sampleswerewithdrawnfromthereaction mix-turesand extractedthree timeswiththesamevolume ofethyl acetate(containing5mMoctanolor5mMdodecaneasinternal standard).Thecombined organicphase wasdriedwith magne-siumsulfateandcentrifuged.ThesupernatantwasanalysedbyGC (seeFig.2foranexemplarychromatogramwithauthentic stan-dards).Concentrationsreportedhavebeendeterminedbasedon calibrationcurvesusingauthenticstandards.
4. Resultsanddiscussion
Thestartingpointofourinvestigationwasatwo-liquidphase systemusingethylbenzeneassubstratereservoirandproductsink. ForinsituH2O2generationwechosethepreviouslydescribed
alco-holoxidase-catalyzedoxidationofmethanol(Scheme1).
As shown in Fig. 3, reactions employing diffusibleenzymes resulted in comparably fast product formation (2.9mMh−1, TF(rAaeUPO)=8.1s−1,TF(PpAOx)=13.4s−1).Wehypothesizethat theoverallproductivityofthereactionsystemmayhavebeen lim-itedbyoxygentransferintotheaqueousreactionmedium.In a previousstudyusingacomparablesetupavolumetric productiv-ityaround2.5mMh−1hadbeenobserved,whichwasattributedto
Scheme1. Enantioselectivehydroxylationofethylbenzeneusingthetwoliquid
phase(2LP)approachandmethanoloxidationforinsituH2O2generation.
Ethyl-benzeneservedasorganicphase(substratereservoirandproductsink).
diffusionlimitationovertheinterphase[20].Alsotherobustnessof thispreliminarysetupwascomparablypoorwithacompleteloss ofethylbenzenehydroxylationactivityafter24h.Atpresentstage wehavenofurtherinsightintothemolecularreasonforthispoor robustness.Possibly,themechanicallydemandingconditions(such asshearstressorthepresenceofahydrophobicinterphase)account forthislowlong-termstabilityandfurtherin-depthinvestigations arenecessarytoclarifythisissue.Nevertheless,rAaeUPOandPpAOx performed206,000and343,000catalyticturnovers,respectively.
To address the poor stability of the biocatalysts under the reaction conditions we evaluated using immobilized enzyme preparations.AsshowninFig.3,thereactionutilizing immobi-lizedrAaeUPOandPpAOxwassignificantlymorerobustbutalso significantlylessactive.Usingthesamenominalenzyme
concen-350 E.Fernández-Fueyoetal./JournalofMolecularCatalysisB:Enzymatic134(2016)347–352
Fig.2.ExemplaryGCchromatogramofauthenticstandards.Temperatureprofile:90◦Cfor5min,20◦C/minto110for15min,25◦C/minto220for1min.Peakassignment:
ethylbenzene(5.0min),acetophenone(11.3min),dodecane(13.2min),(R)-1-phenylethanol(21.8min),(S)-1-phenylethanol(22.3min).
Fig.3.rAaeUPO-catalyzedhydroxylationofethylbenzeneinatwo-liquid-phase system(2LPS)usingfreerAaeUPOandfreePpAOx(䊐)orimmobilizedrAaeUPOand PpAOx().Conditions:2LPS(Vethylbenzene=Vaq=1mL),100mMphosphatebuffer
(pH7.0),[methanol]=200mM,[rAaeUPO]=100nM,[PpAOx]=60nM,T=30◦C.In
bothcases,(R)-1-phenylethanolwasthemajorproduct(>95%,ee>98%)withless than5%oftheoveroxidationproduct(acetophenone,leftoutforreasonsofclarity).
trationsascomparedtothefreeenzymes,theproductformation ratedroppedto0.11mMh−1concomitantlyleadingtoareduction oftheenzymes’performancebymorethan20-fold.Thisisinline withthefindingthattheimmobilized enzymesaresignificantly lessactivethantheirfreependants.
Havingtheimmobilizedenzymesathand,wedrewour atten-tiontotheiruseunder neatconditions(i.e.using ethylbenzene assole reaction medium; in otherwords the aqueouslayer of
Scheme1isreducedtoaminimumoriginatingfromthe
immo-Scheme2.Enantioselectivehydroxylationofneatethylbenzeneusingimmobilized
rAaeUPOandtertBuOOHasoxidant.
bilizationprocedure).Inafirstsetofexperimentsweevaluated thebi-enzymaticcascadeusingPpAOxforinsituH2O2generation.
However,onlytraceamounts ofproductweredetectableunder thesereactionconditions.Possibly,theinsituformedH2O2(and/or
formaldehyde)accumulatedquicklyinthesmallaqueouslayerto criticalconcentrationsandleadtofastenzymeinactivation. There-fore,wedecidedtousetertBuOOHasperoxidedonortopromote
thereaction(Scheme2).
tertBuOOHprovedtobeamilderoxidantascomparedtoH 2O2
astheoxidativeinactivationoftherAaeUPOhemewassignificantly slower(Fig.4).
Thestabilityoftheimmobilizedenzymeinpureethylbenzene wasfoundtobeacceptablewithapproximately46%activityloss over24h(Fig.5).
ThedosageoftertBuOOHhadaverysignificanteffectontherate
androbustness ofthehydroxylationreaction(Fig.6).The over-allreaction rate almostlinearly correlated withthe amountof hydroperoxideaddedwhile therobustnessof thereaction fol-lowedanoppositetrend:addingtertBuOOHat40mMh−1leadto
acompleteceaseofproductformationafter30minatlatest. How-ever,lowerdosingratesof10or20mMh−1resultedinfairlylinear productaccumulationoverthereactiontimeobserved.Mostlikely thisbehavior canbeexplainedbytheincreasingenzyme activ-itywithincreasingavailabilityofthecosubstrate(tertBuOOH)and
theincreasingoxidativeinactivationoftheprostheticheme-group withincreasingperoxideconcentrations.
Fig.4.StabilityofimmobilizedrAaeUPOagainstperoxide.Theimmobilized rAae-UPOwasincubatedinphosphatebuffer(pH5.0)containing10mMtertBuOOH(䊏)
and10mMH2O2(),atintervalssampleswerewithdrawnandsubjectedtoan
activityassay.Errorbarsarenotshownsincethestandarddeviationwasalways below5%.
Fig.5. StabilityofimmobilizedrAaeUPOsuspendedinneatethylbenzene.The resid-ualactivitywasdetermineduponadditionof10mMoftertBuOOHandincubationof
1hat30◦C.Conditions:ethylbenzeneand[rAaeUPO]=400nMat30◦C.Errorbars
arenotshownsincethestandarddeviationwasalwaysbelow5%.
In all reactions a certain amount of overoxidation of (R)-1-phenylethanol toacetophenone wasobserved culminating in 13–21mMofthisundesiredsideproduct.Hereitisinterestingto notethatthisoveroxidationwassignificantinthefirstphaseofthe reactionwhileafterapprox.30–60minthisreactionsloweddown significantlyleading toa relativelystablelevel ofaetophenone. Today,wearelackingaplausibleexplanationforthisobservation.
Fig.6.ComparisonoftheeffectofdifferenttertBuOOHdosingratesonthe
pro-ductivityandrobustnessoftherAaeUPO-catalyzedhydroxylationofethylbenzene. Generalconditions:ethylbenzenewasusedassoleliquidphasesupplemented withimmobilizedrAaeUPO(correspondingtoanoverallconcentrationof35M), T=30◦C, tertBuOOHwasaddedportionwise at30minintervalscorresponding
to䊏:40mMh−1,:20mMh−1,:10mMh−1;straightlinescorrespondto
(R)-1-phenylethanol,dottedlinescorrespondtoacetophenone.
Possibly,theaccumulatingtertBuOHinhibitedtheoveroxidation;
furtherinvestigationsarecurrentlyunderway.
Itshouldbementionedthatintheseexperimentscomparably largenominalrAaeUPOconcentrations(35M)hadbeenemployed tocompensateforthepooractivityoftheimmobilizedenzyme. Hence,thecatalyticperformanceofrAaeUPOwasratherpoor(2700 catalyticturnoversin3h).Furtherexperimentswithsignificantly reducedenzymeconcentrations(400nMand600nm,respectively) wereconducted(Fig.7)revealingtheimportanceoftheenzyme amountontherobustnessoftheoverallreaction:Whileusinglarge amounts(35M)ofenzymeandanominaltertBuOOHdosingrateof
20mMh−1linearproductaccumulationwasobservedthroughout theexperiments.However,usinglow(400nMor600nM) concen-trationsofrAaeUPOproductaccumulationceasedafter1.5hand 2.5h,respectively.Nevertheless,upto40mMofenantiopure (R)-1-phenylethanolwasformedundertheseconditionscorresponding toatotalturnovernumberof67,500fortheenzyme.
Overall we concludethat theratio of tertBuOOH addition to
enzymeconcentrationisthedecisivefactordeterminingthe effi-ciencyoftheoverallreactionintermsofvolumetricproductivity andtotalturnoveroftheenzyme.Furtheroptimizationstudiesare currentlyongoinginourlaboratory.Particularly,controllingthe wateractivityisbeinginvestigatedtooptimizetheenzymeactivity andoptimizationoftheenzymeloadingonthecarrierarecurrently ongoing.
Despitethecomparablyearlystageofdevelopment,we pro-ceeded to semi-preparative scale to demonstrate the practical feasibility of the proposed reaction setup. On 250mL scale a totalamountof1.25gof(R)-phenylethanol(isolatedyield)were obtained within 3h of reaction time yielding a respectable TTN(rAaeUPO)ofmorethan90,000.Hence,weareconvincedthat anoptimizedreactionschemeexhibitssignificantpotentialfor syn-theticorganicchemistry.
352 E.Fernández-Fueyoetal./JournalofMolecularCatalysisB:Enzymatic134(2016)347–352
Fig.7. ComparisonoftheeffectofdifferentconcentrationsofimmobilizedrAaeUPO (400nM,600nM䊐),20mMh−1oftertBuOOHwasaddedat30minintervalsto
pro-moterAaeUPOcatalyzedhydroxylationofethylbenzene.(R)-1-phenylethanol:solid lines,acetophenone:dashedlines.Conditions:ethylbenzeneand[rAaeUPO]=400 or600nMat30◦C.Reactionvolume250mL.Errorbarsarenotshownsincethe
standarddeviationwasalwaysbelow5%.
5. Conclusions
Today,comparablyfewstudiesdealwiththeapplicationof oxi-doreductasesunder non-aqueousconditions. The pooraqueous solubilityofmostreagentsofinteresthowevernecessitates inten-sifiedresearchonbiocatalysisin‘neoteric’solventsi.e.alternative solventsenablinghighersubstratepayloads.Forsynthetic appli-cationsthisisofutmostimportanceaschemistswillacceptand applyonlythosemethodsthatgivethemaccesstopracticalproduct amounts.
In the present study we have taken the firststeps towards usingperoxygenasesundernonaqueousreactionconditions.Using
immobilized rAaeUPO suspended in the neat starting material (ethylbenzene)wecouldprovidethefirstevidenceforthisreaction setup.Thelimitationsidentifiedsofarcomprise(1)thelow resid-ualactivityoftheheterogenizedbiocatalystand(2)thecomparably poorstabilityoftheenzymeundernon-aqueousconditions.Future studies will have to address these shortcomings. Nevertheless, despitetheveryearlystageofdevelopmentofthissystem, prepar-ative, gram-scale synthesis of enantiopure (R)-1-phenylethanol couldbedemonstrated,whichmakesusconfidentthatan opti-mizedreactionsystemmayexhibitsomepreparativerelevance. Acknowledgement
FinancialsupportbytheEuropeanResearchCouncil(ERC Con-solidatorGrantNo.648026)isgratefullyacknowledged.
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