Biomimetic polymers of plant cutin: An approach from molecular modeling

BIOMIMETICPOLYMERSOFPLANTCUTIN:ANAPPROACHFROMMOLECULARMODELLING  aM.A.San‐Miguel*,aJ.Oviedo,bJ.A.Heredia‐Guerrero,cA.HerediaandbJ.J.Benítez  aDepartamentodeQuímicaFísica,UniversidaddeSevilla,E‐41012Sevilla,Spain. bInstitutodeCienciadeMaterialesdeSevilla(ICMS),CSIC‐US,ConsejoSuperiorde InvestigacionesCientíficas(CSIC),IsladelaCartuja,E‐41092Sevilla,Spain. cDepartamentodeBiologíaMolecularyBioquímica,IHSMLaMayoraUMA‐CSIC, UniversidaddeMálaga,E‐29071Málaga(Spain).  *e‐mail:[email protected]s   ABSTRACT Biomimeticsofmaterialsisbasedonadoptingandreproducingamodelinnature withawell‐definedfunctionalityoptimizedthroughevolution.Anexampleisbarrier polymersthatprotectlivingtissuesfromtheenvironment.Theprotectinglayeroffruits, leavesandnon‐lignifiedstemsistheplantcuticle.Thecuticleisacomplexsysteminwhich thecutinisthemaincomponent.Cutinisabiopolyestermadeofpolyhydroxylatedcarboxylic acidsof16and18carbonatoms.Thebiosynthesisofcutininplantsisnotwellunderstood yetbutadirectchemicalrouteinvolvingtheself‐assemblyofeithermoleculesormolecular aggregateshasbeenproposed.Inthiswork,wepresentacombinedstudyusing experimentalandsimulationtechniquesonself‐assembledlayersofmonomersselectively functionalizedwithhydroxylgroups.Ourresultsdemonstratethatthenumberandposition ofthehydroxylgroupsarecriticalfortheinteractionbetweensinglemoleculesandthe furtherrearrangement.Also,thepresenceoflateralhydroxylgroupsreinforceslateral interactionsandfavorsthebi‐dimensionalgrowth(2D),whileterminalhydroxylgroups facilitatetheformationofasecondlayercausedbyhead‐tailinteractions.Thebalanceof 2D/3Dgrowthisfundamentalfortheplanttocreateaprotectinglayerbothlargeenoughin 2Dandthickenoughin3D. KEYWORDS Cutin;Biopolyesters;AFM;MDsimulations.  INTRODUCTION Inthepastdecadestherehasbeenagreatefforttodesignenvironmentalfriendly polymericmaterialsreplacingthoseobtainedfrompetroleum.Asuccessfulexampleare biodegradableshort‐chainpolyesterslikepolylacticacidandpolyhydroxybutyrate,which, basedontheirhydrophobicity,arecurrentlyusedassingle‐usepackingmaterial.However muchlessresearchhasbeenfocusedonlong‐chain(16carbonatomsandabove)polyesters despitetheyarethebaseofubiquitousplantbarrierbiopolymerslikesuberinandcutin. Understandingtheformationandpropertiesofthesenaturalpolymericmaterialsisan interestingroutetodesignnewnon‐toxic,fullybiodegradableandwaterresistantproducts. Cutin,forinstance,isaquitepurepolyesterpresentatthecuticle,theskinoffruits,seeds, leavesandgreenstemsofhigherplants.ChemicallyiscomposedbyamixtureofC16andC18 polyhydroxyacidsbeingthe9(10),16‐dihydroxyhexadecanoicacid,byfar,themostabundant [1].Despiteitsearlycharacterizationinplanttissues,cutinbiosynthesisisnotyetwell understood.Enzymaticandgenetranscriptionroutesarestillunderdiscussion.However,a directchemicalsynthesisfromeitherself‐assembledmoleculesoraggregateshasbeen proposedrecently[2].Thus,theunderstandingoftheintermolecularinteractionbetween monomericunitsisakeyissue.Toaddressthispoint,weproposemodelsystemsinwhich thealiphaticskeletonofthemoleculeisselectivelyfunctionalizedwithhydroxylgroupsin primaryandsecondarypositionsandplacedontopofaninertflatsupportintheformof self‐assembledlayers.Inparticular,wehaveselectedpalmiticacic(PA),16‐ hydroxyhexadecanoicacid(HPA)andthe9,16‐dihydroxypalmiticacid(diHPA)asreference monomerswithout‐OHgroups,withaterminalhydroxylgroupandwithaterminalanda secondary–OHgroups,respectively. Resultsobtainedfrommoleculardynamics(MD)simulationsarecomparedwith experimentalAtomicForceMicroscopy(AFM)datatodeterminetheroleofthespecific hydroxylfunctionalizationinthestructureoftheself‐assembledlayer.Thisinformationwill beusedtodesigntherightmonomericcompositionleadingtoconsistentpolymericfilms. METHODS Moleculardynamics(MD)simulationswerecarriedoutusingDL_POLYcode[3‐4]in thecanonicalensemble(NVT).TheinitialvelocitiesweresetupfromaBoltzmann distribution.Theequationsofmotionwereintegratedbyusingthevelocity‐Verletalgorithm withatimestepof1fs.Thetemperaturewaskeptconstantat300KbyapplyingaNosé– Hooverthermostat[5‐6]. Theinitialconfigurationsforthemonolayersystemswerebuiltbyplacing100 molecules(all‐transconfiguration)withthe–COOHgroupfacingthesurfaceandarrangedin atriangularlatticewithadistanceof5.2Åtothenearestneighborsyieldingasurfacedensity (ρs)of21.4Å2/molecule.PeriodicconditionswereimposedalongtheXandYdirectionsina boxofdimensionsofLx=52.0Å,Ly=45.0ÅandLz=200.0Å. Acutoffradiusof15ÅwasusedfortheLennard‐Jones(6–12)potentialterms.The electrostaticinteractionsweretreatedusingthemethodofHautmanandKleinwhichisan adaptationoftheEwaldmethodforsystemswhichareperiodicintwodimensionsonly.[7] Simulationswereextendedupto10nsforthemonolayersystemsand4nsforthe bilayersystemstoassureequilibriumconditions.Configurationsweresavedevery1000steps (1ps)andthestatisticalanalysiswasdoneforthelastnanosecond. Theinteratomicpotentialparametershavebeenusedalreadyinpreviousstudiesof similarsystems[8‐10].Alkanechainsweremodelledwithaunitedatompotential,basedon CHARMM22butwithparametersadjustedtogiveabetterdescriptionofthecrystalstructure ofn‐alkanes[11].Theotheratomsinthehydroxylandcarboxylicgroupswererepresented explicitly[12‐13]. Thesubstratewascharacterizedasaflatsurface,andtheinteractionbetweeneach particleandthesurfacewascomputedaccordingto    3 0 3 12 0 12 zz C zz C rV     whereCn(σεn)1/2,zisthedistancetothesurface,andz0isalimitapproachdistancefor eachcenter.TheparametersεandσareLennard‐Jonespotentialparameters,andtheyare estimatedbymeansoftheLorentz‐Berthelotcombiningrules.CnparametersforCH2andO weretakenfromtheliterature[14‐15]. RESULTS Monolayerstructures Wehavechosenaseriesofmoleculescontaininghydroxylgroupsindifferent positionsalongthealkylchaintostudytheself‐assemblyprocessonmuscovitemicasurface asareferenceinertflatsupport.Thepalmiticacid(PA)hasbeenselectedasareference moleculewithnohydroxylgroups,the16‐hydroxyhexadecanoicacid(HPA)withaterminal hydroxylgroup(primary‐OHgroup)andthe9,16‐dihydroxypalmiticacid(diHPA)whichhas bothaprimaryandasecondary–OHgroups. TopographicAtomicForceMicroscopyimagesindicatethatPAdoself‐assembleon micaintheformofdendriticmonolayerislandstypically700‐800nmindiameterandabout 1.3‐1.5nmhigh(seeFigure1a).Thisislandheightcorrespondswithatiltedarrangementof moleculesexposingtheirhydrophobicmethyleneend,asinferredfromfrictionandadhesion AFMmapping.Therefore,thePAmoleculesinteractwithmicasurfacethroughthecarboxylic group,likelybyprotonexchangewithK+ionsfrommicaandtheformationofthe correspondingcarboxylate.Obtaininglowsaturationcoverage(10‐15%)despitethepalmitic acidsolutionconcentrationusedsupportsthislaterargument. Hydrophilic–COOHgroupsbondtothesurfacebut,simultaneously,theymayinteract withneighbor–COOHgroupstoformahydrogenbondingnetwork.Moleculardynamics simulationshavebeenconductedtogaininsightintotheroleofsuchhydrogenbondingon theself‐assemblyprocessofpalmiticacidonmica. ResultsfromMDsimulationsindicateacleartendencytoformwellself‐assembled aggregates(orislandsinahigherorderofsizemagnitudewhichisoutofoursimulation scale)togetherwithanunstructuredgroupofpalmiticacidmoleculesconnectedbya hydrogenbondingnetwork(seeFigure2a).Suchunstructuredphaseisconsideredtobe responsibleforthelowcoverage,dendriticandlowheightislandobservedbyAFM. Next,weconsiderthemodificationsinducedbythepresenceofaprimary(terminal) hydroxylgroupinthe16‐hydroxyhexadecanoicacid.Additionofanendhydroxylgrouptothe alkylchaindrasticallychangesthegrowingpatternwhencomparedwithpalmiticacid. Molecularself‐assemblyof16‐hydroxyhexadecanoicacidformselongatedandterraced multilayeredislandswithstepsheightsof2.5nm,whichisquitesimilartotheall‐trans configurationlength(2.2nm),Figure1b. MDsimulationsofa16‐hydroxyhexadecanoicacidmonolayerwerecarriedoutusinga similarprotocolthanforpalmiticacid.Thestructureofanequilibrated16‐ hydroxyhexadecanoicacidisshowninFigure2b.Incorporationofaterminalhydroxylgroup inthemoleculecausestheformationofasecondhydrogenbondnetworkwhichsignificantly reducesthemoleculartiltincreasingsothelayerthicknessinagreementwiththeAFM observations.Thisevidencecanbemonitoredbyinspectingtheangleformedbetweenthe tailvectorandthenormalsurface.Thetailvectorhasbeendefinedastheeigenvector associatedtothesmallesteigenvalueofthemomentofinertiatensorforallcarbonatomsin thealkanechain.Thiscorrespondstothatprincipalaxisaboutwhichrotationistheeasiest. Wehavecomputedthetiltangledistributionforbothpalmiticand16‐hydroxyhexadecanoic acidmonolayersandresultsareplottedinFigure3.Palmiticacidshowsabi‐modal distribution.Onewithmaximalintensityat30°correspondstothosemoleculesincompact aggregates,andasecondoneathightiltangles(closeto90°)arisingfromthose unassembledmolecules.Incontrast,thetiltdistributionfor16‐hydroxyhexadecanoicacidis rangedbetween20and40°asaconsequenceofamoreeffectiveself‐assemblyprocess. Thethirdmoleculestudied(9,16‐dihydroxypalmiticacid,diHPA)isafattyacidthat containsbothaprimary(end)andasecondary(mid‐chain)hydroxylgroups.Thismoleculeis themainmonomerextractedafterplantcutindepolymerization.TheresultsfromMD simulationofamonolayerindicatetheformationoftwohydrogenbondingnetworks:one resultedfrominteractionsbetweensecondaryhydroxylgroupsandanotherbetween primary–OHgroups.Thus,themonolayerexhibitsatiltangledistributionwhich,differently fromthe16‐hydroxydecanoicacid,ispeakedat35°asconsequenceofamorecompact structure.ThisisinremarkableagreementwiththeAFMobservationsindicatingthatthis moleculeformsinterconnectedanduniformislandswithahighsurfacecoverage,Figure1c. Comparedwithotherhydroxylatedfattyacids,thefirstmonolayerofdiHPAdevelops muchfasterandinalargerextentthantheonecorrespondingtopalmiticacid,inwhichno secondaryhydroxylgroupsfavor2Dgrowth.Besides,themonolayerformationstageiswelldefinedindiHPAwhileitisnotobservableinHPA,whichreadilygoestoa3Dgrowing pattern.TheeffectiveformationofthemonolayerindiHPAcanbeexplainedbya cooperationmechanisminvolvingbothsecondaryandprimaryhydroxylgroups.Thenetwork ofhydrogenbondingbetweenprimary–OHgroupsisreinforcedbyasecondnetworkcreated bybridgingsecondaryhydroxylgroups(Figure2c).  Formationofmultilayersystems However,themostimportantfeatureoftheself‐assemblyprocessof16‐ hydroxyhexadecanoicacidonmicaistheformationofmultilayeredislands.Assumingthat 16‐hydroxyhexadecanoicacidbondstomicathroughthecarboxylend,thepresenceof exposedhydroxylgroupscausescompetitionbetweenadsorptiononmicatocreatenew islands(2Dgrowth)andadsorptionontopofanexistingmonolayer(3Dgrowth).Inthe secondcase,theexposedterminalhydroxylgroupstriggermultilayergrowthandadditional headtotail(carboxyltohydroxylgroups)hydrogenbondsarecreated. TopographicAFMmeasurementsdonotprovideinformationaboutthemolecular orientationintheself‐assembledmonolayersof16‐hydroxyhexadecanoicacid,i.e.whether themoleculebondstothesurfacethroughthe–COOHor–OHgroup.PreviousAFMresults showthatfattyacidSAMsonmicaaremorestablethanthosecorrespondingtothe analogousprimaryalcohol[16].Thisfactallowsustoassumereasonablythattheadsorption occurspreferentiallybythecarboxylicgroup.Furthermore,previousSTMstudiesonselfassemblyoffattyhydroxylacidsonHPOGhaveshownthat–COOH•••HOOC–interactionis strongerthan–COOH•••HO–[17‐18].Thisobservationmaycausecompetitionbetween headtohead(–COOH•••HOOC–)andheadtomicabondingandreverseorientation(bonded tomicathroughthehydroxylgroup)ofafractionofmoleculesintheself‐assembledsystem. Asfirstapproximationinoursimulations,wehaveassumedthatmoleculesinthemonolayer adsorbtothesurfaceonlythroughthe–COOH.Then,themoleculesinthesecondlayer interactwiththeexposed–OHgroupsonlybythe–COOHgroup.Thus,tomodelabilayerof bothpalmiticand16‐hydroxyhexadecanoicacids,asecondequilibratedlayerwasplacedat4 Åfromthehighestatomsinthemonolayerpreviouslyequilibrated.Then,simulationswere extendedfor4ns.Figure4showsasideviewofthefinalconfigurationforbothsystems.Itis clearthatthepalmiticacidbilayerishinderedbecauseinteractionbetweenthehydrophobic tailinthefirstlayerandthehydrophilicgroupinthesecondlayerisnoteffective.Avery differentscenarioisobservedfor16‐hydroxyhexadecanoicacid,whereterminalhydroxyl groupsinthefirstlayerinteractwithcarboxylicgroupsinthesecondlayerbyhydrogen bonding,leadingtoawell‐organizedself‐assembledbilayer. Whenbothprimaryandsecondaryhydroxylgroupsarepresent,theself‐assembly processisacombinationofthetwopatternsalreadydescribed.Secondaryhydroxylgroups favour2Dgrowthofislandswhileprimaryhydroxylspromoteformationofmultilayers.Thisis clearlyseeninFigure1c,whereevolutionof9,16‐dihydroxypalmiticacid(diHPA)layersvs. preparationsolutionconcentrationisshown.Atlowconcentrationthefirstmonolayer developswithaminorbutgrowingpresenceofthesecondmonolayerastheconcentrationis raised.Fullcoverageofmicasupportisachievedabove2mM.Thesystemfurtherdevelops untilthefirstmonolayerisalsofullycoveredbyasecondoneand,finally,atthehighest concentration,athreelayersdepositpredominatesbeforelargeparticlesprecipitation occurs. ResultsfromtheMDsimulationsofadiHPAbilayerindicatealsotheformationofa well‐developedsysteminwhichtheinteractionsbetweenthesecondaryhydroxylgroups influencesignificantlyonthemolecularorientation.ItcanbeseenfromFigure4,that despitethediHPAmoleculesdonotshowall‐transconformationsasHPA,theystillselfassembleinacompactstructure. Therefore,forHPAthemultilayersystemisdevelopedfrominteractionsbetweenthe terminal–OHgroupsofaformedlayerandthe–COOHgroupsofnewmolecules approaching.Thus,theemerginghydrogenbondingnetworksareflexibleenoughtoallow thealkanechainstokeepthemostfavorableconformation(all‐trans)andtheeffective packingbetweenthemleadstoatiltangledistributionpeakedat38°(Figure5).Ontheother hand,thesecondary–OHgroupofthediHPAmoleculesintroducesadditionalhydrogen bondingnetworkdominatingthelateralmolecularinteractionsandleadingtoaselfassembledsystemwithamolecularorientationclosetotheverticality(tiltangledistribution c.a.10‐20°).  CONCLUSIONS Wehavereportedacombinedstudyofexperimentalandcomputersimulation techniquesonself‐assembledsystemsofthreedifferentmonomersselectivelyfunctionalized withhydroxylgroupsonmicasurfaceactingasaninertsupport.Ourresultsshowthat palmiticacid(PA)formswellself‐assembledislandstogetherwithunstructuredaggregates whichareresponsibleforthelowcoverageobservedintheAFMimages.Thepresenceofa terminalhydroxylgroupin16‐hydroxyhexadecanoicacid(HPA)allowsmoreefficientlateral molecularinteractionsachievinghighmonolayercoveragesandalsotriggerstheformationof multilayerstructures(3Dgrowth).Theadditionofasecondaryhydroxylgroupinthe9,16‐ dihydroxypalmiticacid(diHPA)allowsforanotherhydrogenbondingnetworkimprovingthe bi‐dimensionalgrowth(2D)andthusthefirstmonolayerdevelopsmuchrapidly. Furthermore,themultilayergrowthalsooccursbutalwaysaccompaniedbyahighcoverage ofthelastlayersbeforeadditionallayersstarttodevelop.Therefore,tocreateaneffective protectinglayerintheplant,theself‐assemblyprocesshastobalancebothgrowths,being largeenoughin2Dandthickenoughin3D.Thefirstoneismainlycontrolledbyinteractions betweensecondaryhydroxylgroupsandthesecondonebyprimaryhydroxylandacid groups.  ACKNOWLEDGEMENTS FundingisprovidedbytheSpanishMinisteriodeEconomíayCompetitividadunderproject CTQ2011‐24299andbyJuntadeAndalucíagrantTEP‐7418undertheMotrizProgram.  REFERENCES [1]Arrieta‐BaezD,Cruz‐CarrilloM,Gómez‐PatiñoMB,Zepeda‐VallejoLG(2011)Derivatives of10,16‐dihydroxyhexadecanoicacidisolatedfromtomato(Solanumlycopersicum)as potentialmaterialforaliphaticpolyesters.Molecules16:4923‐4936.doi: 10.3390/molecules16064923 [2]DomínguezE,Heredia‐GuerreroJA,BenítezJJ,HerediaA(2010)Self‐assemblyof supramolecularlipidnanoparticlesintheformationofplantbiopolyestercutin.MolBioSyst 6:948‐950.doi:10.1039/B927186D [3]SmithW,YongCW,RodgerPM(2002)DL_POLY:applicationtomolecularsimulation.Mol Simul28:385‐471.doi:10.1080/08927020290018769 [4]Availableatwww.ccp5.ac.uk/DL_POLY [5]NoseS(1984)Aunifiedformulationoftheconstanttemperaturemoleculardynamics methods.JChemPhys81:511‐519. [6]NoseS(2002)Amoleculardynamicsmethodforsimulationsinthecanonicalensemble. MolPhys100:191‐198.10.1080/00268970110089108 [7]HautmanJ,KleinML(1992)MolPhys75:379‐395.doi:10.1080/00268979200100301 [8]Heredia‐GuerreroJA,San‐MiguelMA,SansomMSP,HerediaA,BenitezJJ(2009)Chemical Reactionsin2D:Self‐AssemblyandSelf‐Esterificationof9(10),16‐DihydroxypalmiticAcidon MicaSurface.Langmuir25:6869‐6874.doi:10.1021/la9001412 [9]Heredia‐GuerreroJA,San‐MiguelMA,SansomMSP,HerediaA,BenitezJJ(2010)Aleuritic (9,10,16‐trihydroxypalmitic)acidself‐assemblyonmica.PhysChemChemPhys12:10423‐ 10428.doi:10.1039/c0cp00163e [10]Heredia‐GuerreroJA,San‐MiguelMA,LunaM,DomínguezE,HerediaA,BenítezJJ (2011)Structureandsupportinducedstructuredisruptionofsoftnanoparticlesobtained fromhydroxylatedfattyacids.SoftMatter7:4357‐4363.doi:10.1039/c0sm01545h [11]San‐MiguelMA,RodgerPM(2001)SimulationofDepositionofWaxtoIronOxide Surfaces.MolSimul26:193‐216.doi:10.1080/08927020108028293 [12]JorgensenWL(1986)OptimizedIntermolecularPotentialFunctionsforLiquidAlcohols.J PhysChem90:1276‐1284. [13]BriggsJM,NguyenTB,JorgensenWL(1991)MonteCarloSlmulationsofLiquidAcetic AcldandMethylAcetatewiththeOPLSPotentialFunctions.JPhysChem95:3315‐3322. [14]HautmanJ,KleinML(1989)Simulationofamonolayerofalkylthiolchains.JChemPhys 91:4994‐5001. [15]FartariaRPS,FreitasFFM,SilvaFernandesFMS(2005)Astudyof1‐decanethiolselfassemblyongoldelectrodesbycomputersimulation.JElectroanalChem574:321‐331. doi:10.1016/j.jelechem.2004.08.015 [16]BenítezJJ,Heredia‐GuerreroJA,HerediaA(2007)Self‐assemblyofcarboxylicacidsand hydroxylderivativesonmica.AqualitativeAFMstudy.JPhysChemC111:9465‐9470.doi: 10.1021/jp070563y [17]CyrDM,VenkataramanB,FlynnGW(1996)STMinvestigationsoforganicmolecules             Figure4  MICA (c)   Tilt Angle 0 102030405060708090 Probability HPA diHPA     Figure5  