Biocompatible graft copolymers from bacterial poly(gamma-glutamic acid) and poly(lactic acid)

  ARTICLE  Pleasedonotadjustmargins Pleasedonotadjustmargins Received00thJanuary20xx, Accepted00thJanuary20xx DOI:10.1039/x0xx00000x  BiocompatibleGraftCopolymersfromBacterialPoly(‐Glutamic Acid)andPoly(LacticAcid) CristianaL.Zaccaria,aValeriaCedrati,aAndreaNitti,aEnricaChiesa,bAntxonMartinezdeIlarduya,c MonserratGarcia‐Alvarez,cMassimilianoMeli,dGiorgioColombo,a,dDarioPasini*a Wereportonanovelapproachtothemodularandconvergentconstructionofbiocompatiblegraftcopolymersstarting frombacterialpoly(‐glutamicacid)(PGA)andincorporatingpoly(lacticacid)(PLA).Thesyntheticstrategyiscontrolledat differentlevels:a)thechoiceofasuitableinitiatorforthering‐openingpolymerizationoflactide;b)thechemical elaborationofthepolylacticfragments;c)theirconvergent“graftingto”functionalizationofbacterial‐PGApropargyl esterusingcopper(I)‐catalyzedalkyne‐azidecycloaddition(CuAAC)clickchemistry.Thegraftcopolymersarecharacterized intermsoftheirthermalandmacromolecularproperties,theirconformationalpreferencesthroughmolecularmodelling, andtheircytotoxicity. Introduction Poly(‐glutamicacid)(‐PGA),1formallyanylon4derivative,isa bacterialpolymerusedinthecosmeticandfoodindustries.Itis obtainedfromrenewableresources(byfermentation,fromaseries ofbacteriastrains),2‐4anditscharacteristicsareexcellentasa macromolecularbackboneforinnovativebiomaterials.The scientificinterestaround‐PGAhasgreatlyriseninthelast20 years.5‐9 The‐carboxylicgroupof‐PGAcanbederivatized,tomodulate thephysicalandchemicalpropertiesofthenativebiopolymer.10 Severalstrategiesfor‐PGAfunctionalizationhavebeenrecently proposed.11‐14Wehaverecentlyreportedaremarkablyefficient alkylationmethodologyusinghomogeneousreactionconditionsin lowpolarityorganicsolvents.Solubilizationof‐PGAisobtainedby exchangingthecationassociatedtothecarboxylatefunctionalities withalongalkylchainquaternaryammoniumcation.Esterified‐ PGAhomoorcopolymers,containingallylorpropargylgroups,have beenobtainedinhighyields.CuAACclickchemistry15havethen beenused,inthecaseofpropargylesters,toinstallfurther functionalgroupsontothe‐PGAbackbone.16 Blockcopolymers,whichcanbesimplifiedasthecovalentmerging ofdifferenttypesofmacromolecules,havebeenwidelyexplored, forimprovingthepropertiesoftheseparatepolymeric components,andforprovidinginterestingandproperty‐directed conformationsandmorphologies,withrecentoutstanding examplesinthefieldofnanoscienceandnanopatterning.17Inthe contextofbiomaterials,blockcopolymersarecommon,butgraft copolymershavenotbeenaswidelyexploredtodate.Examplesof graftcopolymershavebeenreportedasapotentialstrategyfor enhancingbiostabilityproperties,18orenhancingdrugdelivery performance.19Evenconsideringoneofthemostpopular biocompatiblescaffolds,poly(lacticacid)(PLA),itisperhaps surprisingtofindonlyafewreportsonitsincorporationingraft copolymers:thesereportsaremainlybasedonthepolymerization ofpreformedmethacrylatePLAmacromonomers.20,21 Thesyntheticdifficultiesinaddressingthereliableandcontrolled functionalizationof‐PGAhavehamperedamodularand molecularlypreciseapproachto‐PGAbasedgraftcopolymers.In thefewreportsavailable,‐PGAbasedgraftcopolymersarebuilt withunoptimalcontrolofthedegreeoffunctionalizationandofthe coverageofthependantchainsfromthemain‐PGAbackbone.22,23 Herewereportaboutthesynthesis,characterization,and propertiesofanewfamilyofgraftcopolymersinwhich biocompatiblepolyestersareefficiently“clicked”ontothe‐PGA backbones.Theoverallsyntheticstrategyisschematically illustratedinFigure1.Themodularityofthesynthesistakes advantageofthecontrolledring‐openingpolymerization(ROP)of lactide,sothattheinitiatingfunctionalityiseffectivelyinsertedat onechainend.Theotherchainendischemicallyelaborated,witha simple,innovativeprotocol,tointroducetheazidereactiveterminal functionality,whichisthenclickedontopropargyl‐functionalized‐ PGAester.Themodularityofthesyntheticapproachallowedusto systematicallychangethePLAlengthgraftedontothemain backbone,uncoveringinterestingthermalpropertiesofthegraft copolymers.Structuralcharacterizationthroughconformational searchtechniquesprovidesinitialatomic‐resolutioninsightintothe structuralpreferencesofmodelcopolymers.Citotoxicitytests demonstratedthepreeminentroleoftheinitiatingROP A R 2| fu n de t   Fig u gra f co p R e Sy n of t th e ot h be c Its pr o ext co s bio th e po s fu n as de c ha v ma pr e di m re p lite co m (Sn ha n R TICLE J.Name.,201 2 n ctionality,whi c t erminingthet o u re1.Aschem a f ted‐PGAcop o p olymers(botto m e sultsand D n thesisofthe  t hegraftpoly m e cyclicdimero f h erside.Poly(l a c auseitisam o greennature, o ducedbyfe r remelyappeal s tmakesitve r plastics.PLAc a e useofas p s itioningoft h n ctionalities.O n describedin F c oratethewho v eadecisivei m cromolecular e cursorsisrep o m eroflactica c p ortedbelow. rature,werei n m paredtoeit h (Oct) 2 )asthe n dsusing4‐di m 2 ,00,1‐3 c hdecorateth e o xicityofthefi a ticviewofth e o lymers,andthe m ). D iscussion  ‐PGAgraftco p m ersisachieve d flacticacid(la c a cticacid)(PL A o ngstthemos t mostlyafford e r mentation,a n ingincombin a r ycompetitive a nbeobtaine d p ecificalcohol h einitiatingr e n ceclickedco n F igure1,such lecomb‐likem m pactincontr construct.Th e o rtedinSche m c id)wasused c Severalmeth o n itiallytested b h erdiazabicycl o catalysts,be t m ethylaminopy r e exteriorofth nalgraftcopol y e modularsynt h chemicalstruc t p olymers.The c d infiveoveral c tide)onones i A )waschosen t studiedbioc o e dbythefact t n dthenpoly m a tionwith‐P G andcurrently d throughaRO initiatorall o e sidueason e n vergentlyont o initiatingfun acromolecules , ollingtheove r e initialprep a m e1.LL‐lactide c onsistentlyfo r o dologies,alr e b yusforthe R o undecene(D B t terresultsw e r idine(DMAP) Please d Please d egraftcopoly m y mer. h eticapproach t t ureofthesynt h c onvergentsy n lsteps,startin g i deand‐PGA o aspolyesters c o mpatiblepoly e t hatthemono m erized, 24 ma G A.Itsrelative usedinthe f i P approach,in o wsforthe p e oftheend o the‐PGAsc a ctionalitieswi l , andtherefor e r allproperties a rationofth e (lactideisthe r alltheexperi e adypresent i R OPoflactide. B U)ortinoct a e reobtained i a sthecatalyst . d onotadjus t d onotadjus t m er,in  t oPLA‐ h esized n thesis g from o nthe c affold e sters. meris kesit e lylow i eldof which p recise chain affold, l lfully e could ofthe e PLA e cyclic ments inthe When a noate i nour . 25 The use o adva n proc e pote n DMA solve benz y quan t hexa n ratio integ resid u GPC m confi r relati orde r ares h 5a w brou g Sche m RO 4R 5R  Sche m elabo r  To o intro d inth e leavi n react proc e T t margins t margins o ftheDMAP,a n tages,inter m e ss,andforav n tiallytoxicti Pwasusedi n ntandgentle h y licalcohol1 t itativeyields n es.Goodagr e ofmonomers rationofthe u eandofthe m easurements . r mthecontroll onshipsbetwe r natureofth e h owninFigure w asobtainedi g htthrougho u m e1. 1 or 2 OH OH + RO OO n O 6a-c R= P 7a R=C 2 OOH n+ =PhCH 2 =C 2 H 5 4 a 4 b 4 c 5 a m e1.Synthesis r ationfortheins o urknowledg e d uceatermina e transformati o n ggroup(met h iononthes e durecouldn T hisjournalis© norganocatal y m softheover a o idingcontam n ‐basedcatal y substoichiom e h eatingfor24 h (Scheme1) aswaxesafte r e ementbetwe couldbeobse uniqueprot o repeatingunit . Asreportedi n ednatureoft h e nconversion a e kinetics,and S1.Byusinge nasimilarly u tthefuncti o O O O O D CH 2 4 3 Br O P hCH 2 H 5 N a D M R T 8 85-9 0 Br Br O C a n=5 b n=10 c n=15 a n=5 ofPLAthrou g e rtionoftheazi d ,theonlyk n lazideinvolve s o nofthetermi n h anesulfonate) e condarycar b o tbereprod u TheRoyalSoci e y st,intheROP a llsustainabili t inationofthe y sts.Inour o etricquantitie h .Polymers4 w astheiniti a rpurification b e entheobser v e rvedby 1 HN M o nresonance s t sandvalues w n theliterature , h eROPpolym e andnumbera v thenarrowre e thanol2asth e controlledm a o nalizationse q R O D MAP 2 Cl 2 (dry) 4 5°C 4 5 RO O a N 3 M F T 8a-c 9a R 8 4-95% 0 % R O Et 3 N C H 2 Cl 2 (dry) RT 70-75% g hROP,and s d eterminalunits . nownderivati s atwosteppr o n alsecondary a andsubsequ e b onusingso d u cedreliablyi JournalN a e tyofChemistry possessesob v t yofthesynt h finalproduct w o ptimizedprot sindryCH 2 Cl w ereobtained w torinessen t b yprecipitatio v edandcalcul a M R,bytherel a oftheiniti a w ereconfirme d , 25 wewereab l rization.Theli n v erageM n ,the sultingdispers e initiator,pol y a nner,andit q uenceshow n O OOH n R =PhCH 2 R =C 2 H 5 O nN 3 O R=PhCH 2 R =C 2 H 5 O OO nO 6a-c R=Ph C 7a R=C 2 H 5 ubsequentche m .  zationofPL A o cedure,consi s a lcoholintoa g e ntlyasubstit u d iumazide. 26 nourhands. a me 20xx v ious h etic w ith ocol 2 as w ith t ially nin a ted a tive a ting d by l eto n ear first ities y mer was n in Br C H 2  m ical A to s ting g ood u tion This We JournalName ARTICLE Thisjournalis©TheRoyalSocietyofChemistry20xxJ.Name.,2013,00,1‐3|3 Pleasedonotadjustmargins Pleasedonotadjustmargins thereforesuccessfullydesignedanothertwostepprocedure,which hastheadvantageoftransformingtheterminalsecondaryalcohol ofthePLAchainsinaprimaryterminalalkylbromide,pronetobe transformedintotheazidebyastraightforwardsubstitution reaction(Scheme1).Theacylationreactionwasconductedwith bromoacetylbromideinthepresenceoftriethylamine,toobtain derivatives6and7ingoodyields.Thesubstitutionofbrominewith anazido‐terminalgrouptakesplaceatroomtemperaturewithan excessofNaN3(1.5equivalent)andDMFasasolvent.Polymers8 and9couldbeobtainedinpureformafterprecipitationinhexane asthenonsolvent. Propargylfunctionalizedγ‐PGA10wasobtainedasacopolymer, withahighdegreeofesterification,asdescribedinourprevious paper.16Thelackofcompleteesterificationinthecaseof‐PGAisa consequenceofstericfactors;16severalbatchesofpolymer10 showedconsistentlyandreliablyasubstitutioncoverageof propargylgroupsofaround90%,asindicatedinScheme2.Polymer 10wasreactedwithazidoandbenzylterminatedPLApolymers8a‐c (usedinslightexcess).Thereactionconditionsfortheclick chemistry(CuSO4/NaascorbateinamixedDMF/H2Osolvent system)werepreviouslyoptimizedonmodelsystems,andafforded fullyfunctionalizedgraftcopolymers12a‐cinexcellentyieldsinall cases.Polymers12werecarefullypurifiedbyprecipitationinacidic water,andthenasecondpurificationbydissolvingthecrudeinTHF andreprecipitatingitinhexane.Thefidelityoftheconvergent, “graftingonto”approachusingtheoptimizedCuAACprotocolwas verifiedbyNMRspectroscopy(seebelow).Theprotocolwas equallysuccessfulusingethylesterterminatedazidoPLA9a,to affordpolymer13aingoodyields. Inordertoverifytheefficiencyandthereliabilityofourconvergent functionalizationstrategy,wemadeanexperimentinwhichPLA8c wasusedinlessthanstoichiometricquantities(ca.0.5equivalents). Indeed,terpolymer11c,inwhichca.50%ofalkyneterminal functionalitieswerestillpronetofurtherfunctionalization,was obtainedingoodyieldsusingthepreviouslymentionedCuAAC protocol.Polymer13ccouldbefurtherfunctionalized(Scheme2) withcompound14toobtainfully“clicked”polymer15cinhigh yields.Allpolymerscouldbeobtainedaswhitepowders,after thoroughpurificationproceduresinvolvingatleasttwopurification intononsolvents.  FTIR,NMR,GPCandThermalCharacterizationoftheGraft Copolymers. Infraredspectraof12a‐cand15cgraftcopolymersaredepictedin FigureS2.Thesespectradisplaytwoweakpeaksat1654cm‐1and 1542cm‐1correspondingtothestretchingandbendingvibrationsof amidecarbonylandN‐Hfunctionalgroups,respectively.Astrong absorptionpeakcouldbedetectedat1750cm‐1andcorrespondsto thestretchingvibrationofestercarbonylgroupofPLAsidechains. Asitcanbeobserved,theintensityofthispeakincreaseswiththe lengthofthePLAsidechain.NMRspectroscopywasessentialto confirmthestructureandtodeterminethecompositionofthegraft copolymers.The1HNMR(CDCl3)spectraofrepresentativesamples forpolymers6‐9areshowninFigure2withattributions,and highlighthowthetransformationoftheendchainfunctionalgroups occursquantitatively.Theinsethighlightstheunexpectedsmall changesintheresonancesoftheterminalCH2units(ca.3.91ppmin polymer6aand7ato3.96ppminpolymer8aand9a)uponthe transformationoftheterminalbromideintoazide.Further confirmationofthecompletetransformationtoazidecamefrom theIRspectrum,whichshowsthedistinctiveabsorptionpeakat 2110cm‐1,correspondingtothestretchingofthe‐N3group.  Scheme2.Synthesisofgraft‐PGAPLAcopolymers.EstimatedcopolymercompositiondeterminedbyNMRspectroscopyofthepurifiedproducts.   ARTICLE  Pleasedonotadjustmargins Pleasedonotadjustmargins  Figure2.Comparisonofthe1HNMRspectra(CDCl3)ofpolymers(from bottomtotop:8a,6a,9a,7a).Inset:enlargementofthefullNMRspectraon theleftshowingthe‐COCH2‐Xterminalunitsatca.4ppm.Theasterisks indicatesolventimpurities.  1HNMRand13CNMRspectraofpolymers11c,12a‐c,13aand15c confirmedtheefficiencyoftheclickcouplingandofthepurification procedures.Betterresolutionandreducedbroadeningofthe signalswasachievedat80°CindeuteratedDMSO.  Figure3.1H‐NMRspectraof(a)polymer12a,(b)polymer12b,(c)polymer 12cwithincreasinglengthofPLAsidechainsand(d)polymer15c. Asanexample,the4‐9ppmregionofgraftcopolymersisshownin Figure3(fullspectraaredepictedintheSI).NHprotonsignalsof functionalized(atca.7.9ppm)andunfunctionalized(atca.7.7 ppm)portionsofthecopolymersaredistinguishable. Thedisappearanceofthe1HNMRspectraoftheuniquesignalsof thepropargylestergroups(forexample,theprotonresonancesof theCH2C≡Cgroupsatca.4.7ppm),andfromtheappearanceofthe newlyformedaromatictriazoleprotonresonancesatca.8.1ppm confirmedtheefficiencyoftheCuAACclicksyntheticprotocol.Two signalsinthisregionwereobservedforpolymer15c,duetothe doublefunctionalityofthesidechains(PLAandtert‐butyloxy carbonyl)builton‐PGA.Theabsenceofanyresidualsignalrelated totheCOCH2N3terminalunitsofthePLAreagentatca.4ppm, togetherwiththecoherenceoftheprotonresonanceintegrations withtheproposedstructure,confirmedthatanyexcessor unreactedPLAchainshadbeenefficientlyremoved. Importantinformationwasalsogatheredfromthe13CNMR spectroscopy(FigureS3).Intheregionofaround170ppm,both carbonylsofesterandamidegroupsofthe‐PGAunitscanbe observedat171.5and171.4ppmrespectively.Ontheotherhand, carbonylsofPLAunitscanbeobservedintherange169.1‐169.4 ppm.AsthelengthofPLAsidechainincreases,thesignalsdueto innercarbonyls,whichcanbeobservedat169.1ppm,increasesin intensityoveroutercarbonylswhichappearat166.6and169.4 ppm.Thesesignalsarenotsplit,confirmingthatnoracemizationof theL‐lacticacidunitstookplace,bothduringtheringopening polymerization,aswellasduringtheclickreaction. Thethermalstabilityofgraftcopolymerswereevaluatedby termogravimetricanalysiscarriedoutunderinertatmosphere (Figure4andFigureS4).Grafted‐PGAwithincreasedlengthofPLA sidechains(polymers12a‐c)werestableuptoaround270ºC.The remainingweightat600ºCdecreasedwiththecontentofthese unitsinthecopolymer(TableS1).Itislikelythatunzipping transesterificationreactionsoflactateunitswithgenerationof volatileLL‐lactidecouldberesponsibleforthisthermalbehavior. Forpolymer15c,theslightweightlossataround180ºCis attributabletothepresenceofthethermallylabiletert‐ butoxycarbonylgroups,withagoodmatchbetweenthecalculated (lossofisobutylene,onthebasisofthestructureandthecopolymer compositiondeterminedby1HNMR)andobserved(3.4%calcdvs. 2.4%observed)weightlossstartingat160°Candendingat200ºC. DSCheatingtraces(secondheatingscan)ofpolymers12a‐cand15c showedsecondordertransitions,attributedtotheglasstransition temperatures,andremarkablyinlinewithliteraturevaluesforPLA samples.27Additionally,broadendothermsat118and108ºCwere observedforpolymers15cand12crespectivelyinthefirstheating DSCthermograms(FigureS5),whichweattributedtothemeltingof crystalsmadeofshortPLAgraftedchains.Thesepeakswerenot observedforpolymers12withshorterPLAsidechains.Itisvery 4. 55. 05. 56. 06. 57. 07. 58. 0 (ppm) JournalName ARTICLE Thisjournalis©TheRoyalSocietyofChemistry20xxJ.Name.,2013,00,1‐3|5 Pleasedonotadjustmargins Pleasedonotadjustmargins interestingtonotethatthethermalpropertiesofthegraft copolymersareessentiallydominatedbythePLApendantchains, andthattheinsertionofthePLAchainsofsuitablelength(polymers 12band12c)wasabletoprofoundlymodifythethermalbehavior of‐PGA,sinceglasstransitionsforthegraftmacromolecular constructscanbeobserved.Itmustberemindedthat‐PGAby itselfdoesnotshowanyglassormeltingtransitiontemperatures, butinsteaddecomposesuponheatingaround200°Cthroughan unzippingdepolymerizationmechanismwithreleaseof pyroglutamicacid.28 100 200 300 400 500 600 0 20 40 60 80 100 Remaining Weigth (%) Tem p erature ( ºC ) 12a 12b 12c 15c a) -20 0 20 40 60 80 100 120 140 Endo Tem p erature ( ºC ) 12a 12b 12c 15c b) Figure4.(a)TGAand(b)DSCtraces(secondheatingscan)ofPLAgraft‐PGA copolymers.  PowderX‐raydiffractionmeasurementswereperformedforPLA graftedsamples12a‐cand15c.AsitcanbeobservedinFigure5, sample12cdisplayedrepresentativediffractionpeaksat2theta Bragganglesof14.8,16.7,19.0,22.3correspondingtothe(010), (110/200),(203),(204)planes,respectively,attributedtoPLAside chainscrystallizedinthe‐crystallineform.29,30,31Tocalculatethe phasecrystallinecontent,crystallinepeakswereextractedfromthe diffractionpatternbypeakdeconvolutionandacrystallineindexof 17%wasestimatedforthissample(FigureS6).Thesediffractions werenotdetectedinsample12aandscarcelyobservedinsample 12bindicatingthatthelengthofthePLAsidechainplaysakeyrole inthiscrystallization.Ontheotherhandsample15cdisplayeda diffractionpatternsimilartosample12balthoughabroadmelting peakat118CisobservedbyDSC.Thisdiscrepancycouldbe attributedtoabroadcoldcrystallizationthatcouldhappen betweentheglasstransitionandmeltingduringtheheating process.  5 101520253035 (204) (203) (110/200) (010) 15 c 12 c 12 b Intensity (a.u.) Diffraction angle 2  (º) 12 a  Figure5.PowderWAXSprofilesofPLAgraft‐PGAcopolymers12a‐cand 15c.  SelectedgraftcopolymerswerecharacterizedbyGPC chromatographyusingdimethylacetamide(DMAc)asthesolvent (seeexampleinFigureS7),whichsolubilizedwellallthesamples. TheresultsarereportedinTable1.Aspreviouslyreported,the reactionconditionsusedforsynthesisbroughtasubstantial reductioninthemolecularweightfromthebacterial‐PGAstarting polymertopropargylfunctionalizedγ‐PGA10.Further functionalizationbyclickingdidbringfurtherreductionsbutthe degreeofpolymerizationineither12bor15cremainedhigh.TheĐ valuesremainedremarkablysimilarduringthewholesynthetic process.UncertaintiesintheGPCmeasurementsmaybeseverely enhancedbythecompact,globularnatureofthegraftcopolymers (seebelowmolecularmodelling),asopposedtotherandomcoil conformationsofthePMMAsamplesusedasstandards.  Table1.Molecularweightcharacteristicsofselectedsynthesized copolymersobtainedbyGPCinDMAc.a PolymerMnĐDPb 101210001,8630 12b1880001,5280 15c1820001,7420 aForanalysismethod,seeexperimentalpart.Đ=dispersityindex.bDegree ofpolymerization;calculatedonthebasisofMnvalues,usingtheweighted (accordingtocompositiondeterminedby1HNMRspectroscopy)average repeatingunitmolecularweight.  MolecularModellingandAFMstudies.Theconformational preferencesoftheconstitutingunitscanbeakeydeterminantof thesupramolecularorganizationalbehavior,propensitytofurther modificationandinteractionpropertiesofthe‐PGA‐based copolymers.Tostartsheddinglightontheconformationalbehavior ofthetriazole‐lacticacidcontainingderivatives,wefirststudieda simplifiedsystemcontainingoneunitoflacticacidandoneunitof glutamate(namelyn,m=1).Towardsthisend,weusedtheMonte CarloMolecularMechanics(MCMM)conformationalsearch method,asimplementedintheMaestroSuite ARTICLEJournalName 6|J.Name.,2012,00,1‐3Thisjournalis©TheRoyalSocietyofChemistry20xx Pleasedonotadjustmargins Pleasedonotadjustmargins (www.schrodinger.com),employingasvariablesallrotatablebonds, tosamplethepotentialenergysurfaceofthemoleculeinan octanolsolventmodelabletorecapitulatetypicallaboratory conditions(octanol,organicsolvent).Theensemblesofsampled conformationswerefirstsubjectedtoclusteranalysis,usingan agglomerativemethod,toidentifythemostrepresentative conformations.Thestructuresrepresentativeofthemost populatedclustersarecompact,withatendencytoform intramolecularhydrogenbondinginteractionsandtoexpose hydrophobicgroupstocontactwiththesolvent.Next,wesetoutto characterizetheconformationalpreferencesofalongercopolymer (n=1,m=5,Figure6).   Figure6.Thestructuresofthe2mostpopulatedclustersobtainedfromMD simulationsforthePLA‐PGAcopolymers.Thedottedlinesrepresent intramolecularhydrogenbondinginteractions.Toprow:n=1,m=5.Bottom row:n=5,m=5.  Consideringthelengthofthemolecule,hereweturnedto MolecularDynamicssimulationstosampletheconformational landscape.Simulationswereoncemoreruninoctanol.The trajectorieswereanalyzedbymeansofclusteranalysisandtime evolutionoftheradiusofgyration(FigureS8).Thecopolymer populatescompactconformations,whereby2mainclustersare abletorecapitulatemostoftheconformationaldiversity.Itcanbe noticed,inparticular,thatonefaceofthetriazoleringisshielded fromthesolventbythefoldingontopofitofthelactateunits. Finally,wesetouttosimulateamoreextendedsystem,withn,m= 5(Figure6).Inthissystem,thetwomostpopulatedclustersshow thatthepolymertendstofoldupincompactstructures characterizedbyacorecontainingthetriazolerings,insomecases engagedinintramolecularhydrogenbondinginteractionswiththe amidichydrogens,andanoutsidelayercontainingPLArepetitive units. Thefoldingbehaviourindicatedbythemodellingstudiessuggest thatthesegraftcopolymersdonotphaseseparateintermsof nanoscalemorphology,sincethetwodifferentblocksconstituting themacromolecularstructure(thepolyglutamateandthepolylactic units)areinfacthiddenbyeachotherinclosedstructures.Phase separationisinsteadcommoninwelldefinedblockcopolymers.32 AFMstudieswerethereforecarriedoutinordertoobtain informationaboutthenanoscalemorphology,phasesystemsand possiblephaseseparationsinthinfilmsofcopolymers12a‐c.The observedmorphologiesinallthethreefilmsanalyzedshowedthe absenceofphaseseparationonthenanoscale,andconfirmedthe presenceofessentiallyuniformfilms(FigureS9).  CytotoxicityTestsandHydrophilicityStudies.Inordertoestablish thebiocompatibilityofthesynthesizedgraftcopolymers,3‐(4,5‐ dimehyilthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide(MTT)assays wereundertakenforselectedsamples(12aand13a)atthe concentrationrangeof10–250mg/mL.33Ascanbenotedfrom Figure7,raw‐PGAdoesnotshowanyevidenceofcytotoxicity:the cellsurvivalpercentageisalwayshigherthantherelativeblank control.Moreover,thehighest‐PGAconcentrations(50–100– 250mg/mL)seemtopromotecellviabilityalongthefirst24hof incubationconfirmingthewell‐knownbiocompatibilityoftheraw polymer.2Asbenchmarkvalues,chitosanandhyaluronicacid,two frequentlyusedpolymersrecognizedasGRAS(Generally RecognizedasSafe),showinMTTassaystoxicityeffectsonlyat concentrationshigherthan1mg/mL.34  CTR blank 10 20 25 50 100 250 *** *  Figure7.MTTassayperformedon‐PGA,12aand13aincubatedfor24h withNormalHumanDermalFibroblast(NHDFs).Thedatarepresentsthe mean±SD(n=3independentexperiments).Resultsareexpressedascell survivalpercentagecomparedtotheuntreatedcells(CTR).Blankisthe negativecontrol(solventwithoutpolymers).Statisticaldifferencesforp value<0.05(*),pvalue<0.001(***).  Resultsconcerningthebenzylterminatedgraftcopolymer(12a), showasignificantreductionofcellularsurvivalifcomparedtothe relatedblankcontrol.Thecytotoxicityeffectof12ais concentration‐dependent:thecellsurvivalpercentagedecreases from80±14%to11±2%byincreasingthepolymerconcentration. Statisticalanalysis(Dunnett'smultiplecomparisonstest)reveals significantdifferencesat100and250mg/mLcomparedtothe blankcounterpart.Itisalsoevidentthatpolymer12atriggersa notablereduction(pvalue<0.05)ofthe‐PGAbiocompatibilityat alltheconcentrationstested,andthereforewecanspeculatethat toxicitywasduetotheuseofbenzylalcoholasinitiator. Cytotoxicitydatacollectedforpolymer13a,presentingethyl terminalfunctionalities,showedcellsurvivalvaluesalwayshigher than80%after24hofincubation.Thismeansthatethyltermination isabletoimprovethebiocompatibilityofbenzylterminatedgraft JournalName ARTICLE Thisjournalis©TheRoyalSocietyofChemistry20xxJ.Name.,2013,00,1‐3|7 Pleasedonotadjustmargins Pleasedonotadjustmargins copolymer.Itisfoundtobeasafematerialthatcouldbeusedin thebiomedicalfieldintherangeofconcentration10–250µg/mL. Hydrophilicitystudieswerecarriedoutbymeansofcontactangle measurementsonfilmscastandannealedinthepolymerseries 12a‐c.Itwasfoundthatcontactanglevalueswithwaterwere, respectively,85.7°,85.5°,82.9°for12a‐c.Theresultsuggestsa stablehydrophilicityofthefilmsinthepresenceofincreasing lengthsofthehydrophobicPLAsegments. Conclusions Wehavereportedanovelapproachtothemodularconstructionof biocompatiblegraftcopolymersstartingfrombacterial‐PGAand PLA.Thesyntheticstrategytakesadvantageofaconvergent approach.TheROPoflactideusingeitherbenzylorethylalcoholas theinitiatorisfollowedbytheinnovativepostfunctionalization approachofthePLAterminalends,tointroduceanazide.We demonstratedtheefficiencyandprecisionoftheCuAACclick chemistryforthecouplingoftheazido‐terminatedPLAchainsonto thepropargylterminated‐PGA,toaffordafamilyofgraft copolymersinwhichthePLAchainlengthsweresystematically varied. Theconformationalpreferencesthroughmolecularmodelling indicatedaglobular,compactstructureforthegraftcopolymers. AFMstudiesconfirmedtheabsenceofphase‐separated morphologiesinthinfilmsofthegraftcopolymers,builtfromtwo verydifferentmonomericunits.Theirthermalpropertiesindicatea profoundeffectoftheintroductionofthePLApendantchains,since glassthermaltransitionscouldbeobserved.Inthederivativeswith longerPLAgraftedchains,thereareevidencesthatthePLAside chainsareabletocrystallizeinthecrystallineform.Whenethyl esterfunctionalitiesdecoratetheexteriorofthemacromolecules, cytotoxicityisbroughttovaluescomparabletothoseofcurrently usedpolymersandbiopolymersinthebiomaterialsarena. Consideringthat‐PGAbyitselfdoesnotshowanyglassormelting transitiontemperatures,thedescribedapproachmayopenup possibilitiesinthe3Dprintingand/orbulkprocessingofsuch macromolecularconstructs. ExperimentalSection GeneralExperimental.‐PGAwaspurchasedandcharacterizedas previouslydescribed.16Compounds1016and1435werepreparedas previouslydescribed.LL‐Lactidewasrecrystallizedfromtoluene beforeuse.Allothercommerciallyavailablecompoundswereused asreceived.1Hand13CNMRspectrawererecordedfromsolutions inCDCl3ordimethylsulfoxide(DMSO)‐d6on200,300or400MHz instrumentsandcalibratedwiththesolventresidualprotonsignal. InfraredspectrawererecordedonaFTIRspectrophotometer equippedwithadiffusereflectanceaccessoryusingKBrpowderas theinertsupport. StaticcontactangledeterminationsweremadewithaKSVCAM200 instrument,withthewatersessiledropmethod.Sampleswere preparedbydissolving30mgofpolymerin1mLofCHCl3,then castingthesolutionontoaglasssubstrate.Theresultingthinfilms wereannealedat80°Cfor24hours,afterwhichtheovenwas turnedoffandallowedtocoolslowlytoroomtemperature. WASXdiffractionpatternswererecordedonthePANalyticalX’Pert PROMPDθ/θ diffractometerusingtheCuKα radiationof wavelength0.1542nmfrompowderedsamplessandwiched betweenamorphouspolyesterfilmsof3.6micronthicknesses.  Gelpermeationchromatography(GPC).ForPLAsamplesGPCwas carriedoutonaWaterssystemequippedwithaRIdetector. Sampleswereprefilteredusing0.4mNylonorPTFEfiltersand thendirectlyinjected.ThemobilephasewasTHFstabilizedwith BHT(2,6‐di‐tert‐butyl‐4‐methylphenol)(1mL/min,40˚C)asetof twouniversalcolumns(Styragel4Eand5E)inseries.Low polydispersitypolystyrenestandardswereusedforthecalibration curve(Flukakit).Molecularweightdistributiondata(Mw,Mnand dispersity)areobtainedthroughelaborationwiththesoftware Breeze.For‐PGAderivatives,GPCinDMAcwasperformedusing twouniversalcolumns,thermostatedat30°C,andcalibrated againstPMMAstandards. ComputationalAnalysis.Theconformationalanalysesand MolecularDynamics(MD)simulationswereconductedusingthe MaestroSuiteofProgramsfromSchrodinger (www.schrodinger.com).TheOLPS336forcefieldwiththeoctanol solventmodelwasusedinallphasesofmodeling.37Thetorsional conformationalsearchforthecompoundwithn,m=1with62was carriedoutusingtheMCMMmethod38,39andamaximumnumber ofstepswere30000.Thenon‐bondedcut‐offsweresetto8Åfor VanderWaalsand20Afortheelectrostaticinteractions.Eachnew conformationfoundbytheconformationalsearchwereminimized for2500stepsofconjugategradientmethodbeforethenovelty check.40Themostpopulatedconformationswereidentifiedbythe k‐meansclusteringmethod,usingtheRMSDamongconformations asametricsfordistances.MDsimulationsinimplicitsolventwater andoctanolforthecompoundswithn=1,m=5(98atoms)andn= 5,m=5(387atoms)werestartedfromacompletelyextended conformation.Thesimulationsstartwithaconjugategradient minimizationuntilthethresholdof0.05[kcalmol‐1Å‐1]onenergy gradientisreached.Theequilibrationof1nsandtheproduction phaseof300nsfortheeachsystemwerecarriedoutat300Kwith anintegrationstepof1.5fs.TheSHAKEalgorithm41wasusedto constrainbondlengthstotheiroriginalvalues.Theanalysisonthe trajectoriesandtheconformationalsearchwereconductedby GROMACSanalysistools[www.gromacs.org]. AFManalysis.Allofthefilmswereimagedusinga ThermomicroscopeAutoProbeCPResearchIIAtomicForce Microscopeintapping‐modewithsiliconcantileverswithresonance frequenciesofabout160kHz,springconstantsof5.0N/m,andradii lessthan10nm.Sampleswerepreparedbydissolving30mgof polymerin1mLofCHCl3,thenspincoatingthesolutionontoaglass substrateat1500rpmfor60s.Theresultingthinfilmswere annealedat80°Cfor24hours,afterwhichtheovenwasturnedoff andallowedtocoolslowlytoroomtemperature. MTTassay.NHDFswereculturedinDMEM(Dulbecco'sModified EagleMedium)suppliedwith10%(v/v)FetalBovineSerum(FBS) and1%(v/v)penicillin/streptomycinsolution.Cellsweretrypsinized whensubconfluent,seededina96‐wellplate(seedingdensity= 10,000cells/well)andincubatedat37°Cina5%CO2for24htill ARTICLEJournalName 8|J.Name.,2012,00,1‐3Thisjournalis©TheRoyalSocietyofChemistry20xx Pleasedonotadjustmargins Pleasedonotadjustmargins reaching70%ofcellconfluence.Cytotoxicitytestswereperformed byincubatingNHDFsfor24hat37°Cina5%CO2atmospherewith ‐PGA,12aand13asolutionsattheconcentrationrangeof10‐250 mg/mL.Briefly,theselectedcompounds(‐PGA,12aand13a)were dissolvedinDMSOatdifferentconcentrations,namely0.8,1.6,2,4, 8and20mg/mL.50lofeachsolutionwasdilutedwithDMEM suppliedwith10%(v/v)FBSand1%(v/v)penicillin/streptomycin solutiontoreachafinalconcentrationof10,20,25,50,100,250 mg/mL.50lofDMSOdilutedwithDMEMwasusedasblank.An aliquotof200mLofeachsolutionwasincubatedwithcellsfor24h at37°Cina5%CO2atmosphere.Untreatedcellswereusedas control(CTR).MTTassayevaluatedthecellsviabilityafterincubation andresultswereexpressedasviabilitypercentagecomparedto untreatedcells.  Representativeprocedureforthesynthesisofpolymers4and5 throughROPoflactide.Polymer4a.Initiator‐Lactidemonomer ratio1:2.5.Inanoven‐driedflask,underN2atmosphere,LL‐lactide (5.0g,34.7mmol)andDMAP(3.4g,27.8mmol)weredissolvedin dryCH2Cl2(60mL).Benzylalcohol1(1.5g,13.9mmol)wasthen added.Thereactionmixturewasheatedwithstirringat45°Cfor24 h.ThereactionmixturewaswashedwithH2O(50mL)anddiluted HCl(0.1N,50mL).Thereactionsolventwasremovedinvacuo,the crudereactionwasdissolvedinaminimumamountofCH2Cl2and theproductpurifiedbyprecipitationinhexane(200mL).The precipitatewascooledto4°Cforseveralhours,andthenfiltered anddried,togivepolymer4aasawaxywhitesolid(6.1g,94%).1H NMR(CDCl3,200MHz)=7.34(bs,5H,aromatics),5.25‐5.14(m, 2H+~5H,‐CH2‐benzylicand–CH(CH3)‐lacticpolymerchain),4.42‐ 4.32(q,1H,‐CH(CH3)‐OHterminalunit),3.79(bs,1H,‐OHterminal), 1.60‐1.42(bm,~15H,‐CH3polymerchain).Polymer5a.Initiator‐ Lactidemonomerratio1:2.5.FromLL‐lactide(4.0g,28mmol), DMAP(2.7g,22mmol)andEtOH(0.65mL,11mmol).Polymer5a wasobtainedasawaxywhitesolid(4.0g,85%).1HNMR(CDCl3,200 MHz)=5.25‐5.06(bs,5H,‐CH‐polymerchain),4.42‐4.32(bs,1H,‐ CHOHterminal),4.24‐4.13(bm,2H,‐CH2‐ethyl),1.59‐1.47(bm,ca. 15H,‐CH3polymerchain),1.29‐1.23(bm,3H,‐CH3ethylterminal).  Representativeprocedureforthesynthesisofpolymers6and7. Polymer6a.Polymer4a(1.0g,2.14mmolofterminal functionalities)andEt3N(0.6mL,4.27mmol)weredissolvedindry CH2Cl2(10mL).Bromoacetylbromide(0.74mL,8.5mmol)indry CH2Cl2(5mL)wasaddeddropwiseat0°C.Thereactionmixturewas stirredatroomtemperaturefor24h,andthenwashedwithH2O(3 x15mL),dilutedHCl(0.1M,15mL),Na2CO3(1%,15mL)andagain H2O(15mL).Theorganicphasewasdried(Na2SO4),filteredandthe solventremovedinvacuotogivepolymer6aasawaxybrownsolid (0.9g,72%).1HNMR(CDCl3,200MHz)=7.36(bs,5H,aromatics), 5.24‐5.16(m,2H+~5H,‐CH2‐ benzylicand–CH(CH3)‐ polymer chain),3.92(bs,2H,terminal–CH2Br),1.63‐1.51(bm,~15H,‐ CH(CH3)‐polymerchain).13CNMR(CDCl3,75MHz)=169.4(m,CO lactic),166.5(COterminal),135.0(CquatAr),128.5‐128.2(CHAr), 69.2(m,CHlactic),67.1(CH2Ph),25.1(CH2Br),16.5(m,CH3lactic). Polymer7a.Frompolymer5a(1.5g,3.7mmolterminal functionalities),Et3N(0.87mL,6.28mmol)andbromoacetyl bromide(1.1mL,13mmol).Polymer7awasobtainedasawaxy brownsolid(1.4g,74%).1HNMR(CDCl3,200MHz)=5.31‐5.12 (bs,1H,‐CH‐ polymerchain),4.25‐4.15(bs,2H,‐CH2‐ ethyl),3.92 (bm,2H,‐CH2Brterminal),1.61‐1.5(bm,ca.15H,‐CH3polymer chain),1‐31‐1.24(bm,3H,‐CH3ethyl).  Representativeprocedureforthesynthesisofpolymers8. Polymer8a.Polymer6a(1.0g,1.7mmolofterminalchains)was dissolvedinDMF(6mL).Sodiumazide(NaN3)(0.17g,2.6mmol) wasaddedandthereactionmixturewasstirredatroom temperaturefor24h.Thereactionwasthenpartitionedbyadding NH4Cl1M(8mL)andCH2Cl2astheorganicphase(3x8mL).The organicphasewaswashedonceagainwithNH4Cl1M(3x6mL), dried(Na2SO4)andfiltered.Thepolymerwasfurtherpurifiedby dissolvingitinaminimumamountofCH2Cl2andprecipitationin hexane(200mL).Theprecipitatewascooledto4°Cforseveral hours,andthenfilteredanddried,togivepolymer8aasawaxy solid(0.9g,91%).1HNMR(CDCl3,200MHz)=7.36(bs,5H,H aromatics),5.23‐5.16(m,2H+~5H,‐CH2‐ benzylicand–CH(CH3)‐ polymerchain),3.98‐3.97(d,2H,‐CH2N3terminal),1.63‐1.51(bm, ~15H,‐CH(CH3)‐polymerchain).13CNMR(CDCl3,75MHz)=169.5‐ 169.4(m,COlactic),167.7(COterminal),135.0(CquatAr),128.5‐ 128.1(CHAr),69.4‐68.9(m,CHlactic),67.1(CH2Ph),49.9(CH2N3), 16.6‐16.5(m,CH3lactic).IR(cm‐1)=2108(N3stretching),1760, 1680,1460.Polymer9a.Frompolymer7a(1.2g,2.3mmol),NaN3 (0.21g,3.3mmol),togivepolymer9aasawaxysolid(1.1g,90%). 1HNMR(CDCl3,200MHz)=5.25‐5.11(bs,5H,‐CH‐ polymer chain),4.25‐4.14(bs,2H,‐CH2‐ ethyl),3.98‐3.96(bm,2H,‐CH2N3 terminal),1.6‐1.49(bm,ca.15H,‐CH3polymerchain),1.3‐1.24(bm, 3H,‐CH3ethylterminal).  Representativeprocedurefortheclickchemistrygraftingandthe synthesisofpolymers11c,12a‐c,13aand15c.Polymer11c. Polymer10(260mg,1.5mmol)andpolymer8c(1.4g,0.08mmolof terminalfunctionalities)weredissolvedinDMF(30mL).CuSO4 5H2O(60mg,0.3mmol)andsodiumascorbate(150mg,0.6mmol) areaddedascatalyst.H2O(3mL,10%v/v)wasslowlyaddedtothe solution,andthereactionmixturewasstirredatroomtemperature for24h.Thepolymerispurifiedbyprecipitationinasolutionof dilutedHCl(0.1M,800mL),filteredanddried.Itwasfurther redissolvedinasmallamountofCH2Cl2andprecipitatedinhexane (500mL),filteredanddried,toaffordpolymer11casawhite powder(850mg,51%).1HNMR(CD3SOCD3,200MHz)=8.31(bs, 1H;NH),8.13(bs,1H;‐CH‐triazole),7.35(bs,5H;Haromatic),5.50 (bs,2H;‐NCH2COO‐),5.17(bm,24H,‐CH‐PLAchainand‐COOCH2‐), 4.70(bs,2H;‐CH2propargylCH2),4.21(bs,1H;‐CH‐γ‐PGAchain), 3.50(bs,1H;terminaltriplebond‐CH),2.21‐1.76(bm,4H;‐CH2‐γ‐ PGAchain),1.47‐1.44(bm,66H;‐CH3PLAchain).Polymer12a. FromPolymer10(0.22g,1.3mmol)andpolymer8a(1.0g,1.82 mmolofterminalfunctionalities)toaffordpolymer12aasawhite powder(1.0g,85%).1HNMR(CD3SOCD3,200MHz)=8.26‐8.24 (bs,1H,NH‐PGA),8.12(bs,1H,‐CH‐ triazole),7.34(bs,5H, aromatic),5.5(bs,2H,‐NCH2‐PLAchain),5.2‐5.14(bm,5H,‐CH‐PLA chain,2H,‐COOCH2‐)4.26‐4.18(bs,1H,‐CH‐‐PGAchain),2.23‐1.76 (bm,4H,‐CH2‐‐PGAchain),1.46‐1.12(bm,ca.15H,‐CH3PLA).13C NMR(CDCl3,100MHz)=172.2‐172.0(CO‐PGA),169.5‐169.4(m, COlactic),166.7(COterminal),142.0(Cquattriazole),135.0(CquatAr), 128.5‐128.1(CHAr),126.2(CHtriazole),69.4‐68.0(m,CHlactic), 66.5(CH2Ph),58.2(COOCH2‐PGA),51.8(‐CH‐PGA),50.0 JournalName ARTICLE Thisjournalis©TheRoyalSocietyofChemistry20xxJ.Name.,2013,00,1‐3|9 Pleasedonotadjustmargins Pleasedonotadjustmargins (triazole‐CH2),31.0(‐CH2‐PGA),26.5(‐CH2‐PGA),16.6‐16.5(m, CH3lactic).Polymer13a.Frompolymer10(0.2g,1.16mmol)and polymer9a(0.8g,1.62mmol)toaffordpolymer13aasawhite powder(870mg,87%).1HNMR(CD3SOCD3,200MHz)=8.13(bs, 1H,NH‐PGA),7.94(bs,1H,‐CH‐triazole),5.5(bs,2H,‐NCH2‐PLA chain),5.22‐5.18(bm,5H,‐CH‐PLAchain,2H,‐COOCH2‐,2H,‐CH2‐ ethyl)4.22‐4.09(bs,1H,‐CH‐‐PGAchain),2.23‐1.76(bm,4H,‐CH2‐ ‐PGAchain),1.47‐1.4(bm,ca.15H,‐CH3PLA),1.21‐1.14(bm,3H,‐ CH3ethylterminal).Polymer15c.Frompolymer11c(124mg,0.07 mmolofterminalalkynefunctionalities)andcompound14(14mg, 0.09mmol),toaffordpolymer15casawhitepowder(138mg, quantitative).1HNMR(CD3SOCD3,200MHz)=8.26‐8.24(bs,1H, NH‐PGA),8.12(bs,1H,‐CH‐triazole),7.34(bs,5H,aromatics),5.5 (bs,2H,‐NCH2‐PLAchain),5.2‐5.14(bm,5H,‐CH‐PLAchain,2H,‐ COOCH2‐)4.26‐4.18(bs,1H,‐CH‐‐PGAchain),2.23‐1.76(bm,4H,‐ CH2‐‐PGAchain),1.46‐1.34(bm,ca.15H,‐CH3PLAandt‐butyl).13C NMR(CDCl3,100MHz)=172.2‐172.0(CO‐PGA),169.5‐169.4(m, COlactic),166.7(COterminal),142.0(Cquattriazole),135.0(CquatAr), 128.5‐128.1(CHAr),126.2(CHtriazole),82.0(C(CH3)3),69.4‐68.0 (m,CHlactic),66.5(CH2Ph),58.2(COOCH2‐PGA),51.8(‐CH‐ PGA),51.2‐50.8(triazole‐CH2),31.0(‐CH2‐PGA),27.2(C(CH3)3), 26.5(‐CH2‐PGA),16.6‐16.5(m,CH3lactic). AuthorContributions D.P.designedtheproject,supervisedC.L.Z.,V.C.andA.N,andwrote thefirstdraftofthepaper.C.L.Z.andV.C.performedthe experimentalwork,withsubstantialcontributionsbyA.N.andE.C. A.M.D.I.andM.G.‐A.performedcharacterizationwork.G.C.andM. M.performedthecomputationalanalysis.Resultswerediscussed withthecontributionsofallauthors.Themanuscriptwaswritten throughcontributionsofallauthors. Conflictsofinterest Therearenoconflictstodeclare. Acknowledgements WethankSebastianMunozGuerra(UPC),BiceContiandIdaGenta (UniversityofPavia)forusefuldiscussion,EugenioRoàandAurora Pacini(UNIPV)forearlyinvolvementinthiswork,MaddalenaPatrini (UNIPV)fortheAFMstudies,LorenzodeVitaandPiersandro Pallavicini(UNIPV)forcontactanglemeasurements,andAmitav Sanyal(BogaziciUniversity,Turkey)forselectedGPCexperiments. TheUniversityofPavia(postdoctoralfellowshiptypeAtoAN), INSTM(ConsorzioInteruniversitarioNazionaleperlaScienzae TecnologiadeiMateriali)‐RegioneLombardia(JointCallfor Proposals,GAMMA‐PGAProject,andPGGABIOMATProject),Alma 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