Carbohydrate-resorcinarene complexes involved in the facilitated transport of alditols across a supported liquid membrane

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Journal of Membrane Science

j our na l h o me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / m e m s c i

Carbohydrate–resorcinarene complexes involved in the facilitated transport of alditols across a supported liquid membrane

MiloudiHlaïbi^a,b,∗,NabilaTbeur^a,b,AbdelkhalekBenjjar^a,OussamaKamal^a,LaurentLebrun^b

aLaboratoired’InterfaceMatériauxetChimiedel’Environnement(LIME),UniversitéHassanII,FacultédesSciencesd’AïnChock,BP5366,Maârif,Casablanca,Morocco

bLaboratoire“Polymères,Biopolymères,Surfaces”,UMR6270CNRS,UniversitédeRouen,FacultédesSciences,76821Mont-Saint-Aignan,France

a r t i c l e i n f o

Articlehistory:

Received5May2010 Receivedinrevisedform 24December2010 Accepted27April2011 Available online 5 May 2011

Keywords:

Facilitatedtransport Resorcinarene

Supportedliquidmembrane Molecularrecognition Permeability Flux

Stabilityconstants

Apparentdiffusioncoefficients cisortransconfigurationsite

a b s t r a c t

Asupportedliquidmembrane(SLM)containingaresorcinarenecarrierhasbeenusedfortheselective transportsofarabinitolandperseitolfromconcentratedaqueoussolutions(0.2–0.025M).Maltitoland lactitol,twoalditolsderivedfromdisaccharides,werealsostudied.Themembraneismadeofamicro- porouspoly(tetrafluoroethylene)filmimpregnatedwitha0.01MsolutionofthecarrierinCCl4.The permeabilitiesoftheSLMforallalditolswerecalculated.Foreachalditol,theinitialfluxisrelatedto initialconcentrationinthefeedphasebyasaturationlaw,indicatingthattherate-determiningstepin thetransportmechanismisthemigrationofa(1/1)carrier–carbohydratecomplexintheimmobilized organicphase.Usingamodelpreviouslypublished,theapparentdiffusioncoefficientsD*andthestability constantsKofthecarrier–alditolcomplexesweredetermined.Themagnitudesofthestabilityconstants arerelatedtothestructuresofthealditols,particularlytothenumberandorientationofhydroxylgroups thatinteractwiththecarrier.Insmallalditolsthatcontainanarabinotriolsystem,thesiteofbindingto thecarrieristheerythrodiolgroup.Inthelargemoleculesofdisaccharidesandperseitolinwhichmore OHgroupsareavailable,morestablecarrier–alditolcomplexesareformed:itisbelievedthattworemote sitesofbindinginteractwithtwoindependentrecognitionsitesofthecarrier.

Aim:Tounderstandthenatureofchemicalrecognitionbetweenthecarrierandpolyols.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Noveltechniquesrecentlyintroducedfortheseparationofmix- turesofcarbohydratesarebasedonmembraneprocesses[1a].A discussionoftheirpotentialitieshasbeenreportedearlier [1b].

Selectivetransportofcarbohydratesacrossamembraneisachieved whenthemembranecontainsareagent(thehostorcarrier)that canformhost–guestcomplexeswithcarbohydrates(the guests).

Thefluxofeachcarbohydrateacrossthemembranedependson thestrengthofthehost–guestcomplex,allowingtheseparationof mixturesofcarbohydratesbasedondifferenttransferrates.This processisnamed:facilitated transport,oralsocarrier-mediated transport.

Severaltypesofmembraneshavebeendevelopedandadapted fortheseparationofcarbohydrates[1a].Thefirstgenerationwas basedonion-exchangemembranesfortheselectivetransportof boratecomplexes[2–4].Then,supportedliquidmembranes(SLMs)

∗Corresponding author at: Laboratoire d’InterfaceMatériaux et Chimie de l’Environnement(LIME),UniversitéHassanII,FacultédesSciencesd’AïnChock,BP 5366,Maârif,Casablanca,Morocco.Tel.:+212522230680;

fax:+212522230674.

E-mailaddress:miloudi58@hotmail.com(M.Hlaïbi).

wereproposed,becausetheyallowlargefluxesofcarbohydrates due tothe diffusion of the carrier complexes within their liq- uidorganiclayer.Themostusedcarriersarephenylboronicacids thataresolubleintheorganicsolventofthosemembranes[5–9].

Recently, Smith designed polymer inclusion membranes(PIMs) madeofplasticizedcelluloseacetate,inwhichthecarrierisimmo- bilizedinthepolymericnetwork.Thesemembranesdonotallow largefluxes,butpresentabetterstabilitythanSLMsthatfrequently sufferfromleakingofsolventorcarrierintothesurroundingaque- ousphases[10,11].

RenewedinterestwasfoundinSLMswiththeintroductionof amphiphilic,neutralmacrocyclesascarriers.Whenthesecarriers havealargelipophilicmoiety,theirextractionintotheaqueous phases is negligible,and thecorresponding membranesexhibit outstandingstabilities withrespecttoleakage ofcarrier orsol- vent [12,13].Moreover,these carriers maybedesigned sothat host–guestbindinginvolveselaborateinteractionbetweenthesug- arsand thecarrier thatis thebasis formolecular recognition.A suitablecarrierfortheextractionofunderivatizedcarbohydrates intoanorganicsolventisthelipophiliccompound1(Fig.1)discov- eredbyAoyamaetal.[14–16].

Thishostbelongstothefamilyofresorcinarenes,thatareana- loguesofcalix[4]arenesinwhichthefourphenolunitsarereplaced by resorcinolunits [17,18].Resorcinarene 1 selectively extracts 0376-7388/$–seefrontmatter© 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.memsci.2011.04.055

OH HO

R H

OH

OH R

H HO

HO

R H

HO R

H

OH

Fig.1.Structureofresorcinarene1(R=CH3(CH2)10).

severalsugarsfromconcentratedaqueoussolutionsintoananhy- drousCCl4layer.Whentheorganicsolventissaturatedwithwater, theextractionisstillselective,butthedissolvedspeciesarelikely ternarycarrier–sugar–watercomplexesratherthancarrier–sugar complexes. Selective extraction of sugars with reagent 1 was demonstrated[19].Theadvantagesofusingtheneutralcarrier1 insteadofacidiccompoundssuchasarylboronicacidshavebeen discussedelsewhere[20,7,21,22].

SLMsaremadeofahydrophobicandporouspolymerfilm(the support)impregnatedwithanorganicsolutionofthelipophiliccar- rier.SLMsrequirelittleenergybecausethetransportmechanism isfundamentallyaliquid–liquidextractionprocess.Sincethemain problemassociatedwithliquid–liquidextractionisthemanipula- tionoflargequantitiesofharmfulorganicvolatilesolvents,SLMs, thatusesmallamountsofsolvent,areapreferredoption,because theyareagoodanswertopresentconcernsaboutprotectionofthe environment.

ASLMcontainingcarrier1waspreviouslyusedforthetrans- portofsugars[12]andsmallalditols[13].Akineticstudyindicated thattherate-determiningstepisthemigrationofthecarrier–sugar complex(CS)withintheorganicsolvent.Amodelderivedfromthe Michaelis–Mentenschemeallowedthecalculationoftheapparent diffusioncoefficientD*ofthecomplexandofitsstabilityconstant K.

ThestabilityconstantKreferstotheheterogeneousinterfacial equilibrium:

S(aq)+C(org)CS(org)

whereaqandorgstandforaqueousandorganicphases,respec- tively,CisthecarrierandSisthecarbohydrate(substrate).The magnitudeofthestabilityconstantKofthecomplexesisause- fultoolinordertospecifythenatureofthecarrier–carbohydrate complexes.Thestabilitiesofthecomplexesaregovernedbysev- eralaspectsofthesubstratestructure.Themainoneisthenumber ofhydroxylgroupsofthesugarthatcaninteractwiththoseofthe carrier.Theotheroneistheorientationofthesegroups.Itmay beassumedthatstronginteractiontakesplacewhenbothsetsof hydroxylgroupsofthesugarandcarriercloselymatch.

Ourlong-termprojectisaimingattheseparationofcarbohy- dratesbymeansofSLMprocesses.Forthispurpose,themembrane shouldcontainalipophiliccarrierthatcanspecificallyrecognize sugarsby forminghost–guestcomplexes. Continuing ourprevi- ousstudies ofthe facilitated transport ofcarbohydrates bythe resorcinarene1carrierthroughaSLM,thecaseofhigheralditols wasinvestigated.Resultsforthetransportofsimplealditols[23]

showedthatthetransport isselective,ifthestability constants and/orapparentdiffusioncoefficientsofthecarrier–alditolcom-

Fig.2.Fischerrepresentationofstudiedalditols.

plexes vary with the structure of carbohydrates. The studied alditolswerechosenbasedontheconfigurationoftheirhydroxyl groups.Initially,theresearchfocusedonsmallalditolswithasin- glesite,threoorerythro.Theresultsgavevaluableinformationon thesiteofcomplexation,thestability oftheformedcomplexes, andthefactorsthatcontrolthetransport[13].Thesiteofinter- actionofsmallalditolswithresorcinarene1wasreportedtobe aninternaldiolsystem.Theorderofstabilityofthealditolcom- plexesofcarrier1was:pentitols<tetritolsandthreo<erythro[23].

Thestabilityconstantofthearabinosecomplexwasmuchlower, probablybecausethestiffpyranosering isless flexiblethanan alditolacyclicchain[12].Thedifferencebetweenthealditolsused inthepresentstudy(arabinitol,maltitolis4-␣-d-glucopyranosyl-d- glucitol,lactitolis4-␤-d-galactopyranosyl-d-glucitolandperseitol isd-glycero-d-galacto-heptitol)(Fig.2)andthosepreviouslyused, is that the molecules now contained both erythro and threo diol systems, allowing a competition between severalpossible sites of binding. Our aim was to specify the characteristics of thesites responsiblefor binding withresorcinarene1, through determinationofthestabilityconstantsKandapparentdiffusion coefficients D*of thecomplexes involved inthe transportpro- cess.

2. Experimental

The alditolsand otherchemicals werecommercial products (AldrichorFluka)ofthepurestavailablegradeandwereusedas received.Resorcinarene1wassynthesizedaccordingtoapublished procedure[15,19,24].Its¹Hand¹³CNMRspectra,measuredwith a BrukerARX-400spectrometer,wereidenticalwiththoseof a commercialsample(purchasedfromFluka).

TheSLMsupportwasamicroporousPTFEfilm(Goodfellow)of thickness63␮m.Characteristicvaluesareporosity84%andpore diameter0.45␮m.Themembranesampleswerediscswithdiam- eterlargerthan 5cm, typically about6cm. They wereinserted betweenthecellhalf-compartments,usingtwoTeflonrings,the

internaldiameterofwhichwasexactly5cm.Hence,theeffective membraneareaavailablefordiffusionwas19.6cm².

Thetransportcell[12,13],ismadeoftwocompartmentsofequal volumes(100mL)separatedbytheSLMpreparedbysoakingdur- ing15hasquareportionofthepolymerfilm(8cm×8cm),into a0.01Msolutionofresorcinarene1inpurecarbontetrachloride.

Thecellwasimmersedintoathermostatedbath(T,298K).The solutionsinbothcompartmentswerestirredwithmagneticbars, usingaVariomagapparatus.

TheSLMwasconditioned,during16h,byequilibratingthetwo compartmentsfilledwithpurewaterwiththeSLM,beforereplac- ingwaterbythecarbohydratesolutioninthefeedcompartment.

Webelievethatthisconditioningprocedurewassufficientforequi- libratingwaterofthereceivingcompartmentwithCCl₄,together withsaturatingtheliquidmembranewithwater.

Initially,thefeedcompartmentcontainedthealditolsolution (c₀=0.20–0.0125M) and the receiving compartment contained purewater.Twodifferentprocedureswereused:

-Inthefirstone,mode(A),forthestudyofthelifetimeofthemem- braneandthereproducibilityofresults,weusedforeachofthe experimentsanewalditolsolution.Withthisprocedure,weveri- fiedthattheresultswerereproduciblewhenthesamemembrane samplewasusedduring10days,withoutshowinganysignof failure.

-Inthesecondone(mode(B),thiswork),tostudytheeffectofthe concentrationofthestudiedalditolontheparametersoftrans- port,thesamemembranesampleandthesamesolutionwere usedthroughoutallruns.Atequilibrium,whenthesugarconcen- trationsareequalineachcompartment,attheendofeachrun, thecontentsofbothcompartmentswerewithdrawnandmixed together.Then,100mLoftheresultingsolutionwereintroduced inthefeedcompartmentand100mLofwaterinthereceiving compartmentandthefollowingrunwasstarted.Thisprocedure, thatsaveslargequantitiesofexpensivesubstrates,wastypically repeatedfourorfivetimes.

Inbothprocedures,smallaliquots(v=1.0mL)ofthereceiving phasewerewithdrawnatknownintervals.Someexperimentswere continuedfor24h,when samplesofboth aqueousphaseswere withdrawnandanalyzedtoensurethatequalconcentrationswere present,indicatingthatequilibriumwasreached.

ThesampleswereanalyzedusingaHPLCapparatusequipped witha30-cmPhenomenexRezexcolumnincalciumform,main- tainedinanovenat85^◦C.Theeluentwaspurewater,degassed andfiltratedwithacelluloseestermembrane(Millipore,poresize 0.45␮m). Theflow rate was0.6mL/min. Thepump wasa Shi- madzuLC-9Amodel.DetectionwasachievedwithaVarianRI-4 refractometer. Typical retention times (inmin) for the alditols were13.5min(lactitol), 16.0min(maltitol), 20.0min(arabinitol) and22.0min(perseitol).Theconcentrationsofalditolsweredeter- minedbyanalyzing chromatographicdata withtheVarianStar software.Allexperimentswereduplicatedandwerereproducible with3%asrelativestandarddeviation.

3. Experimentalprocessesandresults 3.1. ConditioningoftheSLM

WhentheSLM wasusedfor transport immediatelyafterits preparation,thefluxoftransportedalditolremainedsmallfora longinitialperiod,typically8–10h.Afterthistime,therateoftrans- portincreased andtheconcentrationof alditolin thereceiving phasec_R increasedrapidlyoverseveralhours.Thisbehaviorwas observedpreviouslywithaSLMcontainingthisresorcinarenecar-

rier[12,13]andwasattributedtotheslowincorporationofwater intothemembranetoformpresumablyacarrier–watercomplex.

Afterthisinitialperiod(inductionperiod),thetransportofalditols increased,probablybecausethesubstraterapidlyexchangedwith water at the interfaces to form the carrier–substrate complex, or possibly a ternary carrier–substrate–water complex that is theactivespeciesforthetransportprocess.Suchaphenomenon appearstobecommonwithmembranesusedforthetransportof carbohydrates.Forexample,thepresenceofwaterinthePIMwas reportedtobeimportantforthetransportofcarbohydratesthrough plasticizedcellulosetriacetatemembranescontainingion-paircar- riers[10,25].Theexistenceofthisinductionperiodcomplicatesthe analysisofthekineticstudies,becauseitmakesdifficulttoapproxi- matetheexactvalueoftheslopeoftheplotsdrawnforafirst-order reaction[13].Inthisstudy,inordertosuppressthisperiodofslow transport,theSLMwasconditionedfor16hinthecellbetween two phasesofpurewater.Afterthis time,theinductionperiod wasnotobservedandthetransportofalditolsbeganjustafterits introductioninthefeedphase.

3.2. Transportexperiments

Variouscausesformembraneinstabilityhavebeendiscussed elsewhere[26].Specifically,theyconsistinlossofcarrierorsolvent, thatareinsufficientlyretainedintheporesofthesupport.However, theSLMstudiedinthisworkseemstorepresentanidealcase,since thetransportedcarbohydratesarealmostinsolubleintheorganic phase(unlessascomplexeswiththecarrier),whereasthecarrieris insolubleinaqueousmedium,avoidingthusitswashing-outfrom theorganiclayer.Thestabilityofthismembranewaspreviously demonstrated:theSLMcouldbeusedfor10daysforthetransport ofsugars[12,13].Inthepresentstudywithalditols,mostexperi- mentswerecarriedoutduring5dayswiththesamemembrane, withoutanyobservationofleaking.AnotheradvantageofthisSLM isthatonlyfacilitatedtransportofthealditolstookplace,aspassive diffusionofthesubstratesacrosstheSLMcouldnotbedetectedin theabsenceofcarrier.Thisresultisconsistentwiththehydropho- bicnatureofthePTFEfilm,andwiththefactthatuncomplexed alditolsarenotdissolvedincarbontetrachloride.

3.2.1. Modeoftransport

Theexperimentsonthetransportofstudiedalditolswerecar- riedoutusingamembraneconsistingofathinandmicroporous polymerfilm,impregnatedwithanorganicsolventcontainingthe lipophiliccarrier.TheSLMispreparedfromasolution(0.01M)of resorcinarene1dissolvedinCCl4,confinedinamicroporoussup- portofpoly(tetrafluoroethylene)(PTFE).Thestabilityofthissystem hasbeencontrolled[12].Itsreproducibilityhasbeenverifiedfrom theresultsobtainedforthetransport ofxylitoland ribitol[13].

Inthiswork,wehaveimprovedthequalityandaccuracyofour experimentsbyeliminatingtheinductionperiod,usingthemode (B)for thetransport ofalditols [13].Thisprocedureconsistsin allowinguptake ofwater bytheSLM for16h, beforeintroduc- ingin thesourcecompartmentthealditoltobetransported.In thiscase,thetransportstartsimmediatelyaftertheintroduction ofthesubstrateandextendstoequalconcentrationsbetweenthe twoaqueousphases,indicatingthatequilibriumwasreached.After reachingequilibriumandasaprevioussteptothenextexperiment, thesolutioninthereceivingphaseisreplacedbypurewaterand thetransportphenomenonstartsagain.Thisproceduremode(B) isrepeatedduringfourtofivedaysusingthesameSLMfortrans- portingthesamealditol,atdecreasingconcentrations(example:

0.2–0.1–0.05–0.025–0.0125M).Becausetheinitialsolutionisstep- wisediluted,thisprocedurealsosaveslargequantitiesofexpensive substrates.

Thismethodhasseveraladvantages.First, itallowstomini- mizelossofsolvent,duringthepreparationoftheSLM,andsmall quantitiesofmembranesandsubstratesareused,whichisparticu- larlyeconomicalinthecaseofrarecompounds.Ontheotherhand, itreducesthetimerequiredtocompletestudyofeach ofstud- iedalditolsto4or5days,whileimprovingthequalityofresults asweproceededin thesameexperimentalconditions.In addi- tion,theeliminationoftheinductionperiodmakesitpossibleto obtainaccurateexperimentaldatanecessaryfordeterminingthe permeabilitiesandthefluxesforthefacilitatedtransportofstudied alditols.

3.3. Determinationofpermeabilities(P)andfluxes(J) (macroscopicparameters)

Theprincipleofthecalculationofpermeabilitiesandfluxeshas beendevelopedelsewhere[12,13].Thetransportrateismeasured bydeterminingtheincreaseofthealditolconcentrationcRinthe receivingphaseversustimet.ThisrateisrelatedtothefluxJof alditolbyEq.(1)

dcR

dt =JS

V (1)

whereSisthemembraneareaandVisthevolumeofthereceiving phase.

Thereportedvalueswerecalculatedusingthemembranearea calculatedfromthediameteroftheTeflonringsthatheldtheSLM andcorrespondtotheoperationalproperties ofthemembrane.

Normalizeddata,usefulforcomparisonwithothertypesofSLMs, wouldrequiretakingintoaccounttheporosityandtortuosityofthe membranesupport.Sincetheaimofthepresentworkwasmainly astudyofthenatureofthecomplexesinvolvedinthetransportof carbohydrateswithresorcinarenecarriers,datanormalizationwas notconsiderednecessary.

Whenthesystem reachesa quasi-steadystate, theflux J is relatedtoc,thedifferencebetweentheconcentrationsofalditol inthefeed(cF)andthereceivingphases(cR),andthemembrane thicknesslbyEq.(2)derivedfromFick’sFirstLaw

J=Pc

l (2)

wherePisthepermeabilityofthemembrane.

Sincethefluxofalditolsisverylarge,theconcentration(c_R)of thereceivingphaseisnotnegligibleversustheconcentration(cF) ofthefeedphase.Thus,ciscalculatedusingEq.(3)wherec0is theinitialconcentrationofalditolinthefeedphase:

c_F=c₀−c_R and c=c₀−2c_R (3) CombiningEqs.(1)–(3)yieldsdifferentialEq.(4):

Pdt= (lV/S)dc_R

c₀−2c_R (4)

IntegrationofbothtermsofEq.(4)yieldsEq.(5):

P(t−t_L)=

lV S

₁

2

ln

_c

0

c0−2cR

(5) whichshowsthat,afteraninductionperiod(t_L)thatmaylastupto severalhours,theterm

−ln(c₀−2c_R)isalinearfunctionoftime(t).Ontheotherhand,if themembranewasequilibratedwithwaterfor16hbeforestarting theexperiment,theinductionperiodcompletelydisappeared.This resultdemonstratesthattheinductionperiodisduetouptakeof waterbythemembraneandtheplots(−ln(c₀−2c_R)=f(t))forthe transportofthealditolswereindeedstraightlines.Thepermeabil-

2 2,5 3 3,5 4 4,5 5

10 8

6 4 2

0 t(h)

[Arabinitol]=0,1M [Lactitol]=0,1M [Maltitol]=0,1M [Perseitol]=0,08M

–Ln (C0-2 CR)

– Ln (C₀- 2 C_R) = f(t)

Fig.3.Plotsof−ln(c0−2cR)vs.timetforthetransportofarabinitol,lactitol,maltitol andperseitolacrosstheSLM.

ityPvaluesforthevariousalditolswerecalculatedfromtheslopes aoftheplots,usingEq.(6):

P=a

lV 2S

(6) Attheinitialtime, thealditolconcentrationsintheaqueous phasesarecF≈c0andcR≈0.Atthisinstant,theinitialvalueofthe flux,J_i,canbecalculatedbyEq.(7)derivedfromEq.(2).

J_i=Pc0

l (7)

3.3.1. Calculationofthepermeabilitiesandfluxes

Asforsimple alditols[13],thepermeabilities andthefluxes weredeterminedandcalculatedfromchangesinconcentrationc_R inthereceivingphase,versustimetforeachalditol.Anexampleof theevolutionofthefunction“−ln(c0−2cR)”versustimet,ispre- sentedbythediagraminFig.3forarabinitolandthreeotherstudied alditols.TheobtainedstraightlinesareinagreementwithEq.(5), drawnfromtheproposedkineticmodelforthetransportofthese compoundsthroughtheSLM.Thus,thepermeabilitiesandfluxes arecalculatedfromtheslopesofthelines,representedbythedia- graminFig.3,accordingtoEqs.(6)and(7).Theobtainedresults fordifferentinitialconcentrationsofstudiedalditolsaregrouped inTables1and2.

Theseresultsclearlyshowthatthepermeabilitiesdecreasewith increasinginitialconcentrationsofalditolsinthefeedphase.Con- sequently,thesaturationlawdescribedinthetransportofsugars [12],alsoappliestoallstudiedalditols.

3.4. Modelinganddeterminationofmicroscopicparameters(D*

andK)

ThemechanismfortheoveralltransportofasubstrateS(alditol) bythecarrierCacrosstheliquidmembraneisknowntoinvolvefive consecutivesteps[12,13].Thereactionbetweenanalditolandthe carrieratthefeedphase-SLMinterface,yieldsacomplexsoluble inthemembraneorganicphase.Thiscomplexwasassumedtobe a(1:1)alditol-resorcinarene1species.Thestoichiometryofsuch complexescouldnotbefoundintheliterature.However,inthecase ofribose,theexistenceofa(1:1)complexwithresorcinarene1in CCl4wasreportedbyAoyamaetal.[15].Theextractionofsugars