A simple preparation of N-acetylated chitosan highly soluble(3)

Althoughvariousmethodsareavailablefordepolymerizationofchitosan,weemployedanoxidativedegradationwithNaBO3tosavetime.Alsoaqueousaceticacidwasusedasthesolventtoachievethereactionunderhomoge-neousconditions.Consequently,themainchainofchitosanseemstoberandomlycut

up.Table1liststhedegradationconditionsalongwiththeweight-averagemolecularweightandthenumber-averagedegreeofpolymerizationforthechitosansamplesob-tained.Theweight-averagemolecularweightvariesaccordingtotheNaBO3concentrationandthereactiontime,andaseriesofchitosansamplesofmolecularweightsfrom8800to600,000wasobtained.

ThedepolymerizedchitosansamplesthusobtainedwereN-acetylatedwithaceticanhy-drideinaqueousaceticacidtoenablethereactiontoproceedundersimpleandhomoge-neousconditions,andthentreatedwithmethanolicpotassiumhydroxide[11].The1HNMRspectrumofthepartiallyN-acetylatedchitosaninD2OisshowninFig.2(top).AlthoughthespectrumgivescomplexsignalsbecausethepartiallyN-acetylatedchitosanisthecopolymerofglucosamine(GlcN)andN-acetylglucosamine(GlcNAc),theresonanceat2.04ppmiseasilyassignedtotheN-acetylprotonsandapparentlytherearenootheracetylprotonssuchasO-acetylprotons.The1

HNMRspectrumwasalsorecordedinthepresenceofCF(bottom).Itcan3COODasshowninFig.2beseenthatthepeaksat2.72,3.53,and4.49ppminDby2Oshiftsigni cantlydown eld,asindicatedthearrows.ThesepeaksareassignedtotheH-2,H-3,andH-1oftheGlcNresidue,respectively.Thepeakat4.56ppmcorrespondstotheH-1signaloftheGlcNAcresidue,andtheresonancesoftheH-2andH-3oftheGlcNAcresidueexistat3.77and3.48ppm,respectively.Theseas-signmentsaresupportedbythe2D1H–1Hchemicalshiftcorrelationspectrareportedpreviously[12].TheH-4,H-5,H-6a,andH-6bwithresonancesbetween3.4and4.0ppmwerenotassigned.

ThedegreeofN-acetylationcanbecalcu-latedfromtheratiooftheintegralintensityoftheH-1oftheGlcNAcresiduetothatoftheGlcNresidueforthe1HNMRspectrainthepresenceofCFN-acetylation3COOD.WecanalsoestimatethedegreefromtheratiooftheintegralintensityoftheN-acetylprotonstothesumofintegralintensitiesoftheH-2,H-3,H-4,H-5,H-6a,andH-6basreportedbyHiraietal.[13].Fig.3showstherelationshipbetweenthedegreeofN-acetylationandthe

N.Kubotaetal./CarbohydrateResearch324(2000)268–274273

molarratioofaceticanhydridetoGlcNunits.ThederivativeswithdifferentN-acetylationdegreeswerepreparedbychangingthequan-tityofaceticanhydride,independentlyofthemolecularweight.However,anexcessivelylargeamountofaceticanhydridewasrequiredwhentheacetylationwascarriedoutinsuchanaqueoussystemasinthisreport;about50%oftheaminogroupsareacetylatedwith8–9equivalentsofaceticanhydride.

Fig.4depictsthemolecular-weightdepen-denceofthetransmittanceof1%aqueoussolutionsoftheN-acetylatedchitosanderiva-tiveswithabout50%degreeofN-acetylation.Thewatersolubilityobviouslydependsonthemolecularweightofchitosan,althoughsomescatterindata,whichispossiblyduetothequalityofsamples,canbeseen.AccordingtoVa rumetal.,theH-1signalofthe1HNMRspectrumisverysensitivetothesequenceoftheGlcNandGlcNAcresiduesanditisfea-sibletodecidethefrequencyofthefourpossibledisaccharides:GlcN–GlcN,GlcN–GlcNAc,GlcNAc–GlcN,andGlcNAc–Glc-NAc[14].Fig.5showsthe1HNMRspectrumintheanomericproton(H-1)regionofthepartiallyN-acetylatedchitosaninDThisspectrum2Ocon-tainingDClat80°C.isessen-tiallysimilartothebottomofFig.2.TheH-1resonanceoftheGlcNresidueappearsastwodoubletpeaks[15,16]andtheycorrespondtotwodisaccharidesGlcN–GlcN(a)andGlcN–GlcNAc(b),respectively.Ontheotherhand,theH-1resonanceoftheGlcNAcresiduealsoshowstwodoubletpeaks,whichcorrespondtotwodisaccharidesGlcNAc–GlcN(c)andGlcNAc–GlcNAc(d),respectively.Thefrac-tionsofGlcN–GlcNAcorGlcNAc–GlcNareallintherange0.24–0.28(Table2).ThehalfN-acetylatedchitosanderivativespreparedherestronglysuggestthattheN-acetylgroupsrandomlydistributeandthedeviationfromrandomnessisverysmall,regardlessofthemolecularweightofchitosan.Accordingly,thedecreaseinwatersolubilityofthehalfN-acetylatedchitosanofhighmolecularweightisprobablyduetothehighmolecularweightitself,nottheblockwisedistributionofN-acetylgroups.

Thereisapossibilitythatthewatersolubil-ityofthehalfN-acetylatedchitosanderiva-tivesobtainedinthiswaymightdecreaseinanalkalinesolution[17],becausetheyincludeabout50%oftheGlcNresidueswhosepK6.6[18].Fig.6showstheaisestimatedaspHdependenceofthetransmittanceofthehalfN-acetylatedchitosansolutions.WhenthehalfN-acetylatedchitosanderivativesaredis-solvedinaqueousaceticacid,theirsolubilityatneutralpHappearstobehigherthanthatinpurewater.Theionicstrengthmightbeacauseforthisphenomenon.Inthecasesofthelower-molecular-weightderivatives,thewatersolubilityishighandretainedoverawidepHrange,whereasinthecasesofthehigher-molecular-weightderivatives,itishighatacidicpHbutabruptlydecreasesatapHalittleoverneutrality.Especially,thederiva-tivesof8.8k-53,19k-51,and33k-47giveveryhighsolubility,butthesolubilityoftherestdecreaseswithincreasingmolecularweightinthealkalineregion.ItseemsthatthehighwatersolubilityofthehalfN-acetylatedchi-tosanderivativesoflowmolecularweightisattributedtothedecreaseofintermolecularinteractions,suchasvanderWaalsforces;thelowerthemolecularweight,thelowertheintermolecularattractionforces.

WealsoinvestigatedthesolubilityofthehalfN-acetylatedchitosanderivativesoflowmolecularweightsinDMAandMeDMAisknowntobeagoodsolventforchitin2SO.andcellulosewhenitisusedwithLiCl[19]andMe2SOisfrequentlyusedinenzymeas-says.AfterthehalfN-acetylatedchitosanwaspreviouslydissolvedindeionizedwater,DMA(Fig.7)orMeadded;thelower2SO(Fig.8)wasgraduallythemolecularweight,thehigherthesolubilityinaqueousDMA.ThesolubilityofthehalfN-acetylatedchitosanderivativesinaqueousMethanthatinaqueous2SOisslightlyhigherDMA.Thederivativeof8.8k-53,inparticular,ismarkedlysolubleinaqueousMequently,reducingthemolecularweight2SO.Conse-priortoN-acetylationisoneofthemosteffectivemethodsofrenderingchitosansolubleinaqueousorganicsolvents.

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