A simple preparation of N-acetylated chitosan highly soluble in water and aqueous organic solvents

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CarbohydrateResearch324(2000)268–274

AsimplepreparationofhalfN-acetylatedchitosanhighlysolubleinwaterandaqueousorganicsolvents

NaojiKubotaa,*,NobuhideTatsumotoa,TakayukiSanoa,KaoriToyab

b

GeneralEducationChemistryandPhysics,OitaMedicalUni6ersity,Hasama-machi,Oita879-5593,JapanDepartmentofAppliedChemistry,FacultyofEngineering,OitaUni6ersity,Dannoharu,Oita870-1192,Japan

Received8June1999;accepted3September1999

a

Abstract

Asimpleandimprovedmethodofpreparinghighlysolublechitosan(halfN-acetylatedchitosan)wasdevelopedusingaseriesofchitosansamplesoflowmolecularweights,andthesolubilityofthehalfN-acetylatedchitosaninwaterandorganicsolventswasinvestigatedindetail.Toreducethemolecularweight,chitosanwastreatedwithNaBO3undertheconditionthatchitosanwashomogeneouslydissolvedinaqueousaceticacid.Weight-averagemolecularweightsoftheobtainedchitosansamplesweredeterminedusingasize-exclusionchromatographysystemequippedwithalow-anglelaserlight-scatteringphotometer.EachchitosansamplewasthenN-acetylatedwithaceticanhydrideundertheconditionthatchitosanwashomogeneouslydissolvedinaqueousaceticacidagain.ThewatersolubilityofthehalfN-acetylatedchitosanthuspreparedincreasedwithdecreasingmolecularweight.From1HNMRspectroscopy,itwassuggestedthatthesequenceofN-acetylglucosamineandglucosamineresidueswasrandom.ThesolubilityofthehalfN-acetylatedchitosanoflowmolecularweightwasratherhigheveninaqueousdimethylacetamideanddimethylsulfoxide.©2000ElsevierScienceLtd.Allrightsreserved.

Keywords:Chitin;Chitosan;HalfN-acetylatedchitosan;Solubility;Molecularweight;DegreeofN-acetylation

1.Introduction

Chitin,poly-b-(1 4)-N-acetyl-D-glucosa-mine,isthesecondmostabundantnaturalpolysaccharideandexistslargelyintheshellsofcrustaceaandinsects.Chitosan,poly-b-(1 4)-D-glucosamine,canbereadilyob-tainedfromchitinbydeacetylationwithalkali.Theuseofchitinandchitosaninvari-ousfunctionalmaterials,includingbiomedicalmaterials,haverecentlybeendeveloped[1,2].Wehavealsoreportedonthemembraneap-plicationsofchitosananditsderivatives

[3–5].

*Correspondingauthor.Tel.:+81-97-586-5605;fax:+81-97-586-5619.

E-mailaddress:nkubota@oita-med.ac.jp(N.Kubota)

However,theapplicationsofchitinandchi-tosaninbiology,inwhichmanyenzymeas-saysareperformedatneutralpH,isquiterestricted,becausetheyareessentiallyinsolu-bleinneutralwater.Ifwater-solublechitinandchitosancouldbepreparedinasimplemanner,theirbiologicalandphysiologicalap-plicationswoulddevelopdramatically.

Chitinandchitosanbecomewatersolublethroughachemicalmodi cationinwhichthedegreeofsubstitutioniscontrolled.Forin-stance,itisknownthatwater-solublechitinwithadeacetylationdegreeofabout50%canbeobtainedfromchitinbyhydrolysiswithalkali(Fig.1)[6,7].Itwassuggestedthatthewatersolubilityresultedfromtherandomdis-tributionofN-acetylgroups.Ontheotherhand,water-solublechitosanwithabouta

PII:S0008-6215(99)00263-3

0008-6215/00/$-seefrontmatter©2000ElsevierScienceLtd.Allrightsreserved.

N.Kubotaetal./CarbohydrateResearch324(2000)268–274269

Fig.1.Syntheticroutestowater-solublechitinandchitosan.

50%degreeofN-acetylationwaspreparedfromchitosanbyN-acetylationwithaceticanhy-dride(Fig.1)[8].However,averylongreactiontime,i.e.,over2days,wasrequiredintheformer,andacomplexsolventsystem,i.e.,aceticacid–water–methanol–pyridine,wasusedinthelatter.Furthermore,Aibareportedthatreductionofthemolecularweightdidnotin uencethewatersolubilityofthepartiallyN-acetylatedchitosan[9].However,therela-tionshipbetweenmolecularweightandsuchwatersolubilitywasnotclear,sinceheusedonlytwodifferentmolecularweights.Inaddi-tion,therearefewreportsonthesolubilityofpartiallyN-acetylatedchitosanincommonor-ganicsolvents.

Inthispaper,therefore,wereportasimpleandimprovedmethodtopreparehighlywater-solublechitosan(halfN-acetylatedchitosan)usingaseriesofchitosansamplesoflowmolecularweights.Chitosanispreviouslyde-gradedbytreatmentwithNaBO3[10]andthenN-acetylatedwithaceticanhydrideinaqueous

aceticacid.TherelationbetweenmolecularweightandwatersolubilityoftheobtainedN-acetylatedchitosanisquantitativelyinvesti-gatedandthesolubilityofthehalfN-acetylatedchitosaninsomeorganicsolventsisexamined.2.Experimental

Materials.—Commerciallyavailablechi-tosanfromTokyoKaseiKogyo,Tokyo,Japan,waspuri edthricebyreprecipitationusingaqAcOH(5%)andaqKOHinadvance.Itwas nallywashedwithdeionizedwaterandace-tone,andthendriedinvacuo.Standardpullu-lansforsize-exclusionchromatography(SEC)werepurchasedfromShowaDenko,Tokyo,Japan.Aceticanhydride,acetone,N,N-dimethylacetamide(DMA),andMe2SOfromWakoPureChemicalIndustries,Osaka,Japan,weredistilledjustbeforeuse.Allotherchemicalswereofreagentgradeandusedasreceived.

Table1

Degradationconditionsandweight-averagemolecularweightofchitosanSamplecode

DegradationconditionNaBO3(%w/v)

600k380k340k230k150k110k78k63k33k19k12k8.8k

a

wM

Temperature(°C)

Time(h)

6.0×1053.8×1053.4×1052.3×1051.5×1051.1×1057.8×1046.3×1043.3×1041.9×1041.2×1048.8×103

naP

Untreated

0.010.010.010.050.51.02.0sat.sat.sat.sat.

r.t.3050505050505050505011111111248

1.4×1039.3×1029.0×1025.9×1024.0×1023.1×1022.0×1021.7×1028.2×105.1×103.0×102.3×10

n;Mn=SHi/S(Hi/Mi),whereHiisthesampleconcentrationandNumber-averagedegreeofpolymerizationcalculatedfromM

MiisthemolecularweightofeachfractionofSEC.

270N.Kubotaetal./CarbohydrateResearch324(2000)268–274

Fig.2.1HNMRspectraofpartiallyN-acetylatedchitosaninD2O(top)andinDsignalsindicatedbyarrows2O-containingCFareassigned3COOD(bottom).ThetotheH-2,H-3,andH-1oftheGlcNresiduefromtheright.

Fig.3.RelationshipbetweenthedegreeofN-acetylationandthemolarratioofAc2OtoGlcNunits.TheerrorbarrepresentsthemaximumandminimumofN-acetylationde-grees,regardlessofmolecularweight.

Degradationofchitosan.—Puri edchitosan(0.5g)wasdissolvedin5%aqAcOH(30mL),andaqNaBOcentrationwas3(100mL)ofaprescribedcon-added.Afterstirringatapre-determinedtemperatureforapredeterminedtime,thereactionmixturewascooled,andaqKOHwasaddeduntilthepHreached10–11.Theprecipitateobtainedwascentrifugedand

washedwithdeionizedwateruntilitwasneu-tral,rinsedwithacetone,driedinvacuoatroomtemperature(rt),anddriedfurtherun-dervacuumat50°C.

Determinationofmolecularweight.—Weight-averagemolecularweightsofthede-gradedchitosansamplesweredeterminedusingaTosohSECsystem(column:TSKgelGMPWXL,7.8mmi.d.×30cm)equippedwithalow-anglelaserlight-scatteringphoto-meter(LS-8000)andadifferentialrefractome-ter(RI-8011).Eachsamplewasdissolvedinacetatebuffer(1M,pH4.8),whichwastheeluent,and lteredthroughamicro lter(0.45

Fig.4.Molecular-weightdependenceofwatersolubilityofhalfN-acetylatedchitosan.Watersolubilitywascharacterizedastransmittanceofthedispersionsat600nm.

Fig.5.1HNMRspectraintheregionforresonancesofanomericprotonsofpartiallyN-acetylatedchitosaninDcontainingDClat80°C:(a)GlcN–GlcN,(b)GlcN–Glc-2ONAc,(c)GlcNAc–GlcN,and(d)GlcNAc–GlcNAc.

N.Kubotaetal./CarbohydrateResearch324(2000)268–274271

Table2

Fractionsoffourpossibledisaccharidesforwater-solublehalfN-acetylatedchitosanSamplecode600k-50380k-54340k-52230k-52150k-50110k-5178k-5163k-4833k-4719k-5112k-488.8k-53

DegreeofN-acetylation(%)505452525051514847514853

FGlcN–GlcN0.240.200.230.220.240.220.230.280.260.220.240.21

FGlcN–GlcNAc(FGlcNAc–GlcN)0.260.260.250.260.260.270.260.240.270.270.280.26

FGlcNAc–GlcNAc0.240.280.260.260.240.230.250.240.200.240.200.27

mm).The nalconcentrationofthesampleswas1.0g/L.Thecolumntemperaturewas40°C,the owratewas1.0mL/min,andtheinjectedsamplesizewas100mL.

N-Acetylationofchitosan.—Chitosan(0.3g)wasdissolvedin10%aqAcOH(50mL),andAc2Owasadded.Afterstirringatambienttemperaturefor5h,aqNaOHwasaddeduntilthepHreached8–9inordertostopthereaction.Thereactionmixturewasdialyzedagainstdeionizedwaterfor2daystoremoveanymicroionsandlyophilized.TheacetylatedchitosanwasthentreatedwithmethanolicKOHfor5hatrtandrepeatedlywashedwithMeOHusingacentrifuge.Finally,itwasdis-solvedindeionizedwaterandlyophilized.NMRspectroscopy.—1HNMRspectrawererecordedonaBrukerARX300spec-trometerinD2Oat25°C.ThepartiallyN-acetylatedchitosanwaslyophilizedtwicefromD2OsolutionbeforepreparingthesamplesforNMRanalysis.Allsamplesweredissolvedin5mmdiametertubes,andthesampleconcen-trationswere20mg/mL.1HNMRspectrawerealsorecordedinthepresenceof6%CF3COOD.ThechemicalshiftsweregivenonthelscalerelativetoMe4Si.Themeasure-mentconditionswereasfollows:aspectralwindowof8064Hz,32kdatapoints,apulseangleof30°,anacquisitiontimeof2.03s,and32scanswithadelayof1sbetweenscans.Inordertodecreasethelinewidthoftheanomericprotonsignals,eachsampleofthehalfN-acetylatedchitosanwasdissolvedinD2Ocontaining1%DClandthe1HNMRspectrawererecordedat80°C.

Estimationofsolubility.—WatersolubilityofthehalfN-acetylatedchitosanwasevalu-atedfromtheturbidity.Afterbeingpulverizedgently,thehalfN-acetylatedchitosan(50mg)wasdissolvedindeionizedwater(5mL),andthetransmittanceofthesolutionwasrecordedonaHitachiU-1000spectrophotometerusingaquartzcellwithanopticalpathlengthof1cmat600nm.ThepHdependenceofthewatersolubilityofthehalfN-acetylatedchi-tosanwasalsoestimatedfromthetransmit-tanceofthesolution,whichwaspreviouslypreparedwithaqAcOH(10%),bythestep-wiseadditionofconcdNaOH.Thesampleconcentrationswere0.5%.ThesolubilityofthehalfN-acetylatedchitosaninaqorganicsolventswassimilarlyestimatedfromthe

Fig.6.pHdependenceofwatersolubilityofhalfN-acetylatedchitosan:( )8.8k-53,( )19k-51,( )33k-47,( )78k-51,( )150k-50,( )340k-52,and( )600k-50.SamplecodescorrespondtothoseinTable2.

272N.Kubotaetal./CarbohydrateResearch324(2000)268–274

Fig.7.DependenceofsolubilityofhalfN-acetylatedchitosanonDMAcontent:( )8.8k-53,( )12k-48,( )19k-51,( )33k-47,and( )63k-48.SamplecodescorrespondtothoseinTable2.

Fig.8.DependenceofsolubilityofhalfN-acetylatedchitosanonMe2SOcontent:( )8.8k-53,( )12k-48,( )19k-51,( )33k-47,and( )63k-48.SamplecodescorrespondtothoseinTable2.

transmittanceofthesolution,whichwaspre-paredinadvancewithdeionizedwater,byaddingorganicsolventsstepwise.Thesampleconcentrationswere1.0%.3.Resultsanddiscussion

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.

Inconclusion,itwasrevealedthathighlysolublehalfN-acetylatedchitosancouldbepreparedthroughasimpleandimprovedmethod:reductionofthemolecularweightof

274N.Kubotaetal./CarbohydrateResearch324(2000)268–274

chitosan,followedbyN-acetylationinaqueousaceticacid.ThehalfN-acetylatedchitosanthusobtainedhadarandomdistribu-tionoftheN-acetylgroups,andthelowerthemolecularweight,thehigherthewatersolubil-ity.Furthermore,thesolubilityofthehalfN-acetylatedchitosaninaqueousorganicsol-ventsalsoincreasedwithdecreasingmolecularweight.References

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