ContentslistsavailableatScienceDirect
Carbohydrate
Polymers
jo u r n al h om ep a ge : w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l
Synthesis
of
an
O-alkynyl-chitosan
and
its
chemoselective
conjugation
with
a
PEG-like
amino-azide
through
click
chemistry
José
Ricardo
Oliveira
a,b,
M.
Cristina
L.
Martins
a,
Luís
Mafra
c,
Paula
Gomes
a,∗aCIQ-UP,DepartamentodeQuímicaeBioquímica,FaculdadedeCiências,UniversidadedoPorto,RuadoCampoAlegre687,4169-007Porto,Portugal bINEB–InstitutodeEngenhariaBiomédica,DivisãodeBiomateriais,UniversidadedoPorto,RuadoCampoAlegre823,4150-180Porto,Portugal cDepartmentofChemistry,CICECO,UniversityofAveiro,3810-193Aveiro,Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received2March2011
Receivedinrevisedform22July2011 Accepted25July2011
Available online 3 August 2011 Keywords: Azide–alkynecoupling Bioadhesion Biomaterials Chitosan clickchemistry FTIR
Huisgen’s1,3-dipolarcycloaddition Size-exclusionchromatography Solid-stateNMR
XPS
a
b
s
t
r
a
c
t
N-Phthaloyl-chitosanO-prop-2-ynylcarbamatewaspreparedasabiopolymeramenabletoundergo chemoselectiveconjugationbyazide–alkynecoupling,whileallowingupturnofchitosan’saminesafter dephthaloylation.N-phthaloylchitosanwaspreparedaccordingtopreviouslydescribedmethodsand,due toitslowsolubilityincurrentorganicmedia,subsequentmodificationswereruninheterogeneous con-ditions.Activationofhydroxylswithcarbonyl-1,1-diimidazoleandcouplingtopropargylamineyielded N-phthaloyl-chitosanO-prop-2-ynylcarbamate,thencoupledtoamodelPEG-likeazidebyazide–alkyne coupling,givingthe expectedtriazolylconjugate.N-Dephthaloylationallowed recoveryofthefree amines,responsibleforchitosan’sbioadhesionandtissue-regenerationproperties.
ThestructuresofallpolymerswereconfirmedbyFourier-transformedinfra-red(FT-IR)andX-ray photoelectron(XPS)spectroscopies,aswellasbysolid-statenuclearmagneticresonance(SSNMR).All chitosanderivativeswerepoorlysolubleinbothaqueousandorganicmedia,whichmakesthem suit-ablefortopicalapplicationsorforremovaloftoxicsubstancesfromeitherthegastricintestinaltractor environmentalsources.
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Some of the most appealing characteristics of chitosan are its bioadhesive properties and its ability to promote cell pro-liferation and, consequently, tissue regeneration (Berger, Reist, Mayer, Felt, &Gurny, 2004; Berger, Reist, Mayer, Felt, Peppas, et al., 2004). These properties of chitosan are of outmost importancefor biomedical engineering and are enhanced upon decreasingthepolymer’sdegreeofacetylation(Amaral,Lamghari, Sousa, Sampaio, & Barbosa, 2005), meaning that such proper-ties are intrinsically related with the polymer’s amino groups thatarecationicinacidic-neutralmedia(Batista,Pinto,Gomes,& Gomes,2006), likeinphysiological fluids.Many chemical mod-ifications have been carried out on chitosan’s d-glucosamine units,conferringtothenewmaterialssurprisingpropertiesand additional possibilities of engaging in a wide range of chemi-cal reactions for diverse applications (Muzzarelli, 1988; Kurita et al., 2002; Holappa, Hjálmarsdóttir, et al., 2006; Holappa, Nevalainen, Soininen, Másson, & Järvinen, 2006b; Rúnarsson, Holappa,Jónsdóttir,Steinsson,&Másson,2008;Rúnarssonetal.,
∗ Correspondingauthor.Tel.:+351220402563;fax:+351220402659. E-mailaddress:pgomes@fc.up.pt(P.Gomes).
2010),e.g.,biomedicalengineeringand/ordrugdelivery(Anitha etal.,2011;Iqbal,Sarti,Perera,&Bernkop-Schnürch,2011;Molnár, Barbu,Lien,Górecki,&Tsibouklis,2010;Muzzarelli,2009,2011). However,mostchitosanmodificationsdescribedintheliterature havebeencarriedoutexclusivelyorinclusivelyatthepolymer’s primary aminogroups, which is undesirablefor applicationsin which the polymer’samine-dependent bioadhesionand/or pH-dependentpropertiesshouldhaveakeyrole.Inviewofthis,we have workedon the synthesis and characterization of a novel chitosan derivative that conserves the primary amine groups unchanged,whilebeingselectivelymodifiedatitsprimaryalcohol functioninordertobearanalkynemoietyamenabletoundergo furthermodificationsbywell-knownorganicreactions,e.g., Huis-gen1,3-dipolarcycloaddition(a“clickreaction”correspondingto anazide–alkynecoupling)(Kolb,Finn,&Sharpless,2001)or Sono-gashiracoupling(Chinchilla&Nájera,2007).Thesereactionsmay serve,amongotherpurposes,forfacilechitosanconjugationwith drugsandotherbioactivemolecules,likeazide-modifiedpeptides orsugars,already availableinthemarketoreasilyproducedby currentorganicsynthesisprocedures(Lahann,2009).Therefore,a selectively N-protectedO-alkynylatedchitosan waspreparedby straightforwardmethodsandsubmittedtoatestazide–alkyne cou-plingreactionwithacommercialazide(PEG-likeazide)toserveas proof-of-concept,ashereindescribed.
0144-8617/$–seefrontmatter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2011.07.043
2. Materialsandmethods 2.1. Chemicals
ChitosanwasobtainedfromFrance-Chitine.Phthalicanhydride, tetrahydrofuran(THF),1,1-carbonyldiimidazole(CDI), propargy-lamine, copper (II) acetate, sodium ascorbate, ethylenediamine tetraaceticacid(EDTA)disodiumsalt,hydrazinemonohydrate,and 11-azido-3,6,9-trioxaundecan-1-aminewerefromSigma–Aldrich. Potassiumbromide(KBr)andallremainingorganicchemicalsand solventswerefromMerck.
2.2. Purificationofcommercialchitosan
Chitosanwaspurifiedbythereprecipitationmethod(Crompton etal.,2007).Briefly,1gofchitosanpowderwaspre-hydratedin 197.92mlofultrapurewater,for24hat4◦Cunderslowmagnetic stirring.Then,aceticacid(2.28ml)wasadded,andtheslurryleft overnightatroomtemperature. Aftercompletedissolution, the resultinggelwasfilteredthrougha 20micrometerporesize fil-tertoremoveundissolvedor gelatinousparticles.Chitosanwas thenprecipitatedthroughdropwiseadditionof1MaqueousNaOH, whilestirring.Finally,theregeneratedchitosanwaswashedwith ultrapurewaterbycentrifugationuntilneutrality,freeze-driedand groundedinalaboratorymilltoyieldafinepowder.
Thedegreeofacetylationandmolecularweightofthepurified chitosanweredeterminedusingFT-IRFouriertransforminfrared spectroscopy(seeSection2.8)andhighperfomancesize-exclusion chromatography(HP-SEC,seeSection2.10),respectively.
2.3. PreparationofN-phthaloyl-chitosan(2)
Thechemicalroutetowardsthetargetchitosan derivativeis giveninScheme1andbeganwiththesynthesisof N-phthaloyl-chitosan(2),accordingtothemethoddevelopedbyKurita,Ikeda, Yoshida,Shimojoh,andHarata (2002).Briefly,purified chitosan, 1(1.0g,6.20mmol)andphthalicanhydride (2.76g,18.60mmol) weresuspendedin30mlDMFcontaining5%(v/v)waterandthe mixtureheatedat120◦Cwithmagneticstirring,underargon atmo-sphere.After8h,themixturewascooledtoroomtemperatureand pouredintoicewater.Theresultingpaletanprecipitatewas col-lectedonaG4filterfunnel,thoroughlywashedwithcoldmethanol, anddriedinairtogive1.4gofN-phthaloyl-chitosan(2).
2.4. PreparationofN-phthaloyl-chitosanO-prop-2-ynyl carbamate(3)
N-phthaloyl-chitosan2(0.6g,2mmol),pre-washedwithTHF, wassuspendedin9mlof30mg/mlCDIinTHF,andtheslurrytaken to40◦Cunderargonatmosphereandgentlemagneticstirring.After 5h,thesolidfractionwascollectedonaG4filterfunnelandwashed thoroughlywithTHFtoremove CDIexcess.Theprecipitatewas transferredtoanewflasktowhichwereadded6mlof30mg/ml propargylamineinTHF.Theslurrywasgentlystirredatroom tem-peraturefor24h,afterwhichthesolidfractionwascollectedon aG4filterfunnel,washedwithmethanolanddriedinairtogive N-phthaloyl-chitosanO-prop-2-ynylcarbamate(3).
2.5. PreparationofN-phthaloyl-chitosan
O-(11-amino-3,6,9-undecan)triazolylcarbamate(4)
N-Phthaloyl-chitosan O-prop-2-ynyl carbamate 3 (0.4g 0.9mmol),copperacetate(0.06g,0.45mmol),sodiumascorbate (0.12g, 0.64mmol) and the PEG-like azide (11-azido-3,6,9-trioxaundecan-1-amine)(0.2g,0.9mmol)weremixedin16mlof tert-butanol/H2O(1:1,v/v)andthemixtureleftovernightunder
gentlemagneticstirring,atroomtemperature.Thesolidfraction, N-phthaloyl-chitosanO-(11-amino-3,6,9-undecan)triazolyl carba-mate4,wascollectedonaG4filterfunnelandthoroughlywashed firstwith0.1MEDTAandthenwithmethanol,andfinallydriedin air.
2.6. RemovaloftheN-phthaloylprotectinggroupfrom4: synthesisofchitosanO-(11-amino-3,6,9-undecan)triazolyl carbamate(5)
N-Phthaloyl-chitosanO-11-amino-3,6,9-undecantriazolyl car-bamate 4 (0.15g, 0.25mmol) was reacted with hydrazine monohydrate (1ml, 18mmol) in 10ml ethanol, at 40◦C with gentle stirring. After 18h, the solid fraction was collected by centrifugation, thoroughlywashed withethanol and water and freeze-driedtogivethedeprotectedchitosan O-(11-amino-3,6,9-undecan)triazolylcarbamate5.
2.7. XPSanalysis
Chitosan,1,anditsderivatives3–5wereanalysedbyXPS. Sam-pleswerepreparedbycompressingthepowderedpolymersinto 1mm-thickpellets.MeasurementswerecarriedoutonaVG Sci-entific ESCALAB 200Aspectrometer usingaluminum (15kV)as a radiation source. Photoelectrons were analysed at a take-off anglenormaltotheinterface.Surveyspectrawerecollectedover the0–1100eV rangewith analyserpass energyof 50eV. High-resolutionC1s,N1sandO1sspectrawerecollectedwithanalyser passenergyof20eV.Thebindingenergyscaleswerereferenced bysettingtheC1sbindingenergyto285.0eV.Spectrawerefitted usingtheXPSPEAK(version4.1)software.
2.8. FT-IRanalysis
FT-IR spectra of chitosan, 1, and its derivatives 3–5, were obtainedusingaPerkin-ElmerSystem2000FT-IRspectrometer. AllthesampleswererecordedasKBrpelletspreparedby blend-ing2mgofthepowderedpolymer,previouslydriedovernightat 60◦Cunderreducedpressure,with200mgKBr,previouslydriedat 105◦Cfor24h.Theinfraredspectrawereobtainedbyaccumulation of100interferograms,at4cm−1spectralresolution,between4000 and400cm−1andafternitrogenpurgeofthesamplecompartment for2mintominimizebandsthatcouldarisefromresidualair com-ponents(CO2andH2O).SpectrawerefittedusingtheSpectrumGX FT-IR(version5.3)software.TheDAwasestimatedaccordingtothe methodproposedbyBrugnerottoetal.(2001),wheretheamideIII (C–Nstretchingvibration)band(1320cm−1)wasusedasthe ana-lyticalbandandthebandat1420cm−1(inplaneO–Hdeformation vibration)wasusedastheinternalreferenceband.
2.9. SSNMRmeasurements
Carbon-13 cross-polarization/magic angle spinning nuclear magneticresonance(13C-CP/MASNMR)spectrawererecordedona 9.4TWB(400MHz,1HLarmorfrequency)BrukerAvance spectrom-eter(DSXmodel).A4mmdouble-resonancemagicanglespinning probewasemployedat400.1(1H)and100.6MHz(13C)Larmor fre-quencies.SampleswerespuninZrO2rotorsusingaspinningrateof 12kHz.13C-CP/MASNMRspectrawererecordedusingarampstep (varyingfrom100%to50%inamplitudeusing100points);contact time:1.5ms;1H90◦excitationpulse:3.75s;1Hand13C radio-frequencyfieldstrengthforcrosspolarizationwassetto87kHz and68kHz,respectively;numberofscans:1k;recycledelay:5s. TPPM-15decoupling(Bennettetal.,1995)wasemployedduring thesignalacquisitionusinga4.8spulselengthforthebasicTPPM
N N N O O O NH2 O HO OH O HO NH O O CH3 O N O O OH n (a) (b) (c) (d) 2 1 3 4 5 R O HO OH O HO NH O O CH3 O NH2 OH n O HO O O HO NH O O CH3 O N O O OR n N H O O HO O O HO NH O O CH3 O N O O OR1 n N H O R1 (b) N N N O O O NH2 O HO O O HO NH O O CH3 O NH2 OR1 n N H O R1
Scheme1.Chemicalroutetowardscationicchitosan5,obtainedafteranazide-alkyneconjugationreactioninvolvingtheO-alkynylatedchitosanderivative3:(a)phthalic anhydride,DMF,120◦C,8h;(b)CDI,THF,40◦C,5h;propargylamine,THF,rt,24h;(c)copper(II)acetate,sodiumascorbate,11-azido-3,6,9-trioxaundecan-1-amine, tert-butanol/H2O,rt,overnight;(d)hydrazinemonohydrate,H2O,100◦C,18h.Forsimplicity,onlymodifiedmonomersof2–5areconsideredintheScheme.
pulseunitalongthe1Hchannel,employinga1Hradio-frequency fieldstrengthof100kHz.
13Cchemicalshiftsarequotedinpartspermillion(ppm)and calibratedwithrespect totheexternal reference,glycine(C O, 176.03ppm).
2.10. HP-SECmeasurements
Themolecularweightofchitosan(1)wasdeterminedbyHP-SEC using a modular system composed of an isocratic pump (K-1001,Knauer),avacuumdegasser(Wellchrom2channelK-5002, Knauer),aviscometer/rightanglelaserlightscattering(RALLS)dual detector(T60AViscotek)andarefractiveindexdetector(K-2301 Knauer)operating thesame wavelength as the RALLSdetector (670nm).PLaquagel–OHmixedcolumnswereusedfor separa-tions.Thesamplesweredissolvedinthemobilephase(0.5Macetic acid/0.2Msodiumacetatewith0.01%(v/v)NaN3)at1mg/ml, fil-teredandinjectedthroughamanualinjectionvalveequippedwith a100lloop.Theflow-ratewasmaintainedat1.0ml/min.
3. Resultsanddiscussion
Chitosan (1) obtained after purification of the commercial powder presented a molecularweight of 283.000–472.000 and a DAof 16%, determinedasdescribed in theprevioussections. Thisstartingmaterialwasthensubjectedtochemical modifica-tionsdepicted in Scheme1. The stepwiseprocedureemployed isnecessarysince thereactivity ofchitosan’sprimaryaminesis considerably higher than that of the primary hydroxyls, so N-protectionfollowedbyO-activationisasuitableapproach.Chitosan aminegroupswereprotectedbyN-phthaloylationtoyield2,as N-phthaloylationisnowacurrentandversatileprocedure when-everchemoselectiveO-modificationofthebiopolymerisdesirable (Kuritaetal.,2002;Kurita, Yoshida,&Umemura,2010).The N-phthaloyl derivative 2 was poorly soluble in a wide range of commonorganicandaqueous/organicsolvents,asexpectedfrom previousfindingsbyKuritaetal.(2002).Theseauthorsascribed suchphenomenon toan increasedcrystallinity of themodified polymer,whichcouldbeconfirmedbyusthrough dephthaloyla-tion(accordingtotheprocedurein2.6)ofN-phthaloylchitosan(2) backtoits“unmodified”form,whichwasfoundtobenolonger solubleinacidicaqueousmedia,aswastheoriginalpolymer(1).
Thelowsolubilityof2 madeuscarryout subsequentreactions underheterogeneousconditions,asrecentliteratureshowsthat chemicalmodificationsofchitosancanbeensuccessfullyachieved undersuchconditions(Elkholy,Khalil,&Elsabee,2011;Li,Yuan, Gu,&Re, 2010;Shen, Ji,Yang, &Zheng, 2007;Wan, Gao,&Li, 2010).
N,N-Carbonyldiimidazole (CDI) was used for activation of hydroxylsin2towardssubsequentcouplingwithpropargylamine toyield3;CDIisacheapandefficientcouplingagentusedin indus-trialproductionofpeptides(Montalbetti&Falque,2005), which hasbeen successfullyemployed inthemodificationof polysac-charidesascellulose,dextranorchitosanformultipleapplications (Chirachanchai,Lertworasirikul,&Tachaboonyakiat,2001;Oh,Lee, &Park,2009;Önal&Telefoncu,2003).Propargylaminewasselected asthealkynedonor,asit providesahydrophilicandstable,yet chemo-reversible,carbamategrouplinkedtotheterminalalkyne throughaflexiblemethylene.Obviously,thisapproachis applica-bletootheralkynyl–aminesfortailoredspacersizeandflexibility. Thestructuresofchitosanderivatives2and3wereconfirmed,as demonstratedthroughtheuseofatoolboxofspectroscopic tech-niques,discussedfurtherbelow.
Toofferproof-of-conceptregardingtheabilityofpolymer3to undergoachemoselectivecouplingtoanazidemoiety,wehave submittedthis polymertoaHuisgen’s1,3-dipolar cycloaddition with11-azido-3,6,9-trioxaundecan-1-amine,takenas representa-tiveofanyazide-modifiedbiomolecule(drug,peptide,etc.).This testazidewaschosengiven itscommercialavailability, reason-ablecost,mediumsizeandoverallhydrophiliccharacter(PEG-like chain witha terminal primary amine). Recent reports describe successfulapplicationoftheazide–alkynecouplingreactionto cre-atenovelchitosan-basedbiomaterials(Lietal.,2010;Yuan,Zhao, Gu,&Ren,2010;Yuan,Li,Gu,Cao,&Ren,2011;Yuan,Zhao,Gu, Ren,&Ren,2011;Zhangetal.,2011).Inmostcases,theclassical Cu(I)/N,N,N,N,N-pentamethyldiethylenetriamine(PMDETA) sys-temhasbeenused(Lietal.,2010;Yuanetal.,2010;Yuan,Li,etal., 2011;Yuan,Zhao,etal.,2011)topromotetheclickreaction,assome authorshavepreviouslyreportedthatinsitugenerationofCu(I)by meansoftheCu(II)/ascorbatesystemleadstosevere depolimeriza-tionofthechitosanbackbone(Lallana,Fernández-Megía,&Riguera, 2009;Kulbokaiteetal.,2009).Interestingly,nosuchproblemwas reportedonalatestworkwheretheCu(II)/ascorbatesystemhas beenusedtocreatechitosan-basedmicrocapsulesthrough multi-layerassembly byclickchemistry(Zhangetal.,2011).Anyway, westartedbyusingtheCu(I)/PMDETAsystem,butsoondiscarded thisoptionduetotheinefficientremovalofexcesscopperfrom thepolymermatrix,evenafterextensiveworkupwithEDTA(data notshown).Thisproblem,alsoreportedbyotherauthors(Lallana, Fernández-Megía&Riguera,2009),wasnotobservedwhenusing Cu(II)/ascorbate,whichthereforebecameoursystemofchoice.In ordertoavoidoratleastminimizethedepolimerizationproblems allegedlyassociatedtoCu(II)/ascorbate,wehaverunthe cycload-ditionreactionatroomtemperaturefornolongerthan12h.
Asdemonstratedbystructuralanalyses(seediscussionbelow), thecycloadditionoccurredasexpected toyield4. Thelow sol-ubility of this product in a wide range of aqueous buffers and aqueous/organiceluents, prevented us toaccurately determine the polymer’s molecular weight by HP-SEC. Nonetheless, the insolubilityof4indirectlysuggeststhatthisisstillahigh molecular-weight polymer. Furthermore, based on a previous work by CabreraandVanCutsem,whousedmatrix-assistedlaser desorp-tionionization–time-of-flightmassspectrometry(MALDI-TOFMS) todetectchitooligosaccharides(Cabrera&VanCutsem,2005),we usedthistechniquetoanalysepolymer4,butnopeakswerefound below100kDa(datanotshown).
N-Deprotectionof4toyieldtheO-modifiedcationicchitosan 5wascarried out usinghydrazine,commonly employedfor
N-Table1
Elementalanalysisdata(%C,N,O)asdeterminedbyXPSanalysisofpolymers1–5.
Polymer Atomic% C(1s) O(1s) N(1s) 1 58.5 33.6 7.9 2 67.4 27.6 5.0 3 66.8 26.3 7.0 4 65.6 25.8 8.7 5 62.3 27.9 9.9
dephthaloylationofN-phthaloyl-chitosanderivatives(Kuritaetal.,
2010).Again,givenpriorreportsonsignificantbreakdownofthe chitosan’sbackboneinthecourseofN-dephthaloylation(Makuˇska &Gorochovceva,2006),wehaverunthedeprotectionfor18hat only40◦C.Structuralanalysesdescribedbelowallowedconfirming fullremovaloftheN-phthaloylprotectinggrouptogivepolymer 5.
Finally,itshouldbenotedthat,inallcases,productpolymers keptthefinepowderstructureofthestartingmaterials,and no grindingwasrequiredinanystep.
3.1. Comparativeanalysisofchitosan(1)anditsderivatives (2–5)byXPS
Table1showstherelativeatomicpercentagesofcarbon,oxygen andnitrogenofunmodifiedchitosan(1)andchitosanderivatives (2–5),calculatedfromhighresolutionXPSspectra.Theresolved XPShighresolutionspectraofC1s,O1sandN1sofunmodified chi-tosanandchitosanderivatescanbevisualizedatFig.1.Therelative atomicpercentagesofcarbonindifferentenvironments,obtained fromtheC1sXPShighresolutionspectraofunmodifiedand modi-fiedchitosan,aredescribedinTable2,whereassimilarinformation referringtoO1sandN1sisgiveninTable3.
Resultsobtainedforunmodifiedchitosanwereasexpectedand agreedwithdata reportedby otherauthors (Amaral, Granja, & Barbosa, 2005; Beamson & Briggs, 1992). The C1s spectrum of unmodifiedchitosanwasfittedusingthreepeaksasdescribed else-where (Amaral, Granja,et al., 2005).The peak at285.0eV was assignedtoC–Cand C–Htypecarbonsmixedwith–CH2–from a surface contaminant (Amaral, Granja, et al., 2005); the peak at 286.4eV wasassigned to C–NH2, C–OHand C–O–C carbons, whereasthepeakat281.1eVwasassigned tocarbonsfromthe O–C–OandN–C Ogroups.RegardingtheresolvedO1sspectrum, threepeakswereindentified:at531.6eV,assignedtoN–C Oin N-acetylatedglucosamineunitsandCO–N–CO;at532.8eV,assigned to C–O–C and C–OH; and at 534.0eV,assigned to O–C–O.The resolvedN1sspectrumrevealedthreepeaks:at399.7eV,assigned tonitrogens in C–N and CO–N bonds; at 400.7eV, assigned to CO–N–COandN–CO–O;andat401.8eV,assignedtoaminogroups intheammoniumform(NH3+).
AfterN-phthaloylation,i.e.,regardingderivative2ascompared tostartingmaterial1,therewasanincreaseintheatomic percent-ageofcarbonaccompaniedbyasmalldecreaseinthepercentages ofnitrogenandoxygen,anexpectedconsequenceofthe incorpora-tionofthephthaloylgroup.Theincreaseofthepeakat285.0eVwas ascribedtotheinsertionofC Ctypecarbonsfromthephthaloyl group.Consequently,thecarbonpeakat286.4eVhasdecreaseddue tothedisappearanceofC–NH2typecarbonsafterN-phthaloylation. Althoughthecarbonpeakat288.1eVdidnotshowanyrelevant changeinintensity,acloseinspectionofthispeakinFig.1showsa smallshiftofthebindingenergythatcouldberelatedwithinsertion ofCO–N–COcarbons.IntheresolvedO1sspectrum,theincreaseof thepeakat531.6eVwasascribedtotheinsertionofCO–N–COfrom thephthaloylgroup.N1sXPSspectrumdemonstratedtheinsertion ofthephthaloylgroupbythedisappearanceofthepeakat401.8eV
Fig.1.XPShighresolutionspectraof:(a–c)unmodifiedchitosan(1);(d–f)N-phthaloyl-chitosan(2);(g–i)N-phthaloyl-chitosanO-prop-2-ynylcarbamate3;(j–m) N-phthaloyl-chitosanO-(11-amino-3,6,9-undecan)triazolylcarbamate4,and(n–p)O-triazolylchitosan5powders.O1s,N1sandC1sregionsareshown.
Table2
C(1s)surfaceatomiccompositionofthedifferentpolymers1–5.
Polymer Atomic%C(1s)
C C C–C/C–H/C C C–NH2/C–OH/C–O–C O–C–O/N–C O/CO–N–CO N–CO–O
283.7eV 285eV 286.4eV 288.1eV 289.2eV 1 – 16 65 19 – 2 – 42 40 18 – 3 3 42 36 15 4 4 – 38 48 11 3 5 – 28 58 12 2 Table3
O(1s)andN(1s)surfaceatomiccompositionofthedifferentpolymers1–5.
Polymer Atomic%O(1s) Atomic%N(1s)
N–C O/CO–N–CO C–O–C/C–OH O–C–O C–N/CO–N CO–N–CO/N–CO–O NH3+
531.6eV 532.8eV 534.0eV 399.7eV 400.7eV 401.8eV 1 6 87 7 93 – 7 2 22 64 14 72 28 – 3 28 57 15 65 35 – 4 17 61 22 52 32 16 5 13 71 16 52 33 15
assignedtoNH3+andtotheemergenceofanewpeakappearedat
400.7eV,assignedtoCO–N–COandN–CO–O(Table3).
Ascomparedtoits N-phthaloylchitosan2 precursor,the O-alkynylderivative3presenteda decreasein thepercentagesof carbonand oxygenalong withasmall increasein the percent-age of nitrogen, compatible with insertion of propargylamine. TheresolvedC1sspectrumof3revealedtwonewpeaks:oneat 283.7eV, assigned tothealkyne group(C C), and theother at 289.2eVascribedtothecarbamategroup(N–CO–O).Thedecrease of thepeak relative intensityat 286.4eV wascompatible with partialmodificationoftheC–OHtype carbons.For theO1s sur-faceatomic percentage,there wasa smallincrease of thepeak at531.6eV,duetoN–C Ofromthecarbamatebondformed.In theresolvedN1sspectrum,theexpectedincreaseofthepeakat 400.7eV, ascribed tothe carbamate group (N–CO–O), wasalso observed.
XPS analysis of N-phthaloyl-chitosan O-(11-amino-3,6,9-undecan)triazolyl carbamate (4) showed a decrease in the percentagesofcarbonandoxygenandconcomitantincreaseinthe percentageofnitrogen,supportingtheoccurrenceofthedesired azide–alkynecouplingtoyieldatriazolestructure.WhentheC1s spectrumof4iscomparedtothatofitsprecursor3,thecarbon peakat283.7eVdisappears,whichbyitselfdemonstratesthe con-versionoftheC Cgroupintosomethingdifferent.Furthermore, anincreaseofthepeakat286.4eVcanbeascribedtotheinsertion ofadditionalC–O–Ctypecarbons,present inthePEG-likechain fromtheazideusedinthecycloaddition.Thiswasalsoobservedin theresolvedO1sspectrum,frompeaksat532.8eVand534.0eV, duetoC–O–CandO–C–Obonds,respectively,whichincreaseupon insertionof thePEG-likechain. Forthe resolvedN1sspectrum, thepeakat401.8eV,duetoNH3+,reappearsasaconsequenceof insertionoftheamino-terminatedligand.
Finally,XPSanalysisrevealedthatthedephthaloylation reac-tiontogivepolymer5ledtoadecreaseintheatomicpercentage ofcarbonalongwithanincreaseintheatomicpercentageofall theotherelements,asexpectedfromtheremovalofthephthaloyl group. This interpretation was supported by the almost com-plete disappearanceof the peak at 285eV (C C type carbons) togetherwithaclearincreaseintherelativeintensityofthepeakat 286.4eV(C–O–C+C–NH2typecarbons).IntheresolvedO1s spec-trum,theexpecteddecreaseofthepeakat531.6eVascribedto
CO–N–COwasobserved,duetophthaloylremoval.Fortheresolved N1sspectrum,onlychangesonrelativeatomicpercentageswere observed, since thetypes ofnitrogen atoms already present in precursor 4 remained the same after dephthaloylation to give 5.
3.2. Analysisofchitosan(1)anditsderivatives(2–5)byFT-IR FT-IRwasalsousedtomonitorthestructuralchangescaused bythestepwisechemicalmodificationofchitosan(1)alltheway throughintermediatepolymers2–4untilthefinalcationicpolymer 5.TheobtainedinfraredspectraarethusdisplayedinFigs.2and3. TheIRspectrum(Fig.2)ofunmodifiedchitosan(1)presented theexpectedcharacteristicbands(Gomes,Gomes,Batista,Pinto, &Silva,2008),like:broadintensebandat3450–3200cm−1 due tooverlappingcontributions ofdifferenthydrogen-bondedX–H stretchingvibrations,asO–Hstretchingat3426cm−1,NH2 asym-metricstretchingat3377cm−1andN–Hstretchingat3303cm−1; C–Hstretchingat2878cm−1;characteristicvibrationmodesfrom GlcNHAcunits,asamideIat1656cm−1(C Ostretchingfrom
sec-Fig.2.FT-IRspectra(KBrpellets)of(1)unmodifiedchitosanand(2)N-phthaloyl chitosanpowders.
Fig.3. FT-IRspectra(KBrpellets)of(3)N-phthaloyl-chitosanO-prop-2-ynyl car-bamate,(4)N-phthaloyl-chitosanO-(11-amino-3,6,9-undecan)triazolylcarbamate and(5)chitosanO-(11-amino-3,6,9-undecan)triazolylcarbamatepowders.
ondaryamides),amideII(inplaneN–Hdeformationcoupledwith C–Nstretchingfromsecondaryamides)andtheN–Hbendingofthe primaryamineat1597cm−1,amideIIIat1322cm−1(C–N stretch-ing)and –CH3 symmetricalangular deformation at1378cm−1; O–H planedeformation(primaryalcohol)at 1421cm−1; C–O–C stretching vibration in the glucopyranose ring at 1032cm−1; and the specific bands of the ˇ(1–4) glycoside bridge at 1153 and 897cm−1 (Socrates, 2001). The IR spectrum (Fig. 2) of N-phthaloylchitosan(2)displayedadditionalpeakswithrespectto theunmodifiedchitosan,characteristicofthephthalimidegroup (Socrates,2001)inserted:twoC Ostretchingbandsat1778and 1709cm−1 andimideC Cstretchingat1551cm−1; C–Houtof planedeformationat720cm−1andaromaticringdeformationat 530cm−1 from thearomaticringof thephthaloylgroup.These observationswereinagreementwithpreviouslyreporteddataon similarN-phthaloylchitosanderivativesdevelopedbyKuritaetal. (2002).
TheIRspectrum(Fig.3)ofN-phthaloyl-chitosanO-prop-2-ynyl carbamate, 3, herein proposed as a novel “clickable” chitosan, exhibitedthefollowingadditionalpeaks: C–Hstretching vibra-tion at 3291cm−1 and C C stretching vibration at 2122cm−1, typicalofmonosubstitutedalkynes(Socrates,2001);absorption due to CHN group (N–H deformation and C–N stretching) at 1530cm−1 and coupled C–N and C–O stretching vibration at 1251cm−1, from the carbamate group. These features were in complete agreementwithformation ofN-phthaloyl-chitosan O-prop-2-ynylcarbamate,3.
Afterreactingpolymer3withthePEG-likeazide under clas-sicalazide–alkynecouplingconditions,thetriazolylderivative4 wasobtained,whoseFT-IRspectrum(Fig.2)displayedtworelevant changes,ascomparedtoitsprecursor(3);abandat830cm−1, char-acteristicofN–C Ninplanebending(Krishnakumar&JohnXavier, 2004),arisingfromformationofthetriazolering;additional evi-denceoftheoccurrenceoftheazide–alkynecouplingwasprovided bythedisappearanceofthetypicalmonosubstitutedalkyne vibra-tionmodesat3291cm−1 ( C–Hstretching)and2122cm−1 (C C stretching).Giventhestructuralnatureoftheazideusedinthe cycloaddition, i.e.,ofits 3,6,9-trioxaundecan-1-aminebackbone, alladditionalvibrationmodesfromthis backbonewere expect-edlyoverlappedtosimilarmodesalreadypresentontheprecursor polymericmatrix.
Finaldeblockingoftheprimaryaminesbydephthaloylationled to5, whose FT-IRspectrum (Fig.2)revealed theexpected fea-tures,i.e.,disappearanceoftheaforementionedphthaloyl-related
bands,i.e.,C Ostretchingabove1700cm−1,imideC C stretch-ingat∼1550cm−1; C–Houtofplanedeformationat720cm−1 andaromaticringdeformationat530cm−1andaswelldescribe previouslytheappearanceofthepeakcharacteristicofNH2 asym-metricstretchingat3368cm−1withpreservationoftheremaining spectralfeatures.
Overall,FT-IRanalysisofthefivepolymerswasinagreement withXPS-derived data,providing further confirmation that the chemicalroutedepictedonScheme1wassuitabletointroduce thedesiredmodificationsontochitosan.
3.3. Characterizationofpolymers1–5bySSNMR
Duetoitsabilitytoprobe locallyand selectivelythe chemi-calenvironmentofeachcarbonnucleus,SSNMRspectroscopywas usedtounambiguouslyidentifyeachchitosanderivativewithhigh accuracy.Fig.4showstheensembleoftheSSNMRresultsusing the13C-CP/MASNMRmethod.The13C-CP/MASNMRspectrumof 1 (Fig. 4a) showsthat the startingbiopolymer has, as itsmain component, chitosan residues identified by the 13C resonances observedatcarbonchemicalshifts,ı∼105.4(C1),57.2(C2),75.1 (C3, C5), 83.1(C4)and 60.8 (C6) ppm. Twoadditional 13C res-onancesmaybeobservedat ı∼173.7and 23.5ppm, whichare assignedtothecarbonyl(C7)andmethyl(C8)functionalgroups typicallypresentinthechitinskeleton. Uponreactionof1with phthalicanhydride,polymer2wasobtained(Scheme1).The struc-tureofpolymer2maybeeasilyconfirmedbytheobservationof three13CresonancestypicaloftheN-phthaloylsubstituent appear-ingatı∼168.9,134.7,131.0and123.4ppm,whichcorrespondto theC7,C10,C8andC9carbonatoms,respectively(Fig.4b),and agreeswithdatapreviouslyreported(Kuritaetal.,2002).Theacetyl 13Cresonancesfromchitinmaystillbeobserved(C7).Polymer 3, obtainedaftercoupling 2 withpropargylamine,maybe also straightforwardlydetected,bythepresenceofthe13Cresonances typicalofthepropargylsubstituent(RgroupinFig.4c).For exam-ple,13Cpeaksobservedatı∼157.0and30.7ppmareassignedto theamidecarbonyl[C(1)]andtotheN–CH2carbon[C(2)], respec-tively.Thecarbonsinvolvedinthetriplebondaresuperimposed withthe13C resonancesfromthechitosanskeletonobservedin theregionbetweenı∼70and85ppm,hamperingtheirindividual assignment.
SSNMRalsoconfirmedthatpolymer 3wasconverted into4 throughtheHuisgen’s1,3-dipolarcycloadditionperformed,with concomitantformationofatriazoleringatpositionsC6andC6. Thisisclearlyshownbythe13Cresonancesatı∼50.9,40.1and 36.8ppm,whichdonotappearintheprevious13C-CP/MASNMR spectraof1–3(Fig.4a–c).Thesethreeresonancesareassignedto C[5],C[2]andC[7]carbonsfromthetriazolesubstituent(R1).Itis worthmentioningthattwoadditional13CpeaksfromtheR
1 moi-ety,resonatingatı∼156.7and145.4ppm,arealsoobservedat higherchemicalshifts.TheformerpeakisassignedtotheC O(C[1]; 156.7ppm)fromtheR1 side-chaininconfigurationA,whilethe latterresonanceisassignedtotheC O(C[3]*;145.4ppm),which belongtotheR1substituentfollowingthearrangementadoptedin configurationB(Fig.4d).SSNMRwas,thus,theonlymethodthat allowedtodiscriminatebetweenbothconfigurationsexistentin polymer4.Thisfactisaconsequenceofthetwopossible orien-tationsatwhichtheazide-alkynecycloadditionmaytakeplace, i.e.,theformationofboth the1,4and1,5cycloaddition adducts (Kolb,Finn&Sharpless,2001;Meldal&Tomøe,2008).This par-ticularfindingfurtherdemonstratestheoccurrenceofthedesired cycloaddition,whichconvertedtheO-alkynylatedchitosan3into theO-triazolylconjugate4.
Finally, SSNMR analysis of polymer 5 showed that the N-phthaloylprotectinggroup,presentin4,wasremovedasdesired.
Fig.4.SSNMRspectraofa)unmodifiedchitosan1,b)N-phthaloyl-chitosan2,c)N-phthaloyl-chitosanO-prop-2-ynylcarbamate3,d)N-phthaloyl-chitosan O-(11-amino-3,6,9-undecan)triazolylcarbamate4ande)chitosanO-(11-amino-3,6,9-undecan)triazolylcarbamate5.Selectedcarbonassignmentsaccordingtonumberinggiventostructures shownontheright.Asterisksdepictspinningsidebands.
Completeremovalofthisgroupmaybeconfirmedbythe disap-pearanceof thefour13C resonances(C7–10)atı∼168.9,131.0, 123.4and134.7ppm(Fig.4b–d).The13Cspectrumof5is essen-tiallythesameasthe13Cspectrumof1excludingtheadditional
peaksobservedin5(Fig.4e)duetotheR1substituent.Noticethat theanomericcarbon(C1)resonancesobservedinthespectrumof thechitosanmonomerin1and5(cf.Fig.4aandeemphasizedbythe reddashedline)havethesamechemicalshifts,whileinalltheother
13Cspectra(Fig.4b–d)thechemicalshiftpositionofC1carbonsare slightlyshiftedtohigherfieldsduetotheeffectoftheN-phthaloyl protectinggroupattachedtothenearbyC2carbonatom.
Inconclusion,itisdemonstratedthatthefinalpolymer,5,is obtainedfreeofanydetectableimpurities,bearingitsR1groupin twodifferentconfigurationsandhavinganaminegroupatposition C2,whichprovesthat5nolongercontainstheN-phthaloylamine protectinggroupatthatposition.
4. Concludingremarks
Straightforwardchemistrywasemployedforthesynthesisof N-phthaloyl-chitosanO-prop-2-ynylcarbamate,proposedasanovel polymer that may be conjugatedto other [bio]molecules by a chemoselectiveazide-alkynecoupling,whilepreserving bioadhe-sionandpH-dependentpropertiesassociatedtochitosan’sprimary amines. This polymer was then conjugated, by means of the Huisgen’s1,3-dipolarcycloaddition(azide-alkynecoupling)with amodelazide(PEG-like azide),producing astabletriazole link-agecompatiblewithsubsequentremovaloftheamine-protecting group,torecovertheoriginalchitosan’sprimaryaminesthatare responsibleformanyoftheattractivefeaturesofthisbiopolymer. Thestructuresofallstarting,intermediateandfinalmaterialswere characterizedandconfirmedbymeansofdifferentanalysis tech-niques,i.e.,XPS,FT-IRandSSNMR,alloftheminperfectagreement witheachother.
Thelowsolubilityofthepolymersobtainedineitheraqueous ororganicmediagivesthempotentialfortopicalapplications,e.g., treatmentofinfectedskinwounds(Batista,Gallemí,Adeva,Gomes, &Gomes, 2009;Dai,Tegos,Burkatovskaya,Castano,&Hamblin, 2009) or transdermal gene delivery (Özbas¸-Turan, Akbu˘ga, & Sezer,2010)orimmunization(Prabuh,Sundaramoorthi,Selvaraju, & Tamilselvi, 2011). Properly modified insoluble chitosans are alsointerestingfor removaloftoxic substancesfromeitherthe body(Park,Kwon,Choi,&Kim,2000)orenvironmentalsources (Bhatnagar&Sillanpää,2009).
Thereisagrowingnumberofcommerciallyavailablemolecules bearing an azide moiety for click conjugations. So, a chitosan derivativeas N-phthaloyl-chitosan O-prop-2-ynyl carbamate, 3, mayserveasapolymertemplateforchemoselectivecouplingto anyazide,fortailoredapplicationsandproperties.
Acknowledgments
TheauthorsthankFundac¸ãoparaaCiênciaeTecnologia(FCT, Portugal)andFEDER(EuropeanUnion)forco-fundingthisproject (ref. PTDC/CTM/65330/2006 and ref. FCOMP-01-0124-FEDER-007135).PGthanksFCTforpluri-annualfundingtoResearchUnit CIQUP.LMacknowledgesFCTforfinancingthePortugueseNMR network(ref.REDE/1517/RMN/2005)and theproject PTDC/QUI-QUI/100998/2008,which allowedtheuseof theNMR facilities, efficiently.TheauthorsarealsoindebtedtoDrs.CristinaBarrias (INEB)andEliandredeOliveira(ParcCientíficdelaUniversitatde Barcelona,Spain)forcollaborationinmolecularweightanalysisby HP-SECandMALDI-TOFMS,respectively.
References
Amaral,I.F.,Granja,P.,&Barbosa,M.A.(2005).Chemicalmodificationofchitosan byphosphorylation:anXPS,FT-IRandSEMstudy.JournalofBiomaterialsScience, PolymerEdition,16,1575–1593.
Amaral,I.F.,Lamghari,M.,Sousa,S.R.,Sampaio,P.,&Barbosa,M.A.(2005).Ratbone marrowstromalcellosteogenicdifferentiationandfibronectinadsorptionon chitosanmembranes:theeffectofthedegreeofacetylation.JournalofBiomedical MaterialsResearch,75A,387–397.
Anitha,A.,Deepa,N.,Chennazhi,K.P.,Nair,S.V.,Tamura,H.,&Jayakumar,R.(2011). Developmentofmuchoadhesive,thiolatedchitosannanoparticlesfor biomedi-calapplications.CarbohydratePolymers,83,66–73.
Batista,M.K.S.,Gallemí,M.,Adeva,A.,Gomes,C.A.R.,&Gomes,P.(2009).Facile regioselectivesynthesisofanovelchitosan–pexigananconjugatewithpotential interestforthetreatmentofinfectedskinlesions.SyntheticCommunications,39, 1228–1240.
Batista,M.K.S.,Pinto,L.F.,Gomes,C.A.R.,&Gomes,P.(2006).Novelhighly-soluble peptide-chitosanpolymers:synthesisandspectralcharacterization. Carbohy-dratePolymers,64,299–305.
Beamson,G.,&Briggs,D.(1992).HighResolutionXPSofOrganicPolymers:TheScientia ESCA300Database.NewYork(USA):JohnWiley&Sons.
Bennett,A.E.,Rienstra,C.M.,Auger,M.,Lakshmi,K.V.,&Griffin,R.G.(1995). Het-eronucleardecouplinginrotatingsolids.TheJournalofChemicalPhysics,103, 6951–6958.
Berger,J.,Reist,M.,Mayer,J.M.,Felt,O.,&Gurny,R.(2004).Structureandinteractions inchitosanhydrogelsformedbycomplexationoraggregationfor biomedi-calapplications.EuropeanJournalofPharmaceuticsandBiopharmaceutics,57, 35–52.
Berger,J.,Reist,M.,Mayer,J.M.,Felt,O.,Peppas,N.A.,&Gurny,R.(2004).Structure andinteractionsincovalentlyandionicallycrosslinkedchitosanhydrogelsfor biomedicalapplications.EuropeanJournalofPharmaceuticsand Biopharmaceu-tics,57,19–34.
Bhatnagar,A.,&Sillanpää,M.(2009).Applicationsofchitin-andchitosan-derivatives forthedetoxificationofwaterandwastewater:ashortreview.Advancesin ColloidandInterfaceScience,152,26–38.
Brugnerotto,J.,Lizardi,J.,Goycoolea,F.M.,Argüelles-Monal,W.,Desbrières,J.,& Rinaudo,M.(2001).Aninfraredinvestigationinrelationwithchitinandchitosan characterization.Polymer,42,3569–3580.
Cabrera,J.C.,&VanCutsem,P.(2005).Preparationofchitooligosaccharideswith degreeofpolymerizationhigherthan6byacidorenzymaticdegradationof chitosan.BiochemicalEngineeringJournal,25,165–172.
Chinchilla,R.,&Nájera,C.(2007).TheSonogashirareaction:aboomingmethodology insyntheticorganicchemistry.ChemicalReviews,107,874–922.
Chirachanchai, S., Lertworasirikul,A., &Tachaboonyakiat, W.(2001). Carbaryl insecticideconjugationontochitosanviaiodochitosanandchitosancarbonyl imidazolideprecursors.CarbohydratePolymers,46,19–27.
Crompton,K.E.,Goud,J.D.,Bellamkonda,R.V.,Gengenbach,T.R.,Finkelstein, D. I., Horne, M. K., et al. (2007). Polylysine-functionalised thermore-sponsive chitosanhydrogelforneural tissueengineering.Biomaterials, 28, 441–449.
Dai,T.,Tegos,G.P.,Burkatovskaya,M.,Castano,A.P.,&Hamblin,M.R.(2009). Chi-tosanacetatebandageasatopicalantimicrobialdressingforinfectedburns. AntimicrobialAgentsandChemotherapy,53,393–400.
Elkholy,S.S.,Khalil,K.D.,&Elsabee,M.Z.(2011).Graftingofacryloyl cyanoaceto-hydrazideontochitosan.JournalofPolymerResearch,18,459–467.
Gomes,P.,Gomes,C.A.R.,Batista,M.K.S.,Pinto,L.F.,&Silva,P.A.P.(2008).Synthesis, structuralcharacterizationandpropertiesofwater-soluble N-(␥-propanoyl-aminoacid)-chitosans.CarbohydratePolymers,71,54–65.
Holappa,J.,Hjálmarsdóttir,M.,Másson,M.,Rúnarsson,Ö.,Asplund,T.,Soininen, P.,etal.(2006).AntimicrobialactivityofchitosanN-betainates.Carbohydrate Polymers,65,114–118.
Holappa, J., Nevalainen, T., Soininen, P., Másson, M., & Järvinen, T. (2006). Synthesis of novel quaternary chitosan derivatives via N-chloroacyl-6-O-triphenylmethylchitosans.Biomacromolecules,7,407–410.
Iqbal,J.,Sarti,F.,Perera,G.,&Bernkop-Schnürch,A.(2011).Developmentand invivoevaluationofanoraldrugdeliverysystemforpaclitaxel.Biomaterials, 32,170–175.
Kolb,H.C.,Finn,M.G.,&Sharpless,K.B.(2001).ClickChemistry:diversechemical functionfromafewgoodreactions.AngewandteChemieInternationalEdition,40, 2004–2021.
Krishnakumar,V.,&JohnXavier,R.(2004).FTRamanandFT-IRspectralstudiesof 3-mercapto-1,2,4-triazole.SpectrochimicaActa–PartA:MolecularandBiomolecular Spectroscopy,60,709–714.
Kulbokaite,R.,Ciuta,G.,Netopilik,M.,&Makuska,R.(2009).N-PEGylationofchitosan viaclickreactions.ReactiveandFunctionalPolymers.,69,771–778.
Kurita,K.,Ikeda,H.,Yoshida,Y.,Shimojoh,M.,&Harata,M.(2002).Chemoselective protectionoftheaminogroupsofchitosanbycontrolledphthaloylation:facile preparationofaprecursorusefulforchemicalmodifications.Biomacromolecules, 3,1–4.
Kurita,K.,Yoshida,Y.,&Umemura, T.(2010).Finelyselectiveprotectionsand deprotectionsofmultifunctionalchitinandchitosantosynthesizekey inter-mediatesforregioselectivechemicalmodifications.CarbohydratePolymers,81, 434–440.
Lahann,J.(2009).ClickChemistryforBiotechnologyandMaterialsScience.Chichester (UK):JohnWiley&Sons.
Lallana,E.,Fernández-Megía,E.,&Riguera,R.(2009).Surpassingtheuseofcopper intheclickfunctionalizationofpolymericnanostructures:astrainpromoted approach.JournaloftheAmericanChemicalSociety.,131,5748–5750.
Li,X.,Yuan,W.,Gu,S.,&Re,J.(2010).Synthesisandself-assemblyoftunable thermosensitivechitosanamphiphiliccopolymersbyclickchemistry.Materials Letters,64,2663–2666.
Makuˇska,R.,&Gorochovceva,N.(2006).Regioselectivegraftingofpoly(ethylene glycol)ontochitosanthroughC-6positionofglucosamineunits.Carbohydrate Polymers,64,319–327.
Meldal,M.,&Tomøe,C.W.(2008).Cu-catalyzedazide-alkynecycloaddition. Chem-icalReviews,108,2952–3015.
Molnár,E.,Barbu,E.,Lien,C.-F.,Górecki,D.C.,&Tsibouklis,J.(2010).Towarddrug deliveryintothebrain:synthesis,characterization,andpreliminaryinvitro
assessmentofalkylglyceryl-functionalizedchitosannanoparticles. Biomacro-molecules,11,2880–2889.
Montalbetti,C.A.G.N.,&Falque,V.(2005).Amidebondformationandpeptide coupling.Tetrahedron,61,10827–10852.
Muzzarelli,R.A.A.(1988).Carboxymethylatedchitinsandchitosans.Carbohydrate Polymers,8,1–21.
Muzzarelli,R.A.A.(2009).Chitinsandchitosansfortherepairofwoundedskin, nerve,cartilageandbone.CarbohydratePolymers,76,167–182.
Muzarelli,R.A.A.(2011).Chitosancompositeswithinorganics,morphogenetic proteinsandstemcells,forboneregeneration.CarbohydratePolymers, 83, 1433–1445.
Oh,J.K.,Lee,D.I.,&Park,J.M.(2009).Biopolymer-basedmicrogels/nanogelsfor drugdeliveryapplications.ProgressinPolymerScience,34,1261–1282. Önal,S.,&Telefoncu,A.(2003).ComparisonofchitinandamberliteIRA-938for
a-galactosidaseimmobilization.ArtificalCells,BloodSubstitutes,andBiotechnology, 31,19–33.
Özbas¸-Turan,S., Akbu˘ga,J.,&Sezer, A.D.(2010).Topicalapplication of anti-senseoligonucleotide-loadedchitosannanoparticlestorats.Oligonucleotides, 20,147–153.
Park,S.O.,Kwon,O.S.,Choi,C.W.,&Kim,C.-J.(2000).Glucosamine-mediated detox-ificationofp-benzoquinoneanditsremovalbychitosan.BiotechnologyLetters, 22,21–24.
Prabuh,K.,Sundaramoorthi,V.,Selvaraju,C.K.,&Tamilselvi,K.K.N.(2011).Topical immunizationandtheuseofchitosanascarrier.InternationalJournalofResearch inAyurvedaandPharmacy,2,90–94.
Shen,X.,Ji,Y.,Yang,Q.,&Zheng,X.(2007).Preparation,characterization,and rhe-ologicalpropertiesofdibutyrylchitin.JournalofMacromolecularScienceB,49, 250–258.
Socrates,G.(2001).InfraredandRamanCharacteristicGroupFrequenciesTablesand Charts(3rdedition).NewYork(USA):JohnWiley&Sons,Ltd.
Rúnarsson,Ö.,Holappa,J.,Jónsdóttir,S.,Steinsson,H.,&Másson,M.(2008). N-selective‘one-pot’synthesisofhighlysubstitutedtrimethylchitosan(TMC). CarbohydratePolymers,74,740–744.
Rúnarsson,Ö.,Holappa,J.,Malainer,C.,Steinsson,H.,Hjálmarsdóttir,M.,Nevalainen, T.,etal.(2010).AntibacterialactivityofN-quaternarychitosanderivatives: syn-thesis,characterizationandstructureactivityrelationship(SAR)investigations. EuropeanPolymerJournal,46,1251–1267.
Wan,A.,Gao,Q.,&Li,H.(2010).Effectsofmolecularweightanddegreeof acetyla-tiononthereleaseofnitricoxidefromchitosan-nitricoxideadducts.Journalof AppliedPolymerScience,117,2183–2188.
Yuan,W., Li, X.,Gu, S., Cao,A., & Ren,J. (2011).Amphiphilic chitosan graft copolymerviacombinationofROP,ATRPandclickchemistry:synthesis, self-assembly,thermosensitivity,fluorescence,andcontrolleddrugrelease.Polymer, 52,658–666.
Yuan,W.,Zhao,Z.,Gu,S.,&Ren,J.(2010).Synthesis,characterization,andproperties ofamphiphilicchitosancopolymerswithmixedsidechainsbyclickchemistry. JournalofPolymerScienceA,48,3476–3486.
Yuan,W.,Zhao,Z.,Gu,S.,Ren,T.,&Ren,J.(2011).Synthesisandself-assemblyof pH-responsivechitosangraftcopolymerbythecombinationofatomtransfer radicalpolymerizationandclickchemistry.MaterialsLetters,65,793–796. Zhang,J.,Li,C.,Xue,Z.-Y.,Cheng,H.-W.,Huang,F.-W.,Zhuo,R.-X.,etal.(2011).
Fab-ricationoflactobionic-loadedchitosanmicrocapsulesaspotentialdrugcarriers targetingtheliver.ActaBiomaterialia,7,1665–1673.