Conformational Flexibility of Chitosan: A Molecular ModelingStudy

Søren Skovstrup,† Signe Grann Hansen, Troels Skrydstrup, and Birgit Schiøtt*

Centre for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Centre (iNANO),Department of Chemistry, Aarhus University, 8000 Aarhus C, Denmark

Received July 2, 2010; Revised Manuscript Received September 24, 2010

Chitin and chitosan are naturally occurring polysaccharides composed of �-(1,4) linked N-acetylglucosamineunits (GlcNAc) and, for chitosan, also glucosamine units (GlcN). In recent years, chitosan has attracted muchinterest because of its special physical and chemical properties related to drug delivery, wound healing, andtissue engineering. However, limited structural knowledge is available for chitosan because of its composition ofthe randomly mixed building blocks, GlcNAc and GlcN. In this study, we present exhaustive combined moleculardynamics and Monte Carlo simulations that unravel the conformational flexibility of the �-(1,4)-linkage in di-,tri-, and tetrasaccharide models of chitin and chitosan. The most flexible disaccharide unit was found to beGlcN-GlcNAc, populating four conformations. Furthermore, it is found that the conformational freedom of aglycosidic bond is independent of the flexibility of the neighboring linkages along the oligomer. The results areinterpreted with respect to hydrogen bond formation and implications for polymer properties.

Introduction

Chitin and chitosan are biopolymers found in various plantsand animals, mainly crustaceans and fungi.1 Chitin is ahomopolymer composed of repeating N-acetylglucosamine(GlcNAc) units linked by �-(1,4) glycosidic bonds, whereaschitosans are polymers of randomly distributed GlcNAc andglucosamine (GlcN) units linked by �-(1,4) glycosidic bonds.(See Figure 1a.) The proportion of GlcNAc units is describedby the degree of acetylation (DA), thus, chitin has a DA of100%. Chitosan has recently shown very promising propertiesfor a range of applications, including biofabrication,2 deliverysystems for macromolecules,3 wound dressing,4 and tissueengineering5 as well as a number of applications in the foodindustry,6 including antimicrobial activity, food additive, andwater purification.

The fully deacetylated chitosan polymer (DA ) 0%) existsin two crystal forms: hydrous and anhydrous.7 Moreover,chitosan forms salts with a range of organic and inorganic acids.8

Most of the known chitosan structures possess a two-fold helix-like structure7,8b,c of the sugar chains with repeating periods ofbetween two and eight sugar units.8-10 However, the most recentchitosan crystal structure revealed a five-fold helical symmetryalong the polymer axis.8d The chains can be linked either directlythrough hydrogen bonds, via structural water molecules, or, asin anhydrous chitosan, through hydrophobic interactions,9 givingrise to sheet-like structures of the crystals.11 For chitosan witha DA different from 0% and for chitosan in solution, much lessdirect structural evidence is found. Structural knowledge hasbeen gathered from other means, and it is known that the DAaffects the structure and the overall conformational dynamicsof the polysaccharide chain.12-16 The available data are brieflyreviewed in the following paragraph.

The large number of primary amines gives chitosan uniquechemical properties. At low pH, chitosan is a water-soluble,

cationic species because of its protonated amines, whereaschitosan is uncharged and insoluble at high pH. Typically, the

* To whom correspondence should be addressed. E-mail: birgit@chem.au.dk. Fax: +45 8619 6199. Tel: +45 8942 3953.

† Current address: Department of Medicinal Chemistry, Faculty ofPharmaceutical Sciences, University of Copenhagen, Denmark.

Figure 1. Dihedral angles and nomenclature used in this study. (a)Schematic representation and shorthand notation of a chitosanfragment; (b) φ, (H1-C1-O4-1-C4-1); (c) ψ, (C1-O4-1-C4-1-H4-1); (d) �, (H1-C1-C4-1-H4-1); (e) ω, (O5-C5-C6-O6);(f) γ, (C1-C2-N-C(dO)), (g) atom labels; and (h) nomenclature ofa oligosaccharide. Residues are labeled i +n toward the nonreducingend and i -n toward the reducing end. The glycosidic bond betweenresidue i n and i n-1 is denoted� n-1. Rings are referred to as A, B, C,and D starting from the nonreducing end.

Biomacromolecules 2010, 11, 3196–32073196

10.1021/bm100736w 2010 American Chemical SocietyPublished on Web 10/20/2010

border between the soluble and insoluble states lies betweenpH 6.0 and 6.5, depending on the DA and the ionic strength ofthe media. The pKa of chitosans in aqueous solutions rangesfrom 6.3 to 7.3 (for 5.2 and 89% DAs, respectively).12a Thesolubility in aqueous media decreases as the DA increases,thereby reducing the amount of cationic sites.12b The electrostaticbehavior of chitosans is divided into three domains: (1) chitosanswith DA < 20% behave as polyelectrolytes and are quite solublein aqueous media; (2) chitosans with 20e DAe 50% constitutea transition domain; and (3) chitosans with DA > 50% exhibitincreasing hydrophobic character due to the lowered numberof cationic sites.12b Similarly, chitosan chains with DA < 25%are quite flexible and independent of the exact DA, whereasthe chains become gradually stiffer for chitosans with DAs of25-50%. For DAs above 50%, the stiffness of the chainincreases, and hence the persistence length becomes more orless constant.13,14 The accessible conformational space of theglycosidic linkage has been reported to be affected by the natureof the substituent at C2 on the nonreducing saccharide unit.15

The distribution of N-acetyl groups along the chain has greatinfluence on the stiffness. Brugnerotto et al.13a have reportedmodeling studies of chitosans with different substitution patterns(GlcN or GlcNAc) by using the four possible disaccharides asthe cores for modeling long chains using coarse techniques.Persistence lengths of 90 Å for 0% DA chitosan and of 125 Åfor chitin were found. For 50% DA chitosan, they found arandom pattern with a persistence length of 115 Å; the alternateABAB pattern had a persistence length of 135 Å, whereas anA2B2 pattern had a persistence length of 97 Å. Furthermore,grouping (from 2 to 20 consecutive units) showed no variationin the predicted persistence length for 50% DA.13a

Chitosan with 0% DA possesses a higher degree of elongationand tensile strength than chitin.16 Chitin favors a more extendedconformation and is a little stiffer than chitosan.13a A recentall-atom MD study of 0% DA chitosan and chitin decamersfocuses on their aqueous structures and how explicit watermolecules can interrupt the important intrachain hydrogen bondbetween OH3 and O5+1. (For notation, see Figure 1.)13c

As indicated above, a number of studies on the physicalproperties of chitosan have been reported. Other studies aredealing with, for example, the tensile strength and elongation,17

the conformation of chitosan docked into a protein,18 theconformation of chitobiose and chitosan chains (using coarsetechniques),15 and the conformation of polysaccharides (usingcoarse techniques).13a However, to the best of our knowledge,there has so far been no systematic all-atom modeling study ofthe conformational flexibility of all combinations of tri- andtetrasaccharides composed of �-(1,4)-linked GlcNAc or GlcNunits including chitosan models with varying DAs, includinganalysis of chitosan with DAs differing from 0%.

The aim of this study is thus to achieve a greater understand-ing of the structural features of chitosan. Through a detailedsystematic evaluation of a total of 31 di-, tri-, and tetrasaccha-rides of �-(1,4)-linked GlcNAc and GlcN units, the flexibilityof chitosan as a function of the acetylation pattern is elucidatedby following the conformational behavior of the five dihedralangles shown in Figure 1. We will focus on the consequencesof the observed dynamics of a glycosidic bond as a result ofN-acetylation of either the reducing or the nonreducing glu-cosamine moiety.

Experimental Section

All calculations were performed using MacroModel (version 9.0)19

and the AMBER* force field with the Senderowitz-Still all-atom

pyranose parameters20 similarly to the protocol developed by Bernardiet al.21 for performing conformational analysis of oligosaccharides. Theoligosaccharides were built in Maestro22 using the fragment libraries(7 disaccharides, 8 trisaccharides, and 16 tetrasaccharides) and modifiedmanually to the desired structures. Water solvation was simulatedimplicitly by MacroModel’s generalized Born continuum solvent model(GB/SA).23 Charges were taken from the force field, and no truncationof the nonbonded interactions was applied. Examination of the structuresand the dynamics of each saccharide was performed in four steps. First,a Monte Carlo/energy minimization (MC/EM) conformational searchwas carried out to locate at least two distinct local minima structuresfor each oligosaccharide studied. The conformational searches werecarried out using 15 000 steps of the MCMM torsional samplingprocedure and applying an energy window of 20 kJ/mol. No ring-opening/closing was allowed, and no endocyclic bonds or hydrogenbonds were used as explicit variables during the Monte Carlo search.All other bonds were sampled between 60 and 180°. The foundstructures were minimized through the PRCG method. Second, adynamic simulation was performed following the MC/SD24 protocolprovided in MacroModel using the GB/SA implicit water model. Thesame degrees of freedom were applied as those in the conformationalsearch, and ring-openings were not allowed. All torsion angles wereallowed to rotate at each MC step, and at least one should change. Thesimulations were run at 300 K, with a dynamic time step of 1.0 fs.Two separate runs were performed for each saccharide, with the startingpoint being the two distinct minimum conformations found in the initialMC/EM stages. Sampling was done for 25 ns for each disaccharide,35 ns for each trisaccharide, and 50 ns for each tetrasaccharide. Eachaccepted MC step was followed by an SD step. Structures were savedevery 5 ps for later evaluation. Convergence was checked by comparingthe two MC/SD runs with regard to the relative energy as a functionof geometrical conformations (in particular, φ, ψ, ω, and � angles).Subsequently, in the third stage of the simulation protocol, the savedsnapshots from the MC/SD dynamics simulations were minimized bythe PRCG protocol. Convergence was determined by gradients with athreshold of 0.05 kJ/mol ·Å. Finally, the minimized structures wereclustered according to the angle distribution and binned in 10° intervalsto form a general overview of the distinct conformations.

A large number of hydrogen bonds were measured and evaluated.A strong hydrogen bond was here defined as a donor-acceptorinteraction where the distance between the acceptor heteroatom andthe hydrogen atom is below 2.1 Å, the donor angle, measured asD-H · · ·A, is larger than 120°, and the acceptor angle, H · · ·A-C, isabove 90°. A weak hydrogen bond was defined as a donor-acceptorrelationship with a distance between the acceptor heteroatom and donorhydrogen between 2.1 and 3.1 Å and with a donor angle >120°.

Results and Discussion

Disaccharides. Initially, seven disaccharides were modeledand studied to decide which structural motifs to include in thestudy of the longer oligomers, and, in particular, to decide howto model the termini of the oligomers. Figure 2 presents themodeled disaccharides along with short-hand notations for thetri- and tetrasaccharides included in the study.

Conformational Analysis. The influence of a methyl groupinstead of a free hydroxyl group at the termini on the con-formational freedom of the glycoside bond is studied in 0%DA chitosan models 1, 2, and 3. Acetylation of both aminogroups gives a chitin dimer, and the conformational dynamicsof the glycosidic bond in chitin is likewise examined withoutand with protection of the termini in 4 and 5, respectively.Finally, models of mixed chitosan oligomers, with DA ) 50%are studied. The effect of having acetyl groups at either thereducing (6) or nonreducing (7) side of the glycoside bond isstudied without protection of the termini.

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The computed average values of the glycosidic bond anglesφ, ψ, and � of the disaccharides are listed in Table S1 in theSupporting Information. All modeled structures have verysimilar average properties of the glycosidic linkage; however,small, yet important, differences are found. The averageglycosidic bond angle, �, shows some variation, 31° in 5 (chitin)to 42° in 1-3 (0% DA chitosan), due to the observation thatthe glycosidic linkage can exist in four conformations with �being approximately -90, -30, 40, and 170°. The conformationwith � ≈ 40° is the most common conformation (as will belater discussed). ψ contributes the most to the differencesobserved in the averages of � because of the exoanomeric effectdamping the fluctuation of φ.25 Structures 1, 2, and 3 havealmost identical glycosidic bond angles considering both theaveraged values and the (φ,ψ)-distribution plots in Figure 3.They are all GlcN-GlcN disaccharides modeling 0% DAchitosan, only differing in the protection of O4+1 and O1. Allthree disaccharides essentially exist in only one conformer with� ≈ 40°. (Frequency plots are provided in Figure S1 of theSupporting Information.) The three structures hereby imply thatprotection of neither the O4+1 nor the O1 hydroxyl group hasa notable impact on the conformational properties of the centralglycosidic linkage.

The conformational freedom of the glycosides is moreprevalent when either one or both of the amines are acetylated,and the most dynamic glycosidic bond is found in structure 5,

MeO-GlcNAc-GlcNAc-OMe, which shows the broadestdistribution in the glycosidic bond angle, �, and thereby hasthe highest population of conformers with � different from 40°,resulting in the lowest average value of �. Please see plots inFigure S1 of the Supporting Information. In structure 6, whichmodels the 50% DA chitosan fragment GlcN-GlcNAc, fourpossible conformations of � are observed, although those with� * 40° are observed in <5% of the total structural population.The overall effect of a methoxy group rather than a hydroxygroup at the terminal O4+1 and O1 is evaluated through thetwo chitin models, 4 and 5. For both models, two conformationsare found. The relative populations are slightly influenced bythe protection groups, and hence the conformations with � *40° are slightly more frequently sampled in the methoxyprotected model, 5, than in the unprotected model, 4. In thiscase, an effect from the protection groups on the populationsof � is observed, although not large. Therefore, to mimic thestructure of an oligo- or polysaccharide, it was decided to model,in the following, tri- and tetrasaccharides with the nonreducingend C4 hydroxyl group and the reducing end C1 (the anomeric)hydroxyl group as methoxy-protected saccharides.

Hydrogen Bonds. It is generally recognized that the C3hydroxyl group of ring B hydrogen bonds to O5 of ring A(nonreducing end) in the two-fold helix-like structures.8b Indeed,the OH3 · · ·O5+1 hydrogen bond is an important and generalmotif in structures of hydrous, anhydrous, as well as salts ofchitosan.7 The existence of the OH3 · · ·O5+1 hydrogen bond wasevaluated for all disaccharides, and particularly the GlcNAc-GlcNAc and GlcN-GlcNAc saccharides, structures 4, 5, and

Figure 2. (a) Modeled disaccharides and short-hand notation for (b)trisaccharides and (c) tetrasaccharides.

Figure 3. Distribution plots of (φ, ψ) for structures 1, 2, and 3.

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6, showed interesting features in the population of the hydrogenbond (data provided in Table S2 of the Supporting Information).Structures 1, 2, 3, and 7, which have GlcN at the reducing end,show strong OH3 · · ·O5+1 hydrogen bonds in >60% of thesimulated structures, and after energy minimization, in practi-cally all structures, indicating that the structures are located inthe same potential energy minimum. Indeed, the (φ, ψ)distribution plots in Figure 3 and the frequency distribution plotsin Figure S1 of the Supporting Information support the pictureof a general overall “single-minimum conformation”. With aGlcNAc unit in the reducing end, the frequency of theOH3 · · ·O5+1 hydrogen bond drops to as low as 41% and, afterminimization in the MeO-GlcNAc-GlcNAc-OMe saccharide,5, 66%. This finding indicates the presence of at least oneadditional conformation of the glycosidic linkage, which is alsoseen in the frequency distribution plots of 4, 5, and 6 (Figure 4and Supporting Information, Figure S1).

Among the nonminimized structures of model 7, with a GlcNunit at the reducing end, a small population with � ≈ -30° isfound (Figure S1 of the Supporting Information). However, theseall minimize to the � ≈ 40° conformation, indicating a verylow energy barrier between the two conformations in 7. Thisbehavior is not observed for structures with GlcNAc at thereducing end, 4-6, which, after minimization, contain asignificant population with � ≈ -30°, as seen in the (φ, ψ)distribution plots of the energy minimized structures resultingfrom the MC/SD trajectories depicted in Figure 4. Clearly, 4-6populates more than one conformation, whereas 1-3 and 7 aremainly found in only one conformation.

Dynamics of the C5 Hydroxymethyl Group. The orientationof the C6 hydroxyl is measured through the ω dihedral angle.Three conformations are possible corresponding to the ( gaucheand the anti conformations, traditionally referred to as gg, gt,and tg, as illustrated in Figure 5a. The distribution of the threeconformations of the C6 hydroxyl groups were measured forω+1 and ω (data in Table S3 of the Supporting Information).The distribution of the conformations of ω+1 was found to beclearly different from the distribution of ω. For ω+1, the gtconformation is the major conformation in all modeled di-saccharides.

The preference for the gt conformation of ω+1 is a result ofthe gauche effect,26 but steric effects and the possibility offorming hydrogen bonds also play a role. The gauche effectorients the two electronegative atoms, O5+1 and O6+1, gaucheto each other because of a presumed σC-H-σ*C-O orbitalinteraction. In addition, the hydroxyl group in the gt conforma-tion is able to form a hydrogen bond to O3, provided that theOH3 · · ·O5+1 hydrogen bond is formed, which orients O3correctly. Therefore, the OH6+1 · · ·O3 hydrogen bond is present,although weak, 30-40% of the time (46-66% after structureminimization). In the gg conformation, the C6 hydroxyl groupis placed between O5 and C4 and is therefore more affected bysteric strain than in the gt and tg position and thus is lessfavorable.

The distribution of ω was found to be very different fromthat of ω+1. For ω, the tg conformation was significantly morepopulated in the models that have a primary amine, GlcN, atthe nonreducing ring, models 1, 2, 3, and 6. The reason is thepossibility of forming a hydrogen bond between the C6 hydroxylgroup and the primary amine at the nonreducing end. (This isshown in Figure 6a for 1 and demonstrated below for trisac-charides.)

The GlcN-GlcN disaccharides 1, 2, and 3 differ in thefunctionality at O4+1 and O1, with 3 resembling a disaccharidefragment of a polysaccharide; that is, the methoxy groups mimiclinking to the neighboring glucosamine units along the polymer.The tg population of ω+1 is 15 and 17% in 1 and 2, respectively,whereas it drops to a practically nonexistent 2% in 3. Obviously,the inclusion of a methoxy group at O4+1 disrupts the possibilityfor OH4+1 to hydrogen bond to O6+1. The hydrogen bondingnetwork favoring the ω+1 tg conformation is shown in Figure6a for 1. In contrast, the C4+1 methoxy group does not havethe same influence on the population of the ω+1 tg conformationin the GlcNAc-GlcNAc models 4 and 5. This is due to thering A acetamide attracting the hydroxyl groups throughcooperative hydrogen bonding in a counter-clockwise manner,as shown in Figure 6b for 4.

Dynamics of the Acetamide Group. Similar to the C6-hydroxyl group, the acetamide group can sample differentconformations through the dihedral angle γ. Because of theplanarity of the amide group, the data (Table S4 and S5 of the

Figure 4. Conformational dynamics of the glycosidic bond throughthe (φ, ψ)-distribution plots of chitin and chitosan disaccharides withvarious DAs, models 1-7. Plots are based on minimized structures.The four major conformations of � are included.

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Supporting Information) revealed that the tg conformation isfurther split into two distinct, yet closely spaced, conformations,denoted tg1 and tg2 (Figure 6c). It is found that the orientationof the acetyl group is neatly tied to the possibilities of forminghydrogen bonds. The populations of the four conformations ofγ+1 in 4 and 7 are virtually the same, with 15% in the ggconformation, 10% in the gt, 0% in tg1, and 75% in tg2. Thedominating conformation is tg2 because of the formation of astrong hydrogen bond from the C3+1 hydroxyl group, as depictedin Figure 6b. Model 5 differs slightly from 4 and 7 because ofthe O4+1 and C1 methoxy groups. Even though compound 5 isable to form a strong hydrogen bond between OH3+1 and theacetamide, the population of the γ+1 tg2 conformation is ∼15%lower than tg2 in 4 and 7. The reason for this difference in thepopulation of the γ+1 tg2 conformation is the breakage of thecooperative hydrogen bonding network depicted in Figure 6b

for 4, which is not possible in 5. In 4 and 7, OH4+1 hydrogenbonds to O3+1, thereby orienting OH3+1 correctly to hydrogenbond to the acetyl group.

The acetamide of ring A is able to hydrogen bond to the C6hydroxymethyl of ring B. In general, these inter-ring NH+1 · · ·O6hydrogen bonds are formed in the presence of a hydrogen bondbetween the C3+1 hydroxyl and the acetamide, OH3+1 · · ·O(dC)+1. Whereas the presence of the C4+1 methoxy groupaffects the distribution of the γ+1 angle moderately, that is,moving 15% of the tg2 conformers in 4 to the gg and gtconformers in 5, the distribution of the γ angle is much moredramatically affected by the presence of the C1 methoxy group.In 4 and 6, the large population of the gt conformation of γ(ca. 85%) is a consequence of a hydrogen bonding interactionbetween OH1 and the acetamide, OH1 · · ·O(dC), as shown inFigure 6b. In 5, C1 is protected with a methoxy group, and

Figure 5. (a) Molecular structures and Newman projections viewedalong the C6-C5 bond direction showing the three distinct conforma-tions of the ω dihedral of the C6 hydroxyl group. The first characterin the conformation refers to the position of the OH group relativelyto O5 and the last character refers to the position of OH relatively toC4. (b) Molecular models and Newman projections showing the fourconformations of the γ angle viewed along the N-C2 bond positioningthe acetamide group relatively to the sugar ring. The first characterdescribes the position of the acetyl relatively to C1, and the secondcharacter describes the position of the acetyl relatively to C3. Forclarity, only polar hydrogens are included in the molecular figures.

Figure 6. (a) In uncapped, 0% DA chitosan, models 1 and 2, ahydrogen bond is present between OH4+1 and O6+1, giving rise toan increased population of the ω+1 tg conformation. (b) The acetamideof ring A in 4 interacting with the hydroxy groups of C3+1 and C4+1

resulting in the γ+1 angle taking the tg2 conformation. Notice that theγ angle is shown in the gt conformation in which a hydrogen bond isformed to the unprotected C1 hydroxyl group. Removing this hydrogenbonding option (by the C1 protection group) results in a dramatic dropin the gt population of γ (from 87% in 4 to 20% in 5). (c) In chitin, 5,the population of the two tg conformations (overlaid in the display) ofγ dominates because of OH3 being able to hydrogen bond to theacetamide oxygen. Thereby, the otherwise persistent OH3 · · ·O5+1

hydrogen bonding interaction is disrupted, which ultimately results ina more flexible glycosidic linkage. Note that the population of thehydrogen bond between OH3 and the adjacent acetamide (shown inthe model with green carbons and a light-green OH3 hydrogen atom)exactly equals the population of tg2 conformers of γ.

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hydrogen bonding between OH1 and the acetamide is no longerpossible, resulting in a drop in the population of the gtconformation of γ to the expected level of below one-third. In5, the tg conformation of γ would be expected to be populatedby circa one-third of the structures in the absence of any stericor hydrogen bonding interactions. However, the ability to forma hydrogen bond between the acetamide and OH3 increases thetg population and rotates some of the existing tg conformersfrom the tg1 to the tg2 position. The hydrogen bond “switching”of OH3 emerges when the frequency of the hydrogen bondinginteraction OH3 · · ·O5+1 is compared with the frequency of theinteraction OH3 · · ·O(dC). Combined, these two interactionsare found in 91% of all minimized structures. Therefore, OH3is hydrogen bonding to either the adjacent acetamide (green inFigure 6c) or O5 of the adjacent sugar ring in the nonreducingdirection (gray in Figure 6c). Note that the tg2 population of γprecisely accounts for the population of hydrogen bondinginteractions with OH3. When comparing 4 and 5, the presenceof the C1 methoxy group (in 5) seems to result in a higherpopulation of the tg2 conformation of γ and thus a lowerpopulation of the OH3 · · ·O5+1 hydrogen bonding interaction.Ultimately, this leads to a destabilization of (and therefore alower population of) the glycosidic linkage conformation at �≈ 40°.

Trisaccharides. Eight trisaccharides (Figure 2b and FigureS2 of the Supporting Information), all bearing a methoxy groupat the C4 position of the nonreducing end and at the anomericcenter of the reducing end, were modeled. For the disaccharidesdiscussed above, the conformational flexibility of the centralglycosidic linkage was only moderately affected by thesefunctionalizations. Therefore, we decided to continue modelingthe methoxylated sugars only, thereby avoiding artifacts in thehydrogen bonding pattern, as described above for the unpro-tected disaccharides. Structure 8 represents the trisaccharidemodel of 0% DA chitosan. The position of the N-acetyl groupis systematically varied in models 9-11 of 33% DA chitosanand in models 12-14 of 67% DA chitosan. The last model,15, mimics chitin being fully N-acetylated.

Glycosidic Linkage. When measuring bond angles for thesaved snapshots from the MC/SD simulations of the modeledtrisaccharides 8-15, it was found that the average glycosidicangles resemble those of the disaccharides. All data are providedin Table S6 of the Supporting Information.

The trends are relatively clear; the presence of an acetamidegroup at the sugar ring at the reducing side of the glycosidiclinkage (� as well as �-1) decreases the average value of thedihedral angle with 5-10° from approximately 42 to 31-37°.When the acetamide is placed at the sugar ring at the nonre-ducing side of a glycosidic linkage, the average of the glycosidebond angle is lowered by only 3 to 4° relative to fullydeacetylated chitosan, model 8. In Figure 7, plots of the twoglycosidic bond angles are shown as (�;�-1) distributions forfour of the trisaccharides 8, 9, 11, and 14, which emphasizesthe trends found. The remaining plots of the glycosidic bondangles can be found in Figure S3 of the Supporting Information.The observed trend for the average values measured can berationalized by viewing these conformational plots. Starting with0% DA chitosan, 8, only one conformation is found for bothglycosidic bond angles, and the distribution is almost sym-metrical around � ) �-1 in the plot. In 8, 98% of the sampledconformers have � ∈ [0°; 75°]; likewise, 98% have �-1 ∈ [0°;75°]. The 33% DA chitosan models 9-11 reveal that theintroduction of a GlcNAc unit practically only influences theglycosidic bond toward the nonreducing site of the GlcNAc unit.

Hence 9, with a GlcNAc in the nonreducing end, shows a slightbroadening of � (toward a conformation of � ≈ -30°) thoughmaintaining 95% of the snapshots within � ∈ [0°; 75°] and 97%within �-1 ∈ [0°; 75°]. This observation implies that theflexibility of a GlcNAc-GlcN glycosidic bond is close toidentical to the flexibility of the GlcN-GlcN linkage. This isnot true for a GlcN-GlcNAc linkage, as revealed in model 10,where the central sugar unit is GlcNAc, and further confirmedin 11, where the GlcNAc unit is positioned at the reducing end.Structures 10 and 11 reveal three new conformations of theGlcN-GlcNAC glycosidic linkage centered around � ≈ -90,

Figure 7. (�; �-1) distribution plots of the glycosidic bond angles ofthe four trisaccharides 8, 9, 11, and 14. (�; �-1) distribution plots forall of the modeled trisaccharides are provided in Figure S3 of theSupporting Information.

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-30, and 170°. In 10, these are populated with 8, 5, and 6%,respectively, whereas the other glycosidic linkage, �-1, is onlyslightly broadened, as was similarly observed for the GlcNAc-GlcN linkage in 9. In 11, the GlcN-GlcNAc linkage ispopulated with 7, 5, 80, and 7% for �-1 ≈ -90, 30, 40, and170°, respectively, whereas �, being a GlcN-GlcN linkage, isunaffected, and similarly distributed as the glycosidic bonds in8. Overall, it can be concluded that the GlcN-GlcN and theGlcNAc-GlcN linkages both sample virtually only one con-formation, whereas the GlcN-GlcNAc linkage is more flexibleand samples four conformations of the glycosidic bond.

In the case of two adjacent GlcNAc units, as found in 67%DA chitosans 12 and 14, the glycosidic bond angle betweenthe two GlcNAc units is affected by the appearance of bothGlcNAc units. Comparing the distribution in � and �-1 ofstructures 9 and 12 it is apparent that the second acetyl groupin 12 induces some flexibility into the GlcNAc-GlcNAclinkage. More specifically, � is practically found in twoconformations, centered at -30 and 40°, with relative popula-tions of 9 and 89%. �-1 is still restricted to the singleconformation observed for a GlcNAc-GlcN linkage describedabove. Moving the second GlcNAc unit to the reducing end(trisaccharide 13) results in a conformational picture in ac-cordance with the previous observations; � is practicallyrestricted to a single conformation, which is a signature of theGlcNAc-GlcN linkage, whereas �-1 is very flexible, showingfour conformations, as seen for all GlcN-GlcNAc linkages sofar. Finally, in model 14, the two acetylated sugar rings areneighbors at the reducing end, resulting in � being very flexible,as seen above for the other GlcN-GlcNAc linkages, and a lessflexible �-1 displaying two conformations, �-1 ≈ -30 and 40°populated with 9 and 89% of the snapshots. A third lowpopulated conformation at �-1 ≈ -90° (populated with 1%)also emerges. This is in full agreement with the GlcNAc-GlcNAclinkage in 12 and also with the linkages found in the 100% DA(chitin) trisaccharide 15.

The most significant difference between the two flexible typesof glycosidic linkages, GlcN-GlcNAc and GlcNAc-GlcNAc, isthat the conformation with � ≈ 170° is only present in the former.Besides this, they sample the same conformations, yet with differentfrequencies, which can best be seen when comparing the plots of10 and 12 in Figure S3 of the Supporting Information. Morespecifically, the conformation with � ≈-90° is only rarely sampledby the GlcNAc-GlcNAc linkage. The most flexible trisaccharidemodeled is thus 14, MeO-GlcN-GlcNAc-GlcNAc-OMe, wherethe very dynamic GlcN-GlcNAc linkage is found as well as theother flexible GlcNAc-GlcNAc linkage. The full statistics are seenin Table S7 of the Supporting Information.

More Detailed Examination of the Dihedral Angles of 14.To gain more knowledge about the commonly occurringconformations, the snapshots of 14 were further studied becausethis was found to be the most flexible trisaccharide. In Figure8, the populated states of the two glycosidic linkages aredisplayed in terms of (φ; ψ) distribution plots (panel a and b)along with the (�; �-1) scatter plot (panel c), all based onminimized structures of the snapshots collected for 14. Ingeneral, conformers centered at (60°; -155°) in the (φ; ψ) graphcorrespond to � values of approximately -90°, conformers at(35°; -65°) correspond to � ≈ -30°, conformers at (50°; 0°)correspond to � ≈ 40°, and conformers at (170°; 3°) correspondto � ≈ 170°. From the (φ; ψ) graphs in Figure 8, it is evident,that φ is very restricted to the interval [25°; 60°]. In fact, theonly conformers outside that area are located in the area of theinfrequently visited conformation at φ ≈ 170°. The very

restricted fluctuation of φ is due to the exoanomeric effect; thatis, the dihedral angle O5+1-C1+1-O4-C4 is slightly smallerthan 60° because of the nO5 f σ*C1-O5 orbital overlap.27 Thedynamics of ψ is quite different from the dynamics of φ. ψfrequently visits two conformations, one conformer in the span[-15°; 5°] and the other conformer around [-70°; -40°].Additionally, a third conformer around [-157°; -152°] isoccasionally found.

The molecular structure of the conformation with (�; �-1)around (40°; 40°) is depicted in Figure 9a. This is the majorconformation in all examined trisaccharides. In 14, it ischaracterized by forming short OH3 · · ·O5+1 hydrogen bondsin 70% of the structures and OH3-1 · · ·O5 hydrogen bonds in75% of the sampled structures. A hydrogen bond between OH3and the adjacent acetamide is found in 22% of the snapshots,whereas OH3-1 hydrogen bonds to the adjacent acetamide in19% of the snapshots (not shown in the Figure). This meansthat in 92% of the sampled structures of this conformation OH3hydrogen bond to either the neighboring O5+1 or the adjacentC2 acetamide (on its own ring); for OH3-1, this is 94%.

The conformation with (�; �-1) ) (-90°; 40°) (Figure 9b) isfound in 8% of the sampled structures. It forms no hydrogenbonding interactions between the nonreducing unit and thecentral GlcNAc. Consequently, the glycosidic linkage betweenrings A and B is exclusively governed by the exoanomeric effect

Figure 8. Plots of the angle distributions of the glycosidic bonds in14 (MeO-GlcN-GlcNAc-GlcNAc-OMe). (a) (φ; ψ) graph of thebond between the nonreducing end and the central sugar unit. Theφ-ψ combinations resulting in the four observed dihedral angles of� are encircled. (b) (φ-1; ψ-1) graph of the bond between the centraland the reducing end sugar unit. (c) (�; �-1) graph of the trisaccharide.All three plots are based on the minimized structures.

3202 Biomacromolecules, Vol. 11, No. 11, 2010 Skovstrup et al.

and steric strain between the two rings, and the two sugar ringsare oriented almost perpendicularly. Because of the absence ofthe OH3 · · ·O5+1 hydrogen bond, OH3 interacts through stronghydrogen bonds with the adjacent acetamide in 80% of thestructures. The other glycosidic linkage is found in the major�-1 ≈ 40° conformation, allowing for the OH3-1 · · ·O5 hydrogenbond to form.

The conformation with (�; �-1) ) (-30°; 40°) (Figure 9c) isfound in 5% of the sampled structures. In this conformation,the OH3 · · ·O5+1 hydrogen bond is present in only 13% of thestructures (not shown in the Figure), whereas OH3 is hydrogenbonding to the adjacent acetamide in 54% of the savedsnapshots. There is no strong, persistent, attractive nonbondedinteraction between rings A and B; that is, the conformation is

largely governed by the exoanomeric effect and steric interac-tions. To reveal the difference in the OH3 · · ·O5+1 hydrogenbonding pattern in the � ≈ -30 (Figure 9c) and 40° (Figure9a) conformations, both with �-1 ≈ 40°, the relationship betweenthe OH3 and O5+1 was studied. The distance between the heteroatoms, O3 and O5+1, is 2.8 Å in both cases. For � ≈ -30°, thedonor angle (O3-H3 · · ·O5+1) is ∼140°, whereas the � ≈ 40°conformer has a 148° donor angle. The acceptor angles,C1+1-O5+1 · · ·H3 and C5+1-O5+1 · · ·H3, are 83 and 91°,respectively, in the � ≈ -30° conformer, whereas they are foundaround 103 and 144° in the � ≈ 40° conformer. Therefore,because of the equatorial configuration of the C3 hydroxy groupof ring B and the puckering of ring A, the conformers with �≈ 40° are able to form strong OH3 · · ·O5+1 hydrogen bondinginteractions between rings B and A. Likewise, the conformerswith � ≈ -30° are suffering from distinctly worse acceptorangles, resulting in the OH3 · · ·O5+1 hydrogen bonding interac-tion being characterized as weak.

The conformation with (�; �-1) ≈ (170°; 40°) (Figure 9d) isfound in 6% of the sampled structures. The stabilizing force isfound to be a persistent, strong hydrogen bonding interactionfrom the primary, cationic amine of ring A to O3 at ring B, thelatter which in 82% of the structures with this conformation isalso hydrogen bonding to the adjacent acetamide at ring B. Inaddition, in 34% of the structures with this conformation thering B acetamide is hydrogen bonding to O6-1 (compared withan overall NH · · ·O6-1 hydrogen bond population of 17% forthe total of the sampled conformations of 14).

The conformation with (�; �-1) ≈ (40°; -30°), Figure 9e, isfound in 7% of the sampled snapshots. It is obviously verysimilar to the (�; �-1) ≈ (-30°; 40°) conformation. However,the (�; �-1) ≈ (40°; -30°) conformation shows a strongOH3-1 · · ·O5 hydrogen bonding interaction in only 5% of thestructures (compared with a population of 13% for theOH3 · · ·O5+1 hydrogen bond in the (�; �-1) ≈ (-30°; 40°)conformation) (the hydrogen bond is not shown in the Figure),whereas OH3-1 is hydrogen bonding with the adjacent aceta-mide in 63% of the sampled structures of this conformer.

Influence of the Acetamide on the Glycosidic Bond. Theconformers of 14 clearly demonstrate that flexibility of a dihedrallinkage, measured through the � angle, is dependent on thenature of the surrounding sugar units. Therefore, some flexibilityis obtained when both of the saccharide units linked by theglycoside bond are N-acetyl glucosamines, whereas greaterflexibility is obtained when only the one toward the reducingsite is acetylated. To point out the difference between the twosituations further, 14, MeO-GlcN-GlcNAc-GlcNAc-OMe,is in the following compared with 8, MeO-GlcN-GlcN-GlcN-OMe, 10, MeO-GlcN-GlcNAc-GlcN-OMe, and 11,MeO-GlcN-GlcN-GlcNAc-OMe.

Comparing the rather rigid structure of the fully deacetylatedchitosan model, 8, with the 33% DA MeO-GlcN-GlcNAc-GlcN-OMe saccharide, 10, it is clear that only the glycosidebond between the nonreducing sugar rings A and B is markedlyaffected by the presence of the acetamide at ring B in 10. Theconformations and populations of this linkage are very similarto those observed for the MeO-GlcN-GlcNAc-GlcNAc-OMesaccharide, 14. (The statistics of all conformations are listed inTable S7 of the Supporting Information.) Therefore, the natureof the unit at the reducing end, ring C, seems to have noinfluence on the bond linking rings A and B. Apparently, thepresence of an acetamide at C2 affects only the nearestglycosidic bond significantly toward the nonreducing side.Similarly, in 11, where only ring C is acetylated, the glycosidic

Figure 9. (a) Major conformation of 14 with (�; �-1) around (40°; 40°).Panels b-e show four less-populated conformations of 14 with: (b)(�; �-1) ) (-90°; 40°), (c) (�; �-1) ) (-30°; 40°), (d) (�; �-1) ) (170°;40°), and (e) (�; �-1) ) (40°; -30°). The most frequently sampledhydrogen bonds in each conformation are shown as black lines, andthe populations of the conformation and the individual hydrogen bondsare noted.

Conformational Flexibility of Chitosan upon Degree of Acetylation Biomacromolecules, Vol. 11, No. 11, 2010 3203

bond between rings B and C is very flexible, whereas theglycosidic bond between rings A and B is not flexible and thusis not affected by the acetamide group at ring C. Introducing asecond acetyl glucosamine on ring B, that is, turning from 11to 14, results in a lower population of the conformation with�-1 ≈ -90°, with a computed change in population from 7%in 11 to 1% in 14. Assuming a simple Boltzmann distributionamong the conformers sampled, this corresponds to a rise inpotential energy of ∼4.3 kJ/mol for visiting this conformationof the �-1 angle. Similarly, conformers with �-1 ≈ 170°completely disappear in 14. In return, the population ofconformers with �-1 ≈ -30° is increased from 5% in 11 to 9%in 14. Apparently, the introduction of the acetamide on ring Brestricts the observed flexible nature of the glycoside bondbetween the two acetylated rings B and C to values around -30and 40°.

The reason that the conformation with �-1 ≈ -30° is morefavorable in 14 compared with that in 11 is a consequence ofthe �-1 ≈ 40° conformation being less stabilized in 14. That is,in 11, the �-1 ≈ 40° conformation is stabilized not only by thestrong OH3-1 · · ·O5 hydrogen bonding interaction but also byhydrogen bonding interactions between the primary cationicamine at ring B and the C5 hydroxymethyl of ring C (presentin 44% of all sampled structures); this is shown in Figure 10a.In 14, ring B bears not the primary cationic amine but anacetamide, resulting in the hydrogen bonding interactionbetween the ring B amine and the ring C hydroxymethyl beingmuch weaker and less-populated (by 17% of the sampledstructures) and thus not stabilizing the �-1 ≈ 40° conformationto the same extent. The loss of the �-1 ≈ 170° conformation in14 is most likely due to loss of the cooperative hydrogenbonding network extending from the amine of ring B to O3-1

onward to the adjacent acetamide on ring C, illustrated for 11in Figure 10b. The reason for the markedly lower populationof �-1 ) -90° in 14 compared with that in 11 is not obviousgiven that there are no important nonbonded, attractive interac-tions between the two rings in either 11 or 14.

Tetrasaccharides. The 16 tetrasaccharides shown in Figure2c were modeled, mimicking 0% DA chitosan (16), 25% DAchitosan (17-20), 50% DA chitosan (21-26), 75% DA chitosan(27-30), and chitin (31). The models thus include all possiblecombinations of the GlcN and GlcNAc building blocks in atetrasaccharide. The conformational analysis (details given inthe Supporting Information in Table S8) reveals that the sametrends are seen for the tetrasaccharides, as was found for thedi- and trisaccharides above. Again, it is evident, that the pre-sence of an acetyl glucosamine unit is only influencing the verynearest glycosidic bonds, primarily toward the nonreducing side.The most flexible glycosidic bond is found in the GlcN-GlcNAclinkage sampling four conformations, followed by the GlcNAc-GlcNAc structure with three possible values of �. The leastdynamic glycoside bonds are present in the GlcNAc-GlcN andGlcN-GlcN linkages sampling only one conformation with �≈ 40°. This confirms that it is the nature of the sugar to thereducing side of the glycoside bond that determines theflexibility of �, whereas the sugar ring at the nonreducing sideof the glycoside bond is less important in this respect. In otherwords, only a GlcNAc unit to the reducing side of the glycosidebond leads to flexibility (with four possible conformations in aGlcN-GlcNAc linkage), whereas a second GlcNAc unit to thenonreducing side slightly reduces this flexibility to two con-formations, with a third conformation being rarely sampled.

Persistence Lengths of Tetrasaccharides. Evidently, theflexibility of the glycosidic linkage is reflected in the persistencelength of the saccharide. The persistence length of a polymer,that is, the length for which the memory of the initial orientationof the polymer persists,28 is, in the worm-like chain model,29

defined as the overall length of the polysaccharide measured asthe sum of the projections of the length of each individual unitonto the direction defined by the first unit.30 However, becausethe longest oligomers in this study are tetramers, a directmeasurement of the end-to-end distance of the tetrasaccharidesis similarly an indication of the flexibility, but the number cannotbe directly converted to persistence length for extended polymers.

The average, minimum, and maximum end-to-end lengthsof the examined tetrasaccharides were measured and are listedin Table S9 of the Supporting Information. The maximum end-to-end length is found to be quite constant, ∼21.6 Å. The longestsaccharide chain is found in saccharide MeO-GlcN-GlcN-GlcNAc-GlcNAc-OMe, 26, which in the extended conforma-tion has glycosidic bond angles: � ) 38.3°, �-1 ) 25.8°, and�-2 ) 26.1°. The shortest end-to-end length is merely 11.9 Å,found in structure 30, MeO-GlcN-GlcNAc-GlcNAc-GlcNAc-OMe, which indeed is a tetrasaccharide made up ofthree of the most flexible linkages. This structure has glycosidicbond angles of � ) -108.7°, �-1 ) -178.6°, and �-2 ) 40.5°and is shown in Figure 11. The theoretically most flexibletetrasaccharide will be the one with the most GlcN-GlcNAc

Figure 10. Important hydrogen bonds stabilizing the specific confor-mation of the glycosidic linkage in a GlcN-GlcNAc fragment in, forexample, structure 11, identified in (a) the conformation at �-1 ≈ 40°and (b) the �-1 ≈ 170° conformation. For clarity, only rings B and Care shown, and only the distances involving the amines and aceta-mides are given.

Figure 11. Conformer of 30 with the shortest measured end-to-endlength.

3204 Biomacromolecules, Vol. 11, No. 11, 2010 Skovstrup et al.

linkages, that is, GlcN-GlcNAc-GlcN-GlcNAc. This corre-sponds to 25, which indeed is found to be the tetrasaccharidewith the shortest average end-to-end length and also containsthe conformation with the second shortest minimum length,12.1 Å.

The average end-to-end length varies by ∼0.5 Å, from 20.2to 20.7 Å. Compound 16 (0% DA chitosan) is the most extendedstructure according to the average end-to-end lengths. Asnapshot of 16 with an end-to-end length of 20.7 Å showsaverage glycosidic bond angles of 52.0 (�), 47.3 (�-1), and 39.6°(�-2). Apparently, the extended, linear saccharides have gly-cosidic linkages, �, around 40°.

In Figure 12, plots of the end-to-end length as a function ofsimulation time are depicted for 16, 25, and 31 (chitin). Theplots illustrate how the majorities of the structures sampled aresimilar in length, in the range of 20.3 to 21.1 Å. This is expectedbecause 25 has 81% of the sampled structures in � ≈ 40°, >99%in �-1 ≈ 40°, and 82% in �-2 ≈ 40°. 16, which mimics 0% DAchitosan, has the longest average end-to-end length, 20.7 Å,and essentially no structures deviate from the extended confor-mation. In 31 (chitin), snapshots occasionally deviate from theaverage length, resulting in the average end-to-end length beingslightly shorter, 20.5 Å. As noted above, 25 is the most flexibleof the examined tetrasaccharides. It has an average end-to-endlength of 20.2 Å, which is 0.5 Å shorter than 16. In 25, manystructures deviate from the average length. In general, snapshotswith end-to-end lengths close to the average length showglycosidic bond angles around -30°, 40°, or both. A short end-

to-end length (<20 Å) results from one or more glycosidic bondangles of either -90 or 170°. Glycosidic bond angles of -90°only occur in ∼1% of the GlcNAc-GlcNAc linkages and ∼9%of the GlcN-GlcNAc linkages, whereas glycosidic bond anglesof ∼170° are only found in 7% of the sampled GlcN-GlcNAcstructures. These findings are in perfect agreement with thedistributions of the conformers of the glycosidic linkages. In16, >99% of the snapshots have �, �-1, and �-2 of ∼40°. Incontrast, 25 is very flexible, with 29% of the sampled structuresshowing values of �, �-2, or both that are different from -30and 40°.

Conclusions

In this study, we have reported exhaustive and systematicevaluations of the conformational dynamics of the glycosidiclinkages in chitin and chitosan with varying DA, as modeled in31 di-, tri-, and tetrasaccharides. The results are clear-cut; it isthe sugar ring to the reducing side that dictates the flexibilityof the glycosidic linkage. Only a GlcNAc unit to the reducingside of the glycoside bond leads to flexibility. Four possibleconformations are found in a GlcN-GlcNAc linkage, whereasa GlcNAc unit at the nonreducing side slightly reduces theglycosidic flexibility of the GlcNAc-GlcNAc linkage to threeconformations, of which one is only rarely sampled. Therefore,the GlcN-GlcNAc linkage was found to be more flexible thanthe GlcNAc-GlcNAc linkage, both of which are much moreflexible than the GlcNAc-GlcN and GlcN-GlcN linkages.However, most importantly, the GlcN-GlcNAc linkage regu-larly visits a conformation significantly different from thosevisited by the others.

Brugnerotto et al.13a,b and Lamarque et al.14 find that extendedchitosan polymers with up to 3000 sugar units having DA <25% are quite flexible and that for DA ) 25-50% the chainsare getting stiffer, whereas DA > 50% results in rather rigidstructures, although it is stated by Lamarque et al. that thesedomains are most pronounced in polymers at a high weight-averaged degree of polymerization.14 Given the results of thework presented in this article, the presence of these threedomains can be rationalized in more detail. The flexibility of achitosan chain is determined by the number of glycosidic bondangles differing from -30 or 40°. Hence, the flexibility is almostexclusively determined by the number of GlcN-GlcNAclinkages,althoughaverysmallfraction(1%)oftheGlcNAc-GlcNAcangles also contributes to flexibility. The other two types ofglycosidic linkage, GlcN-GlcN and GlcNAc-GlcN, do notcontribute to the flexibility of oligo- or polysaccharides becausethey are found only with � ≈ 40°. Therefore, the moreGlcN-GlcNAc linkages found in a chain, the more flexible thechain will appear. Therefore, >50% DA will evidently result infewer GlcN-GlcNAc linkages as the percentage of GlcN unitsdrops with increasing DA and thus less-flexible chains, whichis indeed consistent with the experimentally determined longerpersistence lengths for chitin than those for chitosan with verylow DAs13a and also observed by Franca et al. in their modelingstudy of decamers of chitin and 0% DA chitosan.13c

The GlcN-GlcNAc linkage was found to sample fourconformations of � around -90, -30, 40, and 170°, respectively.The GlcNAc-GlcNAc linkage visited three of these conforma-tions, namely, � ≈ -90, -30, and 40°; however, the -90°conformation was only infrequently visited. The two stifflinkages, GlcNAc-GlcN and GlcN-GlcN, both sampled onlyone conformation of �, which is the conformation around 40°.The reason for this was found to be the differences in hydrogen

Figure 12. Plots of the measured end-to-end lengths of the snapshotssampled from MC/SD as a function of time in 16 (top), 25 (middle),and 31 (bottom).

Conformational Flexibility of Chitosan upon Degree of Acetylation Biomacromolecules, Vol. 11, No. 11, 2010 3205

bonding possibilities, in particular, for OH3. In all fourglycosidic linkages, the major conformation has � ≈ 40°. Thisconformation is largely stabilized by a strong hydrogen bondinginteraction between the two involved sugar rings, namely,between the reducing end OH3 and the nonreducing end O5+1,OH3 · · ·O5+1. When the reducing-end sugar is a GlcN unit, thishydrogen bonding interaction is present in virtually all snapshots,thereby effectively locking the glycosidic linkage of chitosanwith low DAs. However, acetylation of the reducing sugar unitled to another good hydrogen bonding partner for OH3, namely,the carbonyl group of the adjacent acetamide, O(dC), andconsequently, the otherwise persistent hydrogen bond betweenOH3 and the nonreducing sugar ring is now found in only ∼82%of the sampled structures, resulting in the observed much moreflexible nature of these linkages.

Franca et al. studied the effect of explicit water solvation onthe conformational dynamics of decamers of chitin and 0% DAchitosan.13c In line with our observations, they found the stabilityof the intrachain hydrogen bond OH3 · · ·O5+1 to be the maindeterminant of flexibility of the saccharide chains. Franca et al.use explicit solvation in their simulations, which allows forstudying interactions with explicit water molecules. Even thoughour study uses an implicit continuum water model for theaqueous solution, the results from Franca et al.’s study comparevery well to our observation for the two extremes, chitin and0% DA chitosan, with respect to stability, population of certainhydrogen bonds, and so on. One discrepancy, however, is notedbetween the two studies; Franca et al. found an OH3 · · ·O5+1

hydrogen bond population of 53% in 0% DA chitosan at lowpH, whereas we find it to be present in 87 and >99% of thestructures after minimization for our 0% DA models of chitosan,which must be accounted for by a slightly different hydrogenbonding network when explicit water molecules are included.For chitin, it was demonstrated that the explicit water resultedin the coordination of a water molecule to O3 and OH6+1,thereby stabilizing the OH3 · · ·O5+1 hydrogen bonding interac-tion. However, the water molecule was also reported to interactwith OH3 in 19% of the chitin structures and 27% of thechitosan structures when chitosan was modeled as charged atlow pH.13c The adjacent acetyl group was reported not to bedirectly involved in this hydrogen bonding pattern. A chargedprimary amine, as found in chitosan, at this position must beexpected to be directly involved in the hydrogen bonding pattern,thus coordinating a water molecule strongly between the primaryamine and the adjacent OH3 and thereby orienting the OH3group correctly and strongly stabilizing the OH3 · · ·O5+1

interaction. It was shown by Franca et al. that in chitosan atlow pH the orientation of the coordinated water molecule wasquite different and much more specific and rigid than in chitin.Extending this to our results for chitosan with nonzero DA, theinclusion of explicit water and, in particular, a water moleculecoordinating to the OH3, can be expected to perturb the stabilityof the OH3 · · ·O5+1 hydrogen bonding interaction. In the caseof a charged primary amine adjacent to the OH3 (as in 0% DAchitosan) the coordination of the water molecule can bespeculated to result in an overall stabilization of the conforma-tion at � ≈ 40°. With an acetamide adjacent to the OH3, thewater interaction is not as pronounced, and the perturbation ofthe OH3 · · ·O5+1 interaction may only result in an overall slightstabilization of the � ≈ 40° conformation because it simulta-neously coordinates to OH6+1. Such a picture supports the trendsoutlined in this work, that a GlcNAc to the reducing side of thelinkage leads to a less-stabilized � ≈ 40° conformation and thusgreater flexibility, also in water.

The detailed knowledge achieved in this study can be veryvaluable for studying enzymatic degradation products of chiti-nases and chitosanases. The knowledge about conformerdistribution from this study may be compared directly to NMRdata of degraded chitosan units and thereby assist in gettingknowledge of the structure of the original enzymatic substrate.31

Along the same lines, with knowledge of the glycosidicconformational flexibility observed in this study, it is possibleto quantify the composition of structurally unknown chitosanoligomers by NOE experiments across the glycosidic linkage.We foresee that such knowledge will be invaluable whendesigning chitosan variants with tailor-made properties.32

Acknowledgment. The Danish Center for Scientific Comput-ing is acknowledged for resources and computing time alongwith financial support from the Danish Natural Science ResearchCouncil and Research Foundation as well as from the Carlsberg,Lundbeck, and Novo Nordisk Foundations. Jens Ølgaard Duus,Carlsberg Research Center, is acknowledged for inspiration inthe beginning of the project and for discussing the results,progress, and perspectives of the findings.

Supporting Information Available. Tabular material listingthe statistical analysis of populations of the identified conforma-tions for di-, tri-, and tetrasaccharides and figures of the modeledtri- and tetrasaccharides along with graphs and plots showingconformational flexibilty in the models. This material is availablefree of charge via the Internet at http://pubs.acs.org.

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