Conformational Flexibility of Chitosan: A Molecular Modeling Study

3196

Biomacromolecules 2010, 11, 3196–3207

Conformational Flexibility of Chitosan: A Molecular Modeling Study 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 β-(1,4) linked N-acetylglucosamine units (GlcNAc) and, for chitosan, also glucosamine units (GlcN). In recent years, chitosan has attracted much interest because of its special physical and chemical properties related to drug delivery, wound healing, and tissue engineering. However, limited structural knowledge is available for chitosan because of its composition of the randomly mixed building blocks, GlcNAc and GlcN. In this study, we present exhaustive combined molecular dynamics and Monte Carlo simulations that unravel the conformational flexibility of the β-(1,4)-linkage in di-, tri-, and tetrasaccharide models of chitin and chitosan. The most flexible disaccharide unit was found to be GlcN-GlcNAc, populating four conformations. Furthermore, it is found that the conformational freedom of a glycosidic bond is independent of the flexibility of the neighboring linkages along the oligomer. The results are interpreted with respect to hydrogen bond formation and implications for polymer properties.

Introduction Chitin and chitosan are biopolymers found in various plants and animals, mainly crustaceans and fungi.1 Chitin is a homopolymer composed of repeating N-acetylglucosamine (GlcNAc) units linked by β-(1,4) glycosidic bonds, whereas chitosans are polymers of randomly distributed GlcNAc and glucosamine (GlcN) units linked by β-(1,4) glycosidic bonds. (See Figure 1a.) The proportion of GlcNAc units is described by the degree of acetylation (DA), thus, chitin has a DA of 100%. Chitosan has recently shown very promising properties for a range of applications, including biofabrication,2 delivery systems for macromolecules,3 wound dressing,4 and tissue engineering5 as well as a number of applications in the food industry,6 including antimicrobial activity, food additive, and water purification. The fully deacetylated chitosan polymer (DA ) 0%) exists in 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 helixlike structure7,8b,c of the sugar chains with repeating periods of between two and eight sugar units.8-10 However, the most recent chitosan crystal structure revealed a five-fold helical symmetry along the polymer axis.8d The chains can be linked either directly through hydrogen bonds, via structural water molecules, or, as in anhydrous chitosan, through hydrophobic interactions,9 giving rise to sheet-like structures of the crystals.11 For chitosan with a DA different from 0% and for chitosan in solution, much less direct structural evidence is found. Structural knowledge has been gathered from other means, and it is known that the DA affects the structure and the overall conformational dynamics of the polysaccharide chain.12-16 The available data are briefly reviewed in the following paragraph. The large number of primary amines gives chitosan unique chemical properties. At low pH, chitosan is a water-soluble, * 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 of Pharmaceutical Sciences, University of Copenhagen, Denmark.

Figure 1. Dihedral angles and nomenclature used in this study. (a) Schematic representation and shorthand notation of a chitosan fragment; (b) φ, (H1-C1-O4-1-C4-1); (c) ψ, (C1-O4-1C4-1-H4-1); (d) χ, (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 of a oligosaccharide. Residues are labeled i +n toward the nonreducing end and i -n toward the reducing end. The glycosidic bond between residue i n and i n-1 is denotedχ n-1. Rings are referred to as A, B, C, and D starting from the nonreducing end.

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

10.1021/bm100736w  2010 American Chemical Society Published on Web 10/20/2010

Conformational Flexibility of Chitosan upon Degree of Acetylation

border between the soluble and insoluble states lies between pH 6.0 and 6.5, depending on the DA and the ionic strength of the media. The pKa of chitosans in aqueous solutions ranges from 6.3 to 7.3 (for 5.2 and 89% DAs, respectively).12a The solubility in aqueous media decreases as the DA increases, thereby reducing the amount of cationic sites.12b The electrostatic behavior of chitosans is divided into three domains: (1) chitosans with DA < 20% behave as polyelectrolytes and are quite soluble in aqueous media; (2) chitosans with 20 e DA e 50% constitute a transition domain; and (3) chitosans with DA > 50% exhibit increasing hydrophobic character due to the lowered number of cationic sites.12b Similarly, chitosan chains with DA < 25% are quite flexible and independent of the exact DA, whereas the chains become gradually stiffer for chitosans with DAs of 25-50%. For DAs above 50%, the stiffness of the chain increases, and hence the persistence length becomes more or less constant.13,14 The accessible conformational space of the glycosidic linkage has been reported to be affected by the nature of the substituent at C2 on the nonreducing saccharide unit.15 The distribution of N-acetyl groups along the chain has great influence on the stiffness. Brugnerotto et al.13a have reported modeling studies of chitosans with different substitution patterns (GlcN or GlcNAc) by using the four possible disaccharides as the cores for modeling long chains using coarse techniques. Persistence lengths of 90 Å for 0% DA chitosan and of 125 Å for chitin were found. For 50% DA chitosan, they found a random pattern with a persistence length of 115 Å; the alternate ABAB pattern had a persistence length of 135 Å, whereas an A2B2 pattern had a persistence length of 97 Å. Furthermore, grouping (from 2 to 20 consecutive units) showed no variation in the predicted persistence length for 50% DA.13a Chitosan with 0% DA possesses a higher degree of elongation and tensile strength than chitin.16 Chitin favors a more extended conformation and is a little stiffer than chitosan.13a A recent all-atom MD study of 0% DA chitosan and chitin decamers focuses on their aqueous structures and how explicit water molecules can interrupt the important intrachain hydrogen bond between OH3 and O5+1. (For notation, see Figure 1.)13c As indicated above, a number of studies on the physical properties of chitosan have been reported. Other studies are dealing with, for example, the tensile strength and elongation,17 the conformation of chitosan docked into a protein,18 the conformation of chitobiose and chitosan chains (using coarse techniques),15 and the conformation of polysaccharides (using coarse techniques).13a However, to the best of our knowledge, there has so far been no systematic all-atom modeling study of the conformational flexibility of all combinations of tri- and tetrasaccharides composed of β-(1,4)-linked GlcNAc or GlcN units including chitosan models with varying DAs, including analysis of chitosan with DAs differing from 0%. The aim of this study is thus to achieve a greater understanding of the structural features of chitosan. Through a detailed systematic evaluation of a total of 31 di-, tri-, and tetrasaccharides of β-(1,4)-linked GlcNAc and GlcN units, the flexibility of chitosan as a function of the acetylation pattern is elucidated by following the conformational behavior of the five dihedral angles shown in Figure 1. We will focus on the consequences of the observed dynamics of a glycosidic bond as a result of N-acetylation of either the reducing or the nonreducing glucosamine moiety.

Experimental Section All calculations were performed using MacroModel (version 9.0)19 and the AMBER* force field with the Senderowitz-Still all-atom

Biomacromolecules, Vol. 11, No. 11, 2010

3197

pyranose parameters20 similarly to the protocol developed by Bernardi et al.21 for performing conformational analysis of oligosaccharides. The oligosaccharides were built in Maestro22 using the fragment libraries (7 disaccharides, 8 trisaccharides, and 16 tetrasaccharides) and modified manually to the desired structures. Water solvation was simulated implicitly by MacroModel’s generalized Born continuum solvent model (GB/SA).23 Charges were taken from the force field, and no truncation of the nonbonded interactions was applied. Examination of the structures and the dynamics of each saccharide was performed in four steps. First, a Monte Carlo/energy minimization (MC/EM) conformational search was carried out to locate at least two distinct local minima structures for each oligosaccharide studied. The conformational searches were carried out using 15 000 steps of the MCMM torsional sampling procedure and applying an energy window of 20 kJ/mol. No ringopening/closing was allowed, and no endocyclic bonds or hydrogen bonds were used as explicit variables during the Monte Carlo search. All other bonds were sampled between 60 and 180°. The found structures were minimized through the PRCG method. Second, a dynamic simulation was performed following the MC/SD24 protocol provided in MacroModel using the GB/SA implicit water model. The same degrees of freedom were applied as those in the conformational search, and ring-openings were not allowed. All torsion angles were allowed to rotate at each MC step, and at least one should change. The simulations were run at 300 K, with a dynamic time step of 1.0 fs. Two separate runs were performed for each saccharide, with the starting point being the two distinct minimum conformations found in the initial MC/EM stages. Sampling was done for 25 ns for each disaccharide, 35 ns for each trisaccharide, and 50 ns for each tetrasaccharide. Each accepted MC step was followed by an SD step. Structures were saved every 5 ps for later evaluation. Convergence was checked by comparing the two MC/SD runs with regard to the relative energy as a function of geometrical conformations (in particular, φ, ψ, ω, and χ angles). Subsequently, in the third stage of the simulation protocol, the saved snapshots from the MC/SD dynamics simulations were minimized by the PRCG protocol. Convergence was determined by gradients with a threshold of 0.05 kJ/mol · Å. Finally, the minimized structures were clustered according to the angle distribution and binned in 10° intervals to 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-acceptor interaction where the distance between the acceptor heteroatom and the hydrogen atom is below 2.1 Å, the donor angle, measured as D-H · · · A, is larger than 120°, and the acceptor angle, H · · · A-C, is above 90°. A weak hydrogen bond was defined as a donor-acceptor relationship with a distance between the acceptor heteroatom and donor hydrogen between 2.1 and 3.1 Å and with a donor angle >120°.

Results and Discussion Disaccharides. Initially, seven disaccharides were modeled and studied to decide which structural motifs to include in the study of the longer oligomers, and, in particular, to decide how to model the termini of the oligomers. Figure 2 presents the modeled disaccharides along with short-hand notations for the tri- and tetrasaccharides included in the study. Conformational Analysis. The influence of a methyl group instead of a free hydroxyl group at the termini on the conformational freedom of the glycoside bond is studied in 0% DA chitosan models 1, 2, and 3. Acetylation of both amino groups gives a chitin dimer, and the conformational dynamics of the glycosidic bond in chitin is likewise examined without and 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 the reducing (6) or nonreducing (7) side of the glycoside bond is studied without protection of the termini.

3198


Skovstrup et al.

Figure 3. Distribution plots of (φ, ψ) for structures 1, 2, and 3. Figure 2. (a) Modeled disaccharides and short-hand notation for (b) trisaccharides and (c) tetrasaccharides.

The computed average values of the glycosidic bond angles φ, ψ, and χ of the disaccharides are listed in Table S1 in the Supporting Information. All modeled structures have very similar average properties of the glycosidic linkage; however, small, yet important, differences are found. The average glycosidic bond angle, χ, shows some variation, 31° in 5 (chitin) to 42° in 1-3 (0% DA chitosan), due to the observation that the glycosidic linkage can exist in four conformations with χ being approximately -90, -30, 40, and 170°. The conformation with χ ≈ 40° is the most common conformation (as will be later discussed). ψ contributes the most to the differences observed in the averages of χ because of the exoanomeric effect damping the fluctuation of φ.25 Structures 1, 2, and 3 have almost identical glycosidic bond angles considering both the averaged values and the (φ,ψ)-distribution plots in Figure 3. They are all GlcN-GlcN disaccharides modeling 0% DA chitosan, only differing in the protection of O4+1 and O1. All three disaccharides essentially exist in only one conformer with χ ≈ 40°. (Frequency plots are provided in Figure S1 of the Supporting Information.) The three structures hereby imply that protection of neither the O4+1 nor the O1 hydroxyl group has a notable impact on the conformational properties of the central glycosidic linkage. The conformational freedom of the glycosides is more prevalent 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 broadest distribution in the glycosidic bond angle, χ, and thereby has the highest population of conformers with χ different from 40°, resulting in the lowest average value of χ. Please see plots in Figure S1 of the Supporting Information. In structure 6, which models the 50% DA chitosan fragment GlcN-GlcNAc, four possible conformations of χ are observed, although those with χ * 40° are observed in 60% of the simulated structures, and after energy minimization, in practically all structures, indicating that the structures are located in the same potential energy minimum. Indeed, the (φ, ψ) distribution plots in Figure 3 and the frequency distribution plots in Figure S1 of the Supporting Information support the picture of a general overall “single-minimum conformation”. With a GlcNAc unit in the reducing end, the frequency of the OH3 · · · O5+1 hydrogen bond drops to as low as 41% and, after minimization in the MeO-GlcNAc-GlcNAc-OMe saccharide, 5, 66%. This finding indicates the presence of at least one additional conformation of the glycosidic linkage, which is also seen in the frequency distribution plots of 4, 5, and 6 (Figure 4 and Supporting Information, Figure S1).


3199

Among the nonminimized structures of model 7, with a GlcN unit at the reducing end, a small population with χ ≈ -30° is found (Figure S1 of the Supporting Information). However, these all minimize to the χ ≈ 40° conformation, indicating a very low energy barrier between the two conformations in 7. This behavior is not observed for structures with GlcNAc at the reducing end, 4-6, which, after minimization, contain a significant population with χ ≈ -30°, as seen in the (φ, ψ) distribution plots of the energy minimized structures resulting from the MC/SD trajectories depicted in Figure 4. Clearly, 4-6 populates more than one conformation, whereas 1-3 and 7 are mainly found in only one conformation. Dynamics of the C5 Hydroxymethyl Group. The orientation of the C6 hydroxyl is measured through the ω dihedral angle. Three conformations are possible corresponding to the ( gauche and the anti conformations, traditionally referred to as gg, gt, and tg, as illustrated in Figure 5a. The distribution of the three conformations of the C6 hydroxyl groups were measured for ω+1 and ω (data in Table S3 of the Supporting Information). The distribution of the conformations of ω+1 was found to be clearly different from the distribution of ω. For ω+1, the gt conformation is the major conformation in all modeled disaccharides. The preference for the gt conformation of ω+1 is a result of the gauche effect,26 but steric effects and the possibility of forming hydrogen bonds also play a role. The gauche effect orients the two electronegative atoms, O5+1 and O6+1, gauche to each other because of a presumed σC-H-σ*C-O orbital interaction. In addition, the hydroxyl group in the gt conformation is able to form a hydrogen bond to O3, provided that the OH3 · · · O5+1 hydrogen bond is formed, which orients O3 correctly. Therefore, the OH6+1 · · · O3 hydrogen bond is present, although weak, 30-40% of the time (46-66% after structure minimization). In the gg conformation, the C6 hydroxyl group is placed between O5 and C4 and is therefore more affected by steric strain than in the gt and tg position and thus is less favorable. The distribution of ω was found to be very different from that of ω+1. For ω, the tg conformation was significantly more populated in the models that have a primary amine, GlcN, at the nonreducing ring, models 1, 2, 3, and 6. The reason is the possibility of forming a hydrogen bond between the C6 hydroxyl group and the primary amine at the nonreducing end. (This is shown in Figure 6a for 1 and demonstrated below for trisaccharides.) The GlcN-GlcN disaccharides 1, 2, and 3 differ in the functionality at O4+1 and O1, with 3 resembling a disaccharide fragment of a polysaccharide; that is, the methoxy groups mimic linking 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 possibility for OH4+1 to hydrogen bond to O6+1. The hydrogen bonding network favoring the ω+1 tg conformation is shown in Figure 6a for 1. In contrast, the C4+1 methoxy group does not have the same influence on the population of the ω+1 tg conformation in the GlcNAc-GlcNAc models 4 and 5. This is due to the ring A acetamide attracting the hydroxyl groups through cooperative hydrogen bonding in a counter-clockwise manner, as shown in Figure 6b for 4. Dynamics of the Acetamide Group. Similar to the C6hydroxyl group, the acetamide group can sample different conformations through the dihedral angle γ. Because of the planarity of the amide group, the data (Table S4 and S5 of the

3200


Figure 5. (a) Molecular structures and Newman projections viewed along the C6-C5 bond direction showing the three distinct conformations of the ω dihedral of the C6 hydroxyl group. The first character in the conformation refers to the position of the OH group relatively to O5 and the last character refers to the position of OH relatively to C4. (b) Molecular models and Newman projections showing the four conformations of the γ angle viewed along the N-C2 bond positioning the acetamide group relatively to the sugar ring. The first character describes the position of the acetyl relatively to C1, and the second character describes the position of the acetyl relatively to C3. For clarity, only polar hydrogens are included in the molecular figures.

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

Skovstrup et al.

Figure 6. (a) In uncapped, 0% DA chitosan, models 1 and 2, a hydrogen bond is present between OH4+1 and O6+1, giving rise to an increased population of the ω+1 tg conformation. (b) The acetamide of 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 the γ angle is shown in the gt conformation in which a hydrogen bond is formed to the unprotected C1 hydroxyl group. Removing this hydrogen bonding option (by the C1 protection group) results in a dramatic drop in 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) of γ dominates because of OH3 being able to hydrogen bond to the acetamide oxygen. Thereby, the otherwise persistent OH3 · · · O5+1 hydrogen bonding interaction is disrupted, which ultimately results in a more flexible glycosidic linkage. Note that the population of the hydrogen bond between OH3 and the adjacent acetamide (shown in the model with green carbons and a light-green OH3 hydrogen atom) exactly equals the population of tg2 conformers of γ.

for 4, which is not possible in 5. In 4 and 7, OH4+1 hydrogen bonds to O3+1, thereby orienting OH3+1 correctly to hydrogen bond to the acetyl group. The acetamide of ring A is able to hydrogen bond to the C6 hydroxymethyl of ring B. In general, these inter-ring NH+1 · · · O6 hydrogen bonds are formed in the presence of a hydrogen bond between the C3+1 hydroxyl and the acetamide, OH3+1 · · · O(dC)+1. Whereas the presence of the C4+1 methoxy group affects the distribution of the γ+1 angle moderately, that is, moving 15% of the tg2 conformers in 4 to the gg and gt conformers in 5, the distribution of the γ angle is much more dramatically 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 interaction between OH1 and the acetamide, OH1 · · · O(dC), as shown in Figure 6b. In 5, C1 is protected with a methoxy group, and


hydrogen bonding between OH1 and the acetamide is no longer possible, resulting in a drop in the population of the gt conformation of γ to the expected level of below one-third. In 5, the tg conformation of γ would be expected to be populated by circa one-third of the structures in the absence of any steric or hydrogen bonding interactions. However, the ability to form a hydrogen bond between the acetamide and OH3 increases the tg population and rotates some of the existing tg conformers from the tg1 to the tg2 position. The hydrogen bond “switching” of OH3 emerges when the frequency of the hydrogen bonding interaction OH3 · · · O5+1 is compared with the frequency of the interaction OH3 · · · O(dC). Combined, these two interactions are found in 91% of all minimized structures. Therefore, OH3 is hydrogen bonding to either the adjacent acetamide (green in Figure 6c) or O5 of the adjacent sugar ring in the nonreducing direction (gray in Figure 6c). Note that the tg2 population of γ precisely accounts for the population of hydrogen bonding interactions with OH3. When comparing 4 and 5, the presence of the C1 methoxy group (in 5) seems to result in a higher population of the tg2 conformation of γ and thus a lower population of the OH3 · · · O5+1 hydrogen bonding interaction. Ultimately, this leads to a destabilization of (and therefore a lower population of) the glycosidic linkage conformation at χ ≈ 40°. Trisaccharides. Eight trisaccharides (Figure 2b and Figure S2 of the Supporting Information), all bearing a methoxy group at the C4 position of the nonreducing end and at the anomeric center of the reducing end, were modeled. For the disaccharides discussed above, the conformational flexibility of the central glycosidic linkage was only moderately affected by these functionalizations. Therefore, we decided to continue modeling the methoxylated sugars only, thereby avoiding artifacts in the hydrogen bonding pattern, as described above for the unprotected disaccharides. Structure 8 represents the trisaccharide model of 0% DA chitosan. The position of the N-acetyl group is systematically varied in models 9-11 of 33% DA chitosan and 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 the saved snapshots from the MC/SD simulations of the modeled trisaccharides 8-15, it was found that the average glycosidic angles resemble those of the disaccharides. All data are provided in Table S6 of the Supporting Information. The trends are relatively clear; the presence of an acetamide group at the sugar ring at the reducing side of the glycosidic linkage (χ as well as χ-1) decreases the average value of the dihedral angle with 5-10° from approximately 42 to 31-37°. When the acetamide is placed at the sugar ring at the nonreducing side of a glycosidic linkage, the average of the glycoside bond angle is lowered by only 3 to 4° relative to fully deacetylated chitosan, model 8. In Figure 7, plots of the two glycosidic bond angles are shown as (χ;χ-1) distributions for four of the trisaccharides 8, 9, 11, and 14, which emphasizes the trends found. The remaining plots of the glycosidic bond angles can be found in Figure S3 of the Supporting Information. The observed trend for the average values measured can be rationalized by viewing these conformational plots. Starting with 0% DA chitosan, 8, only one conformation is found for both glycosidic bond angles, and the distribution is almost symmetrical around χ ) χ-1 in the plot. In 8, 98% of the sampled conformers have χ ∈ [0°; 75°]; likewise, 98% have χ-1 ∈ [0°; 75°]. The 33% DA chitosan models 9-11 reveal that the introduction of a GlcNAc unit practically only influences the glycosidic bond toward the nonreducing site of the GlcNAc unit.


3201

Figure 7. (χ; χ-1) distribution plots of the glycosidic bond angles of the four trisaccharides 8, 9, 11, and 14. (χ; χ-1) distribution plots for all of the modeled trisaccharides are provided in Figure S3 of the Supporting Information.

Hence 9, with a GlcNAc in the nonreducing end, shows a slight broadening of χ (toward a conformation of χ ≈ -30°) though maintaining 95% of the snapshots within χ ∈ [0°; 75°] and 97% within χ-1 ∈ [0°; 75°]. This observation implies that the flexibility of a GlcNAc-GlcN glycosidic bond is close to identical to the flexibility of the GlcN-GlcN linkage. This is not true for a GlcN-GlcNAc linkage, as revealed in model 10, where the central sugar unit is GlcNAc, and further confirmed in 11, where the GlcNAc unit is positioned at the reducing end. Structures 10 and 11 reveal three new conformations of the GlcN-GlcNAC glycosidic linkage centered around χ ≈ -90,

3202


-30, and 170°. In 10, these are populated with 8, 5, and 6%, respectively, whereas the other glycosidic linkage, χ-1, is only slightly broadened, as was similarly observed for the GlcNAcGlcN linkage in 9. In 11, the GlcN-GlcNAc linkage is populated with 7, 5, 80, and 7% for χ-1 ≈ -90, 30, 40, and 170°, respectively, whereas χ, being a GlcN-GlcN linkage, is unaffected, and similarly distributed as the glycosidic bonds in 8. Overall, it can be concluded that the GlcN-GlcN and the GlcNAc-GlcN linkages both sample virtually only one conformation, whereas the GlcN-GlcNAc linkage is more flexible and 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 between the two GlcNAc units is affected by the appearance of both GlcNAc units. Comparing the distribution in χ and χ-1 of structures 9 and 12 it is apparent that the second acetyl group in 12 induces some flexibility into the GlcNAc-GlcNAc linkage. More specifically, χ is practically found in two conformations, centered at -30 and 40°, with relative populations of 9 and 89%. χ-1 is still restricted to the single conformation observed for a GlcNAc-GlcN linkage described above. Moving the second GlcNAc unit to the reducing end (trisaccharide 13) results in a conformational picture in accordance with the previous observations; χ is practically restricted to a single conformation, which is a signature of the GlcNAc-GlcN linkage, whereas χ-1 is very flexible, showing four conformations, as seen for all GlcN-GlcNAc linkages so far. Finally, in model 14, the two acetylated sugar rings are neighbors at the reducing end, resulting in χ being very flexible, as seen above for the other GlcN-GlcNAc linkages, and a less flexible χ-1 displaying two conformations, χ-1 ≈ -30 and 40° populated with 9 and 89% of the snapshots. A third low populated conformation at χ-1 ≈ -90° (populated with 1%) also emerges. This is in full agreement with the GlcNAc-GlcNAc linkage in 12 and also with the linkages found in the 100% DA (chitin) trisaccharide 15. The most significant difference between the two flexible types of glycosidic linkages, GlcN-GlcNAc and GlcNAc-GlcNAc, is that the conformation with χ ≈ 170° is only present in the former. Besides this, they sample the same conformations, yet with different frequencies, which can best be seen when comparing the plots of 10 and 12 in Figure S3 of the Supporting Information. More specifically, the conformation with χ ≈ -90° is only rarely sampled by the GlcNAc-GlcNAc linkage. The most flexible trisaccharide modeled is thus 14, MeO-GlcN-GlcNAc-GlcNAc-OMe, where the very dynamic GlcN-GlcNAc linkage is found as well as the other flexible GlcNAc-GlcNAc linkage. The full statistics are seen in Table S7 of the Supporting Information. More Detailed Examination of the Dihedral Angles of 14. To gain more knowledge about the commonly occurring conformations, the snapshots of 14 were further studied because this was found to be the most flexible trisaccharide. In Figure 8, the populated states of the two glycosidic linkages are displayed in terms of (φ; ψ) distribution plots (panel a and b) along with the (χ; χ-1) scatter plot (panel c), all based on minimized structures of the snapshots collected for 14. In general, conformers centered at (60°; -155°) in the (φ; ψ) graph correspond to χ values of approximately -90°, conformers at (35°; -65°) correspond to χ ≈ -30°, conformers at (50°; 0°) correspond to χ ≈ 40°, and conformers at (170°; 3°) correspond to χ ≈ 170°. From the (φ; ψ) graphs in Figure 8, it is evident, that φ is very restricted to the interval [25°; 60°]. In fact, the only conformers outside that area are located in the area of the infrequently visited conformation at φ ≈ 170°. The very

Skovstrup et al.

Figure 8. Plots of the angle distributions of the glycosidic bonds in 14 (MeO-GlcN-GlcNAc-GlcNAc-OMe). (a) (φ; ψ) graph of the bond between the nonreducing end and the central sugar unit. The φ-ψ combinations resulting in the four observed dihedral angles of χ are encircled. (b) (φ-1; ψ-1) graph of the bond between the central and the reducing end sugar unit. (c) (χ; χ-1) graph of the trisaccharide. All three plots are based on the minimized structures.

restricted fluctuation of φ is due to the exoanomeric effect; that is, the dihedral angle O5+1-C1+1-O4-C4 is slightly smaller than 60° because of the nO5 f σ*C1-O5 orbital overlap.27 The dynamics of ψ is quite different from the dynamics of φ. ψ frequently visits two conformations, one conformer in the span [-15°; 5°] and the other conformer around [-70°; -40°]. Additionally, a third conformer around [-157°; -152°] is occasionally found. The molecular structure of the conformation with (χ; χ-1) around (40°; 40°) is depicted in Figure 9a. This is the major conformation in all examined trisaccharides. In 14, it is characterized by forming short OH3 · · · O5+1 hydrogen bonds in 70% of the structures and OH3-1 · · · O5 hydrogen bonds in 75% of the sampled structures. A hydrogen bond between OH3 and the adjacent acetamide is found in 22% of the snapshots, whereas OH3-1 hydrogen bonds to the adjacent acetamide in 19% of the snapshots (not shown in the Figure). This means that in 92% of the sampled structures of this conformation OH3 hydrogen bond to either the neighboring O5+1 or the adjacent C2 acetamide (on its own ring); for OH3-1, this is 94%. The conformation with (χ; χ-1) ) (-90°; 40°) (Figure 9b) is found in 8% of the sampled structures. It forms no hydrogen bonding interactions between the nonreducing unit and the central GlcNAc. Consequently, the glycosidic linkage between rings A and B is exclusively governed by the exoanomeric effect


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

and steric strain between the two rings, and the two sugar rings are oriented almost perpendicularly. Because of the absence of the OH3 · · · O5+1 hydrogen bond, OH3 interacts through strong hydrogen bonds with the adjacent acetamide in 80% of the structures. The other glycosidic linkage is found in the major χ-1 ≈ 40° conformation, allowing for the OH3-1 · · · O5 hydrogen bond to form. The conformation with (χ; χ-1) ) (-30°; 40°) (Figure 9c) is found in 5% of the sampled structures. In this conformation, the OH3 · · · O5+1 hydrogen bond is present in only 13% of the structures (not shown in the Figure), whereas OH3 is hydrogen bonding to the adjacent acetamide in 54% of the saved snapshots. There is no strong, persistent, attractive nonbonded interaction between rings A and B; that is, the conformation is


3203

largely governed by the exoanomeric effect and steric interactions. To reveal the difference in the OH3 · · · O5+1 hydrogen bonding pattern in the χ ≈ -30 (Figure 9c) and 40° (Figure 9a) conformations, both with χ-1 ≈ 40°, the relationship between the OH3 and O5+1 was studied. The distance between the hetero atoms, O3 and O5+1, is 2.8 Å in both cases. For χ ≈ -30°, the donor angle (O3-H3 · · · O5+1) is ∼140°, whereas the χ ≈ 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 χ ≈ -30° conformer, whereas they are found around 103 and 144° in the χ ≈ 40° conformer. Therefore, because of the equatorial configuration of the C3 hydroxy group of ring B and the puckering of ring A, the conformers with χ ≈ 40° are able to form strong OH3 · · · O5+1 hydrogen bonding interactions between rings B and A. Likewise, the conformers with χ ≈ -30° are suffering from distinctly worse acceptor angles, resulting in the OH3 · · · O5+1 hydrogen bonding interaction being characterized as weak. The conformation with (χ; χ-1) ≈ (170°; 40°) (Figure 9d) is found in 6% of the sampled structures. The stabilizing force is found to be a persistent, strong hydrogen bonding interaction from the primary, cationic amine of ring A to O3 at ring B, the latter which in 82% of the structures with this conformation is also hydrogen bonding to the adjacent acetamide at ring B. In addition, in 34% of the structures with this conformation the ring B acetamide is hydrogen bonding to O6-1 (compared with an overall NH · · · O6-1 hydrogen bond population of 17% for the total of the sampled conformations of 14). The conformation with (χ; χ-1) ≈ (40°; -30°), Figure 9e, is found in 7% of the sampled snapshots. It is obviously very similar to the (χ; χ-1) ≈ (-30°; 40°) conformation. However, the (χ; χ-1) ≈ (40°; -30°) conformation shows a strong OH3-1 · · · O5 hydrogen bonding interaction in only 5% of the structures (compared with a population of 13% for the OH3 · · · O5+1 hydrogen bond in the (χ; χ-1) ≈ (-30°; 40°) conformation) (the hydrogen bond is not shown in the Figure), whereas OH3-1 is hydrogen bonding with the adjacent acetamide in 63% of the sampled structures of this conformer. Influence of the Acetamide on the Glycosidic Bond. The conformers of 14 clearly demonstrate that flexibility of a dihedral linkage, measured through the χ angle, is dependent on the nature of the surrounding sugar units. Therefore, some flexibility is obtained when both of the saccharide units linked by the glycoside bond are N-acetyl glucosamines, whereas greater flexibility is obtained when only the one toward the reducing site is acetylated. To point out the difference between the two situations further, 14, MeO-GlcN-GlcNAc-GlcNAc-OMe, is in the following compared with 8, MeO-GlcN-GlcNGlcN-OMe, 10, MeO-GlcN-GlcNAc-GlcN-OMe, and 11, MeO-GlcN-GlcN-GlcNAc-OMe. Comparing the rather rigid structure of the fully deacetylated chitosan model, 8, with the 33% DA MeO-GlcN-GlcNAcGlcN-OMe saccharide, 10, it is clear that only the glycoside bond between the nonreducing sugar rings A and B is markedly affected by the presence of the acetamide at ring B in 10. The conformations and populations of this linkage are very similar to those observed for the MeO-GlcN-GlcNAc-GlcNAc-OMe saccharide, 14. (The statistics of all conformations are listed in Table S7 of the Supporting Information.) Therefore, the nature of the unit at the reducing end, ring C, seems to have no influence on the bond linking rings A and B. Apparently, the presence of an acetamide at C2 affects only the nearest glycosidic bond significantly toward the nonreducing side. Similarly, in 11, where only ring C is acetylated, the glycosidic

3204


Skovstrup et al.

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

Figure 10. Important hydrogen bonds stabilizing the specific conformation of the glycosidic linkage in a GlcN-GlcNAc fragment in, for example, structure 11, identified in (a) the conformation at χ-1 ≈ 40° and (b) the χ-1 ≈ 170° conformation. For clarity, only rings B and C are shown, and only the distances involving the amines and acetamides are given.

bond between rings B and C is very flexible, whereas the glycosidic bond between rings A and B is not flexible and thus is not affected by the acetamide group at ring C. Introducing a second acetyl glucosamine on ring B, that is, turning from 11 to 14, results in a lower population of the conformation with χ-1 ≈ -90°, with a computed change in population from 7% in 11 to 1% in 14. Assuming a simple Boltzmann distribution among the conformers sampled, this corresponds to a rise in potential energy of ∼4.3 kJ/mol for visiting this conformation of the χ-1 angle. Similarly, conformers with χ-1 ≈ 170° completely disappear in 14. In return, the population of conformers with χ-1 ≈ -30° is increased from 5% in 11 to 9% in 14. Apparently, the introduction of the acetamide on ring B restricts the observed flexible nature of the glycoside bond between the two acetylated rings B and C to values around -30 and 40°. The reason that the conformation with χ-1 ≈ -30° is more favorable in 14 compared with that in 11 is a consequence of the χ-1 ≈ 40° conformation being less stabilized in 14. That is, in 11, the χ-1 ≈ 40° conformation is stabilized not only by the strong OH3-1 · · · O5 hydrogen bonding interaction but also by hydrogen bonding interactions between the primary cationic amine at ring B and the C5 hydroxymethyl of ring C (present in 44% of all sampled structures); this is shown in Figure 10a. In 14, ring B bears not the primary cationic amine but an acetamide, resulting in the hydrogen bonding interaction between the ring B amine and the ring C hydroxymethyl being much weaker and less-populated (by 17% of the sampled structures) and thus not stabilizing the χ-1 ≈ 40° conformation to the same extent. The loss of the χ-1 ≈ 170° conformation in 14 is most likely due to loss of the cooperative hydrogen bonding network extending from the amine of ring B to O3-1 onward to the adjacent acetamide on ring C, illustrated for 11 in Figure 10b. The reason for the markedly lower population of χ-1 ) -90° in 14 compared with that in 11 is not obvious given that there are no important nonbonded, attractive interactions between the two rings in either 11 or 14.

Tetrasaccharides. The 16 tetrasaccharides shown in Figure 2c were modeled, mimicking 0% DA chitosan (16), 25% DA chitosan (17-20), 50% DA chitosan (21-26), 75% DA chitosan (27-30), and chitin (31). The models thus include all possible combinations of the GlcN and GlcNAc building blocks in a tetrasaccharide. The conformational analysis (details given in the Supporting Information in Table S8) reveals that the same trends are seen for the tetrasaccharides, as was found for the di- and trisaccharides above. Again, it is evident, that the presence of an acetyl glucosamine unit is only influencing the very nearest glycosidic bonds, primarily toward the nonreducing side. The most flexible glycosidic bond is found in the GlcN-GlcNAc linkage sampling four conformations, followed by the GlcNAcGlcNAc structure with three possible values of χ. The least dynamic glycoside bonds are present in the GlcNAc-GlcN and GlcN-GlcN linkages sampling only one conformation with χ ≈ 40°. This confirms that it is the nature of the sugar to the reducing side of the glycoside bond that determines the flexibility of χ, whereas the sugar ring at the nonreducing side of the glycoside bond is less important in this respect. In other words, only a GlcNAc unit to the reducing side of the glycoside bond leads to flexibility (with four possible conformations in a GlcN-GlcNAc linkage), whereas a second GlcNAc unit to the nonreducing side slightly reduces this flexibility to two conformations, with a third conformation being rarely sampled. Persistence Lengths of Tetrasaccharides. Evidently, the flexibility of the glycosidic linkage is reflected in the persistence length of the saccharide. The persistence length of a polymer, that is, the length for which the memory of the initial orientation of the polymer persists,28 is, in the worm-like chain model,29 defined as the overall length of the polysaccharide measured as the sum of the projections of the length of each individual unit onto the direction defined by the first unit.30 However, because the longest oligomers in this study are tetramers, a direct measurement of the end-to-end distance of the tetrasaccharides is similarly an indication of the flexibility, but the number cannot be directly converted to persistence length for extended polymers. The average, minimum, and maximum end-to-end lengths of the examined tetrasaccharides were measured and are listed in Table S9 of the Supporting Information. The maximum endto-end length is found to be quite constant, ∼21.6 Å. The longest saccharide chain is found in saccharide MeO-GlcN-GlcNGlcNAc-GlcNAc-OMe, 26, which in the extended conformation has glycosidic bond angles: χ ) 38.3°, χ-1 ) 25.8°, and χ-2 ) 26.1°. The shortest end-to-end length is merely 11.9 Å, found in structure 30, MeO-GlcN-GlcNAc-GlcNAcGlcNAc-OMe, which indeed is a tetrasaccharide made up of three of the most flexible linkages. This structure has glycosidic bond angles of χ ) -108.7°, χ-1 ) -178.6°, and χ-2 ) 40.5° and is shown in Figure 11. The theoretically most flexible tetrasaccharide will be the one with the most GlcN-GlcNAc



3205

to-end length (99% of the snapshots have χ, χ-1, and χ-2 of ∼40°. In contrast, 25 is very flexible, with 29% of the sampled structures showing values of χ, χ-2, or both that are different from -30 and 40°.

Conclusions

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

linkages, that is, GlcN-GlcNAc-GlcN-GlcNAc. This corresponds to 25, which indeed is found to be the tetrasaccharide with the shortest average end-to-end length and also contains the conformation with the second shortest minimum length, 12.1 Å. The average end-to-end length varies by ∼0.5 Å, from 20.2 to 20.7 Å. Compound 16 (0% DA chitosan) is the most extended structure according to the average end-to-end lengths. A snapshot of 16 with an end-to-end length of 20.7 Å shows average glycosidic bond angles of 52.0 (χ), 47.3 (χ-1), and 39.6° (χ-2). Apparently, the extended, linear saccharides have glycosidic linkages, χ, around 40°. In Figure 12, plots of the end-to-end length as a function of simulation time are depicted for 16, 25, and 31 (chitin). The plots illustrate how the majorities of the structures sampled are similar in length, in the range of 20.3 to 21.1 Å. This is expected because 25 has 81% of the sampled structures in χ ≈ 40°, >99% in χ-1 ≈ 40°, and 82% in χ-2 ≈ 40°. 16, which mimics 0% DA chitosan, has the longest average end-to-end length, 20.7 Å, and essentially no structures deviate from the extended conformation. In 31 (chitin), snapshots occasionally deviate from the average length, resulting in the average end-to-end length being slightly shorter, 20.5 Å. As noted above, 25 is the most flexible of the examined tetrasaccharides. It has an average end-to-end length of 20.2 Å, which is 0.5 Å shorter than 16. In 25, many structures deviate from the average length. In general, snapshots with end-to-end lengths close to the average length show glycosidic bond angles around -30°, 40°, or both. A short end-

In this study, we have reported exhaustive and systematic evaluations of the conformational dynamics of the glycosidic linkages in chitin and chitosan with varying DA, as modeled in 31 di-, tri-, and tetrasaccharides. The results are clear-cut; it is the sugar ring to the reducing side that dictates the flexibility of the glycosidic linkage. Only a GlcNAc unit to the reducing side of the glycoside bond leads to flexibility. Four possible conformations are found in a GlcN-GlcNAc linkage, whereas a GlcNAc unit at the nonreducing side slightly reduces the glycosidic flexibility of the GlcNAc-GlcNAc linkage to three conformations, of which one is only rarely sampled. Therefore, the GlcN-GlcNAc linkage was found to be more flexible than the GlcNAc-GlcNAc linkage, both of which are much more flexible than the GlcNAc-GlcN and GlcN-GlcN linkages. However, most importantly, the GlcN-GlcNAc linkage regularly visits a conformation significantly different from those visited by the others. Brugnerotto et al.13a,b and Lamarque et al.14 find that extended chitosan polymers with up to 3000 sugar units having DA < 25% are quite flexible and that for DA ) 25-50% the chains are getting stiffer, whereas DA > 50% results in rather rigid structures, although it is stated by Lamarque et al. that these domains are most pronounced in polymers at a high weightaveraged degree of polymerization.14 Given the results of the work presented in this article, the presence of these three domains can be rationalized in more detail. The flexibility of a chitosan chain is determined by the number of glycosidic bond angles differing from -30 or 40°. Hence, the flexibility is almost exclusively determined by the number of GlcN-GlcNAc linkages,althoughaverysmallfraction(1%)oftheGlcNAc-GlcNAc angles also contributes to flexibility. The other two types of glycosidic linkage, GlcN-GlcN and GlcNAc-GlcN, do not contribute to the flexibility of oligo- or polysaccharides because they are found only with χ ≈ 40°. Therefore, the more GlcN-GlcNAc linkages found in a chain, the more flexible the chain will appear. Therefore, >50% DA will evidently result in fewer GlcN-GlcNAc linkages as the percentage of GlcN units drops with increasing DA and thus less-flexible chains, which is indeed consistent with the experimentally determined longer persistence lengths for chitin than those for chitosan with very low DAs13a and also observed by Franca et al. in their modeling study of decamers of chitin and 0% DA chitosan.13c The GlcN-GlcNAc linkage was found to sample four conformations of χ around -90, -30, 40, and 170°, respectively. The GlcNAc-GlcNAc linkage visited three of these conformations, namely, χ ≈ -90, -30, and 40°; however, the -90° conformation was only infrequently visited. The two stiff linkages, GlcNAc-GlcN and GlcN-GlcN, both sampled only one conformation of χ, which is the conformation around 40°. The reason for this was found to be the differences in hydrogen

3206


bonding possibilities, in particular, for OH3. In all four glycosidic linkages, the major conformation has χ ≈ 40°. This conformation is largely stabilized by a strong hydrogen bonding interaction 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, this hydrogen bonding interaction is present in virtually all snapshots, thereby effectively locking the glycosidic linkage of chitosan with low DAs. However, acetylation of the reducing sugar unit led to another good hydrogen bonding partner for OH3, namely, the carbonyl group of the adjacent acetamide, O(dC), and consequently, the otherwise persistent hydrogen bond between OH3 and the nonreducing sugar ring is now found in only ∼82% of the sampled structures, resulting in the observed much more flexible nature of these linkages. Franca et al. studied the effect of explicit water solvation on the conformational dynamics of decamers of chitin and 0% DA chitosan.13c In line with our observations, they found the stability of the intrachain hydrogen bond OH3 · · · O5+1 to be the main determinant of flexibility of the saccharide chains. Franca et al. use explicit solvation in their simulations, which allows for studying interactions with explicit water molecules. Even though our study uses an implicit continuum water model for the aqueous solution, the results from Franca et al.’s study compare very well to our observation for the two extremes, chitin and 0% DA chitosan, with respect to stability, population of certain hydrogen bonds, and so on. One discrepancy, however, is noted between the two studies; Franca et al. found an OH3 · · · O5+1 hydrogen bond population of 53% in 0% DA chitosan at low pH, whereas we find it to be present in 87 and >99% of the structures after minimization for our 0% DA models of chitosan, which must be accounted for by a slightly different hydrogen bonding network when explicit water molecules are included. For chitin, it was demonstrated that the explicit water resulted in the coordination of a water molecule to O3 and OH6+1, thereby stabilizing the OH3 · · · O5+1 hydrogen bonding interaction. However, the water molecule was also reported to interact with OH3 in 19% of the chitin structures and 27% of the chitosan structures when chitosan was modeled as charged at low pH.13c The adjacent acetyl group was reported not to be directly involved in this hydrogen bonding pattern. A charged primary amine, as found in chitosan, at this position must be expected to be directly involved in the hydrogen bonding pattern, thus coordinating a water molecule strongly between the primary amine and the adjacent OH3 and thereby orienting the OH3 group correctly and strongly stabilizing the OH3 · · · O5+1 interaction. It was shown by Franca et al. that in chitosan at low pH the orientation of the coordinated water molecule was quite different and much more specific and rigid than in chitin. Extending this to our results for chitosan with nonzero DA, the inclusion of explicit water and, in particular, a water molecule coordinating to the OH3, can be expected to perturb the stability of the OH3 · · · O5+1 hydrogen bonding interaction. In the case of a charged primary amine adjacent to the OH3 (as in 0% DA chitosan) the coordination of the water molecule can be speculated to result in an overall stabilization of the conformation at χ ≈ 40°. With an acetamide adjacent to the OH3, the water interaction is not as pronounced, and the perturbation of the OH3 · · · O5+1 interaction may only result in an overall slight stabilization of the χ ≈ 40° conformation because it simultaneously coordinates to OH6+1. Such a picture supports the trends outlined in this work, that a GlcNAc to the reducing side of the linkage leads to a less-stabilized χ ≈ 40° conformation and thus greater flexibility, also in water.

Skovstrup et al.

The detailed knowledge achieved in this study can be very valuable for studying enzymatic degradation products of chitinases and chitosanases. The knowledge about conformer distribution from this study may be compared directly to NMR data of degraded chitosan units and thereby assist in getting knowledge of the structure of the original enzymatic substrate.31 Along the same lines, with knowledge of the glycosidic conformational flexibility observed in this study, it is possible to quantify the composition of structurally unknown chitosan oligomers by NOE experiments across the glycosidic linkage. We foresee that such knowledge will be invaluable when designing chitosan variants with tailor-made properties.32 Acknowledgment. The Danish Center for Scientific Computing is acknowledged for resources and computing time along with financial support from the Danish Natural Science Research Council and Research Foundation as well as from the Carlsberg, Lundbeck, and Novo Nordisk Foundations. Jens Ølgaard Duus, Carlsberg Research Center, is acknowledged for inspiration in the beginning of the project and for discussing the results, progress, and perspectives of the findings. Supporting Information Available. Tabular material listing the statistical analysis of populations of the identified conformations for di-, tri-, and tetrasaccharides and figures of the modeled tri- and tetrasaccharides along with graphs and plots showing conformational flexibilty in the models. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Agboh, O. C.; Qin, Y. Polym. AdV. Technol. 1997, 8, 355–365. (2) Yi, H.; Wu, L.; Bentley, W. E.; Ghodssi, R.; Robloff, G. W.; Culver, J. N.; Payne, G. F. Biomacromolecules 2005, 6, 2881–2894. (3) Janes, K. A.; Calvo, P.; Alonso, M. J. AdV. Drug DeliVery ReV. 2001, 47, 83–97. (4) Khan, T. A.; Peh, K. C.; Ch’ng, H. S. J. Pharm. Pharm. Sci. 2000, 3, 303–311. (5) Hsu, S.; Whu, S. W.; Tsai, C.; Wu, Y.; Chen, H.; Hsieh, K. J. Polym. Res. 2004, 11, 141–147. (6) Shahidi, F.; Arachchi, J. K. V.; Jeon, Y. Trends Food Sci. Technol. 1999, 10, 37–51. (7) (a) Yui, T.; Imada, K.; Okuyama, K.; Obata, Y.; Suzuki, K.; Ogawa, K. Macromolecules 1994, 27, 7601–7605. (b) Okuyama, K.; Noguchi, K.; Miyazawa, T.; Yui, T.; Ogawa, K. Macromolecules 1997, 30, 5849–5855. (8) (a) Lertworasirikul, A.; Yokoyama, S.; Noguchi, K.; Ogawa, K.; Okuyama, K. Carbohydr. Res. 2004, 339, 835–843. (b) Okuyama, K.; Noguchi, K.; Kanenari, M.; Egawa, T.; Osawa, K.; Ogawa, K. Carbohydr. Polym. 2000, 41, 237–247. (c) Lertworasirikul, A.; Tsue, S.; Noguchi, K.; Okuyama, K.; Ogawa, K. Carbohydr. Res. 2003, 338, 1229–1233. (d) Kawahara, M.; Yui, T.; Oka, K.; Zugenmaier, P.; Suzuki, S.; Kitamura, S.; Okuyama, K.; Ogawa, K. Biosci., Biotechnol., Biochem. 2003, 67, 1545–1550. (9) Okuyama, K.; Noguchi, K.; Hanafusa, Y.; Osawa, K.; Ogawa, K. Int. J. Biol. Macromol. 1999, 26, 285–293. (10) (a) Li, Q. X.; Song, B. Z.; Yang, Z. Q.; Fan, H. L. Carbohydr. Polym. 2006, 63, 272–282. (b) Pedroni, V. I.; Gschaider, M. E.; Schulz, P. C. Macromol. Biosci. 2003, 3, 531–534. (c) Pedroni, V. I.; Schulz, P. C.; Gschaider, M. E. Colloid Polym. Sci. 2003, 282, 100–102. (11) For a deeper discussion of the known crystal structures of chitosans and chitosan salts, please see: (a) Ogawa, K.; Yui, T.; Okuyama, K. Int. J. Biol. Macromol. 2004, 34, 1–8. (b) Mogilevskaya, E. L.; Akopova, T. A.; Zelenetskii, A. N.; Ozerin, A. N. Polym. Sci. Ser. A 2006, 48, 116–123. (12) (a) Sorlier, P.; Denuzie`re, A.; Viton, C.; Domard, A. Biomacromolecules 2001, 2, 765–772. (b) Schatz, C.; Viton, C.; Delair, T.; Pichot, C.; Domard, A. Biomacromolecules 2003, 4, 641–648. (13) (a) Brugnerotto, J.; Desbrie`res, J.; Heux, L.; Mazeau, K.; Rinaudo, M. Macromol. Symp. 2001, 168, 1–20. (b) Brugnerotto, J.; Desbrie`res, J.; Roberts, G.; Rinaudo, M. Polymer 2001, 42, 9921–9927. (c) Franca, E. F.; Lins, R. D.; Freitas, L. C. G.; Straatsma, T. P. J. Chem. Theory Comput. 2008, 4, 2141–2149.

Conformational Flexibility of Chitosan upon Degree of Acetylation (14) Lamarque, G.; Lucas, J. M.; Viton, C.; Domard, A. Biomacromolecules 2005, 6, 131–142. (15) Mazeau, K.; Pe´rez, S.; Rinaudo, M. J. Carbohydr. Chem. 2000, 19, 1269–1284. (16) Yamaguchi, I.; Itoh, S.; Suzuki, M.; Sakane, M.; Osaka, A.; Tanaka, J. Biomaterials 2003, 24, 2031–2036. (17) Tomihata, K.; Ikada, Y. Biomaterials 1997, 18, 567–575. (18) Espinosa, J. F.; Asensio, J. L.; Garcia, J. L.; Laynes, J.; Bruix, M.; Wright, C.; Siebert, H. C.; Gabius, H. J.; Canãda, F. J.; JimeńezBarbero, J. Eur. J. Biochem. 2000, 267, 3965–3978. (19) MacroModel, version 9.0; Schro¨dinger, LLC: New York, 2005. (20) Senderowitz, H.; Still, W. C. J. Org. Chem. 1997, 62, 1427–1438. (21) Brocca, P.; Bernardi, A.; Raimondi, L.; Sonnino, S. Glycoconjugate J. 2000, 17, 283–299. (22) Maestro, version 7.0; Schro¨dinger, LLC: New York, 2005. (23) Senderowitz, H.; Parish, C.; Still, W. C. J. Am. Chem. Soc. 1996, 118, 2078–2086. (24) (a) Guarnieri, F.; Still, W. C. J. Comput. Chem. 1994, 15, 1302–1310. (b) Bernardi, A.; Raimondi, L.; Zanferrari, J. THEOCHEM 1997, 395396, 361–373. (25) (a) Batchelor, R. J.; Green, D. F.; Johnston, B. D.; Patrick, B. O.; Pinto, B. M. Carbohydr. Res. 2001, 330, 421–426. (b) Asensio, J. L.;


(26) (27) (28) (29)

(30) (31) (32)

3207

Canãda, F. J.; Garcia-Herrero, A.; Murillo, M. T.; FernańdezMayoralas, A.; Johns, B. A.; Kozak, J.; Zhu, Z.; Johnson, C. R.; Jimeńez-Barbero, J. J. Am. Chem. Soc. 1999, 121, 11318–11329. Wolfe, S. Acc. Chem. Res. 1972, 5, 102–111. Clayden, J.; Greeves, N.; Warren, S.; Wothers, P. Organic Chemistry; Oxford University Press: New York, 2001. Rivetti, C.; Walker, C.; Bustamante, C. J. Mol. Biol. 1998, 280, 41– 59. (a) Kratky, O.; Porod, G. Recl. TraV. Chim. Pays-Bas 1949, 68, 1106– 1122. (b) Shellman, J. A. Biopolymers 1974, 13, 217–226. (c) Landau, L. D.; Lifshitz, E. M. Statistical Physics, 3rd ed.; Pergamon Press: New York, 1980; Vol. 3. (a) Yamakawa, H. Modern Theory of Polymer Solutions; Harper & Row, New York, 1971. (b) Lee, S. H.; Kapral, R. J. Chem. Phys. 2006, 124, 214901. Leisner, J. J.; Larsen, M. H.; Ingmer, H.; Petersen, B. O.; Duus, J. Ø.; Palcic, M. M. J. Appl. Microbiol. 2009, 107, 2080–2087. Howard, K. A.; Paludan, S. R.; Behlke, M. A.; Besenbacher, F.; Deleuran, B.; Kjems, J. Mol. Ther. 2009, 17, 162–168.

BM100736W

Conformational Flexibility of Chitosan: A Molecular Modeling Study

Recommend Documents