Dynamics of Chitosan by 1H NMR Relaxation - Biomacromolecules

Biomacromolecules 2010, 11, 2079–2086

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Dynamics of Chitosan by 1H NMR Relaxation Ramon Novoa-Carballal, Eduardo Fernandez-Megia,* and Ricardo Riguera* Departamento de Quı´mica Orgańica, Facultad de Quı´mica, and Unidad de RMN de Biomolećulas Asociada al CSIC, Universidad de Santiago de Compostela, Avda. de las Ciencias S.N. 15782 Santiago de Compostela, Spain Received April 23, 2010; Revised Manuscript Received June 18, 2010

The dynamics of chitosan (CS) in solution have been studied by 1H NMR relaxation [longitudinal (T1) and transverse (T2) relaxation times and NOE] as a function of the degrees of acetylation (DA, 1-70) and polymerization (DP, 10-1200), temperature (278-343 K), concentration (0.1-30 g/L), and ionic strength (50-400 mM). This analysis points to CS as a semirigid polymer with increased flexibility at higher DA in agreement with reduced electrostatic repulsions between protonated amino groups.

Introduction Chitosan (CS) is a linear polysaccharide comprising variable proportions of glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) through β(1-4) glycosidic bonds (Figure 1). Thanks to its low toxicity and high biodegradability, biocompatibility, and antimicrobial activity, CS has emerged as an interesting biopolymer with applications in drug delivery, biofabrication, and food industry.1–5 The biological, physical, and physicochemical properties of CS in aqueous solutions are determined by external (pH, ionic strength, temperature) as well as structural parameters [degrees of acetylation (DA) and polymerization (DP)].6–8 CS has been usually described in aqueous solution as a semirigid polymer by viscometry, and dynamic (DLS) and static light scattering (SLS).9–11 Interestingly, the conformation of CS in solution is highly dependent on DA because of the balance between two possible contrasting effects: the electrostatic repulsion between adjacent protonated amino groups, and the steric hindrance associated to the bulkier acetamido groups. Thus, increasing DA values lead on the one hand to a reduction of the electrostatic repulsion (and so, to a higher flexibility), but on the other hand, to an increase in steric hindrance hampering chain rotation (higher chain stiffness).12 Several reports on the study of the influence of DA on the conformation of CS have been published so far with contradictory outcomes. Qin and co-workers described variations in the R and K factors in the Mark-Houwink-Sakurada equation ([η] ) KMwR) when DA increases from 0 to 30.13 In contrast, the groups of Domard, Berth, and Rinaudo found the intrinsic persistence length (Lp) being independent of DA in the range DA 2-60.8,14–16 In a more recent study of a homogeneous series of reacetylated CS with different DP (650-2600) and DA (0-70) by SLS, SEC-MALLS, and capillary viscosity, Domard and co-workers reported an increase in flexibility with DA for CS samples with DP up to a value around 1600, while increasingly stiffer chains at higher DP.10 Vårum and co-workers have reopened the debate by studying a homogeneous series of reacetylated CS with different DP (200-2500) and DA (0-60) by size exclusion chromatography-multiangle laser-light scattering (SEC-MALLS) and viscosity. They found little to no * To whom correspondence should be addressed. E-mail: [email protected] (E.F.-M.); [email protected] (R.R.).

Figure 1. Chitosan (CS).

detectable influence of DA on intrinsic viscosity, radius of gyration (Rg), and Lp.9 Nuclear magnetic resonance (NMR) is a powerful tool for the study of the dynamics and flexibility of macromolecules.17 Information about the motions within these systems is usually extracted by measuring longitudinal (T1) and transverse relaxation times (T2), along with nuclear Overhauser effect (NOE).18,19 13 C relaxation is commonly preferred for quantitative modeling of polymer dynamics including polysaccharides,20–27 as the knowledge of the C-H distance and the absence of spin diffusion allow to probe for local information. In addition, relevant qualitative information on the motions within several polymers has been extracted from 1H relaxation,19 that benefits from a faster data acquisition due to the high natural abundance of the 1H nucleus and its high gyromagnetic ratio. With the aim of studying the effect of DA and DP on the dynamics of CS in aqueous solution, we decided to perform a NMR relaxation study that could shed light on previous conflicting results by viscosity and LS. Because of the large number of samples required (with varying DA and DP) and the typically long NMR acquisition times, a 1H relaxation study suited more convenient for the present purpose. Herein, we describe our results on the dynamics of CS in solution by analyzing 1H NMR relaxation parameters (T1, T2, and NOE) of a collection of samples with varying DA (1-70) and DP (10-1200) at several temperatures, concentrations, and ionic strengths.

Materials and Methods Four commercial CS samples with different DP and DA were purchased. These samples were denoted as Cx-y, where “x” represents the DP and “y” the DA. Samples CS360-7 and CS480-14 were obtained from FMC BioPolymer as their hydrochloride salts: Protasan Cl 110 (batch number 310-490-01) and Protasan Cl 113 (batch number FP110-02), respectively. Samples CS730-17 and CS1200-20 were obtained from Fluka: CS low MW (catalog number 22741, lot 407568/1) and

10.1021/bm100447f  2010 American Chemical Society Published on Web 07/02/2010

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CS high MW (catalog number 22743, lot 371936/1), respectively. Prior to NMR studies, commercial CS samples were dissolved in 0.5% (w/ v) AcOH (5 g/L) and sequentially dialyzed against 10-3 M HCl, 5.5 × 10-3 M NH4OH, and deionized water to obtain pure CS samples in their free amino form. Then, samples were lyophilized. Pullulan samples were purchased from Polymer Standards Service (batch number pulkit02; Mw 342-710000, PDI ∼1.1). pH values were determined using a portable pH meter (Crison PH25) connected to a glass electrode (Crison 5209). The calibration of the pH meter was done with pH 4.01, 7.00, and 9.21 buffer solutions (Crison). pD values were obtained by adding 0.40 units to the pH reading.28 N-Deacetylation of CS. Finely grounded CS was suspended in aqueous 40% (w/v) NaOH at 333-353 K under a N2 stream. After 15-45 min of stirring, the reaction mixture was filtered and thoroughly washed with deionized water (333 K). The resulting powdery residue was dried overnight under vacuum (P2O5). This process was repeated until a desired DA was obtained.29 N-Acetylation of CS. CS samples with DA larger than those present in the commercial samples were prepared by homogeneous Nacetylation.30 Briefly, CS was dissolved (10 g/L) in a 1:1 mixture 0.5% (w/v) AcOH/1,2-propanediol at rt. Then, to reach a certain DA value, a freshly prepared solution of acetic anhydride in 1,2-propanediol was added. The resulting solution was stirred at rt for 2 h. After sequential dialysis against 10-3 M HCl, 5.5 × 10-3 M NH4OH, and deionized water, the CS solution was lyophilized. Depolymerization. CS samples with DP lower than those present inthecommercialsampleswerepreparedbynitrousaciddepolymerization.31–33 Briefly, CS480-14 · HCl was dissolved (10 g/L) in deionized water under magnetic stirring at rt. When CS was completely dissolved, 1 M NaNO2 was added dropwise. After 1 h of stirring, 1 M NaOH was added until pH 10 was reached. The precipitated CS (samples CS60-14 and CS160-14) was recovered by centrifugation, washed several times with deionized water, and lyophilized. Sample CS10-14 was obtained by depolymerization of a solution of CS60-14 in 0.056 M TFA-d (8 g/L) by addition of 0.05 M NaNO2. Determination of Molecular Weight. Weight-average molecular weight (Mw) of CS samples was determined by SEC-MALLS.6–8,10 An Iso Pump G1310A (Hewlett-Packard) was connected to two PSS Novema GPC columns (10 µm, 30 Å, 8 × 300 mm; and 10 µm, 3000 Å, 8 × 300 mm). A PSS SLD7000 MALLS detector (Brookhaven Instruments Corporation) operating at 660 nm and a G1362A refractive index detector (Agilent) were connected on line. A 0.15 M NH4OAc/ 0.2 M AcOH buffer (pH 4.5) was used as eluent. Polymer solutions were filtered through 0.2 µm pore size membranes before injection. Polymer concentrations were in the range 0.1-5 g/L, depending on DP and DA. Refractive index increment dn/dC was taken from the literature.6–8 In the case of CS10-14, as the LS signal was of low intensity, molecular weight was confirmed by NMR [relative integration between the H1 signals of GlcN (4.88 ppm) and the 2,5-anhydro-Dmannose(5.01ppm)obtainedatthereducingendduringdepolymerization].31–33 NMR Spectroscopy. NMR experiments were recorded at Bruker DRX-500 [operating at 11.74 T, equipped with an inverse detected probe (BBI 5 mm with z gradients), and controlled with Topspin 1.3 software], Varian Mercury 300 [operating at 7.04 T, equipped with an autoswitchable direct detected probe (5 mm with z gradients), and controlled with VNMRj 1.1 software], and Bruker DPX-250 [operating at 5.87 T, equipped with a direct detected probe (BBO 5 mm), and controlled with XWINNMR software] spectrometers. NMR experiments were recorded at 298 K, unless otherwise stated. 1H chemical shifts are expressed in ppm and referred to internal sodium 3-trimethylsilylpropane sulfonate (TSP). Mestre-C Software (Mestrelab Research) was used for spectral processing and OriginPro7.5 Software (Originlab) to perform the exponential/linear fittings for T1, T2, and NOESY experiments. CS samples were dissolved in 0.056 M TFA-d in D2O at a concentration of 8 g/L (ionic strength 50 mM). CS samples at 8 g/L with ionic strengths 200 and 400 mM were prepared by adding NaCl dissolved in 0.056 M TFA-d in D2O to a 10 g/L solution of CS in

Novoa-Carballal et al. Table 1. Structural Parameters of the CS Samples Studied CS

Mwa (105 g/mol)

DPwa

DAb

PDIa

CS10-14 CS60-14 CS160-14 CS360-1 CS360-7 CS360-19 CS360-30 CS360-39 CS360-48 CS360-70 CS730-1 CS730-6 CS730-17 CS730-27 CS730-39 CS730-70 CS1200-1 CS1200-3 CS1200-20 CS1200-40 CS1200-50 CS1200-70

0.02 0.10 0.27 0.58 0.57 0.67 0.63 0.63 0.69 0.70 1.23 1.26 1.21 1.35 1.28 1.32 1.89 1.89 2.06 2.17 2.35 2.28

12c 60 160 359 343 365 365 355 369 369 762 769 719 782 721 700 1170 1163 1214 1219 1238 1200

14 14 14 1 7 19 30 39 48 70 1 6 17 27 39 70 1 3 20 40 50 70

1.25 1.21 1.26 1.45 1.61 1.38 1.50 1.35 1.55 1.70 1.43 1.23 1.42 1.31 1.24 1.12 1.20 1.40 1.51 1.37 1.6 2.0

a Determined by SEC-MALLS. by 1H NMR.

b

Determined by 1H NMR.

c

DP ) 10

0.056 M TFA-d. For the experiments carried out at different concentrations, a 350 mM CD3CO2D/135 mM NaOD (D2O) buffer solution, pD 4.5, was used. Characterization of CS. DA values were determined by 1H NMR (10 mg/mL) in 2% DCl at 298 K (DA < 40),34 or 20% DCl at 343 K (DA > 40).35 GlcNAc distribution was determined by analysis of the relative intensities of the diad frequencies in 13C NMR as proposed by Vårum and co-workers, using modified acquisition parameters (62.9 MHz, 200.000 scans, 0.1 s acquisition time, no relaxation delay).36 With this aim, commercial CS samples were first dissolved in 0.16 M TFA-d (30 g/L) and treated with 1 M NaNO2 to reduce the DP.31–33 Measurement of T1 and T2 Relaxation Times. T1 values were determined using a standard inversion-recovery pulse sequence. The recovery delay (τ) between the inversion pulse and the read pulse was varied along 16 values. The number of scans was 16, 32, or 128 depending on sample concentration. T2 values were determined using the Carr-Purcell-Meiboom-Gil pulse sequence using 16 values of τ, with a delay between π pulses of 140 µs. The number of scans was 16, 64, or 256 depending on sample concentration. Given T1 and T2 are averaged values among 3-5 measurements. Estimated uncertainties are in all cases smaller than 2% for T1, and 10% for T2. A recovery delay between scans of 13 s (>5 × T1) was set for all 1H NMR experiments. 1D Selective NOESY and ROESY Experiments. 1D PFGSENOESY and 1D PFGSE-ROESY selective experiments37 were performed for the H2 of GlcN and the NAc group. In both cases, the selection of the signal was achieved with an off-resonance phase-shifted Gaussian pulse truncated at 5% level, and the duration of the pulse was 4 ms. 1D-NOESY experiments were recorded with mixing times 3, 25, 50, 75, 100, 200, and 350 ms. The number of scans was 16. 1D-PFGSE-ROESY experiments were acquired with mixing times 5, 10, 30, 60, and 100 ms, and the spin locking field strength was 2.27 kHz. The number of scans was 128. A recovery delay between scans of 13 s (>5 × T1) was used for all the experiments.

Results and Discussion A whole set of 22 CS samples with DP in the range 10-1200 and DA values 1-70 were prepared from four commercial samples, namely, CS360-7, CS480-14, CS730-17, and CS1200-20 (Table 1). The analysis of the relative intensities of the dyads in the 13C NMR spectra of these commercial samples was in

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Table 2. Representative Relaxation Times of CS360-48 δ (ppm) T1 (s) T2 (ms)

2.05 1.66

3.16 1.57 24

3.56 1.42 21

3.65-3.74 1.39 19

good agreement with a homogeneous distribution of GlcNAc units along the polymer chains.36 From these samples, CS with higher and lower DA were obtained by N-acetylation30 and deacetylation,29,38 respectively, following procedures ensuring a homogeneous distribution of N-acetyl groups. In addition, CS samples of DP 10, 60, and 160 were obtained by nitrous acid depolymerization of CS480-14.31–33 1 H NMR relaxation parameters of the CS samples in Table 1 were recorded in aqueous trifluoroacetic acid (TFA) at a concentration of 8 g/L (0.056 M TFA-d in D2O). Aqueous TFA not only dissolves CS samples faster than hydrochloric acid, but also samples of higher DA. The selection of TFA-d is also supported by the lack of the residual acetyl overlapping signal present in CD3CO2D solutions. In addition, solutions of CS in 0.056 M TFA-d were stable for 1 month at rt with no variation in DA and DP as determined by 1H NMR and SEC-MALLS. Typical 1H NMR spectra of CS samples (DP 360) with various DA are shown in Figure S1 in the Supporting Information (SI). The resonances corresponding to H1 and NAc of GlcNAc and H1 and H2 of GlcN are well separated as previously reported.39,40 The assignment of the remaining resonances within the multiplet at 3.5-4.0 ppm was possible by comparison of their chemical shifts with those of CS and chitin hexamers (Table S1 in the SI).41 Relaxation Data Analysis. In most polymeric materials, the general dependence of the relaxation times (T1 and T2) on correlation time (τ) is similar to the one observed for isotropic motions, even when a variety of motions are present.18 Thus, as τ increases, T1 values decrease monotonically, reaching a minimum followed by an increase in T1 with a further increase in τ. On the contrary, T2 values decrease continually with increasing τ, displaying always smaller values than T1, especially at long τ. In this context, τ should be understood as an effective correlation time (τeff), which represents an average of the correlation times for every motion affecting relaxation. Although 1H dipolar relaxation between neighboring protons is the main relaxation pathway in polymeric systems, crosscorrelation effects, usually leading to nonexponential decays, may also be present.18 Accordingly, the time-dependence of the longitudinal and transverse magnetizations were carefully analyzed for the CS samples in Table 1. Monoexponential decays and magnetization recoveries were typically obtained (in CPMG and inversion recovery experiments) for all the 1H resonances in the whole range of DA and DP analyzed (shown in Figures S2 and S3 in the SI for the H2 of GlcN), with the exception of the NAc group. In this case, a nonexponential T2 decay was always observed, a fact that has been interpreted as due to cross-correlation effects from the free rotation of the methyl group.18 It is worth mentioning that, although nonexponential decays in macromolecules are not exclusive of T2, are known to affect T1 to a lower extent.42–44 Representative T1 and T2 values for CS360-48 are shown in Table 2. T2 values in the range of 20 ms and much larger T1 of around 1.5 s were typically obtained. The existence of these large T1 - T2 differences not necessarily implies relaxation occurring outside the extreme narrowing region (long τ). Thus, in polymers with a wide spectrum of anisotropic motions, T1 senses high frequency motions, while T2 responds to both high and low frequencies.18,19 Interestingly, small but reproducible differences

3.74-3.79 1.44 25

3.79-3.96 1.38 24

4.55 1.44 16

4.88 1.52 17

were observed for the T1 of the sugar ring protons, in agreement with a semirigid character of CS in solution (cross-relaxation not rapid enough to produce complete T1 averaging).45 For comparison purposes, the 1H T1 of hyluronan, pullulan, and dextran were also determined under identical experimental conditions (temperature and spectrometer frequency), revealing 1 H T1 differences for hyaluronan similar to CS (ca. 0.1 s), and larger differences in the range 0.6-0.8 s for the more flexible pullulan and dextran. In order to study the effect of DA, DP, and concentration on the dynamics of CS in aqueous solution, the H2 of GlcN was selected as a probe resonance. This selection is based on the presence of this probe in all CS samples studied independently on DA, and this resonance being well separated in the 1H NMR spectrum, and not suffering from cross-correlation effects. Dependence of 1H Relaxation Times on DP. In modeling the dynamics of polysaccharides, polypeptides, and synthetic polymers, two types of motions are considered: First, the overall rotatory diffusion of the polymer chain as a whole, and second local chain motions. In this context, localized motions are considered the segmental reorientations via conformational transitions, anisotropic internal rotations of pendant groups, and small-amplitude librations.18,19 For sufficiently high molecular weight random coil polymers (above a critical molecular weight), the overall motion is much slower than the chain local motions, and thus makes a negligible contribution to the T1 values of the backbone and side-chain nuclei. Below that critical chain length, relaxation times increase with decreasing molecular weight as overall motions become sufficiently rapid to influence relaxation.18,19 Indeed, this critical chain length depends on chemical structure, showing larger values on increasing chain stiffness. This length is fairly low for flexible polysaccharides (around 7-15 monosaccharide units for pullulan in D2O,46 cellulose triacetate in CDCl3,47 and amylose and dextran in D2O)48 and poly(ethylene glycol) (30 repeating units in D2O), but increases for more rigid polymers up to 100 repeating units for styrene, and nearly 300 for poly(methylmethacrylate) (both in CDCl3).18 Interestingly, extremely rigid polymer chains can exceptionally display relaxation times with extended molecular weight dependence. Thus, poly(γ-benzyl-L-glutamate), a polyaminoacid adopting a highly rigid R-helical conformation in DMF, displays increasing T1 values at DP 137-393 as a result of reduced internal motions and relaxation being controlled by overall tumbling.18,49 Figure 2a shows the 1H T1 dependence of CS on DP (DP 10-1200, samples with average DA 16) as well as for the flexible unbranched polysaccharide pullulan, taken as a representative flexible polysaccharide (2-1265 monosaccharide units, the structure of pullulan is shown in Figure S4 in the SI).50 While 1H T1 in pullulan resulted to be independent of molecular weight above 8 sugar units (in agreement with its known great flexibility and previous 13C T1 measurements),46 CS showed decreasing T1 values up to a critical DP in the range 360-730. This critical DP compares well with polymers such as poly(methylmethacrylate) (critical DP < 300) and poly(dimethyl siloxane) (critical DP < 400),18 and reveals a semirigid character of CS that agrees with previous reports by viscometry and light scattering.9–11

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Figure 2. Dependence of 1H T1 (a) and T2 (b) on DP for CS (H2 of GlcN, samples with average DA 16) and pullulan (H4 of the A unit in the repeating maltotriose) at 8 g/L in 0.056 M TFA-d (298 K, 500 MHz). CS samples: CS10-14, CS60-14, CS160-14, CS360-19, CS730-17, CS1200-20. Lines are guides for the eye.

The dependence of T2 on molecular weight in random coils (at concentrations lower than an average value of 100 g/L) follows a similar trend to T1, showing constant values above a critical DP. At higher concentrations, nevertheless, T2 reveals as a decreasing function of molecular weight. This higher sensitivity of T2 to molecular weight and concentration (compared to T1) results from the contribution of low frequency motions to the J(0) spectral density in the expression for T2. Thus, as macroscopic viscosity and chain entanglements increase with concentration, long-range motions are hindered to a greater extent than segmental motions and so T2 drops while T1 remains unaffected. Similar effects are typically seen also in polymer melts.18,51 Figure 2b shows the variation of 1H T2 with DP for CS and pullulan (8 g/L). While pullulan reached a constant T2 value around a critical size of 70 sugar units (larger critical size than the one obtained for T1 as a result of the contribution of low frequency motions to T2), for CS T2 diminished with molecular weight in the whole range of DP studied. This decrease was initially fast up to DP 160 (from 110 to 27 ms) and slowed down at higher DP values (from 27 to 15 ms), reflecting the restriction of long-range motions on increasing intermolecular interactions at higher DP. The fact that this continuous decrease of T2 with DP is observed at concentrations as low as 8 g/L reinforces the semirigid character of CS. Dependence of 1H Relaxation Times on DA. Having established the usefulness of 1H relaxation measurements in the study of the dynamics of CS and its semirigid nature, a T1 and T2 relaxation study of samples with varying DA (1-70) was addressed with the aim of ascertain the effect of this parameter on the solution properties of CS (DP: 360, 730, 1200; Table 1). As seen in Figures 3 and 4, T1 and T2 revealed a slight increase with DA in the range 1-70, an effect that resulted in a more pronounced increase at higher DP values (increase in

Figure 3. Dependence of 1H T1 on DA for CS (H2 of GlcN) at 8 g/L in 0.056 M TFA-d (298 K, 500 MHz; uncertainty ranges are shown on top of bars).

T1 of 0.06 s for DP 360, and 0.12 s for DP 730 and 1200; increase in T2 of 1 ms for DP 360, and 3 ms for DP 730 and 1200). This picture suggests an increase in flexibility for the more acetylated CS and points to a reduction in electrostatic repulsions between protonated amino groups as the main factor controlling CS flexibility on increasing DA (as opposed to the increasing steric hindrance associated to the bulkier acetamido groups). The 2-fold larger relative increment of T2 with DA compared to T1 reflects again the higher sensitivity of T2 to low frequency motions. In addition, Figures 3 and 4 illustrate that irrespective of DA, the dependence of relaxation times with DP follows the same trend shown in Figure 2. DP 360 shows slightly larger T1 values than DP 730 and 1200 and a monotonic decrease of T2 with DP is observed. Dependence of 1H Relaxation Times on Concentration. In polymers, the variation of 1H T1 and T2 with concentration follows a similar trend as with molecular weight. For this reason, it is usually employed as a source of additional information about the dynamical properties of these species in solution. In general, on increasing the concentration of random coils, and in spite of huge changes in macroscopic viscosity, T1 and T2 remain essentially independent of concentration up to critical values above which relaxation times decrease. Characteristic critical concentrations are approximately 100 g/L for T2 and much larger for T1 (in the range of 10-15% molar fraction of monomer units). This huge difference in critical concentrations for T1 and T2 results from the aforementioned contribution of low frequency motions to the J(0) spectral density in the expression for T2.18,19,51


Figure 4. Dependence of 1H T2 on DA for CS (H2 of GlcN) at 8 g/L in 0.056 TFA-d (298 K, 500 MHz; uncertainty ranges are shown on top of bars).

Figure 5. Dependence of 1H T1 (a) and T2 (b) on concentration for CS (H2 of GlcN) in 350 mM CD3CO2D/135 mM NaOD buffer, pD 4.5 (298 K, 500 MHz).

The dependence of 1H T1 and T2 on concentration for CS (CS1200-3 and CS1200-70) is shown in Figure 5. While T1 was shown to be independent of concentration between 0.1 and 30 g/L for the two limiting DA values studied, T2 remained constant up to 1 g/L, but decreased afterward. This picture is in agreement with T1 relaxation being governed by local motions, while T2 also senses long-range motions that are hindered above the overlap concentration (C*).10 This fairly low critical concentration for T2 in CS reflects again its semirigid character when compared with typical random coils.18,19,51


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Dependence of 1H T1 Relaxation Time on Temperature. The variation of T1 with the temperature has been recognized as an accurate means to ascertain the relative flexibility within polysaccharides. Thus, the position of the T1 minimum (ωo · τ ∼ 1) can be used to estimate an effective correlation time of the local motions involved in relaxation (τeff ∼ 3 × 10-10 s at 500 MHz),45 while differences in the position of this minimum as a qualitative indicator of the relative rates of these motions: the lower the temperature of the T1 minimum, the higher the rate of the local motions.20–24 Four CS samples were selected (CS360-1, CS360-70, CS1200-3, CS1200-70) for studying the variation of T1 with the temperature (278-343 K). Before carrying the T1 measurements, the stability of the samples in 0.056 M TFA-d (8 g/L) at 323 and 343 K was assessed by 1H NMR and SEC-MALLS (looking for possible hydrolysis of the NAc groups and O-glycosidic linkages). It was found that samples with DA 1-3 were completely stable under these conditions, while those with DA 70 were depolymerized at 343 K in less than 15 min.52 Accordingly, the T1 relaxation study of the less acetylated samples covered the range 278-343 K, while those with DA 70 were analyzed up to 323 K. After 1H T1 measurements, the integrity of the samples was confirmed by 1H NMR and SECMALLS. Figure 6a,b shows that, as temperature increases, T1 decreases down to a minimum (between 307 and 323 K) to subsequently increase with further increments of temperature. The position of the T1 minimum is clearly sample-dependent [CS, T1 minimum (K): CS360-1, 313; CS360-70, 307; CS1200-3, 323; CS1200-70, 313], with minima shifted to lower temperatures at higher DA in agreement with the aforementioned faster local dynamics of the more acetylated CS samples (Figures 3 and 4). As longitudinal relaxation in DP 360 is still affected by overall motions at 298 K, DP 360 samples display larger T1 values than DP 1200 (Figure 6c,d). At higher temperatures, larger differences in T1 develop as a result of faster overall motions. On the contrary, at lower temperatures, T1 values converge as slower overall motions stop influencing longitudinal relaxation. Additional information on local motions as responsible of longitudinal relaxation in CS can be obtained by comparing τeff (∼3 × 10-10 s) and the overall motion correlation time (τR) at the temperature of the minimum. An estimation of τR for the four CS samples analyzed resulted from the hydrodynamic equation τR ) 2Mw [η]ηo/3RT (where Mw is the molecular weight, [η] is the intrinsic viscosity of the polymer in solution, and ηo is the solvent viscosity; see the SI).20–24 τR values in the range 10-5-10-6 s were obtained (Table S2 in the SI), much larger than the estimated τeff, suggesting that longitudinal relaxation is dominated by local motions (in fact, as the hydrodynamic equation is applicable for infinite dilution, under the present experimental conditions, τR must be considerably higher than 10-5-10-6 s). Dependence of 1H T1 Relaxation Time on Ionic Strength. Another essential parameter to consider when studying the dynamics of polyelectrolytes such as CS is the ionic strength.6,11,16,53 It is known that electrostatic repulsions are very dependent on ionic strength, so we decided to analyze its influence on the local motions of CS. With this aim, the 1H T1 of two CS samples with limiting DA values (CS1200-1 and CS1200-70) were analyzed at ionic strengths 50-400 mM. As seen in Figure 7, on increasing the ionic strength, larger T1 values resulted for the two samples (similar relative enhancements), suggesting that the electrostatic repulsions are not

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Figure 7. Dependence of 1H T1 on ionic strength for CS (H2 of GlcN) at 8 g/L in 0.056 M TFA-d (500 MHz).

Figure 6. Dependence of 1H T1 on temperature for CS (H2 of GlcN) at 8 g/L in 0.056 M TFA-d (500 MHz). Lines are guides for the eye.

completely inhibited and that the enhanced flexibility for the more acetylated CS operates in the whole range of ionic strengths studied. 1D-NOESY Experiments. Relaxation in macromolecules is usually influenced by extensive cross-relaxation (i.e., spin diffusion).19 This is also the case for CS, as confirmed by selective inversion recovery experiments with samples of various DP and DA (CS360-1, CS360-70, CS1200-3, and CS1200-70; see the SI). In all cases, selective T1 (T1S) has been found to be smaller than T1, in agreement with a faster cross-relaxation following selective inversion. With the aim of ruling out a DA-dependent spin diffusion disturbing the conclusions from the above relaxation studies, NOESY experiments54–56 were carried out on CS samples with

different DA and DP. In kinetic NOESY experiments with sufficiently short mixing times, when a spin S in a multispin IS system is saturated, the intensity of the signal observed for the spin I depends on the mixing time and the cross-relaxation rate of the IS pair (σNOE). The cross-relaxation rate relies on dynamics and the internuclear distance and so, contains qualitative information about the rate of the motions influencing crossrelaxation. Thus, faster NOE growths relate to larger correlation times and slower motions.57,58 1D-NOESY experiments of CS with various DA and DP (CS360-1, CS360-70, CS1200-3, and CS1200-70) were carried out by selective saturation of the H2 of GlcN (3.16 ppm) and analysis of the H1 of GlcN (4.88 ppm; mixing times up to 350 ms, 0.056 M TFA-d, 500 MHz). The selection of the H2/H1 pair of GlcN was based on these peaks being well-separated in the 1H NMR spectrum of CS, and because the internuclear H2-H1 distance can be assumed as constant, independently on DP and DA. In addition, 1D-ROESY experiments provided evidence of the direct nature of the H2/H1 NOE (Figure 8). Thus, ROE experiments are not only less sensitive to spin diffusion than NOE, but also distinguish direct and indirect contributions by their sign.57 As shown in Figure 8, when the H2 of GlcN is saturated in CS360-1, negative NOE peaks appeared at 3.72 (H4 of GlcN), 3.90 (H3 of GlcN), and 4.86 ppm (H1 of GlcN). For CS360-70, a more complex 1D-NOESY spectrum appeared because of NOE also with protons at the GlcNAc units. While the negative NOE of the ring protons denote a reduced mobility, the NAc group showed positive NOE in agreement with the methyl free rotation. Similar results were obtained for the CS samples CS1200-3 and CS1200-70. From the 1D-NOESY spectra, the NOE build up curves for the H2/H1 pair of GlcN showed a linear growth between 3 and 100 ms for all the CS samples studied (Figure 9). Crossrelaxation rates were measured as the slope of the linear fits (Table 3). Results in Figure 9 and Table 3 show that the more acetylated samples have smaller cross-relaxation rates (slower NOE growth) than the less acetylated CS, in agreement with the higher flexibility of CS at higher DA. Similar outcome was obtained from 1D-NOESY experiments recorded at 1 and 8 g/L in a pD 4.5 acetate buffer (Table 3 and Figure S7). These results



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Conclusions The dynamics of CS in solution have been studied for the first time by 1H NMR relaxation (T1, T2, NOE) as a function of DA (1-70), DP (10-1200), concentration, temperature, and ionic strength. This study has revealed T1 as governed by local motions, while T2 as governed by local and long-range motions. The variation of T1 and T2 with DP and concentration at 298 K has shown the semirigid character of CS in solution: T1 remained constant up to 30 g/L and above a critical DP in the range 360-730, while T2 resulted as a decreasing function of DP and concentration. The analysis of the relaxation parameters as a function of DA revealed an increase in flexibility for the more acetylated samples, in agreement with a reduction in electrostatic repulsions between protonated amino groups. On increasing the ionic strength, larger T1 values resulted independently on DA (similar relative enhancements), suggesting that the electrostatic repulsions are not completely inhibited and that the enhanced flexibility for the more acetylated CS operates in the whole range of ionic strengths studied. As mentioned in the introduction, there has been much debate on the flexibility of CS as a function of DA, with conflicting results by different authors and techniques. Considering the most recent reports with homogeneous series of CS, our results agree with those by the group of Domard10 describing an enhanced flexibility for the more acetylated samples, as opposed to the conclusions by the group of Vårum,9 who found little or no detectable dependence on DA. Figure 8. 1D-NOESY (75 ms mixing time) and ROESY (15 ms mixing time) experiments on CS360-1 and CS360-70 obtained after selective inversion of the H2 of GlcN (8 g/L, 0.056 M TFA-d, 298 K, 500 MHz).

Acknowledgment. This work was financially supported by the Spanish Government (CTQ2009-10963) and the Xunta de Galicia (PGIDIT06PXIB209058PR). The authors thank Dr. Manuel Martin-Pastor from the NMR Unit of the USC for helpful discussions. R.N.-C. thanks the Spanish Government for a FPU fellowship. Supporting Information Available. 1H NMR spectra of CS, typical monoexponential decays (CPMG) and magnetization recoveries (inversion recovery), selective T1S experiments, and NOESY build-up in acetate buffer. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes

Figure 9. 1D-NOESY build up for the H2/H1 proton pair of GlcN in CS (8 g/L, 0.056 M TFA-d, 298 K, 500 MHz). Table 3. Experimental σNOE (s-1) for the H2/H1 Proton Pair of GlcN in CS solvent

g/L

CS360-1

CS360-70

CS1200-3

CS1200-70

0.056 M TFA-d acetate buffera acetate buffera

8 8 1

-0.11 -0.09 -0.08

-0.04 -0.05 -0.04

-0.21 -0.22 -0.17

-0.09 -0.09 -0.08

a

Acetate buffer: 350 mM CD3CO2D/135 mM NaOD, pD 4.5.

confirm the validity of the above relaxation studies and reveal 1 H relaxation as a powerful tool for the accelerated study of the dynamical properties of large collections of macromolecular samples.

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