Molecular Dynamics of Amorphous Gentiobiose Studied by Solid

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Molecular Dynamics of Amorphous Gentiobiose Studied by Solid-State NMR Teresa G. Nunes,*,† Hermıńio P. Diogo,† Susana S. Pinto,† and Joaquim J. Moura Ramos‡ Centro de Quı´mica Estrutural, Complexo I, IST, TULisbon, AV. RoVisco Pais, 1049-001 Lisboa, Portugal, and CQFM (Centro de Quı´mica-Fı´sica Molecular) and IN (Institute of Nanoscience and Nanotechnology), Instituto Superior Tećnico, TULisbon, 1049-001 Lisboa, Portugal ReceiVed: July 9, 2010; ReVised Manuscript ReceiVed: October 9, 2010

A solid-state NMR (SSNMR) study is reported on the effect of temperature on the molecular mobility of amorphous gentiobiose, which is complemented with data obtained from crystalline samples. 13C crosspolarization/magic-angle-spinning (CPMAS) spectra and 1H MAS spectra were obtained for gentiobiose at natural abundance, in the amorphous state, from 293 K up to the glass transformation region (Tg ) 359 K). Two well-defined molecular mobility regimes were observed, corresponding to different motional modes. NMR results on molecular dynamics are discussed and compared with those obtained by thermally stimulated depolarization currents (TSDC) and dielectric relaxation spectroscopy (DRS). SSNMR spectra presented evidence for a new polymorphic form of gentiobiose, not yet reported in the literature, which is obtained by slow heating of the amorphous solid up to 364 K inside the NMR zirconia rotor. Introduction Carbohydrates are the most abundant class of organic compounds found in living organisms.1,2 They play key roles like being a major supply of metabolic energy, and being an essential component of the DNA molecule or a structural material (cellulose). The disaccharides are widely applied in the pharmaceutical, medical (as cryoprotectants), and food sciences. The technique thermally stimulated depolarization currents (TSDC) has been recently used to elucidate the features of the molecular mobility in the amorphous solid state of several carbohydrates.3-6 In the first part of this work,7 a molecular mobility study on the disaccharides gentiobiose and cellobiose was performed using TSDC, in the temperature region from 108 to 423 K. The slow molecular mobility was characterized in the crystal and in the glassy state. The features of different motional components of the secondary relaxation have been monitored as a function of time as the glass structurally relaxes on aging. It was concluded that some modes of motion of this mobility are aging independent, while others are affected by aging. In fact, the secondary relaxations in polysaccharides have been widely studied8 and their origins were recently reviewed,9 which were based on mechanical as well as on dielectric spectroscopy observations. However, detailed SSNMR studies are still lacking in order to provide information about the molecular nature of the motions. Two secondary relaxations have been identified in the dielectric spectra of some carbohydrates in the glassy state;10 the faster one (γ) is a common feature of the entire sugar family (from mono- to polysaccharide). Yet, the molecular origin of this process is still not unambiguously identified, but the fact that in some monosaccharides (fructose and ribose) the relaxation time does not depend either on pressure or in thermal history of the sample indicates an involvement of intramolecular degrees of freedom.11 NMR, which is being widely used to obtain structural and molecular dynamics information on these systems,12 enabled to conclude * Corresponding author. † Centro de Quı´mica Estrutural. ‡ CQFM and IN, Instituto Superior Tećnico.

SCHEME 1: Chemical Structure of r- and β-Gentiobiose: R1 ) H, R2 ) OH and R1 ) OH, R2 ) H, Respectively

that γ-relaxation is not related to the motion of the CH2OH group.13 The slower relaxation (labeled β) was identified as intermolecular in origin, involving twisting motion of the monosugar rings around the glycosidic bond;10 the corresponding activation energy provides valuable information about the flexibility of the glycosidic bond, which is essential for the control of the diffusivity of drugs or water entrapped in the sugar matrix, because only rigid molecules can be used as amorphous phase of the drugs.10 Considering the fact that some disaccharides have similar structures, it is reasonable to expect that the same type of motion is responsible for the γ-relaxation. However, it was found that γ-relaxation slows down significantly in sucrose. One can try to find some relation between the relatively greater flexibility of the fructofuranosyl residue compared to glucopyranosyl one and the variations in the dielectric strength of the γ-relaxation peak in some disaccharides.10 Unusual properties are again observed in sucrose and also in trehalose; the shapes of their β-relaxation are quite different from that of other five disaccharides, which present similar shapes.10 In order to get further insight on the molecular dynamics of amorphous solid gentiobiose, namely about the origins of secondary relaxations and molecular mobility at the glass transition temperature, we have now carried out a SSNMR study. Scheme 1 shows the chemical structure of gentiobiose anomers. Because of the anomeric effect, the R-anomer is more stable than the β-anomer in vacuum (thermodynamic preference for

10.1021/jp106371w  2010 American Chemical Society Published on Web 11/10/2010

Molecular Dynamics of Amorphous Gentiobiose an axial position of polar groups bonded to the anomeric carbon C1) but in aqueous solution (and also in the crystalline form), the R/β ratio is reversed due to solvent effects and hydrogen bonding.14 The crystal structure of β-D-glucopyranosyl-(1f6)-β-Dglucopyranose was determined.15 Both glucopyranose residues of the molecule are in the 4C1 chair conformation. All oxygen atoms, except the OH(2′) hydroxyl, the bridge oxygen, and the ring oxygen atoms, are hydrogen bonded, forming an infinite chain, but there are no intramolecular hydrogen bonds. It is well-known that line widths in SSNMR spectroscopy are strongly dependent on dipole-dipole interaction, particularly when protons are involved. Thus, structural and molecular mobility studies may be performed using NMR because dipole-dipole interaction depends on internuclear distances and vector orientations. Moreover, under favorable conditions, chemical shift anisotropy (CSA) also allows getting useful information. Initially developed to suppress dipolar broadening, MAS became a routine technique in solid sate. 13C CPMAS, was also used to probe moving molecular groups or segments; only molecules that are rigid or with restricted mobility contribute significantly to the spectra. Thus, molecular dynamics occurring with frequencies above 10 kHz affects the 13C CPMAS spectra, changing the cross-polarization efficiency16 and the respective spectral resolution. Moreover, 13C CPMAS enables identifying molecules that undergo restricted motions with rates: (a) below 10 kHz (slow exchange, observable in favorable conditions17), (b) between 10 kHz and 1 MHz frequencies (representing the intermediate regime), because they produce a line broadening due to the motion interference with the dipolar decoupling,18 (c) above 1 MHz, since they create partial averaging of the effective dipolar coupling, without decoupling interference, thus producing a spectral renarrowing. This study used (b) and (c) 13C CPMAS spectral effects, complemented with line-width 1H MAS measurements, to probe gentiobiose motions. Experimental Section Different gentiobiose forms were studied: (a) Crystalline (sample CR1), purchased from Acros Organics (Geel, Belgium) with 95% of the β-anomer (and about 5% of the R-anomer), which was kept inside a desiccator over P2O5 several days before NMR measurements. As reported in the first part of this work,7 the DSC melting peak of the sample occurred with an onset at Ton ) 458.6 ( 0.2 K and a maximum intensity at Tmax ) 468.1 ( 0.2 K, at a heating rate of 5 K min-1, which is in good agreement with the literature, Tm ) 469 K.19 The calorimetric glass transition temperature was found to be Tg ) 359 K (on heating at 10 K · min-1). No endothermic event, attributed to residual water, was detected in the thermogram. The absence of a band at 1680 cm-1 in the FT-IR spectra, assigned to water bending, is also an indication of the low water content of the sample. (b) Amorphous (sample AM) that was obtained as follows: (1) freeze-drying a 0.095 M aqueous solution of sample (a) using a Lyoalfa commercial apparatus (model 6-80, Telstar Industrial, Barcelona, Spain), which was first frozen in liquid nitrogen, and the primary drying was carried out for 20 h at 191 ( 5 K under a vacuum of 10-2 mbar, (2) the sample was subsequently dried in an oven at ca. 373 K for 1 day to remove residual water and, finally, kept at room temperature inside a desiccator for 1 week before NMR measurements. (c) Crystalline (sample CR2) which was obtained by gradually heating up sample AM to 364 K (inside the NMR magnet) and

J. Phys. Chem. B, Vol. 114, No. 48, 2010 15977 slowly cooled down to about 293 K, while keeping the sample inside the sealed zirconia rotor used for NMR observations. NMR spectra were recorded on a Bruker MSL-300 P spectrometer operating at 75.47 and 300.13 MHz for 13C and 1 H observations, respectively. Standard 4 and 7 mm ZrO2 rotors and double-bearing probes were used. 13C cross-polarization/ magic-angle-spinning (CPMAS) spectra were obtained with 3.7 kHz rate, 1 or 3 ms contact time (for the observation of crystalline or amorphous samples, respectively), 5 µs rf-pulse duration (90° magnetization tip angle), 1H decoupling rf-field of 50 kHz using continuous wave irradiation at the nominal frequency of protons, 5 s recycling delay and 400 scans; spin-lattice relaxation times (T1) were measured using the Torchia sequence20 (30 s relaxation delay, 1 ms contact time and 5 µs rf pulse duration). 1H spectra were obtained with a MAS rate of 3.7 kHz and also with rates in the range 0.29-9.7 kHz. Glycine (δ(13CO) ) 176.1 ppm) and ethanol (δ(CH3) ) 1.23 ppm) were used as external references for 13C and 1H chemical shifts, respectively. Temperature was controlled with a Bruker variable-temperature unit B-VT 1000E. In general, 10 K increments were selected, and experiments started from the lowest temperature. Thirty minutes waiting time allowed each temperature stabilization; the probe was always tuned before starting any experiment. Results and Discussion 1. 13C CPMAS at 293 K from Crystalline and Amorphous Gentiobiose. Figure 1 shows typical 13C CPMAS spectra which were obtained from different gentiobiose samples: (a) CR1, (b) AM, and (c) CR2. Three frequency regions are identified in the spectra obtained from crystalline samples, which correspond to anomeric, methine, and methylene carbon wellresolved resonances: 90-103, 68-85, and 60-68 ppm, respectively. Table 1 displays the 13C chemical shifts of some gentiobiose carbon nuclei and a tentative assignment of C2,2′, C3,3′, C4,4′, and C5,5′ resonances; Figure 1 shows the full spectral assignment. The chemical shifts of resonances obtained from the solidstate spectra of CR1 and CR2 samples that remain ambiguous are 75.70, 74.42, 73.31, 69.00, and 68.37 ppm. Since the isotropic shifts are influenced by effects like molecular conformation, inter- and intramolecular interactions such as hydrogen bonding, and crystal packing, it is not possible to assign all the resonances unequivocally just by comparison with the chemical shifts in aqueous solution; the complete assignment of 1H and 13 C signals for all 19 R- and β-D-glucosyl-D-glucosides in D2O at 303 K was recently reported.21 The spectra obtained from amorphous gentiobiose show a distribution of chemical shifts, as expected for a disordered system, and several differences may be noticed when comparing with data obtained for the crystalline samples (Figure 1): (a) changes in the relative intensity of resonances, particularly relevant for the most intense signals, which are from methine carbons (68-85 ppm), (b) line broadening, particularly noticeable for C1′ and C6′, in which case the frequency ranges are about 100-110 ppm and 59-65 ppm, respectively, and (c) similar intensity obtained for C1R and C1β signals. These observations are consistent with higher disorder, at least, in the chemical environment of C1′ and C6′ in the amorphous sample; the asymmetric shape of line obtained from C1,1′ was already reported on glassy R-R′ trehalose and the main source of structural disorder was assigned to the distribution of glycosidic torsion angles.22 Moreover, the fact that the signal of C1R is extremely small in the spectrum of the crystalline sample

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Figure 1. Section).

13

Nunes et al.

C (CPMAS) spectra obtained from crystalline (CR1) (a), amorphous (b), and treated gentiobiose (CR2) (c) (see the Experimental

TABLE 1: 13C (CPMAS) Chemical Shifts (ppm) of Gentiobiose, Externally Referenced to Glycine (δ(13CO) ) 176.1 ppm), Obtained for CR1 and CR2 Samplesa gentiobiose R β gentiobiose R β

C1

C2

C3

C4

C5

C6

91.47 73.31 75.70 69.00 74.42 66.61 (92.77) (72.05) (73.30) (70.06) (71.07) (69.28) 96.73 73.31 75.70 69.00 74.42 66.61 (96.61) (74.66) (76.27) (70.11) (75.52) (69.44) C1′

C2′

C3′

C4′

C5′

C6′

100.23 73.31 75.70 68.37 74.42 59.92 (103.30) (73.72) (76.29) (70.24) (76.52) (61.37) 100.23 73.31 75.70 68.37 74.42 59.92 (103.33) (73.72) (76.27) (70.25) (76.56) (61.36)

a The resonances from C2,2′, C3,3′, C4,4′, and C5,5′ nuclei are tentatively assigned. The chemical shifts in D2O are also shown for comparison (from ref 21 in parentheses).

indicates that R-anomers are less stable than β-anomers; both R- and β-anomers appear to be equally stable in amorphous gentiobiose (C1R resonance presents an intensity similar to C1β, as it was already pointed out). The 13C CPMAS spectra of CR1 and CR2 are similar, both displaying good resolution as a consequence of the high rigidity of the molecules, which indicates that gentiobiose crystallizes as a result of the slow cooling down of the amorphous sample. Hence, it indicates that the heating of the amorphous sample up to a temperature higher than Tg liberated a molecular mobility that allowed subsequent crystal formation. Nevertheless, some differences may be noticed in the relative intensity of CR1 and CR2 signals; also, the CR2 spectrum presents a better resolution for C3,3′, C4,4′, and C5,5′ resonances, which, in particular, show a lower signal-to-noise ratio. This observation, above all, is in agreement with the CR2 crystal lattice expansion, as a consequence of the thermal treatment (a lower molecular packing leads to increased C · · · H internuclear distances and, consequently, to less efficient CP). Within the limitations of the present data, these spectral differences point to distinct CR1 and CR2 structures, which look

like not to involve carbon atoms, because similar 13C chemical shifts were obtained in both samples. Therefore, we have used 1 H MAS spectroscopy in order to get further insight on those structural modifications. 2. 1H MAS at 293 K from Crystalline Gentiobiose: Influence of the Spinning Rate. Structural modifications induced by gradually heating amorphous gentiobiose up to a temperature higher than Tg and slowly cooling down were probed using 1H MAS spectroscopy; thus, samples CR1 and CR2 were observed under different spinning rates, at about 293 K. Figure 2 enables comparing 1H MAS spectra recorded from CR1 sample at 3.6 and 9.7 kHz MAS rates. As stated in subsection 1, in the solid-state spectrum it is not possible to identify all the resonances unequivocally just by comparison with the chemical shifts (ppm) obtained in D2O at 303 K (see). Table 221 Due to the fast isotropic tumbling of molecules in liquids, anisotropic interactions such as dipolar couplings and CSA are averaged to zero. Moreover, in a solid, the presence of water molecules (free water, bound water, or water of crystallization) must be taken into account; in D2O, it is not possible to differentiate hydroxyl groups undergoing fast exchange processes with the solvent molecules. Therefore, a tentative spectral assignment was performed here, which concerns mainly the dominant β-anomer, according to the 13C data. But, first, the dominant homonuclear dipolar interaction has to be explained. In solids, the full width at halfmaximum (fwhm) of 1H signals depends mainly on dipolar couplings and is governed by the “static” (or “secular”) part of the dipolar Hamiltonian, energy conserving A and B terms of the “dipolar alphabet”, which is the common designation of A to F terms in the total dipolar Hamiltonian; considering two protons, it characterizes the degree to which spin I affects the magnetic field at another spin S:

ˆ IS ) H

( )

( )

µo 2 -3 h γ r [A + B + C + D + E + F] 4π H IS 4π2 (1)

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Figure 3. 1H spectra obtained at different MAS rate (kHz) from sample CR2: a, static; b, 0.29; c, 0.58; d, 6.05; e, 8.52; and f, 9.71. On the right-hand side is shown a detail of the spectrum b.

Figure 2. (1) 1H spectra obtained from sample CR1 at the indicated MAS rates. (2) 1H spectra obtained at 9.7 kHz MAS rate from gentiobiose samples: (a) CR1 and (b) CR2. TABLE 2 anomer

H1

H1′

R-gentiobiose 5.21 4.48 β-gentiobiose 4.64 4.50

H2,H3,H4;H2′, H3′,H4′ 3.30-3.70 3.24-3.49

H5

H5′

H6,H6′

3.97 3.44 3.71-4.14 3.62 3.45 3.71-4.20

where rIS is the internuclear distance, µ0 the permeability of free space (4π × 10-7 H m-1) and γH the gyromagnetic ratio of proton spin (26.75 × 107 rad T-1 s-1);

A ) (1 - 3 cos2 θ)IˆZSˆZ

and 1 B ) - (1 - 3 cos2 θ)(Iˆ+Sˆ- + Iˆ-Sˆ+) 4

where Îz and Sˆz are the z components of the nuclear spin angular momentum operators (z is the direction of the external static magnetic field B0), Iˆ( ) Iˆx ( iIˆy, θ describes the orientation of the internuclear vector with respect to the orientation of B0 and φ is a phase angle perpendicular to B0. While A can be visualized as a field shift at a nucleus, the B term couples spins of opposite polarization, enabling them to exchange polarization states (“flip-flop” transitions). The homonuclear dipolar coupling, represented by the Hamiltonian in the secular form (as it commutes with the Zeeman Hamiltonian of the spin system) can be written more concisely:

Hhomo ) -D(1/2)(3 cos2 θ - 1)[3IˆzSˆz - Iˆ · Sˆ]

(2)

where Î · Sˆ ) ÎxSˆx + ÎySˆy + ÎzSˆz (Iˆx,y and Sˆx,y are the x,y components of the nuclear spin angular momentum operators) and D (Hz) is the dipolar coupling constant given by

D ) (µ0 /4π)[(h/4π2)γH2rIS-3]

(3)

Complete dipolar broadening suppression requires that the MAS rate be much larger than the magnitude of the static coupling. In the case of CH2 groups, with a short interproton distance (1.7 Å), D is about 25 kHz and the corresponding dipolar coupling splitting (2D) is 50 kHz; thus, in the present study, the corresponding homonuclear dipolar interaction could not be removed, even at the highest spinning rate (9.71 kHz), and the signals were not detectable. A similar remark applies to H5 and H5′ resonances, which have high density of protons in the vicinity, and, to a less extent, to H4 and H4′. Moreover, H2, H3, H2′, and H3′, in CH(OH) groups, are also expected to be involved in strong dipole-dipole interactions. Therefore, beyond protons in hydroxyl groups, only the anomeric protons are expected to experience small homonuclear dipolar interaction. Hence, the narrow resonance at 3.34 ppm is from protons experiencing low dipolar interaction, also with small CSA (no spinning side bands were observed, even at a rate of 3.6 kHz). By increasing the spinning speed to 9.7 kHz, the signals at 3.4 and 3.6 ppm became resolved, showing that the dipole-dipole interaction was averaged out, but the resonance at about 4.5 ppm remained broad; at this stage, it is not possible to unambiguously assign these signals. However, free water molecules, if present in solid gentiobiose samples, are expected to give resonances at about 4.5-4.8 ppm, which should be upfield shifted when occurs hydrogen bond breaking. Then, it is reasonable to consider that a similar trend is followed by OH groups in gentiobiose, that is, OH bound to C2′ (not involved in hydrogen bonding15) should be observed at a magnetic field higher than all the other OH groups. Thus, the three narrow signals recorded from CR1 (Figure 2, panel 1 and panel 2a) could be assigned to hydroxyl groups not forming hydrogen bonds, either in gentiobiose, as OH(2′), or in structural water molecules, like water of crystallization. Figure 2, panel 2, shows 1H spectra obtained at 9.7 kHz MAS rate from CR1 and CR2 samples. Clearly, resonances at lower frequencies are better resolved in the spectrum of CR1 sample; moreover, the CR2 resonance at 4.89 ppm is broad and upfield shifted to 4.6 ppm in CR1 spectrum, while the CR2 signals at 5.58 and 6.06 ppm are not observed from CR1. Figure 3 shows the 1H spectra recorded from sample CR2, at the following MAS rates (kHz): (a) 0 (static), (b) 0.29, (c) 0.58, (d) 6.05, (e) 8.52, and (f) 9.71. While broad signals from the static sample spread over more than 24 ppm, wellresolved resonances are shown between 3.2 and 6.4 ppm in the MAS spectra, namely in spectrum (f), also shown in Figure 2, panel 2. Further information was obtained from low-MAS spectra, which was significant to assign the resonances. While no

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spinning side bands were obtained at 6.05 kHz (Figure 3, line c), at very low-MAS (290 Hz, Figure 3, detail b), were recorded four spinning side bands, which were the result of the frequency modulation of the isotropic signal at 4.89 ppm; subsequent to baseline correction, the intensities of the five signals were obtained by integration of the Gaussian functions used for spectrum deconvolution. These data were the input of a computer program based on the algorithm by Hersfeld and Berger,23 in order to obtain (a) the principal components of the shielding tensor (σ11, σ22, and σ33, respectively, equal to -2.60 ( 0.06, 0.05 ( 0.00 and 2.65 ( 0.05 ppm, referenced to an isotropic shielding, (σ11 + σ22 + σ33)/3, of 0 ppm), (b) the chemical shift anisotropy, CSA ) σ33 - (σ11 + σ22)/2 ) 3.98 ( 0.06 ppm, and (c) the asymmetry factor, η ) 1 - (σ22 - σ11)/ (σ33 - σ22) ) 0.01 ( 0.06. The principal components σ11, σ22, and σ33 are indicated in Figure 3, referenced to the measured isotropic signal (4.89 ppm): 7.49 ( 0.06, 4.93 ( 0.00, and 2.24 ( 0.05 ppm. Noticeably, the tensor is not axially symmetric, which can be concluded by simple spectrum inspection; also, CSA is small. These results favor a preliminary assignment of the resonance at 4.89 ppm to β-anomeric protons. Another peak pattern is shown in the spectrum of Figure 3, detail b); its low intensity is consistent with the presence of the less abundant R-anomer, namely with the corresponding anomeric proton H1. The assignment of those peak patterns to hydroxyl protons in hydrogen bonds, either in gentiobiose or in free water, is ruled out based on low-MAS spectrum data a-c, because then the corresponding shielding tensor should be approximately axially symmetric and the anisotropy very large (>10 ppm).24 Consequently, at this point, signals at 4.89, 5.58, and 6.06 ppm in Figure 3f are tentatively assigned to the anomeric protons (H1,H1′). It should be mentioned here that future research work should include X-ray diffraction studies and less common NMR experiments; 13C-1H MAS-J-heteronuclear multiple-quantum coherence (HMQC) or 13C-1H MAS-J-heteronuclear singlequantum coherence (HSQC) would allow, in particular, to unequivocally assign the chemical shifts of all directly bonded protons through the correct selection of the coupling constant (J) evolution period; such spectra were not run due to experimental constraints. However, it must be pointed out that, conversely to CR1, the resonances of the anomeric protons in CR2 are considerably deshielded if compared to those in solution state. This remark is in agreement with the CR2 crystal lattice expansion, as a consequence of the thermal treatment, as already indicated according to 13C CPMAS observations. Other less-resolved signals observed at 3.3 and 3.8 ppm could be from OH not involved in hydrogen bonding15 (OH(2′), or water molecules, like water of crystallization, as for CR1 sample). Polysaccharides are hygroscopic and this interpretation cannot be discarded because traces of water may be present even in dry samples. In fact, the faster relaxation (γ) was already interpreted as the result of orientation of bonded water25 or confined water26 but, to date, this issue was not clarified. Overall, significant differences were noticed when comparing the spectra obtained from crystalline gentiobiose samples, CR1 and CR2, which must be due to structural parameters (like H-H distances and electronic shielding anisotropies) being changed by gradually heating CR1 up to 364 K and slowly cooling down to 293 K.

Nunes et al.

Figure 4. 13C CPMAS spectra of amorphous gentiobiose obtained at (a) 293, (b) 313, (c) 323, (d) 333, and (e) 343 K.

As already pointed out, in solids the fwhm of 1H signals depends mainly on dipolar couplings, which are mostly homogeneous interactions, according to the definition of Maricq and Waugh;27 the remaining “inhomogeneous” component of the natural line width is confirmed to have the same properties as in dilute-spin NMR. The “homogeneous” component is shown to depend primarily on the ratio between an effective local dipolar coupling strength and the spin rate, modified by geometrical factors which loosely correspond to the “dimensionality” of the coupling network. Variations in the NMR frequency due to chemical shift effects are shown to have a minimal impact on 1H resolution. Different kinds of dipolar interactions have to be considered: those involving nuclei of the same group and nuclei belonging to different nonbonded groups and interactions between rigid segments of a nonrigid molecule. A detailed study of the factors determining the line width (and hence resolution) in 1H solid-state magic-angle spinning NMR was described.28 Although it has been known from the early days of MAS that resolution of spectra for abundant nuclear spins, such as 1H, increases roughly in a linear way with increasing sample rotation rate, the difficulty of describing the dynamics of extended networks of coupled spins has made it difficult to predict a priori the resolution expected for a given sample. 3. 13C CPMAS and 1H MAS from Amorphous Gentiobiose: Influence of the Temperature. 3a. 13C CPMAS at 293-343 K. Figure 4 shows 13C CPMAS spectra of amorphous gentiobiose, obtained over the temperature range 293-343 K. The spectra recorded at temperatures higher than 343 K (not shown here) have a very low signal-to-noise ratio, because the increase in mobility with temperature hampers an effective magnetization transfer from hydrogen to carbon nuclei under the cross-polarization acquisition mode. The CP efficiency of mobile sites is in general rather less than for static sites. In Figure 4, it is clearly observed that, by increasing the temperature, was obtained a narrowing of the broad resonance which, at 293 K, has a maximum intensity at δ ) 75.1 ppm (most probably due to superimposed C2,2′, C3,3′, and C5,5′ signals); consequently, at 343 K, was obtained better resolution for the resonance at about 70 ppm (displayed as a shoulder at 293 K) presumably from C4,4′, as a consequence of the line narrowing of C2,2′, C3,3′, and C5,5′ overlapping resonances. This means that the motion of CH(OH) groups containing C2,2′



TABLE 3: Spin-Lattice Relaxation Times of 13C Nuclei with a Chemical Shift of 75.1 ppm Obtained at the Indicated Temperatures T1/s

293.3 K

313 K

323 K

333 K

14 ( 3

10.4 ( 1.6

6.6 ( 0.3

1.98 ( 0.17

and C3,3′ atoms is fast enough to produce the averaging of the CH dipolar couplings; a similar effect explains the line width decrease of C5,5′ signal, which may be due, in particular, to the mobility increase of the exocycle group, CH2OH, and the glycosidic linkage. In fact, considering that the CH dipolar coupling (DCH) is close to 15 kHz, fast motions at frequency higher than 30 kHz partially average out anisotropic DCH and consequently is observed a line narrowing. However, the motional amplitudes should be restricted because CP is still efficient.16 A 13C T1 experiment, which is particularly important for elucidating localized motions, also showed that T1 (at δ 75.1 ppm) has decreased from 14 ( 3 s at 293 K to 1.98 ( 0.17 s at 333 K (Table 3). In fact, T1 relaxation is predominantly influenced by spin-lattice or motional processes in the MHz frequency regime; that is, spin-spin contributions to T1 are not significant. The variation of T1 with T shown in Table 2 indicates that the motional rate associated with the observed line narrowing should be higher than 1 MHz. In particular, T1 should be minimum at the glass transition temperature; therefore, on the basis of T1 data, it is only possible to conclude that Tg g 333 K. Concerning the intensity variation with T of C1R and C1β signals, the spectra of Figure 4 suggest the presence of molecular motions with rates between 10 kHz and 1 MHz in amorphous gentiobiose because, molecular motions occurring in the intermediate regime, affect the CP efficiency producing a decrease in the overall signal intensity,16 which is particularly noticeable at 333 K. Also, an increasing line broadening with T was observed for the resonance with a maximum at about 62 ppm, which was from C6′. This observation indicates that, in these 1H decoupled 13 C CPMAS spectra, the time scale for CH2OH motions at 343 K is comparable to the inverse of the 1H decoupling frequency (50 kHz), in which condition molecular motions interfere with the decoupling frequency with an ensuing line broadening.18 Overall, the influence of temperature in the 13C CPMAS spectra was detected, in particular, at C1R, C1β, C2,2′, C3,3′, C5,5′, and C6′ sites, and is consistent with motions occurring in different frequency scales. In particular, these results show that motion of the monomeric units, via glycosidic linkage, which was considered to be the origin of β-relaxation in several polysaccharides,9 induce changes in C2,2′, C3,3′, C5,5′, and C6′ dynamics; namely, intermolecular hydrogen bond breaking may occur which involve the hydroxyl groups bonded to C3,3′, C2, and C6′. In fact, each anomer unit has eight OH groups which, according to the structure of the crystalline β-anomer,15 all except the OH(2′) participate in intermolecular hydrogen bonds, forming an infinite chain: O(6′)H f O(2), O(2)H f O(1), O(1)H f O(4), O(4)H f O(4′), O(4′)H f O(3′), O(3′)H f O(3), O(3)H f O(6′), etc. So, because each of the seven sites can act as a proton donor/acceptor, proton exchange may occur between those sites: O(6′) T HO(2), O(2) T HO(1), O(1) T HO(4), O(4) T HO(4′), O(4′) T HO(3′), O(3′) T HO(3), O(3) T HO(6′), etc. According to the 13C CP/MAS data, these are the favored proton transfers: O(6′) T HO(2), O(2) T HO(1), O(3′) T HO(3), O(3) T HO(6′). Such dynamic process, a motion between two sites X(O-H · · · O) f Y(O · · · H-O), was already described as a thermally activated jump between two

Figure 5. 1H spectra obtained at 300 MHz with a MAS rate of 3.7 kHz at temperatures in the range 293-364 K. * indicates spinning side bands.

configurations X and Y in an asymmetric potential well.29 It must be pointed out here that, in amorphous gentiobiose, dynamic processes involving the R-anomer, whose crystalline structure is still unknown, must be also taken into consideration. The 13C CP/MAS data were complemented with 1H MAS spectroscopy experiments described below. 3b. 1H MAS Spectra at 293-364 K. The influence of temperature on molecular mobility of amorphous gentiobiose was also followed up by 1H MAS NMR. Figure 5 shows typical 1 H MAS (Fourier transformed Bloch decays) obtained from sample AM in the temperature range 293-364 K, under a MAS rate of 3.7 kHz. The spectrum obtained at 293 K in static mode (Figure 3a) shows at least two components, which spread over about 14 and 30 kHz, respectively, but strong line narrowing and the appearance of spinning side bands are noticed at higher temperatures in Figure 5. Spectral data in Figure 5 are in agreement with a decrease of the correlation time τC when the sample is heated up (τC basically provides a monitor for the rate of motion, being small for short typical time between changes of molecular position). Fluctuations in the homonuclear dipolar interaction due to molecular motion are described by terms C to F of the “dipolar alphabet” (see eq 1):

3 C ) - sin θ cos θ e-iφ(IˆzSˆ+ + Î+Sˆz), 2 3 D ) - sin θ cos θ e-iφ(IˆzSˆ- + Î-Sˆz) 2 3 3 E ) - sin2 θ e-i2φÎ+Sˆ+, and F ) - sin2 θ e-i2φÎ-Sˆ4 4 The influence of isotropic thermal motion on MAS NMR spectra was described theoretically for dominating (a) inhomogeneous magnetic dipolar interaction and (b) CSA interaction.30 MAS can fail in the presence of molecular mobility, causing line widths to increase dramatically;27 under the fast-spinning regime, the maximum line broadening occurs at about 2πνr/k ≈ 0.55, with k the rate constant (s-1) and νr the spinning rate (Hz), a condition that was not reached in the present study. In fact, we have observed a strong line narrowing with T and it is

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Nunes et al.

Figure 6. (a) Simulations of the variation of fwhmMAS with τC according to eq 5, for wr equal to 23 248 rad · s-1 and M2 equal to 1, 2, 3, 4, 5 (solid line), and 6 × 108 s-2 (from top to bottom). (b) 1H MAS spectrum simulations using eq 4 (and subsequent Fourier transform) and different τC values (1, 3, 5, 7, and 10 µs, from top to bottom), for wr and M2 equal to 23 248 rad · s-1 and 5 × 108 s-2, respectively (see text for details).

reasonable to consider that, under a MAS rate of only 3.7 kHz (Figure 5), this result may be due to a dominant “inhomogeneous” interaction being canceled, most probably from protons in hydroxyl groups because, in the case of bridging OH groups, the dominating line-broadening mechanism is an inhomogeneous magnetic dipolar interaction. The line width in 1H spectra can be significantly reduced when any molecular motion occurs which averages (totally or partially) the dipolar coupling on the NMR time scale, i.e., a motion with a correlation rate τC-1 of the order of the line width or higher. It is well-known that, when the sample is spun rapidly, local fields produced at an observed spin, due to dipolar coupling with neighboring spins and orthogonal to the spinning axis, are rendered time dependent and modulate the NMR signal at multiples of the spin rate. Then, a set of spinning side bands at multiples of the spin rate are produced, and the width of the central band is determined by the distribution of local fields along the rotation axis. This is the case of 1H spectra recorded from gentiobiose at higher temperatures (Figure 5). According to 13C CPMAS data, the influence of temperature was mainly relevant for C1R, C1β, C2,2′, C3,3′, C5,5′, and C6′ sites, and was consistent with motions occurring in different frequency scales. Therefore, it is reasonable to consider that, under similar experimental conditions, the more mobile hydrogen atoms are in the vicinity of those carbon atoms. However, due to the strong homonuclear interactions, the observed signals are thus assigned to protons in hydroxyl groups bonded to C2,2′, C3,3′, and C6′. Because the MAS rate was kept constant, spectra in Figure 5 show that the system has changed from medium to fast motion. An analytical model to illustrate and quantify the influence of motion on line widths in MAS NMR experiments (fwhmMAS) governed by inhomogeneous interactions was introduced and its application was widened.31,32 If the inhomogeneous magnetic dipolar interaction is the dominant contribution for the linebroadening mechanism and assuming that the thermal motion can be described by a single correlation time τC, the envelope of the free induction decay under MAS is given by30

{ ( )[

φMAS(t) ) exp -

F(2wr, t) 2M2 F(wr, t) + 3 2

]}

(4)

where M2 (s-2) is the second moment for the central line, wr (rad · s-1) is the the spinning rate of the sample, and

F(wr, t) ) tτc[1 + (wrτc)2]-2 - [1 - (wrτc)2]τc2 + [1 + (wrτc)2]-2[1 - e(t/τc) cos wrt ] 2wrτc3[1 + (wrτc)2]-2e(t/τc) sin wrt Under the condition t . τC, where t is of the order of the observation time and which is equivalent to (fwhmMAS · π)-1 . τC, eq 4 yields an exponential decay and the corresponding line shape is expressed by a Lorentzian function; hence, the line width under MAS and motion (fwhmMAS) is given by the eq 5:30

fwhmMAS )

( )[

τc M2 2τc + 2 3π 1 + (w τ ) 1 + (2wrτc)2 r c

]

(5)

When wr ) 0 (static mode), fwhmMAS increases monotonically with τC, up to the limit of the validity of eq 5 given by M2τC2 e 1. Under MAS, fwhmMAS exhibits a maximum as a function of τC (as already pointed out) and a monotonic decrease both at smaller (line narrowing caused by thermal motion) and at higher values of τC (influence of MAS). For example, eq 5 was already successfully applied to the mobility study of acidic protons in bridging hydroxyl groups (Si-OH-Al) in zeolite frameworks.33 Figure 6a shows the variation of ln(fwhmMAS)-1 with τC, which was obtained using eq 5, for M2 values in the range 108 to 6 × 108 s-2 and wr equal to 23 248 rad · s-1 (the value selected in this study, see Figure 5).30 As expected, the curves present a minimum as a function of τC, an increase both at smaller and at higher values of τC and also an increase with M2. The influence of τC in the spectral line shape is clearly observed in Figure 6b, which shows 1H MAS spectrum simulations obtained using eq 4, considering τC equal to 1, 3, 5, 7, and 10 µs (from Figure 6a, corresponding to line narrowing induced by thermal motion), the same value of wr and M2 equal to 5 × 108 s-2, which is the value that, according to eq 5 and Figure 6a, gives fwhmMAS similar to the experimental data obtained at higher T (Figure 5). However, in a first approximation, fwhmMAS was assumed here to be described by a single correlation time τC, obeying to the equation

1/fwhmMAS ) constant · τC

(6)



Figure 7. FwhmMAS of the 1H spectra obtained at 300 MHz with a MAS rate of 3.7 kHz over the temperature range 293-364 K.

where the constant may contain terms like ∆νXY2 ) |νX - νY|2, the magnitude of the “frequency jump” resulting from reorientation between sites X(O-H · · · O) and Y(O · · · H-O). The activation energy Ea for proton mobility was calculated from the Arrhenius relation

τC ) τ0 exp(Ea /RT)

(7)

where R is the gas constant. Figure 7 shows a plot of ln(1/ fwhmMAS), obtained for the 1H signals recorded over the temperature range 293-364 K, as a function of 1/T. Figure 7 shows unequivocally that the molecular mobility regime changed at about 345 K, so that two linear functions were needed to fit the whole experimental data. The equations of the two straight lines obtained from the linear regression of the experimental points in Figure 7 are

ln(1/fwhmMAS) ) (-1.1 ( 0.7) - (2280 ( 220)/T (for 292 eT(K) e 343) (8) and

ln(1/fwhmMAS) ) (17.7 ( 1.1) - (8731 ( 380)/T (for 345 eT(K) e 364) (9) The corresponding average activation energies are Ea ) 18.9 ( 1.8 kJ · mol-1 and Ea ) 72.6 ( 3.2 kJ · mol-1. Then, a possible interpretation of the data could implicate two well-defined mobility processes: at lower temperature with lower activation energy, typical of local motions of specific segments (internal rotations, conformational changes, libration motions), and another with higher Ea associated to more hindered molecular motions. Considering that the mobility of hydroxyl groups involved in intermolecular hydrogen bonding is distinct from that of non-hydrogen-bonded hydroxyl groups (OH(2′)), the present data is consistent with motion of both types of hydroxyl groups being observed and, because the frequency being probed is in the kilohertz range, both are assigned to local, noncooperative secondary relaxations. Glucose and galactose have a CH2OH group attached to C5 while xylose and arabinose have not. This

is the main structural difference between those two pairs of carbohydrates. From the dielectric relaxation spectroscopy studies of those substances, it was concluded34 that the CH2OH group strongly influences the γ-process below Tg. A more recent NMR study on glucose13 indicated that the nature of the involved motion is a librational type of motion as suggested earlier by Williams and Watts,35 rather than a nearly free tumbling around the C5′-C6′ bond. In crystalline cellobiose it was found that T1 of protons is dominated by the motions of hydroxyl groups between 120 and 380 K, characterized by an activation energy of about 8.74 kJ.mol-1;36 moreover, the spin-lattice in the rotating frame data suggested that hydroxyl groups have a distribution of dynamics. On the other hand, the average value of Ea ) 72.6 ( 3.2 kJ · mol-1 is small compared with the typical values of the activation energy observed for the glass transition relaxation, so that the mobility observed at temperatures above 345 K seems not to be attributed to the glass transformation but rather to a secondary relaxation, most probably the β-relaxation. That average Ea value agrees with the data reported on the activation energy for β-relaxation in maltose9 (73 kJ · mol-1) and cellobiose37 (81 kJ · mol-1). For example, in the study of amorphous glucose by 2D echo decay 13C NMR,13 an average activation energy Ea ) 480 kJ · mol -1 was found for the rotational motion of the whole ring and exocyclic CH2OH group, in agreement with the values obtained from viscosity and dielectric relaxation data. Furthermore, the average value of the activation energy at Tg obtained for gentiobiose by DSC is Ea ) 412 kJ · mol -1.7 In this context, it is reasonable to consider that the motions that the 1H MAS NMR method detects are not those associated to the structural relaxation at Tg, where main-chain motion occurs, because we did not run experiments that can probe processes occurring in the hertz to kilohertz range (frequency expected for R-relaxation); for this purpose, other methods should be used (e.g., centerband-only detection of exchange38). The mobility observed by NMR in the interval 345 e T (K) e 364, probably corresponds to the sub-Tg process that was observed at ∼330 K by TSDC in gentiobiose and also in cellobiose, with average activation enthalpies distributed between 70 and 120 kJ · mol-1, and with negligible activation entropies.7 Motional processes below Tg (i.e., in the glassy state) involving disruption of some intermolecular hydrogen bonding can be at the origin of this relaxation. A motional process was found by SSNMR in the rubber state (above Tg) of some polymeric systems which was attributed to the randomization of the conformations and isotropization of the molecular orientations.39,40 Such a relaxation process that has a longer time scale and a lower activation energy compared with the glass transition can be considered as an alternative candidate for the attribution of the discussed mobility, observed in gentiobiose near and below the glass transformation. Further research work is needed to clarify this problem. Conclusions 1

H MAS gave evidence for molecular mobility under at least two well-defined mobility regimes below the glass transition temperature and in the glass transformation range. At lower temperature, from 293 to 344 K, the observed average activation energy of the motional modes is Ea ) 18.9 ( 1.8 kJ · mol-1. At higher temperatures, from 344 to 364 K, more hindered molecular motions are activated (Ea ) 72.6 ( 3.2 kJ · mol-1). The attribution of this mobility at the molecular level is discussed. The lower temperature and lower activation energy mobility surely correspond to local, low-amplitude, and non-

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cooperative motional modes, often identified with the γ-relaxation. The higher activation modes, on the other hand, can be reasonably assigned to a β-type or Johari-Goldstein relaxation. The influence of temperature in the 13C CPMAS spectra was followed at different sites and is consistent with motions occurring in different frequency scales, which imply motion of the monomeric units, via glycosidic linkage, with consequent intermolecular hydrogen bond breaking. This observation is in agreement with the fact that the β-relaxation is a precursor of the glass transition. Our NMR results did not reveal the glass transition relaxation of gentiobiose, despite the fact that the experimental measurements have been carried out up to glass transformation region (Tg ) 359 K). It is worth mentioning that a similar effect was observed using the experimental technique of TSDC, which was considered unexpected, since only few systems, like for example trehalose,7 display this intriguing behavior, i.e., the absence of a signal attributed to the glass transition temperature. Moreover, we have obtained 1H MAS evidence for structural differences between untreated sample and gentiobiose submitted to thermal treatment, which were both crystalline forms with similar 13C chemical shifts. This constitutes a new indication of the presence of polymorphism in gentiobiose. References and Notes (1) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321–327. (2) Dwek, R. A.; Butters, T. D. Chem. ReV. 2002, 102, 283–284. (3) Correia, N. T.; Diogo, H. P.; Moura Ramos, J. J. J. Food Sci. 2009, 74, 526–533. (4) Moura Ramos, J. J.; Pinto, S. S.; Diogo, H. P. ChemPhysChem. 2007, 8, 2391–2396. (5) Diogo, H. P.; Moura Ramos, J. J. Carbohydr. Res. 2008, 343, 2797– 2803. (6) Moura Ramos, J. J.; Diogo, H. P.; Pinto, S. S. J. Chem. Phys. 2007, 126, 144506–1/6. (7) Pinto, S. S.; Diogo, H. P.; Nunes, T. G.; Moura Ramos, J. J. Carbohydr. Res. 2010, 345, 1802–1807. (8) Einfeldt, J.; Meissner, D.; Kwasniewski, A. Prog. Polym. Sci. 2001, 26, 1419–1472. (9) Kaminski, K.; Kaminska, E.; Ngai, K. L.; Paluch, M.; Wlodarczyk, P.; Kasprzycka, A.; Szeja, W. J. Phys. Chem. B 2009, 113, 10088–10096. (10) Kaminski, K.; Kaminska, E.; Wlodarczyk, P.; Pawlus, S.; Kimla, D.; Kasprzycka, A.; Paluch, M.; Ziolo, J.; Szeja, W.; Ngai, K. L. J. Phys. Chem. B 2008, 112, 12816–12823. (11) Kaminski, K.; Kaminska, E.; Hensel-Bielowka, S.; Pawlus, S.; Paluch, M.; Ziolo, J. J. Chem. Phys. 2008, 129, 084501-084505.

Nunes et al. (12) Jouanneau, D.; Boulenguer, P.; Mazoyer, J.; Helbert, W. Carbohydr. Res. 2010, 345, 547–551. (13) van Dusschoten, D.; Tracht, U.; Heuer, A.; Spiess, H. W. J. Phys. Chem. A 1999, 103, 8359–8364. (14) Corchado, J. C.; Sanchez, M. L.; Aguilar, M. A. J. Am. Chem. Soc. 2004, 126, 7311–7319. (15) Rohrer, D. C.; Sarko, A.; Bluhm, T. L.; Lee, Y. N. Acta Cryst. B 1980, 36, 650–654. (16) Cobo, M. F.; Malina´kova´, K.; Reichert, D.; Saalwa¨chter, K.; deAzevedo, E. R. Phys. Chem. Chem. Phys. 2009, 11, 7036–7047. (17) Helluy, X.; Kummerlen, J.; Sebald, A. Organometallics 1998, 17, 5003–5008. (18) Rothwell, W.; Waugh, J. J. Chem. Phys. 1981, 74, 2721–2732. (19) Miller, D. P.; de Pablo, J. J. J. Phys. Chem. B 2000, 104, 8876– 8883. (20) Torchia, D. A. J. Magn. Reson. 1978, 30, 613–616. (21) Roslund, M. U.; Ta¨htinen, P.; Niemitz, M.; Sjo¨holm, R. Carbohydr. Res. 2008, 343, 101–112. (22) Zhang, P.; Klymachyov, A. N.; Brown, S.; Ellington, J. G.; Grandinetti, P. J. Solid State NMR 1998, 12, 221–225. (23) Herzfeld, J.; Berger, A. E. J. Chem. Phys. 1980, 73, 6021–6030. (24) Mehring, M. Principles of High Resolution NMR in Solids; Springer: Heidelberg, Germany, 1983. (25) Schartel, B.; Wendling, J.; Wendorff, J. H. Macromolecules 1996, 29, 1521–1527. (26) Cerveny, S.; Schwartz, G. A.; Bergman, R.; Swenson, J. Phys. ReV. Lett. 2004, 93, 245702–245705. (27) Maricq, M. M.; Waugh, J. S. J. Chem. Phys. 1979, 70, 3300–3316. (28) Zorin, V. E.; Brown, S. P.; Hodgkinson, P. J. Chem. Phys. 2006, 125, 144508–144513. (29) Andrew, E. R.; Latanowicz, L. J. Magn. Reson. 1986, 68, 232– 239. (30) Fenzke, D.; Gerstein, B. C.; Pfeifer, H. J. Magn. Reson. 1992, 98, 469–474. (31) Ashbrook, S. E.; Antonijevic, S.; Berry, A. J.; Wimperis, S. Chem. Phys. Lett. 2002, 364, 634–642. (32) Thrippleton, M. J.; Cutajar, M.; Wimperis, S. Chem. Phys. Lett. 2008, 452, 233–238. (33) Baba, T.; Komatsu, N.; Ono, Y.; Sugisawa, H. J. Phys. Chem. B 1998, 102, 804–808. (34) Noel, T. R.; Ring, S. G.; Whittam, M. A. J. Phys. Chem. 1992, 96, 5662–5667. (35) Williams, G.; Watts, D. C. Trans. Faraday Soc. 1971, 67, 1971– 1979. (36) Tang, H. R.; Belton, P. S. Solid State NMR 2002, 21, 117–133. (37) Prevosto, D.; Capaccioli, S.; Sharifi, S.; Kessairi, K.; Lucchesi, M.; Rolla, P. A. J. Non-Cryst. Solids 2007, 353, 4278–4282. (38) Reichert, D.; Pascui, O.; Bonagamba, T.; Belton, P.; Schmidt, A.; Azevedo, E. J. Magn. Reson. 2008, 191, 141–146. (39) Wind, M.; Brombacher, L.; Heuer, A.; Graf, R.; Spiess, H. W. Solid State NMR 2005, 27, 132–139. (40) Wind, M.; Graf, R.; Heuer, A.; Spiess, H. W. Phys. ReV. Lett. 2003, 91, 155702/1–4.

JP106371W

Molecular Dynamics of Amorphous Gentiobiose Studied by Solid

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