Structure of Aqueous Trehalose Solution by Neutron Diffraction and

Article pubs.acs.org/JPCB

Structure of Aqueous Trehalose Solution by Neutron Diffraction and Structural Modeling Christoffer Olsson,† Helén Jansson,‡ Tristan Youngs,§ and Jan Swenson*,† †

Department of Physics and ‡Department of Civil and Environmental Engineering, Chalmers University of Technology, SE-412 96 Göteborg, Sweden § ISIS Pulsed Neutron and Muon Source, STFC Rutherford Appleton Laboratory, Harwell Campus, Oxon, OX11 0QX, Oxfordshire, U.K. ABSTRACT: The molecular structure of an aqueous solution of the disaccharide trehalose (C12H22O11) has been studied by neutron diffraction and empirical potential structure refinement modeling. Six different isotope compositions with 33 wt % trehalose (corresponding to 38 water molecules per trehalose molecule) were measured to ensure that water− water, trehalose−water, and trehalose−trehalose correlations were accurately determined. In fact, this is the first neutron diffraction study of an aqueous trehalose solution in which also the nonexchangeable hydrogen atoms in trehalose are deuterated. With this approach, it was possible to determine that (1) there is a substantial hydrogen bonding between trehalose and water (∼11 hydrogen bonds per trehalose molecule), which is in contrast to previous neutron diffraction studies, and (2) there is no tendency of clustering of trehalose, in contrast to what is generally observed by molecular dynamics simulations and experimentally found for other disaccharides. Thus, the results give the structural picture that trehalose prefers to interact with water and participate in a hydrogen-bonded network. This strong network character of the solution might be one of the key reasons for its extraordinary stabilization effect on biological materials.

1. INTRODUCTION Trehalose (C12H22O11) is a sugar molecule that has claimed the spotlight for substantial research during the past decades due to its well-known extraordinary stabilization effect on biological materials, such as proteins1 and cell membranes.2 It can be found in many different extremophiles (e.g., the tardigrade3 or the Jericho rose4), capable of surviving different environmental stresses, such as desiccation or extreme colds, which has been found to be linked to the special properties of trehalose. However, the exact reason for why trehalose has an extraordinary ability to stabilize biological macromolecules and materials is not fully understood. Thus, further knowledge of the mechanisms behind this stabilization capability could yield great technical advances in areas where biological preservation is important, such as the food or pharmaceutical industry, as well as for long-term storage of, for example, oocytes, sperms, and possibly also human organ transplants. From the literature, it is obvious that there are a multitude of different properties of trehalose that could explain the reason for its stabilization effect. Several studies point out that trehalose exhibits a high glass-transition temperature5,6 compared to that of other comparable disaccharides and also that trehalose has a higher ability to inhibit water crystallization.7 Although these effects are highly important for the stabilization mechanism of trehalose, it has also been © XXXX American Chemical Society

shown that there ought to be further effects behind the unique properties of trehalose (discussed in, e.g., ref 8). For example, when trehalose is used for stabilizing lipid vesicles, it replaces bound water molecules, thus making these less likely to burst or fuse with other vesicles.2 On the other hand, when trehalose is used for stabilizing proteins, most studies indicate that the water molecules become trapped at the protein surface9−11 in an exclusion zone, rather than being replaced by trehalose on the protein surface. To understand such interactions and the effect of them, it is not sufficient to understand only the interactions between trehalose and the biomolecules, but a deeper understanding of the interactions between water and trehalose is also required. One prevailing model regarding the properties of trehalose in water was presented by Branca et al.7 According to this model, trehalose, as compared to other similar disaccharides (such as sucrose and maltose), exhibits a high destructuring effect on water. Thus, it has a high ability to break the network structure of bulk water, due to the extensive ability to form hydrogen bonds (HBs) with water molecules.10,12−16 Thereby, multiple hydration layers are formed, which in turn disturb the network Received: October 19, 2016 Revised: November 18, 2016 Published: November 18, 2016 A

DOI: 10.1021/acs.jpcb.6b10556 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

hydrogenated trehalose powder in D2O and drying the solutions under vacuum at 70 °C. α,α-Trehalose with deuterated carbon-bound hydrogens was purchased from Omicron Biochemicals Inc. as an anhydrous powder. 2.2. Neutron Diffraction Experiments. The neutron diffraction measurements were performed on the near and intermediate range order diffractometer (NIMROD) at the ISIS Pulsed Neutron and Muon Source (STFC Rutherford Appleton Laboratory, U.K.). The aqueous trehalose solutions were placed in standard 1 mm thick Ti0.676Zr0.324 containers and sealed with a PTFE O-ring. Before the measurements, the sample containers were mounted on a sample changer connected to a water bath for temperature control (298 K). Prior to the measurements, data were collected for the empty cans and a vanadium plate for background subtraction and normalization, respectively. Data corrections and reductions were performed using the GUDRUN suite (2015 version). The corrected data obtained is the total differential scattering cross section

formation, leading to the formation of ice crystals. These observations have also been supported by different molecular dynamics (MD) simulation studies.14,17 However, a recent study by Pagnotta et al.18 has indicated the reverse scenario, that is, that there are in fact very few interactions between trehalose and water.18 As a consequence, the extraordinary properties of trehalose have to be explained in a different way. This study aims to investigate and evaluate these two different scenarios using a combination of neutron diffraction and empirical potential structure refinement (EPSR) modeling on aqueous solutions containing 33 wt % trehalose. To the best of our knowledge, this diffraction study is the first one to have used trehalose with deuterated carbon-bound hydrogen atoms in the sample preparation. Thereby it also results in the highest number of reliable contrasting structure factors for this system. By careful analysis of the partial radial distribution functions (RDFs) calculated from the EPSR-produced structural model, both an extensive perturbation of the water structure and a high hydration number of trehalose were found. Thus, the results support the structural models presented in, for example, refs 7, 14, 17, and 19. Moreover, various properties of aqueous trehalose, such as the homogeneous distribution of trehalose molecules in water and the flexibility of the trehalose molecules, have also been investigated. The homogeneity of trehalose solutions (i.e., whether the trehalose molecules form clusters) has previously been studied by, for example, Lerbret et al.14 and Sapir et al.,20 and it has been determined to be highly dependent on the concentration.20 Cluster formation of trehalose has been shown21 to be correlated to the stabilizing effect of trehalose on proteins. Thus, a deeper understanding of whether trehalose forms clusters in the absence of proteins is of great importance for understanding the structural properties of trehalose−water− protein systems. In the present study, it is shown that the trehalose molecules are, in fact, more homogeneously distributed than predicted for a statistical random distribution of molecules. This indicates a far less propensity of cluster formation than previously obtained by MD simulations; see refs 14 and 20.

F (Q ) =

α ,β

Table 1. Isotope Compositions of the Six Different Samplesa sample name

deuteration of M-atoms

deuteration of H-atoms

no no no yes yes yes

no yes half no yes half

(1)

where Q is the momentum transfer of the scattered neutron, cα and bα are the number density and the scattering length of atom type α. cα,β(Q) is the partial structure factor of atom types α and β. The partial structure factors are the Fourier transforms of the partial radial distribution functions (RDFs), gα,β(r), which fully describe the structural correlations in a material. In theory, it would be possible to isotopically label each distinct atom in a sample and thus to directly obtain all of the partial structure factors (and then also all of the partial RDFs). However, in practice this is not a feasible method for a complex molecule, such as trehalose. Instead, six isotopically different samples were prepared (see Table 1), and with the help of the EPSR software, all of the partial RDFs were extracted. 2.3. EPSR Modeling. EPSR is a Monte Carlo-based method to produce the structural models of materials in quantitative agreement with measured neutron diffraction data. With this method, an initial inter- and intramolecular potential is assumed, which during the simulation is modified by the addition of an empirical potential to minimize the difference between the calculated F(Q) of the structural model and the corresponding measured F(Q). Thus, the empirical potential is modified until the total potential produces a structure in quantitative agreement with the measured F(Q). For more details regarding the EPSR method, the reader is referred to, for example, ref 22. For the present simulation, a cubic simulation box containing 2000 water molecules and 52 trehalose molecules with periodic boundary conditions was used. The atomic number density was 0.10677 atoms/Å3, as measured by a density meter (Anton-Paar U-tube). The parameters for the reference potential were adopted from ref 18, in which a modified version of the OPSLAA force field23 was applied for trehalose, and the extended simple point charge (SPC/E) model was used for water24 (as shown in detail in Table 2; Figure 1). The atomic labeling was also set identical to ref 18 to make comparisons to this study easier. After the final potential had been reached, the results were averaged over 10 000 different configurations to obtain good statistics. 2.4. Analysis of the Structural Model. To study the average local structure in the EPSR-produced model, the coordination numbers for different atomic pairs were

2. MATERIALS AND METHODS 2.1. Sample Preparation. As shown in Table 1, six different samples with the same molar concentration of

H-Tre in H2O H-Tre in D2O H-Tre in HDO D-Tre in H2O D-Tre in D2O D-Tre in HDO

∑ cαcβbαbβ*(Sα ,β(Q ) − 1)

a

The so-called M-atoms refer to the nonexchangeable hydrogen atoms, which are bonded to carbon atoms.

trehalose, but with different isotopical compositions, were prepared for the measurements. All samples were composed of 38 water molecules per trehalose (corresponding to 33 wt % trehalose in the nondeuterated case). The hydrogenated α,αtrehalose (>99% purity) was purchased from Sigma-Aldrich in the form of a powdered dihydrate. The deuteration of the hydroxyl groups was performed by repeatedly dissolving the B


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distance of 3.4 Å and with an added restriction of θO−H···O to be at a minimum of 120°. This definition is used in refs 14, 29−31 (although in ref 31, the maximum distance is 3.5 Å, and the reader should thus be reminded that in comparison with this reference a slightly lower number of HBs is expected). • Criterion 4 is the same as criterion 3, except that the angle restriction is 160° and thus represents stronger HBs, as used in ref 14. With these criteria, it is possible to compare our results with previous studies, in which MD simulations or EPSR modeling were used. In addition, when analyzing the cluster-size distribution of trehalose, criterion 1 was used for the intermolecular Ot−Ht pairs (Ot and Ht indicating any trehalose oxygen or hydrogen). Thus, if one or more atoms in two trehalose molecules fulfilled this criterion, they were counted as being located in the same cluster.

Table 2. Lennard-Jones Parameters for the Different Labeled Atoms, where Hw and Ow Represent the Water Atoms in an SPC/E Modela atom label

ε (kJ/mol)

σ (Å)

charge (e)

Hw Ow O O1 O2 O3 H M C

0 0.65 0.71128 0.58576 0.58576 0.71128 0.05 0.12552 0.27614

0 3.166 3.1 2.9 2.9 3.1 1.7 1.7 3.5

0.43238 −0.8476 −0.5 −0.5 −0.5 −0.5 0.3005 0.0 0.258

a

O and O3 are part of hydroxyl groups and bonded to the exchangeable H-atoms (see Figure 1). The nonexchangeable hydrogen atoms, labeled M, are bonded to the carbon atoms, labeled as C. O1 is the oxygen linking the two glycosidic units, and O2 labels the oxygens within each glycosidic ring, as shown in Figure 1.

3. RESULTS AND DISCUSSION In Figure 2, the experimental data and the corresponding EPSR fits are presented for each isotope composition (described in

Figure 1. Pictorial sketch of trehalose with atom labels of the most relevant atoms. Labels not shown here are the M-atoms, representing the hydrogen atoms bonded to the carbon atoms (labeled as C).

determined. Coordination numbers from atoms α to β (nβα) in the range between r1 and r2 were calculated as (2)

Figure 2. Differential scattering cross section data for the six different isotope compositions. The dashed lines represent the EPSR fits to each dataset.

where cβ is the atomic number density of atom β and gα,β is the partial RDF for the α−β atom pair. Thus, the first coordination shell is calculated from r1 = 0 to the minimum that follows the first peak in gα,β(r), the second coordination shell from that point to after the second peak, and so forth. In addition to the coordination number, which merely counts the number of atoms within a shell of another one (or the same type of atom), the number of HBs (nHB) was determined according to different criteria to be able to make direct comparisons with the corresponding values in the literature. For the calculations, the following criteria were used: • Criterion 1 is a simple calculation of the number of potentially H-bonding hydrogens in the vicinity of a hydrogen-accepting oxygen, as determined by eq 2 for different gO,H(r)’s, where the oxygen−hydrogen distance is less than 2.5 Å. This definition is used in ref 25 as well as in similar EPSR studies on other similar systems based on such as glycerol26 and cellobiose.27 • Criterion 2 produces HBs similarly as criterion 1, but with an added angle restriction. With this criterion, the θO−H···O angle is greater than 145°. In addition, the maximum oxygen−hydrogen distance is 2.4 Å, as adopted from ref 28. • Criterion 3 uses the coordination numbers for oxygen− oxygen correlations, with a maximum oxygen−oxygen

Table 1). As can be observed, with the EPSR simulations, the experimental data can be reproduced with a good accuracy. However, some deviations can be observed in the low-Q range, which are partly caused by the finite size of the structural model. Nevertheless, from the figure, it is obvious that the experimental data show no indication of any significant clustering of trehalose. Rather, the lack of any substantial small-angle scattering in the low-Q range shows that the trehalose and water molecules are basically homogeneously mixed. 3.1. Structure of Water. Here, we aim to elucidate how trehalose affects the local structure of water. Therefore, Figure 3 shows a comparison between the partial RDFs of water atoms in the present trehalose solution and literature data for the corresponding RDFs for bulk water at the same temperature (298 K).32,33 Several differences are clearly observed. The first peak in gOwOw(r) at 2.7 Å becomes sharper and more intense for the trehalose solution. Another substantial effect is seen in the second peak of gOwOw(r) in the region of 3.5 < r < 5.5 Å. This peak is often assigned as a signature of tetrahedral structure in water.34 In the case of pure water (the dotted line in the figure), it can clearly be seen as a single broad peak centered around 4.5 Å. This is in contrast to the trehalose solution, where this peak has split into two smaller peaks, one centered around 3.7 Å and another one at 5.2 Å. Thus, the

nαβ

= 4πcβ

∫r

1

r2

2

g α , β (r )r d r

C


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Figure 4. Angular probability distribution for the angle θ formed between three water oxygens, as seen in the sketch within the figure. The bulk water data for this distribution was obtained from ref 37.

Figure 3. Partial RDFs for different water atom pairs. The solid lines correspond to distributions in water−trehalose mixtures, and the dashed black lines are data obtained for bulk water.32

structure, a water molecule has, on average, four first-order neighbors. However, if the tetrahedral structure is perturbed, an additional water may enter the first-order neighboring shell. This can result in a bifurcation of a HB, which leads to the formation of equilateral triangular structures with the central water molecule.36 As seen from the 60° peak, such a formation of Ow···Ow···Ow isosceles is strongly favored in the trehalose solution, compared to that of the tetrahedral structure (as seen from the 109° peak). Moreover, because a peak at 60° has been hypothesized to be associated with a water molecule having a coordination number of 5 or more,37 its appearance may be caused by intermolecular compression. Another angle distribution of interest is the distribution of θOw···Ow−Hw, which is shown in Figure 5. This distribution provides information regarding the degree of linearity of the HB. For the formation of the strongest HB between two water

disappearance (or splitting) of the peak at 4.5 Å clearly shows that the tetrahedral structure of water becomes substantially altered in the presence of trehalose. It should be noted here that this structural alteration shows analogies to the difference between low-density amorphous (LDA) ice and high-density amorphous (HDA) ice.35 The peak at 4.5 Å in gOwOw(r) of liquid water is intact in LDA but is split into two in HDA, although the distance between the two peaks is smaller than that shown in the present trehalose solution. In the case of HDA, the splitting is believed to be caused by an extra water molecule being forced into the first hydration shell, which, in turn, gives a more orientational freedom to the water molecules in the second and third coordination shells.35 A clear difference compared to bulk water can also be noted for the gHwHw(r) distribution. In this case, the first two distinct peaks, which are related to the tetrahedral coordination of water, have been shifted to shorter distances. A similar shift of these two peaks has previously been found to occur by an increase in temperature,15 indicating that the breaking of water structure, due to trehalose, appears to be analogous to an increase in temperature. The angle distribution of the intermolecular bonds between water molecules is another helpful tool for studying the structure of water.36 Of particular interest is the angle distribution of θOw···Ow···Ow, which provides information about how the water molecules are oriented with respect to each other; see Figure 4. In the case of pure water, there is a broad peak with a maximum near the tetrahedral angle of 109°, which shows how, on average, pure water arranges in a tetrahedral structure. The broadness of the peak shows a distorted structure of liquid water compared to that of ice, which displays a much more defined peak for the tetrahedral configuration (see, e.g., ref 36). In the case of the water− trehalose mixture, Figure 4 shows that the 109° peak is significantly reduced, which is another clear indication of the fact that trehalose destructures the tetrahedral network of water. The most pronounced peak for the water−trehalose mixture is instead located near 60°. This peak is also visible in liquid water, albeit with a lower intensity. In a tetrahedral water

Figure 5. Angular probability distribution, probing the linearity of the HBs between water molecules. The definition of angle θ is shown in the sketch inside the figure. The probability distribution for the bulk water data was obtained from ref 36. D


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The Journal of Physical Chemistry B molecules, a sharp peak is expected around θ = 0°, originating from the fact that the hydrogen atom is located linearly between the two water oxygens. With this geometry, another peak originating from the second hydrogen on the HB donor water molecule should be detected around 105°.36 By comparing the angle distribution of the trehalose solution with that of pure water, as is done in Figure 5, it is clear that particularly the second peak becomes weaker and shifted to a lower angle when trehalose is present in the solution. An even more pronounced weakening of the second peak occurs when ice melts to water due to that the location of the second hydrogen atom becomes less defined.36 This implies that the angle distribution shown in Figure 5 provides further evidence for the fact that trehalose perturbs the water structure. Thus, from the above analysis, it is evident that many indicators of “structured” water disappear in the presence of trehalose. Further insights into how the water structure is altered can be gained also by analyzing the coordination number of a water molecule, that is, the average number of molecules surrounding a given water molecule. The coordination number of water in the first shell is determined to be 4.4 by integration of the first gOwOw(r) peak (Figure 3, eq 2), up to a distance of 3.4 Å, which is somewhat lower than 4.7, as previously determined for liquid water using the same criterion32,37,38 (see Table 3). If the

the water obviously loses some of its bulk structure. Therefore, a more detailed picture of how the water and the trehalose molecules interact is necessary for the understanding of the results discussed above. In this section, the different partial RDFs of water−trehalose atom pairs are presented for such an analysis. Figure 6 shows the partial RDFs of the Ow−Ot, Hw−Ot, and Ht−Ow pairs. Note that the first peak in the Ow−Ot correlation is lower and broader for the Ow−O1 and Ow−O2 pairs (Figure 6a) as compared to that of the hydroxyl oxygens (Ow−O and Ow−O3 pairs, Figure 6c). This indicates that the water molecules coordinate more to the eight different hydroxyl groups (six O’s and two O3’s) than to the internal oxygens (O1 and O2). However, the peak positions for these latter oxygens are somewhat closer than for the hydroxyl oxygens (peak position at 2.7 Å for O1 and O2 and at 3.0 Å for O and O3), suggesting a stronger bonding to trehalose. Using eq 2 for the coordination numbers and criteria 1−4 for the number of HBs, the results from this quantitative analysis of the different O−O and O−H partial RDFs are presented in Tables 4 and 5, respectively. The number of HBs as obtained in other studies (using the same criterion) is shown by values in the parentheses in the same tables. 3.2.1. Coordination Number of Water−Trehalose Oxygens. A simple method of counting the number of water molecules surrounding a trehalose molecule is to set up a maximum distance between a trehalose oxygen and a water oxygen and then calculate the coordination number within this distance. Such an approach gives a good indication of the hydration number of trehalose, although the calculated value (as presented in Table 4) does not take into account the possibility that there is no hydrogen bonding involved in the Ot−Ow correlation. Nonetheless, it should be possible to obtain an indication of the most probable Ot−Ow interactions. As previously mentioned, most of the water molecules surrounding a trehalose molecule are oriented around the two different kinds of hydroxyl groups, O and O3 (2.25 and 2.88 H2O per oxygen, respectively). The fact that there is more water surrounding the O3 groups can be seen as a natural consequence of the fact that this moiety is protruding out further from the trehalose molecule than the other hydroxyl groups, thus being more accessible to the surrounding water. A similar argument can be made for why the internal oxygens, O1 and O2, have so few neighboring water molecules (0.31 and 1.31, respectively). These oxygens are in fact sterically hindered to approach the surrounding water. By counting the total number of water oxygens surrounding the trehalose oxygens, it is found that there are about 22 water oxygens in the vicinity of each trehalose molecule, which is low compared to the results obtained in, for example, ref 39 (27.5 H2O/trehalose) and ref 31 (35.9 H2O/trehalose). As mentioned above, the number of water molecules in the first coordination shell does not necessarily reflect the actual hydration number, which is a measure of the number of water molecules that are hydrogen-bonded to the sugar molecule. In the literature, this number is, to no surprise, considerably lower than the Ot−Ow coordination number due to the fact that not every water molecule surrounding a trehalose molecule is hydrogen-bonded to it. Hydration numbers of 12.5,29 13.0,14 18.9,31 and 13.530 H2O/trehalose have been obtained from different MD studies using criterion 3 as the definition of a HB. However, these values are somewhat higher than that (10.5) obtained in this study by the same criterion. If we instead

Table 3. Coordination Numbers (nβα)a α−β Ow−Ow Ow−Hw Hw −Hw

rminb 3.2 2.3 2.9

rpeak 2.7 1.8 2.2

nβα (r < 3.4 Å) 32

4.40 (4.7 )

nβα (r < rminc) 3.75 (4.732,37,38) 3.7 4.7

a

Numbers in parentheses show corresponding data from other studies. rmin was determined as the r value where there is a minimum in gα,β(r) after the first peak. cThese minimum distances are slightly different for different samples. For bulk water, rmin for the Ow−Ow correlation is about 3.4 Å as compared to 3.2 Å in aqueous trehalose. b

number of water molecules in the first coordination shell exceeds 4, then equilateral triangle structures are expected to be formed between water oxygens37 (as discussed above). Considering the increased number of equilateral triangles compared to that for bulk water, as shown in Figure 4, one would expect an increase in the number of water molecules in the first hydration shell, rather than the observed decrease. However, the coordination numbers presented in Table 3 are calculated using the atomic number density of Ow-atoms, which should produce a lower coordination number, considering that a lot of potential water−water correlations are replaced by water−trehalose correlations. Instead, if we use the atomic number density of any other oxygen, including those in trehalose, and calculate the total oxygen−oxygen coordination number, we find that the value in the trehalose solution actually exceeds that in bulk water (nOw Ow = 5.7 and 4.7, respectively). Thus, also from this analysis, it can be concluded that the presence of trehalose promotes a destructuring of the tetrahedral network of water by promoting additional oxygen− oxygen interactions (from either other water molecules or from trehalose itself). 3.2. Intermolecular Structure of Water−Trehalose. The destructuring of the tetrahedral hydrogen-bonded network of water is, of course, a consequence of the water−solute interactions. As one or more HB-sites of a water molecule bind to a trehalose molecule (thus giving away one of its HB-sites), E


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Figure 6. Partial RDFs for water−trehalose atom pairs. (a) Water oxygen−trehalose carbon bound oxygen pairs. (b) Water hydrogen−trehalose hydroxyl group oxygen pairs. (c) Water hydrogen−trehalose carbon bound oxygen pairs. (d) Water oxygen/hydrogen−trehalose hydrogen/hydroxyl group oxygen pairs.

Table 4. Coordination Numbers (nβα) and Number of HBs (nHB), As Determined by Different Criteria for Oxygen−Oxygen Pairs between Water and Trehalosea α−β

rminb (Å)

rpeakc (Å)

O−Ow O1−Ow O2−Ow O3−Ow Ntot

3.8 3.2 3.5 3.9

3.0 2.7 2.7 3.0

nβαd 2.25 0.31 1.31 2.88 22.2

nHB criterion 4

(3.331) (0.631) (1.5) (3.531) (27.5,39 35.931)

0.15 0.04 0.13 0.31 1.8 (6.214)

nHB criterion 3 1.02 0.15 0.54 1.54 10.5

(2.4531) (0.131) (0.631) (2.731) (12.5,29 13.0,14 18.9,31 13.530)

a

Numbers in parentheses showing corresponding data from other studies. The total number of bonds, or coordination numbers, for a single trehalose molecule (Ntot) are calculated by summing up the contributions from six O−Ow, one O1−Ow, two O2−Ow, and two O3−Ow. bFirst minima after first peak in g(r). cPeak position of the first peak. dCoordination number in the first shell; doo < 3.4 Å.

Table 5. Coordination Numbers (nβα) and Number of HBs (nHB) As Determined by Different Criteria, for Oxygen− Hydrogen Pairs between Water and Trehalosea α−β

rmin (Å)b

rpeak (Å)c

nHB criterion 1

nHB criterion 2

H−Ow O−Hw O1−Hw O2−Hw O3−Hw Ntot

2.5 2.5 2.4 2.3 2.2

2.3 2.0 2.0 1.9 2.0

0.55 0.75 0.28 0.77 1.00 12.8 (825)

0.18 0.23 0.19 0.35 0.37 4.44 (11.5,28 1431)

strongly bonded to trehalose and then particularly to the hydroxyl group oxygens. 3.2.2. Coordination Numbers of Trehalose−Water Hydrogens. Another common method of defining a HB between two species (see, e.g., refs 25−27) is criterion 1, in which the coordination number between hydrogen-bonding atoms of trehalose and water is calculated with a maximum distance of 2.5 Å. From the analysis given in Table 5, it is found that the water molecules are more likely to be the hydrogen donors Hw Ow than the trehalose molecules (nHw O = 0.75, nO3 = 1.00, nH = 0.55). Furthermore, the first peaks in gHwO and gHwO3 are located at shorter distances than the peak in gOwH (2.0 Å compared to 2.3 Å), indicating that the water donor scenario yields a stronger bond. Moreover, compared to the internal O1 and O2 oxygens, the hydroxyl groups show again the highest number of HBs with water. However, it should be pointed out that the partial RDF, gHwO2, does show a strong indication of hydrogen bonding through a sharp peak (sharp compared to the first peaks for the hydroxyl oxygens) in its RDF at a relatively short distance (1.9 Å compared to 2.0 Å for the hydroxyl oxygens). This can be an indication of the strongly bonded so-called internal water molecules binding at these sites, as previously predicted by, for example, refs 18, 29, 39.

a

Numbers in parentheses showing corresponding data from other studies. The total number of bonds, or coordination numbers, for a single trehalose molecule (Ntot) are calculated by summing up the contributions from eight H−Ow, six O−Hw, one O1−Hw, two O2− Hw, and two O3−Hw. bFirst minima after the first peak in g(r). cPeak position of the first peak.

sharpen the definition of a HB to criterion 4, as was used in ref 14 to obtain a hydration number of 6.5, a value of 1.8 is obtained. This large drop in HBs, from the 120° criterion to the 160° criterion, indicates that most (∼9 out of 11) trehalose− water HBs are relatively weak, whereas two water molecules are F


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concentrations similar to those investigated in this study. The decrease in hydration number due to the increase in trehalose concentration was hypothesized to be caused by an increase in the number of trehalose−trehalose interactions (leading to a superposition of different trehalose hydration shells). However, as the authors also pointed out, these hydration numbers represent the number of water molecules that are dynamically perturbed by trehalose and do not directly correspond to the number of hydrogen-bonded water molecules, which might be lower as discussed above.19 Therefore, it is difficult to make a direct comparison with the results obtained in this study. Branca et al.41 determined the hydration numbers of trehalose to be 15.2 and 12.1 using acoustic and viscosity measurements, respectively. The latter value is in good agreement with our results obtained using criterion 1. Similarly, using two different calorimetric measurements, hydration numbers of 12.79 and 10.942 were obtained, suggesting, once again, that the hydration numbers obtained here using criteria 1 and 3 are the most representative. 3.3. Intramolecular Structure of Trehalose. The conformation and flexibility of the trehalose molecules, consisting of two glucose rings, are examined by measuring two dihedral angles Φ and Φ′, which are related to the two different dihedral angles around the glycosidic linkage. These are defined as Φ = M1−C1−O1−C2 and Φ′ = C1−O1−C2− M2, as illustrated in Figure 7 (red showing Φ and green

The total number of intermolecular HBs (Ow−Ht, plus Hw− Ot) between trehalose and water using criterion 1 is 12.8, which is again high compared to that in ref 18, where the EPSR method was used as well. Comparing the results with MD studies using criterion 2, it is found that the results in this study yield a lower hydration number (4.44 HBs per trehalose compared to 11.528 and 1431). 3.2.3. Further Comparison to Previous Studies. In conclusion, the number of water molecules in the vicinity of each trehalose molecule appears to be about 22. Out of these 22, there are 10−12 water molecules, which, according to the more generous criteria (1 and 3), can be considered to be directly bonded to each trehalose molecule. Out of these, there are however only around 2−4 water molecules that can be considered strongly bonded according to the stricter requirements of a HB (criteria 2 and 4). The latter number is comparable with the results obtained by Pagnotta et al.18 However, as the present study also shows that the perturbation of the water structure is substantial, it can be concluded that the hydrogen bonding between water and trehalose must be extensive. Therefore, the numbers given by criteria 1 and 3 are better describing the extensive hydrogen bonding between water and trehalose and thus in agreement with the ideas of, for example, Branca et al.7 Furthermore, the extensive hydrogen bonding between trehalose and water is supported by quasielastic neutron scattering measurements12 and MD simulations,40 which have shown that trehalose is able to slow the water dynamics more than other disaccharides.12 The major discrepancy between the present results and those found in the literature is obtained in a comparison with Pagnotta et al.,18 although these authors used the same methods as those used in this study (i.e., neutron diffraction and ESPR modeling). They found that trehalose is barely hydrogen-bonded to water (apart from a couple of so-called “internal water molecules”). The discrepancy between these apparently similar studies is most likely due to the fact that different isotope compositions were used. For a total of five out of eight isotope compositions, Pagnotta et al. used what they called “deuterated” trehalose. These samples were made by exchanging the exchangeable hydrogen atoms with deuterium (D), although neglecting the fact that when the sugar is dissolved in H2O all of the D atoms of trehalose are rapidly exchanged back to the hydrogen provided from H2O. To avoid this effect, some of the samples in the present study were prepared by deuterated trehalose, that is, also the nonexchangeable carbon-bound hydrogen atoms were deuterated. Thus, these samples were prepared such that they ensure that the D atoms remain bonded to trehalose. Furthermore, when trehalose was dissolved in D2O, the exchangeable hydrogen atoms of trehalose were always deuterated to avoid H-exchange with D2O, as shown in Table 1. The different results in this study, compared to the previous findings by Pagnotta et al.,18 show the importance of using appropriate isotope substitutions for the resulting structural model. Here, we will also make a comparison between the different hydration numbers (nHB), as obtained by the different criteria, as well as between different experimental results where the number of HBs are directly or indirectly measured. Such a comparison gives the opportunity to determine the criteria that correspond most accurately to other experimental data. Lupi et al.19 obtained, using terahertz light scattering, a hydration number of 25 water molecules per trehalose for a very dilute solution and about 17 water molecules at

Figure 7. Pictorial sketch of a trehalose molecule with the sequence of atoms defining dihedral angles Φ and Φ′, highlighted in red and green, respectively.

showing Φ′). We find that these two dihedral angles display the mean values of Φ = −51 ± 12° and Φ′ = −39 ± 12°. These values are in relatively good agreement with a previous MD study by Lerbret et al.,14 in which Φ = −50 ± 12° and Φ′ = −51 ± 13° were obtained. In that study,14 it was also shown that trehalose exhibits a larger conformational flexibility in comparison to that of other disaccharides, indicating fewer intramolecular interactions and thus further strengthening the idea that trehalose possesses more available HB-sites than those in similar sugars.14 3.4. Intermolecular Structure of Trehalose. The probability of finding a trehalose cluster of a certain size is shown in Figure 8. The criterion for cluster formation was set to an intermolecular Ot−Ht distance not exceeding 2.5 Å (Ot and Ht belonging to different hydroxyl and hydroxymethyl groups, respectively). From the probability distribution, it can be observed that the probability that a trehalose molecule is not bound to any other trehalose molecule is ∼75% and that the probability for a pair formation (two trehalose molecules together) is ∼15%. Moreover, the probability for larger clusters decreases exponentially with the cluster size and is basically zero for clusters containing more than four trehalose molecules. In fact, by a comparison with the clusters formed in a hardG


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trehalose in biomolecules is beyond the scope of the present study. However, worth noting is that a similar scenario was also observed in aqueous solutions of glycerol,45 which is another well-known stabilizer of biological materials. Therefore, one might speculate that the ability to stabilize biomolecules is related to the homogeneity of the solution. Probably, the network character of homogeneous aqueous trehalose solutions is responsible for both their high glass-transition temperature (as compared to that of solutions of similar solutes) and their ability to prevent ice formation at relatively high water contents. In addition, it is also possible that trehalose solutions allow more water to interact with the biomolecules, compared to other types of sugar, without inducing any detrimental ice formation close to the surface of the biomolecules. Thus, a number of properties related to the destructuring effect of trehalose on the network structure of water might be the explanation for its extraordinary stabilizing and cryoprotective role. Another such property is the substantial amount of HBs formed between water and trehalose. This effect can also be seen in dynamical studies of aqueous trehalose, which have shown that the dynamics of water molecules is strongly slowed down in the presence of trehalose.10,12,40 Furthermore, there have been studies showing that the presence of trehalose does indeed slow the dynamics of proteins (see, e.g., ref 46). This indicates that the slower water dynamics translates into reduced dynamics of the biomolecules. It has also been suggested by Branca and Magazú et al.43,44,47,48 that the higher fragility of trehalose at high dilutions, compared to that of similar disaccharides, is related to its extraordinary stabilization properties. They argue that a more fragile glass former is better to adapt its structure to different complex biological surfaces.10,43,44,47,48 Thus, the stability provided by trehalose can be attributed to a combination of its capability of forming hydrated structures, which are capable of adapting to the biological surfaces, and a subsequent reduction in the dynamics of the biomolecules by these structures.12 However, to clarify the role of biological stabilization by trehalose in further detail, more studies on the structure of aqueous trehalose solutions containing biomolecules are required.

Figure 8. Cluster-size distribution of trehalose molecules. The red line gives statistics from the EPSR model. The black dashed line is obtained from a hard-sphere model made within the EPSR suite. The red line indicates that ∼0.75 of all trehalose molecules do not form HBs with other molecules and that the probability of finding a cluster larger than four molecules is practically zero.

sphere random distribution model (also shown in Figure 8), it is evident that the propensity of trehalose to form clusters is even lower than that in such a hard-sphere random distribution model of the solution. This implies that trehalose prefers to form HBs to water rather than to other trehalose molecules. It is also worth noting that the absence of trehalose clusters is compatible with the qualitative analysis shown in Figure 2, that is, the samples are lacking larger structures due to the lack of substantial scattering in the low-Q region. As suggested by Lerbret et al.,14 one possible reason for why trehalose is able to interact with more water molecules, compared to its homologues (like sucrose or maltose), is that trehalose less likely forms sugar−sugar HBs. Thus, overall, trehalose has a larger fraction of HB-sites available for water interactions compared to those in its homologues.14 However, although several findings in ref 14 are in good agreement with the present results, for example, in the case of water−trehalose interactions and trehalose flexibility, that study showed larger cluster formations of trehalose compared to what is seen in this study. Similarly, other MD studies, for example, ref 20, have also shown that trehalose tends to form larger clusters. This indicates that there exist tendencies of trehalose to cluster in the force fields used in these MD studies, which is in contrast to what is shown by the experimental neutron diffraction data presented in this study. Further experimental support for that clusters of trehalose are unlikely to be formed comes from diffusion43 and viscosity44 measurements of aqueous trehalose solutions. Using the Stokes−Einstein relation and the same approaches as used in ref 45 to estimate the hydrodynamic radius of trehalose at the present concentration, it is evident that these dynamical data are not compatible with the Brownian motion of clusters of trehalose. 3.5. Stabilization of Biological Molecules. How exact the extensive hydrogen bonding between trehalose and water, and the associated homogeneous distribution of trehalose molecules, relates to the stabilizing and cryoprotective role of

4. CONCLUSIONS Using a combination of neutron diffraction and EPSR modeling, the structure of an aqueous solution containing 33 wt % trehalose has been determined. It is found that trehalose forms approximately 11 HBs to water and retains about 22 water molecules in its first hydration shell. Moreover, the HBs formed with trehalose mainly involve the hydroxyl groups and especially the hydroxymethyl group. The study also shows that trehalose substantially alters the hydrogen-bonded network structure of water. The O−O coordination number of water in the aqueous solution is slightly decreased compared to that of bulk water. However, taking into account also the interactions between water and trehalose results in an increase in the total O−O coordination number from 4.7 to 5.7. Hence, the presence of trehalose perturbs the tetrahedral order of bulk water by allowing more oxygen−oxygen interactions. The present study also reveals that the trehalose molecules are homogeneously distributed because trehalose prefers to form HBs with water rather than with other trehalose molecules. This finding is in contrast to what is generally observed by MD simulations of aqueous trehalose solutions as well as experimental studies on other disaccharides, in which H


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(11) Corradini, D.; Strekalova, E. G.; Stanley, H. E.; Gallo, P. Microscopic Mechanism of Protein Cryopreservation in an Aqueous Solution with Trehalose. Sci. Rep. 2013, 3, No. 1218. (12) Magazù, S.; Migliardo, F.; Telling, M. T. F. Study of the Dynamical Properties of Water in Disaccharide Solutions. Eur. Biophys. J. 2007, 36, 163−171. (13) Branca, C.; Maccarrone, S.; Magazù, S.; Maisano, G.; Bennington, S. M.; Taylor, J. Tetrahedral Order in Homologous Disaccharide-Water Mixtures. J. Chem. Phys. 2005, 122, No. 174513. (14) Lerbret, A.; Bordat, P.; Affouard, F.; Descamps, M.; Migliardo, F. How Homogeneous Are the Trehalose, Maltose, and Sucrose Water Solutions? An Insight from Molecular Dynamics Simulations. J. Phys. Chem. B 2005, 109, 11046−11057. (15) Branca, C.; Magazú, V.; Maisano, G.; Migliardo, F.; Soper, A. Study on Destructuring Effect of Trehalose on Water by Neutron Diffraction. Appl. Phys. A: Mater. Sci. Process. 2002, 74, s450−s451. (16) Branca, C.; Magazú, S.; Migliardo, F.; Migliardo, P. Destructuring Effect of Trehalose on the Tetrahedral Network of Water: A Raman and Neutron Diffraction Comparison. Phys. A 2002, 304, 314−318. (17) Lupi, L.; Comez, L.; Paolantoni, M.; Fioretto, D.; Ladanyi, B. M. Dynamics of Biological Water: Insights from Molecular Modeling of Light Scattering in Aqueous Trehalose Solutions. J. Phys. Chem. B 2012, 116, 7499−7508. (18) Pagnotta, S. E.; McLain, S. E.; Soper, A. K.; Bruni, F.; Ricci, M. A. Water and Trehalose: How Much Do They Interact with Each Other? J. Phys. Chem. B 2010, 114, 4904−4908. (19) Lupi, L.; Comez, L.; Paolantoni, M.; Perticaroli, S.; Sassi, P.; Morresi, A.; Ladanyi, B. M.; Fioretto, D. Hydration and Aggregation in Mono- and Disaccharide Aqueous Solutions by Gigahertz-to-Terahertz Light Scattering and Molecular Dynamics Simulations. J. Phys. Chem. B 2012, 116, 14760−14767. (20) Sapir, L.; Harries, D. Linking Trehalose Self-Association with Binary Aqueous Solution Equation of State. J. Phys. Chem. B 2011, 115, 624−634. (21) Lins, R. D.; Pereira, C. S.; Hünenberger, P. H. Trehalose− Protein Interaction in Aqueous Solution. Proteins: Struct., Funct., Bioinf. 2004, 55, 177−186. (22) Soper, A. K. Empirical Potential Monte Carlo Simulation of Fluid Structure. Chem. Phys. 1996, 202, 295−306. (23) Damm, W.; Frontera, A.; Tirado−Rives, J.; Jorgensen, W. L. Opls All-Atom Force Field for Carbohydrates. J. Comput. Chem. 1997, 18, 1955−1970. (24) Berendsen, H.; Grigera, J.; Straatsma, T. The Missing Term in Effective Pair Potentials. J. Phys. Chem. 1987, 91, 6269−6271. (25) Pagnotta, S.; Ricci, M.; Bruni, F.; McLain, S.; Magazú, S. Water Structure around Trehalose. Chem. Phys. 2008, 345, 159−163. (26) Towey, J. J.; Soper, A. K.; Dougan, L. Molecular Insight into the Hydrogen Bonding and Micro-Segregation of a Cryoprotectant Molecule. J. Phys. Chem. B 2012, 116, 13898−13904. (27) O’Dell, W. B.; Baker, D. C.; McLain, S. E. Structural Evidence for Inter-Residue Hydrogen Bonding Observed for Cellobiose in Aqueous Solution. PLoS One 2012, 7, No. e45311. (28) Donnamaria, M. C.; Howard, E. I.; Grigera, J. R. Interaction of Water with α,α-Trehalose in Solution: Molecular Dynamics Simulation Approach. J. Chem. Soc., Faraday Trans. 1994, 90, 2731− 2735. (29) Conrad, P. B.; de Pablo, J. J. Computer Simulation of the Cryoprotectant Disaccharide α,α-Trehalose in Aqueous Solution. J. Phys. Chem. A 1999, 103, 4049−4055. (30) Lee, S. L.; Debenedetti, P. G.; Errington, J. R. A Computational Study of Hydration, Solution Structure, and Dynamics in Dilute Carbohydrate Solutions. J. Chem. Phys. 2005, 122, No. 204511. (31) Bonanno, G.; Noto, R.; Fornili, S. L. Water Interaction with α,αTrehalose: Molecular Dynamics Simulation. J. Chem. Soc., Faraday Trans. 1998, 94, 2755−2762. (32) Soper, A. K. The Radial Distribution Functions of Water and Ice from 220 to 673 K and at Pressures up to 400 Mpa. Chem. Phys. 2000, 258, 121−137.

clustering of sugar molecules is commonly observed. On the other hand, our results support the idea that an important aspect of the cryopreservative ability of trehalose is its excellent tendency to bind to water, as put forth by, for example, Branca and Magazú et al.7 The tendency to form a large number of HBs with water may also be the reason why trehalose shows a great ability to inhibit water crystallization. It might also be the reason for the relatively high glass-transition temperature5 and viscosity41 of aqueous trehalose solutions, that is, all properties that seem to be important for its excellent stabilizing and cryopreservative role in biological macromolecules, such as proteins. Finally, it should be noted that the results obtained in this study are partially contradictory to the findings obtained by Pagnotta et al.18 in a similar study in which neutron diffraction was combined with EPSR. These contradictions are most likely caused by the fact that in that study they did not deuterate the nonexchangeable hydrogens of trehalose and furthermore neglected the rapid exchange of H and D between water and the hydroxyl groups of trehalose.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +4631 772 56 80. ORCID

Jan Swenson: 0000-0001-5640-4766 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Swedish Research Council. We thank the STFC for a beamtime allocation (experiment RB1520094).

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REFERENCES

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Structure of Aqueous Trehalose Solution by Neutron Diffraction and

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