Structural and vibrational characterization of anhydrous and dihydrated species of trehalose based on the FTIR and FTRaman spectra and DFT calculations

4.1 Structural analysis

Table 1 shows the calculated total and relative energies, dipole moments and populations for all the studied trehalose species in gas and aqueous solution phases. Here, we have considered two different structures for the αα-trehalose anhydrous and dihydrated species, named C1 and C2 in order to find the most stable species, as observed in Table 1. The results clearly show that the αβ anhydrous and the C1αα- dihydrated species are the most stables and, hence, the higher populations are found for the αβ- anhydrous species in gas phase with a value of 60% while in solution the value decreases up to 35.33%, then, the population of the ββ species increases drastically in this medium from 0.16% in the gas phase up to 46.30%. In relation to the dipole moment values, in gas phase the three anhydrous species show similar values while their values notably change in solution observing a particular decrease in the value for the ββ species. This notable reduction in the dipole moment value for the anhydrous ββ species could probably be related to their major volume expansion in solution and to their higher solvation energy value, as will see later. The dipole moment values for the two dihydrated species also increase in solution presenting the most stable species the higher value in gas phase but the lower value in solution, in relation to the C2 species. Here, the few variations observed in the dipole moments for the dihydrated species in solution could be related to a small perturbation of water hydration shell and short hydrogen bonds between trehalose oxygens and water hydrogens, as reported by Pagnotta et al. (2008). The αα dimeric species present an energy value of −2901.5588 Hartrees in gas phase with a dipole moment value of 6.05 D. This way, this dimeric species show a higher stability as compared to two units of αα dihydrated species, it is 2 × −1450.7569 = −2901.5138 Hartrees.

A comparison of the calculated geometrical parameters for the anhydrous and dihydrated species of trehalose in gas phase with those corresponding experimental ones for the dihydrated species (Taga et al., 1972 and Brown et al., 1972) by means of the root-mean-square deviation (RMSD) values can be seen in Table 2. The RMSD values show a very good agreement for the bond lengths and angles of all the species and, especially, in the dihedral angles of the dihydrated species with a difference only of 7.4° while for the anhydrous species the RMSD values vary between 65.5 and 38.1°, as expected because the compared experimental species is also dihydrated. Note that the higher differences among the structures are observed in the dihedral C18–C20–C22–O10 angles where only the dihydrated species has a value close to the experimental one, as explained before. In general, the optimized parameters are underestimated by the B3LYP/6-31G^∗ calculations, in reference to the experimental values and, only some calculated distances are approximately similar in all the species such as those C–C bonds belonging to the ring R1. Probably, this observation can be explained because the α-D-glucopyranosyl R1 ring remains practically constant in the αα, αβ and ββ species including the dihydrated one while the other α-D-glucopyranoside ring R2 change in the αβ and ββ species. For this reason, both αα anhydrous and dihydrated species have practically similar bond lengths values with exception of the C13–O1, C20–C22, C21–C23, C14–O4 and C18–O8 bonds which show slightly variations in the values, as indicated in Table 2.

4.2 Volume variations and solvation energies

The molecular volume variations (Ugliengo, 1998) observed in the anhydrous and dihydrated species in solution, in reference to the values in the gas phase and, the calculated solvation energies at the B3LYP/6-31G^∗ calculations level using the polarized continuum (PCM) and solvation (SD) models (Tomasi and Persico, 1994; Miertus et al., 1998; Marenich et al., 2009) are summarized in Table 3. The results for all the trehalose species show expansions of volume in aqueous solution where the value increases according to the following order: αβ-trehalose (1.4 ų) < αα-trehalose (2.1 ų) < ββ-trehalose (2.8 ų). This variation observed could suggest that the position of the OH group also has influence on the volume variation, thus when the position is ββ- the variation is higher. This way, the reduction in their dipole moment value could justify that observation. On the other hand, the volume expansions theoretically observed for trehalose, maltose and sucrose could probably explain that different water molecules are around disaccharide–water mixtures which generate a particular higher ‘‘rigidity’’ in trehalose due to their lower volume variation, as was experimentally observed by Varga et al. (2008). A similar relation is observed in the calculated solvation energy values, as indicated in Table 3. Thus, the species with higher volume variation, it is the ββ-trehalose form, present the higher solvation energy value, probably because only for this species in solution the dipole moment values significantly decrease their value from 2.72 D in gas phase to 0.81 D in solution while for the remain species the values increase in this medium. When these values are compared with the values corresponding to the two maltose anhydrous species we observed a contrary relation because the species with higher volume variation present the lower solvation energy value. On the other hand, when the solvation energies for the trehalose species are compared with the other sugars, we observed that the β anhydrous and monohydrated species have higher values than the corresponding α ones where, in particular, the values of the hydrated sugar species decreasing according to the following order: Sucrose dihydrated > β-maltose > Anhydrous sucrose > Anhydrous β-Lactose > Sucrose pentahydrated > Anhydrous α-Lactose > α-α-trehalose dihydrated > α-Lactose Monohydrated > Anhydrous maltose while the three anhydrous species of trehalose have the lowest solvation energy values, as observed in Table 3. These low solvation energies values observed for trehalose in relation to sucrose and maltose probably can explain in part why a trehalose/water mixture shows a larger structural resistance to temperature changes and a higher ‘‘rigidity’’ in comparison with maltose/H₂O and sucrose/H₂O mixtures, as mentioned by Varga et al. (2008). This property of trehalose could also be related to the most intense disaccharide–water interaction observed for trehalose by Magazù et al. (2007).

4.3 Charges, molecular electrostatic potentials (MEP) and bond orders (BO) studies

For the four trehalose forms, two charge’s types, the atomic MK and NPA charges (Besler et al., 1990; Glendening et al., 1996), were considered using the B3LYP/6-31G^∗Method, as was mentioned in section computational details. These charges for those species in gas phase are summarized in Table S1 while the values in solution are given in Table S2. The exhaustive inspection of the values show that the NPA charges have in general higher values than the MK charges in both media and besides, these charges on the C22 and C23 atoms in the four species have negative signs, as observed in Tables S1 and S2. Note that the MK charge on the C19 atom belonging to the R1 ring of the dihydrated species present negative sign in gas phase and positive in solution while on the other ones are observed positive sings. On the other hand, these charges in solution are observed with negative signs on the C12 and C13 atoms in the dihydrated species while in gas phase on both atoms are observed positive signs. Note that for the dihydrated species in gas phase we observed positive signs in both charges on all the H atoms corresponding to the water molecules. On the contrary, in solution the NPA charges on the H atoms of the dihydrated species are observed with negative signs while the MK exhibits in this media positive signs. These variations in the charges observed for the species in solution could clearly explain the hydration process with water molecules of the solvent. Besides, these changes in the charges possibly will explain the most intense disaccharide–water interaction observed for trehalose in solution by Magazù et al. (2007) that moreover will justify their higher bioprotective effectiveness.

The MEPs were analyzed on all the atoms of the four species in both media and the corresponding values are presented in Table S3. Analyzing the results, we observed that in both media the most negative values are located on the O10 atoms of all the species while the less negative values for the anhydrous species are observed on the O1 atoms and for the dihydrated species on the O3 atom. Thus, these results for the anhydrous species could indicate that the inter-ring C13-O1–C12 angle is independent of the medium employed. Besides, this result probably is also related to that observed experimentally by Taga et al. (1972) for the dihydrated species because neither the ring oxygens nor the glycosidic linkage oxygen accept hydrogen bonds. Later, as it is expected, the lowest negative MEP values for all the trehalose species are observed on the H atoms where the anhydrous species show the highest values on the H34 atoms while the lower values are observed on the H38 and H39 atoms of these species probably because the distances between these atoms are close to the corresponding O1 atoms (2.248–2.241 Å) and, for these reasons, they are the more labile atoms. Analyzing, the values for the dihydrated species, the higher values are also observed on the H34 atoms while the lowest values on the H atoms of the water molecules. The different reaction sites can be obviously observed by the different colorations when the MEP mapped surfaces for the α-α-anhydrous and dihydrated species are compared in Fig. S2. Thus, the blue colors show the electrophilic sites observed on the H atoms while the nucleophilic sites are clearly observed in red color on all the O atoms. Here, it is interesting to note that in the dihydrated species a strong blue color are observed on the H atoms of the water molecules in agreement to the low MEP values, as observed in Table S3.

In relation to the bond order values, Table S4 show these values expressed as Wiberg indexes, for the four trehalose species. The results clearly show that for the four species the higher BO values are observed in the O atoms that belong to the R1 and R2 rings and to the inter-ring O atoms, these are the O1, O2 and O3 atoms, which have values between 2,030 and 1,978 while their values clearly decrease in solution. Here, newly we evidenced that experimental results found by Taga et al. (1972) for the dihydrated species. On the contrary, in general remaining O atoms of all the species belonging to different OH groups in the two rings are observed slight increase in the BO values in solution. These higher values in solution probably indicate that these H atoms linked to those O atoms are less labile. On the contrary, regarding the BO values for the C atoms, the C16 and C17 atoms in αα-trehalose and the C17 atoms in αβ and ββ-trehalose and the C14 and C19 atoms in the dihydrated species present the higher values in gas phase but, some of these values change few in solution. Probably, due to the different positions of the OH groups linked to the C atoms some values for these atoms are different in the four species. Other very important result is observed in the H atoms of the four species because when those atoms are linked to the C atoms have higher values than the other ones, as expected due to that the H atoms bonded to the OH groups are most labile and, as a consequence they have the lowest values. An expected result is observed in the dihydrated species, because the O and H atoms belonging to the water molecules present the lowest BO values but, they increase notably in solution due to the hydration of these atoms with water molecules. These results can explain that the water mobility within the first hydration shell is ca. 25% smaller than that of pure water, as observed by Bonanno et al. (1998) from molecular dynamics simulation studies for the water interaction with αα-trehalose.

4.4 Stability study

The stabilities of all the trehalose’s species in gas and aqueous solution phases were studied using NBO (Glendening et al., 1996) and AIM (Biegler-Köning et al., 2001) calculations. Thus, the donor–acceptor interaction energies obtained from the second order perturbation calculations in those media are presented in Table S5. Here, the main donor–acceptor interaction energies for the anhydrous species in both media are observed from the lone pairs of the O atoms to the antibonding O–C orbitals (LP(2)O1 → σ*O–C) while for the dihydrated species besides these are also observed those from the lone pairs of the O atoms to the antibonding O–H orbitals (LP(2)O1 → σ*O–H), as can be seen in Table S5. Thus, the contributions to the total energy show clearly that the dihydrated species are the most stable in both media than the anhydrous species. The presence of a additional H bonds interaction in solution in the dihydrated species will explain perhaps the role of trehalose in the stabilization of proteins because, as recently was mentioned by Olsson et al. (2016), the protein adsorbs water and thereby reduces the water content in the trehalose–water system.

The stabilities of all the trehalose’s species were also studied by means of the topological properties with the AIM2000 program (Biegler-Köning et al., 2001). Accordingly, the calculated charge electron density, (ρ) and the Laplacian values, ∇²ρ(r) in the bond (BCPs) and ring critical points (RCPs) for the R1 and R2 rings of the three anhydrous and dihydrated species in both media are given in Tables S6 and S7, respectively. Analyzing first the three anhydrous species we observed from Table S6 notable differences between the values in gas phase and those in solution. For instance, the αβ- and ββ- species in gas phase present the two expected RCPs due to the R1 and R2 rings and the H bonds O6–H42 interactions while this latter interaction it is not observed in the αα- species. On the contrary, in solution the αα- and αβ- species have the two RCPs and a new H–H interaction while in the ββ- species there is not observed the latter interaction. This observation can be justified by the large distance between the O6 and H42 atoms of 3.519 Å. On the other hand, the distance between the H33 and H38 atoms of 2.615 Å justifies that in the αα species in gas phase there is not observed interaction between both atoms. For the dihydrated trehalose species a very different situation is found when their topological properties are analyzed in gas and in aqueous solution phases. Thus, Table S7 shows their calculated properties using the B3LYP/6-31G^∗ Method in both media. First, in both media are observed the two RCPs but in gas phase are observed six O–H interactions while in solution are observed only five because the O8–H44 interaction in solution present a distance of 3.139 Å. These results clearly show the high stability of the dihydrated species in both media and, besides, suggest the presence of this species in the solid state, in complete agreement with the NBO results. Here, another very important result is the formation of the H bond O3–H49 interactions in both media observed only for the dihydrated species because this O3 atom belong to the glucopyranose R1 ring. This result is different from that observed experimentally by Taga et al. (1972) for the dihydrated species where neither the ring oxygens nor the glycosidic linkage oxygen accept hydrogen bonds. The AIM study explains the ability of trehalose to form H bonds and, this way, to stabilize the membranes, as reported by Crowe and Crowe (1984).

The high stability of the dihydrated trehalose species predicted using NBO and AIM studies, as compared with the anhydrous species due to the H bonds formation could support: (i) the small perturbation of water hydration shell and short hydrogen bonds between trehalose oxygens and water hydrogens (Pagnotta et al., 2008), (ii) the water dynamics in the presence of bioprotectant systems which is affected by all the disaccharides and particularly by trehalose (Varga et al., 2008; Magazù et al., 2007), (iv) that trehalose is shown to affect the swelling properties of the polymer with temperature, stabilizing its conformation (Magazù et al., 1998), (v) the presence of the disaccharides inhibits the protein dynamical transition (Magazù et al., 2011) and, (vi) only the H bonds intra-molecular, as mentioned by Ballone et al. (2000), because the DFT results for the isolated molecule is a simplified scheme and provides a fairly good description of ground-state geometries. On the contrary, the cohesion of the crystal is mainly due to the formation of several intermolecular hydrogen bonds which involve every hydroxyl group in the system.

4.5 Frontier orbitals and descriptors study

Trehalose is a sugar that acts on the stability of membranes in anhydrobiotic organisms and this effect is only observed in this carbohydrate by Crowe and Crowe (1984), for this reason, it is very important to predict their reactivities and behaviors in gas and in aqueous solution in order to understand their mechanism of bio-protecting. Thus, their reactivities in those media were predicted using the frontier orbitals (Parr and Pearson, 1983; Brédas, 2014; Márquez and Brandán, 2014; Cataldo et al., 2014; Romani and Brandán, 2015a; Romani et al., 2015b), whose values are presented in Table S8, while the chemical potential (μ), electronegativity (χ), global hardness (η), global softness (S), global electrophilicity index (ω) and mucleophilic index (E) descriptors were employed (Márquez and Brandán, 2014; Cataldo et al., 2014; Romani and Brandán, 2015a; Romani et al., 2015b; Márquez et al., 2015a,b) to predict their behaviors in the two media and, they are summarized in Table S9. These properties calculated for the trehalose species were then compared with those reported for maltose and lactose (Iramain et al., 2016). Here, the high gap values observed for the anhydrous and dihydrated species of trehalose could probably explain that it is a no reducing disaccharide of glucose while the similar lower gap values for maltose and lactose probably justify that these carbohydrates are reducing sugars. Note that the reactivity in all the sugars increases in aqueous solution. In particular, if trehalose is less reactive in water in a protein–trehalose–water system the protein adsorbs water decreasing their content from the trehalose–water system, as reported by Olsson et al. (2016). Moreover, the major intensity in the trehalose–water interaction and its higher bioprotective effectiveness, as mentioned by Magazù et al., 2007 could also be justified by the high gap value and low reactivity. On the other hand, the high gap values observed for the anhydrous and dihydrated species of trehalose suggest a reduced reactivity that could support that this species acts as a nonreducing disaccharide of glucose while the similar lower gap values for maltose and lactose probably justify that these carbohydrates are reducing sugars. Here, it is necessary to remember that in a reducing disaccharide of glucose (maltose) one of the two monosacharides has one free hemiacetal unit that generates a reducing aldehyde group while the other one is occupied with the glycosidic bond which cannot act as reducing agent. On the contrary, trehalose is a nonreducing disaccharide of glucose because there is not a free hemiacetal unit but there is an acetal linkage between their anomeric centers, giving a structure without one free hemiacetal unit to act as reducing agent.

In relation to the descriptors, it is necessary to clarify that as their equations are widely known these were not included in the main manuscript but in Table S9. Regarding exhaustively the descriptors, we observed first that all the values observed in gas phase decrease in solution and, then, that the less reactive species (>gap) are the trehalose’s species which have > global hardness (η) and <global softness while the anhydrous species also present > nucleophilic and electrophilic indexes. Analyzing the nucleophilic and electrophilic indexes for the hydrated disaccharides in solution, we observed that the presence of two water molecules in trehalose, in relation to one in maltose and lactose, increase the nucleophilicity thus, the observed tendency is: trehalose > maltose > lactose while the trend change with the electrophilicity to: maltose > trehalose > lactose. These results are in agreement with those MEP values predicted for the nucleophilic sites on all the O atoms including all OH groups and, with the results suggested by Ballone et al. (2000), because the cohesion of the crystal is mainly due to the formation of several intermolecular hydrogen bonds which involve every hydroxyl group in the system. The nucleophilicity index observed especially in the hydrated species could probably be related with that important property to stabilize membranes due to their ability to form H bonds and, hence, to replace water around the head group of a phospholipid, according to the results observed by Crowe and Crowe (1984). In addition, that nucleophilicity index will explain why trehalose inhibits the protein dynamical transition during the addition of disaccharides to hydrated lysozyme and why the of hydrogen bond connectivity has influence on transport properties for the trehalose–water system, as revealed by Magazù et al. (2011) and 1998, respectively.

4.6 Vibrational analysis

All the trehalose species were optimized using B3LYP/6-31G^∗ calculations with C₁ symmetry where the three anhydrous species have 129 normal vibration modes while the dihydrated species present 147 normal vibration modes and, where all these modes show activity in the infrared and Raman spectra. The experimental infrared and Raman spectra of dihydrated trehalose in the solid phase can be seen in Fig. 4 while in Fig. 5 is presented a comparison of the IR spectrum of the αα-dihydrated species with the corresponding predicted for this species and their dimer in gas phase at the B3LYP/6-31G^∗ level of theory. On the other hand, Fig. S3 shows the comparisons among the experimental IR of dihydrated trehalose in the solid phase with the corresponding predicted for the three anhydrous species in gas phase using that level of theory. The comparisons among the experimental IR of dihydrated trehalose in the solid phase in the 4000–2500 cm⁻¹ region with the predicted for this species and their corresponding dimer in gas phase at the same level of theory are given in Fig. S4 while in Fig. S5 are presented these same comparisons but in the 2000–0 cm⁻¹ region. These two comparisons clearly evidence the presence of more than one molecule in the solid state due to the increase in the numbers and intensities of the bands in both spectra for this species in relation to the observed in the dihydrated species. A similar situation is observed when the Raman spectra are compared in those two regions, as can be seen in Fig. S6 and S7. In the 4000–2500 cm⁻¹ region the presence of a dimer is also observed in the form of the bands with higher intensities (Fig. S6, green spectrum) while the quantities and forms of the bands in the 2000–0 cm⁻¹ region could support the presence of the dimer, as those two most intense bands are observed in the Raman spectra at approximately 1500 cm⁻¹. In Table 4 is summarized the observed and calculated wavenumbers and assignments for the three anhydrous and dihydrated species in gas phase using SQM/B3LYP/6-31G^∗ calculations while for the assignments of the dimer the GaussView program (Nielsen and Holder, 2008) was used. We observe from Table 4 that the assignments are clearly different for the αα-, αβ- and ββ-trehalose species indicating, this way, the influence of the position of the OH groups on the bands observed in both spectra. Here, the SQM force fields were calculated with the Molvib program (Sundius, 2002) using scale factors valid for B3LYP/6-31G^∗ calculations (Rauhut and Pulay, 1995a,b). These results can be obtained at the request of the authors. On the other hand, the vibrational assignments for the anhydrous and dihydrated species were performed taking into account those assignments reported for other sugars (Brizuela et al., 2012a, 2014; Márquez et al., 2015a,b; Iramain et al., 2016), those reported by Takahashi et al. (2006) for the low-frequency vibrations of the dihydrated species and, in accordance with ours DFT calculations obtained here. It is necessary to clarify that Ballone et al. (2000) have studied structural properties and harmonic vibrational modes of trehalose in the gas phase and in the monohydrate crystal in order to analyze the local conformation and the relative strength of intra- and intermolecular hydrogen bonds, and the effects of the crystal environment on the molecular geometry and vibrational frequencies. Their results provide a fairly good description of ground-state geometries, while vibrational properties are only in qualitative agreement with those computed by the density functional method.

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Figure 4

Experimental FTIR spectrum of αα-dihydrated in the solid phase in the 4000–400 cm⁻¹ region compared with their corresponding FT-Raman spectrum in the 4000–10 cm⁻¹ region.

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Figure 5

Experimental FTIR spectra of αα-dihydrated in the solid phase compared with the corresponding predicted for this species and their dimer in gas phase at the B3LYP/6-31G^* level of theory.

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Later, the assignments for some groups are presented below.

4.7 Force field

For the three anhydrous species of trehalose and their dihydrated form the force constants were computed using the SQMFF methodology (Rauhut and Pulay, 1995a) with the Molvib program (Sundius, 2002). The results in the gas phase at the B3LYP/6-31G^∗ level of theory are presented in Table 5 together with those values reported for the maltose and lactose species (Márquez et al., 2015a,b; Iramain et al., 2016) at the same level of theory. Comparing first the values for the anhydrous species, we observed that the three species show practically the same values and there are no significant changes among them, however, when these values are compared with those corresponding to the dihydrated species all the values slightly increase for this species in relation to the anhydrous species with exception of the f(νO–H) force constant whose value decrease for the dihydrated species. This observation is probably related with the higher quantity of intra-molecular interactions observed for this species using NBO and AIM studies (Tables S5–S7). On the other hand, the anhydrous species of maltose and lactose show higher values in the f(δC–O–C) force constants related to the glycosidic bonds, between 2.78 and 1.88 mdyn Å⁻¹ as compared with those corresponding to trehalose (1.19–1.14 mdyn Å⁻¹) while the hydrated species of trehalose present a force constant value (2.31 mdyn Å⁻¹) between those corresponding to maltose (2.78–2.27 mdyn Å⁻¹) and lactose (1.92 mdyn Å⁻¹). Here, the f(δC–O–C) force constants related to the glycosidic bonds evidence clearly the different characteristics in the three sugars: maltose > trehalose > lactose but these values are not justified because maltose and lactose are reducing sugars while trehalose is a non-reducing sugar. Hence, the behavior electrophilic of trehalose could also be related to their f(νO–H)_H2O force constant because the value for the dihydrated trehalose is 6.88 mdyn Å⁻¹ while for monohydrated lactose is 6.96 mdyn Å⁻¹ and for monohydrated maltose 7.25–7.05 mdyn Å⁻¹. Here, the low f(νO–H)_H2O force constant value for trehalose are strongly related to the lowest BO values observed in particular for the O–H bonds corresponding to the water molecules. This way, the following relation is observed in the f(νO–H)_H2O force constants: trehalose < lactose < maltose showing a low f(νO–H)_H2O force constant value for trehalose which could justify: (i) the very fragile character from the trehalose–water system to the temperature and concentration changes, as experimentally was reported by Branca et al. (1999) from viscosity and Raman scattering studies, (ii) why the presence of the disaccharides inhibits the protein dynamical transition (Magazù et al., 1998) from studies of the influence of hydrogen bond connectivity on transport properties for the same trehalose–water system, (iii) the non-reducing property of this sugar and, (iv) the ability of trehalose to form H bonds and, this way, to stabilize the membranes, as reported by Crowe and Crowe (1984).