[Frontiers in Bioscience 14, 3523-3535, January 1, 2009]
Thermodynamic, hydration and structural characteristics of alpha,alpha-trehalose
Takao Furuki,1 Kazuyuki Oku2, Minoru Sakurai1
1
TABLE OF CONTENTS
1. ABSTRACT
A nonreducing disaccharide, alpha,alpha-trehalose, accumulates endogenously in diverse anhydrobiotic organisms in their dehydrating process or prior to their desiccation, being thought to have a protective function either as a water replacement molecule or as a vitrification agent in the dry state. Trehalose acts also as a protectant against physiological stress, including freezing, ethanol and oxidation. To elucidate the origin of these different functions of this sugar, it is necessary to obtain a deep insight into the physicochemical properties of trehalose at the molecular level. In this review, we focus our attention on the thermodynamic, hydration and structural properties of carbohydrates, and extract the characteristic feature of trehalose. On the basis of these findings, we subsequently discuss the underlying mechanism for protein stabilization by trehalose in solution and for its anitoxidant function on unsaturated fatty acids.
2. INTRODUCTION
Water is essential for life, but some organisms survive desiccation and the dry state for long periods during which metabolism and life processes come to a halt, but resume on rehydration. Desiccation tolerance, or anhydrobiosis (''life without water''), is found across diverse biological kingdoms, including plants and animals such as mushrooms, yeast, fungi and brine shrimp (1,2). A nonreducing disaccharide, alpha,alpha-trehalose (hereafter trehalose, Figure 1), accumulates in these anhydrobiotic organisms and is thought to have a protective function either as water replacement molecules or vitrification agents in the dry state (1-3). Trehalose acts also as a protectant against other environmental stresses, freezing (4,5), ethanol (6) and oxidation (7-9).
In the food and pharmaceutical industries, trehalose has been utilized as a stabilizer to preserveproteins and enzymes in the dry state (10-13). Recently human platelet was successfully freeze-dried with trehalose (14). In addition to the preservation of such dried biomaterials, this sugar acts a good stabilizer of proteins and membranes in the presence of bulk water. It has been found that trehalose is very effective in stabilizing labile proteins during exposure to high temperatures in solution (15-18). Our early 31P NMR study indicated that trehalose stabilizes unilamellar liposome by increasing the packing density among the constitutive phospholipid molecules, leading to inhibition of the fusion of the liposome (19). A recent finding is that trehalose inhibits the aggregation of proteins associated with Huntington's and Alzheimer's diseases. Tanaka et al. reported that trehalose could be used to inhibit the aggregation of polyglutamine in vivo in a rat model for Huntington's disease (20) while an in vivo study by Liu et al. indicated that this sugar effectively inhibits the aggregation and neurotoxicity of beta-amyloid 40 and 42 (21). Many other studies have been reported focusing on the application of this sugar, including in medical and cosmetic uses (1,3).
As described above, trehalose acts as a water substitute and exhibits a protective effect during various biological stresses. Our goal is to understand why trehalose is superior to other saccharides as such a stress protectant, and why it behaves as a chemical chaperone. To address this problem, it is necessary to gain a deep insight into the physicochemical properties of trehalose at the molecular level. In this review, we first focus our attention on the thermodynamic, hydration and structural properties of carbohydrates, then extract the characteristic features of trehalose. On the basis of these findings, we discuss the underlying mechanism for protein stabilization by trehalose in solution and for its antioxidant function on unsaturated fatty acids. Thus, the major part of this review concerns the biological role of this sugar in aqueous solution. Readers who are interested in the preservation of dry materials by trehalose are referred to excellent reviews by Crowe et al. (1,3).
3. THERMODYNAMIC CHARACTERIZATION OF CARBOHYDRATES
Thermodynamic properties of aqueous carbohydrates have been studied for more than three decades, because of their academic and commercial importance in view of biological processing and storage accompanying water stress such as freezing and desiccation. In particular, the study of glassy property of carbohydrates has made considerable progress in the 1990s'. The glassy property of a given carbohydrate follows the state diagram as shown in Figure 2, where Tg', Tg and Cg' are the glass transition temperatures of the solution and the pure solute, and the solute concentration in the residual unfrozen matrix resulting from a freeze concentration. In Figure 2, Tg'max corresponds to the glass transition temperature at the maximally freeze-concentrated concentration Cg'max and these quantities are regarded as the intrinsic properties of a given carbohydrate. In general, to measure Tg'max, isothermal annealing is required at a temperature above Tg' but below the melting point of a given aqueous carbohydrate (23). Tg' values obtained without such annealing were, however, in good agreement with the corresponding Tg'max, although the observed values for disaccharides were slightly smaller (24). Therefore, Tg' can be used to characterize the glassy property of di- and oligo-saccharides instead of Tg'max. On the other hand, the relationship between Cg'max and Cg' is not so simple. Here we explain it by taking an example of aqueous sucrose. By cooling an aqueous solution of sucrose, water is crystallized and then the amount of ice can be measured from the heat of fusion of ice, Hf. Usually, Hf decreases linearly with the initial sucrose concentration (24). Then Cg' was estimated by extrapolating the linear regression line to Hf = 0. The resulting Cg'value was 69 wt%, which is smaller than the maximally freeze-concentrated concentration, Cg'max, up to ca. 80 wt%. (23,25,26). In general, Cg' evaluated in the above way corresponds to the threshold when neither ice formation on cooling nor melting of ice on heating was observed in differential scanning calorimetry (DSC) measurements (26-29), in other words, the minimum concentration of the carbohydrate necessary for keeping all water unfrozen in the aqueous solution.
To date, there has been growing consensus that the glassy property is one of the key factors that should be considered when developing a method for the effective preservation of biomaterials (30-34). Early studies in the 1990s' found that from aqueous mono- to polysaccharides, there is a close relationship between molecular weighs of carbohydrates and their glass transition temperatures Tg' (30-34). However, some exceptional and unexplainable facts were also found. For example, a large difference up to 40 �C was found even among disaccharides (35): the Tg s of anhydrous alpha,alpha-trehalose and sucrose are 115 �C and 75 �C, respectively (Table 1). Such a discrepancy should be rationalized by factors other than just molecular weight.
To address this problem, one of the authors of the present review (T. F.) investigated the thermodynamic properties of aqueous carbohydrates from mono- to oligosaccharides (Tables 2 and 3) through DSC measurements. Figure 3 shows the plot of Tg' vs. Uw, where Uw is the amount of unfrozen water (given in moles of water per mole of carbohydrate) calculated from the Cg' value at Hf = 0 obtained in the same way as described in the above paragraph. As a whole Tg' is mainly determined by the molecular weight of the carbohydrate dissolved in water, whereas Uw varies from sugar to sugar even among a family with the same molecular weight such as disaccharides. Uw is found, however, to increase linearly with Tg (Figure 4) (24,37). It is noteworthy that the following relationships exist between the molecular structure of carbohydrates and their thermodynamic properties: (1) for gluco-disaccharide (No. 1 to 11), Tg and Uw depends on both the position and type (alpha or beta) of the glycosidic linkage, (2) the disaccharides composed of glucose and fructose residues (No. 12 to 15) have a lower Tg and smaller Uw than other disaccharides, and (3) disaccharides with a galactosyl residue (No. 16 to 18) have a higher Tg and a larger Uw than other disaccharides.
Especially for trehalose, various Tg values have been reported so far from 73 �C (38) to 116.9 �C (35), although the value of 115�2 �C appears to have been widely accepted as the exact Tg of anhydrous trehalose (35, 39-41). The Tg of trehalose (numbered as 1) is highest among the saccharides shown in Figure 4, although the value is not necessarily special, at least not anomalous. In addition, trehalose has another noteworthy glass-forming property in favor of its application as a biological protective agent for long-term storage in dried states. Namely that glassy trehalose is more stable than other vitrified, well-known disaccharides, as demonstrated by an up to 150 kJ mol-1 larger activation energy of the enthalpy relaxation, DErel, for glassy trehalose relative to that of glassy maltose or sucrose (41). DErel is thought to be the activation energy of the translational diffusion of molecules forming the glass of interest, being a direct measure of the chemical and physical stability of the vitrified matrix. In summary, the characteristic physicochemical nature with not only high Tg but also high DErel makes trehalose a superior biological protective agent to other saccharides.
The dependence of unfrozen water content on carbohydrate species is closely related to the extent of forming or breaking hydrogen bonds with water. This view is supported by the correlation found between Uw and DHsol (Figure 5), where DHsol represents the heat of solution of amorphous carbohydrates and can be approximated as the sum of three pair-wise interactive contributions.
DHsol = DHs-s + DHw-w + DHs-w (1)
where DHs-s represents the enthalpy required for breaking the inter- and in some cases intra-molecular interactions in the amorphous carbohydrate to be dissolved (i.e. DHs-s>0), DHw-w the enthalpy associated with breaking hydrogen bonds among water molecules (i.e. DHw-w>0), and DHs-w the enthalpy of forming hydrogen bonds between water and the dissolved carbohydrate (i.e. DHs-w<0). Unlike the amorphous carbohydrates, DHsol for crystalline carbohydrates is positive (35,42), indicating that DHs-s is larger in the latter than in the former. DHsol is a good measure for the capability of hydrogen bond formation of solutes with water, under the assumption that the change of DHs-s+ DHw-w from sugar to sugar is negligible relative to that of their associated DHs-w. In Figure 5 the linear correlation holds true even in a narrow region, the middle region in Figure 5, to cover disaccharides. This suggests that the correlation originates from the difference in stereochemistry among the carbohydrates studied
4. HYDRATION STRUCTURE OF CARBOHYDRATES
A possible factor determining the unfrozen behavior of an aqueous solution of a dissolved solute is the compatibility of the tetrahedral hydrogen bond network of water with the stereochemistry of the solute (43,44). In the field of liquid physics, ultrasound velocity measurements have often been employed to obtain information on the hydration shell structure, providing the isentropic coefficients of compressibility, - (1/v) (∂V/∂P)s, of the aqueous solution (bs) and of pure water (bw), respectively (45). If the solute molecule itself is incompressible, which would be true for small molecules under ordinary pressures, the isentropic apparent partial molar compressibility, KS,2, of the solution is given as follows (46):
KS,2 = 1000 (bs- bw)/md + bs Vc (2)
where m, d, and Vc represent the number of moles of solute per kilogram of water, the density of the solution, and the molar volume at the molarity of solute, respectively. The isentropic partial molar compressibility, K�S,2, can be estimated by extrapolating the molarity, m, to zero, i.e. as the limiting value at an infinite dilution. The values of K�S,2 reflect sensitively the characteristics of the hydration shell structure. When a dissolved solute disturbs or breaks the three dimensional hydrogen-bond network of water surrounding the solute, the water in the hydration shell becomes denser and less compressible than bulk water. This situation leads to a more negative value of K�S,2. Figure 6 shows the correlation between Uw and K�S,2 in the case of carbohydrates. Uw increases linearly with decreasing K�S,2, which indicates that more water remained unfrozen in the presence of a carbohydrate that causes destructuring of the hydrogen bond network of water (24,37).
A similar argument was also made by other experimental and theoretical studies using techniques such as Raman (47-50) or inelastic scattering (49), and molecular dynamics simulation (50,51), where, for example, the relative spectral contribution from bulk and hydration water to the O-H stretching band was comparatively analyzed for the aqueous solutions of trehalose, maltose, and sucrose. Of particular interest, according to ref. 50, is that trehalose exerts a superior, destroying effect - relative to the others - on the hydrogen bond network of water at a sugar concentration above 30 wt%. Moreover, in ref. 51, statistics of cluster sizes made of water molecules via their hydrogen bonds was investigated and it was found that in aqueous trehalose the water cluster size was smaller than in aqueous maltose and sucrose when the concentration of sugar was more than 40 wt%. Based on these results, the remarkable cryoprotective effectiveness of trehalose as compared with other disaccharides was interpreted as due to its poorer fit into the tetrahedral hydrogen bond network of water, which should destroy the pure water cluster and thereby hinder the growth of ice (47-51).
Galema et al. proposed a hydration model for carbohydrates from a detailed inspection of their K�S,2 data (46). One of the features of this model is that the hydration property of carbohydrates is governed by relative orientation of the next-nearest neighbor hydroxyl (OH) groups, such as those in positions 2 and 4 of the pyranose ring (Figure 7). When both of the two OH groups occupy the equatorial position of the ring in a chair conformation, the spacing of their oxygen atoms is ca. 4.85 Ǻ, fitting well into the spacing between the next-nearest neighbor oxygen atoms of water molecules involved in the tetrahedral hydrogen bond network. From such a point of view, monosaccharides are classified into three groups. Group A: OH (2) is equatorial and OH (4) is axial; Group B: OH (2) is either axial or equatorial and OH (4) is equatorial; and Group C: both OH (2) and OH (4) are axial. For D-aldohexoses, D-galactose belongs to Group A, D-glucose and D-mannose to Group B, and D-talose to Group C. On the other hand, disaccharides are classified based upon the combination of their constitutive monosaccharides (Table 2). As far as we know, to date, there is little or no information confirming the conformational preference of carbohydrates in either freeze-concentrated matrix or anhydrous glassy states. However, in view of the good correlation between Uw and K�S,2 (Figure 6), the stereospecific hydration model deduced from dilute aqueous solutions is recognized as a useful frame to rationalize the differences in Uw from sugar to sugar.
Table 4 shows the comparison between Uw and the hydration numbers determined by various methods for four representative disaccharides. Interestingly, Uw and the hydration numbers follow the same order in relative magnitude, lactose > trehalose > maltose > sucrose. Particularly it should be noted that the Uw and hydration number of lactose, composed of Group A and B monosaccharide units, are larger than those of trehalose and maltose, both of which involve only a Group B unit. This result indicates that the hydration properties of disaccharides are governed partially by the stereochemistry of the constitutive unit.
5. DYNAMIC ASPECT OF THE HYDRATION OF CARBOHYDRATES
In order to characterize the hydration phenomena in more detail, it is worthwhile to get information on the mobility of water molecules in the presence of carbohydrates. One of the useful techniques for such a purpose is 17O-NMR (nuclear magnetic resonance) spectroscopy. In the so-called two state model (56), 17O nuclei in the aqueous solution are assumed to be distributed between the following two motional states: the water in the hydration shell and the bulk water. If the exchange rate between hydration and bulk water is fast enough relative to the magnitude of the spin-lattice relaxation times of 17O nucleus, the following relation is satisfied (56,57):
1/T1 = (1-ch)/ T1o + ch/T1h (3)
where T1o and T1h are the spin-lattice relaxation times of the natural abundance 17O nucleus in pure water and in the aqueous solutions of carbohydrates, respectively and ch represents the mole fraction of hydration water. In the extreme narrowing conditions, the correlation time tc of molecular reorientation is obtained as follows (58):
1/T1 = (3/125) (1+ h2/3) (e2qQ/h)2tc (4)
where h and (e2qQ/h) represent an asymmetric parameter of the electrical field gradient and the quadrupole coupling constant, respectively. The value of h is usually small,
being omitted (58). The larger value of tc indicates more restricted molecular mobility. From eqs. (3) and (4),
NDHN ≡ 55.5B = nh (K (tch/tco - 1) - 1) (5)
K = (e2qQh/h)2 / (e2qQo/h)2
where B is the slope of the plot between T1o/T1 and the molarity of the carbohydrate, m. The subscripts h and o represent the hydration water and pure, i.e. bulk water, respectively, and nh represents the hydration number. Both values of (e2qQh/h) and (e2qQo/h) are taken as equal to that of pure water, resulting in K=1 (58). NDHN, or dynamic hydration number (58), is, as defined in eq. (5), the product of the hydration number and the mobility of water molecules. We reported data of NDHN which revealed the high hydration ability of trehalose relative to several gluco-oligosaccharides (59). As shown in Table 5, NDHN lies in the order maltopentaose > trehalose > maltotriose > maltose. In addition, what is more important is that the magnitude of tch/tco being a measure of water molecular mobility in the neighborhood of the dissolved carbohydrate is larger for trehalose than for any other gluco-oligosaccharides studied. This finding suggests that trehalose is the most effective to induce decreased mobility of its surrounding water molecules. Taken together, this disaccharide has the characteristic hydration ability in terms of not only its large hydration number but also the remarkably lowered dynamics of its hydration water.
It has been observed that thermodynamic properties of water like partial molar heat capacity and volume, relative to the structure of aqueous solutions, show a considerable increase in the presence of trehalose (42). These values are higher in magnitude compared with those for several other mono- and disaccharides as well as polyols (42,60). It has also been reported that trehalose has a larger hydrated volume compared with other sugars (61). Combining these results with the above 17O NMR data (59), it is inferred that trehalose could form stronger and more extensive hydrogen bonding with water molecules.
The information from the above experimental data is limited to water dynamics and structure averaged over an inhomogeneous sugar surface in various conformational states, and they do not provide any detailed picture about the origin of water dynamics variations depending on the stereochemistry of carbohydrates (Table 5). To address this issue, molecular dynamics (MD) simulations have been applied to solvated sugar systems. Before discussing those results, however, our attention should be for a while turned to the conformational property of trehalose itself. Molecular mechanics and quantum chemical calculations by French's group indicated that trehalose has only a single energy minimum around the glycosidic bond (62,63): the
minimum is located at the glycosidic dihedaral angles of (f, j)=(-60�, -60�). Figure 8A shows the population density map for the dihedral angle distributions obtained from a MD simulation for trehalose in aqueous solution. As expected, the (f, j) angles are located only around (-60�, -60�), corresponding to the gauche conformation. Consequently, this sugar has a single conformation around the glycosidic bond and its overall shape is clam shell-like (Figure 1). Such conformational rigidity of trehalose comes from the alpha,alpha-1,1 type of glycosidic linkage, which is unique to this sugar among naturally occurring gluco-disaccharides. Indeed, neotrehalose, which has an alpha,beta-1,1 configuration, has more than two stable conformations around the glycosidic linkage (Figure 8B) (62). Similarly, other types of glycosidic linkage, including (1-4), (1-6) bonds and so on, allow for multiple conformers. A recent experimental study on the interaction of trehalose with lipid membrane indicated that unlike other naturally occurring disaccharides, the two glucopyranose rings of trehalose can fit the bilayer surface and hydrogen-bond with the head groups of the phosphoholipid molecules (64). Taken together, the less flexible alpha,alpha-1,1-glycosidic linkage could be an important clue to explain the biological functions of trehalose.To date, the MD simulation for aqueous trehalose has been reported by several groups (50,51,54,65-69). Our early MD study indicated that trehalose can hydrogen-bond with the surrounding water more extensively than maltose, leading to more restrained translational diffusion of water molecules around trehalose (65). A recent remarkable increase in computer performance allows for more rapid and accurate MD calculations for various carbohydrates in solution. Most recently, Choi et al. have performed systematic computational work for a series of disaccharides to obtain an atomic-level insight of unique biochemical role of the alpha,alpha-1,1-linked glucopyranoside dimer over the other glycosidically linked sugars (69). In that study, the following 13 different homodisaccharides with different glycosidic linkages were examined: 1-11 (Table 2), alpha-D-Galp-1,1-alpha-D-Galp, and alpha-D-Manp-1,1-alpha-D-Manp. Analyses of the hydration number and radial distribution function of solvent water molecules showed that there was very little hydration adjacent to the glycosidic oxygen of trehalose and that the dynamic conformation of trehalose was less flexible than any of the other sugars (Figure 8). Due to such properties, a highly anisotropic hydration shell is formed around this sugar in aqueous solution (Figure 9). In addition, they evaluated the number of long-lived hydrogen bonds defined as having a lifetime longer than 20 ps. As a result, trehalose was shown to make an average of 2.8 of long-lived hydrogen bonds with water, which is a much larger number than the average number of hydrogen bonds for the other 12 sugars. The stable hydrogen-bond network was thought to be derived from the formation of long-lived water bridges at the expense of decreasing the dynamics of the water molecules. This dynamic reduction of water by trehalose was also confirmed from data for the lowest translational diffusion coefficients and the lowest intermolecular coulombic energy of the water molecules around trehalose. These results rigorously support our 17O NMR results (59). According to Choi et al., trehalose is a 'dynamic reducer' for solvent water molecules, which comes from its anisotropic hydration and conformational rigidity (69). It should be again stressed that such a peculiar property of trehalose originates from the presence of its alpha,alpha-1,1-linkage.
Taken together with sections 4 and 5, trehalose is a water structure maker in the sense that it forms a highly anisotropic and unmobilized hydration shell around itself. However, this simultaneously means that the tetrahedral hydrogen bond network in water is highly perturbed by the addition of trehalose, as evidenced from the data of the isentropic partial molar compressibility K�S,2. In this sense, trehalose is a water structure breaker as well. Such a dual character is the key to understand various biological roles of this sugar.
6. PROTEIN STABILIZATION BY TREHALOSE
Protein stabilization by trehalose in aqueous solution (15-18) is an example of the biological functions of trehalose which come from its character as a water structure maker. Timasheff and coworkers investigated the stabilization of RNase A by trehalose based on the so-called preferential hydration model (17), which insists that sugars and polyols stabilize the folded structure of proteins in solution as a result of greater preferential hydration of the unfolded state compared with the native state. Wyman linkage analysis (eq. 6) and the data of the transfer free energy on unfolding suggested that at room temperature trehalose stabilizes this protein through the preferential hydration mechanism, although at higher temperatures it tends to preferentially bind to the protein. In general, however, different proteins are expected to interact with cosolvent molecules in varied ways depending on their physicochemical properties. Indeed, trehalose has been observed to provide protection to different proteins to various extents and the efficacy of protection depends on the nature of the protein (15,70). Thus, to understand the mechanism of trehalose-mediated thermal stability of proteins in detail, Kaushik and Bhat have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties: ribonuclease A, lysozyme, cytochrome c, alpha-chymotrypsinogen, and trypsin-inhibitor (18). They also used the Wyman linkage analysis (eq. (6)) to determine the relative preferential interaction of trehalose with the two end states of the proteins,
d (ln K)/d (ln a)= (nD - nN) = Dn (6)
where K is the equilibrium constant for the conversion reaction N→D, a is the cosolvent activity, and nD and nN are the numbers of cosolvent molecules bound to the denatured and the native protein molecules, respectively. The Dn values obtained for the above proteins at 1.5 M trehalose vary from -7 to -4. The negative values indicate the preferential exclusion of trehalose (equivalent to preferential hydration) from the hydration shell of the protein upon denaturation.
Preferential hydration should occur when the interaction of a given cosolvent with water is stronger than its interaction with a protein. In other words, such a cosolvent is a water structure maker whose hydration shell is strongly structured. In this regard, trehalose is a good cosolvent causing preferential hydration. The preferential hydration effect should lead to a loss in the entropy of solvation upon protein denaturation, rendering the unfolded state even more unstable, and resulting in a shift of the equilibrium in favor of the native state. In addition, the water structure maker causes a significant increase of the surface tension of water. For example, at identical concentrations, trehalose increases the surface tension of water by much larger amounts compared with other sugars and polyols (18). Protein denaturation in such a solution would need additional energy to accommodate its increased surface area. Thus, the effect of surface tension is also an important factor contributing to the stabilization of a protein.
According to the preferential hydration model as described above, trehalose is expected to promote the aggregation of unfolded proteins because the aggregated state should have a smaller protein-solvent interface than their isolatedly dissolved state. However, in contradiction to this expectation, trehalose has been shown to suppress aggregation of unfolded proteins both in vivo (20,71) and in vitro (21) states. Although the underlying mechanism for such phenomena is far from being fully understood at present, a key to solve this issue may exist in another peculiar property of this sugar, that is, a specific interaction with hydrophobic compounds as described in the next section.
7. ANTIOXYDANT FUNCTION OF TREHALOSE
In addition to the protective function against water stresses, there is growing evidence that trehalose is capable of protecting proteins and unsaturated fatty acid (UFA) against oxidative damage (8). An in vivo study by Benaroudj et al. indicated that trehalose protects yeast cells and cellular proteins from damage by oxygen radicals (7). The antioxidant function on UFA has been extensively studied by our group from both experimental and theoretical viewpoints as described below (8,72,73).
As shown in Figure 10, the autoxidation of UFA is initialized by the reaction in which activated oxygen or free radicals attract hydrogen atoms from the allyl group of UFA. In the subsequent reaction, the resultant conjugated diene reacts with O2 and another UFA molecule to produce hydroperoxy-fatty acid, followed by degradation or polymerization by the secondary oxidation reaction. The formation of hydroperoxide (HPOD) can be evaluated by monitoring the absorption (233 nm) of the conjugated diene. The effect of trehalose on HPOD formation of linoleic acid (LA) is shown in Figure 11 (73), and indicates that the amount of HPOD produced decreases depending on trehalose concentration and thus that trehalose depresses the reaction rate of the autoxidation step. However, such a phenomenon was not observed for the mixture of trehalose with UFA possessing trans type C=C double bonds such as eladic acid (18:2, trans) and linoeladic acid (18:1, trans). In addition, other disaccharides, such as sucrose, maltose and neotrehalose, showed a negligible effect on HPOD formation. Therefore, trehalose interacts specifically with UFA possessing a cis type C=C double bond (s) such as LA (18:2, cis), resulting in the depression of the formation of HPOD. Stoichiometry of this complex formation was elucidated by observing the 1H-T1 values of olefin and the adjacent methylene protons of LA. As shown in Figure 12 (8), the 1H-T1 vs. trehalose concentration curve reaches a plateau at a 2:1 molar ratio of trehalose to LA. When using oleic acid (18:1, cis) or alpha-linolenic acid (18:3, cis) as
UFA, the curve reached a plateau at a molar ratio of 1:1 and 3:1, respectively. From these results, it was concluded that trehalose and a cis type C=C double bond forms a complex with 1:1 stoichiometry. In addition, the 6,6' site and 3,3' sites of trehalose were shown to participate in the interaction from the observation of 1H-1H NOESY spectrum depicted in Figure 13 (72), where pentadiene was used as a model of UFA and a clear cross-peak was observed between the olefin proton signals (5.3-5.4 ppm) of pentadiene and the 6 (6')-methylene and 3 (3') proton signals (3.8-3.9 ppm) of trehalose.
A theoretical model for the trehalose-cis C=C bond complex is shown in Figure 14 (72), where the OH-6' of trehalose interacts with the pi-orbital at the mid position of the double bond and simultaneously the OH-3 forms the C-H∙∙∙O type of hydrogen bond at a terminal of the double bond. The complex formation energy (stabilization energy) was estimated to be 23.1 and 32.5 kJ mol-1 from the HF/6-31G** and B3LYP/6-31G** calculations, respectively. Moreover, Oku et al. gave a clear answer to the problem whether the autoxidation step in Figure 10 is really suppressed as a result of the above complex formation (8,72). Actual calculations were performed for the hydrogen abstraction reaction from the activated methylene of heptadiene as follows:
-CH2-CH=CH-CH2-CH=CH-CH2-→ -CH2-CH=CH-oCH-CH=CH-CH2-
As a result, when trehalose is not complexed with heptadiene, the activation energies of the reaction initiated by methyl radical are 61.9 kJ mol-1 (UHF/6-31G**) and 38.5 kJ mol-1 (UB3LYP/6-31G**), while upon complexation this increases up to 157.9 kJ mol-1 (UHF) and 161.6 kJ mol-1 (UB3LYP). These results indicate that the OH∙∙∙pi and CH∙∙∙O multiple hydrogen bonds can strongly affect the electronic state of the diene and that the hydrogen abstraction reaction is significantly depressed by the interaction with trehalose.
As described in section 5, trehalose has a single conformation due to the rigidity of the alpha,alpha-1,1 glycosidic bond. As a result of this, once the multiple hydrogen bond (Figure 14) is formed, it is expected to have a substantial lifetime. Such stable complex formation would be impossible in the cases of other disaccharides because of free rotation around the glycosidic bond. Interestingly, the hydrogen bonding pattern of the trehalose/diene complex shown in Figure 14 is similar to that of a theoretical model for a water dimer/ethylene complex given in ref. 74, where one water molecule binds to the pi-bond at the midpoint of the double bond and the oxygen atom of the other does to the CH group through the CH∙∙∙O interaction (see Figure 7b in ref. 74). In spite of such an apparent structural similarity, a C=C double bond is thought to have higher affinity with one trehalose molecule than with two water molecules because the latter case accompanies a larger entropy loss. Thus, it is possible that trehalose preferentially binds to a double bond instead of to solvent molecules.
The finding that trehalose can form intermolecular complexes with various cis double bonds (8,72) leads us to expect that this sugar can also interact with benzene compounds, whose double bonds are cis rather than trans. If this is true, such a peculiar interaction may be related to the suppressive effect of this sugar on the aggregation of peptide and proteins as described in the previous section. Namely, there is a possibility that this sugar binds to aromatic side chains that are exposed to the aqueous phase upon unfolding and consequently act as a spacer to inhibit the direct contact between unfolded protein molecules. This interesting issue is now under investigation in our laboratory.
8. PERSPECTIVE
The uniqueness of trehalose comes from the presence of an alpha,alpha-1,1-linkage, which brings about the rigid conformation with a clam, shell-like shape. Because of its conformational rigidity, trehalose has a unique hydration characteristic: a spatially anisotropic but dynamically stable hydration shell. This in turn brings about several characteristic thermodynamic properties for its aqueous solution: relatively large amount of unfrozen water (Uw), more negative isentropic partial molar compressibility (K�S,2), relatively large values of partial molar heat capacity and volume and larger surface tension. These results reflect the dual character of this sugar as a good water structure maker and breaker. The character as a water structure breaker explains the fact that this sugar acts as a better cryoprotectant than other disaccharides. The larger Uw value is related to a higher Tg (Figure 4), although the molecular origin of such a relation remains unclear at present. The characteristic glassy property with not only high Tg but also high DErel makes trehalose a superior desiccation protectant than other saccharides. The character as a water structure maker contributes to the stabilizing function on proteins in solution. Finally, the conformational rigidity leads to the peculiar interaction with unsaturated compounds with cis type C=C double bond (s) and thus this sugar acts as a protectant against oxidation stress. The unified view obtained here is a significant advance in understanding the limitation and further possibility of this sugar in various applications and in undertaking the molecular design of more effective protectants in the future.
9. ACKNOWLEDGMENTS
This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and also in part by Grants-in-Aid for Scientific Research on Priority Areas (no.18031012) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Send correspondence to: Minoru Sakurai, Center for Biological Resources and Informatics, Tokyo Institute of Technology, B-62 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan, Tel:81-45-924-5795, Fax: 81-45-924-5795, E-mail:msakurai@bio.titech.ac.jp
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