Crystal Structure of an Anhydrous Form of Trehalose: Structure of

(1) Trehalose is found at particularly high concentrations in the so-called ... and a specific structural interaction with water in the crystalline st...
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J. Phys. Chem. B 2008, 112, 9105–9111

9105

Crystal Structure of an Anhydrous Form of Trehalose: Structure of Water Channels of Trehalose Polymorphism H. Nagase,*,† N. Ogawa,† T. Endo,† M. Shiro,‡ H. Ueda,† and M. Sakurai§ Department of Physical Chemistry, Hoshi UniVersity, 2-4-41 Ebara, Shinagawa-ku, Tokyo, 142-8501, Japan, X-ray Research Laboratory, Rigaku Corporation, 3-9-12 Matsubara-cho, Akishima-shi, Tokyo 196-8666, Japan, and Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259 Nagatsuda-cho, Midori-ku, Yokohama 226-8501, Japan ReceiVed: January 31, 2008; ReVised Manuscript ReceiVed: May 25, 2008

R, R-Trehalose (trehalose) is a nonreducing disaccharide of glucose and is accumulated at high concentrations in some anhydrobiotic organisms, which can survive without water for long periods and rapidly resume active metabolism upon hydration. Although it has been proposed that the intriguing mechanism of bioprotection in anhydrobiosis is conferred by a water channel, details of such a channel have yet to be revealed. We determined the crystal structure of a trehalose anhydrate to further understand the relationship between the structure of water channels and the trehalose polymorph. The space group was identical to that of the dihydrate and the lattice constants were also very similar. Among the five intermolecular hydrogen bonds between the trehalose molecules, four were preserved in the anhydrate. If dehydration of the dihydrate is slow and/or gentle enough to preserve the hydrogen bonds, transformation from the dihydrate to the anhydrate may occur. There are two different holes, hole-1 and hole-2, along one crystal axis. Hole-1 is constructed by trehalose molecules with a screw diad at its center, while hole-2 has a smaller diameter and is without a symmetry operator. Because of the screw axis at the center of hole-1, hollows are present at the side of the hole with diameters roughly equal to that of hole-1. Hole-1 and side pockets followed by hollows correspond to the positions of two water molecules of the dihydrate. The side hollows of the water channel are also observed in the waterfilled hole of the dihydrate. Consequently, hole-1 is considered to be a one-dimensional water channel with side pockets. We also calculated molecular and crystal energies to examine the rapid water uptake of the anhydrate. It was demonstrated that the intermolecular interactions in the anhydrate were weaker than in the other anhydrous form, and probably also than those in amorphous trehalose. The anhydrate provides water capture for another solid form and gives protection from water uptake. These structural properties of the anhydrate may elucidate bioprotection in anhydrobiosis. 1. Introduction Trehalose (R-D-glucopyranosyl R-D-glucopyranoside) is a nonreducing disaccharide in which two glucose molecules are linked together in a 1,1-glycosidic linkage. Although there are three possible anomers of trehalose, namely R,β-trehalose (neotrehalose), β,β-trehalose (isotrehalose) and R,R-trehalose (simply called “trehalose”), only the latter of these has been isolated from and biosynthesized in living organisms.1 Trehalose is found at particularly high concentrations in the so-called “anhydrobiotic organisms” that can survive without water for long periods.2 In addition, some anhydrobiotic plants containing trehalose, for example, Selaginella lepidophylla, can repeatedly dehydrate and rehydrate without incurring damage, at drying temperatures of up to 50 °C.3 Trehalose is also accumulated in the larvae of an African chironomid, comprising 18% of its dry body mass, and its accumulation is attributed to the successful induction of cryptobiosis.4 Moreover, trehalose is found in Escherichia coli in cold environments, where its role is to increase cell viability upon further reduction of temperature.5 Trehalose reduces damage to proteins during freeze-drying6–9 * To whom correspondence should be addressed. E-mail: nagase@ hoshi.ac.jp. Tel./fax.: +81-3-5498-5159. † Hoshi University. ‡ Rigaku Corporation. § Tokyo Institute of Technology.

and membranes10,11 during freezing and/or drying;12 it also stabilizes human blood platelets during freeze-drying13,14 and mammalian cells in the dry state.15 Recently, it was suggested that trehalose shows promise as a therapeutic drug for polyglutamine diseases.16 Two hypotheses have been proposed to account for the stabilizing effect of trehalose: (1) “the water replacement hypothesis”, where saccharides are found to interact with the products by hydrogen bonding in a similar manner to the replaced water, and (2) “the vitrification hypothesis”, where saccharides form glassy sugar with extremely high viscosity. These hypotheses are not contradictory, and both glass formation of a saccharide and its direct interaction with proteins and membranes are thought to be required for its stabilizing effect.17 Furthermore, the clam shell conformation of trehalose resulting from R,R-(1-1) glycosidic linkage may be one of the key factors for the bioprotection of anhydrobiosis.18 Proteins and membranes are immobilized in the viscous glass, leading to the extremely high activation energies required for any reaction to occur. The glass-transition temperature of an amorphous sugar, Tg, is the transition temperature at which the amorphous glassy sugar transforms to an amorphous rubbery one. The Tg of trehalose is significantly higher than that of other mono- and disaccharides at all water contents, a phenomenon known as the “trehalose anomaly”,19 although the Tg values of some saccharides at zero

10.1021/jp800936z CCC: $40.75  2008 American Chemical Society Published on Web 07/08/2008

9106 J. Phys. Chem. B, Vol. 112, No. 30, 2008 water content are renewed. Amorphous trehalose not only shows a reduction in Tg with increasing water content,20 but is also converted to the dihydrate by the addition of water under appropriate conditions.21 However, as has been clarified by a study of the adsorption of trehalose,22 it is not consistently converted to the dihydrate by the addition of water. Therefore, it is expected that Tg of amorphous trehalose decreases at low water content. Recently, Kilburn et al. proposed that the mechanism of bioprotection is caused by a combination of the stability of glassy trehalose and a specific structural interaction with water in the crystalline state. This interaction comprises an ability to capture water from amorphous trehalose by locally forming a dihydrate. Water in the dihydrate is organized into confined one-dimensional water channels, which act as both sink and source of water in low-moisture systems.23 The empty and water-filled channels correspond to the crystal structures of an anhydrous form, generally referred to as R-form, and the dihydrate, respectively.23,24 To date, one hydrous form and two anhydrous forms of trehalose have been reported.25 The dihydrate26,27 (designated here as Tre(h)) and one anhydrous form28 (designated here as Tre(β)) have been determined crystallographically; however, the other anhydrous form has not. The undetermined anhydrous form has been reported as form II,29–31 R-form,25,32,33 and κ-form34,35 and has been characterized by powder X-ray diffractometry, differential scanning calorimetry, thermal gravimetry, Fourier transform infrared spectroscopy and so on. This form, herein referred to as Tre(R), shows a hygroscopic nature, and is rapidly converted to the original form of Tre(h) under appropriate conditions.30,34,36 Sussich et al. stated that the formation of Tre(R) and the reversible production of Tre(h) by exposure to humidity may be key mechanisms for the protective action of trehalose.24,36,37 However, the crystal structure needs to be determined to allow a more detailed discussion of the reversible transformations between Tre(R) and Tre(h). In this study, we have revealed the crystal structure of Tre(R) and compared its structure with that of Tre(h). Furthermore, we have calculated the molecular and crystal energies of Tre(R) and Tre(h) and discussed the structure of their respective water channels. 2. Methods 2.1. Experimental Methods. Tre(h), was purchased from Sigma (MO) and was used without further purification. Water was purified using the Milli-Q Gradient (Millipore Ltd.). Tre(h) was dissolved into water, and then nitrogen gas was blown into the trehalose solution (through the tip of a Pasteur pipet) to obtain plate-like thin crystals of Tre(h). A colorless plate-like crystal of Tre(h) was mounted on a glass fiber. Tre(R) was crystallized by dehydration of the crystal of Tre(h) using dry nitrogen gas at 25 °C for approximately 12 h. The transition from Tre(h) to Tre(R) was monitored by the results on the imaging plates of a two-dimensional detector. All measurements were made on a Rigaku RAXIS RAPID imaging plate area detector with graphite monochromated Cu KR radiation (λ ) 1.54187 Å). Indexing was performed from three oscillations that were exposed for 36 and 300 s on Tre(h) and Tre(R), respectively. The crystal-to-detector distance was 127.40 mm. Readout was performed in the 0.100 mm pixel mode. The data for Tre(h) were collected at a temperature of 25(1) °C to a maximum 2θ value of 136.5°. A total of 90 oscillation images were collected. The data for Tre(R) were collected at a temperature of -180(1) °C to a maximum 2θ value of 100.8°. A total of 180 oscillation images were collected.

Nagase et al. Data reduction for Tre(h): Of the 19211 reflections that were collected, 1767 were unique (Rint ) 0.050); equivalent reflections were merged. The linear absorption coefficient, µ, for Cu KR radiation is 12.198 cm-1. An empirical absorption correction was applied, which resulted in transmission factors ranging from 0.625 to 0.941. Data reduction for Tre(R): Of the 12386 reflections that were collected, 923 were unique (Rint ) 0.196); equivalent reflections were merged. The linear absorption coefficient, µ, for Cu KR radiation is 12.116 cm-1. An empirical absorption correction was applied, which resulted in transmission factors ranging from 0.605 to 0.964. The data were corrected for Lorentz and polarization effects. The crystal structure was solved by the direct method (SHELX-9738) and expanded using Fourier techniques.39 The non-hydrogen atoms were refined anisotropically. Hydrogen atoms of hydroxyl groups were refined by a differential Fourier map. The other hydrogen atoms were refined using a riding model. All calculations were performed using the CrystalStructure crystallographic software package40 except for refinement, which was performed using SHELXL-97. Tre(h) was ground in a mortar and pestle and sieved to obtain a particle size of less than 75 µm. Powdery Tre(R) was prepared by heating powdery Tre(h) in the sample holders to 100 at 4.5 °C/min under vacuum (7 torr) and further heating at this temperature for 30 min under vacuum, according to a previously reported method.34 X-ray powder diffraction (XRD) was recorded using a RINT 1400 X-ray diffractometer and rotating anode source of Cu KR (1.54187 Å) (Rigaku Co., Tokyo, Japan). XRD was carried out at 4°/min with a diffraction angle (2θ) from 3 to 50° at 50 kV, 150 mA for the sample holder of the glass plate. To obtain dry conditions, the measurement was performed under a stream of nitrogen gas (5 L/min). 2.2. Theoretical Methods. To obtain the relative position and orientation between trehalose molecules in Tre(R) and Tre(h), molecular overlap was performed according to the method introduced by Horn.41 The molecular energy of trehalose molecules in an isolated system was calculated using the density functional theory (DFT) at the B3LYP/6-311+G(d,p) level and the second-order Møller-Plesset method (MP2) at the 6-311+G(d,p) level, using Gaussian 03.42 Molecular mechanics (MM) calculations with MMFF94s force fields43–49 using CONFLEX6 software50 were also performed for trehalose molecules in the Tre(R), Tre(β), and Tre(h) forms. The crystal energy based on the molecular mechanics (MM) calculation was also performed by CONFLEX6, using the MMFF94s force field. The crystal and lattice energies are given by N N(M-1)

Ecrystal ) Eintra + Elattice

Elattice )

∑ ∑ i

Eijinter

j

with Rij e Rmax (1) where Eintra is the total intramolecular energy that contributed to the original molecules, and Elattice is the total intermolecular energy between atom i of a referenced original molecule and atom j in the surrounding, symmetry-related virtual molecules within Rmax (effective intermolecular interaction distance; the mean value of Ecrystal for Rmax from 40 to 100 Å by 10 Å was used). Rij is the intermolecular distance between atom i and atom j. N is the number of atoms in an original molecule, and M is the number of molecules in the crystal. Intramolecular potential functions are represented by MMFF94s. Intermolecular potential functions are defined by the nonbonded interaction terms of

Crystal Structure of an Anhydrous Form of Trehalose

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Figure 2. X-ray powder diffraction of Tre(R) (a) and simulation patterns of Tre(R) (b) and Tre(h) (c). Full width at half-maximum is 0.35 (b) and 0.1 (c). Arrow indicates the peak at 16.9°.

Figure 1. An imaging plate of Tre(h) measured at 25 °C (A) and Tre(R) measured at -180 °C (B). Intensity of spots in green and yellow rectangles was measured but not in red rectangle.

MMFF94s: van der Waals and electrostatic interactions. 3. Results and Discussion 3.1. Confirmation of r-Form of Trehalose. An imaging plate of Tre(h) measured at 25 °C is shown in Figure 1A. The intensity of the spots surrounded by a green square or rectangle, named “full”, indicates the total intensity of the corresponding reflection. The intensity of the spots surrounded by a yellow square or rectangle, named “partial”, indicates a part of the total intensity of the corresponding reflection. Intensity was not measured in the red square or rectangle. Almost all Tre(h) spots were surrounded by small squares, indicating the quality of the crystal. A reflection image of Tre(R) is shown in Figure 1B. After a number of attempts, suitable lattice constants were determined for Tre(R). Many of the spots were surrounded by rectangles calculated by the lattice constant, indicating that the lattice was appropriate for Tre(R). There were, however, spots that were not indexed by the lattice constants. This suggested that the mosaicity of the crystal was high and that some components of approximately the same size coexist in Tre(R). To confirm that these components are one and the same, we compared the powdery simulation of Tre(R) with the powder diffraction pattern of a sample prepared by the method previously reported.34 As shown in Figure 2, the simulation was equal to the diffraction pattern, except for the peak at 16.9° (2θ). Although the peak at 16.9° in Tre(h) corresponds to the Miller index (1 1 2), it was not caused by the original Tre(h) or the Tre(h) converted from Tre(R), because these forms of Tre(h) do not show a preferred orientation. The origin of this peak cannot be explained at the present stage. 3.2. Molecular Structure in r-Form. An ORTEP drawing of Tre(R) at the 50% probability level is shown in Figure 3. The trehalose molecule has an approximate C2 symmetry in Tre(R) form, as well as in the Tre(h) and Tre(β) forms. Three torsion angles, ψΗ, φΗ, and ω (Table 1), describe the overall

Figure 3. ORTEP drawing of Tre(R). Thermal ellipsoids enclose 50% of electron density.

conformation in a disaccharide. The ω-torsion angles, O5-C5C6-O6 and O5′-C5′-C6′-O6′ are -63.4(11)° and -61.0(12)°, respectively, showing that two C5-C6 rotamers are in the gauche-gauche conformation, while those of both Tre(h) and Tre(β) are in the gauche-trans and gauche-gauche conformations. Linkage angle θ (C1-O-C1′) of the two glucose-rings is 113.0(9)°, which may differ significantly from 115.87(13)° in Tre(h). Two pyranose rings have 4C1 conformation with puckering parameters:51 Q ) 0.548(16) Å, q2 ) 0.036(15) Å, q3 ) 0.547(16) Å, φ2 ) 18(25)° and θ ) 3.8(16)°; Q′ ) 0.523(15) Å, q2′ ) 0.024(14) Å, q3′ ) 0.522(15) Å, φ2′ ) 200(37)° and θ′ ) 2.6(16)°. The ideal chair conformations of the pyranose ring form are represented by puckering parameters, θ and φ. Several ideal conformations are specified: chair for θ ) 0° and φ ) 0 °, boat for θ ) 90° and φ ) 0°, twist boat for θ ) 90° and φ ) 90°, half-chair for θ ) 67.7° and φ ) 90°. The puckering amplitudes (q3. q2 and q3′. q2′) of the two pyranose rings showed a slightly distorted chair conformation. The total puckering amplitude (Q) of Tre(R) is smaller than that of an ideal cyclohexane chair conformation (0.63 Å),51 and is similar to those of Tre(β)28 and Tre(h).28 3.3. Crystal Structure of r-Form. The crystal data of Tre(R) and Tre(h) are summarized in Table 2. We compared the data of Tre(R) at -180 °C with those of Tre(h) at 25 °C, because we have not yet obtained sufficient crystal data of Tre(R) at 25 °C to discuss the structural details. The lattice constants of Tre(h) at 25 °C changed by approximate ∆a ) -0.8 Å, ∆b ) -0.6 Å, ∆c ) +0.7 Å, ∆V ) -191 Å3, due to dehydration and cooling to -180 °C. Molecular overlap or best fit of the two trehalose molecules in Tre(h) and Tre(R) was performed to obtain the relative positions and orientation between them. The molecular overlap of the trehalose molecule in Tre(h) to that in

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TABLE 1: Comparison of Molecular Conformation among Polymorphism of Trehalosea conformation sample

ψH

φH

ω

ω′

θ

Tre(h), in the present study Tre(h)-(01)26 Tre(h)-(02)26 Tre(h)-(10)26 Tre(h)27 Tre(β)28 Tre(R), in the present study trehalose molecule after MMb,c trehalose molecule after MMb,d

-46.91 -39.81 -45.18 -39.79 -36.18 -57.43 -49.35 -40.21 -50.03

-59.54 -61.97 -58.36 -61.08 -69.58 -58.33 -65.26 -40.21 -21.33

69.83(19) 70.1 69.7 69.9 69.8 71.0(6) -63.4(11) -176.68 66.30

-75.67(9) -75.7 -75.7 -75.2 -75.3 -72.1(6) -61.0(12) -176.68 56.74

115.87(13) 115.8 115.9 115.7 115.7(3) 113.3(4) 113.0(9) 111.99 110.82

a ΨH: HC1′-C1′-O1-C1. φH: HC1-C1-O1-C1′. ω: O5-C5-C6-O6. ω′: O5′-C5′-C6′-O6′. θ: C1-O1-C1′. b More stable conformations of trehalose molecule in an isolated system after MM calculation. c MM calculation for trehalose molecule in Tre(R). d MM calculation for trehalose molecule in Tre(h).

TABLE 2: Crystal and Experimental Data of Tre(a) and Tre(h) Tre(R) formula: C12H22O11 formula weight: 342.30 crystal system: orthorhombic space group: P212121 Z ) 4 a ) 6.7999(15) Å b ) 11.638(3) Å c ) 18.583(5) Å V ) 1470.5(6) Å3 Dx ) 1.546 g/cm3 no. of reflections used ) 923 no. of parameters refined ) 209 reflection/parameter ratio ) 4.42 2θmax ) 100.8° with Cu KR R1 (I > 2.00 σ (I)) ) 0.0954 R (all reflections) ) 0.1062 wR2 (all reflections) ) 0.2628 (∆/σ)max ) 0.0010 (∆F)max ) 0.34 e-/Å3 (∆F)min ) -0.30 e-/Å3 deposition number:a CCDC 668079

TABLE 3: Intermolecular Hydrogen-Bond Distance and Angle between Trehalose Molecules in Tre(h) and Tre(r)a

Tre(h) formula: C12H22O11 · 2H2O formula weight: 378.33 crystal system: orthorhombic space group: P212121 Z ) 4 a ) 7.5969(10) Å b ) 12.2305(10) Å c ) 17.887(5) Å V ) 1661.9(5) Å3 Dx ) 1.512 g/cm3 no. of reflections used ) 1767 no. of parameters refined ) 227 reflection/parameter ratio ) 7.78 2θmax ) 136.5° with Cu KR R1 (I > 2.00 σ (I)) ) 0.0282 R (all reflections) ) 0.0288 wR2 (all reflections) ) 0.0749 (∆/σ)max ) 0.0010 (∆F)max ) 0.18 e-/Å3 (∆F)min ) -0.22 e-/Å3 deposition number:a CCDC 668078

a These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/ cif.

Tre(R) was achieved by translation of the centroid of the molecule with the translation vector, and rotation of the molecule around the rotation vector passing through the centroid. The molecular overlap of the backbone of the two pyranose rings and the linkage oxygen atom of the trehalose molecule in Tre(R) and Tre(h) could translate Tre(h) by 0.32 Å with the translation vector (0.174, 0.233, -0.123), and rotate it by 22.0 ° around the rotation vector parallel to the vector (0.858, -0.136, 0.496). The translation and rotation vectors were represented by a rectangular coordinates system constructed from the Tre(h) lattice. No intramolecular hydrogen bonds were found in either Tre(R) or Tre(h). The hydrogen bond scheme was consistent with that reported by Brown et al.;26 twelve hydrogen bonds per molecule were detected in Tre(h). These consisted of seven hydroxyl groups hydrogen-bonded to the crystal waters, and five hydroxyl groups (Table 3) hydrogen-bonded to the hydroxyl groups and to the ring oxygen atom of the adjacent molecule. Four of these hydrogen bonds were preserved by the transformation from Tre(h) to Tre(R) (nos. 1-4 and 1′-4′ in Table 3). In this context, preservation means that the translation and screw operations were unchanged. Among these four bonds, two formed hydrogen bond sequences along the a-axis and the others

no.

D-H · · · A

D · · · · A (Å) D-H (Å) H · · · A (Å)

D-H · · · A (deg)

1 2 3 4 5

O3′-HO3′ · · · O31) O3-HO3 · · · O4′1) O4′-HO4′ · · · O5′2) O6′-HO6′ · · · O43) O6-HO6 · · · O6′4)

2.753(2) 2.906(2) 2.883(2) 2.730(2) 2.702(2)

0.820 0.820 0.820 0.820 0.820

1.978 2.087 2.175 1.937 1.888

157.2 176.7 144.7 162.7 171.6

1′ 2′ 3′ 4′

O3′-HO3′ · · · O31) O3-HO3 · · · O4′1) O4′-HO4′ · · · O5′2) O6′-HO6′ · · · O43)

2.831(12) 2.699(11) 2.674(12) 2.767(11)

0.840 0.840 0.840 0.840

2.025 1.988 1.906 2.459

160.5 141.9 151.5 102.6

6 7 8 9 10

O2-HO2 · · · O45) O2′-HO2′ · · · O21) O6-HO6 · · · O36) O6′-HO6′ · · · O52) O6′-HO6′ · · · O62)

2.828(12) 3.005(11) 2.859(11) 2.830(12) 3.072(12)

0.840 0.840 0.840 0.840 0.840

2.024 2.172 2.254 2.505 2.317

160.0 171.4 129.2 104.1 149.9

a Symmetry operator: (1) 1/2 + x, 3/2 - y, 1 - z; (2) 1 - x, 1/2 + y, 1/2 - z; (3) 3/2 - x, 1 - y, -1/2 + z; (4) 1 - x, -1/2 + y, 1/2 - z; (5) -1 + x, +y, +z; (6) 1/2 + x, 1/2 - y, 1 - z. No. 1-5, intermolecular hydrogen bond (IHB) in Tre(h); no. 1′-10, IHB in Tre(R).

formed hydrogen bond sequences along the b- and c-axes. By transformation from Tre(h) to Tre(R), three hydrogen bond sequences were formed along the a-axis (nos. 6-8 in Table 3) and two sequences along the b-axis (nos. 9 and 10 in Table 3). No hydrogen bond sequences were formed along the c-axis. These results suggest that the growth of nuclei in Tre(R) occurs two-dimensionally in layers along a- and b-axes, as reported previously.35 It is expected that the crystal waters are taken in through a pathway along these layers. 3.4. Structure of Water Channel. Figure 4 panels a and b show the packing diagrams projected along the a-axis of the Tre(R) and Tre(h). W1 and W2 are crystal waters of Tre(h). Yellow and light-green circles with diameters of 4.3 Å in Tre(R) indicate the presence of two different kinds of holes: one large and one small (Figure 4a). The diameter of 4.3 Å corresponds to the radius of the hole calculated, assuming the spherical holes reported previously.15 No hole was observed on the packing diagram projected along the b-axis. Compared with the packing diagram of Tre(h), the large and small holes correspond to the positions of W1 and W2, respectively, and are referred to here as hole-1 and hole-2. Hole-1 has a 21-screw axis at its center, whereas hole-2 has no symmetry operator and is surrounded by trehalose molecules connected by a screw axis (yellow circle). Consequently, hole-1 is considered to be a onedimensional water channel, but hole-2 is relatively narrow and is not considered to be the same water channel as hole-1. Figure

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Figure 4. Packing diagram of (a) Tre(R) and (b) Tre(h) along a-axis. Two different holes in Tre(R), large and small, are indicated by yellow and light green circles, respectively. Diameter of each circle is 4.3 Å. W1 and W2 are crystal water of Tre(h). Trehalose molecules are drawn by a wireframe model with a partial space-filling model. Crystal waters in lattice are drawn by a ball-and-stick model. Green and blue lines indicate band c-axes.

5 panels a and b show the side view of the empty and waterfilled channels of Tre(R) and Tre(h). Due to the screw axis at the center of hole-1, side pockets, followed by a hollow of approximately 4.3 Å diameter, are present at the side of the hole. The passage of the side hollow is not hindered by the hydrogen bonds. Therefore, water molecules in the water channel could easily move to positions corresponding to those of W2 molecules in the side pockets. By means of the side hollows, Tre(R) can have twice the water reserve capacity of the water channel alone. The side hollows of the water channel are also observed in the water-filled hole of Tre(h) (Figure 5b). The nearest distance of W1 · · · W1 is 4.32 Å and W1 · · · W2 passing through a side hollow is 4.06 Å, whereas the nearest distance of W2 · · · W2 along the a-axis is 7.60 Å, suggesting that W2 moves reversibly to the position of W1 passing through a side hollow. 3.5. Energy Calculation. Molecular and crystal energies were calculated to investigate the source of the rapid water uptake by Tre(R) compared with Tre(β). In the MP2 calculation (Table 4), the stability order of the trehalose molecules in an isolated system was found to be, in order from most to least stable, Tre(R) > Tre(h) > Tre(β), and the order was in accordance with the results of DFT and MM calculations. To obtain a more stable conformation of trehalose molecules, MM calculation was performed. Two stable conformations (Table 2) were obtained from trehalose molecules in Tre(h), Tre(R), and Tre(β). One had C2 symmetry and the total steric energy decreased by 191.9 kcal/mol from the initial value (371.3 kcal/ mol) of trehalose molecules in Tre(R). The other had C1 symmetry and the total steric energy decreased by 248.9 and 391.4 kcal/mol from the initial value (426.4 and 570.5 kcal/ mol) of trehalose molecules in Tre(h) and Tre(β), respectively.

For trehalose molecules in Tre(R), the decrease in total steric energy was mainly due to a decrease in bond strain energy (81% of the total steric energy). The maximum difference in C-C and C-O length was 0.067 Å and that in C-H and O-H length was 0.108 and 0.144 Å, respectively. The minimum differences in C-H and O-H lengths were 0.096 and 0.134 Å, respectively. The crystal energies of the trehalose polymorphisms were calculated (Figure 6). Tre(β) is more stable than both Tre(R) and the hypothetical structure of Tre(h) (designated here as Tre(h)-W1-W2), in which two crystal waters are excluded. It is interesting that Tre(R) is less stable than Tre(h)-W1-W2. Crystal energy is the sum of the intra- and intermolecular interactions. In Tre(R), the trehalose molecule is more stable and the crystal less stable than in Tre(h)-W1-W2 and Tre(β). Thus, the intermolecular interactions of the trehalose molecule in Tre(R) are weaker than those in Tre(β) and probably also those in amorphous trehalose, since Tre(R) rapidly reverts to Tre(h), while Tre(β) and amorphous trehalose remain unchanged at 43% relative humidity and 25 °C.34 The weak intermolecular interactions, the structure of the relatively large hole, and the similarity in molecular arrangement between Tre(R) and Tre(h) may allow Tre(R) to be easily rehydrated. Water molecules would be taken into the empty channel more easily than into hole-2 owing to the difference in hole size and would run out to the corresponding W2 position through the side hollows. When water channels are filled with water, the trehalose molecules translate, rotate, and deform, and water is captured in Tre(h). If dehydration of Tre(h) is slow and/or gentle enough to preserve the hydrogen bonds, transformation from Tre(h) to Tre(R) occurs reversibly. Many anhydrobiotic plants and insects accumulate amorphous trehalose in the body during the dry season as protection against

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Figure 5. Side view of empty (a) and water-filled (b) water channels of Tre(R) and Tre(h). Trehalose and water molecules are drawn by wireframe, partial ball-and-stick, and space-filling models. Oxygen and hydrogen atoms of water molecules, W1 and W2, are colored with aqua and tangerine, respectively. Diameter of the yellow circle is 4.3 Å. Water in front of the water-filled hole is W2. The shortest distance between inside water, W1, and outside water, W2, of the hole is 4.06 Å, while that between W2 and W2 is 7.60 Å. Red, green, and blue lines indicate a-, b-, and c-axes, respectively.

TABLE 4: Energy Difference between the Trehalose Molecules in Tre(r), Tre(β), and Tre(h) method MM (Conflex6), difference (kcal/mol) DFT (Gaussian), difference (kcal/mol) MP2 (Gaussian), difference (kcal/mol)

Tre(R) Tre(h)

Tre(h) Tre(β)

Tre(R) Tre(β)

-55.1

-144.2

-199.2

-61.2

-171.2

-232.4

-62.2

-178.8

-241.0

dehydration stress. When anhydrobiotic plants and insects with an accumulation of amorphous trehalose are exposed to small amounts of moisture, this water is used to form Tre(h) in nanoscale.23 When drying of the system occurs, transformation from Tre(h) to Tre(R) is induced. We have reported that if a mixture of Tre(R) and amorphous trehalose is exposed to moisture, Tre(R) is transformed to Tre(h) more rapidly than by water adsorption to amorphous trehalose.34 These rapid changes in Tre(R) are probably due to weak intermolecular interactions, the structure of the relatively large hole, and the similarity in the molecular arrangement with Tre(h). Tre(h) is stable, and amorphous trehalose remains unchanged. Therefore, Tre(R) supplies a water capture method for another solid form and protects it from water uptake. These mechanisms may elucidate bioprotection in anhydrobiosis. 4. Conclusion The trehalose molecule has approximate C2 symmetry in Tre(R) form, as well as in Tre(h) and Tre(β), and two

Figure 6. Crystal energies of trehalose polymorphisms, Tre(h), Tre(R), and Tre (β), and hypothetical structures, Tre(h)-W1, Tre(h)-W2 and Tre(h)-W1-W2 where W1 and W2 indicate the crystal water of Tre(h). Minus sign indicates the exclusion of W1 or/and W2 from Tre(h).

glucopyranose rings have near-ideal 4C1 conformation. The molecular arrangement in Tre(R) was very similar to that in Tre(h) and there are hydrogen bonds preserved in both. Slow dehydration may preserve these hydrogen bonds and induce the transformation from Tre(h) to Tre(R), but not to amorphous trehalose. There are water channels along the a-axis, which is constructed by trehalose molecules with a screw diad at the center. Due to the screw axis at the center of the channels, side pockets followed by a hollow are present at the sides of the channels. The size of the side hollows is roughly equal to that of the channels. The insides of the channels and the side pockets correspond to the positions of two water molecules of Tre(h). When the water channels are filled with water, the trehalose molecules translate, rotate, and deform, and water is captured in Tre(h). The weak intermolecular interactions between tre-

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