Thermodynamic Transitions of

Apr 14, 2009 - Ferro-Pfanstiehl (Ferro-Pfanstiehl Laboratories, Waukegan, IL). TRE was dissolved in ultrapure water (UPW: electrical resistiv- ity > 1...
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J. Phys. Chem. B 2009, 113, 6792–6799

Hydrogen Bonding and Kinetic/Thermodynamic Transitions of Aqueous Trehalose Solutions at Cryogenic Temperatures Jason Malsam and Alptekin Aksan* Biostabilization Laboratory, Department of Mechanical Engineering, UniVersity of Minnesota, Minneapolis, Minnesota 55455 ReceiVed: NoVember 11, 2008; ReVised Manuscript ReceiVed: February 10, 2009

Carbohydrates play important roles in the survival of freeze-tolerant organisms. In order to understand the role of carbohydrates on hydrogen bonding (HB) and thermodynamic/kinetic transitions, aqueous trehalose solutions at cryogenic temperatures were analyzed using FTIR spectroscopy. Distinct changes in water-water and water-carbohydrate HB organization were identified during supercooling, freezing, and vitrification. FTIR spectroscopy revealed the kosmotropic effect of trehalose and the presence of two distinct water families in supercooled carbohydrate solutions, (1) water molecules directly associated with the carbohydrate, forming its hydration layer(s) and (2) water molecules that are involved in water-water HB in small clusters. The latter showed characteristics of water in hydrophilic confinement. 1. Introduction Certain anhydrobiotic and freeze-tolerant organisms synthesize and accumulate large amounts of carbohydrates such as trehalose (TRE), sucrose (SUC), and maltose (MAL) to form intracellular glasses.1,2 Therefore, understanding carbohydrate-water interactions is important for successful biopreservation of macromolecules, cells, and organs,1,3-5 food products,6 and pharmaceuticals7,8 by desiccation, lyophilization, or cryopreservation.9 The physical and chemical properties as well as the thermodynamic and kinetic transitions of aqueous carbohydrate solutions have been extensively investigated.10-15 It has been shown that carbohydrate-water16 and water-water hydrogen bonding (HB)17,18 plays important roles in the freezing and vitrification behaviors of the carbohydrate solutions. However, to date, it has not been possible to establish the exact details of the molecular-level phenomena that govern carbohydrate-water interactions at cryogenic temperatures.19 Carbohydrates are known to increase the residence times of the water molecules in their close proximity (in their hydration layers).20,21 It is also known that high carbohydrate concentrations in solution (φ > 20-30%; φ ) mass of sugar/mass of solution) increase the HB of water (i.e., the kosmotropic effect).13,20-22 The exact molecular basis of the kosmotropic effect is not known. However, it is speculated that it could be due to increased carbohydrate-carbohydrate associations21 that would decrease water-carbohydrate interactions (an effect similar to preferential exclusion23). In this research, we have utilized temperature-ramp FTIR spectroscopy to establish the effects of water HB organization on the thermodynamic and kinetic transitions of aqueous solutions at cryogenic temperatures. 2. Materials and Methods High-purity trehalose (TRE) dihydrate was purchased from Ferro-Pfanstiehl (Ferro-Pfanstiehl Laboratories, Waukegan, IL). TRE was dissolved in ultrapure water (UPW: electrical resistiv* To whom correspondence [email protected].

should

be

addressed.

E-mail:

ity > 18.2 MΩ · cm at 25 °C) and 2% deuterium oxide (D2O) to prepare φ ) 10, 20, 30, 40, or 43% w/w solutions. Note that φ ) 43% w/w is the solubility limit of TRE in water at room temperature.24 UPW was prepared by filtering deionized water through a Milli-Q water purification system (Millipore, Billerica, MA). D2O, at a purity of 99.9%, was purchased from Cambridge (Cambridge Isotope Laboratories, Andover, MA). High-purity sucrose (SUC) and maltose (MAL) were purchased from Fisher (Fisher Scientific, Pittsburgh, PA) and Sigma (Sigma-Aldrich, Saint Louis, MO), respectively. Note that φ ) 68.1% w/w for SUC24 and φ ) 51.9% w/w for MAL25 are the saturation concentrations at room temperature. The mass of water in the TRE dihydrate powder was taken into account in the preparation of the experimental solutions. In a typical experiment, 100 nL of the experimental solution was deposited between two CaF2 windows. The windows were 16 mm in diameter and 0.5 mm in thickness. The windows were sealed with vacuum grease to eliminate evaporation and to generate a thin uniform film of approximately 1 µm thickness. The sealed sample was then transferred to a Fourier transform infrared spectrometer (FTIR: Thermo-Nicolet Continuum equipped with a Mercury Cadmium Telluride detector, Thermo Electron, Waltham, MA) equipped with a FDCS 196 (Linkam Scientific Instruments Ltd., UK) freeze-drying cryostage. The FTIR sampling resolution was 4 cm-1, and 128 IR scans were averaged per spectra in the 9000-900 cm-1 wavenumber range. The cryostage was used for precise control of the sample temperature. The sample was cooled as low as -180 °C and heated back to the room temperature at a constant rate of 2 °C/min. The IR spectra were analyzed using Omnic (Thermo-Nicolet) and Peakfit4 software (Systat Software, Inc., San Jose, CA). 2.1. FTIR Analysis. The ν-OH (∼3000-3700 cm-1)26-28 band has been used extensively to quantify the effects of temperature and solutes on water.27-29 However, due to the significant contributions from the hydroxyl groups of the carbohydrate in this region, an accurate prediction of water behavior was not feasible (especially at high sugar concentrations).30 Therefore, the δ-OH (∼1550-1800 cm-1)11,31 and the (ν2 + ν3) (∼4500-5500 cm-1)32-34 bands of water were

10.1021/jp8099434 CCC: $40.75  2009 American Chemical Society Published on Web 04/14/2009

Aqueous Trehalose Solutions at Cryogenic Temperatures

J. Phys. Chem. B, Vol. 113, No. 19, 2009 6793 attributed to water molecules that have coordination numbers of 3-4 and are simultaneously HB donors and acceptors.32,42 The S1 band (5116 cm-1) is attributed to water molecules with one active HB (single HB donor).42 The highest-frequency band (S0: 5232 cm-1) is attributed to water molecules that are not HB donors.33,42 For example, the spectra in the NIR region collected from neat water confined in hydrophilic silica nanopores (curve 1 in Figure 2A) shows that water molecules are preferentially HB to the hydroxyl groups of the silica (note the location of the peak at 5270 cm-1, which is at a higher frequency than the S0 peak of neat water located at 5232 cm-1 in Figure 2A). 2.2. Gibbs Free-Energy Calculation. The water-water HB enthalpy and entropy were calculated from the Gibbs free-energy (∆G°) of the NHB to HB transition as given by

HB ( NHB )

∆G° ) ∆H° - T∆S° ) -RT ln

(1)

where R is the ideal gas constant, T is the absolute temperature, and ∆H° and ∆S° are the HB enthalpy and entropy, respectively. 3. Results

Figure 1. Deconvolution of the IR spectrum at 23 °C. (A) δ-OH band; (B) (ν2 + ν3) band of UPW. The dashed line shows the IR spectra of the 43% w/w TRE solution.

analyzed to determine the changes in HB organization in aqueous carbohydrate solutions at cryogenic temperatures. All of the water bands (ν-OH, δ-OH, and ν2 + ν3) were deconvolved using Gaussian peaks to reveal distinct water families (clusters of water molecules with different HB organizations). In the ν-OH band, three families were identified, networking water (NW), intermediate water (IW), and multimer water (MW).35 The NW family (3314 cm-1) originates from tetrahedrally bonded water molecules.35 IW (3441 cm-1) is the weakly HB family with energetically unfavorable HBs.35-37 The MW family (3570 cm-1) originates from water monomers and dimers.35 The δ-OH band only originates from water13,19,26 and does not contain any contribution from the hydroxyl groups of carbohydrates. This band was deconvolved to yield two distinct Gaussian peaks (Figure 1A). The first peak at 1671 cm-1 was attributed to an extensively HB water population.10,11,36,38 Note that the location of this peak did not change in the presence of carbohydrates since the peak originated from tetrahedrally HB water. The second peak was located at 1646 cm-1 in UPW26,36,38 and at 1640 cm-1 in the TRE solutions.11 This peak originated from the NHB (non-hydrogen bonded) water population36 that is not tetrahedrally HB. The shift in the location of this peak in the presence of carbohydrates was attributed to the effect of the carbohydrate on the NHB water families. The combination band in the near-infrared (NIR) region consists of the δ-OH (i.e., ν2) and the ν-OH (i.e., ν3) bands of water.33,39 The contribution of carbohydrate hydroxyl groups to this band are negligible.40 In the (ν2 + ν3) band, three families can be distinguished at 4920, 5116, and 5232 cm-1 (Figure 1B).32,33,39,41 The lowest-frequency band (S2: 4920 cm-1) is

Temperature-ramp FTIR spectroscopy experiments were conducted with 10 e φ e 43 w/w TRE, 68.1% w/w SUC, and 51.9% w/w MAL solutions to examine the temperature- and concentration-induced changes in water-water and watercarbohydrate HB during freezing or vitrification of the supercooled solution. 3.1. Changes in Water Structure. 3.1.1. TemperatureInduced Hydrogen Bonding in Water. HB organization of water increases with decreasing temperature.19,36,43,44 The peak maximum of the δ-OH band (1550-1750 cm-1) of neat water gradually shifted (inset in Figure 1A) to higher frequencies with decreasing temperature (e.g., fδ-OH,20C(liquid) ) 1644.1 cm-1, fδ-1 OH,-18C(liquid) ) 1648.2 cm ). The shift implied an increase in the HB population at the expense of the NHB population (Figure 1A).38 The increase in HB with decreasing temperature was also evident in the ν-OH band, which shifted to lower frequencies (showing an increase in the NW population at the expense of the MW and IW populations). The peak maximum of the asymmetrical (ν2 + ν3) water band was located at f(ν2+ν3),20C(liquid) ) 5190 cm-1 at room temperature (curve 2a in Figure 2A). This peak shifted toward lower frequencies (Figure 1B) with decreasing temperature (e.g., f(ν2+ν3),-18C(liquid) ) 5145 cm-1; curve 2b in Figure 2A), also indicating an increase in the HB networking of water.32,34,45 In summary, all of the waterassociated spectral bands (δ-OH, ν-OH, and ν2 + ν3) showed an increase in HB networking in supercooled water with decreasing temperature. Upon freezing of neat water, the δ-OH peak intensity dropped suddenly and remained unchanged with decreasing temperature (inset in Figure 1A), while the (ν2 + ν3) peak became more symmetrical and shifted to lower frequencies32 (e.g., f(ν2+ν3),-20C(ice) ) 5035 cm-1; curve 2c in Figure 2A). Frozen neat water spectra showed very little contribution from the S0 peak, suggesting that all water molecules were extensively HB in the ice phase (i.e., monomeric water, at detectable quantities, was not present in ice).32,45,46 3.1.2. Carbohydrate-Induced Hydrogen Bonding in Water. The presence of TRE in the solution increased the HB of water (the kosmotropic effect21 as observed previously by Branca et al.20,47 and others19). In the saturated TRE solution (43% w/w), the (ν2 + ν3) peak maximum at 20 °C was located at a lower wavenumber (5170 cm-1) than neat water (Figure 2A). This

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Figure 2. NIR spectra of (A) neat water and TRE solutions and (B) TRE, SUC, and MAL solutions.

showed that TRE at this concentration had an effect on water similar to supercooling to approximately T ) -12 °C (i.e., a significant increase in HB). Moreover, in the presence of TRE in the NIR region, a strong secondary peak appeared (S2* at ∼4800 cm-1 at 20 °C; see curve 3a in Figure 2A) close to the location of the S2 family observed in neat water (4920 cm-1 at 20 °C). Note that peaks in the neighborhood of S2* were previously reported in the literature in solutions containing kosmotropes.46 The fact that the S2* peak was located at a lower frequency than the S2 peak suggested that the S2* family had higher degree of HB and also a potentially higher HB strength than the S2 family. Therefore, this new peak (S2*) was attributed to a new organization in water networking caused by the insertion of the TRE molecules into the HB network of water molecules.21 At room temperature, the S2 population in neat water was calculated as 22%. In the presence of 10% w/w TRE, the S2* population increased to 31.6% and then to 42.5% in

the 43% w/w TRE solution. Similarly, analysis of the δ-OH band showed a HB water population of 29% in neat water, which increased to 36 and then to 40% in the presence of 10 and 43% w/w TRE, respectively. 3.1.3. Hydrogen Bonding in Supercooled and Frozen Trehalose Solutions. Cooling experiments were conducted with aqueous TRE, MAL, and SUC solutions that contained 2% D2O (as well as with solutions in pure H2O). This was done to compare the changes in the HB of solutions of identical carbohydrate concentration during supercooling versus freezing. Note that in the presence of D2O, carbohydrate solutions tend to freeze at a lower degree of supercooling (or at lower cooling rates). The δ-OH Band. Similar to what was observed in neat water, HB organization in TRE solutions increased with decreasing temperature. This was indicated by the shift of the δ-OH peak to higher frequencies with reducing temperature. In the saturated

Aqueous Trehalose Solutions at Cryogenic Temperatures TRE solution, the δ-OH peak location reached a plateau value of fδ-OH,43%TRE ) 1662.4 cm-1 (corresponding to a HB population of 62%) at approximately T ) -90 °C. In the lowerconcentration solutions, with freezing, the δ-OH band intensity dropped sharply and remained insensitive to further reduction in temperature. Note that the δ-OH band peak intensity measured after freezing was proportional to the concentration of TRE in the original solution. This was attributed to the increasing amount of unfrozen water at higher TRE concentrations. When the solutions were frozen at T ) -30 °C, the frequency of the δ-OH peak in the 30 and 40% TRE solutions stayed at 1651.4 and 1658.2 cm-1, respectively. (ν2 + ν3) Band. During supercooling of the saturated TRE solution, the S1* peak (note that the S1 family is denoted as S1* in the TRE solutions) shifted toward lower frequencies (e.g., 5110 cm-1 at T ) -100 °C), while the location of the S2* peak remained the same (curve 3b in Figure 2A,B). The shape, spectral features, and temperature dependence of the S1* peak was identical to that of the S1 peak of the supercooled water extrapolated to low temperatures. Moreover, if freezing occurred during cooling, the S1* peak shifted to lower frequencies (curve 3c in Figure 2B), similar in behavior to the S1 peak of the neat water (curve 2c in Figure 2B). However, neither the frequency nor the magnitude of the S2* peak changed upon freezing. The magnitude only increased with increasing TRE concentration (curve 3c in Figure 2B). Note that the arrows in Figure 2B show the spectral traces of the frozen 20 and 30% TRE solutions. The S2* peak was also observed in amorphous saturated MAL and SUC solutions (curves 5a and 5b in Figure 2B). This suggested that in the supercooled solution, in addition to the carbohydrate-associated water molecules (that make up the hydration layers of the carbohydrate21 and give rise to S2*), there were clusters of water molecules (represented by S1*) that were not associated with the carbohydrate. This proposition was further strengthened by the results of the drying experiments conducted with TRE and SUC solutions (dried at 0% RH for 4 or 24 h at room temperature). With increased drying time, the magnitude of the S1* peak decreased continuously while the S2* peak remained unaltered until the S1* peak disappeared completely (curves 4a and 4b in Figure 2A). Only after complete disappearance of the S1* peak did the S2* peak began to decrease in magnitude (i.e., the clusters of water that were not associated with TRE evaporated before the water molecules in the hydration layers of TRE). IR spectra of out-of-the-box TRE powder (TRE dihydrate crystals) did not have the S1* peak but did have a very small, noisy peak in the NIR region at 4730 cm-1, which was attributed to the very tightly bound water present in the dihydrate TRE crystals. In summary, these results support the proposition that there are two distinct populations of water in supercooled amorphous carbohydrate solutions. The first population gives rise to the S2* family, which forms extensively networked HB associations with TRE (with higher HB strength)48 and involves the water molecules in the first few hydration layers of the carbohydrate. The second population (S1*) represents supercooled clusters of water that are not associated with the carbohydrate but are involved in water-water HBs. It is mainly this population of water that is supercooled or, alternatively (depending on the cooling rate), frozen during cooling. 3.2. Changes in Trehalose Structure. In order to investigate the water-carbohydrate (i.e., TRE hydration) and carbohydratecarbohydrate HB interactions, the evolution of the 1370 cm-1 band with concentration and temperature was analyzed. The 1370 cm-1 band is associated with the asymmetric bending of

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Figure 3. Change in the normalized corrected area of the 1370 cm-1 band with respect to temperature. Note: All experimental solutions include 2% D2O.

Figure 4. Change in the glycosidic frequency of 43% w/w TRE in UPW and φ ) 20, 30, 40, and 43% w/w TRE in the presence of 2% D2O.

the OH and CH2 groups of carbohydrates and is known to be strongly affected by hydration.49 In all of the TRE, SUC, and MAL solutions tested, with cooling, the 1370 cm-1 peak gradually increased in magnitude (Figure 3) and became more defined, which was a sign of decreasing HB between water and carbohydrate.49 This was expected since the HB of water increases with decreasing temperature (see previous section); carbohydrate hydration should decrease, and the carbohydratecarbohydrate interactions (intermolecular as well as intramolecular interactions) should increase.12 The peak at 1150 cm-1 in the IR spectra originates from ν(C-O)50,51 of the glycosidic linkage connecting the two glucose pyranose rings in TRE. TRE has high mobility around its glycosidic linkage.49,50,52 The flexibility of TRE around its glycosidic linkage decreased with increasing TRE concentration in the solution (Figure 4). Also, with reduction in temperature in supercooled liquid, TRE glycosidic band shifted to higher frequencies much faster when TRE concentration was lower (Figure 4). It is interesting that the flexibility of the molecule around the glycosidic link was higher with reduced hydration. This can only be explained by the increasing TRE-TRE interactions at low temperatures, which may cause folding of the carbohydrate around its glycosidic linkage. Temperatureand concentration-dependent changes in TRE hydration and structure have been reported in the literature.12,17,20,53 3.3. Hydrogen Bond Strength. It is not known how much the ν-OH band is affected by the presence of high concentrations of carbohydrates in the solution, especially during cooling, freezing, or vitrification. On the other hand, the δ-OH and the

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Figure 5. Change in ∆G° as a function of temperature in neat water, silica nanopore-confined water, φ ) 10, 20, 30, 40, and 43% w/w TRE in the presence of 2% D2O and 43% w/w TRE, 68.1% w/w SUC, and 51.9% w/w MAL in H2O.

(ν2 + ν3) combination bands originate mainly from water. Therefore, these bands were used to calculate the water-water HB strength and entropy by examining the evolution of HB with temperature in different TRE concentration solutions.36 ∆G° of water in different solutions was calculated (eq 1) from the evolution of the fully HB clusters from the weakly HB distorted clusters (NHB family) during supercooling. In dilute solutions (φ < 43%), ∆G° decreased linearly with temperature until freezing commenced (Figure 5). Note that standard deviations are not presented in Figure 5 for clarity. Once freezing occurred, ∆G° became insensitive to temperature decrease since the δ-OH peak stopped changing. At all temperatures above the freezing temperature, ∆G° decreased with increasing concentration of TRE. This was attributed to an increase in the HB population of water with increasing TRE (i.e., the kosmotropic effect). However, the temperature dependence of ∆G° was independent of TRE concentration. This implied that the increase in water HB with decreasing temperature did not depend on the TRE concentration. This was only possible if there were isolated water clusters in the solution that were not affected by the presence of TRE in the solution. In the saturated TRE solution, ∆G° decreased linearly with decreasing temperature until T ∼ -50 °C, where the slope started to change (Figure 5). The change in ∆G° with temperature stopped at the glass transition (Tg ∼ -80 °C). Interestingly, saturated SUC and MAL solutions (as well as silica nanoporeconfined water) showed similar behaviors, experiencing a slope change in ∆G° at T ∼ -50 °C. Note that TRE, SUC, and MAL are disaccharides with identical molecular weights (342.30 g/mol). The change in the slope of ∆G° might be a result of depletion of the NHB water molecules or might even be attributed to the fragile-to-strong transition of water located at T ) -51 °C.43,54 The transition of NHB to HB with temperature was used to calculate the enthalpy and entropy of water-water HB. In neat water, calculations based on ∆G° using δ-OH yielded a HB enthalpy value of ∆H° ) 9.6 ( 0.4 kJ/mol. On the other hand, ∆G° calculated from the ν-OH region (IW to NW transition) yielded ∆H° ) 13.8 kJ/mol. Both of these values were within the range (9.2-18.8 kJ/mol) reported in the literature for water HB.36,55,56 Values calculated for the HB entropy using the two different bands were also similar (∆S°δ-OH ) 41.5 ( 0.8 J/molK and ∆S°ν-OH ) 38.2 J/molK) and were within the range (30-46 J/molK) reported in the literature.55 ∆H° and ∆S° values calculated for different TRE concentrations are presented in

Malsam and Aksan Table 1. In the presence of TRE, water HB strength decreased (∆H° ) 6.9 ( 0.7 kJ/mol for φ ) 10%); however, the HB strength was independent of the concentration of TRE (Table 1). This was also observed in experiments conducted with Brillouin inelastic UV scattering.57 The insensitivity of water HB strength on TRE concentration (Table 1) also supported the proposed scheme that TRE molecules participated in HB networks with a certain number of water molecules in their close proximity while the rest of the water in the solution clustered as confined water (and were not affected by the presence of TRE). The inherent assumption here is that the δ-OH band mainly originates from the confined water clusters (that are not in the hydration layers of TRE), which also explains the relative insensitivity of the δ-OH band to the presence of carbohydrates in the solution.16,19,26 In Table 1, the enthalpy and entropy of transition between the S1* and S2* families of the (ν2 + ν3) band are also presented. These represent the energetic and entropic values for the transition of the water molecules between the hydration layer of TRE and the confined water clusters. For all TRE concentrations, ∆H°S1fS2 < ∆H°, showing that increased HB networking during cooling was energetically possible. Note that in neat water, as expected, these values were equal (∆H° ) 9.6 ( 0.4 kJ/mol and ∆H°S1fS2 ) 9.7 ( 0.2 kJ/mol) since there was no TRE-associated water in the system. The temperature dependence of carbohydrate hydration in supercooled, amorphous (unfrozen) solutions was analyzed using the 1370 cm-1 peak (Figure 6). TRE and SUC solutions exhibited two unique transitions, while MAL exhibited one during supercooling. The lower-temperature transition corresponded to the glass transition of the amorphous solutions, Tg ) -80 °C for TRE,58 -76 °C for SUC,59 and -74 °C for MAL.60 The higher-temperature transitions were at Tj ∼ -55 °C for TRE (Figure 6A) and -45 °C for SUC (Figure 6B). A high-temperature transition was not detected in saturated MAL solution. The high-temperature transitions can be attributed to an increase in carbohydrate-water HB within the first hydration layer or an increase in carbohydrate-carbohydrate interactions (i.e., the percolation). However, more research is required to conclusively determine the exact mechanism of this transition. Note that the transitions observed in the 1370 cm-1 band (Figure 6) are due to carbohydrate-water and carbohydrate-carbohydrate HB, which are different from the water-water HB transitions presented in Figure 5. 4. Discussion According to the mixture theory of water, the population of water molecules organized in a tetrahedrally coordinated (icelike) configuration increases with decreasing temperature.61 We have detected the increase in water-water HB with decreasing temperature through the analysis of all three IR bands (δ-OH, ν-OH, and ν2 + ν3)36 of water. Water-water HB also increased in the presence of carbohydrates in the solution (kosmotropic effect).19,20,47 The detailed analysis presented here revealed the presence of two distinct water populations in carbohydrate solutions, (1) the water molecules that are directly associated with the carbohydrate, forming its hydration layer(s)12,17,20,53 and (2) water molecules in small clusters that are only involved in water-water HB, showing characteristics of water in hydrophilic confinement (Figure 7A).62,63 There is strong evidence for the presence of the first water population. Molecular dynamics simulations show that each TRE molecule interacts with at least eight water molecules,

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TABLE 1: Measured and Calculated Parameters for TRE Solutions TRE concentration [w/w %] 0% (UPW) 10% 20% 30% 40% 43% a

∆H° [kJ/mol]

∆H°S1fS2 [kJ/mol]

∆S° [kJ/mol · K]

δ-OH band

NIR band

δ-OH band

NIR band

measured freezing temperature [°C]

9.6a ( 0.4 6.9 ( 0.7 6.8 ( 0.2 6.7 ( 0.2 6.3 ( 0.1 6.3 ( 0.1

9.7 ( 0.2 5.6 ( 0.7 5.6 ( 0.2 5.8 ( 0.2 5.8 ( 0.5 6.1 ( 0.3

41.5b ( 0.8 28.1 ( 2.5 27.3 ( 0.4 26.6 ( 0.8 24.6 ( 0.1 24.6 ( 0.1

39.1 ( 0.8 20.8 ( 4.2 21.6 ( 1.7 19.9 ( 0.8 21.6 ( 1.9 16.6 ( 2.1

-11.8 ( 0.7 -20.5 ( 4.1 -23.1 ( 1.0 -25.0 ( 1.2 -29.7 ( 3.4 -30.2 ( 0.5

∆S°S1fS2 [kJ/mol · K]

Reported range (9.2-18.8 kJ/mol).36,55,56 b Reported range (30-46 kJ/mol K).55

increasing their residence times.49,57 It is also plausible to have confined water clusters in carbohydrate solutions. If cooled sufficiently fast, all carbohydrate solutions can be vitrified,64 which means that it is possible to obtain glasses of the same chemical composition but different water content. Therefore, direct carbohydrate-carbohydrate interactions (or direct interac-

Figure 6. Change in the normalized corrected area of the 1370 cm-1 band with respect to temperature of (A) 43% w/w TRE, (B) 68.1% w/w SUC + 2% D2O, and (C) 51.9% w/w MAL + 2% D2O solutions.

tions between hydration layers) cannot be a prerequisite for vitrification. The molar ratio of water to TRE at its saturation concentration of 43% w/w is 25.2:1. It is known that the TRE coordination number is approximately 8.17,19 If all of the water in the glass interacted with (or was affected by) the carbohydrate, this would require TRE to have at least three hydration layers. The molar ratio of water to TRE increases to 44.3:1 in a 30% w/w solution, which could also be vitrified if cooled sufficiently fast. If all water molecules interacted with the carbohydrate, this would require at least six hydration layers for TRE. However, is known that solute effects do not extend beyond one to two layers of water,9 Therefore, there should be water molecules present in the glass that do not interact with the carbohydrate. Moreover, it is known that the δ-OH peak of water is not very sensitive to the presence of carbohydrates.16,26 The lack of sensitivity is not easily explained since carbohydrates strongly associate with and alter the mobility of the water molecules in their immediate surroundings.17,53,65 In our experiments, the δ-OH peak location changed only very slightly toward higher frequencies with increasing concentrations of TRE in the solution (Figure 1A). One possible explanation for this behavior (within the framework of our proposition) is that the δ-OH band mainly originates from the confined water clusters in the solution (not from those in the hydration layers of carbohydrates). The ∆G° of the TRE solutions at all temperatures were lower than that of neat water (Figure 5) and also lower than that of water confined in silica nanopores and micelles.63,66 This further supported the hypothesis that the behavior of the δ-OH band was dominated by the confined water clusters in the TRE solution and not by the TRE-associated water molecules. Note that ∆H° in hydrophilic silica-confined water is approximately 7.7 kJ/mol,63 which is much less than the ∆H° of neat water but very close to the values (6.3-6.9 kJ/mol) measured in the TRE solutions (Table 1).55,56 From a water HB perspective, the effect of the presence of TRE at saturation concentrations (φ ) 43% w/w) in water was identical to supercooling of neat water

Figure 7. Water populations in carbohydrate solutions.

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to approximately T ) -12 °C with fδ-OH,43%TRE ) 1648.3 cm-1. Interestingly, when the saturated solution was dried at 0% RH for 24 h (to a water content much less than that of the 43% w/w TRE solution), the δ-OH peak frequency did not change significantly (fδ-OH,DRIED ) 1648.7 cm-1). This showed that at the saturation level, the effect of TRE on water HB had reached its maximum, and further reduction in water content by drying did not change the water-water HB strength.67 This was further evidence that the δ-OH peak was mainly affected by water-water HB in the confined clusters. It is not exactly possible to determine the coordination number of the water molecules that give rise to the S2* peak in the NIR region.32,34 The fact that the S2* peak was located at a lower frequency than the S2 peak suggested that the coordination numbers of the water molecules in this family were higher (according to the mixture theory of water68); however, this shift might also be caused by the increase21 in the water HB strength due to the presence of TRE (according to the continuum model for water69). Irrespective of the model, it was obvious that two distinct water families existed in the solutions with high carbohydrate concentration. Experiments conducted with amorphous MAL and SUC solutions (curves 5a and 5b in Figure 2B) showed that this was not actually unique to TRE. Spectra from both MAL and SUC showed characteristics of confined water clusters distinct from carbohydrate-associated water (Figure 2). It is envisioned that the water clusters in the carbohydrate solution (the S1* family) are surrounded by the hydration layers of the carbohydrates (the S2* family), creating a condition similar to hydrophilic confinement (Figure 7A). It is known that in hydrophilic confinement, the freezing temperature of water is depressed as a function of the pore size.70-72 To avoid crystallization at temperatures as low as -100 °C in the 43% TRE solution, the confined water cluster radius should be on the order of 1 nm71 (implying that the clusters should be 4-5 water molecules in diameter), which, interestingly, corresponds to the size range proposed for heterogeneous mobility regions within glasses.73 Of course, the cluster size would decrease significantly at higher concentrations of sugars.74 It has been suggested that preferential exclusion23,75 is the mechanism of action of carbohydrates in conferring stability to macromolecules at low temperatures. Additionally, it is known that the stability is enhanced if the macromolecules are kept in a glassy medium rather than in a frozen solution.3,4 Both of these well-supported theories are in agreement with what was observed in this study. It can be envisioned that in the presence of high concentrations of carbohydrates at cryogenic temperatures, the carbohydrates are preferentially excluded from the macromolecule surfaces, whereas the macromolecules are dissolved in the supercooled water clusters surrounded by the amorphous sugar matrix (Figure 7B). It is known that confinement in small water clusters entrapped in reverse micelles and in solid hydrophilic nanopores decreases the molecular mobility of macromolecules.66,76-79 Similarly, encapsulation of macromolecules in the confined water clusters of amorphous carbohydrate solutions would reduce molecular motions, therefore conferring stability. Note that Figure 7 is just a schematic to facilitate introduction of our proposition. It is known in amorphous (and in glassy) materials that a long-range order is not present, and therefore, one would expect to see a size distribution of water cluster sizes. In different water content glasses, the average size of the water clusters as well as their distribution would then play roles in determining their effective-

Malsam and Aksan ness in stabilizing the macromolecule.14 Further experimentation is required to determine the validity of the proposition made here. Hydration of the carbohydrates is dominated by hydrogen bonding of their equatorial hydroxyl sites with water,80 while the glycosidic linkage exhibits the lowest level of hydration.17 It is known that TRE does not have any internal HBs,49 giving the molecule high flexibility around its glycosidic linkage. This ability is thought to contribute to its high cryo-/lyoprotective efficiency.81 TRE experiences dehydration82 during cooling and changes its configuration about the glycosidic linkage, potentially forming carbohydrate-carbohydrate HBs.3,17 Increased flexibility around the glycosidic bond may correlate to the hightemperature transition observed in the carbohydrate-water HB (Figure 6). 5. Conclusion We have used FTIR spectroscopy analysis with (10 e φ e 43%) supercooled, frozen, and vitrified TRE solutions to determine the changes in water-water and water-carbohydrate HB with temperature and concentration. The presence of carbohydrates in the solution increased the HB networking of water (i.e., kosmotropic effect). FTIR spectroscopy revealed the presence of two distinct water families in aqueous carbohydrate solutions, (1) water molecules directly associated with the carbohydrate, forming its hydration layer(s)12,17,20,53 and (2) water molecules that are not associated with the carbohydrate but are involved in water-water HB in small clusters. The latter showed characteristics of water in hydrophilic confinement (Figure 7). Acknowledgment. This research is partially supported by University of Minnesota Grant-in-Aid (20838) and NSF (CBET0644784). References and Notes (1) Fuller, B. J.; Lane, A. N.; Benson, E. E. Life in the Frozen State; CRC Press: Boca Raton, FL, 2004. (2) Crowe, J.; Hoekstra, F.; Crowe, L. Annu. ReV. Physiol. 1992, 54, 579. (3) Miller, D. P.; de Pablo, J. J.; Corti, H. Pharm. Res. 1997, 14, 578. (4) Conrad, P. B.; Miller, C. A.; Cielenski, P. R.; de Pablo, J. J. Cryobiology 2000, 41, 17. (5) Carpenter, J. F.; Crowe, J. H. Biochemistry 1989, 28, 3916. (6) Torreggiani, D.; Forni, E.; Guercilena, I.; Maestrelli, A.; Bertolo, G.; Archer, G. P.; Kennedy, C. J.; Bone, S.; Blond, G.; Contreras-Lopez, E.; Champion, D. Food Res. Int. 1999, 32, 441. (7) Roberts, C. J.; Debenedetti, P. G. AIChE J. 2002, 48, 1140. (8) Wang, W. Int. J. Pharm. 2000, 203, 1. (9) Aksan, A.; Toner, M. Roles of Thermodynamic State and Molecular Mobility in Biopreservation. In The Biomedical Engineering Handbook, 3rd ed.; Bronzino, J. D., Ed.; Taylor & Francis: Boca Raton, FL, 2006, Vol. 3. (10) Branca, C.; Magazu, S.; Maisano, G.; Migliardo, P. J. Chem. Phys. 1999, 111, 281. (11) Gharsallaoui, A.; Roge´, B.; Mathlouthi, M. Food Chem. 2008, 106, 1329. (12) Conrad, P. B.; de Pablo, J. J. J. Phys. Chem. A 1999, 103, 4049. (13) Lerbret, A.; Bordat, P.; Affouard, F.; Guinet, Y.; He´doux, A.; Paccou, L.; Pre´vost, D.; Descamps, M. Carbohydr. Res. 2005, 340, 881. (14) Simperler, A.; Kornherr, A.; Chopra, R.; Jones, W.; Motherwell, W. D. S.; Zifferer, G. Carbohydr. Res. 2007, 342, 1470. (15) Gaı¨da, L. B.; Dussap, C. G.; Gros, J. B. Food Chem. 2006, 96, 387. (16) Zelent, B.; Nucci, N. V.; Vanderkooi, J. M. J. Phys. Chem. A 2004, 108, 11141. (17) Choi, Y.; Cho, K. W.; Jeong, K.; Jung, S. Carbohydr. Res. 2006, 341, 1020. (18) Koop, T.; Luo, B.; Tsias, A.; Peter, T. Nature (London) 2000, 406, 611. (19) Gallina, M. E.; Sassi, P.; Paolantoni, M.; Morresi, A.; Cataliotti, R. S. J. Phys. Chem. B 2006, 110, 8856.

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