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FT-IR Study of the Interlamellar Water Confined in Glycolipid Nanotube Walls Yanli Guo,† Hiroharu Yui,*,†,‡ Hiroyuki Minamikawa,‡,§ Mitsutoshi Masuda,‡,§ Shoko Kamiya,‡ Tsuguo Sawada,† Kohzo Ito,† and Toshimi Shimizu*,‡,§ Department of Advanced Materials Science, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561, Japan, CREST, Japan Science and Technology Agency (JST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, and Nanoarchitectonics Research Center (NARC), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan Received December 15, 2004. In Final Form: February 15, 2005 The local hydrogen-bonding environment of water confined in glycolipid nanotubes (LNTs) was investigated by Fourier transform infrared (FT-IR) spectroscopy. Using X-ray diffraction (XRD), we estimated the thickness of an interlamellar water layer, which was confined between the bilayer membranes constructing the walls of the LNTs, to be 1.3 ( 0.3 nm. FT-IR spectroscopic measurement of the confined water showed an obvious reduction in IR absorption in both the low-energy (around 3000 cm-1) and high-energy regions (around 3600 cm-1) of the OH stretching band as compared to bulk water. The reduction around 3000 cm-1 indicated a decrease in the relative proportion of the water molecules with a long-range network structure due to a geometrical restriction. This agrees with the results obtained for other multilamellar systems. On the other hand, the remarkable reduction around 3600 cm-1, which was not observed in the other systems, indicated the absence of weakly hydrogen-bonded water aggregates due to the effect of sugar headgroups.
Introduction Studies on the structure and dynamics of water molecules confined in restricted geometries are of great interest, since the confined water plays an important role in biological and geological systems.1-5 In the past two decades, water confined either in the cores of spherical reversed micelles (Figure 1a) or between the layers of various lamellar structures (Figure 1b,c) has been widely studied by a variety of experimental and computer simulation methods.6-17 Infrared (IR) and Raman spec* To whom correspondence should be addressed. E-mail: yui@ molle.k.u-tokyo.ac.jp (H.Y.);
[email protected] (T.S.). † The University of Tokyo. ‡ Japan Science and Technology Agency (JST). § National Institute of Advanced Industrial Science and Technology (AIST). (1) Marechal, Y.; Chamel, A. J. Phys. Chem. 1996, 100, 8551-8555. (2) Chapman, D. J. Food Eng. 1994, 22, 367-380. (3) Costanzo, P. M.; Giese, R. F.; Lipsicas, M.; Straley, C. Nature 1982, 296, 549-551. (4) Pitteloud, C.; Powell, D. H.; Gonzalez, M. A.; Cuello, G. J. Colloids Surf., A 2003, 217, 129-136. (5) Yui, H.; Guo, Y.; Koyama, K.; Sawada, T.; John, G.; Yang, B.; Masuda, M.; Shimizu, T. Langmuir 2005, 21, 721-727. (6) Macdonald, H.; Bedwell, B.; Gulari, E. Langmuir 1986, 2, 704708. (7) Onori, G.; Santucci, A. J. Phys. Chem. 1993, 97, 5430-5434. (8) Jain, T. K.; Varshney, M.; Maitra, A. J. Phys. Chem. 1989, 93, 7409-7416. (9) Li, Q.; Weng, S. F.; Wu, J. G.; Zhou, N. F. J. Phys. Chem. B 1998, 102, 3168-3174. (10) Zhou, N. F.; Li, Q.; Wu, J. G.; Chen, J.; Weng, S. F.; Xu, G. X. Langmuir 2001, 17, 4505-4509. (11) Brubach, J. B.; Mermet, A.; Filabozzi, A.; Gerschel, A.; Lairez, D.; Krafft, M. P.; Roy, P. J. Phys. Chem. B 2001, 105, 430-435. (12) Umemura, J.; Matsumoto, M.; Kawai, T.; Takenaka, T. Can. J. Chem. 1985, 63, 1713-1718. (13) Lhert, F.; Capelle, F.; Blaudez, D.; Heywang, C.; Turlet, J. M. J. Phys. Chem. B 2000, 104, 11704-11707. (14) Berger, C.; Desbat, B.; Kellay, H.; Turlet, J. M.; Blaudez, D. Langmuir 2003, 19, 1-5. (15) Scott, H. L. Chem. Phys. Lett. 1984, 109, 570-573.
Figure 1. Schematic diagrams of water confined in several representative molecular aggregates: (a) reversed spherical micelle; (b) black soap film; (c) multilamellar structure; (d) glycolipid nanotube (LNT).
troscopic experiments have demonstrated that the characteristics of confined water with dimensions of a few nanometers obviously differ from those of bulk water. The deviation is derived from the size confinement and the effect of the polar headgroups of amphiphiles. Several research groups have examined the effect of size confine(16) Lafleur, M.; Pigeon, M.; Pezolet, M.; Caille, J. P. J. Phys. Chem. 1989, 93, 1522-1526. (17) Boissiere, C.; Brubach, J. B.; Mermet, A.; de Marzi, G.; Bourgaux, C.; Prouzet, E.; Roy, P. J. Phys. Chem. B 2002, 106, 1032-1035.
10.1021/la046906q CCC: $30.25 © 2005 American Chemical Society Published on Web 04/16/2005
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ment on IR spectroscopic characteristics by observing the corresponding spectroscopic change in water when increasing the degree of confinement.11,14,17 A reduction in IR absorption in the low-frequency region of the OH stretching band was observed when the dimension of the confined water was decreased to a few nanometers. On the other hand, the effects of the headgroups on the IR spectra have also been investigated by comparing the spectroscopic characteristics of water confined in various systems with different types of ionic and nonionic headgroups.9,10,14 Although the water confined in a variety of molecular aggregates with ionic headgroups has been studied in terms of the electrostatic effects of the ionic headgroups,6-10,12-17 the water confined in nonionic headgroup systems has not been investigated widely. In the present study, we investigated the IR spectroscopic characteristics of the water confined in a typical nonionic system, glycolipid nanotubes (LNTs) (Figure 1d). The LNT used in this study is formed from glycolipid molecules on the basis of a self-assembling process.18-21 As shown in Figure 1d, the wall of the self-assembled LNTs is composed of several tens of glycolipid bilayer membranes, in which the molecules arrange themselves with polar headgroups outward and with nonpolar tails inward. As a result, the sugar hydroxyl groups surround the interlamellar space between the adjacent bilayer membranes. There should be very small amounts of water confined in the hydrophilic interlamellar space. The confinement and the surrounding sugar headgroups should influence the hydrogen-bonding environment of the interlamellar water. In the present paper, we analyze the hydrogen-bonding features of the interlamellar water based on Fourier transform infrared (FT-IR) spectroscopy and discuss the effects of confinement and the sugar headgroups on the hydrogen-bonding environment. Experimental Section Preparation of LNTs. The preparation of the LNTs using N-(11-cis-octadecenoyl)-β-D-glucopyranosylamine (1) has been described in detail elsewhere.18-21 Glycolipids have recently emerged as potential molecular building blocks for supramolecular aggregates. They can self-assemble into stable tubular structures of nanometer order.22 The synthetic glycolipid 1 used in this study was newly developed and structurally optimized by our group. In this glycolipid, the glucopyranosyl headgroup is linked to the unsaturated C18 hydrocarbon chain via an amide linkage. It can produce well-defined LNTs with uniform diameters in >98% yields. In this study, 5 mg of 1 was added to 100 mL of distilled water and then dispersed at 96 °C for about 2 h. Upon cooling, an aqueous dispersion of white lipid nanotubes was obtained. Scanning Electron Microscopy (SEM). A 5 µL suspension of the LNTs was dropped onto double-sided tape glued to a SEM grid. The sample was dried in air to remove the bulk water. It was then dried in a vacuum for 12 h to remove the water confined in the hollow cylinder (“in-tube” water). XRD Measurements. An X-ray diffraction (XRD) measurement was carried out with a Rigaku R-AXIS IV X-ray diffractometer. A suspension of the LNTs was centrifuged to collect the LNTs. The obtained wet LNTs were then transferred into a quartz (18) Yang, B.; Kamiya, S.; Yui, H.; Masuda, M.; Shimizu, T. Chem. Lett. 2003, 32, 1146-1147. (19) Yang, B.; Kamiya, S.; Yoshida, K.; Shimizu, T. Chem Commun. 2004, 5, 500-501. (20) Yang, B.; Kamiya, S.; Shimizu, Y.; Koshizaki, N.; Shimizu, T. Chem. Mater. 2004, 16, 2826-2831. (21) Kamiya, S.; Minamikawa, H.; Jung, J. H.; Yang, B.; Masuda, M.; Shimizu, T. Langmuir 2005, 21, 743-750. (22) (a) John, G.; Masuda, M.; Okada, Y.; Yase, K.; Shimizu, T. Adv. Mater. 2001, 13, 715-718. (b) Jung, J. H.; John, G.; Yoshida, K.; Shimizu, T. J. Am. Chem. Soc. 2002, 124, 10674-10675. (c) Masuda, M.; Shimizu, T. Langmuir 2004, 20, 5969-5977.
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Figure 2. Scanning electron microscopic image of the LNT, approximately 400 nm in outer diameter and 50 nm in inner diameter. capillary (Glas, 1.5 mm in outer diameter and 0.01 mm in wall thickness). The capillary was flame-sealed to avoid the evaporation of water. The X-ray diffraction experiment was performed with Cu KR radiation (40 kV, 30 mA) monochromated with graded elliptic multilayer optics. The diffraction pattern was recorded with an imaging plate (Fuji-Photo Film) in a flat camera (camera length 145.5 mm). The measurement was carried out at 25 °C under a 30 min exposure. ATR-FT-IR Measurements. All measurements were carried out in an attenuated total reflection (ATR) diamond cell (PIKE Technologies, Inc.) at ambient temperature using an FT-IR spectroscope (FT/IR-680plus, Jasco Co., Japan). A 20 µL suspension of the LNTs was dropped into the liquid-holding cell. Since interlamellar water should be detected without the interference of bulk water, in-tube water, or OH groups in the glycolipid, we removed them sequentially by drying the samples stepwise. We then measured the direct difference in the two FT-IR spectra, which were obtained before and after the interlamellar water was removed. In this study, we initially dried the LNTs in air to remove the bulk water and then in a vacuum to remove the in-tube water. To remove the water confined in the interlamellar space, we then added acetone onto the same sample and decomposed the tubular structure. The added acetone and interlamellar water were finally removed with a gas flow of dried nitrogen. During the drying process in air and vacuum, IR spectra were measured at regular intervals to monitor the extent of drying.
Results and Discussion SEM and XRD Analysis. Figure 2 shows the scanning electron microscopic images of the LNT used in this study. A straight tube, approximately 400 and 50 nm in outer and inner diameter, respectively, is observed clearly to have a rolling-up structure. The SEM image gives direct evidence of the hollow cylindrical structure with open ends. In the one-dimensional X-ray diffraction diagram, the wet LNT sample afforded six diffraction peaks in the small angle region (Figure 3). The x-axis is plotted as the scattering vector q ) 2π/d, where d is a Bragg d spacing value. The six d spacing values give a ratio of 1:1/2:1/3:1/ 1 1 4: /5: /6, indicating that the membranes are in a lamellar phase. The d spacing value of the (001) diffraction, corresponding to the total thickness of a single lipid bilayer membrane and an interlamellar water layer,23 can be calculated by Bragg’s rule as 4.44 nm. Since the wall of the LNT is about 200 nm thick (Figure 2), we can estimate that it consists of approximately 50 lipid bilayer membranes. The thickness of the interlamellar water layer can be estimated by a further analysis of X-ray diffraction. (23) Schneider, M. F.; Zantl, R.; Gege, C.; Schmidt, R. R.; Rappolt, M.; Tanaka, M. Biophys. J. 2003, 84, 306-313.
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Figure 3. X-ray diffractogram of LNTs dispersed in water.
Figure 4. Electron density distributions of glycolipid bilayers in the LNTs.
From the small-angle X-ray diffractogram with six Bragg peaks (Figure 3), we estimated the electron density profile of the glycolipid bilayer.23 On the basis of Fourier transform, the electron density profile, F(z), was calculated as shown in eq 1 below N
F(z) )
∑ f(h)|F(h)| cos h)1
2πhz d
(1)
where h is the diffraction order, f(h) the phase factor, |F(h)| the absolute value of the structure factor, and z the distance from the center of the lipid bilayer. |F(h)| of the nonzero-order diffraction peaks was obtained from eq 2.
|F(h)| ) xh2I(h)
(2)
Here, I(h) is the integral intensity of the diffraction peak. Since the lamellar phase is centrosymmetric, the phase factor, f(h), can be +1 or -1. Assuming that both 1 and -1 are of possibility, 32 (26 × 1/2) electron density profiles could be acquired. Considering that the structure of the glycolipid bilayer used in this study is interdigitated24 and the lowest electron density should not appear in the center, three profiles without a minimum in the center were chosen as the candidates for the electron density distribution of the glycolipid bilayer, as shown in Figure 4. The peak at the center, which shows a higher electron density value than the minimum but is still relatively low, represents the double bonds located around the center of the bilayers. Since water has a higher electron density (24) Adachi, T.; Takahashi, H.; Ohki, K.; Hatta, I. Biophys. J. 1995, 68, 1850-1855.
than the sugar headgroups, the maximum on both sides represents the electron density of the interlamellar water. From the three candidates, we can estimate the relative locations of the nonpolar chains, the polar headgroups, and the interlamellar water region. By measuring the width of the interlamellar water region in Figure 4, the thickness of the interlamellar water was calculated as approximately 1.3 nm. The resolution of the electron density profile depends on the lattice parameter, d (4.44 nm), and the number, N, of the structure factors used in eq 1. In the present paper, the resolution is of the order of [1/(N - 1) - 1/N]2d, approximately 0.3 nm. We found the estimated thickness (1.3 ( 0.3 nm) to be compatible with the interlamellar thickness of the other layered structures.14,16,17 FT-IR Analysis. To investigate the hydrogen-bonding features of the water confined between the bilayer membranes, we acquired the IR spectrum for the interlamellar water by the direct difference method described below. As shown in Figure 5, drying the LNTs in air and a vacuum enabled us to sequentially remove the bulk water and in-tube water. The interlamellar water was removed by a gas flow of dry nitrogen. The drying process in air and vacuum was monitored by measuring the IR absorption at 3380 and 3250 cm-1 at regular intervals. The results are shown in Figure 6. At the beginning of the drying process in air, the absorbance at both 3380 and 3250 cm-1 underwent no remarkable change with time. This finding means that the evaporated water at the beginning was undetected by the attenuated total reflection infrared (ATR) configuration (Figure 5a, I). After 80 min, the bulk water located close to the surface of the ATR diamond began to evaporate (Figure 5a, II), and the corresponding IR absorption decreased rapidly. Over an additional period of 20 min, the IR absorption did not change any further, suggesting that the water outside the LNTs was removed spontaneously (Figure 5a, III). After keeping the samples in air for more 80 min, we dried the LNTs by a vacuum pump to remove the in-tube water. At the beginning, the absorption decreased rapidly and then slowly, and finally, it remained almost constant. Since the inner diameter of the LNT used in this research is approximately 50 nm, a drying process using a vacuum pump allows the water inside the LNT cylinders to be removed. On the other hand, the interlamellar water remained due to the confinement of the multilamellar structure. Decomposing the tubular structure with acetone followed by a drying process with a gas flow of dry nitrogen enabled us to remove the interlamellar water. The dry nitrogen gas flowed through the lipid molecules for 3 h until the IR spectra did not change. The IR spectral change of the LNTs during the drying process in air, vacuum, and nitrogen gas is shown in Figure 7. The three spectra were normalized with the CH2 stretching bands at 2918 to 2852 cm-1, since their intensities are least influenced by change in the structure and the amount of water. As shown in Figure 7, the OH stretching band of the sugar headgroups shows a sharp peak centered at 3432 cm-1. The NH stretching peak of the glycolipid appears at 3323 cm-1. The IR absorption from 3700 to 3000 cm-1 decreases due to the removal of the in-tube water and the interlamellar water. By subtracting the IR spectrum 3 from 2 (Figure 7), we acquired the spectrum for the interlamellar water. We compared the OH stretching band of the interlamellar water (solid line) with that of pure water in bulk (dotted line) (Figure 8). A decrease in IR absorption around 3000 and 3600 cm-1 resulted in a narrower bandwidth than that of bulk water. To explain the spectral characteristics,
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Figure 5. Schematic illustration of the drying processes in air and vacuum. (a) Drying process in air: (I) at the beginning of drying in air, the upper layer of the bulk water began to be removed; (II) drying in air for 80 min, the water which contributes to the IR signal began to evaporate; (III) drying in air for 180 min, the bulk water was removed thoroughly. (b) Drying process in a vacuum: the in-tube water was removed in this process.
Figure 6. Change in the IR absorbance at 3380 and 3250 cm-1 with the drying process.
Figure 7. ATR-FT-IR absorbance spectra for LNTs after (1) drying in air, (2) drying in a vacuum, and (3) drying in nitrogen gas. Figure 9. Schematic diagram of the structure of the LNT.
Figure 8. ATR-FT-IR spectra of interlamellar water (solid line) and bulk water (dotted line).
it is necessary to make assignments for the different energy regions of the OH stretching band. According to previous studies,6,7,11,17 the OH stretching band of bulk water is usually divided into three main components. The assignment for the three components is still controversial. There are mainly two kinds of assumptions.11,25 In the present paper, we used the assignment proposed by Brubach et
al. to explain the spectral characteristics of this system.11,17 Brubach et al. pointed out that three types of water species, including network water, intermediate water, and multimer water in different hydrogen-bonding environments, coexisted in the bulk water. The molecules of the network water are most likely connected tetrahedrally, forming extended water networks such as ice. They correspond to the low-energy component (at ∼3310 cm-1) in the OH stretching band. The molecules of the intermediate water establish, on average, three bonds with the neighboring water molecules, corresponding to the medium-energy component (at ∼3455 cm-1). The multimer water is defined as weakly hydrogen-bonded water aggregates, such as small water aggregates, corresponding to the highestenergy component centered at ∼3580 cm-1. According to the above assumption, in the present study, the OH stretching band of the interlamellar water showed (25) Sokolowska, A.; Kecki, Z. J. Raman Spectrosc. 1986, 17, 29.
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a decrease in the relative proportions of both low-energy (around 3000 cm-1) and high-energy (around 3600 cm-1) components, indicating that the relative proportions of both the strongly hydrogen-bonded network water and the weakly hydrogen-bonded multimer water decreased. A decrease in the relative proportion of the network water is ascribable to the hindrance of the long-range hydrogenbonding network by the confinement. The result agrees with those obtained for other similar systems, such as black soap film,12-14 multilamellar liposome,16 and the lamellar structure of AOT.17 However, the difference between the present system and the other systems is that a remarkable reduction in the high-energy side of about 3600 cm-1 was observed for the present system. This means that few weakly hydrogen-bonded water aggregates exist between the bilayer membranes. By comparing the multilamellar structure of the LNT with those of the other systems, we found that, although the water confined in each system is of similar thickness, the headgroups located at interfaces have different features. We assumed that the absence of weakly hydrogen-bonded water aggregates could be attributed to the effect of sugar headgroups on the structure of the interlamellar water confined in the present system. According to the previous studies,26,27 glucose has been proved to be one kind of nonionic kosmotrope, which can fit into a network structure of water clusters, with hydrogen bonding, by replacing a chair-form water hexamer in a cluster. It may stabilize the hydrogen-bonded network of interlamellar water. As a result, the weakly
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hydrogen-bonded water molecules decreased. On the other hand, as shown in Figure 9, unlike other nonionic headgroups,11,14 between which there is no strong interheadgroup interaction, the sugar groups form hydrogen bonds not only with the water molecules but also with the surrounding sugar groups in the same bilayer.28 Therefore, we assumed that there is not enough space between the neighboring headgroups to trap or accommodate small water aggregates with weak hydrogen bonds. Conclusions The thickness of the interlamellar water between the bilayer membranes in the glycolipid nanotubes was determined to be 1.3 ( 0.3 nm by X-ray diffraction. FT-IR spectroscopy showed that the confinement induced the decrease in the relative proportion of the network water as compared to the bulk water. Furthermore, weakly hydrogen-bonded water aggregates proved to be absent in the interlamellar space of the glycolipid nanotubes. The reason for the characteristic feature of our system was considered from the existence of sugar OH groups that may stabilize the hydrogen-bonded network of interlamellar water. Intralamellar hydrogen bonding between sugar groups decreases the possibility of the presence of small water aggregates. Acknowledgment. The work was supported by JSPS (Japan Society for the Promotion of Science). LA046906Q
(26) Batchelor, J. D.; Olteanu, A.; Tripathy, A.; Pielak, G. J. J. Am. Chem. Soc. 2004, 126, 1958-1961. (27) Oro, J. R. D. J. Biol. Phys. 2001, 27, 73-79
(28) Masuda, M.; Shimizu, T. Carbohydr. Res. 1997, 302, 139-147.
