Received 14 December 2012
A highly hydrated -cyclodextrin/1-undecanol inclusion complex: crystal structure and hydrogen-bond network from high-resolution neutron diffraction at 20 K
Delphine Gallois-Montbrun,a,b Geneviève Le Bas,a* Sax A. Mason,c Thierry Prangéd and Sylviane Lesieura*
aUMR CNRS 8612, LabEx LERMIT Universite Paris-Sud, 5 rue Jean-Baptiste Clément, F-92296 Chatenay-Malabry CEDEX, France,bLaboratoire des Glucides, UMR CNRS 6219, Université de Picardie Jules Verne, 33 rue St. Leu, F-80039 Amiens CEDEX, France,cInstitut Max von Laue-Paul Langevin, 6 rue Jules Horowitz, BP 156, F-38042 Grenoble CEDEX, France, and dLaboratoire de Cristallographie et RMN biologiques, UMR CNRS 8015 Université Paris Descartes, 4 avenue de l'Observatoire, F-75270 Paris CEDEX 06, France
The monoclinic C2 crystal structure of an -cyclodextrin/1-undecanol host-guest inclusion complex was solved using single-crystal neutron diffraction. Large high-quality crystals were specially produced by optimizing temperature-controlled growth conditions. The hydrate crystallizes in a channel-type structure formed by head-to-head dimer units of -cyclodextrin molecules stacked like coins in a roll. The alkyl chain of the guest lipid is entirely embedded inside the tubular cavity delimited by the -cyclodextrin dimer and adopts an all-trans planar zigzag conformation, while the alcohol polar head group is outside close to the -cyclodextrin primary hydroxyl groups. The cyclodextrin dimer forms columns, which adopt a quasi-square arrangement much less compact than the quasi-hexagonal close packing already observed in the less hydrated -cyclodextrin channel-type structures usually found with similar linear guests. The lack of compactness of this crystal form is related to the high number of interstitial water molecules. The replacement of 1-undecanol by 1-decanol does not modify the overall crystal structure of the hydrate as shown by additional X-ray diffraction investigations comparing the two host-guest assemblies. This is the first study that analyses the entire hydrogen-bonding network involved in the formation of a cyclodextrin dimer surrounded by its shell of water molecules.
Native cyclodextrins (CDs) are a family of macrocyclic oligosaccharides most commonly composed of six (-CD), seven (-CD) or eight (-CD) (-1,4)-linked D-glucopyranose units. Due to the hydrophobic character of their inner cavity, these molecules are known to form complexes with a wide variety of guest molecules, notably in aqueous medium, inclusion complexes which can crystallize in the solid state. Native CDs have a truncated conical structure with the primary hydroxyl groups on the narrow side and the secondary ones on the wide side (Fig. 1; Saenger et al., 1998), imparting intensely hydrophilic behaviour. The inclusion phenomena are the result of weak interactions involving both hydrophobic and hydrophilic parts of CDs, such as hydrogen bonds, electrostatic interactions and van der Waals forces rather than strong chemical bonding (Rekharsky & Inoue, 1998). Since the inclusion into the CD macrocycle is non-covalent, CDs have frequently been used as model compounds to study intermolecular interactions involved in molecular recognition processes which are of prime importance in biological structure and function (Lehn, 1990). In addition, the relevance of cyclodextrins and related host-guest inclusion compounds is well established as providing building blocks for the construction of nanoscale supramolecular systems (Chen & Liu, 2010; Chen & Jiang, 2011).
| || Figure 1 |
Schematic representation of the chemical structure of the -CD molecule. (a) Atom-numbering scheme of one glucose unit; (b) cyclic configuration; (c) truncated cone-shape side view.
Stereoselective discrimination, which arises from the ability of the guest molecule to fit inside the CD cavity, is considered to be the most important factor regulating the complexation reaction (Harata, 1998). In addition, in crystallized CD complexes, even though the molecular packing is mainly governed by the arrangement of hydrated CD molecules (because they are responsible for most of the intermolecular contacts), the resulting packing mode can also depend on the guest molecule (McMullan et al., 1973). Three main packing modes have been described: cages, layers and channels, each providing a suitable cavity in which the guest molecule can be accommodated. The channel packing mode is more suited to elongated guests such as linear polymers or alkyl chains. This type of packing can include molecules that are longer than the depth of the CD cavity and hence penetrate two or more CD rings (Harata, 2006) displaying either head-to-head or head-to-tail channels. Head-to-tail channels are formed from a one-dimensional repetition of CD monomers. In head-to-head channels the repetition unit is a CD dimer with the secondary hydroxyl sides facing each other. Moreover, it has been shown that, even within the channel packing mode, the molecular arrangement can be modulated by the geometry of the guest (Harata & Kawano, 2002). Part of the interest in studying CD complexes with linear molecules arises from the possibility of inducing the arrangement of the macrocycle molecules into larger arrays to achieve further organization into higher-order supramolecular systems that could serve as molecular devices such as molecular tubes in solution (Harada, 2001; Wenz et al., 2006) or could yield new porous materials in the solid state (Rusa et al., 2002).
Until now, information about the three-dimensional channel-type structures formed by hydrated -CD inclusion compounds has been provided almost exclusively by X-ray crystallography studies and mostly on powder samples. Furthermore, as obtaining single crystals of good quality is difficult, the number of solved crystal structures of this type is small and the structures are mostly restricted to quasi-hexagonal close-packed columns with low hydration levels (Sicard-Roselli et al., 2001; Noltemeyer & Saenger, 1980; Nicolis et al., 1994; Odagaki et al., 1990; Klingert & Rihs, 1991; Gu et al., 2010). However, in all cases it has been clearly shown that the crystal packing is stabilized by a hydrogen-bond network involving CD hydroxyls and water molecules distributed in the intermolecular space between the CD channels. These observations strongly suggest that the hydration conditions used during complex crystallization should greatly influence the packing mode. The verification of such a hypothesis requires the development of experimental procedures to vary the degree of hydration of the complex and the precise description of the H-atom positions that could not be found by X-ray diffraction. In this respect, neutron diffraction is the best tool to clarify water-mediated hydrogen-bonding interactions since H atoms can be accurately and unambiguously identified, even when disordered.
In a series of preliminary studies, we systematically examined the influence of temperature and hydration on the crystallization of -CD inclusion complexes with single-chain lipids having different polar head groups (Gallois-Montbrun et al., 2005). X-ray diffraction analysis showed that all crystals consist of head-to-head dimers which assemble in columns, but organized in different channel-type structures depending on the guest lipid and crystallization conditions. Four distinct channel-type packing modes were thereby identified, while some of the complexes studied displayed pseudo-polymorphism specifically linked to their degree of hydration.
The present work focuses on one of the newly found structures, a monoclinic C2 head-to-head channel structure, which is typical for highly hydrated host-guest inclusion compounds obtained with long-chain alcohols. While the channel-type assemblies of -CD/monoalkyl amphiphile complexes are mostly characterized by quasi-hexagonal compact packing in the plane perpendicular to the column axis, this monoclinic C2 form is an exception since it adopts a much less compact quasi-square arrangement, no doubt due to a higher number of entrapped water molecules. The role of hydration in the crystal compactness has indeed been noticed by X-ray powder diffraction analysis for analogous -CD inclusion complexes (Uyar et al., 2006) which showed a reversible solid-solid phase transition between tetragonal and pseudo-hexagonal channel crystal structures induced by desorption/(re)sorption of water.
This study is aimed at understanding more fully the water-mediated pseudo-polymorphism of -CD/lipid inclusion compounds by resolving the hydrogen-bond network of the most hydrated channel-type structure, monoclinic C2. For this purpose, temperature-controlled conditions were specially designed to prepare suitable single crystals of -CD/1-undecanol hydrate, chosen because it is possible to obtain as large crystals of several mm3. High-resolution neutron diffraction was then used to determine the complete structure of the complex and in particular to accurately locate the H atoms. The structures of pure hydrated -CD (Klar et al., 1980), -CD (Zabel et al., 1986; Betzel et al., 1984), -CD (Ding et al., 1991) and -CD (Imamura et al., 2001) as well as of -CD/ethanol (Steiner et al., 1990) and -CD/cyclopentanone (Le Bas & Mason, 1994) complexes have already been determined by neutron diffraction, but they result from the packing of monomeric CD molecules. The monoclinic C2 structure herein investigated is the first one solved by neutron diffraction which shows dimeric CD units and furthermore CD dimers in a channel structure. Diffraction data were measured at 20 K by using the D19 instrument at ILL, equipped with a new very large position-sensitive detector. The very low temperature was chosen for elucidation of the hydrogen-bond spatial distribution. The same crystal form was also characterized at 277 K on the W32 beamline of the LURE synchrotron source (Laboratoire pour l'Utilization du Rayonnement Electromagnétique, Orsay, France). We concentrate on the more accurate neutron results and we refer to the X-ray results when they are new or different. Complementary information obtained from additional X-ray diffraction analysis is introduced to give insight into the influence of the hydrocarbon chain length of the lipid guest by replacing 1-undecanol by 1-decanol to form monoclinic C2 crystals.
Precise volumes (Microman®, Gilson, Roissy en France, France) of 1-undecanol or 1-decanol (from Sigma-Aldrich, St Louis, MO, USA; 99% purity) were added to 10 ml of a previously filtered (through 0.20 µm Minisart® High Flow syringe filter, Sartorius Stedim France, Aubagne, France, and 363 K heated) aqueous solution of 0.05 M -cyclodextrin (-CD hydrate powder from Sigma-Aldrich, St Louis, MO, USA; purity > 98%, containing 9.9 to 10.6% water, measured by the provider via Karl-Fisher titration), to obtain final alcohol-to-cyclodextrin molar ratios of 1:2. The resulting mixture was stirred and kept at 363 K for 1 h before being divided into three different test tubes (3 ml aliquots) which were stoppered and immersed in hot water at 363 K filling a specially designed container made of three nested Dewar flasks all placed in a polystyrene box. Then the tubes and their contents were allowed to cool to room temperature at a slow average cooling rate of 4 K per day. Single crystals grew upon cooling over 2-3 weeks. In order to prevent decomposition, the crystals were stored in their mother liquor until X-ray and neutron diffraction analyses.
X-ray diffraction (XRD) structural data were obtained using the DCI Synchrotron source at LURE. Single crystals of 1-undecanol and 1-decanol complexes were mounted in glass capillaries of 1 mm diameter in the presence of their mother liquor and kept at 277 K during the measurements, which were performed on the W32 wiggler beamline at wavelength 0.97 Å. The detector was a Mar 345 image plate (Marresearch, Hamburg, Germany). Complete datasets were collected to 1.0 Å resolution. All data sets were auto-indexed, processed, scaled and merged using DENZO and SCALEPACK from the HKL package (HKL Research, Charlottesville, VA, USA). Crystallographic data and experimental details are given in Table 1 (first two columns).
For neutron diffraction, a well formed transparent single crystal of the 1-undecanol complex of volume about 6 mm3 (1.27 × 1.48 × 3.40 mm) was mounted to prevent dehydration inside a thin-walled quartz tube between two wads of moistened quartz wool, and the tube was attached with an O-ring seal to a purpose-designed Al base. The tube was then mounted on a Displex cryorefrigerator (Archer & Lehmann, 1986) on the ILL thermal-beam diffractometer D19 equipped with a new horizontally curved `banana-shaped' position-sensitive detector (Buffet et al., 2005). This detector is based on multi-wire gas-counter technology (5 atm 3He and 1 atm CF4) and incorporates an electrostatic lens to improve vertical resolution. The detector is mounted symmetrically about the equatorial plane, with a sample-to-detector distance of 76 cm, and subtends 30° vertically and 120° horizontally. The diffraction pattern is read out with 256 × 640 pixels per frame, with pixel spacing of 0.12° vertically and 0.19° horizontally. This new detector assures accurate data even with `small size' crystals ( 1 mm3) and relatively fast collection times, because many reflections can be measured quasi-simultaneously. The crystal was cooled slowly (2 K min-1) to 20 K while monitoring a strong reflection. No change in mosaic spread was observed. Bragg intensity data were measured first at a neutron wavelength of 1.4596 (1) Å from a Cu(220) monochromator in reflection, for 5.5 d; extra scans were then made for 3 d to extend the resolution, at a wavelength of 1.2414 (1) Å from a Ge(115) monochromator. These wavelengths were calibrated by refining the three-dimensional positions of 976 reflections at 1.4596 (1) Å, and 1974 at 1.2414 (1) Å, both from a deuterated potassium dihydrogen phosphate (DKDP) standard crystal. The accessible intensities up to 2 124°, were measured, to pre-set monitor counts, in a series of 80° scans, in steps of 0.07°, and typical counting times of 20.5 s per step at 1.46 Å, and 30 s per step at 1.24 Å. This rather long time-per-frame ensured reasonable counting statistics. Strong reflections were monitored regularly and showed no significant variation. A wide range of crystal orientations (different and positions) was used to cover a significant part of the reciprocal space. Because of its large horizontal opening angle, only one detector position was required. The unit-cell dimensions were calculated (ILL computer program RAFD19) at the end of the data collection from the three-dimensional centroids of 7800 strong reflections at 1.4596 (1) Å as a = 28.7452 (4), b = 27.0276 (4), c = 15.7252 (2) Å, = 95.827 (1)°. Bragg intensities were integrated in three-dimensions using a new version of the computer program RETREAT (Wilkinson et al., 1988) modified for the new detector geometry. For the 7796 strongest reflections the mean positional errors for the centroids were 0.03, 0.03 and 0.04°, in the scan, horizontal and vertical directions, respectively. At 1.24 Å for 1963 strong reflections, mean errors were 0.02, 0.03 and 0.04°. The intensities were corrected (ILL computer program ABSCAN) for attenuation by the inner cylindrical heat shields (minimum and maximum transmission coefficients 0.9048 and 0.9675 at 1.24 Å; 0.8907 and 0.9637 at 1.46 Å); at this stage, reflections for which the diffracted beam was attenuated by passing through the ends of the cylindrical cryorefrigerator cans were rejected. At 1.46 Å, 31 346 usable intensities were recorded, yielding 11 529 unique reflections, Rint = 0.1078, Rsigma = 0.0692. At 1.24 Å, 16 264 observations, yielding 11 457 unique reflections, Rint = 0.0826, Rsigma = 0.0865. For the combined data set, 47 610 usable hkls were recorded, yielding 13 268 unique reflections, Rint = 0.1211, Rsigma = 0.0912. Further crystallographic data and experimental details are given in Table 1 (third column).
For the 277 K X-ray diffraction data, the structure of the -CD/1-undecanol complex was solved by trial and error using a starting model constructed with SYBYL7.3 software (Tripos International, Saint Louis, MO, USA). This model used the atomic coordinates of the skeleton atoms constituting the -CD dimer from the -CD/n-butylisothiocyanate complex (Sicard-Roselli et al., 2001) set in the quasi-tetragonal primitive P1 cell of this -CD/1-undecanol complex: a = 15.838, b = 19.771, c = 19.785 Å, = 86.87, = 85.71, = 85.70°. By analogy with the packing arrangement in the tetragonal -CD/cadmium poly(iodide) complex (Noltemeyer & Saenger, 1980; a = b = 19.93, c = 30. 88 Å), two distinct dimers were set in the P1 unit cell. In the first steps using the program SHELXL (Sheldrick, 2008) the different molecules of this starting model were refined as rigid bodies using low-resolution data. Structure factors and Fourier maps were subsequently computed. Since the missing atoms could be recognized in the difference maps, all `correct' atomic positions of the initial skeleton were redetermined. At this stage all atoms of the -CD molecules and O atoms of some water molecules were located. When the position of the binary axis was clearly defined and as the space group C2 symmetry was obvious, further refinement was performed using the monoclinic C2 cell. Calculated positions of H atoms were then added to the CD molecules.
The -CD/1-decanol complex structure was solved by isomorphous molecular replacement of the -CD glucosidic skeleton atoms of the -CD/1-undecanol complex structure. The structure was then refined by alternating series of difference-Fourier syntheses with full-matrix least-squares refinement cycles using SHELXL.
In both structures, water and guest molecules exhibit high disorder. As a consequence, the electron density of the corresponding atoms appeared at a very low level in the difference maps: 13.5 O atoms of the water molecules distributed over 23 sites for the -CD/1-undecanol complex were located from the difference-Fourier electron density maps and 12.5 O atoms of the water molecules distributed over 21 sites for the -CD/1-decanol complex. The guest alcohol O atom, although disordered in both structures, was distinguishable, but the rest of the aliphatic chain was difficult to follow because of disorder. A molecular model of the guest was first built using SYBYL and was subsequently improved by fitting into the difference electron density corresponding to the guest, using TURBO-FRODO graphics software (Roussel & Cambillau, 1991). Three positions of the guest were found in the -CD/1-undecanol complex structure corresponding to opposite orientations within the dimer cavity: the alcohol O atom was seen at the two extremities of the dimer cylinder, whereas for the -CD/1-decanol complex structure, two guest positions were found. However, in both structures residual densities can be observed in the difference electron-density maps. Non-isotropic refinements were pursued for all the hydroxyl O atoms of host -CD molecules, O atoms of water and alcohol group O atoms of the guest. Considering that the number of reflections is relatively limited for these synchrotron data, only some cyclodextrin C and glycosidic O atoms were refined anisotropically. The position of the aliphatic chain of the guest molecule was refined with constraints applied on 1,2 and 1,3 distances. The refinement converged to R1 = 0.089 for the Fo 4(Fo) reflections for the -CD/1-decanol complex and to R1 = 0.079 for the Fo 4(Fo) reflections for the -CD/1-undecanol complex.
Initial neutron difference-Fourier maps were based on the atomic coordinates of -CD glucosidic skeleton atoms determined by X-ray diffraction structural analysis of the corresponding complex. The structure was then refined by alternating series of difference-Fourier syntheses with full-matrix least-squares refinement cycles using SHELXL. H atoms were located from nuclear density maps using the computer program WINCOOT (Lohkamp et al., 2005; Emsley & Cowtan, 2004). All the H atoms belonging to the -CD macrocycles, to the guest alcohol and to most of the water molecules could be located. The entire asymmetric unit contains two -CD molecules and a total of 21.75 water molecules distributed over 25 positions, of which 17 are fully occupied. One -CD primary hydroxyl C66BH2-O66BH group is twofold disordered as are the terminal H atoms of two other primary hydroxyls O65AH and O63BH. During refinements, the sums of their occupation factors were fixed at 1.0. It is also of note that all the secondary hydroxyl H atoms are twofold disordered and the two positions are well resolved in the density maps. For the guest molecule, although all the atomic positions were clearly observed in the density maps (occupancy factor of 0.8); some residual peaks of low density were also found, suggesting the existence of a minor disordered position (occupancy factor of 0.2). Water H atoms were restrained to chemically reasonable water geometry. Most C and O atoms were refined anisotropically. A total of 2382 atomic parameters were refined against all 13 268 data, the refinement converging to R1 = 0.1237 for the Fo 4(Fo) reflections (Table 1).
The solid-state structure of the hydrated form of the -CD/1-undecanol inclusion complex was fully determined from neutron diffraction. A complete description has been developed at atomic and supramolecular scales of this new channel-type architecture which crystallizes in the monoclinic C2 space group. Fractional atomic coordinates, displacement parameters, occupation factors, interatomic distances and structure factors have been deposited.1
In the following, atom labelling is consistent with that generally used for carbohydrates, i.e. C51 denotes the C5 atom of the glycoside residue 1 of the -CD molecule. An additional letter A or B is used to distinguish the two -CD molecules present in the asymmetric unit. Hydration water O atoms are numbered from OW1 to OW25.
The asymmetric unit consists of two molecules of -CD forming a head-to-head dimer through which one molecule of 1-undecanol threads and where 21.75 water molecules are distributed over 25 sites. Dimers are packed in columns running along the c axis to form a perfect channel-type structure with the dimer periodicity equal to the parameter c (15.73 Å). The guest alkyl chain is sequestered inside the cavity formed by two CD molecules along the column and fits perfectly with the dimer height. Fig. 2 shows the projection of the unit cell on the ab plane. Four dimers of -CD are arranged in the monoclinic C2 unit cell and as Z = 4 there are 87 water molecules distributed over 100 sites. In the ab plane the C2 cell forms a quasi-square with almost equivalent orthogonal parameters a = 28.75 and b = 27.03 Å, which correspond to the width of two cyclodextrin rings arranged side by side. Two neighbouring dimers along the a axis are related by a twofold axis. Along the b axis, two contiguous dimers are related by a helicoidal 21 axis. This quasi-square arrangement formed by four adjacent columns leads to a larger intermolecular volume between -CD molecule stacks compared with the almost hexagonally close-packed stacks found in most known columnar crystal structures of -CD dimers. Indeed, in these latter cases one hydrated cyclodextrin dimer typically crystallizes with eight or nine water molecules (Harata & Kawano, 2002; Sicard-Roselli et al., 2001; Noltemeyer & Saenger, 1980; Klingert & Rihs, 1991), while, in our case, the C2 cell allows the inclusion of 25 water molecules per hydrated dimer. Looking at the projection on the ab plane (Fig. 2), the packing resembles the tetragonal P42212 X-ray crystal structure of the (-CD)2·Cd0.5·I5·27H2O inclusion complex with unit-cell parameters a = b = 19.93 Å (Noltemeyer & Saenger, 1980). The tetragonal two-dimensional (a, b) cell of the latter structure is close to the primitive P1 cell (a + b)/2, (b - a)/2, associated with the C2 centred cell of the -CD/1-undecanol inclusion complex crystal structure) which has two edges of 19.78 Å and an angle = 86.9°.
| || Figure 2 |
Projection of the unit cell on the ab plane. Hydrogen bonds are represented by dashed lines, water molecules are blue. Along a two contiguous dimers are related by a twofold axis; along b two contiguous dimers are related by a helicoidal 21 axis.
Examining the structure projected on the ac and bc planes shows that the -CD packing in the monoclinic C2 space group is completely different from that in the tetragonal P42212 crystal system. There is one dimer in the asymmetric unit instead of half a dimer, and, in projection on the bc plane (Fig. 3), we observe that two adjacent columns of -CD dimers are offset from each other so that the columns are arranged in staggered rows. For the projection onto the perpendicular ac plane viewed along the b axis, there is a huge shift between each pair of dimer columns, of the magnitude of nearly the height of one cyclodextrin molecule (Fig. 4). Interestingly, within the same crystal structure different types of molecular interactions occur and lead to distinct and more or less substantial shifts between columns. These special features have also been observed in crystals of the -CD/decanoic acid complex, despite the limited resolution of the X-ray diffraction data (Rodríquez-Llamazares et al., 2007). This latter complex crystallizes in the triclinic space group P1 in which -CD columns also adopt quasi-square packing, but with even lower crystal symmetry since four different -CD dimers constitute the asymmetric unit. In our case, the high-resolution neutron diffraction analysis of the crystallized -CD/1-undecanol complex enabled us to describe accurately, in addition to the molecular geometry, the hydrogen-bond network in the structure which generates the peculiar channel-type organization.
| || Figure 3 |
Packing mode of the -CD dimers in projection on the bc plane. Two adjacent dimers along the b axis are generated by a parallel helicoidal 21 axis. Hydrogen bonds are represented by dashed lines. Two direct hydrogen bonds between primary hydroxyls are shown (O61A-HO64B, top left and bottom right, and O64A-HO61B, bottom left and top right), as well as one direct hydrogen bond between secondary hydroxyls (O21A-HO24A).
| || Figure 4 |
Packing mode of the -CD dimers in projection on the ac plane. Two contiguous dimers along the a axis are generated by a perpendicular twofold axis. Hydrogen bonds are represented by dashed lines. On the left of the figure, where twofold related dimers are at the same level, one direct hydrogen bond is observed between primary hydroxyls (O65A-HO63B). On the right of the figure, where a shift of one CD molecule occurs between twofold-related dimers, all the hydrogen bonds are mediated by water molecules.
In the head-to-head dimer the -CD molecules are crystallographically independent. The glucose units have the usual 4C1 conformation and both macrocycles form an almost regular hexagon (Table 2). We compare first the overall geometry of this crystalline -CD dimer with that of other known -CD dimeric complexes which have been studied by X-ray diffraction. In this structure the dimer exhibits an almost ideal cylindrical shape (Fig. 5) close to what was found in the X-ray diffraction crystal structures of the (-CD)2·Cd0.5·I5·27H2O (Noltemeyer & Saenger, 1980) and the -CD/decanoic acid complexes (Rodríquez-Llamazares et al., 2007), which both also exhibit the quasi-square arrangement. By contrast, in crystal structures of less hydrated -CD dimers with quasi-hexagonal close packing, all determined by X-ray diffraction, the two -CD molecules constituting each dimer unit are shifted laterally with respect to each other (Harata & Kawano, 2002; Noltemeyer & Saenger, 1980). For the -CD/1-undecanol complex discussed here, the regular shape of the dimer rather resembles that observed for -CD dimers in a number of X-ray crystal structures (Mentzafos et al., 1991). Furthermore, the distance O2O3 between two adjacent glucose residues in the same macrocycle is in the range 2.75 (2)-2.83 (2) Å (Table 3), with an average of 2.79 Å. This is very similar to the distance of 2.78 Å found in -CD dimers (Mentzafos et al., 1991). The situation is quite different in the crystal structures of -CD dimers with quasi-hexagonally packed stacks as in the triclinic (-CD)2·LiI5·8H2O complex (Noltemeyer & Saenger, 1980) or the monoclinic (-CD)2·C6H8N2O8·9H2O one (Harata & Kawano, 2002) characterized by O2O3 distances between 2.78-3.30 and 2.77-3.54 Å, respectively.
| || Figure 5 |
-CD dimer. Hydrogen bonds are represented by green dashed lines. Six intermolecular (intradimer) hydrogen bonds alternately involving all O3-H hydroxyls are observed. In each CD moiety, six intramolecular hydrogen bonds alternately involving the O3-H and the O2-H secondary hydroxyl are observed. All H atoms of the secondary hydroxyls have two positions (s.o.f. = 0.5).
One of the main results of this study is the determination of the H-atom positions including detailed analysis of all the inter- and intra-dimer hydrogen bonds, which is essential to understanding the conformation of the dimer units. Below, a step-by-step description of hydrogen bonds found inside each cyclodextrin molecule and between two cyclodextrin molecules forming the same dimer is first presented before addressing the bonding between dimers.
All the secondary hydroxyl groups belonging to a given -CD molecule are linked by hydrogen bonds to form a large crown. This crown stabilizes each macrocycle, as is seen by neutron diffraction in pure hydrated -CD crystals (Zabel et al., 1986; Betzel et al., 1984), while it remains incomplete in pure hydrated -CD crystals (Klar et al., 1980). With respect to the structure of the -CD/1-undecanol hydrate, all H atoms of the 24 secondary hydroxyl groups of the two -CD molecules of the asymmetric unit were located and all are shown to be twofold disordered. This implies that the H atom of each of the two secondary hydroxyl groups occupies two positions, with s.o.f. = 0.5, and this atom is alternately involved in two different hydrogen bonds. Then, in all intramolecular hydrogen bonds involving interglucose O2O3 atoms, O3-H and O2-H become alternately donor and acceptor (Table 3, Fig. 5).
This finding has already been described in the hydrated -CD monomer structure as a dynamic phenomenon for which the authors introduced the term `flip-flop' hydrogen bonds (Zabel et al., 1986). In our work, the s.o.f. value of the secondary hydroxyl H atoms suggests a possible dynamic phenomenon in the crystal state at higher temperature (i.e. room temperature). However, it is probable that the experimental cooling rate was sufficiently slow (2 K min-1) to allow ordering of dynamically disordered H atoms into one or other static position. Additional thermodynamic studies and X-ray diffuse scattering analyses may provide a definitive answer. Note that the alternative position of the O2-H hydrogen atom points towards water molecule sites or is involved in direct intramolecular hydrogen bonds, whereas the alternative position of the O3-H hydrogen atom is hydrogen bonded to a hydroxyl O3 oxygen atom of the second -CD molecule of the dimer and thus stabilizes the dimeric assembly.
A number of (intermolecular) hydrogen bonds stabilize the -CD dimer; it is noteworthy that all of them are observed uniquely between the O3 secondary hydroxyl groups (Table 4). Once again, this is analogous to what is observed in -CD dimers. This feature has also been described in the X-ray diffraction structure of the dimeric -CD/1,12-dodecanoic acid complex (Makedonopoulou & Mavridis, 2000), but without precise hydrogen positions and has been subject to debate. The interactions connecting -CD macrocycles inside the dimer unit are shown in Fig. 5. It is most likely that O3-H is a donor in an interdimer hydrogen bond stabilizing the dimer when the secondary hydroxyl group O2-H is a donor in an intramolecular (interglucose) hydrogen bond. On the other hand, O3-H is a donor in an intramolecular (interglucose) hydrogen bond stabilizing the macrocycle, when the hydroxyl group O2-H acts as a donor in an `external' hydrogen bond. We also emphasize that all the secondary hydroxyl O3-H atoms are donors only in intramolecular or interdimer hydrogen bonds that hold the dimer together, whereas the secondary hydroxyl O2-Hs are alternatively donors in an intramolecular hydrogen bond and in a hydrogen bond with a water molecule or with an -CD dimer of an adjacent column. For those `external' hydrogen bonds which will be described below, the position of the hydrogen is clearly `outwards' away from the -CD macrocycle.
The column formed by the dimers stacked along the c axis is stabilized by two direct hydrogen bonds between primary hydroxyl groups. Thus, O66A-H acts as a donor in a hydrogen bond formed with the primary hydroxyl O62B-H in the immediately adjacent dimer unit in the same column. Regarding O66B-H, it is bound as a donor to the primary hydroxyl group O62A-H which belongs to the next dimer unit along the c axis (Table 5). Fig. 6 illustrates the packing mode of the -CD dimers in the channel type structure along the c axis. It is interesting that the four primary hydroxyl groups involved in direct intracolumn (interdimer) hydrogen bonds point inwards, whereas all other primary hydroxyl groups point outwards and are clearly involved in hydrogen bonds with water molecules or with primary hydroxyl groups of cyclodextrins forming adjacent columns. The case of O66B-H is special. This primary hydroxyl is twofold disordered and presents an alternative position oriented away from the cavity. For this other conformation the hydroxyl H atom position was not found in the neutron difference maps. Among the 12 primary hydroxyls of the -CD dimer unit, only this primary hydroxyl and associated C66B atom are disordered over two positions. In addition to these direct bonds, two interdimer water-mediated hydrogen bonds also stabilize the column. The primary hydroxyl groups O65A-H and O62B-H which belong to two dimer units placed on top of each other are bound via the water molecule OW5 as well as O63A-H and O65B-H which are bound via OW15 (Tables 6 and 7).
| || Figure 6 |
Column of -CD dimers in projection on the ac plane viewed along the b axis. Hydrogen bonds are represented by dashed lines. Two direct hydrogen bonds are shown between the two dimers involving four primary hydroxyls which point inwards (O66A-H O62B, left, O62A-HO66B, right). The hydrogen bond observed between the guest alcohol (in yellow) and the -CD dimer located just below is also shown. Note a slight shift along the a axis between the two dimer units.
Several direct bonds involving -CD hydroxyls take part in the stabilization of the channel packing, as shown in the perpendicular projections of the structure (Figs. 3 and 4). In the bc plane projection (Fig. 3), two types of direct hydrogen bonds connect the adjacent dimers related by a helicoidal 21 axis: on one hand, hydrogen bonds involving primary hydroxyl groups and on the other hand hydrogen bonds involving secondary hydroxyl groups. The primary hydroxyl group O61A-H is thus directly bound to O64B-H which belongs to the adjacent dimer units, and also along the b axis the primary hydroxyl group O64A-H is involved in a direct hydrogen bond with O61B-H. Although this is not shown in Fig. 3 because only one period along c is represented, the primary hydroxyl group O64B-H is also directly and diagonally linked to the primary hydroxyl group O61B-H of the adjacent column dimer units located below (Table 5). Regarding the direct hydrogen bonds involving secondary hydroxyl groups shown in the projection on the bc plane, we note that the H atom of the O21A-H hydroxyl group in its alternative `outward' position (s.o.f. = 0.5) is bonded to the O24A-H secondary hydroxyl group of the adjacent dimer along the b axis. Alternately, the O24A-H hydrogen atom, when it points `outwards' (s.o.f. = 0.5), is bound to the adjacent dimer unit hydroxyl group, O21A-H. This interdimer hydrogen bond is always formed, and both O21A-H and O24A-H of the two adjacent units along the b axis are either a donor or an acceptor. These direct hydrogen bonds induce the shift between two adjacent dimers observed in the bc plane projection.
In the ac plane projection (Fig. 4) two distinct kinds of interaction between different adjacent columns can be observed, as was already highlighted. First, when twofold-related dimers are at the same level along the c axis, two direct hydrogen bonds are observed. The first of these interactions occurs between the primary hydroxyl groups O65A-H and O63B-H, which belong to two adjacent related dimers (Table 5). Those two primary hydroxyl groups act alternately as a donor or as an acceptor and their H atoms are both twofold disordered. The second one, involving secondary hydroxyl groups, is observed notably between the two symmetrically related O23B-H hydroxyl groups which belong to two related adjacent dimer units along the a axis. This hydrogen bond involves the alternative `outward' position of the H atom of this secondary hydroxyl group. Then, in the same projection when a shift occurs between twofold-related dimers, no more direct hydrogen bonds are observed between hydroxyl groups of the two related dimers, but only water-molecule-mediated hydrogen bonds. These two adjacent dimers along the a axis are connected via one water molecule at least. Furthermore, it is of interest to note that, due to the substantial shift of almost the height of one -CD molecule (Fig. 4), a primary hydroxyl group is bound via a water molecule to a secondary hydroxyl group of the adjacent dimer. Indeed, O62A-H acts as a donor in a hydrogen bond with OW10, which is itself bound as a donor to the secondary hydroxyl group O36-H which belongs to the adjacent dimer unit along the a axis (Tables 6 and 7).
There are 21.75 molecules in the asymmetric unit distributed over 25 sites, of which 17 are fully occupied. Among these 17 fully occupied sites, six water O atoms are twofold disordered. The remaining eight water molecule sites are partially occupied. Nearly all H-atom positions were found in the neutron density maps. For some water molecules the H atoms can occupy two different positions, whereas the O-atom position does not change significantly. Thus, in some cases, three or four partially occupied positions of H atoms were found per water molecule where the water molecules can rotate around the position of the O atom. Occasionally, some H atoms as well as some O atoms were not taken into account when the corresponding peaks in the neutron density maps were badly resolved or had low levels close to the background level. Consequently, some water molecules are seen with only one H atom or without any H atom, or the whole water molecule is missing. These unresolved water positions correspond to small voids accessible to the solvent of volume about 57 Å3, i.e. close to the volume of two water molecules, suggesting that there is room for two more sites in addition to the 25 in the asymmetric unit (Fig. 2). These positions should correspond to water molecules close to the symmetry axis and belonging to the second hydration sphere. All the resolved H atoms found contribute to specific hydrogen bonds maintaining the exceptionally hydrated structure observed here for a complex of -CD.
Tables 6 and 7 present all the hydrogen bonds involving water molecules acting as donors. We can distinguish the water molecules which are hydrogen-bound to the -CD secondary hydroxyls from the water molecules bound to the -CD primary hydroxyls. It is worth emphasizing that, while in less hydrated -CD dimer columnar crystal structures almost all the water molecules are linked to primary hydroxyls (Sicard-Roselli et al., 2001; Noltemeyer & Saenger, 1980), in the present highly hydrated structure many water molecules are also located around secondary hydroxyl groups. Indeed, in this monoclinic C2 structure, at least one of the two secondary hydroxyl groups belonging to each -CD glucose unit binds a water molecule.
In the hydrogen-bonded water network involving secondary hydroxyls, eight water molecules OW1, OW3, OW4, OW10 (and its alternative position O10W), OW11 (and its alternative position O11W), OW14, OW20 and OW25 (and its alternative position O25W) are directly bound to at least one secondary hydroxyl group, among which one molecule, OW10, also interacts with a primary hydroxyl group. Except for the alternative position of OW10, these eight water molecules are directly bound to secondary O2-H hydroxyl groups of eight different glucose units, among the 12 forming one dimer. In five of these hydrogen bonds (those involving OW1, OW3, OW4, OW14 and OW20) the O2-H hydroxyl can be alternatively a donor and an acceptor. In the other three, O2-H is the only donor. Four of the eight water molecules, namely OW4, OW10, O11W and OW25, are also bound to four secondary O3-H hydroxyls and in each case the hydroxyl acts as an acceptor. Note that these four O3-H hydroxyls belong to the remaining four glucose units among the 12 units whose O2-H hydroxyls are not involved in hydrogen bonds with water molecules.
At the level of the primary hydroxyl groups, nine water molecules OW5, OW6, OW8, OW10, OW13, OW15, OW16, OW18 and OW19 (and its alternative position O19W) are directly bound to at least one primary hydroxyl group. In some of those hydrogen bonds, the primary hydroxyl group can be alternately a donor or an acceptor because its H atom is twofold disordered. All but three of the primary hydroxyl groups are bound to at least one water molecule, except O66A-H, O66B-H with its alternative position OB66-H, and O64B-H, which form only direct interdimer hydrogen bonds. For seven primary hydroxyls: O62A-H, O63A-H, O65A-H, O61B-H, O62B-H, O63B-H and O65B-H (Table 6), the H atom, which points away with respect to the cavity, is bound to a water molecule. Four among these seven hydroxyl groups, O63A-H, O62B-H, O63B-H and O65B-H, acting as acceptors, are also bound to a second water molecule (Table 7). The last two primary hydroxyl groups O61A-H and O64A-H are acceptors in hydrogen bonds formed with water molecules, since they are donors in interdimer hydrogen bonds.
Three water molecules OW9, OW16 and OW22 form a hydrogen bond with the O5 atom of the glucose unit of the -CD macrocycle and with other water molecules (Table 7). Seven water molecules OW2, OW7, OW12, OW17, OW21, OW23 (with its alternative position O23W) and OW24 interact only with other water molecules. These are all located in the vicinity of the primary hydroxyl groups, except OW2 which is located not far from the secondary hydroxyl groups. OW2 acts as a link between OW14 bound to a secondary hydroxyl, and OW6 bound to a primary hydroxyl.
In such a highly hydrated crystal, the overall analysis of water molecule positions and related hydrogen-bond networks provides evidence of an exceptional three-dimensional supramolecular structure in which independent tubular water channels are regularly embedded between cyclodextrin dimer columns. The water molecules form two distinct channels in the asymmetric unit outside the CD columns. These infinite water channels are parallel to the CD columns and show a co-axial core-shell organization. The shell of the water channels directly interacts with the neighbouring host columns and constitutes the water coordination sphere of the cyclodextrins. The core corresponds to the second sphere of hydration and is more weakly bound, as shown by the two X-ray diffraction structures described below. It is reasonable to assume that the water molecules in the channel core can easily leave the crystal lattice during the dehydration process. This phenomenon of porosity was also observed in the structures formed by other clathrates (Fucke et al., 2011).
The neutron density maps clearly revealed the atomic positions of the guest molecule including H atoms. The guest molecule spans the entire length of the -CD dimer and its terminal hydroxyl group is located at the primary hydroxyl end. The hydrocarbon chain of the 1-undecanol molecule exhibits an all-trans planar zigzag conformation and is totally embedded inside the tubular cavity formed by the host -CD dimer (Fig. 7). The guest aliphatic chain is in tight van der Waals contact with the inside hydrophobic wall of the -CD dimer cavity. The closest distances are given in Table 8 with cut-off distances smaller than 2.60 and 3.00 Å for HH and HO contacts, respectively. Thus, four short C-HO contacts involve the methylene groups of the guest chain and the cyclodextrin O4 atoms. Among the 25 guest-host HH short contacts (< 2.60 Å), ten involve -CD C3-H groups and 15 involve -CD C5-H groups. The distances between the C5-H groups and the H atoms of the guest aliphatic chain are the shortest. One direct hydrogen bond is clearly observed between a -CD primary hydroxyl and the 1-undecanol hydroxyl, even though the latter is disordered. More precisely, it is hydrogen bonded to the cyclodextrin primary hydroxyl group O66B which then acts as an acceptor (Table 8) and points `inwards' (Fig. 7). Then, within a column the guest molecule binds to the cyclodextrin forming the dimer next to the one in which it is included. The -CD primary hydroxyl group is also disordered and has two distinct conformations inward to and outward from the cavity as described previously. Note that this `inwards'-oriented hydroxyl group is also bound as a donor to the primary hydroxyl group O62A-H which belongs to the next dimer unit along the c axis.
| || Figure 7 |
A view of the nuclear density omit-map that shows the 1-undecanol molecule embedded inside the tubular cavity formed by the -CD dimer. The map was calculated by removing the contribution of the guest molecule. The negative peaks of the H atoms are represented in red, the positive peaks of the C and O atoms are in blue.
X-ray diffraction was independently used to solve the crystal structures of the -CD/1-undecanol complex and the -CD/1-decanol complex differing in guest chain length only by one C atom (Table 1). The crystal structures of these two different inclusion complexes are quasi-isomorphous. The arrangement of the -CD columns is almost identical, as well as the positions of the water molecules. Although the two guest molecules are very similar chemically and geometrically, their crystal structures deserve to be compared in detail, especially the number and positions of the water molecules. Furthermore, neutron and X-ray diffraction structures found for the -CD/1-undecanol complex merit comparison since two different single crystals were analyzed at two different temperatures (20 and 277 K, respectively). As reported below, the two crystal structures differ in small but significant details, the major differences arising from the observed disorder which was amplified by the change in temperature. The number and positions of the water molecules vary slightly. Also owing to the crystal-specific disorder for this kind of inclusion compound, the conformations of cyclodextrin primary hydroxyls as well as the position of the guest molecule, even within the same complex, also showed some differences.
Comparison of the three data sets can be made on the basis of three distinct crystal structures. In the three structures, the number of water molecule sites is almost the same. Over the 25 sites observed at 20 K in the neutron diffraction structure of the -CD/1-undecanol hydrate, 23 are present in the 277 K X-ray diffraction structure of the same complex and 21 in that of -CD/1-decanol hydrate. However, the overall apparent occupancy of water molecules in the asymmetric units of the crystals is different and amounts to 21.75, 13.5 and 12.5, respectively. This underlines greater disorder at 277 K. In fact, all O atoms of the water molecules are located at similar positions in the asymmetric units of the three structures. The two water molecules OW7 and OW24 observed at 20 K by neutron diffraction in the -CD/1-undecanol hydrate structure were not located by X-ray diffraction analysis at 277 K. Regarding the -CD/1-decanol hydrate, the four water molecules OW7, OW9, OW21 and OW24 are not found in the 277 K structure. These water molecules are bound only to other water molecules in the crystal structure and presumably are more subject to thermal motion as a result of weaker binding. Besides, taking into account that at the same temperature the cell volumes found for the two structures obtained with 1-undecanol and 1-decanol as a respective guest are almost identical (Table 1), it is very likely that the hydration is similar in both inclusion compound crystals. Accordingly, while in the neutron diffraction structure at 20 K two water molecules have been described as too diffuse to model (corresponding approximately to the calculated volume of voids accessible to the solvent), in the two X-ray diffraction structures at 277 K the apparent void volumes increase due to the additional unseen water molecules, and then due to entropy gain of water rather than actual dehydration.
In the electron density maps the peaks corresponding to the atomic positions of water molecules remain much less well resolved than those of the neutron diffraction density maps. The occupation factor of each water site in the -CD/1-undecanol complex structure is about 1.6 times higher at 20 K than at 277 K. For both 277 K complex structures (-CD/1-undecanol and -CD/1-decanol), the water molecules are much more disordered than in the 20 K structure. Moreover, many water molecules are twofold disordered at 277 K while these molecules are ordered at 20 K with the O atom occupying a single position. These differences are due only in part to temperature because the X-ray and neutron diffraction analyses were performed on different single crystals and using different experimental setups.
In the three structures, the coordinates of the C and O atoms of -CDs are almost identical, except those of the disordered primary hydroxyl groups O62A, O63A, O66A, O62B and O66B. These five O atoms are disordered at 277 K in the X-ray diffraction crystal structures of the two alcohol complexes while only O66B is disordered at 20 K in the neutron diffraction structure. At 20 K, the conformation of O62A, O66A and O62B is exclusively `inwards' to the cavity, while at 277 K these three primary hydroxyl O atoms present an alternative position which points `outwards' from the cavity. By contrast, though the O63A hydroxyl presents, in the two crystal structures at 277 K, both inwards and outwards positions, only the outward position (pointing towards the OW6 water molecule) is observed in the crystal structure at 20 K.
With respect to the guest molecules, just as for the water molecules, the atomic positions at 277 K are subject to disorder compared with the 20 K neutron diffraction structure. As a result, the refined apparent occupancy factors are lower in the 277 K X-ray diffraction structures than in the 20 K neutron diffraction one. Owing to the high disorder, the guest geometry in the 277 K crystal structures could not be further investigated.
The high intensity of the ILL neutron source, combined with accurate high-resolution data from the very large D19 position-sensitive detector and analysis at very low temperature, made it possible to accurately determine a novel crystal structure with channel-type organization formed by a lipid-based -CD inclusion complex. Furthermore, despite the poor quality crystals of this class of compounds, mainly because of disorder, the H-atom positions and hydrogen-bonding network are fully described for the first time in a dimer of -CD molecules, as were the interactions between the host, guest and water molecules.
The monoclinic C2 crystals are characterized by an exceptionally hydrated quasi-square column packing of head-to-head dimers. The guest lipid is almost `swallowed' by the two bridged macrocycles; only its hydroxyl group extends outside to bind the next dimer. Each -CD macrocycle has a rigid geometry because of tight intramolecular hydrogen bonds between O2 and O3 secondary hydroxyl groups so that the whole dimer adopts a cylindrical shape reminiscent of the typical -CD dimer conformation. The intermolecular association of the two -CD molecules which constitute the dimer unit only occurs through hydrogen bonding of the O3 secondary hydroxyl groups.
The stacking of the dimers along the c axis generates an almost perfect channel with however a very slight distortion due to a small shift along the a axis between two contiguous dimers. This channel is stabilized by two direct hydrogen bonds between four primary hydroxyl groups (adopting a special `inwards' conformation in a very specific way for three of them) and two other hydrogen bonds, each mediated by one water molecule. The long guest molecule is then completely isolated from the polar water environment inside the -CD dimer channel. It spans the entire height of two stacked -CDs and adopts a perfect all-trans planar zigzag conformation. Its alcohol hydroxyl group emerges from the -CD primary hydroxyls and is directly hydrogen bonded to a primary hydroxyl group of the cyclodextrin molecule belonging to the contiguous -CD dimer within the same column. Interestingly, the change in hydrocarbon chain length of the guest by one C atom does not modify the crystal lattice providing hydration conditions applied during the inclusion complex formation are identical.
Of great interest is the high degree of hydration of the crystal structure with the hydrogen-bond network involving 25 water molecules per dimer unit occupying the space between the columns. The presence of a high number of interstitial water molecules explains the lack of compactness of this quasi-square columnar packing and the significant expansion of the crystal structure, compared with the quasi-hexagonal close packing characteristic of other less hydrated -CD channel-type crystal structures usually formed with similar linear guest molecules (Sicard-Roselli et al., 2001; Noltemeyer & Saenger, 1980). In this respect, by varying the hydration level of the -CD/1-undecanol complex, it would be expected that the crystal structure evolves to another crystal form involving fewer water molecules, as already shown in the case of the -CD/1-dodecanol complex (Gallois-Montbrun et al., 2005).
The almost perfectly cylindrical shape of the -CD dimer found in the monoclinic C2 crystal structure is due to the high hydration level that induces the macrocycles to align with each other, whereas, in less hydrated crystal forms as observed with the 1-dodecanol guest, one of the two -CD molecules of the dimer is offset from the other. We note that one or more water molecules are attached to at least one of the two secondary hydroxyls of each -CD glucose unit in the dimer and almost all primary hydroxyl groups are bound to at least one water molecule (nine out of 12). This -CD dimer surrounded by its shell of water molecules and including a linear guest, which, in this case, adopts the exact length of the dimeric cavity, constitutes a tightly hydrogen-bonded chemical entity, even within the columns since two contiguous dimers slightly shift from each other and share relatively few hydrogen bonds. Therefore, from a thermodynamic point of view, we believe that such crystalline entities can serve as an ideal basis to model any pre-existing dimer of -CD molecules, when driven by inclusion complex formation in aqueous solution, and that the dimers behave as single building blocks so they can be considered as effective precursors of the crystallized channel-type assemblies.
This work was supported by a PhD studentship of the Conseil Régional de Picardie (Nord-Pas-de-Calais, France) and the Centre National de la Recherche Scientifique (CNRS, France). The authors especially thank Professor Florence Djedaïni-Pilard (Laboratoire des Glucides, UMR CNRS 6219, Université de Picardie Jules Verne, France) for her valuable assistance.
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