Communication pubs.acs.org/crystal
Hydrophobic Tetramer Structures of Cholic Acid Xudong Wang,*,† Chenxi Li,† Yueqin Duan,‡ and Yan Lu*,‡ †
Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China ‡ School of Materials Science & Engineering, Tianjin University of Technology, Tianjin 300384, China S Supporting Information *
ABSTRACT: Cholic acid (CA) is a primary bile acid which possesses facial amphiphilicity and self-assembly ability. CA is slightly soluble in water, which makes it impossible to form the self-assembled structures spontaneously in aqueous solutions. To increase the water solubility, a cholic acid ester (CA-4EG) with a hydrophilic tail (tetraethyleneglycol, 4EG) was synthesized, which forms self-assembled structures in aqueous solutions. The self-assembled crystals of CA are obtained by acidic hydrolyzing the ester bond of CA-4EG. The rhombic hydrophobic tetramer structure units and fiberlayer structures are found in the self-assembled crystals of CA.
1. INTRODUCTION Bile acids are facial amphiphiles which possess a steroid skeleton with a concave hydrophilic face with one to three hydroxyl groups and a convex hydrophobic face with methyl groups and a side chain with a carboxyl group. The solubility of bile acids is low in water (slightly soluble to hardly soluble), which makes them impossible to form the self-assembled structures or crystals spontaneously in aqueous solution.1 The salts of bile acids (bile salts) self-assemble in aqueous solution. In this process, the hydrophobic interaction between the hydrophobic faces of the facial amphiphiles is the main driving force, and the H-bonds between the OH groups also play an important role in stabilizing the self-assembled structures.2 At present, several self-assembled models (e.g., dimers, disclike micelles, etc.) have been proposed, but the actual structures are still not known due to the limits of the experimental techniques used to study the micelle structures.2−7 Single-crystal diffraction provides the most accurate structure information. Many single crystals of bile salts were prepared, such as sodium cholate,8,9 calcium cholate,10 cesium cholate,11 sodium taurodeoxycholate,12 sodium deoxycholate,13 rubidium deoxycholate, 14 sodium glycodeoxycholate,15 etc., yet the hydrophobic selfassembled structure units are neither found in these crystals nor in the crystals of bile acids from organic solvents.1 To study the unique facial amphiphilicity of bile acids, we select cholic acid (CA), one of the primary bile acids as the representative. CA is slightly soluble in water,16 which makes it impossible to form the self-assembled structures spontaneously in aqueous solution.1 A cholic acid ester (CA-4EG) with a hydrophilic tail (tetraethyleneglycol, 4EG) was synthesized to increase the water solubility.17 CA-4EG forms hydrophobic aggregates above the critical aggregation concentration (0.3 g/ © 2013 American Chemical Society
L). The crystals of CA are obtained by acidic hydrolyzing the aggregates of CA-4EG. The rhombic hydrophobic tetramer units of CA are found in the crystal structure.
2. EXPERIMENTAL SECTION Crystal Preparation. CA-4EG (0.1 g) was dissolved in MeOH (0.5 mL), followed by the addition of water (49.5 mL), then 1 M hydrochloric acid (50 mL) was added. The transparent solutions were sealed. Finally, the crystals of CA were prepared in the acidic aqueous solutions.18 Procedure for Crystal Analysis. Native data sets were collected at the BL17U beamline of the Shanghai Synchrotron Radiation Facility (SSRF) with a MAR 225 CCD detector system. One crystal prism was trapped with a loop and quickly put on the stage at the liquid nitrogen atmosphere (100 K). The wavelength is 0.8856 Å, and the resolution limits are from 50.0 to 0.90 Å. With an oscillation angle of 1° per image, 360 diffraction images were collected and processed with CrystalClear. The structure was solved by direct methods and refined using the full-matrix least-squares technique with SHELXL-97.
3. RESULTS AND DISCUSSION 3.1. Microscopic Observation of CA Crystals. The crystals are long prisms (Figure 1, top). The high aspect ratio of the prisms indicates that the CA molecules have a strong preference for anisotropic unidirectional growth.19,20 A few splits appear on one crystal surface of an air-dry crystal prism (Figure 1, bottom), which indicates there are a large number of solvent molecules in the crystal prism and layer structures may exist. With a comparison of the morphologies of crystal prisms Received: November 5, 2013 Revised: December 3, 2013 Published: December 6, 2013 23
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solvents,21,22 it is almost impossible to provide some valuable information on the self-assembled structures of sodium cholate in water. It is obvious that the use of organic solvents did not favor the hydrophobic interaction between the hydrophobic faces of CA. That is to say, the formation of hydrophobic packing in these crystals may only be attributed to dense packing, while the hydrophobic tetramer structure of CA from water is very likely similar with the self-assembled structures of sodium cholate. 3.3. Proposed Tetramer Structures of CA-4EG, CA, and Cholate. On the basis of the hydrophobic tetramer units of CA in crystals, the similar tetramers of CA-4EG in aqueous solutions are proposed (Scheme 1a). The CA moieties of CA4EG form the middle part, and the 4EG tails stretch out in aqueous solutions. The hydrophobic interaction between the hydrophobic faces of CA-4EG is the initial driving force for the formation of tetramer structures in aqueous solutions, and then two C3−OH groups form H-bonds to stabilize the tetramer structures. The tetramer structures of CA are preserved after acidic hydrolyzing the ester bond of CA-4EG (Scheme 1b). Four CA molecules form the rhombic hydrophobic tetramer through head (C3)-head and tail (C24)-tail arrangement, in which the COOH groups of L/D and R/U (see Scheme 1 caption for abbreviations) adopt a gauche conformation. L/U and R/U form hydrophobic interaction with each other through rings A and so do L/D and R/D; R/U and R/D form hydrophobic interaction with each other through rings B, C, and D and so do L/U and L/D. The cis-fused configuration of rings A and B of CA behaves perfectly in the hydrophobic tetramer structures. Two C3−OH groups form hydrogen bonds with each other and so do two COOH groups (Scheme 1b). The hydrophobic and H-bonding interactions play key roles in stabilizing the tetramer structures of CA in aqueous solutions. It is obvious that the solubility of the tetramers of CA is much higher than that of the individual CA, which is only slightly soluble in aqueous solutions. It is the tetramers of CA rather than the individual
Figure 1. Cryo-SEM (top) and SEM (bottom) images of crystals of CA.
in the frozen and dry states, it is necessary to perform the structure study on crystal prisms in the frozen state. 3.2. Crystal Structure of CA. The crystal belongs to C2 space group. Lattice parameters: a = 40.334(5) Å, b = 7.729(9) Å, c = 15.620(18) Å, β = 90.602(3)o, V = 4869(10) Å3, and Z = 8. Figure 2a shows the existence of fiber-layer structures at the cross-section of prisms. The rhombic tetramer structure units with the hydrophobic faces of CA molecules toward the inner side and the hydrophilic faces toward the outer side are found. The stacking of tetramers along the b axis forms hydrophobic fibers (Figure 1b), which is the length direction of prisms. The array of fibers forms fiber-layer structures, and there exists a 21 screw axis between the adjacent fibers. Meanwhile, two series of the Z-shaped H-bonding interactions are formed between COOH groups and C7 and C12−OH groups along the 21 screw axis (Figure 2 shadow and Figure S1 of the Supporting Information). Although the hydrophobic packing through the hydrophobic faces has been found in crystals of CA from organic
Figure 2. Crystal structure of CA: (a) top view and (b) side view of the fiber-layer structures. H atoms and solvent molecules are omitted for clarity. 24
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Scheme 1. Proposed tetramer structures of (a) CA-4EG, (b) CA, and (c) cholate in aqueous solutionsa
a
L/U, R/U, L/D, and R/D are the abbreviations of left/up, right/up, left/down, and right/down, respectively.
crystals. On the basis of the tetramer units of CA in crystals, the tetramer structures of CA-4EG, CA, and cholate are proposed.
molecules that serve as the basic building blocks to construct the crystal prisms, which can be considered as a kind of selfassembled crystals. With the inspiration of the proposed hydrophobic tetramer structures of CA in aqueous solutions, there are reasons to believe that the sodium cholate also forms similar tetramer structures to avoid the exposure of the hydrophobic faces to the aqueous environment (Scheme 1c). The stepwise deprotonation may form the complex tetramers of CA and cholate (Scheme S1 of the Supporting Information). These primary tetramers may stack to forms octamers, dodecamers, etc., and the rodlike secondary micelles of sodium salts of bile acids have been reported.2,23−29 It is well-known that bile salts play a key role in emulsification and membrane transport of fat-soluble molecules. It is very likely that the hydrophobic tetramers can serve as the carriers to transport these fat-soluble molecules. That is to say the physiological functions of bile salts are based on their tetramer structures.
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ASSOCIATED CONTENT
S Supporting Information *
Cif file of the crystals of CA; H-bonds in crystals of CA, and the proposed tetramer structures. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank X. X. Zhu (University of Montreal) for critical reading of the manuscript. We would like to thank He, Jianhua, Huang, Shen and Liu, Yahui (SSRF) for the help of synchrotron radiation measurements and Song, Haibin for resolving the crystal structure. The authors are grateful for the financial support from National Natural Science Foundation of China (Grants 50703018 and 51373122) and Program for New Century Excellent Talents in University (Grant NCET-121066).
4. CONCLUSIONS Four CA-4EG molecules form tetramer structures through hydrophobic and hydrogen bonding interactions in aqueous solutions, and the tetramers of CA are obtained by acidic hydrolyzing the ester bond of CA-4EG. The tetramers of CA rather than the individual molecules are the basic building blocks to construct the crystal prisms of CA. The stacking of tetramers forms the fibers with the hydrophobic faces of CA molecules toward the inner side and the hydrophilic faces toward the outer side. The array of fibers forms the fiber-layer structures. The crystallization process is hierarchical, and the crystal prism of CA is a kind of self-assembled crystals. The gauche conformation of COOH groups of CA is discovered in
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REFERENCES
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