Orientation of Fluorinated Cholesterol in Lipid Bilayers Analyzed by 19 F Tensor Calculation and Solid-State NMR Nobuaki Matsumori,* ,² Yusuke Kasai, ² Tohru Oishi, ² Michio Murata, ² and Kaoru Nomura ‡ Department of Chemistry, Graduate School of Science, Osaka UniVersity, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan, and Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto-Cho, Mishima-Gun, Osaka 618-8503, Japan Received October 9, 2007; E-mail: matumori@ch.wani.osaka-u.ac.jp Abstract: 6-F-cholesterol was reported to exhibit biological and interfacial properties similar to unmodified cholesterol. We have also found that 6-F-cholesterol mimicked the cholesterol activity observed in the systems of amphotericin B and lipid rafts. However, to use 6-F-cholesterol as a molecular probe to explore molecular recognition in membranes, it is indispensable to have detailed knowledge of the dynamic and orientation properties of the molecule in membrane environments. In this paper, we present the molecular orientation of 6-F-cholesterol (30 mol %) in dimyristoylphosphatidylcholine (DMPC) bilayers revealed by combined use of 19 F chemical shift anisotropy (CSA), 2 H NMR, and C-F rotational echo double resonance (REDOR) experiments. The axis of rotation of 6-F-cholesterol was shown to be in a similar direction to that of cholesterol in DMPC bilayers, which is almost parallel to the long axis of the molecular frame. The molecular order parameter of 6-F-cholesterol was determined to be ca. 0.85, which is within the range of reported values of cholesterol. These findings suggest that the dynamic properties of 6-F-cholesterol in DMPC are quite similar to those of unmodified cholesterol; therefore, the introduction of a fluorine atom at C6 has virtually no effect on cholesterol dynamics in membranes. In addition, this study demonstrates the practical utility of theoretical calculations for determining the 19 F CSA principal axes, which would be extremely difficult to obtain experimentally. The combined use of quantum calculations and solid-state 19 F NMR will make it possible to apply the orientation information of 19 F CSA tensors to membrane systems. Introduction Use of 19 F NMR has been of interest for many years in investigating biological systems, due in large part to the experimentally attractive properties of 19 F, including 100% natural abundance, spin I ) 1/2, large magnetogyric ratio, and low background signals in biological samples. In particular, solid-state 19 F NMR has become an indispensable tool for characterizing the orientation and dynamics of membrane- associated peptides. 1 Subsequently, the need for fluorinated compounds suitable for 19 F NMR measurements has also increased. In the course of our studies on amphotericin B, which is a channel-forming antibiotic, and lipid rafts, we used fluorinated derivatives to investigate molecular recognition in bilayer membranes 2-4 and found 6-F-cholesterol 1 to be a successful molecular mimic of cholesterol for investigating intermolecular interactions involving cholesterol. 6-F-cholesterol was originally reported as a growth factor of yeast that has the activity comparable with cholesterol. 5 Then, Kauffmann et al. studied the air-water interfacial properties of cholesterol and its derivatives 6 and showed the similarity of 6-F-cholesterol to unmodified cholesterol. In our recent experiments, 6-F- cholesterol was shown to reproduce cholesterol activity in the systems of amphotericin B 4 and lipid rafts (to be published in due course). However, to use 6-F-cholesterol as a molecular probe in examining intermolecular recognition in membranes, it is indispensable to have detailed knowledge of its dynamic and orientation properties in membrane environments. Due to its large anisotropic effect, 1 19 F chemical shift anisotropy (CSA) seems to be the best choice for obtaining the orientational information of 6-F-cholesterol in membranes. The CSA tensor is defined by three principal values (δ 11 , δ 22 , and δ 33 ) and three corresponding orthogonal axes, called principal axes. 7 Thus, motion and orientation information with respect to the CSA tensor axes can be obtained by chemical shift measurements. In particular, 13 C and 15 N CSA tensors in peptide bonds are frequently used to assign the alignments of helical ² Osaka University. ‡ Suntory Institute for Bioorganic Research. (1) Ulrich, A. S. Prog. Nucl. Magn. Reson. Spectrosc. 2005, 46,1-21. (2) Matsumori, N.; Umegawa, Y.; Oishi, T.; Murata, M. Bioorg. Med. Chem. Lett. 2005, 15, 3565-3567. (3) Tsuchikawa, H.; Matsushita, N.; Matsumori, N.; Murata, M.; Oishi, T. Tetrahedron Lett. 2006, 47, 6187-6191. (4) Kasai, Y.; Matsumori, N.; Umegawa, Y.; Matsuoka, S.; Ueno, H.; Ikeuchi, H.; Oishi, T.; Murata, M. Chemistry 2008, 14, 1178-1185. (5) Harte, R. A.; Yeaman, S. J.; McElhinney, J.; Suckling, C. J.; Jackson, B.; Suckling, K. E. Chem. Phys. Lipids 1996, 83, 45-59. (6) Kauffman, J. M.; Westerman, P. W.; Carey, M. C. J. Lipid Res. 2000, 41, 991-1003. (7) Duer, M. J. Introduction to Solid-State NMR Spectroscopy; Blackwell Publishing: Oxford, UK, 2004. Published on Web 03/15/2008 10.1021/ja077580l CCC: $40.75 © 2008 American Chemical Society J. AM. CHEM. SOC. 2008, 130, 4757-4766 9 4757