Lipid rafts are membrane microdomains rich in sphingomyelin (SM) and cholesterol (Chol), and their physical properties are grossly different from surrounding lipid bilayers. Although lipid rafts are assumed to play essential roles in biological processes such as intercellular signal transduction, molecular interactions in lipid rafts are hardly understood because lipid molecules in rafts undergo a rapid association and dissociation equilibrium. In this study we attempted to prepare molecular probes for observing the interaction between SM and Chol in rafts. The ^<13>C{^<15>N}REDOR spectra of the membrane composed of 2-^<15>N-SM 6 and 4-^<13>C-Chol did not show any interaction between SM and Chol; the lifetime of their complex was too short to observe the intermolecular interaction due to thier rapid diffusion in the membrane. Then, for stabilizing the interaction, we synthesized SM-Chol conjugate 14 possessing hydroxylamine (-O-NH-) in the linker. Rafts formation by the conjugate 14 was assessed by several experiments, which consequently indicated that microdomains consisting of 14 were similar to those of SM/Chol. In order to examinc whether the conjuage reproduces hydrogen bond as seen for SM/Chol, we ran molecular dynamic simulation of the membrane comprised of the conjugate 14 or of SM/Chol=1/1. The resulte showed that the hydrogen bond was generated between the amide oxygen of SM part and -O-NH- hydrogen of Chol part in conjugate 14 to a comparable extent with SM/Chol=1:1. In summary, conjugate 14 was shown to reproduce the SM/Chol system, and therefore serve a molecular probe for studies of intermolecular interaction in lipid rafts.
