89(P21) フッ素固体NMRを用いたニコチン性アセチルコリンレセプターとリガンドとの相互作用解析(ポスター発表の部)

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The venom of the Egyptian wasp Philanthus triangulum, philanthotoxin 433 (PhTX-433) is a polyamine amide with butyryl/tyrosyl/spermine moieties. It is a noncompetitive antagonist of the nicotinic acetylcholine receptors (nAChR) and various glutamate receptors (GluR). Preliminary photocrosslinking results, coupled with structure/activity relations (SAR), have led to a putative model representing the binding of philanthotoxins in channels gated by nAChR. We now demonstrate the ^<19>F solid state NMR (SS NMR) technique to determine the interaction between various ligands and the 270KDa membrane protein, nAChR. Fluorine has natural abundance of nearly 100% and its sensitivity is as high as proton in NMR. The chemical shifts of fluorine are very sensitive to the chemical environment and the secondary structure induced shifts can be as high as 10ppm. Three fluorinated PhTX analogs were synthesized in order to observe a signal from a bound ligand and there were subjected to the binding studies with nAChR which was prepared from Torpedo californica. To extend this approach fluorinated phencyclinde (PCP), an antagonist of nAChR, was synthesized and binding studies were performed. The fluorine signal of bound CF_3-PCP in nAChR was observed in the SS-NMR spectrum, which disappeared upon adding of a stronger antagonist TCP. This method is highly sensitive requiring only ca. 10μg of the fluorinated ligand. Magnetic dipole-dipole interactions have been used to determine internuclear distances in molecules in SS NMR. Some of the widely used techniques are rotational resonance (RR) and radio-frequency-driven dipolar recouping (RFDR) for homo-nuclear dipolar interactions. Due to much stronger dipolar interaction than carbon, fluorine should offer longer distance and more sensitive measurements. To explore longer distances systematically, polyphenyl compounds were designed and synthesized. The CF_3, and SCF_3 group were selected as fluorine functions and the phenyl, biphenyl, terphenyl and quaterphenyl fluorinated compounds 8, 9, 10, 11 were synthesized mainly by Suzuki coupling. To avoid intermolecular homonuclear dipolar couplings from the neighboring fluorine compounds, all fluorinated compounds were diluted with structurally similar non-fluorinated compounds by more than 100 folds. A clear correlation was observed between the magnetization exchange rate and the internuclear distances up to a F-F distance approaching 20A in both RR and RFDR experiments.
1997-07-20

89(P21) フッ素固体NMRを用いたニコチン性アセチルコリンレセプターとリガンドとの相互作用解析(ポスター発表の部)

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