Molecular-reorientational dynamics investigation of biomolecules in solution by NMR, X-ray and CSI-MS.

I.用PFG核磁共振、CSI-MS和X-射线单晶分析对甾体化合物在稀溶液中和固体中的氢键进行了广泛的分析。虽然结晶状态下的X射线分析和溶液中的CSI-MS测量表明，观察到的甾体化合物的扩散系数D_&lt；obs&gt；可能会随着氢键数目的不同而变化，但从扩散研究中确定的实际观察到的D_&lt；obs&gt；值不断减小，与氢键数目的减少没有相关性。明确地证实了基于甾体化合物分子间氢键的链状结构(簇)的形成。II.为了证实这一在溶液中前所未有的现象，我们用PFG核磁共振进行了扩散研究。由PFG核磁共振测量得到了BG+NBA(n=1~4)的D值，包括BG和BA本身。观察到的D值随着附着的BA(N)数目的增加而减小。这一结果清楚地支持了CSI-MS观察到的配合物在溶液中的有序形成。III.发现^&lt；19&gt；F{1H}NOE和2D^1H-lt；-19&gt；F HOESY实验检测到氟烷^&lt；19&gt；F与HSA质子之间存在分子间的NOE。用~1H和~(19)&GT；F核磁共振测定了氟烷的扩散系数，表明氟烷与人血清白蛋白相互作用。这些结果表明，这些技术非常适合于在药物发现过程中识别与蛋白质受体结合的含氟配体。IV.基于差分扩散的核磁共振技术可以使用简单的脉冲序列直接从络合物中检测配体信号，而不需要同位素标记。目前的结果表明，这些技术非常适合于在药物发现过程中识别与蛋白质受体结合的配体。

I. An extensive analysis of hydrogen bonding of steroid compounds in diluted solution is preformed by PFG NMR, CSI-MS and in the solid state by X-ray crystallographic analysis. Although X-ray analysis in the crystalline state and CSI-MS measurement in solution suggested that the observed diffusion coefficient D_<obs> of the steroid compounds may vary in accordance with the number of hydrogen bonds, the actual observed D_<obs> value determined from the diffusion studies diminished constantly without correlation on the decreasing numbers of hydrogen bonds. Formation of a chain structure (cluster) based on intermolecular hydrogen bonding of the steroid compounds is unambiguously confirmed.II. To confirm this unprecedented phenomenon in solution, we performed diffusion study by PFG NMR. The D values of the complexes BG + nBA (n=1〜4) including BG and BA itself, obtained by PFG NMR measurements. The observed D value decreases in accordance with the increasing number of attached BA (n). This result clearly supports the ordered formation of the complexes in solution as observed by CSI-MS.III. It was found that ^<19>F{^1H} NOE and 2D ^1H-<-19>F HOESY experiments detected intermolecular NOEs between halothane ^<19>F and HSA protons. Measurements of diffusion coefficients for halothane were also carried out by ^1H and ^<19>F NMR, indicating the interaction of halothane with HSA. The present results indicate that these techniques are very suitable for identification of a fluorine containing ligand binding with a protein receptor in the drug discovery process.IV. The difference diffusion-based NMR technique can directly detect the ligand signals from the complex using a simple pulse sequence without the need for isotope labeling. The present results indicate that these techniques are very suitable for identifying a ligand binding with a protein receptor in the drug discovery process.