| Part I. The first half of this thesis describes the development of highly sensitive methods that allow the stereochemical assignments of two classes of natural products with biological significance, namely, sphingoid bases (intracellular signaling mediators) and brassinosteroids (plant growth promoters). Exciton coupled circular dichroic (ECCD) spectra for all stereoisomers of sphingoid bases as well as brassinosteroids with sidechain syn-diol moiety have been prepared. Each spectrum shows characteristic ECCD profile, thus allowing stereochemical assignment of unknown species. In addition, several attempts to lower the analysis scale have been made. A picomole-scale HPLC method was developed for the stereochemical analysis of sphingoid bases, whereas fluorescence detected circular dichroic (FDCD) spectroscopy has been applied to the two classes of compounds.;Part II. Endogenous ouabain is a sodium pump inhibitor that is known to exist in minuscule quantity in mammals. It had been generally believed that endogenous ouabain is a close structural isomer of ouabain, a plant origin cardiac glycoside. Structural and biological characterization of endogenous ouabain, however, has long been hampered by the sample shortage. The second half of this thesis describes the structural characterization of endogenous ouabain from bovine hypothalamus. Series of chemical-spectroscopic analyses revealed that endogenous ouabain was, in fact, ouabain, a well-known cardiac glycoside from plants. It turned out, however, the ouabain molecule easily forms various inorganic complexes through its polyhydroxylated steroid backbone moiety and the molecular structure is dramatically changed upon complexation, which had caused many ambiguities in the past structural analysis of endogenous ouabain. The finding also suggested a possibility that some contradictory results associated with the past biological studies on endogenous ouabain were, at least in part, caused by complexation. of ouabain in the physiological environment. Ouabain 1,5,19- and 1,11,19-phosphates, which represents the two distinct conformers of ouabain-inorganic complexes, were subsequently prepared and their physiological properties were examined with a Madin-Darby canine kidney (MDCK) cell-based assay system using a microphysiometer, which can detect cellular metabolic activity. |
