Structure and dynamic properties of dehydroergosterol, 13-113-113-1

Dehydroergosterol has been widely used as a fluorescent analog of cholesterol for the investigation of lipoprotein, model membrane, and biological membrane structure. Although its synthesis was reported over fifty years ago, the complete structure and assignment of the three double bonds in the rings has not heretofore been firmly established. Therefore, dehydroergosterol was synthesized and purified by reverse phase high performance liquid chromatography. The proposed structure (^{8, 7, 9(11), 22}-ergostatetraen-3-o1), including the location of the double bond at ⁹⁽¹¹⁾, was confirmed by mass spectroscopy,¹H-NMR, and¹³C-NMR. In addition, a convenient assay for determination of impurities in dehydroergosterol preparations utilizing absorbance peak ratios is described. The spectroscopic properties of dehydroergosterol are highly dependent on solvent dielectric constant. Dehydroergosterol was incorporated into sonicated unilamellar vesicles composed of dimyristoylphosphatidylcholine or distearoylphosphatidylcholine. Arrhenius plots of dehydroergosterol fluorescence polarization indicated that the sterol was sensitive to the phase transitions of these phospholipids near 23° and 54°C, respectively. Differential polarized phase fluorescence and lifetime analysis were used to determine the dynamic properties of dehydroergosterol in the vesicles. At 37°C the limiting anisotropy, order parameter, and rotational rate of dehydroergosterol in dimyristoylphosphatidylcholine were 0.162, 0.65, and 0.71 nsec, respectively. The limiting anisotropy and order parameter, but not the rotational rate, of dehydroergosterol were sensitive to the temperature and/or the physical state of the phospholipid.