Abstract
Treatment of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism, with perchloric acid in methanol resulted in its partial isomerization to the beta,gamma-unsaturated 15-ketosterols, 3 beta-hydroxy-5 alpha,14 beta-cholest-8-en-15-one (2) and 3 beta-hydroxy-5 alpha,14 beta-cholest-7-en-15-one (3), which were easily separated from 1 by chromatography. Isomers 1, 2, and 3 could be distinguished by their chromatographic retention times as well as by their physical and spectral properties. Reduction of 2 with sodium borohydride gave 5 alpha,14 beta-cholest-8-ene-3 beta,15 beta-diol (4), for which the C-15 configuration was established from the lanthanide-induced shifts of its 3 beta-tert-butyldimethylsilyl ether. 1H and 13C NMR chemical shift differences between 2, 3, and 4 indicated the involvement of variable populations of conformers that differ in the flexible C-D ring system and in the side chain. Compounds 2, 3, and 4 lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.
Highlights
5a,l4/3-cholest-8-ene-3B,15B-diol(4), for which the C-15 con- Materials and methods figuration was established from the lanthanide-induced shifts of its 3/3-rLrt-butyldimethylsilyl etlier
We report the isolation and identification of these sterols, along with their chromatographic and spectral properties and their effects on the Abbreviations: BSTFA, N,O-bis(trimethylsily1) trifluoroacetamide; CHO, Chinese hamster ovary; COSY, 'H-'H shift-correlated spectroscopy; DEFT, distortionless enhancement by polarization transfer; GC, gas chromatography;HETCOR, 'H-"C heteronuclear shift-correlated spectroscopy; hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), 3-hydroxy-3-methylglutarylcoenzyme A; High performance liquid chromatography (HPLC), high performance liquid chromatography:MS, mass spectrometry or mass spectrum; LIS, lanthanide-induced shifts; Medium pressure liquid chromatography (MPLC), medium pressure liquid chromatography; NMR, nuclear magnetic resonance; phosphate-buffered saline (PBS), phosphate-bufferedsaline; SC, side chain; TLC, thin-layer chromatography; TBDMS, tmf-butyldimethylsilyl; TMS, trimethylsilyl; TMSOH, trimethylsilanol; tR, retention time; Yb(fod),tris
The effects of 5cr,14~-cholest-8-ene-3P,15P-dio(l4) on the levels of HMG-CoA reductase activity in CHO-K1 cells are presented in Table 8.The A8-3&15-diol 4 appeared to be slightly more active than the A8(14)-15-ketosterol 1 in blocking the rise in HMG-CoA reductase activity induced by transfer of the cells to lipid-deficient media
Summary
5a,l4/3-cholest-8-ene-3B,15B-diol(4), for which the C-15 con- Materials and methods figuration was established from the lanthanide-induced shifts of its 3/3-rLrt-butyldimethylsilyl etlier. We report the isolation and identification of these sterols, along with their chromatographic and spectral properties and their effects on the Abbreviations: BSTFA, N,O-bis(trimethylsily1) trifluoroacetamide; CHO, Chinese hamster ovary; COSY, 'H-'H shift-correlated spectroscopy; DEFT, distortionless enhancement by polarization transfer; GC, gas chromatography;HETCOR, 'H-"C heteronuclear shift-correlated spectroscopy; HMG-CoA, 3-hydroxy-3-methylglutarylcoenzyme A; HPLC, high performance liquid chromatography:MS, mass spectrometry or mass spectrum; LIS, lanthanide-induced shifts; MPLC, medium pressure liquid chromatography; NMR, nuclear magnetic resonance; PBS, phosphate-bufferedsaline; SC, side chain; TLC, thin-layer chromatography; TBDMS, tmf-butyldimethylsilyl; TMS, trimethylsilyl; TMSOH, trimethylsilanol; tR, retention time; Yb(fod),,tris-. The sterols were added as ethanolic solutions to Ham's F12 medium supplemented with 5% delipidated (10) fetal calf serum (lipid-deficient medium) and allowed to equilibrate for at least 6 h at room temperature prior to storage at 4°C. YHighresolution MS data indicted the following formulas: m/z 325, CZ,H3,; m/z 281, CZ1H29
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