Inhibitors of sterol synthesis. Characterization of trimethylsilyl dienol ethers of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one. Applications in the analysis of mitochondrial metabolites of the 15-ketosterol by gas chromatography-mass spectrometry.
Derivatization of delta 8(14)-15-ketosterols as bis-TMS dienol ethers facilitates their analysis by gas chromatography-mass spectrometry (GC-MS). Conditions are presented for the preparation of each of the three possible bis-TMS dienol ethers of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism. Treatment of 1 with N,O-bis(tri-methylsilyl)-trifluoroacetamide (BSTFA) and pyridine for 20 h at 100 degrees C produced primarily 3 beta,15-bis(trimethylsilyloxy)-5 alpha-cholesta-7,14-diene. Treatment of 1 with BSTFA-pyridine in the presence of 0.1% perchloric acid for 20 h at 22 degrees C gave mainly the delta 8(14)15, dienol ether. Heating this reaction mixture at 100 degrees C for 4 days gave mainly the delta 8,14 ether. The structures of the three dienol ethers were established by GC-MS and 1H and 13C nuclear magnetic resonance spectroscopy. The three TMS dienol ethers of 1 were resolved by capillary GC and gave very simple mass spectra upon electron impact with fragmentation limited almost exclusively to the elimination of trimethylsilanol, methyl, and the side chain. The TMS dienol ethers showed reduced artefact formation, improved chromatographic resolution, and increased sensitivity relative to the delta 8(14)-15-ketosterols, properties that improve the detection and identification of minor components in analyses of complex biological mixtures. The utility of this derivatization is illustrated for the delta 7,14 TMS dienol ethers of the 3-deoxy, 3-keto, 3 alpha-hydroxy, and 3 beta-methoxy analogs of 1 and for delta 8(14)-15-ketosterols in mixtures obtained from incubations of 1 with rat liver mitochondria in the presence of NADPH.
St Pyrek
J
J
Department of Biochemistry, Rice University, Houston, TX 77251-1892.
Wilson
W K
WK
Numazawa
S
S
Schoepfer
G J
GJ
D000818:Animals; D002247:Carbon Isotopes; D002783:Cholestenones / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002849:Chromatography, Gas / Q000379:methods*; D007553:Isotope Labeling; D008099:Liver / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism*; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D013261:Sterols / Q000096:biosynthesis*; D014297:Trimethylsilyl Compounds / Q000737:chemistry*
D002247:Carbon Isotopes; D002783:Cholestenones; D013261:Sterols; D014297:Trimethylsilyl Compounds; C015073:cholest-8(14)-en-3-ol-15-one; D009249:NADP