Abstract
4,4-Dimethyl-5α-cholesta-8,14,24-trien-3β-ol (I) from human follicular fluid and 4,4-dimethyl-5α-cholesta-8,24-dien-3β-ol (II) from bull testes have been reported to activate meiosis in mouse oocytes (Byskov et al., 1995. Nature. 374: 559–562). Described herein are new chemical syntheses of I, II, and the Δ8(14),24 analog XXII. A critical step in these syntheses was a remarkably high yield side chain oxidation of 3β-acetoxy-4,4-dimethyl-5α-cholest-8(14)-en-15-one to the corresponding C24 24-hydroxy compound VI. Oxidation of VI to the aldehyde, followed by Wittig olefination gave 3β-acetoxy-4,4-dimethyl-5α-cholesta-8(14),24-dien-15-one. Reduction with sodium borohydride to the 15β-hydroxysteryl ester, dehydration with sulfuric acid in CHCl3, and saponification furnished I in high purity. Reduction of VI with sodium borohydride to the 15-hydroxysteroid followed by dehydration gave 3β-acetoxy-4,4-dimethyl-5α-chola-8,14-dien-24-ol. Hydrogenation over Raney nickel gave the monounsaturated Δ8(14) and Δ8 compounds. Oxidation to the corresponding aldehydes followed by Wittig olefination and saponification gave II and XXII. Chromatographic, mass spectral, and 1H and 13C nuclear magnetic resonance spectral data have been presented for the synthetic sterols and their derivatives. I, II, XXII, and their Δ8,14 and Δ7,14 analogs, at 3 μg per ml, caused a resumption of meiosis in mouse oocytes in the presence of hypoxanthine (3.5 mm). Under the same conditions, Δ5 and Δ5,7 sterols were inactive.—Ruan, B., S. Watanabe, J. J. Eppig, C. Kwoh, N. Dzidic, J. Pang, W. K. Wilson, and G. J. Schroepfer, Jr. Sterols affecting meiosis: novel chemical syntheses and the biological activity and spectral properties of the synthetic sterols. J. Lipid. Res. 1998. 39: 2005–2020.
Highlights
4,4-Dimethyl-5␣-cholesta-8,14,24-trien-3-ol (I) from human follicular fluid and 4,4-dimethyl-5␣-cholesta8,24-dien-3-ol (II) from bull testes have been reported to activate meiosis in mouse oocytes
I and II were samples isolated from human follicular fluid and bull testicular tissue, respectively, with chemical characterization based on gas chromatography–mass spectrometry (GC–mass spectral (MS)) and, in the case of II, 13C nuclear magnetic resonance (NMR) spectroscopy in the absence of authentic standards
Trimethylsilyl ether derivatives were prepared by treatment of the steroids with a 1:1 mixture of bis(trimethylsilyl)trifluoroacetamide and pyridine for 2 h under nitrogen at 40ЊC, followed by evaporation to dryness at 40ЊC under nitrogen
Summary
Melting points (MP) were measured in sealed, evacuated capillary tubes using a Thomas-Hoover apparatus. Agϩ-HPLC was carried out as described previously (20). In the current study 5 m Nucleosil SA cation exchange (100 Å pore size) columns (250 mm ϫ 4.6 mm, 300 mm ϫ 3.2 mm, 300 mm ϫ 4.6 mm, and 250 mm ϫ 10 mm) were obtained from Alltech Associates (Deerfield, IL), and they were charged with silver ion and prepared for use as described previously (20). 4,4-Dimethylcholest-5-en-3-ol, 4,4-dimethylcholesta-5,7dien-3-ol, 4,4-dimethyl-5␣-cholesta-8,14-dien-3-ol (III), and 4,4-dimethyl-5␣-cholesta-7,14-dien-3-ol (IV) were prepared as described previously (21) and, after chromatographic purification, showed purities of Ͼ99%, ϳ97%, ϳ95%, and ϳ96%, respectively, as judged by capillary GC and 1H NMR. (20R,22R)-cholest-5-ene-3,20,22-triol and (20S,22S)-cholest5-ene-3,20,22-triol were prepared as described previously (22), and each showed a purity Ͼ99% on the basis of TLC, Agϩ-HPLC, and 1H NMR. Bovine serum albumin (crystallized) was obtained from ICN Biochemical (Aurora, OH) and equine chorionic gonadotropin was from Dyosynth (Oss, Holland)
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