Mechanistic studies on C-19 demethylation in oestrogen biosynthesis.
Journal: 1982/August - Biochemical Journal
ISSN: 0264-6021
PUBMED: 7092812
Abstract:
Mechanistic aspects of the biosynthesis of oestrogen have been studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves three steps using O(2) and NADPH, in which the C-19 methyl group of an androgen is oxidised to formic acid with concomitant production of the aromatic ring of oestrogen: [Formula: see text] To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labelled precursors were synthesized. Notable amongst these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labelled with (2)H in addition to (18)O. In order to follow the fate of the labelled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analysed by mass spectrometry. Experimental procedures were devised to minimize the exchange of oxygen atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[(18)O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-oestratriene-17-one (V, oestrone), it was found that the formic acid from C-19 retained the original substrate oxygen. When the equivalent (16)O substrates were aromatized under (18)O(2), the formic acid from both substrates contained one atom of (18)O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that one atom of oxygen from O(2) is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into oestrogen. This conclusion was further substantiated by demonstrating that in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10beta-Hydroxy-4-oestrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into oestrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10-C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into oestrogen are discussed.
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Biochem J 201(3): 569-580

Mechanistic studies on C-19 demethylation in oestrogen biosynthesis

Abstract

Mechanistic aspects of the biosynthesis of oestrogen have been studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves three steps using O2 and NADPH, in which the C-19 methyl group of an androgen is oxidised to formic acid with concomitant production of the aromatic ring of oestrogen: [Formula: see text] To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labelled precursors were synthesized. Notable amongst these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labelled with H in addition to O. In order to follow the fate of the labelled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analysed by mass spectrometry. Experimental procedures were devised to minimize the exchange of oxygen atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-oestratriene-17-one (V, oestrone), it was found that the formic acid from C-19 retained the original substrate oxygen. When the equivalent O substrates were aromatized under O2, the formic acid from both substrates contained one atom of O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that one atom of oxygen from O2 is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into oestrogen. This conclusion was further substantiated by demonstrating that in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10β-Hydroxy-4-oestrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into oestrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10–C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into oestrogen are discussed.

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Selected References

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  • Akhtar M, Skinner SJ. The intermediary role of a 19-oxoandrogen in the biosynthesis of oestrogen. Biochem J. 1968 Sep;109(2):318–321.[PMC free article] [PubMed] [Google Scholar]
  • Akhtar M, Alexander K, Boar RB, McGhie JF, Barton DH. Chemical and enzymic studies on the characterization of intermediates during the removal of the 14alpha-methyl group in cholesterol biosynthesis. The use of 32-functionalized lanostane derivatives. Biochem J. 1978 Mar 1;169(3):449–463.[PMC free article] [PubMed] [Google Scholar]
  • Akhtar M, Calder M, Smith T, Wright JN. Mechanistic and stereochemical studies on 3-oxo steroid delta 4-delta 5-isomerase from human placenta. Biochem J. 1980 Feb 1;185(2):411–421.[PMC free article] [PubMed] [Google Scholar]
  • Braselton WE, Jr, Orr JC, Engel LL. The twin ion technique for detection of metabolites by gas chromatography-mass spectrometry: intermediates in estrogen biosynthesis. Anal Biochem. 1973 May;53(1):64–85. [PubMed] [Google Scholar]
  • Braselton WE, Jr, Engel LL, Orr JC. The flux of intermediates and products in aromatizaton of C19 steroids by human placental microsomes. Eur J Biochem. 1974 Oct 1;48(1):35–43. [PubMed] [Google Scholar]
  • Brodie HJ, Kripalani KJ, Possanza G. Studies on the mechanism of estrogen biosynthesis. VI. The stereochemistry of hydrogen elimination at C-2 during aromatization. J Am Chem Soc. 1969 Feb 26;91(5):1241–1242. [PubMed] [Google Scholar]
  • Corina DL. Determination of the 18O content of formic acid as benzyl formate. Anal Biochem. 1977 Jun;80(2):639–642. [PubMed] [Google Scholar]
  • Corina DL, Dunstan PM. Benzyl esters in the gas-chromatographic purification of radioactive acetic acid from bacteria, and for the possible analysis of other short-chain acids. Anal Biochem. 1973 Jun;53(2):571–578. [PubMed] [Google Scholar]
  • Fishman J, Raju MS. Mechanism of estrogen biosynthesis. Stereochemistry of C-1 hydrogen elimination in the aromatization of 2 beta-hydroxy-19-oxoandrostenedione. J Biol Chem. 1981 May 10;256(9):4472–4477. [PubMed] [Google Scholar]
  • Fishman J, Guzik H, Dixon D. Sterochemistry of estrogen biosynthesis. Biochemistry. 1969 Nov;8(11):4304–4309. [PubMed] [Google Scholar]
  • Hosoda H, Fishman J. Unusually facile aromatization of 2 beta-hydroxy-19-oxo-4-androstene-3, 17-dione to estrone. Implications in estrogen biosynthesis. J Am Chem Soc. 1974 Nov 13;96(23):7325–7329. [PubMed] [Google Scholar]
  • MEYER AS. Conversion of 19-hydroxy-delta 4-androstene-3,17-dione to estrone by endocrine tissue. Biochim Biophys Acta. 1955 Jul;17(3):441–442. [PubMed] [Google Scholar]
  • Morand P, Williamson DG, Layne DS, Lompa-Krzymien L, Salvador J. Conversion of an androgen epoxide into 17beta-estradiol by human placental microsomes. Biochemistry. 1975 Feb 11;14(3):635–638. [PubMed] [Google Scholar]
  • Osawa Y, Shibata K, Rohrer D, Weeks C, Duax WL. Letter: Reassignment of the absolute configuration of 19-substituted 19-hydroxysteroids and stereomechanism of estrogen biosynthesis. J Am Chem Soc. 1975 Jul 23;97(15):4400–4402. [PubMed] [Google Scholar]
  • RYAN KJ. Biological aromatization of steroids. J Biol Chem. 1959 Feb;234(2):268–272. [PubMed] [Google Scholar]
  • Skinner SJ, Akhtar M. The stereospecific removal of a C-19 hydrogen atom in oestrogen biosynthesis. Biochem J. 1969 Aug;114(1):75–81.[PMC free article] [PubMed] [Google Scholar]
  • Thompson EA, Jr, Siiteri PK. Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J Biol Chem. 1974 Sep 10;249(17):5364–5372. [PubMed] [Google Scholar]
  • Townsley JD, Brodie HJ. Studies on the mechanism of estrogen biosynthesis. 3. The stereochemistry of aromatization of C19 and C18 steroids. Biochemistry. 1968 Jan;7(1):33–40. [PubMed] [Google Scholar]
Department of Biochemistry, University of Southampton, Southampton SO9 3TU, U.K.
Abstract
Mechanistic aspects of the biosynthesis of oestrogen have been studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves three steps using O2 and NADPH, in which the C-19 methyl group of an androgen is oxidised to formic acid with concomitant production of the aromatic ring of oestrogen: [Formula: see text] To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labelled precursors were synthesized. Notable amongst these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labelled with H in addition to O. In order to follow the fate of the labelled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analysed by mass spectrometry. Experimental procedures were devised to minimize the exchange of oxygen atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-oestratriene-17-one (V, oestrone), it was found that the formic acid from C-19 retained the original substrate oxygen. When the equivalent O substrates were aromatized under O2, the formic acid from both substrates contained one atom of O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that one atom of oxygen from O2 is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into oestrogen. This conclusion was further substantiated by demonstrating that in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10β-Hydroxy-4-oestrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into oestrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10–C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into oestrogen are discussed.
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