Microbial hydroxylation of steroids. 7. Hydroxylation of B-nortestosterone and related compounds by Rhizopus arrhizus ATCC 11145, and 13C nuclear magnetic resonance spectra of some B-norsteroids

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A series of A"3-ketosteroids has been incubated with the C-6P hydroxylating fungus Rhizopus arrhizus ATCC 1 1145. Ring A aromatic steroids are not metabolized by this fungus, nor are 3-chloro-and 3-(N-pyrrolideno)-androsta-3,Sdienes.A'.'-3-~etosteroids are epoxidized at the 6.7 position by R. arrhizus with P stereochemistry; in contrast, both 5a-and 5P-A"3-ketosteroids are epoxidized with a stereochemistry by fungal and peracid reagents. The results of this and previous studies now make it possible to define the nature of the hydroxylating/epoxidizing species of the 6P hydroxylase enzyme of R. arrhizrts. Evidence is presented for a stepwise reaction mechanism in both cases. Improved methods of synthesis are described for 6-methylene-testosterone and estra-4,6-dien-l7P-ol-3-one.HERBERT L. HOLLAND, P. CHINNA CHENCHAIAH, EVERTON M. THOMAS, BEATRIX MADER et MICHAEL J. DENNIS. Can. J. Chem. 62, 2740Chem. 62, (1984.On a procede i l'incubation d'une serie de Ah ceto-3 steroi'des en presence du champignon Rhizopus arrhizus ATCC 1 1 145 qui provoque des rtactions d'hydroxylation en C-6P. Les steroi'des comportant un cycle A aromatique ainsi que les chloro-3 ou (N-pyrro1ideno)-3 androstadiknes-3,5 ne sont pas mCtabolisCs par ce champignon. Sous I'influence du R. arrhizus, les A4.6 ctto-3 steroi'des subissent une reaction d'epoxydation en position 6,7 et la sterkochimie de la reaction est P; par opposition, les Ah ctto-3 steroi'des tant de la serie 5 a que de la serie 5P subissent des rkactions d'epoxydation dont la stdrtochimie est a tant sous I'action des champignons que des peracides. Grice aux resultats de la presente etude ainsi que ces obtenus anttrieurement, il est maintenant possible de definir la nature des espkces, presentes dans I'enzyme hydroxylase 6P du R. arrhizus, qui provoquent les reactions d'hydroxylation et d'epoxydation. Dans chacun de ces cas, on presente des donnkes qui suggkrent un micanisme rkactionnel par Ctapes. On decrit des methodes ameliorees de synthkse pour la mCthylkne-6 testosterone ainsi que pour I'estradiene-4,6 01-17P one-3.[Traduit par le journal]

Section: Discussionmentioning

confidence: 51%

“…W e have studied the latter reaction, performed by the fungus Rhizopus arrhizus, and have demonstrated that the substrate is hydroxylated in its A3,'-dienol form (eq. [I]) (3)(4)(5)(6)(7)(8).…”

Section: Introductionmentioning

confidence: 99%

Microbial hydroxylation of steroids. 9. Epoxidation of Δ⁶-3-ketosteroids by Rhizopusarrhizus ATCC 11145, and the mechanism of the 6β hydroxylase enzyme

Holland¹,

Chenchaiah²,

Thomas³

et al. 1984

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“…R . arrhizus is capable of hydroxylation at both C-6a and C-6P of B-norandrostanes (24). None of the incubations with R. arrhizus produced detectable amounts of the ketosteroids 4d or 7 d .…”

Section: Introductionmentioning

confidence: 91%

“…The product was then extracted into methylene chloride and the extract dried and evaporated. The residue was crystallized from methanol to give the title compound These compounds were obtained by incubation of B-norandrost-4-ene-3.17-dione (7a) with Rhizopus arrhizus ATCC 11 145 as described (24). Physical and spectral data were as previously reported (24).…”

Section: -Methylpregn-4-ene-320-dime (9 10)mentioning

confidence: 99%

Microbial hydroxylation of steroids. 8. Incubation of Cn halo- and other substituted steroids with Cn hydroxylating fungi

Holland¹,

Thomas²

1982

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A series of C-21 substituted progesterone derivatives (R = F, CI, Br, CH,) has been prepared, and incubated the C-21 hydroxylating fungus A. niger. No biotransformation was observed where R = Cl, Br, or CH,, but a minor amount of hydroxylation occurred at an unidentified site where R = F. Two C-1lP substituted progesterone derivatives (R = F, OH) were converted to the corresponding C-11 ketone by the C-1 l a hydroxylator R. stolonifer, but a C-11P hydroxylator (C. lunata) was unable to perform a similar transformation with a C -l l a hydroxy substrate. C. lunata hydroxylated a C-1 I P fluoro substrate at C-14a and C-21 in low yield. C-7a and -7P-hydroxy androst-4-ene-3.17-diones were prepared and incubated with C-7P hydroxylating (R. stolonifer) and C-7u hydroxylating (M. griseocyanus) fungi respectively. No ketone formation was observed. Similarly, the C-60 hydroxylator R. arrhizus was unable to transform a C-6a-hydroxy substrate or C-6a-or C-613-hydroxy-B-norsteroids. No oxidation at halogen was observed for any of the substrates used. I3C-I9F coupling constants for 1 lp-fluoroprogesterone suggest the presence of through space interactions between fluorine and both C-18 and C-19. Introduction for hydroxylation, but has little effect in cases The widespread use of organohalides .in both where the favoured hydroxylation Site is remote industry and medicine has led to concern about from C,. Similarly, the metabolism of a fluorogibtheir toxic and/or carcinogenic effects (1). This in berellin derivative by Gibberella fugikuroi octurn stimulated research into their metabolic fate, curred largely independently of the Presence of which has indicated that, in many cases, bioactiva-fluorine (71, although the same group (8) found that tion of organohalides by a cytochrome p-450 de-Aspergillus aLienaceous could not produce an opendant rnixed-function oxygenase enzyme is a fluoro analog of avenaciolide when given the corkey step in their degradation, leading in some responding fluorinated Precursors. instances to the formation of toxicologically significant metabolites (2-4). Possible oxidative routesResults and discussion which have been considered (5) for this degradationThe substrates were prepared as described in the include hydroxylation at saturated carbon, oxida-Experimental. The use of tetraethyl tin (9) to tion of the halogen to a hypervalent state, and produce 21-methylprogesterone (le) from the acid epoxidation of C=C bonds. In this study, we have chloride of 8 is preferable to the existing methods utilized halosteroids, and fungi known to hydrox-(10, 11) using diethyl cadmium. The halosteroids ylate steroids at specific sites, in order to investi-were prepared by unexceptional procedures. The gate the first of these proposed modes of degrada-synthesis of 1 lp-fluoroprogesterone (3a) proceedtion.ed via the 9a-bromo compound 9 (12); we were Previous work with fluorosteroids and a series of unable to reproduce reports of the direct producfungi by the Oxford group (6) has demonstrated tion of 3a by reaction of 1la-...

“…The conversion of 19-dl-2b into 19-dl-& was performed by a published procedure (51). The product was purified by preparative layer chromatography and obtained in 60% yield.…”

Section: Androst-4-ene-17p-ol-3-one 19-d(19-dl-2a)mentioning

confidence: 99%

Enzymic aromatization of deuterium labelled testosterone and androst-4-ene-3,17-dione

Holland¹,

Taylor²

1981

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The preparation and aromatization by human placental microsomes of androst-4-ene-3-ones labelled with deuterium at C-4, C-6α, C-6β, C-16, and C-19 is described. Deuterium nmr spectra are reported for these compounds and for a sample of a 1,2-dideuterated androst-4-ene-3-one; the latter is formed nonstereospecifically by reduction of the C-1(2) double bond of a Δ1,4-diene-3-one. Aromatization by placental microsomes occurs with retention of deuterium at C-4 and C-6, but with some loss from C-16. The aromatization of 19-d1 and 19,19,19-d3 steroids in the presence of 16,16-d2 steroids has been carried out to determine the deuterium isotope effect for the oxidative removal of C-19. The values obtained (kH/kD for 19-d1 = 2.3, kH/kD for 19,19, 19-d3 = 3.2) are a combination of primary and secondary effects, but suggest that oxidation at C-19 is a rate-limiting reaction of the biosynthetic sequence.The fungus Pellicularia filamentosa NRRL 2727, reported to hydroxylate androst-4-ene-3,17-dione at C-19, gave only the products of hydroxylation at C-11α, C-11β, and C-14.