The Mechanism of The Microbial Hydroxylation of Steroids. Part 1. The C-21 Hydroxylation of Progesterone by Aspergillusniger ATCC 9142

Abstract

. Can. J. Chem. 53,845 (1975). The mechanism of the C-21 hydroxylation of progesterone (la) by Aspergillus niger ATCC 9142 to give 11deoxycorticosterone (lb) has been studied by the use of progesterone derivatives and of C-21 deuterium labelled progesterones. The requirement of the C-21 hydroxylase system for a C-20 carbonyl group is demonstrated and the possiblity of the involvement of this group, in the C-20,21 enol form, in the C-21 hydroxylation reaction has been discussed. However, on the basis of the observed deuterium isotope effect (KHIKD = 1.25), a mechanism for the microbial C-21 hydroxylation reaction is proposed in which the rate-determining step is the direct insertion of oxygen into a C-21 carbon-hydrogen bond and not one involving enolization of the C-20 carbonyl.In addition, C-1 l a and C-15P hydroxylation of both 20a-and 20P-hydroxypregn-4-ene-3-one (2a and 2b) by A. niger has been observed.HERBERT L. HOLLAND et BARBARA J. AURET. Can. J. Chem. 53,845(1975).On a etudit le mtcanisme de l'hydroxylation (en position C-21) de la progesttrone ( l a ) par I'Aspergill~rs niger ATCC 9142 conduisant B la dtoxy-11 corticosttrone (lb); cette Ctude a t t t effectute i I'aide de dtrivts de la progesterone et de progesttrone marqute au deuterium en position C-21. On a dtmontre la ntcessitt d'un groupe carbonyle en C-20 pour rendre efficace le systkme d'hydroxylase en C-21; on discute aussi de la possibilitt de I'implication du groupement carbonyle en position 20 sous forme d'tnol C-20,21 lors d e la riaction d'hydroxylation en C-21. Toutefois, sur la base de I'observation d'un effet isotopique ( K H / K~ = 1.25), on propose un micanisme pour la rkaction d'hydroxylation microbiologique en C-21 dans lequel I'itape dtterminante est I'insertion directe de I'oxygene dans le lien carbone-hydrogene en C-21 et on suggtre que cette reaction n'implique pas une tnolisation du groupement carbonyle en position C-20.De plus I'Aspergillus niger provoque I'hydroxylation en C-1 l a et en C-15P des hydroxy-2Oaet -20P pregene-4 one-3 (2a et 2b).[ Traduit par lejournal] In spite of the wide application of microbial steroid hydroxylation in both industry and research (1) and a thorough investigation of related biochemical processes (2), comparatively little is known about the chemistry of this reaction.The proposal that microbial hydroxylation at saturated carbon proceeds by direct electrophilic insertion of oxygen into a C-H bond followed from the discovery of a relationship between microbial hydroxylation of steroids at C, and microbial epoxidation of steroids unsaturated at C,, demonstrating the electrophilic nature of microbial hydroxylation (3, 4) and the observation that microbial hydroxylation at saturated carbon proceeds with retention of configuration (5-7).The question of enzymic substrate activation has received little consideration. For hydroxylation at saturated carbon, chemical analogy suggests that activation is unnecessary : oxidation of steroids at saturated carbon has been achieved under electrophilic conditio...

The mechanism of the microbial hydroxylation of steroids. Part 4. The C-6 β hydroxylation of androst-4-ene-3,17-dione and related compounds by Rhizopusarrhizus ATCC 11145

“…Circumstantial evidence f o r this was obtained (2) by incubation of 4a-methyl-4,4a,5,6,7,8-hexahydro-2(3H)-naphthalenone ( l a ) with Rhizopus arrhizus, a microorganism known t o hydroxylate A4-3-ketosteroids at C-6P (3). Both C-8P a n d C-8a substituted products (2u a n d 3u, respectively) were obtained, a n d their formation rationalized by stereoelectronically controlled electrophilic addition of oxygen t o the s-trans en01 40.…”

“…T h e observation (4) t h a t androst-4-ene-3,17-dione-6a-T (5b) was hydroxylated a t C-6P by R. urrhizus without loss of label has been interpreted (4) in terms of a direct hydroxylation with retention of configuration, following the pattern f o r enzymic hydroxylation a t saturated c a r b o n (5-8), b u t since subsequent investigation of the enolization of A4-3-ketosteroids (9-12) has established t h a t formation of t h e A39' en01 occurs with stereoelectronically controlled preference f o r the loss of the C-6P hydrogen relative t o t h a t at C-6a of 53: 1 (2), data such as t h a t obtained f r o m hydroxylation of 5 b at C-6P are n o t amenable t o unambiguous mechanistic interpretation. This paper describes the synthesis a n d incubation with R. urrhizus of a series of specifically deuterated androst-4-ene-3,17-diones (5c-5g) a n d of the 19-norsteroid analogue l b a n d estr-4-ene-3,17-dione (6u).…”

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

“…The 21-halosteroids lb-ld were incubated with Aspergillus niger ATCC 9142, a fungus known to hydroxylate l a at C-21 in good yield (16). The 21-methyl derivative l e was also studied for comparative purposes.…”

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

“…The mechanism of the hydroxylation of androst- ' 4-ene-3,17-dione (la) at C-6$ by the fungus Rhizopus arrhizus ATCC 11 145 has been studied in some detail (1)(2)(3)(4)(5)(6). The reaction proceeds by enzymic enolization of the A4-3-ketone, followed by tion at C-6 of the product is determined largely by conventional stereoelectronic interactions during the oxidation process, rather than being dictated by constraints placed upon the reactants by their interaction with the enzyme.…”

“…The reaction of a A3s5 en01 acetate or en01 ether with m-chloroperoxybenzoic acid has been used successfully as a model system to study the stereoelectronic control of product formation during electrophilic oxidation at C-6 in the normal steroid series (3) and related bicyclic enones (1,3). In an attempt to use this reaction in the B-nor series, the preparation of compounds 8a and 8b was undertaken, using standard procedures for the preparation of A3,5 en01 ethers (13) and acetates (14) from A4-3-ketosteroids.…”