Microbiological hydroxylation. Part XV. Hydroxylation in the terminal rings of mono- and di-oxygenated 5alpha;-androstanes with the fungusDaedalea rufescens

摘要

1976Microbiological Hydroxylation. Part XV.l Hydroxylation in the Ter-minal Rings of Mono- and Di-oxygenated 5a-Androstanes with the FungusDaedalea rufescensBy Alan M. Bell, Sir Ewart R. H. Jones, G. Denis Meakins,' John 0. Miners, and Anthony Pendlebury,Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QYMono- and di-ketones and keto-alcohols derived from 5a-androstane have been incubated with the fungusDaedalea rufescens. a Basidiomycete species not previously reported as a steroid hydroxylator. All but one ofthe dioxygenated substrates, and the monoketones with the keto-group in a central ring, are hydroxylated fairlyrapidly. The cleanest hydroxylations occur with 7-oxygenated androstanes and lead to the 3p,16P-dihydroxy-derivatives.Incubation of 3,3-ethylenedioxy-5o-androstan-7-one (in which hydroxyiation is accompanied by reductionof the 7-keto-group) followed by hydrolysis of the product gives 7a.l GP-dihydroxy-5a-androstan-3-one in 57yield.IN the preceding Part the hydroxylation of certainoxygenated 5u-androstanes with Wojnowicia graminisand Ophiobolus herpotrichus, two Ascomycete fungi, wasreported; the main feature was the ability of thesemicro-organisms to hydroxylate the terminal rings ofsome substrates.Efficient hydroxylation occurred onlywith dioxygenated androstanes , a result which imposesa severe limitation on the usefulness of these fungi. Insearching for micro-organisms which are less demandingin their substrate requirements, we screened a number ofBasidiomycete species. Of these Daedalea rufescens (notpreviously reported as a steroid hydroxylator 2, ap-peared one of the most promising, and was thereforeselected for systematic investigation.Table 1 summarises the microbiological results ob-tained by incubating some monoketones, diketones, andderived mono-acetals, and keto-alcohols derived from5a-androstane with vegetative cell cultures of Daedalearufescens.Table 2 lists the n.m.r. spectra of the steroids,substrates, and products, involved here for whichspectroscopic data have not appeared in earlier public-t For details of Supplementary Publications, see Notice toAuthors No. 7 in J.C.S. Perkin I, 1974, Index issue.1 Part XIV, V. E. M. Chambers, Sir Ewart R. H. Jones, G. D.Meakins, J. 0.Miners, and A. L. Wilkins, J.C.S. Perkin I, 1975,66.ations : the arabic serial number sequence of steroids dis-cussed earlier is used in this Table, which containssteroids nos. 732-752. The structures of new com-pounds follow, as usual,3 from a combination of spectro-metric and chemical methods. For new compounds then.m.r. signals appear in Table 2, and the other inform-ation required for their characterisation is given inTable 3. As with the earlier paper,l and for reasons dis-cussed there, the whole of the Experimental section 4 hasbeen deposited as Supplementary Publication No. SUP21218 (10 pp., 1 microfiche).?A striking feature is the contrast between the slowhydroxylation of the monoketones having the carbonylgroup in a terminal ring and the much faster substitutionof the isomers with the group in a central ring.Whilethe latter (the 6-, 7-, and ll-ketones) behave similarly,in that hydroxylation (possibly followed by oxidation ofa new hydroxy-group) occurs in both rings A and D, theprocesses differ markedly in specificity: the 6- and 11-ketones give several products whereas with the 7-ketone2 W. Charney and H. L. Herzog, ' Microbial Transformationsof Steroids,' Academic Press, New York, 1967.8 A. M. Bell, P. C. Cherry, I. M. Clark, W. A. Denny, SirEwart R. H. Jones, G. D. Meakins, and P. D. Woodgate, J.C.S.Pevkivt I, 1972, 2081.4 Full details of the microbiological and chemical operationsare recorded by A. Pendlebury, D.Phil. Thesis, Oxford, 1972368 J.C.S. Perkin Iremarkably selective 3P,lGP-dihydroxylation is accom-panied by reduction of the carbonyl function, mainly t othe 7a-alcohol.All the dioxygenated androstanes apart from the 3-oxo-7-acetal are hydroxylated fairly rapidly.The clean-est processes (again leading to 3,16-disubstitution or tohydroxylate positions 3 and 16 appears to operate, butless clearly, with 11-oxo-substrates. Thus 15-hydroxyl-ation of the 3,ll-dione and of the 3p-hydroxy-ll-ketonecompetes more evenly with 16-hydroxylation, and 3-substitution does not dominate the situation with the11,17-diketone.TABLE 1Hydroxylations with Daedalea rufescensH5a-AndrostaneThe substrates, all derivatives of 5a-andros tane are indicated by trivial names, e.g. 3P-OH-17-CO represents 3P-hydroxy-Sa-androstan-17-one. In the lsquo;Productsrsquo; columns those oxygen functions introducd during the incubation are in bold type, and n.i.indicatesthat no product was isolated. The substrates were introduced as solutions in ethanol and incubated for the times (usually 4 or6 days) specified in the Experimental section. The yields are calculated after making allowance for recovered starting material.Substrate Main hydroxylationSubstrate recovered () product (s) Other products3-CO6-CO7-CO1 l-co7,7::1-3-c03,3:E -7-co7a-OH-3-CO7P-OH-3-CO3,11-(CO),3P-OH-ll-CO3,17-(CO),17P-OH-3-CO3P-OH-17-CO7,l l-(CO),7,17-(CO),1 1,17-(CO) 27,16-(CO),902120106860936019283360031600007- 1SP,Ga, lflP-(OH),11.1.16411612392612333066632818281517244239321676 4276310.613632.62.6167863.61311697monosubstitution if one of these positions is blocked)occur with substrates having a 7-0x0- or a 7a-hydroxy-group; the 7p-hydroxy-compound and the 7-acetals areutilised less efficiently.The tendency of D. rzcfescens toThe results can be interpreted along lines similar tothose adumbrated in the preceding Part for the fungiW . graminis and 0. herpotrichus. Thus D. rufescens hasa propensity for attacking certain positions, chiefly 3, 71975TABLE 2N.m.r. signalsThe results, presented in the form used earlier, a were obtained by examining solutions in CDQ, at 100 MHz.The weak signals re-corded with saturated solutions of some relatively insoluble triols are not given: each of these triols is followed by an entry forthe corresponding acetate.No.732733734735736737738739740741742743744745746747748749760751752Compound7,7-Ethylenedioxy-5a-androstan-3-oneBa-Androstane-7,16-dione5a-Androstane-2,11,16-trione6a-Androstane-3,11,15-trione5a- Androst- 14-ene-3,11,16-trione7,7-Ethylenedioxy-5a-androstan-3B-ol14a-Hydroxy-5a-androstane-3,11,16-trione5a-Androstane-7a, 16P-diolSp, 16p-Dihydroxy-5a-androstane-1 l-one3p, 6a-Dihydroxy-5a-androstane- 16-one3p, 15P-Dihydroxy- 5ac-androstan-6-one3p, 15P-Dihydroxy-5a-androstan- 1 l-one:3,3-Ethylenedioxy-5a-androstane-7a, 16P-diolTia, 17P-Dihydroxy-5a-androstan-3-one3@,7a, 16a-Triacetoxy-5a-androstane313,7@, 16P-Triacetoxy-5a-androstane3p, 7a.lG~-Trihydroxy-5a-androstan- 1 l-one3p, 7a, 16P-Triacetoxy-5a-androstan- 1 l-one3p, 7 @, 17 @-Trihydroxy-5a-androstan- 1 l-one 3@,14a, 16P-Trihydroxy-5a-androstan- 1 l-one3p, 1 6P-Diacetoxy- 14a-hydroxy-5a-androstan-1 l-one191819181918191819181918191819181918191819181918191819181918191819181918191878-959-278.929.138.969.168.779-218.658-759.169-308-759.009.189.038-749.099.129-149-218.998.949-059-169-058.999-249-149.129.119.098-959-168.929-288-969.03T~ (calc.)8-959.268.909-138-999-168-759.228-638.759-169.298-729.019-209.058.729.089.129-139.229.008-959.059.179.068.989.239.149-139.119.098.929.148.909308.939.04a Ref.3. Not fully characterised.and 16, of suitable substrates. Three sites, so disposedas to correspond closely to positions 3, 7, and 16 of thesteroid nucleus, are thought to be present on the enzymesurface (Scheme). While each site is regarded as beingcapable of binding to a suitable oxygenated group of thesubstrate and of hydroxylating a carbon centre of thesteroid molecule which becomes close to it in the enzyme-steroid complex, the sites are presumed to differ in theirmain tendencies, viz., binding at the central site andhydroxylation at the terminal sites.The orientation onthe enzyme surface taken up by substrates having a 7-oxo- or a 7a-hydroxy-group is then peculiarly suited toclean hydroxylation. With 1 l-ketones, binding in thereverse mode5 does not produce so close a fit betweenparticular carbon centres and the hydroxylating sites,V. E. M. Chambers, W. A. Denny, J. M. Evans, Sir EwartR. H. Jones, A. Kasal, G. D. Meakins. and J. Pragnell. J.C.S.Perkin I , 1973, 1500.,bsol;CH-OR and othercharacteristic signals( Acetal)H-15H-3(Acetal)H-7H-16H-2H-16H-6H-3H-16H-3H-15H-7H-16(Acetal)H-7H-17H-3H-7H-1633-3H-7H-16H-3H- 7H-16H-3H-7)H-17H-3H-166.033.996.426.036-165.565.925.536-56.445.756.435.596.155.546.056-146.325.275.074.835.275.404.835.305.004.776-46.175.344-61557 (10, 10, 6 , 6)5binding bsol;Normal modeHO- @ 16 --OHbinding 9Reverse modeSCHEME Model for hydroxylations with D.rufescenJ.C.S. Perkin Iand substitution occurs at several positions. The lackof reactivity of the terminal ring monoketones is againconsidered to indicate that binding of the oxo-groups tothe terminal sites is weak, or that the complex produceddifficult to obtain by normal chemical methods, theefficient hydroxylations of the 7-oxo-3-acetal and the 7a-hydroxy-3-ketone are noteworthy. These (followed inthe former case by hydrolysis of the acetal group) lead toTABLE 3Characterisation of new compoundsCompound7,7-Ethylenedioxy-5a-androstan-3-oneBa-Androstane-7,16-dione6a-Androstane-2,11,16-trioneBa-Androstane-3,11, 15-trione5a-Androst-14-ene-3,11,16-trione14a-Hydroxy-5a-androstane-3,11,16-trione5a-Androstane-7e 16P-diol2@, 168-Dihydroxy-5a-androstan-1 l-one3p,tla-Dihydroxy-5o-androstan- 16-one3f3,168-Dihydroxy-5a-androstan-6-one3p, 15p-Dihydroxy-5a-androstan-1 l-one3,3-Ethylenedioxy-5a-androstane-7a, 16p-diol3p, 7a, 16~-Triacetoxy-5a-androstane38,7a, 16~-Trihydroxy-5a-androstan-l l-one3p,7a, 16P-Triacetoxy-5a-androstan-1 l-one38,7@, 17P-Trihydroxy-5a-androstan-1 l-one3@,14a, 16P-Trihydroxy-5a-androstan-11-oneM.p.("C) *160-152174-175222-224 $152-154 8120-122281-283174--175.5185-1 87235-238228-229243-244205-206.5151-153283-285244-246236-237 (1209-6-270 t t3@,16@-Diacetoxy- 14u-hydroxy-6a-androstan-1 l-one 324-226FoundFoundFoundFoundFoundFoundFoundFoundFoundFoundFoundFoundFoundFoundFoundFoundFoundFoundC21H320S req*C19H2802 req.C19H2603 req*C19H2603 req-C1SH2403 req*CIBHZ~04 req.C19H3202 req*C19H3003 req.C1SH3003 req.C1SH3003 req*C1SH3003 req.C21H34O4 req.C25H3806 req*C19H3004 req*C25H3,Oi req.ClBH3004 req.C19H3004 req.CPi3H3406 rq.Analytical figures (OA)C75.875.979.079.175.575.576.875.575.876.071.671.777.878.074.274.574-374.574.574.574.674.571.77 1.9569.369-170.870.867-166.967.367.067-167.068.068.0H9.79.79.79.88.78.78-68.78.38.058.18.210.711.09.99.99.79.99.89.99.69-99.69.88.78.89.49.48.28-19.69.59.39.58.48.4* From acetone-hexane unless otherwise specified. t Rotations determined with CHCl, as solvent unless otherwise indicated.$ From acetone.5 From EtOH. 7 Rotation determined with dioxan as solvent. 11 From CHC1,-MeOH. ** Rotation deter-mined with EtOH as solvent. t i From MeOH.does not bring other steroid centres close enough to the the 3-oxo-7a,16P-diol and thence, by Huang-Minlonhydroxylating sites. Although this pictorial approach reduction, to 5a-andros t ane-7 a, 16 p-diol.is not sufficiently precise for a detailed treatment of the we thank the S.R.C. for a studentship (to A. pa) and aresdb it Serves auseful model in Planning Preparative fellowship (to J. 0. M.), and Glaxo Research Ltd., for asequences involving micro-organisms. grant and gifts of chemicals.In connection with the synthesis of steroids which are 4/1963 Received, 24th Sefltembev, 1974