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A series of alkyl phenyl selenides has been incubated with the fungi Aspergillus niger, Aspergillus foetidus, Mortierella isabellina, and Helminthosporium sp. These fungi oxidize the corresponding sulfides to sulfoxides efficiently, but in no case was any evidence obtained that the microbial oxidation of selenide to selenoxide was occurring. The fate of methyl phenyl selenide following incubation with M. isabellina was investigated using methyl-14C-labeled substrate, and by quantitative selenium analysis. These techniques indicate that the selenide is taken into the fungal cell efficiently and that some metabolic cleavage of the selenium-methyl carbon bond may occur.

The fungus Gnomoniafructicola ATCC 11430 hydroxylates testosterone at C-2β in 40–60% yield. The enzyme involved has been shown to be an inducible CO sensitive, cyanide insensitive oxygenase, with a requirement for theΔ4-3-ketosteroid substrate functionality. The use of 2α- and 2β-deuterium labelled substrates has shown that hydrogen loss from C-2 during 2β hydroxylation is isotope dependent and non-stereospecific. This is interpreted in terms of a Δ2,4-dienolic enzyme-bound intermediate form of the substrate. Keywords: Gnomoniafructicola, hydroxylation, steroids, testosterone.

The biosynthesis of berberine and hydrastine (in Hydrastis canadensis), corydaline and protopine (in Corydalis solida), and ochotensimine and protopine (in C. ochotensis), has been investigated by the administration of [3-14C]-3′,4′-dihydroxyphenylalanine ([3-14C]DOPA). In all cases, incorporation of label was predominantly into the isoquinoline portion of the alkaloid. The role of DOPA in the early stages of isoquinoline alkaloid biosynthesis in these plants is discussed in the light of this and other relevant data. In addition, the later stages of corydaline biosynthesis have been studied by the administration of [9-methoxy-14C]palmatine and -tetrahydropalmatine to C. solida, and the origin of the exocyclic carbons of ochotensimine further verified by feeding [methyl-14C,3H]methionine to C. ochotensis.

The indolo[2:3,3':4']pyrido[1,2-b]naphthyridine ring system, which is present in several alkaloids, has been prepared in a single reaction between the lithio derivative of a 3-cyano-4-methylpyridine and N-benzyl-3,4-dihydro-β-carboline in the presence of trimethylsilyl trifluoromethanesulfonate. Using appropriately substituted starting materials, N-benzyl-13b,14-dihydronauclefine and N-benzyl-13b,14-dihydroangustine have been made in this way. A new preparation of the synthetic intermediate 9-benzyl-3,4-dihydro-β-carboline is also described.

The isomeric 5,6α- and 5,6β-epoxycholestanes, in addition to an analogous series of compounds substituted at C-3 with hydroxy (α or β stereochemistry) or ethylene ketal groups, have been treated with aluminum isopropoxide or tert-butoxide. The latter series of reactions did not give identifiable material, but aluminum isopropoxide gave products derived from epoxide opening and rearrangement in all cases. With epoxides unsubstituted at C-3, aluminum isopropoxide functioned as a Lewis acid in promoting epoxide rearrangements. In the presence of a C-3 alcohol function, additional products were obtained arising from fragmentation of the C-4,C-5 bond, or from β-elimination of the epoxide involving the loss of a C-7 hydrogen. Meerwein–Pondorff reduction of product carbonyl groups was also observed. C-3 ketal substituted epoxides were rearranged cleanly to 6-hydroxy-Δ4-3-ketones. Cyclohexene oxide reacted with aluminum isopropoxide (but not with tert-butoxide) to give two products arising from epoxide addition reactions. Structures for these products are proposed based on their 13C nmr spectra, and a possible route for their formation is presented. None of the epoxides examined in this study reacted with magnesium methoxide.

The natural abundance 13C nuclear magnetic resonance spectra of a number of spirobenzylisoquinoline alkaloids and related model compounds have been recorded. The carbon resonances of the alkaloids were assigned by comparison with the spectra of other isoquinoline alkaloids and with those of the model compounds. It has been shown that 13C nmr spectroscopy may be used to differentiate between diastereomers in this series.

The incubation of B-norandrost-4-ene-3-ones and B-nor-3β-hydroxyandrost-5-enes with Rhizopus arrhizus ATCC 11145 has been described. The products are consistent with a mechanism of oxidation at C-6 in which the stereochemistry of substitution at C-6 is controlled by stereoelectronic interactions in the substrate, and is not dictated by enzymic constraint during the reaction. The carbon-13 nuclear magnetic resonance spectra of several B-nor-Δ4 and Δ5 steroids have been presented.

The steroid analogue 4,4a,5,6,7,8-hexahydro-2(3H)-naphthalenone was hydroxylated at C-8α, C-8β, and C-4a by Rhizopusarrhizus. Similar products were obtained by peracid oxidation of the corresponding enol ethers: hydroxylation of estr-4-ene-3,17-dione by the same fungus occurred at the analogous C-6 and C-10 positions. These results are consistent with a mechanism of microbial hydroxylation involving the enol form of the Δ4-3-ketone. Data from the incubations with R. arrhizus of androst-4-ene-3,17-dione specifically labelled with deuterium at C-4, C-6α, or C-6β and from those of other deuterium labelled substrates have been interpreted in terms of a mechanism of C-β hydroxylation involving a rate-determining step before enolization of the ketone, followed by rapid enolization and oxidation of the enol to give the 6β-hydroxy-Δ4-3-ketone. The kinetic isotope effect, kH/kD, for the hydroxylation of androst-4-ene-3,17-dione at C-6β has been found to be 1.2 ± 0.1.

Anions derived from furo[3,4-c]pyridin-3(1H)-one, by treatment with lithium diisopropylamide, react with substituted, 3,4-dihydroisoquinolines and 2-methyl-3,4-dihydroisoquinolinium salts yielding nitrogen analogues of the protoberberine and phthalideisoquinoline alkaloids, respectively.

Tritium labelling is crucial to the investigation of the metabolic effects of norethynodrel and norethindrone--2 widely used progestogen components of oral contraceptives. However, preparation of labelled samples of these progestins is generally a complex chemical process involving numerous steps. The authors report a 1-step method for the preparation of deuterium or tritium-labelled norethynodrel, norethindrone, and 6-methyleneandrostenedione that uses a modified phase transfer catalyzed exchange procedure. The exchange agent (deuterium oxide and lithium or sodium hydroxide) is crucial to this process. Norethynodrel is efficiently exchanged by either sodium or lithium hydroxide, while norethindrone is poorly exchanged by either sodium or lithium hydroxide, while norethindrone is poorly exchanged under these conditions. Tritium-labelled specimens of all 3 hormones can be obtained through use of tritium-labelled water. In addition to providing a simple means for the preparation of tritium-labelled keto steroids whose chemical reactivity rules out the use of normal enolic exchange procedures, this method can be used to label other compounds that contain carbonyl.

A series of unsaturated steroids has been incubated with fungi which are known to hydroxylate at a site corresponding to the allylic position in the analogous saturated steroids. In some cases, the anticipated hydroxylation occurred without rearrangement of the double bond. In a number of instances, however, products were obtained whose structures implied that allylic rearrangement had occurred during the reaction. The formation of these products is consistent with a stepwise mechanism of enzymatic oxidation. Possible routes for product formation are presented which incorporate this proposal.

The products of the incubation of androst-5-ene-3,17-dione (2a), 3β-hydroxyandrost-5-ene-17-one (2b), and androsta-3,5-diene-17-one (3) with Rhizopus arrhizus ATCC 11145 under a variety of conditions have been identified and the mechanisms of their formation discussed. In addition, several C-(4,5)- and C-(5,6)-epoxyandrostanes have been incubated with R. arrhizus, the products identified and possible pathways for their formation presented.

The testosterone derivatives A-nortestosterone acetate and B-homo-Δ1-testosterone acetate have been incubated with Rhizopusarrhizus, a fungus which hydroxylates testosterone at the C-6β and C-11 α positions. These modes of hydroxylation were also observed for the A-nor steroid. The B-homo steroid, however, was oxidized at C-2β and concurrently hydroxylated at C-6β and C-7aβ (but not at C-11) by R. arrhizus. Δ1 steroids of conventional skeleton were hydroxylated at C-11α by R. arrhizus; in addition, Δ1-androstenedione was epoxidized at the 1,2β position. No C-6 hydroxylation of conventional Δ1,4-3-ketosteroids was observed.

The 13Cmr spectra of 21-haloprogesterones have been assigned, and chemical shifts compared with those of simple α-haloketones. In addition, the 13Cmr spectra of some C-5α and C-6β halosteroids are presented, and long range 13C—19F coupling observed between C-19 and the C-6β fluorine in some cases. The spectra of several oxygenated analogues and of a series of 5α-hydroxy-6-ketosteroids are also discussed.

Seven alkaloids have been isolated from Lycopodium paniculatum Desvaux. Three of these, lycopodine, dihydrolycopodine, and acetyldihydrolycopodine are alkaloids of established structure. Of the four new alkaloids the structure of one, paniculatine, has been clarified. Its structure, elucidated through an X-ray crystallographic study of its hydrobromide, is different from other alkaloids of the Lycopodium family. The spectral properties of paniculatine are interpreted in the light of its structure.

A series of C-3 oxygenated and unsubstituted (4,5)α, (4,5)β, (5,6)α, and (5,6)β epoxy steroids has been prepared, and the reactions of these compounds with strong bases (potassium tert-butoxide, LDA, and LDEA) were investigated. Only LDEA gave rise to product formation; β elimination of the epoxide to give a β-hydroxy olefin was observed in this case. The regioselectivity of product formation is consistent with a mechanism of rearrangement involving removal of a hydrogen located syn to the epoxide oxygen. In some cases, a directing influence from a polar substituent (OH) of the starting material was also apparent. The 13C nmr spectra of the steroidal epoxides used in this study were assigned; these data are diagnostic of the conformation of ring A of (4,5)α and (4,5)β epoxy steroids.

The reduction product of dehydrocordrastine, 6 , with di-isobutylaluminum hydride spontaneously rearranged to the spirobenzylisoquinolines 7a and 7b , analogous to the alkaloids sibiricine ( 5a ), corydaine ( 5b ), and yenhusomidine ( 5c ).

The fungus Mortierella isabellina converts ethyl benzene and a number of parasubstituted derivatives to the corresponding optically active 1-phenylethanols with enantiomeric excesses between 5 and 40%. Hydrogen removal from the substrate preceeds product formation and is stereochemically independent of it.

The products arising from the incubations of C-6α and C-6β chloro-, fluoro-, and methyl-substituted Δ4-3-keto steroids with the C-6β hydroxylating fungus Rhizopusarrhizus ATCC 11145 have been identified, and their formation rationalised in terms of Δ3,5 enolic intermediates. The incubations of C-6 chloro and methyl Δ3,5 enol acetates with R. arrhizus, and the oxidations by m-chloroperoxybenzoic acid of the corresponding enol ethyl ethers have also been described. The relevance of the results so obtained to the mechanism of C-6β hydroxylation of Δ4-3-keto steroids by R. arrhizus is discussed.

A series of para-substituted alkyl aryl sulphides has been transformed stereoselectively to the sulphoxide by incubation with the fungus Mortierellaisabellina. The enantiomeric purity of the products was dependent on the nature of substitution in the aromatic ring and at sulphur. Comparisons of the enantiomeric purities obtained from sulphides with para substituents of the same steric size but different electronic properties indicate that the stereoselectivity of S oxidation is susceptible to electron withdrawing or donating factors. This conclusion lends credence to a stepwise oxidation mechanism involving an electron deficient sulphur intermediate.

A two-step synthesis of the title compound from [cyano-14C]-copper(I) cyanide is described. The method is extensible to the preparation of carbonyl-labelled 3,4-dihydroxybenzaldehyde.

3-18O-Testosterone (1) is transformed to estradiol (4) by human placental microsomes, and to the 6β- and 11α-hydroxy derivatives 2 and 3 by Rhizopusarrhizus, without significant loss of label. Neither process is therefore one which involves reversible Schiff base formation at C-3 at any stage.

The C-21 hydroxyl group of C-17- and C-21-deuterium labelled tetrahydrodeoxycorticosterone is removed by the anaerobic intestinal bacterium Eubacteriumlentum ATCC 25559 with retention of deuterium label at C-17α and loss of one of the two deuterium atoms originally present at C-21. Dehydroxylation at C-21 proceeds with an apparent primary kinetic isotope effect, KH/KD, at C-21 of 2.6. These data suggest a route for C-21 dehydroxylation involving a C-20(21)-enediol intermediate.

Seven alkaloids have been isolated from Lycopodium paniculatum Desvaux. Three of these, lycopodine, dihydrolycopodine, and acetyldihydrolycopodine are alkaloids of established structure. Of the fou...

Incubation of the oxathiolanes (I) with the fungus Mortierella isabellina leads to sulfoxidation yielding the products (II) as single diastereomers, whose relative stereochemistry is assigned by 1H NMR analysis.

Incubations of several polycyclic aromatic hydrocarbons (PAHs) and heteroaromatic compounds with a series of common micro-organisms have been performed. The PAHs were not metabolized by any of the fungi studied. The sulphur-containing heterocyclic aromatic compounds dibenzothiophene, thioxanthone and thiochromanone were oxidized at sulphur by C. elegans. Other fungi are capable of oxidation at the sulphur atom of dibenzothiophene and thioxanthone. C-1 and C-3 methyl substituted thioxanthones are hydroxylated at the methyl group by C. elegans.

Bei der Persaureoxidation der Steroide (I) werden die im Formelschema angegebenen Oxidationsprodukte erhalten.

3-18O-Testosterone (1) is transformed to estradiol (4) by human placental microsomes, and to the 6β- and 11α-hydroxy derivatives 2 and 3 by Rhizopusarrhizus, without significant loss of label. Neither process is therefore one which involves reversible Schiff base formation at C-3 at any stage.

HERBERT L. HOLLAND and BARBARA J. AURET. 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 20aand 20P-hydroxypregn-4-ene-3-one (2a and 2b) by A. niger has been observed.

Biotransformation by resting cultures of the aerobic fungi Mortierella isabellina NRRL 1757 and Helminthosporium species NRRL 4761 can be used as method for the removal of O- and N-benzyl groups, respectively, under neutral, room temperature conditions.