Biotransformation of beta-ketosulfides to produce chiral beta-hydroxysulfoxides.

The biotransformations of a series of substituted phenylthio-2-propanone and benzylthio-2-propanone were carried out using Helminthosporium sp. NRRL 4671, Mortierella isabellina ATCC 42613, or Rhodococcus erythropolis IGTS8. Several products gave microbial oxidation of sulfide to sulfoxide and reduction of carbonyl to secondary alcohol, producing beta-hydroxysulfoxides in medium to high enantiomeric and diastereomeric purities. Fungal biotransformations using Helminthosporium sp. and M. isabellina resulted in the opposite sulfoxide configurations of various beta-hydroxysulfoxide products.

Enzymatic hydroxylation reactions.

During the past 18 months, considerable progress has been made in the understanding of the key enzyme-substrate interactions that control the regioselectivity and stereoselectivity of the hydroxylation reaction performed by cytochrome-P450-dependent enzymes of mammalian origin. The manipulation of microbial hydroxylating enzymes, in both whole-cell and cell-free environments, has also been examined in the context of controlling the regioselectivity and stereoselectivity of the hydroxylation reaction. Several new applications for hydroxylating enzymes have been reported, and the construction of chimeric hydroxylating enzymes has been used both for mechanistic studies and for the production of enzymes with high hydroxylating activity for a defined substrate.

Recent advances in applied and mechanistic aspects of the enzymatic hydroxylation of steroids by whole-cell biocatalysts.

Recent advances in microbial steroid hydroxylation are covered, including new biocatalysts and substrate groups and new methodologies such as the use of low-water systems, immobilised biocatalysts, genetically constructed biocatalysts and enzyme mimics. Mechanistic factors that control the regiochemistry and stereochemistry of steroid hydroxylation are also discussed.

Enantioselective synthesis and pharmacological evaluation of a new type of verapamil analog with hypotensive and calcium antagonist activities.

PURPOSE: The syntheses and evaluation for cardiovascular activity in the rat of both enantiomers of a verapamil analog in which the cyano group has been replaced by hydroxyl. METHODS: (+)- and (-)-alpha-[3-[[2-(3,4-Dimethoxyphenyl)ethyl]methylamino]propyl]- 3,4-dimethoxy-alpha-(1-methyl ethyl)benzyl alcohol were prepared from chiral sulfoxides produced by microbial biotransformations using Mortierella isabellina ATCC 42613 or Helminthsporium species NRRL 4671, and were examined for hypotensive and calcium antagonist activity using anaesthetized normotensive rats and isolated rat aorta and atria. RESULTS: The analogs showed a pharmacological profile similar to that exhibited by verapamil, possessing a remarkable hypotensive activity, accompanied by a significant bradycardia, in anaesthetized normotensive rats. In vitro, these analogs displayed clear inhibitory effects: in isolated rat aorta they inhibited, in a concentration-dependent fashion, the contractions and 45Ca2+ uptake induced by norepinephrine and high KCl, and in isolated rat atria the analogs considerably decreased the rate of contraction (negative chronotropic effects). No significant differences between the quantitative cardiovascular effects produced by the two enantiomers of the verapamil analogs were observed. CONCLUSIONS: The results suggest that, like that of verapamil, the cardiovascular activity exhibited by the new compounds seems to be due, at least in part, to a blockage of transmembrane calcium channels present in vascular smooth muscle cells.

C-H activation.

Although the biocatalytic activation of carbon-hydrogen bonds by oxidation to alcohols was one of the first biotransformations to be exploited by the chemical industry, this remarkable process continues to be the subject of intense research activity. Recent developments in this area have centred on the use of new biocatalysts and substrates for C-H activation, in particular on the use of recombinant strains of yeast and bacteria expressing useful hydroxylase enzymes, on mechanistic work using hydroxylase enzymes that relies on the tools of molecular biology to address outstanding questions, particularly those of substrate selection and product predictability, and on the application of these systems for hydroxylation and heteroatom dealkylation reactions.

Microbial hydroxylation of acetylaminosteroids.

An investigation of the microbial biotransformation of a range of 3 beta-, 17 beta-, and 20-acetylamino C18 to C21 steroids by microorganisms known to hydroxylate conventional steroids has been undertaken, using Aspergillus ochraceus, Bacillus megaterium, Curvularia lunata, and Rhizoputus arrhizus. A. ochraceus and B. megaterium gave products of 11 alpha- and 15 beta-hydroxylation, respectively. In the case of C. lunata, the products were predominantly those of this organism's normal C-11 beta- and C-14 alpha-hydroxylating pathways, but in one instance, 3 beta-acetylamino-7 alpha-hydroxy-5 alpha-androstan-17-one, appeared to results from direction of the site of hydroxylation by the substitution pattern of the substrate. The products from R. arrhizus generally corresponded to those previously obtained from normal steroids of similar skeleton, with 6 beta- 11 alpha-hydroxylation predominating, but again the sites of hydroxylation and the range of hydroxylated products were found to depend on the substitution pattern of the substrate.

Microbial transformations.

Although the use of microbial biocatalysts for chemical reactions pre-dates by a considerable margin the application of isolated enzymes for this purpose, considerable progress continues to be made in the use of whole-cell catalysts. The application of whole-cell biocatalysts in oxygenase-catalysed reactions, the reductions of carbonyl and nitro groups, and the hydrolysis of nitriles and epoxides, are all areas of continued development. In addition, the use of microbial transformations in the production of drug metabolites, carbohydrates, and amino acids is expanding. Methodological development such as the application of novel immobilisation techniques or the use of non-aqueous solvents continues to enhance the usefulness of microbial biocatalysts.

Biotransformation of corticosteroids by Penicillium decumbens ATCC 10436.

The biotransformation of a series of corticosteroids by the fungus Penicillium decumbens ATCC 10436 has been investigated. Conversion to the corresponding 5 alpha-dihydrosteroid was observed for all the delta 4-3-ketosteroids studied with the exception of deoxycorticosterone, which was converted to a delta 1.4-diene. Deoxycorticosterone acetate was, however, converted to a 5 alpha-dihydro product concomitant with ester hydrolysis. Other substrates carrying a C-21 acetoxy group were also hydrolyzed to the alcohol. In two cases (resulting from deoxycorticosterone acetate and 11-deoxycortisone) the 5 alpha-3-keto product was further reduced to the 3 beta-alcohol. No reduction of delta 1.4-dienes was observed.

Formation of 5 alpha steroids by biotransformation involving the 5 alpha-reductase activity of Penicillium decumbens.

The biotransformation of a series of delta 4-3-ketosteroids by the fungus Penicillium decumbens ATCC 10436 has been investigated. Conversion to the 5 alpha-dihydrosteroid was observed for several substrates of the androstene and pregnene series: the reaction is tolerant of non-polar substituents (Cl and CH3) at C-4 of the substrate, but does not occur in the presence of a 4-hydroxyl group, or with additional unsaturation at the delta 1 or delta 6 positions. A-nor-, B-nor-, 3-deoxy-, and 3,5-cycloandrostanes are not reduced, but 6-methylenetestosterone is converted to a 6-methylene-5 alpha-dihydro derivative. Several biotransformations are reported which involve oxidoreductase activity at C-3 and/or C-17, either concomitant or independent of delta 4 reduction: the substrate specificity of the oxidoreductase processes has been examined and defined by the use of 3 alpha-hydroxy, 3 beta-hydroxy, 3-keto, 17 beta-hydroxy and 17-keto substituted steroids. In this way, the existence in P. decumbens of 3 beta-hydroxy-3-keto and 17 beta-hydroxy-17-keto oxidoreductases has been demonstrated.

Biotransformation of organic sulfides--IV. Formation of chiral benzyl alkyl and phenyl alkyl sulfoxides by Helminthosporium species NRRL 4671.

The fungus Helminthosporium species NRRL 4671 has been used for the biotransformation of a series of phenyl alkyl sulfides with alkyl groups ranging from methyl to n-hexyl, and benzyl alkyl sulfides with alkyl groups from methyl to n-nonyl. Several 2-phenylethyl and 3-phenylpropyl sulfides have also been examined as substrates, together with cyclohexyl methyl sulfide and 1- and 2-naphthyl methyl sulfides. For the majority of substrates, sulfoxide formation occurred in moderate yield and with predominant (S) chirality at sulfur; lesser amounts of sulfone product were also obtained in some cases. The data so obtained have been used to define the preparatively useful limits of S-oxidation of phenyl alkyl sulfides and benzyl alkyl sulfides by biotransformation using Helminthosporium.

2-Methoxycanthin-6-one: A New Alkaloid from the Stem Wood of Quassia amara.

A new alkaloid, 2-methoxycanthin-6-one ( 1) has been isolated from the methanol extract of the stem wood of QUASSIA AMARA. In addition quassin ( 6) was also isolated. The structure of 1 was determined by spectroscopic methods.

Fungal biotransformation of organophosphines.

1. The biotransformations of triphenyl phosphite and of triphenyl-, tri-n.-butyl-, diethylphenyl-, and ethylmethylphenyl-phosphines by the fungi Mortierella isabellina ATCC 42613, Helminthosporium species NRRL 4671, and Aspergillus foetidus ATCC 10254, have been examined. 2. The triaryl and tri-n.-butyl substrates underwent oxidation at phosphorus in low yield, a process attributed largely to auto-oxidation under the experimental conditions used. 3. The alkyl aryl phosphines were enzymically oxidized at phosphorus, but concurrent auto-oxidation also occurred. 4. Incubation of ethylmethylphenyl phosphine with M. isabellina gave the corresponding S(-) phosphine oxide in a 92% yield with an enantiomeric purity of 13%.

Effect of cell immobilization and organic solvents on sulfoxidation and steroid hydroxylation by Mortierella isabellina.

The effects of calcium alginate bead immobilization and the presence of organic solvents on two bioconversion reactions carried out by Mortierella isabellina ATCC 42613 have been investigated. These reactions, the 14 alpha-hydroxylation of progesterone and the sulfoxidation of thioanisole, both proceed in high yield using resting-cell bioconversions, but are not carried out by alginate bead preparations in the absence of an organic co-solvent, the best results being obtained with 5 or 10% aqueous methanol. The stereoselectivity of sulfoxidation of thioanisole was found to be dependent upon the nature and concentration of organic co-solvent.

Preparation of deuterium and tritium labelled norethynodrel, norethindrone, and 6-methyleneandrostenedione.

PIP: 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.^ieng

Biotransformation of polycyclic aromatic compounds by fungi.

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.