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A comparative molecular field analysis (CoMFA) was used to model the efficacy with which the Rhodococcus erythropolis mono-oxygenase, DszC, catalyzes the enantioselective sulfoxidation of a broad range of substrates. Experimentally determined values of both the yield and enantiomeric excess for this reaction were employed to create these CoMFA models. A highly predictive CoMFA model was constructed for the prediction of enantiomeric excess of the sulfoxide product. The predictive ability of the model was demonstrated by both cross-validation of the training set ( q 2 = 0.74) and for an external test set of substrates. The enantiomeric excesses of the members of the test set, which also included two amino acid sulfides that were structurally distinct from the membership of the training set, were predicted well by the CoMFA model. Product yield was not modelled well by any CoMFA model. Different models comparing the likely bioactive conformations of the substrates suggest that most compounds assume an ‘extended’ conformation upon binding. Contour diagrams illustrating significant substrate–enzyme interactions suggest that the model, which predicts the enantiomeric excess, is consistent with previous conclusions regarding the effect of various substrate substitutions on the enantiopurity of the product of the biotransformation.

A version of Rhodococcus erythropolis IGTS8 BKO-53, designed as a model system for the biodesulfurization of crude oil with a high conversion activity of dibenzothiophenes to the corresponding sulfoxides has been used for oxidation of a large number of simple sulfides. A large variety of sulfides were converted to chiral sulfoxides in good yield. Sulfoxide stereoisomers were generally formed as (R) configuration in moderate stereochemical purity, but the sulfoxide diasteromers of methionine amino acid derivatives were produced at >90% optical purity.

Treatment of N -methoxycarbonyl C -carboxylate ester derivatives of S -methyl- l -cysteine by chloroperoxidase (CPO)/hydrogen peroxide resulted in oxidation at sulfur to produce the ( R S ) sulfoxide in moderate to high diastereomeric excess (DE). The ( S S ) natural product sulfoxide chondrine was obtained via biotransformation of the N -t.boc derivative of l -4-S-morpholine-2-carboxylic acid using Beauveria bassiana or Beauveria caledonica .

Treatment of N-methoxycarbonyl C-carboxylate ester derivatives of L- and D-methionine and L-ethionine by chloroperoxidase–hydrogen peroxide resulted in oxidation at sulfur to produce the (RS) sulfoxide in moderate to high diastereomeric excess. The (RS) sulfoxide of methionine was also obtained in moderate to high diastereomeric excess from (±)SO-N-methoxycarbonyl-L-methionine methyl ester sulfoxide by ester hydrolysis using α-chymotrypsin, Aspergillus sp. protease or subtilisin Carlsberg. Key words: amino acid oxidation, biocatalysis, biotransformation, chloroperoxidase, enzyme catalysis, lipase, sulfoxidation.

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.

It has been shown previously that the vanadium bromoperoxidase from Ascophyllum nodosum mediates the production of ( R )-methyl phenyl sulfoxide with 91% enantiomeric excess from the corresponding sulfide in the presence of hydrogen peroxide. Investigation of the sulfoxidation activity of this enzyme shows that activating substituents at the para -position of the aromatic ring of methyl phenyl sulfide positively influence the selectivity of the reaction, whereas strongly electron-withdrawing groups cancel the catalyzed sulfoxidation reaction. The first evidence is presented that the vanadium bromoperoxidase catalyzes the sulfoxidation of racemic non-aromatic cyclic thioethers with high kinetic resolution.

Biotransformations of the N -phthaloyl derivatives of d - and l -methionine and of d - and l -ethionine by Beauveria bassiana ATCC 7159 or Beauveria caledonica ATCC 64970 produce the corresponding ( S S ) sulfoxides in good yield and diastereomeric excess. Pure ( S S S C ) diastereomers can be obtained from l -series substrates by crystallisation of the biotransformation extract, and the corresponding ( S S R C ) products obtained from d -series substrates by chromatography of the biotransformation extract. Hydrogen peroxide-catalysed oxidation of the N -phthaloyl derivatives of d - and l -methionine and of d - and l -ethionine gives diastereomeric mixtures from which the ( S S S C ) and ( R S R C ) diastereomers can be obtained by crystallisation, and the ( S S R C ) and ( R S S C ) diastereomers obtained by chromatography. N- Cbz- and N - t -Boc methionines are also converted to sulfoxides with predominant ( S S ) configuration by both B. bassiana and B. caledonica , but the isolated yields and d.e. of products were generally lower than those obtained from the N- phthaloyl substrates. Removal of the N- phthaloyl group from diastereomerically pure methionine and ethionine sulfoxides gave the corresponding amino acid sulfoxides in high yield; removal of N- Cbz and N - t -Boc groups from protected methionine sulfoxides was also achieved without loss of configuration at sulfur.

The biohydroxylation of a series of amides and related amino, keto and hydrocarbon substrates by the fungal biocatalyst Beauveria bassiana ATCC 7159 has been examined. The product distributions, together with data obtained from selective inhibition experiments using the cyt.P-450 inhibitors isosafrole, 1-aminobenzotriazole and phenylacetylene, suggest that B. bassiana contains a range of hydroxylase enzymes with different substrate specificities. A paradigm is presented for the interpretation of the results of microbial hydroxylation and for the application of existing active site models for B. bassiana.

Benzyl methyl sulfides substituted with methyl, chloro, cyano, bromo, methoxy, nitro and amino groups in the ortho or meta positions of the aromatic ring have been converted to ( S ) sulfoxides by biotransformation using the fungal biocatalyst Helminthosporium species NRRL 4671. The enantiomeric excesses for meta -substituted examples were high in those cases where the substituent was of a polar nature, and comparable to those observed for the corresponding para -substituted substrates. With one exception ( o -amino), the ortho -substituted examples gave sulfoxides of lower enantiomeric purity. The role of a suitably located polar substituent on an aryl ring of the substrate in ensuring a high enantiomeric excess in sulfoxidation by Helminthosporium species has been confirmed by the biotransformations of 4-(methylthiomethyl)benzyl alcohol and 2-(4-nitrophenyl) ethyl methyl sulfide, which give sulfoxides of much higher optical purity than those obtained from the corresponding unsubstituted substrates.

The enantioselective oxidation of a series of heterocyclic prochiral sulfides to chiral sulfoxides has been examined using the fungal biocatalysts Helminthosporium species NRRL 4671 and Mortierella isabellina ATCC 42613. Methylthiofuranyl and -thiophenyl substrates gave (S)-configuration products in low to moderate enantiomeric purity on biotransformation with H. species, but pyridyl sulfides with the nitrogen atom located at an optimal distance of 8-10 Å from the sulfur centre gave (S) sulfoxides of high enantiomeric purity. The biotransformation of appropriately substituted benzothiane substrates by M. isabellina also gave products of high enantiomeric purity, but with (R) configuration at sulfur. The acceptability of the substrate and the configuration of sulfur oxidation by both H. species and M. isabellina for the range of substrates examined were found to be consistent with predictions based on cubic space models for these oxidations.Key words: bioconversion, biotransformation, Helminthosporium, Mortierella isabellina, sulfide, sulfoxide.

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.

The enantioselective oxidation of a series of heterocyclic prochiral sulfides to chiral sulfoxides has been examined using the fungal biocatalysts Helminthosporium species NRRL 4671 and Mortierella isabellina ATCC 42613. Methylthiofuranyl and -thiophenyl substrates gave (S)-configuration products in low to moderate enantiomeric purity on biotransformation with H. species, but pyridyl sulfides with the nitrogen atom located at an optimal distance of 8-10 A from the sulfur centre gave (S) sulfoxides of high enantiomeric purity. The biotransformation of appropriately substituted benzothiane substrates by M. isabellina also gave products of high enantiomeric purity, but with (R) configuration at sulfur. The acceptability of the substrate and the configuration of sulfur oxidation by both H. species and M. isabellina for the range of substrates examined were found to be consistent with predictions based on cubic space models for these oxidations.

An investigation has been undertaken of the microbial biotransformation of 2-oxatestosterone by microorganisms known to hydroxylate conventional steroids, using Aspergillus ochraceus, Bacillus megaterium, Curvularia lunata and Rhizopus arrhizus. A. ochraceus and B. megaterium gave products of 11α- and 15β-hydroxylation, respectively. Biotransformation by C. lunata gave C-11β- and C-14α-hydroxylated products, whereas R. arrhizus produced only the 6β-hydroxy derivative.

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.

Para-substituted phenyl 3-chloropropyl and phenyl 3-hydroxypropyl sulfides have been oxidised to the corresponding (R)-sulfoxides by biotransformation with Mortierella isabellina ATCC 42613, and to the (S)-sulfoxides by biotransformation with Helminthosporium species NRRL 4671 or Acinetobacter calcoaceticus NCIMB 9871. The stereoselectivity of these oxidations was consistent with that predicted by examination of previously published models for sulfoxidation by these biocatalysts. In several cases ((R)- and (S)-p-cyanophenyl 3-chloropropyl sulfoxide, (S)-p-cyanophenyl 3-hydroxypropyl sulfoxide, and (R)-p-methoxyphenyl 3-chloropropyl sulfoxide) enantiopure materials were obtained by crystallisation of the biotransformation products. The latter are valuable chiral starting materials for the synthesis of analogues of the cardiovascular agent verapamil.

Washed-cell preparations of recombinant Escherichia coli JM109(pDTG141), engineered to express the naphthalene dioxygenase (NDO) gene from Pseudomonas sp. NCIB 9816-4, have been used to biooxidise a range of aryl alkyl-, dialkyl- and bicyclic sulfides. A series of 16 phenyl alkyl sulfides was oxidised to equivalent sulfoxides, typically with moderate to high (>90%) yield and high enantioselectivity (>85% ee), the ( S )-enantiomer being the predominant product, with little if any further oxidation. The addition of some electron-donating or electron-withdrawing groups to the phenyl ring decreased yield and/or stereoselectivity of the NDO-catalysed biotransformation, whereas increasing the size of the alkyl chain ( n C 3 H 7 , i C 3 H 7 and n C 4 H 9 ) resulted in a notable inversion in selectivity to yield ( R )-series sulfoxides (>74% ee) as the predominant products. The addition of one or more methylene groups between the phenyl ring and sulfur atom resulted in notable reductions in both the yield and stereoselectivity of the ( S )-predominant sulfoxidations. With the exception of cyclohexyl- and n -hexyl methyl sulfide which both gave ( S )-sulfoxides with good stereoselectivity and yield, other dialkyl- and bicyclic sulfides were poor substrates for sulfoxidation by NDO. Both the close agreement with data obtained using purified NDO and the absence of stereoselective sulfoxidation in equivalent controls with the E. coli JM109 host support the contribution made by the cloned NDO carried on the pDTG141 plasmid.

Biocatalytic or chemical oxidations can be used in a complementary manner for the preparation of all four diastereomers of methionine sulfoxide with high diastereomeric purity in overall isolated yields of 20–55% from methionine. The N-phthaloyl derivatives of L- and D-methionine were selectively oxidised to the ( S S S C ) and ( S S R C ) sulfoxides respectively by biotransformation using the fungus Beauveria bassiana ATCC 7159. Hydrogen peroxide oxidation of the same materials gave mixtures from which the ( S S S C ) and ( R S R C ) isomers can be readily isolated by crystallisation. Chromatography of the residual material then afforded the ( R S S C ) and ( S S R C ) isomers.

The fungus Mortierella isabellina ATCC 42613 catalyses the biotransformation of substituted-aryl methyl sulfides to give sulfoxides of predominantly (R) configuration; of aryl-substituted alkenes to give chiral vicinal diols; and of various phenylalkanes and phenylcycloalkanes to give (R)-configuration benzylic alcohols. The nature of the products from all these processes may be accounted for by a single model based on restrictive space descriptors that can be used to rationalise these reactions, and which is proposed as a predictor of the outcome of the M. isabellina-catalyzed oxidations of similar substrates.

The fungus Helminthosporium species NRRL 4671 converts a wide range of prochiral sulfides to the corresponding chiral sulfoxides, the majority of which have ( S ) configuration at sulfur. The formation of a series of chiral cyclopentyl alkyl, cyclohexyl alkyl, benzyl alkyl, and methyl alkyl sulfoxides by biotransformation of the corresponding sulfides using Helminthosporium species is described. The analysis of over 90 such biotransformations has resulted in the development of a model based on restrictive space descriptors that has been used to rationalize these reactions, and that is proposed as a predictor of the outcome of Helminthosporium -catalyzed sulfoxidations.

The stereopreference of cyclohexanone monooxygenase for a series of benzyl methyl sulfides bearing in the p-position groups with different σp values has been rationalized by locating inside the enzyme active site model electron-rich and electron-poor regions.

Phenyl methyl sulfides substituted in the para position with methyl, fluoro, chloro, bromo, cyano, nitro, amino, acyl, methoxy, thiomethyl and methylsulfinyl groups have been converted to ( S ) sulfoxides by biotransformation using Helminthosporium species NRRL 4671. The highest yields and enantiomeric excesses were obtained with bromo, cyano, methoxy, thiomethyl and methylsulfinyl substituents and in two cases ( para -Br and -CN) the products could be crystallized to give ( S ) sulfoxide of ≥ 96% ee.

Several steroidal [4,6-b,c]benzothiazepines have been prepared via base-catalyzed Michael addition of 2-aminothiophenol to 3β,17β-diacetoxyandrost-4-en-6-one. The product of this reaction has the 4β,5α stereochemistry, but cyclizes to a benzothiazepine with the steroidal 4β,5β configuration, confirmed by X-ray crystallographic analysis. 2-Aminothiophenol reacts with 3β,17β-diacetoxyandrost-4-en-6-one under acidic conditions to give a steroidal 6-spiro-benzothiazole. An androst-4-ene-3,17-dione-based [4,6]benzothiazepine has been shown to be a moderate competitive inhibitor of the human placental aromatase enzyme with IC50 = 42.3 µM. Keywords: steroid, aromatase, benzothiazepine.

1-Methoxy-2-naphthol was converted by MOM ether formation, lithiation at C-3, reaction with 3,3-dimethylacryloyl chloride, cyclization, and oxidation using cerium ammonium nitrate to 4-keto-α-lapachone (2,2-dimethyl-4-keto-1-oxa-1,2,3,4-tetrahydroanthra-5,10-quinone). Reduction by biotransformation using Mortierellaisabellina ATCC 42613 gave (S)-4-hydroxy-α-lapachone in high (> 95%) optical purity. Reduction of several 2,2-dimethyl-4-chromanones to the corresponding (S)-alcohols by M. isabellina is also reported. Keywords: biotransformation, chemoenzymatic, 4-hydroxy-α-lapachone, Mortierellaisabellina.

The fungus Helminthosporium species NRRL 4671 has been used for the biotransformation of a series of para -alkylbenzyl sulfides with alkyl groups consisting of methyl, ethyl, n.propyl, isopropyl, n.butyl, and t.butyl. 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. For substrates with alkyl groups other than methyl, hydroxylation of the alkyl substituent at the terminal carbon atom was also observed. The latter reaction was investigated by the use of isopropylbenzene, t.butylbenzene, and (±) para n.butylbenzyl methyl sulfoxide as substrates. The data so obtained suggest that, during biotransformation of para -alkyl benzyl methyl sulfides by Helminthosporium , S -oxidation precedes carbon hydroxylation.

The fungi Helminthosporium species NRRL 4671 and Mortierella isabellina ATCC 42613 have been used for the biotransformation of a series of isothiocyanatoalkyl methyl sulfides and their synthetic precursors, ω-(methylthio)alkylphthalimides. H . species gave predominantly ( S ) sulfoxides in all cases; M. isabellina gave ( R ) isothiocyanatoalkyl methyl sulfoxides, but in the case of two ω-(methylthio)alkylphthalimides substantial conversion of sulfoxide to sulfone resulted in the isolation of the former with predominat ( S ) configuration. A correction is made of the previously reported configurations of two biotransformation products (Tetrahedron: Asymmetry, 1994 , 5 , 1129).

An active site model to explain and predict the stereoselectivity of the sultoxidation of organic sulfides to optically active sulfoxides catalyzed by cyclohexanone monooxygenase from Acinetobacter NCIB 9871 is proposed. The model is based on cubic-space descriptors and derives from the results obtained with over 30 different sulfides. The model, which also appears to be suitable for explaining the stereoselectivity of cyclohexanone monooxygenase-catalyzed Baeyer-Villiger reactions, is consistent with and expands on the features of other previously described models for the latter process.

Benzyl alkyl sulfides with alkyl groups from methyl to hexyl, para -alkylbenzyl groups from methyl to butyl, 2-phenylethyl methyl sulfide and 3-phenylpropyl methyl sulfide have been oxidized by cyclohexanone monooxygenase from Acinetobacter NCIB 9871. Sulfoxides with enantiomeric excesses ranging from 80% for the ( R )-configuration to 96% ee for the ( S )-configuration were obtained.

The use of ( S )- α -methoxyphenyl acetic acid (MPAA) as a general chiral 1 H NMR shift reagent for the stereochemical analysis of sulfoxides is demonstrated. Using this methodology, both the enantiomeric purity and the absolute configuration of a wide variety of sulfoxides can be determined.

We have investigated the use of lipoprotein lipase enzymes from Pseudomonas sp. and Pseudomonas fluorescens for the enantioselective resolution and desymmetrization of racemic binaphthols. The reactions were carried out using a non-aqueous environment (iPr 2 O/acetone/vinyl acetate), and yielded mono-acetate ester products of the parent unsubstituted substrate, the 6,6′-dibromo-substrate, and the 6,6′-dimethoxy-substrate with high enantiomeric selectivity.

The various models that have been proposed for microbial biotransformations involving carbon hydroxylation and sulfide oxidation are reviewed. From these data, those substrate features that are important in controlling the regiochemistry and stereochemistry of these oxidative bioconversions are identified.

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.

The fungus Mortierella isabellina ATCC 42613 converts phenyl-substituted olefins to vicinal diols. The substrate selectivity and product stereochemistry of this biotransformation can be rationalized by application of the model developed for benzylic hydroxylation of aromatic hydrocarbons by M isabellina.

The 15-thia-15-deoxy analogue of prostaglandin E1 methyl ester has been prepared by the zirconocene chloride mediated conjugate addition of an n-pentyl ethynyl sulfide derived anion to an appropriately substituted cyclopentenone. Similar methodology has also been used to prepare other 3-(3′-thia-1′-octenyl)-cyclopentanones.

Single biotransformations of 1-(phenylthio)-2-propanone and 1-(p-methoxyphenylthio)-2-propanone by the fungus Helminthosporium species NRRL 4671 resulted in both sulfur oxidation to the sulfoxide and carbonyl reduction to the alcohol. The corresponding (SS,SC)-1-sulfinyl-2-propanols were obtained as single diastereomers following simple crystallization.Key words: biocatalysis, biotransformation, carbonyl reduction, Helminthosporium sp. NRRL 4671, sulfoxidation.

The active site topography of the hydroxylase enzyme of Mortierella isabellina ATCC 42613, which carries out the benzylic hydroxylation of toluene, ethylbenzene, and related compounds, has been explored. Operating in a whole cell biotransformation mode, this enzyme shows selectivity in substrate processing based on the nature, position and size of substituent side chains close to the site of hydroxylation. The result of determination of the yield and stereochemistry of hydroxylation of over twenty substrates and potential substrates, together with previously reported data, have been used to propose an active site model for the benzylic hydroxylase enzyme.

(E)-3β,17β-Dihydroxyandrost-4-en-6-one oxime has been converted into (E)-6- hydroximinotestosterone and (E)-6-hydroximinoandrost-4-ene-3,17-dione by chemical methods, and into (E)-6-hydroximinoandrosta-1,4-diene-3,17-dione by biotransformation using Arthrobacter simplex ATCC 6946.

An efficient method for the preparation of multi-gram quantities of ethynyl sulfides is exemplified by the synthesis of ethynyl n.pentyl sulfide. Treatment of the latter with zirconocene chloride hydride/N-iodosuccinimide gave E-2-iodoethenyl n.pentyl sulfide in high yield and stereoselectivity. The latter compound rearranged to the Z isomer on storage at −20°C.

The 15-thia-15-deoxy analogue of prostaglandin E1 methyl ester has been prepared by the zirconocene chloride mediated conjugate addition of an n-pentyl ethynyl sulfide derived anion to an appropriately substituted cyclopentenone. Similar methodology has also been used to prepare other 3-(3′-thia-1′-octenyl)-cyclopentanones.

The fungus Helminthosporium species NRRL 4671 oxidizes 1-isothiocyanato-4-(methylthio)butane to (−)-1-isothiocyanato-(4R)-(methylsulfinyl)butane (sulforaphane). Helminthosporium also converts 1-methylthio-4-(N-phthalimidyl)butane to the (R) sulfoxide.

Benzyl alkyl sulfides with alkyl groups from methyl to hexyl, para -alkylbenzyl groups from methyl to butyl, 2-phenylethyl methyl sulfide and 3-phenylpropyl methyl sulfide have been oxidized by cyclohexanone monooxygenase from Acinetobacter NCIB 9871. Sulfoxides with enantiomeric excesses ranging from 80% for the ( R )-configuration to 96% ee for the ( S )-configuration were obtained.

The fungus Mortierella isabellina ATCC 42613 converts phenyl-substituted olefins to vicinal diols. The substrate selectivity and product stereochemistry of this biotransformation can be rationalized by application of the model developed for benzylic hydroxylation of aromatic hydrocarbons by M isabellina.

The fungi Helminthosporium species NRRL 4671 and Mortierella isabellina ATCC 42613 have been used for the biotransformation of a series of isothiocyanatoalkyl methyl sulfides and their synthetic precursors, ω-(methylthio)alkylphthalimides. H . species gave predominantly ( S ) sulfoxides in all cases; M. isabellina gave ( R ) isothiocyanatoalkyl methyl sulfoxides, but in the case of two ω-(methylthio)alkylphthalimides substantial conversion of sulfoxide to sulfone resulted in the isolation of the former with predominat ( S ) configuration. A correction is made of the previously reported configurations of two biotransformation products (Tetrahedron: Asymmetry, 1994 , 5 , 1129).