Herbert L. Holland

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.

The biocatalytic reactions of Beauveria spp.

Fungi of the Beauveria spp., typified by Beauveria bassiana ATCC 7159, are among the most frequently used whole cell biocatalysts. They have been reported to catalyse many different reactions, including oxidative, reductive and hydrolytic transformations, of a wide range of substrates. This review covers the range and application of biocatalytic reactions of Beauveria spp., with emphasis on the scope and utility of Beauveria-catalysed reactions for preparative biotransformations.

A predictive active site model for the cyclohexanone monooxygenase catalyzed oxidation of sulfides to chiral sulfoxides

Abstract 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.

(S)-α-methoxyphenyl acetic acid : a new NMR chiral shift reagent for the stereochemical analysis of sulfoxides

Abstract 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.

A new paradigm for biohydroxylation by Beauveria bassiana ATCC 7159

Abstract 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.

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.

Biotransformation of organic sulfides. Part 7. Formation of chiral isothiocyanato sulfoxides and related compounds by microbial biotransformation

Abstract 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).

Biocatalytic and chemical routes to all the stereoisomers of methionine and ethionine sulfoxides

Abstract 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 (SS) sulfoxides in good yield and diastereomeric excess. Pure (SSSC) diastereomers can be obtained from l -series substrates by crystallisation of the biotransformation extract, and the corresponding (SSRC) 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 (SSSC) and (RSRC) diastereomers can be obtained by crystallisation, and the (SSRC) and (RSSC) diastereomers obtained by chromatography. N-Cbz- and N-t-Boc methionines are also converted to sulfoxides with predominant (SS) 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.

Biotransformation of organic sulfides—VII. A predictive model for sulfoxidation by Helminthosporium species NRRL 4671

Abstract 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.