Anna de Raadt

Oxidative biotransformations using oxygenases

Considerable progress has been made in manipulating oxidative biotransformations using oxygenases. Substrate acceptance, catalytic activity, regioselectivity and stereoselectivity have been improved significantly by substrate engineering, enzyme engineering or biocatalyst screening. Preparative biotransformations have been carried out to synthesize useful pharmaceutical intermediates or chiral synthons on the gram to several-hundred-gram scale, by use of whole cells of wild type or recombinant strains. The synthetic application of oxygenases in vitro has been shown to be possible by enzymatic or electrochemical regeneration of NADH or NADPH.

Selective transformation of nitriles into amides and carboxylic acids by an immobilized nitrilase

Abstract Using an immobilized nitrilase from Rhodococcus sp. mild and selective hydrolysis of nitriles can be achieved even in the presence of acid or base sensitive groups under neutral conditions. This method is applicable to a broad range of substrates as exemplified by aliphatic, alicyclic, heterocyclic and carbohydrate type nitriles.

The use of substrate engineering in biohydroxylation.

In the biohydroxylation of nonactivated carbon atoms, substrate engineering has been found to be a very useful and simple means to influence substrate acceptance and the regioselectivity and stereoselectivity of this transformation. Recently, this methodology has been applied to the hydroxylation of a large number of compounds including cycloalkane carboxylic acids, ketones, amines, amides and alcohols.

The Concept of Docking and Protecting Groups in Biohydroxylation

The hydroxylation of unactivated carbon atoms employing methods developed in the realms of classical organic chemistry is difficult to achieve and the processes available lack the degree of chemo-, regio- and enantioselectivity required for organic synthesis. To improve this situation, the concept of docking/protecting groups should enable the organic chemist to employ biohydroxylation as an easy tool for preparative work. Similar to the common practice of using protective groups in organic chemistry, a docking/protecting (d/p) group is introduced first, then the biotransformation is performed, and finally the d/p group is removed. The aim of this concept is not only to avoid time consuming microorganism screening methods, but also to improve hydroxylation position predictability, prevent undesired side reactions, aid substrate detection, and product recovery. This approach is successfully applied to carboxylic acids, ketones, aldehydes, and alcohols.

The Concept of Docking/Protecting Groups in Biohydroxylation

A general principle for biohydroxylation, in which time-consuming screening and enrichment techniques are avoided, is demonstrated by the introduction of a docking/protecting group into the substrate. This facilitates acceptance by the microorganism and allows the use of a narrow range of microorganisms, for example Beauveria bassiana ATTC 7159 (B. b.), for the hydroxylation of compounds with diverse structures. After the biohydroxylation, the docking/protecting group is removed (see scheme).

A novel synthetic application of glucose isomerase: Quantitative conversion of D-glucose and L-idofuranose, derivatives modified at position five into the corresponding D-fructo- and L-sorbopyranoses.

Abstract Glucose isomerase (E.C. 5.3.1.5) quantitatively converts a wide range of D -glucose as well as L -idose derivatives modified at position five, such as 5-O-benzyl-, 5-azido-5-deoxy-, 5-deoxy-5-fluoro analogues into the corresponding 2-ketosugars. The reaction works on a preparative scale with isolated yields ranging between 50 and 80%. Products obtained are a new deoxyfluoro sugar, potential non-nutrative as well as intermediates for the synthesis of glycosidase inhibitors and highly functionalized C-glycosides.

Stereoselective hydroxylation of an achiral cyclopentanecarboxylic acid derivative using engineered P450s BM-3

Substrate engineered, achiral carboxylic acid derivative was biohydroxylated with various mutants of cytochrome P450 BM-3 to give two out of the four possible diastereoisomers in high de and ee. The BM-3 mutants exhibit up to 9200 total turnovers for hydroxylation of the engineered substrate, which without the protecting group is not transformed by this enzyme.

Chiral auxiliaries as docking/protecting groups: biohydroxylation of selected ketones with Beauveria bassiana ATCC 7159

Abstract The concept of chiral docking/protecting groups for biohydroxylation was extended from cyclopentanone to other ketones. Reaction of cyclohexanone, (R)-3-methylcyclohexanone, cycloheptanone, 5-methyl-2-hexanone and 4-methyl-2-pentanone with (R)-2-amino-1-propanol and subsequent in situ benzoylation afforded the corresponding N-benzoylated oxazolidine derivatives. All substrates were hydroxylated with the fungus Beauveria bassiana ATCC 7159, one of which was diastereoselectively hydroxylated with a d.e. of 99%. In this manner, access to the corresponding hydroxylated ketones was provided.

Chemoselective enzymatic hydrolysis of aliphatic and alicyclic nitriles

Mild and selective hydrolysis of aliphatic and alicyclic nitriles leading to carboxylic acids and amides was achieved under neutral conditions by an immobilized enzyme preparation from Rhodococcus sp. This method is particularly useful for the transformation of compounds containing other acid- or base-sensitive groups.

Chiral Auxiliaries as Docking/Protecting Groups in Biohydroxylation: The Hydroxylation of Enantiopure Spirooxazolidines Derived from Cyclopentanone UsingBeauveria bassiana ATCC 7159

The aim of this work was to explore the scope and limitations of chiral docking/protecting groups as chiral auxiliaries in the biohydroxylation of unactivated methylene groups. As a model compound, cyclopentanone 1 was reacted with a range of enantiomerically pure amino alcohols 2a−n as well as 7a and b, varying substituents R1 and R2. The resulting chiral spirooxazolidines 3a−n as well as 8a and b were exposed to the fungus Beauveria bassiana ATCC 7159 and the resultant hydroxylated products were characterised. Introducing chirality into the substrate before the fermentation was found to have a major effect on the course of the biohydroxylation relative to the achiral analogue 3a (Table 1, entry 1). The nature of R1/R2 influenced both the product yield and the optical purity of the products (e.g. Table 1, entry 2). In addition, the absolute configuration of the final product 6 could be dictated solely by the nature of the docking/protecting group used (compare entry 8 with entry 9). Concerning the chain length of R1/R2, it was found that hydroxylation only took place in the cyclopentane ring when the heterocyclic ring was substituted with a methyl, ethyl or isopropyl (entries 2−5, 8, 9, 15, and 16). With increasing chain length, where R1/R2 are propyl, isobutyl or sec-butyl groups, a mixture of products was obtained in which the hydroxyl group was either on the cyclopentane ring or on the side-chain (entries 10−14).