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.

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.

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.