Abstract

A group of convenient chiral intermediates for the production of pharmaceuticals are epoxides and ß-substituted alcohols. Environmentally safe preparation of these compounds is preferably done via biocatalysis. However, the required enzymes are often not available. Therefore, we will develop new biocatalysts and explore new biocatalytic strategies for the preparation of these chiral compounds. For this, we will use innovative methods: screening of genomic databases for homologes of known useful enzymes; directed evolution with high-throughput screening for improved enantioselectivity; and genetic construction of biocatalysts in which different conversion steps are integrated. We aim to develop new synthetic routes exploiting novel concepts of enantiocomplementarity and enantioconvergence, thus minimizing the cost of resources and the production of waste.

Description of the work

• We will identify, by genomic database analysis, and over-express new useful enzymes for biocatalysis (monooxygenases, epoxide hydrolases, and halohydrin lyases), using suitable host organisms for optimised enzyme production and for obtaining integrated biocatalysts in which consecutive steps can be carried out without the need for intermediate product isolation.
• New epoxide-converting enzymes will be isolated and characterised, including establishing the substrate range, the substrate-conversion kinetics, product effects, and the kinetic and catalytic mechanisms.
• The properties of epoxide-producing and -metabolizing enzymes will be improved by directed evolution and site-directed mutagenesis. Directed evolution will use semi-random banks of mutant genes and novel high throughput MS methods for screening for improved enzyme enantioselectivity.
• The biocatalytic scope and performance of the engineered biocatalysts will be established (regio- and enantioselectivity, range of conversions, scale-up issues, performance under operating conditions).
• We will develop new synthetic strategies that make use of combinations of enzymes to obtain enantioconvergent and enantiocomplementary conversion, thus minimising waste production and use of resources. The target compounds will be epoxides and related compounds of pharmaceutical interest. A toolbox of enzymes will be generated for use in the pharmaceutical industry.

• The availability of a toolbox of 5-10 engineered biocatalysts that can be used for the preparation of enantiopure epoxides and related compounds. The kits should be suitable for testing by industrial partners and for commercial development (enzyme kits).
• New biocatalytic reactions that use these improved enzymes and engineered cells, including a description of their substrate range, kinetic properties, enantioselectivity, conditions for use, and application range.
• A description of useful strategies for the enantioconvergent synthesis of enantiopure pharmaceuticals, starting from racemic mixtures of epoxides, haloalcohols, and related cheap substrates.

Contacts

Coordinator

• D.B. JANSSEN / University of Groningen Groningen Biomolecular Sciences and / NETHERLANDS

Participant

• R. WOHLGEMUTH / Fluka GmbH Biochemistry Department / SWITZERLAND
• David J. LEAK / Imperial College of Science Technology and Medicine / UNITED KINGDOM
• M.T. REETZ / Max-Planck Institut für Kohlenforschung GERMANY
• M. ARAND / University of Mainz Institute of Toxicology / GERMANY
• R. FURSTOSS / Université de la Méditerranée Faculté des Sciences de Luminy / FRANCE