Engineered aldolase biocatalysts for the stereoselective synthesis of fluorinated small molecules

The presence of fluorine atoms can have a profound effect on the biological activity of small molecules (Science 2007, 317, 1881), and the wide range of fluorinated drugs include the antidepressant, Prozac, the cholestrol-lowering drug, Lipitor, and the antibiotic, Ciprobay. Fluorination affects nearly all physical and adsorption, distribution, metabolism, and excretion properties of small molecules: for example, bioavailability may be enhanced through the modulation of basicity of amine substituents. The effect of fluorination on fundamental protein-ligand interactions is less well understood, and may stem from direct contact with a target protein, or through stereoelectronic effects on the ligand's conformation. This proposal concerns the application of engineered biocatalysts in the stereoselective synthesis of fluorinated small molecules. The proposal, therefore, clearly falls within the remit of BBSRC, particularly the Biocatalysis aspect of 'Bioengineering for industry and the environment', a theme of the EBS committee. There is a remarkably limited armoury of synthesis methods for controlling the configuration of chiral centres bearing a fluorine atom. Most established methods rely on controlling the stereochemistry of the C-F bond directly, either by stereospecific displacement of an alcohol, or by electrophilic reaction with an activited alkene. These methods generate many by-products. In this project, an entirely different approach will be exploited: the configuration of F-bearing stereocentres will be controlled through the stereoselective construction of a neighbouring C-C bond. The 'atom economic' synthetic approach will yield highly functionalised, chiral products which would be flexible precursors for the synthesis of a wide range of fluorinated heterocycles. The project will exploit engineered aldolase enzymes which will catalyse the attack of fluoropyruvate onto a range of aldehyde substrates. An unsual property of sialic aldolase is that it is able to accept a few donors other than its natural donor (pyruvate): we will therefore exploit aldolases whose ability to accept fluoropyruvate has been optimised by directed evolution. We have previously expanded the range of aldehydes that are accepted by sialic acid aldolse using directed evolution (see: Angew. Chem., Int. Ed. 2005, 44, 2109; Prot. Eng. Des. Sel. 2005, 18, 239). The condensation of fluoropyruvate with an aldehyde inevitably leads to the formation of two new stereogenic centres. There are, therefore, four possible stereoisomeric products which could, in principle, be formed. We have previously used directed evolution to modify, for the first time, the stereochemical course of enzyme-catalysed carbon-carbon bond formation (Proc. Natl. Acad. Sci. USA 2003, 100, 3143). Furthermore, we have created a complementary pair of aldolase enzymes which catalyse the condensation of the same starting materials to yield two different stereoisomeric products (J. Am. Chem. Soc. 2006, 128, 16238). We will extend this approach to the problem of controlling the condensation of fluoropyruvate with aldehydes. The engineered aldolase enzymes may be exploited in the stereoselective synthesis of fluorinated sialic acid derivatives. Such chiral products are valuable precursors of a wide range of fluorinated heterocyclic products. The project will take advantage of the complementary skills of the academic and industrial collaborators. Professor Nelson and Dr Berry have established a powerful collaboration in which directed evolution is used to solve problems in synthetic chemistry. Dr Wells brings huge experience in the exploitation of biocatalysts in the synthesis of functionalised small molecules on an industrial scale. The collaboration will thus yield biocatalytic processes which exploit enzymes whose properties have been optimised for the industrial scale, stereoselective synthesis of chiral fluorinated small molecule building blocks.