Galman, J.; (2010) Biocatalytic and organocatalytic approaches to ketodiol synthesis. Doctoral thesis , UCL (University College London).

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Abstract

The enzyme transketolase (TK) (EC2.2.1.1) catalyses a reversible asymmetric carboncarbon bond forming reaction, where a two carbon ketose donor is transferred to an aldose acceptor. The use of hydroxypyruvate (HPA), a non-phosphorylated ketol donor with subsequent loss of carbon dioxide renders the reaction irreversible generating the dihydroxyketone product. Several years ago the TK gene from the plasmid of E. coli (BJ502/pKD112A) was incorporated into a high copy plasmid leading to the overexpression of the protein in E. coli, a suitable host for industrial processes. The TK condensation of HPA with glycoladehyde and propanal, in analogous but separate experiments, has been described in the literature. The on-going development of the enzyme TK as a practical biocatalyst performed on a small scale recently led to the discovery of the first biomimetic TK reaction. The aims of the PhD project were principally two-fold; to explore both biomimetic and biocatalytic routes to α,α’-dihydroxyketones. Firstly, investigation of the new one pot synthesis of racemic dihydroxyketones; in terms of substrate scope of the reaction, including aromatic, aliphatic hydrophobic aldehydes as substrates. Donors other than hydroxypyruvate (HPA) were also investigated to assess the general synthetic utility of the reaction. Reaction optimisation studies and preliminary mechanistic studies were performed. In addition, investigations into the development of an asymmetric organocatalytic reaction were pursued. Secondly, libraries of TK mutants were also screened against a range of non-natural substrates. A colorimetric assay for screening active mutants in a 96 well plate format was used to identify sucessfully a number of active mutants with enhanced substrate specificity towards novel cyclic and aromatic acceptor molecules, as well as being used for determining initial rate velocities. A chiral assay as a means to determine the absolute stereochemistry, using chiral derivatising agents (CDAs) was established which was used with HPLC for successful characterisation of the dihydroxyketone motifs. Chapter 1 of this thesis is an introduction to the important role of thiamine dependent enzymes in synthesis, in particular transketolase: the structure, mechanism and its usage in industry is covered. Chemical synthetic routes to ketodiol motifs are also mentioned. Chapter 2 presents the use of a novel chiral assay to determine absolute stereochemistries achieved in biocatalytic or biomimetic reactions. Chapter 3 describes studies of stereoselectivities with active point single TK mutants on cyclic aldehydes. Chapter 4 covers the use of aromatic acceptor substrates with transketolase. Chapter 5 describes investigation into novel asymmetric biomimetic routes to ketodiol synthesis and finally Chapter 6 concludes the studies and encourages further research.

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