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