The Escherichia coli glucuronylsynthase is an engineered
glycosynthase enzyme derived from wild-type E. coli
β-glucuronidase where a single-point mutation has changed the
function of the enzyme from a hydrolase to a synthase. The
glucuronylsynthase catalyses the formation of a glycosidic bond
between a glucopyranuronic acid moiety and an alcohol acceptor to
yield a glucuronide compound. Glucuronides are an important class
of compounds in pharmacology and toxicology, in medicinal
chemistry and in sports anti-doping. Previous work on the
glucuronylsynthase elucidated conditions for the synthesis of
some simple glucuronides and provided possibilities for further
improvement.
This thesis covers work undertaken to further develop and
understand the glucuronylsynthase enzyme as a viable synthetic
protocol, especially in the context of steroid glucuronide
synthesis for anti-doping purposes. This was approached from
three different routes, these being the synthesis of a range of
steroid glucuronides, the synthesis of an 18O-labelled steroid
glucuronide as an alternative to current labelling protocols, and
an investigation into the Michaelis-Menten kinetics of the
glucuronylsynthase to determine the effect of tert-butanol on the
enzyme. An additional study into the stability of the
glucopyranuronyl donor under the glucuronylsynthase conditions
was performed to resolve an unanswered question from previous
work where additional equivalents of sugar appeared to be
required for good yields of glucuronide product.
A set of revised conditions for the synthesis of steroid
glucuronides using the glucuronylsynthase was developed using
tert-butanol as a co-solvent for improved reaction rates and
solubility of the steroid substrates. A rapid purification method
using weak-anion exchange (WAX) solid-phase extraction (SPE) was
developed for the direct purification of crude glucuronylsynthase
reactions. A screen of a library of steroid substrates was
performed and the conversions quantified by 1H NMR integration of
the relevant protons to provide a clearer understanding of how
well the glucuronylsynthase is able to process such steroid
substrates. Some non-steroidal glucuronides were also prepared,
hinting at a broader substrate scope beyond steroids and simple
alcohols.
To achieve the second goal a new synthesis of the α-D-glucuronyl
fluoride 29 was developed excluding ordinary water for maximum
incorporation of 18O-labelled water. This sugar was then used to
synthesise a labelled steroid glucuronide with characterisation
performed by 1H NMR and mass spectrometry.
The Michaelis-Menten kinetics of the glucuronylsynthase were
investigated using dehydroepiandrosterone O-(carboxymethyl)oxime
33 as the steroid substrate of choice due to its improved aqueous
solubility and the variables of tert-butanol concentration and
temperature were investigated to uncover the effect of
tert-butanol on the glucuronylsynthase.
The hydrolysis of the α-D-glucuronyl fluoride 29 was studied
using a fluoride-selective electrode for higher sensitivity over
previous analytical methods such as a zirconium-based
fluorescence quenching approach. The initial rates of general
base-catalysed hydrolysis in various buffers at 37 °C including
with and without tert-butanol were measured and the corresponding
first-order rate constants for hydrolysis were calculated.
The advancement of the Escherichia coli glucuronylsynthase
protocol was thus carried out on multiple fronts and this thesis
describes such efforts from synthetic and analytical points of
view
