Advancing the Escherichia coli Glucuronylsynthase: Kinetics and Synthetic Applications
Creators: name: Ma, Paul
Publisher: name: The Australian National University
Year: 2016
Subjects: Glucuronylsynthase
glycosynthase
glucuronide
solid-phase extraction
enzyme kinetics
Michaelis-Menten

description: 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.