On the mechanism of Dehydroquinate synthase : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Chemistry

The aim of this thesis is to investigate the influence of fluorine substitution on the second reaction of the shikimate pathway catalysed by the enzyme 3-dehydroquinate synthase. The shikimate pathway is an essential pathway that is required for the synthesis of aromatic compounds in bacteria, microbial eukaryotes and plants. The enzyme, 3-dehydroquinate synthase, catalyses the second step of the shikimate pathway, the conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) into 3-dehydroquinate (DHQ). Item_03771-1.jpg It has been reported that when (3S)-3-fluoro DAHP (where fluorine occupies the C3 axial position) is treated with the enzyme 3-dehydroquinate synthase, two products, the expected (6S)-6-fluorodehydroquinate (5) and its C1 epimer, (6S)-6-fluoro-1-epideydroquinate (6) are formed in a ratio of 2 : 1. Item_03771-2.jpg The C1 epimer of 3-dehydroquinate was reported to be formed from the natural substrate DAHP in a solution reaction, but not in the enzyme catalysed reaction. Therefore, it has been suggested that fluorine substitution at the axial position on C3 stabilises the fluoroenolpyranose intermediate allowing the intermediate to dissociate from the enzyme and cyclise to complete the formation of (6S)-6-fluoro-1-epideydroquinate free in solution. The results reported in this thesis are from an investigation carried out to understand further the influence of fluorine orientation on the stereochemical outcome of the products in the dehydroquinate synthase reaction. (3S)-3-Fluoro DAHP was synthesised in large amounts using both chemical and enzymatic synthesis. This was achieved by treating the isomers of 3-fluoro phosphoenolpyruvate and D-erythrose 4-phosphate with DAHP synthase, the first enzyme of the shikimate pathway. The erythrose 4-phosphate was prepared by lead tetraacetate oxidation of D-glucose 6-phosphate. The isomers of 3-fluoro phosphoenolpyruvate were prepared from 3-bromo, 3-fluoropyruvic acid by the Perkow reaction. Then (3S)-3-3-fluoro DAHP was purified by anion exchange chromatography. The chemical synthesis of erythrose 4-phosphate and the isomers of 3-fluoro phosphoenolpyruvate and the enzymatic synthesis of (3S)-3- fluoro DAHP and its purification are discussed in Chapter Two. A recombinant Escherichia coli strain (pJB 14) was used to over-express the enzyme dehydroquinate synthase, and partial purification of the enzyme was achieved by anion exchange chromatography. Chapter Three describes the production and purification of the enzyme 3-dehydroquinate synthase. Purified (3S)-3-fluoru DAHP was treated with the E. coli enzyme 3-dehydroquinate synthase. Formation of both (6S)-6-fluorodehydroquinate and its C1 epimer was observed. The reaction was followed at different pH and temperature values. The ratio of products produced in the enzyme-catalysed reaction was monitored by 19 F NMR spectroscopy. No significant change in the ratios was observed with the different conditions employed. The results from these experiments are discussed in Chapter Four. Our results are consistent with the hypothesis that the fluoroenolpyranose intermediate is released to the solution, where it cyclises without the constraint of an enzymatic template. To test this hypothesis unequivocally, further investigations are required and these are discussed in Future Directions. [NB: Mathematical/chemical formulae or equations have been omitted from the abstract due to website limitations. Please read the full text PDF file for a complete abstract.

Two-Dimensional FTIR as a Tool to Study the Chemical Interactions within Cellulose-Ionic Liquid Solutions

In this study two-dimensional FTIR analysis was applied to understand the temperature effects on processing cellulose solutions in imidazolium-based ionic liquids. Analysis of the imidazolium ion νC2–H peak revealed hydrogen bonding within cellulose solutions to be dynamic on heating and cooling. The extent of hydrogen bonding was stronger on heating, consistent with greater ion mobility at higher temperature when the ionic liquid network structure is broken. At ambient temperatures a blue shifted νC2–H peak was indicative of greater cation-anion interactions, consistent with the ionic liquid network structure. Both cellulose and water further impact the extent of hydrogen bonding in these solutions. The FTIR spectral changes appeared gradual with temperature and contrast shear induced rheology changes which were observed on heating above 70°C and cooling below 40°C. The influence of cellulose on solution viscosity was not distinguished on initial heating as the ionic liquid network structure dominates rheology behaviour. On cooling, the quantity of cellulose has a greater influence on solution rheology. Outcomes suggest processing cellulose in ionic liquids above 40°C and to reduce the impacts of cation-anion effects and enhance solubilisation, processing should be done at 70°C

Generation of Spherical Cellulose Nanoparticles from Ionic Liquid Processing via Novel Nonsolvent Addition and Drying

A novel method to prepare spherical cellulose nanoparticles has been developed using imidazolium ionic liquid processing and regeneration from controlled acetonitrile nonsolvent addition and drying. Nanoparticles ranging from 100 to 400 nm have been prepared with high uniformity. Minimisation of moisture via solvent exchange drying led to discrete nanoparticles, whereas the presence of ambient moisture during regeneration contributed to aggregated morphologies. Chemical analyses of the spherical cellulose nanoparticles reveal a high-amorphous cellulose content. Furthermore, the range of particle sizes achieved with acetonitrile nonsolvent fractionation and solvent exchange drying suggest the size and uniformity of nanoparticle distributions reflect the fractionated cellulose weight fractions. This ionic liquid method is simple, energy efficient, and likely to have wide applicability across other biopolymers as well as potential to prepare surface functionalized spherical cellulose nanoparticles